Letter Cite This: Nano Lett. 2019, 19, 4527−4534
pubs.acs.org/NanoLett
Carbogenic Nanozyme with Ultrahigh Reactive Nitrogen Species Selectivity for Traumatic Brain Injury Xiaoyu Mu,†,∇ Hua He,‡,∇ Junying Wang,†,∇ Wei Long,§ Qifeng Li,∥ Haile Liu,† Yalong Gao,∥ Lufei Ouyang,† Qinjuan Ren,† Si Sun,† Jingya Wang,§ Jiang Yang,⊥ Qiang Liu,§ Yuanming Sun,§ Changlong Liu,† Xiao-Dong Zhang,*,†,# and Wenping Hu†,#
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Tianjin Key Laboratory of Low Dimensional Materials Physics and Preparing Technology, School of Sciences, Tianjin University, Tianjin 300350, China ‡ State Key Laboratory of Heavy Oil Processing and Center for Bioengineering and Biotechnology, China University of Petroleum (East China), Qingdao 266580, China § Tianjin Key Laboratory of Molecular Nuclear Medicine, Institute of Radiation Medicine, Chinese Academy of Medical Sciences and Peking Union Medical College, Number 238, Baidi Road, Tianjin 300192, China ∥ Department of Neurosurgery and Key Laboratory of Post-trauma Neuro-repair and Regeneration in Central Nervous System, Tianjin Medical University General Hospital, Tianjin 300052, China ⊥ State Key Laboratory of Oncology in South China, Collaborative Innovation Center for Cancer Medicine, Sun Yat-sen University Cancer Center, Guangzhou 510060, China # Tianjin Collaborative Innovation Center of Chemical Science and Engineering, Tianjin 300072, China S Supporting Information *
ABSTRACT: Reactive oxygen and nitrogen species (RONS), especially reactive nitrogen species (RNS) are intermediate products during incidence of nervous system diseases, showing continuous damage for traumatic brain injury (TBI). Here, we developed a carbogenic nanozyme, which shows an antioxidant activity 12 times higher than ascorbic acid (AA) and behaves as multienzyme mimetics. Importantly, the nanozyme exhibits an ultrahigh scavenging efficiency (∼16 times higher than AA) toward highly active RNS, such as •NO and ONOO− as well as traditional reactive oxygen species (ROS) including O2•−, H2O2, and •OH. In vitro experiments show that neuron cells injured by H2O2 or lipopolysaccharide can be significantly recovered after carbogenic nanozyme treatment via scavenging all kinds of RONS. Moreover, the carbogenic nanozyme can serve as various enzyme mimetics and eliminate the harmful peroxide and glutathione disulfide from injured mice, demonstrating its potential as a therapeutic for acute TBI. KEYWORDS: Traumatic brain injury, reactive oxygen and nitrogen species, nanozyme, catalytic selectivity
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radicals under the NADPH oxidase catalysis by mechanical damage to mitochondria. These free radicals can subsequently damage cellular membranes by producing excessive lipid peroxidation and consumption of superoxide dismutase (SOD), leading to loss of function.4 The •NO free radical is the primary RNS after TBI with one unpaired electron and it can form various isoforms upon nitric oxide synthase catalysis in endothelial cells and neurons. In the biosystem, the •NO can be utilized as a signaling molecule with diverse functions and plays an important role in several important biological processes, including cerebral vasodilation, neurotransmission, and synaptic plasticity. After TBI, however, •NO can react with O2•− and then form ONOO−, which is detrimental to proteins,
raumatic brain injury (TBI) is a major disease with millions of people suffering from traffic accidents, sports, and military conflicts around the world every year.1,2 TBI can trigger many complications, and one of the most prominent is neuroinflammation.1 Damage to the central nervous system (CNS) can elicits inflammatory responses from neuron, microglia, and astrocytes.3 These neuron cells can immediately respond to injury by signaling translations and thus induce a series of chemical and biochemical reactions in vivo.4 Consequently, these reactions lead to significant tissue necrosis, apoptosis, and ferroptosis. Therefore, the major challenge in TBI therapy remains how to decrease the immune responses to brain injuries.5 Reactive oxygen species (ROS) and reactive nitrogen species (RNS) with unpaired electrons triggered by inflammation are among the most important elements for TBI.1 Brain ischemia following TBI can generate ROS such as O2•‑ and •OH © 2019 American Chemical Society
Received: April 1, 2019 Revised: June 15, 2019 Published: June 18, 2019 4527
DOI: 10.1021/acs.nanolett.9b01333 Nano Lett. 2019, 19, 4527−4534
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Figure 1. Illustration and characterization of carbogenic nanozyme. (a) Schematic illustration of nanozyme. (b) TEM image of the nanozyme. (c,d) C 1s and N 1s XPS spectra for the nanozyme, respectively. (e) Raman spectrum of the nanozyme. (f) FTIR spectra of lysine, ascorbic acid, and carbogenic nanozyme.
