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Multifunctional gene carriers with enhanced specific penetration and nucleus accumulation to promote neovascularization of HUVECs in vivo Xuefang Hao, Qian Li, Jintang Guo, Xiang-Kui Ren, Yakai Feng, Changcan Shi, and Wencheng Zhang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b11615 • Publication Date (Web): 26 Sep 2017 Downloaded from http://pubs.acs.org on September 28, 2017

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ACS Applied Materials & Interfaces

Multifunctional gene carriers with enhanced specific penetration and nucleus accumulation to promote neovascularization of HUVECs in vivo Xuefang Hao1, Qian Li1, Jintang Guo1,2, Xiangkui Ren1,2, Yakai Feng*1,2,3, Changcan Shi*4,5, Wencheng Zhang*6 Corresponding Author: Y. Feng, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300350, China Email:

[email protected]

(Y.

Feng),

[email protected]

(C.

Shi),

[email protected] (W. Zhang) 1 School of Chemical Engineering and Technology, Tianjin University, Yaguan Road 135, Tianjin 300350, China 2 Collaborative Innovation Center of Chemical Science and Chemical Engineering (Tianjin), Tianjin 300350, China 3 Key Laboratory of Systems Bioengineering (Ministry of Education), Tianjin University, Tianjin 300072, China 4 School of Ophthalmology & Optometry, Eye Hospital, School of Biomedical Engineering, Wenzhou Medical University, Wenzhou, Zhejiang 325011, China 5 Wenzhou Institute of Biomaterials and Engineering, CNITECH, CAS, Wenzhou, Zhejiang 325011, China 6 Department of Physiology and Pathophysiology, Logistics University of Chinese People’s Armed Police Force, Tianjin 300309, China

ACS Paragon Plus Environment


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Abstract Recently, gene therapy has attracted much attention especially for the treatment of vascular disease. However, it’s still challenging to develop the gene carriers with high biocompatibility as well as highly efficient gene delivery to overcome multiple barriers. Herein, a frequently-used cell-penetrating peptide PKKKRKV (TAT) was selected as a functional sequence of the gene carrier with distinctive cell-penetrating ability. REDV peptide with selectively targeting function for endothelial cells (ECs) and nuclear localization signals (NLS) were integrated with this TAT peptide to obtain a highly efficient gene delivery system with ECs specificity and nucleus accumulation capacity. Besides, the glycine sequences with different repeat numbers were inserted into the above integrated peptide. These glycine sequences acted as a flexible spacer arm to exert the targeting, cell-penetrating and nucleus accumulation functions of each functional peptides. Three tandem peptides REDV-Gm-TAT-Gm-NLS (m = 0, 1 and 4) complexed with pZNF580 plasmid to form gene complexes. The results of hemocompatibility and cytocompatibility indicated that these peptides and gene complexes were non-toxic and biocompatible. The internalization efficiency and mechanism of these gene complexes were investigated. The internalization efficiency was improved as the introduction of targeting REDV and glycine sequence, and the REDV-G4-TAT-G4-NLS/pZNF580 (TP-G4/pZNF580) complexes showed the highest cellular uptake among the gene complexes. The TP-G4/pZNF580 complexes also presented significantly higher internalization efficiency (about 1.36 times) in human


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umbilical vein endothelial cells (HUVECs) than human umbilical artery smooth muscle cells. TP-G4/pZNF580 complexes substantially promoted the expression of pZNF580 by confocal live cell imaging, gene delivery efficiency and HUVECs migration assay. The in vitro and in vivo revascularization ability of transfected HUVECs was further enhanced obviously. In conclusion, these multifunctional REDV-Gm-TAT-Gm-NLS peptides offer a promising and efficacious delivery option for neovascularization to treat vascular diseases.

Keywords: Gene delivery, Nuclear localization signals, Cell-penetrating peptide, REDV peptide, HUVECs

1 Introduction Nowadays, tissue engineering has obtained quite great progress via biomaterial modification and transplantation of cultured cells etc.1-5 However, for the peripheral vascular diseases and critical limb ischemia, it is still limited by its inadequate ability of vascularizing tissues in vitro or in vivo. So, angiogenesis is still a severe challenge for the therapy of vascular diseases, especially for the regeneration of damaged vascular tissue and reconstruction of blood vessels.6 Studies have proved that the proliferation, migration and differentiation of endothelial cells (ECs) are key steps for angiogenesis.7 Besides, the formation of vascular rings is a very critical point for angiogenesis. Now gene therapy has attracted much attention for neovascularization and has been considered as an extra tool for the treatment of critical limb ischemia with a tailored



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therapeutic method.8,9 Efficient remedial gene and gene carriers with high safety and efficiency attract researchers’ much attention.10-13 Recently, Park et al. fabricated vehicles carrying an angiogenesis-related peptide (apelin) and VEGF165 gene, which were taken up by human mesenchymal stem cells (hMSCs), and results demonstrated that hMSCs could differentiate into ECs and accelerate neovascularization of ECs.14 In our previous studies, multifarious nanoparticles (NPs), micelles or mixed micelles were prepared from linear, star-shaped or comb-like amphiphilic polymers comprising polyethylenimine (PEI), and used as pZNF580 carriers for rapid endothelialization and neovascularization.15-19 Moreover, REDV and CAG peptides were conjugated to the outmost surface of carriers to endow them with targeting function, which can selectively adhere to ECs over smooth muscle cells (SMCs) via a receptor-ligand binding affinity.20-26 REDV peptide can mediate ECs adhesion and migration by recognizing integrin α4β1 on ECs.27 Ji et al. prepared REDV and PEG-modified surface and proved the high selectivity of REDV peptide for ECs.28 In order to improve the transfection efficiency, TAT-NLS peptide with both cell-penetrating ability and nuclear localization capacity was used to cooperatively condense DNA with PEI-based amphiphilic polymer.29 These carriers possessed enhanced transfection efficiency, high protein

expression,

accelerated

wound

healing

ability as

well

as

good

neovascularization in vitro and in vivo. However, gene delivery efficiency is still hindered by some barriers (such as low biocompatibility, cell membrane, endo/lysosome and cell nuclear etc.) for successful gene therapy. Cell-penetrating peptides (CPPs) have received great attention and been intensively


