PEG-mCeramide修饰的阿霉素脂质体的制备及其抗肿瘤作用

发布时间：2018-05-26 22:10

本文选题：阿霉素脂质体 + GCS　；参考：《第二军医大学》2014年硕士论文

【摘要】：多药耐药(MDR)，，是肿瘤细胞对多种化疗药物耐药的种常见的耐药形式，也是导致肿瘤化疗失败和肿瘤复发的重要原因，成为当前研究肿瘤治疗的热点话题。纳米技术应用于临床制药是化疗研究的项重大突破，而且利用纳米技术克服肿瘤细胞耐药引起了广泛关注。本文结合了肿瘤耐药细胞的自身代谢特点，采用乙醇注入法制备PEG-mCeramide修饰的脂质体，以硫酸铵梯度法包裹阿霉素，得到PEG-mCeramide修饰的阿霉素脂质体（Lipo-ADR-Cer-）。本项研究展示了，Lipo-ADR-Cer-能够降低肿瘤耐药细胞对阿霉素的IC50，体外明显抑制肿瘤耐药细胞MCF-7-ADR和HL-60-ADR的细胞活性，体内具备强的抗肿瘤效果。通过检测糖基化ceramide合酶（GCS）在乳腺癌和白血病细胞株中的表达发现，在MCF-7-ADR和HL-60-ADR细胞株上GCS的表达均比MCF-7和HL-60敏感肿瘤细胞株增多。我们考虑GCS的过表达与肿瘤细胞的耐药相关，这些实验结果为利用外源性的ceramide来克服肿瘤耐药增加可能。同时，体外游离药物细胞杀伤实验表明PEG-mCeramide比对照PEG-DSPE的细胞毒性明显增强，不同浓度的游离的PEG-mCeramide与游离阿霉素联合给药有增敏效果，增敏指数随着PEG-mCeramide浓度的增高而不断增加，而且我们发现在耐药细胞株上，低剂量的PEG-mCeramide联合阿霉素的增敏效果比敏感细胞强。本课题中我们采用乙醇注入法制备PEG-mCeramide修饰的空白脂质体和无修饰的对照空白脂质体，以硫酸铵梯度法包裹化疗药物阿霉素，制备PEG-mCeramide修饰的阿霉素脂质体（Lipo-ADR-Cer-）和对照阿霉素脂质体（Lipo-ADR-）。对纳米脂质体的粒径、Zeta电位、载药量，包封率等基本表征进行检测，结果表明两种阿霉素脂质体的基本表征无明显差异，而且两种阿霉素脂质体的不同PH条件下的体外释放实验结果亦无明显差异。我们通过cck8法检测Lipo-ADR-Cer-、Lipo-ADR、游离ADR和游离ADR+PEG-mCeramide在不同给药时间点对MCF-7、MCF-7-ADR、HL-60和HL-60-ADR细胞株的体外杀伤效果，结果展示了在两株敏感肿瘤细胞株（MCF-7和HL-60）上,Lipo-ADR-Cer-和Lipo-ADR-的杀伤效果无统计学差异，而在两株耐药的肿瘤细胞株（MCF-7-ADR和HL-60-ADR）上, Lipo-ADR-Cer-的杀伤效果明显强于Lipo-ADR-。为探讨Lipo-ADR-Cer-杀伤效果增强的机制，我们利用流式细胞术和共聚焦显微镜技术对比了两种脂质体在细胞转染和内吞的差异，结果显示两者均无统计学差异。故我们考虑Lipo-ADR-Cer-杀伤效果增强的机制在于耐药细胞自身的代谢特点。在体内实验中，我们构建了乳腺癌腋下负荷裸鼠的动物模型和白血病裸鼠动物模型，通过系统观察了肿瘤小鼠的体重变化和肿瘤大小，定期定量对小鼠尾静脉注射Lipo-ADR-Cer-、Lipo-ADR-、游离ADR和游离ADR+PEG-mCeramide以及PBS后，评价乳腺癌小鼠的体内肿瘤抑制效果和白血病小鼠的生存状况。体内抗肿瘤实验表明，在两种肿瘤细胞的动物模型上，PEG-mCeramide修饰的阿霉素脂质体组的体内抗肿瘤效果比对照阿霉素脂质体组的效果强，游离的阿霉素联合PEG-mCeramide体内抗肿瘤效果比游离阿霉素组的效果强，但两组游离药物组的治疗效果均不及两组纳米药物组，而且游离阿霉素组与PBS组的效果相近。结论：与敏感的肿瘤细胞株相比，GCS在耐药肿瘤细胞株上的表达增多；对比对照阿霉素脂质体，PEG-mCeramide修饰的阿霉素脂质体的基本表征、体外释放过程、体外转染效率以及细胞的内吞过程均无统计学差异；在耐药细胞株上，对比对照阿霉素脂质体，PEG-mCeramide修饰的阿霉素脂质体的体外杀伤效果和体内抗肿瘤效果增强。本课题结合肿瘤耐药细胞的自身特点，利用具有杀伤效应PEG-mCeramide修饰纳米脂质体载体，制备PEG-mCeramide修饰的阿霉素脂质体，为探讨克服肿瘤细胞耐药提供了新的证据，为治疗MDR提供了条新的途径。
[Abstract]:Multidrug resistance (MDR) is a common form of drug resistance to a variety of chemotherapeutic drugs. It is also an important cause of tumor chemotherapy failure and tumor recurrence. It has become a hot topic in the study of tumor treatment. The application of nanotechnology to clinical pharmacy is a major breakthrough in the study of chemotherapy and the use of nanotechnology to overcome cancer. In this paper, PEG-mCeramide modified liposomes were prepared by ethanol injection, and adriamycin (Lipo-ADR-Cer-) modified by PEG-mCeramide was obtained by the ammonium sulfate gradient method. This study shows that Lipo-ADR-Cer- can reduce the swelling. Tumor resistant cells to adriamycin IC50 significantly inhibited the cell activity of tumor resistant cells MCF-7-ADR and HL-60-ADR in vitro, and had strong anti-tumor effect in vivo.
The expression of glycosylated ceramide synthase (GCS) in breast cancer and leukemia cells showed that the expression of GCS on MCF-7-ADR and HL-60-ADR cell lines increased more than that of MCF-7 and HL-60 sensitive tumor cells. We consider that the overexpression of GCS is associated with the drug resistance of the tumor cells. These experimental results are the use of exogenous ceramide to a gram. At the same time, the cytotoxicity test of free drug cells in vitro showed that the cytotoxicity of PEG-mCeramide was significantly higher than that of the control PEG-DSPE. The combination of free PEG-mCeramide and free adriamycin in different concentrations had a sensitizing effect, and the sensitization index increased with the increase of PEG-mCeramide concentration, and we found that In drug-resistant cell lines, low dose of PEG-mCeramide combined with adriamycin has stronger sensitivity than sensitive cells.
