Abstract
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Objectives: Colon cancer is a good candidate for image-guided PTT (photothermal therapy) with the application of colonoscopy. Exs (exosomes) emerged as a promising nanocarrier for image-guided PTT due to their biological origin, nontoxic and highly efficient targeting. In this study, we engineered an Exs-based theranostic nanoprobe in order to investigate the feasibility for image-guided PTT of colon cancer based on PET/CT and NIRF (near-infrared fluorescence) imaging.
Methods: Exs were obtained by differential ultracentrifugation from goat’s milk and examined by TEM (transmission electron microscopy), DLS (dynamic light scattering), and WB (western blot analysis). The ability of Exs to bind tumor cells was verified by flow cytometry and confocal microscopy. Exs@ICG (indocyanine green) was obtained by co-incubation ICG and exosomes under ultrasound and examined by UV (ultraviolet specrophotometer). Then DSPE-PEG2000-N3 was inserted into Exs@ICG and N3-Exs@ICG was gained. The toxicity and antitumor effect of N3-Exs@ICG in vitro were assessed by CCK8 assay. N3-Exs@ICG was injected into the tumor-bearing nude mice through tail veins for NIRF imaging at different times (2h, 6h, 12h, 24h, 48h). 68Ga-NETA-DBCO was injected into the tumor-bearing mice for PET imaging based on “click chemistry” at 24h after the injection of N3-Exs@ICG. The in vivo anti-tumor activity of N3-Exs@ICG was investigated in subcutaneous MC38 tumor models. The models were randomly allocated into eight groups and each group include 7 models. When all subcutaneous tumors reached the acceptable sizes (about 7days, approximately 100-200 mm3), the mice were treated. The treatment protocols for each group were as follows: NS(normal saline), free ICG ( 20mg ICG/kg body weight), Exs (50mg Exs/kg body weight), N3-Exs-ICG (20mg ICG/kg body weight), NS + laser (2 w/cm2, 2 min), free ICG + laser (2 w/cm2, 2 min), Exs (50mg Exs/kg body weight) + laser, N3-Exs-ICG + laser. At the end of experiment, all mice were sacrificed, and the tissues, including heart, liver, spleen, lung and kidney, were collected for H&E staining.
Results: Membrane vesicles were observed under TEM. DLS detected that the hydrodynamic diameters of the Exs were 73.87 ± 0.73 nm and the zeta potential was -7.36 ± 0.53. WB confirmed the expression of two Exs markers: CD9 and CD63. Confocal microscopy showed that Exs had a strong binding ability to tumor cells. Exs@ICG and ICG had different UV spectrums. After incubating different concentrations of N3-Exs@ICG with MC38 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 24h after administration of N3-Exs@ICG. The orthotopic colon cancer could be visualized preoperatively with PET/CT when the pretargeting time is 24 h, and the imaging time is 2 h after the injection of 68Ga-NETA-DBCO. Among eight groups, the tumors in N3-Exs-ICG + laser group had the minimum size and weight. The mean tumor volume in this group on day 14 was 110.00 ± 24.25 mm3 (n = 7), which was significantly reduced when compared with the NS group (1879.90 ± 809.66 mm3, n = 7) and other groups. The mean tumor weight in this group on day 14 was 72.44 ± 19.81 mg (n = 7), which was significantly reduced when compared with the NS group (2954.75± 460.58 mg, n = 7) and other groups. In addition, no significant mice weight changes and organ damages were observed in each group. Conclusion: The research successfully engineered an Exs-based theranostic nanoprobe. These promising results showed that this biological nanoprobe can be used for multimodality PET/CT and NIRF imaging of colon cancer and holds a promising potential for image-guided PTT. Funding: This work was supported by the National Natural Science Foundation of China (No. 81873904).