Review

PEGylation in anti-cancer therapy: An overview

Prajna Mishra a, Bismita Nayak b, R.K. Dey a,*a Centre for Applied Chemistry, Central University of Jharkhand, Ranchi 835 205, Jharkhand, Indiab Department of Life Science, National Institute of Technology, Rourkela 769 008, Odisha, India

A R T I C L E I N F O

Article history:

Received 1 July 2015

Received in revised form 20 August

2015

Accepted 26 August 2015

Available online 14 September 2015

A B S T R A C T

Advanced drug delivery systems using poly(ethylene glycol) (PEG) is an important devel-

opment in anti-cancer therapy. PEGylation has the ability to enhance the retention time of

the therapeutics like proteins, enzymes small molecular drugs, liposomes and nanoparticles

by protecting them against various degrading mechanisms active inside a tissue or cell, which

consequently improves their therapeutic potential. PEGylation effectively alters the phar-

macokinetics (PK) of a variety of drugs and dramatically improves the pharmaceutical values;

recent development of which includes fabrication of stimuli-sensitive polymers/smart poly-

mers and polymeric micelles to cope of with the pathophysiological environment of targeted

site with less toxic effects and more effectiveness. This overview discusses PEGylation in-

volving proteins, enzymes, low molecular weight drugs, liposomes and nanoparticles that

has been developed, clinically tried for anti-cancer therapy during the last decade.

© 2016 The Authors. Production and hosting by Elsevier B.V. on behalf of Shenyang Phar-

maceutical University. This is an open access article under the CC BY-NC-ND license

(http://creativecommons.org/licenses/by-nc-nd/4.0/).

Keywords:

PEG

PEGylation

Targeted drug delivery

Polymers

1. Introduction

Cancer is one of the leading causes of death worldwide. Me-tastases are the primary cause of death from cancer. Cancercells proliferate at much faster rate than the normal cells. Theavailable traditional cancer chemotherapy is not essentially se-lective as it depends on the kinetics of the cell growth.Targetedcancer therapies are expected to be more effective and ben-eficiary in comparison to available conventional treatmentprocedures.

The last few decades of research in the particular area arefocused on exploring the treatment of cancer at its molecu-lar level.This will be helpful in developing better therapeutics.Polymer therapeutics is establishing as an innovative and

reliable method for its ability to conjugate with protein,enzymes, nanoparticles, liposomes and low molecular weightdrugs. In this regard, polyethylene glycol (PEG), a water-soluble and biocompatible polymer, is the most commonly usednon-ionic polymer in the field of polymer-based drug deliv-ery [1]. Passive targeting with PEGs in combination with activetargeting (entry into the tumor cell via ligand receptor, antigenantibody interaction) delivery systems has been effectively em-ployed to achieve better therapeutic index of anti-cancer drugs.Further, with the introduction of stimuli-responsive chemi-cal moiety in PEGylated prodrugs, the sensitiveness of the drugmolecule toward the pathophysiological environment of tumorcells in vivo evolved as a new era of site-specific targeted drugdelivery system. Hence this overview deals with the develop-ment and recent advancement in various aspects of PEGylation

* Corresponding author. Centre for Applied Chemistry, Central University of Jharkhand, Ranchi 835 205, Jharkhand, India.Tel.: +91 8987727378;fax: +91-671-2306624.

E-mail address: rkdey@rediffmail.com; ratan.dey@cuj.ac.in (R.K. Dey).Peer review under responsibility of Shenyang Pharmaceutical University.

http://dx.doi.org/10.1016/j.ajps.2015.08.0111818-0876/© 2016 The Authors. Production and hosting by Elsevier B.V. on behalf of Shenyang Pharmaceutical University. This is anopen access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

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in cancer treatment and its future prospective in a compre-hensive way.

