pH Triggered Size Increasing Gene Carrier for Efficient Tumor

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pH Triggered Size Increasing Gene Carrier for Efficient Tumor Accumulation and Excellent Anti-tumor Effect Zhaopei Guo, Jie Chen, Lin Lin, Xiuwen Guan, Pingjie Sun, Meiwan Chen, Huayu Tian, and Xuesi Chen ACS Appl. Mater. Interfaces, Just Accepted Manuscript • Publication Date (Web): 20 Apr 2017 Downloaded from http://pubs.acs.org on April 23, 2017

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ACS Applied Materials & Interfaces

pH Triggered Size Increasing Gene Carrier for Efficient Tumor Accumulation and Excellent Antitumor Effect Zhaopei Guo,†, ‡, ║ Jie Chen, †, ║ Lin Lin, † Xiuwen Guan, § Pingjie Sun, † Meiwan Chen, *,‡ Huayu Tian*,† and Xuesi Chen*,† †

Key Laboratory of Polymer Ecomaterials; Changchun Institute of Applied Chemistry, Chinese

Academy of Sciences, Changchun 130022, China ‡

State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical

Sciences, University of Macau, Taipa, Macao 999078, China §

University of Chinese Academy of Sciences, Beijing 100049, P. R. China

ABSTRACT: High efficiency and serum resistant capacity are important for gene carrier in vivo usage. In this study, transfection efficiency and cell toxicity of polyethylenimine (PEI) (branched, Mw=25K) was remarkably improved, when mixed with polyanion (polyethylene glycolpolyglutamic acid (PEG-PLG) or polyglutamic acid (PLG)). Different composite orders of PEI, polyanion, and gene, for example, PEI is first complexed with DNA, and then with polyanion, or PEI is first complexed with polyanion, and then with DNA, were studied. Results showed that only the polyanion/PEI complexes exhibited additional properties, such as decreased pH, resulting in increased particle size, as well as enhanced serum resistance capability and improved

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tumor accumulation. The prepared gene carrier showed excellent antitumor effect, with no damage on major organs, which is suitable for in vivo gene antitumor therapy. KEYWORDS: PEI, zwitterionic, polyglutamic acid, gene carriers, shielding

INTRODUCTION Gene therapy is among the most efficient methods to cure various human diseases, including cancer. It acts by delivering nucleic acids into ill cells to correct protein expression profiles.1-2 Nonviral gene carriers, particularly cationic polymers, are considered safe and efficient candidates for successful gene therapy, because of their potential safety usage in clinical application, high gene loading capacity, flexible design, and large-scale preparation.3-6 Hyperbranched polyethylenimine (PEI25K), as the “golden standard” for polymeric gene carriers,7-10 has more efficient gene delivery capability than other cationic polymer carriers, such as poly (L-lysine), polyamidoamine, chitosan and polyphosphoester.11-13 As gene carrier, PEI exhibits several beneficial properties, such as high gene transfection efficiency, low cytotoxicity, long circulation, and more effective accumulation in tumor in vivo. Although PEI has excellent gene delivery capacity in vitro, the shortage is significant when it is used in vivo because of the following reasons. First, the cytotoxicity caused by high grate of hemolysis limit their usage in large amount.14 Second, to attain good gene transfection, a large number of cationic polymer, is often needed, thus, excessive free cationic polymers exist in the system,15-16 diluting the polymers in the blood and leading to low gene transfection efficiency. To improve the cytotoxicity and gene transfection efficiency, PEI has been subjected to some modifications. Grafting biocompatible materials to PEI is considered as among the most efficient methods. Based on this, some researchers grafted polyamino acid to PEI, which can effectively


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reduce the PEI’s charge density, enhance biocompatibility, and achieve more gene transfection efficiency.14,

17-20

Another method is reducing the amount of cationic polymers. You and

coworkers grafted fluorocarbon chains to PEI, largely reducing the N/P ratio (nitrogen (in PEI) to phosphorus (in DNA or RNA) ratio) compared with PEI at optimum transfection effect.21 The reduced N/P ratio means less free cationic polymer exists in the transfection system, thus, reducing its cytotoxicity. For in vivo therapy via systemic injection of gene-loaded cationic polymers, PEGylation has shown tremendous potential for enhancing long circulation. However, PEGylation of cationic polymers can enhance nanoparticle circulation time, but reduce the internalization of cancer cells.22 To address this problem, researchers prepared conjugated PEG via acidity-sensitive linker to shield the cationic polymers. At tumor acidic environment, PEG detached and exposed the cationic nanoparticles, helping to enhance cellular uptake and improve tumor therapy efficiency.23 However, this single strategy cannot deal with complicated tumor environment. Another reported method for in vivo usage of cationic/DNA polyplexes is shielding polyanion to the polyplexes, and this method can significantly decrease their zeta potential24-25 and improve their in vivo application.26 However to our knowledge, the shielded polyanion cannot increase the tumor therapy efficiency compared with the unshielded one.27 Therefore, combining the cationic/DNA polyplexes while simultaneously reducing cytotoxicity, enhancing transfection efficiency, and developing easy-to-use PEI in vivo has become a challenge. Aside from gene carriers, therapeutic gene is also important in gene therapy. The main mechanisms of tumor cells are angiogenesis and proliferation.28 Rapid and unending tumor cell proliferation consumes high amount of nutrients and oxygen, tumors have to stimulate the formation of new blood vessels through processes driven primarily by vascular endothelial


