Reactive Oxygen Species-Biodegradable Gene Carrier for the

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ROS-Biodegradable Gene Carrier for Targeting Therapy of Breast Cancer Chunhui Ruan, Lisha Liu, Qingbing Wang, Xinli Chen, Qinjun Chen, Yifei Lu, Yu Zhang, Xi He, Yujie Zhang, Qin Guo, Tao Sun, and Chen Jiang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b01712 • Publication Date (Web): 02 Mar 2018 Downloaded from http://pubs.acs.org on March 2, 2018

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ROS-Biodegradable Gene Carrier for Targeting Therapy of Breast Cancer Chunhui Ruan,1 Lisha Liu,1 Qingbing Wang,2 Xinli Chen,1 Qinjun Chen,1 Yifei Lu,1 Yu Zhang,1 Xi He,1 Yujie Zhang,1 Qin Guo,1 Tao Sun,*,1 Chen Jiang1 1

Key Laboratory of Smart Drug Delivery, Ministry of Education, State Key

Laboratory of Medical Neurobiology, Department of Pharmaceutics, School of Pharmacy, Fudan University, Shanghai 200032, China 2

Department of Interventional Radiology, Ruijin Hospital Shanghai Jiao Tong

University School of Medicine Shanghai 200025, PR China

1

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Abstract An ideal gene-carrying vector is supposed to exhibit outstanding gene condensing capability with positively charged macromolecules to protect the carried gene during in vivo circulation, and a rapid dissociation upon microenvironmental stimuli at the aimed sites to release the escorted gene. Currently, it still remains a challenge to develop an ideal gene carrier with efficient transfection ability and meanwhile low toxicity

for

clinical

applications.

Herein,

we

innovatively

introduced

a

ROS-biodegradable boric acid ester linkage in elaborating the design of gene carrier. In virtue of the featured intracellular characteristics such as the high level of reactive oxygen species (ROS) in tumor cells, a ROS-biodegradable electropositive polymer derived from branched polyethyleneimine (BPEI) with low molecular weight (1.2 K) through a crosslinking reaction by boric acid ester bond was developed in this study to achieve condensation and escorting of carried genes. Furthermore, the polymer was modified with substance P (SP) peptide as the targeting ligand through polyethylene glycol (PEG). The final fabricated SP-crosslinked BPEI/pDNA nanoparticles (NPs) exhibit favorable biocompatibility, ROS-cleavability, fine targeting ability, as well as high transfection efficiency compared with parental BPEI1.2 K both in vitro and in vivo. SP-crosslinked BPEI/siRNA (Plk1) polyplex possesses favorable gene-silencing effects in vitro and satisfactory antitumor ability in vivo. Hopefully, this novel crosslinked electropositive polymer may serve well as a safe and efficient gene delivery vehicle in clinic.

Keywords: Gene delivery; Polyethyleneimine; ROS-responsive; Substance P; Plk1

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Introduction Malignant tumors have undoubtedly become the most fatal pathema and the most serious threat to human health normally with unsatisfactory treatment effect as well as the mounting incidence rate. Conventional treatments including surgery and following radio/chemotherapy suffer from their own limitations: surgery can hardly eradicate the whole bulk of tumor which causes the inescapable risk of relapse; radiotherapy may induce the damage of normal organs and immune system; chemotherapy potentially results in a systemic toxicity and tumor’s drug resistance.1,2 Under the circumstances, gene therapy represents a promising strategy, which is characterized by counteracting or replacing aberrant gene adversely affected by the condition in a precise and specific manner.3-5 Encouragingly, the prospect of gene therapy has been cheerful since the market authorization of many oligonucleotides-based drugs such as Gendicine, Glybera and Strimvelis.6,7 The recent development of CRISPR/Cas9 system definitely casts a new light on the precise gene-based cancer therapy.8,9 The therapeutic effect of gene therapy greatly relies on the gene-delivery vehicle that mediates the transfection, no matter for viral or non-viral vehicles. High efficient viral vectors commonly generate immune reactions, which greatly limits their clinical application. In this context, non-viral gene-delivery strategies have attracted much attention and have been a hotspot during the past few years.10-12 An ideal non-viral gene delivery vehicle is supposed to exhibit the following traits: i) favorable biocompatibility; ii) outstanding gene condensing capability with positively charged macromolecules to condense and protect escorted genes during in vivo circulation; iii) fine targeting ability. To some extent, the first two characteristics above are relatively indispensable and somehow contradictory in view of the fact that the electropositive macromolecular materials hold fine condensing efficiency but are bio-refractory into fragments to release the escorting cargo as well as to reduce the toxicity.13,14 Herein, developing a polymer with high positive density that can bio-degrade at focal sites is still challenging. The high level of reactive oxygen species (ROS) is a unique hallmark associated with pathological process in many diseases including cancer,15 Alzheimer’s disease,16 3

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inflammation,17 reperfusion injury18 and cardiovascular disease.19 For instance, cancer cells constantly generate high level of ROS (up to 100 µM and 1 mM), much higher in comparison with normal cells (~ 0.02 µM).20,21 Therefore, the distinct high level of ROS can be utilized as a unique trigger for delivery systems to distinguish focal and normal sites. There are numerous well-studied tailoring strategies employing lower intracellular tumor pH, matrix metalloproteinases (MMPs) level or hypoxia condition. During recent couples of years, ROS-triggered gene vector is increasingly attractive with fine distinguished responsiveness between focal and normal cells. For instance, in the pioneering work reported by Y. Shen, they developed a boronic ester-based charge-reversal gene carrier, where, however, the ROS-responsive procedure is relatively long, even exposed to high ROS level (80 mM).22-23 Prof. N. Murthy also prepared a termed thioketal nanoparticles for ROS-initiated siRNA orally delivery to intestinal inflammation.24 More sophisticated ROS-triggered gene carriers with higher sensitivity should be developed. The aryl borate esters can be cracked by ROS species, where the reaction condition is mild and rapid.25-28 Besides that, both the borate esters itself and the cleavage product-boronic acids cause unconspicuous toxicity to human tissues.26 Thus the aforementioned information inspired us to explore the arlyboronic esters as the ROS-responsive linkage to develop bio-degradable gene vector. Herein, in this study, we innovatively developed a ROS-biodegradable electropositive and biocompatible polymer derived from branched polyethylenimine (BPEI) with low molecular weight (1.2 K) through a photo-crosslinking reaction to achieve stable condensing and escorting ability of genes as well as the efficient gene release at the aimed sites. We further functionalized this polymer with a tumor-targeting substance P (SP) peptide (with

a

sequence

as

Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met)

via

polyethylene glycol (PEG). The SP peptide can specifically recognize and combine with the neurokinin-1 (NK-1) receptors that are found to be selectively overexpressed in several malignant tumors including breast cancer.29 The plasmid red fluorescence protein DNA (pRFP) was first employed to evaluate the transfection efficiency and safety issue with the polymer. The final constructed SP-crosslinked BPEI/pDNA 4

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nanoparticles (NPs) exhibit low toxicity, ROS-cleavability and as well as much higher transfection efficiency compared with parental BPEI1.2 K both in vitro and in vivo. Finally Plk1 siRNA was selected as the therapeutic gene and loaded by the crosslinked BPEI polymer to treat breast cancer. The fabricated SP-crosslinked BPEI/siRNA polyplex exhibited efficient delivery of siRNA into cancer cells, and remarkably specific gene-silencing effects to induce cancer cell apoptosis and significantly suppressed tumor growth following systemic administration. Hopefully this novel ROS-responsive crosslinked electropositive polymer may serve well as a promising tool for the safe and efficient gene delivery vehicle in clinic.

