Chitosan rate of uptake in HEK293 cells is influenced by soluble versus microparticle state and enhanced by serum-induced cell metabolism and lactate-based media acidification.
Chitosan rate of uptake in HEK293 cells is influenced by soluble versus microparticle state and enhanced by serum-induced cell metabolism and lactate-based media acidification.
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HEK293细胞中的壳聚糖摄取速率受到可溶性与微粒状态的影响，并通过血清诱导的细胞代谢和基于乳酸的培养基酸化增强。
DOI：
10.3390/molecules18011015
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发表时间：
2013-01-15
期刊：
影响因子：
--
通讯作者：
Lavertu M
中科院分区：
文献类型：
--
作者：
Hoemann CD;Guzmán-Morales J;Tran-Khanh N;Lavallée G;Jolicoeur M;Lavertu M
壳聚糖是由葡萄糖胺和N-乙酰葡萄糖胺组成的生物相容性多糖。该聚合物具有在pH 6的可溶性链和pH 7的不溶性微粒之间波动的独特行为。本研究的目的是测试假设，壳聚糖的结构，溶解状态，和血清影响细胞摄取率。脱乙酰度为80%和95%的壳聚糖（中等和低粘度）用罗丹明标记，并使用活体共聚焦显微镜和流式细胞术分析颗粒大小、介质溶解度和HEK 293上皮细胞的摄取。在含有或不含10%血清的pH 7.4培养基中，壳聚糖完全沉淀成直径为0.5至1.4 µm的微颗粒，带有轻微的负电荷。在无血清培养基中培养24小时期间，壳聚糖颗粒保持在细胞外。在含血清的培养物中，颗粒以血清剂量依赖性方式被摄取到细胞内囊泡中。用血清调理壳聚糖或用表皮生长因子（EGF）替代血清未能介导无血清壳聚糖颗粒摄取。血清刺激细胞酸化培养基，部分是通过乳酸的产生。通过7 mM乳酸盐酸化至pH 6.5的培养基在可溶性级分中保持50%的壳聚糖，并导致小囊泡中轻微均匀的无血清摄取。结论：培养基酸化介导非生物污染的可溶性壳聚糖链的少量体外摄取，而血清-生物污染的不溶性壳聚糖微粒需要持续的血清暴露以产生巨胞饮所需的能量。
Chitosan is a biocompatible polysaccharide composed of glucosamine and N-acetylglucosamine. The polymer has a unique behavior of fluctuating between soluble chains at pH 6 and insoluble microparticles at pH 7. The purpose of this study was to test the hypothesis that chitosan structure, solubility state, and serum influence the rate of cell uptake. Chitosans with 80% and 95% degree of deacetylation (medium and low viscosity) were tagged with rhodamine and analyzed for particle size, media solubility, and uptake by HEK293 epithelial cells using live confocal microscopy and flow cytometry. In media pH 7.4 with or without 10% serum, chitosans fully precipitated into 0.5 to 1.4 µm diameter microparticles with a slight negative charge. During 24 h of culture in serum-free medium, chitosan particles remained extracellular. In cultures with serum, particles were taken up into intracellular vesicles in a serum dose-dependent manner. Opsonization of chitosan with serum, or replacement of serum by epidermal growth factor (EGF) failed to mediate serum-free chitosan particle uptake. Serum stimulated cells to acidify the media, partly by lactate generation. Media acidified to pH 6.5 by 7 mM lactate maintained 50% of chitosan in the soluble fraction, and led to minor uniform serum-free uptake in small vesicles. Conclusion: Media acidification mediates minor in vitro uptake of non-biofouled soluble chitosan chains, while serum-biofouled insoluble chitosan microparticles require sustained serum exposure to generate energy required for macropinocytosis.
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