【作者】 王晓辉；

【导师】 张亚宏； 张杰；

【作者基本信息】 河南大学 ， 药理学， 2020， 硕士

【摘要】 目前,临床上治疗癌症主要依赖抗癌药物,其中从天然产物中开发出的抗癌药约为抗癌药总量的三分之一。自然界中的天然抗癌药种类繁多、获取资源丰富,但天然抗癌药有着自身的不足之处,比如水溶性差、靶向性差、生物利用度低等,极大地限制了其在临床中的应用。因此将天然抗癌药与新型纳米材料相结合构建出靶向纳米载药体系,可显著提高天然抗癌药的水溶性以及生物利用度,并且赋予天然抗癌药靶向性,有利于药物浓度在癌变区域的富集,减少对正常组织的损伤。纳米材料还可凭借自身的EPR效应被动靶向癌变组织,进一步增强了药物的靶向性。新型纳米材料量子点合成方法简便,并具有出色的荧光性能与极低的生物毒性,因此可用于纳米载药的荧光成像研究,实现药物在细胞内荧光成像功能,有助于直观、快速、准确地获取纳米药物在细胞内分布、释放和药效机制等重要信息,为设计和开发临床应用纳米药物提供实验基础。据报道,三萜类化合物及其衍生物具有诸多的药理活性,如抗肿瘤,抗炎,抗艾滋病HIV蛋白酶等。熊果酸(UA)是一种常见的五环三萜类化合物,并具有抗菌、抗炎、抗病毒、抗肿瘤等药理性质,其对多种肿瘤细胞具有较强的抑制活性,科研人员已经把熊果酸作为一种癌症预防和治疗药物展开相关的研究。熊果酸具有良好的抗肿瘤活性,机理是它可以阻断G1期的细胞周期进程并诱导细胞凋亡。尽管熊果酸能显著抑制肿瘤细胞的增殖,但由于其水溶性差、生物利用度极低限制了其在临床的应用。基于此本文构建了两种熊果酸靶向荧光载药体系,并对两种体系进行了详细结构表征,评价了其体外抗癌活性,具体研究内容分为以下两个部分:1.本文通过溶胶-凝胶法成功合成出Zn O QDs并且量子点表面富含氨基官能团。该方法合成出的Zn O QDs不仅具备优异的荧光性能和良好的水溶性,同时还具有p H敏感性。采用共沉淀法制备出磁性Fe3O4纳米粒子,该纳米粒子经过油酸分子吸附,以及KMn O4氧化后,使其表面富含羧基官能团。随后,通过TEM、SEM、FTIR、UV、XRD等一系列手段分别对两种纳米材料进行了详细表征。Zn O QDs与Fe3O4纳米粒子通过化学键偶联构建出具有荧光性能的磁性复合纳米粒子(Fe3O4@Zn O),并在复合纳米粒子的表面负载天然抗癌药熊果酸,建立了p H响应型的磁靶向纳米载药体系(Fe3O4@Zn O-UA)。通过MTT法检测熊果酸和纳米载药体系对肿瘤细胞的抑制效果,将熊果酸和纳米载药体系分别作用于Hep G2、SMMC-7721、MCF-7、A549四种肿瘤细胞,结果表明纳米载药体系对肿瘤细胞的抑制活性优于单独使用的熊果酸。细胞摄取实验证明,纳米载药体系可穿越细胞膜进入细胞质,在荧光显微镜下呈现出亮黄色荧光,成功的实现了细胞成像功能,为进一步研究纳米载药体系的作用机制提供了基础。最后通过荧光显微镜、高内涵成像系统对纳米载药体系进行一系列检测,发现纳米载药体系能促使线粒体膜电位下降,活性氧(ROS)升高,促使肿瘤细胞发生凋亡。2.本文通过高温热解法制备出Zn-CQDs,该量子点具有优异的荧光性能。通过TEM对Zn-CQDs进行形貌表征可知量子点的粒径为5.16±0.32 nm。天然抗癌药熊果酸经过聚乙二醇以及叶酸的修饰,不仅改善了水溶性并使其赋予靶向性;为进一步增强抗癌活性,对其进行连接天然抗癌药紫杉醇(PTX),形成UA-PEG-FA-PTX,有利于延长在体内滞留时长。将Zn-CQDs通过静电力作用与UA-PEG-FA-PTX进行物理吸附,构建出具有荧光性能的纳米载药体系Zn-CQDs@UA-PEG-FA-PTX。通过TEM、SEM、FTIR、UV、XRD、1H NMR等仪器对该载药体系进行了详细表征。通过MTT法检测纳米载药体系Zn-CQDs@UA-PEG-FA-PTX对肿瘤细胞的抑制活性,结果表明纳米载药体系对肿瘤细胞的抑制活性优于单独使用的熊果酸。细胞摄取实验证明,纳米载药体系可进入到细胞质内,实现了细胞成像功能。最后通过荧光显微镜、高内涵成像系统对纳米载药体系进行了细胞凋亡的相关实验,发现纳米载药体系可诱导细胞凋亡,同时使线粒体膜电位下降,活性氧升高。更多还原

