Xuefang Hao

CREDVW-Linked Polymeric Micelles As a Targeting Gene Transfer Vector for Selective Transfection and Proliferation of Endothelial Cells

Nowadays, gene transfer technology has been widely used to promote endothelialization of artificial vascular grafts. However, the lack of gene vectors with low cytotoxicity and targeting function still remains a pressing challenge. Herein, polyethylenimine (PEI, 1.8 kDa or 10 kDa) was conjugated to an amphiphilic and biodegradable diblock copolymer poly(ethylene glycol)-b-poly(lactide-co-glycolide) (mPEG-b-PLGA) to prepare mPEG-b-PLGA-g-PEI copolymers with the aim to develop gene vectors with low cytotoxicity while high transfection efficiency. The micelles were prepared from mPEG-b-PLGA-g-PEI copolymers by self-assembly method. Furthermore, Cys-Arg-Glu-Asp-Val-Trp (CREDVW) peptide was linked to micelle surface to enable the micelles with special recognition for endothelial cells (ECs). In addition, pEGFP-ZNF580 plasmids were condensed into these CREDVW-linked micelles to enhance the proliferation of ECs. These CREDVW-linked micelle/pEGFP-ZNF580 complexes exhibited low cytotoxicity by MTT assay. The cell transfection results demonstrated that pEGFP-ZNF580 could be transferred into ECs efficiently by these micelles. The results of Western blot analysis showed that the relative ZNF580 protein level in transfected ECs increased to 76.9%. The rapid migration of transfected ECs can be verified by wound healing assay. These results indicated that CREDVW-linked micelles could be a suitable gene transfer vector with low cytotoxicity and high transfection efficiency, which has great potential for rapid endothelialization of artificial blood vessels.

Biodegradable PEI modified complex micelles as gene carriers with tunable gene transfection efficiency for ECs

In recent years, gene therapy has evoked an increasing interest in clinical treatments of coronary diseases because it is a potential strategy to realize rapid endothelialization of artificial vascular grafts. The balance of high transfection efficiency and low cytotoxicity of nonviral gene carriers is an important issue to be solved. In this study, we aim to establish a gene delivery system offering an elegant way to tune the transfection activity and cytotoxicity. Biodegradable complex micelles were prepared from polyethylenimine-b-poly(lactide-co-3(S)-methyl-morpholine-2,5-dione)-b-polyethylenimine (PEI-b-PLMD-b-PEI) and methoxy-poly(ethylene glycol)-b-poly(lactide-co-3(S)-methyl-morpholine-2,5-dione) (mPEG-b-PLMD) copolymers by a co-assembly method. Then the ZNF580 gene plasmid (pDNA) was encapsulated into the complex micelles. The hydrodynamic size and zeta potential of these complex micelles and micelles/pDNA complexes indicated that they were feasible for use in cellular uptake and gene transfection. As expected, the transfection efficiency and cytotoxicity of these micelles/pDNA complexes could be conveniently tuned by changing the mass ratio of mPEG-b-PLMD to PEI-b-PLMD-b-PEI (3/1, 2/2, 1/3 and 0/4) in the mixed mPEG/PEI shell. The transfection efficiency increased as the mass ratio of mPEG-b-PLMD/PEI-b-PLMD-b-PEI decreased from 3/1 to 0/4, while the cytotoxicity showed an opposite tendency. Moreover, ZNF580 protein expression determined by Western blot analysis and the migration of transfected endothelial cells (ECs) by wound healing assay were consistent with the result of transfection efficiency. All these results indicated that the co-assembled complex micelles could act as suitable gene carriers with tunable gene transfection efficiency and cytotoxicity, which should have great potential for the transfection of vascular ECs.

CAGW Peptide- and PEG-Modified Gene Carrier for Selective Gene Delivery and Promotion of Angiogenesis in HUVECs in Vivo

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 CAG- and PEG-modified copolymers could form nanoparticles (NPs) by self-assembly method and were used as gene carriers for the pEGFP-ZNF580 (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.

