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Advances in the synthesis of amphiphilic block copolymers via RAFTpolymerization: Stimuli-responsive drug and gene delivery

Adam W. York, Stacey E. Kirkland, Charles L. McCormick

Department of Polymer Science, The University of Southern Mississippi, Hattiesburg, MS 39406, USA

Received 10 September 2007; accepted 14 February 2008Available online 26 February 2008

Abstract

Controlled/living radical polymerization methods, including the versatile reversible additionfragmentation chain transfer (RAFT)polymerization process, are rapidly moving to the forefront in construction of drug and gene delivery vehicles. The RAFT technique allows anunprecedented latitude in the synthesis of water soluble or amphiphilic architectures with precise dimensions and appropriate functionality forattachment and targeted delivery of diagnostic and therapeutic agents. This review focuses on the chemistry of the RAFT process and its potentialfor preparing well-defined block copolymers and conjugates capable of stimuli-responsive assembly and release of bioactive agents in thephysiological environment. Recent examples of block copolymers with designed structures and segmental compositions responsive to changes inpH or temperature are reviewed and hurdles facing further development of these novel systems are discussed. 2008 Elsevier B.V. All rights reserved.

Keywords: Water soluble polymers; Controlled release; Targeted delivery; Bioconjugation; Interpolyelectrolyte complexes; Cross-linked micelles; Stimuli-responsivepolymers

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10192. RAFT polymerization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1019

2.1. RAFT CTAs, initiators, and monomers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10213. Polymeric prodrugs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1024

3.1. Polymer backbone conjugation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10243.2. End-group conjugation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1025

4. Polymeric micelle delivery systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10264.1. Stimuli-responsive micelles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10264.2. Shell cross-linked micelles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1027

5. Polyelectrolyte complexes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10306. Conclusions and outlook . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1032Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1032References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1033List of abbreviations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0

Available online at www.sciencedirect.com

Advanced Drug Delivery Reviews 60 (2008) 1018 1036www.elsevier.com/locate/addr

This review is part of the Advanced Drug Delivery Reviews theme issue on Design and Development Strategies of Polymer Materials for Drug and Gene DeliveryApplications. Paper number 133 in a series by McCormick Research Group entitled Water Soluble Polymers. Corresponding author. Tel.: +1 601 266 4872; fax: +1 601 266 5504.

E-mail address: [email protected] (C.L. McCormick).

1036

0169-409X/$ - see front matter 2008 Elsevier B.V. All rights reserved.doi:10.1016/j.addr.2008.02.006
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1. Introduction

Recent advances in controlled/living radical polymeriza-tion (CLRP) methods are certain to impact future approaches todiagnosis and treatment of infectious and genetic diseases.Specifically, one such technique, reversible additionfragmen-

tation chain transfer (RAFT) polymerization, allows unprece-dented latitude in synthesis of water soluble architectures with

precise dimensions and appropriate functionality for conjuga-tion to and targeted delivery of diagnostic and therapeuticagents. Although to date only limited reports have appearedregarding the construction of such novel drug delivery vehicles,it is apparent that the synthetic control afforded by the RAFT

process and the resulting facile post-polymerization functiona-lization, cross-linking, and bioconjugation are advantageouswhen compared to more conventional processes currentlyutilized. In the following review, the RAFT process will bediscussed, demonstrating its utility for polymerizing a variety of

functional monomers, often directly in water and not requiringprotecting group chemistry. Selected examples of smartpolymeric systems prepared by RAFT or other CLRPtechniques and designed with precise nanoscale dimensions,narrow molecular weight distributions, reactive pendant orterminal moieties and surface functionality will be presented. Insome cases, for example in drug delivery from shell cross-linked micelles or in delivery of polynucleotides frominterpolyelectrolyte complexes, significant advancements in

protection of the active agent, carrier stabilization, andcontrolled release have been realized, although formidablechallenges remain.

Impetus for research on polymeric delivery vehicles has

come from consideration of technical, economic and safetyissues. The fact that viral vectors are very effective at targetingand delivering therapeutic agents to the cytoplasm or the nuclearenvelope of the cell has been well established. However,shortcomings of viral vectors include the invocation of animmunogenic response by the host, the high cost of manu-facturing, and the lack of cell specificity [1]. Thus a number ofnon-viral delivery systems with low immunogenicity yetspecific targeting and controlled activity have been considered.For example, conventional liposomes have been utilized tosequester and transport chemotherapeutics [2], polynucleotides[3], and proteins [4]. Although such systems have been shown

to possess many requisite requirements [1,5] for in vivoapplication including: protection of cargo from degradation,ease of surface modification for targeting, and reasonablemanufacturing costs, disadvantages often are reported such aslimited solubility and partitioning, less than optimal pharmo-kinetics, and liposome instability in the physiological environ-ment[6].

More recently research has centered on polymer basedcarrier systems including polymerdrug conjugates, micelles,

polymersomes, and nanoparticles. A major goal of this work isto mimic the highly evolved trafficking and delivery efficiencyof viral vectors while avoiding the non-specific toxicity andimmunogenicity issues discussed above. Several significant

barriers are currently being addressed. The non-viral carrier

must be able to degrade or dissociate into low molecular weightspecies capable of excretion through the kidneys. In the case ofnon-degradable polymeric carriers, the carrier must be of lowmolecular weight to be efficiently excreted. Both extracellularand intracellular factors present significant challenges in thesuccessful delivery of active agents via non-viral vectors.

Extracellular barriers include, but are not limited to, packagingof the active agents by the carrier, stability and circulation in the

bloodstream, and specific cellular binding; intracellular barriersinclude endosomal release, cytoplasm transport, and release ofthe active species [1].

Prior to the mid 1980s only limited synthetic tools wereavailable to polymer chemists for construction of deliveryvehicles, although Ringsdorf and others, for example, hadeloquently proposed model targeting systems [7,8]. Control of

polymer architecture, molecular weight and molecular weightdistribution, placement of reactive structopendant or structo-terminal functionality, and solubility/dispersion in biologically

relevant media were obstacles inherent to existing polymertechnology. For example, block and star copolymer architec-tures with appropriate functionality could only be achieved withanionic, cationic, or group transfer chain growth polymerizationtechniques with limited types of monomers, often requiring

protecting group chemistry, under stringent conditions in theabsence of water. Fortunately rapid developments in two majorareas: dendrimer synthesis and CLRP now allow control over anumber of necessary design criteria. Although we focus on thelatter area here, excellent reviews on the chemistry and

biological applications of dendrimers are found in recentliterature [9,10].

