目的 通过将肉桂醛（CA）共价接枝于壳寡糖（COS）侧链，并进一步自组装形成纳米粒（COS-CA-NPs），探讨其在肿瘤微酸环境下对乳腺癌转移的抑制效应及潜在作用机制。方法 采用席夫碱反应合成不同接枝度的COS-CA衍生物，分别命名为低接枝产物（COS-CA_L）和高接枝产物（COS-CA_H）；通过水相诱导将两种衍生物自组装为纳米粒子（COS-CA_LNPs、COS-CA_H-NPs）。采用核磁共振氢谱（¹H-NMR）、傅里叶变换红外光谱（FT-IR）表征COS-CA衍生物的化学结构；通过粒径、多分散性指数（PDI）、ζ电位评估纳米粒在不同pH环境下的理化特性，并采用透射电镜观察两种纳米粒的外观形态；采用MTT法检测纳米粒对MDA-MB-231人乳腺癌细胞增殖的抑制效应；通过细胞划痕和Transwell实验评价纳米粒对该细胞迁移、侵袭的影响；利用免疫荧光法和蛋白质免疫印迹法，检测纳米粒对核因子（NF）-κB及基质金属蛋白酶-9（MMP-9）表达水平的调控效应。结果 成功合成COS-CA_L、COS-CA_H两种衍生物；两种接枝比的COS-CA-NPs均呈球形，粒径均小于200 nm，且在低p H时可逐渐解离并释放CA。其中，与COS-CA_H-NPs相比，COS-CA_L-NPs对弱酸环境更为敏感，在pH 6.5时可发生质子化并携带正电荷。中性环境下，两种纳米粒对MDA-MB-231细胞的增殖抑制效果略低于游离CA；而在pH 6.5条件下，COS-CA_L-NPs显著降低了CA对该细胞的半数抑制浓度(IC₅₀)值。此外，游离CA、COS-CA_LNPs、COS-CA_H-NPs均能有效抑MDA-MB-231细胞的迁移与侵袭，其中COS-CA_L-NPs的抑制效果最优（P<0.01）。其抑制乳腺癌转移的作用机制主要通过CA抑制NF-κB活性、下调MMP-9表达水平实现。结论 COS-CA接枝物可自组装形成纳米药物粒子，其中COS-CA_L-NPs在肿瘤微酸环境中更易被乳腺癌细胞摄取并释放CA，进而通过调控NF-κB/MMP-9信号通路，有效抑制乳腺癌细胞的增殖、迁移及侵袭过程。

Objective By covalently grafting cinnamaldehyde（CA） onto the side chains of chitosan oligosaccharides（COS） and further self-assembling to form nanoparticles（COS-CA-NPs）, and investigating the inhibitory effect of these nanoparticles on breast cancer metastasis under the micro-acidic environment of tumors and their potential mechanism of action. Methods Different grafting degrees of COS-CA derivatives were synthesized via the Schiff base reaction and named as low grafting product（COS-CA_L） and high grafting product（COS-CA_H）. The two derivatives were self-assembled into nanoparticles（COS-CA_L-NPs and COS-CA_H-NPs） through aqueous phase induction. The chemical structure of COS-CA derivatives was characterized by ¹H-NMR and FT-IR. The physicochemical properties of the nanoparticles at different pH environments were evaluated by particle size, polydispersity index（PDI）, and ζ potential. The morphology of the two nanoparticles was observed by transmission electron microscopy（TEM）. The inhibitory effect of the nanoparticles on the proliferation of MDA-MB-231 human breast cancer cells was detected by MTT assay. The effects of the nanoparticles on cell migration and invasion were evaluated by cell scratch and Transwell assays. The regulatory effects of the nanoparticles on the expression levels of nuclear factor（NF）-κB and matrix metalloproteinase-9（MMP-9） were detected by immunofluorescence and Western blotting. Results COS-CA_L and COS-CA_H derivatives were successfully synthesized. Both COSCA-NPs with different grafting ratios were spherical, with particle sizes less than 200 nm, and could gradually dissociate and release CA at low pH. Compared with COS-CA_H-NPs, COS-CA_L-NPs were more sensitive to micro-acidic environments and could be protonated and carry positive charges at pH 6.5. Under neutral conditions, the proliferation inhibitory effects of the two nanoparticles on MDA-MB-231 cells were slightly lower than that of free CA. However, at pH 6.5, COS-CA_L-NPs significantly reduced the half maximal inhibitory concentration(IC₅₀) value of CA for this cell line. Additionally, free CA, COS-CA_L-NPs, and COS-CA_H-NPs could all effectively inhibit the migration and invasion of MDA-MB-231 cells, with COS-CA_L-NPs showing the best inhibitory effect（P＜0.01）. The mechanism of inhibiting breast cancer metastasis mainly involves CA inhibiting NF-κB activity and down-regulating MMP-9 expression levels. Conclusion COS-CA grafts can self-assemble into nanoparticles. Among them, COS-CA_L-NPs are more easily taken up by breast cancer cells and release CA in tumor micro-acidic environments, thereby effectively inhibiting the proliferation, migration, and invasion of breast cancer cells through the regulation of the NF-κB/MMP-9 signaling pathway.

R979.1

国家自然科学基金资助项目(32301126); 安徽省教育厅优秀青年基金资助项目(2024AH030039); 安徽省中青年教师培育项目(YQYB2024037); 安徽省卫健委重点项目(AHWJ2024Aa20195); 安徽省博士后科研项目(2024C857)