【作者】 徐涛；

【导师】 杨红；

【作者基本信息】 苏州大学 ， 药剂学， 2020， 硕士

【摘要】 恶性肿瘤是全球主要的死亡原因。常规肿瘤治疗方法的固有局限性促使纳米药物在更有效、更安全的肿瘤治疗中快速发展。然而在纳米药物的开发中,可控制和可重复的纳米药物合成、纳米载体材料生物相容性、安全性等问题还需解决。基于蛋白质模板法仿生合成的纳米药物递送系统具有合成简单、可控、生物相容性好的优点,是理想的纳米药物载体,可有效提高肿瘤治疗效果。两种或多种药物的联合治疗可促进不同药物之间的协同作用从而降低药物毒性和克服耐药性,在肿瘤治疗中有巨大的潜力。联合治疗和纳米药物递送在肿瘤治疗中显示出重大前景。本研究将化学疗法和光动力疗法(Photodynamic therapy,PDT)通过纳米给药系统联合,以实现协同抗肿瘤能力,从而改善肿瘤治疗效果。本文使用人血清白蛋白(Human serum albumin,HSA)作为药物递送载体,通过配位诱导的沉淀反应模拟白蛋白诱导无机离子生物矿化的原理,共同包载顺铂前体离子和光敏剂二氢卟吩e6(Chlorine 6,Ce6),制得Pt/Ce6@HSA纳米粒(Pt/Ce6@HSA NPs)。通过优化蛋白纳米粒的粒径、释药行为等理化特性,Pt/Ce6@HSA纳米粒可以有效被肿瘤细胞摄取,在低强度660 nm激光照射下,在肿瘤细胞内产生更多单线态氧,增强药物抗肿瘤活性并实现肿瘤的高效协同治疗。本文研究表明,基于蛋白仿生合成的联合疗法具有较大的潜力,可用于化学联合光动力疗法以达到有效的抗肿瘤效果。具体内容概述如下:第一章:简要阐述了肿瘤及其治疗方法。着重介绍了铂类配合物作为化疗药物和光敏剂及光动力治疗等新型肿瘤治疗手段,对化疗和光动力治疗的联合治疗进行了简要综述,然后介绍了白蛋白纳米药物载体的研究进展,进而提出本文的课题设计思路及研究内容。第二章:通过条件筛选和优化,制备合成了粒径合适的Pt/Ce6@HSA纳米粒,并对其进行结构和理化性质表征。实验结果表明,Pt/Ce6@HSA纳米粒透射电镜大小为4.9±1.0 nm,水合粒径30 nm左右,粒径均一、分散性良好;与游离药物相比,具有较优的稳定性及较好的单线态氧产生能力,纳米粒的单线态氧量子产率为游离Ce6的2倍;EDX、XPS结果证明铂以正二价形式存在于上述纳米粒;药物释放结果发现,与游离顺铂及Ce6相比,蛋白纳米粒48 h的累积释放显著小于游离药物,具有明显的缓释作用。第三章:考察了 Pt/Ce6@HSA纳米粒在细胞水平的抗肿瘤效应。通过细胞摄取、亚细胞定位实验、MTT实验、2’,7’-二氯荧光黄双乙酸盐(DCFH-DA)、吖啶橙(AO)、线粒体膜电位荧光探针(JC-1)等细胞染色实验、凋亡以及Western blot等实验考察了纳米粒对肿瘤细胞的杀伤作用和凋亡机理。实验结果表明,纳米粒通过网格蛋白介导的内吞作用有效进入细胞并主要分布在细胞溶酶体中;加上PDT效果后,细胞内单线态氧水平显著增加,能够破坏溶酶体膜,促进纳米粒进入细胞核中发挥化疗作用,增加细胞毒性作用,使IC50降至1.91 μmol·L-1,降低了 21.5倍;化疗联合光动力治疗时,线粒体膜电位下降明显,细胞凋亡增多,凋亡相关蛋白含量变化明显,证明所构建的纳米粒可有效进入肿瘤细胞,并通过化疗以及光动力治疗的协同效应显著提高顺铂和Ce6对肿瘤细胞的损伤作用。第四章:在细胞水平实验的基础上,进一步考察了 Pt/Ce6@HSA纳米粒在动物体内的抗肿瘤效果和安全性评价。主要通过药代动力学、组织分布、抑瘤、生化指标等实验考察。结果表明,与游离顺铂相比,纳米粒血液循环更长,消除半衰期时间为游离顺铂的7倍,AUC为顺铂的90倍;纳米粒在4T1皮下及原位肿瘤模型上均具有良好的肿瘤靶向性,静脉注射12 h后肿瘤部位铂的含量能够达到12 ID%g-1且Ce6能够在肿瘤部位有效产生单线态氧,从而通过化疗和光动力治疗的联合作用实现皮下肿瘤的完全消除,并有效抑制原位肿瘤转移;初步安全性结果表明,纳米粒纳米粒可以从组织内逐渐清除,与游离顺铂相比,Pt/Ce6@HSA纳米粒无明显肝肾损伤,具有更低的毒副作用。总之,利用蛋白质模板的仿生合成法构建了具有化疗和光动力治疗效果的纳米粒,提升药物治疗效果并能够有效降低药物毒性,显示出对肿瘤细胞的协同杀伤作用和抑制肿瘤生长的作用。更多还原

