Fluolab【Adv.Mater.】华南理工张云娇团队|双效纳米片：100%清除ROS、逆转肝纤维化！一体化破解胆汁淤积性肝炎精准诊疗难题
文章标题：ALP-Responsive Luminescent Nanosheets Promote Precise Hepatitis Theragnostic by Co-Targeting NLRP3 and Cholestasis
通讯作者：Hao Zhang, Yunjiao Zhang
文章概要
引言
胆汁淤积性肝炎是一种渐进性的炎症性肝脏疾病，其临床特征表现为胆汁酸稳态被破坏、活性氧（ROS）过度产生以及长期的慢性炎症。由于早期症状缺乏特异性且缺乏高效的靶向治疗手段，该病在临床上面临着巨大的诊疗挑战。从发病机制来看，过量的胆汁酸滞留会通过双信号通路激活NLRP3炎性小体，进而放大肝细胞焦亡并形成炎症恶性循环。虽然一些小分子抑制剂在临床前模型中展现出了一定效果，但往往受限于系统性毒性和缺乏肝细胞特异性。此外，碱性磷酸酶（ALP） 作为胆汁淤积性肝损伤的敏感生物标志物，其在体内的高表达对疾病监控至关重要，但传统的检测方法难以实现体内实时特异性成像。为了同时解决肝脏炎症、胆汁酸失衡以及实时诊断监测的难题，研究团队创新性地开发出了一种集治疗与疾病监控于一体的多功能纳米诊疗平台。
Schematic of CyP-CuGA-UDCA NSs synergistically regulates NLRP3 inflammasome activity, restores bile acid homeostasis, and enables ALP-triggered in vivo imaging of cholestatic hepatitis.
主要实验及结论
研究人员通过简便的一步水热法成功合成了铜-没食子酸纳米片（CuGA NSs）。各项表征实验表明，该纳米片展现出规整的二维片状结构，流体力学粒径约为100纳米，且在水、PBS以及含有血清的培养基中均表现出优异的生理稳定性和分散性。在针对催化活性的系统评估中，CuGA NSs展现出了强大的广谱ROS清除能力。它具有高效的超氧化物歧化酶（SOD）样活性、过氧化物酶（POD）样活性以及过氧化氢酶（CAT）样催化功能，不仅能以浓度依赖的方式高效清除超氧阴离子，还可以在生理及酸性微环境中触发快速的氧气释放，从而有效淬灭高细胞毒性的羟基自由基。
Synthesis and characterization of CuGA NSs. (A) Size distribution of CuGA NSs in water as measured by dynamic light scattering (DLS). (B) Zeta potential measurement of CuGA NSs. (C) TEM image of CuGA NSs, showing a well-defined two-dimensional sheet-like morphology. Scale bar, 100 nm. (D) High-angle annular dark-field scanning TEM (HAADF-STEM) image and corresponding elemental mapping for carbon (C), oxygen (O), and copper (Cu) of CuGA NSs. Scale bar, 20 nm. (E) Surface elemental percentage composition of CuGA NSs. (F) The total XPS survey spectrum and (G) individual elemental high-resolution Cu 2p spectra of CuGA NSs. (H) Fourier-transform infrared (FTIR) spectra of free gallic acid (GA) and CuGA NSs. (I) Ultraviolet–visible (UV–vis) absorption spectra of GA and CuGA NSs (J) Hydrodynamic diameter distribution of CuGA NSs measured over seven days in different solutions (n = 3 for each group).
在体外细胞实验中，研究团队深入探讨了该纳米片的细胞摄取行为和抗炎机制。实验结果证实，巨噬细胞能够对该纳米片进行高效的细胞内吞。面对由脂多糖和尼日利亚菌素诱导的巨噬细胞炎症模型，CuGA NSs表现出了显著的双阶段炎症抑制效应。在启动阶段，它通过下调磷酸化NF-κB和IKKβ的表达来阻断转录，显著减少了下游促炎细胞因子TNF-α的释放；在激活阶段，它通过清除线粒体活性氧（mtROS）并维持线粒体膜电位的稳定性，选择性地破坏了炎性小体的组装，进而大幅度削减了成熟IL-1β、IL-18和IL-6等促炎因子的分泌。更重要的是，这种对炎性小体组装的阻断作用，从根本上抑制了细胞焦亡关键蛋白GSDMD的剪切，使乳酸脱氢酶的释放量大幅降低，为细胞提供了强有力的细胞保护屏障。
Multiple nanozyme-mimicking activities of CuGA NSs. (A) Schematic illustration of enzyme-mimicking activities of CuGA NSs. (B) SOD-like activity of CuGA NSs measured by a Total SOD Activity Assay Kit (WST-8 method, n = 3 for each group). (C) Evaluation of concentration-dependent superoxide anion (O2−) scavenging activity of CuGA NSs using electron spin resonance (ESR) spectroscopy with DMPO spin trapping. (D) UV absorbance values of TMB solution at 652 nm under different conditions. (E) Time-dependent spectrum of absorbance values at 652 nm for a mixture of TMB, H2O2, and CuGA NSs. (F) Evaluation of concentration-dependent hydroxyl radicals (·OH) scavenging activity of CuGA NSs using ESR spectroscopy. (G,H) The CAT-like activity of CuGA NSs was determined by O2 generation at various CuGA NSs (G) and H2O2 (H) concentrations over time. (I) Relative CAT-like activity of CuGA NSs at different pH (n = 3 for each group).
