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
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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