Dipartimento di Fisica e Chimica-Emilio Segré, Università degli Studi di Palermo, 90128 Palermo, Italy.
Dipartimento di Scienze e Tecnologie Biologiche Chimiche e Farmaceutiche, Università degli Studi di Palermo, 90128 Palermo, Italy.
Biomolecules. 2023 Mar 7;13(3):492. doi: 10.3390/biom13030492.
Continuous progress has been made in the development of new molecules for therapeutic purposes. This is driven by the need to address several challenges such as molecular instability and biocompatibility, difficulties in crossing the plasma membrane, and the development of host resistance. In this context, cell-penetrating peptides (CPPs) constitute a promising tool for the development of new therapies due to their intrinsic ability to deliver therapeutic molecules to cells and tissues. These short peptides have gained increasing attention for applications in drug delivery as well as for their antimicrobial and anticancer activity but the general rules regulating the events involved in cellular uptake and in the following processes are still unclear. Here, we use fluorescence microscopy methods to analyze the interactions between the multifunctional peptide Transportan 10 (TP10) and the giant plasma membrane vesicles (GPMVs) derived from cancer cells. This aims to highlight the molecular mechanisms underlying functional interactions which bring its translocation across the membrane or cytotoxic mechanisms leading to membrane collapse and disruption. The Fluorescence Lifetime Imaging Microscopy (FLIM) method coupled with the phasor approach analysis proved to be the winning choice for following highly dynamic spatially heterogeneous events in real-time and highlighting aspects of such complex phenomena. Thanks to the presented approach, we were able to identify and monitor TP10 translocation into the lumen, internalization, and membrane-induced modifications depending on the peptide concentration regime.
在治疗目的的新分子开发方面不断取得进展。这是由于需要解决分子不稳定性和生物相容性、跨膜困难以及宿主抗性发展等挑战。在这种情况下，细胞穿透肽（CPP）由于其内在的将治疗分子递送到细胞和组织的能力，构成了开发新疗法的有前途的工具。这些短肽因其在药物输送中的应用以及其抗菌和抗癌活性而受到越来越多的关注，但调节细胞摄取和随后过程中涉及的事件的一般规则仍不清楚。在这里，我们使用荧光显微镜方法分析多功能肽 Transportan 10（TP10）与源自癌细胞的巨大质膜囊泡（GPMVs）之间的相互作用。这旨在突出其跨膜易位或导致膜崩溃和破坏的细胞毒性机制背后的功能相互作用的分子机制。荧光寿命成像显微镜（FLIM）方法与phasor 分析相结合被证明是实时跟踪高度动态的空间异质事件并突出此类复杂现象的方面的最佳选择。通过所提出的方法，我们能够根据肽浓度范围识别和监测 TP10 向腔内化和膜诱导修饰的易位。