1. Materials and methods 1.1. Materials Branched polyethyleneimine (Mw = 1.2 K and Mw = 25 K) (BPEI1.2 K and BPEI25 K) were purchased from Sigma-Aldrich (St. Louis, USA). 4-Vinylphenylboronic acid, 3-allyloxy-1,2-propanediol,

propylene

sulfide

and

2,2-dimethoxy-2-phenylacetophenone (DMPA) were supplied by Energy Chemical (Shanghai, China), and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) was

purchased

from

Aladdin

Industrial

Corporation

(Shanghai,

China).

ω-Succinimidyl carbonate-polyethylene glycol-maleimide (NHS-PEG3.5 K-Mal) and methoxy-PEG3 K-NHS (MeO-PEG3 K-NHS) were synthesized by Jenkem Technology Co., Ltd. (Beijing, China). Substance P (SP) peptide with thiol group modification was synthesized by China Peptides Co., Ltd. (Suzhou, China). siRNA targeting Polo-like

Kinase

1

(Plk1)

mRNA

5’-UAAGGAGGGUGAUCUUCUUCAdTdT-3’)

(siPlk1, and

antisense

strand,

Cy5-labeled

siRNA

(Cy5-siRNA, antisense strand, 5’-AACCACTCAACTTTTTCCCAAdTdT-3’) were synthesized by GenePharma (Suzhou, China). The red fluorescence protein (RFP) plasmid (pRFP) (GeneChem Co., Ltd., Shanghai, China) and pGL3-control vector (Promega, Madison, WI, USA) were purified using QIAGEN Plasmid Mega Kit (Qiagen GmbH, Hilden, Germany). Monoclonal antibody against Plk1 was purchased from Abcam (Cambridge, UK) and actin antibody was obtained from Beyotime 5

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Institute of Biotechnology (Shanghai, China). Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were purchased from Gibco BRL (Carlsbad, CA, USA). All other chemicals were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). MDA-MB-231 and HEK 293 cell lines were purchased from Shanghai Institutes for Cell Resource Center, Chinese Academy of Sciences (Shanghai, China). MDA-MB-231/luci cell line was purchased from Cell Biolabs, Inc. (San Diego, USA). Female nude mice (20-25 g) were supplied by the Experimental Animals Department of Fudan University (Shanghai, China). All animal experiments were carried out strictly in accordance with guidelines approved by Fudan University' ethics committee.

1.2. Synthesis of the SP-crosslinked BPEI 1.2.1. Synthesis of the boronic ester diene crosslinker 4-Vinylphenylboronic acid (300 mg, 2 mmol) and 3-allyloxy-1,2-propanediol (396mg, 3 mmol) were stirred in dry dichloromethane (DCM) (20 mL) with anhydrous sulfate sodium (2 g) at 40 °C overnight. After complete reaction, the redundant sulfate sodium was eliminated through suction filtration and the ultimate compound 4-((Allyloxy)methyl)-2-(4-vinylphenyl)-1,3,2-dioxaborolane (VPBE) was purified by the column chromatography over silica gel (petroleum ether/ethyl acetate = 3/1, v/v) to obtain the final pale brown oily liquid (370 mg, 1.5 mmol, 75%). The compound was solubilized in dimethyl sulfoxide-d6 (DMSO-d6) and characterized by nuclear magnetic resonance (NMR) spectrometer (600 MHz, Bruker, Billerica, MA, USA) at 25 °C.

1.2.2. Synthesis of the crosslinked BPEI First, 0.5 M HCl was added dropwise to the aqueous branched polyethylene amine (BPEI, Mw = 1.2 K, 1 g) to adjust pH to 7.2, and water was removed by freeze-drying. Next, the obtained solid BPEI (700 mg, 1 eq) was dissolved in methanol (MeOH) under N2, and propylene sulfide (90 µL, 2 eq) was added into the above solution via a 6

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syringe. The whole solution was stirred under 60 °C for 12 h. After that, the solvent was removed and the residue was precipitation in cold ether for three times to give thiol-modified BPEI. The amount of thiol was determined by the Ellman’s method. Briefly, the precipitate was redissolved in 10 mL MeOH again, 10 µL of which was added into 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer (1.9 mL) to react with Ellman’s reagent (80 µL) for 0.5 h. The concentration of thiol group was calculated through the absorbance at 412 nm with a UV detector. As for the crosslinking reaction, excessive crosslinker VPBE (5eq thiol mol) and catalytic amount of 2,2-dimethoxy-2-phenylacetophenone (DMPA) were added into the above thiol-modified BPEI solution and then sonicated until a clear solution was formed. Then, the solution was irradiated under 365 nm for 12 h, with rotating periodically to ensure uniform irradiation. Finally, the solvent was removed and the crosslinked BPEI was precipitated in cold ether for three times. The crosslinked BPEI was then purified by a dialysis method (Mw = 10 K) to obtain the macromolecular polymer. The thiol amount after crosslinking reaction was analyzed using Ellman’s reagent.

1.2.3. Synthesis of PEGylated and SP crosslinked BPEI First, the sulfydryl modified SP peptide was reacted with NHS-PEG3.5K-Mal with the ratio of 1.5:1 (mol/mol) in PBS buffer (pH 7.0) overnight at room temperature and followed by dialysis (Mw = 3.0 K) to obtain the pure product SP-PEG-NHS. The MAL groups of PEG were specifically reacted with the thiol groups of peptide. SP-PEG-NHS was then solubilized in D2O and characterized by NMR spectroscopy. For the synthesis of PEGylated and SP peptide modified crosslinked BPEI (SP crosslinked BPEI), briefly, the crosslinked BPEI was reacted with MeO-PEG-NHS and SP-PEG-NHS respectively with the mole ratio of 1:1 in PBS buffer (pH 7.0) for 12 h at room temperature and purified by dialysis (Mw = 10 K).

1.3. Plasmid DNA (pDNA) polyplexes 1.3.1 Characterization of pDNA polyplexes For the preparation of different pDNA-loaded nanoparticles (NPs), various N/P ratios 7

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of BPEI1.2 K, PEG-crosslinked BPEI and SP-crosslinked BPEI solutions were added into DNA solutions (200 µg/ml), immediately vortexed for 30 s and incubated at room temperature for 0.5 h. The size and zeta potential of different NPs were measured by dynamic light scattering (DLS) (Zetasizer Nano-ZS, Malvern, UK). The morphology of SP-crosslinked BPEI/pDNA NPs was performed using transmission electron microscope (TEM, JEOL JEM-1200EX, Tokyo, Japan). To confirm the degradation of the crosslinked BPEI/pDNA NPs, 30% H2O2 was added into the NPs with a final concentration of 1 mM and the size variation was observed at different selected time. The degradation of crosslinked BPEI/pDNA under other ROS molecules including hydroxyl radical (OH•), hypochlorous acid/hypochlorite (HOCl/−OCl)30 was also similarly examined via DLS assay for 2 h. Hydroxyl radical was produced by mixing CuCl2 with H2O2.21 Size observation of crosslinked BPEI/pDNA NPs in serum at different time was applied to evaluate the serum stability. Various N/P ratios of different NPs were freshly prepared with the aforementioned method. Each sample was then added with loading buffer, and 0.7% agarose gel electrophoresis with 0.5 mg/ml of ethidium bromide (EB) was then conducted at 100 V in Tris-acetate-EDTA (TAE) buffer (40 mM Tris, 20 mM acetic acid and 1 mM EDTA, pH 8.0) for 45 min to evaluate the DNA encapsulation effect compared with naked DNA. For the ROS-responsive gene release assay, the sodium heparin was added into the H2O2 treated SP-crosslinked BPEI/pDNA NPs with a final concentration of 10 mg/mL and incubated at room temperature for 2 h to release the DNA from the NPs. Next the samples were analyzed by 0.7% agarose gel electrophoresis.