We used ethanol injection to prepare PEG-mCeramide modified blank liposomes and unmodified control blank liposomes, encapsulated adriamycin by ammonium sulfate gradient method, and prepared PEG-mCeramide modified adriamycin liposomes (Lipo-ADR-Cer-) and control adriamycin liposomes (Lipo-ADR-). The particle size of nano liposomes, Zet The basic characterization of a potential, drug loading and encapsulation efficiency showed that there was no significant difference in the basic characterization of two adriamycin liposomes, and there was no significant difference in the experimental results of two kinds of adriamycin liposomes under different PH conditions. We detected Lipo-ADR-Cer-, Lipo-ADR, free ADR and free ADR+PEG-mCerami by CCK8 method. The killing effect of De on MCF-7, MCF-7-ADR, HL-60 and HL-60-ADR cell lines at different time points showed that there was no significant difference in the killing effect of Lipo-ADR-Cer- and Lipo-ADR- on two sensitive tumor cell lines (MCF-7 and HL-60), and the killing of Lipo-ADR-Cer- was on the two drug-resistant swollen cell lines (MCF-7-ADR and HL-60-ADR). The effect of the injury was significantly stronger than that of Lipo-ADR-. as a mechanism for enhancing the effect of Lipo-ADR-Cer- killing. We compared the difference in cell transfection and endocytosis between two liposomes by flow cytometry and confocal microscopy. The results showed that there was no statistical difference between the two. Therefore, we consider that the mechanism of the enhancement of Lipo-ADR-Cer- killing effect is tolerance. The metabolic characteristics of the drug cell itself.
In the experiment in vivo, we constructed the animal model of breast cancer axillary load nude mice and the nude mouse model of leukemic nude mice. The weight change and tumor size of the tumor mice were observed by the system. Lipo-ADR-Cer-, Lipo-ADR-, free ADR, free ADR+PEG-mCeramide and PBS were regularly injected into the tail vein of mice to evaluate the small breast cancer. The tumor inhibition effect in vivo and the survival status of leukemia mice in vivo. In vivo antitumor experiments showed that in the animal models of two tumor cells, the anti tumor effect of PEG-mCeramide modified adriamycin liposome group was better than that of the adriamycin liposome group. Free adriamycin combined with the anti tumor effect of PEG-mCeramide in vivo. The results were better than that in the free adriamycin group, but the effect of the two groups of free drug groups was less than that of the two groups of nanoscale drugs, and the effect of the free adriamycin group was similar to that in the PBS group.
Conclusion: compared with the sensitive tumor cell lines, the expression of GCS in the resistant tumor cell lines increased; compared with the control of adriamycin liposomes, the basic characterization of the adriamycin liposomes modified by PEG-mCeramide, the release process in vitro, the transfection efficiency in vitro and the endocytosis process of the cells were not statistically different. The effects of PEG-mCeramide modified adriamycin liposomes in vitro and in vivo anti-tumor effect are enhanced according to the liposomes of adriamycin. This subject combines the characteristics of the tumor resistant cells and uses the PEG-mCeramide modified nano liposome to prepare the adriamycin liposomes modified by PEG-mCeramide. Cell resistance provides new evidence, which provides a new way for the treatment of MDR.
【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R943;R96

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