review of literature review of...

Review of Literature

Dept. Of Pharmaceutics, JSSCP, Mysore 7

4. REVIEW OF LITERATURE

4.1.Vaccination

Vaccination as a deliberate attempt to protect human beings against disease

has a long history and more widespread use of vaccines could prevent 1.6 million

deaths a year, among children less than five years of age. Over the next few years a

new generation of vaccines will become available that could save the lives of up to 10

million individuals: e.g. vaccines against diarrhoeal diseases, hepatitis C, malaria,

acquired immunodeficiency syndrome (AIDS), sexually transmitted and other

diseases8. Current development efforts seek combination vaccines that protect against

multiple pathogens, in keeping with the ultimate goal of combining all the antigens

recommended for routine immunization into a single multivalent product 9.

Immunization has been the most important way of combating viral and

bacterial infections. Traditional vaccines are mainly composed of heat-inactivated

bacteria or virus. This vaccine concept has been proved effective in controlling and

even eradication of many infectious diseases.

Still newer approaches to vaccine development are urgently needed for two

reasons. First there are several infectious diseases for which the traditional approaches

have failed and for which vaccines still needed to be developed as shown in Table 1

and second, the regulatory authorities now require vaccines to meet extremely high

standards of safety and chemical-physical characterization.

Newer trends for vaccine designing include sequencing of bacterial genome

using genome technology followed by proteomics, which involves identification of

whole sets of proteins encoded by an organism.



In-vivo expression technology (IVET) and signature tagged mutagenesis

(STM) are the two new methods that have been formulated to isolate genes that are

specifically induced during infection.

These above mentioned modern techniques have given rise to effacious

vaccines like hepatitis B vaccine, Bordetella pertusis vaccine and vaccine against

Helicobacter pylori Serogroup B by reverse vaccinology 10.

However, the success of immunization is not only dependent on nature of

immunogenic components, but also on the preparation form. Newly developed

vaccine formulations have to satisfy detailed criteria to guarantee the highest possible

quality, safety and efficacy standards.

The majority of pathogens invade the body via one or more of the mucosal

routes. Oral, nasal, pulmonary, and urino-genital routes are the most common

pathways for entry of infectious pathogens into the human host. Therefore, the

importance of generating a �first-line of defense� at the site of entry has been well

recognized. In order to have adequate mucosal protection, there are several factors

that can influence the effectiveness of vaccines and amongst them, the most critical

factor in mucosal vaccine effectiveness is the route of administration and potential for

the antigen to be processed by the antigen-presenting immune cells, such as

macrophages and dendritic cells. Presently, most vaccines are administered via the

parenteral route or via other invasive routes. It is critically important to examine the

development of mucosal vaccination strategies that can effectively trigger systemic as

well as mucosal immunity.

Polymeric nanoparticles are also actively being investigated for vaccine

delivery. For instance, Estevan and coworkers have encapsulated extracts of

Salmonella enterica serovar Abortusovis in polymeric nanoparticles 11. Upon



subcutaneous injection in Balb/c mice, the nanoparticles conferred a significant

protection and the antibody titer levels were similar to those induced by the attenuated

commercial vaccine Rv6. In another study, ovalbumin was encapsulated in alginate-

coated chitosan nanoparticles and delivered to Peyer�s patches 12.

4.2. Nanotechnology

Nanotechnology refers broadly to a field of applied science and technology

whose unifying theme is the control of matter on the molecular level in scales smaller

than 1 µm, normally in size ranges from 1-100 nm, and the fabrication of particles

within that size range. Nanotechnology is expected to bring a fundamental change in

manufacturing in the next few years and will have an enormous impact on life

sciences including drug delivery, diagnostics, nutraceuticals and production of

biomaterials. Engineered nanoparticles (NP) (<100nm) are an important tool to realize

a number of these applications. Conceptually nanoparticle is a collective name for

both nanospheres and nanocapsules. Drugs may be absorbed at the sphere surface or

encapsulated within the particle. Nanoparticles now days are receiving considerable

attention for the delivery of therapeutic drugs. Uptake of nanoparticles prepared from

hydrophobic polymers seems to be higher than that of particles with more hydrophilic

surfaces 13, thus more hydrophilic particles may be rapidly eliminated.

The potential advantages of nanoparticles as oral drug carriers are enhancement of

bioavailability, delivery of vaccine antigens to the gut-associated lymphoid tissues

(GALT), controlled release, and reduction of the gastrointestinal irritation caused by

drugs 14.

Depending on their composition and intended use, nanoparticles can be

administered orally, parenterally, or locally 15-18. A number of different strategies have



been proposed in order to modify the physicochemical characteristics of the

nanoparticles and, thus, their interactions within a biological environment. For

example, it is possible to change the chemical nature of the polymeric matrix of the

nanoparticles and thereby alter certain biological phenomena such as biorecognition,

biodistribution, bioadhesion, biocompatibility, and biodegradation. Some polymeric

materials used for this purpose are gelatin, chitosan, sodium alginate, poly (alkyl

cyanoacrylates), poly- (lactic acid), poly(lactic-co-glycolic acid), poly[ethylene

glycol- co-(lactic-glycolic acid)], poly(-caprolactone), and poly- (methyl

methacrylate) 19-20.

It is well known that the reticuloendothelial system, mainly the liver and

spleen, is a major obstacle to active targeting because of its ability to recognize these

systems, remove them from systemic circulation, and, consequently, avoid the

effective delivery of the nanospheres to organs other than those of the

reticuloendothelial system 21. Surface characteristics of nanoparticles can be easily

manipulated to achieve both passive and active drug targeting. Site-specific targeting

can be also be achieved by attaching targeting ligands to surface of particles or use of

magnetic support.

Knowledge of the fundamental relationships would allow nanoparticles to be

designed with defined size and surface characteristics for delivery to specific cells or

organs in body. On the other hand, polymeric nanoparticles offer some specific

advantages over liposomes. For instance, they help to increase the stability of

drugs/proteins and possess useful controlled release properties 22, 23.

4.2.1. Channels of Uptake

Nanoparticles have to cross the gastrointestinal barrier either by passive

diffusion via transcellular or paracellular pathways to deliver their drug content in the



blood, lymph, or target organs. Another possible mechanism for the transport of

nanoparticles across intestinal cells is paracellular uptake via aqueous channels. In

humans, the equivalent pore diameter has been estimated to be between 4 and 8 A°

and about 10�15 A° in rat and rabbit 24. The epithelial transport of larger molecules or

particles can be increased by reversibly increasing the permeability of the tissue by

opening the tight junctions under the influence of some mucoadhesive polymers and

penetration enhancers 25.

4.2.2. Methods of preparation

Many methods have been developed for preparing nanoparticles; these

methods can be classified into two main categories according to formulation need

whether the formulation requires a polymerization reaction or is achieved directly

from a macromolecule or preformed polymer 26.

4.2.2.1. Interfacial polycondensation

Polymeric nanoparticles can be also prepared by the interfacial

polycondensation of the lipophilic monomer, such as phtaloyldichloride and the

hydrophilic monomer, diethylenetriamine, in the presence and absence of the

surfactant 27. These nanoparticles were smaller than 500 nm.

4.2.2.2. Emulsification/solvent evaporation

Emulsification-solvent evaporation involves two steps. The first step requires

emulsification of the polymer solution into an aqueous phase. During the second step

polymer solvent is evaporated, inducing polymer precipitation as nanospheres. A

polymer organic solution containing the dissolved drug is dispersed into nanodroplets,

using a dispersing agent and high-energy homogenization 28, in a nonsolvent or

suspension medium such as chloroform (ICH, class 2) or ethyl acetate (ICH, class 3).



Polymer get precipitated in form of nanospheres in which drug is finely dispersed in

the matrix of polymer. Further to evaporate the solvent, temperature is increased

under pressure or by continuous stirring. The size of nanospheres can be controlled by

adjusting the stir rate, type and amount of dispersing agent, viscosity of organic and

aqueous phases, and temperature.

4.2.2.3. Solvent displacement and interfacial deposition

Solvent displacement method is based on principle of spontaneous

emulsification of the organic internal phase in which polymer had been already

dissolved into the aqueous external phase. However, there is difference in products

achieved. Solvent displacement leads to formation of nanospheres or nanocapsules.

Solvent displacement involves the precipitation of a preformed polymer from an

organic solution and the diffusion of the organic solvent in the aqueous medium in the

presence or absence of a surfactant 29-32.

The solvent displacement technique allows the preparation of nanocapsules when a

small volume of nontoxic oil is incorporated in the organic phase. Considering the oil-

based central cavities of the nanocapsules, high loading efficiencies are generally

reported for lipophilic drugs when nanocapsules are prepared 33.

4.2.2.4.Emulsification/solvent diffusion

Emulsification/solvent diffusion (ESD) usually emphasizes the use of organic

solvents. In this method the encapsulating polymer is first dissolved in a partially

water soluble solvent (propylene carbonate) which is further saturated with water (to

maintain initial thermodynamic equilibrium of both liquids).

For the formation of nanoparticles, it is necessary to promote the diffusion of the

solvent of the dispersed phase by dilution with an excess of water when the organic

solvent is partly miscible with water. Subsequently, the polymer-water saturated



solvent phase is emulsified in an aqueous solution containing stabilizer, leading to

solvent diffusion to the external phase and the formation of nanospheres or

nanocapsules, according to the oil-to-polymer ratio. Finally, the solvent is eliminated

by evaporation or filtration, depending upon its boiling point.

Several drug-loaded nanoparticles were produced by the ESD technique, including

mesotetra(hydroxyphenyl) porphyrin-loaded PLGA (p-THPP) nanoparticles34,35

doxorubicin-loaded PLGA nanoparticles36, plasmid DNA-loaded PLA

nanoparticles37, coumarin-loaded PLA nanoparticles38, indocyanine39, cyclosporine

(Cy-A)-loaded gelatin and cyclosporin (Cy-A)-loaded sodium glycolate

nanoparticles40.

4.2.2.5. Salting out method

Salting -out method is based on the separation of a water miscible solvent

from aqueous solution via a salting-out effect with the help of a salting out agent

(electrolytes, such as magnesium chloride, calcium chloride, and magnesium acetate,

or non- electrolytes such as sucrose).The salting-out procedure can be considered as a

modification of the emulsification/solvent diffusion. Polymer and drug are first

dissolved in a solvent such as acetone, which is emulsified into an aqueous gel

containing the salting-out agent and a colloidal stabilizer such as

polyvinylpyrrolidone. This oil/water emulsion is sufficiently diluted with water or

aqueous solution to enhance the diffusion of acetone into the aqueous phase, thus

inducing the formation of nanospheres.

The selection of the salting-out agent is important, because it can play an

important role in the encapsulation efficiency of the drug. Both the solvent and the

salting-out agent are then eliminated by cross-flow filtration 33.



4.3.Applications of Nanoparticles

Table 1 enumerates various drugs which have been used in nano drug delivery

systems.

Table 1: List of drugs used in nanoparticles drug delivery

Drug Carrier System References

Antifungal drugs Submicronized emulsion 41

Camptothecin Solid lipid nanoparticles 42

B-Cyclodextrin Nanosphere 43

Danazol Lipid-based emulsion 44

Flurbiprofen Nanosuspension 45

Mitoxantrone Magnetic nanoparticles 46

UCB-35440-3 Nanocrystal 47

Cephalosporin Nanoconugates 48

Betamethasone CaCo3 nanoparticles 49

Table 2 Marketed nanomedicines developed using Nanocrystal Technology.

S No Brand Name Active Ingredient Marketed places

1 Avinza Morphine sulphate Marketed in US

2 Emend Aprepitant, poorly water soluble

compound

Marketed in US

3 Herbesser Diltiazem Marketed in Japan

4 Naprelan Naproxen sodium Marketed in US

5 Rapamume Sirolimus (immunosuppressant) Marketed in US

6 Theodur Theophyyline Marketed in Japan

7 Verelan PM Verapamil Marketed in US

8 TriCor Fenofibrate Marketed in US



Table 3: List of Nanoparticluates medicine approved by FDA.

Brand Name Active Ingredient Developed by

Company

Indications

Triglide Nanocrstalline

fenofibrate

SkyePharma First

Horizon

Lipid disorders

Estrasorb

Estradiol

hemihydrate

micellar

Nanoparticles

Novavax Reduction of

vasomotor

symptoms

Adagen

Pegylated

adenosine

deaminase

Enzon Enzyme

replacement

therapy

Amphotec Colloidal

suspension of lipid

based amphotericin

B

Sequus For invasive

aspergillosis

pateints

Oncaspar

Pegasparginase Enzon Leukemia

Elestrin Estradiol gel

containing calcium

phosphate

nanoparticles

BioSante Treatment for

moderate to severe

hot flashes in

menopausal

womens



Table 4: List of Nanoparticluates medicine under clinical trials by FDA 50.

Brand Name Active

Ingredient

Developed by

Company

Indications Status

Vivagel

Dendrimer gel

StarPharma

Holdings

Vaginal

microbicide for

the

preventionof

HIV and

genital herpes

Phase II

NB-00X

Nanoemuslion

droplets

Nanobio Herpes labialis

caused ny

herpes simplex

I virus

Phase II

Aurimmune

(CYT-6091)

Colloidal gold

nanoparticles

CytImmune

Sciences

Solid tumours Phase II

INGN-401 Liposomes

FUS-1

Introgen

Therapeutics

Metastatic, non

small cell lung

cancer

Phase I

SGT-53

p-53 liposomes Synergene

Therapeutics

Solid tumours Phase I

4.3.1. Nanoparticles for oral delivery of peptides and proteins

Protein drug-delivery technologies are of ever increasing importance because

of new therapeutic proteins and peptides which are being discovered. Significant

advances in biotechnological and biochemistry aspects have led to the discovery of a

large number of bioactive molecules and vaccines based on peptides and proteins.

