Redox Dual-Responsive Polyplex with Effective Endosomal

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pH/Redox Dual-Responsive Polyplex with Effective Endosomal Escape for Co-Delivery of siRNA and Doxorubicin against Drug-Resistant Cancer Cells Yan Gao, Li Jia, Qi Wang, Haiyang Hu, Xiuli Zhao, Dawei Chen, and Mingxi Qiao ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.9b02016 • Publication Date (Web): 18 Apr 2019 Downloaded from http://pubs.acs.org on April 18, 2019

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ACS Applied Materials & Interfaces

pH/Redox Dual-Responsive Polyplex with Effective Endosomal Escape for Co-Delivery of siRNA and Doxorubicin against Drug-Resistant Cancer Cells

Yan Gao†, Li Jia†, §, Qi Wang†, Haiyang Hu†, Xiuli Zhao†, Dawei Chen†, Mingxi Qiao*, †, ‡

* Corresponding author: Email: [email protected] †

Department of Pharmaceutics, School of Pharmacy, Shenyang Pharmaceutical University, 103

Wenhua Road, Shenyang 110016, P. R. China ‡

Shenyang Key Laboratory of Functional Drug Carrier Materials, Shenyang Pharmaceutical

University, 103 Wenhua Road, Shenyang 110016, P. R. China § Department

of Pharmacy, Heze Medical College, Heze 274000, P. R. China

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Abstract The enhanced endo-lysosomal sequestration still remains a big challenge in overcoming multidrug resistance (MDR). Herein, a dual-responsive polyplex with effective endo-lysosomal escape based on methoxypoly (ethylene glycol)-polylactide-polyhistidine-ss-oligoethylenimine (mPEG-bPLA-PHis-ssOEI) was developed for co-delivering MDR1 siRNA and doxorubicin (DOX). The polyplex showed good encapsulation of DOX and siRNA as well as triggered payloads release in response to pH/redox stimuli because of the PHis protonation and the disulfide bond cleavage. The polyplex at N/P ratio of 7 demonstrated much higher payloads delivery efficiency, MDR1 gene silence efficiency, cytotoxicity against MCF-7/ADR cell, and stronger MCF-7/ADR tumor growth inhibition than the polyplexes at higher N/P ratios. This was attributed to the stronger electrostatic attraction between siRNA and OEIs at higher N/P ratios that suppressed the release of MDR1 siRNA and OEIs, which played a dominant role in overcoming payloads endo-lysosomal sequestration by OEIs induced membrane permeabilization effect. Consequently, the polyplex with effective endo-lysosomal escape provides a rational approach to co-delivery siRNAs and chemotherapy agents for MDR reversal. Key words: endosomal escape, MDR, co-delivery, siRNA delivery, pH sensitive, redox sensitive 1. Introduction Multidrug resistance (MDR) remarkably compromises the cancer chemotherapy and effective MDR reversal remains a significant challenge.1-3 Recently, co-delivery of siRNAs with chemotherapy drugs by nanocarriers has demonstrated great potential in combating MDR due to the synergistic effect derived from gene silencing and chemotherapy.4-9 The most distinct feature of this approach is that two therapeutic agents with a specific ratio can be delivered into the same cancer cell simultaneously, thereby creating enhanced MDR reversal effect via combination therapeutic effects of siRNAs and cytotoxic drugs.10-14 Despite the challenge of co-delivery of siRNAs and chemotherapy drugs, impressive progress has been made in this area by the development of various nanoparticles for MDR reversal.15 Some studies aimed to overcome MDR by suppressing the ATP-binding cassette (ABC) transport proteins mediated drug efflux via co-delivery of siRNAs and chemotherapy drugs.16-17 For example, downregulation of P-gp expression by MDR1 siRNA led to the enhanced intracellular accumulation of DOX in the drug-resistance cells and thus a significantly increased toxicity.18-20 2 ACS Paragon Plus Environment

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Other studies aimed to overcome MDR by co-delivery of chemotherapy drugs and siRNAs suppressing over-expression of anti-apoptotic proteins such as Bcl-214, 21-23 and Bcl-xL24. In order to achieve better synchronized release of siRNAs and chemotherapy drugs with remarkably different physiochemical properties, more advanced nanocarriers were developed by integrating intracellular stimuli-responsive function.25-31 For example, the pH responsive micelle responding to endo-lysosomal pH based on N-succinyl chitosan–poly-L-lysine–palmitic acid (NSC–PLL–PA) was developed for co-delivery of DOX and P-gp siRNA against MDR.32 A dually reduction and pH sensitive micelle based on the copolymer of PEG-PAsp (AED)-PDPA was developed to codeliver DOX and Bcl-2 siRNA for an improved MDR reversal effect.33 The dual responsive design of the nanocarriers offered more efficient release of both chemotherapy drugs and siRNAs in the acidic endo-lysosomes, leading to enhanced MDR reversal. Although the stimuli-responsive design has been integrated to optimize the co-delivery of siRNAs and chemotherapy drugs, the key intracellular barrier of the MDR cancer cells remains to be overcome. In terms of siRNA delivery, endosomal entrapment has been considered as the uppermost intracellular barrier for siRNA delivery.34-35 The siRNAs being entrapped in endosomal vesicles will be decomposed by the highly acidic environment and lysosomal hydrolases activated as endosomal vesicles go through a maturation process, leading to an extremely low translocation of siRNA into the cytoplasm.36-38 This becomes even more challenging when it comes to MDR cells because many MDR cancer cells demonstrate an enhanced sequestration of therapeutic agents in endo-lysosomes arising from the augmented volume, number and membrane area of endolysosomal vesicles.39-40 In addition, endocytosis and exocytosis rate are usually accelerated in MDR cells.41 The enhanced sequestration and exocytosis via endo-lysosomal vesicles constituted another important mechanism of drug resistance besides the overexpression of multidrug resistance protein.42-44 Therefore, developing rational nanocarriers which can overcome endolysosomal barrier is highly demanded for effectively co-delivering siRNAs and chemotherapy drugs to the MDR cancer cells. Herein, a pH/redox dually responsive methoxy-poly (ethylene glycol)-polylactidepolyhistidine-ss-oligoethylenimine (mPEG-b-PLA-PHis-ss-OEI) based polyplex with enhanced endo-lysosomal escape design was constructed to co-deliver siRNAs/chemotherapy drugs for MDR reversal. The polyplex consisted of a pH-sensitive PEG-b-PLA-PHis linked with OEI via redox cleavable disulfide bond. The amphiphilic copolymer self-assembled into micelles with 3 ACS Paragon Plus Environment


hydrophobic core for accommodation of water-insoluble drugs as well as the hydrophilic palisade providing the enhanced permeation and retention (EPR) mediated targeting effect. The palisade of the micelles contained cationic OEI, which was capable of forming siRNA polyplexes. The distinctive design of the copolymer was applied to provide the polyplex with accelerated release of payloads in response to endo-lysosomal environment and enhanced endo-lysosomal escape for effective co-delivery against MDR cells (Fig. 1). After being internalized, the acidity and reduction potential inside the endo-lysosomes would induce the protonation of PHis and cleavage of disulfide bonds, eventually resulting in burst release of payloads. Moreover, redox cleavable disulfide bonds design would create the enhanced endo-lysosomal escape of the payloads by producing free OEI that could permeabilize endosomal membrane besides the “proton sponge” effect.45-46 As a proof-of-concept research, siRNA targeting MDR1 gene (MDR1 siRNA) and DOX were selected for a co-delivery study on MCF-7/ADR cancer. In vitro evaluations and in vivo antitumor studies were carried out to elucidate the effective co-delivery of MDR1 siRNA/DOX with the pH/redox dual-responsive polyplex in combating MDR.


