聚乙烯亚胺修饰白蛋白微泡转染真核细胞的实验研究
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摘要
目的:探讨聚乙烯亚胺修饰白蛋白微泡转染真核细胞的方法,从而提高白蛋白微泡转基因效率。
方法:(1)分别将25%人体白蛋白、葡萄糖和甘露醇等按一定的质量比置于10 ml瓶中,混合后用超声振动仪声振20 S(电压100 V、电流0.5 A)制得白蛋白微泡(Albumin Microbubbles,AMB);(2)将微泡包裹气体由空气换为全氟丙烷气体,并在微泡组分中添加聚乙二醇,以提高微泡稳定性;(3)在微泡组分中添加聚乙烯亚胺,获得聚乙烯亚胺修饰包裹全氟丙烷气体微泡(PAMB),通过转染CHO细胞,36 h后消化细胞流式细胞仪检测GFP阳性细胞比例,最大转基因效率时质量比即为最佳白蛋白与聚乙烯亚胺质量比;(4)使用纳米激光粒度仪和Zeta电位分析仪检测微泡表面电位;(5)将质粒DNA分别与AMB和PAMB按一定的比例混合后,加入SYBR Green,SYBR Green与DNA结合后可被激发出绿色荧光,在荧光显微镜下观察,结合质粒DNA的微泡可发出绿色荧光;如果微泡未与质粒DNA结合则只能看到绿色荧光而无微泡形态;(6)转染CHO, 293T, M231, 293, SK-Hep-1, SKBR-3, MCF-7, COS 8种细胞,使用流式细胞仪检测GFP阳性细胞比例以比较PEI、白蛋白+PEI、Lipofectamine 2000和PAMB的转基因效率;(7)使用CCK-8试剂盒检测PEI, Lipofectamine 2000和PAMB细胞毒性及转染后细胞增值能力,细胞活性= PEI组, Lipofectamine 2000组和PAMB组吸光值/未处理组吸光值,细胞增殖指数=加入转染试剂之前和加入转染试剂作用6 h之后0 h, 24 h, 48 h, 72 h吸光值/加入转染试剂之前吸光值;(8)使用COS细胞比较AMB,SonoVue微泡和PAMB在超声介导下转基因效率,超声强度为0.5 W/cm2、1.0 W/cm2和2.0 W/cm2,时间分别为30 S和60 S;(9)探讨超声在微泡转基因中的作用,制备CHO细胞爬片,分别按如下分组加入试剂:①只加入DNA荧光染料NA-Green(不能主动进入细胞);②AMB+DNA+NA-Green;③AMB+DNA+NA-Green+超声(1.0 W/cm2, 30 S) ;④PAMB+ NA-Green ;⑤PAMB+DNA+NA-Green ;⑥PAMB+DNA+ NA-Green+超声(1.0 W/cm2, 30 S),37℃、5% CO2培养6 h后吸取培养液,使用PBS冲洗三遍,每孔加入1ml 1%多聚甲醛固定15 min,用PBS冲洗两遍,然后使用共聚焦荧光显微镜观察拍照。
结果:(1)将微泡包裹气体由空气换为全氟丙烷气体,并在微泡组分中添加聚乙二醇后,微泡稳定性大大提高,半衰期由原来的半小时至数天延长到6个月;(2)通过转染CHO细胞,得到最佳白蛋白与聚乙烯亚胺的质量比为125:1,此时PAMB转染CHO细胞最大效率可达55%左右;(3)纳米激光粒度仪检测显示: AMB平均直径1400 nm左右,而PAMB平均直径在600 nm左右,Zeta电位分析仪检测结果示: AMB的表面电位为(-59.28±3.48)mV,而PAMB的表面电位为(-44.56±0.75)mV;(4)结合质粒DNA的微泡(PAMB)在荧光显微镜下可发出绿色荧光, AMB可见大量绿色荧光而未见微泡形态,未添加DNA的PAMB未见荧光;(5)通过8种细胞比较PEI、白蛋白+PEI、Lipofectamine 2000和PAMB的转基因效率,发现PAMB组明显高于单纯PEI组和白蛋白+PEI组(P<0.05),而在293T、COS、SKBR-3和SK-Hep-1细胞PAMB的转基因效率高于Lipofectamine 2000,在CHO、293、M231和MCF-7细胞转基因效率低于Lipofectamine 2000(P>0.05),白蛋白+PEI的转基因效率低于单纯PEI的转基因效率(P<0.05);(6)PEI,PAMB和Lipofectamine 2000均具有细胞毒性,PAMB的细胞毒性低于PEI和Lipofectamine 2000(P<0.05),且使用PAMB转染后细胞增殖能力明显高于使用PEI和Lipofectamine 2000转染的细胞(P<0.05);(7)通过实施不同强度和时间的超声,聚乙烯亚胺修饰白蛋白微泡微泡转基因效率在各种强度和时间下均明显高于白蛋白微泡和SonoVue微泡(P<0.05),不同超声强度和时间下聚乙烯亚胺修饰白蛋白微泡微泡转基因效率在COS和CHO两种细胞均没有显著差异,且与未超声组相比略有下降,但无统计学意义(P>0.05);(8)超声介导AMB和SonoVue微泡破裂转基因时,质粒DNA依靠空化效应在细胞膜上形成的微孔进入细胞,而PAMB可携带质粒DNA主动进入细胞。
结论:本研究制作的聚乙烯亚胺修饰包裹全氟丙烷气体微泡可将其携带的外源基因转入真核细胞,且体外实验证实其具有低细胞毒性和较高的转基因效率,是一种有效的体外基因转染方法,为PAMB作为体内转基因载体提供实验依据。
Objective:To improve the transgene efficiency of albumin microbubbles,we investigated a method of transgene with polyethylenimine coated albumin microbubbles.
