A Reactive Oxygen Species (ROS)-Degradable Polymeric

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A Reactive Oxygen Species (ROS)-Degradable Polymeric Nanoplatform for Hypoxia-Targeted Gene Delivery: Unpacking DNA and Reducing Toxicity Yuxin Zhang, Jie Zhou, Shengnan Ma, Yiyan He, Jun Yang, and Zhongwei Gu Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.9b00054 • Publication Date (Web): 28 Mar 2019 Downloaded from http://pubs.acs.org on March 29, 2019

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Biomacromolecules

A Reactive Oxygen Species (ROS)-Degradable Polymeric Nanoplatform for Hypoxia-Targeted Gene Delivery: Unpacking DNA and Reducing Toxicity

Yuxin Zhanga, Jie Zhoua, Shengnan Maa, Yiyan Hea,b,*, Jun Yangc, and Zhongwei Gua,b,*

a National

Engineering Research Center for Biomaterials, Sichuan University, No. 29 Wangjiang

Road, Chengdu 610064, P. R. China.

b College

of Materials Science and Engineering, Nanjing Tech University, No. 30 Puzhu

Road(S), Nanjing 211816, P. R. China.

c The

Key Laboratory of Bioactive Materials, Ministry of Education, College of Life Science,

Nankai University, Tianjin 300071, P. R. China.

* Corresponding

author

E-mail: [email protected] (Y. He), [email protected] & [email protected] (Z. Gu)

ACS Paragon Plus Environment

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ABSTRACT: Smart polymers as ideal gene carriers have drawn increasing attentions due to the effective DNA release once triggered by intrinsic stimuli, as well as reduced cytotoxicity. Herein, a stimulus-responsive, positively-charged and water-soluble polymer (OEI-TKx) was facilely engineered by cross-linking low molecular weight oligoethylenimine (OEI) via thioketal (TK) linkages that would cleave selectively in reactive oxygen species (ROS)-rich environments induced by hypoxia. Agarose gel electrophoresis assay demonstrated that the threshold N/P ratios for complete retardation of negatively-charged DNA migration were above 5 for OEI-TKx. The reduction in DNA-condensing capability and the changes in particle size, size distribution and particle morphology all illustrated that OEI-TKx possessed excellent ROS responsiveness. OEITKx/DNA polyplexes showed lower toxicity and higher gene transfection efficiency compared with PEI/DNA polyplexes. The optimum formulation, OEI-TKx/DNA polyplexes (N/P = 40), showed a little better performance than PEI/DNA polyplexes in cellular uptake profile. Furthermore, OEI-TKx/DNA polyplexes could escape from endosomes to the cytosol as efficiently as PEI/DNA polyplexes. Confocal images confirmed that OEI-TKx/DNA polyplexes could more effectively release DNA than PEI/DNA polyplexes, mainly owing to the valid cleavage of thioketal linkages induced by characteristic rich-ROS in Hela cells. These results suggested that OEI-TKx could represent an on-demand stimulus-responsive gene delivery platform.

Keywords: gene delivery; hypoxia-targeted; DNA release; reactive oxygen species; thioketal linkage


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INTRODUCTION Gene therapy represents a novel therapeutic procedure which shows great promise in treating patients with inherited1 and acquired diseases.2 One major setback in achieving satisfactory curative efficacy may be due to the lack of efficient gene delivery systems.3 Especially when it comes to cancer therapy, various hurdles of effective gene transfection include targeting the intended tumor tissue, penetrating the cell membrane, escaping from the endosome, releasing therapeutic gene and so on.4-6 Since virus-associated biohazards restrict the routine clinical application of viral vectors,7 the synthetic nonviral vectors for gene delivery including cationic lipid-based vector, polymeric vector, dendrimers, micelles and peptides have particularly caused increasing attentions in recent years in terms of their easier synthetic route and chemical modification, more convenient large-scale manufacturing and lower biosafety concerns.8-9 During gene delivery process by nonviral vectors, cationic polymers are widely adopted to condense DNA into nanosized polymers/DNA polyplexes for easier cellular internalization. However, the thermodynamic stability and intrinsic resistance to disassembly of fabricated polyplexes impede the release of DNA for further transcription to occur once inside the cell. Among various synthetic polymers used for nonviral gene delivery including polyethylenimine (PEI), poly(L-lysine) (PLL), poly(beta-amino ester)s10 and so on, PEI has been widely used as one of the most potent cationic polymers for gene delivery in both preclinical and clinical studies.11-12 In particular, 25 kDa branched PEI is considered as a gold standard in gene delivery because of its effective endosomal escape by proton sponge effect.13 However, the molecular weight of PEI can strongly influence its transfection efficiency and cytotoxicity. For example, PEI with a high molecular weight represents significant gene transfection but severe cytotoxicity, while low molecular weight PEI has good biocompatibility but poor transfection efficiency.14 To deal with



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such critical contradiction, it is thus an urgent demand to develop a gene delivery carrier that possesses sufficient molecular weight and charge density but could degrade into small segments during the transfection process to obtain lower toxicity but high gene transfection efficiency.15-16 Inspired by stimuli-responsive technologies based on a solid understanding of tumor microenvironment, the most current researches on establishing smart or intelligent materials in the rational design of the delivery system are becoming increasingly important due to their outstanding performance in facilitating payload release only in response to a specific stimulus that is linked to certain diseases.17-18 Therefore, many efficient nonviral gene delivery vectors have been proposed for the treatment of cancer relying on the use of unique endogenous stimuli, such as lower pH values,19 elevated glutathione levels,20-21 increased oxidative stress,22 hypoxia23 and specific overexpressed enzymes.24 It is of note that there are only a tiny minority of reports with respect to stimuli-responsive materials based on reactive oxygen species (ROS) including H2O2, superoxide anions (O2-) and hydroxyl radicals, which are overproduced as inherent factors distinctively associated with various diseases including cancer, diabetes, inflammation and cardiovascular diseases, in comparison with normal cells.25-26 Recent reports on advanced materials with ROSsensitivity explored in drug/gene deliery applications are based on their functional groups including boronic acid groups, sulfide groups, thioether groups, thioketal groups, selenium/tellurium and proline oligomers.27 A study reported the synthesis and application of a ROS-responsive PATK polymer based on thioketal linkage for safe and effective gene delivery.28 In this study, the rich-ROS in cancer cells were exploited to trigger the degradation of DNA/PATK complexed NPs and induce an efficient intracellular nucleic acids release. Keeping these in mind, a viable strategy is to prepare ROS-responsive high molecular weight OEI-TKx from low molecular weight oligoethylenimine (OEI) utilizing the biodegradable


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thioketal (TK) linkages that are prone to cleave triggered by high level of intracellular ROS in cancer hypoxic cells for the intracellular gene delivery, which has been proven to not only allow reduced toxicity but also effective DNA release and high gene transfection efficiency (Scheme 1A). EXPERIMENTAL SECTION Materials. Branched 800 Da oligoethylenimine (OEI) and 25 kDa polyethylenimine (PEI) were bought from Sigma-Aldrich. 3-mercaptopropionic acid was bought from Aladdin Industrial Corporation. J&K Chemical CO., Ltd was the source of 1-ethyl-3-[3-(dimethylamino)-propyl] carbodiimide (EDC). N-hydroxysuccinimide (NHS) was bought from Sigma-Aldrich. All the anhydrous solvents were from J&K Chemical CO., Ltd. Regenerated Cellulose Dialysis Membrane (MWCO = 1,000, 6,000-8,000 Da) were commercially available from Spectrum/Por. Diphenyleneiodonium chloride (DPI) was purchased from Sigma-Aldrich. Reactive Oxygen Species Assay Kit (ROS Assay Kit) and Hoechst 33342 (bisBenzimide H 33342) were from Beyotime® Biotechnology. Dojindo Laboratories commercially provided Cell Counting Kit-8 (CCK-8). The reporter plasmids, including pEGFP and pGL3-Luc, were from Promega. A BCA protein assay kit was purchased from Pierce. The Reporter Lysis 5X Buffer and Luciferase Assay System, 10-Pack were purchased from Promega. Intracellular Nucleic Acid Localization Kit, Label IT® Tracker™ Cy®5 was commercially available from Mirus Bio Corporation. LysoGreen was supplied by KeyGEN BioTECH. Cell Culture. Hela, HepG2, HEK293T, and L929 cells were commercially provided by the Cell Bank of Type Culture Collection of Chinese Academy of Sciences (ATCC) and were routinely maintained in DMEM (Gibco®) medium plus 10% (v/v) heat-inactivated FBS (Gibco®) and 1% streptomycin/penicillin (Hyclone) at 37 °C with 5% CO2 in a humidified incubator.



