Highly efficient conversion of superoxide to oxygen using hydrophilic carbon clusters Errol L. G. Samuela,1, Daniela C. Marcanoa,b,1, Vladimir Berkac,1, Brittany R. Bitnerd,e, Gang Wuc, Austin Pottera, Roderic H. Fabianf,g, Robia G. Pautlerd,e, Thomas A. Kentd,f,g,2, Ah-Lim Tsaic,2, and James M. Toura,b,2 aDepartment of Chemistry and bSmalley Institute for Nanoscale Science and Technology, Rice University, Houston, TX 77005; cHematology, Internal Medicine, University of Texas Houston Medical School, Houston, TX 77030; dInterdepartmental Program in Translational Biology and Molecular Medicine and Departments of eMolecular Physiology and Biophysics and fNeurology, Baylor College of Medicine, Houston, TX 77030; and gCenter for Translational Research in Inflammatory Diseases, Michael E. DeBakey Veterans Affairs Medical Center, Houston, TX 77030 Edited* by Robert F. Curl, Rice University, Houston, TX, and approved January 12, 2015 (received for review September 8, 2014) Many diseases are associated with oxidative stress, which occurs these data, we estimate that there are 2,000–5,000 sp2 carbon when the production of reactive oxygen species (ROS) over- atoms on each PEG-HCC core. We have demonstrated the •− whelms the scavenging ability of an organism. Here, we evaluated efficacy of PEG-HCCs for normalizing in vivo O2 in models the carbon nanoparticle antioxidant properties of poly(ethylene of traumatic brain injury with concomitant hypotension. Si- • glycolated) hydrophilic carbon clusters (PEG-HCCs) by electron multaneously, we observed normalization in NO levels in paramagnetic resonance (EPR) spectroscopy, oxygen electrode, these experiments (26, 27). A better understanding of these and spectrophotometric assays. These carbon nanoparticles have 1 materials is necessary to potentially translate these thera- •− equivalent of stable radical and showed superoxide (O2 ) dismu- peutic findings to the clinic. • tase-like properties yet were inert to nitric oxide (NO ) as well as In the present work, we evaluated antioxidant properties of − peroxynitrite (ONOO ). Thus, PEG-HCCs can act as selective anti- PEG-HCCs. Using spin-trap EPR spectroscopy, we demonstrate •− oxidants that do not require regeneration by enzymes. Our steady- that PEG-HCCs scavenge O2 with high efficiency. X-ray state kinetic assay using KO2 and direct freeze-trap EPR to follow photoelectron spectroscopy (XPS) indicates that covalent addi- its decay removed the rate-limiting substrate provision, thus en- tion of ROS to the PEG-HCCs is not responsible for the ob- •− abling determination of the remarkable intrinsic turnover numbers served activity. Direct measurement of O concentration using •− > −1 2 of O2 to O2 by PEG-HCCs at 20,000 s .Themajorproductsof freeze-trap EPR demonstrates that PEG-HCCs behave as cata- this catalytic turnover are O2 and H2O2, making the PEG-HCCs lysts, and measurements made with a Clark oxygen electrode a biomimetic superoxide dismutase. during the reaction reveal that the rate of production of O2 is •− above that expected due to self-dismutation of O2 in water. An superoxide | antioxidant | carbon nanoparticles | equivalent amount of H2O2 is also simultaneously produced. hydrophilic carbon clusters | superoxide dismutase mimetic Finally, selectivity for ROS is confirmed using a hemoglobin and • a pyrogallol red assay; PEG-HCCs are unreactive to both NO CHEMISTRY •− − eactive oxygen species (ROS), such as superoxide (O2 ), and ONOO . These results clarify the fundamental processes Rhydrogen peroxide (H2O2), organic peroxides, and hydroxyl involved in the previously observed in vivo protection against • radical ( OH), are a consequence of aerobic metabolism (1, 2). oxygen damage (26, 27). These ROS are necessary for the signaling pathways in biological processes (3, 4) such as cell migration, circadian rhythm, stem Significance cell proliferation, and neurogenesis (5). In healthy systems, ROS are efficiently regulated by the defensive enzymes superoxide Mechanistic studies of nontoxic hydrophilic carbon cluster dismutase (SOD) and catalase, and by antioxidants such as glu- nanoparticles show that they are able to accomplish the direct tathione, vitamin A, ascorbic acid, uric acid, hydroquinones, and conversion of superoxide to dioxygen and hydrogen peroxide. vitamin E (6). When the production of ROS overwhelms the This is accomplished faster than in most single-active-site scavenging ability of the defense system, oxidative stress occurs, – enzymes, and it is precisely what dioxygen-deficient tissue causing dysfunctions in cell metabolism (7 16). needs in the face of injury where reactive oxygen species, In addition to ROS, reactive nitrogen species (RNS) such as • particularly superoxide, overwhelm the natural enzymes re- nitric oxide (NO ), nitrogen dioxide, and dinitrogen trioxide can • quired to remove superoxide. We confirm here that the hy- befoundinallorganisms.NO can act as an oxidizing or re- drophilic carbon clusters are unreactive