The potential and advances in RNAi therapy: chemical and structural modifications of siRNA molecules and use of biocompatible nanocarriers.

COREL-07203; No of Pages 9 Journal of Controlled Release xxx (2014) xxx–xxx

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The potential and advances in RNAi therapy: Chemical and structural modifications of siRNA molecules and use of biocompatible nanocarriers Min Kyung Joo, Ji Young Yhee, Sun Hwa Kim ⁎, Kwangmeyung Kim ⁎ Center for Theragnosis, Biomedical Research Center, Korea Institute of Science and Technology (KIST), Hwarangno 14-gil 6, Seongbuk-gu, Seoul 136-791, South Korea

a r t i c l e

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Article history: Received 14 March 2014 Accepted 17 May 2014 Available online xxxx Keywords: Chemical and structural modifications Gene delivery Polymerized siRNA RNA interference

a b s t r a c t Small interfering RNA (siRNA) has attracted great attention as a potential new drug due to its highly sequencespecific gene silencing ability and generality in therapeutic target. However, the medical applications of siRNA have been severely hindered by the lack of an optimal systemic delivery methodology. This poor delivery performance of siRNA is mainly caused by its inherent physicochemical properties including short and stiff structure, low charge density and vulnerability to nuclease cleavage. Thus, the successful development of efficient systemic delivery platform for siRNA is a fundamental requirement necessary to bring siRNA-based drugs to the market. Herein, we describe some siRNA delivery methods based on the chemical and structural modifications of delivery materials and siRNA itself to carry siRNA therapeutics safely to the targeted place without adverse effects. This review particularly explains the latest progress of chemically and structurally modified siRNA polymer (polysiRNA)-based delivery systems. The stable and compact siRNA polyplexes, which are formed by poly-siRNA and different types of biocompatible materials, can enhance serum stability and target delivery efficiency in vitro and in vivo. In addition, this review provides specific information on poly-siRNA delivery systems from basics to therapeutic applications in different animal disease models. © 2014 Elsevier B.V. All rights reserved.

1. Introduction In the past decade, RNA interference (RNAi) has drawn a lot of interest due to its great potentials for therapeutic applications [1–5]. Fundamentally, RNAi is induced by 21–25 nucleotide double-stranded RNA fragment (called small interfering RNA, siRNA), which makes RNA-induced silencing complexes (RISCs) and finally leads to endonucleolytic cleavage of the complementary target mRNA [6,7]. After the first observation of the RNAi mechanism in transgenic plants in the early 1900s [8], its specific gene silencing ability allows the RNAi technology a wide range of applications not only in functional genomics analysis and but also in the development of novel therapeutic drugs to treat various diseases [9,10]. In particular, the potential advantages of specific gene silencing at extremely low intracellular concentrations, few toxic effect and wide application make siRNA more attractive to the pharmaceutical industry. Despite these promising benefits, siRNA has a lot of technological barriers to be widely used in clinical therapy settings due to the lack of efficient delivery methodologies. In general, the synthetic siRNA molecules show low stability in physiological fluids, inefficient cellular uptake, poor tissue/cell specificity, and rapid clearance [11]. Therefore, successful siRNA therapeutics requires effective and safe carrier systems to overcome the inherent defects of siRNA and achieve maximum gene ⁎ Corresponding authors. Tel.: +82 958 6639. E-mail addresses: [email protected] (S.H. Kim), [email protected] (K. Kim).