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growth factor (VEGF).29 VEGF is overexpressed and secreted mostly by tumor cells, which could stimulate proliferation of endothelial cells, causing angiogenesis in tumor tissue.30 RNA interference (RNAi) is a promising technique in treating various genetic diseases,31-32 and RNAimediated silencing VEGF expression has been proven to successfully inhibit VEGF expression, resulting in decreased blood vessel density and delayed tumor growth.33-35 In this study, the antiVEGF agent was used as shVEGF for tumor therapy. Based on the abovementioned problems, we designed a pH-triggered size increase nanocarrier for gene delivery. This design can overcome main limitations in meeting a whole deliver process. The polyanion PEG-PLG was first complexed with PEI to form zwitterionic surface (positive and negative charged) nanocomplexes, which was used as the gene carrier. PLG as polyanion can reduce toxicity and PEG can efficiently bypass the plasma compartment, thus prolonging the circulation time for in vivo applications. Furthermore, the zwitterionic particles also help to prolong the in vivo circulation time.36-37 The pH-dependent size changeable characteristic diminished the sizes in physiological pH 7.4, which is suitable for blood circulation,38 and enlarged the sizes in tumor acidic pH help to accumulate in tumor

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(Scheme 1). The novel

delivery system showed substantially higher transfection capacity than PEI in vitro and superior antitumor therapeutic efficacy in vivo. This delivery system would be regarded as the competitive candidate for future cancer therapy. EXPERIMENTAL SECTION Materials. . Hyperbranched polyethylenimine (Mn=10kDa and Mw=25kDa) was purchased from Sigma-Aldrich. O-(2-Aminoethyl)-O’-(2-methyl) polyethylene glycol (PEG-NH2, Mw = 2000) was purchased from jenkem Beijing. N-carboxyanhydride of γ-benzyl-L-glutamate (BLG-NCA) and PLG were prepared according to previously method.24, 39 Glutamate and trifluoroacetic acid


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were purchased from GL Biochem Ltd. (Shanghai, China). Hydrobromic acid in acetic acid 33% (v/v) was purchased from ACROS. Luciferase plasmid (pGL3-control), cell lysate, and luciferase reporter gene assay kit were purchased from Promega (Mannheim, Germany). Luc siRNA (sense:

5'-CUUACGCUGAGUACUUCGAdTdT-3'

UCGAAGUACUCAGCGUAAGdTdT-3') AGCUUCAUGAGUCGCAUUCdTdT-3'

and

Rev and

and

antisense:

siRNA antisense:

(sense:

5'5'5'-

GAAUGCGACUCAUGAAGCUdTdT-3') duplexes were purchased from GenePharma Co., Ltd (Shanghai, P. R. China). Cy5-DNA was purchased from RIBOBIO Co., Ltd. (Guangzhou, P. R. China). The control sequence does not match any human genome sequence. MTT was purchased from Amresco (Solon, Ohio, USA). A bicinchoninic acid (BCA) protein assay kit was purchased from Pierce (Rockford, USA). Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were purchased from Gibco (Grand Island, USA). Calf thymus DNA, Methyl-βcyclodextrin (M-β-CD) and cytochalasin D were purchased from Sigma (St-Louis, MO, USA). Lyso Tracker Green were purchased from Molecular Probes, chlorpromazine was purchased from TCI. The coding region (5′-AUGUGAAUGCAGACCAAAGAA3′) of the VEGF gene was targeted in this study. Oligonucleotides that encoded the corresponding small hairpin RNA (shRNA) were synthesized commercially in GenePharma Co. Ltd. (Shanghai, China) namely shVEGF. Synthesis and Characterization of PLG and PEG-PLG. PLG was prepared according to previously reported method.24 Generally, hexylamine was used as the initiator to ring opening BLG-NCA polymerization, then deprotective benzyl groups. PEG-PLG was prepared using similar method as preparation of PLG, here, PEG-NH2 was used as the initiator. 1H NMR spectra of PLG and PEG-PLG were recorded on a Bruker AV-400 spectrometer (Bruker,


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Ettlingen, Germany). The measurements were carried out at room temperature, and CF3COOD was used as the solvent. Preparation of Carriers/DNA or RNA Polyplexes. . For preparing PLG/PEI (GP) and PEGPLG/PEI (PGP) complexes, a solution of PEI, PLG and PEG-PLG at certain concentration, PLG or PEG-PLG was added to PEI and vortexed at different weight ratios, such as 1/1, 0.5/1 and 0.25/1. For preparing PEI/DNA or PEI/RNA, GP/DNA or GP/RNA and PGP/DNA or PGP/RNA complexes, a solution of PEI, GP and PGP was complexed with DNA or RNA at different weight ratios, such as 10/1, 5/1, 2.5/1 and 1.25/1(ratios of GP and PGP to DNA or RNA were calculated according to the amount of PEI containing in the complex). Cell Culture and Plasmid Purification. . Cells were cultured in DMEM high glucose supplemented with 10% (v/v) heat-inactivated FBS, 100 units/mL penicillin, and 100 µg/mL streptomycin in a 5% CO2 incubator at 37 °C under 95% humidity. The 5.3-kilobase expression vector pGL3 (Promega, Madison, WI) coding for the luciferase gene driven by the SV40 promoter and enhancer, was grown in DH5α E. coli and purified with a commercial plasmid purification kit (Bio-Rad, Hercules, CA) Cytotoxicity Assay. . The cytotoxicity of the complexes was assessed using the MTT assay. HeLa cells were seeded at 1.0×104 cells/well in 96-well plates, cultured for 24 h. For the cytotoxicity study of the polyplexes, the culture medium was aspirated and replaced with 180 µL of DMEM. Subsequently, 20 µL of PEI/DNA, GP/DNA, and PGP/DNA complexes at different weight ratios were added to each well. The plates were returned to the incubator for an additional 48 h. At the end of the experiments, 20 µL of MTT solution (5 mg/mL in PBS) was added to each well. The plates were returned to the incubator for 4h. Then, the MTT solution was carefully removed from each well, and 200 µL of DMSO was added to dissolve the MTT