The major CLRP techniques include atom transfer radical

polymerization (ATRP) [11], stable free radical polymerization(SFRP) [12], reversible additionfragmentation chain transfer(RAFT) polymerization [13,14], and the specialized area ofaqueous RAFT polymerization [15,16]. Although a recentreport[17] suggests some opportunities for SFRP, most effortstoward construction of polymeric delivery vehicles withcontrolled structures and molecular weight have utilizedATRP or RAFT. While each of the techniques has its inherentadvantages and limitations, the RAFT process is arguably themost amenable to controlled delivery/controlled activity in the

physiological environment due to the variety of functionalmonomers that can be polymerized directly in water without

requiring protecting group chemistry and the facility by whichstructopendant and structoterminal (both and ) functionalitycan be placed for subsequent conjugation to synthetic or

biological molecules. Additionally cross-linking and clickchemistry are easily conducted on RAFT-synthesized polymersunder facile reaction conditions. In fact, it is likely that manycontrolled delivery polymers previously made by conventionaltechniques or more tedious CLRP processes will be prepared byRAFT polymerization in the future.

2. RAFT polymerization

RAFT polymerization was first reported by the AustralianCSIRO group in 1998 [13,14,18] and later adapted to aqueous

1019A.W. York e t al. / Advanced Drug Delivery Reviews 60 (2008) 10181036



media in our laboratories and others [15,16]. Many of the

specifics of this process including the fate of intermediateradical species and competing side reactions are still not totallyresolved. However, the reader is referred to the above reviewsfor further discussion. The mechanism of RAFT polymerizationas originally proposed operates as a degenerative chain transfer

process illustrated in Scheme 1. The RAFT process is similar toconventional free radical polymerization but incorporates achain transfer agent (CTA). The CTA typically contains athiocarbonylthio moiety that is reactive towards radicals,subsequently facilitating the fragmentation of the resultingintermediate radical species (Scheme 1, step II) [13,15]. Thefunctional species R then reacts with monomer creating RM

which can reversibly add to another thiocarbonylthio group.Eventually, the main equilibrium is reached between the

propagating polymeric radical and the dormant macroCTA;the intermediate radical can then fragment in either directiongiving all polymer chains equal chances to grow, resulting inuniform chain growth and thus narrow polydispersities.Excellent control over the molecular weight can be achievedif CTA, monomer, initiator, reaction conditions, and conversionare appropriately chosen. Stopping the polymerization atmoderate conversions lowers the chance of termination, thus

preserving the thiocarbonylthio chain end. The retention of thisliving chain end allows the polymer, referred to as amacroCTA, to be isolated and subsequently chain extendedwith a second monomer (Scheme 1, step III) [13,15,16,19].

The living nature of the RAFT process and the ability to

polymerize a wide variety of vinyl monomers allow forsynthesis of an extensive library of functional blockcopolymers.

As in all free radical polymerizations, a source of initiatingradicals is required to start the polymerization (Scheme 1, stepI). In a RAFT polymerization, the ratio of [CTA]0/[I]0 isgenerally greater than one, ensuring that there are a greaternumber of CTA molecules in solution than free radicals. Theconcentration of free radicals in the system is dictated by thedegree of initiator dissociation, while the number of chains is

predominantly controlled by the CTA concentration in solution.The reversible equilibrium allows activation of a large number

of CTA molecules by a small number of initiator fragments.Activation requires the addition of primary radical from theinitiator to a CTA that subsequently fragments releasing a new

primary radical R in Scheme 1, step II. The R group of theCTA must efficiently fragment and reinitiate polymerizationand, in RAFT polymerization, nearly all chains are initiated bythe R group; thus, initiator generated chains are few. Under suchconditions, the molecular weight of the polymerization iscontrolled by the [monomer] ([M]) to [CTA] ratio and thetheoretical molecular weight is given by

Mn;

theoretical

M 0 MW monomer q

CTA 0 MWCTA 1

Scheme 1. The proposed RAFT mechanism for homopolymerization (I and II) and chain extension of a macroCTA (III).

1020 A.W. York et al. / Advanced Drug Delivery Reviews 60 (2008) 10181036



where [M]0 is the initial monomer concentration, MW monomeris the molecular weight of the monomer, is the conversion,

MW CTA is the molecular weight of the CTA, and [CTA]0 is theinitial concentration of the CTA [13,16]. It is apparent from thisequation that a specific molecular weight or chain length can betargeted by stopping the polymerization at a predeterminedtime. This control allows the synthesis of homopolymers or

block copolymers with tuned dimensions, making RAFT veryattractive for drug or gene delivery applications. In addition to

controlling the chain length, RAFT polymerization can also beused to synthesize polymers with advanced architecturesincluding multi-block, star, graft, statistical, alternating, aswell as gradient (co)polymers [13,16,20] (Fig. 1).

2.1. RAFT CTAs, initiators, and monomers

The key to the RAFT process and subsequently control overmolecular weight is the thiocarbonylthio moiety of the CTA.Various thiocarbonylthio groups have been reported in theliterature and can be classified in one of the following categories:trithiocarbonates [2124], dithioesters [2535], xanthates[13,19,36], and dithiocarbamates [37]. The CTA consists of astabilizing or destabilizing Z group and the previously mentioned

R group that must efficiently reinitiate the polymerization. Thechoice of the Z group, R group, and monomer is not arbitrary andmust be carefully made in order to achieve a successful RAFT

polymerization. For further discussion on CTA and monomer

Fig. 1. Polymer architectures available through controlled/living radical polymerization.

Fig. 2. Carboxylic acid containing chain transfer agents (CTAs).




selection, the reader is referred to an extensive review by Favierand Charrerye [38].

CTAs of particular interest for drug and gene delivery areones that allow for facile pre- or post-polymerization conjuga-tion to biological compounds. Five examples of such CTAs,all of which contain a carboxylic acid, are the dithioester

4-cyanopentanoic acid dithiobenzoate (CTP) [18] and the fourtrithiocarbonates 2-(1-carboxy-1-methyl-ethylsulfanylthiocar-

bonylsulfanyl)-2-methylpropionic acid (CMP) [21], 2-ethylsul-fanylthiocarbonylsulfanyl-2-methyl propionic acid (EMP) [39],2-dodecylsulfanylthiocarbonylsulfanyl-2-methyl propionicacid (DMP) [21], and 4-cyano-4-(dodecylsulfanylthiocarbo-nyl)sulfanyl pentanoic acid (CDMP) [40] shown in Fig. 2.Polymers and copolymers prepared with the above CTAs have-carboxylic and -thiocarbonylthio functionality which areeasily derivatized. The -carboxylic acid allows for carbodii-mide mediated functionalization with bioactive compounds, forexample those containing a primary amine. However it should

be noted that protection of the -thiocarbonylthio group isnecessary [19,32]. The presence of the carboxylic acid in CTP,CMP and EMP, allows these CTAs to be dissolved directly inaqueous solutions, facilitating aqueous RAFT polymerizations.Furthermore, the carboxylic acid of the R group can be activatedusing carbodiimide chemistry and N-hydroxysuccinimide (NHS)

prior to polymerization, allowing for facile bioconjugation, asdemonstrated by D'Agosto and coworkers [41]. CTAs have also

been synthesized with functionalities other than carboxylic acids.For example Thomas [42] in our group synthesized a sulfonate-containing CTA which allowed polymerizations of monomersover a wide pH range, extending the utility of RAFT. Davis,Bulmus and coworkers [43] synthesized a novel trithiocarbonate

that contained a polyethylene glycol-substituted (PEGylated) Zgroup and a pyridyldisulfide R group that could be easilyfunctionalized with thiol containing compounds, in this case

bovine serum albumin (BSA). It should be noted that althoughpolyethylene glycol (PEG) and polyethylene oxide (PEO) havethe same repeat structure, the former is polymerized by conden-sation polymerization of ethylene glycol and the latter by ringopening polymerization of ethylene oxide.