【Abstract】 At present,the clinical treatment of cancer mainly depends on anti-cancer drugs,and the anti-cancer drugs developed from natural products are about one-third of the total anti-cancer drugs.There are many types of natural anticancer drugs in nature and abundant resources,but natural anticancer drugs have their own shortcomings,such as poor water solubility,poor targeting,and low bioavailability,which greatly limits their clinical application..Therefore,the combination of natural anticancer drugs and new nanomaterials to build a targeted nano drug delivery system can significantly improve the water solubility and bioavailability of natural anticancer drugs,and give natural anticancer drugs targeting,which is conducive to drug concentration Enrichment in cancerous areas reduces damage to normal tissues.Nanomaterials can also passively target cancerous tissues by virtue of their own EPR effect,further enhancing the targeting of drugs.The new nanomaterial quantum dot synthesis method is simple,and has excellent fluorescence performance and very low biological toxicity.Therefore,it can be used in nano-drug-loaded fluorescence imaging research and realize the fluorescence imaging function of drugs in cells,which is intuitive,fast and accurate To obtain important information such as the distribution,release and pharmacodynamic mechanism of nano-drugs in cells to provide an experimental basis for the design and development of clinical nano-drugs.According to reports,triterpenoids and their derivatives have many pharmacological activities,such as anti-tumor,anti-inflammatory,anti-AIDS HIV protease and so on.Ursolic acid(UA)is a common pentacyclic triterpenoid compound and has pharmacological properties such as antibacterial,anti-inflammatory,anti-viral,and anti-tumor.It has strong inhibitory activity on a variety of tumor cells.Research on ursolic acid as a cancer prevention and treatment drug.Ursolic acid has good anti-tumor activity,the mechanism is that it can block the cell cycle progression of G1 phase and induce apoptosis.Although ursolic acid can significantly inhibit the proliferation of tumor cells,its poor water solubility and extremelylow bioavailability limit its clinical application.Based on this,two ursolic acid-targeted fluorescent drug delivery systems were constructed,and the two systems were characterized in detail to evaluate their anti-cancer activity in vitro.The specific research content is divided into the following two parts:1.In this paper,Zn O QDs were successfully synthesized by sol-gel method and the surface of quantum dots was rich in amino functional groups.The Zn O QDs synthesized by this method not only have excellent fluorescence properties and good water solubility,but also have p H sensitivity.Co-precipitation method was used to prepare magnetic Fe3O4 nanoparticles.After being adsorbed by oleic acid molecules and oxidized by KMn O4,the nanoparticles were enriched in the surface with carboxyl functional groups.Subsequently,the two nanomaterials were characterized in detail by a series of means such as TEM,SEM,FTIR,UV,XRD and so on.Zn O QDs and Fe3O4 nanoparticles are chemically coupled to construct magnetic composite nanoparticles with fluorescent properties(Fe3O4@Zn O),and the surface of the composite nanoparticles is loaded with the natural anticancer drug ursolic acid to establish a p H-responsive magnetic target Nanometer drug loading system(Fe3O4@Zn O-UA).The inhibitory effect of ursolic acid and nano drug-carrying system on tumor cells was detected by MTT method,and the ursolic acid and nano drug-carrying system were applied to Hep G2,SMMC-7721,MCF-7 and A549 tumor cells respectively.The results showed that The inhibitory activity of the nano drug-carrying system on tumor cells is superior to ursolic acid used alone.Cell uptake experiments proved that the nano drug-carrying system can cross the cell membrane and enter the cytoplasm,showing bright yellow fluorescence under a fluorescent microscope,successfully achieving the cell imaging function,and providing a basis for further research on the mechanism of action of the nano drug-carrying system.Finally,a series of tests were carried out on the nano drug-loading system through a fluorescence microscope and a high-content imaging system,and it was found that the nano drug-loading system can cause the mitochondrial membrane potential to decrease,and the reactive oxygen species(ROS)to increase,which promotes tumor cell apoptosis.2.In this paper,Zn-CQDs were prepared by high-temperature pyrolysis.The quantumdots have excellent fluorescence properties.The morphological characterization of Zn-CQDs by TEM shows that the particle size of quantum dots is 5.16 ± 0.32 nm.The natural anticancer drug ursolic acid is modified with polyethylene glycol and folic acid,not only to improve water solubility and to make it targeted;to further enhance anticancer activity,it is connected to the natural anticancer drug paclitaxel(PTX),The formation of UA-PEG-FA-PTX helps to prolong the residence time in the body.The Zn-CQDs were physically adsorbed with UA-PEG-FA-PTX by electrostatic force,and the nano-drug loading system Zn-CQDs@UA-PEG-FA-PTX with fluorescent properties was constructed.The drug-carrying system was characterized in detail by TEM,SEM,FTIR,UV,XRD,1H NMR and other instruments.The inhibitory activity of nano-drug system Zn-CQDs@UA-PEG-FA-PTX on tumor cells was detected by MTT method.The results showed that the inhibitory activity of nano-drug system on tumor cells was superior to ursolic acid alone.Cell uptake experiments prove that the nano drug-loading system can enter the cytoplasm and realize the function of cell imaging.Finally,a fluorescence microscope and a high-content imaging system were used to conduct apoptosis-related experiments on the nano-drug-loaded system.It was found that the nano-drug-loaded system could induce apoptosis,meanwhile,the mitochondrial membrane potential decreased and the reactive oxygen species increased.更多还原