Oxidized Carbon Nanoparticles Enhance Cellular Energetics With Application to Injured Brain

Abstract

Pro-energetic effects of functionalized, oxidized carbon nanozymes (OCNs) are reported. OCNs, derived from harsh acid oxidation of single-wall carbon nanotubes or activated charcoal are previously shown to possess multiple nanozymatic activities including mimicking superoxide dismutase and catalyzing the oxidation of reduced nicotinamide adenine dinucleotide (NADH) to NAD⁺. These actions are predicted to generate a glycolytic shift and enhance mitochondrial energetics under impaired conditions. Impaired mitochondrial energy metabolism is increasingly recognized as an important facet of traumatic brain injury (TBI) pathophysiology and decreases the efficiency of electron transport chain (ETC)-coupled adenosine triphosphate (ATP) and NAD⁺ regeneration. In vitro, OCNs promote a pro-aerobic shift in energy metabolism that persists through ETC inhibition and enhances glycolytic flux, glycolytic ATP production, and cellular generation of lactate, a crucial auxiliary substrate for energy metabolism. To address specific mechanisms of iron injury from hemorrhage, OCNs with the iron chelator, deferoxamine (DEF), covalently-linked were synthesized. DEF-linked OCNs induce a glycolytic shift in-vitro and in-vivo in tissue sections from a rat model of TBI complicated by hemorrhagic contusion. OCNs further reduced hemorrhage volumes 3 days following TBI. These results suggest OCNs are promising as pleiotropic mediators of cell and tissue resilience to injury.

Keywords: Mitochondria; Oxidized carbon nanozyme; bioenergetics; lactate; traumatic brain injury.

Figure 1

OCNs induce pro‐aerobic and pro‐glycolytic shifts in bEnd.3 cell energy metabolism. A) Oxygen consumption rate (OCR) and b) extracellular acidification rate (ECAR) are concurrently increased in bEnd.3 brain endothelial cells pretreated with PEGylated hydrophilic carbon clusters (PEG‐HCC) at therapeutically relevant dosages of 4 and 16 µg mL⁻¹ versus untreated control (CTL). OCR is a measure of mitochondrial oxidative metabolism and ECAR is a measure of glycolytic rate. Higher OCR values indicate an aerobic shift in cellular energy metabolism, reflected in c) higher basal respiratory rate (difference between initial baseline and minimum OCRs following the addition of rotenone and antimycin A), d) higher proton leak (a protective adaptation against mitochondrial ROS generation, calculated as the difference between minimum OCR following oligomycin injection and OCR after the addition of rotenone/antimycin A) and e) maximal respiratory rate (difference between the maximal OCR following the addition of the uncoupling reagent FCCP and OCR after the addition of rotenone/antimycin A). Higher ECAR values indicate a glycolytic shift in energy metabolism, which is reflected in f) higher extracellular lactate levels versus CTL in 4 independent assays (represented by connected dots) following 24 h treatment of bEnd.3 cells with PEG‐HCCs. Specialized extracellular flux assays for the measurement of cellular ATP bioenergetics further reveal that PEG‐HCC treatment increased g) glycolytic ATP production, h) mitochondrial ATP production, and i) total (mitochondrial + glycolytic) cellular ATP production in a dose‐dependent manner. These findings suggest that OCNs induce pro‐aerobic and pro‐glycolytic shifts in cellular energy metabolism that persist through insults to mitochondrial function. Mean + SEM, one‐way ANOVA; *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 2

Synthesis and characterization of DEF‐PEG‐cOACs. a) Reaction scheme for the synthesis of DEF‐PEG‐cOACs from cOACs derived from fuming nitric acid oxidation of sieved cocoanut shell‐derived activated charcoal. b) Representative high‐resolution transmission electron microscopy of cOACs (scale bar 20 nm). (c) Size distribution of cOAC particles (mean: 3–3.5 nm; maximum: 7 nm; n = 360 particles). d) Thermogravimetric analysis of cOACs showed a mass loss of 56% (dashed line), PEG‐cOACs (blue line; 91%), and DEF‐PEG‐cOACs showing a mass loss of 78% (green line). e) Nanoparticle tracking analysis of PEG‐cOACs (mean: 89 nm; mode: 78 nm) and DEF‐PEG‐cOACs (mean: 94 nm; mode: 89 nm). f–h) High‐resolution X‐ray photoelectron spectroscopy C 1s spectrum of cOACs, PEG‐cOACs, and DEF‐PEG‐cOACs.