Method:(1) Albumin microbubbles (AMB) were prepared by sonicating the mixture of human albumin, manose and glucose; (2) Air filled in albumin microbubbles was exchanged with perfluoropropane and polyethylene glycol (PEG) was added as one gradient of microbubbles to improve the stability of albumin microbubbles; (3) Polyethylenimine (PEI) coated albumin microbubbles (PAMB) was prepared by sonicating the mixture of human albumin, PEI, polyethylene glycol (PEG), glucose, manose and perfluoropropane. Through transfecting CHO cell line with plasmid pIRSE2-EGFP, the optimal weight ratio of albumin and PEI was insured by measureing the percentage of GFP with flow cytometry; (4) Mean particle diameter and surface potential of AMB and PAMB were determined by photon cross correlation spectroscopy with a NANOPHOX particle size analysis system and ZetaProbe 7020; (5) To identify PAMB combined with pDNA, PAMB and pDNA with addition of SYBR green were mixed in serum-free Opti-MEM?Ⅰmedium in an Eppendorf tube. When SYBR green was combined with plasmid DNA, it could be excited by blue light and emitted green fluorescence. Thereafter, if PAMB combined with pDNA, it could emit green fluorescence. Green microbubbles could be inspected with a fluorescence microscrope; (6) Through transfecting CHO, 293T, M231, 293, SK-Hep-1, SKBR-3, MCF-7 and COS cell lines with plasmid pIRSE2-EGFP, we compared the transgene efficiency of PEI, albumin+PEI, Lipofectamine 2000 and PAMB; (7) The cytotoxicity and cell proliferation ablity of PEI, Lipofectamine 2000 and PAMB were measured by cell counting kit-8. Cell proliferation index was calculated by the OD value prior to Transfection, and 0 h, 24 h, 48 h, 72 h posttransfection / OD value prior to transfection; (8) COS cell line was transfected by AMB, SonoVue and PAMB mediated by ultrasound with different ultrasound (U) intensity and time; (9) The effect of ultrasound on microbubbles transgene was investigated. After CHO cell sections were prepared, reagents were added as follows in each group:①o nly NA-Green (a kind of DNA fluorescence dye which can not enter cells actively);②AMB+DNA+NA-Green;③AMB+DNA+NA-Green+U(1.0 W/cm2, 30 S);④P AMB+ NA-Green;⑤PAMB+DNA+NA-Green;⑥P AMB+DNA+ NA-Green+U (1.0 W/cm2, 30 S). After incubation at 37℃, 5% CO2 for 6 h; the medium was removed. The sections were rinsed for three times and immobilized with 1% paraformaldehyde, then were inspected with confocal microscoup and photos were taken.
Results:(1) The half time of albumin microbubbles was extended from half an hour or several days to 6 months after air that filled in albumin microbubbles was exchanged with perfluoropropane and polyethylene glycol (PEG) was added as one gradient of microbubbles; (2) The optimal weight ratio of albumin and PEI was 125:1, which was obtained through transfecting CHO cell line with plasmid pIRSE2-EGFP, and the highest transfection efficiency of PAMB was about 55% on CHO cell line; (3) The particle size analysis showed the average particle diameter of AMB and PAMB was about 1400nm and 600nm, and the surface potentials of PAMB and AMB were -44.56±0.75 mV and -59.28±3.48 mV, respectively; (4) AMB could not combine with pDNA and only emitted plenty of fluorescence without outlook of microbubbles, PAMB combined with pDNA emitted fluorescence; (5) The transgene efficiency in PAMB group was higher than that in PEI group and albumin + PEI group in each cell line (P<0.01) and there was no statistical difference between PAMB group and Lipofectamine 2000 group; (6) Cytotoxicity of PAMB was lower than PEI and Lipofectamine 2000(P < 0.01), the proliferation index in no treatment group and PAMB was higher than that in PEI and Lipofectamine 2000 group (P < 0.01), there was no statistical difference PAMB group and no treatment group (P>0.05); (7) COS cells were transfected with AMB, SonoVue and PAMB mediated by ultrasound with different ultrasound intensity and time, the results showed that the transfection efficiency of PAMB was higher than the efficiency of AMB and SonoVue (P < 0.01), there was no statistical difference between AMB and SonoVue, and there were no statistical differences between different ultrasound intensity and time, and compared with PAMB alone; (8) Destruction of AMB and SonoVue mediated by ultrasound could increase the permeability of membrane, plasmid DNA enter cells through these micropores, while PAMB could bring plasmid DNA into cells actively.
Conlusion:In summary, PAMB is very useful and low toxicity gene delivery method with high transfection efficiency in vitro. Further studies are necessary to examine the detailed mechanism of the effect of ultrasound on PAMB and transfection efficiency in vivo.