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studied as carriers for various therapeutic drugs or genes due to their attractive potential for membrane permeability.30-34 HIV-1 TAT (47-57) peptide (TAT, Tyr-Gly-Arg-LysLys-Arg-Arg-Gln-Arg-Arg-Arg, YGRKKRRQRRR) is one of the most widely used CPPs for crossing cellular membrane.35-37 Saltzman et al38 prepared a kind of cellpenetrating peptide modified poly(lactic-co-glycolic acid) NPs, and cell internalization efficiency was dramatically increased compared with unmodified NPs. However, the intelligent CPP-based strategies were restricted by the non-specificity of CPPs because of their strong cell penetrating effect. It was alleviated by conjugating cyclic RGD peptide or other specific peptides to octaarginine via an amide bond, and a tandem peptide R8-RGD [RRRRRRRR-c(RGDfK)] was obtained.39 The R8-RGD served as a promising gene carrier for tumor therapy by simultaneously having the specificity of RGD and the strong cell-penetrating ability of R8.40,41 But scarce research focused on neovascularization by using these CPPs as gene carriers and their transmembrane mechanism or endocytic pathways for ECs. It’s well known that the efficient nuclear accumulation of therapeutic genes is one of the most critical steps in the process of gene delivery. Nuclear localization signal (NLS, (Pro-Lys-Lys-Lys-Arg-Lys-Val, PKKKRKV), originated from the large T antigen of the SV40 virus, possesses endosomal buffering function and nuclear localization capacity via the classical nuclear import pathway.42,43 Enhanced gene delivery has been reported as the introduction of NLS.44,45 Zhang et al. designed TAT-NLS peptide as the carrier of VEGF165 gene for neovascularization in ischemic tissue, and obvious enhancement of vascularization was obtained.46 Besides, the spacer arm length between a CPP and a



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NP was very critical for the promotion of cellular uptake, and a suitable spacer arm length is needed for CPPs fully exerting their function.47 Therapeutic genes play an important role in successful gene therapy. ZNF580 gene, as a gene of C2H2 zinc finger transcription factor, is very important for the migration and proliferation of ECs.48 Our previous studies also demonstrated that pZNF580 could promote the proliferation and migration of ECs as well as vascularization property.24-26 Herein, we integrated TAT and NLS sequences as a gene carrier with cell-penetrating ability and nuclear accumulation capacity in order to achieve efficient gene carriers with good biocompatibility for ECs. Besides, REDV peptide was also conjugated to this integrated TAT and NLS peptide to obtain a three tandem peptide REDV-TAT-NLS. Furthermore, the glycine (G) sequences with different repeat numbers were inserted into the sequence of REDV-TAT-NLS for the three parts, namely REDV, TAT and NLS, exerting their functions well. These peptide sequences were denoted as REDV-Gm-TATGm-NLS (m = 0, 1 and 4), and they were used as the gene carriers for pZNF580 plasmid. The formation of gene complexes, the process of gene delivery and the promotion of human umbilical vein endothelial cells (HUVECs) neovascularization were shown in Scheme 1. The biocompatibility, cellular uptake efficiency, transmembrane mechanism, gene delivery efficiency and protein expression of these gene complexes were investigated. Moreover, the proliferation, migration and neovascularization capacity of HUVECs treated with these gene complexes were also evaluated in vitro and in vivo.


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Scheme 1 Preparation process of pZNF580 complexes, selective gene delivery and neovascularization of HUVECs in vivo

2 Experiments 2.1 Materials Arg-Glu-Asp-Val

(REDV),

Tyr-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-Pro-

Lys-Lys-Lys-Arg-Lys-Val (YGRKKRRQRRR-PKKKRKV, TAT-NLS), Arg-Glu-AspVal-Tyr-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-Pro-Lys-Lys-Lys-Arg-Lys-Val (REDV-YGRKKRRQRRR-PKKKRKV, REDV-TAT-NLS), Arg-Glu-Asp-Val-GlyTyr-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-Gly-Pro-Lys-Lys-Lys-Arg-Lys-Val (REDV-G-YGRKKRRQRRR-G-PKKKRKV, REDV-G-TAT-G-NLS) and Arg-GluAsp-Val-Gly-Gly-Gly-Gly-Tyr-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-GlyGly-Gly-Gly-Pro-Lys-Lys-Lys-Arg-Lys-Val

(REDV-G4-YGRKKRRQRRR-G4-

PKKKRKV, REDV-G4-TAT-G4-NLS) peptides were purchased from GL Biochem. (Shanghai) Ltd. 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide



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(MTT) was obtained from Ding Guo Chang Sheng Biotech. Co., Ltd. (Beijing, China). Dulbecco's modified eagle medium (DMEM) and fetal bovine serum (FBS) were purchased from Invitrogen Corporation (Carlsbad, CA). A BCA protein assay kit was supplied by Solarbio Science and Technology Co., Ltd (Beijing, China). Rabbit antihuman ZNF580 polyclonal antibody, goat anti-rabbit IgG and amiloride hydrochloride were purchased from Abcam Ltd. (Shanghai, China). Rabbit anti-beta-actin antibody was obtained from Beijing Biosynthesis Biotechnology Co., Ltd. (Beijing, China). Cy5 labeled oligonucleotide (Cy5-oligonucleotide) was purchased from Sangon Biotech Co., Ltd. (Shanghai, China). LysoTracker Green (DND-26, Life Technologies) and Hoechst 33342 were obtained from Shanghai Invitrogen Biotechnology Co., Ltd. (Shanghai, China). Chlorpromazine hydrochloride was purchased from Sigma-Aldrich (St. Louis, USA). Filipin III was purchased from Cayman Chemical (Michigan, USA). TransScript First-Strand cDNA Synthesis SuperMix and TransStartTM Top Green qPCR SuperMix were provided by Transgen Biotech Co., Ltd. (Beijing, China). HUVECs were obtained from the Cell Bank of Typical Culture Collection of Chinese Academy of Sciences (Shanghai, China). The pEGFP-ZNF580 plasmid (pZNF580), human umbilical artery smooth muscle cells (HUASMCs) and male mice were supplied by Department of Physiology and Pathophysiology, Logistics University of Chinese People’s Armed Police Force. Matrigel (Cat. Nos. 356234) was purchased from Corning Company. 2.2 Preparation and characterization of TAT-NLS (NTP) and REDV-Gm-TAT-GmNLS (TP-Gm) complexes with pZNF580 The gene complexes at different w/w ratios of peptides and pZNF580 were prepared by


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mixing appropriate amount of NTP or TP-Gm solutions (1 mmol L-1) and pDNA (50 μg mL-1). Then, the mixed solutions incubated at 37 °C for 1 h to form stable gene complexes. The hydrodynamic diameter and zeta potential of NTP/pZNF580 and TPGm/pZNF580 complexes at different w/w ratios (1, 2, 3, 4 and 5) were measured by using a Zetasizer Nano ZS (Malvern Instrument, Inc., Worcestershire, UK). The condensing pDNA ability of all gene complexes was evaluated by agarose gel electrophoresis assay and the procedure was the same as previous studies.24 2.3 Hemocompatibility of NTP and TP-Gm Hemolysis assay was performed to evaluate the hemocompatibility of the NTP and TPGm solutions. Healthy and fresh human blood was collected into heparin sodium vacuum tubes and centrifuged at 3000 rpm for 5 min. After washing with PBS (pH = 7.4) until the supernatant fluid was clear and transparent, the red blood cells (RBCs) were suspended in 0.01 M PBS (pH = 7.4). The NTP and TP-Gm solutions (1000 μL, 0.5 mg mL-1) were added into the RBCs solution (25 μL) and incubated for 24 h. PBS (pH = 7.4) and purified water as negative and positive controls were set as 0% and 100% (absorbance value), respectively. The absorbance of supernatant was measured at 540 nm. The hemolysis of the NTP and TP solutions was calculated using Equation (1).

