The efficiency of maltodextrin and porous silicon nanoparticles for the in vitro and in vivo stabilization of tyrosine hydroxylase
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- Department of Chemistry 
Parkinson's disease is a severe neurodegenerative disease, which causes deterioration of the dopaminergic neurons of the substantia nigra. Lack of dopamine causes motor function problems and cognitive decline. Due to unwanted side effects resulting from the traditional treatment of the disease by L-DOPA, the use of formulations of tyrosine hydroxylase (TH), the rate limiting enzyme in the biosynthesis of dopamine, could yield an effective treatment with less side effects. This is due to the high level of regulation TH is subject to compared to L-DOPA. To further increase delivery to the target site, the use of nanoparticles as nanocarriers can be used. Maltodextrin nanoparticles (MDNPs) and porous silicon nanoparticles (pSiNPs) were investigated to this respect. DLS was used as a simple method of confirming or debunking loading of NPs, since TH and each NP originated distinct size peaks (approximately 13 nm for TH, 90 nm for MDNPs and 120 nm for pSiNPs) when measured. Loading was easily confirmed for the TH:MDNPs complex, but not for the pSiNPs, where DLS measurement in the buffer (50 mM HEPES, pH 6, 50 mM NaCl) and concentrations (0.1 mg/ml in 1:10 000 pSiNPs) that no longer showed free TH, but we could not distinguish between pSiNPs peaks and aggregates of TH. A Thioflavin-T assay was performed in order to find out whether the presence of pSiNPs promoted the aggregation of TH, or if it hindered it. Through these experiments it became clear that the pH of the buffer was the most significant factor associated to aggregation, but that this effect could be avoided by altering the pH of the complex from pH 6 to pH 7. Since MDNPs loading was the most successful, the stability of TH:MDNPs was studied in vitro by using DLS to look at the diameter of the average size of particles in the sample solution over time and at different temperatures. In both cases it was found that a 1:1 w/w ratio of TH:MDNPs would prevent aggregation, and also that a 2:1 w/w ratio TH:MDNP would at least delay the effects of aggregation. Activity measurements also revealed that TH retained activity in both complexes, but more so in MDNPs. In vivo injection of TH:MDNPs was performed in order to find out if the complex could be taken up by brain cells. Fluorescent tags were used, and TH:MDNP-Atto488 and TH-Alexa568:MDNP was injected, and was located in tissues near the injection site using fluorescence microscopy. Further investigation using confocal microscopy showed that the complexes were very likely to have been taken up by the cells.