领域：学术论文 · 自然科学

Spinal cord injury (SCI) is a complex and dynamic pathological condition characterized by disrupted lipid metabolism and neuroinflammatory responses, posing significant therapeutic challenges. To address these, biomimetic bacterial outer membrane nanoparticles (BM-NPs) are designed by integrating the precise targeting capability of detoxified outer membrane vesicles (dOMVs) with the efficient drug-loading properties of liposomes. BM-NPs exhibit superior targeting efficiency toward peripheral neutrophils and macrophages, enabling spatiotemporal drug delivery via immune cells. 

An innovative “Tortoise and Hare” dynamic adaptive delivery strategy is introduced, where neutrophils facilitate rapid drug transport during the acute phase of SCI, while macrophages ensure sustained delivery during the subacute phase. This strategy aligns with the dynamic pathological progression of SCI, offering precision targeting tailored to different stages of injury. BM-NPs demonstrate multifaceted therapeutic effects, including the suppression of foam cell formation through coordinated enhancement of lipid droplet autophagy and cholesterol efflux. Furthermore, they modulate the inflammatory microenvironment, preserve myelin integrity, and significantly promote neural functional recovery post-SCI. By overcoming the limitations of conventional delivery systems in targeting and timeliness, BM-NPs offer an innovative, highly efficient, and clinically translatable platform for SCI treatment and other acute inflammatory disorders of the central nervous system.

1. Introduction
The pathological features of spinal cord injury (SCI) encompass inflammatory responses, foam cell formation, myelin degradation, and neurological dysfunction. Among these, resident microglia and peripheral macrophages in the spinal cord play dual roles: they participate in the clearance of myelin debris while serving as pivotal mediators in the inflammatory response. However, their phagocytic activity can inadvertently lead to foam cell formation, exacerbating lipid metabolism dysregulation and inflammatory cascades, thereby posing a major barrier to effective treatment. Moreover, the presence of the blood-spinal cord barrier (BSCB) significantly hampers drug delivery efficiency, further limiting the therapeutic outcomes. Although these nanotechnologies have improved drug targeting and therapeutic efficacy to some extent, they lack active cellular functionality, which limits their timeliness and delivery efficiency in the complex pathological environment of SCI. Conversely, cell-based drug delivery systems present a promising alternative. Leveraging the innate chemotactic abilities of intact cells, these systems enable precise and dynamic drug delivery.[7] In our previous study, we employed engineered macrophages for drug delivery in the treatment of SCI and revealed a significantly improved functional recovery in a rat SCI model. However, we also observed that blood-derived macrophages only began to appear at the injury site at ≈3 days post-SCI. This indicates the existence of “treatment void” period before macrophages reach the injury site, where no effective therapeutic intervention occurs. Therefore, it is critical to develop more timely and efficient therapeutic strategies to address this unmet need. 

To address the aforementioned challenges, in this study, we developed biomimetic bacterial outer membrane nanoparticles (BM-NPs) derived from detoxified outer membrane vesicles (dOMVs) of msbB-deficient Escherichia coli. By knocking out the msbB gene, the toxicity of lipopolysaccharide (LPS) in dOMVs was significantly reduced while retaining the functional outer membrane protein A (OmpA), enabling efficient immune cell targeting. Through fusion with drug-loaded liposomes, BMNPs not only exhibited excellent targeting specificity and 
 drugloading capacity but also achieved dual-drug synergistic regulation: rapamycin (Rapa) induced autophagy to promote lipid droplet degradation, while LXR-623 enhanced the free cholesterol efflux. This synergistic effect effectively inhibited foam cell formation and improved the inflammatory microenvironment. Instead of simply decorating liposomes with isolated OmpA proteins, we adopted a fusion strategy with dOMVs to construct BMNPs. This approach is expected to better preserve the native conformation and biological functionality of outer membrane proteins within a physiological lipid environment. In contrast, direct anchoring of membrane proteins onto synthetic liposomes may increase the risk of conformational instability, functional loss, and batch-to-batch variability. Therefore, the biomimetic fusion-based design may offer improved nanoparticle stability, immune cell targeting, and therapeutic potential for SCI treatment.

