Comprehensive Insights into the Multi-Antioxidative Mechanisms of Melanin Nanoparticles and Their Application To Protect Brain from Injury in Ischemic Stroke

Abstract

Nanotechnology-mediated antioxidative therapy is emerging as a novel strategy for treating a myriad of important diseases through scavenging excessive reactive oxygen and nitrogen species (RONS), a mechanism critical in disease development and progression. However, similar to antioxidative enzymes, currently studied nanoantioxidants have demonstrated scavenging activity to specific RONS, and sufficient antioxidative effects against multiple RONS generated in diseases remain elusive. Here we propose to develop bioinspired melanin nanoparticles (MeNPs) for more potent and safer antioxidative therapy. While melanin is known to function as a potential radical scavenger, its antioxidative mechanisms are far from clear, and its applications for the treatment of RONS-associated diseases have yet to be well-explored. In this study, we provide for the first time exhaustive characterization of the activities of MeNPs against multiple RONS including O₂^•-, H₂O₂, ^•OH, ^•NO, and ONOO^-, the main toxic RONS generated in diseases. The potential of MeNPs for antioxidative therapy has also been evaluated in vitro and in a rat model of ischemic stroke. In addition to the broad defense against these RONS, MeNPs can also attenuate the RONS-triggered inflammatory responses through suppressing the expression of inflammatory mediators and cytokines. In vivo results further demonstrate that these unique multi-antioxidative, anti-inflammatory, and biocompatible features of MeNPs contribute to their effective protection of ischemic brains with negligible side effects.

Figure 1

(A) TEM image of PEG-MeNPs. (B) Effect of PEG, SOD, MeNPs, and PEG-MeNPs on EPR signals of DEPMPO-OOH. (C) O₂ production from the KO₂ solution (100 μM) with vs. without PEG-MeNPs. The insert is the digital picture of the PEG-MeNPs solution before vs. after addition of KO₂. (D) PEG-MeNP-catalyzed dismutation of O₂^•− under different concentrations of KO₂. (E) Michaelis-Menten kinetic plot of the initial reaction rate vs. the KO₂ concentration for PEG-MeNP-catalyzed dismutation of O₂^•−. (F) O₂ production from the KO₂ solution (100 μM) catalyzed by SOD vs. PEG-MeNPs. The concentration of PEG-MeNPs for catalysis is 0.1 nM. Assay was performed in triplicate.

Figure 2

(A) EPR spectra of PEG-MeNPs under different pH values, in the presence of GSH, or after reaction with KO₂ for 5 times (5 min each). (B) Absorbance changes in the mixture of Amplex Red and HRP induced by KO₂ with or without PEG-MeNPs. The insert is the digital picture of Amplex Red and HRP solution before vs. after addition of KO₂ and PEG-MeNPs. (C) The ratio between H₂O₂ and O₂ produced by PEG-MeNP-catalyzed dismutation of KO₂. (D) pH values of KO₂ and KOH solutions with vs. without PEG-MeNPs. The concentration of PEG-MeNPs is 0.1 nM. Assay was perfomed in triplicate.

Figure 3

(A) Schematic illustration of RONS metabolism. (B) EPR spectra of DEPMPO-OH obtained by trapping ^•OH with spin-trap reagent DEPMPO in the absence vs. presence of MeNPs or PEG-MeNPs. The ^•OH was generated by the Fenton reaction between H₂O₂ and Cu⁺ ions. For reaction #2 and #3, MeNPs and PEG-MeNPs were, respectively, added to the mixture of DEPMPO and H₂O₂, followed by the addition of Cu⁺ ions. In reaction #4, PEG-MeNPs were pre-incubated with DEPMPO and Cu⁺, followed by the addition of H₂O₂. (C) Digital picture of the carboxy-PTIO solution alone (#1) vs. the carboxy-PTIO solutions with ^•NO-generating NOC7 and PEG-MeNPs (#2–5: 0, 1, 2, and 4 nM). (D) EPR spectra of the five samples from (C). (E) ONOO⁻ scavenging effect of PEG-MeNPs.

Figure 4

(A) Cytotoxicity of PEG-MeNPs. (B) Intracellular O2^•− scavenging by PEG-MeNPs in AMA-treated Neuro 2A cells. (C) Confocal fluorescence images of O₂^•− levels in the AMA-treated Neuro 2A cells. Scale bar is 50 μm. (D) ROS levels in non-treated and PEG-MeNP-treated Neuro 2A cells under CoCl₂-induced hypoxic conditions. (E) Western blot analysis of the expression of Bax and Bcl-2 in the CoCl₂-stimualted Neuro 2A cells with vs. without PEG-MeNPs, as well as in the cells only treated with PEG-MeNPs.

Figure 5

(A) Schematic illustration of inflammatory mechanism triggered by the elevated RONS in non-neuronal cells. (B) Western blot analysis of COX-2 expression in LPS-stimulated macrophages. (C) Immunofluorescence images of the expression of COX-2 in LPS-stimulated macrophages with vs. without pretreatment of PEG-MeNPs. Scale bar is 10 μm. Levels of (D) ROS and (E) RNS in LPS-stimulated macrophages with different concentrations of PEG-MeNPs.

Figure 6

(A) Schematic representation of the ischemic stroke model. (B) Representative images of TTC-stained brain slices from different groups. The corresponding (C) infarct areas and (D) O₂^•− levels in brain tissues of the three groups (*p<0.05 and **p<0.01 vs. saline control).