Reactive Oxygen Species-Biodegradable Gene Carrier for the

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

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Reactive Oxygen Species-Biodegradable Gene Carrier for the 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† †

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 ‡ Department of Interventional Radiology, Ruijin Hospital Shanghai Jiao Tong University School of Medicine Shanghai 200025, PR China S Supporting Information *

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 low toxicity for clinical applications. Herein, we have innovatively introduced a reactive oxygen species (ROS)-biodegradable boric acid ester linkage in elaborating the design of a gene carrier. In virtue of the featured intracellular characteristics such as the high level of ROS in tumor cells, an ROS-biodegradable electropositive polymer derived from branched polyethylenimine (BPEI) with a low molecular weight (1.2k) through a cross-linking reaction by the 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. The final fabricated SP-cross-linked BPEI/plasmid DNA nanoparticles exhibit favorable biocompatibility, ROS-cleavability, and fine targeting ability as well as high transfection efficiency compared with parental BPEI1.2k both in vitro and in vivo. SP-cross-linked BPEI/small interfering RNA (pololike kinase 1) polyplex possesses favorable gene-silencing effects in vitro and satisfactory antitumor ability in vivo. Hopefully, this novel cross-linked electropositive polymer may serve well as a safe and efficient gene-delivery vehicle in the clinic. KEYWORDS: gene delivery, polyethylenimine, ROS-responsive, substance P, Plk1 manner.3−5 Encouragingly, the prospect of gene therapy has been cheerful since the market authorization of many oligonucleotide-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 nonviral vehicles. Highly efficient viral vectors commonly generate immune reactions, which greatly limit their clinical application. In this context, nonviral gene-

1. INTRODUCTION Malignant tumors have undoubtedly become the most fatal pathema and the most serious threat to human health because of unsatisfactory treatment effect as well as the mounting incidence rate. Conventional treatments, including surgery and the 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 the 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 © XXXX American Chemical Society

Received: January 29, 2018 Accepted: March 2, 2018 Published: March 2, 2018 A

DOI: 10.1021/acsami.8b01712 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces

cancer cells and remarkably specific gene-silencing effects to induce cancer cell apoptosis and to significantly suppress tumor growth following systemic administration. Hopefully, this novel ROS-responsive cross-linked electropositive polymer may serve well as a safe and efficient gene-delivery vehicle in the clinic.

delivery strategies have attracted much attention and have been a hotspot in the past few years.10−12 An ideal nonviral 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 the escorted genes during in vivo circulation; (iii) fine targeting ability and (iv) efficient gene release character in cytoplasm. To some extent, the electropositive macromolecular materials hold fine condensing efficiency but are biorefractory into fragments to release the escorting cargo as well as to reduce the toxicity.13,14 Herein, developing a polymer with a high positive density that can biodegrade at focal sites is still challenging. The high level of reactive oxygen species (ROS) is a unique hallmark associated with the pathological process in many diseases including cancer,15 Alzheimer’s disease,16 inflammation,17 reperfusion injury,18 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 wellstudied tailoring strategies employing lower intracellular tumor pH, matrix metalloproteinase level, or hypoxia condition. In the recent couple of years, ROS-triggered gene vector has become increasingly attractive with fine distinguished responsiveness between focal and normal cells. For instance, in the pioneering work reported by Shen, they developed a boronic ester-based charge-reversal gene carrier, where the ROS-responsive procedure is relatively long, even when exposed to a high ROS level (80 mM).22,23 Prof. Murthy also prepared thioketal nanoparticles (NPs) for ROS-initiated small interfering RNA (siRNA) oral delivery for intestinal inflammation.24 More sophisticated ROS-triggered gene carriers with higher sensitivity should be developed. The aryl borate esters can be cracked by the ROS species, where the reaction condition is mild and rapid.25−28 Besides that, both the borate ester itself and the cleavage product, boronic acids, cause unconspicuous toxicity to human tissues.26 Thus, the aforementioned information inspired us to explore the arylboronic esters as the ROS-responsive linkage to develop a biodegradable gene vector. Herein, we innovatively developed a ROS-biodegradable electropositive and biocompatible polymer derived from branched polyethylenimine (BPEI) with a low molecular weight (1.2k) through a photo-cross-linking reaction to achieve stable condensing and escorting of genes as well as for 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-GlnPhe-Phe-Gly-Leu-Met) via polyethylene glycol (PEG). The SP peptide can specifically recognize and combine with 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 (pRFP) DNA was first employed to evaluate the transfection efficiency and safety issue with the polymer. The final constructed SP-crosslinked BPEI/plasmid DNA (pDNA) NPs exhibit low toxicity and ROS-cleavability as well as a much higher transfection efficiency compared with parental BPEI1.2k both in vitro and in vivo. Finally, pololike kinase 1 (Plk1) siRNA was selected as the therapeutic gene and loaded in the cross-linked BPEI polymer to treat breast cancer. The fabricated SP-cross-linked BPEI/ siRNA polyplex exhibited efficient delivery of siRNA into