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Synthesis of ROS-cleavable Thioketal (TK) Linkage. Dried hydrogen chloride was bubbled into a reaction between anhydrous 3-mercaptopropionic acid (5.20 g, 49.05 mmol) and anhydrous acetone (5.69 g, 98.10 mmol) for 6 h of continuous stirring at 25 ℃. Then, a cold icesalt mixture was used to quench the reaction until no crystal was precipitated. Next, the crystals were filtered, washed three times using cold hexane and cold deionized water, respectively. Finally, ROS-cleavable thioketal (TK) linkage containing carboxyl-terminal residues was acquired as a white powder after drying in a vacuum desiccator. The structure of the synthetic product was confirmed by ESI-MS and 1H NMR (Bruker AV II-400 MHz) using CD3OD as the solvent. In order to investigate the degradation of thioketal linkage under ROS conditions, thioketal linkage was treated with 100 mM H2O2 containing 1.6 μM CuCl2 and 400 mM H2O2 containing 1.6 μM CuCl2 at 37 ℃ for 12 h, respectively, followed by using 1H NMR to analyze its structural changes. Synthesis of Thioketal Cross-linked OEI (OEI-TKx). Before cross-linked, OEI (3.20 g, 4.00 mmol) dissolved in water was accurately adjusted to pH 8.5 by diluted HCl solution and lyophilized for further ease of use. Then, carboxyl groups in ROS-cleavable thioketal linkage were firstly activated by NHS in the presence of EDC to form NHS esters which were then reacted with amine groups in the lyophilized OEI. To be specific, thioketal (0.35 g, 1.39 mmol) and NHS (0.39 g, 3.39 mmol) were dissolved in 5.0 mL of anhydrous THF and stirred in the dry nitrogen protection environment. Then, the 5.0 mL of anhydrous THF solution of EDC (0.53 g, 3.41 mmol) was added dropwise into the aforementioned mixture at 0 ℃. Thereafter, the solution was then allowed to proceed with magnetic stirring for an additional 24 h at 25 ℃. After filtration and evaporation, the reactive intermediate (TK-NHS) was obtained. To synthesize thioketal cross-linked OEI (OEI-TKx), 2.2 mL of anhydrous DMSO solution of lyophilized OEI (0.80 g, 1.00 mmol) was obtained to achieve a 25% of the final mass


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concentration in the reaction mixture. Meantime, the reactive intermediate (TK-NHS) was redissolved in 2.0 mL of anhydrous dichloromethane (DCM, CH2Cl2) to obtain the active ester solution which was then added dropwise into the OEI solution. After DCM was removed by evaporation under reduced pressure, the solution was persistently mixed for 48 h at 35 ℃ in the presence of dry nitrogen and then purified by extensive dialysis against deionized water by means of a dialysis membrane (MWCO 6,000-8,000 Da) for 2 days. After freeze-drying, the OEI-TKx was obtained as light yellow viscous gel that would be used for further study about its characterizations and applications. The structure of the obtained product was characterized by 1H NMR using D2O as the solvent. The molecular weight (Mw) and the corresponding molecular weight distribution (PDI) of cross-linked polymer were monitored via a gel permeation chromatography (GPC) instrument (Waters, Milford, MA). To prove the ROS responsiveness of thioketal linkage, OEI-TKx was treated with various ROS conditions. As regards 1H NMR detection, OEI-TKx was subjected to 100 mM H2O2 containing 1.6 μM CuCl2 for 12 h or 72 h at 37 ℃, respectively. For GPC analysis, OEI-TKx was incubated with 100 mM H2O2 containing 1.6 μM CuCl2 for 12 h or 72 h, 400 mM H2O2 containing 1.6 μM CuCl2 for 72 h at 37 ℃, respectively. PEI and OEI served as control groups. Assembly and Characterization of OEI-TKx/DNA Polyplexes. Polyplexes were formulated by adding an equal volume of cationic polymer to nucleic acid at the desired molar ratio of cationic polymer amine groups to nucleic acid phosphate groups (N/P ratio). The calculation of the N/P ratio is based on that a DNA subunit featuring one phosphate corresponds to 330 g/mol,29 and one subunit of 25 kDa PEI or 800 Da OEI containing one nitrogen corresponds to 43 g/mol. According to the chemical structure of OEI-TKx, the average molar mass of repeating unit in OEI-TKx is 720 g/mol which contains 12 nitrogens. Therefore, 60 g/mol is used as an



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average molar mass per nitrogen for OEI-TKx. To calculate the N/P ratios for 25 kDa PEI or 800 Da OEI, the equation is as follows: N:P = (weights of PEI or OEI (g)/43 (g/mol)):(weights of DNA (g)/330 (g/mol)) To calculate the N/P ratios for OEI-TKx, the equation is as follows: N:P = (weights of OEI-TKx (g)/60 (g/mol)):(weights of DNA (g)/330 (g/mol)) The OEI-TKx/DNA polyplexes were achieved by a gentle mixture of various amounts of OEITKx and the same dosage of DNA to reach varying N/P ratios. The obtained solution proceeded another 30 min of incubation at room temperature prior to use. Unless otherwise stated or agreed upon, the following OEI-TKx/DNA polyplexes used in our experiments were fabricated at the appropriate N/P ratio of 40. Gel electrophoresis assay was performed to confirm efficient DNA complexation by cationic OEI-TKx. In brief, the OEI-TKx/DNA polyplexes were freshly prepared by mixing various amounts of OEI-TKx with DNA (200 ng/well) to reach different N/P ratios ranging from 0.5 to 100. PEI group (N/P = 10) and naked DNA were served as positive and negative controls, respectively. To observe the dissociation of DNA from the polymer after exposure to ROS, the OEI-TKx/DNA polyplexes at varying N/P ratios were incubated under indicated ROS environment (25 mM H2O2 and 1.6 μM of CuCl2 solution) for 12 h at 37 ℃ in the presence of 0.3 M of sodium chloride. Then, ten microliters of the resulting polyplexes solutions were loaded on an agarose gel (1%, w/v) in standard Tris-acetate (TAE) running buffer at 85 V for 50 min, followed by the gel stained for 20 min with EtBr. After that, the DNA motion patterns were analyzed by visualizing DNA bands by Molecular Imager ChemiDoc XRS+ (Bio-Rad, USA). The number mean hydrodynamic diameter and zeta potential measurements of fabricated OEI-TKx/DNA polyplexes were made using Zetasizer Nano ZS90 (Malvern Instruments Ltd, UK).


