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Pegylated poly-l -arginine derivatives of chitosan for effective delivery of siRNA
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Journal of Controlled Release j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j c o n r e l
Pegylated poly-L-arginine derivatives of chitosan for effective delivery of siRNA Sang Myoung Noh a,1, Myung Ok Park b,1, Gayong Shim c, Su Eun Han c, Han Young Lee c, Jun Hyuk Huh c, Myoung Suk Kim b, Jin Joo Choi b, Kwangmeyung Kim d, Ick Chan Kwon d, Jin-Seok Kim e, Kwang-Hyun Baek f, Yu-Kyoung Oh a,⁎ a
College of Pharmacy, Seoul National University, Sinlim-dong, Gwanak-gu, Seoul, South Korea Biopolymed Inc., Seoul, South Korea School of Life Sciences and Biotechnology, Korea University, Anam-dong, Seungbuk-gu, Seoul, South Korea d Biomedical Research Center, Korea Institute of Science and Technology, Seoul, South Korea e School of Pharmacy, Sookmyung Women's University, Seoul, South Korea f Department of Biomedical Science, CHA University, Seoul, South Korea b c
a r t i c l e
i n f o
Article history: Received 20 May 2009 Accepted 5 April 2010 Available online 10 April 2010 Keywords: Chitosan Polyarginine Polyethylene glycol siRNA Serum stability
a b s t r a c t For delivery of siRNA, chitosan (CS) was derivatized with poly-L-arginine (PLR) and polyethylene glycol (PEG). The formation of polyplexes with siRNA was confirmed by gel retardation. The PLR-grafted CS formed nanosized particles with siRNA. PLR-grafted CS showed higher cellular delivery efficiency of siRNA than did CS, pegylated CS, PLR, or pegylated PLR. The extent of reduction in the expression of fluorescent proteins was highest following treatment of the cells using PLR derivatives of CS in complexes with specific siRNAs. Cell viability was greater in populations treated with pegylated CS–PLR than in those treated with PLR. Hemolysis of erythrocytes was reduced upon conjugation of PLR with CS. The delivery of siRNAs via pegylated CS–PLR revealed little dependence on serum. Molecular imaging techniques revealed that the intratumoral administration of red fluorescent protein-specific siRNA in complexes with pegylated CS–PLR significantly silenced the expression of red fluorescent proteins in tumor tissues in vivo. These results indicate that pegylated CS–PLR might be useful for in vivo delivery of therapeutic siRNAs. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Small interfering RNAs (siRNAs) are emerging as a new class of therapeutics. In less than one decade after the discovery of RNA interference in mammalian cells, several siRNAs are in clinical trials for treatment of disease [1]. However, most siRNAs in clinical trials are administered in naked forms for local delivery into the eye or lung [2]. siRNA applications have been restricted because of their limited penetration of cell membranes [3]. Several nonviral delivery systems for siRNA have been studied, including the use of cationic polymers such as polyethylenimine (PEI) and chitosan (CS) [4]. Among several cationic polymers, CS has been widely studied in delivery systems of negatively charged nucleic acidbased medicines [5]. Its advantages are biocompatibility, biodegradability, low cost, and positive charge, which allows it to easily form polyelectrolyte complexes [6]. However, the siRNA transfection efficiency of CS is still limited, and further development of improved CS-based delivery systems by grafting on other materials is required. In this study, we derivatized CS with a biodegradable cationic polymer, poly-L-arginine (PLR), and further pegylated the derivatives ⁎ Corresponding author. Tel.: + 82 2 880 2493; fax: + 82 2 882 2493. E-mail address: [email protected] (Y.-K. Oh). 1 Both authors contributed equally to this manuscript. 0168-3659/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.jconrel.2010.04.005
of CS. Here, we report that CS grafted with PLR and polyethylene glycol (PEG) was effective in cellular delivery of siRNA with low cytotoxicity and hemolysis. Moreover, this delivery system using a CS derivative was able to produce silencing of target proteins in tumor tissues in vivo. 2. Materials and methods 2.1. Materials Low molecular weight chitosan (CS, m.w. 50–150 kD; deacetylation degree, 87%) was purchased from Fluka (Buchs, Switzerland). Poly-L-arginine (PLR, m.w. 15–70 kD), was from Sigma-Aldrich (St. Louis, MO, USA). A luciferase mRNA-specific siRNA (siGL2) and a murine survivin-specific siRNA (siSVN) were from Samchully Pharm. Co. (Seoul, South Korea). A green fluorescent protein-specific siRNA (siGFP) and a red fluorescent protein-specific siRNA (siRFP) were from Bioneer Co. (Daejeon, South Korea). 2.2. Gel permeation chromatography (GPC) The molecular weights (Mn, Mw) and distributions (Mw / Mn) of CS and CS derivatives were determined by GPC. GPC was carried out in a column (7.8 mm × 300 mm) of 2 × Jordi gel polar pack wax mixed-bed
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equipped with a Waters 2410 differential refractometer and Waters 515 pump (Waters, Milford, MA, USA).
software (version 3.5, Systat Software, Richmond, CA, USA) was used for all analyses. A p value of less than 0.05 was considered significant.
2.3. Particle size and zeta-potential measurements
3. Results
The sizes and zeta potentials of the complexes of polymers and siRNA were measured using an ELS-8000 instrument (Photal, Osaka, Japan). Ten micrograms of siSVN and 120 µg of each polymer were complexed in 3 mL of phosphate-buffered saline (pH 7.4). The diameters were determined via He–Ne laser (10 mW) light scattering.