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Fig. 1 (a) Schematic illustration of the pH/redox dual-responsive mPPP-ssOEI/DOX/siRNA codelivery polyplex with effective endo-lysosomal escape. (b) Schematic illustration of the multidrug resistant reversal of the polyplex in vivo. The polyplexes are administered through intravenous injection and accumulated in tumor tissue with enhanced permeation and retention (EPR) mediated targeting effect. After being internalized (ⅰ), the acidic and reduction potential environment of endo-lysosome triggers the payloads release and the subsequent endo-lysosomal escape (ⅱ). The free siRNA and DOX will exert the synergistic effect of gene silencing and cell apoptosis to the drug resistant cells (ⅲ). 5 ACS Paragon Plus Environment


2. Methods and materials 2.1 Materials The raw materials used in the synthesis experiment were provided by the following companies: Bioglaco (Beijing, China) [D, L-Lactide]，Sigma (St. Louis, MO) [Poly (ethylene glycol) methyl ether (mPEG, MW 2000 g/mol), OEI (Mw 1.8 k) and Tin (II) 2-ethylhexanoate ((Sn (Oct)2)], J&K Ltd [N, N-Carbonyldiimidazole (CDI), Succinimidyl 3- (2-pyridyldithio) propionate (SPDP), DL-Dithiothreitol (DTT)], Top-peptide (Beijing, China) [Poly-L-histidine (PHis) polypeptide (customized, purity: 96.58%)]. The model drug doxorubicin hydrochloride was purchased from Meilun Biotechnology (Dalian, China). Reagents used for cell culturing were offered by Beyotime Biotechnology Co. Ltd (Nantong, China). FITC Mouse Anti-Human Pglycoprotein(P-gp/FITC) was purchased from BD Biosciences. The antibodies used in western blot experiment were all provided by Abcam (Cambridge, England), and the other related agents were purchased from Thermo Fisher Scientific. The other chemical reagents were analytical pure provided by Concord Technology Co. Ltd. (Tianjin, China). NC-siRNA, FAM siRNA, Cy5siRNA and MDR1 siRNA were customized by GenePharma (Shanghai) (The specific sequences of siRNAs were given in the Supporting Information). 2.2 Synthesis of mPEG-PLA-PHis-ssOEI (mPPP-ssOEI) copolymer The synthesis procedures of mPPP-ssOEI copolymer were based on our previous research.25 In simple terms, mPEG-b-PLA copolymer was firstly synthesized through a ring-opening polymerization reaction initiated by Sn (Oct)2. The end hydroxyl groups of the mPEG-b-PLA were activated with CDI for a coupling reaction with poly-L-histidine to obtain mPEG-b-PLA-PHis-ssPHis-PLA-b-mPEG. After the disulfide bonds was cut off by DTT, the polymer (mPEG-b-PLAPHis-SH) had reducing group-sulfhydryl (-SH) was obtained. mPEG-b-PLA-PHis-ssOEI (mPPPssOEI) was synthesized with thiolated OEI and mPEG-b-PLA-PHis-SH by means of a thioldisulfide exchange reaction. Details of synthesis were shown in Fig. S1. 2.3 Characterizations of the copolymer of mPPP-ssOEI The chemical structures of the intermediates and mPPP-ssOEI were characterized by 1H NMR spectroscopy (Bruker DRX-600) and IR spectrometry, respectively. To determine the molecular weight and its distribution, gel permeation chromatography (GPC) was applied, the optimum conditions were as follows: column: waters Styragel™ HT3; detector: Waters 2414 6 ACS Paragon Plus Environment

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refractive index detector; pump: Waters 1515 HPLC Pump; mobile phase: tetrahydrofuran; flow rate: 1 mL/min; temperature: 35C. The buffering capacity of mPPP-ssOEI was measured by acid-base titration method. In brief, accurately measured 50.0 mg of mPPP-ssOEI copolymer, and dissolved it in 10.0 mL of 0.1 mol/L NaCl solution. Firstly, the pHs of copolymer solutions were adjusted to 12.0 by adding 0.1 mol/L NaOH solution. Then, titrated the solutions with 0.01 mol/L HCl solution to pH 3.0. The 0.1 mol/L NaCl solution served as control. The titration curve was drawn with the pH value as the abscissa and HCl volume being consumed as the ordinate. 2.4 Preparation of mPPP-ssOEI/DOX/MDR1 siRNA polyplexes 2.4.1

Preparation of DOX base Accurately measured 20.0 mg of doxorubicin hydrochloride, and dissolved it in 10.0 mL of

deionized water. After appreciated volume of chloroform was added, triethylamine (molar ratio, triethylamine: DOX = 1.5) was added drop wisely to the aqueous phase to form DOX base followed by portioning into the chloroform phase. The organic phase was evaporated at 37 C under rotary vacuum, and then reconstituted with an appropriate amount of methanol solution to prepare a DOX base stock solution (1 mg/mL). 2.4.2

Preparation of mPPP-ssOEI/DOX/MDR1 siRNA polyplexes Preparation of polyplexes adopted the thin-film dispersion method. 15.0 mg of mPPP-ssOEI

was accurately measured and dissolved in methylene dichloride (10.0 mL). After that, 1.0 mL of DOX base stock solution was added and shook well. The solvent was evaporated at 40C until a uniform thin film was obtained, and evaporated for one night to remove the residual solvent in the film at 25 C. Then, the film was hydrated with 10.0 mL of DEPC-treated deionized water for 30 min followed by sonication (45 kHz) in ice bath for 10 min. The mPPP-ssOEI/DOX micelle and MDR1 siRNA were mixed and incubated for 0.5 h at 25 C to obtain mPPP-ssOEI/DOX/MDR1 siRNA polyplex. The polyplexes at different N/P ratios were prepared by adjusting the proportion of mPPP-ssOEI to siRNA. 2.5 Characterizations of mPPP-ssOEI/DOX/MDR1 siRNA polyplexes 2.5.1 Physicochemical properties of the polyplexes The morphology of mPPP-ssOEI/DOX/MDR1 siRNA polyplex was observed using transmission electron microscopy (TEM, JEM-1230, Japan) with an accelerating voltage of 100 7 ACS Paragon Plus Environment