Thermal initiators, such as azo-based initiators (Fig. 3), aretypically used to initiate RAFT polymerizations; however otherinitiators such as organic peroxides or UV initiators can also be

utilized [13,19]. Even though a small number of chains in RAFTare initiator derived chains, it can be advantageous to use aninitiator that has identical functionality to that of the R group ofthe CTA. For example, our group frequently uses this strategy,employing the water soluble azo initiator 4,4-azobis(4-cyano-

pentanoic acid) (V-501) and CTP, which assures only carboxylic

acid functionality at the chain end of the resulting polymers[26,44,45]. 2,2-Azobis[2-(2-imidazolin-2-yl)propane] dihy-drochloride (VA-044) is water soluble and possess a lowdecomposition temperature allowing for polymerization oftemperature-responsive polymers directly in water without

precipitation. For example, Convertine [39] in our group recentlyreported the room temperature aqueous RAFT synthesis of themicelle forming block copolymer, poly(N,N-dimethylacryla-mide-block-N-isopropylacrylamide) (PDMA-b-PNIPAM), thathas potential for the uptake and release of hydrophobic drugs.Such thermally reversible micelles are of particular interest to the

biomedical field since PNIPAM possesses a lower critical

solution temperature (LCST) near that of the human body.PNIPAM will be discussed later in further detail in the stimuli-responsive micelle section.

Reports of RAFT polymers for biomedical and pharmaceu-tical applications have increased dramatically over the last fewyears, likely due to facile synthesis of biologically relevantarchitectures with water solubility [15,16] under mild conditions.Though both ATRP and RAFT may be conducted at mildtemperatures in organic media, a wider range of monomers has

been successfully polymerized at room temperature in aqueousmedia utilizing RAFT polymerization techniques. This allows, in

principle, direct block copolymer formation from thiocarbo-nylthio macroCTAs derived from biopolymers, for example,

polysaccharides or polypeptides without degradation or dena-turation expected at elevated temperatures. In addition, RAFT

polymerization does not require the potentially toxic transitionmetal species and coordinating ligands required by ATRP.However, it has recently been shown that the thiocarbonylthiomoiety of the chain transfer agent can be toxic [46,47]. This iseasily circumvented by the removal of the thiocarbonylthiomoiety through one of several post-polymerization modificationsoutlined in references [19,48]. Also of great utility in preparingtargeted, controlled activity polymers is the aforementioned widerange of monomers that can be polymerized by RAFT including

Fig. 3. Diazo initiators commonly used in RAFT polymerization.




Fig. 4. Selected monomers polymerized via CLRP.




neutral [2123,3437,30,31,45,4954], cationic[2426,5557],anionic [27,29,55,58,59], and zwitterionic [30,31,60] monomers(Fig. 4).

Poly(N-(2-hydroxypropyl)methacrylamide) (PHPMA) is a pri-mary example of an important polymer extensively studied as anon-viral carrier for anticancer drugs, particularly doxorubicin[6168]. Although HPMA polymers have been shown to benonimmunogenic, they necessarily possessed broad polydispersi-

ties because they were previously synthesized by conventional freeradical polymerization techniques. In 2005 we reported the prepa-ration of a well-defined near-monodisperse PHPMA via aqueousRAFT polymerization [45]. Several potentially useful blockcopolymers synthesized from monomers listed in Fig. 4 will bediscussed in later sections; for example, DMA/NIPAM blockcopolymers form responsive micelles and HPMA/N-[3-(dimethy-lamino)propyl]methacrylamide (DMAPMA) block copolymersform interpolyelectrolyte complexes with RNA.

3. Polymeric prodrugs

Polymeric prodrugs have been studied for over three decades

for delivery of therapeutic agents. As early as 1975, Ringsdorf[7]suggested design criteria for such a system having four maincomponents including a water soluble, biocompatible backbone,a therapeutic agent, a spacer to separate thetherapeutic agent fromthe backbone, and a targeting moiety (Fig. 5). Polymeric

prodrugs, as well as other synthetic carriers, can be passively oractively targeted. Passively targeted carriers do not contain atargeting moiety but rely on increased circulation time, provided

by the polymeric carrier. Untargeted carriers are usually used fortreating specific types of tumoral tissues and rely on the enhanced

permeability and retention (EPR) effect[6872]. The EPR effectoccurs because capillaries around tumors have enhanced vascular

permeability and limited lymphatic drainage [69], leading to theaccumulation of macromolecules in tumor cells. Because normaltissue can remove macromolecules by lymphatic drainage,accumulation of macromolecules does not occur. Activelytargeted polymeric carriers have a directing moiety, or moieties,conjugated along the backbone or to the end group of the polymerchain. Targeting moieties include, but are not limited to,antibodies, antibody fragments, folate, and carbohydrates.

Polymeric prodrug activity requires that the linked therapeuticagent either remains active while conjugated or be released fromthe backbone once the carrier has reached the targeted site. Untilrecently polymeric prodrugs were synthesized by non-controlled

polymerization methods, resulting in poorly defined polymerswith broad molecular weight distributions. This may notnecessarilybe a problem, providing the polymer is biodegradable;otherwise the polymer must be sufficiently small to allowexcretion through the kidneys over time. It has been shown thatlinear polymers below 40 kDa, or approximately 5 nm in dia-meter, are cleared readily through renal excretion [73]. Thereforeit is desirable to produce non-biodegradable linear polymers nearthis limit to increase circulation, but excretion can occur over

time. The RAFT techniques offer unprecedented opportunities forsize selection since polymers with predetermined molecularweights can be easily synthesized. Selecting monomers withappropriate biocompatibility and CTAs with reactive function-ality alsoallowsfacile bioconjugationto drugs, peptides, proteins,targeting moieties, fluorescent dyes, etc.

3.1. Polymer backbone conjugation

Attachment of drugs or other biological species directly to apolymer backbone has been the subject of extensive investiga-tion. A primary example is the work published by Kopeek

Fig. 5. Design criteria for polymeric prodrugs as suggested by Ringsdorf [7].

Scheme 2. Conjugation of an anthrax inhibitor peptide to a RAFT-synthesized HPMA/NMS copolymer [82].




et al., Ulbrich et al., and others who focused on the delivery ofanticancer therapeutics using the biocompatible polymer,PHPMA [6469,7479]. A potential problem with convention-ally polymerized HPMA is again that of polydispersity, whichcan now be circumvented by RAFT polymerization [45].