3.1. Synthesis of CS-based cationic polymers
2.4. Assessment of siRNA cellular uptake The cellular uptake of siRNA delivered by various cationic polymers was measured using flow cytometry. At 12 h before transfection, 1 × 105 cells were seeded. Next, Block-IT™ (20 pmole) complexed to each polymer was added to Hepa 1–6 cells and incubated for 24 h. For flow cytometry, the cells were analyzed by a BD FACS Calibur using Cell Quest Pro software (BD Bioscience, San Jose, CA, USA). In some experiments, the intensity of cellular fluorescence was assessed after incubation in various serum concentrations, and measured using a Zenyth 3100 Microplate Reader (Anthos Co., Salzburg, Austria) at excitation and emission wavelengths of 485 and 535 nm, respectively. 2.5. Western blot analysis The siRNA-mediated reduction of target gene expression was detected at the protein level by western blotting. Hepa 1–6 cells, A549 cells, or VK2 cells were treated for 48 h with various complexes of siRNA with cationic polymers. Lysates of whole cells (30 µg of total protein) were separated on a 10% SDS-PAGE gel. Western blots were performed with specific antibodies to survivin (1:1000, sc-10811, Santa Cruz Biotechnology, CA, USA) and β-actin (1:2500, sc-47778, Santa Cruz Biotechnology). 2.6. Cell viability and in vitro hemolysis assays An MTT assay was used to measure the cytotoxicity of cationic polymers as described previously [7]. An in vitro hemolysis assay was performed to estimate the toxicity of cationic polymers in blood as described previously [8]. 2.7. In vivo molecular imaging Female nu/nu mice (Orient Bio Inc., Seungnam, Gyeonggi-do, South Korea) were used for the induction of red fluorescent protein (RFP)-expressing tumors. The mice were subcutaneously injected with 1 × 106 B16F10-RFP cells at left and right flanks. At 8 days after the inoculation, the right flank tumors were treated with siRNA in naked or complexes. The left flank tumors were untreated in all mice. The polyplexes were prepared at the polymer/siRNA weight ratio of 12/1. Treatment of mice involved three intratumoral injections of the complexes at a dose of 120 µg of cationic polymers (CS–PLR, PEG–CS– PLR) and 10 µg of siRNA per mouse. Three days after treatment, in vivo imaging was performed on the B16F10-RFP tumor-bearing mice using the Kodak Image Station 4000MM (Eastman Kodak Co., Rochester, NY, USA) equipped with band-pass excitation and long-pass emission filters at 535 nm and 600 nm. The images were processed with Kodak molecular imaging software version 4.0. 2.8. Statistics Statistical analysis of data was performed using ANOVA with the Student–Newman–Keuls test employed as a post-hoc test. SigmaStat
The synthesis schemes of CS–PLR and PEG–CS–PLR conjugates are summarized in Suppl. Fig. 1. PLR was covalently linked to CS via formation of amide bonds between the amine groups (–NH2) of CS and the carboxyl groups (–COOH) of PLR. The formation of amide bonds between the activated carboxyl groups (–COOH) of PLR and amine groups (–NH2) of CS resulted in the conjugates of CS–PLR. The conjugation of PLR to CS and the pegylation of CS–PLR conjugates were confirmed by 1H NMR (data not shown). The grafting of PLR and PEG to CS was tested by FI-IR spectra. PEG–CS–PLR showed the characteristic absorption band around 2900 cm− 1 and 1650 cm− 1, resulting from the increased C–H bond and amide (C O) bond formation by grafting of PEG to CS–PLR. GPC results (Table 1) indicate that PEG–CS with a number-average molecular weight (Mn) of ca. 78,000 and a polydispersity index of 1.4 was obtained. Mn of PEG–CS–PLR was ca. 90,000 with a polydispersity index of 1.4. The average molecular weights indicate that the average numbers of PEG per CS in PEG–CS conjugates were 4. Moreover, the average numbers of PEG and PLR per CS in PEG–CS conjugates were calculated as 4 and 1, respectively. 3.2. Characterization of siRNA and cationic polymer complexes All cationic polymers tested in this study formed complexes with negatively charged siRNA. However, the types of cationic polymers affected the minimal complexation ratios at which the complexes showed the gel retardation. The polymers CS, PEG–PLR, CS–PLR, and PEG–CS–PLR showed complete gel retardation starting at the polymer/siRNA weight ratio of 5:1 (Suppl. Fig. 2). Meanwhile, PEG– CS and PLR formed complexes with siRNA from the weight ratios of 50:1 and 0.5:1, respectively. Table 2 shows the sizes and zeta-potential values on the complexes between each polymer and siRNA at the complexation weight ratio of 12/1. Among the groups tested, CS/siRNA complexes showed the smallest particle size and the lowest zeta potential. The sizes of the particles did not significantly differ between CS–PLR and PEG–CS–PLR, showing less than 400 nm sizes upon complexation with siRNAs. As compared to CS, CS–PLR and PEG–CS–PLR showed significantly higher zeta potential upon complexation with siRNAs. 3.3. Intracellular delivery of dsRNA by cationic polymers FACS analysis (Fig. 1) revealed that the PLR conjugates of CS increased the intracellular delivery of siRNA as compared with CS. To quantitate the intracellular uptake of siRNA, fluorescent dsRNA was complexed to various cationic polymers with polymer/siRNA weight ratio of 12:1. Fig. 1 shows that the fluorescence-positive populations of the cells were larger with delivery of siRNA using CS–PLR and PEG– CS–PLR than using other cationic polymer groups. CS–PLR (Fig. 1H) and PEG–CS–PLR (Fig. 1I) showed 78% and 79% positive cell populations,
Table 1 Molecular characteristic of the CS and CS derivatives. Polymers
Mn × 103
Mw × 103
Mw / Mn
CS PEG–CS PEG–CS–PLR
70.0 78.0 90.0
140.0 107.2 122.7
2.0 1.4 1.4
Mn: number-average molecular weight, Mw: weight-average molecular weight, Mw / Mn: polydispersity index.
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Table 2 Particle sizes and zeta potentials of polymers and siRNA complexes. Polymer/siRNA (12/1, w/w)
Particle size (nm)
Zeta potential (mV)
CS/siRNA CS–PLR/siRNA PEG–CS–PLR/siRNA
117.5 ± 4.9 307.6 ± 118.3 375.1 ± 188.4
+ 15.6 ± 6.8 + 35.3 ± 4.5 + 23.9 ± 3.6
whereas CS alone (Fig. 1D) and PEG–CS (Fig. 1E) resulted in 1.5% and 2.6% positive populations, respectively. Widely used transfection agent, Lipofectamine™2000 (L2K), was used for comparison with the cationic polymers synthesized in this study. The delivery efficiency of CS–PLR or PEG–CS–PLR was comparable to that of L2K (Fig. 1C). 3.4. Reduction of target gene expression The treatment of siRNAs differentially reduced the expression of target protein, depending on the delivery systems. As target proteins, survivin and GFP were tested using siSVN and siGFP, respectively. Western blot data reveals that the cellular levels of survivin reduced after treatment of siSVN complexed with L2K, CS–PLR, or PEG–CS–PLR at all the complexation ratios tested. Indeed, we tested the silencing of target protein after treatment with PLR/siRNA complexes. However, due to the high cytotoxicity of PLR, PLR was excluded from the comparison groups. A549 (Fig. 2B) and VK2 (Fig. 2C) cells showed that the reduction of survivin levels were dependent on the complexation ratios, resulting in the highest reduction at the ratio of 12:1. In addition to survivin-silencing, the reduction of GFP expression was tested using siGFP complexed with various delivery systems. Consistent to siSVN treatment, the PLR conjugates of CS were effective in siGFP-mediated silencing of target protein expression. For polyplexes, the complexation ratio between polymer and siGFP was 12:1 (w:w). When GFP-expressing cells were treated with the complexes of siGFP and CS, the expression level of GFP was similar to the untreated control. Following siGFP treatments of the cells with CS–
Fig. 2. Silencing of target protein expression after treatment with siRNAs. (A) Hepa 1–6, (B) A549, (C) VK2 cells were treated with siSVNs alone or using various delivery systems. The protein extracts were analyzed by western blot. (D) 293 T-GFP cells were treated with siGFP alone or in complexes with various delivery systems. The complexation ratio between polymer and siGFP was 12:1 (w:w). After 72 h, the fluorescence was observed under a fluorescence microscope. Scale bar is 100 µm.
