siRNA-Loaded Polyion Complex Micelle Decorated with Charge

Nov 30, 2015 - Small interfering RNA (siRNA) needs an efficient delivery vehicle to reach the cytoplasm of target cells for successful RNA interferenc...
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siRNA-Loaded Polyion Complex Micelle Decorated with Charge-Conversional Polymer Tuned to Undergo Stepwise Response to Intra-Tumoral and Intra-Endosomal pHs for Exerting Enhanced RNAi Efficacy Montira Tangsangasaksri,† Hiroyasu Takemoto,‡ Mitsuru Naito,§ Yoshinori Maeda,§ Daiki Sueyoshi,† Hyun Jin Kim,§ Yutaka Miura,§ Jooyeon Ahn,∥ Ryota Azuma,∥ Nobuhiro Nishiyama,‡,⊥ Kanjiro Miyata,*,§,⊥ and Kazunori Kataoka*,†,§,∥,⊥ †

Department of Bioengineering, Graduate School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan ‡ Polymer Chemistry Division, Chemical Resources Laboratory, Tokyo Institute of Technology, R1-11, 4259 Nagatsuta, Midori-ku, Yokohama 226-8503, Japan § Center for Disease Biology and Integrative Medicine, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan ∥ Department of Materials Engineering, Graduate School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan ⊥ Innovation Center of NanoMedicine, Institute of Industry Promotion-Kawasaki, 3-25-14 Tonomachi, Kawasaki-ku, Kawasaki 210-0821, Japan S Supporting Information *

ABSTRACT: Small interfering RNA (siRNA) needs an efficient delivery vehicle to reach the cytoplasm of target cells for successful RNA interference (RNAi) therapy. This study aimed to develop an siRNA-loaded polyion complex (PIC) micelle equipped with a smart polymeric shell featuring tumor targetability and endosome escapability for enhanced RNAi activity in cancer cells. To this end, an acidic pH-responsive polypeptide was designed to exert a stepwise change in its charged state from negative to modestly positive and highly positive in response to slightly acidic environment of tumor (pH ∼6.7) and further lowered-pH condition of late endosomal compartments (pH ∼5.0), respectively, for selective binding to cancer cell surface and subsequent endosome disruption. This polypeptide, termed PAsp(DET-CDM/DBCO), was synthesized by introducing acid-labile carboxydimethyl maleate (CDM) and dibenzylcyclooctyne (DBCO) moieties into a polyaspartamide derivative bearing two-repeated aminoethylene side chains (PAsp(DET)). Then, PAsp(DET-CDM/DBCO) was installed on the surface of disulfide cross-linked PIC micelles prepared from cholesterol-modified siRNA (Chol-siRNA) and azide-poly(ethylene glycol)-b-poly[(3-mercaptopropylamidine)-L-lysine] (N3-PEG-b-PLys(MPA)) through the copper-free click reaction. Successful PAsp(DET-CDM/DBCO) coverage of PIC micelles was confirmed by a significant decrease in ζ-potential as well as a narrowly distributed size of 40 nm. The PAsp(DET-CDM/ DBCO)-installed micelles significantly improved the gene-silencing efficiency in cultured lung cancer cells, compared with nonmodified control micelles, especially after incubation at pH 6.7. This improved silencing activity was nicely correlated with the facilitated cellular uptake of siRNA payloads at the acidic pH and the efficient endosomal escape. These results demonstrate that the acidic pH-responsive polypeptide shell is a promising design strategy for tumor-targeted siRNA delivery.

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INTRODUCTION Small interfering RNA (siRNA)-mediated gene silencing, termed RNA interference (RNAi), has become a potential therapeutic strategy since its discovery in 1998.1 siRNA, a short doublestranded oligonucleotides with ∼21-nt, assembles into RNAinduced silencing complex (RISC) in the cytoplasm, allowing the selective cleavage of complementary mRNA;1,2 however, the poor bioavailability of siRNA, which is mainly due to susceptibility to enzymatic degradations, inefficient cellular uptake, and endosomal entrapment (or digestion) in the cells, generates the need © XXXX American Chemical Society

of a delivery carrier to protect fragile siRNA molecules and improve the target tissue accumulation, cellular uptake, and intracellular trafficking. The development of efficient carriers is therefore a crucial issue for successful RNAi therapies.3,4 Polyion complex (PIC) micelles, which can be prepared from an oppositely charged pair of nucleic acids and block catiomers, Received: October 5, 2015 Revised: November 24, 2015

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DOI: 10.1021/acs.biomac.5b01334 Biomacromolecules XXXX, XXX, XXX−XXX

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Figure 1. Schematic illustrations for preparation of smart polymeric shell on Chol-siRNA-loaded/disulfide cross-linked PIC micelle (A) and two-step acidic pH-responsive functions for tumor-targeted siRNA delivery (B).

pH-responsive, zwitterionic polypeptide (Figure 1A). In particular, this polypeptide was engineered by introducing a carboxydimethyl maleate (CDM) moiety into an endosome-disrupting polyaspartamide derivative with twice-repeated aminoethylene units (PAsp(DET)) through maleic acid amide bond (PAsp(DETCDM)).18,19 As illustrated in Figure 1B, PAsp(DET-CDM) was assumed to gradate its charged state in response to two intravital acidic conditions, that is, tumor environments20,21 and late endosomal/lysosomal compartments.22,23 At neutral pH, PAsp(DET-CDM) may be biologically inert because of its zwitterionic hydrophilicity, which can suppress nonspecific adsorption of blood components and normal tissue. Once reaching the lowered-pH environment of tumor (pH ∼6.7), PAsp(DET-CDM) undergoes acid hydrolysis of CDM moieties to generate the parent, monoprotonated PAsp(DET) for electrostatic binding to negatively charged cancer cell surface. After endocytosis by cancer cells, PAsp(DET) adopts the diprotonated state in late endosomal compartments (pH ∼5.0), destabilizing the endosomal membrane through their charged interactions.6,24 Herein, PAsp(DET-CDM) was integrated into the reversibly stabilized PIC micelle featuring

have been developed as a promising platform of nucleic acid carriers.5−11 They are composed of two main structures, the PIC core, and the outer shell. The PIC core can sequester fragile nucleic acid payloads from enzymatic degradation and immune recognition, while the outer shell contributes to avoid nonspecific adsorption of biomacromolecules, such as serum proteins, directed toward the longevity under harsh in vivo condition.10−12 Nonionic and hydrophilic poly(ethylene glycol) (PEG) has been most extensively utilized as a shell-forming block; however, the PEG shell concurrently lessens cellular uptake of micelles by target cells, compromising the gene silencing efficacy.6,13,14 To circumvent this drawback of PEG shell, targeting ligand molecules or PEG-detachable functionalities have been installed into PEGylated nanocarriers.6,10,11,15−17 Nevertheless, further improvements in the material design are still required for more efficient RNAi activity in target cells. Herein, we report a novel design of smart polymeric shell that can overcome the PEG dilemma for tumor-targeted cellular uptake and subsequent endosomal escape of siRNA payloads. To this end, PEG shell was functionalized with an acidic B