[Abs(sample)−Abs(negative control)]

Hemolysis (%) = [Abs(positive control)−Abs(negative control)]×100% Equation (1)

2.4 Cytocompatibility of NTP, TP-Gm and their gene complexes



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The cytotoxicity of NTP, TP-Gm (m = 0, 1 and 4) and their gene complexes for HUVECs were evaluated by MTT assay. Cells (1 × 104 cells/well) were seeded onto a 96-well plate and cultured for 24 h to 80% confluence. Then the cells were serumstarved overnight and treated with NTP, TP-Gm and their gene complexes at different concentrations ranging from 5 to 120 μg mL-1. After incubation for 48 h, MTT reagent (20 μL, 5 mg mL-1) was added to each well and cultured for 4 h. The optical density (OD) at 490 nm was measured using a microplate reader (BIO-RAD, iMarkTM, USA). 2.5 Cellular uptake and transmembrane mechanism of Cy5-oligonucleotide complexes Different Cy5-oligonucleotide complexes were prepared to quantitatively evaluate their cellular uptake in HUVECs by a flow cytometry (Beckman MoFlo XDP, USA). Cells were seeded into 6-well plates (3 × 105 cells/well) and transfected with these Cy5oligonucleotide complexes (3 μg Cy5-oligonucleotide for each well, w/w ratio = 5). After 4 h incubation, cells were washed three times with 0.01 M PBS (pH = 7.4) and trypsinized with 0.25% trypsin. Then, the cells were centrifuged, harvested and resuspended in 300 μL PBS (pH = 7.4). The suspension of cells was analyzed with a flow cytometer. The transmembrane mechanism was investigated by NTP and TP-Gm/Cy5oligonucleotide complexes. Cells were seeded onto 6-well plates, cultured as described above and pretreated in DMEM with various endocytic inhibitors: chlorpromazine (30 μM), amiloride hydrochloride (30 μM) and filipin III (5 μg mL-1) at 37 °C for 1 h. REDV peptide was also used to pretreat the cells and then cultured for 1 h. Then the


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NTP/pZNF580 and TP-Gm/pZNF580 complexes were added into each well for 4 h incubation. Subsequently, the cells were washed three times with 0.01 M PBS (pH = 7.4) and resuspended in 300 μL PBS (pH = 7.4) after trypsinization and centrifugation. The result was analyzed by a flow cytometry (Beckman MoFlo XDP, USA). 2.6 Intracellular trafficking of Cy5-oligonucleotide complexes The intracellular tracking study of different complexes was carried out by a confocal laser scanning microscope (CLSM). HUVECs were seeded in confocal dish for livecell imaging at 1 × 105 cells per dish and then cultured for 24 h. The cells were transfected by various Cy5-oligonucleotide complexes and washed twice with PBS (pH = 7.4) after 24 h incubation. Subsequently, the endo/lysosomes were stained with pHsensitive dye LysoTracker Green (75 nM) for 1 h and the cellular nuclei were stained with Hoechst 33342 for 20 min. Thereafter, the cells were washed twice with PBS (pH = 7.4) before observation. The fluorescence was analyzed by a CLSM (Olympus FV1000, Japan) at 649 nm, 504 nm and 350 nm excitation wavelengths for Cy5 (red), LysoTracker Green (green) and Hoechst 33342 (blue), respectively. The co-localization rate (CLR) was calculated by Equation (2) via the Image-Pro Plus 6.0 software: Co-localization rate =

Co-localization area Cy5 channel area

× 100%

Equation

(2) 2.7 In vitro transfection HUVECs and HUASMCs were seeded in 24-well plates (1 × 105 cells/well) and incubated in DMEM containing 10% FBS until 70%-80% confluence. The cells were starved for 12 h and then NTP/pZNF580 and TP-Gm/pZNF580 complexes at w/w ratio



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of 5 were added into each well (3 μg pZNF580 per well). After 4 h incubation, the medium was replaced with fresh DMEM containing 10% FBS and the cells were cultured for another 24 h. The expressed green fluorescent proteins (GFPs) were observed by an inverted fluorescent microscope (Fluorescence OLYMPUS U-RFLT50, microscopy Olympus DP72). 2.8 In vitro quantitative real-time PCR assay The gene delivery efficiency of different carriers was evaluated at mRNA level of pZNF580 plasmid expression by in vitro quantitative real-time PCR assay. HUVECs were incubated with various complexes for 24 h. Total RNA was extracted from the transfected cells using TRIzol reagent and then reverse-transcribed into cDNA by TransScript First-Strand cDNA Synthesis SuperMix according to the manufacture’s protocol. As templates, the resulting cDNAs were quantified via a SYBR Green on ABI 7300 stepone sequence detection PCR system (Applied Biosystems) for quantitative real-time PCR. The sequences of ZNF580 primers were as follows: forward 5’AAAAAGCTTGTGGAGGCGCACGTGCTG-3’,

reverse

5’-

AAAAAGATCTTGCCCGGAGTGCGCCCGTG-3’. The mRNA level of GAPDH gene was measured as an internal normalization standard and its forward and reverse primers

sequences

were

5’-AGGTGAAGGTCGGAGTCAAC-3’

and

5’-

CGCTCCTGGAAGATGGTGAT-3’, respectively. The PCR was carried out at the conditions of 95 °C for 10 min, 40 cycles at 95 °C for 15 s, 60 °C for 1 min, and 72 °C for 20 s. The results were analyzed by using StepOne software v2.1. 2.9 Western blot assay


