Cell membrane disruption and recovery for intracellular delivery

项目摘要 细胞内递送在生物学研究和治疗应用中起着重要作用，然而，有效的细胞内递送是不可能的。 外源化合物和大分子货物的细胞内递送仍然是一个长期存在的挑战。 已建立的方法的复杂机制及其对细胞行为的通常不可预测的影响， 极大地限制了生物学实验的范围，降低了潜在的有前途的细胞疗法的功效 理念的基于膜破坏的方法已经成为快速、直接和有效地利用膜的关键策略。 通用的细胞内递送，因为它们较少依赖于货物性质和细胞类型，能够 以提供几乎任何分散在溶液中的亚微米材料。快速切换膜扰动的能力 效果的打开和关闭提供了额外的控制级别，可以进行时间操作， 即时交付。然而，膜破坏策略的关键挑战是：1）不一致 质膜损伤水平（这将导致低活力和效率）; 2）差的通量或 可扩展性（如显微注射）; 3）对质膜破裂和恢复的理解不足 反应PI在科罗拉多大学博尔德分校的研究小组以跨学科研究为中心， 生物学、医学、物理学和微/纳米工程的前沿。PI小组的主要研究方向是 是开发新的技术来定量地了解细胞膜的破坏和恢复，并探索 其在下一代精确细胞内药物递送中的应用。今后五年的目标是：（一） 开发一种新型的微流体平台，包括纳米工程表面技术和声流体装置， 其能够精确地在细胞膜上产生均匀、同质、数量可控的破裂， 孔的大小，以定量地了解细胞膜的破坏和恢复动力学在分子， 细胞、蛋白质组学和高通量水平; ii）证明了精确的细胞内药物递送系统， 可控剂量，最小毒性，最大效率和高通量，提供洞察力或促进 下一代细胞内药物输送。到目前为止，生物学家们还没有应用 从膜破裂和修复研究中收集到的工程细胞渗透性。拟议 研究计划将弥合这两个完全不同的领域之间的科学差距： 递送方法;以及质膜破坏和修复反应的细胞机械生物学。的 生物医学研究界将从更机械和透明的理解中受益匪浅 细胞内递送，以进一步发展更强大的技术，并实现关键的医疗 和工业应用。 1

PROJECT ABSTRACT Intracellular delivery plays an essential role in biological research and therapeutic applications, however, efficient intracellular delivery of exogenous compounds and macromolecular cargo remains a long-standing challenge. The complex mechanisms of established methods and their often unpredictable impact on cell behaviour have dramatically limited the scope of biological experiments and reduced efficacy of potentially promising cell therapy concepts. Membrane disruption-based approaches have emerged as key strategies for rapid, direct and universal intracellular delivery because they are less dependent on cargo properties and cell types, being able to deliver almost any submicron material dispersed in solution. The ability to rapidly switch membrane-perturbing effects on and off provides an additional level of control, enabling temporal manipulation and rapid, almost instantaneous delivery. However, key challenges of membrane disruption strategies have been: 1) inconsistent level of plasma membrane injury (which would lead to low viability and efficiency); 2) poor throughput or scalability (e.g. microinjection); and 3) inadequate understanding of plasma membrane disruption and recovery response. The PI's research group at University of Colorado Boulder centers on interdisciplinary research at the frontiers of Biology, Medicine, Physics, and Micro/Nano Engineering. The main research thrust in the PI's group is to develop new technologies to quantitatively understand cell membrane disruption and recovery, and explore its application for next generation precise intracellular drug delivery. The goals for the next five years are to i) develop a novel microfluidic platform, including NanoEngineered Surface Technology and Acoustofluidic devices, that can precisely generate uniform and homogenous disruptions at cell membrane with controllable number and size of the pores, to quantitatively understand cell membrane disruption and recovery dynamics at molecular, cellular, proteiomic and high throughput level; ii) demonstrate a precise intracellular drug delivery system with controllable dose, minimum toxicity, maximum efficiency, and high throughput, providing insight for or promoting the next generation intracellular drug delivery. So far, biologists have not applied the fundamental insights gleaned from membrane disruption and repair studies toward engineering cell permeability. The proposed research grogram will bridge the scientific gap between these two disparate fields: the engineering of intracellular delivery approaches; and the cellular mechanobiology of plasma membrane disruption and repair response. The biomedical research community would benefit greatly from a more mechanistic and transparent understanding of intracellular delivery, both to further the development of more robust techniques and to realize key medical and industrial applications. 1