Cationic Lipid and Peptide Synthesis for Co-delivery and Gene Therapy

Samin Fathali Nejad

Studenteropgave: Speciale

Abstrakt

Gene therapy is an intracellular delivery of genetic materials into specific cells to generate a therapeutic effect by correcting an existing abnormality or providing the cells with a new function. A major obstacle has been the delivery of genes to the appropriate cell affected by the disorder, without triggering unwanted side effects like cytotoxicity or unsettling the immune balance. Liposomes as non-viral gene delivery vectors have been to some extent successful in avoiding these side-effects. This study has employed novel liposomal gene delivery vehicles by incorporating short-chained (C11) cationic saturated and unsaturated dimethylamine (SDMA and USDMA) lipids, cationic innate defense regulatory (IDR) peptides, IDR-1018 and IDR-HH2 and neutral helper lipids, cholesterol (Chol) and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), in their formulations. These 3-component liposomes (peptide/lipid/co-lipid) were made to investigate their effects on delivering DNA in terms of cytotoxicity and transfection efficiency. This was done through formulating liposomes with DNA, forming lipoplexes, in four different molar charge ratios, (+/-) 1.5:1, 3:1, 5:1 and 10:1. The so-called lipoplexes were characterized using particle sizing and DNA-binding assays and then transfected into Chinese hamster ovarian cells (CHO-K1) with the aid of Lipofectamine 2000TM. Their relative transfection efficiency and cytotoxicity were evaluated using β-galactosidase assay and MTS assay, respectively. The comparisons of USDMA versus SDMA, IDR-1018 versus IDR-HH2 and Chol versus DOPE were evaluated. The mean values of the results were calculated and the relative p-values from the student’s t-tests were measured to observe any significant differences between these constituents. Although the results from the gel retardation assay did not appear to be successful in showing any clear trends between bindings of the DNA to the proteins, the hydrodynamic diameter measurements showed reasonable size variations, between 200-900 nm, at <1 Polydispersity, which are relatively optimal for lipoplexes prepared without extrusion. The results showed that all of the lipoplexes were transfected into the cells. The lipoplexes at molar charge ratios 3:1 and 5:1 showed higher transfection efficiencies than the rest. The results from the student’s t-test revealed that USDMA performed better than SDMA, Chol performed better than DOPE and the two peptides equally as good, in the relative transfection efficiencies. The cytotoxicity measurements showed a concentration dependent cytotoxicity. The results indicated a high cell viability at low molar charge ratios (1.5:1), with all of them exceeding ≥ 115% cell viability. The p-values showed no overall significant difference between the cytotoxicity of the two peptides, the two lipids and the two co-lipids. The transfection efficiency as well as the cell viability results from the hybrid of cationic peptides and cationic lipids to form a liposome for non-viral DNA delivery vector showed better outcomes compared to the results obtained from using the same cationic lipids (alone) as the DNA delivery vectors. This suggests that incorporation of cationic peptides in the vector could potentially have a positive impact on gene therapy. Further analysis and investigations are, however, necessary to prove these results.

UddannelserKemi, (Bachelor/kandidatuddannelse) Kandidat
SprogEngelsk
Udgivelsesdato28 apr. 2017
Antal sider76
VejledereHåvard Jenssen & William Goldring