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DNA (3 μg) were gently mixed with various amounts of OEI-TKx in 50 μL of HEPES buffer (pH 7.4) at room temperature for 30 min to obtain desired N/P ratios and then were diluted to 1 mL with HEPES buffer. The final concentration of DNA was 3 μg/mL for dynamic light scattering (DLS) measurements. To observe particle size alterations of OEI-TKx/DNA polyplexes after ROS exposure, the OEI-TKx/DNA polyplexes (N/P = 40) were subjected to incubation with an aqueous solution containing 400 mM H2O2 and 1.6 μM CuCl2 at different time points (0 - 72 h) for detection and analysis by DLS. The morphology of OEI-TKx/DNA polyplexes was visualized using transmission electron microscopy (TEM, Tecnai G2 F20 S-TWIN, FEI, USA). 30 μL of DNA solution (100 ng/μL) was mixed with 24 μL of OEI-TKx solution (1 mg/mL) at the N/P ratio of 40 for measurement. Data were analyzed with Gatan DigitalMicrograph software. In order to investigate the morphological changes of OEI-TKx/DNA polyplexes when exposed to ROS conditions, the OEI-TKx/DNA polyplexes (N/P = 40) were incubated at a final H2O2 concentration of 400 mM containing 1.6 μM CuCl2 for 24 h, 48 h or 72 h at 37 ℃, respectively. Ethidium Bromide (EtBr) Exclusion Assay. Abilities of OEI-TKx and PEI for plasmid DNA decomplexation under various concentrations of H2O2 (100 μM, 10 mM and 50 mM) were examined to measure the shielded amount of DNA in the OEI-TKx/DNA polyplexes or PEI/DNA polyplexes by standard EtBr exclusion assay. In brief, 5 μg/mL of EtBr solution with further addition of 5 μg/mL of DNA was incubated for 15 min at room temperature, and then the DNAEtBr solution was added stepwise to pre-determined amounts of the cationic polymers at room temperature to reach the N/P ratio of 40 for OEI-TKx/DNA polyplexes and the N/P ratio of 10 for PEI group for additional half an hour of incubation. The corresponding fluorescence intensities of polyplexes solutions were measured at the 510 nm of excitation wavelength and 590 nm of



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emission wavelength using a fluorescence spectrophotometer (Hitachi F-7000, Japan). Destabilization of OEI-TKx/DNA polyplexes and PEI/DNA polyplexes in response to ROS conditions was quantified by the measurement of the fluorescence intensity recovery after treatment with varying concentrations of H2O2 solutions at 37 ℃ for 12 h. A pure EtBr solution and the DNA/EtBr solution were used as negative and positive controls, respectively. The DNA condensation efficiency was defined as following: DNA Condensation Efficiency (%) = (1 - (FFEB) / (F0-FEB)) × 100%, where F0, FEB and F denoted the fluorescence intensity of DNA/EtBr solution, pure EtBr solution and DNA/EtBr solution with OEI-TKx or PEI polymer, respectively. The ROS-sensitivity of OEI-TKx/DNA polyplexes under various ROS, including hydroxyl radical and H2O2 was also evaluated by EtBr exclusion assay. The OEI-TKx/DNA polyplexes were exposed to various ROS conditions to obtain a final concentration of 50 mM. H2O2 was mixed with CuCl2 to form hydroxyl radicals. Then the DNA condensation efficiency of the OEITKx/DNA polyplexes (N/P = 40) after 12 h of treatment under various ROS conditions was evaluated as described above. Intracellular ROS Generation Levels in Different Cell Lines. Intracellular ROS generation in different cell lines was quantitatively detected using a ROS Assay Kit. Briefly, HEK293T, L929, Hela and HepG2 cells (1 × 105 cells/well) resuspended in 1 mL of complete DMEM were respectively seeded in 12-well culture plates and allowed to incubate at 37 ℃ for 24 h to adhere. And then cold PBS was used to wash the cells prior to dye staining. The cells were then incubated with a final 10 μM of 2′-7′-dichlorodihydrofluorescein diacetate (DCFH-DA) dye for 30 min at 37 °C in serum-free medium, followed by harvested, washed with cold PBS three times and dispersed in 0.3 mL of PBS. The mean fluorescence intensity (MFI) of 2′-7′dichlorodihydrofluorescein (DCF: Excitation (Ex) 488 nm/Emission (Em) 525 nm) from the


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suspensions of cells were quantified by flow cytometry via BD Accuri C6 (USA) and the results were analyzed with BD Accuri C6 and Flowjob. Each assay was conducted in triplicate. For the qualitative observation of intracellular ROS generation, Hela cells (1 × 105 cells/well) in 2 mL of DMEM with 10% FBS were plated in a 6-well plate and allowed to adhere for 24 h. And then cold PBS was used to wash the cells prior to dye staining. Then Hela cells were incubated with a final 10 μM of DCFH-DA dye for 15 min at 37 °C in 2 mL of serum-free medium and washed three times with cold PBS, followed by 5 μg/mL of Hoechst 33342 staining of the cellular nuclei for twenty minutes. Green and blue fluorescence from the stained cells were respectively observed by an inverted fluorescence microscope using fluorescence microscope (Leica, Germany). For the qualitative observation of intracellular ROS level in HEK293T or Hela cells under hypoxic conditions or treated with 200 μM of vitamin C (VC), HEK293T or Hela cells (1 × 105 cells/well) in 1 mL of DMEM with 10% FBS were plated in a 12-well plate and allowed to adhere for 24 h. Cells were pretreated with various concentrations of CoCl2 (0, 25 μM, 50 μM and 100 μM) or 200 μM of VC for 24 h. And then cold PBS was used to wash the cells prior to dye staining. Then cells were incubated with a final concentration of DCFH-DA dye at 10 μM for 30 min at 37 °C in 1 mL of serum-free medium and washed three times with cold PBS. Green fluorescence from the stained cells was observed by an inverted fluorescence microscope using fluorescence microscope (Leica, Germany). In Vitro Gene Transfection Study. GFP as a reporter gene was used to qualitatively evaluate the gene transfection of HEK293T, L929, Hela and HepG2 cell lines in vitro. Firstly, cells (8 × 103 cells/well) dispersed in 100 μL of complete culture medium were plated in 96-well culture plates to attach for 24 h. Then the medium was replaced with 90 μL of fresh serum-free culture



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medium before transfection. Subsequently, the cells were treated with 10 μL of each fabricated OEI-TKx/DNA polyplexes (200 ng of DNA per well) at the desired final N/P ratios. PEI group (N/P = 10) was set as a positive control group. The medium was changed to 100 μL of fresh complete one without samples 4 h of post-transfection. The expression of EGFP in HEK293T, L929, Hela and HepG2 cell lines were directly detected and imaged 48 h of post-transfection via an inverted fluorescence microscope (Leica, Germany). Luciferase expression of Hela and HEK293T cells was measured using luciferase as a reporter gene. PEI and OEI served as controls. In brief, cells (8 × 103 cells/well) in 100 μL of complete culture medium were plated in 96-well culture plates to attach for 24 h. Then the medium was changed to 90 μL of fresh serum-free culture medium before transfection. Subsequently, the cells were exposed to 10 μL of each fabricated OEI-TKx/DNA polyplexes and OEI group (200 ng of DNA per well) at the desired final N/P ratios. PEI group (N/P = 10) was set as a positive control group. Then the growth medium was changed to 100 μL of fresh complete DMEM 4 h of posttransfection, and the cells were maintained for an additional 20 h under normal growth conditions. Thereafter, the transfected cells were washed with 100 μL of cold PBS three times, followed by lysed with 80 μL of 1 × reporter lysis buffer 24 h of post-transfection. The evaluation of luciferase activity was performed by the Luciferase Assay System as per the standard protocol described in manufacture manual using a microplate reader (Thermo Scientific, USA) and was normalized against the protein concentration according to a BCA protein assay kit, which was ultimately presented as the relative light units per milligram protein (RLU/mg protein). Luciferase gene transfection was also performed to evaluate the level of luciferase expression under hypoxic conditions. Cells were prepared as described above. Then the medium was changed to 90 μL of fresh serum-free culture medium before transfection. Subsequently, the cells were


