2.3. Incorporation of spacers in prodrug conjugates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 366

Prog. Polym. Sci. 31 (2006) 359397

www.elsevier.com/locate/ppolysci0079-6700/$ - see front matter q 2006 Elsevier Ltd. All rights reserved.Polymerdrug conjugates: Progress in polymeric prodrugs

Jayant Khandare a, Tamara Minko a,b,c,*

a Department of Pharmaceutics, Ernest Mario School of Pharmacy, Rutgers, The State University of New Jersey,

160 Frelinghuysen Road, Piscataway, NJ 08854-8020, USAb The Cancer Institute of New Jersey, 195 Little Albany Street, New Brunswick, NJ 08903, USA

c New Jersey Center for Biomaterials, 610 Taylor Road, Piscataway, NJ 08854, USA

Received 13 May 2005; received in revised form 30 August 2005; accepted 12 September 2005

Abstract

Polymers are used as carriers for the delivery of drugs, proteins, targeting moieties, and imaging agents. Several polymers,

poly(ethylene glycol) (PEG), N-(2-hydroxypropyl)methacrylamide (HPMA), and poly(lactide-co-glycolide) (PLGA) copolymers

have been successfully utilized in clinical research. Recently, interest in polymer conjugation with biologically active components

has increased remarkably as such conjugates are preferably accumulated in solid tumors and can reduce systemic toxicity. Based on

the site and the mode of action, polymer conjugates possess either tuned degradable or non-degradable bonds. In order to obtain

such bonds, most of the strategies involve incorporation of amino acids, peptides or small chains as spacer molecules through

multiple steps to include protections and deprotections. There is a need to design efficient synthetic methods to obtain polymeric

conjugates with drugs and other bioactive components. Designs should aim to decrease the steric hindrance exhibited by polymers

and the biocomponents. In addition, the reactivity of polymer and drug must be enhanced. This is especially true for the use of high

molecular weight linear polymers and bulkier unstable drugs such as steroids and chemotherapeutic agents. Further, it is essential

to elucidate the structure activity relationship (SAR) of a drug when it is conjugated with a polymer using different conjugation

sites, as this can vary the efficacy and mechanism of action when compared with its free form. This review will discuss the current

synthetic advances in polymer-conjugation with different bioactive components of clinical importance. In addition, the review will

describe the strategies for reduction of steric hindrance and increase in reactivity of the polymers, drugs and bioactive agents and

highlight the requisite structure activity relationship in polymerdrug bioconjugates. Finally, we will focus on passive and active

targeting of polymeric drug delivery systems to specific site of drug action.

q 2006 Elsevier Ltd. All rights reserved.

Keywords: Polymer; Conjugation; Drug delivery; Prodrug; Enhanced permeability and retention; In vivo and in vitro

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 360

2. Design and synthesis of polymeric prodrugs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 362

2.1. Polymeric drug delivery system (PDDS) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 364

2.2. n-hydroxysuccinimide (NHS) ester and coupling methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 365doi:10.1016/j.progpolymsci.2005.09.004

* Corresponding author. Address: Department of Pharmaceutics, Ernest Mario School of Pharmacy, Rutgers, The State University of New Jersey,

160 Frelinghuysen Road, Piscataway, NJ 08854-8020, USA. Tel.:C1 732 445 3831x214; fax: C1 732 445 3134.E-mail address: minko@rci.rutgers.edu (T. Minko).

cross-linkers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 368

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 369

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 369

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 372

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 378

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 378

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 380

opolymer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 383

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 385

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 388

R) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 388

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 388

n of spacers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 391

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 392

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 392

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 393

. . . .

. . . .

. . . .

J. Khandare, T. Minko / Prog. Polym. Sci. 31 (2006) 359397360polymer forms so-called polymeric prodrug. Poly-

meric conjugates of conventional drugs (polymeric

prodrugs) have several advantages over their low

molecular weight precursors. The main advantages

include: (1) an increase in water solubility of low

soluble or insoluble drugs, and therefore, enhancement

of drug bioavailability; (2) protection of drug from

deactivation and preservation of its activity during

circulation, transport to targeted organ or tissue and

intracellular trafficking; (3) an improvement in phar-

macokinetics; (4) a reduction in antigenic activity of the

drug leading to a less pronounced immunological body

response; (5) the ability to provide passive or active

targeting of the drug specifically to the site of its action;

(6) the possibility to form an advanced complex drug

delivery system, which, in addition to drug and polymer

carrier, may include several other active components

that enhance the specific activity of the main drug. Due

to these advantages over to free form of a drug, the

Various architectures of polymers have been used as

vehicles to deliver drugs, along with relevant targeting

agents [2,3]. Depending on the nature and site of action

of a drug, either homopolymers, or graft or block

Table 1

Current status of polymeric bioconjugates in cancer therapy

Polymer Drug/biomolecules

in conjugate

Current

phase

Name of the

company

PEGa Paclitaxel I Pharmacia

P-HPMAb Doxorubicin (DOX)

micelle and Platinate

II/III Access Pharma

P-HPMA Paclitaxel I Pharmacia

P-HPMA Camptothecin I Pharmacia

Poly

(glutamate)

Camptothecin III Cell

Therapeutics

PEG Camptothecin II Enzon

PEG Aspartic acid I NCI

P-HPMA DOX II Pharmacia

P-HPMA DOX-Galactosamine II Pharmacia

a Polyethylene glycol (PEG).2.4. Carbodiimide coupling reactions or zero lengths

3. Design and synthesis of polymeric conjugates . . . . . .

3.1. Dextran . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

3.2. Poly(ethylene glycol) (PEG) and conjugates . . .

3.3. PEGHIV agent conjugates . . . . . . . . . . . . . . .

3.4. PEGdrug and targeted delivery . . . . . . . . . . .

4. Poly-amino acid conjugates . . . . . . . . . . . . . . . . . . . .

4.1. N-(2-hydroxypropyl)methacrylamide (HPMA) c

5. Dendrimer and its conjugates . . . . . . . . . . . . . . . . . . .

6. Critical aspects of polymer conjugation . . . . . . . . . . .

6.1. Structureactivity relationship of conjugates (SA

6.2. Steric hindrance . . . . . . . . . . . . . . . . . . . . . . .

6.3. Enhanced reactivity of polymers by incorporatio

6.4. Targeting of polymeric drugs . . . . . . . . . . . . .

6.4.1. Passive tumor targeting . . . . . . . . . . . . . . . . . .

6.4.2. Active targeting . . . . . . . . . . . . . . . . . . . . . . .

7. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . .

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction

A prodrug is a form of a drug that remains inactive

during its delivery to the site of action and is activated

by the specific conditions in the targeted site. In other

words, a prodrug is an inactive precursor of a drug.

Prodrug reconversion (i.e. its conversion into its active

form) occurs in the body inside a specific organ, tissue

or cell. In most cases, normal metabolic processes such

as the cleavage of a bond between a polymer and a drug

by specific cellular enzymes are utilized to achieve

prodrug reconversion. A conjugation of a drug with apolymeric prodrug conjugates has lead into a new era of. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 394

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 394

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 394

polymeric drug delivery systems (PDDS). The task of

obtaining a versatile polymer as an ideal candidate in

drug delivery apparently seems to be intricate, as it has

to undergo several vigorous clinical barriers. Therefore,

many researchers rely on the polymers which have been

approved and well established. For decades, the

delivery of biomolecules using polymeric materials

has attracted considerable attention from polymer

chemists, chemical engineers and pharmaceutical

scientists [1]. However, the design and synthesis of

new polymeric candidates in view of their biological

implications has just been initiated.b Poly N-(2-Hydroxypropyl) methacrylamide (P-HPMA).

polymers are being extensively used in bioconjugates.

In general, a biologically active component undergoes

numerous physio-chemical barriers, which can be

further complicated by converting it into a polymeric

prodrug. In addition, due to their higher molecular

weight, polymers are known to dominate the physical

properties of the bioconjugated moiety. It has been well

established that polymers can enhance the aqueous

solubility of drug molecules by conjugation. Moreover,

polymers are being used for the preparation of various

formulations such as liposomes, microparticles or

nanoparticles [4]. Along with the polymer, the

physico-chemical properties of the drug or biomolecule

to be conjugated are equally important. The following

properties of the drug molecules make it suitable as an

ideal candidate to form the polymeric conjugate: (1)

lower aqueous solubility, (2) instability at varied

physiological pHs, (3) higher systemic toxicity, and

(4) reduced cellular entry. Numerous polymeric

prodrugs are in clinical phases (Table 1) and several

others have been approved (Table 2).

Despite progress, the delivery of active components

using clinically approved polymers remains a challenge.