CuGA NSs inhibited NLRP3 inflammasome activation and reduced pyroptosis in vitro. (A) Fluorescence microscopy images of bone marrow-derived macrophages (BMDMs) after co-incubation with Cy5-labeled CuGA NSs (red) for different times (0–4 h). Nuclei were stained with DAPI (blue). Scale bar, 20 µm. (B) Western blot analysis showing the expression levels of key proteins in the NF-κB signaling pathway and NLRP3 in BMDMs pretreated with different concentrations of CuGA NSs (0, 5, 10, 20, 40 µg/mL) (C) ELISA detecting the secretion level of TNF-α in the supernatant of BMDMs treated with different concentrations of CuGA NSs (0, 5, 10, 20, 40 µg/mL). TNF-α production (as measured by ELISA) in supernatant (SN). (D) Fluorescence microscopy analysis in LPS-primed BMDMs treated with or without CuGA NSs and then stimulated with nigericin, followed by staining with DCFH-DA probe. Scale bar, 100 µm. (E) Western blot analysis of mature IL-1β (mIL-1β), cleaved caspase-1 (p20) in SN and pro-IL-1β, pro-caspase-1, NLRP3, ASC in lysates (Input) of BMDMs. (F) ELISA quantification of IL-1β, IL-18, and IL-6 levels in culture supernatants (SN). (G) The release of LDH in the BMDM cell supernatants was detected by the LDH cytotoxicity assay kit after indicated treatment (n = 3). (H) Western blot analysis of pyroptosis-related proteins (mIL-1β, p20, GSDMD) in BMDMs treated with LPS (100 ng/mL) for 3 h, followed by co-incubation with CuGA NSs for 4 h, 5 µM nigericin was added for an additional 30-min treatment. (I) Fluorescent microscopic images of BMDM cells with different treatments stained by Calcein-AM (live cells, green)/PI (dead cells, red) double-staining. Scale bar for 50 µm. (J) Fluorescence microscopy images using the MitoSOX probe (red) to assess mitochondrial ROS in BMDMs primed with LPS (100 ng/mL, 3 h), with or without pretreatment with CuGA NSs (20 µg/mL) for 4 h, followed by stimulation with nigericin (5 µM) for 30 min. Scale bar, 20 µm. The data are presented as mean ± SEM from triplicates (one-way ANOVA with Dunnett's post-hoc test).
为了实现对胆汁淤积性肝炎的协同治疗与实时监控，研究人员将临床常用的胆汁酸调节药物熊去氧胆酸（UDCA） 接枝到纳米框架上，并进一步偶联了对碱性磷酸酶敏感的近红外荧光探针（CyP），最终构建出CyP-CuGA-UDCA纳米诊疗系统。体外与体内荧光成像实验表明，该探针能够对ALP展示出极高的敏感性和特异性，其荧光激活强度与酶浓度呈现出完美的线性关系。在小鼠模型试验中，通过尾静脉注射该多功能纳米片后，小鼠肝脏部位呈现出强烈的近红外荧光信号。随着治疗的推进，肝脏部位的荧光信号在第五天显著减弱，并在第十天几乎完全消失，这种动态的荧光消退过程与体内ALP活性的下降高度吻合，成功实现了对疾病进展与治疗效果的非侵入性实时跟踪监测。
CyP-CuGA-UDCA NSs monitored cholestatic hepatitis disease progression. (A) Zeta potential of CuGA NSs, UDCA, and CuGA-UDCA NSs. (B) FTIR spectrum of CuGA NSs, UDCA, and CuGA-UDCA NSs. (C) UV–vis absorption spectrum analysis of CuGA NSs, UDCA, and CuGA-UDCA NSs. (D) Schematic illustration of the CyP probe's luminescent response to ALP. (E) Kinetic curve of fluorescence activation over time (0–60 min) for the CyP probe (10 µM) in the presence of ALP (0–256 U/L). (F) Fluorescence intensity of the CyP probe (10 µM) after incubation with different concentrations of ALP (0–256 U/mL) at 37°C for 60 min. (G) HPLC monitoring of CyP probe response to ALP activation. (H) Detection of CyP-CuGA-UDCA NSs in response to ALP-released fluorescence by fluorescence spectrophotometry. (I) Fluorescence in vivo imaging system (IVIS) monitors CyP-CuGA-UDCA NSs in response to ALP-released fluorescence. (J) In vivo fluorescence imaging of cholestatic hepatitis mice at days 0, 5, and 10 post-treatment, acquired 1 h after tail vein injection of CyP-CuGA NSs(10 mg/kg) or CyP-CuGA-UDCA NSs (10 mg/kg) using in vivo imaging system. (K) Ex vivo fluorescence images of major organs (heart, liver, spleen, lung, kidney) harvested 1 h after tail vein injection of CyP-CuGA-UDCA NSs (10 mg/kg) or CyP-CuGA NSs (10 mg/kg) on treatment day 12. (L) Quantitative analysis of the ex vivo organ fluorescence signals in (K).
