背景：结直肠癌(Colorectal Cancer, CRC)是全球第三大常见癌症（9.6%）和第二大致死癌症（9.3%），严重威胁民众健康。肝转移是结直肠癌最常见的远端转移。约30%-50%的结直肠癌患者会发生肝转移。尽管手术技术和治疗方法不断发展，但是转移性结直肠癌患者的预后仍然很差，5 年生存率不到15%。合理有效的治疗仍是一大挑战，亟需立足肿瘤基础研究开发更加精准有效的治疗技术和方案。

近年来，随着16s rRNA和宏基因组检测技术的发展和对肿瘤微环境的深入了解，越来越多的证据证实了多种瘤内菌的存在。其中，具核梭杆菌（Fusobacterium nucleatum，Fn）是消化道肿瘤尤其是CRC中最具代表性的肿瘤相关菌，在结直肠癌及肝转移部位高度富集并且提示预后不良。研究表明Fn可通过诱导DNA损伤、介导相关信号通路以及促进免疫抑制性免疫微环境的形成进而促进结直肠癌的发生发展、转移与复发。此外，Fn可通过调节自噬或焦亡通路促进对结直肠癌的化疗耐药性。目前，临床上尚无靶向清除瘤内菌，进而抑制结直肠癌及其肝转移进展，同时不影响机体正常微生物群的药物。因此，研究靶向清除肿瘤微生物的纳米药物，有助于解除化疗抵抗，为提升结直肠癌及其肝转移治疗效果的提供创新性辅助策略。

目的：利用纳米技术构建具有主动靶向功能的用于治疗结直肠癌及其肝转移的抗生素化疗药一体化纳米药物。探究其体内外抗菌抗肿瘤作用，评价其肿瘤多重靶向能力、探究抑制结直肠癌及其肝转移进展的机制，评估肿瘤免疫微环境重塑、抗菌抗肿瘤免疫反应激活的效果，分析抗菌治疗和化疗的协同关系。

方法：本研究收集36例本院结直肠癌患者癌、癌旁及其肝转移组织标本，通过荧光原位杂交技术检测组织中的Fn的浸润程度，同时收集其临床病理相关信息。针对具核梭杆菌在结直肠癌及其肝转移部位富集的特点，本研究设计通过乳化法将甲硝唑（Metronidazole, MTI）和奥沙利铂（Oxaliplatin, OXA）封装在聚乳酸-羟基乙酸共聚物（poly (lactic-co-glycolic acid), PLGA）纳米载药系统中,构建了PLGA-MTI-OXA纳米药物。随后通过超声法将中性粒细胞膜囊泡包覆在PLGA-MTI-OXA上，形成具有主动靶向肿瘤能力的中性粒细胞膜仿生纳米药物NM@PLGA-MTI-OXA。通过高效液相色谱和 UV-Vis等方法验证 NM@PLGA-MTI-OXA有效包载了MTI和OXA并确认了最佳比例。通过透射电镜和粒径分析仪观察和检测了纳米颗粒的形貌、粒径和电位。在体内外探究了中性粒细胞靶向肿瘤部位及与肿瘤细胞结合的机制。通过构建Fn浸润的急性炎症模型以及皮下瘤模型探究NM@PLGA-MTI-OXA对于炎症部位和肿瘤部位的靶向能力。通过纸片扩散药敏实验、细菌扫描电镜、细菌死活染色以及平板菌落形成检测纳米药物对于细菌增殖的抑制作用。通过CCK-8检测纳米药物对于肿瘤细胞存活的影响，使用Annexin V/PI双染探究纳米药物对肿瘤细胞凋亡的影响，通过划痕实验探究其对肿瘤细胞迁移能力的影响。通过构建3种Fn浸润的肿瘤模型，用于评价纳米药物的体内抑菌和抑瘤能力。通过16s rRNA对小鼠粪便微生物组测序分析纳米药物对肠道菌群平衡的影响。通过免疫组化检测EMT相关蛋白的表达变化用来评估纳米药物对于上皮-间充质转化进展的抑制作用。免疫组化和流式细胞术检测小鼠的免疫细胞水平，酶联免疫吸附试验检测纳米药物对外周血中炎性免疫相关因子的表达水平的影响。

