【作者】 周伟；

【导师】 黄世文；

【作者基本信息】 武汉大学 ， 化学、高分子化学与物理， 2017， 博士

【摘要】 癌症的化学疗法是现阶段临床上用于癌症治疗的主要手段之一。但传统的化疗方法是一种系统性全身治疗手段,不具有癌细胞特异性识别能力,治疗过程通常带来较强的副作用。随着纳米技术的发展,人们发现运用纳米载体进行药物递送能够在保护药物活性的同时,使药物尽可能多的在肿瘤部位富集,提高药物的疗效,减小药物带来的副作用。随着抗癌药物纳米载体研究的不断深入,人们意识到仅仅通过纳米粒子的被动靶向能力提升药物在肿瘤部位的富集达到治愈癌症的目的难度很大。这些仅具有被动靶向能力的载药纳米粒子虽然在一定程度上提高了药物在癌细胞内的富集,但一方面仍有大量药物未能被癌细胞摄取,另一方面,即便药物进入到癌细胞中,某些在细胞特定亚器官中作用的药物并不能被准确的送到作用位点处,这往往会降低药物的治疗效果,极大的削弱抗癌效率。为了进一步提高抗癌药物在作用位点处的药物浓度,提升药物的抗癌效率,一系列具有主动靶向功能的抗癌药物纳米载体应运而生。而随着主动靶向性纳米载体技术的不断发展和对于药物作用位点研究的不断深入,人们对纳米材料在药物递送过程中的精确性要求也不断提高。除了多种具有癌细胞靶向功能的纳米载体材料被开发并应用于癌症治疗,具有更精准主动靶向功能的亚细胞器靶向性纳米载体材料也受到越来越多的关注。本文设计、合成了一系列具有亚器官(线粒体)靶向性的纳米粒子,用于化疗药物精准递送到癌细胞线粒体中进行癌症治疗的研究。具体的研究工作如下:论文第一章主要介绍了癌症治疗的现状及不同种类的主动靶向性材料的研究进展。重点介绍了新型的以线粒体为靶点的亚细胞器靶向纳米粒子在癌症治疗方面的应用,对这类线粒体靶向性纳米粒子在癌症治疗过程中所起的作用和存在的问题进行了分析。第二章中,我们制备了一种长度为40-50 nm,宽度为20-30 nm的多孔棒状纳米三氧化二铁,同时合成了一种线粒体靶向性高分子TPP-PEG-NH2,通过硅烷化试剂将上述高分子键接到多孔纳米铁的表面,得到了具有线粒体靶向性的多孔纳米铁。利用这种新型的线粒体靶向性纳米粒子装载了三氧化二砷,得到了 ATO@TPP-MNRs载药纳米粒子。我们通过红外,透射电镜,动态光散射等多种表征方法对纳米粒子的外貌,形态,尺寸等进行了表征。运用MTT法,流式细胞仪检测等分析方法进行了载药纳米粒子对人类乳腺癌细胞MCF-7的毒性评估。此外,我们还通过激光共聚焦对纳米粒子在细胞内的分布情况进行了定位分析,证明了带有TPP(三苯基膦)基团的纳米粒子的线粒体靶向性增强。此外,我们通过不同的实验方法对细胞中细胞色素c,caspase-3和caspase-9进行了评估,进一步证明了,载药纳米粒子对癌细胞毒性的增强是通过增强药物在癌细胞线粒体中的富集,造成癌细胞线粒体的损伤程度增大引起更多的癌细胞凋亡。虽然在第二章中通过体外的毒性实验证明了当材料表面增加了线粒体靶向性基团时,载药纳米粒子将药物精准递送到癌细胞线粒体中,使药物对癌细胞的杀伤能力明显增强,但是一旦纳米粒子进入体内后,由于线粒体靶向性基团给纳米粒子所带来的高的表面正电荷,使其在体内循环过程中更容易吸附血清蛋白,这种额外的蛋白吸附一方面会降低纳米粒子的体内循环能力,另一方面由于血清的覆盖会一定程度的削弱纳米粒子的线粒体靶向能力,影响载药纳米粒子的体内抗癌效率。同时,这种线粒体靶向性并不具有癌细胞特异性,一旦纳米粒子进入到正常细胞中,药物在正常细胞的线粒体中富集,对正常细胞的毒性增强,最终导致癌症治疗过程中的副作用变大。为了消除在体内循环过程中线粒体靶向性基团高的正电荷所带来的负面影响,第三章中,我们制备了一种具有还原响应性的癌细胞内可活化线粒体靶向性纳米粒子PLGA/CPT/DSSP,用于改善紫杉醇的体内抗癌效率。这种可活化的LPNPs在体内循环过程中,通过长的PEG4000链段有效的屏蔽了线粒体靶向基团TPP带来的高正电荷,解决了高正电荷带来的血清过度吸附问题,确保了纳米粒子通过被动靶向能力尽可能多的在肿瘤组织处富集。而一旦纳米粒子进入癌细胞中,在肿瘤细胞内高浓度GSH的作用下,DSSP(DLPE-S-S-PEG4000)链段能够迅速发生断裂,TPP基团被暴露出来,纳米粒子恢复强的线粒体靶向能力,将抗癌药物精准的递送到癌细胞线粒体中。此外,由于DSSP只在癌细胞中存在响应能力,这种载药纳米粒子即使进入到正常细胞,DSSP链段也不会发生断裂,载药纳米粒子的线粒体靶向功能始终被屏蔽,药物不会在线粒体中产生富集,这有效的减小了具有线粒体靶向功能的药物体系对正常细胞的毒性,降低了化疗过程中的副作用。我们通过一系列表征手段对纳米粒子的粒径,表面电荷等性质进行了表征。在高浓度GSH条件下研究了纳米粒子的电荷变化。载药纳米粒子的体内外毒性实验结果均证明了这种可活化的线粒体靶向性载药纳米体系很好的解决了传统线粒体靶向性纳米载体高的正电荷在体内循环中的不利影响,并能显著的提高化疗药物的抗癌效率。第三章中设计合成的可活化纳米粒子虽然能够显著性提高药物在癌细胞线粒体中的富集,但是由于纳米粒子在血液循环过程中,仅通过被动靶向性在肿瘤组织处富集的纳米粒子仍然有限,有部分药物并未被癌细胞摄取。而这些未被癌细胞摄取的药物,通常会在癌症治疗过程中带来较严重的副作用。为了进一步提升癌细胞对载药纳米粒子的摄取,提升抗癌药物的利用率,降低化疗带来的副作用,我们在纳米粒子表面引入了癌细胞靶向性基团,设计合成了一种新型的具有双靶向功能的纳米粒子。这种新型双靶向纳米粒子与传统的双靶向载体具有显著性差异,其双靶向性具有程序化的特点,细胞靶向性与亚细胞器靶向性依次展现,互相之间并无干扰。此外,细胞靶向性分子DSPB(DLPE-S-S-Biotin)在引入癌细胞靶向性的同时,同样有效解决了线粒体靶向性基团所带来的高正电荷的问题。通过体内外实验的测试发现,这种程序性双靶向性载药纳米粒子在体外毒性和体内抗肿瘤效率方面均表现出了很好的效果,相较于单靶向性载药纳米粒子的抗肿瘤效率均有了明显的提升,是一种潜在的可用于临床抗肿瘤治疗的药物体系。更多还原