Methods such as encapsulation, complexation or

covalent conjugation are routinely used in drug delivery

research [5]. But, the resulting complexes formed are

often unstable in a physiological environment. Various

bioconjugate methodologies that would form a stable

bond are being reported. Covalent conjugation of

biomolecules, e.g. protein drugs to synthetic polymers,

particularly poly(ethylene glycol) (PEG) does increase

their plasma residence, reduces protein immunogenicity

and can increase therapeutic index [6]. Successful

bioconjugation depends upon the chemical structure,

molecular weight, steric hindrance and the reactivity of

the biomolecule as well as the polymer. In order to

synthesize a bioconjugate, both chemical entities need to

posses a reactive or functional groups such as COOH,

OH, SH or NH2. However, the presence of multiple

reactive groups makes the task a bit complex. Therefore,

the synthetic methodology to form a conjugate involves

either protection or deprotection of the groups. There is

Table 2

Polymeric drug delivery systems (PDDS) that have received regulatory approval. [17]

Polymer/Formulation Drug or Active agent Brand/Trade

name/s

Manufacturer Therapeutic Indication Approved

Year/Country

Liposomal Amphotericin B AmBisome Gilead, Fujisawa Fungal infection 1990 (Europe),

1997

J. Khandare, T. Minko / Prog. Polym. Sci. 31 (2006) 359397 361PEG Adenosine deaminase Adagen

Styrene maleic acid and

neocarzinostatin

copolymer in Ethiodol

Zinostatin stimalamer SMANCS/

Lipiodol

Stealth (PEG-stabilized)

liposomal

Doxorubicin Doxil/Caelyx

Liposomal Doxorubicin Myocet

Liposomal cytosine Arabinoside DepoCyt

PEG Interferon a-2b PEG-Intron

Liposomal Verteporfin Visudyne

PEG Granulocyte colony

stimulating factor or

pegfilgrastim

Neulasta

PEG L-asparaginase Oncaspar/

pegaspargase

PEG Adenosine deaminase. AdagenLeishmaniasis 2000

Enzon Severe combined

immunodeficiency

1990

Yamanouchi Hepatocellular carcinoma 1993 1996

(Japan)

Schering Plough,

Alza

Kaposis sarcoma 1995

Refractory ovarian cancer 1999

Refractory breast cancer 2003 (Europe,

Canada)

Elan Metastatic breast cancer in

combination with

cyclophosphamide

2000 (Europe)

SkyePharma Lymphomatous meningitis 1999

Neoplastic meningitis Phase IV

Enzon, Schering-

Plough

Hepatitis C 2001

QLT, Novartis Wet macular degeneration

in conjunction with laser

treatment

2000 2001

(Japan)

Amgen Reduction of febrile

neutropenia associated with

chemotherapy

2002

Enzon Antineoplastic 1997

Enzon immunodeficiency disease 1993

type of prodrugthe drug delivery system (DDS).

Such a system can be constructed not only to target a

desired organ as a whole, its cells or specific organelles

inside certain cells but also to release a specified

amount of the drug at specific times. The polymeric

prodrug conjugate can also increase aqueous solubility,

enhance biodistribution and retain the inherent phar-

macological properties of the drug intact [11]. Three

major types of polymeric prodrugs are currently being

used [12]. Prodrugs of the first type are broken down

inside cells to form active substance or substances. The

second type of prodrug is usually the combination of

two or more substances. Under specific intracellular

conditions, these substances react forming an active

drug. The third type of prodrug, targeted drug delivery

systems, usually includes three components: a targeting

moiety, a carrier, and one or more active component(s).

The targeting ability of the delivery system depends on

the several variables including: receptor expression;

ligands internalization; choice of antibody, antibody

fragments or non-antibody ligands; and binding affinity

of the ligand [13]. Therefore, the selection of a suitable

polymer and a targeting moiety is vital to the

effectiveness of prodrugs.

J. Khandare, T. Minko / Prog. Polym. Sci. 31 (2006) 359397362further need to design simple and yet appropriate

polymeric-conjugation methodology. Many of the

most commonly used strategies involve use of both

coupling agents such as dicyclohexyl carbodiimide

(DCC) and 1-ethyl-3-(3-dimethylaminopropyl)carbo-

diimide or use of N-hydroxysuccinimide esters.

Chemical conjugation of drugs or other biomole-

cules to polymers and its modifications can form stable

bonds such as ester, amide, and disulphide. The

resulting bond linkage should be relatively stable to

prevent drug release during its transport before the

cellular localization of the drug. Covalent bonds (e.g.

ester or amide) are stable and could deliver the drug at

the targeted site, but such bonds may not easily release

targeting agents and peptides under the influence of

acceptable environmental changes [7]. In the past, most

of the polymeric prodrugs have been developed for the

delivery of anticancer agents. High molecular weight

prodrugs containing cytotoxic components have been

developed to decrease peripheral side effects and to

obtain a more specific administration of the drugs to the

cancerous tissues [8,9]. Favorably, a macromolecular

antitumor prodrug is expected to be stable in circulation

and should degrade only after reaching the targeted

cells or tissues. Polymerdrug conjugates can therefore

be tailored for activation by extra- or intracellular

enzymes releasing the parent drug in situ.

In this review, our focus is on the most promising

polymer candidates that are being used to form

bioconjugates. There is a necessity to decrease the

steric hindrance and increase the reactivity of polymers

as well as biomolecules to be chemically conjugated. In

this regard, we will discuss the current progress and

advances with various methodologies to obtain poly-

meric prodrugs.

2. Design and synthesis of polymeric prodrugs

Over the last decade, polymer chemists have been

actively involved in designing polymeric materials for

biomedical applications. One particular approach

towards an improved use of drugs for therapeutic

applications is to design polymeric prodrugs. A

prodrug is a chemical entity of an active parent drug

with altered physico-chemical properties [10]. In a

prodrug, a drug precursor remains inactive during

delivery to the site of action and is specifically activated

at the target site. The utilization of prodrugs, to a certain

extent, allows for the preservation of specific activity of

a drug and targets its release to certain cells or their

organelles. The most complete realization of theprodrug approach is possible by the use of an advancedFig. 1. Schematic presentation for (a) polymeric prodrug with

targeting agent and (b) hyperbranched polymer conjugate withtargeting and imaging agent.

Fig. 2. Schemes of various polymer architectures showing steric hindrance and crowding effect for chemical conjugation of drug molecules

(a) linear polymer, (b) copolymer, (c) branched polymer, and (d) hyperbranched polymer.

J. Khandare, T. Minko / Prog. Polym. Sci. 31 (2006) 359397 363

J. Khandare, T. Minko / Prog. Polym. Sci. 31 (2006) 359397364Numerous polymeric conjugation methods have

been attempted since 1950s. In 1955, Jatzkewitz

reported peptaminpolyvinylpyrrolidone conjugates

improve the efficacy of the drug [14]. Subsequential

biological aspects of polymeric prodrugs were not

previously taken into consideration. However, the

conjugation methodology would be more applied in

the forthcoming years. In 1975, a rational model for

pharmacologically active polymers was proposed by

Ringsdorfconsidered to be the pioneer of polymeric

prodrug research [15]. In general, an ideal polymeric

prodrug model consists mainly of a combination of one

or more components: (a) a polymeric backbone as a

vehicle, (b) one or more drugs of the biological active

components, (c) spacer(s) for hydrolysis of the

biomolecule and versatility for conjugation, (d) an

imaging agent and (e) targeting moiety (Fig. 1a and b).

The drug delivery carrier can be either biocompa-

tible or an inert biodegradable polymer. The drug is

coupled directly or via a spacer arm onto the polymer

backbone. Selection of the spacer arm is critical as it

opens the possibility of controlling the site and the rate

of release of the active drug from the conjugates either

by hydrolysis or by enzymatic degradation. The most

challenging aspect of this protocol is the possibility of

altering the body distribution and the cellular uptake by

cell-specific or non-specific uptake enhancers.

Due to current interdisciplinary research, molecular

biologists and organic, polymer chemists can now

design tailor-made polymeric carriers with different

structural architecture. Soluble polymers as potential

drug carriers have been reviewed in detail [16]. The

polymers selected for preparing macromolecular pro-

drugs can be categorized according to: (a) chemical

nature (vinylic or acrylic polymers, polysaccharides,

poly(a-amino acids), etc., (b) biodegradability, (c)origin (either natural polymers or synthetic polymers)

and (d) molecular weight (oligomers, macromers and

polymers). Below are the schematic polymeric archi-

tectural structures in conjugated form with their

bioactive components. Most of the polymers possess

crowding effect for chemical conjugation (Fig. 2).

Further synthetic strategies will need to be designed to

reduce such effects that will improve the conjugation

ratio and payload of component with the polymer.

2.1. Polymeric drug delivery system (PDDS)

During the last decade, polymer chemistry was

dedicated to synthesis, derivatization, degradation,

characterization, application, and evaluation, fornewer biocompatible and biodegradable polymers, allof which were used as carriers for polymeric drug

delivery systems. These systems possess unique roles in

enhanced physiological drug distribution, bioavailabil-

ity, drug targeting, time-controlled release, sensor-

responsive release, etc. The emergence of polymer

therapeutics has instigated research interfacing polymer

chemistry and the biomedical sciences including

initiation of nanosized (5100 nm) polymer-based

pharmaceuticals [6]. Polymer therapeutics include:

rationally designed macromolecular drugs, polymer

drug and polymerprotein conjugates, polymeric

micelles containing covalently bound drug, and

polyplexes for DNA delivery. The clinical applications

of polymerprotein conjugates and their resulting

outcomes seem to be promising, especially for

polymeranticancer-drug therapeutics [6].

Many of the pharmacological properties of conven-

tional free drugs can be improved through the use of

polymeric drug delivery systems (PDDS), which

include particulate carriers composed primarily of

lipids and/or polymers, and their associated thera-

peutics [17]. Several PDDS have already reached the

market (Table 2). The majority of the PDDS approved

are mainly targeted for parenteral administration and

include either liposomal or therapeutic molecules

linked to poly(ethylene glycol) (PEG) polymers. The

pegylation process is being used extensively in prodrug

conjugates and the synthesis approach involves

attachment of repeating units of poly(ethylene glycol)

to a polypeptide drug. More than 30 years, scientists

have developed various techniques to build PEG

polymers and attach them to a drug of choice. Recently,

pegylated drug forms in therapeutics of hepatitis C,

acromegaly, rheumatoid arthritis, neutropenia, various

cancers, wound healing, and other disorders either have

been approved or are undergoing clinical trials [18].

Proteins and peptides hold great promise as therapeutic

agents; however, they are prone for degradation by

proteolytic enzymes. These bioactives can be rapidly

cleared by the kidneys, generate neutralizing anti-

bodies, and have a short circulating half-life. Therefore,

pegylation of such drugs can overcome these and other

shortfalls. In addition, increased molecular mass of

proteins and peptides shields them from proteolytic

enzymes and enhances pharmacokinetics [18].