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exposed to 10 μL of OEI-TKx/DNA polyplexes at the N/P ratio of 60 in HEK293T cells and N/P ratio of 40 in Hela cells. Then the growth medium was changed to 100 μL of fresh complete DMEM 4 h of post-transfection. For the creation of hypoxic conditions, various concentrations of CoCl2 (0, 25 μM, 50 μM and 100 μM) were added to the cells and the cells were maintained for an additional 20 h under hypoxic conditions. Thereafter, RLU/mg protein was determined as described above. Luciferase expression levels were calculated as the percent luc expression present relative to normoxic conditions. In addition, luciferase gene transfection was performed in the treatment with extra 200 μM of VC, which could inhibit the production of intracellular ROS. Cells were prepared as described above. Cells were alternatively pretreated with 200 μM of VC for 12 h. Transfection was again carried out with the same protocol, and gene transfection process was maintained at VC concentration of 200 μM to block ROS until the end of the cell transfection. Thereafter, RLU/mg protein was determined as described above. Luciferase expression levels were calculated as the percent luc expression present relative to normoxic conditions. Furthermore, luciferase gene transfection was performed in the treatment with extra diphenyleneiodonium (DPI), which could inhibit the production of intracellular ROS. Before gene transfection protocol, Hela cells were pre-treated with desired concentrations of DPI (0 - 10 μM) for 30 min at 37 ℃ in serum-free medium, followed by incubated with 10 μL of OEI-TKx/DNA polyplexes (200 ng of DNA per well) at the optimal N/P ratio of 40 with indicated DPI concentrations at 37 ℃. Then the growth medium was changed to fresh complete DMEM and DPI at the pre-determined concentrations 4 h of post-transfection. The luciferase expression was determined under normal growth conditions 10 h of post-transfection as described above.



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In Vitro Cytotoxicity Assay. The evaluation of the cell viability of OEI-TKx and OEITKx/DNA polyplexes was performed by CCK-8 assay. OEI, OEI/DNA polyplexes, PEI and PEI/DNA polyplexes were analyzed as controls. Briefly, Hela and HEK293T cells (8 × 103 cells/well) resuspended in 100 μL of culture medium supplemented with serum were plated in 96well culture plates to attach for 24 h, respectively. Then cells were exposed to the corresponding amounts of OEI-TKx or OEI-TKx/DNA polyplexes to achieve various final concentrations of polymers or desired N/P ratios of polyplexes for additional 24 h of incubation. At 24 h of posttransfection, cells were incubated with 10% CCK-8 in fresh serum-free culture medium for an additional 1.5 h at 37 ℃ in the dark. The UV absorbance of each well was measured by a microplate reader at the 450 nm of wavelength. The relative cell viability was accurately normalized based on the formula: Cell viability (%) = (ODsample - ODblank) / (ODcontrol - ODblank) × 100% Cell viability under hypoxic conditions or treated with 200 μM of VC were also determined by CCK-8 assay. Cells were pretreated with various concentrations of CoCl2 (0, 25 μM, 50 μM and 100 μM) or 200 μM of VC for 24 h. CCK-8 was added to the cells at the end of cell incubation and the cell viability was calculated with the same protocol. Cellular Uptake Study. Confocal laser scanning microscopy (CLSM, Leica TCS SP5, Germany) and flow cytometry (BD Accuri C6, USA) were used to measure the cellular uptake level of OEI-TKx. DNA was labelled by Cy®5 (Ex 633 nm/Em 670 nm), an Intracellular Nucleic Acid Localization Kit. In a qualitative study, Hela cells (1 × 104 cells/dish) in 1 mL of 10% FBScontaining cell culture medium were plated in 35 mm confocal dishes (Ф = 15 mm) for 24 h to attach. After the medium was removed, the cells continued to incubate with 10 μL of OEITKx/DNA polyplexes (N/P = 40) containing 50% Cy5-labeled DNA (Cy5DNA, 300 ng) along with


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190 μL of fresh serum-free culture medium for 1 h, 3 h or 6 h (4 h of transfection followed by rinsed and another 2 h of incubation in fresh complete medium), respectively. PEI/Cy5DNA polyplexes (N/P = 10) were set as a positive control group. The cells were then washed three times, followed by 5 μg/mL of Hoechst 33342 (Ex 350 nm/Em 461 nm) staining for twenty minutes before observation. After washed thrice with cold PBS, the intracellular localization of the polyplexes was visualized by CLSM. To further quantitatively analyze the cellular uptake efficiency, Hela cells (1 × 104 cells/well) in 1 mL of complete culture medium were plated in 12-well plates to attach for 24 h. Thereafter, the medium was changed to fresh serum-free culture medium, into which OEI-TKx/DNA polyplexes (N/P = 40) containing 1 μg of 50% Cy5DNA per well were added for 1 h, 3 h or 6 h (4 h of transfection followed by rinsed and another 2 h of incubation in fresh complete medium). PEI/Cy5DNA polyplexes (N/P = 10) were set as a positive control group. After the cells were rinsed thrice with cold PBS, harvested by trypsin treatment, collected by centrifuged for 3 min at 1200 rpm, washed with cold PBS for another time and dispersed in cold PBS, the fluorescence intensity of Cy5DNA was quantified by flow cytometry (Cy5: Ex 633 nm/Em 670 nm) using BD Accuri C6 and the results were analyzed with the relative BD Accuri C6 software. Each assay was conducted in triplicate. Determination of Endosomal Escape and DNA Release. To illustrate the endosomal disruption capacity of different polyplexes by CLSM, intracellular tracking study was performed on Hela cells using LysoGreen (Ex 443 nm/Em 505 nm) following manufacturer’s protocol. Briefly, Hela cells (1 × 104 cells/dish) were plated in 35 mm confocal dishes (Ф = 15 mm) in complete DMEM for 24 h at 37 ℃, followed by treated with 10 μL of different polyplexes containing 300 ng of 50% Cy5DNA at their optimal N/P ratios in 200 μL of fresh serum-free culture



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medium for 1 h, 3 h or 6 h (4 h of transfection followed by rinsed and another 2 h of incubation in fresh complete medium). PEI/Cy5DNA polyplexes (N/P = 10) were set as a positive control group. After three more washes with cold PBS, the cells were maintained in serum-free medium containing 2 μM of LysoGreen for 40 min to label lysosomes, followed by 5 μg/mL of Hoechst 33342 staining the nuclei for twenty minutes prior to observation by CLSM. The degree of colocalization of red (Cy5DNA) and green (LysoGreen-stained lysosomes) fluorescence was quantified using the Overlap Coefficient R by Image-Pro Plus Version 6.0 software. The Intracellular Dissociation of

FITCOEI-TK /Cy5DNA x

Polyplexes. In order to visualize

the intracellular dissociation of polyplexes by CLSM, FITC (Ex 495 nm/Em 525 nm) and Cy5 (Ex 633 nm/Em 670 nm) were used to label cationic polymers (PEI and OEI-TKx) and DNA, respectively. In brief, Hela cells (1 × 104 cells/dish) were plated in 35 mm confocal dishes (Ф = 15 mm) for 24 h in complete culture medium prior to use. Then 300 ng of 50%

Cy5DNA

was

complexed with OEI-TKx labelled by FITC (FITCOEI-TKx) at room temperature for 30 min to achieve


polyplexes (N/P = 40). Next, the resulting polyplexes were added

into the fresh serum-free culture medium for 1 h, 3 h, 6 h or 24 h (4 h of transfection followed by rinsed and another 2 h and 20 h of incubation in fresh complete medium).

FITCPEI/Cy5DNA

polyplexes (N/P = 10) were set as a positive control group. At desired time points, the cells were washed with cold PBS thrice, followed by 5 μg/mL of Hoechst 33342 (Ex 350 nm/Em 461 nm) staining the nuclei for twenty minutes. After unreacted dyes were removed by three more washes with cold PBS, the intracellular dissociation of the polyplexes in the cells was immediately observed by CLSM. The degree of colocalization of red (Cy5DNA) and green (FITCOEI-TKx or FITCPEI)

fluorescence was quantified using the Overlap Coefficient R by Image-Pro Plus Version

6.0 software.


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Statistical Analysis. All average results were expressed as mean values ± standard deviations (± S.D.) from at least three independent measurements or otherwise indicated. The areas of microscopic observation were selected at random. Statistical significance between data sets was determined by two-tailed, unpaired t-tests. In all experiments, P < 0.05 was regarded as statistically significant.



