López Rubén R, Ben El Khyat Chaymaa Zouggari, Chen Yunxi, Tsering Thupten, Dickinson Kyle, Bustamante Prisca, Erzingatzian Armen, Bartolomucci Alexandra, Ferrier Sarah Tadhg, Douanne Noélie, Mounier Catherine, Stiharu Ion, Nerguizian Vahé, Burnier Julia V
Cancer Research Program, Research Institute of the McGill University Health Centre, 1001 Decarie Blvd, Montreal, QC, H4A 3J1, Canada.
Department of Pathology, McGill University, Quebec, Canada.
Sci Rep. 2025 Mar 11;15(1):8430. doi: 10.1038/s41598-025-91873-5.
Extracellular vesicles (EVs) are secreted by most cell types and play a central role in cell-cell communication. These naturally occurring nanoparticles have been particularly implicated in cancer, but EV heterogeneity and lengthy isolation methods with low yield make them difficult to study. To circumvent the challenges in EV research, we aimed to develop a unique synthetic model by engineering bioinspired liposomes to study EV properties and their impact on cellular uptake. We produced EV-like liposomes mimicking the physicochemical properties as cancer EVs. First, using a panel of cancer and non-cancer cell lines, small EVs were isolated by ultracentrifugation and characterized by dynamic light scattering (DLS) and nanoparticle tracking analysis (NTA). Cancer EVs ranged in mean size from 107.9 to 161 nm by NTA, hydrodynamic diameter from 152 to 355 nm by DLS, with a zeta potential ranging from - 25 to -6 mV. EV markers TSG101 and CD81 were positive on all EVs. Using a microfluidics bottom-up approach, liposomes were produced using the nanoprecipitation method adapted to micromixers developed by our group. A library of liposome formulations was created that mimicked the ranges of size (90-222 nm) and zeta potential (anionic [-47 mV] to neutral [-1 mV]) at a production throughput of up to 41 mL/h and yielding a concentration of 1 × 10 particles per mL. EV size and zeta potential were reproduced by controlling the flow conditions and lipid composition set by a statistical model based on the response surface methodology. The model was fairly accurate with an R-squared > 70% for both parameters between the targeted EV and the obtained liposomes. Finally, the internalization of fluorescently labeled EV-like liposomes was assessed by confocal microscopy and flow cytometry, and correlated with decreasing liposome size and less negative zeta potential, providing insights into the effects of key EV physicochemical properties. Our data demonstrated that liposomes can be used as a powerful synthetic model of EVs. By mimicking cancer cell-derived EV properties, the effects on cellular internalization can be assessed individually and in combination. Taken together, we present a novel system that can accelerate research on the effects of EVs in cancer models.
细胞外囊泡（EVs）由大多数细胞类型分泌，在细胞间通讯中起核心作用。这些天然存在的纳米颗粒与癌症尤其相关，但EV的异质性以及产量低的冗长分离方法使其难以研究。为了规避EV研究中的挑战，我们旨在通过设计受生物启发的脂质体来开发一种独特的合成模型，以研究EV的性质及其对细胞摄取的影响。我们制备了模仿癌症EVs物理化学性质的类EV脂质体。首先，使用一组癌细胞系和非癌细胞系，通过超速离心分离小EVs，并通过动态光散射（DLS）和纳米颗粒跟踪分析（NTA）进行表征。通过NTA测定，癌症EVs的平均大小在107.9至161nm之间，通过DLS测定的流体动力学直径在152至355nm之间，ζ电位在-25至-6mV之间。所有EVs上的EV标志物TSG101和CD81均呈阳性。采用微流控自下而上的方法，使用适用于我们小组开发的微混合器的纳米沉淀法制备脂质体。创建了一个脂质体制剂库，其模仿了大小范围（90 - 222nm）和ζ电位（阴离子[-47mV]至中性[-1mV]），生产通量高达41mL/h，每毫升产生1×10个颗粒的浓度。通过控制基于响应面方法的统计模型设定的流动条件和脂质组成，再现了EV的大小和ζ电位。该模型相当准确，目标EV与所得脂质体之间这两个参数的决定系数R²均>70%。最后，通过共聚焦显微镜和流式细胞术评估荧光标记的类EV脂质体的内化情况，并将其与脂质体大小减小和ζ电位负性降低相关联，从而深入了解关键EV物理化学性质的影响。我们的数据表明脂质体可用作强大的EV合成模型。通过模仿癌细胞衍生的EV性质，可以单独和联合评估其对细胞内化的影响。综上所述，我们提出了一种新系统，可加速对EVs在癌症模型中作用的研究。
