| Multidrug resistance (MDR) is a major obstacle in the chemotherapy of malignant cancer. Tumor cells will be resistant to different kinds of cytotoxic agents after a period of treatment. However, the mechanisms of MDR have not reached an agreement. P-glycoprotein (P-gp)-mediated drug efflux is regarded as a popular opinion. Therefore, a large amount of researchers all over the world are trying to co-encapsulate P-gp inhibitors (also known as a kind of chemosensitizer) such as verapamil, PSC833et al with cytotoxic agents in nano drug delivery systems, which can not only antagonize or cancel the effect caused by active efflux of MDR cancer cells but also decrease the side effect of these antitumor drugs. As we know, the first, second, third generations of P-gp inhibitors are reported to be non-selective. When the P-gp in tumor cells are inhibited, those in normal tissues are blocked, too, which will lead to an accumulation of cytotoxic agents in normal cells.Folic acid is proved to possess high affinity with the plasma membranes of many kinds malignant tumor cells. Vincristine will induce the patients to be cross-resistant to doxorubicin, taxol, colchicine during the chemotherapy. Patients will also be cross-resistant to vincristine when treated with doxorubicin, taxol, et al, correspondingly. Therefore, we aim to construct an active targeting nano drug delivery system encapsulated with vincristine without P-gp inhibitors-folic acid-PEG335o-liposome/vincristine (FA-PEG-LS/VCR), in order to enhance the target-non-target tissue ratio and the Therapeutic Efficacy and improve the multidrug resistance to vincristine.Chapter one consisted of two parts. The first part focused on the synthesis and characterization of the liposomal functional membrane material, folic acid-PEG3350-DSPE (FA-PEG-DSPE). In the second part, we constructed and characterized the folic acid modified liposomal drug delivery system. The dehydration condensation reaction via DCC was conducted to form an amide linkage which linked the folic acid to one terminal of the bifunctional PEG. Another terminal was linked with SUC, which introduced a carboxyl. Another dehydration condensation reaction was conducted between the products and the amide linkages on DSPE, which led to the final products, FA-PEG-DSPE. The structural characterization of the above mentioned products was confirmed by means of HPLC,!H-NMR and other technical methods. Folic acid-conjugated liposomes loaded with VCR (FA-PEG-LS/VCR), FAM (FA-PEG-LS/FAM), DiR (FA-PEG-LS/DiR) were prepared via thin films hydration method, respectively. They were also characterized of their vesicle sizes and size distributions, physical appearances, encapsulation efficiency (EE), drug-loading efficiency (DL), in vitro leakage rate and other attributes. The results showed that FA-PEG3350-DSPE was successfully synthesized with the purity of95%plus. The prepared liposomes,including FA-PEG-LS/VCR, PEG-LS/VCR, FA-PEG-LS/FAM, PEG-LS/FAM, FA-PEG-LS/DiR, PEG-LS/DiR, have narrowly distributed vesicle sizes of70-90run, which were suitable for the research work. The VCR-loaded liposomes were spherical particles as characterized by TEM and AFM. EE and DL of VCR loaded liposomes were above95%and5%, respectively. The leakage rates of all kinds of liposomes are no more than2%after4-72hours at4℃. Folic acid modification had no obvious influence on the properties of liposomes. The above mentioned attributes changed little after concentration. This chapter is the basis for the following work.Chapter two consisted of three parts. The first part focused on the targeting ability to human nasopharyngeal epidermoid carcinoma cell KBv200of liposomes modified by folic acid in vivo and in vitro. The second part focused on the therapeutic efficacy to KBv200of FA-PEG-LS/VCR drug delivery system in vivo and in vitro. In the last part, we verified and discussed the MDR mechanism of this kind of cell. Firstly, the in vitro targeting ability of the PEGylated liposomes modified by folic acid to KBv200MDR tumor cells was assessed by cellular uptake test (flow cytometry assay). Nude mice models of MDR tumors were constructed by s.c. inoculation near axillary of KBv200cells and based on these models, the in vivo targeting ability of FA-PEG-LS to the solid tumors were investigated. Secondly, the in vitro growth inhibition of FA-PEG-LS/VCR on KBv200cells was investigated by MTT assay. The pharmacodynamic study of inhibition effect of folic acid-conjugated VCR-loaded liposomes on tumors was carried on the above mentioned nude mice models. HE and TUNEL staining methods were adopted to assess the pro-apoptotic efficacy of FA-PEG-LS/VCR, quantitatively. Thirdly, the first generation P-gp inhibitor, verapamil was used to inhibit the MDR cell KBv200and the sensitive cell KB to investigate the growth inhibition ratio before and after P-gp inhibition. Results indicated that FAM or DiR loaded liposomes modified by folic acid were uptaken by KBv200cells most in vitro or in vivo compared with other drug delivery systems, respectively, showing its good targeting ability. The cellular uptake assay showed that the targeting efficacy of FA-PEG-LS was2.96-fold as PEG-LS. Meanwhile, the in vivo imaging test showed that the targeting efficacy of FA-PEG-LS was1.68-fold as PEG-LS. FA-PEG-LS/VCR inhibited the growth of tumors on the KBv200nude mice models effectively and the tumor-bearing mice lived better than other testing groups. Results revealed that the tumor inhibitory rates (TIR) are60.11%in volume and59.98%in weight, respectively. When the dosage was trebled, the TIR in volume and weight became74.75%and69.55%, respectively. Tumor section results indicated that FA-PEG-LS/VCR induced apoptosis more effectively than PEG-LS/VCR, free VCR and physiological saline. The apoptotic index (AI) of the above mentioned treatments were24.1±3.1%,14.4±3.2%,11.8±4.4%,5.6±2.2%, respectively. The depolarization ability of a drug delivery system is characterized by the mitochondrial membrane-potential, which was also an index to evaluate the apoptosis-inducing ability. After P-gp inhibited by verapamil, the cellular growth inhibiting ability of FA-PEG-LS/VCR, PEG-LS/VCR and free VCR were enhanced significantly, whose IC50values changed from26.92nmol/L,398.11nmol/L,1584.89nmol/L to8.66nmol/L,123.03nmol/L,288.40nmol/L, respectively. The largest increase amplitude reached5.5-fold. Meanwhile, the above mentioned three treatments did not show great difference in the cellular growth inhibition test of KB, with no significant change in IC50values, which indicated that P-gp played a vital role in the acceptance of MDR of the cell. It was also revealed that the folic acid-conjugated PEGylated liposomes loaded with vincristine were able to improve the therapeutic effect of MDR tumors.In summary, in this study, we successfully constructed a targeting liposomal drug delivery system loaded by vincristine modified by folic acid. It demonstrated a good targeting ability and tumor growth inhibiting efficacy to MDR solid tumors via intravenous injection administration. FA-PEG-LS/VCR proved a promising active targeting drug delivery system for the treatment of MDR tumors. |
