Geng Tianjiao, Tian Lei, Paek Song Yee, Leung Euphemia, Chamley Lawrence W, Wu Zimei
School of Pharmacy, Faculty of Medical and Health Sciences, University of Auckland, Auckland 1010, New Zealand.
Department of Pharmacy, Renji Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200127, China.
Pharmaceutics. 2024 Sep 13;16(9):1206. doi: 10.3390/pharmaceutics16091206.
Extracellular vesicles (EVs) have attracted great attention as promising intracellular drug delivery carriers. While the endocytic pathways of small EVs (sEVs, <200 nm) have been reported, there is limited understanding of large EVs (lEVs, >200 nm), despite their potential applications for drug delivery. Additionally, the low yield of EVs during isolation remains a major challenge in their application. Herein, we aimed to compare the endocytic pathways of sEVs and lEVs using MIA PaCa-2 pancreatic cancer cell-derived EVs as models and to explore the efficiency of their production. The cellular uptake of EVs by MIA PaCa-2 cells was assessed and the pathways were investigated with the aid of endocytic inhibitors. The yield and protein content of sEVs and lEVs from the Integra CELLine culture system and the conventional flasks were compared. Our findings revealed that both sEVs and lEVs produced by the Integra CELLine system entered their parental cells via multiple routes, including caveolin-mediated endocytosis, clathrin-mediated endocytosis, and actin-dependent phagocytosis or macropinocytosis. Notably, caveolin- and clathrin-mediated endocytosis were more prominent in the uptake of sEVs, while actin-dependent phagocytosis and macropinocytosis were significant for both sEVs and lEVs. Compared with conventional flasks, the Integra CELLine system demonstrated a 9-fold increase in sEVs yield and a 6.5-fold increase in lEVs yield, along with 3- to 4-fold higher protein content per 10 EVs. Given that different endocytic pathways led to distinct intracellular trafficking routes, this study highlights the unique potentials of sEVs and lEVs for intracellular cargo delivery. The Integra CELLine proves to be a highly productive and cost-effective system for generating EVs with favourable properties for drug delivery.
细胞外囊泡（EVs）作为一种有前景的细胞内药物递送载体，已引起了极大关注。虽然小细胞外囊泡（sEVs，<200 nm）的内吞途径已有报道，但对于大细胞外囊泡（lEVs，>200 nm）的了解仍然有限，尽管它们在药物递送方面具有潜在应用价值。此外，EVs分离过程中的低产量仍然是其应用中的一个主要挑战。在此，我们旨在以源自MIA PaCa-2胰腺癌细胞的EVs为模型，比较sEVs和lEVs的内吞途径，并探索其产生效率。评估了MIA PaCa-2细胞对EVs的细胞摄取情况，并借助内吞抑制剂研究了其途径。比较了Integra CELLine培养系统和传统培养瓶中sEVs和lEVs的产量及蛋白质含量。我们的研究结果表明，Integra CELLine系统产生的sEVs和lEVs均通过多种途径进入其亲本细胞，包括小窝蛋白介导的内吞作用、网格蛋白介导的内吞作用以及肌动蛋白依赖性吞噬作用或巨胞饮作用。值得注意的是，小窝蛋白和网格蛋白介导的内吞作用在sEVs摄取中更为突出，而肌动蛋白依赖性吞噬作用和巨胞饮作用对sEVs和lEVs均很重要。与传统培养瓶相比，Integra CELLine系统的sEVs产量提高了9倍，lEVs产量提高了6.5倍，每10个EVs的蛋白质含量高出3至4倍。鉴于不同的内吞途径导致不同的细胞内运输途径，本研究突出了sEVs和lEVs在细胞内货物递送方面的独特潜力。Integra CELLine被证明是一个高效且具有成本效益的系统，可用于生成具有有利于药物递送特性的EVs。
