携Gelsolin单抗载紫杉醇靶向高分子超声微泡对肿瘤转移成像及抑制作用的研究

Tumor metastasis is the most common phenomenon for carcinoma which is closely related to the poor prognosis because of lack of effective treatments. It is essential to identify and give warnings earlier for tumor metastasis in order to carry out effective clinical interventions. In our previous studies, Gelsolin was proved to locate at cytoskeleton and cytoplasm while its expression on the membrane was frequently accompanied with lymph node metastasis. This result showed that it is possibly a molecular target for identification and treatment of tumor lymphatic metastasis. In this project, gelsolin-targeted paclitaxel-loaded PLGA contrast microbubbles will be developed. The targeting function and imaging capacities in vitro will be studied first. The lymphatic metastasis animal model will be constructed for further experiments in vivo. The active targeting function in vivo will be detected by real-time quantitative fluorescence imaging system of live animals. The effects of gelsolin-targeted PLGA contrast microbubbles on the contrast-enhanced ultrasound imaging in vivo will be evaluated further. Then the release of paclitaxel in the target tissues will be analyzed by the liquid mass spectrometry and the inhibition of lymph node metastasis in vivo will be investigated. We hope that the gelsolin-targeted, paclitaxel-loaded PLGA contrast microbubbles could be helpful for early diagnosis of tumors and lymph node metastasis by real time contrast-enhanced ultrasound imaging as well as for targeting treatment under the real-time visualized ultrasound monitoring. A new method for molecular imaging and targeted therapy of the lymphatic metastasis will be provided in this study.

肿瘤转移与不良预后密切相关，早期识别肿瘤转移并采取有效的干预措施已成为目前临床亟待解决的问题。我们在前期研究中发现：凝溶胶蛋白（Gelsolin）作为细胞骨架和胞浆蛋白，其在细胞膜上的表达常常与肿瘤细胞淋巴道转移相伴随，因此有望成为识别及预测肿瘤淋巴道转移的分子靶标。在此基础上，本研究拟制备基于Gelsolin靶向的、载紫杉醇的新型生物纳米材料PLGA高分子超声微泡，观察其体外寻靶能力、显影能力；构建小鼠肝癌淋巴道转移动物模型，实时、定量检测其在动物模型体内的主动寻靶能力，超声造影评价其体内显影能力，液相质谱分析紫杉醇的靶向释放能力以及其对体内淋巴道转移的抑制能力。本项目所设计制备的靶向载药超声微泡既有针对肿瘤淋巴道转移的实时动态分子成像能力，又具有可视化超声实时监测下化疗药物的靶向释放能力，为肿瘤淋巴道转移的分子显像及靶向性治疗提供新方法。

淋巴道转移与肿瘤患者不良预后密切相关，如何早期识别、预警肿瘤淋巴道转移并采取有效的干预措施已成为临床亟待解决的关键问题。我们前期研究发现凝溶胶蛋白（Gelsolin）可能成为识别及预测肿瘤淋巴道转移的分子靶标。在本研究中，我们成功制备了携Gelsolin单抗的PLGA高分子靶向超声微泡，GSN-PLGA靶向超声造影剂平均粒径为（575.67±4.71）nm，表面电位(－11.46±1.19)mV，造影剂表面GSN单抗结合率达96.93%。体外摄取实验显示细胞表面高表达Gelsolin抗体的小鼠腹水型肝癌高淋巴转移细胞株（Hca-F细胞）可较多摄取GSN-PLGA靶向造影剂。体外显像实验显示靶向超声造影剂体外显影效果较好，但是进一步体内研究表明GSN-PLGA靶向造影剂体内显影能力不佳；为进一步改善靶向造影剂的显影能力，我们进一步制备了携Gelsolin单抗靶向相变型超声造影剂，并进行及体外靶向和显影实验，结果显示GSN-PLGA-PFP造影剂表面GSN单抗连接率高达98.31%，且于43.5℃时纳米粒开始发生相变；体外摄取实验显示Hca-F细胞可较多地摄取GSN-PLGA-PFP，体外行LIFU辐照后可观察到显影效果明显增强，表明其热致相变效果明显，可与Hca-F细胞特异性结合且体外显影效果较好。为进一步研究靶向载药高分子超声微泡在体内的靶向分布，我们建立了荷瘤小鼠模型，并进行了小动物活体荧光显像实验，结果表明在注射等剂量的荧光造影剂的条件下，荧光靶向超声造影剂(DiI-GSN-PLGA)较非靶向超声造影剂（DiI-PLGA）在荷瘤小鼠肿瘤区域内荧光汇聚明显，离体后小鼠组织经荧光照射显示荧光靶向超声微泡组小鼠的肿瘤组织及同侧淋巴结可见高浓度的荧光汇聚，非靶向组小鼠荧光汇聚较弱。此外，我们还研究了携Gelsolin单抗的载紫杉醇PLGA高分子靶向超声微泡对体内淋巴道转移的抑制机制和规律，给药4周后GSN-PTX-PLGA微球组与PTX-PLGA 微球组肿瘤生长曲线平缓，与对照组比较肿瘤体积较小，小鼠淋巴结转移率（30%）显著降低，这表明我们制备的靶向载药微球GSN-PTX-PLGA与非靶向载药微球PTX-PLGA对淋巴道高转移Hca-F细胞荷瘤小鼠的治疗有明显抑瘤效果。由于PLGA成膜材料超声分子成像能力较弱，体内超声分子显像能力未达到预期目标，还有待进一步探索。