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Polydopamine Nanoparticles as Efficient Scavengers for Reactive Oxygen Species in Periodontal Disease Xingfu Bao, Jiahui Zhao, Jian Sun, Min Hu, and Xiurong Yang ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.8b04022 • Publication Date (Web): 20 Jul 2018 Downloaded from http://pubs.acs.org on July 20, 2018

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Polydopamine Nanoparticles as Efficient Scavengers for Reactive Oxygen Species in Periodontal Disease

Xingfu Bao,†,‡ Jiahui Zhao,†,§Jian Sun,† Min Hu,‡ and Xiurong Yang *,†

† State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, Jilin 130022, China ‡ School of Stomatology, Jilin University, Changchun 130021, China § University of Chinese Academy of Sciences, Beijing 100049, China

E-mail: [email protected]

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Abstract Antioxidative therapy has been considered as an efficient strategy to treat a series of excessive reactive oxygen species (ROS)-triggered diseases including oxidative stress-induced periodontal disease. However, current natural enzymes and nanozymes often show their high specificity towards given ROS and appear insufficient antioxidative effects against multiple ROS generated in the diseases process. Meanwhile, multi-enzyme-based antioxidant defense systems are usually confined by the complicated synthesis, as well as potential unwanted residue and toxicity. Various supports are highly needed to immobilize natural enzymes and antioxidants during the bio-related usages due to their low operational stability and difficulty of reuse. To overcome these limitations, we develop a high-performance platform by using biodegradable polydopamine nanoparticles (PDA NPs) as smart ROS scavengers in oxidative stress-induced periodontal disease. Although PDA-based materials are well known to eliminate ROS both in vitro and in vivo, their antioxidative performance in periodontal disease and relative mechanisms have yet to be well explored. In this study, PDA NPs can act as ROS scavengers in dental specialties with ideal outcomes. Spectroscopic and in vitro experiments provide strong evidences for the roles of PDA NPs in scavenging multiple ROS and suppressing ROS-induced inflammation reaction. In addition to above investigations, results of a murine periodontitis model clearly demonstrate the feasibility of PDA NPs as robust antioxidants to remove ROS and decrease the periodontal inflammation without any side effects. Taking together, our present study will provide valuable insight into the development of safe and efficient antioxidant defense platforms for further biomedical usages.

Keywords: antioxidant defense system · polydopamine nanoparticles · reactive oxygen species · periodontal disease · inflammation · long-term toxicity


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As one of the most common oral diseases worldwide, periodontal disease has been considered as the main cause of tooth loss in adults and a major danger against human health. According to a recent clinical survey, the incidence of periodontal disease has increased over 40% among the population in developed countries while it may be much higher in developing countries owing to the worse dental treatment conditions.1-3 The primary predisposing causes of periodontal disease can be ascribed to the imbalance between the amount of bacterial pathogen and the host immune response towards infection. In detail, the formation of dental plaque facilitates the growth of pathogens and promotes the release of toxins during the initiation and progression of periodontal disease. Then, immune cells are recruited and activated, followed by the up-regulation of pro-inflammatory cytokines and release of reactive oxygen species (ROS). Finally, the degradation of periodontal fibers and bone occur, leading to serious soft and hard tissue destruction.4-6 Nowadays, methods including mechanical debridement for dental plaque, antibiotics and anti-inflammatory drug for pathogen infection, as well as surgery techniques for severe cases have addressed the crucial aspect of periodontal disease with admirable outcomes.7-9 Despite their usefulness, surgical manipulation and mechanical debridement often comprises with complicated series of steps and exposure risk. Current antibiotics and anti-inflammatory medication usually cause potential adverse effects and unwanted systemic immune response. In this regard, innovatory non-surgical yet efficient alternatives towards periodontal disease with insignificant side effects are highly desired. Owing to the persistent inflammation in response towards bacterial pathogens, the excessive production of ROS from immune cells in periodontal disease, which oversteps the intracellular antioxidant defense capacity, can induce oxidative damage, interfere with cell cycle progression, and lead to irreversible tissue injury.10-12 Significantly, oxidative stress-induced detrimental influences on periodontal cells via lipid peroxidation, protein denaturation, or DNA damage has been considered as one of the most important issues associated with periodontal inflammation. In that case, we envision that efficient local ROS scavenging may alter the periodontal micro-environment and relieve its pathological proceeding from inflammatory conditions. Recent studies demonstrate that various antioxidative defense strategies based on natural enzymes, nanozymes, and antioxidants can efficiently ACS Paragon Plus Environment

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maintain the intracellular redox balance and protect the cells against oxidative damage.13-37 For example, various antioxidants and natural enzymes can eliminate excessive intracellular ROS and work together as medicines for anti-inflammation therapy.14, 18-20 With higher stability than natural enzymes, a series of nanomaterials including CeO2, MnO2-x, Fe3O4, and prussian blue, as well as their composites, have been developed as nanozymes to exhibit admirable antioxidative enzyme-like activity.22,

24, 27, 28, 31, 32, 34

However, supports such as hydrogels, organic medium, and inorganic materials are highly needed to immobilize these natural enzymes and antioxidants during the bio-related usages because of their low operational stability and difficulty of reuse. Single component nanozymes and natural enzymes often fail to sufficiently prevent oxidative damage due to their high specificity of antioxidative activity against ROS and the presence of multiple ROS generated in the diseases progress. In addition, multi-nanozymebased antioxidative composites often require complicated synthesis. More importantly, the potential side effects associated with various exogenous nanoagents may bring other intractable problem in practical usages and confine their clinical translation.38-40 Accordingly, innovative antioxidative strategies together with ideal antioxidants to efficiently scavenge pathogenic ROS in periodontal disease must meet the following criteria: (1) robust antioxidative activity against multiple ROS, (2) great ability to relieve ROS-triggered inflammatory reaction, as well as (3) possible degradability and low systemic toxicity after subgingival administration. However, it is still a challenge for currently reported antioxidants to meet all above requirements in the treatment of periodontal disease. Arising from their excellent near-infrared absorbance, strong chelating capacity towards metal ions, and high biocompatibility, polydopamine (PDA) and their derivatives have been well employed as photoacoustic contrast agents, photothermal agents, and chelating agents for biomedical usages.41-48 Moreover, as naturally occurring biopolymers, PDA-based materials also show their capacities in deactivating radical species generated by ultraviolet light, protecting brain from ROS-induced injury in ischemic stroke, as well as treating acute inflammation-induced injury.49-53 All these evidences indicate that PDA-based materials with abundant reductive functional groups such as catechol and imine exhibit more promising in scavenging multiple ROS both in vitro and in vivo.14, 19, 41 Herein, we develop an ACS Paragon Plus Environment