silencing effect. Trying to develop efficient delivery systems, in the last decade, two different approaches for siRNA carriers have been developed: viral and non-viral vectors. In particular, the advantages of nonviral vectors including low immunogenicity, relatively low production cost and reproducibility potentially make them promising methods for delivering siRNA drugs [12–14]. For instance, the enhanced stability and intracellular delivery of siRNA have been investigated by using various non-viral gene delivery agents, such as dendrimers [14,15], lipidbased agents [16–19], cationized gelatin [20] and protamine–antibody fusion protein [21]. A gold standard in non-viral gene delivery, polyethylenimine (PEI) and its derivatives have been also widely applied to in vitro and in vivo siRNA delivery due to its outstanding transfection efficiency and endosomal proton sponge effect [22–25]. Likewise, up to date many studies have been focused on using conventional delivery strategies designed for existing nucleic acid drugs (such as plasmid DNA or antisense oligonucleotide) to develop siRNAbased therapeutics [15,26,27]. Unfortunately, however, the conventional delivery approaches didn't completely satisfy the clinical quality requirement for siRNA drugs, particularly due to the stiff structure and low charge density of short double-stranded siRNA. This is the main reason that synthetic siRNA molecules have difficulties in making condensed and stable nano-complexes with general cationic gene carriers such as cationic lipids and polymers via electrostatic interactions alone. More recently, chemical and structural modifications of siRNA itself and delivery materials have been suggested to overcome the innate structural problem of siRNA; chemical crosslinking of siRNA monomers,

http://dx.doi.org/10.1016/j.jconrel.2014.05.030 0168-3659/© 2014 Elsevier B.V. All rights reserved.

Please cite this article as: M.K. Joo, et al., The potential and advances in RNAi therapy: Chemical and structural modifications of siRNA molecules and use of biocompatible nano..., J. Control. Release (2014), http://dx.doi.org/10.1016/j.jconrel.2014.05.030

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M.K. Joo et al. / Journal of Controlled Release xxx (2014) xxx–xxx

chemical conjugations of siRNA to the non-viral delivery reagents, DNAtetrahedron-based siRNA structure, etc. In this article, we review recent siRNA delivery approaches focusing on chemically and structurally modified siRNA polymer (poly-siRNA)-based delivery systems, having nano-sized and compact poly-siRNA complexes with biocompatible gene carriers. Furthermore, the specific information on poly-siRNA delivery systems will be provided from basic to therapeutic applications in different animal disease models. 2. Chemical and structural modifications of siRNA and delivery materials The critical problems of in vivo siRNA drug administration are divided into two main issues: plasma stability and pharmacokinetic issues. Forming compact and stable nanoparticles can increase the stability of siRNA against enzymatic degradation inside the blood stream, thus leading to enhanced pharmacokinetic properties of siRNA molecules. However, due to the low charge density and stiff structure of siRNA, it is hard to produce compact nano-sized complexes between siRNA molecules and cationic condensing reagents. In the case of the enlarged siRNA molecules through the chemical and structural modifications, it allows siRNA to obtain enough intermolecular physical forces, such as electrostatic force, to be condensed with various polymeric carriers.

Thus, the structurally and chemically modified siRNA molecules can offer a wide variety of vector choices for siRNA delivery, because they exhibit a high binding affinity for the gene carriers. 2.1. Various attempts of chemical and structural modifications of siRNA nanocarriers Recently, the attempts to overcome the inherent problems of siRNA were done by various chemical and structural modifications of siRNA molecules themselves [28–30] (Fig. 1). The fundamental concept of these researches is that increasing the molecular weight and charge density of siRNA via siRNA modifications can induce the formation of stable nanoparticle structures leading to enhanced stability and delivery efficiency. The very first attempt for siRNA structural modification was the sticky siRNA (ssiRNA), generated with siRNA monomers having short complementary 3′-overhangs through siRNA hybridization under general annealing conditions [31]. Specifically, the modified siRNA has short complementary A5-8/T5-83′ overhangs which induce structurization of monomeric siRNA (mono-siRNA). The successfully hybridized ‘gene-like’ structure of siRNA leads to form stable and condensed nano-complexes of siRNA/PEI. The in vitro gene silencing experiments suggest that the structured siRNA has the same efficiency

Fig. 1. Schematic representation of various chemical and structural modifications of siRNA.



