Elaboration on the Distribution of Hydrophobic Segments in the

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Elaboration on the distribution of hydrophobic segments in the chains of amphiphilic cationic polymers for siRNA delivery Changrong Wang, Lili Du, Junhui Zhou, Lingwei Meng, Qiang Cheng, Chun Wang, Xiaoxia Wang, Deyao Zhao, Yuanyu Huang, Shuquan Zheng, Huiqing Cao, Jianhua Zhang, Liandong Deng, Zicai Liang, and Anjie Dong ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b07337 • Publication Date (Web): 01 Sep 2017 Downloaded from http://pubs.acs.org on September 1, 2017

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ACS Applied Materials & Interfaces

Elaboration on the Distribution of Hydrophobic Segments in the Chains of Amphiphilic Cationic Polymers for siRNA Delivery Changrong Wang a, c, 1, Lili Du b, 1, Junhui Zhou a, Lingwei Meng b, e, Qiang Cheng b, Chun Wang a

, Xiaoxia Wang b, Deyao Zhao b, Yuanyu Huang b,d , Shuquan Zheng b, Huiqing Cao b, Jianhua

Zhang a, Liandong Deng a, Zicai Liang b, c* , Anjie Dong a, c,* a

Department of Polymer Science and Technology, School of Chemical Engineering and

Technology, Key Laboratory of Systems Bioengineering (Ministry of Education), Tianjin University, Tianjin 300072, China b

Laboratory of Nucleic Acid Technology, Institute of Molecular Medicine, Peking University,

Beijing 100871, China c

Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Tianjin

300072, China d

Advanced Research Institute for Multidisciplinary Science, Beijing Institute of Technology,

Beijing 100081, China e

Peking-Tsinghua Center for Life Science, Academy for Advanced Interdisciplinary Studies,

Peking University, Beijing 100871, China

ACS Paragon Plus Environment

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KEYWORD: siRNA delivery; delivery efficiency; amphiphilic cationic polymers; distribution of the hydrophobic segments; gene silence. ABSTRACT

Hydrophobization of cationic polymers, as an efficient strategy, had been widely developed in structure of cationic polymeric micelles to improve the delivery efficiency of nucleic acids. However, the distribution of hydrophobic segments in the polymer chains is rarely considered. Here, we elaborated three types of hydrophobized PEG-blocked cationic polymers with different distribution of the hydrophobic segments in the polymer chains, respectively, PEG-PAM-PDP (E-A-D), PEG-PDP-PAM (E-D-A), and PEG-P(AM/DP) (E-(A/D)), which were synthesized by RAFT (reversible addition-fragmentation chain transfer) polymerization

of methoxy

polyethylene glycol (PEG), cationic monomer aminoethyl methacrylate (AM) and pH-sensitive hydrophobic monomer 2-diisopropylaminoethyl methacrylate (DP). In aqueous solution, all of the three copolymers, E-A-D, E-D-A and E-(A/D), were able to spontaneously form nano-sized micelles (100~150nm) (ME-A-D, ME-D-A and ME-(A/D)), and well incorporated siRNA into complex micelles (CMs). The effect of distributions of the hydrophobic segments on siRNA delivery had been evaluated in vitro and in vivo. Compared with ME-D-A and ME-(A/D), ME-A-D showed best siRNA binding capacity to form stable ME-A-D/siRNA CMs less than 100 nm, mediated best gene silence efficiency and inhibition effect of tumor cell growth in vitro, and showed better liver gene silencing effect in vivo. In the case of ME-(A/D) with a random distribution of cationic and hydrophobic segment, higher gene silence efficiency than Lipo2000 but less than ME-A-D and MED-A

was got. As the mole ratio of positive and negative charges increasing, ME-D-A/siRNA and

ME-A-D/siRNA presented similar performances in size, zeta potential, cell uptake and gene silence, but ME-(A/D)/siRNA showed reversed performances. In addition, ME-A-D as the best


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siRNA carrier was evaluated in tumor tissue in the xenograft murine model, and showed good anticancer capacity. Obviously, the distribution of the hydrophobic segments in the amphiphilic cationic polymer chains should be seriously considered in the design of siRNA vectors.

INTRODUCTION

RNAi technology has captured broad attention among industrial and academic investigators for its potential to treat an incalculable of diseases.1-2 Small interfering RNA (siRNA) introduces exogenous synthetic molecules for biomedical research.3 As a hopeful candidate for the treatment of multitudinous diseases (e.g., cancer, neurodegenerative disorders and infectious diseases), siRNA has been promptly developed.4-8 However, because of its negative charge, naked siRNA is vulnerable to degradation by nucleases in blood serum and incapable to cross the cell membrane.9-10 Therefore, viral and non-viral carriers are broadly employed for systematic gene delivery. Viral gene carriers have been criticized for their potential risk of immunogenicity and toxicity, which limited their clinical usage.11 Non-viral systems may be safer and offer more biocompatibility, which indicates great potential in systematic gene delivery. Primarily due to its inoperative delivery mechanisms, favorable clinical outcomes are still a long way off.11-12 Among the non-viral systems, cationic polymers have become one of the most broadly accepted tactics for siRNA delivery due to their limited immunogenicity, biocompatibility, and excellent ability to condense siRNA to nanosized polyion complex (PIC) micelle for protecting siRNA payloads from enzymatic degradations and delivering siRNA into cells, and the flexible potential for structure modification13-16, such as polyamidoamine (PAMAM) dendrimer17-18, polyethylenimine (PEI)19, poly (L-lysine) (PLL)20, chitosan21, poly (2-dimethylaminoethyl methacrylate) (PDMAEMA)22-25 and 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP)26


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and their derivatives. To improve the stability and biocompatibility, and to prolong the circulation of PIC micelles in blood, A-B type block copolymer with polyethylene glycol (PEG) (A block) and polycations (B block) had been widely accepted as an indispensable delivery platform in construction of siRNA vectors.27-28 However, several disadvantages were induced by the PEGylation, called ‘PEG dilemma’ phenomenon, such as hampering the electrostatic interaction between cationic polymer and siRNA, inhibiting the uptake by cells and efficient escape from early endosome, which leaded to the low transfection efficacy.25,

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In addition,

although with the protection of PEG shell, the stability of the electrostatic complexes between siRNA and polycationic segments are inevitably interfered by the counterpart negative charged biomacromolecules in the biological conditions. So far, several approaches for overcoming the ‘PEG dilemma’ and further stabilizing the PIC micelles of siRNA/PEG-blocked polycation have been explored, such as introducing tumorspecific ligands30, incorporate sheddable PEG29, integrating disulfide crosslinking31 and hydrophobization by randomly integrating hydrophobic moieties (C) into the polycationic segments to construct A-(B/C) type copolymer32, or by introducing hydrophobic C block to construct A-B-C and A-C-B types of triblock copolymers. A lot of studies found that those A-BC, A-C-B or A-(B/C) types of hydrophobized PEG-blocked polycations not only stabilized siRNA loaded PIC micelles but also improved cell uptake, resulting highly improvement in gene silence efficiency.33-43 Particularly, the advanced hydrophobization strategy recently focused on introduction of the pH-sensitive hydrophobic moieties to enhance endosome escape and cytosolic siRNA release via the disassembly of the PIC micelles triggered by the hydrophilization of ultra pH-sensitive hydrophobic inner core in acidic endosome condition.38-39, 44

