| Resveratrol(RES) has excellent performance of anti-cancer or tumor, and it can be used as health food and the development of new drugs, especially anti-cancer drugs. RES has been hailed as the new green anti-cancer drugs following the taxol. But resveratrol is a fat-soluble drugs, with poor water solubility and poor oral absorption, and there is no targeting. Target site of treatment is almost impossible to achieve an effective concentration, with unstable metabolism in vivo, it is very limited in clinical application. In this paper, the development of the RES liposome and liposome freeze-dried powder was researched, so as to achieve half-life extension of RES, to enhance anti-tumor effects of RES, to improve the therapeutic index of RES, and to reduce drug side effects, with a purpose to enhance clinical application of RES.In the study, HPLC determination was developed for RES in vitro, we got the standard curve of the concentration of methanol solution of RES: A=164.9C-14.34(R2=0.999); and it was validated by characterization of linearity, precision as well as accuracy. pH 7.4 PBS was used as the dialysis medium to separate liposomes and free drug. And methanol solution was applied to destroy the structure of liposomes. The RES in methanol solution of liposomes was determined by HPLC and the encapsulation efficiency(EE) was calculated. Through inspection the dialysis of blank liposomes and free drug, got the proof of the separation of liposomes and free drugs using dialysis is a reliable method.This study developed a reverse-phase evaporation method for encapsulating RES into liposomes. The effect of different formulation factors on the encapsulation efficiency(EE) and the size of RES liposome was investigated. These factors included the amount of phospholipids, cholesterol phospholipid ratio, phospholipid drug ratio, organic phase and water phase volume ratio. By the orthogonal design, the EE(95%) was obtained when using RES 10mg, SPC 400mg, Chol 40mg, Anhydrous ether 20mL and pH7.4 PBS lOmL. Through the adjustment of process conditions on the reverse phase evaporation method, such as the rate of volatile ether the steep of pear-shaped bottle or the power and time of cell ultrasonic crushing device, we achieved a process optimization. Freeze-drying with 2.5% lactose and 2.5% trehalose as cryoprotectant was carried out to achieve long-term stability.First of all, the establishment of a method using ultraviolet spectrophotometry at different pH (5.5,6.5,7.4) of phosphoric buffer solution to determine the concentration of RES. The drug release studies were performed in vitro simulating the desired application conditions, such as pH7.4, pH6.5 and pH5.5. As a result, the release of RES from the liposomes is not pH-dependent, the speed of release in the three pH conditions was close. Compare the release of RES liposome with the release of free RES, we can see that RES liposome has obvious Sustained-release effect. Free RES solution, RES liposome, and RES-PEG liposome were administrated intravenously via the rat tail vein. HPLC system was employed, using genitein as the internal standard, to detect the concentrations of drugs in plasma. DAS2.0 program, a standard software for pharmacokinetic analysis was introduced to calculate the pharmacokinetic parameters. The pharmacokinetics results showed that liposome group plasma half-life is significantly increased under the same dosage, as compared to the solution of free drug, with AUC values 4 times higher than the free drug group, indicating the clearance rate of the free drug is decreased by the liposomes, also suggesting the longer blood exposure time. RES has acute toxicity, however when RES are encapsulated into liposome, the acute toxicity maybe be favorably attenuated. |
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