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Research Article Cite This: ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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Improved Cell Transfection of siRNA by pH-Responsive Nanomicelles Self-Assembled with mPEG‑b‑PHis‑b‑PEI Copolymers Bin Xu, Yuan-Jun Zhu, Cheng-Han Wang, Chong Qiu, Jing Sun, Yi Yan, Xin Chen, Jian-Cheng Wang,* and Qiang Zhang Beijing Key Laboratory of Molecular Pharmaceutics and New Drug Delivery Systems, State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Xueyuan Road 38, Beijing 100191, China

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S Supporting Information *

ABSTRACT: Here, the novel pH-responsive nanomicelles self-assembled with amphipathic meo-poly(ethylene glycol)-bpoly(L-histidine)-b-polyethylenimine (mPEG-b-PHis-b-PEI, EHE) copolymers based on hydrophobic interaction of PHis with deprotonation of imidazoles were developed for siRNA transfection. The cationic nanomicelles could electrostatically compact siRNA into stable EHE/siRNA nanoplexes with a hydrodynamic diameter of ∼190 nm and present a low toxicity in normal physiological condition (pH ∼ 7.4). Different from pH-irresponsive ECE/siRNA nanoplexes based on mPEG-b-poly(ε-caprolactone)-b-PEI (ECE), the EHE/siRNA nanoplexes exhibited a higher cellular uptake along with an increased ζ-potential (from +18 to +32 mV) when the pH changed from 7.4 to 6.8 (extracellular acidic microenvironments). After cell internalization, the EHE/siRNA nanoplexes also exhibited an enhanced nanostructural disassembling and release of siRNA from lysosomal acidic microenvironments (pH ∼ 5.5). Furthermore, it was demonstrated that the EHE/siEGFR nanoplexes downregulated the expression levels of the corresponding mRNA and protein more efficiently than ECE/siEGFR in HeLa cells. The improved siRNA silencing effects of EHE/siEGFR nanoplexes resulted from the higher cellular uptake and enhanced endosomal/lysosomal escape, which is associated with the pH-responsive disassembly of nanostructure as well as the synergistic “proton sponge” effects of PHis and PEI in EHE copolymers. Therefore, the pH-responsive EHE nanomicelles would be promising and potential carriers for cell transfection of siRNA. KEYWORDS: siRNA delivery, poly(l-histidine), pH-responsive triblock copolymer, disassembly, endosomal escape

1. INTRODUCTION Over the past decade, small interfering RNA (siRNA) has been increasingly demonstrated as a promising and potential gene drug in refractory disease treatment owing to its specific silencing effects on target messenger RNA (mRNA).1−3 However, naked siRNA with a negative charge and a high molecular weight is not suitable for application in vivo because of nuclease degradation and inability to cross cellular membranes.4,5 Up to now, various viral and nonviral delivery systems have been developed for enhancing the siRNA therapy efficiency.6,7 In spite of high transfection efficiency, the practical use of viral vectors was restricted by insertional mutagenesis, immunogenicity, and high toxicity.8,9 The nonviral delivery system including liposomes, micelles, and nanoparticles (NPs) might offer a safer alternative for siRNA delivery.10,11 Generally, several biological barriers would be encountered during siRNA delivery in vivo, including safety in blood circulation, specific tumor accumulation, endosomal/lysosomal escape of siRNA in cytoplasm.12 To overcome these barriers, stimuli-responsive multifunctional nanocarriers responding to endogenous signals in tumor microenvironment (TME) (such © XXXX American Chemical Society

as acidic pH, hypoxia, overexpressed enzymes, and cytokines) or intracellular signals (such as lower pH in endosome, reductive environment) have been developed.13−17 Among these, pH-responsive functional nanocarriers were considered as the most commonly used drug delivery systems. Typically, normal blood remains well buffered and constant around pH 7.4, whereas acidification happens in tumor microenvironment (pH ∼ 6.8).18 After internalization into cancer cells, drugloaded nanocarriers will confront pH changes from early endosomes (pH 5.5−6.0)19,20 to later lysosomes (pH 5.0− 5.5).21 It was reported that a dual pH-responsive siRNAloaded nanoparticles22 self-assembled by the poly(ethylene glycol)-carboxyl-ended polybutadiene-polyethylenimine, PEGCPB-PEI (PCPP) polymer at pH 7.4, which had a long-term blood circulation. In this nanoparticle, enhanced cell uptake and transfection efficiency were attributed to the detachment of outer PEG shell at tumor extracellular pH condition and disassembly of nanoparticles along with pH-responsive Received: March 15, 2018 Accepted: June 8, 2018 Published: June 8, 2018 A

DOI: 10.1021/acsami.8b04301 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces Scheme 1. Schematic Illustration of pH-Responsive EHE/siRNA Nanoplexesa

a

(A) The EHE copolymers could self-assemble into cationic micelles and compact siRNA into EHE/siRNA nanoplexes at the physiological condition (pH 7.4). (B) When reaching tumor acidic microenvironments (pH ∼ 5.0−6.8), the EHE/siRNA nanoplexes could exhibit a higher cellular uptake and an enhanced escape of siRNA from endosome/lysosomes, owing to the synergistic proton sponge effects of polyethyleneimine (PEI) and pH-responsive PHis segments in EHE polymers.

displays the hydrophilic and electropositive properties after protonation of imidazole in acidic environments, whereas it changes in the hydrophobic and electroneutral property under deprotonation of imidazole at neutral pH (∼7.4). In previous researches, some PHis-based polymers have been used to establish nanoparticles for delivering hydrophobic drugs into cells (such as paclitaxel and doxorubicin) and improve the intracellular drug release based on the pH-triggered disassembly of the hydrophobic core.29−31 However, PHisbased cationic polymers for siRNA delivery have been rarely reported until now. These polymers may contribute to form a stable siRNA-loaded cationic nanoparticles and enhance the lysosomal escape via the synergistic proton sponge effects32 of pH-responsive PHis imidazole groups and cationic polymeric fragment. Considering pH-responsive structural changes and synergistic proton sponge effects of PEI and PHis, a novel amphipathic meo-poly(ethylene glycol)-b-poly(L-histidine)-bpolyethylenimine triblock copolymer (EHE) with lowmolecular-weight branched polyethyleneimine (LMw-PEI1.2k) was synthesized in the present study. The EHE copolymers could self-assemble into cationic micelles and compacted siRNA into EHE/siRNA nanoplexes at physiological condition (pH ∼ 7.4) based on the pH-responsive deprotonation property of PHis as well as the electrostatic adsorption of PEI (Scheme 1A). The hydrophilic poly(ethylene glycol)