Transmission electron microscopy (TEM) images of the nanozyme exhibit an average diameter of ∼2.7 nm (Figures 1b and S1a−c), consistent with AFM results (Figure S1d,e). The hydrodynamic size is ∼3.1 nm from dynamic light scattering (Figure S1f), below the generally agreed renal clearance cutoff of 5.5 nm.37,38 The intralayer spacing of 2.6 Å corresponds to the graphitic (100) plane (Figure 1b). As shown in Figures 1c and S2, the nanozyme possesses CO (288.1 eV), CN (287.5 eV), CO (286.2 eV), and CC (284.6 eV). Meanwhile, pyridinic N (398.8 eV), pyrrolic N (399.8 eV), and graphitic N (400.9 eV) can be found in the N 1s band (Figure 1d), indicating that intermolecular cyclization and condensation reactions occurred during formation of the nanozyme. Moreover, the nanozyme displays the main Raman features of D band at 1350 cm−1 and G band at 1588 cm−1, further confirming the formation of graphitic structure (Figure 1e). The peak intensity ratios of D and G bands (ID/IG) is 0.921. In addition, a weak Raman peak at around 1450 cm−1 is also detected, which could be ascribed to the N doping of the graphitic structure such as the formation of NN aromatic. For the FTIR spectrum of nanozymes (Figure 1f), the broad peak at 3300−3500 cm−1 can be assigned to OH or NH in-plane stretching of the amine groups, and the peak at 1610− 1670 cm−1 could be overlapping with two peaks, ∼1650 and ∼1617 cm−1, corresponding to CC or −NHCO− and NH out-of-plane stretching vibrations, respectively. This fact
membrane lipids, and DNA, causing extensive oxidative cellular damage. Unfortunately, the RNS following TBI is highly active and toxic and is difficult to be cleared.1,4 Nanozymes with high catalytic activities provide a potential solution for treatment of CNS and ROS related diseases.6−20 The catalytic CeO2 and redox metal oxides have shown protective effects against brain injuries through scavenging ROS21−26 but they suffered slow hepatobiliary excretion.27 Organic materials,20,28 such as polymers29−33 and carbon dots,34−36 can be used to reduce neurodegeneration via their antioxidant activities. However, catalytic nanozymes with high selectivity, especially for RNS, are still scarce, limiting further implementation for TBI therapy. In this work, we reported an ultrasmall fluorescent carbogenic nanozyme (CN) with ultrahigh RNS selectivity. It can catalyze the highly active •NO and ONOO− in vitro, remove the harmful peroxide and superoxide in vivo, and reduce immune responses of TBI mice. Unlike conventional inorganic nanozymes, the reported carbogenic nanozyme shows efficient renal clearance without any side effects at high doses.
