Extracellular Vesicles-based Nanoprobe For Multi-modal Imaging Of Colon Cancer

Background: Extracellular vesicles from tumor cells(TEV)have emerged as promising nanocarriers for drug delivery.Noninvasive multimodality imaging for tracing the in vivo trafficking of TEV may accelerate their clinical translation.In this study,we developed a TEV-based nanoprobe via hydrophobic insertion mechanism and evaluated its performance in dual single-photon emission computed tomography(SPECT)and near-infrared fluorescence(NIRF)imaging of colon cancer.Furthermore,tumor-targeting ability of TEV was evaluated with extracellular vesicles from adipose stem cells used as control.Methods: TEV were isolated from HCT116 tumor cell supernatant by the classical differential hypercentrifugation method and characterized using transmission electron microscopy(TEM)and dynamic light scattering(DLS).Tumor cell binding ability was evaluated by flow cytometry analysis and confocal imaging.TEV were modified with Cyanine7(Cy7)-terminated 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000](DSPE-PEG2000)and hydrazinonicotinic acid(HYNIC)-tagged DSPE-PEG2000,and followed by 99 mTc labeling to obtain 99mTc-TEV-Cy7.In order to investigate the targeting ability of the TEV-based nanoprobe and its application in visualizing colon cancer,a series of in vitro and in vivo studies including cell uptake SPECT/NIRF imaging and ex vivo biodistribution were performed.To confirm the specificity of 99mTcTEV-Cy7,the modified AEV(99mTc-AEV-Cy7)were also developed and investigated as a nonspecific control.Results: TEV were successfully isolated from HCT116 supernatants,and their membrane vesicle structure was confirmed by TEM.The average hydrodynamic diameter and zeta potential of TEV were 110.87 ± 4.61 nm and –9.20 ± 0.41 m V,respectively.Confocal microscopy and flow cytometry findings confirmed the high tumor binding ability of TEV.The uptake rate of 99mTc-TEV-Cy7 by HCT116 cells increased over time,reaching 14.07 ± 1.31% at 6 h of co-incubation.NIRF and SPECT imaging indicated that the most appropriate imaging time was 18 h after the injection of 99mTc-TEV-Cy7 when the tumor uptake(1.46% ± 0.06% ID/g)and tumor-to-muscle ratio(8.22 ± 0.65)peaked.Compared with radiolabeled extracellular vesicles from adipose stem cells(99mTc-AEV-Cy7),99mTc-TEV-Cy7 exhibited a significantly higher tumor accumulation in tumor-bearing mice.Conclusion: Hydrophobic insertion-based engineering of TEV may represent a promising approach to develop and label extracellular vesicles for use as nanoprobes in dual SPECT/NIRF imaging.Our findings confirmed that TEV has a higher tumor-targeting ability than AEV and highlight the potential usefulness of extracellular vesicles in biomedical applications.Background: Colon cancer contributes to high mortality rates as the result of incomplete resection in tumor surgery.Multimodal imaging can provide preoperative evaluation and intraoperative image-guiding.As biocompatible nanocarriers,extracellular vesicles hold great promise for multimodal imaging.In this study,we aim to synthesized an extracellular vesicles-based nanoprobe to visualize colon cancer with positron-emission tomography/computed tomography(PET/CT)and near-infrared fluorescence(NIRF)imaging,and investigated its utility in image-guided surgery of colon cancer in animal models.Methods: Extracellular vesicles was isolated from adipose stem cells(AEV)supernatant by the classical differential hypercentrifugation method and characterized.PET imaging with short half-life radionuclide was successfully achieved by click chemistry-based pre-targeting strategy to tracking the distribution of AEV in vivo.1,2-distearoyl-sn-glycero-3-phosphoethanola-mine-N-[Cyanine7(polyethyleneglycol)-2000](DSPE-PEG2000-Cy7)was modified onto the surface of AEV for NIRF imaging.PET/CT and NIRF imaging of tumor bearing nude mice were performed at different time.The image-guided surgery was performed under real-time NIRF imaging.Results: AEV were successfully isolated from ASCs supernatants.TEM confirmed the membrane vesicles.Western blot identified the expression of two AEV makers,CD9 and CD63.The average hydrodynamic diameters of AEV detected by DLS was 136.5 ± 2.5 nm,and the zeta potential was-7.93 ± 0.24.The PET images and biodistribution data showed that the optimized lag time for the AEV was 20 h before the injection of radiotracer,and the best imaging time was 2 h after injection of radiotracer.NIRF images were gained with clear tumor visibility at 10,20 and 30 h after the injection of modified AEV.Both PET/CT and NIRF imaging clearly visualized the orthotopic colon transplantation tumor,providing cross-validation by multimodality.Under the real-time NIRF imaging,the preoperative tumor boundary and the postoperative residual tumor tissue could be clearly observed.Conclusion: A novel nanoprobe based AEV was successfully isolated and modified with N3 and Cy7.This extracellular vesicle-based nanoprobe can be used for multimodality imaging of colon cancer and NIRF image-guided surgery.In addition,the developed extracellular vesicles-based nanoprobe has great potential to be multimodal nanocarriers.