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formazan crystals. The plate was incubated for an additional 10 min before the absorbance at 492 nm was recorded by an ELISA microplate reader (Bio-Rad). The cell viability (%) was calculated according to the following equation: Cell viability (%) = (Asample/Acontrol) × 100 where Asample is the absorbance of the ternary complex-treated cells and Acontrol is the absorbance of the untreated cells. Each experiment was done in quadruplicate and repeated at least thrice. In Vitro Transfection. To evaluate the transfection efficiency of the carriers, HeLa, CHO, MCF7 and CT26 cells were seeded in 96-well plates, with the density of 1.0 × 104 cells/well, in 200 µL growth medium (90% DMEM and 10% FBS). All cell lines were incubated at 37°C for 24 h. To evaluate the transfection efficiency affected by different amount of serum content, after cells were seeded for 24h, media were replaced with cultures containing 0%, 10%, 20% , 30%, 40%, and 50% FBS). Then, 20 µL of GP/DNA and PGP/DNA (wt/wt) polyplexes were added. After 48 h transfection, the media was removed, and the cells were gently washed thrice with PBS, then, thoroughly lysed with cell lysis buffer (Promega, 50 µL/well). The luciferase activity was determined by detecting the light emission from an aliquot of cell lysate incubated with 100 µL of luciferin substrate (Promega) in a luminometer (GloMax 20/20, Promega). The protein content of the cell lysate was determined by a BCA protein assay kit (Pierce, Rockford, IL, USA). All experiments were performed in triplicate to ascertain reproducibility. To study the gene silencing, Huh-7 (1.0×104 cells/well) cells were seeded in a 96-well plate prior to transfection, and then the medium was replaced with 200 µL of the fresh growth medium. Polymer/siRNA (200 ng of siRNA and 20µL of sterile water) complexes with different mass ratios were added to each well, and then the cells were incubated for 48 h at 37°C. Then the cells were washed three times with PBS. After thorough lysis of cells by cell lysate buffer (Promega,


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50µL/well), luciferase gene expression was quantified using a luciferase reporter gene assay kit and photon counting with a luminometer (GloMax™ 20/20, Promega), adding 100 µL of luciferin to 20 µL of cell lysate in each well. The protein content was measured using BCA protein assay kit (Pierce, Rockford, IL, USA). Luciferase activity was expressed as LUC/mg protein. The relative luciferase activity was related to untreated control cells. Zeta Potential and Particles Size Analysis. Zeta potential and particles sizes were measured using a Zeta Potential/BI-90Plus particle size analyzer (Brookhaven, USA) at room temperature. For preparing of the complexes, a solution of PLG, or PEG-PLG was mixed with PEI at certain weight ratio and vortexed, a solution of PEI and the complexes of GP, or PGP was mixed with DNA (Calf thymus DNA) at certain weight ratio and vortexed, then all the complexes were incubation at room temperature for 30 min before measured. Uptake Efficiency and Endocytosis Inhibition. The internalization efficiency of the complexes was evaluated using Cy5-labeled pDNA (3 µg of pDNA/well). Experiments were performed in six-well plates at a density of 1.0×105 cells/well in 2 mL of the growth medium (90% DMEM and 10% FBS). After 24 h, complexes were added with different weight ratios and incubated at 37 °C for 4 h. Then, the cells were harvested and washed with PBS, detached with 0.25% trypsin and then resuspended in 500µL of PBS (pH 7.4). Finally, internalization efficiency was carried out using a flow cytometer (FACS Caliber, Becton-Dickinson, San Jose, CA, USA) For the endocytosis inhibition assay, the inhibitor M-β-CD, chlorpromazine and cytochalasin D was added with concentration 10µg/mL, 6µg/mL, and 3.5µg/mL, respectively. After 40 min incubated at 37°C, the complexes were added and additionally incubated for 4 h. Then, the transfected culture was washed once with PBS. Subsequently, the cells were detached with


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0.25% trypsin and then resuspended in 500µL of PBS. Finally, the internalization efficiency was evaluated using a FACS Calibur System from Becton-Dickinson (San Joes, CA). Intracellular Trafficking. To observe intracellular trafficking, Cy5-DNA was used. Cells were seeded in six-well plates at an initial density of 5.0×104 cells/well. Before the cells were fixed with 3.7% paraformaldehyde, they were incubated with the complexes for 4 h. After immobilization, the cover slips were washed thrice with PBS, then, treated with 75 nM of Lysotracker for 30 min at 37°C, again, the cover slips were washed thrice with cold PBS, and the cell nuclei were stained with 2 mL DAPI (0.5µg/mL) for 10 min. The cover slips were washed several times with PBS, enclosed in glycerol, and then visualized by CLSM (Leica TCS SP2; Leica Microsystems, Wetzlar, Germany). Protein BSA Adsorption. 0.5mL of polymers solution (1mg/mL) and 0.5mL of BSA solution (2mg/mL) were mixed. The mixed solution was shaken and kept at 37°C for 30 min. Then the supernatant was carefully collected after centrifugation. BSA concentration in the supernatant was calculated using a previously established calibration curve, from the UV absorbance at 280 nm. The protein adsorbed on the polyplexes was calculated according to: q= (Ci - Cs) V/m Ci is the initial BSA concentration, Cs is the BSA concentration in the supernatant after adsorption experiments, V is the total volume of the mixture solution (1 mL), and m is the weight of the polymer (0.5 mg) in the mixed solution. Tumor Accumulation. Four- to five-week-old female BALB/C nude mice were obtained from Vital River Company in Beijing and were housed under specific pathogen-free conditions. All experimental procedures were in accordance with the guidelines for laboratory animals established by the Animal Care and Use Committee of Northeast Normal University. BALB/C