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Scheme 1. (A) Schematic illustration of OEI-TKx/DNA polyplexes for intracellular gene delivery. (B) Synthetic route of ROS-responsive OEI-TKx.

RESULTS AND DISCUSSION Design and Characterization of OEI-TKx. Basic design route of ROS-cleavable OEI-TKx was illustrated in Scheme 1B and described in the experimental details. Briefly, the thioketal linkage was prepared by a reaction between the anhydrous 3-mercaptopropionic acid and anhydrous acetone in the presence of dry hydrogen chloride. Subsequently, the TK-NHS as an active ester was obtained by a reaction that carboxyl groups at the terminal end of TK were activated by NHS, using EDC as the condensation agent. Finally, the TK-NHS reacted with the primary amine group of OEI in anhydrous DMSO to obtain the end product of OEI-TKx for future applications. The detailed structure and synthesis procedure of OEI-TKx were confirmed by ESIMS and 1H NMR. In Figure S1A, the mass-to-charge ratio (m/z) of 251.33 [M-H]+ was ascribed to thioketal linkage. In addition, 1H NMR spectroscopy (Figure 1A) of the product showed that the peaks at 1.58, 2.58, and 2.85 ppm were assigned to Ha, Hb, and Hc moieties of thioketal linkage, respectively. Compared with OEI, there was a new peak appeared at 1.58 ppm in the 1H NMR spectroscopy of OEI-TKx which could be the signal of ROS-cleavable thioketal linkage. Therefore, we have successfully synthesized OEI-TKx which was proved by the 1H NMR spectra. The GPC result determined that the molecular weights of OEI-TKx were Mw = 10.0 kDa and Mn = 4.2 kDa with a polydispersity index of 2.4 which were shown in Table 1. Characterization of OEI-TKx/DNA Polyplexes. In the process of effective gene delivery, the capability of vectors to bind and condense DNA is a beneficial and advantageous prerequisite to prevent degradation by enzymes and endosomal acidic environments before internalization.


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Based on such considerations, gel electrophoresis assay was performed to confirm efficient DNA complexation by cationic OEI-TKx, utilizing PEI and Naked DNA as positive group and negative group, respectively. The results (Figure 1B) showed that the DNA migration was completely retained by OEI-TKx on the gel at the N/P ratios of 10 and higher.

Figure 1. Characterization of OEI-TKx by 1H NMR and OEI-TKx/DNA polyplexes by agarose gel electrophoresis, DLS and TEM. (A) 1H NMR spectrum of TK, OEI and OEI-TKx. (B) Gel retardation assay of DNA complexed with OEI-TKx at the increased N/P ratios. (C) DLS measurements of OEI-TKx/DNA polyplexes at varying N/P ratios in number mean hydrodynamic diameter and zeta potential. (D) TEM images of OEI-TKx/DNA polyplexes (N/P = 40).



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Table 1. Molecular weights of various polymers detected using GPC. Molecular Weights

Polymers

Mw/103

Mn/103

PDI

OEI-TKx

10.0

4.2

2.4

OEI-TKx 100 mM H2O2 (12 h)

7.8

3.1

2.5


2.5

1.6

1.6


1.1

0.8

1.3

PEI

22.1

8.8

2.5

OEI

1.4

1.3

1.1

We also carried out the hydrodynamic size, size distribution and zeta potential measurements via DLS to evaluate the DNA compaction ability of OEI-TKx. According to the DLS measurement (Figure 1C), the surface charge of OEI-TKx/DNA polyplexes (N/P = 5) was characterized by a ζpotential of -6.6 mV, which might be due to a greater inclination towards aggregation and the existence of larger particles, while OEI-TKx/DNA polyplexes presented higher positive surface charge but smaller particle sizes at the increased N/P ratios.30-31 It should be pointed that there were no significant zeta potential changes despite the N/P ratio boosting up to 250 when a plateau value (+30 mV) was attained at the N/P ratio of 40. Additional analysis by DLS confirmed that the fabricated OEI-TKx/DNA polyplexes (N/P = 40) were well dispersed with average size about 110 nm, indicating that OEI-TKx polymer could efficiently condense DNA to form stable nanoparticles with good uniform dispersity at the N/P ratios above 40. In addition, TEM images showed that the OEI-TKx/DNA polyplexes were visible as uniform and compact spheres in shape with size around 70 nm at the N/P ratio of 40 (Figure 1D), which is smaller than the result of DLS (~110 nm). The certain difference in the characterization of particle size for the same sample between the two measurement methods could be due to the evaporation of water in the TEM experiments, leading a shrinkage effect.32 ACS Paragon Plus Environment

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Figure 2. Analysis of the responsiveness of the thioketal linkage to ROS. (A) 1H NMR spectrum of OEI-TKx before and after incubation with 100 mM H2O2 and 1.6 μM CuCl2 at 37 ℃ for 12 h or 72 h. (B) GPC traces of OEI-TKx before and after degradation in ROS conditions (100 mM H2O2 and 1.6 μM CuCl2 or 400 mM H2O2 and 1.6 μM CuCl2) for 12 h or 72 h at 37 ℃. (C) The release of DNA from OEI-TKx/DNA polyplexes at the increased N/P ratios before and after incubation with 25 mM H2O2 containing 1.6 μM CuCl2 for 12 h at 37 ℃, as measured by agarose gel



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electrophoresis assay. (D) Particle size and (E) particle dispersion index (PDI) variations of OEITKx/DNA polyplexes (N/P = 40) after incubation with or without the treatment of 400 mM H2O2 and 1.6 μM CuCl2. (F) TEM images of OEI-TKx/DNA polyplexes (N/P = 40) after treatment with 400 mM H2O2 containing 1.6 μM CuCl2 for 0, 24 h, 48 h or 72 h at 37 ℃.

ROS-responsive Behavior of OEI-TKx. The degradation behavior of OEI-TKx after treatment with simulated ROS was detected by 1H NMR spectroscopy. Accordingly, OEI-TKx was exposed to 100 mM H2O2 plus 1.6 μM CuCl2 for 12 h or 72 h at 37 ℃, respectively.28 As depicted in Figure 2A, the thioketal linkages in the OEI-TKx (δ(c) 1.58 ppm) could be cleaved by ROS stimuli, generating acetone (δ(c1 and c2) 2.16 ppm) in the process of cleavage as a by-product. The degradation profile of thioketal linkage was also detected by 1H NMR spectroscopy (Figure S1B). These results were consistent with a previous dethioacetalization study using H2O2.33 The time-dependent or H2O2-concentration-dependent changes in molecular weight of OEITKx were also confirmed by GPC (Figure 2B). When incubated with 100 mM H2O2 containing 1.6 μM CuCl2, the molecular weight of OEI-TKx dropped significantly with the extending of incubation time. Remarkably, the molecular weight of degradation product of OEI-TKx was approximately equal to that of OEI after exposure to 400 mM H2O2 containing 1.6 μM CuCl2 for 72 h at 37 ℃, indicating a more significant decrease even complete degradation. These above results demonstrated that the thioketal linkage in the OEI-TKx possessing time-dependent and H2O2-concentration-dependent responses could be used for a corresponding ROS-sensitive gene delivery system. Efficient dissociation of DNA from OEI-TKx/DNA polyplexes triggered by ROS was detected by an agarose gel electrophoresis assay (Figure 2C). The DNA migration could be


