【作者】 陈铭；

【导师】 李辉；

【作者基本信息】 中国地质大学 ， 材料科学与工程， 2025， 博士

【摘要】 肿瘤作为一种全球性的重大健康问题,严重威胁着人类的生命和健康。近年来,随着分子生物学和纳米医学等多学科交叉发展,新型核酸药物靶向治疗为肿瘤患者带来了新的希望。通过纳米载体可以有效实现核酸药物的精准递送,能特异性地抑制肿瘤细胞的关键分子信号通路,从而实现抑制肿瘤细胞的生长和增殖,同时减少药物在正常组织中的分布,降低副作用。球形核酸（SNAs）由纳米颗粒核心和一层密集排列的寡核苷酸壳组成,具有优异的抗核酸酶降解能力、低免疫刺激性以及无需转染试剂即可实现高效细胞摄取,成为理想的肿瘤治疗药物,可以实现高效且持久的抗肿瘤效果。然而,SNAs的进一步成药应用仍然面临诸多挑战,包括构效关系不明确、集成功能困难、纳米核心的生物安全性等问题。近期,研究者们实现了以C₆₀、蛋白分子等单个大分子为核心,化学修饰DNA序列,精准合成分子量明确的第二代球形核酸（m-SNAs）,系统的研究了它们的构效关系问题,却存在核酸序列单一、功能拓展困难的问题。如何通过简单且合理路径设计,借助分子组装原理,构建结构明确、功能多样的新型纳米核酸药物,是当前亟待解决的关键科学问题。针对上述难点及实际需求,我们将分子球形核酸的核心置换为具有超分子拓展、组装能力的环糊精分子,构筑了一种新型超分子球形核酸药物（Supra-SNAs）。Supra-SNAs的分子结构可以由合成、分离条件等进行调控,从而影响其生物学效能,同时环糊精的主体空腔可以进一步结合功能化的核酸序列,有效提升其递送和靶向特性。在本论文中,我们构建了一系列新型Supra-SNAs分子,通过模块化的设计实现了对Supra-SNAs结构和功能的精细调控,系统考察了多种合成、组装条件,对于Supra-SNAs性能的影响。本论文的主要工作内容包括如下要点:1.具有精确结构的超分子球形核酸合成及核仁素靶向基因递送应用为了深入探究SNAs构效关系并实现其多功能修饰,我们采用分子内调控的策略。通过点击化学反应,在七取代叠氮化β-环糊精表面精准修饰了1至7条DNA反义核酸分子,合成了一系列m-SNAs化合物。同样地,在金刚烷分子（Ada）一端修饰了AS1411适配体（靶向细胞核仁素受体）,获得Ada-AS1411功能序列。借助环糊精和金刚烷的超分子组装特性,将m-SNAs与Ada-AS1411组合,成功构建了具有结构精确、靶向细胞核仁素的Supra-SNAs药物。凝胶电泳分析、原子力显微镜（AFM）表征、基质辅助激光解吸/电离飞行时间（MALDI-TOF）质谱等多种手段,证明了Supra-SNAs分子量明确、结构明晰。SKOV3细胞实验结果表明,随着DNA接枝密度的增加,细胞摄取效率也得到逐渐提升;在引入靶向适配体AS1411后,细胞摄取效率进一步提高约2.5倍。蛋白组学研究表明,Supra-SNAs药物通过抑制细胞内磷脂酰肌醇3-激酶（PI3K）/蛋白激酶B（AKT）信号通路,诱导细胞凋亡,从而有效杀伤肿瘤细胞。荷瘤小鼠实验进一步表明,Supra-SNAs在肿瘤区域展现出更优异的肿瘤滞留效果以及良好的抗肿瘤效果,对正常组织未表现明显的毒性。2.多级精确可控组装的超分子球形核酸用于高效核酸递送前面对于Supra-SNAs的构效关系研究初步表明,DNA接枝量和接枝密度可以有效的提升其性能,为了进一步调控体系DNA接枝密度,我们在分子内调控的基础上,进一步引入分子间的调控策略,考察这种双重调控作用对于Supra-SNAs性能的影响。我们通过点击化学反应,分别在七取代叠氮化β-环糊精表面精准修饰单链DNA序列和Y型DNA序列,成功获得了携带有修饰7条和14条DNA链的两种m-SNAs。同样,我们设计合成了负载有不同数量金刚烷分子的一系列线形、Y形、四边形和星形多臂客体分子,并进一步利用超分子组装将这些多臂客体分子与m-SNAs结合,成功构建了不同尺寸大小和DNA接枝密度的八种Supra-SNAs分子。它们的尺寸从20到78 nm,接枝密度1.6到1.9 pmol/cm²。通过荧光实验,我们研究了它们在溶液中的组装动力学和热力学,滴定确定了金刚烷客体分子与两类m-SNAs之间的组装比例,分别为1:2、1:3、1:4和1:8,验证了分子设计。进一步的细胞实验表明,随着DNA接枝密度的增加,细胞摄取效率逐渐提高,并在四个金刚烷组装（Tetra-Supra-SNA-7）时增幅最强。然而,当用八个金刚烷复合体组装时（Octa-Supra-SNA-7）,细胞摄取效率提升有限。此外,基于以Y型DNA构建的Supra-SNAs,其细胞摄取效率要明显优于以单链Supra-SNAs体系。在亚细胞器精细分析中,我们发现Supra-SNAs在进入细胞后,会优先聚集在溶酶体中,随后再进入线粒体,并最终造成线粒体损伤。在体内荧光成像实验中,我们也可以观察到Tetra-Supra-SNA-7在肿瘤区域有更好的富集效果,优于其他组装形式的Supra-SNAs。3.多功能修饰的超分子球形核酸实现对PD-L1/CD47蛋白的靶向降解前序的研究表明,Supra-SNAs体系具有良好的模块化能力,我们进一步引入多肽序列,构建了一种多功能修饰的Supra-SNAs结构,可以特异性结合PD-L1与CD47蛋白双免疫检查点,从而实现更有效的靶向降解和肿瘤治疗。具体来说,我们通过点击反应在七取代叠氮化β-环糊精表面分别修饰了7条PD-L1核酸适配体与7条CD47结合多肽,得到两种不同的m-SNAs（m-SNA-P7和m-SNA-C7）。紧接着,我们设计了两条氨基修饰的溶酶体靶向适配体链,其间断区为两条互补序列组成,并通过酰胺键连接在金刚烷一端。利用超分子组装技术,我们将这两条靶向适配体链分别与两个m-SNAs组装形成Supra-SNAs。通过碱基互补配对技术,将两个Supra-SNAs组装成一个具有三种功能修饰的复合体Supra-SNAs。通过一系列表征手段如凝胶电泳分析、AFM表征等证明了复合体Supra-SNAs的成功构建。体外细胞实验表明,对比单一蛋白降解功能的Supra-SNAs,该复合体Supra-SNAs不仅表现出优异的蛋白降解能力,能同时抑制PD-L1与CD47蛋白两个免疫检查点,有效抑制肿瘤细胞的生长。更多还原

【Abstract】 Tumors,as a major global health problem,have seriously threatened human life and health.In recent years,with the cross-disciplinary development of molecular biology and nanomedicine,new nucleic acid drug targeted therapy has brought new hope to cancer patients.Through nanocarriers,nucleic acid drugs can be effectively delivered accurately,which can specifically inhibit the key molecular signaling pathways of tumor cells,thereby inhibiting the growth and proliferation of tumor cells,while reducing the distribution in normal tissues and reducing side effects.Spherical nucleic acids(SNAs),composed of a nanoparticle core and a layer of densely arranged oligonucleotide shells,have become ideal tumor therapeutic drugs for efficient and lasting anti-tumor treatment because of its excellent resistance to nuclease degradation,low immunostimulation,and efficient cell uptake without transfection reagents.However,the further drug application of SNAs still faces many