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nude mice injected subcutaneously with CT26 single-cell suspension in PBS medium. Ten days later, CT26 tumor-bearing (0.5 cm in diameter) BALB/C nude mice were injected with complexes of PEI/cy5-DNA, GP/cy5-DNA and PGP/Cy5-DNA via the tail vein. After 12h, 24h, 48h, 72h, 96h and 144h, the accumulation of Cy5-DNA was observed by an in vivo imaging system (CRI Maestro 500FL) after the nude was anesthetized. In Vivo Tumor Treatment. CT26 cells suspension in PBS medium was injected subcutaneously of the BALB/C mice. Tumor size was measured using a vernier caliper across its longest (a, mm) and shortest (b, mm) diameters and the tumor volumes were calculated using the following equation: Tumor volume (mm3) = 0.5ab2 After ten days, when the tumor size became approximately 70mm3, the animals were divided randomly into five groups and given a tail vein injection of PBS, shVEGF, PEI/shVEGF, GP/shVEGF and PGP/shVEGF complexes, respectively. Histopathology Evaluation. The heart, lung, liver, spleen, kidney and tumor were dissected from the mice (after 14 days treatment) for histopathological analysis. The tissues were paraffinembedded and cut at 5 mm thickness. Then, tissues were stained using hematoxylin and eosin to assess histological alterations with a microscope. Intratumoral microvessel density was also evaluated. The excised tumors were fixed with 4% paraformaldehyde. 8 mm thickness histological sections were obtained using a Freezing Microtome (Leica, Germany) and treated with paraformaldehyde for further fixation. After blocking with goat serum (10% in PBS), sections were further treated with CD31 (PECAM-1) antibody (Epitomics, America) followed by a fluorescent secondary antibody and DAPI.


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Statistical Analysis. All of the data presented as a mean value ± standard deviation of independent measurements. Statistical significance when the P values were less than 0.05. RESULTS AND DISCUSSION Synthesis and Characterization of Polymers. The synthesis of PLG and its structure analysis was according to previous publication.27 PEG-PLG was prepared using PEG-NH2 to initiate BLG-NCA ring opening polymerization, then deprotective benzyl groups. Comparing the 1H NMR spectra shown in Figure 1, peaks of a, b, and c were assigned to PLG and PEG-PLG. The peaks at 0.7, 1.2, and 1.5 ppm were assigned to initiator hexylamine, and the peak at 3.8 ppm was assigned to initiator PEG. The number-average molecular weight (Mn) of PLG was 2.1 kDa, with 16° of polymerization as previously reported. The Mn of PEG-PLG was 4.2 kDa, calculated by the integral area of “a” and “PEG” (known as Mn 2 kDa). Meanwhile the degree of polymerization of PLG in PEG-PLG was 17, which was near to previously used PLG. In Vitro Transfection and Cytotoxicity. In vitro transfection efficiency of PLG/PEI (GP) and PEG-PLG/PEI (PGP) were firstly studied in HeLa and CT26 cells. Different weight ratio of PLG or PEG-PLG to PEI was mixed to form nano particles, which was used as the gene carrier (weight ratio of PLG or PEG-PLG to PEI are 1:1, named as GP1 or PGP1. 0.5:1 named as GP2 or PGP2. 0.25:1 named as GP3 and PGP3). Then, the nano particles complexed with pGL3, with the mass ratio of carriers to pGL3 was 10, 5, 2.5 and 1.25 (calculated by the amount of PEI content in the particles, because DNA compressing and cytotoxicity are mainly associated with PEI. PLG or PEG-PLG showed no cytotoxicity (Figure S1)). Transfection result and corresponding cell toxicity was shown in Figure 2. Using GP or PGP as the gene carrier to deliver pGL3, there was a notable enhancement of the transfection efficiency compared to PEI in HeLa and CT26 cells (Figure 2A and Figure 2B), despite the PLG or PEG-PLG content in GP


