cationic polymer nanoparticles and nanogels: from synthesis to biotechnological applications

Cationic Polymer Nanoparticles and Nanogels: From Synthesis toBiotechnological ApplicationsJose Ramos,† Jacqueline Forcada,*,† and Roque Hidalgo-Alvarez*,‡

†POLYMAT, Bionanoparticles Group, Departamento de Química Aplicada, UFI 11/56, Facultad de Ciencias Químicas, Universidaddel País Vasco UPV/EHU, Apdo. 1072, 20080 Donostia-San Sebastian, Spain‡Grupo de Física de Fluidos y Biocoloides, Departamento de Física Aplicada, Facultad de Ciencias, Universidad de Granada, 18071Granada, Spain

CONTENTS

1. Introduction A2. Designing the Cationic System C

2.1. Cationic Monomers and Polymers C2.2. Cationic Initiators D2.3. Cationic Surfactants D

3. Cationic Latexes E3.1. Synthesis Strategies To Produce Cationic

Latexes F3.1.1. Unseeded Emulsion Polymerization Pro-

cesses F3.1.2. Seeded Emulsion Polymerization Pro-

cesses P3.1.3. Other Polymerization Processes in Dis-

persed Media T3.2. Characterization of Cationic Latexes T3.3. Applications of Cationic Latexes V

3.3.1. Adsorption of Proteins on CationicLatexes V

3.3.2. Latex Immunoassay Aggregation Z3.3.3. Adsorption of Polyelectrolytes and

Surfactants on Cationic Latexes AB3.3.4. Heteroaggregation of Colloidal Disper-

sions AG3.3.5. Colloidal Monolayer Formed by Cationic

Latex Particles at the Air−Water Inter-face AL

3.3.6. Deposition of Cationic Latexes AL3.3.7. Cationic Latexes as Catalyst Supports AN3.3.8. Film Formation with Cationic Latexes AN

4. Cationic Micro/Nanogels AO4.1. Strategies To Produce Cationic Micro/Nano-

gels, Characterizations, and Applications AP

4.1.1. Conventional Production of Micro/Nanogels AP

4.1.2. Nonconventional Production of Micro/Nanogels AV

5. Conclusions and Future Perspectives BAAuthor Information BC

Corresponding Authors BCNotes BCBiographies BC

Acknowledgments BCDedication BDReferences BD

1. INTRODUCTION

In the past several decades, aqueous polymeric dispersionsprepared by means of polymerization processes in dispersedmedia to produce polymeric particles having diameters in thecolloidal range, have garnered increasing interest from bothacademic and industrial points of view.1 These nanoparticlesare used in a large variety of applications, e.g., adhesives, water-based coatings, textile, paper, additives, and flocculants. Theyare also suitable for use as fine or highly added value polymericmaterials for medical diagnostic tests, antibody purifications,drug delivery systems, and material for calibrations.Monodisperse polymer colloids have proved to be very useful

model systems for studying various colloidal phenomena anddeveloping different technological applications. Most of theexperiments reported have been performed with negativelycharged particles, and relatively little attention has been paid topositively charged samples. It is usual in the literature ofpolymer colloids to use the expression “cationic latexes” todesignate the cationic polymeric particles. In this review thesynthesis of cationic polymer particles and nanogels byemulsion polymerization will be comprehensively revised. Anin-depth study on the kinetics of cationic systems will bedetailed and compared with that of well-known anionicsystems. Then polymeric and colloidal features of the cationicparticles/nanogels will be revised, and finally, some biotechno-logical applications of cationic particles/nanogels will bedescribed in detail.Nowadays, in the field of cancer therapies, the idea of a

carrier going to the specific target, passively or actively, isabsolutely critical for the effective drug doses to reach the

Received: July 2, 2012

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pathological region of interest without damaging the surround-ing health cells or tissues. This is the idea of the “magic bullet”.2

This could consist of a delivery platform with nanometric sizeable to be specifically targeted to the tumor tissue, avoidingpremature fragmentation and degradation3 and helping thetransfer of a more concentrated drug load through the cellularmembrane. This integral system could demonstrate a controlleddelivery by activation by means of one or more stimuli such astemperature, pH, light, etc. It should be taken into account that,to release concentrated drug loads in nonhealthy tissues, it isnecessary to have a robust delivery platform with the ability tocontrol the release of the drug in a precise way with previousactivation by an external stimulus. Another aspect to take intoaccount is that particle sizes and their functional groups have ahigh impact on biodistribution and pharmacokinetics. As anexample, nanoparticles with positive charge have a higherremoval velocity than those with negative charge, and withrespect to the size, particles with sizes between 100 and 200nm, if unprotected, are quickly removed from the bloodstreamby the mononuclear phagocyte system (MPS).4 However, theaddition of biocompatible polymers such as polyethylene glycol(PEG) onto their surfaces (in the case of hard nanoparticles)avoids the actuation of the phagocytes as a result of the stericinterferences provoked by the PEG chains on the nanoparticlesurface and increases considerably the average circulation life inblood (from 1/2 to 5 h). The liver and spleen trap particlespoorly or in a less covered manner. This type of surface-modified nanoparticle can be considered as stealth for theimmune system, and it is used to help in diminishing theremoval velocity, inhibiting opsonization (labeling of nano-particles with opsonin proteins which the macrophagerecognizes, rejecting the foreign body).5 A longer circulationtime, and therefore an increase in bioavailability, is veryimportant because most of the nanocarriers operate bytargeting tumors passively and taking advantage of theirpermeable vascularization and poor lymphatic drainage as aresult of the rapid and active tumor angiogenesis. Thisphenomenon allows the nanocarriers to cross the endothelialbarrier and accumulate in tumor tissues while they leave thesurrounding healthy tissue intact.6

Cationic vectors facilitate cellular uptake. This has beenproven in the case of the use of liposomes and cationic micellesin gene therapy. The use of cationic nanocarriers will facilitateendocytosis and allow loads that are nonpermeable to the cellmembrane, such as hydrophobic drugs or DNA molecules, tobe transported and released from the endosomes to travel(endosomal escape) to the chosen place.Nowadays, polymer particles and nanogels with cationic

charge are being used in emerging biomedical technologies dueto the strong interaction between DNA and cationic polymercolloids, the acid-swellable behavior of the nanoparticle/nanogel, and the ability to form oriented bonds with proteins,among other aspects.On one hand, polycation−DNA complexes are called

polyplexes. These polyplexes are very useful in gene therapyto improve the delivery of the new DNA into the cell. DNAmust be protected from damage such as its rapid enzymaticdegradation in serum conditions, and its entry into the cellmust be facilitated. To this end, new polyplexes have the abilityto protect DNA from undesirable degradation during thetransfection process. On the other hand, tumor tissues exhibit alower extracellular pH than normal tissues together with aslightly higher temperature.

Drug delivery systems have been designed to use pH as amechanism to improve delivery of chemotherapeutics.Improved delivery mechanisms may reduce side effects andincrease the quality of life of patients. Synthesized polymericdelivery vehicles based on particles/nanogels from conventionalanionic pH-sensitive polymers exhibit swelling behavior at highpH. This mechanism is not useful for delivery to the acidicenvironments present in tumor tissues. Thus, the drug deliveryvehicles must have a reverse acid swelling behavior, which isachieved with cationic monomers.The monodispersity of the particles/nanogels is one of the

main requirements from the point of view of theirbiotechnological applications. For the objective of findingsuitable experimental conditions for obtaining particles of agiven size, having a narrow particle size distribution (PSD), thetype of polymerization process is important. The most suitableprocesses found in the literature to obtain polymeric colloidalparticles are those related to heterophase polymerizationprocesses, mainly emulsion polymerization (seeded or un-seeded, batch or semicontinuous). To the best of ourknowledge,7 the emulsion polymerization technique is themost efficient and profitable polymerization method to producepolymeric nanoparticles (latex particles). As a result of thecompartmentalization of the polymerization reaction in thenanoparticles dispersed in the continuous aqueous medium,reaction times are short and high conversions of monomer topolymer are obtained, and consequently, high reaction rates,good heat transfer (the continuous medium is water), and thepossibility of controlling both particle size distributions (andtherefore their monodispersity) and, if necessary, molecularweight distributions, comonomer compositions, and surfacefunctionalization are achieved. Apart from these characteristicsbecause it is a polymerization process in dispersed media, inemulsion polymerization nucleation processes, latex particlesare formed and the monomer(s) needed for their growth istransported through the continuous phase. Another advantagein terms of versatility is that the monomer(s) can be fedcontinuously into the reactor either as a neat monomer or as anemulsion and the monomer to water ratio can be adjusted toobtain the desired solids content.Applications of latex particles made by emulsion polymer-

ization in the biomedical field were concentrated initially in thearea of in vitro immunoassays. Polymer particles have beenextensively used in this field, starting in 1956 with thedevelopment of the latex agglutination test.8 After this, asignificant number of applications of polymer particles in thebiomedical field emerged in a combination of new biotechno-logical developments.Polymeric colloids synthesized by emulsion polymerization

are currently finding new applications in the biomedical field.By controlling the experimental conditions of the emulsionpolymerization process, one can obtain colloidal systems withparticle sizes, monodispersities, and specific surface character-istics required for their use in biomedical applications.Moreover, colloidal systems comprise small polymeric particlessuspended in aqueous medium, having a high surface area. Forall these reasons, polymeric particles are being used as carriersof biomolecules, such as proteins, enzymes, etc. Among thevariety of applications of supported biomolecules on the surfaceof polymeric particles, there are immunodiagnosis tests,labeling, identification, quantification, and separation of cells,and drug delivery systems.9

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2. DESIGNING THE CATIONIC SYSTEMCationically charged nanoparticles and nanogels can beprepared by several polymerization processes, but alwaysusing cationic reagents able to be (i) covalently bonded to ananoparticle or nanogel (monomers, polymers, and initiators)or (ii) physically adsorbed onto the nanoparticle surface(surfactants). It is impossible to revise, and even to mention,the huge amount of cationic compounds commerciallyavailable. However, the design of a cationic system with theright choice of cationic reagents is of paramount importancefrom all points of view (from synthesis to final application).Therefore, in this review the most remarkable cationiccompounds, used in several polymerization processes describedbelow, are classified as cationic monomers and polymers,cationic initiators, and cationic surfactants.2.1. Cationic Monomers and Polymers

Scheme 1 shows the main cationic monomers used in thesynthesis of cationic colloids by emulsion polymerization. Ascan be seen, different families of cationic monomers can beused depending on the type of cationic charge required. On onehand, cationic nanoparticles with pH-dependent surface chargedensities are obtained if neutral forms of vinylpyridines [2-vinylpyridine (2VP) or 4-vinylpyridine (4VP)] and(dialkylamino)ethyl methacrylates [2-(dimethylamino)ethylmethacrylate (DMAEMA) or 2-(diethylamino)ethyl methacry-late (DEAEMA)] are used. On the other hand, cationicnanoparticles with constant surface charge densities areobtained when quaternary ammonium cationic monomers areused. These cationic monomers are obtained by quarternizationof neutral amine-containing monomers:

(i) Quaternization of vinylpyridines such as 1-methyl-4-vinylpyridinium bromide (MVPC), 1-methyl-4-vinyl-pyridinium iodine (MVPI), 1,2-dimethyl-5-vinylpyridi-nium methyl sulfate (DMVP), and 1-ethyl-2-methyl-5-vinylpyridinium bromide (EMVP).

(ii) Quaternization of (dialkylamino)ethyl methacrylatessuch as [2-(methacryloyloxy)ethyl]trimethylammoniumchloride (MATMAC), [2-(methacryloyloxy)ethyl]-trimethylammonium iodine (MATMAI), N,N-dimethyl-N-butyl-N-ethyl methacrylate ammonium bromide(DMBEMAB), and N ,N -d imethyl -N -buty l -N -(methacrylamidinopropyl)ammonium bromide(DMBMAPAB)

(iii) Quaternization of other amine-containing monomerssuch as (vinylbenzyl)trimethylammonium chloride(VBTMAC), [3-(methacry loylamino)propyl] -trimethylammonium chloride (MAPTMAC), and dia-llyldimethylammonium chloride (DADMAC).

Other cationic monomers used in emulsion polymerizationare (vinylbenzyl)isothiouronium chloride (VBIC) and (4-vinylbenzyl)hydrazine (VBH). Special attention is focused onprimary amino-functionalized monomers such as vinylbenzyl-amine hydrochloride (VBAH) and aminoethyl methacrylatehydrochloride (AEMH) because a primary amino functionalitycan react directly with a high variety of biomolecules, bindingthem onto the surface of cationic nanoparticles.The choice of an adequate cationic monomer will depend on

the polymerization process used and the final application of thecationic system. As a general rule, the increase in the cationicmonomer concentration leads to a faster polymerization rate,higher polymerization conversions, a smaller particle size, and

Scheme 1. Cationic Monomersa

aVinylpyridines and their quaternary ammonium salts: 2VP = 2-vinylpyridine; 4VP = 4-vinylpyridine; MVPC = 1-methyl-4-vinyl-pyridinium chloride; MVPI = 1-methyl-4-vinylpyridinium iodine;DMVP = 1,2-dimethyl-5-vinylpyridinium methyl sulfate; EMVP = 1-ethyl-2-methyl-5-vinylpyridinium bromide. (Dialkylamino)ethyl meth-acrylates: DMAEMA = 2-(dimethylamino)ethyl methacrylate; DEAE-MA = 2-(diethylamino)ethyl methacrylate. Quaternary ammoniumcationic monomers (QACMs): VBTMAC = vinylbenzyl trimethylam-monium chloride; MATMAC = [2-(methacryloyloxy)ethyl] trimethy-lammonium chloride; MATMAI = [2-(methacryloyloxy) ethyl]trimethylammonium iodine; MAPTMAC = [3-(methacryloyl-amino)propyl] trimethylammonium chloride; DADMAC = diallyldimethy-


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higher water-soluble polymer (polyelectrolyte) formation in abatch emulsion polymerization process. To favor surfaceincorporation of a cationic monomer, reducing the formationof polyelectrolytes, other polymerization processes such assemicontinuous, seeded, or seeded shot-growth can be used.10

Related to monomer reactivity, differences will be given by thetype of reactive double bond of the cationic monomer.However, it can be said without doubt that the pH of thereaction medium will be the key parameter for success of thepolymerization process and the stability of the nanoparticlesnucleated.To much less extent than cationic monomers, cationic

polymers can also be used in the synthesis of cationic colloidsby emulsion polymerization. Scheme 2 shows the main cationic

polymers used. As can be seen, all the cationic polymersdepicted are primary amino-functionalized polymers: poly-ethylenimine (PEI), poly(allylamine) (PAAm), and poly-(vinylamine) (PVAm).2.2. Cationic Initiators

Scheme 3 shows the cationic azo compounds, which can beused as thermal initiators in the synthesis of cationic polymericcolloids by emulsion polymerization. As can be seen, the mostcommon cationic initiators are the family of azobisami-dines:11,12 2,2′-azobisisobutyramidine dihydrochloride or 2,2′-azobis(2-methylpropionamidine) dihydrochloride (AIBA or V-50), 2,2′-azobis(N,N′-dimethyleneisobutyramidine) dihydro-chloride or 2,2′-azobis[2-(2-imidazolin-2-yl)propane] dihydro-chloride (ADIBA or VA-044), 2,2′-dimethyl-2,2′-azobis(N-benzylpropionamidine) dihydrochloride (VA-552), 2,2′-azobis-(1-imino-1-pyrrolidino-2-ethylpropane) dihydrochloride (VA-067), and 2,2′-azobis[2-[1-(2-hydroxyethyl)-2-imidazolin-2-yl]-propane] dihydrochloride (VA-060). Amidines are monoacidstrong bases which hydrolyze in the presence of both acid andbase catalysis.13 The hydrolysis of amidines is a two-stepreaction. The first step is the most rapid and results in theformation of an amine (ammonia form unsubstitued amidines)and an amide. The second step is the hydrolysis of the amide toa carboxylic acid, and since amides are generally stable, thesecond step is slow. However, the sensitivity to hydrolysis isvery dependent on the substituents. AIBA is the most commoncationic initiator used in the synthesis of cationic nanoparticles

and nanogels, followed by ADIBA. However, the otherbisamidines (VA-552, VA-067, and VA-060) have rarely beenused. AIBA initiator contains primary amidines, while ADIBAcontains cyclic disubstituted amidines. This difference inchemical structure makes ADIBA, which is the cyclic analogueof AIBA, more hydrolytically stable; no hydrolysis productswere observed during its homolytic decomposition.14 However,as can be observed in Scheme 4, during the homolytic processof AIBA, hydrolysis of the primary amidine groups takes place,giving amide products such as 2,2′-azo-2-carbamyl-2′-amidino-bispropane hydrochloride (ACAP) and 2,2′-azobis(2-carbamyl-propane) (ACP). The amide products essentially do notundergo homolysis under the conditions that AIBA does. Thestudy of the competitive rates of hydrolysis and thermaldecomposition of AIBA in aqueous solution was published byIto15 in 1973 and by Wahl et al.14 in 1998. They bothconcluded that the steady-state concentration of radicalsformed by AIBA is relatively low because hydrolysis productsare stable toward decomposition and do not contribute toradical formation. Furthermore, during the course of anemulsion polymerization using AIBA, the amidine hydro-chloride function is partitioned between undissociated initiator,free radicals in water solution, byproducts from couplingreactions, and polymer/oligomer chain ends. With respect tothe hydrolysis process, the first and the last of these sites(undissociated initiator and polymer/oligomer chain ends) arethe most important, so it is on the polymer/oligomer chainends where the hydrolysis affects the colloidal stability of theparticles formed. The pH is the parameter which controls therelationship between the homolysis and the hydrolysisprocesses in the AIBA initiator. Increasing the pH increasesthe hydrolysis of the primary amidines because the hydroxyl ionacts as a catalyst. Conversely, the homolysis of AIBA decreases.At 60 °C Ito15 found that the ratio kh′/kd for AIBA goes from17 at pH 10 to 1.11 at pH 7 and that hydrolysis at lower pH ismuch less important than homolysis. For that reason, anemulsion polymerization using AIBA as the cationic initiatormust be carried out at pH lower than 7 to minimize thehydrolysis process.On the other hand, cationic initiators containing quaternized

nitrogens, such as N,N′-dimethyl-4,4′-azobis(4-cyano-1-meth-ylpiperidine) dinitrate (DACMP), have been used as analternative to cationic bisamidines.16

2.3. Cationic Surfactants

Scheme 5 shows the main cationic surfactants used in thesynthesis of cationic polymer colloids by emulsion polymer-ization. The most common cationic surfactants used arequaternary ammonium salts such as hexadecyltrimethylammo-nium bromide or cetyltrimethylammonium bromide (HDTABor CTAB), cetyltrimethylammonium chloride (CTAC),dodecyltrimethylammonium bromide (DTAB), and tetradecyl-dimethylbenzylammonium chloride (TBAC). However, in afew studies other types of cationic surfactants can be found,such as dodecylpyridinium chloride (DPC), octadecylpyridi-nium chloride (OPC), and Hyamine 1622 (HY). One of themost important roles that a surfactant has during the synthesisof latex particles by means of a conventional emulsionpolymerization process is helping the solubilization of themonomer in the aqueous phase in the form of swollen micelles,increasing in this way the availability of the monomer in thecontinuous phase. Another function is to stabilize the newparticles as they are formed. By increasing the amount of

Scheme 1. continued

lammonium chloride; DMBEMAB = N,N-dimethyl-N-butyl-N-ethylmethacrylate ammonium bromide; DMBMAPAB = N,N-dimethyl-N-butyl- N-methacrylamidino propyl ammonium bromide. Othercationic monomers: VBIC = vinylbenzyl isothiouronium chloride;VBH = 4-vinylbenzyl hydrazine. Primary amino-functionalizedmonomers: VBAH = vinylbenzylamine hydrochloride; AEMH =aminoethyl methacrylate hycrochloride.

Scheme 2. Cationic Synthetic Polymersa

aPEI = polyethylenimine; PAAm = poly(allylamine); PVAm =poly(vinylamine).


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surfactant, the colloidal stability of the particles increases andcoagulation decreases. The surfactant also has a stronginfluence on the particle size. It is well-known that both thetype and amount of surfactant determine the nucleation stageof an emulsion polymerization. When the amount of surfactantis below its critical micellar concentration (CMC), thesurfactant molecules are preferably in the aqueous phase ratherthan forming micelles, and in this way, only homogeneousnucleation takes place. On the other hand, when the amount ofemulsifier is above its CMC, the surfactant forms micelles andis also present in the aqueous phase. In this case, micellarnucleation is the most important mechanism that takes place inan emulsion polymerization when slightly water-solublemonomers, such as styrene, are polymerized, and the

concentration or number of micelles determines the numberof particles formed throughout interval I of the emulsionpolymerization.17 The choice of an adequate cationic surfactantwill be critical in synthesizing colloidally stable nanoparticles.

3. CATIONIC LATEXES

Up to now the main and fundamental research in emulsionpolymerization has been based on anionic systems, because inalmost all the applications negatively charged particles arerequired. However, during the past decade the mechanismsgoverning cationic emulsion polymerization have been ex-plored. It was found that the knowledge of well-studied anionicsystems could not be extrapolated to cationic systems. In ourwork,18−20 the batch cationic emulsion polymerization of

Scheme 3. Cationic Radical Initiatorsa

aAIBA or V-50 = 2,2′-azobisisobutyramidine dihydrochloride or 2,2′-azobis(2-methylpropionamidine) dihydrochloride; ADIBA or VA-044 = 2,2′-azobis(N,N′-dimethyleneisobutyramidine) dihydrochloride or 2,2′-azobis[2-(2-imidazolin-2-yl)propane] dihydrochloride; VA-552 = 2,2′-dimethyl-2,2′-azobis(N-benzylpropionamidine) dihydrochloride; VA-067 = 2,2′-azobis(1-imino-1-pyrrolidino-2-ethylpropane) dihydrochloride; VA-060 =2,2′-azobis[2-[1-(2-hydroxyethyl)-2-imidazolin-2-yl]propane] dihydrochloride; DACMP = N,N′-dimethyl-4,4′-azobis(4-cyano-1-methylpiperidine)dinitrate.

Scheme 4. Homolysis and Hydrolysis of AIBA Initiatora

aAIBA = 2,2′-azobisisobutyramidine dihydrochloride; ACAP = 2,2′-azo-2-carbamyl-2′-amidinobispropane hydrochloride; ACP = 2,2′-azobis(2-carbamylpropane).


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styrene was compared with the anionic one. The maindifference found was that, under the experimental conditionsstudied, the kinetics of cationic systems were affected by theparticle size, while in the anionic system, due to the lowerparticle size and lower initiation rates, the rate of polymer-ization was not dependent on the volume of the latex particles.Furthermore, in the cationic systems a dependence on theparticle size of the rate of polymerization per particle togetherwith the average number of radicals per particle was found.These differences were explained taking into account thelimited particle coagulation observed with cationic surfactantsand the high rate of radical formation of cationic initiators.

3.1. Synthesis Strategies To Produce Cationic Latexes

Cationic nanoparticles are obtained by using a combination ofseveral cationic reagents such as cationic initiators, cationicmonomers, cationic polymers, and/or cationic surfactants. Toproduce cationic nanoparticles, the most suitable processesfound in the literature are those related to heterophasepolymerization processes, mainly emulsion polymerization.This technique is observed as the most efficient and profitablepolymerization process to produce polymeric nanoparticles.3.1.1. Unseeded Emulsion Polymerization Processes.

As commented in the Introduction, among the various types ofheterophase polymerization techniques, there is emulsionpolymerization, which is the most relevant to produce polymernanoparticles or latexes in the colloidal range.One type of emulsion polymerization process is the

unseeded or ab initio emulsion polymerization process, whichis divided into three intervals encompassing the particleformation stage, called “interval I” according to Harkins’s

theory,21,22 and particle growth stages, intervals II and III. Inthis kind of polymerization process, the particle nucleationperiod is short, thereby giving rise to distinct particle formationand growth periods. However, if the initiator concentration islow and the surfactant concentration is high, it is possible forparticle nucleation and particle growth to proceed simulta-neously for a significant period of the polymerization.In ab initio polymerizations, the particle nucleation stage

often is a source of batch-to-batch variability. The limitedcontrol that can be exerted over polymer and latex propertiesgreatly restricts the commercial utility of unseeded processes.Nevertheless, ab initio processes are of great importance tostudy and compare nucleation (interval I) and growth stages(intervals II and III) using different amounts and types ofmonomers, initiators, and surfactants.

3.1.1.1. Conventional Emulsion Polymerization Processes.The birth of emulsion polymerization can be dated to 1909,when the idea of mimicking the conditions used by MotherNature to synthesize the natural latex of Hevea brasiliensis andGuyanensis rubber trees was attempted to improve theproperties of synthetic rubbers produced by bulk polymer-ization.23 In 1912, a patented idea of using an aqueousemulsion of a monomer to carry out polymerization appearedfor the first time.24 This was the birth of heterophasepolymerization. For the next 20 years, the importance ofemulsion polymerization increased due to the activities ofcompanies in Germany and the United States and subsequentsupport and sponsoring by both governments.25 During theseyears a large number of patents accumulated, but in the sameperiod (1930−1940) only very few papers were published inscientific journals. While the mechanism of emulsion polymer-

Scheme 5. Cationic Surfactantsa

aHDTAB or CTAB = hexadecyltrimethylammonium bromide or cetyltrimethylammonium bromide; CTAC = cetyltrimethylammonium chloride;DTAB = dodecyltrimethylammonium bromide; TBAC = tetradecyldimethylbenzylammonium chloride; DPC = dodecylpyridinium chloride; OPC =octadecylpyridinium chloride; HY = Hyamine 1622.


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ization was briefly discussed in the scientific literature duringthis period, the large number of patents filed in many countriestake into account the extensive work which was carried outduring the same period in the research departments of variousindustrial companies.26 In 1945, Hohenstein et al.27 published apaper on the polymerization of styrene in agitated soapemulsions.In 1947, a group from Dow Chemical Corp. reported for the

first time the synthesis of monodisperse polystyrene latexes.28

The same year, Harkins22 developed a general and qualitativedescription of emulsion polymerization having two mainfeatures: there are two loci for particle formation or nucleation,the monomer-swollen micelles and the aqueous phase, and themonomer-swollen polymer particles are the locus in whichnearly all of the polymer is formed. Smith and Ewart29

published in 1948 the most important contribution to theemulsion polymerization theory, developing a quantitativetheory of the radical polymerization kinetics in the monomer-swollen polymer particles, considering that free radicals comingfrom the aqueous phase are supplied to the particles. Theypresented the equation for the calculation of the number ofparticles containing a given number of growing radicals anddefined the different cases depending on the average number ofradicals per particle. Since then, very well-known authors in thefield have reported their interesting contributions to theknowledge of emulsion polymerization kinetics. Among them,there are fundamental contributions to the knowledge ofemulsion polymerization kinetics, such as that of monomers oftechnical interest by Gerrens,30 the proposed mechanism onthe precipitation of an insoluble growing radical forming aparticle,31 and the quantitative theory of nonmicellar particlenucleation presented by Fitch and Tsai.32 These contributionstogether with the theoretical and experimental contributions onhomogeneous particle nucleation in emulsion polymerizationby Hansen and Ugelstad,33 nowadays called HUFT theory, forHansen, Ugelstad, Fitch, and Tsai, were the basis for thedevelopment of this polymerization technique in dispersedmedia.Since 1976, the contributions have become more and more

important for the understanding of emulsion polymerizationkinetics, among them the calculation of the average number ofradicals per particle taking into account radical entry, exit,initiation, and termination in the aqueous phase34 and the roleof coagulation of primary particles during the nucleationperiod.35

Each year during the past 50 years, an extensive literatureincluding books,36,37 reviews, papers, and patent applicationson this field have been published.The main and fundamental research in conventional

emulsion polymerization is based on anionic systems, andone can only find a few studies on cationic systems.There is some controversy about the first time the synthesis

of cationic latexes was reported because several patents claimedtheir preparation during the 1960s. However, in the openliterature the first studies were published in the 1970s. In 1970,Breitenbach et al.38 compared the kinetics of the cationicemulsion polymerization of styrene with that of thehomologous anionic emulsion polymerization. They suggestedthat one of the important parameters for the kinetics ofemulsion polymerization was the rate of formation of freeradicals in the emulsion system. The persulfate ion generallyused as a source of radicals is strongly influenced in its rate ofdecomposition by different additives present in an emulsion

polymerization system. However, the cationic azo initiatorAIBA is very little affected by the presence of monomers,emulsifiers, and salts. The only restriction is that the cationicinitiator should only be used with cationic emulsifiers. In thissense, they used a cationic system composed of AIBA as thecationic initiator and CTAC as the cationic surfactant. Theyfound that at least in the beginning of the reaction seriousdiscrepancies existed between the experimental data andSmith−Ewart theory.29 An electrostatic effect in cationicsystems was found because with the addition of KBr orCaCl2 the rate of polymerization and the number of particleswent through a maximum. Furthermore, the cationic latexparticles formed were 1 order of magnitude larger than thoseobtained in the anionic emulsion polymerization and had a verynarrow size distribution. There seemed to be a very shortperiod of particle formation in the cationic system.The emulsion copolymerization of styrene with 4VP at

different monomer ratios in the presence of poly(oxyethyleneoctylphenyl ether) with 19−20 oxyethylene units as thenonionic surfactant at pH 2 and 11 was reported by Ohtsukaet al.39 They studied the effect of 4VP on the kinetics of theemulsion polymerization of styrene and on the distribution ofpolymeric VP in the cationic latex. A bimodal distribution ofthe particle diameter was obtained in the polymerization underthe conditions of low surfactant concentration and high 4VPfraction in the monomer feed. This was caused by insufficientstabilization of the resulting particles and by some changes inthe character of growing radicals in the aqueous phase withconversion. Polymerization under acidic conditions was affectedby the amphiphilicity of 4VP-rich radicals, which depended onthe 4VP fraction in the monomer feed. In latex particlesprepared at pH 2, the 4VP units were located preferentially onthe surface, whereas the latex particles prepared at pH 11 had anearly statistical distribution of 4VP on their surface.Wieboldt et al.40 studied the emulsion polymerization of

styrene by using different cationic surfactants and ADIBA as theinitiator. The cationic surfactants used were DPC, DTAB, HY,OPC, and TBAC. For all these surfactants, the authorsproposed a coagulative mechanism. The surface activity andconcentration of the emulsifier were rate-determining factors.At very low emulsifier concentration the particle surface chargemainly arose from initiator fragments and was independent ofthe emulsifier concentration. The coagulation rate was constantand also independent of the emulsifier concentration, so that arelatively small number of particles were formed. When theemulsifier concentration was increased, the surface chargedensity rose rapidly, the rate of coagulation decreased, andmost of the small particles grew to larger ones. At still highersurfactant concentration, the particle surface became saturatedand the surface charge density was independent of theemulsifier concentration. Consequently, the rate of coagulationand the number of particles were independent of the emulsifierconcentration. However, heterocoagulation (i.e., coagulationbetween large and small particles) could occur during thegrowing period. Larger particles could capture small particles,and dispersions of particles with large polydispersity could beformed. At lower polymerization temperature, the adsorption ofthe emulsifier was enhanced, the period of seeding increased, asmaller number of primary seed particles coagulated, and theparticle size distribution became broader.Ramos et al.18,19 studied the batch emulsion polymerization

of styrene using different cationic surfactants (DTAB andHDTAB) and initiators (AIBA and ADIBA). First, the best


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conditions to obtain stable cationic latexes at high conversionswere identified. When the surfactant concentration was aboveits CMC, latexes with high conversions were achieved for thetwo surfactants studied (DTAB and HDTAB). Cationic latexeswith less coagulum were obtained using ADIBA as the cationicinitiator due to its superior resistance to hydrolysis. AIBA ishydrolyzed to amide at basic pH values, and in this way, theconcentration of radicals formed in the aqueous phasedecreases. Subsequently, the kinetics of the batch cationicemulsion polymerization of styrene was studied in-depth andcompared with its homologous anionic case. Scheme 6 showsthe dependences of the surfactant and initiator concentrationson the rate of polymerization (Rp) and the number of polymerparticles (Np) for the different systems. As can be seen, theexponents obtained for both the rate of polymerization and thenumber of particles with respect to the sodium dodecyl sulfate(SDS) and potassium persulfate (KPS) concentrations in theanionic system were similar to those obtained for Smith−Ewart’s case II29 (Rp ≈ Np ≈ [SDS]0.6[KPS]0.4). These resultswere also in agreement with those proposed by Gardon41 forlow particle size systems at low initiation rates and corroboratethe linear dependence of the rate of polymerization on thenumber of particles, which means that for a conventionalanionic emulsion polymerization of styrene the rate ofpolymerization is not dependent on the volume of the latexparticles. However, the kinetics of the cationic systems was verydifferent mainly due to the different properties of cationicinitiators and surfactants. Regarding cationic systems in whichDTAB was used as the cationic surfactant,18 the exponentsobtained by fitting the experimental data with respect to boththe cationic surfactant (DTAB) and initiators (ADIBA orAIBA) were much higher for the number of particles than thoseobtained for the rate of polymerization (see Scheme 6). Thesecationic systems represent a typical case of the so-called“limited particle coagulation” that is said to control the particlepopulation in the nucleation step.42 When the primary particlesare formed, they may start to coagulate with each other. Thestability of the particles will be dependent on their surfacecharge and size and the electrolyte concentration (and valency).When the particles coagulate, the surface charge will increase, asmost of the surface-active groups stay on the surface. When theparticles become sufficiently large, they will have enoughcharged groups to prevent further coagulation. This is due tothe simple picture of the limited particle coagulation. Underthese conditions, the number of particles formed will be afunction of the surfactant concentration and also the type of

surfactant. The rate of surfactant adsorption on primaryparticles relative to the coagulation rate of these particlesmust be important, and surfactants that adsorb fast will givehigher particle numbers than surfactants that adsorb at a slowerrate. At low surfactant concentration, SDS has a faster rate ofadsorption than its cationic homologue DTAB, nucleating moreparticles. However, at high surfactant levels DTAB adsorptionis competitive with particle growth, so a larger number ofparticles can be stabilized, achieving a higher number ofparticles than with its anionic homologue. In addition, cationicinitiators (ADIBA and AIBA) have a higher decomposition ratewith respect to KPS. In this way, cationic systems have a fasterinitial rate of radical formation (26 and 5 times higher forADIBA and AIBA, respectively), so the number of nucleatedparticles suffers slight changes with increasing concentration ofthe cationic initiator because the amount of initial radicalsneeded to promote the nucleation has been exceeded.Therefore, the main difference found between anionic andcationic emulsion polymerization of styrene was that thekinetics of cationic systems were affected by the particle size.Furthermore, a dependence of the particle size on the rate ofpolymerization per particle together with the average numberof radicals per particle was found in cationic systems. As can beseen in Scheme 6, this dependence was stronger with variationof the cationic initiator concentration (ADIBA or AIBA) thanwith variation of the cationic surfactant DTAB.On the other hand, regarding cationic systems in which

HDTAB was used as the cationic surfactant,19 lower depend-ences of the rate of polymerization and the number of particleson the HDTAB surfactant concentration were found than byusing DTAB (see Scheme 6), but a higher effect of the size ofthe particles on the rate of polymerization per particle and onthe average number of particles was observed. Furthermore,different kinetic behaviors were observed with the two cationicinitiators used (ADIBA and AIBA), and they were due to thelower stabilizing effect of the cationic radicals provided byAIBA, which means that a lower amount of particles werenucleated. Using ADIBA, the same number of particles wasobtained by increasing the initiator concentration, but a fasterpolymerization rate was observed. This was due to the strongdependence of the average number of radicals per particle onthe initiator concentration at the same particle size. UsingAIBA, higher dependences of the rate of polymerization andnumber of particles were obtained, and a double effect on theaverage number of particles was observed: the effect of theparticle size and the effect of the amount of initiator added.

