Shell Gene Carriers with Different Lengths of PLGA Chains to

Article Cite This: Langmuir 2017, 33, 13315-13325

pubs.acs.org/Langmuir

Core/Shell Gene Carriers with Different Lengths of PLGA Chains to Transfect Endothelial Cells Xinghong Duo,†,‡,§ Qian Li,†,§ Jun Wang,†,§ Juan Lv,†,§ Xuefang Hao,†,§ Yakai Feng,*,†,§,∥,⊥ Xiangkui Ren,*,† Changcan Shi,*,#,∇ and Wencheng Zhang^ †

School of Chemical Engineering and Technology, Tianjin University, Yaguan Road 135, Tianjin 300350, China School of Chemistry and Chemical Engineering, Qinghai University for Nationalities, Bayi middle Road 3, Xining, Qinghai 810007, China § Collaborative Innovation Center of Chemical Science and Chemical Engineering (Tianjin), Weijin Road 92, Tianjin 300072, China ∥ Joint Laboratory for Biomaterials and Regenerative Medicine, Tianjin University-Helmholtz-Zentrum Geesthacht, Yaguan Road 135, Tianjin 300350, China ⊥ Key Laboratory of Systems Bioengineering of Ministry of Education, Tianjin University, Yaguan Road 135, Tianjin 300350, China # Wenzhou Institute of Biomaterials and Engineering, CNITECH, CAS, Wenzhou, Zhejiang 325011, China ∇ Institute of Biomaterials and Engineering, Wenzhou Medical University, Wenzhou, Zhejiang 325011, China ^ Department of Physiology and Pathophysiology, Logistics University of Chinese People’s Armed Police Force, Tianjin 300162, China

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‡

ABSTRACT: In order to improve the transfection efficiency and reduce the cytotoxicity of gene carriers, many strategies have been used to develop novel gene carriers. In this study, five complex micelles (MSP(2 k), MSP(4 k), MSP(6 k), MSP(8 k), and MSP(10 k)) were prepared from methoxy-poly(ethylene glycol)-bpoly(D,L-lactide-co-glycolide) (mPEG-b-PLGA) and sorbitol-poly(D,L-lactide-coglycolide)-graf t-PEI (sorbitol-PLGA-g-PEI, where the designed molecular weights of PLGA chains were 2 kDa, 4 kDa, 6 kDa, 8 kDa, and 10 kDa, respectively) copolymers by a self-assembly method, and the mass ratio of mPEG-b-PLGA to sorbitol-PLGA-g-PEI was 1/3. These complex micelles and their gene complexes had appropriate sizes and zeta potentials, and pEGFP-ZNF580 (pDNA) could be efficiently internalized into EA.hy926 cells by their gene complexes (MSP(2 k)/ pDNA, MSP(4 k)/pDNA, MSP(6 k)/pDNA, MSP(8 k)/pDNA, and MSP(10 k)/ pDNA). The MTT assay results demonstrated that the gene complexes had low cytotoxicity in vitro. When the hydrophobic PLGA chain increased above 6 kDa, the gene complexes showed higher performance than that prepared from short hydrophobic chains. Moreover, the relative ZNF580 protein expression levels in MSP(6 k)/pDNA, MSP(8 k)/pDNA, and MSP(10 k)/pDNA) groups were 79.6%, 71.2%, and 73%, respectively. These gene complexes could promote the transfection of endothelial cells, while providing important information and insight for the design of new and effective gene carriers to promote the proliferation and migration of endothelial cells.

1. INTRODUCTION

improved safety profile and ease of preparation and manipulation, nonviral gene carriers have been extensively used in gene delivery systems. Cationic polymers have a pivotal status in nonviral gene carriers, which have received a lot of attention. Among cationic polymers, polyethylenimine (PEI) is well-known for its high transfection efficiency. It could condense negatively charged DNAs through electrostatic interaction to form complexes and shows a unique proton-sponge effect within a wide pH range. Due to its flexible structure, PEI can condense DNA and

In the past few years, vascular disease has been one of the most serious and deadly diseases in the world.1 Gene therapy offers a promising way to successfully treat vascular disease, which is the primary objective of science and research.1−3 Gene therapy involves the transfer of gene fragments into target cells by means of gene carriers. Accordingly, developing safe and effective gene carrier systems is a key issue in gene therapy. Gene delivery carriers have been classified into two major categories, namely viral vectors and nonviral carriers.4−7 A major obstacle associated with viral vectors is the immune response of the body to them. During the past few years, the substantial development in nonviral carriers has proven that these gene carriers are reasonably reliable.6,8 As a result of the © 2017 American Chemical Society

