Study On Aggregation Of Ionic Liposomes Induced By Small Molecules And Its Mechanism

In the present research, the aggregation of ionic liposome was induced by small molecular weight compounds, and its mechnism was discussed.According to literatures, three lipids were synthesized through using 1-hexadecylamine and 1-bromohexadecane as starting materials. Passing eight steps, including substitution and condensation reaction, the desired products, cationic peptide lipid N,N-dihexadecyl-N^α- [6-(trimethylammonio) hexanoyl]alaninamide bromide (N⁺C₅Ala2C₁₆),ceramic lipid N,N- dihexadecyl -N^α-[6-[(3-triethoxysilyl)-propyldimethylammonio)hexanoyl]alaninamide bromide((EtO)₃SiC₃N⁺C₅Ala2C₁₆) and hydroxyl lipid N,N- dihexadecyl - N^α- [(3-hydroxyl)-propyldimethylammonio]hexanoyl alaninamide bromide (HOC₃N⁺C₅Ala2C₁₆) were obtained, the yields 4.75%,3.45% and 5.32% respectively.They were characterized using NMR.The aggregation behavior of cationic liposomes was estimated through mesuring the turbidity at 500nm using UV / Vis spectrophotometer. EDTA affecting on the aggregation behavior of cationic liposomes formed from a lipid (N⁺C₅Ala2C₁₆) 1.0 mmol/L at pH 12.00 buffer, was investigated, comparing with that of ceramic liposome (cerasome). The results showed that in a cationic liposome suspension, the turbidity of vesicular suspension obviously increased on addition of EDTA concentration up to 0.25 mmol/L, for the aggregation of the liposomes occurred; the aggregation was redispersed by addition of metal ions such as Ca²⁺,Mg²⁺,Cu²⁺, whereas it wasn't done by Na⁺or K⁺; EDTA combined with a cationic liposome was 0.18 mmol/L by using ultrafiltration and sequentially high performance liquid chromatography;when the aggeration occurred on cerasome was 0.1 mmol/L, but not being redispersed by the metal ions Ca²⁺,Mg²⁺,Cu²⁺,Na⁺,K⁺. In addition, effect of small moleculars such as EDTA,citric acid with multi-carboxyl groups on the aggregation behavior of cationic liposome at pH range from 2.00 to 12.00 was also investigated. The results showed that the turbidity of liposome suspension was obviously increased with the dissociation numbers of carboxyl groups in both of compounds more than or equal to three at a contain pH, the aggregation of liposomes occurring, and conversely no aggregation was observed when the number was less than three. Judging from the above resulrs, the aggregation of liposomes were formed through electrostatic charge neutralization and adsorption between the negative charges of dissociated carboxyl groups and positive charges of the liposome and using small molecular compound as bridging molecules. Effect of small molecular compound, diguanidine ethane, diguanidine hexane and guanidine ethanol synthesized through two steps including substitution and condensation reaction at a yield of 73%, 65% and 62% and confirmed through NMR, on the aggeration of the liposomes 2.0 mmol/L mixed from DMPCrDHP (1:1) were investigated. It was evident from the results that diguanidine ethane1.0mmol/L, diguanidine hexane 4.0mmol/L induced the occurrence of the liposome aggregation, no aggregation was formed on addition of guanidine ethanol up to 20mmol/L. Liposome functionalized with phosphate groups and bridging molecules with guanidy were employed as the recognizable moieties, liposomes aggregation induced by the bridging molecules through molecular recognition. The molecular recognition interaction was further investigated at different molar ratio of cholesterol molecules to DMPC (20% -100%) as a function of the cholesterol content in mixed liposomes (DMPC-cholesterol-DHP). And the results showed that cholesterol incorporated into liposomes significantly modifies their molecular recognition performance.