cleavage of phenylborate in lysosomes. Therefore, siRNAloaded pH-responsive nanocarriers may provide an attractive targeting therapeutic approach for tumor treatment owing to their structural change in mildly acidic microenvironments both in the interstitium of solid tumors and endosome/ lysosome of tumor cells. In terms of pH-sensitive biomaterials, polyethyleneimine (PEI) and poly(L-histidine) (PHis) are two important polymers commonly used to fabricate pH-responsive functional nanocarriers. Branched PEI containing primary, secondary, and tertiary amine groups has aroused huge interest in siRNA delivery because of its significant pH-responsive “proton sponge” effects.23 Although high-molecular-weight branched PEI25k was quite effective in delivering nucleic acids, its inherent cytotoxicity caused by the destructive interaction with biomembrane has confined its practical applications.24,25 Low-molecular-weight branched polyethyleneimine (LMwPEI1.2k) can reduce the toxicity significantly, but bring down the transfection efficiency at the same time.26 In previous studies, LMw-PEI1.2k conjugated with hydrophobic polymer chains could be assembled into nanoscale carriers for siRNA delivery.27,28 Poly(L-histidine) (PHis) is of great significance in pHsensitive materials. The lone-pair electrons in the unsaturated nitrogen of the imidazole ring endow PHis (pKa ∼ 6.0) with a pH-responsive protonation−deprotonation property. PHis B


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ACS Applied Materials & Interfaces

dine) (1.17 g, 0.18 mmol), succinic anhydride (36.0 mg, 0.36 mmol), and 4-dimethylaminopyridine (DMAP) (43.9 mg, 0.36 mmol) were dissolved in 30 mL DMSO and stirred at 40 °C for 1 day under nitrogen. After dialyzing against DMSO for 2 days and against water for 1 day (MWCO: 3.5 kDa), the product was collected via lyophilization. 2.2.4. Synthesis of Meo-poly(ethylene glycol)-b-polyhistidine-bpolyethyleneimine. Meo-poly(ethylene glycol)-b-poly(Nim-DNP-histidine)-COOH (0.86 g, 0.13 mmol), N-hydroxysuccinimide (NHS) (30 mg, 0.26 mmol), and 1,3-dicyclohexylcarbodiimide (DCC) (54 mg, 0.26 mmol) were dissolved in 8 mL DMSO and stirred at room temperature for 1 day under nitrogen. Then, branched polyethyleneimine (LMw-PEI1.2K) (0.936 g, 0.78 mmol) was suspended in 4 mL DMSO and injected into the solution of PEG−PHis. Then, the mixture was stirred for 1 day. Next, 2-mercaptoethanol (6.5 mL, 88 mmol) was added into the mixture drop by drop and the solution was stirred overnight. After that, the mixture was dialyzed against DMSO for 3 days with five changes and against water for 2 days with four changes (MWCO: 3.5 kDa). The final product was collected after lyophilization. 2.3. Preparation and Characterization of siRNA-Loaded NPs. Meo-poly(ethylene glycol)-b-poly(L-histidine)-b-polyethylenimine (EHE) was dissolved in acidic deionized water (pH = 4.5) to form a homogeneous solution with a concentration of 1 mg/mL (whereas ECE was dissolved in DMSO). Subsequently, a certain volume of EHE solution was taken and mixed with alkaline deionized water (pH = 9.5) at the volume ratio of 1:1 and blank micelle solution was formed after ultrasound. The siRNA aqueous solution (10 μL, 10 μM) was added into the blank micelle solution (90 μL) and incubated at 37 °C for 40 min. After EHE/siRNA nanoplexes were obtained, size and ζ-potential were determined at room temperature by dynamic light scattering (DLS, Malvern Zetasizer Nano ZS, Malvern, U.K.). Furthermore, the morphologies of EHE/siRNA nanoplexes were analyzed by scanning electron microscope (SEM). As a control, meo-poly(ethylene glycol)-b-poly(ε-caprolactone)-bpolyethylenimine (mPEG-b-PCL-b-PEI, ECE) was also synthesized and used to form ECE micelle according to the similar method for the preparation of EHE micelles except that the ECE copolymer was dissolved in DMSO before self-assembly. 2.4. Gel Retardation Assay. The siRNA-loaded NPs (N/P = 20) were mixed with FBS at the volume ratio of 1:1 and incubated for different times at 37 °C. The samples were kept in −20 °C before gel retardation assay and tackled with 3% sodium dodecyl sulfate (SDS). Briefly, the samples were mixed with loading buffer and electrophoresed on a 1% agarose gel containing 0.1‰ GelRed (a special luminant dye for siRNA staining). Electrophoresis was performed at 80 mV for 3 min and subsequently at 100 mV for 12 min, and the final gels were photographed under UV illumination. Free siRNA was used as the control. 2.5. Hemagglutination Assay. Erythrocytes (red blood cells, RBC) were isolated from Sprague-Dawley rat blood by centrifugation at 1500g for 5 min at 4 °C and washed with physiological saline solution (0.9% sodium chloride, w/v). The cell pellet was suspended in a 2% erythrocyte suspension with a prechilled phosphate-buffered saline (PBS) buffer (pH = 7.4). Different siRNA-loaded NP solution was mixed with 0.2% erythrocyte suspension at N/P = 20, and the final concentration of siRNA was 100 nM. The mixed solution was incubated at 37 °C for 30 min, followed by observation under a microscope. Five percent Glu was used as a negative control. 2.6. Investigation of pH-Responsive Characters of EHE/ siRNA Nanoplexes. EHE/siRNA and ECE/siRNA nanoplexes were diluted (1:2, v/v) in 20 mM Hepes buffer solution with different pH (7.4, 6.8, 6.0, or 5.5) and incubated for 20 min at room temperature. The N/P of nanoplexes was 20 and the final concentration of siRNA was 100 nM. Then, the size and ζ-potential were monitored by dynamic light scattering (Malvern Zetasizer Nano ZS, Malvern, U.K.). 2.6.1. Observation of Structure Change by Transmission Electron Microscopy (TEM). Briefly, 10 μL mixture samples were applied on a copper grid, followed by removing excess solution with filter paper and then adding 10 μL staining solution (2% uranyl

(PEG) shells render the NPs a prolonged blood circulation time and an enhanced tumor accumulation through the enhanced permeability and retention effects. When entering in tumor acidic microenvironments (pH ∼ 5.0−6.8), the EHE/ siRNA nanoplexes would exhibit a higher cellular uptake, a reinforced disassembling, and a rapid escape of siRNA from lysosomes by the virtue of the synergistic proton sponge effects of polyethyleneimine (PEI) along with the pH-responsive PHis segments in EHE polymers (Scheme 1B).