2. MATERIALS AND METHODS 2.1. Materials. BPEI (Mw = 1.2k and Mw = 25k) (BPEI1.2k and BPEI25k) was purchased from Sigma-Aldrich (St. Louis, USA). 4Vinylphenylboronic 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)-1piperazineethanesulfonic acid (HEPES) was purchased from Aladdin Industrial Corp. (Shanghai, China). ω-Succinimidyl carbonate-PEGmaleimide (NHS-PEG3.5k-Mal) and methoxy-PEG3k-NHS (MeOPEG3k-NHS) were synthesized by Jenkem Technology Co., Ltd. (Beijing, China). SP peptide with thiol group modification was synthesized by China Peptides Co., Ltd. (Suzhou, China). siRNA targeting Plk1 messenger RNA (mRNA) (siPlk1, antisense strand, 5′UAAGGAGGGUGAUCUUCUUCAdTdT-3′) and Cy5-labeled s i R N A ( C y 5 - s i R N A , a n ti s e n s e s tr a n d , 5 ′ - A A C C A C T CAACTTTTTCCCAAdTdT-3′) were synthesized by GenePharma (Suzhou, China). pRFP (GeneChem Co., Ltd., Shanghai, China) and pGL3-control vector (Promega, Madison, WI, USA) were purified using the 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 Institute of Biotechnology (Shanghai, China). Dulbecco’s modified Eagle’s medium and fetal bovine serum 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 the guidelines approved by the Fudan University Ethics Committee. 2.2. Synthesis of SP-Cross-Linked BPEI. 2.2.1. Synthesis of the Boronic Ester Diene Cross-Linker. 4-Vinylphenylboronic acid (300 mg, 2 mmol) and 3-allyloxy-1,2-propanediol (396 mg, 3 mmol) were stirred in dry dichloromethane (20 mL) with anhydrous sulfate sodium (2 g) at 40 °C overnight. After the reaction was complete, 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 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 a nuclear magnetic resonance (NMR) spectrometer (600 MHz, Bruker, Billerica, MA, USA) at 25 °C. 2.2.2. Synthesis of Cross-Linked BPEI. First, 0.5 M HCl was added dropwise to the aqueous BPEI (Mw = 1.2k, 1 g) to adjust the pH to 7.2, and water was removed by freeze-drying. Next, the obtained solid BPEI (700 mg, 1 equiv) was dissolved in methanol (MeOH) under N2, and propylene sulfide (90 μL, 2 equiv) was added into the above solution via a syringe. The whole solution was stirred under 60 °C for 12 h. After that, the solvent was removed, and the residue was precipitated in cold ether 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 of MeOH again, 10 μL of which was added into HEPES buffer (1.9 mL) to react with Ellman’s reagent (80 μL) for 0.5 h. The concentration of the thiol group was calculated through absorbance at 412 nm with an ultraviolet (UV) detector. As for the cross-linking reaction, excessive cross-linker VPBE (5 equiv thiol mol) and a catalytic amount of 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, B


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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 preincubated with the SP peptide at 37 °C for 15 min and then added to SP-cross-linked BPEI/pDNA NPs for 0.5 h. After the incubation, the cells were washed with D-Hank’s buffer three times and photographed under a 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 BPEI1.2k/pDNA, BPEI25k/pDNA, cross-linked BPEI/pDNA, and SPcross-linked BPEI/pDNA NPs (N/P = 10) containing 5 μg pDNA in a serum-free medium at 37 °C for 4 h. After incubation, the medium was replaced with a complete serum-containing medium, and the cells were cultured for another 44 h. The expression of RFP was visualized under a 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 the luciferase activity assay, after transfection, the cells were washed with PBS three times and then treated with 150 μL of lysis reagent provided in the Promega Luciferase Assay Kit (Promega, Madison, WI, USA). The lysis solution was centrifuged at 12 000 rpm for 5 min at 4 °C. Luciferase activity in the supernatant was quantified by a Luciferase Assay System (Promega, Madison, WI, USA), and the total protein amount was determined by a Bradford protein assay. The final data were determined by the light unit of each sample minus the blank group, and the results were expressed as light units/mg protein. 2.3.4. 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, the mice were anesthetized; the tumors were dissected and fixed in 4% paraformaldehyde for 24 h for frozen sections. The obtained sections were then stained with 4′,6diamidino-2-phenylindole (DAPI) for 10 min at room temperature, washed with PBS (pH 7.4), and immediately photographed under a fluorescence microscope. 2.4. siPlk1 Polyplexes. 2.4.1. Characterization of siPlk1 Polyplexes. To determine the optimal N/P ratio between the BPEI polymer and siRNA, various N/P ratios of PEG-cross-linked BPEI and SP-cross-linked 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 series of different NPs were measured by DLS to confirm the optimized ratio. The morphology of SP-cross-linked BPEI/siRNA NPs was also captured using TEM. The ROS-responsive assay of cross-linked BPEI/ siRNA NPs was the same as that of the above cross-linked BPEI/ pDNA NPs via DLS results. 2.4.2. Plk1 Gene-Silencing Assay in Vitro. To evaluate the silencing capability of hybrid NPs, MDA-MB-231 cells were seeded into 6 well plates at a 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 the 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′-AGCCTGAGGCCCGATACTACCTAC3′ (Plk1-forward) and 5′-ATTAGGAGTCCCACACAGGGTCTTC3′ (Plk1-reverse). All operations were completed by Servicebio Company (Wuhan, China). For the western blot analysis, the cells were lysed with 150 μL of immunoprecipitation cell lysis buffer, which was freshly added to phenylmethanesulfonyl fluoride at a concentration of 1 mM on ice for 15 min. The lysates were then centrifuged for 10 min at 12 000 rpm, and the concentration of the total protein in the supernatant was