方法:(1)分别将25%人体白蛋白、葡萄糖和甘露醇等按一定的质量比置于10 ml瓶中,混合后用超声振动仪声振20 S(电压100 V、电流0.5 A)制得白蛋白微泡(Albumin Microbubbles,AMB);(2)将微泡包裹气体由空气换为全氟丙烷气体,并在微泡组分中添加聚乙二醇,以提高微泡稳定性;(3)在微泡组分中添加聚乙烯亚胺,获得聚乙烯亚胺修饰包裹全氟丙烷气体微泡(PAMB),通过转染CHO细胞,36 h后消化细胞流式细胞仪检测GFP阳性细胞比例,最大转基因效率时质量比即为最佳白蛋白与聚乙烯亚胺质量比;(4)使用纳米激光粒度仪和Zeta电位分析仪检测微泡表面电位;(5)将质粒DNA分别与AMB和PAMB按一定的比例混合后,加入SYBR Green,SYBR Green与DNA结合后可被激发出绿色荧光,在荧光显微镜下观察,结合质粒DNA的微泡可发出绿色荧光;如果微泡未与质粒DNA结合则只能看到绿色荧光而无微泡形态;(6)转染CHO, 293T, M231, 293, SK-Hep-1, SKBR-3, MCF-7, COS 8种细胞,使用流式细胞仪检测GFP阳性细胞比例以比较PEI、白蛋白+PEI、Lipofectamine 2000和PAMB的转基因效率;(7)使用CCK-8试剂盒检测PEI, Lipofectamine 2000和PAMB细胞毒性及转染后细胞增值能力,细胞活性= PEI组, Lipofectamine 2000组和PAMB组吸光值/未处理组吸光值,细胞增殖指数=加入转染试剂之前和加入转染试剂作用6 h之后0 h, 24 h, 48 h, 72 h吸光值/加入转染试剂之前吸光值;(8)使用COS细胞比较AMB,SonoVue微泡和PAMB在超声介导下转基因效率,超声强度为0.5 W/cm2、1.0 W/cm2和2.0 W/cm2,时间分别为30 S和60 S;(9)探讨超声在微泡转基因中的作用,制备CHO细胞爬片,分别按如下分组加入试剂:①只加入DNA荧光染料NA-Green(不能主动进入细胞);②AMB+DNA+NA-Green;③AMB+DNA+NA-Green+超声(1.0 W/cm2, 30 S) ;④PAMB+ NA-Green ;⑤PAMB+DNA+NA-Green ;⑥PAMB+DNA+ NA-Green+超声(1.0 W/cm2, 30 S),37℃、5% CO2培养6 h后吸取培养液,使用PBS冲洗三遍,每孔加入1ml 1%多聚甲醛固定15 min,用PBS冲洗两遍,然后使用共聚焦荧光显微镜观察拍照。
结果:(1)将微泡包裹气体由空气换为全氟丙烷气体,并在微泡组分中添加聚乙二醇后,微泡稳定性大大提高,半衰期由原来的半小时至数天延长到6个月;(2)通过转染CHO细胞,得到最佳白蛋白与聚乙烯亚胺的质量比为125:1,此时PAMB转染CHO细胞最大效率可达55%左右;(3)纳米激光粒度仪检测显示: AMB平均直径1400 nm左右,而PAMB平均直径在600 nm左右,Zeta电位分析仪检测结果示: AMB的表面电位为(-59.28±3.48)mV,而PAMB的表面电位为(-44.56±0.75)mV;(4)结合质粒DNA的微泡(PAMB)在荧光显微镜下可发出绿色荧光, AMB可见大量绿色荧光而未见微泡形态,未添加DNA的PAMB未见荧光;(5)通过8种细胞比较PEI、白蛋白+PEI、Lipofectamine 2000和PAMB的转基因效率,发现PAMB组明显高于单纯PEI组和白蛋白+PEI组(P<0.05),而在293T、COS、SKBR-3和SK-Hep-1细胞PAMB的转基因效率高于Lipofectamine 2000,在CHO、293、M231和MCF-7细胞转基因效率低于Lipofectamine 2000(P>0.05),白蛋白+PEI的转基因效率低于单纯PEI的转基因效率(P<0.05);(6)PEI,PAMB和Lipofectamine 2000均具有细胞毒性,PAMB的细胞毒性低于PEI和Lipofectamine 2000(P<0.05),且使用PAMB转染后细胞增殖能力明显高于使用PEI和Lipofectamine 2000转染的细胞(P<0.05);(7)通过实施不同强度和时间的超声,聚乙烯亚胺修饰白蛋白微泡微泡转基因效率在各种强度和时间下均明显高于白蛋白微泡和SonoVue微泡(P<0.05),不同超声强度和时间下聚乙烯亚胺修饰白蛋白微泡微泡转基因效率在COS和CHO两种细胞均没有显著差异,且与未超声组相比略有下降,但无统计学意义(P>0.05);(8)超声介导AMB和SonoVue微泡破裂转基因时,质粒DNA依靠空化效应在细胞膜上形成的微孔进入细胞,而PAMB可携带质粒DNA主动进入细胞。
结论:本研究制作的聚乙烯亚胺修饰包裹全氟丙烷气体微泡可将其携带的外源基因转入真核细胞,且体外实验证实其具有低细胞毒性和较高的转基因效率,是一种有效的体外基因转染方法,为PAMB作为体内转基因载体提供实验依据。
Objective:To improve the transgene efficiency of albumin microbubbles,we investigated a method of transgene with polyethylenimine coated albumin microbubbles.
Method:(1) Albumin microbubbles (AMB) were prepared by sonicating the mixture of human albumin, manose and glucose; (2) Air filled in albumin microbubbles was exchanged with perfluoropropane and polyethylene glycol (PEG) was added as one gradient of microbubbles to improve the stability of albumin microbubbles; (3) Polyethylenimine (PEI) coated albumin microbubbles (PAMB) was prepared by sonicating the mixture of human albumin, PEI, polyethylene glycol (PEG), glucose, manose and perfluoropropane. Through transfecting CHO cell line with plasmid pIRSE2-EGFP, the optimal weight ratio of albumin and PEI was insured by measureing the percentage of GFP with flow cytometry; (4) Mean particle diameter and surface potential of AMB and PAMB were determined by photon cross correlation spectroscopy with a NANOPHOX particle size analysis system and ZetaProbe 7020; (5) To identify PAMB combined with pDNA, PAMB and pDNA with addition of SYBR green were mixed in serum-free Opti-MEM?Ⅰmedium in an Eppendorf tube. When SYBR green was combined with plasmid DNA, it could be excited by blue light and emitted green fluorescence. Thereafter, if PAMB combined with pDNA, it could emit green fluorescence. Green microbubbles could be inspected with a fluorescence microscrope; (6) Through transfecting CHO, 293T, M231, 293, SK-Hep-1, SKBR-3, MCF-7 and COS cell lines with plasmid pIRSE2-EGFP, we compared the transgene efficiency of PEI, albumin+PEI, Lipofectamine 2000 and PAMB; (7) The cytotoxicity and cell proliferation ablity of PEI, Lipofectamine 2000 and PAMB were measured by cell counting kit-8. Cell proliferation index was calculated by the OD value prior to Transfection, and 0 h, 24 h, 48 h, 72 h posttransfection / OD value prior to transfection; (8) COS cell line was transfected by AMB, SonoVue and PAMB mediated by ultrasound with different ultrasound (U) intensity and time; (9) The effect of ultrasound on microbubbles transgene was investigated. After CHO cell sections were prepared, reagents were added as follows in each group:①o nly NA-Green (a kind of DNA fluorescence dye which can not enter cells actively);②AMB+DNA+NA-Green;③AMB+DNA+NA-Green+U(1.0 W/cm2, 30 S);④P AMB+ NA-Green;⑤PAMB+DNA+NA-Green;⑥P AMB+DNA+ NA-Green+U (1.0 W/cm2, 30 S). After incubation at 37℃, 5% CO2 for 6 h; the medium was removed. The sections were rinsed for three times and immobilized with 1% paraformaldehyde, then were inspected with confocal microscoup and photos were taken.