Different pH-sensitive hydrophobic moieties had been installed into A-B-C35,

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, A-C-B41


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triblock copolymers, and randomly hydrophobized diblock copolymers A-(B/C)38-39, and were certificated to result in considerable gene delivery efficiency. Our previous work, by randomly installing a certain ratio of D, L-lactide (DLLA) moieties into the hydrophobic polycaprolactone (PCL) segments of PEG-b-PCL-g-PDMAEMA (mPEGblock-PCL-graft-poly(dimethylamino ethyl methacrylate)), best gene silencing effect was obtained but with the lowest cellular uptake capacity in vitro.45 In our other work, we found the triblock copolymer micelle of PG-PDPA-PCB was inability in siRNA delivery on cell lines, which constructed with poly(carboxybetaine) (PCB) as the antifouling hydrophilic segments, guanidinated poly(aminoethyl methacrylate) (PG) as a polycation and the pH-sensitive hydrophobic poly(diisopropylethyl methacrylate) (PDPA) as the core-formed segments. While, ultra-strong ability in siRNA delivery was obtained even on the hard-to-transfect human acute monoblastic leukemia cell line U937 (more than 98% gene silence efficiency) just by randomly copolymerized some DMAEMA moieties into the core segments, which were able to adjust the initial disassembly pH of the core near to 6.8 and improve the cytosolic siRNA release.46 These results indicated the component of the hydrophobic segments as the inner core was a key issue for the siRNA delivery efficiency, which, beside the stabilization function for PIC micelles, also affected the cell uptake, endosome escape and cytosolic siRNA release behaviors. Then, what’s the effect of the distribution of hydrophobic segments in the chains of amphiphilic cationic polymers on siRNA delivery, i.e. what is the difference for A-B-C, A-C-B and A-(B/C) in siRNA delivery potential? Rarely information about this is known.33 Herein, our interest focused on the effect of the distribution of hydrophobic segments in the chains of amphiphilic cationic polymers on siRNA delivery. To elaborate this issue, we prepared A-B-C, A-C-B and A-(B/C) types of amphiphilic block polycations with similar compositions


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but different distribution of hydrophobic moieties in the chains, respectively PEG-PAM-PDP (EA-D), PEG-PDP-PAM (E-D-A) and PE-P(AM/DP) (E-(A/D)). As shown in Scheme 1, three copolymers all could self-assembly into micelles with PEG (as the stable shell), aminoethyl methacrylate (AM) (as the cationic moieties to binding siRNA), and 2-diisopropylaminoethyl methacrylate (DP) (as the pH-sensitive hydrophobic core moieties). The physicochemical properties (including siRNA binding ability, particle size distribution and zeta potential, and the stability of the siRNA-loaded micelles) were characterized. Also, the cell toxicity, intracellular uptake, endosome escape ability, and gene silencing efficacy were evaluated in vitro and in vivo to reveal the structure-function relationship of the distribution of hydrophobic segments in the amphiphilic cationic polymer micelles. The results of this study may provide significant suggestion in designing cationic polymer vector of siRNA. EXPERIMENTAL SECTION Materials Methoxy poly(ethylene glycol) (mPEG2000, purified in diethyl ether), 2-(Diisopropyl amino) ethyl methacrylate (DPA-MA), azobisisobutyronitrile (AIBN), di-tert-butyl dicarbonate (Boc), 2aminoethanol, methacryloylchloride, ethidium bromide, dimethyl sulfoxide (DMSO), Trizol Reagent, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) , and RNAlater were bought from Sigma-Aldrich. Lipofectamine 2000, Dulbecco’s modified Eagles’s medium (DMEM), Opti-MEM, fetal bovine serum (FBS), penicilin-streptomycin and trypsin were purchased from Thermo Fisher. Agarose was got from GEN TECH (Hong Kong, China). All of the siRNA used in this paper, including Cy5-labeled siRNA (Cy5-siRNA), siFL, siApoB, siPLK1, and siRRM2, were provided by Suzhou Ribo Life Science Co., Ltd. (Jiangsu, China).


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The sequences were listed in Table S2. Antibody for GADPH was purchased from Zhongshan Goldenbridge Biotechnology Co. Ltd. (Beijing, China) and antibody for PLK1 was purchased from cell Signaling Technology, Inc. (Danvers, MA). Synthesis of N-(tert-butoxycarbonyl) aminoethyl methacrylate N-(tert-butoxycarbonyl) aminoethyl methacrylate was prepared according to the procedure reported previously.47 Methacryloyl chloride (6.7 mL, 68 mmol) was dropwise added to tertbutyl N-(2-hydroxyethyl) carbamate (10 g, 62 mmol) and Triethylamine (12.9 mL, 93 mmol) in CH2Cl2 (100 mL) under Ar, and cooled in an 0 ºC bath. One hour later, the mixture solution was removed to room temperature and reacted overnight. The CH2Cl2 solution was washed with double-distilled water, 10% HCl, 10% K2CO3, sat NaHCO3 and brine. Then the organic layer was dried and evaporated. At last, the solid product was purified by recrystallization from CH2Cl2/hexane. Synthesis of S-dodecyl-S'-(α, α'-dimethyl-α''-acetic acid) trithiocarbonate (DDMAT) and PEG-DDMAT The synthesis of RAFT chain transfer agent was done as previously described, and the DDMAT was subsequently conjugated to PEG.48-49 In brief, tricaprylylmethylammonium chloride (2.43 g, 0.006 mol), 1-Dodecanethiol (30.285 g, 0.15 mol) and acetone (72.15 g, 1.231 mol) were added into a three round bottom flask under a nitrogen atmosphere and cooled to 10 ºC with vigorous stirring. And 50% (12.57 g, 0.1575 mol) sodium hydroxide solution was dropwise added slowly. Carbon disulfide (11.4075 g, 0.15 mol) dissolved in acetone (15.135 g, 2.58 mol) was dropwise added over 20 min. After that, chloroform (26.73 g, 0.225 mol) was dropwise added in on portion, and then 50% (60 g, 0.75 mol) sodium hydroxide solution was


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added slowly and stirred overnight. 225 mL of double-distilled water and concentrated HCl (37.5 mL) were added separately, and evaporating off acetone. The solid was dispersed in 100 mL of 2-propanol. The undissolved solid was removed. The 2-propanol solvent was evaporated, and the crude product was recrystallized to obtain yellow crystalline solid. The mPEG-based macro-RAFT agent was synthesized by esterification reaction of mPEG2000 (10 g, 5 mmol) and DDMAT. Dicyclohexylcarbodimide (2.05 g, 10 mmol) was added to the dichloromethane (75 mL) solution of mPEG2000 (10 g, 5 mmol), DDMAT (3.65 g, 10 mmol), and DMAP (25 mg). After 48 h, the cyclohexyl urea was removed, and the mixture was concentrated and precipitated into diethyl ether. The precipitated mPEG-DDMAT was dried under vacuum. Polymer synthesis and characterization The two triblock copolymers E-A-D and E-D-A were synthesized via RAFT polymerization by sequential monomer addition method.46 The random diblock copolymer E-(A/D) was synthesized via RAFT by one-pot. The polymerization reaction using AIBN as the initiator was achieved at 70 ºC for 24 h with a molar ratio of 5:1 [macro-DDMAT]: [Initiator]. A monomer feed ratio of [AMA]:[DPA]:[macro-DDMAT] was 50:50:1 (mol %). The reactions were stopped and further dialyzed for 24 h, and lyophilized. After that, the next reaction was carried out. Deprotection of Boc-protected polymers: TFA was used as deprotection agent. In briefly, the Boc-protected polymer was deprotected in TFA, dialyzed for 24 h, and lyophilized. The characterization of polymers was performed by 1H NMR (Varian Unity-Plus INOVA 400). Preparation of copolymer micelles and siRNA loading


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The copolymer micelles (ME-A-D, ME-D-A and ME-(A/D)) were prepared and obtained by nanoprecipitation technology. In details, each copolymer (15 mg) was dissolved in TFE (trifluoroethanol, 1mL), and added dropwise into double-distilled water (5 mL) under stirring. Subsequently, the polymer solution was dialyzed in double-distilled water by regularly exchanging water for 24 h to completely remove the TFE. The final volume of polymer solution was adjusted to 15 mL with double-distilled water. Before complexing with siRNA, the concentration of amine groups in copolymer micelle solutions was adjusted to 1 mM. And then the copolymer micelles were added into equal volume of siRNA solution, incubated for 20 min at 25 ºC, and then used for the following experiments. Agarose gel electrophoresis assay siRNA loaded CMs were prepared similarly with previous work from our laboratory.46 Briefly, ME-A-D, ME-D-A and ME-(A/D) were mixed with 0.3 µg NC siRNA at different N/P ratios of 1, 2, 5, and 8 for 20 min at 25 ºC. After the volume was adjusted to 16 µL, 4 µL of 6× loading buffer was added into the siRNA loaded CMs, and then the mixtures was totally loaded onto 2% agarose gel with the concentration of ethidium bromide of 5 µg/mL. At last, electrophoresis was carried out in 1×TAE running buffer at 120 V for 20 min. Eventually, the results were detected and analyzed at UV light wavelength of 254 nm using image master VDS thermal imaging system (Bio-Rad, Hercules, CA). Physicochemical Properties Before using in transfection, the physicochemical properties of CMs were evaluated. Samples of siRNA loaded CMs were prepared by adding 4 µg of siRNA with ME-A-D, ME-D-A and ME-(A/D) at various N/P ratios of 5, 10, and 15. After adjusting the volume to 1 mL with DEPC water, the