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Western blot analysis was performed to evaluate gene delivery efficiency at the level of protein expression. HUVECs were seeded into 6-well plates and transfected with NTP/pZNF580 and TP/pZNF580 complexes for 24 h. Then the transfected cells were washed with cold 0.01M PBS (pH = 7.4) for three times, and followed by extracting the total protein with RIPA lysis buffer containing 1% volume of PMSF. The resulting lysates were centrifugated (12000 rpm) at 4 °C for 10 min. Thereafter, the total proteins were quantified by BCA protein assay kit and denatured after adding 5 × SDS. Approximately 80 μg protein per lane was obtained by using 10% SDS-PAGE and transferred onto polyvinylidene difluoride (PVDF) membrane. The proteins were blocked in TBST containing 8% defat milk for 1 h and then immunoreacted with rabbit anti-human ZNF580 polyclonal antibody overnight. The obtained protein blots were incubated 1 h with horseradish peroxidase conjugated anti-rabbit secondary antibody and developed by the standard enhanced chemiluminescence (ECL) kit. The result was observed through a gel image analysis system. Housekeeping endogenous protein βactin was used as internal standard. All belts were quantitatively analyzed by Image J software. 2.10 EC migration assays To evaluate the migration capability of the transfected HUVECs by different complexes, the transwell migration assay and wound healing assay were performed. The transwell migration assay was carried out in transwell chambers with 8.0 μm pore size gelatinized polycarbonate membrane. The transfected cells were starved with serum-free medium for 12 h and then seeded in the upper transwell chambers (1.2 × 105 cells/well). The



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lower transwell chambers were filled with fresh medium containing 10% FBS. After 6 h of incubation, the upper chambers were washed twice with 0.01 M PBS (pH = 7.4) and fixed with 4% paraformaldehyde for 10 min. The cells on the inside surface of the upper chamber were removed, while the cells on the lower surface of the upper chambers were stained with eosin at 37 °C for 8 min and observed by an inverted fluorescent microscope (Fluorescence OLYMPUS U-RFLT50 and microscopy Olympus DP72). The number of migrated cells in three random fields from each well was counted by Image-Pro Plus 6.0 software to evaluate migration ability. The wound healing assay was performed by scratching at the middle of each well to form a linear wound after 24 h transfection. Then the cells were washed with D-Hanks to remove the cell debris, and incubated in culture medium without serum at 37 °C. The migration of the cells was monitored and photographed at different time points (0, 6 and 12 h) via an inverted microscope (OLYMPUS U-RFLT50 and microscopy Olympus DP72). The migration ability was further evaluated by calculating relative recovered area (%) of cells after 12 h using Image J software by Equation (3). The recovered area

Relative recovered area (%) = The total wounded area × 100%

Equation (3)

2.11 In vitro tube formation assay The in vitro tube formation assay was performed according to the Corning Matrigel Matrix instruction. Growth factor-reduced Matrigel was dissolved at 4 °C overnight and 50 μL of the Matrigel was coated on a pre-cooled 96-well plate, followed by incubation at 37 °C for 1 h. Subsequently, HUVECs transfected with various complexes were seeded onto the layer of Matrigel (4 × 104 cells/well) and cultured for 6 h. HUVECs


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treated with pDNA were used as a control. Photographs of the formed tubule-like structures at five randomly fields were obtained via an optical microscope and the number of the tubules in each photograph was counted by Image-Pro Plus 6.0 software. 2.12 In vivo angiogenesis assay Male mice (6 weeks old, 20-25 g) were used for in vivo angiogenesis assay. It was performed as follows: HUVECs were pretreated with gene complexes for 4 h and cultured for another 24 h. The transfected cells mixed with 800 μL matrigel at a final cell concentration of 1 × 106 cells/mL after being trypsinized with 0.25% trypsin. The mixture was subcutaneously injected into the mice. Four days later, the mice were sacrificed by euthanasia, and the matrigel implants were sliced into thick sections after being fixed with formalin and embedded in paraffin. The sections were stained with hematoxylin and eosin (H&E), and the luminal structure was observed using a microscope. In addition, the sections were also immunohistochemically stained with mouse anti-CD31 antibody (diluted in PBS (pH = 7.4) at 1: 20) for 1 h, and followed by staining with goat anti-mouse IgG H&L secondary antibody (Alexa Fluor 594). DAPI was used for cellular nuclei staining. Then the stained sections were observed by a fluorescence microscope to further determine the formation of micro-vessel structure. The protocol of in vivo study was approved by Armed Police Logistics College, and conformed to the “Guide for the protection and use of experimental animals” of American National Institutes of Health. 2.13 Statistical analysis All experiments were performed three times and the results were presented as mean ±



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standard deviation (SD). Statistical significance of differences values were calculated by a Student’s t-test. P values less than 0.05 were considered to be statistically significant.

3 Results 3.1 Preparation and characterization of NTP, TP-Gm and their gene complexes TAT and NLS have been considered as typical peptides facilitating cellular internalization and nuclear accumulation, respectively.46 Meanwhile, REDV peptide is proved to selectively adhere to ECs instead of smooth muscle cells (SMCs).25,49 In order to combine the special functions of these peptides, TAT, NLS and REDV sequences were integrated together to form a three block tandem peptide REDV-TAT-NLS. Moreover, a glycine sequence was inserted into this REDV-TAT-NLS sequence as a flexible segment to provide them a spacer arm for exerting multifunction well. The spacer arm length was controlled by varying the number of glycine residue, and the final designed sequences were denoted as REDV-Gm-TAT-Gm-NLS (m=0, 1 and 4). They could complex with pZNF580 plasmid because of their positive charge in physiological condition. These multifunctional peptide sequences with different m values of 0, 1 and 4 were abbreviated as TP-G0, TP-G1 and TP-G4, respectively. TATNLS/pZNF580 (NTP/pZNF580) complexes with non-targeting function was also prepared as the control group. Nanoscale gene complexes and suitable positive surface charges are necessary for nonspecific cellular uptake to enhance gene delivery efficiency.50 The size and surface


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charge of these complexes were determined by a Zetasizer (Malvern Instruments, Malvern, UK). The size distribution of the complexes ranged from 128 to 256 nm and no significant regularity in size was observed among different groups (Figure 1(1)). The surface charge of the complexes was slightly influenced by the introduced glycine sequence (Figure 1(2)). As the repeat number of glycine sequences increased, the corresponding complex surface charge reduced at the same w/w ratio. The NTP/pZNF580, TP-G0/pZNF580, TP-G1/pZNF580 and TP-G4/pZNF580 complexes showed positive surface charge at w/w ratio above 2, and their zeta potentials increased with the increase of w/w ratio. The complexes with adjustable surface charge via changing w/w ratio of TP-Gm and pZNF580 benefited for low cytotoxicity and also for high cellular uptake and efficient gene delivery. Generally, cationic polymers could complex with negatively charged DNA via electrostatic interaction. The lysine/arginine-enriched peptides as gene carries were positive-charged and their DNA condensing ability was evaluated by agarose gel retardation assay (Figure 2). In consistent with the results of surface charge, the NTP, TP-G0, TP-G1 and TP-G4 could inhibit pZNF580 migration at w/w ratio of 2, 2, 3 and 3, separately. These results demonstrated that these designed peptides could complex with pZNF580 plasmid and protect it from damage during the process of circulation in blood.