1.3.2. Cytotoxicity assay of pDNA polyplexes The cytotoxicity of different NPs with various N/P ratios was evaluated through the classic MTT assay. Briefly, HEK 293 and MDA-MB-231 cells were seeded into 96-well plates at the density of 8 × 103 cells per well. Cells reaching 80% confluence were exposed to 100 µL different NPs containing 0.5 µg pDNA with N/P ratios from 10 to 30. After incubation for 6 h, cells were rinsed by PBS and incubated for another 8

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18 h. After that, the medium was removed and 100 µL of MTT solution (5 mg/mL) was added to each well and incubated for an additional 4 h. At last, the supernatants were totally replaced by 150 µL of DMSO per well and shaken for 10 min at 37 °C. Absorbance was measured at 570 nm using a microplate reader. The cytotoxicity of crosslinked BPEI and BPEI1.2 K was also evaluated similarly on MDA-MB-231 cells at different concentrations ranging from 5 to 80 µg/mL.

1.3.3. Cellular uptake and gene transfection assay of pDNA polyplexes in vitro To visualize the cellular uptake of pDNA NPs under a fluorescence inverted microscope (Leica, Wetzlar, Germany), BPEI materials were pre-labelled with the NHS ester-Bodipy in PBS buffer (pH 8.0). MDA-MB-231 cells were seeded in 24-well plates (Corning-Coaster, Tokyo, Japan) at a density of 5 × 104 cells per well. When achieving 80% confluence, both cells were incubated with crosslinked BPEI/ pDNA, SP-crosslinked BPEI/ pDNA NPs at the concentration of 5 µg pDNA in 200 µL per well of the serum-free medium for 0.5 h. To investigate the mechanism of internalization, the cells were first pre-incubated with SP peptide at 37 °C for 15 min and then added with SP-crosslinked BPEI/pDNA NPs for 0.5 h. After the incubation, cells were washed with D-Hank’s buffer for three times and photographed under fluorescence microscope. For the gene transfection assay, MDA-MB-231 cells were seeded into 24-well plates at a density of 5 × 104 cells per well and grown to reach 80% confluence prior to transfection. The cells were treated with BPEI1.2

K/pDNA,

BPEI25

K/pDNA,

crosslinked BPEI/pDNA and SP-crosslinked BPEI/pDNA NPs (N/P = 10) containing 5 µg pDNA in serum-free medium at 37 °C for 4 h. After incubation, the medium was replaced with complete serum-containing medium and cells were cultured for another 44 h. The expression of red fluorescence protein was visualized under fluorescence microscope. The plasmid pGL3-control luciferase reporter vector was introduced to evaluate the quantitative study of transfection efficiency. The transfection process was the same as above. For luciferase activity assay, cells after transfection were washed with PBS for three times and then treated with 150 µL lysis reagent supplied by the 9

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Promega Luciferase Assay Kit (Promega, Madison, WI, USA). The lysis solution was centrifuged at 12000 rpm for 5 min at 4 °C. Luciferase activity in the supernatant was quantified by a Luciferase Assay System (Promega, Madison, WI, USA) and total protein amount was determined by a Bradford Protein Assay. The final data was determined by the light unit of each sample minus the blank group and the results were expressed as light units/mg protein.

1.3.4. Qualitative evaluation of reporter gene transfection of pDNA polyplexes in vivo Female BALB/C nude mice (20-25 g) were xenografted with 1 × 106 MDA-MB-231/luci cells, which were suspended in 100 µL of Matrigel solution (5 mg/mL in PBS) by subcutaneous injection into the second right mammary fat pad of nude mice. The different pDNA polyplexes (N/P = 10) were injected through the tail vein of tumor-bearing nude mice at the dose of 50 µg pDNA per mouse. Post 48-h injection, mice were anesthetized and tumors were dissected, fixed in 4% paraformaldehyde for 24 h for frozen sections. The obtained sections were then stained with DAPI for 10 min at room temperature, washed with PBS (pH 7.4) and immediately photographed under the fluorescence microscope.

1.4. siPlk1 polyplexes 1.4.1 Characterization of siPlk1 polyplexes To determine the optimal N/P ratio between the BPEI polymer and siRNA, various N/P ratios of PEG-crosslinked BPEI and SP-crosslinked BPEI solutions were added into siPlk1 solutions (200 µg/ml), immediately vortexed for 30 s and incubated at room temperature for 30 min. The size and zeta potential of a serial different NPs was measured by DLS to confirm the optimized ratio. The morphology of SP-crosslinked BPEI/siRNA NPs was also captured using TEM. The ROS-responsive assay of crosslinked BPEI/siRNA NPs was the same as the above crosslinked BPEI/pDNA NPs via DLS results. 1.4.2. Plk1 gene silencing assay in vitro 10

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To evaluate the silencing capability of hybrid NPs, MDA-MB-231 cells were seeded into 6-well plates at the density of 5 × 105 cells and incubated for 24 h. The cells were transfected with various formulations at a final siRNA dose of 5 µg per well (N/P = 3) for 4 h. After further incubation for 20 h (for mRNA isolation) or 44 h (for protein extraction) at 37 °C, the levels of Plk1 mRNA and protein expression were measured using quantitative reverse transcriptase (qRT-PCR) and western blot, respectively. For qRT-PCR assay, total RNA was extracted using an RNeasy mini kit (Qiagen, CA, USA) according to the manufacturer's protocol. The primer for Plk1 was 5’-AGCCTGAGGCCCGATACTACCTAC-3’

(Plk1-forward)

and,

5’-ATTAGGAGTCCCACACAGGGTCTTC-3’ (Plk1-reverse). All the operations were completed by Servicebio Company (Wuhan, China). For the western blot analysis, the cells were lysed with 150 µL IP cell lysis buffer which freshly added with phenylmethanesulfonyl fluoride (PMSF) at the concentration of 1 mM on ice for 15 min. The lysates were then centrifuged for 10 min at 12000 rpm and the concentration of total protein in supernatant was determined through the BCA Protein Assay Kit (Beyotime Biotechnology, Beijing, China). 40 µg of total protein was separated on 12% polyacrylamide-SDS gels and then transferred to PVDF membranes (300 mA for 2 h). The membranes were blocked in 5% nonfat milk in phosphate buffered saline with Tween-20 (TBST, pH 7.4) for 2 h and incubated with monoclonal antibodies against Plk1 and Actin overnight at 4 °C. After three washes with TBST, the membranes were incubated with goat anti-rabbit or anti-mouse IgG-HRP secondary antibodies for 2 h at room temperature. Finally, the bands were visualized under the gel imager (Bio-Rad, CA, USA).

1.4.3. Bio-distribution and therapeutic efficacy of siPlk1 polyplexes in vivo Cy5 labelled siRNA (Cy5-siRNA) was used to investigate the bio-distribution of siRNA-loaded NPs. Briefly, tumor-bearing mice with MDA-MB-231/luci cells were injected with crosslinked BPEI and SP-crosslinked BPEI polmer/Cy5-siRNA NPs (20 µg siRNA per mouse) through the tail vein. The mice were anesthetized post 24 h and visualized under non-invasive In-Vivo Imaging System (IVIS) (Caliper, Newton, MA, 11

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USA). Next, the mice were sacrificed, and the tumors plus other main organs (heart, liver, spleen, lung, and kidney) were excised for further visualization by IVIS.