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size distribution and zeta potential of CMs with and without siRNA were measured by dynamic laser scattering (DLS, Zetasizer Nano ZS, Malvern, UK). In vitro gene silencing measurement For luciferase assay, MDA-MB-231-luc cells were used and seeded in 24-well plates (5×104 cells each well). After 24 h, the incubation medium was removed and Opti-MEM (600 µL) containing 0.4 µg of siFL loaded CMs at N/P ratios of 5, 10 or 15 was added instead. After transfected for 4 h, the transfection mixture was replaced with fresh complete DMEM medium (1 mL per well), and further incubated to 20 h. Subsequently, cells were washed with 1×PBS for three times, and lysed for 30 min using 100 µL 1× passive lysis buffer (Promega Co., Madison, WI) with violently shaking to ensure complete lysis. After the cell lysate was centrifuged (30 s, 12,000 rpm), the relative luminescence units (RLUs) of the supernatant were measured by a fluorometer (Synergy HT, BioTek, USA). To detect the gene silencing efficiency in mRNA level by RT-PCR assay, MDA-MB-231 cells were plated in 6-well plates (2 × 105 cells each well). After 24 h, the cells were transfected with ME-A-D/siPLK1, ME-D-A/siPLK1, and ME-(A/D)/siPLK1. After 24 h of transfection, total RNA was extracted using TRIzol Reagent (Thermo Fisher) and converted to cDNA by reverse transcription. Then RT-PCR system was performed by using SYBR Green PCR Master Mix with GAPDH as the internal control. To confirm the gene silencing efficiency in protein level by western blot assay, 6-well plates were used to plate MDA-MB-231 cells (2 × 105 cells each well). 24 h later, cells were transfected with siRNA loaded ME-A-D, ME-D-A and ME-(A/D) CMs at different N/P ratios of 5, 10, and 15 with siRNA concentration of 50 nM. After transfected for 48 h, the total protein


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concentration was measured by BCA protein assay kit. Then 50 µg protein was loaded on the gel, separated by SDS-PAGE, transferred to PVDF membranes at 100 V for 1 h in transfer buffer, blocked with 5% BSA for 1 h, incubated with rabbit polyclonal PLK1 primary antibody (diluted 1:1000 in 5% BSA) overnight at 4 ºC, washed with 1×TBST, and finally cultivated with (HRP)labeled goat anti-rabbit secondary antibody (diluted 1:2000 in 5% BSA) for 1 h at 25 ºC. At last, the membranes were treated using an ECL kit and exposed using Bio-Rad UNIVERSAL HOOD II (Bio-Rad Laboratory, Bossier City, LA). Cell viability 96-well plates were used to seed MDA-MB-231 cells (1×104 cells per well) 24 h before transfection. Then cells were transfected with siRNA-loaded CMs (N/P=5, 10, and 15; siNC dose: 50 nM) for 1 day. After 1 day incubation, MTT solution (10 µL, 5 mg/mL in PBS) was added into each well with the final MTT concentration of 0.5 mg/mL, and incubated for 4 h. The medium was removed gently and replaced with DMSO (50 µL/well). After incubated for 30 min at 37 ºC to completely dissolve the formazan crystals, the absorbance at 540 nm was measured by Multi-Mode Microplate Reader (650 nm used as the reference wavelength). Untreated cells were marked as control (mock). Cell viability was calculated as follows: Cell viability (%) = ((OD540(sample) -OD650(sample))/(OD540(mock) -OD650(mock)))×100 Fluorescence-activated cell sorting (FACS) To study the cellular uptake behavior of micelles, MDA-MB-231 cells were plated in 6-well plates (2 × 105 cells each well). 24 h later, the cells were transfected with Cy5-siRNA-loaded ME-A-D, ME-D-A and ME-(A/D) CMs at different N/P ratios (Cy5-siRNA: 50 nM). After uptake for 4


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h, the cells were digested by trypsin, washed with 1×PBS, and subsequently suspended in 400 µL cold 1×PBS. At last, FACS Calibur flow cytometer (Becton Dickinson, San Jose, CA, USA) was used to detect the fluorescence signal. Confocal observation For studies of the subcellular localization of siRNA-loaded micelles, MDA-MB-231 cells were plated into 35 mm dishes (2×105 cells each well). 1 day later, cells were transfected with ME-A-D/Cy5-siRNA, ME-(A/D)/Cy5-siRNA, and ME-D-A/Cy5-siRNA (siRNA: 50 nM). After 4 h for transfection, cells were washed with 1×PBS (three times) and stained with 1 mL fresh DMEM containing 5 µL Hoechst 33342 (1 mg/mL, indicated nucleus) and 1 µL Lysotracker Green (1:1000, indicated endosome/lysosome). After stained for 30 min, cells were washed with 1×PBS (three times). At last, cells were imaged by Zeiss confocal microscope (LSM700, Carl Zeiss, Germany). In vivo distribution and gene silencing efficiency All involved animals in this work were maintained in Peking University Laboratory Animal Center. All procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of Peking University. Female C57BL/6 mice (18-20 g) were bought from VitalRiver Co (Beijing, China). To assess the in vivo distribution of siRNA-loaded micelles, Cy5-labled siRNA was used. Four groups were randomly divided, and treated with PBS, naked Cy5-siRNA, ME-A-D/Cy5-siRNA and ME-DA/Cy5-siRNA

by intravenous injection (Cy5-siRNA dose: 1 mg/kg). After 5 h and 24 h, main


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organs were harvested and imaged using Kodak in vivo imaging system (Kodak In-vivo Imaging System FX Pro, Carestream Health, USA). To assess in vivo gene silencing efficiency in liver tissue, female C57BL/6 mice were treated with siApoB loaded ME-A-D and ME-D-A CMs (N/P=10, siApoB dose: 1 mg/kg). After 48 h, blood sample was collected to detect the total CHO concentration in serum. Then mice were sacrificed and liver tissues were taken out. Liver ApoB mRNA level was detected by RT-PCR. In vivo tumor accumulation and tumor inhibition activity To evaluate in vivo tumor accumulation, xenograft murine tumor model was established on female BALB/c nude mice (16-18 g). In detail, MDA-MB-231 cells (5×106 cells) were suspended in 1×PBS (100 µL), and then subcutaneously injected in the right side of axillary fossa. When the tumor grew to about 300 mm3, the mice were randomly separated into three groups, and treated with 1×PBS, naked Cy5-siRNA and Cy5-siRNA loaded ME-A-D CMs by intravenously inject in (siRNA dose: 1 mg/kg). Cy5 signals of mice whole body was captured using animal imaging system among 24 h. Finally, mice were disposed to harvest the tumor, and Cy5 signals in tumor tissues were further captured. To evaluate in vivo tumor growth inhibition of ME-A-D formulation, tumor model were prepared as described above. When the tumor grew to about 40 mm3, tumor bearing mice were separated into four groups randomly, and treated with 1×PBS, naked siRRM2, ME-A-D/siNC (N/P=10) and ME-A-D/siRRM2 (N/P=10) by intravenous injection, respectively. Mice were injected every three days for 4 times at the siRRM2 dose of 1 mg/kg. During the entire experiment, tumor volume was recorded every day using caliper until day 12 and calculated