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completely retarded by OEI-TKx at the N/P ratios of 10 and higher without H2O2 treatment. However, the DNA condensation level of OEI-TKx was decreased when the samples were treated with 50 mM H2O2 containing 1.6 μM CuCl2 at 37 ℃ for 12 h, as confirmed by the migration of DNA in the agarose gel. The variations of particle size and particle dispersion index (PDI) in terms of OEI-TKx/DNA polyplexes were monitored by DLS. Before ROS exposure, OEI-TKx/DNA polyplexes could maintain a stable formation with the diameter around 110 nm (Figure 2D) and a relative low PDI (~0.15) (Figure 2E) for 72 h. However, after exposure to 400 mM H2O2 containing 1.6 μM CuCl2 at 37 ℃, OEI-TKx/DNA polyplexes dramatically swelled from 106 nm to 596 nm at 24 h time point, even reached up to ~1500 nm after 48 h and reduced to 791 nm after 72 h of incubation. The particle size of OEI-TKx/DNA polyplexes experienced a process from small to large and again to small, which indicated that the cleavage of thioketal linkages caused continuous swelling even complete destruction of stable nanoparticles. In addition, the PDI of OEI-TKx/DNA polyplexes also changed with the extension of incubation time in the ROS environment. For instance, PDI increased from 0.14 to 0.48 after incubation for 24 h, reached 0.91 at 48 h time point and changed to 0.44 after 72 h of treatment, which indicated the damage of stable formation. The particle size increase of OEI-TKx/DNA polyplexes resulted from the ROS-responsible cleavage of the thioketal linkage, which could provide the prerequisite for the cargo release in the package. TEM images also clearly demonstrated ROS-induced destabilization of the OEI-TKx/DNA polyplexes (Figure 2F). After exposure to 400 mM H2O2 and 1.6 μM CuCl2 at desired time points (0, 24 h, 48 h or 72 h) at 37 ℃, OEI-TKx/DNA polyplexes changed significantly, expanding in diameter with irregular and loose morphology even with aggregating of fragments at 72 h time point, which coincided well with the size measurement by DLS (Figure 2D).



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In addition, EtBr exclusion assay was adopted to demonstrate the disassembly of the OEITKx/DNA polyplexes stimulated by biological concentrations of H2O2 (e.g., 0.1 mM and 1 mM)34 (Figure 3A). Increased fluorescence intensity of OEI-TKx/DNA polyplexes under various ROS levels was represented as a quantified indicator for the destabilization of the polyplexes because of more efficient embeddedness of EtBr into DNA bases. Upon exposure to H2O2, the DNA condensation efficiency of OEI-TKx was significantly reduced from 81% to 35% along with the increase of H2O2 concentration up to 50 mM. In contrast, non-responsive PEI showed a trivial change in terms of the DNA package after the same concentrations of H2O2 treatment. The EtBr exclusion assay also confirmed the degradation selectivity of the OEI-TKx/DNA polyplexes under various ROS types, including hydroxyl radical and H2O2 (Figure 3B). Among the tested ROS types, OEI-TKx showed the highest sensitivity to the hydroxyl radical, leading to the lowest DNA packaging ability in return.

Figure 3. ROS-responsive DNA release. (A) DNA condensation levels of OEI-TKx/DNA (N/P = 40) and PEI/DNA (N/P = 10) under various concentrations of H2O2 (0, 0.1 mM, 1 mM and 50 mM) confirmed by EtBr exclusion assay. (B) Selectivity of ROS-triggered DNA release from OEITKx/DNA (N/P = 40) under various ROS types, including H2O2 and hydroxyl radical as detected by EtBr exclusion assay, using distilled water as a negative control. (n = 3, ∗ p < 0.05, ∗ ∗ p < 0.01). ACS Paragon Plus Environment

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Intracellular ROS Generation Levels in Different Cell Lines. Encouraged by the ROSresponsive properties of the designed OEI-TKx, we investigated intracellular ROS levels spontaneously generated in different cell lines (HEK293T, L929, Hela and HepG2 cells) by using a ROS Assay Kit. In the present studies, DCFH-DA, a cell membrane permeable fluorescent dye, was used as a probe for intracellular ROS, which was rapidly oxidized to a strongly fluorescent DCF adduct with ROS. The fluorescence intensity of DCF is a good indicator of ROS level in cells. As depicted in Figure 4A, the intracellular ROS levels of HEK293T and Hela cells were significantly higher in comparison with HepG2 and L929 cells. Furthermore, images by fluorescence microscopy of Hela cells double-stained by Hoechst 33342 and DCFH-DA validated the inherently high level of intracellular ROS in Hela cell (Figure 4B).

Figure 4. (A) ROS levels in HEK293T, L929, Hela and HepG2 cells quantified using DCFH-DA reagent by flow cytometry. ∗∗ p < 0.01. (B) Observation of intracellular ROS accumulation in Hela cells detected by inverted fluorescence microscopy. The cellular nuclei were dyed blue with Hoechst 33342. Scale bars: 75 μm.



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In Vitro Gene Transfection Study. GFP as a reporter gene was used to qualitatively evaluate the superior overall gene transfection in vitro of HEK293T (ROS-high), L929 (ROS-low), Hela (ROS-high), and HepG2 (ROS-low) cells treated with OEI-TKx/DNA polyplexes in the serumfree medium. It should be noted that OEI-TKx/DNA polyplexes showed larger areas of green fluorescent dots in HEK293T (Figure 5A) and Hela cells (Figure 5B), compared with L929 (Figure S2A) and HepG2 (Figure S2B) cells. Particularly, the highest green fluorescent protein expression of OEI-TKx group (N/P ratios of 40, 50, 60 and 30) was stronger than that of PEI group at the N/P ratio of 10 used as a positive control in Hela, HepG2, HEK293T and L929 cells, respectively (Figure 5C and Figure S2C).

Figure 5. Gene transfection of various polyplexes in HEK293T, Hela, L929, and HepG2 cells. EGFP expression of (A) HEK293T and (B) Hela cells treated with OEI-TKx/DNA polyplexes at various N/P ratios and PEI group at the N/P ratio of 10 observed by inverted fluorescence ACS Paragon Plus Environment

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microscopy. (C) GFP expression and optical images of different cells after transfected with OEITKx/DNA polyplexes at optimal ratios. After 4 h of transfection without serum, the cells were exposed to another 44 h of incubation with fresh complete DMEM.

Figure 6. Luciferase gene expression of (A) HEK293T and (B) Hela cells transfected with OEITKx/DNA polyplexes for 4 h without 10% FBS and an additional 20 h of culture with fresh complete cell culture medium. PEI group at the N/P ratio of 10 and OEI group at different N/P ratios were used as positive and negative controls, respectively. (n = 6, ∗∗ p < 0.01).

In addition, the method of the luciferase reporter gene was also used to quantitatively assess the gene transfection efficiency of HEK293T (Figure 6A) and Hela cells (Figure 6B) treated with OEI-TKx/DNA polyplexes. The expression of the luciferase gene in Hela and HEK293T cells added by OEI-TKx/DNA polyplexes cultured with the serum-free medium reached the highest spots at the N/P ratios of 40 and 60, respectively. However, OEI/DNA polyplexes presented the lowest expression of luciferase at the same N/P ratios in Hela and HEK293T cells, respectively. It was also noteworthy that the corresponding luciferase gene expression of OEI-TKx/DNA polyplexes was 7 times higher in Hela cells and 28 times higher in HEK293T cells than that of PEI group at the N/P ratio of 10. ACS Paragon Plus Environment


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Figure 7. Observation of intracellular ROS accumulation in (A) HEK293T and (B) Hela cells under conditions of 100 μM of CoCl2-induced hypoxia or treated with 200 μM of VC to deplete the generated ROS detected by inverted fluorescence microscopy. Scale bars: 100 μm. Cell viability of HEK293T and Hela cells in the presence of (C) various concentrations of CoCl2 (0, 25 μM, 50 μM and 100 μM) or (D) 200 μM of VC for 24 h. Luciferase expression of HEK293T and Hela cells in the presence of (E) various concentrations of CoCl2 (0, 25 μM, 50 μM and 100 μM) or (F) 200 μM of VC. (n = 6, ∗∗ p < 0.01).


