CAGW Peptide- and PEG-Modified Gene Carrier for Selective Gene

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CAGW Peptide- and PEG-Modified Gene Carrier for Selective Gene Delivery and Promotion of Angiogenesis in HUVECs in Vivo Jing Yang,†,‡,▽ Xuefang Hao,†,‡,▽ Qian Li,†,‡ Mary Akpanyung,† Abdelilah Nejjari,† Agnaldo Luis Neve,† Xiangkui Ren,†,§ Jintang Guo,†,§ Yakai Feng,*,†,‡,§,∥ Changcan Shi,*,⊥,# and Wencheng Zhang¶ †

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

ABSTRACT: Gene therapy is a promising strategy for angiogenesis, but developing gene carriers with low cytotoxicity and high gene delivery efficiency in vivo is a key issue. In the present study, we synthesized the CAGW peptide- and poly(ethylene glycol) (PEG)modified amphiphilic copolymers. CAGW peptide serves as a targeting ligand for endothelial cells (ECs). Different amounts of CAGW peptide were effectively conjugated to the amphiphilic copolymer via heterofunctional poly(ethylene glycol). These CAGand PEG-modified copolymers could form nanoparticles (NPs) by self-assembly method and were used as gene carriers for the pEGFPZNF580 (pZNF580) plasmid. CAGW and PEG modification coordinately improved the hemocompatibility and cytocompatibility of NPs. The results of cellular uptake showed significantly enhanced internalization efficiency of pZNF580 after CAGW modification. Gene expression at mRNA and protein levels demonstrated that EC-targeted NPs possessed high gene delivery efficiency, especially the NPs with higher content of CAGW peptide (1.16 wt %). Furthermore, in vitro and in vivo vascularization assays also showed outstanding vascularization ability of human umbilical vein endothelial cells treated by the NP/pZNF580 complexes. This study demonstrates that the CAGW peptide-modified NP is a promising candidate for gene therapy in angiogenesis. KEYWORDS: peptide, angiogenesis, HUVECs, pZNF580, gene carriers, PEG, CAG and formation of vascular rings.11 On the other hand, gene therapy has attracted significant attention as an effective method for the therapy of cardiovascular diseases, especially for neovascularization.12,13 Many research groups have focused on the gene delivery method for promoting capillary growth and formation of vascular rings. Zhang et al. investigated the effect of VEGF165 gene delivered by TAT-NLS peptide on neovascularization in ischemic tissue, and obvious enhancement of vascularization was observed.14 More recently, a kind of dual combinatorial pVEGF/pBMP2 (plasmid of vascular endothelial growth factor and bone morphogenetic protein 2) geneactivated scaffold was prepared, and this scaffold demonstrated superior MSC-mediated osteogenesis in vitro and elevated

1. INTRODUCTION Currently, cardiovascular disease is one of the highest global killers that threaten human health. Artificial vascular grafts and stents have attracted increasing attention to treat this disease.1−5 Although vascular grafts combining polymeric scaffolds and cells have made significant progress, the actual effect of clinical treatment is still not satisfactory, and no true meaning of tissue system has been obtained except for some parenchyma tissues, such as skin and cartilage.6,7 Neovascularization is still a severe challenge for three-dimensional vascular grafts because it is difficult to form a functional vascular network system in a short time.8 Therefore, angiogenesis is a key factor in the regeneration of damaged vascular tissue, and the vascularization is very important for the reconstruction of blood vessels.9,10 The vascularization process is closely related to the migration and proliferation of endothelial cells (ECs), capillary growth, © XXXX American Chemical Society

Received: November 17, 2016 Accepted: January 12, 2017

A

DOI: 10.1021/acsami.6b14769 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces

Scheme 1. Preparation Process of NP/pZNF580 Complexes and Selective Gene Delivery as Well as Promotion of Neovascularization of HUVECs

vascularization by host cells in vivo.15 In the process of regulating angiogenesis, the pEGFP-ZNF580 (pZNF580) gene has also been proven to be able to promote vascularization of endothelial progenitor cells.16 In our previous studies, we investigated and verified that pZNF580 could promote the proliferation and migration ability of ECs.17−20 However, to the best of our knowledge, no research to date has focused on the effect of pZNF580 on angiogenesis. The gene carrier plays an important role in DNA protection and delivery because naked DNA is easily degraded by enzymes.21,22 Therefore, developing ideal gene carriers with low cytotoxicity and high transfection efficiency is urgent.23−27 Polyethylenimine (PEI) is widely used as a gene carrier due to its transfection efficiency being fairly higher than that of other polycations. However, evident cytotoxicity of the high molecular weight PEI cannot be avoided.28−30 For compromising between cytotoxicity and transfection effects, various biodegradable amphiphilic polymers based on PEI were prepared, such as polyethylenimine-g-poly(lactide-co-glycolide)-g-polyethylenimine (PEI-PLGA), polyethylenimine-gpoly[( L -lactide)-co-(3(S)-methyl-2,5-morpholinedione)], methoxypoly(ethylene glycol)-b-poly(3(S)-methyl-morpholine2,5-dione-co-lactide)-g-polyethylenimine, and so forth, which were used as gene carries and obtained efficient gene delivery and expression.18,31,32 For enabling gene carriers with cell-type selective function, the strategy of specific adhesion on ECs is a good choice for delivering therapeutic genes into ECs and then improving the vascularization ability of ECs by gene complexes. As described in previous studies, REDV peptide-modified gene carriers could selectively adhere on ECs and improve the transfection efficiency of ECs.33−36 However, there are some

