PEGylation of polyamidoamine dendrimer and the properties for gene vectors / 药学学报

Polyamidoamine-polyethylene glycol (PAMAM-PEG) copolymers were synthesized using IPDI as coupling reagent by two-step method. The copolymers were characterized by IR spectrum and 1H NMR spectrum, and the PEG conjugating ratios of the copolymers were calculated equal to 10% and 30% separately. MTT assay indicated that after PEGylation a lower cytotoxicity of the copolymers could be found, and with increasing PEG conjugating ratio the cytotoxicity decreased obviously. Agarose gel retardation assay demonstrated that PAMAM-PEG copolymers could be combined with DNA and PAMAM-PEG/DNA complexes were prepared by self-assembly. DLS measurement showed that when N/P > or = 50, the particle size of copolymer/ gene complexes was in a range of 150-200 nm, and the zeta potential was in a range of 10-25 mV. In vitro gene transfection illustrated that when N/P < or = 50, the gene transfection efficiency of PAMAM-PEG copolymers was a little less than that of PAMAM-G5, but the transfection efficiency can be raised by increasing N/P ratio or transfection time. Considering both cytotoxicity and transfection efficiency aspects PAMAM-PEG-13 was more effect than PAMAM-PEG-39 in PEGylation.

Experimental study on prevention of postsurgical adhesions by using O-carboxymethylchitosan solution after an abdominal operation / 生物医学工程学杂志

O-Carboxymethylchitosan (O-CMC) in 1000 g batch was prepared from chitin as starting material and its chemical structure was confirmed by analysis of IR and NMR. O-CMC solution, sodium hyaluronate (HA) solution and physiological saline were used in Sprague-Dawley rat model for prevention of postsurgical adhesions; after 7 days of an abdominal operation, the 3 groups were evaluated according to Belluco standard, the mean scores of O-CMC group, HA group and physiological saline group were 2.5 +/- 3.1, 3.3 +/- 3.6 and 10.3 +/- 1.0, respectively. Histological inspection showed that in O-CMC group, mesothelial cells on peritonaeum or cecum surfaces were almost restored; in HA group the injured surface of peritonaeum was mostly repaired, but in physiological saline group the injured surface of cecum was just a little repaired; there were extensive adhesions between peritonaeum and cecum, and inflammatory response was quite serious. Experimental results indicated that O-CMC and HA had excellent efficiency and O-CMC was slightly better than HA for the prevention of postsurgical adhesions.

In vitro study of regulation of shear stress on antithrombogenic potentials of endothelialized polyurethane small diameter artificial blood vessel / 生物医学工程学杂志

This study was designed to investigate the changes of prostaglandin I2 (PGI2) and nitric oxide (NO) secreted by endothelialized polyurethane small diameter artificial blood vessel. The peripheral blood mononuclear cells of healthy adult were separated and induced into endothelial progenitor cells (EPCs), which were identified by the methods of discrepancy microphage and fluorescent immunology labeling. After the induced cells being seeded on the polyurethane small-diameter artificial vessels, the endothelialized polyurethane small diameter artificial blood vessels were divided into four different experimental groups, including stationary group, low-flow shear stress group (5 dynes/cm2), medium-flow shearstress group (15 dynes/cm2), and high-flow shear stress group (25 dynes/cm2). Then, the levels of 6-ketoprostaglandin F1alpha (6-keto-PGF1alpha) and NO of different time were measured by enzyme-linked immunosorbent assay and nitrate reductase methods. The peripheral blood mononuclear cells differentiated into EPCs. They presented typical "spindie-shaped" appearance, and they were positively labeled by fluorescent acetylated-LDL, lectin, FLK-1 and vWF. Shear stress enhanced the production of NO and 6-keto-PGF1alpha by EPCs in a dose-dependent manner. Therefore, shear stress increases the secretion of NO and PGI2 by EPC, which suggests that shear stress can improve the antithrombogenic potentials of endothelialized polyurethane small diameter artificial blood vessel.

