| Abstract |
[Truncated abstract] Nascent research into nanomaterial toxicity indicates significant deviations from the parent material, with concerns regarding the potential of nanomaterials to cross biological barriers and enter into cells and organelles due to their small size. This thesis sets out to prove the hypothesis that chitosan nanoparticles could induce cytotoxicity in human intestinal and liver cells. Chitosan nanoparticles were evaluated because extensive research into their biomedical applications was based largely on the biodegradable and biocompatible profile of chitosan. In order to evaluate cytotoxicity, a novel method for the reproducible fabrication of a continual supply of chitosan nanoparticles (NP) was first developed using the spinning disc processing (SDP) technology. NP with a diameter of 20 ± 3 nm and zeta potential of 53.3 ± 4.3 mV were successfully produced, and were comparable to those produced by the conventional method. Particle size was modulated by the type and concentration of acid used in the chitosan feed solution. NP following dialysis and lyophilisation were stable in size when stored in the dry state at -20 C for up to 2 months. Dispersion of the lyophilised NP into various biorelevant media affected particle size, zeta potential and aggregation status. NP particle size was preserved in Hanks balanced salt solution and supplemented culture media, although protein adsorption in the latter caused the NP to assume a negative zeta potential. Evidence of particle aggregation was noted in phosphate buffered saline. NP toxicity against human intestinal cells was studied using the Caco-2 cell model and chitosan (Cs) as control at pH 7.4 (physiological) and pH 6.0 (upper GI). |
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At corresponding concentrations (0.025 % to 0.1 %), NP at pH 6.0 caused greater decreases in Caco-2 cell viability than NP at pH 7.4, or Cs at both pH. The activity of alkaline phosphatase, a brush border enzyme, was enhanced by more than 1.5 folds by both Cs and NP at pH 6.0. However, only NP was internalised by the cells, and this was associated with cell membrane damage and nuclear fragmentation suggestive of necrotic cell death. NP but not Cs also facilitated the paracellular transport of fluorescein, with sensitisation of effects seen with the 2nd NP dose compared to the 1st dose. Removal of NP effectively reversed the effects for both doses. NP toxicity against liver cells was conducted on the novel BHAL human liver cell line, although the murine BMOLTAT liver cells were used initially when BHAL was not yet available. The BMOLTAT cells showed greater resilience to Cs and NP, emphasising the importance of selecting representative human cells for in vitro cytotoxicity evaluation of nanoparticles. The BHAL cells were resilient to Cs, but cellular uptake of NP at pH 6.0 inflicted cell membrane damage, leading to reduction in cell viability and proliferation. There were significant nuclear and cytoplasmic damage consistent with necrotic cell death. CYP3A4-mediated activity in the BHAL cells was, however, induced by both Cs and NP. Compared with Caco-2, the BHAL cells exhibited greater resilience to NP... |