2. PEGylation and its significance

The technique of covalently attaching polyethylene glycol (PEG)to a given molecule is known as “PEGylation” and is now a well-established method in the field of targeted drug deliverysystems. The general structure of monomethoxy PEG (mPEG)can be represented as CH3O–(CH2-CH2O)n–CH2-CH2–OH. At thebeginning of PEG chemistry, in the late1970s, Professor FrankDavis and his colleagues had shown that the immunologicalproperties as well as the stability of bovine serum albumin andbovine liver catalase can be successfully altered by cova-lently linking them to methoxy PEG (mPEG) [2,3] using cyanuricchloride as an activating agent. The process of PEGylation canbe extended to liposomes, peptides, carbohydrates, enzymes,antibody fragments, nucleotides, small organic molecules andeven to different nanoparticle formulations [4–8]. mPEG is themost useful unit for polypeptide modification [9]. Now severalderivatives of PEG molecules are available that vary in mo-lecular weight and structure, such as linear, branched, PEGdendrimers and more recently multi-arm PEGS [9].The first stepof PEGylation is to activate PEG by conjugating a functional de-rivative of PEG at one or both the terminals of PEG chain.

PEGylation conjugation techniques can be classified into twocategories: i) first-generation random PEGylation, and ii) second-generation site-specific PEGylation. Thanks to the secondgeneration PEGylation processes that resulted in well-definedconjugated products with improved product profiles over thoseobtained through non-specific random conjugations.

Irreversibly conjugating PEGs had some adverse effects onthe specific biological activity of many therapeutics. Thus, tominimize the loss of activity, a reversible (or releasable prodrug)PEGylation concept has been formulated. Reversible PEGylationconcept deals with attachment of drugs to PEG derivativesthrough cleavable linkages (Fig. 1). The release of drug occursby therapeutic agents through enzymatic, hydrolytic cleav-age or reduction in vivo at a predetermined kinetic rate overa time period [10].

The objective of most PEG conjugation techniques aims atincreasing the circulation half-life without affecting activity.It is to be noted that the distinct advancement in the PEG con-jugation processes and diversity in the nature of the PEGs usedfor the conjugation has attributed to the increased demand forPEGylated pharmaceutical products.

PEGylation enhances the therapeutic efficacy of the drugsby bringing in several advantageous modifications over the non-PEGylated products.The systematic classification is illustratedin Fig. 2. Increase in the serum half-life of the conjugate is themajor way of enhancing therapeutic potential of the PEGylatedconjugate. PEGylation prolongs the circulation time of conju-gated therapeutics by increasing its hydrophilicity and reducingthe rate of glomerular filtration [11,12]. Few factors such as pro-tection from reticuloendothelial cells, proteolytic enzymes anddecreased formation of neutralizing antibodies against theprotein by masking antigenic sites by formation of a protec-tive hydrophilic shield are the key components of PEG moleculethat attributes to the improved pharmacokinetic profile (PK)of the conjugates [13–16]. It has also been reported thatPEGylation increases the absorption half-life of subcutane-ously administered agents and is associated with a decreasedvolume of distribution [11]. PEG is a non-biodegradable polymer

PEG SPACER/LINKER Drug PEG Linker Drug

(PEG-Prodrug)

Fig. 1 – PEG-prodrug.

Fig. 2 – Significance of PEGylated therapeutics.

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that puts limits on its use. It has been shown that PEGs (upto molecular weight 20 kDa) is primarily excreted through therenal system, whereas higher molecular weight PEG chains geteliminated by fecal excretion [17]. PEGylation proved to be themost promising approach for increasing the serum half-life ofthe conjugated therapeutics, which is related to enhance-ment of efficacy of the conjugate. However, PEGylation imposescertain disadvantages on liposomes, especially for the deliv-ery of genes and nucleic acids in anti-cancer therapy as itssurface hydrophilic shield reduces the cellular uptake and im-proves the stability of the lipid envelope, and the process resultsin poor endosomal escape via membrane fusion and degra-dation of cargos in lysosomes [18,19]. Hence the use of PEG ingene and nucleic acid delivery to cancer cell is referred to as“PEG dilemma” [20]. The issue can be efficiently addressed bydesigning pH-sensitive and tumor-specific targeted PEGylatedtherapeutics [21–23].

