Polymer–drug conjugates: Progress in polymeric prodrugs
Jayant Khandarea, Tamara Minkoa,b,c,*
aDepartment of Pharmaceutics, Ernest Mario School of Pharmacy, Rutgers, The State University of New Jersey,
160 Frelinghuysen Road, Piscataway, NJ 08854-8020, USA
bThe Cancer Institute of New Jersey, 195 Little Albany Street, New Brunswick, NJ 08903, USA
cNew 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
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
hasincreased remarkablyas suchconjugates are preferably accumulated insolid tumors andcan reducesystemic 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 polymer–drug 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
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Design and synthesis of polymeric prodrugs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.1.Polymeric drug delivery system (PDDS) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.2. n-hydroxysuccinimide (NHS) ester and coupling methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.3. Incorporation of spacers in prodrug conjugates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Prog. Polym. Sci. 31 (2006) 359–397
0079-6700/$ - see front matter q 2006 Elsevier Ltd. All rights reserved.
*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: email@example.com (T. Minko).
Design and synthesis of polymeric conjugates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.1. Dextran . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.2. Poly(ethylene glycol) (PEG) and conjugates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.3. PEG–HIV agent conjugates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.4. PEG–drug and targeted delivery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Poly-amino acid conjugates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.1.N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Dendrimer and its conjugates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Critical aspects of polymer conjugation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.1. Structure–activity relationship of conjugates (SAR) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.2.Steric hindrance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.3. Enhanced reactivity of polymers by incorporation of spacers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.4. Targeting of polymeric drugs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.4.1. Passive tumor targeting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.4.2.Active targeting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Carbodiimide coupling reactions or zero lengths cross-linkers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .368
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 a
polymer 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 reductionin antigenic activityof 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
polymeric prodrug conjugates has lead into a new era of
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 . However, the design and synthesis of
new polymeric candidates in view of their biological
implications has just been initiated.
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
Current status of polymeric bioconjugates in cancer therapy
Name of the
micelle and Platinate
aPolyethylene glycol (PEG).
bPoly N-(2-Hydroxypropyl) methacrylamide (P-HPMA).
J. Khandare, T. Minko / Prog. Polym. Sci. 31 (2006) 359–397360
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 . 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
Methods such as encapsulation, complexation or
covalent conjugation are routinely used in drug delivery
research . 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
and can increase therapeutic index . 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
OH, –SH or –NH2. However, the presence of multiple
the synthetic methodology to form a conjugate involves
either protection or deprotection of the groups. There is
Polymeric drug delivery systems (PDDS) that have received regulatory approval. 
Polymer/Formulation Drug or Active agentBrand/Trade
Manufacturer Therapeutic IndicationApproved
Liposomal Amphotericin BAmBisome Gilead, Fujisawa Fungal infection1990 (Europe),
PEG Adenosine deaminaseAdagenEnzon
Styrene maleic acid and
copolymer in Ethiodol
Doxorubicin Doxil/Caelyx Schering Plough,
Kaposi’s sarcoma 1995
Refractory ovarian cancer
Refractory breast cancer
2000 (Europe) Liposomal DoxorubicinMyocetElanMetastatic breast cancer in
Liposomal cytosineArabinoside DepoCytSkyePharma1999
2001 PEGInterferon a-2bPEG-Intron Enzon, Schering-
QLT, NovartisLiposomal VerteporfinVisudyne Wet macular degeneration
in conjunction with laser
Reduction of febrile
neutropenia associated with
PEG Granulocyte colony
stimulating factor or
Neulasta Amgen 2002
PEGAdenosine deaminase.Enzon immunodeficiency disease1993
J. Khandare, T. Minko / Prog. Polym. Sci. 31 (2006) 359–397361
further 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 . 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. Polymer–drug 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-
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 . 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 the
prodrug approach is possible by the use of an advanced
type of prodrug—the 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 . Three
major types of polymeric prodrugs are currently being
used . 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 . Therefore, the selection of a suitable
polymer and a targeting moiety is vital to the
effectiveness of prodrugs.
Fig. 1. Schematic presentation for (a) polymeric prodrug with
targeting agent and (b) hyperbranched polymer conjugate with
targeting and imaging agent.
J. Khandare, T. Minko / Prog. Polym. Sci. 31 (2006) 359–397362
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) 359–397363
Numerous polymeric conjugation methods have
been attempted since 1950s. In 1955, Jatzkewitz
reported peptamin–polyvinylpyrrolidone conjugates
improve the efficacy of the drug . 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
Ringsdorf—considered to be the pioneer of polymeric
prodrug research . 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 . 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, for
newer biocompatible and biodegradable polymers, all
of 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 (5–100 nm) polymer-based
pharmaceuticals . Polymer therapeutics include:
rationally designed macromolecular drugs, polymer–
drug and polymer–protein conjugates, polymeric
micelles containing covalently bound drug, and
polyplexes for DNA delivery. The clinical applications
of polymer–protein conjugates and their resulting
outcomes seem to be promising, especially for
polymer–anticancer-drug therapeutics .
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 . 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 .
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 .
