聚焦超声介导靶向EGFR载药氟碳纳米粒定位释放治疗胰腺癌的实验研究

Gemcitabine was first-line drugs against pancreatic cancer. The main clinical formulations was gemcitabine hydrochloride and lyophilized powder for injection. Due to its fast metabolism in the body and short half-life in plasma， continual intravenous administration must be given to maintain its toxicity to cancer cells.It lead to increase systemic toxicity and side-effects significantly. Therefore, to find a new targeted drug delivery systems and positioning release technology was neccecary for improving the prognosis of patients. In previous work, the research group has been successful preparing for gemcitabine fluorocarbon nanoparticles. The drug delivery system was nano-scale which colud make it reach outside the blood vessels through vascular endothelial gap by passive targeting mechanism. The phase transition was another characteristics of fluorocarbon nanoparticles. And it could be ruptured by the pulsed focused ultrasound. This study on the basis of the biotin-avidin connection methods prepared to make a kind of gemcitabine loaded and EGFR targeted fluorocarbon nanoparticles.The EGFR monoclonal antibody on the surface of fluorocarbon nanoparticles could be connected with EGFR on the surface of pancreatic cancer cell and inhibit the tumor angiogenesis. The elective uptake of local tumor,arised from active targeting effect, could overcome the defects of micron-level ultrasound targeted drug delivery system.The study would like to evaluate the target effect by pancreatic carcinoma BXPC-3 cell assay and nude mouse back skin wing window model. And the study also prepared to evaluate the pharmacokinetics and tumor location positioning release by the orthotopic pancreatic cancer model in nude mice. To explore its synergistic mechanism of the treatment of pancreatic cancer mediated positioning release by pulsed focused ultrasound. And to unify the passive targeting, active targeting and physical targeting together. It provides a new idea and method for the comprehensive treatment of pancreatic cancer.

吉西他滨是目前抗胰腺癌一线化疗药物，由于副作用大，靶向性差，临床疗效受到限制。寻找穿透力强、稳定性好、特异性强的靶向载药系统和精确定位释放技术对于改善胰腺癌预后具有重要意义。本课题组在前期工作中已成功制备载吉西他滨氟碳纳米粒，可通过被动靶向作用透过血管内皮间隙到达血管外，且可在超声作用下产生液气相转变并破裂。本研究拟在此基础上采用尼妥珠单克隆抗体作为配体制备包裹吉西他滨的靶向EGFR氟碳纳米粒载药系统，可通过主动靶向作用与细胞表面EGFR特异性结合，选择性浓聚于肿瘤局部并抑制肿瘤血管生成；本研究还将通过胰腺癌BXPC-3细胞试验和裸鼠脊背皮翼视窗模型评价其靶向性；拟采用裸鼠原位胰腺癌模型评价其药代动力学，探索其在脉冲型聚焦超声介导下定位释放治疗胰腺癌的最优参数和增效机制，将被动靶向、主动靶向与物理靶向作用相结合，为胰腺癌综合治疗提供一种新的思路和方法。

【研究背景】胰腺癌恶性程度高、预后极差。寻找稳定性好、特异性强的靶向载药系统和精确定位释放技术对于改善胰腺癌预后具有重要意义。.【研究内容与方法】采用双乳化法制备载吉西他滨高分子靶向氟碳纳米粒，制备靶向EGFR载吉西他滨介孔氧化硅材料，制备靶向有机硅氟碳荧光纳米粒，评价安全性及各自优劣。利用胰腺癌BXPC-3细胞试验、裸鼠脊背皮翼视窗模型评价材料的体外及体内靶向性。制备裸鼠原位胰腺癌模型及皮下移植瘤模型，通过超声、磁共振及病理检测评价脉冲型聚焦超声联合靶向材料的增效机制。.【重要结果及关键数据】所制备未载药脂膜氟碳乳剂的平均粒径为1.24μm,其中99%的微滴粒径小于5μm。所制备的MSNs分散性较好，分布均一，粒径平均为30nm，Zeta电位为-26.47mv。其表面介孔的孔间距为2 nm。靶向有机硅氟碳荧光纳米粒平均直径为100nm，电位-20.8mv，低浓度材料对细胞没有毒性，高浓度MSNs明显抑制细胞的增殖，导致细胞坏死增加，G1期细胞比例增大，动物毒性实验ALT和AST显著升高。靶向材料诱导胰腺癌BxPC-3细胞凋亡，凋亡率为(4.52士2.57)％，HIFU联合靶向性有机硅氟碳纳米粒组凋亡率为(21.75士0.32)％。荧光显微镜下可以观察到FITC标记的绿色携尼妥珠靶向介孔二氧化硅材料靶向性好，围绕在红色的细胞膜周围。携尼妥珠靶向介孔二氧化硅载药系统联合脉冲型聚焦超声治疗后，造影剂主要在肿瘤周边出现灌注，肿瘤内部则呈现不规则的灌注缺损区域。超声造影参数如峰值强度（PI）及曲线下面积（AUC），在聚焦超声照射后24小时后较照射前减少；在照射后48小时和2周时较照射前则表现为增加。达峰时间（TP）和曲线斜率（sharpness）在聚焦超声照射后24小时较照射前都表现为增加。而达峰时间在照射后48小时和2周时都较照射前短。病理可见联合组所形成的肿瘤凝固性坏死体积最大，凝固性坏死区呈灰白色，镜下可见红染区明显增加，周围组织与坏死区分界清晰，肿瘤的边缘区域也明显可见细胞核固缩，细胞间隙增大，TUNEL 染色消融灶内部分细胞发生凋亡。.【科学意义】脉冲型聚焦超声联合靶向载药氟碳纳米粒治疗裸鼠胰腺癌，将被动靶向、主动靶向与物理靶向作用相结合，为临床胰腺癌综合治疗奠定了良好的动物实验基础。