【Abstract】 Cancer is the leading cause of death worldwide.The inherent limitations of conventional cancer treatment methods have prompted the rapid development of nanomedicines in more effective and safer cancer treatments.However,in the development of nanomedicine,the controllable and repeatable nanomedicine synthesis,the biocompatibility and safety of nanocarrier materials need to be solved.The nano-drug delivery system based on the biomimetic synthesis of the protein template method has the advantages of simple synthesis,controllability,and good biocompatibility.It is an ideal nano-drug carrier and can effectively improve the effect of tumor treatment.The combination therapy of two or more drugs can promote the synergistic effect between different drugs to reduce drug toxicities and overcome drug resistance,which has great potential in tumor treatment.Therefore combination therapy and nano-drug delivery show great promise in cancer treatment.In this study,chemotherapy and photodynamic therapy(PDT)were combined through a nano drug delivery system to achieve a synergistic anti-tumor ability,thereby improving the effectiveness of tumor treatment.Using human serum albumin(HSA)as a drug delivery vehicle,the principle of albumin-induced biomineralization of inorganic ions was simulated by coordination-induced precipitation reactions.Cisplatin precursor ion and photosensitizer chlorine 6(Ce6)were co-loaded to provide dual drug loading delivery system Pt/Ce6@HSA nanoparticles(Pt/Ce6@HSA NPs).By optimizing the physical and chemical properties of this protein nanoparticles,such as particle size and drug release behavior,Pt/Ce6@HSA nanoparticles can be effectively taken up by tumor cells.After 660nm low intensity laser irradiation,the cellular singlet oxygen level in Pt/Ce6@HSA nanoparticles treated cells was elevated dramatically,which can enhance the anti-tumor activity of the drug and achieve efficient treatment of tumors.This study showed that albumin biomimetic synthsis-based nanotherapeutics has great potential and can be used in combination with chemical-photodynamic therapy to achieve promise antitumor effects.The specific content was summarized as follows:Chapter 1:A brief description of cancer and its treatment methods were first introduced.The platinum chemotherapeutic drugs,photosensitizers and novel photodynamic therapy were emphatically introduced.The combined treatment of chemotherapy and photodynamic therapy was briefly reviewed.Then the research progress of albumin nano-drug carriers was introduced,after that the project of design ideas and research content were proposed.Chapter 2:By screening and optimization of preparation and synthesis conditions,Pt/Ce6@HSA nanoparticles with appropriate size were prepared,and characterized its structure and physicochemical properties.The experimental results indicated that the TEM size of the nanoparticles was 4.9 ± 1.0 nm and the hydrated particle size was about 30 nm,the particle size was uniform and the dispersion wass good;compared with free drugs,they have better stability and better singlet oxygen generation ability,the singlet oxygen yield of the nanoparticle was 2 times that of the free Ce6;EDX and XPS results indicated that platinum was bivalent form existing in the above-mentioned nanoparticles;the drug release results founded that compared with free cisplatin and Ce6,the cumulative release of protein nanoparticles at 48 h was significantly smaller than that of free drugs,indicating a significant sustained release effect.Chapter 3:The antitumor effect of Pt/Ce6@HSA nanoparticles at the cellular level was investigated.Celluar uptake,subcellular localization experiments,MTT experiment and cell staining experiments including 2,7-Dichlorodihydrofluorescein diacetate(DCFH-DA),acridine orange(AO),mitochondrial membrane potential statining experiments(JC-1)and so on,apoptosis,Western blot and other experiments have carried on to examine the cell-killing effect and apoptosis mechanism of nanoparticles on tumor cells.The experimental results showed that the nanoparticles effectively entered cells through clathrin-mediated endocytosis and were mainly distributed in the lysosome;after adding the PDT effect,the singlet oxygen level in the cell was significantly increased,which can destroy the lysosomal membrane and then promote the nanoparticles to enter the nucleus to play the role of chemotherapy and increase the cytotoxic effect,leading to the IC50 was reduced to 1.91 μmol·L-1,which was a decrease of 21.5 times;when chemotherapy combined with photodynamic therapy,mitochondrial membrane potential decreased significantly,apoptosis increased and apoptosis-related protein content changed significantly,which proved that the constructed nanoparticles can effectively enter tumor cells,and the synergistic effect of chemotherapy and photodynamic therapy can significantly improve the damage to tumor cells.Chapter 4:On the basis of cell-level experiments,the antitumor effect and safety evaluation of Pt/Ce6@HSA nanoparticles at the animal level were further investigated.Pharmacokinetics,tissue distribution,tumor suppression,biochemical indicators and other experimental were mainly carried on.The results showed that compared with free cisplatin,nanoparticles have longer blood circulation,the elimination half-life time was 7 times that of free drugs,and the AUCwas 90 times that of cisplatin;nanoparticles had great tumor targeting in 4T1 subcutaneous and orthotropic tumor models,the content of Pt in the tumor site can reach 12ID%g-1 after 12 hours of intravenous injection,and Ce6 can effectively produce singlet oxygen in the tumor site,thereby achieving the complete elimination of subcutaneous tumors and effective inhibition of tumor metastasis in situ through the combined effect of chemotherapy and photodynamic therapy;preliminary safety results showed that nanoparticles can be gradually cleared from the tissue and compared with free cisplatin,Pt/Ce6@HSA nanoparticles had no significant liver and kidney damage indicating lower toxic side effects.In conclusion,the biomimetic synthesis of protein templates was used to construct nanoparticles with the effects of chemotherapy and photodynamic therapy,which can improve the drug treatment effect and effectively reduce drug toxicity,showing a synergistic killing effect on tumor cells and a tumor growth inhibition effect.更多还原