Preparation And Characterization Of MePEG-PLA Polymeric Micelles And Their Celluar Uptake

As a biocompatible and slowly degradable polymer, amphilphilic block copolymer MePEG-PLA has been widely used in drug delivery systems, especially in drug-controlled release and targeting delivery system. Due to the numerous advantages of polymeric micelles and the anticancer activity of paclitaxel, the MePEG-PLA polymer micelle of paclitaxel is considered to be important theoretically and practically. In this article, we prepared paclitaxel-loaded MePEG-PLA polymer micelles. It can enhance both the solubilization of paclitaxel and the ability to kill tumor cells, thereby decrease the side effects of paclitaxel. What's more, by modifying quantums dots with MePEG-PLA, the oil-soluble quantum dots become water-soluble. So it can be used for cell labelling. The main works were as follows.(1) The structure of the MePEG-PLA was investigated in detail by using FT-IR, 1H-NMR, and GPC. Fluorometry was used to determine the Critical Micelle Concentration (CMC) with pyrene as the fluorescent probe. The results showed that the CMC value of the amphiphilic polymer was 1.5 mg/L. The film formation method and co-solvent evaporation method were used to prepare PTX-loaded MePEG-PLA polymer micelles. The results showed that the size of the micelles was approximately 200 nm and the size distribution was not equality. While the PTX-loaded MePEG-PLA polymer micelles prepared by co-solvent evaporation method displayed small particle size(27±3 nm) and narrow size distribution. Moreover, the morphology of the micelles was investigated by transmission electron microscopy (TEM). The result showed that the particles were spherical in shape without aggregation. The envelope efficiency measured by HPLC was 4.23%. The average size of fluorescent labled MePEG-PLA polymer micelles prepared by co-solvent evaporation method was 40±4 nm and the TEM results showed the particles were spherical in shape with inner core and outer shell .(2) In vitro releasing property of PTX-loaded MePEG-PLA polymer micelles was studied and the drug releasing performance could be described by first-order-model and could be described by the following equation: Q = 126.60-131.67e-0.044t. The inhibitory of the PTX-loaded MePEG-PLA polymeric micelles to the human ovarian cancer cells A2780 was determined with the method of MTT. The results showed that the blank MePEG-PLA was nearly nontoxic but the PTX-loaded MePEG-PLA polymeric micelles prepared by co-solvent evaporation method showed stronger anti-tumor efficiency than free paclitaxel. Because of the larger size, the anti-tumor efficiency of PTX-loaded MePEG-PLA polymeric micelles prepared by film formation method showed little difference with free paclitaxel.(3) For the cellular uptake study, fluorescent marker Nile Red was loaded into polymer micelles. Using fluorescence microscopy, fluorescence spectrophotometry, confocal fluorescence microscopy and total internal reflection fluorescence microscope (TIRFM), the uptake mechanism of polymer micelles by A2780 cell lines was investigated qualitatively and quantitatively. The results suggested that the uptake process is concentration, time and energy-dependent. Moreover, the microphotographs from TIRFM showed the emergence and disappearance of swallowing vesicles. These results suggested that at least endocytosis is involved during the process. Double-labeling confocal microscopy in live cells reavealed the localization of polymeric micelles in several cytoplasmic organelles, including mitochondria /ER and partially lysosomes. Some fluorescent labed micelles were escaped from lysosomes to cytoplasmic. The stability of fluorescent labed micelles was also studied by testing the size distribution and fluorescent intensity. After storing at 4℃for a month, the fluorescence intensity showed no significant change while the size distrubtion changed from single narrow peak to bimodal peak suggesting the aggregates of polymer micelles.(4) MePEG-PLA modified quantum dots were studied. Prepared by improved co-solvent evaporation method, the CdSe / ZnS QDs-loaded polymer micelle showed that the sizes were around 100 nm, the PDI varied from 0.14 to 0.40, the fluorescence intensity were approximately 60 and the zeta potential were around -20 mV. Additionally, the morphology of the QDs-loaded micelle was investigated by TEM. The results showed the micelles were vesicle in shape without aggregation. The emission wavelength of the MePEG-PLA modified quantum dots showed 2 nm red shift compared with free quantum dots. Moreover, In vitro fluorescence stability test showed that the modified quantum dots still had strong fluorescence after storing at 4℃for a week. The fluorescence microscopy showed that the celluar uptake of micellar modified quantum dots were concentration and time dependent. Under high concentration of quantum dots with long period time (8 h), it can cause cell death. Cells were effectively labed by MePEG-PLA modified quantum dots according to confocal fluorescence microscopy. So, MePEG-PLA modified quantum dots is a potential labeling material which can be used for biological fluorescence imaging.