超声联合新型脑靶向免疫脂质体-微泡载药体系提高跨BBB给药效率的实验研究

The blood brain barrier (BBB) has been a bottleneck in the treatment of central nervous system (CNS) disease. Exploring a drug delivery system which can not only through the systemic circulation, but also across the BBB is our aim.The applicant proposed to build a new brain targeting immunoliposome microbubble drug delivery system（Tf- DOX-PLs microbubble complexes）with target identification, the effective drug delivery and acoustic controllable based on the characteristics of microbubble and liposome. We can exploit that the combination of ultrasound and drug delivery system mediated BBB disrupt as well as the biological targeting of immunoliposome to improve the efficiency of drug delivery crossing the BBB. To investigate the acoustic target，receptor-mediated biological targeting and the efficiency of drug administration of the Tf- DOX-PLs microbubble complexes with the help of ultrasonication on C6 glioma in rats, based on the successful construction of the drug delivery system. The study not only expands the research of Ultrasound biological effects and ultrasound/microbubble therapy, but also reveals the mechanism of ultrasound/immunoliposome microbubbles drug delivery system mediated BBB disrupted, particularly the receptor-mediated transcellular pathway. The study will provide new evidences to ultrasound in the treatments in CNS disease.

血脑屏障（BBB）是中枢神经系统疾病治疗的瓶颈，探索一种既能通过体循环，又能跨越BBB的脑靶向载药体系是业者追求的目标。申请人提出联合超声微泡和免疫脂质体各自优点，构建一种具备靶向识别、有效载药和声学可控的新型脑靶向脂质体微泡载药体系（载药免疫脂质体-微泡复合物）。利用免疫脂质体生物学靶向性使复合物富集在靶区，以超声辐照靶区，使微泡破裂靶向开放BBB，同时促使药物释放、弥散和内皮细胞胞吞增加提高跨BBB给药效率。在已成功构建载药脑靶向脂质体微泡复合物基础上，以C6胶质瘤大鼠模型为研究对象，采用激光共聚焦、电镜、RT-PCR和western-blot等技术，重点对超声介导的声学靶向、BBB受体介导的生物学靶向、总体给药效率及治疗效果进行研究，其结果不仅拓展超声生物学效应和超声/微泡治疗学的研究，还揭示超声/微泡促进BBB开放机制，尤其跨细胞通路机制，为超声在CNS疾病治疗提供新的依据。

背景：血脑屏障的存在阻碍药物到底脑实质，严重影响脑恶性肿瘤的治疗，因此探讨一种能有效通过血脑屏障的载药系统将有助于药物通过血脑屏障到达脑组织，从而起到抗肿瘤的作用。研究内容：搜集人胶质瘤标本和非肿瘤脑组织，通过免疫组化检测转铁蛋白受体的表达差异；构建血脑屏障体外模型，大鼠颅内肿瘤模型，为将化疗药物通过血脑屏障及靶向胶质瘤治疗打下基础。采用双乳化法及碳二亚胺法制备以液态氟碳PFP为内核的，以PLGA为壳层材料的，以转铁蛋白受体为靶点的新型多功能靶向相变型液态氟碳载药纳米粒（Tf-PFP-DOX-PLGA）,并对其外观、粒径、电位、载药量、包封率及连靶情况等进行测定。同时，通过加热、低强度聚焦超声(LIFU)等手段检测其相变发生情况。重要结果：免疫组化结果证实转铁蛋白受体在胶质瘤组织中表达明显高于非肿瘤脑组织，因此转铁蛋白可作为靶向分子，使化疗药物靶向胶质瘤细胞；培养原代大鼠脑微血管内皮细胞和星形胶质细胞，培养大鼠C6胶质瘤细胞，并利用细胞插入器，成功构建血脑屏障和血瘤屏障体外模型；检测跨内皮细胞电阻，荧光素钠和菊芬渗透系数，紧密连接蛋白的表达，证实血脑/血瘤屏障体外模型构建成功;利用C6胶质瘤细胞构建大鼠颅内肿瘤模型，MRI和HE染色证实建模成功。成功制备纳米级载药体系Tf-PFP-DOX-PLGA, 平均粒径168.6±23.3nm、平均电位-23.5±4.6mV、载药量为32.5±15.8μg/mg、包封率为27.93±5.12%、Tf连接率97.8±2.2%，并可在加热（48℃）或LIFU(5W-3min)作用下成功实现液气相变。