recent progress in copolymer-mediated sirna delivery

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Introduction

RNAi-mediated gene silencing is a promising tool for treating various diseases, including cancer. It entails a short, double-stranded nucleotide with 21–23 base pairs that degrades pathogenic mRNA to suppress protein translation (Ge et al., 2004; Schiffelers et al., 2005; Aagaard & Rossi, 2007; Martin & Caplen, 2007; Shen, 2008; Oh & Park, 2009). In principle, the machinery of exogenous siRNA is very similar to that of endogenous dsRNA: it is integrated with various proteins to form an RNA-induced silencing complex and subsequently triggers cleavage of mRNA in the cytoplasm (Liu & Paroo, 2010). The cleaved mRNA is then degraded by intracellular nucleases and is no longer available for protein translation.

One of the most promising strategies to efficiently deliver the unstable siRNA while preventing its degradation is to develop various encapsulation carriers based on biopolymers and synthetic polymers. Biopolymers, such as polypeptides and proteins, have been applied to siRNA delivery (Putnam, 2006; Mintzer & Simanek, 2009; Winkler,

2011), but they are less efficient than synthetic polymers because they cannot escape the endosomal pathway. Many synthetic polymers used for gene delivery have received considerable attention as potential siRNA vectors because they are able to use mechanisms different from those of bacteria and viruses to penetrate targeted cell membranes and escape the endosomal pathway (Varkouhi et al., 2011). This pathway refers to the action of vesicles known as endosomes that have an internal pH of approximately 5. These dynamic organelles mature irreversibly from early to late endosomes and then form lysosomes, which contain digestive enzymes. Thus, polymer–siRNA complexes entering target cells via endocytosis are entrapped in endosomes that become lysosomes and are then actively degraded by digestive enzymes. This process prevents polymer–siRNA complexes from arriving at their intracellular targets. To facilitate early release of therapeutic cargos into the cytosol, these synthetic polymers must include either a proton-sponge moiety or pH-responsive components as endosomal-releasing agents.

MInI RevIew

Recent progress in copolymer-mediated siRNA delivery

Zong-Wei Wu, Chih-Te Chien, Chia-Yeh Liu, Jia-Ying Yan, and Shu-Yi Lin

Center for Nanomedicine Research, National Health Research Institutes, Zhunan, Taiwan

AbstractRNAi-mediated gene silencing has great potential for treating various diseases, including cancer, by delivering a specific short interfering RNA (siRNA) to knock down pathogenic mRNAs and suppress protein translation. Although many researchers are dedicated to devising polymer-based vehicles for exogenous in vitro siRNA transfection, few synthetic vehicles are feasible in vivo. Recent studies have presented copolymer-based vectors that are minimally immunogenic and facilitate highly efficient internalizing of exogenous siRNA, compared with homopolymer-based vectors. Cationic segments, organelle-escape units, and degradable fragments are essential to a copolymer-based vehicle for siRNA delivery. The majority of these cationic segments are derived from polyamines, including polylysine, polyarginine, chitosan, polyethylenimines and polyamidoamine dendrimers. Not only do these cationic polyamines protect siRNA, they can also promote disruption of endosomal membranes. Degradable fragments of copolymers must be derived from various polyelectrolytes to release the siRNA once the complexes enter the cytoplasm. This review describes recent progress in copolymer-mediated siRNA delivery, including various building blocks for biocompatible copolymers for efficient in vitro siRNA delivery, and a useful basis for addressing the challenges of in vivo siRNA delivery.Keywords: Vector, polyplex, copolymer, endosomolysis

Address for Correspondence: Shu-Yi Lin, Center for Nanomedicine Research, National Health Research Institutes, 35 Keyan Road, Zhunan 35053, Taiwan. Tel: 886-37-246-166. Ext: 38127. E-mail: [email protected]

(Received 13 March 2012; revised 23 May 2012; accepted 29 May 2012)

Journal of Drug Targeting, 2012; 20(7): 551–560© 2012 Informa UK, Ltd.ISSN 1061-186X print/ISSN 1029-2330 onlineDOI: 10.3109/1061186X.2012.699057