DOI: 10.1021/acs.biomac.5b01334 Biomacromolecules XXXX, XXX, XXX−XXX

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dialysis against 0.01 N HCl (aq) 4 times and deionized water twice. The solution was subsequently lyophilized to obtain N3-PEG-b-PLys with chloride salt as white powder (466 mg, 88.9% yield). The deprotection of TFA group was confirmed by the generation of methylene protons adjacent to the ε-amino group (−(CH2)3CH2NH3Cl, δ 2.9) in a 1H NMR spectrum (400 MHz, D2O, 25 °C). Synthesis of N3-PEG-b-PLys(MPA). DTBP/HCl was reacted with the primary amine groups of N3-PEG-b-PLys, generating N3-PEG-bPLys possessing thiol groups at the side chains (N3-PEG-b-PLys(MPA)) as previously described.11 In brief, N3-PEG-b-PLys (50 mg, 0.12 mmol amine) was dissolved in 100 mM borate buffer (5 mL, pH 9.0). DTBP (73 mg, 0.24 mmol) was dissolved in chilled water (1 mL) and mixed with the polymer solution. The mixture was stirred at 25 °C for 45 min, then dialyzed (3.5K MWCO) against 10 mM phosphate buffer containing 150 mM NaCl (pH 7.4) three times to remove excess DTBP. DTT (50 mg, 0.32 mmol) was added to the dialyzed solution and the mixture was incubated for 30 min at room temperature to cleave the disulfide linkage derived from DTBP. The reduced polymer solution was dialyzed against 0.01 N HCl (aq) and deionized water, followed by filtration and lyophilization to obtain N3-PEG-b-PLys(MPA) as white powder (55 mg, 91.5% yield). The complete introduction of DTBP was confirmed by 1H NMR spectrum (400 MHz, D2O/DCl, 25 °C), as calculated from the peak intensity ratio of propylene protons in PLys side chains (−(CH2)3−, δ 1.3−1.9) to protons in mercaptoethyl groups in DTBP (HS(CH2)2−, δ 2.7−2.9). Synthesis of Poly(β-benzyl L-aspartate) (PBLA). PBLA was synthesized through the ring-opening polymerization of BLA-NCA, initiated by n-butylamine as previously described.19,24 In brief, n-butylamine (3.36 μL, 0.034 mmol) in DCM (340 μL) was added to BLA-NCA (1 g, 4.02 mmol) dissolved in the mixture of DMF (2 mL) and DCM (18 mL) under an argon atmosphere. The reaction continued to stir for 48 h at 35 °C. The reaction solution was poured in an excess amount of the mixture of n-hexane/ethyl acetate (v/v 6/4), followed by filtration to collect the precipitate. The precipitate was dried under reduced pressure overnight to obtain PBLA as white powder (637 mg, 77% yield). The DP of PBLA was calculated to be 110 by 1H NMR spectrum (400 MHz, DMSO-d6, 80 °C), as calculated from the peak intensity ratio of methyl protons at the C-terminus (CH3CH2CH2CH2−, δ 0.8) to the benzyl protons of the side chain (C6H5CH2−, δ 7.3). Synthesis of Poly{N′-[N-(2-aminoethyl)-2-aminoethyl]aspartamide} (PAsp(DET)). PAsp(DET) was synthesized through aminolysis reaction of benzyl groups of PBLA with DET. In brief, 400 mg (0.018 mmol) of PBLA (DP = 110) was lyophilized from the mixture of benzene (10 mL) and DCM (2 mL). The obtained powder and DET (9.66 mL, 97.6 mmol, 50 equiv to benzyl group of PBLA) were separately dissolved in NMP containing 0.5 M thiourea (20 mL for PBLA and 10 mL for DET), followed by cooling to 15 °C under an argon atmosphere. The PBLA solution was dropwise added to DET solution with stirring and reacted for 1 h at 15 °C. The mixture was dropwise poured in 64.15 mL of 5 N HCl (aq) for neutralization. The neutralized solution was dialyzed (6K-8K MWCO) against 0.01 N HCl (aq) four times and deionized water twice at 4 °C, then lyophilized to obtain PAsp(DET) with chloride salt as white powder (349 mg, 80% yield). The complete introduction of DET moiety into the side chains was confirmed by 1H NMR spectrum (400 MHz, D2O, 25 °C), as calculated from the peak intensity ratio of methyl protons at the C-terminus (CH3CH2CH2CH2−, δ 0.8) to the ethylene protons in the DET moiety (H2N(CH2)2NH(CH2)2NH−, δ 3.1−3.6). Synthesis of PAsp(DET) Modified with CDM and Dibenzocyclooctyne (PAsp(DET-CDM/DBCO)) and PAsp(DET) Modified with SUC and Dibenzocyclooctyne (PAsp(DET-SUC/DBCO)). PAsp(DET) (20 mg, 0.67 μmol) was dissolved in 100 mM NaHCO3 buffer (5 mL, pH 9) at 4 °C. DBCO-NHS ester (1.9 mg, 4.7 μmol) was dissolved in DMF (1 mL) and was added to polymer solution, followed by stirring at 4 °C for 1 h. Then, to introduce CDM or SUC to the PAsp(DET) side chains, CDM (87 mg, 0.472 mmol) or SUC (35 mg, 0.350 mmol) was dissolved in ethanol (6 and 3 mL, respectively) and was directly added into the reaction solution. The mixture was further stirred at 4 °C for 3 h. The reaction solution was purified by ultracentrifugation using Amicon Ultra-15 centrifugal filter units (10K MWCO,

a disulfide cross-linked core with cholesterol-modified siRNA (Chol-siRNA), which can permit selective payload release in cytosolic reductive environments.9,11 The functionality of smart polymeric shell on PIC micelles is validated by physicochemical and biological evaluations, demonstrating its potential for tumortargeted siRNA delivery.