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or PGP, and there were 4 to 6 times higher than that of PEI at optimum transfection efficiency. When the weight ratio of PLG or PEG-PLG to PEI was 0.5, the transfection efficiency showed more superior, so GP2 or PGP2 (weight ratio of PLG or PEG-PLG to PEI was 0.5) was selected and used in the following experiment. From the MTT results, we found that mingling PLG or PEG-PLG in to PEI had remarkably decreased the cytotoxicity, even at the ratio of carriers/pGL3 at 10/1, the cell viability was nearly 70%, while there was only 30% cell viability of PEI, both in HeLa and CT26 cells (Figure 2C and Figure 2D). Therefore, introducing of PLG or PEG-PLG could effectively reduce toxicity of PEI, accordingly, the transfection efficiency was enhanced. Transfection efficiency with different mixing order of PLG or PEG-PLG, PEI and DNA was also studied (the optimized ratio of PLG or PEG-PLG to PEI was selected at 0.5:1). Different mixing order of PLG or PEG-PLG, PEI and DNA had no big difference in in vitro transfection efficiency, and they all showed superiority than PEI (Figure S2). This can attributed to introducing of PLG or PEG-PLG largely decreasing cytotoxicity of PEI. The transfection efficiency with optimum selected GP2 and PGP2 were also assessed in CHO and MCF7 cell lines, using pGL3 as the reporter gene (Figure 3A and Figure 3B), and RNAinduced gene silencing in Huh7 cells (Figure 3C). In these cell lines GP2 and PGP2 still showed excellent luciferase expression and gene silencing effect. With optimum transfection ratio, GP2 and PGP2 showed 4 to 7 time higher luciferase expression than PEI. The luciferase knockdown efficiency in Huh7 cells was nearly 70% for PGP2 and 61% for GP2, which was higher than PEI only about 37%. Therefore, introducing of PLG or PEG-PLG to PEI would also enhance DNA or RNA transfection capacity in many types of cells. The cell toxicity caused by PEI/DNA, GP2/DNA, and PGP2/DNA in these cell lines was also investigated, and the similar results with


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that in HeLa and CT26, was obtained, GP2/DNA and PGP2/DNA showed lower cytotoxicity than PEI/DNA, at polymer/DNA ratio 10/1(Figure S3). Particle Size, Zeta Potential, Uptake Efficiency, Endocytosis Inhibition, and Intracellular Trafficking. Typically, nanoparticles can achieve intracellular uptake by cells even up to several hundred nanometers.40 The complex particles of DNA with PEI, GP2, and PGP2 were lower than 130 nm in diameter. The sizes of DNA/ GP2 and DNA/ PGP2 distributed between 80 and 100 nm at different weight ratios was lower than that of DNA/PEI complex (Figure 4A). The zeta potential of DNA/ GP2 and DNA/ PGP2 at different weight ratios was lower than that of DNA/PEI, and they were all positively charged (Figure 4B). At optimum transfection ratio, such as the ratio of DNA/PEI, DNA/ GP2, and DNA/ PGP2 being 0.4, 0.4 and 0.4, respectively, zeta potential of DNA/ PGP2 was much lower than the others. Particle surface with less positive charge will help to decrease cytotoxicity. Transfection efficiency is affected by cellular uptake efficiency. In general, when more polymer/DNA complex particles are uptake by cells, high transfection efficiency would be observed. Uptake efficiency of PEI, GP2, and PGP2 complex with Cy5-DNA by CT26 cells was measured using flow cytometry. The uptake efficiency of GP2 and PGP2 was similar, but they were much higher than PEI (Figure 5A). The high uptake efficiency of GP2 and PGP2 than PEI was consistent with their high transfection efficiency (Figure 3). Endocytic pathway of complexes by CT26 cells was demonstrated by endocytosis inhibition assay. First, the chemical inhibitors M-β-CD, chlorpromazine and cytochalasin D, which are known to inhibit caveolae-mediated

endocytosis, clathrin-mediated endocytosis, and

macropinocytosis, respectively, were added to inhibit cell uptake pathway. Then, the complexes were added, and the mixture was incubated for 4 hours in CT26 cells. The uptake efficiency


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decreased sharply with the chemical inhibitor chlorpromazine, whereas minimal effect was noted with M-β-CD and cytochalasin D. Thus, GP2 and PGP2 had the same uptake pathway with PEI, and was mainly affected by clathrin-mediated endocytosis, and minimally affected by caveolaemediated endocytosis or macropinocytosis (Figure 5B). Confocal laser scanning microscopy (CLSM) assay was used to observe the intracellular trafficking of PEI, GP2 and PGP2. CT26 cells were treated with Cy5-labeled DNA/carrier complexes, and then stained with Lysotracker. As shown in Figure 5C, the red dots of Cy5labelded complexes could be observed in all groups, and they were colocated with lysosome (green dots) and showed yellow fluorescence. More dots were found in GP2 and PGP2 than in PEI, and this result can be attributed to their higher uptake efficiency. We found that the complexes, particularly PGP2, were very close to the nuclei, and this was important for DNA to enter into nuclei and successfully transfect. Protein BSA Adsorption and Serum-tolerant Assay. For in vivo usage, polycation mediated transfection was largely affected by the presence of serum,41 because positive polymers could easily form large particles with negatively-charged protein in serum, leading to decreased transfection efficiency.42 In this study, BSA was used as model protein to demonstrate electrostatic adsorption. The experimental procedure was conducted according to previous report.43-44 We found that PEI had the highest amount of protein adsorption, whereas GP2 and PGP2 only adsorbed approximately 1/3 and 1/8 of protein compared with that of PEI (Figure 6A). The results indicated that adding PLG to PEI could induce tolerance to protein adsorption, and a PEG-PLG complex would farther decrease protein adsorption. The transfection efficiency of PEI, GP2, and PGP2 was also studied with different serum contents in CT26 cells (Figure 6B). With serum content enhanced, transfection efficiency of PEI rapidly decreased, and a sharp


