肿瘤新生血管靶向微泡-双配体修饰的载药纳米粒复合物体系构建及其抗肿瘤性能评价

Ultrasound targeted microbubble destruction (UTMD) could increase endothelial gap and membrane permeability and drug loaded nanoparticles (NP) are of efficient payload capacity, therefore by combining advantages of the two systems, the complexes of microbubbles and nanoparticles will have a promising application prospect in drug delivery. However, there is an urgent problem needs to be studied whether the NP could be effectively uptaken by cancer cells in vivo and avoid the uptake by reticuloendothelial systems (RES). In previous studies, by utilizing folate targeting effect and Tat transmembrane ability we have designed folate and cell penetrating peptide modified paclitaxel loaded NP successfully and confirmed higher cellular uptake efficiency in tumor tissue. Herein, we aimed to develop a novel drug delivery and imaging system by conjugating to tumor-neovascular targeted microbubbles to NP through avidin-biotin chemistry. This system could accumulate in tumor vascular and achieve targeted drug delivery by utilizing dual ligand effects and US effect, hence enhance targeted celluar uptake in tumor endothelial cells and tumor cells. The conjugation of targeted microbubbles to NP can attribute to reduce unspecific accumulation of nanoparticles , increase enhance specific intracellular drug delivery and perform molecular imaging at the same time. DSPE-PEG2000-COOH, one of film materials chosen to prepare microbubbles, is able to reject NP uptake in RES and provide functional group for ligand link chemistry.

微泡爆破能增加内皮间隙和胞膜通透性，而纳米粒具有较好的生物相容性、载药量高、性能稳定，故将两种体系优势组合，构建微泡-载药纳米粒复合物，较具应用前景。但体内环境下如何促进纳米粒被肿瘤组织有效摄取并减少网状内皮系统(RES)吞噬等非特异性聚集，是亟待解决的问题。前期研究中利用叶酸受体的内吞作用及穿膜肽主动穿膜的协同效应，我们已制备出叶酸、穿膜肽双配体载紫杉醇纳米粒，并证实双配体修饰对肿瘤细胞内纳米粒摄取有增效作用。本课题拟将靶向肿瘤新生血管的微泡通过生物素-亲和素桥与双配体纳米粒偶联构建复合物，该体系借助靶向微泡引导首先聚集于肿瘤血管，并通过超声定向爆破和双配体作用实现药物靶向输送，最终促进肿瘤血管内皮细胞及肿瘤细胞有效药物摄取。微泡成膜材料中DSPE-PEG2000-COOH的加入有利于体系逃逸RES同时为配体化学连接提供了功能基团。靶向微泡引入体系有助于减少非特异聚集，且兼具分子探针潜能

在本研究中，我们制备了酸敏双靶向阿霉素前药肝素纳米粒-微泡复合物，并探讨其联合超声辐照治疗肿瘤的疗效。阿霉素通过酸敏键与叶酸、cRGD肽双靶向修饰的肝素纳米粒结合形成阿霉素前药（DP），再通过生物素-亲和素桥联与微泡形成复合物（DPMC）。超声空化及声孔效应作用促进DPMC在细胞内蓄积，能够提高治疗效率。DP粒径不均一，呈双峰（149.6±29.8和1036.2±38.8 nm,PDI:1.0），而DPMC粒径分布均一（5.804±2.1μm），且超声辐照后变成粒径均一的小粒子（128.6±42.3 nm, PDI: 0.21)）。DPMC经αvβ3介导与肿瘤新生血管内皮细胞靶向结合，超声辐照后形成的小粒子经叶酸介导结合于肿瘤细胞，便于药物递送。体外实验显示，相对于游离DOX组和DP组，DPMC组对乳腺癌MCF-7细胞表现出更强的肿瘤特异性和杀伤能力。.活体荧光成像和超声分子成像技术显示DPMC联合超声辐照能够特异性的聚集在肿瘤组织内，表明其具有诊疗一体化功能。在MCF-7小鼠移植瘤模型中，DPMC联合超声辐照通过促进细胞凋亡、抑制细胞增殖及抗血管生成多种机制抑瘤效果明显优于游离DOX组和DPMC组，且未明显减轻小鼠体重。我们提供了一种方法，能够把大粒径或者聚合的粒子递送到肿瘤位置，进而发挥其治疗作用。

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