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MATERIALS AND METHODS

Materials. ε-Trifluoroacethyl-L-lysine N-carboxy anhydride (Lys(TFA)-NCA) was synthesized by the Fuchs-Farthing method,25,26 and β-benzyl-L-aspartate N-carboxy anhydride (BLA-NCA) was obtained from Chuo Kaseihin (Tokyo, Japan). Trinitrobenzenesulfonate (TNBS), succinic anhydride (SUC), thiourea, glycine, D2O (99.9%), DCl, (35% in D2O), dibenzocyclooctyne-N-hydroxysuccinimidyl ester (DBCO-NHS ester), 0.4% trypan blue solution, and RPMI-1640 medium were purchased from Sigma-Aldrich (St. Louis, MO). Dithiothreitol (DTT) and diethylenetriamine (DET) were purchased from Wako Pure Chemical Industries (Osaka, Japan). DET was distilled before use. Anhydrous dichloromethane (DCM) and anhydrous N,N-dimethylformamide (DMF) were purchased from Kanto Chemical (Tokyo, Japan) and used after passing through two neutral alumina columns (Glass Contour, Irvine, CA). Dimethyl 3,3′-dithiobispropionimidate/2HCl (DTBP/HCl), Slide-A-Lyzer dialysis cassettes (3.5K molecular weight cut off (MWCO)), and RPMI-1640 medium, powder (without HEPES and sodium bicarbonate) were purchased from Thermo Scientific (Rockford, IL). Benzene was purchased from Nacalai Tesque (Kyoto, Japan). Diethyl ether was obtained from Showa Ether (Kanagawa, Japan). Carboxy-dimethyl maleic anhydride (CDM) was synthesized as previously described.19 Cell Counting Kit 8 (CCK-8) was purchased from Dojindo Laboratories (Kumamoto, Japan). Fetal bovine serum (FBS) was purchased from Dainippon Sumitomo Pharma (Osaka, Japan). Human lung carcinoma cell line, A549, was purchased from the American Type Culture Collection (Manassas, VA). A series of siRNAs were synthesized by Hokkaido System Science (Hokkaido, Japan), and their sequences were as follows: (1) PLK1 siRNA (siPLK1): 5′-AGA uCA CCC uCC UuA AAu AUU-3′ (sense) and 5′-UAU UUA AgG AGG GUG AuC UUU-3′ (antisense), where capital and lowercase letters represent RNA and 2′-O-methylated RNA, respectively,27 and (2) nontargeted control sequence (siCont): 5′-UUC UCC GAA CGU GUC ACG UdTdT-3′ (sense) and 5′-ACG UGA CAC GUU CGG AGA AdTdT-3′ (antisense). Cy3 dye and Chol moiety were introduced to the 5′-end of the antisense strand and the sense strand, respectively. 1 H NMR spectra were measured on JNM-ECS400 (JEOL, Tokyo, Japan). Synthesis of Azide-poly(ethylene glycol)-block-poly(L-lysine) (N3-PEG-b-PLys). N3-PEG-NH2 (Mw = 12 000) was synthesized as previously described.28 N3-PEG-b-PLys(TFA) was synthesized by a ring opening polymerization of Lys(TFA)-NCA with N3-PEG-NH2 as a macroinitiator. In brief, N3-PEG-NH2 (300 mg, 25 μmol) was lyophilized from benzene. N3-PEG-NH2 and Lys(TFA)-NCA (369 mg, 1.38 mmol, 55 equiv to the macroinitiator) were separately dissolved in anhydrous DMF containing 1 M thiourea (3 and 2.5 mL, respectively). The Lys(TFA)-NCA solution was added to N3-PEG-NH2 solution and the mixture was reacted at 25 °C for 3 days. The reactant solution was poured into excess amount of the mixture of diethyl ether/methanol (v/v 9/1) to precipitate N3-PEG-b-PLys(TFA) as white powder (596 mg). The molecular weight distribution of the polymer was confirmed by size exclusion chromatography (HLC-8220, TOSOH Corporation, Tokyo, Japan) equipped with two TSK gel columns (TSK-gel Super AW4000 and Super AW3000) using DMF containing LiCl (10 mM) as carrier solvent at 0.8 mL/min and was determined to be 1.08. The degree of polymerization (DP) of Lys(TFA) units was calculated to be 45 by 1 H NMR spectrum (400 MHz, DMSO-d6, 80 °C), as calculated from the peak intensity ratio of the β-, γ-, and δ-methylene protons of lysine (−(CH2)3−, δ 1.3−1.9) to protons in oxyethylene units in PEG chain (−CH2CH2O−, δ 3.5). Deprotection of TFA group was further carried out to yield N3-PEG-b-PLys. In brief, N3-PEG-b-PLys(TFA) (596 mg) was dissolved in the mixture of MeOH (59.6 mL) and 1 N NaOH (5.96 mL). The solution continued to stir at 35 °C for 8 h, followed by C

