| Paclitaxel is a potent antitumor drug that is widely used in clinical,but its low solubility and high toxicity limit its application.Currently,clinical used polyoxyethylene castor oil and anhydrous ethanol mixed solution as paclitaxel injection solution are highly allergenic and toxic.Paclitaxel liposomes can effectively improve its water solubility,reduce cytotoxicity and solvent sensitization.However,due to the structure complexity and forms diversity,and,the preparation process is manifold.Therefore,to establish a quality analysis method with good stability,good repeatability has become a problem.To establish a quality analysis method suitable for paclitaxel liposomes,in this paper,paclitaxel liposome was prepared by conventional method,a method for the determination of important quality analysis indexes such as content and entrapment efficiency was explored by high performance liquid chromatography(HPLC).A method for the determination of the content and entrapment efficiency of high performance liquid chromatography was established.The reliability and stability of paclitaxel liposome content and entrapment efficiency were determined.Novel paclitaxel-liposome modified by mPEG-curcumin was prepared and the content,entrapment efficiency,in vitro release and in vivo tissue distribution were determined by the established method.The results showed that the method were stable and reproducible for the modified liposome.The established method in this study can be used for the quality analysis of mPEG-curcumin-modified paclitaxel liposomes(mPEG-cur/pac/lip).The preparation method of paclitaxel liposomes was carried out by membrane hydration,and the hydration solvent was 5% glucose solution containing 1% Tween-80.The method for the determination of paclitaxel liposomes was explored,The chromatographic conditions were as follows: methanol/water=78/22,flow rate: 1.0 m L·min-1,injection volume: 10 ?L,detection wavelength: 228 nm,column temperature: 35 °C.The concentration of paclitaxel in the range of 1-160 ?g·m L-1 and 20-800 ng·m L-1 has a good linear relationship,R2?0.999.The average recoveries were 101.1%,the precision and reproducibility were good,the RSD values were less than 2,the quantitation limit QL was 29.86 ng·mL-1,and the detection limit MDL was 9.86 ng·m L-1.Filtration,dialysis diffusion and protamine were used to evaluate the entrapment efficiency of paclitaxel liposome and protamine method was chosen in this study.The protamine methods were as follows: protamine concentration: 10 mg·mL-1,protamine and liposome volume ratio:2.5:10.The entrapment efficiency of paclitaxel liposomes measured by this method was 97% or more.To verify the applicability of this method,membrane hydration method was used to prepare a novel paclitaxel liposome(mPEG-cur/pac/lip)with long circulation and anti-multidrug resistance.The prescription was as follows: paclitaxel: soybean phosphatidylcholine: methoxy polyethylene glycol-phosphatidylethanolamine: cholesterol: methoxy polyethylene glycol-curcumin=2:24:2:12:3.Mannitol(8%)was selected as the freeze-dried protective agent.The content and entrapment efficiency of the prepared liposome were evaluated using the analysis method established in this study.The results showed that the separation of paclitaxel in mPEG-cur/pac/lip was good,and the retention time of other excipients did not interfere with the determination of the content.The content of paclitaxel was 969.66 ?g·mL-1 and the drug loading content was 4.65%.The entrapment efficiency of mPEG-cur/pac/lip before and after freeze-drying was determined by the protamine method.The entrapment efficiency of mPEG-cur/pac/lip decreased slightly after freeze-drying,but still more than 90%.The method was also applied to the determination of drug release in mPEG-cur/pac/lip in vitro.The results showed that the method was effective for the evaluation of release profile of mPEG-cur/pac/lip no interference was observed for paclitaxel.In vitro release results showed that mPEG-cur/pac/lip had a sustained release effect.The particle size was detected by laser particle size analyzer with an average size of 840 nm and the distribution of liposomes was uniform.Zeta potential is less than-30 mV,the low potential(less than-30 mV or higher than 30 mV)suggested that the mPEG-cur/pac/lip were stable in the aqueous solution.The sphere-like morphology was observed by transmission electron microscopy(TEM),and the structure of the bimolecular layer can be observed in some particles.The mPEG-cur/pac/lip and conventional paclitaxel were injected intravenously into healthy Kunming mice.The results showed that the toxicity and sensitization of liposomes were significantly decreased.The established quality analysis method can also be used for the determination of mouse tissue samples.The results showed that the impurities in the tissue samples were well separated from the peak of paclitaxel,and the retention time of paclitaxel was not disturbed in the determination of tissue samples such as heart,liver,spleen,lung and kidney.A certain sustained release was observed in the mPEG-cur/pac/lip group.MPEG-cur/pac/lip was targeted to spleen and lung compared to common paclitaxel injection.In this paper,a set of paclitaxel liposome quality analysis method based on high performance liquid chromatography(HPLC)was developed.And the method was applied to the quality analysis of the novel liposome mPEG-cur/pac/lip with anti-multidrug resistance.The results showed that this method can be used for the determination of the content,entrapment efficiency,release and tissue distribution of mPEG-cur/pac/lip.The method has good reproducibility and high reliability,indicating that the method can be used for the quality analysis of mPEG-cur/pac/lip.The results showed that the prepared mPEG-cur/pac/lip has high entrapment efficiency,good stability and spleen,lung targeting.The method established for the determination of liposomes and the preparation of mPEG-cur/pac/lip have laid a good foundation for the further development and utilization of paclitaxel. |
PDF Full Text Request