PLR and PEG–CS–PLR, the cellular levels of GFP were significantly reduced. Representative fluorescence microscopy pictures of siGFPtreated 293 T-GFP cells were taken at 72 h post-transfection (Fig. 2D). 3.5. Cytotoxicity and hemolysis of cationic polymers PLR conjugates of CS provided higher cell viability than did PLR. The use of PEG–CS–PLR for delivery of siGL2 (polymer/siRNA weight ratio, 12/1) into A549 cells (Fig. 3A) yielded viability of 83.5 ± 2.0% at the concentration of 15 µg/mL. Since the cytotoxicity was tested at the increasing concentration of polymers, we could not make direct comparison with L2K, and excluded L2K from the comparison group. The treatment of PLR showed 22.1 ± 2.7 % at the concentration of 15 µg/mL. In order to evaluate the blood compatibility of cationic polymers, a hemolysis assay was performed. The polyplexes of CS and siGL2 did not cause significant levels of hemolysis. Among the cationic polymers tested in this study, PLR and siGL2 complexes showed the highest degree of hemolysis. At 5 mg/mL concentrations, PLR revealed 51.3 ± 1.1% of hydrolysis. Regardless of pegylation, the PLR conjugates of CS showed significantly less hemolysis than did PLR (Fig. 3B). 3.6. Serum stability of CS derivatives
Fig. 1. FACS analysis of cells after treatment with fluorescent dsRNA using various cationic polymers. Hepa 1–6 cells were treated with fluorescent dsRNA (Block-IT™) naked (B) or in complexes with L2K (C), CS (D), PEG–CS (E), PLR (F), PEG–PLR (G), CS–PLR (H), or PEG–CS–PLR (I). The complexation ratio of polymer/siRNA was 12:1 (w/w). After 24 h, the fluorescence-positive populations were analyzed by FACS. (A) Untreated cells were used as a negative control. The graphs were expressed as the percentage of fluorescencepositive cells versus forward scatter (FSC-H).
The serum stability of PEG–CS–PLR and CS–PLR was compared with that of L2K. The presence of serum did not significantly affect the intracellular delivery efficiency of PEG–CS–PLR (Fig. 4). Fluorescent dsRNA was complexed to PEG–CS–PLR or CS–PLR at polymer/siRNA weight ratio of 12:1. Following the delivery of fluorescent dsRNA using L2K or CS–PLR, the cellular intensity of fluorescence reduced as the concentrations of serum increased. At the serum concentrations
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no significant reduction in RFP fluorescence was observed in tumor tissues upon molecular imaging 3 days after the treatment (Fig. 5A). The intratumoral injection of siGL2 using CS–PLR or PEG–CS–PLR did not reduce the RFP fluorescence in tumor (Fig. 5A). In contrast, the PEG–CS–PLR-mediated delivery of siRFP produced a significant reduction in levels of the target protein, RFP, in tumor tissues. Similarly, siRFP delivered by CS–PLR provided a significantly higher reduction of the RFP expression as compared to siGL2 delivered by the same carrier. As compared to that in untreated tumor tissues, the expression of RFP in siRFP-treated tumor tissues was 16.9 ± 1.7% and 10.4 ± 4.5%, following treatment with CS–PLR and PEG–CS–PLR, respectively (Fig. 5A). Unlike naked siRFP (Fig. 5B,E), siRFP complexed to CS–PLR (Fig. 5C,F) or to PEG–CS–PLR (Fig. 5D,G) produced a significant reduction in RFP fluorescence after molecular imaging of the whole mouse body. 4. Discussion In this study, we demonstrated that pegylated PLR conjugates of CS would be effective in the delivery of siRNA both in vitro and in vivo. Moreover, the transfection efficiency with PEG–CS–PLR was not dependent on serum levels in the media.
Fig. 3. Cell viability and induction of hemolysis after treatment with polyplexes. Cationic polymers and siGL2 were complexed at a ratio of 12:1 and added to A549 cells (A). The viability of the cells was measured by an MTT assay. (B) The degree of hemolysis was expressed relative to that in untreated cells. The results are expressed as the mean ± S.E.M. of four independent experiments. *: Significantly different from other groups (p b 0.05).
higher than 30%, the cellular intensity of fluorescence was significantly higher in PEG–CS–PLR group than other groups. 3.7. In vivo silencing of target protein CS–PLR and PEG–CS–PLR were tested for in vivo delivery of siRNA. When the B16F10-RFP tumor tissues were injected with naked siRFP,
Fig. 4. Uptake of fluorescent dsRNA in the presence of serum. Fluorescent dsRNA was complexed to L2K, CS–PLR, or PEG–CS–PLR at polymer/siRNA weight ratio of 12:1, and delivered to Hepa 1–6 cells in media with different serum content. The fluorescencepositive populations were analyzed by FACS. The results are expressed as the average of four independent experiments. *: Significantly different from other groups (p b 0.05).
Fig. 5. Molecular imaging of tumor-bearing mice after siRNA treatment. siRNAs in naked form, CS–PLR, or PEG–CS–PLR complexes were intratumorally administered to the right flank (R) of mice bearing B16F10-RFP tumors in each side. In all mice, tumors in the left flank (L) were untreated as a control. Three days post-treatment of siRNAs, in vivo imaging was done. (A) The expression of RFP in siRNA-treated tumors relative to the untreated tumors were quantitatively expressed (n = 4). The representative images of the groups treated with naked siRFP (B and E), siRFP complexed to CS–PLR (C and F), or to PEG–CS–PLR(D and G) were obtained under the whole body white-light (B, C, and D) and NIRF (E,F, and G) conditions. *: Significantly different from the group treated with siGL2 delivered by the same carrier (p b 0.05).
