Oligohistidine and targeting peptide functionalized TAT-NLS for enhancing cellular uptake and promoting angiogenesis in vivo
Gene therapy has been developed and used in medical treatment for many years, especially for the enhancement of endothelialization and angiogenesis. But slow endosomal escape rate is still one of the major barriers to successful gene delivery. In order to evaluate whether introducing oligohistidine (Hn) sequence into gene carriers can promote endosomal escape and gene transfection or not, we designed and synthesized Arg-Glu-Asp-Val (REDV) peptide functionalized TAT-NLS-Hn (TAT: typical cell-penetrating peptide, NLS: nuclear localization signals, Hn: oligohistidine sequence, n: 4, 8 and 12) peptides with different Hn sequence lengths. pEGFP-ZNF580 (pZNF580) was condensed by these peptides to form gene complexes, which were used to transfect human umbilical vein endothelial cells (HUVECs).
MTT assay showed that the gene complexes exhibited low cytotoxicity for HUVECs. The results of cellular uptake and co-localization ratio demonstrated that the gene complexes prepared from TAT-NLS-Hn with long Hn sequence (n = 12) benefited for high internalization efficiency of pZNF580. In addition, the results of western blot analysis and PCR assay of REDV-TAT-NLS-H12/pZNF580 complexes showed significantly enhanced gene expression at protein and mRNA level. Wound healing assay and transwell migration assay also confirmed the improved proliferation and migration ability of the transfected HUVECs by these complexes. Furthermore, the in vitro and in vivo angiogenesis assay illustrated that these complexes could promote the tube formation ability of HUVECs.
The above results indicated that the delivery efficiency of pZNF580 and its expression could be enhanced by introducing Hn sequence into gene carriers. The Hn sequence in REDV-TAT-NLS-Hn is beneficial for high gene transfection. These REDV and Hn functionalized TAT-NLS peptides are promising gene carriers in gene therapy.
KeywordsGene carrier Peptide Histidine REDV Targeting HUVECs pZNF580
Nowadays, small-diameter artificial blood vessels (< 6 mm) have been used in clinical treatment of cardiovascular diseases, but their long-term patency rate remains low [1, 2]. To solve this problem, many surface modification strategies have been developed to enhance the hemocompatibility of artificial blood vascular materials, such as grafting zwitterionic polynorbornene, phosphorylcholine, heparin, gelatin or silk fibroin to avoid platelet adhesion and aggregation [3, 4, 5, 6, 7].
The re-endothelialization of small-diameter artificial blood vessels is beneficial for high long-term patency. Surface modification with bioactive peptides, such as Arg-Gly-Asp (RGD) , Cys-Ala-Gly (CAG)  and Arg-Glu-Asp-Val (REDV), can promote the attachment to endothelial cells (ECs). Among these peptides, REDV peptide can be specially recognized by α4β1 integrin, which is enriched in ECs but lacked in smooth muscle cells (SMCs) [10, 11]. Ji et al. proved that the combination of rapamycin-loaded polymer base layer and REDV peptide tethered top layer could promote the competitive adhesion of human umbilical vein endothelial cells (HUVECs) over human aortic smooth muscle cells, and enhanced in situ endothelialization . Recently, our studies demonstrated that REDV-modified gene carriers could specially recognize ECs and selectively enhance transfection efficiency by transferring DNA into ECs so as to promote their proliferation and migration as well as vascularization in vitro and in vivo [13, 14, 15, 16].
Gene therapy has been considered as an effective method for EC migration and proliferation, which benefits for rapid endothelialization and neovascularization . For successful gene therapy, the major key task is to develop effective gene carriers. Compared with viral vectors, the synthesized gene carriers have gained much attention owing to their high safety, low cost and convenient preparation [18, 19, 20]. However, there still exist some biological barriers in gene delivery , such as low biocompatibility, DNA packaging ability, cellular uptake, endosomal escape  and DNA release .