discrepancies between reports about the effect of REDV peptide, probably due to differences in the strategies.37,38 Moreover, tripeptide Cys-Ala-Gly (CAG) is a newfound peptide with quite high selectivity for ECs over smooth muscle cells (SMCs) and specific adhesion on ECs.39,40 It was isolated from collagen type IV, which is the major component of extracellular matrix (ECM) in the basement membrane of blood vessels and could distinguish ECs from SMCs.39 The electrospun fibers of polycaprolactone (PCL) blended with CAG peptide could significantly enhance the adhesion and proliferation of ECs on them. The endothelialization degree of the vascular scaffold is 1.3-times as high as that of the control group (without CAG peptide).41 In addition, our previous study reported poly(carbonate urethane) vascular material was cooperatively modified by CAG peptide and hydrophilic poly(ethylene glycol) methacrylate. The combination of ECspecific adhesion (CAG peptide) and nonspecific rejection (PEG component) endowed the surface of vascular materials with efficient anticlotting properties.42 It is hypothesized that CAG peptide should benefit for specific transfection of ECs and enhance angiogenesis if this peptide is linked on gene carriers. However, little research has focused on CAG peptide-guided gene delivery to ECs, especially for vascular reconstruction. Herein, the CAGW peptide was conveniently conjugated to the amphiphilic PEI-PLGA polymer via a heterofunctional poly(ethylene glycol) (NHS-PEG-OPSS) to obtain the CAGW-PEG-PEI-PLGA polymer. This polymer could form CAGW-PEG-PEI-PLGA nanoparticles (NPs) in aqueous phase and complex with pZNF580 through electrostatic interaction. CAGW and PEG modification could improve the hemocompatibility and cytocompatibility of the NPs and NP/pZNF580 B


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spectrometer (Varian Cary Eclipse fluorescence spectrometer). A series of CAGW solutions with different concentrations (1.0−10 mg L−1) were prepared in PBS (pH 7.4) to obtain standard curves for CAGW quantification. The fluorescence intensities of PEI-PLGA and PEG-PEI-PLGA NPs were also detected and used as controls. The diameter and zeta potential of NPs and NP/pZNF580 complexes with different N/P molar ratios (1, 5, 10, 15, 20, and 25) were measured by a Zetasizer 3000HS (Malvern Instrument, Inc., Worcestershire, UK) using a procedure described previously.33 All of the NPs were examined for their ability to condense and load pZNF580 plasmid by agarose gel electrophoresis assay as in previous studies.35,43 2.4. Hemocompatibility of NPs. Healthy, fresh human blood was collected into vacuum tubes (EDTA-2K), and 5 mL of fresh blood was mixed with 10 mL of PBS (pH 7.4). Then, the mixture was centrifuged at 3000 rpm for 5 min. The obtained red blood cells (RBCs) were washed with PBS (pH 7.4) until the supernatant fluid was clear and transparent. Then, the red blood cells were diluted to 8 × 109 cell/mL. Twenty-five microliters of the cell suspension was gently mixed with 1 mL of NP solution (0.5 mg mL−1), and the mixture was incubated at 37 °C for 24 h. Two microliters of the mixture was dripped on a slide and then observed by an optical microscope. The residual mixture solution after centrifugation (3000 rpm, 6 min) was observed and photographed. Ultrapure water and PBS (pH 7.4) were used as positive and negative controls, respectively. 2.5. Cytocompatibility of NP/pZNF580 Complexes. The cytocompatibility of NP/pZNF580 complexes was tested by MTT assay. HUVECs were seeded into a 96-well plate (1 × 104 cell/well) and cultured in an incubator overnight until 70% confluence. After 12 h of starvation, the NP/pZNF580 complexes were added to the well as predetermined concentrations of pZNF580 (1, 3, 5, 8, and 10 μg mL−1). When the cells were incubated for 4 h, the medium was replaced to remove the complexes, and the cells were further incubated for another 24 h. Then, 20 μL of MTT solution (5 mg/mL dissolved in 0.01 M PBS) was added to each well and cultured for 4 h. The medium in each well was removed, and 150 μL of DMSO was added. After 10 min oscillation, determination of absorbance at 590 nm was monitored by an enzyme-labeled instrument, and the HUVECs without any treatment were used as control group to calculate the relative cell viability. 2.6. In Vitro Transfection of NP/pZNF580 Complexes. Transfection assays were performed using pZNF580 as a reporter gene according to previous studies.35−37 HUVECs were seeded in a 6well tissue culture plate with a density of 4 × 105 cells per well and incubated until 50−70% confluence. After starvation using serum-free medium for 12 h, NP/pZNF580 complexes at an N/P molar ratio of 15 were added to each well (1.5 μg of pZNF580 per well) and incubated for 4 h. Then, the culture medium was exchanged with complete medium, and the cells continued to be incubated. The green fluorescence protein (GFP) expressed in the cells was recorded by an inverted fluorescence microscope (Fluorescence, OLYMPUS URFLT50; microscopy, Olympus DP72) at 24 h. The transfection efficiency was given as the percentage of positive cells (with GFP) in total cells and was also determined by a flow cytometer (Beckman MoFlo XDP). PEI (25 kDa)/pZNF580 complex was used as a control group. 2.7. Real-Time Quantitative PCR. The transfection efficiency of various NP/pZNF580 complexes for HUVECs could be studied at the level of mRNA expression of the ZNF580 gene by real-time quantitative PCR analysis technology. After transfection for 24 h as described above, total RNA in cells was extracted with 1 mL of TRIzol reagent (Invitrogen). First strand cDNA was obtained by reverse transcription from 1 mg of total RNA in a 20 mL reaction system (CWBIO, CW2569) on the basis of the manufacturer’s protocol. The resulting cDNAs were used as templates for quantitative real-time PCR using SYBR Green on an ABI 7300 stepone sequence detection PCR system (Applied Biosystems). The reaction mixture was acquired according to the manufacturer’s protocol (TransStartTM Top Green qPCR SuperMix AQ 131-02). After denaturation at 95 °C for 10 min, the mixture was treated at 95 °C for 15 s, 60 °C for 1 min, and 72 °C

complexes. The CAGW peptide-functionalized carriers were used for specific delivery of pZNF580 plasmid into human umbilical vein endothelial cells (HUVECs), and their internalization efficiency, transfection efficiency, and ZNF580 mRNA expression were studied by cellular uptake assay, in vitro transfection, and real-time quantitative PCR, respectively. In addition, in vitro and in vivo vascularization of HUVECs transfected by the NP/pZNF580 complexes was also investigated to explore the application potential of these NPs with specific HUVEC target functions (Scheme 1).