Received: August 18, 2017 Revised: October 27, 2017 Published: November 3, 2017 13315

DOI: 10.1021/acs.langmuir.7b02934 Langmuir 2017, 33, 13315−13325

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Scheme 1. Self-Assembly of Amphiphilic Copolymers and the Condensation with pDNA and the Delivery of pDNA to EA.hy926 Cells by Gene Complexes

adequately transfer it to avoid endosomal disruption.9−11 On the other hand, its transfection efficiency increases with the increase of molecular weight; unfortunately its toxicity also increases.12,13 In order to improve the transfection efficiency and reduce cytotoxicity, many modification strategies have been developed, such as PEG,14,15 nanoparticle,16,17 and zwitterion polymer modification.18 Hydrophobic chain modification of PEI is also an effective method,19 particularly the hydrophobic segment can improve the adsorption mediated endocytosis.20,21 Poly(ε-caprolactone) (PCL),22 poly(lactic acid) (PLA),23 poly(γ-benzyl L-glutamate) (PBLG),24 etc. can act as hydrophobic core directly to enhance the delivery capability of gene carriers.25 Owing to its biocompatible and biodegradable properties, poly(D,L-lactide-co-glycolide) (PLGA) has been widely used as biomaterials.26 Jeong et al. prepared dextranPLGA copolymers, which could form a core−shell structure with a narrow size distribution.27 In our previous studies, we used mPEG and PLGA linear diblock copolymers to modify PEI (1.8 and 10 kDa) and prepared micelles in an aqueous environment. These micelles were proven to be safe and effective when used as gene carriers to transfect EA.hy926 cells.28−36 Hydrophobic PLGA segments formed the inner core to increase the biodegradability of the polymeric micelles and PEG was beneficial for prolonging the circulation time in vivo.37−39 In addition, polymeric micelles had a specific ability to escape capture by the reticuloendothelial system (RES), and the hydrophobic core could enhance cell interactions and tissue permeability.40 Moreover, star-shaped polycations have low cytotoxicity and high transfection efficiency compared with linear polycations. For example, a star-shaped polymeric micelle

which was prepared from eight-armed polymers exhibited much low cytotoxicity and substantially high transfection efficiency.41 Sorbitol is an organic osmolyte that occurs widely in plants, especially in the Rosaceae family. It is produced commercially by reduction of D-glucose or D-glucono-1,4-lactone and is extensively used in the food industry because of its complete water solubility and lack of any discernible toxicity.42 Higashi et al. investigated lipidated sorbitol-based molecular transporters for gene carriers.43 Kaustabh et al. showed that sorbitol-based transporters have high intracellular selectivity toward mitochondria.44 Usually PEI molecular weight affects gene transfection efficiency and cytotoxicity.29−31,33 In our previous work, the complex micelles based on PEI and biodegradable copolymers could act as suitable gene carriers with acceptable gene transfection efficiency and cytotoxicity.35,39 We synthesized a cationic copolymer of poly(lactide-co-3(S)-methyl-morpholine2,5-dione)-graf t-PEI (PLMD-g-PEI) and a diblock copolymer of methoxy-poly(ethylene glycol)-block-poly(lactide-co-3(S)methyl-morpholine-2,5-dione) (mPEG-b-PLMD). They formed a series of microparticles with a biodegradable poly(lactide-co-3(S)-methyl-morpholine-2,5-dione) (PLMD) core and a mixed shell, which consisted of different ratios of mPEG/PEI in mass and were used to prepare complex micelles as gene carriers for the purpose of reducing the cytotoxicity of PEI and improving the proliferation of EA.hy926 cells in vitro.39 The complex micelles had low cytotoxicity and could effectively enhance the proliferation and migration of endothelial cells (ECs).35,39 In present study, the graft copolymers sorbitol-poly(DLlactide-co-glycolide)-graf t-PEI (sorbitol-PLGA-g-PEI) (the mo13316