Activated monomers have also been polymerized by CLRP

methods and provide pendant functionality for attachment ofdrugs, targeting moieties, and compatible entities. The polymer-ization ofN-acryloxysuccinimide (NAS) orN-methacryloxysuc-cinimide (NMS), for example, has been achieved by both RAFT

polymerization [8082] and ATRP [83]. Mller and coworkers[83] successfully polymerized NMS via ATRP in organic media.The pendant NMS units were reacted with specified quantities ofthe model compound, glycineglycine--naphtylamide hydro-

bromide. The remaining NMS units were then functionalizedwith1-amino-2-propanol, resulting in a water soluble PHPMA copoly-mer conjugate. Recently, Kaneet al. [82] successfully synthesizednarrowly dispersed HPMA/NMS random copolymer via RAFT

polymerization in organic media (Scheme 2). NMS had to beadded gradually to the polymerization due to the difference inreactivity ratios between HPMA and NMS to ensure uniformdistribution of the NMS monomer. The incorporation of the NMSmonomer was kept between 20 and 28%. Bioconjugation of a

peptide that inhibits the assembly of anthrax toxin to PHPMA-co-PNMS was demonstrated by the authors who reported a threeorders of magnitude improvement in inhibition by the polymer

bioconjugate relative to the free peptide.

3.2. End-group conjugation

Haddleton et al. [84] have recently reviewed literature

reporting bioconjugation to polymers prepared by CLRP. Herewe have selected a few systems to illustrate the potential ofthese methods. Maynard's group [8587] utilized ATRP to

produce bioconjugates with controlled molecular weights andnarrow polydispersities. They functionalized ATRP initiatorswith biotin, BSA, and lysozyme and subsequently polymerized

N-isopropylacrylamide (NIPAM) from modified ATRP initia-

tors. The resulting systems retained biological activity even afterconjugation. At elevated temperatures the PNIPAM segmentscollapse and the covalently attached bioconjugates are renderedinactivate. Possible applications of this and related work includeenzyme regulation or recovery [86,88,89]. Stayton, Hoffmanand coworkers [9092] have also reported the bioconjugation of

biotin to RAFT-synthesized PNIPAM. They successfullyconjugated PEGylated biotin to the -terminal chain end [91]

by carbodiimide chemistry and to the -terminal chain end[90,92] by thiolmaleimide chemistry (Scheme 3). Hong et al.[93] reported the synthesis of a biotinylated CTA that was thenused to directly polymerize HPMA and NIPAM, by RAFT

polymerization. The resulting biotinylated, temperature-respon-sive, water soluble block copolymer was shown to self-assembleinto polymeric micelles. Although bioconjugates synthesized bythese groups have yet to be used for drug delivery, the potentialof such modified polymers for drug or gene delivery is apparent.

End-group modification of RAFT-generated polymers is

facilitated by the CTA employed during synthesis. Reduction ofthe -terminal thiocarbonylthio chain end yields thio-terminalpolymers easily modified by chemistry commonly used in thebiosciences. For example, our group recently reported primaryamine functionalization of poly(N-(2-hydroxypropyl)methacry-lamide-block-N-[3-(dimethylamino)propyl] methacrylamide)(PHPMA-b-PDMAPMA) block copolymer at the -terminalchain end after reduction of the thiocarbonylthio moiety bysodium borohydride (NaBH4) [94]. Utilizing a disulfide ex-change with cystamine, the tertiary polymeric thiol with lowreactivity was functionalized yielding a primary amine with highreactivity. Such primary amines are used extensively throughout

biochemistry to react with activated carboxylic acids. In our

work, the amine functionalized polymer was successfullyconjugated to an activated fluorescent compound, 6-(fluores-cein-5-carboxamido)hexanoic acid, succinimidyl ester (5-SFX)(Scheme 4). It is easily imaginable that such techniques could

be used to produce bioconjugates such as peptides, proteinsor targeting moieties for drug or gene delivery. This methodalso allows RAFT polymers to be functionalized with biological

Scheme 3. Biotinylation pathway of RAFT-synthesized polymers at the -terminal chain end (upper) and -terminal chain end (lower) [9092].




motifs at the -terminal chain end, a process that has met withlimited success in the past. Our group first reported the in situreduction of RAFT-generated polymers, directly in water, tostabilize transition metal nanoparticles which have potentialfor biodiagnostics [95,96]. The facile reduction of the thiocar-

bonylthio moiety to a polymeric thiol was also recently exploitedby Oupicky and coworkers [97] for the direct conjugation ofa maleimide functionalized biotin to a temperature-responsive(PEO-b-NIPAM) block copolymer.

4. Polymeric micelle delivery systems

The self-assembly of macromolecularspecies into micelles hasbeen studied since 1984 [8] in hopes of developing therapeuticcontrolled release systems. Polymeric micelles typically consistof diblock or triblock copolymers. In stimuli-responsive micellesone of the hydrophilic blocks is renderedhydrophobic in responseto an external stimulus [15,98] which is usually a change intemperature, pH, or salt concentration. The evolution of CLRPmethods has prompted the synthesis of a wide variety of stimuli-responsive block copolymers with controlled block lengths.CLRP allows for the facile tuning of the hydrodynamic size, animportant parameter when designing micelles for drug deliveryapplications. Dimensions are typically in the nanometer range

which is suggested to be ideal for drug delivery throughmicrometer sized capillaries [73]. Classical polymeric micellesare less than ideal, however, for delivery applications since theydissociate into unimers when diluted below the critical micelleconcentration (CMC). Under physiological conditions, at highdilution, release kinetics for micelle-entrapped active agentswould depend on the rate of the micelle-to-unimers transition. Aswill be shown in the following sections, micelle cross-linking canlead to greater control of pharmokinetic release.

4.1. Stimuli-responsive micelles

Stimuli-responsive block copolymers contain a permanentlyhydrophilic block and a stimuli-responsive block which canundergoa conformationalchange, promoting the self-assembly ofthe block copolymers into micelle-like structures with ahydrophobic core and a hydrophilic corona (Scheme 5). Thesestructures can sequester hydrophobic molecules that can bereleased in response to changes in the surrounding environment.Moststimuli-responsive block copolymers havebeensynthesized

by RAFT or ATRP polymerization due to versatility in monomerselection and mild reaction conditions these techniques offer.

The first successful pH-responsive block copolymerssynthesized by aqueous RAFT polymerization were reported

Scheme 4. -Terminal chain end functionalization of HPMA-block-DMAPMA RAFT polymer. Reduction of the polymeric end group followed by the disulfideexchange with cystamine provides a primary amine functionalized polymer which readily reacts with activated esters [94].