Low cellular uptake is a major barrier in gene delivery because cell membrane acts as a significant physical obstacle for gene carriers. In recent years, gene delivery systems based on cationic peptides have been widely studied owing to their high biocompatibility and specific functions . Compared with polymeric gene carriers, these peptide carriers exhibit nearly no cytotoxicity for cells. Cell-penetrating peptides (CPPs), a series of short arginine-rich cationic peptides, have the specific ability to cross cell membrane. They serve as a flexible strategy for overcoming the first barrier and further improving the gene delivery effect [25, 26, 27, 28, 29]. Tyr-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg–Arg-Arg (YGRKKRRQRRR, TAT peptide) is one of non-specific selectively CPPs , which was derived from human immunodeficiency viruses 1 (HIV-1) . TAT has been proven to promote cellular uptake and gene delivery because of its high cell membrane penetration. Gao et al. modified the poly(N-isopropylacrylamide) (PNIPAM) microgel particles with TAT peptide to form PNIPAM-FL-TAT particle, which showed significantly high cellular internalization compared with the PNIPAM group .
Another key issue in gene delivery is whether therapeutic DNAs can effectively cross the nuclear pore complexes (NPCs) on the nuclear membrane into nucleus. Nuclear access is limited by small nuclear pore diameter (only 9 nm), so only small molecules can be directly transported into nucleus through NPCs [33, 34]. Several methods have been investigated to specially translocate DNAs into nucleus, for example, gene carriers containing nuclear localization signal (NLS) peptides [35, 36]. One of the most well-known NLS peptides is Pro-Lys-Lys–Lys-Arg-Lys-Val sequence (PKKKRKV), which is derived from the large T antigen of the SV40 virus and has been reported to improve the nuclear access and enhance gene delivery successfully. Zhang et al. used TAT-PKKKRKV peptide to carry VEGF165 plasmid to promote the expression of VEGF165 protein and angiogenesis . TAT-NLS can transfer various cell styles without specificity and selectivity because the TAT peptide sequence possesses the ability to quickly enter into almost all live cells .
After cellular uptake, the gene complexes should rapidly escape from endosomal/lysosomal compartment [39, 40]. Many studies focused on the promotion of endosomal/lysosomal escape. Polycations, such as polyethyleneimine and polyamidoamine dendrimers, have the ability to destroy the endosomal membrane and escape from endosomes owing to the proton sponge effect. Polycations can catch large amount of protons and cause the influx of Cl−, leading to the disruption of endosome and release of DNAs into cytoplasm [41, 42, 43, 44]. Moreover, histidine (H)-enriched gene carriers can also break the endosomal membrane due to the protonation of its imidazole ring. Imidazole ring facilitates gene delivery from endosome into cytosol because of its pH proton sponge effect [45, 46]. It exhibits hydrophobic under physiological condition (pH = 7.4) and becomes hydrophilic via protonating the unsaturated nitrogen in imidazole group in an acidic environment, such as endosome/lysosome [47, 48]. At low pH value (pH < 7.4), H-enriched gene carriers rupture the endosomal membrane and escape from endosomes . Herein, in this paper, we aimed to design and prepare the EC targeting gene carriers based on CPPs, REDV and oligohistidine (Hn, n = 4, 8 and 12) to overcome the multiple barriers in the process of gene delivery. These multifunctional carriers possessed EC targeting function, high internalization efficiency, enhanced endosome/lysosome escape capacity as well as nucleus translocation ability.
Peptides named REDV, REDV-YGRKKRRQRRR-PKKKRKV (abbreviated as REDV-TAT-NLS-H0), REDV-YGRKKRRQRRR-PKKKRKV-HHHH (abbreviated as REDV-TAT-NLS-H4), REDV-YGRKKRRQRRR-PKKKRKV-HHHHHHHH (abbreviated as REDV-TAT-NLS-H8) and REDV-YGRKKRRQRRR-PKKKRKV-HHHHHHHHHHHH (abbreviated as REDV-TAT-NLS-H12) were synthesized by GL Biochem. Ltd. (Shanghai, China). 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (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). Transwell chambers and Matrigel (Cat. Nos. 356234) was obtained from Corning Incorporated (New York, USA). TransScript First-Strand cDNA Synthesis SuperMix and TransStart™ Top Green qPCR SuperMix were purchased from Transgen Biotech Co., Ltd. (Beijing, China). BCA protein assay kit was obtained from Solarbio Science and Technology Co., Ltd. (Beijing, China). Rabbit anti-human ZNF580 polyclonal antibody, goat anti-rabbit IgG, amiloride hydrochloride, mouse anti-CD31 antibody and goat anti-mouse IgG H&L secondary antibody (Alexa Fluor® 594) 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 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). Human umbilical vein endothelial cells (HUVECs) were obtained from the Cell Bank of Typical Culture Collection of Chinese Academy of Sciences (Shanghai, China). The pZNF580-ZNF580 plasmid (pZNF580), smooth muscle cells (SMCs) and male mice were preserved by department of physiology and pathophysiology, Logistics University of Chinese People’s Armed Police Force.