rotating periodically to ensure uniform irradiation. Finally, the solvent was removed, and the cross-linked BPEI was precipitated in cold ether three times. The cross-linked BPEI was then purified by a dialysis method (Mw = 10k) to obtain the macromolecular polymer. The thiol amount after the cross-linking reaction was analyzed using Ellman’s reagent. 2.2.3. Synthesis of PEGylated and SP Cross-Linked BPEI. First, the sulfydryl-modified SP peptide was reacted with NHS-PEG3.5k-Mal in the ratio of 1.5:1 (mol/mol) in phosphate-buffered saline (PBS) buffer (pH 7.0) overnight at room temperature, followed by dialysis (Mw = 3.0k) to obtain the pure product SP-PEG-NHS. The Mal groups of PEG were specifically reacted with the thiol groups of the peptide. SPPEG-NHS was then solubilized in D2O and characterized by NMR spectroscopy. For the synthesis of PEGylated and SP peptide-modified cross-linked BPEI (SP-cross-linked BPEI), briefly, the cross-linked BPEI was reacted with MeO-PEG-NHS and SP-PEG-NHS, respectively, in the mole ratio of 1:1 in PBS buffer (pH 8.0) for 12 h at room temperature and purified by dialysis (Mw = 10k). 2.3. pDNA Polyplexes. 2.3.1. Characterization of pDNA Polyplexes. For the preparation of different pDNA-loaded NPs, various N/P ratios of BPEI1.2k, PEG-cross-linked 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-cross-linked BPEI/pDNA NPs was performed using transmission electron microscopy (TEM, JEOL JEM-1200EX, Tokyo, Japan). To confirm the degradation of the cross-linked 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 times. The degradation of cross-linked BPEI/pDNA under other ROS molecules, including the hydroxyl radical (OH•) and hypochlorous acid/hypochlorite (HOCl/−OCl),30 was also examined via DLS assay for 2 h. Hydroxyl radical was produced by mixing CuCl2 with H2O2.21 Size observation of cross-linked BPEI/pDNA NPs in serum at different times 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 the loading buffer, and 0.7% agarose gel electrophoresis with 0.5 mg/mL of ethidium bromide was then conducted at 100 V in Tris-acetateethylenediaminetetraacetic acid (EDTA) 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 ROSresponsive gene release assay, sodium heparin was added into the H2 O2 -treated SP-cross-linked 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. 2.3.2. 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 a density of 8 × 103 cells per well. Cells reaching 80% confluence were exposed to 100 μL of different NPs containing 0.5 μg pDNA with N/P ratios from 10 to 30. After incubation for 6 h, the cells were rinsed by PBS and incubated for another 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 cross-linked BPEI and BPEI1.2k was evaluated in a similar manner on MDA-MB-231 cells at different concentrations ranging from 5 to 80 μg/mL. 2.3.3. 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 prelabelled 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 and SP-cross-linked BPEI/pDNA NPs at a C


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Scheme 1. (A) Synthesis of Cross-Linked BPEI and SP-Cross-Linked BPEI. (B) Scheme Illustration of the SP Peptide-Mediated Cellular Uptake and ROS-Biodegradable SP-Cross-linked BPEI/siRNA in Tumor Cells

and observed under the fluorescent microscope. At the end of the tumor-treatment assay, the main organs (heart, liver, spleen, lung, and kidney) were excised for hematoxylin and eosin (H&E) staining. 2.5. 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).

determined through the BCA Protein Assay Kit (Beyotime Biotechnology, Beijing, China). The total protein (40 μg) was separated on 12% polyacrylamide-sodium dodecyl sulfate gels and then transferred to polyvinylidene fluoride membranes (300 mA for 2 h). The membranes were blocked in 5% nonfat milk in PBS with Tween-20 [Tris-buffered saline (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 a gel imager (Bio-Rad, CA, USA). 2.4.3. Biodistribution and Therapeutic Efficacy of siPlk1 Polyplexes in Vivo. Cy5-siRNA was used to investigate the biodistribution of siRNA-loaded NPs. Briefly, the tumor-bearing mice with MDA-MB-231/luci cells were injected with cross-linked BPEI and SP-cross-linked BPEI polymer/Cy5-siRNA NPs (20 μg siRNA per mouse) through the tail vein. The mice were anesthetized post 24 h and visualized under a noninvasive in vivo imaging system (IVIS) (Caliper, Newton, MA, 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. 2.4.4. Antitumor Efficacy in Vivo. Tumor-bearing mice with MDAMB-231/luci cells were randomly divided into four groups (n = 8) and treated with saline, free siPlk1, cross-linked 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 2 days. For the detection of Plk1 protein in tumor tissues, the tumor was removed after the last injection and homogenized in radioimmunoprecipitation assay lysis buffer for 2 min. The lysates were then centrifuged for 10 min at 12 000 rpm. The BCA method was used to determine the concentration of the total protein in the supernatant. Finally, the proteins were detected by the 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; then, they were stained with DAPI