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according to the follows: tumor volume (mm3) = 1/2 × length × width2. To assess the biocompatibility and safety, body weight was monitored every day. At the end of experiment, RRM2 mRNA level in tumors were tested via RT-PCR and three kinds of serum cytokines (IL-2, IL-6 and TNF-α) were measured using Luminex Technology (4A Biotech Co., Ltd., Beijing, China). RESULTS AND DISCUSSION Synthesis and characterization of block copolymers and siRNA-loaded micelle The two triblock copolymers E-A-D and E-D-A were synthesized via RAFT polymerization (shown in Scheme S1) by sequential monomer addition method. As shown in Figure S1, the E-A and E-D were characterized by 1H NMR (Varian Unity-Plus INOVA 400). The diblock copolymer E-(A/D) with random hydrophobic moieties was synthesized via RAFT polymerization by one-pot (Scheme S1). The mPEG characteristic peaks at 3.37 ppm, PAM moieties at 8.34 ppm and PDP moieties at 1.29 ppm were displayed clearly in 1H NMR spectrum (Figure S1). According to the 1H NMR spectra, three copolymers with consistent composition were synthesized, respectively as PEG45-PAM46-PDP45 (E-A-D), PEG45-PDP45-PAM44 (E-D-A) and PEG45-P(AM42/DP42) (E-(A/D)). The mean number molecular weight of E-A-D, E-D-A and E-(A/D) were determined by 1H NMR and PDI was measured by GPC using PS as standards (Table S1). As shown in Figure 1, all the polymers could form nano-sized micelles. ME-A-D, ME-D-A showed the similar sizes (~120 nm) and zeta potentials (~10 mV), but ME-(A/D) showed larger size about 170 nm but lower zeta potential about 5 mV (Figure 1B and C). siRNA-loaded micelles, ME-A-D/siRNA, ME-D-A/siRNA and ME-(A/D)/siRNA, were prepared respectively by electrostatic


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interaction of ME-A-D, ME-D-A or ME-(A/D) with siRNA. Agarose gel electrophoresis was used to investigate the siRNA loading capacity. ME-A-D could completely bind siRNA at N/P = 2, but N/P = 5 was needed for ME-D-A or ME-(A/D) (Figure 1A), which meant ME-A-D presented the best ability in binding siRNA. To further characterize the interaction between the three amphiphilic polymer micelles and siRNA, the size distribution and zeta potential of siRNA-loaded CMs at various N/P ratios were measured. For the two triblock copolymer micelles ME-A-D and ME-D-A, when siRNA was bound on the micelles by electronic interaction with the cationic moieties, the micelles were nearly half condensed to about 60 nm with a decrease in zeta potential which increased again with N/P ratio going up (Figure 1B). Conversely, with siRNA binding, the size and zeta potential of ME-(A/D) increased at N/P=5, then both decreased in reverse proportion to N/P ratio. Furthermore, the stability of three cationic polymers after complexing with siRNA was also evaluated and the results indicated that ME-A-D/siRNA, ME-D-A/siRNA kept good stability in size with time, but growing trend and vibration was observed for ME-(A/D)/siRNA micelles (Figure S2). Altogether, the obtained results indicated the distribution of hydrophobic moieties in the polymer chains obviously affected the siRNA binding and the stability of the formed complex micelles because of the structural difference of the polymer micelles as our assumption in Scheme 1. It could be concluded that the triblock copolymer micelles, as ME-A-D and ME-D-A, featuring a tightly packed hydrophobic core were preferable chose, and especially, ME-A-D may be the best optimal structure for siRNA delivery, whose intermediate cationic layer with an outside PEG layer performed best siRNA binding capacity, supposed as the less disturbance of PEG chains on the interaction between cationic segments and siRNA. In vitro cellular siRNA delivery of siRNA-loaded micelles


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Luciferase assay was utilized in MDA-MB-231-Luc cells to assess the three cationic polymer micelles, and the results were shown in Figure 2A. It can be seen that all of the siRNA-loaded CMs efficiently mediated high luciferase gene silencing efficiency (>90%). Further, more than 92% cell viability was observed for all the siRNA-loaded micelles (Figure 2B), indicating the excellent safety and biocompatibility of the three polymers. We also evaluated the cytotoxicity of the polymers and found that all the polymers showed little cytotoxicity at concentration of 10 µg/mL, which was much higher than transfection concentration about 3 µg/mL (Figure S3). RT-PCR assay was performed using siPLK1, which was in consistent with the results of luciferase assay. The PLK1 knockdown results were shown in Figure 2C. The results confirmed that the three kinds of micelles showed considerable knockdown efficiency compared with Lipo 2000. Especially, the PLK1 knockdown efficiency of ME-A-D/siPLK1 and ME-D-A/siPLK1 CMs sensitively relied on the N/P ratio. Besides, ME-A-D/siPLK1 CMs reached the highest knockdown efficiency (85%) at N/P =15, far beyond Lipo 2000 (54%), ME-D-A (70%) and ME-(A/D) (64%). Western blot was applied in MDA-MB-231 cells to evaluate the gene knockdown efficiency on PLK1 protein level (Figure 2D). Western blot data also showed that the three CMs had high gene-silencing efficacy in protein level. Similar to PLK1 mRNA expression level, the PLK1 knockdown efficiency of ME-A-D/siPLK1 in protein level was also associated with the N/P ratio, and reached the highest knockdown efficiency at N/P=15. FACS was used to evaluate the internalization of Cy5-siRNA-loaded micelles (N/P=10) after incubation for 4 h (Figure 3A.). The mean fluorescence intensities of Cy5-siRNA-loaded ME-A-D, ME-D-A and ME-(A/D) CMs at various N/P ratios were detected and analyzed for quantification (Figure 3B). Compared to naked Cy5-siRNA, ME-A-D, ME-D-A and ME-(A/D) showed extremely


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higher uptake efficiencies and showed different dependences on N/P ratio, which were related to the zeta potentials in Figure 1C, i.e. the higher zeta potential led to higher cell uptake. Among the three kinds of micelles, ME-A-D/siRNA presented the highest cellular uptake efficiency, and ME-(A/D)/siRNA showed the lowest value. After transfection, the subcellular localizations of siRNA-loaded CMs at various N/P ratios were analyzed by CLSM. Cy5 was used to mark siRNA, hoechst 33342 was used to mark nuclei, and lysotracker green was used to mark the endosome/lysosome to identify the localization of siRNA-loaded CMs. As shown in Figure 3C, it can be seen that all the three CMs showed visible fluorescence signal in the cytoplasm. Corresponding with Figure 3B, ME-A-D/siRNA CMs displayed stronger signal than ME-(A/D)/siRNA CMs and ME-D-A/siRNA CMs. The co-localization ratio of Cy5 siRNA and lysotracker green was calculated. As shown in Figure 3C, it could be observed the dependence of the co-localization ratio of red signals (Cy5-siRNA) in endosome on the N/P also related to the zeta potential, which indicated high zeta potential could improve the endosome escape. By comparison, ME-(A/D) presented best endosome escape ability among the three kinds of micelles but lowest cell uptake, which was supposed due to the looser structure of the core. The TEM also showed the ME-(A/D) had looser structure of the core (Figure S4). It is noteworthy that ME-(A/D)/siRNA at lower N/P ratio (N/P=5) behaved comparable gene silence efficiency with ME-A-D/siRNA and ME-D-A/siRNA at N/P=15. Unfortunately, serious hemolysis in phosphate buffered saline was observed for ME-(A/D), but not any for ME-A-D and ME-D-A (shown in Figure S5). Therefore, ME-(A/D) was not considered in the further evaluation below. In vitro tumor cell growth inhibition