Scheme 5. Reversible micellization in response to an external stimulus.




in 2001 by our laboratory [99]. Poly(sodium styrene sulfonate)(PNaSS) macroCTA was successfully chain extended withsodium 4-vinylbenzoic acid (VBA) yielding a pH-responsive

block copolymer. At low pH, the carboxylic acid groups ofPVBA are protonated rendering the PVBA block hydrophobic.This pH change induces micellization that is fully reversible

when the pH is increased. The reversible micellizationmonitored by dynamic light scattering (DLS) indicatedindividual block copolymer chains (unimers) with hydrody-namic diameters of 8 nm at high pH and micelles at low pH withhydrodynamic diameters of 38 nm. Other pH-responsive

block copolymers synthesized by RAFT include poly(sodium2-acrylamido-2-methylpropanesulfonate-block-sodium3-acry-lamido-3-methylbutanoate) (PAMPS-b-PAMBA) [27,28], poly(sodium 2-acrylamido-2-methylpropanesulfonate- block-sodium 6-acrylamido hexanoate) [29], poly(N,N-dimethylacry-lamide- block-N,N-dimethylbenzylvinylamine) (PDMA-b-PDMBVA) [35], poly(2-vinylpyridine-block-4-vinylpyridine)

(P2VP-b-P4VP) [100], and poly(N,N-dimethylacrylamide-block-[N-isopropylacrylamide-stat-N-acryloylvaline]) (PDMA-b-(PNIPAM-stat-PAVAL)) [101].

A number of thermoresponsive polymers have been studied aspotential pharmaceutical delivery agents. Most thermoresponsivepolymers are synthesized from N-alkyl acrylamides, the mostresearched being PNIPAM since it possesses a LCST of 32 C,close to physiological temperature, 37 C. Above the LCST,PNIPAM becomes hydrophobic, a result of a gain in entropy dueto the disruption of the water shell associated with the isopropylgroups of NIPAM. For detailed information, the reader is directedto work by Winnik and coworkers [102107] who havepublishedextensively on the behavior of conventionally and RAFT-

polymerizedPNIPAM. Other thermoresponsive polymers includepoly(N-acryloylpiperidine) (PNAPi), poly(N-n-propylacryla-mide) (PnPA), and poly(N-acryloylpyrrolidine) (PAPy).

In 2000, Ganachaud and coworkers [34] reported the RAFTpolymerization of NIPAM in 1,4-dioxane. Subsequently a largenumber of reports of RAFT polymerization of this monomer inorganic media with various CTAs have appeared [16]. Using anappropriate CTA and initiator, we reported the first roomtemperature RAFT polymerization of NIPAM directly in water,utilizing the diazo initiator VA-044 and EMP or CMP as chaintransfer agents [39]. Significantly, this procedure allows polymer-ization at temperatures below the LCST of PNIPAM which

prevents polymer aggregation. In addition we have prepared, di-and triblock copolymers by first polymerizing the hydrophilicmonomer DMA and subsequently chain extending the resultingmacroCTA with NIPAM. ABA triblocks were synthesized in twosteps using CMP, a difunctional chain transfer agent, while ABdiblocks were synthesized using the monofunctional chain transferagent EMP. The micellization behavior of the block copolymerswas studied via DLS and static light scattering (SLS). It was foundthat as the NIPAM block length increased, the critical micelletemperature (CMT) decreased. Reversible micellization, shownin Fig. 6, was also demonstrated for PDMA100-b-PNIPAM460showing the transition from unimers to assembled micelles as thetemperature was cycled between 25 C and 45 C in 30 minintervals. This study shows the potential of RAFT polymerization

in providing non-viral carriers with tunable size that displayreversible micellization. Importantly, reversible micellization holds

promisefor the uptake and release of active agents and the eventualelimination of the carrier from circulation provided the micelle-to-unimer process can be controlled.

Oupicky and coworkers [97] reported the temperature-mediated association behavior of heterobifunctional copolymersin aqueous solution. PEG-b-PNIPAM block copolymers weresynthesizedvia organic RAFTpolymerization utilizing a modifiedDMP CTA. A lysine terminal PEG was then conjugated to the

carboxylic terminus of DMP resulting in a PEGylated macroCTA.After the successful polymerization of NIPAM, the thiocarbo-nylthio was reduced using excess hexylamine and the resultingthiol was then conjugated to maleimide derivatized biotin. The

binding efficiency of biotin with avidin was monitored attemperatures below and above the LCST of the block copolymer.Above the LCST, it was found that biotin is less accessible tointeract with avidin, while below the LCST biotin is readilyavailable. It is noteworthy that such temperature-responsivesystems allow the selective presentation of ligands that may be

beneficial in biomedical applications. Several other researchgroups including Yusa and Morshima [108], Liu and Perrier[109],

and Voit et al. [110] have reported PNIPAM-based block copoly-mers for temperature controlled assembly.

4.2. Shell cross-linked micelles

A fundamental problem affecting pharmokinetics of drugrelease from the thermo-reversible systems, discussed in the

previous section, is spontaneous dissociation of block copoly-mer micelles at concentrations below the CMC. To circumventthis issue, chemical or physical cross-linking techniques have

been developed which lock the micellar structure. A numberof significant advances have been made in the field since theseminal reports by Wooley's group in 1996 [111]. An extensivereview of these structures generally referred to as shell cross-

Fig. 6. Thermo-reversible association of PDMA100-b-PNIPAM460 as measured bydynamic light scattering (DLS) as the temperature is cycled from 25 to 45 C [39].




linked (SCL) micelles has recently been published by Armesand Read [112].

Armes and coworkers [113] were the first to report SCLmicelles that incorporated stimuli-responsive monomers. Blockcopolymers consisting of 2-[(dimethylamino)ethyl]methacry-late (DMAEMA) and 2-[(N-morpholino)ethyl] methacrylate

(MEMA) were synthesized via group transfer polymerizationthat formed inverse micelles in the presence of water at 60 C,due to the hydrophobicity of MEMA at elevated temperatures.The partially quaternized DMAEMA shell was subsequentlycross-linked using 1,2-bis-(2-iodoethoxy)ethane (BIEE).Although group transfer polymerization is inherently limitedin scope, this work showed that a stimuli-responsive SCLmicelle system with tunable properties was possible. Develop-ments in CLRP techniques, especially RAFT and ATRP, haveresulted in a wider range of block copolymers which easily self-assemble into micelles directly in water in response to externalstimuli. A number of recent examples with chemical or physical

locking

of SCL micelles are discussed below.SCL micelles can be prepared from di- (AB) or triblock (ABC)copolymers. Generally, SCL systems utilizing diblocks must beformed in very dilute solutions in order to prevent inter-micellarcross-linking [112]. Armes and coworkers [114] found a way of

preventing this problemby cross-linking the inner block of an ABCtriblock copolymer. The outer hydrophilic A block provides stericstabilization to the micellar system, thus preventing inter-micellarcross-linking and ensuring that cross-linking only occurs in theinner shell (B block). Their ABC triblock copolymer was synthe-sized using ATRP and a PEO macro-initiator to subsequently

polymerize DMAEMA followed by MEMA. Micelles wereformed by salting out the MEMA block from aqueous solution.

BIEE was then used to cross-link the inner DMAEMA shell of themicellar system and the resulting SCL micelles were characterizedvia NMR, DLS, and TEM. The PEO block was sufficiently long to

prevent inter-micellar cross-linking at concentrations as high as10 wt%.