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efficient antioxidant defense platform for ROS removal in oxidative stress-induced periodontal disease by using PDA nanoparticles (NPs) as smart scavengers. Our data provide strong evidences for the roles of PDA NPs in scavenging multiple ROS and suppressing ROS-induced inflammation reaction in dental specialties. In vivo studies based on a murine periodontitis model clearly demonstrate the feasibility of PDA NPs as robust antioxidants to remove ROS and decrease the periodontal inflammation. Moreover, investigations focused on the long-term toxicity and possible metabolic pathway of PDA NPs after subgingival injection indicate their high biocompatibility and biodegradable behavior. We expect that this study will yield precious insight into the development of safe and efficient antioxidant defense platforms for wide biomedical usages, which are not limited to the present periodontal disease. Results and discussion Figure 1A illustrated the rational synthesis of PDA NPs via a classical Stöber method, as well as their further usages as efficient ROS scavengers in periodontal disease. In detail, PDA NPs were well yielded via a self-polymerization manner at room temperature while aqueous solution containing ethanol and ammonia was selected as a reaction medium. Typical images from scanning electron microscopy (SEM) and transmission electron microscopy (TEM) indicated that the resultant PDA NPs held a monodisperse spherical structure with an average diameter of 160 nm (Figure 1b and Figure 1c). Compared with some naturally resulting melanin particles with a diameter ranging from 50 nm to 200 nm, these PDA NPs held a more uniform characteristics in both size and shape. Results of Fourier transform infrared (FT-IR) spectra provided additional evidence of the successful formation of PDA NPs (Figure 1D). Absorption band between 3500 cm-1 and 3200 cm-1 could be detected in both dopamine and PDA NPs due to the stretching vibration of O-H, N-H, and NH2. However, intense absorption peak around 1600 cm-1 in PDA NPs was ascribed to the formation of indole-related structure after the oxidation and self-polymerization of dopamine.43, 49 A weaker absorption band between 800 cm-1 and 700 cm-1 could be clearly found in PDA NPs than that of dopamine, indicating the decrease of aromatic hydrogen and aromatic nucleus, as well as the successful formation of polymer structure in PDA NPs. Because there were still some residual functional groups on the surface of PDA NPs, which were similar with those of dopamine, PDA ACS Paragon Plus Environment

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NPs dispersed well in various physiological solutions such as 0.9% NaCl solution, fetal bovine serum (FBS), and DMEM (inset of Figure 1C). Results of time-dependent UV-vis absorbance spectra and size change of PDA NPs in 0.9% NaCl solution over 1 d further revealed that PDA NPs could remain their monodispersity and stability for a long period without any detectable agglomeration and deposition, which was highly similar with a previous study (Figure S1).43 In addition, zeta-potential of PDA NPs was measured to be about -25 mV. To sum up, the great colloidal stability of PDA NPs in aqueous solution therefore endowed them with high potential in bio-related usages. Prior to using PDA NPs as ROS scavengers in periodontal disease both in vitro and in vivo, it was essential to explore their cytotoxicity and blood compatibility. Human gingival epithelial (HGE) cells were achieved from orthodontic patients with informed consent and identified via immunohistochemical (IHC) staining at first. Detailed operation was provided in Figure S2A and experimental section. With the assistance of careful incubation, cells could grow out from the tissue pieces (Figure S2B). Moreover, the attachment and spread of these cells held a paving stone manner (Figure S2C). Subsequently, IHC staining for cytokeratin was used to process the identification of these cells. Similar with some previous studies, these cells stained with cytokeratin antibody showed a positive result, indicated the achievement of HGE cells (Figure S2D).54, 55 Standard methyl thiazolyl tetrazolium (MTT) assay was carried out to determine the relative viabilities of HGE cells towards PDA NPs. As expected, no significant cytotoxicity induced by PDA NPs was observed for all the groups even upon a high concentration of 1 mg/mL (Figure 2A). Calcein AM and propidium iodide (PI) were used to carry out live-dead cell staining. In detail, dead cells stained with PI and live cells stained with calcein AM exhibited a red color emission and a green color emission upon the fluorescence excitation, respectively. Results of live/dead staining demonstrated that only green staining could be detected from the fluorescence images, indicating that PDA NPs with high bio-compatibility did not alter the morphology and relative viability of HGE cells (inset of Figure 2A, Figure 2B, and Figure S2E). To further check any potential cell damage induced by PDA NPs, the release of lactate dehydrogenase (LDH) was investigated after the coincubation process. As shown in Figure 2C and Table S1, it was found that all the leakage percentages ACS Paragon Plus Environment

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of LDH from above samples were lower than 5%, indicating that PDA NPs did not induce obvious cell membrane damage. Moreover, no differences in cellular ATP levels were detected between the control group and the PDA NPs-treated groups, demonstrating that PDA NPs did not affect the mitochondrial function after cellular uptake (Figure 2D). Hemolytic assay was used to explore the interaction between PDA NPs and blood components. No obvious hemolysis after the 3 h co-incubation process occurred even upon a high concentration of 1 mg/mL (Figure 2E and Table S2). Prothrombin time (PT) and activated partial thromboplastin time (APTT) were considered as two essential factors to estimate the extrinsic and intrinsic coagulation routes, respectively. Results from the effect of blood coagulation time revealed that there were no obvious differences between the control group and the PDA NPs-treated groups, and the effect of PDA NPs on the blood coagulation time could be negligible (Figure 2F). As previously mentioned, all these necessary exploration indicated the overall safety of PDA NPs in vitro. Having successfully understanding the in vitro toxicity of PDA NPs, we then estimated their ROS removal ability. In this study, the scavenging efficiency of hydroxyl radicals (HO·) and superoxide radicals (O2·-) by using PDA NPs as scavengers was explored in detail.26, 34 HO· scavenging efficiency was obtained by measuring the presence of fluorescent 2-hydroxyterephthalic acid. A dramatically decrease of fluorescence signal around 425 nm in the fluorescence spectra was detected in the presence of PDA NPs with various concentrations, which confirmed that PDA NPs could efficiently eliminate HO· (Figure 3A and Figure S3A). The scavenging efficiency of PDA NPs towards HO· followed a concentration-dependent manner and could be highly enhanced with the increasing of PDA NPs’ concentrations. While the concentration of PDA NPs was 0.1 mg/mL, the scavenging ratio of HO· could reach 90%, which was sensitized from H2O2 with a concentration of 10 mM. When the concentration of PDA NPs was increased to 0.125 mg/mL, nearly all the HO· were removed from the system based on our present design. In addition, O2·- scavenging efficiency was acquired by measuring the inhibition ratio of photo-reduction of NBT. Typically, a strong absorbance signal could be detected after ultraviolet (UV) radiation in the presence of riboflavin, methionine, and NBT, demonstrating the high levels of O2·-. Once solution containing PDA NPs was added into above mixture, the signal around 560 nm in the ACS Paragon Plus Environment

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absorption spectra significantly decreased. More than 80% O2·- could be removed from the solution in the presence of PDA NPs with a concentration of 0.05 mg/mL, indicating that PDA NPs possessed excellent removal capacity towards O2·- (Figure 3B and Figure S3B). When the concentration of PDA NPs was increased to 0.1 mg/mL, O2·- were totally cleaned up from the system as expected. All above results demonstrated the admirable removal capacity of PDA NPs towards various ROS. Above successful verification of PDA NPs’ antioxidant capacity further inspired us to explore their potential usages as scavengers against intracellular ROS. Prior to the investigation of the intracellular ROS removal capacity of PDA NPs, the uptake kinetics of HGE cells towards PDA NPs was explored by using Cu2+-modified PDA NPs as probes. As expected, the uptake efficiency showed a incubation period-dependent manner and the uptake efficiency was higher than 50% when the incubation period was increased to 12 h, indicating that PDA NPs could be efficiently uptaken by HGE cells (Figure 4A). Subsequently, fluorescence microscope observation together with flow cytometry were used to detect the intracellular ROS removal capacity of PDA NPs. Firstly. HGE cells were treated with PDA NPs to allow the efficient cellular uptake. Secondly, exogenous Rosup reagent was added into the culture medium to sensitize the generation of intracellular ROS. Thirdly, dichlorofluorescein diacetate (DCFHDA) was added into above culture medium to determine the ROS production and relative ROS removal capacity via PDA NPs. As shown in Figure 3C, negligible fluorescence could be detected from both the control group and the PDA NPs-treated group, indicating that our nanoparticles could not lead to the generation of ROS. However, a high fluorescence signal occurred in the Rosup-treated group, which demonstrated that intracellular non-fluorescent DCFH could be oxidized by ROS to fluorescent 2, 7dichlorofluorescein (DCF). With the assistance of PDA NPs, the fluorescence intensity from the Rosuptreated group decreased significantly. Results based on flow cytometry also suggested that nearly 80% ROS could be removed by PDA NPs upon a co-incubation concentration of 0.1 mg/mL (Figure S4B). For a comparison, uric acid (UA) as an endogenous antioxidant scavenger against ROS was selected in this study.56, 57 As shown in Figure S5, PDA NPs with a concentration of 0.1 mg/mL held a similar ROS removal capacity with that of UA with a concentration of 0.25 mM, indicating that our PDA NPs could ACS Paragon Plus Environment