Results:(1) The half time of albumin microbubbles was extended from half an hour or several days to 6 months after air that filled in albumin microbubbles was exchanged with perfluoropropane and polyethylene glycol (PEG) was added as one gradient of microbubbles; (2) The optimal weight ratio of albumin and PEI was 125:1, which was obtained through transfecting CHO cell line with plasmid pIRSE2-EGFP, and the highest transfection efficiency of PAMB was about 55% on CHO cell line; (3) The particle size analysis showed the average particle diameter of AMB and PAMB was about 1400nm and 600nm, and the surface potentials of PAMB and AMB were -44.56±0.75 mV and -59.28±3.48 mV, respectively; (4) AMB could not combine with pDNA and only emitted plenty of fluorescence without outlook of microbubbles, PAMB combined with pDNA emitted fluorescence; (5) The transgene efficiency in PAMB group was higher than that in PEI group and albumin + PEI group in each cell line (P<0.01) and there was no statistical difference between PAMB group and Lipofectamine 2000 group; (6) Cytotoxicity of PAMB was lower than PEI and Lipofectamine 2000(P < 0.01), the proliferation index in no treatment group and PAMB was higher than that in PEI and Lipofectamine 2000 group (P < 0.01), there was no statistical difference PAMB group and no treatment group (P>0.05); (7) COS cells were transfected with AMB, SonoVue and PAMB mediated by ultrasound with different ultrasound intensity and time, the results showed that the transfection efficiency of PAMB was higher than the efficiency of AMB and SonoVue (P < 0.01), there was no statistical difference between AMB and SonoVue, and there were no statistical differences between different ultrasound intensity and time, and compared with PAMB alone; (8) Destruction of AMB and SonoVue mediated by ultrasound could increase the permeability of membrane, plasmid DNA enter cells through these micropores, while PAMB could bring plasmid DNA into cells actively.
Conlusion:In summary, PAMB is very useful and low toxicity gene delivery method with high transfection efficiency in vitro. Further studies are necessary to examine the detailed mechanism of the effect of ultrasound on PAMB and transfection efficiency in vivo.
引文
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20 Messai I, Munier S, Ataman-Onal Y, et al. Elaboration of poly(ethyleneimine) coated poly(d,l-lactic acid) particles. Effect of ionic strength on the surface properties and DNA binding capabilities. Colloids Surf B Biointerfaces 2003; 32: 293-305.
21 Edelstein ML, Abedi MR, Wixon J. Gene therapy clinical trials worldwide to 2007: an update. J Gene Med 2007; 9: 833-842.
22 Kichler A, Leborgne C, Coeytaux E, et al. Polyethylenimine-mediated gene delivery: a mechanistic study. J Gene Med 2001; 3: 135-144.
23 Wells DJ. Electroporation and ultrasound enhanced non-viral gene delivery in vitro and in vivo. Cell Biol Toxicol 2010; 26: 21-28.
24 Sumiyama K, Kawakami K, Yagita K. A simple and highly efficient transgenesis method in mice with the Tol2 transposon system and cytoplasmic microinjection. Genomics 2010; In press.
25 Lin CT, Yen CF, Shaw SW, at el. Gene gun administration of therapeutic HPV DNA vaccination restores the efficacy of prolonged defrosted viral based vaccine. Vaccine 2009; 2752: 7352-7358.
26 Rezende FC, Gomes HC, Lisboa B, at el. Electroporation of vascular endothelial growth factor gene in a unipedicle transverse rectus abdominis myocutaneous flap reduces necrosis. Ann Plast Surg 2010; 64:242-246.
27 Ren JL, Xu CS, Zhou ZY, et al. A Novel Ultrasound Microbubble Carrying Gene and Tat Peptide: Preparation and Characterization. Acad Radiol 2009; 16:1457-1465.
28 Li HL, Zheng XZ, Wang HP, et al. Ultrasound-targeted microbubble destruction enhances AAV-mediated gene transfection in human RPE cells in vitro and rat retina in vivo. Gene Ther 2009; 16: 1146-1153.
29 Li W, L Su, Ren JL, et al. Gene Transfection to Retinal Ganglion Cells Mediated by Ultrasound Microbubbles in vitro. Acad Radiol 2009; 16:1086-1094.
30 Xenariou S, Griesenbach U, Liang HD, et al. Use of ultrasound to enhance nonviral lung gene transfer in vivo. Gene Ther 2007; 14: 768-774.
31 Meijering BD, Juffermans LJ, Wamel AV, et al. Ultrasound and microbubble - targeted delivery of macromolecules is regulated by induction of endocytosis and pore formation. Circ Res 2009; 104: 679-687.
32 Liang HD, Blomley MJK. The role of ultrasound in molecular imaging. Br J Radiol 2003; 76: 140-150.
33 Bekeredjian R, Chen S, Frenkel PA, et al. Ultrasound-targeted microbubble destruction can repeatedly direct highly specific plasmid expression to the heart. Circulation 2003; 108:1022-1026.
34 Guo DP, Li XY, Sun P, et al. Ultrasound/microbubble enhances foreign gene expression in ECV304 cells and murine myocardium. Acta Biochim Biophys Sin (Shanghai) 2004; 36: 824-831.
35 Guo DP, Li XY, Sun P, et al. Ultrasound-targeted microbubble destruction improves the low density lipoprotein receptor gene expression in HepG2 cells. Biochem Biophys Res Commun 2006; 343: 470-474.
36李肖蓉,邵力正,王强,等.新型白蛋白微泡经超声介导破裂促进EGFP基因在Cos-7细胞的表达.中华超声影像学杂志2003; 12:236-239.
1 Enomoto S, Yoshiyama M, Omura T, et al. Microbubble destruction with ultrasound augments neovascularisation by bone marrow cell transplantation in rat hind limb ischaemia. Heart 2006; 92:515-520.
2 Wang ZG, Ling ZY, Ran HT, et al. Ultrasound-mediated microbubble destruction enhances VEGF gene delivery to the infarcted myocardium in rats. Clin Imag, 2004; 28:395-398.
3 Guo DP, Li XY, Sun P, et al. Ultrasound/microbubble enhances foreign gene expression in ECV304 cells and murine myocardium. Acta Biochim Biophys Sin (Shanghai) 2004; 36: 824-831.
4 Guo DP, Li XY, Sun P, et al. Ultrasound-targeted microbubble destruction improves the low density lipoprotein receptor gene expression in HepG2 cells. Biochem Biophys Res Commun 2006; 343: 470-474.
5李肖蓉,邵力正,王强,等.新型白蛋白微泡经超声介导破裂促进EGFP基因在Cos-7细胞的表达.中华超声影像学杂志2003; 12:236-239.
6 Li T, Tachibana K, Kuroki M, et al. Gene transfer with echo-enhanced contrast agents: comparison between Albunex, Optison, and Levovist in mice—initial results. Radiology 2003; 229: 423-428.
7 Watanabe A, Otake R, Nozaki T,et al. Effects of microbubbles on ultrasound -mediated gene transfer in human prostate cancer PC3 cells: Comparison among Levovist, YM454, and MRX-815H . Cancer Lett 2008; 265: 107-112.
8 Li YS, Davidson E, Reid CN, et al. Optimising ultrasound-mediated gene transfer (sonoporation) in vitro and prolonged expression of a transgene in vivo: Potential applications for gene therapy of cancer. Cancer Lett 2009; 273: 62-69.
