Jintang Guo

Hemocompatible surface of electrospun nanofibrous scaffolds by ATRP modification

The electrospun scaffolds are potential application in vascular tissue engineering since they can mimic the nano-sized dimension of natural extracellular matrix (ECM). We prepared a fibrous scaffold from polycarbonateurethane (PCU) by electrospinning technology. In order to improve the hydrophilicity and hemocompatibility of the fibrous scaffold, poly(ethylene glycol) methacrylate (PEGMA) was grafted onto the fiber surface by surface-initiated atom transfer radical polymerization (SI-ATRP) method. Although SI-ATRP has been developed and used for surface modification for many years, there are only few studies about the modification of electrospun fiber by this method. The modified fibrous scaffolds were characterized by SEM, Fourier transform infrared (FTIR), and X-ray photoelectron spectroscopy (XPS). The scaffold morphology showed no significant difference when PEGMA was grafted onto the scaffold surface. Based on the water contact angle measurement, the surface hydrophilicity of the scaffold surface was improved significantly after grafting hydrophilic PEGMA (P=0.0012). The modified surface showed effective resistance for platelet adhesion compared with the unmodified surface. Activated partial thromboplastin time (APTT) of the PCU-g-PEGMA scaffold was much longer than that of the unmodified PCU scaffold. The cyto-compatibility of electrospun nanofibrous scaffolds was tested by human umbilical vein endothelial cells (HUVECs). The images of 7-day cultured cells on the scaffold surface were observed by SEM. The modified scaffolds showed high tendency to induce cell adhesion. Moreover, the cells reached out pseudopodia along the fibrous direction and formed a continuous monolayer. Hemolysis test showed that the grafted chains of PEGMA reduced blood coagulation. These results indicated that the modified electrospun nanofibrous scaffolds were potential application as artificial blood vessels.

Grafting of phosphorylcholine functional groups on polycarbonate urethane surface for resisting platelet adhesion

In order to improve the resistance of platelet adhesion on material surface, 2-methacryloyloxyethyl phosphorylcholine (MPC) was grafted onto polycarbonate urethane (PCU) surface via Michael reaction to create biomimetic structure. After introducing primary amine groups via coupling tris(2-aminoethyl)amine (TAEA) onto the polymer surface, the double bond of MPC reacted with the amino group to obtain MPC modified PCU. The modified surface was characterized by Fourier transform infrared (FTIR) spectroscopy and X-ray photoelectron spectroscopy (XPS). The results verified that MPC was grafted onto PCU surface by Michael reaction method. The MPC grafted PCU surface had a low water contact angle and a high water uptake. This means that the hydrophilic PC functional groups improved the surface hydrophilicity significantly. In addition, surface morphology of MPC grafted PCU film was imaged by atomic force microscope (AFM). The results showed that the grafted surface was rougher than the blank PCU surface. In addition, platelet adhesion study was evaluated by scanning electron microscopy (SEM) observation. The PCU films after treated with platelet-rich plasma demonstrated that much fewer platelets adhered to the MPC-grafted PCU surface than to the blank PCU surface. The antithrombogenicity of the MPC-grafted PCU surface was determined by the activated partial thromboplastin time (APTT). The result suggested that the MPC modified PCU may have potential application as biomaterials in blood-contacting and some subcutaneously implanted devices.

Electrospun hemocompatible PU/gelatin-heparin nanofibrous bilayer scaffolds as potential artificial blood vessels

Abstract

Targeting REDV peptide functionalized polycationic gene carrier for enhancing the transfection and migration capability of human endothelial cells