1.4.4. Antitumor efficacy in vivo Tumor-bearing mice with MDA-MB-231/luci cells were randomly divided into four groups (n = 8) and treated with saline, free siPlk1, crosslinked BPEI/siPlk1 and SP-crosslinked BPEI/siRNA at a dose of 20 µg siPlk1 per mouse through the tail intravenous injection every other day. The body weight and tumor volume were recorded every two days. For the detection of Plk1 protein analysis in tumor tissues, the tumor was removed after the last injection and homogenized in RIPA lysis buffer for 2 min. The lysates were then centrifuged for 10 min at 12000 rpm. BCA method was used to determine the concentration of total protein in the supernatant. Finally proteins were detected by western blot assay as described above. Besides that, TUNEL assay was further applied to further validate the apoptotic efficacy. After treatment with different NPs, the mice were killed and tumors were excised for frozen sections. The sections were performed according to the instructions of the kit to detect the broken nuclear DNA fragments, stained with DAPI, and observed under the fluorescent microscope. At the end of the tumor treatment assay, main organs (heart, liver, spleen, lung, kidney) were excised for hematoxylin and eosin (H & E) staining.

1.5. Statistical analysis All data were represented as means ± SD. Analysis was performed with the computer program GraphPad Prism software. The statistical significance of relevant results was assessed using Student's t test; p < 0.05 was considered statistically significant in all analyses (95% confidence level).

2. Results and discussion 2.1. Synthesis of the SP-crosslinked BPEI 12

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The specific synthesis route of SP-crosslinked BPEI was shown in Scheme 1A. To fabricate a positive-charged polymer, we successfully synthesized a borate-based crosslinker VPBE of which the structure was confirmed by 1H NMR spectra (Figure S1). The specific crosslinking reaction was then performed through the “click reaction” between the thiol groups pre-modified on the BPEI and the two olefin bonds of VPBE crosslinker. The BPEI1.2 K monomer was first reacted with propylene sulfide and the amount of sulfydryl groups was calculated by Ellman’s reagent. The methyl peak of propylene sulfide (δ = 1.2-1.5 ppm) appeared in 1H NMR spectra (Figure S2) indicated the successful modification. The next click reaction was a consumption process of thiol groups irradiated by UV365 nm. Table S1 showed that the concentration of thiol groups decreased from 0.213 ± 0.03 mmol/g to 0.085 ± 0.02 mmol/g after the crosslinking treatment. The benzene ring peak of VPBE crosslinker appeared in the final crosslinked BPEI compound (Figure S3) and the sharp decline of thiol amount after irradiation indicated the successful crosslinking reaction with preferable efficiency After that, the SP peptide was conjugated onto the crosslinked BPEI polymer via a PEG moiety. In the spectrum of Figure S4, the multiple characteristic peaks of SP peptide were found in SP-crosslinked BPEI molecules, suggesting a successful connection. During the synthesis process of crosslinked BPEI, we also optimized the crosslinking degree by adjusting the ratio of modificatory thiol groups. Different ratios of propylene sulfide (2 eq, 4 eq) relative to BPEI1.2 K monomer were investigated. Disappointedly, the increased amount of propylene sulfide (4 eq) resulted in elevated thiol modification of BPEI, eventually leading higher crosslinking degree in the final crosslinking reaction. And due to the aromatics structure among the VPBE crosslinker, the solubility crosslinked BPEI was not favorable (Figure S7A). Hence, we finally selected the relatively low ratio of propylene sulfide (2 eq).

13

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Scheme 1. (A) The synthesis process of Crosslinked BPEI and SP-crosslinked BPEI. (B) Scheme illustration of the SP peptide mediated cellular uptake and ROS-biodegradable of the SP-crosslinked BPEI/siRNA in tumor cells.

2.2. pDNA polyplexes 2.2.1. Characterization of pDNA polyplexes The red fluorescence protein (RFP) plasmid (pRFP), a reporter gene was employed to evaluate the gene delivery efficiency of the crosslinked BPEI vehicle. The condensation efficiency was evaluated through the gel electrophoresis assay. Figure S5 showed that the crosslinked BPEI exhibited strong interaction ability with pDNA and could restrain its retardation at a crosslinked BPEI weight/pDNA weight (w/w) ratio of 0.6. As for the uncrosslinked BPEI1.2 K monomer, a higher ratio (0.8) was appeared to prevent the movement of pDNA. The gel electrophoresis assay only indicated the positive charge of BPEI could offset the negative charge of the packaged pDNA. To obtain compacted and uniform-sized NPs, the specific N/P ratio was further optimized according to the DLS results. As shown in Figure 1A, the 14

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crosslinked BPEI can efficiently condense pDNA to form uniform NPs with the diameter of about 151.83 ± 15.03 nm at a low N/P ratio of 10. In comparison, the uncrosslinked BPEI1.2 K showed relatively large size distribution (over 200 nm) even at high ratios, which indicated its weak DNA binding affinity due to its low molecular weight. Due to the modification of SP peptide, the mean size of SP-crosslinked BPEI/pDNA was slightly larger than crosslinked BPEI/pDNA. In addition, the zeta potential of different NPs with various N/P ratios were also measured (Figure 1B). When the N/P ratio increased from 5 to 10, all zeta potential values dramatically augmented and reached a plateau at 10 mV. The electric property of BPEI1.2 K/pDNA was about -12.31 ± 3.37 mV at the beginning, which performed absolute negative charge, while the crosslinked group exhibited a positive electricity about 11.47 ± 1.43 mV. Even at the high N/P ratio of 20, the crosslinked BPEI/pDNA (about 25 mV) held much more electropositivity density than the BPEI1.2 K/pDNA (about 15mV). By the way, we also investigated the zeta potential of various simple BPEI materials. Table S2 implied that the crosslinked BPEI evidently possessed much more powerful positive charge (55.9 ± 1.28 mV) than the pure BPEI1.2 K monomer (16.20 ± 1.90 mV). The PEG and SP peptide conjugation onto the crosslinked BPEI mildly reduced the positive density (about 40 mV). After complexing with pDNA, the electric charge was partly neutralized and thus weakened. All these above results substantiated that the chemical crosslinking strategy increased the Mw and enriched the positive charge density of BPEI, consequently enhanced the condensation ability to capture gene more efficiently and more tightly. Balancing the above DLS and zeta potential results, the optimized N/P ratio was confirmed at 10 considering that excessive positive charge of NPs would generate cellular toxicity and non-specific distribution.31,32 Furthermore, TEM in Figure 1D showed a spherical and uniform morphology of SP-crosslinked BPEI/pDNA NPs at N/P ratio of 10 and also exhibited a core-shell structure with the PEG conjunction as the hydrophilic stabilization shell.33 The serum stability assay in Figure S8 showed that the size of crosslinked BPEI/pDNA NPs still maintained the relatively stable nanostructure only with slightly larger even at the existence of serum for 20 h, which indicated that our constructed gene delivery 15

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system was resistant to the abominable blood environment.

Figure 1. Characterization of different pDNA polyplexes. (A-B) The mean size (A) and zeta potential (B) of different NPs. (C-D) DLS (C) and TEM (D) result of SP-crosslinked BPEI/pDNA at the N/P ratio of 10.

