Chitosan-based nanoparticles (NPs) are widely used in drug delivery, device-based therapy, tissue engineering, and medical imaging. In this aspect, a clear understanding of how physicochemical properties of these NPs affect the cytological response is in high demand. The objective of this study is to evaluate the effect of surface charge on cellular uptake profiles (rate and amount) and intracellular trafficking. We fabricate three kinds of NPs (∼ 215 nm) with different surface charge via SPG membrane emulsification technique and deposition method. They possess uniform size as well as identical other physicochemical properties, minimizing any differences between the NPs except for surface charge. Moreover, we extend our research to eight cell lines, which could help to obtain a representative conclusion. Results show that the cellular uptake rate and amount are both positively correlated with the surface charge in all cell line. Subsequent intracellular trafficking indicates that some of positively charged NPs could escape from lysosome after being internalized and exhibit perinuclear localization, whereas the negatively and neutrally charged NPs prefer to colocalize with lysosome. These results are critical in building the knowledge base required to design chitosan-based NPs to be used efficiently and specifically.

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Fluorescent microspheres are widely used as biological tracers. In this study, uniformly sized chitosan microspheres crosslinked with glutaraldehyde (CG microspheres) and formaldehyde (CF microspheres) are successfully prepared by the Shirasu Porous Glass (SPG) membrane emulsification technique. Selectively reduced CG microspheres (SRCG microspheres) are obtained by NaBH4 reduction. These chitosan microspheres are found to exhibit fluorescent properties without conjugation to any fluorescent agent. The fluorescence color varies with different crosslinkers and can be modulated by further chemical reduction, whereas the fluorescence intensity can be controlled by tuning the particle size and degree of crosslinking. The autofluorescence of the microspheres is applied to study the phagocytosis of HepG2 cells using the microspheres as novel tracers. Quantitative and qualitative evaluations show that these chitosan microspheres serve as bright, inert, durable, and extremely photostable tracers.

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Monodisperse chitosan microspheres with different structures are prepared and loaded with proteins, as exemplified in the figure. The different types of microspheres show different protein release profiles, which implies that that their properties can be adjusted to fit the needs of different therapeutic applications. The structural properties of the microspheres are varied by adjusting the surface charge, cavity size, and wall porosity.

Clinical application of paclitaxel (PTX) is limited because of its poor solubility in aqueous media. To overcome this hurdle, we devised an oral delivery system by encapsulating PTX into N-((2-hydroxy-3-trimethylammonium) propyl) chitosan chloride (HTCC) nanoparticles. These nanoparticles were small (~130 nm), had a narrow size distribution, and displayed high loading efficiency owing to the homogeneous distribution of PTX nanocrystals. The matrix hydrophilicity and porous structure of the obtained nanoparticles accelerated their degradation and improved drug release. In vitro and in vivo transport experiments had proved that the presence of positive charges enhanced the intestinal permeability of these nanoparticles. Further in vitro experiment of cytotoxicity showed that the PTX-loaded HTCC nanoparticle (HTCC-NP:PTX) was more effective than native PTX owing to enhanced cellular uptake. Drug distribution in tissues and in vivo imaging studies confirmed the preferred accumulation of HTCC-NP:PTX in subcutaneous tumor tissue. Subsequent tumor xenograft assays demonstrated the promising therapeutic effect of HTCC-NP:PTX on inhibition of tumor growth and induction of apoptosis in tumor cells. Additional investigation into side effects revealed that HTCC-NP:PTX caused lower Cremophor EL-associated toxicities compared with Taxol. These results strongly supported the notion that HTCC nanoparticle (HTCC-NP) is a promising candidate as an oral carrier of PTX for cancer therapy.

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