Biomaterials 35 (2014) 7970e7977

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Targetable micelleplex hydrogel for long-term, effective, and systemic siRNA delivery Young-Min Kim a, b, Soo-Chang Song a, b, * a b

Center for Biomaterials, Korea Institute of Science & Technology, Seoul 130-650, Republic of Korea Department of Biomolecular Science, University of Science and Technology (UST), Daejeon 305-350, Republic of Korea

a r t i c l e i n f o

a b s t r a c t

Article history: Received 4 April 2014 Accepted 23 May 2014 Available online 18 June 2014

We developed a targetable micelleplex hydrogel as a new efficient systemic siRNA delivery material that functions as a targetable gene carrier, and a hydrogel capable of controlled release to overcome drawbacks of multiple administrations of systemic siRNA carriers due to decreased fluctuation of them in the serum. The micelleplexes, complexes between polymeric micelles and siRNAs could turn into gel after subcutaneous injection and be slowly released from the gel. The released micelleplexes selectively accumulated in the tumor and showed anti-tumor effect due to gene silencing for an extended period of time with only one injection in anywhere in vivo model. Moreover, the duration of therapy can be controlled by adjusting the amount and properties of the hydrogel. Therefore, this micelleplex hydrogel is expected to be a new effective siRNA delivery material for systemic long-term gene silencing. © 2014 Elsevier Ltd. All rights reserved.

Keywords: Gene therapy Nanoparticle Hydrogel Drug delivery

1. Introduction RNA interference (RNAi) is a mechanism to reduce the level of a specific protein by inhibition of gene expression. RNAi-induced therapy, especially siRNA (small interference RNA)-mediated treatment, is attractive because it can be used to treat various diseases by inhibiting specific proteins at a relatively lower cost than other biological methods. However, RNAi has limited applications in clinical fields due to the inability of siRNA to enter the cell membrane [1,2]. To enhance therapeutic efficacy, various siRNA carriers including liposomes, polyplexes, and micelles with nano-sized diameters, have been studied, especially in the context of tumors, because carriers with size of 20 nme200 nm easily accumulate at the tumor site owing to an enhanced permeation and retention (EPR) effect. Moreover, ligands were also attached to these carriers to enhance the selective delivery of siRNAs only to the target cells [3]. Although many materials were tested in most cases, only a small percentage of the injected dose was delivered to the tumor after intravenous injection [4]. In the treatment of tumors, because the serum level of the carriers is not enough to cure tumor by one injection, multiple * Corresponding author. Center for Biomaterials, Korea Institute of Science & Technology, Seoul 130-650, Republic of Korea. Tel.: þ82 2 958 5123; fax: þ82 2 958 5308. E-mail address: [email protected] (S.-C. Song). http://dx.doi.org/10.1016/j.biomaterials.2014.05.070 0142-9612/© 2014 Elsevier Ltd. All rights reserved.

injections are often necessary to maintain effective carrier concentrations. However, fluctuations in carrier concentration due to multiple injections induce many drawbacks, such as decreased selectivity of the elicited pharmacological effect, promotion of counter-regulatory processes, and side effects due to high doses of drugs. These drawbacks result in low efficacy and a need to increase the dosage of drugs for treatment finally [5,6]. To overcome the drawbacks of multiple injections and enhance delivery efficacy, a controlled release system having long-term effects after a single injection was developed. Some studies on controlled release systems focused on intradermal injection of nanoparticle and lipid carriers. The specificity of the dermis which is located far from the blood vessels, ensured a sustained and localized effect of nanoparticles within the injection site for 11 days, however, the therapeutic potential could be decreased by the long distance from the blood vessels [7]. In contrast to intradermal injections, subcutaneous injection showed better therapeutic potential and a rapid release of nanoparticles within 10 h [8]. As an alternative to nanoparticle solutions, subcutaneous injections of hydrogels showing solegel transition after the injection were studied in terms of prolonged release periods and easy handling. However, the addition of poly(3-hydroxybutyrateco-3-hydroxyhexanoate (PHBHHx) nanoparticles to injectable alginate hydrogel as a controlled release system showed the release for only 3 days [9]. The targetable micelleplex hydrogel was suggested as a new way to achieve efficient siRNA delivery material because of its dual

