Functionalized polymersomes for biomedical applications

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Polymer Chemistry c3py00023k

REVIEW

1Functionalized polymersomes for biomedicalapplications

Prasad V. Pawar, Shalini V. Gohil, Jay Prakash Jainand Neeraj Kumar*

Functionalized polymersomes are the latest drug carrierusing polymeric vesicular architecture to achieve delivery oftherapeutic agents to specific tissues/organs. This reviewfocuses on their development along with their biomedicalapplications.

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REV � C3PY00023K_GRABS

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PolymerChemistry

REVIEW

Functionalized po

PMNDNftftftra

including polymersomes and nanfor delivery of “difficult to deliver”

aDepartment of Pharmaceutics, National I

Research (NIPER), S.A.S. Nagar, Mohali 1

niper.ac.in; Fax: +91-172-2214692bDepartment of Orthopaedic Surgery, Institut

of Connecticut Health Center, 263, FarmingcDepartment of Drug Metabolism and Pharm

Biomedical Research (NIBR), Hi Tech City,

Pradesh, India

Cite this: DOI: 10.1039/c3py00023k

Received 5th January 2013Accepted 4th February 2013

DOI: 10.1039/c3py00023k

www.rsc.org/polymers

This journal is ª The Royal Society of

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lymersomes for biomedicalapplications

Prasad V. Pawar,a Shalini V. Gohil,b Jay Prakash Jainc and Neeraj Kumar*a

Polymersomes are latest entry to the drug carrier systems and have proven their utility to deliver

therapeutic agents to specific tissues/organs due to their versatile polymeric architectures and self-

assembly of polymeric chains into vesicular containers of therapeutic agents. Recent research has

focused on the development of multi-functional polymersomes as targeted drug delivery systems for

combined therapeutic applications and theranostic applications where therapeutic delivery via passive

or active targeting can be simultaneously combined with diagnostic capabilities. Functionalized

polymersomes have been prepared by (a) conjugation of functional ligands to preformed polymeric

vesicles, (b) self-assembly of end-group functionalized block copolymers, or (c) use of polymers with

functionalized hydrophilic blocks. This review focuses on various strategies used for developing

functionalized polymersomes, as a means to achieve multiple goals of therapy as well as diagnosis by

use of specific ligands, while maintaining their ability to encapsulate and deliver desired therapeutic or

diagnostic principles. Various ligands used for such functionalization have been discussed with a focus

on cellular/tissue targeting as well as other biomedical applications of the same.

rasad V. Pawar completed his.S. (Pharmaceutics) with Dreeraj Kumar in 2010 from theepartment of Pharmaceutics,IPER-S.A.S. Nagar, India. Heurther continued his Ph.D. inhe same lab. His Ph.D. workocuses on the oral delivery ofailor-made nano-polymersomesor anti-cancer agents for thereatment of breast cancer. Hisesearch interests are indvanced drug deliveryotechnology based approachesmolecules.

nstitute of Pharmaceutical Education &

60062, Punjab, India. E-mail: neeraj@

e for Regenerative Engineering, University

ton Avenue, Farmington, CT, 06030, USA

acokinetics (DMPK), Novartis Institute of

Madhapur, Hyderabad-500033, Andhra

Chemistry 2013

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1 Introduction

Polymersomes are one of the most interesting and versatilevesicular architectures among various self-assembled systemsfor emerging applications in elds ranging from drug deliveryto diagnostics. These carrier systems are prepared by the self-assembly of amphiphilic block copolymers containing two ormore chemically distinct monomer sequences joined bya covalent bond that prevents blocks from macrophase

Shalini V. Gohil received herM.S. (Pharmaceutics) in 2006and Ph.D. in 2011 under theguidance of Dr Neeraj Kumar,from NIPER-S.A.S. Nagar, India.Her Ph.D. project focused ondevelopment of an injectablebiomimetic composite gra forbone regeneration. She hascontributed 14 articles includingpatents, original research arti-cles, reviews and book chapters.She is currently working as

a postdoctoral fellow in Department of Orthopaedic Surgery,Institute for Regenerative Engineering, University of ConnecticutHealth Center, CT, USA. Her research interests include injectablebone gras, biomaterials for drug delivery and regenerativemedicine and nano-assisted regenerative medicine.

Polym. Chem., 2013, xx, 1–18 | 1

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Polymer Chemistry Review

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separation upon dissolution. Such a “polymeric approach” tovesicle formation helps to broaden the range of achievabledesired physicochemical properties by variation of the amphi-philic copolymer chemistry that enables tunable design ofpolymersomes.1,2 The dimensions and morphologies of thesestructures can be controlled by varying the chemical constitu-tion and size of the copolymer, the preparation methods andthe solution properties such as initial copolymer concentration,pH, temperature, and solvent type.3–7 Due to their hollow andspherical morphology, polymersomes are capable of encapsu-lating various agents within the vesicle core or in the hydro-phobic bilayer depending on the characteristics of theencapsulant. The aqueous core of polymersomes is separatedfrom the outside medium by a hydrophobic membrane whichmakes them a vesicle-like structure with an aqueous inner coresurrounded by a hydrophobic periphery and thus makes themsuitable as drug carriers for hydrophobic, hydrophilic andamphoteric drug molecules. In general, hydrophilic moleculesare encapsulated within the central aqueous core, whereashydrophobic molecules are entrapped in the hydrophobicbilayer membrane.8 Previously published literature may beconsulted for details on basic aspects of “polymersomes”including requirements of polymer composition for self-assembly, their advantages, preparation methods (Table 1) andapplications as a drug delivery system as well as their charac-terization techniques.9–12

Polymersomes may be formulated in the size range of tens ofnanometers to several micrometers in diameter, and their sizecan be optimized for specic applications. In the case ofnanosized drug carriers including nanopolymersomes, theunique size scale of the particles enables achievement of anenhanced-permeability-and-retention (EPR) effect in tumortargeting. Utilization of the unique tissue physiology of thetarget for delivering active compounds is termed as passivetargeting and is used to enhance the therapeutic effect of a drug

Jay Prakash Jain completed hismasters and Ph.D. in 2005 and2010 respectively from NIPER-S.A.S. Nagar, India under theguidance of Dr Neeraj Kumar.He has worked on novel drugdelivery system using tailor-made biodegradable polymersand on the development andevaluation of amphotericin-Bloaded nano-polymersomeformulations. His work involveddiverse area of chemistry to

preclinical evaluations of DDS. He has contributed 21 articlesincluding patents, original research articles, reviews and bookchapters. In 2010, he joined Novartis Institute of BiomedicalResearch (NIBR) and is involved in clinical development of drugsfrom drug metabolism and pharmacokinetics (DMPK) perspective.

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with minimum adverse effects.13–16 Targeting may also be ach-ieved by various means such as localized external energy acti-vation, for instance pH17 or temperature change in the tumormicroenvironment.18 Further, active targeting may be accom-plished by specic recognition of target cells by carrier-conju-gated molecules. In the case of cancer, a biomarker expressedon the surface of cancer cells may be identied and its cognatebinding vector may be loaded onto probes/carriers to achieverecognition and tumor targeting. A wide variety of targetingligands have been used to guide nanoparticles toward the site ofinterest, including small organic molecules, oligosaccharides,aptamers, peptides, antibodies and other proteins, withmolecular weights ranging from a few hundred to tens ofthousands of Daltons.

Recent research in the eld of polymersomes has focused ondevelopment of multi-functional polymersomes for targetingspecic tissues/organs, combination therapeutic applications,and for theranostic applications which simultaneously providetherapeutic delivery via passive or active targeting as well asdiagnostic capability. These multi-functional vesicles arecapable of simultaneously loading therapeutic agents alongwith agents including but not limited to biomarkers, anti-bodies, optical imaging agents etc. into their hydrophobic andhydrophilic regions. Multi-functional polymersomes may beprepared by any of the following methods: (a) conjugation offunctional ligands to preformed vesicles, (b) self-assembly ofend-group functionalized block copolymers, or (c) use of poly-mers with functionalized hydrophilic blocks.19

The current review focuses on recent developments in thepreparation methods and applications of functionalized poly-mersomes as a means to achieve multiple goals of therapy aswell as diagnosis by specically targeting, binding, or adheringto certain surfaces or cells while maintaining their ability toencapsulate and deliver desired therapeutic or diagnosticprinciples. The strategies as well as the ligands used for such

Dr Neeraj Kumar studied at IIT-Roorkee, (India) and obtainedhis Ph.D. in 1999. He was asso-ciated with Professor Avi Dombfor 2 years as Lady Davis fellowat HUJI (Israel) and one yearwith Professor Ram Mahato atUTHSC (USA). He joined NIPER-S.A.S. Nagar, India as assistantprofessor of Pharmaceutics inJuly 2003. He has guided 7 Ph.D.students and 39 masterstudents. He has contributed

one edited book and one journal issue “Advanced Drug Deliverysystems”, 85 articles including research articles, reviews, book-chapters and led 13 patents based on the technologies developedin his lab. His research interests are in biomaterials, drug delivery,polymersomes-based carrier systems, nanotechnology and tissueengineering.