kV. The polyplex samples were placed on the copper mesh and sustained for 1 min, removed the excessive solution by filter paper. After that, negatively stained by 2% phosphotungstic acid for 3 min, removed the excessive dye solution filter paper, and dried at 25 C. The zeta potentials and particle sizes of the blank polyplexes and the drug loaded polyplexes (N/P ratios: 1, 2, 3, 4, 6, 8, 10, 15 and 20) were determined by Zetasizer Nano ZS (Malvern, UK). 2.5.2 Encapsulation efficiency (EE) and drug loading (DL) of payloads The EE and DL of DOX were determined by ultracentrifugation method. The polyplexes solutions (3.0 mL) were placed in centrifuge tubes and centrifugated (speed: 80000 rpm; time: 2 h), then 2.5 mL of supernatants were collected into 10 mL volumetric flask and dilute with methanol to the volume. The amount of free DOX was determined by the Varioskan Flash multifunctional microplate reader (Thermo Scientific, USA) (Ex: 497nm; Em: 588 nm). 2.5 mL of polyplexes solutions were transferred into a 10 mL volumetric flask and diluted to the volume with methanol to determine the total amount of DOX. Then, the EE and DL of polyplexes were calculated by the following formulas. EE (%) = DL (%) =

ntotal ― nfree ntotal

× 100%

wDOX wDOX + wcopolymer

× 100%

ntotal and nfree were defined as the amount of total and unencapsulated DOX, wDOX and wcopolymer were represented the weight of encapsulated DOX and weight of copolymer, respectively. The amount of FAM-siRNA complexed by the copolymer was determined by the ultrafiltration method. The polyplexes solutions at different N/P ratios (1, 2, 3, 7, 10, 15, 20 and 30) were added into Amicon Ultra-4 centrifugal filter devices (50 kD, Millipore, Massachusetts) and centrifugated (speed: 4000 rpm, time: 10 min). Solutions being centrifugated which contained uncomplexed FAM-siRNA were collected and quantified by microplate reader (Ex: 480 nm; Em: 520 nm). The EE of siRNA was calculated as follows. EE (%) =

ntotal ― nfree ntotal

× 100%

ntotal and nfree represented the amount of total and uncomplexed siRNA, respectively. 2.6 Agarose gel electrophoresis analysis 2.6.1

Binding ability of the mPPP-ssOEI copolymer 8 ACS Paragon Plus Environment

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The mPPP-ssOEI/ siRNA polyplexes solutions at different N/P ratios (0, 1, 2, 3, 4, 5, 6, 7, 8, 10, 15 and 20) were sampled in agarose gel plate (2% (w/v), ethidium bromide (0.5 mg/mL)) which was submerged in electrophoresis tank filled with appropriate amount of Tris-acetate-EDTA buffer. The system was running for 20 min at 100 volts. Then, a Tanon 2500 R automatic digital gel image analysis system was used to obtain visual result. 2.6.2

Resistance to heparin replacement Polyplexes solutions at different N/P ratios (0, 1, 2, 3, 4, 5, 6, 7, 8, 10, 15 and 20) were mixed

with heparin (heparin/siRNA (IU/mg) = 2) by votexing for 10 min. After that, the mixtures were incubated at 25C for 60 min. Then the samples were electrophoresed at 100 volts for 20 min. 2.6.3

Serum stability Fetal bovine serum (FBS) was added into polyplexes solutions at N/P ratios of 7, 10 and 20

and incubated in shaker at 37 C. The polyplexes were sampled at 0 h, 1 h, 2 h, 4 h, 6 h, 8 h,12 h and 24 h and mixed with heparin (heparin/siRNA (IU/mg) = 10) followed by incubation for 1 h at 25 C. Then the samples were electrophoresed at 100 volts for 20 min. 2.7 In vitro release of payloads in response to pH/redox stimuli The phosphate buffer at different pHs (7.4, 6.5, 5.5) with/without 10 mmol/L DTT were selected as the media to simulate biological and early/late endosomal conditions. The polyplexes solutions at different N/P ratios (7, 10, 15, 20) were mixed with release media and incubated for 120 min at 37 C followed by testing in vitro release of siRNA with agarose gel electrophoresis method. For quantitation of siRNA, the samples were ultra-filtrated and analyzed by microplate reader as described before. The in vitro release of free DOX from polyplexes was measured by dialysis method. The dialysis bags containing polyplexes solutions were immersed into the conical flasks with different release media. Then, the conical flasks were putted into a shaking incubator (speed: 100 rpm; temperature: 37 °C), and sampled at 0.5 h,1 h, 2 h, 3 h, 4 h, 5 h, 6 h, 8 h, 10 h, 12 h and 24 h, respectively. The DOX was assayed by the microplate reader. 2.8 Cytological evaluation 2.8.1 Cytotoxicity of the polyplexes against MCF-7/ADR cell The MCF-7/ADR cell line was used to evaluate the cytotoxicity of the polyplexes by MTT assay. The cells were cultivated with RPMI-1640 medium (10% FBS, 1% penicillin- streptomycin). 9 ACS Paragon Plus Environment


To keep the resistance of the cell line to DOX, kept the culture medium containin 1 g/mL of DOX. The cells were inoculated in 96-well plates (5000 cells/well) and incubated for one night. The blank polyplex, free DOX, mPPP-ssOEI/DOX, mPPP-ssOEI/DOX/NC siRNA and mPPPssOEI/DOX/MDR1 siRNA (DOX: 5μg/mL; siRNA: 50 nmol/L) were added and incubated for 48 h, respectively. The medium was set as control group. After that, 20 μL of MTT solution (5 mg/mL) was added and allowing for another incubation for 4 h. Then, the culture media were replaced by 150 μL of DMSO and the optical density (OD) values at 570 nm were measured by the microplate reader. Cell viability (CV) was calculated as follows. CV (%) =

ODsample ― ODblank ODcontrol ― ODblank

× 100%

Where, the ODblank, ODsample and ODcontrol represented the OD values of blank group, treatment group and control group, respectively. The IC50 of free DOX solution and mPPP-ssOEI/DOX/MDR1 siRNA were further investigated by using of MTT assay method. The MCF-7/ADR cells were inoculated in 96-well plates (5000 cells/well) and incubated for one night. A series of DOX solution (1 μg/mL, 5 μg/mL, 10 μg/mL, 50 μg/mL, 100 μg/mL, 150 μg/mL, 200 μg/mL and 300 μg/mL) and mPPPssOEI/DOX/MDR1 siRNA solution (DOX: 1 μg/mL, 3 μg/mL, 6 μg/mL, 10 μg/mL, 20 μg/mL, 40 μg/mL and 80 μg/mL; siRNA: 50 nmol/L) were added and cultured for 48 h, respectively. Then, manifested as described above and the IC50 values were calculated according to nonlinear regression analysis based on the OD values. 2.8.2 Cellular uptake of the polyplexes Confocal laser scanning microscopy (CLSM) and flow cytometry (FCM) were used to investigate the internalization and intracellular release of DOX as well as fluorescence-labeled FAM-siRNA from the polyplexes (DOX: 5 μg/mL; FAM siRNA: 50 nmol/L). MCF-7/ADR cells (1×105 cells/well) were seeded on the coverslips in 6-well plates and incubated for one night. The original media were discarded, and replaced by serum-free medium (SFM). The polyplexes solutions were added and incubated for 1 h, 2 h, 3 h, 4 h and 6 h, respectively. After that, stained the cell nucleus by Hoechst 33258 (50 μg/mL), sealed the coverslips and microscope slides with a glycerin-water mixture followed by observation with CLSM. As for flow cytometry detection, the cells (1×106 cells/well) were inoculated in 6-well plates and incubated for one night. Then, the polyplexes solutions were added and incubated for 1 h, 2 h, 3 h, 4 h and 6 h, respectively. At each 10 ACS Paragon Plus Environment

