Structural and Functional Tailoring of Melanin-Like Polydopamine Radical Scavengers

Abstract

Open AccessCCS ChemistryRESEARCH ARTICLE1 Apr 2020Structural and Functional Tailoring of Melanin-Like Polydopamine Radical Scavengers Peng Yang†, Zhipeng Gu†, Fang Zhu and Yiwen Li Peng Yang† College of Polymer Science and Engineering, State Key Laboratory of Polymer Materials Engineering, Sichuan University, Chengdu 610065 (China) †P. Yang and Z. Gu contributed equally to this work.Google Scholar More articles by this author , Zhipeng Gu† College of Polymer Science and Engineering, State Key Laboratory of Polymer Materials Engineering, Sichuan University, Chengdu 610065 (China) †P. Yang and Z. Gu contributed equally to this work.Google Scholar More articles by this author , Fang Zhu College of Polymer Science and Engineering, State Key Laboratory of Polymer Materials Engineering, Sichuan University, Chengdu 610065 (China) Google Scholar More articles by this author and Yiwen Li *Corresponding author: E-mail Address: [email protected] College of Polymer Science and Engineering, State Key Laboratory of Polymer Materials Engineering, Sichuan University, Chengdu 610065 (China) Google Scholar More articles by this author https://doi.org/10.31635/ccschem.020.201900077 SectionsSupplemental MaterialAboutAbstractPDF ToolsAdd to favoritesTrack Citations ShareFacebookTwitterLinked InEmail Melanin is a ubiquitous but poorly understood polymeric, dark brown to black pigment biomaterial found both in nature and by organic synthesis. Its structural heterogeneity and disordered covalent/noncovalent interactions make it a grand challenge to tune and improve the physical and biological properties of both melanin sources. Herein, we report a facile one-pot fabrication approach for synthetic melanins with controlled size and composition via copolymerization of dopamine and basic amino acid such as arginine in an aqueous solution. The resulting arginine-doped polydopamine melanin-like nanoparticles possess less ordered nonplanar microstructures within the particles, whereas the conventional polydopamine-based melanins contain a variety of compact π-stacked microstructures. The assembly of these distinct polymeric microstructures could lead the preceding ones for greater accessibility to free radicals, with resultant stronger free-radical scavenging effect, and thus, exhibit superior antioxidant performance toward cellular oxidative stress. This work has inspired us to continue to fabricate new synthetic melanin pigments with tunable and improved functions by rational structural tailoring. Download figure Download PowerPoint Introduction Melanin refers to a family of biopigments distributed mostly in animals, in particular, on the human body, and also, from some plant sources. The pigment possesses many intriguing physicochemical and biological functions, including photoprotection, photothermal conversion, energy dissipation, metal ion-chelation capacity, and free-radical scavenging properties.1–6 Despite its ubiquitous existence in nature, it remains part of a class of enigmatic materials. Unlike other types of biomacromolecules such as proteins or nucleic acids, the sophisticated biosynthetic pathways toward natural melanin formation, particularly, the mechanisms that govern the polymerization and the self-assembly of the disordered oligomers, have not been fully understood yet.7–13 Notably, such a poor understanding substantially limits the rational strategies for controlling and fine-tuning of the electronic, optical, and free-radical properties of melanin-inspired synthetic biopolymers. Therefore, the current resurgence of interest in this area is to explore melanin in both natural and synthetic systems to elucidate their structure-function relationships, mimic, and improve the emergent functionalities. One of the essential features of body melanin or its synthetic analogues [i.e., polydopamine (PDA) nanospheres] is the balance between its generation and the presence of reactive oxygen species (ROS), which, otherwise might lead to oxidative stress and consequently, serious disease states in the human body.14–21 Recently, a couple of studies have illustrated that melanin-like PDA nanomaterials could be employed as a class of intelligent free-radical scavengers for the treatment of a series of diseases, including reactive oxygen and nitrogen species-induced ischemic stroke, acute inflammation-induced injury, oxidative stress-induced periodontal diseases, and osteoarthritis.22–25 Thus, further tailoring and improvement of the free-radical scavenging activities of melanin and its polymeric derivatives seem