2. EXPERIMENTS 2.1. Materials. Branched PEI (10 and 25 kDa) was purchased from Sigma-Aldrich (Beijing, China). Orthopyridyl disulfide-polyethylene glycol-N-hydroxysuccinimide (OPSS-PEG-NHS, Mn = 2294 Da (the Mn of PEG is 2000 Da), 99%) was obtained from JenKem (Beijing) Technology Co., Ltd. The CAGW peptide was supplied by GL Biochem (Shanghai) Ltd. Ketamine, xylazine, and pZNF580 plasmid were preserved by the Department of Physiology and Pathophysiology, Logistics University of Chinese People’s Armed Police Force. FITC-CD31 was purchased from ZhenJiang HouPu Biotech Co., Ltd. (China). Hematoxylin (H), eosin (E), 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT), and dimethyl sulfoxide (DMSO) were purchased from DingGuo ChangSheng Biotech Co., Ltd. (Beijing, China). Matrigel (Cat. no. 356234) was obtained from Corning Company. Male mice were supplied by the Department of Physiology and Pathophysiology, Logistics University of Chinese People’s Armed Police Force. Rabbit antihuman pZNF580 polyclonal antibody and goat antirabbit IgG were supplied by Abcam (HK) Ltd. (Hong Kong, China). BCA protein assay kit was purchased from Solarbio Science and Technology Co., Ltd. (Beijing, China). HUVECs were obtained from the Cell Bank of Typical Culture Collection of Chinese Academy of Sciences (Shanghai, China). 2.2. Synthesis of CAGW Peptide-Modified NPs and NP/ pZNF580 Complexes. 2.2.1. Synthesis of CAGW Peptide-Modified NPs. The PEI (10 kDa)-PLGA copolymer was synthesized as in previous studies.33,34 CAGW peptide was conjugated to PEI-PLGA copolymer through linker NHS-PEG-OPSS to obtain CAGW-PEGPEI-PLGA (CAGW-PPP) polymers. These polymers with different CAGW peptide contents were prepared by altering the molar ratios of PEI (in PEI-PLGA copolymer), PEG and CAGW (PEI/PEG/CAGW = 1:5:1 or 1:5:5). PEI-PLGA copolymer (5 mg) and NHS-PEG-OPSS were dissolved in 2 mL of DMSO and stirred at room temperature for 2 h. Then, the CAGW peptide was dissolved in PBS (pH 7.4) and slowly added to the DMSO solution under constant stirring in the dark overnight. The CAGW-PPP polymer was purified and obtained by dialysis and lyophilization. CAGW-modified NPs with core−shell−corona structure were prepared as follows: 1 mL of CAGW-PPP DMSO solution (5 mg mL−1) was slowly added dropwise to 10 mL of PBS (pH 7.4) under strong stirring. Then, the mixed solution was dialyzed against PBS (pH 7.4) to remove DMSO, and CAGW-PPP1 NP (PEI/PEG/CAGW = 1:5:1) was obtained. CAGW-PPP2 NP (PEI/PEG/CAGW = 1:5:5) with high CAG content was prepared by an analogous method. 2.2.2. Synthesis of NP/pZNF580 Complexes. The NP/pZNF580 complexes were prepared by mixing pZNF580 plasmid solution (50 μg mL−1 in PBS (pH 7.4)) and NP solutions (0.5 mg mL−1) under stirring to achieve the desired N/P molar ratios (1, 5, 10, 15, 20, and 25). All of the mixtures were incubated for 30 min at room temperature. The N/P molar ratio was calculated from N content in polymer and P content in plasmid. 2.3. Physicochemical Characterization of NPs and NP/ pZNF580 Complexes. 1H NMR spectrum was performed to characterize the resultant CAGW-PPP polymer with a Bruker Avance spectrometer (AV-400, Bruker, Karlsruche, Germany) operating at 400 MHz in DMSO-d6. For determining the content of CAGW in CAGW-PPP polymers, the fluorescence intensities of NPs at 360 nm emission wavelength were monitored by excitation at 280 nm using a fluorescence C