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time point, the cells were rinsed with precooled PBS thrice, trypsinized and collected, followed by suspending in 500 μL precooled PBS and filtering by a 200-mesh copper screen to ensure the single dispersed state. The mPPP-ssOEI/DOX and mPPP-ssOEI/FAM-siRNA polyplexes were set as fluorescence compensation. In addition, the influence of N/P ratio was also investigated. In brief, the polyplexes solutions at different N/P ratios (7, 10, 15, 20) were incubated with cells seeded into 6-well plates for 4 h, followed by manipulated as described before and detected by FCM. 2.8.3 Intracellular trafficking and subcellular localization The intracellular transport pathway of polyplexes was studied by CLSM with the help of FAM-siRNA, Lyso-tracker Red DND and Hoechst 33258. MCF-7/ADR cells (1×105 cells/well) were seeded on the glass coverslips in 6-well plates and incubated for one night. On the second day, the original culture media were replaced by 2.0 mL of SFM contained polyplexes (FAMsiRNA: 50 nmol/L) at N/P ratio of 7, 10 and 15. The proton pump inhibitor bafilomycin A1 was added to every group, serving as the control group to inspect the mechanism of endo-lysosomal escape.47-48 After incubation for 6 h, the endosome was stained with Lyso-tracker Red DND, fixed (4% paraformaldehyde) for 0.5 h and stained cell nucleus by Hoechst 33258. The images were captured using CLSM. 2.8.4 P-gp silencing efficiency and DOX accumulation inside the cells Gene silencing effects of polyplexes were investigated by utilizing specific binding of P-gp with P-gp/FITC. MCF-7/ADR cells (1×106 cells/well) were inoculated in 6-well plates and incubated for one night. Then, different polyplexes solutions (mPPP-ssOEI/DOX, mPPPssOEI/MDR1 siRNA and mPPP-ssOEI/DOX/MDR1 siRNA (DOX: 5μg/mL; MDR1 siRNA: 50 nmol/L)) were added and co-incubated with the cells for 4 h. Verapamil group and culture medium group were served as the positive control and negative control, respectively. After incubated for another two days, discarded the media and rinsed the cells with PBS and collected. 20 μL of Pgp/FITC was added and allowed to react for 30 min at 4 ℃ in the dark place. After that, the cells were collected and resuspended in 500 μL PBS and detected by FCM. The accumulation of DOX inside MCF-7/ADR cells co-delivering with/without MDR1 siRNA was also studied by detecting red fluorescence by FCM. 2.8.5 Diffusion of propidium iodide (PI) through the permeabilized membrane The SFM at different pHs (7.4, 6.5, 5.5) with/without 10 mmol/L DTT were prepared. MCF7/ADR cells (1×106 cells/well) were inoculated in 6-well plates and incubated for one night. After 11 ACS Paragon Plus Environment


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that, the media were replaced by those prepared before. Then, polyplexes solutions at different N/P ratios (7, 10, 15, 20), OEI solution and 0.17 mmol/L Triton X-100 were added 49-50. Then, 50 µL of PI/H2O solution (5 mg/mL) were added, respectively. After cultured for 1 h, cells were processed as described before and detected by FCM. All the data was normalized to Triton X-100. 2.8.6 Hemolytic effects of the polyplexes Rabbit erythrocyte was used to investigated the hemolytic effects of the polyplexes.51 To isolated the erythrocyte, after the blood was collected, centrifugated (speed: 2000 rpm; time: 10 min; temperature: 4C) and washed until the supernatant was colorless. The cell pellets were diluted with different media (0.1 mol/L PBS buffer solutions at pH 7.4, 6.5 and 5.5, with/without 10 mmol/L DTT) to the erythrocyte concentration of 0.2%. Then, the polyplexes solutions at different N/P ratios (7, 10, 15, 20), OEI solution and 0.17mmol/L Triton X-100 were mixed with the erythrocyte suspension, separately, and incubated at 37 °C for 60 min. The supernatants post centrifugation were translocated into 96-well plates, and the OD values were detected by microplate reader (540nm) to determine released hemoglobin. The relative hemolysis rates were calculated as follows and all the data was normalized to Triton X-100. Relative hemolysis rate (%) =

ODsample ― ODPBS buffer ODTriton ― ODPBS buffer

× 100%

Where, the ODPBS buffer, ODsample and ODTriton represented the OD values of blank group, treatment group and positive control group, respectively. 2.8.7 Western blot In the western blot analysis, MCF-7/ADR cells (1×105 cells/well) were inoculated in 6-well plates and incubated for one night. DOX + MDR1 siRNA solution and polyplexes solutions (DOX, 5μg/mL; MDR1 siRNA, 50 nmol/L) at different N/P ratios were added and incubated for 4 h. Then, original media were discarded and replaced by fresh RPMI-1640, and further incubated for two days. Then, cells were rinsed with precooled PBS and collected, lysed on ice for 0.5 h. The supernatants post centrifugation (speed: 16000 rpm; time: 5 min) were isolated and quantified by the BCA protein assay kit. Proteins were sampled and separated on the SDS-PAGE in equal amount loading method, transferred onto the polyvinylidene difluoride (PVDF) membranes, blocked with skim milk, and incubated with P-gp and -actin primary antibody for one night at 4C, respectively. After washed by TBST, PVDF membranes were incubated with the horseradish peroxidase (HRP)-conjugated secondary antibody for 2 h at 25 C and washed by TBST again. 12 ACS Paragon Plus Environment

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After that, let the protein on PVDF membrane interact with ECL western blotting substrate for appreciated time, and visualized the bands under MicroChemi 4.2 Gel Imager (DNR, Israel). 2.9 In vivo study in MCF-7/ADR tumor xenograft nude mouse model 2.9.1 Biodistribution in nude mouse model The tumor-targeting ability of the polyplexes was assessed on the MCF-7/ADR tumor xenograft nude mouse model. MCF-7/ADR cells (1×107 cells) were injected subcutaneously into female BALB/c nude mice in right axillary fossa to establish the tumor model. The mice (18~22 g) were purchased from the Shenyang Pharmaceutical University (Shenyang, China) Experimental Animal Center. All animal experiments were carried out in accordance with the National Institutes of Health guide for the care and use of Laboratory animals (NIH Publications No. 8023, revised 1978).