This journal is ª The Royal Society of Chemistry 2013

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Table 1 Methods for polymersome preparation along with their properties

Method Description Polymersome properties Ref.

1. Solvent free methods The amphiphilic polymer is broughtin contact with the aqueousmedium in its dry state and issubsequently hydrated to yieldvesicles

— 21

1.1. Film rehydration method Polymeric lm is formed on theglass surface and then aqueousbuffer is added for the hydration ofthe lm

Small multilamellar structures areformed having broad sizedistribution

2, 22

1.2. Polymer hydration method Amphiphilic polymer is nothydrated as a thin lm on a surfacebut is directly hydrated as bulkpowder in aqueous buffer solution

Small multilamellar structures areformed having broad sizedistribution

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1.3. Electroformation method The polymer lm is spread on a pairof electrodes and electric current isapplied aer addition of aqueousbuffer to facilitate hydration

Polymersomes having diameter inmicrometer range are formed withexcellent monodispersity. However,low yield is obtained

2, 22, 23

2. Solvent displacement methods Amphiphilic polymer is rstdissolved in an appropriate organicsolvent and then mixed with water.The organic phase is subsequentlyremoved with an appropriatetechnique

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2.1. Solvent injection method Amphiphilic polymer is dissolved inan appropriate organic solvent orsolvent mixture which is then addeddropwise to an aqueous bufferunder vigorous stirring

Polymersomes with broad sizedistribution are formed

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2.2. Double emulsion method Uniform double emulsion is formedusing microuidic technique,wherein droplet-in-drop or core–shell structures (water dropletssurrounded by a layer of organicsolvent) are dispersed ina continuous water phase

Both unilamellar and multilamellarvesicles are formed with highlyuniform size

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Review Polymer Chemistry

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functionalization have been discussed with a focus on the drugdelivery as well as diagnostic applications of the same.

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2 Ligands/molecules used forfunctionalized polymersome

A wide variety of targeting molecules have been assessed, withvarying degrees of success, for their potential application indrug delivery. These include antibodies, peptides, carbohy-drates and small organic molecules.

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2.1 Antibodies

Antibodies were initially believed as “targeting missiles” whichhit their specic biological targets. However, their use is muchmore complex for targeting and biological properties than theexpected approach by the researchers. There are various anti-bodies which have been found to be very effective and suitablefor the treatment of different malignancies. Various types ofmonoclonal antibodies have been approved by FDA and haveemerged as important therapeutic agents as they have bothtargeting ability and anticancer activity. Antibodies such as


trastuzumab, gemtuzumab and bevacizumab show their tar-geting ability by interacting either with tumor antigens to altertheir cell signaling pathway or by binding with overexpressedreceptors present on the tumor cells.

Trastuzumab is an anti-HER2 monoclonal antibody whichinhibits cell proliferation by acting on HER2 oncoproteinreceptors overexpressed on the tumor cells.20 Bevacizumab isa humanizedmonoclonal antibody which has been used to treatadvanced stages of cancer. It specically inhibits vascularendothelial growth factor (VEGF), an endothelial-cell-specicmitogen or VEGF receptor signaling which acts as the keyregulator in tumor angiogenesis, thereby reducing cell prolif-eration.26 Various antibodies have been used for the preparationof functionalized polymersomes to target different diseasessuch as inammation, memory impairment and cancer (Table2). Anti-EGFR antibody-coupled polymersomes were used fortargeting cancer cells as they are a very attractive candidate ofEGF receptors which are present on normal cell surfaces but areoverexpressed on the surface of different cancer cells includinglung, colorectal and breast cancer.27 These antibodies facilitateendocytic uptake of polymersomes to improve their antitumor

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Table 2 Ligands and their targets used for enhanced therapeutic efficacy of functionalized polymersomes

Ligand Target for ligandDiseaseindications Advantages Disadvantages References

AntibodiesOX26 Transferrin

receptorsMemoryimpairment

(1) Have a high degree of specicity for thetarget tissue

(1) Expensive and time-consuming toproduce

27, 28, 36

Anti-EGFR EGF receptor Tumortargeting

(2) Wide range of binding affinities can beachieved

(2) Suffer from various stability issues

(3) Have intrinsic cytotoxicity due to theirability to interfere with molecules thatstimulate cell proliferation anddifferentiation leading to improved anti-tumor efficacy

(3) Highly immunogenic and showsdeleterious side effects(4) Poor in vivo mobility and thereforedelay/reduced uptake over desired target

(4) The cells are targeted in two distinctways: (a) altering the signaling pathway ofcell and (b) showing cytotoxic activity onthe cell

(5) Large size and high molecular weight,hence attaching multiple ligandS is notpossible

PeptidesRGD Integrin avb6

adhesion receptorsTumortargeting

(1) Rapid blood clearance (1) Very susceptible to in vivo proteolyticdegradation

32, 33,37–41

Tet1 Trisialogangliosideclostridial toxinreceptor

Sensorineuralhearing Loss

(2) Ease of penetration of the tumor’svascular endothelium

Tat Dendritic cells Neurons (3) Increased diffusion rates in tissuePR_b Integrins expressed

on cancer cellsProstatecancer

(4) Low immunogenicity

Lactoferrin Low-densitylipoproteinreceptor-relatedprotein (LRP)

Alzheimer’sdisease

Transferrin Transferrinreceptors

Braintargeting

CarbohydratesMannose Lectin

concanavalin A— (1) Specically interact with only lectins (1) Weak binding affinity 42–44

(2) Targeted to whole organ, so somenormal cells can be affectedGlucose Protein receptors —

Small organic moleculesFolate Folate receptors Breast cancer (1) Absence of immunogenicity (1) Lack specicity in targeting 36, 45–53Biocytin Scavenger receptor

A1 (SRA-1)Macrophagetargeting

(2) Better penetration into the tumor cellsas the size is very small

(2) Ligands such as folate and transferrincan be found in signicant levels in bodyuids and the free ligand will compete forbinding with the targeted therapy

(3) Small molecules are oen chemicallystable and inexpensive to manufacture

Biotin Scavenger receptorA1 (SRA-1)

Macrophagetargeting

(4) Attaching multiple ligands is possible(5) Readily available and easy to handle


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efficacy. Polymersomes functionalized with mouse-anti-ratmonoclonal antibody OX26 have been used for brain targetingas they specically bind to transferrin receptor sites present inthe brain.28
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2.2 Peptides

Although antibodies are found to be an interesting and viableoption for targeting nanocarrier systems, they are highlyimmunogenic and produce deleterious side effects. They showpoor in vivomobility and therefore delay/reduce uptake in to thedesired target.29,30 Further, they have relatively large size andhence it is difficult to attach multiple molecules onto eachnanocarrier. This in turn indicates that ligand molecules

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should have small size and peptides fulll this requirementremarkably. Further, as compared to antibodies, peptides notonly target the specic surface receptors but can also penetrateinto cells or tissue to cause cell toxicity. These peptides may bepre-designed to target specic antigen epitopes, as an alterna-tive to antibodies. In general, peptides show their action byacting as specic ligand for receptors present on cells/organssuch as tumor cells, stroma or neovasculature. They can also acton specic surface epitopes or with subcellular component likemitochondria.