with 10-fold lower doses of mono-siRNA. Interestingly, under in vivo conditions, the structured siRNA treated groups exhibited much higher gene silencing efficiency (78%) than the classical mono-siRNA treated ones (54%). A chemical polymerization method was also used to increase the size of siRNA molecules [32]. A crosslinker containing a cleavable disulfide bond was utilized to obtain multimerized siRNA (multisiRNA). The single-stranded sense and antisense siRNA dimers were prepared by conjugating the 3′-thiol-modified sense and antisense mono-siRNA strands individually via a cleavable crosslinker, dithiobismaleimidoethane (DTME). The next step led to the hybridization process, of annealing between dimerized sense and antisense strands of siRNA to produce multi-siRNA. The increasing chain length and charge density of multi-siRNA resulted in the formation of more compact and condensed polyplexes with linear PEI. Furthermore, the multi-siRNA/PEI polyplexes exhibited higher stability under both serum and heparin competitive conditions compared to the monosiRNA/PEI polyplexes. For the disulfide linkage inserted as a crosslinker, the multi-siRNA molecules could be rapidly degraded into siRNA monomers in the highly reductive intracellular region to potentiate the RNAi process. As a result, the multi-siRNA/PEI polyplexes could more efficiently suppress targeted gene (GFP and VEGF) expression in vitro and in vivo than the mono-siRNA/PEI polyplexes. There were also structural modification approaches using DNA/RNA nanostructures for siRNA delivery. Focusing on the molecularly selfassembled nucleic acid nanoparticles such as tetrahedral oligonucleotide nanoparticles (ONPs) [33], therapeutic siRNA with targeting moiety, which was bound to each side of the nanostructured ONPs consisting of six short DNA fragments, obtained efficient targeted siRNA delivery in vivo. Specifically, the ONPs consist of six DNA strands having complementary overhangs with therapeutic siRNA at the 3′-ends. These molecularly structured ONPs have a narrow size distribution (~ 28.6 nm) which is an ideal form as a tumor targeting nanocarrier. The easy design of DNA sequences can control the physiochemical properties of the nanoparticles. Furthermore, the monodisperse ONPs offer complete control over the conjugation site and conjugation ratio of both siRNA and tumor-targeting ligands. For the construction of condensed RNA structures, the rolling circle transcription (RCT) technique was used as enzymatic siRNA polymerization method [34]. The elongated pure hairpin-siRNA strands led to self-assembly into organized nano- and micro-structures. Because RNAi-microsponges alone showed very low cellular uptake due to their strong negative net charge and micro-scale size, PEI formulation was applied to improve RNA condensation and stability resulting in enhanced intracellular delivery efficiency in vitro and in vivo. The RNAi-microsponge technique is expected to overcome the significant difficulties of obtaining both high loading efficiency and delivery efficiency of siRNA for RNAi therapy. 2.2. Disulfide crosslinking of siRNA for the synthesis of polymerized siRNA (poly-siRNA) Introducing the functional group ‘thiol’ to the siRNA molecules has a lot of merits. It can be easily complexed with the thiol modified carriers, and the formed disulfide linkages can be broken under reductive conditions like cell cytosol. Based on this concept, the polymerization of siRNA via disulfide crosslinking of thiolated siRNA monomers was designed for the development of efficient in vivo siRNA delivery systems [35]. The resulted poly-siRNA conjugates containing a broad range of base pairs (50–300 bps and greater than 300 bps) was synthesized via oxidation of pre-thiolated mono-siRNA to facilitate reducibility (Fig. 2A). The ladder-like pattern shown by PAGE data indicates the efficiency of polymerization depending on disulfide bond formation (Fig. 2B). The synthesized poly-siRNA conjugates were easily degraded into mono-siRNA fragments after treatment with 1,4-dithiothreitol (DTT),