结果：具核梭杆菌在结直肠患者肿瘤组织及肝转移组织高度浸润，其富集程度与远端转移相关。本课题成功设计并构建了形状大小均一的NM@PLGA-MTI-OXA，能够高度靶向Fn浸润的炎性肿瘤部位。NM@PLGA-MTI-OXA表面保留了中性粒细胞膜表面蛋白，能够与Fn诱导的肿瘤细胞上表达升高的粘附分子VCAM1、CD44和ICAM1结合，促进肿瘤细胞的摄取，高效清除胞内菌，促进细胞凋亡并抑制细胞迁移。在体内，NM@PLGA-MTI-OXA对于Fn浸润的动物模型均有明显的抑制作用，有效清除肿瘤内浸润细菌，同时不影响肠道菌群平衡，具有良好的生物安全性。机制上，NM@PLGA-MTI-OXA可以降低肿瘤组织EMT相关蛋白N-cadherin、Vimentin、Slug 和 Snail的表达，逆转Fn介导的EMT进展。同时，NM@PLGA-MTI-OXA可提高肿瘤内与外周血中CD4+T细胞和CD8+T细胞比例，抑制外周血中MDSCs富集，下调外周血中炎性因子IL-6、IL-10和IL-22的表达。

结论：本研究提出一种新型的抗肿瘤策略，旨在通过破坏CRC与Fn之间的肿瘤-病原共生关系，抑制肿瘤的进展与转移。成功设计并构建中性粒细胞膜仿生的纳米药物 NM@PLGA-MTI-OXA。该纳米系统能够高度靶向Fn浸润的CRC组织，进而被肿瘤细胞高效摄取，实现肿瘤细胞内外的Fn的有效清除与肿瘤细胞的杀伤。另外，NM@PLGA-MTI-OXA可逆转Fn介导的EMT进展，重塑肿瘤免疫微环境，同时不会破坏肠道微生物群的稳态，具有良好的生物安全性。本研究为临床病理明确有瘤内菌浸润的结直肠及肝转移患者治疗策略提供了新思路，聚焦于靶向并清除肿瘤微环境中的微生物，解除化疗抵抗，提高肿瘤部位药物富集浓度，具有显著的临床应用潜力。

Background:

Colorectal cancer (CRC) ranks as the third most prevalent malignancy and the second leading cause of cancer-related mortality worldwide, posing a significant threat to public health. Liver metastasis is the most common form of distant metastasis in CRC, occurring in approximately 30%–50% of patients. Despite advances in surgical techniques and treatment strategies, the prognosis for patients with metastatic CRC remains poor, with a 5-year survival rate of less than 15%. There remains an urgent need for effective and rational therapeutic strategies, underscoring the significance of developing more precise and efficient treatments grounded in tumor biology.

In recent years, with the development of 16s rRNA and metagenomic sequencing technologies and the growing understanding of the tumor microenvironment, increasing evidence has confirmed the presence of intra-tumoral microbiota. Fusobacterium nucleatum (Fn), a representative tumor-associated bacterium particularly enriched in gastrointestinal malignancies, has been found to accumulate in primary colorectal tumors and liver metastases and is associated with poor prognosis. Several studies have suggested that Fn could promote CRC progression by inducing DNA damage, modulating key signaling pathways, and fostering the immunosuppressive tumor microenvironment. Moreover, Fn has been shown to mediate chemotherapy resistance by regulating autophagy and pyroptosis pathways. Currently, there is no clinically available drugs selectively target and eliminate intratumoral bacteria to suppress CRC and liver metastasis progression while preserving the normal microbiota. Thus, the development of nanomedicines that specifically target and eliminate tumor-resident bacteria offers a promising strategy to overcome chemoresistance and improve the efficacy of CRC treatment.

