PEGylation in anti-cancer therapy:An overview

Prjn Mishr,Bismit Nyk,R.K.Dey,*

aCentre for Applied Chemistry,Central University of Jharkhand,Ranchi 835 205,Jharkhand,India

bDepartment of Life Science,National Institute of Technology,Rourkela 769 008,Odisha,India

Review

PEGylation in anti-cancer therapy:An overview

Prajna Mishraa,Bismita Nayakb,R.K.Deya,*

aCentre for Applied Chemistry,Central University of Jharkhand,Ranchi 835 205,Jharkhand,India

bDepartment of Life Science,National Institute of Technology,Rourkela 769 008,Odisha,India

A R T I C L EI 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

PEG

Advanced drug delivery systems using poly(ethylene glycol)(PEG)is an important development 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 pharmacokinetics(PK)of a variety of drugs and dramatically improves the pharmaceutical values; recent development of which includes fabrication of stimuli-sensitive polymers/smart polymers and polymeric micelles to cope of with the pathophysiological environment of targeted site with less toxic effects and more effectiveness.This overview discusses PEGylation involving 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 Pharmaceutical University.This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

1.Introduction

Cancer is one of the leading causes of death worldwide.Metastases are the primary cause of death from cancer.Cancer cells proliferate at much faster rate than the normal cells.The available traditional cancer chemotherapy is not essentially selective as it depends on the kinetics of the cell growth.Targeted cancer therapies are expected to be more effective and benefciary in comparison to available conventional treatment procedures.

The last few decades of research in the particular area are focused on exploring the treatment of cancer at its molecular 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 weight drugs.In this regard,polyethylene glycol(PEG),a watersoluble and biocompatible polymer,is the most commonly used non-ionic polymer in the feld of polymer-based drug delivery[1].Passive targeting with PEGs in combination with active targeting(entry into the tumor cell via ligand receptor,antigen antibody interaction)delivery systems has been effectively employed to achieve better therapeutic index of anti-cancer drugs. Further,with the introduction of stimuli-responsive chemical moiety in PEGylated prodrugs,the sensitiveness of the drug molecule toward the pathophysiological environment of tumor cellsin vivoevolved as a new era of site-specifc targeted drug delivery system.Hence this overview deals with the development and recent advancement in various aspects of PEGylationin cancer treatment and its future prospective in a comprehensive way.

2.PEGylation and its signifcance

The technique of covalently attaching polyethylene glycol(PEG) to a given molecule is known as“PEGylation”and is now a wellestablished method in the feld of targeted drug delivery systems.The general structure of monomethoxy PEG(mPEG) can be represented as CH3O–(CH2-CH2O)n–CH2-CH2–OH.At the beginning of PEG chemistry,in the late1970s,Professor Frank Davis and his colleagues had shown that the immunological properties as well as the stability of bovine serum albumin and bovine liver catalase can be successfully altered by covalently linking them to methoxy PEG(mPEG)[2,3]using cyanuric chloride as an activating agent.The process of PEGylation can be extended to liposomes,peptides,carbohydrates,enzymes, antibody fragments,nucleotides,small organic molecules and even to different nanoparticle formulations[4–8].mPEG is the most useful unit for polypeptide modifcation[9].Now several derivatives of PEG molecules are available that vary in molecular weight and structure,such as linear,branched,PEG dendrimers and more recently multi-arm PEGS[9].The frst step of PEGylation is to activate PEG by conjugating a functional derivative of PEG at one or both the terminals of PEG chain.

PEGylation conjugation techniques can be classifed into two categories:i)frst-generation random PEGylation,and ii)secondgeneration site-specifc PEGylation.Thanks to the second generation PEGylation processes that resulted in well-defned conjugated products with improved product profles over those obtained through non-specifc random conjugations.

Irreversibly conjugating PEGs had some adverse effects on the specifc biological activity of many therapeutics.Thus,to minimize the loss of activity,a reversible(or releasable prodrug) PEGylation concept has been formulated.Reversible PEGylation concept deals with attachment of drugs to PEG derivatives through cleavable linkages(Fig.1).The release of drug occurs by therapeutic agents through enzymatic,hydrolytic cleavage or reductionin vivoat a predetermined kinetic rate over a time period[10].

The objective of most PEG conjugation techniques aims at increasing the circulation half-life without affecting activity. It is to be noted that the distinct advancement in the PEG conjugation processes and diversity in the nature of the PEGs used for the conjugation has attributed to the increased demand for PEGylated pharmaceutical products.

PEGylation enhances the therapeutic effcacy of the drugs by bringing in several advantageous modifcations over the non-PEGylated products.The systematic classifcation is illustrated in Fig.2.Increase in the serum half-life of the conjugate is the major way of enhancing therapeutic potential of the PEGylated conjugate.PEGylation prolongs the circulation time of conjugated therapeutics by increasing its hydrophilicity and reducing the rate of glomerular fltration[11,12].Few factors such as protection from reticuloendothelial cells,proteolytic enzymes and decreased formation of neutralizing antibodies against the protein by masking antigenic sites by formation of a protective hydrophilic shield are the key components of PEG molecule that attributes to the improved pharmacokinetic profle(PK) of the conjugates[13–16].It has also been reported that PEGylation increases the absorption half-life of subcutaneously administered agents and is associated with a decreased volume of distribution[11].PEG is a non-biodegradable polymerthat puts limits on its use.It has been shown that PEGs(up to molecular weight 20 kDa)is primarily excreted through the renal system,whereas higher molecular weight PEG chains get eliminated by fecal excretion[17].PEGylation proved to be the most promising approach for increasing the serum half-life of the conjugated therapeutics,which is related to enhancement of effcacy of the conjugate.However,PEGylation imposes certain disadvantages on liposomes,especially for the delivery of genes and nucleic acids in anti-cancer therapy as its surface hydrophilic shield reduces the cellular uptake and improves the stability of the lipid envelope,and the process results in poor endosomal escape via membrane fusion and degradation of cargos in lysosomes[18,19].Hence the use of PEG in gene and nucleic acid delivery to cancer cell is referred to as“PEG dilemma”[20].The issue can be effciently addressed by designing pH-sensitive and tumor-specifc targeted PEGylated therapeutics[21–23].