2.1. Role of PEGylation in passive and active targeting ofdrugs

Passive targeting drug delivery technique, as illustrated in Fig. 3,mostly depends upon the concentration gradient between theintracellular and extracellular space, created due to high con-centration of the drug in the tumor area [24]. PEG conjugatestakes the advantage of enhanced permeation and retention(EPR) effect executed by the tumors and gets accumulated inthe pathophysiological environment of tumor vessels throughleaky vasculature and poor lymphatic drainage. However, thiseffect cannot be studied with low molecular weight drugs thatfreely extravagate causing systemic toxicity, and this is a size-dependent effect. PEGylation increases the solubility, size,molecular mass and serum stability of the drugs. For all these

reasons, PEGylation is considered to be one of the best methodsfor passive targeting of anticancer therapeutics.

The concept of active targeting of drugs is based on the ideaof conjugating drug molecules to targeting entities (antibod-ies, ligands, etc.) for specific interaction with the structurespresent on the cell surface for targeted delivery of the anti-cancer agent [25,26]. The fate of the pro-drug is dictated bythe targeting molecule and the linker molecule present on thepro-drug. The targeting moiety essentially decides the type ofcancer cell for the act of therapeutics. Further, depending onthe linker molecule, the drug gets entry into the tumor cell byeither of the two ways: (i) receptor-mediated internalizationof the whole pro-drug by endocytosis and subsequent degra-dation by endosomal/lysosomal pathway (Fig. 4), or (ii) receptor-independent internalization of the drug into targeted cells afterextracellular cleavage of the pro-drug (Fig. 5) [27]. PEGylatedpro-drugs can be efficiently conjugated to targeting moietiesby different conjugation chemistry in order to achieve the goalof active targeting.The targeted delivery of the PEGylated drugsat the desired site causes high bioavailability and low sys-temic toxicity.

3. PEGylated proteins in anti-cancer therapy

PEGylation of proteins is a well-established method in the phar-maceutical field, but the significance of PEGylated peptides andproteins for anti-cancer therapy has only been realized in thelast several years as more and more PEG conjugates make itto late-phase clinical trials. Enzymes, monoclonal antibodiesand cytokines are the three major class of proteins used in an-ticancer therapy or as adjuvant therapy (Table 1).

Fig. 3 – A schematic illustration of passive targeting with acid-sensitive PEG-prodrugs that cleave in the extracellular space.

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3.1. PEGylated monoclonal antibody fragment

In the field of anti-cancer therapy, monoclonal antibodies rep-resent the major class of protein therapeutics. Antibodies actby binding to the specific antigens/cell surface receptors. Thistask is taken care by the fragment antigen-binding (Fab’) regionon an antibody. Depending upon the receptor and the bindingsite on the receptor against which the antibody is designed,it can either activate cellular signaling pathways leading toapoptosis, cell growth arrest, or block the pathways leading tocell growth that eventually causes tumor cell death (apopto-sis). This event is illustrated pictorially in Fig. 6. The major

drawback associated with Fab’s antibody fragment is its shortserum half-life as it lacks the Fc region of the antibody thatlimits its potential as a therapeutic agent. Hence, suitablePEGylation methods and PEGs are used to ensure minimal lossof the antibody-antigen/cell surface receptor interaction keepingin view the enhancement of serum half-life. It has been re-ported that the hinge region cysteine residues on immuno-globulin G (IgG antibody isotype) Fab’ antibody fragments cantolerate attachment of one or two PEG moieties (up to a totalof 40 kDa molecular weight) with little effect on antigen bindingaffinity. This process also enables significant increase in thehalf-life of the circulating plasma antibodies by reducing the

Fig. 4 – A schematic illustration of receptor mediated internalization and endosomal/lysosomal degradation of the pro-drugduring active targeting.

Fig. 5 – A schematic illustration of Active Targeting with acid sensitive PEG- prodrugs which cleaves in the extracellularspace.

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glomerular filtration and lower immunogenicity than the parentIgG [28].

The example of use of PEG-antibody fragment angiogen-esis inhibitor (CDP791) is illustrated as follows: CDP791PEGylated diFab antibody fragment antagonizes the effect ofvascular endothelial growth factor receptor-2 (VEGFR-2), whichis a prominent angiogenesis stimulatory molecule respon-sible for tumor progression. The short plasma half-life of theunmodified CDP791 antibody fragment, which lacks Fc region,has a low molecular weight and responsible for low therapeu-tic index. PEGylation of the cysteine amino acid present at theC-terminus of the native antibody could be able to resolve thisissue by reducing its kidney clearance. This is demonstratedby the clinical studies for patients with colorectal, ovarian, renalcancer or other tumors [29].