9 Suzuki R, Takizawa T, Negishi Y, et al. Gene delivery by combination of novel liposomal bubbles with perfluoropropane and ultrasound. J Control Release 2007; 117: 130-136.
10 Suzuki R, Takizawa T, Negishi Y, et al. Effective gene delivery with novel liposomal bubbles and ultrasonic destruction technology. Int J Pharm 2008; 354: 49-55.
11 Moore NM, Sheppard C L, Sakiyama-Elbert SE. Characterization of a multifunctional PEG-based gene delivery system containing nuclear localization signals and endosomal escape peptides. Acta Biomaterialia 2009; 5: 854-864.
12 Godbey WT, Wu KK, Mikos AG. Poly(ethylenimine) and its role in gene delivery. J Control Release 1999; 60: 149-160.
13 Kircheis R, Wightman L, Wagner E. Design and gene delivery activity of modified polyethylenimines. Adv Drug Deliv Rev 2001; 53: 341-358.
14 Kichler A, Leborgne C, Coeytaux E, et al. Polyethylenimine-mediated gene delivery: a mechanistic study. J Gene Med 2001; 3: 135-144.
15 Itaka K, Harada A, Yamasaki Y, et al. In situ single cell observation by fluorescence resonance energy transfer reveals fast intra-cytoplasmic delivery and easy release of plasmid DNA complexed with linear polyethylenimine. J Gene Med 2004; 6: 76-84.
16 Burke RS, Pun SH. Extracellular Barriers to in ViWo PEI and PEGylated PEI Polyplex-Mediated Gene Delivery to the Liver. Bioconjugate Chem 2008; 19: 693-704.
17 Mao HQ, Leong KW. Design of polyphosphoester-DNA nanoparticles for non-viral gene delivery. Adv Genet 2005; 53: 275-306.
18 Grosse S, Thévenot G, Aron Y, et al. In vivo gene delivery in the mouse lung with lactosylated polyethylenimine, questioning the relevance of in vitro experiments. Journal of Controlled Release 2008; 132: 105-112.
19 Krajewski, W.A., Effect of in vivo histone hyperacetylation on the state of chromatin fibers. J Biomol Struct Dyn 1999; 16: 1097-1106.
20 Luthman, H., and Magnusson, G., High efficiency polyoma DNA transfection of chloroquine treated cells. Nucleic Acids Res 1983; 11: 1295-1308.
1 Burke RS, Pun SH. Extracellular Barriers to in ViWo PEI and PEGylated PEI Polyplex-Mediated Gene Delivery to the Liver. Bioconjugate Chem 2008; 19: 693-704.
2 Mao HQ, Leong KW. Design of polyphosphoester-DNA nanoparticles for non-viral gene delivery. Adv Genet 2005; 53: 275-306.
3 Wen YT, Pan SR, Luo X, et al. A biodegradable low molecular weight polyethylenimine derivative as low toxicity and efficient gene vector. Bioconjugate Chem 2009; 20: 322-332.
4李经忠,王青青,余海,等. PEI转基因影响因素的测定及其优化.中国生物化学与分子生物学报2004; 20: 234-240.
5 Rudolph C, Schillinger U, Plank C, et al. Nonviral gene delivery to the lung with copolymer-protected and transferrin-modified polyethylenimine. Biochim Biophys Acta-Gen Subj 2002; 1573: 75-83.
6 Ahn CH, Chae SY, Bae YH, et al. Biodegradable poly(ethylenimine) for plasmid DNA delivery. J Control Release 2002; 80: 273-282.
7 Liang B, He ML, Chan CY, et al. The use of folate-PEG-grafted-hybranched-PEI nonviral vector for the inhibition of glioma growth in the rat. Biomaterials 2009; 30: 4014-4020.
8 Kichler A, Leborgne C, Coeytaux E, Danos O. Polyethylenimine-mediated gene delivery: a mechanistic study. J Gene Med 2001; 3: 135-144.
9 Mao HQ, Leong KW. Design of polyphosphoester-DNA nanoparticles for non-viral gene delivery. Adv Genet 2005; 53: 275-306.
10 Paris S, Burlacu A, Durocher Y. Opposing Roles of Syndecan-1 and Syndecan-2 in Polyethyleneimine-mediated Gene Delivery. J Biol Chem 2008; 283: 7697-7704.
11 Kurosaki T, Kitahara T, Fumoto S, et al. Ternary complexes of pDNA, polyethylenimine, and g-polyglutamic acid for gene delivery systems. Biomaterials 2009; 30: 2846-2853.
12 Paris S, Burlacu A, Durocher Y. Opposing roles of syndecan-1 and syndecan-2 in polyethyleneimine-mediated gene delivery. J Biol Chem 2008; 283: 7697-7704.
13 Kurosaki T, Kitahara T, Fumoto S, et al. Ternary complexes of pDNA, polyethylenimine, and g-polyglutamic acid for gene delivery systems. Biomaterials 2009; 30: 2846-2853.
14 Kleemann E, Neu M, Jekel N, et al. Nano-carriers for DNA delivery to the lung based upon a TAT-derived peptide covalently coupled to PEG-PEI. J Control Release 2005; 109: 299-316.
1 Enomoto S, Yoshiyama M, Omura T, et al. Microbubble destruction with ultrasound augments neovascularisation by bone marrow cell transplantation in rat hind limb ischaemia. Heart 2006; 92:515-520.
2 Hu YZ, Zhu JA, Jiang YG, et al. Ultrasound microbubble contrast agents: Application to therapy for peripheral vascular disease. Adv Ther 2009; 26:425-434.
3 Landini L, Santarelli MF, Landini L, et al. Ultrasound techniques for drug delivery in cardiovascular medicine. Curr Drug Discov Technol 2008; 5:328-332.
4 Kaddur K, Palanchon P, Tranquart F, et al. Sonopermeabilization: therapeutic alternative with ultrasound and microbubbles. J Radiol 2007; 88:1777-1786.
5 Husseini GA, Diaz MA, Richardson ES, et al.The Role of Cavitation in Acoustically Activated Drug Delivery. J Control Release 2005; 107:253-261.
6 Mayer CR, Geis NA, Katus HA, et al. Ultrasound targeted microbubble destruction for drug and gene delivery. Expert Opin Drug Deliv 2008; 5:1121-1138.
7刘国通,杨成明,王旭开,等.超声微泡介导转染FKBP12.6基因对小鼠H9c2(221)心肌细胞结构和功能的影响.中华超声影像学杂志,2007,16:163-166.