We simply verified the ROS-responsive characteristic of pure crosslinked BPEI polymer by 1H NMR spectroscopy. Upon treated with 1 mM H2O2, the phenyl borate ester part will be cleaved into boric acid and glycol components.34,35 In 1H NMR spectrum shown in Figure 2A, the peak position of four hydrogen in benzene ring varied from 7.5-7.9 ppm to 6.5-7.0 ppm. Besides that, we observed a phenomenon that the suspension of water-insoluble BPEI with higher crosslinking degree would turn into transparent solution when treated with H2O2. In Figure S7, under the presence of H2O2, the original phenylo boric acid ester bond in crosslinked BPEI was broken up into hydrophilic hydroxyl and carboxyl groups, followed by the crosslinked BPEI dissociated into the soluble single BPEI, which eventually resulted in the water-soluble clear solution. Inspired by the results of ROS-responsive feature of this crosslinked gene delivery polymer, we examined the degradation profile of crosslinked BPEI/pDNA under simulated ROS conditions. In Figure 2C-D, we could

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see that the mean diameter of NPs gradually became larger with H2O2 incubation time prolonged and the size exceeded more than 1 µm when treated for 20 h, which indicated the fracture, expansion and disruption of NPs, while PBS group consistently maintained the same size. Consistent with the H2O2-triggered degradation of NPs, DNA unpackaging from NPs was further proved by heparin replacement electrophoresis assay. Heparin loading with negative electric charge was selected as the DNA competitor. When only treated with heparin, the packaged DNA still settled at the origin hole despite the positive charge of BPEI was partly deprived, which was suffice to show the abundant positive charge of the crosslinked BPEI. As expected, after pre-incubated with H2O2 for 12 h and then heparin, the obvious DNA separation and movement visually demonstrated the ROS-responsive characteristic of this novel gene vector again. It was noteworthy that we observed no DNA release from NPs when directly treated with H2O2, which is probably because though the crosslinked BPEI cleaved into the low molecular fragments, DNA can be still attached onto the cleaved cationic materials and hardly freely move in the electric field (Figure 2B). Figure S9 showed the degradation of NPs under other ROS molecules including OH• and –OCl for only 2 h, which further suggested the ROS-responsive characteristic. Among these ROS stimulis, OH• exhibited the strongest effect and the influence of catalyzer Cu2+ was excluded.

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Figure 2. Evaluation of the ROS-responsive characteristic of the crosslinked BPEI. (A) The 1H NMR of the crosslinked BPEI before and after treated with 1mM H2O2 for 12 h. (B) The heparin replacement result of crosslinked BPEI/pDNA NPs. (C-D) The size change of the crosslinked BPEI/pDNA NPs after treated with 1mM H2O2 for different time (C) and the DLS result of NPs 24 h in PBS and H2O2 (D).

2.2.2. Cytotoxicity assay of pDNA polyplexes The cytotoxicity of different NPs with a series of N/P ratios were evaluated via MTT assay on both MDA-MB-231 and HEK293 cell lines (Figure 3). HEK293 cell in this experiment is one of the human renal epithelial cells and regarded as the representative normal cell to check the possible toxicity and side effect of crosslinked BPEI. Compared with the BPEI1.2 K, crosslinked BPEI polyplexes presented more favorable biocompatibility on both two cells and the cell viability exceeded 50% even at the N/P ratio of 30 on MDA-MB-231 cells. Pure crosslinked BPEI also had low toxicity even at the concentration 40 µg/mL, while the BPEI1.2

K

caused serious

damage to cells at the beginning concentration 5 µg/mL (Figure S6). It could be explained that the ROS-cleaved trait of the crosslinked BPEI in favor of the 18

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degradation into small fragments with much lower toxicity.

Figure 3. The cell viability of HEK293 (A) and MDA-MB-231 (B) cells after incubated with different NPs at various N/P ratios.

2.2.3. Cellular uptake and gene transfection assay of pDNA polyplexes in vitro We evaluated the cellular uptake characteristics of the SP-crosslinked BPEI/pDNA to prove the targeting capacity on MDA-MB-231 cells (Figure 4A). Graphics captured by fluorescence microscope indicated that the SP group exhibited obviously enhanced cellular uptake behavior comparing with the non-targeted crosslinked BPEI/pDNA, ascribed to the high-expressed NK-1 receptor mediated endocytosis. Besides that, the cellular uptake of SP-crosslinked BPEI/pDNA was significantly restricted when the receptors were pre-saturated with the excessive free SP peptide, which further indicated the enhanced accumulation of SP-crosslinked BPEI/pDNA resulted from the specific binding ability of SP peptide to the NK-1 receptors. Then, the gene transfection experiments were performed on MDA-MB-231 cells. Figure 4C exhibited the RFP qualitative expression of different NPs. The BPEI25 K/pDNA

was employed as the positive control. The crosslinked BPEI/pDNA showed

much stronger fluorescence intensity compared with uncrosslinked BPEI1.2 K/pDNA group. This difference was mainly due to the efficient gene separation from cationic carrier after cytosolic delivery. The crosslinked BPEI took an advantage over the simple BPEI thanks to the broken fragments by the high level of ROS in tumor cells, which is expected to release the carried gene efficiently in cytosol to take effect.36 The

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enhanced cellular uptake rooting from the SP peptide also produced the more favorable transfection result of SP-crosslinked BPEI/pDNA group. The PGL-3 plasmid stably expressing the luciferase was further employed to determine the quantitative transfection efficiency of various NPs (Figure 4D). Consistent with the images of qualitative transfection assay, the luciferase activity of cells treated with crosslinked BPEI, especially the SP targeted group (5.94 ± 0.53 × 105) was significantly higher than that of uncrosslinked BPEI/pDNA (1.08 ± 0.13 × 105). It is reasonable that the transfection effect of the synthesized crosslinked BPEI polymer was not as good as the positive control BPEI25 K, which possesses more positive electricity and meanwhile without PEG modification. Meanwhile, the crosslinked BPEI was conjugated with a certain number of PEG for reduced toxicity, but PEGylation might somehow influence the transfection efficiency in vitro compared with the BPEI25 K.37,38 However, another fact is that the efficacy and toxicity of BPEI are strongly connected with its Mw, and the application of BPEI25 K is mainly restricted with its massive molecules and abundant positive charge.

2.2.4. Qualitative evaluation of reporter gene transfection of pDNA polyplexes in vivo Encouraged by the transfection efficiency results in vitro, we evaluated the packaged gene delivery ability of this fabricated vehicle to tumor site on MDA-MB-231 cell bearing nude mice model. Different pDNA-loaded NPs were injected through the tail vein at the dose of 50 µg pRFP/mouse, after two days of expressing the protein, mice were anaesthetized and tumors were excised for frozen section. The images under fluorescence microscope in Figure 4C showed that the RFP expression intensity of crosslinked BPEI group was significantly higher than that of uncrosslinked BPEI, which is due to the better stabilization ability resistant the complicated blood circumstance and efficient separation for the gene to take the following effect originated by the ROS-responsive feature. Besides that, the modified SP peptide ligand on the crosslinked BPEI indeed realized the more precise tumor delivery capability, thus creating the maximum RFP fluorescence intensity in the tumor site. 20

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Figure 4. Cellular uptake and gene transfection efficiency of different formulations. (A) Cellular uptake of Crosslinked BPEI/pDNA and SP-crosslinked BPEI/pDNA on MDA-MB-231 cells 30min after incubation (100 ×, scale bar indicates 50 µm). (B) Qualitative transfection results of different NPs in vitro (100 ×, scale bar indicates 50 µm). (C) The fluorescence images of RFP expression in the tumor site (100 ×, scale bar indicates 50 µm). (D) Quantitative transfection efficiency of different NPs in vitro (n = 3, **p < 0.01 and ***p < 0.001).

