Cellular Uptake Of Several Colloidal Particles And Its Influences On Cell Functions

The colloidal particles have been widely used in biological and biomedical field. However, the interaction between colloidal particles and cells, such as internalization process and influence on cell functions, which are of paramount importance, is still not fully addressed. This study is focused on the internalization process and intracellular fate of the particles and their influences on cells. Metal oxide nanoparticles, poly (lactide-co-glycolide) (PLGA) colloidal particles, ployelectrolyte microcapsules were used to investigate the interaction between colloidal particles and cells. The influences on cell viability and important physiological functions were also detected.Three types of metal oxide nanoparticles (NPs), i.e., TiO2, Fe3O4and TiO2, were employed to investigate the NPs health effects on biological organisms at the cellular level. The physical and chemical properties of these NPs such as size, specific surface area, surface charge, solubility and protein adsorption capacity were characterized. The cellular loading of TiO2 and Fe3O4 NPs increased with the incubation time, while that of ZnO NPs became undetectable after 12 h. The TiO2 and Fe3O4 NPs mainly located inside the vesicles in the cytoplasm, but no ZnO NPs was observed. Influences of the metal oxide NPs on cytotoxicity and functions were then studied in terms of cell viability, mitochondrial membrane potential (MMP), total and released lactate dehydrogenase (LDH) activity and intracellular level of reactive oxygen species (ROS). These results demonstrate that the cell uptake, intracellular dissolution and thereafter release of Zn2+ are the intrinsic reasons for the high toxicity of ZnO NPs.Poly(D,L-lactide-co-glycolide) (PLGA) NPs loaded with lamivudine and coated with bovine serum albumin (BSA) were prepared via a double emulsion method. The influences of experimental parameters such as volume of inner aqueous phase, concentration of organic phase and ultrasonication time on the particle size and drug entrapment efficiency were investigated, obtaining the PLGA particles with a diameter of ～260 nm and drug entrapment efficiency of ～35%. The PLGA particles were readily internalized into the human liver cells within a short time and increased gradually with the prolongation of incubation time regardless of the loading of lamivudine. The particles either resided within lysosomes or transferred to cytoplasm, but could not enter into the cell nucleus. The cell viability was not significantly influenced in the presence of the particles, suggesting that this kind of particles may be a good candidate for the intracellular anti-hepatitis B drug delivery.The covalently assembled microcapsules with poly(allylamine hydrochloride) (PAH) and glutaraldehyde as building blocks were prepared via layer-by-layer (LbL) assembly on spherical dextran sulfate-doped CaCO3 template, followed by removal of the template. The microcapsules were easily ingested by SMCs mainly through macropinosis and caveolae-mediated endocytosis pathways. No exocytosis was observed. The capsules mainly dispersed in cytoplasm without colocalization in early endosomes and cell nucleus within 48 h culture in vitro. The results of gene chip revealed that 886 genes were down-regulated and 758 genes were up-regulated more than 2 folds after the cells were exposed to microcapsules, suggesting substantial and profound alternation of phenotypes and functions. Influences of the capsule uptake on cell toxicity and functions were then studied in terms of cell viability, cytoskeleton organization, cell cycle, cell adhesion and migration. Although uptake of the microcapsules only caused slightly decrease of cell viability, it did cause significant alternation of cytoskeleton organization and cell cycle. The cell adhesion and migration ability were largely impaired too, demonstrating again the comprehensive impacts of capsule uptake on cell functions.PAH layer which assembled on MnCO3 microparticles was cross-linked with a dicarboxy linker 3,3’-Dithiodipropionic acid (DPA) through amino-carboxyl reaction in the presence of 1-hydroxy-2,5-pyrrolidinedione (NHS) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC). Following dissolution of the template, stable, cross-linked (PAH/DPA)5 microcapsules were obtained. MTT and LDH assays showed that the microcapsules had weak effect on the cell viability and membrane integrity, indicating negligible cytotoxicity toward the SMC cells. In the presence of L-glutathione (GSH), the sulfhydryl-disulfide exchange reaction occurred between the disulfide cross-linked microcapsules and GSH, leading to degradation of the microcapsules. As a result, the permeability of microcapsules changed and the fluorescent rhodamine B isothiocyanate-dextran (RITC-dextran) can diffuse into the interior of the microcapsules. Further more, FCM results showed that double stained microcapsules showed FRET positive and experienced energy transfer decrease with the prolongation of incubation time. Confocal Raman spectroscopy confirmed the dissociation of disulfide bond and degradation of intracellular microcapsules. The cytoskeleton of SMC cells was disrupted with the internalization of microcapsules, and could not be recovered spontaneously after the partial degradation of intracellular microcapsules.