3.2. PEGylated cytokines

Cytokines represent another class of protein therapeutics em-ployed mainly as adjuvant therapy in classical anti-cancer

chemotherapy protocols either to control or bring improve-ments in patient conditions. These small secreted proteinsbelong to the immunotherapy category and mobilizes the body’simmune system to fight cancer. The process is illustrated pic-torially in Fig. 7.

3.2.1. PEG-interferon-alpha conjugatesThis process is illustrated as PEG-interferon-α2b (PEG-INTRON®/Sylatron™) and PEG-interferon-α2a (Pegasys®), which arediscussed as follows:

3.2.1.1. PEG-interferon-α2b (PEG-INTRON®/Sylatron™). ThePEGylated version of interferon-α2b was synthesized by con-jugating interferon-α2b, with a single chain 12 kDa PEG-SC viaa urethane bond [30]. It displayed a half-life of 27–37 h with10-fold lower clearance and minor change in the volume of dis-tribution in comparison to native form [31]. Based upon theoutcome of clinical studies in the year 2011, the PEGylated drugpeginterferon alfa-2b (PEG-IFN) got FDA approval for adju-vant treatment of melanoma patients with microscopic or gross

Table 1 – PEG protein/enzyme conjugates in clinical development as anticancer therapy.

Trade name Conjugate Protein/Enzyme FDA approved date/clinical trial status and use

Sylatron™ PEG-interferon α-2b PEG-interferon α-2b March 9, 2011, approved as adjuvant therapy for resected stage IIImelanoma

Pegasys® PEG-interferon α-2a Interferon α-2a Phase I for melanoma and phase II as for chronic myelogenous leukemiaNeulasta® PEG-filgrastim G-CSF(granulocyte-colony

stimulating factor)2002, to treat neutropenia during chemotherapy

Oncaspar® PEG-asparaginase Asparaginase February 1994, acute lymphoblastic leukemia, and in July 24, 2006 first-linetreatment for acute lymphoblastic leukemia

Fig. 6 – (a) IgG full length antibody and (b) PEGylated IgG Fab’ antibody fragment–cell surface receptor interaction and itsoutcome.

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nodal involvement following definitive surgical resection in-cluding complete lymphadenectomy [32]. Sylatron™ is anotherbrand name for peginterferon alfa-2b exclusively approved byFDA for adjuvant therapy in cancer treatment.

3.2.1.2. PEG-interferon-α2a (Pegasys®). Another PEGylated in-terferon, Pegasys®, is prepared by mono-PEGylation ofinterferon-α-2a with an N-hydroxysuccinimide (NHS) acti-vated 40 kDa branched PEG molecule [33]. PEGylation prolongedthe serum half-life from 3.8 to 65 h, slowing down the clear-ance by more than 100-fold.This has also reduced the volumeof distribution to fivefold with respect to the native interferon-α2b [31]. PEGASYS® was efficient in improving the patientcompliance by enabling once-weekly dosing while maintain-ing acceptable safety, tolerability, and activity profiles in clinicalstudies [34]. Currently, PEGASYS® is under evaluation as ad-juvant therapy for patients with intermediate and high-riskmelanomas [35].

3.2.2. PEG-granulocyte colony stimulating factor(PEG-filgrastim)PEG-filgrastim was synthesized by conjugating a linear 20 kDamPEG-aldehyde derivative to an N-terminal methionine residueof filgrastim through reductive alkylation under mild acidic con-ditions [36,37]. It is to be noted that a single dose of PEG-filgrastim per chemotherapy cycle could be able to reduce therisk of febrile neutropenia significantly with respect to the nativeprotein (11% vs. 19%) [38–40]. Currently, PEGylated-G-CSF(Pegfilgastrim, Neulasta®) is used as an adjuvant therapy forpatients with non-myeloid malignancies receiving myelo-suppressive chemotherapy (bone marrow suppression as a sideeffect of chemotherapy) associated with a 20% risk of febrileneutropenia [41,42].

