Engineered Trehalose Permeable to Mammalian Cells
Engineered Trehalose Permeable to Mammalian Cells
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DOI：
10.1371/journal.pone.0130323
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发表时间：
2015-06-26
期刊：
影响因子：
3.7
通讯作者：
Toner, Mehmet
中科院分区：
文献类型：
--
作者：
Abazari, Alireza;Meimetis, Labros G.;Toner, Mehmet
海藻糖是一种天然存在的二糖，与某些单细胞和多细胞生物物种非凡的应激耐受能力有关。在哺乳动物细胞中，细胞内和细胞外海藻糖的存在已被证明可以提高对冷冻和干燥的耐受性。由于哺乳动物细胞不合成也不输入海藻糖，因此开发有效的细胞内递送海藻糖的新方法一直是一个持续的研究。在此，我们研究了工程化的海藻糖亲脂衍生物的膜渗透性。与普通海藻糖、海藻糖二乙酸酯 (2-O-Ac-Tre) 和海藻糖四乙酸酯 (4-O-Ac-Tre) 相比，与 6 个乙酰基缀合的海藻糖（海藻糖六乙酸酯或 6-O-Ac-Tre）在大鼠肝细胞中表现出优异的通透性。一旦进入细胞，细胞内酯酶就会水解 6-O-Ac-Tre 分子，将游离海藻糖释放到细胞质中。细胞内海藻糖（加上乙酰化变体）的总浓度高达细胞外 6-O-Ac-Tre 浓度的 10 倍，达到适合生物保存应用的浓度。为了描述这种积累现象，提出了扩散反应模型，并通过拟合实验数据确定了 6-O-Ac-Tre 的渗透性和反应动力学。进一步的研究表明，细胞内海藻糖的负载和存在对细胞活力和功能的影响可以忽略不计。海藻糖化学结构的改造而不是操纵细胞，是一种无害、细胞友好的海藻糖递送方法，已证明在不同类型的细胞和细胞系中负载海藻糖的潜力，并且可以促进海藻糖作为细胞内保护剂在生物保存研究中的广泛应用。
Trehalose is a naturally occurring disaccharide which is associated with extraordinary stress-tolerance capacity in certain species of unicellular and multicellular organisms. In mammalian cells, presence of intra- and extracellular trehalose has been shown to confer improved tolerance against freezing and desiccation. Since mammalian cells do not synthesize nor import trehalose, the development of novel methods for efficient intracellular delivery of trehalose has been an ongoing investigation. Herein, we studied the membrane permeability of engineered lipophilic derivatives of trehalose. Trehalose conjugated with 6 acetyl groups (trehalose hexaacetate or 6-O-Ac-Tre) demonstrated superior permeability in rat hepatocytes compared with regular trehalose, trehalose diacetate (2-O-Ac-Tre) and trehalose tetraacetate (4-O-Ac-Tre). Once in the cell, intracellular esterases hydrolyzed the 6-O-Ac-Tre molecules, releasing free trehalose into the cytoplasm. The total concentration of intracellular trehalose (plus acetylated variants) reached as high as 10 fold the extracellular concentration of 6-O-Ac-Tre, attaining concentrations suitable for applications in biopreservation. To describe this accumulation phenomenon, a diffusion-reaction model was proposed and the permeability and reaction kinetics of 6-O-Ac-Tre were determined by fitting to experimental data. Further studies suggested that the impact of the loading and the presence of intracellular trehalose on cellular viability and function were negligible. Engineering of trehalose chemical structure rather than manipulating the cell, is an innocuous, cell-friendly method for trehalose delivery, with demonstrated potential for trehalose loading in different types of cells and cell lines, and can facilitate the wide-spread application of trehalose as an intracellular protective agent in biopreservation studies.