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different time periods (1, 3, 6, 9, and 12 h). The Cy3 fluorescence was detected using a confocal laser scanning microscope, LSM510 (Carl Zeiss, Oberkochen, Germany) equipped with Confocor3 module and a C-Apochromat 40× water objective (Carl Zeiss, Oberkochen, Germany). The detection was carried out using a 543 nm He−Ne laser for excitation and a 580 nm long pass filter for emission. Autocorrelation curves obtained from 10 measurements at a sampling time of 20 s were converted into diffusion coefficient using Rhodamine 6G as a standard with Zeiss Confocor3 software. In Vitro Gene Silencing Assay by Quantitative Real-Time PCR (qRT-PCR). Endogenous gene-silencing activities of nonmodified PIC micelles and CDM- (or SUC-) micelles were investigated by qPCR for polo-like kinase 1 (PLK1) as a targeted gene. For surface modification ratio optimization, A549 cells were seeded in a 24-well plate (25 000 cells/well) in growth medium (RPMI-1640 containing 10% FBS) and were incubated for 24 h. Then, the cells were incubated with the nonmodified PIC micelles and the CDM-micelles loaded with Chol-siPLK1 or Chol-siCont at pH 7.4 for 48 h (final siRNA concentration: 200 nM). After 48 h, the cells were washed with PBS and the total RNA was extracted using RNeasy mini kit (Qiagen, Valencia, CA). Reverse transcription was performed using ReverTra Ace (Toyobo, Osaka, Japan) and qPCR was carried out by Taqman Gene Expression Assays Protocol with an ABI 7500 Fast Real-Time PCR System (Applied Biosystems, Foster City, CA). Human β-actin was employed as an endogenous house-keeping gene to normalize the PLK1 mRNA amount. The mRNA expression level was calculated based on comparative Ct method (2−ΔΔCt) with Applied Biosystems 7500 Software v2.0. The results are presented as mean and standard error of mean obtained from four samples. To elucidate the effect of pH, A549 cells were seeded into 24-well plate (25,000 cells/well) in growth medium (RPMI-1640 containing 10% FBS) and were incubated for 24 h. Then, the cells were incubated with the nonmodified PIC micelles and the CDM- (or SUC-) micelles loaded with Chol-siPLK1 or Chol-siCont at pH 7.4 or 6.7 for 48 h (final siRNA concentration: 200 nM). For incubation at pH 6.7, RPMI-1640 medium powder was dissolved in milli-Q water and the pH was adjusted to be 6.7 by adding HCl (aq). The medium was further adjusted to possess 0.15 g/L NaHCO3 and 10% FBS, and preincubated in the presence of 5% CO2 at 37 °C overnight to maintain the pH. Then, the cells were washed with PBS and the total RNA was extracted and qPCR was carried out following the above protocol. Human β-Actin was employed as an endogenous house-keeping gene to normalize the PLK1 mRNA amount. The mRNA expression level was calculated based on comparative Ct method (2−ΔΔCt) with Applied Biosystems 7500 Software v2.0. The results are presented as mean and standard error of mean obtained from 4 samples. Cellular Uptake. A549 cells were seeded into 96-well plate (10 000 cells/well) and were incubated for 24 h in the growth medium. Nonmodified PIC micelles and CDM- (or SUC-) micelles were prepared with Cy3-labeled Chol-siRNA. Then, the micelle solution (10 μL per 90 μL of growth medium) was added to the cells at pH 7.4 or 6.7 for 3 h (final siRNA concentration: 200 nM). The medium was replaced with fresh growth medium containing Hoechst 33342 (Dojindo Laboratories), followed by the incubation at 37 °C for 5 min for staining nuclei. The growth medium was further replaced with fresh growth medium containing 0.4% trypan blue solution (1 μL per 100 μL growth medium) for quenching the Cy3 dye on cellular membrane before the measurement. The Cy3 fluorescence inside the cells was measured and analyzed by IN Cell Analyzer 1000 (GE Healthcare, Buckinghamshire, U.K.) according to the manufacturer’s protocol. The results are presented as mean and standard error of mean obtained from four samples. Confocal Laser Scanning Microscope (CLSM). A549 cells were seeded into 8-well Lab-Tek chambered borosilicate cover-glass (20 000 cells/well) in the growth medium and were incubated for 24 h. Then, the cells were incubated with the nonmodified PIC micelles and the CDM- (or SUC-) micelles loaded with Cy3-labeled Chol-siRNA at pH 6.7 for 2 h (final siRNA concentration: 200 nM). The growth medium was replaced with fresh growth medium, followed by the incubation for 3, 6, and 12 h. Before each observation, late endosomes/lysosomes

Millipore, Billerica, MA) against 10 mM NaHCO3 buffer (pH 9) eight times and deionized water twice (3000g, 15 min for each centrifugation). The purified solution was lyophilized to obtain PAsp(DET-CDM/ DBCO) or PAsp(DET-SUC/DBCO) as white powder (27 and 18 mg, respectively). The introduction ratios of DBCO and CDM (or SUC) were determined from 1H NMR spectrum (400 MHz, D2O, 70 °C). The introduction ratio of DBCO was calculated to be ∼5% for both PAsp(DET-CDM/DBCO) and PAsp(DET-SUC/DBCO), as calculated from the peak intensity ratio of methyl protons at the C-terminus (CH3CH2CH2CH2−, δ 0.8 ppm) and benzyl protons of DBCO ((C4H4)2C7H2N−, δ 7.1−7.7). The complete introduction of CDM and SUC into residual primary amino groups of PAsp(DET) side chains was confirmed from the peak intensity ratios of methyl protons at the C-terminus (CH3CH2CH2CH2−, δ 0.8) to methyl protons in CDM (C = C−CH3, δ 1.8−2.0) for PAsp(DET-CDM/DBCO) and to methylene protons in SUC (COCH2CH2COONa, δ 2.4−2.6) for PAsp(DET-SUC/DBCO), respectively. Quantitative Analysis of Exposed Primary Amines. The hydrolysis property of maleic acid amide in PAsp(DET-CDM/ DBCO) was investigated by detecting the exposure of primary amine using TNBS. In brief, PAsp(DET-CDM/DBCO) or PAsp(DET-SUC/ DBCO) was dissolved in distilled water (1 mg/mL). Each polymer solution (10 μL) was mixed with 10 mM phosphate buffer pH 6.7 or 7.4 (90 μL), and the mixtures were incubated at 37 °C for 10 min, 30 min, 1 h, 2 h, 3 h, and 6 h. After the incubation, the solution was further mixed with 10 μL of TNBS (5 mg/mL in 100 mM borate buffer pH 9), followed by the incubation at room temperature for another 15 min. UV absorbance at 420 nm was measured for each solution to calculate the amount of exposed primary amines using the standard curve based on glycine solution. PIC Micelle Formation with Chol-siRNA. N3-PEG-b-PLys(MPA) was dissolved in 10 mM HEPES buffer (pH 7.4) at 5 mg/mL. The polymer solution was reduced with DTT (100 mM) at 25 °C for 15 min to cleave the preformed disulfide bonds. The reduced polymer solution was mixed with Chol-siRNA solution in 10 mM HEPES (15 μM siRNA concentration, pH 7.4) at [amidine]/[phosphate] = 1.4, as previously described to obtain PIC micelles.11 The PIC micelle solution was dialyzed (3.5K MWCO) against 5 mM HEPES buffer (pH 7.4) containing 0.5% DMSO for 1 day to form disulfide bonds within the micelles and 5 mM HEPES buffer (pH 7.4) for 2 days to remove DMSO. Surface Modification of PIC Micelles. The azide groups on the PIC micelles were subjected to copper-free click conjugation with PAsp(DET-CDM/DBCO) (or PAsp(DET-SUC/DBCO)) for surface modification. For optimization of surface modification ratio, PAsp(DET-CDM/DBCO) was dissolved in 10 mM HEPES buffer (pH 7.4) and added to PIC micelle solution at [DBCO in PAsp(DET-CDM/ DBCO)]/[N3 on the micelle] ratios of 0, 0.8, 1.5, 3, 6, and 9, followed by stirring at 4 °C for ∼2 h. The modified micelles were immediately applied to experiments without any purification. Similarly, PAsp(DETSUC/DBCO) was used as an acidic pH-unresponsive control polymer. The resulting PIC micelles were termed CDM- (or SUC)-micelles. Dynamic Light Scattering (DLS) and ζ-Potential Measurement. The hydrodynamic size and ζ-potential of the nonmodified PIC micelles and the CDM-micelles were analyzed by Zetasizer Nano ZS (Malvern Instruments, Worcestershire, U.K.) equipped with a standard λ = 633 nm laser as the incident beam. The measurements were carried out at 25 °C with a detection angle of 173°. The solutions were loaded into low-volume cuvette (ZEN2012) or disposable capillary cell (DTS1070) for DLS and ζ-potential measurements, respectively. The obtained data were analyzed based on cumulative method and calculated by the Stokes−Einstein equation to determine hydrodynamic diameter and polydispersity index (PDI). Fluorescence Correlation Spectroscopy (FCS). The stability of the nonmodified PIC micelles and the CDM-micelles in the presence of FBS was investigated by using FCS. PIC micelles loaded with Cy3-labeled Chol-siRNA (1 μM siRNA concentration, 10 μL) were mixed with 10 mM HEPES (pH 7.4) containing 50 and 90% FBS (90 μL) (final siRNA concentration: 100 nM). The prepared sample (100 μL) was added to 8-well Lab-Tek chambered borosilicate coverglass (Nalge Nunc International, Rochester, NY) and was incubated at D