Encapsulation of bioactive molecules in polymeric nanoparticles protects them

against enzymatic and hydrolytic degradation. For instance, it has been found that



insulin-loaded nanoparticles have preserved insulin activity and produced blood

glucose reduction in diabetic rats for up to 14 days following the oral administration51.

There are three possible pathways for protein and peptide drug absorption

through the GI tract. The first is via the M-cells of Payer�s patches, the second via a

transcellular route involving enterocytes, and the third via paracellular avenues

through tight junctions 52, 53. The nanosystems are providing a viable alternative for

these drugs such as liposomes, polymeric micelles, and NPDDSs (Nanoaprticles drug

delivery systems). Rodrigues et al. have reported an interesting work on lectin

nanocarrier conjugate. They used dextran/poly(e-caprolactone) polyester polymers

and conjugated with three different proteins, lectins from leaves of Bauhinia

monandra and Lens culinaris, and bovine serum albumin (BSA). The nanoparticles

having a size around 200 nm could be used for delivering proteins 54.

4.3.2. Nanoparticles as a vaccine adjuvant

There are at least three reasons that nanoparticles might have an advantage

compared to microparticles. The first reason is that nanoparticles have increased

surface area for adsorption allowing for a higher antigen: polymer ratio. The second

reason is possible enhanced immunogenicity, although the evidence is conflicting. It

is well established for vaccine delivery that < 5 mm particles offer enhanced immune

responses compared to larger particles. Particle uptake by macrophages has been

found to be optimal for smaller microparticles (< 3 mm). There is evidence that even

smaller nanoparticles have increased uptake, which suggests that nanoparticles may

enhance immune responses compared to microparticles. The third reason

nanoparticles have advantage over microparticles is preparation and processing

simplifications for nanoparticles. For this to be an advantage compared to

microparticles, the exact preparation method for the nanoparticles is important.



Microparticles are typically prepared by the double-emulsion solvent

emulsification evaporation method. These methods uses water/ oil/water double

emulsion that is formed by high shear homogenization. Nanoparticles of approximate

size 200 nm can also be created by a modified solvent emulsification evaporation

method, the same method had been used to create microparticles also 55 however this

has no advantage over microparticles. An alternate nanoparticle preparation method

that offers a wide range of particle size is the solvent displacement method and was

first described earlier 29. This technique uses a water miscible solvent to produce a

one-phase mixture by magnetic stirring. These nanoparticles, depending on the

solvent, polymer type, polymer concentration, and addition of emulsifiers, can range

in size from 50 to 500 nm 56, 57. Various alternative approaches for nanoparticle

preparation have also been described based on emulsions, including spray drying, 58

and phase separation, 59, 60 however the solvent displacement method is the simplest.

The solvent displacement method has clear advantages compared to the other method

to make microparticles because it is single phase that does not require high shear

homogenization.

Couvreur and coworkers have developed insulin loaded poly

(isobutylcyanoacrylate) nanocapsules that upon oral administration, gave results

suggesting that nanocapsules could deliver insulin directly to the blood 61 and cause a

dramatic reduction of blood glycaemia, following oral administration to diabetic rats

51. Alphandary and coworkers had shown, based on TEM observations, that the same

nanocapsules were absorbed by the intestinal epithelial cells, leading to the transport

of insulin through the intestinal mucosa. However, nanocapsules were highly

degraded upon transport across M cell containing epithelium 62.



Peptide and protein encapsulation in polymeric nanocarriers have also been

applied to oral vaccination applications. Locally produced secretory IgA constitutes

over 80% of all antibodies produced in mucosa associated tissues 53, 64 and are

considered to be among the most important protective humoral immune factors.

Furthermore, a fascinating feature of mucosal immunology is that

administration of antigen in one mucosal site can lead to generation of immune

responses not only locally but also at distant mucosal sites, a phenomenon referred to

as common mucosal immune system. Finally, mucosal immunization has also the

potential to elicit an immune response against infectious diseases for which current

parenteral vaccines either have a low efficiency or are inexistent, such as vaccines

against HIV and tuberculosis 65, 66.

Eldridge and coworkers have asserted that microspheres smaller than 5 ìm in

diameter were transported by M cells through the efferent lymphatic macrophages,

numerous microparticulate systems were developed to reach the exiting goal of oral

immunization 67, 68. Among the vehicles developed during these last years, polymeric

biodegradable microparticles, mainly composed of PLA or PLGA, have been

probably the most extensively studied. A great number of proteins have been

successfully encapsulated in PLGA microparticles with a full maintenance of

structural and immunologic integrity 69-73.

As discussed above, particles in the nanoscale size rather than microscale size

are more adapted for cellular uptake in the GI tract. Up to now, few studies have

examined the capacity of biodegradable loaded-nanoparticles to induce an in-vivo

immune response after oral administration. Jung et al. 74 have used a poly (vinyl

alcohol)-co-PLGA to reach a high level of tetanus toxoid loading by adsorption.

Nanoparticles given per os to mice induced seric IgG and IgA immune responses, the



IgA titers being significantly higher than the control (intra peritoneal administration).

Particle size was found to significantly affect the induction of antibody production,

smaller particles inducing higher titers. In addition, cholera toxin B subunit (CTB)

entrapped in nanoparticles (420 nm) caused comparable immunogenicity than the

potent oral adjuvant, cholera toxin 75. The influence of the dose on the serum IgG

response after oral delivery of BSA at a dosing range from 50 to 200 ìg, entrapped in

1 ìm PLGA nanoparticles has elicited a systemic IgG dose/response relationship76.

The influence of particle size on the immune response after oral delivery of

BSA entrapped in 200, 500 and 1000 nm PLGA particles have also been studied 77. In

contrast to the results found in the literature over the more extensive intestinal

absorption of smaller particles, a higher serum IgG antibody levels and a similar

IgG2a/IgG1 ratio have been observed working with 1ìm particles than with the 200

and 500 nm particles.

Development of vaccine or antigen engineered nanocarriers are expected to be

immunologically more effective over conventional dosage forms since, they can be

fabricated to specifically target and be retained at the desired site of action. Vaccines

are still in need for some of the pathogenic diseases as shown in Table 5.



Table 5: Vaccines are needed for following pathogens.

Viral Bacterail Parasitic

HIV Mycobacterium leprae Ascaris

Cytomegalovirus Gonococcus Plasmodium

Chikenguniya Escherichia coli and

Proteus

Herpes simplex (urinary tract infection) Schistosomes

Herpes Zoster Enterotoxigenic Trypanosomes

HCV Escherichia coli Ancyclostoma

Para influenza virus Streptococcus pyogenes

Respioratory synctial virus Duodenale

Lassa virus Streptococcus mutans Nactar americanus

Filoviruses Klebsiella Trichuris

(malburg ebola) Pseudomonas Filaris

Papillomavirus Shigella Giardia

Campulobacter,

Helicobacter pylori,

Staphylococcus aureus

Leishmania

4.3.3. Ocular Applications of Nanoparticulate Drug-Delivery System

Due to cornea�s low permeability to drugs and non corneal factors (rapid tear

turnover, nasolacrimal drainage, and systemic absorption) topical ophthalmic drugs

face poor absorption in the eye. Major problem in ocular delivery is to maintain an

adequate concentration of the therapeutic agent in the precorneal area. Topical drop

administration of ophthalmic drugs in aqueous solutions results in extensive drug loss

due to tear fluid and eyelid dynamics 78-80. Polymeric nanoparticles are attractive

colloidal systems because they demonstrate increased stability and have a longer

elimination half-life in tear fluid (up to 20 min), than do conventional drugs applied

topically to the eye, which have half-lives of just one to three minutes. Nanoparticles



drug delivery system have been evaluated for ocular applications to enhance

absorption of thera-peutic drugs, improve bioavailability, reduce systemic side effects,

and sustain intraocular drug levels 81. PLGA has been evaluated and proved to be a

very useful biodegradable polymer for nanoparticluates formulation due to its medical

use, biocompatibility, and safety 82.

4.3.4. Nanoparticulate Drug-Delivery Systems for Pulmonary Treatment

Several studies have exhibited the absorption of high-molecular-weight drugs

such as insulin, heparin, and GCSF (recombinant human granulocyte colony

stimulating factor) through pulmonary drug delivery system 83. As these peptides have

a short life, the development of delivery systems with sustained pharmacological

action would be very useful. An enormous diversity of therapeutic agents is currently

administered to the patients via aerosol inhalation, and the number of potential drug

candidates for pulmonary application increase daily. The major areas of research and

therapeutic applications are asthma 84, cystic fibrosis 85, lung cancer 86, tuberculosis 87,

pulmonary hypertension 88, and diabetes 89. Some of the polymers such as PLGA,

protamine, thiomer, and lipid-based particles can be loaded by VIP or new designed

analogs. Insulin-loaded PBCA NPs were studied by Zhang Q. 90; they demonstrated

that the pulmonary administration of these nanoparticles could significantly prolong

the hypoglycemic effect of insulin.

4.3.5. Nanoparticulate Drug-Delivery Systems for Central Nervous System

Hydrophilic substances with high molecular weight have minimal passive

permeation because BBB (blood brain barrier) favors lipid soluble molecules via

passive diffusion. Transport across BBB is additionally regulated by a number of

transporters including very effective efflux transporters such as multidrug resistance-

associated protein or p-glycoprotein. Several strategies have been tried to cross the



BBB; one alternative strategy is to use drug-carrier systems such as liposomes,

antibodies, and nanoparticles 91. Numerous studies have shown the applications of

nanoparticles for brain targeting 92. It has been reported poly(butylcyanoacrylate)

nanoparticles was able to deliver hexapeptide dalargin, doxorubicin and other agents

into the brain which is significant because of the great difficulty for drugs to cross the

BBB 93.

4.3.6. Mucoadhesive Nanoparticulate Drug-Delivery Systems

Mucosal surfaces are the most common and convenient routes for delivering

drugs to the body. However, macromolecular drugs such as peptides and proteins are

unable to overcome the mucosal barrier and are degraded before reaching the

bloodstream. Nanoparticles drug delivery systems show a promising strategy for

delivering drugs through mucosa. Studies shows that mucosal surfaces are most

common and offers ease of delivering drugs to the body however, it was found that

macromolecular drugs such as peptides and proteins are unable to overcome the

mucosal barrier and are degraded before reaching the systemic circulation.

Polysaccharide Chitosan (CS) is mucoadhesive and CS nanoparticles, CS-coated oil

nanodroplets (nanocapsules), and CS-coated lipid NPs have shown interesting

possibilities for this purpose 94.

Nanoprecipitation techniques using PLGA and PLA polymers were found to

be useful for nanoparticulate delivery of proteins and have shown more versatility and

flexibility in the formulation for protein delivery 95.



4.3.7. Nanoparticulate Drug Delivery in Cancer Treatment

Several nanoaprticle drug delivery system are reported for the application in

cancer therapy, transferring conjugated paclitaxel-loaded nanoparticles 96,

nanovaccines 97, adriamycin-loaded nanoparticles for hepatoma treatment 98, magnetic

PBCA nanospheres with aclacinomycin A in gastric cancer 99, near-infrared

absorption nanospheres 100, polypropylenimine dendrimer nanoparticles for

oligonucleotides 101, lytic-peptide-bound magnetite nanoparticles for breast cancer

treatment 102, ceramic-based nanoparticles entrapping water-insoluble

photosensitizing anticancer drugs 103, and poly(epsilon-caprolactone) nanoparticles for

the delivery of Tamoxifen for breast cancer treatment 104.

4.3.8. Nanoparticles for gene delivery

Polynucleotide vaccines work by delivering genes encoding relevant antigens

to host cells where they are expressed, producing the antigenic protein within the

vicinity of professional antigen presenting cells to initiate immune response. Such

vaccines produce both humoral and cell-mediated immunity because intracellular

production of protein, as opposed to extracellular deposition, stimulates both arms of

the immune system. Nanoparticles loaded with plasmid DNA could also serve as an

efficient sustained release gene delivery system due to their rapid escape from the

degradative endo-lysosomal compartment to the cytoplasmic compartment 105. Hedley

et al. 106 reported that following their intracellular uptake and endolysosomal escape,

nanoparticles could release DNA at a sustained rate resulting in sustained gene

expression. This gene delivery strategy could be applied to facilitate bone healing by

using PLGA nanoparticles containing therapeutic genes such as bone morphogenic

protein.



4.4.Characterization of Nanoparticles

Characterization of nanoparticles is an important prerequisite before drug

delivery take place and many of the tools employed for their characterization are the

same as those used for similar analysis of other submicrometer colloids such as

micelles, liposomes, and emulsions.

4.4.1. Dynamic Light Scattering (DLS)

DLS, also known as photon correlation spectroscopy (PCS) or quasi-elastic

light scattering (QELS) records the variation in the intensity of scattered light on the

microsecond time scale 107, 108. This variation results from interference of light

scattered by individual particles under the influence of Brownian motion, and is

quantified by compilation of an autocorrelation function. This function is fit to an

exponential, or some combination or modification thereof, with the corresponding

decay constant(s) being related to the diffusion coefficient (s). Using standard

assumptions of spherical size, low concentration, and known viscosity of the

suspending medium, particle size is calculated from this coefficient.

