Multi-modality Imaging Of Tumor And Plaque Angiogenesis Using GEBP11 Targeted Magnetic Nanoparticles

Background:Cancer and atherosclerosis, that are associated with angiogenesis, remain the major causes of morbidity and mortality worldwide. Considerable studies have demonstrated that angiogenesis plays a critical role in the development of tumor and atherosclerotic plaque. Neovascularization is necessary for tumor invasion and metastasis. Clinical reports have shown that anti-angiogenesis treatment can suppress tumor growth and prolong patients’ survival time. Plaque angiogenesis has been suggested to promote plaque progress and lesion instability, while angiogenesis inhibitors help to stabilize paque. Therefore, it is very important to improve the diagnosis and treatment of angiogenesis-associated diseases.Previously,we identified a 9-amino acid cyclic peptide(CTKNSYLMC), named GEBP11, which holds high affinity and specificity for neovascularization in gastric cancer. Furthermore, GEBP11 peptide could be served as a potential candidate for tumor imaging and targeted radionuclide therapy by radiolabeled with 131 I. Like many other small molecular weight peptides, GEBP11 has the advantages of straightforward synthesis, good tissue penetration and less or non-immunogenic feature, but further modification is still needed to improve its pharmacokinetic for better therapeutic efficacy.Iron oxide magnetic nanoparticles(MNPs) have been widely used as an effective carrier for peptides to prolong their circulation half-life. MNPs can be employed for both magnetic resonance imaging(MRI) and radio/optical imaging to monitor the localization of peptides in target tissues after their conjugation with radionuclides or fluorophores. Moreover, drug-loaded MNPs can be also used for therapeutic application.In this study, we tried to manufacture a vascular targeted magnetic nanoparticle system for angiogenesis imaging.Aims:1. To construct Cy5.5-GEBP11-DMSA-MNPs and characterize their physicochemical characteristics, binding affinity and cytotoxicity in vitro.2. To assess the targeting efficacy of Cy5.5-GEBP11-DMSA-MNPs as a fluorescence/MR dual-modality imaging probe in vivo.3. To prepare 68Ga-NOTA-GEBP11-DMSA-MNPs, characterize their physicochemical characteristics, binding affinity and cytotoxicity in vitro and assess the targeting efficacy of 68Ga-NOTA-GEBP11-DMSA-MNPs and GEBP11-DMSA-MNPs as a PET or MR imaging probe in vivo.Methods:1. Oleylamine coated Fe3O4 nanoparticles were synthesized by thermal decomposition of ferric acetylacetonate in organic solvent and then DMSA-MNPs were synthesized via a ligand exchange reaction. The physicochemical characteristics of DMSA-MNPs were characterized by TME, DLS and VSM.2. GEBP11 peptide and Cy5.5 were covalently conjugated to DMSA-MNPs in the presence of EDC and NHS. The physicochemical characteristics of Cy5.5-GEBP11-DMSA-MNPs were characterized by TME, DLS and VSM.3. GEBP11 peptide and NOTA were covalently conjugated to DMSA-MNPs in the presence of EDC and NHS and then the size of NOTA-GEBP11-DMSA-MNPs were characterized by TME and DLS.4. The fluorescence property and the relaxivity of Cy5.5-GEBP11-DMSA-MNPs were tested by fluorescence imaging and MR T2-weighted imaging at different iron concentrations..5. The Co-HUVECs were incubated with Cy5.5-GEBP11-DMSA-MNPs for 12 h. The cellular uptake of nanoparticles was detected by Prussian blue staining, confocal fluorescence imaging.6. The Co-HUVECs were incubated with NOTA-GEBP11-DMSA-MNPs. The cellular uptake of nanoparticles was detected by Prussian blue staining. The apoptosis and cell cycle of HEVECs were measured by flow cytometry.7. In vivo fluorescence imaging and MRI were performed at different time points(0, 6,12, 24, 36 and 48 h) before and after administration of nanoparticles. Prussian blue staining and CD31 immunohistochemical staining were performed on tumor sections.8. New Zealand White rabbits were fed high fat diet for 12 weeks after abdominal aorta balloon injury. Ultrosound and IVUS were applied to confirm the plaque formation. Histological staining was used to identify the different plaque components.9. PET scan was performed after the injection of 68Ga-NOTA-GEBP11-DMSA. In vivo 3.0 T MRI was performed at 4h after the ear vein injection of 5 mg Fe/Kg body weight GEBP11-DMSA-MNPs. The lacation of nanoparticles in aorta section from atherosclerotic rabbits was analysed by Prussian blue staining.Results:1. DMSA-MNPs were well dispersed with an average iron core size of 10 nm and an hydrodynamic size of 63.2 ± 4.1 nm respectively. Their saturation magnetization value was 62.7 emu/g at room temperature.2. Cy5.5-GEBP11-DMSA-MNPs dispersed in distilled water had an hydrodynamic diameter of 82.8 ± 6.5 nm and their saturation magnetization value was 50.3 emu/g at room temperature.3. The size of NOTA-GEBP11-DMSA-MNPs had an average particle size of 12 ± 0.7 nm and an hydrodynamic size of 143.2 ± 7.1 nm. They could retain stable for at least a month in water.4. The MRI results suggested that Cy5.5-GEBP11-DMSA-MNPs could significantly reduce the T2 relaxation time and there was a strong linear correlation between the nanoparticles concentration and the signal intensity change. The fluorescent intensity was also linearly proportional to the particle concentration. Furthermore, the similar trend remained in spite of the cellular uptake of Cy5.5-GEBP11-DMSA-MNPs.5. Both Cy5.5-GEBP11-DMSA-MNPs and GEBP11-DMSA-MNPs showed a significant increase in uptake compared with DMSA-MNPs or URP conjugated MNPs. The Cy5.5-GEBP11-DMSA-MNPs was generally localized in the cytoplasm of Co-HUVECs.6. The cellular uptake of NOTA-GEBP11-DMSA-MNPs was significantly higher campared with DMSA-MNPs. The apoptosis and cell cycle of HUVECs were unaffected by the internalization of NOTA-GEBP11-DMSA-MNPs.7. Fluorescence signal from Cy5.5-GEBP11-DMSA-MNPs significantly increased at the tumor site during 12-24 h post-injection, exhibited excellent tumor-to-background contrast from 24 h post-injection, reached a maximum at 36 h post-injection and then slowly washed out over time. MR T2-weighted imaging showed that a dark negative contrast in the tumor regions increased gradually. Compared with pre-injection, the tumor site had a significant 18.3%, 24.7% decrease in MR signal intensity at 24 h or 36 h post-injection respectively.8. Atherosclerotic plaque model was successfully induced by the high-fat diet plus balloon injury in rabbits..9. PET imaging could identify the atherosclerotic plaque when used with 68Ga-NOTA-DMSA-MNPs. After administration of GEBP11-DMSA-MNPs, the area of atherosclerotic plaque in abdominal aorta exhibited significant signal loss on T2-weighted images. Tthe presence of MNPs was confirmed by Prussian blue staining.Conclusion :GEBP11 conjugated DMSA-MNPs, as a novel molecular imaging probe, provides a potential approach for noninvasive assessment of angiogenesis in gastric cancer and atherosclerotic plaque by multi-modality imaging. Moreover, with a good histocompatibility, GEBP11-DMSA-MNPs could be a potential targeted drug delivery vector for angiogenesis-targeted therapy.