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Figure 2. 1H NMR spectrum of PAsp(DET-CDM/DBCO) recorded in D2O at 70 °C (concentration: 5 mg/mL). This polyaspartamide derivative has the mixed sequence of α and β isomers, and its ω-end may be modified with either CDM or DBCO moieties. Only α isomer and CDM-modified ω-end are depicted in this figure for simplicity. and nuclei were stained with LysoTracker Green (Molecular Probes, Eugene, OR) and Hoechst 33342, respectively. The growth medium was further replaced with fresh growth medium containing 0.4% trypan blue solution (1 μL per 100 μL growth medium) prior to the CLSM observation. CLSM images were recorded using LSM510 equipped with a Plan-Apochromat 40× oil immersion objective (Carl Zeiss, Jena, Germany) at excitation wavelengths of 488 (Ar laser), 543 (He−Ne laser), and 710 nm (MaiTai laser) for LysoTracker Green, Cy3, and Hoechst 33342, respectively. The colocalization ratio of Cy3 and LysoTracker Green was determined by ImarisColoc software following the equation: colocalization ratio = Cy3 pixelscolocalization/Cy3 pixelstotal, where Cy3 pixelscolocalization represents the pixel number of Cy3 colocalized with LysoTracker green, and Cy3 pixelstotal represents the total pixel number of Cy3. The results are presented as mean and standard error of mean obtained from 15 cells. Cell Viability Assay. A549 cells were seeded into 96-well plate (2,500 cells/well) and were incubated for 24 h in the growth medium. Then, the cells were incubated with nonmodified PIC micelles and the CDM- (or SUC-) micelles loaded with Chol-siCont at pH 7.4 for 48 h (final siRNA concentration: 200 nM). After 48 h, the Cell Counting Kit-8 solution (CCK 8, Dojindo Laboratories, Kumamoto, Japan) was added to the medium (1 μL/10 μL media) and allowed to incubate at 37 °C for 1 h. The absorbance of extracellular matrix was measured at 450 nm using a microplate reader (model 680, BIO-RAD, Hercules, CA). The cell viability in each well was calculated relative to the nontreated control well. The results are presented as mean and standard error of mean obtained from six samples. Statistical analysis. The statistical analysis was determined by Student’s t test with two-tailed distribution. The data were considered to be significant difference at p < 0.05.

surface, as shown in Figure 1A. PAsp(DET) was prepared from PBLA (DP of PLBA = 110, Mw/Mn = 1.1) through the aminolysis reaction with excess amount of DET (Scheme S1). The quantitative conversion of BLA to Asp(DET) was confirmed by 1H NMR (data not shown). Then, DBCO moiety was introduced into primary amines in the side chain of PAsp(DET) through amide bond formation, followed by introduction of CDM moiety to the residual primary amines (Scheme S1). The substitution degrees of DBCO and CDM moieties were calculated to be ∼5 and ∼95% from the 1H NMR spectrum by the peak intensity ratio of methyl protons at the C-terminus (δ 0.8) to benzyl protons of DBCO (δ 7.2−7.8) and methyl protons in CDM (δ 1.8 to 2.0), respectively (Figure 2). Similarly, an acidic pHnonresponsive control polypeptide, PAsp(DET-SUC/DBCO), was synthesized by introducing succinic (SUC) moiety into PAsp(DET-DBCO).18 The substitution degrees of DBCO and SUC were confirmed to be ∼5 and ∼95%, respectively, from 1 H NMR spectrum by the peak intensity ratio of methyl protons at the C-terminus to benzyl protons of DBCO and methylene protons in SUC (δ 2.4−2.6) (Figure S1). Next, the acidic pH-responsiveness of PAsp(DET-CDM/ DBCO) was examined by the detection of exposed primary amines after acid hydrolysis of CDM moieties and compared with PAsp(DET-SUC/DBCO). These polypeptides were incubated at 37 °C for varying periods of time in 10 mM phosphate buffer at pH 7.4 and 6.7, which mimic the pH in the bloodstream and the tumor environment, respectively. Thereafter, sample solutions were reacted with TNBS, which forms chromogenic N-substituted 2,4,6-trinitroaniline derivatives after reaction with primary amines for detection by the UV absorbance at ∼420 nm. Whereas the increase in the amount of exposed amines of PAsp(DET-CDM/DBCO) was observed at both pH values, the acidic pH obviously facilitated the generation of primary amines with an increase in incubation time compared with the neutral pH (Figure 3A). It should be noted that the exposed amine content at pH 6.7 was calculated to be ∼34% after 6 h of incubation, demonstrating the acid-labile nature of PAsp(DET-CDM/DBCO). In contrast, PAsp(DET-SUC/DBCO)