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In vitro inhibition of ME-A-D /siRNA and ME-D-A /siRNA CMs (N/P = 10) on tumor growth were evaluated on MDA-MB-231 cell line. Ribonucleotide reductase M2 (RRM2), which acts a crucial role in DNA synthesis and repair, is an important target for tumor gene therapy.50 RRM2 overexpression acts a dynamic role in the cellular response to DNA damage and in tumor progression.51-52 Here, we used ME-A-D and ME-D-A as delivery system of RRM2 siRNA (siRRM2), which was determined to the reduction of the RRM2 expression. As shown in Figure 4A, the ME-A-D/siRRM2 CMs showed higher knockdown efficiency in RRM2 mRNA expression level than ME-D-A/siRRM2 CMs. It was consistent with the result of cell viability (Figure 4B). Colony formation assay indicated that both siRRM2-loaded micelles could efficiently inhibit the formation of tumor cell colony, but ME-A-D/siRRM2 showed better efficiency compared with MED-A/siRRM2

(Figure 4C). Then we evaluated the cell apoptosis efficiency of the ME-A-D/siRRM2

CMs and ME-D-A/siRRM2 CMs. After 48 h of transfection, both of the siRRM2-loaded CMs could significantly induce apoptosis (Figure 5). The apoptosis efficiency of ME-A-D/siRRM2 and ME-D-A/siRRM2 CMs were 49.2% and 59.6% respectively. This indicated that ME-A-D/siRRM2 CMs had better efficiency in inducing apoptosis compared with ME-D-A/siRRM2 CMs, which was consistent with the above results. In vivo distribution and in vivo gene silencing efficiency In vivo distribution analysis of siRNA was carried out through intravenous injection by using Cy5-siRNA. As shown in Figure 6A, Cy5 fluorescence signals became weaker from 5 h to 24 h both in siRNA-loaded micelle groups and naked siRNA group, and signals dominant accumulated in kidney. But stronger accumulation and more long-term release displayed for MEA-D/siRNA

and ME-D-A CMs in liver, spleen, and lung (Figure S6). Through liver cryosections, we

further detected siRNA subcellular location (Figure 6B), which showed that both ME-A-D/siRNA


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and ME-D-A/siRNA groups accumulated in liver cells after 5 h, but more fluorescence signals were found in ME-A-D/siRNA group after 24 h. siApoB was used to evaluate the ability of CMs to mediate gene silencing efficiency in vivo. ApoB acts as the scaffold in liver to solubilize fatty acids and cholesterol for secretion into the blood for circulation.53 Here, normal injection of PBS, naked siApoB, siNC-loaded micelles and two ME-A-D/siApoB and ME-D-A/siApoB CMs at N/P = 10 were administered by intravenous injection (siRNA dose : 1 mg/kg). Two days later, the ApoB mRNA expression level treated with ME-A-D/siApoB and ME-D-A/siApoB CMs largely reduced. And ME-A-D/siApoB showed higher knockdown efficiency than ME-D-A/siApoB (Figure 6C). Consistently, liver coefficient, CHO (cholesterol) in serum was elevated. It can be seen that CHO concentration in serum treated with ME-A-D/siApoB was lower than ME-D-A/siApoB (Figure 6D). In summary, in vivo biodistribution and functional study investigated that ME-A-D and ME-D-A, especially ME-A-D have potential applications in delivering siRNA to cure diseases. In vivo tumor accumulation and tumor inhibition activity Above results indicated that ME-A-D was the best choice for potential gene delivery carrier. So, the in vivo tumor accumulation and tumor inhibition activity were evaluated on ME-A-D. After i.v. injection of PBS, naked siRNA and ME-A-D/siRNA, fluorescence images of whole-body were collected at different time points (Figure 7A). Attributed to the long circulation time, the ME-AD/siRNA

CMs could be gradually eliminated from the blood and resulted in the accumulation in

the tumor tissues. 24 h later, mice were sacrificed, and the tumor tissues were taken out for quantification analysis (Figure 7B). The mice treated with ME-A-D/Cy5-siRNA group showed


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remarkably stronger Cy5 fluorescence signals in tumor tissues compared with naked Cy5-siRNA group (Figure 7C). And then the in vivo antitumor activity of ME-A-D/siRRM2 was further investigated. As shown in Figure 8, ME-A-D/siRRM2 CMs treated mice exhibited remarkably smaller tumor volume compared to the other group (Figure 8A). The degrees of limited inhibition between naked siRRM2 and ME-A-D/siNC had no difference due to rapid siRRM2 degradation and release of inefficient siNC. The mice body weight was recorded every day to evaluate the general toxicity (Figure 8B). The ME-A-D/siRRM2 CMs treated groups all maintained healthy body weight. The levels of relative RRM2 mRNA expression in tumor tissues were detected. The ME-A-D/siRRM2 CMs exhibited distinctly higher knockdown efficiency than the others (Figure 8C). The concentration of IL-2, IL-6 and TNF-α in blood serum showed no difference (Figure 8D-F), which illustrated that the ME-A-D/siRRM2 CMs had no immunotoxicity in vivo. CONCLUSION In this study, by using aminoethyl methacrylate (AM) as cationic moieties and

2-

diisopropylaminoethyl methacrylate (DP) as pH-sensitive hydrophobic moieties, we synthesized three kinds of hydrophobized PEG-blocked cationic copolymers, PEG-PAM-PDP (E-A-D), PEG-PDP-PAM (E-D-A), and PEG-P(AM/DP) (E-(A/D)), which have the similar composition but different distribution of hydrophobic segments in the polymer chains. The three copolymers all can self-assemble into micelles (ME-A-D, ME-D-A and ME-(A/D), respectively) but with different structures due to the different distribution of hydrophobic DP segments. Comparing with ME-D-A and ME-(A/D), ME-A-D showed superior abilities in binding siRNA, introducing cell internalization and endosome escaping in cells, mediating gene silencing in MDA-MB-231 cells in vitro, and


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more efficiency in delivering siRNA to liver in vivo. That is to say, the distribution of the hydrophobic moieties should be considered as a crucial issue in design of cationic polymer vectors. Besides, ME-(A/D) could effectively accumulate in tumor and inhibit tumor growth in vivo. Therefore we believe that E-A-D with the optimal segment distribution may be an efficient suggestion for developing new polycations for siRNA delivery, in vitro and in vivo. ASSOCIATED CONTENT Supporting Informaton Schemaic illustration of synthetic routes of E-A-D, E-D-A and E-(A/D). 1H NMR spectrum of E-A-D, E-D-A and E-(A/D). The stability of siRNA-loaded micelles at N/P=10. Cell cytotoxicity of nano-sized micelles.TEM images of ME-A-D, ME-D-A, ME-(A/D). Hemolysis assay of micelles in PBS at pH=7.4. Quantitative analysis of different organs in vivo. siRNA sequences. AUTHOR INFORMATION Corresponding Author *Zicai Liang; E-mail: [email protected] *Anjie Dong; E-mail: [email protected] Author Contributions Changrong Wang managed the design, synthesis, and characterization of three polymers, and was involved in manuscript preparation and revision. Lili Du managed to design and analyze the following biological comparision of three polymers, and was also involved in the manuscript editing and revision. Junhui Zhou was involved in the detection of chemical characterization of polymers by 1H NMR. Lingwei Meng was involved in the detection of physicochemical properties, RT-PCR assay, and in vivo experiments. Qiang Cheng was involved in the literature research, structure designation, flow cytometry assay, and manuscript preparation. Chun Wang was involved in the detection of TEM image and GPC. Xiaoxia Wang was involved in the


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literature research, cell apoptosis assay, and statistical analysis. Deyao Zhao was involved in the cell incubation and western blot assay. Yuanyu Huang was involved in luciferase assay and confocal laser scanning microscopy assay. Shuquan Zheng was involved in the in vivo distribution and gene silencing efficiency in liver tissue. Huiqing Cao was involved in the manuscript preparation, manuscript editing, and statistical analysis. Jianhua Zhang was involved in the design and characterization of polymers. Liandong Deng was involved in the synthesis process and characterization of the polymers. Zicai Liang managed the literature research, the designation, statistical analysis, manuscript editing, and manuscript revision. Anjie Dong managed the synthesis, manuscript preparation, manuscript editing, and manuscript revision. Notes The authors declare no competing financial interest. ACKNOWLEDGMENTS This work was supported by grants from the National Natural Science Foundation of China (No. 31671021, 81473128, and 81502586), the National Basic Research Program of the Chinese Ministry of Science and Technology (No. 2013CB531202), and the National Drug Program of China (No. 2012ZX09102301-006) . REFERENCES [1] Yin, H.; Kanasty, R. L.; Eltoukhy, A. A.; Vegas, A. J.; Dorkin, J. R.; Anderson, D. G. NonViral Vectors for Gene-Based Therapy. Nature Reviews Genetics. 2014, 15, 541-555. [2] Sun, W.; Ji, W.; Hall, J. M.; Hu, Q.; Wang, C.; Beisel, C. L.; Gu, Z. Self-Assembled DNA Nanoclews for the Efficient Delivery of CRISPR–Cas9 for Genome Editing. Angewandte Chemie. 2015, 127, 12197-12201. [3] Wilson, R. C.; Doudna, J. A. Molecular Mechanisms of RNA Interference. Annual review of biophysics. 2013, 42, 217-239.