Targeting gene engineering should be considered as an effective method for promoting endothelialization of vascular grafts. Herein, we developed a targeting REDV peptide functionalized polycationic gene carrier for carrying the pEGFP-ZNF580 plasmid with the aim of enhancing the transfection and migration capability of human endothelial cells. This polycationic gene carrier with the REDV peptide (mPEG-P(LA-co-CL)-PEI-REDV) was prepared by the conjugation of the Cys-Arg-Glu-Asp-Val-Trp (CREDVW) peptide with the amphiphilic block copolymer methoxy poly(ethylene glycol) ether-poly(l-lactide-co-ε-caprolactone)-poly(ethyleneimine) (mPEG-P(LA-co-CL)-PEI). mPEG-P(LA-co-CL)-PEI nanoparticles (NP) and mPEG-P(LA-co-CL)-PEI-REDV nanoparticles (REDV-NP) were formed by the self-assembly of the corresponding polycationic polymers, and then their pEGFP-ZNF580 complexes were prepared via the electrostatic interaction with pEGFP-ZNF580 plasmids, respectively. Gel electrophoresis results show that the targeted REDV-NPs could compress pEGFP-ZNF580 plasmids into stable complexes and protect the plasmids against desoxyribonuclease degradation. MTT assay indicates that these targeted REDV-NP/pEGFP-ZNF580 complexes exhibit better cyto-compatibility than the non-targeted NP/pEGFP-ZNF580 complexes and the control PEI 1800 Da/pEGFP-ZNF580 complexes. In vitro transfection experiments and western blot analysis of EA.hy926 endothelial cells show that the pEGFP-ZNF580 plasmid expression and the relative protein level transfected by targeted REDV-NP/pEGFP-ZNF580 complexes are roughly consistent with that transfected by PEI 25 kDa/pEGFP-ZNF580 complexes. More importantly, the scratch wound assay results demonstrate that the migration capability of EA.hy926 cells has been improved significantly by the expression of the pEGFP-ZNF580 plasmid. Our results indicate that the polycationic polymer with functional REDV peptides can be a potential candidate as a pEGFP-ZNF580 plasmid delivery carrier and may be used in the endothelialization of vascular grafts.

Functionalization of polycarbonate surfaces by grafting PEG and zwitterionic polymers with a multicomb structure.

The hemocompatibility of polycarbonateurethane (PCU) surfaces is improved by decoration with poly(poly(ethylene glycol) methacrylate) (poly(PEGMA)) and zwitterionic poly(3-((2-(methacryloyloxy)ethyl)dimethylammonio)propane-1-sulfonate) (poly(DMAPS)) blocks providing a novel multicomb structure obtained by application of surface-initiated atom transfer radical polymerization (s-ATRP) conditions. The PCU-poly(PEGMA-g-DMAPS) surface shows high hydrophilicity with a low contact angle of 20.6 ± 1.8°, while PCU-poly(PEGMA-b-DMAPS) surface exhibitsed a contact angle of 30.5 ± 2.6°. Furthermore, PCU-poly(PEGMA-g-DMAPS) surface shows very low platelet adsorption indicating that multicomb structure modified PCUs are preferred candidate materials for blood-contacting materials.

Modification of polycarbonateurethane surface with poly (ethylene glycol) monoacrylate and phosphorylcholine glyceraldehyde for anti-platelet adhesion

Poly(ethylene glycol) monoacrylate (PEGMA) is grafted onto polycarbonateurethane (PCU) surface via ultraviolet initiated photopolymerization. The hydroxyl groups of poly(PEGMA) on the surface react with one NCO group of isophorone diisocyanate (IPDI) and another NCO group of IPDI is then hydrolyzed to form amino terminal group, which is further grafted with phosphorylcholine glyceraldehyde to establish a biocompatible hydrophilic structure on the surface. Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy confirm the successful grafting of both PEG and phosphorylcholine functional groups on the surface. The decrease of the water contact angle for the modified film is caused by synergic effect of PEG and phosphorylcholine, which both have the high hydrophilicity. Furthermore, the number of platelets adhered is relative low on the synergetically modified PCU film compared with the PCU film modified only by poly(PEGMA). Our synergic modification method using both PEG and phosphorylcholine may be applied in surface modification of blood-contacting biomaterials and some relevant devices.