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decrease was noted when serum content reached 20%. By comparison, transfection efficiency of GP2 only decreased minimally with serum content of up to 30%, and PGP2 had no affect even at serum content of 30%. The results proved that introducing PLG, particularly PEG-PLG, to PEI could substantially enhance the tolerance of protein adsorption and retain the gene transfer capacity of polycation carriers, making GP2 and PGP2 more easily used in vivo. Transfection efficiency affected by serum was also observed with different complex orders of PLG or PEGPLG to PEI. Although the in vitro transfection efficiency had no significant difference (Figure S2), the serum-induced effect was substantial. When PEI that was first complexed with DNA, and then coated with PLG or PEG-PLG exhibited little serum resistance capacity, which was close to PEI (Figure S4). Thus, this PEI would have low therapeutic efficiency when used in vivo, and this result had been proven in our previous reports.27 Tumor accumulation and complex sizes changed with pH value. When used in vivo, most positive polymers or particles might cause severe serum inhibition and were rapidly cleared from the plasma, thus, their further applications were seriously hindered. GP2 and PGP2 could effectively decrease protein adsorption, and obtain improved transfection efficiency even at serum content of up to 30%. Further experiment was conducted to study the DNA delivery capacity of GP2 and PGP2 into tumors in vivo, compared with PEI. Four- to five-week-old female BALB/C nude mice with CT26 tumor were injected with Cy5-DNA/PEI, Cy5-DNA/GP2 and Cy5-DNA/PGP2 via tail vein. After one-time injection, the fluorescence of Cy5 was detected at different time points. Before 24 h, fluorescence was found in tumors of all the three groups, but the intensity of PEI-mediated Cy5-DNA was significantly lower than the others, and PGP2 showed the highest fluorescence intensity (Figure 7A). This result meant that PGP2 could effectively enhance the amount of DNA entering into tumor. The tumor fluorescence of PEI


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group had almost disappeared, at 48 h after injection, however, for GP2 and PGP2 groups, fluorescence intensity only minimally decreased, and the fluorescence was still clear even after 144 h postinjection. This result indicated that GP2- and PGP2-mediated Cy5-DNA could more efficiently accumulate and maintain in tumor. More fluorescence was observed for PGP2 in tumor than GP2, indicating that PGP2 was more effective than GP2 when used in vivo. Size was reported to play an important role for nanoparticle accumulation in tumors.45 Nanoparticles could more efficiently enter into tumor tissues when their diameters are less than 200 nm, particularly below 100 nm. The particle sizes of GP2 and PGP2 were measured at pH values of 7.4, 6.5, and 6.0. We found that the sizes increased from nearly 100 nm to more than 200 nm when the pH value decreased from 7.4 to 6.0 (Figure 7B and Figure 7C). This may be because low pH value could reduce the amount of negative charge of PLG or PEG-PLG, and the reduced negative charge would weaken the electrostatic attraction between PLG and PEI, resulting in enlarged particle sizes (Figure 7D). Therefore, when the GP2 or PGP2 particles circulated into tumor tissues, the acidic environment

46

enlarges their sizes, and the large sizes were helpful for the

accumulation and maintenance of these particles in tumors. In vivo antitumor therapy. The antitumor efficiency of GP2 and PGP2 was investigated in vivo. The BALB/C mice were injected subcutaneously with CT26 cell suspension in PBS to form CT26 tumor. When the tumor size became approximately 70 mm3, the mice were randomly divided into five groups, and each group were injected with 180 µL PBS, shVEGF, PEI/shVEGF, GP2/shVEGF and PGP2/shVEGF complexes through tail vein every two days containing 15 µg shVEGF. Simultaneously, the body weight and tumor volume were measured every two days. After six treatments, we found that the growth rate of tumors was significantly restrained in the PGP2/shVEGF group (Figure 8A and Figure 8C). GP2/shVEGF and


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PEI/shVEGF also showed tumor-inhibitory effects compared with the control, but they were less than that of PGP2/shVEGF. The more efficient tumor restraining capability of PGP2/shVEGF could be attributed to their easier accumulation and maintenance in tumor. The mice body weight did not significantly change (Figure 8B), indicating that the mice had well body condition during treatment. The content of intratumoral VEGF was associated with neovascularization, thus, the microvessel density in tumors was studied by using the CD31 antibody (green fluorescence) and the nuclear counterstain, DAPI (blue fluorescence). As shown in Figure 8D, the lower microvessel density indicates better treatment effect. We observed a substantially decreased microvessel density in the tumors treated with PGP2/shVEGF compared with the control and shVEGF groups. A significant treatment effect was also noted for PGP2/shVEGF than PEI/shVEGF and GP2/shVEGF. These results were according with tumor treatment effect. The safety of treatment materials was evaluated via hematoxylin and eosin (H&E) staining (Figure 9). The harvested major organs, including heart, liver, spleen, lung, and kidney, and tumors from mice were sliced and stained with H&E. All the therapy groups showed no mutative morphology, implying that the carriers could not hurt these organs during tumor treatment. For tumors, after treatment by GP2/shVEGF and PGP2/shVEGF, particularly the latter, majority of cell nuclei was dissolved. Therefore, they are more efficient in reducing tumor cells. Decreasing cytotoxicity and enhancing transfection efficiency are important for further application of polycationic carrier in gene delivery. This aim was usually achieved by grafting biocompatible materials, such as poly (amino acid) and PEG.14, 17-19, 47 More direct method was shielding the cationic carriers by polyanions,26 and the shielded cationic polymer/DNA polyplexes showed increased efficiency in gene delivery. In this study, we focus on the


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biocompatible polyanion materials, PLG and PEG-PLG, giving a more detailed investigation and making these materials more suitable for in vivo application. For preparation of gene carrier, we first combined polyanion with PEI to form electrostatic complex and load the gene (Figure 10). The decrease in PEI cytotoxicity through negatively charged PLG is important for in vivo application. However, too much PLG in the polyplexes would lead to negatively-charged particle surface, sharply reducing their gene transfection efficiency24,