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Copolymer-mediated siRNA delivery

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Synthetic polymers have been extensively studied, in part because of their well-defined chemistries and physical characteristics and their high degree of molecu-lar diversity, which can be modified to fine-tune their physicochemical properties. Cationic polymers can associate with negative siRNA to form small and com-pact structures, spontaneously forming polyplexes (i.e. vector–siRNA complexes). The resulting particles are capable of protecting siRNA from nucleolytic enzymes by steric blocking, thereby substantially prolonging the half-life of siRNA. In addition, completely encapsulated siRNA is capable of avoiding recognition by Toll-LR 3, which is an important facilitator of off-target immune activation (Jackson & Linsley, 2010; Merkel et al., 2011), through various pathways such as endocytosis and phagocytosis. Furthermore, studies have demonstrated that the delivery vehicle can activate NF-κB (an onco-gene) of p53-deficient cells to cause cell death (Merkel et al., 2011). Interestingly, these polymer-related off-target effects appear to be cell line dependent. Thus, the design of cationic polymers needs to be re-evaluated with regard to unexpected toxicity, and their efficiency can be improved by altering side-chain composition or molecu-lar weight of polymers, or by using a copolymer.

In general, polymers comprising cationic amine groups have a strong buffering capacity and can induce an extensive flow of ions and water into the endosomal environment, leading to rupture of the endosomal mem-brane and release of its entrapped contents. For example, polyethylenimines (PEI) with branched-like structures and high molecular weights have a strong buffering effect, because they contain protonatable amine groups, which increase osmotic pressure in the endosome and disrupt the endosomal membrane (Merdan et al., 2002; Zhang et al., 2007). This strategy can be implemented with various endosomal escape enhancers: hydrophilic and hydrophobic electrolytes within the side chains of copo-lymers such as poly–propylacrylic acid (PPAA) (Hoffman et al., 2002; De Paula et al., 2007).

To our knowledge, no review has been devoted to addressing copolymeric siRNA vectors that are enhanced with endosomal escape mechanisms. Thus, we focus on recent progress in copolymer-mediated siRNA delivery, including the development of various vectors, and provide a useful basis for addressing the challenges of modulating efficient siRNA association and dissociation with copoly-mers. Given the exploding interest in RNAi, we expect that advances in siRNA delivery will improve many copolymeric delivery systems. We first briefly introduce the homopoly-meric vectors and then describe endocytosis-disrupting copolymers that have been applied in siRNA delivery.

various well-known homopolymeric vectors

Several cationic polymers, including polylysine (PLL), polyarginine, chitosan, PEI, and polyamidoamine (PAMAM) dendrimers, have been thoroughly inves-tigated as non-viral siRNA and plasmid vectors (Gary

et al., 2007; Zhang et al., 2007; Aigner, 2008; Fattal & Bochot, 2008; Morille et al., 2008; Oh & Park, 2009; Kim et al., 2010d; Duncan, 2011; Bruno, 2011). Protonatable polymers can associate with siRNA through electro-static interactions between positive charges exposed on the vector and negative phosphates on the RNA back-bone, thus spontaneously forming small and compact polyplexes.

PLL was one of the first polymers used for non-viral gene delivery (Wu & Wu, 1987), and its efficiency depen-dents on its molecular weight (Wolfert et al., 1999). Although PLL has protonatable amines, its building blocks cannot support the buffer capacity needed for endosomolysis, resulting in lower RNA silencing effi-ciency (Varkouhi et al., 2011). A similar limitation was observed in chitosan (a food additive), which is sig-nificantly more biocompatible than PEI. Thus, chitosans are interesting candidates for gene vectors. However, they still lack the buffer capacity needed for endoso-molysis, which is essential to siRNA release from the endosome. With regard to PLL analogues, modification of PLL with histidine or other imidazole groups signifi-cantly improves endosomal escape, with no significant increase in toxicity (Cho et al., 2003). Nevertheless, this has not yet been applied to siRNA delivery. Polyarginine has efficiently delivered siRNA for tumour suppression in a mouse model (Kim et al., 2010d) and was conju-gated with a specific antibody to target leukaemia cells (Lee et al., 2010b).