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function as a gene carrier with passive & active targeting ability and as a hydrogel showing controlled release of only one material. Targetable micelleplexes were firstly created by mixing polymeric micelles and siRNAs. After injection into the body, the targetable micelleplex solution turned into a targetable micelleplex hydrogel due to an increase in temperature. Degradation and dissolution of the hydrogel caused the release of the targetable micelleplexes that easily circulated through lymphatic or blood vessels because of their nano-sizes. Accumulation via the EPR effect at the tumor site and receptor-mediated endocytosis via targeting moiety occurred sequentially. Here, FPP, [folate-polyethyleneimine(PEI)-conjugated poly(organophosphazene)], a biodegradable polymer, amphiphilic forming micelles, showing solegel transition in solution as a function of the temperature, was used to form a targetable micelleplex hydrogel for the treatment of tumors. The PEI in FPP can form a micelleplex with anionic siRNAs by ionic interaction and turn into gel after

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subcutaneous injection due to the properties of poly(organophosphazene). The micelleplexes were released slowly by dissolution and degradation of the gel into the blood stream directly or through lymphatic vessels. Because the micelleplexes contained folate, a specific targeting moiety, they selectively entered the folate receptor (FR)-over-expressing cancer cells, but not normal cells through the blood. Furthermore, the release time of the micelleplexes from the hydrogel could also be easily tuned by adjusting the amount of hydrogel. To validate this model, we investigated the formation of nano-sized micelleplexes of FPP with siRNAs, their selective delivery in vitro, and their therapeutic effect in an in vivo tumor model, and analyzed the potential for controlling the release rate (Scheme 1). 2. Materials and methods 2.1. Materials Hexachlorocyclotriphosphazene was acquired from SigmaeAldrich (St. Louis, MO, USA) and sublimated for purification at 55  C under vacuum (about

Scheme 1. Structural and schematic diagrams of folate-poly(ethyleneimine)-conjugated poly(organophosphazene) (FPP). a) Structure of FPP. b) Schematic diagram of selective RNA  interference by micelleplex hydrogel. The micelleplex was obtained by simply mixing an FPP solution with siRNA at 4 C. After injection into the body, solegel transition occurred due to hydrophobic interactions. The hydrogel releases dissociated micelleplexes having folate as the targeting moiety according to time dependent degradation and dissolution. The released micelleplexes enter the circulation pathway, accumulates in tumor region via EPR effect, and enter only the tumor cells via receptor-mediated endocytosis. Gene suppression is induced by cleavage of the target mRNA only in tumor cells.