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a reducing agent. The major percentage (ca. 66%) of the poly-siRNA part was observed in the mixture of oligomeric siRNA (up to 300 base pairs) and the rest was detected in the polymeric siRNA part (above 300 base pairs). The enlarged poly-siRNA conjugates exhibited greatly enhanced ionic interaction with the cationic substances due to the higher anionic charge density, more than 14 times greater than that of mono-siRNA. The compact form of the poly-siRNA polyplexes effectively shielded siRNA molecules against the enzymatic degradation in 50% rat serum conditions for 1 h, suggesting the stability of poly-siRNA nanocarriers under physiological conditions (Fig. 2C). The gene delivery efficiency of poly-siRNA was investigated in vitro using red fluorescent protein (RFP) targeting siRNA in murine melanoma cells (RFP/ B16F10) expressing RFP (Fig. 2D and E). Compared with the monosiRNA system, the poly-siRNA polyplexes showed higher RFP inhibition (80% reduction in RFP level) in RFP/B16F10 cells. Using this poly-siRNA delivery system, the promising results in various in vivo disease models demonstrate the potential of siRNA in clinical applications [36], which will be discussed in more detail at the final section of this article. 3. Natural polymer and protein-based nanocarriers for poly-siRNA delivery The modified self-crosslinked poly-siRNA have been suggested as a new approach for in vivo siRNA delivery. With different nanocarriers, the poly-siRNA delivery systems can be used more efficiently for cancer treatments. In general, natural polymers and proteins are interesting materials for gene carrier due to their safety. In addition to the low toxicity, low levels of reticuloendothelial system (RES) clearance due to the aqueous steric barrier of protein-based carriers leads to better pharmacokinetic properties [37,38]. Thus, it was also anticipated that the natural polymer and protein-based nanocarriers could enhance in vivo siRNA delivery efficiency particularly with poly-siRNA conjugates. In this section, we introduce some natural polymer and proteinbased gene carrier systems combined with poly-siRNA for cancer therapy. 3.1. Glycol chitosan nanoparticles As a natural polymer, chitosan is one of the promising materials for using in nanoparticle formation. Chitosan is a biocompatible, biodegradable and low immunogenetic polysaccharide polymer. Especially, chitosan has many functional groups to enable various chemical modifications and produce various derivatives. Unfortunately, however, the poor water solubility of chitosan still limits its wide applications in nanomedicine. Among various chitosan derivatives, thus glycol chitosan (GC) has attracted significant attention in the drug and gene formulations, particularly due to the complete water solubility and the residual functional groups for further chemical modifications. To use as poly-siRNA carriers, GC backbone was further modified with thiol groups. The thiolated glycol chitosan (tGC) polymer was used as a novel self-crosslinked nanocarrier for the poly-siRNA delivery [36]. tGC formed a stable nanoparticle with poly-siRNA through enhanced charge–charge interaction and self-crosslinking mechanism (Fig. 3A). The self-crosslinked poly-siRNA/tGC nanoparticles (psi-tGC) were prepared by adding poly-siRNA slowly to the tGC polymer solution. They showed enhanced complexation stability and importantly, they were successfully degraded and separated into monomeric double-stranded siRNA in the presence of dithiothreitol (DTT). It suggests that psi-tGC can release free siRNA monomers under reductive conditions found in cell cytosol. Moreover, psi-tGC showed a stable and condensed nanoparticle form due to the inter- and intra-chemical crosslinking leading to increased serum stability. This result supports the hypothesis that psi-tGC is stable in physiological conditions, but is degraded into monomeric siRNAs after cellular uptake in the cytosol for efficient gene silencing. psi-tGC showed a rapid internalization and localization in cytosol within 1 h. In general, glycol chitosan


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Fig. 2. (A) Schematic diagram of the formation of poly-siRNA conjugates and poly-siRNA/PEI complexes. (B) PAGE gel electrophoresis of mono-siRNA, poly-siRNA and poly siRNA incubated with and without DTT. (C) Stability test of mono- and poly-siRNA/PEI complexes under serum conditions. (D) Fluorescence microscopic images of RFP/B16F10 cells transfected by mono-siRNA, poly-siRNA, mono-siRNA/PEI and poly-siRNA/PEI. Reprinted with permission from Journal of Controlled Release 141(3):339–346. Copyright (2010) Elsevier.