challenges,including unclear structure-activity relationship,difficulty in integrating functions,and biosafety of nanocores.Recently,researchers have achieved the precise synthesis of second-generation spherical nucleic acids(m-SNAs)with clear molecular weights by chemically modifying DNA sequences with single macromolecules such as C60 and protein molecules as the core,and systematically studied their structure-activity relationship,but there are still some problems such as single nucleic acid sequences and difficulty in function expansion.Therefore,how to construct new nano-nucleic acid drugs with clear structures and diverse functions through simple and reasonable path design and with the help of molecular assembly principles is a key scientific problem that needs to be solved urgently.In response to the above difficulties and practical needs,we replaced the core of molecular spherical nucleic acids with cyclodextrin molecules with supramolecular expansion and assembly capabilities,and constructed a new type of supramolecular spherical nucleic acid drug(Supra-SNAs).The structure of Supra-SNAs can be regulated by synthesis and separation conditions,which affecting their biological efficacy.At the same time,the main cavity of cyclodextrin can further combine with functionalized nucleic acid sequences to effectively improve its delivery and targeting properties.In this thesis,we constructed of a series of new Supra-SNAs molecules,and achieved fine regulation of the structure and function of Supra-SNAs through modular design.We systematically investigated the effects of various synthesis and assembly conditions on the Supra-SNAs system.The main work of this thesis includes the following points:1.Precisely Structured Supramolecular Spherical Nucleic Acids for Nucleolin-Targeted Gene DeliveryTo deeply explore the structure-activity relationship of SNAs and realize their multifunctional modification,we firstly adopted the strategy of intramolecular regulation.Through click chemistry reaction,1 to 7 DNA antisense nucleic acid molecules were precisely modified on the surface of hepta-substitutedβ-cyclodextrin to synthesize a series of m-SNAs compounds.AS1411 aptamer(targeting nucleolin receptor)was modified at one end of the adamantane molecule(Ada)to obtain the Ada-AS1411functional sequence.With the help of self-assembly characteristics of cyclodextrin and adamantane,m-SNAs were combined with Ada-AS1411 to successfully construct Supra-SNAs with precise structure and targeting nucleolin.The clear molecular weight and structure of Supra-SNAs were proved by gel electrophoresis analysis,atomic force microscopy(AFM)characterization,matrix-assisted laser desorption/ionization time-of-flight(MALDI-TOF)mass spectrometry and other methods.The SKOV3 cell experiments results showed that as the density of DNA grafting increased,the cell uptake efficiency was gradually improved.With the introduction of the targeted aptamer AS1411,the cell uptake efficiency was further increased by about 2.5 times.Proteomics studies have shown that Supra-SNAs induce cell apoptosis by inhibiting the intracellular phosphatidylinositol 3-kinase(PI3K)/protein kinase B(AKT)signaling pathway,effectively killing tumor cells.Tumor-bearing mice further demonstrated that Supra-SNAs exhibited superior tumor retention and antitumor effects in tumor region,and did not show obvious toxicity to normal tissues.2.Controllable Multilevel Precise Structures of Supramolecular Spherical Nucleic Acids for Efficient Antisense DeliveryPrevious studies on the structure-activity relationship of Supra-SNAs have shown that the amount