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0.1 mmHg). Poly(dichlorophosphazene) was prepared using sublimated hexachlorocyclotriphosphazene as described previously. Also a-Amino-u-methoxypoly(ethylene glycol) with molecular weight of 750 Da (AMPEG750) was prepared by a published method [9]. L-Isoleucine ethyl ester hydrochloride (IleOEtHCl) were purchased from A&Z food additives (HangZhou, China). 2-aminoethanol (AEtOH), folic acid and polyethyleneimine with molecular weights of 800 Da (PEI 800: branch type) was obtained from SigmaeAldrich. Tetrahydrofuran (THF) and triethylamine (TEA) were dried under dry nitrogen by reflux over sodium metal/ benzophenone and barium oxide, respectively. All other reagents were purchased from commercial suppliers and used as received. All animal experiments were approved by Animal Care Ethnic Committee (ACEC) of Korea Institute of Science and Technology (KIST). siRNAs of green fluorescent protein (GFP) and vascular endothelial growth factor (VEGF) were obtained from Samchully Pharmaceutical Company (Daejeon, Korea). GFP sense ¼ 50 -GUUCAGCGUGUCCGGCGAGTT-30 GFP anti-sense ¼ 50 -CUCGCCGGACACGCUGAACTT-30 VEGF sense ¼ 50 -GGAGUACCCUGAUGAGAUCTT-30 VEGF anti-sense ¼ 50 -GAUCUCAUCAGGGUACUCCTT-30 2.2. Synthesis of folate-PEI-poly(organophosphazene) conjugate (FPP) All reactions were processed under an atmosphere of dry nitrogen by using standard Schlenk-line techniques. L-PEI-poly(organophosphazene) conjugate (PP) was firstly prepared and detail procedure of synthesis is in previous paper [10]. Folic acid (0.20 g, 0.46 mmol) was dissolved in anhydrous DMF and dried TEA (46.71 mg, 0.46 mmol) was added. Dicyclohexylcarbodiimide, DCC (0.20 g, 0.92 mmol) and nhydroxysuccinimide, NHS (0.11 g, 0.92 mmol) in dried DMF at each flask were added to folic acid solution and incubated for 18 h to activate the carboxyl group of folic acid. The mixture was transferred to PP (6.03 g, 3.98 mmol) in anhydrous DMF. The reaction mixture was stirred at 37  C for 6 h. After the reaction, the solution was filtered, concentrated, and purified by precipitation with 1 M KF solution. The precipitate was dialyzed with a dialysis membrane (Spectra/Por, MWCO:10e12 kDa) against distilled water for 3 days at 4  C and the dialyzed solution was freeze-dried to obtain the final product. Yield: 81%. 1H NMR (CDCl3), d (ppm): 0.8e1.0 (s, 6H), 1.1e1.3 (b, 3H), 1.3e1.6 (b, 2H), 1.6e1.9 (b, 1H), 2.5e2.8 (b, 2H), 2.6e2.9 (b, 74H), 2.8e3.3 (b, 2H), 3.4e3.8 (b, 73H), 3.9 (s, 1H), 4.0e4.3 (b, 3H), 6.4e6.8 (b, 4H),.7.4e7.8 (b-4H), 8.6e8.8 (b-3H). 2.3. Characterization of poly(organophosphazenes) The structures of prepared polymers were estimated by measuring 1H NMR (Varian Gemini-300 spectrometer operating at 300 MHz in the Fourier transform mode with CDCl3) and HPLC (Accela, Thermo scientific). The substituted amounts of folate in FPP were determined by HPLC. The viscosity of the aqueous FPP solutions were performed on a Brookfield RVDV-III þ viscometer between 5 and 70  C under a fixed shear rate of 0.1 se1. The measurements were processed with a set spindle speed of 0.2 rpm and with a heating rate of 0.33  C/min. Molecular weight (MW) of FPP was calculated by gel permeation chromatography (GPC) system (Waters 1515) with a refractive index detector (Waters 2410) and two styragel columns (Waters styragel HR 4E and HR 5E) connected in line at a flow rate of 1 ml/min at 35  C. THF containing 0.1 wt% of tetrabutylammonium bromide was used as a mobile phase. Polystyrenes (MW: 1270; 3760; 12,900; 28,400; 64,200; 183,000; 658,000; 1,050,000; 2,510,000; 3,790,000) were used as standards. 2.4. Formation of micelleplexes between siRNAs and FPP micelles The micelleplexes were induced by adding FPP solution to siRNA solution with a gentle shaking and incubated at 4  C for 30 min. All micelleplexes were induced in DEPC water and freshly prepared before use.