Figure 3

Deferoxamine‐conjugated oxidized carbon nanozymes (DEF‐PEG‐cOACs) demonstrate iron chelation effect in bEnd.3 cells. Mouse brain endothelial (bEnd.3) cells were cultured in media with and without iron (III) chloride (50 µm), DEF‐PEG‐cOACs (4 µg mL⁻¹), and the iron chelator deferoxamine (50 µm). Free deferoxamine was estimated as 100X higher molarity than when particle bound. Intracellular free iron was measured using fluorescence microscopy with FerroOrange, a fluorescent probe sensitive to unbound Fe²⁺. Treatment of iron cells with DEF‐PEG‐cOACs and deferoxamine in the presence of iron (III) chloride significantly decreases FerroOrange fluorescence, reflecting decreased intracellular free iron. DEF‐PEG‐cOACs are ≈63‐fold more potent per mole of deferoxamine than free deferoxamine. No significant difference in fluorescence between DEF‐PEG‐cOAC and deferoxamine‐treated cells was observed in the absence of media iron supplementation, suggesting that DEF‐PEG‐cOACs and deferoxamine do not significantly interfere with FerroOrange fluorescence. N = 3 technical replicates. One‐way ANOVA. *p < 0.05; **p < 0.01; ***p < 0.001; “ns” indicates a non‐significant comparison.

Figure 4

Pro‐energetic effects of deferoxamine‐functionalized OCNs a) Synthesis schematic for PEGylated oxidized carbon nanozymes synthesized from coconut‐derived activated charcoal (PEG‐cOACs) and PEG‐cOACs functionalized with the iron chelator deferoxamine (DEF‐PEG‐cOACs). PEG‐cOACs and DEF‐PEG‐cOACs increase bEnd.3 cell b) oxygen consumption rates (OCR) and c) extracellular acidification rates (ECAR), indicative of pro‐aerobic and pro‐glycolytic shifts in energy metabolism respectively. Of note, the increase in OCR versus untreated (CTL) cells is greater with DEF‐PEG‐cOAC treatment, suggesting an improvement upon the pro‐aerobic shift facilitated by PEG‐cOACs. d) Higher OCR in PEG‐ and DEF‐PEG‐cOAC‐treated bEnd.3 cells after the addition of the ATP synthase inhibitor oligomycin (2 µm) suggests that the pro‐aerobic effects of PEG‐ and DEF‐PEG‐cOACs persist through negative stressors of mitochondrial respiration. e) Higher extracellular lactate release as a result of 24 h PEG‐cOAC and DEF‐PEG‐cOAC treatment of bEnd.3 cells support a pro‐glycolytic role for these OCNs at a comparable magnitude to that of PEG‐HCCs. Mean + SEM, one‐way ANOVA; *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 5

Effect of DEF‐PEG‐cOACs on mitochondrial respiration following traumatic brain injury. a) Schematic of rat model of in vivo contusion TBI. Cortical punch biopsy sections from rats subject to injury and treated with DEF‐PEG‐cOACs (2 mg kg⁻¹, intravenously 15 min and intraperitoneally 2‐ and 22 h post‐injury) trended toward b) higher maximal oxygen consumption rates (OCR) and c) significantly higher extracellular acidification rates (ECAR) versus saline vehicle control (CTL, Veh.), indicating a concurrent aerobic and glycolytic shift in energy metabolism that may signify resilience to the effects of external trauma on cellular energy metabolism. Mean + SEM, one‐way ANOVA; * p < 0.05; ** p < 0.01; *** p < 0.001. Figure using Biorender.com.

