【JACS】东南大学梁高林、周思思|非侵入性光声与荧光双模态成像区分不同时期易损动脉粥样硬化斑块：实现光声4.7倍与荧光3.9倍的阶段动态转换
【JACS】东南大学梁高林、周思思|非侵入性光声与荧光双模态成像区分不同时期易损动脉粥样硬化斑块：实现光声4.7倍与荧光3.9倍的阶段动态转换
文章标题：Noninvasive Photoacoustic and Fluorescence Bimodality Imaging Differentiation of Vulnerable Atherosclerotic Plaques at Different Stages
文章作者：Qiaochu Jiang, Penghao Zhen, Xinru Kong, Zihan Yuan, Yu Ma, Hai-Dong Xu, Xiaoyang Liu, Fan Xing, Wenjun Zhan, Xianbao Sun, Gaolin Liang, Sisi Zhou
临床瓶颈与阶段特异性分子诊断的研究动因
易损动脉粥样硬化斑块破裂是诱发急性心肌梗死等心血管急症的主要诱因，且超过半数的易损斑块在破裂前并无明显管腔狭窄。当前临床主要依赖血管造影等管腔狭窄检测手段，无法识别非狭窄性炎性斑块，更难以动态监测炎症及酶学活性来对斑块破裂风险进行分层。易损斑块风险分层的核心在于精准鉴别病程早期与晚期阶段，然而现有成像技术尚缺乏能够同时靶向不同病理时期特异性生物标志物的智能分子探针。针对这一关键短板，本文设计了一种可激活的有机双模态分子探针，旨在通过早期血管新生标志物与晚期炎性基质金属蛋白酶的时序响应，实现非侵入性光声与荧光双模态成像分期诊断，为高危斑块的早期识别与临床干预提供全新策略。
Fig.1 Design and mechanisms of noninvasive probe RGD-IR-Dimer for fluorescence/photoacoustic (FL/PA) bimodality imaging to differentiate early- and late-stage atherosclerotic vulnerable plaques. (a) Chemical structure of RGD-IR-Dimer and schematic illustration of its behavior: upon self-assembly, it aggregates to activate photoacoustic signals; after MMP-8-mediated enzymatic cleavage, it is converted to RGD-IR-Cleaved to turn on fluorescence signals. (b) Schematic illustration of the working mechanism of fluorescence/photoacoustic (FL/PA) bimodal imaging using RGD-IR-Dimer to distinguish vulnerable atherosclerotic plaques at different stages of progression.
双靶向自组装探针设计与体外及体内多维验证
本文首次提出了一种基于“分子积聚原位自组装—酶解触发解组装”动态级联机制的双靶向激活型分子探针（ RGD-IR-Dimer）。该探针由两个近红外发色团IR780、两组基质金属蛋白酶-8（MMP-8）特异性剪切多肽GILG序列以及两组整合素靶向环肽cRGDfK组成。其工作逻辑在于：探针静脉注射后经介导在病灶富集，局部高浓度驱动其原位自组装为纳米纤维，通过聚集诱导猝灭（ACQ）抑制荧光，同时开启光声（PA）“开”信号；在晚期斑块中，高表达的MMP-8特异性剪切多肽骨架，促使纳米纤维解聚，从而恢复发色团的荧光（FL）“开”信号并伴随光声信号“关”**，构建出由时序生物标志物调控的信号双向切换闭环体系。
实验系统评估了探针的理化性质、细胞分期识别能力与动物模型成像表现。理化测试显示该探针的临界聚集浓度为55.4 μM，组装形成的纳米纤维直径约为40 nm；酶解产物较组装态探针呈现18倍的荧光信号恢复，而组装态的光声信号强于酶解态70倍以上，同时探针在1000 μM浓度下未表现出明显溶血毒性，巨噬细胞存活率超过89%。在体外细胞模型中，模拟早期病灶的脑内皮细胞（高表达、低表达MMP-8）表现为光声信号随时间持续增强而荧光保持沉默；模拟晚期病灶的巨噬细胞源泡沫细胞（高表达MMP-8）中，探针的光声信号在1小时达峰后迅速衰减，荧光信号则持续增强，成功实现了细胞水平上的早晚期鉴别。
在体内验证环节，研究团队在动脉粥样硬化ApoE基因敲除小鼠模型中进行了验证。尾静脉注射探针后，模型小鼠颈动脉区域的光声信号在2小时达到峰值，强度为非靶向对照组的4.7倍，并在8小时回落至基线；荧光信号则在0.5小时即可检出，在6小时达到峰值（较对照组高出3.9倍）并稳定持续长达72小时。这一时间依赖性的“光声达峰衰减—荧光持续点亮”动态特征，在活体水平上验证了探针响应晚期易损斑块微环境的信号转换能力。
Fig.2 Characterization of IR-Dimer and RGD-IR-Dimer. (a) Chemical structures of IR-Dimer and IR780. (b) TEM image of 100 μM IR-Dimer in PBS at pH 7.4. (c) TEM image of 100 μM RGD-IR-Dimer in PBS at pH 7.4. (d) Fluorescence of 100 μM IR-Dimer, 100 μM RGD-IR-Dimer, and 100 μM RGD-IR-Cleaved in PBS at pH 7.4. (e) Photoacoustic imaging of IR-Dimer, RGD-IR-Dimer, and RGD-IR-Cleaved at different concentrations. The compounds were dispersed in PBS (pH 7.4), mixed with 40 wt % agarose solution, and then fixed in capillaries. (f) Hemolysis rates of IR780, IR-Dimer, and RGD-IR-Dimer at different concentrations after incubation at 37 °C for 6 h. RBCs in PBS or water were set as the negative control and positive control, respectively. Wavelength for detection: 450 nm.