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emerge as a high-performance nanoparticulate ROS scavenger. Though promising, PDA NPs held more advantages over small molecular antioxidants rooted from their specific nanoscale size distribution. For example, PDA NPs not only could be efficiently uptaken by HGE cells via endocytosis instead of the concentration-dependent diffusion of small molecules, but also exhibited a sustained antioxidative activity during the interaction period against the flashy effect of molecular antioxidants. In addition, PDA NPs processed better performance compared with natural antioxidant enzymes. Different from the high specificity towards substrates of single natural antioxidant enzymes and complicated synthetic approaches of systems including several natural antioxidant enzymes, multi-enzymatic activity towards the decomposition of O2·- and H2O2 and the efficient antioxidant activity based on reducing polyphenol structure of PDA NPs thus endowed them more potentials for further usages.50, 53 To re-confirm the ROS removal capacity of PDA NPs, Hela cells were selected as another cell type. The fluorescence intensity from the Rosup-treated group decreased sharply after the addition of PDA NPs and the quantitative results indicated that over 75% ROS were cleaned up by PDA NPs (Figure S6). These results revealed that PDA NPs held a broad-spectrum ROS removal capacity in various cell types treated with ROSsensitized reagents. Excessive pro-inflammatory cytokines and some inflammatory mediators were found in periodontal disease including tumor necrosis factor alpha (TNF-α), interleukins (IL-1β and IL6), and inducible nitric oxide synthase, which also acted as biomarkers to describe the levels of periodontal inflammation.3, 4, 54 As shown in Figure 3D and Figure 3E, the expression of TNF-α and IL1β in LPS-treated HGE cells revealed that PDA NPs could efficiently alleviate the activation of these inflammatory mediators and lead to significant reduction of intracellular ROS levels after LPS treatment. Therefore, PDA NPs could efficiently remove ROS from HGE cells sensitized by both Rosup and LPS, further promoting us to explore their ROS removal capacity in a murine periodontitis model. As illustrated in Figure 4A, LPS-induced periodontal disease in mice was rationally developed firstly.20 Secondly, PDA NPs were subgingivally administrated to efficiently remove ROS. Thirdly, DCFH-DA was injected in situ, and the fluorescence signal from DCF was detected. Prior to the development of animal model, in vivo imaging system was used to re-confirm our detection of the ACS Paragon Plus Environment

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intracellular ROS removal efficacy by using PDA NPs as scavengers (Figure 4B). As expected, high fluorescence signal could be found in the Rosup-treated group while no fluorescence signals were detected in the control group and PDA NPs-treated group. Noticeably, HGE cells treated with both PDA NPs and Rosup showed a relative lower fluorescence signal compared with those of the Rosup-treated group, indicating that PDA NPs could efficiently remove intracellular ROS. Moreover, the quantitative information achieved via Image J software indicated that the ROS removal capacity of PDA NPs showed a concentration-dependent manner. Fluorescence imaging combined with X-ray imaging was then used to evaluate the ROS removal efficacy in vivo. As shown in Figure 4C, high fluorescence signal caused by the LPS-induced local inflammation was detected around the DCFH-DA injection site in the LPS-treated group. Meanwhile, no fluorescence signals were found in the control group and the PDA NPs-treated group with a dosage of 0.2 mg/site. While PDA NPs were injected in situ in the LPStreated group, the fluorescence signal gradually decreased due to the great ROS removal capacity of PDA NPs. Additional quantitative information based on Image J software re-confirmed our above results. In addition, photographs of mouse faces were taken after various treatments. As shown in Figure 4D and Figure S7, the gum and lip of mice became red and swollen after LPS treatment compared with those of the control group and the PDA NPs-treated group. As expected, above mentioned inflammation reaction gradually relieved after the administration of PDA NPs, which could help to promote the functional recovery from periodontal disease. Significantly, nearly all the local inflammation together with redness and swollen disappeared after the double administration of PDA NPs. These exciting results indicated that LPS-induced local inflammation could be eased with our PDA NPs, and these PDA NPs-based antioxidants held a great ROS removal capacity in a murine model of periodontal disease. Although promising, only PDA NPs with an average diameter of 160 nm were selected and used as the typical ROS scavengers in periodontal disease according to our present design. To achieve more precise results and practical applied values, further comprehensive explorations focused on the size distributiondependent ROS removal capacity of PDA NPs should be more significant and carried out.


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Considering the high ROS removal efficacy of PDA NPs, we further explored the possible removal mechanism in vitro and metabolic pathway in vivo of these nanoparticles. In this study, concentrations of H2O2 were selected in accordance with the common H2O2 concentrations in macrophage or induced by inflammatory reaction in vivo. As shown in Figure 5A and Figure 5B, PDA NPs lost their UV-visNIR absorbance in the presence of H2O2, and the aqueous solution containing PDA NPs showed a H2O2 concentration-dependent color fading. Correspondingly, our PDA NPs-based ROS removal approach also followed a time-dependent manner, which was similar with some recent studies.50, 53 Photographic observation indicated that more PDA NPs could react with H2O2 along with the increasing of coincubation periods and nearly all the PDA NPs could reacted with H2O2 after 24 h co-incubation. Moreover, TEM image of PDA NPs after H2O2 treatment showed that severe morphological changes occurred after the mild oxidized process (Figure 5C). We attributed this color-fading oxidation to the transform from brown PDA NPs to colorless or light-colored oxidation products including pyrrole-2, 3dicarboxylic acid, indicating that PDA NPs were highly biodegradable in the presence of ROS.41-43 Gingival crevicular fluid (GCF) from volunteers was used as the typical periodontal tissue-related biological fluid to explore the effect of biomolecular corona in the degradation process of PDA NPs.58, 59 At first, PDA NPs treated with GCF were defined as PDA NPs@GCF. Figure S8A illustrated the preparation of PDA NPs@GCF and H2O2-induced degradation process. Figure 1C and Figure S8B revealed that there were no differences in morphology and size between PDA NPs@GCF and PDA NPs. UV-vis absorption spectra demonstrated that PDA NPs@GCF held a certain resistance against H2O2 with a low concentration but could be degraded by H2O2 with a relative high concentration (Figure S8C). Significantly, PDA NPs@GCF exhibited a similar morphological change with that of PDA NPs treated with H2O2 (5 mM) based on TEM image (Figure S8D). These results indicated that biomolecules in GCF could reduce the rapid degradation degree of PDAs but could not avoid their degradation possibility in the presence of H2O2. Therefore, the antioxidative mechanisms of PDA NPs in periodontal disease could be ascribed to their high performance as both antioxidants and biocatalysts according to the previously mentioned insights of PDA NPs in scavenging ROS and our present study.49, 50, 52, 53 ACS Paragon Plus Environment

