Ramon Jana, Pinheiro Cláudio, Vandendriessche Charysse, Lozano-Andrés Estefanía, De Keersmaecker Herlinde, Punj Deep, Fraire Juan C, Geeurickx Edward, Wauben Marca H M, Vader Pieter, Vandenbroucke Roosmarijn E, Hendrix An, Stremersch Stephan, De Smedt Stefaan C, Raemdonck Koen, Braeckmans Kevin
Laboratory of General Biochemistry and Physical Pharmacy, Faculty of Pharmaceutical Sciences, Ghent University, Ghent, Belgium.
Biophotonics Research Group, Ghent University, Ghent, Belgium.
J Nanobiotechnology. 2025 Aug 8;23(1):556. doi: 10.1186/s12951-025-03640-3.
Despite the natural capacity of extracellular vesicles (EVs) to encapsulate intracellular compounds and transfer these to nearby or distant recipient cells, the intentional loading of EVs with cargo molecules remains a challenging endeavor. Pre-formation EV loading (i.e., during EV biogenesis), offers advantages compared to post-formation loading (i.e., after EV isolation), as EV integrity and composition are minimally perturbed. Pre-formation EV loading is primarily achieved through the genetic engineering of the producer cell, which is time consuming and not very flexible regarding the types of molecules that can be incorporated into EVs. In this work, we investigated the possibility of loading cargo molecules into EVs by delivering the cargo directly into the cytosol of the producer cells, which can subsequently be encapsulated into EVs as they are formed. For the cytosolic delivery of cargo molecules, we evaluated the use of photoporation. This membrane disruption technology has been demonstrated to successfully deliver a broad range of cargo molecules into virtually any cell type, while minimally impacting the cell's normal functioning and homeostasis. As a proof-of-concept, we delivered fluorescently labeled dextran macromolecules and anti-EGFP nanobodies into HEK293T cells genetically engineered with gag-EGFP fusion proteins, which are shuttled into EVs. Colocalization of cargo and EGFP fluorescence in secreted EVs can then serve as a convenient readout for successful EV loading. We established that photoporation had minimal impact on EV characteristics such as concentration, size, zeta potential and the enrichment of EV tetraspanin membrane surface molecules. We found that using EGFP-targeted nanobodies resulted in up to 53% loaded EVs (relative to the amount of EGFP EVs), while non-targeted dextran molecules produced on average 12% loaded EVs (relative to the amount of EGFP EVs). These results highlight the promise of photoporation for pre-formation loading of EVs.
尽管细胞外囊泡（EVs）具有天然的能力来包裹细胞内化合物并将其转移到附近或远处的受体细胞，但有意将货物分子装载到EVs中仍然是一项具有挑战性的工作。与形成后装载（即EV分离后）相比，形成前EV装载（即在EV生物发生过程中）具有优势，因为EV的完整性和组成受到的干扰最小。形成前EV装载主要通过对产生细胞进行基因工程来实现，这既耗时，而且在可纳入EVs的分子类型方面也不太灵活。在这项工作中，我们研究了通过将货物直接递送到产生细胞的细胞质中来将货物分子装载到EVs中的可能性，这些货物随后在形成时可被包裹到EVs中。对于货物分子的细胞质递送，我们评估了光穿孔的用途。这种膜破坏技术已被证明能成功地将广泛的货物分子递送到几乎任何细胞类型中，同时对细胞的正常功能和内环境稳定影响最小。作为概念验证，我们将荧光标记的葡聚糖大分子和抗EGFP纳米抗体递送到用gag-EGFP融合蛋白进行基因工程改造的HEK293T细胞中，这些融合蛋白会被转运到EVs中。然后，分泌的EVs中货物与EGFP荧光的共定位可作为成功进行EV装载的便捷读数。我们确定光穿孔对EV特性（如浓度、大小、zeta电位和EV四跨膜蛋白膜表面分子的富集）的影响最小。我们发现，使用靶向EGFP的纳米抗体可产生高达53%的装载EVs（相对于EGFP EVs的量），而未靶向的葡聚糖分子平均产生12%的装载EVs（相对于EGFP EVs的量）。这些结果突出了光穿孔用于EV形成前装载的前景。
