Abstract: Objective　Construction of a specific probe targeting triggering receptor expressed on myeloid cells-1 (TREM-1) for noninvasive in vivo precise identification of vulnerable plaques in mouse carotid arteries by fluorescence imaging.Methods　Synthesized specific nanoprobes targeting TREM-1. Thirty male apolipoprotein E knockout (ApoE-/-) mice were divided into carotid artery vulnerable plaque model group (AS group,n=20) and control group (n=10) based on whether unilateral carotid artery stenosis ring placement was performed and the type of diet administered. AS group mice underwent unilateral carotid artery stenosis ring placement and were fed a high-fat diet for 20-24 weeks. Afterward, six mice were randomly selected for in vivo fluorescence imaging. Based on the injected probe type, these mice were further divided into the AS+non-targeted nanoparticle group (AS+nNPs, n=3) and the AS+TREM 1-targeted nanoparticle group (AS+fNPs, n=3). Control mice received standard maintenance diet for the same duration. Subsequently, three mice were randomly selected for TREM-1-targeted nanoparticle injection (control+fNPs) followed by in vivo fluorescence imaging. This validated whether our constructed specific probes could accurately identify vulnerable plaques in vivo.Results　A specific molecular probe was successfully constructed, exhibiting uniform morphology under electron microscopy with a near-circular shape and a diameter of approximately 65 nm. In vitro fluorescence imaging demonstrates that the probe exhibits excellent near infrared region-Ⅱ (NIR-Ⅱ) luminescence capability. Following euthanasia, aortic vessels from AS group mice underwent gross oil red O staining.Results revealed extensive lipid plaques in the carotid arteries. Hematoxylin-eosin (HE), Masson’s trichrome, and oil red O staining of carotid arteries indicated substantial plaque formation beneath the intima, characterized by lipid-rich necrotic cores and reduced collagen content near the intima, meeting diagnostic criteria for vulnerable plaques. In vivo fluorescence imaging results demonstrated that, compared with the AS+nNPs group, mice in the AS+fNPs group exhibited significantly increased probe accumulation in the carotid artery region and markedly enhanced fluorescence signal intensity.The probe exhibited peak fluorescence intensity 3 h post-injection, with gradual attenuation thereafter,while faint fluorescence remained detectable at 24 h. In ApoE-/- mice fed standard chow and injected with TREM-1 targeted nanoprobes, no significant fluorescence signals were observed in the carotid artery region. The probes demonstrated no apparent toxic side effects on liver and kidney function, cardiac enzymes, or major organs.Conclusion　In vivo fluorescence imaging experiments in mice demonstrate that the specific probes in this study can accurately recognize arterial vulnerable plaques, exhibiting high sensitivity, a prolonged observation window, and excellent biosafety.

Key words: nanoprobe, vulnerable plaque, carotid sleeving vulnerable plaque model

摘要： 目的　构建靶向髓系细胞触发受体-1(TREM-1)的特异性探针,通过荧光成像实现无创在体精准识别小鼠颈 动脉易损斑块。方法　合成靶向TREM-1的特异性纳米探针。根据是否行单侧颈动脉缩窄环放置术及给予饲料 的不同,将30只雄性载脂蛋白E基因敲除(ApoE-/-)小鼠分为颈动脉易损斑块模型组(AS组,n=20)及对照组 (control组,n=10)。AS组小鼠行单侧颈动脉缩窄环放置术并予高脂饲料喂养20~24周,之后随机选取6只小鼠 行在体荧光成像,根据注射探针类型进一步分为AS+非靶向性纳米探针组(AS+nNPs组,n=3)及AS+靶向TREM-1 纳米探针组(AS+fNPs组,n=3)。Control组小鼠给予普通维持饲料喂养相同时间,之后随机选取3只小鼠注射靶 向TREM-1的纳米探针(control+fNPs)进行在体荧光成像。验证本研究所构建的特异性探针能否在体准确识别易 损斑块。结果　成功构建了特异性分子探针,电镜下形态均一,呈类圆形,直径约65 nm。体外荧光成像显示探针 具有良好的近红外二区(NIR-Ⅱ)发光能力。AS组小鼠处死后取主动脉血管行大体油红O染色,结果提示小鼠颈 动脉血管处可见大量脂质斑块;颈动脉血管苏木精-伊红(HE)、马松(Masson)和油红O染色提示内膜下有大量含 脂质坏死核心的斑块形成,且靠近内膜处胶原含量低,符合易损斑块诊断标准。在体荧光成像结果表明,与AS+ nNPs组相比,AS+fNPs组小鼠的颈动脉区域探针富集显著增加,荧光信号强度显著增强。探针在进入体内3 h时 信号最强,此后逐渐衰减,24 h时仍可见微弱荧光信号。Control+fNPs组小鼠未在颈动脉区域观测到明显的荧光信 号。探针对肝肾功能、心肌酶等指标及主要器官无明显毒副作用。结论　小鼠在体荧光成像实验表明本研究构建 的特异性探针能够精准识别动脉易损斑块,具有良好的灵敏度、较长的观测窗口及生物安全性。

关键词: 纳米探针, 易损斑块, 颈动脉套扎易损斑块模型