(1)

300

Size (nm)

250 200 150 100 50 0

1 2 3 4 5

1 2 3 4 5

1 2 3 4 5

1 2 3 4 5

w/w ratio

w/w ratio

w/w ratio

w/w ratio

A

B

C

D

1 2 3 4 5

1 2 3 4 5

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30 20 10 0 -10 -20 A

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Figure 1 Hydrodynamic diameters (1) and zeta potentials (2) of NTP/pZNF580 and TP-Gm/pZNF580 complexes at various w/w ratios (1, 2, 3, 4 and 5). (A) NTP/pZNF580, (B) TP-G0/pZNF580, (C) TP-G1/pZNF580, (D) TP-G4/pZNF580. Data are presented


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as mean ±SD (n = 3).

Figure 2 Agarose gel retardation assay of NTP/pZNF580 and TP-Gm/pZNF580 complexes at various w/w ratios ranging from 0 to 5. (A) NTP/pZNF580, (B) TPG0/pZNF580, (C) TP-G1/pZNF580, (D) TP-G4/pZNF580.



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3.2 Hemocompatibility of NTP and TP-Gm The gene carriers usually come in contact with blood in the process of gene delivery. Therefore, well hemocompatibility is essential for designing safe and efficient gene carriers.24,51 The hemocompatibility of NTP and TP-Gm was investigated by hemolysis assay using fresh human red blood cells (RBCs). In Figure 3(1), the samples showed little hemoglobin leakage, meaning little RBCs were destroyed. Moreover, complete cell morphology was observed in the groups (A, B, C, D) similar to the result of PBS (pH = 7.4) group. As listed in Figure 3(3), all samples exhibited sufficiently low hemolysis rate (< 5%) of RBCs for gene delivery and considered to be well hemocompatible according to the medical standards of hemolysis rate < 5% as the safe value for biomaterials.52-54 In addition, different samples showed reduced hemolysis rate as the conjugation of targeting peptide REDV and insertion of glycine sequence. The TP-G4, integrated REDV peptide and 4 repeat glycine residue in peptide sequence, showed the lowest hemolysis rate (0.18 ± 0.07%) and exhibited extremely good hemocompatibility, which was the prerequisite for gene carriers in vivo.


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Figure 3 Photographs (1), microscope images (2) of RBCs hemolysis results, and the hemolysis values by using ultrapure water and PBS (pH = 7.4) groups as controls (3) in the presence of NTP and TP solutions after 24 h incubation. (A) NTP, (B) TP-G0, (C) TP-G1, (D) TP-G4. Data are presented as mean ± SD (n = 3). 3.3 Cytocompatibility of NTP, TP and their gene complexes



Cytotoxicity is another important factor which must be considered to design and prepare gene carriers with good biocompatibility. The cytocompatibility of NTP, TP and their gene complexes toward HUVECs was measured by MTT assay and the results were shown in Figure 4. All samples showed no reduction in relative cell viability along with increasing concentration. Moreover, the relative cell viability of all samples were higher than 75% even at high concentration of 120 μg/mL, which was much higher than the micelles based on PEI and their gene complexes in our previous studies.26,55 Besides, gene complexes displayed higher relative cell viability than corresponding carriers due to the effect of pZNF580 on HUVECs proliferation and migration.26 In line with the results of hemocompatibility, these peptide-based gene carriers showed very high cytocompatibility.

120

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Figure 4 Relative cell viability of NTP, TP-Gm and their gene complexes treated HUVECs. Data are presented as mean ±SD (n = 3).


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3.4 Cellular uptake and transmembrane mechanism of complexes Cell membrane is the first barrier in gene delivery. The promotion of membranepenetration activity has a great effect on transfection efficiency.56 In order to improve the internalization of gene complexes, TAT peptide with specific function of membranepenetration was used as gene carriers. Simultaneously, REDV peptide was also conjugated to the N-terminal sequence of TAT to achieve targeting ability for HUVECs. Cy5-oligonucleotide was used as a reporter gene to determine the cellular uptake of complexes in HUVECs and HUASMCs. Flow cytometry data (Figure 5(1)) showed that the cellular uptake of all groups were close to 100%, while the mean fluorescence intensity (MFI) was significantly different. As the repeat number of glycine sequences increased, the MFI value demonstrated a rising tendency, and the TP-G4/Cy5oligonucleotide group showed the highest MFI among all groups owing to its high cellpenetrating activity and nuclear translocation ability. In addition, the cellular uptake of TP-G4/Cy5-oligonucleotide group in HUASMCs was also measured to prove its target function for ECs (Figure 5(3)). As shown in Figure 5(3), the cellular uptake efficiency of TP-G4/Cy5-oligonucleotide group in HUVECs was about 1.36 times higher than HUASMCs. These results proved that the TP-G4/Cy5-oligonucleotide complexes preferred to adhere on HUVECs rather than HUASMCs. Previous studies demonstrated that CPPs could be taken up by cells via different endocytic pathways, such as macropinocytosis, endocytic uptake mediated by clathrin or caveolae, and other endocytic pathways.57 The internalization mechanism of arginine-rich CPPs including TAT has been well studied. However, this mechanism is