Various peptides such as Tat, Tet1, RGD and PR_b peptideshave been used for targeted delivery of polymersomes fordifferent applications. Tat peptide has been used as a targetingligand due to its ability to penetrate cells and hence is known as



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cell penetrating peptide (CPP). Christian et al. prepared Tatfunctionalized polymersomes for theranostic application toachieved combined dendritic-cell-based immunotherapy and invivo uorescence imaging.31 Tet1 functionalized PEG-b-PCLpolymersomes were used for the treatment of SensorineuralHearing Loss (SNHL) and showed strong affinity to PC12 cells,dorsal root ganglion cells and primary motor neurons; andmore specically interacted with trisialoganglioside clostridialtoxin (GT1b) receptors expressed on cochlea.32 Tripeptide RGDfunctionalized nanocarriers are most oen used for neo-vasculature delivery of therapeutic agents. They have also beenused for cell and diagnostic imaging and in gene therapy. RGDinteracts with avb6 receptors which are overexpressed oncancerous endothelial cells. Although peptides have manyadvantages in comparison to antibodies, they are very suscep-tible to in vivo proteolytic degradation. They undergo cyclizationif their NH2 and COOH terminals are unprotected.33

2.3 Carbohydrates

Carbohydrates play a vital role in both physiological and path-ological events such as cell growth and differentiation, cell–cellcommunication, inammatory response, tumor metastasis andviral infection. They show highly specic interactions withendogenous lectin, a carbohydrate binding glycoprotein whichis expressed on mammalian cell surfaces.34 For instance,galactose and mannose could recognize the asialoglycoproteinreceptor present only on hepatocytes and thereby serve aseffective liver targeting ligands.35

In a study, mannose functionalized polymersomes wereprepared with both short and long rod coil amphiphilic blockcopolymers to assess the effect of supramolecular architectureon the binding activity of polymersomes against the proteinreceptors. It was observed that short rod coil block copolymerself-assembles into vesicular structures leading to the forma-tion of polymersomes whereas long coil block copolymer formscylindrical micellar structures. This difference in the formationof supramolecular architecture had a signicant effect on thebinding activity against protein receptors as determined byhemagglutination inhibition assay. Further, it was alsoobserved that mannose functionalized polymersomes whenincubated with Escherichia coli specically bind to the bacterialpili of the ORN 178 strain.43 Glucose conjugated polymersomes/glycosomes composed of polybutadiene-block-polystyrene werealso prepared by conjugating the thiol derivative of glucose withthe double bonds of the copolymer through photoadditionreaction.44

2.4 Small organic molecules

Small organic molecules have also been an important class oftargeting moieties which have great potential as targetingligands. These molecules have very diverse structures andproperties which make them very much suitable for function-alization of polymersomes. The small size and low molecularweight of these molecules allow the attachment of multipleligands onto polymersomes. Folic acid is one of the most widelyused targeting ligands due to its high binding affinity toward


receptors that are overexpressed on cancer cells.45 There aremany examples of folate functionalized nanocarrier systemsused for drug targeting, including polymeric nanoparticles,53

dendrimers,48 liposomes54 and polymersomes.46,47

Yang et al. developed pH responsive multifunctional poly-mersomes for combined tumor targeting of doxorubicin andsuperparamagnetic iron oxide (SPIO) nanoparticles. The tri-block copolymer poly(glutamate hydrozone doxorubicin)-poly(-ethylene glycol)-acrylate was functionalized at the acrylateterminal with folic acid (FA) to achieved active tumor targeting.The results of cellular uptake studies reveal the higher uptake ofFA-functionalized polymersomes in comparison to FA-free pol-ymersomes eventually leading to higher cytotoxic activity.46

Similar results were observed for FA-functionalized polymer-somes composed of PEG-PLA-PEG-acrylate triblock copolymer.These FA-functionalized polymersomes are taken up by thefolate receptor-mediated endocytosis process, leading to highercellular uptake and cytotoxicity in the HeLa human cervicaltumor cell line in comparison to FA-free polymersomes.47

3 Strategies for functionalization ofpolymersomes

Functionalization of polymersomes with different ligandsspecically aims to increase the accumulation of drug at thetargeting sites while simultaneously reducing the amount ofdrug reaching normal cells. This leads to reduction in dose,improves the efficacy and reduces its side effects. Functionali-zation of polymersomes can be achieved in three major ways: (a)conjugation of functional ligands to preformed vesicles, (b) self-assembly of end-group functionalized block copolymers, or (c)use of polymers with biofunctional hydrophilic blocks. A briefoverview of the various functional group/molecules used forfunctionalization of polymersomes with regards to both copol-ymers and ligands is summarized in Table 3.

3.1 Conjugation of functional ligands to preformedpolymersomes

Conjugation of ligands onto the surface of preformed poly-mersomes may be done either through a non-covalent bindingapproach or by a covalent binding approach. Both theseapproach have their merits and demerits as discussed below.

3.1.1 Non-covalent binding approach. This approachenables attachment of multiple ligands onto the surface ofpolymersomes with higher density due to the rearrangement ofligands and functional attached groups.

3.1.1.1 Biotin–streptavidin binding approach. A schematicrepresentation of the biotin–streptavidin binding approach isshown in Fig. 1. This approach has been used to functionalizepolymersomes with oligonucleotide poly(guanylic acid) (poly-G)which is a specic ligand for the SRA1 receptor present on themacrophage cells. The ends of PMOXA–PDMS–PMOXA triblockcopolymer were functionalized with biotin in the presence ofdicyclohexylcarbodiimide (DCC) and 4-(dimethylamino)pyri-dine (DMAP). These functionalized polymersomes along withbiotinylated-polyG ligand were incubated in a slight excess of

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Table 3 Functional groups/molecules utilized for functionalization of polymersomes

Method offunctionalization

Required molecule/functional group

Implication for targeted therapeutics ReferencesCopolymer Ligand

Non-covalent binding approachBiotin–streptavidinbinding approach

Biotin Streptavidin Targeting to SRA1 receptor present on the macrophage cells 51

NTA metal complexationapproach

Ni2+ NTA Histidine Targeting to oligohistidine sequence

Adamantane cyclodextrinbinding approach

Cyclodextrin Adamantane Attachment to enzymes on the polymersome surface for performing catalyticreaction

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Covalent binding approachClick chemistryconjugation reaction

Alkyne/azide Alkyne/azide Attaching peptides and enzymes on the surface of the polymersomes 56–59Used for uorescent tags in theranostic applications

Conjugation reaction viabis-arylhydrazone bond

4-Formylbenzoicamide

6-Hydrazino-nicotinicamide

Conjugating antibodies such as anti-biotin IgG and trastuzumab topolymersomes surface to target biotinylated surface and HER2 receptorsexpressed on breast cancer cells SKBR3

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Fig. 1 Schematic representation of the coupling of polyG with polymersomesvia biotin–streptavidin affinity interaction: (a) coupling of biotinylated polymer-somes with streptavidin, (b) subsequent incubation with biotinylated polyguanilicacid3 to render ligand-labeled polymersomes, (c) schematic representation of themode of action by receptor–ligand targeting.


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streptavidin to form a biotin–streptavidin–biotin bridge acrossthe polyG ligand and polymersome surfaces.51

3.1.1.2 Nitrilotriacetic acid (NTA) metal complexationapproach. The NTAmetal complexation approach has been usedto conjugate various proteins onto the surface of polymersomes.Polymersomes were prepared using an amphiphilic PBD-b-PEGdiblock functionalized with NTA end groups. Aer the forma-tion of polymersomes, the surface functionalized NTA endgroup was reacted with Ni2+ or Cu2+ ions to form a NTA metalcomplex. This complex specically binds to oligohistidinesequences of proteins. Taking advantage of this specic inter-action with the NTA metal complex, maltose binding proteincontaining a decahistidine moiety at its terminal and a His-tagged enhanced green uorescent protein were conjugatedonto the surface of polymersomes. The conjugation of proteinonto the surface of polymersomes was conrmed by analyzing

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the NTAmetal complex using UV-Vis and electron paramagneticresonance spectroscopy.60 This approach of conjugatingproteins has also been used for liposomes.61

3.1.1.3 Adamantane cyclodextrin binding approach. Thehost–guest interaction between adamantane and cyclodextrinfor surface functionalization of polymersomes was used byFelici et al.55 The polystyrene homopolymer containing per-methylated b-cyclodextrin (b-CD) as a terminal group was usedfor formation of polymersomes and surface functionalizationwas done by conjugating the enzyme Horse Radish Peroxidase(HRP). HRP was modied by tagging with adamantine usingPEG as a spacer. Specic interaction betweenmodied HRP andpolymersomes was investigated by measuring the catalyticactivity of HRP enzyme. Catalytic activity was observed only inthe case of polymersomes conjugated to adamantane modiedHRP and was absent in the control experiment wherein HRPwas not modied with adamantane. The catalytic activity ofpolymersomes coated with HRP enzyme was assessed aercarrying out multiple steps. The catalytic activity of polymer-somes was found to decrease as the amount of HRP enzyme onthe surface of polymersomes and as the number of washingsteps increased. This indicated the interaction between cyclo-dextrin and adamantane is too weak to withstand the washingprocedure.55

3.1.2 Covalent conjugation approach. The principleadvantage of this approach includes increased ligand bindingstability with improved site specicity and reproducibility.