nanoparticles can be taken up by cells by different endocytotic and macropinocytosis pathways [39], which support the fast cellular uptake of psi-tGC. After systemic tail vain injection, psi-tGC was preferentially accumulated in the tumor regions in a mouse xenograft model compared to naked poly-siRNA or poly-siRNA/PEI polyplexes (Fig. 3B), suggesting the selectivity to tumor tissues of the psi-tGC formulation. In addition, the therapeutic potential of psi-tGC was tested by targeting the vascular endothelial growth factor (VEGF) (Fig. 3C and D). psi(VEGF)-tGC showed also high efficacy of VEGF gene silencing of about 95% in PC3 cancer in vivo. As a result, the tumortargeted delivery of psi-tGC could enhance target gene (VEGF) silencing in a sequence-specific manner leading to successful tumor suppression. 3.2. Gelatin nanoparticles Easy modification with various functional groups [40], low toxicity and antigenicity as a denatured form of collagen make gelatin a potential siRNA carrier [41]. In particular, a complete lack of any serious side effects is the primary merit of gelatin to make it a latent candidate for

multiple administrations of drugs in clinical applications. Although gelatin can be injected intravenously for specific therapeutic purposes [42], it is commonly administered subcutaneously. The loose complexes between natural siRNA and gelatin, which can be easily degraded in the blood before RNAi performance, was the main barrier to overcome in achieving efficient gene silencing with siRNA gelatin nanocarriers. To improve the complexation ability between siRNA and gelatin, both molecules were modified using sulfhydryl groups to produce thiolated gelatin (tGel) and poly-siRNA. poly-siRNA was encapsulated into the self-crosslinked tGel nanoparticles resulting in the formation of stable and compact poly-siRNA-tGel NPs (psi-tGel NPs) [43]. The psi-tGel NPs with a size of average 145 nm effectively protected the siRNA molecules from enzymatic degradation. The functional monomeric siRNA molecules could be released under reductive conditions like inside the cell cytosol. The systemically delivered psi(RFP)-tGel NPs showed an effective down-regulation of targeted RFP gene expression in a mouse xenograft model with murine melanoma cells (RFP/B16F10). It is particularly notable that the psi-tGel NP formation showed no toxicity even with the high transfection dose of 125 μg/ml psi-tGel including 1 μM of siRNA molecules.



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Fig. 3. (A) Schematic diagram for the preparation of poly-siRNA/tGC nanoparticles for siRNA delivery. (B) In vivo real-time NIRF imaging of psi-tGC in SCC-7 tumor-bearing mice after intravenous injection of FPR675-labeled nanoparticles. As control groups, mice were injected with poly-siRNA and psi-PEI. Red circle = site of tumor. (C), (D) In vivo tumor therapeutic effects of psi(VEGF)-tGC in PC-3 tumor-bearing mice. (C) Blood vessel formation of control (CTRL) and psi(VEGF)-tGC treated tumor. (D) Tumor growth of control, free poly-siRNA, psi(sc)-tGC and psi(VEGF)-tGC treated tumor-based mice. Reprinted with permission from Angewandte Chemie 51:7203–7207. Copyright (2012) John Wiley and Sons.

3.3. Serum albumin nanoparticles Human serum albumin (HSA) is also an attractive natural proteinbased gene carrier due to its outstanding physical and biological properties. Different types of drug formulations, such as Abraxane® and Albunex® have been already commercialized by using HSA-based delivery systems. HSA is a main energy source for growth and maintenance under stress-induced conditions [44]. It has been well known that HSA shows relatively high uptake in tumor and inflamed tissue [45]. In particular, HSA-based nanoparticles efficiently accumulate at the tumor site due to the enhanced permeability and retention (EPR) effect [46]. Since the albumin receptors (pg60) contribute to transcytosis, HSA-based drug delivery systems can increase the efficiency of drug transport directly into tumor cells [47]. However, the slightly negative charge of HSA is the barrier to overcome in forming stable complexes with siRNA. For in vivo siRNA delivery, thus HSA was chemically modified using sulfhydryl groups. In this case, the thiolated HSA (tHSA) formed stable nanoparticles particularly with poly-siRNA through