Objective:
This study aims to develop a nanotechnology-based platform integrating antibiotics and chemotherapeutics into a single nanoparticle system with multiple tumor-targeting capabilities for the treatment of CRC and liver metastases. We investigated its antibacterial and antitumor efficacy both in vitro and in vivo and evaluated its multiple tumor-targeting ability. We further explored the underlying mechanisms by which it suppresses tumor progression, assesses its effects on remodeling the tumor immune microenvironment and activating antitumor immune responses, and analyzes the synergistic effect between antimicrobial therapy and chemotherapy.

Methods:
We collected tumor, adjacent normal, and liver metastasis tissues from 36 CRC patients. The infiltration of Fn was detected using fluorescence in situ hybridization (FISH), and corresponding clinicopathological data were collected. A PLGA-based nanoparticle (PLGA-MTI-OXA) was constructed by encapsulating metronidazole (MTI) and oxaliplatin (OXA) using the emulsification method. Then, neutrophil membrane vesicles were coated onto PLGA-MTI-OXA via ultrasonication, forming NM@PLGA-MTI-OXA, a neutrophil neutrophil-mimicking nanomedicine with active tumor-targeting capability. HPLC and UV-Vis spectroscopy were used to confirm successful drug loading and determine the optimal formulation ratio. Transmission electron microscopy and particle size analysis were used to characterize morphology, size, and surface charge. Tumor-targeting and cellular uptake were explored. Antibacterial activity was assessed using disk diffusion assays, scanning electron microscopy, bacterial viability staining, and colony formation assays. Antitumor effects were evaluated using CCK-8 assays for cell viability, Annexin V/PI staining for apoptosis, and wound healing assays for migration. In vivo antibacterial and antitumor efficacy was assessed using three animal models. 16s rRNA sequencing of fecal microbiota was conducted to evaluate changes in gut microbiota diversity and abundance. Immunohistochemistry (IHC) was used to detect EMT-related protein expression, while immune cell infiltration in tumor tissues and peripheral blood was assessed using IHC and flow cytometry. ELISA was used to measure changes in inflammatory cytokines in peripheral blood.

Results:
Fn was found to be highly enriched in CRC tumor tissues and liver metastases which was associated with distant metastasis. We successfully constructed uniform NM@PLGA-MTI-OXA nanoparticles capable of targeting Fn-enriched inflammatory tumor regions and releasing both MTI and OXA. The neutrophil membrane coating retained key surface proteins, which enabled the nanoparticles to bind to upregulated adhesion molecules (VCAM1, CD44, ICAM1) on tumor cells induced by Fn, enhancing cellular uptake.

In vitro, NM@PLGA-MTI-OXA effectively disrupted Fn membranes, was internalized by tumor cells, and efficiently eliminated intracellular bacteria, while promoting apoptosis and inhibiting migration of CRC cells. In vivo, the nanoparticles significantly inhibited Fn infiltration and tumor progression in animal models without disrupting the balance of intestinal microbiota. Mechanistically, NM@PLGA-MTI-OXA downregulated the expression of EMT- related proteins (N-cadherin, Vimentin, Slug, Snail), reversing Fn-induced EMT progression. Furthermore, it enhanced infiltration level of CD4⁺ and CD8⁺ T cells in tumor tissues and peripheral blood, suppressed MDSC infiltration, and reduced expression of inflammatory cytokines (IL-6, IL-10, IL-22) in the peripheral blood.

Conclusion:
This study proposes a novel strategy to disrupt the pathogen–tumor symbiosis between CRC and Fn, thereby inhibiting tumor progression and metastasis. The engineered NM@PLGA-MTI-OXA nanoparticles, cloaked with neutrophil membranes, significantly enhance tumor-targeted delivery and cellular internalization within inflamed tumor microenvironments. This system increases intratumoral accumulation of metronidazole and oxaliplatin, enabling effective eradication of both intracellular and extracellular Fn. NM@PLGA-MTI-OXA reversed the EMT process of tumor cells and reshaped the tumor immune microenvironment while minimizing broad-spectrum damage to the commensal microbiota. This study provides a novel therapeutic strategy for colorectal cancer patients with liver metastases who have confirmed intratumoral bacterial infiltration. By focusing on the targeted elimination of microorganisms within the tumor microenvironment, it aims to overcome chemoresistance and enhance drug accumulation at tumor sites, demonstrating significant potential for clinical application.