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ACS Applied Materials & Interfaces Scheme 2. Synthesis Route of CAGW Functionalized Copolymers of CAGW-PPP

for 20 s for 40 cycles. The sequences of forward and reverse PCR primers were 5′-AAAAAGCTTGTGGAGGCGCACGTGCTG-3′ and 5′-AAAAAGATCTTGCCCGGAGTGCGCCCGTG-3′. The expression of the housekeeping GAPDH gene was measured as an internal normalization standard, and the sequences of its forward and reverse PCR primers were 5′-AGGTGAAGGTCGGAGTCAAC-3′ and 5′CGCTCCTGGAAGATGGTGAT-3′. The data were calculated via relative quantification by using a threshold curve value (2−ΔΔCt). 2.8. Cellular Uptake of Cy5-Labeled NP/pZNF580 Complexes. HUVECs were seeded in a 6-well tissue culture plate (4 × 105 cell/well), cultured to 70−80% confluence, and starved overnight. Cy5-labeled NP/pZNF580 complexes (N/P = 15) containing 2 μg of pZNF580 were added to the culture medium. The cells were washed with PBS (pH 7.4) three times to remove residual complex suspensions, and then cells were collected after 4 h of incubation. The cells were centrifuged and resuspended with PBS several times and then suspended in 400 mL of PBS (pH 7.4). The cellular uptake efficiency of the NP/pZNF580 complexes was analyzed using a flow cytometer (Beckman MoFlo XDP). 2.9. In Vitro Tube Formation Assay. Vascularization capacity of HUVECs after transfection by NP/pZNF580 complexes was evaluated by in vitro tube formation assay. The procedure was performed according to the Corning Matrigel Matrix instructions. Briefly, a 50 μL volume of Matrigel was coated on a precooled 96-well plate and incubated at 37 °C for 1 h. Subsequently, HUVECs transfected with NP/pZNF580 complexes were seeded on the surface of solidified Matrigel at a density of 4 × 104 cells/well and then cultured in low glucose DMEM for 6 h. Then, tubular networks were observed and photographed by an optical microscope. Three parallel experiments were carried out for each sample, and the number of tubular networks was quantified by Image-Pro Plus v 6.0 software. Cells treated by PEI (25 kDa)/pZNF580 complexes and pZNF580 were used as positive and negative controls, respectively. 2.10. In Vivo Angiogenesis Assay. For further evaluating the effect of NP/pZNF580 complexes on vascularization ability of HUVECs, in vivo angiogenesis assays were performed. Male mice (6 weeks old; body weight, 20−25 g) were used as the experimental subjects. HUVECs were transfected with different NP/pZNF580 complexes. After 24 h of transfection, the cells were trypsinized and mixed with 800 μL of matrigel (final concentration of cells, 1 × 106 cells/mL). Before surgery, the mice were anesthetized with ketamine (80 mg/kg of body weight) and xylazine (2.3 mg/kg of body weight). Then, matrigel and the cell suspension were subcutaneously injected into mice using a 1 mL syringe. After 4 days, the mice were sacrificed by euthanasia, and the gelation vascular grafts were taken out, paraffinembedded, and sliced into 4 μm thick sections as previous described.44 The slides were immunohistochemically stained with hematoxylin and eosin (H&E) to evaluate inflammatory lesions and observed by optical microscope. The special CD31 antibody on the surface of HUVECs was stained as follows: the sections were baked at 65 °C for 2 h and dewaxed by

treating with dimethylbenzene I (15 min), dimethylbenzene II (15 min), absolute ethyl alcohol I (10 min), absolute ethyl alcohol II (5 min), 90% alcohol (5 min), 80% alcohol (5 min), and 70% alcohol (3 min) successively. For eliminating the endogenous peroxidase activity, the sections were treated with 3% H2O2, incubated at 25 °C for 5−10 min, washed with ultrapure water, and immersed in PBS (pH 7.4) for 5 min three times. The sections were treated with 0.01 M citrate buffer (pH 6.0) for 1 min. After being cooled to room temperature, they were treated with normal serum diluted in PBS (pH 7.4) and incubated at 25 °C for 10 min, and then the serum was discarded. FITC-CD31 (diluted in PBS (pH 7.4) at 1:100) was added dropwise onto the sections, incubated at 37 °C for 1−2 h, then washed with PBS (pH 7.4) for 5 min three times, and finally blocked. The immunofluorescence was observed by a fluorescence microscope. Animal studies were performed in the Department of Physiology and Pathophysiology, Logistics University of Chinese People’s Armed Police Force, which has an SPF fully enclosed animal feeding room and standard animal experimental facilities. All treatments for animals were kept to the animal protection guidelines of Armed Police Logistics College and conformed to the “Guide for the protection and use of experimental animals” of the American National Institutes of Health. 2.11. Statistical Analysis. All of the experiments were performed at least three times, and the data were shown as mean ± SD (standard deviation). Comparisons were achieved by using one-way ANOVA tests via SPSS 11.5 software. P values less than 0.05 were considered as statistically significant differences.

3. RESULTS AND DISCUSSION 3.1. Synthesis and Characterization of CAGW-PPP Copolymers. CAG-functionalized CAGW-PPP copolymers were prepared from PEI-PLGA copolymer, NHS-PEG-OPSS as linker, and CAGW.33,34 The synthesis route is shown in Scheme 2. The polymers with different CAGW contents were synthesized by altering the molar ratio of PEI-PLGA, NHSPEG-OPSS, and CAGW (PEI/PEG/REDV = 1:5:1 and 1:5:5). The CAGW-PPP copolymer was characterized by 1H NMR spectrum, and the peaks at 1.0−1.5 ppm (OOCCHCH3, 3H), 5.2 ppm (OOCCHCH3, 1H), and 4.8 ppm (OOCCH2, 2H) were assigned to LA and GA residues in this copolymer, respectively (Figure 1). Moreover, the characteristic peaks of d (NHCH2CH2), e (CH2CH2O), and f were assigned to PEI, PEG, and CAGW, respectively, which proved to be the successful synthesis and structure of the CAGW-PPP copolymer. Moreover, the CAGW sequence was designed to contain a tryptophan residue (W) to verify and quantify peptide conjugation by fluorescence method. The fluorescence intensity D


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Figure 1. 1H NMR spectrum of CAGW-PPP copolymer using DMSO-d6 as solvent.