2. MATERIALS AND METHODS 2.1. Materials and Cell Lines. Boc-His(DNP)-OH-IPA was purchased from Energy Chemical Ltd. (Shanghai, China). LMwPEI1.2k was purchased from Sigma-Aldrich (Shanghai, China). mPEG2000−amine were purchased from Beijing Beiluo Biotechnology Co. Ltd. (Beijing, China). mPEG2000−PCL2500-COOH was purchased from Jinan Daigang Biomaterial Co. Ltd. (Jinan, China). Hydroxyethyl piperazine ethanesulfonic acid (Hepes) was purchased from J&K Scientific Ltd. (Beijing, China). OPTI-MEM and LysoTracker Red DND-99 were purchased from Invitrogen (NY). Agarose was obtained from Gene Company (Hong Kong, China). Hoechst 33258 and Hoechst 33342 were purchased from Molecular Probes Inc. (OR). 3-(4,5-Di-methylthiazolyl-2)-2,5-diphenyltetrazolium bromide was purchased from Sigma-Aldrich. Co. (St. Louis, MO). Antiepidermal growth factor receptor (EGFR) siRNA (sense strand: 5′AGG AAU UAA GAG AAG CAA CAU dTdT-3′ and antisense strand: 5′-AUG UUG CUU CUC UUA AUU CCU dTdT-3′, named as siEGFR), negative control siRNA (sense strand: 5′-UUC UCC GAA CGU GUC ACG UTT-3′; antisense strand: 5′-ACG UGA CAC GUU CGG AGA ATT-3′, named as siNC), and fluorescein-labeled siRNA (5′ end of the sense strand, FAM-siRNA and Cy5-siRNA) were synthesized and purified with HPLC by GenePharma Co. Ltd. (Shanghai, China). The EGFR rabbit antibody and β-actin mouse antibody were purchased from Abcam (Shanghai, China). Human EGFR ELISA Kit was purchased from Rockland (ME). Human cervical carcinoma HeLa cells were obtained from the Institute of Basic Medical Science, Chinese Academy of Medical Sciences (Beijing, China). The cells were cultured in Roswell Park Memorial Institute-1640 medium supplemented with 10% fetal bovine serum (FBS, PAN, Germany), 100 U/mL penicillin, and 100 μg/mL streptomycin at 37 °C in a humidified atmosphere containing 5% CO2. The cells for all the experiments were in the logarithmic phase of growth. 2.2. Synthesis Procedures of mPEG-b-PHis-b-PEI (EHE) Copolymer. 2.2.1. Synthesis of Nim-DNP-Histidine Carboxyanhydride Hydrochloride (DNP-NCA·HCl). Boc-His(DNP)-OH isopropanol (3.5 g, 7.28 mmol) was dissolved in 50 mL 1,4-dioxane. Then, 3.6 mL of thionyl chloride (50.96 mmol) was added dropwise to the solution under nitrogen atmosphere. After 1 h, the precipitate was filtered, washed with 1,4-dioxane and diethyl ether, and dried in vacuo. To remove impurity, the product was dissolved in 20 mL acetone. After filtration, the acetone solution was poured into cold diethyl ether. The precipitate was collected by filtration and dried in vacuo. 2.2.2. Synthesis of Meo-poly(ethylene glycol)-b-poly(Nim-DNPhistidine). DNP-NCA·HCl (2.0 g, 5.22 mmol) and sodium carbonate (1.11 g, 10.44 mmol) were added into 10 mL dimethylformamide (DMF). The mixture was stirred at room temperature (rt) for about 1 h under nitrogen atmosphere. Then, meo-PEG2000-NH2 (0.35 g, 0.174 mmol) dissolved in 5 mL DMF was added into the mixture and then stirred at 40 °C under reduced pressure for 3 days. After filtration, the mixture was poured into an ethanol/ether mixture (1:1, v/v). The precipitate was collected by filtration, washed with ethanol and diethyl ether, and dried in vacuo. After dialyzing against dimethyl sulfoxide (DMSO) for 2 days and against water for 1 day (molecular weight cut-off (MWCO): 3.5 kDa), the product was collected via lyophilization. 2.2.3. Synthesis of Meo-poly(ethylene glycol)-b-poly(Nim-DNPhistidine)-COOH. Meo-poly(ethylene glycol)-b-poly(Nim-DNP-histiC


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ACS Applied Materials & Interfaces Scheme 2. Synthesis Procedure of EHE via Ring-Opening Polymerization (ROP)a

a (i) SOCl2, 1,4-dioxane, rt, 1 h; (ii) NaCO3, DMF, rt, 1 h; (iii) mPEG2000-NH2, DMF, 313 K, reduced pressure, 3 days; (iv) succinic anhydride/ DMAP, DMSO, 313 K, 24 h; (v) NHS/DCC, DMSO, rt, 24 h; (vi) sbPEI1.2k, DMSO, rt, 24 h; (vii) 2-mercaptoethanol, DMSO, rt, overnight.

acetate, Sigma-Aldrich) for 2 min. After wicking away the staining solution, the sample grids were dried on a copper grid or a silicon pellet (25 mm2) under vacuum overnight. Then, a JOEL 100CX transmission electron microscope (100 kV) was used to observe the samples. 2.6.2. Hemolytic Effects of siRNA-Loaded NPs. Erythrocytes (RBC) were isolated from the Sprague-Dawley rat blood by centrifugation at 1500g for 5 min at 4 °C and washed with physiological saline solution (0.9% sodium chloride, w/v). The cell pellet was suspended into a 2% erythrocyte suspension with a prechilled different PBS buffer solution (0.1 M, pH = 7.4, 6.8, or 5.5). Different siRNA-loaded NP solutions were mixed with 0.2% erythrocyte suspension and the final concentration of siRNA was 100 nM. Then, the mixed solution was incubated at 37 °C for 2 h. The 5% Glu was used as a negative control and Triton X-100 (2%, v/ v) was used as a positive control. After centrifugation, the supernatant from each sample was transferred to a new 96-well plate and the absorbance was measured at 540 nm using a microplate reader. The relative hemolysis rate = ([Abs]sample − [Abs]buffer)/([Abs]Triton − [Abs]buffer) × 100%. 2.6.3. pH-Responsive Release of siRNA. The Cy5-siRNA-loaded NPs were transferred to a dialysis device (MWCO: 30 kDa) that was immersed in a PBS buffer solution (pH = 7.4 or 5.5) at room temperature. The N/P of the nanoplexes was 20 and the final concentration of siRNA was 1 μM. During stirring, 100 μL of the PBS solution was withdrawn at different time points. The fluorescence intensity of Cy5-siRNA in the PBS solution was determined by a multimode microplate reader (FlexStation 3, Molecular Devices). 2.7. Cellular Uptake Assay. HeLa cells were seeded 3 × 105 per well in six-well plates and confocal dishes (15 mm). After 24 h proliferation, FAM-siRNA-loaded nanoplexes were diluted by OPTIMEM of different pH values (7.4 or 6.8) and the final concentration of siRNA was 100 nM. Then, FAM-siRNA-loaded nanoplexes were exposed to HeLa cells and incubated for 4 h at 37 °C. After that, the