【Abstract】 The chemotherapy of cancer is one of the most effective methods used in the clinical treatment of cancer currently.However,the traditional chemotherapy method is a kind of systemic treatments with poor specificity,which will produces serious toxicity and side effects.With the development of nanotechnology,people find that nanoparticles can be used for drug delivery to protect the drug activity and improve the efficacy of drug.Meanwhile,people also realize that it is difficult to achieve desired effect of cancer treatment only rely on the passive targeting of nanoparticles.The undecorated nanoparticles can increase the drug concentration in cancer cells partly,however,on the one hand,there is still a large number of drug can not be uptaken by cancer cells,on the other hand,it is hard to deliver drug to the acting site after endocytosis of cells,both of which tend to reduce the effect of treatment.In order to increase the drug concentration of anti-cancer drug in the tumor cells and improve the anti-cancer efficiency,a series of active targeting nanoparticles for drug delivery have been exploited.Along with the development of nanotechnology of targeting nanoparticles and the in-depth study of drug acting sites,the precision of drug delivery has been gotten more and more attention,while many suborgan-targeted nanoparticles have been synthesized for the treatment of cancer.In this paper,sevel kinds of mitochondrial targeting nanoparticles were designed and synthesized,which can be used to deliver drug that can induce cells apoptosis through the damage of mitochondria in cancer cells.The main research includes:Chapter 1 mainly introduces the current situation of cancer treatment,and the research progress of targeting nanoparticles acting on different targets.This chapter also focuses on the application and disadvantages of new suborgan targeting nanoparticles in cancer treatment.In chapter 2,we prepare a kind of mesoporous iron-oxide nanorods(MNRs),while mitochondrial targeting polymer TPP-PEG-NH2 is synthesised and decorated on to the surface of MNRs to form TPP-PEG-MNRs.ATO is loaded into the mitochondrial targeting TPP-PEG-MNRs.The appearance and morphology of nanoparticles are characterized by infrared,transmission electron microscopy,dynamic light scattering and so on.The toxicity of ATO-MNRs to human breast cancer MCF-7 is evaluated by MTT and flow cytometry.In addition,CLSM is used for observing the localization of nanoparticles in the cancer cells and confirming the enhancement of mitochondrial targeting of nanoparticles.The release of cytochrome c,caspase-3 and caspase-9 in the cancer cells are also measured,and all results show that ATO@TPP-PEG-MNRs increase the cells apoptosis by enhancing the mitochondrial damage.In the chapter 2,we can find that the toxicity of ATO to cancer cells can be significantly enhanced when ATO is deliverd into the mitochondria in vitro.However,once mitochondrial targeting nanoparticles entrance into the body,the highly positive surface charge of TPP-containing nanocarriers lead to rapid clearance in blood and decrease accumulation in tumor tissue after i.v.injection,which limite the application in vivo.In order to eliminate the negative effects of TPP groups,in the third chapter,we