Most literature reports have focused on non-reversiblecovalent cross-linking utilizing BIEE [114116], carbodiimides[117122], activated esters [80], divinyl sulfone [123], orclickchemistry [124]. However, permanent cross-linked systems aredisadvantageous because most SCL micelles are larger than therenal threshold limit,preventing the excretion of the SCL micelles

through the kidneys. Reversibly cross-linked systems are morepromising because the cross-links can be cleaved by an externalstimulus or reagent causing molecular dissolution of the micelles,thus making excretion more probable. In that regard we recentlyreported the synthesis of reversible SCL micelles utilizing RAFT-synthesized triblock copolymers [81]. The activated monomer

NAS, which can readily react with primary amines, wasincorporated into the inner block. The disulfide containingdiamine cystamine was used to cross-link the inner blockresulting in reversible SCL micelles. The RAFT-synthesized

poly(ethylene oxide)- block-[N,N-dimethylacrylamide-stat-NAS]-block-N-isopropylacrylamide, (PEO-b-(PDMA-sta t-NAS)-b-PNIPAM), triblock copolymers formed micelles atelevated temperatures. The shell of the micelle was then cross-linked through the amidation reaction of cystamine with thestatistically incorporated NAS groups (Scheme 6). The reversi-

bility of the polymer system was monitored by DLS and thereduction of the cystamine disulfide bond was achieved by

addition of a reducing agent such as tris(2-carboxyethyl)-phosphine (TCEP) or dithiothreitol (DTT). After reduction, thesystem could be cross-linked again by the addition of excesscystamine through a thioldisulfide exchange mechanism. It wasshown that the rate of release of the model cardiovascular drugcompound dipyridamole (DIP) from these SCL micelles can beeasily controlled by addition of a reducing agent at temperatures

below the LCSTof the PNIPAM block(Fig.7). When no reducingagent was present, the rate of release of the cross-linked systemwas slower than that of the uncross-linked system at 37 C.

Wooley and coworkers [125] also demonstrated the ability topackage DNA and protect it from degradative enzymes using acationic SCL micelle. The poly(styrene-block-4-vinylpyridine)

(PS-b-P4VP) cationic copolymer self-assembled in aqueoussolution creating a hydrophobic PS core and a solvated P4VPhydrophilic shell [126]. The resulting micelle was cross-linkedthrough a radical oligomerization process locking the micelleconformation. The cationically charged SCL micelles were thenloaded with plasmid DNA and studied using atomic forcemicroscopy (AFM), DLS and enzymatic degradation studies.Although these polymers were synthesized using anionic

polymerization, the same polymers could be synthesized inless stringent conditions using CLRP methods and should have

potential applications in gene delivery.

Scheme 6. Idealized representation of the reversible cross-linking of PEO-b-(PDMA-stat-NAS)-b-PNIPAM SCL micelle [81].




In addition to cross-linking reagents that form covalentlinkages, polyelectrolytes can be used to cross-link systemsthrough electrostatic interactions between the cross-linker (ionichomopolymer) and the oppositely charged shell of the micelles.Advantages of using polyelectrolytes cross-linkers rather thansmall molecular species are that there are no by-product,

polyelectrolytes tend to have low cytoxicity, and the cross-linking can be reversed upon addition of salt[112]. Armes andcoworkers [127] first reported the use of polyelectrolytes tocross-link SCL micelles. A poly(ethylene oxide)-block-2-

[(dimethylamino)ethyl]methacrylate-block-2-[(diethylamino)ethyl]methacrylate) (PEO-b-PDMAEMA-b-PDEAEMA) catio-nic triblock copolymer was synthesized via ATRP and thePDMAEMA inner block was rendered permanently cationic byselective methylation of the tertiary amine group with methyliodide. Above a critical pH, the triblock formed PDEAEMAcore micelles that were subsequently cross-linked by theaddition of the anionic polymer PNaSS. However, unwantedaggregation between micellar species occurred. This wassubsequently prevented using a PEO-block-PNaSS cross-linker.As in the case of ABC SCL micelles, the PEO block providessufficient steric stabilization to prevent inter-micellar bridging.

Our group recently reported the successful cross-linking of athermoresponsive triblock copolymer system utilizing a homo-

polyelectrolyte as a cross-linker [58]. Poly(N,N-dimethylacry-lamide)-block-(N-acryloylalanine)-block-(N-isopropylacryla-mide) (PDMA- b-PAAL-b-PNIPAM) was successfullysynthesized by RAFT polymerization and formed micelles inresponse to an increase in temperature (above the LCST of thetriblock system), as shown by DLS. The micelles were then

cross-linked through polyelectrolyte complexation upon theaddition of the cationic homopolymer poly[(ar-vinylbenzyl)trimethylammonium chloride] (PVBTAC) (Scheme 7). In thiscase the PDMA block serves as a steric stabilizer preventinginter-micellar cross-linking. In addition, these SCL micelles arereversible upon addition of 0.4 M NaCl.

In the processof synthesizing responsive diblock and triblockcopolymers forming stimulus-reversible micelles in water, bothour group and the Armes group have found compositions ofthe constituent corona forming and hydrophobic blocks thatyield vesicles rather than micelles. Armes et al. [128] reported

pH-responsive vesicle formation of an ATRP-synthesized

copolymer, poly(ethylene oxide)-block-poly[2-(diethylamino)ethyl]methacrylate-stat-3-[(trimethoxysilyl)propyl]methacry-late] (PEO-b-PDEAEMA-stat-PTMSPMA). The PDEAEMAresidues serve as base catalysts for the cross-linking of Si(OCH3)3 pendant groups to siloxanes, resulting in the lockingof vesicles morphologies. In addition, it was reported that whenHAuCl4 served as a proton source for PDEAEMA, it could

be reduced in situ with NaBH4 to produce zero valent goldnanoparticles.

Recently our group reported interpolyelectrolyte com-plexation or locking of vesicles based on the RAFT-syn-thesized, thermally responsive poly(N-(3-aminopropyl)methacrylamide-block-N-isopropylacrylamide) (PAPMA-b-

PNIPAM) and anionic cross-linker PAMPS (Fig. 8a) [129].In related work, we recently reported the in situ reduction of

NaAuCl4 in the presence of poly(2-[(dimethylamino)ethyl]methacrylate-block-N-isopropylacrylamide) (PDMAEMA-b-PNIPAM) above the LCST to form gold decorated vesicles(Fig. 8b) [130]. In each of the above cases the resultingvesicles, sometimes referred to as polymersomes, are quitestable for extended periods in aqueous media, indicating their

potential for diagnostics and targeted delivery of therapeuticagents. Current research by several research groups is beingdirected toward understanding precisely how the segmentallengths and compositions of diblock and triblock copolymer

effect formation of micellar versus vesicular structures[131,132].

Scheme 7. Thermo reversible formation of multimeric micelles and subsequent interpolyelectrolyte cross-linking of the RAFT-synthesized PDMA- b-PAAL-b-PNIPAM with PVBTAC [58].

Fig. 7. A release study of the model cardiovascular drug dipyridamole fromreversible SCL micelles PEO-b-(PDMA-s-NAS)-b-PNIPAM sensitive to

reducing environments [81].