4.4.2. Static Light Scattering/Fraunhofer Diffraction

Static light scattering (SLS) is an ensemble method in which the pattern of

light scattered from a solution of particles is collected and fit to fundamental

electromagnetic equations in which size is the primary variable 109.

Fraunhofer (light, laser) diffraction is frequently employed as a sizing method for

nanoparticles. As size drops into the submicrometer regime the differences in the

scattering pattern occur primarily at high angles, so collecting such data becomes

critical�an ability that varies widely among commercial instruments. The



approximations implemented in Fraunhofer theory are acceptable for particles of

diameter 2 mm and higher 110.

4.4.3. Acoustic Methods

Another ensemble approach, acoustic spectroscopy, measures the attenuation

of sound waves as a means of determining size through the fitting of physically

relevant equations 111. In addition, the oscillating electric field generated by the

movement of charged particles under the influence of acoustic energy can be detected

to provide information on surface charge. This is termed electroacoustic spectroscopy

and can also be reversed so that sound waves generated by the oscillatory motion of

charged particles in a varying electric field are observable 112.

4.4.4. Nuclear Magnetic Resonance

Nuclear magnetic resonance (NMR) can be used to determine both the size

and the qualitative nature of nanoparticles. The selectivity afforded by chemical shift

complements the sensitivity to molecular mobility to provide information on the

physicochemical status of components within the nanoparticle 113.

4.4.5. Single-Particle Optical Sensing (SPOS)

A particle counting method, SPOS, which is also known as optical particle

counting, involves recording the obscuration or scattering of a beam of light that

results from the passage of individual particles through a sensor 114. Particles of

diameter less than 1 mm are largely undetected, thus making SPOS very useful in the

determination of the few large particles in a population that may represent a safety

concern, indicate a problem in production, or be harbingers of instability. Drawbacks

include the possible dissolution of analyte during analysis, the large dilution required,

and the need for low backgrounds.



4.4.6. Optical Microscopy

Most nanoparticles are below the resolution limit (ca. 0.5 mm) of direct optical

imaging, though microscopy is still useful to get an estimate of size and crystallinity

of starting materials, as might be desirable in the instance of comminution or

homogenization processing, or other larger particles 115.

4.4.7. Electron Microscopy

Scanning and transmission electron microscopy, SEM and TEM, respectively,

provide a way to directly observe nanoparticles, with the former method being better

for morphological examination 116-119. TEM has a smaller size limit of detection, is a

good validation for other methods, and affords structural information via electron

diffraction, but staining is usually required, and one must be cognizant of the

statistically small sample size and the effect that vacuum can have on the particles.

4.4.8. Atomic Force Microscopy (AFM)

In this technique, a probe tip with atomic scale sharpness is rastered across a

sample to produce a topological map based on the forces at play between the tip and

the surface. The probe can be dragged across the sample (contact mode), or allowed to

hover just above (noncontact mode), with the exact nature of the particular force

employed serving to distinguish among the subtechniques. That ultrahigh resolution is

obtainable with this approach, which along with the ability to map a sample according

to properties in addition to size, e.g., colloidal attraction or resistance to deformation,

makes AFM a valuable tool. However, size and shape has been the most common

application to date 120-121. Nanoparticles are typically presented as an evaporated

suspension on a smooth silicon or mica surface, though not without the possibility of

deformation 122.



4.4.9. Hydrophobic Interaction Chromatography

In this method the analyte is first adsorbed onto a chromatographic stationary

phase using a high concentration of an antichaotropic salt 123. Elution occurs using a

gradient in which the salt concentration is decreased, so that those materials eluting

first are the least hydrophobic because the salt concentration did not need to be

decreased much before the analyte desorbed. Originally developed for proteins,

hydrophobic interaction chromatography has been pressed into service as a means of

characterizing the hydrophobicity of nanoparticle surfaces, a property influenced by

the choice of surfactant and/or polymer and also a key parameter in determining their

in vivo fate 124, 125.

4.4.10. Ispycnic Centrifugation

Another bioanalytical method applied to nanoparticles is centrifugation of

analyte using a sucrose gradient as the suspending media. Under the influence of

Stokes� laws, sedimenting particles will settle until they reach a point where their

density matches that of the gradient. This self-focusing separation allows nanoparticle

density to be determined, which along with particle size and bulk substituent

concentration can in turn be used to calculate a number concentration 126.

4.4.11. Zeta Potential

Zeta potential is used as a surrogate for surface change, and is often measured

by observing the oscillations in signal that result from light scattered by particles

located in an electric field 127, 128.

4.4.12. Differential Scanning Calorimetry (DSC)

Another method that is a little different from its implementation with bulk

materials, DSC can be used to determine the nature and speciation of crystallinity



within nanoparticles through the measurement of glass and melting point temperatures

and their associated enthalpies 129.

4.5. DIARRHEAGENIC ENTEROTOXIGENIC ESCHERICHIA COLI (ETEC)

E. coli are motile Gram-negative bacteria that occupy the intestines of humans

and animals as commensals or as pathogens. The genome consists of a singular

circular DNA molecule of 4 x 106 base pairs with a molecular weight of ~ 2.5 x 109

Daltons. E. coli are sero typed based on their O lipopolysaccharide (LPS) and H

(flagellar) surface antigen (Ag) profiles. ETEC possess five different fimbrial

subtypes, F4 (K88), F18, F5 (K99), F6 (987P), and F41, F5, F6, and F41 fimbriae are

predominantly associated with ETEC that cause scours in neonatal pigs, whereas the

F4 and F18 adhesins are found on ETEC that predominantly cause post-weaning coli

bacillosis (PWC). Once colonization is established, ETEC can elaborate heat-labile

(LT), heat-stable (ST), and East-1 enterotoxins that act upon intestinal enterocytes

causing secretory diarrhea. A variant of two ETEC known as ETEC/STEC (shiga

toxin E. coli) can produce, in addition to LT and ST enterotoxins, Shiga-toxin that

allows STEC to cause edema disease 130-132.

4.5.1. Enterotoxins of ETEC F4 responsible for post-weaning diarrhea in pigs

ETEC F4 infections are responsible for significant death and morbidity in

neonatal and post-weaned pigs. ETEC expressing F4 fimbriae adhere to F4R on

intestinal epithelial cells of F4R positive pigs (F4Rpos) only, resulting in colonization

and subsequent enterotoxin secretion that leads to osmotic diarrhea in piglets.

Depending upon the strain, ETEC F4 express heat-labile (LT), heat-stable (ST), and

or EAST 1 enterotoxins which cause diarrhea 133. ETEC produce one or both of two

enterotoxins, heat-labile enterotoxin (LT) and heat-stable enterotoxin (ST). LT was

found to be similar to cholera toxin physiologically, structurally, and antigenically



and have a similar mode of action. The molecular mass (84 kDa) and the subunit

structure of the two toxins were essentially identical, with an active (A) subunit

surrounded by five identical binding (B) subunits. Following colonization of the small

intestine by ETEC and further release of the LT, the LTB subunits bind irreversibly to

GM1 ganglioside, and the A subunit activates adenylate cyclase, which results in

increases in cyclic AMP, which stimulates chloride secretion in the crypt cells and

inhibits neutral sodium chloride in the villus tips. When these actions exceed the

absorptive capacity of the bowel, purging of watery diarrhea results 134, 135.

ST is a non antigenic low-molecular-weight peptide, consisting of 18 to 19

amino acids. There are two variants, STp and STh, named from their initial discovery

from pigs and humans, respectively, and which have identical mechanisms of action.

Released in the small intestine, ST binds reversibly to guanylate cyclase, resulting in

increased levels of cyclic GMP. ST has also been implicated in the control of cell

proliferation via elevation of intracellular calcium levels. ST is small, monomeric

toxins containing multiple cysteine residues, whose disulfide bonds were mainly

responsible for the heat stability of these toxins. As with LT, chloride secretion by the

crypt cells is then increased and inhibition of neutral sodium chloride absorption

occurs, leading to outpouring of diarrheal stool. Since ETEC must be recognized by

the enterotoxins it produces, diagnosis must depend upon identifying either LT and/or

ST 136.

4.5.2. The fimbriae of the enterobacteriaciae

Fimbriae are 0.5-1.5 microns long proteinaceous appendages peritrichously

distributed at the surface of the bacterium that allow adherence of the bacteria to

fimbriae-specific receptors. Between 100-300 fimbriae are present per bacterium.

Fimbriae are classified according to the presence and position of various amino acids



(aa) in the primary sequence of their major fimbrial subunit. The F4 fimbriae are

placed into a class that do not contain the amino acid cysteine and contain a unique

penultimate tyrosine at their carboxyl terminal end 137. The F4 fimbriae are lectins that

bind to glycoprotein receptors located on the villi lining the small intestine of pigs.

The genes faeA to faeJ are located on a plasmid and give rise to the corresponding

proteins FaeA to FaeJ whose functions range from chaperones to building blocks for

the F4 fimbriae. The F4 has a molecular weight of approximately 26 kDa as

determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-

PAGE). F4 fimbriae are ~ 1 um in length and are primarily composed of hundreds of

repeating major subunits known as FaeG with a relatively small number of minor

subunits interspersed throughout the structure. The minor subunits are not only part of

the fimbrial structure but have important functions as well. The assembly of the F4

fimbriae begins with the expression of genes faeB through faeJ, whose rate of

transcription is influenced by the level of the repressor FaeA. FaeC initiates F4

production as it acts as the nidus to which copious amounts of FaeG subunits are

added with the minor subunits FaeH and FaeF added at integral locations throughout

the structure to permit proper elongation 138.

Figure 1: Genetic organization of the F4 gene cluster; genes are represented by boxes, the designations of the genes are given in the boxes and the molecular mass of the proteins are given in kDa; a short description of the function of the different proteins is given (according to Mol et al., 1994, 139).



4.6.Approaches to study the pathogenicity of an enteric bacterial toxin

4.6.1. In vivo assays

4.6.1.1.Ligated intestinal loops or segments.

In this approach, small intestinal (jejunal or ileal) or colonic ligated intestinal

segments are inoculated with toxin preparations or bacterial cultures and the

subsequent presence or absence of secretion is assessed at time points up to 18 h.

Nishibuchi M et al., 140 demonstrated the use of ligated segments in which isogenic

strains of Vibrio parahaemolyticus differing only in production of the thermostable

direct hemolysin (TDH) were tested in order to report the importance of TDH in

causing the secretion.

4.6.1.2.In vivo perfusion studies.

The above method was just a modification of ligated intestinal loop technique

but allows a more precise and accurate measurement of net fluid movement across the

intestinal epithelium as well as measurements of the net change in specific ions in the

intestinal fluid over time. Practically, a ligated intestinal segment is cannulated with a

multiperforated tube and cleansed by flushing prior to initiation of the experiment 141.

Samples can be analyzed sequentially for ionic content or osmolarity to measure net

changes over time using polyethylene glycol or phenolsulfonphthalein 142 as a volume

marker. The only limitation for this method is that measurements are typically made

over only a few hours.

4.6.1.3.Oral inoculation.

Another approach that came into existence is the oral inoculation that causes

diarrhea or intestinal fluid accumulation in response of to secretion of an enteric

bacterial toxin. Oral inoculation of animal models with isogenic strains can help in

establishing the importance of a toxin for the disease to occur. Isogenic strains of



Yersinia enterocolitica have been used to establish the relevance of heat-stable

enterotoxin that contributes to the severity of disease in a rabbit animal model 143.

4.6.1.4.Ritard model.

The reversible ileal tie adult rabbit diarrheal disease model (RITARD model)

is useful in studying the pathophysiolgy of enteric bacteria in an uninterrupted

intestine. It has been widely used because iit resembles or mimics native disease than

do some of the above approaches 144. Cecum is ligated permanently and the bacteria

are injected into the intestine at a site proximal to a temporary ileal ligature. After a

short incubation period (2 h) in order to allow the infection to become established, the

ligature is removed and the animals are observed for the development of diarrhea

(days). This model has been particularly useful in studying the immune responses to

ETEC 145 and V. cholerae 146.

4.6.2. In vitro assays.

4.6.2.1.Ussing chambers.

The Ussing chamber is a valuable and in vitro experimental approach that has

been used extensively to identify the specific changes in active ion transport

stimulated by enteric bacterial toxins 147. Briefly, intestinal epithelium or polarized

monolayers of cultured intestinal epithelial cells are mounted between Lucite

chambers under conditions of ionic, osmotic, and electrical equilibrium. Ability of an

enteric toxin to stimulate anion (usually chloride) secretion and/or to inhibit NaCl

absorption, both potentially contributing to net intestinal secretion, can be easily

measured under these conditions.



4.6.2.2.Tissue culture assays.

These assays are another experimental approach to identify the role of enteric

bacterial toxins in causing diarrhea using wide variety of non intestinal cell lines.

Changes in shape of cells or by cytotoxicity in response to treatment with the enteric

toxin will give information about the activity of the toxin. Chinese hamster ovary

(CHO) and Y-1 adrenal cells have proven useful for identifying toxins that increase

intracellular cyclic AMP levels with respect to the changes in shape. In response to an

increase in intracellular cyclic AMP levels, CHO cells elongate 148 and Y-1 adrenal

cells become round 149.