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Langmuir Scheme 2. Synthesis Route of mPEG-b-PLGA and Sorbitol-PLGA-g-PEI Polymers

lecular weight of each PLGA chain was designed as 2, 4, 6, 8, and 10 kDa, while the hydrophilic PEI block was 10 kDa) and the liner diblock copolymer mPEG-block-poly(D,L-lactide-coglycolide) (mPEG-b-PLGA) were self-assembled to form complex micelles mPEG-b-PLGA/sorbitol-PLGA-g-PEI (MSP). The complex micelles self-assembly process, the formation of MSP/pEGFP-ZNF580 (MSP/pDNA) gene complexes, and their delivery of pDNA into EA.hy926 cells are shown in Scheme 1. These complex micelles have a hydrophobic biodegradable PLGA core and a hydrophilic mPEG/PEI mixed shell. They were used to deliver pEGFPZNF580 plasmid (pDNA)45 into EA.hy926 cells by endocytosis methods, and then endosomal escape by protonation effect. We investigated the effect of different lengths of PLGA chains, using a constant weight ratio of mPEG-b-PLGA to sorbitolPLGA-g-PEI of 1/3 on cell proliferation and migration in vitro. DNA condensation ability and cytotoxicity of these complex micelles were characterized by agarose gel electrophoresis and MTT. The transfect efficiency of MSP/pDNA gene complexes

was evaluated by transfect assay, wound healing assay, and Western blot analysis in vitro.

2. EXPERIMENTAL SECTION 2.1. Materials. Polyethylenimine (branched PEI, Mw = 10 kDa), sorbitol, methoxy-poly(ethylene glycol) (mPEG) (Mw = 5 kDa, PDI = 1.05), and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma-Aldrich (St. Louis, MO, U.S.A.). Other chemicals and biological products were supplied by the companies referred in our previous study.35 2.2. Preparation of mPEG-b-PLGA−OH Diblock Copolymer. The diblock copolymer was synthesized by a previously reported method.35 2.3. Preparation of Sorbitol-PLGA-g-PEI Graft Copolymers. Sorbitol-PLGA(2 kDa)-OH, sorbitol-PLGA(4 kDa)-OH, sorbitolPLGA(6 kDa)-OH, sorbitol-PLGA(8 kDa)-OH, and sorbitol-PLGA(10 kDa)-OH star-shaped copolymers were prepared by ROP of D,Llactide (DL-LA) and glycolide (GA) using sorbitol as a macroinitiator. The synthesis method was the same as above. The molecular weights of each PLGA chain of these copolymers were designed as 2, 4, 6, 8, and 10 kDa, respectively. Then the sorbitol-PLGA-COOH and 13317


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Figure 1. 1H NMR spectra of diblock copolymer, star-shaped copolymers, and graft copolymer in CDCl3. (a) mPEG-b-PLGA-OH, (b) sorbitolPLGA-OH, (c) sorbitol-PLGA-COOH, and (d) sorbitol-PLGA-g-PEI. 2.5.2. Preparation of Gene Complexes. pEGFP-ZNF580 plasmid (pDNA, 400 μg/mL) was diluted to 50 μg/mL with PBS (pH 7.4). The pDNA was separately added into MSP(2 k), MSP(4 k), MSP(6 k), MSP(8 k), and MSP(10 k) complex micelles at various of N/P molar ratios (N/P = 0.2, 0.5, 1, 2, 5, and 10). The N/P molar ratios were calculated from the N content in PEI and the P content in pDNA. 2.5.3. Size Distribution and Zeta Potential of Complex Micelles and Gene Complexes. The size and zeta potential of MSP(2 k), MSP(4 k) MSP(6 k), MSP(8 k) and MSP(10 k) complex micelles and their gene complexes were measured using the Zetasizer 3000 HS (Malvern Instrument, Inc., Worcestershire, U.K.) at the wavelength of 677 nm with a constant angle of 90°. 2.6. Agarose Gel Electrophoresis. Agarose gel electrophoresis is used to evaluate the pDNA condensation ability of the MSP(2 k), MSP(4 k), MSP(6 k), MSP(8 k), and MSP(10 k) complex micelles.29,30 The specific experiments were performed according to a previous reference.39 2.7. In Vitro Cytotoxicity of Complex Micelles and Gene Complexes. 2.7.1. Cell Culture. EA.hy926 cells were cultured in high glucose DMEM, supplemented 10% FBS, in a 5% CO2 atmosphere at 37 °C. After 24 h, the adherent cells were cultured to 80−90% confluence. 2.7.2. Cytotoxicity. The cytotoxicity of the MSP(2 k), MSP(4 k), MSP(6 k), MSP(8 k), and MSP(10 k) complex micelles and their gene