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In order to understand the metabolic pathway of PDA NPs after subgingival injection, Cu2+modified PDA NPs were prepared at first to facilitate the following testing. Previous studies demonstrated that dopamine could serve as robust capturers towards metal ions due to the intense interaction between dopamine residues and metal ions.47, 48 Based on the ICP-MS results, the loading amount of Cu2+ was calculated to be approximately 80 µg of Cu2+ per mg of Cu2+-modified PDA NPs. These nanoparticles held a spherical structure with an average diameter of 160 nm (inset of Figure 5D). After subgingival injection of Cu2+-modified PDA NPs, tissues around the injection sites were separated at each expected time points, and the remaining percentages of nanoparticles were quantified via the amounts of Cu2+. It was worth noting that Cu2+-modified PDA NPs with a relatively larger dosage of 2 mg/site were administrated in our design in order to achieve accurate results of their subgingival residuals due to the low loading amount of Cu2+ in these nanoparticles. As shown in Figure 5D, the amounts of Cu2+-modified PDA NPs in tissues decreased over time, indicating that these nanoparticles could be eliminated from the mouse bodies with a step-by-step manner. Moreover, Cu2+-modified PDA NPs could be efficiently engulfed by the macrophages, and the uptake amounts held a concentrationdependent manner based on ICP-MS results (Figure 5E and Figure S9A). All the uptake efficiencies of Cu2+-modified PDA NPs by the macrophages were over 60% after 24 co-incubation (Figure S9B). In view of these exciting results, the possible metabolic pathway of PDA NPs after subgingival injection could be ascribed to the uptake-transport-release character of the macrophages.41, 42 Despite some recent studies focused on the development of PDA-based nanocomposites indicted their excellent biocompatibility and low long-term toxicity after intravenous injection, the systemic investigation of these PDA NPs after subgingival administration was still not achieved.42-48 To explore their long-term toxicity, a series of in vivo experiments were designed and carried out. Observation including behavior and body weight was the most straightforward method to evaluate the toxicity of nanomaterials. Assessments of hematological and biochemical markers could quantify the acute or chronic inflammatory responses and the potential toxicity after the administrations of nanoparticles. Histological analysis of exposed tissues could determine the tissue damages from toxic exposure ACS Paragon Plus Environment

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induced by the nanoparticles.42, 50, 60-62 Results based on both in vivo fluorescence imaging of ROS scavenging and antioxidant therapy indicated that PDA NPs with a dosage of 0.2 mg/site after subgingival injection could efficiently remove the local ROS and certainly relieve the periodontal inflammation, thus a relatively larger dosage of 2 mg/site was selected to explore the long-term safety of our PDA NPs. If experimental mice bore above toxic exposure and exhibited normal behavior, any dosages lower than this value could be suggested as safe exposure ones. As shown in Figure 6A, no noticeable differences in the animal behavior and body weight were found between the control group and the test group. To obtain quantitative evaluation, cytokine response assay and blood biochemical assay were investigated, respectively. Results shown in Figure 6B indicated that PDA NPs did not cause significant systemic cytokine responses in mice at 24 h, 30 d and 60 d post injection, and all the serum levels of TNF-α, IFN-γ, and IL-1β were within the normal values. Significantly, the serum levels of above mentioned cytokines were usually selected as essential indicators to explore the acute or chronic injuries induced by nanomaterials.50, 53 Moreover, PDA NPs did not alter the serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), as well as alkaline phosphatasein (ALP) 30 d and 60 d after subgingival injection, and all the parameters of the test group fell in the reference normal ranges (Figure 6C). Last but not least, hematoxylin and eosin (H&E) histology analysis of main organs 30 d and 60 d after administration of PDA NPs indicated that no morphological changes or signs of inflammation occurred after subgingival injection of PDA NPs (Figure 6D, Figure S10, Table S3, and Table S4). It was worth noting that a previous study indicated that PDA NPs synthesized via a similar method had a LD50 value as high as 483.95 mg/kg after intravenous injection.50 For a healthy Kunming mouse (25 g) in this study, above LD50 value after mass conversion should be 12.1 mg/mouse, which was much higher than current dosage of 2 mg/site. More importantly, some previous studies and our results demonstrated that PDA NPs and PDA-based materials could be biodegraded within 60 d, which promoted the selection of 30 d and 60 d as our investigation periods.42, 44, 50 Therefore, all these results revealed that PDA NPs at the present given dosage held extremely low systemic toxicity. Conclusion ACS Paragon Plus Environment

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In summary, we designed and constructed an efficient antioxidant defense platform for ROS removal in oxidative stress-induced periodontal disease by using PDA NPs as intelligent scavengers. A series of systemic investigations were performed in this study to improve our comprehensive understanding of the antioxidative effect of PDA NPs and their relative mechanisms. Spectroscopy results indicated that PDA NPs held broad antioxidative activities against toxic ROS, highlighting their potential as efficient ROS scavengers. In vitro experiments demonstrated that PDA NPs held admirable ROS removal ability and anti-inflammatory activity, which could protect HGE cells against oxidative stress and inflammation reaction. Using a murine periodontitis model, we further evidenced that post-subgingival injection of PDA NPs could efficiently remove ROS in vivo and decrease local periodontal inflammation. Moreover, our well-prepared PDA NPs exhibited biodegradable behavior after ROS treatment, and the possible metabolic pathway of PDA NPs after subgingival injection could be ascribed to the uptake-transportrelease effect of the macrophages. Last but not least, both in vitro and in vivo side effect studies demonstrated the high biocompatibility and low systemic toxicity of PDA NPs. Taking together, our present approach not only provided detailed insight to understand the antioxidant effect of PDA NPs in periodontal disease and relative mechanisms, but also would be highly beneficial in the development of safe and effective antioxidant defense platforms for wide biomedical usages. Experimental section Chemical and materials. Dopamine hydrochloride, CuCl2, terephthalic acid (TA), nitro blue tetrazolium (NBT), chloral hydrate, propidium iodide (PI), calcein AM, and lipopolysaccharide (LPS) were obtained from Sigma-Aldrich. Dulbecco’s modified Eagle’s medium (DMEM), Trypsin, and fetal bovine serum (FBS) were achieved from Sangon. Synthesis of PDA NPs and Cu2+-modified PDA NPs. PDA NPs were prepared via a classical Stöber method with some modifications. Typically, a mixture containing concentrated NH4OH (2 mL), ethanol (40 mL), and water (90 mL) was stirred at room temperature for 0.5 h. Dopamine hydrochloride (0.5 g) in water (10 mL) was added into above mixture, and the reaction was allowed to proceed under stirring for 1 d. Then, PDA NPs were isolated via centrifugation, washed with water, and dried overnight. For ACS Paragon Plus Environment

