Fliposomes: pH-controlled Release from Liposomes Containing New trans-2-Morpholinocyclohexanol-based Amphiphiles that Perform a Conformational Flip and Trigger an Instant Cargo Release upon Acidification

Abstract

A new type of pH-sensitive liposome (fliposomes) was designed based on the amphiphiles that are able to perform a pH-triggered conformational flip (flipids). This flip disrupts the liposome membrane and causes rapid release of the liposome cargo, specifically in the areas of increased acidity. The flipids (1-3) are equipped with a trans-2-morpholinocyclohexanol conformational switch. pH-Sensitive fliposomes containing one of these flipids, POPC and PEG-ceramide (molar ratio 50/45/5) were constructed and characterized. These compositions were stable at 4 o C and pH 7.4 for several months. Fliposomes loaded with ANTS/DPX demonstrated an unusually quick content release (in a few seconds) at pH below 5.5, which was more efficient in the case of flipid 1 with the shorter linear C 12-tails. The pH-titration curve for the fliposome leakage paralleled the curve for the acid-induced conformational flip of 1-3 studied by 1 H NMR. A plausible mechanism of the pH-sensitivity starts with an acid-triggered conformational flip of 1, 2 or 3, which changes the molecular size and shape, shortens the lipid tails, and perturbs the liposome membrane resulting in the content leakage.

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

“…1) [21][22][23][24]. All the lipids (1-8) share the common structural features of a trans-2-aminocyclohexanol (TACH) moiety as the polar headgroup and two trans-alkoxycarbonyl groups at the 4-and 5-positions of the cyclohexane ring as the lipid tails.…”

“…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).…”

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

“…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].…”

“…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).…”