Construction And In Vitro Study Of PIRES-tPA-Dsred Express2Loaded PLGA Nanoparticles-microbubble Complexes

Objective To construct a eukaryotic expression vector pIRES-tPA-Dsred Express2which carried the gene of human tissue-type plasminogen activator. Verify theimproved expression of tissue-type plasminogen activator while human umbilical veinendothelial cell line EA.hy926were transfected by pIRES-tPA-Dsred Express2. Andthe biological activity of the product protein was investigated.Method Access to the tPA gene sequence and insert it into the multiple clone sitesof pIRES-Dsred Express2. DNA sequencing was used to affirm construction of therecombinant. The vector, pIRES-tPA-Dsred Express2, was transfected into humanumbilical vein endothelial cell line EA.hy926cells by Lipofectamine LTX. Reversetranscription real-time fluorescence quantitative PCR was performed to detect mRNAlevels at48hours after transfection. Western blot was taken for the target proteinexpression. Application of ELISA was to assay tPA concentration in the supernatant.The activity of tPA in the supernatant was quantified by enzymatic reactions. Theconcentration and activity of the secreted tPA versus time relationships wasinvestigated.Results The eukaryotic expression vector pIRES-tPA-Dsred Express2wassuccessfully constructed which verified by DNA sequencing. At48hours aftertransfection, significantly red fluorescence can be observed in EA.hy926. Thetransfection efficiency was (20.7±4.2)%. Real-time reverse transcriptase quantitativePCR analysis showed that relative mRNA content of the target protein tPA and redfluorescent protein Dsred in pIRES-tPA-Dsred Express2group was769.21±35.11and1164.26±82.85, significantly higher than that in control. Western Blot showed thattPA and Dsred content was significantly higher in pIRES-tPA-Dsred Express2plasmid group. The concentration and activity of tPA in cell supernatant of pIRES-tPA-DsredExpress2plasmid 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 concentrationand activity of the secreted tPA versus time relationships showed the peakconcentration and activity appeared at24hours after transfection. The relationshipsversus time of tPA activity, the concentration of tPA and PAI-1was associated butdifferent.Conclusion We successfully constructed the eukaryotic expression vector,pIRES-tPA-Dsred Express2, which carrying gene sequence of tissue-type plasminogenactivator. And verify that the vector can correctly guide the synthesis and secretion oftissue-type plasminogen activator, showing a significant biological activity in vitrotransfection. Objective pIRES-tPA-DsRed-Express2-lipidsome complexes was admitted toendothelial cells in vitro mediated by ultrasound microbubble targeted destruction. Theeffection of ultrasound-mediated gene therapy in the plasmid transfection forendothelial cells was investigated.Method The perfluoropropane ultrasound microbubbles were prepared bythin-film hydration. The pIRES-tPA-DsRed Express2-liposome complexes weretransfected into EA.hy926cells mediated by ultrasound microbubble destruction. Celltransfection efficiency, the target protein mRNA relative content in cells, tPA contentand 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 ultrasoundmicrobubble-mediated group (UM+LTX) was (27.3±3.6)%, while (20.6±2.0)%inlipofectamine LTX group (LTX). The relative content of tPA mRNA wasrespectively (953.15±92.77) and (721.32±68.31) in (UM+LTX) and LTX group. Therelative 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 supernatantof (UM+LTX) group, is significantly higher then that of LTX group.Conclusion The perfluoropropane Ultrasound microbubbles were successfullyprepared. Mediated by ultrasound microbubble targeted destruction, the transfectionefficiency of the plasmid-liposome complexes for endothelial cells was further improved. Thereby the expression and secretion of tissue-type plasminogen activatorwas enhanced, as a result the fibrinolytic activity was improved. Objective To construct pIRES-tPA-DsRed Express2loaded PLGA nanoparticles-ultrasound microbubble complexes. To investigate physical and chemical properties, invitro plasmid release manner, Cytotoxicity and cellular uptake ofnanoparticles-microbubble complexes mediated by ultrasound destruction.Method pIRES-tPA-DsRed Express2loaded PLGA nanoparticles were preparedby double emulsion method. Cationic ultrasound microbubble were prepared bythin-film hydration method. Nanoparticles-microbubble complexes were created byelectrostatic adsorption. The plasmid loading of the PLGA nanoparticles andnanoparticles-microbubble complexes was assayed. In vitro release manner and thecytotoxicity of PLGA nanoparticles and nanoparticles-microbubble complexes wasInvestigated. The phagocytic manner for nanoparticles-microbubble complexes afterultrasound 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 thecationic microbubbles was (3.2±1.5)μm and (13.66±2.05)mV. The concentration is(4.3±1.1)×108/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)×108/ml.1ml nanoparticles-microbubble complexes contained (20.5±2.7)μgplasmid. And PLGA nanoparticles contained (42.3±2.1)μg plasmid per mg.Nanoparticles and nanoparticles-microbubble complexes were both shown a burstrelease 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 highestconcentration group appeared mild reduced cell activity. Nanoparticles-microbubblecomplexes group showed a higher cellular uptake efficiency after ultrasound mediatedmicrobubble destruction.Conclusion The nanoparticles-microbubble complexes with a good release pattern,low toxicity, enhanced phagocytic efficiency.