智能声动力学治疗增效剂用于HepG 2肝肿瘤治疗机制研究

Liver tumor is one of the most common highly-lethal tumors, and conventional treatment methods, e.g., surgery, chemotherapy, confronted extremely severe risks in treatment safety and side effects. Thus, sonodynamic therapy (SDT) of high efficiency and high safety holds great potentials in liver tumor therapy. However, the disputed principle of SDT remains unresolved, resulting in restriction of SDT towards clinical application. In previous work, basing on principles of ‘continuous cavitaion’ and ‘reversible absorption’, a novel radiofrequency ablation enhancement agents and a smart CO2 ‘nanobomb’ have been successfully obtained, but both are not applicable to principle investigation of SDT. In this project, Basing on the theoretical basis of previous work, a type of novel SDT enhancement agents that encapsulates sonosensitizers of chemotherapy property and chemically adsorbs CO2 via bubbling route will be constructed, wherein mesoporous organicsilica nanoparticles (MON) are employed as the main building blocks, and their surfaces are chemically chelated by RGD ligands capable of modified agents capable of reversibly adsorbing CO2 and targeting vascular endothelial growth factor (VEGF) receptor in sequence. Influences from variation of the parameters of low-intensity focused ultrasound (LIFU) and pH on the number of CO2 bubbles, cavitation dose and SDT outcomes can be explored via a series of methods, e.g., confocal, mass spectrometer, steady state and transient state fluorescence spectrometer, electron spin resonance, etc., ultimately validating the principle of SDT. The mechanism of marriage targeting consisting of LIFU-mediated passive targeting and RGD-mediated active targeting will be explored, and afterwards basing this, the principle of synergistic treatment of SDT and chemotherapy will be evaluated and investigated via profile monitoring of tumor growth, pathological examination, molecular characterization, paving a new route to SDT for liver tumor.

肝肿瘤是最常见恶性肿瘤之一，传统的手术、化疗均存在风险，因此高效、安全的声动力学治疗（SDT）成为极具潜力的治疗手段，但SDT机制尚未清晰。前期基于“连续空化”原理和可逆吸附原理分别制备了新型射频增效剂和CO2纳米炸弹，但两者均不适用于SDT机制研究。本项目以前期工作为基础，以介孔有机硅为载体，表面化学螯合靶向VEGF受体的靶向配体和可逆吸附CO2的改性剂，同时介孔中担载具有化疗特性的声敏剂，并进一步鼓泡吸附CO2，以期构建一种新型SDT增效剂。通过共聚焦、质谱等手段考察低强度聚焦超声（LIFU）以及pH变化对CO2气泡数量、空化剂量以及SDT效果的影响，明确SDT机制。通过考察SDT增效剂在HepG 2肝肿瘤中富集量研究LIFU介导的被动靶向和RGD主动靶向联合靶向机制；进一步利用肿瘤生长曲线、病理学、分子生物学等手段研究SDT与化疗协同治疗肝癌机制，为临床肝癌SDT研究提供新思路。

尽管免疫治疗方法已经获得了长足的进步，但固有的肿瘤内的免疫抑制微环境和致密的肿瘤内基质屏障仍然阻碍T细胞免疫疗法的效果。因此，我们建立了基于声动力学疗法的纳米平台，以实现连续的二氧化碳增强超声触发的惯性空化核，并在一次注射后实现SDT的多次增强。体外和体内结果表明，连续的超声空化，可以打开肿瘤内的基质屏障，并允许更多基于SDT的纳米平台进入肿瘤内部。它还可以促进大量活性氧（ROS）的产生和增强抗原的暴露，诱导肿瘤细胞高表达钙网蛋白，高迁移率族蛋白B1以及分泌热休克蛋白，通过高效活化效应CD8 + T细胞和减轻免疫抑制性微环境，从而引起强烈的免疫原性细胞死亡。此外，开放肿瘤内基质屏障，还可以使更多活化的效应CD8 + T细胞和与免疫原性死亡相关的成熟DC细胞浸润到肿瘤中，以进一步增强免疫原性死亡和触发免疫记忆效应，有助于显着提高原发肿瘤治疗效果和抑制远处肿瘤的转移。因此，MON-PpIX-LA-CO2增强的声动力和免疫原性死亡，为增强T细胞的免疫疗法提供了新的策略。