Multimodality PET/CT and NIRF imaging for Image-Guided Surgery of Colon Cancer with Exosomes Based Nanoprobe

Objectives: Exosomes, known as nano-sized extracellular vesicles, emerged as a promising carrier for multimodality imaging due to their biological origin, non-toxic and highly efficient targeting. In this study, we engineered an exosome-based multimodal nanoprobe in order to investigate the feasibility for image-guided surgery based on PET/CT and near-infrared fluorescence (NIRF) imaging.

Methods: Exosomes (Exs) were isolated by differential ultracentrifugation from adipose stem cells supernatant and examined by transmission electron microscopy (TEM), dynamic light scattering (DLS), and western blot analysis (WB). DSPE-PEG₂₀₀₀-RGD and DSPE-PEG₂₀₀₀-FITC were inserted into the exosomes under co-incubation at 37 ℃, and the ability of exosomes to bind tumor cells was verified by flow cytometry and confocal microscopy. The toxicity was assessed by CCK8 assay. An exosome-based nanoprobe, Cy7(RGD)-PEG₂₀₀₀-Exs-PEG₂₀₀₀-N_3, was obtained by co-incubation of DSPE-PEG₂₀₀₀-Cy7, DSPE-PEG₂₀₀₀-RGD, DSPE-PEG₂₀₀₀-N_3, and exosomes at 37 ℃. The nanoprobe was injected into the tumor-bearing nude mice through tail veins for NIRF imaging at different times (1, 5, 10, 20, 30, 50, and 70 h). According to the NIRF images, ⁶⁸Ga-NOTA-DBCO was injected into the tumor-bearing nude mice for PET imaging and biodistribution based on “click chemistry” at suitable time points after the injection of Cy7(RGD)-PEG₂₀₀₀-Exs-PEG₂₀₀₀-N₃. Then the best pretargeting and imaging time have been confirmed. Based on the previous results, we introduced the nanoprobe into the orthotopic colon cancer model for PET/CT scans. After preoperative evaluation of tumor lesions by PET/CT, image-guided surgery of the tumor lesions was performed. First, the exact tumor location was identified by NIRF imaging. Subsequently, these tumor lesions were resected. Finally, NIRF and PET/CT imaging was repeated to ensure radical surgical resection.

Results: Membrane vesicles were observed under TEM. DLS detected that the hydrodynamic diameters of the exosomes were 89.43 ± 1.34 nm and the zeta potential was -9.86 ± 1.06. WB confirmed the expression of two exosomes markers: CD9 and CD63. Flow cytometry and confocal microscopy showed that exosomes had a strong binding ability to tumor cells and had a certain degree of saturation. After incubating different concentrations of exosomes with colon cancer cells for 24 h, the cell viability was nearly 100% detected by CCK8 assay, which suggested the nanoparticles had no cytotoxicity. The NIRF images showed that the tumor had clear images at 10, 30 and 50 h after administration of nanoparticle. PET/CT images and the biodistribution results showed that the best pretargeting time is 30 h, and the best imaging time is 2 h after the injection of ⁶⁸Ga-NOTA-DBCO. The orthotopic colon cancer could be visualized preoperatively with PET/CT and could subsequently be resected by image-guided surgery using intraoperative NIRF imaging. In addition, NIRF and PET/CT imaging confirmed no residual tumor tissue was left in situ. Conclusion: The research successfully engineered multimodality PET/CT and NIRF imaging for image-guided surgery of colon cancer with exosomes based nanoprobe. This study also confirmed that exosomes, as natural nanoparticles, are potential high-quality carriers for multimodal imaging and have broad application prospects. Funding: This work was supported by the National Natural Science Foundation of China (No. 81873904).