Although CS has cationic and biocompatible properties, the nucleic acid transfection efficiency of CS has been known to be lower as compared to other cationic molecules such as PEI and cationic dendrimers. To enhance the cationic charge of CS, the additional cationic moieties were linked to CS. CS-graft-PEI were studied to enhance the transfection efficiencies of gene [9,10] and siRNAs [11]. As compared to PEI, PLR used to increase the cationic charge of CS in this study may have merits of biodegradable and biocompatible properties, consistent with the properties of CS. To screen the weight ratios where siRNA can form complexes with cationic polymers, we performed gel retardation assays over three orders of magnitude ranges, from 0.05:1 to 50:1. Gel retardation of siRNA occurred at the weight ratios higher than 5:1 (Suppl. Fig. 2). To find out the optimal weight ratios, the silencing effects of siRNA polyplexes were tested in narrow ranges of 4:1 to 12:1. Our observation of the highest silencing effects at 12:1 (Fig. 2) led us to use the weight ratio of 12:1 throughout the experiments. Despite the biodegradable characteristics, PLR alone could not be used for delivery systems of gene and siRNAs due to substantial cytotoxicity. To reduce the cytotoxicity of PLR, several attempts have been made. The conjugation of poly(N-isopropylacrylamide) to PLR was shown to reduce the cytotoxicity of PLR [12]. The complexation of hyaluronic acid to PLR could reduce the cytotoxicity of PLR more than 20-fold [7]. We observed that the conjugation of PLR to CS could also reduce the cytotoxicity of PLR while enhancing the siRNA delivery efficiency of CS. Pegylation has been widely used to enhance the stability of the nucleic acid delivery systems. Cationic polymers such as poly-L-lysine [13] and PEI [14] have been linked to PEG for delivery of plasmid DNA and siRNAs, respectively. However, the extensive pegylation of cationic polymers can reduce the charge of the polymers and limit the interaction with cell membranes. Recent study reported that the conjugates of PEG–PEI provided lower liver cell uptake of siRNAs than did PEI in vivo [15]. In this study, PEG–CS–PLR did not significantly reduce the cellular delivery of siRNAs as compared to CS–PLR, indicating that the extent of pegylation was proper to allow the interaction of siRNA polyplexes with the cells. We observed that the PLR conjugates of CS provided an enhanced delivery of siRNA compared with CS alone. The use of CS alone did not provide significant cellular delivery of siRNA. Similar to our observation, a previous study reported that the complex of siRNA with low molecular weight CS (about 10 kD) showed little knockdown of endogenous enhanced green fluorescent protein in human lung carcinoma cells [16]. The mechanisms by which PLR derivatization of CS increased the cellular delivery of siRNA require further study. PLR has been reported to enter the cells via interaction with sulfated proteoglycans and cholesterol [17]. Nona-arginine has been shown to enter living cells after binding to heparin sulfate [18]. The possibility exists that the interaction of PLR moieties in CS derivatives with negatively charged components on cell membranes may have resulted in the more effective delivery of siRNA. We observed that PEG–CS–PLR provided siRNA delivery comparable to CS–PLR and L2K, but excelled both in serum stability. The silencing of target genes was similarly observed following the delivery of fluorescent siRNA using PEG–CS–PLR, CS–PLR, and L2K (Fig. 2). However, the cellular delivery of siRNA in the presence of serum (Fig. 4) showed significant differences between PEG–CS–PLR and other groups. Unlike L2K and CS–PLR which showed the decreased siRNA delivery in proportion to the serum content, PEG–CS–PLR provided the siRNA delivery efficiency in serum-independent manner. The mechanisms by which PEG–CS–PLR showed little dependency on serum need to be further studied. In case of cationic lipid-based delivery systems such as L2K, their strong cationic charges are known to induce non-specific binding of serum proteins and subsequent clearance from circulation [19]. Previously, we reported that the use of cholesteryloxypropan-1-amine, a cationic cholesterol derivative,
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could increase delivery of siRNA in serum [20]. The lack of dependence of PEG–CS–PLR transfection efficiency on the presence of serum might be due in part to the limited binding of serum proteins to the complexes between siRNA and pegylated CS polymers. Moreover, the resistance of siRNA polyplexes to RNase digestion could be modified by pegylation. It has been reported that protection of siRNA against RNase digestion increased for pegylated PEI in comparison with PEI alone [14]. A recent study suggested that siRNA-mediated gene targeting was much less dependent than DNA transfection efficacy on the pegylation of the cationic carrier PEI [21]. The serum stability of PEG–CS–PLR would be a valuable property for future application for in vivo administration of siRNAs. We observed that the intratumoral administration of polyplexes between siRFP and PEG–CS–PLR resulted in the silencing of RFP proteins in tumor tissues. The in vivo functionality of siRNA indicates that PEG–CS–PLR might be useful for in situ delivery of various siRNAs. Although RFP-targeted siRNA was used in this study to visualize the silencing effect of siRNA in tumor tissues, oncogene-specific siRNA might be delivered into tumor sites for therapeutic outcomes in the near future. 5. Conclusion In conclusion, the use of pegylated CS–PLR might be of use for enhancing cellular delivery with increased serum stability and low cytotoxicity. The PEG–CS–PLR-mediated delivery of siRNA will be widely applicable for silencing abnormally overexpressed genes in vivo. Acknowledgement This work was supported by the research grants from the Ministry of Education, Science and Technology (F104AA010003-08A0101-00310; 2009-0081879), the Small and Medium Business Administration in Korea (S6070094111), and the Bio-Green 21 program (Code No. 20070501-034-001-009-03-00), Rural Development Administration, South Korea. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.jconrel.2010.04.005. References [1] N. Hokaiwado, F. Takeshita, A. Banas, T. Ochiya, RNAi-based drug discovery and its application to therapeutics, IDrugs 11 (2008) 274–278. [2] E. Fattal, A. Bochot, Ocular delivery of nucleic acids: antisense oligonucleotides, aptamers and siRNA, Adv. Drug. Deliv. Rev. 58 (2006) 1203–1223. [3] Y.K. Oh, T.G. Park, siRNA delivery systems for cancer treatment, Adv. Drug Deliv. Rev. 61 (2009) 850–862. [4] M. Breunig, C. Hozsa, U. Lungwitz, K. Watanabe, I. Umeda, H. Kato, A. Goepferich, Mechanistic investigation of poly(ethylene imine)-based siRNA delivery: disulfide bonds boost intracellular release of the cargo, J. Control. Release 130 (2008) 57–63. [5] S.T. Kim, C.K. Kim, Water-soluble chitosan-based antisense oligodeoxynucleotide of interleukin-5 for treatment of allergic rhinitis, Biomaterials 28 (2007) 3360–3368. [6] C.R. Dass, P.F. Choong, The use of chitosan formulations in cancer therapy, J. Microencapsul. 25 (2008) 275–279. [7] E.J. Kim, G. Shim, K. Kim, I.C. Kwon, Y.K. Oh, C.K. Shim, Hyaluronic acid complexed to biodegradable poly L-arginine for targeted delivery of siRNAs, J. Gene. Med. 11 (2009) 791–803. [8] K. Luo, J. Yin, Z. Song, L. Cui, B. Cao, X. Chen, Biodegradable interpolyelectrolyte complexes based on methoxy poly(ethylene glycol)-b-poly(#, l-glutamic acid) and chitosan, Biomacromolecules 9 (2008) 2653–2661. [9] K. Wong K, G. Sun, X. Zhang, H. Dai, Y. Liu, C. He, K.W. Leong, PEI-g-chitosan, a novel gene delivery system with transfection efficiency comparable to polyethylenimine in vitro and after liver administration in vivo, Bioconjug. Chem. 17 (2006) 152–158. [10] H.L. Jiang, Y.K. Kim, R. Arote, J.W. Nah, M.H. Cho, Y.J. Choi, T. Akaike, C.S. Cho, Chitosan-graft-polyethylenimine as a gene carrier, J. Control. Release 117 (2007) 273–280. [11] B. Ghosn, S.P. Kasturi, K. Roy, Enhancing polysaccharide-mediated delivery of nucleic acids through functionalization with secondary and tertiary amines, Curr. Top. Med. Chem. 8 (2008) 331–340.
