【作者】 汪洋；

【导师】 李新松；

【作者基本信息】 东南大学 ， 材料与化工（专业学位）， 2023， 硕士

【摘要】 癌症是一个全球性公共卫生问题,癌症的发病率和死亡率逐年上升,传统的化疗方法副作用明显,并常出现耐药性。化学动力学疗法（CDT）由于副作用小,能够诱导癌细胞内氧化还原稳态失衡而成为一种有潜力的肿瘤治疗方案。但内源性H₂O₂不足,低p H条件和抗氧化防御上调极大地限制了CDT疗效。青蒿琥酯（ART）是经典抗疟药物青蒿素的一种著名临床衍生物,其独特的内过氧化物桥键结构能够在亚铁血红素作用下通过类芬顿反应产生有毒自由基,发挥强大的抗疟活性,还表现出独特的抗肿瘤特性。青蒿琥酯二倍体磷脂（ART-GPC）是ART的一种两亲性衍生物,能够自组装成脂质体（AD-LP）,具有优异的抗疟药效和生物相容性,甚至可以作为纳米载体用于递送各种药物。ART-GPC及AD-LP具有作为一款抗疟新药的临床应用潜力,但其抗肿瘤作用至今尚未明了。为此,本文首先针对ART-GPC开展初步新药质量分析研究,并开发规模化制备AD-LP方法,进一步利用ART-GPC设计一种不依赖于内源性H₂O₂和p H的CDT新策略,探究ART-GPC抗肿瘤作用,具体研究内容如下:第一部分:利用HPLC完成ART-GPC含量测定、有关物质分析及稳定性研究,并开发一种简便的改进薄膜法（MTFH）用于AD-LP的规模化生产。首先利用IR、NMR和MS对放大合成的ART-GPC进行结构确证,随后建立色谱检测方法,确立ART-GPC标准曲线及一定浓度范围内的线性关系。然后测定三批次放大合成ART-GPC的含量均高于98%,利用LC-MS初步确定有关杂质为DHA异构体,通过加速破坏实验评估ART-GPC的稳定性。最后,开发一种简便的MTFH法用于AD-LP的规模化生产,即通过真空干燥在西林瓶内形成前体脂质体薄膜,并以干膜状态储存,经原位水化后形成脂质体。通过DLS和TEM检测MTFH法与传统方法制备的脂质体的理化性质,证实MTFH法制备的AD-LP性能更佳。通过Qb D策略确定MTFH法制备AD-LP的最佳工艺参数,随后在GMP条件下完成AD-LP的规模化生产,并验证MTFH法的可扩展性和重复性。因此,本实验为ART-GPC含量及有关物质检测提供了参考,为确立ART-GPC的生产、贮藏和运输条件提供了科学依据,MTFH方法的建立为AD-LP规模化制备提供了可行性。第二部分:设计了一种负载脂质体的原位海藻酸盐（ALG）水凝胶用于癌症治疗的策略,即使用负载血红素的青蒿琥酯二倍体磷脂脂质体（HAD-LP）作为级联触发的C-中心自由基纳米发生器,实现不依赖于H₂O₂和p H的CDT。首先,以ART-GPC为原料通过薄膜法制备HAD-LP,利用DLS和TEM表征HAD-LP的理化性质。通过亚甲基蓝降解法研究HAD-LP生成C-中心自由基的过程,表明在GSH的作用下,血红素被还原为亚铁血红素,催化ART-GPC水解产物DHA的内过氧化物桥键断裂,以不依赖于H₂O₂和p H的方式生成有毒C-中心自由基。随后通过UV和CLSM监测细胞内GSH和自由基水平的变化,证实血红素的还原能够诱发细胞内GSH耗竭和自由基水平升高,破坏细胞内氧化还原稳态。与MDA-MB-231或4T1细胞共孵育后,HAD-LP表现出高细胞毒性,利用IVIS观察到水凝胶体系在小鼠体内具有良好的滞留作用。最后,建立4T1荷瘤小鼠肿瘤模型,证实含有HAD-LP的水凝胶抗肿瘤效果最佳,肿瘤生长抑制率达到72.6%。因此,HAD-LP结合ALG水凝胶作为C-中心自由基发生器能够诱导细胞凋亡,具有良好的抗肿瘤效果,并且HAD-LP能够实现不依赖于内源性H₂O₂和p H的CDT,有望成为一种新型化学动力学抗肿瘤治疗的临床候选药物。更多还原

