Preparation, Properties Detection And Analysis Of PcDNA3.1-Myc-His B(-)/tPA Plasmid DNA-Cross Linking Gelatin Microspheres Complex

Experiment I Preparation of Cross Linking Gelatin Microspheres, Detection and Analysis of Physical PropertiesObjective To prepare cross linking gelatin microspheres (CLG microspheres), analyze and detect physical properties.Method Modified diphasic emulsified cold-condensation method was adapted to prepare CLG microspheres, with basic gelatin as material and 25% glutaral solution as cross linking agent. SEM was used to observe morphology and detect the average diameter of CLG microspheres before and after fully swelling in physiologic saline at 37?C. The maximum swelling radio of CLG microspheres was also evaluated. Microelectrophoresis was used to detect the surface charge of CLG microspheres. The ability of binding and delayed releasing dose-time profile of pcDNA3.1-Myc-His B(-)/tPA plasmid DNA by CLG microspheres was also detected by ultraviolet spectrophotometry.Results CLG microspheres were uniform spheroids with small pores on surface and sporadically dispersed without conglutination. The average diameter of CLG microspheres was 21.13±1.42μm and 26.72±1.74μm before and after fully swelling in physiologic saline at 37?C, and there was statistical difference between the average diameter of microspheres before and after fully swelling. The maximum swelling radio was 27.32±0.45% in physiologic saline at 37?C. The surface charge was positive. When the mass proportion of plasmid DNA vs. CLG microspheres reached 1:10, the absorption for plasmid DNA by CLG microspheres reached saturation, with entrapment radio of 62.75±3.31%. pcDNA3.1-Myc-His B(-)/tPA plasmid DNA could be continuously delayed released by CLG microspheres over 4 weeks in vitro.Conclusion CLG microspheres possessed good ability of binding pcDNA3.1-Myc-His B(-)/tPA plasmid DNA to form pcDNA3.1-Myc-His B(-)/tPA plasmid DNA-CLG microspheres complex, and pcDNA3.1-Myc-His B(-)/tPA plasmid DNA could be delayed released continuously from the complex over 4 weeks in vitro.Experiment II Research of Cytotoxicity and Biocompatibility of Cross Linking Gelatin Microspheres Objective To detect cytotoxicity and biocompatibility of CLG microspheres in order to evaluate safety of CLG microspheres used in vivo.Method MTT assay was used to detect cytotoxicity of CLG microspheres. Haemolysis test, prothrombin time and recalcification time experiment were used to evaluate influence on erythrocyte and blood coagulation function by CLG microspheres. The influence on ability of adhesion and migration on Dacron fibers surface of human umbilical vein endothelium cells (HUVECs) by CLG microspheres were also evaluated through SEM, adhesion cells numbers counting and cell migration test.Results Cytotoxicity of CLG microspheres retained as grade 1, and would not increase along with degradation time and concentration of CLG microspheres. The Haemolysis degree was 2.27±0.34%, which indicated that CLG microspheres would not destroy erythrocytes. Prothrombin time was 16.0±0.7 s, and there was no statistical difference with that of negative control group (17.2±0.8 s) (P >0.05). Recalcification time was 165.4±4.5 s, and there was no significantly statistical difference with that of common gelatin (169.2±5.4 s) or negative group (181.2±3.4 s) (P >0.05). Those results indicated that CLG microspheres would not facilitate exogenous and intrinsic coagulation. The amount of HUVECs on the surface of Dacron fibers coated with CLG microspheres solution was (1.47±0.30)×106 and (65.34±11.25)×106 at the end of week 1 and 4 respectively, and there were no statistical difference between that of untreated Dacron fibers, (1.63±0.26)×106 and (62.43±10.06)×106 respectively (P >0.05). The migration distance in CLG microspheres group was 29.61±3.54μm, and there was no statistical difference between that of negative control group (32.41±4.31μm) or common gelatin group (30.55±5.08μm) (P >0.05). Those results indicated that CLG microspheres would not inhibit the ability of adhesion and migration of HUVECs.Conclusion CLG microspheres had little cytotoxicity and would not facilitate coagulation, or inhibit the ability of adhesion and migration of HUVECs, which indicated that CLG microspheres possessed good biocompatibility.Experiment III Experimental Research of Gene Transfection of pcDNA3.1-Myc-His B(-)/tPA Plasmid DNA-Cross Linking Gelatin Microspheres Complex in vitroObjective To detect transient gene transfection efficiency of pcDNA3.1-Myc-His B(-)/tPA plasmid DNA being transferred into HUVECs, which was released from pcDNA3.1-Myc-His B(-)/tPA plasmid DNA-CLG microspheres complex in vitro, and gene expression after transfection.Method Immunofluorescence staining was used to detect the transient gene transfection efficiency of pcDNA3.1-Myc-His B(-)/tPA plasmid DNA-CLG microspheres complex. RT-PCR was used to detect tPA mRNA and Western blotting was used to detect tPA protein synthesized in HUVECs after transfection. ELISA was used to evaluate tPA content in culture medium supernatant, which was secreted by HUVECs after transfection.Results pcDNA3.1-Myc-His B(-)/tPA plasmid DNA, which was released from pcDNA3.1-Myc-His B(-)/tPA plasmid DNA-CLG microspheres complex, could be successfully transferred into HUVECs in vitro, with transient transfection efficiency of 12.74±2.53%, and it was significantly higher than that of simple pcDNA3.1-Myc-His B(-)/tPA plasmid DNA solution (4.63±1.75%) (P <0.05), but lower than pcDNA3.1-Myc-His B(-)/tPA plasmid DNA-liposome complex (26.49±5.23%) (P <0.05). The integral optical density ratio value of tPA mRNA electrophoresis strip in pcDNA3.1-Myc-His B(-)/tPA plasmid DNA-CLG microspheres complex group was 1.65±0.27, which was higher than simple pcDNA3.1-Myc-His B(-)/tPA plasmid DNA solution group (1.22±0.23) (P <0.05), but lower than pcDNA3.1-Myc-His B(-)/tPA plasmid DNA-liposome complex group (2.07±0.25) (P <0.05). The integral optical density ratio values of those 3 groups were all significantly higher than that of blank pcDNA3.1 plasmid DNA vector group (0.86±0.21) and HUVECs in control culture (0.91±0.18) (P <0.05). tPA expression could be detected in HUVECs after transfection by pcDNA3.1-Myc-His B(-)/tPA plasmid DNA-CLG microspheres complex, however it was expressed in trace level in blank pcDNA3.1 plasmid DNA vector group and HUVECs in control culture. tPA could be secreted into culture medium supernatant by HUVECs after transfection, and tPA content in pcDNA3.1-Myc-His B(-)/tPA plasmid DNA-CLG microspheres complex grorp was 497.2±61.9 ng/ml, which was higher than simple pcDNA3.1-Myc-His B(-)/tPA plasmid DNA solution group (295.7±49.1 ng/ml) (P <0.05), but lower than pcDNA3.1-Myc-His B(-)/tPA plasmid DNA-liposome complex group (862.1±96.8 ng/ml) (P <0.05).Conclusion pcDNA3.1-Myc-His B(-)/tPA plasmid DNA which was released from pcDNA3.1-Myc-His B(-)/tPA plasmid DNA-CLG microspheres complex could be successfully transferred into HUVECs and tPA synthesized in HUVECs could be secreted extracellularly after transfection.