Nanopartículas de ouro biofuncionalizadas com glutationa: uma nova abordagem neuroprotetora no estudo da isquemia cerebral

Abstract

Gold nanoparticles (AuNP) have garnered attention as potential neuroprotective agents in animal models of brain diseases, including ischemic stroke, which is characterized by the occlusion or blockage of a blood vessel. Studies have already shown a decrease in infarct volume, functional recovery, and attenuation of oxidative stress and inflammation in animals treated with AuNPs and submitted to ischemia. In addition to acting as a treatment, these nanomaterials also act as nanocarriers, increasing the biodistribution and effectiveness of substances that are rapidly degraded by the body, such as glutathione (GSH). GSH is the peptide in the greatest amount in the body and is a key element in redox balance. Thus, this study aimed to evaluate the effectiveness of treatment with glutathione-functionalized gold nanoparticles in the effects of cerebral ischemia. Gold nanoparticles were synthesized and then biofunctionalized with GSH (GSH-AuNP). For characterization, an analysis of the shape and mass of gold was performed by Surface Plasmonic Resonance (SRP), measured by UV-VIS spectrophotometry; their charge (zeta potential, ZP) was measured. For the cerebral ischemia model, hippocampal slices from 36 male CD1 mice aged 9-11 weeks were used (CEUA/UFMG: 270/2020). The slices were divided into two groups: 1) control, with aCSF (Artificial Cerebrospinal Fluid) containing glucose and bubbled with O2 and CO2, and 2) ischemia with ACSF without glucose and bubbled with N2 and CO2. Each experimental group was further subdivided into PBS control, GSH (100ug/mL), AuNP (8 or 800 ug/mL) and GSH-AuNP concentration x response curve (0,08 - 800ug/mL). The ischemia period lasted 60 minutes, followed by a120 minutes reperfusion period Cell damage was assessed by lactate dehydrogenase (LDH) release, and neurotoxicity by glutamate release. Brain, kidney, and liver were dissected 24h after intravenous injection of 1mg/kg Au for biodistribution analysis. Results were analyzed using Anova Two-Way (p<0,05). The AuNP showed a λmax of 530nm and a charge of -31.4mV, while the GSH- AuNP had a 520nm and -27mV, their spherical shape being inferred and showing the shift of the maximum peak and the charge caused by the modification of the surface by GSH binding. The mass of gold in the AuNP solution was 82mg/L, while that of GSH- AuNP was 44mg/L likely due to centrifugation of the latter. Among control groups, it was observed greater cellular damage and glutamate release in the slices submitted to ischemia (5,132 vs 15,933 U/mg proteín, p<0,0001). It was observed neuroprotection in the treatment group with GSH-AuNP compared to the control group in LDH analysis (3,629 vs 15,933 U/mg protein, p<0,001) and a significant decrease in glutamate release (14,912 vs 28,280 nmol/mg protein, p=0,028) during the ischemic process. It was possible to find AuNP and GSH-AuNP in the liver, while only the GSH-AuNP group was found in the kidney. The successful synthesis and modification of AuNP with GSH, as demonstrated by the characterization results, as well as decrease of LDH and glutamate release, highlight the potential of GSH-AuNP as a protective agent against cellular toxicity