Modification of a polymer to form a conjugate with a

biomolecule depends upon two interrelated chemical

reactions: (1) reactive functional groups present in the

polymer and (2) functional groups present on the

biological component. In general, most of the biomo-

lecules such as ligands, peptides, proteins, carbo-hydrates, lipids, polymers, nucleic acid and

oligonucleotide possess combinations of these

functional groups. Selection of a suitable method,

process, and reagents are crucial for successful

chemical conjugation.

Polymers may undergo several structural changes

with solvents, coupling agents, and reactants. Peptide

and protein PEGylation problems and solutions have

cross-linking which will lead to an unstabilized form

and undesired products.

Modification of the polymer or its bioconjugate can

provide increased biocompatibility, reduced immune

response, enhanced in vivo stability, and passive tumor

targeting for anticancer DDS. In addition, modification

can significantly increase the water solubility of the

S lea

J. Khandare, T. Minko / Prog. Polym. Sci. 31 (2006) 359397 365been reviewed [19]. A detail of bioconjugation

strategies involving PEG and other polymers with

various biomolecules has been discussed briefly in

Section 3.2. Most of the bioconjugation strategies

involve coupling reactive nucleophiles with the

following order of reactivity: thiol, a-amino groups,epsilone amino group, carboxyl and hydroxyl. The

order of reactivity depends upon the pH in the reaction

and presence of steric hindrance on the coupling

moiety. Recent advances in bioconjugation use homo-

bifunctional amine or heterobifunctional coupling

reagents. A number of electrophilic groups are capable

of reacting with amines and other nucleophiles, e.g.

epoxides, vinylsulphones, and aziridines. In addition,

sulphonyl chlorides also react with amines; however,

they are generally water-insoluble and may get

hydrolyzed. Homobifunctional compounds can

form proteinprotein linkages such as N,N 00-ethylenei-minoyl-1-6-diaminohexane, bis-aziridin, divinyl sul-

phone (DVS), nitrogen mustard and bis-sulphonyl

chloride.

On the other hand, heterobifunctional reagents are

useful to couple amines with other functional groups.

The most important amine reacting heterobifunctional

compounds are used in protein chemistry; e.g.

photoactivation, biotinylation and thiolation reactions.

Reactive groups in proteincarboxyl functions offer

alternating thiol reactions as a site for heterobifunc-

tional coupling with amines. The linkages between

these two functions are formed without incorporation of

additional atoms by dehydration to an amide. A class of

such reagents contain compounds with an amine at one

end to allow a reaction with an activated carboxyl

function and an amine reactive moiety at the other.

However, such reactions may undergo cyclization or

R-NH2 +

= OR'

Amine component NHS ester derivative

Fig. 3. NHS esters compounds react with nucleophiles to release the NHto form COOH group, which can further form amide or ester bond with blysine residues involves use of a heterobifunctional

reagent comprising of a N-hydroxysuccinimide

functional group, together with a maleimide or

= O

R' NHR

Amide bond

+

H

NHS leaving group

ving group and form an acetylated product. PEG can be succiniylatedinsoluble component. The following are common

strategies adapted to obtain a polymeric drug delivery

system as biologically active prodrug conjugates.

2.2. N-hydroxysuccinimide (NHS) ester and coupling

methods

Due to their higher reactivity at physiological pH

makes NHS a choice for amine coupling reactions in

bioconjugation synthesis [20]. NHS ester compounds

react with nucleophiles to release the NHS leaving

group and form an acylated product (Fig. 3). NHS ester

is the most common activation chemical agent used to

form reactive acylating agent. Carboxyl groups acti-

vated with NHS esters are highly reactive with amine

nucleophiles. Polymers containing hydroxyl groups

(e.g. PEG) can be modified to obtain anhydride

compounds. PEG or mPEG can be acetylated with

anhydrides to form an ester terminating to free

carboxylate groups (Fig. 4). PEG and its succinimidyl

succinate and succinimidyl glutarate derivative can be

further used for conjugation with drugs or proteins.

The differences in the mode of action of conjugates

have created awareness of the importance in coupling

protocols appropriate for different components. The

successful application of conjugates for therapeutic

implication requires conjugates of defined composition

and of low molecular weight using heterobifuntional

coupling reagents under controlled and optimized

conditions [21]. Among the commonly used coupling

procedures, a heterobifunctional reagent is used to

couple modified lysine residues on one protein to

sulphydryl groups on the second. The modification ofiomolecules.

45% of scFv-c protein was conjugated as PEG(scFv-c)2using the smallest PEG(Mal)2 (2 kDa). However, no

significant increase in scFv-c conjugation was observed

spacer arm can enhance ligandprotein binding and has

-NH2

+

EDC

OOO

H3Cn

NH=O

mPEG with amidebond=O

R

R-OH

+

or EDC.HClnic Solvent

OOO

H3Cn

o=

O

mPEG with ester bond

=OR

sing a carbodiimide as coupling agent to form (a) amide bond and (b) ester

J. Khandare, T. Minko / Prog. Polym. Sci. 31 (2006) 359397366protected sulphydryl group. The linkage formed is one

of two basic types, a disulphide bridge or a thioether

bond (Fig. 5); the difference depends on whether the

introduced group is a sulphydryl or maleimide,

respectively. The thiol group on the second protein

may be an endogenous free sulphydryl or chemically

introduced by modification of lysine residues. NHS is

widely used as an acylating agent and is preferred for

conjugation with amine terminal compounds.

To optimize PEGylation, scFvs have been recombi-

nantly developed in a vector that adds an unpaired

cysteine (c) near the scFv carboxy terminus (scFv-c), to

provide a specific site for thiol conjugation (Fig. 6).

Random PEG conjugation (PEGylation) of small

molecules via amine groups demonstrated variations in

structural conformation and binding affinity [22]. The

authors evaluated applicability of unpaired cysteine for

PEGylation of scFv-c. Conjugation efficiency was

determined for four different scFvs along with several

PEG molecules having thiol reactive groups. ScFvs

R

OOO

H3Cn

OH=

O

Succinylated mPEG

=O(a)

DCCOrga

OOO

H3Cn

OH=

O

Succinylated mPEG

=O(b)

Fig. 4. Succinylated mPEG coupled to amine terminated component u

bond conjugate.produced as scFv-c and purified by anti-E-TAG affinity

chromatography were conjugated using PEG molecules

with maleimide (Mal) or o-pyridyl disulfide (OPSS).

Conjugations were carried out at pH 7.0, with 2 M

excess tris(2-carboxyethyl)phosphine hydrochloride

(TCEP)/scFv and PEGMal or PEGOPSS, using 5:1

(PEG:scFv). PEGMal conjugation efficiency was

further evaluated with 1:5 (PEG:scFv). PEGylation

efficiency was determined for each reaction by

quantitation of the products on SDS-PAGE. ScFv-c

conjugation with unifunctional maleimide PEGs

resulted in PEG conjugates incorporating 3080% of

the scFv-c with a consistent average above 50%. The

efficiency of scFv-c conjugation to both functional

groups of the bifunctional PEG(Mal)2 varied between

the PEG and scFv-c molecules studied. A maximum ofapplication in prodrug conjugates and in biotechnology

[24]. Ideal linkers possess the following characteristics:

(1) stability in the physiological pH if the drug is to be

O

NH-C-X-by use of a greater than 5 M excess of PEG/scFv-c.

Specific examples related to the coupling reactions have

been described in Section 3.

2.3. Incorporation of spacers in prodrug conjugates

Various spacers have been incorporated along with

the polymers and copolymers to decrease the crowding

effect and steric hindrance [23]. The incorporation of aNO

S

O

(a) Thioether linkage (x = Spacer)

Toxin- NH-C-CH2-CH2-S-S-

AntibodyEnzyme

(b) Disulphide linkage

O

Fig. 5. Types of proteinprotein linkages used for synthesis of

antibodyenzyme or antibodytoxin conjugates. The positions of the

proteins with respect to the linkage may be varied (modified from

Ref. [21]).

delivered to the tumor vasculature and (2) they release

the bioactive agent at an appropriate site of action.

For example, amino acid spacers such as glycine,

alanine, and small peptides are preferred due to their

chemical versatility for covalent conjugation and

biodegradability. Heterobifunctional coupling agents

containing succinimidyl have also been used exten-

sively as spacers (Fig. 7). Therapeutic potential of a

carboxypeptidase monoclonal antibody conjugate were

reported using N-succinimidyl anhydrides [2529].

The higher conjugation ratio of an antibody with a

drug can result in a decrease in the ability of the

antibody to bind to its specific receptor. This could be

overcome by introducing a polymer spacer between the

targeting antibody and the drug. The use of an

intermediate polymer with drug molecules carried in

Fig. 6. PEGmaleimide (PEGMal) structures and the maleimide

(Mal) thiol conjugation reaction with scFv-c (scFvSH): (a) methoxy-

PEGMal; (b) PEG(Mal)2; (c) branched methoxy-PEGMal; (d)

thioether bond formation between maleimide and cysteine of scFv-c

(reproduced from Ref. [22]).

Fig. 7. Structures of commonly used heterobifunctional coupling

agents with spacer lengths N-succinimidyl-4-(N-maleimidomethyl)-

cyclohexane-l-carboxylate (SMCC), N-succinimidyl-4-(p-maleimi-

dophenyl)-butyric acid (SMPB), N-maleimidobenzoyl-N-hydroxy-

succinimide (MBS) (reproduced from Ref. [29]).

J. Khandare, T. Minko / Prog. Polym. Sci. 31 (2006) 359397 367Fig. 8. Structure of (a) acid-sensitive bifunctional reagents used for

coupling of anthracycline drugs with polymers or antibodies and (b)

antibodydrug and antibodypolymerdrug conjugates (reproducedfrom Ref. [28]).

its side chains increases the potential number of drug

molecules able to attach to that antibody by modifi-

cation of only a minimum amount of existing amino

acid residues (Fig. 8a and b) [29].