The mice were divided into three groups randomly after the tumor volume reached

appreciated size. Then naked Cy5-siRNA, the polyplexes solutions at N/P ratio of 7 and 15 (DOX: 5mg/kg; Cy5-siRNA: 1mg/kg) were administered by tail vein injection, respectively. The fluorescence signals were recorded at 2 h, 4 h, 8 h, 12 h and 24 h after administration by Carestream FX PRO Image System (Carestream Health, USA) (channel was limited to Cy5 (Ex: 630; Em: 670), therefore DOX fluorescence would not influence the results). The mice were sacrificed at the last timepoint and the tumors, hearts, livers, spleens, lungs and kidneys were isolated for observation. The fluorescence intensities of Cy5 signals were analyzed with Carestream MI SE. To verify the in vivo co-deliver ability of polyplex, the biodistribution of DOX was detected as well. The tumor bearing mice were divided into ten groups randomly, and administered free DOX solution and polyplex solution (N/P 7; DOX: 5mg/kg; MDR1 siRNA: 1mg/kg), respectively. At 2 h, 4 h, 8 h, 12 h and 24 h after administration, the mice were sacrificed, and the main organs and tumor tissues were collected and detected the DOX fluorescence signals (Ex: 497 nm; Em: 588 nm). 2.9.2 Tumor suppression efficiency The antitumor effects of the polyplexes were assessed on the MCF-7/ADR tumor xenograft nude mouse model. After the tumor reached about 100 mm3, the mice were randomized to receive 0.2 mL of saline, DOX + MDR1 siRNA solution and polyplexes solutions (DOX: 5mg/kg; siRNA: 1mg/kg) at different N/P ratios (7, 10, 15, 20) through tail vein injection, respectively. The day 13 ACS Paragon Plus Environment


was denoted Day 0. After that, the mice were administered once every three days and three times in a row. The tumor volumes (length × (width)2/2) and body weights of the mice were monitored every other day. After the last recording (Day 22), the mice were sacrificed and the tumors were isolated and weighed. The relative tumor volume (RTV) and the ratio of tumor weight to body weight (Tumor/Body weight) were calculated by the following formulas. The in vivo gene silencing efficiency analysis was conducted by western blot as described in section of 2.8.7. RTV =

Vn V0

Tumor Bodyweight =

Wtumor Wbody

Where, V0 and Vn were defined as the tumor volume on Day 0 and the day being recorded, Wtumor and Wbody represented the weight of tumor and mice after experiment, respectively. 2.10 Statistical analysis All the experiments were carried out three times in parallel at least. The data was presented as 𝑋 ± 𝑆𝐷 form. Student’s t-test and one-way ANOVA were used to statistical analysis * P < 0.05, ** P < 0.01, and *** P < 0.001. 3

Results and Discussion

3.1 Characterization of the copolymer and mPPP-ssOEI/DOX/MDR1 siRNA polyplexes In the 1H NMR spectrum (DMSO-d6) of copolymer (Fig. 2a), the zero point was defined as tetramethylsilane signal, and chemical shifts of the peaks were expressed in parts per million (δ). The characteristic peaks of mPPP-ssOEI including the followings: mPEG-b-PLA: δ a 3.31 ppm (－OCH3)，δ b 3.61 ppm (－OCH2CH2O－), δ c 5.20 ppm (－COCH (CH3) O－) and δ d 1.46 ppm (－COCH (CH3) O－) ; PHis: δ e 4.91 ppm (－CH－), δ g 7.67 ppm (N－CH＝C) and δ h 7.91 ppm (N－CH＝N), as well as the characteristic peaks of OEI at 2.29-2.72 ppm. According to the GPC result (Fig. 2b), the copolymer showed a unimodal distribution and possessed a small PDI (＜1.1), and weight-average molecular weight (Mw) and number-average molecular weight (Mn) were 7660 Da and 7114 Da, respectively. The acid−base titration curve of the copolymer was 14 ACS Paragon Plus Environment

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presented in Fig. 2c. Comparing with control group (NaCl solution), the pH value of copolymer group declined gradually between 7.4 and 4.0 as the volume of HCl increase, indicating that the copolymer possessed obvious buffering capacity which could binding protons under acidic environment.

Fig. 2 The characterization results of synthesized copolymer of mPPP-ssOEI, (a) 1NMR spectrum, (b) GPC spectrum and corresponding result and (c) Acid-base titration profile. The mPPP-ssOEI copolymer was constructed to be amphiphilic and cationic by integrating hydrophilic PEG, hydrophobic PLA and cationic OEI, which facilitated the self-assembly into the polyplex with negative MDR1 siRNA and accommodating hydrophobic DOX. The complexation ability of the copolymer against MDR1 siRNA was studied by measuring the zeta potential and 15 ACS Paragon Plus Environment


particle size of the polyplexes (Fig. 3a). The particle size of polyplexes significantly declined from 658 ± 22.74 nm to 116 ± 13.80 nm as N/P ratio augmented from 1 to 8 due to the increasing of the electrostatic attractive force. The particle size of the polyplexes reached plateau about 120 nm when the N/P ratio higher than 6, which was consistent with TEM result (Fig. 3b). The EE of siRNA increased as N/P ratio raised, achieving more than 80% at N/P ratio of 3 and with the maximum encapsulation efficiency for 95.69 ± 2.22% (Fig. 3c). The EE and LD of DOX was 80.32 ± 1.44% and 4.81 ± 0.95%, respectively. These results suggested the copolymer featured excellent ability to complex siRNAs and encapsulate hydrophobic chemotherapeutic agents.


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Fig. 3 The characterization results of mPPP-ssOEI/DOX/MDR1 siRNA polyplex. (a) Morphology image of mPPP-ssOEI/DOX/MDR1 siRNA polyplexes captured by TEM, scale bar represents 200 nm. (b) Zeta potential values and particle sizes of polyplexes at different N/P ratios. (c) Encapsulation efficiency of siRNA at different N/P ratios (1, 2, 3, 7, 10, 15, 20 and 30). (d), (e) and (f) Agarose gel electrophoresis analysis results for complexation ability of the copolymer to siRNA, ability to resist anion decomplexation and ability to protect from RNase degradation. (n = 3). The gel retardation assay, heparin decomplexation and serum stability assay were conducted to evaluate the stability of the polyplexes. The gel retardation assay result indicated that when N/P ratio greater than 4, migration of the siRNA could be the effectively retarded by the copolymer. As expected, the polyplexes with higher N/P ratios exhibited stronger complex ability due to the stronger electrostatic interactions (Fig. 3d). The heparin decomplexation assay demonstrated that when N/P ratio was higher than 6, polyplexes could entirely withstand the replacement of anionic heparin (Fig. 3e). With respect to the serum stability test, after co-incubated with serum for 4 h, almost all of the naked siRNA was degraded, while all the polyplexes could protect the siRNA from the nuclease degradation until 12 h (Fig. 3f), demonstrating the polyplexes possessed a good siRNA protection ability. It was worth to note that the excellent siRNA complex ability of the copolymer was achieved at relative low N/P ratios with oligoethylenimine (OEI) which was usually referred to the PEIs with molecular weight less than 2kDa. This could be given the credit to PLA-PHis block involved in copolymer which interacted with siRNA through the nonelectrostatic force, leading to enhanced stability of the polyplexes.52 The use of OEI at low N/P ratio to complex siRNA holds great significance in reducing the cytotoxicity of high molecular weight PEIs, which being suffered from severe limitations.53 3.2 In vitro DOX/siRNA release in response to pH and redox potential Rationally designed nanocarriers offer an approach to synchronize the delivery and the release profiles of chemotherapeutics and siRNAs despite their distinct difference in physical, chemical, and biological characteristics.4 This synchronization allows a certain amount of chemotherapeutics and siRNAs to be delivered into the same cancer cell simultaneously, therefore producing synergistic effect.10,

