单细胞水平实时可视研究超声联合微泡介导质膜穿孔后修复的机制

Sonoporation refers to the temporal cell membrane opening caused by the interaction between the low intensity ultrasound pulses and microbubbles, resulting in facilitation of the transport of membrane impermeable macromolecular into living cells. The merits of non-invasion, dual-targeting to malignant tissues, safety and low cost make this physical delivery approach promising for clinical drug delivery.The resealing of the transient membrane poration induced by ultrasound and microbubbles is closely correlated with the fate of targeted cells, and therefore is important for the efficiency and biosafety of drug delivery. Based on the establishment of a real-time visualization platform at the single-cell level, diverse tumor cell lines were adopted to reveal the kinetics and microscopic process of membrane repair after acoustic cavitation in a spatial and temporal manner.Based on the above system, various experimental technologies will be utilized to study the following scientific problems. (1) The spatiotemporal relation in the cytoskeleton response, mechanosensitive channels and exocytosis after stable cavitation induced by sonoporation will be analyzed; and specific mechanotransduction pathways responsive for the membrane repair after stable cavitation will be studied. (2) The role of the intracellular calcium transient, lysosome exdocytosis and cytoskeleton dynamic behaviors in the membrane resealing after inertial cavitation induced membrane damage will be revealed. (3) The cell fate in the long period after treatment with different acoustic cavitation doses will be determined.These studies will provide us a detailed understanding of the repair mechanisms after the membrane perforations induced by ultrasound and microbubbles, and will propose the strategies for promoting the membrane resealing and improving delivery efficiency and biosafety of ultrasound induced drug delivery. These findings will also establish a solid theoretical foundation for clinical drug delivery by the ultrasound with combination of microbubbles.

低强度超声联合微泡能够介导质膜穿孔，递送大分子药物进入活细胞，其无创安全、低成本、双靶向性的特点彰显了这一物理技术用于药物传输的优势。质膜穿孔后的修复过程和细胞命运息息相关，对药物传输效率及生物安全性至关重要。本研究以多种细胞系为模型，创建单细胞水平上实时可视平台，从时空域揭示超声联合微泡致膜穿孔后修复这一短暂快速、微观复杂的生物过程。基于此，采取多种生物学实验技术进行以下研究：（1）稳态空化致膜穿孔和细胞骨架响应、机械敏感蛋白及胞吞的时空域关系，确定参与质膜修复的机械传导通路；（2）确定胞内钙离子暂流、溶酶体胞吐及细胞骨架动态行为在瞬态空化致膜穿孔后修复的作用机理；（3）确定不同空化剂量致膜穿孔后细胞的命运。通过该项研究，期望深入揭示超声联合微泡介导质膜穿孔后修复的生物学机理，并提出促进质膜修复，改进药物传输效率，提高生物安全性的策略，为其走向临床应用奠定可靠、充分的理论基础。

在为期四年的研究中，本项目在声学领域Top 1期刊 Ultrasonics Sonochemistry发表论文3篇，药物传输领域Top 1期刊 Journal of Controlled Release发表论文1篇，在 Ultrasound in Medicine and Biology发表论文1篇，同时在顶级超声大会IUS及治疗超声大会ISTU上发表会议论文9篇，另有两篇论文发表在声学学报。主要成果为：.一方面，建立了单细胞水平声空化处理及细胞层响应显微实时观测平台，确定了非声学参数（微泡个数、大小以及微泡到细胞距离）和多样性声致穿孔之间的相关关系，为取得可预测及可操控的声致穿孔提供了指导。在此基础上，确定了在可恢复性声致穿孔细胞内，胞内ROS水平首先降低，然后迅速增加，且降低和增加水平和质膜穿孔程度相关。而在不可恢复性穿孔的细胞中，细胞内的ROS水平持续缩减，直至耗竭。这些结果表面不同声致穿孔细胞中不同程度的ROS响应可能诱导其它生化信号发生，且和细胞长期命运相关，从而影响治疗效率和生物安全。另外，我们也发现在质膜穿孔后修复的过程中，细胞骨架Actin发生瞬间破坏且损伤处Actin迅速解聚，然后损伤部分迅速修补，Actin重新聚合塑形，恢复到初始水平，在这一过程中Actin单体复合物形成，并向损伤部位定向迁移和聚合，在形成新的Actin骨架中起了重要的作用。这些线索初步暗示Actin骨架在质膜穿孔后修复直至恢复到初始状态中有重要作用。.另一方面，建立了仿体组织中静态和流动微泡群的声空化特性测量系统，在静态条件下确定微泡声空化的演化历程主要由负声压决定，但也依赖于脉冲长度，且声空化剂量和脉冲长度及重复频率正相关。而在流动条件下，当脉重复频率(PRF)小于临界重复频率(CPRF)时，峰值负压(PNP)和脉冲长度(PL)共同决定微泡群稳态和瞬态空化的阈值声压。当PRF小于CPRF时，PL对流动微泡群稳态空化强度的时域分布无影响；但PRF大于CPRF时，由于存在声驱动下微泡破裂，PL对稳态空化有较大影响。当PRF小于CPRF时，瞬态空化特性与PRF无关；但PRF大于CPRF后，瞬态空化强度逐渐下降且时域分布不均。在未来的声空化临床应用中，这些结果有助于合理选择声学参数，获取可预测可控的空化活动，提升治疗效率。