胆固醇基普鲁兰自组装纳米粒与肝癌细胞相互作用的研究

发布时间：2018-04-25 21:07

本文选题：胆固醇基普鲁兰 + 异硫氰酸荧光素　；参考：《北京协和医学院》2013年博士论文

【摘要】：作为天然高分子材料之一的多糖具有良好的生物相容性和安全性,疏水改性多糖通过自组装方式形成具有独特的“核壳结构”的纳米粒,在体内具有长循环、主体稳定性和被动靶向性等特点,在药物传输领域有着潜在的应用前景。了解纳米粒与细胞的相互作用对于在细胞层次上理解生命体的生理过程、药物的作用机制、基因治疗的机理等具有重要的科学意义和实用价值,同时也可为构建更加安全有效的纳米药物载体提供依据。本研究采用异硫氰酸荧光素(FITC, fluorescein isothiocyanate)标记胆固醇基普鲁兰(CHSP, cholesterol-modified pullulan)并制备纳米粒,讨论了纳米粒浓度、孵育时间以及温度对细胞摄取的影响,研究了CHSP纳米粒的体外HepG2细胞的摄取机制以及亚细胞分布。同时,评价了CHSP纳米粒作为阿霉素(DOX, doxorubin)药物载体的细胞毒性,研究了HepG2细胞对载药纳米粒的摄取以及对药物的亚细胞分布进行定位和定量分析,为肝癌治疗提供了研究基础和理论依据。主要研究内容及结果如下： 1. FITC标记的CHSP (FITC-CHSP)合成及自组装纳米粒的制备和表征合成了FITC-CHSP并通过红外进行表征,结果表明,FITC实现了接枝成功。采用透析法制备了FITC-CHSP自组装纳米粒,并利用透射电镜(TEM)和动态光散射粒度分析仪(DLS)对纳米粒的形态、粒径及粒径分布进行了表征,研究结果表明,制备的FITC-CHSP纳米粒呈规则的球形,平均粒径为63.0±1.9nm。 2. FITC-CHSP自组装纳米粒的体外HepG2细胞摄取机制以及亚细胞分布 CHSP纳米粒的体外细胞毒实验表明：CHSP纳米粒对HepG2细胞无明显的细胞毒性。分别采用激光扫描共聚焦显微镜(CLSM)和荧光光度法对HepG2细胞摄取CHSP纳米粒进行观察和定量,系统研究了纳米粒浓度、孵育时间、孵育温度对纳米粒摄取的影响。结果表明,HepG2细胞摄取纳米粒的过程是能量依赖的过程,呈现出浓度依赖性、时间依赖性和温度依赖性。内吞抑制实验表明：网格蛋白介导的内吞途径以及巨胞饮途径共同参与了CHSP纳米粒的入胞过程。纳米粒的亚细胞分布实验表明：在研究的孵育时间(4h)内,并没有发现CHSP纳米粒进入高尔基体和内质网。当纳米粒与细胞孵育30min时,没有纳米粒定位于溶酶体中。但是,随着孵育时间的延长,大量纳米粒分布于溶酶体中,且定位于溶酶体中的纳米粒逐渐向细胞核周区域移动。 3.载阿霉素CHSP纳米粒的制备及体外药物释放以DOX为模型药物,采用透析法制备了载DOX的CHSP纳米粒,并用TEM和DLS对载药纳米粒进行表征,结果证实,载药纳米粒呈球形,随着药物与载体投料比的增加,纳米粒的粒径也增大(185.6-226.4nm)。采用紫外-可见分光光度法测得载药纳米粒的载药量在10.31-30.79%范围内,可通过调整起始药物投料比对载药量调控,包封率为71.2-88.3%。载药纳米粒的体外释放与释放介质的pH值有关,在低pH环境(pH5.0)中药物释放较快,72h内的累积释放量为62.0%,但在pH6.8或pH7.4的释放介质中,药物释放依次减慢,72h的累积释放量分别为46.4%和30.1%。 4.载阿霉素CHSP纳米粒的体外HepG2细胞摄取研究载药纳米粒的体外细胞毒实验结果表明,不同浓度的游离DOX和载药纳米粒与HepG2细胞分别孵育72h,二者对HepG2细胞的生长均显示出一定的抑制作用,并且随着DOX浓度的增加,二者对HepG2细胞生长的抑制作用均增强。根据量效曲线计算得到游离DOX的IC50为2.36μg/mL,而载DOX的纳米粒的IC50为0.99μg/mL表明,载DOX的纳米粒对HepG2细胞的毒性更大。采用流式细胞仪对HepG2细胞摄取的DOX进行定量,结果表明,随着孵育时间的增加,HepG2细胞摄取DOX的量也增大。当孵育时间大于2h,HepG2细胞摄取载药纳米粒的量要多于游离DOX。采用CLSM以及定量分析软件分别对药物的亚细胞分布进行定位和定量分析结果表明,由于游离药物与载药纳米粒的摄取机制不同,孵育30min,游离DOX主要分布在细胞核中,并且随着孵育时间的增加,细胞以及细胞核中的DOX也增多。孵育30min,载药纳米粒主要分布在胞浆中；1h后部分载药纳米粒分布于溶酶体中,细胞核中DOX显著增加,并且随着孵育时间的增加,细胞、溶酶体以及细胞核中的DOX也增加。孵育4h时,载药纳米粒实验组细胞以及细胞核中的DOX均多于游离DOX实验组。在研究的孵育时间(4h)内,载药纳米粒实验组以及游离DOX实验组细胞分布在高尔基体、内质网以及线粒体中的DOX量很少,随着孵育时间的增加,在上述细胞器中的含量无明显变化。综上所述,CHSP纳米粒具有良好的生物相容性,能通过网格蛋白介导的内吞以及巨胞饮途径快速进入HepG2细胞,同时HepG2细胞对载DOX的CHSP纳米粒的摄取能力要比游离DOX强,载药纳米粒对HepG2细胞的毒性明显大于游离DOX,提示与游离DOX相比,载药纳米粒可在较低剂量下达到较高疗效。CHSP纳米粒有望成为一种新型的肝癌治疗药物载体。
[Abstract]:As one of the natural polymer materials, polysaccharides have good biocompatibility and safety. Hydrophobic modified polysaccharides form a unique "nuclear shell structure" nanoparticles by self-assembly. They have a long cycle in the body, the stability of the body and the passive targeting, and have potential applications in the field of drug delivery. The interaction between nanoparticles and cells is of great scientific and practical value for understanding the physiological processes of the life body at the cellular level, the mechanism of drug action, the mechanism of gene therapy and so on. It also provides a basis for the construction of a more safe and effective nano drug carrier.
