Preparation And Evaluation Of Hydroxycamptothecin Loaded Mixed Micelles

Hydroxycamptothecin (HCPT), a plant alkaloid extracted from Camptotheca acuminate demonstrated strong antitumor activity against lung, ovarian, breast, pancreas, and stomach cancers by targeting intracellular topoisomerase I, a nuclear enzyme that reduces the torsional stress of supercoiled DNA. However, HCPT'is insoluble in water and exists in two forms depending on the pH value:the active lactone forms at pH below 5 and the inactive open ring-carboxylated HCPT-Na+form, which present at neutral pH values. Although the lactone form of HCPT is crucial for its anticancer activity, at physiological pH values most HCPT molecules exist in the inactive carboxylate form. Thus, sparing solubility and labile lactone ring hinder the clinical application of HCPT.For these reasons,various formulation strategies, such as prodrug, solid lipid nanoparticles, micelles, microparticles, liposomes and emulsion, have been designed to increase the solubility of HCPT, stabilize the lactone ring, and reduce the systemic toxicity of this drug.The purpose of this project is to prepare micellar drug delivery system which is stable in circulatory system in the body and resistant to dilute. The micelles were made with phospholipids (PC) and D-a-vitamin E polyethylene glycol 1000 succinate (TPGS) and insoluble anti-cancer drug, HCPT as a model drug. Two methods were used to prepare HCPT-loaded micelles. For one method, hydroxycamptothecin phospholipid complex was prepared firstly and then the mixed micelles were made by mixing the HCPT phospholipid complex and TPGS. For second method, the mixed micelles were made by mixing HCPT, phospholipid and TPGS directly. The physicochemical properties such as drug loading, encapsulation efficiency and in vitro release behavior of micelles were determined for a comprehensive evaluation of two methods. The optimal formulation was obtained and the in vitro release and cytotoxicity test against HeLa cancer cell were also determined in vitro. These strategies provided useful ways to build targeted micelles.The subject specific research process and results are as follows:1,The assay to measure the drug loading content was established. Preparate of standard curve,and take the drug loading coefficient and entrapping ratio as main criterions.2. A novel method to prepare HCPT loaded mixed micelles was established. For this method, hydroxycamptothecin phospholipid complex was prepared firstly and then the mixed micelles were made by mixing the HCPT phospholipid complex and TPGS. The drug loading and entrapment efficiency were taken as criterions to evaluate the effects of the amount of TPGS, the amount of phospholipids, hydration, the hydration volume of the medium, the hydration temperature of the medium, pH and mixing time. The uniform experimental design was applied to optimize the formulation based on the drug loading and entrapment efficiency of HCPT loaded micelles. The optimal formulation is as follows:the ratio of hydroxycamptothecin:phospholipid= 5mM:5mM (mass ratio= 9mg:19mg); the amount of TPGS is 15mM (quality 113.5mg), hydration volume of 5ml, hydration medium for the optimal pH of 4. the drug loading of optimal formulation was 2.92%; encapsulation efficiency was 44.3%.3,Another method to prepare the HCPT loaded mixed micelles was established. For this method, the mixed micelles were made by mixing HCPT, phospholipid and TPGS directly. The physicochemical properties such as drug loading, encapsulation efficiency and in vitro release behavior of micelles were determined. The drug loading and entrapment efficiency were taken as criterions to evaluate the effects of the amount of TPGS, the amount of phospholipids, hydration, the hydration volume of the medium, the hydration temperature of the medium, pH and mixing time. The uniform experimental design was applied to optimize the formulation based on the drug loading and entrapment efficiency of HCPT loaded micelles. The optimal formulation is as follows:The amount of HCPT was 11mg, the amount of PC was 11.4mg, TPGS was 15mM (quality 113.5mg), hydration volume was 10ml, hydration medium pH was 4. The drug loading of optimal formulation was 1.28%; encapsulation efficiency was 25.6%.4,The effects of HCPT solution and HCPT loaded mixed micelles on the growth of hepatoma carcinoma cell Hela cells in vitro were investigated using MTT colorimetric method to evaluate the antineoplasmic activity in vitro.