Among these cationic polymers, PEI has been inten-sively investigated with regard to siRNA delivery. PEI has many amine groups to effectively bind and protect siRNA, and its excellent buffer capacity, due to the proton-sponge effect, promotes endosomal escape. Once the polyplexes have entered the cell via endocytosis, acidification of the endosome enhances the positive charge density of the polymer. This leads to an enhanced influx of chloride and water, which interact with the cationic polymers to swell and burst the endosomes, thus releasing the payload into the cytosol. PEI has two conformations: linear (termed LPEI) and branched (termed BPEI). BPEI appears to deliver siRNA more efficiently than LPEI (Fischer et al., 1999). However, most commercially available PEIs are BPEIs because these are much easier to synthesize than LPEI; thus, relatively few studies have examined the deliv-ery efficiency of LPEI. In addition, BPEI is generally more soluble in water than LPEI. Overcoming the difficulties of synthesizing LPEI is essential, because it may lead to the development of LPEIs with various molecular weights; such variety is already available for BPEIs. Furthermore, the relative efficiencies of BPEI and LPEI as siRNA vec-tors have only been evaluated in vitro, and cytotoxicity is their major drawback. PEI may not be biodegradable.

PAMAM dendrimers represent other potential polymers with strong buffer capacities. They are composed of two simple monomers, such as ethylene diamine and methyl acrylate, that form a branched globular shape in the dendritic cavity via the tertiary amino group, and they

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Copolymer-mediated siRNA delivery 553


have abundant functional terminal groups in their outer shells (Tomalia et al., 1986). The primary amino groups on the surface of PAMAM dendrimers enable RNA binding, while the tertiary amino groups inside the cavity act as a proton sponge in endosomes and promote release into the cytoplasm. PAMAM dendrimers have been explored for siRNA delivery (Zhou et al., 2006), but studies have shown that they are toxic (Lee et al., 2009). Various modifications have been introduced to reduce the cytotoxicity of PAMAM dendrimers without compromising gene silencing. For example, the surface amines of a fourth-generation dendrimer (PAMAM) were methylated to form QPAMAM, which has been further evaluated for siRNA delivery (Tamura et al., 2010). Cyclodextrins (CDs) can be integrated with PAMAM dendrimers to reduce cytotoxicity, because they have a hydrophobic central cavity for drug loading and their outer surface with positive charges can interact with cell membranes (Uekama et al., 1998). Thus, CDs and their derivatives are very suitable for increasing the transfection efficiency of siRNA delivery systems (Arima et al., 2011; Kim et al., 2011).

Copolymers for endosomolysis

Endosomal escape is one of the barriers to efficient siRNA delivery. Once copolymer–siRNA complexes are internalized by endocytosis, the fate of the siRNA is either degradation by lysosomal enzymes or escape from the endosome, which is dependent on the buffering capacity of the copolymer (Kichler et al., 2001). Cationic segments, organelle-escape units, and degradable fragments are essential to excellent copolymer-based vehicles for siRNA delivery. The majority of these cationic segments are derived from polyamines, including PLL, polyarginine, chitosan, PEI, and PAMAM dendrimers. Not only do these cationic polyamines protect siRNA, they can also promote disruption of endosomal membranes. Additionally, many degradable copolymers can be derived from polyamines to graft various polyelectrolytes, such as protonatable and/or degradable fragments, with excellent endosomolysis for efficient siRNA condensation (Table 1). After introduction of a protonatable fragment, the polyamines can easily escape the endosomes. The degradable fragments can then be hydrolysed to facilitate release of the siRNA after entering the cytoplasm. For example, there are numerous reports of polyamines consisting of various functional fragments, such as pH-disrupting polyesters (Nguyen et al., 2008; Xiong et al., 2009; Remaut et al., 2010) and polyketals (Shim & Kwon, 2011), hydrazone linkers (Lin et al., 2010), redoxable polydisulfides (Jeong et al., 2009; Jere et al., 2009b; Matsumoto et al., 2009; Sun et al., 2009; Park et al., 2010; Rahbek et al., 2010; Cho et al., 2011), hydrolytic hydrogels (San Juan et al., 2009; Singh et al., 2009), reactive oxygen species–responsive polythioketal (Wilson et al., 2010), and pH-labile polymers (Wolff & Rozema, 2008). These combinations allow fast release of siRNA after cellular uptake.

