叶酸介导的载药CNTs体系在多功能超声造影剂中的应用及作用机制研究

At present, the ultrasound contrast agent (UCA) used by each big hospital in our country is SonoVue that is mainly imported from foreign, its value is expensive. With the UCA development, the multifunctional UCA with both observation lesions and targeted therapy shows the incomparable advantages in comparison with the traditional single-function UCA.From building multifunctional UCA and improving the ultrasound imaging effect, a Span-PEG multifunctional UCA was designed and prepared by the supercritical CO2 technology and acoustic cavitation method on the basis of CNTs delivery system mediated folic acid in this project. This Span-PEG multifunctional UCA integrated targeting, heat treatment and chemotherapy. The dual targeting effect was achieved by targeting enrichment and directional initiation under the supplement of UTMD technology. The targeting ultrasound-imaging function and antitumor effect were researched for Span-PEG multifunctional UCA combined FA-CNTs-PTX. The synthetic mechanism of functional UCA was put forward. The roles of CNTs delivery system mediated by folic acid in the targeted delivery drug, thermal therapy, chemotherapy and ultrasonic imaging mechanism were clarified. The interaction principle of CNTs and paclitaxel was revealed under the SC -CO2. The above work and achievements will enrich the variety of multifunctional UCA and provide theoretical basis for new CNTs function materials.

目前，我国各大医院使用的超声造影剂主要是进口的声诺维，价格比较贵。随超声造影剂的发展，集观察病灶和靶向治疗同时进行的多功能超声造影剂显示出传统单一功能超声造影剂无法比拟的优势。本项目从构建多功能超声造影剂以及提高超声显影效果思想出发，基于叶酸介导的载药CNTs体系，采用超临界CO2技术和声振空化法设计和制备集靶向-热疗-化疗于一体的Span-PEG多功能超声造影剂，辅以UTMD技术，实现超声造影剂微泡靶向富集和定向引爆的双重靶向作用。研究复合FA-CNTs-PTX的Span-PEG多功能超声造影剂的靶向超声成像功能和抗肿瘤作用，提出功能性造影剂的合成机理，阐明叶酸介导的载药CNTs体系在靶向递药、热疗、化疗和超声成像中的作用机制，并揭示SC-CO2条件下CNTs与紫杉醇的相互作用原理。该项目的设立将丰富多功能超声造影剂的种类，并为开发新的CNTs功能材料提供理论基础。

首先以壳聚糖为偶联剂将叶酸（FA）连接到碳纳米管（CNTs）上得到CNTs-FA复合物，再利用π-π效应将抗肿瘤药物紫杉醇（PTX）负载于CNTs-FA上得到靶向载药复合物PTX-CNTs-FA，采用声振空化法以司盘-聚乙二醇（Span-PEG）作为膜材，并加入叶酸-碳纳米管-紫杉醇（PTX-CNTs-FA）复合物，制备复合PTX-CNTs-FA的Span-PEG超声造影剂微泡。通过单因素试验对复合PTX-CNTs-FA的Span-PEG微泡的形貌和粒度进行优化；以昆明小鼠为模型，通过急性毒性试验确定复合微泡对小鼠的最大耐受量；以接种乳腺癌细胞（MCF-7）的裸鼠移植瘤为体内抗肿瘤的研究对象，通过记录裸鼠体重和肿瘤体积大小，分析复合微泡对肿瘤增长的抑制作用；采用HE染色法、免疫组化法和原位末端转移酶标记技术（TUNEL），从裸鼠乳腺癌肿瘤内肿瘤细胞病理检查结果、PCNA表达情况和细胞凋亡结果，探讨复合微泡对乳腺癌肿瘤的抑制作用；通过多普勒彩色超声仪考察复合微泡的体内外超声显影效果，及治疗前后造影峰值强度变化。实验结果表明，复合PTX-CNTs-FA的Span-PEG微泡的最佳制备工艺参数为Span与PEG膜层比例为1:1、制备温度70℃、超声功率900W、超声时间5min，该条件下复合微泡为中空结构、内部包裹N2，微泡粒径约410 nm，复合微泡中CNTs含量为1.42%、PTX含量为5.06%；复合微泡最大耐受剂量是3500 mg·kg-1，昆明小鼠在该剂量下没有出现死亡和不良反应，并且最大耐受剂量为50 kg成人的350倍，这表明复合微泡具有较低毒性和良好的生物相容性；复合PTX-CNTs-FA的Span-PEG微泡能靶向抑制肿瘤体积增长、抑制肿瘤细胞增值并诱导肿瘤细胞凋亡；复合PTX-CNTs-FA的Span-PEG微泡具有很好的稳定性，微泡中的CNTs具有增强超声造影的功能，此复合超声造影剂微泡可靶向增强超声信号，延长造影时间，提高超声影像的清晰度及分辨率，同时，裸鼠经复合微泡治疗后，肿瘤造影峰值明显降低。