Scheme 6. Kinetics of the Batch Cationic Emulsion Polymerization of Styrene18,19


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Ramos and Forcada20 also studied the kinetics of cationicemulsion polymerization of styrene in the presence of smallamounts of cationic monomers (VBTMAC and MATMAC)using HDTAB as the surfactant and AIBA as the initiator.Polymerizations using the more hydrophobic cationic mono-mer (VBTMAC) showed higher conversions due to the in situcreation of an amphiphilic copolymer with styrene, improvingparticle stability, and faster rates of polymerization wereobserved by increasing the cationic comonomer concentration.With the more hydrophilic cationic comonomer (MATMAC),the same behavior was observed up to 0.012 M MATMAC. Athigher concentrations most of the MATMAC homopolymer-ized in the water phase, and therefore, the ionic strengthcontrolled the colloidal stability of the system occurringcoagulation.3.1.1.2. Surfactant-Free Emulsion Polymerization Pro-

cesses. The majority of the syntheses of cationic nanoparticlesfound in the literature were carried out by means of surfactant-free emulsion polymerization processes. From the point of viewof using cationic nanoparticles in biotechnological applications,the main advantage of these processes is the lack of surfactantin the final latex. Table 1 shows the most representative worksfound in the literature dealing with surfactant-free cationicemulsion polymerization. As can be seen, both cationicinitiators and cationic comonomers are responsible forconferring cationic charge on nanoparticles.By Using Cationic Initiators. In a first simple approach, the

cationic initiator is the only component providing cationiccharge onto the nanoparticle surface. In this way, Sakota and

Okaya43 obtained a stable cationic polystyrene latex in theabsence of surfactants using AIBA as the initiator. Theformation of the particles was attributed to the precipitationof growing radicals produced in water, similar to that proposedfor the polymerization of styrene initiated with KPS. However,the cationic latex was stabilized with the fragments of AIBAchemically bound to the surface of the particles. Later on,Goodwin et al.44 also reported the synthesis of monodispersecationic polystyrene latexes by surfactant-free emulsionpolymerization of styrene using, as cationic initiators, AIBAand ADIBA. In this case, the particle diameters of the latexescovered the range from ca. 83 nm to 1.0 μm, a useful range forexperiments in the colloidal domain. In addition, a dimensionalanalysis of the variables involved gave the following equation,which represents the experimental data over a wide range ofpreparative conditions:

= + −⎧⎨⎩

⎫⎬⎭dT

log( ) 0.384 log[M] [IS]

[In]2563

0.19p

1.099

0.833(1)

where dp is the final diameter of the particles (nm), [M] is theinitial monomer (styrene) concentration, [IS] is the initial ionicstrength (including the initiator), [In] is the initial initiator(AIBA) concentration, and T is the absolute temperature (K).As can be observed, similar to anionic systems, the particle

size increased with an increase of the ionic strength andmonomer concentration and with a decrease of the temperatureand initiator concentration.

Table 1. Cationic Latexes Obtained by Surfactant-Free Emulsion Polymerizationa

main monomer cationic comonomer initiator reaction conditions particle diameter (nm) conversion (%) ref

S AIBA, ADIBA 50−95 °C, 350 rpm 191−1059 16−94 44S DACMP 70−80 °C, 250 rpm 771−834 45VBC, DVB AIBA 50−65 °C, 350 rpm 348−1154 97−98 46MS AIBA 60−80 °C, 250−350 rpm 476−683 45−88 47S DMVP AIBA 65 °C 180−570 48

EMVP 210−570S 4VP KPS 70 °C, 300 rpm 97 (pH 2), 450 (pH 11) 60−99 49S MVPB, MVPI AIBA 60 °C, 300 rpm 105−285 70−99 50S DMAEMA AIBA 60−80 °C 73−134 20−99 43

DEAEMA 77−212S, S-BD DEAEMA H2O2 + Fe(NO3)3 60 °C, ∼50 rpmb 120−160 <60 51, 52S DEAEMA, MAAc KPS 70 °C, ∼50 rpmb 185 >99 53S DMAEMA AIBA 70 °C, 350 rpm 572−699 54S DMAEMA AIBA 70 °C, 300 rpm 148−184 55

MATMAI 119−170S, DVB MAPTMAC AIBA 60 °C, 300 rpm 151−317 40−90 56S VBIC AIBA 70 °C, 350 rpm 102−362 10−99 57S, DVB VBTMAC AIBA 70 °C, 200 rpm 100−114 40−96 58S-BA DMBEMAB AIBA 70 °C 207−242 90−99 59S-BA DMBMAPAB AIBA 70 °C 211−312 80−99 60S, AAm DADMAC AIBA, KPS 70 °C, 400 rpm 426 60−99 61

MAPTMAC 149MATMAC 92−234VBTMAC 87−215

S MATMAC AIBA 70 °C, 400 rpm, EtOHd 86−620 78−99 62S, DVB VBAH AIBA 70−80 °C, 350 rpm 200−1000 47−97 63S VBAH AIBA 70 °C, 300 rpm 126−285 88−96 64, 65

AEMH 100−560 90−100aS = styrene; VBC = vinylbenzyl chloride; DVB = divinylbenzene; BD = butadiene; BA = butyl acrylate; MS = methylstyrene; AAm = acrylamide.bTumbled end-over-end (∼50 rpm). cMAA = methacrylic acid (anionic comonomer). dEtOH = ethanol as cosolvent.


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They observed that when the latexes prepared with AIBAwere subjected to pH values higher than 11 at roomtemperature, coagulation of the latex occurred. When thelatexes were held at 90 °C or a higher temperature for a periodof 16 h, coagulation of the latexes also occurred. Thetemperature instability region appeared to be a consequenceof hydrolysis, but provided high pH and high temperatureswere avoided, the latexes were stable for periods exceeding ayear. On the other hand, in an attempt to prepare a more stablecationic end group, they used ADIBA as the cationic initiator,obtaining a latex with a smaller particle size due to the fasterrate of decomposition of ADIBA, thus increasing the radicalflux at the beginning of the reaction and hence more effectivelystabilizing the particles formed. Furthermore, this latex was lessprone to hydrolysis than those prepared with AIBA as theinitiator.Although AIBA and ADIBA led to polystyrene latexes with

cationic amidine groups on the particle surface, their surfacecharge densities were strongly dependent on the pH. For thisreason, Blaakmeer and Fleer45 synthesized monodispersesurfactant-free polystyrene latex particles with a positivelycharged surface density almost independent of the pH in the4−10 range by using DACMP as the cationic initiator.However, the synthesis described was reproducible with respectto the particle size but not with respect to the surface chargedensity.Cationic poly(vinylbenzyl chloride) (PVBC) latexes were

also prepared by surfactant-free emulsion polymerization usingAIBA as the initiator.46 An unexpected influence of thetemperature on the particle size was found in comparison withwhat is well-known in the case of styrene. The particle size ofpolystyrene latexes increased by a factor of 2.25 when thetemperature decreased from 65 to 50 °C, as reported byGoodwin et al.44 In the case of PVBC latexes, decreasing thetemperature from 65 to 60 °C clearly resulted in a bimodal sizedistribution. The size distribution of the cationic latex obtainedat 65 °C is broader than that generally obtained for a surfactant-free emulsion polymerization; thus, the bimodal size distribu-tion observed at 60 °C was already present at 65 °C, but thedifference between the two populations was not very great.However, at 50 °C, a quite monodisperse and small size latexwas obtained, which did not follow the above-mentioned trend.These results were explained through the side reactionsoccurring during polymerization: hydrolysis of vinylbenzylchloride (VBC) into vinylbenzyl alcohol and cross-linkingreaction by a transfer reaction onto the polymer chain. Suchreactions interfered with the polymerization mechanism.Consequently, the formation of more or less hydrophilic chainsdefinitively accelerated homogeneous nucleation and, sub-sequently, the apparition of a new crop of particles. Thesepolymer chains also acted as either stabilizing or flocculatingagents with respect to the pre-existing particles. Furthermore,the introduction of divinylbenzene (DVB) caused more cross-linking than expected, together with the beneficial effect ofsuppressing VBC hydrolysis. In addition, these cationic PVBClatexes were reacted with an excess of trimethylamine (TMA)at room temperature. The conversion was completed within 2h. These cationic polymer latexes exhibit various physicochem-ical properties according to the extent of cross-linking in theinitial latex particles: (i) In the case of a high-cross-linkingdegree, cationic polymers give cloudy and slightly viscousaqueous solutions, which can be settled upon centrifugation;latex particles retain their structure. They are still suspended in

the medium, but being considerably swollen by water, theyform a microgel. (ii) In the case of a lower cross-linking degree,the aqueous solutions of polycationic chains have a bettertransparency, but a higher viscosity; their sedimentation abilityupon centrifugation strongly decreases. They behave as truesolutes, and the particle structure has totally disappeared.Highly monodisperse cationic poly(methylstyrene) (PMS)

latex particles were also prepared via surfactant-free emulsionpolymerization in the presence of AIBA as the cationicinitiator.47 Because methylstyrene exhibits reactivity analogousto that of styrene, the surfactant-free emulsion polymerizationof methylstyrene also proceeds through a similar nucleationmechanism of styrene. However, in comparison with thestyrene system, a bigger particle size and a lower conversionwere observed using methylstyrene under the same initiatorconcentration. The conversion increased with an increase of theinitiator concentration, and 88% conversion was achieved whenthe methylstyrene to AIBA ratio was 20/1. This ratio was muchhigher than the styrene to AIBA ratio reported by Goodwin etal.,44 only 300/1 being required. This effect was attributed tothe presence of the methyl group, which stabilized the benzylicradical. Furthermore, the particle size was found to decreasewith an increase of the initiator concentration and reactiontemperature. On the other hand, the increase of the ionicstrength of the aqueous phase led to the formation of largerparticles, but had little effect on the particle size distributionand conversion. The agitation speed was found to have aremarkable influence on the particle size distribution. A lowagitation speed (<250 rpm) gave a bimodal particle sizedistribution, while a speed of 350 rpm resulted in nearlymonodisperse latex particles.

By Using Cationic Comonomers. The copolymerizationwith a cationic monomer in the absence of surfactant is a betterand reliable approach to obtain more stable cationic nano-particles. By this approach, a main monomer, in most casesstyrene, is copolymerized with small amounts of a cationicmonomer with only the help of an initiator, which can becationic or not.The mechanism of surfactant-free emulsion polymerization

of styrene in the presence of a cationic comonomer is quitecomplex. The incorporation of a part of the cationic monomerin the particles by copolymerization can occur during thenucleation step, where copolymerization of styrene with thecomonomer takes place in the water phase. Precursors areformed as soon as (co)oligomers reach a critical chain length(usually noticed as a critical degree, zcrit) and then precipitate;these precursors coagulate with each other until matureparticles reach an efficient stabilization through the ionicsurface charges provided by both the initiator and the cationicmonomer. Then, in the growth step, the particles grow until themonomer droplets disappear. During the last step (terminationperiod), the main monomer polymerizes within the monomer-swollen polymer particles while the remaining cationicmonomer mostly homopolymerizes in the aqueous phase,then producing polyelectrolytes. However, the chemicalstructure and properties of the different cationic comonomersused have a strong influence on the polymerization mechanismand on the final latex particle properties.

Family of Vinylpyridines. Cationic comonomers DMVP andEMVP were used together with AIBA initiator in the surfactant-free emulsion polymerization of styrene.48 However, in thiswork the effect of the comonomer content differed stronglyfrom the behavior shown by anionic systems. The particle


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diameter initially decreased with the comonomer content,passed through a minimum, and then increased. Because it ishighly soluble in water, the comonomer reacted with theinitiator to produce many oligomeric radicals and therebyincreased the rate of primary particle formation. An increase inthe comonomer concentration also provided the primaryparticles with greater electrostatic charge, thereby reducingtheir rate of coagulation. This favored a small particle size. Withhigher comonomer concentrations, however, the oligomericradicals contained more comonomer units, thus increasing theirsolubility and their chances of being captured by the existingparticles. This favored a large particle size. The net result wasthe observed initial decrease in the particle diameter withincreasing comonomer, followed by an increase in the particlediameter at high comonomer content.Ohtsuka et al.49 studied the surfactant-free emulsion

copolymerization of styrene with 4VP at various monomerratios and at pH 2 (acid) or 11 (basic). At acidic pH, theaddition of cationic 4VP caused an increase in the rate ofpolymerization and in the number of particles, whereas at basicpH free-base-type 4VP did not contribute to any increase in thenumber of particles. Under basic conditions, the instantaneouscopolymer composition was almost the same as the monomercomposition in the feed throughout the polymerization. Underacidic conditions, on the other hand, 4VP polymerized inpreference to styrene and the reaction course was divided intotwo stages in terms of the main polymerization loci.Furthermore, the distribution of polymerized 4VP on thepolymer particle significantly depended on the pH at which thepolymerization was carried out. More than half of thepolymerized 4VP was exposed on the particle surface in thelatex prepared at pH 2, although a nearly statistical amount of4VP was exposed on the surface in the latex prepared at pH 11.Stable cationic latexes were also prepared by surfactant-free

emulsion polymerization of styrene with MVPC or MVPI asthe comonomer using AIBA as the cationic initiator.50 Theeffects of the comonomer concentration, the initiatorconcentration, and methanol addition were studied, showingthat they were versatile tools controlling the particle size andpolydispersity of the process. Increasing initiator andcomonomer concentrations initially reduced the particlediameter because of the increase in the number of particlesformed during the nucleation period. For both parameters, anincrease in the particle diameter was observed at highconcentrations. The increase in ionic strength, whichaccompanied increasing initiator and comonomer concentra-tions, was believed to dominate the initial concentration effects.Coagulation of primary particles resulted in an enhancement ofthe number-average particle diameter. At comonomer concen-trations exceeding 25 × 10−3 mol/dm,3 bridging was observed,resulting in heterogeneous latexes and thus setting a maximumto the use of MVPB to control particle properties. Addition ofmethanol resulted in increased styrene incorporation in thewater-soluble oligomeric radicals, caused by increased styrenesolubility in the continuous phase. Polyelectrolyte formationwas therefore partially prevented. The change in solubility ofthe formed polymers in the presence of methanol resulted information of an increased number of particles. The increase inthe polymerization rate (and decrease in the particle diameter)lasted up to a concentration of about 3.1 mol/dm3.Furthermore, a clear difference was found between the use ofMVPB and that of MVPI as the comonomer. Conversionmeasurements showed retardation of polymerization when

MVPI was used because AIBA initiator reacted with iodide,forming iodine, which is known to be a radical scavenger.

Family of (Dialkylamino)ethyl Methacrylates. The copoly-merization of styrene with small amounts of (dialkylamino)-ethyl methacrylates and using AIBA as the cationic initiator wasstudied by Sakota and Okaya.43 They studied the effect of thedegree of neutralization of DMAEMA or DEAEMA, observingthat the rate of polymerization increased with an increase in thedegree of neutralization of DMAEMA or DEAEMA, which alsocorresponded to an increase in the number of particles.However, the substitution of DMAEMA for DEAEMA broughtabout a lowering of the rate of polymerization and a decrease inthe number of particles, which was attributed to the differencein the monomer reactivity ratio or solubility in water betweenthese two monomers. DMAEMA is freely soluble in water,whereas DEAEMA is sparingly soluble in water.Alince et al.51,52 prepared cationic latexes of different

styrene/butadiene ratios by surfactant-free emulsion polymer-ization. The cationic charge was supplied by 1% DEAEMAquaternized with dimethyl sulfate, and the initiator mixture wasH2O2 and Fe(NO3)3. Latexes with different softnesses andreasonably monodisperse with particle sizes between 120 and160 nm were obtained.Homola and James53 prepared amphoteric polystyrene

latexes in the absence of surfactants with DEAEMA as thecationic comonomer and methacrylic acid (MAA) as theanionic comonomer and using KPS as the anionic initiator.They observed that the pH had a critical influence on thestability and particle uniformity of the latexes. The stability andthe monodispersity decreased with a pH increase in excess of1.2 due to an increase in the degree of ionization of MAA withpH that subsequently resulted in a decrease in the net positivecharge. The amphoteric latexes prepared consisted of sphericalparticles of high monodispersity, and their isoelectric pointcould be varied in the pH 5.5−8.5 range, depending on theratio of acid to amine in the polymerization.Cationic polystyrene latexes with larger particle diameters

were obtained by Tamai et al.54 using DMAEMA and AIBA asthe cationic comonomer and initiator, respectively.Brouwer et al.55 prepared permanently charged cationic

polystyrene latexes using MATMAI. The cationic comonomercontent was varied over the 0−20 wt % range. Upon theaddition of small amounts of ionic comonomer, the particle sizedropped markedly. However, when the ionic comonomercontent increased, the size slightly dropped further and thenslightly increased. Also, at higher MATMAI content, thepolydispersity increased. Heterodispersity was attributed to thelonger nucleation period along with the high polar monomercontent, similar to cationic DMVP.48 Furthermore, they alsosynthesized cationic polystyrene latexes using protonatedDMAEMA, obtaining larger particle sizes, but the same trendwith respect to the cationic comonomer content as thatobserved using quaternary MATMAI.

Miscellaneous Monomers. Van Streun et al.56 studied theeffect of varying the amount of MAPTMAC on the surfactant-free emulsion copolymerization of styrene and MAPTMACwith AIBA as the cationic initiator. The addition ofMAPTMAC accelerated the polymerization and decreasedthe particle size. Furthermore, with an increase of the cationiccomonomer concentration, more polyelectrolyte was formed.Decreasing the critical chain length of the growing water-soluble oligomeric radicals might decrease the formation ofnonanchored polyelectrolyte. Thus, DVB was added as a cross-


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linker to reduce the critical chain length without affecting thecharge density. However, the addition of DVB caused abroadening of the particle size distribution due to a dualparticle-formation mechanism.Delair et al.57 synthesized monodisperse cationic latex

particles by surfactant-free emulsion polymerization of styreneand VBIC as the cationic comonomer using AIBA as theinitiator. It was shown that the ionic strength played adeterminant role in the particle size, whereas other factors, suchas initiator and VBIC concentrations, mainly influenced thepolymerization yield and the particle stability. The higher theionic strength, the smaller the particle number and thereforethe longer the reaction time.Bon et al.58 prepared cationic latexes by surfactant-free

emulsion (co)polymerization of styrene and DVB monomerwith the use of VBTMAC as the cationic comonomer. At lowDVB/styrene ratios, monodisperse latexes with a highconversion and a negligible amount of pendant vinyl groupswere obtained. On the other hand, a higher DVB/styrene ratioled to incomplete conversion, increasing the amount of residualdouble bonds; however, latexes with only moderate colloidalstability and broad particle size distributions, consisting of verysmall particles (<100 nm) and larger agglomerated particles,were obtained.Xu et al.59 reported the synthesis of cationic latexes by

surfactant-free emulsion polymerization of styrene and n-butylacrylate (BA) in the presence of DMBEMAB using AIBA as theinitiator. As was observed with other cationic comonomers, theconversion rate of the monomer increased with increasingcomonomer DMBEAMB and AIBA concentrations. However,in this work the dependences of the cationic comonomer andinitiator concentrations on the rate of polymerization (Rp) wereobtained: Rp ≈ [DMBEMAB]0.64[AIBA]0.67. Furthermore,under constant ionic strength, the average diameter of cationicnanoparticles decreased with increasing AIBA and DMBEMABconcentrations and also with increasing temperature. On theother hand, under constant concentrations of the comonomerand initiator as well as a constant monomer composition, theaverage diameters showed a change process of increase−decrease−increase in the ionic strength plot. This implies thatthe rate of polymerization and the number of particles showedthe inverse process, i.e., decrease−increase−decrease. The samestudy was carried out using DMBMAPAB as the cationiccomonomer.60 The only differences with respect to usingDMBEMAB as the cationic comonomer were the dependencesof the cationic comonomer and initiator concentrations on therate of polymerization (Rp) obtained: Rp ≈ [DMBEMA-B]2.35[AIBA]1.32.Surfactant-free emulsion copolymerizations of styrene with

four types of quaternary ammonium cationic monomers(QACMs), DADMAC, MAPTMAC, MATMAC, andVBTMAC, were studied by Liu et al.61 For comparison,copolymerizations of styrene (St) with the four types ofQACMs were initially conducted at the same monomer feedratio of St/QACM = 9/1 (mol) using AIBA as the cationicinitiator. They presented the conversion curves of styrene forthe four copolymerizations and also for styrene homopolyme-rization (Figure 1). It was evident that the presence ofVBTMAC, MATMAC, and MAPTMAC significantly enhancedthe consumption rate of styrene. However, the effect ofDADMAC was not significant. They also presented theindividual monomer conversion curves of VBTMAC, MAT-MAC, and MAPTMAC (Figure 2). This was the first time the

consumption of a QACM in surfactant-free emulsion polymer-ization was determined. As can be seen, VBTMAC andMATMAC had higher polymerization rates than MAPTMAC.The conversion of DADMAC could not be determined.Furthermore, Figures 1 and 2 also indicated that QACMswere consumed at relatively lower rates than styrene. Thisimplies a favorable polymerization of styrene in thecopolymerizations. On the other hand, the three copolymeriza-tions with VBTMAC, MATMAC, and MAPTMAC led to theformation of particles with smaller sizes than the particles ofstyrene homopolymer. However, significant agglomerates weredetected in the VBTMAC- and MATMAC-containing latexes.In contrast, only slight changes in the particle characteristicswere induced by copolymerization with DADMAC. Becausethe four types of QACMs have the same quaternary ammoniumcationic group, their repeat units should have almost identicalcontributions to particle stabilization. In addition, althoughthere might be slight differences in hydrophilicity between thefour types of QACM repeat units, the variation in thehydrophilic−hydrophobic property of the oligomer/copolymerchains would be predominantly determined by the molarfraction of the incorporated QACM units. Therefore, thedifferences in the characteristics of the final particles betweenthe four cationic copolymerizations were mainly attributed totheir different copolymerization behaviors. The faster incorpo-ration of QACM repeat units into copolymer chains led to theformation of smaller particles. However, agglomeration of tinyunstable particles could occur at high QACM concentration,

Figure 1. Styrene conversion curves for surfactant-free emulsionhomopolymerization and copolymerization with QACMs using AIBAinitiator. Reprinted with permission from ref 61. Copyright 2000 JohnWiley and Sons.

Figure 2. Conversion curves of QACMs for surfactant-free emulsioncopolymerizations using AIBA initiator. Reprinted with permissionfrom ref 61. Copyright 2000 John Wiley and Sons.


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leading to polydispersity. Nevertheless, lowering the initialQACM concentration prevented the agglomeration. It was alsoobserved that continuous nucleation took place during thecopolymerizations with VBTMAC and with MATMAC whenusing AIBA initiator. In the case of using KPS, VBTMAC-containing particles grew continuously to a mean size muchlarger than that of the corresponding particles initiated byAIBA. Adding acrylamide (AAm) as an extra comonomerinduced various changes in the different systems. For thecopolymer containing a low fraction of the cationic monomerrepeat units (St/DADMAC/AAm system), the incorporation ofacrylamide into the copolymer chains reduced the final particlesize possibly by an enhancement in particle stabilization. In thecase of a copolymer containing a high fraction of the cationicmonomer repeat units (St/VBTMAC/AAm and St/MAT-MAC/AAm systems), the presence of acrylamide suppressedthe formation of agglomerates.Surfactant-free emulsion copolymerizations of styrene with

MATMAC were performed both in aqueous medium and inthe mixture of water and ethanol.62 The objective was tocontrol the characteristics of cationic polymer particles byvarying the properties of the dispersing medium. For thesurfactant-free emulsion copolymerization in water medium, onincreasing the MATMAC/St molar ratio from 0/90 to 5/90,the particle size decreased first and then increased. Theincorporation of MATMAC repeat units in the copolymerchains significantly enhanced particle stabilization and thereforedrastically reduced the particle size and size distribution.Nevertheless, the increase in the monomer feed ratio ofMATMAC to St resulted in a progressive increase in theamount of water-soluble polyelectrolytes. At a molar ratio ofMATMAC/St = 5/90, significant agglomerates were formedprobably due to the bridging flocculation of the primaryparticles by the water-soluble polyelectrolytes. On the otherhand, using ethanol as a cosolvent effectively restricted theformation of the water-soluble polyelectrolytes. When morethan 10% (vol) ethanol was used, spherical particles withoutsignificant agglomerates were successfully obtained at a molarratio of MATMAC/St = 5/90. The particles obtained in thepresence of ethanol were larger than that in the absence ofethanol. These results were due to the complex influences ofethanol on both chemical and physical aspects: (i) increasingstyrene solubility in the continuous phase and hence alteringthe chemical composition of the growing oligoradicals, (ii)decreasing the range of electrostatic repulsive forces because ofthe reduced dielectric constant of the dispersing media, (iii)facilitating the nucleation and the adsorption of the oligomers,and (iv) promoting the coagulation of primary particles.Primary Amino-Functionalized Monomers. Amino-func-

tionalized latex particles were obtained by surfactant-freeemulsion copolymerization of styrene and VBAH using AIBAas the initiator.63 The objective of this work was the preparationof latex particles with sizes ranging between 300 and 500 nm. Inthis sense, magnesium sulfate heptahydrate was added to thereaction medium to increase the ionic strength and thus toincrease the particle size. According to Goodwin et al.,44

cationic monodisperse and monopopulated particles wereobtained by increasing the ionic strength with sodium chloride.However, under the same conditions with magnesium sulfatesalt and, to a lesser extent, VBAH, two different populations ofparticles were nucleated. Furthermore, decreasing the amountof functional monomer and/or magnesium sulfate salt reducedthe heterodispersity of the particles, but even with the smallest

amount of functional monomer, there were still a few smallerparticles besides a majority of larger monodisperse particles. Onthe other hand, it was found that by using 2% molar DVB and apolymerization temperature of 80 °C, the reaction time wasreduced with no effect on the particle size, but monodispersitywas improved. However, the surface of the particles obtainedwith DVB was “rough”, whereas without DVB the surface wassmooth. In contrast, for the emulsion copolymerization ofcationic MAPTMAC with styrene, Van Streun et al.56 obtainedpolydisperse latexes by addition of 1−3% DVB. This differencewas due to the greater reactivity of MAPTMAC than that ofVBAH.Surfactant-free emulsion copolymerization of styrene was

studied in the presence of two amino-containing monomers,AEMH and VBAH, using AIBA as the cationic initiator.64,65

The two cationic monomers were found to similarly affect thekinetics of the emulsion copolymerization of styrene: theoverall polymerization rate and particle number increaseddramatically upon increasing the functional monomer concen-tration. The authors found a relationship between the numberof particles (Np) and the cationic monomer concentration: Np≈ [VBAH or AEMH]1.45. Such a large value indeed confirmedthe strong influence of the cationic monomer concentration onthe nucleation step. However, the final conversion wassignificantly enhanced upon increasing the cationic monomerconcentration, with some difference according to the type ofmonomer; a maximum conversion of 96% was attained withVBAH instead of nearly 100% with AEMH. On the other hand,the final particle variation with the cationic monomerconcentration was found to follow the same trend whetherVBAH or AEMH was used; the particle size significantlydecreased when the cationic monomer concentration increased.As already found by other authors,44 the log−log plots ofparticle diameter (Dw) vs cationic monomer concentrationexhibited two straight lines (Figure 3) with a slope slightly

different for both amino-containing monomers. VBAH andAEMH monomers strongly affected the final particle size with apower variation of 0.64 and 0.53, respectively, indicating thatthe latter did more significantly influence the particle nucleationcompared to the former monomer. AEMH had a morepronounced effect on the particle size, especially at lowconcentration. In addition, the average number of radicals perparticle (n ) was also found to be drastically affected by thecationic monomer concentration in relation to the volume ofthe particle (Figure 4). Except for a low particle size (100 nm),that is, when high concentrations of cationic monomer were

Figure 3. Log−log scale of diameter variation vs cationic monomerconcentration (●, AEMH; ○, VBAH). Reprinted with permissionfrom ref 65. Copyright 1997 John Wiley and Sons.


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used, the obtained curve suggested that n was more likely todepend on the particle volume. This behavior was consistentwith the occurrence of two types of mechanisms during theparticle growth: pseudobulk kinetics would prevail for lowVBAH (or AEMH) concentrations (providing n values ≥1),whereas zero-one kinetics (Smith−Ewart case 2)29 would onlytake place for higher monomer concentrations (giving n = 0.5).Furthermore, the transfer rate constant for the two cationicmonomers at 70 °C, determined with the knowledge of themolecular weight distribution (MWD), were found to be verylarge, suggesting that chain transfer reaction occur from thelabile hydrogen coming from the partially unprotected aminofunction.3.1.1.3. Semicontinuous Emulsion Polymerization Pro-

cesses. Voorn et al.66 reported a systematic investigation oncontrolling cationic charge densities of latex particles in therange of 10−100 μequiv/g by semicontinuous or starved-feedemulsion polymerization. The semicontinuous surfactant-freeemulsion polymerization of isobutyl methacrylate (iBMA)initiated by cationic initiators (AIBA and ADIBA) enables theformation of cationic latex particles (100−250 nm in diameter)with moderate charge densities. By adjusting the initiatorconcentration in the recipe, a maximum charge density of 55μequiv/g can be reached. Semicontinuous emulsion copoly-merization of iBMA and cationic (MATMAC and MAPTAC)or amine-containing (DMAEMA or N-[3-(dimethylamino)-propyl] methacrylamide (DMA)) monomers renders polymerparticles with a much higher cationic charge density up to 97μequiv/g. By variation of the concentration of the cationicmonomer or amine-containing monomer, a good control ofcharge density is possible. However, in both cases the formationof disadvantageous water-soluble polyelectrolytes is significantlyincreased. The aminolysis of the epoxy groups at the surface ofcopolymer latex particles of butyl methacrylate (BMA) andglycicyl methacrylate (GMA) results in the formation of highlycharged (up to 100 μequiv/g) cationic latex particles withnegligible formation of water-soluble polyelectrolytes. Carryingthe extent of conversion of epoxy groups into quaternaryammonium groups can easily control the charge formation.This two-step approach has proved to be the most successfulone in producing highly charged cationic latexes.By using a nonionic surfactant (Emulan NP3070), all three

above-mentioned methods have been successfully used toobtain nanosized (20−40 nm), cationic latex particles. Thesurface charge densities can be controlled similarly to thesurfactant-free emulsion polymerizations.

The epoxy groups at the surface react first, and the reactiongradually proceeds to the interior of the particle. For all theGMA copolymers a conversion of more than 12 mol % epoxygroups, based on the total amount of BMA and GMA present,into quaternary ammonium groups led to the formation of acompletely transparent aqueous solution of cationic GMA/BMA copolymer. Stable latex particles were preserved when theconcentration of TMA and hydrochloric acid (HCl) wasdecreased and less hydrophilic quaternary ammonium groupswere formed. As shown in Figures 5 and 6, as the amount of

quaternary ammonium varied, the surface charge density of theparticles varied in a wide range of 30−100 μequiv/g for cationiclatexes from both surfactant-free and nonionically stabilizedsemicontinuous emulsion polymerization, even though theparticle size differs a lot (210 and 25 nm, respectively). Thepresence of the hairy polyelectrolyte (chains containingquaternary ammonium) did not enlarge the hydrodynamic

Figure 4. Average number of radicals per particle (n ) versus particlesize (●, AEMH; ○, VBAH). Reprinted with permission from ref 64.Copyright 1997 John Wiley and Sons.

Figure 5. Particle size by DLS and particle surface charge density forGMA/BMA copolymer latex from surfactant-free emulsion polymer-izations: (■) particle size of the latex after aminolysis; (□) particle sizeof the same latex treated with 0.1 M NaCl; (●) evolution of thesurface charge density with increasing amount of quaternaryammonium groups. Reprinted from ref 66. Copyright 2005 AmericanChemical Society.

