【作者】 王楠；

【导师】 喻长远；

【作者基本信息】 北京化工大学 ， 生物工程， 2025， 硕士

【摘要】 雷公藤红素（Celastrol,Cel）,是一种来源于中药雷公藤根部的五环三萜类天然化合物,能够有效抑制抗氧化蛋白酶过氧化还原蛋白2（PRDX2）的活性,从而诱导肿瘤细胞凋亡,在多种肿瘤类型中表现出显著的抗肿瘤活性。因其优异的药理潜力,被《Cell》杂志评为最具有潜力开发为现代药物的五种天然小分子之一。然而,其临床转化仍面临水溶性差和系统毒性高等关键障碍。本研究针对上述问题,构建了一种基于铁离子(Fe³⁺)配位自组装的肿瘤微环境响应型纳米递送系统（Celastrol-Fe Nanoparticals,CFeNPs）。该体系通过Fe³⁺介导Cel与功能化聚合物多巴胺-甲基聚乙二醇5000(DOPA-mPEG₅₀₀₀)之间的分子自组装,不仅显著提高了Cel的水溶性,同时有效降低了非靶向毒性。在肿瘤组织的酸性及高ATP浓度条件下,CFeNPs能够实现精准释放Cel和Fe³⁺,协同抑制PRDX2抗氧化通路并诱导铁死亡,同时激发免疫原性细胞死亡效应,显著增强抗肿瘤免疫应答。首先,本研究通过配位自组装策略优化CFeNPs的制备工艺。经正交实验确定最佳摩尔比为Cel:Fe³⁺:DOPA-mPEG₅₀₀₀=1:2:1。在水溶液中,CFeNP可通过自组装形成粒径为85.3 nm、PDI为0.165的纳米粒子。紫外-可见光谱与傅里叶变换红外光谱验证了Cel的酚羟基与DOPA-mPEG₅₀₀₀邻苯二酚基团形成Fe³⁺介导的配位键,X射线光电子能谱进一步确认了三价铁的配位中心。该体系表现出良好的制剂稳定性,冻干后复溶的粒径变化率低于5%（PDI<0.2）,药物包封率达70.0±2.0%。体外释放动力学结果表明,在模拟肿瘤微环境（pH 6.5+10 mM ATP）条件下,24小时内Cel的累积释放率达65%,而在中性pH（7.4）条件下几乎无释放,证实其具备良好的环境响应性。进一步的细胞实验表明,CFeNPs对乳腺癌细胞（4T1）具有显著的选择性杀伤能力,其半数抑制浓度(IC₅₀)为4.38μM,显著低于对正常成纤维细胞（3T3）的IC₅₀（17.61μM）;相比之下,游离Cel在两种细胞中的杀伤效应无显著差异。机制研究显示,CFeNPs在高ATP环境中释放Cel和Fe³⁺,Cel抑制PRDX2介导的抗氧化通路,Fe³⁺诱导铁死亡,两者协同作用增强肿瘤细胞死亡。此外,CFeNPs还促进免疫原性细胞死亡和树突状细胞（DCs）成熟,进一步激活抗肿瘤免疫反应。在体内实验中,CFeNPs在荷瘤小鼠模型中显著抑制肿瘤生长,使肿瘤体积较对照组减少65.2%,且未观察到明显的系统毒性。联合抗PD-1抗体（αPD-1）治疗进一步增强了抗肿瘤效果,展现出良好的协同治疗潜力。综上所述,本研究构建了基于金属配位介导的雷公藤红素纳米递送系统（CFeNPs）,其技术优势体现在以下三方面:（1）采用Fe³⁺配位自组装策略实现70.0±2.0%的高效药物包封,较传统物理包封体系显著提升物理稳定性（冻干复溶后粒径变化率<5%）;（2）通过DOPA-mPEG₅₀₀₀的分子屏蔽作用在提高Cel水溶性,并有效降低系统性毒性;（3）结合Cel和Fe³⁺诱导的“铁死亡-免疫调控”协同机制,在肿瘤微环境中实现PRDX2通路抑制与免疫原性细胞死亡的双重激活。该研究为Cel的靶向递送与铁死亡-免疫联合治疗策略提供了新思路和理论依据。更多还原

