| GP-2was the first semi-synthesized new target compound using the active ingredient of Rabdosia rubescens as lead compound in China, which has a high anti-tumor activity, low toxicity and fine biological tolerance. However, initial pharmacological studies indicate that GP-2functions better for non-gastrointestinal drug delivery than gastrointestinal, and is of relatively poor solubility in water and some other physiologically acceptable solvents. How to enhance its solubility and achieve non-gastrointestinal administration are the main difficulties for its clinical application. The aim of this study is to develop GP-2loaded noisomes to increase the solubility for injection and may improve the clinical efficacy and safety.This study established the HPLC analysis method for GP-2, determined its solubility and apparent distribution coefficient, and provided the methodological basis and theoretical evidence for the prescription design and preparation conditions. The concentrations and entrapment efficiency of GP-2were measured by HPLC. Analytical methods above were proved to meet the needs of vitro sample analysis.GP-2loaded niosomes was prepared by a thin film dispersion-ultrasonic method. The formulation and preparation techniques of GP-2niosomes such as the concentration of lecithin, concentration of tween-80,the ratio of lecithin to drug, the ratio of lecithin to cholesterol, hydration medium, hydration time, hydration temperature, sonication power and sonication time were optimized by signal factors investigation based on particle size, distribution, drug loading and entrapment efficiency. Due to its poor stability at25℃, the formulation factors were optimized again by adding vitamin C as an antioxidant. Finally, the optimized formulation and preparation techniques were as follows:the concentration of lecithin was24g·L-1, the concentration of Tween80and vitamin C were2%and0.2%, the ratio of lecithin to cholesterol was10:1, the ratio of drug to lecithin was1:24, hydration medium was ultra pure water, hydration time was40min, hydration temperature was55℃, sonication power was300W, sonication time was6min. The physicochemical properties of GP-2loaded niosomes were evaluated, the appearance is light blue, the average particle size and distribution were66.16±1.97nm and0.150±0.002, the drug loading and entrapment efficiency were1.95%±0.02%(W/W) and72.6%±0.78%(W/W). Analyzed by differential scanning calorimetry(DSC), the drugs exist in the state of amorphous in the niosomes. Dialysis method was applied into study the vitro release of GP-2niosomes, the cumulative percentages was27.73%in30min, up to92.41%in6h. It followed Higuchi release models. The stability study indicated that GP-2loaded niosomes was suitable kept at4℃.The degradation of GP-2and GP-2loaded niosomes in vitro were studied by means of incubation in homogenized liver tissues. The results showed that GP-2loaded niosomes group had a much slower degradation rate than GP-2control group, and the difference was statistically significant using SPSS19.0software. Models fitting results showed that the degradation of GP-2loaded niosomes in vitro followed the first-order kinetics process.The preliminary safety of GP-2loaded niosomes was evaluated by the abnormal toxicity test, hemolysis test and vascular irritation test. The results indicated that the niosomes had no obvious toxicity, hemolysis and irritation, metting the regulations of the Ⅳ.The effects of the GP-2loaded niosomes on the EC9706and4T1cells pharmacodynamics were investigated by means of MTT, showing that it had obvious inhibition effect on the two cells and the inhibition effect was enhanced with time and dose increment. IC50values of GP-2niosomes towards EC9706cells at24h,48h and72h were10.53μmol·L-1,4.19μmol·L-1and2.63μmol·L-1, respectively,and values towards4T1cells were9.89μmol·L-1,5.23μmol·L-1and3.23μmol·L-1, respectively. |
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