3. RESULTS AND DISCUSSION 3.1. Synthesis of SP-Cross-Linked BPEI. The specific synthesis route of SP-cross-linked BPEI is shown in Scheme 1A. To fabricate a positive-charged polymer, we successfully synthesized a borate-based cross-linker VPBE, the structure of which was confirmed by 1H NMR spectra (Figure S1). The specific cross-linking reaction was then performed through the “click reaction” between the thiol groups premodified on BPEI and the two olefin bonds of the VPBE cross-linker. The BPEI1.2k 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) that appeared in the 1H NMR spectra (Figure S2) indicated the successful modification. The next click reaction was a consumption process of thiol groups irradiated by UV365nm. Table S1 shows that the concentration of thiol groups decreased from 0.213 ± 0.03 to 0.085 ± 0.02 mmol/g after the cross-linking treatment. The benzene ring peak of the VPBE cross-linker appeared in the final cross-linked BPEI compound (Figure S3), and the sharp decline of the thiol amount after irradiation indicated the successful cross-linking reaction with preferable efficiency. After that, the SP peptide was conjugated onto the cross-linked BPEI polymer via a PEG moiety. In the spectrum of Figure S4, the multiple characteristic peaks of the SP peptide were found in SP-cross-linked BPEI molecules, suggesting a successful connection. During the synthesis D


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

Figure 2. Evaluation of the ROS-responsive characteristic of cross-linked BPEI. (A) 1H NMR of the cross-linked BPEI before and after treating with 1 mM H2O2 for 12 h. (B) Heparin replacement result of cross-linked BPEI/pDNA NPs. Size change of the cross-linked BPEI/pDNA NPs after treating with 1 mM H2O2 for different times (C) and the DLS result of NPs 24 h in PBS and H2O2 (D).

process of cross-linked BPEI, we also optimized the crosslinking degree by adjusting the ratio of modificatory thiol groups. Different ratios of propylene sulfide (2, 4 equiv) relative to the BPEI1.2k monomer were investigated. Disappointedly, the increased amount of propylene sulfide (4 equiv) resulted in an elevated thiol modification of BPEI, eventually leading to a higher cross-linking degree in the final cross-linking reaction. Because of the aromatic structure among the VPBE crosslinker, the solubility of cross-linked BPEI was not favorable (Figure S7A). Hence, we finally selected the relatively low ratio of propylene sulfide (2 equiv). 3.2. pDNA Polyplexes. 3.2.1. Characterization of pDNA Polyplexes. pRFP, a reporter gene, was employed to evaluate the gene-delivery efficiency of the cross-linked BPEI vehicle. The condensation efficiency was evaluated through the gel

electrophoresis assay. Figure S5 shows that the cross-linked BPEI exhibited a strong interaction ability with pDNA and could restrain its retardation at a cross-linked BPEI weight/ pDNA weight (w/w) ratio of 0.6. As for the uncross-linked BPEI1.2k monomer, a higher ratio (0.8) appeared to prevent the movement of pDNA. The gel electrophoresis assay only indicated that 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, cross-linked BPEI can efficiently condense pDNA to form uniform NPs with a diameter of about 151.83 ± 15.03 nm at a low N/P ratio of 10. In comparison, uncross-linked BPEI1.2k showed a relatively large size distribution (over 200 nm) even at high ratios, which indicated its weak DNA binding affinity E


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Figure 3. Cell viability of HEK 293 (A) and MDA-MB-231 (B) cells after incubation with different NPs at various N/P ratios.

original phenyl boric acid ester bond in cross-linked BPEI was broken up into hydrophilic hydroxyl and carboxyl groups, followed by the cross-linked BPEI dissociated into the soluble single BPEI, which eventually resulted in the water-soluble clear solution. Inspired by the results of the ROS-responsive feature of this cross-linked gene-delivery polymer, we examined the degradation profile of cross-linked BPEI/pDNA under simulated ROS conditions. In Figure 2C,D, we could see that the mean diameter of NPs gradually became larger with prolonged H2O2 incubation time, and the size exceeded more than 1 μm when treated for 20 h, which indicated the fracture, expansion, and disruption of NPs, whereas the PBS group consistently maintained the same size. Consistent with the H2O2-triggered degradation of NPs, DNA unpackaging from NPs was further proved by the heparin replacement electrophoresis assay. Heparin loading with a 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 being partly deprived, which was suffice to show the abundant positive charge of the crosslinked BPEI. As expected, after preincubation 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 cross-linked BPEI cleaved into 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 shows the degradation of NPs under other ROS molecules including OH• and −OCl for only 2 h, which further suggested the ROSresponsive characteristic. Among these ROS stimuli, OH• exhibited the strongest effect, and the influence of catalyzer Cu2+ was excluded. 3.2.2. 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 HEK 293 cell lines (Figure 3). HEK 293 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 cross-linked BPEI. Compared with BPEI1.2k, crosslinked BPEI polyplexes presented more favorable biocompatibility on both the 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 a low toxicity even at the concentration 40 μg/mL, whereas BPEI1.2k caused serious damage to cells at the beginning concentration of 5 μg/mL (Figure S6). It could be explained that the ROS-cleaved trait of cross-linked BPEI was

