Abstract
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.
Highlights
Chitosan (Figure 1) is a mucoadhesive polysaccharide with wide-ranging applications in drug delivery, gene delivery, vaccines, and tissue engineering [1,2]
This study reports novel data showing that ≥2% serum promotes significant uptake of biofouled biodegradable and non-biodegradable insoluble chitosan microparticles in HEK293 cells, after enough time has elapsed to allow the particles to fully settle on the cell monolayer
Data generated by this study show that the volume of culture media controls the rate of uptake, because it takes longer for chitosan particles to settle in a thick layer of media
Summary
Chitosan (Figure 1) is a mucoadhesive polysaccharide with wide-ranging applications in drug delivery, gene delivery, vaccines, and tissue engineering [1,2]. The mode of delivery, site of action, biodegradation, and cell/tissue responses to chitosan are tightly linked to the polymer structure. Chitosan is derived from chitin, (1-4)-O-linked N-acetyl-β-D-glucosamine (GlcNA), the insoluble backbone component of shrimp and crab shells, by chemical deacetylation. Depending on the degree of deacetylation (DDA), chitosan can have an average glucosamine content that ranges from ~45% to. Enzymatic degradation of chitosan by serum lysozyme requires three consecutive GlcNA residues to hydrolyze the sugar linkages [7]. Deacetylated chitosans (>90% DDA) are non-biodegradable [8]
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