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Furthermore, the method of the luciferase reporter gene was also used to quantitatively assess the gene transfection efficiency of HEK293T (N/P = 60) and Hela cells (N/P = 40) cultured with OEI-TKx/DNA polyplexes under conditions of CoCl2-induced hypoxia or pretreated with VC to deplete the generated ROS. As shown in Figure 7A and Figure 7B, the green fluorescence in the cells became stronger under hypoxic conditions in the presence of 100 μM of CoCl2, indicating the inherently higher level of intracellular ROS. However, the green fluorescence became weaker in the presence of 200 μM of VC, which demonstrated that the generation of intracellular ROS was blocked. Figure 7C and Figure 7D showed that luciferase expression was not associated with CoCl2 or VC toxicity. As depicted in Figure 7E, the production of luciferase was increased approximately 100% compared to that in the absence of CoCl2 in HEK293T and Hela cells at a concentration of 100 μM of CoCl2. However, the luciferase expression levels in HEK293T and Hela cells pretreated with 200 μM of VC dropped to 68% or 28%, respectively, compared to normoxic conditions. These results indicated that the luciferase expression was enhanced in response to CoCl2-induced hypoxia which could cause hypoxia-triggered ROS enhancement, while the luciferase expression was decreased in the presence of VC which could inhibit the generated ROS.



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Figure 8. Luciferase gene expression of Hela cells treated with OEI-TKx/DNA polyplexes (N/P = 40) in serum-free medium at indicated concentrations of DPI. After pretreated with desired concentrations of DPI for 0.5 h, the cells were transfected with OEI-TKx/DNA polyplexes at the same concentrations of DPI for 10 h (4 h of transfection followed by another 6 h of incubation in fresh complete medium). (n = 6, ∗∗ p < 0.01).

To further investigate whether the enhanced gene transfection ability of OEI-TKx/DNA polyplexes is selectively related to high level of intracellular ROS, the relevant inhibitor DPI was used to artificially reduce the concentration of intracellular ROS in Hela cells. The luciferase gene expression of Hela cells pretreated with DPI decreased with the increasing of DPI concentration and reduced by almost two orders of magnitude in comparison with that of the DPI-free cells (Figure 8). Thus, it could be concluded that the OEI-TKx/DNA polyplexes could not efficiently release the DNA without a high level of intracellular ROS, owing to the inefficient cleavage of thioketal linkages and limited disassembly of the polyplexes. In Vitro Cytotoxicity Assay. It is an essential factor for the clinical success of synthetic gene delivery carriers to meet an acceptable safety profile, which had a vital influence on the gene


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transfection.35 In this study, the cytotoxicity of OEI-TKx with various concentrations in vitro was investigated by means of CCK-8 assay to detect the cell viability of HEK293T and HeLa cells. OEI and PEI were used as control groups, respectively. Figure 9A and Figure 9B clearly demonstrated that cationic OEI-TKx and PEI polymers induced dose-dependent effects on the cell survival rate in both HEK293T and Hela cells. It was worth mentioning that the cell viability of the tested cells treated with 20 μg/mL of PEI dramatically decreased to about 15%, which was ascribed to its high cationic charge density.36 However, less cytotoxicity of OEI-TKx was observed at the same concentration in both cell lines. For example, the cell viability of Hela and HEK293T cell lines exposed to 20 µg/mL of OEI-TKx in the complete cell culture medium for 24 h of incubation was over 80%, while the cell viability in the case of PEI sharply dropped to 16% and 15%, respectively.



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Figure 9. The cytotoxic assay of OEI, OEI-TKx and PEI to (A) HEK293T and (B) Hela cells after 24 h of treatment at various polymer concentrations from 2 to 40 μg/mL. The cytotoxic assay of (C) HEK293T and (D) Hela cells treated with OEI/DNA polyplexes, OEI-TKx/DNA polyplexes and PEI/DNA polyplexes at desired N/P ratios (10 to 100) 24 h of post-transfection. (n = 6, ∗∗ p < 0.01).

Furthermore, we investigated the cytotoxicity of OEI-TKx/DNA polyplexes by CCK-8 assay using OEI/DNA polyplexes and PEI/DNA polyplexes as control groups. As shown in Figure 9C and Figure 9D, OEI-TKx/DNA polyplexes exhibited the similar remarkably high cell viability to OEI group in both cell lines even at the N/P ratio of 50. Conversely, PEI/DNA polyplexes presented the highest cell cytotoxicity. For example, cell viability still remained around 100% in


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HEK293T cells after treatment with OEI-TKx/DNA polyplexes, while only 19% viability was found for PEI/DNA polyplexes. These above results illustrated that the higher cell viability of OEI-TKx and OEI-TKx/DNA polyplexes in comparison with PEI and PEI/DNA polyplexes in Hela and HEK293T cell lines could be attributed to higher level of intracellular ROS, which could cleave the thioketal linkages and facilitate the rapid biodegradation of OEI-TKx into low molecular weight OEI fragments that were less toxic. As a consequence, cytotoxicity was significantly reduced. However, it is well known that the non-degradable PEI is cytotoxic in many cell lines. Indeed, it is beneficial to crosslink oligocations with low molecular weight for high molecular weight polycations via ROSresponsive thioketal linkage in consideration of reducing toxicity.37 Inspired by the previous studies which illustrated that the improvement of transfection efficiency might be related to the increased interaction with the cellular membrane, effective endosome/lysosome escape and moderate binding between vectors and DNA, we also investigated the cellular uptake, endo/lysosomal escape, polyplexes disassembly and DNA release after the cleavage of thioketal linkages of OEI-TKx/DNA polyplexes in Hela cells, with PEI/DNA polyplexes serving as control group. Cellular Uptake Study. It is necessary for efficient nonviral gene delivery to overcome several biological barriers, including successful cell surface attachment, plasma membrane penetration, lysosomal escape, DNA release and so on. Hence, the intracellular uptake efficiency of the fabricated polyplexes is of great importance in gene delivery, which is the first step for gene transfection. The cellular uptake profiles of OEI-TKx/DNA polyplexes in Hela cells were qualitatively and quantitatively measured by CLSM (Figure 10A) and flow cytometry (Figure 10B and Figure 10C), respectively.



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Figure 10. Cellular uptake of OEI-TKx/Cy5DNA polyplexes (N/P = 40) and PEI/Cy5DNA polyplexes (N/P = 10) in Hela cells with serum-free medium. (A) Confocal images, (B) Mean fluorescence intensity (MFI) and (C) Representative histograms by flow cytometry of Hela cells added by OEI-TKx/Cy5DNA polyplexes and PEI/Cy5DNA polyplexes 1 h, 3 h, and 6 h of posttransfection (4 h of transfection followed by rinsed and another 2 h of incubation in fresh complete medium). The cellular nuclei were dyed blue with Hoechst 33342, and DNA (red) was labelled with Cy5. All scale bars are 25 µm. ∗∗ p < 0.01.