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, nucleicacid and
J. Khandare, T. Minko / Prog. Polym. Sci. 31 (2006) 359–397 364
oligonucleotide possess combinations of these
functional groups. Selection of a suitable method,
process, and reagents are crucial for successful
Polymers may undergo several structural changes
with solvents, coupling agents, and reactants. Peptide
and protein PEGylation problems and solutions have
been reviewed . 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
form protein–protein linkages such as N,N00-ethylenei-
minoyl-1-6-diaminohexane, bis-aziridin, divinyl sul-
phone (DVS), nitrogen mustard and bis-sulphonyl
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 protein–carboxyl 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
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
insoluble 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
Due to their higher reactivity at physiological pH
makes NHS a choice for amine coupling reactions in
bioconjugation synthesis . 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 . 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 of
lysine residues involves use of a heterobifunctional
reagent comprising of a N-hydroxysuccinimide
functional group, together with a maleimide or
Amine componentNHS ester derivativeAmide bond
NHS leaving group
Fig. 3. NHS esters compounds react with nucleophiles to release the NHS leaving group and form an acetylated product. PEG can be succiniylated
to form –COOH group, which can further form amide or ester bond with biomolecules.
J. Khandare, T. Minko / Prog. Polym. Sci. 31 (2006) 359–397 365
protected 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 . 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
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 PEG–Mal or PEG–OPSS, using 5:1
(PEG:scFv). PEG–Mal 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 30–80% 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)2varied between
the PEG and scFv-c molecules studied. A maximum of
using the smallest PEG–(Mal)2(2 kDa). However, no
significant increase in scFv-c conjugation was observed
by use of a greater than 5 M excess of PEG/scFv-c.
Specificexamples relatedtothe 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 . The incorporation of a
spacer arm can enhance ligand–protein binding and has
application in prodrug conjugates and in biotechnology
. Ideal linkers possess the following characteristics:
(1) stability in the physiological pH if the drug is to be
mPEG with amidebond
DCC or EDC.HCl
mPEG with ester bond
Fig. 4. Succinylated mPEG coupled to amine terminated component using a carbodiimide as coupling agent to form (a) amide bond and (b) ester
(a) Thioether linkage (x = Spacer)
(b) Disulphide linkage
Fig. 5. Types of protein–protein linkages used for synthesis of
antibody–enzyme or antibody–toxin conjugates. The positions of the
proteins with respect to the linkage may be varied (modified from
J. Khandare, T. Minko / Prog. Polym. Sci. 31 (2006) 359–397366
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 [25–29].
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. PEG–maleimide (PEG–Mal) structures and the maleimide
(Mal) thiol conjugation reaction with scFv-c (scFv–SH): (a) methoxy-
PEG–Mal; (b) PEG–(Mal)2; (c) branched methoxy-PEG–Mal; (d)
thioether bond formation between maleimide and cysteine of scFv-c
(reproduced from Ref. ).
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. ).
Fig. 8. Structure of (a) acid-sensitive bifunctional reagents used for
coupling of anthracycline drugs with polymers or antibodies and (b)
antibody–drug and antibody–polymer–drug conjugates (reproduced
from Ref. ).
J. Khandare, T. Minko / Prog. Polym. Sci. 31 (2006) 359–397367
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) .
In most of the bioconjugates, the NHS ester
anhydride is reacted with primary –NH2of 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
from 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 . The applications of
spacers to form a polymer conjugate are discussed in
the polymer conjugate text.
2.4. Carbodiimide coupling reactions or zero lengths
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 . Modification of these con-
jugates depends upon the following two interrelated
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.
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. ).
Fig. 10. Schemes for two N-hydroxysuccinimide-based reagents commonly used for insertion of thiol residues into proteins (reproduced from
J. Khandare, T. Minko / Prog. Polym. Sci. 31 (2006) 359–397368
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 polymer–polymer 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 50phosphate of oligonucleotide can be
conjugated 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 –NH2terminal 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
Several natural and synthetic polymers have
attracted the attention as prodrugs in biomedical
Fig. 11. Structure of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide
hydrochloride (EDC) coupling agent.
o-acylisourea active intermediate
Amide bond formation
Amide bond formation
Fig. 12. Formation of amide or ester bond. EDC$HCl (a) and DCC (b) activates carboxylic acid and further reacts with amine or hydroxyl group to
form amide or ester bond, respectively.
J. Khandare, T. Minko / Prog. Polym. Sci. 31 (2006) 359–397 369
applications. 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 hydroxylated
cyclohexyl 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 . 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
dextran–anticancer conjugate . Conjugates of
dextrans with corticosteroids have been evaluated
previously for the local delivery of steroids in colon
as anti-inflammatory agents . A macromolecular
prodrug of methylprednisolone (MP) was synthesized
by conjugating MP with dextran using succinic acid (S)
as a spacer . MP-succinate (MPS) conjugate was
prepared using 1,10-carbonyldidmidazole as a coupling
agent with succinic acid as a spacer (Fig. 15). The
hydrolysis of dextran MP (DMP) conjugate in rat blood
was achieved with a half-life of w25 h. The hydrolysis
of MPS to MP was proved to occur in the liver
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 . The
dextran was chemically conjugated with the amine
containing drugs or proteins using the periodate method
The method to obtain b-(4-hydroxy-3,5-di-tert-
butylphenyl) propionic acid (PA)–dextran conjugates
(PADC) was reported . 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),
FK506–dextran conjugate, was developed and its
physico-chemical, biological and pharmacokinetic
Fig. 15. Dextran–methylprednisolone conjugate. MP-succinate
(MPS) conjugate was prepared using 1,10-carbonyldidmidazole as a
coupling agent with succinic acid as a spacer (reproduced from
Fig. 14. Structure of dextran.
5' phosphate oligonucleotide
Fig. 13. Phosphate oligonucleotide-polymer conjugation using EDC
as couplng agent.
J. Khandare, T. Minko / Prog. Polym. Sci. 31 (2006) 359–397370
characteristics were studied . 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
[3H]FK506–dextran conjugate by chemical hydrolysis,
with a half-life of 150 h in phosphate buffer. The
dextran–FK506 conjugate was synthesized using a
solution of carboxy-n-pentyl-dextran (C6D-ED) in
phosphate buffer with activated ester of FK506 in di-
Potent 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 .