Figure 3. Hydrodynamic diameters (column chart) and zeta potentials (curve chart) of NPs and NP/pZNF580 complexes at various N/P molar ratios (1, 5, 10, 15, 20, and 25) for PEI-PLGA NPs and PEIPLGA/pZNF580 complexes (A), PEG-PEI-PLGA NPs and PEG-PEIPLGA/pZNF580 complexes (B), CAGW-PPP1 NPs and CAGWPPP1/pZNF580 complexes (C), and CAGW-PPP2 NPs and CAGWPPP2/pZNF580 complexes (D).

peptide. Meanwhile, the contents of CAGW peptide in CAGWPPP1 and CAGW-PPP2 were 0.66 and 1.16 wt %, respectively (standard curve: y = 38.50×C (μg/mL) + 18.28 (R2 = 0.999)). 3.2. Zeta Potential and Hydrodynamic Diameter of NPs and NP/pZNF580 Complexes. The amphiphilic copolymers self-assembled into NPs with a core−shell−corona structure in aqueous phase, and these NPs could complex and condense pZNF580 to form NP/pZNF580 complexes through electrostatic neutralization. The size and zeta potential of NP/ pZNF580 complexes at different N/P molar ratios were measured by dynamic light scattering method (DLS). As shown in Figure 3, comparing PEI-PLGA/pZNF580 (A) and PEG-PEI-PLGA/pZNF580 (B) groups at the same N/P ratio, PEG modification led to a decrease of zeta potential, whereas no significant difference in the size of the gene complexes was observed after the conjugation of CAGW. In addition, the size of NP/pZNF580 complexes approximately reduced with the increase of N/P molar ratios, but the zeta potential increased. All four kinds of complexes showed the same trend. When the molar ratio of N/P was 1, most positive charges of the NPs were neutralized with the negative charges of the plasmid; thus, the zeta potential was near zero. As the N/P molar ratio increased from 5 to 25, the zeta potential increased due to excessive positive charges, whereas the sizes of NP/pZNF580 complexes were reduced because of the close binding effect between NPs and pZNF580 plasmid. When the N/P molar ratio was equal to or higher than 15, the size of NP/pZNF580 complexes was below 250 nm and the zeta potential was higher than 10 mV, which were beneficial for endocytosis and transfection. Thus, we used the NP/pZNF580 complexes at N/P molar ratio = 15 for the following studies. 3.3. Agarose Gel Electrophoresis Assay to Evaluate DNA Binding Ability of NPs. It is well-known that efficient DNA binding ability is essential for gene carriers. Generally, cationic polymers could load and condense negatively charged DNA via electrostatic neutralization. The binding abilities of PEI-PLGA, PEG-PEI-PLGA, CAGW-PPP1, and CAGW-PPP2 NPs with pZNF580 were investigated by agarose gel electrophoresis assay. Positively charged polymers could protect DNA after complexation and inhibit DNA migration toward the

Figure 2. (1) Fluorescence emission spectrum of the polymer solutions PEI-PLGA (A), PEG-PEI-PLGA (B), CAGW-PPP1 (C), and CAGW-PPP2 (D). (2) Standard curve of the CAGW peptide (■) and fluorescence intensity (○) of CAGW-PPP1 (C) and CAGWPPP2 (D).

at 360 nm was detected when excited at 280 nm wavelength, and the content of the CAGW peptide in these polymers was determined. As shown in Figure 2, compared with the negative control of PEI-PLGA, a strong fluorescence spectrum at 360 nm could be observed for the CAGW-PPP copolymer solutions, which demonstrated the successful conjugation of E


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Figure 4. Agarose gel retardation assay of NP/pZNF580 complexes at various N/P molar ratios (0, 1, 5, 10, 15, 20, and 25) for PEI-PLGA/ pZNF580 (A), PEG-PEI-PLGA/pZNF580 (B), CAGW-PPP1/pZNF580 (C), and CAGW-PPP2/pZNF580 complexes (D).

anode in the electric field. The results of agarose gel electrophoresis assay are shown in Figure 4. For the PEIPLGA group, the migration of pZNF580 could be fully inhibited at an N/P molar ratio of 1, whereas other groups with PEG modification could condense and load pZNF580 completely at an N/P molar ratio of 5. These results were attributed to the core−shell structure of PEI-PLGA beneficial for high condensation of pZNF580, whereas PEG modification decreases this condensation ability. After introducing PEG to the NPs, the positive charges on the surface of the NPs decreased due to the shielding effect of PEG, leading to completely inhibited plasmid migration only at the higher N/P molar ratio.45,46 Moreover, CAGW peptide conjugation showed no evident difference in the ability of binding pZNF580 plasmid by comparing (B−D) groups. Despite different NPs displaying different N/P molar ratios for sufficient complexation of pZNF580, all of these NPs could completely complex and condense plasmid at N/P molar ratios ≥5. 3.4. Hemocompatibility of NP/pZNF580 Complexes. Blood components tend to adhere onto the surface of foreign materials, especially positively charged materials, which may cause inefficient gene delivery. Therefore, high hemocompatibility is a prerequisite for NPs serving as efficient gene carriers.47 For evaluating hemocompatibility of the NPs, a hemolysis test was performed as previous described,48−50 and the results are shown in Figure 5. It could be observed that PEIPLGA NPs destroyed the largest number of RBCs compared with that of other NPs, and a large amount of hemoglobin leaked out from the RBCs (Figure 5(1, A)). This result could be explained by membrane damage induced by positive charges of PEI on the surface of PEI-PLGA NPs, leading to hemolysis.51 Microscope image (A) in Figure 5(2) shows lots of deformed RBCs and cell debris. Alternatively, PEG-PEIPLGA (B), CAGW-PPP1 (C), and CAGW-PPP2 (D) NPs showed less hemoglobin leak, and most cells retained their complete cell morphology, similar to the result of the PBS negative control. These NPs exhibited very good hemocompatibility after PEG modification. They were suitable and superior for their application in blood systems. 3.5. Cytocompatibility of NP/pZNF580 Complexes. The cytotoxicity of NP/pZNF580 complexes on HUVECs was investigated by MTT assay, and the results are shown in Figure 6. At an N/P molar ratio of 15, the concentration of NPs increased with the gradually increasing concentration (from 1 to 10 μg/mL) of the pZNF580 plasmid. The cell viability of