8 Datta S, Coussios CC, McAdory LE, et al. Correlation of cavitation with ultrasound enhancement of thrombolysis. Ultrasound Med Biol 2006; 32:1257-1267.
9 Ohl CD, Arora M, Ikink R, et al. Sonoporation from Jetting Cavitation Bubbles. Biophys J 2006; 91:4285-4295.
10 Cochran SA, Prausnitz MR. Sonoluminescence as an indicator of cell membrane discruption by acoustic cavitation. Ultrasound Med Biol 2001; 27: 841-850.
11 Enomoto S, Yoshiyama M, Omura T, et al. Microbubble destruction with ultrasound augments neovascularisation by bone marrow cell transplantation in rat hind limb ischaemia.Heart,2006, 92:515-520.
12 Wang ZG, Ling ZY, Ran HT, et al. Ultrasound-mediated microbubble destruction enhances VEGF gene delivery to the infarcted myocardium in rats. Clin Imag 2004; 28:395-398.
13 Hu YZ, Zhu JA, Jiang YG, et al. Ultrasound microbubble contrast agents: Application to therapy for peripheral vascular disease. Adv Ther 2009; 26: 425-434.
14 Landini L, Santarelli MF, Landini L, et al. Ultrasound techniques for drug delivery in cardiovascular medicine. Curr Drug Discov Technol 2008; 5: 328-32.
15 Lu QL, Liang HD, Partridge T. Microbubble ultrasound improves the efficiency of gene transduction in skeletal muscle in vivo with reduced tissue damage. Gene Therapy 2003; 10: 396-405.
16 Yang LJ, Shirakata YJ, Tamai K, et al. Microbubble-enhanced ultrasound for gene transfer into living skin equivalents. Journal of Dermatological Science 2005; 40: 105-114.
17 Haag P, Frauscher F, Gradl J, et al. Microbubble-enhanced ultrasound to deliver an antisense oligodeoxynucleotide targeting the human androgen receptor into prostate tumours. Journal of Steroid Biochemistry & Molecular Biology 2006; 102: 103-113.
18 Hernot S, Klibanov AL. Microbubbles in ultrasound-triggered drug and gene delivery. Advanced Drug Delivery Reviews 2008; 60: 1153-1166.
19 Lavorini-Doyle C, Gebremedhin S, Konopka K, et al. Gene delivery to oral cancer cells by nonviral vectors: why some cells are resistant to transfection. J Calif Dent Assoc 2009; 37: 855-858.
1. Otani K, Yamahara K, Ohnishi S, et al.Nonviral delivery of siRNA into mesenchymal stem cells by a combination of ultrasound and microbubbles. J Control Release 2009; 133:146-153.
2. Greenleaf WJ, Bolander ME, Sarkar G, et al. Artificial cavitation nuclei significantly enhance acoustically induced cell transfection.Ultrasound Med Biol 1998; 24: 587-595.
3. Watanabe Y, Aoi A, Horie S, et al.Low-intensity ultrasound and microbubbles enhance the antitumor effect of cisplatin. Cancer Sci 2008; 99: 2525-2531.
4. Kaddur K, Palanchon P, Tranquart F, et al. Sonopermeabilization: therapeutic alternative with ultrasound and microbubbles. J Radiol 2007; 88: 1777-1786.
5. Husseini GA, Diaz de la Rosa MA, Richardson ES, et al.The Role of Cavitation in Acoustically Activated Drug Delivery. J Control Release 2005; 107: 253-261.
6. Mayer CR, Geis NA, Katus HA, et al. Ultrasound targeted microbubble destructionfor drug and gene delivery. Expert Opin Drug Deliv 2008; 5:1121-1138.
7.刘国通,杨成明,王旭开,等.超声微泡介导转染FKBP12.6基因对小鼠H9c2(221)心肌细胞结构和功能的影响.中华超声影像学杂志2007; 16:163-166.
8. Datta S, Coussios CC, McAdory LE, et al. Correlation of cavitation with ultrasound enhancement of thrombolysis. Ultrasound Med Biol 2006; 32:1257–1267.
9. Ohl CD, Arora M, Ikink R, et al. Sonoporation from Jetting Cavitation Bubbles. Biophys J 2006; 91:4285-4295.
10. Cochran SA, Prausnitz MR. Sonoluminescence as an indicator of cell membrane discruption by acoustic cavitation. Ultrasound Med Biol 2001; 27: 841-850.
11. Enomoto S, Yoshiyama M, Omura T,et al. Microbubble destruction with ultrasound augments neovascularisation by bone marrow cell transplantation in rat hind limb ischaemia.Heart 2006; 92:515-520.
12. Wang ZG, Ling ZY, Ran HT, et al. Ultrasound-mediated microbubble destruction enhances VEGF gene delivery to the infarcted myocardium in rats. Clin Imag 2004; 28: 395-398.
13. Hu YZ, Zhu JA, Jiang YG,et al. Ultrasound microbubble contrast agents: Application to therapy for peripheral vascular disease. Adv Ther 2009; 26: 425-434.
14. Landini L, Santarelli MF, Landini L, et al. Ultrasound techniques for drug delivery in cardiovascular medicine. Curr Drug Discov Technol 2008; 5: 328-32.
15.李肖蓉,邵力正,王强,等.新型白蛋白微泡经超声介导破裂促进EGFP基因在Cos-7细胞的表达.中华超声影像学杂志2003; 12: 236-239.
16. Guo DP, Li XY, Sun P, et al. Ultrasound/microbubble enhances foreign gene expression in ECV304 cells and murine myocardium. Acta Biochim Biophys Sin (Shanghai) 2004; 36: 824-831.
17. Guo DP, Li XY, Sun P, et al. Ultrasound-targeted microbubble destruction improves the low density lipoprotein receptor gene expression in HepG2 cells. Biochem Biophys Res Commun 2006; 343: 470-474.
18. Takahashi M, Kido K, Aoi A, et al. Spinal gene transfer using ultrasound and microbubbles. J Control Release 2007; 117: 267-272.
19. Kondo I, Ohmori K, Oshita A, et al. Treatment of acute myocardial infarction by hepatocyte growth factor gene transfer. J Am Coll Cardiol 2004; 44: 644-653.
20. Li T, Tachibana K, Kuroki M, et al. Gene transfer with echo-enhanced contrast agents: comparison between Albunex, Optison, and Levovist in mice-initial results. Radiology 2003; 229: 423-428.
