AIM： To explore the effects of ultrasound exposure combined with microbubble contrast agent （SonoVue） on the permeability of the cellular membrane and on the expression of plasrnid DNA encoding enhanced green fluor...AIM： To explore the effects of ultrasound exposure combined with microbubble contrast agent （SonoVue） on the permeability of the cellular membrane and on the expression of plasrnid DNA encoding enhanced green fluorescent protein （pEGFP） transfer into human umbilical vein endothelial cells （HUVECs）. METHODS： HUVECs with fluorescein isothiocyanatedextran （FD500） and HUVECs with pEGFP were exposed to continuous wave （1.9 MHz, 80.0 mW/cm^2） for 5 min, with or without a SonoVue. The percentage of FD500 taken by the HUVECs and the transient expression rate of pEGFP in the HUVECs were examined by fluorescence microscopy and flow cytornetry, respectively. RESULTS： The percentage of FDS00-positive HUVECs in the group of ultrasound exposure combined with SonoVue was significantly higher than that of the group of ultrasound exposure alone （24.0%± 5.5% vs 66.6% ± 4.1%, P 〈 0.001）. Compared with the group of ultrasound exposure alone, the transfection expression rate of pEGFP in HUVECs was markedly increased with the addition of SonoVue （16.1% ± 1.9% vs 1.5% ± 0.2%, P 〈 0.001）. No statistical significant difference was observed in the HUVECs survival rates between the ultrasound group with and without the addition of SonoVue （94.1% ± 2.3% vs 91.1% ± 4.1% ）. CONCLUSION： The cell membrane permeability of HUVECs and the transfection efficiency of pEGFP into HUVECs exposed to ultrasound are significantly increased after addition of an ultrasound contrast agent without obvious damage to the survival of HUVECs. This non- invasive gene transfer method may be a useful tool for clinical gene therapy of hepatic tumors.展开更多
Background It has been proven that ultrasonic destruction of microbubbles can enhance gene transfection efficiency into the noncardiac cells, but there are few reports about cardiac myocytes. Moreover, the exact mecha...Background It has been proven that ultrasonic destruction of microbubbles can enhance gene transfection efficiency into the noncardiac cells, but there are few reports about cardiac myocytes. Moreover, the exact mechanisms are not yet clear; whether the characteristic of microbubbles can affect the gene transfection efficiency or not is still controversial.This study was designed to investigate whether the ultrasound destruction of gene-loaded microbubbles could enhance the plasmids carried reporter gene transfection in primary cultured myocardial cell, and evaluate the effects of microbubbles characteristics on the transgene expression in cardiac myocytes.Methods The β-galactosidase plasmids attached to the two types of microbubbles, air-contained sonicated dextrose albumin （ASDA） and perfluoropropane-exposed sonicated dextrose albumin （PESDA） were prepared. The gene transfection into cardiac myocytes was performed in vitro by naked plasmids, ultrasound exposure, ultrasonic destruction of gene-loaded microbubbles and calcium phosphate precipitation, and then the gene expression and cell viability were analyzed.Results The ultrasonic destruction of gene-loaded microbubbles enhanced gene expression in cardiac myocytes compared with naked plasmid transfection （（51.95±2..41） U/g or （29.28±3.65） U/g vs. （0.84-0.21） U/g, P ＜0.01）, and ultrasonic destruction PESDA resulted in more significant gene expression than ASDA （（51.95e2.41） U/g vs. （29.28±3.65）U/g, P ＜0.05）. Ultrasonic destruction of microbubbles during calcium phosphate precipitation gene transfection enhanced 3-galactosidase activity nearly 8-fold compared with calcium phosphate precipitation gene transfection alone （（111.35±11.21） U/g protein vs. （14.13±2.58） U/g protein, P＜0.01）. Even 6 hours after calcium phosphate precipitation gene transfection, ultrasound-mediated microbubbles destruction resulted in more intense gene expression （（35.63±7.65）U/g vs. （14.13±2.58） U/g, P＜0.05 ）.Conclusions Ultrasonic destruction of microbubbles might be a promising method for the delivery of non-viral DNA into cardiac myocytes, and the gene tranfection is related to the characteristics of microbubbles.展开更多
To optimize the operating variables that affect the transfection of antisense oligodeoxyribonucleotide（AS-ODNs） by insonated gas-filled lipid microbubbles,SF6-filled microbubbles were prepared by sonication-lyophili...To optimize the operating variables that affect the transfection of antisense oligodeoxyribonucleotide（AS-ODNs） by insonated gas-filled lipid microbubbles,SF6-filled microbubbles were prepared by sonication-lyophilization method.An AS-ODNs sequence and a breast cancer cell line SK-BR-3 were used to define the various operating variables determining the transfection efficiency of SF6-filled microbubbles.Three levels of mixing speed,different durations of mixing and various delay time before ultrasound were examined,separately.Transfection efficiency was detected by fluorescence microscopy.Transfection results with and without incubation of AS-ODNs and microbubbles before mixing cells were compared.From the results,there is no significant difference between the transinfection efficiency with or without incubation of AS-ODNs and microbubbles before mixing cells.AS-ODNs transfection efficiency showed an increasing trend with mixing speed and mixing duration,but there is a negative relationship with delay time before ultrasound.The optimum parameters for AS-ODNs transfection by SF6-filled microbubbles were found at a mixing speed of 40-50 r·min-1 for 30-60 s with less than 60 s delay before ultrasound.For a successful transfection,long time of incubation with gene is essential for normal nonviral vectors such as liposomes or cationic lipid-polymer hybrids,because these vectors depend on endocytosis and membrane fusion to realize transfection.Unlike liposomes and cationic lipid-polymer hybrids,gas-filled lipid microbubbles depend on sonorporation effect to realize transfection.Therefore,the incubation of gene and microbubbles before mixing cells may not be necessary.Ultrasound-mediated AS-ODNs transfection enhanced by gas-filled lipid microbubbles represents an effective avenue for gene transfer.展开更多