3.1.2.1 Click chemistry conjugation reaction. The concept ofclick chemistry was introduced by Sharpless and coworkers in200162 which involves the generation of large substances byadjoining small units together with heteroatom links (C–X–C) inbetween. For click chemistry, the reaction should:

(a) have simple reaction conditions but with high thermo-dynamic driving force;

(b) be insensitive to oxygen and water;(c) allow efficient and easy transformation of starting mate-

rials into new substances with useful properties;



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(d) enable easy product isolation: if purication is required itmust be by nonchromatographic methods, such as crystalliza-tion or distillation, and the product must be stable underphysiological conditions;

(e) generate only innocuous waste products that can beremoved by nonchromatographic methods;

(f) give very high yield with the product being stable atphysiological conditions.

van Hest and co-workers used click chemistry reactions forconjugating different ligands on the surface of polymersomes.The polymersomes composed of azide terminated poly(styrene)-block-poly(acrylic acid) (PS-b-PAA) diblock copolymers wereconjugated to ligands such as an alkyne bearing uorescentdansyl probe, biotin and eGFP.56 The reaction was carried out bysubjecting polymersomes to an aqueous solution of acetylene-functionalized dansyl probe in combination with a coppercatalyst using tris(benzyltriazolylmethyl)amine (TBTA) asa ligand (Fig. 2). Aer completion of reaction within 24 h, theunreacted dansyl probe and catalyst were removed by extensivedialysis against a 0.55 mM solution of EDTA. A referencecompound was made by attaching dansyl probe to PS-b-PAA tomeasure the degree of functionalization of polymersomes. Theresults showed that 23% of azide moieties present on poly-mersomes were functionalized. The probable reason forachieving a relatively low degree of functionalization wasreported as inaccessibility of the azide functionalities present inthe interior of the polymersomes.

Azide–alkyne click chemistry was also used in polymersome–protein conjugates which act as nanoreactors to perform

Fig. 2 Surface functionalization of azide terminated polymersomes with analkyne bearing fluorescent dansyl probe and biotin.56


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cascades of enzymatic reactions. Polymersome encapsulatedwith two enzymes, viz. glucose oxidase (GOx) and Candida ant-arctica lipase B (CalB), was functionalized by conjugating HRPon its surface. Azido groups were introduced on the HRPenzymes by reacting them with imidazole-1-sulfonyl azidehydrochloride. This reaction was carried out in aqueous envi-ronment in the presence of Cu(II) which acts as a catalyst. Theazido-HRP was conjugated to the acetylene anchors present onthe surface of polymersomes.57

Martin et al. also used a click chemistry approach to func-tionalize poly(butadiene-block-ethylene oxide) polymersomeswith dendritic and nondendritic mannose derivatives, a well-known multivalent ligand (Fig. 3). They make use of alkynefunctionalities present on the mannosylated dendritic structureand the azide functional group on the polymersome surface.The functionalized polymersomes were assessed for theirbinding affinity towardsmannose binding protein concanavalinA using hemagglutination assay. The experimental resultsrevealed that polymersomes functionalized with dendriticderivatives of mannose showed around 2-fold enhancement inthe relative binding affinity towards concanavalin A incomparison to the nondendritic derivatives.58

Azide–alkyne “click” chemistry reaction was also used tofunctionalized performed polymersomes with targetingpeptide, viz. GRGDSP and PR_b. Polymersomes composed ofpoly(ethylene oxide)-b-poly(1,2-butadiene) diblock copolymerconsisting of either hydroxyl or azide functionality specicallyreact with the N-terminal of targeting peptide. This specicreaction allowed the targeting peptide to orient on the surface ofthe polymersomes.59

3.1.2.2 Conjugation reaction via bis-arylhydrazone bond.Proteins can also be conjugated onto the surface of polymer-somes via formation of covalent aliphatic imine bonds.Conjugation chemistry of the bis-arylhydrazone bond was usedto specically conjugate oligonucleotides to antibodies63 and tobind protein to other proteins.64 An imine bond was formed bythe reaction between the amino group of protein and thealdehyde group produced on the surface of the polymersomes(Fig. 4). However, this method of conjugating protein to poly-mersomes led to the formation of large aggregates. The iminebond formed in this reaction was unstable due to hydrolysis inthe aqueous environment. As compared to aliphatic imines,aromatic imines based on bis-arylhydrazone bonds were very

Fig. 3 Polymersome functionalized with nondendritic and dendritic biologicalligands.

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4 5

Fig. 4 Stable bis-arylhydrazone bond formation in the specific reaction between 4FB modified polymersomes and HyNic modified ligands.


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stable in aqueous solution due to their large delocalized elec-tron system. A bis-arylhydrazone bond was formed between twoheterobifunctional linkers: (1) aromatic hydrazine and (2)aromatic aldehyde. This type of conjugation reaction can becarried out in aqueous environment without using any catalystin contrast to click chemistry reactions which require toxiccatalysts such as copper (Cu(II)).

The strategy of conjugating proteins to polymersomesurfaces with stable bis-arylhydrazone bonds was rst used byEgli and co-workers.52 Polymersomes composed of poly(-dimethylsiloxane)-block-poly(2-methyloxazoline) diblock copol-ymers were functionalized with 4-formylbenzoate (4FB) to getaldehyde groups on the surface. Simultaneously, the targetingantibodies were functionalized with 6-hydrazinonicotinateacetone hydrazone (HyNic) to get hydrazone groups (Fig. 4). Egliintroduced complementary reactive functional groups in poly-mersomes as well as in the targeting ligand. The functionali-zation of polymersomes and ligand with NHS-4FB and (NHS-HyNic) respectively was conrmed by a colorimetric reactionwith 2-hydrazinopyridine and by gel permeation chromatog-raphy of the polymer. Both these conjugation reactants wereallowed to incubate for 16 h in phosphate buffer (pH 6) to formstable bis-arylhydrazone bonds. The approximate number ofeYFP molecules binding per polymersome was determinedusing the FCS autocorrelation curve of the eYFP–polymersomeconjugate. Using the same strategy, various antibodies such asanti-biotin IgG and trastuzumab were conjugated to polymer-some surfaces to target biotinylated surfaces and HER2 recep-tors expressed by breast cancer cells SKBR3.52 Targeted uptakeof polymersome–trastuzumab conjugates was studied by incu-bating SKBR3 cells for 2 h with sulforhodamine-B-containingpolymersome–trastuzumab conjugates and sulforhodamine-Bcontaining polymersomes without trastuzumab.52

3.1.3 Miscellaneous covalent conjugation reactions. Thereare various other strategies for functionalization of polymer-somes which make use of different functional groups. Theseinclude thiol, vinyl sulfone, amine hydroxyl, amine, and alde-hyde which are either present on the surface of the polymer-somes or on the targeting ligand.

8 | Polym. Chem., 2013, xx, 1–18

3.1.3.1 Thiol and vinyl groups. Petersen and coworkerssynthesized and characterized vinyl sulfone-terminated poly(-ethylene oxide)-block-poly(g-methyl-3-caprolactone) amphi-philic diblock copolymer.65 In an aqueous environment, theself-assembling behavior of this copolymer leads to formationof polymersomes with highly reactive vinyl sulfone groups ontheir surfaces. Targeting is achieved by conjugating thiol groupsof the targeting peptide to the vinyl sulfone present on thesurface of polymersomes. This conjugation reaction is veryeffective as the vinyl sulfone group, being highly reactive, reactsvery rapidly (within 1 h) with the thiol group via a Michaeladdition reaction.

3.1.3.2 Hydroxyl and amino groups. Egli et al. used amineand hydroxyl end groups present on the surface of PDMS-b-PMOXA polymersomes for conjugating uorescent dye AlexaFluor 633 on the surface of polymersomes to understand theeffects of functional group concentration on polymersomesurface modication.52 They measured the number of uo-rophores attached per polymersome using FCS. Conjugation ofuorescent dye to polymersomes showed a linear correlation atlow functional group concentration on the polymersomesurface. However, at higher concentrations, saturation wasobserved aer the uorescent dye caused steric hindrance forfurther attachment on the polymersome surface.

3.1.3.3 Aldehyde and amine groups. Foster and co-workersperformed immobilization of polymersomes on aminated glass

Fig. 5 Aldehyde tailored polymersomes covalently immobilized on an aminatedglass surface.



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surfaces by making use of aldehyde groups present on thepolymersome surface (Fig. 5). Aminated glass surface wasprepared by immersing glass plates (cover slides) for 2 h ina solution of 3-aminopropyltriethoxysilane (APTS) in dry THF (1mmol mL�1) to generate free amino groups on the glass surface.Different block copolymer (PCL-b-PEG, PLA-b-PEG, and PI-b-PEG) polymersomes having aldehyde groups on their surfaceswere investigated for the morphological stability when cova-lently attached onto the aminated glass surface. All the poly-mersomes showed different footprint areas and differentshapes due to differences in their bilayer stiffness.66

3.2 Self-assembly of end-group functionalized blockcopolymers

This strategy involves graing of targeting ligand at the endgroup of an amphiphilic block copolymer before the formationof polymersomes.19 The main advantage of this strategy is thatthe purication and characterization of th efunctionalizedpolymer is possible without any difficulty. Further, the end-group functionalized polymer can be mixed with non-func-tionalized polymer in an appropriate ratio to control the surfacedensity of the targeting ligand on polymersomes. However,attachment of the targeting ligand to the amphiphilic blockcopolymer may lead to the disturbance in hydrophilic tohydrophobic balance essentially required for the formation ofpolymersomes.19

Mannose functionalized at the terminal end of tetra(p-phe-nylene)-block-PEG polymer was synthesized for targeting E. coli.The polymersomes formed showed 800-fold increase in thebinding affinity towards pili of ORN 178 E. coli strain.42 Inanother study, galactose-terminated amphiphilic polymers weresynthesized, but the polymersomes formed did not show anybinding affinity to pili of E. coli.43 In the former, mannoserecognized the specic lectin binding protein (concanavalin A)and showed high inhibitory potency, whereas in the latter studygalactose was unable to recognize the specic lectin bindingprotein. This signies the importance of ligand specicity usedfor end-group functionalization of polymersomes.

