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Influence of VHH on cellular uptake of LNPs and transfection in joint cells

Verhoeven, M. (2024) Influence of VHH on cellular uptake of LNPs and transfection in joint cells.

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Abstract:Osteoarthritis (OA) and rheumatoid arthritis (RA) are the most common chronic degenerative joint diseases, that both primarly occur in the elderly population. In these diseases, synovitis occurs, in other words the inflammation of the synovium membrane. During inflammation, the synovium is a source of pro-inflammatory and catabolic products, leading to an imbalance between the cartilage synthesis and degradation. Currently there are two treatment pathways: non-pharmaceutical and pharmaceutical. The first involves weight loss and exercises. The second may involve surgery; this is usually done only in cases of meniscal damage. Intra-articular glucocorticoid injections can be given, but mainly relieve pain. Finally, NSAIDs can be prescribed to inhibit inflammation, but this is strongly discouraged in people with concurrent conditions, such as heart problems. The aim for the future is provide balance between the cartilage synthesis and degradation. This can potentially be achieved by target M1 macrophages using VHHs as targeting ligands on the LNPs containing miRNA, in which the miRNA causes reduced inflammation. This study will focus on a proof of concept, by looking at cellular uptake of LNPs containing plasmid DNA, in joint cells. The used VHH will target the IR-1L1 receptor. Therefore, chondrocytes known to contain IR-1L1 receptors were used for the proof of concept. First, the LNPs were made with a microfluidic chip. Then three cell experiments were done. First, to look at cellular uptake and transfection in chondrocytes, with LNPs without VHH. Then also to look at cellular uptake and transfection with LNPs without VHH, but in macrophages. And lastly to look at specific cellular uptake and transfection in chondrocytes, with LNPs with VHH. In all three experiments, cellular uptake and transfection was looked at with EVOS microscopy and these photos were quantified with MATLAB. A presto blue assay was also done, to look at the toxicity of the LNPs. Metabolic activity assays showed that both chondrocytes and macrophages retained similar activity up to 24 hours, but showed reduced activity after 96 hours, with transfection further reducing activity in chondrocytes. Cellular uptake shows higher uptake of LNPs in chondrocytes compared with macrophages, and higher concentration of LNPs led to higher uptake. However, addition of VHH to LNPs resulted in reduced uptake in chondrocytes. Transfection was observed in chondrocytes but not in macrophages, with higher LNP concentrations leading to higher transfection signals. Although, LNPs with VHH did not lead to transfection. Overall, VHHs affected the uptake of LNPs into joint cells opposite to expectations, suggesting that further research is needed to fully understand their influence on cellular uptake. Furture research should take a look into another quantification of the EVOS microscopy picture, for example flow cytometry. In addition, the binding of VHH to cell receptors could be examined. In which it would be examined whether there is binding to the specific receptor first and then uptake, for example using a timelapse with EVOS microscopy. It would also be beneficial to look at the low uptake of LNPs with VHH by examining the properties of LNPs with compared to without VHH. Lastly, the future aim is to delivery drugs with the LNPs to specific cells. In this study the loaded LNPs seems to be more toxic to the cells than the non loaded LNPs. Follow-up studies should look into the toxicity of miRNA when inside the LNP.
Item Type:Essay (Bachelor)
Faculty:TNW: Science and Technology
Programme:Biomedical Technology BSc (56226)
Link to this item:https://purl.utwente.nl/essays/103596
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