22

Page 23 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


[4] Hannon, G. J.; Rossi, J. J. Unlocking the Potential of the Human Genome with RNA Interference. Nature. 2004, 431, 371-378. [5] Davis, M. E.; Zuckerman, J. E.; Choi, C. H. J.; Seligson, D.; Tolcher, A.; Alabi, C. A.; Yen, Y.; Heidel, J. D.; Ribas, A. Evidence of RNAi in Humans from Systemically Administered siRNA via Targeted Nanoparticles. Nature. 2010, 464, 1067-1070. [6] Kumar, P.; Ban, H. S.; Kim, S. S.; Wu, H.; Pearson, T.; Greiner, D. L.; Laouar, A.; Yao, J. H.; Haridas, V.; Habiro, K.; Yang, Y. G.; Jeong, J. H.; Lee, K. Y.; Kim, S. W.; Peipp, M.; Fey, G. H.; Manjunath, N.; Shultz, L. D.; T Cell-Specific siRNA Delivery Suppresses HIV-1 Infection in Humanized Mice. Cell. 2008, 134, 577-586. [7] Thi, E. P.; Mire, C. E.; Lee, A. C.; Geisbert, J. B.; Zhou, J. Z.; Agans, K. N.; Snead, N. M.; Deer, D. J.; Barnard, T. R.; Fenton, K. A.; MacLachlan, I.; Geisbert, T. W. Lipid Nanoparticle siRNA Treatment of Ebola Virus Makona Infected Nonhuman Primates. Nature. 2015, 521, 362365. [8] Raoul, C.; Abbas-Terki, T.; Bensadoun, J. C.; Guillot, S.; Haase, G.; Szulc, J.; Henderson, C. E.; Aebischer, P. Lentiviral-Mediated Silencing of SOD1 through RNA Interference Retards Disease Onset and Progression in A Mouse Model of ALS. Nature medicine. 2005, 11, 423-428. [9] Truong, N. P.; Gu, W.; Prasadam, I.; Jia, Z.; Crawford, R.; Xiao, Y.; Monteiro, M. J. An Influenza Virus-inspired Polymer System for the Timed Release of siRNA. Nature Communications. 2013, 4, 1-7. [10] Semple, S. C.; Akinc, A.; Chen, J.; Sandhu, A. P.; Mui, B. L.; Cho, C. K.; Sah, D. W. Y.; Stebbing, D.; Crosle, E. J.; Yaworski, E.; Hafez, I. M.; Dorkin, J. R.; Qin, J.; Lam, K.; Rajeev, K.


23


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 24 of 40

G.; Wong, K. F.; Jeffs, L. B.; Nechev, L.; Eisenhardt, M. L.; Weinstein, M. J.; Chen, Q. M.; Alvarez, R.; Barros, S. A.; De, S.; Klimuk, S. K.; Borland, T.; Kosovrasti, V.; Cantley, W. L.; Tam, Y. K.; Manoharan, M.; Ciufolini, M. A.; Tracy, M. A.; Fougerolles, A. D.; Maclachlan, L.; Cullis, P. R.; Madden, T. D.; Hafez, I. M. Rational Design of Cationic Lipids for siRNA Delivery. Nature biotechnology. 2010, 28, 172-176. [11] Mokhtarzadeh, A.; Alibakhshi, A.; Yaghoobi, H.; Hashemi, M.; Hejazi, M.; Ramezani, M. Recent Advances on Biocompatible and Biodegradable Nanoparticles as Gene Carriers. Expert opinion on biological therapy. 2016, 16, 771-785. [12] Luo, C.; Sun, J.; Sun, B.; He, Z. Prodrug-Based Nanoparticulate Drug Delivery Strategies for Cancer Therapy. Trends in pharmacological sciences. 2014, 35, 556-566. [13] Kozielski, K. L.; Tzeng, S. Y.; Hurtado De Mendoza, B. A.; Green, J. J. Bioreducible Cationic Polymer-Based Nanoparticles for Efficient and Environmentally Triggered Cytoplasmic siRNA Delivery to Primary Human Brain Cancer Cells. ACS nano. 2014, 8, 3232-3241. [14] Davis, M. E. The First Targeted Delivery of siRNA in Humans via A Self-Assembling, Cyclodextrin Polymer-Based Nanoparticle: from Concept to Clinic. Molecular pharmaceutics. 2009, 6, 659-668. [15] Kim, H. J.; Takemoto, H.; Yi, Y.; Zheng, M.; Maeda, Y.; Chaya, H.; Hayashi, K.; Mi, P.; Pittella, F.; Chrise, R. J.; Toh, K.; Matsumoto, Y.; Nishiyama, N.; Miyata, K.; Kataoka, K. Precise Engineering of siRNA Delivery Vehicles to Tumors Using Polyion Complexes and Gold Nanoparticles. ACS nano. 2014, 8, 8979-8991.


24

Page 25 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


[16] Lin, D.; Jiang, Q.; Cheng, Q.; Huang, Y.; Huang, P.; Han, S.; Guo, S.; Liang, Z.; Dong, A. Polycation-Detachable Nanoparticles Self-Assembled from mPEG-PCL-g-SS-PDMAEMA for in Vitro and in Vivo siRNA Delivery. Acta biomaterialia. 2013, 9, 7746-7757. [17] Liu, X.; Liu, C.; Laurini, E.; Posocco, P.; Pricl, S.; Qu, F.; Rocchi, P.; Peng, L. Efficient Delivery of Sticky siRNA and Potent Gene Silencing in A Prostate Cancer Model Using A Generation 5 Triethanolamine-Core PAMAM Dendrimer. Molecular pharmaceutics. 2012, 9, 470-481. [18] Yu, T.; Liu, X.; Bolcato-Bellemin, A. L.; Wang, Y.; Liu, C.; Erbacher, P.; Qu, F.; Rocchi, P.; Behr, J. P.; Peng, L. An Amphiphilic Dendrimer for Effective Delivery of Small Interfering RNA and Gene Silencing in Vitro and in Vivo. Angewandte Chemie. 2012, 124, 8606-8612. [19] Günther, M.; Lipka, J.; Malek, A.; Gutsch, D.; Kreyling, W.; Aigner, A. Polyethylenimines for RNAi-Mediated Gene Targeting in Vivo and siRNA Delivery to the Lung. European Journal of Pharmaceutics and Biopharmaceutics. 2011, 77, 438-449. [20] Kano, A.; Moriyama, K.; Yamano, T.; Nakamura, I.; Shimada, N.; Maruyama, A. Grafting of Poly (ethylene glycol) to Poly-Lysine Augments Its Lifetime in Blood Circulation and Accumulation in Tumors without Loss of the Ability to Associate with siRNA. Journal of controlled release. 2011, 149, 2-7. [21] Howard, K. A.; Paludan, S. R.; Behlke, M. A.; Besenbacher, F.; Deleuran, B.; Kjems, J. Chitosan/siRNA Nanoparticle–Mediated TNF-α Knockdown in Peritoneal Macrophages for Anti-Inflammatory Treatment in A Murine Arthritis Model. Molecular therapy. 2009, 17, 162168.