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Journal of Controlled Release j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j c o n r e l
Pegylated poly-L-arginine derivatives of chitosan for effective delivery of siRNA Sang Myoung Noh a,1, Myung Ok Park b,1, Gayong Shim c, Su Eun Han c, Han Young Lee c, Jun Hyuk Huh c, Myoung Suk Kim b, Jin Joo Choi b, Kwangmeyung Kim d, Ick Chan Kwon d, Jin-Seok Kim e, Kwang-Hyun Baek f, Yu-Kyoung Oh a,⁎ a
College of Pharmacy, Seoul National University, Sinlim-dong, Gwanak-gu, Seoul, South Korea Biopolymed Inc., Seoul, South Korea School of Life Sciences and Biotechnology, Korea University, Anam-dong, Seungbuk-gu, Seoul, South Korea d Biomedical Research Center, Korea Institute of Science and Technology, Seoul, South Korea e School of Pharmacy, Sookmyung Women's University, Seoul, South Korea f Department of Biomedical Science, CHA University, Seoul, South Korea b c
a r t i c l e
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Article history: Received 20 May 2009 Accepted 5 April 2010 Available online 10 April 2010 Keywords: Chitosan Polyarginine Polyethylene glycol siRNA Serum stability
a b s t r a c t For delivery of siRNA, chitosan (CS) was derivatized with poly-L-arginine (PLR) and polyethylene glycol (PEG). The formation of polyplexes with siRNA was confirmed by gel retardation. The PLR-grafted CS formed nanosized particles with siRNA. PLR-grafted CS showed higher cellular delivery efficiency of siRNA than did CS, pegylated CS, PLR, or pegylated PLR. The extent of reduction in the expression of fluorescent proteins was highest following treatment of the cells using PLR derivatives of CS in complexes with specific siRNAs. Cell viability was greater in populations treated with pegylated CS–PLR than in those treated with PLR. Hemolysis of erythrocytes was reduced upon conjugation of PLR with CS. The delivery of siRNAs via pegylated CS–PLR revealed little dependence on serum. Molecular imaging techniques revealed that the intratumoral administration of red fluorescent protein-specific siRNA in complexes with pegylated CS–PLR significantly silenced the expression of red fluorescent proteins in tumor tissues in vivo. These results indicate that pegylated CS–PLR might be useful for in vivo delivery of therapeutic siRNAs. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Small interfering RNAs (siRNAs) are emerging as a new class of therapeutics. In less than one decade after the discovery of RNA interference in mammalian cells, several siRNAs are in clinical trials for treatment of disease [1]. However, most siRNAs in clinical trials are administered in naked forms for local delivery into the eye or lung [2]. siRNA applications have been restricted because of their limited penetration of cell membranes [3]. Several nonviral delivery systems for siRNA have been studied, including the use of cationic polymers such as polyethylenimine (PEI) and chitosan (CS) [4]. Among several cationic polymers, CS has been widely studied in delivery systems of negatively charged nucleic acidbased medicines [5]. Its advantages are biocompatibility, biodegradability, low cost, and positive charge, which allows it to easily form polyelectrolyte complexes [6]. However, the siRNA transfection efficiency of CS is still limited, and further development of improved CS-based delivery systems by grafting on other materials is required. In this study, we derivatized CS with a biodegradable cationic polymer, poly-L-arginine (PLR), and further pegylated the derivatives ⁎ Corresponding author. Tel.: + 82 2 880 2493; fax: + 82 2 882 2493. E-mail address: [email protected] (Y.-K. Oh). 1 Both authors contributed equally to this manuscript. 0168-3659/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.jconrel.2010.04.005
of CS. Here, we report that CS grafted with PLR and polyethylene glycol (PEG) was effective in cellular delivery of siRNA with low cytotoxicity and hemolysis. Moreover, this delivery system using a CS derivative was able to produce silencing of target proteins in tumor tissues in vivo. 2. Materials and methods 2.1. Materials Low molecular weight chitosan (CS, m.w. 50–150 kD; deacetylation degree, 87%) was purchased from Fluka (Buchs, Switzerland). Poly-L-arginine (PLR, m.w. 15–70 kD), was from Sigma-Aldrich (St. Louis, MO, USA). A luciferase mRNA-specific siRNA (siGL2) and a murine survivin-specific siRNA (siSVN) were from Samchully Pharm. Co. (Seoul, South Korea). A green fluorescent protein-specific siRNA (siGFP) and a red fluorescent protein-specific siRNA (siRFP) were from Bioneer Co. (Daejeon, South Korea). 2.2. Gel permeation chromatography (GPC) The molecular weights (Mn, Mw) and distributions (Mw / Mn) of CS and CS derivatives were determined by GPC. GPC was carried out in a column (7.8 mm × 300 mm) of 2 × Jordi gel polar pack wax mixed-bed
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equipped with a Waters 2410 differential refractometer and Waters 515 pump (Waters, Milford, MA, USA).
software (version 3.5, Systat Software, Richmond, CA, USA) was used for all analyses. A p value of less than 0.05 was considered significant.
2.3. Particle size and zeta-potential measurements
3. Results
The sizes and zeta potentials of the complexes of polymers and siRNA were measured using an ELS-8000 instrument (Photal, Osaka, Japan). Ten micrograms of siSVN and 120 µg of each polymer were complexed in 3 mL of phosphate-buffered saline (pH 7.4). The diameters were determined via He–Ne laser (10 mW) light scattering.