cells in six-well plates were harvested and washed three times with prechilled PBS. The intracellular fluorescence intensities were detected by a FACS Calibur flow cytometry (Becton Dickinson, San Jose, CA) immediately. Besides, the cells in confocal dishes were washed three times with prechilled PBS followed by fixation with 4% paraformaldehyde for 15 min at room temperature. After incubation of Triton X-100, F-actin and nucleus were individually stained by rhodamine-labeled phallacidin and Hoechst 33258. The cells were visualized under a Leica TCS SP8 confocal laser scan microscope (CLSM, Leica Microsystems, Heidelberg, Germany). 2.8. Co-localization Assay of siRNA-Loaded NPs. HeLa cells were plated in confocal dishes (15 mm) at 2.5 × 105 cells per well for 24 h proliferation. The cell culture medium was replaced with OPTIMEM containing various concentrations of different indicators including TRF (Alexa Fluor-488 conjugated transferrin, 10 μg/mL), CT-B (Alexa Fluor-488 conjugated cholera toxin subunit B, 2 μg/ mL), and dextran (fluorescein isothiocyanate (FITC) conjugates, 1 mg/mL). Then, the cells were incubated with Cy5-siRNA-loaded nanoplexes (siRNA final concentration of 100 nM) in OPTI-MEM (pH = 6.8) for 3 h at 37 °C, and the nucleus was stained by Hoechst 33342 (10 μg/mL) for another 15 min. After incubation, the cells were washed three times with precooled PBS solution and fixated with 4% paraformaldehyde for 15 min. The cells were visualized via a Leica TCS SP8 confocal fluorescence microscope (Leica Microsystems, Heidelberg, Germany). 2.9. Endosomal Escape Assay. HeLa cells were seeded 3 × 105 per well and plated in confocal dishes (15 mm). After 24 h, the culture medium was displaced by OPTI-MEM (pH = 7.4 or 6.8) with FAM-siRNA-loaded nanoplexes, in which the final concentration of siRNA was 100 nM. The cells were incubated at 37 °C for 4 or 5 h. After removing the medium and washing with PBS buffer thrice, the endosomes and nuclei were stained with Lysotracker red and Hoechst 33342, respectively. The cells were visualized under a Leica TCS SP8 D


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Figure 1. (A) 1H NMR (400 MHz) spectra of mPEG-b-PHis in DMSO-d6. (B) 1H NMR (400 MHz) spectra of mPEG-b-PHis-b-PEI in DMSO-d6. confocal laser scan microscope (Leica Microsystems, Heidelberg, Germany). 2.10. Real-Time Polymerase Chain Reaction (RT-PCR) Assay. The cells were seeded into six-well plates (3 × 105 cells per well). After 24 h proliferation, various nanoparticles containing siEGFR (100 nM) were exposed to the cells and incubated in OPTI-MEM (pH = 7.4 or 6.8) for 4 h at 37 °C. The cells were incubated for another 24 h in a 1640 complement medium for proliferation. The total RNA was extracted using the TRIzol reagent and then reverse transcribed with a GoScriptTM Reverse Transcription System (A5001) (Promega). PCR was performed on cDNA pretreated with GoTaq qPCR Master Mix (A6002) (Promega) on a real-time PCR amplifier (MX3005P, Stratagene). All the quantitation was normalized to an endogenous control GAPDH. The relative quantitation value for each target gene compared to the calibrator is expressed as 2−(Ct−Cc) (Ct and Cc are the mean threshold cycle differences after normalizing to GAPDH). 2.11. Western Blot and Enzyme-Linked Immunosorbent Assay (ELISA). The cells were cultured in six-well plates (3 × 105 cells per well). After 24 h proliferation, nanoparticles (100 nM of siEGFR) were exposed to the cells and incubated in OPTI-MEM (pH = 7.4 or 6.8) for 4 h at 37 °C. After refreshing the medium to 1640 complement medium, the cells were incubated for another 48 h. The proteins of the cells were extracted and separated by SDSpolyacrylamide gel electrophoresis and then transferred to poly(vinylidene difluoride) membranes, followed by incubation with EGFR rabbit antibody and β-actin mouse antibody (abcam, Shanghai, China), separately. Then, the blots were incubated with the corresponding secondary antibody (horseradish peroxidase-conjugated antirabbit and antimouse IgG; cell signaling) at room temperature for 2 h and visualized using an enhanced chemiluminescence detection system (Pierce). Meanwhile, the quantitative analysis of proteins was detected by ELISA Kit (Rockland, ME). Briefly, after washing with PBS for three

times, the EGFR antibody was added into the 96-well plate and incubated for 1 h. Then, ABC working solution was added and incubated for 30 min. After incubation with TMB substrate reagent, the absorbance was measured at 450 nm using a microplate reader. 2.12. Statistical Analysis. All the results were expressed as mean ± standard deviation (SD). For comparisions between two groups, unpaired Student’s t-test (two-tailed) was used. For statistical analysis between multiple groups, one-way analysis of variance with Tukey tests was applied. All the statistical analyses were performed by GraphPad Prism Software (Version 5.0, GraphPad Software, San Diego, CA). A value of p < 0.05 was considered as statistically significant difference.

