| Background:The project aims to study how to improve the intake of ATP in the state of tissue ischemia and hypoxia, and prepars the new formulation which is intravenous or external use for clinical application; explore a high bioavailability, low toxicity and stability drug delivery systems. Liposome is the most important and most widely used nano-drug delivery systems in the world, which is non-toxic, non-immunogenicity, and good tissue compatibility, it can improve the bioavailability of drugs, and enhance the organ targeting. At present, only the liposome dilivery system is used in clinical,and the vast majority of other delivery system are still in pre-clinical research stage.Methods:In the project we choose soybean Phophatidylcholine and cholesterol as the main membrane materials and ATP as a model drug, to regulate Zeta potential of liposomes by octadecylamine.We prepared ATP liposomes in the way of reverse phase evaporation, and optimize the formulation though the orthogonal test in the index of entrapment efficiency. The factors contain phospholipid concentration,phospholipids and cholesterol ratio, drug and lipids ratio, water temperature, spin speed and and so on, we establish the ATP assay method in the liposomes and the method of entrapment efficiency determination, and we also study the physical and chemical properties of ATP liposomes, in vitro release properties, the short-time stability, which provide the experimental and theoretical basis for the development and application of liposomes.Results:1. Establishment of the ATP assay method and liposome encapsulation efficency method.The liposomes and free drug are separated by high speed centrifugation.and the entrapment effieieney and drug concentration were determined by the method of HPLC. Results:There was a good linearity of the drug concentration with in the range of 20-640ug/ml and the linear equation was A= 0.1602C+7.3767, r=0.9997. The recovery rate was more than 98% in the middle and high concentration,but there is large deviations in the low concentration, which may be related to instrumental error, and all of the RSD (intra-and inter-day) were less than 2.0%.2. Preparation of ATP liposomes, and the physical and chemical propertiesWith the encapsulation efficiency as judgment index. The effects of various factors on entrapment efficiency were investigated by single factor and orthogonal experients. The factors contain phospholipid concentration,phospholipids and cholesterol ratio, drug and lipids ratio, water temperature, spin speed and and so on.The ATP liposome obtained after optimized prescription was multi-layer balls cystic body in the transmission electron microscopy.We regulate the zeta potential of liposomes by octadecylamine,and phospholipids and cholesterol in a fixed prescription,change the amount of octadecylamine added in the formulation at a ratio of 0:1,0.05:1,0.10:1 to lipids,the zeta potential was 2.1±0.4mv,12.3±L2mv,20.4±2.6mv.At a ratio of 0.05to l,the ATP liposome diameter we obtained is 810.9nm,and the polydispersity is 0.543.The optimized fotmulation is as follows:the phospholipid concentration of 10mg/ml, phospholipids and cholesterol mass ratio of 4:1, lipid and drug ratio is 1:2, the speed of rotary evaporator is 30r/min, octadecylamine and phospholipid mass ratio is 0.05:1, the average encapsulation efficiency in this formulation is (56.99±4.91)%, drug loading rate is (21.81±1.89)%.3. The release kinetics of ATP liposomes in vitro and the short-time stability.We use the pH= 7.4 phosphate buffer isotonic with human plasma as the release medium, which contains 137 mmol/L NaCl,3 mmol/L KC1,8 mmol/L Na2HPO4, and 1 mmol/L KH2PO4. The in vitro release of ATP from the medium were evaluated using the dialysis bag diffusion technique.ATP-loaded liposomes all presented controlled release properties, and nonlinear fits of ATP release from liposomes was modeled. The in vitro release behavior of ATP from liposomes could be described by Weibull kineties model and expressed by the following equations:Lnln (1/(1-Q))= 0.5871nt-1.656.The stability results show that the leakage rate of ATP liposomes is 27.74% at 4℃storage for 15 days, therefore, in order to increase the stability of liposome we can transfer ATP liposome into ATP proliposomes by using freeze-drying method next step.Conclutions:This project developes a liposome with high encapsulation efficiency successfully, and the processing technology is simple, feasible and reproducible; liposomes as carrier of ATP can significantly increase the half-life of ATP in vitro; are conducive to injection or topical administration and facilitate the clinical application.At the same time, this study finds that with the time goes, the leakage rate of ATP liposome increases, the next step we must solve is to further improve the stability of liposomes, so the lyophilized ATP liposomes study is needed. |
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