Preparation and characterization of complexes
Preparation of REDV-TAT-NLS-Hn/pZNF580 complexes
The pZNF580 plasmid was diluted to 50 μg mL−1 with PBS (pH = 7.4) buffer. The REDV-TAT-NLS-Hn/pZNF580 complexes were prepared by mixing REDV-TAT-NLS-Hn solution (0.5 mg mL−1) and plasmid solution with various weight ratios ranging from 0.5 to 5. The gene complex solutions were stirred for 30 min at room temperature and used for following experiments.
Particle size and zeta potential measurements
The average particle size and zeta potential of REDV-TAT-NLS-Hn and REDV-TAT-NLS-Hn/pZNF580 complexes were measured using a Zetasizer Nano ZS (Malvern Instrument, Inc., Worcestershire, UK) with a constant angle of 173°.
Agarose gel electrophoresis
The condensing ability of different pZNF580 complexes was evaluated by agarose gel electrophoresis assay. Briefly, the gene complexes were mixed with 6× loading buffer and electrophoresed on the 0.8% agarose gel containing 5 μL GoldView with 1× TAE buffer at 120 V for 25 min. Naked DNA was used as control. A UV illuminator was used to visualize the location of DNA bonds.
Human umbilical vein endothelial cells (HUVECs) and human umbilical artery smooth muscle cells (HUASMCs) were, respectively plated in cell culture flasks and incubated with DMEM containing 10% FBS at 37 °C in humidified atmosphere with 5% CO2. The cells were cultured to reach 80–90% confluence before use.
In vitro cytotoxicity
The cytotoxicity of REDV-TAT-NLS-Hn and REDV-TAT-NLS-Hn/pZNF580 complexes for HUVECs was evaluated by MTT assay. Briefly, cells (1 × 104 cells/well) were plated in 96-well plate and cultured in DMEM containing 10% FBS for 24 h to achieve 80% confluence. Then the medium was removed and serum-free medium was added to each well and cultured overnight. After that, various REDV-TAT-NLS-Hn solutions and pZNF580 complexes at various concentrations ranging from 5 to 120 μg mL−1 were added and incubated for 48 h. Then, MTT reagent (20 μL, 5 mg mL−1) was added to each well and cultured for another 4 h to form formazan crystals. Then, the medium was removed, followed by adding 150 μL DMSO to dissolve the formazan crystals. The optical density was measured at 490 nm using a microplate reader (BIO-RAD, iMark™, USA). The relative cell viability was calculated as: relative cell viability (%) = (OD490(sample)/OD490(control)) × 100%, where OD490(sample) represents the absorbance value of experimental wells minus zero wells, and OD490(control) represents the absorbance value of untreated cell wells minus zero wells).
In vitro transfection
The transfection efficiency of REDV-TAT-NLS-Hn/pZNF580 complexes was evaluated by HUVECs and HUASMCs. In brief, HUVECs and HUASMCs were seeded in 24-well plates at a density of 1 × 105 cells per well and cultured with DMEM containing 10% FBS until 70–80% confluence. Before transfection, the cells were starved with serum-free medium for 12 h. REDV-TAT-NLS-Hn/pZNF580 complexes 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 in CO2 incubator. To detect the expression of pZNF580, green fluorescent protein was observed via an inverted fluorescent microscope (Fluorescence OLYMPUS U-RFLT50, microscopy Olympus DP72) at 24 h point.
Cell migration assay
The migration ability of the transfected cells was also evaluated using transwell chambers with 8.0 μm pore sized, gelatinized polycarbonate membrane. The upper transwell chambers were pre-treated with serum-free medium at 37 °C with 5% CO2 in incubator for 2 h. The transfected cells were starved with serum-free medium for 12 h, and then seeded in the upper transwell chambers (1.2 × 105 cells per well). At the same time, the lower transwell chambers were added with fresh medium containing 10% FBS, followed by incubating the transwell system for 6 h. The upper chambers were washed twice with 0.01 M PBS (pH = 7.4) and fixed with 4% paraformaldehyde/PBS (pH = 7.4) at room temperature for 10 min. Sterilized cotton swabs were used to remove the cells inside the chambers that didn’t pass through the 8.0 μm pore. The cells on the lower surface of the upper chambers were stained with eosin at 37 °C for 8 min. Migrating cells were observed under an inverted fluorescent microscope, and the number of migrated cells was counted by Image-Pro Plus 6.0 software.