4.7.DIAGNOSIS, TREATMENT AND MANAGEMENT

4.7.1. Laboratory Assays

Since ETEC must be recognized by the enterotoxins it produces, diagnosis

must depend upon identifying either LT and/or ST. Assays such as staphylococcal

coagglutination 150, passive latex agglutination 151, immunoprecipitation in agar, and

the Biken test 152 were found to be specific but were not used widely for diagnostic

purposes. Enzyme-linked immunosorbent assays became a widely used method for

detecting LT, particularly using microtiter GM1 ganglioside methods 153, 154.

Subsequently, combined GM1 enzyme-linked immunosorbent assays for ST and LT

were developed 154, 155. During recent years DNA probes, with either radioactive or

nonradioactive detections or GM1 enzyme-linked immunosorbent assays using

monoclonal antibodies against ST or LT have been the most widely used methods for

detection of ETEC toxins 156.

The more recently developed DNA probe methods have the capacity to detect

the structural genes for toxins and CFs and thus have the advantage of detecting

ETEC from samples which have been stored for long periods of time and where



phenotypic changes may have taken place. These procedures are more difficult to

adapt to field sites in developing countries, where laboratory facilities may be

inadequate for molecular microbiological methods. Furthermore, in some instances

ETEC CFs can only be detected by molecular but not phenotypic methods, since they

are not exposed on the bacterial surfaces due to mutation of genes required for surface

expression 157.

The treatment of diarrheal disease due to ETEC is the same as that for cholera

or any other acute secretory diarrheal disease.

4.7.2. Rehydration

Rapid rehydration using intravenous fluids (such as Ringer�s lactate) is

required initially for all patients with severe dehydration. After restoration of blood

pressure and major signs of dehydration, patients can be put on oral rehydration

solutions for the remainder of therapy.

4.7.3. Antimicrobials

The use of antimicrobials in the treatment of ETEC diarrhea is problematic,

since an etiologic diagnosis cannot be made rapidly. This differs from the treatment of

cholera, an epidemic disease, where clinical findings and rapid laboratory tests can

readily lead to correct diagnosis. It has been difficult to study the effect of

antimicrobials in children with ETEC disease and antimicrobials are not used

routinely in treatment of childhood diarrhea. Antimicrobials that have been used in

effective treatment include doxycycline, trimethoprim-sulfamethoxazole,

erythromycin, norfloxacin, ciprofloxacin, ofloxacin, azithromycin, and rifamycin.



4.7.4. Multidrug Resistance Patterns

Due to increasing microbial resistance of ETEC, newer drugs have been used.

A fluoroquinolone such as ciprofloxacin, levofloxacin, or ofloxacin is currently the

drug of choice, since no significant resistance to these drugs has yet developed 158-159.

A newer nonabsorbed drug, rifaxamin, has also been shown to be as effective as a

fluoroquinolone and has only recently been approved for use in the United States 160.

Multidrug resistance is increasing in ETEC due to the widespread use of

chemotherapeutic agents in countries where diarrhea is endemic.

4.7.5. PREVENTION

4.7.5.1.Vaccine Development

Prevention of ETEC infection is clearly related to water and sanitation,

including food preparation and distribution. Other methods on a microscale are

presently being done: building safe-water tube wells, chlorination/filtration/heating of

drinking water, and building and improving latrines. These attempts to block

transmission are certainly effective if implemented but cannot solve the problem

quickly. Therefore, there is much interest in the development of vaccines for

prevention of ETEC disease 161.

4.7.5.2.Purified CFs and Enterotoxoids

Various purified CFs have been tested as oral immunogens but have been

considered less suitable since they are expensive to prepare and sensitive to

proteolytic degradation 162. To protect the fimbriae from degradation in the stomach,

purified CFs have been incorporated into biodegradable microspheres. An alternative

administration route that has been considered is to give an ETEC vaccine by the

transcutaneous route. Such administration of E. coli CS6 together with LT has



induced immune responses against CS6 in about half of the volunteers and anti-LT

responses in all of them 163.

4.7.5.3.Inactivated Whole-Cell Vaccines

Another approach that has been extensively attempted is to immunize orally

with killed ETEC bacteria that express then most important CFs on the bacterial

surface together with an appropriate LT toxoid, i.e., cholera toxin B subunit or LTB

164.

4.7.5.4.Live Oral ETEC Vaccines

The potential of live ETEC vaccines has been suggested based on previous

findings in human volunteers that a live vaccine strain expressing different CSs

afforded highly significant protection against challenge with wild-type ETEC

expressing the corresponding CS factors 165.

4.8.Porous dosage forms

Researchers are now focusing on development of porous nano materials for

controlled drug delivery because of many advantages like porous structure, high

surface area, tunable pore sizes with narrow distribution and well defined surface

properties 166-167. Porous carriers is widely used in pharmaceutical as it proves to be

promising in development of novel drug delivery systems such as floating drug

delivery systems, sustained drug delivery systems and improvement of solubility of

many poorly water soluble drugs 168-171. Because of enormous porous structure these

systems allows the incorporation of variety of drugs as they get easily adsorb and

release them in a more predictable and reproducible manner. An extensive work has

been done on use of microporous, mesoprosous and nanoporous carriers as a drug

delivery tool 172-173. However liquid penetration into these systems is dependent on

both molecular and bulk property of liquid and surface property of porous medium. It



was reported that drug release from these porous systems can be completed within

few minutes and can be varied to upto several hours or days 174-175. Drug can be

loaded in these porous materials by various methods like simple mixing, solvent

evaporation, loading under high pressure, stirring in drug solution. Porous

nanoparticles can be made up of diverse materials in a variety of different conditions

that can be targeted to different parts of body with various size ranges. These porous

nanoparticles prove to be robust tool for drug delivery and proved to be useful to

encapsulate large variety of drugs of different chemistry and molecular weight 176.

Porous nanoparticles offer various advantages like ease of flow, processing,

aerolisation and provide a paradigm shift in drug delivery. In the coming years there

is growing interest in porous carriers as a adjuvant for drug delivery systems.

Illum L et al., 65 had described variety of different types of nasal vaccine

systems to include cholera toxin, microspheres, nanoparticles, liposomes, attenuated

virus and cells and outer membrane proteins (proteosomes). He discussed work on the

use of the cationic polysaccharide, chitosan as a delivery system for nasally

administered vaccines. Several animal studies have been carried out on influenza,

pertussis and diphtheria vaccines with good results. After nasal administration of the

chitosan-antigen nasal vaccines it was generally found that the nasal formulation

induced significant serum IgG responses similar to and secretory IgA levels superior

to what was induced by a parenteral administration of the vaccine. It was found that

animals vaccinated via the nasal route with the various chitosan-antigen vaccines

were protected against the appropriate challenge. So far the nasal chitosan vaccine

delivery system has been tested for vaccination against influenza in human subjects.

Study results revealed that the nasal chitosan influenza vaccine was both effective and

protective according to the CPMP requirements.



Eldridge JH et al., 67 have investigated the use of biodegradable and

biocompatible microspheres as a vaccine delivery system for both parenteral and

enteral immunization. Microspheres composed of poly (DL-lactide-co-glycolide)

which contained a toxoid vaccine of Staphylococcal enterotoxin B were found to

strongly potentiate the circulating anti-toxin antibody response following

intraperitoneal injection. Following oral administration, microspheres less than 10

microns in diameter were specifically taken up into the Peyer's patches of the gut-

associated lymphoid tissue, where those greater than or equal to 5 microns remained

fixed for an extended period. Microspheres less than 5 microns were disseminated

within macrophages to the mesenteric lymph nodes, blood circulation and spleen. It

was found that oral immunization with enterotoxoid-containing microspheres induced

circulating toxin-specific antibodies and a concurrent secretory IgA anti-toxin

response in saliva, gut wash fluids and bronchial-alveolar wash (BAW) fluids. In

contrast, soluble enterotoxoid was completely ineffective as an oral immunogen.

Attarki K et al., 74 have studied the encapsulation of vaccines in

biodegradable microspheres and reported them as an excellent mucosal immunogens

with a high potential for immunization against bacterial infections. They tested the

protective immunity elicited by intragastric vaccination with phosphorylcholine (PC)

encapsulated in poly (DL-lactide-co-glycolide) (DL-PLG) microspheres against

Salmonella typhimurium in a mouse model of invasive intestinal infection. Mice were

primed intragastrically on days 1, 2, and 3 and boosted on days 28, 29, and 30 with

PC (280 microg) coupled to porcine thyroglobulin (PC-thyr) encapsulated in DL-PLG

microspheres, free PC-thyr, or blank microspheres. A significant rise in anti-PC

immunoglobulin A (IgA) titers, as measured by an enzyme-linked immunosorbent

assay, was observed in the intestinal secretions after immunization with PC-loaded



microspheres, compared to the titers of mice immunized with free PC-thyr or blank

microspheres. Control mice were primed intraperitoneally on day 1 with 15 microg of

PC in complete Freund's adjuvant and boosted on days 10, 14, and 20 with the same

dose without adjuvant but via the same route. In these mice, the levels of anti-PC IgA

in intestinal secretions were equivalent to those of the mice intragastrically

immunized with PC-loaded microspheres, but protection was significantly weaker,

suggesting that either the IgAs were not functional or that other immune mechanisms

are important in protection. Results proved the potential of antigen encapsulation in

DL-PLG microspheres for eliciting protective immunity against invasive intestinal

bacterial diseases and suggest that a similar strategy could be used against diseases

caused by other PC-bearing microorganisms.

Gutierro I et al., 82 have entrapped BSA in particles of different sizes (200,

500 and 1000 nm) prepared from poly (D,L-lactic-co-glycolic) acid by a double

emulsion method. The particles were given, either intranasally, orally or

subcutaneously, to Balb/c mice and the serum IgG, IgG1 and IgG2a response elicited

was compared to that obtained by the subcutaneous administration of either free

antigen, free antigen emulsified 1:1 with Freund's Complete Adjuvant (FCA), or free

antigen administered with Al(OH)(3). The administration of 1000 nm particles

generally elicited a higher serum IgG response than that obtained with the

administration of 500 or 200 nm sized nanospheres, the immune response for 500 nm

particles being similar than that obtained with 200 nm by the subcutaneous and the

oral route, and higher by the intranasal route. It was found that the route of

administration influences the serum IgG2a/IgG1 ratio after the administration of free

antigen, but not after the administration of the particles. Therefore, differences on the

total serum IgG response induced by particles of different sizes do not result in



differences on the IgG1 or IgG2a-type immune responses, suggesting that the antigen

processing and presentation is similar in all cases tested for PLGA particles.

Sher P et al., 166 have studied the use of low density porous carriers in the

pharmaceutical applications. Response surface methodology, using 3(2) factorial

design was used to study drug adsorption on and its release patterns from microporous

polypropylene (Accurel MP 1000) in the absence of additives. Ibuprofen, as model

drug, was adsorbed on the polymer by solvent evaporation using two organic solvents

methanol (M) and dichloromethane (DCM). The amount of carrier (100 mg) and its

particle size range (250-350 microm) were kept invariant while solvent volume (X1)

and drug amount (X2) were taken as variables. It was found that drug adsorption

pattern depended on the type and amount of solvent used. DSC, XRD, FTIR and

TGA, predict crystalline nature and physical form of adsorption. SEM showed the

penetration and adsorption of the drug in and on the microporous polymer. Accurel

MP 1000 had a pore volume of 1.992 g/cm3 and surface area of 55.9855 m2/g as

detected by mercury porosimetery. On drug adsorption, pore volume ranged from

0.413 to 1.198 g/cm3 for methanol and 0.280-0.759 g/cm3 for DCM. Similarly

surface area was in the range 38.445-25.497 m2/g for methanol and 18.710-

32.528m2/g for DCM. The drug release was investigated in phosphate buffer pH 7.2.

All batches showed excellent in vitro floating property. Drug release was partial with

recovery to complete dependent on type and volume of solvent. Effect of solvent

properties shows a positive influence on drug adsorption and release.

Byrne RS et al., 173 have investigated three commercially available

microparticulate porous ceramics, N-light N3, Starlight SLK1000 and Carbolite

16/20. Starlight SLK1000 and Carbolite 16/20 were principally composed of mullite,



while N-light N3 was principally composed of quartz. Each porous ceramic was

partly open-cell with varying porosities and pore size distributions. Using a novel

vacuum loading technique, N-light N3 was loaded with benzoic acid, sodium

benzoate and diltiazem HCl, while Starlight SLK1000 and Carbolite 16/20 were

loaded with diltiazem HCl. The drug loading was influenced by the solution

concentration and by the porosity and bulk density of the ceramic. In vitro dissolution

testing of the loaded porous microparticles showed an initial burst release of each

drug followed by sustained release. Study results revealed that drug release was

influenced by the surface pore size distribution of the ceramic and by electrostatic

interactions between the interior and exterior microparticle surfaces and the drug.

Catarina et al., 177 have extensively studied polymeric nanoparticles as

particulate carriers in the pharmaceutical and medical fields, because they show

promise as drug delivery systems as a result of their controlled- and sustained-release

properties, subcellular size, and biocompatibility with tissue and cells. Several

methods to prepare nanoparticles have been developed during the last two decades,

classified according to whether the particle formation involves a polymerization

reaction or arises from a macromolecule or preformed polymer. In this review the

most important preparation methods are described, especially those that make use of

natural polymers. This study laid emphasis on advantages and disadvantages so as to

facilitate selection of an appropriate nanoencapsulation method according to a

particular application.