In most of the bioconjugates, the NHS ester

anhydride is reacted with primary NH2 of the peptide

at slightly higher pH (7.5) to form an amide bond which

links the maleimide group to the protein and releases

NHS (Figs. 9 and 10). N-hydroxysuccinimide released

2.4. Carbodiimide coupling reactions or zero lengths

cross-linkers

Coupling and condensation reactions are unique to

obtain chemical conjugates involving drugs or other

biocomponents with polymers. The smallest possible

reagents for bioconjugate synthesis are called zero

length cross-linkers [30]. Modification of these con-

jugates depends upon the following two interrelated

Fig. 9. Scheme for protein coupling using N-hydroxysuccinimide ester/maleimide heterobifunctional agents. X represents the spacer groups of

varying chain lengths (reproduced from Ref. [29]).

J. Khandare, T. Minko / Prog. Polym. Sci. 31 (2006) 359397368from the protein can be easily removed either by

dialysis or by gel filtration using Sephadex columns

such as G10 or G25. Thereafter, the maleimide group

can be further reacted with the thiol containing moieties

or proteins to form a thioether bond in the presence of a

slightly acidic or neutral pH [29]. The applications of

spacers to form a polymer conjugate are discussed in

the polymer conjugate text.Fig. 10. Schemes for two N-hydroxysuccinimide-based reagents commonl

Ref. [29]).chemical reactions; the reactive functional groups on

the different cross-linking or deriavatizing reagents and

the functional groups present on the target biomolecule

to be modified. Coupling agents mediate the conju-

gation of the two molecules by forming a bond with no

additional spacer atom. Therefore, one atom of the

molecule is covalently linked to an atom of the second

molecule with no additional linker or spacer needed.y used for insertion of thiol residues into proteins (reproduced from

Carbodiimides (Figs. 11 and 12a and b) are most

commonly used as coupling reagents to obtain amide

linkage between a carboxylate and an amine or

phosphoramidate linkage between a phosphate and an

amine. They are unique due to their efficiency and

versatility to form a conjugate between two polymers,

between protein molecules, between a peptide and a

drug molecule, or between a peptide and a protein plus

any combination of these small molecules.

The carbodiimides are either water-soluble or water-

insoluble. The water-soluble carbodiimides are used for

biomacromolecular conjugation with variations of

buffer solutions. Water-insoluble carbodiimides are

used in presence of organic solvents for conjugation of

polymers with drugs and imaging agents, polymers and

peptides, and even polymerpolymer conjugates.

Usually, a byproduct is formed; which is mostly

insoluble in the solvent medium and facilitates easier

purification (Fig. 12).

Biomacromolecules containing phosphate groups

such as the 50 phosphate of oligonucleotide can beconjugated to amine containing molecules by using a

carbodiimide mediated reaction (e.g. EDC). Carbodii-

mide activates the phosphate to an intermediate phos-

phate ester, identical to its reaction with carboxylates.

Further, in the presence of an amine on a polymer

containing NH2 terminal groups, carbodiimide can be

conjugated to form a stable phosphoramidate bond

(Fig. 13). The application of carbodiimide as a coupling

agent to form a polymeric bioconjugate, with examples,

has been discussed in the subsequent polymeric text.

3. Design and synthesis of polymeric conjugates

3.1. Dextran

Several natural and synthetic polymers have

attracted the attention as prodrugs in biomedical

CH3

NC

N

NH

Cl

CH3

+

CH3

Fig. 11. Structure of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide

hydrochloride (EDC) coupling agent.

ON

CCl-

CH3O

R

o-acylisourea active intermediate

HCl

NH

+

(a)

H

-acyli

J. Khandare, T. Minko / Prog. Polym. Sci. 31 (2006) 359397 369CH3

NC

N

CH3 N

Amide bond formation

EDC.

R-COOH+

R'-NH2

R-COO-NHR'

NH-C-N

O

R-COOH+

o(b)CN

DCCR'-NH2

R-COO-NHR'Amide bond formation

Fig. 12. Formation of amide or ester bond. EDC$HCl (a) and DCC (b) activaform amide or ester bond, respectively.Cl-CH3

CH3

CH3

Esterbondformation

NH

+ R-COOH+

R'-OH

R-COO-R'

NR-COOH

+

R'-OH

R-COO-R'

sourea intermediate

Esterbondformation

tes carboxylic acid and further reacts with amine or hydroxyl group to

lysosomal fraction, but not in the control samples

lacking lysosomes. The hydrolysis rate constants for

DMP conjugate over to MP and MPS in the lysosomal

fraction were not significantly different from those in

the control samples. The authors also delineated the

applications of dextrans for targeted and sustained

delivery of therapeutic and imaging agents [35]. The

dextran was chemically conjugated with the amine

containing drugs or proteins using the periodate method

(Fig. 16).

The method to obtain b-(4-hydroxy-3,5-di-tert-butylphenyl) propionic acid (PA)dextran conjugates

+ R'-NH2O

O

O-

POH EDC

O

O

O-

PNH

R'

Phosphoramidate bond5' phosphate oligonucleotideR'~

J. Khandare, T. Minko / Prog. Polym. Sci. 31 (2006) 359397370applications. In particular, natural biopolymers, e.g.

dextran and chitosan have been used extensively for

prodrug conjugation research. Dextran is a natural

polysaccharide containing monomers of simple sugar

glucose (Fig. 14). This polyglucose biopolymer is

characterized by a-1,6 linkages, with hydroxylatedcyclohexyl units and generally produced by enzymes

from certain strains of Leuconostoc or Streptococcus.

However, dextran has more compact structure than any

other polymers. The chemical properties of dextran can

be modified chemically for the specific applications.

Dextran possesses multiple primary and secondary

hydroxyl groups and therefore can be easily conjugated

with drugs and proteins with reactive groups either by

direct conjugation or by incorporation of a spacer arm.

After oral administration, the polymer is not signifi-

cantly absorbed. Therefore, most of the effective

applications of dextran as polymeric carriers are

through injections. A few studies have reported the

potential of dextran in colon-specific delivery of drugs

via the oral route [31]. On the other hand by systemic

administration, the pharmacokinetics of the dextran

conjugates along with therapeutic agents are signifi-

cantly affected by the kinetics of the dextran. Animal

and human studies have shown that both the distri-

bution and elimination of dextrans are dependent on

molecular weight and the net charge.

Dextran has been extensively evaluated as a

polymeric vehicle for delivery of anticancer drugs to

the tumor tissue through a passive accumulation of the

dextrananticancer conjugate [32]. Conjugates of

dextrans with corticosteroids have been evaluated

Fig. 13. Phosphate oligonucleotide-polymer conjugation using EDC

as couplng agent.previously for the local delivery of steroids in colon

as anti-inflammatory agents [33]. A macromolecular

O

O

CH2

OH

OHOHO

O

OH

OHHOO

Fig. 14. Structure of dextran.prodrug of methylprednisolone (MP) was synthesized

by conjugating MP with dextran using succinic acid (S)

as a spacer [34]. MP-succinate (MPS) conjugate was

prepared using 1,1 0-carbonyldidmidazole as a couplingagent with succinic acid as a spacer (Fig. 15). The

hydrolysis of dextran MP (DMP) conjugate in rat blood

was achieved with a half-life ofw25 h. The hydrolysisof MPS to MP was proved to occur in the liver

OCH2

O

OHO

OH

OGlu

CO

H2C

H2C C

OO

C=OOH

O

HO

CH3Methylprednisolone

Succinate linker

Glu

Dextran

H2C

Fig. 15. Dextranmethylprednisolone conjugate. MP-succinate

(MPS) conjugate was prepared using 1,1 0-carbonyldidmidazole as acoupling agent with succinic acid as a spacer (reproduced from

Ref. [34]).(PADC) was reported [36]. The reaction was carried

out using dicyclohexyl carbodiimide (DCC) as a

coupling agent (Fig. 17). In most of the coupling

reactions, either pyridine or p-dimethylaminopyridine

(DMAP) was used as a catalyst. Dicyclohexylurea was

formed as a byproduct in the form of precipitate during

the reaction, easily removed by filtration.

A macromolecular prodrug of Tacrolimus (FK506),

FK506dextran conjugate, was developed and its

physico-chemical, biological and pharmacokinetic

drug

J. Khandare, T. Minko / Prog. Polym. Sci. 31 (2006) 359397 371characteristics were studied [37]. The conjugate was

estimated to have 0.45% of Tacrolimus (FK506) agent

and the coupling molar ratio was approximately 1:1

(dextran to FK506) (Fig. 18). Low molecular weight

radioactive compound(s) which eluted in the same

fractions as [3H]FK506 were released from

[3H]FK506dextran conjugate by chemical hydrolysis,

with a half-life of 150 h in phosphate buffer. The

dextranFK506 conjugate was synthesized using a

solution of carboxy-n-pentyl-dextran (C6D-ED) in

phosphate buffer with activated ester of FK506 in di-

oxane.

Fig. 16. Dextran was chemically conjugated with the amine containingPotent anticancer agents, such as camptothecin have

been conjugated to dextran to form prodrugs (Fig. 19).

Carrier and dose effects on the pharmacokinetics of

T-0128, a camptothecin analogue-carboxymethyl dex-

tran conjugate, were reported in control and tumor

bearing rats [38].

Conjugation of drugs with dextrans has exhibited

prolonged effect, reduced toxicity, and immunogeni-

city. Most of the studies have been carried out in

animals, however, with only a few experiments being

performed on humans [35]. The multiple OH groups

Fig. 17. Synthetic scheme for sterically hindered phenoon the polymeric dextran backbone provides possible

functional sites for drug conjugation. Dextran of Mw70,000 Da was conjugated to doxorubicin via an acid-

labile linkage for intratumoral delivery [39]. Dextran is

considered as a polymeric carrier because of its

biocompatibility and biodegradability [40].

