【作者】 彭川；

【导师】 丁明明；

【作者基本信息】 四川大学 ， 生物医学工程， 2023， 博士

【摘要】 疾病的诊断、治疗和发病机制的探究一直是生物医用高分子领域的研究重点。在疾病诊断方面,具有高灵敏度、选择性和非侵入性等优点的荧光成像在生物医学和传感等领域得到了广泛应用。然而,目前大多数已报道的荧光成像分子都需要复杂的合成过程,这会造成成本增加和生物相容性较差等问题,进而影响其在生物医学成像和治疗等方面的应用。在疾病治疗方面,高分子治疗试剂一直受到科研工作者的广泛关注,相比于小分子治疗试剂,高分子治疗试剂具有高疗效、低毒副作用的优势。当疾病发生时,必要的诊断和治疗至关重要,但是对于疾病的发病机制的研究也不可忽视,这将有效预防疾病的发生。例如,高度组织化的淀粉样纤维的产生与许多构象疾病相关,这种疾病的特征是错误折叠的蛋白质失去了正常的生理作用而获得了毒性。因此,利用合成高分子来模拟蛋白质复杂有序的高级结构将有助于理解蛋白质的结构和功能,从而为构象疾病的发生和治疗提供理论依据。其中,聚氨基酸材料因其优异的生物相容性、生物可降解性、易于修饰和保留了蛋白质特有的二级构象等性能而被广泛地用于生物医用材料的研究。基于此,本研究设计合成了一系列仿蛋白聚氨基酸衍生物,探究其在疾病诊断、治疗方面的应用,并通过二级结构的调控为进一步理解构象疾病的发生提供了新的见解。本文主要取得的研究结论如下:1.前期工作通过二硝基苄基（ONB）保护二硫苏糖醇（DTT）上的巯基制备了光敏小分子ONB-DTT,这种光敏小分子能够通过光控原位产生含活性巯基且具有还原性的小分子物质,实现了聚合物材料的还原降解和超快药物释放。受此启发,我们将ONB基团引入聚半胱氨酸中以保护侧链巯基,利用光控实现侧链巯基的可控脱保护和大分子还原剂的高效合成。本章首先设计合成了一系列具有不同分子量和手性且含巯基的大分子还原剂聚半胱氨酸（L-SH和DL-SH）,通过核磁共振氢谱（¹H NMR）、傅里叶变换红外光谱（FTIR）和凝胶渗透色谱（GPC）等手段证明了材料的合成成功。研究表明,大分子还原剂L-SH和DL-SH均表现出优异的荧光性能且生色团为巯基簇,具有目前报道的硫原子基发光物质中最高的荧光量子产率。结合实验和计算机模拟阐明了巯基簇的发光机制。同时,发现这种含大分子还原剂表现出激发依赖的多色发光性能,能够实现活细胞的多色成像。此外,该大分子还原剂可以实现可逆荧光开关性能,不仅能够对胞内活性氧（ROS）和谷胱甘肽（GSH）的含量进行定量检测,而且能够在小鼠体内实现骨关节炎（OA）的诊疗一体化,为新型荧光材料的设计和氧化物质过表达疾病的治疗提供了新平台。2.基于大分子还原剂L-SH和DL-SH上的巯基可以配位金属离子,同时在氧化还原条件下可以实现巯基和二硫键的可逆切换,我们制备出了含Cu²⁺和二硫键的聚半胱氨酸金属药物,期望得到一种在还原条件下能够实现快速高效释药的聚合物囊泡。通过FTIR、动态光散射（DLS）、透射电镜（TEM）、X射线光电子能谱（XPS）等手段证明了金属药物载体的成功制备,并测得自组装体形貌均为囊泡。紫外和荧光测试证明了该金属药物载体可发生类芬顿反应,载药实验表明我们发现了一种提高姜黄素（Cur）载药量的新方法,通过包载光敏小分子ONB-DTT,实现了光控制“特洛伊木马”策略原位产生还原性物质的超快药物释放。细胞实验表明金属药物载药系统具有优异的生物相容性并表现出对肿瘤细胞的特异性杀伤能力,并通过还原响应实现了胞内成像。氧化应激、脂质过氧化、钙网蛋白（CRT）、高迁移率族蛋白B1（HMGB1）和三磷酸腺苷（ATP）实验表明该金属药物载药系统具有引起免疫原性细胞死亡（ICD）的潜力。通过建立小鼠肿瘤模型证明了金属药物载药系统具有良好的肿瘤靶向性,同时能够通过提高抗肿瘤免疫原性,实现对肿瘤的有效抑制和杀伤,表明该金属药物载药系统在肿瘤的临床治疗中具有巨大潜力。3.通过先修饰再聚合和先聚合再修饰的方法合成了一系列烷基链修饰的聚半胱氨酸,¹H NMR、FTIR等手段证明了材料的合成成功,FTIR和圆二色谱（CD）测试证明纯烷基链修饰的聚半胱氨酸在氧化门控作用下无法实现构象的有序转变（β折叠到无归卷曲）,而含氨基甲酸酯键烷基链修饰的聚半胱氨酸在氧化门控作用下实现了构象的有序转变（β折叠到α螺旋）,证明了氨基甲酸酯键是实现聚半胱氨酸构象有序转变的关键,这为理解构象疾病的发生和治疗提供了新见解。更多还原