supernatant was observed. One drop of sample solution was placed on a copper grid and negative staining was done with 2 wt% uranyl acetate. 2.7. In vitro serum stability For serum stability assay, 0.2 mg of siRNA was used for complexation with conjugates at N/P ¼ 20. The prepared micelleplexes were incubated in 50% fetal bovine serum (FBS) for predetermined period in a water bath at 37  C with a slow motion (50 rpm). After incubation, 2 ml of 2% sodium dodecyl sulfate (SDS) were treated to each samples and loaded them on a 12% polyacrylamide gel. After running the gel during 1 h, the gels were stained by EtBr and observed remained siRNA by GelDoc (MiniBIS Pro, DNR Bioimaging system, Israel). 2.8. In vitro selective cellular uptake test using cy3-tagged siRNA MDAMB231, breast cancer cells and A549, human lung carcinoma cells were purchased from Korea Cell Line Bank (KCLB). The cells (2.5  104 cells/well) were seeded in 6 well plates and incubated overnight in complete RPMI1640 culture media. And then, the culture media were replaced with serum free media, contained different micelleplexes, which was prepared using 2.5 mg of siRNA. After 5 h, each well was washed with PBS and filled with cold PBS. The intensity of cy3 was checked by Kodak Image Station 4000MM digital imaging system (Carestream Health, New Haven, CT, USA). 2.9. In vitro transfection assay by checking the reduction of VEGF level MDAMB231 cells (5  104 cells/well) were seeded in 24-well plates (SPL) and incubated overnight in complete RPMI1640 culture media. And then, the culture media were replaced with serum free media, contained different micelleplexes, which was prepared using 0.5 mg of siRNA. After 5 h, the media were changed to fresh media contained FBS, and incubated for 6 h. Thereafter, the media were replaced with fresh media contained FBS and incubated for further 16 h. The VEGF levels of each group were quantified by commercial enzyme-linked immunosorbent assay (ELISA) kit (R&D systems, Minneapolis, MN, USA) using cell culture supernatants. The percentage of VEGF levels were normalized with the amounts of total protein, which was quantified by bicinchoninic acid (BCA) assay. 2.10. In vivo micelleplexes distribution test The 1  107 cells of MDAMB231 in 100 ml of PBS were injected into right dorsal subcutis of Balb/c nude mice (6 weeks, male, from Orient Bio, Korea). When the mean volume of tumors reached approximately 100 mm3 (Length  Width  Height  p/6), the solutions of FPP/cy3-tagged VEGF siRNA micelleplexes at N/P ¼ 20, 40 ml were injected into left dorsal subcutis of mice. The intensity of cy3 was checked at predetermined time intervals by IVIS spectrum (Caliper Life Sciences Inc., Hopkinton, MA, USA). At day 10, organs were dissected from sacrificed mice after perfusion and intensity of cy3 was also checked by KODAK imaging system. 2.11. Tissue staining Dissected organs were moved to a mold and freezed in OCT compound. After section of organs in 10 mm diameter, tissues were fixed and blocked. VEGF monoclonal antibody (Santacruz biotechnology, California, USA) was treated onto tissues and incubated at RT for 1 h. Alexa 488 tagged anti-mouse secondary antibody (Santacruz biotechnology, California, USA) for detecting primary antibody was treated after rinsing (green). Also DAPI staining was also conducted for observation of nuclei (blue). After dehydration and mounting, fluorescent intensities of tissue samples were checked by Zeiss confocal microscope (LSM pascal/Beam splitters: HFT 488,543,633/Filters: BP 505e530, LP 560). 2.12. In vivo anti-tumor activity test

