| Targeted chemotherapy of cancer is a hotspot in recent years. Targeted drug delivery means that the specific drugs can reach organs, tissues or cells selectively for treatment in the target area. The concentration and the therapeutic index of specific drugs in target site would increase through selective administration. And the toxicity of non-target site would decrease.Targeted therapy of drugs consist of active targeting and passive targeting.In recent years.The targeted drug delivery system of polymer micelles prepared by the amphiphilic block copolymers received extensive attention,and become the most important direction in the field of paclitaxel delivery system.The amphiphilic block copolymers can self-assemble into a unique "core - shell" structure of micelles in aqueous solutiondue to the different solubility of its hydrophilic and hydrophobic chains. The hydrophobic segments aggregate into nuclear to increase the solubility of liposoluble drugs; and the ydrophilic segments assemble together into a shell for the stability and protection of the micelles.Compared with other delivery systems, polymer micelle drug delivery system has many advantages:such as small size and hydrophilic shell of polymer micelles makes the drug-loaded micelles can avoid the recognition of human reticuloendothelial system (RES) for prolonged metabolic period in human blood circulation. The polymer micelles can accumulated in tumor tissue because of the high permeability and high retention in its vascular endothelial (EPR effect), which is beneficial to enhance the effect of specific drugs. Micelles as targeted drug carrier materials is a new drug delivery systems which can be transported to the diseased region selectively to improve the bioavailability of drugs and to reduce the side effects.Although the EPR effect of the nanoparticles with PEG, they can not deliver drugs to any specific target cells specifically.In order to further improve the drug concentration in targeted tumor region, People started to pay attention to ligand, protein and other special materials conjugated with the nanoparticles to achieve the purpose of active targeting in recent years.Folate receptor expression is highly conserved in normal tissues, which is only expressed on certain epithelial cells but highly expressed in most of the tumor cells from epithelial tissue.Compared with other receptor-mediated drug delivery system, the unique advantages of folate receptor system are as follows:①Without synthesis and purification of ligand protein, so the micelles are non-immunogenic compared with other protein ligand;②Relatively simple structure and easy synthesis;③Smaller, better pharmacokinetic characteristics, repeatable administration of drugs;④Only highly expressed in tumor cells, so it has a good tumor-specific effect;⑤The high expression of folate receptor in human tumors. Based on the above characteristics, the Folate-mediated active targeting therapy has become a research focus.But there are more active targeting drugs using liposomes as carriers so far. Polymer nanoparticles mediated as active targeting carriers is not very common, especially the polymer micelles. The biggest problem of liposomes is fragile to oxidation, as well as poor stability, medication leakage and low drug loading capacity.Although both the polymer nanoparticles and micelles are belong to colloidal systems,the former has good stability and high drug loading capacity and the surface can be easily modified for specific requirementscomparison with liposomes.Paclitaxel (PTX) as a natural anti-cancer active substance is isolated from the Taxus species and then made into liquid preparations by the application of advanced technology Which has a good therapeutic effect for a variety of malignant tumors. Because of its low water solubility, the paclitaxel is a mixture substances dissolved by polyoxyethylene castor oil and ethanol for clinical injection, which have a strong adverse reaction to the body, so its clinical application is greatly limited.Lung cancer is one of the most common malignant tumor, as a serious threat to human health and life. In chemotherapy program of lung cancer, the Taxol is a drug of first choice.To reduce the adverse reaction, and increase drug concentration in target site, people actively use related biological materials for its modification.In this study amphiphilic biodegradable polymer MPEG-b-P (LA-co-MCC) congugated with paclitaxel (PTX) to produce polymer prodrugs, then assemble into micelles with a polymer carrier MPEG-b-P (LA-co-DHP/FA) with folic acid ligand. Then the micelles as spherical particles is produced composed of hydrophilic shell and lipophilic core. Taxol as a liposoluble drug is encapsulated in the nucleus, and its solubility in water will increase as micelles with prolonged circulation in vivo as well as increased bioavailability and reduced toxicity and a targeted effect to tumor cells with overexpression of folate ligands.To investigate the tumor inhibition effect, Lewis lung cancer mice models were established in this experiment followed by associated detection to provide the theoretical basis for clinical application.The main results of this study are as follows:1. Conjugated the amphiphilic biodegradable polymer MPEG-b-P (LA-co-MCC) with paclitaxel (PTX) to produce prodrug of polymer micelle paclitaxel (PTX Micelle) MPEG-b-P (LA-co-MCC/PTX). Another amphiphilic biodegradable polymer MPEG-b-P (LA-co-DHP) conjugated with folic acid to produce the polymer micelleswith folic acid (FA-micelles) MPEG-b-P (LA-co-DHP/FA); so that the two mixed-assemble into micelles. Thus the folic acid-paclitaxel micelles (FA-PTX micelles) is prepared.