3.3. PEGylated enzymes in anti-cancer therapy

Therapeutic enzymes represent a growing class of bio-pharmaceuticals, and PEGylation has played a major role in

improving several of these products [43]. Many depletingenzymes are active against tumors. Enzymes’ intrinsic prop-erty of degrading amino acids is essential for cancer cellsexistence. The fate of the tumor cell is dictated by the differ-ent cellular pathways regulated by the substrate (amino acid)to be degraded, and the process of degradation is illustratedin Fig. 8.The normal cells are not affected because the normalcells can synthesize the amino acids for their growth.This situ-ation is particularly the most advantageous aspect of usingdepleting enzymes in cancer therapy (Table 1).Therefore, duringPEGylation procedure, a combination of these enzymes, lowmolecular weight (5–10 kDa) PEGs and random amine conju-gation strategies are employed.

3.3.1. PEG-arginine depleting enzymesArginine is a nonessential amino acid in humans. It has beenreported that arginine deficiency inhibits tumor growth, an-giogenesis and nitric oxide synthesis [44].Two types of argininedegrading enzymes, i.e. i) arginine deiminase (ADI) and ii) ar-ginase (ARG), which can be utilized as antitumor agents, arediscussed below:

3.3.1.1. PEG-arginine deiminase. The PEGylation of argininedeiminase proved to be a better therapeutic approach foranticancer treatment. Among the several PEGylated ADI for-mulations the ADI-PEG20000, formulated by conjugating10–12 chains of 20 kDa PEG with ADI by using the succinimidylsuccinate linker, is proved to be the acceptable one fromin vivo study results [45]. Clinical studies have shown better ef-ficacy of ADI PEG 200,000 in terms of antitumor activity andtolerability [46,47]. Currently, ADI PEG 200,000 versus placebois under phase III clinical trial for advanced hepato-cellular carcinoma. Further, Phase II for acute myeloidleukemia/non-Hodgkin’s lymphoma and Phase I (for meta-static melanoma in combination with cis-platin; for solidtumors in combination with docetaxel) are under trial[48].

Fig. 7 – Representing PEGylated cytokines in anticancer therapy.

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3.3.1.2. PEG-arginase. The depleting enzyme arginase is an en-dogenous protein expressed in humans. The conjugate, PEG-rhArg, has 10 to 12 polymer chains of PEG 5000 per proteinmolecule that is covalently attached via a succinamide pro-pionic acid (SPA) linker. This conjugate remains in fully activecondition [49]. The PEGylated form executes sufficient cata-lytic activity at physiological pH with a prolonged plasma half-life of 3 days in comparison to the native form, which has ahalf-life of several minutes only. Currently, this conjugate isunder phase I/II clinical trials.

3.3.2. PEG-asparagine depleting enzyme(PEG-L-asparaginase)Depletion of asparagine eventually results in leukemic celldeath. Leukemic cells lack the enzyme asparagine synthe-tase, an enzyme required for asparagine synthesis, and dependon the exogenous supply of asparagine for their growth andsurvival.Therefore, asparaginase, the depleting enzyme for as-paragine, plays a critical role as a therapeutic enzyme in treatingacute lymphoblastic leukemia (ALL). Oncaspar is a modifiedform of the enzyme L-asparaginase approved by FDA in 1994.Oncaspar consists of tetrameric enzyme L-asparaginase derivedfrom E. coli, and it is covalently conjugated with approxi-mately 69–82 molecules of monomethoxy polyethylene glycol(mpeg), each having molecular weight of 5 kDa [50]. Oncaspar

proved to be a better treatment option for patients who wereallergic to the native form of the drug. The U.S. Food and DrugAdministration granted approval to pegaspargase (Oncaspar,Enzon Pharmaceuticals, Inc) in July 2006 for the first-line treat-ment of patients with acute lymphoblastic leukemia (ALL) asa component of a multi-agent chemotherapy regimen [51].

4. PEGYLATED low molecular weightanticancer drugs

Various PEGylated low molecular weight anti-cancer drugs arecurrently under development. For example, topoisomerase I in-hibitor camptothecin-based drugs (irinotecan, topotecan, SN38,exetecan, etc.) is reported to be useful in the treatment of manysolid tumors. However, the hydrophobicity of such materiallimits their therapeutic efficacy. Few of the examples, listed inTable 2, are discussed below:

4.1. PEG-SN38 (EZN-2208)

EZN-2208, the product Enzon Pharmaceuticals, Inc, is aPEGylated SN38 (10-hydroxy-7-ethyl-camptothecin (a deriva-tive of camptothecin)). SN38 is the active moiety of CPT-11

Fig. 8 – Representing PEGylated depleting enzymes in anticancer therapy.