Employing this simple strategy, two research groups have used a poly(β-amino ester) composed of PEI and poly(ethylene glycol) to facilitate siRNA release (Jere et al., 2008; Vandenbroucke et al., 2008). Various endosome-disrupting fragments grafted to carrier sys-tems have been developed for this purpose (Wang et al., 2009), and they allow the vectors to change their pH-sensitive amphiphilic structure at the endosomal pH. In addition, other copolymers composed of chi-tosan and PEI have been prepared, and these are able to protect the siRNA in the extracellular environment and effectively deliver it to the site of interest. Another example, poly(latic-co-glycolic acid) (PLGA) is an older polymer that has been approved by the US Food and Drug Administration. Recently, PLGA has been used to mediate siRNA delivery (Campolongo & Luo, 2009). Unlike the cationic polymers, PLGA is biodegradable, decomposing into lactic acid and glycolic acid, mak-ing it biocompatible. Studies have shown that PLGA can be densely loaded with siRNA for local administra-tion, such as intravaginal gene silencing (Katas et al., 2009; Woodrow et al., 2009). However, it is essential to improve the siRNA-encapsulation efficiency of PLGA by using polyamines with low molecular weight, such as PEI. Release of siRNA is significantly more efficient when the PLGA–siRNA complex is subjected to acidic conditions (very similar to the endosomal environment) instead of physiological conditions (pH 7.4). The release mechanism involves pores that form on the surface of decomposing PLGA (Woodrow et al., 2009).

Polycarboxylate polymers, including poly(ethylacrylic acid), PPAA, and poly(butylacrylic acid), are pH-respon-sive polymers that are able to disrupt the endosomal membrane for efficient siRNA release into the cyto-plasm (Hoffman et al., 2002; El-Sayed et al., 2005). The carboxylate group of these polymers can be transformed to carboxylic acid, altering their polarity to make them hydrophobic instead of hydrophilic and thus increas-ing their interaction with the endosomal membrane. However, polycarboxylate polymers cannot encapsulate siRNA and must be combined with cationic polymers for siRNA association. Table 1 summarizes endosome-disrupting copolymers that were used in siRNA delivery from 2004 through 2011.

Challenges and perspectives

Safety will be a primary concern in future studies, fol-lowed by the rate of siRNA transfection. Like viral vec-tors, these polymeric and copolymeric vehicles may have safety issues. Evidence of biocompatibility is lack-ing for the copolymers designed so far. Novel polymers with excellent biocompatibility are needed to improve transfection efficiency. In addition, it is important to study whether these polyplexes release siRNA efficiently enough for mRNA knockdown. However, we still lack the convincing data and controllable parameters necessary for in vivo studies of mRNA silencing with polymer–siRNA

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Table 1. Copolymer-based siRNA vectors recently tested in vitro and in vivo.Polymer typesa In vitro In vivo ReferencesPLGA/PEI Mouse Khan et al., 2004Polypeptide MDA-MB-435, C6, C6/lacZ, SVR-bag4 Leng et al., 2005BPEI-PEG-FOL KB, A549 Sun et al., 2005Dextran, PEG Hepa-luc Mouse Wong et al., 2006R8-PEG-PE, pNP-PEG-PE SK-MES-1 Zhang et al., 2006Polydisulfide amine COS7, MDA-MB-231, U373 MG Wang et al., 2007CDM-PEG Mouse Rozema et al., 2007RGD-PEG-BPEI, PEG-LPEI, PDMAEA-ppz Mouse de Wolf et al., 2007Poly(TETA/CBA) PC-3 Hoon Jeong et al., 2007LPEI, Lipid A549 Mouse Bolcato-Bellemin et al., 2007PEI-HD-1 HuH7, HuH7eGFPLuc Tarcha et al., 2007Chitosan H1299 Liu et al., 2007CDP, PEG-AD, Tf-PEG-AD Monkey Heidel et al., 2007dendrimer-cyclodextrin NIH3T3 Tsutsumi et al., 2007CDP-Im, PEG-AD, Tf-PEG-AD HeLa Bartlett and Davis, 2007THCO HeLa, U87 Wang et al., 2008PEG, BPEI, PLL, PEI-PEG-DMMAn-Mel Neuro 2A-eGFPluc Meyer et al., 2008Poly (DAH/CBA) H9C2 Kim et al., 2008Lyophilized chitosan H1299 Andersen et al., 2008DEAPA(68)–PVA–PLGA H1299 Nguyen et al., 2008PEG-PCL/DM, PEG-PLGA MCF-7 Mouse Bouclier et al., 2008LPEI-PEG A549 Jere et al., 2008PbAE HuH-7 Vandenbroucke et al., 2008PEG-b-PCL-b-PPEEA HEK293 Sun et al., 2008Pluronic-PEI PC-3, HeLa Lee et al., 2008PEG-DMA, lipid HeLa, HUVEC Auguste et al., 2008EHCO-BN-PEG CHO-d1 Wang et al., 2009