due to its low molecular weight. Because of the modification of the SP peptide, the mean size of SP-cross-linked BPEI/pDNA was slightly larger than cross-linked BPEI/pDNA. In addition, the zeta potentials 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 about 20 mV. The electric property of BPEI1.2k/pDNA was about −12.31 ± 3.37 mV at the beginning, which exhibited an absolute negative charge, whereas the crosslinked group exhibited a positive electric property of about 11.47 ± 1.43 mV. Even at the high N/P ratio of 20, cross-linked BPEI/pDNA (about 25 mV) held much more electropositive density than BPEI1.2k/pDNA (about 15 mV). By the way, we also investigated the zeta potential of various simple BPEI materials. Table S2 implied that cross-linked BPEI evidently possessed a much more powerful positive charge (55.9 ± 1.28 mV) than the pure BPEI1.2k monomer (16.20 ± 1.90 mV). The PEG and SP peptide conjugation onto cross-linked 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 cross-linking strategy increased the Mw and enriched the positive charge density of BPEI and consequently enhanced the condensation ability to capture the gene more efficiently and more tightly. Balancing the above DLS and zeta potential results, the optimized N/P ratio was confirmed to be 10, considering that excessive positive charge of NPs would generate cellular toxicity and nonspecific distribution.31,32 Furthermore, TEM in Figure 1D shows a spherical and uniform morphology of SP-cross-linked BPEI/pDNA NPs at an N/P ratio of 10 and also exhibits a core−shell structure with the PEG conjunction as the hydrophilic stabilization shell.33 The serum stability assay in Figure S8 shows that the size of cross-linked BPEI/pDNA NPs still maintained a relatively stable nanostructure, only slightly larger, even at the existence of serum for 20 h, which indicated that our constructed genedelivery system was resistant to the abominable blood environment. We simply verified the ROS-responsive characteristic of the pure cross-linked BPEI polymer by 1H NMR spectroscopy. Upon treatment with 1 mM H2O2, the phenyl borate ester part will be cleaved into boric acid and glycol components.34,35 In the 1H NMR spectrum shown in Figure 2A, the peak position of four hydrogens in the benzene ring varied from 7.5−7.9 to 6.5−7.0 ppm. Besides that, we observed a phenomenon that the suspension of water-insoluble BPEI with a higher cross-linking degree would turn into a transparent solution when treated with H2O2. In Figure S7, under the presence of H2O2, the F


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Figure 4. Cellular uptake and gene transfection efficiency of different formulations. (A) Cellular uptake of cross-linked BPEI/pDNA and SP-crosslinked BPEI/pDNA on MDA-MB-231 cells 30 min after incubation (100×, scale bar indicates 50 μm). (B) Qualitative transfection results of different NPs in vitro (100×, scale bar indicates 50 μm). (C) 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).

transfection efficiency in vitro compared with BPEI25k.37,38 However, another fact is that the efficacy and toxicity of BPEI are strongly connected with its Mw, and the application of BPEI25k is mainly restricted to its massive molecules and abundant positive charge. 3.2.4. 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 the tumor site on the 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 2 days of expressing the protein, the mice were anaesthetized and tumors were excised for frozen section. Figure 4C showed that the RFP expression intensity of the cross-linked BPEI group was significantly higher than that of uncross-linked BPEI. Because cross-linked BPEI/pDNA NPs were more resistant to the complicated blood circumstance and held efficient gene separation ability to take the following effect originated by the ROS-responsive feature. Besides that, the modified SP peptide ligand on cross-linked BPEI indeed realized the more precise tumor-delivery capability, thus creating the maximum RFP fluorescence intensity in the tumor site. 3.3. siPlk1 Polyplexes. 3.3.1. Characterization of siPlk1 Polyplexes. Because we have demonstrated that this designed novel cross-linked BPEI polymer exhibited the favorable genedelivery potential via the reporter gene both in vitro and in vivo, Plk1 was selected as a model therapeutic siRNA for 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 siPlk1 into uniform-sized NPs, just as the same procedure as for pDNA. Table 1 shows that both the mean diameter (90.30 ± 5.87 nm) and polydispersity index (PDI) (0.190 ± 0.021) were perfect at the N/P ratio of 3. The electric property of cross-linked BPEI/siRNA was about −11.83 ± 3.15 mV at the N/P ratio of 1, which had an absolute negative charge, whereas the zeta potential reversed from negative to

in favor of degradation into small fragments with much lower toxicity. 3.2.3. Cellular Uptake and Gene Transfection Assay of pDNA Polyplexes in Vitro. We evaluated the cellular uptake characteristics of the SP-cross-linked BPEI/pDNA to prove the targeting capacity on MDA-MB-231 cells (Figure 4A). Graphics captured by a fluorescence microscope indicated that the SP group exhibited an obviously enhanced cellular uptake behavior compared with the nontargeted cross-linked BPEI/pDNA, ascribed to the high-expressed NK-1 receptor-mediated endocytosis. Besides that, the cellular uptake of SP-cross-linked BPEI/pDNA was significantly restricted when the receptors were presaturated with the excessive free SP peptide, which further indicated that the enhanced accumulation of SP-crosslinked BPEI/pDNA resulted from the specific binding ability of the SP peptide to the NK-1 receptors. Then, the gene transfection experiments were performed on MDA-MB-231 cells. Figure 4C exhibits the RFP qualitative expression of different NPs. BPEI25k/pDNA was employed as the positive control. Cross-linked BPEI/pDNA showed a much stronger fluorescence intensity compared with the uncrosslinked BPEI1.2k/pDNA group. This difference was mainly because of the efficient gene separation from the cationic carrier after cytosolic delivery. Cross-linked BPEI took 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 enhanced cellular uptake rooting from the SP peptide also produced the more favorable transfection result of the SP-cross-linked 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 the qualitative transfection assay, the luciferase activity of cells treated with cross-linked BPEI, especially the SP-targeted group (5.94 ± 0.53 × 105), was significantly higher than that of uncross-linked BPEI/pDNA (1.08 ± 0.13 × 105). It is reasonable that the transfection effect of the synthesized cross-linked BPEI polymer was not as good as the positive control BPEI25k, which possesses more positive electricity and without PEG modification. Meanwhile, the cross-linked BPEI was conjugated with a certain amount of PEG for reduced toxicity, but PEGylation might somehow influence the G