【Abstract】 Celastrol（Cel）,a pentacyclic triterpenoid natural compound derived from the root of the traditional Chinese herb Tripterygium wilfordii,effectively inhibits the activity of the antioxidant enzyme peroxiredoxin 2（PRDX2）,thereby inducing tumor cell apoptosis and demonstrating significant antitumor activity across multiple cancer types.Recognized by Cell as one of the top five natural small molecules with the highest potential for modern drug development,Celastrol faces critical challenges in clinical translation due to its poor aqueous solubility and systemic toxicity.To address these limitations,this study developed a tumor microenvironment-responsive nanodelivery system based on iron ion(Fe³⁺)-mediated coordination self-assembly,termed Celastrol-Fe Nanoparticles（CFeNPs）.This system leverages Fe³⁺-coordinated molecularself-assemblybetweenCelandfunctionalized dopamine-methoxypolyethylene glycol 5000（DOPA-mPEG5000）,significantly enhancing Cel’s solubility while reducing off-target toxicity.Under acidic and high ATP conditions in tumor tissues,CFeNPs enable precise release of Cel and Fe³⁺,synergistically inhibiting the PRDX2 antioxidant pathway to induce ferroptosis and triggering immunogenic cell death（ICD）,thereby amplifying antitumor immune responses.First,the preparation process of CFeNPs was optimized using a coordination self-assembly strategy.Orthogonal experiments determined the optimal molar ratio of Cel:Fe³⁺:DOPA-mPEG5000 as 1:2:1.In aqueous solution,CFeNPs self-assembled into nanoparticles with an average diameter of 85.3 nm and a polydispersity index（PDI）of 0.165.Ultraviolet-visible（UV-Vis）spectroscopy and Fourier transform infrared（FTIR）spectroscopy confirmed the formation of Fe³⁺-mediated coordination bonds between Cel’s phenolic hydroxyl groups and the catechol moieties of DOPA-mPEG5000,while X-ray photoelectron spectroscopy（XPS）verified the trivalent iron coordination center.The system exhibited excellent formulation stability,with less than 5%variation in particle size（PDI<0.2）after lyophilization and reconstitution,achieving a drug encapsulation efficiency of 70.0±2.0%.In vitro release kinetics revealed that under simulated tumor microenvironment conditions（pH 6.5+10 mM ATP）,cumulative Cel release reached 65%within 24 hours,whereas minimal release（<5%）occurred at neutral pH（7.4）,confirming its robust environmental responsiveness.Further cellular experiments demonstrated that CFeNPs exhibited selective cytotoxicity toward breast cancer cells（4T1）,with a half-maximal inhibitory concentration（IC50）of 4.38μM,significantly lower than that for normal fibroblasts（3T3,IC50=17.61μM）.In contrast,free Cel showed comparable cytotoxicity in both cell types.Mechanistic studies revealed that under high ATP conditions,CFeNPs released Cel and Fe³⁺:Cel inhibited the PRDX2-mediated antioxidant pathway,while Fe³⁺induced ferroptosis,synergistically enhancing tumor cell death.Additionally,CFeNPs promoted immunogenic cell death and dendritic cell（DC）maturation,further activating antitumor immunity.In in vivo studies,CFeNPs significantly suppressed tumor growth in a murine xenograft model,reducing tumor volume by 65.2%compared to the control group,with no observed systemic toxicity.Combination therapy with anti-PD-1 antibodies（αPD-1）further amplified antitumor efficacy,highlighting its potential for synergistic treatment.In summary,this study established a Celastrol nanodelivery system（CFeNPs）based on metal coordination-mediated self-assembly,with three key technological advantages:（1）High encapsulation efficiency（70.0±2.0%）via Fe³⁺coordination self-assembly,significantly improving physical stability compared to traditional physical encapsulation methods（particle size variation<5% post-lyophilization）;（2）Enhanced solubility and reduced systemic toxicity through molecular shielding by DOPA-mPEG5000;（3）Synergistic"ferroptosis-immunomodulation"mechanism combining Cel and Fe³⁺,enabling dual activation of PRDX2 pathway inhibition and immunogenic cell death in the tumor microenvironment.This work provides novel insights and a theoreticalfoundationfortargetedCeldeliveryand ferroptosis-immunotherapy combination strategies.更多还原