Preparation And Anti - Tumor Effect Of Methotrexate Liposomes

Liposomes have several advantages of good biocompatibility,biodegradability and non-toxicity,which are readily to be phagocytized by mononuclear phagocyte system in the blood. Tumor-associated macrophages(TAMs) are major constituents within tumor microenviroment,which origin from blood mononuclear phagocyte system and could promote tumor progression and metastasis. It’s reported that methotrexate(MTX) showed specific toxicity to mononuclear phagocytes. We prepared conventional MTX-loaded liposomes and PEG-modified MTX-loaded stealth liposomes,and investigated the differences of cellular toxicity on macrophages, and that of pharmacokinetics following i.v. injection for both liposomes. This study aims to confirm that whether MTX-loaded liposomes have anti-tumor activity by preferentially damaging TAMs or not. In the contrary,it will also give us some clues about the feasibility of TAMs as an adjuvant therapeutic target in cancer. The thesis consists of four chapters, and is shown as follows.1) Preparation and characterization of conventional MTX liposomesConventional MTX liposomes were prepared by thin film dispersion method. G-50 mini-column spin method was used to purify conventional MTX liposomes and the recoveries of liposomes was more than 90%. The size and morphology of liposomes were measured by dynamic light scattering (DLS) and scanning electron microscope (SEM),and the results showed that the size peak of these liposomes were around 1 μm and the shape was spherical. The entrapment efficiency of MTX liposomes were determimated by high-performance liquid chromatography(HPLC),which was about 4%.2) The cytotoxic effects of conventional MTX liposomesL929 supernatent and M-CSF were used to induce BDMC from bone marrow cell progenitors and the purity of BMDM were identified by acid phosphatase enzyme staining,and the results showed that the morphology of BMDM induced by M-CSF were more uniform than that by L929 supernatent and had higer cell viability. High-content analysis was used to evaluate the uptake activity of macrophages and MTS method was used to evaluate the cytotoxity of MTX liposomes,and the results showed that both BMDM and RAW 264.7 can internalized the liposomes and this activity was time- and dose-dependent.In addition,the phagocytosis ability of BMDM was significantly higher than RAW 264.7. MTX liposomes were toxic to both cell lines and had significantly higher toxicity than the same dose of free MTX.3) In vivo imaging of conventional and PEG-modified long circulating liposomesCT 26 cells and 3-MCA were used to prepare tumor models in BALB/C and C57BL/6 mice respectively and the rate of tumor formation was above 90%. DiR-Labeled conventional liposomes and PEG-modified long-circulating liposomes were used to investigate the pharmacokinetics of liposomes,and the results showed that both liposomes were accumulated in liver,spleen and tumor.4) Preparation of long-circulating MTX liposomes and anti-tumor activity in MG-63The MTX-LIP were prepared using film dispersion method,and the PEG2000-MTX-LIP were added using the post-insertion method. The ultracentrifugation method and mini-column spin method with Sephadex G-10 or G-50 as packing were employed to separate the free drug from the MTX-loaded liposomes. Their recovery,size,morphology,encapsulation efficiency and drug-to-lipid ratio were evaluated. The cytotoxity of PEG2000-MTX-LIP purified with ultracentrifugation method and spin column G-50 method under three dose levels were determined by MTS. The results showed that spin column G-50 was suited for purifying the PEG2ooo-MTX-LIP,and the liposomes were about 200 nm in size,with a narrow particle size distribution and spherical or ellipsoidal under TEM. The PEG2000-MTX-LIP prepared in this experiment showed much higher anti-tumor activity than free MTX in MG-63 in vitro,providing the basis for further investigation of its anti-tumor effect for human osteosarcoma in vivo.In conclusion,MTX traditional liposomes prepared in this experiment had significant toxicity to BMDM and RAW 264.7. DiR-Labeled conventional liposomes and PEG-modified long-circulating liposomes exhibited different pharmacokinetic properties,the former were swallowed by macrophages preferentially and the latter could enter the tumor partially,which might show different anti-tumor activity.