University of Twente Student Theses
VHH-Mediated targeting of osteoarthritic Synovium by synthesizing functional lipid nanoparticles using post-insertion
Mourik, C.M. van (2024) VHH-Mediated targeting of osteoarthritic Synovium by synthesizing functional lipid nanoparticles using post-insertion.
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| Abstract: | Osteoarthritis is the most common type of arthritis and one of the primary causes of disability worldwide. This disease is characterized by synovitis, the inflammation of the synovial membrane of the joint. The inflamed membrane secretes catabolic and pro-inflammatory products, this results in an imbalance between M1(pro-inflammatory) and M2(anti-inflammatory) macrophages. The imbalance eventually causes cartilage degradation in the joint. It is a chronic disease with no effective treatments so far. A potential treatment option is to use targeted drug delivery. In this way, drugs can be transported to specific target cells. Using siRNA as the drug has proven to be effective. The drugs will inhibit M1 or activate M2 macrophages. For the transport and encapsulation of the drugs, ionizable lipid nanoparticles (LNPs) can be used. LNPs are neutral at physiological pH in the body but become cationic when a low pH is reached in cells. Resulting in the breakdown of the LNP and the release of its content. To target specific types of macrophages, the LNPs need to be coated with biological ligands. For this, VHHs show promise to be used since they are small antibody fragments of approximately 15 kDa with the same binding affinity as monoclonal antibodies. This thesis focuses on the synthesis of the functional LNPs. The method of post-insertion of lipids is tested, and thereby, the lipids are first conjugated to the functional biological ligand. After conjugation, the functional lipid is inserted into the LNP, for this, three molar ratios of lipids are tested: 0.1, 0.2 and 0.3%. The biological ligand in this report is first a fluorescent Cy3 dye to confirm conjugation, and later a VHH that targets IL-1R receptors on cells. Two reactive groups, DBCO and Maleimide, and two PEG spacer lengths, 2000 and 5000 Da, are tested for the lipids. LNPs are successfully synthesized with mCherry and tRNA as drugs, with the help of a microfluidic chip with a herringbone structure. The conjugation of the lipids(DSPE-PEG-Mal/DBCO) to the Cy3-PEG-SH/Azide-PEG2000-Cy3 dye is analyzed with fluorescence measurements and confirmed for both reactive groups and PEG spacers. A higher conjugation is achieved with the 5000 Da PEG spacers than the 2000 Da PEG spacers. The conjugation of DSPE-PEG-Mal to VHH with a 5000 Da PEG spacer is confirmed by a quantification by SDS-page. The post-insertion of DSPE-PEG-Mal-Cy3 and DSPEPEG-Mal-VHH showed inconclusive results. The post-insertion of DSPE-PEG-DBCO-Cy3 succeeded for both PEG spacers, analyzed by fluorescence measurements. A higher molar ratio of DSPE-PEG-DBCO-Cy3 leads to a higher fluorescence signal, so more lipids are inserted into the LNPs. DSPE-PEG-DBCO-Cy3 with 5000 Da PEG has a higher fluorescence than with 2000 Da PEG, so more lipids are inserted into the LNPs. All post-inserted LNPs are measured on size and PDI with DLS. Post-insertion leads to a higher size and PDI of the LNPs, a higher molar ratio does not influence the size and PDI. |
| Item Type: | Essay (Bachelor) |
| Faculty: | TNW: Science and Technology |
| Subject: | 44 medicine |
| Programme: | Biomedical Technology BSc (56226) |
| Link to this item: | https://purl.utwente.nl/essays/102560 |
| Export this item as: | BibTeX EndNote HTML Citation Reference Manager |
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