3.3 Use of polymers with biofunctional hydrophilic blocks

In this strategy, the amphiphilic block copolymers consist ofbiomolecules which act as a hydrophilic block.19 This hydro-philic block can be glycopolymers, linear carbohydrate chains,peptides, proteins or oligonucleotides.67,68 These biomoleculescan also be graed on a polymer backbone as side chain groups.Since these biomolecules act either as hydrophilic blocks orside chain groups, a high degree of functionalization can beachieved at both the inner and outer surface of polymersomes.19

This may enhance the cell interaction in comparison to end-group functionalized block copolymers.

Amphiphilic block copolymers comprising polystyrene asthe hydrophobic block and poly(2-(b-D-glucopyranosyloxy)ethyl)acrylate as the hydrophilic block were synthesized by Liang et al.using atom transfer radical polymerization (ATRP).69,70 Theseblock copolymers self-assemble in an aqueous environment toform different structures including micelles, hollow tubules,


polymersomes, and porous spheres. Further, Lecommandouxand coworkers synthesized poly(g-benzyl L-glutamate)-block-hyaluronan (PBLG-b-HYA) copolymer using the azide–alkyneclick chemistry reaction.71 The block copolymer was used toformulate doxorubicin loaded polymersomes using the nano-precipitation method. Polymersomes were investigated for theirintracellular drug delivery potential onto cancerous cell models,viz. MCF 7 and U87 CD44 expressing cells. Flow cytometry andmicroscopic imaging data suggested that hyaluronan basedpolymersomes efficiently delivered doxorubicin intracellularlyin CD44 overexpressing cancer cells resulting in a higher cyto-toxicity in targeted tissues and reduced cardiotoxicity in tumorbearing rats.72

Lecommandoux and coworkers also reviewed the use ofpolypeptides as the hydrophilic block in the formation ofvesicular structures.73 Polypeptide (hydrophilic block) linked toa hydrophobic polymer can form an amphiphilic block copol-ymer which can self-assemble in an aqueous environment toform polymersomes. Polypeptide based vesicular systems areused for passive targeting by exploiting physiological conditionssuch as temperature and pH prevailing at a disease site.73 Anamphiphilic galactose containing polypeptide block copolymerhas been reported by the group of Lecommandoux and Heise.The sequential ring opening polymerization of benzyl-L-gluta-mate and propargylglycine (PG) N-carboxyanhydrides wascarried out to obtain varying ratios of block lengths. Azidefunctionalized galactose was linked to a poly(PG) block by usingthe Huisgen cycloaddition click reaction. The bioactivity ofglycopeptide polymersomes formed from these copolymers wasassessed by carbohydrate–lectin binding experiments.74 Theblock copolymer with longer glycopeptide blocks showedgreater lectin binding activity as compared to the control group.

4 Functionalized polymersomes inbiomedical applications

From the past few decades, polymersomes have becomeprominent nanocarrier systems in the eld of biomedicalscience. They are of great interest and have the potential forbiomedical applications including targeted drug delivery,cellular targeting, cell imaging and theranostics (Table 4).

4.1 Targeted drug delivery

The current focus in development of drug delivery is on targeteddrug delivery which provides a higher concentration of thera-peutic agents at the site of action while sparing normal tissue.Targeted drug delivery is achieved by functionalization ofnanocarriers with different targeting ligands which are specicto the particular organ, tissues or cells. Functionalized poly-mersomes have many applications in targeting different organs/cells including brain, cochlea, macrophages, and tumors.

4.1.1 Brain targeting. The blood brain barrier (BBB) is themost formidable physiological barrier encountered in thetreatment of cerebral diseases such as epilepsy, meningitis,AIDS, encephalopathy, brain tumors such as glioblastoma, andneurodegenerative diseases such as Alzheimer’s and

Polym. Chem., 2013, xx, 1–18 | 9

Table 4 Various ligands used for functionalization of polymersomes for different applications

Ligand class Type of system Details References

Targeted drug deliveryAntibody Poly(ethylene glycol)-poly(3-caprolactone) (PEG-PCL)

polymersomes conjugated with mouse-anti-rat monoclonalantibody OX26

A novel drug (model peptide) carrier for braindelivery

28

Glycoprotein Self-assembled poly(ethylene glycol)-b-poly(D,L-lactic-co-glycolicacid) (PEG-PLGA) polymersomes conjugated with lactoferrin (Lf-POS)

A uorescent probe (6-coumarin) anda neuroprotective peptide (S14G-humanin)incorporated into Lf-POS for brain delivery

39

Glycoprotein Lf-conjugated (Lf-PS) and Tf-conjugated (Tf-PS) polymersomesprepared using a blend of poly(butadiene-b-ethyleneoxide) : poly(ethylene glycol-b-lactic acid) : maleimide-PEG-PLAin the ratio 7 : 2 : 1

Comparison of Lf-PS and Tf-PS in bEnd.3 cells asthe model of the blood brain barrier (BBB), Tf wasfound to be more effective than Lf in braintargeting

41

Antibody (anti-EGFR)

mPEG-peptide-PDLLA polymersomes immobilized with anti-epidermal growth factor receptor (anti-EGFR) antibody (abEGFR);where peptide sequence is Gly-Phe-Leu-Gly-Phe (GFLGF)

Systemic cancer chemotherapy, enhanced cellularuptake, lysosomal targeting as the peptide linkeris cleavable by the lysosomal enzyme cathepsin B

27

Oligonucleotide polyG ligand functionalized poly(2-methyloxazoline)-b-poly(dimethylsiloxane)-b-poly(2-methyloxazoline) (PMOXA-b-PDMS-b-PMOXA) vesicles with encapsulated pravastatin

PolyG shows affinity to macrophage scavengerreceptor A1 (SRA-1) and helps in cell specicdelivery of pravastatin into macrophage-richatherosclerotic plaques for atherosclerosistreatment

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Peptide (RGDpeptide)

Peptide functionalized poly(ethylene oxide)-b-poly(g-methyl-3-caprolactone) (PEO-b-PMCL) polymersomes

End functionalization with vinyl sulphoneelectrophile at the poly(ethylene oxide) coronaterminus used for site-selective attachment toa thiol containing targeting peptide for targeteddrug delivery

65

Peptide (Tet1peptide)

Tet1 functionalized PEG-b-PCL polymersomes for targeteddelivery of rat cochlear nerve

Polymersomes provide targeting specically totrisialoganglioside clostridial toxin receptorpresent on the cochlear nerve in the inner ear

32

Peptide (PR_b) Tumor necrosis factor loaded poly(ethylene oxide)-b-poly(butadiene) polymersomes functionalized with PR_btargeting peptide

PR_b peptide mimics the cell adhesion bindingsite in bronectin, and adheres effectively tointegrins expressed on the surface of prostatecancer cells

38

Peptide (PR_b) siRNA loaded poly(ethylene oxide)-b-poly(butadiene)polymersomes functionalized with PR_b targeting peptide

PR_b-functionalized polymersomes effectivelydeliver siRNA to both cell lines, viz. T47D andMCF10A cells

75

Peptides (G23,P50)

PBD-PEG polymersomes functionalized with GM1- and prion-targeting peptides for brain targeting

G 23 functionalized polymersomes provide higherbrain targeting in comparison to P 50functionalized polymersomes

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Peptides (A665and A666)

Peptide functionalized PEG-b-PCL polymersomes for effectivetargeting to outer hair cells (OHCs) in rat cochlear explants

Functionalized polymersomes showed abundantand specic internalization in OHCs incomparison to nonfunctionalized polymersomes

77

Enzymes,peptides

Isocyanopeptide-styrene polymersomes with enzymes anchoredat three different locations

Positional assembly of three enzymes inpolymersomes at three different locations,namely, in their lumen (glucose oxidase, GOx), intheir bilayer membrane (Candida antarctica lipaseB, CalB) and on their surface HRP

57, 78

Carbohydrates(mannose)