Figure 5. Photographs of hemolysis results of RBCs in the presence of NPs after 24 h incubation for PEI-PLGA (A), PEG-PEI-PLGA (B), CAGW-PPP1 (C), and CAGW-PPP2 (D); purified water (+) and PBS (−) were the positive and negative controls, respectively. The presence of red hemoglobin in the supernatant indicates damaged RBCs.

Figure 6. Relative cell viability of HUVECs in the presence of PEIPLGA/pZNF580 complexes (A), PEG-PEI-PLGA/pZNF580 (B), CAGW-PPP1/pZNF580 (C), and CAGW-PPP2/pZNF580 complexes (D) with various pZNF580 concentrations (1, 3, 5, 8, and 10 μg/mL) based on MTT assays. PEI(25 kDa)/pZNF580 complexes (E) were used as control. (*, statistically significant difference from the A group with P < 0.05, #, statistically significant difference from the E group with P < 0.05.).

F


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Figure 7. Fluorescence images (dark-field (left image) and corresponding bright-field (right image)) (1) and transfection efficiency (2) of HUVECs transfected for 24 h by PEI-PLGA/pZNF580 (A), PEG-PEI-PLGA/pZNF580 (B), CAGW-PPP1/pZNF580 (C), and CAGW-PPP2/pZNF580 complexes (D). PEI (25 kDa)/pZNF580 complexes (E) served as the positive control. HUASMCs (F) transfected by CAGW-PPP2/pZNF580 complexes were also used as a control group for selectivity. Scale bar is 100 μm.

pZNF580 plasmid to targeted cells was investigated by in vitro transfection of HUVECs and quantified by measuring GFP expression via a live-cell flow cytometry. As shown in Figure 7, the PEI-PLGA/pZNF580 complex (A) had high transfection efficiency of HUVECs (3.97%), but it was not suitable for in vivo gene delivery systems considering its serious cytotoxicity and low hemocompatibility. The introduction of PEG significantly improved the biocompatibility of the gene complexes, but its transfection efficiency of HUVECs was not satisfactory (2.68%, Figure 7(B)). The CAGW peptide possessed specific target function for HUVECs, and CAGWPPP/pZNF580 complexes functionalized by the CAGW peptide can effectively transfect HUVECs in comparison with that of PEG-PEI-PLGA/pZNF580 complexes. Simultaneously, the content of the CAGW peptide in the polymer demonstrated a significant impact on transfection efficiency. When the content of CAGW peptide was low (transfection efficiency = 3.78%, Figure 7(C)), no significant improvement of transfection efficiency was obtained compared with that of the

HUVECs decreased with the increase of pZNF580 concentration. Compared with the PEI (25 kDa)/pZNF580 control group (F), the other four groups showed much higher cell viabilities. The PEG-PEI-PLGA/pZNF580 complexes (B), CAGW-PPP1/pZNF580 complexes (C), and CAGW-PPP2/ pZNF580 complexes (D) could still maintain 90% of the relative cell viability even at 10 μg/mL of pZNF580 concentration, demonstrating that these complexes have less cytotoxicity to HUVECs. However, for the PEI-PLGA/ pZNF580 complexes without the PEG component (A), the relative cell viability was less than 50% when the pZNF580 concentration was higher than 8 μg/mL. These results prove that PEG modification can form a hydrophilic protective layer on the outermost surface of NPs, which is beneficial for reducing the cytotoxicity of NP/pZNF580 complexes. The NPs and gene complexes with a PEG-hydrated layer displayed excellent cytocompatibility.52 3.6. In Vitro Transfection of NPs/pZNF580 Complexes in HUVECs. The efficiency of different NPs delivering the G


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3.7. Real-Time Quantitative PCR. The HUVEC transfection efficiency of various NP/pZNF580 complexes was further verified by PCR analysis technique at the level of ZNF580 gene transcription. The measurement results of ZNF580 mRNA in HUVECs after 24 h transfection are shown in Figure 8. The relative ZNF580 mRNA contents of PEI-PLGA/pZNF580, CAGW-PPP2/pZNF580, and PEI (25 kDa)/pZNF580 complex groups were much higher than those of other groups. Moreover, the CAGW-PPP2/pZNF580 complex group showed the optimal result, much higher (∼2times) than that of the negative control group (Figure 8(F)) and slightly higher than that of the positive control group (Figure 8(E)). These results are in accordance with the transfection in vitro results, which highlights the effect of PEG and CAGW peptide modification at the level of ZNF580 mRNA expression. 3.8. Cellular Uptake of Cy5-Labeled NP/pZNF580 Complexes. The cellular uptake capacity of the Cy5-labeled NP/pZNF580 complexes in HUVECs was assessed by flow cytometry, and the results are shown in Figure 9. The values of cellular uptake were close to 100% (Figure 9(2)), which showed that almost all cells were taken up the Cy5-labeled NP/ pZNF580 complexes. However, the mean fluorescence intensity (MFI) of each group was entirely different (Figure 9(3)). The MFIs of the PEI-PLGA/pZNF580 and PEG-PEIPLGA/pZNF580 complex groups were 367 and 267, respectively, which showed that the PEG shielding layer not only effectively reduced the cytotoxicity of the NP/pZNF580 complex on HUVECs but also had a negative effect on cellular uptake. In addition, no significant difference was observed in the MFI between PEG-PEI-PLGA/pZNF580 and CAGWPPP1/pZNF580 (MFI = 281) complex groups, whereas the CAGW-PPP2/pZNF580 complex group evidently showed the highest MFI (461), which was even higher than the positive

