血管内皮生长因子受体2单抗的靶向超声造影剂制备及体外评价
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摘要
目的探讨携血管内皮生长因子受体2(KDR)单抗的靶向脂质微泡的制备方法,并对其物理特性、生物活性、靶向黏附稳定性进行体外测定。方法采用声振仪制备生物素化脂质微泡(MB-B),再应用生物素-亲和素桥连技术构建携KDR单抗的靶向微泡(MB-BAB-KDR)。荧光显微镜下观察MB-B、单纯单抗微泡(MB-B-KDR)、MB-BAB-KDR与荧光二抗孵育后荧光强度。利用平行板流动腔技术体外模拟生理血流剪切力条件,评价靶向微泡的黏附效能。结果制备的靶向微泡MB-BAB-KDR呈圆球形,粒度分布均匀。与荧光二抗孵育后,MB-BAB-KDR发出明亮的绿色荧光(Ⅱ级),而MB-B-KDR显示微弱的绿色荧光(Ⅰ级)、MB-B无荧光(0级)。体外黏附实验显示,MB-BAB-KDR结合数目随着小鼠KDR Fc包被浓度的增高而增加(P<0.05),相同浓度下,随着时间的增加,MB-BAB-KDR结合数目增加。结论成功构建生物素-亲和素桥连的靶向脂质体微泡MB-BABKDR,体外黏附实验显示MB-BAB-KDR具有黏附稳定性,且与时间、浓度呈正相关。
Objective To prepare a new targeted liposome ultrasonic contrast agent with anti-KDR antibody that binds specifically with KDR as the main receptor of VEGF and evaluate its physical characteristics,biological activity and specific binding capability in vitro.Methods A sonicator was used to prepare the biotinylated lipid micro-bubbles(MB-B),and biotinavidin-mediated technique was used for attachment of anti-mouse KDR monoclonal antibody to the micro-bubble shell to generate MB-BAB-KDR.MB-BAB-KDR was incubated with fluorescent second antibody to assess the link condition,and the control groups were the MB-B and micro-bubbles with the antibody alone(MB-B-KDR).A parallel plate flow chamber system was used to characterize micro-bubbles attachment efficiency to KDR Fc.Results The surface of the micro-bubbles could carry KDR antibody through the biotin-avidin bridge and MB-BAB-KDR were spherical and well-distributed.After incubation with the second antibody,MB-BAB-KDR could be observed to emit bright green fluorescence(Grade II) as compared with little or weak fluorescence in the control MB-B group(Grade 0) and MB-B-KDR group(Grade I).Targeted micro-bubbles bound to KDR Fc increased as the KDR Fc concentration increased(P<0.05).Conclusion The targeted liposome contrast agent linked with KDR antibody by biotin-avidin bridge we prepared shows an increased binding number as the KDR Fc concentration increases,which provides a novel approach to molecular imaging study of endometrial receptivity.
Objective To prepare a new targeted liposome ultrasonic contrast agent with anti-KDR antibody that binds specifically with KDR as the main receptor of VEGF and evaluate its physical characteristics,biological activity and specific binding capability in vitro.Methods A sonicator was used to prepare the biotinylated lipid micro-bubbles(MB-B),and biotinavidin-mediated technique was used for attachment of anti-mouse KDR monoclonal antibody to the micro-bubble shell to generate MB-BAB-KDR.MB-BAB-KDR was incubated with fluorescent second antibody to assess the link condition,and the control groups were the MB-B and micro-bubbles with the antibody alone(MB-B-KDR).A parallel plate flow chamber system was used to characterize micro-bubbles attachment efficiency to KDR Fc.Results The surface of the micro-bubbles could carry KDR antibody through the biotin-avidin bridge and MB-BAB-KDR were spherical and well-distributed.After incubation with the second antibody,MB-BAB-KDR could be observed to emit bright green fluorescence(Grade II) as compared with little or weak fluorescence in the control MB-B group(Grade 0) and MB-B-KDR group(Grade I).Targeted micro-bubbles bound to KDR Fc increased as the KDR Fc concentration increased(P<0.05).Conclusion The targeted liposome contrast agent linked with KDR antibody by biotin-avidin bridge we prepared shows an increased binding number as the KDR Fc concentration increases,which provides a novel approach to molecular imaging study of endometrial receptivity.
引文
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[12]Yasu T,Greener Y,Jablonski E,et al.Activated leukocytes and endothelial cells enhance retention of ultrasound microspheres containing perfluoropropane in inflamed vemules[J].Int J Cardiol,2005,98(2):245-52.