Mannose conjugated rod coil polymersomes for targeting proteinreceptors

Short and long rod coil mannosylatedamphiphiles were synthesized to formsupermolecular architectures as polymersomesand micellar structures respectively. The bindingactivity towards protein receptors is signicantlyinuenced by the shape and size ofsupermolecular materials

43

Carbohydrates(glucose)

Glucose conjugated polybutadiene-block-polystyrenepolymersomes

Thiol derivative of glucose was conjugated crossthe double bonds of a 1,2-polybutadiene-block-polystyrene through photoaddition reaction

44

Hyaluronan Doxorubicin (DOX)-loaded poly(g-benzyl-glutamate)-block-hyaluronan (PBLG-b-HYA) based polymersomes

Enhanced drug accumulation at the tumor siteowing to passive accumulation (EPR effect) andactive targeting (CD44 mediated endocytosis) inEAT bearing mice

71, 79

Hyaluronan Doxorubicin (DOX)-loaded poly(g-benzyl-glutamate)-block-hyaluronan (PBLG-b-HYA) based polymersomes

Polymersomes efficiently delivered doxorubicinintracellularly in CD44 overexpressing receptorcancer cells resulting in a higher cytotoxicity andreduced cardiotoxicity in tumor bearing rats

72

10 | Polym. Chem., 2013, xx, 1–18 This journal is ª The Royal Society of Chemistry 2013


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Table 4 (Contd. )

Ligand class Type of system Details References

Hyaluronan Docetaxel loaded poly(g-benzyl L-glutamate)-block-hyaluronanbased polymersomes

Increased t1/2 and enhanced uptake of docetaxelloaded polymersomes in Ehrlich Ascites Tumor(EAT) tumor bearing mice

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Protein Bacterial channel forming protein LamB inserted in poly(2-methyloxazoline)-poly(dimethyl-siloxane)-b-poly(2-methyl-oxazoline) polymersomes. LamB serves as a receptor for l phageto trigger the ejection of phage DNA

Such phage mediated DNA loading inpolymersomes provides a stable model system toinvestigate the transport of foreign genes intoeukaryotic cells and has potential applications ingene delivery

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Cellular targetingAntibody Anti-intercellular adhesion molecule-1 (ICAM-1) antibody

functionalized poly(ethylene oxide)-polybutadiene polymersomesVascular targeting where the adhesion propertiesof functionalized polymersomes were examinedunder ow conditions

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Adhesion moiety(biocytin)

Poly(ethylene oxide)-polybutadiene (PEO-PBD) diblock copolymerwhere terminal hydroxyl of the hydrophilic block is modied withbiotin-lysine (biocytin)

Biocytin imparts specic adhesiveness toa polymer colloid coated with avidin henceprovides an opportunity to investigate the role ofavidin–biotin ligation on adhesive strength

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Adhesion moiety(biotin)

Biotin-functionalized poly(2-methyloxazoline)-b-poly(dimethylsiloxane)-b-poly(2-methyloxazoline) triblockcopolymer

Fluorescent-labeled nanocontainers targetedagainst the scavenger receptor A1 (SRA-1) frommacrophages, showing receptor-specic bindingfollowed by vesicular uptake for diagnostic ortherapeutic medical use

51

Dendritic cell imagingPeptide and NIRemissiveuorophores

Cell permeable peptide, Tat, conjugated to NIR emissive PEO-b-PBD polymersomes

Enable efficient intracellular delivery for trackingdendritic cells and may be used as optical probes

37

Theranostic applicationsGlycoprotein Lactoferrin-conjugated polymersomes prepared using blend of

methoxy poly(ethylene glycol)-b-poly(3-caprolactone) (MPEG-PCL)and a-carboxyl poly(ethylene glycol)-poly(3-caprolactone) (COOH-PEG-PCL) holding doxorubicin and tetrandrine

The design integrates both a BBB and glioma-targeting moiety and a MDR inhibitor forchemotherapy for glioma treatment and is alsocombined with a NIR probe for in vivo imaging

82

Tat peptide Conjugation of Tat peptide to the terminal hydroxyl of NIRemissive polymersomes composed of poly(ethylene glycol)-poly(butadiene)

Combined dendritic-cell-based immunotherapyand in vivo uorescence imaging

31

Folate astargeting ligand

Doxorubicin and superparamagnetic iron oxide nanoparticle(DOX/SPIONP) loaded polymersomes consisting of FA-PEG-P(Glu-Hyd-DOX)-PEG-acrylate)

SPIONPs were entrapped in a hydrophilic core toachieve ultrasensitive magnetic resonanceimaging (MRI) detection, and DOX wasconjugated to the pH sensitive polyglutamatesegment via hydrazone bond for antitumorefficacy

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Folate astargeting ligand

Doxorubicin and superparamagnetic iron oxide nanoparticle(DOX/SPIONP) loaded polymersomes consisting of FA-PEG-PLA-PEG-acrylate

SPIONPs were encapsulated in a hydrophilic coreto achieve ultrasensitive MRI detection, and DOXin the hydrophobic membrane (PLA) forantitumor efficacy

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Parkinson’s. It limits the access of a large number of drugmolecules, including antibiotics, anticancer agents anda variety of central nervous system (CNS)-active drugs, especiallyneuropeptides. Therefore, there is a need to develop improveddrug delivery strategies that will allow the passage of drugmolecules through the BBB in a safe and effective manner.Some of the developed strategies include disruption of the BBBby osmotic and chemical means as well as use of intracerebralimplants. However, these strategies lead to complications suchas cellular stress or injury in neurons and glial cells which isexpressed by the induction of heat shock protein. Moreover,these strategies are only able to deliver drugs across the BBB butare unable to allow the drug to access relevant target sites within


the brain. By using nanotechnology, it is possible to deliver thedrug to the targeted tissue across the BBB while avoidinginjuries to the surrounding brain tissue.

Surface functionalization of polymersomes with variousantibodies such as transferrin (Tf), lactoferrin (Lf) and anti-ratmonoclonal antibody (OX-26) has been reported to improve thedelivery of therapeutic agents to the brain. Tf and Lf are glyco-proteins which have a broad spectrum of functions particularlyin host defence mechanisms against infection and in severeinammation. This broad spectrum of biological functionsrelies on their interactions with numerous cells such as plate-lets, endothelial cells of mesencephalic microvessels anddopaminergic neurons.40 Both these molecules have also been

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proved to cross the BBB via transferrin receptor-mediatedtranscytosis using an in vitro model of the BBB (bovine braincapillary endothelial cells and astrocytes).

Pang and coworkers compared the relative superiority of Tfand Lf conjugated polymersomes (Tf and Lf-PS) in brain drugtargeting. The results of in vitro uptake studies in mice revealedhigher brain uptake of Tf-PS as compared to Lf-PS. A pharma-cokinetics study showed a similar plasma concentration timeprole for both the conjugated ligands. However, Tf-PS showeda 1.84-fold increase in AUC in comparison to Lf-PS. Further, theelimination and distribution rate constants for Lf-PS were 3.12-and 2.11-fold higher in comparison to Tf-PS.83

In another study, Yu et al. developed lactoferrin conjugatedself-assembled poly(ethylene glycol)-poly(D,L-lactic-co-glycolicacid) (PEG-PLGA) polymersomes. They investigated the braintargeting ability of Lf-PS by varying the densities of lactoferrinon the surface of the polymersomes. Three different formula-tions with varying densities of lactoferrin (number of lactoferrinmolecules ranging from 59–268) conjugation per polymersomewas tested intravenously in mice by using 6-coumarin asa uorescent probe. The polymersomes with 101 lactoferrinmolecules were found to be optimal as demonstrated by theBBB permeability surface area. Further, the plasma clearance ofpolymersomes was found to be inversely proportional to thelactoferrin density conjugated on the polymersome surface.Other parameters, such as AUC and percentage of injected doseper gram of brain tissue, were found to increase with increasedin surface lactoferrin densities. However, at higher surfacedensity, a saturation phenomenon was observed resulting indecreased brain tissue accumulation of polymersomes.39 Thereported reason was saturation of lactoferrin specic receptorspresent in the brain.

