Ultrabright NIR-II Fluorescent Nanoprobes Chaperoned by Albumin Enable Pan-Ovarian Imaging in Multi-Scale Cancer Models

Introduction: : Ovarian cancer is among the most aggressive gynecological malignancies, with a high mortality rate largely due to late-stage diagnosis. By the time most patients are diagnosed, the cancer has often metastasized extensively throughout the abdominal cavity. Currently, the primary treatment is cytoreductive surgery guided by visual inspection and palpation, aiming to remove as much tumor mass as possible. However, this method is invasive, carries significant risk, and can severely affect patients' quality of life. Fluorescence image-guided surgery (FIGS) has emerged as a promising technique, offering real-time, high-contrast visualization of tumor tissue during surgery. Unlike conventional imaging modalities such as MRI or CT, FIGS uses non-ionizing near-infrared (NIR) light (700–900 nm), which minimizes tissue autofluorescence, enhances tissue penetration, and reduces scattering. Despite its advantages, current FIGS imaging agents suffer from limitations, including low fluorescence quantum yield, non-specific protein adsorption, rapid blood clearance, and insufficient tumor specificity, limiting their clinical utility in complete ovarian cancer resection. To overcome these challenges, we developed a novel endogenous albumin-based semiconducting polymer nanoparticle probe (BSA-FA@SPs) for comprehensive ovarian cancer imaging. This nanoprobe is designed to target tumors across all stages—from primary to metastatic. By incorporating a bovine serum albumin (BSA) coating, we aim to leverage the natural affinity for SPARC (SPARC stands for Secreted Protein Acidic and Rich in Cysteine) receptors, which are overexpressed in metastatic ovarian cancer. Additionally, folic acid functionalization enhances targeting of early-stage ovarian tumors. Consequently, these nanoprobes allow pan-ovarian imaging across multi-scale models including on-chip and ex vivo models.

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 ovaries were acquired from Nasco Education. The synthesis of BSA@SP and DSPE-PEG@SP was performed using a modified nanoprecipitation technique, followed by ultracentrifugation to purify the nanoprobes from any unreacted dyes, proteins, or surfactants. To modify BSA with folic acid, it was achieved via carbodiimide (EDC-NHS) crosslinking and then subsequently used for the nanoprecipitation to make BSA-FA@SP nanoprobes. The nanoprobes were subjected to ultracentrifugation to purify the nanoprobes from any unreacted dyes, proteins, folic acids, or surfactants. The nanoprobes were characterized using dynamic light scattering and zeta potential measurements, UV-Vis-NIR absorption spectra, NIR fluorescence spectra, and transmission electron microscopy images. Fluorescence imaging was conducted with an IVIS Imager from PerkinElmer. Molecular docking studies were performed using Chimera and Auto Dock Vina. Cytotoxicity assays, including MTT and live/dead staining, as well as in vitro cellular uptake studies of BSA-FA@SP, BSA@SP, and DSPE-PEG@SP nanoprobes, were conducted in various in vitro models—2D cancer cell cultures, 3D ovarian cancer spheroids, and on-chip systems—using OVCAR3, SKOV3, and OVCAR8 cell lines obtained from ATCC. 3D bio-printed ovarian cancer phantoms were used to evaluate ex vivo porcine tissue permeation of BSA-FA@SP nanoprobes and the FDA-approved dye, indocyanine green (ICG), and to perform both afterglow-guided and fluorescence-guided visualization on 3D bio-printed ovarian cancer phantoms in ex vivo porcine ovaries.

Results, Conclusions, and Discussions:: Using molecular docking studies, we first investigated how SP probes interact with BSA protein. The results showed that the hydrophobic SP dye preferentially binds to the hydrophobic domains of BSA (Fig. 2a), supported by hydrophobic amino acid residues at the binding site (Data not shown). In contrast, other functional proteins exhibited significantly lower binding affinities towards the SP probes (Fig. 2a, b). We then optimized the BSA concentration (ranging from 10 to 200 μM) and successfully formed BSA@SP nanoprobes via nanoprecipitation (Fig. 2c-e). Folic acid was conjugated to BSA at the optimized concentration using carbodiimide chemistry, and the BSA-FA conjugates were purified by dialysis. Successful conjugation was confirmed by absorption spectroscopy and mass spectrometry. These BSA-FA conjugates were used to prepare BSA-FA@SP nanoprobes, while DSPE-PEG@SP nanoprobes, lacking affinity for SPARC or folate receptors, served as controls. Physicochemical characterization, including dynamic light scattering and zeta potential measurements, confirmed that the nanoprobes maintained long-term colloidal stability under physiologically relevant conditions, such as tumor-mimicking pH and serum-containing solutions. Optical assessments using the IR VIVO imager showed that BSA significantly enhanced the NIR-II luminescence of SP probes under 808 nm excitation, an effect not seen with the DSPE-PEG coating (Fig. 2f). This amplification is attributed to hydrophobic interactions between SP and BSA’s hydrophobic domains, which stabilize the excited state of the dye, amplifying the NIR-II emission. We then performed in vitro assessments of cell viability and cellular uptake in SKOV-3 ovarian cancer cells using 2D cultures, 3D spheroids, and microfluidic on-chip models, with the on-chip data presented as a representative example (Fig. 3). IVIS imaging revealed that BSA-FA@SP nanoprobes were more efficiently internalized into SKOV-3 tumor clusters compared to DSPE-PEG@SP (Fig. 3e-g), likely due to targeting both folate and SPARC receptors. Finally, we explored the potential of BSA-FA@SP nanoprobes in image-guided surgeries using 3D tumor-mimicking phantoms in porcine ovaries, demonstrating superior performance over fluorescence-guided surgery (Data not shown). Overall, our study introduces a promising strategy for protein-based nanoprobes capable of providing pan-ovarian cancer imaging and aiding in pre-operative, intraoperative, and post-surgery monitoring.

Acknowledgements and/or References (Optional):: We thank Texas Tech University for financial support. Imaging studies on the IR VIVO system was funded by the Core Facility Support Award (grant number RP200572) from the Cancer Prevention and Research Institute of Texas (CPRIT) to the Imaging Core, Texas Tech University Health Sciences Center at Amarillo.