nontrivial to promote their ultimate technological utilization. d’Ischia’s group26 pioneered the elucidation of structure–property relationship of 5,6-dihydroxyindole-2-carboxylic acid (DHICA)-based and 5,6-dihydroxyindole (DHI)-based melanins. They observed the former compound could exhibit a superior free-radical scavenging property since the extra carboxylate group was able to twist the inter-ring oligomeric angles and induced the formation of nonplanar microstructures with weak aggregating interactions, consequently accounting for greater accessibility of free radicals in DHICA-based melanin, compared with the DHI-based melanin-containing compact π-stacked microstructures. Although this model was successfully proposed almost 7 years ago, to the best of our knowledge, minimal stimulated work has been documented.27 Therefore, there is still an urgent need to adopt simple, versatile, and robust methods, aimed at assessing and improving the antioxidant properties of melanin as free-radical scavengers. In this paper, we sought to report our first effort toward this goal through rational structural and functional tailoring of melanin-like PDA for enhanced free-radical scavenging. We achieved this process directly by doping additive molecules into PDA microstructure networks during the self-polymerization of dopamine monomer under mild conditions. We hypothesized that the doped molecular segments might destroy the compact microstructures of dopamine oligomers within PDA,28 and further, lead to the formation of weak aggregate structures for strong accessibility of free radicals (Scheme 1). The model additive molecules used in the study were amino acids, which contained an amine group that could react readily with many occurring intermediates during the dopamine polymerization via Michael addition reaction and/or Schiff base reaction.29 We believed that this series of melanin-like amino acid-doped PDA nanoparticles could demonstrate superior antioxidant effects and improved free-radical scavenging capabilities both in vitro and in vivo, compared with conventional PDA nanoparticles. Scheme 1 | The structure–antioxidant property relationships proposed for conventional polydopamine (PDA-0) (a) and amino acid-doped PDA-1, 2, or 3 (b). Download figure Download PowerPoint Experimental Method Materials Dopamine hydrochloride (98%) was purchased from J&K Chemical Ltd (Shanghai, China). All the amino acids and 5,5-dimethyl-1-pyrroline N-oxide (DMPO) (97%) were purchased from Shanghai Aladdin Biochemical Technology Co. Ltd (China). FeCl2 (98%) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) (95%) were purchased from Shanghai Titan Scientific Co. Ltd. H2O2 (30%), ethanol, and ammonium hydroxide (28–30%) were purchased from Chengdu Kelong Chemical Reagent Co. Ltd (Sichuan, China). All reagents were freshly used as received in the studies. Amino acid copolymerization screening and testing Briefly 6.25 mg of amino acid was dissolved in 15 mL of deionized water and stirred mildly for 10 min. Then, 12.5 mg of dopamine hydrochloride was added to the solution. An optical image was taken 4 h after the start of the reaction. The products were obtained by centrifugation (26800 g for 10 min) and three times washes with water followed by repeated centrifugations. Synthesis of arginine-doped melanin-like PDA nanoparticles In a typical synthesis of melanin-like PDA nanoparticles, we dissolved 100 mg dopamine hydrochloride in 110 mL deionized water. Arginine was dissolved in 10 mL deionized water and then injected into the solution at room temperature (25 °C) with vigorous stirring. The solution turned into pale brown color gradually and subsequently changed to dark brown. After 4 h reaction time, the melanin-like PDA nanoparticles were obtained by centrifugation and washed three times with deionized water. The size control of melanin-like PDA nanoparticles was achieved by adjusting the arginine dosage. Time-dependent experiments Exactly 100 mg of dopamine hydrochloride was dissolved in 110 mL deionized water. Then 50 mg of arginine was dissolved in 10 mL deionized water and injected into the solution at room temperature (25 °C) with vigorous stirring. The melanin-like PDA products were collected at different reaction times (0.5, 2, 4, 8, 12, 24 h) after quick centrifugation and several washes with deionized water with repeated centrifugations. Chirality-dependent experiments Exactly 100 mg dopamine hydrochloride was dissolved in 110 mL deionized water. Then 50 mg of D-arginine was dissolved in 10 mL deionized water and injected into the dopamine solution at room temperature (25 °C) with vigorous