2.5. Gel retardation assay 0.5 mg of siRNA was used for complexation with conjugates at predetermined N/ P ratios. The induced micelleplexes were loaded on 12% polyacrylamide gels and electrophoresis was carried out at 100 mV. After 1 h, the gels were stained by ethidium bromide (EtBr) and observed retardation of siRNA by GelDoc (MiniBIS Pro, DNR Bioimaging system, Israel). 2.6. Measurement of size and z-potential The sizes and z-potentials of FPP/siRNA polyplexes were measured by a Zetasizer Nano ZS (Malvern Instruments Ltd., Malvern, UK) at room temperature. The final concentration of siRNA was 10 mg/ml and polymer was (7.9 mge223 mg/ml as a function of N/P ratio). The samples were measured in triplicate. Transmission electron microscopy(TEM) and cryoTEM: The sizes and shapes of FPP/VEGF siRNA micelleplexes at N/P 20 were observed by TEM (CM30 electron microscope, Philips, CA), cryoTEM (Tecani G2 F20, Philips, CA) with same concentration (10 mg/1 ml siRNA). In addition to the freshly prepared micelleplexes, we also confirmed whether the micelleplexes hydrogel could release micelleplexes or not. To confirm that, the micelleplexes hydrogel (0.1 g) was incubated in 0.9 ml of DEPC water for 7 days at 37  C in a water bath with slow motion (50 rpm), and the taken

The 1  107 cells of PC-3 in 100 ml of PBS were injected into right dorsal subcutis of Balb/c nude mice (8 weeks, male, from Orient Bio, Korea). When the volume of tumors reached around 100 mm3 (length  width  height  p/6), the mice were divided into four groups (1. PBS only/2. VEGF siRNA only/3. FPP þ GFP siRNA/4. FPP þ VEGF siRNA). Each experimental group (40 ml, N/P ¼ 20, n ¼ 5) was injected into right dorsal subcutis of mice, and body weights and tumor volumes were monitored for three weeks at predetermined time intervals. The sizes of the tumors were measured using caliper and tumor volumes were calculated by length  width  height  p/6. At day 21, the mice were sacrificed and tumor tissues were dissected. The tumor tissues were took a picture, homogenized and centrifuged to obtain supernatant for further analysis. Total proteins were quantified by BCA assay. For Western-blot, 20 mg of protein was separated on SDS-PAGE, transferred onto nitrocellulose membrane, blocked and incubated overnight with VEGF mouse monoclonal antibody and b-actin mouse monoclonal antibody (Santacruz Biotechnology, California, USA) each of them. After washing, the membrane was incubated with horseradish peroxidase-labeled goat anti-mouse IgG secondary andibody (Santacruz Biotechnology) and the bands were visualized using the ECL western blotting detection reagents (GE Healthcare, Buckinghamshire, UK). The VEGF expression was normalized with b-actin and the band intensities quantified by Image J software.

Y.-M. Kim, S.-C. Song / Biomaterials 35 (2014) 7970e7977 2.13. In vivo toxicity test The FPP solution (7 wt%, 100 mL) was injected into the dorsal subcutis of balb/c mice (6 weeks, female, from Orient Bio, Korea). These mice were sacrificed, and the existence of the gel was checked at predetermined time intervals. The tissue where the FPP was injected was dissected and liver stained with hematoxylin and eosin.

3. Results and discussion

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0.03 mol% for the best targeting efficiency and 0.06 mol%, the folate content in FPP was also enough to induce targeted delivery [10,11]. The conjugation of folate to PP was characterized by 1H nuclear magnetic resonance (1H NMR) and high performance liquid chromatography (HPLC) (Fig. S2). The aqueous solution of FPP exhibited temperature-dependent solegel transition with same mechanism in previous paper (Fig. S3) [9]. In brief, gelation of FPP is induced by hydrophobic interaction due to increased temperature.

3.1. Synthesis and characterization of FPP hydrogel First, FPP was synthesized by amide coupling between the terminal carboxyl group of the folate and the primary amine group of PP (Fig. S1). The synthesis of PP was performed as previously described. The optimum folate content in folate-PEG-liposome was

3.2. Formation and properties of micelleplexes between FPP micelles and siRNAs The complexation between siRNAs and FPP via ionic interaction was confirmed using a gel retardation assay and zeta nanosizer

Fig. 1. a) Gel retardation test of micelleplexes as a function of the N/P ratio to confirm the complexation between micelles and siRNAs. b, c) Sizes and zeta potentials of micelleplexes as a function of the N/P ratio and FPP micelles by dynamic light scattering (DLS). d, e) CryoTEM image of FPP micelles and micelleplexes. f) TEM image of micelleplexes. g) TEM image of released micelleplexes from the micelleplex hydrogel at day 14. h, i) In vitro stability tests of siRNAs only and micelleplexes groups as a function of time.