Mixed micelles obtained by co-assembling comb-like and grafting copolymers as gene carriers for efficient gene delivery and expression in endothelial cells

Gene delivery can enhance the endothelialization of biomaterial surfaces. However, the lack of efficient target function is still the major concern that hinders the clinical application of gene therapy. With the aim to develop a specific targeting gene carrier for endothelial cells (ECs), the Cys-Arg-Glu-Asp-Val-Trp (CREDVW) peptide was linked to the comb-like copolymer of poly(lactide-co-3(S)-methyl-morpholine-2,5-dione)-poly(poly(ethylene glycol) monomethacrylate) (PLMD-PPEGMA) to form the CREDVW modified copolymer PLMD-PPEGMA-CREDVW, which could enhance the special recognition of ECs. Mixed micelles were then prepared by co-assembling this comb-like copolymer and the amphiphilic grafting copolymer poly(lactide-co-3(S)-methyl-morpholine-2,5-dione)-g-polyethylenimine (PLMD-g-PEI). These mixed micelles with the CREDVW-functional peptide exhibited good pEGFP-ZNF580 (pDNA) binding ability and could condense it into complexes with proper size and positive zeta potential. The MTT results demonstrated the low cytotoxicity of the CREDVW-modified mixed micelle/pDNA complexes. The internalization efficiency of the CREDVW-modified complexes with targeting function was about two times higher than the dysfunctional CREVDW-modified complexes. Besides, the transfection efficiency of these complexes was more pronounced, compared to the control group, PEI(10 kDa)/pDNA, as detected by means of in vitro transfection studies. Western blot analysis demonstrated relatively high protein levels in the transfected cells by CREDVW-modified mixed micelle/pDNA complexes, up to 75%, in comparison to the control group (26%). In addition, the cell migration ability was significantly improved as demonstrated by the wound healing assay. These results indicated that the mixed micelles could act as an active targeting gene carrier, having both tunable gene transfection efficiency and low cytotoxicity, which are beneficial for the endothelialization of biomaterial surface.

Co-self-assembly of cationic microparticles to deliver pEGFP-ZNF580 for promoting the transfection and migration of endothelial cells.

The gene transfection efficiency of polyethylenimine (PEI) varies with its molecular weight. Usually, high molecular weight of PEI means high gene transfection, as well as high cytotoxicity in gene delivery in vivo. In order to enhance the transfection efficiency and reduce the cytotoxicity of PEI-based gene carriers, a novel cationic gene carrier was developed by co-self-assembly of cationic copolymers. First, a star-shaped copolymer poly(3(S)-methyl-morpholine-2,5-dione-co-lactide) (P(MMD-co-LA)) was synthesized using D-sorbitol as an initiator, and the cationic copolymer (P(MMD-co-LA)-g-PEI) was obtained after grafting low-molecular weight PEI. Then, by co-self-assembly of this cationic copolymer and a diblock copolymer methoxy-poly(ethylene glycol) (mPEG)-b-P(MMD-co-LA), microparticles (MPs) were formed. The core of MPs consisted of a biodegradable block of P(MMD-co-LA), and the shell was formed by mPEG and PEI blocks. Finally, after condensation of pEGFP-ZNF580 by these MPs, the plasmids were protected from enzymatic hydrolysis effectively. The result indicated that pEGFP-ZNF580-loaded MP complexes were suitable for cellular uptake and gene transfection. When the mass ratio of mPEG-b-P(MMD-co-LA) to P(MMD-co-LA)-g-PEI reached 3/1, the cytotoxicity of the complexes was very low at low concentration (20 μg mL−1). Additionally, pEGFP-ZNF580 could be transported into endothelial cells (ECs) effectively via the complexes of MPs/pEGFP-ZNF580. Wound-healing assay showed that the transfected ECs recovered in 24 h. Cationic MPs designed in the present study could be used as an applicable gene carrier for the endothelialization of artificial blood vessels.