Poly-β-peptides Zhang et al., 2009

Poly(CBA-SP) A549 Mouse Jere et al., 2009b

N,N″-dioleylglutamide A549, HeLa, WM266.4, B16F10-RFP Mouse Suh et al., 2009

PEO-b-(PCL-g-SP) PEO-b-(PCL-g-TC) PEO-b-(PCL-g-DP)

MDA435/LCC6 Xiong et al., 2009

Chitosan-g-BPEI A549 Jere et al., 2009aPullulan Rabbit San Juan et al., 2009PDMAEMA-co-PAA-co-BMA HeLa Convertine et al., 2009PLGA/spermidine Mouse Campolongo and Luo, 2009PDMAEMA-MAPEG PC-3 Kong et al., 2009PEG-b-PDEAMA HuH-7 Tamura et al., 2009PEG-b-(PLL-IM) HuH-7 Matsumoto et al., 2009BPEI-PLGA, DextranVS C2C12, APCs, HDF Singh et al., 2009PLGA/spermidine HepG2, HeLa Mouse Woodrow et al., 2009PEI-lipid-PEG HT-29 Liu et al., 2009PEG-PCL, PEG-PLA, PEG-PBD A549, C2C12 Mouse Kim et al., 2009BPEI-PLGA CHO K1, HEK 293 Katas et al., 2009PEO-PAA-PNIPAM Xu et al., 2009PEI-PEG HeLa Mouse Merkel et al., 2009PEG-PLL-DMMAn-Mel, PEG-PLL-PDP Neuro2A Mouse Meyer et al., 2009G3-[PEG-RGD]-[DOX] U87 Kaneshiro and Lu, 2009Poly(CBA-DAH-Arg) PC-3, HeLa, KB, A2780 Sun et al., 2009DEAPA-PVA SKOV Kissel et al., 2010Chitosan PC12 Mittnacht et al., 2010Poly(EAA-co-BMA/HMA)-b-BLA-b-NASI-NCA

MCF-7 Lin et al., 2010

PbAE A549 Remaut et al., 2010PEAMA-PEG HuH-7 Tamura et al., 2010

(Continued)

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PAsp(DET)-Stearoyl, PLL-Stearoyl Panc-1 H.J. (Kim et al., 2010b)PLL-PEG, DMMAn HuH-7 Buyens et al., 2010PPADT RAW 264.7 Mouse Wilson et al., 2010PAMAM Pavan et al., 2010Chitosan-PLR-PEG A549, Hepa 1–6, VK2 Mouse Noh et al., 2010PDMAEMA-b-(BMA)-b-(PAA) HeLa Convertine et al., 2010PDMAEMA-PSMA/PDbB NCI-ADR/RES Benoit et al., 2010PLL Singh et al., 2010PAsp(DET) B16F10 Takemoto et al., 2010PLGA Cun et al., 2010Poly(CBA-DAH) NIH3T3, H9C2 Nam et al., 2010PHPMA-s-PAPMA York et al., 2010Poly(CBA-DAH) PC-3 S.H. (Kim et al., 2010c)PEI-PLA Mouse Brunner et al., 2010PLGA H1299 Jensen et al., 2010PEI B16F10 S.Y. (Lee et al., 2010a)BPEI-b-HA B16F1 Mouse Park et al., 2010BPEI-StA B16 Mouse Alshamsan et al., 2010PNIPAAm-biotin MDA-MB-435 C. (Kim et al., 2010a)Chitosan-BPEI B16F10 Mouse Huh et al., 2010Acetal-PEO-b-PCL-g-SP/DP Peptide-PEO-b-PCL-g-SP