【Abstract】 Cancer is a global public health problem,and its incidence and mortality are increasing year by year.Traditional chemotherapy has limited therapeutic effect of cancer due to drug resistance,side effects and other problems.Chemodynamic therapy（CDT）is becoming a powerful tumor treatment option because of its low side effects and redox homeostasis imbalance in cancer cells.However,low p H condition,insufficient endogenous H₂O₂ and up-regulated intracellular antioxidant defense greatly limited the therapeutic effect of CDT.Artesunate（ART）is a well-known clinical derivative of the classic antimalarial drug artemisinin.The unique internal peroxide bridge structure of ART can generate C-center free radicals through Fenton-like reaction under the action of heme.ART exerts strong antimalarial activity and exhibits potential anti-tumor properties.Artesunate dimer glycerophosphocholine（ART-GPC）is an amphiphilic derivative of artesunate invented in our laboratory.ART-GPC can self-assemble into liposome（AD-LP）with excellent antimalarial efficacy and biocompatibility.AD-LP can even be used as a nanocarrier to deliver various drugs.ART-GPC and AD-LP have the potential of clinical application as a new antimalarial drug,but their antitumor effect is unclear.Therefore,a preliminary quality analysis on ART-GPC is firstly carried out in this report,and a large-scale preparation method of AD-LP is developed.Furthermore,ART-GPC is used to design a new CDT strategy being independent of endogenous H₂O₂ and p H to explore its anti-tumor effect.The main contents are as follows.In the first part,ART-GPC content determination,related substance analysis and stability study were performed by HPLC.And a modified thin film method（MTFH）was developed for the large-scale production of AD-LP.Firstly,IR,NMR and MS were used to confirm the structure of the amplified ART-GPC.A chromatographic detection method was established.ART-GPC standard curve and the linear relation within a certain concentration range were established.Subsequently,the content of synthetic ART-GPC in three batches was all higher than 98%after measurement.MS was used to determine the related impurity as DHA isomer,and the stability of ART-GPC was evaluated by accelerated failure experiment.Moreover,a simple MTFH method was developed for the large-scale production of AD-LP.Preliposome film was formed in injection bottle through vacuum drying and stored in dry film state.Liposomes were fabricated after in situ hydration.The physicochemical properties of liposomes prepared by MTFH method and traditional methods were detected by DLS and TEM.The optimum process parameters of AD-LP prepared by MTFH method were determined by Qb D strategy.Finally,the mass production of AD-LP was carried out under GMP conditions,and the expansibility and repeatability of MTFH method were verified by various physicochemical properties.Therefore,this experiment provided a reference for the detection of ART-GPC content and related substances,a scientific basis for establishing the production,storage and transportation conditions of ART-GPC.And the development of MTFH method provided feasibility for large-scale preparation of AD-LP.In the second part,a liposome-incorporated in-situ alginate（ALG）hydrogel locoregional treatment strategy was developed,which involves using hemin-loaded artesunate dimer liposomes（HAD-LP）as cascade-triggered self-amplified C-center free radical nanogenerator to enhance chemodynamic therapy（CDT）in a H₂O₂ and p H-independent manner.Firstly,HAD-LP was prepared by thin film method using ART-GPC as raw material.DLS and TEM were used to characterize the physicochemical properties of HAD-LP.The generation of C-center free radicals from HAD-LP was carefully evaluated by using methylene blue degradation method.The results suggested that the hemin was reduced to heme under the action of GSH,which could catalyze the breakage of endoperoxide of ART-GPC derived DHA to generate toxic C-centered free radicals in a H₂O₂ and p H-independent manner.Moreover,the change of intracellular GSH and free radical level was monitored through UV and CLSM.It was revealed that the hemin reduction induced GSH depletion and elevated free radical level,disrupting cellular redox homeostasis.After coincubation with MDA-MB-231 or 4T1 cells,HAD-LP were found to be highly cytotoxic.The IVIS was used to observe that the hydrogel system had a great retention effect in mice.Finally,the tumor model of 4T1 tumor-bearing mice was established.The HAD-LP incorporated ALG hydrogel system achieved the best antitumor effect with the growth inhibition rate of 72.6%.Together,the hemin-loaded artesunate dimer liposome-incorporated alginate hydrogel possessed effective antitumor activity through redox-triggered C-center free radical generation induced apoptosis in a H₂O₂ and p H-independent manner,which might be a promising candidate in the application of chemodynamic anti-tumor therapy.更多还原