(1) The characterization of Polymer-paclitaxel Conjugate Micelles (PTX-micelles and FA-PTX micelles collectively called) are as follows: These two micelles are spherical with distribution in uniform size and good dispersibility observed by Electron microscopy, compared with non-load particlesobtained with the same preparation conditions, Drugs has little effec on size and morphology of drug-loaded nanoparticles. Observation of the characteristics of size distribution in solution by dynamic light scattering of polymer conjugated paclitaxel nanoparticles show that "PTX micelle" and FA-PTX micelles are on a basis of a single symmetric distribution. The average size of the particles were 43.6nm, 58.3nm. which can avoid the monitor of reticuloendothelial system to achive the passive and active targeting effect; (2) The drug release behavior of the micelles in PBS detected by the HPLC show that PTX micelle release fast within 24 hours and the cumulative release rate reached to 40% followed by a continuous slow release. The cumulative release in 6 days reached to 80%.Paclitaxel has a low CMC with more stable molecular chains for a longer release time.2. In vitro (1) Detection of proliferation effect of chemical drugs on Lewis lung cancer cells by MTT method,most of the drugs got statistically significant inhibition on Lewis lung cancer cell lines and the inhibition is concentration and time-dependent. (2) Observation of the effect of chemical drugs on Lewis lung cancer cells under the microscope showed the adherent lost, the pseudopodia disappeared with rounded cells and slow growth with split-phase reduction or disappearance and the accumulation of rough particles within the cytoplasm; Integrity destruction, disintegration and rupture, and the cells crack into pieces. The same concentration (including PTX) with the same time, PTX, PTX micelles, FA-PTX micelles observed abnormal cell morphology became more and more clear with gradually increased degree of multi-cellular debris.(3) Flow cytometry detection of the chemical drugs on Lewis lung cancer cells show that the effect of PTX, PTX micelles, FA-PTX Micellar with 1μg/ml concentration of PTX on Lewis lung cancer cells after 72 hours, the apoptosis rate increased gradually with significantly difference compared with the negative control group (P﹤0.05), as well as the PTX micelles, FA-PTX with significant differences between the micelle (P﹤0.05). Show that the conjugated polymer-paclitaxel micelles can induce apoptosis and to achieve tumor inhibition.3. In vivo C57BL / 6 mice were inoculated with Lewis lung cancer cells for preparation of tumor-bearing mice models, with FA-PTX micelles, PTX micelles, PTX treatment respectively, with normal saline as the control group to detecte the efficacy; 7 and 14 days after drug administration, the relevant indicators detected as follows; (1) The general state of mice after drug administration: when injection in the mice tail vein, they showed irritability and screaming in PTX group; followed by side effects such as malaise, dark coat and significantly reduced diet 2 days after administration. All the other groups showed no significant adverse reactions. (2) 7 days after the dministration, three drugs of PTX, PTX micelles and FA-PTX micelles significantly inhibited tumor, compared with the control group (P<0.01) by mice weighing. There were statistically significant difference between FA-PTX micelle group and the other two drugs groups (PTX,PTX micelles) (P<0.05). 14 days after the administration, There were statistically significant difference between FA-PTX micelle group and the other two drugs groups (PTX,PTX micelles) and the control group (P<0.05). (3) TUNEL method to detect apoptosis. The results showed that 7 days, 14 days after the administration, the comparison of gray value of each drug group and control group had statistically differences (P<0.01). The grey values followed the order of Control > Taxol > PTX micelles > FA-PTX micelles. (4) The apoptosis rates of the tumor cells were evaluated by flow cytometry using Annexin V?FITC and PI double staining. All the Annexin V positive cells were considered as apoptotic ones. As shown in Figure 3 and Table 3, the apoptosis rates of the control group were significantly lower than those of the drug groups on the 7th and 14th day after drug administration (P<0.05), and there were also significant differences in apoptosis rate between the FA-PTX micelles group and the other two drug groups (P<0.05). Moreover, on the 7th day after drug administration, the percentage of apoptosis were in the order of Control < Taxol < PTX micelles < FA-PTX micelles, while the order was Control < Taxol |
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