Amphiphilic Block Copoplymers in Strimuli Responsive Drug Delivery - REVIEW - 1-s2.0-S0169409X08000550-Main

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Advanced Drug Delivery Reviews 60 (2008) 1018 1036 www.elsevier.com/locate/addr
Advances in the synthesis of amphiphilic block copolymers via RAFT polymerization: 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 2008 Available 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 an unprecedented latitude in the synthesis of water soluble or amphiphilic architectures with precise dimensions and appropriate functionality for attachment and targeted delivery of diagnostic and therapeutic agents. This review focuses on the chemistry of the RAFT process and its potential for preparing well-defined block copolymers and conjugates capable of stimuli-responsive assembly and release of bioactive agents in the physiological environment. Recent examples of block copolymers with designed structures and segmental compositions responsive to changes in pH 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-responsive polymers
Contents Introduction . . . . . . . . . . . . . . . . . . RAFT polymerization . . . . . . . . . . . . . 2.1. RAFT CTAs, initiators, and monomers 3. Polymeric prodrugs . . . . . . . . . . . . . . 3.1. Polymer backbone conjugation . . . . . 3.2. End-group conjugation . . . . . . . . . 4. Polymeric micelle delivery systems . . . . . 4.1. Stimuli-responsive micelles . . . . . . 4.2. Shell cross-linked micelles . . . . . . . 5. Polyelectrolyte complexes . . . . . . . . . . . 6. Conclusions and outlook . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . List of abbreviations . . . . . . . . . . . . . . . . 1. 2. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1019 . 1019 . 1021 . 1024 . 1024 . 1025 . 1026 . 1026 . 1027 . 1030 . 1032 . 1032 . 1033 . . 1036 . 0
This review is part of the Advanced Drug Delivery Reviews theme issue on Design and Development Strategies of Polymer Materials for Drug and Gene Delivery Applications. 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: Charles.McCormick@usm.edu (C.L. McCormick). 0169-409X/$ - see front matter 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.addr.2008.02.006
A.W. York et al. / Advanced Drug Delivery Reviews 60 (2008) 10181036
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1. Introduction Recent advances in controlled/living radical polymerization (CLRP) methods are certain to impact future approaches to diagnosis and treatment of infectious and genetic diseases. Specifically, one such technique, reversible additionfragmentation chain transfer (RAFT) polymerization, allows unprecedented latitude in synthesis of water soluble architectures with precise dimensions and appropriate functionality for conjugation to and targeted delivery of diagnostic and therapeutic agents. Although to date only limited reports have appeared regarding 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 functionalization, cross-linking, and bioconjugation are advantageous when compared to more conventional processes currently utilized. In the following review, the RAFT process will be discussed, demonstrating its utility for polymerizing a variety of functional monomers, often directly in water and not requiring protecting group chemistry. Selected examples of smart polymeric systems prepared by RAFT or other CLRP techniques and designed with precise nanoscale dimensions, narrow molecular weight distributions, reactive pendant or terminal moieties and surface functionality will be presented. In some cases, for example in drug delivery from shell crosslinked micelles or in delivery of polynucleotides from interpolyelectrolyte complexes, significant advancements in protection of the active agent, carrier stabilization, and controlled release have been realized, although formidable challenges remain. Impetus for research on polymeric delivery vehicles has come from consideration of technical, economic and safety issues. The fact that viral vectors are very effective at targeting and delivering therapeutic agents to the cytoplasm or the nuclear envelope of the cell has been well established. However, shortcomings of viral vectors include the invocation of an immunogenic response by the host, the high cost of manufacturing, and the lack of cell specificity [1]. Thus a number of non-viral delivery systems with low immunogenicity yet specific targeting and controlled activity have been considered. For example, conventional liposomes have been utilized to sequester and transport chemotherapeutics [2], polynucleotides [3], and proteins [4]. Although such systems have been shown to possess many requisite requirements [1,5] for in vivo application including: protection of cargo from degradation, ease of surface modification for targeting, and reasonable manufacturing costs, disadvantages often are reported such as limited solubility and partitioning, less than optimal pharmokinetics, and liposome instability in the physiological environment [6]. More recently research has centered on polymer based carrier systems including polymerdrug conjugates, micelles, polymersomes, and nanoparticles. A major goal of this work is to mimic the highly evolved trafficking and delivery efficiency of viral vectors while avoiding the non-specific toxicity and immunogenicity issues discussed above. Several significant barriers are currently being addressed. The non-viral carrier
must be able to degrade or dissociate into low molecular weight species capable of excretion through the kidneys. In the case of non-degradable polymeric carriers, the carrier must be of low molecular weight to be efficiently excreted. Both extracellular and intracellular factors present significant challenges in the successful delivery of active agents via non-viral vectors. Extracellular barriers include, but are not limited to, packaging of the active agents by the carrier, stability and circulation in the bloodstream, and specific cellular binding; intracellular barriers include endosomal release, cytoplasm transport, and release of the active species [1]. Prior to the mid 1980s only limited synthetic tools were available to polymer chemists for construction of delivery vehicles, although Ringsdorf and others, for example, had eloquently proposed model targeting systems [7,8]. Control of polymer architecture, molecular weight and molecular weight distribution, placement of reactive structopendant or structoterminal functionality, and solubility/dispersion in biologically relevant media were obstacles inherent to existing polymer technology. For example, block and star copolymer architectures with appropriate functionality could only be achieved with anionic, cationic, or group transfer chain growth polymerization techniques with limited types of monomers, often requiring protecting group chemistry, under stringent conditions in the absence of water. Fortunately rapid developments in two major areas: dendrimer synthesis and CLRP now allow control over a number of necessary design criteria. Although we focus on the latter area here, excellent reviews on the chemistry and biological applications of dendrimers are found in recent literature [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 of aqueous RAFT polymerization [15,16]. Although a recent report [17] suggests some opportunities for SFRP, most efforts toward construction of polymeric delivery vehicles with controlled structures and molecular weight have utilized ATRP or RAFT. While each of the techniques has its inherent advantages and limitations, the RAFT process is arguably the most amenable to controlled delivery/controlled activity in the physiological environment due to the variety of functional monomers that can be polymerized directly in water without requiring protecting group chemistry and the facility by which structopendant and structoterminal (both and ) functionality can be placed for subsequent conjugation to synthetic or biological molecules. Additionally cross-linking and click chemistry are easily conducted on RAFT-synthesized polymers under facile reaction conditions. In fact, it is likely that many controlled delivery polymers previously made by conventional techniques or more tedious CLRP processes will be prepared by RAFT polymerization in the future. 2. RAFT polymerization RAFT polymerization was first reported by the Australian CSIRO group in 1998 [13,14,18] and later adapted to aqueous