MDA435/LCC6 Xiong et al., 2010

PEG-b-PCL-b-PPEEA BT474 Mouse Mao et al., 2011Lipid, PG, AA, DS, HS, PAA, CMC B16 Mouse Schlegel et al., 2011BPEI-PCL-PEG HeLa Liu et al., 2011Chitosan-g-(PEI-b-cyclodextrin) HEK293, COS7, L929 Ping et al., 2011BPEI-StA HuH-7 Mouse Huang et al., 2011PLL HEK 293 Chang Kang and Bae, 2011PEO-amine, extremely large library HeLa Mouse Siegwart et al., 2011Polypeptide SKOV-3 Canine et al., 2011Poly(MAG-b-AEMA) HeLa Smith et al., 2011Lipid, PLGA-PEG HeLa Mouse Shi et al., 2011PSKE, PSE, PSKE-PSE E.G7-OVA, NIH 3T3 Mouse Shim and Kwon, 2011PEG-b-PLL(N2IM-IM), PEG-b-PLL(MPA) B16F10 Mouse Christie et al., 2011LPEI-AScMs-PEG U87 Sparks et al., 2011PEG-PAsp(DET-Aco), PEG-PAsp(DET-Car) PanC-1 Pittella et al., 2011PLGA, lipid-PEG-Fol KB Mouse Cheng and Saltzman, 2011OEI-HD, OEI-HD-suc-PEG Neuro2A Philipp et al., 2011PEI-lipid(CA, MA, PA, StA, OA, LA) MDA-MB-435 Aliabadi et al., 2011PEI-lipid(CA, PA, OA, LA) MDA-MB-231 Montazeri Aliabadi et al., 2011PLA-PDX-PEG Mouse Manaka et al., 2011PAMAM-PLL-PEG A2780 Patil et al., 2011PSTEMPO-g-PEG HepG2 Ikeda et al., 2011PCL Mouse Kriegel and Amiji, 2011PEG-BPEI L929, NCI-H1299 Merkel et al., 2011PEG-b-(PCL-g-PDMAEMA) HeLa Mouse Lin et al., 2011PEG-PDMAEMA-co-SS MC3T3-E1.4 Cho et al., 2011Fol-PDMAEMA-co-PAA-co-BMA Hela Benoit et al., 2011LPEI-b-PPG-b-LPEI HepG2, 911 Brissault et al., 2011LPEI-PCL, Fol-PEG-PGA Bel-7402 Cao et al., 2011Acetal-PEO-b-PCL-g-SP/DP, PEO-b-PCL-Hyd-DOX

MDA-MB-435 Mouse Xiong and Lavasanifar, 2011

Dendrimer-cyclodextrin PC12 Kim et al., 2011Dendrimer-cyclodextrin NIH3T3 Arima et al., 2011LPEI-s-PEG A549, H460, MDA-MB-231 Mouse Tsai et al., 2011aAbbreviations are defined in the appendix.

Table 1. (Continued).Polymer typesa In vitro In vivo References

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complexes. It is too soon to draw conclusions about the rate of siRNA transfection via polymer-based vehicles. In order to elevate the transfection level, the efficiency of siRNA transfection by many polyplexes must be verified in biological fluids (i.e. serum) rather than in non-biolog-ical media or media containing only a small percentage of serum. The stability of polyplexes is affected by many factors, such as ionic strength. Once these formidable obstacles are overcome, polyplexes will be a powerful tool for therapeutic sequence-specific inhibition of gene expression.

Declaration of interest

The authors declare no conflicts of interest.

