Fluolab【Adv.Mater.】中科院化学所白春礼院士联合北大口腔医学院张学慧|2合1靶向策略！全盛富勒烯智能凝胶双管齐下，攻克糖尿病骨再生医学难题
文章标题：A Dual‐Targeting Strategy Against ROS and GSK‐3β With a PEGylated Fullerene/Smart Hydrogel Synergistic System for Diabetic Bone Regeneration
通讯作者：Jie Li, Xuehui Zhang, Chunru Wang, Chunli Bai
文章概要
引言
在临床医学中，糖尿病环境下的骨再生一直是一项巨大的挑战。由于长期处于高血糖状态，患处往往伴随着严重的局部氧化 stress 和持续性炎症，这种恶劣的微环境会导致巨噬细胞异常极化为促炎的 M1 型，从而破坏骨愈合级联反应并抑制间充质干细胞的成骨分化。虽然传统的生物材料或局部释放抗炎分子的疗法被广泛应用，但由于无法精准调控这种复杂的免疫微环境，修复效果往往不尽人意。富勒烯纳米材料因其卓越的活性氧（ROS）清除能力而在抗炎领域展现出巨大的应用潜力，但市面上大多数水溶性富勒烯衍生物多为结构复杂的异构体混合物，难以明确其结构与活性之间的关系，从而阻碍了其临床转化。为了打破这一瓶颈，研究团队成功开发出一种结构明确的五聚乙二醇化富勒烯衍生物（FPEG₅），并将其融入具有智能响应性的水凝胶中，旨在通过双靶向策略同时解决氧化应激与病理性免疫极化两大难题，为糖尿病骨修复开辟了全新的途径。
Schematic diagram illustrating the glucose/ROS-responsive release strategy of FPEG5-PVA-TSPBA hydrogel and the mechanism of FPEG5-induced diabetic bone regeneration through dual ROS scavenging and GSK-3β-targeted immune-osteo cascade reprogramming.
主要实验及结论
研究人员首先通过精心设计的一步反应成功合成了高纯度且结构明确的富勒烯衍生物 FPEG₃、FPEG₄ 和 FPEG₅。实验结果表明，FPEG₅ 在各种 pH 环境下均能保持优异的水溶性和单分散状态，具备良好的药物样潜能。在随后的活性氧清除能力测试中，电子顺磁共振波谱明确证实了 FPEG₅ 能够高效清除羟基自由基和超氧阴离子。在体外糖尿病巨噬细胞模型中，FPEG₅ 不仅显著降低了细胞内的 ROS 水平，还展现出强大的免疫调节功能。它能够以浓度依赖的方式将促炎的 M1 型巨噬细胞重新编程为促再生的 M2 型巨噬细胞，大幅下调了白介素-1β、诱导型一氧化氮合酶以及白介素-6等促炎基因的表达，同时显著提升了抗炎基因 Ym-1 的水平。在与其他几种传统水溶性富勒烯衍生物的对比中，FPEG₅ 在清除活性氧和驱动巨噬细胞抗炎极化两方面均表现出了最强劲的综合性能。
Synthesis of PEGylated fullerene derivatives and their antioxidant and immunomodulatory capability. (A) Schematic illustration of the synthetic route to FPEG3/4/5. (B) Hydrodynamic diameter distribution of FPEG5 under varying pH conditions. (C) Zeta potential measurements of FPEG3/4/5 (n = 3). (D) Electron paramagnetic resonance (EPR) spectra showing the concentration-dependent scavenging of •OH and (O2•−) by 50 µM FPEG5. (E) Viability of BMDMs after 48 h treatment with FPEG3/4/5 (n = 3). (F) Flow cytometry of intracellular ROS levels in BMDMs. (G) Flow cytometry analysis of the M2 marker CD206 and the M1 marker CD86 in BMDMs. (H) Representative immunofluorescence images of BMDMs stained for CD86 and CD206 (green), F-actin (red), and Dapi (blue). Scale bars: 25 µm. (I) RT-qPCR analysis of pro-inflammatory (IL-1β, iNOS, IL-6) and anti-inflammatory (Ym-1) gene expression in BMDMs (n = 3). Data are presented as mean ± SD. Statistical significance was determined by one-way ANOVA with Tukey's post hoc test; Significance is indicated relative to the DM group (# indicated): ***p < 0.001.
