【作者】 周强；

【导师】 赵语；

【作者基本信息】 重庆医科大学 ， 临床药学， 2025， 硕士

【摘要】 研究背景:恶性黑色素瘤是一种由黑素细胞恶变形成的皮肤肿瘤,发病率和死亡率呈快速增长趋势,但现有治疗手段十分有限且耐药率高。吴茱萸碱(EVO)是一种效果显著的抗肿瘤新药,可以通过多途径杀伤黑色素瘤细胞,但因强疏水性导致抗肿瘤效果受限。利用纳米技术构建药物递送系统能够显著提高EVO溶解度,改善其生物利用度,增加EVO在肿瘤部位的富集程度并增强抗肿瘤效果。目的:本研究旨在设计和制备EVO@ZIF-8/HA(EZH)纳米粒,评估其增强EVO抗黑色素瘤的效果以及生物安全性,为EVO的临床应用提供新的策略。方法:1.纳米粒的制备与表征:利用“一步合成法”合成EVO@ZIF-8(EZ),通过静电吸附作用将透明质酸(HA)修饰到其表面得到EVO@ZIF-8/HA(EZH)纳米粒,随后对粒径、Zeta电位、PDI、形态进行表征,考察稳定性、体外释放及体外溶血情况。2.体外抗肿瘤效果研究:倒置荧光显微镜观察B16F10细胞摄取纳米粒,评估纳米粒被摄取以及主动靶向能力;通过细胞毒性实验(MTT)、细胞活死染色、集落形成实验及细胞凋亡实验考察EZH增强EVO对B16F10细胞的杀伤和抑制增殖效果。3.体内抗肿瘤效果及生物安全性评价:利用小鼠活体成像和器官离体成像考察纳米粒在B16F10荷瘤小鼠体内的分布情况;进行体内药效学实验,记录给药后肿瘤体积、体重变化,评估抗肿瘤效果;通过血常规、血生化及H&E染色评估体内安全性。结果:1.成功制备纳米粒EZH,平均水合粒径为175.4±0.7 nm,PDI为0.09±0.01,Zeta电位为-36.2±0.5 mV。EZH具有高载药量(6.2±0.6%)、良好的稳定性、pH响应性体外释放效果以及血液相容性。2.体外抗肿瘤实验结果显示,HA修饰的纳米粒具有主动靶向能力,细胞摄取效率更高;细胞毒性、活死染色、集落形成和凋亡实验均显示EZH可增强EVO的抗肿瘤效果。3.体内抗肿瘤效果及生物安全性评价结果显示:小鼠活体及离体成像显示HA修饰纳米粒可快速高效聚集于肿瘤部位,体现其主动靶向性。体内药效学实验结果显示EZH组肿瘤体积的大小仅为对照组的25.5%,EVO组的45.4%,显著增强了EVO的抗肿瘤效果,抑制了B16F10肿瘤的生长。血常规、血生化及器官H&E染色分析显示纳米粒体内安全性良好,未见明显毒性。结论:本研究成功开发了一种兼具主动靶向肿瘤组织和pH响应性的纳米粒EZH。EZH具有良好的形态、药物负载效率、稳定性等,解决了EVO疏水性强的难题,使之能在生理状态下稳定存在。EZH能将EVO高效递送到肿瘤部位并显著增强EVO的抗肿瘤作用,为恶性黑色素瘤的临床治疗提供了新的策略。更多还原

【Abstract】 BackgroundMelanoma,a cutaneous malignancy arising from the neoplastic transformation of melanocytes,is experiencing a rapid increase in incidence and mortality.However,the currently available therapeutic options are limited and frequently hampered by substantial drug resistance.Evarine(EVO)is an efficacious anti-tumor agent capable of inducing melanoma cell death through multiple pathways;however,its therapeutic efficacy is constrained by its pronounced hydrophobicity and low bioavailability.The application of nanotechnology to construct drug delivery systems can markedly enhance its solubility,and increase drug concentration at the tumor site,thereby improving bioavailability and enhancing its anti-tumor activity.ObjectiveThe objective of this study was to design and prepare EVO@ZIF-8/HA(EZH)nanoparticles,evaluate their enhanced anti-melanoma efficacy and biosafety,and provide novel strategies for the clinical application of EVO.Methods1.Preparation and characterization of nanoparticles:EVO@ZIF-8(EZ)was synthesized via a one-step synthesis method,and EVO@ZIF-8/HA(EZH)nanoparticles were obtained through electrostatic adsorption-mediated surface modification with hyaluronic acid(HA).Subsequently,particle size,Zeta potential,PDI,and morphology were characterized.Moreover,the in vitro stability,release kinetics,and hemolytic activity were also examined.2.In vitro anti-tumor effect study:The uptake of nanoparticles by B16F10cells was visualized using an inverted fluorescence microscope to assess nanoparticle uptake efficiency and active targeting capability.Cytotoxicity assays(MTT),cell death staining,colony formation assays,and apoptosis analyses were conducted to evaluate the effects of EZH on EVO-induced cytotoxicity and proliferation inhibition in B16F10 cells.3.In Vivo anti-tumor effect and biosafety evaluation:The biodistribution of nanoparticles in B16F10 tumor-bearing mice was examined using both in vivo imaging and ex vivo organ imaging.Pharmacodynamic experiments were performed in vivo to monitor changes in tumor volume and body weight post-administration,thereby evaluating anti-tumor efficacy.In vivo safety was assessed via blood routine tests,biochemical analyses,and histopathological H&E staining.Results1.EZH nanoparticles were successfully fabricated with an average hydrated particle size of 175.4±0.7 nm,PDI of 0.09±0.01,and Zeta potential of-36.2±0.5 mV.EZH demonstrated high drug loading(6.2±0.6%),excellent stability,pH-responsive in vitro release characteristics,and favorable blood compatibility.2.In vitro anti-tumor experiments revealed that HA-modified nanoparticles exhibited active targeting ability and enhanced cellular uptake efficiency.Cytotoxicity,living/death staining,colony formation,and apoptosis analyses confirmed that EZH significantly augmented the anti-tumor efficacy of EVO.3.In vivo anti-tumor effect and biosafety evaluations indicated that both in vivo and ex vivo imaging of mice demonstrated rapid and efficient aggregation of HA-modified nanoparticles at tumor sites,reflecting their active targeting capability.In vivo pharmacodynamic results showed that the tumor volume in the EZH group was only 25.5%of the control group and45.4%of the EVO group,indicating a substantial enhancement of EVO’s anti-tumor efficacy and inhibition of B16F10 tumor growth.Blood routine,biochemical,and organ H&E staining analyses confirmed the in vivo safety of the nanoparticles,with no evident toxicity observed.ConclusionIn this study,we successfully developed EZH nanoparticles with dual functionalities of active tumor targeting and pH responsiveness.EZH exhibited desirable morphology,drug loading efficiency,and stability,addressing the hydrophobicity challenge of EVO and ensuring its stability under physiological conditions.EZH efficiently delivered EVO to the tumor site,significantly enhancing its anti-tumor efficacy,and providing a promising strategy for the clinical treatment of malignant melanoma.更多还原