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Appendix: Components of polymer-based vectorsAbbreviation PolymerAA Alginic acidAcetal 1,1-diethoxy-3-methoxyl-Aco Cis-aconitic anhydrideAEMA N-(2-aminoethyl) methacrylamideAScMs Mucic acid-derivatized polymerBLA Β-benzyl l-aspartateBMA Butyl methacrylateBN BombesinBPEI Branched poly(ethylenimine)CA Caprylic acidCar Carballylic anhydrideCBA Cystamine bis-acrylamideCDM Carboxy dimethylmaleic anhydrideCDP Cyclodextrin derivativesCMC Sodium carboxymethyl celluloseDAH 1,6-diaminohexaneDEAPA DiethylaminopropylamineDET DiethylenetriamineDM Dodecyl-malateDMA Poly[2-(dimethylamino)ethyl methacrylate]DMMAn Dimethylmaleic anhydrideDOX DoxorubicinDP N,N-dimethyldipropylenetriamineDS Dextran sulfateEAA Ethyl acrylic acidEHCO N-(1-aminoethyl)iminobis[N-(oleicyl-cysteinyl-

histinyl-1-aminoethyl)propionamide]FOL FolateG3 Poly(l-lysine) dendrimers with a silsesquioxane

cubic coreHA Hyaluronic acidHD HexanedioldiacrylateHMA Hexyl methacrylateHS Heparan sulfateHyd Hydrazone bondsIM 2-iminothiolaneLA Linoleic acidLPEI Linear poly(ethylenimine)MA Myristic acidMAG 2-deoxy-2-methacrylamidoglucopyranoseMAPEG Poly(ethylene glycol-methyl ether,

ω-methacrylate)Mel MelittinNASI N-acryloxy succinimideNCA N-carboxy-anhydrideOA Oleic acidOEI Oligoethylenimine

PA Palmitic acidPAA Poly(acrylic acid)PAMAM Poly(amidoamine)PAPMA Poly[N-(3-aminopropyl) methacrylamide]PAsp PolyaspartatePbAE Poly(β-amino esters)PBD Poly(butadiene)PCL Poly(ε-caprolactone)PDbB Poly(dimethylaminoethyl

methacrylate-block-butylmethacrylate)PDEAMA Poly[2-(N,N-diethylaminoethyl)

methacrylate]PDMAEA Poly(dimethylaminoethyl acrylate)PDMAEMA Poly(dimethylaminoethyl methacrylate)PDP Succinimidyl 3-(2-pyridyldithio) propionatePDX PolydioxanonePE 1,2-dipalmitoyl-sn-glycero-3-

phosphoethanolaminePEAMA Poly[2-(N,N-diethylaminoethyl) methacrylate]PEG Poly(ethylene glycol)PEO Poly(ethylene oxide)PG Poly(l-glutamic acid)PGA Poly(glutamic acid)PHPMA Poly[N-(2-hydroxypropyl)methacrylamide]PLA Poly(lactic acid)PLGA Poly(lactic-co-glycolic acid)PLL Poly-l-lysinePLR poly(l-arginine)PNIPAAm Poly(N-isopropylacrylamide)pNP p-nitrophenyl carbonatePPADT poly-(1,4-phenyleneacetone dimethylene

thioketal)PPEEA Poly(2-aminoethyl ethylene phosphate)PPG Poly(propylene glycol)

ppz Phosphazene

PSE Poly(spermine ester)

PSKE Poly(spermine ketal ester)

PSMA Poly(styrene-alt-maleic anhydride)

PSTEMPO p-N-TEMPO-aminomethylstyrene

PVA Poly(vinyl alcohol)

R8 Arginine octamer

RGD Arg-Gly-Asp peptide

SP Spermine

SS Bis(2-methacryloyloxyethyl)disulfide

StA Stearic acid

TC Tetraethylenepentamine

TETA Triethylenetetramine

THCO 1,4,7-triazanonylimino-bis[N-(oleicyl-cysteinyl-histinyl)-(1-aminoethyl)propionamide]

VS Vinyl sulfone

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recent progress in copolymer-mediated sirna delivery

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