3.1. Synthesis of CS-based cationic polymers
2.4. Assessment of siRNA cellular uptake The cellular uptake of siRNA delivered by various cationic polymers was measured using flow cytometry. At 12 h before transfection, 1 × 105 cells were seeded. Next, Block-IT™ (20 pmole) complexed to each polymer was added to Hepa 1–6 cells and incubated for 24 h. For flow cytometry, the cells were analyzed by a BD FACS Calibur using Cell Quest Pro software (BD Bioscience, San Jose, CA, USA). In some experiments, the intensity of cellular fluorescence was assessed after incubation in various serum concentrations, and measured using a Zenyth 3100 Microplate Reader (Anthos Co., Salzburg, Austria) at excitation and emission wavelengths of 485 and 535 nm, respectively. 2.5. Western blot analysis The siRNA-mediated reduction of target gene expression was detected at the protein level by western blotting. Hepa 1–6 cells, A549 cells, or VK2 cells were treated for 48 h with various complexes of siRNA with cationic polymers. Lysates of whole cells (30 µg of total protein) were separated on a 10% SDS-PAGE gel. Western blots were performed with specific antibodies to survivin (1:1000, sc-10811, Santa Cruz Biotechnology, CA, USA) and β-actin (1:2500, sc-47778, Santa Cruz Biotechnology). 2.6. Cell viability and in vitro hemolysis assays An MTT assay was used to measure the cytotoxicity of cationic polymers as described previously [7]. An in vitro hemolysis assay was performed to estimate the toxicity of cationic polymers in blood as described previously [8]. 2.7. In vivo molecular imaging Female nu/nu mice (Orient Bio Inc., Seungnam, Gyeonggi-do, South Korea) were used for the induction of red fluorescent protein (RFP)-expressing tumors. The mice were subcutaneously injected with 1 × 106 B16F10-RFP cells at left and right flanks. At 8 days after the inoculation, the right flank tumors were treated with siRNA in naked or complexes. The left flank tumors were untreated in all mice. The polyplexes were prepared at the polymer/siRNA weight ratio of 12/1. Treatment of mice involved three intratumoral injections of the complexes at a dose of 120 µg of cationic polymers (CS–PLR, PEG–CS– PLR) and 10 µg of siRNA per mouse. Three days after treatment, in vivo imaging was performed on the B16F10-RFP tumor-bearing mice using the Kodak Image Station 4000MM (Eastman Kodak Co., Rochester, NY, USA) equipped with band-pass excitation and long-pass emission filters at 535 nm and 600 nm. The images were processed with Kodak molecular imaging software version 4.0. 2.8. Statistics Statistical analysis of data was performed using ANOVA with the Student–Newman–Keuls test employed as a post-hoc test. SigmaStat
The synthesis schemes of CS–PLR and PEG–CS–PLR conjugates are summarized in Suppl. Fig. 1. PLR was covalently linked to CS via formation of amide bonds between the amine groups (–NH2) of CS and the carboxyl groups (–COOH) of PLR. The formation of amide bonds between the activated carboxyl groups (–COOH) of PLR and amine groups (–NH2) of CS resulted in the conjugates of CS–PLR. The conjugation of PLR to CS and the pegylation of CS–PLR conjugates were confirmed by 1H NMR (data not shown). The grafting of PLR and PEG to CS was tested by FI-IR spectra. PEG–CS–PLR showed the characteristic absorption band around 2900 cm− 1 and 1650 cm− 1, resulting from the increased C–H bond and amide (C O) bond formation by grafting of PEG to CS–PLR. GPC results (Table 1) indicate that PEG–CS with a number-average molecular weight (Mn) of ca. 78,000 and a polydispersity index of 1.4 was obtained. Mn of PEG–CS–PLR was ca. 90,000 with a polydispersity index of 1.4. The average molecular weights indicate that the average numbers of PEG per CS in PEG–CS conjugates were 4. Moreover, the average numbers of PEG and PLR per CS in PEG–CS conjugates were calculated as 4 and 1, respectively. 3.2. Characterization of siRNA and cationic polymer complexes All cationic polymers tested in this study formed complexes with negatively charged siRNA. However, the types of cationic polymers affected the minimal complexation ratios at which the complexes showed the gel retardation. The polymers CS, PEG–PLR, CS–PLR, and PEG–CS–PLR showed complete gel retardation starting at the polymer/siRNA weight ratio of 5:1 (Suppl. Fig. 2). Meanwhile, PEG– CS and PLR formed complexes with siRNA from the weight ratios of 50:1 and 0.5:1, respectively. Table 2 shows the sizes and zeta-potential values on the complexes between each polymer and siRNA at the complexation weight ratio of 12/1. Among the groups tested, CS/siRNA complexes showed the smallest particle size and the lowest zeta potential. The sizes of the particles did not significantly differ between CS–PLR and PEG–CS–PLR, showing less than 400 nm sizes upon complexation with siRNAs. As compared to CS, CS–PLR and PEG–CS–PLR showed significantly higher zeta potential upon complexation with siRNAs. 3.3. Intracellular delivery of dsRNA by cationic polymers FACS analysis (Fig. 1) revealed that the PLR conjugates of CS increased the intracellular delivery of siRNA as compared with CS. To quantitate the intracellular uptake of siRNA, fluorescent dsRNA was complexed to various cationic polymers with polymer/siRNA weight ratio of 12:1. Fig. 1 shows that the fluorescence-positive populations of the cells were larger with delivery of siRNA using CS–PLR and PEG– CS–PLR than using other cationic polymer groups. CS–PLR (Fig. 1H) and PEG–CS–PLR (Fig. 1I) showed 78% and 79% positive cell populations,
Table 1 Molecular characteristic of the CS and CS derivatives. Polymers
Mn × 103
Mw × 103
Mw / Mn
CS PEG–CS PEG–CS–PLR
70.0 78.0 90.0
140.0 107.2 122.7
2.0 1.4 1.4
Mn: number-average molecular weight, Mw: weight-average molecular weight, Mw / Mn: polydispersity index.
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Table 2 Particle sizes and zeta potentials of polymers and siRNA complexes. Polymer/siRNA (12/1, w/w)
Particle size (nm)
Zeta potential (mV)
CS/siRNA CS–PLR/siRNA PEG–CS–PLR/siRNA
117.5 ± 4.9 307.6 ± 118.3 375.1 ± 188.4
+ 15.6 ± 6.8 + 35.3 ± 4.5 + 23.9 ± 3.6
whereas CS alone (Fig. 1D) and PEG–CS (Fig. 1E) resulted in 1.5% and 2.6% positive populations, respectively. Widely used transfection agent, Lipofectamine™2000 (L2K), was used for comparison with the cationic polymers synthesized in this study. The delivery efficiency of CS–PLR or PEG–CS–PLR was comparable to that of L2K (Fig. 1C). 3.4. Reduction of target gene expression The treatment of siRNAs differentially reduced the expression of target protein, depending on the delivery systems. As target proteins, survivin and GFP were tested using siSVN and siGFP, respectively. Western blot data reveals that the cellular levels of survivin reduced after treatment of siSVN complexed with L2K, CS–PLR, or PEG–CS–PLR at all the complexation ratios tested. Indeed, we tested the silencing of target protein after treatment with PLR/siRNA complexes. However, due to the high cytotoxicity of PLR, PLR was excluded from the comparison groups. A549 (Fig. 2B) and VK2 (Fig. 2C) cells showed that the reduction of survivin levels were dependent on the complexation ratios, resulting in the highest reduction at the ratio of 12:1. In addition to survivin-silencing, the reduction of GFP expression was tested using siGFP complexed with various delivery systems. Consistent to siSVN treatment, the PLR conjugates of CS were effective in siGFP-mediated silencing of target protein expression. For polyplexes, the complexation ratio between polymer and siGFP was 12:1 (w:w). When GFP-expressing cells were treated with the complexes of siGFP and CS, the expression level of GFP was similar to the untreated control. Following siGFP treatments of the cells with CS–
Fig. 2. Silencing of target protein expression after treatment with siRNAs. (A) Hepa 1–6, (B) A549, (C) VK2 cells were treated with siSVNs alone or using various delivery systems. The protein extracts were analyzed by western blot. (D) 293 T-GFP cells were treated with siGFP alone or in complexes with various delivery systems. The complexation ratio between polymer and siGFP was 12:1 (w:w). After 72 h, the fluorescence was observed under a fluorescence microscope. Scale bar is 100 µm.