toward nitric oxide ducing agent depending on the environment (17), is more sta- radical, which is a potent vasodilator that also has an impor- ble than other radicals (half-life 4–15 s) (18), and is synthesized • tant role in neurotransmission and cytoprotection. The mech- in small amounts in vivo (17–22). NO is a potent vasodilator anistic results help to explain the preclinical efficacy of these and has an important role in neurotransmission and cytopro- carbon nanoparticles in mitigating the deleterious effects of tection (17, 18, 22, 23). Owing to its biological importance and the low concentration found normally in vivo, it is often im- superoxide on traumatized tissue. • portant to avoid alteration of NO levels in biological systems Author contributions: D.C.M. designed research; E.L.G.S., D.C.M., V.B., B.R.B., G.W., A.P., to prevent aggravation of acute pathologies including ischemia R.H.F., and R.G.P. performed research; E.L.G.S., V.B., and G.W. contributed new reagents/ and reperfusion. analytic tools; T.A.K., A.-L.T., and J.M.T. directed research; E.L.G.S., V.B., B.R.B., R.H.F., R.G.P., One way to treat these detrimental pathologies is to supply T.A.K., A.-L.T., and J.M.T. analyzed data; and E.L.G.S., D.C.M., V.B., G.W., A.P., T.A.K., A.-L.T., antioxidant molecules or particles that renormalize the disturbed and J.M.T. wrote the paper. oxidative condition. We recently developed a biocompatible The authors declare no conflict of interest. carbon nanoparticle, the poly(ethylene glycolated) hydrophilic *This Direct Submission article had a prearranged editor. carbon cluster (PEG-HCC), which has shown ability to scavenge 1E.L.G.S., D.C.M., and V.B. contributed equally to this work. oxyradicals and protect against oxyradical damage in rodent 2To whom correspondence may be addressed. Email: [email protected], Ah-Lim.Tsai@uth. models and thus far has demonstrated no in vivo toxicity in tmc.edu, or [email protected]. – laboratory rodents (24 27). The carbon cores of PEG-HCCs This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. are ∼3 nm wide and range from 30 to 40 nm long. Based on 1073/pnas.1417047112/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1417047112 PNAS | February 24, 2015 | vol. 112 | no. 8 | 2343–2348 Downloaded by guest on September 29, 2021 •− Reaction of PEG-HCCs with O Is Catalytic. Two mechanisms were AB 2 •− DEPMPO-OOH DEPMPO-OH considered for the observed O2 antioxidant activity: (i) radical annihilation owing to the covalent bond formation between the PBS PBS radical and the graphitic domains of the PEG-HCCs and/or •− (ii) transformation of O2 to O2 by the PEG-HCCs. Because PEG PEG XPS only detected a slight oxygen increase (<10%, Fig. S2)inthe PEG-HCCs after KO2 treatment, covalent oxygen addition to the PEG-HCC PEG-HCC PEG-HCCs cannot be the main mechanism. EPR amplitude EPR amplitude To test the transformation hypothesis, we established a man- •− ual freeze-trap EPR steady-state kinetic assay for O2 con- 3220 3280 3340 3400 3220 3280 3340 3400 •− sumption using KO2 to provide excess O2 and therefore shift Magnetic Field (G) Magnetic Field (G) the rate-limiting step to the intrinsic capability of PEG-HCCs in •− •− • turning over O2 . This approach helped us to avoid the dis- Fig. 1. Effect of PBS, PEG, and PEG-HCCs on O2 and OH radicals. (A) EPR •− spectra obtained from the O system or DEPMPO-OOH adduct at pH 7.4 advantages of commonly used spin-trap EPR methods, which 2 suffer from unfavorable trapping efficiencies and the loss of di- and room temperature. Spectra were recorded after 70 s of the KO2 addi- tion. The PEG-HCCs spectrum was corrected by subtracting the signals of the rect structural and kinetic information (17). • •− PEG-HCCs alone. (B) EPR spectra obtained from the OH system or DEPMPO- The typical EPR spectrum of 15-s freeze-trapped O2 is OH. Spectra were recorded after 90 s of the H2O2 addition. The adduct characterized by the axial symmetry of its three principle g values • •− stability was followed for 30 min. No correction was necessary for the OH (17, 38), and as Fig. 3 shows, the O2 EPR signal decreased in scavenging experiments. the presence of PEG-HCCs. The rate of second-order self-dis- •− mutation of O2 is very pH-sensitive and decreases exponen- tially from its pKa (4.8) to pH 11 owing to increased charge Results and Discussion repulsion between substrate molecules (39). Efforts to circum- • •− PEG-HCCs Scavenge OH and O2 . The scavenging capacity of vent this at pH 8 by reducing [KO2] to as low as 0.1 mM failed, PEG-HCCs was evaluated by EPR with the spin trap 5-(dieth- because we could not freeze-trap any EPR-detectable radical. oxyphosphoryl)-5-methylpyrrole-N-oxide (DEPMPO). DEPMPO Therefore, to achieve sufficient and reliable concentrations of •− produces relatively stable paramagnetic adducts upon reaction O2 in solution, the quenching experiments are best carried out • •− with OH (3, 4, 28, 29) and O (6, 29, 30) at room temperature in 50 mM NaOH (pH 13).