sorbitol-PLGA-g-PEI polymers were synthesized according to the previous studies.35 2.4. Characterization of Copolymers. 1H NMR spectra of the synthesized copolymers were recorded with a Bruker Avance spectrometer (AV-400, Bruker, Karlsruhe, Germany) operating at 400 MHz in deuterated chloroform (CDCl 3 ) solvent and tetramethylsilane (TMS) as the internal standard. The numberaverage molecular weight (Mn), weight-average molecular weight (Mw), and PDI were determined by gel permeation chromatography (GPC, Malvern Viscotek, U.K.) in THF. 2.5. Preparation and Characterization of Complex Micelles. 2.5.1. Preparation of Complex Micelles. As an example, mPEG-bPLGA-OH and sorbitol-PLGA(6 kDa)-g-PEI(10 kDa) copolymers were dissolved in DMSO to obtain two copolymer solutions with the concentration of 5 mg/mL, respectively. Subsequently, the copolymer solutions were mixed with the weight ratio of mPEG-b-PLGA to sorbitol-PLGA-g-PEI of 1/3, and then 2.0 mL of this mixed copolymer solution was added dropwise to 20 mL of phosphate buffer saline (PBS, pH 7.4) in a conical flask with stirring case. The mPEG-bPLGA/sorbitol-PLGA(6 kDa)-g-PEI(10 kDa) complex micelles (MSP(6 k)) were dialyzed against PBS for 12 h (MWCO = 14 kDa) at a low speed (500 rpm), and PBS was exchanged every 1 h to remove DMSO. Similarly, MSP(2 k), MSP(4 k), MSP(8 k), and MSP(10 k) complex micelles were prepared by an analogous method. 13318


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Langmuir complexes were evaluated by the MTT assay using PEI (10 kDa) as a control.28,33 2.8. In Vitro Transfection. EA.hy926 cells was transfected with the MSP(2 k)/pDNA, MSP(4 k)/pDNA, MSP(6 k)/pDNA, MSP(8 k)/pDNA, and MSP(10 k)/pDNA gene complexes (N/P = 5) by a similar method in previous studies.33,36 Green fluorescence protein (GFP) was recorded by an inverted fluorescent microscope. 2.9. Wound Healing Assay. We assessed the migration capability of EA.hy926 cells transfected by gene complexes using a scratch wound healing assay28,36 following a protocol previously reported.36,46 EA.hy926 cells were transfected by MSP(2 k)/pDNA, MSP(4 k)/ pDNA, MSP(6 k)/pDNA, MSP(8 k)/pDNA, and MSP(10 k)/pDNA gene complexes at the N/P molar ratio = 5. The migration process at different time points (0, 6, and 12 h) was monitored and the migration area was calculated by ImageJ 2.0 as previous study.47 2.10. Western Blot Analysis. Western blot analysis was used to measure the expression of ZNF580 gene at the protein level in EA.hy926 cells as described in a previous study.28 EA.hy926 cells was transfected with different gene complexes, and the β-actin antibody was used as the control. The band was analyzed via ImageJ 2.0. 2.11. Statistical Analysis. All experiments were performed at least three times. Quantitative data are presented as the mean ± SD. Statistical comparisons were made with the Student’s t test. p values of 80%. Therefore, we used this concentration for the following studies. 3.5. In Vitro Transfection. As shown in Scheme 1, when the MSP/pDNA gene complexes were cultured with EA.hy926 cells, they entered into cells via endocytosis. After MSP/pDNA gene complexes escaped from endosomes, pDNA was carried into the nucleus, translated, and expressed. The migration and proliferation of transfected cells were promoted accompanied by the expression of pDNA. The transfection of EA.hy926 cells by gene complexes was evaluated at the N/P molar ratio for 5. The PEI concentration of gene complexes was 20 μg/mL. pEGFP-ZNF580 plasmid was used as the positive control. After 24 h, a substantial amount of green fluorescence could be observed under an inverted fluorescence microscope (Figure 6(1)), which indicated that the pEGFP-ZNF580 plasmid loaded by MSP(2

