Bioimaging plays a crucial role in modern medicine and research. It allows for the non-invasive, real-time observation of tissues and cellular activities within living organisms. This technology provides powerful support for cancer diagnosis, drug development, and studies of various biological mechanisms.<sup><ahref="#re_1">1</a>,<ahref="#re_2">2</a>,<ahref="#re_3">3</a></sup>
<p><span>Timeline of Major Advances in Intraoperative Imaging Technologies</span></p>
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Among the different bioimaging techniques, fluorescence imaging is one of the most widely used.<sup><ahref="#re_1">1</a>,<ahref="#re_3">3</a></sup>Fluorescence-Guided Surgery (FGS) has become a key tool in modern surgical procedures.<sup><ahref="#re_2">2</a></sup>By using specific fluorescent probes, surgeons can view tumor margins, blood vessel structures, and lymph node locations in real time during surgery. This significantly improves surgical precision, reduces damage to healthy tissues, and enhances patient outcomes.
While fluorescence imaging has been widely applied across multiple disciplines, particularly excelling in its high sensitivity and superior spatiotemporal resolution, it still faces significant challenges in real-time deep tissue imaging. The optical opacity of biological tissues limits the penetration depth of light signals, reducing imaging quality, especially in clinical and biomedical applications.<sup><ahref="#re_4">4</a>,<ahref="#re_5">5</a>,<ahref="#re_6">6</a></sup>
<p><span>Comparison of Penetration Depth between Lower-wavelength Fluorescence and NIR Fluorescence</span></p>
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Near-infrared II (NIR-II) imaging offers several key advantages over other imaging techniques, particularly in the context of biological and medical applications. The NIR-II window (900 nm to 1700 nm) allows for deeper tissue penetration, reduced scattering, and lower levels of autofluorescence, all of which contribute to significantly enhanced imaging resolution and clarity.<sup><ahref="#re_8">8</a>,<ahref="#re_9">9</a></sup>
<b>ICG (Indocyanine Green)</b> is a notable example of a standalone NIR probe that has been approved by the FDA for clinical use. It is the only NIR-I dye currently authorized for applications like fluorescence-guided surgery and vascular imaging. Despite its regulatory approval and widespread use, ICG also suffers from many of the limitations inherent to NIR probes. Its fluorescence signal can be weak, and its poor stability in vivo leads to rapid clearance by the liver and kidneys, which restricts its imaging potential. Additionally, ICG lacks strong target specificity, resulting in low accumulation at target sites and high background signals, which can impair the quality of imaging.
To overcome the limitations of NIR probes in vivo, we propose a delivery system based on genetically engineered human serum albumin (HSA). By encapsulating and delivering NIR-II probes using HSA, we can effectively prevent the formation of a protein corona, extend the circulation time of the probes in the body, and enhance their bioavailability. The formation of a protein corona usually leads to rapid clearance of the probe, reducing its targeting efficiency. As a naturally occurring protein in the bloodstream, HSA not only covalently binds with the probe but also significantly improves the probe's stability and targeting ability.
Through site-directed mutagenesis of HSA (e.g., modification of the Cys476 site), we disrupted disulfide bonds to further enhance the covalent binding between HSA and the probe. This optimization ensures that the probe remains stable and active for extended periods in vivo, while accurately targeting specific tissues. The encapsulation by HSA reduces interactions between the probe and the immune system, minimizing the risk of rapid clearance and improving the overall imaging efficiency and effectiveness.