Figure 6. Particle size by DLS and particle surface charge density forGMA/BMA copolymer latex from nonionically stabilized emulsionpolymerizations: (▼) particle size of the latex after aminolysis; (■)particle size of the same latex treated with 0.1 M NaCl; (●) evolutionof the surface charge density with increasing amount of quaternaryammonium groups. Reprinted from ref 66. Copyright 2005 AmericanChemical Society.


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diameter (measured by dynamic light scattering (DLS)) of thelatex particles slightly as can be seen in Figures 5 and 6.Addition of 1 mL of 0.1 M NaCl was used to collapse the layerof polyelectrolyte on the particle surface to determine the actualparticle size, which revealed the actual particle size of themodified latex particles was about 205 nm for the surfactant-free latex and 25 nm for the nonionically stabilized latex as alsoshown in Figures 5 and 6, respectively.3.1.1.4. Emulsion Polymerization with Cationic Polymers

as Stabilizers. Cationic polymers as stabilizers in emulsionpolymerization have been only used in a few works. Van Streunet al.67 prepared a cationic latex by an emulsion copolymeriza-tion of styrene and divinylbenzene in the presence of the blockcopolymer polystyrene-block-poly(4-vinylpyridine) previouslyquaternized with methyl iodine (PS-b-qPVP). The blockcopolymer served as a surfactant, stabilizing the latex particlesby its charged hydrophilic qPVP part. The hydrophobicpolystyrene block acted as an anchoring group. This anchoringwas achieved by insertion of the hydrophobic polystyreneblocks into the latex particles. The resulting product was a latexstabilized by immobilized cationic qPVP.Campbell et al.68 used poly[(vinyl alcohol)-co-(vinylamine)]

as the stabilizer for the emulsion polymerization of styreneusing ammonium and persulfate (APS) and KPS as initiators.Stable and relatively uniform cationic polystyrene latexes,ranging from 100 to 500 nm, were obtained using a lowmolecular weight cationic copolymer as the stabilizer, withadjustment of the pH to 2−7, and stirring at 700 rpm to formthe emulsion. The reaction was carried out at 70 °C and 200rpm. The predominant mechanism of stabilization of thepolystyrene particles by the poly[(vinyl alcohol)-co-(vinyl-amine)] was physical adsorption rather than grafting of thestabilizer to the particle. Most of the stabilizer was removed by

serum replacement. The adsorption occurred by coupling ofopposite charges, e.g., sulfate end groups of the polystyrenechains with the vinylamine groups of the stabilizer orhydrophobic or hydrogen bonding of the backbone chains ofthe stabilizer with the polystyrene surface.In the past decade, the group of Li has been focusing on the

development of simple and versatile routes to produce well-defined cationic amphiphilic core−shell particles based on theredox initiation between alkyl hydroperoxide and the aminegroup of a water-soluble polymer in water.69−75 Scheme 7illustrates the formation mechanism of amphiphilic core−shellparticles.74,75 Alkyl hydroperoxides (ROOH) such as tert-butylhydroperoxide (t-BuOOH) primarily interact with the aminogroup of the polymer backbone, forming a redox pair.Subsequent electron transfer and loss of a proton result inthe formation of an amino radical and an alkoxy radical (RO•).The amino radical is capable of initiating graft polymerizationof the vinyl monomer in water. The resulting amphiphilicmacroradicals can self-assemble to form polymeric micelle-likemicrodomains, which become loci for the subsequent polymer-ization of the monomer: similar to emulsion polymerization. Atthe same time, the generated RO• radical can either initiatehomopolymerization of the vinyl monomer inside the micelleor abstract a hydrogen atom from the polymer backbone tocreate a backbone radical that can also initiate graft polymer-ization of the vinyl monomer. As a result, core−shell particleswith hydrophobic polymer cores and water-soluble polymers asthe shells can be produced with a narrow particle sizedistribution and a well-defined core−shell nanostructure.A variety of core−shell particles have been prepared by this

approach through selection of different shell materials, vinylmonomers, and appropriate reaction conditions. For example,the hydrophilic shell materials can range from natural

Scheme 7. Formation Mechanism of an Amphiphilic Core−Shell Particle via Graft Polymerization of a Vinyl Monomer from aWater-Soluble Polymer Containing an Amino Group in Aqueous Mediuma

aReprinted with permission from ref 75. Copyright 2010 Springer-Verlag.


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biopolymers such as albumin bovine serum, casein, chitosan,cellulase, and gelatin to synthetic polymers such as PEI, PAAm,and PVAm. Vinyl monomers can be styrene, methylmethacrylate (MMA), BA, and 2-hydroxypropyl methacrylate(HPMA). Figure 7 displays the morphologies of variousparticles containing biopolymers shells such as poly(methylmethacrylate) (PMMA)/chitosan, PMMA/casein, poly(n-butylacrylate) (PBA)/chitosan, and PMMA/cellulase and particleswith synthetic polymer shells such as PMMA/PEI, PMMA/poly(allylamine), and PBA/PMMA/PVAm. This approach hasthe unique feature of combining graft polymerization, in situself-assembly of the resulting amphiphilic graft copolymer, andemulsion polymerization in one-pot synthesis. This versatileand simple process enables preparation of a wide variety ofamphiphilic core−shell particles with different chemicalstructures, compositions, sizes, and functionalities. Somedistinctive advantages of this synthetic process and its productsinclude the following: (i) the particles have a well-definedcore−shell nanostructure with particle sizes ranging from 60 to500 nm in diameter and a narrow particle size distribution; (ii)the core diameter, shell thickness, and surface functionality canbe easily altered through control of the reaction conditions;(iii) the core properties can be varied from hard or soft tohollow; (iv) the shell component can use a wide range ofcommercially available and inexpensive amine-containingwater-soluble polymers, particularly biopolymers; (v) theprocess uses aqueous-based chemistry, which is environ-mentally benign; (vi) the particles are easy to synthesize in ahigh solids content (up to 30%) in the absence of surfactant.

3.1.2. Seeded Emulsion Polymerization Processes.Cationic latex particles with well-defined characteristics, suchas desired and uniform particle size, amount of cationicfunctional groups, and location of these functional groupswithin the particles, are of great interest in biotechnologicalapplications. By using a seeded emulsion polymerizationprocess, these characteristics can be easily controlled becausethe seed particles avoid the particle nucleation stage, achievingconsistent control of the particle number and particle sizedistribution (PSD).

3.1.2.1. Shot-Growth Cationic Emulsion PolymerizationProcess. As was explained above, the addition of a cationiccharged comonomer results in latexes having a higher solidscontent and a greater surface charge density than in the absenceof a cationic monomer. However, a seeded-growth process andthe so-called shot-growth emulsion polymerization may furtherincrease the surface charge, reducing the formation of water-soluble polymers. The difference between these two processesis that, in the latter case, a second charge of monomer is addedto the emulsion while the first charge is still reacting. In aseeded-growth process the latex seed is purified before a secondemulsion polymerization process is carried out. It is claimedthat a better morphology as well as an increase of surface chargedensity can be achieved in the case of the shot-growth processcompared with the seeded-growth process. As in the case ofusing anionic comonomers, this phenomenon was explained bythe difference in swelling behavior between purified (i.e.,monomer-free) and nonpurified latexes or by the presence ofoligomeric radicals in the slightly monomer-swollen particles.

Figure 7. Examples of core−shell particles produced using water-soluble polymers containing amino groups. Reprinted with permission from ref 75.Copyright 2010 Springer-Verlag.


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Van Streun et al.56 studied the shot-growth process using aninitial charge of styrene as the main monomer, MAPTMAC asthe cationic comonomer, and AIBA as the cationic initiator.After 3 h (conversion >80%), a second charge of monomer,comonomer, initiator, and water was added. They concludedthat the surface charge density increased slightly by using theshot-growth process. In addition, for a high concentration ofMAPTMAC, the resulting latex had a higher polydispersity.However, when the second charge was added at lowconversion, latexes with a relatively high polydispersity wereproduced.Ganachaud et al.76 prepared functionalized polystyrene latex

particles either by a seeded-growth process or by a shot-growthprocess using AIBA as the cationic initiator and VBAH as thecationic monomer. The capabilities of both processes toproduce cationic particles in terms of functionalization yieldswere compared. Functionalizing seed particles gave poorfunctionalization yields. However, with the shot-growthprocess, the yields were improved but the concentrationrange in the feed was limited due to a secondary nucleationtaking place at functional monomer concentrations as low as21% in the feed. This process required, as the first step, thesynthesis of particles with low amounts of functional monomerto set the particle size. The remaining amounts of styrene andVBAH were added in a second step at about 80% conversion, ahigh enough conversion to avoid a secondary nucleationprocess. This procedure allowed them to synthesize sphericalmonodisperse particles with differing charge densities and withfairly good yields.3.1.2.2. Batch-Seeded Cationic Emulsion Polymerization

Process. Bon et al.58 prepared cationic latexes by using asequence of different batch surfactant-free emulsion polymer-ization steps. First, slightly cross-linked and monodispersepolystyrene latex particles were prepared by batch surfactant-free emulsion polymerization using AIBA as the cationicinitiator and DVB as the cross-linker. Then these particles wereused for the seeded emulsion polymerization of DVB. Thepresence of monomer droplets was avoided by keeping themonomer/polymer-swelling ratio below its maximum value,and therefore, polymerization starts in emulsion polymerizationinterval III. From this seeded process, monodisperse “network-containing” latexes with a considerable amount of residual vinylgroups were obtained (40% of the DVB molecules thatpolymerized reacted once). Therefore, these latexes are suitablefor graft polymerization of VBC, despite the fact that aconsiderable amount of residual double bonds present in thelatexes cannot serve as reactive sites for grafting because of theshielding effect, caused by both steric hindrance and highviscosity in the inner regions of the polymer network. The graftpolymerizations were carried out at 25 °C by using a redoxinitiator system with sodium formaldehyde sulfoxylate (SFS) asthe reducing agent, an Fen+ (ethylenediaminetetraacetic acid,EDTA) complex as the mediator with EDTA as thesequestering agent, and cumene hydroperoxyde (CHP) as theoxidizing agent. Under the experimental conditions, the VBCmonomer and the CHP initiator are both imbibed in the seedparticles. The remaining two components of the redox initiatorsystem are mainly present in the aqueous phase. It is assumedthat radical decomposition of the CHP takes place near thesurface of the particle, almost immediately followed by radicalentry. However, due to the highly cross-linked nature of theseed particle, the mobility of the radical was restricted, resultingin an enhanced average time required for the growing radical to

diffuse to the interior of the particle. The high viscosity andsteric hindrance inside the particle, as well as the anchoring ofthe growing radical by polymerization with a pendant vinylgroup, “excluded” reaction loci in the inner regions; therefore,polymerization took place mainly in the shell region of the latexparticle. The particles obtained from the graft polymerization ofVBC were nonspherical, consisting of a well-defined,monodisperse core with a spherical shape whose size wasabout the same as the size of the original seed latex particlesand a sharp cockled shell of grafted VBC. The final cationicpolymer colloid was obtained after amination of thechloromethyl groups of the polymer particle, grafted withVBC, using a 10-fold excess of TMA. These modified cationiclatex particles had well-defined monodisperse, sphericallyshaped cores, which are composed of poly(styrene−DVB)surrounded by a grafted “shell” layer of poly(trimethyl-(vinylbenzyl)ammonium salt). The aminated polymer colloidshad a very high surface charge of 1.14 mequiv/g (2.19 C/m2).In a first approach and to obtain amino-functionalized latex

particles, Miraballes-Martinez and Forcada77 synthesizedmonodisperse latex particles with surface amino groups by atwo-step emulsion polymerization process. In the first step,anionic seeds were prepared by batch emulsion polymerizationof styrene using Aerosol MA-80 as the anionic surfactant andKPS as the anionic initiator, and in the second step twodifferent cationic amino-containing monomers (VBAH andAEMH) were copolymerized with styrene onto the anionicseeds using anionic initiators and surfactants. In this seededprocess, monodisperse amphoteric amino sulfate latex particleswere obtained. The surface amino groups conferred the cationiccharacter, while the emulsifier and initiator systems suppliedthe anionic charge, resulting in latexes with very poor colloidalstability. The most stable latexes were obtained using thesmallest anionic seed and the cationic VBAH monomer.In a following work, cationic latex particles with surface

amino groups were prepared by a multistep batch emulsionpolymerization.78 In the first one or two steps, monodispersecationic latex particles to be used as the seed were synthesizedusing styrene and MAPTMAC as the cationic comonomer, andin the third step, AEMH or VBAH was used to prepare the finalfunctionalized latex particles. AIBA was used as the cationicinitiator, and different concentrations of two quaternaryammonium emulsifiers with hydrophobic chains of differentlengths were examined (DTAB and HDTAB). The amino,amidine, and quaternary ammonium surface groups providedthe cationic character to the particles. The colloidal stabilitybehavior of the prepared latexes was compatible with theircationic character and was clearly higher when compared withthat of the previous amphoteric amino sulfate latexes.Looking for more advanced synthesis strategies, Ramos et

al.79 prepared cationic latex particles by several multistepemulsion polymerization processes. The main objective of thiswork was the preparation of amino-functionalized latex particlesto develop a new immunoreagent to detect serum ferritin. Tosucceed in this objective, monodisperse particle sizes from 200to 500 nm were well designed by several batches and shot-growth processes, always using AEMH as the cationicmonomer.Van Berkel et al.80 studied radical entry in the batch-seeded

emulsion polymerization of styrene for four different systems,incorporating an anionic and a cationic initiator (KPS and V-50) into both anionically and cationically charged seed latexes.Throughout this work, “zero−one” conditions were employed


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because they offer the advantage that the particle size issufficiently small (∼70 nm diameter) that intraparticletermination is not rate-determining. Presenting data in theform of the entry efficiency factor ( fentry) as a function of theradical flux permitted direct comparison, independent of anyeffects from differences in the rate of initiation and particleconcentration, between polymerization systems. Figure 8 shows

fentry data obtained with KPS as the anionic initiator and usingAN01 as the anionic latex seed (system KPS/AN01) orCATH03 as the cationic latex seed (system KPS/CATH03).The trends seen in both systems were very similar, with fentrydecreasing as the initiator concentration was increased. As canbe seen, changing the nature of the latex particle surface chargehad a minimal effect on the entry efficiency, suggesting thatcharge interactions between the particle surface and enteringradicals have little or no impact on the entry process, consistentwith the entry model of Maxwell et al.81 However, changing theinitiator charge had a significant effect on the initiatorefficiency. Figure 9 shows fentry data obtained with V-50 asthe cationic initiator and using CATH03 as the cationic latexseed (system V-50/CATH03) or AN01 as the anionic latexseed (system V-50/AN01). In both systems, the cationicinitiator had efficiencies that were relatively invariant withchanging radical flux. However, entry efficiencies for system V-50/AN01 (cationic entry into an anionic latex) were low, falling

in the range of 0.2−0.4. These results demonstrated theimportance of the nature of the initiator (in the sense of theinitiating radicals it gives rise to) in defining the mechanism forentry in emulsion polymerization.The experimental results in Figures 8 and 9 were also

compared with results calculated under the same conditionsusing the entry model of Maxwell et al.81 According to thismodel, the rate-determining steps in entry are assumed to beonly aqueous-phase propagation and termination to formsurface-active z-meric oligomeric radicals; entry is solely by z-mers, for which actual entry into the particle is so fast as not tobe rate-determining. As can be seen in Figure 9, this model wasin excellent accord with experiments where KPS was theinitiator with z = 2; i.e., a primary KPS radical must undergotwo propagation events before a species capable of entry isformed, the first being rapid. Where V-50 was used (see Figure9), comparison with model results was somewhat veryambiguous. Entry efficiency for this cationic initiator washigh. A value of z = 1 (i.e., virtually every radical enters aparticle without undergoing aqueous-phase termination)appears most likely, although further experiments may berequired to verify this.

3.1.2.3. Semicontinuous Seeded Cationic Emulsion Poly-merization Process. Ramos and Forcada82 studied the effectsof the concentration and type of cationic surfactants on thekinetic and colloidal features in the semicontinuous seededcationic emulsion polymerization of styrene using ADIBA asthe cationic initiator and both DTAB and HDTAB as cationicsurfactants. The features of the cationic seeded polymerizationswere analyzed by consideration of the concentration of theemulsifier and the final number of latex particles (Np). Theevolution of the instantaneous conversions and Np values of thedifferent reactions were related to the nucleation, growth, andcoagulation processes occurring in these seeded polymer-izations. In addition, the evolution of the average number ofradicals per particle (n ) was also calculated. In all the reactions,except for reactions with the highest concentration of DTAB,the value of n increased during the polymerization, reflecting atransition from a zero−one-type to pseudobulk kinetics. Whenthe amount of DTAB was high, zero−one-type kinetics wereobserved throughout the polymerization because new particleswere generated continuosly throughout the reaction. Reactionswithout emulsifier or with the lowest amount of HDTABshowed the highest value of n from the beginning to endtogether with the lowest values of Np because of thecoagulation processes. Figure 10 shows the PSDs of the finallatexes obtained in the different seeded polymerizations. Thevertical line pointed out by the arrow represents the expectedvolume-average diameter determined by transmission electronmicroscopy (TEM) (d v) that would be obtained if the totalparticle number remained constant. As can be seen, thediameters of the latex particles obtained with HDTAB werecloser to that reference. This fact indicated that this emulsifierwas more efficient in stabilizing the growing particles of theseed during the semicontinuous emulsion polymerization. Ifonly monodisperse latexes are considered, a d v value mostsimilar to the expected theoretical one was obtained forreaction H4 carried out with 8 × 10−3 M HDTAB (8.7 timesCMCHDTAB). PSDs obtained from reactions carried out withDTAB emulsifier, at concentrations equal to its CMC and 2times its CMC, presented more and smaller particles than thoseobtained by the addition of HDTAB to the polymerizationrecipe. At lower emulsifier concentrations equal to half the

Figure 8. Comparison of fentry as a function of the radical flux for entryof an anionic initiator into anionic and cationic latexes: points,experimental, KPS/AN01 (tilted squares), KPS/CATH03 (squares);lines, modeled values calculated using Maxwell et al., z = 1 (), z = 2(−−−), z = 3 (---); parameter values in text. Reprinted from ref 80.Copyright 2003 American Chemical Society.

Figure 9. Comparison of fentry as a function of the radical flux for entryof a cationic initiator into anionic and cationic latexes: points,experimental, V-50/CATH03 (triangles), V-50/AN01 (circles); lines,modeled values calculated using Maxwell et al., z = 1 (), z = 2(−−−), z = 3 (---); parameter values in text. Reprinted from ref 80.Copyright 2003 American Chemical Society.


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CMC, the system had lower colloidal stability with DTAB.Furthermore, the parking areas of the two emulsifiers justifiedand corroborated the results obtained. At equal emulsifierconcentrations, HDTAB was able to cover 7.5 times more seedparticles than DTAB.Ramos and Forcada83 also synthesized monodisperse latex

particles with different amounts of surface amino and amidinegroups by means of a semicontinuous seeded cationic emulsionpolymerization of styrene, AEMH as the cationic monomer,and ADIBA as the cationic initiator. In this work, theconversion of the primary amino monomer AEMH wasreported for the first time. High partial conversions for styreneand limited ones for the cationic monomer were achieved. Theevolution of the AEMH partial overall conversions is shown inFigure 11 for latexes AE2, AE4, AE6, AE8, and AE10,

synthesized with 2, 4, 6, 8, and 10 wt % AEMH with respectto styrene, respectively. As can be seen, the conversionincreased when the amount of AEMH in the feed increased,but in all reactions a limited conversion was achieved. Thislimited conversion was due to the chain transfer reactions ofthe deprotonated cationic monomer aminoethyl methacrylate(AEM). Sauzedde et al.65 found a strong chain transferringactivity of the nonprotected form, even if there was a smallamount of AEM in the system (pH 3). The transfer to AEMoccurs in the water phase and at the particle surface, becausethe cationic monomer is predominantely soluble in water. Thisexplains that styrene conversion was not affected by transferreactions, and in this way, only polymerization in the waterphase and on the particle surface were affected. A reliablemethod for the quantification of surface amidine and amino

groups was developed by means of conductometric titrationswith and without glutaraldehyde. The amount of surfaceamidine groups provided by the cationic initiator was higherwhen the amount of cationic monomer added increased. Inaddition, the partition coefficient of AEMH was calculated as0.91, which means that the cationic monomer polymerizes withthe same probability in the aquous phase and in the particles.A mathematical model for the semicontinuous seeded

cationic emulsion polymerization of AEMH and styrene waspresented by Ramos and Forcada.84 The model includes themost distinctive features of this copolymerization. It takes intoconsideration the polymerization and partitioning of thecationic monomer AEMH in the aqueous phase and in theouter shell of the growing polymer particles and the possibilityof having radical concentration profiles in the polymer particles.Furthermore, the main effort in this model was focused on theprediction of the thickness of the outer shell (δ) and the totalsurface charge density (σ0) during the reaction and thedistinction between the surface charges provided by thecationic monomer from that given by the cationic initiator.As can be seen in Figure 12, the model predicted the

experimental data for both monomers quite well. With respectto δ, the value increased when the amount of AEMH fedincreased, which means that, on increasing the cationicmonomer concentration, the copolymerization extent in theouter shell is higher. However, the thickness of the outer shellwas much smaller than the diameter of the particles. Thisimplies that most of the styrene homopolymerizes in the coreand only a small amount copolymerizes in the outer shell.The noncommercial functional monomer VBH was synthe-

sized and subsequently copolymerized with styrene by means ofa semicontinuous seeded emulsion polymerization process toobtain cationic hydrazine-functionalized latex nanoparticles.85

The effects of the pH, cationic initiator and surfactant amounts,VBH/styrene ratio, reaction temperature, and acetone/waterratio on the kinetics and colloidal features of the cationic latexeswere studied. VBH monomer behaved as an additionalstabilizer. This behavior was due to the in situ creation of anamphiphilic copolymer with styrene at acidic pH. At highconcentrations of VBH new nucleations took place even whenthe surfactant concentration was below its CMC, making itimpossible to keep the number of particles provided by theinitial seed constant. The stabilizing effect of VBH hindered the

Figure 10. PSDs of the final lattices obtained in the different seededpolymerizations: (■) D0, (●) D1, (◆) D2, (▲) D3, (○) H1, (◇)H2, and (Δ) H4. Reprinted with permission from ref 82. Copyright2003 Wiley Periodicals, Inc.

Figure 11. Evolution of the AEMH partial overall conversions of thecationic polymerizations: (Δ) AE2; (◇) AE4; (Δ) AE6; (▽) AE8;(□) AE10. Reprinted with permission from ref 83. Copyright 2003Wiley Periodicals, Inc.

Figure 12. Partial overall conversions for the semicontinuous seededcationic emulsion copolymerization of styrene and AEMH in reactionAE10. Styrene data: (■) experimental and (―) model prediction.AEMH data: (□) experimental and (---) model prediction. Reprintedwith permission from ref 84. Copyright 2006 Elsevier Ltd.


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control of the semicontinuous polymerizations. Due to theamphiphilic character of VBH at acidic pH, the hydrazinegroups of the functionalized monomer were masked withacetone to form hydrazone groups, as shown in Scheme 8. The

main advantage of using masked VBH with acetone was theminimization of secondary nucleations in the semicontinuousseeded emulsion polymerizations. However, the formedhydrazone group decreased the radical’s efficiency, achievinglimited conversions due to the formation of less reactiveradicals, which were not able to propagate. This problem wasovercome by adding more initiator. By controlling cationicinitiator concentration, complete conversions were obtainedwithout particle coagulation. In addition, the use of maskedVBH allows preservation of the hydrazine groups protected inthe form of hydrazone groups on the particle surface untilfurther use.3.1.3. Other Polymerization Processes in Dispersed

Media. Cationic nanoparticles can also be prepared by meansof other polymerization processes in dispersed media such asmicroemulsion polymerization86−91 and miniemulsion poly-merization.92−101 Nevertheless, the main requirements ofnanoparticles for biomedical applications (desired and uniformparticle size, amount of cationic functional groups, and locationof these functional groups within the particles) make emulsionpolymerization processes much more versatile.3.2. Characterization of Cationic Latexes

Latex characterizations are carried out once the latex samplesare treated using different cleaning methods, which can includestream stripping to remove the residual monomer, dialysis orserum replacement to displace the salt and other species of lowmolecular weight, centrifugation to remove both oligomers andionic species, and ion exchange over a mixed bed of ionexchange resins to set the latex surface into the appropriateionic form. In some cases and due to the nature of the processitself, cleaning may alter the basic characteristics of the polymerlatex: this can be morphologicalas in the case of extraction ofthe monomer and/or low molecular weight oligomers from theparticle dispersionor related to the surfaceas in the casewhere ionogenic surface groups are altered. In many cases thisis unavoidable. Cleaning may also lead to loss of stability if thestabilizing moiety is an adsorbed surface-active agent.102

Focusing on the surface characteristics of latex particles, theyhave charges or hydrophilic groups on their surfaces thatprovide dispersion stability in aqueous media. Furthermore, aslatex particles are polymeric, and polymers can have hydro-phobic nature, their surfaces have both hydrophilic andhydrophobic natures. The amphiphilic character of the particlesurfaces depends on the polarity of the particle core, functionalgroups on the surface, type of charges, and surface chargedensities.The cationic latex samples are usually characterized via the

following steps:

(a) Experimental determination of the particle sizedistribution using dynamic light scattering (photon correlationspectroscopy, PCS) and/or TEM and scanning electronmicroscopy (SEM). The first technique is quite appropriatefor getting rapid information on the particle size evolution as afunction of the experimental conditions (monomer concen-tration, kinetics, latex stability, etc.); it is also a sensitive tool toidentify the formation of aggregates coming from a lack ofstabilization during the synthesis of polymer colloids or for thedetection of very tiny particles generated by secondarynucleation. The second is definitely more reliable for checkingsize homogeneity; such a technique is also quite powerful forthe observation of anomalous particle shapes.(b) Measurement of the total concentration of bound surface

charge groups that is usually called surface charge density (σ0)and is obtained by potentiometric and/or conductometrictitration. This technique permits strong and weak acid or basegroups to be distinguished. Charged surface groups arise frominitiator fragments, chemisorbed surfactant, and ionic como-nomers included in the synthesis recipes.103 It must beobserved that, in general, the area occupied by the chargedsurface groups represents only a few percent of the total area.The best picture of the surface of a latex particle is a barepolymer surface (hydrophobic or hydrophilic depending on thepolymer(s) used in the polymerization reaction) with somebound ionic groups distributed randomly on it. In some cases, acomplete characterization of the surface charge density iscarried out using the conductometric titration of amine oncationic50,55,104−106 or amphoteric107−109 latexes. In a few cases,the surface charge density is examined using 1H NMR,conductometry,110 and spectroscopy methods (UV, fluores-cence).63,111 Whereas the first method only provides semi-quantitative information on the overall surface charge density,the latter two were more accurate and suitable fordiscriminating amidine (coming from the initiator) and amine(arising from the cationic monomer) surface ionic groups.65

Cationic latexes with strong electrostatic repulsions betweenthe particles can give rise to colloidal crystals regardless of theuniformity of the particle shape.112

(c) Electrokinetic characterization, which usually consists ofthe calculation of the ζ-potential (i.e., the potential at theslipping plane) or the effective charge (Zeff) on cationic latexes.The various electrokinetic techniques available for character-ization of electrical double layers in cationic latexes usuallyconsist of measurements of the electrophoretic mobility (μe)because this technique is well established and easily applicableto dilute dispersions. For that reason, electrophoresis is themost commonly used method for obtaining the ζ-potential orthe effective charge of cationic latex particles. It is essential thatthe cationic latex concentration exceed a certain value ofparticle concentration, since otherwise the results of electro-phoretic mobility are erratic and even come to exhibit anegative mobility at neutral pH using the normal glass flat cellof any electrophoresis apparatus,113,114 an observation alsonoted by Pelton et al.115 At lower latex concentrations theparticles are probably contaminated by small amounts ofdissolved polysilicate anions leaching from the glass intosolution and adsorbing onto the cationic latex. These effects aremost pronounced at high pH values (see Figure 13). In general,μe is pH-dependent, which is in agreement with the weak basischaracter of the surface ionic groups (amidine, for example) ofcationic latexes (Figure 14). A rather puzzling aspect of theelectrokinetic behavior of cationic latexes is that the calculated

Scheme 8. Synthesis of (4-Vinylbenzyl)acetone Hydrazone


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ζ-potentials pass through a maximum as a function ofincreasing ionic strength, whereas current double layer modelsunequivocally predict a continuous decrease. This phenomenonis quite general, since it is always observed in experimentsdealing with electrophoretic mobilities of bare cationiclatexes.116−120 Various explanations are being proposed forthis behavior. The first is an enhancement of the interfacialconcentration of ions from the electrolyte. This can result fromspecific adsorption, or from a so-called “co-ion enrichment”, inwhich co-ions have an enriched population at the interface,creating a concentration-dependent charging of the surface asobserved electrophoretically. Above a certain threshold electro-lyte concentration, the surface is saturated, and the mobilitydecreases with electrolyte concentration owing to classicaldouble layer compression. The second explanation suggeststhat the surface is somewhat rough or hairy,121,122 and as aresult, the location of the shear plane varies with the electrolyteconcentration. At low electrolyte concentrations, the shearplane is a bit farther from the surface than at higher electrolyteconcentrations, resulting in a lower ζ-potential and thus a lowervalue for the mobility than would be expected in the context ofclassical theory, which assumes a fixed shear plane. In this

sense, Midmore and Hunter123 indicated that the most likelyexplanation for the mobility-salt concentration maximum seemsto be the shear plane moving away from the surface as the saltconcentration decreases. This phenomenon results in twoeffects. First, it lowers the ζ-potential in the usual way, andsecond, it lowers the mobility by a much larger percentage byintroducing ionic conduction in the diffuse layer but inside theshear plane. This conduction is taken into account by thetheory of Dukhin and Semenikhin,124 and effectively, itremoves the ζ-potential maximum. The ζ-potential valuescalculated by the nonequilibrium theory developed byDukhin−Semenikhin are adequate because the agreementbetween diffuse-layer potential (ψδ) and ζ-potential values isquite good. In conclusion, it can be affirmed that theconversion of electrophoretic mobility into ζ-potential shouldbe carried out by means of a nonequilibrium that includes theinherent anomalous surface conductance of the cationic latexparticle−electrolyte solution interface. This is partly satisfied bythe Dukhin−Semenikhin theory, since it takes into account theanomalous surface conductance associated with the presence ofa boundary layer. The third explanation suggests that themobility maximum arises owing to electrophoretic relaxation, adeformation of the ionic atmosphere around the colloidalparticles during electrophoretic motion that has the effect ofcreating a small steady-state dipole in the diffuse layer, whichacts to oppose the applied field and thus diminish the observedelectrophoretic mobility. However, Yezek and Rowell125

demonstrated that the maximum in the ζ-potential is removed,giving a continuous decrease as a function of 1/1 electrolyteconcentrations when the ζ-potential is calculated using thetheory of O’Brien and White,126 which accounts for electro-phoretic relaxation. It is evident that the explanation of theelectrokinetic behavior of cationic latexes is still an openquestion. Nevertheless, a very recent seminal work by Calero etal.127 has shed light on this controversial subject, demonstratingthat the effective charge of colloids is mainly dominated bysolvation thermodynamics, that is, the chaotropic/kosmotropiccharacter of ions and the hydrophilic and hydrophobiccharacter of surfaces. This could be a good explanation toclarify the puzzle of very different results obtained with cationiclatexes of similar surface charge densities but different chemicalsurface structures.128 From the present results, it is obviousthat, in electrokinetic investigations, positively charged latexsamples behave similarly to negatively charged latex samples129

and that all discussions about the surface structure of the latterapply to the first as well. Nevertheless and as was commentedbefore, it should be noted that the dilute samples of cationiclatexes are easily contaminated by ionic impurities dissolvedfrom the glassware and this can even produce a chargeinversion at Zeff.

117 To avoid ionic contamination, the use ofplastic containers in the handling of cationic latexes is indeedrecommended. When the cationic latex is composed of a hardcore and a soft shell (e.g., made of poly(N-isopropylacrylamide)(PNIPAM)), the conversion of electrophoretic mobility intoeffective charge or potential is not an easy task and othertheoretical treatments are preferred. Ohshima et al.130,131

proposed a theoretical analysis of the electrophoretic mobilityof the so-called “soft particles” in those cases where themobility is insensitive to the position of the slipping plane andto the thickness of the polyelectrolyte layer. This implies thatfor such cases ζ-potential loses its meaning. According toOhshima’s theory, the electrophoretic mobility of a particlebearing a shell of polyelectrolyte depends on (a) the Donnan

Figure 13. Electrophoretic mobility of cationic polystyrene particlesversus the latex concentration for two storage times: (●) 0.5 h; (○) 24h. Reprinted with permission from ref 117. Copyright 1990 Springer.

Figure 14. Electrophoretic mobility of sample H5 as a function of thepH in 10−3 M KBr. Reprinted with permission from ref 116. Copyright1986 Elsevier Ltd.


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potential of the polyelectrolyte surface layer, (b) the potentialat the boundary between the surface layer and the surroundingsolution, (c) the density of the fixed charges, and (d) thefriction parameter. This theory was successfully applied toanalyze the electrokinetic behavior of soft cationic latex-es.130Furthermore, the cationic latexes are being used ascolloidal models to test sophisticated theories about the roleof ionic size correlations and ion condensation at the electricdouble layer (edl) of colloids.132,133

(d) Determination of the latex surface polarity. Thedetermination of the polymer surface tension can be considereda tool complementary to surface analysis. For this purpose,contact angle measurement appears to be a convenient method.The surface hydrophilicity/hydrophobicity ratio reflects thelatex surface polarity, which may be different from that of thebulk. The contact angle method has been used to analyze thesurface polarity of cationic amino latexes, which were preparedby emulsifier-free emulsion copolymerization of styrene withVBAH using V-50 as the initiator.111 Contact angle measure-ments were carried out by dropping two test liquids(diiodomethane and water) on a latex pellet. Various modelshave been proposed for determining the surface energy fromcontact angle measurements. The polarity factor (Xp) isobtained as a combination of the two contributions (dispersiveand polar interactions) to the surface tension. Experimentally, aslight increase of Xp upon increasing the amine surface chargedensity was obtained. This factor is, however, unable todiscriminate between amidine and amine ionic groups on thelatex surface.(e) Characterization of the stability in the colloidal sense.