Fig.1–PEG-prodrug.

Fig.2–Signifcance of PEGylated therapeutics.

2.1.Role of PEGylation in passive and active targeting of drugs

Passive targeting drug delivery technique,as illustrated in Fig.3, mostly depends upon the concentration gradient between the intracellular and extracellular space,created due to high concentration of the drug in the tumor area[24].PEG conjugates takes the advantage of enhanced permeation and retention (EPR)effect executed by the tumors and gets accumulated in the pathophysiological environment of tumor vessels through leaky vasculature and poor lymphatic drainage.However,this effect cannot be studied with low molecular weight drugs that freely extravagate causing systemic toxicity,and this is a sizedependent 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 methods for passive targeting of anticancer therapeutics.

The concept of active targeting of drugs is based on the idea of conjugating drug molecules to targeting entities(antibodies,ligands,etc.)for specifc interaction with the structures present on the cell surface for targeted delivery of the anticancer agent[25,26].The fate of the pro-drug is dictated by the targeting molecule and the linker molecule present on the pro-drug.The targeting moiety essentially decides the type of cancer cell for the act of therapeutics.Further,depending on the linker molecule,the drug gets entry into the tumor cell by either of the two ways:(i)receptor-mediated internalization of the whole pro-drug by endocytosis and subsequent degradation by endosomal/lysosomal pathway(Fig.4),or(ii)receptorindependent internalization of the drug into targeted cells after extracellular cleavage of the pro-drug(Fig.5)[27].PEGylated pro-drugs can be effciently conjugated to targeting moieties by different conjugation chemistry in order to achieve the goal of active targeting.The targeted delivery of the PEGylated drugs at the desired site causes high bioavailability and low systemic toxicity.

3.PEGylated proteins in anti-cancer therapy

PEGylation of proteins is a well-established method in the pharmaceutical feld,but the signifcance of PEGylated peptides and proteins for anti-cancer therapy has only been realized in the last several years as more and more PEG conjugates make it to late-phase clinical trials.Enzymes,monoclonal antibodies and cytokines are the three major class of proteins used in anticancer 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.

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

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

3.1.PEGylated monoclonal antibody fragment

In the feld of anti-cancer therapy,monoclonal antibodies represent the major class of protein therapeutics.Antibodies act by binding to the specifc antigens/cell surface receptors.This task is taken care by the fragment antigen-binding(Fab’)region on an antibody.Depending upon the receptor and the binding site on the receptor against which the antibody is designed, it can either activate cellular signaling pathways leading to apoptosis,cell growth arrest,or block the pathways leading to cell growth that eventually causes tumor cell death(apoptosis).This event is illustrated pictorially in Fig.6.The major drawback associated with Fab’s antibody fragment is its short serum half-life as it lacks the Fc region of the antibody that limits its potential as a therapeutic agent.Hence,suitable PEGylation methods and PEGs are used to ensure minimal loss of the antibody-antigen/cell surface receptor interaction keeping in view the enhancement of serum half-life.It has been reported that the hinge region cysteine residues on immunoglobulin G(IgG antibody isotype)Fab’antibody fragments can tolerate attachment of one or two PEG moieties(up to a total of 40 kDa molecular weight)with little effect on antigen binding affnity.This process also enables signifcant increase in the half-life of the circulating plasma antibodies by reducing theglomerular fltration and lower immunogenicity than the parent IgG[28].

The example of use of PEG-antibody fragment angiogenesis inhibitor(CDP791)is illustrated as follows:CDP791 PEGylated diFab antibody fragment antagonizes the effect of vascular endothelial growth factor receptor-2(VEGFR-2),which is a prominent angiogenesis stimulatory molecule responsible for tumor progression.The short plasma half-life of the unmodifed CDP791 antibody fragment,which lacks Fc region, has a low molecular weight and responsible for low therapeutic index.PEGylation of the cysteine amino acid present at the C-terminus of the native antibody could be able to resolve this issue by reducing its kidney clearance.This is demonstrated by the clinical studies for patients with colorectal,ovarian,renal cancer or other tumors[29].

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

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

3.2.PEGylated cytokines

Cytokines represent another class of protein therapeutics employed mainly as adjuvant therapy in classical anti-cancer chemotherapy protocols either to control or bring improvements in patient conditions.These small secreted proteins belong to the immunotherapy category and mobilizes the body’s immune system to fght cancer.The process is illustrated pictorially in Fig.7.

3.2.1.PEG-interferon-alpha conjugates

This process is illustrated as PEG-interferon-α2b(PEG-INTRON?/ Sylatron?)and PEG-interferon-α2a(Pegasys?),which are discussed as follows:

3.2.1.1.PEG-interferon-α2b(PEG-INTRON?/Sylatron?).The PEGylated version of interferon-α2b was synthesized by conjugating interferon-α2b,with a single chain 12 kDa PEG-SC via a urethane bond[30].It displayed a half-life of 27–37 h with 10-fold lower clearance and minor change in the volume of distribution in comparison to native form[31].Based upon the outcome of clinical studies in the year 2011,the PEGylated drug peginterferon alfa-2b(PEG-IFN)got FDA approval for adjuvant treatment of melanoma patients with microscopic or grossnodal involvement following defnitive surgical resection including complete lymphadenectomy[32].Sylatron?is another brand name for peginterferon alfa-2b exclusively approved by FDA for adjuvant therapy in cancer treatment.

Fig.7–Representing PEGylated cytokines in anticancer therapy.