stirring for 4 h. The melanin-like PDA nanoparticles were obtained by centrifugation and washed several times with deionized water, followed by repeated centrifugations to retrieve the nanoparticles. DPPH free-radical scavenging testing We used standard DPPH assay to evaluate the radical scavenging activities of the different melanin-like PDA samples. The following describes a typical procedure: A fresh DPPH/ethanol (0.1 mM) solution was used for the measurement. A certain amount of sample (∼0.25 mg) was added to the DPPH solution. The total volume of the reaction system was 12 mL. After mixing each sample with DPPH, the absorbance was measured at 517 nm over 25 min time course to evaluate the scavenging activities. Hydroxyl radical scavenging testing A spin-trapping reaction mixture was prepared consisting of the following: 100 μL of DMPO (80 mM), 50 μL of H2O2 (10 mM), 50 μL of melanin-like PDA sample solution, or 50 μL deionized water as control, and lastly, 50 μL of FeCl2 (20 mM) was added. An equal volume of each sample was transferred into a quartz capillary tube (inside diameter = 2 mm), and after 4.5 min incubation period, the electron paramagnetic resonance (EPR) was recorded. The instrument settings used for the measurement were microwave power: 15.89 mW, modulation amplitude: 1 G, and scan range: 100 G. Mouse fibroblasts and dental pulp stem cells culture NIH mouse embryonic fibroblast 3T3 cells obtained from Sun Yat-sen University, Guangdong, China, were used as the cell line for 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra-zolium bromide (MTT) assay. The cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM; Gibco-BRL, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS), maintained at 37 °C in 5% CO2 and a humidified atmosphere. Also, we isolated and collected human dental pulp stem cells (hDPSCs), as reported previously.30 Briefly, healthy human third molars were extracted from 12 adults (six males and six females of ages 18–20 years) at the Department of Oral and Maxillofacial Surgery, the Affiliated Stomatological Hospital of Sun Yat-sen University. Each participant provided informed consent, and approval was granted by the Sun Yat-sen University Research Ethics Committee. Cells from pulp tissues obtained from each participant were isolated by digestion with type I collagenase (3 mg/mL) and 4 mg/mL dispase II (Gibco-BRL) and incubated at 37 °C for 30 min and 5% CO2. After that, the cells were cultured in α-MEM (Gibco-BRL) supplemented with 10% of fetal calf serum (Gibco-BRL), 100 μg/mL streptomycin, 100 U/mL penicillin (HyClone, Logan, UT, USA), and 5 mmol/L glutamine (Gibco-BRL) and incubated at 37 °C until time of experiment. All the hDPSCs isolated from the 12 adults were mixed for all related experiments. In vitro proliferation studies of NIH 3T3 cells We performed MTT assay to evaluate the cell viability of each sample. Briefly, NIH 3T3 cells were incubated in 96-well plates at a density of 1000 cells per well for 24 h. Then the cells were treated with different melanin-like PDA samples at different concentrations for another 24 or 48 h. The corresponding cell viability was evaluated for each sample using the MTT assay. Intracellular oxidative stress of hDPSCs The level of ROS was measured by 2′,7′-dichlorofluorescin diacetate (DCFH-DA) staining. Briefly, incubated hDPSCs cells were pretreated with PDA-0 or PDA-2 (50 or 80 μg/mL) for 24 h and then treated with 100 μM H2O2 at 37 °C for 24 or 48 h. Cells were stained with DCFH-DA (10 μM) for 10 min at 37 °C in the dark. Samples were analyzed by flow cytometry and fluorescence microscopy (Carl Zeiss, Munich, Germany). Additionally, the oxidative stress indicators [glutathione (GSH) and malondialdehyde (MDA)] were measured using their respective assay kits (Beyotime, Shanghai, China), according to the manufacturer’s instructions. Intracellular antioxidant protection of hDPSCs DPSCs with a density of 1000 cells per well were seeded in 6-well plates for 24 h and pretreated with PDA-0 and PDA-2 at 50 and 80 μg/mL for another 24 h. Subsequently, the cells were treated with 100 μM hydrogen peroxide (H2O2), followed by incubation for 24 h and the determination of cells’ viability using the MTT assay. Also, the cells were evaluated employing an acridine orange/ethidium bromide (AO/EB) double-staining assay by treating the cells with AO (5 μg/mL) and EB (5 μg/mL) for 10 min. Then images were taken by confocal fluorescence microscopy (Zeiss LSM 900, Munich, Germany). In vivo cutaneous wound model The protocols for all the experiments performed were approved by the Animal Ethics Committee of Sun Yat-sen