BM-NPs exhibited precise spatiotemporal targeting capabilities through a novel “Tortoise and Hare” collaborative delivery strategy. This approach emphasizes the complementary roles of neutrophils and macrophages in stage-specific drug delivery during SCI treatment. In the acute phase, neutrophils rapidly recognize and internalize BM-NPs via their innate chemotactic properties, acting as “Trojan horse” carriers preferentially recruited to the injury site. These neutrophils, akin to the swift hare, promptly release neutrophil extracellular traps (NETs) upon stimulation from the injury microenvironment, triggering the early release of drug-loaded NPs to regulate microglial function.

Approximately 3 days post-injury, monocyte-derived macrophages carrying BM-NPs gradually infiltrate the injury site, functioning as the enduring tortoise. These macrophages provide sustained drug delivery, thus extending the therapeutic effects. This collaborative delivery strategy enables BM-NPs to achieve stage-specific regulation across different pathological phases of SCI, effectively inhibiting foam cell formation, ameliorating the inflammator microenvironment, and ultimately promoting significant neurological recovery.
In this study, we propose an immune cell-mediated dynamic adaptive delivery strategy that precisely aligns with the pathological stages of SCI, offering a novel and promising solution for SCI treatment.

2. Results and Discussion
 2.1. Exacerbation of Foam Cell Formation Following SCI
During SCI, acute mechanical compression and subsequent inflammatory stimuli lead to significant demyelination. The resident microglia and blood-derived macrophages are the primary phagocytes responsible for clearing myelin debris in the spinal cord. Due to similarities in morphology, gene expression, and surface protein markers, their specific roles have often been conflated in early studies. However, these cells exhibit distinct differences in activation timing, spatial distribution, and myelin debris clearance capacity.  Following injury, the resident microglia are the first responders, rapidly initiating the phagocytosis of tissue debris. In contrast, blood-derived macrophages are typically recruited to the injury site on approximately day 3 and gradually assume the primary role in clearing the myelin debris. While transient myelin uptake directs these cells toward resolving disease phenotypes, persistent intracellular myelin accumulation induces foam cell formation. To investigate the temporal dynamics of foam cell development post-SCI, we analyzed a single-cell RNA sequencing dataset from Li et al. Cell type annotation based on specific markers identified distinct populations of microglia and macrophages (Figure 1A,B). Temporal expression dynamics of foam cell-associated genes revealed a significant upregulation in both microglia and macrophages at various post-SCI time points (Figure 1C).

Immunofluorescence staining of spinal cord tissues pre- and post-injury revealed co-localization of the microglialmarker Iba-1 with the myelin basic protein (MBP) as early as day 1 and 3 post SCI (Figure 1D). This finding underscores the role of microglia, the resident immune cells ofthe central nervous system, as “pioneers” during the early stages of SCI. Microglia rapidly transition to a reactive phenotype and actively participate in myelin debris clearance. By day 7, substantial myelin debris was detected within the CD68+ cells (marking the activated microglia and macrophages), and this phenomenon persisted until day 28, reflecting the prolonged burden of phagocytes in clearing debris post-SCI (Figure 1D). Further analysis of lipid accumulation using BODIPY fluorescence staining revealed the formation of lipid droplets as early as day 3 post-injury. This accumulation intensified progressively on days 7 and 28 (Figure 1E,F).

2.2. Autophagy and Cholesterol Efflux in Foam Cell Formation 
The dynamic changes in foam cell-associated gene expression and lipid droplet accumulation in the injury site post-SCI highlight the necessity for early and effective therapeutic intervention. In recent years, metabolic reprogramming has emerged as a promising strategy to modulate the cellular metabolic states, offering new perspectives on foam cell formation and inflammation regulation. The complex environment of the injury site is characterized by high levels of reactive oxygen species (ROS), inflammatory cytokines, and myelin debris. Therefore, there is a pressing need for a therapeutic approach that not only inhibits lipid accumulation in foam cells but also promotes their reparative phenotype. To identify effective strategies for mitigating foam cell formation, we investigated the impact of synergistic regulation of macrophage metabolism on lipid accumulation in foam cells.

