【作者】 胡智渊；

【导师】 阮景德；

【作者基本信息】 中南大学 ， 生物医学工程， 2007， 硕士

【摘要】 目的：拓宽表多柔比星纳米粒的制剂学种类，制备了表多柔比星聚PLA-PEG纳米粒。考察PLA-PEG纳米颗粒作为一种药物载体与培养细胞及其器官的生物相溶性。分析小鼠肝脏在注射大剂量空白PLA-PEG纳米粒(42.04 mg／kg，i．v．)之后药物代谢基因表达谱的变化。分析ATP结合盒转运蛋白在PLA-PEG纳米粒肝脏代谢和细胞膜转运中起何种作用。并提出肝细胞对PLA-PEG纳米粒的向外排泄机制。方法：本试验采用均匀设计方法设计实验，制备出表多柔比星PLA-PEG纳米粒，考察纳米粒的形态学，理化性质，药学指标。MTT还原法体外测定了PLA-PEG纳米粒对培养细胞HepG2生存力的影响。细胞原位凋亡检测试验考察大剂量空白PLA-PEG纳米粒是否会对几个重要脏器(肝脏，心脏和肾脏)存在潜在性的损伤作用。SOD、NOS酶活性以及MDA水平检测考察大剂量PLA-PEG纳米颗粒是否影响小鼠肝脏，心脏和肾脏细胞和血清的抗氧化能力。我们通过小鼠肝细胞对PLA-PEG纳米颗粒的体外摄取和外排泄试验考察肝细胞对PLA-PEG纳米颗粒的摄取程度及其排泄机制。我们随后使用小鼠药物代谢cDNA芯片和RT-PCR试验检测了96个药物代谢基因的表达谱。并通过试验组和对照组样品间基因转录的相对丰度分析，筛选对PLA-PEG纳米颗粒肝脏代谢过程中扮演重要角色的基因。并根据ATP-结合盒传递蛋白的药理学功能及其本试验的结果，提出肝细胞向外排泄细胞内PLA-PEG纳米颗粒的机制模型。结果：本试验制备出表多柔比星PLA-PEG纳米粒。得到的载药纳米粒平均粒径为174.5nm，包封率为90.42％，载药率为8.22％。试验结果证明以PLA-PEG为载体材料，以溶剂挥发法制备表多柔比星PLA-PEG纳米粒的工艺是可行的。MTT还原法结果表明与未加纳米颗粒的对照组相比，在0.001-0.1mg／ml纳米颗粒使用浓度范围内，HepG2细胞在与纳米颗粒共同培养24h之后，纳米粒对细胞活力的影响与对照组相比无统计学意义(P＜0.05)。同时，细胞凋亡检测证实，昆明小鼠在注射大剂量(42.04 mg／kg，i．v．)空白PLA-PEG纳米颗粒之后，纳米颗粒不会诱导肝脏、肾脏及心脏细胞的凋亡，大剂量PLA-PEG纳米颗粒对肝细胞不存在着潜在性的损伤作用。与未注射纳米颗粒的对照组相比，在大剂量作用的几个组织(肝，心，肾)和血清样品中，SOD、NOS酶活性以及MDA水平与空白对照组比较均没有出现统计学意义上的差异(P＜0.05)，大剂量PLA-PEG纳米颗粒不会影响小鼠肝细胞的抗氧化能力。体外小鼠肝细胞对PLA-PEG纳米颗粒的摄取试验结果表明该种细胞对PLA-PEG纳米颗粒的摄取在750μg／ml到1000μg／ml的浓度范围之内会达到饱和吸收，体外小鼠肝细胞对PLA-PEG纳米颗粒的外排试验表明大约51％-52％(51.5％和52.0％)被小鼠肝细胞摄取的PLA-PEG纳米颗粒会被肝细胞外排。小鼠药物代谢cDNA芯片分析和RT-PCR检测结果表明与空白对照组(注射生理盐水)相比，在注射大剂量纳米颗粒(42.04 mg／kg，i．v．)的昆明小鼠的肝细胞内，出现了许多ATP-结合盒传递蛋白(ABCA8，ABCD3，ABCD4和ABCA5)表达的上调和GSTP1的下调，特别是ABCA8和ABCC5的表达上调尤为突出。结论：以PLA-PEG为载体材料，以溶剂挥发法制备表多柔比星PLA-PEG纳米粒药物工艺是可行的。PLA-PEG作为一种抗癌药物载体，应用于体内具有很好的生物相溶性。但是小鼠肝细胞对PLA-PEG纳米颗粒的吸收能力是有限的，小鼠肝细胞对PLA-PEG纳米颗粒的吸收在达到饱和之后会外排一定比率的纳米颗粒。我们根据试验结果及其前人的研究成果提出两个细胞外排泄细胞内PLA-PEG纳米颗粒的模型，模型一：我们认为这些ATP结合盒运载体，特别是ABCA8在小鼠肝细胞外排PLA-PEG纳米颗粒水解之后的低聚物过程中扮演了重要的角色。模型二：我们认为ABCC5表达多药耐药蛋白质5(MDR5)参与向外运输PLA-PEG纳米粒与GSH的共轭物GSX的过程。大剂量注射PLA-PEG纳米颗粒(42.04mg／kg，i．v．)之后，小鼠肝细胞对PLA-PEG纳米粒产生了耐药抗药性。两个重要的运载体基因ABCA8，ABCC5和GSTP1与这种抗药机制有密切的关系。更多还原