Wendorf et al., 178 have prepared nanoparticles ranging in size from 110 to

230 nm using Poly (lactide-co-glycolide) (PLG) polymers. The objective of this work

was to obtain a nanoparticle formulation that could be sterile filtered, lyophilized, and



resuspended to the initial size with excipients appropriate for use as a vaccine

formulation. Protein antigens were adsorbed to the particles; the protein-nanoparticles

were then lyophilized with the excipients. Vaccine compatible excipient combinations

of sugars alone, surfactants alone, and sugars and surfactants were tested to find

conditions where initial particle size was recovered. It was reported that nanoparticle

formulations in vivo were either similar or had enhanced immunogenicity compared

to aluminum hydroxide formulations. It is concluded that a lyophilized nanoparticle

formulation with adsorbed protein antigen and minimal excipients is an effective

vaccine delivery system.

Borges et al., 179 have studied that the adsorption of antigens onto chitosan

particles is an easy and unique mild loading process suitable to be used with vaccines.

In order to increase the stability of this particles and to prevent an immediate

desorption in gastrointestinal fluids, a coating process with sodium alginate was

developed. One of the challenges of this developing process was to keep the particles

in the nanosized range in order to be taken up by M-cells of the Peyer�s patches. The

observed inversion of the particles� zeta potential values after coating suggested the

presence of an alginate coating layer. These results were confirmed by FTIR and DSC

techniques. Additionally, in vitro release studies showed that the presence of the

alginate layer around the particles was able to prevent a burst release of loaded

ovalbumin and to improve the stability of the nanoparticles in simulated intestinal

fluid at 37 ◦C. The optimization of the coating process resulted in 35% (w/w) for the

loading capacity of the coated particles. SEM investigations confirmed a suitable size

of the coated nanoparticles for the uptake by M cells.



Rieux et al., 180 have laid emphasis on peptides and proteins that remain

poorly bioavailable upon oral administration. One of the most promising strategies to

improve their oral delivery relies on their association with colloidal carriers, e.g.

polymeric nanoparticles, stable in gastrointestinal tract, protective for encapsulated

substances and able to modulate physicochemical characteristics, drug release and

biological behavior. The mechanisms of transport of these nanoparticles across

intestinal mucosa are reviewed. In particular, the influence of size and surface

properties on their non specific uptake or their targeted uptake by enterocytes and/or

M cells is discussed. Enhancement of their uptake by appropriate cells, i.e. M cells by

(i) modeling surface properties to optimize access to and transport by M cells (ii)

identifying surface markers specific to human M cell allowing targeting to M cells

and nanoparticles transcytosis is illustrated. Encouraging results upon in vivo testing

are reported but low bioavailability and lack of control on absorbed dose slow down

products development. It was reported that vaccines are certainly the most promising

applications for orally delivered nanoparticles.

Arayne et al., 181 have laid emphasis mainly on fabrication of porous

nanoparticles, its characterization and its use for controlled release of drug. It also

encompasses the strategies that have been used to translate and fabricate a wide range

of particulate carriers e.g., nanospheres, liposomes, micelles, oil-in-water emulsions,

with prolonged circulation and/or target specificity. Sol-gel technique is one of the

most widely used techniques to fabricate porous nanoparticles within the polymer.

Such nanoparticles have also applications in vascular drug delivery and release, site-

specific targeting, as well as transfusion medicine. This article will highlight rational

approaches in design and surface engineering of nanoscale vehicles and entities for

site-specific drug delivery.



Huyghebaert N et al., 182 have developed a multi-particulate formulation of

F4 fimbriae for oral vaccination of suckling piglets against enterotoxigenic

Escherichia coli infections. From the economical point of view, a pellet formulation

was optimized to decrease vaccine dose and dosing frequency. It was found after

disintegration testing, pellets consisting of lactose (a-lactose monohydrate 90

mesh/lactose 75/25 (w/w)) and microcrystalline cellulose in a ratio of 80/20 (w/w)

showed a sponge-like structure from which F4 fimbriae could be released. Coating of

these pellets resulted in good enteric properties. Dissolution test showed that F4

fimbriae were released from the optimized enteric-coated pellets but interaction

between F4 fimbriae and the coating polymer was seen. This incompatibility leads to

unpredictable in vitro quantification of F4 biological activity.

Broeck W V et al., 183 have studied different procedures for preparing and

purifying F4ac fimbriae of the enterotoxigenic Escherichia coli strain GIS 26

(O149:K91:F4ac LT+Sta+STb+) and the purity and yield of F4ac were compared.

Fimbriae were prepared by either mechanical shearing or heat shock treatment of

concentrated bacterial suspensions (1011 bacteria/ml). The mechanical shearing

procedure resulted in approximately 1.7 mg fimbriae (i.e. 74.4% of the isolated

protein) and 0.6 mg (25.6%) contaminating proteins per 1012 bacteria, whereas the

yield of fimbriae following heatshock treatment was lower (0.3 mg per 1012 bacteria,

i.e. 26.2%) and the relative contamination higher (1.0 mg per 1012 bacteria, i.e.

73.8%). A further purification consisted of either anion exchange chromatography

(AEC) or electro-elution from SDS-polyacrylamide gels. It was concluded that native

fimbriae as well as major subunits were able to bind to the receptors, and the

specificity of the binding was demonstrated by blockage with F4ac-specific MAb.



Verdonck F et al., 184 have studied that receptor-mediated uptake of orally

administered antigen can lead to an antigen-specific immune response, whereas oral

administration of most other non-replicating soluble antigens results in the induction

of oral tolerance. In the present study, it is shown that fimbriae purified from an F4

(K88) + enterotoxigenic Escherichia coli strain can function as a mucosal carrier

molecule for the model antigen human serum albumin (HSA). Study indicates that F4

fimbriae as mucosal carrier and CT as adjuvant synergistically improve the induction

of a HSA-specific immune response following oral immunization of pigs. These

results could open new perspectives in the development of vaccines against

enteropathogens.

Broeck V W et al., 185 have developed an effective way of stimulating the

mucosal immune system which was examined in piglets, using F4 fimbriae of

enterotoxigenic Escherichia coli (ETEC). It was demonstrated that purified F4

fimbriae, as opposed to oval albumin (OVA), are powerful oral immunogens.

Furthermore, a priming of the mucosal immune system is better obtained by oral

infection (ASC localized in mesenteric LN) than by IM F4 injection (ASC localized

in spleen and retropharyngeal LN) since an oral boost with purified F4 induced a

secondary response in the orally infected animal (mainly IgA and IgG ASC, rapid

increase of IgA antibodies) while in the IM primed animal a secondary (more

circulating antigen- specific ASC than in the unprimed animal) as well as a primary

IgM and IgA response (mainly IgM ASC, slow increase of IgA antibodies),

suggesting a primary mucosal response, were seen. An oral challenge of the naive

control displayed a primary response (mainly IgM ASC, slow increase of IgA and IgG

antibodies). It was concluded that the capacity of purified F4 to activate the mucosal

Dept. Of Pharmaceutics, JSSCP, Mysore

immune system on oral administration, is of importance for the development of oral

vaccines against ETEC infections.

Verdonck F et al

available against intestinal infections since the induction of a protective intestinal

immune response is difficult to achieve. For instance, oral administration of most

proteins results in oral tolerance instead of an antigen

shown before that as a result of oral immunization of piglets with F4 fimbriae purified

from pathogenic enterotoxigenic

receptor (F4R) in the intestine and induce a protective F4

Data suggest and concluded that the mucosal immunogenicity of soluble virulence

factors can be increased by the construction of stable polymeric structures and

therefore help in the development of effective mucosal vaccines.

Murillo M et al.,

Brucella ovis by the spray

glycolide RG502H [PLGA], and blends with poly

obtain microparticles smaller than 5 ìm. Microparticles were

efficiency, release studies, acidification of the in vitro release medium, and in vitro

J744-macrophage experiments (phagocytosis and toxicity of the preparations) to

determine the optimal formulation for vaccination purposes. All

suggest that the microparticulated antigenic formulation containing the higher ratio of

PEC is susceptible to be used in animal vaccination studies.

Haining et al., 188

that exploits the ability of APCs to cross

engulfed in the low pH environment

Review o



vaccines against ETEC infections.

Verdonck F et al., 186 have laid emphasis on vaccines that are commercially

inal infections since the induction of a protective intestinal


proteins results in oral tolerance instead of an antigen-specific immune response. It is

a result of oral immunization of piglets with F4 fimbriae purified

from pathogenic enterotoxigenic Escherichia coli (ETEC), the fimbriae bind to the F4

receptor (F4R) in the intestine and induce a protective F4-specific immune response.

ncluded that the mucosal immunogenicity of soluble virulence


therefore help in the development of effective mucosal vaccines.

., 187 have encapsulated the antigenic extract Hot Saline from

by the spray-drying technique with different polyesters (poly

glycolide RG502H [PLGA], and blends with poly- -caprolactone [PEC]) in order to

obtain microparticles smaller than 5 ìm. Microparticles were tested for encapsulation


macrophage experiments (phagocytosis and toxicity of the preparations) to

determine the optimal formulation for vaccination purposes. All these characteristics


PEC is susceptible to be used in animal vaccination studies.

8 have reported the use of a novel pH-triggered

that exploits the ability of APCs to cross-present MHC I-restricted cells that have been

engulfed in the low pH environment of the phagosome. A model MHC class I


47


laid emphasis on vaccines that are commercially

inal infections since the induction of a protective intestinal


specific immune response. It is

a result of oral immunization of piglets with F4 fimbriae purified

(ETEC), the fimbriae bind to the F4

specific immune response.

ncluded that the mucosal immunogenicity of soluble virulence


enic extract Hot Saline from

drying technique with different polyesters (poly-lactide-co-

caprolactone [PEC]) in order to

tested for encapsulation


macrophage experiments (phagocytosis and toxicity of the preparations) to

these characteristics


microparticle

that have been

of the phagosome. A model MHC class I-



restricted peptide Ag from the influenza A matrix protein was encapsulated in spray-

dried microparticles composed of dipalmitoylphosphatidylcholine and the pH-

sensitive polymethacrylate Eudragit E100. Encapsulation of the peptide in the

microparticles resulted in efficient presentation of the peptide to CD8+ T cells by

human dendritic cells in vitro, and was superior to unencapsulated peptide or peptide

encapsulated in an analogous pH-insensitive particle. These microparticles can be

modified to coencapsulate a range of adjuvants along with the Ag of interest.

Encapsulation of MHC I epitopes in pH-triggered microparticles increases Ag

presentation and may improve CD8+ T cell priming to peptide vaccines against

viruses and cancer.

Baras B et al., 189 have worked on the immunogenicity of a single nasal or

oral administration of recombinant 28-kDa glutathione S-transferase of Schistosoma

mansoni (rSm28GST) entrapped by poly (lactideco- glycolide) (PLG)- or

polycaprolactone (PCL) biodegradable microparticles. Whatever the polymer and the

route of administration used, the equivalent of 100 mg of entrapped rSm28GST

induced a long-lasting and stable antigen-specific serum antibody response, with a

peak at 9 to 10 weeks following immunization. Pooled 10-week sera from mice

receiving PLG microparticles by the nasal or oral route neutralized the rSm28GST

enzymatic activity, whereas sera of mice receiving either PCL microparticles, free

rSm28GST, or empty microparticles inefficiently neutralized this enzymatic activity.

Study shows that a single administration of these microparticles could provide distinct

and timely release pulses of microencapsulated antigen, which might greatly facilitate

future vaccine development.



Gregory et al., 190 have prepared starch based microporous microparticles by

spray drying techniques and had discussed its properties and applications.

Gupta et al., 191 have prepared prostaglandin E1 (PGE1) encapsulated large

porous microparticles for sustained pulmonary vasodilation and hypothesized that

incorporation of an effervescent porogen, ammonium bicarbonate (NH4HCO3), in

internal aqueous phase results in development of large and highly porous

microparticles ideal for deep lung deposition of the inhaled drug at a controlled rate.

Jain et al.,192 have prepared porous carrier based floating orlistat microspheres

for gastric delivery using calcium silicate as porous carrier, orlistat, an oral anti-

obesity agent; and eudragit S as polymer, by solvent evaporation method and to

evaluate their gastro-retentive and controlled-release properties. The effect of various

formulation and process variables on the particle morphology, micromeritic

properties, in vitro floating behavior, percentage drug entrapment, and in vitro drug

release was studied. The gamma scintigraphy of the optimized formulation was

performed in albino rabbits to monitor the transit of floating microspheres in the

gastrointestinal tract. The orlistat-loaded optimized formulation was orally

administered to albino rabbits, and blood samples collected were used to determine

pharmacokinetic parameters of orlistat from floating microspheres. The microspheres

were found to be regular in shape and highly porous. Microsphere formulation CS4,

containing 200 mg calcium silicate, showed the best floating ability (88% +/- 4%

buoyancy) in simulated gastric fluid as compared with other formulations. It was

found that release pattern of orlistat in simulated gastric fluid from all floating

microspheres followed Higuchi matrix model and Peppas-Korsmeyer model.

Prolonged gastric residence time of over 6 hours was achieved in all rabbits for



calcium silicate-based floating microspheres of orlistat. The enhanced elimination

half-life observed after pharmacokinetic investigations in the present study is due to

the floating nature of the designed formulations.

Gu et al., 193 have demonstrated magnetic manipulation, fluorescent tracking,

and localized delivery of a drug payload to cancer cells in vitro using nanostructured

porous silicon microparticles as a carrier. The multifunctional microparticles are

prepared by electrochemical porosification of a silicon wafer in a hydrofluoric acid-

containing electrolyte, followed by removal and fracture of the porous layer into

particles using ultrasound. The intrinsically luminescent particles are loaded with

super paramagnetic iron oxide nanoparticles and the anti-cancer drug doxorubicin.