3.2.1.2.PEG-interferon-α2a(Pegasys?).Another PEGylated interferon,Pegasys?,is prepared by mono-PEGylation of interferon-α-2a with an N-hydroxysuccinimide(NHS)activated 40 kDa branched PEG molecule[33].PEGylation prolonged the serum half-life from 3.8 to 65 h,slowing down the clearance by more than 100-fold.This has also reduced the volume of distribution to fvefold with respect to the native interferonα2b[31].PEGASYS?was effcient in improving the patient compliance by enabling once-weekly dosing while maintaining acceptable safety,tolerability,and activity profles in clinical studies[34].Currently,PEGASYS?is under evaluation as adjuvant therapy for patients with intermediate and high-risk melanomas[35].

3.2.2.PEG-granulocyte colony stimulating factor

(PEG-flgrastim)

PEG-flgrastim was synthesized by conjugating a linear 20 kDa mPEG-aldehyde derivative to an N-terminal methionine residue of flgrastim through reductive alkylation under mild acidic conditions[36,37].It is to be noted that a single dose of PEG-flgrastim per chemotherapy cycle could be able to reduce the risk of febrile neutropenia signifcantly with respect to the native protein(11%vs.19%)[38–40].Currently,PEGylated-G-CSF (Pegflgastrim,Neulasta?)is used as an adjuvant therapy for patients with non-myeloid malignancies receiving myelosuppressive chemotherapy(bone marrow suppression as a side effect of chemotherapy)associated with a 20%risk of febrile neutropenia[41,42].

3.3.PEGylated enzymes in anti-cancer therapy

Therapeutic enzymes represent a growing class of biopharmaceuticals,and PEGylation has played a major role in improving several of these products[43].Many depleting enzymes are active against tumors.Enzymes’intrinsic property of degrading amino acids is essential for cancer cells existence.The fate of the tumor cell is dictated by the different cellular pathways regulated by the substrate(amino acid) to be degraded,and the process of degradation is illustrated in Fig.8.The normal cells are not affected because the normal cells can synthesize the amino acids for their growth.This situation is particularly the most advantageous aspect of using depleting enzymes in cancer therapy(Table 1).Therefore,during PEGylation procedure,a combination of these enzymes,low molecular weight(5–10 kDa)PEGs and random amine conjugation strategies are employed.

3.3.1.PEG-arginine depleting enzymes

Arginine is a nonessential amino acid in humans.It has been reported that arginine defciency inhibits tumor growth,angiogenesis and nitric oxide synthesis[44].Two types of arginine degrading enzymes,i.e.i)arginine deiminase(ADI)and ii)arginase(ARG),which can be utilized as antitumor agents,are discussed below:

3.3.1.1.PEG-arginine deiminase.The PEGylation of arginine deiminase proved to be a better therapeutic approach for anticancer treatment.Among the several PEGylated ADI formulations the ADI-PEG20000,formulated by conjugating 10–12 chains of 20 kDa PEG with ADI by using the succinimidyl succinate linker,is proved to be the acceptable one fromin vivostudy results[45].Clinical studies have shown better effcacy of ADI PEG 200,000 in terms of antitumor activity and tolerability[46,47].Currently,ADI PEG 200,000 versus placebo is under phase III clinical trial for advanced hepatocellular carcinoma.Further,Phase II for acute myeloid leukemia/non-Hodgkin’s lymphoma and Phase I(for metastatic melanoma in combination withcis-platin;for solid tumors in combination with docetaxel)are under trial [48].

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

3.3.1.2.PEG-arginase.The depleting enzyme arginase is an endogenous protein expressed in humans.The conjugate,PEG-rhArg,has 10 to 12 polymer chains of PEG 5000 per protein molecule that is covalently attached via a succinamide propionic acid(SPA)linker.This conjugate remains in fully active condition[49].The PEGylated form executes suffcient catalytic activity at physiological pH with a prolonged plasma halflife of 3 days in comparison to the native form,which has a half-life of several minutes only.Currently,this conjugate is under phase I/II clinical trials.

3.3.2.PEG-asparagine depleting enzyme (PEG-L-asparaginase)

Depletion of asparagine eventually results in leukemic cell death.Leukemic cells lack the enzyme asparagine synthetase,an enzyme required for asparagine synthesis,and depend on the exogenous supply of asparagine for their growth and survival.Therefore,asparaginase,the depleting enzyme for asparagine,plays a critical role as a therapeutic enzyme in treating acute lymphoblastic leukemia(ALL).Oncaspar is a modifed form of the enzyme L-asparaginase approved by FDA in 1994. Oncaspar consists of tetrameric enzyme L-asparaginase derived fromE.coli,and it is covalently conjugated with approximately 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 were allergic to the native form of the drug.The U.S.Food and Drug Administration granted approval to pegaspargase(Oncaspar, Enzon Pharmaceuticals,Inc)in July 2006 for the frst-line treatment of patients with acute lymphoblastic leukemia(ALL)as a component of a multi-agent chemotherapy regimen[51].

4.PEGYLATED low molecular weight anticancer drugs

Various PEGylated low molecular weight anti-cancer drugs are currently under development.For example,topoisomerase I inhibitor camptothecin-based drugs(irinotecan,topotecan,SN38, exetecan,etc.)is reported to be useful in the treatment of many solid tumors.However,the hydrophobicity of such material limits their therapeutic effcacy.Few of the examples,listed in Table 2,are discussed below:

4.1.PEG-SN38(EZN-2208)

EZN-2208,the product Enzon Pharmaceuticals,Inc,is a PEGylated SN38(10-hydroxy-7-ethyl-camptothecin(a derivative of camptothecin)).SN38 is the active moiety of CPT-11(Camptosar?,irinotecan)and reported to be a potent topoisomerase I inhibitor.In this PEGylated product,the 20-OH group of SN38 was selectively coupled with a 4 arm PEG of 40 kDa through a glycine spacer to preserve the E ring of SN38 in the active lactone form while leaving the drug 10-OH free[52].PEGylation was able to enhance the solubility of SN38 by about 1000-fold.In fact,EZN-2208 showed a 207-fold higher exposure to SN38 compared to irinotecan in treated mice[53]. The conjugate showed promising antitumor activity bothin vitroandin vivo.However,following phase II trial,Enzon Pharmaceuticals,Inc.announced the discontinuance of its EZN-2208 clinical program[54].