Capillary-like tube formation
The formation ability of capillary-like tube structure was evaluated by HUVECs in vitro. Matrigel was dissolved at 4 °C overnight, then each well of the pre-cooling 96-well plate was coated with 50 μL growth factor-reduced Matrigel, and followed by incubation at 37 °C for 1 h. Subsequently, HUVECs were transfected with various REDV-TAT-NLS-Hn/pZNF580 complexes. The transfected cells were trypsinized and seeded on the Matrigel (4 × 104 cells per well), followed by being cultured for 6 h. The cells treated with pZNF580 were used as the negative control. Images of the formation of capillary-like structure at five randomly fields were obtained by using a microscope. The number of the formed tubes in each image was counted manually.
Western blot analysis
HUVECs were plated on a 6-well plate and transfected with REDV-TAT-NLS-Hn/pZNF580 complexes for 24 h. The transfected cells were washed three times with cold 0.01 M PBS (pH = 7.4), and followed by extracting the total protein with RIPA lysis buffer containing 1% volume of PMSF. After 30 min on ice, the lysates were centrifuged at 12,000 rpm at 4 °C for 10 min. The total protein was quantified by BCA protein assay kit and denatured by adding 5× SDS. The same amount of each sample (approximately 80 μg protein) was loaded into each lane, separated by 10% polyacrylamide SDS-PAGE gel and transferred onto polyvinylidene difluoride (PVDF) membranes. The membranes were cultured with 8% defat milk in TBST for 1 h and incubated with rabbit anti-ZNF580 polyclonal antibody in TBST overnight. Thereafter, blots were incubated with horseradish peroxidase conjugated anti-rabbit secondary antibody for 1 h and washed twice with TBST. Protein bands were developed by a standard enhanced chemiluminescence (ECL) kit, and observed via a gel image analysis system.
Quantitative real-time PCR assay
HUVECs were treated with various REDV-TAT-NLS-Hn/pZNF580 complexes and cultured in an incubator. The cells which were treated with pZNF580 were used as a negative control. After 24 h transfection, the total RNA was extracted from cells with TRIzol reagent, and reverse-transcribed into cDNA using TransScript First-Strand cDNA Synthesis SuperMix. The resulting cDNAs were used as templates for quantitative real-time PCR using TransStart™ Top Green qPCR SuperMix, and detected with a SYBR Green on ABI 7300 stepone sequence detection PCR system (Applied Biosystems). The PCR primer sequences of ZNF580 were as follows: ZNF580 forward 5′-AAAAAGCTTGTGGAGGCGCACGTGCTG-3′, and ZNF580 reverse 5′-AAAAAGATCTTGCCCGGAGTGCGCCCGTG-3′. The expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control. The forward and reverse primer sequences of GAPDH were 5′-AGGTGAAGGTCGGAGTCAAC-3′, 5′-CGCTCCTGGAAGATGGTGAT-3′, respectively. The results were analyzed using StepOne software v2.1.
Cellular uptake and confocal laser scanning microscopy (CLSM) assay
The cellular uptake and mean fluorescence intensity (MFI) in HUVECs were quantitatively evaluated by a flow cytometry. Cy5-oligonucleotide was mixed with unlabeled oligonucleotide at a 1:1 ratio. Cells were seeded into 6-well plates at 3 × 105 cells per well and transfected with various REDV-TAT-NLS-Hn/Cy5-oligonucleotide complexes. After 4 h incubation, cells were washed three times with 0.01 M PBS (pH = 7.4) and trypsinized with 0.25% trypsin. Subsequently, the cells were centrifuged, collected and re-suspended in 300 μL PBS (pH = 7.4), followed by analyzing with a flow cytometer (Beckman MoFlo XDP, USA). HUASMCs treated with REDV-TAT-NLS-H12/Cy5-oligonucleotide complexes was used as a control group.
