多光谱光声示踪策略用于炎症反应中巨噬细胞的大视野实时监测

炎症是血管系统常见的防御反应，其中涉及免疫细胞的招募激活和干细胞归巢。研究这些功能细胞在体内向炎症部位的动态迁移和富集可为疾病诊断和治疗提供相关有效信息，具有重要的意义。然而，现有的光学手段很难实现在大视野下以理想的分辨率示踪和分析这些细胞类群在炎症微环境中的实时状态。

基于这个问题，我们设计并合成了一种近红外吸收的半导体聚合物p-BBT-TQP，并将其包覆为穿膜肽修饰功能化纳米粒子Tat-p-BBT-TQP NPs （Tat-BTNPs），以对炎症反应中发挥重要作用的巨噬细胞进行标记与体内光声示踪。Tat-BTNPs的光物理性质表征结果显示其具有近红外二区荧光以及良好的光声特性。体外细胞实验证明了Tat-BTNPs可长时间稳定标记巨噬细胞且不影响其细胞活性，表明Tat-BTNPs具有体内长期示踪的潜力。利用该纳米探针的光声特性，本工作揭示了巨噬细胞、中性粒细胞和间充质干细胞对小鼠炎症部位的趋向行为。借助多光谱光学分辨率光声显微镜（Optical-resolution photoacoustic microscopy, ORPAM），我们可以在大视野范围（直径为9 mm的圆形视野）中以细胞尺度分辨率实时监测炎症部位的光声信号变化，用于判断标记细胞在该部位的动态富集过程，且光声结果的准确性可以通过活体近红外二区和双光子荧光结果进行双重验证。此外，对血管微观结构和标记细胞的高灵敏检测可以揭示各种细胞类型在对炎症部位的时间依赖性富集行为上的相关性。综上，本研究提供了一种有效且具有广阔应用前景的光声示踪策略，以分析趋向病变/受损区域功能细胞的体内行为和命运。

Inflammation is a common defense response of the vascular system and involves the recruitment and activation of immune cells as well as the homing of stem cells. It is of great significance to study the dynamic migration and aggregation of these functional cells to inflammatory sites in vivo, which can provide useful information for diagnosis and treatment. However, existing optical methods are difficult to track and analyze the real-time status of these cell types in the inflammatory microenvironment with ideal resolution in a large field of view. 

Based on this challenge, we designed and synthesized the semiconductor polymer p-BBT-TQP with near infrared absorption and coated it as peptide functionalized nanoparticles Tat-p-BBT-TQP NPs (Tat-BTNPs) to track macrophages, an important cell type in inflammatory response in vivo. The photophysical properties of Tat-BTNPs showed that it had fluorescence in the second near-infrared window (NIR-II) and great photoacoustic properties. In vitro cellular experiments demonstrated that macrophages can be labeled by Tat-BTNPs for a long time with no toxicity, indicating that Tat-BTNPs has the potential of long-term labeling in vivo. This study revealed the targeting behavior of macrophages, neutrophils and mesenchymal stem cells to inflammatory sites in mice, utilizing the photoacoustic properties of Tat-BTNPs. Facilitated by a multispectral optical-resolution photoacoustic microscopy (ORPAM), we can continuously monitor the in vivo photoacoustic signals of labeled cells at inflammatory sites with cellular resolution in a wide field (a circular field with a diameter of 9 mm), to determine the dynamic enrichment process of labeled cells. The accuracy of the obtained photoacoustic results can be verified by in vivo NIR-II and two-photon fluorescence results. In addition, highly sensitive detection of vascular microstructure and labeled cells can reveal correlations between different cell types in time-dependent enrichment behavior toward inflammatory sites. Therefore, our study provides an effective and promising photoacoustic tracking strategy to analyze the in vivo behavior and fate of functional cells targeting diseased or damaged regions.

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