CuGA-UDCA NSs ameliorate cholestasis and liver injury in mice fed 0.1% DDC. (A) Body weight change curves of mice during the experimental period. (n = 6 per group). (B) Representative photographs of livers from each group at the end of treatment. (C) Liver weight statistics of mice in different treatment groups (PBS, DDC, CuGA NSs, UDCA, and CuGA-UDCA NSs). Data are presented as mean ± SEM (n = 6). (D) The serum ALT, AST, ALP, TBA, and TBIL levels were measured in mice from different treatment groups (n = 4). (E) H&E staining in each treatment group showed the therapeutic effect of CuGA-UDCA NSs; scale bar = 50 µm. (F, I) Representative MASSON showed that CuGA-UDCA NSs alleviated liver fibrosis (F) and the quantitative statistics of liver fibrosis area in different treatment groups (I). Data are presented as mean ± SEM (n = 3). Scale bar, 200 µm. (G, J) Immunofluorescence co-staining images of the cholangiocyte marker K19 (green) and NF-κB-inducing kinase (NIK, red) in liver sections, indicating intrahepatic bile duct injury status (G) and the quantitative statistics of NIK+ cells in the livers of mice from different treatment groups (J). Nuclei were counterstained with DAPI (blue). Scale bar, 100 µm. Data are presented as mean ± SEM (n = 3). (H) Quantitative statistics of bile salt precipitation in the livers of mice from different treatment groups. (K) Liver injury scores of mice in different treatment groups. The data are presented as mean ± SEM from triplicates (one-way ANOVA with Dunnett's post-hoc test).
血清生化与组织病理学评估结果进一步证实了该一体化平台的卓越疗效。在连续给药后，模型小鼠的体重表现出明显的恢复趋势，原本因组织水肿而导致的肝脏肿大症状得到了彻底扭转。生化指标显示，血清中的丙氨酸氨基转移酶（ALT）、天冬氨酸氨基转移酶（AST）、碱性磷酸酶（ALP）以及总胆汁酸和总胆红素水平较未治疗组均出现了断崖式下跌，且治疗效果明显优于单一药物治疗。组织学切片及马松三色染色结果表明，该纳米平台不仅显著减少了肝脏内巨噬细胞的浸润和胆汁盐沉淀，保护了肝内胆管免受损伤，更大幅度地减轻了肝组织的纤维化重塑和肝细胞凋亡。各项体内生物安全指标也明确指出，该纳米制剂对心、肾等核心器官无任何毒副作用，具备极佳的生物相容性。
CuGA-UDCA NSs ameliorate DDC-induced inflammatory infiltration and apoptosis. (A) ELISA detection of serum levels of inflammatory factors IL-1β, TNF-α, IL-18, and IL-6 in different treatment groups (PBS, DDC, CuGA NSs, UDCA, and CuGA-UDCA NSs). Data are presented as mean ± SEM (n = 4). (B) Immunohistochemical staining images for the macrophage marker F4/80 in liver sections. Scale bar, 50 µm. (C) Quantification of F4/80 expression levels in liver tissues across different treatment groups in mice. (D,E) Representative DHE fluorescence staining images (D) and quantitative analysis (E) of ROS fluorescence intensity in liver sections from different treatment groups in mice. Scale bar, 100 µm. (F,G) TUNEL staining (green) to assess hepatocyte apoptosis. Nuclei were counterstained with DAPI (blue). Scale bar, 100 µm. (F) and quantitative statistical analysis (G) in liver sections from different treatment groups in mice. The data are presented as mean ± SEM from triplicates (one-way ANOVA with Dunnett's post-hoc test).
总结及展望
这项研究成功构建了一种具有多重酶学模拟活性的新型铜-没食子酸纳米平台，通过将抗炎纳米酶、胆汁酸调节剂以及酶响应性近红外荧光探针完美整合在单一种类的二维纳米材料中，实现了ROS清除、炎性小体抑制、胆汁酸调节和实时病理成像的四位一体化协同精准诊疗。这种将精准治疗与多模态疾病追踪相结合的设计理念，彻底攻克了传统疗法在应对多因素复杂疾病时顾此失彼的短板。该多功能纳米片所展现出的优异的协同治疗效果、灵敏的实时病理响应能力以及出色的临床转化安全性，不仅为胆汁淤积性肝病的精准干预提供了全新的靶向递送范式，也为未来设计其他复杂的炎症性疾病或者恶性肿瘤的智能化纳米医药平台提供了极具启发性的通用型蓝图。