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the preparation of Cu2+-modified PDA NPs, above PDA NPs (10 mg) were labeled with Cu2+ by the addition of CuCl2 (10 mg/mL, 0.2 mL) in acid buffer (10 mL, pH = 5.5). 1 h later, Cu2+-modified PDA NPs were separated via centrifugation, washed with water, and dried for further use. Cell cultures. HGE cells were achieved from clinical patients with informed consent. Buccal gingiva specimen about 1-2 mm was extracted from orthodontic patients with mean age of 16 years upon sterile condition, as well as immersed into D-Hanks solution with penicillin and streptomycin. The epithelium and lamina propria were carefully separated from each other via microsurgical forceps. All tissue was cut into small blocks (0.5 mm×0.5 mm), washed repeatedly, and inoculated with a uniform spacing dispersion (0.5 mm) close to the basolateral membrane of the bottle wall. Above dispersed tissue was then placed in a humidified incubator (37 ºC, 5% CO2). 1 h later, DMEM containing FBS (15%) was added. All the tissue pieces were removed when fibrous cells grew out. After digesting, cells were harvested, washed, and cultured in DMEM containing FBS (10%). Then, immunohistochemical staining for cytokeratin was used to identify HGE cells. Hela cells were supplied by ATCC (American Type Culture Collection) and cultured in DMEM containing FBS (10%) for further usages. Cytotoxicity studies. HGE cells were cultured in 96-well plates with a density of 104 per well for 6 h. PDA NPs with different concentrations were added into the medium. After incubation for 24 h or 48 h, cells were treated with MTT to allow the formation of formazan crystal. The purple formazan product was dissolved with DMSO, and measured on a Bio-Rad microplate reader. The viability was normalized to the viability without any treatment (n=6). Cellular viability observation. HGE cells were cultured in 6-well plates with a density of 104 per well for 12 h. PDA NPs with different concentrations were added into the medium. 24 h later, cells were washed with 0.9% NaCl solution, as well as stained with calcein AM and PI. Fluorescence images were collected on an Olympus imaging system. Release of LDH. Supernatants were harvested 24 h after the incubation with PDA NPs and assayed for LDH activity via a homogeneous membrane integrity assay kit. Released LDH was expressed as a percentage of the maximum release induced by the incubation of HGE cells with Triton X-100. NonACS Paragon Plus Environment

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treated medium was used as the background, and the measurements were performed on a Bio-Rad microplate reader (n=4). ATP assay. HGE cells were cultured in 96-well plates with a density of 104 per well for 24 h. PDA NPs were added into above wells. 24 h later, ATP levels were measured using a mitochondrial toxicity assay according to the manufacturer’s instructions (n=4). Hemolysis and coagulation assays. Red blood cells were isolated from serum, and diluted to 1/4 of their volumes with 0.9% NaCl solution. Diluted red blood cells (0.2 mL) were mixed with 0.9% NaCl solution (0.8 mL) as negative control, water (0.8 mL) as positive control, and 0.9% NaCl solution containing PDA NPs (0.8 mL) as experimental samples. 3 h later, the absorbance of supernatants at 541 nm was monitored on a UV-vis spectrometer (n=4). For coagulation assay, plasma (0.5 mL) was mixed with 0.9% NaCl solution containing PDA NPs (0.5 mL) for 5 min. After centrifugation, a fullyautomatic blood coagulation analyzer was used to confirm APTT and PT from the supernatants (n=4). Scavenging of HO·. HO· scavenging efficiency of PDA NPs was achieved by measuring the presence of fluorescent 2-hydroxyterephthalic acid. Solutions containing TA (0.5 mM), H2O2 (10 mM), and PDA NPs were prepared in PBS (25 mM, pH 7.4). TA as a non-fluorescent compound could capture HO· to produce 2-hydroxyterephthalic acid (Ex: 320 nm, Em: 425 nm). The mixtures were incubated for another 12 h and measured via fluorescence analysis. Scavenging of O2·-. O2·- scavenging efficiency of PDA NPs was achieved by measuring the inhibition ratio of photo-reduction of NBT. Solutions containing riboflavin (20 µM), methionine (12.5 mM), NBT (75 µM), and PDA NPs were prepared in PBS (25 mM, pH 7.4). The mixtures were illuminated upon ultraviolet radiation for 15 min. After illumination, the absorbance of mixtures were measured. Sample containing riboflavin, methionine, and NBT was defined as negative control. Sample containing riboflavin, methionine, and NBT after illumination was defined as positive control. All the experiments were carried out in dark without illumination. Inhibition percentage was calculated as the following formula: inhibition ratio = [(A0-An)/(Ap -An)]×100%. A0, An, Ap were the absorbance of the treated samples, negative control, and positive control, respectively. ACS Paragon Plus Environment

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Uptake kinetics of HGE cells towards Cu2+-modified PDA NPs. HGE cells were plated in a 6-well plate with a density of 104 per well for 12 h. Cu2+-modified PDA NPs with a concentration of 0.1 mg/mL were added into above medium with a volume of 1 mL. To confirm the uptake kinetics of HGE cells towards Cu2+-modified PDA NPs, cells were digested after the removal of medium at each expected time points, washed with 0.9% NaCl solution, harvested after centrifugation, treated with aqua regia, and quantified via inductively coupled plasma-mass spectrometry (ICP-MS) by detecting the amounts of Cu2+ (n=3). Intracellular ROS scavenging. HGE cells were plated in a 6-well plate with a density of 104 per well for 12 h. After medium was removed, cells were incubated with DMEM containing FBS (10%) and PDA NPs (0.1 mg/mL) for 6 h. Cells were washed with 0.9% NaCl solution to remove excess PDA NPs and then incubated with DMEM containing FBS (10%) and Rosup. 1 h later, cell medium was replaced with DCFH-DA, followed by incubation for 0.5 h at 37 ºC in dark. Group without any treatment was defined as control. Fluorescence microscopy images were collected on an Olympus imaging system. For the quantitative results, trysinized HGE cells were subjected to flow cytometry (n=4). For a comparison, UA was selected as another ROS scavenger in this study. Briefly, plated HGE cells were cultured in DMEM containing FBS (10%) and uric acid (0.25 mM) for 20 min, followed by the addition of Rosup. 1 h later, cell medium was replaced with DCFH-DA, followed by incubation for 0.5 h at 37 ºC in dark. Fluorescence microscopy images were collected on an Olympus imaging system. Moreover, the ROS removal capacity of PDA NPs in Hela cells was explored. Experimental detail was similar with that in the treatment of HGE cells. In vitro fluorescence imaging of ROS scavenging. HGE cells were plated in a 6-well plate with a density of 104 per well. Cells without any treatment and treated with Rosup were defined as negative control and positive control, respectively. Cells treated with PDA NPs (1 mg/mL) and positive controls treated with PDA NPs with different concentrations were defined as the experimental groups. The incubation periods of PDA NPs were defined as 6 h. Above samples were mixed with DCFH-DA, and


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fluorescence imaging was performed on a small animal imaging system with an excitation wavelength of 490 nm (n=3). Fluorescence signal intensity was quantified via Image J software. Expression of inflammatory mediators in LPS-treated HGE cells. HGE cells were plated in 6-well plates with a density of 106 per well for 24 h. After medium was removed, cells were incubated in DMEM containing FBS (10%) and LPS. PDA NPs were then added into above medium with a final concentration of 0.1 mg/mL. 6 h later, levels of TNF-α and IL-1β in supernatants were measured via an enzyme-linked immunosorbent assay (n=4). Animal administration. Kunming mice (25 g) and BALB/c nude mice (20 g) were purchased from Laboratory Animal Center of Jilin University. All animal procedures were processed according to the guidelines of the Institutional Animal Care and Use Committee. In vivo fluorescence imaging of ROS scavenging. To contrast LPS-induced periodontal disease in BALB/c nude mice, LPS (1 mg/mL, 0.02 mL) was administrated via subgingival injection every day. 3 d later, PDA NPs were injected in situ with a dosage of 0.2 mg/site. 24 h later, DCFH-DA was injected to detect the remaining ROS. Whole body fluorescence imaging was recorded on a small animal imaging system (n=3). Fluorescence signal intensity was measured quantified via Image J software. Mice without any treatment and treated with LPS were defined as negative control and positive control, respectively. In addition, Kunming mice were subgingivally administrated with LPS (1 mg/mL, 0.02 mL) for 3 d in a row (n=3). PDA NPs were then injected in situ with a single dosage of 0.2 mg/site at each expected time points. 3 d after various treatments, photographs of mouse faces were achieved. Degradation of PDA NPs with the addition of H2O2. PDA NPs with a concentration of 2 mg/mL were treated with H2O2 in dark. UV-vis absorption spectra of all the supernatants were obtained after H2O2 treatments. Meanwhile, TEM image of PDA NPs 12 h after the treatment of H2O2 (5 mM) was collected. Degradation of PDA NPs@GCF in the presence of H2O2. GCF was extracted from volunteers. Typically, sterilized GCF collection paper was gently inserted into the periodontal pocket until there was a slight resistance. 30 s later, collection paper containing GCF was removed and sealed in an eppendorf tube. PBS (100 mM, 120 µL, pH 7.4) was added and the sample was eluted for 1 h under vibration. ACS Paragon Plus Environment