Pang et al. developed a novel drug carrier for brain delivery,poly(ethylene glycol)-poly(3-caprolactone) (PEG-PCL) polymer-somes conjugated with mouse-anti-rat monoclonal antibodyOX26 (OX26-PO). Pharmacokinetics of 6-coumarin loadedOX26-PO (IV route) reveal a 2.62-fold increase in 6-coumarinconcentration per gram of brain tissue as compared to the non-conjugated PEG-PCL polymersomes, suggesting signicantenhancement of delivering 6-coumarin to the brain for reducingscopolamine induce memory impairment. These results werefurther conrmed using NC1900 (a peptide) loaded OX26-PO.The concentration of NC 1900 per gram of brain tissue wasincrease by 2.36-fold when OX26-PO was employed OX26-PO ascompared to the PEG-PCL polymersomes. Moreover, theyfurther proved that the surface density of conjugated antibodiesalso plays an important role in improving the permeability ofpolymersomes across the BBB.28

Recently Stojanov et al. demonstrated the brain targetingpotential of polymersomes functionalized with ganglioside (G23) and prion (P 50) targeting peptides. Biodistribution studiesin mice show signicantly higher accumulation of G 23 func-tionalized polymersomes in the brain in comparison to P 50functionalized polymersomes. This could be due to strongpromotion of transcytosis of polymersomes acrossthe BBB by G23 peptide.76

12 | Polym. Chem., 2013, xx, 1–18

4.1.2 Cochlea targeting. Traditional strategies such asintravenous or oral administration of therapeutic agents for thetreatment of inner ear diseases is primarily limited due to thepresence of blood-labyrinthine and low circulatory distributionability of the delivery system to the cochlea cells.84 Developmentof multifunctional nano-formulations holds a promising meansfor drug delivery to treat sensorineural hearing loss (SNHL) andMeniere’s disease which are associated with cochlea cells.Functionalization is achieved by coupling ligands onto itssurface which are specic to the various receptors expressed oncochlea. These receptors include tyrosine kinase receptors, tri-sialoganglioside clostridial toxin receptors and neurotrophinreceptors.85

Functionalized poly(ethylene glycol)-block-poly(3-capro-lactone) methyl ether polymersomes were functionalized usingNerve Growth Factor-derived peptide (hNgf EE) to target cells ofthe inner ear. The targetability of functionalized polymersomeswas analyzed on complex multi-cellular in vitro environmentcultures from mouse cochlea and PC-12 cells. The functional-ized polymersomes showed prominent targeting ability asindicated by their higher uptake within spiral ganglion neurons(SGNs) and Schwann cells and nerve bers as compared to non-functionalized polymersomes. However, there was no apparenthair cell loss indicating no toxicity caused due to polymer-somes. Moreover, the uptake of polymersomes was mostly inthe cytoplasmic region but was not in the nucleus thus pre-venting uncontrolled penetration through the nuclearmembrane which might cause toxicity by damaging DNA.Though there is lack of in vivo data, targetability of hNgf EEfunctionalized polymersomes was further conrmed on PC-12cells which express hNgf EE specic tyrosine kinase receptors. Alower level of tyrosine kinase receptor expression minimizes theuptake of functionalized targeted polymersomes.85

Zhang et al. functionalized polymersome surfaces with theTet1 peptide sequence with the aim of targeting polymersomesto cochlear nerve for the treatment of hearing loss and otherneurological diseases. Tet1 peptide specically binds to thetrisialoganglioside clostridial toxin receptor expressed on thecochlea. Intracochlear administration of Tet1 functionalizedpolymersomes showed specic localization into the neurola-ments and migration to the tractus spiralis foraminous regionpresent in cochlea. They were also observed in spiral ganglionsatellite cells of the spiral ganglion as well as in the nerve bersof CNS.32

Peptide functionalized polymersomes composed of poly(-ethylene glycol)-block-poly(caprolactone) (PEG-b-PCL) wereinvestigated as a delivery system for targeting the inner ear.77 Apeptide which specically binds to prestin, a unique protein inthe inner ear that is solely expressed in sensorineural hearingloss, was used to functionalize polymersomes. The targetingability of these peptide functionalized polymersomes wasassessed on outer hair cells (OHCs). Functionalized polymer-somes showed abundant and specic internalization in OHCsand were sparsely internalized in the other cells. In contrast, thenon-functionalized polymersomes were non-specically inter-nalized by different type of cells present in cochlear explants.77



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4.1.3 Macrophage targeting. Targeted drug delivery to themacrophages has been utilized for the treatment of a wide rangeof disease states, including infection, autoimmune disease,cancer and atherosclerosis. Various receptors present on thesurface of macrophages have been explored for drug targeting.These receptors include scavenger receptor A1, complement,bronectin lipoprotein, mannosyl, galactosyl, etc.

Broz et al. functionalized polymersomes with biotinylatedpolyG ligand which is very specic to the scavenger receptor A1present on macrophages. This functionalization was carried outusing biotin–streptavidin coupling agents. Ligand–receptorinteraction studies were carried out by performing cell culturestudies. PolyG functionalized polymersomes showed strikingreceptor–ligand binding interaction with COS-7 cells having theSRA-1 receptor. At low temperature, the polymersomes wereable to bind to the cell surface but were not internalized in thecells. However, at 37 �C the polymersomes were completelytaken up by the cells suggesting active transport processes forpolymersome uptake. Similar results were observed in the caseof THP-1 cells expressing SRA-1 receptors on their surface.However, cells such as COS-7, which is devoid of SRA1 receptor,showed no cell binding and uptake of functionalizedpolymersomes.50

4.1.4 Tumor targeting. Polymersomes composed of mPEG-pep-PDLLA block copolymer were functionalized across thecarboxyl groups of peptide (pep) with anti-EGFR antibodiesusing N-ethyl-N0-(3-(dimethylamino)propyl)carbodiimide/N-hydroxysuccinimide (EDC/NHS) chemistry. Anti-EFGR antibodyis used for targeting since it is very specic to EGF receptors.These receptors are present on normal cell surfaces, howeverthey are overexpressed on different human cancer cell linesincluding breast, colorectal and lung cancer cells. In cellularuptake studies conducted on SKBR3 cells, it was found that anti-EFGR antibody conjugated polymersomes showed enhancedendocytosis within 3 days as compared to the polymersomeswithout the anti-EGFR antibody. Polymersomes without anti-body were also internalized but at a lower rate which might bedue the reduction in the cell membrane interaction because ofthe presence of high PEG content on the surface ofpolymersomes.27

Peptide functionalized polymersomes for the treatment ofprostate cancer were also explored by Kokkoli and coworkers.PR_b is a highly effective integrin targeting peptide that mimicsthe cell adhesion binding site in bronectin. This PR_b peptidewas conjugated onto the surface of poly(ethylene oxide)-b-poly(butadiene) (PEO-PBD) polymersomes. PR_b-functionalizedpolymersomes were found to effectively internalize withinprostate cancer cells aer adhering specically to a5b1 integ-rins expressed on the surface of prostate cancer cells. Inter-nalization of polymer vesicles was found to be dependent on thesurface concentration of PR_b. Cytotoxicity studies for 0%, 1%,5%, 10% and 50% PR_b-functionalized polymersomes wereperformed. It was found for the rst time that internalization ofpolymersomes within the cancer cells was dependent on thesurface concentration of PR_b with the largest increase incytotoxicity occurring when proceeding from 1% PR_b to 5%


PR_b. Polymer vesicles functionalized with only 5% (w/w) PR_bpeptide achieved a 4.4-fold increase in the observed cytotoxicitycompared to non-functionalized polymersomes.38

Recently, Kokkoli’s group utilized the same PR_b peptidefunctionalized PEO-PBD polymersomes for delivery of siRNA tobreast cancer cells in vitro.75 The studies were carried out on twodifferent cell lines, MCF10A and T47D breast cancer cells. PR_bpeptide functionalized polymersomes undergo more effectiveinternalization in both cell lines. In cancerous T47D cellspeptide functionalized polymersomes were 7-fold more effec-tively delivered than non-functionalized polymersomes. More-over, peptide functionalized polymersomes showedsignicantly greater levels of delivery in the cancerous T47Dcells as compared to the non-cancerous MCF10A cells.75

Lecommandoux and coworkers developed hyaluronanconjugated poly(a-benzyl L-glutamate) polymersomes for tar-geting cancer cells expressing CD44 receptor on their surface.Selective targeting of hyaluronan conjugated doxorubicin-loaded polymersomes was investigated on Ehrlich ascites tumor(EAT) bearing mice. Conjugation of hyaluronan on the surfaceof polymersomes was found to increase the circulation time ofpolymersomes as compared to free drug. The percent tumorinhibition rate was increased 2-fold in the case of polymer-somes in comparison to free doxorubicin and control groups,over a period of 30 days aer treatment. Moreover, the life spanof mice treated with polymersomes was increased by 6 times ascompared to those treated with the free drug.79

Polymersomes conjugated with anti-biotin and trastuzumabantibodies showed signicantly faster cell uptake as comparedto polymersomes without trastuzumab which showed very lowcell uptake. Further, polymersome–trastuzumab conjugatesshowed very specic cell attachment and inhibited cell prolif-eration in the range of 14–21% as compared to the polymer-somes without trastuzumab which show inhibition of cells in 7–11%.52

4.2 Cellular targeting

Cellular targeting is of increasing interest in delivering biolog-ically relevant molecules including drugs, genes and proteins.Functionalization of polymersomes with cell adhesion mole-cules such as biotin and biocytin enables the delivery of thera-peutic agents at the desired sites thereby decreasing the toxiceffect on normal cells.