oxidative intermolecular disulfide crosslinking (Fig. 4A) [48]. The stable nanosized complexes of poly-siRNA and tHSA (psi-tHSA) were made after self-assembly and chemical crosslinking. The poly-siRNA conjugates were successfully encapsulated and were tightly bound to the tHSA carriers which lead to stable nanoparticle formation (Fig. 4B and C). Compared to the commercial transfection agent Lipofectamine, the psi-tHSA system showed comparable in vitro target gene silencing activity without remarkable cytotoxicity. Using the psi-tHSA formulation, poly-siRNA could be protected against enzymatic degradation under serum conditions and enhance the blood circulation time and tumor accumulation (Fig. 4D). In murine melanoma cells (RFP/B16F10) grafted mouse model, the tumor-localized psi(RFP)-tHSA showed successful in vivo target gene silencing in a sequence specific manner (Fig. 4E). 3.4. Transferrin nanoparticles Transferrin (TF) is well known as a representative blood plasma glycoprotein. In general, continuously proliferating cells such as malignant


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Fig. 4. (A) Schematic diagram of condensed poly-siRNA/tHSA complexes. (B) Gel retardation assay of psi-tHSA (left) and Coomasie blue-staining of protein after gel electrophoresis assay of tHSAs (right). (C) Transmission electron microscopy (TEM) images of psi-tHSA. (D) In vivo real-time NIR fluorescence images of the SCC7 tumor-bearing mice after intravenous injection of psi and psi-tHSA. (E) In vivo gene silencing effects of psi(RFP)-tHSA in RFP/B16F10 bearing mice. Ex vivo red fluorescence images of the excised tumors after intravenously administration of psi(RFP)-tHSA and the semi-quantitative reverse transcriptase-PCR for evaluating RFP mRNA levels. Reprinted with permission from Biomaterials 34(37):9475–9485. Copyright (2013) Elsevier.

cancer cells overexpress TF receptors on their cell surface [49]. Through the TF receptor-mediated endocytosis mechanism, transferrin-based formulations could more effectively deliver anti-cancer drugs to target cancer sites [50,51]. In addition to unique tumor-targeting ability, biocompatibility and prolonged blood circulation of TF make it a potential siRNA carrier. However, natural TF has low binding affinity with negative nucleic acids. Therefore, both TF and siRNA molecules were chemically modified with sulfhydryl groups to overcome this problem [52]. The resulting thiolated TF (tTF) self-crosslinked into TF nanoparticles by encapsulating the poly-siRNA conjugates to be used as a biocompatible and tumor-targetable siRNA carrier in cancer therapy. The polysiRNA/tTF nanoparticles (psi-tTF NPs) protected siRNA from enzymatic degradation and delivered active siRNA molecules safely to the cytoplasm of the cancer cells in a TF receptor dependent manner. The psi-tTF NPs showed very low toxicity with a siRNA dose of 200 nM, which is the concentration of Lipofectamin formulation showing poor

cell viability (below 50%). Notably, the psi-tTF NPs showed also long blood circulation and high accumulation at the tumor site in vivo. 4. Therapeutic applications of poly-siRNA nanocarriers As mentioned above, the modified self-crosslinked poly-siRNA conjugates showed promising results by using various natural polymer and protein-based nanocarriers. Importantly, the effective in vivo gene silencing of poly-siRNA nanocarriers went over without remarkable toxicity. These potential poly-siRNA nanocarriers can be used as a general systemic delivery platform for siRNA as both a diagnostic and therapeutic tool by enhancing the strength of each carrier. The time-dependent biodistribution of poly-siRNA/tGC nanoparticles (psi-tGC) was evaluated using fluorescent imaging after intravenous injection into SCC-7 tumor bearing mice to investigate the enhanced stability and tumor targeting ability in vivo. The result