Fig.3 In vitro photoacoustic and fluorescence imaging of the early-stage atherosclerotic vulnerable plaque model. (a) Schematic illustration of RGD-IR-Dimer self-assembly in bEnd.3 cells and the subsequent “turn-on” of photoacoustic signals. bEnd.3 cells incubated with 10 μM probes at 37 °C for different time points; then, cells were collected and fixed in capillaries for imaging. (b) PA images and (c) the corresponding quantified PA signal intensities of cells treated with IR-Dimer and RGD-IR-Dimer at different time points (mean ± SD, n = 3). (d) FL images and (e) corresponding quantified FL signal intensities of cells treated with IR-Dimer and RGD-IR-Dimer at different time points (mean ± SD, n = 3) (for PA imaging, wavelength = 780 nm; for FL imaging, λex = 780 nm, λem= 845 nm).
Fig.4 In vitro photoacoustic and fluorescence imaging of the late-stage atherosclerotic vulnerable plaque model. (a) Schematic illustration of RGD-IR-Dimer self-assembly in foam cells (a late-stage plaque model recapitulating an MMP-8-rich microenvironment) followed by enzyme-instructed disassembly, which turns on photoacoustic signals during the assembled state and switches to fluorescence signals after cleavage. Macrophages and foam cells were incubated with 10 μM probes at 37 °C for different time points, then collected, fixed in capillaries, and imaged. (b) PA images and corresponding quantified PA signal intensities of cells treated with (c) IR-Dimer and (d) RGD-IR-Dimer at different time points (mean ± SD, n = 3). (e) FL images and corresponding quantified FL signal intensities of cells treated with (f) IR-Dimer and (g) RGD-IR-Dimer at different time points (mean ± SD, n = 3) (for PA imaging, wavelength = 780 nm; for FL imaging, λex = 780 nm, λem= 845 nm).
Fig.5 In vivo photoacoustic and fluorescence imaging of vulnerable atherosclerotic (AS) plaques in an ApoE–/– mouse model. (a) PA images and (b) FL images of the carotid artery region at various time points after intravenous injection of 3 mg/kg RGD-IR-Dimer into healthy C57BL/6 mice, 3 mg/kg IR-Dimer, or 3 mg/kg RGD-IR-Dimer into AS model mice. (c) Quantified PA signal intensities corresponding to a (mean ± SD, n = 2). (d) Quantified FL signal intensities corresponding to (b) (mean ± SD, n = 2) (for PA imaging, wavelength = 780 nm; for FL imaging, λex = 780 nm, λem= 845 nm).
核心机制创新与病理分期性能的量化提升
本研究的核心创新在于打破了传统单一靶点或预制纳米探针的局限，首次利用单分子二聚体原位组装与解聚的物理化学转变，将早期血管新生标志物（）与晚期破裂核心酶（MMP-8）的时序表达转化为“光声独亮”到“光声转荧光”的明确模态指纹。在性能上实现了4.7倍光声增强、3.9倍靶向荧光富集以及18倍荧光开启信噪比，有效攻克了传统造影无法识别非狭窄性早期及晚期炎性斑块的临床痛点。论文同时指出，由于现有小鼠模型缺乏模拟人类早期斑块血管新生的病理结构，体内直接早晚期分期对照仍待适配动物模型建立后深入探索，但该原位组装转换范式已为肿瘤侵袭、慢性炎症等时序性疾病的早期预警提供了极具前景的通用化设计框架。
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