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RESULTS AND DISCUSSION The carbogenic nanozyme was prepared by a simple microwave-heating methord of lysine and ascorbic acid (AA). Figure 1a shows the general scheme of carbogenic nanozyme. 4528
DOI: 10.1021/acs.nanolett.9b01333 Nano Lett. 2019, 19, 4527−4534
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Figure 2. RONS scavenging activities of carbogenic nanozyme. (a) Illustration of RONS scavenging of nanozyme. Total antioxidant capacity against (b) ROS and (c) RNS of the nanozyme. (d) •OH, (e) O2•−, (g) •NO, and (h) ONOO− scavenging activities of the nanozyme, respectively. General (f) ROS and (i) RNS scavenging activities of the nanozyme compared with AA.
may imply the formation of amide groups through interactions with the carboxylic groups as amine groups. Additionally, three peaks at 3063, 2945−2863, 1323, and 1145 cm−1 can be attributed to CH, aliphatic CH, CN and phenolic/alcoholic CO stretching vibrations, respectively. Therefore, the surface functional groups of nanozymes could contain a hydroxyl and an amide/amino group which induces the zeta potential of the nanozyme and is characterized as −14.4 mV. In addition, the nanozyme exhibits an absorption peak at 266 nm assigned to the π−π* transition of the aromatic CC bond and an emission center at ∼417 nm under 335 nm excitation attributed to nonradiative electron− hole recombination by the surface functional groups (Figure S3). Matrix-assisted laser desorption/ionization time-of-flight mass spectra (MALDI-TOF MS) show the molecular weights of nanozymes are mainly between 1420 and 1580 Da with equal intervals of m/z 24 (Figure S4a−e), implying unique edge structures. Gel permeation chromatography (GPC) analysis was employed to further demonstrate the nanozyme with an average relative molecular weight of ∼1507 g/mol (Figure S4f). Moreover, the nanozymes exhibit good stability in physiological conditions during a week (Figure S5). Therefore, the nanozymes contain graphitic structures with N doping as the center and pyrrolic ring, and hydroxy and amide/amino groups as the surface functional groups. Figure 2 shows the enzyme-mimicking properties of carbogenic nanozyme for all RONS during TBI. The mechanistic diagram for scavenging RONS with the nanozyme is shown in Figure 2a. The general antioxidative properties
against ROS were determined using the ABTS method (Figure 2b). The nanozyme exhibits strong scavenging activities for free radicals, about 25 times higher than lysine. Furthermore, compared with antioxidant AA, the nanozyme shows a ∼12fold better scavenging activity (Figure 2f), and the antioxidant capacity exhibits no decrease during 7 days (Figure S6). Meanwhile, the general RNS scavenging capability of the nanozyme was evaluated using representative DPPH, which is an ideal compound due to the abundant N radicals with unpaired electrons. Figures 2c and S7 reveal that the nanozyme can effectively and continuously eliminate DPPH, superior to natural enzymes. We further investigated the RONS scavenging of the nanozyme in detail by monitoring individual group reactions. Figures 2d and 2e show the scavenging capacity of •OH and O2•− using the electron spin resonance (ESR). With increasing nanozyme concentrations, the ESR signals from the spin adducts, BMPO/•OH and DEPMPO/ •OOH, significantly decrease, indicative of increasing scavenging efficiency toward •OH and O2•−, respectively. In addition, the nanozyme exihibits catalase-like activity for H2 O 2 scavenging and follows typical Michaelis−Menten kinetics with the affinity (KM) of 393.3 mM (Figure S8). Previous work reported that Prussian blue nanoparticles (PBNPs) as multienzyme mimetics can scavenge ROS, 39 but the scavenging capacity of the nanozyme for •OH and O2•− here is nearly stronger 2 orders of magnitude than PBNPs (Figures S9 and 10). Next, we tested the scavenging capacity of the nanozyme for •NO and ONOO−. Importantly, as shown in Figure 2g, the ESR signal from carboxy-PTI produced by 4529
DOI: 10.1021/acs.nanolett.9b01333 Nano Lett. 2019, 19, 4527−4534
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Figure 3. In vitro RONS scavenging activity of carbogenic nanozyme and cell apoptosis in H2O2-treated N2a cells. (a,c,e,g) Fluorescent images and (b,d,f,h) corresponding statistical histograms of intracellular general ROS, O2•−, •OH/ONOO−, and •NO level under different condition via staining and flow cytometry, respectively. (i) Cell apoptosis test using FITC-Annexin V and PI staining. (j) Statistical diagram of early apoptosis and late apoptosis obtained from cell apoptosis test.
