实时可视化研究超声联合微泡诱导内质网损伤发生的机制和规律

Sonoporation-mediated delivery of drugs and genes has been challenged by its cytotoxic effects, such as anti-proliferation and apoptosis induction. To minimize these effects that compromise the safety and effectiveness of sonoporation, it is essential to understand the biophysical mechanism of cavitation-induced intracellular organelle damage. This project seeks to unveil how the endoplasmic reticulum (ER) damage occurs during sonoporation in direct and indirect manners. First, we will establish a real-time confocal imaging method for studying sonoporation-induced ER damage, based on the well-defined experimental model with single-cell and single-microbubble that is triggered by a single ultrasound pulse. Then, this project will focus on the temporal and spatial characteristics of ER structure damage directly caused by acoustic cavitation, identify biophysical parameters that play critical roles in achieving successful ER recovery, and explore the potential relationship between ER structure damage and cell apoptosis. Also, this project will investigate functional impairment of the ER organelle indirectly caused by calcium influx, and reveal the dose-response relationship between these two events. Particularly, intracellular calcium chelation will be conducted to exploit potential ER protection strategies during sonoporation. The outcomes of this project will contribute to the basic understanding of cavitation-induced cellular damage and, as well as parameter optimization of sonoporation-mediated intracellular delivery.

声致穿孔的生物负效应限制了此项技术应用于药物/基因输送的细胞类型和治疗效率，从重要细胞器结构和功能损伤的新角度来研究声致穿孔负效应产生的生物物理原理，可为解决上述难题提供相应的理论依据和方法指导。本申请拟针对超声联合微泡诱导内质网损伤发生的机制和规律展开实验研究。为此，通过设计单细胞单微泡单超声脉冲的精准化实验模型，建立实时可视化监测内质网声致穿孔损伤的研究方法。从而揭示超声空化效应对内质网结构的直接损伤机制，阐明内质网结构损伤形成与修复的空间和时间规律，明确实现可逆化内质网声致穿孔方案的关键生物物理参数，以及探索内质网结构损伤与细胞凋亡发生之间的潜在联系；此外，揭示声致穿孔过程中钙内流现象对内质网功能的间接损伤机制，明确胞内钙升高幅度与内质网功能损伤程度之间的剂量规律，探索预载胞内钙螯合剂法对内质网功能损伤的保护作用。本项目对推动声致穿孔药物释放过程的基础研究和应用优化有重要意义。

如何在提高细胞生存率同时显著提升基因转染效率一直是超声联合微泡诱导基因递送（声致穿孔）领域所面临的主要科学问题。本项目从细胞器内质网的结构和功能发生损伤和修复的动态过程出发，发现并验证了内质网膜结构对大分子质粒入胞扩散的阻碍作用，以及内质网压力对细胞凋亡发生的诱导作用。此外，本项目从声致穿孔基因转染的工程化应用角度出发，探索并验证了钙离子螯合剂法对内质网损伤应激压力的保护作用，以及内质网离散化处理对基因表达水平的提高作用。更进一步地，在本项目的开展过程中，项目组成员将声致穿孔凋亡诱导关键参数用于脑胶质瘤治疗和皮肤疫苗递送，探索声致穿孔效应的工程应用前景。目前，本项目的研究工作仍在进行中，我们将继续针对内质网胞内扩散阻碍和压力凋亡保护这两个重要的声致穿孔问题进行深入研究，为声致穿孔技术的药物递送和凋亡诱导应用提供更多实时观测细胞数据。