Cellular uptake mechanism
To further elucidate the cellular uptake mechanism of REDV-TAT-NLS-Hn/Cy5-oligonucleotide complexes, HUVECs were pretreated with different inhibitors before transfection . Briefly, cells were seeded onto the 6-well plates and cultured as described above. To probe the cellular uptake mechanism of REDV-TAT-NLS-Hn/Cy5-oligonucleotide complexes, the cells were pretreated in DMEM with various endocytic inhibitors including chlorpromazine (30 μM), amiloride hydrochloride (30 μM) and filipin III (5 μg mL−1) at 37 °C for 1 h, which were used to inhibit the clathrin-mediated endocytosis, micropinocytosis and caveolae-mediated endocytosis, respectively. REDV peptide was also used to pretreat the cells and cultured for 1 h before transfection in order to evaluate the REDV function in endocytic pathway. The REDV-TAT-NLS-H12/Cy5-oligonucleotide complexes were added into each well for 4 h incubation. Subsequently, cells were washed three times with 0.01 M PBS (pH = 7.4) followed by trypsinization and centrifugation. Cells were then re-suspended in 300 μL PBS (pH = 7.4) and analyzed by a flow cytometry (Beckman MoFlo XDP, USA).
In vivo angiogenesis assay
To evaluate the angiogenesis ability of the transfected HUVECs by REDV-TAT-NLS-Hn/pZNF580 complexes, in vivo angiogenesis assay was performed as previously described . Male mice (6 weeks old, 20–25 g) were used as an animal experimental model. HUVECs were pretreated with different REDV-TAT-NLS-Hn/pZNF580 complexes for 4 h and cultured for another 24 h. Then the transfected cells were trypsinized with 0.25% trypsin and mixed with 800 μL matrigel at a final concentration of 1 × 106 cells mL−1. Before surgery, male mice were treated with chloral hydrate (300 mg/kg) for anesthesia. The mixture was subcutaneously injected in mice using a 1 mL syringe with a 25-gauge needle. Four days later, the mice were injected with excess chloral hydrate to euthanasia. Matrigel implants were removed, fixed with formalin, embedded in paraffin, and sliced into thick sections. Then the sections were stained with hematoxylin and eosin (H&E) and the luminal structures were observed using a microscope. In addition, the sections were immunohistochemically stained with mouse anti-CD31 antibody [diluted in PBS (pH = 7.4) at 1:20] for 60 min and followed with goat anti-mouse IgG H&L secondary antibody (Alexa Fluor® 594). The cell nuclei were stained with DAPI. Immunohistochemically stained sections were used to further determine the formation of microvessel structure which was observed by a fluorescence microscope for each section.
Male mice were preserved by Department of Physiology and Pathophysiology, Logistics University of Chinese People’s Armed Police Force and hosted in SPF room of animal house. All animals were treated following the protocol approved by Armed Police Logistics College and conformed to the “Guide for the protection and use of experimental animals” of the American National Institutes of Health.
All statistical analyses were performed with the one-way ANOVA with a P < 0.05 being considered significant.
Particle size and zeta potential of REDV-TAT-NLS-Hn micelles and their gene complexes
Size and zeta potential of REDV-TAT-NLS-Hn micelles
Zeta potential (mV)
228.1 ± 5.3
0.30 ± 0.04
27.8 ± 1.8
142.3 ± 4.3
0.32 ± 0.02
25.8 ± 1.7
151.7 ± 2.5
0.30 ± 0.03
26.7 ± 0.4
136.6 ± 1.7
0.24 ± 0.06
26.3 ± 0.3
Hemocompatibility of REDV-TAT-NLS-Hn peptides
pDNA binding ability of REDV-TAT-NLS-Hn peptides
In vitro cytotoxicity
In vitro transfection
Cell migration assay
In vitro tube formation
Western blot analysis
Quantitative real-time RT-PCR assay
Cellular uptake and CLSM
Besides, the MFI of transfected HUASMCs was also evaluated to verify the target function of the gene complexes. In Fig. 12(3), the results showed that the MFI of transfected HUVECs was significantly higher than in HUASMCs, which demonstrated the targeting ability of REDV-TAT-NLS-H12/Cy5-oligonucleotide complexes to HUVECs.