In this study, FITC (fluorescein isothiocyanate) was used to mark the cholesterol Kip Ruland (CHSP, cholesterol-modified pullulan) and prepare nanoparticles. The effect of nanoparticle concentration, incubation time and temperature on cell uptake was discussed. The uptake mechanism of HepG2 cells in vitro and subcells of CHSP nanoparticles were studied. At the same time, the cytotoxicity of CHSP nanoparticles as a drug carrier for DOX (doxorubin) was evaluated. The uptake of drug loaded nanoparticles by HepG2 cells and the location and quantitative analysis of the subcellular distribution of drugs were studied. The basis and theoretical basis for the treatment of liver cancer were provided. The main contents and results were as follows:
Preparation and characterization of 1. FITC labeled CHSP (FITC-CHSP) synthesized and self assembled nanoparticles
The FITC-CHSP was synthesized and characterized by IR. The results showed that the grafting was successful by FITC. The FITC-CHSP self assembled nanoparticles were prepared by dialysis method. The morphology, particle size and particle size distribution of the nanoparticles were characterized by transmission electron microscopy (TEM) and dynamic light scattering particle size analyzer (DLS). The results showed that the prepared FITC-CHSP was prepared. The nanoparticles were regular spheres with an average particle size of 63 + 1.9nm..
HepG2 cell uptake mechanism and subcellular distribution of 2. FITC-CHSP self assembled nanoparticles in vitro
In vitro cytotoxicity test of CHSP nanoparticles showed that CHSP nanoparticles had no obvious cytotoxicity to HepG2 cells. The HepG2 cells were observed and quantified by laser scanning confocal microscopy (CLSM) and fluoropphotometry respectively. The concentration of nanoparticles, incubation time and incubation temperature on nanoparticles uptake were studied. The results showed that the process of HepG2 cells uptake of nanoparticles was an energy dependent process, showing a concentration dependence, time dependence and temperature dependence. Endocytosis inhibition experiments showed that the endocytic pathway mediated by the gridin and the mega drink pathway participated in the cellular process of CHSP nanoparticles. The subcellular distribution of nanoparticles In the incubation time (4h), no CHSP nanoparticles were found to enter the Golgi bodies and endoplasmic reticulum. When the nanoparticles and cells incubated 30min, no nanoparticles were located in the lysosomes. However, a large number of nanoparticles were distributed in the lysosomes with the prolongation of incubation time, and the nanoparticles located in the lysosome gradually turned to the nucleus of the nucleus. Regional movement.
Preparation of 3. adriamycin CHSP nanoparticles and drug release in vitro
Using DOX as the model drug, the DOX loaded CHSP nanoparticles were prepared by dialysis method, and the drug loaded nanoparticles were characterized by TEM and DLS. The results showed that the drug loaded nanoparticles were spherical. With the increase of the ratio of drug to carrier, the particle size of nanoparticles increased (185.6-226.4nm). The loading of drug loaded nanoparticles was measured by UV visible spectrophotometry. In the range of 10.31-30.79%, the drug delivery ratio can be adjusted by adjusting the initial drug delivery ratio. The release of the encapsulated 71.2-88.3%. drug loaded nanoparticles in vitro is related to the pH value of the release medium. The release of drugs in the low pH environment (pH5.0) is faster and the cumulative release amount in 72h is 62%, but the release of drugs in the release medium of pH6.8 or pH7.4 is dependent on the release of drugs. The cumulative release of 72h was 46.4% and 30.1%. respectively.