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ACS Applied Materials & Interfaces Table 1. Mean Size and PDI Results of Cross-Linked BPEI/ siPlk1 with Various N/P Ratios N/P ratio mean size (nm) PDI zeta potential (mV)

1 134.30 ± 12.24 0.256 ± 0.013 −11.83 ± 3.15

3 90.30 ± 5.87 0.190 ± 0.021 16.03 ± 3.07

6 303.32 ± 15.21 0.273 ± 0.026 24.30 ± 3.67

positive (16.03 ± 3.07 mV) when the N/P ratio increased to 3. Figure 5 shows the spherical and uniform morphology of SPcross-linked 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 cross-linked BPEI/siRNA NPs in Figure S10 gradually became larger when treated with H2O2 and exhibited the same ROS-responsive character. 3.3.2. Plk1 Gene-Silencing Assay in Vitro. We evaluated the gene-silencing effect of the siPlk1-loaded NPs on MDA-MB231 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, whereas the total protein was extracted after 48 h for the western blot assay. As shown in Figure 6, both cross-linked BPEI/siPlk1 and SP-cross-linked BPEI/siPlk1 complexes resulted in the downregulation of Plk1 mRNA and protein expression, especially for the SP peptide-targeting group. Because of the NK-1 receptor-mediated enhanced cellular uptake of SP-cross-linked BPEI/siPlk1 NPs, nearly 60% of Plk1 mRNA expression was suppressed and there was a very distinct inhibition of protein expression. 3.3.3. Biodistribution and Therapeutic Efficacy of siPlk1 Polyplexes in Vivo. Cy5-siRNA was packaged into different complexes to observe the biodistribution in the breast tumor model nude mice. Twenty-four hours after intravenous injection 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 cross-linked BPEI/ siRNA and SP-cross-linked BPEI/siRNA. For the cross-linked BPEI/siRNA group, the distribution ability into the tumor is analyzed mainly because of the enhanced permeability and retention (EPR) effect and PEG shielding effect (passive targeting); whereas for SP-cross-linked BPEI/siRNA, besides the above two factors, the positive breast cancer-targeting function of the 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, and lung was similar in both the 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

Figure 6. Gene downregulation results of various formulations. 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.

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

the SP-cross-linked BPEI vehicle. Because of the NK-1 receptor, the SP specific binding receptor is also widely

Figure 5. Characterization of SP-cross-linked BPEI/siRNA polyplexes. DLS (A) and TEM (B) result of SP-cross-linked BPEI/siRNA at the N/P ratio of 3. H


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

distributed in the renal pelvis besides the tumor.43 We inferred that the amount of SP group in mice kidneys after 24 h was remarkably high, which might be due to 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. 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 received an intravenous injection of saline, free siPlk1, cross-linked BPEI/ siPlk1, and SP-cross-linked 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 groups sharply increased along with the monitoring time. While siPlk1 was packaged into the stable complexes through the cross-linked BPEI gene vector and injected to the tumor-bearing nude mice, the tumor growth was found greatly suppressed and the SP-cross-linked 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 the 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 the cross-linked BPEI/siPlk1 and SP-cross-linked BPEI/siPlk1 groups, which was consistent with the tumor volume results. To further confirm the relationship between the suppressed tumor growth and the downregulation of Plk1 protein in tumor cells, the tumor was excised 24 h post the last injection, and the total protein was extracted for the western blot assay. Mice treated with saline and free siPlk1 exhibited little reductions in the Plk1 protein level. By contrast, there was a significant downregulation both in cross-linked 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 that should be

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: DAPIlabeled nucleus (100×, scale bars indicate 80 μm).

taken into consideration when evaluating a satisfactory genedelivery vehicle is the biocompatibility and safety. Although the current clinical trials with lipid-based nanocarriers have high efficiency, the toxicity, especially at high administration dosage, greatly led to its clinical failure.44 From the normal daily observation of the body weight and the living state of mice during the treatment period, little side effects were observed. 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 the treated groups (Figure S11). In conclusion, we present 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 I