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 . The multiple –OH groups
on the polymeric dextran backbone provides possible
functional sites for drug conjugation. Dextran of Mw
70,000 Da was conjugated to doxorubicin via an acid-
labile linkage for intratumoral delivery . Dextran is
considered as a polymeric carrier because of its
biocompatibility and biodegradability .
An example of tumor-targeted dextran-based drug
delivery system has been described by Chau et al. .
A dextran–peptide–methotrexate 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
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
Fig. 16. Dextran was chemically conjugated with the amine containing drugs or proteins using the periodate method (reproduced from Ref. ).
Fig. 17. Synthetic scheme for sterically hindered phenol–dextran conjugate (reproduced from Ref. ).
J. Khandare, T. Minko / Prog. Polym. Sci. 31 (2006) 359–397371
gained 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 reach
intracellular targets. Covalent conjugation of synthetic
polymers—particularly poly(ethylene glycol) (PEG)—
to bioactive components increases plasma residence
time 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 entered
routine clinical use. PEG-modified adenosine de-
(ONCASPARw) were the first PEG modified enzymes
being used in early 1990s.
Most commonly used, monomethoxy poly(ethylene
glycol) (mPEG–OH), can be functionalized and
conjugated with drug and other biological components.
Having only one or two terminal functional groups at
Fig. 18. Scheme for FK506–dextran conjugate. The dextran–FK506 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. ).
Fig. 19. Synthesis of FITC-labeled CPT (T-2513)–dextran conjugate (reproduced from Ref. ).
J. Khandare, T. Minko / Prog. Polym. Sci. 31 (2006) 359–397372
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
Fig. 20. Structures for (a) monomethoxy-poly(ethylene glycol) and
(b) di hydroxyl terminated poly(ethylene glycol).
3 x =O,n=2
4 x =O, n =2
5 x = NH, n = 2
1 R = Cl
2 R = mPEG-O
7 R = Imidazole
8 R = O-TCP
9 R = O-pNP
10 R = O-Su
11 AA=Gly,Ala etc
R = Cl
1 or 2
x = O2C (CH2)n , OCH2,
O, or an amino acid residue
13 and NaCN BH3
Fig. 21. (a) mPEG-based protein-modifying methods. Protein modification with all of these agents results in acylated amine-containing linkages:
amides,derivedfrom active esters3–6 and 11, orcarbamates,derivedfrom 7 to 10. Alkylating reagents (12and 13)both react withproteins forming
secondaryamine conjugation with amino-containing residues.As representedin (b)tresylate (12)alkylates directly, whileacetaldehyde (13) is used
in reductive alkylation reactions. The numbering (1–13) represent to the order in which these activated polymers were introduced (reproduced from
J. Khandare, T. Minko / Prog. Polym. Sci. 31 (2006) 359–397 373
Coupling reactions between –NH2 groups of
proteins and mPEG with an electrophilic functional
group have been used in most cases for preparation of
PEG–protein conjugates . 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 (–NH2and 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 mPEG–OH polymer conjugates. Derivatives
1and 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 (3–11) contain reactive acyl groups refer-
enced as acylating agents. Protein modification with all
of these agents results in acylated amine-containing
linkages: amides—derived from active esters (3–6 and
11)—or carbamates—derived from (7–10). 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 (1–13) rep-
resent the order in which these activated polymers were
It has been established that PEGylation greatly
enhances water solubility and decreases immuno-
genicity . 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 been
reported . 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 PEG’s
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
incorporating the dendrimer arms with an amino
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 PEG–Ara-
C conjugates for controlled release were reported .
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 polymer’s
terminals. Furthermore, to increase the drug loading of
the polymer, the –hydroxyl of PEG was functionalized
Fig. 22. PEG-carbamate-paclitaxel (PCT) conjugate (reproduced from Ref. ).
J. Khandare, T. Minko / Prog. Polym. Sci. 31 (2006) 359–397374
MW 10000 Da
1) Amino adipic acid
EDC / NHS
AD= Amino adipic acid
Amino adipic acid
EDC / NHS
AD= Amino adapic acid
Fig. 23. Synthetic schemes for PEG10000–AD2–Ara-C4(7) (a) and PEG10000–AD2–AD4–Ara-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. ).
J. Khandare, T. Minko / Prog. Polym. Sci. 31 (2006) 359–397375
with 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
PEG–Ara-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 acidspacer
(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 PEG–p-
nitrophenyl carbonate after alkaline hydrolysis. Acti-
vated PEG was further coupled with amino acid and the
intermediate PEG–amino acid was linked to Ara-C by
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 [48–51]. PEG activated with phosgene
and N-hydroxysuccinimide was reacted with –NH2
groups 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 . Conjugates
of adriamycin with PEG–poly(aspartamide) block
copolymers forms micelles . Adriamycin (ADR),
an anthracycline anticancer drug, was bound to the
CH2-CH2-CH2-CO Ara- C
CH2-CO - Ara -C
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)2conjugate. The two carboxylic acid functions
are emphasized by space filling, and the distance (A˚) between them is
reported (reproduced from Ref. ).
J. Khandare, T. Minko / Prog. Polym. Sci. 31 (2006) 359–397376
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
(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
demonstrated 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 polymer–drug conjugates are
considered to be most promising with unique delivery
systems [54–59]; 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 . 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). rPEG–lysozyme conjugate is relatively stable
in 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 .
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,
is first activated at the –OH terminal of either diol or
mono methoxy PEG. Modification of a branched PEG
Fig. 25. Scheme for multi-block PEG–Dox (hydrazone) with antibody conjugate (reproduced from Ref. ).