3. RESULTS 3.1. Synthesis and Characterizations of EHE Copolymers. The synthetic procedure of EHE is illustrated in Scheme 2. Ring-opening polymerization (ROP) was employed to synthesize the pH-responsive triblock polymer mPEG-b-PHisb-PEI (EHE). The degree of PHis was 16, which could be calculated from the 1H NMR spectra (Figure 1A). As shown in Figure 1B, the PEI1.2k block was successfully connected to PHis block and the characterization of EHE could be identified: 1H NMR (400 MHz, DMSO-d6) δ 7.50 (NCH−N), 6.75 (C CH−N), 4.44 (OC−CH−N−), 3.51 (−CH2−CH2−O−), 3.35 (meo-), 3.12 (−CH2− imidazole), 3.07 (−CH2− imidazole), 2.55−2.98 (CH, CH2 from PEI), and 2.33 (O C−CH2−CH2−CO). The pH-irresponsive triblock meopoly(ethylene glycol)-b-poly(ε-caprolactone)-b-polyethylenimine (mPEG-b-PCL-b-PEI, ECE) was also synthesized and used as the control material (detail synthesis procedure seen from Figure S1 in the Supporting Information). The spectra of E


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ACS Applied Materials & Interfaces Table 1. Molecular Structure, Molecular Weight, and Polydispersity Index (PDI) of Copolymers Mnc (103 g/mol) a

b

sample

structure

EHE ECE

mPEG45-b-PHis16-b-PEI2 mPEG45-b-PCL21-b-PEI2

Mwd (103 g/mol)

PEG

PHis/PCL

PEI

total

total

PDI

2.0 2.0

2.2 2.5

1.2 1.2

5.3 5.7

5.5 6.5

1.44 1.29

a

EHE: meo-poly(ethylene glycol)-b-poly(L-histidine)-b-polyethylenimine, ECE: meo-poly(ethylene glycol)-b-polycaprolactone-b-polyethylenimine. bThe molecular structure of the polymer was determined by 1H NMR. cThe mean molecular weight (Mn) of each block was estimated by 1H NMR. dThe mean molecular weight (Mw) of the copolymers was measured by GPC, PDI = Mw/Mn measured by GPC.

Figure 2. Characterization of EHE/siRNA nanoplexes at N/P of 20. (A) Size distribution of EHE/siRNA nanoplexes in 5% HBG (5% glucose with 10 mM Hepes buffer, pH = 7.4). (B) ζ-Potential of EHE/siRNA nanoplexes in 5% HBG. (C) SEM images of EHE/siRNA. (D) Analysis of erythrocyte aggregation after treatment with different siRNA complexes, physiological saline as control. (E) siRNA-protecting effects of different siRNA complexes by gel retardation assay.

the EHE/siRNA nanoplexes showed a relatively low ζpotential (approximately +18.6 mV) at N/P = 20 (Figure 2B) and a uniform dry diameter (∼40 nm, observation by SEM) (Figure 2C). Meanwhile, it was demonstrated that the EHE nanomicelles could compact the siRNA effectively when the N/P ratio was above 4:1 (Figures S4 and S5 in the Supporting Information). 3.3. Stability and Safety Evaluation of EHE/siRNA NPs. As shown in Figure 2E, free siRNAs were degraded completely within 3 h in FBS due to nuclease enzymes in serum.33 The PEI1.2k/siRNA complexes showed a weak extension of siRNA degradation (6 h). The degradation of siRNA in ECE/siRNA delayed to 12 h. Noteworthy, most of the siRNA in EHE/siRNA nanoplexes kept intact till 48 h. In addition, EHE caused little aggregation of serum proteins (Figure S8 in the Supporting Information) compared with PEI1.2k and PEI25k. These results suggested that the EHE/

Fourier transform infrared (FTIR) and gel permeation chromatography (GPC) of EHE and ECE polymers are shown in Figures S2 and S3 in the Supporting Information. The degree of polymerization and molecular weight of the polymers are shown in Table 1. 3.2. Characterization of siRNA-Loaded NPs. The PHis block converts to hydrophilic from hydrophobic after complete protonation of imidazole (Scheme 1A). After alkalinization of acidic aqueous, the EHE polymer self-assembled into micelles. Meanwhile, the ECE micelles were prepared in deionized water. The siRNA-loaded nanoplexes were obtained by the electrostatic interaction between the negatively charged siRNA and the positively charged micelles. The size and ζ-potential of PHis based the pH-responsive siRNA-loaded NPs were observed by dynamic light scattering (DLS). As shown in Figure 2A, EHE/siRNA nanoplexes had a fairly uniform hydrodynamic diameter (189.5 nm) in Hepes buffer with a low polydispersity index (PDI 0.15). In addition, F


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Figure 3. pH-responsive characterization of siRNA-loaded NPs at N/P of 20. (A) Size distribution of EHE/siRNA and ECE/siRNA in Hepes buffer (pH = 7.4, 6.8, 6.0, and 5.5). (B) ζ-Potential of EHE/siRNA and ECE/siRNA in Hepes buffer (pH = 7.4, 6.8, 6.0, and 5.5). (C) TEM images of different nanoplexes in Hepes buffer. (a) EHE/siRNA and (b) ECE/siRNA. (D) The schematic illustration of PHis changes at different pH conditions. (E) In vitro release of Cy5-siRNA from EHE/siRNA and ECE/siRNA at pH 7.4 and 5.5. (F) Hemolysis of erythrocyte after incubation with siRNA-loaded NPs at different pH (7.4 and 5.5). (G) Schematic illustration of pH-responsive changes in EHE/siRNA nanoplexes at different pH (7.4, 6.8, 6.0, and 5.5). The data are shown as mean ± SD (n = 3). **p < 0.01. G


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Figure 4. Cellular uptake of siRNA-loaded NPs in HeLa cells. (A) CLSM images of HeLa cells transfected with FAM-siRNA-loaded NPs (green) diluted with OPTI (pH = 6.8 or 7.4) at 37 °C for 4 h at a 100 nM siRNA dose. The Rhodamine-labeled phalloidin (red) was used to show cell skeleton and Hoechst 33258 (blue) for cell nucleus. (B) The quantitative intracellular fluorescence intensities were detected by flow cytometry after 4 h incubation of different nanoparticles at different N/P (FAM-siRNA: 100 nM) in OPTI (pH = 6.8 or 7.4). The data are shown as mean ± SD (n = 3).

