pH-Tolerant giant vesicles composed of cationic lipids with imine linkages and oleic acids

Giant vesicles (GVs) have attracted attention as functional materials because they can encapsulate both hydrophilic and hydrophobic compounds. For next generation functional GVs, both tolerance and stimuli-sensitivity are needed. So far, vesicles tolerant to acidic or basic conditions were generated using a mixture of cationic lipids and fatty acids. Here, to create functional GVs that are tolerant to a wide pH range but sensitively respond at below a specific pH, the behaviour of GVs composed of a cationic lipid with an imine bond and oleic acid was investigated. Even though the GVs prepared by the film swelling method were tolerant to strongly acidic conditions, GVs without oleic acid gradually shrank, accompanied by the generation of oil droplets at the same pH. 1H NMR analysis revealed that during hydration of the film, the imine bond hydrolysed to provide a cationic surfactant and an oil component in the presence of oleic acid due to its own Lewis basicity, suggesting the dissociation of oleic acid. The results of fluorescence spectroscopy using an environment-responsive probe and IR spectroscopy indicated that the GV tolerance originated from the intermolecular interactions of cationic lipids and anionic oleate.


Figures
p. 5 Scheme S1. Synthesis of Im having a hydrolysable group.

Synthesis of (E)-2-((4-(heptyloxy)benzylidene)amino)ethylene-N,N-dimethylamine(A1)
n-Heptyloxybenzaldehyde (HBA) (2.20 g, 10 mmol) and N,N-dimethylethylenediamine (1.32 g, 15 mmol) were added to MeCN (20 mL). A catalytic amount of acetic acid (1 drop) was added, and then the mixture was stirred at 80 °C for 12 h. After the reaction, the solvent was removed by evaporation under reduced pressure. The obtained crude product dissolved in 15 mL of ethyl acetate was washed three times with a 5% aqueous NaHCO 3 solution, and then dried over anhydrous magnesium sulfate.
The solvent of filtrate was evaporated under reduced pressure to obtain the mixture of A1 and HBA (2.84 g), as yellow viscous liquid, whose ratio was 1/0.07 (mol/mol) analyzed by 1 H NMR spectrum using CDCl 3 .

Synthesis of N-(E)-((2-((4-(heptyloxy)benzylidene)amino)ethyl)-N-hexadecyl-N,Ndimethylammonoum bromide (Im)
A mixture of A1 including HBA (2.84 g) and 1-bromohexadecane (2.68 g, 7.76 mmol) was reacted in anhydrous MeCN (20 mL) with stirring at 70 °C for 24 h. After the reaction, the solvent was removed by evaporation under reduced pressure. The crude product was dissolved in chloroform (10 mL) at 25 °C, and then ethyl acetate (50 mL) was added at room temperature to reprecipitate to obtain Im (3.36 g), as a white crystal, in a yield of 56% in a two-step reaction.

S3
Synthesis of the amphiphile having an amine group (Am) [1] Am was synthesized according to the procedure of Scheme S2 reported by Chen et al 1 .

Synthesis of N-(2-aminoethyl)-N-hexadecyl-N,N-dimethylammoinium bromide(Am)
A mixture of B2 (29.4 mg, 0.0629 mmol) and 47% aqueous HBr solution (500 µL) was reacted in a screw-capped tube at 100 ° C for 3 days. After the reaction, the solvent was removed by evaporation under reduced pressure. Purification was carried out by recrystallization using ethanol/diethyl ether

S4
(1/1, v /v, 1.0 mL) to obtain Am (25. 3mg), as a brown crystal, in a yield of 98%.   Calculation of the f-test was confirmed that 0% and 50% was an unequal variance because the p value was <0.05. From the calculation of the t-test, it was also confirmed that T was significant because the p value was < 0.05. (b) T of 10 or more GVs composed of 1 mM Im (red) and Im/Am/HBA = 0.50 mM/0.5 mM/0.5 mM (blue) (n ≥ 10). There was no difference between 0% and 50% depending on the initial size. From these, the statistical difference in GVs radius changes in the difference lipid composition was more clearly confirmed.   Table   Table S1. Microscopic observation of samples composed of Im, HBA, Am, and oleic acid.