Fliposomes: pH-Sensitive Liposomes Containing a trans-2-morpholinocyclohexanol-Based Lipid That Performs a Conformational Flip and Triggers an Instant Cargo Release in Acidic Medium

Abstract

Incorporation of a pH-sensitive conformational switch into a lipid structure enables a drastic conformational flip upon protonation that disrupts the liposome membrane and causes rapid release of cargo specifically in areas of increased acidity. pH-sensitive liposomes containing the amphiphile (1) with trans-2-morpholinocyclohexanol conformational switch, a phospholipid, and a PEG-lipid conjugate were constructed and characterized. The optimized composition—1/POPC/PEG-ceramide (50/4/5)—could be stored at 4 °C and pH 7.4 for up to 1.5 years, and was stable in blood serum in vitro after 48 h at 37 °C. Liposomes loaded with ANTS/DPX or methotrexate demonstrated an unusually quick content release (in a few seconds) at pH below 5.5, which was independent of inter-liposome contact. The pH-titration curve for the liposome leakage paralleled the curve for the acid-induced conformational flip of 1 studied by 1H-NMR. Freeze-fracture electron microscopy images showed budding and division of the bilayer at pH 5.5. A plausible mechanism of pH-sensitivity involves an acid-triggered conformational flip of 1, shortening of lipid tails, and membrane perturbations, which cause the content leakage. The methotrexate-loaded liposomes demonstrated much higher cytotoxicity in HeLa cells than the free drug indicating that they can serve as viable drug delivery systems.

trans-2-Aminocyclohexanol-based amphiphiles as highly efficient helper lipids for gene delivery by lipoplexes

“…The amphiphiles 1-3, 6, and 9, and the corresponding intermediates were prepared as described earlier [21][22][23][24]. Other TACH-lipids ( Fig.…”

“…The spin-spin coupling constants between several pairs of vicinal protons attached to the cyclohexane moiety are strongly conformationdependent, which allowed the assignment of a predominant conformation and the estimation of a position of the conformational equilibrium as described previously [21][22][23][24]31,32]. To characterize the pH-induced change of conformational equilibrium, a TACH-lipid was dissolved in CD 3 OD (0.02-0.03 M), and the changes of its 1 H NMR spectrum (600 MHz) were monitored during titration of the solution with d-trifluoroacetic acid (d-TFA) and with a base: 1,8-diazabicyclo [5.4.0]undec-7-ene (DBU).…”

“…We recently designed a novel type of amphiphiles based on trans-2-aminocyclohexanol (TACH) and introduced these TACH-lipids as a pHsensitive conformational switch of liposome bilayer [21][22][23][24] (Scheme 1). In the original conformational equilibrium A ⇆ B (Scheme 1), the amine and the hydroxy groups of TACH-lipid are predominantly in axial positions while both lipidic trans-ester groups adopt equatorial positions (A).…”

“…This impulse is mechanically transmitted via the conformational flip of the cyclohexane ring to force both ester groups into axial positions (BH + on Scheme 1), thereby drastically increasing the spatial separation of the lipid tails and perturbing the bilayer structure. After incorporation into liposome membranes, TACH-lipids significantly enhanced the release from both the traditional liposomes based on phospholipids and the sterically hindered liposomes containing mPEG2000-ceramide (PEG-ceramide) upon exposure to lowered pH [21][22][23][24]. To highlight the key role of the conformational flip in the pHtriggered liposome leakage, we introduced the terms "flipids" for the amphiphiles containing a pH-sensitive conformational switch and "fliposomes" for the liposomes comprising these amphiphiles [22][23][24].…”

“…1). The analogous amphiphiles 9 [21,22] and 10 ( Fig. 1) were also synthesized as controls that do not perform pH-triggered conformational switch.…”