ECE/siRNA at pH 5.5, indicating that the pH-responsive disassembly of EHE/siRNA could greatly promote the release of free siRNA. Furthermore, siRNA/polymer nanoplexes were evaluated for their ability to induce red blood cell (RBC) hemolysis at different pH (7.4 or 5.5), which is relevant to the endosomal/ lysosomal escape capacity.39 As shown in Figure 3F, a slight hemolysis was caused by EHE/siRNA (1%), ECE/siRNA (2%), and PEI1.2k/siRNA (3%). It was interesting that EHE/ siRNA showed a more significant hemolysis (40%) than ECE/ siRNA (17%), PEI1.2k/siRNA (15%), or even PEI25k/siRNA (30%) at the late endosome/lysosome acidity (pH 5.5). 3.5. pH-Responsive Cell Uptake. To investigate the pHresponsive cell uptake of EHE/siRNA, culture media with different pH were used to simulate normal physiological condition (pH ∼ 7.4) or tumor microenvironments (pH ∼ 6.8) tissue along with FAM-labeled siRNA serving as a fluorescent indicator. After incubation with FAM-siRNAloaded nanoplexes for 4 h, the intracellular fluorescence distribution (green signal) in HeLa cells was observed by CLSM. As shown in Figure 4A, the cells treated with ECE/ siRNA NPs showed similar distribution of green fluorescence dots between pH 7.4 and 6.8 at N/P of 10, although brighter green fluorescence dots were observed at N/P of 20. However, the cells treated with EHE/siRNA (N/P of 10 or 20) showed more obvious green fluorescence dots at pH 6.8 than at pH 7.4. The intracellular fluorescence intensities were further examined quantitatively by flow cytometer. As shown in Figure 4B, higher intracellular fluorescence intensities were found in the cells treated with EHE/siRNA than with ECE/siRNA in any N/P ratio at pH 6.8, whereas lower fluorescence intensities were shown in EHE/siRNA than in ECE/siRNA at pH 7.4. Furthermore, a significant difference in the intracellular fluorescence intensities between pH 7.4 and 6.8 was observed in PHis-based EHE/siRNA nanoplexes at all N/P ratios, about an average increase (∼150%) of cellular uptake at pH 6.8 compared with pH 7.4. However, the ECE/siRNA nanoplexes exhibited similar cellular uptake between pH 7.4 and 6.8 at any N/P ratio. 3.6. Endocytosis Pathway. The fate of siRNA-loaded NPs after internalization can be largely affected by the endocytosis pathways, which mainly include clathrin-mediated endocytosis (CME), caveolae-mediated endocytosis (CvME),

siRNA nanoplexes could keep stable during blood circulation.34,35 Cationic polymer-based siRNA delivery systems usually caused damage to cell membrane integrity and induced erythrocytes aggregation.36 As shown in Figure 2D, PEI1.2k/ siRNA caused a slight erythrocyte aggregation. In contrast, little erythrocyte aggregation was caused by EHE/siRNA and ECE/siRNA. Furthermore, there was no significant hemolytic activity in the EHE/siRNA group at any concentration (siRNA < 500 nM) (Figure S7 in the Supporting Information). These results collectively indicated that EHE micelle could be a stable and safe carrier for siRNA delivery. 3.4. pH-Responsive Structural Changes of EHE/siRNA NPs. To investigate the pH-responsive structural changes of EHE/siRNA nanoplexes, the changes of size/ζ-potential and siRNA release rates at different pH conditions were examined while the ECE/siRNA nanoplexes were used for comparison. As shown in Figure 3A, the ECE/siRNA nanoplexes revealed stable hydrodynamic diameters (∼94 nm) at different pH conditions. However, the size of EHE/siRNA nanoplexes become bigger with the increase in acidity (from ∼184 nm at pH 7.4 to ∼342 nm at pH 6.0) and reach the maximum (∼791 nm) at pH 5.5. Moreover, as the pH changed from 7.4 to 6.8, EHE/siRNA showed a sharp increase in ζ-potential (from +18 to +32 mV), whereas a modest increase was observed in the ECE/siRNA group (from +22 to +23 mV) (Figure 3B). Additionally, the morphologies of EHE/siRNA and ECE/ siRNA nanoplexes were observed by TEM at different pH (Figure 3C). There was no significant difference between ECE/siRNA nanoplexes under various pH conditions (average dry diameters of 40 nm). On the contrary, the EHE/siRNA nanoplexes evidently swelled (average dry diameters increasing from 40 to 80 nm) at pH 6.0 (the early endosome acidity). When the pH decreased to 5.5, no intact nanoparticle was observed. Generally, the disassembly of nanoplexes could enhance the intracellular drug release.37,38 Here, around 90% of the loaded Cy5-siRNA of EHE/siRNA was released within 4 h at pH 5.5, whereas less than 30% of the loaded siRNA was released at pH 7.4 (Figure 3E). For ECE/siRNA, the release of Cy5-siRNA had no obvious difference between pH 5.5 (less than 40%) and 7.4 (around 30%). More importantly, EHE/siRNA released siRNA into the cytoplasm in a larger amount and speed than H


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Figure 5. Endocytosis pathway of siRNA-loaded NPs in HeLa cells. (A) Qualitative analysis of endocytosis pathways is observed by CLSM after incubation with EHE/Cy5-siRNA and ECE/Cy5-siRNA nanoplexes in OPTI (pH = 6.8) for 3 h at a 100 nM siRNA dose. Hoechst 33342 (blue) for cell nucleus. The yellow dots represent the co-localization (white arrows) of pathway markers (green) and Cy5-siRNA (red). (a) EHE/siRNA and (b) ECE/siRNA. (B) Co-localization of siRNA and pathway markers observed by CLSM. (c) EHE/siRNA and (d) ECE/siRNA. (C) Semiquantitative analysis of endocytosis pathways of EHE/siRNA and ECE/siRNA. The data are shown as mean ± SD (n = 3). **p < 0.01.