An example of tumor-targeted dextran-based drug

delivery system has been described by Chau et al. [41].

A dextranpeptidemethotrexate conjugate for tumor-

targeted delivery of chemotherapeutics contains Pro-

Val-Gly-Leu-Ile-Gly peptide linker cleavable by matrix

metalloproteinase II (MMP-2) and IX (MMP-9). Both

s or proteins using the periodate method (reproduced from Ref. [35]).enzymes are over expressed in tumors. The linker

chemistry and the backbone charge were optimized to

allow high sensitivity of the conjugates toward the

targeted enzymes. In the presence of the targeted

enzymes, the peptide linker was cleaved and peptidyl

methotrexate (anticancer drug) was released. Satisfac-

tory stability of the new conjugates was demonstrated

in serum containing conditions, suggesting that the

conjugates can remain intact in systemic circulation.

Apart from natural polymers such as dextran, other

synthetic polymers such as PEG conjugates have

ldextran conjugate (reproduced from Ref. [36]).

intracellular targets. Covalent conjugation of synthetic

Fig. 18. Scheme for FK506dextran conjugate. The dextranFK506 conjugate was synthesized using a solution of carboxy-n-pentyl-dextran (C6D-

ED) in phosphate buffer with activated ester of FK506 in di-oxane (reproduced from Ref. [37]).

J. Khandare, T. Minko / Prog. Polym. Sci. 31 (2006) 359397372polymersparticularly poly(ethylene glycol) (PEG)

to bioactive components increases plasma residencegained interest for numerous therapeutic applications

and will be discussed in Section 3.2.

3.2. Poly(ethylene glycol) (PEG) and conjugates

PEG conjugates are classical prodrugs with an

enhanced permeability and retention (EPR) effect, and

which can accumulate significantly into tumor mass

and cross the cell membranes by endocytosis to reachFig. 19. Synthesis of FITC-labeled CPT (T-2513)dtime and the therapeutic index yet protein immuno-

genicity is reduced. Several PEGylated enzymes

(adenosine deaminase, L-asparaginase) and cytokines

(including interferon a and G-CSF) have now enteredroutine clinical use. PEG-modified adenosine de-

aminase (ADAGENw) and PEG-L-asparaginase

(ONCASPARw) were the first PEG modified enzymes

being used in early 1990s.

Most commonly used, monomethoxy poly(ethylene

glycol) (mPEGOH), can be functionalized and

conjugated with drug and other biological components.

Having only one or two terminal functional groups atextran conjugate (reproduced from Ref. [38]).

the end of polymer chain, PEG has a limitation with a

poor loading capacity. PEG has a linear or branched

polyether terminated with hydroxyl groups and is

synthesized by an anionic ring opening polymerization

of ethylene oxide initiated by a nucleophilic attack of a

hydroxide ion on the epoxide ring. Most used PEGs for

prodrug modification are either monomethoxy PEG

(Fig. 20a) or di-hydroxyl PEG (Fig. 20b). High aqueous

solubility makes PEG polymer a versatile candidate for

the prodrug conjugation. PEG is also considered to be

somewhat hydrophobic due to its solubility in many

organic solvents.

O OHO

n

H3C(a)

HOO

OHn

PEG

(b)

Fig. 20. Structures for (a) monomethoxy-poly(ethylene glycol) and

(b) di hydroxyl terminated poly(ethylene glycol).

X C

O

H2C

nC

O

O SumPEG

3 x =O,n=24 x =O, n =25 x = NH, n = 2

mPEG O C

O

O Su

6

mPEG-OH

mPEG O CO

AA

mPEG O C

O

O SumPEG O C

O

O Su

mPEG O 2HCOF3

O N

NN

Cl

R

1 R = Cl2 R = mPEG-O

O Su

7 R = Imidazole8 R = O-TCP9 R = O-pNP10 R = O-Su

11 AA=Gly,Ala etc13

SO O

12

mPEG

(a)

mPEG O N

NN

NH

R

R = Clor mPEG-O

Protein

1 or 2

3 -11H2N

12 13 and NaCN BH3

HN Protein

Protein

mPEG-

(b)

Fig. 21. (a) mPEG-based protein-modifying methods. Protein modification w

amides, derived from active esters 36 and 11, or carbamates, derived from 7

secondary amine conjugation with amino-containing residues. As represented

in reductive alkylation reactions. The numbering (113) represent to the orde

Ref. [42]).

J. Khandare, T. Minko / Prog. Polym. Sci. 31 (2006) 359397 373X C

O

mPEG HN Protein

x = O2C (CH2)n , OCH2,O, or an amino acid residue

ith all of these agents results in acylated amine-containing linkages:

to 10. Alkylating reagents (12 and 13) both react with proteins forming

in (b) tresylate (12) alkylates directly, while acetaldehyde (13) is used

r in which these activated polymers were introduced (reproduced from

T) co

J. Khandare, T. Minko / Prog. Polym. Sci. 31 (2006) 359397374Coupling reactions between NH2 groups of

proteins and mPEG with an electrophilic functional

group have been used in most cases for preparation of

PEGprotein conjugates [42]. Such reactions usually

result in formation of conjugates composed of a

globular protein in its core to which several polymer

chains are covalently linked. The composition of such a

graft copolymeric system is dependent on the number

of available attachment sites (NH2 and other nucleo-

philic groups) on the reactivity of the mPEG, and

reaction conditions. Fig. 21a and b illustrates the most

commonly used methods of mPEG-based protein

modifying reagents. Figure represents multiple com-

ponents of mPEGOH polymer conjugates. Derivatives

1 and 2 contain a reactive aryl chloride residue, which is

displaced by a nucleophilic amino group by a reaction

with peptides or proteins, as shown in Fig. 21b.

Derivatives (1 and 2) are acylating reagents, whereas

derivatives (311) contain reactive acyl groups refer-

enced as acylating agents. Protein modification with all

of these agents results in acylated amine-containing

linkages: amidesderived from active esters (36 and

Fig. 22. PEG-carbamate-paclitaxel (PC11)or carbamatesderived from (710). Alkylating

reagents (12 and 13) both react with proteins forming

secondary amine conjugation with amino-containing

residues. As represented in Fig. 21b, tresylate (12)

alkylates directly, while acetaldehyde (13) is used in

reductive alkylation reactions. Numbers (113) rep-

resent the order in which these activated polymers were

introduced [42].

It has been established that PEGylation greatly

enhances water solubility and decreases immuno-

genicity [43]. However, bioactive compounds con-

jugated with PEG polymers must be chemically

stable in its conjugated form until released.

Successful applications of covalently bonded PEG

with proteins and anticancer agents have beenreported [44]. The authors designed and evaluated

water-soluble mPEG5000 paclitaxel-7-carbamates

conjugates (Fig. 22).

PEG has severe limited conjugation capacity since

only two terminal functional groups exist at the end of

the polymer chain (or just one in the case of the most

used monomethoxy poly(ethylene glycol) (mPEG

OH)), which can be functionalized and conjugated to

the biocomponents. Recently, this limitation of PEG

was overcome by coupling amino acids, such as

bicarboxylic amino acid and aspartic acid, to the PEG

[45,46]. Such derivatization doubled the number of

active groups of the original molecule of PEG. Using

the same method with recursive derivatization, den-

drimeric structures were achieved at each PEGs

extremity. However, the authors encountered some

problems in this study, namely the lower reactivity of

the bicarboxylic acids groups towards Ara-C binding. It

was inferred that low reactivity is a result of steric

hindrance between two molecules of Ara-C when they

are conjugated to neighboring carboxylic moieties. It

was suggested that this effect might be overcome by

njugate (reproduced from Ref. [44]).incorporating the dendrimer arms with an amino

alcohol (H2N[CH2CH2O]2H).

PEG polymers with hydroxyl terminals can be

modified easily using small amino acids or other

aliphatic chains molecules. For example, linear or

branched PEG of varying molecular weight PEGAra-

C conjugates for controlled release were reported [47].

The antitumor agent 1-b-D-arabinofuranosilcytosyne(Ara-C) was covalently linked to varying molecular

weight OH terminal PEGs through an amino acid

spacer in order to improve the in vivo stability and

blood residence time. Conjugation was carried out with

one or two available hydroxyl groups at the polymers

terminals. Furthermore, to increase the drug loading of

the polymer, the hydroxyl of PEG was functionalized

HO-PEG-OH + Cl C O

O

NO2

CH2Cl2MW 10000 Da

PEG-O CO

O

NO2

13

H2O/CH2CN 1) Amino adipic acid

2

PEG-O CHN

O

CH

14

COOH

CH2-CH2-CH2-COOH 2

CH2Cl2

EDC / NHSPEG- O C

HN

O

CH

15

CONHS

CH2-CH2-CH2-CONHS

2Ara CPyridine

PEG-AD2-Ara-C7

2) Et3N

Et3N

AD= Amino adipic acid

(a)

H2O/CH2CNPEG-O C

HN

O

CH

16

CONH

CH2-CH2-CH2-

2

PEG -O CHN

O

CH

15

CONHS

CH2-CH2-CH2-CONHS

2

Et3NAmino adipic acid CH

COOH

CH2-CH2-CH2-COOH

CONH

CHCOOH

CH2-CH2-CH2-COOHEDC / NHS

CH2Cl2

PEG-O CHN

O

CH

17

CONH

CH2-CH2-CH2-

2

CHCONHS

CH2-CH2-CH2-CONHS

CONH

CHCONHS

CH2-CH2-CH2-CONHSAra-C

Pyridine

PEG-A2-AD4-Ara-C8

AD= Amino adapic acid

8

(b)

Fig. 23. Synthetic schemes for PEG10000AD2Ara-C4 (7) (a) and PEG10000AD2AD4Ara-C8 (8) conjugates (b). The antitumour agent 1-b-D-

arabinofuranosilcytosyne (Ara-C) was covalently linked to varying molecular weight OH terminal PEGs through an amino acid spacer in order to

improve the in vivo stability and blood residence time (reproduced from Ref. [47]).