Table 2 – PEGylated low molecular weight drugs/liposomal derivatives/thermo-sensitive conjugates and nanoparticles inclinical development as anticancer therapy.

Trade name Conjugate Parent drug FDA approved date/clinical trial status

EZN-2208 PEG-SN38 SN38 (camptothecinderivative)

Phase II, various cancer

NKTR-102Irinotecan

PEG-irinotecan Irinotecan Phase III, metastatic or locally recurrent breast cancer and Phase II,solid tumor malignancies, including ovarian, colorectal, glioma,small cell and non-small cell lung cancers

Doxil (Caelyx) PEG-liposomal doxorubicin Doxorubicin November 1995 for ovarian/breast cancer and Kaposi’s sarcomaThermoDox PEG-thermosensitive

liposomal doxorubicinDoxorubicin Phase III, hepatocellular carcinoma

CALLA 01 Pegylated cyclodextrinnanoparticle

SiRNA In clinical phase I for solid tumors

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(Camptosar®, irinotecan) and reported to be a potenttopoisomerase I inhibitor. In this PEGylated product, the 20-OH group of SN38 was selectively coupled with a 4 arm PEGof 40 kDa through a glycine spacer to preserve the E ring ofSN38 in the active lactone form while leaving the drug 10-OHfree [52]. PEGylation was able to enhance the solubility of SN38by about 1000-fold. In fact, EZN-2208 showed a 207-fold higherexposure to SN38 compared to irinotecan in treated mice [53].The conjugate showed promising antitumor activity both in vitroand in vivo. However, following phase II trial, Enzon Pharma-ceuticals, Inc. announced the discontinuance of its EZN-2208clinical program [54].

4.2. PEG-irinotecan (NKTR-102, Etirinotecan pegol)

NKTR-102 was developed by NektarTherapeutics as a PEGylatedformulation for the treatment of colorectal cancer and othersolid tumors using the architecture of new multi-arm PEGs. Itis the first long-acting topoisomerase I inhibitor. It is synthe-sized by covalently conjugating irinotecan to a four-arm PEG[55]. SN38 (10-hydroxy-7-ethyl-camptothecin, a derivative ofcamptothecin) is the active metabolite of NKTR-102. It is re-ported in both Phase I and II studies for NKTR-102 thatsustained exposure of the active drug was associated withpromising anti-tumor activity [56–58]. Currently NKTR-102 isin Phase III clinical trial for patients with metastatic or locallyrecurrent breast cancer and Phase II clinical trial for patientswith solid tumor malignancies that include ovarian, colorectal,small cell and non-small cell lung cancers [59].

5. PEGylated nanoparticles in anti-cancertherapy

Nanoparticles (NPs) are synthetic materials with dimensionsfrom 1 to 1000 nano-meters. NPs have large payloads, stabil-ity and the capacity for multiple, simultaneous applicationsdue to their unique size and high surface area:volume ratio[60]. Despite these advantages, the major drawbacks associ-ated with NP drug delivery system for clinical studies areassociated with short circulating half-life due to uptake by thereticuloendothelial system (RES) for larger NPs, whereas smallerNPs are subjects to tissue extravazations and renal clearance[61]. Liposomes, solid lipids nanoparticles, dendrimers, poly-mers, silicon or carbon materials, and gold and magneticnanoparticles are examples of nano-carriers that have beenstudied as drug delivery systems in cancer therapy. There-fore, surface modification of the nanoparticles with PEGs ofvarious chain length, shape, density, molecular weight and in-corporation of different targeting moieties (ligands, antibodies,etc.) is emerging as a more promising and technologically ad-vanced drug delivery system in anti-cancer therapy [62–64].There are currently more than 35 US FDA-approved PEGylatedNPs, with a larger number in preclinical studies for both imagingand therapy [8]. Among several PEGylated nanoparticle for-mulations for anticancer therapy, liposomes have been mostextensively studied.

