Synergistic Organelle-Microenvironment Regulation Strategy for Intervertebral Disc Degeneration Therapy by Reinforcing Mitochondrial Outer Membrane Stability and Sequestering exDNA

Free extracellular DNA (exDNA) accumulation within the cytoplasm is closely associated with chronic inflammation in intervertebral disc degeneration (IVDD). While numerous minimally invasive treatments targeting the various etiologies of IVDD have been developed, there remains a lack of systematic approaches to address the chronic inflammation caused by both intracellular and extracellular nucleic acids. In this study, we propose a novel strategy for treating IVDD that involves dual nucleic acid clearance. This approach targets and regulates mitochondrial DNA (mtDNA) release while clearing exDNA from the microenvironment, working synergistically to block the inflammatory cascade. We first analyzed the mechanism of chronic inflammation mediated by intracellular and extracellular nucleic acids during the progression of IVDD using single-cell sequencing and clinical sample analysis. Next, we developed a composite delivery system consisting of engineered nanovesicles with mitochondrial targeting functions (BNPMT) and a nucleic acid clearance hydrogel (H-P gel). BNPMT achieves targeted delivery to nucleus pulposus cells via membrane modification with triphenylphosphonium (TPP), while encapsulated BAI1, a small molecule drug, specifically inhibits mitochondrial outer membrane permeabilization (miMOMP). This reduces mitochondrial DNA (mtDNA) leakage into the cytoplasm, thereby blocking the mtDNA-cGAS/STING signaling pathway-mediated inflammatory response in nucleus pulposus cells. The H-P gel, crosslinked by Schiff base reactions and using G3-PAMAM dendrimers as functional crosslinkers, efficiently captures exDNA from the intervertebral disc microenvironment. This inhibits cfDNA-induced M1 macrophage polarization and the release of inflammatory cytokines. In an IVDD rat model, this dual-modal system demonstrated synergistic therapeutic advantages, significantly reversing the progression of IVDD over 8 weeks. This targeted nucleic acid clearance strategy provides a novel approach for treating IVDD and offers a theoretical framework for addressing other nucleic acid-related inflammatory diseases.细胞质内游离细胞外DNA（free extracellular DNA, exDNA）的蓄积，与椎间盘退变（intervertebral disc degeneration, IVDD）中的慢性炎症密切相关。尽管目前已开发出多种针对椎间盘退变不同病因的微创治疗手段，但仍缺乏系统性方法来解决由细胞内与细胞外核酸共同介导的慢性炎症。本研究提出一种全新的椎间盘退变治疗策略，即双核酸清除疗法。该策略可靶向调控线粒体DNA（mitochondrial DNA, mtDNA）的释放，同时清除微环境中的exDNA，通过协同作用阻断炎症级联反应。本研究首先通过单细胞测序（single-cell sequencing）与临床样本分析，阐明了椎间盘退变进程中细胞内与细胞外核酸介导的慢性炎症机制。随后，我们开发了一种复合递送系统，该系统由具备线粒体靶向功能的工程化纳米囊泡（BNPMT）与核酸清除水凝胶（H-P凝胶）组成。BNPMT通过三苯基膦（triphenylphosphonium, TPP）修饰细胞膜，实现对髓核细胞的靶向递送；其包裹的小分子药物BAI1可特异性抑制线粒体外膜透化（mitochondrial outer membrane permeabilization, miMOMP）。此举可减少线粒体DNA（mtDNA）向细胞质的渗漏，从而阻断髓核细胞中mtDNA-cGAS/STING信号通路介导的炎症反应。H-P凝胶以席夫碱反应进行交联，并以G3代聚酰胺-胺树枝状大分子（G3-PAMAM dendrimers）作为功能交联剂，可高效捕获椎间盘微环境中的exDNA，进而抑制游离DNA诱导的M1型巨噬细胞极化及炎症细胞因子释放。在椎间盘退变大鼠模型中，该双模式递送系统展现出协同治疗优势，可在8周内显著逆转椎间盘退变的进程。这种靶向核酸清除策略为椎间盘退变的治疗提供了全新思路，也为其他核酸相关性炎症性疾病的治疗提供了理论框架。