J. Khandare, T. Minko / Prog. Polym. Sci. 31 (2006) 359397 375

J. Khandare, T. Minko / Prog. Polym. Sci. 31 (2006) 359397376PEG

(a)

-O CHN

O

CH

CONH CHCO-Ara-C

CH2-CH2-CH2-COOHwith a bicarboxylic amino acid to form a tetrafunctional

derivative. Finally, the conjugates with four or eight

Ara-C molecules for each PEG chain were prepared

(Fig. 23). The authors investigated steric hindrance in

(b)

CH2-CH2-CH-

HC COOH

CH2-CH2-CH2-CO Ara- C

CONH

PEG-O CHN

O

CH

CONH

-H2C

CHCO-Ara-C

CH2-COOH

HC COOH

CH2-CO - Ara -C

CONH

Fig. 24. (a) Most stable molecular structure of PEG(aminoadipic

acid)(Ara-C)2 conjugate. The two carboxylic acid functions are

emphasized by a space filling representation, and the distance (A)

between them is reported. (b) Most stable conformation of PEG

(aspartic acid)(Ara-C)2 conjugate. The two carboxylic acid functions

are emphasized by space filling, and the distance (A) between them is

reported (reproduced from Ref. [47]).PEGAra-C conjugates using molecular modeling to

investigate the most suitable bicarboxylic amino acid

with the least steric hindrance (Fig. 24a and b).

Computer aided design suggested that aminoadipic

acid was most suitable, because the carboxylic groups

are sufficiently separated to accommodate Ara-C

without the necessity to incorporate spacer arms. The

theoretical findings were confirmed and supported by

the experimental conjugation results. PEG conjugates

with Ara-C were prepared through an amino acid spacer

(e.g. non-leucine or lysine). Hydroxyl groups of PEG

were activated by p-nitrophenyl chloroformate to form

a stable carbamate linkage between PEG and the amino

acid. The degree of PEG hydroxyl group activation

with p-nitrophenyl chloroformate was determined by

UV analysis of the p-nitrophenol released from PEGp-

nitrophenyl carbonate after alkaline hydrolysis. Acti-

vated PEG was further coupled with amino acid and the

intermediate PEGamino acid was linked to Ara-C by

EDC/NHS activation.

Design and synthesis of non-targeted or antibody-

targeted biodegradable PEG multi-block coupled with

N2,N5-diglutamyllysine tripeptide with doxorubicin

(Dox) attached through acid-sensitive hydrazone bond

was reported [4851]. PEG activated with phosgene

and N-hydroxysuccinimide was reacted with NH2groups of triethyl ester of tripeptide N2,N6-diglutamyl-

lysine to obtain a degradable multi-block polymer. The

polymer was then converted to the corresponding

polyhydrazide by hydrazinolysis of the ethyl ester

with hydrazine hydrate. The non-targeted conjugate

was prepared by direct coupling of Dox with the

hydrazide PEG multi-block polymer (Fig. 25). Whereas

the antibody-targeted conjugates, a part of the polymer-

bound hydrazide group was modified with succinimidyl

3-(2-pyridyldisulfanyl) propanoate to introduce a

pyridyldisulfanyl group for subsequent conjugation

with a modified antibody. Dox was coupled to the

remaining hydrazide groups using acid-labile hydra-

zone bonds to obtain a polymer precursor.

In addition, human immunoglobulin IgG modified

with 2-iminothiolane was conjugated to the polymer by

substitution of the 2-pyridylsulfanyl groups of the

polymer with SH groups of the antibody. It was

demonstrated that Dox was rapidly released from the

conjugates when incubated in phosphate buffer at

lysosomal pH 5 and 7.4 (blood).

PEG has been used to modify a number of

therapeutically interesting proteins [52]. Conjugates

of adriamycin with PEGpoly(aspartamide) block

copolymers forms micelles [53]. Adriamycin (ADR),an anthracycline anticancer drug, was bound to the

ne) w

J. Khandare, T. Minko / Prog. Polym. Sci. 31 (2006) 359397 377poly(aspartic acid) chain of poly(ethylene glycol)

poly(aspartic acid) block copolymer by amide bond

formation between an amino group of adriamycin and

the carboxyl groups of the poly(aspartic acid) chain

(Fig. 26). The hydrophilic PEG chains form the outer

shell and the hydrophobic poly(aspartic acid)doxor-

ubicin components form the inner core. It was

Fig. 25. Scheme for multi-block PEGDox (hydrazodemonstrated that these systems have very high

in vivo antitumor activity and show a reduced non-

specific accumulation in the heart, lungs and liver.

Water-soluble PEG polymerdrug conjugates are

considered to be most promising with unique delivery

systems [5459]; especially PEG polymer which is the

most utilized carrier to deliver drugs in cancer therapy.

In tumor tissue, spacers are necessary to be incorpor-

tated between the drug and its carrier in order to enable

the release drug from that carrier either in slightly

acidic extracellular fluids or, after endocytosis, in

endosomes or lysosomes of cancer cells [60]. Acid-

sensitive hydrazone bond was formed between the C13

carbonyl group of anthracyclines (Dox, daunomycin

(Dau) and polymer hydrazides or amide bond of a cis-

aconityl residue containing spacer. Structure of Dox

bound to the polymer via pH-sensitive trityl spacer is

shown in Fig. 27.

It is worthwhile to note that even the larger proteins

or peptides have been successfully conjugated with

linear polymers. Various amounts of a releasable PEG

linker (rPEG) were conjugated to the protein lysozyme

(Fig. 28). rPEGlysozyme conjugate is relatively stablein pH 7.4 buffer for over 24 h. However, regeneration

of native protein from the rPEG conjugates occurred, as

expected, in a higher pH buffer of rat plasma [61].

Lysozyme is released more rapidly from the mono-

substituted conjugate than from the di-substituted

conjugate, suggesting possibility of steric hindrance

for the enzyme cleavage. PEG, being a linear polymer,

ith antibody conjugate (reproduced from Ref. [50]).is first activated at the OH terminal of either diol or

mono methoxy PEG. Modification of a branched PEG

Fig. 26. Adriamycin-conjugated poly(ethylene glycol)poly(aspartic

acid) block copolymer. Adriamycin (ADR), an anthracycline antic-

ancer drug, was bound to the poly(aspartic acid) chain of

poly(ethylene glycol)poly(aspartic acid) block copolymer by

amide bond formation between an amino group of adriamycin and

the carboxyl groups of the poly(aspartic acid) chain (reproduced from

Ref. [53]).

aqueous solubility of drugs [6466]. Recently, HIV

J. Khandare, T. Minko / Prog. Polym. Sci. 31 (2006) 359397378prodrug conjugates were designed, synthesized, and

evaluated to overcome several biopharmaceutical

challenges associated with HIV-1 protease inhibitorchain can provide an umbrella-like covering (U-PEG,

PEG 2) and has demonstrated applications in protein or

peptide conjugation [62,63].

3.3. PEGHIV agent conjugates

PEGylation significantly enhances biopharmaceuti-

cal characteristics such as circulation half-life and

Fig. 27. Structure of Doxpolymer conjugate with a pH-sensitive

trityl spacer (reproduced from Ref. [60]).(PIs) and prodrugs [67]. Various PEG-based prodrug

conjugates of the HIV-1 (PI) saquinavir (SQV) were

synthesized using different chemical groups having the

capability to modify pharmacokinetic properties. The

prodrug conjugates included SQVcysteinePEG3400,

SQVcysteinePEG3400biotin, SQVcysteine

Fig. 28. Scheme for PEGylation of lysozyme with PEG-BE linker

(reproduced from Ref. [61]).(R.I.CK-Tat9) [a cationic retro-inverso-cysteine-

lysine-Tat nonapeptide]PEG3400, and SQVcysteine

(R.I.CK(stearate)-Tat9)PEG3400. In all the conju-

gates, SQV was linked with cysteine to form a

degradable SQVcysteine ester bond. In addition, the

amino group of the cysteine moiety provided an

attachment site for degradation of the amide bond

with N-hydroxysuccinimide-activated forms of PEG

and PEGbiotin (Fig. 29).

3.4. PEGdrug and targeted delivery

Targeting of the cancer drug and other anti-

neoplastic agents is critical to the specific sites as it

can maximize cell-death effect during the tumor

growth phase during which majority of the cells remain

sensitive to pharmacotherapy. Also, healthy cells are

protected from exposure to the cytotoxic agent [68].

Recently, a two-tier approach was demonstrated using

the drug, camptothecin (CPT), and two different

targeting agentsluteinizing hormone-releasing hor-

mone (LHRH) and BCL2 homology 3 (BH3) peptide.

LHRH peptide was targeted to extra-cellular LHRH

receptors overexpressed in several cancer cells in order

to: increase the cancer specificity of the drug, reduce

adverse drug side effects, and enhance drug uptake by

cancer cells. BH3 peptide was targeted to intracellular

controlling mechanisms of apoptosis used to suppress

the cellular anti-apoptotic defense to enhance drug

anticancer activity [69]. CPT was first coupled to an

amino acid via a biodegradable ester bond to the

hydroxyl group at position 20, using Boc-Cys (Trt)

amino acid. Diisopropylcarbodiimide, was used as a

coupling agent and protecting groups were removed

using trifluoroacetic acid in methylene chloride. The

prodrug conjugate, CPTcysteine ester, had two

potential, orthogonal conjugation sitesthe amino

group and the thiol group (Fig. 30a).