The drug-containing particles are delivered to human cervical cancer (HeLa) cells in

vitro, under the guidance of a magnetic field. It was found that the high concentration

of particles in the proximity of the magnetic field results in a high concentration of

drug being released in that region of the Petri dish, and localized cell death is

confirmed by cellular viability assay.

Rawat et al., 194 have investigated the feasibility of large porous particles as

long-acting carriers for pulmonary delivery of low molecular weight heparin

(LMWH). Microspheres were prepared with a biodegradable polymer, poly(lactic-co-

glycolic acid) (PLGA), by a double-emulsion�solvent-evaporation technique. The

drug entrapment efficiencies of the microspheres were increased by modifying them

with three different additives�polyethyleneimine (PEI), Span 60 and stearylamine.

The resulting microspheres were evaluated for morphology, size, zeta potential,

density, in vitro drug-release properties, cytotoxicity, and for pulmonary absorption in

vivo. Scanning electron microscopic examination suggests that the porosity of the



particles increased with the increase in aqueous volume fraction. It was found that the

amount of aqueous volume fraction and the type of core-modifying agent added to the

aqueous interior had varying degrees of effect on the size, density and aerodynamic

diameter of the particles. Study results showed that when PEI was incorporated in the

internal aqueous phase, the entrapment efficiency was increased from 16.22 ± 1.32%

to 54.82 ± 2.79%. The amount of drug released in the initial burst phase and the

release-rate constant for the core-modified microspheres were greater than those for

the plain microspheres. After pulmonary administration, the half-life of the drug from

the PEI- and stearylamine-modified microspheres was increased by 5- to 6-fold

compared to the drug entrapped in plain microspheres. The viability of Calu-3 cells

was not adversely affected when incubated with the microspheres. It was found that

newly developed porous microspheres of LMWH have the potential to be used in a

form deliverable by dry-powder inhaler as an alternative to multiple parenteral

administrations of LMWH.

Gupta et al., 195 have investigated the hypothesis of large porous poly (lactic-

co-glycolic acid) (PLGA) microparticles modified with polyethyleneimine (PEI) are

viable carriers for pulmonary delivery of prostaglandin E1(PGE1) used in the

treatment of pulmonary arterial hypertension (PAH), a pulmonary vascular disorder.

The particles were prepared by a double-emulsion solvent evaporation method with

PEI-25 kDa in the internal aqueous phase to produce an osmotic pressure gradient.

Polyvinyl alcohol (PVA) was used for external coating of the particles. The particles

were examined for morphology, size, aerodynamic diameter, surface area, pore

volume and in-vitro release profiles. Particles with optimal properties for inhalation

were tested for in-vivo pulmonary absorption, metabolic stability in rat lung

homogenates, and acute toxicity in rat bronchoalveolar lavage fluid and respiratory



epithelial cells, Calu-3. The micromeritic data indicated that the PEI-modified

particles of PGE1 are optimal for inhalation. It was found that incorporation of PEI in

the formulations resulted in an increased entrapment efficiency 83.26 ± 3.04% for

particles with 1% PVA and 95.48±0.46% for particles with 2% PVA. The amount of

cumulative drug released into the simulated interstitial lung fluid was between

50.8±0.76% and 55.36±0.06%. A remarkable extension of the circulation half-life up

to 6.0�6.5 hours was observed when the formulations were administered via the

lungs. The metabolic stability and toxicity studies showed that the optimized

formulations were stable at physiological conditions and relatively safe to the lungs

and respiratory epithelium. Overall, this study demonstrates that large porous

inhalable polymeric microparticles can be a feasible option for non-invasive and

controlled release of PGE1 for treatment of PAH.

Borges et al., 196 have found that the adsorption of antigens onto chitosan

particles is an easy and unique mild loading process suitable to be used with vaccines.

In order to increase the stability of this particles and to prevent an immediate

desorption in gastrointestinal fluids, a coating process with sodium alginate was

developed. One of the challenges of this developing process was to keep the particles

in the nanosized range in order to be taken up by M-cells of the Peyer�s patches. The

observed inversion of the particles� zeta potential values after coating suggested the

presence of an alginate coating layer. These results were confirmed by FTIR and DSC

techniques. In vitro release studies showed that the presence of the alginate layer

around the particles was able to prevent a burst release of loaded ovalbumin and to

improve the stability of the nanoparticles in simulated intestinal fluid at 37 ◦C. The

optimisation of the coating process resulted in 35% (w/w) for the loading capacity of



the coated particles. SEM investigations confirmed a suitable size of the coated

nanoparticles for the uptake by M-cells.

Janet et al., 197 have prepared a nanoparticle formulation that could be sterile

filtered, lyophilized, and resuspended to the initial size with excipients appropriate for

use as a vaccine formulation. Poly (lactide-co-glycolide) (PLG) polymers were used

to create nanoparticles ranging in size from 110 to 230 nm. Protein antigens were

adsorbed to the particles; the protein-nanoparticles were then lyophilized with the

excipients. Vaccine compatible excipient combinations of sugars alone, surfactants

alone, and sugars and surfactants were tested to find conditions where initial particle

size was recovered. Sterile filtration of smaller nanoparticles led to minimal PLG

losses and allowed the particle preparation to be a nonaseptic process. We found that

the smaller nanoparticles of size -120 nm required higher surfactant concentration to

resuspend post lyophilization than slightly larger (-220 nm) particles. To resuspend

120 nm nanoparticles formulations of poly (vinyl alcohol) (PVA) with

sucrose/mannitol or dioctyl sodium sulfosuccinate (DSS) with trehalose/mannitol

were sufficient. The protein nanoparticles resuspension with the same excipients was

dependent on the protein and protein loading level. It was found that nanoparticle

formulations in vivo were either similar or had enhanced immunogenicity compared

to aluminum hydroxide formulations. A lyophilized nanoparticle formulation with

adsorbed protein antigen and minimal excipients is an effective vaccine delivery

system.

Koh et al., 198 have reported that enterotoxigenic Escherichia coli (ETEC)

infections result in large economic losses in the swine industry worldwide. The

organism causes diarrhea by adhering to and colonizing enterocytes in the small



intestines. While much progress has been made in understanding the pathogenesis of

ETEC, no homologous intestinal epithelial cultures suitable for studying porcine

ETEC pathogenesis have been described prior to this report. In the current study, we

investigated the adherence of various porcine ETEC strains to two porcine (IPEC-1

and IPEC-J2) and one human (INT-407) small intestinal epithelial cell lines. Each cell

line was assessed for its ability to support the adherence of E. coli expressing fimbrial

adhesins K88ab, K88ac, K88ad, K99, F41, 987P, and F18.Wild-type ETEC

expressing K88ab, K88ac, and K88ad efficiently bound to both IPEC-1 and IPEC-J2

cells. An ETEC strain expressing both K99 and F41 bound heavily to both porcine

cell lines but an E. coli strain expressing only K99 bound very poorly to these cells. E.

coli expressing F18 adhesin strongly bound to IPEC-1 cells but did not adhere to

IPEC-J2 cells. It was found that the E. coli strains G58-1 and 711 which express no

fimbrial adhesins and those that express 987P fimbriae failed to bind to either porcine

cell line. Only strains B41 and K12:K99 bound in abundance to INT-407 cells. The

binding of porcine ETEC to IPEC-J2, IPEC-1 and INT-407 with varying affinities,

together with lack of binding of 987P ETEC and non-fimbriated E. coli strains,

suggests strain-specific E. coli binding to these cell lines. These findings suggest the

potential usefulness of porcine intestinal cell lines for studying ETEC pathogenesis.

Moon et al., 199 have demonstrated the use of fimbrial vaccines given

parenterally to pregnant cattle, sheep and swine to protect suckling newborn calves,

lambs and pigs against enterotoxigenic Escherichia coli (ETEC) infections. Such

vaccines are practical and effective because (1) most fatal ETEC infections in farm

animals occur in the early neonatal period when the antibody titres in colostrum and

milk are highest, (2) more than 90% of the ETEC in farm animals belong to a small

family of fimbrial antigen types, (3) fimbriae consist of good protein antigens on the



bacterial surface where the)' are readily accessible to antibody, (4)fimbriae are

required or a critical step (adhesion-colonization) early in the pathogenesis of the

disease. ETEC infections continue to be a significant clinical problem in farm

animals' in spite of extensive use of fimbriae-based vaccines. Definitive data on the

efficacy of the commercial vaccines infield use are not available. The prevailing

perception among animal health professionals is that the vaccines are effective, that

the problem occurs chiefly among non-vaccinated animals and that in some herds

vaccination moves peak prevalence of disease from the first to the second or third

week after birth, when mortality is lower. It has been suggested that extensive use of

vaccines will rapidly select for the emergence of novel or previously low prevalence

fimbrial antigen types. There is no evidence that this has happened after a decade of

routine vaccine use in the United States. However, there is no active direct

surveillance for such emergence. In contrast to the rational development of vaccines

to provide passive lacteal protection against ETEC in suckling neonates,

comparatively little progress has been made in providing the knowledge required .for

development of vaccines to protect against postweaning ETEC infections in swine.

Li et al., 200 had reported that escherichia coli expressing F4 fimbriaeis the

major pathogenic bacteria that causes diarrhea in piglets before weaning. The

adhesion of E. coli to the brush borders of the epithelial cells of piglets is the

precondition leading to diarrhea, which in turn is due to the presence of the F4

receptors determined by an autosomal recessive gene on the brush borders of the

epithelial cells. In order to clarify the genetic mechanism of the adhesion, an in vitro

adhesion experiment was carried out for three variants of E. coli F4 (ab, ac, and ad) in

366 piglets of three pig breeds [Landrace (LR), Large White (LW), and Songliao

Black (SB)]. The results showed that there existed significant differences (P<0.001)



in the adhesion percentage among the three breeds. Most SB piglets were nonadhesive

for all the three variants, whereas most LR piglets were adhesive. Within each breed

except for LR, the propor-tions of the three F4 variants adhering to the brush borders

differed significantly. According to the patterns of the adhesion of the three F4

variants in the three breeds, it is very likely that the three F4 variants F4ab, F4ac, and

F4ad have different receptors that are controlled by three different loci.

Verdonck et al., 201 have reported that only a few vaccines are commercially

available against intestinal infections since the induction of a protective intestinal


proteins results in oral tolerance instead of an antigen-specific immune response. It

was shown before that as a result of oral immunization of piglets with F4 fimbriae

purified from pathogenic Enterotoxigenic Escherichia coli (ETEC), the fimbriae bind

to the F4 receptor (F4R) in the intestine and induce a protective F4-specific immune

response. F4 fimbriae are very stable polymeric structures composed of some minor

subunits and amajor subunit FaeG that is also the fimbrial adhesin. In the present

study, the mutagenesis experiments identified FaeG amino acids 97 (N to K) and 201

(I to V) as determinants for F4 polymeric stability. The interaction between the FaeG

subunits in mutant F4 fimbriae is reduced but both mutant and wild type fimbriae

behaved identically in F4R binding and showed equal stability in the gastro-intestinal

lumen. Oral immunization experiments indicated that a higher degree of

polymerisation of the fimbriae in the intestinewas correlated with a better F4-specific

mucosal immunogenicity. These data suggest that the mucosal immunogenicity of

soluble virulence factors can be increased by the construction of stable polymeric

structures and therefore help in the development of effective mucosal vaccines.



Snoeck et al., 202 have developed oral immunization model in pigs in which

F4 (K88) fimbriae of enterotoxigenic Escherichia coli are administered to induce a

protective intestinal immunity, was used to determine the optimal inductive sites of

the F4-specific intestinal immune response. Hereto, pigs were immunised with F4

orally, in the lumen of the mid-jejunum, ileum or mid-colon. Throughout the small

intestine, the highest number of ASC was found following jejunal immunisation,

followed by ileal, oral and colonic immunisation. To determine the significance of

Peyer�s patches in the induced immune response, F4 was injected into the jejunal

Peyer�s patches (JPP), lamina propria (LP) and ileal Peyer�s patches (IPP).

Immunisation in the JPP induced the highest number ASC in the small intestine,

whereas immunisation in the LP and IPP resulted in lower intestinal antibody

responses. In conclusion, we have shown that the JPP are the major inductive sites of

the F4-specific intestinal antibody response. This knowledge could be important when

using the pig as an animal model for vaccination studies.

Fairbrother et al., 203 have reported that escherichia coli is one of the most

important causes of postweaning diarrhea in pigs. This diarrhea is responsible for

economic losses due to mortality, morbidity, decreased growth rate, and cost of

medication. The E. coli causing postweaning diarrhea mostly carry the F4 (K88) or

the F18 adhesin. Recently, an increase in incidence of outbreaks of severe E. coli-

associated diarrhea has been observed worldwide. The factors contributing to the

increased number of outbreaks of this more severe form of E. coli-associated diarrhea

are not yet fully understood. These could include the emergence of more virulent E.

coli clones, such as the O149:LT:STa:STb:EAST1:F4ac, or recent changes in the

management of pigs. Development of multiple bacterial resistance to a wide range of

commonly used antibiotics and a recent increase in the prevalence and severity of the



postweaning syndromes will necessitate the use of alternative measures for their

control. New vaccination strategies include the oral immunization of piglets with live

avirulent E. coli strains carrying the fimbrial adhesins or oral administration of

purified F4 (K88) fimbriae. Other approaches to control this disease include

supplementation of the feed with egg yolk antibodies from chickens immunized with

F4 or F18 adhesins, breeding of F18- and F4-resistant animals, supplementation with

zinc and/or spray-dried plasma, dietary acidification, phage therapy, or the use of

probiotics. To date, not a single strategy has proved to be totally effective and it is

probable that the most successful approach on a particular farm will involve a

combination of diet modification and other preventive measures.