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Scheme 1. The proposed RAFT mechanism for homopolymerization (I and II) and chain extension of a macroCTA (III).
media in our laboratories and others [15,16]. Many of the specifics of this process including the fate of intermediate radical species and competing side reactions are still not totally resolved. However, the reader is referred to the above reviews for further discussion. The mechanism of RAFT polymerization as originally proposed operates as a degenerative chain transfer process illustrated in Scheme 1. The RAFT process is similar to conventional free radical polymerization but incorporates a chain transfer agent (CTA). The CTA typically contains a thiocarbonylthio moiety that is reactive towards radicals, subsequently facilitating the fragmentation of the resulting intermediate radical species (Scheme 1, step II) [13,15]. The functional 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 direction giving all polymer chains equal chances to grow, resulting in uniform chain growth and thus narrow polydispersities. Excellent control over the molecular weight can be achieved if CTA, monomer, initiator, reaction conditions, and conversion are appropriately chosen. Stopping the polymerization at moderate conversions lowers the chance of termination, thus preserving the thiocarbonylthio chain end. The retention of this living chain end allows the polymer, referred to as a macroCTA, to be isolated and subsequently chain extended with 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 for synthesis of an extensive library of functional block copolymers. As in all free radical polymerizations, a source of initiating radicals is required to start the polymerization (Scheme 1, step I). In a RAFT polymerization, the ratio of [CTA]0/[I]0 is generally greater than one, ensuring that there are a greater number of CTA molecules in solution than free radicals. The concentration of free radicals in the system is dictated by the degree 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 the initiator to a CTA that subsequently fragments releasing a new primary radical R in Scheme 1, step II. The R group of the CTA must efficiently fragment and reinitiate polymerization and, in RAFT polymerization, nearly all chains are initiated by the R group; thus, initiator generated chains are few. Under such conditions, the molecular weight of the polymerization is controlled by the [monomer] ([M]) to [CTA] ratio and the theoretical molecular weight is given by M 0 MW monomer q MW CTA CTA0
Mn;
theoretical
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Fig. 1. Polymer architectures available through controlled/living radical polymerization.
where [M]0 is the initial monomer concentration, MW monomer is the molecular weight of the monomer, is the conversion, MW CTA is the molecular weight of the CTA, and [CTA]0 is the initial concentration of the CTA [13,16]. It is apparent from this equation that a specific molecular weight or chain length can be targeted by stopping the polymerization at a predetermined time. This control allows the synthesis of homopolymers or block copolymers with tuned dimensions, making RAFT very attractive for drug or gene delivery applications. In addition to controlling the chain length, RAFT polymerization can also be used to synthesize polymers with advanced architectures including multi-block, star, graft, statistical, alternating, as well 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 over molecular weight is the thiocarbonylthio moiety of the CTA. Various thiocarbonylthio groups have been reported in the literature 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 a stabilizing or destabilizing Z group and the previously mentioned R group that must efficiently reinitiate the polymerization. The choice of the Z group, R group, and monomer is not arbitrary and must be carefully made in order to achieve a successful RAFT polymerization. For further discussion on CTA and monomer
Fig. 2. Carboxylic acid containing chain transfer agents (CTAs).
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selection, the reader is referred to an extensive review by Favier and Charrerye [38]. CTAs of particular interest for drug and gene delivery are ones that allow for facile pre- or post-polymerization conjugation 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 four trithiocarbonates 2-(1-carboxy-1-methyl-ethylsulfanylthiocarbonylsulfanyl)-2-methylpropionic acid (CMP) [21], 2-ethylsulfanylthiocarbonylsulfanyl-2-methyl propionic acid (EMP) [39], 2-dodecylsulfanylthiocarbonylsulfanyl-2-methyl propionic acid (DMP) [21], and 4-cyano-4-(dodecylsulfanylthiocarbonyl)sulfanyl pentanoic acid (CDMP) [40] shown in Fig. 2. Polymers and copolymers prepared with the above CTAs have -carboxylic and -thiocarbonylthio functionality which are easily derivatized. The -carboxylic acid allows for carbodiimide mediated functionalization with bioactive compounds, for example those containing a primary amine. However it should be noted that protection of the -thiocarbonylthio group is necessary [19,32]. The presence of the carboxylic acid in CTP, CMP and EMP, allows these CTAs to be dissolved directly in aqueous solutions, facilitating aqueous RAFT polymerizations. Furthermore, the carboxylic acid of the R group can be activated using carbodiimide chemistry and N-hydroxysuccinimide (NHS) prior to polymerization, allowing for facile bioconjugation, as demonstrated 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 sulfonatecontaining CTA which allowed polymerizations of monomers over 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) Z group and a pyridyldisulfide R group that could be easily functionalized with thiol containing compounds, in this case bovine serum albumin (BSA). It should be noted that although polyethylene glycol (PEG) and polyethylene oxide (PEO) have the same repeat structure, the former is polymerized by condensation polymerization of ethylene glycol and the latter by ring opening polymerization of ethylene oxide. Thermal initiators, such as azo-based initiators (Fig. 3), are typically used to initiate RAFT polymerizations; however other initiators such as organic peroxides or UV initiators can also be
