Study On Long Circulating Liposomes Loaded With Total Salvianolic Acids

In the present study, long circulating liposomes loaded with total salvianolic acids, lipophilic agents from traditional Chinese medicine danshen, were prepared in the aim of overcoming the limitations of traditional danshen preparations, such as instability, short circulation time and short effect time in vivo. This study may provide some references for developing new preparations of danshen with long circulation time, high bioavailability and long effect time. Also, this study was an exploration for new drug delivery system for traditional Chinese medicine.First of all, a UV method for total salvianolic acids and a HPLC method for salvianolic acid B were established. The stability and octanol-water partition coefficient of total salvianolic acids were studied. It was shown that the influence of temperature, illumination and sonication on the content of total salvianolic acids and salvianolic acid B was very small. But the pH has effect on salvianolic acid B while no effect on total salvianolic acids. Salvianolic acid B was instability when the pH of solution was lower than 4.0 and higher than 7.0. The pH lower, the Octanol-water partition coefficient of salvianolic acid B higher.Then PEG-DSPE used to prepare long circulating liposomes was synthesized and identificated by infrared scanning. Different methods for determining entrapment efficiency were compared and the rapid and convenient method of ultrafiltration was chosen. The total salvianolic acid in liposomes was detected by UV after the liposomes was dissolved in alcohol, slvianolic acid B was detected by HPLC after liposomes was dissolved in TritonX-100 solution. After free total salvianolic acid and free salvianolic acid B were separated by ultrafiltration and detected, the entrapment efficiency was calculated.Reverse-phase evaporation technique was used to prepare the long-circulating liposomes. By the single factor evaluation, chloroform and physiologic saline were chosed to be organic solution and solvent, respectively. Sonication time was also chosed. It was found that the entrapment efficiency could be increased using acid buffer solution to prepare liposome. So the acid buffer solution was further studied. The glycine-hydrochloric acid buffer showed the best effect. The single factor evaluation showed that pH of acid buffer, ratio of cholesterol to phospholipids and ratio of total salvianolic acid to phospholipids influenced the entrptment efficiency obviously. These three factors were studied in the formulation optimization with central composite design. Using the optimized formulation, liposomes and long-circulating liposomes with high entrapment efficiency, high loading amount and small particle size were prepared.The investigation of stability of liposomes and long-circulating liposomes showed that the entrapment efficiency of liposomes and long-circulating liposomes decreased a little after 10 days, so freeze drying technology was used to increase the stability of liposomes. Different supporting agents and different concentration was compared and 10% sucrose was chosed. The particle size and shape didn't change in the freeze drying process. The lyophilized liposomes and long-circulating liposomes were stable at 4℃. The entrapment efficiency, drug content and particle size didn't change in 3 months. Macrophage was used to study the effect of different ratio of PEG-DSPE to phospholipids on phagocytosis. The result showed that long-circulating liposomes which contain 5% PEG-DSPE decreased the phagpcytosis most significantly. In vitro release revealed that liposomes and long-circulating liposomes could prolong the release of total salvionolic acid. Serum could accelerated the release of conventional liposomes, but had no effect on long-circulating liposomes.The in vivo behavior of total salvianolic acid in long-circulating liposomes was studied by intravenous administration to rats. Total salvianolic acid in solution, liposomes and long-circulating liposomes all fit two compartment model. Encapsulation of total salvianolic acids in liposomes and long circulating liposomes produced a significant change in drug pharmacokinetic parameters. The elimination half life of liposomes was 4.7 times longer than total salvianolic acids solution. For long-circulating liposomes, the changes were more significant, the elimination half life was 16.5 times longer than total salvianolic acids solution and 3 times longer than liposomes. The advantages of liposomes and long-circulating liposomes were confirmed by the AUC and MRT values. The AUC of liposomes and long-circulating liposomes increased more than 6-fold and 13.5-fold versus the total salvianolic acid solution, respectively. For MRT value, the liposomes and long-circulating liposomes were 8.2 times and 35.6 times compared to solution, respectively. The results of pharmacokinetics showed that liposomes and long-circulating liposomes could prolong the circulating time of SalB and increase its bioavailability. Long-circulating liposomes were better than liposomes. The tissue distribution showed that liposomes and long-circulating liposomes could increase the distribution of Sal B in different tissues. In short time after administration, the distribution of liposomes were higher than long-circulating liposomes, but the distribution of the long-circulating liposomes became higher than liposomes following time prolonging.Pharmacokinetics and pharmacodynamics of liposomes and long-circulating liposomes in Beagle dogs were further studied. The analytical method of blood concentration was established and evaluated. Pharmacokinetic parameters in dogs also showed that liposomes and long-circulating liposomes could prolong the circulation time of SalB, increase its bioavailability. Long-circulating liposomes were better than liposomes. The pharmacodynamic result showed that all of total salvianolic acid solution, liposomes and long-circulating liposomes decreased the whole blood viscosity obviously. The solution decreased the viscosity most obviously and quikly, but 2 hours after administration, the viscosity began to increase, then it decreased again. The time of decreasing viscosity of liposomes and long-circulating liposomes were longer than solution. Their effect could last even 10 hours after administration. The decline of viscosity of liposomes was obviously than long-circulating liposomes in 2-6 hours, but the superiority of long-circulating liposomes displayed after 8 hours.Our present study showed that long circulating liposomes loaded with total salvianolic acids could prolong the circulating time of drug, increase its bioavailability and prolong its effect time. Our study may provide some references for developing new preparations of danshen with long circulation time, high bioavailability and long effect time. Also, this study was an exploration for new drug delivery system for traditional Chinese medicine.