目的 通過將肉桂醛（CA）共價接枝于殼寡糖（COS）側(cè)鏈，并進(jìn)一步自組裝形成納米粒（COS-CA-NPs），探討其在腫瘤微酸環(huán)境下對乳腺癌轉(zhuǎn)移的抑制效應(yīng)及潛在作用機(jī)制。方法 采用席夫堿反應(yīng)合成不同接枝度的COS-CA衍生物，分別命名為低接枝產(chǎn)物（COS-CA_L）和高接枝產(chǎn)物（COS-CA_H）；通過水相誘導(dǎo)將兩種衍生物自組裝為納米粒子（COS-CA_LNPs、COS-CA_H-NPs）。采用核磁共振氫譜（¹H-NMR）、傅里葉變換紅外光譜（FT-IR）表征COS-CA衍生物的化學(xué)結(jié)構(gòu)；通過粒徑、多分散性指數(shù)（PDI）、ζ電位評估納米粒在不同pH環(huán)境下的理化特性，并采用透射電鏡觀察兩種納米粒的外觀形態(tài)；采用MTT法檢測納米粒對MDA-MB-231人乳腺癌細(xì)胞增殖的抑制效應(yīng)；通過細(xì)胞劃痕和Transwell實驗評價納米粒對該細(xì)胞遷移、侵襲的影響；利用免疫熒光法和蛋白質(zhì)免疫印跡法，檢測納米粒對核因子（NF）-κB及基質(zhì)金屬蛋白酶-9（MMP-9）表達(dá)水平的調(diào)控效應(yīng)。結(jié)果 成功合成COS-CA_L、COS-CA_H兩種衍生物；兩種接枝比的COS-CA-NPs均呈球形，粒徑均小于200 nm，且在低p H時可逐漸解離并釋放CA。其中，與COS-CA_H-NPs相比，COS-CA_L-NPs對弱酸環(huán)境更為敏感，在pH 6.5時可發(fā)生質(zhì)子化并攜帶正電荷。中性環(huán)境下，兩種納米粒對MDA-MB-231細(xì)胞的增殖抑制效果略低于游離CA；而在pH 6.5條件下，COS-CA_L-NPs顯著降低了CA對該細(xì)胞的半數(shù)抑制濃度(IC₅₀)值。此外，游離CA、COS-CA_LNPs、COS-CA_H-NPs均能有效抑MDA-MB-231細(xì)胞的遷移與侵襲，其中COS-CA_L-NPs的抑制效果最優(yōu)（P<0.01）。其抑制乳腺癌轉(zhuǎn)移的作用機(jī)制主要通過CA抑制NF-κB活性、下調(diào)MMP-9表達(dá)水平實現(xiàn)。結(jié)論 COS-CA接枝物可自組裝形成納米藥物粒子，其中COS-CA_L-NPs在腫瘤微酸環(huán)境中更易被乳腺癌細(xì)胞攝取并釋放CA，進(jìn)而通過調(diào)控NF-κB/MMP-9信號通路，有效抑制乳腺癌細(xì)胞的增殖、遷移及侵襲過程。

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

國家自然科學(xué)基金資助項目(32301126); 安徽省教育廳優(yōu)秀青年基金資助項目(2024AH030039); 安徽省中青年教師培育項目(YQYB2024037); 安徽省衛(wèi)健委重點項目(AHWJ2024Aa20195); 安徽省博士后科研項目(2024C857)