Figure 3. Zeta potential of complex micelles and gene complexes at different N/P ratios. (A) MSP(2 k) complex micelles and MSP(2 k)/ pDNA gene complexes; (B) MSP(4 k) complex micelles and MSP(4 k)/pDNA gene complexes; (C) MSP(6 k) complex micelles and MSP(6 k)/pDNA gene complexes; (D) MSP(8 k) complex micelles and MSP(8 k)/pDNA gene complex; (E) MSP(10 k) complex micelles and MSP(10 k)/pDNA gene complexes.

condense anionic pDNA, the complex micelles should contain enough cationic PEI chains. Here, we prepared complex micelles with mPEG-b-PLGA/sorbitol-PLGA-g-PEI weight ratio of 1/3. These complex micelles possessed a positive charge because of the cationic PEI in the shell. The zeta potentials of their gene complexes gradually increased along with increases of the N/P molar ratio. The length of the hydrophobic chain did not affect the zeta potential. 3.3. Agarose Gel Electrophoresis. An important prerequisite for gene carrier employing polycations is the condensation ability of polyanionic DNA into small particles.

Figure 4. Agarose gel electrophoresis of pDNA and gene complexes at different N/P molar ratios (0.2, 0.5, 1, 2, 5 and 10). (a) MSP(2 k)/pDNA gene complexes, (b) MSP(4 k)/pDNA gene complexes, (c) MSP(6 k)/pDNA gene complexes, (d) MSP(8 k)/pDNA gene complexes, and (e) MSP(10 k)/pDNA gene complexes. 13320


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k), MSP(4 k), MSP(6 k), MSP(8 k), and MSP(10 k) complex micelles had been transfected and successfully expressed in cells. When the length of the hydrophobic chain increased, the green fluorescence spots increased. Particularly, MSP(6 k)/ pDNA, MSP(8 K)/pDNA, and MSP(10 k)/pDNA gene complexes showed much higher transfection efficiency (Figure 6(2)). PLGA chains with these chain lengths could form tightly hydrophobic core and exhibited a delicate balance between the hydrophilic and hydrophobic chains. This effect benefits for efficiently gene delivery and cell transfection. 3.6. Wound Healing Assay. Summary Investigation of ECs migration is important to understand the pathophysiology of illnesses.63 The wound healing assay has been used to study migration properties of cell lines. In this assay, pDNA was used as the control group. EA.hy926 cells were transfected with MSP(2 k)/pDNA, MSP(4 k)/pDNA, MSP(6 k)/pDNA, MSP(8 k)/pDNA, and MSP(10 k)/pDNA gene complexes at the N/P molar ratio of 5. After 48 h, a monolayer of EA.hy926 cells was formed in a 6-well plate. An artificial scratch with parallel borders was mechanically created as shown in Figure 7(1) at 0 h. Cells treated with pDNA migrated much slower

Figure 5. Relative cell viability of EA.hy926 cells after 48 h of treatment with different PEI concentrations of complex micelles and gene complexes at the N/P molar ratio of 5. Cells treated with PEI(10 kDa) served as the control group. (1) Complex micelles groups and (2) gene complexes groups.

Figure 7. Migration process of EA.hy926 cells at different time points and migration area after 12 h. (1) Migration process of EA.hy926 cells at 0, 6, and 12 h; (2) migration area (%) after 12 h calculated by ImageJ 2.0. (A) Cells treated with pDNA served as the negative control group; (B) cells treated with MSP (2 k)/pDNA gene complexes; (C) cells treated with MSP (4 k)/pDNA gene complexes; (D) cells treated with MSP(6 k)/pDNA gene complexes; (E) cells treated with MSP (8 k)/pDNA gene complexes; (F) cells treated with MSP(10 k)/pDNA gene complexes. (Mean ± SD, n = 3, *p < 0.05 vs control group.)

than the transfected cells in the gene complexes groups. Compared with the control group, cells treated with gene complexes showed higher migration rate. The migration area (recover area) was quantitatively calculated by ImageJ 2.0, and the results are shown in Figure 7(2). The MSP(6 k)/pDNA, MSP(8 k)/pDNA, and MSP(10 k)/pDNA gene complexes groups were relative high in migration area with the values of 77.1%, 70.1%, and 73.6%, respectively, implying that the migration ability of transfected cells was relatively high. Both transfection and wound healing assay have demonstrated that the gene complexes obviously enhanced the migration of