Figure 6

Protective effects of deferoxamine‐functionalized OCNs in a rat model of contusion‐based hemorrhagic traumatic brain injury (TBI) a) Parietal lobe hemorrhagic lesions in PEG‐ and DEF‐PEG‐cOAC‐treated rats post‐cortical injury exhibited significantly reduced b) area (mm²) relative to saline vehicle‐treated animals, suggesting that PEG‐ and DEF‐PEG‐cOAC treatment confers protection and reduces the deleterious effects of contusion TBI, with a non‐significant trend toward increased efficacy with DEF‐PEG‐cOAC treatment (48.7% vs 68.2% PEG‐cOAC vs DEF‐cOAC‐PEG respective reduction in hematoma area; NS difference). N = 4–5 animals/group. One‐way ANOVA. * p < 0.05; ** p < 0.01; *** p < 0.001.

Figure 7

Proposed mechanism of OCN‐catalyzed electron transfers within mitochondrial respiration. a) OCNs oxidize the reduced electron carrier NADH to NAD+ and facilitate the transfer of electrons to oxidized cytochrome c (CytC_ox). Reduced cytochrome c (CytC_red) is an important mitochondrial electron carrier protein that transfers electrons between coenzyme Q‐cytochrome c reductase (mito. respiratory complex III) and cytochrome c oxidase (mito. respiratory complex IV), thereby promoting the formation of a proton gradient between the mitochondrial intermembrane space and the matrix. b) OCN‐catalyzed electron transfer between NADH and CytC may bypass inhibited respiratory complexes, thus preserving the intermembrane proton gradient necessary for ATP generation. Electron transfer to OCNs may also enable OCN‐mediated dismutation of mitochondrially‐toxic reactive oxygen species (ROS).

Figure 8

PEG‐OCNs increase the rate of lactate production by altering cytosolic metabolism. NADH metabolism in glycolysis is facilitated by glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) and lactate dehydrogenase (LDH). GAPDH produces NADH from the oxidation of glyceraldehyde‐3‐phosphate to 1,3‐phosphoglycerate, and LDH reversibly reduces pyruvate to lactate with NADH, and vice versa. The flux of pyruvate into the mitochondria is regulated by the tricarboxylic acid cycle which inhibits the pyruvate dehydrogenase complex when TCA intermediates increase beyond a threshold. In this report, we observed a large increase in extracellular acidification with a small change in mitochondrial oxygen consumption rate (OCR) in cells treated with PEG‐OCNs. The increase in extracellular acidification of PEG‐OCN‐treated cells can be in part accounted for by an increase in glycolytic rate caused by PEG‐OCNs oxidizing the NADH to NAD+ produced by GAPDH and increasing the rate of intermediate flux by increasing the concentration of substrate (NAD⁺) for GAPDH. The tricarboxylic acid cycle (TCA) rate limits mitochondrial pyruvate flux which leads to an accumulation of lactate and consequent H⁺ release because of pyruvate availability for LDH and a small increase in (OCR). The relative differences in intermediate flux and production are shown as changes in line width and font size, respectively.

Figure 9

Proposed schematic of pleiotropic OCN‐mediated protection during hemorrhagic traumatic brain injury (TBI). Disruption of cerebral vasculature as a result of injury results in extravascular leakage and pooling of blood. Iron released from the breakdown of hemoglobin in blood generates cytotoxic reactive oxygen species (ROS) via the Fenton reaction, exacerbating cellular injury and decreasing the efficiency of neuronal energy metabolism. Deferoxamine‐functionalized OCNs may mediate cell and tissue protection during hemorrhagic TBI by i): chelating free iron, thereby limiting ROS generation, ii): catalyzing the dismutation of ROS, iii): rescuing injury‐inhibited aerobic (mitochondrial) respiration, and iv): stimulating glycolytic energy metabolism in neurons, astrocytes and endothelial cells, thus ensuring the generation of lactate that is transported to neurons via the endothelial/astrocyte‐neuron lactate shunt and utilized as an important adjunct energy source for mitochondrial ATP and NAD+ generation.