carboxy-PTIO and •NO completely disappears after the addition with 6 μM nanozyme, indicating thorough scavenging of the highly active •NO and even the more stable carboxyPTIO. This result reveals that the nanozyme exhibits high scavenging activity for •NO, better than the reported melanin nanoparticles at the same mass concentration.28 The absorption peak at 302 nm from ONOO− appreciably decreases with increasing nanozyme concentrations and time (Figures 2h and S11). The steady-state kinetic assay determined by varying the concentration of ONOO− follows a Michaelis−Menten kinetics with KM of 383.3 μM, higher than the affinity for H2O2 and indicating significant scavenging capacity for ONOO−. Because ONOO− is the intermediate product from •NO and O2•− during the oxidative stress and can cause extensive cellular damage following TBI, it is important for TBI treatment. The nanozyme possesses a high scavenging capability for RNS, ∼16 times higher than AA
(Figure 2i). The antioxidant activity of AA is dependent on the active sites of enol groups. Flavonoids, a group of natural antioxidants, exhibited good scavenging activities attributed to the catechol moiety on ring B.40 Especially, quercetin and anthocyanidin contained the combination of the catechol moiety with a CC and a 3-OH, acting as extremely active scavengers. In addition, melatonin can scavenge a variety of free radials including NO and ONOO− due to active pyrrolic ring in the indole moiety.41,42 Resveratrol as a functional scavenger was originated from the reactive center of phenolic moiety.43 Therefore, based on available and reliable structural information on nanozyme, the much better antioxidant activity of the nanozyme versus AA (see in Figure S12) could be attributed to the synergistic effect of active sites and surface functional groups including hydroxyl groups, CC bonds, pyrrolic ring, and phenolic structure. 4530
DOI: 10.1021/acs.nanolett.9b01333 Nano Lett. 2019, 19, 4527−4534
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Figure 4. In vivo treatment of carbogenic nanozyme to TBI mice. (a) Brain optical images and (b) quantitative analysis of BBB permeability by EB staining and spectrophotometry, respectively. * indicates p < 0.05 compared with the TBI group. (c,d) MMP-9 level in hippocampus. (e,f) Astrocytes activation level. (g−j) Oxidative stress-related indicators, including SOD, H2O2, lipid peroxidation, and GSSG of TBI mice with or without nanozyme treatment. DG: dentate gyrus.
staining, respectively. H2O2 treatment leads to strong fluorescence and produces lots of RNS. After nanozyme treatment, the fluorescence from injured cells is almost recovered to the healthy level, indicating highly efficient clearance of •NO and ONOO− (Figure 3e−h). Because of high toxicity, •NO and ONOO− can always damage cells and trigger a series of chemical and biochemical reactions, and thereby the high selectivity of carbogenic nanozyme for RNS suggests more efficient treatment for TBI. Meanwhile, lipopolysaccharide (LPS)-induced neuron cell injury was investigated, because LPS is one of the most intriguing neuroprotective stimuli. The nanozyme treatment can decrease RONS levels close to a normal level in LPS-induced neuron cells, especially sensitive for RNS (Figure S13). Moreover, cell survival of H2O2- or LPS-stimulated neural cells were implemented by MTT assay (Figure S14), confirming the strong in vitro neuron protection of nanozyme. For further analyses, cell apoptosis and cell cycle of H2O2- or LPSstimulated neural cells were performed by flow cytometry. For the cell apoptosis (Figure 3i), carbogenic nanozymes only induce negligible apoptosis level compared with the control normal group, revealing no significant toxicity of the nanozyme. However, cell apoptosis increases sharply after H2O2 stimulation, with the apoptotic population accounted for