Figure 6. (1) Fluorescence images of EA.hy926 cells transfected by gene complexes with the N/P molar ratio of 5 with concentration of 40 μg/mL at time intervals of 12 and 24 h, and pEGFP-ZNF580 plasmid as a control; (2) transfection efficiency at 24 h. (A) Cells treated with pDNA served as the positive control; (B) cells treated with MSP(2 k)/pDNA gene complexes; (C) cells treated with MSP(4 k)/pDNA gene complexes; (D) cells treated with MSP(6 k)/pDNA gene complexes; (E) cells treated with MSP(8 k)/pDNA gene complexes; (F) cells treated with MSP(10 k)/pDNA gene complexes. (Mean ± SD, n = 3, *p < 0.05 vs control group.)

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modification of the liposoma surface. The introduction of a PEG segment onto the surface of gene carriers can increase the high storage stability and long-circulating ability,65 because PEG shields the liposomes against destructive mechanisms in body.66,67 In our previous work, we have developed the micelles with a biodegradable hydrophobic core (PLGA, PLMD, etc.), a PEI shell, and a PEG corona (or a mixed shell of PEI and PEG) as gene carriers.28,29,35,36,68−70 Owing to the long-circulating characteristic of PEG, these gene carriers are beneficial to cell transfection.35,36,69 Besides linear polymer carriers, star-shaped multiarm polymers have been developed and used as gene carriers.41,69,71 In present study, PEI was grafted onto the star-shape copolymer sorbitol-PLGA-COOH with different lengths of PLGA hydrophobic chains (2, 4, 6, 8, and 10 kDa) to obtain sorbitol-PLGA-g-PEI. Then, the diblock copolymer of mPEGPLGA was mixed with sorbitol-PLGA-g-PEI grafting copolymer to form complex micelles by self-assembly at a weight ratio of 1:3. These complex micelles consisted of a biodegradable PLGA core and a PEG/PEI mixed shell (Scheme 2). During the self-assembly process, the hydrophobic chains can aggregate to form the core. This hydrophobic PLGA core enables them with the biodegradability, and it could also enhance the stabilization of micelles.35,37,38,72,73 In the PEG/PEI mixed shell, the hydrophilic PEG stretched preferentially at the outmost of the micelles, thus shielding the positive charge of PEI to decrease the cytotoxicity of the complex micelles. Meanwhile, PEG benefited for stability and long-circulation time of gene carriers. From the results shown in Figure 2, the size of complex micelles increased from 162.32 ± 7.36 to 185.33 ± 3.36 nm when the length of the PLGA chain increased from 2 to 10 kDa. However, the size of their gene complexes did not show this tendency. These complex micelles had strong pDNA compression ability; they could inhibit pZNF580 migration at low N/P molar ratio (N/P = 1 or 2; Figure 4). These gene complexes possessed proper zeta potential for cell uptake. Hydrophilic PEG can form a hydrated layer on the biomaterial surface, effectively inhibiting the adsorption of proteins and platelets. Besides, PEG can also improve the biocompatibility and hemocompatibility of biomaterial35,74 The shielding effect of PEG on the surface of gene complexes could decrease the cytotoxicity of PEI.35,75 At a PEI concentration of 60 μg/mL, cells treated with these gene complexes exhibited high relative cell viability (>60.2%), while only 20.8% in PEI/pDNA control group (Figure 5 (2)). These gene complexes could deliver gene into cells effectively and enhance cell proliferation and migration. Interestingly, the gene complexes with longer PLGA chain length, such as MSP(6 k)/ pDNA, MSP(8 k)/pDNA, and MSP(10 k)/pDNA, exhibited a relatively higher transfection efficiency than those with shorter chain length (MSP(2 k)/pDNA and MSP(4 k)/pDNA). In the wound healing assay, the migration area of these longer chain gene complexes was also relatively high (Figure 7). Compared with the control group, the gene complexes with longer PLGA chain length (MSP(6 k)/pDNA, MSP(8 k)/pDNA, and MSP(10 k)/pDNA) could promote cell proliferation and migration effectively and also induce high ZNF580 protein expression (Figure 8). The in vitro transfection, wound healing, and Western blot assay results proved that amphiphilic copolymers with properly long hydrophobic chain benefited for high transfection efficiency and migration of ECs.