[13]Biyiksiz PC,Filiz S,Vural B.Is sildenafil citrate affect endometrial receptivity?An immunohistochemical study[J].Gynecol Endocrinol,2011,27(10):767-74.
[14]Baston-Buest DM,Porn AC,Schanz A,et al.Expression of the vascular endothelial growth factor receptor neuropilin-1 at the human embryo-maternal interface[J].Eur J Obstet Gynecol Reprod Biol,2011,154(2):151-6.
[15]Sidell N,Feng Y,Hao L,et al.Retinoic acid is a cofactor for translational regulation of vascular endothelial growth factor in human endometrial stromal cells[J].Mol Endocrinol,2010,24(1):148-60.
[16]Senqupta J,Lalitkumar PG,Najwa AR,et al.Immunoneutralization of vascular endothelial growth factor inhibits pregnancy establishment in the rhesus monkey(Macaca mulatta)[J].Reproduction,2007,133(6):1199-211.
[17]Blackwell JE,Dagia NM,Diekerson JB,et al.Ligand coated nanosphere adhesion to E-and P-selectin under statie and flow eondition[sJ].Ann Biomed Eng,2001,29(6):523-33.
[2]Kaczmarek MM,Waclawik A,Blitek A,et al.Expression of the vascular endothelial growth factor-receptor system in the porcine endometrium throughout the estrous cycle and early pregnancy[J].Mol Reprod Dev,2008,75(2):362-72.
[3]Hwu YM,Chen CP,Li SH,et al.Expression of vascular endothelial growth factor messenger ribonucleic acid and protein in human preimplantation embryo[sJ].Fertil Steril,2006,85(6):1830-2.
[4]Linder JR.Evolving applicationgs for contrast ultrasound[J].Am J Cardiol,2002,90(10A):72J-82J.
[5]Lawrence MB,Melntire LV,Eskin SG.Effect of flow on polymorphonuclear leukocyte/endothelial cell adhesion[J].Blood,1987,70(5):1284-90.
[6]郑道文,杨莉,宾建平,等.亲和素桥连构建携抗P选择素单抗靶向超声微泡及体外荧光鉴定[J].中国医学影像技术,2008,24(8):1182-5.
[7]纪丽景,燕翼,宾建平.靶向超声微泡靶点和配体的研究进展[J].中国医学影像学杂志,2009,17(4):300-2.
[8]Willmann JK,Paulmurugan R,Chen K,et al.US imaging of tumor angiogenesiswith microbubbles targeted to vascular endothelial growth factor receptor type 2 in mice[J].Radiology,2008,246(2):508-18.
[9]Rychak JJ,Graba J,Cheung AM,et al.Microultrasound molecular imaging of vascular endothelial growth factor receptor 2 in a mouse model of tumor angiogenesi[sJ].Mol Imaging,2007,6(5):289-96.
[10]Kaczmarek MM,Blitek A,Schams D,et al.Effect of luteinizing hormone and tumour necrosis factor-alpha on VEGF secretion by cultured porcine endometrial stromal cells[J].Reprod Domest Anim,2010,45(3):481-6.
[11]Allegra A,Marino A,Coffaro F,et al.Is there a uniform basal endometrial gene expression profile during the implantation window in women who became pregnant in a subsequent ICSI cycle[J]?Hum Reprod,2009,24(10):2549-57.
[12]Yasu T,Greener Y,Jablonski E,et al.Activated leukocytes and endothelial cells enhance retention of ultrasound microspheres containing perfluoropropane in inflamed vemules[J].Int J Cardiol,2005,98(2):245-52.
[13]Biyiksiz PC,Filiz S,Vural B.Is sildenafil citrate affect endometrial receptivity?An immunohistochemical study[J].Gynecol Endocrinol,2011,27(10):767-74.
[14]Baston-Buest DM,Porn AC,Schanz A,et al.Expression of the vascular endothelial growth factor receptor neuropilin-1 at the human embryo-maternal interface[J].Eur J Obstet Gynecol Reprod Biol,2011,154(2):151-6.
[15]Sidell N,Feng Y,Hao L,et al.Retinoic acid is a cofactor for translational regulation of vascular endothelial growth factor in human endometrial stromal cells[J].Mol Endocrinol,2010,24(1):148-60.
[16]Senqupta J,Lalitkumar PG,Najwa AR,et al.Immunoneutralization of vascular endothelial growth factor inhibits pregnancy establishment in the rhesus monkey(Macaca mulatta)[J].Reproduction,2007,133(6):1199-211.
[17]Blackwell JE,Dagia NM,Diekerson JB,et al.Ligand coated nanosphere adhesion to E-and P-selectin under statie and flow eondition[sJ].Ann Biomed Eng,2001,29(6):523-33.