Efficacy of redox nanoparticles for improving survival of transplanted cells in a mouse model of ischemic stroke.

The success of cell transplantation therapy for ischemic stroke is hindered by the low cell survival rate in poststroke brain, due in part to high free radical production and ensuing oxidative stress. We have developed redox nanoparticles to eliminate reactive oxygen species. In this study, we tested the protective efficacy of these redox nanoparticles in cell culture and a mouse model of ischemic stroke. Induced human dental pulp stem cells were subjected to oxygen-glucose deprivation and reoxygenation to recapitulate ischemia and reperfusion in the penumbra surrounding a cerebral infarct. Cell viability using WST-8 assay, apoptosis using TUNEL, free radicals using MitoSOX, and inflammatory cytokines using ELISA kit were measured in the presence and absence of redox nanoparticles after oxygen-glucose deprivation and reoxygenation. The scavenging activity of redox nanoparticles against reactive oxygen species was detected by electron spin resonance. Moreover, induced cells were transplanted intracerebrally into to the distal middle cerebral artery occlusion model with and without redox nanoparticles, and the survival rate measured. Cell viability was enhanced, while apoptosis, free radical generation, and inflammatory cytokine expression levels were reduced in cultures with redox nanoparticles. Further, reduced redox nanoparticles were detected in the cytoplasm, indicating free radical scavenging. Addition of redox nanoparticles also improved the survival rate of transplanted cells after 6 weeks in vivo. These redox nanoparticles may increase the applicability and success of induced stem cell therapy for ischemic stroke patents by promoting long-term survival.

Antioxidant nanomedicine with cytoplasmic distribution in neuronal cells shows superior neurovascular protection properties.

This study investigated whether nitroxide radical (4-amino-TEMPOL)-containing nanoparticles (RNPs; antioxidant nanomedicine) can prevent neurovascular unit impairment caused by reactive oxygen species (ROS) after cerebral ischemia-reperfusion. C57BL/6J mice underwent transient middle cerebral artery occlusion (tMCAO). The mice were randomly divided and administered intra-arterial RNPs injection (9 mg/kg, 7 µM/kg), edaravone (3 mg/kg, 17 µM/kg), or phosphate-buffered saline (control group). Survival rate and neurological score were evaluated 24 h post-injection. RNPs distribution was determined using immunofluorescence staining and blood-brain barrier (BBB) disruption using Evans blue extravasation assay. Effect of RNPs and edaravone on microglia polarization into microglia M1 and M2 was evaluated. We also determined multiple ROS-scavenging activities in brain homogenates of RNPs- and edaravone-treated animals using an electron spin resonance-based spin-trapping method. Compared with edaravone, RNPs significantly improved the survival rate and neurological deficit, inhibited BBB disruption and supported polarization of microglia into M2 microglia. RNPs were localized in endothelial cells, the perivascular space, neuronal cell cytoplasm, astrocytes, and microglia. Scavenging capacities of hydroxyl, alkoxyl, and peroxyl radicals were significantly higher in the RNPs-treated group. RNPs show promising results as a future neuroprotective nanomedicine approach for cerebral ischemia-reperfusion injury.

Novel neuroprotection using antioxidant nanoparticles in a mouse model of head trauma.

INTRODUCTION: Free radicals and reactive oxygen species are related to deteriorating pathological conditions after head trauma because of their secondary effects. 2,2,6,6-Tetramethylpiperidine-1-oxyl (TEMPO) scavenges free radicals; however, this molecule is also toxic. Here, we have evaluated the neuroprotective effect of antioxidant nanoparticles, which consisted of a novel core-shell type nanoparticle containing 4-amino-TEMPO, that is, redox-active nitroxide radical-containing nanoparticles (RNPs). METHODS: Institute of Cancer Research mice were subjected to a head-impact procedure, randomly divided into four groups and intravenously (3 mg/kg) administered phosphate-buffered saline, TEMPO, micelle (a self-assembling block copolymer micelle without a TEMPO moiety), or RNP through the tail vein immediately thereafter and intraperitoneally at days 1, 3, and 5 after traumatic brain injury (TBI). The RNP distribution was detected by rhodamine labeling. Cognitive behavior was assessed using the neurological severity score and a rotarod test at days 1, 3, and 7 following TBI, and contusion volume was measured at day 7 after TBI. Free radical-scavenging capacity was analyzed by electron paramagnetic resonance on day 1 after TBI, and immunostaining was used to observe mobilization of microglia (Iba-1) and rescued neuronal cells (NeuN). RESULTS: Redox-active nitroxide radical-containing nanoparticle was detected in the microvessels around the injured area in the brain. Cognitive behavior assessment was significantly better, and contusion volume was significantly smaller in the RNP group compared with the other groups. Superoxide anion scavenging capacity was significantly higher in the RNP group, and neuronal loss was significantly suppressed around the injured area at day 7 after TBI. Furthermore, in the RNP group, neurodegenerative microglia production was suppressed at days 3 and 7 after TBI, whereas neuroprotective microglia production was higher at day 7 after TBI. CONCLUSION: The RNP administration after TBI improved cognitive behavior and reduced contusion volume by improving reactive oxygen species scavenging capacity. Therefore, RNP may have a neuroprotective effect after TBI. LEVEL OF EVIDENCE: Therapeutic test.

Neurovascular Unit Protection From Cerebral Ischemia-Reperfusion Injury by Radical-Containing Nanoparticles in Mice.

BACKGROUND AND PURPOSE: Reperfusion therapy by mechanical thrombectomy is used to treat acute ischemic stroke. However, reactive oxygen species generation after reperfusion therapy causes cerebral ischemia-reperfusion injury, which aggravates cerebral infarction. There is limited evidence for clinical efficacy in stroke for antioxidants. Here, we developed a novel core-shell type nanoparticle containing 4-amino-4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (nitroxide radical-containing nanoparticles [RNPs]) and investigated its ability to scavenge reactive oxygen species and confer neuroprotection. METHODS: C57BL/6J mice underwent transient middle cerebral artery occlusion and then received RNPs (9 mg/kg) through the common carotid artery. Infarction size, neurological scale, and blood-brain barrier damage were visualized by Evans blue extravasation 24 hours after reperfusion. RNP distribution was detected by rhodamine labeling. Blood-brain barrier damage, neuronal apoptosis, and oxidative neuronal cell damage were evaluated in ischemic brains. Multiple free radical-scavenging capacities were analyzed by an electron paramagnetic resonance-based method. RESULTS: RNPs were detected in endothelial cells and around neuronal cells in the ischemic lesion. Infarction size, neurological scale, and Evans blue extravasation were significantly lower after RNP treatment. RNP treatment preserved the endothelium and endothelial tight junctions in the ischemic brain; neuronal apoptosis, O2- production, and gene oxidation were significantly suppressed. Reactive oxygen species scavenging capacities against OH, ROO, and O2- improved by RNP treatment. CONCLUSIONS: An intra-arterial RNP injection after cerebral ischemia-reperfusion injury reduced blood-brain barrier damage and infarction volume by improving multiple reactive oxygen species scavenging capacities. Therefore, RNPs can provide neurovascular unit protection.