Huang Yishu, Stankevych Mariia, Gujrati Vipul, Klemm Uwe, Mohammed Azeem, Wiesner David, Saccomano Mara, Tost Monica, Feuchtinger Annette, Mishra Kanuj, Bruns Oliver, Geerlof Arie, Ntziachristos Vasilis, Stiel Andre C
Institute of Biological and Medical Imaging, Bioengineering Center, Helmholtz Zentrum München, Neuherberg, Germany; Chair of Biological Imaging, Central Institute for Translational Cancer Research (TranslaTUM), School of Medicine and Health & School of Computation, Information and Technology, Technical University of Munich, Munich, Germany.
Institute of Biological and Medical Imaging, Bioengineering Center, Helmholtz Zentrum München, Neuherberg, Germany.
Acta Biomater. 2025 Mar 15;195:536-546. doi: 10.1016/j.actbio.2025.02.002. Epub 2025 Feb 4.
Optoacoustic imaging (OAI) is a unique in vivo imaging technique combining deep tissue penetration with high resolution and molecular sensitivity. OAI relying on strong intrinsic contrast, such as blood hemoglobin, already shows its value in medical diagnostics. However, OAI sensitivity to current extrinsic contrast agents is insufficient and limits its role in detecting disease-related biomarkers. The recently introduced concept of photoswitching and temporal unmixing techniques for OAI allows detecting extrinsic contrast with high sensitivity, allowing the visualization of small populations of cells labeled with photoswitching proteins deep within the tissue. However, transgene modification might not be permitted in some cases, such as for diagnostic use. Therefore, it is desirable to leverage the concept of photoswitching OAI towards injectable formulations. Since photoswitchable synthetic dyes are mainly excited by blue wavelengths unsuited for imaging in tissue, we propose exploiting the addition of XTENs to photoswitching proteins towards yielding injectable agents. The addition of XTEN to a protein enhances its plasma half-life and bioavailability, thus allowing its use, for example, in targeted labeling approaches. In this pilot study, we show that intravenously injected near-infrared absorbing photoswitchable proteins, ReBphP-PCM, coupled to XTEN, allow highly sensitive optoacoustic visualization of a tumor xenograft in vivo. The sensitivity to XTENs-ReBphP-PCM determined by ex vivo analysis of labeled cells is one to two orders of magnitude beyond conventional synthetic dyes used currently in OAI. The enhanced sensitivity afforded by photoswitching OAI, in combination with the increased bioavailability and biocompatibility of XTENs-ReBphP-PCM, makes this fusion protein a promising tool for facilitating sensitive detection of biomarkers in OAI with a potential for future use in diagnostics. STATEMENT OF SIGNIFICANCE: Optoacoustic imaging (OAI) is a unique in vivo imaging technique that combines deep tissue penetration with high resolution. OAI, which relies on intrinsic contrast, such as blood hemoglobin, could already be valuable in medical diagnostics. However, the use of extrinsic contrast agents to augment disease-related biomarkers in research and diagnostics suffers from very limited sensitivity of the generated contrast agent. We present an intravenously injected photoswitchable protein, ReBphP-PCM, coupled to XTEN, allowing highly sensitive OAI. The sensitivity is one to two orders of magnitude greater than that of conventional synthetic dyes used currently in OA imaging. The high sensitivity afforded by photoswitching together with the enhanced bioavailability and biocompatibility of the XTENs-ReBphP-PCM make this a standard agent for high-quality detection of OAI with potential for clinical use.
光声成像（OAI）是一种独特的体内成像技术，它将深层组织穿透性与高分辨率及分子敏感性相结合。依赖于血液血红蛋白等强大内在对比度的OAI，已在医学诊断中展现出其价值。然而，OAI对当前外源性造影剂的敏感性不足，限制了其在检测疾病相关生物标志物方面的作用。最近引入的用于OAI的光开关和时间解混技术概念，能够高灵敏度地检测外源性造影剂，使得在组织深处标记有光开关蛋白的少量细胞得以可视化。然而，在某些情况下，如用于诊断用途时，转基因修饰可能不被允许。因此，期望将光开关OAI的概念应用于可注射制剂。由于光开关合成染料主要由不适用于组织成像的蓝光波长激发，我们提议通过向光开关蛋白添加XTENs来开发可注射制剂。向蛋白质添加XTEN可延长其血浆半衰期并提高生物利用度，从而使其可用于例如靶向标记方法。在这项初步研究中，我们表明静脉注射与XTEN偶联的近红外吸收光开关蛋白ReBphP - PCM，能够在体内对肿瘤异种移植进行高灵敏度的光声可视化。通过对标记细胞的离体分析确定，对XTENs - ReBphP - PCM的灵敏度比目前OAI中使用的传统合成染料高1至2个数量级。光开关OAI提供的增强灵敏度，与XTENs - ReBphP - PCM提高的生物利用度和生物相容性相结合，使这种融合蛋白成为促进OAI中生物标志物灵敏检测的有前景工具，具有未来用于诊断的潜力。重要性声明：光声成像（OAI）是一种独特的体内成像技术，它将深层组织穿透性与高分辨率相结合。依赖于血液血红蛋白等内在对比度的OAI，在医学诊断中已具有价值。然而，在研究和诊断中使用外源性造影剂来增强疾病相关生物标志物时，所产生的造影剂灵敏度非常有限。我们展示了一种静脉注射的与XTEN偶联的光开关蛋白ReBphP - PCM，可实现高灵敏度的OAI。其灵敏度比目前OA成像中使用的传统合成染料高1至2个数量级。光开关提供的高灵敏度与XTENs - ReBphP - PCM增强的生物利用度和生物相容性相结合，使其成为高质量OAI检测的标准试剂，具有临床应用潜力。