showed high accumulation of psi-tGC in tumor tissue and its colocalization with SCC-7 tumor cells, indicating the cellular penetration through leakage from tumor blood vessels and quick uptake into tumor cells. The in vivo therapeutic efficacy of psi(VEGF)-tGC were investigated in tumor bearing mice. The psi(VEGF)-tGC treatment could significantly reduce the microvessel formation in tumor tissue. As a result, the tumor volume of the psi(VEGF)-tGC treated mice was 80% reduced compared to that of the controlled mice. The psi-tGC formulations showed not only effective tumor targeting siRNA delivery, but also low toxicity which makes this system more attractive. In the case of the poly-siRNA/tGel nanoparticles (psi-tGel NPs), they showed 2.8 times higher tumor accumulation than naked poly-siRNA based on the EPR effect, leading to efficient gene silencing in tumors after intravenous injection. Moreover, the complexes of tHSA and poly-siRNA (psi-tHSA) accumulated specifically at the tumor sites in tumor bearing mice after intravenous injection with efficient gene silencing. The growth of PC-3 tumor in PC-3 tumor xenografts was also successfully suppressed by inhibition of tumor-related angiogenesis by using therapeutic VEGF siRNA loaded psi-tHSAs. These promising results indicate that the poly-siRNA

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nanocarrier systems including psi-tGC, psi-tGel and psi-tHSA can be used as systemic siRNA carriers for cancer therapy. Another approach is using the self-crosslinked poly-siRNA nanocarriers for the treatment of rheumatoid arthritis (RA) (Fig. 5) [53]. Tumor necrosis factor (TNF)-α plays a pivotal role in the release of other cytokines and the induction of chronic inflammation, which are involved in the pathogenesis of RA. The treatment of anti-TNF-α monoclonal antibodies, such as infliximab and adalimumab, was limited by the risk of injection site reactions, infusion-related reactions and infection. The poly-siRNA-tGC nanoparticles (psi-tGC-NPs) have already shown promising results as siRNA nanocarrier for cancer treatment reported before [36]. Since the stable psi(TNF-α)-tGC nanoparticles with an average diameter of 370 nm also showed rapid cellular uptake and admirable TNF-a gene silencing efficacy in the macrophage culture system. Likewise, the active siRNA monomers released from the psitGC-NPs could efficiently knock-down the target mRNA in a sequencespecific manner. In particular, the psi-tGC-NPs accumulated with enhanced properties in arthritic joints in collagen-induced arthritis (CIA) mice. Based on the in vivo treatment monitoring data with the

Fig. 5. (A) Schematic diagram of TNF-α gene knockdown after uptake of psi-tGC-NPs by activated macrophage cells in RA model. (B) TNF-α mRNA levels obtained from LPS-activated RAW 264.7 cells treated with poly-siRNA, psi(scramble)-tGC-NPs, psi(TNF-α)-LF and psi TNF-α-tGC-NPs. (C) In vivo NIRF images of arthritic joints after intravenous injection of free psi(TNF-α) and psi(TNF-α)-tGC-NPs. (D) Monitoring of RA progression in CIA mice of control (CTRL), MTX and psi(TNF-α)-tGC-NPs. Mouse paw images of control and psi(TNF-α)-tGC-NP-treated groups (upper). Three-dimensional reconstruction of paws using Micro-CT (lower). Reprinted with permission from Molecular Therapy http://dx.doi.org/10.1038/mt.2013.245. Copyright (2013) Nature Publishing Group.


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matrix metalloproteinase 3-specific nanoprobe and micro-computed tomography, the intravenous injection of psi(TNF-α)-tGC significantly inhibited inflammation and bone erosion in CIA mice, comparable to methotrexate (5 mg/kg) which is considered the standard treatment for RA. These results suggest the possibility of using the poly-siRNA nanocarriers as in vivo siRNA delivery platforms for the treatment of different systemic diseases.