Given the need for targeted drug or gene delivery systemsand the fact that SCL micelles deliver active agents, a number ofresearch groups have considered modifying surfaces of SCLmicelles by conjugating targeting moieties, peptides, proteins,or other biological motifs. Wooley's research group has madesignificant contributions to this area in recent years and has

synthesized SCL micellar systems that have great potential forincreasing drug delivery efficacy. For example they reported thesurface modification of poly(-caprolactone-block-acrylic acid)(PCL-b-PAA) SCL micelles synthesized by a combination ofring opening polymerization and ATRP, followed by hydrolysisto obtain PAA from poly(tert-butylacrylate) [118]. The PAAshell was approximately 50% cross-linked using carbodiimidechemistry leaving the remaining carboxylic groups available forfurther modification. Later this group functionalized these co-

polymers with both a fluorescent label and a protein transduc-tion domain (PTD) peptide [133]. Despite the low percent ofPTD peptide conjugation, as might be expected from this solid

phase reaction, the SCL micelles displayed the ability to bind tocellular surfaces and subsequently cross the cellular membrane,as shown by fluorescent confocal microscopy. Wooley andcoworkers have also successfully polymerized numerous diblockcopolymer systems via ATRP or SFRP capable of forming SCLmicelles that have been surface modified with various bioconju-gates including folate [134], antigens [135], and integrin bindingligands [136].

5. Polyelectrolyte complexes

Polyelectrolyte complexes resulting from the electrostaticinteractions between two oppositely charged polyelectrolytes are

being intensely pursued for gene delivery applications. The mostpromising examples involve complexation between cationichomopolymers or hydrophilic-block-cationic block copolymersandpolynucleotides such as DNA or RNA. The functional groupsof the cationic polymer or copolymer associate with the nega-tively charged phosphate backbone of polynucleotides. Polyelec-trolyte complexes, sometimes referred to as interpolyelectrolyte

complexes (IPECs) or block ionomer complexes (BICs), providethe complexed polynucleotide protection from degradativeenzymes, while remaining in the aqueous phase. In order tomaintain solubility or dispersability the complex must maintain anet positive or negative charge or be stabilized via a hydrophilic-co-cationic polymer.

The non-viral carrier must overcome several other barriers inorder to efficiently deliver polynucleotides (genes). These

barriers can be broken into two general categories: extracellularbarriers which encompass all obstacles encountered beforereaching the targeted cell membrane and intracellular barrierswhich encompass obstacles encountered from the point ofcellular uptake to the cytoplasmic release or nuclear localizationof the gene. Major extracellular barriers include packaging ofthe active agents by the carrier, stability and circulation in the

bloodstream, and specific cellular binding. Packaging andserum stability are interrelated because gene packaging resultsin the steric shielding of polynucleotides from nucleases in the

blood stream. Serum stability is also affected by the net chargeof the complex. Neutral complexes often aggregate when placedin physiological conditions. Conversely, net anionic andcationic polyelectrolyte complexes remain soluble but introduce

problems in the intracellular delivery of the gene (see below).Once the target cell internalizes the polyelectrolyte complexthrough endocytosis, new hurdles are presented, includingendosomal release, cytoplasmic transport, and release of thegene. For further information on cellular barriers the reader isreferred to an excellent review by Pack, Hoffman, et al. [1].

The polymeric carrier plays a pivotal role in overcoming theabove mentioned barriers as well providing a route for endosomalescape and polynucleotide release. Until recently most studies of

IPECs for gene delivery have used commercially available cationicpolymers such as poly(L-lysine) (PLL) or poly(ethylenimine) (PEI)[1,5]. PEI is able to escape the endosome via the proton spongeeffect,a phenomenon thatultimately results in theosmotic swellingof the endosome causing it to rupture. Though PEI-based com-

plexes are able to escape the endosome, these polymers were notoriginally designed for gene delivery and hence have suboptimal

Fig. 8. Thermally responsive vesicle structures from a) PAPMA-b-PNIPAM and b) PDMAEMA-b-PNIPAM. The vesicular structure shown in a) was subsequentlycross-linked with the anionic polymer PAMPS [129]. Structure b) was locked by in situ reduction of NaAuCl4 yielding gold nanoparticle decorated vesicles [130].




properties, including high cytotoxicity, inherently broad molecularweight distributions, and little structural uniformity (e.g. linear or

branched). If other architectures are desired, such as blocks, extrasynthetic steps are required. For further information regardingcommercially available polymers for gene delivery the reader isreferred to reviews by Pack et al. [1], Kissel et al. [76], and Kim et

al. [5]. Rapid advancements in polymerization techniques,including CLRP, have provided researchers with the toolsnecessary to synthesize potential gene carriers. It is anticipatedthat both extra- and intracellular barriers can be overcome throughthe intelligent and rational design of polymeric carriers.

One current area of scrutiny is the effect of the polycation/polynucleotide ratio on the size, stability, and formation ofpolyelectrolyte complex. Typically the N/P (nitrogen tophosphate) ratio is used to determine the net charge of thecomplex. N/P ratios are often less than or greater than one inorder to confer water solubility. However, electrostatic repul-sions between the negatively charged complexes and the cell

membrane (also negatively charged) prevent transfection.Conversely, positively charged complexes associate with cellmembranes via strong electrostatic interactions leading to non-specific cellular uptake through adsorptive endocytosis [137].Furthermore, this strong interaction with the cellular membraneoften leads to membrane disruption resulting in cell death. Evenif positive complexes could be targeted to specific cells,negative proteins found in the blood can associate with thecomplex resulting in precipitation and leading to its clearance

by phagocytic cells [1]. Neutral complexes (N/P=1) can inprinciple circumvent the problems associated with negative andpositive complexes, but neutral complexes often have lowsolubility leading to precipitation.

An attractive alternative to simple complexes is utilization ofhydrophilic-block-cationic copolymers for polynucleotide com-

plexation. Electrostatic interactions through the cationic blockstabilize the polynucleotide while the hydrophilic block

provides steric stabilization of the entire complex. The additionof the hydrophilic block makes it possible to form neutralcomplexes, N/P=1, that may solve stability and circulation

problems seen in the abovementioned polyelectrolyte com-plexes. The water soluble, biocompatible, and nonimmunogenicPHPMA or PEO is usually used as the hydrophilic block, whilethe cationic block generally consists of tertiary or quaternaryamines. In principle, the binding/release properties of the carrier

can be tailored by choice of cationic monomer and polymer

block architecture. For example, quaternization of tertiaryamines with varying alkyl groups or the random copolymeriza-tion of the cationic block with a neutral monomer will result in

polymers with different binding strengths.Though promising, the reports of CLRP polymers synthe-

sized specifically for use in gene delivery are limited. In 2006,

we reported the RAFT synthesis of a series of PHPMA-b-PDMAPMA copolymers for the complexation and potentialdelivery of small interfering RNA (siRNA) (Scheme 8) [44].The ability of the block copolymers to stabilize the complexeswas studied while maintaining a N/P ratio of one. The effect ofDMAPMA block length on complexation properties couldalso be studied due to the high level of control using RAFT

polymerization. Results indicated that the DMAPMA blocklength was the major factor effecting the stabilization of the

bound siRNA (43 nucleotides). The block copolymers withDMAPMA block lengths of 13 and 23 performed the best andalso displayed the ability to protect siRNA from enzymatic

degradation under physiological conditions as shown by moni-toring the absorbance at 260 nm in the presence of nucleaseRNase A (Fig. 9). The unprotected control was rapidly hydro-lyzed as shown by an increase in the absorbance while the

polymer-bound siRNA showed little degradation.Oupicky and coworkers [138] synthesized PNIPAM, via