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data. In the gel retardation assay, the formation of polyplexes was observed over N/P ¼ 10. The N/P value is the ratio of moles of the amine groups of cationic polymers to phosphate groups of RNA (Fig. 1a). The sizes of the polyplexes decreased as a function of the N/P ratio from 150 nm to 50 nm and, according to the zeta nanosizer data, the surface charges increased from 5 mV to 15 mV due to the complexation with the cationic FPP (Fig. 1bec). Here, we focused only on the sizes of the polymer group. Compared to previously reported results, the size of FPP decreased from 300 nm to 150 nm after folate conjugation to PP, and the polydispersity index(PDI) also decreased from 0.544 to 0.423, indicating that the self-assembled polymer was more condensed than previously reported polymers [12]. Therefore, we thought that FPP had a more stable structure than PP due to increased hydrophobicity after folate conjugation. This was demonstrated by the increase of the viscosity after conjugation when the temperature was decreased  from 66.8  C to 41.8 C, because the gelation property of the thermosensitive phosphazene is mediated by hydrophobic interactions [13]. We assumed that the increased hydrophobicity could affect the formation of amphiphilic polymers such as spherical micelles, cylindrical micelles and polymersomes in solution. According to cryotransmission electron microscopy (cryo-TEM) measurements, FPP,

the amphiphilic polymer formed spherical micelles in solution (Fig. 1d) [14]. The complexation between micelles and siRNAs by ionic interaction after simple mixing generated micelleplexes (Fig. 1eef). The release of 50e100 nm sized micelleplexes from the micelleplex hydrogel at day 14 was confirmed by TEM (Fig. 1g), suggesting that there was no difference between the original micelleplexes and those released from the hydrogel. The stability of micelleplexes was evaluated at certain time points by observing the denaturation of the siRNAs. siRNA alone showed rapid degradation within 6 h, whereas the micelleplexes between FPPs and siRNAs showed superior stability up to 2 weeks (Fig. 1hei). These results suggest that the presence of micelleplexes protects the siRNAs in the plasma after their release from the hydrogel within at least 2 weeks. 3.3. In vitro selective siRNA delivery and gene silencing efficiency The selective cellular uptake of micelleplexes containing fluorescent dye (cy3)-tagged siRNA in the FR-over-expressing cell line and FR-deficient cell line was observed by using a fluorescence microscope (Fig. 2a). A549 (human lung carcinoma cell line, FRdeficient) and MDA-MB-231 (human breast cancer cell line, FRexpressing) were used for this experiment [15,16]. As expected, the

Fig. 2. a) In vitro cellular uptake of micelleplexes in different cell lines expressing different folate-receptor levels (FR over-expressed or deficient). b) In vitro gene silencing effect of micelleplexes for quantification of the inhibition effect (Student's t test, *p < 0.001 versus cell only).

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group containing only the cells did not show any fluorescence. Polyethyleneimine (PEI) 25K, one of the major gene carriers, showed high fluorescence intensity within both A549 and MDAMB-231 cell lines, meaning that it delivered siRNAs into the cells without any FR selectivity. In contrast to PEI 25K, the FPP micelleplex group showed high fluorescence intensity only in MDA-MB231 cells and very low fluorescence intensity in A549 cells. These results indicate that FPP micelleplexes entered FR-over-expressing cells via receptor-mediated endocytosis with selectivity and correlated with previous researches regarding folate targeting in same cell line [17]. The selectivity of the micelleplexes would decrease the side effects due to random endocytosis and relatively increase the efficacy of the delivery. And the transfection efficacy of FPP micelleplexes was quantified by measuring the reduction of the vascular epithelial growth factor (VEGF) expression levels in the cells after treatment with micelleplexes carrying a VEGF-specific siRNAs by using a VEGF ELISA kit. From the above two experiments, we concluded that the FPP polyplexes selectively entered