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(3) Zhu, L.; Simpson, J. M.; Xu, X.; He, H.; Zhang, D.; Yin, L. Cationic Polypeptoids with Optimized Molecular Characteristics Toward Efficient Nonviral Gene Delivery. ACS Appl. Mater. Interfaces 2017, 9, 23476−23486. (4) Naldini, L. Gene Therapy Returns to Centre Stage. Nature 2015, 526, 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, 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 Delivery 2017, 14, 685−696. (7) Stein, C. A.; Castanotto, D. FDA-Approved Oligonucleotide Therapies in 2017. Mol. Ther. 2017, 25, 1069−1075. (8) Mout, R.; Ray, M.; Tonga, G. Y.; Lee, Y.-W.; Tay, T.; Sasaki, K.; Rotello, V. M. Direct Cytosolic Delivery of CRISPR/Cas9Ribonucleoprotein for Efficient Gene Editing. ACS Nano 2017, 11, 2452−2458. (9) Yang, J.; Meng, X.; Pan, J.; Jiang, N.; Zhou, C.; Wu, Z.; Gong, Z. CRISPR/Cas9-Mediated Noncoding RNA Editing in Human Cancers. RNA Biol. 2017, 13, 35−43. (10) Li, S.-D.; Huang, L. Gene Therapy Progress and Prospects: Non-Viral Gene Therapy by Systemic Delivery. Gene Ther. 2006, 13, 1313−1319. (11) Schaffert, D.; Wagner, E. Gene Therapy Progress and Prospects: Synthetic Polymer-Based Systems. Gene Ther. 2008, 15, 1131−1138. (12) He, H.; Zheng, N.; Song, Z.; Kim, K. H.; Yao, C.; Zhang, R.; Zhang, C.; Huang, Y.; Uckun, F. M.; Cheng, J.; Zhang, Y.; Yin, L. Suppression of Hepatic Inflammation via Systemic siRNA Delivery by Membrane-Disruptive and Endosomolytic Helical Polypeptide Hybrid Nanoparticles. ACS Nano 2016, 10, 1859−1870. (13) He, H.; Bai, Y.; Wang, J.; Deng, Q.; Zhu, L.; Meng, F.; Zhong, Z.; Yin, L. Reversibly Cross-Linked Polyplexes Enable CancerTargeted Gene Delivery via Self-Promoted DNA Release and SelfDiminished Toxicity. Biomacromolecules 2015, 16, 1390−1400. (14) Deng, Q.; Li, X.; Zhu, L.; He, H.; Chen, D.; Chen, Y.; Yin, L. Serum-Resistant, Reactive Oxygen Species (ROS)-Potentiated Gene Delivery in Cancer Cells Mediated by Fluorinated, DiselenideCrosslinked Polyplexes. Biomater. Sci. 2017, 5, 1174−1182. (15) Benz, C. C.; Yau, C. Ageing, Oxidative Stress and Cancer: Paradigms in Parallax. Nat. Rev. Cancer 2008, 8, 875−879. (16) Sayre, L. M.; Perry, G.; Smith, M. A. Oxidative Stress and Neurotoxicity. Chem. Res. Toxicol. 2008, 21, 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, 12462−12465. (18) Zweier, J. L.; Talukder, M. A. H. The Role of Oxidants and Free Radicals in Reperfusion Injury. Cardiovasc. Res. 2006, 70, 181−190. (19) Tsutsui, H.; Kinugawa, S.; Matsushima, S. Mitochondrial Oxidative Stress and Dysfunction in Myocardial Remodeling. Cardiovasc. Res. 2009, 81, 449−456. (20) Xu, X.; Saw, P. E.; Tao, W.; Li, Y.; Ji, X.; Bhasin, S.; Liu, Y.; Ayyash, D.; Rasmussen, J.; Huo, M.; Shi, J.; Farokhzad, O. C. ROSResponsive Polyprodrug Nanoparticles for Triggered Drug Delivery and Effective Cancer Therapy. Adv. Mater. 2017, 29, 1700141. (21) Shim, M. S.; Xia, Y. A Reactive Oxygen Species (ROS)Responsive Polymer for Safe, Efficient, and Targeted Gene Delivery in Cancer Cells. Angew. Chem., Int. Ed. Engl. 2013, 52, 6926−6929. (22) Liu, X.; Xiang, J.; Zhu, D.; Jiang, L.; Zhou, Z.; Tang, J.; Liu, X.; Huang, Y.; Shen, Y. Fusogenic Reactive Oxygen Species Triggered Charge-Reversal Vector for Effective Gene Delivery. Adv. Mater. 2016, 28, 1743−1752. (23) Li, Y.; Bai, H.; Wang, H.; Shen, Y.; Tang, G.; Ping, Y. Reactive Oxygen Species (ROS)-Responsive Nanomedicine for RNAi-Based Cancer Therapy. Nanoscale 2017, 10, 203−214. (24) Wilson, D. S.; Dalmasso, G.; Wang, L.; Sitaraman, S. V.; Merlin, D.; Murthy, N. Orally Delivered Thioketal Nanoparticles Loaded with

the in vitro and in vivo results into account, such a genedelivery platform in this study put forward a new orientation for the future gene therapy.

4. CONCLUSIONS Since the discovery in 1990s, gene therapy has attracted tremendous interest to open a new therapeutic field. However, there are still many challenges and barriers to overcome the limitations of safety and for the effectiveness of the carriers for a wider application. In this work, a cross-linked BPEI polymer modified with the SP peptide-targeting ligand (SP-cross-linked BPEI) and an ROS-cleavable characteristic was developed as a novel, safe, and efficient gene-delivery platform. Both pDNA and 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 the ROS abundant environment endowed the complexes with an 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-cross-linked BPEI/siPlk1 exhibited a relatively strong tumor growth inhibition efficacy. Additionally, cross-linked BPEI caused low systemic toxicity. In summary, this work presented a potent and promising siRNA delivery strategy, which provided a basis for future gene therapy.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.8b01712. 1 H NMR spectra, gel electrophoresis assay of different NPs, amount of thiol groups on BPEI, zeta potential of BPEI, cell viability of MDA-MB-231 cells, water solubility of cross-linked BPEI, size of cross-linked BPEI/pDNA NPs in serum, size of cross-linked BPEI/ pDNA NPs under different ROS molecules, size of crosslinked BPEI/siRNA NPs treated with 1 mM H2O2, and H&E images of major organs (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: +86-21-5198-0187. ORCID

Chen Jiang: 0000-0002-4833-9121 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Natural Science Funds of China (21602030 and 81172993), the Shanghai Sailing Program (16YF1400900), the Scientific Research Foundation of Fudan University for Talent Introduction (JJF301103), and the National Science Fund for Distinguished Young Scholars (81425023).