RAFT polymerization, utilizing a CTA with -terminalcarboxylic functionality. The carboxylic acid was activated bycarbodiimide chemistry and subsequently linked to branchedPEI. The ability of the PNIPAM-co-PEI copolymer to complexwith plasmid DNA and subsequently transfect cells was studied.The complexation between the copolymer and the plasmidDNA at various N/P ratios was monitored by ethidium bromide

exclusion assay. It was found that as the N/P ratio was increasedabove a ratio of 2.5, no further decrease was observed; thefluorescence intensity decreased indicating that ethidium

bromide was unable to intercalate the condensed DNA. Whencompared to conventionally synthesized PNIPAM, the RAFTPNIPAM exhibited wider phase transitions. The authorsattributed this to the aminolysis of the thiocarbonylthio groupwhen in the presence of the amine containing cell culturemedium or amine-functional PEI. Therefore, PNIPAM synthe-sized by conventional free radical polymerization was used forthe remainder of the studies. The copolymer complexes hadcytoxicity similar to that of the PEI/DNA complexes at

temperatures above the LCST, while cytotoxicity decreased at

Scheme 8. Idealized representation of the interpolyelectrolyte complexation of PHPMA- b-PDMAPMA with siRNA [44].




temperatures below the LCST. The PNIPAMPEI/DNAcomplexes had lower levels of cellular uptake and transfectionas compared to the PEI/DNA complexes when temperatureswere below the LCST and increased to PEI/DNA complexlevels above the LCST. Even though the remainder of thestudies used a conventionally polymerized PNIPAM, the -terminal thiocarbonylthio of the RAFT-generated polymermight be easily modified in future work. In this regard, variousmethods for removing the thiocarbonylthio functionality areoutlined in references [19,48].

Phosphorylcholine-based polymers reduce non-specific proteinadsorption [139] and have therefore been used as biomaterials

[140,141]. Stolnik et al. [142] synthesized a series of block copoly-mers via ATRP comprised of the cationic monomer DMAEMAand the zwitterionic monomer 2-(methacryloyloxyethyl phosphor-ylcholine) (MPC) capable of condensing DNA. An optimal blockcomposition of the PDMAEMA-block-PMPC copolymers wasfound necessary in order to obtain stable colloids and prevent non-specific cellular binding even though the MPC moiety slightlyinhibited DNA binding.

Overall uptake of polyelectrolyte complexes with low trans-fection efficiencies can be increased by exploiting the over-expression of folate receptors on cancer cells. In order to main-tain high transfection efficiency while eliminating non-specific

uptake, Armes and coworkers [143] synthesized folic acidconjugated PDMAEMA-block-PMPC copolymers through a

post-polymerization modification. Preliminary studies showedthat the folic acid conjugated block copolymer was preferen-tially taken up in specific cell lines. Although there are minimalreports on the conjugation of biologically active species toRAFT-synthesized polymers, the techniques mentioned earlierfor polymeric prodrugs could be easily utilized to target genecarriers to specific sites. The reaction pathways illustrated inScheme 3 can be employed to conjugate targeting moieties to

polymeric carriers to increase the efficacy of delivery. Forexample two routes that can be easily adapted for the facilemodification of RAFT-synthesized polymers include reaction ofthe carboxylic group built in to the CTAs shown in Fig. 2 or the

reduction of the thiocarbonylthio -terminal chain end to athiol. Carboxylic groups and thiols are widely used in

biochemistry and can react readily with primary amines, thiols,maleimides, as well as other reactive groups.

Mallapragada and coworkers [144] synthesized a pentablockcopolymer utilizing ATRP. A difunctional PEO-block-poly(propy-

lene oxide)-block-PEO (PEO-b-PPO-b-PEO) macro-initiator wasused in the polymerization of the cationic monomer 2-[(diethyla-mino)ethyl]methacrylate(DEAEMA). The pentablock was capableof condensing plasmid DNA. The polyelectrolyte complexdisplayed lower cytotoxicity as compared to linear PEI, whilemaintaining the ability to protect the plasmid DNA from enzymaticdegradation. At low N/P ratios (e.g. 3:1 and 4:1) the polymer/DNAcomplex was able to transfect cells with efficiencies similar to thatof linear PEI, but at higher ratios the complex became morecytotoxic. In addition, the pentablock copolymer displayed thermo-reversible gelation behavior due to the presence of the PPO block.This thermoresponsive behavior may be advantageous for local

delivery of genes or other therapeutic agents administered bysubcutaneous injections.The systems mentioned above have shown promise for

future delivery of polynucleotides, but many unresolved issuesmust be addressed. These include: development of successfulconjugation techniques for attaching targeting moieties to thecarriers, enhancing cellular uptake, and optimizing interpolye-lectrolyte binding strength, and endosomal release.

6. Conclusions and outlook

In this report we have examined the utility of CLRP techniques,particularly RAFT and aqueous RAFT technology in providing

amphiphilicblock copolymer architectures appropriatefordrug andgene delivery platforms. Proper selection of CTAs, monomers, andreaction conditions allows for unprecedented opportunities in notonly controlling block copolymer structure and polydispersity butalso in providing reactive structopendant or structoterminalfunctional groups for further extension, copolymerization, post-

polymerization conjugation or cross-linking. The RAFT processalso can proceed directly in water without requiring protectinggroup chemistry providedthemonomer andCTA arewater solubleand competing hydrolysis mechanisms are minimized. We havehighlighted several literature examples of systems prepared byRAFTor closely related CLRP techniques fordelivery of drugsand

polynucleotides. Although in their infancy relative to conventionaltechniques, RAFT and aqueous RAFT procedures appear to havegreat potential for preparation of controlled delivery systems under

physiological conditions. One major challenge for syntheticchemists will be understanding parameters that control selforganization of assembled delivery vehicles, for example micelles,vesicles, interpolyelectrolyte complexes, etc. Once such morphol-ogies can be constructed reproducibly, delivery and release under

biologically relevant conditions canbe pursuedin a systematicway.

Acknowledgements

The authors would like to acknowledge the US Departmentof Energy (DE-FC26-01BC15317), the Robert M. Hearin

Fig. 9. Enzymatic degradation of free siRNA and PHPMA258-b-PDMAPMA23complexed with siRNA [44].




Foundation, and the MRSEC program of the National ScienceFoundation (DMR-0213883) for financial support.

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