Mice after TBI can produce excessive H2O2 in mitochondria, inducing cell apoptosis by RONS invasion, and thus it is important to investigate the selectivity of free radicals in vitro. Figure 3a exhibits the fluorescence images of H2O2 treated N2a cells with and without nanozyme by 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) staining. H2O2 treated cells show stronger fluorescence than healthy cells, implying excessive ROS generation (Figure 3a). After nanozyme treatment, the fluorescence signal remarkably decreases, owing to effective ROS scavenging. Quantitative measurements by flow cytometry demonstrate that general ROS level can increase to 4.25-fold upon H2O2 stimulation, but the excessive ROS in cells can be eliminated to reach close to a healthy level with nanozyme treatment (Figure 3b). We subsequently investigated important ROS, O2•− in cells by dihydroethidium (DHE) staining (Figure 3c,d). Similarly, the nanozyme treatment induces a remarkable decrease of fluorescence signal from H2O2 stimulation, indicating attenuation of O2•−, consistent with flow cytometry (Figure 3d). The O2•‑ clearance is directly related to CNS diseases and melanin NPs have previously shown potential advantages in treating a stroke.28 Next, RNS levels including •NO and ONOO− were evaluated in cells using 3-amino,4-aminomethyl-2′,7′-difluorescein diacetate (DAF-FM DA) and hydroxyphenyl fluorescein (HPF) 4531
DOI: 10.1021/acs.nanolett.9b01333 Nano Lett. 2019, 19, 4527−4534
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Figure 5. Spatial learning and memory assessments by Morris water maze 14 days post injury. (a) Model diagram of Morris water maze test. (b) Searching time for the platform and (c) swimming distance to the platform at the last trial each day during 5 days training. (d) Duration time in the platform area, (e) swimming distance to the platform, and (f) frequency on the platform.
damage, and mice following TBI can activate microglia and astrocytes.4 As shown in Figures 4e,f and S18, lots of microglia and astrocytes are produced after TBI, originating from strong neuron inflammation. These cells, however, are rescued by nanozyme treatment, suggesting reduction of pro-inflammatory immune responses. Moreover, we investigated oxidative stressrelated indicators ex vivo, including SOD, lipid peroxidation, H2O2, and glutathione disulfide (GSSG) in TBI mice after nanozyme treatment. SOD is an enzyme that alternately catalyzes O2− into O2 or H2O2, and TBI can stimulate the immune cells and consume lots of SOD, leading to decrease of SOD activity and increase of H2O2 amount (Figure 4g,h). Meanwhile, lipid peroxidation and GSSG are important bioindicators of cellular states, and TBI can increase their expression levels, signifying severe oxidative stress (Figure 4i,j). After nanozyme treatment, SOD activity increases, while the amount of H2O2, lipid peroxidation, and GSSG decreases. Therefore, the carbogenic nanozyme could confer therapeutic effects for acute TBI by clearing RONS. Finally, the behavior tests were conducted to evaluate the spatial learning and memory abilities by Morris water maze (Figure 5a). Figures 5b and 5c show the searching time for the platform and swimming distance to the platform at the last trial each day during 5 days training, respectively. The two main parameters indicate spatial learning ability of TBI mice treated with nanozymes. Compared to the TBI mice, both searching time and swimming distance to the platform gradually decrease with training days after nanozyme treatment, suggesting that the nanozymes could effectively improve the learning ability of TBI mice. After 5 days training, the platform was removed and each mouse kept swimming for 60 s to evaluate the spatial memory capacity of TBI mice treated with nanozymes. As shown in Figure 5d−f, the duration time in the platform area, swimming distance to the platform, and frequency on the platform are analyzed as the spatial memory indicators. For the nanozyme-treated TBI mice, duration time and frequency on the platform increase close to the normal level, and searching