21. Watanabe A,Otake R,Nozaki T,et al. Effects of microbubbles on ultrasound-mediated gene transfer in human prostate cancer PC3 cells: Comparison among Levovist, YM454, and MRX-815H . Cancer Lett 2008; 265: 107-112.
22. Li YS, Davidson E, Reid CN,et al. Optimising ultrasound-mediated gene transfer (sonoporation) in vitro and prolonged expression of a transgene in vivo: Potential applications for gene therapy of cancer. Cancer Lett 2009; 273: 62-69.
23. Suzuki R, Takizawa T, Negishi Y, et al. Gene delivery by combination of novel liposomal bubbles with perfluoropropane and ultrasound. J Control Release 2007; 117: 130-136.
24. Suzuki R, Takizawa T, Negishi Y,et al. Effective gene delivery with novel liposomal bubbles and ultrasonic destruction technology. Int J Pharm 2008; 354: 49-55.
25. Tsunoda S, Mazda O, Oda Y, et al. Sonoporation using microbubble BR14 promotes pDNA/siRNA transduction to murine heart. Biochem Biophys Res Commun 2005; 336: 118-127.
26. Meijering BD, Juffermans LJ, van Wamel A, et al. Ultrasound and microbubble-targeted delivery of macromolecules is regulated by induction of endocytosis and pore formation. Circ Res 2009; 104: 679-687.
27.彭幼玲,郭爱林,肖敏,等.超声造影剂促进野生型p53基因转染抑制大鼠卵巢癌生长的实验研究.中华超声影像学杂志2006; 15: 61-64.
28.陈云超,张青萍,LIANG HD,等.超声和微泡造影剂介导细胞基因转染的实验研究.中华超声影像学杂志2006; 15: 864-868.
29. Alter J, Sennoga CA, Lopes DM, et al. Microbubble stability is a major determinant of the efficiency of ultrasound and microbubble mediated in Vivo gene transfer. Ultrasound Med Biol 2009; 35: 976-984.
30. Hashiya N, Aoki M, Tachibana K,et al. Local delivery of E2F decoy oligodeoxynucleotides using ultrasound with microbubble agent (Optison) inhibits intimal hyperplasia after balloon injury in rat carotid artery model. Biochem Biophys Res Commun 2004; 317:508-514.
31. Yang L, Shirakata Y, Tamai K, ,et al. Microbubble-enhanced ultrasound for gene transfer into living skin equivalents. J Dermatol Sci 2005; 40:105-114.
32. Sonoda S, Tachibana K, Uchino E, et al. Gene transfer to corneal epithelium and keratocytes mediated by ultrasound with microbubbles. Invest Ophthalmol Vis Sci 2006; 47: 558-564.
33. Haag P, Frauscher F, Gradl J,et al. Microbubble-enhanced ultrasound to deliver an antisense oligodeoxynucleotide targeting the human androgen receptor into prostate tumours. J Steroid Biochem Mol Biol 2006;102:103-113
34. Chen S, Ding JH, Bekeredjian R,et al. Efficient gene delivery to pancreatic islets with ultrasonic microbubble destruction technology.Proc Natl Acad Sci USA 2006; 103:8469-8474.
2 Edelstein ML, Abedi MR, Wixon J. Gene therapy clinical trials worldwide to 2007: an update. J Gene Med 2007; 9: 833-842.
3 Brenner MK, Okur FV. Overview of gene therapy clinical progress including cancer treatment with gene-modified T cells. Hematology Am Soc Hematol Educ Program 2009; 675-681.
4 Kaiser J. Gene therapy. Beta-thalassemia treatment succeeds, with a caveat. Science 2009; 326: 1468-1479.
5 Buch PK, Bainbridge JW, Ali RR. AAV-mediated gene therapy for retinal disorders: from mouse to man. Gene Ther 2008; 15: 849-857.
6 Cheung W, Pontoriero F, Taratula O, et al. DNA and carbon nanotubes as medicine . Adv Drug Deliv Rev 2010; In press.
7 Baker AH. Designing gene delivery vectors for cardiovascular gene therapy. Prog Biophys Mol Biol 2004; 84:279-299.
8 Li YS, Davidson E, Reid CN, et al. Optimising ultrasound-mediated gene transfer (sonoporation) in vitro and prolonged expression of a transgene in vivo: Potential applications for gene therapy of cancer. Cancer Lett 2009; 273: 62-69.
9 Brake O, Westerink JT, Berkhout B. Lentiviral vector engineering for anti-HIV RNAi gene therapy. Methods Mol Biol 2010; 614: 201-213.
10 Heilbronn R, Weger S. Viral vectors for gene transfer: current status of gene therapeutics. Handb Exp Pharmacol 2010; 197: 143-170.
11 Zhou C, Yu B, Yang X, et al. Lipid-coated nano-calcium-phosphate (LNCP) for gene delivery. Int J Pharm 2010; In press.
12 Chirmule N, Propert K, Magosin S, Qian Y, Qian R,Wilson J. Immune responses to adenovirus and adenoassociated virus in humans. Gene Ther 1999; 6: 1574-1583.
13 Manno CS, Pierce GF, Arruda VR, Glader B, Ragni M, Rasko JJ. Successful transduction of liver in hemophilia by AAV-Factor IX and limitations imposed by the host immune response. Nat Med 2006; 12: 342-347.
14 Zhang CM, Zhang XJ, Liu CB, et al. Expression of endostatin mediated by a novel non-viral delivery system inhibits human umbilical vein endothelial cells in vitro. Mol Biol Rep 2010; 37: 1755-1762.
15 Phillips LC, Klibanov AL, Bowles DK, et al. Focused in vivo delivery of plasmid DNA to the porcine vascular wall via intravascular ultrasound destruction of microbubbles. J Vasc Res 2010; 47: 270-274.
16 Halama A, Kuliński M, Librowski T, et al. Polymer-based non-viral gene delivery as a concept for the treatment of cancer. Pharmacol Rep 2009; 61: 993-999.
17 Zou S, Scarfo K, Nantz MH, et al. Lipid-mediated delivery of RNA is more efficient than delivery of DNA in non-dividing cells. Int J Pharm 2010; 389: 232-243.
18 Holladay CA, O'Brien T, Pandit A. Non-viral gene therapy for myocardial engineering. Wiley Interdiscip Rev Nanomed Nanobiotechnol 2009; In press.
19 Oku N, Yamazaki Y, Matsuura M, et al. A novel non-viral gene transfer system, polycation liposomes. Advanced Drug Delivery Reviews 2001; 52: 209-218.