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

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

NKTR-102 was developed by NektarTherapeutics as a PEGylated formulation for the treatment of colorectal cancer and other solid tumors using the architecture of new multi-arm PEGs.It is the frst long-acting topoisomerase I inhibitor.It is synthesized by covalently conjugating irinotecan to a four-arm PEG [55].SN38(10-hydroxy-7-ethyl-camptothecin,a derivative of camptothecin)is the active metabolite of NKTR-102.It is reported in both Phase I and II studies for NKTR-102 that sustained exposure of the active drug was associated with promising anti-tumor activity[56–58].Currently NKTR-102 is in Phase III clinical trial for patients with metastatic or locally recurrent breast cancer and Phase II clinical trial for patients with solid tumor malignancies that include ovarian,colorectal, small cell and non-small cell lung cancers[59].

5.PEGylated nanoparticles in anti-cancer therapy

Nanoparticles(NPs)are synthetic materials with dimensions from 1 to 1000 nano-meters.NPs have large payloads,stability and the capacity for multiple,simultaneous applications due to their unique size and high surface area:volume ratio [60].Despite these advantages,the major drawbacks associated with NP drug delivery system for clinical studies are associated with short circulating half-life due to uptake by the reticuloendothelial system(RES)for larger NPs,whereas smaller NPs are subjects to tissue extravazations and renal clearance [61].Liposomes,solid lipids nanoparticles,dendrimers,polymers,silicon or carbon materials,and gold and magnetic nanoparticles are examples of nano-carriers that have been studied as drug delivery systems in cancer therapy.Therefore,surface modifcation of the nanoparticles with PEGs of various chain length,shape,density,molecular weight and incorporation of different targeting moieties(ligands,antibodies, etc.)is emerging as a more promising and technologically advanced drug delivery system in anti-cancer therapy[62–64]. There are currently more than 35 US FDA-approved PEGylated NPs,with a larger number in preclinical studies for both imaging and therapy[8].Among several PEGylated nanoparticle formulations for anticancer therapy,liposomes have been most extensively studied.

5.1.PEGylated liposomes in anticancer therapy

Liposomes are spherical,self-closed structures formed by one or more concentric lipid bilayers with an encapsulated aqueous phase in the center and between the bilayers composed of natural or synthetic lipids[65].The development of longcirculating liposomes with inclusion of the synthetic polymer poly-(ethylene glycol)(PEG)in liposome composition could be able to solve the issue of low serum half-life associated with liposomes.PEG can be incorporated on the liposomal surface in a number of ways.However,anchoring the polymer in the liposomal membrane via a cross-linked lipid,PEG-distearoylphosphatidylethanolamine[DSPE],is reported to be the most widely accepted method[66,67].Preclinical studies with PEGylated liposomes reported that the cytotoxic agents entrapped in PEGylated liposomes tend to accumulate in tumors [68].However,recent preclinical studies of anti-cancer drug enclosed in PEGylated liposomes in rodents and dogs have shown the rapid blood clearance of the pegylated drug carrier system due to the increased anti-PEG-IgM production[69,70].An example of PEGylated liposomal formulations,PEGylated liposomal doxorubicin(PLD),and most extensively studied,is discussed below:

5.1.1.Doxil(PEGylated liposomal doxorubicin)

DOXIL is the trade name for PEGylated liposomal doxorubicin formulated to achieve better drug effcacy for cancer chemotherapy.This product contains doxorubicin(Adriamycin) enclosed in an 80–90 nm size uni-lamellar liposome coated with PEG.The modifcation increases the circulatory half-life of the drug leading to its enhanced bioavailability at the tumor site [71,72].PEGylated liposomal doxorubicin has fewer side effects on healthy cells than regular doxorubicin[73,74].PLD has improved pharmacokinetic features,such as long circulation time of about 60–90 h for doses in the range of 35–70 mg/m2in patients 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 clearance is dramatically slower and its volume of distribution remains very small,which is roughly equivalent to the intravascular volume[75–77].After obtaining approval from FDA, PEGylated liposomal doxorubicin(PLD)(DOXIL/Caelyx)is currently used to treat Kaposi’s sarcoma and recurrent ovarian cancer(Table 2).

6.PEGylated smart polymers in anticancer therapy

Smart polymers are defned as polymers that undergo reversible large,physical or chemical changes in response to small external changes in the environmental conditions,such as temperature,pH,light,magnetic or electric feld,ionic factors, biological molecules,etc.Smart polymers show promising applications in the biomedical feld as delivery systems of therapeutic agents[78].Among various smart polymers currently in use in biomedical feld of research,the temperature sensitive systems are the most studied systems.The greater therapeutic index of the targeted drug delivery systems canbe achieved by adjusting the transition temperature(Tt)of thermally responsive polymers,i.e.,between body temperature (37°C)and the temperature approved for mild clinical hyperthermia(42°C)[79].Within the temperature ranges,these polymers facilitate tissue accumulation by localizing the aggregation of systemically delivered carriers to the heated tumor volume[80,81].For example,ThermoDox,a temperaturesensitive doxorubicin-loaded PEGylated liposome(DPPC), releases encapsulated doxorubicin at elevated tissue temperature.DPPC has a transition temperature of 41.5°C,which makes it suitable for temperature-sensitive technology[82].The temperature can be achieved by radiofrequency ablation technique. ForThermoDox,the concentration of the drug is up to 25 times more in the treatment area than IV doxorubicin,and several fold the concentration of other liposomally encapsulated doxorubicins[82,83].Currently,it is under phase III clinical trial for hepatocellular carcinoma(Table 2).