Lin et al. prepared functionalized polymersomes by linkingadhesion molecule biocytin directly to the hydrophilic terminalof PEO-PBD block copolymer. Biocytin is an amide compoundformed from the vitamin biotin and the amino acid L-lysine,which imparts specic adhesiveness to a polymer coated withavidin. The adhesive properties of biotinylated polymersomeswere measured using a micropipette aspiration technique. Thiswas done by measuring the relative adhesive strength of func-tionalized polymersomes toward avidin coated microspheres.The polymersomes in which the length of functionalized poly-mer is greater than the surrounding length of polymer, showedthe greatest adhesion strength. The critical tension of adhesionwas found to be maximum when the length of functionalized

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polymer was the same as that of the surface brush of the poly-mersomes membrane, i.e. at 10 mol% functionalized polymerconcentration. When the length of the functionalized polymerwas found to be greater than the surrounding polymer, themaximal critical tension was found to be 55 mol%. This indi-cates the functionalized polymer length controls the interfacialadhesion of polymersomes to the avidin coated microspheres.49

Further, Lin et al. functionalized polymersomes with an anti-ICAM-1 antibody, using modular biotin–avidin chemistry. Theadhesiveness between an anti-ICAM-1-functionalized polymer-some and ICAM-1-functionalized polystyrene microspheres wasmeasured using the micropipette aspiration technique byquantifying the critical tension for detachment. It was foundthat the adhesion strength increases in proportion to thesurface density of anti-ICAM-1 molecules, suggesting thepossible application of functionalized polymersomes in target-ing inammatory vascular endothelium cells.36

The surface functionalization of HRP on porous polymer-somes was achieved by click chemistry between the acetylenegroup present on the polymersome surface and azido functionsof HRP. The azido group was introduced into HRP by applyinga diazo transfer reaction on the amines of the lysine residuesand on the amino group present at the terminal of HRP. Alongwith HRP , two other enzymes ,viz. glucose oxidase (GOx) andCandida antarctica lipase B (CalB), were incorporated in theaqueous core and lipid membrane with efficiencies of 25% and17% respectively. Polymersomes containing all these threeenzymes act as a nanoreactor for performing the three stepcascade reaction in the oxidation of glucose.57 CalB wasresponsible for deprotecting monoacetylated glucose whereasGOx converted glucose to gluconolactone and hydrogenperoxide. The hydrogen peroxide thus formed was utilized byHRP to convert 2,2-azinobis(3-ethylbenzothiazoline-6-sulfonicacid) (ABTS) to ABTS_+. The HRP conjugation efficiency wasdetermined by covalently linking a commercially availableruthenium complex to free amine of the protein. The quanti-tative detection of ruthenium and, hence, of proteins was doneusing inductively coupled plasma-mass spectrometry (ICP-MS).The conjugation efficiency was found to be more than 90%indicating almost all the surface of polymersomes was occupiedby HRP enzymes.57

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4.3 Dendritic cell imaging

Dendritic cells (DCs) play a crucial role in regulating innate andadaptive immune responses against foreign antigens. Owing tothis, these cells have become prominent targets for the treat-ment of inammatory diseases such as arthritis, psoriasis,Crohn’s disease, cancer and autoimmune diseases. They alsoplay a pivotal role in capturing, presenting and processing ofantigens as well as in controlling an array of responses.86–88 Theability to track DCs in vivo is imperative for the development ofDC-based cellular therapies. Cellular tracking of DCs helps inunderstanding numerous aspects of DC therapy and in evalu-ating DC sources, maturation states, antigen delivery methods,activation stimuli, and the doses and frequencies of DC vacci-nations. Various noninvasive imaging techniques such as

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magnetic resonance imaging (MRI), scintigraphic imaging and19F-MRI have been use for tracking dendritic cells. However,these techniques are not quantitative in nature and requirevarious harmful chemicals such as contrast agents and radio-isotopes. Fluorescence-based imaging is an alternative nonin-vasive technique which makes use of near-infrareduorophores (NIRFs). This approach helps in detecting in vivocells at centimeter tissue depths repeatedly without disturbingthe physiological functions of cells. Another benet of usingNIRF based imaging is that it is extremely sensitive and requiressub-nanomolar concentrations of exogenous contrast agents.

NIR emissive polymersomes functionalized with cellpermeable cationic peptide Tat demonstrated signicantlyenhanced, time dependent uptake by the dendritic cells. Thecontrol NIR emissive polymersomes reach half-maximal uo-rescent intensity at 7 h as against 5 h required for Tat-conju-gated NIR emissive polymersomes. Moreover, the Tat-conjugated NIR emissive polymersomes were found to remainlocalized within the cells for at least three days.37

4.4 Theranostic applications

Theranostics can be dened as the design and fabrication ofmultifunctional integrated nanocarrier systems which candiagnose, deliver targeted therapy and monitor the response tothe therapy.89 Diagnosis is done by imaging to characterize thediverse phenotypes of disease cells whereas targeted delivery oftherapeutic agents eradicates all of the diverse phenotypes.

Among all the nanocarrier systems, nanoparticles90 andpolymersomes82 have been used for theranostic applications.Lactoferrin conjugated NIR emissive polymersomes encapsu-lating doxorubicin and tetrandrine showed enhanced intracel-lular delivery and cytotoxic activity at a signicantly lowconcentration of both doxorubicin and tetrandrine against C6glioma cells compared to the native drug. Enhanced delivery oflactoferrin conjugated polymersomes (loaded with NIR dye) tothe brain was also demonstrated in glioma model rats using invivo imaging. These polymersomes were found to cross the BBBand accumulate at the tumor site in the brain and this wassubstantiated by the tissue distribution of drug which showedthat conjugation of lactoferrin onto the surface of polymer-somes increases its brain uptake compared to non-conjugatedpolymersomes.82 NIR emissive polymersomes functionalizedwith Tat peptide were also prepared for in vivo dendritic celltracking and for dendritic-cell-based immunotherapy.31

Doxorubicin and superparamagnetic iron oxide nanoparticle(DOX/SPIONP) loaded polymersomes consisting of folate (FA)-PEG-(P(Glu-Hyd-DOX)-PEG-acrylate) were developed whereSPIONPs were entrapped in the hydrophilic core to achieveultrasensitive MRI detection and DOX was conjugated to the pHsensitive polyglutamate segment via a hydrazone bond forantitumor efficacy.46 A similar application was reported withpolymersomes composed of a PLA chain as the hydrophobicblock instead of a polyglutamic hydrazone chain.47


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5 Conclusions

This review summarizes the various ligands/moleculesincluding antibodies, peptides, carbohydrate and small organicmolecules and strategies used for functionalization of poly-mersomes. Functionalization of polymersomes can be doneeither using preformed polymersomes or by graing the tar-geting ligand to the amphiphilic polymer prior to the formationof polymersomes. Each of these strategies has its own advan-tages along with certain limitations which need to be overcome.Functionalized polymersomes have been used in many appli-cations including targeted drug delivery. In drug delivery, theyhave shown a reduction in cytotoxicity of many therapeuticagents due to direct delivery of the active moiety to the diseasedtissues/organ. They are also very useful carrier systems forcellular targeting and diagnosis. In theranostic applications,functionalized polymersomes have tried to blur the line ofdemarcation between diagnosis and therapy; however, muchmore progress still needs to be made.

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6 Future directions

“Magic bullets” have long been a vision for drug deliveryscientists, and polymersomes may provide unique opportuni-ties to step towards this Holy Grail due to tailor-made possi-bilities of their structural design. Various approaches andadvancements to augment the delivery of polymersomes tospecic anatomical/physiological sites have been discussed.Although, polymersomes are exible for different methods fortargeting, identication of specic targets and respectiveligands is critical to obviate off-target toxicity and betterpersonalized medicine (for specic groups of patients express-ing the target—especially in oncology). Establishing risk/benetanalysis in relation to long term safety (specically for chronicuse, slightly relaxed for diagnostic applications), efficacy andpharmacokinetics (more importantly toxicokinetics) for theseadvanced systems would be the challenging but most vital stepfor evolving them to clinical use. Testing the formulation inmost relevant pre-clinical species or multiple species would berequired to get better insight into its in vivo behavior inhumans. Another important aspect for drug delivery scientistsis to meet manufacturing (scale-up) and quality guidelines toproduce consistently performing quality products. For diag-nostic applications the road may not be as challenging as fortherapeutic applications; nevertheless, an integrated multi-modality approach is required for development of polymersomeformulations, which because of its exibility provides a uniqueopportunity to achieve this.

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