HepG2 uptake of 4. adriamycin CHSP nanoparticles in vitro
In vitro cytotoxicity test of drug loaded nanoparticles showed that different concentrations of free DOX and drug loaded nanoparticles incubated 72h with HepG2 cells respectively. The two had a certain inhibitory effect on the growth of HepG2 cells, and with the increase of DOX concentration, the inhibition of the growth of HepG2 cells increased by the increase of the concentration of DOX. According to the volume effect curve, the results were calculated. The IC50 of free DOX was 2.36 mu g/mL, while IC50 loaded with DOX nanoparticles showed that the DOX loaded nanoparticles were more toxic to HepG2 cells. The flow cytometry was used to quantify DOX for HepG2 cells. The results showed that the HepG2 cells increased the amount of DOX as the incubation time increased. The amount of drug loaded nanoparticles was more than that of free DOX. using CLSM and quantitative analysis software to locate and quantify the subcellular distribution of drugs. The results showed that the free DOX was mainly distributed in the nucleus because of the different uptake mechanism between the free drug and the drug loaded nanoparticles, and the free DOX was mainly distributed in the nucleus, and as the incubation time increased, it was thinner. The number of DOX in cell and nucleus was also increased. The drug loaded nanoparticles were mainly distributed in the cytoplasm. After 1h, some drug loaded nanoparticles were distributed in the lysosome, and the DOX in the nucleus increased significantly. And as the incubation time increased, the cells, lysosomes and the DOX in the nucleus were also increased. When incubating 4h, the drug loaded nanoparticles experiment group cells and the incubating 4H. The DOX in the nucleus was more than that in the free DOX experiment group. In the incubation time (4h), the cells of the drug loaded nanoparticles and the free DOX experimental group were distributed in Golgi body, and the amount of DOX in the endoplasmic reticulum and mitochondria was very little.
To sum up, CHSP nanoparticles have good biocompatibility, can quickly enter HepG2 cells through the endocytic endocytosis and megagocytosis pathway, and the uptake of DOX CHSP nanoparticles by HepG2 cells is stronger than that of free DOX. The toxicity of drug loaded nanoparticles to HepG2 cells is significantly greater than that of free DOX, suggesting that compared with free DOX, the drug loaded nanoparticles are more toxic than free DOX. Drug loaded nanoparticles can achieve high efficacy at lower doses..CHSP nanoparticles are expected to become a new drug carrier for the treatment of liver cancer.

【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R318.08

【参考文献】