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influenced by many factors (e.g. cargos, specific stimuli and other conditions). In order to investigate the endocytic pathways of TP-G4/Cy5-oligonucleotide complexes, chlorpromazine (CPZ), filipin (Filip), and amiloride (Amil) were chosen as inhibitors of chathrin-mediated, caveolae-mediated endocytic pathways and macropinocytosis, respectively (Figure 5(4)). Besides, free REDV was also used to evaluate the receptormediated cellular uptake. In Figure 5(4), significant decrease (about 84.52%) in cellular uptake of TP-G4/Cy5-oligonucleotide complexes by CPZ inhibition was observed, while Amil (about 1.30% reduction) and Filip (about 5.89% reduction) inhibitors displayed a little effect on cellular uptake. So, clathrin-mediated endocytosis was a major endocytic pathway of TP-G4/Cy5-oligonucleotide complexes, which was consistent with previous studies.41 Besides, the cellular uptake of TP-G4/Cy5oligonucleotide complexes was also decreased (about 16% reduction) in the presence of free REDV peptide. The result further proved that the integration of REDV domain into peptide sequence facilitates the internalization of its complexes via specifically binding to integrin receptors on HUVECs. This kind of receptor-mediated cellular uptake can be inhibited by free REDV peptide binding to the targeting sites on HUVECs. After adding three inhibitors of CPZ, Amil and Filip, 15.09% cellular uptake was still remained and the value was close to 16% reduction inhibited by free REDV peptide. This phenomenon verified that the endocytic pathways of TP-G4/Cy5oligonucleotide complexes mainly include not only clathrin-mediated endocytosis, but also receptor-mediated cellular uptake. Moreover, 80% clathrin-mediated endocytosis, about 15% receptor-mediated cellular uptake and some other endocytic pathways were


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involved in the endocytic pathways of TP-G4/Cy5-oligonucleotide. Therefore, the cellular uptake efficiency of the three tandem peptide TP-G4 was improved via mutiple endocytic pathways and had great potential for targeting gene delivery.

(2) *

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Figure 5 Cellular uptake of complexes in HUVECs (1), mean fluorescence intensity measured by flow cytometry (2), cellular uptake of TP-G4/ZNF580 in HUVECs and



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HUASMCs (3) and effects of inhibitors on the internalization of TP-G4/Cy5oligonucleotide in HUVECs (4). (A) NTP/Cy5-oligonucleotide, (B) TP-G0/Cy5oligonucleotide, (C)TP-G1/Cy5-oligonucleotide, (D) TP-G4/Cy5-oligonucleotide complexes (E) Cy5-oligonucleotide. (n = 3, mean ± SD, *p < 0.05, statistically significant difference between these two groups).

3.5 Intracellular trafficking of Cy5-oligonucleotide complexes For efficient gene delivery, the internalized complexes localize in endo/lysosomes, and they should quickly escape from lysosome and finally traverse into nucleus. Confocal live cell imaging was performed by a CLSM to investigate the distribution of Cy5oligonucleotide in the cytoplasm. As depicted in Figure 6, Cy5-oligonucleotide displayed red fluorescence, and endo/lysosomes were labeled by Lysotracker to show green fluorescence. The overlay of the two fluorescence in the merged images showed yellow spots, which illustrated the complexes were taken up by HUVECs and formed endo/lysosomes. Besides, the overlap of red fluorescence and blue fluorescence (pink) suggests the complexes escaped from endo/lysosomes, and gene entered into cell nucleus efficiently owing to the nucleus localization capacity of NLS. According to the data listed in Figure 6, the lysosome CLR represents the complexes entrapped in endo/lysosomes at the time point and the lowest lysosome CLR and nucleus CLR of NTP/Cy5-oligonucleotide group may be caused by low cellular uptake and low nucleus accumulation compared with other groups. These results verified that the targeting function of REDV sequence in TP/Cy5-oligonucleotide groups may affect their cellular


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uptake efficiency. The nucleus CLR value was elevated with increasing the repeat number of glycine sequences in the peptides because the complexes exert more efficient function of selectivity for ECs, membrane penetration and nucleus translocation due to the insertion of this flexible segment. In brief, the TP-G4/Cy5-oligonucleotide possessed the highest nucleus CLR (about 19.8%), which is beneficial for efficient gene delivery.

Figure 6 CLSM of different gene complexes at 24 h. (A) Cells treated with NTP/Cy5oligonucleotide, (B) cells treated with TP-G0/Cy5-oligonucleotide, (C) cells treated with TP-G1/Cy5-oligonucleotide, (D) cells treated with TP-G4/Cy5-oligonucleotide. The lysosome and cell nucleus were stained by LysoTracker Green (green) and Hoechst (blue), respectively. 3.6 Gene delivery efficiency The efficiency of gene delivery was evaluated at the level of mRNA and protein expression by transfection in vitro, quantitative real-time PCR assay and western blot assay. After taken up by HUVECs, the complexes could escape from endo/lysosomes



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and release pZNF580 plasmid. Then the plasmid entered into cell nucleus efficiently via nucleus translocation capacity of NLS segment in the complexes. As shown in Figure 7, green fluorescence protein could be observed in the images (A, B, C, D), demonstrating the pEGFP gene in pZNF580 plasmid transcripted and expressed into proteins successfully. But the HUVECs treated by pZNF580 group and the HUASMCs treated by TP-G4/pZNF580 group did not observed green fluorescence proteins, which further proved efficient gene delivery by these peptides and their targeting function for HUVECs. In vitro quantitative real-time PCR assay and western blot assay were performed to investigate the efficiency of gene delivery quantificationally. The results of quantitative real-time PCR (Figure 8) showed that the TP-Gm/pZNF580 complexes group had higher mRNA expression than the NTP/pZNF580 complexes and the control groups. The mRNA expression of TP-G4/pZNF580 complexes group was the highest (2.1 folds relative to non-treated group), and the tendency with increasing glycine number in TPGm/pZNF580 complexes was the same as the results of cellular uptake and intracellular distribution. The efficiency of gene delivery was also confirmed by western blot assay and the data was shown in Figure 9. Similar result was obtained and the TPG4/pZNF580 complexes group presented the optimal protein expression owing to the synergistic effect of REDV peptide (targeting function), TAT (membrane-penetration activity) and NLS peptides (nucleus accumulation capacity). These results demonstrated that the TP-G4 exhibited great potential for efficient gene delivery in HUVECs.


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Figure 7 Fluorescence images of HUVECs transfected for 24 h by (A) NTP/pZNF580, (B) TP-G0/pZNF580, (C) TP-G1/pZNF580, (D) TP-G4/pZNF580 and (E) pZNF580, and fluorescence images of HUASMCs transfected for 24 h by (F) TP-G4/pZNF580. A, B, C, D, E and F were the fluorescence images, and A’, B’, C’, D’, E’ and F’ were the corresponding bright-field images.


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ZNF580 mRNA expression (Folds VS. non-treated)


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* 2.0

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B

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Figure 8 Quantitative ZNF580 transfection efficiency of HUVECs transfected by (A) NTP/pZNF580, (B) TP-G0/pZNF580, (C) TP-G1/pZNF580, (D) TP-G4/pZNF580 and (E) only pZNF580 as the control by qPCR. (n = 3, *statistically significant different from the E group with p < 0.05).