25


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 26 of 40

[22] Zhu, C.; Jung, S.; Luo, S.; Meng, F.; Zhu, X.; Park, T. G.; Zhong, Z. Co-Delivery of siRNA and Paclitaxel into Cancer Cells by Biodegradable Cationic Micelles based on PDMAEMA– PCL–PDMAEMA Triblock Copolymers. Biomaterials. 2010, 31, 2408-2416. [23] Guo, S.; Huang, Y.; Wei, T.; Zhang, W.; Wang, W.; Lin, D.; Zhang, X.; Kumar, A.; Du, Q.; Xing, J.; Deng, L.; Liang, Z.; Wang, P. C.; Dong, A.; Liang, X. Amphiphilic and Biodegradable methoxy Polyethylene glycol-Block-(Polycaprolactone-Graft-Poly (2-(dimethylamino) ethyl methacrylate)) as An Effective Gene Carrier. Biomaterials. 2011, 32, 879-889. [24] Guo, S.; Huang, Y.; Zhang, W.; Wang, W.; Wei, T.; Lin, D.; Xing, J.; Deng, L.; Du, Q.; Liang, Z.; Liang, X.; Dong, A. Ternary Complexes of Amphiphilic Polycaprolactone-Graft-Poly (N, N-dimethylaminoethyl methacrylate), DNA and Polyglutamic acid-Graft-Poly (ethylene glycol) for Gene Delivery. Biomaterials. 2011, 32, 4283-4292. [25] Huang, Y.; Lin, D.; Jiang, Q.; Zhang, W.; Guo, S.; Xiao, P.; Zheng, S.; Wang, X.; Chen, H.; Zhang H.; Deng, L.; Xing, J.; Du, Q.; Dong, A.; Liang, Z. Binary and Ternary Complexes Based on Polycaprolactone-Graft-Poly (N, N-dimethylaminoethyl methacrylate) for Targeted siRNA Delivery. Biomaterials. 2012, 33, 4653-4664. [26] Wilson, D. S.; Dalmasso, G.; Wang, L.; Sitaraman, S. V.; Merlin, D.; Murthy, N. Orally Delivered Thioketal Nanoparticles Loaded with TNF-α–siRNA Target Inflammation and Inhibit Gene Expression in the Intestines. Nature materials. 2010, 9, 923-928. [27] Guo, S.; Huang, L. Nanoparticles Escaping RES and Endosome: Challenges for siRNA Delivery for Cancer Therapy. Journal of Nanomaterials. 2011, 11, 1687-4110.


26

Page 27 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


[28] Liu, Y.; Xu, C. F.; Iqbal, S.; Yang, X. Z.; Wang, J. Responsive Nanocarriers as An Emerging Platform for Cascaded Delivery of Nucleic Acids to Cancer. Adv. Drug Deliv. Rev. 2017, http://dx.doi.org/10.1016/j.addr.2017.03004. [29] Li, S, D.; Huang, L. Stealth Nanoparticles: High Density but Sheddable PEG is A Key for Tumor Targeting. Journal of controlled release: official journal of the Controlled Release Society. 2010, 145, 178-181. [30] Benns, J. M.; Mahato, R. I.; Kim, S. W. Optimization of Factors Influencing the Transfection Efficiency of Folate–PEG–Folate-Graft-Polyethylenimine. Journal of controlled release. 2002, 79, 255-269. [31] Christie, R. J.; Matsumoto, Y.; Miyata, K.; Nomoto, T.; Fukushima, S.; Osada, K.; Halnaut, J.; Pittella, F.; Kim, H. J.; Nishiyama, N.; Kataoka, K. Targeted Polymeric Micelles for siRNA Treatment of Experimental Cancer by Intravenous Injection. Acs Nano. 2012, 6, 5174-5189. [32] Kim, H. J.; Ishii, T.; Zheng, M.; Watanabe, S.; Toh, K.; Matsumoto, Y.; Nishiyama, N.; Miyata, K.; Kataoka, K. Multifunctional Polyion Complex Micelle Featuring Enhanced Stability, Targetability, and Endosome Escapability for Systemic siRNA Delivery to Subcutaneous Model of Lung Cancer. Drug delivery and translational research. 2014, 4, 50-60. [33] Kim, H. J.; Miyata, K.; Nomoto, T.; Zheng, M.; Kim, A.; Liu, X.; Cabral, H.; Christie, R. J.; Nishiyama, N.; Kataoka, K. siRNA Delivery from Triblock Copolymer Micelles with Spatiallyordered Compartments of PEG Shell, siRNA-loaded Intermediate Layer, and Hydrophobic Core. Biomaterials. 2014, 35, 4548-4556.


27


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 28 of 40

[34] Sun, T. M.; Du, J. Z.; Yao, Y. D.; Mao, C. Q.; Dou, S.; Huang, S. Y.; Zhang, P.; Leong, K. M.; Song, E.; Wang, J. Simultaneous Delivery of siRNA and Paclitaxel via A “Two-in-One” Micelleplex Promotes Synergistic Tumor Suppression. ACS nano. 2011, 5, 1483-1494. [35] Qian, J.; Berkland, C. pH-Sensitive Triblock Copolymers for Efficient siRNA Encapsulation and Delivery. Polymer Chemistry. 2015, 6, 3472-3479. [36] Sun, T. M.; Du, J. Z.; Yan, L. F.; Mao, H. Q.; Wang, J. Self-Assembled Biodegradable Micellar Nanoparticles of Amphiphilic and Cationic Block Copolymer for siRNA Delivery. Biomaterials. 2008, 29, 4348-4355. [37] Endres, T. K.; Beck-Broichsitter, M.; Samsonova, O.; Renette, T.; Kissel, T. H. SelfAssembled Biodegradable Amphiphilic PEG–PCL–lPEI Triblock Copolymers at the Borderline between Micelles and Nanoparticles Designed for Drug and Gene Delivery. Biomaterials. 2011, 32, 7721-7731. [38] Xu, X.; Wu, J.; Liu, Y.; Saw, P. E.; Tao, W.; Yu, M.; Zope, H.; Si, M.; Victorious, A.; Rasmussen, J.; Ayyash, D.; Farokhzad, O. C.; Shi, J. Multifunctional Envelope-Type siRNA Delivery Nanoparticle Platform for Prostate Cancer Therapy. ACS nano. 2017, 11, 2618-2627. [39] Xu, X.; Wu, J.; Liu, Y.; Yu, M.; Zhao, L.; Zhu, X.; Bhasin, S.; Li, Q.; Ha, E.; Shi, J.; Farokhzad, O. C. Ultra-pH-Responsive and Tumor-Penetrating Nanoplatform for Targeted siRNA Delivery with Robust Anti-Cancer Efficacy. Angewandte Chemie International Edition. 2016, 55, 7091-7094.


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[40] Chen, W.; Yuan, Y.; Cheng, D.; Chen, J.; Wang, L.; Shuai, X. Co-Delivery of Doxorubicin and siRNA with Reduction and pH Dually Sensitive Nanocarrier for Synergistic Cancer Therapy. Small. 2014, 10, 2678-2687. [41] Blanazs, A.; Massignani, M.; Battaglia, G.; Armes, S. P.; Ryan, A. J. Tailoring Macromolecular Expression at Polymersome Surfaces. Advanced Functional Materials. 2009, 19, 2906-2914. [42] Gary, D. J.; Lee, H.; Sharma, R.; Lee, J. S.; Kim, Y.; Cui, Z. Y.; Jia, D.; Bowman, V. D.; Chipman, P. R.; Wan, L.; Zou, Y.; Mao, G.; Park, K.; Herbert, B.; Konieczny, S. F.; Won, Y. Y. Influence of Nano-Carrier Architecture on in Vitro siRNA Delivery Performance and in Vivo Biodistribution: Polyplexes vs Micelleplexes. ACS nano. 2011, 5, 3493-3505. [43] Nelson, C. E.; Kintzing, J. R.; Hanna, A.; Shannon, J. M.; Gupta, M. K.; Duvall, C. L. Balancing Cationic and Hydrophobic Content of PEGylated siRNA Polyplexes Enhances Endosome Escape, Stability, Blood Circulation Time, and Bioactivity in Vivo. ACS nano. 2013, 7, 8870-8880. [44] Yu, H.; Zou, Y.; Wang, Y.; Huang, X.; Huang, G.; Sumer, B. D.; Boothman, D. A.; Gao, J. Overcoming Endosomal Barrier by Amphotericin B-Loaded Dual pH-Responsive PDMA-bPDPA Micelleplexes for siRNA Delivery. ACS nano. 2011, 5, 9246-9255. [45] Han, S.; Cheng, Q.; Wu, Y.; Zhou, J.; Long, X.; Wei, T.; Huang, Y.; Zheng, S.; Zhang, J.; Deng, L.; Wang, X.; Liang, X.; Cao, H.; Liang, Z.; Dong, A. Effects of Hydrophobic Core Components in Amphiphilic PDMAEMA Nanoparticles on siRNA Delivery. Biomaterials. 2015, 48, 45-55.