Enhancing cholesterol efflux is widely recognized as an effective intervention strategy. This process primarily relies on the function of the ATP-binding cassette transporter A1 (ABCA1), which facilitates the transport of free cholesterol to the extracellular space, thereby reducing the intracellular cholesterol burden. However, the initial step of cholesterol efflux involves the release of cholesterol from lipid droplets. However, the initial step of cholesterol efflux involves the release of cholesterol from lipid droplets. Consequently, promoting the efficient hydrolysis of cholesterol esters in lipid droplets is critical for achieving effective cholesterol efflux.  Lipophagy, the autophagy-mediated degradation of lipid droplets, has been proven pivotal for lipid droplet processing within cells. Based on this, we explored the synergistic regulation of foam cell formation using the autophagy inducer Rapa and the liver X receptor agonist LXR-623. 
The cytotoxicity of the drugs was assessed first (Figure S1A,B, Supporting Information). Subsequently, a foam cell model was established in vitro using purified myelin debris, and the intracellular total cholesterol content was measured post-treatment to optimize the drug combination ratio (Figure S1C, Supporting Information). At a Rapa: LXR-623 ratio of 1:5 (nmol/nmol, with Rapa concentration set as a reference at 40 nmol mL−1), the intracellular cholesterol levels were significantly reduced. Although a further reduction in the cholesterol content was observed at a ratio of1:8, the encapsulation of such a high drug ratio within the NPs posed technical challenges. Therefore, a ratio of 1:5 was selected. To evaluate whether the combination therapy could synergistically inhibit foam cell formation, macrophages were stimulated with myelin debris (0.5 mg mL−1) for 72 h to establish a foam cell model. Western blot analysis revealed that, compared to the PBS group, the combination treatment significantly increased the LC3B-II/LC3B-I ratio and downregulated the expression of the autophagy-inhibitory protein P62 (Figure S2A,B, Supporting Information). Compared to the PBS group, immunofluorescence analysis further indicated that the combination treatment notably increased the number ofLC3B-positive puncta (Figure S3A,C, Supporting Information), while reducing the number ofP62-positive puncta (Figure S3B,D, Supporting Information). 

Such a finding suggests that the combination treatment restored the autophagic flux in foam cells. Additionally, fluorescence imaging demonstrated that the combination treatment significantly reduced the BODIPY fluorescence intensity in cells (Figure S3A,E, Supporting Information), indicating a reduction in the intracellular lipid burden. The treatment also markedly upregulated the expression of LXR𝛼 andABCA1,suggestingan enhanced cholesterol efflux capacity. Quantitative analysis of the intracellular total cholesterol and extracellular free cholesterol in the culture supernatant (Figure S4A,B, Supporting Information) showed that the PBS group exhibited significantly elevated intracellular cholesterol levels and reduced extracellular free cholesterol. Conversely, the combination treatment group exhibited significantly reduced intracellular cholesterol levels with increased extracellular free cholesterol levels, thereby significantly improving the cholesterol efflux efficiency (Figure S4C, Supporting Information). These findings demonstrate that promoting autophagy and cholesterol efflux synergistically inhibits foam cell formation.

翻译考点：
考点 1："biomimetic bacterial outer membrane nanoparticles (BM-NPs)" 只能译为 "仿生细菌外膜纳米颗粒 (BM-NPs)"，因为这是已经研究出来的医学成果，期刊以及新闻上都采取此翻译
考点2："spatiotemporal drug delivery" 推荐译为 "时空药物递送"
考点 3："detoxified outer membrane vesicles (dOMVs)" 必须译为 "低毒细菌外膜囊泡 (dOMVs)"，因为这是已经研究出来的医学成果，期刊以及新闻上都采取此翻译
考点 4："msbB-deficient Escherichia coli" 应该译为 "msbB基因缺陷型大肠杆菌"