5. Perspectives Recently, gene therapy has drawn much wider attention as a new therapeutic tool for various genetic disorders. Especially, siRNA drugs are extremely promising RNAi therapeutics due to its specific gene knockdown ability with a wide range of genetic targets. Besides the amazing potentials as a future genetic medicine, a lot of barriers are accompanied leading to the need of improvement for the delivery carriers and siRNA activity itself. To overcome the major delivery obstacles, until now different types of delivery approaches have been attempted using viral and non-viral vectors. Recently, several siRNA-based drugs already entered clinical trials and are in the developmental pipeline [54]. Although the siRNA-based therapeutics prefer the intravenous injection method for practical reasons, most of them are not injected intravenously but rather into the target tissues directly. Thus, the development of safe and effective systemic siRNA delivery systems is still considered to be crucial for a wide range of clinical applications of siRNA. The focus at the early tries was to design different delivery vehicles by designing the materials with different size, shape, structure, chemistry and mechanisms of the delivery system. Currently, another focus was to improve the inherent poor pharmacokinetic properties of siRNA itself. Different chemical and structural modifications of siRNA molecules could change the size and nanostructure of siRNA and improve its complexation ability and in vitro/in vivo delivery efficiency. In order to develop effective in vivo siRNA delivery systems, thus siRNA therapeutics requires not only the novel design of vehicles but also diverse modifications of siRNA structure, consequently leading to their successful translation from bench to bed side.

Acknowledgements This work was supported by grants (NRF-2012K1A1A2A01056095, NRF-2009-0081876 and NRF-2013K1A1A2A02050115) of National Research Foundation of Korea (NRF) and the Intramural Research Program of KIST.

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Recent advances in biocompatible nanocarriers for delivery of chemotherapeutic cargoes towards cancer therapy.

Liposomal siRNA nanocarriers for cancer therapy.

Chemical and structural effects of base modifications in messenger RNA.

Advances in the use of nanocarriers for cancer diagnosis and treatment.

Mucus barrier-triggered disassembly of siRNA nanocarriers.

Design of potential RNAi (miRNA and siRNA) molecules for Middle East respiratory syndrome coronavirus (MERS-CoV) gene silencing by computational method.

Design of potential RNAi (miRNA and siRNA) molecules for Middle East respiratory syndrome coronavirus (MERS-CoV) gene silencing by computational method.

siRNA Delivery by Stimuli-Sensitive Nanocarriers.

Biocompatible colloidal dispersions as potential formulations of natural pyrethrins: a structural and efficacy study.

Advances in adjuvant therapy: potential for prognostic and predictive biomarkers.

Size, charge, and stability of fully serine-based catanionic vesicles: towards versatile biocompatible nanocarriers.

Chemical modifications of proteins: history and applications.

A Light-Driven Therapy of Pancreatic Adenocarcinoma Using Gold Nanorods-Based Nanocarriers for Co-Delivery of Doxorubicin and siRNA.

Oncogene dependency and the potential of targeted RNAi-based anti-cancer therapy.

Structural analysis of a group II intron by chemical modifications and minimal energy calculations.

Toward optimization of AgoshRNA molecules that use a non-canonical RNAi pathway: variations in the top and bottom base pairs.

Synthesis of a stabilized 177Lu-siRNA complex and evaluation of its stability and RNAi activity.

A RNA nanotechnology platform for a simultaneous two-in-one siRNA delivery and its application in synergistic RNAi therapy.

Biocompatible nanocarriers that respond to oxidative environments via interactions between chitosan and multiple metal ions.

Poly(amidoamine) dendrimer nanocarriers and their aerosol formulations for siRNA delivery to the lung epithelium.

Development of a simple, biocompatible and cost-effective Inulin-Diethylenetriamine based siRNA delivery system.

L Overlapping Region of Respiratory Syncytial Virus: In Silico Design.

deoxycholic acid-modified polyethylenimine complexes for myocardial infarction.

Recent advances in nanoparticle-mediated siRNA delivery.