Hybrid magneto-fluorescent nano-probe for live apoptotic cells monitoring at brain cerebral ischemia

Abstract

α-Fe₂O₃ Magnetic nanoparticles (MNPs) have been synthesized, functionalized at silica that ends up with -NH₂ group to form FMNPs. Conjugation of FMNPs with a fluorescently-labeled poly-caspase inhibitor valylalanylaspartic acid fluoromethyl ketone (SR-FLICA) which serves as a pan-caspase inhibitor was carried out to form finally a hybrid probe SR-FLICA-FMNPs. This probe could be used as a multimodal magneto-fluorescent platform for live apoptotic cells monitoring using MR and fluorescence imaging techniques. Characterization results of the as-synthesized MNPs and functionalized FMNPs by SEM, TEM, N₂ isotherm, XRD and magnetic VSM showed that, a controlled morphological structure of MNPs could be synthesized with cubic-shaped, ferromagnetic, base-centered orthorhombic space group R3c and average size of 45.8 ± 3.2 and 50.3 ± 1.6 nm for MNPs and FMNPs, respectively. Phantom MRI experimental results of the examined MNPs and FMNPs confirmed the concentration dependency nature of T₂ signal reduction. In addition, in vitro and in vivo sensing studies on our conjugated hybrid multifunctional probe SR-FLICA-FMNPs using 9 L gliosarcoma cells confirmed that; it could positively intact within the astrocytes and the nuclei of the apoptotic cells taking into account the starting material's cytotoxicity. Several histo-chemical protocols could be examined to confirm such behavior. Confocal and fluorescence microscopes' results of the histological stained apoptotic cells confirmed positive and specific expressions of our designed probe. MRI monitoring results of apoptotic rat models after focal brain transient cerebral ischemia showed a remarkable time-dependent reduction of T₂* weighted signal up to 4 h indicating that our newly designed hybrid probe has long blood circulation and could be used as a future contrasting agent. Moreover, the distribution of our probe was evaluated by subtracting the T₂* signal images before and after injection with SR-FLICA-FMNPs and was significantly correlated with the histological findings by staining via terminal deoxynucleotidyl transferase-mediated uridine 5'-triphosphate-biotin nick end labeling. Moreover, clearance study confirmed that; our magneto-fluorescent hybrid probe could be cleared through liver Kupffer cells. Thus; the newly developed SR-FLICA-FMNPs could be considered as a future multifunctional probe for in vitro and in vivo apoptotic cells monitoring.

Keywords: Fluorescently-labeled poly-caspase inhibitor (SR-FLICA); Functionalized magnetic nanoparticles (FMNPs); Live apoptotic cells; Magnetic resonance imaging (MRI).