Figure 8. Quantitative pZNF580 transfection efficiency of HUVECs transfected by PEI-PLGA/pZNF580 (A), PEG-PEI-PLGA/pZNF580 (B), CAGW-PPP1/pZNF580 (C), and CAGW-PPP2/pZNF580 complexes (D) with PEI (2.5 kDa)/pZNF580 complexes (E) as the positive control and only pZNF580 (F) as the negative control by PCR. (*, statistically significant difference from the F group with P < 0.05).

PEI-PLGA/pZNF580 complexes group (Figure 7(A)). However, the complexes with higher content of CAGW peptide (Figure 7 (D)) showed evident promotion of the expression of GFP in HUVECs (4.88%), which was a match for the transfection efficiency of the positive control group (5.30%, Figure 7(E)). In addition, for demonstrating the selectivity of CAG peptide, transfection of human umbilical artery smooth muscle cells (HUASMCs) by CAGW-PPP2/pZNF580 complexes was also attempted (Figure 7(F)). The low transfection efficiency of HUASMCs further proves that the CAGW peptide-modified gene complexes could specifically increase the transfection effect of HUVECs.

Figure 9. Cellular uptake of PEI-PLGA/pZNF580 (A), PEG-PEI-PLGA/pZNF580 (B), CAGW-PPP1/pZNF580 (C), and CAGW-PPP2/pZNF580 complexes (D) with PEI (25 kDa)/pZNF580 complexes (E) and nontreated group (F) as the positive and negative controls, respectively. (1) Intracellular fluorescence intensities, (2) percentages of cellular uptake, (3) mean fluorescence intensity were measured by flow cytometry (n = 3; *, statistically significant difference from the F group with P < 0.05). H


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Figure 10. In vitro tube formation assay evaluating the effect of NP/pZNF580 complexes on capillary tube formation of HUVECs. (1) Microscopy images of HUVECs after incubation on Matrigel for 6 h at 37 °C. Before seeding on Matrigel, the HUVECs were respectively cultured with different NP/pZNF580 complexes for 24 h. PEI-PLGA/pZNF580 (A), PEG-PEI-PLGA/pZNF580 (B), CAGW-PPP1/pZNF580 (C), and CAGW-PPP2/ pZNF580 (D) with PEI (25 kDa)/pZNF580 complexes (E) as the positive control and nontreated HUVECs (F) or only pZNF580-treated HUVECs (G) as the negative control. Scale bar = 100 μm. (2) Tube number corresponding to different groups. A black circle and an arrow were used to point the tube ring as an example shown in 1(A). (*, statistically significant difference from the F group with P < 0.05).

control group PEI (25 kDa)/pZNF580 (MFI = 415). These results indicated that the CAGW peptide was indeed able to enhance the internalization of NP/pZNF580 complexes, especially for high CAGW content. 3.9. In Vitro Tube Formation Assay. For investigating the effect of NP/pZNF580 complexes on vascularization in defective vascular tissue, the in vitro tube formation of HUVECs transfected by the gene complexes was assessed by Corning Matrigel.53 HUVECs treated with different NP/ pZNF580 complexes proliferated and migrated on matrigel without any growth factors, and finally, a vascular ring could form (Figure 10). All of the groups showed tube formation after 6 h. The HUVECs transfected by NP/pZNF580 complexes had stronger vascularization ability than that of the negative control group (Figure 10 (F)), especially the CAGWPPP2/pZNF580 complex group (Figure 10(D)). This group displayed ∼2-times the vascular ring number in each field compared with the negative control group and also more than the positive control group (Figure 10(E)). This indicated that the tube formation ability was significantly increased when HUVECs were transfected with the CAGW-PPP2/pZNF580 complex, whereas the CAGW-PPP1/pZNF580 complex group

(Figure 10 (C)) showed relatively lower tube-forming ability. It could be concluded that the content of the CAGW peptide in the complexes had a significant effect on vascularization of HUVECs. The reason is that the specific adhesion capacity for HUVECs was enhanced by the content of the CAG peptide; thus, the efficiency of cellular uptake and transfection efficiency was improved. The overexpression of pZNF580 plasmid promoted the proliferation, migration, and vascularization of HUVECs. 3.10. In Vivo Angiogenesis Assay. For investigating the revascularization ability of HUVECs in vivo after being transfected by NP/pZNF580 complexes, HUVECs treated with gene complexes were mixed with Corning Matrigel and directly transplanted into the subcutaneous tissue of mice. According to the Matrigel instructions, the scaffold formed by HUVECs and Matrigel displayed initial vascularization after 3 days in culture, and then the scaffold was sectioned and stained with H&E and anti-CD31 vessel staining after transplantation for 4 days for insurance purposes. As shown in Figure 11, revascularization was hardly observed in the negative control group (Figure 11(F)) and only a small number of individually dispersed cells could be observed, which demonstrated very I


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Figure 11. In vivo neovascularization assay evaluating the effect of NP/pZNF580 complexes on HUVEC capillary tube formation. HUVECs were first transfected by PEI-PLMD/pZNF580 (A), PEG-PEI-PLGA/pZNF580 (B), CAGW-PPP1/pZNF580 (C), or CAGW-PPP2/pZNF580 complexes (D) with PEI (25 kDa)/pZNF580 complexes (E) as the positive control and only pZNF580-treated HUVECs as the negative control (F). Then, the HUVECs were mixed with Matrigel for subcutaneous injection. After 4 days of culture, the implants were sectioned and stained with H&E (1) and immunohistochemical staining with anti-CD31 (2). Arrows indicate vessel-like structures. Scale bar is 100 μm.

