Abstract：
Electroporation techniques have emerged as attractive tools for intracellular delivery, rendering promising prospects towards clinical therapies. Transient disruption of membrane permeability is the critical process for efficient electroporation-based cargo delivery. However, smart nanotools for precise characterization of transient membrane changes induced by strong electric pulses are extremely limited. Herein, multivalent membrane-anchored fluorescent nanoprobes (MMFNPs) that take advantages of flexible functionalization and spatial arrangement of DNA frameworks are developed for in situ evaluation of electric field-induced membrane permeability during reversible electroporation . Single-molecule fluorescence imaging techniques are adopted to precisely verify the excellent analytical performance of the engineered MMFNPs. Benefited from tight membrane anchoring and sensitive adenosine triphosphate (ATP) profiling, varying degrees of membrane disturbances are visually exhibited under different intensities of the microsecond pulse electric field (µsPEF). Significantly, the dynamic process of membrane repair during reversible electroporation is well demonstrated via ATP fluctuations monitored by the designed MMFNPs. Furthermore, molecular dynamics (MD) simulations are performed for accurate verification of electroporation-driven dynamic cargo entry via membrane nanopores. This work provides an avenue for effectively capturing transient fluctuations of membrane permeability under external stimuli, offering valuable guidance for developing efficient and safe electroporation-driven delivery strategies for clinical diagnosis and therapeutics.
摘要：
电穿孔技术已经成为细胞内递送的有吸引力的工具。为临床治疗提供有希望的前景。膜渗透性的瞬时破坏是有效的基于电穿孔的货物递送的关键过程。然而,用于精确表征由强电脉冲引起的瞬时膜变化的智能纳米工具非常有限。在这里，开发了利用DNA框架的灵活功能化和空间排列的多价膜锚定荧光纳米探针（MMFNP），用于在可逆电穿孔过程中原位评估电场诱导的膜通透性。采用单分子荧光成像技术来精确验证工程MMFNP的优异分析性能。受益于紧密的膜锚定和敏感的三磷酸腺苷（ATP）分析，在不同强度的微秒脉冲电场（µsPEF）下，视觉上表现出不同程度的膜干扰。重要的是,通过设计的MMFNP监测的ATP波动，可以很好地证明可逆电穿孔过程中膜修复的动态过程。此外，进行分子动力学（MD）模拟，以准确验证电穿孔驱动的动态货物通过膜纳米孔进入。这项工作提供了一种途径，有效地捕获膜通透性在外部刺激下的瞬时波动，为开发有效和安全的电穿孔驱动的临床诊断和治疗策略提供有价值的指导。
