Although gene therapy holds great promise for the treatment of both acquired and genetic diseases, its development has been limited by practical considerations. Although many novel routes of gene delivery are under investigation, efficacy of delivery remains quite poor particularly with primary cells, with viral vectors still most efficient means of delivery. Safety concerns and immunogenicity greatly limit the clinical potential of viral vectors. The aim of this project was to explore the efficacy of a novel, safe, lipid-based delivery system, cochleates (Biodelivery Sciences International). These highly stable lipid bilayer structures are non-toxic and non-inflammatory. The molecules housed within the lipid bilayers, and the cochleates themselves, are protected from degradation by this unique tightly-packed, multilayer structure. Encochleated amphotericin B remained biologically active when delivered by various routes, including oral administration. Rhodamine-labelled cochleates (10 μg/ml) were incubated with a murine mammary adenocarcinoma cell line (4T1), a macrophage-hybrid cell line (H36.12) and primary monocytes/macrophages. Cochleates entered the 4T1 cells more readily than the H36.12 cells. At 6 hours 39% of the 4T1 cells were positive for rhodamine and 23.1% of the H36-12 cells were rhodamine- positive at this time. 7.24% +/− 3.77 and 33.3% of human monocyte derived macrophages were rhodamine positive after 1 or five hours respectively reaching 48+/− 5.48% after 24 hours. 51.45+/− 15.62% peritoneal murine macrophages incubated for 1 hour in vitro were rhodamine positive. After 3 hours 38.07 +/− 14.95% were rhodamine positive. 4T1 cells were incubated in vitro with cochleates containing GFP-expression plasmid for up to 7 days and viewed daily to monitor GFP expression. From 48 hours GFP expression was observed. This GFP expression was sustained for up to 7 days, with maximum transfection levels of 19.49+/−10.12% seen 96 hours after addition of 37.5μg/ml cochleates to cells. Macrophages were elicited to the peritoneal cavity of BALB/c mice by thioglycollate injection and 500μg of cochleates were injected to this area. Six hours after intra-peritoneal (i.p.) injection of rho-cochleates 25.69 +/− 0.1273% of recovered macrophages were rhodamine positive. 24 hours post i.p. injection of cochleates, 18.55+/−2.5% of recovered macrophages were rhodamine positive. Intra-tumour injection of GFP cochleates (150μg) to Lewis Lung flank tumours on C57BL/6 mice (n=2) resulted in 4.06+/−1.03% GFP positive cells 3 days after injection. In contrast to the cationic lipid transfection reagents, in vitro cochleate uptake is associated with little or no cytotoxicity. Our in vivo studies suggest that cochleates may also be an effective method of in vivo transgene delivery. Further studies are required to optimise their ability to transfect a range of primary cells and tumours in the in vivo setting.
[Proc Amer Assoc Cancer Res, Volume 45, 2004]