54

The polyplexes were designed to integrate endo-lysosomal

stimuli triggered payloads release because most of nanocarriers are internalized by endocytosis pathway, ending up in endo-lysosomes. The in vitro release profiles of the payloads from 17 ACS Paragon Plus Environment


polyplexes were evaluated under the conditions which could simulate the biological and endolysosomal environments. To guarantee the rationality of the release test, sink condition and free diffusion of DOX from the dialysis membrane was confirmed previously. As shown in Fig. 4a, the release of DOX from polyplexes at endo-lysosomal pH (~5.0) was much faster than that at physiological pH (7.4). Within 12 hours, the polyplexes released 39.12 ± 1.06% of DOX at pH 7.4, indicating the good DOX encapsulation and low DOX leakage in the physiological environment. In contrast, the polyplexes released about 89.24 ± 1.18% and 70.33 ± 1.63% of DOX at pH 5.5 and 6.5 in the same period, respectively. In addition, the DOX release from the polyplexes was not affected by the presence of DTT, this suggested that the DOX was incorporated in hydrophobic PHis-PLA domain and release of the DOX from the polyplexes was accelerated by the protonation of PHis blocks under the acidic pH. The protonation degree of the PHis blocks increased as pH decline, resulting more DOX release at lower pH.

Fig. 4 The influence of pH/redox stimuli on the in vitro release profiles of payloads from polyplexes. (a) The release profiles of DOX from the polyplex (N/P 7) assessed by microplate reader. (b) The release profiles of siRNA from polyplex (N/P 7) investigated by fluorescent 18 ACS Paragon Plus Environment

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intensity determination. (c) and (d) The release profiles of siRNA from polyplexes at different N/P ratios (7, 10, 15 and 20) investigated by agarose gel electrophoresis and fluorescent intensity determination, respectively. * P < 0.05, ** P < 0.01, and *** P < 0.001 (n = 3). Furthermore, the release of siRNA from the polyplexes under the stimulation of pH/redox was investigated. The polyplexes displayed pH triggered siRNA release profiles. The release of siRNA from polyplex (N/P 7) was significantly augmented from 8.30 ± 0.63% to 25.64 ± 1.41% and further increased to 40.41 ± 1.33% when the pH decreased from 7.4 to 6.5 and further down to 5.5, respectively (Fig. 4b). It’s worth to note that the presence of DTT further contributed to siRNA release from polyplexes under low pHs (pH 5.5 and pH 6.5). The siRNA polyplex was constructed at low N/P ratio (7) with OEI with molecular weight 1.8 kDa in order to reduce the effect of electrostatic interactions on the siRNA release. The siRNA polyplex was stabilized by the combination of electrostatic interactions provided from OEI and non-electrostatic interactions provided from PHis-PLA blocks. The acidic pH triggered the protonation of PHis, making it more hydrophilic. The protonation of PHis would compromise the structure integrity of the polyplex by hydrophobic-hydrophilic transition and translocation of PHis blocks, as well as the inducement of water molecules in the polyplex. This change would diminish the stabilization effect of nonelectrostatic interactions provided from PHis-PLA blocks.25, 55 Moreover, PHis blocks showed no electrostatic binding with siRNA under pH of 5.5 despite the increased protonation because of the low cationic charge density (Fig. S2). OEI protonation was increased by about 10% as pH declined from 7.4 to 5.556, demonstrating a slight increase of electrostatic interactions with siRNA. Therefore, under the acidic pH, the polyplex was deprived of the stabilization interactions from PHis-PLA blocks with almost unchanged electrostatic interactions, causing the burst release of siRNA. The protonation of PHis also resulted in the exposure of the disulfide bonds for cleavage in presence of DTT, further triggering the siRNA release.25 The accelerated siRNA release profile was verified dependent on N/P ratio. Stimuli triggered siRNA release was markedly restrained when the N/P ratio was raised above 7 because the strong electrostatic attractive force between siRNA and the cationic copolymer, which could not be overcome by the pH/redox potential induced polyplex structure change (Fig. 4c, d). The accelerated release of siRNA from the carrier in the endo-lysosomes is highly expected for efficient siRNA delivery to the MDR cells because of the enhanced sequestration and exocytosis via the endo-lysosomal vesicles. 19 ACS Paragon Plus Environment


3.3 In vitro cytotoxicity It could be seen from Fig. 5a, comparing to blank control group, the free DOX solution treatment group showed cell viability higher than 80% and revealed no significant difference, suggesting that DOX could not exerted effective antitumor effect to drug resistant cells. The mPPP-ssOEI/DOX groups exhibited moderate cytotoxicity against MCF-7/ADR cells due to different uptake mechanism of polyplex with free DOX, which could bypass the P-gp pumps. However, without the help of MDR1 siRNA, the intracellular delivery of DOX could be blocked by resistant cells with mediation of active functioning P-gp pumps before exerting cytotoxicity effect. Comparing to mPPP-ssOEI/DOX, codelivery of DOX and MDR1 siRNA by the polyplexes at N/P ratios lower than 15 exhibited significantly lower cell viability (P < 0.05). The cytotoxicity of the polyplex at N/P of 7 (47.11 ± 2.19%) was strongest and markedly decreased as the N/P ratio augmented (Fig. 5a). The cell viability increased to about 80% when the N/P ratio got higher than 20 indicating N/P ratio dependent cytotoxicity of the polyplex. The markable difference between mPPP-ssOEI/DOX/NC siRNA and mPPP-ssOEI/DOX/MDR1 siRNA indicated that the cytotoxicity of siRNA was sequence specific. The intracellular uptake of DOX and P-gp expression measurements verified that higher cytotoxicity of polyplexes at lower N/P ratios were attributed to the higher MDR1 gene silence efficiency in the MCF-7/ADR cells (Fig. 5b, c, d), leading to higher DOX accumulation inside the drug resistant cells (Fig. 5e). The IC50 result (Fig. S3) could further confirm this characteristic, comparing to the IC50 value of free DOX solution (122.60 ± 3.61 μg/mL) against MCF-7/ADR cells, the polyplex with N/P ratio at 7 exhibited the lowest IC50 value (12.97 ± 2.43 μg/mL) and the reversal factor was approximately ten.


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Fig. 5 (a) In vitro cytotoxicity of polyplexes at different N/P ratios (7, 10, 15, 20 and 30) against MCF-7/ADR cells. (b) P-gp gene silence efficiency of different formulations against MCF-7/ADR cell line after 48 h co-incubation investigated by western blot analysis. (c) P-gp expression in MCF-7/ADR cells detected by FCM and presented in the form of histogram profile. (d) The quantitively result of P-gp gene silence efficiency calculated based on gray value in western blot result. (e) DOX accumulation inside MCF-7/ADR cells detected by FCM. * P < 0.05, ** P < 0.01, and *** P < 0.001 (n = 3).