7.PEGylated polymeric micelles in anticancer therapy

Polymeric micelles are colloidal dispersions prepared by blockcopolymers,consisting of hydrophilic and hydrophobic monomer units.Self-assembling amphiphilic polymeric micelles represents an effcient drug delivery system for poorly soluble or insoluble drugs[84].Different varieties of amphiphilic polymeric micelles(i.e.diblock AB type,triblock ABA type or graft copolymers)can be designed by arranging the monomeric units in different ways and orders[85,86].The hydrophobic block constitute the core and the hydrophilic block makes the corona of the micelles.The water-soluble PEG blocks with a molecular weight from 1 to 15 kDa are considered as the most suitable hydrophilic corona-forming blocks[87].Various preclinical and clinical studies have shown the potential use of PEGylated polymeric micelles with different hydrophobic blocks such as PLGA poly(D,L-lactide-co-glycolide)[88–90],poly aspartate[91],γ-benzyl-L-glutamate[92],polyglutamate(Pglu) [93],and poly(D,L-lactic acid)[94]in anti-cancer therapy.It is to be noted that fve different PEGylated polymeric micellar formulations,as enlisted in Table 3,are currently under clinical trials for possible anticancer treatment[95,96].

8.PEGylated nanoparticles for SiRNA delivery in anticancer therapy

RNA interference is a natural phenomenon employed to selectively turn off the genes expressed in some diseases. Molecular therapy using small interfering RNA(siRNA)has shown great therapeutic potential for tumors and other diseases caused by abnormal gene over-expression or mutation. It is a highly specifc process for gene silencing.However,naked molecules of siRNA are vulnerable to premature renal clearance and nuclease degradation.The negative charge and hydrophilicity of siRNA also limit its permeability through cellular and endolysosomal membranes.Therefore,in order to overcome these issues,siRNA requires a carrier system for effective delivery[97].Modifcation of drug delivery systems with PEGs of suitable chain length,molecular weight and percent composition was proven to be effcient in overcoming intracellular and systemic siRNA delivery barriers[98].CALLA 01, a nanoparticle formulation(Calando Pharmaceuticals)formulated by using cyclodextrin nanoparticles conjugated to transferrin and coated with PEG,is the frst one to enter under phase I clinical trials for solid tumors[44,99],in addition to few more which are currently under development(Table 2).

9.Conclusions

PEGylation offers a great advantage for bioactive molecules in pharmaceutical and biological applications by way of reducing protein immunogenicity and increased serum half-life of the drugs.This overview highlighted on the use of PEGylated proteins,low molecular weight drugs and PEG micelles. PEGylation improves the therapeutic effcacy of a drug by passive targeting in a novel way.The process can also be combined effectively with active targeting and stimuli-responsive targeted therapies for the development of new methodologies for the treatment of cancer.It is important to note that the effcacy of PEGylated drugs depends on overall exposure and its relationship to the pharmacodynamics of the drug.Molecular weight of PEG chain and its structural modifcations carries strategic importance for conjugation with drug molecule for effective PEGylation process.The research in this direction shall be helpful in effective cancer treatment process in near future.

Acknowledgements

The authors wish to acknowledge Dr.Rajakishore Mishra from Central University Jharkhand,India,and Dr.Basabi Rana from Loyola 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 Prajna Mishra for pursuing her research work is acknowledged.

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

R E F E R E N C E S

[1]Knop K,Hoogenboom R,Fischer D,et al.Poly(ethylene glycol)in drug delivery:pros and cons as well as potential alternatives.Angew Chem Int Ed Engl 2010;49:6288–6308.

[2]Abuchowski A,Van Es T,Palczuk NC,et al.Alteration of immunological properties of bovine serum albumin by covalent attachment of polyethylene glycol.J Biol Chem 1977;252:3578–3581.

[3]Abuchowski A,McCoy JR,Palczuk NC,et al.Effect of covalent attachment of polyethylene glycol on immunogenicity and circulating life of bovine liver catalase.J Biol Chem 1977;252:3582–3586.

[4]Immordino ML,Dosio F,Cattel L.Stealth liposomes:review of the basic science,rationale,and clinical applications, existing and potential.Int J Nanomedicine 2006;1:297–315.

[5]Matsushima A,Kodera Y,Hiroto M,et al.Bioconjugates of proteins and polyethylene glycol:potent tools in biotechnological processes.J Mol Catal B Enzym 1996;2:1–17.

[6]Eugenia Giorgi M,Agusti R,de Lederkremer Beilstein RM. Carbohydrate PEGylation,an approach to improve pharmacological potency.J Org Chem 2014;10:1433–1444.

[7]Riley T,Riggs-Sauthier J.The benefts and challenges of PEGylating small molecules.Pharm Technol 2008;32:88–94.

[8]Jokerst JV,Lobovkina T,Zare RN,et al.Nanoparticle PEGylation for imaging and therapy.Nanomedicine 2011;6:715–728.

[9]Roberts MJ,Bentley MD,Harris JM.Chemistry for peptide and protein PEGylation.Adv Drug Deliv Rev 2002;54:459–476.

[10]Veronese FM,Pasut G.PEGylation:posttranslational bioengineering of protein bio therapeutics.Drug Discov Today Technol 2008;5:57–64.

[11]Caliceti P,Veronese FM.Pharmacokinetic and biodistribution properties of poly(ethylene glycol)-protein conjugates.Adv Drug Deliv Rev 2003;55:1261–1277.

[12]Bhat R,Timasheff SN.Steric exclusion is the principal source of the preferential hydration of proteins in the presence of polyethylene glycols.Protein Sci 1992;1:1133–1143.

[13]Bailon P,Berthold W.Polyethylene glycol-conjugated pharmaceutical proteins.Pharm Sci Technol Today 1998;1:352–356.

[14]Monfardini C,Schiavon O,Caliceti P,et al.A branched monomethoxypoly(ethylene glycol)for protein modifcation.Bioconjug Chem 1995;6:62–69.