In vitro study of seeding of peripheral blood endothelial progenitor cells on endothelialized polyurethane small diameter artificial blood vessel and shear stress treatment / 生物医学工程学杂志

In this study, the peripheral blood mononuclear cells of healthy adult were acquired and inducted by vascular endothelial growth factor, et cetera. The differentiated endothelial cells were observed and identified as EPCs by the double positive staining of fluorescent labeled acetylated-LDL and lectin, seeded on the polyurethane small-diameter artificial vessels, treated by shear stress of 15 dyn/cm2, and observed by scanning electronic microscopy. As a result, the peripheral blood mononuclear cells differentiated into EPCs. They were positively stained by labeled acetylated-LDL and lectin. Under observation of scanning electronic microscope, the unseeded polyurethane small-diameter artificial vessel being suited for the growth and spreading of the cells; the cell lineage on surface of artificial vessels of stationary group being arrayed in chaos, and that of shear stress group being arrayed in direction. Therefore, the peripheral cells can differentiate into EPCs, and EPCs can be used as novel source cells for the accelerated endothelialization of small diameter artificial vessel. Shear stress contributes to the mechanic moulding of cell lineage on the surface of artificial vessel.

Compliance of small diameter polyurethane artificial vascular graft / 生物医学工程学杂志

The radical compliance of small diameter artificial vascular grafts was measured by a device made of laser micrometer, pressure transducer, A/D card, micro computer and pulsed circulation loop, the volumetric compliance was measured by a device made of micro-syringe and pressure transducer; the longitudinal compliance was calculated from volumetric compliance and radical compliance. The research results showed that the radical compliance, volumetric compliance and longitudinal compliance would rise not only with the increase in polyurethane (PU) elasticity and salt/polymer ratio (or porosity), but also with the decrease in dipping layers (or wall thickness). Circumferential moduli E zeta and longitudinal moduli E theta could be calculated from volumetric compliance and longitudinal compliance respectively; E theta and E zeta would decrease with the increase in PU elasticity and salt/polymer ratio, but was independent on number of dipping layers. Small diameter PU artificial vascular grafts with compliance close to natural vein or artery can be prepared by choosing of more elastic PU materials (Chro or PCU1500), optimization of salt/polymer ratio (6:1), and the number of dipping layers (4-6 layers).

In vitro induction, differentiation and seeding of source cells for the endothelialization of small-diameter artificial blood vessel of polyurethane / 中国组织工程研究

BACKGROUND: At present, after transplantation of small diameter artificial blood vessel, long-term patency rate is low due to being lacking of endothelial cells for lining and anti-thrombus characters. In some studies,mature endothelial cells were tried to be seeded in the artificial vessel to boost up its anti-thrombus capability so as to improve the long-term patency rate, but we got unsatisfied effect due to the defects of seed cells and scaffolds. Therefore, in clinic, proper seed cells and vascular scaffolds have been searched for improving the long-term low pateney rate in transplantation of small diameter artificial blood vessel.OBJECTIVE: To investigate the feasibility that differentiation of bone marrow mononuclear cells induced in vitro into endothelial-progenitor cells (EPCs) and seed polyurethane small diameter artificial blood vessel so as to provide proper seed cells for endotheliazation of polyurethane small diameter artificial blood vessel.DESIGN: Observation experiment SETTING: Cardivascular Medical Department and Staff Room of Immunology, First Hospital Affiliated to Sun Yat-sen University MATERILAS: This experiment was carried out at the First Hospital Affiliated to Sun Yat-sen University from September 2004 to May 2005. About 10 mL of bone marrow from healthy adult volunteers (n=7) was used in this experiment.METHODS: Bone marrow mononuclear cells of healthy adult were collected and put in the fibronectin pre-coated DMEM culture medium, then induced by vascular endothelial growth factor and basic fibroblast growth factor. Induced cells were observed under fluorescence microscope and identified with immunohistochemical staining. The induced and proliferated EPCs were seeded onto the surface of polyurethane small diameter artificial blood vessel. Morphological change was observed under scanning electron microscope.MAIN OUTCOME MEASURES: ① Cellular morphological change.② Staining results of immunohistochemical VWF and CD 34 antibody . ③ Adhesive growth status of EPCs on the polyurethane small diameter artificial blood vessel RESULTS: ① In the vascular endothelial growth factor and basic fibroblast growth factor and other inducers , bone marrow mononuclear cells differentiated into EPCs , presenting typical "spindle-shaped" appearance under an inverted fluorescence microscope and became to form a monolayer that arrayed in "cobblestone-like" ② Immunohistochemical staining showed von willebrand factor(VWF) and CD34 antigen stained positive. ③ Under the scanning electron microscope, surface of polyurethane small diameter artificial blood vessel without seeded cells presented typical polyporous honeycomb-like structure , and the size of hole suited the crawling of EPCs. After seeding the cells, we observed the adhesion, crawling and spreading of the EPCs on the surface of polyurethane small diameter artificial blood vessel. Some EPCs grew into the honeycomb-like holes were seen occasionally.CONCLUSION: Bone marrow mononuclear cells can be induced and differentiated into EPCs, while induced and differentiated EPCs well grow adhesively in the polyurethane small diameter artificial vessels, suggesting that differentiation of bone marrow mononuclear cells induced in vitro into EPCS, which can be used as seed cells for endothelialization of polyurethane small diameter artificial blood vessels.