utilized [13,19]. Even though a small number of chains in RAFT are initiator derived chains, it can be advantageous to use an initiator that has identical functionality to that of the R group of the CTA. For example, our group frequently uses this strategy, employing the water soluble azo initiator 4,4-azobis(4-cyanopentanoic 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] dihydrochloride (VA-044) is water soluble and possess a low decomposition temperature allowing for polymerization of temperature-responsive polymers directly in water without precipitation. For example, Convertine [39] in our group recently reported the room temperature aqueous RAFT synthesis of the micelle forming block copolymer, poly(N,N-dimethylacrylamide-block-N-isopropylacrylamide) (PDMA-b-PNIPAM), that has 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 stimuliresponsive micelle section. Reports of RAFT polymers for biomedical and pharmaceutical applications have increased dramatically over the last few years, likely due to facile synthesis of biologically relevant architectures with water solubility [15,16] under mild conditions. Though both ATRP and RAFT may be conducted at mild temperatures in organic media, a wider range of monomers has been successfully polymerized at room temperature in aqueous media utilizing RAFT polymerization techniques. This allows, in principle, direct block copolymer formation from thiocarbonylthio macroCTAs derived from biopolymers, for example, polysaccharides or polypeptides without degradation or denaturation expected at elevated temperatures. In addition, RAFT polymerization does not require the potentially toxic transition metal species and coordinating ligands required by ATRP. However, it has recently been shown that the thiocarbonylthio moiety of the chain transfer agent can be toxic [46,47]. This is easily circumvented by the removal of the thiocarbonylthio moiety through one of several post-polymerization modifications outlined in references [19,48]. Also of great utility in preparing targeted, controlled activity polymers is the aforementioned wide range of monomers that can be polymerized by RAFT including
Fig. 3. Diazo initiators commonly used in RAFT polymerization.
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Fig. 4. Selected monomers polymerized via CLRP.
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Fig. 5. Design criteria for polymeric prodrugs as suggested by Ringsdorf [7].
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 primary example of an important polymer extensively studied as a non-viral carrier for anticancer drugs, particularly doxorubicin [6168]. Although HPMA polymers have been shown to be nonimmunogenic, they necessarily possessed broad polydispersities because they were previously synthesized by conventional free radical polymerization techniques. In 2005 we reported the preparation of a well-defined near-monodisperse PHPMA via aqueous RAFT polymerization [45]. Several potentially useful block copolymers synthesized from monomers listed in Fig. 4 will be discussed in later sections; for example, DMA/NIPAM block copolymers form responsive micelles and HPMA/N-[3-(dimethylamino)propyl]methacrylamide (DMAPMA) block copolymers form 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 main components including a water soluble, biocompatible backbone, a therapeutic agent, a spacer to separate the therapeutic agent from the backbone, and a targeting moiety (Fig. 5). Polymeric prodrugs, as well as other synthetic carriers, can be passively or actively targeted. Passively targeted carriers do not contain a targeting moiety but rely on increased circulation time, provided by the polymeric carrier. Untargeted carriers are usually used for treating specific types of tumoral tissues and rely on the enhanced permeability and retention (EPR) effect [6872]. The EPR effect occurs because capillaries around tumors have enhanced vascular
permeability and limited lymphatic drainage [69], leading to the accumulation of macromolecules in tumor cells. Because normal tissue can remove macromolecules by lymphatic drainage, accumulation of macromolecules does not occur. Actively targeted polymeric carriers have a directing moiety, or moieties, conjugated along the backbone or to the end group of the polymer chain. Targeting moieties include, but are not limited to, antibodies, antibody fragments, folate, and carbohydrates. Polymeric prodrug activity requires that the linked therapeutic agent either remains active while conjugated or be released from the backbone once the carrier has reached the targeted site. Until recently polymeric prodrugs were synthesized by non-controlled polymerization methods, resulting in poorly defined polymers with broad molecular weight distributions. This may not necessarily be a problem, providing the polymer is biodegradable; otherwise the polymer must be sufficiently small to allow excretion through the kidneys over time. It has been shown that linear polymers below 40 kDa, or approximately 5 nm in diameter, are cleared readily through renal excretion [73]. Therefore it is desirable to produce non-biodegradable linear polymers near this limit to increase circulation, but excretion can occur over time. The RAFT techniques offer unprecedented opportunities for size selection since polymers with predetermined molecular weights can be easily synthesized. Selecting monomers with appropriate biocompatibility and CTAs with reactive functionality also allows facile bioconjugation to drugs, peptides, proteins, targeting moieties, fluorescent dyes, etc. 3.1. Polymer backbone conjugation Attachment of drugs or other biological species directly to a polymer backbone has been the subject of extensive investigation. A primary example is the work published by Kopeek
Scheme 2. Conjugation of an anthrax inhibitor peptide to a RAFT-synthesized HPMA/NMS copolymer [82].
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et al., Ulbrich et al., and others who focused on the delivery of anticancer therapeutics using the biocompatible polymer, PHPMA [6469,7479]. A potential problem with conventionally polymerized HPMA is again that of polydispersity, which can now be circumvented by RAFT polymerization [45]. Activated monomers have also been polymerized by CLRP methods and provide pendant functionality for attachment of drugs, targeting moieties, and compatible entities. The polymerization of N-acryloxysuccinimide (NAS) or N-methacryloxysuccinimide (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 of the model compound, glycineglycine--naphtylamide hydrobromide. The remaining NMS units were then functionalized with 1-amino-2-propanol, resulting in a water soluble PHPMA copolymer conjugate. Recently, Kane et al. [82] successfully synthesized narrowly dispersed HPMA/NMS random copolymer via RAFT polymerization in organic media (Scheme 2). NMS had to be added gradually to the polymerization due to the difference in reactivity ratios between HPMA and NMS to ensure uniform distribution of the NMS monomer. The incorporation of the NMS monomer was kept between 20 and 28%. Bioconjugation of a peptide that inhibits the assembly of anthrax toxin to PHPMA-coPNMS was demonstrated by the authors who reported a three orders 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. Here we have selected a few systems to illustrate the potential of these methods. Maynard's group [8587] utilized ATRP to produce bioconjugates with controlled molecular weights and narrow polydispersities. They functionalized ATRP initiators with biotin, BSA, and lysozyme and subsequently polymerized N-isopropylacrylamide (NIPAM) from modified ATRP initia-