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REFERENCES

(1) Lin, G.; Zhang, H.; Huang, L. Smart Polymeric Nanoparticles for Cancer Gene Delivery. Mol. Pharm. 2015, 12, 314−321. (2) Chen, Y.; Gao, D.-Y.; Huang, L. In Vivo Delivery of miRNAs for Cancer Therapy: Challenges and Strategies. Adv. Drug Delivery Rev. 2015, 81, 128−141. J


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(44) Zhou, J.; Patel, T. R.; Fu, M.; Bertram, J. P.; Saltzman, W. M. Octa-Functional PLGA Nanoparticles for Targeted and Efficient siRNA Delivery to Tumors. Biomaterials 2012, 33, 583−591.

TNF-α-siRNA Target Inflammation and Inhibit Gene Expression in the Intestines. Nat. Mater. 2010, 9, 923−928. (25) Jourden, J. L. M.; Daniel, K. B.; Cohen, S. M. Investigation of Self-Immolative Linkers in the Design of Hydrogen Peroxide Activated Metalloprotein Inhibitors. Chem. Commun. 2011, 47, 7968−7970. (26) Kuang, Y.; Balakrishnan, K.; Gandhi, V.; Peng, X. Hydrogen Peroxide Inducible DNA Cross-Linking Agents: Targeted Anticancer Prodrugs. J. Am. Chem. Soc. 2011, 133, 19278−19281. (27) Broaders, K. E.; Grandhe, S.; Fréchet, J. M. J. A Biocompatible Oxidation-Triggered Carrier Polymer with Potential in Therapeutics. J. Am. Chem. Soc. 2011, 133, 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. (29) Muñoz, M.; Coveñas, R.; Esteban, F.; Redondo, M. The Substance P/NK-1 Receptor System: NK-1 Receptor Antagonists as Anti-Cancer Drugs. J. Biosci. 2015, 40, 441−463. (30) Lou, Z.; Li, P.; Han, K. Redox-Responsive Fluorescent Probes with Different Design Strategies. Acc. Chem. Res. 2015, 48, 1358−1368. (31) An, S.; Jiang, X.; Shi, J.; He, X.; Li, J.; Guo, Y.; Zhang, Y.; Ma, H.; Lu, Y.; 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, 1670−1690. (33) Gu, Q.; Xing, J. Z.; Huang, M.; He, C.; Chen, C. SN-38 Loaded Polymeric Micelles to Enhance Cancer Therapy. Nanotechnology 2012, 23, 205101. (34) de Gracia Lux, C.; Joshi-Barr, S.; Nguyen, 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, 15758−15764. (35) Jourden, J. L. M.; Daniel, K. B.; Cohen, S. M. Investigation of Self-Immolative Linkers in the Design of Hydrogen Peroxide Activated Metalloprotein Inhibitors. Chem. Commun. 2011, 47, 7968−7970. (36) An, S.; He, D.; Wagner, E.; Jiang, C. Peptide-Like Polymers Exerting Effective Glioma-Targeted siRNA Delivery and Release for Therapeutic Application. Small 2015, 11, 5142−5150. (37) Wang, W.; Xiong, W.; Wan, J.; Sun, X.; Xu, H.; Yang, X. The Decrease of PAMAM Dendrimer-Induced Cytotoxicity by PEGylation via Attenuation of Oxidative Stress. Nanotechnology 2009, 20, 105103. (38) Huang, R.; Liu, S.; Shao, K.; Han, L.; Ke, W.d.; Liu, Y.; Li, J.; Huang, S.; Jiang, C. Evaluation and Mechanism Studies of PEGylated Dendrigraft Poly-L-Lysines as Novel Gene Delivery Vectors. Nanotechnology 2010, 21, 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, 4955−4965. (40) Jiang, Y.; Tang, R.; Duncan, B.; Jiang, Z.; Yan, B.; Mout, R.; Rotello, V. M. Direct Cytosolic Delivery of siRNA Using NanoparticleStabilized Nanocapsules. Angew. Chem., Int. Ed. Engl. 2015, 54, 506− 510. (41) Alex, M. R. A.; Nehate, C.; Veeranarayanan, S.; Kumar, D. S.; 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.; Edsbäcker, E.; Björklund, A.-C.; Hamil, A. S.; Goroshchuk, O.; Grandér, D.; Dowdy, S. F.; Palm-Apergi, C. RNAi Prodrugs Targeting Plk1 Induce Specific Gene Silencing in Primary Cells from Pediatric T-Acute Lymphoblastic Leukemia Patients. J. Controlled Release 2017, 261, 199−206. (43) Boer, P. A.; Gontijo, J. A. R. 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, 191−207. K


Reactive Oxygen Species-Biodegradable Gene Carrier for the

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