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
J. Khandare, T. Minko / Prog. Polym. Sci. 31 (2006) 359–397377
chain can provide an umbrella-like covering (U-PEG,
PEG 2) and has demonstrated applications in protein or
peptide conjugation [62,63].
3.3. PEG–HIV agent conjugates
PEGylation significantly enhances biopharmaceuti-
cal characteristics such as circulation half-life and
aqueous solubility of drugs [64–66]. Recently, HIV
prodrug conjugates were designed, synthesized, and
evaluated to overcome several biopharmaceutical
challenges associated with HIV-1 protease inhibitor
(PIs) and prodrugs . 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 SQV–cysteine–PEG3400,
(R.I.CK-Tat9) [a cationic retro-inverso-cysteine-
lysine-Tat nonapeptide]–PEG3400, and SQV–cysteine
(R.I.CK(stearate)-Tat9)–PEG3400. In all the conju-
gates, SQV was linked with cysteine to form a
degradable SQV–cysteine 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 PEG–biotin (Fig. 29).
3.4. PEG–drug 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 .
Recently, a two-tier approach was demonstrated using
the drug, camptothecin (CPT), and two different
targeting agents—luteinizing 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 . 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, CPT–cysteine ester, had two
potential, orthogonal conjugation sites—the amino
group and the thiol group (Fig. 30a).
CPT-Gly ester was first reacted with bifunctional
reagent, NHS–PEG–Vinyl sulphone (VS), where the
(N-hydroxysuccinimide ester of PEG (Fig. 30b). An
analog of BH3 (Ac-Met-Gly-Gln-Val-Gly-Arg-Gln-
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-
Trp-Ser-Tyr-DLys-Leu-Arg-Pro-NH-Et), which had a
Fig. 27. Structure of Dox–polymer conjugate with a pH-sensitive
trityl spacer (reproduced from Ref. ).
Fig. 28. Scheme for PEGylation of lysozyme with PEG-BE linker
(reproduced from Ref. ).
J. Khandare, T. Minko / Prog. Polym. Sci. 31 (2006) 359–397378
at position 6, was first reacted with one equivalent of
NHS–PEG–VS 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
CPT–PEG–BH3 and CPT–PEG–LHRH conjugates was
higher than the non-targeted PEG–CPT 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 . In this study,
human ovarian carcinoma cells were incubated with
free CPT, CPT–PEG, CPT–PEG–BH3 or CPT–PEG–
LHRH conjugates and the mixture of CPT–PEG–
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
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 CPT–PEG–BH3 and LHRH peptide in a CPT–
PEG–LHRH conjugates and their mixture. These
results were consistent with the cytotoxicity and
end labeling (TUNEL)
Fig. 29. Scheme to prepare saquinavir (SQV) conjugates. SQV was linked with cysteine to form a degradable SQV–cysteine ester bond. In addition,
the amino group of the cysteine moiety provided an attachment site for steady sate degrading amide bond with N-hydroxysuccinimide-activated
forms of PEG and PEG–biotin (reproduced from Ref. ).
J. Khandare, T. Minko / Prog. Polym. Sci. 31 (2006) 359–397379
gene 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);especiallythe use
of PEG 40,000 Da which is estimated to have a plasma
circulating half-life of approximately 8–9 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 .
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
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 .
In the past, various reviews have been published
predicting poly(amino acids) as promising polymeric
candidates [72–74]. However, only few polymers such
as poly(g-substituted glutamates) and copolymers have
been 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
Fig.30. Syntheticschemes for(a) CPT–PEG–BH3conjugate. CPTwasfirstcoupledto an aminoacid via a biodegradable ester bondto the hydroxyl
group at position 20, using Boc-Cys (Trt) amino acid and (b) LHRH–PEG–CPT conjugate (reproduced from Ref. ).
J. Khandare, T. Minko / Prog. Polym. Sci. 31 (2006) 359–397 380
molecules (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)
tetrapeptides to link drugs to a macromolecular carrier
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
very rapidly in the presence of both lysosomal enzymes
and collagenase IV. Biological experiments indicate that
PHEG–MMC 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
. 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 charge
neutrality. a-Methoxy-u-carboxy–poly(ethylene oxide)
with 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
reduction of the Schiff base. In addition, a-
Fig. 31. Typical fluorescence microscopy images of TUNEL labeled A2780 human ovarian carcinoma cells after exposure to CPT, CPT–PEG,
CPT–PEG–BH3, CPT–PEG–LHRH and the mixture of CPT–PEG–BH3 and CPT–PEG–LHRH. Cells were incubated 48 h with equivalent CPT
concentration of 3 nM (modified from Ref. ).
Fig. 32. Structures of (a) polylysine and (b) poly-N-(2-hydroxyethyl-
J. Khandare, T. Minko / Prog. Polym. Sci. 31 (2006) 359–397 381
was prepared by radical solution polymerization in the
presence of 3-mercaptopropionic acid (MPA) as a chain
transfer agent. The PLL and PHPMA–COOH 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 .
Whereas certain polycations can transfect mammalian
cells, these vectors can be cytotoxic and are much less
efficient than their viral counterparts .
A polycation gene delivery vector, polylysine-graft-
imidazoleacetic acid (Fig. 35), has recently been shown
to deliver genes in vitro with low cytotoxicity . The
polycation imidazole groups were conjugated to the
3-amines of Mw 34,300 Da poly-L-lysine in three
different 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 structure–activity relationships of
these polymers. Amidation of the 3-amine of poly-L-
lysine with 4-imidazoleacetic acid was achieved usinga
EDAC/NHS coupling conjugation method.