The stability of a colloidal dispersion is usually expressed interms of the stability ratio (W) defined as the reciprocalfraction of successful (leading to permanent contact) collisions.The stability ratio is given by the ratio of the fast rate constant(kf) and the k rate constant at the conditions of interest. Thestability ratio is close to unity in the fast regime, increases in theslow regime, and becomes very large when the suspension isstable (W = kf/k). The stability domains in the colloidal senseare found using different optical,134,135 electrical, or rheo-logical.136 techniques. The most common way to determine thecolloidal stability of cationic latexes in aqueous or nonaqueousmedia is by means of the experimental determination of thecoagulation critical concentration (CCC), which is defined asthe electrolyte concentration where W = 1.78,134,137,138

Nevertheless, the forces acting between two cationic latexspheres in aqueous media can be directly measured usingatomic force microscopy (AFM).139 This technique is veryimportant to carry out the measurements in degassed water toavoid the appearance of anomalous attractive forces thatdepend on the amount of gas dissolved in the water.

3.3. Applications of Cationic Latexes

Positively charged latexes have been used in diverseapplications where their positive charge can give rise to anattractive electrostatic interaction with polyeletrolytes such asproteins or surfactants or where a symmetric effect due toelectrical charges is sought as in the self-assembly of colloidparticles oppositely charged by heteroaggregation. Also, theselatexes have been applied in cases with different morphologies(dimensionalities) where bulk and surface forces are the drivinginteractions of the structures formed in 3 and 2 dimensions,respectively.

3.3.1. Adsorption of Proteins on Cationic Latexes.3.3.1.1. Adsorption of Immunoglobulin G. The adsorption ofimmunoglobulin G (IgG) molecules on cationic latexes is ofconsiderable interest in the field of medical diagnosis, as theIgG−latex system is widely used for macroscopic detection ofan antigen−antibody reaction. The main advantages of thismethod are rapidity, low cost, simplicity, and easy determi-nation by direct visual control or by analysis with an opticalreading device (spectrophometer, nephelometer, autoanalyzer,etc). Singer and Plotz8 reported the first latex immunoassay.The basis for this reaction is the physical or chemicaladsorption of IgG molecules on monodisperse latex particlesand the colloidal stabilization of the sensitized latex particles soformed. In this way, the presence of an antigen recognized bythe antibody will produce an immunological reaction with thesubsequent aggregation of the latex particles. It is evident thatthe attached antibody molecule will determine the specificimmunoreaction, and there are two types of IgG that can beused for this purpose depending on the method of obtention:polyclonal IgG, which is separated as the immunologicalfraction from the serum of a sensitized animal, or monoclonalIgG, obtained from only one T-cell type artificially reproducedin ascites. The first type is less specific as it is a mixture ofmultiple slightly different IgG molecules, but it is cheaper andrecognizes different antigen epitopes. Monoclonal IgG, never-theless, is a very homogeneous sample; thus, it is moreadequate for a basic investigation of the mechanism involved inthis process. Different authors studied the adsorption ofpolyclonal and monoclonal IgG molecules on cationic latexparticles.140−144 Hidalgo-Alvarez and Galisteo-Gonzalez145

comprehensively reviewed the adsorption characteristics ofimmunoglobulins on different types of surfaces. The analysisincluded surfaces for adsorption, monolayer adsorption,thermodynamics, parameters that influence adsorption, andmodels incorporating structural rearrangements (heterogene-ity) either in the immunoglobulins or in the surface sites.Special attention was paid to determining factors such as ionicstrength, pH, isoelectric point and solubility of immunoglobu-lins, and type of adsorbent. The role played by the hydrophobicand electrostatic forces in the adsorption processes wasdiscussed, and the electrokinetic behavior and colloidal stabilityof the protein−surface complexes were analyzed. Sometechniques that help in providing estimates of the qualitativenature of immunoglobulin adsorption, such as ellipsometry,total internal reflection fluorescence, quasielastic light scatter-ing, surface force microscopy, and scanning probe microscopy,were presented. Also, the uses and applications of immuno-globulin adsorption were considered briefly.In the protein adsorption processes the most influential

factors are (a) hydrophobic dehydration of parts of theadsorbent and/or protein molecules, (b) the structural stabilityand size of the protein molecule, and (c) electrostaticinteraction between the protein and adsorbent.142,143 Themajority of the protein adsorption studies simulate physio-logical conditions, implying that the ionic strength is relativelyhigh. Under those experimental conditions the electrostaticforces between the protein and adsorbent are negligible. Anumber of studies140−142 have carefully examined the physicalchemistry of the interactions of proteins (BSA and IgG, mainly)with cationic latexes. Buffers used by Elgersma et al.140,141 werephosphates, which had a dramatic effect on the effective chargeof the cationic latex particles. They can also affect electrostatic


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interaction between the protein and cationic latex due to thecoadsorption of low molecular weight ions.To establish the role played by the electrostatic interactions

between rabbit polyclonal IgG molecules (isoelectric point(iep) values ranged between 6.1 and 8.7) and a cationic latex(particle diameter of 246 ± 6 nm and surface charge density of204 ± 5 mC/m2 due to amidine groups), Galisteo-Gonzalez etal.142 studied the adsorption of these immunoglobulins ontocationic polystyrene particles, the electrokinetic behavior of thesensitized cationic latex particles at low ionic strength, and thecolloidal stability of particles coated with IgG molecules. In thiscase, organic buffers (bis-Tris, Tris, and AMP (2-amino-2-methyl-1-propanol)) were used to determine the adsorptionisotherms of IgG molecules onto cationic latex particles. Thesalt concentrations were calculated to give a final ionic strengthof 2 mM.From the adsorption viewpoint, the stability of IgG is of

capital importance because its concentration in the supernatantis utilized in the depletion method to calculate the amount ofIgG adsorbed onto cationic latex particles. After 2 h, for IgGsolutions at 2 mM and pH 5, 7, and 9, the IgG denaturationwas negligible in all three cases studied, and hence, this was theoptimal value of the residence time to perform the adsorptionisotherms. The isotherms had the following features incommon:

(i) In all cases they showed well-defined plateaus at high IgGconcentrations.

(ii) Even when the IgG molecules had the same charge sign

as the cationic latex, adsorption occurred spontaneously.

This is due to the fact that in protein adsorption the most

influential factor is the hydrophobic dehydration of parts

of the latex surface and/or IgG molecules.140

Isoelectric focusing (IEF) measurements performed with thesupernatants after IgG adsorption onto cationic latex particlesshowed that at pH 9 the preferential adsorption is partlydetermined by electrostatic factors; IgG molecules with thelowest IEP are preferentially adsorbed on positively chargedlatex particles, whereas at pH 5 and 7 no preferential adsorptionis observed (see Figure 15). However, it must be noted thatpreferential adsorption was less clear in this case than that ofthe supernatants of IgG suspensions in contact with negativelycharged latex particles. This behavior of polyclonal IgGmolecules against opposite sign surface charge density latexparticles can be due to several factors: different polymerpolarity and degree of hydrophobicity of both latexes due to alocal effect of the ionic surface groups (amidine or sulfate),heterogeneous distribution of the different IgG fractions, etc.The differences between cationic and anionic latexes become

more evident when measuring the plateau values of adsorption(Γpl) as a function of the pH. The occurrence of a maximum inthe Γpl(pH) curves around the average value of the iep of thepolyclonal IgG may have several reasons. According to severalauthors,140,141 the most important contribution is thedecreasing conformational stability of the IgG with increasingnet charge on it. This implies a greater tendency to structuralrearrangement of the adsorbing molecules, resulting in a largesurface area per molecule and therefore a smaller amountadsorbed. Also, it might be due to an increase of lateralelectrostatic repulsion between adsorbed molecules, which maylead to a smaller amount adsorbed at pH values different fromthe average iep of the IgG.146

It must be noted that cationic latexes show higher adsorptionof IgG at pH values higher than 7 (see Figure 16).142 This

finding constitutes additional experimental evidence of the roleplayed by the electrostatic forces in the adsorption of polyclonalIgG on cationic latexes at low ionic strength. The electrostaticforces are not important for the occurrence of IgG adsorption,but nevertheless, they influence adsorption significantly.IgG molecules adsorbed on cationic latex particles are in a

dynamic state. Although generally they are not desorbed as aresult of a simple dilution, they can be displaced by an increasein ionic strength. Figure 16 also shows the remaining amountafter subsequent displacement by resuspension in 0.5 mol/dm3

NaCl. The ease of displacement should give some indication ofthe amount of IgG bound electrostatically to the cationic latexparticles, which at intermediate pH values becomes almost 50%of the IgG adsorbed at low ionic strength.Additionally, electrophoretic mobility measurements can be

made to provide more information on the electrostatic forces

Figure 15. IEF of supernatants after rabbit IgG adsorption ontopositively charged PS beads at an ionic strength of 2 mM: (a, b, e) IgGbefore adsorption, (a′) IgG adsorbed at pH 5; (b′) IgG adsorbed atpH 7; (c′) IgG adsorbed at pH 9 (m indicates molecular weightmarker). Reprinted with permission from ref 142. Copyright 1994Elsevier Ltd.

Figure 16. Plateau adsorption of IgG on PS beads as a function of theadsorption pH: (◇) amount of IgG on anionic PS at 2 mM; (□)amount of IgG on cationic PS at 2 mM; (◆) amount of IgG remainingon anionic PS after desorption at 500 mM; (■) amount of IgGremaining on cationic PS after desorption at 500 mM. Reprinted withpermission from ref 142. Copyright 1994 Elsevier Ltd.


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between the IgG molecules and the cationic latex particles. InFigure 17 mobilities at complete coverage of the IgG−cationic

latex complexes are shown. It must be noted that the mobilitiesof the bare cationic latex particles are virtually independent ofthe pH in the 4−9 range. Besides, there is a significantdifference between bare and covered cationic latex particles.This difference can be related to the electrostatic interactionbetween IgG and latex particles. The iep of the latex particle−IgG complexes is located at pH 8. This difference between theiep ranges of the IgG molecules and the IgG−latex complexesindicates that the cationic surface charge must compensate, atleast partly, the charge distribution on the protein. Never-theless, one must keep in mind that in the adsorption of apolyclonal IgG onto differently charged latex particles apreferential adsorption of different fractions is feasible. Also, adecrease in the mobility of the IgG−latex complexes incomparison with the mobility of the bare cationic latex particlescan be observed, which could explain the extremely lowcolloidal stability at physiological pH of the sensitized latexparticles. The dependence of the mobility on the ionic strengthis shown in Figure 18. Cationic latex particles with differentdegrees of coverage of IgG at pH 7 were used in those

electrophoretic experiments. The most striking features of theseresults are the appearance of a maximum in the mobility as afunction of the ionic strength and the displacement of themaximum at lower ionic strengths as the degree of coverageincreases. The occurrence of a maximum in the mobility isprobably due to the effect of the anomalous surfaceconductance between the slipping plane and the latex particlesurface.123 With increasing degree of coverage, the value of themobility decreases to reach very low values at 50 mM andcomplete coverage by IgG molecules. Besides chargeneutralization in the vicinity of the latex and IgG, the adsorbingmolecules will cause a shift in the shear plane farther out fromthe latex surface, leading to a lowering of the electrophoreticmobility. Consequently, the adsorption of IgG molecules onthe cationic latex particle−aqueous solution interfaces changesdramatically the electrokinetic behavior of such interfaces. Thestructure of the electrical double layer of IgG moleculesimmobilized on cationic latex particles was studied by Galisteo-Gonzalez et al.147 Determination of this structure is of primaryimportance in problems related to stability in latex agglutina-tion immunoassays. That work was an attempt to know thepotential distribution parameters around these structuredinterfaces. The conversion of electrophoretic mobility datainto ζ-potential was carried out by different theoreticalapproaches developed by Smoluchowski (classical equation),O’Brien−White (ζO−W), and Dukhin−Semenikhin (ζD−S). Theζ-potential calculated with allowance for edl polarization wassubstantially larger than that calculated according to theclassical electrokinetic theory. The larger values of ζD−S incomparison with ζO−W were readily explained on the basis that,in the first theory, the contribution to polarization from all ionsof the diffuse layer was taken into account, whereas O’Brien−White accounted for only the ions corresponding to thehydrodynamically mobile part of the edl. This is an indicationthat the anomalous surface conductance of cationic latexparticles coated by IgG molecules is much larger than that ofbare latex particles.The coagulation of cationic latex particles at high IgG surface

coverage is probably the result of an interaction between thehydrophobic domains of the adsorbed IgG molecules. Thisattractive interaction is presumably much more powerful thanthe electrostatic repulsion between charged sensitized cationiclatex particles. The use of these particles in latex immunoassayrequires improving their stability in the colloidal sense.Ortega-Vinuesa et al.148 carried out a comprehensive

experimental study on the affinity of polyclonal IgG andF(ab′)2 fragments to latex particles with different hydro-phobicity degrees, surface charge densities, and electrical chargesigns (positive or negative). The most important results of thisstudy are the following:

(1) The most hydrophobic latex, that is, the cationic one,adsorbs the maximum amounts of both antibodymolecules.

(2) The highest affinity shown by antibodies for sorbentsurfaces is found when both have opposite charge signs,that is, under favorable electrostatic interaction con-ditions.

(3) When adsorbing IgG and F(ab′)2 as a function of pH inlow ionic strength media, a maximum amount ofadsorbed antibodies is observed near the isoelectricpoint of the protein−latex complex.

Figure 17. Electrophoretic mobility vs resuspension pH: (□) barecationic PS beads; (■) saturated cationic PS beads; (◇) bare anionicPS beads; (◆) saturated anionic PS beads. Reprinted with permissionfrom ref 142. Copyright 1994 Elsevier Ltd.

Figure 18. Electrophoretic mobility vs ionic strength: (◇) barecationic PS beads; (Δ) 15% coverage; (□) 30% coverage; (hourglass)60% coverage, (○) 90% coverage; (▽) 100% coverage. Reprinted withpermission from ref 142. Copyright 1994 Elsevier Ltd.


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3.3.1.2. Strategies To Improve the Colloidal Stability ofSentitized Latex Particles. To increase the colloidal stability ofthe IgG−cationic latex particles, the coadsorption of albuminmolecules149,150 or nonionic surfactants is proposed by differentauthors. The first option is adequate for physically adsorbedIgG, and the second strategy requires the protein molecules tobe chemically bound to the cationic latex surface.Elgersma et al.149 completed a very interesting study on the

competitive adsorption between bovine serum albumin (BSA)and monoclonal immune γ-globulins (IgG’s) by sequential andsimultaneous addition of the proteins to differently chargedpolystyrene (PS) latexes as the adsorbates. These authors paidspecial attention to the role played by the electrostaticinteractions in the adsorption process and performed experi-ments with (i) IgG’s of different isoelectric points, (ii)positively and negatively charged latexes, and (iii) differentpH values. Furthermore, they studied the displacement of thepreadsorbed protein by the second added protein in sequentialadsorption experiments. It was observed that BSA more easilydisplaces the preadsorbed IgG than the converse. In this case,the electrostatic interactions did not play a major role, and evenmore, under certain conditions their effect was absent.Concerning competitive adsorption, the displacement of thefirst protein is not always a prerequisite for the adsorption ofthe second protein. However, in simultaneous adsorptionexperiments from binary and ternary mixtures of BSA (iep =4.7−5.0) and four monoclonal IgG’s (iep from 4.9−5.2 to 7.9−8.1), the enrichment of one of the proteins at the expense ofthe other(s) was studied by these authors as a function of thesupply from the solution and the electrostatic interactionbetween the proteins and latex. The most important conclusionis that under electrostatically repulsive conditions competitiveadsorption is strongly influenced by the electrostatic interactionbetween the adsorbent surface and the respective proteins. Thepreference for one protein or another became less pronouncedwith increasing adsorption time. However, when the proteinswere electrostatically attracted to the adsorbent, the influenceof electrostatics on preferential adsorption was hardlydiscernible. Perhaps, the most relevant conclusion of thiswork is that with both sequential and simultaneous(competitive) addition of the proteins the results indicatedthat the conformational rearrangements in adsorbates of BSAare faster than in those of the IgG’s. Elgersema et al.151

confirmed this important discovery in a work on the kinetics ofsingle and competitive protein adsorption studied byreflectometry and streaming potential measurements.3.3.1.3. Adsorption of F(ab′)2. It is well-known144,152,153 that

the use of an F(ab′)2 fragment instead of the whole IgGantibody for antigen−antibody sites eliminates false positivesdue to the presence of the rheumatoid factor, when anti-antibody is used as the final reagent,154 and that protein bindsto the Fc portion of the whole IgG antibody. The adsorption ofan F(ab′)2 fragment on cationic latexes was experimentallystudied by Ortega-Vinuesa et al.155 and Buijs et al.144 TheF(ab′)2 fragment was obtained by pepsin digestion of rabbitpolyclonal IgG and two monoclonals, both mouse anti-hCG(human chorionic gonadotropin) from isotype IgG-1, followedby different purification chromatography processes to removeundigested IgG. In the first case, the iep values obtained for thisfragment were in the 4.6−6.0 range. The molecular weight was102 000. The cationic latex particles were prepared usingADIBA initiator as previously described.116 The particlediameter and surface charge density (amidine groups) were

191 ± 5 nm and 53.0 ± 1.4 mC/m2, respectively. Consideringthe size of an F(ab′)2 molecule, a compact monolayer of thisprotein fragment is assumed to be about 3.2 mg of F(ab′)2/m2

of latex surface. The adsorption was performed at low ionicstrength (0.002 mol/dm3) and using organic buffers. TheΓpl(pH) curves exhibit a maximum at pH 7 with an F(ab′)2maximum amount adsorbed of 6.4 mg of protein fragments/m2

of latex surface, which corresponds to the formation of a bilayeron the latex particles. In comparison with the results obtainedby others on Fab adsorption, F(ab′)2 adsorption is much lesspH-dependent, which implies that in the case of the F(ab′)2molecules the main driving force for the adsorption on thepolymer surfaces is the hydrophobic attractive force. In anattempt to distinguish more effectively between hydrophobicand electrostatic interaction in the adsorption of F(ab′)2molecules on cationic latexes, Ortega-Vinuesa et al.155,156

studied the desorption process caused by an increase in thesolution ionic strength (up to 0.5 mol/dm3, pH 7). Therefore,all molecules adsorbed by electrostatic effects should beremoved from the positively charged latex surface. The resultof this experiment was that the final adsorbed amount was 3.8mg of F(ab′)2/m2, which is close to the theoretical value of apacked monolayer of F(ab′)2 molecules. This confirms that onthe cationic latex surface an extra adsorption of F(ab′)2molecules occurs at low ionic strength and pH 7, which isdue to an attractive electrostatic interaction between thenegatively charged F(ab′)2 molecules and the positively chargedlatex surface.The electrokinetic characterization of the latex particles

coated by F(ab′)2 molecules enables more insight to be gainedinto the electrostatic interaction between the protein fragmentand the latex surface and correlation of their values ofelectrophoretic mobility with the colloidal stability of theF(ab′)2−latex complexes. With increasing adsorbed amountfrom 0 to 3.6 mg/m2, the electrophoretic mobility decreasesfrom 3.5 × 10−8 to 0.5 × 10−8 m2/(V s).157,158 Hence, thecolloidal stability of these complexes should be very low at ahigh degree of protein fragment coverage. The colloidalstability of the F(ab′)2−latex complexes was assessed bymeasuring the time dependency of the turbidity with aspectrophotometer. The CCC was determined at threedifferent pH values, and it decreased monotonously with theadsorbed F(ab′)2 amount on the cationic latex, which is inagreement with the variation of the electrophoretic mobility.However, for the same amount of adsorbed F(ab′)2, the CCCvalues are in the order CCC(pH 5) > CCC(pH 7) > CCC(pH9), which cannot be explained by the mobility values at thosepH values. Hence, the stability of the F(ab′)2−latex complexesis not controlled by the electrostatic interaction energy sincethe highest mobility values are found at pH 9 whereas the CCCis almost zero at this basic pH. This suggests that the stability atpH 5 is probably due to a steric effect between the adsorbedF(ab′)2 molecules.The adsorption and electrophoresis experiments carried out

with F(ab′)2 coming from monoclonal antibodies144 onpositively charged latex particles demonstrated that the affinityof F(ab′)2 molecules for the latex particles was barelyinfluenced by electrostatic interactions. At the saturation level,however, the adsorbed amounts were dependent on the overallelectrostatic interaction, resulting in a maximum amountadsorbed when the charge of the protein fragment is partlycompensated by the sorbent surface charge. The trends in theadsorption of the monoclonal antibodies (IgG) and the


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corresponding F(ab′)2 molecules were similar, although therewas some evidence that hydrophobic interactions and/orconformational changes were less important for F(ab′)2adsorption. Also, it was shown that the orientation (end-onor side-on) of the F(ab′)2 molecules at the solid−liquidinterface can be determined by the sorbent surface charge.To ensure the correct orientation of the Fab fragments,

Delair et al.57 synthesized cationic latex particles bearingsulfhydryl groups, which permit a covalent coupling betweenthe protein fragment and the functionalized latex particles.Nakamura et al.159 have questioned the use of the ζ-potential

as a parameter for predicting the colloidal stability of sensitizedlatex particles. In the same sense, the studies of Ohshima andKondo160−162 revealed that the ζ-potential loses its meaning forsoft colloidal particles with a structured interface, since theelectrophoretic mobility is insensitive to the precise position ofthe slipping plane; thus, a different approach must be used fordescribing the electrokinetic behavior of F(ab′)2 moleculesadsorbed onto cationic polymer surfaces. The theoretical modeldeveloped by the above-mentioned authors gives the electro-phoretic mobility for structured solid−liquid interfaces as afunction of three parameters: N (charged group density in theprotein layer, assuming a homogeneous charge distribution); λ,which gives an idea about the frictional force that the proteinlayer exerts on the surrounding liquid; d (depth of the proteinlayer from the latex surface). In the case of latex particles coatedwith increasing amounts of F(ab′)2 molecules, the best fittingbetween experimental and theoretical electrophoretic mobilitydata is obtained when λ is 0.3 nm−1 and N is 0.025 mol−1. Thedepth of the bound protein layer, d, is 8 nm. The value of d forF(ab′)2 bound to the latex surface obtained by these authorsseems to be reasonable because it falls in the 4.4−14.3 nmrange reported by several authors.153,163 The density of chargedgroups (N) in the surface region of the F(ab′)2−latex complexobtained in that work is identical to that obtained by Nakamuraet al.159 for human serum albumin (HSA)−latex complexes,which reinforces the great resemblance between the electro-kinetic behavior of albumin and F(ab′)2 molecules adsorbed onlatex particles. The depth of the bound protein fragmentF(ab′)2 layer (8 nm) is, however, between the values obtainedby those authors for the IgG−latex (10 nm) and HSA−latex (6nm) complexes, which are in agreement with the molecularsizes of IgG, F(ab′)2, and HAS.To increase the colloidal stability of F(ab′)2−cationic latex

particles, the coadsorption of albumin molecules was completedby Ortega-Vinuesa et al.156 This coadsorption process ofF(ab′)2 and monomeric bovine serum albumin (m-BSA) isusually carried out by means of a sequential process. Sequentialprotein adsorption is a two-step process. First, one type ofprotein (F(ab′)2) is adsorbed on the latex particles. It is leftthere for a certain time, after which a second protein (BSA) isadded to this protein−adsorbent complex. Adsorption of thesecond protein may involve partial or complete displacement ofthe preadsorbed protein. Besides, the adsorption of this secondprotein could be desirable to increase the colloidal stability ofthe immunolatex, because BSA is a highly charged protein atneutral pH.The ability of adsorbed protein to be displaced was

monitored as a possible indicator of adsorbed protein−surfaceinteractions. F(ab′)2 was adsorbed on cationic latex for 4 h. Thelatex-bound F(ab′)2 was then incubated with m-BSA for 20 h.Besides, the sequential F(ab′)2 and m-BSA adsorption wasperformed at two different degrees of coverage of F(ab′)2. The

fraction of F(ab′)2 displaced is significant: 23%, 29%, and 31%at the three pH values studied (5, 7, and 9), which could beexplained by taking into account the electrostatic interactionbetween the positively charged polymer surface and the netnegative charge on the albumin molecules. This effect was alsoobserved by Elgersma et al.141 in single BSA adsorption oncationic latex. As the amount of preadsorbed F(ab′)2 increases,the protein fragment is progressively easier to displace by m-BSA and the amount of the latter that can reach the surfacedecreases. The results seem to indicate that single F(ab′)2adsorption on cationic latex takes place with the formation of atleast one bilayer, which is broken by the BSA molecules in thesecond step of the sequential adsorption. The displacement ofF(ab′)2 occurs only when the preadsorbed amounts are largerthan a certain critical value, which depends on the adsorption/desorption pH. The main factor in the desorption of F(ab′)2 onthe cationic latex is an increase of the ionic strength and thepresence of BSA. However, the colloidal stability of theF(ab′)2−cationic latex complex was significantly improved byBSA adsorption.Also, F(ab′)2 molecules were coadsorbed with a cationic

commercial lipid, namely, distearoyldimethylammonium bro-mide (DSDMA),164 on a cationic latex sample. The cationiclipid was adsorbed (≅0.4 μmol m−2) on the cationic latex onlywhen the content of ethanol in the media was very low (1%, v/v) at pH 7. However, in a sequential adsorption the amount ofthe cationic lipid adsorbed on a previously F(ab′)2 coatedcationic latex slightly increased. In any case, the electrophoreticmobility and CCC of the F(ab′)2−DSDMA−cationic latexcomplexes decreased with increasing amount of adsorbedF(ab′)2. Therefore, it was not possible to stabilize cationiclatex−F(ab′)2 complexes under physiological conditionsadsorbing a cationic lipid, and this is why there are notdeveloped immunodiagnostic tests based on latex immunoassayaggregation (LIA) reactions with these systems.

3.3.2. Latex Immunoassay Aggregation. LIA proce-dures use submicrometer polymer particles as substrates forantigen−antibody reactions to measure certain analytes. Thistype of immunoassay offers a double advantage, since itcombines high sensitivity with simplicity and inexpensivenonhazardous reagents (in comparison with any radioimmuno-assay method). To improve the detection limit of the latex-based immunoassays, an optical instrumental method isrequired. As discussed by Newman et al.,165 LIA is based onthe formation of a particle-enhanced immune complex and,subsequently, detection using transmitted light, which dependson the following:

(1) The diameter, concentration, and refractive index of thepolymer carriers and surface charge density.

(2) The concentration and surface density of protein.(3) The wavelength and intensity of the light source.(4) The pH, ionic strength, and temperature.(5) The presence of additives in the reaction medium.(6) The colloidal stability of the protein−latex complexes.Cationic latexes are being used in the development of

immunoassays based on the antigen−antibody reaction.However, basic studies of the method are not as numerous interms of the effects on the sensitivity of factors such as the sizeof the latex particles, the wavelength used in light absorptionmeasurements, the pH and ionic strength of the reactionmedium, and the amount of protein bound to the latexnanoparticle surface. An immunoturbidimetric quantitation by


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latex agglutination requires that consideration be given to thefollowing:

(1) The characteristics of the polymer colloids in terms ofionic properties, hydrophilicity, functional groups react-ing with proteins, etc.

(2) The conditions for sensitization, which determine theamount and mode of attachment of antibody or antigen.

(3) The conditions for agglutination tests, which influencethe efficiency of the reaction between antigen andantibody, regardless of which is attached to the particlesurface.

(4) The characteristics of the apparatus used for theturbidimetric assay.

The first two aspects were analyzed by Ortega-Vinuesa andHidalgo-Alvarez.156 Ortega-Vinuesa et al.166 studied the thirdand fourth aspects. In this case, F(ab′)2 antibody fragmentsfrom anti-C-reactive protein (CRP) rabbit polyclonal IgG wereused. These fragments were obtained by pepsin digestion ofIgG and purified by gel filtration chromatography followed byprotein A chromatography to remove undigested IgG. Puritywas checked by SDS−PAGE (SDS−PAGE stands for sodiumdodecyl sulfate−polyacrylamide gel electrophoresis and is amethod used to separate proteins according to their size), andthe molecular weight was found to be 102 000. No IgGcontamination was detected. The isoelectric points of F(ab′)2were in the 4.7−6.0 range.To suppress nonspecific interactions of the complementary

antigen and to increase the colloidal stability of the sensitizedparticles (immunolatex) in the reaction medium, nonoccupiedsites on the cationic latex particles were coated with BSA. Theprocedure used was similar to that employed with IgGmolecules.167 In brief, the reactivity of the immunolatex wasmeasured by turbidimetry after 5 min of immunoaggregationwith human CRP in a spectrophotometer.As a general rule, the sensitized cationic latexes had a

relatively higher colloidal stability than the sensitized anioniclatexes, and hence, they provided reagents with a better opticalresponse. Less than 0.025 μg/mL C-reactive protein wasdetected using cationic latex particle enhanced opticalimmunoassay. The sensitivity, reproducibility, and detectionlimit of these latex agglutination immunoassays depend on thetechnique used to detect the aggregated product.There are a number of instruments that permit full

quantification of the extent of colloidal particle aggregation,thus avoiding the subjectiveness of manual detection. Theseinstrumental methods are far more sensitive than visualdetection methods. Moreover, such instruments are commer-cially available. The following optical methods are the mostused:

(1) Turbidimetry, which relies on the absorbance of acationic latex suspension before and after sample isadded.

(2) Nephelometry, a method based on the intensity of thescattered light at a determined angle. The readings ofagglutinated samples are compared with the results of ablank test.

(3) Angular anisotropy, in which scattered light is measuredat two angles, usually one above and one below 90°.

(4) Photon correlation spectroscopy or dynamic lightscattering, an assay method based on the principle thatwhen laser light is directed onto a particle, the frequencychange of the scattered light will be related to the speed

of the particle. A single particle will move (diffusion)relatively faster than two adhering particles.

Ortega-Vinuesa et al.168 carried out a comparative study ofthese optical techniques applied to particle-enhanced assays ofC-reactive protein using cationic (amidine groups) latexes. Foreach optical technique the following aspects were studied:sensitivity, detection limit, reaction time, amount of sampleused, and availability of the required detection device. Theresults obtained by these authors showed that both angularanisotropy and photon correlation spectroscopy offered lowerdetection limits (near 1 ng/mL CRP) and used little reagent,but had longer assay times than the classical optical techniquesof turbidimetry and nephelometry.In the previously mentioned latex aggregation immunoassays

the antibodies or fragments of IgG molecules were physicallyadsorbed onto cationic (amidine group) latexes. However,antibodies can be covalently bound to the cationic latex withamino groups, which must be positioned on the surface of thelatex particles (see Figures 19 and 20).

Figure 19. Angular anisotropy. Ratio between the intensity of lightscattered to 30° and 70° (open symbols) and 90° (solid symbols) andthe CRP concentration. Triplicate experiments are shown (first,squares; second, circles; third, tilted squares). Reprinted withpermission from ref 168. Copyright 1997 Elsevier Ltd.

Figure 20. Photon correlation spectroscopy. Average sizes of theaggregates as a function of the antigen concentration. Triplicateexperiments are shown: first (■); second (●); third (⧫). The dashedline represents the average diffusion coefficient of these aggregates.Reprinted with permission from ref 168. Copyright 1997 Elsevier Ltd.


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Several reports7,169,170 have indicated that immunoreagentsare more stable if functionalized latexes bind antibodiescovalently because the chemical attachment is very stableover time. The use of amino-functionalized latexes has severaladvantages over that of other functionalities (the binding agent,glutaraldehyde, is more stable than carbodiimide, and it can beused to form spacer arms and allows a good antibodyorientation).171

Ramos et al.79 have prepared amino-functionalized latexesusing a multistep method with the purpose of developing a newimmunoreagent. Since the synthesis of these amino latexes hasalready been described in section 3.1.2.2 of this review, in thispart the focus is on the application of this type of cationic latexin the development of a new immunoreagent to measure theserum ferritin concentration. The antihuman ferritin IgG wascovalently coupled to the amino-modified latexes with aprocedure consisting of three steps. The first one was theactivation of particle amino groups with glutaraldehyde. Latexparticles at a final concentration of 5 mg of latex/mL of 10−2

mol/dm3 (pH 6.8) phosphate buffer were incubated withglutaraldehyde solutions at different concentrations for 4 h atroom temperature with continuous mixing. The residualaldehyde groups of the latex particles could then be used forcovalent binding of amino ligands.After removal of unreacted glutaraldehyde by repeated

centrifugation and washing with the starting buffer, the particleswere resuspended in 0.1 mol/dm3 (pH 9.5) carbonate buffer,and antibody solutions were added and incubated overnight at4 °C. The imine bond formed was unstable because it had thetendency to hydrolyze with time, again giving amine andaldehyde groups.172 To overcome this inconvenience, theparticles were incubated with sodium borohydride in differentsolutions for 1 h at room temperature with continuous mixing.After removal of the reducing agent, the particles were washedwith 0.1 mol/dm3 (pH 7.4) phosphate-buffered saline (PBS),1% Tween 20 solution, to elute antibodies not covalentlyattached to the particles. Finally, the coated particles wereresuspended (0.2 g/dm3) in glycine-buffered saline−BSAcontaining 0.17 mol/dm3 NaCl, 0.1 mol/dm3 glycine, 1 g/dm3 BSA, 1% Tween 20, and 40 mg/dm3 N3Na (at pH 7.4),kept at 4 °C, and sonicated briefly to provide a working latexreagent.To optimize the coupling procedure, different parameters

were varied: concentration of glutaraldehyde (1−0.125%),concentration of antibody added (2.5−0.15 mg/m2), andconcentration of reducing agent (10−0.1 g/dm3).The immunoaggregation reaction was carried out in a

CobasMira Plus clinical chemistry analyzer by turbidimetricassay.The following parameters were studied:

(1) Concentration of glutaraldehyde (the best reactivity wasobtained with the lowest concentration (0.125%)).