CPT-Gly ester was first reacted with bifunctional

reagent, NHSPEGVinyl sulphone (VS), where the

amino group formed an amide bond with the active ester

(N-hydroxysuccinimide ester of PEG (Fig. 30b). An

analog of BH3 (Ac-Met-Gly-Gln-Val-Gly-Arg-Gln-

Leu-Ala-Ile-Ile-Gly-Asp-Asp-Ile-Asn-Arg-Arg-Tyr-

CysNH2), containing an extra residue of cysteine at the

C-terminus, was synthesized by the solid phase peptide

method. The previous reaction mixture was coupled to

the thiol group of BH3, which formed a thioether bond

with the VS group on the PEG. The LHRH analog-

LHRHLys6des-Gly10Pro9-ethylamide (Gln-His-

Trp-Ser-Tyr-DLys-Leu-Arg-Pro-NH-Et), which had areactive amino group only on the side chain of the lysine

J. Khandare, T. Minko / Prog. Polym. Sci. 31 (2006) 359397 379at position 6, was first reacted with one equivalent of

NHSPEGVS in DMF. CPT-Cys was then added to

obtain the thioether bond formation between the VS

group and the thiol group. The efficacy of the targeted

CPTPEGBH3 and CPTPEGLHRH conjugates was

higher than the non-targeted PEGCPT conjugate.

The feasibility of a two tier targeting of CPT

PEG conjugates to LHRH receptors and cellular anti-

apoptotic defense using LHRH and BH3 peptides,

respectively, was investigated [69]. In this study,

human ovarian carcinoma cells were incubated with

free CPT, CPTPEG, CPTPEGBH3 or CPTPEG

LHRH conjugates and the mixture of CPTPEG

BH3 or CPTPEGLHRH conjugates. Two

Fig. 29. Scheme to prepare saquinavir (SQV) conjugates. SQV was linked wi

the amino group of the cysteine moiety provided an attachment site for stea

forms of PEG and PEGbiotin (reproduced from Ref. [67]).approaches were used to assess the induction of

apoptosis: (1) measurement of the enrichment of cell

cytoplasm by histone-associated DNA fragments

(mono- and oligonucleosomes) and (2) the detection

of single- and double-stranded DNA breaks (nicks)

using terminal deoxynucleotidyl transferase mediated

dUTP-fluorescein nick end labeling (TUNEL)

method. The results obtained in these experiments

demonstrated that conjugation of CPT to PEG

increased its proapoptotic activity (Fig. 31). Further

enhancement was achieved by using BH3 peptide in

a CPTPEGBH3 and LHRH peptide in a CPT

PEGLHRH conjugates and their mixture. These

results were consistent with the cytotoxicity and

th cysteine to form a degradable SQVcysteine ester bond. In addition,

dy sate degrading amide bond with N-hydroxysuccinimide-activated

J. Khandare, T. Minko / Prog. Polym. Sci. 31 (2006) 359397380gene expression analysis results and showed that

simultaneous targeting and suppression of cellular

anti-apoptotic defense substantially increased antic-

ancer activity of camptothecin.

Recent development has lead to the use of higher

molecular weight PEG (O20,000 Da); especially the useof PEG 40,000 Da which is estimated to have a plasma

circulating half-life of approximately 89 h in a mouse.

Conjugation of small organic molecules with high

molecular weight PEG conjugates to form a prodrug

has proven to be a successful polymeric candidate [70].

4. Poly-amino acid conjugates

Due to demand of new biomaterials for a variety of

biomedical application, new potential candidates are

being designed and synthesized. Various amino acid

based polymers and their complex forming nature are

Fig. 30. Synthetic schemes for (a) CPTPEGBH3 conjugate. CPT was first c

group at position 20, using Boc-Cys (Trt) amino acid and (b) LHRHPEGbeing explored to deliver drugs to genetic materials. Since

poly(amino acid)s are structurally related to natural

proteins, the synthesis of amino acid-based polymers is

explored as a potential source of new biomaterials [71].

In the past, various reviews have been published

predicting poly(amino acids) as promising polymeric

candidates [7274]. However, only few polymers such

as poly(g-substituted glutamates) and copolymers havebeen screened as promising materials for biomedical

applications. Cationic polymers such as poly(L-lysine)

and their complexes with DNA have increased attention

as synthetic vectors for the delivery of genes. Synthetic

poly(a-amino acids) like poly(L-lysine), poly(L-gluta-mic acid), and poly((N-hydroxyalkyl) glutamines) can

be synthesized by ring-opening polymerization of the

N-carboxyanhydride monomers. These polymers have

functionalities in their side groups (amine, hydroxyl,

and carboxyl) that allow covalent coupling with drug

oupled to an amino acid via a biodegradable ester bond to the hydroxyl

CPT conjugate (reproduced from Ref. [69]).

A2780 human ovarian carcinoma cells after exposure to CPT, CPTPEG,

3 and CPTPEGLHRH. Cells were incubated 48 h with equivalent CPT

J. Khandare, T. Minko / Prog. Polym. Sci. 31 (2006) 359397 381molecules (Fig. 32a and b). In general, poly(L-amino

acids) are biocompatible and biodegradable.

Poly-N-(2-hydroxyethyl)-L-glutamine (PHEG) pro-

drugs of the antitumor antibiotic mitomycin C (MMC)

were synthesized using peptidyl spacers along with tri and

tetrapeptides to link drugs to a macromolecular carrier

(Fig. 33) [75]. Conjugates having a terminal glycine in the

spacer are less stable to hydrolysis than those with a

terminal hydrophobic amino acid both in buffer and in

serum. The Gly-Phe-Ala-Leu conjugate released MMC

Fig. 31. Typical fluorescence microscopy images of TUNEL labeled

CPTPEGBH3, CPTPEGLHRH and the mixture of CPTPEGBH

concentration of 3 nM (modified from Ref. [69]).very rapidly in the presence of both lysosomal enzymes

and collagenase IV. Biological experiments indicate that

PHEGMMC conjugates act as prodrugs of MMC.

Cytotoxicity was observed after hydrolytic release of

the active compound in vitro. MMC was coupled to the

Fmoc-protected oligopeptide pentafluorophenyl ester.

After deprotection, the amine-containing spacer-MMC

derivatives and tyrosinamide were coupled with 4-nitro-

phenyl carbonate containing PHEG (Fig. 33).

Delivery of genetic materials is a long-term goal for

which researchers continually strive. Vectors for gene

delivery formed by self-assembly of DNA with poly

(L-lysine) grafted with hydrophilic polymers was reported

[76]. Poly(L-lysine) (PLL) grafted with range of

hydrophilic polymer blocks, including poly(ethylene

glycol) (PEG), dextran and poly[N-(2-hydroxypropyl)-

methacrylamide] (PHPMA) showed efficient binding to

DNA. PEG-containing complexes increased transfection

activity against cells in vitro. Complexes formed with all

polymer conjugates exhibit greater aqueous solubility

than simple PLL/DNA complexes, particularly at chargeneutrality. a-Methoxy-u-carboxypoly(ethylene oxide)(PEGCOOH) was prepared by the reaction of PEGNH2with succinic anhydride (Fig. 34). Conversely, PLL-g-

PEG copolymers were synthesized by partially coupling

amino functions of PLL with a-methoxy-u-carboxy-poly(ethyleneoxide) using EDC as a coupling agent.

Further, dextran-grafted PLL was prepared by reductive

coupling of NH2 groups of PLL with the terminal

aldehyde groups of dextran using NaCNBH3 for selective

reduction of the Schiff base. In addition, a-n

(a)

-NH-CH-Cn

O=C-NHCH2CH2OH

(CH2)2

[ ]

O(b)

Fig. 32. Structures of (a) polylysine and (b) poly-N-(2-hydroxyethyl-

L-glutamine).

J. Khandare, T. Minko / Prog. Polym. Sci. 31 (2006) 359397382(carboxyethylenethio)poly(HPMA) (PHPMACOOH)

was prepared by radical solution polymerization in the

presence of 3-mercaptopropionic acid (MPA) as a chain

transfer agent. The PLL and PHPMACOOH were

dissolved in water and adjusted to pH 5.0 by dropwise

addition of HCl (0.5%). 1-Ethyl-3-[(dimethylamino)pro-

pyl]carbodiimide hydrochloride (EDC) was added as

coupling agent and the pH was maintained at 5.

Despite the advances in the field of therapeutics, the

delivery of nucleic acids and other genetic materials

remains a challenge [77,78]. Due to their intrinsic

ability, viral vectors introduce exogenous DNA into

host cells and have had limited success in delivery of

therapeutic genes due to immunogenicity. In this

regard, synthetic cationic polymers are a promising

alternative to viral vectors, e.g. polycations [79].

Whereas certain polycations can transfect mammalian

cells, these vectors can be cytotoxic and are much less

efficient than their viral counterparts [80].

A polycation gene delivery vector, polylysine-graft-

imidazoleacetic acid (Fig. 35), has recently been shown

to deliver genes in vitro with low cytotoxicity [81]. The

polycation imidazole groups were conjugated to the

Fig. 33. Mitomycin-C (MMC) poly-N-(2-hydroxyethyl)-L-glutamine)

(PHEG) conjugate. MMC was coupled to the Fmoc-protected

oligopeptide pentafluorophenyl ester. After deprotections the amine-

containing spacer-MMC derivatives and tyrosinamide were coupled

with 4-nitrophenyl carbonate containing PHEG (reproduced from

Ref. [75]).3-amines of Mw 34,300 Da poly-L-lysine in threedifferent molar ratios. It was observed that the reporter

gene expression increased non-linearly with the

increasing imidazole content in polycations, providing

an example of the structureactivity relationships of

these polymers. Amidation of the 3-amine of poly-L-lysine with 4-imidazoleacetic acid was achieved using a

EDAC/NHS coupling conjugation method.

Fig. 34. Structures of the grafted copolymers (a) PLL-g-PEG, (b)

PLL-g-dextran and (c) PLL-g-PHPMA (reproduced from Ref. [76]).