Furthermore, the intercellular distribution of Cy5-oligonucleotide in transfected HUVECs was evaluated by CLSM. The intracellular yellow pixels represented the entrapment of REDV-TAT-NLS-Hn/Cy5-oligonucleotide complexes in the endosome/lysosome, whereas the intracellular pink pixels illustrated their presence in nucleus. As shown in Fig. 12(4), the nucleus CLR of REDV-TAT-NLS-Hn/Cy5-oligonucleotide complexes increased with increasing Hn sequence length, while the lysosome CLR decreased. These results demonstrated that more complexes with longer Hn sequence entered into HUVECs, escaped from endosome/lysosomes into cytoplasm and entered into nucleus. As aforementioned, the Hn sequence was hydrophobic at pH 7.4, which was advantageous for cellular uptake. Besides, this Hn sequence could change to hydrophilic and positive-charged under acid condition in endosome/lysosome. Thus, it could help endosomal/lysosomal membrane rupture and escape owing to the pH buffer capacity of imidazole ring in Hn residues [59, 60]. In addition, the nucleus CLR of REDV-TAT-NLS-H12/Cy5-oligonucleotide complexes was 58.5 ± 4.0%, which was about sixfold as high as REDV-TAT-NLS-H0/Cy5-oligonucleotide complexes group. These results indicated that these peptides containing Hn, REDV and NLS sequences, especially the REDV-TAT-NLS-H12 peptide can promote the cellular uptake efficiency, endosome/lysosome escape ability and nuclear location capacity, which could improve the efficiency of gene delivery.
Cellular uptake mechanism
Gene carriers can enter into cells via several pathways, such as micropinocytosis, clathrin-mediated endocytosis, caveolae-mediated endocytosis and other endocytic pathways [61, 62, 63, 64]. To study the cellular uptake mechanism of these REDV-TAT-NLS-Hn/pZNF580 complexes, different inhibitors were used for particular endocytic pathways. In this paper, three typical endocytosis pathways including clathrin-mediated endocytosis, caveolae-mediated endocytosis and micropinocytosis were studied. Amiloride (Amil) is reported to inhibit the micropinocytosis by blocking the Na+/H+ channels, chlorpromazine (CPZ) can inhibit clathrin-mediated endocytosis by the interruption of clathrin, while filipin (Filip) can inhibit the caveolae-mediated endocytosis via combination with cholesterol .
In vivo angiogenesis assay
In recent years, gene therapy offers a promising alternative for the treatment of cardiovascular diseases. The development of non-virus gene carriers with low cytotoxicity and high safety is especially necessary for vascularization. Cationic peptides are synthesized from natural amino acids, which are widely used as gene carriers. Compared with other gene carriers, the peptide-based gene carriers exhibit relatively low cytotoxicity and good biocompatibility. As a kind of widely used CPPs, the arginine-rich TAT peptide shows high ability of crossing cell membrane to overcome the first barrier of gene delivery. Herein, three peptides with integrated sequence of REDV-YGRKKRRQRRR-PKKKRKV-Hn (REDV-TAT-NLS-Hn, n = 4, 8 and 12) were designed as the gene carrier for pZNF580 plasmid, and REDV-YGRKKRRQRRR-PKKKRKV-H0 without oligohistidine sequence was used as a control. In order to deliver genes into HUVECs selectively, REDV peptide was inserted into the peptide sequence due to its specific recognition of α4β1 integrin on the membrane of ECs. To increase the endosomal escape ability of the peptide gene carrier, oligohistidine Hn sequences were used to acquire cationic ions under acid condition especially in endosome/lysosome.
The REDV-TAT-NLS-Hn/pZNF580 complexes were prepared by adding pZNF580 plasmid to REDV-TAT-NLS-Hn solution. The size of REDV-TAT-NLS-Hn/pZNF580 complexes ranged from 117 to 288 nm and their zeta potential could be regulated by changing the weight ratio of peptide and pZNF580. According to the results of agarose gel electrophoresis, REDV-TAT-NLS-H0, REDV-TAT-NLS-H4, REDV-TAT-NLS-H8 and REDV-TAT-NLS-H12 peptides could completely condense the negative-charged pZNF580 plasmid to form gene complexes at the w/w ratio of 2, 2, 2 and 3, respectively. In addition, these peptides exhibited good blood compatibility and less destructed RBCs. The H residue possesses pH buffer capacity and hydrophobicity at pH 7.4. Thus, the positive charge of these peptides decreased when Hn sequence increased from 4 to 12. According to the cytotoxicity assay, the relative cell viability of the peptides and their gene complexes was higher than 80% even at a high concentration (120 μg mL−1), which showed a brilliant low cytotoxicity compared with PEI (25 kDa) . The cellular uptake and MFI of the complexes increased when the Hn sequence length increased from 4 to 12. The reason is that the hydrophobicity of gene carriers expedites the hydrophobic interaction with cell membrane, which can promote their cellular internalization . Moreover, the MFI of REDV-TAT-NLS-H12/Cy5-oligonucleotide complexes in HUVECs was about twofold as much as that in HUASMCs, which exhibited the specific recognition ability to HUVECs due to REDV in peptide sequence. Compared with the REDV-TAT-NLS-H0/pZNF580 complexes, REDV-TAT-NLS-Hn/pZNF580 complexes showed higher gene delivery and transfection efficiency due to the synergistic effects of the peptide sequences to enhance cellular uptake, endosome/lysosome escape and nucleus translocation. The results of western blot analysis and PCR analysis also demonstrated the same tendency of ZNF580 expression at the protein level and mRNA level. For the HUVEC migration assay, the migration rate of REDV-TAT-NLS-Hn/pZNF580 complexes groups was much higher than that of the REDV-TAT-NLS-H0/pZNF580 group because of their higher transfection efficiency. REDV-TAT-NLS-H12/pZNF580 group showed the highest migration rate (224 migrated cells), which was higher than the standard transfection reagent PEI 25 kDa/pZNF580 group . Furthermore, according to the intercellular distribution assay, the REDV-TAT-NLS-H12/Cy5-oligonucleotide complexes showed the highest ability of endosome/lysosome escape.