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After centrifugation, supernatant containing GCF (100 µL) was collected for further usage. Freeze-dried PDA NPs (2 mg) were dispersed in above supernatant and incubated for 12 h. PDA NPs@GCF were obtained after centrifugation and re-dispersed in H2O2. TEM observation and UV-vis measurement were used to explore the physicochemical properties of PDA NPs@GCF after the treatment of H2O2. Remaining amount of PDA NPs in mouse gingivae. Cu2+-modified PDA NPs was administrated via subgingival injection (2mg/site). Tissues around the injection sites from Kunming mice were collected at each expected time points. The remaining amount of PDA NPs was quantified via ICP-MS by detecting the amount of Cu2+ (n=4). Cellular uptake of PDA NPs. Raw 264.7 macrophages were plated in a 6-well plate with a density of 104 per well for 12 h. Cu2+-modified PDA NPs were added into above medium with a volume of 1 mL. 24 h later, cells were washed with 0.9% NaCl solution and observed under an Olympus imaging system. To confirm the uptake amounts and relative uptake efficiencies of Cu2+-modified PDA NPs by the macrophages, cells were digested after the removal of medium, washed with cool 0.9% NaCl solution, harvested after centrifugation, treated with aqua regia, and quantified via ICP-MS by detecting the amounts of Cu2+ (n=3). Long-term toxicity. Kunming mice after subgingival injection of PDA NPs (2 mg/site) was defined as the test group and mice without any treatment was denoted as the control group, respectively. Mouse body weights of above two groups were recorded for 60 d (n=6). Blood was collected at each expected time points and serum was isolated. Levels of TNF-α, IFN-γ, and IL-1β were measured via an enzymelinked immunosorbent assay (n=4). At every expected periods, mice were sacrificed. Main organs were harvested, fixed in buffered formalin, embedded in paraffin, sectioned, and stained with hematoxylin and eosin. Blood from above two groups was collected and investigated via clinical route (n=6). Associated content Supporting information The supporting information is available free of charge on the ACS Publications website at DOI: . Figure S1-S10 and Table S1-S4. ACS Paragon Plus Environment

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Author information Corresponding authors *E-mail: [email protected] Orcid Jian Sun: 0000-0001-8964-6227 Xiurong Yang: 0000-0003-0021-5135 Acknowledgment Financial support was provided by National Natural Science Foundation of China (21435005, 21627808), Key Research Program of Frontier Sciences of CAS (QYZDY-SSW-SLH019), Jilin Province Science and Technology Development Plan Project (20160520156JH), and Norman Bethune Program of Jilin University (2015321). References 1. Kinane, D.; Stathopoulou, P.; Papapanou, P. Periodontal Diseases. Nat. Rev. Dis. Primers 2017, 3, 17038. 2. Gross, A.; Paskett, K.; Cheever, V.; Lipsky, M. Periodontitis: a Global Disease and the Primary Care Provider’s Role. Postgrad. Med. J. 2017, 93, 560-565. 3. Albandar, J. Aggressive and Acute Periodontal Diseases. Periodontol. 2000 2014, 65, 7-12. 4. Masi, S.; Salpea, K.; Li, K.; Parkar, M.; Nibali, L.; Donos, N.; Patel, K.; Taddei, S.; Deanfield, J.; D’Aiuto, F.; Humphries, S. Oxidative Stress, Chronic Inflammation, and Telomere Length in Patients with Periodontitis. Free Radical Bio. Med. 2011, 50, 730-735. 5. Darveau, R. Periodontitis: A Polymicrobial Disruption of Host Homeostasis. Nat. Rev. Microbiol. 2010, 8, 481-490. 6. Hajishengallis, G. Periodontitis: from Microbial Immune Subversion to Systemic Inflammation. Nat. Rev. Immunol. 2015, 15, 30-44. 7. Leresche, L.; Dworkin, S. The Role of Stress in Inflammatory Disease, Including Periodontal Disease: Review of Concepts and Current Findings. Periodontol. 2000 2002, 30, 91-103. ACS Paragon Plus Environment

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8. Slots, J. Periodontitis: Facts, Fallacies and the Future. Periodontol. 2000 2017, 75, 7-23. 9. Pihlstrom, B.; Michalowicz, B.; Johnson, N. Periodontal Diseases. Lancet 2005, 366, 1809-1820. 10. Hirschfeld, J.; White, P.; Milward, M.; Cooper, P.; Chapple, I. Modulation of Neutrophil Extracellular Trap (NET) and Reactive Oxygen Species (ROS) Release by Periodontal Bacteria. Infect. Immun. 2017, 85, IAI.00297-17. 11. Kanzaki, H.; Wada, S.; Narimiya, T.; Yamaguchi, Y.; Katsumata, Y.; Itohiya, K.; Fukaya, S.; Miyamoto, Y.; Nakamura, Y. Pathways that Regulate ROS Scavenging Enzymes, and Their Role in Defense against Tissue Destruction in Periodontitis. Front. Physiol. 2017, 8, 351. 12. Liu, C.; Mo, L.; Niu, Y.; Li, X.; Zhou, X.; Xu, X. The Role of Reactive Oxygen Species and Autophagy in Periodontitis and Their Potential Linkage. Front. Physiol. 2017, 8, 439. 13. Tomofuji, T.; Ekuni, D.; Sanbe, T.; Irie, K.; Azuma, T.; Maruyama, T.; Tamaki, N.; Murakami, J.; Kokeguchi, S.; Yamamoto, T. Effects of Vitamin C Intake on Gingival Oxidative Stress in Rat Periodontitis. Free Radical Bio. Med. 2009, 46, 163-168. 14. Jodko-Piórecka, K.; Litwinienko, G. Antioxidant Activity of Dopamine and L-DOPA in Lipid Micelles and Their Cooperation with an Analogue of α-Tocopherol. Free Radical Bio. Med. 2015, 83, 111. 15. Lian, X.; Fang, Y.; Joseph, E.; Wang, Q.; Li, J.; Banerjee, S.; Lollar, C.; Wang, X.; Zhou, H. Enzyme-MOF (Metal-Organic Framework) Composites. Chem. Soc. Rev. 2017, 46, 3386-3401. 16. Feng, D.; Liu, T.; Su, J.; Bosch, M.; Wei, Z.; Wan, W.; Yuan, D.; Chen, Y.; Wang, X.; Wang, K.; Lian, X.; Gu, Z.; Park, J.; Zou, X.; Zhou, H. Stable Metal-Organic Frameworks Containing Single-Molecule Traps for Enzyme Encapsulation. Nat. Commun. 2015, 6, 5979. 17. Liu, Y.; Du, J.; Yan, M.; Lau, M.; Hu, J.; Han, H.; Yang. O.; Liang, S.; Wei, W.; Wang, H.; Li, J.; Zhu, X.; Shi, L.; Chen, W.; Ji, C.; Lu. Y. Biomimetic Enzyme Nanocomplexes and Their Use as Antidotes and Preventive Measures for Alcohol Intoxication. Nat. Nanotechnol. 2013, 8, 187-192. 18. Lee, Y.; Kim, H.; Kang, S.; Lee. J.; Park, J.; Jon, S. Bilirubin Nanoparticles as a Nanomedicine for Anti-Inflammation Therapy. Angew. Chem. Int. Ed. 2016, 55, 7460-7463. ACS Paragon Plus Environment

