J10 - Ultrabright NIR-I Afterglow Nanoprobes Amplified via Protein Chaperoning Enable Image-Guided Resection of Deep-Seated Tumors

Introduction: : Fluorescence imaging using near-infrared (NIR) light in the first window (650-900nm) has become a vital tool in oncological procedures, providing real-time feedback for tumor identification during surgery. However, the use of clinically approved fluorophores like 5-aminolevulinic acid, methylene blue, and indocyanine green (ICG) is limited by tissue autofluorescence, which results in low signal-to-background ratios (SBRs), poor sensitivity, and shallow imaging depths (~2-3mm). Additionally, prolonged light exposure can cause photobleaching of the dye. Afterglow imaging, utilizing persistent luminescence after light removal, has emerged as a promising solution to overcome the limitations of NIR fluorescence imaging. However, its adoption is limited by the availability of efficient NIR-responsive afterglow materials, which often suffer from low brightness and lack of cyclic modulation in afterglow emission. To address these challenges, we developed an ultrabright, NIR-responsive afterglow nanoprobe composed of functional proteins as encapsulation matrices coated with a weakly NIR afterglow polymer (SP), named Protein@SP. This nanoprobe shows a five-fold increase in NIR afterglow luminescence compared to polymer-lipid coated SP (DSPE-PEG@SP) under the same conditions. It also provides extended luminescence for up to 20 minutes and is capable of imaging at depths of 10-20 mm. We hypothesize that this enhancement of afterglow luminescence attributes is due to the electron-rich environment provided by the protein matrices, which stabilize the SP and promote the generation of singlet oxygen (¹O₂) radicals, boosting the SP's afterglow properties. Our results highlight the potential of NIR-I afterglow imaging for deep tumor resection during surgery.

Materials and

Methods: : All materials were purchased from Sigma-Aldrich unless otherwise specified. Porcine tissues were obtained from local grocery stores, and dry-preserved porcine lungs were acquired from Nasco Education. The Singlet Oxygen Sensor Green (SOSG) test kit was supplied by Lumiprobe USA. The synthesis of Protein@SP and DSPE-PEG@SP was performed using a modified nanoprecipitation technique (Figure 2a), followed by ultracentrifugation to purify the nanoprobes from any unreacted dyes, proteins, or surfactants. The nanoprobes were characterized using dynamic light scattering and zeta potential measurements, as well as UV-Vis-NIR absorption spectra, NIR fluorescence spectra, and transmission electron microscopy images. Afterglow imaging were conducted with an IVIS Imager from PerkinElmer under the bioluminescence mode and ICG channel. Molecular docking studies were performed using Chimera and AutoDock Vina. To evaluate ¹O₂ radicals generation upon light excitation for Protein@SP nanoprobes and DSPE-PEG@SP nanoprobes, SOSG and electron spin resonance spectroscopy was used. Cytotoxicity assays and in vitro cellular uptake experiments of the nanoparticles were carried out using 4T1 cancer cells. Tumor cell-mimicking phantoms were prepared following a protocol previously established by our team (Srivastava, I. et al., ACS Nano, 2023). These phantoms were used to evaluate ex vivo porcine tissue permeation of Protein@SP nanoprobes and the FDA-approved dye indocyanine green (ICG), and to perform both afterglow-guided and fluorescence-guided surgical procedures on ex vivo porcine lungs.

Results, Conclusions, and Discussions:: Using bovine serum albumin (BSA) as a model protein matrix, we successfully developed BSA@SP nanoprobes and corresponding control DSPE-PEG@SP nanoprobes. Comprehensive physicochemical characterization and optical assessments were performed. The hydrodynamic diameter of the DSPE-PEG@SP nanoprobes was measured at 108 nm (Figure 2b), while BSA@SP nanoprobes exhibited a diameter of approximately 125 nm (Figure 2c). Additionally, when imaged using the bioluminescence mode of the IVIS imager, the BSA@SP nanoprobes displayed significantly stronger afterglow luminescence in the ICG channel compared to the DSPE-PEG@SP nanoprobes (Figure 2d). To further assess their stability, long-term colloidal stability tests were conducted under physiologically relevant conditions such as tumor pH or serum. (Data not shown). We also evaluated their biocompatibility and cellular uptake in metastatic tumor cell lines, including 4T1 and A549 cells (Data not shown). Our preliminary molecular docking studies provided insights into the interaction between BSA and SP, revealing that the hydrophobic amino acid residues on BSA are responsible for binding with the hydrophobic SP molecules (Figure 2e, f). Next, we demonstrated the superior tissue penetration and afterglow signal profiles of BSA@SP nanoprobes compared to the fluorescence signals from the FDA-approved ICG, using ex vivo tumor-mimicking phantoms and various porcine tissue types (skin, muscle, and fat) (Figure 3a, b). Building on this, we explored BSA@SP's potential in image-guided surgeries by implanting tumor-mimicking phantoms within porcine lungs. We then conducted direct comparisons between the clinical standard of fluorescence-guided surgery and afterglow-guided surgery, highlighting the latter's superior performance (Figure 3c, d). Overall, our study introduces a promising strategy for enhancing current afterglow materials through protein complexation, resulting in ultrahigh signal-to-background ratios and cyclic afterglow signals.

Acknowledgements and/or References (Optional):: The authors thank Texas Tech University for financial support. We thank American Heart Association for partial funding of this work.