Fig. 35. Polylysine-graft-imidazoleacetic acid conjugates (reproduced

from Ref. [81]).

J. Khandare, T. Minko / Prog. Polym. Sci. 31 (2006) 359397 383C

CH3

CO

HN

CH2

CHHO

CH3

C

CH3

CO

NH

CH2

C=O

H2C

H2C

CH2-CH-CONH-CH-CONH-CH2-CO

NH CH2

CH

CH3H3C

HOH2CC O

O

O

O

CH3

OHNH

OCH3OH

HO

HO

O

Fig. 36. Structure of PHMPA copolymeradriamycin conjugate4.1. N-(2-hydroxypropyl)methacrylamide (HPMA)

copolymer

Since 1973, HPMA is the most investigated and

advanced polymer used in therapeutics due to its

versatility as a vehicle. Other clinically established

polymers in terms of biocompatibility and biodegrada-

tion kinetics are linear poly(ethylene glycol) and PLGA

copolymers. HPMA homopolymer was designed and

synthesized in Czechoslovakia as a plasma expander

[82]. HPMA being hydrophilic, increases water

solubility of the drugs and has proven to be non-toxic

in rats. Currently, antitumor agent HPMADox

conjugate is under clinical trials.

An HPMA copolymer with adriamycin conjugated

with the peptidyl linker Gly-Phe-Leu-Gly (PK1)

(Fig. 36), has been developed [83]. It was demonstrated

that HPMAadriamycin conjugates are less toxic than

the free drug and can accumulate inside solid tumor

models. HPMA drug conjugates are known to induce

endocytosis for the delivery of drugs into the cells.

PHPMA hydrazides were synthesized by modifying it

with N-succinimidyl 3-(2-pyridyldisulfanyl) propano-

ate (SPDP) to introduce the pyridyldisulfanyl groups

(reproduced from Ref. [83]).for subsequent conjugation with a modified antibody

(Fig. 37). Dox was bound to the remaining hydrazide

groups via an acid-labile hydrazone bond [84,85].

Human immunoglobulin IgG was modified with

2-iminothiolane by conjugating to the HPMA polymer

by substitution of the 2-pyridylsulfanyl groups of the

polymer with SH groups of the antibody. Another type

of the conjugate used a hydrazone linkage formed by

direct coupling of the periodate-oxidized antibody with

hydrazide groups remaining in the PHPMA-hydrazide

polymer after Dox attachment.

Anticancer drug, Paclitaxel, due to its low water

solubility, has been an ideal candidate for preparing

water-soluble prodrugs. It has been established that the

2 0- or 7-hydroxy group of Paclitaxel is suitable forstructure modification. Many attempts have been made

to couple low-molecular-weight solubilizing moieties

at the C2 0 or C7 position. These prodrugs are mainlyester derivatives including succinate, sulfonic acid, and

amino acid/phosphate derivatives [86,87].

Polymerdrug conjugates have demonstrated high

potential for targeting drugs to a cancerous tumor [88].

Apart from the selection of the ideal polymer to form a

prodrug, there are several other important aspects that

govern the success of polymeric conjugation. Most of

the carriers selected for covalent conjugation of drugs

with polymers must be water-soluble, non-toxic, and

possess a degree of compatability for chemical

conjugation. Advanced study of the acid-sensitive

DoxHPMA copolymer conjugates was reported

[8991]. PolymerDox conjugates containing side

chains of hydrazone-bound Dox moieties were attached

via single-amino-acid or the longer oligopeptide

spacers. Enzymatically degradable Gly-Phe-Leu-Gly

or non-degradable Gly, Gly-Gly, b-Ala, 6-aminohex-anoyl (AH) or 4-aminobenzoyl (AB) spacers were used.

Also HPMA-based conjugates with Dox attached

through Gly-Phe-Leu-Gly, Gly-Gly, and AH

spacerscontaining cis-aconityl residue at the spacer

endwere synthesized and studied (Fig. 38). It was

shown that the rate of Dox released from all the

conjugates under study was pH-dependent, with highest

rates obtained at pH 5.

A detailed investigation showed that mechanisms of

anticancer action of HPMA-copolymer bound antic-

ancer drug, Doxorubicin, differ substantially from those

of free drugs (Fig. 39) [9296]. It was shown that high

molecular weight HPMA-copolymer bound drugs

accumulated preferentially in solid tumors, with only

a trace amount of drugs detected in healthy organs. In

contrast, significant amount of free drugs were alsofound in the liver, heart, lungs, spleen and kidneys.

J. Khandare, T. Minko / Prog. Polym. Sci. 31 (2006) 359397384C

CH3

CO

HN

CH2

CHHO

CH3

C

CH3

CO

X

NH

N

H2C

CCH2OHO

OOCH3 OH

OH

OH

C

CH3

CO

X

NH

H2C

C=O

(H2C

CH2

CH2Therefore, conjugation of the anticancer drug to HPMA

copolymer substantially limited adverse side effects to

healthy organs imposed upon by free drugs.

In addition, the distribution of polymeric drugs

within the tumor was considerably more uniform when

compared with that of free drugs, which accumulated

mainly in the tumor regions with maximal permeability

of the vascular endothelium. HPMA-copolymer bound

anticancer drugs overcame existing drug efflux pumps

located in the plasma membrane of cancer cells and

prevented de novo development of multi-drug resist-

ance during repeated chemotherapy. In contrast, free

low molecular weight drugs activated existing multi-

drug resistance and initiated its de novo development

after prolonged treatment. The polymeric drugs

internalized into the cancer cells by endocytosis were

transported through the cellular cytoplasm in

O

O

H3C

HONH2 . HCl

NH

S

S

N

+

S

NH

CHN (CH2)3 SH

Fig. 37. Scheme of the synthesis of HPMA copolymerDox-antibody con

N-succinimidyl 3-(2-pyridyldisulfanyl) propanoate (SPDP) to introduce the

antibody (reproduced from Ref. [84]).C

CH3

CO

HN

CH2

CHHO

CH3

C

CH3

CO

X

NH

N

H2C

CCH2OHO

OOCH3 OH

OH

OH

C

CH3

CO

X

NH

H2C(H2C

NH

C=O

CH2membrane limited organelles. This protected the

drugs from cellular detoxification enzymes and pre-

served their high anticancer activity. Moreover,

HPMA-copolymer bound drugs suppressed the activity

of cellular detoxification enzymes. In contrast, free

drugsin most casesactivated cellular detoxification

mechanisms, antioxidant defense systems, and other

non-specific cellular defensive mechanisms. Finally,

the polymeric drugs activated caspase-dependent cell

death signaling pathways as they induced both

apoptosis and necrosis in tumor cells. Simultaneously,

HPMA-polymer bound drugs suppressed cell death

dependent mechanisms. In contrast, free low molecular

weight anticancer drugs activated cellular cell death

defensive mechanisms. As a result, cell death induction

by polymeric drugs was more pronounced when

compared with the free drugs. These data justify that

O

O

H3C

HONH2 .HCl

CH2

S

NH

CHN (CH2)3 S

jugates. PHPMA hydrazides were synthesized by modifying it with

pyridyldisulfanyl groups for subsequent conjugation with a modified

HO NH2 . HCl

J. Khandare, T. Minko / Prog. Polym. Sci. 31 (2006) 359397 385C

CH3

CO

(a)

HNCH2CHHOCH3

C

CH3

CO

X

NH

N

H2C

CCH2OHO

O

O

O

H3C

OCH3 OH

OH

OH

C

CH3

CO

X

NH

H2C

NH2

(H2Cconjugation of low molecular weight drugs to HPMA

copolymer carrier substantially increased its antitumor

activity.

5. Dendrimer and its conjugates

Dendritic polymers are emerging as potentially ideal

drug delivery vehicles because they are easily

manipulated and they provide a large density of

functional groups [2,9799]. Dendrimers possess

unique properties and are considered to be most

promising candidates for delivery of drug and

bioactives [100104]. The following characteristics of

dendrimers makes them ideal candidates for conju-

gation with biomolecules. Dendrimers are (1) mono-

disperse (w1.0), (2) nanosize (w20 nm), (3) havemultiple functional groups at the terminal (generation

28), and (4) possess end group tailorability for

conjugation. Due to these unique properties, dendri-

mers can be modified to obtain a high payload of

Fig. 38. Structure of HPMA copolymerDox conjugates: (a) hyrazone bond-

Dox conjugates containing side chains of hydrazone-bound Dox moieties

(reproduced from Ref. [89]).(b)H2CC

CH3

CO

HNCH2CHHOCH3

C

CH3

CO

X

NH

(CH2)2

H2C

NH

C

CH3

CO

X

NH

NH

(H2C

(CH2)2

NH2

O CH3

OH

C=O

CH2

HC COOH

C=O

NHbioactive components. So far they have been primarily

functionalized to deliver anticancer agents along with

targeting moieties. Recently, polyether dendritic

compounds with folate residues on their surface were

prepared as model drug carriers with potential tumor

cell specificity [2]. Although the dendrimers have

capability to conjugate high amounts of drug mol-

ecules, most of the reports imply conjugation of only 4

5 molecules.

In general, the low conjugation ratio could be a net

effect of all of the following: (1) lower reactivity, (2)

higher steric hindrance exhibited by the biomolecule as

well as the dendrimer, (3) small radius of gyration (Rh)

for chemical conjugation, and (4) crowding effect of the

functional groups at the terminals. Recently, nanoscale

dendritic drug delivery was targeted to the tumor cells

by using the folate receptor [102]. The nanodevice was

ethylenediamine core polyamidoamine (PAMAM)

dendrimer of generation 5. Folic acid, fluorescein, and

methotrexate were covalently attached to the surface to

HOH2COCO

OO

OCH3OH

OH

HO

contain