[15]Delgado C,Francis GE,Fisher D.The uses and properties of PEG linked proteins.Crit Rev Ther Drug Carrier Syst 1992;9:249–304.

[16]Nucci ML,Shorr R,Abuchowski A.The therapeutic value of poly(ethylene glycol)-modifed proteins.Adv Drug Deliv Rev 1991;6:133–151.

[17]Yamaoka T,Tabata Y,Ikada Y.Distribution and tissue uptake of poly(ethylene glycol)with different molecular weights after intravenous administration to mice.J Pharm Sci 1994;83:601–606.

[18]Mishra S,Webster P,Davis ME.PEGylation signifcantly affects cellular uptake and intracellular traffcking of nonviral gene delivery particles.Eur J Cell Biol 2004;83:97–111.

[19]Remaut K,Lucas B,Braeckmans K,et al.PEGylation of liposomes favours the endosomal degradation of the delivered phosphodiester oligonucleotides.J Control Release 2007;117:256–266.

[20]Hatakeyama H,Akita H,Harashima H.The polyethyleneglycol dilemma:advantage and disadvantage of PEGylation of liposomes for systemic genes and nucleic acids delivery to tumors.Biol Pharm Bull 2013;36:892–899.

[21]Sato Y,Hatakeyama H,Sakurai Y,et al.A pH-sensitive cationic lipid facilitates the delivery of liposomal siRNA and gene silencing activityin vitroandin vivo.J Control Release 2012;163:267–276.

[22]Hatakeyama H,Akita H,Harashima H.A multifunctional envelope type nano device(MEND)for gene delivery to tumours based on the EPR effect:a strategy for overcoming the PEG dilemma.Adv Drug Deliv Rev 2011;63:152–160.

[23]Hatakeyama H,Akita H,Kogure K,et al.Development of a novel systemic gene delivery system for cancer therapy with a tumor-specifc cleavable PEG-lipid.Gene Ther 2007;14:68–77.

[24]Seymour LW,Miyamoto Y,Maeda H,et al.Infuence of molecular weight on passive tumour accumulation of a soluble macromolecular drug carrier.Eur J Cancer 1995;31A:766–770.

[25]Schrama D,Reisfeld RA,Becker JC.Antibody targeted drugs as cancer therapeutics.Nat Rev Drug Discov 2006;5:147–159.

[26]Allen TM.Ligand-targeted therapeutics in anticancer therapy.Nat Rev Cancer 2002;2:750–763.

[27]Banerjee SS,Aher N,Patil R,et al.Poly(ethyleneglycol)-prodrug conjugates:concept,design,and applications.J Drug Deliv 2012;2012:Article ID 103973.

[28]Chapman AP.PEGylated antibodies and anti-body fragments for improved therapy:a review.Adv Drug Deliv Rev 2002;54:531–545.

[29]Ton NC,Parker GJ,Jackson A,et al.Phase I evaluation of CDP791,a PEGylated di-Fab’conjugate that binds vascular endothelial growth factor receptor 2.Clin Cancer Res 2007;13:7113–7118.

[30]Wang YS,Youngster S,Grace M,et al.Structural and biological characterization of pegylated recombinant interferon alpha-2b and its therapeutic implications.Adv Drug Deliv Rev 2002;54:547–570.

[31]Zeuzem S,Welsch C,Herrmann E.Pharmacokinetics of peg interferons.Semin Liver Dis 2003;23:23–28.

[32]Herndon TM,Demko SG,Jiang X,et al.Peginterferon-alfa-2b for the adjuvant treatment of patients with melanoma. Oncologist 2012;17:1323–1328.

[33]Reddy KR,Modi MW,Pedder S.Use of peginterferon α2a (40KD)(Pegasys?)for the treatment of hepatitis C.Adv Drug Deliv Rev 2002;54:571–586.

[34]Lipton JH,Khoroshko N,Golenkov A,et al.Pegasys CML Study Group.Phase II,randomized,multicenter,comparative study of peginterferon-alpha-2a(40kD)(Pegasys)versus interferon alpha-2a(Roferon-A)in patients with treatmentna?ve,chronic-phase chronic myelogenous leukemia.Leuk Lymphoma 2007;48:497–505.

[35]Loquai C,Nashan D,Hensen P,et al.Safety of pegylated interferon-alpha-2a in adjuvant therapy of intermediate and high-risk melanomas.Eur J Dermatol 2008;18:29–35.

[36]Veronese FM,Mero A.The impact of PEGylation on biological therapies.Biodrugs 2008;22:315–329.

[37]Kinstler O,Molineux G,Treuheit M,et al.Mono-N-terminal poly(ethylene glycol)-protein conjugates.Adv Drug Deliv Rev 2002;54:477–485.

[38]Green MD,Koelbl H,Baselga J,et al.A randomized doubleblind multi center phase III study of fxed-dose singleadministration pegflgrastim versus daily flgrastim in patients receiving myelosuppressive chemotherapy.Ann Oncol 2003;14:29–35.

[39]Holmes FA,Jones SE,O’Shaughnessy J,et al.Comparable effcacy and safety profles of once-per-cycle pegflgrastim and daily injection flgrastim in chemo-therapy-induced neutropenia:a multi center dose-fnding study in women with breast cancer.Ann Oncol 2002;13:903–909.

[40]Grigg A,Solal-Celigny P,Hoskin P,et al.Open-label, randomized study of pegflgrastim vs.daily flgrastim as anadjunct to chemotherapy in elderly patients with non-Hodgkin’s lymphoma.Leuk Lymphoma 2003;44:1503–1508.

[41]Vogel CL,Wojtukiewicz MZ,Carroll RR,et al.First and subsequent cycle use of pegflgrastim prevents febrile neutropenia in patients with breast cancer:a multi center, double-blind,placebo-controlled phase III study.J Clin Oncol 2005;23:1178–1184.

