透明质酸修饰羽扇豆醇沸石咪唑骨架材料制备、表征及抗肝纤维化研究

Preparation, characterization and anti-hepatic fibrosis study of hyaluronic acid-modified psoraleol ZIF-8

摘要

目的 构建一种透明质酸(hyaluronic acid,HA)修饰、pH响应的羽扇豆醇(Lupeol)沸石咪唑骨架(zeolitic imidazolate framework,ZIF-8)递药系统,并进行理化性质和抗肝纤维化评价。方法 采用单因素法优化ZIF-8的合成,通过一步合成法制备Lupeol@ZIF-8,经HA修饰得HA/Lupeol@ZIF-8。采用透射电镜(transmission electron microscope,TEM)、动态光散射(dynamic light scattering,DLS)、傅里叶红外光谱法(fourier transform infrared spectroscopy,FT-IR)、X射线衍射法(X-Ray diffraction,XRD)进行表征。评估纳米颗粒在不同pH磷酸盐缓冲液的药物释放特性及其生理环境稳定性。通过细胞毒性实验(cell counting kit-8,CCK-8)、活性氧和细胞摄取检测分析HA/Lupeol@ZIF-8对HSC-T6细胞的增殖抑制、氧化应激和靶向细胞效应,并采用Western blot技术检测相关蛋白表达水平。建立肝纤维化(hepatic fibrosis,HF)大鼠模型,考察HA/Lupeol@ZIF-8的体内组织分布特征。结果 ZIF-8最佳处方工艺为25 ℃、Zn²⁺/2-甲基咪唑(2-Methylimidazole,2-Mim)摩尔比1∶8反应24 h。Lupeol@ZIF-8最佳载药方法为一步合成法,2-Mim∶Lupeol=1∶8为合成最佳比例,该条件下所得颗粒包封率为97.36%,载药率为23.75%。TEM表明ZIF-8与Lupeol@ZIF-8呈均匀多面体结构,而HA/Lupeol@ZIF-8形态接近球形;DLS测得三者粒径为(72.81±0.43)nm、(80.29±1.03)nm和(125.7±0.16)nm,分布均一(PDI<0.4);Zeta电位为(15.8±0.57)mV、(-7.06±0.02)mV和(-13.2±0.64)mV。XRD与FT-IR分析证实纳米粒成功构建。体外释药表明HA/Lupeol@ZIF-8在模拟纤维化酸性环境中可快速释药(释放率为中性条件4倍),其血浆稳定性、溶血安全性及储存稳定性均良好。体内/外实验结果表明,HA/Lupeol@ZIF-8可有效抑制HSC-T6细胞增殖、诱导氧化应激和靶向活化肝星状细胞(activated hepatic stellate cells,aHSCs),并调控相关蛋白表达。结论 成功制备一种兼具主动靶向(HA)与pH响应释药(ZIF-8)的双靶向纳米系统HA/Lupeol@ZIF-8,显著增强了其抗HF活性与肝脏靶向效率。

Abstract

Objective To construct a hyaluronic acid-modified, pH-responsive zeolitic imidazolate framework-8(ZIF-8) nano-drug delivery system for Lupeol and to evaluate its physicochemical properties and anti-hepatic fibrosis effects. Methods The synthesis of ZIF-8 was optimized using a single-factor method. Lupeol@ZIF-8 was prepared via a one-pot synthesis method and further modified with HA to obtain HA/Lupeol@ZIF-8. The nanoparticles were characterized by transmission electron microscopy (TEM), dynamic light scattering (DLS), Fourier transform infrared spectroscopy (FT-IR) and X-ray diffraction (XRD). The drug release profile in phosphate buffers at different pH values and the stability under physiological conditions were evaluated. The inhibitory effect of HA/Lupeol@ZIF-8 on HSC-T6 cell proliferation and its reactive oxygen species (ROS) induction effect were analyzed using CCK-8, ROS measurement, and cell uptake detection. Related protein expression levels were detected by Western blot. A rat model of HF was established to investigate in vivo tissue distribution of HA/Lupeol@ZIF-8. Results The optimal preparation conditions for ZIF-8 were determined as reaction at 25 ℃ for 24 h with Zn²⁺/2-Mim ratio of 1∶8. The optimal drug loading method for Lupeol@ZIF-8 was the one-pot synthesis with a 2-Mim∶Lupeol ratio of 1∶8, yielding an encapsulation efficiency of 97.36% and a drug loading capacity of 23.75%. TEM showed that ZIF-8 and Lupeol@ZIF-8 exhibited uniform polyhedral structures, while HA/Lupeol@ZIF-8 appeared nearly spherical. DLS measurements indicated particle sizes of (72.81±0.43)nm, (80.29±1.03)nm and (125.7±0.16)nm for ZIF-8, Lupeol@ZIF-8 and HA/Lupeol@ZIF-8, with uniform distributions (PDI<0.4). Zeta potentials were (15.8±0.57)mV, (-7.06±0.02)mV and (-13.2±0.64)mV. XRD and FT-IR analyses confirmed the successful construction of the nanoparticles. In vitro release studies demonstrated that HA/Lupeol@ZIF-8 rapidly released the drug in a simulated acidic fibrotic environment (release rate was 4 times higher than under neutral conditions) and it exhibited good plasma stability, hemocompatibility and storage stability. Cell experiments revealed that HA/Lupeol@ZIF-8 effectively inhibited HSC-T6 cell proliferation, induced oxidative stress, and regulated the expression of related proteins. In vitro and in vivo experiments demonstrated that HA/Lupeol@ZIF-8 effectively inhibited the proliferation of HSC-T6 cells, induced oxidative stress, and targeted aHSCs, while regulating the expression of related proteins. Conclusion A dual-targeting nanosystem HA/Lupeol@ZIF-8, which combines active targeting (HA) with pH-responsive drug release (ZIF-8), was successfully prepared. This study demonstrates that the HA/Lupeol@ZIF-8 nanosystem effectively addresses the bottleneck issues of poor solubility and insufficient targeting of lupanol, significantly enhancing its anti-hepatic fibrosis activity and liver targeting efficiency. This provides a novel strategy for efficient delivery and treatment of hepatic fibrosis based on lupanol.

关键词

羽扇豆醇 / ZIF-8 / 透明质酸 / 肝纤维化 / pH释放

Key words

lupeol / ZIF-8 / hyaluronic acid / hepatic fibrosis / pH release

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基金资助

国家自然科学基金项目(82160742);新疆生产建设兵团重点领域科技攻关计划项目(2023CB008-25)