ABSTRACT
Autophagy-the lysosomal degradation of cytoplasmic components via their sequestration into double-membraned autophagosomes-has not been detected non-invasively. Here we show that the flux of autophagosomes can be measured via magnetic resonance imaging or serial near-infrared fluorescence imaging of intravenously injected iron oxide nanoparticles decorated with cathepsin-cleavable arginine-rich peptides functionalized with the near-infrared fluorochrome Cy5.5 (the peptides facilitate the uptake of the nanoparticles by early autophagosomes, and are then cleaved by cathepsins in lysosomes). In the heart tissue of live mice, the nanoparticles enabled quantitative measurements of changes in autophagic flux, upregulated genetically, by ischaemia-reperfusion injury or via starvation, or inhibited via the administration of a chemotherapeutic or the antibiotic bafilomycin. In mice receiving doxorubicin, pre-starvation improved cardiac function and overall survival, suggesting that bursts of increased autophagic flux may have cardioprotective effects during chemotherapy. Autophagy-detecting nanoparticle probes may facilitate the further understanding of the roles of autophagy in disease.
Subject(s)
Autophagy , Fluorescent Dyes , Nanoparticles , Spectroscopy, Near-Infrared , Animals , Anti-Bacterial Agents/administration & dosage , Anti-Bacterial Agents/pharmacology , Arginine/chemistry , Autophagy/drug effects , Carbocyanines/chemistry , Cathepsins/chemistry , Doxorubicin/administration & dosage , Doxorubicin/pharmacology , Fluorescent Dyes/chemistry , Macrolides/administration & dosage , Macrolides/pharmacology , Magnetic Resonance Imaging/methods , Mice , Nanoparticles/chemistry , Spectroscopy, Near-Infrared/methodsABSTRACT
Delivery of antibodies to monitor key biomarkers of retinopathy in vivo represents a significant challenge because living cells do not take up immunoglobulins to cellular antigens. We met this challenge by developing novel contrast agents for retinopathy, which we used with magnetic resonance imaging (MRI). Biotinylated rabbit polyclonal to chick IgY (rIgPxcIgY) and phosphorylthioate-modified oligoDNA (sODN) with random sequence (bio-sODN-Ran) were conjugated with NeutrAvidin-activated superparamagnetic iron oxide nanoparticles (SPION). The resulting Ran-SPION-rIgPxcIgY carries chick polyclonal to microtubule-associated protein 2 (MAP2) as Ran-SPION-rIgP/cIgY-MAP2, or to rhodopsin (Rho) as anti-Rho-SPION-Ran. We examined the uptake of Ran-SPION-rIgP/cIgY-MAP2 or SPION-rIgP/cIgY-MAP2 in normal C57black6 mice (n = 3 each, 40 µg/kg, i.c.v.); we found retention of Ran-SPION-rIgP/cIgY-MAP2 using molecular contrast-enhanced MRI in vivo and validated neuronal uptake using Cy5-goat IgPxcIgY ex vivo. Applying this novel method to monitor retinopathy in a bilateral carotid artery occlusion-induced ocular ischemia, we observed pericytes (at d 2, using Gd-nestin, by eyedrop solution), significant photoreceptor degeneration (at d 20, using anti-Rho-SPION-Ran, eyedrops, P = 0.03, Student's t test), and gliosis in Müller cells (at 6 mo, using SPION-glial fibrillary acidic protein administered by intraperitoneal injection) in surviving mice (n ≥ 5). Molecular contrast-enhanced MRI results were confirmed by optical and electron microscopy. We conclude that chimera and molecular contrast-enhanced MRI provide sufficient sensitivity for monitoring retinopathy and for theranostic applications.