PEGylated fullerene drives macrophage repolarization through direct targeting of GSK-3β. (A) Schematic workflow of the PELSA. (B) Local stability profiles identifying ligand-binding regions. The gray-shaded area represents a log2 fold-change (FC) window between +0.3 and −0.3. The x-axis corresponds to the protein sequence from the N- to C-terminus. (C) Microscale thermophoresis (MST) dose–response curves showing the binding of FPEG5 to purified GSK-3β (n = 3). (D) Thermal shift binding assay (CETSA) evaluating the interaction between FPEG5 (50 µM) and GSK-3β. (E) Left: The optimal docking model of FPEG5 in GSK-3β. FPEG5 and amino acid residues are shown in yellow and blue, respectively. Key hydrogen bonds (blue solid lines) and hydrophobic interactions (magenta dashed lines) are indicated. Right: The binding mode of FPEG5 in GSK-3β. The active-site pocket is displayed as an electrostatic surface. (F) Western blot analysis of inflammatory signaling following FPEG5 treatment through the GSK-3β pathway. (G) Co-immunoprecipitation (Co-IP) /Western blot analysis of assessing the interaction between GSK-3β and β-catenin in lysates from DM and DM + FPEG5 groups. (H) Western blot analysis of GSK-3β pathway activity following pharmacological activation and FPEG5 intervention. (I) Flow cytometry analysis of the M2 marker CD206 and the M1 marker CD86 in BMDMs after treatment with an agonist and FPEG5. (J) Flow cytometry analysis of the M2 marker CD206 and the M1 marker CD86 after siCtrl and siGSK-3β conditions and FPEG5 treatments. (K) Western blot evaluation of NF-κB pathway modulation by FPEG5 under DM conditions. (L) The proposed mechanism that FPEG5 inhibits the NF-κB pathways to promote macrophage repolarization via dual-targeting GSK-3β and ROS.
为了深入探究 FPEG₅ 驱动巨噬细胞重编程的内在分子机制，研究团队采用了基于肽段的局部稳定性分析（PELSA）技术来进行全蛋白质组尺度的靶点筛选。令人惊喜的是，除了传统的活性氧清除通路外，GSK-3β 被成功鉴定为 FPEG₅ 的直接结合靶点。微尺度热泳动实验和细胞热移位分析一致证实了 FPEG₅ 与 GSK-3β 之间存在高亲和力的直接相互作用，并且其结合位点与分子对接模拟预测的激酶结构域口袋高度吻合。在功能层面上，FPEG₅ 的结合选择性地阻断了 GSK-3β 与 β-catenin 的相互作用，从而抑制了 β-catenin 的磷酸化降解，促使其在细胞内稳定积累。这种稳定的 β-catenin 能够与高效率清除 ROS 的过程产生协同效应，共同增强 IκBα 的稳定性，强力抑制 NF-κB 炎症信号通路的激活，进而从根本上逆转了高血糖微环境带来的病理性炎症。
PEGylated fullerene-induced macrophage reprogramming enhances osteogenic differentiation in vitro. (A) Schematic of the Transwell coculture system used to evaluate the osteoinductive potential of FPEG5-mediated macrophage reprogramming. (B,C) Quantitative RT-qPCR analysis of osteogenic marker genes (BMP-2, RUNX-2, COL1, OPN, and SP7) in mBMSCs under the indicated conditions at day 4 (B) and day 7 (C) (n = 3). (D) Representative immunofluorescence images of mBMSCs stained for OPN (green), F-actin (red), and Dapi (blue) after different treatments at day 4. (E) Representative immunofluorescence images of mBMSCs stained for BMP-2 (green), F-actin (red) and Dapi (blue) at day 4. (F) Alkaline phosphatase (ALP) activity staining (day 7) and Alizarin red (ARS) staining for mineralized matrix deposition (day 14) in mBMSCs. Scale bars: 25 µm (D, E); 400 µm (F). Data are presented as mean ± SD. Statistical significance was determined by one-way ANOVA with Tukey's post hoc test; Significance is indicated relative to the DM group (# indicated). ns, not significant; *p < 0.05, **p < 0.01, ***p < 0.001.