tors. The resulting systems retained biological activity even after conjugation. At elevated temperatures the PNIPAM segments collapse and the covalently attached bioconjugates are rendered inactivate. Possible applications of this and related work include enzyme regulation or recovery [86,88,89]. Stayton, Hoffman and coworkers [9092] have also reported the bioconjugation of biotin to RAFT-synthesized PNIPAM. They successfully conjugated 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 then used to directly polymerize HPMA and NIPAM, by RAFT polymerization. The resulting biotinylated, temperature-responsive, water soluble block copolymer was shown to self-assemble into polymeric micelles. Although bioconjugates synthesized by these groups have yet to be used for drug delivery, the potential of 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 of the -terminal thiocarbonylthio chain end yields thio-terminal polymers easily modified by chemistry commonly used in the biosciences. For example, our group recently reported primary amine functionalization of poly(N-(2-hydroxypropyl)methacrylamide-block-N-[3-(dimethylamino)propyl] methacrylamide) (PHPMA-b-PDMAPMA) block copolymer at the -terminal chain end after reduction of the thiocarbonylthio moiety by sodium borohydride (NaBH4) [94]. Utilizing a disulfide exchange with cystamine, the tertiary polymeric thiol with low reactivity was functionalized yielding a primary amine with high reactivity. Such primary amines are used extensively throughout biochemistry to react with activated carboxylic acids. In our work, the amine functionalized polymer was successfully conjugated to an activated fluorescent compound, 6-(fluorescein-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, proteins or targeting moieties for drug or gene delivery. This method also 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].
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Scheme 4. -Terminal chain end functionalization of HPMA-block-DMAPMA RAFT polymer. Reduction of the polymeric end group followed by the disulfide exchange with cystamine provides a primary amine functionalized polymer which readily reacts with activated esters [94].
motifs at the -terminal chain end, a process that has met with limited success in the past. Our group first reported the in situ reduction of RAFT-generated polymers, directly in water, to stabilize transition metal nanoparticles which have potential for biodiagnostics [95,96]. The facile reduction of the thiocarbonylthio moiety to a polymeric thiol was also recently exploited by Oupicky and coworkers [97] for the direct conjugation of a maleimide functionalized biotin to a temperature-responsive (PEO-b-NIPAM) block copolymer. 4. Polymeric micelle delivery systems The self-assembly of macromolecular species into micelles has been studied since 1984 [8] in hopes of developing therapeutic controlled release systems. Polymeric micelles typically consist of diblock or triblock copolymers. In stimuli-responsive micelles one of the hydrophilic blocks is rendered hydrophobic in response to an external stimulus [15,98] which is usually a change in temperature, pH, or salt concentration. The evolution of CLRP methods has prompted the synthesis of a wide variety of stimuliresponsive block copolymers with controlled block lengths. CLRP allows for the facile tuning of the hydrodynamic size, an important parameter when designing micelles for drug delivery applications. Dimensions are typically in the nanometer range
which is suggested to be ideal for drug delivery through micrometer sized capillaries [73]. Classical polymeric micelles are less than ideal, however, for delivery applications since they dissociate into unimers when diluted below the critical micelle concentration (CMC). Under physiological conditions, at high dilution, release kinetics for micelle-entrapped active agents would depend on the rate of the micelle-to-unimers transition. As will be shown in the following sections, micelle cross-linking can lead to greater control of pharmokinetic release. 4.1. Stimuli-responsive micelles Stimuli-responsive block copolymers contain a permanently hydrophilic block and a stimuli-responsive block which can undergo a conformational change, promoting the self-assembly of the block copolymers into micelle-like structures with a hydrophobic core and a hydrophilic corona (Scheme 5). These structures can sequester hydrophobic molecules that can be released in response to changes in the surrounding environment. Most stimuli-responsive block copolymers have been synthesized by RAFT or ATRP polymerization due to versatility in monomer selection and mild reaction conditions these techniques offer. The first successful pH-responsive block copolymers synthesized by aqueous RAFT polymerization were reported
Scheme 5. Reversible micellization in response to an external stimulus.
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in 2001 by our laboratory [99]. Poly(sodium styrene sulfonate) (PNaSS) macroCTA was successfully chain extended with sodium 4-vinylbenzoic acid (VBA) yielding a pH-responsive block copolymer. At low pH, the carboxylic acid groups of PVBA are protonated rendering the PVBA block hydrophobic. This pH change induces micellization that is fully reversible when the pH is increased. The reversible micellization monitored by dynamic light scattering (DLS) indicated individual block copolymer chains (unimers) with hydrodynamic diameters of 8 nm at high pH and micelles at low pH with hydrodynamic diameters of 38 nm. Other pH-responsive block copolymers synthesized by RAFT include poly(sodium 2-acrylamido-2-methylpropanesulfonate-block-sodium3-acrylamido-3-methylbutanoate) (PAMPS-b-PAMBA) [27,28], poly (sodium 2-acrylamido-2-methylpropanesulfonate- block sodium 6-acrylamido hexanoate) [29], poly(N,N-dimethylacrylamideblock-N,N -dimethylbenzylvinylamine) (PDMA- b PDMBVA) [35], poly(2-vinylpyridine-block-4-vinylpyridine) (P2VP-b-P4VP) [100], and poly(N,N-dimethylacrylamideblock-[N-isopropylacrylamide-stat-N-acryloylvaline]) (PDMAb-(PNIPAM-stat-PAVAL)) [101]. A number of thermoresponsive polymers have been studied as potential pharmaceutical delivery agents. Most thermoresponsive polymers are synthesized from N-alkyl acrylamides, the most researched 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 due to the disruption of the water shell associated with the isopropyl groups of NIPAM. For detailed information, the reader is directed to work by Winnik and coworkers [102107] who have published extensively on the behavior of conventionally and RAFTpolymerized PNIPAM. Other thermoresponsive polymers include poly(N-acryloylpiperidine) (PNAPi), poly(N-n-propylacrylamide) (PnPA), and poly(N-acryloylpyrrolidine) (PAPy). In 2000, Ganachaud and coworkers [34] reported the RAFT polymerization of NIPAM in 1,4-dioxane. Subsequently a large number of reports of RAFT polymerization of this monomer in organic media with various CTAs have appeared [16]. Using an appropriate CTA and initiator, we reported the first room temperature RAFT polymerization of NIPAM directly in water, utilizing the diazo initiator VA-044 and EMP or CMP as chain transfer agents [39]. Significantly, this procedure allows polymerization at temperatures below the LCST of PNIPAM which prevents polymer aggregation. In addition we have prepared, diand triblock copolymers by first polymerizing the hydrophilic monomer DMA and subsequently chain extending the resulting macroCTA with NIPAM. ABA triblocks were synthesized in two steps using CMP, a difunctional chain transfer agent, while AB diblocks were synthesized using the monofunctional chain transfer agent EMP. The micellization behavior of the block copolymers was studied via DLS and static light scattering (SLS). It was found that as the NIPAM block length increased, the critical micelle temperature (CMT) decreased. Reversible micellization, shown in Fig. 6, was also demonstrated for PDMA100-b-PNIPAM460 showing the transition from unimers to assembled micelles as the temperature was cycled between 25 C and 45 C in 30 min intervals. This study shows the potential of RAFT polymerization