Deprez et al., 204 have studied the post weaning excretion rate of hemolytic E.

coli was determined in piglets from herds affected with edema disease and on control

farms. No distinct difference in rate of excretion was observed. A split litter trial was

set up to evaluate the importance of sow's milk in the postweaning rise of fecal

hemolytic E. coli. 525 ml of sow's milk a day, mixed with the feed, completely

inhibited that postweaning rise, even after oral challenge with a pathogenic strain.

Rasschaert et al., 205 have analyzed quantitatively the mucin 4 polymorphism

for determining the F4ac/ab receptor status of a total of 63 pigs by comparing it with

the in vitro villous adhesion assay. The probability of a susceptible genotype for the

mucin 4 increases significantly with increasing F4ab or F4ac ETEC adhesion per 250

microm villi (P=0.029 for F4ab, P=0.030 for F4ac), with the odds ratio for each unit

increase of F4ab or F4ac equal to, respectively, 1.036 (95% CI [1.004-1.069]) and

1.018 (95% CI [1.002-1.034]). In the phenotypic in vitro villous adhesion test, a cut-

off value of 5 bacteria was chosen as criteria for the distinction between an F4R



positive and F4R negative pig. The sensitivity and specificity for the in vitro villous

adhesion test, with the genotyping test for mucin 4 as golden standard, is 100% and

24%, respectively, for F4ab as well as F4ac. Absence of adhesion of F4ac and F4ab

ETEC to the villous brush borders was not associated with genotypic resistance

suggesting that there is at least one other receptor for F4ab/ac Escherichia coli. As a

consequence, not only mucin 4 gene polymorphism but also expression of these other

receptor(s) has to be included in a screening assay for F4ac/ab receptor negative pig.

Verdonck et al., 206 have reported that piglets are susceptible to F4 (K88) +

enterotoxigenic Escherichia coli (ETEC)-induced neonatal and post-weaning

diarrhea. The F4 fimbriae are composed of some minor subunits and the major

subunit FaeG that also constitutes the adhesin. Parenteral vaccination of sows with an

F4-containing vaccine protects the suckling piglets against neonatal F4+ ETEC-

induced diarrhea, but no commercial (mucosal) vaccine exists against F4+ ETEC-

induced weaning diarrhea. To develop a vaccine, a bioactive F4-receptor (F4R)

binding FaeG molecule is required that binds to the F4R following oral immunization

and induces a FaeG-specific immune response. The present study reports the altered

binding of the FaeG-specific monoclonal antibody IMM01 with bioactive versus non-

bioactive F4 fimbrial adhesin FaeG. The correlation of altered IMM01 binding with

altered FaeG bioactivity permits the use of an IMM01-based ELISA as a fast, specific

and sensitive in vitro selection for potent F4 or (recombinant) FaeG antigen

formulations, useful in an F4+ ETEC vaccine.

Broeck et al., 207 have demonstrated an effective way of stimulating the

mucosal immune system was examined in piglets, using F4 fimbriae of

enterotoxigenic Escherichia coli (ETEC). It was demonstrated that purified F4



fimbriae, as opposed to ovalbumin (OVA), are powerful oral immunogens. Indeed,

oral administration of purified F4 induced antigen-specific antibody-secreting cells

(ASC) in the Peyer's patches, mesenteric lymph nodes (LN), blood and lamina propria

4, 7, 9 and 11 days postimmunization, respectively, indicating a stimulation of the

mucosal immune system, whereas upon oral administration of OVA, no immune

response was observed. Moreover, the induced F4-specific IgA and IgG antibody

responses were comparable with those obtained upon oral infection with viable E. coli

and intramuscular (i.m.) F4 injection, respectively. Furthermore, a priming of the

mucosal immune system is better obtained by oral infection (ASC localized in

mesenteric LN) than by i.m. F4 injection (ASC localized in spleen and

retropharyngeal LN) since an oral boost with purified F4 induced a secondary

response in the orally infected animal (mainly IgA and IgG ASC, rapid increase of

IgA antibodies) while in the i.m. primed animal a secondary (more circulating

antigen-specific ASC than in the unprimed animal) as well as a primary IgM and IgA

response (mainly IgM ASC, slow increase of IgA antibodies), suggesting a primary

mucosal response, were seen. An oral challenge of the naive control displayed a

primary response (mainly IgM ASC, slow increase of IgA and IgG antibodies). It was

found that the capacity of purified F4 to activate the mucosal immune system on oral

administration is of importance for the development of oral vaccines against ETEC

infections.

Broeck et al., 208 have demonstrated F4 receptor-positive (F4R+) and F4

receptor-negative (F4R-) pigs by orally vaccinating them with purified F4 fimbriae of

enterotoxigenic Escherichia coli (ETEC). Serum immunoglobulin G (IgG) and IgA

responses were readily detected in F4R+ animals, whereas immune responses were

not detected in F4R- animals. Even after a subsequent oral infection with virulent



F4(+) ETEC and a booster immunization with F4, the F4R- animals remained F4

seronegative whereas the unvaccinated F4R+ pigs exhibited clear IgA and IgG

responses. These results clearly demonstrate that F4Rs are a prerequisite for an

immune response following oral immunization. Furthermore, indications that oral F4

vaccination can induce mucosal protection were obtained, since the experimental

ETEC infection did not induce a systemic booster response or fecal ETEC excretion

in orally vaccinated F4R+ pigs, in contrast to the clear immune response and ETEC

excretion of unvaccinated F4R+ animals. F4-specific IgA antibodies could be found

in the feces of the vaccinated F4R+ pigs. They are secreted at the intestinal mucosal

surface and appear to prevent ETEC infection. It was reported that F4R-dependent

induction of a mucosal immune response can be used as a model to better understand

mucosal immunization and mucosal immune responses and can contribute to the

development of oral vaccines in veterinary as well as in human medicine.

Rasschaert et al., 209 have quantitatively analyzed the mucin 4 polymorphism

for determining the F4ac/ab receptor status of a total of 63 pigs by comparing it with

the in vitro villous adhesion assay. The probability of a susceptible genotype for the

mucin 4 increases significantly with increasing F4ab or F4ac ETEC adhesion per 250

mm villi (P = 0.029 for F4ab, P = 0.030 for F4ac), with the odds ratio for each unit

increase of F4ab or F4ac equal to, respectively, 1.036 (95% CI [1.004�1.069]) and

1.018 (95% CI [1.002� 1.034]). In the phenotypic in vitro villous adhesion test, a cut-

off value of 5 bacteria was chosen as criteria for the distinction between an F4R

positive and F4R negative pig. The sensitivity and specificity for the in vitro villous

adhesion test, with the genotyping test for mucin 4 as golden standard, is 100% and

24%, respectively, for F4ab as well as F4ac. It was reported that not only mucin 4



gene polymorphism but also expression of these other receptor(s) has (have) to be

included in the screening assay for F4ac/ab receptor negative pigs.

Devriendt et al.,210 have demonstrated in his study that polymeric fimbriae

are essential for adhesion to porcine intestinal epithelial cells (IEC) and the secretion

of IL-6 and IL-8 by IEC. Infections with F4 (+) enterotoxigenic Escherichia coli

(ETEC) cause severe diarrhea in piglets, resulting in morbidity and mortality. F4

fimbriae are the key virulence factors mediating the attachment of F4 (+) ETEC to the

intestinal epithelium. Intestinal epithelial cells (IEC) are recently being recognized as

important regulators of the intestinal immune system through the secretion of

cytokines. Results of the study revealed that a potential mucosal adjuvant capacity of

ETEC-derived flagellin and may improve rational vaccine design against F4 (+)

ETEC infections.

Verdonck et al.,211 emphasized on developing vaccine for the treatment of

enterotoxigenic E. coli (ETEC) infection. Fimbriae-specific antibody response of

newly weaned pigs following infection with the Shiga-like toxin type II variant (SLT-

IIv) producing F18+ verotoxigenic E. coli (VTEC) (strain 107/86) was compared with

the response following an infection with LT producing F4+ enterotoxigenic E. coli

(ETEC) (strain GIS 26). It was reported that F4+ ETEC were able to colonize the gut

very soon after infection, as peak excretion of F4+ E. coli bacteria was seen 2 days

post-infection (dpi), but had already disappeared 7 dpi. On the other hand, F18+

VTEC infection resulted in a slower colonization of the gut as the peak excretion of

F18+ E. coli was observed between 3 and 5 dpi, but this colonization remained longer

as F18+ E. coli were detected till 9 dpi in feces. Furthermore, this fast colonization

pattern of F4+ ETEC is accompanied with the presence of F4-specific antibodies in



mucosal tissues and serum from 4 dpi onward, with maximal amounts of F4-specific

IgA in the jejunal lamina propria and serum 7 dpi.

Verdonck et al.,212 have reported the importance of adhesins in the

pathogenicity of several bacteria on their usefulness in vaccines. Gene of the F4

(K88)-fimbrial adhesin FaeG of the pathogenic enterotoxigenic Escherichia coli

(ETEC) strain GIS26 was cloned in the pET30Ek-LIC vector and expressed with an

N-terminal His- and S-tag in the cytoplasm of BL21 (DE3). Recombinant FaeG

(rFaeG) subunits were isolated from insoluble cytoplasmic aggregates and refolded

into a native-like F4 receptor (F4R)-binding conformation. Indeed, the presence of

conformational epitopes was shown by ELISA and the ability to bind the F4R was

observed by inhibiting the adhesion of F4+ ETEC to F4R+ villi with increasing

concentrations of native-like re-folded rFaeG subunits. The rFaeG subunits appear as

monomers, whereas the purified F4 fimbriae are multimers. It was reported that oral

immunization of newly weaned piglets with native-like rFaeG induced a mucosal and

systemic F4-specific immune response, significantly reducing F4+ E. coli excretion

from 2 till 5 days following challenge infection. However, improvement of stability

and immunogenicity of rFaeG is necessary since a higher F4-specific response was

obtained following immunization with purified F4 fimbriae. Furthermore, the N-

terminal fusion of a His- and S-tag was not detrimental for binding the F4R,

supporting the use of FaeG as mucosal carrier. Results of the study revealed that oral

immunization with a recombinant fimbrial adhesin subunit of Escherichia coli

induces a mucosal and systemic fimbriae-specific immune response.

Rasschaert et al.,213 have demonstrated the internalization of F4 fimbriae in

the porcine intestinal epithelial cell line IPEC-J2. Enterotoxigenic Escherichia coli



(ETEC) cause severe diarrhea in neonatal and recently weaned piglets. It was reported

that oral immunization of F4 receptor positive piglets with purified F4 fimbriae

induces a protective F4-specific intestinal immune response. However, in F4 receptor

negative animals no F4-specific immune response can be elicited, indicating that the

induction of an F4-specific mucosal immune response upon oral immunization is

receptor-dependent. Although F4 fimbriae undergo transcytosis across the intestinal

epithelium in vivo, the endocytosis pathways used remain unknown. The results in

the present study demonstrate that F4 fimbriae are internalized through a clathrin-

dependent pathway. It was reported that F4 fimbriae are transcytosed across

differentiated IPEC-J2 cells. This receptor-dependent transcytosis of F4 fimbriae may

explain the immunogenicity of these fimbriae upon oral administration in vivo.


4.9.5-Fluorouracil 214, 215

Figure 2: Structure of 5

Molecular formula

Molecular weight

Melting point

Category

Description

Dissociation Constant (pK

Solubility

Storage

Formulations

Identification

pH

Mechanism of action

Pharmacokinetic profile of 5

Half life

Volume of distribution

Clearance

Protein binding

Therapeutic uses

Review o


5

Figure 2: Structure of 5-fluorouracil.

: C4H3FN2O2

: 130.1

: 282° to 283° C

: Anti-cancer.

: White to practically white, odourless, crystalline

powder

Dissociation Constant (pKa ): : 8.0

: Sparingly soluble in water, slightly soluble in

alcohol, practically insoluble in CHCl

: Stored in air tight containers

light.

: Intravenous route, capsules.

: Light absorption in the range 200

µg/ml solution in 7.4 pH buffer

absorption maximum at 267 nm

: 4.5 to 5.0

: The nucleotide of 5-FU, 5-Fluoro

thymidylate synthase blocks the conversion of

deoxyuridic acid to deoxy thymidylic acid

Pharmacokinetic profile of 5-FU

: 15 to 20 min

: 17.5 L/70kg

: 63 L/h

: 94%

: Anti neoplastic and immunosuppressive agent


65

practically white, odourless, crystalline

Sparingly soluble in water, slightly soluble in

alcohol, practically insoluble in CHCl3

Stored in air tight containers protected from

Light absorption in the range 200-400 nm of a 10

µg/ml solution in 7.4 pH buffer exhibits

Fluoro-2-deoxy-

thymidylate synthase blocks the conversion of

deoxyuridic acid to deoxy thymidylic acid

Anti neoplastic and immunosuppressive agent


4.10. Metoprolol tartrate

Figure3

Molecular formula

Molecular weight

Melting point

IUPAC Name

Category

Dose

Description

Solubility

Formulations

Pharmacokinetic profile

Half life

Absorption

Bioavailability

Therapeutic uses

Review o


Metoprolol tartrate 216

Figure3: Structure of Metoprolol tartarate.