[42]Smith TJ,Khatcheressian J,Lyman GH,et al.Update of recommendations for the use of white blood cell growth factors:an evidence-based clinical practice guideline.J Clin Oncol 2006;24:3187–3205.

[43]Vellard M.The enzyme as drug:application of enzymes as pharmaceuticals.Curr Opin Biotechnol 2003;14:444–450.

[44]Cheng PN,Leung YC,Lo WH,et al.Remission of hepatocellular carcinoma with arginine depletion induced by systemic release of endogenous hepatic arginase due to trans hepatic arterial embolisation,augmented by high-dose insulin:arginase as a potential drug candidate for hepatocellular carcinoma.Cancer Lett 2005;224:67–80.

[45]Holtsberg FW,Ensor CM,Steiner MR,et al.Poly(ethylene glycol)(PEG)conjugated arginine deiminase:effects of PEG formulations on its pharmacological properties.J Control Release 2002;80:259–271.

[46]Glazer ES,Piccirillo M,Albino V,et al.Phase II study of pegylated arginine deiminase for non resectable and metastatic hepatocellular carcinoma.J Clin Oncol 2010;28:2220–2226.

[47]Ascierto PA,Scala S,Castello G,et al.Pegylated arginine deiminase treatment of patients with metastatic melanoma: results from phase I and II studies.J Clin Oncol 2005;23:7660–7668.

[48]＜https://clinicaltrials.gov＞.

[49]Cheng PN,Lam T,Lam W,et al.Pegylated recombinant human arginase(rhArg-peg5,000mw)inhibits thein vitroandin vivoproliferation of human hepatocellular carcinoma through arginine depletion.Cancer Res 2007;67:309–317.

[50]Pillai G.Nanomedicines for cancer therapy:an update of FDA approved and those under various stages of development.SOJ Pharm Pharm Sci 2014;1(2):1–13.

[51]Dinndorf PA,Gootenberg J,Cohen MH,et al.FDA drug approval summary:pegaspargase(Oncaspar?)for the frstline treatment of children with acute lymphoblastic leukemia(ALL).Oncologist 2007;12:991–998.

[52]Zhao H,Rubio B,Sapra P,et al.Novel prodrugs of SN38 using multi arm poly(ethylene glycol)linkers.Bioconjug Chem 2008;19:849–859.

[53]Sapra P,Zhao H,Mehlig M,et al.Novel delivery of SN38 markedly inhibits tumor growth in xenografts,including a Camptothecin-11–refractory model.Clin Cancer Res 2008;14:1888–1896.

[54]＜http://enzon.com/posts/view/42＞.

[55]Hoch U,Staschen CM,Johnson RK,et al.Nonclinical pharmacokinetics and activity of etirinotecan pegol(NKTR-102),a long-acting topoisomerase 1 inhibitor,in multiple cancer models.Cancer Chemother Pharmacol 2014;74:1125–1137.

[56]Jameson GS,Hamm JT,Weiss GJ,et al.A multicenter,phase I,dose-escalation study to assess the safety,tolerability,and pharmacokinetics of etirinotecan pegol in patients with refractory solid tumors.Clin Cancer Res 2013;19:268–278.

[57]Vergote IB,Garcia A,Micha J,et al.Randomized multicenter phase II trial comparing two schedules of etirinotecan pegol (NKTR-102)in women with recurrent platinum-resistant/ refractory epithelial ovarian cancer.J Clin Oncol 2013;31:4060–4066.

[58]Awada A,Garcia AA,Chan S,et al.Two schedules of etirinotecan pegol(NKTR-102)in patients with previously treated metastatic breast cancer:a randomised phase 2 study.Lancet Oncol 2013;14:1216–1225.

[59]＜http://www.nektar.com＞.

[60]Jain PK,Huang X,El-Sayed IH,et al.Noble metals on the nano-scale:optical and photo thermal properties and some applications in imaging,sensing,biology,and medicine.Acc Chem Res 2008;41:1578–1586.

[61]Cole AJ,Yang VC,David AE.Cancer theranostics:the rise of targeted magnetic nanoparticles.Trends Biotechnol 2011;29:323–332.

[62]Cruza LJ,Tackena PJ,Fokkinkb R,et al.The infuence of PEG chain length and targeting moiety on antibody-mediated delivery of nanoparticle vaccines to human dendritic cells. Biomaterials 2011;32:6791–6803.

[63]Bao W,Liu R,Wang Y,et al.Plga-Pll-Peg-Tf-based targeted nanoparticles drug delivery system enhance antitumor effcacy via intrinsic apoptosis pathway.Int J Nanomedicine 2015;10:557–566.

[64]Zhang F,Zhang S,Pollack SF,et al.Improving paclitaxel delivery:in vitroandin vivocharacterization of PEGylated polyphosphoester-based nanocarriers.J Am Chem Soc 2015;137:2056–2066.

[65]Pisal DS,Kosloski MP,Balu-Iyer SV.Delivery of therapeutic proteins.J Pharm Sci 2010;99:2557–2575.

[66]Allen TM,Hansen C,Martin F,et al.Liposomes containing synthetic lipid derivatives of poly(ethylene glycol)show prolonged circulation half-livesin vivo.Biochim Biophys Acta 1991;1066:29–36.

[67]Allen C,Dos SN,Gallagher R,et al.Controlling the physical behavior and biological performance of liposome formulations through use of surface grafted poly(ethylene glycol).Biosci Rep 2002;22:225–250.

[68]Newman MS,Colbern GT,Working PK,et al.Comparative pharmacokinetics,tissue distribution,and therapeutic effectiveness of cisplatin encapsulated in long-circulating pegylated liposomes(SPI-077)in tumor-bearing mice. Cancer Chemother Pharmacol 1999;43:1–7.

[69]Yang Q,Ma Y,Zhao Y,et al.Accelerated drug release and clearance of PEGylated epirubicin liposomes following repeated injections:a new challenge for sequential low-dose chemotherapy.Int J Nanomedicine 2013;8:1257–1268.

