Engineering Success

Issues with the Probe Molecule:

When used independently, NIR-II probes such as CO-1080 face several challenges that significantly limit their effectiveness for in vivo imaging. One of the primary issues is their rapid degradation within biological environments, which reduces the probe's functional lifespan and makes long-term imaging difficult. Additionally, the probes are prone to being quickly cleared from the body by the liver and kidneys, resulting in short circulation times. Another major challenge is the formation of a protein corona—a layer of proteins that adsorbs onto the surface of the probe upon its introduction into the bloodstream. This protein corona can alter the surface properties of the probe, impacting its ability to target specific tissues and diminishing its imaging efficiency. These limitations make it difficult for standalone NIR-II probes to perform effectively in complex biological systems, particularly for deep tissue imaging and long-duration studies.

Preliminary Solution:

To overcome these challenges, we propose encapsulating the NIR-II probe within human serum albumin (HSA). HSA is a highly biocompatible protein naturally present in the human body, and it serves as an ideal carrier to improve the in vivo behavior of the probe. By encapsulating the probe within HSA, we aim to enhance the probe’s stability, protecting it from rapid degradation.

Furthermore, the HSA coating can help prevent the probe from being rapidly cleared from circulation, thereby extending its half-life and allowing it to remain in the bloodstream for longer periods. The HSA encapsulation also acts as a protective barrier against the formation of a protein corona, preserving the probe's surface properties and maintaining its targeting efficiency. Ultimately, this step is expected to significantly improve the biocompatibility, circulation time, and overall imaging performance of the probe, enabling more reliable and effective in vivo imaging applications, especially in complex biological environments.

Fluorescence spectra of CO-1080 in DMSO solution, CO-1080 in PBS solution, and HSA@CO-1080 FPs under 1064 nm excitation

Photostability of CO-1080 and HSA@CO-1080 FPs under 1064 nm excitation (mean ± SD, n = 3 independent samples per group).

Objective:

Building on the encapsulation of the CO-1080 NIR-II probe with HSA, we sought to further investigate the molecular interactions between CO-1080 and HSA to enhance their binding efficiency. The ultimate goal was to improve the performance of the probe in biological environments, particularly in NIR-II imaging applications, by optimizing the binding affinity between CO-1080 and HSA through site-directed mutagenesis. This iterative design process aims to create a more robust system, where enhanced protein-probe interactions improve the probes' stability and effectiveness for bioimaging.

CO-1080 and HSA Binding Analysis:

Previous studies have suggested that CO-1080 tends to covalently bind to domain III (DIII) of HSA. Proteomic analysis identified Cys477 and Cys487 as potential covalent binding sites for CO-1080. Among these, Cys477 showed a higher binding score, making it the most likely covalent binding site for CO-1080. The presence of reversible disulfide bonds between Cys476 and Cys487, as well as between Cys461 and Cys477, provided an opportunity to explore mutations that could break these bonds, release free –SH groups, and potentially accelerate the binding of CO-1080 to HSA.

gel electrophoresis analysis (n = 4 independent experiment) of the binding ability between CO-1080 chromophores and HSA, DI, DII, DIII.

To determine whether such mutations would affect the non-covalent affinity between CO-1080 and HSA, we performed molecular docking simulations using the Glide software. These simulations allowed us to predict the non-covalent interactions between CO1080 and recombinant HSA. The mutation of Cys476 to Gly (C476G) was introduced, and docking scores and binding energies were calculated using the MM/GBSA method. Results demonstrated that all single cysteine mutations enhanced the non-covalent binding affinity of recombinant HSA to CO-1080, with the Cys476G mutation showing the most significant improvement.

Mutation Strategy:

Based on the results of the molecular docking simulations, we focused on site-directed mutagenesis of key cysteine residues in HSA, specifically Cys476 and Cys477, to enhance CO-1080 binding efficiency. The hypothesis was that breaking the disulfide bonds between Cys476 and Cys477, or Cys461 and Cys487, would free –SH groups that could enhance covalent binding with CO1080. The primary mutation strategy involved replacing Cys476 with Gly (C476G), which was predicted to expand the protein cavity, expose more binding sites, and increase the overall affinity between the probe and the protein.

Experimental Validation:

Following the docking simulations, we produced recombinant HSA (rHSA) with the Cys476-to-Gly mutation using site-directed mutagenesis (SDM). Experimental validation confirmed that the C476G mutation led to an expansion of the protein cavity, which facilitated easier access of CO-1080 to the binding site. The mutation also exposed free –SH groups that accelerated the covalent binding reaction between CO-1080 and rHSA, particularly under mild conditions at room temperature. These findings confirmed the success of the C476G mutation in improving both non-covalent and covalent binding interactions.

Gel electrophoresis results of CO-1080@albumin incubated at room temperature for 2 h

Optimization and Further Exploration:

Further validation was performed using electrophoresis and mass spectrometry to quantify the binding efficiency of the mutated rHSA. The results demonstrated significant improvements in both non-covalent and covalent binding efficiency for the C476G mutation compared to wild-type HSA. Moving forward, we plan to use GaMD (Gaussian Accelerated Molecular Dynamics) or meta dynamics simulations to explore the binding pathway of the CO-1080 probe molecule into the HSA binding site.

These simulation techniques will help identify key residues involved in the binding pathway and provide detailed insights into their roles during the binding process. We will also investigate the spatial and electronic effects of nearby residues along the binding pathway, assessing their potential impact on binding efficiency. Through this analysis, we aim to further optimize the interaction between the probe and protein, ultimately improving the binding efficiency of the CO-1080@rHSA complex and enhancing its potential for NIR-II bioimaging applications