血管内皮生长因子受体2及整合素双靶向超声微泡的制备及其体外成像与寻靶能力检测

Preparation of vascular endothelial growth factor receptor 2/Integrinαvβ3 dual-targeted contrast ultrasound agent and its ultrasound imaging features and targeted ability: a in vitro study

目的制备以肿瘤血管内皮生长因子受体2(VEGFR2)及整合素αvβ3(Integrinαvβ3)为靶点的双靶向超声造影剂,评价造影剂的物理性质,并检测其超声成像能力及体外寻靶能力。方法利用生物素-亲和素桥接法,将未接靶的超声造影剂USphere LA分别与VEGFR2单抗、Integrinαvβ3单抗及VEGFR2单抗+Integrinαvβ3单抗结合,制备以VEGFR2、Integrinαvβ3为靶点的单靶向造影剂及同时以二者为靶点的双靶向造影剂,以未连接抗体的非靶向微泡USphere LA作为对照组。观察所得微泡形态并测量微泡大小,将微泡置于4℃保存,并在不同时间节点(1 h、3 h、12 h、3 d、5 d、7 d、14 d)观察微泡并评价其稳定性。对比VEGFR2/Integrinαvβ3及SonoVue在制备时及制备后3 d超声成像特点,计算其灰度值。将非靶向、单靶及双靶造影剂分别与细胞表面高表达VEGFR2及Integrinαvβ3的小鼠肝癌细胞Hepa1-6及两者低表达的小鼠纤维细胞C3H10结合,观察各组细胞与微泡结合情况;采用抗体阻断试验重复以上操作,再次观察细胞与微泡结合情况。结果新鲜制备的双靶向造影剂平均粒径为1 289 nm,4℃条件下保存的微泡在制备后3 d内浓度下降缓慢,5 d后浓度下降明显。VEGFR2/Integrinαvβ3双靶向微泡初制备时与相同浓度SonoVue超声成像灰度值差异无统计学意义(P=0.113),3 d后双靶向微泡灰度值高于SonoVue(P<0.001)。Hepa1-6细胞与微泡结合量呈现以下趋势:双靶向造影剂>单靶向造影剂>非靶向造影剂(均P<0.05),C3H10细胞与各组造影剂结合量差异无统计学意义(均P>0.05);预先行抗体阻断后,单靶及双靶向造影与Hepa1-6细胞结合量较抗体阻断前减低(均P<0.05),各组细胞与微泡结合数量差异无统计学意义(P>0.05)。结论以VEGFR2及Integrinαvβ3为靶点的双靶向造影剂物理性质稳定,在体外有良好的超声成像能力及寻靶能力。

Objective To prepare the vascular endothelial growth factor receptor 2(VEGFR2)/Integrinαvβ3 dual-targeted contrast ultrasound agent, and further evaluating the physical properties, imaging characteristics and targeted ability in vitro. Methods VEGFR2 targeted microbubble (MBV), Integrinαvβ3 targeted microbubble (MBI) and VEGFR2/Integrinαvβ3 dual targeted microbubble (MBD)were prepared by attaching VEGFR2 antibody, Integrinαvβ3 antibody and both of VEGFR2/Integrinαvβ3 antibody with no targeted USphere LA respectively, using biotin-avidin linkage method. USphere LA with no antibody attached were used as non-targeted microbubble(MBN). Microbubble′s physical properties were observed, and its stability was detected by caculating bubble′s concentration at different time points. MBD′s ultrasound imaging characteristics were evaluated by comparing its grey level of ultrasound imaging with SonoVue at the time of preparation and 3 days after preparation. To detect the targeted ability of microbubble, different types of microbubble were added into Hepa 1-6 and C3H10 cells, respectively. The above procedure was repeated using the pre-antibody blocking test in Hepa 1-6 cell. Results MBD had a mean size of 1 256 nm.The concentration of microbubble in the first duration of three days was declining slowly, and its speed was accelerated after five days of preparation. The grey level of new prepared MBD was similar to that of SonoVue in the same concentration (P=0.113), while the level was higher than that of SonoVue within 3 days after preparation(P<0.001). The number of microbubble binding to Hepea1-6 led a tendency of MBD>single targeted microbubble >MBN(all P<0.05). The number of C3H10 and pre-blocked Hepa1-6 cell attached to each group of microbubble had no statistical difference (all P>0.05). In addition, following the pre-blocked precedure, the number of Hepa 1-6 cell attached to each group of microbubble had no statistical difference either (all P>0.05). Conclusions VEGFR2/Integrinαvβ3 dual-targeted contrast ultrasound agent is a stable microbubble and it has excellent ultrasound imaging and targeting ability in vitro.