Nano-liposome mediated delivery of the anti-diabetic protein E4orf1
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Objectives: E4orf1 is an adenoviral-derived protein that promotes cellular glucose uptake by up-regulating AKT phosphorylation. Proof of concept studies show that the transgenic or viral-vector mediated expression of the E4orf1 remarkably enhances glycemic control in animal models. However, a system suitable for delivering the E4orf1 protein in humans is needed for extending the findings to clinical setting. As the first step, here we show construction and testing of nano-liposome, a well-known nano-particle, as a carrier to deliver the E4orf1 protein to 3T3-L1 cells in-vitro Method: GST- tagged E4orf1 protein was encapsulated in nano-liposomes which were prepared using Soy phosphatidylcholine and labeled with Rhodamine-PE (Phosphoethanolamine). The size and polydispersity index (PI) of nano-liposomes were measured using a Brookhaven BI-MAS particle size analyzer. To test the delivery efficacy of the nano-liposomes, murine 3T3-L1 cells were treated with E4orf1-containing nano-liposomes (E4 group) or void nano-liposomes (Void group) and expression determined over time by immunofluorescence. Cell lysates from treated cells were used to examine changes in molecular signaling. Results: The diameter of nano-liposomes was 133.5 nm and 90.7 nm and the PI was 0.2 and 0.1 for E4 and void nano-liposomes respectively. The average encapsulation efficiency was 99% as measured by the Enzyme Immuno Assay using anti-E4orf1 antibodies. The E4 group of cells showed maximum GST and Rhodamine expression at 24 h compared to 2, 4 and 12 h. The Void group showed the expression of Rhodamine, but not GST. The expression of E4orf1 in E4 group of cells was confirmed at 24, 48 and 72 h. Western blot analysis in cells treated for 24, 48 and 72 h showed significant increase in p-AKT expression at 72 h in E4 cells compared to the Void group. Conclusions: We report the first successful delivery of E4orf1 encapsulated liposomes and the expected effect of E4orf1 on cell signaling. It provides the proof of concept for extending the use of nano-particle-mediated delivery system to investigate the anti-diabetic potential of E4orf1 in humans.