5.1. PEGylated liposomes in anticancer therapy

Liposomes are spherical, self-closed structures formed by oneor more concentric lipid bilayers with an encapsulated aqueousphase in the center and between the bilayers composed ofnatural or synthetic lipids [65]. The development of long-circulating liposomes with inclusion of the synthetic polymerpoly-(ethylene glycol) (PEG) in liposome composition could beable to solve the issue of low serum half-life associated withliposomes. PEG can be incorporated on the liposomal surfacein a number of ways. However, anchoring the polymer inthe liposomal membrane via a cross-linked lipid, PEG-distearoylphosphatidylethanolamine [DSPE], is reported to bethe most widely accepted method [66,67]. Preclinical studieswith PEGylated liposomes reported that the cytotoxic agentsentrapped in PEGylated liposomes tend to accumulate in tumors[68]. However, recent preclinical studies of anti-cancer drug en-closed in PEGylated liposomes in rodents and dogs have shownthe rapid blood clearance of the pegylated drug carrier systemdue to the increased anti-PEG-IgM production [69,70]. Anexample of PEGylated liposomal formulations, PEGylated li-posomal doxorubicin (PLD), and most extensively studied, isdiscussed below:

5.1.1. Doxil (PEGylated liposomal doxorubicin)DOXIL is the trade name for PEGylated liposomal doxorubi-cin formulated to achieve better drug efficacy for cancerchemotherapy.This product contains doxorubicin (Adriamycin)enclosed in an 80–90 nm size uni-lamellar liposome coated withPEG. The modification increases the circulatory half-life of thedrug leading to its enhanced bioavailability at the tumor site[71,72]. PEGylated liposomal doxorubicin has fewer side effectson healthy cells than regular doxorubicin [73,74]. PLD has im-proved pharmacokinetic features, such as long circulation timeof about 60–90 h for doses in the range of 35–70 mg/m2 in pa-tients with solid tumors. After PLD administration, nearly 100%of the drug in the plasma remains in the encapsulated form.Moreover, in comparison to free doxorubicin PLD, plasma clear-ance is dramatically slower and its volume of distributionremains very small, which is roughly equivalent to the intra-vascular volume [75–77]. After obtaining approval from FDA,PEGylated liposomal doxorubicin (PLD) (DOXIL/Caelyx) is cur-rently used to treat Kaposi’s sarcoma and recurrent ovariancancer (Table 2).

6. PEGylated smart polymers in anticancertherapy

Smart polymers are defined as polymers that undergo revers-ible large, physical or chemical changes in response to smallexternal changes in the environmental conditions, such as tem-perature, pH, light, magnetic or electric field, ionic factors,biological molecules, etc. Smart polymers show promising ap-plications in the biomedical field as delivery systems oftherapeutic agents [78]. Among various smart polymers cur-rently in use in biomedical field of research, the temperaturesensitive systems are the most studied systems. The greatertherapeutic index of the targeted drug delivery systems can

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be achieved by adjusting the transition temperature (Tt) of ther-mally responsive polymers, i.e., between body temperature(37 °C) and the temperature approved for mild clinical hyper-thermia (42 °C) [79]. Within the temperature ranges, thesepolymers facilitate tissue accumulation by localizing the ag-gregation of systemically delivered carriers to the heated tumorvolume [80,81]. For example, ThermoDox, a temperature-sensitive doxorubicin-loaded PEGylated liposome (DPPC),releases encapsulated doxorubicin at elevated tissue tempera-ture. DPPC has a transition temperature of 41.5 °C, which makesit suitable for temperature-sensitive technology [82]. The tem-perature can be achieved by radiofrequency ablation technique.For ThermoDox, the concentration of the drug is up to 25 timesmore in the treatment area than IV doxorubicin, and severalfold the concentration of other liposomally encapsulated doxo-rubicins [82,83]. Currently, it is under phase III clinical trial forhepatocellular carcinoma (Table 2).