In order to investigate the cellular uptake mechanism of the REDV-TAT-NLS-H12/pZNF580 complexes, cells were pre-treated with different inhibitors before transfection. The result demonstrated that clathrin-mediated endocytosis was the main pathway for the cellular uptake of REDV-TAT-NLS-H12/Cy5-oligonucleotide complexes, and other pathways including caveolae-mediated endocytosis, micropinocytosis and other endocytosis pathways also contributed to the cellular uptake. In addition, targeting REDV peptide in REDV-TAT-NLS-H12/Cy5-oligonucleotide complexes also benefited for the cellular uptake.
To evaluate the tube formation ability of HUVECs treated by REDV-TAT-NLS-Hn/pZNF580 complexes, the angiogenesis assay was performed both in vitro and in vivo. The cells transfected with the REDV-TAT-NLS-Hn/pZNF580 complexes could enhance the tube formation ability compared with the naked pZNF580 group. The angiogenesis ability of HUVECs transfected by REDV-TAT-NLS-H12/pZNF580 complexes was the highest among all of the groups. These complexes could efficiently deliver gene into cells.
According to the above results, the multifunctional REDV-TAT-NLS-Hn micelles could efficiently condense pZNF580, enhance gene delivery ability, and promote the migration, proliferation as well as neovascularization ability of HUVECs, which have great potential in the application of therapeutic vascularization for various vascular diseases.
In summary, this paper designed the multifunctional targeting peptide sequences of REDV-YGRKKRRQRRR-PKKKRKV-Hn (n = 4, 8 and 12) as a gene carrier for improving the cellular uptake and gene delivery. TAT peptide and targeting REDV peptide benefited for the cellular uptake of pZNF580 plasmid in HUVECs. And the Hn sequence simultaneously improved the internalization efficiency and endosomal escape of REDV-TAT-NLS-Hn/pZNF580 complexes, which further enhanced the expression of ZNF580 as well as the proliferation and migration ability of HUVECs. The expression of ZNF580 could enhance the tube formation ability of transfected HUVECs in vivo. Overall, these multifunctional peptide gene carriers provide an outstanding platform for rapid endothelialization and revascularization.
QL designed the experiments, performed the cell experiments including MTT, transfection assay, cell migration assay, tube formation assay, cellular uptake and CLSM, performed molecular biology experiments including western blot and quantitative real-time PCR, did animal experiments, and drafted manuscript. XFH prepared peptides and characterized peptides. YKF conceived research, participated in experimental design, and participated in manuscript writing. JTG, XKR, CCS and WCZ helped to improve the manuscript. All authors read and approved the final manuscript.
This project was supported by National Key R&D Program of China (Grant No. 2016YFC1100300), National Natural Science Foundation of China (Grant Nos. 31370969 and 51673145), International Science & Technology Cooperation Program of China (Grant No. 2013DFG52040).
The authors declare that they have no competing interests.
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All data generated or analyzed during this study are included in the article.
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All the co-authors were aware of this submission and approve for publication.
Ethical approval and consent to participate
This study was performed with the “Guide for the protection and use of experimental animals” and was approved by the Committee of Ethics of Armed Police Logistics College.
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