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Figure 9 Western blot analysis for pZNF580 protein expression in HUVECs transfected by different complexes after 24 h. (A) NTP/pZNF580, (B) TP-G0/pZNF580, (C) TPG1/pZNF580, (D) TP-G4/pZNF580. (mean ±SD, n = 3,*statistically different from (A) group (p < 0.05)). 3.7 EC migration assay Cell migration is an essential capacity for the wound healing process, formation of vascular rings and further neovascularization.20 To investigate the effect of complexes on HUVECs migration, wound healing assay and transwell migration assay were performed. The results of wound healing assay were shown in Figure 10. The complexes groups recovered much larger wound area than the control group, and the



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wound recovered area of NTP/pZNF580, TP-G0/pZNF580, TP-G1/pZNF580 and TPG4/pZNF580 complexes groups increased successively. The TP-G0/pZNF580 group showed a little larger recovered area than the NTP/pZNF580 group because the REDV peptide was beneficial for EC adhesion so as to improve pZNF580 delivery efficiency. The TP-G4/pZNF580 group recovered significant larger area than other groups, which proved that TAT and NLS peptides could exert membrane penetration function and nucleus translocation capacity well after the integration of glycine sequence as flexible segments. The efficiency of gene delivery was enhanced in this TP-G4/pZNF580 group. The images of transwell migration assay were shown in Figure 11(1), and the number of migrated cells was calculated via Image-Pro Plus 6.0 software (Figure 11(2)). In consistent with the results of wound healing assay, the number of migrated cells for different groups increased by REDV conjugation and glycine insertion. A maximum migrated cell number of 179 was obtained for HUVECs treated with TP-G4/pZNF580 group. The migration results of HUVECs treated with different complexes verified the effect of REDV and glycine on HUVECs migration, and the consequence was also in agreement with previous studies.20,47


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Figure 10 Wound recovery of HUVECs at 0, 6 and 12 h time points (1) and the relative recovered area after 12 h calculated by the Image-J software (2). (A) NTP/pZNF580 complexes treated group, (B) TP-G0/pZNF580 complexes treated group, (C) TPG1/pZNF580 complexes treated group and (D) TP-G4/pZNF580 complexes treated group with (E) only pZNF580 treated as control (mean ± SD, n = 3, * statistically different from the control group (only pZNF580) with p < 0.05).



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Figure 11 Transwell migration assay of HUVECs after 6 h (1) and the average migrating cell number (2). (A) NTP/pZNF580 treated group, (B) TP-G0/pZNF580 treated group, (C) TP-G1/pZNF580 treated group, (D) TP-G4/pZNF580 treated group, (E) pZNF580 treated as a control (mean ± SD, n = 3, *statistically different from E group with p < 0.05). 3.8 In vitro tube formation assay The formation of tubule-like structure is very crucial for the neovascularization of


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critical limb ischemia.58 To investigate the effect of different complexes on neovascularization in defective vascular tissue, in vitro tube formation assay was performed by using Corning Matrigel. In Figure 12(1), different sizes of tubule-like structures were observed in all images, which demonstrated that HUVECs transfected by different samples proliferated and migrated on matrigel without any growth factors. HUVECs only treated with pZNF580 were used as a control group, and about 9 tubes were achieved, much less than the groups of HUVECs transfected with gene complexes. The NTP/pZNF580 group displayed less number of tubes than the TP-G0/pZNF580 group, verifying the introduced REDV peptide facilitating tubule-like structures formation. The tube number of TP-G4/pZNF580 group was about 25 and far higher than other groups. This can be explained by its good biocompatibility and high gene delivery efficiency. These results demonstrated that the peptide-based gene carries with multifunctions exhibited great potential on neovascularization in defective vascular tissues.



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Figure 12 The effect of different complexes on pZNF580-mediated HUVECs tube formation in vitro. (1) Microscopy images of HUVECs after incubation on Matrigel for 6 h at 37 °C. (A) NTP/pZNF580 treated HUVECs, (B) TP-G0/pZNF580 treated HUVECs, (C) TP-G1/pZNF580 treated HUVECs, (D) TP-G4/pZNF580 treated HUVECs, (E) only pZNF580-treated HUVECs. (2) Tube number corresponding to different groups. (n = 3, *statistically significant different from the E group with p < 0.05).


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3.9 In vivo angiogenesis assay Neovascularization can improve blood circulation in defective vascular tissues, and is considered to be the crucial step for the therapy of vascular diseases, especially for limb ischemia.10 Therefore, to evaluate the in vivo angiogenesis effect of different gene complexes, HUVECs was transfected with these complexes and in vivo transplantation was performed after mixed with Corning Matrigel. As illustrated in Figure 13(1), revascularization was occurred and tubule-like structures (arrows indicated the formed tubule-like structures) were observed in all of the groups except the control through H&E staining, which demonstrated HUVECs treated with different complexes exhibited neovascularization ability. The tubule-like structure number of A, B, C, D groups was 2, 2, 3 and 4, respectively. The HUVECs transfected by TP-G4/pZNF580 complexes presented relatively more tubule-like structure number than other groups. In Figure 13(2), the tubule-like structure number showed a similar result as Figure 13(1). It illustrated stronger revascularization capacity that may be caused by more efficient pZNF580 plasmid delivery and expression of TP-G4/pZNF580 complexes. These results proved that efficacious pZNF580 expression delivered by TP-G4 can mediate the formation of tubule-like structure, which is very beneficial for the treatment of vascular diseases.



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Figure 13 In vivo angiogenesis assay to evaluate neovascularization ability of HUVECs transfected by different complexes. (1) Microscopy images of the HUVECs and Matrigel mixed implants sectioned and stained with H&E. (2) Microscopy images of the implants immunohistochemical stained with anti-CD31. Arrows indicate vessel-like structures. (A) NTP/pZNF580 treated HUVECs, (B) TP-G0/pZNF580 treated HUVECs, (C) TP-G1/pZNF580 treated HUVECs, (D) TP-G4/pZNF580 treated HUVECs, (E) pZNF580-treated HUVECs as control group.

