The Biological Implications of Sialic Acid Binding Nanoparticles in T cells

Examensarbete för masterexamen
Master's Thesis
Biomedical engineering (MPBME), MSc
de Carvalho, Viktoria
The immune system is heavily regulated and affected by changes in the composition of cell surface molecules, such as glycans. One sugar usually located at the end of glycans is sialic acid (SA). Due to its negative charge and location, it is involved in many immune processes, such as immune cell migration and maturation, but many functions of SA are still unknown. The position and charge provide us with an easy target for engineered particles with SA affinity. Seeing that immune cells are sialylated, we can use this to target the immune system and possibly modulate it. This project has focused on polymeric SA affine nanoparticles (NPs) and T cells. The project primarily worked with the Jurkat E6-1 cell line of CD4+ T cells. Peripheral blood mononuclear cells (PBMCs) purified from buffy coat were also used. Cytotoxicity of NPs was investigated with the Alamar Blue assay. Activation markers and NP-binding to cells were investigated with flow cytometry and the cytokine profile was evaluated with enzyme-linked immunosorbent assay (ELISA). Confocal microscopy was used to qualitatively evaluate NP binding specificity. The results show that the NPs are not cytotoxic to T cells in tested concentrations ranging up to 500 μg/mL. Flow cytometry revealed that the NPs bind less to activated Jurkat cells and CD4+ primary cells, but more to activated CD8+ primary cells, compared to the respective unactivated cells. Confocal microscopy confirmed that NPs bind to unactivated cells to a higher extent. The NPs decreased the cellular expression of CD62L, which could be due to some activation asserted by the NPs, or steric hindering of the anti-CD62L antibody. ELISA showed that the IL-10 concentration in supernatant from PBMCs was lower for cells exposed to NPs, but further research is needed to confirm whether the NPs have a significant effect on the IL-10 profile of PBMCs. The IL-10 profile of Jurkats was not changed between particle concentrations. Finally, flow cytometry showed that the level of activation markers CD69 and CD25 were not affected by the particle concentrations, nor was the IL-8 concentration after evaluation with ELISA, leading to the conclusion that the activation level in T cells is not affected by the SA affine polymeric NPs. Thus, the NPs prove promising for future use in immunomodulation, with diagnostic or T cell activation-based targeting possibilities, and as a tool for understanding SA expression.
T cells , polymeric nanoparticles , sialic acid , Jurkat , PBMC , glycans , CD69 , CD62L
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