Project Description

Importance of Bioimaging

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.^1,2,3

Timeline of Major Advances in Intraoperative Imaging Technologies

Among the different bioimaging techniques, fluorescence imaging is one of the most widely used.^1,3Fluorescence-Guided Surgery (FGS) has become a key tool in modern surgical procedures.²By 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.

In addition to clinical applications, bioimaging is also advancing fundamental life science research. Researchers can use imaging technologies to explore the intricacies of biological processes such as cell division, molecular interactions, and gene expression. The rapid development of modern bioimaging techniques enables us to explore the mysteries of life with higher resolution, deeper tissue penetration, and longer imaging durations.

Limitations of Current Bioimaging Techniques

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.^4,5,6

Comparison of Penetration Depth between Lower-wavelength Fluorescence and NIR Fluorescence

Current near-infrared (NIR-I) fluorescence imaging, though used for in vivo imaging, falls short in terms of penetration depth and resolution, often failing to meet the demands for deeper tissue observation. NIR-II imaging, with its reduced scattering and deeper tissue penetration, has garnered attention. However, existing NIR-II small-molecule fluorescent dyes exhibit limitations in optical performance and pharmacokinetics. These dyes have limited emission wavelengths, insufficient photostability, and poor biocompatibility, which restricts their practical clinical applications.

Furthermore, current NIR probes often have low targeting efficiency, short circulation times, and are quickly cleared from the body, limiting their utility. These technical challenges need to be addressed by improving probe design and developing more efficient fluorescent dyes to enhance imaging quality and clinical potential.^5,6,7

Advantages of NIR-II Imaging

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.^8,9

In traditional imaging methods, biological tissues create substantial scattering and absorption, limiting the ability to visualize deep structures. However, the longer wavelengths used in NIR-II imaging result in reduced light scattering, enabling clearer and deeper tissue imaging. This makes NIR-II imaging particularly effective for in vivo imaging, allowing researchers and clinicians to capture detailed images of deep-seated tissues without the need for invasive procedures.^{9,10,11,12,13}

Additionally, NIR-II imaging produces lower levels of autofluorescence compared to NIR-I and visible light imaging. This reduced background noise improves the contrast and specificity of the images, making it easier to distinguish between different tissue types and detect subtle changes within the body.

These advantages make NIR-II imaging a promising tool for applications such as real-time surgical guidance, early disease detection, and dynamic biological imaging.⁸

Limitations of Standalone NIR Probes

Standalone NIR probes, while promising for various imaging applications, face several challenges when used in vivo. One of the primary issues is their rapid degradation and poor bioavailability. NIR probes, when introduced into the body, are often quickly cleared by the liver and kidneys, limiting their ability to remain in circulation long enough to achieve effective imaging. This short circulation time reduces the probe's chances of accumulating at the target site, leading to lower imaging efficacy.

Additionally, many NIR probes suffer from a lack of target specificity. Without the use of targeting agents like antibodies or peptides, these probes may diffuse indiscriminately throughout the body, increasing background signals and reducing the overall signal-to noise ratio in the images. Even when targeting agents are used, they can be susceptible to enzymatic degradation or immune system responses, further compromising the probe's effectiveness in vivo.¹⁴

ICG (Indocyanine Green) 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.

Furthermore, ICG is prone to photobleaching and has limited tissue penetration, making it less effective for deep-tissue imaging. Although it is currently the gold standard for NIR-I imaging in clinical settings, its shortcomings highlight the need for more advanced probes with enhanced pharmacokinetics, deeper penetration, and better target specificity.

Our Solution

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.

This genetically engineered HSA delivery system addresses the challenges of short circulation time and low targeting efficiency typically seen with NIR probes. It opens new possibilities for NIR-II probes in deep tissue imaging, offering great potential for longer imaging applications and advancing the future of clinical imaging.

Reference

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