3.4 Cellular uptake and endo-lysosomal escape of the payloads For the sake of investigating mechanism underlying the impact of N/P ratio on cytotoxicity of polyplex, the characteristic of cellular internalization and the mechanism of endo-lysosomal escape of payloads were studied, respectively. The cellular uptake behaviors of payloads were studied using both CLSM and FCM. The CLSM analysis (Fig. 6) revealed cellular uptake of FAM siRNA (green) and DOX (red) were both time-dependent. During 1 h, both of DOX and FAM fluorescence intensity were very weak and increased with the incubation time. The overlap of the two appeared yellow indicating that effective co-delivering DOX and siRNA. The appearance of diffused distribution of green and red signal as well as the approaching of red signal to the nucleus indicated the translocation of siRNA and DOX from the endo-lysosomes into the cytoplasm. Additionally, as time proceeded, the purple signal (overlap of DOX and Hoechst 33258) increased gradually, suggesting the DOX released from the polyplex and migrated into the nuclear region.


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Fig. 6 The cellular uptake behavior of polyplex (N/P 7) against MCF-7/ADR cells detected by CLSM.

Images were captured at the time point of 1 h, 2 h, 3h, 4 h, and 6 h post the co-incubation

of mPPP-ssOEI/DOX/FAM siRNA polyplexes with MCF-7/ADR cells. Nucleus was stained by Hoechst 33258 (blue) and siRNA labelled by FAM (green). We further used FCM to quantitatively estimate the transfection efficiency. As illustrated in Fig. 7a, the cellular uptake behavior presented time-dependent characteristic which was in accordance with CLSM result. In addition, because of the negative potential of the cell membrane, the delivery systems with positive charge will be more likely internalized by cells due to the 23 ACS Paragon Plus Environment


enhanced electrostatic interactions. The cellular uptake behavior of polyplex showed N/P ratio dependence (Fig. 7b, c). As N/P ratio got higher, both DOX and FAM fluorescence intensity inside the cells were strengthened because of the higher zeta potential (Fig. 3b).

Fig. 7 (a) Internalization behavior of DOX and FAM siRNA against MCF-7/ADR cells detected by FCM shown in the form of scatter diagram of two parameter histogram. The mPPP-ssOEI/DOX and mPPP-ssOEI/FAM-siRNA polyplexes were set as fluorescence compensation. (b) and (c) The influence of N/P ratio (7, 10, 15 and 20) on cell uptake of polyplexes determined by FCM, fluorescence signals of DOX and FAM were presented by histogram in different channels, respectively. To investigate the transportation of the payloads in the cells and its endo-lysosomal escape, the fluorescence signals of FAM-labeled siRNA inside cells were detected by CLSM with Lipo2000 as a negative control. As shown in Fig. 8, the polyplex (N/P 7) treated cells presented 24 ACS Paragon Plus Environment

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much broader distribution of green fluorescence signal (FAM siRNA) and much less distribution of orange fluorescence signal (overlap of FAM siRNA and Lysotracker Red) than the other three polyplexes (N/P 10, 15 and 20). This indicated that the endo-lysosomal escape of the payloads became worse as the N/P ratio raised. It should be noticed, as N/P ratio increased, the polyplex facilitated endo-lysosomal escape was not reinforced as expected. This seemed to be contradicted to the “proton sponge” effect, in which the endo-lysosomal escape was supposed to be enhanced due to more nitrogen available for protonation. It could be assumed that other mechanism than “proton sponge” effect was involved in endo-lysosomal escape of the payloads. Recent studies indicated that PEI facilitated the endo-lysosomal escape by inducing membrane destabilization effect.45-46

Fig. 8 Intracellular trafficking of mPPP-ssOEI/FAM siRNA at different N/P molar ratios (7, 10, 15, 20) were examined by CLSM. The nucleus and endosome were stained by Hoechst 33258 and Lysotracker Red which showed blue and red fluorescence signal, respectively. siRNA was labelled by FAM which gave green signal fluorescence. To prove the endosomal membrane destabilization effect, bafilomycin A1, a proton pump inhibitor was used to shut off the “proton sponge” effect facilitated endo-lysosomal escape. As it could be seen in Fig. 8, the addition of bafilomycin A1 slightly affected the endo-lysosomal escape 25 ACS Paragon Plus Environment


behavior of polyplex at N/P ratio of 7, which still presented most broadly distribution of FAM siRNA in cytoplasm. In contrast, endo-lysosomal escape was remarkedly suppressed by bafilomycin A1 in polyplexes at N/P ratios of 10, 15 and 20, indicating that “proton sponge” effect assisted endo-lysosomal escape of these polyplexes, primarily. Considering in vitro release result that polyplex at N/P ratio of 7 favored cutoff OEI blocks via disulfide bonds cleavage, the enhanced endo-lysosomal escape could be attributed to the effect exerted by the free OEI blocks. To confirm the contribution of OEI facilitating endo-lysosomal escape, the membrane destabilizing activity of the polyplex was further investigated by measuring the red blood cell hemolysis and fluorescence intensity of PI inside the cells, an indicator of diffusion-in. Erythrocyte has been considered as a more biologically relevant model to study membrane property of the carriers.57-58 By detecting the lysis of erythrocyte and release of hemoglobin in the medium, the membrane destabilizing effect of the polyplex could be elucidated. Therefore, the hemolytic activities of the polyplexes formulations was assessed by measuring the erythrocyte lysis, with saline and OEI solution as negative control and positive control, respectively. As it displayed in Fig. 9a, the polyplexes showed pH and DTT dependent hemolysis. The polyplexes exhibited comparable hemolysis with negative control at pH 7.4, indicating the good biocompatibility. However, the hemolysis of the polyplexes significantly increased under acidic conditions. Moreover, the presence of DTT further increased the hemolysis of the polyplexes at the same pH condition. The hemolysis ratios of polyplexes to free OEI (positive control) indicated that about 70% and 50% OEI were cut off under pH 5.5 + DTT and pH 6.5 + DTT conditions, respectively, which was in accordance with the in vitro release data (Fig. 4d). Furthermore, N/P ratio had a remarkable influence on hemolysis of the polyplexes (Fig. 9b). With N/P ratio augmented from 7 to 10, hemolysis rate declined sharply from 50.74 ± 4.59% to 15.40 ± 3.15%. Further increasing N/P ratio showed moderate effect on the hemolysis of the polyplexes. PI is a kind of nuclear stain reagents which can pass through the permeabilized membrane but intact one, means that the dead cells or cells with destabilized membrane can be stained but the living cells with integrated membrane.59-60 Therefore, the enhanced fluorescence intensity of PI indicates an augment in membrane permeability. The PI signal intensity showed right shifts after incubation of MCF-7/ADR cells with the polyplex (N/P 7) under acidic conditions in presence of DTT. The largest fluorescence shift was observed under pH 5.5 + DTT condition, followed by pH 5.5, pH 6.5 + DTT and pH 6.5 (Fig. 9c). As expected, the N/P ratio had a remarkable influence 26 ACS Paragon Plus Environment

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on the fluorescence intensity of PI (Fig. 9d), indicating the membrane destabilization effects of the polyplexes was N/P ratio dependent. These results suggested that the redox potential cutoff OEI blocks from the polyplexes played a dominant role in facilitating the membrane destabilization and the following payloads endo-lysosomal escape. Lower N/P ratio favored payloads endolysosomal escape due to the higher cutoff efficiency of OEI under the endo-lysosomal environment. The augment of N/P ratio of polyplex led to an enhance in electrostatic attractive force between OEI and siRNA, which compromised the cleavage of OEI from the copolymer and the following endo-lysosomal escape, resulting in lower delivery efficiency of DOX and siRNA. Because of the enhanced sequestration of drug resistant cells, nanocarriers equipped with effective endolysosomal escape are highly expected for successful delivery of therapeutic agents. In this study, the polyplexes were integrated a triggered OEI release in endo-lysosomes for enhanced endolysosomal escape of the payloads via the combination effect of OEI induced membrane destabilization and “proton sponge” effect.