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
Fig. 34. Structures of the grafted copolymers (a) PLL-g-PEG, (b)
PLL-g-dextran and (c) PLL-g-PHPMA (reproduced from Ref. ).
Fig.35. Polylysine-graft-imidazoleacetic acid conjugates(reproduced
from Ref. ).
J. Khandare, T. Minko / Prog. Polym. Sci. 31 (2006) 359–397382
4.1. N-(2-hydroxypropyl)methacrylamide (HPMA)
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
. HPMA being hydrophilic, increases water
solubility of the drugs and has proven to be non-toxic
in rats. Currently, antitumor agent HPMA–Dox
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 . It was demonstrated
that HPMA–adriamycin 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
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
20- or 7-hydroxy group of Paclitaxel is suitable for
structure modification. Many attempts have been made
to couple low-molecular-weight solubilizing moieties
at the C20or C7 position. These prodrugs are mainly
ester derivatives including succinate, sulfonic acid, and
amino acid/phosphate derivatives [86,87].
Polymer–drug conjugates have demonstrated high
potential for targeting drugs to a cancerous tumor .
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
Dox–HPMA copolymer conjugates was reported
[89–91]. Polymer–Dox 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
spacers—containing cis-aconityl residue at the spacer
end—were 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) [92–96]. 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 also
found in the liver, heart, lungs, spleen and kidneys.
Fig. 36. Structure of PHMPA copolymer–adriamycin conjugate
(reproduced from Ref. ).
J. Khandare, T. Minko / Prog. Polym. Sci. 31 (2006) 359–397383
Therefore, 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
membrane 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
drugs—in most cases—activated 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
NH2 . HCl
Fig. 37. Scheme of the synthesis of HPMA copolymer–Dox-antibody conjugates. PHPMA hydrazides were synthesized by modifying it with
N-succinimidyl 3-(2-pyridyldisulfanyl) propanoate (SPDP) to introduce the pyridyldisulfanyl groups for subsequent conjugation with a modified
antibody (reproduced from Ref. ).
J. Khandare, T. Minko / Prog. Polym. Sci. 31 (2006) 359–397384
conjugation of low molecular weight drugs to HPMA
copolymer carrier substantially increased its antitumor
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 [2,97–99]. Dendrimers possess
unique properties and are considered to be most
promising candidates for delivery of drug and
bioactives [100–104]. 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) have
multiple functional groups at the terminal (generation
2–8), and (4) possess end group ‘tailorability’ for
conjugation. Due to these unique properties, dendri-
mers can be modified to obtain a ‘high payload’ of
bioactive 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 . Although the dendrimers have
capability to conjugate high amounts of drug mol-
ecules, most of the reports imply conjugation of only 4–
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 . The nanodevice was
ethylenediamine core polyamidoamine (PAMAM)
dendrimer of generation 5. Folic acid, fluorescein, and
methotrexate were covalently attached to the surface to
NH2 . HCl
Fig. 38. Structure of HPMA copolymer–Dox conjugates: (a) hyrazone bond-containing spacer; (b) cis-aconityl group-containing spacer. Polymer–
Dox conjugates containing side chains of hydrazone-bound Dox moieties were attached via single-amino-acid or longer oligopeptide spacers
(reproduced from Ref. ).
J. Khandare, T. Minko / Prog. Polym. Sci. 31 (2006) 359–397385
provide targeting, imaging, and intracellular drug
In other research, PAMAM dendrimer generation 3
(G3) with 32 amino terminal groups and 5-acetyl
salicylic acid (ASA) pro-moiety containing two
different azo linkers PABA or PAH were synthesized
. The release of drug from the conjugates in vitro
was compared to sulfasalazine (Fig. 40). Various
applications of dendrimers have yet to be explored for
a very broad range of therapeutic areas. Simultaneous
conjugation of drug and targeting moiety on dendrimers
has been popular. Ester-terminated polyether hydrazide
groups were introduced on to the surface of the
dendrimers by reacting with the hydrazine moiety
(Fig. 41). In addition, folate residues were conjugated
to the hydrazide chain ends of the dendrimers by direct
condensation with folic acid in the presence of a
condensing agent or by reaction with an active ester
derivative offolic acid. Complete fictionalization of the
terminal hydrazide groups was achieved for both the
first and the second generation dendrimers with four
and eight hydrazide groups. These functionalized
dendrimer conjugates were prepared as model drug
carriers with potential tumor cell specificity.
One of the key features of the dendrimer is the
nanosize vehicle, which can be conjugated to the drug
or bioactive agents to obtain a high payload per mole
of the dendrimer. This created device has the
potential to ‘bombard’ multiple drug molecules and
other agents simultaneously inside the cell. Recently,
the high payload of an average of w50 molecules of
ibuprofen were conjugated per dendrimer. In addition,
the pharmacological activity was measured by
inhibition of prostaglandin secretion . In spite
of the high payload capacity of conjugation,
dendrimers seem to conjugate with only 4–5 drug
As previously discussed, lower drug conjugation
ratios can also be obtained with most of the drug
molecules . As many as 12 steroid molecules of
methylprednisolone (MP) per molecule of generation 4
(PAMAM) were obtained (Fig. 42). In the first
experiment,glutaric acid was coupled to MP to enhance
the reactivity of the drug and as a spacer molecule. This
modified MP was further conjugated with –hydroxyl
terminal dendrimer using DCC coupling agent. By
lowering the steric hindrance and using a spacer
payload of the drug conjugation was increased. Even
at short intervals of time, this conjugate demonstrated
comparable therapeutic activity as to that of the free
Fig. 39. Comparison of main mechanisms of antitumor action of high molecular weight HPMA-copolymer bound and low molecular weight free
J. Khandare, T. Minko / Prog. Polym. Sci. 31 (2006) 359–397 386
Fig. 40. StructuresofPAMAMdendrimerG3 (a), PAMAM–PABA–SAconjugate(b), andPAMAM–PAH–SAconjugate (c) (reproducedfrom Ref.