26

because of the negatively-

charged cell membrane could not easily adhere and internalize these particles.48-49 Grafting of PEG to cationic carrier would reduce transfection efficiency because of the decreased cellular uptake efficiency. Interestingly, the PGP2 and GP2 showed high transfection efficiency than PEI in multicell lines, and this result might be attributed to their different composite orders. The high transfection efficiency of gene vectors is mostly attributed to their high cell uptake efficiency. The uptake pathway by cells was also important because one of the barriers for nonviral gene delivery was escaping from lysosomes.50 Pinocytosis, which is further divided into clathrin-dependent endocytosis, caveolae-mediated endocytosis, clathrin-/caveolin-independent endocytosis, and macropinocytosis is the most common endocytosis. Moreover, clathrindependent endocytosis has been proven to be most efficient because provides an acidic environment which is helpful for inducing proton-sponge effect of cationic vectors to disrupt the endosome. This uptake pathway can also move transgene cargo very closely to nuclear region, subsequently increasing the nuclear import.51 Comparing with PEI, GP2 and PGP2 showed more efficiency in cell internalization, and they were proven to be the same uptake pathway with PEI (Figure 5A and 5B). Therefore, mixing PLG or PEG-PLG with PEI would allow easy cell uptake of PEI, and retain the PEI uptake pathway, and this phenomenon was also observed directly by CLSM (Figure 5C).


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The mainly problem with polycationic vectors used in vivo was their nonspecific adsorption with negative-charged protein in serum, known as opsonization.52 The opsonized polyplexes could be easily captured by mononuclear phagocytic system (MPS), or the reticuloendothelial system (RES), resulting in rapid clearance from the blood. Thus, the circulating time and entrance of polyplexes into tumor tissue is shortened. A recent study found that carriers modified with PEG 53

or zwitterionic groups are as the most effective method for avoiding clearance by RES, and

zwitterionic is more efficient than PEG for in vivo usage.36-37 We mixed PLG or PEG-PLG with PEI to form nanoparticles, and these nanoparticles were composed of negatively-charged PLG or PEG-PLG and positively-charged PEI. They could be regarded as zwitterionic particles. Particularly, PGP2 not only contained zwitterionic properties but also consisted of PEG, making it better BSA protein resistance than PEI and GP (Figure 6 and Figure S4). When used in vivo, GP2 and PGP2 showed significant tumor cumulated effect, in particular, PGP2 showed strong fluorescence signal in tumor even after 144 h (Figure 7). For in vivo antitumor therapy, shVEGF was used as anti-VEGF agent. Anti-VEGF therapy inhibited tumor growth via anti-angiogenesis mechanisms rather than killing off tumor cells directly. Given the increased transfection and tumor accumulation, GP2, particularly PGP2, showed highly efficiency tumor inhibition. The tumors treated with PGP2/shVEGF were restrained at very small size, and the CD31 antibody-labeled neovascularization was significantly less than the other groups. As shown in Figure 9, almost no damage was incurred on the major organs, implying that the vectors had almost no toxicity on the tissues of major organs and that the delivery of shVEGF did not affect any on major organs. All these results showed that the vectors were beneficial for use in vivo. CONCLUSIONS


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The novel zwitterionic gene carriers were prepared by mixing PLG or PEG-PLG with PEI. The carriers showed higher DNA expression in several cell lines, such as HeLa, CHO, MCF7 and CT26, and better siRNA transfer efficiency in Huh7 cells than PEI, as well as significantly decreased cell toxicity. When used in vivo, the gene-loaded polyplexes could effectively accumulate in CT26 model tumor. Moreover, shVEGF was carried by these polyplexes, which showed significant effect on CT26 tumor suppression. Therefore, the novel zwitterionic polyplexes are promising gene carriers for tumor therapy. ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website, Cell viability with different concentration of PLG or PEG-PLG, Transfection efficiency of PLG or PEG-PLG, PEI and DNA with different composites order (the weight ratio of PLG or PEGPLG to PEI was optimal proportion 0.5:1, the red color words stand for priority compound and then complex with the other ingredient), Relative cell viability of PEI/DNA GP2/DNA, and PGP2/DNA at different weight ratio, in CHO, MCF7, and Huh7 cells, Transfection efficiency of PLG or PEG-PLG, PEI and DNA at different composites order, with different FBS content (the weight ratio of PLG or PEG-PLG, PEI and DNA was selected at 1.25:2.5:1) AUTHOR INFORMATION Corresponding Authors *(M.C.) E-mail: [email protected]. *(H.T.) E-mail: [email protected]. *(X.C.) E-mail: [email protected]. Author Contributions


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║

Z.G. and J.C. contributed equally to this work.