(2) Particle size and surface charge density (the immunor-eactivity decreases with the size and the particle electricalcharge).

(3) Antibody concentration in the activation step (at an IgGconcentration of 0.9 mg/m2, the best analytical measure-ment and a high covalent coupling efficiency wereobtained).

(4) Sodium borohydride concentration. The effect ofborohydride was assayed to reduce the imine bond,and an excess of reducing agent improved the

immunoreactivity of the reagent prepared by theseauthors.

(5) Detection limit (the detection limit for ferritin was 3.5ng/mL). It should be noted that in a comparison studyby Sanz-Izquierdo et al.173 between latex particles withdifferent functionalized surface groups (amino, acetal,and chloromethyl) the lowest detection limit was foundin the case of the amino-modified particles.

3.3.3. Adsorption of Polyelectrolytes and Surfactantson Cationic Latexes. In the past few decades, syntheticmonodisperse colloids, such as cationic latexes, have beenwidely employed as a solid substrate in polyelectro-lyte113,174−185 and surfactant164,184,186−188 adsorption experi-ments. In general, surface-adsorbed polyelectrolytes increaseparticle coagulation rates by two distinct mechanisms: chargeneutralization and/or polymer bridging. In the first case, theadsorption of polyelectrolyte segments to oppositely chargedsites on the particle surface results in a decrease in the repulsiveelectrostatic interactions between approaching particles andsubsequently an increase in particle coagulation rates. Chargeneutralization has been documented in the coagulation ofpositively charged particles by anionic polyelectrolytes.189

Polymer binding occurs when polymer molecules simulta-neously attach to two or more molecules, leading to particledestabilization at intermediate surface coverage. In other cases,depending on the conformation adopted by the polymer at thesolid−liquid interface, polyelectrolytes can improve the stabilityor rheological properties of colloidal dispersions by means of asteric hindrance mechanism.175,181

In that context, there are several studies on the adsorption ofnucleic acid probes (deoxyribonucleic acid (DNA), single-stranded DNA (ssDNA) fragments, oligodeoxyribonucleotides(ODNs) or, more simply, oligonucleotides) on cationiclatexes.176,190−193 In general, they have the goal of elucidatingthe adsorption/desorption mechanism of ssDNA molecules onaminated latex particles or biodegradable polymer-basedparticles.194

Perhaps it is opportune now to remember that DNA is madeup by two single strands, which are in turn composed of a chainof nucleotides. Each nucleotide consists of a phosphate group, adesoxyribose sugar, and one of the four following nucleic bases:thymine (T), cytosine (C), adenine (A), and guanine (G). Thephosphate and deoxyribose groups constitute a skeletoncommon to all DNA. On the contrary, the order of basesalong the chain is specific to each DNA molecule andconstitutes the genetic code of each organism.ODNs are fragments of small ssDNA (generally less than 200

nucleotides) that can be considered as small polyelectrolytes.Automatic synthesis of ODNs permits their chemicalmodification, and they can be matched with an ssDNAfragment, so they are of interest for different biologicalapplications.It is quite difficult to set up a general rule on the adsorption

mechanism of ssDNA molecules on cationic latex particlesbecause the chain length is instrumental in the role played bythe hydrophobic interactions and the structural changessuffered by ssDNA molecules at the solid−liquid interface.Nevertheless, in general, the attractive electrostatic interactionis the predominant force in the adsorption of acidic ssDNA oncationic latex particles. Morevover, a linear correlation wasfound between the amount of an oligonucleotide, poly(TGC),adsorbed onto a cationic (amine) latex and the ζ-potential ofthe latex particles.190 This is experimental evidence that the


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adsorption behavior of ODNs on cationic latex particles can beexplained using the following equation related to the adsorptionof polyelectrolytes:195

= −N KC nEexp( )S f (2)

where n is the number of nucleotides, E the average adsorptionenergy per base, Cf the equilibrium ODN concentration, and Ka particular constant depending on the nature of the systembeing investigated and experimental conditions. The adsorptionenergy E is usually assumed as the sum of two contributingfactors (electrostatic and nonelectrostatic interactions):

≈ +nE E Eelectrostatic nonelectrostatic (3)

The electrostatic contribution can be determined from theODN charge (σODN) and the net surface charge of the latex(σlatex) and expressed as the product of these two charges, i.e.

σ σ≈Eelectrostatic latex ODN (4)

This electrostatic energy is thus expected to vary linearly withrespect to the surface charge density (or the ζ-potential). Asmentioned above, different authors experimentally found thislinear dependency.190,191

The nonelectrostatic contribution to the overall energy (E)might be caused by the interactions between the aromatic basesof the ODNs and hydrophobic patches on the latex particles(hydrophobic forces) and/or hydrogen binding between theamine groups of the latex particles (at basic pH) and theoxygen groups of the ODNs. In general, this contribution issmall, but in some cases seems to not be negligible.191

The hydrophobic interactions and the conformation changesof the ssDNA molecules become more important as the chainlength increases. For instance, the driving force for theadsorption of a double-stranded DNA (dsDNA) (2000 basepairs) on a supposed cationic latex (with negative electro-phoretic mobility!) is not of electrostatic origin but rather dueto a hydrophobic effect.196

The maximal adsorbed amount (Ns,max) of ODNs adsorbedas a function of the surface charge density of the latex particlescan be quantitatively predicted using the general approach ofHesselink197,198 proposed for the adsorption of polyelectrolytesand expressed according to the following equation:

ε κπα

σα

= −Nk T u

e eln( )

4s,maxB

2 2latex

(5)

where αe is the charge per ODN chain, e is the elementarycharge, α > 0.5 for a highly charged polyelectrolyte, ε is thedielectric constant, κ is the reciprocal Debye length, T is theabsolute temperature, kB is the Boltzmann constant, and ln(u)is the nonelectrical free energy gain. Ganachaud et al.,191 whenadsorbing an ODN (dC12G5T10) on an aminated latex, foundreasonable agreement between the calculated and theexperimental values of Ns,max.As a general trend, ODN desorption from the surface of

cationic latexes is easier at pH values where the attractiveelectrostatic interactions between both components are weaker,i.e, pH 9.0.176 Furthermore, ODN release can be induced bythe addition of an anionic surfactant (SDS) or by increasing thepH of the dispersion medium.199 To avoid the desorption ofthe ODN caused, e.g., by an increase in the ionic strength orpH changes, Delair et al.200 developed a covalent procedure ofODN immobilization onto amino-containing hydrophobicpolystyrene cationic and hydrophilic PNIPAM latexes. Theobtained conjugates were used as diagnostic tests with

enhanced sensitivity. Also, cationic latex particles have beenemployed to detect nucleic acids (dsDNA fragments) by meansof an affinity sensor based on surface plasmon resonance(SPR).201

As a model anionic polyelectrolyte, ssDNA molecules havebeen used to elucidate the factors that influence the aggregationof cationic latex particles by negatively charged polyelectro-lytes.178,179,177 These authors studied the destabilization ofcationic latex with amidine groups caused by the adsorption ofssDNA of different chain lengths (from 3 to 1400 nucleotides)with a thymine base composition. The goal of this study was toexplain how polymer flexibility and polymer−surface inter-actions affected the coagulation process. Under the neutral pHconditions employed, ssDNA molecules are acidic polyelec-trolytes with a negative charge per nucleotide (DNAmonomer). The following effects were experimentally studied:(i) polymer chain length on adsorption; (ii) DNA coatings oncoagulation rates; (iii) aggregate structure; (iv) polymer surfaceconformation.In relation to the chain length effect of the ssDNA molecules

on their adsorption on a cationic latex (480 nm in size, 131mC/m2 surface charge density due to amidine groups, 1.19 nmmean distance between charges), in general, the adsorptionisotherms exhibited a high-affinity character in which theadsorbed amount rapidly approaches saturation with increasingssDNA concentration in solution.177,191 Importantly, the massof ssDNA adsorbed at saturation coverage appears to beindependent of the chain length for molecules at least 10nucleotides in length. This phenomenon is commonly observedfor fully charged polyelectrolytes adsorbed to oppositelycharged particles and is typically interpreted to mean that thepolyelectrolyte in question is adsorbing in a flat conforma-tion.202,203 For saturating levels of the different samples ofssDNA used, the average area occupied by each ssDNAsegment was approximately 0.84 nm2, and there are 1.5 times asmany polyelectrolyte charges as particle charges. The measure-ments of the thickness of the adsorbed ssDNA layer by PCS atlow ionic strength (0.005 M NaCl) established that thesepolyelectrolytes are adsorbed on the latex with all segments intrains.Concerning the effect of ssDNA coatings on the coagulation

rates of a cationic latex (120 nm in size, 81 mC/m2 surfacecharge density due to amidine groups, 1.5 nm mean distancebetween charges), Walker and Grant177 found that the particlesare stable at low and high polyelectrolyte doses and rapidlycoagulate at an intermediate dose. These experiments werecarried out with an ssDNA sample 40 nucleotides in length, butsimilar results were obtained with other samples. The particleswere destabilized at some critical polyelectrolyte concentration(CPC) and were stable at polyelectrolyte doses above andbelow the CPC. Moreover, coagulation of ssDNA-coatedcationic particles at the CPC is diffusion-limited. Also, in thiscase, coagulation is due to charge neutralization since particledestabilization occurs when precisely the right amount ofssDNA is added to completely neutralize the surface charge. Ata higher ssDNA dose, the net charge on the latex particles wasreversed and the particle suspension was stable. Polymerbridging did not occur in these systems, even when ssDNAmolecules employed to destabilize the suspension were aboutthe same size as the particles.177 Gotting et al.204 observedsimilar behaviors with a model ODN (phosphorothioateoligonucleotide, PTO 16-mer) with the sequence 5′-ACG


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GAA ACC GTA GCT G-3′ adsorbed onto a cationic latex withimidazolinium end groups.The aggregate structure was obtained by examining how the

average hydrodynamic radius changes over long time scalesusing dynamic scaling theory, and the fractal dimension df =1.61 is solely dictated by the nature of the coagulation kinetics,in this case diffusion-limited cluster aggregation (DLCA).With respect to the polymer−surface conformation, as

Walker and Grant178,179 demonstrated the conformation ofmodified ssDNA molecules (these molecules contain ethyl-phosphonate linkages in which the negative charge on thephosphorus was turned off and replaced with a hydrophobicethyl group) at the solid−liquid interface can be determinedusing a biochemical technique called hydroxyl radical foot-printing (HRF). This consists of using OH radical to cleave theODN part not in contact with the surface. ssDNA moleculesare adsorbed on the surface of latex particles, and then hydroxylradicals are generated by the Fenton reaction.205 These resultsprovide important new insight into the relationship betweenthe structure of adsorbed polyelectrolyte layers and the stabilityof aqueous colloidal dispersions. The ssDNA moleculesadsorbed on a positively charged latex are highly protectedfrom hydroxyl radical attachment, suggesting that the sugarmoieties in the ssDNA molecules interact directly with the latexsurface. Because ssDNA adsorbs close to the cationic latexsurface, this molecule influences colloid stability by altering theelectrostatic character of the cationic latex surface. In this case,steric and bridging forces are of secondary importance. Itshould be noted that with anionic latex particles the results arecompletely different.179 An experimental study carried out byCharreyre et al.206 succeeded in determining the conformationof the ODN chemically bound by its 5′-end on a cationic latexby fluorescence energy transfer (FET). This method consists ofusing a couple of fluorescent molecules, the fluorescein (as adonor) bound to the 3′-end of the ODN and tetramethylrhod-amine (as an acceptor) immobilized on the latex surface. Theefficiency of this method varies as a function of the meandistance between the two fluorophores (i.e., between the ODNand the surface).193 Fluorescence energy transfer studies ofODNs bound to the surface of an amine-containing latex in thepresence of a nonionic surfactant (Triton X-405) providedinteresting information about the interfacial conformation ofimmobilized ODNs under different experimental conditions. A“brush”-type structure was observed at pH 10, whereas atneutral or weakly acidic pH the conformation was mostlyflat.206 Using a completely different technique (small-angleneutron scattering), Elaissari et al.192 tried to find the structureof adsorbed and covalently bound ssDNA fragments (poly-thymidylic acid, dT35) on aminated latex particles. Also, in thiscase, the adsorbed molecules lie close in a flat conformation onthe surface of the cationic latex particles, irrespective of the pHand ionic strength. Again, this can be attributed to strongattractive electrostatic interactions between the negativelycharged oligonucleotides and the positively charged latexparticles. The covalently bound dT35 molecules, however, atbasic pH (9.2) and high surface coverage (0.8 mg/m2) extendmore radially into the solvent, giving rise to a thicker layer(from 6 to 8 nm) in comparison to the case of physicaladsorption (from 3 to 6 nm).To optimize the adsorption of genomic DNA (250−300 base

pairs) molecules on cationic latexes, Guven et al.207,208 haverecently prepared monodisperse cationic nanoparticles byemulsifier-free microemulsion polymerization with a minimum

size of ∼78 nm and a positive ζ-potential in a very broad rangeof pH values. Special attention was paid to the pH effect onDNA adsorption; it was found that below pH 8 the adsorbedamount was quite large (about 204−215 mg of DNA/g of latexnanoparticles). However, at pH 9 and 10 the adsorbed amountdecreased dramatically to about 100 mg of DNA/g of latex.Once again, the electrostatic interaction between acidic DNAmolecules and positively charged latex particles is the keystoneaccounting for this behavior.The adsorption of polypeptides on cationic latexes has been

used as a test to explore different electrokinetic theories toconvert electrophoretic mobilities into ζ-potentials182 or toexplain the electrokinetic data in terms of conformationalchanges of the adsorbed polypeptides chains.183 In other cases,to avoid desorption of the adsorbed molecules from the solidphase, covalent grafting was long investigated, leading to anirreversible binding of peptide onto functional latex particles.209

There are very few studies on the adsorption of positivelycharged polyelectrolytes on cationic latex particles. Rustemeirand Killmann180 have studied the adsorption isotherms of thepH-dependent positively charged polyelectrolyte polylysine(PLL) on negatively and positively charged polystyrene latexes.Obviously, we are particularly interested in reviewing thefindings obtained with the cationic latex. In this case, the resultsobtained were very clear; there is no adsorption of the PLL(molecular weight ∼557 000) on the cationic latex at pH 6−7and an ionic strength of 0 or 0.5 M NaBr. The repulsiveelectrostatic force is enough to avoid the adsorption of PLL ona positively charged interface.To date, there have been relatively few studies re-

ported113,174,175,181,210 on the adsorption/desorption of anegatively charged polyelectrolyte on a positively chargedsolid−liquid interface. Such studies have determined system-atically the effect of the adsorption procedure and pH values,113

ionic strength,174 and degree of ionization and molecularweight181 on polyelectrolyte adsorption characteristics. In thesestudies, Meadows et al. demonstrated using electron spinresonance (ESR) spectroscopy how the equilibrium adsorbedlayer concentration and configuration of the hydrolyzedpolyacrylamide (PAA) on a cationic latex (amidine) can besignificantly manipulated by control of the adsorptionprocedure. For example, while adsorption from a lowelectrolyte concentration (direct adsorption) results inpredominantly flat adsorbed layer configurations, adsorptionfrom a high electrolyte concentration followed by redispersioninto the low electrolyte medium (indirect adsorption) gives riseto enhanced levels of adsorption and more extended (thicker)adsorbed layer configurations. The same authors175 analyzedthe colloidal stability of the above-mentioned dispersionsthrough observation of the interparticle repulsive forces using asurface balance technique and examined the stability/redispersibility of the dispersions in the presence of addedelectrolyte. Briefly, these authors found that the dispersionprepared by an indirect adsorption procedure exhibitedmarkedly increased interparticle repulsions compared to thoseof its directly prepared counterpart. In addition, the indirectlyprepared dispersion was considerably more stable (stericcontribution) toward the addition of 1/1 electrolyte, with theconcentration of added NaCl necessary to produce aggregationof the dispersion being over 10 times that required foraggregation of the directly prepared counterpart. In conclusion,these works reveal the instrumental role played by theconformation (trains, loops, and tails) of the anionic


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polyelectrolyte adsorbed onto cationic latex particles. Thesmallest amount of polyelectrolyte adsorbed (0.18 mg/m2,direct adsorption) is a good fit with the fact that the polymersegments are adsorbed almost entirely in trains close to thelatex surface, whereas the biggest one (0.32 mg/m2, indirectadsorption) agrees with a high proportion (75%) of adsorbedpolymer segments present in the form of loops and tails.Nevertheless, in the case of strongly charged polyelectrolytes(poly(styrenesulfonate), PSS), the repulsive steric forcesbetween the adsorbed polyelectrolyte layers remain unim-portant in stabilizing these systems.211

The most important weak anionic polyelectrolyte in industryis poly(acrylic acid) (PAAc). However, little is known on howPAAc influences the stability of cationic latexes. To fill this gapand clarify how the degree of ionization and the molecular massof PAAc influence the stability of (amidine) cationic latexes,Sadeghpour et al.181 have recently studied both effects. Ingeneral, the results obtained are in agreement with the well-known correlation that exists between the polyelectrolyteconformation and colloidal stability of polyelectrolyte-coatedlatexes.The stabilization of bubbles and foams by adsorbed particles

has been known for over a century.212 However, currently thereis a growing interest in the use of cationic or modified cationiclatex particles as stabilizers of aqueous foams because recentstudies suggest that the air−water interface exhibits anioniccharacter.213,214 Fujii et al.215 have prepared PS particlescarrying pH-responsive poly(2-(diethylamino)ethyl methacry-late) (PDEA), which is covalently bound to the particle core.These hairy cationic latex particles can act as pH-responsivestabilizers of aqueous foams by adsorption at the air−watersurface. A similar procedure was previously employed byKettlewell et al.216 and Hunter et al.217 but using in these casesa poly(ethylene glycol) monomethacrylate macromonomer(PEGMA) and cationic polystyrene latexes prepared with anazo initiator (AIBA). The PEGMA−AIBA−PS latex proved tobe the best foam stabilizer even at relatively low latexconcentrations (3.0 wt %), with long-term foam stabilitiesbeing obtained after drying. In this context, Yang andPelton218,219 have very recently developed a new technologyto facilitate the froth flotation of hydrophilic glass beads using acationic PS latex with controlled hydrophobicity.In the case of the surfactant adsorption, experiments on

cationic latexes could also supply some interesting informationabout the surface characteristics. In fact, Vijayendran220 haveshown that polymer polarity exerts a considerable influence onthe Gibbs energy of adsorption of SDS at latex−water interfacesand developed an adsorption model that relates the saturationadsorption of the surfactant molecules to the polarity of thepolymer surface.Surfactants are used as adsorbates at the solid−liquid

interface to control the surface charge and/or the hydro-phobic−hydrophilic character of the surface. Also, in this case,most of the experiments reported on surfactant adsorptionwere performed with anionic latexes, and relatively littleattention was paid to cationic latexes.Galisteo-Gonzalez et al.186 studied experimentally the effect

of the alkyl chain length of some anionic surfactants such asdocecanesulfonate (SDSo), tetradecanesulfonate (STSo), andhexadecanesulfonate (SHSo) on cationic latexes with amidineionic superficial groups (712 ± 12 nm in diameter and 327mC/m2 surface charge density). The adsorption isothermsindicate that the adsorption mechanism of alkanesulfonate

molecules on the cationic latex particles involves mainlyhydrophobic interactions between both components, althoughthe attractive Coulomb interaction might also play a certain role(see Figure 21). The plateau value of 10.1 μmol/m2 obtained

by these authors for the adsorption of SDSo corresponds to acharge density of 890 mC/m2, reflecting the formation ofmultilayers of surfactant on the latex surface, because thesurface charge density is 327 mC/m2. This seems to againsupport the idea of an adsorption mainly due to thehydrophobic interactions between the n-alkyl chains and thepositively charged latex surfaces, which are strongly hydro-phobic. The high value of the surface charge density of somecationic latexes, unlike that of the surfactant, may induce theformation of clusters of vertically oriented surfactant moleculesheld together in part by hydrophobic interactions between theirlong chains. Such clusters can occur at surfactant concen-trations well below the bulk CMC (SDSo, 9.8 × 10−3 M; STSo,2.6 × 10−3 M; HSTo, 7.0 × 10−4 M),221 and this phenomenonis usually called “hemimicelle formation”.222 The adsorbedamounts of n-alkanesulfonate anions on amidine polystyreneparticles are much larger than those reported by Connor andOttewill223 for the adsorption of n-alkyltrimethylammoniumcations on carboxylate polystyrene particles. The variationtrends, however, are in both cases very similar. The adsorptionprocess occurs in two main steps. First, a well-defined jump isfound, which corresponds to the adsorption of the surface-active anions onto the cationic groups of the surface with thealkyl chains lying flat on the surface. Second, a more gradualadsorption occurs with the alkyl chains adsorbing onto a surfacewith a net negative charge. The hydrophobic character of thecharge-determining groups on the positively charged poly-styrene surface also favors this second step.Different authors188,224−227 examined the single and

sequential adsorption of an anionic surfactant (e.g., SDS) anda nonionic surfactant (e.g., Triton X-100 (p-(1,1,3,3-tetramethylbutyl)phenyl polyethylene glycol)) on cationiclatexes.Romero-Cano et al.224 accomplished some years ago a

complete adsorption study on Triton X-100 onto cationiclatexes having amidine ionic superficial groups and a surfacecharge density of 92 mC/m2. The pH of the aqueous solution

Figure 21. Isotherms for the adsorption of SDSo, STSo, and SHSo atpH 6.0 in 10−3 M KBr solution on cationic polystyrene particles.Reprinted with permission from ref 186. Copyright 1990 Springer.


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controls the surface charge, and hence, this factor is consideredan important variable in that study. The adsorption isothermsshowed that an increase in the surface charge yielded a decreasein the amount of the adsorbed nonionic surfactant. Theexperimental results were explained using a descriptivemechanism of the adsorption process, which considers thepresence of holes in the layer of adsorbed surfactant. Theadsorption isotherms were analyzed with two classicaladsorption theories, those of Langmuir228,229 and Kronberg etal.230−232 Both theories gave a good description of the results,but the latter offered more information on the adsorptionphenomena. In a subsequent work, the same authors studiedthe desorption of Triton X-100 from cationic latexes due to adiscontinuous method based on the wash step (centrifugation−removal−redispersion) or a continuous method based on thereplacement of the dispersed media. The desorption resultsshowed that under all the experimental conditions utilized aresidual amount of the nonionic surfactant (∼1.3 μmol/m2)remains adsorbed, indicating irreversibility of the nonionicsurfactant adsorption.The addition of a nonionic surfactant to colloidal dispersions

is widely employed to modify the colloidal stability. Once thesurfactant molecules are adsorbed onto the particle surface,three different types of stabilization can be distinguished as aconsequence of the relation between the van der Waalsattraction energy (VA) and the steric interaction energy (VS).The colloidal stability of cationic latexes before and afteradsorption of Triton X-100 were studied by Romero-Cano etal.226 using optical methods to determine the stability factor,W,previously defined. Whatever the theoretical expression of W, itdepends on the interaction potential between the interactingparticles (V(H), where H is the surface to surface distance ofthe two approaching particles).233−236 The interaction potentialV(H) can be separated into three contributions: electrostaticrepulsion (VR), attractive interaction (VA), and stericinteraction (VS):

= + +V H V H V H V H( ) ( ) ( ) ( )R A S (6)

VR(H) and VA(H) can be calculated using the classicalexpressions of the Derjaguin−Landau−Verwey−Overbeek(DLVO) theory:237,238

πκ

γ κ= −V Hank T

H( )64

exp( )RB

22

(7)

where a is the particle radius, n is the number of ions per unitvolume, κ is the Debye reciprocal length, kB is the Boltzmannconstant, and T is the absolute temperature. The factor γ isrelated to the diffuse-layer potential, ψδ, through

γψ ψ

ψ= ≈ →δ δδ

ze

k T

ze

k Ttanh

4 4(when 0)

B B (8)

and the nonsimplified expressions for the attractive Hamaker239

interaction is

= −+

++

+ ++

⎛⎝⎜

⎞⎠⎟

V HA a

H aHa

H a

H aHH a

( )6

24

2( 2 )

ln4

( 2 )

A

2

2

2

2

2

2(9)

where A is the Hamaker constant.If the Stern layer thickness is considered, eq 9 must be

modified by shifting the reference plane for repulsive energy

outward over a distance corresponding to the thickness (Δ) ofthe Stern layer. The final expression is

πκ

γ κ=+ Δ

− − ΔV Ha nk T

H( )64 ( )

exp( ( 2 ))RB

22

(10)

The steric repulsion due to a layer of polymer adsorbed onto acationic latex can be calculated using the expressions obtainedby Vincent et al.240 According to this DLVO extended theory,the steric stabilization effect is usually due to two contributions,osmotic and elastic:

= +V H V H V H( ) ( ) ( )S osm elas (11)

If there are polymeric chains covering the external surface of ananoparticle and δ is the average thickness of such coils, thenan osmotic effect will appear when the two particles are nearerthan a distance equal to 2δ. The osmotic pressure of the solventin the overlap zone will be less than that in the regions externalto it, leading to a driving force for the spontaneous flow ofsolvent into the overlap zone, which pushes the particles apart:

π ϕ χ δ= − −⎜ ⎟⎜ ⎟⎛⎝

⎞⎠⎛⎝

⎞⎠V H

av

H( )

4 12 2osm

12

22

(12)

where v1 is the molecular volume of the solvent, ϕ2 is theeffective volume fraction of segments in the adsorbed layer, andχ is the Flory−Huggins solvency parameter.However, if the two particles are closer than a distance equal

to δ, at least some of the polymer molecules will be forced toundergo elastic compression. Thermodynamically, this com-pression corresponds to a net loss in configurational entropy.This effect gives rise to a new repulsion potential, Velas(H),related to the restriction of the movement of the hydrophiliccoils extended toward the solvent.

π ϕ δ ρδ δ

δ

δδ

= −

− + + +

⎜ ⎟

⎜ ⎟

⎛⎝⎜

⎞⎠⎟⎛⎝⎜⎜

⎡⎣⎢

⎛⎝

⎞⎠

⎤⎦⎥⎞⎠⎟⎟

⎡⎣⎢

⎤⎦⎥

⎛⎝

⎞⎠

V Ha

MH H H

H H

( )2

ln3 /

2

6 ln3 /

23 1

elasw

22

2

2

(13)

where Δ2 and Mw are the density and the molecular weight ofthe adsorbed polymer. This effect modifies the osmoticpotential, which is now given by

π ϕ χ δδ δ

= − − − ⎜ ⎟⎜ ⎟⎛⎝

⎞⎠

⎡⎣⎢

⎛⎝

⎞⎠⎤⎦⎥V H

av

H H( )

4 12 2

14

lnosm1

22 2

(14)

For the electrosteric stabilization mechanism, both effects(electrostatic repulsion and steric stabilization) must becombined. Normally, the total interaction energy is assumedto be the sum of all attractive and repulsive potentials (eq 6)(see Figure 22).226,241

Experimental log W versus log(electrolyte concentration)plots for the bare cationic latex were fitted using the DLVOtheory, and values of the diffuse-layer potential (ψδ ≈ 18 mV)and the Hamaker constant (A ≈ 7 × 10−21 J) were obtained. Itshould be noted that ψδ and A are related to VR and VA (theLondon−van der Waals interaction), respectively (see Figure23). These values of ψδ and A are in agreement with typicalvalues of both parameters for polystyrene latex particles.In the same way, log W versus log(electrolyte concentration)

plots for cationic latex particles covered with the above-mentioned nonionic surfactant were fitted using the extended


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DLVO theory, and reasonable agreement was found assuming athickness of ∼1.0 nm for the surfactant layer around latexparticles (see Figure 24). Although Triton X-100 (TX100) is anonionic surfactant, the electrical state of the cationic latexparticle−electrolyte solution changes slightly when surfactantmolecules are present at that interface.227

Also, the adsorption of Triton X-100 is used to estimate thehydrophobic character of the cationic latex−solution interface.Typically the maximum amount of nonionic surfactantadsorbed on cationic latexes with amidine groups on theexternal surface is around 2.24 μmol/m2,155,164 which isequivalent to an area per molecule of Triton X-100 on alatex surface of 74 Å2. These values are quite different fromthose obtained with an anionic latex with sulfate anionic groupson the surface, 1.25 μmol/m2 and 135 Å2, respectively. Thisresult indicates that the cationic latex with amidine groups onthe external surface is more hydrophobic than the anionic latexhaving sulfate groups. The cationic latex has two types of sitesfor adsorption, charge sites that interact with the anionic

headgroup of the surface-active agent and hydrophobic sites onwhich the alkyl chains adsorb.Concerning the sequential adsorption of nonionic and

anionic surfactants, Porcel et al.188 accomplished an exper-imental study on the sequential adsorption of SDS and TritonX-100 onto cationic latexes. A comparison between the twosurfactants showed that SDS was more easily replaced thanTriton X-100 when sequential adsorption on the cationic latexwas studied. The electrical state (electrophoretic mobility) ofthe solid−liquid interface depends on the addition sequenceorder of both surfactants. In relation to colloidal stability, whena layer of nonionic surfactant is adsorbed on the surface of thecationic latex, the electrosteric mechanism explains theexperimental results. If SDS is adsorbed, the stabilization orcoagulation of the coated cationic latex particles is aconsequence of the changes in the electrical repulsion betweenparticles. However, when both surfactants are adsorbed, theassumption of additivity is not correct; that is, the electrostaticrepulsion and the steric stabilization (osmotic and elasticcompression) are not totally independent.

3.3.4. Heteroaggregation of Colloidal Dispersions.Heteroaggregation is the aggregation of mixed particle systemswhere the colloidal particles may differ in charge, size, andchemical composition. The phenomenon of heteroaggregationis shown to be important in applications such as mineralflotation, cell recovery, stability of emulsions, paper and cementadditives, or retention aids, and synthesis of engineeringceramics, among others.242−247

Heteroaggregation, however, is not as extensively studied ashomoaggregation, i.e., the aggregation of monocomponentcolloidal dispersions. This may be mainly due to the relativelycomplex interactions between dissimilar particles that theclassical Derjaguin−Landau−Verwey−Overbeek theory cannotaccount for.129,248 While the overlapping of the electricaldouble layers surrounding two like particles is alwaysunfavorable, this is not necessarily the case when unlikeparticles approach each other. At low electrolyte concen-trations, attractive interactions between oppositely chargedparticles can even increase the aggregation rate to values abovethe diffusion limit.249,250 The Hogg−Healy−Fuerstenau (HHF)theory for two dissimilar spheres is based on two assumptions:

Figure 22. Net interaction energy as a function of distance for thePSHEMA latex: (◆) Velas; (+) Vosm; (□) VR; (×) VA; (Δ) V.Reprinted with permission from ref 241. Copyright 1996 Elsevier Ltd.

Figure 23. Stability of cationic latex PS-CAT at pH 6 (□), 8 (Δ), 9(○), and 10 (*). The line represents the theoretical fitting curve.Reprinted from ref 226. Copyright 2001 American Chemical Society.

Figure 24. Theoretical dependence of W on [NaCl]: experimentaldata for bare particles (□) and 2.0 μmol/m2 PS-CAT/TX100 complex(*) at pH 6. Reprinted from ref 226. Copyright 2001 AmericanChemical Society.