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RESULTS AND DISCUSSION Synthesis and Characterization of Acidic pH-Responsive and Unresponsive Polymers. To construct the smart polymeric shell, an acidic pH-responsive zwitterionic polypeptide, PAsp(DET-CDM/DBCO), was synthesized to consist of three functional parts: (i) polyaspartamide backbone bearing twice-repeated aminoethylene units (PAsp(DET)) for endosome disruption, (ii) carboxydimethyl maleate (CDM) moiety as a negatively charged acid-labile structure, and (iii) dibenzocyclooctyne (DBCO) moiety for click conjugation to micellar E

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Figure 3. Quantitative analysis of exposed primary amines of PAsp(DET-CDM/DBCO) (A) and PAsp(DET-SUC/DBCO) (B) at pH 7.4 (open circle) and at pH 6.7 (closed circle), determined by a colorimetric assay using TNBS.

cells for RNAi cancer therapy.29 The CDM-modified micelles (CDM-micelles) prepared at DBCO/N3 = 0.8 and 1.5 induced modest sequence-specific gene silencing, yet comparable to the nonmodified micelles. In contrast, the CDM-micelles prepared at DBCO/N3 = 3 and higher significantly improved the gene silencing efficiency compared with those at the lower mixing ratios, indicating that the surface modification with a certain amount of PAsp(DET-CDM/DBCO) should be required for the enhanced gene silencing activity of PIC micelles. Also, no further increase in the gene silencing efficiency was observed beyond DBCO/N3 of 3, possibly due to the saturation in surface modification with PAsp(DET-CDM/DBCO), as suggested by the ζ-potential change (Figure 4C). On the basis of these results, the CDM-micelles prepared at DBCO/N3 = 3, including the smallest amount of PAsp(DET-CDM/DBCO) for the significantly enhanced gene silencing, was selected for further investigations described later. The acidic pH-responsiveness of CDM-micelles was then evaluated in terms of the change in ζ-potential because the ζ-potential of CDM-micelles was assumed to increase with the degradation of anionic CDM moieties at pH 6.7 and the facilitated amine protonation in side chains of PAsp(DET) at pH 5.0 as illustrated in Figure 1B. Figure 5A displays the positive shift in ζ-potential of CDM-micelles after 1 h of incubation under the acidic conditions. Notably, considerably higher ζ-potential was observed at pH 5.0, probably due to both the degradation of CDM moieties and the facilitated amine protonation. These results demonstrate the two-step acidic pH responsiveness of CDM-micelles. Next, stability of CDM-micelles was investigated in the presence of FBS, as it is required for effective siRNA delivery in vitro as well as in vivo. Herein, the diffusion coefficient of Cy3-labeled Chol-siRNA (Cy3/Chol-siRNA)-loaded PIC micelles was determined as an indicator for size change in 50% FBS-containing medium by FCS. Nonmodified micelles showed a negligible change in diffusion coefficient over the incubation period (Figure 5B), indicating that the initial micelle structure was maintained without aggregation and dissociation (or siRNA release), even in the presence of 50% FBS. This structural durability was presumably due to the stabilized PIC core through hydrophobic association of Chol moieties and disulfide crosslinking, associated with sterically stabilized PEG shell. Similarly, the CDM-micelles also maintained their initial diffusion coefficients in the serum-containing medium, suggesting that the zwitterionic polypeptide coverage did not compromise the colloidal stability of PEG shell. It should be also noted that the nonmodified and CDM-micelles maintained the similar diffusion

exhibited almost no increase in the amount of exposed amines regardless of pH (Figure 3B), confirming its pH-unresponsive nature as a control polypeptide. PIC Micelle Formation, Modification, And Characterizations. For preparation of N3-functionalized disulfide crosslinked PIC micelles, N3-PEG-b-PLys(MPA) was synthesized using N3-PEG-NH2 (Mw = 12 000), as previously described.11 A narrow molecular weight distribution (Mw/Mn = 1.08) was confirmed for the parent N3-PEG-b-PLys by size exclusion chromatography (data not shown). Also, the DP of PLys segment was determined to be 45 from the 1H NMR spectrum of N3-PEG-b-PLys (data not shown). The quantitative introduction of MPA moiety into the side chains of PLys segment was confirmed from 1H NMR spectrum of N3-PEG-b-PLys(MPA) (Figure S2). Then, the PIC micelles were prepared from N3-PEG-b-PLys(MPA) and Chol-siRNA at [amidine in N3PEG-b-PLys(MPA)]/[phosphate in Chol-siRNA] = 1.4, according to the previous optimization.11 The micelle formation was confirmed from the size of ∼40 nm with a narrow distribution (polydispersity index (PDI) < 0.1), associated with a slightly positive ζ-potential of 4 mV. The prepared PIC micelle was subjected to the surface modification with PAsp(DET-CDM/DBCO) through copper-free click conjugation between DBCO and N3 moieties, generating a stable covalent bond between PAsp(DET-CDM/DBCO) and PEG shell. To optimize the surface modification rate, we mixed PAsp(DET-CDM/DBCO) with the micelle solution at different mixing ratios. Regardless of the mixing ratio, the size and PDI of PIC micelles were kept almost constant (Figure 4A), indicating that the original micelle structure should be maintained after surface modification. As shown in Figure 4B, the narrowly dispersed nature of PIC micelles was confirmed at a residual molar ratio of DBCO/N3 = 3 (or a polymer molar ratio of PAsp(DET-CDM/ DBCO)/N3-PEG-b-PLys(MPA) = 0.6) in the DLS histogram. On the contrary, the gradual decrease in ζ-potential was clearly observed with an increase in the mixing ratio (Figure 4C), consistent with the coverage of micellar surface with the negatively charged PAsp(DET-CDM/DBCO). Also, the decrease in ζ-potential apparently leveled off between 3 and 6 in DBCO/N3 ratio (or 0.6 and 1.2 in PAsp(DET-CDM/DBCO)/N3-PEG-b-PLys(MPA)), suggesting saturation of the conjugation sites. Then, the effect of the surface modification rate was validated by in vitro gene silencing assay using a human lung carcinoma cell line, A549 (Figure 4D). In this assay, polo-like kinase 1 (PLK1) responsible for the cell cycle regulation was selected as a target gene because PLK1 silencing can induce the apoptosis of cancer F