【Abstract】 Objective: In order to enlarge galenic pharmacy of epirubicin PLA—PEG nanoparticles, we prepared epirnbicin-loaded PLA-PEGnanoparticles. In order to investigate the biocompatibility of PLA-PEGnanoparticles with cultured cells and organs. We analyzed the changes ofgene profile of mouse liver after a large dose injection of blank PLA-PEGnanoparticles (42.04 mg/kg, i.v.). We analyzed the effect of ATP-bindingcassette (ABC) transporters on nanoparticles hepatic metabolism and cellmembrane transport. Also we produce the mechanism of cell effiux ofPLA-PEG nanoparticles by hepatic cells.Methods: In our experiments, we prepared doxorubicin-loadedPLA-PEG nanoparticles by the method of uniform design, also analyzedthe morphology, physico-chemical property and pharmaceutic index, . Weanalyzed the effect ofPLA-PEG nanoparticles on cell viability by themethod of MTT assay. In situ cell apoptosis assay was used to investigatethe potential damage of blank PLA-PEG nanoparticles to importantorgans. SOD, NOS, and MDA activity assay was used to investigate theantioxygen ability afeter large dose injection.Then Kunming mice weretreated with large dose of PLA-PEG nanoparticles (42.04mg/kg, i.v) andafter 4 days total RNA was isolated to elucidate patterns of gene expression using a mouse cDNA-microarray (Super Array).Results: Blank PLA-PEG nanoparticles have been successfullyprepared and MTT assay suggested that the nanoparticles with HepG2cell co-culture model do not cause significant changes in membraneintegrity in controlled concentration range (0.001 mg/ml to 0.1 mg/ml).Immunohistochemical analysis demonstrated that large dose of PLA-PEGnanoparticles injection (42.04mg/kg, i.v) didn’t induce hepatic cellsapoptosis. From biochemical assay experiments, although the levels ofSOD decreased and that of MDA, NOS increased after treatment withlarge dose of PLA-PEG nanoparticles injection (42.04mg/kg, i.v), butthey were all not significant (p＞0.05). Results of in vitro accumulationand efflux experiments indicated that about 51%-52 %( 51.5% and 52.0%)intracellular PLA-PEG nanoparticles was expulsed aider mouse primaryhepatocytes reached a saturation uptake of nanoparticles during theconcentration range of 750μg/ml to 1000μg/ml. Treatment withnanoparticles resulted in over-expression of a lot of ATP-binding cassette(ABC) transporters, especially two ABC transporters (ABCA8 andABCC5/MRP5) and down-regulation of GSTP1, in comparison with thecontrol. ABCA8 could extrude low molecular weight polymers afterPLA-PEG nanoparticles hydrolysis outside the cells. We also discoveredthat ABCC5 expressed multidrug resistance protein 5 (MRP5) to pumpout conjugate (GS-X) of PLA-PEG nanoparticles with GSH. The results were confirmed by RT-PCR.Conclusion: The biocompatibility of PLA-PEG nanoparticles withcultured cells and organs is very well. The uptake of naoparticles byhepatic cells is limited. The nanoparticles in cells are pumped bytransporters after saturation uptake. The results suggested that ABCtransporters (especially ABCA8) pump out the polymers after hydrolysisfrom mouse hepatic cells and large dose of PLA-PEG nanoparticles makemouse hepatic cells gain drug resistance to PLA-PEG nanoparticles.更多还原