1887

Abstract

Abstract: Background: While promising, cationic lipid-mediated gene delivery can still benefit from improvements in lipid design and lipid-DNA (lipoplex) formulation. The putative mechanism of cellular lipoplex uptake is believed to occur by endocytosis, where the key influential factors are lipoplex size and morphology; lamellar and inverted hexagonal. Lamellar lipoplexes offer superior protection to the DNA cargo, while the inverted hexagonal phase best facilitates endosomal escape. Ideally, the initial lipoplex packaging would have the lamellar phase upon uptake, followed by a phase transition to hexagonal, facilitating cargo release into the cytosol. The cationic lipid structure defines its molecular packing parameter, S, which in turn controls the lipid phase transition. A molar weighted average packing parameter (Smix) for the overall cationic and neutral co-lipid mixture within a lipoplex formulation is predictive of a lamellar (S<1) or hexagonal (S>1) phase lipoplex. Objectives: Pyridinium-based cationic lipids represent a class of non-viral vectors that have shown promise in gene delivery. The objective of this study was to test the influence of lipid shape on lipoplex phase structure through small-angle x-ray diffraction (SAXD), and correlate shape with transfection efficiency. Lipoplexes co-formulated with pyridinium lipid, (16:0)(11:1) having a calculated shape parameter, S, of 1.08 and the commercial cationic vector, EPC (S=0.94) were predicted to undergo a packing transition from lamellar to hexagonal as the ratio of (16:0)(11:1) / EPC is increased. A fixed amount of neutral co-lipid was employed (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, DOPE: S=1.01; or cholesterol, Chol: S=1.20). Given that cholesterol is a higher-S lipid than DOPE, the lipoplex phase transition from lamellar to hexagonal was anticipated to occur at a lower ratio of (16:0)(11:1) to EPC. Methods: Liposomes were prepared from pyridinium-based cationic lipids in combination with EPC and co-lipid, DOPE or cholesterol. Lipoplexes were then formulated by incubating the liposomes with plasmid DNA at various N/P (+/-) molar charge ratios, and subsequently characterized by gel retardation, DNAse I degradation, biocompatibility and β-galactosidase (β-gal) transfection assays using Chinese Hamster Ovarian (CHO-K1) cells. Lastly, lipoplexes at N/P molar charge ratio 3 (only) were analyzed by SAXS at the synchrotron in Grenoble, France. Results: The SAXD results revealed that the (16:0)(11:1)/EPC/DOPE-DNA lipoplex formulations underwent a lamellar to hexagonal packing transition when the (16:0)(11:1)/EPC molar ratio was increased from 1:2 to 1:1, where the Smix increased from 1.01 to 1.04. However, (16:0)(11:1)/EPC/Chol-DNA lipoplex formulations underwent a lamellar to hexagonal transition when the (16:0)(11:1)/EPC molar ratio increased from 1:1 to 2:1; when Smix increased from 1.11 to 1.13. For both DOPE and Chol containing lipoplexes, the greatest transfection was found at (16:0)(11:1) to EPC ratios below 2:1, at an N/P molar charge ratio of 3. Conclusion: The lamellar to hexagonal packing transition, as determined by SAXD, for the lipid-DNA lipoplexes composed of the (16:0)(11:1)/EPC mixture occurred as predicted by our Smix calculations when DOPE was employed as co-lipid. The same was not observed when cholesterol was employed co-lipid. Finally, superior lipoplex transfection correlated with lamellar packing.

Loading

Article metrics loading...

/content/papers/10.5339/qfarf.2013.BIOP-0141
2013-11-20
2020-09-27
Loading full text...

Full text loading...

http://instance.metastore.ingenta.com/content/papers/10.5339/qfarf.2013.BIOP-0141
Loading
This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error