and the grafting density of DNA can effectively improve its performance.In order to further regulate the DNA grafting density,we introduced intermolecular and intramolecular regulations strategy to investigate these dual regulations effect on the performance of Supra-SNAs.We used click-chemistry to precisely modify single-stranded and Y-shaped DNA sequences on the surface of hepta-substitutedβ-cyclodextrin,respectively,and successfully obtained two m-SNAs with 7 and 14 modified DNA chains.Similarly,we designed and synthesized a series of linear,Y-shaped,quadrilateral and star-shaped multi-arm guest molecules loaded with different numbers of adamantane molecules,and further used supramolecular assembly to combine these guest molecules with m-SNAs,successfully constructing eight Supra-SNAs with different sizes and DNA grafting densities.These sizes range from 20 to 78 nm,and the grafting density is 1.6 to1.9 pmol/cm~2.Through fluorescence experiments,we investigated their assembly kinetics and thermodynamics in solution,and determined the assembly ratios between adamantane guest molecule and m-SNAs,which were 1:2,1:3,1:4 and 1:8,respectively.Further cell experiments showed that with the increase of DNA grafting density,the cellular uptake efficiency gradually increased,with the strongest enhancement observed when assembled with four adamantane complexes(Tetra-Supra-SNA-7).However,when assembled with eight adamantane complexes(Octaa-Supra-SNA-7),the cellular uptake efficiency was limited.It’s worth noting that the cellular uptake efficiency of Supra-SNAs constructed based on Y-shaped DNA was significantly better than that of the single-stranded Supra-SNAs.Intriguingly,subcellular analysis revealed that upon entering cells,Supra-SNAs first aggregated in lysosomes before migrating to mitochondria,ultimately causing mitochondrial damage.In vivo fluorescence imaging experiments also demonstrated that Supra-SNAs assembled with four adamantanes exhibited superior tumor enrichment in the tumor region compared to other assembly forms.3.Multifunctionally modified Supramolecular Spherical Nucleic Acids for Targeting and Degradation of PD-L1/CD47 ProteinsPrevious studies have shown that the Supra-SNAs have good modularity.We further introduced peptide sequences and constructed a multifunctional modified Supra-SNAs that can specifically bind to the dual immune checkpoints of PD-L1 and CD47 proteins,achieving effective targeted degradation and tumor treatment.Specifically,we modified seven PD-L1 nucleic acid aptamers and seven CD47 binding peptides on the surface of hepta-substitutedβ-cyclodextrin by click reaction to obtain two different m-SNAs(m-SNA-P7 and m-SNA-C7).Similarly,we designed two amino-modified lysosomal targeting aptamer chains,which interrupter regions consisted of two complementary sequences and were connected to adamantane by amide bonds.By supramolecular assembly technology,we assembled these two targeting aptamer chains with two m-SNAs to form Supra-SNAs.The final complex Supra-SNAs were constructed by the assembly of two Supra-SNAs through base complementary pairing.The successful construction of the complex Supra-SNAs was demonstrated through a series of characterization methods such as gel electrophoresis analysis and AFM characterization.In vitro cell experiments showed that compared with Supra-SNAs with a single protein degradation function,the complex Supra-SNAs not only exhibited excellent protein degradation ability,but also could simultaneously inhibit two immune checkpoints,PD-L1 and CD47 proteins,effectively inhibiting the growth of tumor cells.更多还原