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(i) the linear Debye−Huckel (D−H) approximation in whichthe electrostatic potential is assumed to obey the linearizedform of the Poisson−Boltzmann (P−B) equation and (ii) theDerjaguin approximation237 in which the sphere−sphereinteraction energy is obtained from a knowledge of thedistance dependence of the energy per unit area forcorresponding parallel plates by an integration over the surfaceof one sphere. The D−H approximation yields the interactionenergy to quadratic order in the surface potential, andtherefore, the HHF formula can only be applied for sufficientlylow surface potentials.The expressions derived by Hogg, Healy, and Fuerstenau for

the London−van der Waals and electrostatic interactionbetween two dissimilar particles are

= −− +

+− −

+− +− −

⎛⎝⎜

⎞⎠⎟

V rA a a

r a aa a

r a a

r a ar a a

( )6

2( )

2( )

ln( )( )

A1 2

21 2

21 2

21 2

2

21 2

2

21 2

2(15)

επ ψ ψ

ψ ψ

=+

+ +

+ − −

κ

κ

− − −

− − −

V ra a

a a( ) {( ) ln(1 e )

( ) ln(1 e )}

r a a

r a a

E1 2

1 201 02

2 ( )

01 022 ( )

1 2

1 2 (16)

a1 and a2 are the particle radii, ψ01 and ψ02 are the surfacepotentials, r is the center to center distance, and κ is the Debyereciprocal length, which depends on the solvent ionicconcentration.Ohshima et al.251 have extended the HHF results to

moderate potentials by first obtaining an expression for theinteraction between plates correct to the sixth power in thesurface potentials and then using Derjaguin’s method to givethe result for the interaction between spheres. Derjaguin’smethod, however, is applicable only for large particle radii andfor small particle separations. Using an integral equationmethod devised by McCartney and Levine252 for theinteraction between two similar spheres, Bell et al.253 derivedan improved expression for the interaction between dissimilarspheres, which tends to the HHF formula at small separationsand has the correct asymptotic behavior at large separations.The utility of the improvement of Bell et al. is that it isuniformly valid for both small and large separations providedthe surface potential is low. Although curvature corrections areincluded to some extent in their treatment, Ohshima et al.254

have derived the exact expression for correction terms due tocurvature effects, without using Derjaguin’s method, butconsidering that the surface potentials on the particles remainconstant during interaction and are small enough to apply thelinear D−H approximations to the P−B equation. Nevertheless,the differences between the exact and approximated (HHF)solutions are habitually within the experimental errors of themeasurements involved in any heteroaggregation experiment.Probably, it is due to the fact that between interacting spheresthe real interaction potentials are the “effective” and not thesurface potentials. However, there are some doubts about thevalidity of the HHF theory when the sphere radii are verydifferent in magnitude.255 The HHF approximation is accuratefor large κa (κ is the Debye screening parameter, and a is thesphere radius) and small κh (h is the distance of closestapproach between the spheres), but Sader et al.256 havepresented a modified HHF approximation which is accurateeven for moderated κa values and for all κh ranges of practical

interest. The formulas derived by these authors are very simple,but accurate, analytic expressions for the electrical double layerand interaction free energy between two spherical colloidalparticles valid up to the moderate- to high-potential regime andall κh values.There are many interesting phenomena in electrostatic

heteroaggregation which were discovered in the past decadeusing simulation methods257−262 and single-cluster detectiontechniques263 for monitoring the time evolution of the clustersize distribution. Puertas et al.264 reported an interesting effectarising at very low electrolyte concentration in simulated 1/1mixtures of equally sized particles with opposite electric surfacecharge. They found that the cluster concentration profilesexhibit a noncontinuous behavior at relatively long aggregationtimes. In other words, clusters differing by only one constituentparticle behave quite differently. They named this effect clusterdiscrimination. The first experimental support for thishypothesis was reported a few years ago.265 Clusterdiscrimination was found experimentally in heteroaggregationprocesses arising in 1/1 mixtures of positive and negativepolymer colloids at low and very low ionic concentrations.Monomer discrimination could be detected already at 10−2

mol/dm3 KBr, while dimer discrimination started to appearonly for electrolyte concentration smaller than 10−3 mol/dm3

(see Figure 25). This shows that cluster discrimination is not an

intrinsic property of pure heteroaggregation processes since it isnot fully developed as soon as homoaggregation processes arecompletely absent. Furthermore, it is observed that fordecreasing ionic concentrations dimer discrimination isbecoming more pronounced. This finding implies that clusterdiscrimination is most likely related to the range of theattractive electrostatic interactions between the oppositelycharged colloids. The experimental results were also comparedwith the Brownian dynamics simulations (BDSs) performed byPuertas et al.264 Not only qualitative but also quantitativeagreement was observed when the adequate normalizationswere performed. Especially, the onset and the increasingstrength of dimer discrimination were predicted quitesatisfactorily by the BDS. In their simulations, Puertas et al.found that cluster discrimination gives rise to an odd−evenbehavior in the cluster concentration profiles; i.e., odd size

Figure 25. Cluster concentration profiles at fixed time (t0 ≈ 2 × 104s)for different KBr concentrations: 1.0 M (left-pointing triangle) 10 mM(◇); 1.0 mM (▽); 0.1 mM (Δ); 0.01 mM (○); no added KBr (□).Reprinted with permission from ref 265. Copyright 2004 AmericanPhysical Society.


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clusters become dominant in the cluster size distributions(CSDs). Experimental data confirmed this prediction formonomers and dimers. Hence, the good agreement betweenexperiments and simulations supports the hypothesis that thecluster discrimination phenomenon originates mainly from thelong-range electrostatic interactions.Other interesting phenomena are predicted to occur in

electrostatic heteroaggregation processes as a function of therelative concentration of the two reacting species. For instance,Lopez-Lopez et al.266 have studied by means of off-latticesimulations the binary diffusion-limited cluster−cluster aggre-gation (BDLCA) processes. The fundamental role played bythe relative concentration, x, was investigated for both shortand long aggregation times.At short aggregation times, the predominant reaction is

dimer formation due to bond formation between two unlikeparticles. In this region, the effective dimer formation rateconstant, kS(x), follows the parabolic behavior predicted by theHHF theory.At long aggregation times, the aggregation behavior is highly

dependent on x. For x > xc ≈ 0.15, aggregation continues untila single cluster is formed. In this region, the time evolution ofthe CSD is somewhat similar to the well-known diffusion-limited cluster aggregation (DLCA) processes. The maindifference was found to be an excess of monomers that isobserved even for x = 1/2. This monomer excess seems to beidentical to the monomer discrimination mentioned above. Inother words, these BDLCA simulations show that monomerdiscrimination may occur even in the absence of any particle−particle interaction (see Figure 26).At x values close to xc, these authors found an atypical time

evolution for oligomers composed of 8−10 particles. Theirnumber reached two maxima corresponding to two differentcompositions: several minority particles per cluster at shorttimes and just one minority particle per cluster at long times(see Figure 27). This behavior was not reported for on-latticeBDLCA simulations.257,267,268

At relative concentrations below xc, stable aggregatescontinue to diffuse in the system and a single cluster is neverformed. In summary, the proposed scheme for BDLCAprocesses for relative concentrations below xc comprises thefollowing five stages:

(1) HHF stage. Fast reactions between unlike monomersform dimers.

(2) Seed formation stage. Dimers continue to be formed.They also grow by adding further majority particles andtherefore become first-order seeds. This stage ends whenall free minority monomers have disappeared.

(3) Seed aggregation stage. Some first-order seeds reactamong them, forming higher order seeds. These seedskeep growing by adding majority monomers.

(4) Seed completion stage, The seeds are so highly coveredthat they cannot react any longer among themselves.Nevertheless, they still can grow by adding majoritymonomers.

(5) Stable aggregate stage. All clusters are completely coatedby majority particles. Aggregation comes to an end.

This aggregation scheme is representative of all the simulatedBDLCA processes for relative concentrations clearly below xc.However, the moments at which these stages start and enddepend on the initial relative concentrations.

Theoretical models based on Smoluchowski’s theory269 weredemonstrated again to be an important tool to rationalize thekinetic properties in the heteroaggregation of equimolarmixtures.270 The situation changes completely if the binarycharged colloidal system possesses large asymmetries betweenthe numbers of positive and negative particles. Recent studieson the heteroaggregation of these type of systems reveal theexistence of novel, interesting phenomena, such as theformation of clusters with a high stability and the appearanceof two peaks in the time evolution of the concentration ofcertain sorts of clusters (two-hump effect).271 The appearance

Figure 26. Cluster size distribution up to 10-mers, ni(t) (thin dashedlines for odd i and thin solid lines for even i), and the overall numberof aggregates, M1(t) (thick solid line), at initial relative concentrationsof (a) x = 0.50, (b) x = 0.15, and (c) x = 0.05. The numbers indicatethe number of constituent particles of the clusters. Reprinted withpermission from ref 266. Copyright 2005 American Physical Society.

Figure 27. Time evolution of the composition detailed cluster sizedistribution for octamers, n8

l(t), at x = 0.15 and l = 1 (thin solid line), l= 2 (dashed line), and l = 3 (dotted line). The number of octamers,n8(t), is also plotted (thick solid line). Reprinted with permission fromref 266. Copyright 2005 American Physical Society.


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of the two-hump effect in the cluster size distribution isobserved in electrostatic heteroaggregation processes at lowelectrolyte concentration with large asymmetries in the relativeparticle concentration (but similar sizes and surface potentials)by means of Brownian dynamics computer simulations andexperimental measurements. The study of the time evolution ofthis type of aggregating system demonstrates that severalcluster formation mechanisms with different characteristics timescales are involved.272 The results obtained by these authorsshow that the internal composition of the clusters is the mainaspect determining the time evolution of the process. Theheteroaggregation of binary charged colloidal systems at highparticle concentration asymmetry leads to new, fascinatingphenomena with no analogues in the homoaggregationprocesses: the formation of clusters of high stability and theappearance of multiple peaks in the cluster size distribution.The heteroaggregation between particles with different

particle sizes and chemical compositions is also possible.Different authors using thermodynamics and kinetic ap-proaches have studied the colloidal stability of binary latexdispersions of similar chemistry but varying size. Shenoy etal.273 derived a formally correct expression for the effectivedoublet stability ratio (Weff) of a bimodal system 100 and 200nm in diameter. It accounts for the difference in particle sizeand extends the HHF theory. Others authors, such as Borkovecet al.,274−276 studied the early stages of heteroaggregation oflatexes with different sizes experimentally. A novel multianglestatic and dynamic light scattering device was used as apowerful tool to probe the particle aggregation process in situ.These authors were able to discriminate the rate constants forA−A, B−B, and A−B particle aggregation.274 In the first stageof their study, the more general situation, where homoag-gregation and heteroaggregation occur simultaneously, wasexcluded, but a little later Borkovec et al.276 also addressed thissituation.The values of the apparent and absolute heteroaggregation

rate constants, kAB, obtained by static light scattering (SLS) andDLS were equal within experimental error for the three types ofbinary samples used.274 The mixing ratio of the particle radiiranged from 0.5 to 0.8, the cationic latexes being of smaller size.In their experiments, the ionic strength was 10−4 mol/dm3 andthe pH was adjusted to 4 by adding HCl. Under theseexperimental conditions, only heteroaggregates were formed,and no homoaggregation took place. The values of the absoluteheteroaggregation rate constants obtained by multiangle DLSare within the range of 5.28 × 10−18 and 6.01 × 10−18 m3 s−1.These values can be considered as independent of the mixingratio. Subsequent technical improvements in the opticaltreatments of the experimental data did not significantlychange the values of kAB obtained by the same authors.276

Time-resolved multiangle SLS and DLS are able to measure theheteroaggregation rate despite the simultaneous formation ofhomoaggregates. Yu and Borkovec277 measured the early-stageaggregation process between positively charged amidineparticles 67 nm in radius (particle A) and negatively chargedsulfate particles 84 nm in radius (particle B) dispersed in KClelectrolyte solutions at pH 4. This means that kAA, kBB, and kABwere simultaneously determined. At 10−4 mol/dm3 KCl thehomoaggregation rate constants were negligible, whereas kABbecame 5.42 × 10−18 m3 s−1. At 0.3 mol/dm3, however, kAA andkBB were 4.90 × 10−18 and 3.80 × 10−18 m3 s−1, respectively,and kAB became 3.21 × 10−18 m3 s−1. In a very interestingexperimental work, Olsen et al.277 observed that in the

aggregation of cationic latex particles (R2) by the addition ofsmaller anionic latex particles (R1) the size ratios (R2/R1) candetermine the monomodal or bimodal (two peaks in theaggregation rate) aggregation rate response. The expectedmonomodal response is only found for smaller particle sizeratios (R2/R1 ≤ 0.13), whereas the bimodal response is mostnotable for intermediate particle size ratios, R2/R1= 0.36 and0.49 being the clearest. Probably, the explanation of this curiousbehavior is given by the different weights of the collision rateconstant and collision efficiency in the rate of aggregate growthdepending on the particle size ratios.Concerning heteroaggregation processes due to differences

in the chemical composition, we can distinguish three differenttypes of studies:(1) Heteroaggregation between hard and soft particles.278,279

An interesting case of heteroaggregation between hard and softparticles was studied by Islam et al.,280 where a variation oftemperature is the cause of the heteroaggregation between acationic PNIPAM microgel and an anionic polystyrene latex.The mixed dispersions are colloidally stable at 20 °C, whereasat elevated temperatures (50 °C) a heteroaggregation of thedispersion takes place within certain concentration ranges ofmicrogel particles. By using a surface-masking technique, basedon the heteroaggregation of small (thermosensitive microgel)and large colloidal (latex) particles, Bradley and Rowe281 haveprepared Janus (two faces with different surface properties)microgels.(2) Heteroaggregation between hard and hard par-

ticles.282,283 The attractive interaction between hard particleswith opposite charges is used for monolayer formation uponself-assembly of monodisperse anionic latex particles andmultilayer formation upon alternating self-assembly of cationicand anionic latex particles at positive glass supports.284 The self-assembled multilayers were highly porous and exhibited a verylarge surface area, which makes them attractive as separationlayers, filters, and supports for catalysis. With growth of thepolyelectrolyte and adsorbance of neutral polymer on latexparticles, the hard behavior of the colloidal particles can bechanged; this is the way to systematically prepare hairy colloids.The most interesting aspect of the heteroaggregation of hairycolloids is their fully reversible assembly in aqueoussolutions.285

(3) Heteroaggregation between soft and soft particles.286,287

Probably, microgels are the best model soft particles given thatthey show soft repulsive interactions arising from repulsionbetween hydrated polymer hairy chains located at the exteriorof the particles. In comparison with the heteroaggregationbetween hard particles, binary mixtures of soft particles havepossibilities of exhibiting phase behaviors that have never beenobserved before using hard particles. In this sense, Suzuki andHorigome288 have very recently reported the phase behaviorsof binary mixtures composed of temperature-sensitive cationicand anionic gels. Both microgels were synthesized by aqueousfree radical precipitation polymerization using N-isopropyla-crylamide and N,N′-methylenebisacrylamide but using differenttypes of water-soluble initiators and comonomers. The mostinteresting result found by these authors is that the presence ofa small amount of electrolyte altered the dispersing behavior ofthe binary mixture when each microgel was in its hydratedswollen state. Furthermore, the addition of a small amount ofsalt prevented the binary mixtures from flocculating, resulting innon-close-packed structures on a planar substrate in the drystate, which is similar to single-species microgels. Adding a


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small amount of salt and gently stirring could redisperse theslow flocculation of microgels. These tunable properties areascribed to the existence of electrostatic interactions and sterichindrance of hydrated polymer chains at the exterior of themicrogels.Also, Hou et al.289 have investigated the effects of cations on

the sorting of oppositely charged microgels. They usedthermally sensitive anionic microgels of poly(N-isopropylacry-lamide-co-sodium acrylate) and cationic microgels of poly(N-isopropylacrylamide-co-(vinylbenzyl)trimethylammoniumchloride) as a simple model to understand the role of ions oncell-sorting. The most relevant conclusion of this study on theheteroaggregation of those microgels in the presence ofdifferent cations is that the adhesive property plays animportant role in the sorting of oppositely charged microgels.Microgels can be considered as stimulus-responsive, or smart,

materials. According to Bradley et al.,290 a smart material is onethat can sense a stimulus from its surroundings, e.g.,temperature or light, and react to it in a useful, reliable,reproducible manner. Smart materials therefore respond tochanges in the environment in a rather predictable manner, andsome have a memory as they revert back to their original stateonce the stimulus is removed. The dispersion behavior of smartparticles can be manipulated by external stimuli such astemperature, pH, electric/magnetic fields, or light. The above-mentioned authors have recently reviewed the studies onheteroaggregation where at least one of the particles is astimulus-responsive smart colloid.A new challenge in the use of oppositely charged microgels is

the preparation of pH-triggered physical gels. McParlane etal.291 were the first to investigate the formation of dual pH-triggered physical gel (soft particle glass) using concentratedmixed dispersions of heteroaggregated pH-responsive microgels(poly(ethyl acrylate−methacrylic acid−1,4-butanediol diacry-late) (PEAMAA) and poly(2-nonylpyridine−divinylbenzene(PVP)). The phase diagram for the mixed PEAMAA/PVPheteroaggregates showed that glasses were obtained at bothhigh and low pH values. From pH 4.0 to pH 6.4, theheteroaggregated microgels flowed after tube inversion andwere weak gels. As these authors emphasize, the experimentaldifferences between these particle gels and glasses were theability of the particle gels to flow and low elasticities. It shouldbe noted that the phase diagram of the heterodispersion is thesum of the individual phase diagrams for each microgel. Thisimplies that it is the pH-triggered swelling of the microgelparticles that is responsible for the onset of gel formation forthe mixed systems.In the first case of heteroaggregation between hard and soft

particles, negatively charged polystyrene latex and positivelycharged microgel particles of similar size and surface chargewere used. The structures of the aggregates as well as theaggregation kinetics were investigated.278 Perhaps the mostinteresting aspect of this investigation is that the differences inthe homoaggregation rates of both types of particles permit theformation of large clusters formed by microgel particles (ormicrogel−polystyrene particles), while the smaller clusters arecomposed of polystyrene particles.In the second case, the structure of the heteroaggregates was

studied as a function of the mixing ratio.For the third case, the use of thermosensitive microgels287

adds temperature as a new variable in the formation ofheteroaggregates. Some authors have prepared, using thestepwise heterocoagulation concept, nanocomposites292 and

fluorescent and highly magnetic core−shell polymer par-ticles.293 Obviously, this technique relies on electrostaticinteractions to induce the coating of small particles ontolarge ones. In the same direction, Han et al.294 have recentlystudied the effect of the surface charge density and particle sizeof both opposite latex particles on the formation ofheteroaggregates which are used as anionic ion exchangeresins. On one hand, Vincent et al.295 and Goodwin andOttewill et al.296 were the pioneers in the study of theadsorption of small cationic polystyrene particles onto largeanionic polystyrene ones. On the other hand, Furusawa etal.297,298 were the first researchers to apply this procedure to theelaboration of silica−latex composites and magnetic particles.An alternative strategy to insert mutual interactions among thecolloidal components was used by Bayer et al.299 This strategyis based on the coverage of colloidal particles withcomplementary H-bond patterns. The simplest complementaryH-bonding motif in this sense is to cover one colloidalcomponent with an H-bond donor and the other colloidalcomponent with a H-bond acceptor. They synthesized 4-hydroxylstyrene-functionalized cross-linked colloids as onecomponent and 4-vinylpyridine-functionalized cross-linkedpolystyrene colloids as the second component. Polymerizationwas carried out by means of surfactant-free emulsionpolymerization. If both latexes are directly combined as asuspension in CHCl3, a fast heteroaggregation is observed.In heteroaggregation of binary particle systems an exciting

challenge is the determination of the cluster composition. Thiscan be achieved if particle populations are marked (e.g.,fluorescently labeled). Rollie and Sundmacher300 determineddynamically by flow cytometry the cluster composition of abinary particle mixture of oppositely charged polystryreneparticles and rhodamine B-labeled melamine−formaldehydeparticles. The cluster composition is mainly dependent on theionic strength and particle number ratios.Studies on heteroaggregation processes in two-dimensional

systems are very scarce301 in comparison with the number ofworks published on this topic for three-dimensional systems.Only a few simulations have been done regarding the effect ofdifferent particle sizes (binary colloidal monolayers) on two-dimensional heteroaggregation.302−304 There are practically noexperimental studies on this topic; only Ristenpart et al.305 usedan ac electric field to assemble planar superlattices of binarycolloidal suspensions on an electrode. They observed triangularor square-packed arrays depending on the field frequency andrelative particle concentrations. The structure formation inbinary colloids observed by Ristenpart et al. was studiedtheoretically by Varga et al.306 These authors found that thetotal concentration of particles, the relative concentration, andthe relative dipole moment of the components determine thestructure of the colloid. At a low concentration of particles,aggregation leads to fractal structures. For increasingconcentrations, a crossover to lattice structures was observed.The results obtained by these authors at high concentration arein good agreement with those found experimentally.It is worth pointing out that heteroaggregation is an

emerging scientific topic, and much work still remains to bedone in this field. Some important subjects involving theaggregation of binary colloidal systems are, for instance, theheteroaggregation of nonspherical particles, the aggregation ofpatchy colloids, and the exploration of the phase diagrams ofbinary colloidal systems with opposite charges at differentnumber ratios, x. The experimental observation of the BDLCA


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regime and the heteroaggregation of charge-asymmetriccolloidal mixtures in two dimensions (particles trapped at theliquid−fluid interface or confined between walls) are also ofgreat interest.271 On the other hand, the heteroaggregation withor the deposition onto natural colloids (inorganic solids, smallorganic compounds, and larger rigid biopolymers), followed bytheir sedimentation from the water phase, is the main removalmechanism of nanoparticles (CeO2, for example) in naturalwater.307

3.3.5. Colloidal Monolayer Formed by Cationic LatexParticles at the Air−Water Interface. Recently, the behaviorof colloidal dispersions confined in 2-D geometry has drawnwide interest. From an experimental point of view, there aredifferent forms of 2-D systems. For example, a dispersion ofcharged particles bound by two charged plates of the same signconstitutes a 2-D system since the electrostatic repulsion makesthe particles remain confined at the intermediate plane betweentwo walls.308 Also, a system consisting of particles trapped atthe air−liquid or liquid 1−liquid 2 interface can be consideredto be a 2-D system.309−311 The formation of colloidalmonolayers is especially interesting because of the ability ofparticles to affect the stability of emulsions, foams, andinterfacial properties. Also, in this case, the number of workswith cationic latex particles is much less than that with anioniclatex particles.312,313

Once cationic latex particles are dispersed at the interface(air−water, usually), they remain trapped because of the surfacetension effect and electrostatic forces. In two-dimensionalaggregation, additional theoretical and experimental complex-ities appear in comparison with coagulation of bulk colloidalsuspensions (see Figure 28).314 On one hand, since the

particles are trapped at the interface, the interaction forcebetween them depends on their degree of wetting.315

Consequently, the interaction between the wet parts of thecolloidal particles is quite different from the interactionbetween the external parts, and the theoretical treatment fordetermining the energy requires numerical computation.314

Moreover, from an experimental point of view, the depositionprocedure of colloidal particles at the interface is a delicateprocess due to the difficulty of avoiding initial heterogeneitiesand fluctuations in the particle surface dispersion.In some works, the kinetic and morphological behavior of

colloidal aggregation for cationic latex particles confined at theair−water interface is studied as a function of the saltconcentration (KBr, Na2SO4, and Na3PO4).

312,313 The so-called CCC, i.e., the salt concentration at the transition fromslow coagulation (many collisions are necessary for twoaggregates to stick together; this is the reaction-limited clusteraggregation (RLCA) regime) to rapid coagulation (aggregationcontrolled only by the diffusion time of the aggregates, theDLCA regime), was determined experimentally by Moncho-Jorda et al.313 from both kinetic and structural properties, and agood accordance between both results was achieved. Theseexperiments showed that the valence of the counterions did notaffect the qualitative behavior of the aggregation properties, butit already changed the CCC inversely to the value of thevalence (see Figure 29).Although similar aggregation behavior for the CCC is

expected in the two-dimensional case, actually there is a bigdifference in relation to three dimensions. In two dimensions, alarge amount of salt is necessary for inducing particlecoagulation (1 mol/dm3 vs 0.15 mol/dm3 KBr, for instance).313

To explain the large stability of colloidal monolayers observedeven for very high salt concentrations, Robinson andEarnshaw315 proposed the existence of dipole−dipole repulsiveinteractions. According to them, the dipoles are surface chargesat the top of the particle that have trapped a counterion fromthe solvent during the initial turbulent spreading. Followingthese authors, this interaction is responsible for the non-isotropic forces between clusters formed in aggregationprocesses in 2-D. However, other experimental results311

showed that 2-D structured colloidal monolayers formed atthe air−water and oil−water interfaces cannot be explainedonly considering dipolar repulsive interactions; therefore, alonger range interaction potential is necessary. Quesada-Perezet al.316 carried out experiments on stable colloidal monolayersat the air−water interface, and they showed that the particleinteraction potential manifests a long-range repulsive barrier(close to 7 times the particle diameter). In that study, they alsosuggested the possibility of Coulombic electrostatic forces (i.e.,monopolar forces) as well as dipolar forces. This suggestion wasconfirmed317 using molecular dynamics to show that the long-range repulsive interaction between particles at the oil−waterinterface is principally caused by monopole−monopoleCoulombic interactions. In a theoretical model developed byMoncho-Jorda et al.,312 long-range interactions are accountedfor by means of two different interactions: (i) dipole−dipoleforces that control the aggregation at high ionic strength and(ii) monopole−monopole Coulombic interactions that governstability at salt concentrations lower than the CCC. Moreover,the results showed that the fraction of monopoles ( fmon) is themain parameter controlling kinetics in 2-D aggregation, andhence, a CCC can be defined from the salt concentration atwhich fmon becomes zero.

3.3.6. Deposition of Cationic Latexes. In general,deposition318 plays a vital role in many technological andnatural processes, such as thoses involving drugs, cosmetics,detergents, paper making,319 bacterial adhesion, carrierflotation, colloidal contaminant transport, filtration, andsemiconductors. The initial deposition process is generally

Figure 28. Sketch of the colloidal particle arrangement at the interfacebetween phases 2 and 3. Calculations of the fraction included in eachphase are different for the θ < π/2 (A) and θ > π/2 (B) cases. The fatblack line (C) indicates the wetted part of the particle when r > l.Reprinted with permission from ref 314. Copyright 2000 Elsevier Ltd.


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described by a set of transport equations, also known asFokker−Planck equations, which take into account particle−substrate surface interactions. During this stage a constant rateof particle deposition is generally observed. In contrast, thelater stages of deposition are thought to be governed bydesorption, surface heterogeneity, blocking of deposition sites,and excluded area effects. Colloidal forces dominatingdeposition processes and kinetic processes involved have

been widely reviewed.320 However, the role of some importantfactors, such as discreteness of the charges and their possiblerearrangement, in deposition has been scarcely considered.Only Somasundaran et al.318 studied the influence of thesurface heterogeneous nature of the latex particles andsubstrates (frosted glass slides) and the dynamics involved indeposition under free settling conditions using different typesof latex particles (including cationic and zwitterionic latexes)and discussed the results in terms of relevant colloidal forces(electrical double layer and van der Waals interactions,gravitational force). Conventional deposition was found foramidine latex particles, with electrostatic forces playing adominant role. This means that the presence of an attractiveelectrostatic force led to a favorable deposition of amidine latexparticles, whereas that of a repulsive electrostatic force led tothe absence of deposition. Nevertheless, a good deposition ofzwitterionic particles was observed in spite of the presence ofan apparent electrostatic repulsion. The authors explain thisanomalous deposition as due to the reconformation of themixed hairy charged groups.Aizenberg et al.321 have used substrates chemically micro-

patterned with anionic and cationic regions to govern thedeposition of charged colloidal particles (negatively andpositively charged latex particles of around 1 μm). Thismethod of self-assembly of colloidal particles onto a patterned(e.g., lithographically modified) substrate is named “colloidalepitaxy”. According to these authors, the direct observation ofthe colloidal assembly suggests that this process includes twosteps: an initial patterned attachment of colloids to thesubstrate and an additional ordering of the structure upondrying. This approach to the colloidal epitaxy makes it possibleto fabricate complex, high-resolution two-dimensional arrays ofcolloidal particles. Revut and Us’yarov322 have studied the roleplayed by different electrolytes in the deposition of cationiclatex particles on flat surfaces; divalent counterions were foundto have a stronger influence on this process than univalentcounterions. The results indicated that the duration of theadhesion bond is determined by the concentration and natureof the ions in the solution.AFM can be used to investigate the adsorption behavior of

cationic latex particles on mica.323 Particularly, this techniquepermits useful information on the initial kinetics of theadsorption and microstructure of adsorbed particles to beobtained.Alince et al.324 studied the deposition of cationic latex

particles on cellulose fibers. This process may be described asmutual interaction of unlike particles (heterocoagulation) interms of the HHF theory for sphere−plate interaction. The rateof deposition of colloidal particles on a solid substrate in theabsence of a potential barrier (i.e., when particles are ofopposite sign or uncharged) is closely controlled by diffusion.However, differences are noticed in the colloidal behavior ofhard, nonfilming (polystyrene) and soft, film-forming (poly-styrene−butadiene) latexes. The hard latex deposits asindividual particles at a rate that is apparently diffusioncontrolled. The deposition of the sof t latex indicates twoconcurrent mechanisms. The initial deposition of individualparticles is accompanied by aggregation of the depositing latex.The rate is likely affected by the latex’s tendency to coagulate,and full deposition of the homocoagulating latex is observed.The explanation of this different behavior is sought in interplayof attraction forces acting on deposited latexes. The hard latexis apparently not in intimate contact with the fiber surface, and

Figure 29. Kinetic exponent, z, as a function of the salt concentrationfor (a) KBr, (b) Na2SO4, and (c) Na3PO4. The CCC is obtained fromthese plots as the salt concentration at which z reaches its maximumvalue, close to 0.6. Reprinted with permission from ref 313. Copyright2002 Elsevier Ltd.


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when the attractive double layer interaction is diminished dueto the salt effect, the attraction is outbalanced by thermalmotion and fluid shear. The soft latex, however, can establish abetter contact with fibers, and the attraction force over a shorterseparation distance resists the hydrodynamic forces.3.3.7. Cationic Latexes as Catalyst Supports. Catalysts

play a major role in many biochemical reactions and industrialprocesses. To allow continuous operation, an importantrequirement for industrial processes, many catalysts havebeen immobilized onto insoluble, often porous, supportingparticles. In addition, economics and environmental legislationfavor such immobilization. However, the catalytic activity oftendecreases on immobilization as a result of mass transportlimitations. Recently, much attention has been paid to theimmobilization of catalysts without the loss of reactivity.325

Polymer colloids (latexes) offer interesting possibilities ofcombining these two requirements. Obviously, our attention isfocused on the use of cationic latexes as catalyst supports.Catalysis can be carried out in or on cationic latexes. In the firstcase, cationic latexes promote hydrolyses and oxidation oforganic compounds in aqueous dispersions by concentratingreactants and catalysis into the small volume of the latex phaseand by increasing the intrinsic rate constants.326 However, inthe second case, cationic latexes are used as simple supports,which exhibit a high surface area due to the small particlesize.104,325,327−329 Polymer colloids were used as catalystsupports for the air oxidations of phenols, mercaptans, andalkyl aromatic hydrocarbons, for various oxidations of alkenes,and for the decarboxylation of 6-nitrobenzisoxazole-1-carbox-ylate.330,331

Polymer colloids substituted with quaternary ammoniumions are highly active supports for catalysis of reaction oforganic compounds when one of the reactants or a catalyst is ananion that binds strongly to the particles.332,333 These swollencationic latexes are used in aqueous dispersions as supports foro-iodosobenzoate (IBA)-catalyzed hydrolysis of p-nitrophenyldiphenyl phosphate (PNP-DPP), and the highest second-orderrate constant exceeded by a factor of about 2 the maximumvalue reported for IBA in CTAC micelles.334,332,335 All theresults obtained in these studies are qualitatively consistent withan ion exchange model of catalysis in which IBA competes withchloride ion and buffer anion for polymer binding sites and thecatalysis reaction rates depend primarily on the intrapolymerconcentrations of PNP-DPP and IBA catalyst.Also, cationic latexes prepared by emulsifier-free polymer-

ization of styrene and 1-methyl-4-vinylpyridinium bromide(qVPBr) have been used as cocatalysts in the autoxidation of 2-mercaptoethanol in the presence of cobalt(II) phthalocyaninetetrasodium sulfonate (CoTSPc). It was found that all systemsstudied enhanced the catalytic activity compared with thepolymer-free CoTSPc-containing system.325 To improve thecatalyst activities of CoTSPc on cationic latexes, Twigt et al.104

prepared them by the shot-growth method and using the ioniccopolymer poly(styrene-co-1-methyl-4-vinylpyridinium bro-mide) (PS−qVPBr) as the emulsifier. Effectively, an increasein the cocatalytic activity of these hairy latexes in the CoTSPc-catalyzed oxidative coupling of 2-mercaptoethanol was foundcompared with that of the latex prepared batchwise. Schipper etal.329 found an improved method for the mercaptoethanolautoxidation using cationic latexes with short ionene blockswith seven quaternary ammonium groups at their particlesurface.

Furthermore, the cationic latex-bound cobalt(II) complex ofN,N′-ethylenebis(salicylaldimine-5-sodium sulfonate) showedhigh catalytic activity in the autoxidation of 2,6-dibutylphenol inwater in comparison with the conventional polymer-freesystem.327 Furthermore, the complex was useful in thepromotion of nucleophilic displacements of carboxylate anionon alkyl halides in aqueous medium.327 Nevertheless, thecolloidal catalyst showed some loss of activity after successiveruns, which is probably associated with the coagulation of thelatexes used.