60.94% (Figure 3j). After the treatment of nanozymes, apoptotic cell population decreases dramatically to 8.03%. For the cell cycle (Figure S15a−d), the cells treated with only nanozymes are maintained in regular cell cycle progress. However, the cell cycle is arrested largely in G2/M phase after being incubated with H2O2 (41.36%, Figure S15e). The abnormality of cell phase ratio suggests the DNA damages and failure of passing through checkpoints. In contrast, the treatment of nanozymes recovers cell cycle G2/M phase to 18.53%. The similar phenomena were observed for the nanozyme treatment of LPS-stimulated neural cells (Figure S16). These facts indicate that nanozymes are not only safe to neural cells under physiological environment, but also possess strong scavenging abilities for redundant ROS and RNS to achieve the neuron protection. Furthermore, we performed in vivo treatment of nanozyme in TBI mouse models by evaluating oxidative stress, blood− brain barrier (BBB) recovery, key enzymes, and tissue staining. Primary mechanical injury of TBI to the CNS can cause cell membrane disruption and BBB damage and thus we tested the BBB permeability by Evans blue (EB) staining.44 As shown in Figure 4a, BBB permeability significantly increases after TBI, indicative of direct cerebrovascular damage. After nanozyme treatment, the BBB permeability decreases with increasing post injection time, but injured mice without treatment still show distinct BBB damage. Quantitative results show a 30% decrease of BBB permeability by the nanozyme (Figure 4b). Matrix metalloproteinases (MMPs) are also associated with BBB opening and the pathophysiology of acute TBI. Therefore, MMP-9 levels were investigated in cerebral cortex and hippocampus (Figures 4c,d and S17). In a healthy brain, the MMP-9 level is low (red staining), but the TBI mice show gradually increasing expression levels of MMP-9. We conclude that the nanozyme can decrease the MMP-9 levels of injured mice, suggesting effective repair of BBB disruption and subsequent brain edema. The microglia and astrocytes are the key cellular mediators of inflammasome-mediated tissue 4532
DOI: 10.1021/acs.nanolett.9b01333 Nano Lett. 2019, 19, 4527−4534
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distance to the platform shortens than the untreated TBI mice. These facts reveal that the nanozyme treatment could efficiently improve the spatial memory capacity of TBI mice. Further, the pharmacokinetics, biodistribution, and in vivo toxicity were investigated. The half-life of the nanozyme is ∼16 min for first phase and the kidney shows the highest uptakes with the average urine excretion of ∼84.2% during 48 h (Figure S19). Hematology, biochemistry, and pathology evaluations indicate the nanozyme without significant toxicity (Figures S20−22). At present, clinically available TBI therapeutic agents are relatively scarce, and there is an unmet need to develop enzyme-mimetic molecules for TBI treatment.45,46 Considering the safety of nanomaterials,47−49 the carbogenic nanozyme reported here displays an irreplaceable advantage in the excretion route. In the future, highly active nanozymes with specific neuron-targeting capability and multifunctional properties are still desired.
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CONCLUSION In summary, we developed a small multienzyme mimicking carbogenic nanozyme, showing significant free radical scavenging capability. Importantly, the nanozyme exhibits ultrahigh selectivity to RNS, such as •NO and ONOO−. Cell viability of LPS- and H2O2-injured neuron cells shows great improvement after nanozyme treatment via scavenging all kinds of RONS. The excessive RONS in brain tissues induced by acute TBI exhibit potent elimination after nanozyme treatment by intravenous injection. Moreover, the nanozyme treatment could effectively improve the spatial learning and memory abilities of TBI mice. Present work provides a potential route for treatment of central nervous system diseases.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.nanolett.9b01333. Experimental procedures; Figures S1−22 (PDF)
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Phone/Fax: +86-22-2740 4118. ORCID
Hua He: 0000-0002-6084-1034 Xiao-Dong Zhang: 0000-0002-7212-0138 Wenping Hu: 0000-0001-5686-2740 Author Contributions ∇
X.M., H.H., and J.W. contributed equally.
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We gratefully acknowledge the financial support from the National Natural Science Foundation of China (No. 21874154, No. 81471786 and No. 91859101), and the Independent Innovation Foundation of Tianjin University.
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REFERENCES
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DOI: 10.1021/acs.nanolett.9b01333 Nano Lett. 2019, 19, 4527−4534
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DOI: 10.1021/acs.nanolett.9b01333 Nano Lett. 2019, 19, 4527−4534