4 Discussion It is very critical for gene therapy to overcome cellular barriers and various physiological environments.59 For vascular diseases, especially the peripheral vascular


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diseases and critical limb ischemia, gene therapy has been considered as a potential method to induce therapeutic angiogenesis.10,60 Therefore, developing efficient gene delivery systems with high biocompatibility is the crux of gene therapy. Nowadays, cationic polymer gene delivery vehicles can resist serum degradation and provide very high transfection efficiency.61 However, high cytotoxicity and low biocompatibility prove to be a dilemma for efficient gene therapy and need to be addressed urgently. Many studies have devoted to overcome these problems, but low cytotoxicity usually means low gene delivery efficiency.21,24 What’s more, the cytotoxicity is inevitable due to their special structure, especially for the delicate cells, such as ECs etc. Recently, peptide-based gene carriers provide an alternative strategy due to their high biocompatibility. CPPs, known for their specific cell-penetrating activity, serve as a powerful tool to improve internalization efficiency, while gene delivery is hampered by their non-specificity. Besides, nucleus translocation is another limitation for gene delivery and only very small molecules can directly translocate into nucleus via nuclear pore. Therapeutic genes are very difficult to enter into nucleus. NLS sequence is reported to enhance preferential accumulation in nucleus relative to other organelles.62 So, we designed a series of multifunctional peptides via integrating REDV targeting peptide, TAT peptide and NLS sequence in this paper. In addition, 1 and 4 glycinesequences as a spacer arm were inserted into peptide sequence for exerting multiple functions well. For these peptide-based gene carriers, their condensing pDNA ability was influenced by glycine-sequence length. 4 glycine-sequence was usually chosen as the flexible segment in previous studies and showed good effect.47,63 So we investigated



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1 and 4 glycine-sequences as a spacer arm in this study. These peptides can condense pZNF580 gene and form complexes with proper size and zeta potential by altering w/w ratios of peptides and pZNF580. The hemocompatibility and cytocompatibility assay demonstrated that these peptides and their complexes were nontoxic for HUVECs (relative cell viability ＞ 75% at 120 μg/mL) and possessed significantly high hemocompatibility (hemolysis rate ＜ 5%) compared with other cationic polymers, such as gene carriers based on PEI.26,55 These peptide sequences benefit for low cytotoxicity. Importantly, these complexes demonstrated high cellular uptake and internalization efficiency, especially the TP-G4/pZNF580 group showed significantly higher (1.36 times) cellular uptake in HUVECs than HUASMCs due to the specificity of REDV peptide for ECs. The cellular uptake mechanism of TP-G4/pZNF580 was investigated by using different inhibitors for various endocytic pathways. Moreover, free REDV peptide was also used to treat HUVECs before transfecting by Cy5-oligonucleotide complexes. The free REDV peptide occupied α4β1 integrin recognition site on HUVECs and inhibited receptor-mediated endocytosis of TP-G4/pZNF580 complexes. Results showed that the transmembrane mechanism mainly consists of more than 80% clathrin-mediated endocytosis and about 15% receptor-mediated cellular uptake. In addition, the result of confocal live cell imaging showed that pZNF580 plasmid could be internalized by these peptide carriers via forming endosomes, and the plasmid escaped from endo/lysosomes successfully. Then the NLS sequence in the cytoplasm was recognized by importin α and linked to β-karyopherin (importin β).64 Ultimately,


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the plasmid entered nucleus efficiently via the interaction between importin β and nuclear pore complex (NPC), and the TP-G4/pZNF580 complexes displayed optimal nucleus accumulation because NLS sequence was integrated and glycine sequence was inserted as a spacer arm to fully exert its functions (Scheme 1). The gene delivery efficiency of these peptides was evaluated by in vitro transfection, in vitro quantitative real-time PCR assay and western blot assay. The results illustrated that these multifunctional peptides exhibited a tremendous advantage for gene delivery and expression in HUVECs, which highlighted the potential of our designed multifunctional peptides for gene delivery. HUVECs migration assay was also performed to investigate the potential therapeutic effect of these complexes on neovascularization for vascular disease treatment. The HUVECs treated with TPG4/pZNF580 (179 migrated cells) showed the highest migration ability, which was close to that of the optimized carriers based on PEI.20 The consequence can be explained by their high biocompatibility and efficient gene delivery effect compared with other cationic polymer carriers. A significant difference between NTP/pZNF580 and pZNF580 treated groups was observed in cellular uptake and migration assays, but not in ZNF580 mRNA expression. The expression levels of NTP/pZNF580 and pZNF580 treated groups were 1.08 and 1.02, respectively. Cellular uptake is the first step in gene delivery and expression, and the ZNF580 mRNA expression level is affected by many factors, such as endosome escape, nucleus accumulation and gene expression etc. The migration ability may be affected by many other factors except of ZNF580 mRNA expression level. It is still not



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clear why NTP/pZNF580 treated group showed relatively higher migration ability than pZNF580 treated group. In vitro and in vivo angiogenesis assays showed enhanced neovascularization ability of HUVECs transfected by TP-G4/pZNF580 complexes and manifested the application potential of the complexes on neovascularization. In TP-G4 peptide (REDV-G4-TATG4-NLS sequence), three functional peptide sequences could exert their functions owing to the flexible spacer arm of G4. Besides, REDV peptide can selectively adhere onto ECs27,65,66, which benefits for targeting transfection through REDV-mediated endocytosis during the transfection. The TAT sequence also enhances the endocytosis facilitating cellular uptake. NLS can mediate the nuclear accumulation after endo/lysosomes escape of gene complexes. The cellular barriers are overcome by these multifunctional peptides. The synergistic effects of these peptide sequences benefit for highly efficient gene delivery with ECs specificity and nucleus accumulation capacity. Meanwhile, the high biocompatibility and gene delivery efficiency enhance the proliferation, migration and neovascularization of HUVECs in vitro and in vivo. In a word, the above results demonstrated TP-G4 gene carrier possessed efficacious target function for HUVECs, membrane-penetrating ability and nucleus translocation capacity simultaneously, and promising for the therapy of vascular diseases.

5 Conclusions In summary, we designed a series of peptide-based gene carriers with multifunction as an efficient gene delivery system. These tandem peptides REDV-Gm-TAT-Gm-NLS


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(TP-G0, TP-G1 and TP-G4) were designed by integrating REDV, TAT and NLS peptides and introducing different repeat number of glycine as flexible segments. These peptides and their pZNF580 complexes had excellent biocompatibility and efficient gene delivery ability with high internalization efficiency and efficient nucleus accumulation. Moreover, they were preferentially taken up by HUVECs instead of HUASMCs owing to the introduced REDV peptide. HUVECs treated by these complexes showed enhanced migration capacity and neovascularization ability in vitro or in vivo, which could ultimately be used as a novel gene delivery platform for the therapy of vascular diseases.

Acknowledgements This project was supported by National Key R&D Program of China (grant No. 2016YFC1100300), National Natural Science Foundation of China (Grant No. 31370969 and 51673145), International Science & Technology Cooperation Program of China (Grant No. 2013DFG52040), Wenzhou government’s startup fund (Grant No. WIBEZD2014005-03 and WIBEZD2015009-03), and Wenzhou Science and Technology Bureau (Grant No. Y20150088).

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