PLR and PEG–CS–PLR, the cellular levels of GFP were significantly reduced. Representative fluorescence microscopy pictures of siGFPtreated 293 T-GFP cells were taken at 72 h post-transfection (Fig. 2D). 3.5. Cytotoxicity and hemolysis of cationic polymers PLR conjugates of CS provided higher cell viability than did PLR. The use of PEG–CS–PLR for delivery of siGL2 (polymer/siRNA weight ratio, 12/1) into A549 cells (Fig. 3A) yielded viability of 83.5 ± 2.0% at the concentration of 15 µg/mL. Since the cytotoxicity was tested at the increasing concentration of polymers, we could not make direct comparison with L2K, and excluded L2K from the comparison group. The treatment of PLR showed 22.1 ± 2.7 % at the concentration of 15 µg/mL. In order to evaluate the blood compatibility of cationic polymers, a hemolysis assay was performed. The polyplexes of CS and siGL2 did not cause significant levels of hemolysis. Among the cationic polymers tested in this study, PLR and siGL2 complexes showed the highest degree of hemolysis. At 5 mg/mL concentrations, PLR revealed 51.3 ± 1.1% of hydrolysis. Regardless of pegylation, the PLR conjugates of CS showed significantly less hemolysis than did PLR (Fig. 3B). 3.6. Serum stability of CS derivatives
Fig. 1. FACS analysis of cells after treatment with fluorescent dsRNA using various cationic polymers. Hepa 1–6 cells were treated with fluorescent dsRNA (Block-IT™) naked (B) or in complexes with L2K (C), CS (D), PEG–CS (E), PLR (F), PEG–PLR (G), CS–PLR (H), or PEG–CS–PLR (I). The complexation ratio of polymer/siRNA was 12:1 (w/w). After 24 h, the fluorescence-positive populations were analyzed by FACS. (A) Untreated cells were used as a negative control. The graphs were expressed as the percentage of fluorescencepositive cells versus forward scatter (FSC-H).
The serum stability of PEG–CS–PLR and CS–PLR was compared with that of L2K. The presence of serum did not significantly affect the intracellular delivery efficiency of PEG–CS–PLR (Fig. 4). Fluorescent dsRNA was complexed to PEG–CS–PLR or CS–PLR at polymer/siRNA weight ratio of 12:1. Following the delivery of fluorescent dsRNA using L2K or CS–PLR, the cellular intensity of fluorescence reduced as the concentrations of serum increased. At the serum concentrations
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no significant reduction in RFP fluorescence was observed in tumor tissues upon molecular imaging 3 days after the treatment (Fig. 5A). The intratumoral injection of siGL2 using CS–PLR or PEG–CS–PLR did not reduce the RFP fluorescence in tumor (Fig. 5A). In contrast, the PEG–CS–PLR-mediated delivery of siRFP produced a significant reduction in levels of the target protein, RFP, in tumor tissues. Similarly, siRFP delivered by CS–PLR provided a significantly higher reduction of the RFP expression as compared to siGL2 delivered by the same carrier. As compared to that in untreated tumor tissues, the expression of RFP in siRFP-treated tumor tissues was 16.9 ± 1.7% and 10.4 ± 4.5%, following treatment with CS–PLR and PEG–CS–PLR, respectively (Fig. 5A). Unlike naked siRFP (Fig. 5B,E), siRFP complexed to CS–PLR (Fig. 5C,F) or to PEG–CS–PLR (Fig. 5D,G) produced a significant reduction in RFP fluorescence after molecular imaging of the whole mouse body. 4. Discussion In this study, we demonstrated that pegylated PLR conjugates of CS would be effective in the delivery of siRNA both in vitro and in vivo. Moreover, the transfection efficiency with PEG–CS–PLR was not dependent on serum levels in the media.
Fig. 3. Cell viability and induction of hemolysis after treatment with polyplexes. Cationic polymers and siGL2 were complexed at a ratio of 12:1 and added to A549 cells (A). The viability of the cells was measured by an MTT assay. (B) The degree of hemolysis was expressed relative to that in untreated cells. The results are expressed as the mean ± S.E.M. of four independent experiments. *: Significantly different from other groups (p b 0.05).
higher than 30%, the cellular intensity of fluorescence was significantly higher in PEG–CS–PLR group than other groups. 3.7. In vivo silencing of target protein CS–PLR and PEG–CS–PLR were tested for in vivo delivery of siRNA. When the B16F10-RFP tumor tissues were injected with naked siRFP,
Fig. 4. Uptake of fluorescent dsRNA in the presence of serum. Fluorescent dsRNA was complexed to L2K, CS–PLR, or PEG–CS–PLR at polymer/siRNA weight ratio of 12:1, and delivered to Hepa 1–6 cells in media with different serum content. The fluorescencepositive populations were analyzed by FACS. The results are expressed as the average of four independent experiments. *: Significantly different from other groups (p b 0.05).
Fig. 5. Molecular imaging of tumor-bearing mice after siRNA treatment. siRNAs in naked form, CS–PLR, or PEG–CS–PLR complexes were intratumorally administered to the right flank (R) of mice bearing B16F10-RFP tumors in each side. In all mice, tumors in the left flank (L) were untreated as a control. Three days post-treatment of siRNAs, in vivo imaging was done. (A) The expression of RFP in siRNA-treated tumors relative to the untreated tumors were quantitatively expressed (n = 4). The representative images of the groups treated with naked siRFP (B and E), siRFP complexed to CS–PLR (C and F), or to PEG–CS–PLR(D and G) were obtained under the whole body white-light (B, C, and D) and NIRF (E,F, and G) conditions. *: Significantly different from the group treated with siGL2 delivered by the same carrier (p b 0.05).