Fig. 6. Thermo-reversible association of PDMA100-b-PNIPAM460 as measured by dynamic light scattering (DLS) as the temperature is cycled from 25 to 45 C [39].
in providing non-viral carriers with tunable size that display reversible micellization. Importantly, reversible micellization holds promise for the uptake and release of active agents and the eventual elimination of the carrier from circulation provided the micelle-tounimer process can be controlled. Oupicky and coworkers [97] reported the temperaturemediated association behavior of heterobifunctional copolymers in aqueous solution. PEG-b-PNIPAM block copolymers were synthesized via organic RAFT polymerization utilizing a modified DMP 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 thiocarbonylthio was reduced using excess hexylamine and the resulting thiol was then conjugated to maleimide derivatized biotin. The binding efficiency of biotin with avidin was monitored at temperatures below and above the LCST of the block copolymer. Above the LCST, it was found that biotin is less accessible to interact with avidin, while below the LCST biotin is readily available. It is noteworthy that such temperature-responsive systems allow the selective presentation of ligands that may be beneficial in biomedical applications. Several other research groups including Yusa and Morshima [108], Liu and Perrier [109], and Voit et al. [110] have reported PNIPAM-based block copolymers for temperature controlled assembly. 4.2. Shell cross-linked micelles A fundamental problem affecting pharmokinetics of drug release from the thermo-reversible systems, discussed in the previous section, is spontaneous dissociation of block copolymer micelles at concentrations below the CMC. To circumvent this issue, chemical or physical cross-linking techniques have been developed which lock the micellar structure. A number of significant advances have been made in the field since the seminal reports by Wooley's group in 1996 [111]. An extensive review of these structures generally referred to as shell cross-
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linked (SCL) micelles has recently been published by Armes and Read [112]. Armes and coworkers [113] were the first to report SCL micelles that incorporated stimuli-responsive monomers. Block copolymers consisting of 2-[(dimethylamino)ethyl]methacrylate (DMAEMA) and 2-[(N-morpholino)ethyl] methacrylate (MEMA) were synthesized via group transfer polymerization that 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 subsequently cross-linked using 1,2-bis-(2-iodoethoxy)ethane (BIEE). Although group transfer polymerization is inherently limited in scope, this work showed that a stimuli-responsive SCL micelle system with tunable properties was possible. Developments in CLRP techniques, especially RAFT and ATRP, have resulted in a wider range of block copolymers which easily selfassemble into micelles directly in water in response to external stimuli. 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 be formed in very dilute solutions in order to prevent inter-micellar cross-linking [112]. Armes and coworkers [114] found a way of preventing this problem by cross-linking the inner block of an ABC triblock copolymer. The outer hydrophilic A block provides steric stabilization to the micellar system, thus preventing inter-micellar cross-linking and ensuring that cross-linking only occurs in the inner shell (B block). Their ABC triblock copolymer was synthesized using ATRP and a PEO macro-initiator to subsequently polymerize DMAEMA followed by MEMA. Micelles were formed by salting out the MEMA block from aqueous solution. BIEE was then used to cross-link the inner DMAEMA shell of the micellar system and the resulting SCL micelles were characterized via NMR, DLS, and TEM. The PEO block was sufficiently long to prevent inter-micellar cross-linking at concentrations as high as 10 wt%. Most literature reports have focused on non-reversible covalent cross-linking utilizing BIEE [114116], carbodiimides [117122], activated esters [80], divinyl sulfone [123], or click chemistry [124]. However, permanent cross-linked systems are disadvantageous because most SCL micelles are larger than the renal threshold limit, preventing the excretion of the SCL micelles
through the kidneys. Reversibly cross-linked systems are more promising because the cross-links can be cleaved by an external stimulus or reagent causing molecular dissolution of the micelles, thus making excretion more probable. In that regard we recently reported the synthesis of reversible SCL micelles utilizing RAFTsynthesized triblock copolymers [81]. The activated monomer NAS, which can readily react with primary amines, was incorporated into the inner block. The disulfide containing diamine cystamine was used to cross-link the inner block resulting in reversible SCL micelles. The RAFT-synthesized poly(ethylene oxide)- block-[ N,N-dimethylacrylamide- stat NAS]- block- N-isopropylacrylamide, (PEO- b-(PDMA- statNAS)-b-PNIPAM), triblock copolymers formed micelles at elevated temperatures. The shell of the micelle was then crosslinked through the amidation reaction of cystamine with the statistically incorporated NAS groups (Scheme 6). The reversibility of the polymer system was monitored by DLS and the reduction 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, the system could be cross-linked again by the addition of excess cystamine through a thioldisulfide exchange mechanism. It was shown that the rate of release of the model cardiovascular drug compound dipyridamole (DIP) from these SCL micelles can be easily controlled by addition of a reducing agent at temperatures below the LCST of the PNIPAM block (Fig. 7). When no reducing agent was present, the rate of release of the cross-linked system was slower than that of the uncross-linked system at 37 C. Wooley and coworkers [125] also demonstrated the ability to package DNA and protect it from degradative enzymes using a cationic SCL micelle. The poly(styrene-block-4-vinylpyridine) (PS-b-P4VP) cationic copolymer self-assembled in aqueous solution creating a hydrophobic PS core and a solvated P4VP hydrophilic shell [126]. The resulting micelle was cross-linked through a radical oligomerization process locking the micelle conformation. The cationically charged SCL micelles were then loaded with plasmid DNA and studied using atomic force microscopy (AFM), DLS and enzymatic degradation studies. Although these polymers were synthesized using anionic polymerization, the same polymers could be synthesized in less 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].