membrane and the delivery of their cargos to lysosomes.41,42 Similar results were observed in the ECE/siRNA group (Figure 5). 3.7. Endosomal/Lysosomal Escape. After confirming the endocytosis pathway of nanoplexes, we further examined whether EHE/siRNA and ECE/siRNA nanoplexes could escape from endosomes/lysosomes following internalization. As shown in Figure 6C, a strong co-localization (yellow) of FAM-siRNA (green) and late endosome/lysosome (red) could be detected after treatment with either EHE/siRNA or ECE/ siRNA for 4 h. Moreover, a semiquantitative analysis of the colocalization rate (%) between FAM-siRNA-loaded nanoplexes and late endosome/lysosome showed that there was no significant difference in the co-localization between EHE/ siRNA group (41.96 ± 1.69%) and ECE/siRNA group (45.05 ± 1.59%) at 4 h time point (Figure 6A). More interesting, a host of green fluorescence dots of FAM-siRNA were diffusely distributed in the cytoplasm, whereas little co-localization

and macropinocytosis (MP). To determine the internalization pathway of pH-responsive EHE/siRNA and pH-irresponsive ECE/siRNA, we used three kinds of known fluorescent-labeled channel markers:40 Alexa 488-Tf (CME), Alexa 488-CTB (CvME), and FITC-dextran (MP). As seen in Figure 5A,B, a strong co-localization (yellow) of Cy5-siRNA (red) and Alexa 488-Tf (green) could be detected in EHE/siRNA group, indicating that CME was a major pathway of EHE/siRNA. Additionally, some co-localization (yellow) of Cy5-siRNA (red) and Alexa 488-CTB (green) suggested the CvME was also involved in the internalization of EHE/siRNA. On the contrary, macropinocytosis was rarely involved during the cellular uptake suggested by a little overlap (yellow) between Cy5-siRNA (red) and FITC-dextran (green). Semiquantitative results further confirmed that CME was the major endocytosis pathway for EHE/siRNA (Figure 5C), which was widely accepted to involve the compartment acidification by proton pumps located on the endosome I


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Figure 6. Endosomal/lysosomal escape of siRNA-loaded NPs in HeLa cells. (A) The quantitative analysis of the co-localization rate (%) performed by the co-localization computation module of Leica SP8 CLSM. (B) The schematic illustration of late endosome/lysosome escape of siRNA in a pH-dependent manner. (C) Co-localization of siRNA and late endosome/lysosomes in HeLa cells observed by CLSM after incubation with EHE/ FAM-siRNA and ECE/FAM-siRNA nanoplexes (green) in OPTI (pH = 6.8) for 4 and 5 h periods at a 100 nM siRNA dose. The LysoTracker Red (red) is used to show the late endosome/lysosome and Hoechst 33342 (blue) for cell nucleus. Co-localization (yellow) is indicated by white arrow. The data are shown as mean ± SD (n = 3). **p < 0.01. N.S., no significance.

(8.47 ± 1.08%) was observed at 5 h time point in the EHE/ siRNA-treated cells (Figure 6A,C), indicating that a large percentage of EHE/siRNA were released from late endosome/ lysosome into cytoplasm. However, in the ECE/siRNA group, a strong co-localization (30.59 ± 2.94%) was still observed at 5 h time point, which suggested that small percentages of ECE/ siRNA was released from the lysosomes. These results suggested a more powerful late endosomal/lysosomal escape ability of EHE/siRNA.

3.8. In Vitro Gene-Silencing Efficacy. Epidermal growth factor receptor (EGFR)-targeted gene downregulation is effective for cancer therapy.43 Herein, anti-EGFR siRNA (siEGFR) was selected as a model drug to evaluate the delivery efficiency of EHE/siRNA nanoplexes. The EGFR mRNA level in the cells was detected by RT-PCR. When the N/P of the nanoplexes was 10 (Figure 7A), both EHE/siEGFR and ECE/siEGFR exhibited a slight inhibition on the mRNA level at pH 7.4. However, the mRNA level was much lower at pH 6.8 than that at pH 7.4 for EHE/siEGFR, whereas this J


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Figure 7. In vitro gene-silencing efficacies of siRNA-loaded NPs in HeLa cells. (A) Relative expression levels of EGFR mRNA detected by RT-PCR after incubation with siEGFR-loaded NPs in OPTI (pH = 7.4 or 6.8) for 4 h and further incubation in 1640 complete medium for 24 h. The final concentration of siRNA in OPTI is 100 nM. (B) The expression of EGFR protein detected by Western blot assay after incubation with OPTIcontaining siEGFR-loaded NPs for 4 h and further 1640 complete medium for 48 h. (C) Relative expression levels of EGFR protein detected by ELISA after incubation with siEGFR-loaded NPs in OPTI (pH = 7.4 or 6.8) for 4 h and further 1640 complete medium for 48 h. The final concentration of siRNA in OPTI is 100 nM. The data is shown as mean ± SD (n = 3). *p < 0.05, **p < 0.01. N.S., no significance.

EGFR protein expression by ELISA (Figure 7C) also confirmed the results of RT-PCR and Western blot.

difference was not observed in the ECE/siEGFR group. Furthermore, the mRNA level in EHE/siEGFR was significantly lower than that in ECE/siEGFR at pH 6.8. Similar but stronger inhibition was observed when the N/P increased to 20 (Figure 7A). Especially at pH 6.8, EHE/siRNA (N/P = 20) exhibited a much lower mRNA level (54.5 ± 3.3%) than ECE/ siEGFR (73.8 ± 3.8%), even near to Lipo2000/siEGFR (43.4 ± 2.7%). Furthermore, Western blot analysis proved that both ECE/ siEGFR and EHE/siEGFR could decrease the expression level of EGFR protein at N/P of 10 or 20 (Figure 7B). More importantly, under pH 6.8 condition, EHE/siEGFR performed a significantly better silencing efficiency than ECE/siRNA at both N/P of 10 and 20. In addition, the quantitative analysis of

4. DISCUSSION A novel pH-responsive triblock EHE copolymer composed of poly(L-histidine) (PHis), polyethylenimine (PEI1.2k), and poly(ethylene glycol) (PEG) was synthesized in this study. It was demonstrated that amphipathic EHE copolymers could self-assemble into nanomicelles and condensed siRNA into EHE/siRNA nanoplexes with an average hydrodynamic diameter of 189.5 nm at the N/P ratio of 20 (Figures 2A and S4) based on the hydrophobic and electroneutral property of PHis with the deprotonation of imidazoles under neutral pH condition (pH 7.4). As a valuable control, the hydrophobic PCL-based ECE copolymer could self-assemble into stable K