have successfully developed a strategy for improving the anticancer efficacy of paclitaxel via redox-triggered intracellular activation of mitochondria-targeting in vivo.The positive charges of TPP in PLGA/CPT/DSSP are shielded by the longer PEG4000 chains coated on the surfaces of LPNPs outside the cancer cells.The loss of highly positive charge in PLGA/CPT/DSSP is advantageous to prevent the nanocarriers from the rapid clearance from the bloodstream after injection,which ensures the high accumulation of PTX-loaded LPNPs in tumor tissue.After entrance into cancer cells,the mitochondria-targeting of PTX-PLGA/CPT/DSSP is activated by detachment of DSSP under intracellular reduction conditions to enhance mitochondria-targeting,mitochondria dysfunctions and anticancer effect.This kind of activatable mitochondria-targeting nanoplatform,combining the advantages of the EPR effect and mitochondria-targeting of nanocarriers in drug delivery,has showed great potential in the delivery of PTX for cancer treatment with high efficacy.The nanoparticle size,surface charge and other properties of the nanoparticles are characterized by a series of characterization methods.The change of surface charge of nanoparticles under the condition of high concentration of GSH is also observed.The anticancer results of PTX-loaded nanoparticles in vivo show that the activated mitochondria-targeting nanoparticles can effectively solve the adverse effects of the high positive charge of the traditional mitochondrial targeting nanoparticles in systemic circulation,and can significantly improve the anti-cancer efficiency of the anticancer drugs in vivo.In the third chapter,the synthesis of activable mitochondrial targeting nanoparticle can increase the concentration of drugs in the mitochondria of cancer cells.However,in the blood circulation,it is finitely that nanoparticle accumulate in tumor sites only through EPR effect.It can be deduced that majority of drug can not entrance into the cancer cells,which led to serious side-effects.To improve the efficacy of anti-cancer drugs,we designe and synthesize a novel dual-targeting nanocarrier by introducing targeting group of cancer cells on the surface of activable mitochondrial targeting nanoparticle.Different from the traditional dual-targeting nanoparticles,the targeting of this new carrier is defined as programmed targeting.There is no interference between cellular and subcellular targeting,cell-targeted polymer DSPB not only introduce cancer targeting,but also effectively solve the problem of the highly positive charge caused by mitochondrial targeting group TPP.Through the results of in vitro and in vivo anticancer experiments,we find that this programmed dual-targeting nanoparticles exhibit good effect of anti-cancer.We think this programmed dual-targeting nanoparticle is a potential drug delivery system of the clinical anti-tumor treatment in the future.更多还原