Transfection of Vascular Endothelial Growth Factor Gene VEGF165 Mediated With PEG-PEI Copolymers and Its Effect on The Growth of Endothelial Cells / 生物化学与生物物理进展

In order to investigate the ability of PEG-PEI copolymers as gene carriers for delivery of VEGF165. A series of PEG-PEI copolymers with different PEG grafting was prepared and the cytotoxicity was evaluated. Simultaneously,the VEGF165 gene segment with HindⅢ and BamHⅠ site was obtained by PCR, which was cloned into pEGFP-C1. PEG-PEI/ pEGFP-VEGF165 complexes were formed by self-assembly and transfected HUVEc. Transfection efficiency was evaluated by measuring the percentage of cells expressing green fluorecensce protein. The VEGF expression was detected by ELISA, RT-PCR, and the effect of transfection on growth of endothelial cell was evaluated by MTT. The results suggested that the formation of PEG-PEI copolymers could help to reduce the cytotoxicity of PEI. After transfection, the strong expression of green fluorescence protein was observed by fluorescence microscopy. The transfection efficiency was influenced by the number of PEG side chains and N/P ratio. Of all copolymers tested, the transfection efficiency of PEG-PEI(5-25-1) at N/P = 30 reached a maximum, which was much higher than that of PEI. The expression of VEGF protein and mRNA increased significantly, and HUVEc proliferation was accelerated after transfection.These results indicates PEG-PEI copolymers can be used as effective gene carriers for delivery of pEGFP-VEGF165 gene.

Blood compatibility of block copolymer membranes of poly(benzyl L-glutamate)/poly(ethylene glycol) / 生物医学工程学杂志

The blood compatibility of block copolymer membranes of poly(benzyl L-glutamate)/poly(ethylene glycol) and the effect of on the blood compatibility of copolymer were evaluated by the clotting time test, the platelet adhesion and deformation test, and the protein adsorption test. The results showed that in terms of blood compatibility, homopolymer was better than glass and silicone, copolymer was better than homopolymer, and the more the PEG in the copolymer, the better the blood compatibility.

Study on adhesion of platelets to surface of copoly(benzyl glutamate-hydroxy ethyl glutamine) / 生物医学工程学杂志

Copoly (benzyl glutamate-hydroxy ethyl glutamine) were prepared by partially aminolysis of poly benzyl glutamate with hydroxyl ethylamine at 60 degrees C for predeternined period. As aminolysis was proceeding, the content of hydroxy ethyl glutamine in copolymer increased and the yield of aminolysis decreased. When aminolysis time reached 16.5 hours, a copolymer with 0.357 mole fraction of hydroxy ethyl glutamine was obtained. After 16.5 hours a soluble yellow viscous product of reaction was obtained. The aminolysized specimens displayed more swelling degree in water, that is, more hydrophilits. The meassurement of solid-liquid contact angles showed that an increase in critical surface tention with content of hydroxy ethyl glutamine in copolymer was observed. Likewise a obvious increase in polar component and a slight decrease in dispersive of surface free energy, thus an increase in total surface free energy with content of hydroxy ethyl glutamine was found. The platelet adhesion and deformation test indicated that less platelets were adhered to surfaces of all aminolysized specimens than that of both poly benzyl glutamate and polydimethylsilicone. The least adhered platelets on surface of copolymer with 0.133 mole fraction of content of hydroxy ethyl glutamine were observed. The partiall aminolysis of poly benzyl glutamate films is an effective method to improve its hydrophility and antithrombogenicity.