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[46] Zhou, J.; Wu, Y.; Wang, C.; Cheng, Q.; Han, S.; Wang, X.; Zhang, J.; Deng, L.; Zhao, D.; Du, L.; Cao, H.; Liang, Z.; Huang, Y.; Dong, A. pH-Sensitive Nanomicelles for High-Efficiency siRNA Delivery in Vitro and in Vivo: An Insight into the Design of Polycations with Robust Cytosolic Release. Nano Letters. 2016, 16, 6916-6923. [47] Kuroda, K.; DeGrado, F. W. Amphiphilic Polymethacrylate Derivatives as Antimicrobial Agents. J Am Chem Soc. 2005, 127, 4128-29. [48] Lai, J. T.; Filla, D. S. R. Functional Polymers from Novel Carboxyl-Terminated Trithiocarbonates as Highly Efficient RAFT Agents. Macromolecules. 2002, 35, 6754-56. [49] Chiefari, J.; Chong, Y. K.; Ercole, F.; Krstina, J.; Jeffery, J.; Le, T. P.; Mayadunne, R. T.; Meijs, G. F.; Moad, C. L.; Moad, G.; Rizzardo, E.; Thang, S. H. Living Free-Radical Polymerization by Reversible Addition-Fragmentation Chain Transfer: the Raft Process. Macromolecules. 1988, 31, 5559-5562. [50] Shao, J.; Zhou, B.; Chu, B.; Yen, Y. Ribonucleotide Reductase Inhibitors and Future Drug Design. Currr Cancer Drug Targets. 2006, 6, 409-431. [51] Zhang, Y. W.; Jones, T. L.; Martin, S. E.; Caplen, N. J.; Pommier, Y. Pommier. Implication of Checkpoint Kinase-Dependent Up-Regulation of Ribonucleotide Reductase R2 in DNA Damage Response. J. Biol. Chem. 2009, 284, 18085-18095. [52] Furuta, E.; Okuda, H.; Kobayashi, A.; Watabe, K. Metabolic Genes in Cancer: Their Roles in Tumor Progression and Clinical Implications. Biochim. Biophys. Acta. 2010, 1805, 141-152. [53] Cheng, Q.; Huang, Y.; Zheng, H.; Wei, T.; Zheng, S.; Huo, S.; Wang, X.; Du, Q.; Zhang, X.; Zhang, H.; Liang, X.; Wang, C.; Tang, R.; Liang, Z. The Effect of Guanidinylation of PEGylated


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Poly (2-aminoethyl methacrylate) on the Systemic Delivery of siRNA. Biomaterials. 2013, 34, 3120-3131.

Scheme 1. Molecular structures of E-A-D, E-D-A and E-(A/D). The three polymers can form micelles and bind siRNA.


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Figure 1. Physicochemical properties of siRNA-loaded micelles. (A) Agarose gel retardation assay of siRNA-loaded micelles (B) micelle sizes and (C) zeta potentials of siRNA-loaded micelles at different N/P ratios. The suffix numbers on the micelle name refer to the N/P values.


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Figure 2. In vitro cell viability and gene silencing effect of ME-A-D, ME-D-A and ME-(A/D) /siRNA micelles. (A) Luciferase knockdown efficiency was tested on MDA-MB-231-Luc cells, and (B) cytotoxicity was measured using MTT assay. (C) RT-PCR and (D) western blot assay were used to detect the gene silencing efficiency of NPs/siPLK1 complexes at different N/P ratios. In all tests, cells were treated with different formulations for 24 h except for western blot (48 h), and the final siRNA transfection concentration was 50 nM. Each bar represents the mean ± S.E. of three experiments. *P < 0.05, **P < 0.01, and ***P < 0.001 vs. Naked siRNA.


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Figure 3. (A) Intracellular fluorescence intensities of ME-A-D, ME-D-A and ME-(A/D) complexes with Cy5-labeled siRNA determined by flow cytometry at N/P=10. (B) Mean fluorescence intensity (MFI) at different N/P ratio. (C) Intracellular distribution of siRNA-loaded micelles in MDAMB-231 cells at different N/P ratio, determined by confocal laser scanning microscopy. (D) Statistics of co-located ratio of Cy5-siRNA and lysotracker green. Each bar represents the mean ± S.E. of six experiments. *P < 0.05, **P < 0.01, and ***P < 0.001 vs. N/P=5.


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Figure 4. In vitro tumor cell growth inhibition assay. (A) RRM2 mRNA level and (B) cell viability after treatment with lipo/siRRM2, ME-A-D/siNC, ME-A-D/siRRM2, ME-D-A/siNC, ME-DA/siRRM2

complexes for 48 h. (C) Colony formation assay after treatment with siRRM2-loaded

micelles for 2 weeks. (N/P=10, siRNA 50 nM, MDA-MB-231 cells).


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Figure 5. Cell apoptosis assay after treatment with siRRM2-loaded micelles for 48 h. (N/P=10, siRNA 50 nM, MDA-MB-231 cells).


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Figure 6. In vivo distribution of siRNA-loaded micelles in C57BL/6 mice at 5 h and 24 h (1 mg/kg for siRNA). (A) Fluorescence detection of isolated main organs at 5 h and 24 h after tail vein injection. (B) Cryosections of liver tissues observed by confocal microscopy. DAPI and fluorescein isothiocyanate-labeled phalloidin were labeled to stain nuclei and F actin (to show the rough cell outline). Scale bar: 10 µm. (C) ApoB mRNA expression level and (D) CHO concentration in serum after administration for 48 h. Each bar represents the mean ± S.E. of six experiments. *P < 0.05, **P < 0.01, and ***P < 0.001 vs. naked siRNA. (N/P=10, siRNA 50 nM)

Figure 7. Tumor tissue targeted delivery of siRNA mediated by ME-A-D. (A) Fluorescence imaging of MDA-MB-231 bearing mice at different time points (2, 5 and 24 h). (B) Fluorescence images of Cy5-siRNA accumulated in isolated tumor tissues at 24 h after injection at 1 mg/kg. (C) Quantification of siRNA accumulated in tumor tissues using imaging software. Date was normalized to the tumor from PBS treated animal. Each bar represents the mean ± S.E. of three experiments. ***P < 0.001 vs. naked siRNA.


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Figure 8. In vivo tumor growth inhibition. Tumor bearing BALB/c nude mice were i.v. injected with PTMS/siRRM2 every three days for 4 times (n=5). (A) Tumor volumes and (B) body weight were measured every day until day 12. At the end of experiment, (C) RRM2 mRNA level of tumor was tested using RT-PCR. The concentration of (D) Interleukin-2 (IL-2), (E) Interleukin-6 (IL-6), and (F) Tumor Necrosis Factor-α (TNF-α) in blood serum were detected using Luminex Technology. Each bar represents the mean ± S.E. of five experiments. *P < 0.05 and **P < 0.01 vs. naked siRNA.


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Elaboration on the Distribution of Hydrophobic Segments in the

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