Analysis of the interaction of cyclosporine congeners with cell membrane models.

Owing to the relatively high molecular weight of macrocyclic peptides, investigation of the cellular uptake mechanism is required for the efficient design of macrocyclic peptides as potential drugs. We have previously reported, using HPLC, that cyclosporine A, a model macrocyclic peptide, and its congeners B, C, and D had different lipophilicity despite differing by only one amino acid. In the present study, we investigated how this difference in lipophilicity affected the interaction of the congeners with cell membranes. The circular dichroism spectra showed that the secondary structures were similar between the four congeners even at high temperature. The molar ellipticity of the four congeners in the presence of liposomes, as a cell membrane model, differed, and cyclosporines D and A showed lower molar ellipticity, while cyclosporine C exhibited higher molar ellipticity. Fluorescent spectra analysis using Laurdan indicated that liposome hydration was decreased in the presence of the cyclosporines, especially cyclosporines D and A. HPLC analysis also quantitatively showed that the amount of cyclosporine molecules internalized in HpG2 cells was the largest for cyclosporine D. We determined, using spectroscopy and HPLC, that the intensity of the interaction of the congeners with cell membranes was overall correlated with the lipophilicity derived from the side chains of each congener. Our results will contribute to the design of new macrocyclic peptides with favorable drug properties.

Acidic pH-induced changes in lipid nanoparticle membrane packing.

To enable the release of the encapsulated nucleic acids into the cytosol of targeted cells, the interaction of lipid nanoparticles (LNPs) with endosomes is critical. We investigated changes in the physicochemical properties of LNPs containing ionizable cationic lipids that were induced by acidic pH, which reflects the conditions in the maturation of endosomes. We prepared a LNP containing an ionizable cationic lipid. The laurdan generalized polarization values, which are related to the hydration degree of the lipid membrane interface and are often used as an indicator of membrane packing, decreased with a decrease in pH value, showing that the membrane packing was decreased under acidic conditions. Furthermore, the pH-induced variation increased with an increasing percentage of ionizable cationic lipids in the LNPs. These results indicated that electrostatic repulsion between lipid molecules at acidic pH decreased the packing density of the lipids in the LNP membrane. Reducing the order of lipids could be a trigger to form a non-bilayer structure and allow fusion of the LNPs with the membrane of maturing endosomes in an acidic environment. The LNPs were used to incorporate and transport small interfering RNA (siRNA) into cells for knockdown of the expression of ß-galactosidase. The knockdown efficiency of siRNA encapsulated in LNPs tended to increase with the ratio of KC2. These results, which demonstrate the underlying phenomena for the fusion of membranes, will help clarify the mechanism of the release of encapsulated nucleic acids.