Liposome Aggregation And Redispersion Coupled With An Enzymatic Reaction

Based on an enzymatic reaction by lactate dehydrogenase (LDH), aggregation and/ordispersion of vesicles (liposome) were coupled with it under certain conditions. This processis of value in constructing an artificial-multicell system.In the present work, two lipids were synthesized through using 1-hexadecylamine and1-bromohexadecane as starting materials. After seven steps, including substitution andcondensation reaction, the desired products, cationic peptide lipid N,N-dihexadecyl-N^a-[6-(trimethylammonio)hexanoyl]alaninamide bromide (N⁺C₅Ala2C₁₆) and ceramic lipidN,N-dihexadecyl-N^a-[6-[(3-triethoxysilyl)-propyldimethylammonio)hexanoyl]alaninamidebromide((EtO)₃SiC₃N⁺C₅Ala2C₁₆), were obtained, the yields 4.4% and 3.5%.The experimental conditions, sodium pymvate1.0 mmol/L and LDH 2.24 nmol/L, wereconfirmed through investigating the effect of sodium pyruvate, LDH in a 10mM, pH7.0 bufferof 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES) on the aggregationbehavior of vesicles formed from a peptide lipid (N⁺C₅Ala2C₁₆) containing alanine residue,by reduced coenzyme I. Effect of oxidated coenzyme I (NAD⁺) and reduced coenzyme I(NADH) on the aggregation of the cationic vesicles was studied. The results showed that in acationic vesicular suspension formed from the surfactant 0.50 mmol/L, no obvious change ofthe suspension appearance was observed on addition of NAD⁺ carrying one net positivecharge, concentration from 0 to 2.40 mmol/L in the vesicle suspension, and no aggregationwas formed; while the turbidity of vesicular suspension was obviously increased on additionof NADH carrying two net positive charges, concentration from 0.25 to 0.80 mmol/L, and theaggregation of the vesicles occurred; the aggregation was redispersed by the enzyme with theconversion of NADH to NAD⁺ in the enzymatic reaction. The particles of vesiclesaggregation and redispersed vesicles by the enzyme were observed with an electronmicroscopy. The maximum binding concentration 0.33 mmol/L of NADH was confirmedwith an ultrafiltration method.Through investigating the ability of lactate dehydrogase to convert NAD⁺ to NADH inthe presence of lactate acid in Tris buffers at defferent pH, the conditions pH9.06, NAD⁺2mmol/L, Lactate acid 10mmol/L, were confirmed. Upon addition of LDH in the systemformed by vesicles formed from ceramic lipid under above conditions, quick absorbance increase of vesicular suspension at 500nm showed aggregation of the vesicles. It wassurported by atomic force microscopy. In a control experiment, the cationic vesicles formedfrom (N⁺C₅Ala2C₁₆) didn't aggregate in such conditions.