Confocal images in Figure 10A markedly showed that slight red fluorescence was observed in Hela cells treated with OEI-TKx/Cy5DNA polyplexes (N/P = 40) 1 h of post-transfection, while more red fluorescent dots was widely distributed in the cytoplasm and nuclei at 6 h time point. Likewise, the internalization of PEI/Cy5DNA polyplexes (N/P = 10) was also positively associated with the incubation time. However, Figure 10B and Figure 10C revealed that the MFI of internalized OEI-TKx/Cy5DNA polyplexes was up to about 2 times than that of PEI/Cy5DNA ACS Paragon Plus Environment

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polyplexes after 6 h of incubation. Based on previous reviews, we predicted that the possible reason for this phenomenon might be the increasing hydrophobicity of the polyplexes due to the addition of acetyl groups to the PEI, leading to the stronger association of polyplexes with the cell surface and higher cellular uptake efficiency.38 Determination of Endosomal Escape and DNA Release. After successful uptake into cells via endocytosis,39 it is absolutely essential for the internalized molecules getting out of endosomes to have access to the cytosol or the nucleus to maximize their benefits. Therefore, escaping from endosomes successfully is a necessary task for efficient gene transfection.40



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Figure 11. (A) Confocal images of endosomal disruption of Hela cells cultured with OEITKx/Cy5DNA polyplexes and PEI/Cy5DNA polyplexes in serum-free medium for 0.5 h, 1 h, 3 h, and 6 h (4 h of transfection followed by rinsed and another 2 h of incubation in fresh complete medium). DNA (red) was labelled with Cy5, and lysosomes were dyed green with LysoGreen. (B)


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Confocal images of intracellular dissociation of FITCPEI/Cy5DNA


polyplexes and

polyplexes of Hela cells in the serum-free medium after 1 h, 3 h, 6 h and 24 h of

incubation (4 h of transfection followed by rinsed and another 2 h and 20 h of incubation in fresh complete medium). The cellular nuclei were dyed blue with Hoechst 33342, DNA (red) was labelled with Cy5, OEI-TKx and PEI were labelled green with FITC, colocalized

FITCOEI-

TKx/Cy5DNA polyplexes and FITCPEI/Cy5DNA polyplexes were represented as yellow dots. Scale bars: 10 μm. (C) The degree of colocalization of red (Cy5DNA) and green (LysoGreen-stained lysosomes) fluorescence using the Overlap Coefficient R by Image-Pro Plus Version 6.0 software as calculated from confocal images in A. (D) The degree of colocalization of red (Cy5DNA) and green (FITCOEI-TKx or

FITCPEI)

fluorescence using the Overlap Coefficient R from confocal

images in B.

To examine the subsequent intracellular fate of OEI-TKx/DNA polyplexes in Hela cells, endosomal escape was observed by CLSM using

Cy5DNA

(red dots) and LysoGreen (green

fluorescence) which could be used to stain acid compartments (endo/lysosomes). As depicted in Figure 11A, Figure S3A and Figure S3B, the OEI-TKx/DNA polyplexes (N/P ratio of 40) containing

Cy5DNA

firstly interacted with the cell membrane and internalized within roughly

taking 1 h, which could be attributed to the electrostatic interaction between electropositive polyplexes and electronegative cell surface.41 After 3 h of treatment, Cy5DNA was overlapped with endo/lysosomes dyed green with LysoGreen as appreciably yellow fluorescent dots, which demonstrated the colocalization of the Cy5DNA-containing polyplexes with the LysoGreen-stained endo/lysosomes, as evidenced by the strong overlapping between red and green fluorescence at the Overlap Coefficient R of ~0.62 (Figure 11C and Figure S5A). However, following another 3



h of incubation, a large amount of

Cy5DNA

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successfully distributed in the cytoplasm, and the

Overlap Coefficient R dropped to ~0.19, which indicated that the polyplexes efficiently escaped from the endo/lysosomes. Figure 11A, Figure S3A and Figure S3B also showed that PEI/ Cy5DNA polyplexes (N/P = 10) could successfully escape from endo/lysosomes 6 h of post-transfection because of its inherent ability to cause endosomal release.42 Therefore, we could ascribe the similar capacity of avoiding lysosomal degradation of OEI-TKx and PEI polymers to their similar chemical structures with the intrinsic proton sponge effect. It was worth noting that the green fluorescence of OEI-TKx group and PEI group attenuated weakly, which could be due to the swelling and rupture of the acid compartments (endo/lysosomes) via the proton sponge mechanism.43 The Intracellular Dissociation of


Polyplexes. To demonstrate

whether DNA could be released from OEI-TKx/DNA polyplexes efficiently triggered by high ROS level in Hela cells, the intracellular polyplexes dissociation in Hela cells was detected by CLSM using double-labelled polyplexes composed of

Cy5DNA

(red dots) and

fluorescence) (Figure 11B). For comparison, non-degradable

FITCOEI-TK

FITCPEI/Cy5DNA

x

(green

polyplexes were

incubated with Hela cells for desired times. After 6 h of incubation with Hela cells, only a limited part of free

Cy5DNA

(red dots) was disassembled from

FITCOEI-TK

x

(green fluorescence), which

indicated an incomplete dissociation of the FITCOEI-TKx/Cy5DNA polyplexes, as evidenced by the strong overlapping between red and green fluorescence at the Overlap Coefficient R of ~0.69 (Figure 11D). However, almost no yellow fluorescence but a remarkable amount of red dots were obviously observed in

FITCOEI-TK /Cy5DNA-treated x

Hela cells 24 h of post-transfection (Figure

11B and Figure S4A) and the Overlap Coefficient R dropped to ~0.36, confirming efficient intracellular dissociation of the OEI-TKx/DNA polyplexes in ROS-rich Hela cells. In stark contrast,


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Biomacromolecules

an extremely limited amount of Cy5DNA was released from FITCPEI/Cy5DNA polyplexes until 24 h of incubation with Hela cells (Figure 11B and Figure S4B), and the Overlap Coefficient R still remained at ~0.62 (Figure 11D and Figure S5B). These results confirmed regulating the release of DNA by controlling the disassembly of fabricated polyplexes via thioketal linkages cleavage during gene transfection is a feasible strategy for efficient gene delivery. We have demonstrated a thioketal-based cationic polymer OEI-TKx for effective gene delivery, which could efficiently condense DNA into nanosized polyplexes but disassemble once stimulated by abundant intracellular ROS in hypoxic Hela cells. Fracture of thioketal linkages in OEI-TKx gave rise to the degradation of OEI-TKx under hypoxia-induced rich-ROS conditions, leading to an efficient intracellular DNA release in Hela cells. As a consequence, the OEITKx/DNA polyplexes possessed lower toxicity but high gene transfection efficiency in Hela cells. CONCLUSIONS In summary, we have established a positively-charged polymer OEI-TKx containing thioketal linkages for effective gene delivery in hypoxia-induced ROS-rich cells. Due to the introduction of thioketal linkages, OEI-TKx/DNA polyplexes possess lower toxicity and higher cellular uptake efficiency than traditional gold standard (PEI). Nevertheless, there is the same significant effect of OEI-TKx/DNA polyplexes and PEI/DNA polyplexes in endosomal escape profile because of their similar chemical structures. As a result, the higher gene transfection efficiency of fabricated ROSresponsive OEI-TKx/DNA polyplexes in comparison with PEI/DNA polyplexes is mainly owing to valid cleavage of thioketal linkages, efficient disassembly of OEI-TKx/DNA polyplexes and successful intracellular release of complexed DNA once triggered by intracellular ROS mediated by hypoxia. These features demonstrate the distinctive properties of thioketal linkages can



Page 40 of 44

potentially enable novel and versatile perspectives of multifunctional stimulus-responsive materials for on-demand efficient gene delivery. ASSOCIATED CONTENT Supporting Information ESI-MS of thioketal linker (TK), 1H NMR spectrum of TK before and after exposure to ROS, EGFP gene expression of different cells treated with OEI-TKx/DNA polyplexes and PEI/DNA polyplexes, enlarged view of full confocal images of endosomal disruption and intracellular dissociation of Hela cells cultured with OEI-TKx/DNA polyplexes and PEI/DNA polyplexes in serum-free medium at desired time points, quantitative analysis of degree of colocalization of red (Cy5DNA) and green (LysoGreen-stained lysosomes) fluorescence and degree of colocalization of red (Cy5DNA) and green (FITCOEI-TKx or FITCPEI) fluorescence. AUTHOR INFORMATION Corresponding Authors *E-mail: [email protected]. *E-mail: [email protected] & [email protected]. ORCID Yiyan He: 0000-0002-1568-6047 Zhongwei Gu: 0000-0003-1547-6880 Notes The authors declare no competing financial interest. ACKNOWLEDGEMENTS The National Natural Science Foundation of China (31871000, 31500810, 81621003 and 31771067), the National Key Research and Development Program of China (2017YFC1103501),


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Biomacromolecules

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Table of Contents Graphic


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A Reactive Oxygen Species (ROS)-Degradable Polymeric

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