Investigation of Cubosome Interactions with Liposomal Membranes Based on Time-Resolved Small-Angle X-ray Scattering...

Nanosized dispersions of the bicontinuous cubic phase (cubosomes) are emerging carriers for drug delivery. These particles possess well-defined internal structures composed of highly-curved lipid bilayers that can accommodate significant drug payloads. Although cubosomes present promising potential for drug delivery, their physicochemical properties and interactions with cell membranes have not yet been fully understood. To clarify the interactions of the cubosomes with cell membranes, we investigated the changes in the structural and cubic membranes of monoolein (MO) cubosomes when mixed with model cell membranes at different phase states using time-resolved small-angle X-ray scattering (TR-SAXS), cryogenic transmission electron microscopy (cryo-TEM), and fluorescence spectroscopy. TR-SAXS results showed that the cubosomes gradually transitioned from the Im3m phase to the lamellar phase after interacting with the liposomes. The time of the structural change of the cubic phase to the lamellar phase was influenced by the fluidity of the liposome bilayers. Mixing the cubosomes with fluid membrane liposomes required less time to transition to the lamellar phase and vice versa. Cryo-TEM images showed that the well-defined internal structure of the cubosomes disappeared, leaving behind lamellar vesicles after the interaction, further confirming the TR-SAXS results. Laurdan fluorescence probe was used to assess the membrane polarity changes occurring to both the cubosomes and liposomes during the interaction. Examination of the normalized fluorescence intensity of the probed cubosomes showed decreasing intensity, followed by a recovery of intensity, which could indicate the disintegration of the cubic membrane and the formation of a mixed membrane. Also, the kinetics of the disintegration of the cubic phase did not seem to be influenced by the composition of the liposomes, which was in line with the normalized SAXS intensity results. Assessing the generalized polarization (GP₃₄₀) values of the cubosomes and liposomes after mixing revealed that the fluidity and membrane hydration states of the cubosomes and liposomes transitioned to resemble their counterpart, confirming the exchange of material between the particles. Over time, the hydration states of the cubosomes and liposomes equilibrated toward an intermediate state between the two. The time needed to reach the final intermediate state was influenced by the membrane fluidity and hydration of the liposomes, more particularly the difference in GP₃₄₀ values and their membrane phase state. These results highlight the importance of examination of the cubic membrane conditions, such as membrane polarity, and their implications on the changes in the cubic structure during the interaction with liposomal membranes.