恶性肿瘤的主要治疗仍然是通过手术移除患病组织。肿瘤边缘存在或不存在残留患病组织是术后预后和复发的最强预测因素。因此，外科医生通过实时明确地视觉区分患病组织和健康组织的能力至关重要。近红外一（NIRI）荧光发射靶向生物分子构建物，如抗癌抗体-荧光素结合物，即西妥昔单抗-IRDye® 800CW（CTB-IRDye® 800CW），经FDA批准用于临床试验，以荧光引导的切除病变组织，因为与标准白光照射下的裸眼手术技术或非靶向荧光素使用相比，能够提供更好的肿瘤组织直接可视化。然而，虽然有帮助，但CTB-IRDye® 800CW的局限性在于产生次优的肿瘤-背景比率（TBRs），因其光谱/光物理特性未能与预设的临床设置的固定光学窗口完全吻合，导致其对病变组织识别和肿瘤边缘描绘的能力有限，更容易导致切除不完全。为了准确识别深部患病组织，我们采用了光声（PA）断层扫描技术，同时结合CTB-IRDye® 800CW使用，以产生可能导致更高的TBRs的PA信号。然而，在临床试验中，使用IRDye® 800CW进行PA成像也会产生低TBRs的结果。为了克服这些限制，我们开发了NIRDye 812，这是IRDye® 800CW的结构修改拓扑等效物，赋予它比等效CTB-结合物和荧光素IRDye® 800CW自身更高的TBRs和更优越的PA信号能力。为此，我们用硫代替其介位上的氧原子。CTB-NIRDye 812在796nm处表现出红移吸收波长，并在820nm处拥有峰值的近红外一荧光发射波长，更好地将其峰值与商用的预安装固定发射滤光片窗口结合起来，以充分展现其在预-/临床NIRI荧光成像器械上的应用。总的来说，在体内CTB-NIRDye 812的TBRs比CTB-IRDye® 800CW大约提高了2倍。此外，NIRDye 812的PA信号比IRDye® 800CW高约60%。综上所述，通过对其电子核心进行微小修改，我们达成了改进IRDye® 800CW光谱/光物理和PA特性的目标，并使其CTB免疫结合物潜在获得FDA的快速通道或突破性认可，因为其结构近乎相同，显示出明显的改善效果。版权所有 © 2023 Elsevier B.V. All rights reserved.

The primary treatment for malignant tumors remains to be surgical removal of the diseased tissue. The presence or absence of residual diseased tissue at the tumor margin is the strongest predictor of postoperative prognosis and recurrence. Accordingly, reliance on the ability of surgeons to visually distinguish diseased tissue from healthy tissue unambiguously in real time is crucial. Near infrared-I (NIRI) fluorescence-emitting targeting biomolecular constructs such as anticancer antibody-fluorophore conjugates, namely cetuximab-IRDye® 800CW (CTB-IRDye® 800CW), are FDA-approved for clinical trial usage in the fluorescence-guided resection of diseased tissue due to affording improved direct visualization of tumor tissue when compared to the use of either the unaided eye under standard white light illumination (WLI) surgical techniques or non-targeting fluorophores. Unfortunately, though helpful, CTB-IRDye® 800CW affords limited (i) identification of diseased tissue and (ii) tumor margin delineation, because the immunoconjugate generates suboptimal tumor-to-background ratios (TBRs) as a result of its spectral/photophysical profiles poorly aligning with the fixed optical windows of pre-/clinical setups. As such, CTB-IRDye® 800CW is more prone to affording incomplete resection compared to if TBRs were higher due to otherwise. To aid in accurately identifying deep-seated diseased tissue, photoacoustic (PA) tomography has been implemented alongside CTB-IRDye® 800CW to achieve PA signals that could result in higher TBRs. However, in clinical trial practice, using IRDye® 800CW for PA imaging also yields subpar TBRs due to it affording low PA signals. To overcome such limitations, we developed NIRDye 812, a structurally-modified topological equivalent of IRDye® 800CW, to confer it the capability to yield both higher TBRs and superior PA signal than that of the equivalent CTB-conjugate and fluorophore IRDye® 800CW itself, respectively. To do so, we substituted the oxygen atom at its meso-position with a sulfur atom. CTB-NIRDye 812 demonstrated a red-shifted absorption wavelength at 796 nm and a peak NIR-I fluorescence emission wavelength at 820 nm, which better dovetails with the fixed windows of preinstalled fixed emission filters within commercial pre-/clinical NIR-I fluorescence imaging instruments. Overall, CTB-NIRDye 812 provided a ∼ 2-fold increase in TBRs compared to those of CTB-IRDye® 800CW in vivo. Also, NIRDye 812 displayed an ∼60% higher PA signal than that of IRDye® 800CW. Collectively, we achieved our goal of improving upon the spectral/photophysical and PA properties of IRDye® 800CW via introducing a subtle modification to its electronic core such that its CTB immunoconjugate could potentially allow for fast track or breakthrough designation by the FDA due to its near-identical structure displaying considerably improved efficacy.Copyright © 2023 Elsevier B.V. All rights reserved.