Abstract
1064
Objectives: Malignant gliomas are the most common primary malignant brain tumor. Restriction of drugs by the blood-brain barrier (BBB) from entering the brain makes gliomas insensitive to chemotherapy. The T7 peptide (sequence: HAIYPRH) is a heptapeptide that can be used to target gliomas. Exosomes have a strong cargo-loading capacity and have ability to cross the BBB. Goat milk-derived exosomes (GEXs), due to their bulk, safe and cost-effective production, are widely used as a carrier for chemotherapeutic agents. However, there is no good methods to monitor the distribution of exosomes in vivo. In this study, we constructed a molecular probe using GEXs as carriers which modified by T7 peptide and labeled with 68Ga to investigate the application of PET/CT imaging based on GEXs in gliomas which has potential to provide a non-invasive monitoring method for GEXs-based drug delivery system.
Methods: GEXs were extracted from goat’s milk by differential ultracentrifugation and characterized by dynamic light scattering (DLS), transmission electron microscope (TEM), and Western blot (WB). The toxicity was assessed by CCK8 assay. DSPE-PEG2000-T7 and DSPE-PEG2000-FITC were inserted into the GEXs under co-incubation at 37 ℃, and the ability of the GEXs to bind C6 glioma cells was verified by confocal laser scanning microscope (CLSM). A GEXs-based nanoprobe, T7-PEG2000-GEXs-PEG2000-N3, was obtained by co-incubation of DSPE-PEG2000-T7, DSPE-PEG2000-N3 and GEXs at 37 ℃ for 30 min. Cultured C6 glioma cells implanted in the striatum of Sprague-Dawley rat brains were used as the orthotopic glioma rat models. The injection location is 1 mm anterior and 3 mm lateral to the bregma on the right site of skull at a depth of 4.5 mm from the brain surface. Ten days after implantation, 18F-FDG was injected into the rats to verify the establishment of models by PET/CT. The day after injection of 18F-FDG, the nanoprobe was injected into the C6 orthotopic glioma rat models via tail veins previously. 68Ga-L-NETA-DBCO was injected into C6 orthotopic glioma rat models (n=3) based on “click chemistry” and pretargeting technology at a suitable time point after the injection of GEXs-based nanoprobe. According to the previous research of our group, the best pretargeting time was 30 h. A group of C6 orthotopic glioma rat models (n=3) without intravenous injection of the GEXs-based nanoprobe was used as a control. All C6 rats were imaged by PET/CT at 1, 2 and 4 h after injection of 68Ga-L-NETA-DBCO via tail vein.
Results: The hydrodynamic diameters and the zeta potential of GEXs were 73.87 ± 0.86 nm and -11.00 ± 0.71 mV respectively measured by DLS. TEM showed membrane vesicles. WB confirmed the expression of two exosomes markers: CD9 and CD63. After incubating different concentrations of exosomes with C6 glioma cells for 24 h, the cell viability was nearly 100% detected by CCK8 assay, which suggested that the nanoparticles had no cytotoxicity. CLSM showed that GEXs had a strong binding ability to C6 glioma cells. The PET/CT images illustrated that tumors had significant higher radioactivity accumulation in group with pre-injection of GEXs-based nanoprobe at each time point comparing to the group without pre-injection (Figure).
Conclusions: The research successfully used the radionuclide 68Ga to label the GEXs by the pretargeting technique. By means of the ability of GEXs to cross the BBB and high targeting property of the T7 peptide with the gliomas, PET/CT imaging of orthotopic gliomas was achieved. This study also confirmed that GEXs, as safe nanoparticles, are potential to provide PET/CT monitoring for exosomes-based drug delivery system in gliomas therapy. Funding: This work was supported by the National Natural Science Foundation of China (No. 81873904).