Developing a New Drug Delivery System for the Brain

Parkinson’s Disease (PD) is a neurodegenerative disease with limited, symptomatic treatment options. The gradual progression of PD is characterised by the death of dopaminergic neurons in the substantia nigra; the slow progression of PD ultimately resulting in loss of movement and co-ordination is responsible for its high socioeconomic burden. A significant barrier to PD research is the lack of animal or cell-based models for the disease which limits the development of effective treatment. The SH-SY5Y cell line is frequently used for PD research due to its catecholaminergic phenotype and ease of maintenance. SH-SY5Y cells can be differentiated to a neuronal phenotype using differentiating agents including retinoic acid (RA) and Brain Derived Neurotrophic Factor (BDNF). Cells were differentiated to a neuronal phenotype through the addition of RA and gradual Foetal Calf Serum (FCS) starvation from the cell media. The process of cell differentiation was assessed and imaged using inverted microscopy with an attached camera. The SH-SY5Y cells were successfully differentiated to a neuronal phenotype using FCS depravation and addition of RA, protocol demonstrated to be consistently reproducible producing mature, differentiated SH-SY5Y cells within 14 days. For SH-SY5Y cells to be reflective of the substantia nigra and act as an effective PD model the cells must be differentiated to a neuronal phenotype; however, many research papers do not provide information regarding the dopaminergic phenotype of the cells or explain why the cell line was selected as a model. In this research project we set out to investigate the reproducibility of SH-SY5Y cell differentiation protocols using RA, to assess mitochondrial activity of differentiated cells using cell viability assays and to confirm that differentiated SH-SY5Y cells can successfully synthesise dopamine. 3-(4,5-dimethyldiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and PrestoBlue cellular viability assays were conducted to assess cellular metabolism and enzyme activity for the cell population. The ability of differentiated cells to produce dopamine was assessed by dopamine ELISA. The results of the MTT and PrestoBlue cell viability assays confirmed that undifferentiated and differentiated SH-SY5Y cells are metabolically active, however fully differentiated cells proliferate at a reduced rate, approximately one fifth of that of their undifferentiated counterparts, indicating that differentiation is stressful to cells and can result in cell death. ELISA results demonstrate that both undifferentiated and differentiated SH-SY5Y cells synthesise dopamine with the production being significantly upregulated, by an average of six-fold, after differentiation. The findings of this investigation demonstrate that SH-SY5Y cells can be used as an effective model for PD, providing a suitable differentiation protocol has been used to drive the cells towards a neuronal, dopaminergic phenotype. However, due to a gap in the literature the level of dopamine production by differentiated SH-SY5Y cells cannot be compared against in vivo neurons found in the substantia nigra. Results indicate that SH�SY5Y cells, differentiated by FCS deprivation and addition of RA, can be used as a PD model however the dopaminergic phenotype of cells should be assessed on a batch-by-batch basis to ensure that cells continue to act as an effective mod