EA.hy926 cells when the length of PLGA chains increased from 2 to 10 kDa. 3.7. Western Blot Analysis. Western blot analysis was used to quantitatively evaluate the expression of ZNF580 protein in EA.hy926 cells. The EA.hy926 cells were transfected by the pEGFP-ZNF580 plasmid and MSP(2 k)/pDNA, MSP(4 k)/pDNA, MSP(6 k)/pDNA, MSP(8 k)/pDNA, and MSP(10 k)/pDNA gene complexes. The ZNF580 relative protein level (%) was evidently elevated by the expression of pEGFPZNF580 in the transfected cells compared with the control group (p < 0.05). All of these gene complexes obviously promoted the ZNF580 protein expression in EA.hy926 cells, as shown in Figure 8. The cells treated with MSP(6 k)/pDNA,

Figure 8. Western blot analysis of pEGFP−ZNF580 protein expression in EA.hy926 cells transfected with the gene complexes after 48 h. (A) pDNA as the control group; (B) cells transfected with MSP(2 k)/pDNA gene complexes; (C) cells transfected with MSP(4 k)/pDNA gene complexes; (D) cells transfected with MSP(6 k)/ pDNA gene complexes; (E) cells transfected with MSP (8 k)/pDNA gene complexes; (F) cells transfected with MSP (10 k)/pDNA gene complexes. (Mean ± SD, n = 3, *p < 0.05 vs control group.)

MSP(8 k)/pDNA, and MSP(10 k)/pDNA gene complexes showed high protein level with values of 79.6%, 71.2%, and 73%, respectively. These results are consistent with the transfection results, indicating that the suitable chain length of PLGA can benefit for ZNF580 gene release and expression.

4. DISCUSSION The bioactive EC layer on the surface of artificial vascular graft can simulate healthy vascular intimal and enhance its long-term patency. If this layer is deficient, the patency rate is gradually reduced and eventually leads to vascular blockage.64 The transfected ECs with high proliferation and migration benefits for forming this layer quickly, thus preventing thrombosis and restenosis effectively.28,32 However, ECs are considered as one of the most difficult transfected cell types. It has been demonstrated that nano- and microparticles with core−shell structures have an advantage for gene delivery. So many studies have designed and developed this kind of gene delivery system. The development of nontoxic and high transfection efficient carriers is of major importance for gene delivery. The selfassembled micelles of amphiphilic block copolymers have been demonstrated to have unique characteristics such as nanoscale, core−shell structure, low critical association concentration, and good thermodynamic stability.29,30,49 So they could be used as potential drug and gene carriers.49 Besides, the prolongation of liposome circulation in blood was achieved by PEG 13322


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5. CONCLUSION The hydrophobicity of gene carriers affects their delivery efficiency. We prepared a series of amphiphilic grafting copolymers sorbitol-PLGA-g-PEI with different lengths of hydrophobic blocks (2, 4, 6, 8, and 10 kDa). The complex micelles were formed via self-assembly with diblock copolymer and the grafting copolymer at the weight ratio of mPEG-bPLGA/sorbitol-PLGA-g-PEI = 1/3. With the increase of hydrophobic chain length, the size increased from 162.32 ± 7.36 to 185.33 ± 3.36 nm. Their gene complexes could effectively deliver the pEGFP-ZNF580 gene into EA.hy926 cells. They exhibited lower cytotoxicity than the PEI/pDNA control group. When the chain length of the hydrophobic PLGA increased, MSP/pDNA gene complexes could significantly transfect EA.hy926 cells facilitating their proliferation and migration. The ZNF580 protein was highly expressed by these gene complexes. The appropriate hydrophobic chain length is beneficial to cell transfection, proliferation, and migration.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Yakai Feng: 0000-0002-4511-0874 Xiangkui Ren: 0000-0001-7894-9361 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This project was supported by National Key R&D Program of China (Grant No. 2016YFC1100300), National Natural Science Foundation of China (Grant Nos. 31370969 and 51673145), International Science & Technology Cooperation Program of China (Grant No. 2013DFG52040), Tianjin University−Qinghai University for Nationalities of independent innovation fund cooperation project, and the Ministry of Education “Chun Hui plan” cooperation project (No. Z2015049).

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Shell Gene Carriers with Different Lengths of PLGA Chains to

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