heterofunctional NHS-PEG-OPSS. Tryptophan (W) in the CAGW sequence was used to quantify the content of functional CAG peptide conjugated to the copolymer. Then, NPs were prepared from these polymers by the self-assembly method.38 In the present study, the ZNF580 gene was used as the therapeutic gene for angiogenesis. It is a newly found C2H2 zinc finger gene that plays a critical regulatory role in the proliferation and migration of ECs.55,56 It is also an important mediator for H2O2-induced proinflammatory responses in ECs by augmenting the release of proinflammatory cytokine IL-8.57 Moreover, the expression of the ZNF580 gene could be upregulated in injured arterial tissue, chronic hepatitis, hypoxia, and other diseases. However, the mechanism of ZNF580 gene to affect the proliferation and migration of ECs and other potential issues deserves further investigation. The negatively charged pZNF580 gene was condensed by the prepared NPs to form gene complexes. DLS results showed that NPs and gene complexes possessed proper size and zeta potential for endocytosis. These NPs could inhibit pZNF580 migration at a relatively lower N/P molar ratio (N/P = 5) by agarose gel electrophoresis. In addition, the PEG modification could efficiently improve the hemocompatibility and cytocompatibility of NPs. The efficiency of cellular uptake showed that PEG modification could decrease cellular uptake,45 and CAGW peptide modification was beneficial for the cellular uptake. This could be explained by the shielding effect of PEG and specific

weak vascularization ability. However, HUVECs transfected with the NP/pZNF580 complexes showed obvious formation of a vascular ring, especially the CAGW-PPP2/pZNF580 (Figure 11(D)) and PEI (25 kDa)/pZNF580 complex groups (Figure 11(E)). These results indicated that pZNF580 overexpression could enhance the formation of HUVECs vascular lumen in a Matrigel scaffold. The formation of capillary tubelike structures could be observed more intuitively by immunofluorescence staining with anti-CD31 for HUVECs (Figure 11(2)), which proved that HUVECs transfected with CAGW-PPP2/pZNF580 complexes could obviously enhance the vascularization.

4. DISCUSSION It is a challenge to develop safe and efficient gene carriers for angiogenesis, and many aspects, such as biocompatibility, transfection efficiency, in vivo therapeutic effects, and so forth, should be simultaneously considered for designing gene delivery systems. In our previous studies, we designed and prepared various gene carriers based on amphiphilic block copolymers including PEI, PEG, and biodegradable hydrophobic components.18,19,33−38,54 Herein, the copolymer PEIPLGA was synthesized, modified, and used as a targeting gene carrier. For selectively delivering genes to HUVECs, the CAGW peptide was conjugated to the copolymer by J



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adhesion for HUVECs of the CAGW peptide. Moreover, the content of the CAGW peptide also influenced the efficiency of cellular uptake, and the NPs with high content of peptide showed high efficiency of cellular uptake. The in vitro transfection and PCR analysis displayed the same trend in the protein and mRNA expression. This revealed that PEG and the appropriate amount of CAGW peptide conjugation could improve biocompatibility and acquire efficient gene delivery at the same time. For investigating the effect of NP/pZNF580 complexes on vascularization, in vitro tube formation and in vivo angiogenesis assays were performed, and the results demonstrated that HUVECs transfected by NP/pZNF580 complexes could form a vascular ring in vitro and in vivo. Moreover, the HUVECs treated with high CAG complexes demonstrated high vascularization ability similar to that of the PEI (25 kDa)/pZNF580 complex group and much higher than that of the PEI-PLGA/pZNF580 complex group. The pZNF580 plasmid could be efficiently delivered into HUVECs by the CAGW peptide-functionalized CAGW-PPP2 NPs. Then, the plasmid pZNF580 was expressed into protein and enhanced the proliferation and migration ability of HUVECs as well as revascularization ability. Consequently, this strategy of CAG and PEG-modified NPs as a gene carrier has great potential for gene delivery and angiogenesis.

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5. CONCLUSIONS In summary, CAG peptide-modified nanoparticles with specific adhesion for HUVECs were synthesized and used as gene carriers for targeted delivery of the pZNF580 plasmid to HUVECs. High CAG peptide content of gene complexes greatly improved the transfection efficiency of the plasmid in HUVECs. In vitro and in vivo angiogenesis assays revealed that overexpression of pZNF580 plasmid delivered by CAG complexes could significantly enhance the ability of HUVECs for revascularization. Heterofunctional NHS-PEG-OPSS could conveniently conjugate quantitative CAG peptide to a gene carrier and formed a core−shell−corona structure for targeted delivery of genes. This simple and quick preparation strategy provides a new platform for revascularization by targeted gene therapy.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Xiangkui Ren: 0000-0001-7894-9361 Yakai Feng: 0000-0002-4511-0874 Author Contributions ▽

J.Y. and X.H. contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This project was supported by State Key Project of Research and Development (Grant 2016YFC1100300), the National Natural Science Foundation of China (Grants 31370969 and 51673145), International Science & Technology Cooperation Program of China (Grant 2013DFG52040), Wenzhou government’s startup fund (Grants WIBEZD2014005-03 and WIBEZD2015009-03), and Wenzhou Science and Technology Bureau (Grant Y20150088). K


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CAGW Peptide- and PEG-Modified Gene Carrier for Selective Gene

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