Notes The authors declare no competing financial interest. ACKNOWLEDGMENTS The authors are thankful to the National Natural Science Foundation of China 51403205, 21474104, 51520105004, 51390484, 51503200, 51303173, 2014AA020708, 51233004, National program for support of Top-notch young professionals, Jilin province science and technology development program (20160204032GX). REFERENCES (1) Dachs, G. U.; Dougherty, G. J.; Stratford, I. J.; Chaplin, D. J. Targeting Gene Therapy to Cancer: A Review. Oncol. Res. 1997, 9 (6-7), 313-325. (2) Wirth, T.; Parker, N.; Yla-Herttuala, S. History of Gene Therapy. Gene 2013, 525 (2), 162-169. (3) Rahbek, U. L.; Howard, K. A.; Oupicky, D.; Manickam, D. S.; Dong, M. D.; Nielseni, A. F.; Hansen, T. B.; Besenbacher, F.; Kjems, J. Intracellular siRNA and Precursor miRNA Trafficking Using Bioresponsive Copolypeptides. J. Gene Med. 2008, 10 (1), 81-93. (4) Chen, G. H.; Chen, W. J.; Wu, Z.; Yuan, R. X.; Li, H.; Gao, J. M.; Shuai, X. T. MRIVisible Polymeric Vector Bearing CD3 Single Chain Antibody for Gene Delivery to T Cells for Immunosuppression. Biomaterials 2009, 30 (10), 1962-1970. (5) Tian, H. Y.; Chen, J.; Chen, X. S. Nanoparticles for Gene Delivery. Small 2013, 9 (12), 2034-2044. (6) Tian, H. Y.; Guo, Z. P.; Chen, J.; Lin, L.; Xia, J. L.; Dong, X.; Chen, X. S. PEI Conjugated Gold Nanoparticles: Efficient Gene Carriers with Visible Fluorescence. Adv. Healthcare Mater. 2012, 1 (3), 337-341. (7) Godbey, W. T.; Wu, K. K.; Mikos, A. G. Poly(ethylenimine) and its Role in Gene Delivery. J. Controlled Release 1999, 60 (2-3), 149-160. (8) Bragonzi, A.; Boletta, A.; Biffi, A.; Muggia, A.; Sersale, G.; Cheng, S. H.; Bordignon, C.; Assael, B. M.; Conese, M. Comparison between Cationic Polymers and Lipids in Mediating Systemic Gene Delivery to the Lungs. Gene Ther. 1999, 6 (12), 1995-2004. (9) Chen, J.; Liang, H.; Lin, L.; Guo, Z. P.; Sun, P. J.; Chen, M. W.; Tian, H. Y.; Deng, M. X.; Chen, X. S. Gold-Nanorods-Based Gene Carriers with the Capability of Photoacoustic Imaging and Photothermal Therapy. Acs Appl. Mater. Interfaces 2016, 8 (46), 31558-31566. (10) Guan, X. W.; Li, Y. H.; Jiao, Z. X.; Lin, L. L.; Chen, J.; Guo, Z. P.; Tian, H. Y.; Chen, X. S. Codelivery of Antitumor Drug and Gene by a pH-Sensitive Charge-Conversion System. Acs Appl. Mater. Interfaces 2015, 7 (5), 3207-3215.


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Scheme 1. Schematic of the ultrasensitive pH triggered size enhanced gene delivery system


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Figure 1. 1H NMR spectra of PLG and PEG-PLG in TFA.


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Figure 2. In vitro gene transfection efficiency of PEI/pGL3, GP/pGL3, PGP/pGL3 in HeLa cell (A) and CT26 cell (B), and their relative cell viability in HeLa cell (C) and CT26 cell (D).


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Figure 3. In vitro transfection efficiencies of GP2, PGP2 and PEI with pGL3 in (A) CHO, (B) ＊

MCF7 and (C) with siRNA in Huh7 cells, P ＜0.001.


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Figure 4. Particle size (A) and Zeta potentials (B) of DNA/PEI, DNA/GP2 and DNA/PGP2 at different weight ratios, DNA/PEI was 0.4, DNA/GP2 and DNA/PGP2 were 0, 0.8, 0.4, 0.2, 0.1.


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Figure 5. Cellular uptake (A), endocytosis inhibition (B) and CLSM images (C) of PEI/Cy5DNA=2.5/1, GP2/ Cy5-DNA=2.5/1 and PGP2/ Cy5-DNA=2.5/1 in CT26 cells. (Nuclei of the cells stained with DAPI (blue), lysosome labled with LysoTracker Green DND (green), Cy5DNA (red), and the merged picture, scale bar is 20µm).


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Figure 6. Protein adsorption of PEI, GP2 and PGP2 (A). 1mL of polymer solution (1mg/mL) was added to 1mL BSA solution (2mg/mL) and incubated with shaking at 37oC for 30 min prior to measurement, and (B) the transfection efficiency affected by different serum content in CT26 cells, P＊＜0.001.


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Figure 7. Tumor accumulation of PEI, GP2 and PGP2 complex with Cy5-DNA (A). Cy5 labeled complexes was injected by tail vein, and observed at 12h, 24h, 48h, 72h, 96h and 144h. The white circles showed the position of CT26 tumors. (B) and (C) were the particles of GP2 and PGP2 changed with different pH value. (D) Showed the possible mechanism that pH value caused particle size enhancing.


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Figure 8. (A) Tumor volume changes during treatment (red arrow represent the injection times, ＊ P ＜0.05). (B) The body weight changes during treatment. (C) Tumors were excised from each

group after the sacrifice of mice. (D) is the representative photographs of immunofluorescence staining of vascular endothelial cells with the CD31 antibody. The blue and green fluorescence represented the nuclei of cells and the CD31-positive vessel, respectively. Photographs were taken at an original magnification of ×200.


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Figure 9. HE stained images of heart, lung, liver, spleen, kidney and tumor collected from shVEGF, PEI/shVEGF, GP2/shVEGF and PGP2/shVEGF injected mice and control treated mice with PBS.


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Figure 10. Schematic for the forming of zwitterionic PEI/polyanion vector and their complexing with gene.


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Table of Contents (TOC)


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pH Triggered Size Increasing Gene Carrier for Efficient Tumor

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