J. Khandare, T. Minko / Prog. Polym. Sci. 31 (2006) 359–397387
Dendrimers have also been considered to be
prevalent nanovehicles for the delivery of bioactives.
Further research is needed to design, synthesize and
evaluate these nanovehicles to measure their biode-
gradable ability, cellular uptake, release response,
hemolytic effect, and protein interactions.
6. Critical aspects of polymer conjugation
6.1. Structure–activity relationship of conjugates (SAR)
The structure–activity relationship (SAR) means the
effect of a drug, in its conjugated form, on an animal,
plant or the environment as it relates to its molecular
structure. This type of relationship may be assessed by
considering a series of molecules, making gradual
changes to them, then noting the effect on the biological
activity of each change. Alternatively, it may be
possible to evaluate a large body of toxicity data
using in vitro and in vivo assays. Very few reports
suggest the differences in SAR due to variations of the
conjugated sites of a drug with the polymer. Such
studies are possible if a drug candidate has different
sites for conjugation and their activity mechanisms are
established. The drug, Methotrexate (MTX), is an ideal
candidate for these studies, as it has two –COOH
groups available for the covalent linkage with the
polymeric carrier. The drug activity is relatively
maintained when the gamma-carboxyl is chemically
modified, whereas the alpha-carboxyl has much less
bulk tolerance .
Recently, design and synthesis of dextran–pep-
tide–MTX conjugates for tumor-targeted delivery of
chemotherapeutics via the mediation of matrix
metalloproteinase II (MMP-2) and matrix metallo-
proteinase IX (MMP-9) was reported . The
linker chemistry and the backbone charge were
optimized to allow high sensitivity of the conjugates
toward the targeted enzymes (Fig. 43). The Pro-Val-
Gly-Leu-Ile-Gly conjugate carriers were used as the
peptide linker. The charge on the dextran backbone
was neutralized. In the presence of enzymes, the
peptide was cleaved and peptidyl methotrexate was
released. Cytotoxicity assays concluded that the
released product was effective in inhibiting the
proliferation of tumor cells but the potency was
lower than the free MTX. It was assumed that the
drug was not released in the original free form.
Further study is necessary for the SAR of drugs in
conjugated form with polymers.
6.2. Steric hindrance
Steric hindrance describes how molecular groups
interfere with other groups in the structure or other
molecules during chemical conjugation. This effect is
Fig. 41. Scheme for conjugation of folic acid to hydrazide-terminated dendritic macromolecules (reproduced from Ref. ).
J. Khandare, T. Minko / Prog. Polym. Sci. 31 (2006) 359–397 388
Fig. 42. Schematic for dendrimer–glutaric acid–methylprednisolone conjugate. As much as 12 steroid molecules of methylprednisolone (MP) per
molecule of generation 4 polyamidoamine–OH terminated dendrimer (PAMAM) were obtained (reproduced from Ref. ).
J. Khandare, T. Minko / Prog. Polym. Sci. 31 (2006) 359–397389
Fig. 43. Schematic synthesis procedure of dextran–peptide–methotrexate (MTX) conjugate (i) methotrexate with alpha carboxyl protected MTX
(OtBu); (ii) peptide on O-bis-(aminoethyl)ethylene glycol trityl resin; (iii) jeffamine–peptide–MTX (OtBu); (iv) carboxymethyl dextran with
degree of substitution of CM groups at w50% and nominal Mw70,000; (v) ethanolamine; (vi) modified dextran–peptide–MTX (OtBu) with degree
of modification at w50% and degree of drug loading; (vii) modified dextran–peptide–MTX with EDC in H2O; 2B: charge neutralization by
EDC/ethanolamine in excess .
J. Khandare, T. Minko / Prog. Polym. Sci. 31 (2006) 359–397390
due to the interaction of molecules as dictated by their
shape and/or spatial relationships. For example,
molecular atoms that have affinity for one another
may not be at an appropriate distance to attract each
other due to their shape, or they may have other atoms
blocking them. The macroscale architecture of poly-
mers causes steric hindrance for covalent conjugation
with drugs in general, and large peptides molecules in
particular. Steric hindrance drives chemical confor-
mations and may affect the chemical conjugation with
bulkier, unstable molecules. Therefore, a conjugation
reaction involving polymers, peptides and unstable
molecules requires methodologies to reduce this effect.
The most preferred method to decrease steric
hindrance has been to alter the synthesis approach
either by incorporating a spacer arm or by increasing
the reactivity of the polymer or biomolecules ,
including examples cited in the following. An efficient
method for synthetic carbohydrate conjugate vaccine
was reported for the synthesis of sialyl Tn (STn)–
keyhole limpet hemocyanin (KLH) conjugate using a 4-
hydrazide (MMCCH) as a spacer arm to decrease the
steric hindrance during conjugation . It was
observed by the authors that STn(c) conjugation by
direct reductive amination, only 30–100 STn(c) haptens
molecules were conjugated per KLH. This was
attributed to the steric hindrance caused by the large
STn(c) molecules. To over come this problem the
authors introduced an MMCCH spacer arm using the
bifunctional cross-linker molecule MMCCH. The
STn(c)–MMCCH–KLH conjugate resulted to form a
higher epitope density than was possible with STn(c)–
KLH without the spacer arm. When the two conjugates
were used to immunize mice, the STn(c)–MMCCH–
KLH conjugate was found to elicit a higher titer
antibody response than STn(c)–KLH. The higher
antibody titer was probably due to the higher epitope
ratio but may also be due to qualitative issues related to
the carbohydrate epitopes available to the immune
During bioconjugation, high molecular weight
biomolecules and polymers exhibit steric hindrance
for the reactions. This is especially true for the linear
polymers, in general, and dendrimers in particular.
