脂质体作为一种药物递送系统具有生物相容性好、可降解和无毒性的特点，对单核巨噬细胞有着天然的靶向性。肿瘤相关巨噬细胞（tumor-associated macrophages, TAMs）是肿瘤微环境中的重要组成部分，来自于血液单核巨噬细胞，可通过多种机制促进肿瘤的发展与转移。甲氨喋呤（methotrexate, MTX）作为一种常用抗癌药物，有研究显示其对单核巨噬细胞具有特异性毒性。本文将MTX包载入脂质体中，制备了载MTX的传统脂质体和聚乙二醇（Polyethylene Glycol, PEG）修饰长循环脂质体，考察前者在体外对巨噬细胞的毒性作用，并初步考察了二者的体内药动学差异，为进一步研究其在TAMs含量高的小鼠3-甲基胆（3-methylcholanthrene, 3-MCA）骨肉瘤和TAMs含量低的小鼠CT 26结肠癌模型的抗瘤活性提供基础，从而最终确定MTX脂质体能否通过作用于TAMs而发挥抗肿瘤活性，反过来也能进一步验证TAMs是否适宜用于抗肿瘤辅助靶点。

具体研究内容及结果如下：

MTX传统脂质体的制备及表征

采用薄膜分散法制备出了MTX传统脂质体，通过浊度和MTX吸附率的测定，确定了MTX脂质体纯化的工艺条件；通过动态光散射法（DLS）及扫描电子显微镜（SEM）对MTX脂质体的粒径及形态进行表征，并测定了脂质体的包封率和药脂比。实验结果显示，G-50微柱对传统脂质体的回收率和游离MTX的吸附率都达到了90%以上，适合分离脂质体与游离MTX；传统脂质体的粒径分布主峰约在1 μm，脂质体呈圆形，包封率约4%。

MTX脂质体对巨噬细胞的毒性效应研究

利用L929上清和巨噬细胞集落刺激因子（macrophage colony stimulating factor, M-CSF）成功诱导出骨髓来源的巨噬细胞（Bone Marrow-Derived Macrophage, BMDM）；利用酸性磷酸酶法对培养出的BMDM进行鉴定；使用高内涵显微镜测定细胞对脂质体的吞噬，利用MTS对细胞毒性进行测定。结果表明，M-CSF诱导的BMDM比L929上清诱导的BMDM细胞形态更均一，细胞活性更高，细胞纯度达到90%以上；BMDM和RAW 264.7对脂质体都能高效摄取，且呈明显的时间和剂量依赖性，BMDM的摄取能力明显高于RAW 264.7； MTX脂质体对BMDM和RAW 264.7细胞都存在明显的毒性作用，且显著高于同剂量的游离MTX。

传统脂质体和PEG修饰的长循环脂质体的初步药动学研究

制备了传统空白脂质体和PEG修饰的长循环空白脂质体并用荧光染料DiR进行标记。尾静脉分别注入CT 26结肠癌移植瘤模型和3-甲基胆蒽诱发瘤模型小鼠体内后，利用活体成像仪观察了两种脂质体的生物分布情况。研究结果表明，CT 26细胞和3-甲基胆蒽均能成功诱导小鼠产生肿瘤，成瘤率分别在100%和90%；传统脂质体更容易被肝脾巨噬细胞所吞噬，而PEG修饰的脂质体则通过EPR效应进入肿瘤。

MTX长循环脂质体的制备和对人骨肉瘤细胞的体外抗瘤活性研究

采用薄膜分散法制备传统甲氨蝶呤脂质体（MTX-LIP），后插入法制备甲氨蝶呤长循环脂质体（PEG₂₀₀₀-MTX-LIP），并用超速离心法、G-10微柱离心法和G-50微柱离心法去除未包载的游离药物，对三种方法纯化后脂质体的回收率、粒径、形态、包封率及药脂比等理化性质进行表征；用MTS法研究经超速离心法和G-50微柱法纯化后的PEG₂₀₀₀-MTX-LIP对人骨肉瘤细胞MG-63的毒性。研究结果表明，G-50微柱离心法是最合适的MTX脂质体纯化方法，具有回收完全和快速的优点；制备的MTX长循环脂质体粒径约在200 nm，粒径分布窄，粒径均匀，各种纯化方法对脂质体粒径影响较小；相比游离MTX，MTX脂质体对MG-63具有显著增强的体外抗瘤活性，在人骨肉瘤的化疗上有潜在的应用前景。

综上所述，实验中制备的传统MTX脂质体对BMDM和RAW 264.7细胞都具有明显毒性，DiR标记的传统脂质体和PEG修饰的长循环脂质体在体表现出了不同的药动学特性，前者优先被血液循环中单核巨噬细胞吞噬，后者则部分被血液循环中单核细胞吞噬、部分进入肿瘤组织，从而可能表现出不同的抗肿瘤活性。

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 liposomes

Conventional 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 liposomes

L929 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 liposomes

CT 26 cells and 3-MCA were used to prepare tumor models in BALB/C and C57BL/6 mice, and the rate of tumor formation was 100% and 90%, respectively.  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-63

The 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 PEG₂₀₀₀-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.