Fig. 9 (a) and (b) The effect of stimuli and N/P ratio to hemolytic activities of polyplexes by measuring lysis of erythrocyte. (c) The influence of stimuli on the membrane destabilization effects of polyplexes through detecting fluorescence intensity of PI inside MCF-7/ADR cells by FCM. (d) Representative histogram profiles of fluorescence signal of PI being taken up by MCF-7/ADR cells after interacted with polyplex (N/P 7). (e) The influence of N/P ratio on the membrane destabilization effects of polyplexes through detecting fluorescence intensity of PI inside MCF28 ACS Paragon Plus Environment

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7/ADR cells by FCM. * P < 0.05, ** P < 0.01, and *** P < 0.001 (n = 3). 3.5 Biodistribution and tumor suppression efficiency of different polyplexes The tumor targeting abilities of systemically-delivered polyplexes were investigated on MCF7/ADR tumor xenograft nude mouse by Carestream FX PRO Image System. As illustrated in Fig. 10a, naked siRNA was primarily distributed in liver and was quickly metabolized. In contrast, the polyplexes exhibited effective accumulation at tumor site and the fluorescence in tumor was sustained for more than 24 h. Two polyplexes showed similar biodistribution behavior in mice. The images (Fig. 10b) of isolated organs and tumor tissues further demonstrated that the polyplexes were primarily accumulated in tumors. These results indicated the good tumor targeting abilities of the polyplexes, arising from the suitable particle size (Fig. 3b), good stability and “stealth effect” from PEGylation.61 The biodistribution of DOX was also investigated (Fig. S4), and the result was consistent, demonstrated that the copolymer possessed the ability to co-deliver MDR1 siRNA and DOX in vivo.

Fig. 10 (a) In vivo ﬂuorescence signal changes of MCF-7/ADR tumor-bearing nude mice as the time passed away after intravenous injected naked Cy5 siRNA and polyplexes (DOX: 5mg/kg; MDR1 siRNA: 1mg/kg) at N/P ratios of 7 and15. (b) The fluorescence signals of the tumor tissues 29 ACS Paragon Plus Environment


and major organs at 48 h post-injection. To assess the antitumor efficacy in vivo, control formulations (DOX + MDR1 siRNA, polyplex (DOX + NC siRNA) and saline) and polyplexes (DOX: 5mg/kg; Cy5-siRNA: 1mg/kg) at different N/P ratios (7, 10, 15, 20) were administered to the MCF-7/ADR tumor xenografted nude mice, respectively. As shown in Fig. 11a, compared with control treatment groups which relative tumor volume reached about 4.5, co-delivery of DOX and MDR1 siRNA by the polyplexes exhibited much stronger tumor inhibition effect. Among the different polyplexes, polyplex at N/P ratio of 7 (1.22 ± 0.17) demonstrated the strongest tumor inhibition followed by the polyplexes at N/P ratios of 10 (2.50 ± 0.46), 15 (3.33 ± 0.24) and 20 (3.60 ± 0.29). The N/P ratio dependent tumor inhibition was in accordance with the in vitro result (Fig. 5a), which could be attributed to the compromised siRNA delivery efficiency in polyplexes with higher N/P ratio. In addition, polyplex (DOX + NC siRNA) and polyplex (DOX + MDR1 siRNA) at N/P ratio of 7 existed significant difference, indicated that co-delivery MDR1 siRNA with DOX produced synergistic effect in reverting the multidrug resistance. The data of Tumor/Body weight (Fig. 11b) was in consistent with the result. Moreover, the similar weight changes of nude mice (Fig. 11c) in polyplexes treated groups and saline control group demonstrated the preferable compatibility of the polyplexes. P-gp expression in tumor tissues treated by different formulations were shown in Fig. 11e and Fig. 11f. The polyplex (N/P 7, DOX + MDR1 siRNA) treated group showed the lowest P-gp expression (35.15 ± 2.17%) followed by the polyplexes at N/P ratio of 10 (65.27 ± 3.37%), 15 (88.14 ± 4.09%) and 20 (92.88 ± 2.51%). The higher P-gp knockdown efficiency certainly contributed to the better sensibilization of DOX and better tumor multidrug reversal.


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Fig. 11 (a) The relative tumor volumes as the tumor inhibition experiment progressing. (b) Tumor/Body weight of nude mice calculated based on the corresponding data at the end of study. (b) The body weight changes of nude mice. (d) The images of tumor bearing nude mice and isolated tumor tissues in each group at the end of experiment. (e) and (f) P-gp expression in tumor tissues analyzed by western blot and the corresponding quantitative data figured according to gray value. * P < 0.05, ** P < 0.01, and *** P < 0.001 (n = 6). 31 ACS Paragon Plus Environment


Conclusion The pH/redox dual responsive mPEG-PLA-PHis-ssOEI copolymer based polyplex with effective endo-lysosomal escape was designed to co-deliver siRNA and DOX for MDR reversal. The polyplex exhibited excellent ability in complexing siRNA and encapsulating Dox. Polyplex at N/P ratio of 7 showed more efficient pH/redox triggered release and more effective endolysosomal escape of the payloads than the polyplexes at higher N/P ratios, thereby higher intracellular payloads delivery efficiency. This was attributed to the more effective endolysosomal escape facilitated by the redox potential cutoff OEI blocks from the copolymer, which permeabilized the endosomal membrane. The augment of N/P ratio led to over strong electrostatic interaction between siRNA and OEI which resulted the triggered release of payloads and subsequent endo-lysosomal escape being impeded. In vivo evaluation further confirmed that polyplex at N/P ratio of 7 exhibited the strongest tumor growth inhibition and the highest efficiency in downregulation of P-gp expression of MCF-7/ADR cell. Overall, polyplex with effective endolysosomal escape has been demonstrated as a rational delivery system to conquer the multidrug resistant cancers with enhanced endo-lysosomal sequestration and to achieve efficient co-delivery of chemotherapeutics and siRNAs. Acknowledgements The authors are grateful to the National Natural Science Foundation of China for financial support (No. 81573372) and Career Development Support Plan for Young and Middle-aged Teachers in Shenyang Pharmaceutical University (ZQN2014A03). Supporting information The sequences of siRNAs involved in the study. The synthesis flow chart of mPEG-b-PLAPHis-ssOEI copolymer and the exactly synthesis steps. The complexation ability of mPEG-PLAPHis copolymer against siRNA. Result for the IC50 determination. In vivo distribution of DOX. References (1) Livney, Y. D.; Assaraf, Y. G. Rationally Designed Nanovehicles to Overcome Cancer Chemoresistance. Adv. Drug Delivery Rev. 2013, 65 (13-14), 1716-1730. 32 ACS Paragon Plus Environment

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