7. PEGylated polymeric micelles in anti-cancer therapy

Polymeric micelles are colloidal dispersions prepared by block-copolymers, consisting of hydrophilic and hydrophobicmonomer units. Self-assembling amphiphilic polymeric mi-celles represents an efficient drug delivery system for poorlysoluble or insoluble drugs [84]. Different varieties of amphi-philic polymeric micelles (i.e. diblock AB type, triblock ABA typeor graft copolymers) can be designed by arranging the mono-meric units in different ways and orders [85,86]. Thehydrophobic block constitute the core and the hydrophilic blockmakes the corona of the micelles.The water-soluble PEG blockswith a molecular weight from 1 to 15 kDa are considered asthe most suitable hydrophilic corona-forming blocks [87].Variouspreclinical and clinical studies have shown the potential useof PEGylated polymeric micelles with different hydrophobicblocks such as PLGA poly(D,L-lactide-co-glycolide) [88–90], polyaspartate [91], γ-benzyl-L-glutamate [92], polyglutamate (Pglu)[93], and poly(D,L-lactic acid) [94] in anti-cancer therapy. It isto be noted that five different PEGylated polymeric micellar for-mulations, as enlisted in Table 3, are currently under clinicaltrials for possible anticancer treatment [95,96].

8. PEGylated nanoparticles for SiRNA deliveryin anticancer therapy

RNA interference is a natural phenomenon employed to se-lectively turn off the genes expressed in some diseases.

Molecular therapy using small interfering RNA (siRNA) hasshown great therapeutic potential for tumors and other dis-eases caused by abnormal gene over-expression or mutation.It is a highly specific process for gene silencing. However, nakedmolecules of siRNA are vulnerable to premature renal clear-ance and nuclease degradation. The negative charge andhydrophilicity of siRNA also limit its permeability through cel-lular and endolysosomal membranes. Therefore, in order toovercome these issues, siRNA requires a carrier system for ef-fective delivery [97]. Modification of drug delivery systems withPEGs of suitable chain length, molecular weight and percentcomposition was proven to be efficient in overcoming intra-cellular and systemic siRNA delivery barriers [98]. CALLA 01,a nanoparticle formulation (Calando Pharmaceuticals) formu-lated by using cyclodextrin nanoparticles conjugated totransferrin and coated with PEG, is the first one to enter underphase I clinical trials for solid tumors [44,99], in addition to fewmore which are currently under development (Table 2).

9. Conclusions

PEGylation offers a great advantage for bioactive molecules inpharmaceutical and biological applications by way of reduc-ing protein immunogenicity and increased serum half-life ofthe drugs. This overview highlighted on the use of PEGylatedproteins, low molecular weight drugs and PEG micelles.PEGylation improves the therapeutic efficacy of a drug bypassive targeting in a novel way.The process can also be com-bined effectively with active targeting and stimuli-responsivetargeted therapies for the development of new methodolo-gies for the treatment of cancer. It is important to note thatthe efficacy of PEGylated drugs depends on overall exposureand its relationship to the pharmacodynamics of the drug. Mo-lecular weight of PEG chain and its structural modificationscarries strategic importance for conjugation with drug mol-ecule for effective PEGylation process. The research in thisdirection shall be helpful in effective cancer treatment processin near future.

Acknowledgements

The authors wish to acknowledge Dr. Rajakishore Mishra fromCentral University Jharkhand, India, and Dr. Basabi Rana fromLoyola University Chicago, USA, for their valuable suggestions.The grant in aid from DST, New Delhi (Project No. SR/S1/PC-24/2009) is duly acknowledged. University fellowship to PrajnaMishra for pursuing her research work is acknowledged.

Table 3 – PEGylated polymeric micelles in clinical development as anticancer therapy.

Trade name for polymeric micelle Block copolymer Parent drug Clinical trial status

NK105 PEG-P(aspartate) Paclitaxel Paclitaxel Phase II, advanced stomach cancerNK012 PEG-PGlu(SN-38) SN-38 Phase II, breast cancerNC-6300 PEG-P(aspartate) Epirubicin Epirubicin Phase I, breast cancer, stomach cancer, lymphomaNC-6004 PEG-PGlu(cisplatin) Cisplatin Phase I/II, solid tumorsGenexol-PM PEG-P(D,L-lactide) Paclitaxel Paclitaxel Phase IV, breast cancer

Phase II, pancreatic cancer

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