20 Messai I, Munier S, Ataman-Onal Y, et al. Elaboration of poly(ethyleneimine) coated poly(d,l-lactic acid) particles. Effect of ionic strength on the surface properties and DNA binding capabilities. Colloids Surf B Biointerfaces 2003; 32: 293-305.
21 Edelstein ML, Abedi MR, Wixon J. Gene therapy clinical trials worldwide to 2007: an update. J Gene Med 2007; 9: 833-842.
22 Kichler A, Leborgne C, Coeytaux E, et al. Polyethylenimine-mediated gene delivery: a mechanistic study. J Gene Med 2001; 3: 135-144.
23 Wells DJ. Electroporation and ultrasound enhanced non-viral gene delivery in vitro and in vivo. Cell Biol Toxicol 2010; 26: 21-28.
24 Sumiyama K, Kawakami K, Yagita K. A simple and highly efficient transgenesis method in mice with the Tol2 transposon system and cytoplasmic microinjection. Genomics 2010; In press.
25 Lin CT, Yen CF, Shaw SW, at el. Gene gun administration of therapeutic HPV DNA vaccination restores the efficacy of prolonged defrosted viral based vaccine. Vaccine 2009; 2752: 7352-7358.
26 Rezende FC, Gomes HC, Lisboa B, at el. Electroporation of vascular endothelial growth factor gene in a unipedicle transverse rectus abdominis myocutaneous flap reduces necrosis. Ann Plast Surg 2010; 64:242-246.
27 Ren JL, Xu CS, Zhou ZY, et al. A Novel Ultrasound Microbubble Carrying Gene and Tat Peptide: Preparation and Characterization. Acad Radiol 2009; 16:1457-1465.
28 Li HL, Zheng XZ, Wang HP, et al. Ultrasound-targeted microbubble destruction enhances AAV-mediated gene transfection in human RPE cells in vitro and rat retina in vivo. Gene Ther 2009; 16: 1146-1153.
29 Li W, L Su, Ren JL, et al. Gene Transfection to Retinal Ganglion Cells Mediated by Ultrasound Microbubbles in vitro. Acad Radiol 2009; 16:1086-1094.
30 Xenariou S, Griesenbach U, Liang HD, et al. Use of ultrasound to enhance nonviral lung gene transfer in vivo. Gene Ther 2007; 14: 768-774.
31 Meijering BD, Juffermans LJ, Wamel AV, et al. Ultrasound and microbubble - targeted delivery of macromolecules is regulated by induction of endocytosis and pore formation. Circ Res 2009; 104: 679-687.
32 Liang HD, Blomley MJK. The role of ultrasound in molecular imaging. Br J Radiol 2003; 76: 140-150.
33 Bekeredjian R, Chen S, Frenkel PA, et al. Ultrasound-targeted microbubble destruction can repeatedly direct highly specific plasmid expression to the heart. Circulation 2003; 108:1022-1026.
34 Guo DP, Li XY, Sun P, et al. Ultrasound/microbubble enhances foreign gene expression in ECV304 cells and murine myocardium. Acta Biochim Biophys Sin (Shanghai) 2004; 36: 824-831.
35 Guo DP, Li XY, Sun P, et al. Ultrasound-targeted microbubble destruction improves the low density lipoprotein receptor gene expression in HepG2 cells. Biochem Biophys Res Commun 2006; 343: 470-474.
36李肖蓉,邵力正,王强,等.新型白蛋白微泡经超声介导破裂促进EGFP基因在Cos-7细胞的表达.中华超声影像学杂志2003; 12:236-239.
1 Enomoto S, Yoshiyama M, Omura T, et al. Microbubble destruction with ultrasound augments neovascularisation by bone marrow cell transplantation in rat hind limb ischaemia. Heart 2006; 92:515-520.
2 Wang ZG, Ling ZY, Ran HT, et al. Ultrasound-mediated microbubble destruction enhances VEGF gene delivery to the infarcted myocardium in rats. Clin Imag, 2004; 28:395-398.
3 Guo DP, Li XY, Sun P, et al. Ultrasound/microbubble enhances foreign gene expression in ECV304 cells and murine myocardium. Acta Biochim Biophys Sin (Shanghai) 2004; 36: 824-831.
4 Guo DP, Li XY, Sun P, et al. Ultrasound-targeted microbubble destruction improves the low density lipoprotein receptor gene expression in HepG2 cells. Biochem Biophys Res Commun 2006; 343: 470-474.
5李肖蓉,邵力正,王强,等.新型白蛋白微泡经超声介导破裂促进EGFP基因在Cos-7细胞的表达.中华超声影像学杂志2003; 12:236-239.
6 Li T, Tachibana K, Kuroki M, et al. Gene transfer with echo-enhanced contrast agents: comparison between Albunex, Optison, and Levovist in mice—initial results. Radiology 2003; 229: 423-428.
7 Watanabe A, Otake R, Nozaki T,et al. Effects of microbubbles on ultrasound -mediated gene transfer in human prostate cancer PC3 cells: Comparison among Levovist, YM454, and MRX-815H . Cancer Lett 2008; 265: 107-112.
8 Li YS, Davidson E, Reid CN, et al. Optimising ultrasound-mediated gene transfer (sonoporation) in vitro and prolonged expression of a transgene in vivo: Potential applications for gene therapy of cancer. Cancer Lett 2009; 273: 62-69.
9 Suzuki R, Takizawa T, Negishi Y, et al. Gene delivery by combination of novel liposomal bubbles with perfluoropropane and ultrasound. J Control Release 2007; 117: 130-136.
10 Suzuki R, Takizawa T, Negishi Y, et al. Effective gene delivery with novel liposomal bubbles and ultrasonic destruction technology. Int J Pharm 2008; 354: 49-55.
11 Moore NM, Sheppard C L, Sakiyama-Elbert SE. Characterization of a multifunctional PEG-based gene delivery system containing nuclear localization signals and endosomal escape peptides. Acta Biomaterialia 2009; 5: 854-864.
12 Godbey WT, Wu KK, Mikos AG. Poly(ethylenimine) and its role in gene delivery. J Control Release 1999; 60: 149-160.
13 Kircheis R, Wightman L, Wagner E. Design and gene delivery activity of modified polyethylenimines. Adv Drug Deliv Rev 2001; 53: 341-358.
14 Kichler A, Leborgne C, Coeytaux E, et al. Polyethylenimine-mediated gene delivery: a mechanistic study. J Gene Med 2001; 3: 135-144.
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