2.3. siPlk1 polyplexes 2.3.1. Characterization of siPlk1 polyplexes Since we have demonstrated that this designed novel crosslinked BPEI polymer exhibited the favorable gene delivery potential via the reporter gene both in vitro and in vivo, the Polo-like Kinase 1(Plk1) was selected as a model therapeutic siRNA to take a further evaluation of this gene delivery platform for the treatment of breast cancer. Plk1 is a key regulator for the mitotic progression of mammalian cells and its increased activity has been reported to be linked with the tumor progression. siRNA targeting Plk1 has been widely represented as an ideal protein-kinase target for cancer drug development in a wide range of studies.39-42 During the following siRNA experiments, we first optimized the appropriate N/P ratio to condense the siPlk1 into the uniform-sized NPs just as the same procedure as the pDNA. Table 1 showed that both the mean diameter (90.30 ± 5.87 nm) and the PDI (0.190 ± 0.021) were perfect at the N/P ratio of 3. And the electric property of crosslinked BPEI/siRNA was about 21

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-11.83 ± 3.15 mV at the N/P ratio of 1, which performed absolute negative charge, while the zeta potential reversed from negative to positive (16.03 ± 3.07 mV) when the N/P ratio increased to 3. Figure 5 showed a spherical and uniform morphology of SP-crosslinked BPEI/siRNA NPs with the size of about 60 nm. Thus in the subsequent experiments, the ideal formulation was defined as the N/P ratio of 3. Consistent with the pDNA part, the size of crosslinked BPEI/siRNA NPs in Figure S10 gradually became larger when treated with H2O2 and exhibited the same ROS-responsive character. Table 1 The mean size and PDI results of crosslinked BPEI/siPlk1 with various N/P ratios. N/P ratio

1

3

6

Mean Size (nm)

134.30 ± 12.24

90.30 ± 5.87

303.32 ± 15.21

PDI

0.256 ± 0.013

0.190 ± 0.021

0.273 ± 0.026

Zeta Potential (mV)

-11.83 ± 3.15

16.03 ± 3.07

24.30 ± 3.67

Figure 5. Characterization of SP-crosslinked BPEI/siRNA polyplexes. (A-B) DLS (A) and TEM (B) result of SP-crosslinked BPEI/siRNA at the N/P ratio of 3. 2.3.2. Plk1 gene silencing assay in vitro We evaluated the gene silencing effect of the siPlk1-laoded NPs on MDA-MB-231 cells. The cells were incubated with hybrid NPs carrying siPlk1 (5 µg) for 4 h, the mRNA expression was analyzed after 24 h for qRT-PCR, while the total protein was extracted after 48 h for western blot assay, respectively. As shown in Figure 6, both crosslinked BPEI/siPlk1 and SP-crosslinked BPEI/siPlk1 complexes resulted in the downregulation of Plk1 mRNA and protein expression, especially for the SP 22

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peptide-targeting group. Due to the NK-1receptor mediated enhanced cellular uptake of SP-crosslinked BPEI/siPlk1 NP, nearly 60% of Plk1 mRNA expression was suppressed and the very distinct inhibition of protein expression.

Figure 6. Gene downregulation results of various formulations. (A-B) The Plk1 mRNA expression by qRT-PCR (A) and protein expression by western blot (B) (n = 3, **p < 0.01). Actin was used as an internal control.

2.3.3. Bio-distribution and therapeutic efficacy of siPlk1 polyplexes in vivo Cy5 labelled siRNA was packaged into different complexes to observe the bio-distribution in breast tumor model nude mice. After 24 h injected intravenously with 20 µg Cy5-siRNA loaded NPs, from the fluorescence images in Figure 7, certain amount of fluorescence could be observed in the tumor site of both crosslinked

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BPEI/siRNA or SP-crosslinked BPEI/siRNA. For crosslinked BPEI/siRNA group, the distribution ability into tumor is analyzed mainly due to the EPR and PEG shielding effect (passive targeting); while for SP-crosslinked BPEI/siRNA, besides the above two factors, the positive breast cancer targeting function of SP peptide contributed to the outstanding tumor accumulation. After that, major organs were excised for ex vivo imaging to reveal the tissue distribution of NPs. The distribution of NPs in the heart, liver, spleen, lung was similar in both two groups, revealing that SP peptide modification rarely affected the distribution behavior of complexes in vivo. The in vivo distribution results further confirmed the fine siRNA delivery ability to the tumor site of the SP-crosslinked BPEI vehicle. Due to the NK-1 receptor, the substance P specific binding receptor is also widely distributed in the renal pelvis besides tumor.43 We inferred that the amount of SP group in mice kidneys after 24 h was remarkably high might be the combined action of the basic excretory function of kidney and target accumulation caused by specific recognition of NK-1 receptors distributed on the renal.

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Figure 7. In vivo distribution of BPEI derivatives/siRNA NPs. (A) In vivo fluorescence images at 24 h after intravenous injection of Crosslinked BPEI/siRNA and SP-crosslinked BPEI/siRNA. (B) Ex vivo fluorescence images of major organs excised 24 h after injection. Fluorescence signal was from Cy5-labeled siRNA.

Finally, we examined the antitumor effect of this siPlk1 delivery platform. Mice bearing MDA-MB-231/luci cells were divided into four groups and each group were received intravenous injection of saline, free siPlk1, crosslinked BPEI/siPlk1 and SP-crosslinked BPEI/siPlk1 at the siPlk1 dose of 20 µg/mouse every other day. The tumor volume and body weight were recorded every other day. As shown in Figure 8, the tumor volume in the saline and free siPlk1 group sharply increased along with the monitoring time. While upon the siPlk1 was packaged into the stable complexes through the crosslinked BPEI gene vector and injected to the tumor-bearing nude mice, the tumor growth was found greatly suppressed and the SP-crosslinked 25

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BPEI/siPlk1 group exhibited the most favorable antitumor effect (P < 0.05) compared with the free siPlk1 group. After the whole treatment, the tumor was removed for the frozen section to evaluate the tumor apoptosis among different groups using TUNEL assay. As shown in figure 9, compared with the negligible apoptotic signal of the saline and free siPlk1 groups, the apoptotic fluorescence intensity was strongly enhanced in crosslinked BPEI/siPlk1 and SP-crosslinked BPEI/siPlk1 groups, which was consistent with the tumor volume results. To further confirm the relationship between suppressed tumor growth and the downregulation of Plk1 protein in tumor cells, the tumor were excised 24 h post the last injection and the total protein was extracted for western blot assay. Mice treated with saline and free siPlk1 exhibited little reductions in Plk1 protein level. In contrast, there was a significant downregulation both in crosslinked BPEI/siPlk1 and SP-crosslinked BPEI/siPlk1 groups (Figure 8C). These results demonstrated that the Plk1 protein downregulation indeed led to the tumor growth suppression after treatment with the siPlk1 packaged polyplexes. Another vital factor should be taken into consideration when evaluating a satisfactory gene delivery vehicle is the biocompatibility and safety. The current clinical trials such as the lipid-based nanocarriers though with high efficiency, the toxicity especially at high administration dosage greatly led to its clinical failure.44 From the normal daily observation of body weight and living state of mice during the treatment period, there was little side effect. Moreover, we investigated the histological status of the heart, liver, spleen, lungs, and kidneys through H & E staining, which indicated that there was indeed no obvious toxic pathological damage in treated groups (Figure S11). In conclusion, we presented a robust strategy of the crosslinked method to fabricate a novel ROS-responsive electropositive polymer for gene specific precise delivery with good biocompatibility and favorable transfection efficiency. Taking the in vitro and in vivo results into account, such a gene delivery platform in this study put forward a new orientation for the future gene therapy.

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Figure 8. Antitumor efficacy of different siRNA-loaded formulations on MDA-MB-231 cell bearing nude mice. (A-B) Body weight (A) and tumor volume (B) of mice after injection with different formulations (n = 8, *p < 0.05 and **p < 0.01). (C) The Plk1 protein expression results in tumor of different groups. Actin was used as an internal control.