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siRNA nanoparticles enter into an acidifying lysosomal compartment, these unsaturated amino groups are capable of sequestering protons through the proton pump along with an extensive inflow of Cl− ions and water into the lysosomal environment.44−47 Consequently, the lysosomes swell and their membranes are disrupted so that the entrapped components are released (Figure 6B). As seen from Figure 3A−C, PCLbased ECE/siRNA nanoplexes just exhibited a modest increase in the ζ-potential as the pH decreased, which was attributed to the weak proton buffer effect of PEI1.2k.48 However, a series of changes in EHE/siRNA were followed with the changes of pH values (Figure 3G). When the pH changed from 6.8 to 5.5, the size of EHE/siRNA nanoplexes gradually increased to ∼342 nm at pH 6.0, and a complete disassembly at pH 5.5; meanwhile, the ζ-potential was also significantly increased. These changes in the EHE/siRNA nanoplexes at different pH probably came from the partial deprotonation imidazoles in PHis segments. As we know, deprotonated imidazole is hydrophobic and electroneutral, but reverses into hydrophilic and electropositive after protonation (Figure 3D). When the pH (endosomal pH ∼ 6.0) reached to the pKa of PHis, the large loss of hydrophobic interaction and the positive charge repulsion make the nanoplexes unstable because of the fast and large protonation of imidazoles. As the pH decreased to the late endosome/lysosome acidity (∼5.5), the EHE/siRNA nanoplexes were completely disassembled with the further protonation of imidazoles, which could benefit the release of siRNA. As seen in Figure 3E, about 90% of the loaded Cy5siRNA of EHE/siRNA was released within 4 h at pH 5.5. Above all, it was suggested that the improved siRNA-silencing effects were probably related to the improved endosomal/ lysosomal escape of siRNA which is induced by the pHresponsive complete disassembly of EHE nanoplexes as well as the synergistic proton sponge effects between PHis and PEI segments. Based on the above-mentioned discussion about the results of stability and safety evaluation, higher cellular uptake, enhanced endosomal/lysosomal escape capacity, and improved gene-silencing efficiency ECE/siRNA, we believe the EHE micelles would be potential nanocarriers for the in vivo siRNA delivery.

nanomicelles and compact siRNA into nanoplexes with electrostatic interaction. Moreover, it was shown that the EHE/siRNA nanoplexes could protect siRNA from degradation in serum (Figure 2E) and had no obvious aggregations of serum proteins (Figure S8) and erythrocyte (Figure 2D). These results collectively suggested that EHE micelle could be a stable and safe vector for in vivo siRNA delivery. More interestingly, the EHE/siEGFR nanoplexes showed a significantly stronger gene-silencing efficacy on both mRNA and protein levels of EGFR than those of ECE/siRNA, especially performing superior silencing efficiency, similar to Lipo2000/ siRNA at N/P of 20 (Figure 7). High cellular uptake of siRNA nanoparticles is a prerequisite for gene transfection. As shown in Figure 4, higher intracellular fluorescence intensities were found in the cells treated with EHE/siRNA than in those treated with ECE/siRNA in any N/ P ratio at pH 6.8, although lower fluorescence intensities were shown in EHE/siRNA than in ECE/siRNA at pH 7.4. Furthermore, significantly different cell uptake of siRNA between pH 7.4 and 6.8 was observed in PHis-based EHE/ siRNA nanoplexes at all N/P ratios, about an average increase (∼150%) in cellular uptake at pH 6.8 compared with that at pH 7.4. It was demonstrated that the partial protonation of PHis at pH 6.8 could support much more positive charges (from +18 to +32 mV) of EHE/siRNA nanoplexes and contribute to the enhancement of cell membrane interaction as well as promoting cellular uptake of siRNA in acidic TME. When the pH value (∼6.8) is slightly above the pKa (∼6.0) of PHis, partial imidazoles of PHis get protonated and provide the redundant positive charge for the nanoplexes, which could strengthen the interaction with negatively charged siRNA and further compress the nanoplexes. Meanwhile, most imidazoles of PHis are still deprotonated and provide sufficient hydrophobic interaction to stabilize the nanoplexes. This pHdependent cell internalization could give EHE/siRNA nanoplexes the ability to avoid uptake by nontarget tissues and promote the uptake by tumor cells, which would benefit the siRNA delivery in an acidic tumor microenvironment and thus provide an attractive targeting therapeutic approach for tumor treatment. However, for ECE/siRNA nanoplexes, the stronger hydrophobic interaction provided by pH-independent PCL block made the ECE/siRNA nanoplexes have a stable nanostructure with a tight hydrophobic core, show no significant charge changes (from +22 to +23 mV) (Figure 3A), and exhibit similar cellular uptake between pH 7.4 and 6.8 at any N/P ratio (Figure 4). Besides a higher cellular uptake, the enhanced endosomal/ lysosomal escape capacity is crucial for siRNA transfection. Although the similar clathrin-mediated endocytosis pathway was found in EHE/siRNA and ECE/siRNA (Figure 5), significant difference in the endosomal/lysosomal escape of siRNA was observed in both nanoplexes following endocytosis, in which the EHE/siRNA nanoplexes exhibited a significantly stronger endosomal/lysosomal escape ability (Figure 6). Obviously, the weak proton-sponge effects of low-molecularweight branched polyethyleneimine (PEI1.2k) limited the escape capacity of ECE/siRNA nanoplexes from endosomes/ lysosomes (Figure 6A,C). The enhanced endosomal/lysosomal escape ability of EHE/siRNA nanoplexes probably resulted from the synergistic proton sponge effects of PHis and PEI. It is worthy noting that there are many pH-responsive protonable imidazole rings in PHis segments as well as multitype amine groups in PEI segments. Once these cationic PHis-based EHE/

5. CONCLUSIONS In summary, the novel mPEG-b-PHis-b-PEI (EHE) copolymers were successfully synthesized and used as nanocarriers for siRNA transfection. It was demonstrated that the siEGFRloaded EHE nanoplexes could exhibit higher gene-silencing effects than ECE/siEGFR nanoplexes in HeLa cells, probably resulting from the higher cellular uptake and enhanced endosomal/lysosomal escape, which is associated with the pH-responsive disassembly of nanostructure as well as the synergistic proton sponge effects of PHis and PEI.

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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/acsami.8b04301. Detailed experimental procedures; synthetic procedure 1 H NMR spectrum, GPC and FTIR spectroscopy for EHE and ECE copolymers; gel retardation assay of EHE and ECE; cell viability; hemolytic activity; aggregation of serum proteins (PDF) L


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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: 86-10-82805932. ORCID

Jian-Cheng Wang: 0000-0002-1651-8977 Qiang Zhang: 0000-0002-8862-3098 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors acknowledge financial support from the projects of National Natural Science Foundation of China (Grant Nos.81473158, 81690264, and 81773650), the New Drug R&D program of China (Grant No. 2018ZX09721003-004) and the Opening Project of Key Laboratory of Drug Targeting and Drug Delivery System, Ministry of Education (Sichuan University).

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