HYBRIDIZATION FLUORESCENCE STUDY OF LIPID MEMBRANES

Nanotechnology has been an ally in drug delivery system research. Liposomes are lipid nanostructures used for drug delivery but are quickly captured by the immune system. Biomimetic nanovesicles (BNVs) are nanostructures that include components of biological origin in their composition, such as purified cell membranes. A recent strategy involves hybridizing purified cell membranes with liposome membranes to obtain a hybrid nanostructure. This strategy offers advantages, such as better process standardization and cost savings on biological materials. Furthermore, they show promising capabilities in crossing biological barriers and delivering drugs to the target site. In this study, liposomes were produced via a microfluidic route using a "simple T" microdevice with an Flow Rate Ratio (FRR) of 13, a Total Flow Rate (TFR) of 200 µL/min, and a lipid composition of Egg phosphatidylcholine (EPC)/Cholesterol in a molar ratio of 66:34, resulting in liposomes (8 mM lipid concentration) with an average diameter of approximately 75.79 ± 4.79 nm, a polydispersity index (PDI) of 0.18 ± 0.03 and a zeta potential of -9,11 ± 3,70 mV. We also used L-α-Phosphatidylethanolamine-N-(lissamine rhodamine B sulfonyl) (Rhod-PE) and L-α-Phosphatidylethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl) (NBD-PE) to study membrane hybridization using the Fluorescence Resonance Energy Transfer (FRET) method. A fusion study was conducted involving liposome-liposome hybridization and membranes extracted from SH-SY5Y cells. A fluorescence emission analysis of EPC/COL liposomes with varying molar percentages of fluorophores in the lipid composition was performed to achieve the optimal configuration, ranging from 1% to 0.25%. These results were used to select the best concentration of fluorophores for hybridization studies and evaluation via %FRET. Readings were taken with excitation at 460 nm, allowing NBD and Rhodamine to emit fluorescence at 535 nm and 583 nm, respectively, to visualize fluorescence intensity curves indicating that the fluorophores are closer together in the bilayer. Fusion between liposomes via sonication resulted in a %FRET of 82.72, and the fusion between liposomes and SH-SY5Y cell membranes was 84.60 and 85.50 using sonication and freeze-thaw methods, respectively. The FRET study confirmed membrane fusion with a decrease in %FRET. This study may offer scalable and standardized alternatives for the future synthesis of hybrid BNVs.