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Figure 9. TUNEL results of frozen sections of tumors excised from MDA-MB-231 cell bearing nude mice after treatment with different formulations. Green: TUNEL-stained apoptosis cells. Blue: DAPI-labeled nucleus (100 ×, scale bars indicate 80 µm).

3. Conclusion Since the discovery in 1990s, gene therapy has attracted tremendous interests to open a new therapeutic field. However, there are still many challenges and barriers to overcome the limitations of safety and effectiveness of the carriers for the wider application. In this work, a crosslinked BPEI polymer modified with SP peptide targeting ligand (SP-crosslinked BPEI) and ROS-cleavable characteristic was developed as a novel, safe and efficient gene delivery platform. Both the pDNA and

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siPlk1 could be packaged into the delivery vehicle to form stable and compact complexes with uniform sizes. Moreover, the degradation of phenyl borate ester linkages in ROS abundant environment endowed the complexes with efficient gene release, and the NK-1 receptor mediated enhanced cellular uptake all together realized a favorable gene transfection including the RFP protein expression and the Plk1 downregulation both in vitro and in vivo. The SP-crosslinked BPEI/siPlk1 exhibited relatively strong tumor growth inhibition efficacy. Additionally, the crosslinked BPEI caused low systemic toxicity. In summary, this work presented a potent and promising siRNA delivery strategy which provided a basis for the future gene therapy. ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: XXX The 1H NMR spectra, gel electrophoresis assay, the amount of thiol groups on the BPEI, Zeta potential of BPEI, cell viability, water solubility of crosslinked BPEI, size of crosslinked BPEI/pDNA NPs in serum, size of crosslinked BPEI/pDNA NPs under different ROS molecules, size of crosslinked BPEI/siRNA NPs treated with 1mM H2O2 and H & E images of major organs. (PDF) Corresponding Authors *Phone: +86-21-5198-0187; e-mail: [email protected] (T.S.). Notes No competing financial interest was declared. Acknowledgments This work was supported by the National Natural Science Funds of China (21602030 and 81172993), Shanghai Sailing Program (16YF1400900), Scientific Research Foundation of Fudan University for Talent Introduction (JJF301103), National Science Fund for Distinguished Young Scholars (81425023).

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Reference (1) Lin, G. M.; Zhang, H.; Huang, L. Smart Polymeric Nanoparticles for Cancer Gene Delivery. Mol. Pharm. 2015, 12 (2), 314-321. (2) Chen, Y. C.; Gao, D. Y.; Huang, L. In Vivo Delivery of miRNAs for Cancer Therapy: Challenges and Strategies. Adv. Drug Deliv. Rev. 2015, 81, 128-141. (3) Zhu, L. P.; Simpson, J. M.; Xu, X.; He, H.; Zhang, D. H.; Yin, L. C. Cationic Polypeptoids with Optimized Molecular Characteristics Toward Efficient Nonviral Gene Delivery. ACS Appl. Mater. Interfaces. 2017, 9 (28), 23476-23486. (4) Naldini, L. Gene Therapy Returns to Centre Stage. Nature 2015, 526 (7573), 351-360. (5) Ginn, S. L.; Alexander, I. E.; Edelstein, M. L.; Abedi, M. R.; Wixon, J. Gene Therapy Clinical Trials Worldwide to 2012 – An Update. J. Gene Med. 2013, 15 (2), 65-77. (6) Shorter, S. A.; Gollings, A. S.; Gorringe-Pattrick, M. A. M.; Coakley, J. E.; Dyer, P. D. R.; Richardson, S. C. W. The Potential of Toxin-Based Drug Delivery Systems for Enhanced Nucleic Acid Therapeutic Delivery. Expert Opin. Drug Deliv. 2017, 14 (5), 685-696. (7) Stein, C. A.; Castanotto, D. FDA-Approved Oligonucleotide Therapies in 2017. Mol. Ther. 2017, 25 (5), 1069-1075. (8) Mout, R.; Ray, M.; Yesilbag Tonga, G.; Lee, Y. W.; Tay, T.; Sasaki, K.; Rotello, V. M. Direct Cytosolic Delivery of CRISPR/Cas9-Ribonucleoprotein for Efficient Gene Editing. ACS Nano 2017, 11 (3), 2452-2458. (9) Yang, J.; Meng, X. D.; Pan, J. C.; Jiang, N.; Zhou, C. W.; Wu, Z. H.; Gong, Z. H. CRISPR/Cas9-Mediated Noncoding RNA Editing in Human Cancers. RNA Biol. 2017, 13, 1-9. (10) Li, S. D.; Huang, L. Gene Therapy Progress and Prospects: Non-Viral Gene Therapy by Systemic Delivery. Gene Ther. 2006, 13 (18), 1313-1319. (11) Schaffert, D.; Wagner, E. Gene Therapy Progress and Prospects: Synthetic Polymer-Based Systems. Gene Ther. 2008, 15 (16), 1131-1138. (12) He, H.; Zheng, N.; Song, Z. Y.; Kim K. H.; Yao, C.; Zhang, R. J.; Zhang, C. L.; 30

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Huang, Y. H.; Uckun, F. M.; Cheng, J. J.; Zhang, Y. F.; Yin, L. C. Suppression of Hepatic Inflammation via Systemic siRNA Delivery by Membrane-Disruptive and Endosomolytic Helical Polypeptide Hybrid Nanoparticles. ACS Nano 2016, 10 (2), 1859-1870. (13) He, H.; Bai, Y. G.; Wang, J. H.; Deng, Q. R.; Zhu, L. P.; Meng, F. H.; Zhong, Z. Y.; Yin, L. C. Reversibly Cross-Linked Polyplexes Enable Cancer-Targeted Gene Delivery

via

Self-Promoted

DNA Release

and

Self-Diminished

Toxicity.

Biomacromolecules 2015, 16 (4), 1390-1400. (14) Deng, Q. R.; Li, X. D.; Zhu, L. P.; He, H.; Chen, D. L.; Chen, Y. B.; Yin, L. C. Serum-Resistant, Reactive Oxygen Species (ROS)-Potentiated Gene Delivery in Cancer Cells Mediated by Fluorinated, Diselenide-Crosslinked Polyplexes. Biomater. Sci. 2017, 5 (6), 1174-1182. (15) Benz, C. C.; Yau, C. Ageing, Oxidative Stress and Cancer: Paradigms in Parallax. Nat. Rev. Cancer. 2008, 8 (11), 875-879. (16) Sayre, L. M.; Perry, G.; Smith, M. A. Oxidative Stress and Neurotoxicity. Chem. Res. Toxicol. 2008, 21 (1), 172-188. (17) Chung, M. F.; Chia, W. T.; Wan, W. L.; Lin, Y. J.; Sung, H. W. Controlled Release of an Anti-Inflammatory Drug Using an Ultrasensitive ROS-Responsive Gas-Generating Carrier for Localized Inflammation Inhibition. J. Am. Chem. Soc. 2015, 137 (39), 12462-12465. (18) Zweier, J. L.; Talukder, M. A. The Role of Oxidants and Free Radicals in Reperfusion Injury. Cardiovasc. Res. 2006, 70 (2), 181-190. (19) Tsutsui, H.; Kinuqawa, S.; Matsushima, S. Mitochondrial Oxidative Stress and Dysfunction in Myocardial Remodeling. Cardiovasc. Res. 2009, 81 (3), 449-456. (20) Xu, X. D.; Saw, P. E.; Wei, T.; Li, Y. J.; Ji, X. Y.; Bhasin, S.; Liu, Y. L.; Ayyash, D.; Rasmussen, J.; Huo, M.; Shi, J. J.; Farokhzad, O. C. ROS-Responsive Polyprodrug Nanoparticles for Triggered Drug Delivery and Effective Cancer Therapy. Adv. Mater. 2017, 29 (33), 201700141. (21) Shim, M. S.; Xia, Y. N. A Reactive Oxygen Species (ROS)-Responsive Polymer for Safe, Efficient, and Targeted Gene Delivery in Cancer Cells. Angew. Chem. Int. Ed. 31