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Fig. 7. A release study of the model cardiovascular drug dipyridamole from reversible SCL micelles PEO- b-(PDMA-s-NAS)-b-PNIPAM sensitive to reducing environments [81].
In addition to cross-linking reagents that form covalent linkages, polyelectrolytes can be used to cross-link systems through electrostatic interactions between the cross-linker (ionic homopolymer) and the oppositely charged shell of the micelles. Advantages of using polyelectrolytes cross-linkers rather than small molecular species are that there are no by-product, polyelectrolytes tend to have low cytoxicity, and the crosslinking can be reversed upon addition of salt [112]. Armes and coworkers [127] first reported the use of polyelectrolytes to cross-link SCL micelles. A poly(ethylene oxide)-block-2[(dimethylamino)ethyl]methacrylate- block-2-[(diethylamino) ethyl]methacrylate) (PEO-b-PDMAEMA-b-PDEAEMA) cationic triblock copolymer was synthesized via ATRP and the PDMAEMA inner block was rendered permanently cationic by selective methylation of the tertiary amine group with methyl iodide. Above a critical pH, the triblock formed PDEAEMA core micelles that were subsequently cross-linked by the addition of the anionic polymer PNaSS. However, unwanted aggregation between micellar species occurred. This was subsequently prevented using a PEO-block-PNaSS cross-linker. As in the case of ABC SCL micelles, the PEO block provides sufficient steric stabilization to prevent inter-micellar bridging. Our group recently reported the successful cross-linking of a thermoresponsive triblock copolymer system utilizing a homo-
polyelectrolyte as a cross-linker [58]. Poly(N,N-dimethylacrylamide)- block-( N-acryloylalanine)- block-( N-isopropylacrylamide) (PDMA- b -PAAL- b -PNIPAM) was successfully synthesized by RAFT polymerization and formed micelles in response to an increase in temperature (above the LCST of the triblock system), as shown by DLS. The micelles were then cross-linked through polyelectrolyte complexation upon the addition of the cationic homopolymer poly[(ar-vinylbenzyl) trimethylammonium chloride] (PVBTAC) (Scheme 7). In this case the PDMA block serves as a steric stabilizer preventing inter-micellar cross-linking. In addition, these SCL micelles are reversible upon addition of 0.4 M NaCl. In the process of synthesizing responsive diblock and triblock copolymers forming stimulus-reversible micelles in water, both our group and the Armes group have found compositions of the constituent corona forming and hydrophobic blocks that yield 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]methacrylate] (PEO-b-PDEAEMA-stat-PTMSPMA). The PDEAEMA residues serve as base catalysts for the cross-linking of Si (OCH3)3 pendant groups to siloxanes, resulting in the locking of vesicles morphologies. In addition, it was reported that when HAuCl4 served as a proton source for PDEAEMA, it could be reduced in situ with NaBH4 to produce zero valent gold nanoparticles. Recently our group reported interpolyelectrolyte complexation or locking of vesicles based on the RAFT-synthesized, thermally responsive poly( N -(3-aminopropyl) methacrylamide- block- N-isopropylacrylamide) (PAPMA- bPNIPAM) 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-bPNIPAM) above the LCST to form gold decorated vesicles (Fig. 8b) [130]. In each of the above cases the resulting vesicles, sometimes referred to as polymersomes, are quite stable for extended periods in aqueous media, indicating their potential for diagnostics and targeted delivery of therapeutic agents. Current research by several research groups is being directed toward understanding precisely how the segmental lengths 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-bPNIPAM with PVBTAC [58].
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Given the need for targeted drug or gene delivery systems and the fact that SCL micelles deliver active agents, a number of research groups have considered modifying surfaces of SCL micelles by conjugating targeting moieties, peptides, proteins, or other biological motifs. Wooley's research group has made significant contributions to this area in recent years and has synthesized SCL micellar systems that have great potential for increasing drug delivery efficacy. For example they reported the surface modification of poly(-caprolactone-block-acrylic acid) (PCL-b-PAA) SCL micelles synthesized by a combination of ring opening polymerization and ATRP, followed by hydrolysis to obtain PAA from poly(tert-butylacrylate) [118]. The PAA shell was approximately 50% cross-linked using carbodiimide chemistry leaving the remaining carboxylic groups available for further modification. Later this group functionalized these copolymers with both a fluorescent label and a protein transduction domain (PTD) peptide [133]. Despite the low percent of PTD peptide conjugation, as might be expected from this solid phase reaction, the SCL micelles displayed the ability to bind to cellular surfaces and subsequently cross the cellular membrane, as shown by fluorescent confocal microscopy. Wooley and coworkers have also successfully polymerized numerous diblock copolymer systems via ATRP or SFRP capable of forming SCL micelles that have been surface modified with various bioconjugates including folate [134], antigens [135], and integrin binding ligands [136]. 5. Polyelectrolyte complexes Polyelectrolyte complexes resulting from the electrostatic interactions between two oppositely charged polyelectrolytes are being intensely pursued for gene delivery applications. The most promising examples involve complexation between cationic homopolymers or hydrophilic-block-cationic block copolymers and polynucleotides such as DNA or RNA. The functional groups of the cationic polymer or copolymer associate with the negatively charged phosphate backbone of polynucleotides. Polyelectrolyte complexes, sometimes referred to as interpolyelectrolyte
complexes (IPECs) or block ionomer complexes (BICs), provide the complexed polynucleotide protection from degradative enzymes, while remaining in the aqueous phase. In order to maintain solubility or dispersability the complex must maintain a net positive or negative charge or be stabilized via a hydrophilicco-cationic polymer. The non-viral carrier must overcome several other barriers in order to efficiently deliver polynucleotides (genes). These barriers can be broken into two general categories: extracellular barriers which encompass all obstacles encountered before reaching the targeted cell membrane and intracellular barriers which encompass obstacles encountered from the point of cellular uptake to the cytoplasmic release or nuclear localization of the gene. Major extracellular barriers include packaging of the active agents by the carrier, stability and circulation in the bloodstream, and specific cellular binding. Packaging and serum stability are interrelated because gene packaging results in the steric shielding of polynucleotides from nucleases in the blood stream. Serum stability is also affected by the net charge of the complex. Neutral complexes often aggregate when placed in physiological conditions. Conversely, net anionic and cationic polyelectrolyte complexes remain soluble but introduce problems in the intracellular