Gharehchelou Behnaz, Mehrarya Mehrnoush, Sefidbakht Yahya, Uskoković Vuk, Suri Fatemeh, Arjmand Sareh, Maghami Farnaz, Siadat Seyed Omid Ranaei, Karima Saeed, Vosough Massoud
Protein Research Center, Shahid Beheshti University, Tehran, Iran.
Department of Cell and Molecular Biology, Faculty of Life Sciences and Biotechnology, Shahid Beheshti University, Tehran, Iran.
PLoS One. 2025 Jan 13;20(1):e0315168. doi: 10.1371/journal.pone.0315168. eCollection 2025.
Exosomes are natural membrane-enclosed nanovesicles (30-150 nm) involved in cell-cell communication. Recently, they have garnered considerable interest as nanocarriers for the controlled transfer of therapeutic agents to cells. Here, exosomes were derived from bone marrow mesenchymal stem cells using three different isolation methods. Relative to filtration and spin column condensation, the size exclusion chromatography led to the isolation of exosomes with the highest purity. These exosomes were then hybridized with liposomes using freeze-thaw cycles and direct mixing techniques to evaluate whether this combination enhances the transfection efficiency of large plasmids. The efficiency of these hybrids in transferring the Cas9-green fluorescent protein plasmid (pCas9-GFP) into the human embryonic kidney 293T (HEK293T) cells was evaluated compared to the pure exosomes. Both Cas9-GFP-loaded exosomes and exosome-liposome hybrids were taken up well by the HEK293T cells and were able to transfect them with their plasmid loads. Meanwhile, the treatment of the cells with plasmids alone, without any vesicles, resulted in no transfection, indicating that the exosome and exosome-liposome hybrids are essential for the transfer of the plasmids across the cell membrane. The pure exosomes and the hybrids incorporating liposomes obtained by the heating method transfected the cells more efficiently than those containing liposomes obtained by the thin film hydration technique. Interestingly, the method of combining exosomes with liposomes (freeze-thaw vs. direct mixing) proved to be more decisive in determining the size of the vesicular hybrid than their composition. In contrast, the liposome component in the hybrids proved to be decisive for determining the transfection efficiency.
外泌体是参与细胞间通讯的天然膜包纳米囊泡（30 - 150纳米）。最近，它们作为治疗剂向细胞的可控转移的纳米载体引起了广泛关注。在这里，使用三种不同的分离方法从骨髓间充质干细胞中提取外泌体。相对于过滤和离心柱浓缩，尺寸排阻色谱法分离出的外泌体纯度最高。然后使用冻融循环和直接混合技术将这些外泌体与脂质体杂交，以评估这种组合是否能提高大质粒的转染效率。与纯外泌体相比，评估了这些杂交体将Cas9 - 绿色荧光蛋白质粒（pCas9 - GFP）转移到人胚肾293T（HEK293T）细胞中的效率。负载Cas9 - GFP的外泌体和外泌体 - 脂质体杂交体都能被HEK293T细胞很好地摄取，并能够用其携带的质粒转染细胞。同时，单独用质粒处理细胞（无任何囊泡）未导致转染，这表明外泌体和外泌体 - 脂质体杂交体对于质粒跨细胞膜的转移至关重要。通过加热法获得的含脂质体的纯外泌体和杂交体比通过薄膜水化技术获得的含脂质体的外泌体更有效地转染细胞。有趣的是，事实证明，外泌体与脂质体结合的方法（冻融法与直接混合法）在决定囊泡杂交体的大小方面比其组成更具决定性。相比之下，杂交体中的脂质体成分在决定转染效率方面起决定性作用。
