A series of pyridinium cationic lipids was designed, synthesized and characterized. These lipids varied in the lipophilic part, bearing C9 to C20 saturated, unsaturated, straight and branched hydrocarbon chains.
The lipid shape parameter was calculated from the molecular structure of these lipids based on the partial molar volumes of the atoms, and standard bond lengths and bond angles, using fragment additive methods. The shape parameter controls the lamellar/hexagonal phase balance in lipoplexes of the lipid with deoxyribonucleic acid (DNA). The lipid phase behaviour of the lipoplexes was derived from small-angle X-ray scattering experiments and was successfully correlated with the calculated lipid shape parameter.
The synthesized pyridinium lipids were co-formulated (1:1) with 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (EPC) as the co-cationic lipid in 1:1 ratio, and the mixed cationic lipids were co-formulated (3:2) with the neutral lipids 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE) or cholesterol. The effect of variation in cationic lipid structure and lipoplex formulation on the transfection of nucleic acid (β-galactosidase and green fluorescent protein (GFP)) into CHO-K1 cells and the cytotoxicity of these formulations was assessed.
Initial studies on the synthesized lipids bearing saturated and terminally unsaturated C16 chains showed that a Transfection Index (TIPSV) which encompasses the variation in the lipid shape parameter, the phase packing in a hexagonal lipoplex and the partition of these lipids into the lipoplex successfully correlated with transfection efficiency.
To further investigate the effect of the variation of the partition of these lipids to the lipoplex, transfection studies were performed on a series of pyridinium lipids with straight saturated and unsaturated chains of varied lengths, with similar shape parameters but varied partition coefficients (clogP). The correlation of these experimental transfection data with the initial TIPSV was unsuccessful, but the data suggested that chain length as it relates to chain mixing and chain melting behaviours of pure lipids played a role in transfection. A refined transfection index (TIPSVM) was proposed which contained terms for the lipid shape parameter, the phase packing into a hexagonal lipoplex, the partition of these lipids into the lipoplex and a chain melting term. TIPSVM gave an acceptable correlation with the experimental transfection efficiency for the range of compounds. Additional experimental transfection data were obtained for compounds with widely variable lipid shape parameters, either as pure compounds, blends of two pure compounds, or statistically produced mixtures of mixed-chain compounds. Although very short-chain compounds (C9) and very lipophilic compounds (C20) performed poorly, the results from the blends allow the assessment of the role of the shape parameter in the TI. Since the shape parameter and the volume filling term are both calculated with the same molecular parameter, the experimental work demonstrated that only one of these terms is required. Thus a three parameter transfection index (TIPVM) was proposed and found to correlate with the entire set of comparable data.
A Quantitative structure–activity relationship (QSAR) study was done on the cytotoxicity of the transfection formulations utilized. The toxicity of the synthesized pyridinium lipids was shown to correlate with the shape parameter, the lipid mixture partition co-efficient (clogP) and the charge ratio of the lipoplex formulation.
Taken together, the developed transfection index TIPVM and the cytotoxicity correlation uncovered can be used in the design of low-toxicity, high activity pyridinium lipids for transfection of DNA. / Graduate / pariapz@uvic.ca
| Identifer | oai:union.ndltd.org:uvic.ca/oai:dspace.library.uvic.ca:1828/5842 |
| Date | 05 January 2015 |
| Creators | Parvizi, Paria |
| Contributors | Fyles, Thomas M. |
| Source Sets | University of Victoria |
| Language | English, English |
| Detected Language | English |
| Type | Thesis |
| Rights | http://creativecommons.org/licenses/by-nc-nd/2.5/ca/, Available to the World Wide Web |
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