[70]Suzuki T,Ichihara M,Hyodo K,et al.Accelerated blood clearance of PEGylated liposomes containing doxorubicin upon repeated administration to dogs.Int J Pharm 2012;436:636–643.

[71]Krown SE,Northfelt DW,Osoba D,et al.Use of liposomal anthracyclines in Kaposi’s sarcoma.Semin Oncol 2004;31:36–52.

[72]Lao J,Madani J,Puértolas T,et al.Liposomal doxorubicin in the treatment of breast cancer patients:a review.J Drug Deliv 2013;2013:Article ID 456409.

[73]Rahman AM,Yusuf SW,Ewer MS.Anthracycline-induced cardiotoxicity and the cardiac sparing effect of liposomal formulation.Int J Nanomedicine 2007;2:567–583.

[74]O’Brian ME,Wigler N,Inbar M,et al.Reduced cardiotoxicity and comparable effcacy in a phase III trial of pegylated liposomal doxorubicin HCL(CAELYX/DOXIL)versus conventional doxorubicin for frst-line treatment of metastatic breast cancer.Ann Oncol 2004;15:440–449.

[75]Gabizon A.Applications of liposomal drug delivery systems to cancer therapy.In:Nanotechnology for cancer therapy. New York:CRC Press;2006.p.595–611.

[76]Gabizon A,Catane R,Uziely B,et al.Prolonged circulation time and enhanced accumulation in malignant exudates of doxorubicin encapsulated in polyethyleneglycol coated liposomes.Cancer Res 1994;54:987–992.

[77]Amantea MA,Forrest A,Northfelt DW,et al.Population pharmacokinetics and pharmacodynamics of pegylatedliposomal doxorubicin in patients with AIDS-related Kaposi’s sarcoma.Clin Pharmacol Ther 1997;61:301–311.

[78]Aguilar MR,Elvira C,Gallardo A,et al.Smart polymers and their applications as biomaterials.Top Tissue Eng 2007;3:1–27.

[79]Jones E,Thrall D,Dewhirst MW,et al.Prospective thermal dosimetry:the key to hyperthermia’s future.Int J Hyperthermia 2006;22:247–253.

[80]Meyer DE,Kong GA,Dewhirst MW,et al.Targeting a genetically engineered elastin-like polypeptide to solid tumors by local hyperthermia.Cancer Res 2001;61:1548–1554.

[81]Zhang J,Chen H,Xu L,et al.The targeted behavior of thermally responsive nano hydrogel evaluated by NIR system in mouse model.J Control Release 2008;131:34–40.

[82]Slingerland M,Guchelaar HJ,Gelderblom H.Liposomal drug formulations in cancer therapy:15 years along the road. Drug Discov Today 2012;17:160–166.

[83]Yarmolenko PS,Zhao Y,Landon C,et al.Comparative effects of thermo sensitive doxorubicin-containing liposomes and hyperthermia in human and murine tumors.Int J Hyperthermia 2010;26:485–498.

[84]Torchilin VP.Micellar nanocarriers:pharmaceutical perspectives.Pharm Res 2007;24:1–16.

[85]Jones M,Leroux J.Polymeric micelles–a new generation of colloidal drug carriers.Eur J Pharm Biopharm 1999;48:101–111.

[86]Torchilin VP.Structure and design of polymeric surfactantbased drug delivery systems.J Control Release 2001;73:137–172.

[87]Kwon GS.Polymeric micelles for delivery of poorly watersoluble compounds.Crit Rev Ther Drug Carrier Syst 2003;20:357–403.

[88]Yoo HS,Park TG.Folate receptor targeted biodegradable polymeric doxorubicin micelles.J Control Release 2004;96:273–283.

[89]Cheng J,Teply BA,SherifI,et al.Formulation of functionalized PLGA–PEG nanoparticles forin-vivotargeted drug delivery.Biomaterials 2007;28:869–876.

[90]Esmaeili F,Ghahremani MH,Ostad SN,et al.Folate-receptortargeted delivery of docetaxel nanoparticles prepared by PLGA-PEG-folate conjugate.J Drug Target 2008;16:415–423.

[91]Matsumura Y.Poly(amino acid)micelle nano-carriers in preclinical and clinical studies.Adv Drug Deliv Rev 2008;60:899–914.

[92]Plummer R,Wilson RH,Calvert H,et al.A phase I clinical study of cisplatin-incorporated polymeric micelles(NC-6004) in patients with solid tumours.Br J Cancer 2011;104:593–598.

[93]Matsumura Y,Kataoka K.Preclinical and clinical studies of anticancer agent-incorporating polymer micelles.Cancer Sci 2009;100:572–579.

[94]Kim TY,Kim DW,Chung JY,et al.Phase I and pharmacokinetic study of Genexol-PM,a cremophor-free, polymeric micelle-formulated paclitaxel,in patients with advanced malignancies.Clin Cancer Res 2004;10:3708–3716.

[95]Oerlemans C,Bult W,Bos M,et al.Polymeric micelles in anticancer therapy:targeting,imaging and triggered release. Pharm Res 2010;27:2569–2589.

[96]Matsumura Y.The drug discovery by nano medicine and its clinical experience.Jpn J Clin Oncol 2014;44:515–525.

[97]Kanasty RL,Whitehead KA,Vegas AJ,et al.Action and reaction:the biological response to siRNA and its delivery vehicles.Mol Ther 2012;20:513–524.

[98]Miteva M,Kirkbride KC,Kilchrist KV,et al.Tuning PEGylation of mixed micelles to overcome intracellular and systemic siRNA delivery barriers.Biomaterials 2015;38:97–107.

[99]Lee JM,Yoon TJ,Cho YS.Recent developments in nanoparticle-based siRNA delivery for cancer therapy. Biomed Res Int 2013;2013:Article ID 782041.

*< class="emphasis_italic">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.2015.08.011

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PEGylation

Targeted drug delivery

Polymers