Therefore, the hindrance must be reduced either by
incorporation of the spacer molecule or by increasing
the reactivity of the bioconjugating moiety. Instead of
conjugating two large molecules directly, one may be
reacted first with small, reactive spacer arm moiety to
increase the final reactivity. Further, the resultant
conjugate can be coupled with the second molecule
. Schematic steric hindrance exhibited by different
architectures of polymers is represented by Fig. 2a–d.
Few researchers have studied the effect of steric
hindrance in conjugation of biomolecules to polymers.
Several studies do suggest the elucidation of spacer
effects in ligand–protein binding . Crowding of
functional groups and steric hindrance may lead to
lower conjugate ratios with unreacted polymers. There
is further need to understand the role of steric hindrance
in bioconjugation to enhance the efficiency of
6.3. Enhanced reactivity of polymers by incorporation
The reactivity of functional polymers to couple with
other biomolecules, which may be low, could be
enhanced by first conjugating the polymer with reactive
bis functional molecules. The resulting polymer–spacer
conjugate moiety often enhances the reactivity and
decreases steric hindrance for further coupling with
drugs or biomolecules . Commonly used as
spacers for conjugating polymers with drugs and
other biomolecules include a-amino acids such as
glycine, alanine, and serine. The a-amino acids in
peptides and proteins (excluding proline) consist of a
carboxylic acid (–COOH) and an amino (–NH2)
functional group attached to the same tetrahedral
carbon atom which extends diversity for conjugation
with hydroxyl, carboxyl or amino groups of polymer or
biomolecule. Moreover, amino acid based spacers are
short-chained, reactive, biocompatible and may release
the active agent from the conjugate. Di-functional
amino acids such as 6-amino caproic acid (6-ACA) or 4
amino butyric acids (4 ABA) have been used as spacer
arms between the polymers and the ligands for
applications in biotechnology . Polymer carriers
used for conjugation with anticancer drugs are often
linked by polypeptides [111,112]. The authors reported
conjugates of alkylating agent mitomycin C (MMC)
with poly[N5-(2-hydroxyethyl)-L-glutamine] (PHEG)
using oligopeptide spacers designed predominantly
for enzymatic degradation. Hydrolytic stability studies
carried out in buffers at pH 5.5 and 7.4, in serum,
demonstrated that MMC was released from the
conjugates with a rate dependant on the detailed
structure of the spacer (Fig. 44). Most of the
conjugation methods involve the use of spacers,
which provide chemical flexibility for coupling
biological compounds to the polymers.
J. Khandare, T. Minko / Prog. Polym. Sci. 31 (2006) 359–397391
6.4. Targeting of polymeric drugs
Conjugation of low molecular weight drugs to high
molecular weight carriers results in high molecular
weight prodrugs. Such conjugation substantially
changes the mechanisms of cellular drug entrance.
While small molecular weight drugs enter cells
primarily by diffusion, high molecular weight drugs
are internalized mainly by endocytosis. Endocytosis is
a much slower internalization process when compared
with simple diffusion and generally requires much
higher drug concentration outside the cell to produce
the same cellular effect as corresponding low molecular
weight drug. Therefore in most cases, excluding those
when conjugation of a drug to a polymer improves its
bioavailability (i.e. increasing drug solubility), high
molecular weight drugs displayed lower specific
activity, compared to low molecular weight drugs, yet
required a higher dose. For example, polymeric
anticancer drugs are generally less toxic when
compared with free drugs yet require substantially
higher concentrations inside the tumor to kill the same
amount of cancer cells as low molecular weight drugs.
Compensation for this decrease in drug efficacy can
be achieved by targeting a polymeric drug to the
specific organ, tissue and/or cell. The main advantages
of such targeting are the prevention of negative side
effects of regular drugs with an enhancement of the
drug uptake by the targeted cells. Targeting a drug
specifically to certain type of cells and/or specific
organelles inside the cells permits the internalization of
substances with low cellular permeability by endocy-
tosis and drug release in targeted organelles (e.g.
lysosomes, mitochondria, nucleus, etc.). As a result, the
targeted drug acts like a ‘magic bullet’ selectively
killing the villain and sparing the innocent. The
targeting of a drug delivery system is especially
important for cancer chemotherapy, mainly because
of the high toxicity of anticancer drugs currently being
used. Two approaches are generally used to target
polymeric anticancer drugs to the tumor or cancer cells:
(1) passive and (2) active targeting [113,114]. Ligand-
targeted therapeutics (LTTs) is a considered to be a
successful mean for improving the selective toxicity of
anticancer therapeutics. A radioimmunotherapy, an
immunotoxin and an immunoconjugate have each
received clinical approval and over 100 ligand-targeted
therapeutics are currently in clinical trials .
6.4.1. Passive tumor targeting
Passive targeting approaches include: (1) the
enhanced permeability and retention (EPR) effect, (2)
the use of special conditions in tumor or tumor-bearing
organ and (3) topical delivery directly to the tumor. The
EPR effect was first described by Maeda and co-
workers [115–118]. The EPR effect is the result of the
increased permeability of the tumor vascular endo-
thelium to circulating macromolecules combined with
limited lymphatic drainage from the tumor interstitium.
