| OBJECTIVEBased on the pre-prescription study of resveratrol, the study was aim to prepare resveratrol liposome composed of two phospholipids, and establish a systematic quality evaluating system of it in vitro. We concluded the advantages of resveratrol liposome composed of two phospholipids of stability and other properties.To conduct detailed research on its in vitro antitumor activities and provide a reference for the pharmacokinetic studies in the future.Finally to lay new ways and research methods for resveratrol.METHODS1.The pre-formulation study of resveratrol was carried out to establish the HPLC method for resveratrol analysis, and to determine oil-water distribution coefficient and method of encapsulation efficiency.2. After several commonly-used methods of liposome preparation, the thin-film dispersion method was finalized as the preparation method. A single factor and orthogonal design optimization were utilized to optimize the best preparation process with encapsulation efficiency as the indicator.3. Scanning Electronic Microscopy was used to assess the morphology and Malvern ZEA3600nano-granulometer was used to examine the particle size distribution and zeta potential. The physical stability evaluation was done by the iv vitro release rate.4. The growth inhibition of Hepatic carcinoma HepG2and Lung cancer A549cells was determined by MTT assay and the IC50was calculated afterwards. The effect of resveratrol on the cell cycle phase distribution of cancer cells was analyzed using flow cytometry with a propidium iodide method, and the apoptosis morphology was observed by a Hochest33342single staining method.RESULTS1. The results showed that the resveratrol solution appeared a good linear relationship in the range of1～25μg/mL, and the excipients did not interfere with the determination practically. Resveratrol composite phospholipid liposomes and free resveratrol could be well separated by Sephadex filtration method with PH7.4PBS solution as the eluent, the oil-water distribution coefficient was1.50at37℃.2. The thin-film dispersion method can get a high encapsulation efficiency, the ultimum prescription of resveratrol liposomes composed of two phospholipids as follows:the phospholipid concentration was4%, the ratio of HSPC and SPC was1/6, the drug to lipid ratio was1/15, the ratio of cholesterol and phospholipid was1/3. The average encapsulation efficiency was82.47%, no significant difference between different beaches was found.3. Liposomes had uniform particle size, good dispersion under the SEM, the average particle size was89nm and the potential was-43.2mv. The release achieved balance after36h and the cumulative release rate was81.3%, the liposome achieved a sustained release effect and more stable in the plasma.4. The resveratrol liposomes inhibited the proliferation of tumor cells in a concentration-and time-dependent manner, and the resveratrol treatment resulted in a potent effect in S phase in tumor cells.CONCLUSIONS1. HPLC method was accurate, sensitive, easy to operate, improved the efficiency of the detection of resveratrol, the oil-water partition coefficient measurement results showed that resveratrol was a high hydrophobic drug with low polarity which made it suitable for preparing liposomes.2. The thin-film dispersion method was simple and easily carried out in laboratory, SEM results showed that nanosized liposome was uniform and round with smooth surfaces. 3. MTT experiments showed that resveratrol composite liposome presents a slightly better antiproliferative potential than common resveratrol liposome for human lung adenocarcinoma A549cells and human hepatic carcinoma HepG2cells, it inhibited the proliferation of tumor cells in a time and dose dependent manner, and acted as a cycle-specific way. |
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