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19. Alonso, C.; Rubio, L.; Touriño, S.; Martí, M.; Barba, C.; Fernández-Campos, F.; Coderch, L.; Parra, J. Antioxidative Effects and Percutaneous Absorption of Five Polyphenols. Free Radical Bio. Med. 2014, 75, 149-155. 20. Saita, M.; Kaneko, J.; Sato, T.; Takahashi, S.; Wada-Takahashi, S.; Kawamata, R.; Sakurai, T.; Lee, M.; Hamada, N.; Kimoto, K.; Nagasaki, Y. Novel Antioxidative Nanotherapeutics in a Rat Periodontitis Model: Reactive Oxygen Species Scavenging by Redox Injectable Gel Suppresses Alveolar Bone Resorption. Biomaterials 2016, 76, 292-301. 21. Cheng, Z.; Zhou, H.; Yin, J.; Yu, L. Electron Spin Resonance Estimation of Hydroxyl Radical Scavenging Capacity for Lipophilic Antioxidants. J. Agric. Food Chem. 2007, 55, 3325-3333. 22. Bhowmick, D.; Srivastava, S.; D’Silva, P.; Mugesh, G. Highly Efficient Glutathione Peroxidase and Peroxiredoxin Mimetics Protect Mammalian Cells against Oxidative Damage. Angew. Chem. Int. Ed. 2015, 54, 8449-8453. 23. Zhang, C.; Pan, T.; Salesse, C.; Zhang, D.; Miao, L.; Wang, L.; Gao, Y.; Xu, J.; Dong, Z.; Luo, Q.; Liu, J. Reversible Ca2+ Switch of an Engineered Allosteric Antioxidant Selenoenzyme. Angew. Chem. Int. Ed. 2014, 53, 13536-13539. 24. Zhang, Y.; Wang, Z.; Li, X.; Wang, L.; Yin, M.; Wang, L.; Chen, N.; Fan, C.; Song, H. Dietary Iron Oxide Nanoparticles Delay Aging and Ameliorate Neurodegeneration in Drosophila. Adv. Mater. 2016, 28, 1387-1393. 25. Ge, C.; Fang, G.; Shen, X.; Chong, Y.; Wamer, W.; Gao, X.; Chai, Z.; Chen, C.; Yin, J. Facet Energy versus Enzyme-Like Activities: the Unexpected Protection of Palladium Nanocrystals against Oxidative Damage. ACS Nano 2016, 10, 10436-10445. 26. Li, W.; Liu, Z.; Liu, C.; Guan, Y.; Ren, J.; Qu, X. Manganese Dioxide Nanozymes as Responsive Cytoprotective Shells for Individual Living Cell Encapsulation. Angew. Chem. Int. Ed. 2017, 56, 1366113665.


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27. Kim, C.; Kim, T.; Choi, I.; Soh, M.; Kim, D.; Kim, Y.; Jang, H.; Yang, H.; Kim, J.; Park, H.; Park, S.; Park, S.; Yu, T.; Yoon, B.; Lee, S.; Hyeon, T. Ceria Nanoparticles that Can Protect against Ischemic Stroke. Angew. Chem. Int. Ed. 2012, 51, 11039-11043. 28. Soh, M.; Kang, D.; Jeong, H.; Kim, D.; Kim, D.; Yang, W.; Song, C.; Baik, S.; Choi, I.; Ki, S.; Kwon, H.; Kim, T.; Kim, C.; Lee, S.; Hyeon, T. Ceria-Zirconia Nanoparticles as an Enhanced MultiAntioxidant for Sepsis Treatment. Angew. Chem. Int. Ed. 2017, 56, 11399-11403. 29. Li, Y.; He, X.; Yin, J.; Ma, Y.; Zhang, P.; Li, J.; Ding, Y.; Zhang, J.; Zhao, Y.; Chai, Z.; Zhang, Z. Acquired Superoxide-scavenging Ability of Ceria Nanoparticles. Angew. Chem. Int. Ed. 2015, 54, 18321835. 30. Gao, L.; Fan, K.; Yan, X. Iron Oxide Nanozyme: a Multifunctional Enzyme Mimetic for Biomedical Applications. Theranostics 2017, 7, 3207-3227. 31. Chen, Z.; Yin, J.; Zhou, Y.; Zhang, Y.; Song, L.; Song, M.; Hu, S.; Gu, N. Dual Enzyme-like Activities of Iron Oxide Nanoparticles and Their Implication for Diminishing Cytotoxicity. ACS Nano 2012, 6, 4001-4012. 32. Zhang, W.; Hu, S.; Yin, J.; He, W.; Lu, W.; Ma, M.; Gu, N.; Zhang, Y. Prussian Blue Nanoparticles as Multienzyme Mimetics and Reactive Oxygen Species Scavengers. J. Am. Chem. Soc. 2016, 138, 5860-5865. 33. Chong, Y.; Ge, C.; Fang, G.; Tian, X.; Ma, X.; Wen, T.; Wamer, W.; Chen, C.; Chai, Z.; Yin, J. Crossover between Anti- and Pro-oxidant Activities of Graphene Quantum Dots in the Absence or Presence of Light. ACS Nano 2016, 10, 8690-8699. 34. Huang, Y.; Liu, Z.; Liu, C.; Ju, E.; Zhang, Y.; Ren, J.; Qu, X. Self-assembly of Multi-Nanozymes to Mimic an Intracellular Antioxidant Defense System. Angew. Chem. Int. Ed. 2016, 55, 6646-6650. 35. Samuel, E.; Marcano, D.; Berka, V.; Bitner, B.; Wu, G.; Potter, A.; Fabian, R.; Pautler, R.; Kent, T., Tsai, A.; Tour, J. Highly Efficient Conversion of Superoxide to Oxygen Using Hydrophilic Carbon Clusters. P. Natl. Acad. Sci. USA 2015, 112, 2343-2348.