Low molecular drugs coupled with high molecular
weight carriers are inefficiently removed by lymphatic
drainage and therefore accumulate in tumors.
Fig. 44. Structure of the biodegradable PHEG carrier bearing mitocin (MMC) bound via an oligopeptide spacer (reproduced from Ref. ).
J. Khandare, T. Minko / Prog. Polym. Sci. 31 (2006) 359–397 392
Theoretically, any high molecular water-soluble drug
carrier should show such an effect, including water-
soluble polymers, liposomes, etc. The existence of the
EPR effect was experimentally confirmed for the many
types of macromolecular anticancer drug delivery
systems . Passive targeting increases the concen-
tration of the conjugate in the tumor environment and
therefore ‘passively’ forces the polymeric drug to enter
the cells by means of the concentration gradient
between the intracellular and extracellular spaces and
therefore is not very efficient. The more efficient way to
provide targeting is so-called ‘active targeting’ .
6.4.2. Active targeting
Active targeting of a drug delivery system is
usually achieved by adding, to DDS, a targeting
component that provides preferential accumulation of
the whole system or drug in a targeted organ, tissue,
cells, intracellular organelles or certain molecules in
specific cells. The active targeting approach is based
on the interactions between a ligand and a receptor or
between a specific biological pair (e.g. avidin–biotin,
antibody–antigen, lectin–carbohydrate, etc.). In most
cases, a targeting moiety in a polymeric drug delivery
system is focused on the specific receptor or antigen
overexpressed in the plasma membrane or intracellu-
lar membrane of the targeted cells. The targeted
anticancer LHRH–PEG–CPT conjugate described
above is an example of such targeted anticancer
drug delivery system . In this system, LHRH
peptide is used as a targeting moiety to the
corresponding receptors overexpressed in several
cancer cells, PEG polymer—as a carrier and CPT—
as an anticancer drug. The use of a targeting moiety
not only provides targeting of the whole system to the
targeted cells limiting adverse side effects, but also
facilitates cellular uptake of the whole conjugate by
receptor-mediated endocytosis. The interaction of
such molecules with their receptor initiates receptor-
mediated endocytosis; an active process that requires
a significantly lower gradient of internalized sub-
stance across the plasma membrane when compared
with simple endocytosis. By conjugating the drug
delivery system with molecules that are recognized
by extracellular plasma membrane receptors, it is
possible to enhance the influx of whole conjugate by
receptor-mediated endocytosis. This process can be
divided into several distinct steps as schematically
presented in Fig. 45. Interaction of a targeted carrier
with a corresponding receptor leads to the engulfing
of the plasma membrane inside the cells and the
formation of a coated pit. The pit then pinches off
from the plasma membrane and forms an endocytic
vesicle and endosomes-membrane-limited transport
vesicles with a polymeric delivery system inside.
Transport inside the membrane-coated endosome
prevents drugs from degradation by cellular detox-
ification enzymes and therefore preserves its activity.
Endosomes move deep inside the cell and fuse with
lysosomes forming secondary lysosomes. If the bond
Increase in cytotoxicity
Fig. 46. Cytotoxicity of CPT (1), CPT–PEG (2), CPT–PEG–LHRH
(3) in A2780 human ovarian carcinoma cells. IC50 doses were
measured using a modified MTT assay. Cells were incubated 48 h
with 45 different equivalent CPT concentrations. MeansGSD are
shown (modified from Ref. ). *P!0.05 when compared with
CPT.†P!0.05 when compared with CPT–PEG.
Fig. 45. Main stages of receptor-mediated endocytosis: (1) interaction
of targeting moiety with corresponding receptor and internalization of
drug delivery system (DDS); (2) transport of DDS in membrane-
limited organelles; (3) fusion with lysosomes; (4) degradation of a
spacer and release of the drug into cellular cytoplasm.
J. Khandare, T. Minko / Prog. Polym. Sci. 31 (2006) 359–397393
between the targeting moiety (or targeted carrier) or
spacer is designed in such a way that lysosomal
enzymes are capable of breaking it, the drug is
released from the drug delivery complex and might
exit a lysosome by diffusion. As a result of receptor-
mediated endocytosis and transport inside cells in
membrane-limited organelles, targeted polymeric
drugs no longer require a higher concentration
gradient across the plasma membrane. They are
protected from degradation inside the cell and
therefore the specific efficacy is substantially higher
than non-targeted conjugates and free drugs. For
instance, targeted LHRH–PEG–CPT conjugate exhib-
ited several order of magnitudes higher cytotoxicity
against cancer cells when compared with non-targeted
CPT–PEG conjugate and free Camptothecin (Fig. 46)
. Analysis of receptor mediated endocytosis
showed two main limiting critical points of this
process, namely interaction of receptors with corre-
sponding ligands and drug release from drug delivery
system and lysosomes. Both of them should be
addressed when developing a targeted drug delivery
system for effective treatment.
Use of polymeric materials and their implications in
delivering biocomponents seems to be crucial in
therapeutics. It is well accepted that the bioactive
components can be delivered more efficiently by being
converted into a prodrug form. However, such
polymeric bioconjugates need extensive structural and
clinical studies. To date, poly(ethylene glycol) con-
tinues to be a highly investigated polymer for the
covalent modification of biological macromolecules
and has evolved as a successful candidate for many
pharmaceutical and biotechnical applications. Modifi-
cation of biological macromolecules, anticancer drugs,
peptides, and proteins in the form of prodrugs are of
extreme importance in therapeutics. Selection of a
suitable polymer and methodology to conjugate the
same with different bioactive components is a critical
step. The prodrug conjugation approach is a fascinating
field and appears to have a bright future in therapeutics.
This research was supported in part by National
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