3.3.8. Film Formation with Cationic Latexes. Theprocess of transforming a stable dispersion of colloidal particlesinto a continuous film with the same cohesive strength of thebulk material is complex and is usually described in threesequential steps: drying, particle deformation, and diffusion.Particle coalescence and film formation occur if the dryingtemperature is above the polymer glass transition (Tg) or if asmall amount of coalescing solvent is present.336 Therefore, thephysical properties of the film develop after dehydration forcesthe particles into contact. The particle compaction ordeformation step in latex polymer film formation during dryinghas received continuous theoretical and experimental attentionsince the seminal work of Bradford and co-workers337,338 in theearly 1950s that modeled film formation as a Frenkel viscousflow of contacting polymer spheres under polymer−air and/orpolymer−aqueous-phase interfacial tension. Shortly thereafter,Brown339 made compelling arguments that the role of liquidwater was not only contributory but also central to thedeformation process. The principal force was proposed to becapillary compression (which is proportional to the water−airinterfacial tension and inversely proportional to the radius ofthe spheres) of the particle assemblage with water evaporation,controlled by the latex serum−air surface tension, against thedeformation resistance of the polymer characterized by itsviscoelasticity. Virtually all work since has comprised attemptsto variously refine, extend, verify, or refute Brown’s theory andpremises and to propose alternatives, but forces arising fromsurface energies involving aqueous latex serum persist in themodels.340 Vanderhoff et al.,341,342 however, consider that thedriving force for coalescence under the limit conditions for filmformation is the particle−water interfacial tension. Laplace’sequation is used to show that a pressure gradient exists, whichpushes matter from the central part of the particle to theinterparticle contact area. This is due to the very small radius ofcurvature at the edges of the contact zone. The third importanttheory is one proposed by Sheetz.343 According to this author, athin layer of coalesced particles is formed closer to the surfaceof the drying latex. The remaining water evaporates afterdiffusion through this polymer layer, and the packing ofparticles is compressed as if by a piston. Capillary forces ensurethe coalescence of the surface layer. The results obtained byDobler et al.344 support Sheetz’s theory of coalescence wheredeformation of particles is due to compression of the packing ofspheres by evaporation of water through a continuous polymersurface layer permeable only to water vapor.In the particle deformation and compaction stage of latex

polymer film formation, the principal variables are (i) thepolymer composition, (ii) the particle size, (iii) time, and (iv)the water content of the deposited film and in the dryingenvironment.The literature on polymer film formation is extensive, and a

recent review summarizes the works on this.345 Also, in thiscase, the use of cationic latexes is relatively novel. The


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minimum film formation temperature (MFFT) is a much-employed technique to investigate latex polymer filmformation. MFFT is usually defined as the higher value of thetemperature of the transition from a turbid or cracked film to aclear and coherent film.Latex-based formulations that rapidly develop mechanical

integrity before appreciable dehydration has occurred are oftendesirable to prevent flow after application. One way to reducethe flow upon application is to reduce the amount of water inthe latex formulation; however, this also increases the viscosityof the formulation, which quickly leads to undesirablerheological properties for the application. Therefore, latexcompositions that are stable in storage and rapidly “set” at theappropriate time without the addition of other materials(“single-pot” systems) are desirable to simplify the use of theseproducts.Several methods of decreasing the set time using pH changes

have been demonstrated. Rose et al.336 have introduced acontrolled ionic coacervation (CIC) process that rapidly formsuniform, gel-like latex films with significant mechanical integritywithout loss of water from the film. This process uses latexparticles that contain both strong cationic charges and weakprotonated acid groups. The coacervation is defined as anyprogress that causes the particles of a dispersed system toagglomerate in large numbers, which includes precipitation,gelation, flocculation, and coagulation.346 Schmidt et al.347

introduced the concept of a “controlled ionic coacervation” andthey defined CIC as “a controlled aggregation of solublepolymer molecules without precipitation to yield clear, rapid-setfilms”. This concept was extended by Rose et al.336 to latexparticles containing both acidic and cationic functionalities, soCIC is a “controlled aggregation of latex particles withoutcoagulation or phase separation”. Ionization of the weak acidgroups initiates the CIC process for these latexes. Thus, anylatex whose particles have fixed cationic charges and weak acidgroups is potentially a “coacervate latex”. After ionization of theacid groups, the coacervating latex rapidly develops mechanicalintegrity even before significant water loss. Furthermore, duringthis CIC process and throughout the dehydration process, thelatex maintains a homogeneous, opaque, gel-like appearancethat, upon drying, yields a clear, uniform film. This wasdemonstrated experimentally by Rose et al.,336 where the CICprocess does not require a water-soluble polymer to obtain therapid-set film properties. Also, the mechanism for the CICprocess is consistent with models for rapid, irreversible,particle−particle aggregation.The self-organization of cationic latex particles on different

substrates was reported by Watanabe.348 Annealing at temper-ature above the Tg of the latex particles enhanced the adhesivestrength of particle monolayers.

4. CATIONIC MICRO/NANOGELSAnother type of nanoparticle is the microgel or nanogel. Micro/nanogels are cross-linked colloidal particles which can swell byabsorption (uptake) of large amounts of solvent, but they donot dissolve due to the constituent structure of the polymericnetwork, physically or chemically cross-linked.349 Some authorscall them “smart” gels, but their behavior is governed by thesolution thermodynamics of the cross-linked polymeric chains.In this way, their “intelligence” is relative, and in general, theirbehavior (swelling/deswelling) depends on the constituentcomponents and interactions. This behavior is governed by thebalance of the attractive and repulsive forces acting in the

particles: swelling occurs when the ionic repulsion and theosmotic forces are higher than the attractive forces, i.e.,hydrogen bonds and van der Waals and hydrophobicinteractions. The multifunctional properties of nanogels canbe achieved by altering the cross-linking density, chemicalfunctional groups, and surface-active and stimulus-responsiveconstituents.350

The ability the nanogels have to undergo large reversiblechanges in volume make them interesting and suitable materialsto be used as carriers for the uptake and release of compoundsor other materials. Nanogels exhibit a behavior that goes from apolymeric solution (swell form) to a hard particle (collapsedform). As previously commented, they can be consideredstimulus-responsive materials.290 Nanogels can respond tophysical stimuli (temperature, ionic strength, magnetic orelectric fields, ...), chemical stimuli (pH, ions, specificmolecules, ...), and biochemical stimuli (enzymatic substrates,affinity ligands, ...).351 Among them, temperature is moststudied because it is an effective stimulus in a number ofapplications. Nanogels which are able to undergo a volumetricphase change by changing the temperature of the dispersionmedium are very interesting in biotechnological applicationsneeding the delivery of an active compound, molecule, ormaterial in media in which the main variable to consider is thetemperature (see Figure 30). Another type of sensitivity with

interest in biomedical applications is the response to pHchanges. This is the case for pH-sensitive nanogels (they swellwhen the pH approximates the pKa of the ionic monomerincorporated by copolymerization in the cross-linked chainsconstituting the particles); they are useful in the case ofreleasing a biologically active compound in a physiologicalmedium in which the main characteristic is the change in pH.From the biotechnological application point of view, the

interest in nanogel particles comes from their stimulus-responsive nature, i.e, from their ability to suffer reversiblephase transitions in response to stimuli or changes in themedium. Moreover, nanogel particles can respond to changesin the medium more quickly than macroscopic gels due to theirnanometric small size. The nanogels’ sensitivity to the mediumconditions is an advantageous property if the applicationconsists of drug delivery because a response under physiologicalpH and temperature can be obtained. Nanogels are beingproposed as new carriers for the delivery of active ingredientsor drugs due to the possibility of encapsulation of thosecompounds in an aqueous environment and under relativelysoft conditions. An ideal nanogel drug delivery carrier should

Figure 30. PNIPAM vs PVCL thermosensitive nanogels. Differingphase transitions. Reprinted with permission from ref 363. Copyright2012 Royal Society of Chemistry.


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have a few common features, including, but not limited to, asmaller particle size (10−200 nm), biodegradability and/orbiocompatibility, a prolonged blood circulation time, a higheramount of drug or enzyme loading, and/or entrapment andprotection of molecules from the immune system of thebody.352 Some of the active components are extremelyhydrophobic, without cellular permeability, and susceptible tometabolic degradation; because of this, their use is limited. Thistype of agent can be transported without any problem throughphysiological media by using this type of nanoparticle. On theother hand, the size of the particles forming the nanogel is animportant parameter because it governs the efficiency of thedelivery system. For this type of application, particles having adiameter smaller than 1 μm are especially useful.353 The drugsare taken up into the polymeric nanoparticles by adsorption,absorption or “entrapment”, or covalent bonding, and they aredelivered by desorption, diffusion, polymer degradation, or acombination of these mechanisms.354 Different authors indicatethat the objectives to reach in the future design anddevelopment of delivery systems based on nanogels for invivo applications require a high degree of control of theirproperties,355 among them an excellent stability for theircirculation in blood, new functionalities for subsequentbioconjugation/biovectorization, nanometric dimensions indiameter, biocompatibility and/or biodegradability for theireasy expulsion, and drug sustained delivery. Another objectiveshould be an improvement in the design of nanogels withspecific groups which permit a selective absorption into specificcells.Different polymerization methods or techniques in dispersed

media are being used for the preparation of nanogels, amongthem emulsion polymerization, inverse microemulsion poly-merization, anionic copolymerization, cross-linking betweenneighboring chains, and others. At this point it is necessary tocomment that some authors prefer to apply the terminology ofpolymers accepted in IUPAC Recommendations 2011,356 andthey use “precipitation polymerization” for the polymerizationprocess used to produce nanogels instead of using “emulsionpolymerization” due to the high monomer solubility in watercompared to that of the produced polymer. This IUPACrecommendation proposes the use of “precipitation polymer-ization” for a polymerization in which monomer(s), initiator(s),and colloidal stabilizer(s) are dissolved in a solvent, and thiscontinuous phase is a nonsolvent for the formed polymerbeyond a critical molecular weight.Special interest is focused on micro/nanogels based on

polymers which have a lower critical solution temperature(LCST) near the physiological temperature. In the case ofsensitive polymer-based nanogels, the phase transition of thenanoparticles is observed as a volume phase transitiontemperature (VPTT). The most frequently used family ofpolymers in the synthesis of sensitive nanogels is that oftemperature-sensitive poly(alkylacrylamides), more specificallyPNIPAM. However, its toxicity prevents its use in biomedicalapplications. Nevertheless, during the past few years a numberof papers and patents have appeared on this type of nanogel.Among biocompatible and temperature-sensitive monomersthere is N-vinylcaprolactam (VCL), which is a water-solublemonomer.357 The corresponding polymer (poly(N-vinylcapro-lactam), PVCL) combines useful and important propertiesbecause together with its biocompatibility,358 it has a phasetransition in the physiological temperature region (32−38°C).359−362 This combination of properties allows it to be

considered as an adequate material for the design of biomedicaldevices and useful in drug delivery systems.363 With respect topH-sensitive nanogels,364 the choice of polymer depends on thephysiological conditions of the target in which the delivery isneeded.

4.1. Strategies To Produce Cationic Micro/Nanogels,Characterizations, and Applications

Searching in the databases for the synthesis, characterization,and applications of cationic nanogels led us to a few referenceson this type of nanogel. Therefore, it can be said that, as in thecase of cationic latexes, at the moment they are less studiedthan anionics. However, they are very useful as drug deliverysystems to introduce drugs or biomolecules into cells. As wascommented previously, cationic vectors facilitate cellularuptake, and cationic character for the carriers is necessary tocross the cell membrane.On the other hand, by reviewing in detail the literature on

cationic micro/nanogels, it seems that, to date, the interest ismore focused on the bioapplication than on the synthesisprocceses to obtain them. Because of this, in this part, thesyntheses, characterizations, and applications of cationic micro/nanogels are considered together. An exception is made withthe part dedicated to the characterization of PNIPAM-basedmicro/nanogels, which is presented separate from the rest duethem being a more studied type of micro/nanogel.

4.1.1. Conventional Production of Micro/Nanogels.4.1.1.1. PNIPAM-Based Micro/Nanogels. Special attention hasbeen paid to temperature-sensitive aqueous microgels sincePelton and Chibante prepared cross-linked PNIPAM particlesin 1986.365 As commented previously, PNIPAM particlesexhibit a temperature-induced volume transition. It is generallybelieved that hydration of the PNIPAM chains originates localordering in the water molecules around the amide group bymeans of hydrogen bonding. An increase in temperature,however, increases molecular agitation, which in turn causes adisruption of the H-bonding between water and the amidegroups. This leads to a breakdown of local water structurearound the PNIPAM chains that triggers hydrophobicattraction among isopropyl groups. This feature causeshydration of polymer chains below the LCST, andconsequently, microgel particles are swollen, while above theLCST the particles collapse. The LCST for PNIPAM in wateris around 32 °C.366,367

For many years, the group of Pichot and Elaissari at theCNRS in Lyon, France, has been active in producing and usingPNIPAM-containing cationic latexes useful in differentbioapplications. Although these nanoparticles are not reallynanogels, their works are commented on this part of the reviewdue to the thermosensitive nature of the PNIPAM polymer.They started in 1995 by using cationic PNIPAM-based latex

particles for covalent immobilization of oligonucleotideprobes,368 followed by an analysis of the particle size andmorphology vs polymerization process369 together with adiscussion on the surface and colloidal characteristics ofcationic amino-containing PNIPAM−styrene copolymer par-ticles.370 The application of amino-containing cationic latexesbased on polystyrene and PNIPAM to diagnostic test sensitivityenhancement200 and the use of a hydrophilic and cationic latexbased on PNIPAM particles for the specific extraction ofnucleic acids371 were reported the following year.Keeping in mind the increasing interest in the application of

magnetic particles in the biomedical field, these French authors


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prepared monodisperse hydrophilic functional magneticparticles also based on PNIPAM.372,373 The adsorption ofmagnetic iron oxide nanoparticles onto various cationic latexesand the encapsulation of adsorbed iron oxide nanoparticleswere analyzed and discussed. The particles were presented asgood candidates for solid phases in immunoassays. Thepresence of a PNIPAM shell should avoid nonspecificimmobilization of proteins, and the presence of carboxylicgroups provided by the functional monomer (itaconic acid)would allow, after activation, covalent binding of proteins.In 2001, the same group reported the synthesis and

properties of functional polystrene−PNIPAM core−shellparticles or poly(N-isopropylmethacrylamide) (PNIPMAM)microgel particles together with some biomedical applications(enhancing the sensitivity of genetic tests, for the concentrationof proteins or nucleic acids, among others) of these stimulus-responsive particles.374 Focusing their interest on core−shelllatex particles, the adsorption/desorption behavior and covalentgrafting of an antibody onto cationic amino-functionalizedpoly(styrene−N-isopropylacrylamide) core−shell latex particleswas investigated as a function of the temperature, pH, andsalinity.375 The synthesis of cationic poly(MMA)−PNIPAMcore−shell latexes prepared by a two-stage emulsion copoly-merization was also analyzed in terms of the influence of thecross-linker (N,N′-methylenebisacrylamide, MBA) and como-nomer (AEMH) concentrations on the thickness and swellingcapacity of the PNIPAM-based shell layer.376

Another active group working on cationic nanogels based onPNIPAM is that of Kokufuta at the Graduate School of Life andEnvironmental Sciences, University of Tsukuba, Japan. In 2006they published a work on light scattering studies ofpolyelectrolyte complex formation between anionic andcationic nanogels in an aqueous salt-free system,377 followedby a study by dynamic and static light scattering of thegeometrical characteristics of polyelectrolyte nanogel particlesand their polyelectrolyte complexes378 and a paper on theelectrochemically induced aggregation of intraparticle cationicnanogel complexes with a stoichiometric amount of boundpolyanions.379 In these studies PNIPAM-based anionic,cationic, and neutral nanogels were used.More recently, the same group analyzed the water

dispersibility of complexes formed between cationic polyelec-trolyte nanogels and anionic polyions and their complexationby conductometric and light scattering studies, respec-tively.380,381

In addition, Hu et al.382 published the synthesis andphysicochemical properties of cationic microgels based onPNIPMAM. Microgels were synthesized by surfactant-freeradical precipitation polymerization of NIPMAM and thecationic comonomer N-(3-aminopropyl)methacrylate hydro-chloride (APMH). The resultant amine-laden microgels showthe expected swelling properties of thermoresponsive cationicmicrogels as a function of temperature, pH, and ionic strength,as well as reactivity in standard amine bond-forming reactions.Characterization of PNIPAM-Based Micro/Nanogels.

There are numerous papers which refer to the characterizationof anionic PNIPAM particles. Characterization can involve gelstructure, swelling, surface activity, rheology, electrical proper-ties, colloidal stability, and interactions with other molecules(surfactants, drugs, proteins, etc.). Characterization of cationicPNIPAM microgels, however, is not so usual in the literature, asthese types of particles have appeared in the past decade.Cationic PNIPAM microgels are usually obtained by adding a

positive comonomer together with the N-isopropylacrylamide(NIPAM) in the polymerization reaction,383 as well as acationic initiator, commonly V-50.368,384,385 A number oftechniques have been extensively used to characterize thesemicrogels, including PCS,386 potentiometric titrations,385

differential scanning calorimetry (DSC),386,387 isothermaltitration calorimetry,388−391 rheology,392 nuclear magneticresonance spectroscopy,393 fluorescent probes,394 and scanningtransmission X-ray microscopy.395

It is very usual to examine the particle size (particle swellingratio), electrokinetic properties, and colloidal stability of thecationic PNIPAM microgels as a function of the salinity,temperature, and ionic specificity.396,397 In some cases, a goodcorrelation between the charge of the cationic microgelnetwork and its size is found.398

In a comparison study on anionic and cationic microgels,Lopez-Leon et al.396 found that the main difference is in theelectrokinetic behavior of both types of microgels. Theelectrophoretic mobility of the cationic PNIPAM microgelshows a linear dependence on rh

−2 (rh is the hydrodynamicparticle radius), whereas two-step linearity is found for theanionic PNIPAM microgel. This is due to differences in theinternal structure between both microgel samples.396 For thatreason, in a subsequent work, the same authors studied theionic specificities on the electrokinetic behavior of bothnegative and positive PNIPAM microgels. The term“Hofmeister effects” 399 is broadly used to refer to ionicspecificities in many different physical, chemical, and biologicalphenomena. More precisely, Hofmeister effects, series, orsequences refer to the relative effectiveness of anions or cationsin specifically modifying diverse properties of a wide range ofphenomena: cloud points of nonionic surfactants, CMCs,solubilities of salts, surface tensions, pH measurements, ζ-potentials, molecular forces, colloidal stabilities, proteinsolubilities, fluid viscosities, etc. Different authors have studiedthe Hofmeister effects on the electrokinetic behavior of cationicmicrogels,397,400,401 on other cationic polymer nanopar-ticles,402−405 and on an IgG-coated cationic latex.406,407 Twomechanisms underlining Hofmeister effects are the ionaccumulation at the particle surface and water structurealterations, and they are present in the behavior of the cationicmicrogels. Although both mechanisms act together, they exertdifferent effects on the properties of these smart systems. Dueto the extraordinary property of solvency of PNIPAM, whichmanifests polymer as well as hard-sphere behavior, it is possibleto investigate both mechanisms independently. Hydrodynamicdiameter measurements prove to be sensitive to ionicspecificities associated with changes in the structure of thewater molecule, while electrophoretic mobility data are relatedto ionic accumulation exclusion processes. It is quite strikingthat a concentration of 0.01 mol/dm3 NaSCN is enough toreverse the electrophoretic mobility of the cationic PNIPAMparticles.400

Another interesting aspect is the solubility of PNIPAM inalcoholic mixtures. At room temperature PNIPAM is soluble inboth water and alcohols with low molecular weight but tends tobecome insoluble in mixtures of the two solvents at the sametemperature.401 This is termed “cononsolvency” and gives riseto a re-entrant-phase diagram; i.e., when the solventcomposition is varied systematically, the gel undergoes twotransitions: a discontinuous collapse followed by a discontin-uous swelling. The re-entrant transition defines a closed-loopinstability phase boundary having both upper and lower critical


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points.408 One of them was realized by changing thecomposition of the solvent (water−methanol mixture) andthe other by applying the osmotic pressure to a gel network,which was generated by adding large molecules to the outersolution.409 PNIPAM cononsolvency in water−alcohol mix-tures has been extensively studied with microgels and chainsadsorbed onto latex particles.408−411 Despite the number ofworks published on this subject, the molecular origin of thisphenomenon is still controversial. Nowadays, there are threeexplanations for the PNIPAM cononsolvency in alcohol−watermixtures:

(1) The earliest and most widespread interpretation involvescomplexation between the two solvents. Calculationsbased on the Flory−Huggins thermodynamic theorysuggest that the re-entrant behavior results from theperturbation of the alcohol−water interaction parameter(χ12) in the presence of PNIPAM. Consequently, theformation of alcohol−water complexes would bedominant over the hydrogen bonds between PNIPAMand water.408,409,412 This model, however, has problemsexplaining why cononsolvency is also observed in highlydilute PNIPAM solutions where the effect of the polymeron the solution properties should be negligible.

(2) Schild et al.413 suggested that any mechanism to explainthe re-entrant transition must involve local solvent−polymer interactions. Such a collapse transition mecha-nism based on local concentration fluctuations waspreviously proposed by de Gennes414 and would requirethe preferential adsorption of alcohol on PNIPAM. Thevalidity of this theory for these phenomena remains to beexperimentally confirmed.

(3) Recently, Tanaka et al.415 have proposed a thirdalternative according to which cononsolvency resultsfrom the competition between PNIPAM−water andPNIPAM−alcohol hydrogen bonding and a cooperativesolvation mechanism. Their results, however, contrastwith other experimental findings that argue for a re-entrant transition fully controlled by the water−alcoholcomplexation.416,417 The exact nature of the PNIPAMcononsolvency in alcoholic solutions thus remains anopen question.

In summary, we can conclude that the cononsolvencyphenomenon observed for PNIPAM chains in alcohol−watermixtures therefore involves an intricate balance between theattractive and the repulsive components of several differenttypes of forces. Also in this case the cationic systems look tohave a behavior different from that shown by anionic systems. Acomparison study was accomplished by Mielke and Zimehl418

on the behavior of negatively and positively charged PNIPAMparticles in alcohol−water mixtures at a temperature (20 °C)below their phase transition (∼32 °C). Methanol, ethanol, 1-propanol, and 2-propanol were used between 0 and 100 wt %.In general, the results obtained by these authors with anionicPNIPAM showed a minimum in particle diameter betweenalcohol concentrations of 20 and 40 wt %. At low alcoholfractions deswelling of the anionic PNIPAM particles canalways be observed. Alcohol weight fractions larger thanapproximately 80% cause the microgel particles to take upliquid in excess: the microgel particles swell considerably. Thegeneral trend did not appear to be altered using alcohols ofgreater chain length. On the other hand, for cationic PNIPAMparticles, the above-mentioned behavior can only be observed

in methanol. For ethanol−water mixtures the change indiameter of these particles showed a slight maximum at 20wt %. This maximum (swelling effect) increases for 1-propanoland is even more pronounced for 2-propanol. Clearly, in analcohol of higher chain length the properties of cationicPNIPAM show reverse behavior of the anionic PNIPAM. Atalcohol contents above 50 wt % the cationic microgel deswells.Thus, in these alcohol−water mixtures containing low weightfractions of alcohol (ethanol, 1-propanol, and 2-propanol)anionic and cationic PNIPAM particles exhibit the oppositeswelling and shrinking behavior at 20 °C. This reverse behaviorof crude cationic PNIPAM particles in alcohols with two ormore CH3 groups can be explained by specific alcoholadsorption, which produces swelling of the PNIPAM even atvery low alcohol contents. Cononsolvency cannot take place. Atslightly greater alcohol fractions (above 40 wt %), free alcoholmolecules appear in solution competing for water. In this case,deswelling occurs. The behaviors of cationic and anionicmicrogel particles are then similar in nature. At a temperature(40 °C) above their phase transition, the cationic PNIPAMparticles behave similarly to those at 20 °C.Moreover, very recently, Lopez-Leon et al.401 have studied

the effect of salt on the cationic PNIPAM cononsolvency inwater−ethanol mixtures. The results obtained with NaSCN areespecially interesting. SCN− has a strong chaotropic character;i.e., this ion interacts with water weaker than water with itself(structure-breaker). The effect of ions in the cononsolvency ofPNIPAM microgels depends obviously on the molar fraction ofthe alcohol in the binary mixtures. In the intermediate region ofethanol volume fractions, as the concentration of this ion wasincreased, the PNIPAM solvation was first controlled bypolymer−water interactions and second by polymer−ethanolinteractions.The ethanol−water mixtures have also been used to prepare

novel cationic pH-responsive poly((N,N′-dimethylamino)ethylmethacrylate) microgels. In this case, the maximum ratio ofvolume change of the prepared microgels in response to pHvariation was more than 11-fold.419

The following components have been adsorbed onto acationic microgel:

(1) Magnetic particles.372,373

(2) Proteins.420,421

(3) Heavy metals.422

(4) Surfactants.423

In general, the microgel particles can be envisaged asspongelike materials having a spherical conformation consistingof numerous interstitial spaces where these compounds can beadsorbed.

4.1.1.2. PDMAEMA and PDEAEMA-Based Micro/Nanogels.Poly(2-(N,N′-dimethylamino)ethyl methacrylate) (PDMAE-MA) and poly(2-(N,N′-diethylamino)ethyl methacrylate)(PDEAEMA) are pH-, temperature-, and ionic strength-responsive polymers with interesting and potential uses inbiomedical applications due to these sensitive properties.The group of Nagasaki at the Department of Material

Science & Technology at the Tokyo University of Science,Japan, has been very active in synthesizing different DMAEMA-based nanogels. In 2004, using DMAEMA with ethylene glycoldimethacrylate as the cross-linking agent and α-(vinylbenzyl)-ω-carboxy-PEG as the stabilizer, they reported the synthesis viaemulsion polymerization of pH-sensitive DMAEMA-basednanogels with controllable diameters in the range of 50−680


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nm.424 These nanogels have a PEG shell layer with a carboxylacid group at the distal end of each PEG strand confirmed by ζ-potential measurements at varying pH values. This locationcould be an available conjugation site of various ligands. Thesefeatures indicate the potential utility of these nanogels inapplications such as diagnostics and controlled drug-releasingdevices.Taking into account that PEGylated nanogels show unique

properties and functions in synchronizing with the reversiblevolume phase transition of PDEAEMA in response to variousstimuli, such as pH, ionic strength, and temperature, the samegroup proposed the synthesis and characterization of stimulus-responsive PEGylated nanogels composed of a cross-linkedPDEAEMA core and PEG-tethered chain that bear a carboxylicacid group as a platform moiety for the installation ofbiotags.425 Some of the potential biotechnological applicationsof the nanogels are listed, among them, as endosomolyticagents for nonviral gene delivery, drug delivery carriers,nanoreactors, and skin-specific nanocatalysts for reactiveoxygen species (ROS), thermosensitive drug and gene carriers,and ion sensors.The loading of the anticancer drug doxorubicin (DOX) was

carried out in the pH-sensitive PEGylated nanogels based alsoon DMAEMA and prepared by emulsion polymerization havingbiheterofunctional PEG as in the first work presented in2004.426 The loading by solvent evaporation method yielded26% DOX in the PDMAEMA core. The DOX-loaded, pH-sensitive PEGylated nanogel showed almost no initial burstrelease of DOX under physiological pH, whereas significantrelease of DOX was observed at endosomal pH. The antitumoractivity of the loaded nanogels against a human breast cancer

cell line and a human hepatoma cell line, which is a naturaldrug-resistant tumor line, was analyzed, showing that the newnanogels have higher antitumor activity than both free DOXand the DOX-loaded, pH-insensitive, PEGylated nanogels.These findings suggest that these nanogels are promisingnanosized carriers for anticancer drug delivery systems in vivo.The same group reported the synthesis of pH-responsive

PEGylated nanogel platinum particles, which can be utilized inskin-specific ROS scavengers for skin aging,427 by using similarPDMAEMA-based nanogels. Platinum nanoparticles with a sizeof less than 2 nm were synthesized through the reduction ofK2PtCl6 within the PEGylated nanogels. The resulting nanogelsshowed significant catalytic activity for ROS in response to theskin environmental pH (acidic), whereas almost no catalyticactivity for ROS was observed at physiological pH due to theVPTT of the PDMAEMA core.Similar PEGylated nanogels based on PDMAEMA-contain-

ing gold nanoparticles were synthesized through the autor-eduction of HAuCl4, which gives an average number of 10 Aunanoparticles per nanogel particle having a 6 nm diameter.428

The surface plasmon band of the Au nanoparticles containingnanogels was shifted in response to the pH, indicating that thecross-linked PDMAEMA core of the nanogels acts not only as ananoreactor but also as a pH-sensitive matrix.Another bioapplication was reported with these pH-

responsive PEGylated PDMAEMA-based nanogels consistingof their use as targetable and low invasive endosomolytic agentsto induce the enhanced transfection efficiency of nonviral genevectors.429 Polyplexes composed of PEG-block-poly(L-lysine)copolymer and plasmid DNA exhibited a far more efficient

Figure 31. Schematic illustration of the pH-responsive PEGylated nanogel and endosomal escape mechanism. Reprinted with permission from ref429. Copyright 2007 Springer-Verlag.


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transfection ability in the presence of the nanogels without anycytotoxicity (see Figure 31).In 2009, the group of Nagasaki published the enhanced

cytoplasmic delivery of small interfering RNA (siRNA) using astabilized polyion complex based on PEGylated nanogels withcross-linked PDMAEMA-based structure.430 The nanogel−siRNA complex was observed to undergo a remarkableenhancement of the gene-silencing activity against a fireflyluciferase gene expressed in HUH-7 cells. By using confocalfluorescence microscopy, they demonstrated an efficientendosomal escape capability for the transportation of siRNAsinto the cytoplasm, probably due to the buffering effect of thePDMAEMA core, showing the potential of these nanogels to beeffective siRNA carriers for the development of in vivotherapeutic applications of siRNA (see Figure 32).

Thermoresponsive cationic nanogels based on NIPAM,DMAEMA, and quaternary alkylammonium halide salts ofDMAEMA were synthesized by dispersion polymerization.431

The thermoresponsive characteristics of the nanogels andpolyplexes obtained by complexation of these nanogels withsalmon sperm DNA were analyzed, and the results demon-strated that these nanogels, with controllable responsiveproperties determined by the nature of the cationic chargeincorporated, may have potential as vehicles for DNA delivery.Recently, Marek et al.432 reported the synthesis of cationic

nanogels based on DMAEMA by a new inversion emulsion(water-in-oil) polymerization method. Nanogels of PDEAEMAand polyethylene glycol-n monomethyl ether monomethacry-late (P(DEAEMA-g-EGn)) were synthesized. The effects of thisnovel nanoparticle synthesis route of PDEAEMA on severalpolymer properties, including surface charge and swellingresponse, together with the effects of the cross-linking ratio andPEG tether length on the physical properties were alsoexamined. The network morphology was also studied, andthe potential for these systems to be used as drug deliveryagents was evaluated (see Figure 33).4.1.1.3. Use of Living Radical Polymerizations To

Synthesize Cationic Micro/Nanogels. As can be seen in thedifferent works reviewed, generally, the synthesis of nanogels iscarried out in diluted monomer solutions or in heterogeneousmedia, such as a microemulsion or emulsion, under theassistance of a surfactant.Living radical polymerizations such as atom transfer radical

polymerization (ATRP),433 nitroxide-mediated polymerization(NMP),434 and reversible addition−fragmentation transfer(RAFT)435 have also been used for the synthesis of a smallamount of nanogels. As examples, the following two works arebriefly discussed.

Cationic nanogels based on PEGylated PDMAEMA (PEG−PDMAEMA) with potential application in gene delivery weresynthesized via the in situ formation of micelles by anamphiphilic trithiocarbonate macro-RAFT agent by Yan andTao436 by a one-step surfactant-free RAFT process. Nanogelparticles of about 20 nm and +30 eV ζ-potential, which are apotential gene delivery system for further biomedicalapplications, were synthesized.In the second work, new thermoresponsive and acid-

degradable poly(methoxydiethylene glycol methacrylate (MeO-DEGM)−2-aminoethyl methacrylamide hydrochloride(AEMA))-based nanogels via RAFT polymerization usingpoly([2-(methacryloyloxy)ethyl]phosphorylcholine (MPC))macro-RAFT agent were synthesized.437 The sizes of thesenanogels can be tuned by varying the amount of cross-linkerand MeODEGM chain length. AEMA provides the cationiccharacter to the nanogel core, which facilitates the encapsula-tion of oppositely charged proteins (insulin, BSA, and β-galactosidase). The loading efficiency of these proteins dependson the pore size of the nanogels, the cationic component, andthe size of the protein. Degradation and controlled releaseprofiles were analyzed, concluding the existence of promisingapplications of these nanogels for targeted drug deliverysystems and controlled release (see Figure 34).

4.1.1.4. Micro/Nanogels Based on Miscellaneous Mono-mers. Polyampholyte cationic gel particles were synthesized byaqueous redox polymerization in the presence of sodiumdodecylbenzenesulfonate as the surfactant using 1-vinyl-imidazole as the cationic monomer, incorporated into thenetwork of NIPAM cross-linked with MBA. A detailedcomparison of experimental mobilities with theoreticalcalculations was made in terms of three different models.438

The same group439 studied the formation of intra-interparticlepolyelectrolyte complexes between cationic nanogels and astrong polyanion, potassium poly(vinyl alcohol) sulfate(KPVS), demonstrating that the cationic nanogels form

Figure 32. Schematic illustration of the nanogel−siRNA polyioncomplex. Reprinted from ref 430. Copyright 2009 American ChemicalSociety.

Figure 33. SEM micrographs of different PDEAEMA-g-PEG nanogels.Reprinted with permission from ref 432. Copyright 2010 Elsevier Ltd.


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polyelectrolyte complexes with the strong polyanion in theaqueous KCl-free and KCl-containing systems and dividing theresulting complexes into two types: intra- and intercomplexcolloid systems in which the complex formation takes placewith bound KPVS and through electrostatic forces, respectively.In the same year, 2005, during the AIChE Annual Meeting

Conference, Nichenametla et al.440 presented the work “Novelnanoparticles for controlled drug delivery across the blood-brain barrier”, analyzing some interesting aspects, including thefollowing: the cost of developing an average new therapeuticagent, which is approximately $150 million. However, the useof these therapeutic agents is frequently still hampered by thelack of an effective route and mode of delivery. The reasons forthis reduced efficacy are that many of these therapeutic agents,especially therapeutic proteins and peptides, have very shorthalf-lives, do not cross biological barriers, and are metabolizedat other tissue sites. Therefore, improving the effectiveness oftherapeutic agents by optimizing their delivery and dosage andminimizing side effects may be a better investment and morebeneficial to patients than creating entirely new pharmaceut-icals. The blood−brain barrier (BBB) is a dynamic and complexstructure composed principally of specialized capillaryendothelial cells held together by highly restrictive tight

junctions. As the name implies, the BBB serves as a barrierthat prevents the passage of cells and proteins, includingtherapeutic agents, present in the bloodstream from gainingaccess to the central nervous system (CNS). They developednanoparticles, including dendritic nanoparticles, cationic f″-cyclodextrin, and polysaccharide-based nanogels, for controlleddrug delivery across the BBB. The permeability of thenanoparticles with/without model protein drugs, BSA andnerve growth factor (NGF), through a bovine retina endothelialcell (BREC) monolayer, an in vitro BBB model, wasinvestigated. The authors’ hope is that their work will have asignificant impact on the treatment of neurological disorders inthe brain.Sahiner et al. reported the synthesis and characterization of

microgel, nanogel, and hydrogel−hydrogel semi-interpenetrat-ing polymer network (semi-IPN) composites for biomedicalapplications.441 In this work, quaternary ammonium salthydrogels from a cationic monomer, (3-acryamidopropyl)-trimethylammonium chloride ((APTMA)Cl), in a variety ofsizes such as bulk, micro, and nano, are synthesized by water-in-oil microemulsions using lecitin and dioctyl sulfosuccinatesodium salt (AOT). Hydrogel−hydrogel composite semi-IPNswere synthesized by dispersing previously prepared micro/

Figure 34. Core cross-linked micelles with thermoresponsive and degradable cores. Reprinted from ref 437. Copyright 2011 American ChemicalSociety.