: (C15H25NO3)2,C4H6O6

: 684.82.

: 282° to 283° C

: Anti-cancer.

: Beta- adrenoceptor antagonist.

: 100 to 450 mg daily, in divided dos

dose should not exceed 100 mg daily

: White crystalline powder.

: Very soluble in water; freely soluble in ethanol

(95%), in Chloroform and in dichloromethane;

slightly soluble in acetone; practically insoluble

in ether.

: Metoprolol tablets, Metoprolol SR tablets,

Metoprolol ER Capsules.

Pharmacokinetic profile

Half life : 3-4 hrs

Absorption : Completely absorbed from the GI tract.

Bioavailability : Oral bioavailability is about 40 - 50%.

Therapeutic uses : It is an effective â- adrenoceptor antagonist, used

in the treatment of angina, hypertension and

myocardial infarction.


66

100 to 450 mg daily, in divided doses; the initial

exceed 100 mg daily.

Very soluble in water; freely soluble in ethanol

(95%), in Chloroform and in dichloromethane;

slightly soluble in acetone; practically insoluble

Metoprolol SR tablets,

the GI tract.

50%.

adrenoceptor antagonist, used

in the treatment of angina, hypertension and



4.11. Eudragit S-100 217

Eudragit S100 is methacrylic acid copolymers are anionic co-polymeric

products of methacrylic acid and methyl methacrylates and other neutral methacrylic

acid esters. The ratio of the free carboxyl groups to the ester groups is approx. 1:2 in

EUDRAGIT® S 100.

CH3 CH3 CH3 CH3 CH3

| | | | | - [----C------------CH2------------C------------CH2-------------C----] N-

| | | | | COOH COOCH3 COOH COOCH3 COOH

Figure 4: Structure of Eudragit S-100.

Non-proprietary name : Methacrylic acid copolymer.

Synonyms : Eudragit, polymeric methacrylates

Description : white free flowing powders with at least 95% dry

polymer.

Molecular weight : ≥ 100000

Chemical name : Poly methacrylic acid, methyl methacrylates

Viscosity : 50-200 mPas

Solubility : Soluble in acetone, alcohol and 1N NaOH and

insoluble in dichloromethane, water, ethyl acetate

and petroleum ether.

Density : 0.83-0.85 g/cm3

Applications : Eudragit S100 is used as enteric coating agents

since they are resistance to gastric fluid also used

as a binder in both aqueous and organic weight

granulation processes. Also used to form the

matrix layers of transdermal delivery system and

It is soluble in > pH 7.0.



4.12. Eudragit L 100 218

Eudragit® L 100 is anionic copolymers based on methacrylic acid and methyl

methacrylate ratios. The ratio of free carboxyl groups to ester groups is approximately

1:1 in Eudragit® L 100.

Chemical structure

Figure 5: Structure of Eudragit L-100.

Average molecular weight : Approx. 135,000 g/mol

Chemical/IUPAC name : Poly (methacrylic acid-co-methyl methacrylate)

1:1

Targeted drug release area : Jejunum

Dissolution Dissolution above pH 6.0

Description : White free-flowing powder with a faint

characteristic odour.

Functional Category : Enteric coating polymer and for preparation of

controlled release dosage form

Solubility : Practically insoluble in water, freely soluble in

ethanol and in 2-propanol, practically insoluble

in ethyl acetate. It is freely soluble in a 40 g/l

solution of sodium hydroxide.

Particle size: : At least 95 % less than 0.25 mm.

Film formation : Place 1ml of the solution prepared for the

viscosity test on a glass plate and allow drying. A

clear brittle film is formed.

Storage : Protect from warm temperature. Protect against

moisture.


4.13.Chitosan 219

Synonyms

2-Amino-2-deoxy

glucosamine.

Chemical name

Poly-ß-(1,4)-2-Amino-2-deoxy

Chitosan is natural polysaccharide compr

and N- acetyl glucosamine and can be derived by partial deacetylation of chitin

from crustacean shells. Chitin is second most abund

cellulose.

Structural formulae

Functional category

Coating agent; disintegrant; film forming agent; mucoadhesive; tablet binder;

viscosity increasing agent.

Description

Chitosan occurs as odorless, white or cream white powder or flakes.

Properties

Chitosan is a cationic polyamine with a high charge density at pH<6.5; and so

adheres to negatively charged surfaces and chelates metal ions. It is linear

polyelectrolyte with reactive with reactive hydroxyl and amino group. The presence

Review o


deoxy-(1,4)-ß-D-glucopyranan; deacetylated chitin; poly

deoxy-d-glucose

Chitosan is natural polysaccharide comprising of copolymers of glucosamine

acetyl glucosamine and can be derived by partial deacetylation of chitin

from crustacean shells. Chitin is second most abundant natural polysacchride after

Figure 6: Structure of Chitosan.


viscosity increasing agent.






69

glucopyranan; deacetylated chitin; poly-d-

ising of copolymers of glucosamine

acetyl glucosamine and can be derived by partial deacetylation of chitin

ant natural polysacchride after








of a number of amino group, allows chitosan to react chemically with anionic system.

Almost all functional properties of chitosan depend on the chain length, charge

density, and charge distribution.

Acidity/alkalinity

pH = 4.0-6.0 (1 % w/v aqueous solution)

Moisture content

Chitosan absorbs moisture from atmosphere depending on initial moisture

content and temperature and humidity of surrounding air.

Properties of Chitosan:

Physical Properties:

Particle size <30 nm

Density 1.35 � 1.49 g/cc

Solubility: Insoluble in water but soluble in acids.

Chemical properties:

Cationic polyamine

High charge density at pH < 6.5

Adheres to negatively charged surfaces.

Forms gel with polyanions.

High molecular weight, linear polyelectrolyte

Incompatibilities:

Chitosan is incompatible with strong oxidizing agents.



4.14.Triethyl citrate 219 Nonproprietary Names

BP: Triethyl citrate; PhEur: Triethylis citras; USPNF: Triethyl citrate

Synonyms

Citric acid, ethyl ester; Citroflex 2; Citrofol AI; E1505; Hydagen CAT; TEC.

Chemical Name

2-Hydroxy-1,2,3-propanetricarboxylic acid, triethyl ester

Figure 7: Structure of Triethyl citrate.

Empirical Formula and Molecular Weight

C12H20O7 276.29

Functional Category

Plasticizer.

Description

Triethyl citrate is a clear, odorless, practically colorless, oily liquid.

Typical Properties

Acid value: 0.02

Boiling point: 288°C (decomposes)

Flash point: 155°C

Pour point: -45°C

Solubility: soluble 1 in 125 of peanut oil, 1 in 15 of water.



Miscible with ethanol (95%), acetone, and propan-2-ol.

Viscosity (dynamic): 35.2 mPa s (35.2 cP) at 25°C.

Stability and Storage Conditions

Triethyl citrate should be stored in a closed container in a cool, dry location.

When stored in accordance with these conditions, triethyl citrate is a stable product.

Applications in Pharmaceutical Formulation or Technology

Triethyl citrate and the related esters acetyltriethyl citrate, tributyl citrate, and

acetyltributyl are used to plasticize polymers in formulated pharmaceutical coatings.

The coating applications include capsules, tablets, beads, and granules for taste

masking, immediate release, sustainedrelease, and enteric formulations. Triethyl

citrate is also used as a direct food additive for flavoring, for solvency, and as a

surface active agent.



4.15.Polyvinyl Alcohol 220, 221 Nonproprietary Names:

PhEur: Poly (Vinyl Alcohol): USP: Polyvinyl Alcohol

Synonyms

Airvol; Alcotex; Celvol; Elvanol; Gelvatol; Gohsenol; Lemol; Mowiol; poly(alcohol

vinylicus); Polyvinol; PVA; vinyl alcohol polymer.

Chemical Name

Ethenol, homopolymer

Empirical Formula and Molecular Weight

(C2H4O)n 20 000�200 000

Polyvinyl alcohol is a water-soluble synthetic polymer represented by the formula

(C2H4O)n. The value of n for commercially available materials lies between 500 and

5000, equivalent to a molecular weight range of approximately 20 000�200 000.

Figure 8: Structure of Polyvinyl Alcohol.

Description

Polyvinyl alcohol occurs as an odorless, white to cream-colored granular powder.

Typical Properties

Melting point

228°C for fully hydrolyzed grades;

180�190°C for partially hydrolyzed grades.



Solubility

Soluble in water; slightly soluble in ethanol (95%); insoluble in organic

solvents. Dissolution requires dispersion (wetting) of the solid in water at room

temperature followed by heating the mixture to about 908C for approximately 5

minutes. Mixing should be continued while the heated solution is cooled to room

temperature


Polyvinyl alcohol is used primarily in topical pharmaceutical and ophthalmic

formulations It is used as a stabilizing agent for emulsions (0.25�3.0% w/v).

Polyvinyl alcohol is also used as a viscosity-increasing agent for viscous

formulations such as ophthalmic products. It is used in artificial tears and contact lens

solutions for lubrication purposes, in sustained-release formulations for oral

administration and in transdermal patches. Polyvinyl alcohol may be made into

microspheres when mixed with a glutaraldehyde solution.


Polyvinyl alcohol is stable when stored in a tightly sealed container in a cool,

dry place. Aqueous solutions are stable in corrosion resistant sealed containers.

Preservatives may be added to the solution if extended storage is required. Polyvinyl

alcohol undergoes slow degradation at 100°C and rapid degradation at 200°C; it is

stable on exposure to light.



4.16.Lutrol F68 222-223 Nonproprietary Names

BP: Poloxamers; PhEur: Poloxamera; USPNF: Poloxamer 188

Synonyms

Lutrol; Monolan; Pluronic; poloxalkol; polyethylene�propylene glycol copolymer;

polyoxyethylene�polyoxypropylene copolymer; Supronic; Synperonic.

Chemical Name

á-Hydro-ù-hydroxypoly(oxyethylene)poly(oxypropylene) poly(oxyethylene) block

copolymer

The poloxamer polyols are a series of closely related block copolymers of

ethylene oxide and propylene oxide conforming to the general formula

HO(C2H4O)a(C3H6O)b(C2H4O)aH.

Figure 9: Structure of LUTROL F68.

Description

Poloxamers generally occur as white, waxy, free-flowing prilled granules, or as cast

solids. They are practically odorless and tasteless. At room temperature, poloxamer

124 occurs as a colorless liquid.

Functional Category

Dispersing agent; emulsifying and coemulsifying agent; solubilizing agent; tablet

lubricant; wetting agent.



Typical Properties

Acidity/alkalinity: pH = 5.0�7.4 for a 2.5% w/v aqueous solution

Density: 1.06 g/cm3 at 25°C

Flash point: 260°C

Flowability: solid poloxamers are free flowing.

HLB value: 0.5�30; 29 for poloxamer 188.

Melting point: 52�57°C


Poloxamers are nonionic polyoxyethylene�polyoxypropylene copolymers used

primarily in pharmaceutical formulations as emulsifying or solubilizing agents. The

polyoxyethylene segment is hydrophilic while the polyoxypropylene segment is

hydrophobic. Poloxamers are used as emulsifying agents in intravenous fat

emulsions, and as solubilizing and stabilizing agents to maintain the clarity of elixirs

and syrups. Poloxamers may also be used as wetting agents; in ointments, suppository

bases, and gels; and as tablet binders and coatings. Poloxamer 188 has also been used

as an emulsifying agent for fluorocarbons used as artificial blood substitutes and in

the preparation of solid-dispersion systems. Therapeutically, poloxamer 188 is

administered orally as a wetting agent and stool lubricant in the treatment of

constipation; it is usually used in combination with a laxative such as danthron.

Poloxamers may also be used therapeutically as wetting agents in eye-drop

formulations, in the treatment of kidney stones, and as skin-wound cleansers.


Poloxamers are stable materials. Aqueous solutions are stable in the presence of acids,

alkalis, and metal ions. However, aqueous solutions support mold growth. The bulk

material should be stored in a well-closed container in a cool, dry place.


4.17.Ammonium carbonate

Chemical formula

CH6N2

CH8N2

CH5NO

Structural

formula NH2COONH(NH4)2

NH4HCO

Figure 10

Description White powder or hard, white or translucent masses of crystals with an odor

ammonia. On exposure to air

porous lumps or powder.

Functional uses: acidity regulator, raising agent Typical Properties

Solubility: soluble in water

Review o


Ammonium carbonate 224

2O2,

2O3 NO3

COONH4

2HCO3 HCO3

Figure 10: Structure of Ammonium carbonate.

White powder or hard, white or translucent masses of crystals with an odor

ammonia. On exposure to air it become opaque and is finally converted into white

porous lumps or powder.

acidity regulator, raising agent

Solubility: soluble in water


77

White powder or hard, white or translucent masses of crystals with an odor of

it become opaque and is finally converted into white



4.18. Strain

ATCC® Number : 35401� 225

Organism : Escherichia coli (Migula) Castellani and Chalmers

Designations : H10407

Isolation : human feces

Depositor : S Formal

History : ATCC <<--S Formal<<--H.R. DuPont

Biosafety Level : 2

Shipped : freeze-dried

Growth Conditions : ATCC medium3: Nutrient agar or nutrient broth

Temperature: 37°C

Atmosphere: Aerobic

Antigenic Properties: serotype O78:H11

Applications: detection of LT toxin

detection of ctx A11

produces heat stable toxin

production of heat labile and heat stable

enterotoxins

review of literature review of...

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