Heparosan及其构建的纳米载体的内吞途径与药物传递研究

发布时间：2018-05-14 12:15

本文选题：肝素前体 + 内吞途径　；参考：《江南大学》2017年硕士论文

【摘要】：细胞对纳米药物传递系统(NDDSs)内摄的差异会影响药物递送效率及药效发挥。我们在前期用肝素前体(heparosan)构建NDDSs的研究中发现,其会选择性被肿瘤细胞高效且快速摄取,实验结果表明这可能与其内摄途径有关。为阐明原因,本课题分别对heparosan及用其构建的纳米载体在不同类型细胞中的内吞途径、内摄效率、胞内分布及药物命运进行了研究,主要包括如下三方面内容:(1)Heparosan的内吞途径及其胞内分布研究用荧光探针标记heparosan得到HB,并以葡聚糖(Dextran)为对照。细胞内摄化结果显示,较COS7细胞(经SV40病毒转化的非洲绿猴肾成纤维细胞)而言,MCF-7(人乳腺癌细胞)、和A549细胞(人非小肺癌细胞)和B16(小鼠黑色素瘤细胞)三种肿瘤细胞对HB的内摄化具有特异性,且与浓度和孵育时间正相关。HB进入胞内为能量依赖方式,HB在三种肿瘤细胞中主要通过小窝蛋白和巨胞饮途径被细胞摄取,HB进入细胞后主要分布于溶酶体中。(2)Heparosan纳米粒子的内吞途径及其胞内分布研究用疏水性脱氧胆酸修饰heparosan,并通过荧光标记制备得到HD纳米粒子,粒径为170 nm。细胞内摄化实验结果表明,正常细胞和肿瘤细胞均能高效摄取HD,但其进入肿瘤细胞的能力更强。HD的内摄化属于能量依赖方式,并且主要通过网格蛋白介导的内吞和巨胞饮途径被肿瘤细胞摄取。进入细胞后,HD主要分布于正常细胞和肿瘤细胞的溶酶体中。(3)Heparosan纳米载体的药物传递研究分别用脱氧胆酸和维生素E修饰heparosan,得到两亲性载体HepD和HepV,其能有效包载阿霉素(DOX)得到HDD和HVD,载药量分别为4.3%和4.7%。细胞实验显示,HDD和HVD均能快速传递DOX进入B16细胞,且随时间延长,细胞中DOX的量增加,能够有效抑制癌细胞生长。综上所述,heparosan及其构建的纳米载体在不同种类细胞中具有不同的内吞途径和内摄效率,但均分布并聚集在正常细胞和肿瘤细胞中。这一结论对设计溶酶体微环境敏感的heparosan纳米药物传递系统提供了实验参考。
[Abstract]:The difference in cellular uptake of NDDSs may affect drug delivery efficiency and drug efficacy. In our previous study using heparosan to construct NDDSs, we found that it could be selectively ingested by tumor cells efficiently and quickly, and the results suggested that it might be related to its internal pathway. In order to elucidate the reasons, the endocytosis pathway, uptake efficiency, intracellular distribution and drug fate of heparosan and its nanocarriers in different types of cells were studied. The endocytosis pathway and its intracellular distribution were studied in the following three aspects: heparosan was labeled with fluorescent probe and Dextranan was used as control. The results of cellular internalization showed that, MCF-7 (human breast cancer cell), A549 cell (human non-small lung cancer cell) and B16 (mouse melanoma cell) were more specific to HB than COS7 cells (African green monkey renal fibroblasts transformed by SV40 virus). There is a positive correlation between the concentration and incubation time. HB enters the cell in an energy-dependent manner. HB is mainly absorbed by cells into the cell through the pathway of nest protein and giant cell drink, and mainly distributes in lysosome. Studies on endocytosis pathway and intracellular distribution of particles; modified with hydrophobic deoxycholic acid to prepare HD nanoparticles by fluorescence labeling. The particle size is 170 nm. The results showed that both normal cells and tumor cells could ingest HDin highly, but their ability to enter tumor cells was stronger. HD was an energy-dependent way. And mainly through the griddle protein mediated endocytosis and megalocytosis pathway by tumor cells. Studies on Drug transfer of Heparosan Nano-carriers mainly distributed in lysosomes of normal cells and tumor cells after entering cells; modified with deoxycholic acid and vitamin E to obtain amphiphilic vectors HepD and HepV, which can effectively encapsulate Arabidopsis. HDD and HVD were obtained, the drug loading was 4.3% and 4.7%, respectively. Cell experiments showed that both HDD and HVD could transfer DOX into B16 cells quickly, and the amount of DOX in B16 cells increased with time, which could effectively inhibit the growth of cancer cells. In conclusion, the endocytosis pathway and uptake efficiency were different in different types of cells, but they were distributed and clustered in normal cells and tumor cells. This conclusion provides an experimental reference for the design of heparosan nanopharmaceutical delivery system sensitive to lysosomal microenvironment.
【学位授予单位】：江南大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R943

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