Immobilized Bioactive Agents onto Polyurethane Surface with Heparin and Phosphorylcholine Group

AbstractHeparin (HEP) and phosphorylcholine groups (PC) were grafted onto the polyurethane (PU) surface in order to improve biocompatibility and anticoagulant activity. After the surface grafting sites of PU were amplified with the primary amine groups of polyethylenimine (PEI), heparin was covalently linked onto the surface by the reaction between the amino group and the carboxyl group. PC groups were covalently immobilized on the PU-PEI surface through the reaction between the amino group and the aldehyde group of phosphorylcholine glyceraldehyde (PCGA). The surface density of primary amine groups was determined by a ninhydrin assay. The amino group density reached a maximum of 0.88 μmol/cm2 upon incorporation of 10 wt% PEI. The amount of heparin covalently immobilized on the PU-PEI surface was determined by the toluidine blue method. The grafting chemistry resulted in the comparatively dense immobilization of HEP (2.6 μg/cm2) and PC to the PU-PEI surfaces. The HEP and PC modified surfaces were characterized by water uptake (PU 0.15 mg/cm2, PU-PEI 3.54 mg/cm2, PU-HEP 2.04 mg/cm2, PU-PC 2.38 mg/cm2), water contact angle (PU 95.3°, PU-PEI 34.0°, PU-HEP 39.5°, PU-PC 37.2°), attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR), X-ray photoelectron spectroscopy (XPS), atomic force microscopy (AFM), and scanning electron microscope (SEM). The results demonstrated that the PUPEI surface was successfully grafted with HEP and PC. The hydrophilicity and hemocompatibility of these grafted surfaces were significantly improved. These results suggested that the PU-HEP and PU-PC composite films are promising candidates for blood contacting tissue engineering.

Electrospun scaffolds of silk fibroin and poly(lactide-co-glycolide) for endothelial cell growth

Abstract Electrospun scaffolds of silk fibroin (SF) and poly(lactide-co-glycolide) (PLGA) were prepared to mimic the morphology and chemistry of the extracellular matrix. The SF/PLGA scaffolds were treated with ethanol to improve their usability. After ethanol treatment the scaffolds exhibited a smooth surface and uniform fibers. SF transformed from random coil conformation to β-sheet structure after ethanol treatment, so that the SF/PLGA scaffolds showed low hydrophilicity and dissolving rate in water. The mechanical properties and the hydrophilicity of the blended fibrous scaffolds were affected by the weight ratio of SF and PLGA. During degradation of ethanol-treated SF/PLGA scaffolds in vitro, the fibers became thin along with the degradation time. Human umbilical vein endothelial cells (HUVECs) were seeded onto the ethanol-treated nanofibrous scaffolds for cell viability, attachment and morphogenesis studies. These SF/PLGA scaffolds could enhance the viability, spreading and attachment of HUVECs. Based on these results, these ethanol-treated scaffolds are proposed to be a good candidate for endothelial cell growth.

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.

Comb-shaped polymer grafted with REDV peptide, PEG and PEI as targeting gene carrier for selective transfection of human endothelial cells

If plasmid complexes prepared from targeting carriers have endothelial cell selectivity and high transfection efficiency, they can specifically enhance rapid endothelialization by delivering the corresponding gene plasmids. A high content of functional groups in the carriers benefits high selectivity efficiency. Herein, we have synthesized a comb-shaped polymer bearing several Arg-Glu-Asp-Val (REDV) peptides and poly(ethylene glycol) as a pEGFP-ZNF580 gene carrier with cell-type recognition of human endothelial cells. An amphiphilic block copolymer of poly(2-hydroxyethyl methacrylate)-block-poly(ε-caprolactone)-graft-poly(ethylene glycol)-graft-poly(ethyleneimine) conjugated with REDV peptide (PHEMA-b-PCL-g-PEG-g-PEI-REDV) is synthesized, and nanoparticles of it are prepared by polymer self-assembly. This polycationic PHEMA-b-PCL-g-PEG-g-PEI-REDV carrier effectively condenses pEGFP-ZNF580 plasmid to form REDV-targeted complexes and protects pEGFP-ZNF580 integrity from enzymatic hydrolysis. These REDV-targeted complexes exhibit low cytotoxicity but high transfection efficiency to EA.hy926 cells compared with non-targeted complexes (PHEMA-b-PCL-g-PEG-g-PEI/pEGFP-ZNF580) as demonstrated by MTT assay, fluorescence-activated cell sorting analysis and fluorescence analysis. Furthermore, the relative protein level of endothelial cells transfected by REDV-targeted complexes is higher than that by non-targeted complexes. Therefore, REDV-bearing carriers may have potential as effective and targeting transfer carriers for pEGFP-ZNF580 plasmid, and their complexes can be used in the endothelialization of artificial vascular grafts.