delivery of the gene (see below). Once the target cell internalizes the polyelectrolyte complex through endocytosis, new hurdles are presented, including endosomal release, cytoplasmic transport, and release of the gene. For further information on cellular barriers the reader is referred to an excellent review by Pack, Hoffman, et al. [1]. The polymeric carrier plays a pivotal role in overcoming the above mentioned barriers as well providing a route for endosomal escape and polynucleotide release. Until recently most studies of IPECs for gene delivery have used commercially available cationic polymers such as poly(L-lysine) (PLL) or poly(ethylenimine) (PEI) [1,5]. PEI is able to escape the endosome via the proton sponge effect, a phenomenon that ultimately results in the osmotic swelling of the endosome causing it to rupture. Though PEI-based complexes are able to escape the endosome, these polymers were not originally 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 subsequently cross-linked with the anionic polymer PAMPS [129]. Structure b) was locked by in situ reduction of NaAuCl4 yielding gold nanoparticle decorated vesicles [130].
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properties, including high cytotoxicity, inherently broad molecular weight distributions, and little structural uniformity (e.g. linear or branched). If other architectures are desired, such as blocks, extra synthetic steps are required. For further information regarding commercially available polymers for gene delivery the reader is referred 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 tools necessary to synthesize potential gene carriers. It is anticipated that both extra- and intracellular barriers can be overcome through the 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 of polyelectrolyte complex. Typically the N/P (nitrogen to phosphate) ratio is used to determine the net charge of the complex. N/P ratios are often less than or greater than one in order to confer water solubility. However, electrostatic repulsions between the negatively charged complexes and the cell membrane (also negatively charged) prevent transfection. Conversely, positively charged complexes associate with cell membranes via strong electrostatic interactions leading to nonspecific cellular uptake through adsorptive endocytosis [137]. Furthermore, this strong interaction with the cellular membrane often leads to membrane disruption resulting in cell death. Even if positive complexes could be targeted to specific cells, negative proteins found in the blood can associate with the complex resulting in precipitation and leading to its clearance by phagocytic cells [1]. Neutral complexes (N/P = 1) can in principle circumvent the problems associated with negative and positive complexes, but neutral complexes often have low solubility leading to precipitation. An attractive alternative to simple complexes is utilization of hydrophilic-block-cationic copolymers for polynucleotide complexation. Electrostatic interactions through the cationic block stabilize the polynucleotide while the hydrophilic block provides steric stabilization of the entire complex. The addition of the hydrophilic block makes it possible to form neutral complexes, N/P = 1, that may solve stability and circulation problems seen in the abovementioned polyelectrolyte complexes. The water soluble, biocompatible, and nonimmunogenic PHPMA or PEO is usually used as the hydrophilic block, while the cationic block generally consists of tertiary or quaternary amines. 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 tertiary amines with varying alkyl groups or the random copolymerization of the cationic block with a neutral monomer will result in polymers with different binding strengths. Though promising, the reports of CLRP polymers synthesized specifically for use in gene delivery are limited. In 2006, we reported the RAFT synthesis of a series of PHPMA-bPDMAPMA copolymers for the complexation and potential delivery of small interfering RNA (siRNA) (Scheme 8) [44]. The ability of the block copolymers to stabilize the complexes was studied while maintaining a N/P ratio of one. The effect of DMAPMA block length on complexation properties could also be studied due to the high level of control using RAFT polymerization. Results indicated that the DMAPMA block length was the major factor effecting the stabilization of the bound siRNA (43 nucleotides). The block copolymers with DMAPMA block lengths of 13 and 23 performed the best and also displayed the ability to protect siRNA from enzymatic degradation under physiological conditions as shown by monitoring the absorbance at 260 nm in the presence of nuclease RNase A (Fig. 9). The unprotected control was rapidly hydrolyzed 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 -terminal carboxylic functionality. The carboxylic acid was activated by carbodiimide chemistry and subsequently linked to branched PEI. The ability of the PNIPAM-co-PEI copolymer to complex with plasmid DNA and subsequently transfect cells was studied. The complexation between the copolymer and the plasmid DNA at various N/P ratios was monitored by ethidium bromide exclusion assay. It was found that as the N/P ratio was increased above a ratio of 2.5, no further decrease was observed; the fluorescence intensity decreased indicating that ethidium bromide was unable to intercalate the condensed DNA. When compared to conventionally synthesized PNIPAM, the RAFT PNIPAM exhibited wider phase transitions. The authors attributed this to the aminolysis of the thiocarbonylthio group when in the presence of the amine containing cell culture medium or amine-functional PEI. Therefore, PNIPAM synthesized by conventional free radical polymerization was used for the remainder of the studies. The copolymer complexes had cytoxicity 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].
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Fig. 9. Enzymatic degradation of free siRNA and PHPMA258-b-PDMAPMA23 complexed with siRNA [44].
temperatures below the LCST. The PNIPAMPEI/DNA complexes had lower levels of cellular uptake and transfection as compared to the PEI/DNA complexes when temperatures were below the LCST and increased to PEI/DNA complex levels above the LCST. Even though the remainder of the studies used a conventionally polymerized PNIPAM, the terminal thiocarbonylthio of the RAFT-generated polymer might be easily modified in future work. In this regard, various methods for removing the thiocarbonylthio functionality are outlined in references [19,48]. Phosphorylcholine-based polymers reduce non-specific protein adsorption [139] and have therefore been used as biomaterials [140,141]. Stolnik et al. [142] synthesized a series of block copolymers via ATRP comprised of the cationic monomer DMAEMA and the zwitterionic monomer 2-(methacryloyloxyethyl phosphorylcholine) (MPC) capable of condensing DNA. An optimal block composition of the PDMAEMA-block-PMPC copolymers was found necessary in order to obtain stable colloids and prevent nonspecific cellular binding even though the MPC moiety slightly inhibited DNA binding. Overall uptake of polyelectrolyte complexes with low transfection efficiencies can be increased by exploiting the overexpression of folate receptors on cancer cells. In order to maintain high transfection efficiency while eliminating non-specific uptake, Armes and coworkers [143] synthesized folic acid conjugated PDMAEMA-block-PMPC copolymers through a post-polymerization modification. Preliminary studies showed that the folic acid conjugated block copolymer was preferentially taken up in specific cell lines. Although there are minimal reports on the conjugation of biologically active species to RAFT-synthesized polymers, the techniques mentioned earlier for polymeric prodrugs could be easily utilized to target gene carriers to specific sites. The reaction pathways illustrated in Scheme 3 can be employed to conjugate targeting moieties to polymeric carriers to increase the efficacy of delivery. For example two routes that can be easily adapted for the facile modification of RAFT-synthesized polymers include reaction of the carboxylic group built in to the CTAs shown in Fig. 2 or the