【Abstract】 The diagnosis,treatment and pathogenesis of disease have always been the focus of biomedical polymer research.In the field of disease diagnosis,fluorescence imaging with high sensitivity,selectivity and non-invasive has been widely used in biomedicine and sensing.However,most of the reported fluorescence imaging molecules require complex synthesis processes,which can cause problems such as increased cost and poor biocompatibility,which in turn affect their applications in biomedical imaging and therapy.In the treatment of disease,polymeric therapeutic reagents have been of great interest to researchers.Compared to small-molecule therapeutic reagents,polymer therapeutic reagents have the advantages of high efficacy and low toxicity.When a disease occurs,the necessary diagnosis and treatment are crucial,but research into the pathogenesis of the disease cannot be ignored,which will effectively prevent the occurrence of the disease.For example,the formation of highly organised amyloid fibres is associated with many conformational diseases,which are characterised by the loss of normal physiological functions of misfolded proteins,resulting in toxicity.Therefore,the use of synthetic polymers to simulate the complex and ordered high-level structure of proteins will help to understand the structure and function of proteins,thereby providing a theoretical basis for the occurrence and treatment of conformational diseases.Polyamino acid materials are widely used in biomedical materials research due to their excellent biocompatibility,biodegradability,ease of modification and retention of the unique secondary conformation of proteins.Based on this,a series of protein-like polyamino acid derivatives were designed and synthesised in this study to explore their application in disease diagnosis and treatment,and to provide new insights for further understanding the occurrence of conformational diseases through secondary structure regulation.The main research conclusions of this paper are as follows:1.In the previous work,we prepared a photosensitive small molecule ONB-DTT by protecting the sulfhydryl group on dithiothreitol（DTT）with dinitrobenzyl（ONB）.This photosensitive small molecule can generate a small molecule DTT with active sulfhydryl group and reducibility in situ by light control,realize the reduction degradation of polymer materials and ultra-fast drug release,and achieve good tumour chemotherapy and immunosynergistic therapy in mice.Inspired by this,we propose to introduce ONB group into polycysteine to protect side-chain thiols and realize controllable deprotection of side-chain thiols by UV light control.Polycysteine（L-SH and DL-SH）with different molecular weights,chirality and containing sulfhydryl groups were synthesised using small cysteine molecules as starting materials by rational design.The successful synthesis of the materials was demonstrated by ¹H NMR,FTIR,GPC and other methods.Studies have shown that both macromolecular reducing agents（L-SH and DL-SH）exhibit excellent fluorescence properties and the chromophore is a thiol cluster,which has the highest fluorescence quantum yield among the currently reported sulfur-based luminescent materials.The luminescence mechanism of thiol clusters has been elucidated by experiments and computer simulations.At the same time,it was found that this macromolecular reducing agent exhibited excitation-dependent multicolour luminescence properties and could achieve multicolour imaging of living cells.In addition,the macromolecular reducing agent can achieve reversible fluorescence switching performance.It can not only quantitatively detect the levels of intracellular reactive oxygen species（ROS）and glutathione（GSH）,but also realize the integration of diagnosis and treatment of osteoarthritis（OA）in mice,which provides a new platform for the design of new fluorescent materials and the treatment of diseases that overexpress oxidizing substances.2.Based on the fact that the sulfhydryl groups on the macromolecular reducing agents（L-SH and DL-SH）can coordinate metal ions,and the reversible switching of sulfhydryl groups and disulfide bonds can be achieved under redox conditions,we prepared polycysteine metal drugs containing Cu²⁺and disulfide bonds.It is expected that a polymeric vesicle can be obtained that can also achieve rapid and efficient drug release under reducing conditions.The results of FTIR,XPS,DLS and TEM showed that the metal drug carriers were successfully prepared and the morphology of the carrier was vesicle.The UV and fluorescence tests proved that the metal drug carrier could undergo a Fenton-like reaction.The drug loading experiment showed that we have found a new method to improve the drug loading of curcumin（Cur）.By loading the photosensitive small molecule ONB-DTT,the ultra-fast drug release of the photo-controlled“Trojan horse”strategy to generate reducing substances in situ was realised.Cell experiments have shown that metal drug delivery systems have excellent biocompatibility and exhibit specific cytotoxicity against tumour cells,and also achieve intracellular imaging through reduction response.The results of oxidative stress,lipid peroxidation,CRT,HMGB1 and ATP experiments showed that the metal drug delivery system has the potential to induce immunogenic cell death（ICD）.The establishment of a mouse tumour model has demonstrated that the metal drug delivery system has good tumour targeting and can now achieve effective inhibition and killing of tumours by enhancing anti-tumour immunogenicity,indicating that the metal drug delivery system has great potential in the clinical treatment of tumours.3.A series of alkyl chain modified polycysteines have been synthesised by modification followed by polymerisation and polymerisation followed by modification.¹H NMR,FTIR and other methods demonstrated the successful synthesis of the materials.FTIR and CD tests have shown that the pure alkyl chain-modified polycysteine could not achieve an ordered conformational transition（β-sheet to random coil）under oxidative gating,while the alkyl chain-modified polycysteine with carbamate bond achieved an ordered conformational transition（β-sheet toα-helix）under oxidative gating,proving that carbamate bonds are the key to achieving an ordered conformational transformation of polycysteine and providing new insights into the occurrence and treatment of conformational diseases.更多还原