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36. Wei, H.; Wang, E. Nanomaterials with Enzyme-Like Characteristics (Nanozymes): Next-Generation Artificial Enzymes. Chem. Soc. Rev. 2013, 42, 6060-6093. 37. Yao, J.; Cheng, Y.; Zhou, M.; Zhao, S.; Lin, S.; Wang, X.; Wu, J.; Li, S.; Wei, H. ROS Scavenging Mn3O4 Nanozymes for In Vivo Anti-Inflammation. Chem. Sci. 2018, 9, 2927-2933. 38. Pelaz, B.; Jaber, S.; Aberasturi, D.; Wulf, V.; Aida, T.; Fuente, J.; Feldmann, J.; Gaub, H.; Josephson, L.; Kagan, C.; Kotov, N.; Liz-Marzán, L.; Mattoussi, H.; Mulvaney, P.; Murray, C.; Rogach, A.; Weiss, P.; Willner, I.; Parak, W. The State of Nanoparticle-Based Nanoscience and Biotechnology: Progress, Promises, and Challenges. ACS Nano 2012, 6, 8468-8483. 39. Wang, B.; He, X.; Zhang, Z.; Zhao, Y.; Feng, W. Metabolism of Nanomaterials in Vivo: Blood Circulation and Organ Clearance. Accounts Chem. Res. 2013, 46, 761-769. 40. Rivera-Gil, P.; Aberasturi, D.; Wulf, V.; Pelaz, B.; Pino, P.; Zhao, Y.; Fuente, J.; Larramendi, I.; Rojo, T.; Liang, X.; Parak, W. The Challenge to Relate the Physicochemical Properties of Colloidal Nanoparticles to Their Cytotoxicity. Accounts Chem. Res. 2013, 46, 743-749. 41. Liu, Y.; Ai, K.; Lu, L. Polydopamine and Its Derivative Materials: Synthesis and Promising Applications in Energy, Environmental, and Biomedical Fields. Chem. Rev. 2014, 114, 5057-5115. 42. Bettinger, C.; Bruggeman, J.; Misra, A.; Borenstein, J.; Langer, R. Biocompatibility of Biodegradable Semiconducting Melanin Films for Nerve Tissue Engineering. Biomaterials 2009, 30, 3050-3057. 43. Liu, Y.; Ai, K.; Liu, J.; Deng, M.; He, Y.; Lu, L. Dopamine-melanin Colloidal Nanospheres: an Efficient Near-Infrared Photothermal Therapeutic Agent for In Vivo Cancer Therapy. Adv. Mater. 2013, 25, 1353-1359. 44. Zhong, X.; Yang, K.; Dong, Z.; Yi, X.; Wang, Y.; Ge, C.; Zhao, Y.; Liu, Z. Polydopamine as a Biocompatible Multifunctional Nanocarrier for Combined Radioisotope Therapy and Chemotherapy of Cancer. Adv. Funct. Mater. 2015, 25, 7327-7336.


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Nanoparticles

Having

an

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Biomacromolecules 2011, 12, 625-632. 50. Liu, Y.; Ai, K.; Ji, X.; Askhatova, D.; Du, R.; Lu, L.; Shi, J. Comprehensive Insights into the MultiAntioxidative Mechanisms of Melanin Nanoparticles and Their Application to Protect Brain from Injury in Ischemic Stroke. J. Am. Chem. Soc. 2017, 139, 856-862. 51. Panzella, L.; Gentile, G.; D’Errico, G.; Vecchia, N.; Errico, M.; Napolitano, A.; Carfagna, C.; d’Ischia, M. Atypical Structural and p-Electron Features of a Melanin Polymer that Lead to Superior Free-Radical-Scavenging Properties. Angew. Chem. Int. Ed. 2013, 52, 12684-12687. 52. Lin, J.; Wang, M.; Hu, H.; Yang, X.; Wen, B.; Wang, Z.; Jacobson, O.; Song, J.; Zhang, G.; Niu, G.; Huang, P.; Chen, X. Multimodal-Imaging-Guided Cancer Phototherapy by Versatile Biomimetic Theranostics with UV and γ-Irradiation Protection. Adv. Mater. 2016, 28, 3273-3279.


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53. Zhao, H.; Zeng, Z.; Liu, L.; Chen, J.; Zhou, H.; Huang, L.; Huang, J.; Xu, H.; Xu, Y.; Chen, Z.; Wu, Y.; Guo, W.; Wang, J.; Wang, J.; Liu, Z. Polydopamine Nanoparticles for the Treatment of Acute Inflammation-Induced Injury. Nanoscale 2018, 10, 6981-6991. 54. Carro, O.; Evans, S.; Leone, C. Effect of Inflammation on the Proliferation of Human Gingival Epithelial Cells In Vitro. J. Periodontol. 1997, 68, 1070-1075. 55. Altman, L.; Baker, C.; Fleckman, P.; Luchtel, D.; Oda, D. Neutrophil-Mediated Damage to Human Gingival Epithelial Cells. J. Periodont. Res. 1992, 27, 70-79. 56. Ju, E.; Liu, Z.; Du, Y.; Tao, Y.; Ren, J.; Qu, X. Heterogeneous Assembled Nanocomplexes for Ratiometric Detection of Highly Reactive Oxygen Species In Vitro and In Vivo. ACS Nano 2014, 8, 6014-6023. 57. Liu, Z.; Liu, X.; Du, Y.; Ren, J.; Qu, X. Using Plasmonic Copper Sulfide Nanocrystals as Smart Light-Driven Sterilants. ACS Nano 2015, 9, 10335-10346. 58. Johnson, R.; Streckfus, C.; Dai, X.; Tucci, M. Protein Recovery from Several Paper Types Used to Collect Gingival Crevicular Fluid. J. Periodont. Res. 1999, 34, 283-289. 59. Curtis, M.; Griffiths, G.; Price, S.; Coulthurst, S.; Johnson, N. The Total Protein Concentration of Gingival Crevicular Fluid. J. Clin. Periodontol. 1988, 15, 628-632. 60. Zhou, J.; Liu, Z.; Li, F. Upconversion Nanophosphors for Small-Animal Imaging. Chem. Soc. Rev. 2012, 41, 1323-1349. 61. Tsoi, K.; Dai, Q.; Alman, B.; Chan, W. Are Quantum Dots Toxic ? Exploring the Discrepancy Between Cell Culture and Animal Studies. Accounts Chem. Res. 2013, 46, 662-671. 62. Sun, Y.; Feng, W.; Yang, P.; Huang, C.; Li, F. The Biosafety of Lanthanide Upconversion Nanomaterials. Chem. Soc. Rev. 2015, 44, 1509-1525.


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Figure 1. Schematic illustration of the typical synthesis of PDA NPs and their usages as efficient ROS scavengers in periodontal disease (A). SEM image (B), TEM image (C), and FT-IR spectra (D) of PDA NPs. Inset of C: photographic image of PDA NPs with a concentration of 2 mg/mL in various physiological solutions.


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Figure 2. Viability (A) and dual-stained fluorescence images (B) of HGE cells incubated with PDA NPs. Inset of A: dual-stained fluorescence image without any treatment. Scale bar equaled 100 µm. Results of LDH release (C), ATP levels (D), hemolysis (E), and blood coagulation (F) after PDA NPs-treated experiments. Inset of E: photographic image of hemolysis.


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Figure 3. Scavenging efficiencies of HO· (A) and O2·- (B) with PDA NPs. Fluorescence images of HGE cells upon various treatments (C). Scale bar equaled 100 µm. Expression of inflammatory mediators in LPS-treated HGE cells in the absence or presence of PDA NPs (D and E). ***P < 0.001.


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Figure 4. Schematic illustration of LPS-induced periodontal disease in BALB/c nude mice and relative experimental design of ROS scavenging (A). In vitro (B) and in vivo (C) fluorescence imaging of ROS removal capacity and relative quantitative information via PDA NPs by using in vivo imaging system as the test facility. Photographic images of Kunming mice 3 d after various treatments (D). ***P < 0.001.


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Figure 5. UV-vis absorption spectra (A) and relative photographic image (B) of solution containing PDA NPs after the addition of H2O2. TEM image (C) of PDA NPs 12 h after the addition of H2O2 (5 mM). Remaining amounts of Cu2+-modified PDA NPs in mouse gingivae (D). Inset of D: TEM image of Cu2+-modified PDA NPs. Uptake of Cu2+-modified PDA NPs in Raw 264.7 macrophages (E). Scale bar equaled 100 µm.


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Figure 6. Changes in mouse body weights (A), cytokine response in mice (B), serum levels of ALT, AST, and ALP in mice (C), as well as histological images of main organs post treatments (D). Mice received subgingival administration of PDA NPs with a dosage of 2 mg/site were defined as the test group and mice without any treatment were denoted as the control group. Scale bar equaled 200 µm.


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ToC


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Polydopamine Nanoparticles as Efficient ... - ACS Publications

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