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Figure 4. Surface modification of PIC micelles with PAsp(DET-CDM/DBCO) at various mixing (DBCO/N3) ratios. (A) Size (closed circle) and PDI (closed square), (B) DLS histogram of CDM-micelles prepared at DBCO/N3 = 3, (C) ζ-potential, and (D) gene silencing efficiency of PIC micelles incorporating siPLK1 (closed circle) or siCont (open circle) against cultured A549 cells incubated at pH 7.4. Data represent the means ± SEM (n = 4).

Figure 5. (A) ζ-potential histograms of CDM-micelles after 1 h of incubation at pH 7.4, 6.7, and 5.0. Micelle samples prepared in 10 mM HEPES buffer (pH 7.4) were ultrafiltrated using Vivaspin columns (MWCO: 30 000), followed by dilution with 10 mM HEPES buffer (pH 7.4), 10 mM MES buffer (pH 6.7), or 10 mM MES buffer (pH 5.0) for pH adjustment (final siRNA concentration: 3.5 μM). (B) Stability of nonmodified micelles (open diamond) and CDM-micelles (open square) in 50% FBS-containing HEPES buffer (pH 7.4), determined by FCS (siRNA concentration = 100 nM).

(DTT), which, respectively, mimics extracellular or intracellular reductive conditions.30 The obtained result (Figure S4) reveals the facilitated siRNA release from all micelle formulations (nonmodified micelles, CDM-micelles, and PAsp(DET-SUC/ DBCO)-modified micelles (SUC-micelles)) under the stronger reductive condition. Thus, it is demonstrated that the reductive environment-responsiveness of disulfide cross-linked micelles can be maintained after the modification with PAsp(DETCDM/DBCO). In Vitro siRNA Delivery. The cellular uptake of PIC micelles was assumed to accelerate by the acid hydrolysis of CDM

coefficients even in the presence of 90% FBS (Figure S3), suggesting their excellent structural durability in proteinous in vivo milieu. Meanwhile, siRNA delivery carriers need to ultimately release the payloads in the cytoplasm for effective gene silencing. In this regard, the disulfide cross-linking in the micellar core was expected to be cleaved in the cytoplasmic reductive environment, directed toward the preferential siRNA release (Figure 1B).11 Accordingly, the siRNA release profile was further examined by agarose gel electrophoresis after mixing each micelle sample with a strong polyanion, dextran sulfate, in the presence of 10 μM or 10 mM concentration of dithiothreitol G

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Figure 6. (A) Cellular uptake efficiency of Cy3/Chol-siRNA-loaded micelles in cultured A549 cells (siRNA concentration = 200 nM, incubation time = 3 h). Data represent the means ± SEM (n = 4). (B) Colocalization ratios of Cy3/Chol-siRNA with LysoTracker-stained late endosome/lysosome in cultured A549 cells (siRNA concentration = 200 nM). Nonmodified micelles (open diamond), CDM-micelles (open square), and SUC-micelles (open circle). Data represent the means ± SEM obtained from 15 cells. *: p < 0.05, **: p < 0.005.

the cells treated with CDM-micelles. Thus, the colocalization of Cy3/Chol-siRNA with late endosome/lysosome was quantitatively analyzed by pixel counting in the CLSM images, as described in the Methods section. Figure 6B displays that the colocalization ratio of CDM-micelles was significantly lower than those of the other control micelles, nonmodified micelles, and SUC-micelles after 6 and 12 h incubation, consistent with the facilitated endosomal escape of CDM-micelles. Thus, the smart polymeric shell comprising PAsp(DET-CDM/DBCO) was demonstrated to accelerate the cellular internalization of PIC micelles preferably at slightly acidic pH of 6.7 and, once internalized by cancer cells, further facilitate the endosomal escape of siRNA payloads to the cytosol. Because the surface modification with PAsp(DET-CDM/ DBCO) successfully enhanced cellular internalization at slightly acidic pH and endosomal escape of PIC micelles, the effect of medium pH was ultimately investigated for the gene silencing activity of CDM-micelles in cultured A549 cells. To evaluate the PLK1 silencing ability by qRT-PCR, we treated A549 cells with the PIC micelles loading Chol-siPLK1 or Chol-siCont at neutral pH 7.4 and acidic pH 6.7 for 48 h. All Chol-siPLK1-loaded micelles significantly decreased the PLK1 mRNA level compared with Chol-siCont-loaded micelles (Figure 7), indicating their sequence-specific gene silencing activity. It is worth mentioning that the CDM-micelles elicited the greatest gene-silencing efficiency among the tested micelles at pH 7.4, and the genesilencing efficiency of CDM-micelles was further enhanced at pH 6.7. This enhanced gene-silencing activity of CDM-micelles is nicely correlated with their efficient cellular internalization and endosomal escape observed in the cells incubated at pH 6.7 (Figure 6). Additionally, the significant role of covalent conjugation between PAsp(DET-CDM/DBCO) and N3functionalized micelles was validated for the gene-silencing ability. In this experiment, the PIC micelle was prepared from methoxy-terminated PEG-b-PLys(MPA), mixed with PAsp(DET-CDM/DBCO), and compared with the CDM-micelle prepared from azide-terminated PEG-b-PLys(MPA). The genesilencing efficiency of the former micelle was significantly lower than that of the latter CDM-micelle but similar to that of the nonmodified micelle (Figure S5). This result indicates that the covalent conjugation between PAsp(DET-CDM/DBCO) and PEG shell should be crucial for the enhanced gene silencing ability of CDM-micelles. Of note, all tested PIC micelles loading Chol-siCont showed no significant cytotoxicity under the same transfection condition (Figure S6).