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the target cells and successfully inhibited the expression of the target protein (Fig. 2b). 3.4. In vivo release of micelleplexes from hydrogel and their targetability in long-term Then, we analyzed the effects of the targetable micelleplex hydrogel in vivo system. After subcutaneous injection of the hydrogel, released 60 nm-sized micelleplexes from the hydrogel could enter the blood and lymphatic vessels. Oussoren et al. studied the size-dependent movement of liposomes after subcutaneous injection, noting that 70% of injection the injection volume were disappeared within 10 h and entered the circulation pathway via the lymphatic and blood vessels in case of 70 nm-sized liposome, suggesting that our micelleplexes could enter the circulation pathway [8]. To evaluate the targetability and therapeutic potential of the nanopolyplexes released from the hydrogel in vivo, cy3-tagged

Fig. 3. a) In vivo real time imaging of distribution of cy3-tagged siRNAs after s.c. injection into tumor-bearing mice by IVIS. b) Ex vivo NIRF images of dissected organs 10 days post injection. c, d, e) Confocal microscope image of dissected tumor tissue of the cy3-siRNA group, PP þ cy3-siRNA, and FPP þ cy3-siRNA group. Red ¼ cy3-tagged siRNAs, Green ¼ VEGF expression, Blue ¼ nuclei. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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siRNAs were used to generate the micelleplex solution. This solution was administrated to the right dorsal subcutis of mice bearing a tumor on the opposite side, and the results were observed by using an in vivo imaging system (IVIS) at certain time points (Fig. 3a). The fluorescent intensity of the group treated only with siRNA disappeared after 6 h. In the case of the groups treated with PP and FPP, the hydrogel held the cy3-tagged siRNAs at the injection sites and the fluorescence was prolonged up to 10 days. Interestingly, the tumor size decreased only in the FPP group, even though fluorescence intensity was observed only at the injection site and not tumor site. It was suspected that the absence of fluorescence at the tumor site was related to the fact that only a small amount of siRNA was present in the tissues and their fluorescence signals weakened with time. Therefore, the organs from sacrificed mice were dissected and observed using a KODAK imaging instrument to verify that the decreased tumor size was due to the released micelleplexes (Fig. 3b). In the groups treated with PP and only with siRNA, the fluorescence intensity at the tumor site was significantly weaker than that in other organs such as the liver and kidney. Strong fluorescence intensity in the tumor tissue was observed only in the FPP group, indicating that the micelleplexes released from the hydrogel accumulated at the tumor site via receptor-mediated endocytosis and inhibited the tumor growth. The distributions of the released micelleplexes in the FPP group were observed after cryosection of the tumor tissues from the sacrificed mice at day 10 (Fig. 3cee). The tissues were stained to detect the expression of VEGF and the presence of siRNAs. The cell nuclei (blue, DAPI) and VEGF (green, Alexa 488) were observed in both siRNA, PP, and FPP groups, even though siRNAs (red, cy3) was present only in the FPP group. Interestingly, the region where the siRNAs were located showed low VEGF expression. These results suggest that FPP micelleplexes from hydrogel selectively targeted the tumor tissues, entered into the tumor cells and successfully inhibited VEGF expression in vivo.