Multi-targeting peptides for gene carriers with high transfection efficiency

Non-viral gene carriers for gene therapy have been developed for many years. But the gene transfection is generally limited by deficient cellular uptake, low endo/lysosome escape, and weak nuclear translocation. Some targeting peptides have been conjugated onto gene carriers for highly efficient gene delivery. These targeting carriers can overcome some of these limitations to efficiently deliver therapeutic genes into desired cells. In this review, we will summarize the recent development of multi-targeting peptide immobilized non-viral gene carriers for efficient gene therapy, especially for the targeting and suppression of tumor cells, and the transfection and proliferation of endothelial cells. The peptide functionalization of gene carriers is a promising strategy to promote the elimination of solid tumors and the rapid endothelialization of artificial blood vessels.

Oligohistidine and targeting peptide functionalized TAT-NLS for enhancing cellular uptake and promoting angiogenesis in vivo

BackgroundGene 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).ResultsMTT 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.ConclusionThe 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.

Electrospun PCL-PIBMD/SF blend scaffolds with plasmid complexes for endothelial cell proliferation

Tissue engineering scaffolds with gene delivery function play an important role in DNA-based vascular tissue engineering. In the present work, we used biodegradable polyester–polydepsipeptide, silk fibroin (SF) and gene complexes to prepare electrospun scaffolds encapsulating gene complexes in order to enhance the proliferation of endothelial cells. A series of nanofibrous scaffolds with different properties including fiber diameter, hydrophilicity, porosity and mechanical properties were prepared by electrospinning technology with adjusting the weight ratio of poly(e-caprolactone)-b-poly(isobutyl-morpholine-2,5-dione) (PCL-PIBMD) and SF. PCL-PIBMD/SF blend scaffolds were optimized to obtain the scaffolds with a weight ratio of 90/10 to have superior mechanical performance and good biocompatibility. pEGFP-ZNF580 plasmid (pZNF580) complexes were electrosprayed onto these PCL-PIBMD/SF blend scaffolds to promote the proliferation of endothelial cells. In order to maintain the stability and integrity of plasmid complexes loaded in scaffolds, the composite scaffolds were fabricated by alternatively layer-by-layer electrospinning and electrospraying techniques. These composite scaffolds showed obviously low platelet adhesion and good histocompatibility. They could effectively enhance the adhesion, spreading and proliferation of human umbilical vein endothelial cells. These results indicated that the composite scaffolds could serve as an attractive platform to deliver therapeutic genes for vascular tissue engineering.

Core/Shell Gene Carriers with Different Lengths of PLGA Chains to Transfect Endothelial Cells

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

Red-blood-cell-mimetic gene delivery systems for long circulation and high transfection efficiency in ECs

Recently, the red blood cell (RBC) membrane has been used as a mimetic nanocoating for nanoparticles for drug delivery systems to promote their biocompatibility. In the present study, the nano-sized RBC membrane was coated on the surface of gene complexes through electrostatic interactions to prepare biomimetic gene delivery systems so as to improve their biocompatibility and prolong their circulation time in vivo. The structure of the biomimetic gene delivery systems was determined by transmission electron microscopy (TEM) and confocal laser scanning microscopy (CLSM). They exhibited low cytotoxicity and high transfection efficiency in endothelial cells (ECs), which could improve the migration ability of ECs. Besides, the biomimetic gene delivery systems exhibited strong immune evasion and long in vivo circulation time. The phagocytic rate of these biomimetic gene delivery systems reduced 52% compared with that of the PLGA-PEI/pZNF580 control group (without RBC membrane modification). Their circulation time in vivo was more than 2 times higher than that of the control group. Consequently, we provide a simple method for the preparation of camouflaged gene delivery systems, which can further facilitate the development of a gene delivery platform for the therapy of vascular diseases via enhancing EC transfection. This strategy will open up a new avenue for gene delivery systems by RBC membrane camouflage.