Fabrication and characterization of a glucose/ROS-responsive hydrogel system loaded with PEGylated fullerene. (A) Schematic illustration of the degradation and drug release mechanism of the FPEG5-PVA-TSPBA hydrogel in response to elevated ROS and glucose. (B) Gelation kinetics measured for PVA solutions at varying concentrations mixed with 2.5% (w/v) TSPBA cross-linker (n = 3). (C) Schematic demonstrating the injectability of the pre-gel solution using a dual-barrel syringe, where one barrel contains FPEG5-loaded PVA and the other contains TSPBA. (D) Photographs showing the macroscopic degradation of FPEG5-PVA-TSPBA hydrogels incubated for 14 days in PBS, high-glucose (25 mM), high-H2O2 (1 mM), or combined diabetic-mimicking (DM) medium. (E) Cumulative release profile of FPEG5 from the hydrogel under the different conditions over 14 days, measured by UV–vis spectroscopy. (F) Representative SEM images of the internal microstructure of PVA-TSPBA and FPEG5-PVA-TSPBA hydrogels. Scale bars: 2 µm. (G) Compression strength (Left) and Elastic modulus (Right) for PVA-TSPBA and FPEG5-PVA-TSPBA hydrogels (n = 3). Data are presented as mean ± SD. Statistical significance was determined by one-way ANOVA with Tukey's post hoc test; Significance is indicated relative to the PVA-TSPBA group (# indicated). ***p < 0.001.
在成骨分化及体内修复实验中，该系统同样交出了优异的答卷。通过透气小室共培养系统证明，经 FPEG₅ 调控后的促再生型巨噬细胞能够显著上调骨髓间充质干细胞中成骨标记物基因的表达，矿化结节及钙沉积的形成量明显增加。为了实现药物在体内的时空可控释放，研究人员利用聚乙烯醇与苯硼酸类交联剂混合，构建了一种兼具高注射性与优秀机械强度的智能双响应水凝胶（FPEG₅-PVA-TSPBA）。该水凝胶在病理性的高血糖和高活性氧环境下能够发生选择性降解，实现对 FPEG₅ 的按需释放。在糖尿病大鼠颅骨临界尺寸缺陷模型中，微颅骨 CT 成像以及组织学染色结果表明，该智能水凝胶在植入后迅速响应局部环境，在早期炎症窗口期精准释放药物并成功重构局部免疫微环境。在术后八周时，治疗组大鼠展现出极为稳固的骨缺损桥接修复效果，新生骨组织与周围天然骨完美融合，同时展现出卓越的体内生物安全性。
FPEG5-PVA-TSPBA drives diabetic bone regeneration. (A) Schematic of the surgical procedure for creating a critical-sized calvarial defect in a diabetic rat or db/db mice, followed by hydrogel implantation. (B) Representative three-dimensional micro-CT reconstructions of the defect sites at 4 and 8 weeks post-implantation. (C) Histological sections of the defect area stained with H&E and Masson's trichrome staining at 4 and 8 weeks. (D) Flow cytometric analysis of M2 (CD206+) and M1 (CD86+) macrophage populations isolated from the defect site at 1 week post-surgery. (E) Representative immunofluorescence staining for CD86 and CD206 in the defect region at 1 week. Scale bars: 100 µm (C); 10 µm (E).
总结及展望
这项研究成功构建了集成了创新型靶点明确的富勒烯药效团与疾病适应性智能递送系统。FPEG₅ 不仅可以通过清除活性氧来缓解氧化应激，更能够作为精准的免疫调节剂直接靶向 GSK-3β 激酶，双管齐下地阻断了糖尿病微环境下的恶性炎症循环。这种将生物活性纳米材料与病理响应性智能水凝胶相结合的综合治疗策略，不仅大幅加速了糖尿病高难度骨缺损的组织修复进程，也为未来开发更多针对复杂慢性炎症疾病的智能化、靶向性组织工程工程化平台提供了极具临床转化价值的新思路。