Construction And In Vitro Study Of Pires-tpa-dsred Express2Loaded Plga Nanoparticles-microbubble Complexes

Part I:Construction of pIRES-tPA-Dsred Express-2Eukaryotic expression vector and expression in vitroObjective To construct a eukaryotic expression vector pIRES-tPA-Dsred Express2which carried the gene of human tissue-type plasminogen activator. Verify the improved expression of tissue-type plasminogen activator while human umbilical vein endothelial cell line EA.hy926were transfected by pIRES-tPA-Dsred Express2. And the biological activity of the product protein was investigated.Method Access to the tPA gene sequence and insert it into the multiple clone sites of pIRES-Dsred Express2. DNA sequencing was used to affirm construction of the recombinant. The vector, pIRES-tPA-Dsred Express2, was transfected into human umbilical vein endothelial cell line EA.hy926cells by Lipofectamine(?) LTX. Reverse transcription real-time fluorescence quantitative PCR was performed to detect mRNA levels at48hours after transfection. Western blot was taken for the target protein expression. Application of ELISA was to assay tPA concentration in the supernatant. The activity of tPA in the supernatant was quantified by enzymatic reactions. The concentration and activity of the secreted tPA versus time relationships was investigated.Results The eukaryotic expression vector pIRES-tPA-Dsred Express2was successfully constructed which verified by DNA sequencing. At48hours after transfection, significantly red fluorescence can be observed in EA.hy926. The transfection efficiency was (20.7±4.2)%. Real-time reverse transcriptase quantitative PCR analysis showed that relative mRNA content of the target protein tPA and red fluorescent protein Dsred in pIRES-tPA-Dsred Express2group was769.21±35.11and1164.26±82.85, significantly higher than that in control. Western Blot showed that tPA and Dsred content was significantly higher in pIRES-tPA-Dsred Express2plasmid group. The concentration and activity of tPA in cell supernatant of pIRES-tPA-Dsred Express2plasmid group was (4.73±0.02)ng/hour-(105cells) and (9.48±0.12) IU/hour(105cells), which is significantly higher than that in control. The concentration and activity of the secreted tPA versus time relationships showed the peak concentration and activity appeared at24hours after transfection. The relationships versus time of tPA activity, the concentration of tPA and PAI-1was associated but different.Conclusion We successfully constructed the eukaryotic expression vector, pIRES-tPA-Dsred Express2, which carrying gene sequence of tissue-type plasminogen activator. And verify that the vector can correctly guide the synthesis and secretion of tissue-type plasminogen activator, showing a significant biological activity in vitro transfection. Part II:In vitro transfection of pIRES-tPA-Dsred Express2-liposome complexes mediated by ultrasound microbubble and expression in endothelial cellsObjective pIRES-tPA-DsRed-Express2-lipidsome complexes was admitted to endothelial cells in vitro mediated by ultrasound microbubble targeted destruction. The effection of ultrasound-mediated gene therapy in the plasmid transfection for endothelial cells was investigated.Method The perfluoropropane ultrasound microbubbles were prepared by thin-film hydration. The pIRES-tPA-DsRed Express2-liposome complexes were transfected into EA.hy926cells mediated by ultrasound microbubble destruction. Cell transfection efficiency, the target protein mRNA relative content in cells, tPA content and activity in the supernatant was detected.Results The average size of perfluoropropane ultrasound microbubbles was (3.5±1.4)μm. The concentration was (3.3±1.2)×108/ml. The zeta potential was (-2.2±1.5) mV. The microbubbles were stable within12hours after preparation. At48hours after transfection, the transfection efficiency of ultrasound microbubble-mediated group (UM+LTX) was (27.3±3.6)%, while (20.6±2.0)%in lipofectamine(?)LTX group (LTX). The relative content of tPA mRNA was respectively (953.15±92.77) and (721.32±68.31) in (UM+LTX) and LTX group. The relative content of fluorescence protein Dsred mRNA was (1191.22±109.31) and (1092.15±102.71) in each group. The concentration and activity of tPA in supernatant of (UM+LTX) group, is significantly higher then that of LTX group.Conclusion The perfluoropropane Ultrasound microbubbles were successfully prepared. Mediated by ultrasound microbubble targeted destruction, the transfection efficiency of the plasmid-liposome complexes for endothelial cells was further improved. Thereby the expression and secretion of tissue-type plasminogen activator was enhanced, as a result the fibrinolytic activity was improved. Part III:Construction of pIRES-tPA-DsRed Express2loaded PLGA nanoparticles-microbubble complexes and in vitro phagocytosis studyObjective To construct pIRES-tPA-DsRed Express2loaded PLGA nanoparticles-ultrasound microbubble complexes. To investigate physical and chemical properties, in vitro plasmid release manner, Cytotoxicity and cellular uptake of nanoparticles-microbubble complexes mediated by ultrasound destruction.Method pIRES-tPA-DsRed Express2loaded PLGA nanoparticles were prepared by double emulsion method. Cationic ultrasound microbubble were prepared by thin-film hydration method. Nanoparticles-microbubble complexes were created by electrostatic adsorption. The plasmid loading of the PLGA nanoparticles and nanoparticles-microbubble complexes was assayed.In vitro release manner and the cytotoxicity of PLGA nanoparticles and nanoparticles-microbubble complexes was Investigated. The phagocytic manner for nanoparticles-microbubble complexes after ultrasound mediated microbubble destruction was studied.Results The size and zeta potential of the plasmid loading PLGA nanoparticles was (217.2±2.2)nm and (-15.24±0.83)mV. The average size and zeta potential of the cationic microbubbles was (3.2±1.5)μm and (13.66±2.05)mV. The concentration is (4.3±1.1)×10/ml. The average size of the nanoparticles-microbubble complexes was (4.6±1.7)μm and zeta potential was (2.23±1.45)mV, and the concentration was (3.0±1.3)×10/ml.1ml nanoparticles-microbubble complexes contained (20.5±2.7)μg plasmid. And PLGA nanoparticles contained (42.3±2.1)μg plasmid per mg. Nanoparticles and nanoparticles-microbubble complexes were both shown a burst release in the initial segment, and then a trend of stable release instead. The total (57±3)%plasmid encapsulated were released in the first seven days. Nanoparticles and nanoparticles-microbubble complexes showed little cytotoxicity. Only the highest concentration group appeared mild reduced cell activity. Nanoparticles-microbubble complexes group showed a higher cellular uptake efficiency after ultrasound mediated microbubble destruction.Conclusion The nanoparticles-microbubble complexes with a good release pattern, low toxicity, enhanced phagocytic efficiency.