基金Supported by grants from the Nationl Natural Scientific Foundation of China, No.30300082, 30470467, and Scientific Foundation Committee of Guangdong Province, China, No.04009360
文摘AIM： To explore the effects of ultrasound exposure combined with microbubble contrast agent （SonoVue） on the permeability of the cellular membrane and on the expression of plasrnid DNA encoding enhanced green fluorescent protein （pEGFP） transfer into human umbilical vein endothelial cells （HUVECs）. METHODS： HUVECs with fluorescein isothiocyanatedextran （FD500） and HUVECs with pEGFP were exposed to continuous wave （1.9 MHz, 80.0 mW/cm^2） for 5 min, with or without a SonoVue. The percentage of FD500 taken by the HUVECs and the transient expression rate of pEGFP in the HUVECs were examined by fluorescence microscopy and flow cytornetry, respectively. RESULTS： The percentage of FDS00-positive HUVECs in the group of ultrasound exposure combined with SonoVue was significantly higher than that of the group of ultrasound exposure alone （24.0%± 5.5% vs 66.6% ± 4.1%, P 〈 0.001）. Compared with the group of ultrasound exposure alone, the transfection expression rate of pEGFP in HUVECs was markedly increased with the addition of SonoVue （16.1% ± 1.9% vs 1.5% ± 0.2%, P 〈 0.001）. No statistical significant difference was observed in the HUVECs survival rates between the ultrasound group with and without the addition of SonoVue （94.1% ± 2.3% vs 91.1% ± 4.1% ）. CONCLUSION： The cell membrane permeability of HUVECs and the transfection efficiency of pEGFP into HUVECs exposed to ultrasound are significantly increased after addition of an ultrasound contrast agent without obvious damage to the survival of HUVECs. This non- invasive gene transfer method may be a useful tool for clinical gene therapy of hepatic tumors.
文摘Background It has been proven that ultrasonic destruction of microbubbles can enhance gene transfection efficiency into the noncardiac cells, but there are few reports about cardiac myocytes. Moreover, the exact mechanisms are not yet clear; whether the characteristic of microbubbles can affect the gene transfection efficiency or not is still controversial.This study was designed to investigate whether the ultrasound destruction of gene-loaded microbubbles could enhance the plasmids carried reporter gene transfection in primary cultured myocardial cell, and evaluate the effects of microbubbles characteristics on the transgene expression in cardiac myocytes.Methods The β-galactosidase plasmids attached to the two types of microbubbles, air-contained sonicated dextrose albumin （ASDA） and perfluoropropane-exposed sonicated dextrose albumin （PESDA） were prepared. The gene transfection into cardiac myocytes was performed in vitro by naked plasmids, ultrasound exposure, ultrasonic destruction of gene-loaded microbubbles and calcium phosphate precipitation, and then the gene expression and cell viability were analyzed.Results The ultrasonic destruction of gene-loaded microbubbles enhanced gene expression in cardiac myocytes compared with naked plasmid transfection （（51.95±2..41） U/g or （29.28±3.65） U/g vs. （0.84-0.21） U/g, P ＜0.01）, and ultrasonic destruction PESDA resulted in more significant gene expression than ASDA （（51.95e2.41） U/g vs. （29.28±3.65）U/g, P ＜0.05）. Ultrasonic destruction of microbubbles during calcium phosphate precipitation gene transfection enhanced 3-galactosidase activity nearly 8-fold compared with calcium phosphate precipitation gene transfection alone （（111.35±11.21） U/g protein vs. （14.13±2.58） U/g protein, P＜0.01）. Even 6 hours after calcium phosphate precipitation gene transfection, ultrasound-mediated microbubbles destruction resulted in more intense gene expression （（35.63±7.65）U/g vs. （14.13±2.58） U/g, P＜0.05 ）.Conclusions Ultrasonic destruction of microbubbles might be a promising method for the delivery of non-viral DNA into cardiac myocytes, and the gene tranfection is related to the characteristics of microbubbles.
文摘To optimize the operating variables that affect the transfection of antisense oligodeoxyribonucleotide（AS-ODNs） by insonated gas-filled lipid microbubbles,SF6-filled microbubbles were prepared by sonication-lyophilization method.An AS-ODNs sequence and a breast cancer cell line SK-BR-3 were used to define the various operating variables determining the transfection efficiency of SF6-filled microbubbles.Three levels of mixing speed,different durations of mixing and various delay time before ultrasound were examined,separately.Transfection efficiency was detected by fluorescence microscopy.Transfection results with and without incubation of AS-ODNs and microbubbles before mixing cells were compared.From the results,there is no significant difference between the transinfection efficiency with or without incubation of AS-ODNs and microbubbles before mixing cells.AS-ODNs transfection efficiency showed an increasing trend with mixing speed and mixing duration,but there is a negative relationship with delay time before ultrasound.The optimum parameters for AS-ODNs transfection by SF6-filled microbubbles were found at a mixing speed of 40-50 r·min-1 for 30-60 s with less than 60 s delay before ultrasound.For a successful transfection,long time of incubation with gene is essential for normal nonviral vectors such as liposomes or cationic lipid-polymer hybrids,because these vectors depend on endocytosis and membrane fusion to realize transfection.Unlike liposomes and cationic lipid-polymer hybrids,gas-filled lipid microbubbles depend on sonorporation effect to realize transfection.Therefore,the incubation of gene and microbubbles before mixing cells may not be necessary.Ultrasound-mediated AS-ODNs transfection enhanced by gas-filled lipid microbubbles represents an effective avenue for gene transfer.
