| As a new drug delivery system, the nanotechnology-based delivery systems demonstrate a broad varietyh of useful properties, such as controlled release or sustained-release, with a higher biocompatibility and with the ability of passive or active targeting. Paclitaxel was a diterpenes compound isolated from the bark of the Pacific yew tree, which can stabilizes microtubules againse depolymerization. In clinical, paclitaxel is usually used for the treatment of breast and ovarian cancer. Poly(lactic-co-glycolic acid)(PLGA) is a copolymer which is synthesized by glycolic acid and lactic acid. The final metabolic products of PLGA in vivo is water and carbon dioxide. PLGA is approved therapeutic devices owing to its biodegradability and biocompatibility. PLGA-COOH are the PLGA polymers that are end-capped with free carboxylic acid, which can be used for further chemical modification. Folic acid is an essential vitamin which can not be synthesized by the body and must be obtained from the food. Folate receptor is a membrane glycoprotein protein. Folic acid enter the cell through the endocytosis pathway after it combing wih the folate receptor. The folate receptor is expressed at a low level in the normal tissue and organ such as the lung, breast and kidney. But it was expressed at a high level in tumor tissue. Use folic acid for the modification of nanotechnology-based delivery systems can offer the system the functiong of active targeting.This research selected PLGA-COOH as the carrier and prepared the paclitaxel-loaded PLGA nanoparticles. Selecte the drug loading, encapsulation efficiency and the size of the nanoparticles as the factor investigated and opimizied the formulation and preparation technology of Paclitaxel-loaded PLGA nanoparticles. First we used the emulsified solvent-evaporation method to prepare the nanoparticles and used the single factor experiment for the optimization of the formulation and preparation technology of paclitaxel PLGA nanoparticles. The optimized formulation and preparation technology was:the average size and the Zeta potential of the nanoparticles was:lmg paclitaxel and50mgPLGA dissolved in2.5mLCH2Cl2, and use30mL of2%PVA as the emulsifier, the organic phase was added in the emulsifier and then sonicated for10min (work for2s and then stop for2s) under ice-bath. After sonication, the emulsion was then poured into deionized water with the same volume under stirring at400r·min-1. Stir for4h to evapor the CH2Cl2.The size of the nanoparticles obtain is about284nm. The dug loading was1.13%±0.09%and the encapsulation efficiency was57.77%±1.16%. Then we tried the interfacial depositon method for the preparation of paclitaxel-loaded PLGA nanoparticles and tried the factorial design/response surface methodology for the optimization of the formulation and preparation technology of Paclitaxel-loaded PLGA nanoparticles. The optimized formulation and preparation technology was:2mg of paclitaxel and60mg of PLGA dissolved in6ml acetone as the organ phase. The organ pahse was added dropwise into30mL1%PVA solution under agitation at800r· min-1, after agitate for10minute, reduce the rate of agitation to400r· min-1and agitate for5h to evaporate the acetone. A transparent solution with blue opalescence was obtained. Filtered through0.45μm membrane filter and the nanospheres suspension was obtained.The average size and the Zeta potential of the nanoparticles was:281±nm,-30.1±0.3mV. Because the interfacial depositon method was convenient and lead to a higher encapsulation efficiency, it was selected for the preparation of paclitaxel-loaded PLGA nanoparticles.Using carbodiimide as the catalyst for the synthesis of folic acid modified chitosan oligosaccharide:the carboxyl of folic acid was activated by1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide and was then connected with the amino group of chitosan oligosaccharide. Used the ultraviolet spectrum and infrared spectrum for the corroboration of the chemical reaction. The standard curve was established between the concentration of folic acid and the respective absorbance at363nm. The standard curve was: A=8.1789C+0.0681, R=0.9999(the unit of the concentration was mg·ml-1). Certain weight of chitosan oligosaccharide that modified by folic acid was dissolved in water and the respective absorbance at363nm was measured for the calculation of the conjugation rate. The reaction conditions was determined as:220mg of folic acid and192mg of EDC· HC1was dissolved in30mL sodium hydroxide solution at the concentration of0.1mol·L-1, and then525mg of chitosan oligosaccharide was added in under agitation. The solution was stirred at room temperature in dark for16h. The solution was brought to pH7.0by drop wise addition of HC1solution at the concentration of0.1mol/L. The solution was then filtrated and the filtrate was dialysised in water for24h and then lyophilized. The conjugation rate of the folic acid modified chitosan oligosaccharide was about24.39%.Use the folic acid modified chitosan oligosaccharide for the modification of PLGA nanopartivcles. First, the carboxyl on the surface of PLGA nanoparticles was activated by EDC, and then connected with the residual amino of folic acid modified chitosan oligosaccharide. The reaction conditions were as follow:30mg folic acid modified chitosan oligosaccharide and about2mg EDC was dissolved in10mL deionized water, and then added into the suspension of nanoparticles under stirring. The reaction was last for3h under room temperature. The size and Zeta potential of the surface modified nanoparticles was321±0.76nm,22.6±0.26mV。 The drug loading was5.1±0.25%and the encapsulation efficiency was41.96±1.96%, The conjugation rate of the folic acid on the modified nanoparticles was about8.87%.The SKOV-3ovarian cancer cell lines was selected for the experiments in vitro. MTT assay was used to examine the inhibitory effect of paclitaxel nanoparticles and paclitaxel on cell. The concentration and reaction time was selected by experiments and the best paclitaxel concentration was0.1μg/ml, the reaction time was48h. The experiments showed that the cell inhibitory rate was highest when the reaction time was48h. And after48h, the cell inhibitory rate began to decline. However, the cell inhibitory rate became higher as the reaction time prolonged. When the reaction time was96h, the cell inhibition rate of modified nanoparticles was close to the cell inhibition rate of paclitaxel. The cell inhibition rate of the modified nanoparticles was higer than the unmoidified nanoparticles at different reaction time. |
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