moieties in PAsp(DET-CDM/DBCO) conjugated on the PEG shell. This is because the exposed, protonated amines were expected to electrostatically bind to negatively charged cytoplasmic membrane (or anionic glycosaminoglycans on the membrane),31 thereby facilitating the adsorptive endocytosis of PIC micelles (Figure 1B). To validate this assumption, we performed the cellular uptake study at neutral pH of 7.4 and slightly acidic pH of 6.7 by quantifying the fluorescence intensity derived from Cy3/CholsiRNA in cultured A549 cells. The cells were treated with Cy3/ Chol-siRNA-loaded micelles for 3 h prior to the observation. As shown in Figure 6A, the CDM-micelles showed a 2-fold increase in fluorescence intensity under the acidic condition, compared with the neutral condition, demonstrating the acidic pH-accelerated cellular uptake of CDM-micelles. In contrast, the nonmodified and SUC-micelles as pH-unresponsive controls did not elicit such an increase in fluorescence intensity at acidic pH. Thus, the crucial role of acid hydrolyzable CDM moiety was confirmed for the accelerated cellular uptake of CDM-micelles. After endocytosis, nanoparticles, including PIC micelles, are generally entrapped within the endosomal compartment, followed by enzymatic degradation in late endosome/lysosome. To bypass this undesired digestion of siRNA, the acid-labile CDM moiety was installed into the endosome-disrupting polyaspartamide derivative, PAsp(DET). Note that our previous studies have demonstrated that diprotonated diaminoethane units in PAsp(DET) can elicit the endosomal pH-triggered membrane destabilization through the direct interaction with the membrane, facilitating the endosomal escape of nucleic acid payloads.6,24 Thus, the endosome-escapability of CDM-micelles was verified here by CLSM observation of their intracellular distribution. A549 cells were incubated with Cy3/Chol-siRNA-loaded micelles at pH 6.7 for 2 h, followed by further incubation with fresh medium (without micelles) at pH 6.7 for 3, 6, and 12 h. The yellow spots that come from the colocalization of Cy3/CholsiRNA (red) with LysoTracker-stained late endosome/lysosome (green) were clearly observed in the representative CLSM images (Figure S4). In particular, more yellow spots as well as red spots were observed for CDM-micelles (Figure S4B) compared with nonmodified (Figure S4A) and SUC-micelles (Figure S4C), consistent with the enhanced cellular internalization of CDM-micelles under the acidic condition. These results indicate that considerable amounts of siRNA (or micelles) were entrapped in late endosome/lysosome yet associated with a portion of siRNA translocated to the cytoplasm, especially in H

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Figure 7. Gene-silencing ability of nonmodified micelles, CDM-micelles, and SUC-micelles in A549 cells cultured at pH 7.4 and 6.7 for 48 h (siRNA concentration = 200 nM). Data represent the means ± SEM (n = 4). **: p < 0.01.

Recently, several clinical studies suggest the potential safety concerns in RNAi therapeutics because adverse events including fatigue, chills, fever, hypersensitivity, and infusion reactions are noted, possibly derived from the delivery formulations.32,33 In addition, the accumulative toxicity of delivery vehicles is also a potential issue particularly in repetitive administration.33 With regard to these concerns, the present formulation of CDM-micelle may be tolerable because its main polymeric components, PAsp(DET-CDM) and PLys, are biodegradable poly(amino acid)s and also PEG is known to be one of the most biologically inert polymers. It should be noted that the parent structure of PAsp(DET-CDM) surrounding the micellar surface is confirmed to elicit minimal inflammatory cytokine induction to a level comparable to that treated with normal saline, probably due to the self-catalytic rapid degradation as well as the distinctive monoprotonated structure in the side chain at neutral pH.6,34

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CONCLUSIONS In this work, the smart polymeric shell was developed for facilitating the cellular uptake and endosomal escape of siRNAloaded PIC micelles in cancer cells under the condition mimicking the lowered-pH environment of tumor tissues. This smart shell was engineered by covalently conjugating the acidic pH-labile, endosome-disrupting polypeptide PAsp(DET-CDM/ DBCO) onto the disulfide cross-linked/Chol-siRNA-loaded PIC micelles. The successful conjugation of PAsp(DET-CDM/ DBCO) was indicated by the decreased ζ-potential and enhanced gene silencing activity of PIC micelles. The resulting CDM-micelles showed the acidic pH-accelerated cellular uptake of siRNA payloads as well as the facilitated endosomal escape, eliciting the strong gene-silencing in the cancer cells cultured at pH 6.7. These results demonstrate that the smart shell structure should be a promising strategy to overcome the PEG dilemma, directed toward cancer-targeted siRNA delivery.

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spectrum of N3-PEG-b-PLys(MPA) in D2O at 25 °C. Figure S3. Stability of non-modified micelles and CDMmicelles in 90% FBS-containing HEPES buffer, determined by FCS. Figure S4. siRNA release profiles of nonmodified micelle, CDM-micelle, and SUC-micelle under two varying reductive conditions. Figure S5. CLSM images of A549 cells after 12 h incubation with nonmodified micelles, CDMmicelles, and SUC-micelles at pH 6.7. Figure S6. Gene silencing efficiencies of nonmodified micelles prepared from N3-PEG-b-PLys(MPA) without PAsp(DET-CDM/DBCO), CDM-micelles prepared from N3-PEG-b-PLys(MPA) with PAsp(DET-CDM/DBCO), and MeO-micelles prepared from MeO-PEG-b- PLys(MPA) with PAsp(DET-CDM/ DBCO) in A549 cells cultured at pH 6.7. Figure S7. Viability of A549 cells after 48 h incubation with nonmodified micelles, CDM-micelles and SUC-micelles at pH 7.4. (PDF)

AUTHOR INFORMATION

Corresponding Authors

*K.M.: E-mail: [email protected]. *K.K.: E-mail: [email protected]. Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was financially supported by the Funding Program for World-Leading Innovative R&D in Science and Technology (FIRST, JSPS), Grants-in-Aid for Scientific Research of MEXT (JSPS KAKENHI Grant Numbers 25000006 and 25282141), the Center of Innovation (COI) Program (JST), and the Mochida Memorial Foundation for Medical and Pharmaceutical Research.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.biomac.5b01334. Scheme 1. Synthesis procedure of PAsp(DET-CDM/ DBCO). Figure S1. 1H NMR spectrum of PAsp(DETSUC/DBCO) in D2O at 70 °C. Figure S2. 1H NMR

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