Figure 35. Schematic diagrams for (a) the release of PrHy-loaded nanogel under different pH values and (b) the release of IMI-loaded nanogelunder different pH values. Reprinted with permission from ref 443. Copyright 2007 Elsevier Ltd.


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nanogels into neutral monomers such as AAm or 2-hydroxyethyl methacrylate (HEMA) before network formation.Hydrogel swelling and pH response behaviors were investigatedfor bulk gels. Using TEM, SEM, and AFM the morphology,structure, and size of nanomaterials, micromaterials, and bulkmaterials were analyzed. It was confirmed by gel electrophoresisthat a completely charged nanogel forms a strong complex withDNA.At the previously cited 55th Society of Polymer Science

Japan Annual Meeting in Nagoya (2006), the design of a core−shell-type nanogel for high-performance bionanospheres waspresented.442 The lactose-installed core−shell-type nanogelswere prepared via emulsion copolymerization of DEAEMAwith CH2CHPhCH2-PEG−lactose in the presence of cross-linking agent in an aqueous medium. The core of the obtainednanogels showed a drastic volume phase transition in the pHregion at around 7. The cytotoxicity of the obtained nanogelsdecreased with increasing chain length of the PEG on thenanogel surface. The longer PEG chain improved thecompartmentalization of the PDEAEMA gel core to preventexposure of the cationic charge to the outside. Notably, thecytotoxicity of the nanogel did not change regardless of thelactose installation on the surface. Cellular uptake of Texas Redlabeled dextran in HuH7 cells was increased in the presence ofthe lactose-installed nanogel, but not in the presence of thatwithout a lactose moiety at the PEG chain end.Taking into account that delivery systems based on pH-

responsive nanoparticles can control the release of rapidlymetabolized drugs and/or have the ability to protect sensitivedrugs, Tan et al.443 presented comparative drug release studiesof two cationic drugs from pH-responsive nanogels. Thesenanogels consisted of methacrylic acid−ethyl acrylate (MAA−EA) cross-linked with diallyl phtalate (DAP) and weresynthesized by emulsion polymerization. The release of twodifferent drugs (procaine hydrochloride (PrHy) and imipr-amine hydrochloride) loaded via two distinctly differentinteraction forces (hydrophobically bound and electrostaticallybound enhanced by hydrogen bonding, respectively) wasanalyzed under different pH values, MAA−EA molar ratios, andDAP contents by using a drug-selective electrode (DSE) tomeasure the concentrations released from the MAA−AEnanogels (see Figure 35).The same group proposed the following year (2008) how to

avoid and control the undesirable burst release phenomenoncommonly encountered in nanostructured delivery systems.444

For that, the layer-by-layer assembly technique was proposed.Drug (PrHy)-loaded MAA−EA-based nanogels of sizes smallerthan 200 nm were coated with alternating layers of poly-(allylamine hydrochloride) (PAH; catonic) and poly(sodium 4-styrenesulfonate) (PSS; anionic) polyelectrolytes. With everylayer of polyelectrolyte, the radius increased by 2 nm, and the ζ-potential alternated between positive and negative values. PSS-coated nanogels were stable at all pH values, while PAH-coatednanogels were stable up to pH 8. By using a DSE, theconcentration of PrHy released was measured. The high burstrelease was reduced or minimized when the number of layers ofpolyelectrolyte was increased.Recently, Kokufuta and Doi380,381 reported a study on the

water dispersibility of a 1/1 stoichiometric complex between acationic nanogel and linear polyanion (SPENC) composed of across-linked (using MBA as the cross-linker) copolymer of 1-vinylimidazole and NIPAM and discussed in terms of theassociation−dissociation reactions between both of the

polyelectrolyte components. The results indicated that thewhole and a part (segment) of the complexed polyanionsundergo dissociation−association reactions on the surface of aSPENC particle, depending on the ionization state of thecationic gel component. These reactions seem to be a keyfactor for the water dispersibility of the SPENC. The secondwork was focused on understanding the water dispersibility oftheir stoichiometric nanogel complex having a core−shell orcorona structure in terms of its uptake of counterions byconductometric and light scattering studies.For finishing with this part devoted to synthetic micro/

nanogels, it is interesting to comment that a very recent workon self-assembly of biodegradable polyurethanes445 declaresthat the use of these materials in controlled drug deliveryapplications constitutes an important area of research for thedevelopment of polymeric materials in biomedicine. Inparticular, colloidal polyurethane assemblies can increase thesolubility and stability of hydrophobic compounds and improvethe specificity and efficiency of drug action. Their nanoscalesize and modular functionality make them promising for theinjectable, targeted, and controlled delivery of varioustherapeutic agents and imaging probes into required cells.Additionally, cationic polyurethanes are able to self-assemblewith nucleic acids into nanoparticles to enter cells for efficientgene transfection. These emerging nanocarriers open the doorfor addressing the failure of traditional localized deliverysystems and present a compelling future opportunity to achievepersonalized therapy as versatile candidates. This review paperhighlights the research progress in the self-assembly ofbiodegradable polyurethanes for controlled delivery applica-tions, with particular attention being paid to some representa-tive vehicles such as self-assembled polyurethane micelles,nanogels, and polyurethane−DNA complexes, which haveemerged as the focus of interest in recent years.

4.1.2. Nonconventional Production of Micro/Nano-gels. 4.1.2.1. PEI-Based Micro/Nanogels. PEI is a well-knowncationic polymer that has previously been shown to havesignificant potential to deliver genes in vitro and in vivo. PEI iswidely used to prepare different nanogels useful for nonviralgene delivery strategies. PEI is employed as a DNA-compactingmolecule because of high cationic charge and high transfectionefficiency. Upon contact with DNA, PEI condenses bulky DNAinto a nanosized complex ranging from 50 to 200 nm by ionicinteractions between amine groups in PEI and phosphategroups in the DNA backbone.The group of Vinogradov at the Center for Drug Delivery

and Nanomedicine in Nebraska, published in 2005 twopapers446,447 on polyplex PEI-based nanogel formulations fordrug delivery of cytotoxic nucleoside analogues and the role ofthe cellular membrane in drug release. The data demonstratethat the carrier-based approach presented for delivery ofcytotoxic drugs may enhance tumor specificity and significantlyreduce side effects usually observed in cancer chemotherapy.On the other hand, cationic nanogels synthesized canencapsulate large amounts of nucleoside analogues. Thecomplexes were evaluated as potential cytotoxic drugformulations for breast carcinoma cells. A substantial releaseof encapsulated drug was observed following interactions ofdrug-loaded nanogels with cellular membranes (see Figure 36).In 2006, Xu et al.,448 keeping in mind the idea that cationic

polymer nanogels, positively charged submicrometer polymericparticles that swell in water, have attracted increasing researchattention in recent years because of their potential applications


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as gene carriers, presented the synthesis via photochemistry insurfactant-free aqueous solution of novel PEI nanogels to beused as potential gene carriers. In this work, a novel method tosynthesize PEI nanogels with sizes ranging from 80 to 200 nmvia UV irradiation at room temperature in aqueous solutionwithout addition of any kind of surfactant is presented. Themorphology of the nanoparticles is determined to be spherical.The nanogels are of high stability, high transfection efficiency,low toxicity, and low immunogenicity, as has been confirmed byin vivo tests with mice as an animal model and by in vitro testswith human lung and liver cancer cells as well.In 2006, Vinogradov et al.449 presented the synthesis and

characterization of new cationic nanogels composed ofamphiphilic polymers and cationic PEI for encapsulation anddelivery of cytotoxic nucleoside analogues 5′-triphosphates(NTPs) into cancer cells. Nanogels were synthesized by a novelmicellar approach and compared with carriers prepared by theemulsification/evaporation method. Complexes of nanogelswith NTP were prepared; the particle size and in vitro drugrelease were characterized. Resistance of the nanogel-encapsulated NTP to enzymatic hydrolysis was analyzed byion pair high-performance liquid chromatography (HPLC).Binding to isolated cellular membranes, cellular accumulation,and cytotoxicity were compared using some breast carcinomacell lines (see Figure 37). In vivo biodistribution of labeledNTP encapsulated in different nanogels was evaluated incomparison to that of the injected NTP alone. The results showthat formulations of nucleoside analogues in active NTP formwith these nanogels will improve the delivery of these cytotoxicdrugs to cancer cells and the therapeutic potential of thisanticancer chemotherapy.

In 2007, the same group reported a work on formulations ofbiodegradable nanogel carriers with NTPs of nucleosideanalogues that display a reduced cytotoxicity and enhanceddrug activity. Nanogels were synthesized by using two methodsproposed by the same group: the emulsification−evaporationmethod447 and the micellar approach.449 PEI-based biodegrad-able nanogels were synthesized by the same methods but usingbiodegradable PEI. The results show that the toxicity ofnanogels was clearly dependent on the total positive charge ofcarriers and was 5−6-fold lower for carriers loaded with NTP.The toxicity of drug-loaded nanogels focusing on respiratorychain components of cells was evaluated and compared withcell viability assays for drug or drug formulations. Vinogradov etal.450 also presented in 2010 the use of nanogel−nucleoside 5′-triphosphate formulations for the treatment of drug-resistanttumors (see Figure 38).Lee and Yoo451 prepared the so-called DNA nanogels by

chemically conjugating Pluronic (to be defined later) to thesurface of a PEI−DNA complex to prepare thermoresponsivenanogels with endosomal disrupting abilities. The sizes and ζ-potentials of these nanogels changed significantly when thetemperature was increased from 20 to 37 °C. The cytotoxicityand transfection efficiency of the nanogel were also affected bytemperature changes. The results indicate that surfacemodification of nanogels can be potentially applied tothermoresponsive gene carriers, where temperature-responsivecytotoxicities or transfection efficiencies are required (seeFigure 39).Delivery of synthetic siRNA remains the major obstacle to

the therapeutic application of RNA interference. To overcomethis problem, PEI is also useful as reported by Laisheng et al.452

In this work, the synthesis and characterization of PEI−polyethylene glycol diacrylate nanogel useful as an siRNAcarrier is reported. A new nanogel composed of hydrophilicpolyethylene glycol and cationic polyethylenimine for siRNAdelivery was synthesized via inverse microemulsion polymer-ization and characterized. Such a noncytotoxic nanogel issuitable for siRNA loading by electrostatic interaction. ThesiRNA delivery efficiency of the nanogel was evaluated, and theresults indicated that the nanogel induced a high gene-silencingeffect. With the proper particle size, a strong siRNA bindingability, and an efficient gene-silencing function, this nanogel−siRNA formulation has potential for efficient siRNA delivery intherapeutical application.Wei and co-workers synthesized PEI-based nanogels for the

delivery of specific genes to inhibit cell proliferation in coloncarcinoma453 and antitumoral efficacy in lung metastasis.454

Heparin−PEI (HPEI) nanogels were prepared through amidebond heparin-conjugated PEI molecules and used as a nonviralgene vector. HPEI−selected gene complexes were formed ineach different case. Efficient growth inhibition of ovarian cancer

Figure 36. Cellular trafficking of drug-loaded nanogels analyzed byconfocal microscopy in MCF-7 cells after 30 min of incubation (A, D),60 min of incubation (B, E), and 120 min of incubation (C,F) withNG(PEG) (A−C) or NG(P85) (D−F) at concentrations of 0.01 mg/mL NG(PEG) or 0.005 mg/mL NG(P85). Rhodamine-labelednanogels (red) contained encapsulated BODIPY FL ATP (green).Pictures are superimposed bright field and fluorescence images at100× magnification. Reprinted from ref 446. Copyright 2005American Chemical Society.

Figure 37. Nanogel synthesis using the micellar approach. Activated Pluronic block copolymers (A) formed micelles (B) in aqueous solutions, whichcould be covered with a layer of PEI (C) cross-linked by activated PEG molecules (D). Reprinted with permission from ref 449. Copyright 2006Springer Science + Business Media, Inc.


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is also reported455,456 in which the tumor weight decreased byalmost 72% and 87% in the treatment group compared withthat in the empty-vector control group. Meanwhile, decreasedcell proliferation, increased tumor cell apoptosis, and reductionin angiogenesis were observed compared with those in thecontrol groups. All these results indicated that HPEI nanogelsdelivering specific genes inhibiting tumor growth might be ofvalue in the treatment against human colon, lung, and ovariancancers (see Figure 40).Wei et al.457 also published the use of HPEI nanogels for in

vivo pulmonary metastasis therapy. Taking into account firstthat the clinical application for systemic administration ofadenoviral (Ad) vectors is limited, as these vectors do notefficiently penetrate solid tumor masses, and second that PEI isnondegradable and exhibits a high cytotoxicity as its molecularweight increases, low molecular weight PEI (Mn = 1800) wasconjugated to heparin (Mn = 4000−6000) to produce a newtype of cationic degradable nanogel (HPEI), which was thenused to modify Ad vectors. The resulting HPEI−Ad complexes

were used to infect CT26 and HeLa cells in vitro. Additionally,the HPEI−Ad complexes were administrated in vivo via

Figure 38. A cationic network of the nanogel decorated with multiple small-tumor-specific peptide ligands is capable of attracting oppositely chargedtriphosphate drug molecules from aqueous solution and binding them with the formation of compact drug nanoformulations for systemicadministration. Reprinted with permission from ref 450. Copyright 2010 Elsevier Ltd.

Figure 39. Intracellular delivery of a polycation−DNA complex modified with Pluronic: (A) non-cold-shock treatment (only proton sponge effects);(B) cold-shock treatment (proton sponge effect plus disruption by extended Pluronic). Reprinted with permission from ref 451. Copyright 2008Elsevier Ltd.

Figure 40. Preparation scheme of the heparin−PEI nanogel. Reprintedwith permission from ref 454. Copyright 2011 Wiley Periodicals, Inc.


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intravenous injection, and tissue distribution was assessed usingluciferase assays; the therapeutic potential of HPEI−Adcomplexes for pulmonary metastasis mediated by CT26 cellswas also investigated. In vitro, HPEI−Ad complexes enhancedthe transfection efficiency in CT26 cells, reaching 36.3%compared with 0.1% for the native adenovirus. In vivo, HPEI−Ad complexes exhibited greater affinity for lung tissue than thenative adenovirus and effectively inhibited the growth ofpulmonary metastases mediated by CT26 cells. The resultsindicate that Ad vectors modified by HPEI nanogels to formHPEI−Ad complexes enhanced transfection efficiency in CT26cells, targeted to the lung, and demostrated a potential therapyfor pulmonary metastasis.4.1.2.2. Pluronic-Based Micro/Nanogels. Besides the work

of Lee and Yoo previously cited,451 there are some othersfocusing on the use of Pluronics to synthesize nanogels.Pluronic is a triblock copolymer composed of poly(ethyleneoxide) (PEO)−poly(propylene oxide) (PPO)−PEO. Pluroniccopolymers have been extensively explored for controlled drugdelivery applications, especially in a form of micelles. Thehydrophobic PPO segments comprise a hydrophobic core,which is a microenvironment for the incorporation of lipophilicdrugs. The hydrophilic PEO corona prevents aggregation,protein adsorption, and recognition by the reticuloendothelialsystem (RES).458 Low cytotoxicity and weak immunogenicitygive Pluronic copolymers the possibility of topical and systemicadministrations.459 Pluronic copolymers have been studied topromote active membrane transport of numerous anticancerdrugs because they can overcome the multidrug resistance(MDR) effect. The complex mechanisms of Pluronic effects inMDR cells were throroughly reviewed460 and mainly attributed

to inhibiting drug efflux transporters, such as P-glycoprotein(Pgp). Pluronic has been widely applied also to drug deliveryfor its unique thermosensitive gelation property. Above theLCST, it forms physical gels by a hydrophobic interactionbetween hydrophobized PPO blocks. Many studies haveemployed this thermogelation property to control the releaseof many bioactive molecules in response to temperaturemodulations. Among those, Pluronic was conjugated to PEI toprepare thermosensitive gene carriers in an aim to modulatetransfection efficiencies according to temperature changes.461

Wang and co-workers used Puronic F127, one of thepolymers that can inhibit drug efflux transporters in cancertherapy, to produce amphiphilic nanocarriers for DOX.462

Folate-mediated chondroitin sulfate-decorated Pluronic nano-gels via a simple free radical reaction were synthesized, and afurther grafting of a tumor-targeting moiety onto the exteriorshell was prepared. The loading efficiency and release behaviorof DOX and the cellular uptake of the nanogels in cells wereanalyzed (see Figure 41).

4.1.2.3. Cationic Micro/Nanogels for DNA and siRNADelivery. In the context of gene therapy, several colloidal drugcarriers have been proposed to improve nucleic acid tumorlocalization and bioavailability, while reducing toxicity.McAllister et al.463 produced via inverse microemulsion

polymerization nanogels for cellular gene and antisensedelivery. Monodisperse anionic and cationic nanogels wereproduced with controllable sizes ranging from 40 to 200 nm indiameter by using (2-acryloxyethyl)trimethylammonium chlor-ide (AETMAC), 2-hydroxyethyl acrylate (HEA), and poly-ethylene glycol diacrylate (PEGdiA). The polymeric andcolloidal characterizations, cell viability, uptake, and physical

Figure 41. Formation of FA-CS-PF127 (folate-mediated chondroitin sulfate-decorated Pluronic) nanogels. Reprinted with permission from ref 462.Copyright 2009 Elsevier Ltd.


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stability of nanogel−DNA complexes were evaluated underphysiological conditions. The nanogels demonstrated extendedstability in aqueous media and exhibited low toxicity in cellculture. Cationic nanogels formed monodisperse complexeswith nucleotides and showed enhanced oligonucleotide uptakein cell culture. The nanogels synthesized demonstrate potentialutility as carriers of oligonucleotides and DNA for antisense andgene delivery (see Figure 42).

Hasegawa et al.464 reported a novel carrier for quatum dots(QDs) for intracellular labeling. Nanogels of cholesterol-bearing pullulan (CHP) modified with amino groups (CHP-NH2) were mixed with QDs and internalized into the variouscells examined. The efficiency of cellular uptake was muchhigher than that of a conventional carrier (cationic liposome),and the hybrid nanoparticles could be a promising fluorescentprobe for bioimaging (see Figure 43).

From 2005 to now, the group of Akiyoshi at the Institute ofBiomaterials and Bioengineering, Tokyo Medical and DentalUniversity, in Japan, has been one of the most active in thesynthesis and characterizations of cationic nanogels and theirbioapplications.465 They presented in 2005 the design andfunctional evaluation of a new nanogel carrier. In this study,CHP modified with amino groups was synthesized bycarbonyldiimidazole (CDI) activated synthesis. Using 1HNMR and elemental analysis, the numbers of cationic groupsper 100 glucose units were calculated. CHP derivatives formednanogels by self-associations.

This research group presented in the same symposium twomore studies.One study was on the characteristics of nanogel−DNA

complexes for gene carriers,466 reporting that nanogels wereformed by the self-assembly of CHPs. They synthesized variouscationic groups bearing CHPs. (Diethylamino)ethyl (DEAE),(ethylamino)ethyl (EEAE), and spermine groups were selected.In this study, they investigated the interaction between cationicCHP nanogel−plasmid DNA complexes and cells. Theyselected the plasmid DNA to express the green fluorescentprotein (GFP) in mammalian cells for use as a transfectionmarker. The cells that were incubated with cationic CHPnanogel−plasmid DNA complexes expressed GFP. The resultsindicate that CHP−spermine nanogel acts as efficient DNAcarriers compared with CHP−DEAE and CHP−EEAE.They also presented another study dealing with the

characteristics of nanogel−DNA complexes.467 In this work,nanogels were also produced by the self-assembly of CHPs. Inthis case, CHP-NH2 was synthesized and CHP-NH2 formedmonodisperse cationic nanogels (∼20 nm) in water. CHP-NH2nanogels formed complexes with DNAs. The interactionbetween CHP-NH2 nanogel−plasmid DNA complexes andcells was studied. They selected the plasmid DNA to expressthe GFP in mammalian cells for use as a transfection marker.The cells that were incubated with CHP-NH2 nanogel−plasmidDNA complexes expressed GFP. This result indicates that theCHP-NH2 nanogel could be used as an efficient DNA carrier.In the 55th Society of Polymer Science Japan Annual

Meeting in Nagoya (2006), the group of Akiyoshi presented astudy on the design of a new nanogel carrier as a proteincarrier.468 In this work, nanogels of CHP with a cationic chargewere used as intracellular protein carriers. Proteins such asfluorescein isothiocyanate (FITC)−BSA and FITC−antibodywere effectively internalized to HeLa cells in the presence of thenanogels even in the serum medium, showing that theefficiency is higher than that of conventional carriers such ascationic liposome or peptide-based carriers. They alsopresented the work “Functional nanogel as nucleic acidcarrier”,469 reporting that hydrogel nanoparticles (nanogels)were formed by self-assembly of CHPs. They performed thesynthesis of various cationic groups bearing CHPs. The cationicCHPs formed monodisperse cationic nanogels (30−40 nm) inwater. The characteristic of cationic nanogel−nucleic acidcomplexes together with the interaction between cationicnanogel−nucleic acid complexes and cells were analyzed. Theresults indicate that various cationic nanogels and nucleic acidswere complexed and formed nanosize particles. The CHPnanogel, which modified spermine, acts as an efficient nucleicacid carrier in cells.It is well-known that charge is a key parameter of the

polymer for DNA binding, interaction with the cell surface,endosomal escape, and subcellular localization. The nature ofthe polymer charge can enhance the transfection efficiency butmay also result in undesirable cytotoxicity. Both cationicpolymer charge and polymer degradability play a crucial role inpackaging and delivering plasmid DNA. High-density cationiccharge enhances the transfection efficiency but may give rise toundesirable cytotoxicity by destabilizing the plasma membrane,interacting with cellular components, and inhibiting normalcellular processes.470 Taking all these facts into account,Khondee et al.471 synthesized PVAm nanogels bearing discreteamounts of surface charge and used them to examine thebalance between transfection efficiency and cytotoxicity.

Figure 42. Toxicity study indicating the percentage of HeLa cellsliving after incubation with nanogels: solid bar, MTS assay (16 hincubation at 0.125 mg/mL); cross-hatched bar, dye exclusion assay(40 h incubation at 0.25 mg/mL). Nanogel samples contain 12 wt %PEGdiA with 0 wt % AETMAC (a), 12 wt % AETMAC (b), and 25%AETMAC (c). Positive control: polylysine (d). Negative control:blank (e). Reprinted from ref 463. Copyright 2002 American ChemicalSociety.

Figure 43. CLSFM images of cells labeled with CHP-NH2(15)−QDnanoparticles: (A) TIG-3, (B) MRC-5, (C) MCF-7, and (D) YKG-1.All pictures were taken at a magnification of 400×. Reprinted withpermission from ref 464. Copyright 2005 Elsevier Ltd.


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Poly(N-vinylformamide) (PNVF) nanogels were prepared byan inverse emulsion polymerization process using an acid-labilecross-linker. Nanogels were hydrolyzed to yield varying degreesof primary amines. The degree of conversion from PNVF toPVAm was controlled using different concentrations of NaOHand hydrolysis times. Low charge degradable nanogels reducecytotoxicity without compromising the overall transfectionefficiency.The group of Akiyhoshi472 recently reported the develop-

ment of a new gene delivery system capable of endosomedisruption using a polysaccharide-based cationic nanogel. Thesystem is composed of a hexadecyl group-bearing cationiccycloamylose nanogel and a lipolytic enzyme (phospholipase)to hydrolyze membrane phospholipids. The nanogel is able toencapsulate phospholipase to provide an endosome escapefunction. The nanoparticles complexed with pDNA (plasmidDNA) and enhanced its expression. Codelivery of phospholi-pase and pDNA using a nanogel is a novel concept for genedelivery with enhancement of endosomal escape.siRNAs show potential for the teatment of a wide variety of

pathologies with a known genetic origin through squence-specific gene silencing. However, siRNAs do not have favorabledruglike properties and need to be loaded into nanocarriersdesigned to cross the intracellular barriers and deliver siRNA tothe cytoplasm of the target cell (see Figure 44).

From 2008 to 2011, the active group of Akiyoshi referencedbefore reported several works on cationic nanogels useful forintracellular protein delivery,473 nanogels self-assembled witholigo-DNA and their function as artificial nucleic acidchaperones,474 nanogels of cholesterol-bearing cationic cyclo-amilose for siRNA delivery,475 and a nanogel antigenic proteindelivery system for intranasal vaccines;476 here cycloamilose(CA) was used as a new polysaccharide-based biomaterial toattach catonic spermine groups to take advantage of thesuperior activity for the transfection of siRNA shown byspermine derivatives. They also reported works on usingnanogels to deliver proteins to myeloma cells and primary Tlymphocytes477 and on a polysaccharide nanogel gene deliverysystem with an endosome escape function.472 In the majority ofthese works, except for the cases using CA, CHP is used toform nanoparticles by self-assembly in water.

Raemdonck et al.478,479 synthesized cationic dextran nanogels(dex-HEMA-co-TMAEMA) using a UV-induced emulsionpolymerization. They used photochemical internalization(PCI), a method that employs amphiphilic photosensitizersto destabilize endosomal vesicles to liberate a fraction of thesiRNA−nanogels trapped in endocytic vesicles, resulting in anadditional siRNA dose that is released in the cell cytoplasm,prolonging the therapeutic effect (see Figures 45 and 46).

Polymer siRNA complexes (siRNA polyplexes) are beingactively developed to improve the therapeutic application ofsiRNA, but the major limitation for many siRNA polyplexes isthe insufficient mRNA suppression. Given that modifying thesense strand of siRNA with 3′-cholesterol (chol-siRNA)increases the activity of free-nuclease-resistant siRNA in vitroand in vivo, Ambardekar et al.480 proposed the complexation ofchol-siRNA to increase mRNA suppression by siRNApolyplexes. The characteristics and siRNA activity of self-assembled polyplexes formed with chol-siRNA or unmodifiedsiRNA were compared using three types of cationic polymers: aPEI-based biodegradable nanogel synthesized using PEG as across-linker through carbamate bonds, a PEI−PEG-based graftcopolymer, and two PEG-based linear block copolymers. Theresults indicate that chol-siRNA increases nuclease protectionand mRNA suppression at the target tissue by select siRNApolyplexes.

5. CONCLUSIONS AND FUTURE PERSPECTIVESAs was commented previously in the Introduction, cationicpolymer particles and nanogels are being used in emergingbiomedical technologies due to the strong interaction betweennucleic acids and cationic polymer colloids, the acid-swellable

Figure 44. Functional cycloamylose nanogel−PLA2−DNA deliverysystem. Reprinted with permission from ref 472. Copyright 2011Elsevier Ltd.

Figure 45. Chemical structure of dextran hydroxyethyl methacrylate(dex-HEMA), [2-(methacryloyloxy)ethyl]trimethylammonium chlor-ide (TMAEMA), and dextran methacrylate (dex-MA). Dex-HEMA,supplemented with 0.1% photoinitiator (irgacure 2959) andTMAEMA, is emulsified in mineral oil/ABIL EM 90 by ultrasonicationto obtain a water-in-oil emulsion. UV irradiation initiates thepolymerization of dex-HEMA and TMAEMA in the nanodroplets.The image on the right represents an AFM image of dex-HEMA-co-TMAEMA nanogels (DS 21). The scale bar equals 1 mm. Reprintedwith permission from ref 478. Copyright 2009 Wiley-VCH VerlagGmbH & Co. KGaA.


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behavior of the nanoparticle/nanogel, and the ability to formoriented bonds with proteins, among other aspects.Regarding the cationic latexes, in the future the use of new

techniques will be able to achieve a more detailed character-ization of their interfacial morphology, which is scarcely known.In this sense, the role played by the water molecules aroundions of the electrical double layer and the interaction ofhydrated ions with positively charged interfaces will have to befound at the 1 nm scale. It will be possible to prepare hairy,grafted, or hard latex particles with a known and controlledbehavior at solid−liquid, liquid−liquid, and liquid−gasinterfaces.The preparation of Janus particles with positively and

negatively charged faces will be a new challenge.In the next decades, there will be a growing interest in the

use of cationic latexes in practical biomedical applications suchas immunoassays, vaccines, and so on.Although throughout this review several new features of the

cationic nanogels are envisaged for future bioapplications ofthese nanoparticles, numerous challenges are still open inoptimizing their synthesis strategies, characterization, and uses.One of the most challenging applications is the use of

cationic nanogels as carriers or vectors for in vivo siRNAdelivery. As commented before, the use of RNA interference to

treat or prevent a variety of diseases, including cancer, isnowadays a challenge in the biomedical field of therapeutics.The demands to be fulfilled by siRNA nanocarriers comprise

the following:

(1) Adequate siRNA protection by nanogels during circu-lation, preventing clearance, aggregation, degradation,and premature release. New nanogel arquitectures ornanostructures should be envisaged for the adequate andcontrolled cell-specific siRNA release.

(2) Improved stealth properties for the nanogels, avoidingrecognition by the immune system (phagocytes), andenhancement of tumor accumulation, taking advantage ofthe EPR (enhanced permeability and retention) effect intumors. To overcome these challenges, adequate nanogelsizes and shells are required.

(3) Nanogel penetration into dense tissues. To reach cells, itbecomes necessary to pass through the extracellularmatrix of the targeted tissues. The stimulus-sensitivecharacter of the nanogels enables their mobility in thesespecific locations. In this way, the design of new nanogelssensitive to or able to respond to other in vivo stimulidifferent from the pH, temperature, or ionic strengh isneeded.

Figure 46. Gene-silencing kinetics by siRNA-loaded dex-HEMA-co-TMAEMA nanogels as a function of the degree of substitution of the nanogels.Reprinted with permission from ref 479. Copyright 2010 Elsevier Ltd.


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(4) Optimization of cell targeting and endosomal escape

mechanisms for in vivo transfection, proposing different

and new moiety-targeted cationic nanogels.

(5) Avoiding toxicity. Biocompatibility is the first character-

istic to be fulfilled and biodegradability is the next for the

in vivo use of nanogels. Biocompatible and biodegradable

polymer-based cationic nanogels are needed, the

degradation pathway being enzymatic or hydrolytic

depending on the target.As a general conclusion and taking into account the variety of

features required to use cationic nanogels for in vivo siRNA

delivery, the synthesis of new stimulus-responsive nanoparticles

with new abilities to overcome the challenges listed above is a

broad and open path for the near future.

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected].*E-mail: [email protected].

Notes

The authors declare no competing financial interest.

Biographies

Jose Ramos obtained his B.Sc. in 2000 and his Ph.D. in 2005, both

from the University of the Basque Country. His Ph.D. thesis focused

on the synthesis and characterization of cationic latex particles and

microgels for biomedical applications under the supervision of Prof.

Jacqueline Forcada. He spent six months of his Ph.D. study at the

University of Bristol with Prof. Brian Vincent. In 2008 he obtained a

postdoctoral position in the group of Prof. Roque Hidalgo-Alvarez.

Currently, he is at the University of the Basque Country, and his

research interests include the design of polymer and hybrid

nanoparticles and temperature- and pH-responsive biocompatible

and biodegradable nanogels.

Jacqueline Forcada is an Associate Professor of Chemical Engineeringat the University of the Basque Country. She received her Ph.D. inChemistry from the University of the Basque Country in 1987 underthe supervision of Prof. Jose Maria Asua. Her research focuses on thesynthesis, characterization, modeling, and biotechnological applica-tions of functionalized polymeric and hybrid nanoparticles andbiocompatible and biodegradable nanogels. She has published morethan 70 scientific papers in international high-ranking journals, and sheis the author of 7 patent applications (ES, EP, PCT, and US). She hassupervised 10 Ph.D. theses.

Roque Hidalgo-Alvarez is a Professor of Applied Physics at GranadaUniversity in Spain. He was born in La Carolina (Jaen), Spain, in 1952and received his M.S. in Chemistry (1975) and Ph.D. (1979) inPhysics from Granada University under the supervision of ProfessorFernando Gonzalez-Caballero. During 1984 and 1985, he spent a yearat Wageningen Agricultural University as a visiting scientist, workingwith Professor Bert Bijsterbosch. He was promoted to a fullprofessorship in 1992 at Granada University. His research andteaching interests lie in the general area of colloid and interfacesciences with a special emphasis on electrokinetic phenomena andcolloidal stability. He has published 235 scientific papers ininternational journals and has supervised 21 Ph.D. theses. He hasbeen recently appointed as a member of the Academy of Sciences.

ACKNOWLEDGMENTS

In the past several years a great number of colleagues have beenhelpful in discussing the preparation and characterization ofcationic polymer particles and nanogels. R.H.-A. particularlythanks Professor Bert Bijsterbosch and Mr. Ab van der Linde,who were with the Laboratory for Physical and ColloidChemistry at the Wageningen Agricultural University (TheNetherlands). J.R. and J.F. thank Dr. Ainara Imaz for herfrienship and for sharing her work during the past 10 years. We


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mailto:[email protected]

mailto:[email protected]




thank the Spanish Plan Nacional de Materiales (GrantsMAT2009-13155-C04-01, MAT2012-36270-C04-01, andMAT2010-15101). J.R. and R.H.-A. acknowledge ProjectP10-FQM-5977 from “Junta de Andalucia”. R.H.-A. alsoacknowledges funding received from CEIBioTic Granada20F12/16.

DEDICATION

We dedicate this work to Purificacion Escribano. We havemissed her since November 2011.

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cationic polymer nanoparticles and nanogels: from synthesis to biotechnological applications

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