FPP þ siVEGF showed anti-tumor activity until day 21 without any weight change after only one injection (Fig. 4a and Fig. S4). To confirm that the anti-tumor effect was due to decreased VEGF expression following the delivery of siRNA, we measured VEGF expression levels in the tumor tissues (Fig. 4b). Only FPP/siVEGF showed down-regulated VEGF expression level, suggesting that the FPP/siVEGF successfully delivered the siRNAs into the tumor tissues and induced anti-tumor effect in 2 weeks after one injection. Interestingly, we observed the tumor volume in the FPP/siRNA group increased from day 14 to 21, which correlated with the disappearance of fluorescence of siRNAs at day 10 in IVIS data. This result suggests that the inhibitory effect of FPP/siVEGF was reduced after day 14 because the hydrogel was dissolved and degraded (Fig. 4a). The in vivo biocompatibility of the hydrogel was also evaluated to conclude it did not affect to the anti-tumor effect as a toxic material (Fig. S5). 3.6. In vivo gene silencing test To achieve a prolonged therapeutic period of the micelleplexes hydrogel, we adjusted the hydrogel volume and tested the modified conditions in an in vivo tumor-xenograft model (Fig. 5a). In case of same amount of hydrogel (100 mg siRNA) with previous experiment, anti-tumor effect was maintained for 14 days; however, when we injected double the volume (200 mg siRNA) with same N/P ratio, the effect was maintained until 30 days. Gel strength is also additional factor that affect the dissolution and degradation properties of hydrogel [18]. The gel strength is easily adjusted by controlling the gel percentage; thus, we checked that the viscosity was proportional to the concentration of the polymer in the hydrogel (Fig. 5b). Therefore, our results show that the targetable micelleplex hydrogel has the advantage that the therapeutic time and extent are easily controllable by varying the concentration and amount of hydrogel.

3.5. In vivo gene silencing effect mediated anti-tumor effect of hydrogel

4. Conclusion

The therapeutic effects of the FPP micelleplex hydrogel were also examined after injection at the opposite site of the tumor on the dorsal part of the mice. Among the groups including phosphate-buffered saline (PBS), siVEGF (VEGF siRNA), FPP þ siGFP(green fluorescence protein siRNA), and FPP þ siVEGF, only

In summary, we developed a targetable, injectable, and noncytotoxic, micelleplex hydrogel having dual functions: effective systemic targetable siRNAs delivery carrier via passive/active targeting, and hydrogel for tunable therapeutic time by long-term, controlled release of the delivery carrier after only one injection

Fig. 4. Therapeutic effect of micelleplex hydrogel a) In vivo anti-tumor activity of micelleplex hydrogel in tumor xenografts after s.c. injection (*p < 0.05 versus PBS). b) Western blot analysis for intratumoral VEGF expression levels (*p < 0.001 versus PBS).

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Fig. 5. a) In vivo anti-tumor activity of micelleplex hydrogel in tumor xenografts after s.c. injections with different volumes of hydrogel (*p < 0.06 versus PBS). b) Relation between viscosity and concentration of FPP in the hydrogel.

of one material. We expect that this material could serve as a new effective siRNA delivery material for systemic long-term gene silencing. Acknowledgements This research is financially supported by Korea Institute of Science and Technology and the National Research Foundation of Korea. The author thanks to Dr. Jang Il Kim and Jung-Kyo Cho, especially. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.biomaterials.2014.05.070. References [1] Davidson BL, McCray BL. Current prospects for RNA interference-based therapies. Nat Rev Genet 2011;12:329e40. [2] Kanasty R, Dorkin JR, Vegas A, Anderson D. Delivery materials for siRNA therapeutics. Nat Mater 2013;12:967e77. at V. To exploit the tumor microenvironment: passive [3] Danhier F, Feron O, Pre and active tumor targeting of nanocarriers for anti-cancer drug delivery. J Control Release 2010;148:135e46. [4] Bae YH, Park K. Targeted drug delivery to tumors: myths, reality and possibility. J Control Release 2011;153:198e205. [5] Hoffman A. Pharmacodynamic aspects of sustained release preparations. Adv Drug Deliv Rev 1998;33:185e99.

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Targetable micelleplex hydrogel for long-term, effective, and systemic siRNA delivery.

We developed a targetable micelleplex hydrogel as a new efficient systemic siRNA delivery material that functions as a targetable gene carrier, and a ...
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