Although CS has cationic and biocompatible properties, the nucleic acid transfection efficiency of CS has been known to be lower as compared to other cationic molecules such as PEI and cationic dendrimers. To enhance the cationic charge of CS, the additional cationic moieties were linked to CS. CS-graft-PEI were studied to enhance the transfection efficiencies of gene [9,10] and siRNAs [11]. As compared to PEI, PLR used to increase the cationic charge of CS in this study may have merits of biodegradable and biocompatible properties, consistent with the properties of CS. To screen the weight ratios where siRNA can form complexes with cationic polymers, we performed gel retardation assays over three orders of magnitude ranges, from 0.05:1 to 50:1. Gel retardation of siRNA occurred at the weight ratios higher than 5:1 (Suppl. Fig. 2). To find out the optimal weight ratios, the silencing effects of siRNA polyplexes were tested in narrow ranges of 4:1 to 12:1. Our observation of the highest silencing effects at 12:1 (Fig. 2) led us to use the weight ratio of 12:1 throughout the experiments. Despite the biodegradable characteristics, PLR alone could not be used for delivery systems of gene and siRNAs due to substantial cytotoxicity. To reduce the cytotoxicity of PLR, several attempts have been made. The conjugation of poly(N-isopropylacrylamide) to PLR was shown to reduce the cytotoxicity of PLR [12]. The complexation of hyaluronic acid to PLR could reduce the cytotoxicity of PLR more than 20-fold [7]. We observed that the conjugation of PLR to CS could also reduce the cytotoxicity of PLR while enhancing the siRNA delivery efficiency of CS. Pegylation has been widely used to enhance the stability of the nucleic acid delivery systems. Cationic polymers such as poly-L-lysine [13] and PEI [14] have been linked to PEG for delivery of plasmid DNA and siRNAs, respectively. However, the extensive pegylation of cationic polymers can reduce the charge of the polymers and limit the interaction with cell membranes. Recent study reported that the conjugates of PEG–PEI provided lower liver cell uptake of siRNAs than did PEI in vivo [15]. In this study, PEG–CS–PLR did not significantly reduce the cellular delivery of siRNAs as compared to CS–PLR, indicating that the extent of pegylation was proper to allow the interaction of siRNA polyplexes with the cells. We observed that the PLR conjugates of CS provided an enhanced delivery of siRNA compared with CS alone. The use of CS alone did not provide significant cellular delivery of siRNA. Similar to our observation, a previous study reported that the complex of siRNA with low molecular weight CS (about 10 kD) showed little knockdown of endogenous enhanced green fluorescent protein in human lung carcinoma cells [16]. The mechanisms by which PLR derivatization of CS increased the cellular delivery of siRNA require further study. PLR has been reported to enter the cells via interaction with sulfated proteoglycans and cholesterol [17]. Nona-arginine has been shown to enter living cells after binding to heparin sulfate [18]. The possibility exists that the interaction of PLR moieties in CS derivatives with negatively charged components on cell membranes may have resulted in the more effective delivery of siRNA. We observed that PEG–CS–PLR provided siRNA delivery comparable to CS–PLR and L2K, but excelled both in serum stability. The silencing of target genes was similarly observed following the delivery of fluorescent siRNA using PEG–CS–PLR, CS–PLR, and L2K (Fig. 2). However, the cellular delivery of siRNA in the presence of serum (Fig. 4) showed significant differences between PEG–CS–PLR and other groups. Unlike L2K and CS–PLR which showed the decreased siRNA delivery in proportion to the serum content, PEG–CS–PLR provided the siRNA delivery efficiency in serum-independent manner. The mechanisms by which PEG–CS–PLR showed little dependency on serum need to be further studied. In case of cationic lipid-based delivery systems such as L2K, their strong cationic charges are known to induce non-specific binding of serum proteins and subsequent clearance from circulation [19]. Previously, we reported that the use of cholesteryloxypropan-1-amine, a cationic cholesterol derivative,
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could increase delivery of siRNA in serum [20]. The lack of dependence of PEG–CS–PLR transfection efficiency on the presence of serum might be due in part to the limited binding of serum proteins to the complexes between siRNA and pegylated CS polymers. Moreover, the resistance of siRNA polyplexes to RNase digestion could be modified by pegylation. It has been reported that protection of siRNA against RNase digestion increased for pegylated PEI in comparison with PEI alone [14]. A recent study suggested that siRNA-mediated gene targeting was much less dependent than DNA transfection efficacy on the pegylation of the cationic carrier PEI [21]. The serum stability of PEG–CS–PLR would be a valuable property for future application for in vivo administration of siRNAs. We observed that the intratumoral administration of polyplexes between siRFP and PEG–CS–PLR resulted in the silencing of RFP proteins in tumor tissues. The in vivo functionality of siRNA indicates that PEG–CS–PLR might be useful for in situ delivery of various siRNAs. Although RFP-targeted siRNA was used in this study to visualize the silencing effect of siRNA in tumor tissues, oncogene-specific siRNA might be delivered into tumor sites for therapeutic outcomes in the near future. 5. Conclusion In conclusion, the use of pegylated CS–PLR might be of use for enhancing cellular delivery with increased serum stability and low cytotoxicity. The PEG–CS–PLR-mediated delivery of siRNA will be widely applicable for silencing abnormally overexpressed genes in vivo. Acknowledgement This work was supported by the research grants from the Ministry of Education, Science and Technology (F104AA010003-08A0101-00310; 2009-0081879), the Small and Medium Business Administration in Korea (S6070094111), and the Bio-Green 21 program (Code No. 20070501-034-001-009-03-00), Rural Development Administration, South Korea. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.jconrel.2010.04.005. References [1] N. Hokaiwado, F. Takeshita, A. Banas, T. Ochiya, RNAi-based drug discovery and its application to therapeutics, IDrugs 11 (2008) 274–278. [2] E. Fattal, A. Bochot, Ocular delivery of nucleic acids: antisense oligonucleotides, aptamers and siRNA, Adv. Drug. Deliv. Rev. 58 (2006) 1203–1223. [3] Y.K. Oh, T.G. Park, siRNA delivery systems for cancer treatment, Adv. Drug Deliv. Rev. 61 (2009) 850–862. [4] M. Breunig, C. Hozsa, U. Lungwitz, K. Watanabe, I. Umeda, H. Kato, A. Goepferich, Mechanistic investigation of poly(ethylene imine)-based siRNA delivery: disulfide bonds boost intracellular release of the cargo, J. Control. Release 130 (2008) 57–63. [5] S.T. Kim, C.K. Kim, Water-soluble chitosan-based antisense oligodeoxynucleotide of interleukin-5 for treatment of allergic rhinitis, Biomaterials 28 (2007) 3360–3368. [6] C.R. Dass, P.F. Choong, The use of chitosan formulations in cancer therapy, J. Microencapsul. 25 (2008) 275–279. [7] E.J. Kim, G. Shim, K. Kim, I.C. Kwon, Y.K. Oh, C.K. Shim, Hyaluronic acid complexed to biodegradable poly L-arginine for targeted delivery of siRNAs, J. Gene. Med. 11 (2009) 791–803. [8] K. Luo, J. Yin, Z. Song, L. Cui, B. Cao, X. Chen, Biodegradable interpolyelectrolyte complexes based on methoxy poly(ethylene glycol)-b-poly(#, l-glutamic acid) and chitosan, Biomacromolecules 9 (2008) 2653–2661. [9] K. Wong K, G. Sun, X. Zhang, H. Dai, Y. Liu, C. He, K.W. Leong, PEI-g-chitosan, a novel gene delivery system with transfection efficiency comparable to polyethylenimine in vitro and after liver administration in vivo, Bioconjug. Chem. 17 (2006) 152–158. [10] H.L. Jiang, Y.K. Kim, R. Arote, J.W. Nah, M.H. Cho, Y.J. Choi, T. Akaike, C.S. Cho, Chitosan-graft-polyethylenimine as a gene carrier, J. Control. Release 117 (2007) 273–280. [11] B. Ghosn, S.P. Kasturi, K. Roy, Enhancing polysaccharide-mediated delivery of nucleic acids through functionalization with secondary and tertiary amines, Curr. Top. Med. Chem. 8 (2008) 331–340.
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