Marquant Antonin, Berthelot Jade, Bich Claudia, Elfekih Zeineb Ibn, Simon Laurianne, Robin Baptiste, Chopineau Joël, Wang David Tianpei, Emerson Samuel Jay, Wang Aijun, Benedetti Clément, Langlois Simon, Guglielmi Laurence, Martineau Pierre, Aubert-Pouëssel Anne, Morille Marie
ICGM Montpellier University, CNRS, ENSCM Montpellier France.
Institut des Biomolécules Max Mousseron (IBMM) Montpellier France.
J Extracell Biol. 2025 May 14;4(5):e70048. doi: 10.1002/jex2.70048. eCollection 2025 May.
Despite biomolecule delivery is a natural function of extracellular vesicles (EVs), low loading of exogenous macromolecules such as proteins into EVs limits their interest as convincing protein delivery systems for health applications. In this context, lipid-anchorage of exogenous cargo into EV membrane recently emerged as a promising option to enable their vectorisation into cells. Nevertheless, this option was not explored for protein intracellular delivery, and further characterisation of critical parameters governing the association of a lipid-anchored cargo protein to EVs is still needed to confirm the relevance of this anchorage strategy. Therefore, we sought to identify these parameters in a precise and quantitative manner, using bulk and single nanoparticle analysis methods to identify protein loading capacity and subsequent intracellular delivery. We identified incubation temperature, cargo concentration, lipid anchor (LA) structure (lipid nature, linker) and EV origin as critical factors influencing maximal EV loading capacity. Precise control of these parameters enabled to load cargo protein close to EV saturation without hindering cellular delivery. The structural properties of LA influenced not only cargo protein/EV association but also intracellular delivery into different carcinoma cell lines. By thoroughly characterising Lipid-PEG-protein anchorage, this study evidences the interest of this tunable and controllable approach for efficient EV protein delivery.
尽管生物分子递送是细胞外囊泡(EVs)的天然功能,但诸如蛋白质等外源性大分子在EVs中的低负载量限制了它们作为用于健康应用的可靠蛋白质递送系统的价值。在这种背景下,将外源性货物脂质锚定到EV膜上最近成为一种有前景的选择,以使其能够递送至细胞内。然而,这种方法尚未用于蛋白质的细胞内递送,并且仍需要进一步表征控制脂质锚定货物蛋白与EVs结合的关键参数,以确认这种锚定策略的相关性。因此,我们试图以精确和定量的方式确定这些参数,使用整体和单纳米颗粒分析方法来确定蛋白质负载能力和随后的细胞内递送。我们确定孵育温度、货物浓度、脂质锚(LA)结构(脂质性质、连接子)和EV来源是影响最大EV负载能力的关键因素。对这些参数的精确控制能够在不阻碍细胞递送的情况下将货物蛋白负载至接近EV饱和状态。LA的结构特性不仅影响货物蛋白/EV的结合,还影响其向不同癌细胞系的细胞内递送。通过全面表征脂质-聚乙二醇-蛋白锚定,本研究证明了这种可调节和可控方法对于高效EV蛋白递送的价值。
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