reduction of the thiocarbonylthio -terminal chain end to a thiol. 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 pentablock copolymer utilizing ATRP. A difunctional PEO-block-poly(propylene oxide)-block-PEO (PEO-b-PPO-b-PEO) macro-initiator was used in the polymerization of the cationic monomer 2-[(diethylamino)ethyl]methacrylate (DEAEMA). The pentablock was capable of condensing plasmid DNA. The polyelectrolyte complex displayed lower cytotoxicity as compared to linear PEI, while maintaining the ability to protect the plasmid DNA from enzymatic degradation. At low N/P ratios (e.g. 3:1 and 4:1) the polymer/DNA complex was able to transfect cells with efficiencies similar to that of linear PEI, but at higher ratios the complex became more cytotoxic. In addition, the pentablock copolymer displayed thermoreversible 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 by subcutaneous injections. The systems mentioned above have shown promise for future delivery of polynucleotides, but many unresolved issues must be addressed. These include: development of successful conjugation techniques for attaching targeting moieties to the carriers, enhancing cellular uptake, and optimizing interpolyelectrolyte 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 amphiphilic block copolymer architectures appropriate for drug and gene delivery platforms. Proper selection of CTAs, monomers, and reaction conditions allows for unprecedented opportunities in not only controlling block copolymer structure and polydispersity but also in providing reactive structopendant or structoterminal functional groups for further extension, copolymerization, postpolymerization conjugation or cross-linking. The RAFT process also can proceed directly in water without requiring protecting group chemistry provided the monomer and CTA are water soluble and competing hydrolysis mechanisms are minimized. We have highlighted several literature examples of systems prepared by RAFT or closely related CLRP techniques for delivery of drugs and polynucleotides. Although in their infancy relative to conventional techniques, RAFT and aqueous RAFT procedures appear to have great potential for preparation of controlled delivery systems under physiological conditions. One major challenge for synthetic chemists will be understanding parameters that control self organization of assembled delivery vehicles, for example micelles, vesicles, interpolyelectrolyte complexes, etc. Once such morphologies can be constructed reproducibly, delivery and release under biologically relevant conditions can be pursued in a systematic way. Acknowledgements The authors would like to acknowledge the US Department of Energy (DE-FC26-01BC15317), the Robert M. Hearin
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Foundation, and the MRSEC program of the National Science Foundation (DMR-0213883) for financial support. References
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List of abbreviations
N-acryloylalanine Sodium3-acrylamido-3-methylbutanoate N-(3-aminopropyl)methacrylamide Sodium 2-acrylamido-2-methylpropanesulfonate Atomic force microscopy N-acryloylpyrrolidine N-acryloylvaline atom transfer radical polymerization block ionomer complexes 1,2-bis-(2-iodoethoxy)ethane bovine serum albumin 4-cyano-4-(dodecylsulfanylthiocarbonyl)sulfanyl pentanoic acid controlled/living radical polymerization 2-(1-carboxy-1-methyl-ethylsulfanylthiocarbonylsulfanyl)-2-methylpropionic acid CMC critical micelle concentration CMT critical micelle temperature CTA chain transfer agent CTP 4-cyanopentanoic acid dithiobenzoate DIP dipyridamole DEAEMA 2-[(diethylamino)ethyl]methacrylate DLS dynamic light scattering DMA N,N-dimethylacrylamide DMAEMA 2-[(dimethylamino)ethyl]methacrylate DMAPMA N-[3-(dimethylamino)propyl]methacrylamide DMP 2-dodecylsulfanylthiocarbonylsulfanyl-2-methyl propionic acid DMBVA N,N-dimethylbenzylvinylamine DTT dithiothreitol EMP 2-ethylsulfanylthiocarbonylsulfanyl-2-methyl propionic acid IPECs interpolyelectrolyte complexes EPR enhanced permeability and retention HPMA N-(2-hydroxypropyl)methacrylamide LCST lower critical solution temperature MEMA 2-[(N-morpholino)ethyl] methacrylate MPC 2-(methacryloyloxyethyl phosphorylcholine) NAPi N-acryloylpiperidine NAS N-acryloxysuccinimide NaSS Sodium styrene sulfonate NHS N-hydroxysuccinimide NIPAM N-isopropylacrylamide NMS N-methacryloxysuccinimide nPA N-n-propylacrylamide PAA poly(acrylic acid) PCL poly(-caprolactone) PEI poly(ethylenimine) PEG poly(ethylene glycol) PLL poly(L-lysine) PEO poly(ethylene oxide) PPO poly(propylene oxide) PS poly(styrene) PTD protein transduction domain RAFT reversible additionfragmentation chain transfer SCL shell cross-linked SFRP stable free radical polymerization 5-SFX 6-(fluorescein-5-carboxamido)hexanoic acid, succinimidyl ester SLS static light scattering siRNA small interfering RNA TCEP tris(2-carboxyethyl)-phosphine TMSPMA 3-[(trimethoxysilyl)propyl]methacrylate V-501 4,4-azobis(4-cyanopentanoic acid) VA-044 2,2-Azobis[2-(2-imidazolin-2-yl)propane] dihydrochloride VBA sodium 4-vinylbenzoic acid VBTAC (ar-Vinylbenzyl)trimethylammonium chloride 2VP 2-vinylpyridine 4VP 4-vinylpyridine AAL AMBA AMPA AMPS AFM Apy AVAL ATRP BICs BIEE BSA CDMP CLRP CMP

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

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

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

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

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

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

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

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

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

Fig. 4. Selected monomers polymerized via CLRP.

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

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].

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

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

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

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

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

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

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

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

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

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

[73] [74] [75] [76]

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

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