The Studies On Hyaluronic Acid Coated Paclitaxel Loaded Nanostructured Lipid Carriers

Tumor was a serious threat to human health and so far, it was the second leading cause of death among devastating human diseases. Therefore, the prevention and treatment of tumor was an urgent task, currently, chemotherapy was was one of the most effective treatments of tumor.Paclitaxel (PTX) was a compound extracted from the bark of species yew, Taxus baccata, and it is one of the first choices of anti-cancer drugs. Paclitaxel promoted microtubule polymerization and inhibited microtubule depolymerization during mitosis of cells, therefore, the mitosis was inhibited and cells remained in G2and M phase, and finally, the tumor growth was inhibited. Paclitaxel was hydrophic drug, which limited its clinical application. Selecting appropriate carriers, increasing the solubility of paclitaxel and reducing the toxicity of drug delivery system were the primary problems to be solved. The lack of active targeting to tumor caused damage to normal cells, resulting adverse reactions. Increasing the solubility of paclitaxel and designing active tumor target delivery system were the two main issues on paclitaxel utilization. Based on the two aspects, we built the drug delvery system-hyaluronic acid modification paclitaxel loaded nanostructured lipid carriers, which could increase the solubility of paclitaxel and realize active tumor targeting. Firstly, nanostructured lipid carrier (NLC) was chosed to load paclitaxel, which could increase the solubility of paclitaxel, avoid the use of solubilizing agent, and reduce the toxicity of drug delivery system. Secondly, hyaluronic acid, selected as the active targeting agent, was modified to the surface of paclitaxel loaded NLC via electrostatic attraction, achieving the purpose of active tumor target. This drug delivery system showed some advantages:1) the carriers were composed of lipid materials, therefore it was biocompatible and biodegradable;2) nanostructured lipid carriers were with higher drug loading, small particle size and passive tumor targeting;3) hyaluronic acid was the extracellular matrix components in human cells, therefore, it was biocompatible and biodegradable;4) hyaluronic acid targeted to CD44which was over-expressed on the surface of tumor, achieving active tumor target. The main contents in our study were as follows:1. The measurement of paclitaxel content and entrapment efficiency of NLCHPLC was chosed for the determination of paclitaxel content in vitro. A method which was established by our laboratory, solution-ultracentrifugation, was chosed to separate paclitaxel from paclitaxel loaded NLC. Then, HPLC was used for the determination of entrapment efficiency of NLC.2. The preparation of paclitaxel loaded nanostructured lipid carriers (PTX-NLC)PTX-NLC was prepared by melt emulsification. The freshly prepare PTX-NLC was lyophilized to improve the stability of PTX-NLC. Transmission electron microscopy was used to observe the particle morphology of the PTX-NLC, and Zetasizer3000was used to study the particle size and zeta potential. The results showed that PTX-NLC was approximately spherical, the particle size and zeta potential of the PTX-NLC were102.63±7.36nm and70.57±2.20mV, respectively, and the EE%and DL%were (91.99±0.71)%and (4.18±0.03)%, respectively. The particle size of freeze-dried PTX-NLC was140.9±7.51nm and zeta potential was68.83±1.81mV. Therefore, the lyophilized PTX-NLC was stable, the physical and chemical properties had no change comparing with the fresh preparations.3. The preparation of hyaluronic acid coated paclitaxel loaded nanostructured lipid carriers (HA-NLC)The hyaluronic acid coated paclitaxel loaded nanostructured lipid carriers (HA-NLC) was prepared via electrostatic attraction between anionic hyaluronic acid and cationic PTX-NLC. Two different molecular weight hyaluronic acid-100,0000and30,0000were selected respectively. The results showed that hyaluronic acid with molecular weight30,0000was the optimal one. The particle size and zeta potential of HA-NLC were173.83±2.82nm and-40.25±0.36mV, respectively. The lyophilized HA-NLC was stable, the physical and chemical properties had little change comparing with the fresh preparations..4. The in vitro study of HA-NLCThe release of PTX from Taxol(?), PTX-NLC and HA-NLC were measured at37 ℃under sink conditions, respectively.. The results showed that the drug release from PTX-NLC and HA-NLC was slower than from Taxol(?) and both followed the Bioexponential, while Taxol(?) followed the Weibull equation. HCT116, CT26and B16cell lines were selected for in vitro cytotoxicity study. First, FITC-labeled CD44antibody was used to determine quantity of CD44on cell surface, then the vitro cytotoxicity of Taxol(?), PTX-NLC and HA-NLC were investigated. The results showed that CD44was over expressed in the three cell lines. Compared with Taxol(?), PTX-NLC revealed more cytotoxicity in three cell lines, HA-PTX revealed the most cytotoxicity, indicating that HA-PTX could effectively target to tumors in vitro. The blank NLC revealed some degree cytotoxicity, which may contributed to the cationic surfactants CTAB, while blank HA-NLC showed no cytotoxicity, which indicated that HA was successfully coated on the surface of NLC and reduced the cytotoxicity of CTAB.5. The pharmacodynamics and pharmacokinetic study in vivoB16tumor-bearing mice were used to investigate the pharmacodynamics and pharmacokinetic study in vivo. The pharmacodynamics results showed that compared with the Taxol(?) group, HA-NLC had better antitumor effect in vivo. The phannacokinetic results showed that HA-NLC could prolong drug circulation time in blood, and increase the accumulation drug concentration in tumor, indicating that HA-PTX could effectively target to tumors in vivo.In summary, PTX-NLC was successfully prepared, which increased the loading of PTX, and PTX-NLC was biodegradability and biocompatible, which reduced the toxicity of drug delivery system. HA-NLC which was successfully prepared via electrostatic attraction, could actively target to tumors, realizing the efficiency, low toxicity and active tumor target. Therefore, HA-NLC was a promising drug delivery system.