Abstract
Whilst there is a large body of evidence looking at the design of cationic liposomes as transfection agents, correlates of formulation to function remain elusive. In this research, we investigate if lipid packaging can give further insights into transfection efficacy. DNA lipoplexes composed of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) or 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) in combination with 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) or 1,2-stearoyl-3-trimethylammonium-propane (DSTAP) were prepared by the lipid hydration method. Each of the formulations was prepared by hydration in dH2O or phosphate buffer saline (PBS) to investigate the effect of buffer salts on lipoplex physicochemical characteristics and in vitro transfection. In addition, Langmuir monolayer studies were performed to investigate any possible correlation between lipid packaging and liposome attributes. Using PBS, rather than dH2O, to prepare the lipoplexes increased the size of vesicles in most of formulations and resulted in variation in transfection efficacies. However, one combination of lipids (DSPE:DOTAP) could not form liposomes in PBS, whilst the DSPE:DSTAP combination could not form liposomes in either aqueous media. Monolayer studies demonstrated saturated lipid combinations offered dramatically closer molecular packing compared to the other combinations which could suggest why this lipid combination could not form vesicles. Of the lipoplexes prepared, those formulated with DSTAP showed higher transfection efficacy, however, the effect of buffer on transfection efficiency was formulation dependent.
Highlights
In the development of new healthcare strategies, correction of underlying pharmacology of diseases instead of alteration of symptoms remains a key goal
Amongst the two classic groups of vectors, viral vectors provide high transfection efficiency but these systems are still limited by the dogma of toxicity and immunogenic reactions and much research has focused on the use of non-viral vectors, given their superior safety profile despite their lower efficacy
The ideal extrapolated area per molecule was calculated based on taking the average area for the lipid combination, such that the calculated area could be compared to the actual area per molecule of the mixture
Summary
In the development of new healthcare strategies, correction of underlying pharmacology of diseases instead of alteration of symptoms remains a key goal. Developments in gene therapy remain slow and the major unresolved concern with gene therapy is how to effectively deliver the gene to the target site. It has been known for some time that direct injection of ‘naked’ DNA allows transgene expression in muscle [2], in most of the cases ‘naked’ DNA molecules are not able to enter cells efficiently due to their large size, negative charge and nuclease mediated degradation in vivo [3]. An ideal vector should be stable and have high efficiency, with minimal toxicity and unrestricted size limitation for genetic payload (which can include DNA, siRNA etc.).
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