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Page 32 of 35

Engl. 2013, 52 (27), 6926-6929. (22) Liu, X.; Xiang, J. J.; Zhu, D. C.; Jiang, L. M.; Zhou, Z. X.; Tang, J. B.; Liu, X. R.; Huang, Y. Z.; Shen, Y. Q. Fusogenic Reactive Oxygen Species Triggered Charge-Reversal Vector for Effective Gene Delivery. Adv. Mater. 2016, 28 (9), 1743-1752. (23) Li, Y.; Bai, H. Z.; Wang, H. B.; Shen, Y. Q.; Tang, G. P.; Ping, Y. Reactive Oxygen Species (ROS)-Responsive Nanomedicine for RNAi-Based Cancer Therapy. Nanoscale 2017, 10 (1), 203-214. (24) Wilson, D. S.; Dalmasso, G.; Wang, L. X.; Sitaraman, S. V.; Merlin, D.; Murthy, N. Orally Delivered Thioketal Nanoparticles Loaded with TNF-α-siRNA Target Inflammation and Inhibit Gene Expression in the Intestines. Nat. Mater. 2010, 9 (11), 923-928. (25) Jourden, J. L.; Daniel, K. B.; Cohen, S. M. Investigation of Self-Immolative Linkers in the Design of Hydrogen Peroxide Activated Metalloprotein Inhibitors. Chem. Commun. 2011, 47 (28), 7968-7970. (26) Kuang, Y. Y.; Balakrishnan, K.; Gandhi, V.; Peng, X. Hydrogen Peroxide Inducible DNA Cross-Linking Agents: Targeted Anticancer Prodrugs. J. Am. Chem. Soc. 2011, 133 (48), 19278-19281. (27)

Broaders,

K.

E.;

Grandhe,

S.;

Frechet,

J.

M.

A

Biocompatible

Oxidation-Triggered Carrier Polymer with Potential in Therapeutics. J. Am. Chem. Soc. 2011, 133 (4), 756-758. (28) Noh, J.; Kwon, B.; Han, E.; Park, M.; Yang, W.; Cho, W.; Yoo, W.; Khang, G.; Lee, D. Amplification of Oxidative Stress By a Dual Stimuli-Responsive Hybrid Drug Enhances Cancer Cell Death. Nat. Commun. 2015, 6, 6907-6916. (29) Munoz, M.; Covenas, R.; Esteban, F.; Redondo, M. The Substance P/NK-1 Receptor System: NK-1 Receptor Antagonists as Anti-Cancer Drugs. J. Biosci. 2015, 40 (2), 441-463. (30) Lou, Z. R.; Li, P.; Han, K. L. Redox-Responsive Fluorescent Probes with Different Design Strategies. Acc. Chem. Res. 2015, 48 (5), 1358-1368. (31) An, S.; Jiang, X. T.; Shi, J. S.; He, X.; Li, J. F.; Guo, Y. B.; Zhang, Y.; Ma, H. J.; 32

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ACS Applied Materials & Interfaces

Lu, Y. F.; Jiang, C. Single-Component Self-Assembled RNAi Nanoparticles Functionalized with Tumor-Targeting iNGR Delivering Abundant siRNA for Efficient Glioma Therapy. Biomaterials 2015, 53, 330-340. (32) Stace, T. M.; Damiano, E. R. An Electrochemical Model of the Transport of Charged Molecules Through the Capillary Glycocalyx. Biophys. J. 2001, 80 (4), 1670-1690. (33) Gu, Q. R.; Xing, J. Z.; Huang, M.; He, C.; Chen, C. SN-38 Loaded Polymeric Micelles to Enhance Cancer Therapy. Nanotechnology 2012, 23 (20), 205101. (34) de Gracia Lux, C.; Joshi-Barr, S.; Nquyen, T.; Mahmoud, E.; Schopf, E.; Fomina, N.; Almutairi, A. Biocompatible Polymeric Nanoparticles Degrade and Release Cargo in Response to Biologically Relevant Levels of Hydrogen Peroxide. J. Am. Chem. Soc. 2012, 134 (38), 15758-15764. (35) Jourden, J. L.; Daniel, K. B.; Cohen, S. M. Investigation of Self-Immolative Linkers in the Design of Hydrogen Peroxide Activated Metalloprotein Inhibitors. Chem Commun. 2011, 47 (28), 7968-7970. (36) An, S.; He, D. S.; Wagner, E.; Jiang, C. Peptide-Like Polymers Exerting Effective Glioma-Targeted siRNA Delivery and Release for Therapeutic Application. Small 2015, 11 (38), 5142-5150. (37) Wang, W.; Xiong, W.; Wan, J. L.; Sun, X. H.; Xu, H. B.; Yang, X. L. The Decrease of PAMAM Dendrimer-Induced Cytotoxicity by PEGylation via Attenuation of Oxidative Stress. Nanotechnology 2009, 20 (10), 105103. (38) Huang, R. Q.; Liu, S. H.; Shao, K.; Han, L.; Ke, W. L.; Liu, Y.; Li, J. F.; Huang, S. X.; Jiang, C. Evaluation and Mechanism Studies of PEGylated Dendrigraft Poly-L-Lysines as Novel Gene Delivery Vectors. Nanotechnology 2010, 21 (26), 265101. (39) Yang, X. Z.; Dou, S.; Wang, Y. C.; Long, H. Y.; Xiong, M. H.; Mao, C. Q.; Yao, Y. D.; Wang, J. Single-Step Assembly of Cationic Lipidpolymer Hybrid Nanoparticles for Systemic Delivery of siRNA. ACS Nano 2012, 6 (6), 4955-4965. (40) Jiang, Y.; Tang, R.; Duncan, B.; Jiang, Z. W.; Yan, B.; Mout, R.; Rotello, V. M. Direct Cytosolic Delivery of siRNA Using Nanoparticle-Stabilized Nanocapsules. 33

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Angew. Chem. Int. Ed. Engl. 2015, 54 (2), 506-510. (41) Aji Alex, M. R.; Nehate, C.; Veeranarayanan, S.; Sakthi Kumar, D.; Kulshreshtha, R.; Koul, V. Self-Assembled Dual Responsive Micelles Stabilized with Protein for Co-Delivery of Drug and siRNA in Cancer Therapy. Biomaterials 2017, 133, 94-106. (42) Kolosenko, I.; Edsbacker, E.; Bjorklund, A. C.; Hamil, A. S.; Goroshchuk, O.; Grander, D.; Dowdy, S. F.; Palm-Aperqi, C. RNAi Prodrugs Targeting Plk1 Induce Specific Gene Silencing in Primary Cells from Pediatric T-Acute Lymphoblastic Leukemia Patients. J. Control. Release. 2017, 261, 199-206. (43) Boer, P. A.; Gontijo, J. A. Nuclear Localization of SP, CGRP, and NK 1 R in a Subpopulation of Dorsal Root Ganglia Subpopulation Cells in Rats. Cell Mol. Neurobiol. 2006, 26 (2), 191-207. (44) Zhou, J. B.; Patel, T. R.; Michael, F.; Bertram, J. P.; Saltzman, W. M. Octa-Functional PLGA Nanoparticles for Targeted and Efficient siRNA Delivery to Tumors. Biomaterials 2012, 33 (2), 583-591.

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