University. Fifteen healthy male Sprague-Dawley rats (obtained from Sun Yat-sen University) weighing ∼160 g were anesthetized. Then the back skin of each animal was shaved and disinfected. Full-thickness skin round wounds of 15 mm diameter were prepared by excising the dorsum of the animals, and the wounds were photographed with a ruler to measure the wound area. The animals were divided randomly into 3 groups (five rats per group) for control, PDA-0, and PDA-2 treatments in each group. The wounds were smeared with PDA-0 or PDA-2 for the different groups twice a day from the first day using the same dose, respectively. The control group was treated with PBS (vehicle). All animals were sacrificed after wound closure, and the wounds were removed from the animals for histopathological examination. Percent wound size reduction for rats in each group was estimated as follows: % = (A0 − At)/A0 × 100, where A0 is the initial wound area, At the open area of the wound on the first, fifth, ninth, and 17th day accordingly. Histological and immunohistochemical assessment Excised wound site from each group was collected and fixed with 4% paraformaldehyde for 24 h, then embedded in paraffin. The sections of 5 μm thickness were stained with Hematoxylin–Eosin (H&E) dye, Masson’s trichrome staining, and immunohistochemical (IHC) staining, to define the wound area. The IHC technique was utilized for neovascularization evaluation by treating and allowing the sections to react with primary anti-vascular endothelial growth factor antibody (anti-VEGF, [VG76e]; Novus Biologicals, Shanghai, China). All sections were analyzed using light microscopy and evaluated by two blinded experienced investigators. Results and Discussion The newly designed melanin-like PDA nanoparticles were fabricated directly via copolymerization of an amino acid and dopamine molecules in an aqueous solution (Figure 1a). Among different types of natural amino acids, our study found that only basic amino acids such as arginine and lysine could successfully induce the self-polymerization of dopamine monomer under the typical conditions (pH > 8.5; room temperature) (), during which the reaction solution gradually turned yellow and ultimately, black after 4 h reaction (Figure 1b). For example, an arginine-doped PDA nanoparticles with uniform size could be prepared by 4 h coincubation of arginine and dopamine monomers in water (Figure 1c). Note that the size of the resulting arginine-doped PDA could be rigorously controlled and finely tuned from 80 nm to 240 nm by adjusting different experimental parameters, evident by the results obtained from scanning electron microscopy (SEM). In particular, we observed that the size of the resulting arginine-doped PDA nanoparticles decreased with increasing arginine concentration using our system (Figure 1d; ), while the particle size gradually increased as the reaction time was prolonged (). These observations were consistent with the size tuning of conventional PDA nanoparticles.31 Additionally, the chirality of the arginine used did not influence the size of nanoparticles formed (). Notably, the promising stability of the prepared arginine-doped PDA nanoparticles in aqueous solution was confirmed further by the results of the zeta potential (), reaffirming further, their potential in biomedical applications. Figure 1 | (a) Synthetic scheme of amino acid-doped PDA nanoparticles. (b) Optical image of dopamine solution after 4 h incubation with different amino acid types. (c) SEM image of the resulting arginine-doped PDA. (d) The effect of arginine concentration on the particle size of resulting arginine-doped PDA nanoparticles (dopamine concentration: 0.83 mg/mL; reaction time: 4 h; room temperature). The particle size data were obtained from the SEM images. (e) Transmission electron microscopy image and relevant element mapping images of the arginine-doped PDA with an average diameter of ∼220 nm. Spectra was obtained from high-resolution X-ray photoelectron spectroscopy of C 1s (f), O 1s (g), and N 1s (h) regions for arginine-doped PDA nanoparticles. Fitted curves are labeled with the corresponding species. The red lines in the spectra represent the global envelopes corresponding to the sum of the Gaussian–Lorentzian peaks used to fit the spectra. Download figure Download PowerPoint Then we performed a chemical structure analysis of arginine-doped PDA nanoparticles and detailed investigation of their polymerization mechanism. We found that the existence of C, N, and O elements within arginine-doped PDA nanoparticles (noted in Figure 1c) was supported fully by the electron energy-loss spectroscopy (EELS) mapping analysis (Figure 1e), with these results matching well with the observation made from an X-ray photoelectron spectroscopy (XPS) survey data (Figure 1f–h; ). The C 1s regions for the testing nanoparticles contained four peaks: C−C (284.1 eV), C−O/C−N (286.5 eV), and C=O/C=N (288.5 eV) species from PDA structure,32–34 and −C−H/−C−C (283.5 eV) species from arginine segment (Figure 1f).35 Additionally, the N 1s regions for the sample committed into two contribution peaks, corresponding to =N−R (398.6 eV) and aromatic N (399.5 eV) species (Figure 1g).36,37 Also, the O 1s regions could be assigned into two peaks, attributed to C=O (531.0 eV) and O–H (532.1 eV) species (Figure 1h).38,39 Collectively, these pieces of evidence suggested the successful doping of the arginine unit into the molecular structure of PDA within the final arginine-doped PDA product. Moreover, this conclusion was confirmed by analyzing the polymerization mechanism further during the particles formation process by electrospray ionization mass spectrometry (ESI–MS) ().40 For example, several characteristic mass-to-charge ratio (m/z) peaks were apparent under typical experimental conditions for arginine-doped PDA at the 20 min reaction time point, as shown by the possible corresponding chemical structures of the intermediates proposed in . Our analytical results suggested that different types of intermediates conjugated between the arginine and dopamine monomer/oligomers, again demonstrating the successful doping nature of the arginine molecules within the PDA microstructures. Subsequently, we investigated carefully the physical properties and free-radical scavenging activities of our newly designed melanin-like PDA nanoparticles. We eliminated the particle size effect by synthesizing a specific series of similar-sized (∼130 nm) arginine-doped PDA nanoparticles (PDA-1, PDA-2, and PDA-3) with different doping content (from 0.21% to 8.69%) (Figure 2a, ), and PDA-0, the conventional PDA nanoparticle, used as a control sample (). Note that the degree of arginine doping within the PDA was determined by measuring the nitrogen content in the testing sample, which could be measured directly by organic elemental analysis. All of our arginine-doped samples exhibited similar characteristics as the conventional PDA-0, as revealed by Fourier-transform infrared spectroscopy (FTIR; ), indicating that the main chemical composition of these fabricated samples was still intact. Further, we speculated that the doped arginine might hinder the formation of compact inter-unit π-conjugation aggregate structures in the conventional PDA nanoparticles (PDA-0), which could increase the free-radical concentration within the particles, thereby, accounting for stronger accessibility of free radicals. This hypothesis was supported clearly by the density measurements of the samples (Figure 2a). For instance, the density of PDA-0 was 1.58 ± 0.10 g/cm3, while that of PDA-1 (only at 0.21% arginine doping) was a little bit lower (1.47 ± 0.02 g/cm3), probably owing to the presence of less compact structures within the particles. Meanwhile, the particle density continued to decrease with increasing arginine doping content up to 3.22% (e.g., the density of PDA-2 was 1.29 ± 0.02 g/cm3). However, as the content of the doped arginine was increased to 8.69%, the trend reverted inducing the density of PDA-3 to increase to 1.35 ± 0.01 g/cm3, which might have been contributed by the high doping content of the arginine molecules of the PDA, resulting in high density (∼1.46 g/cm3). Recently, a cation–π interaction effects were observed in the classic PDA system,41,42 in which it was demonstrated that the synergistic relationship between aromatic and cationic amino acids could ultimately enhance underwater adhesion by cation–π interactions, which could dissociate by pH increase. Here, we also performed simple testing to rapidly check a plausible existence of cation–π interaction effect within our system by dissolving 2 mg PDA-0 and PDA-1 in 4 mL NaOH solution at varying OH− concentrations. After 2 h incubation, we measured the absorption from 200 to 800 nm by UV–vis spectrometry of the filtered solution to determine the degree of dissociation of the testing samples. We observed that the PDA-1 showed less degree of dissociation, compared with the PDA-0 control (), suggesting an enhancement of cation–π interaction effects via the arginine doping within our fabricated PDA system. Then we employed EPR to evaluate the free-radical concentration within melanin-like PDA nanoparticles to further verify our hypothesis (Figure 2b). The EPR spectra of our entire PDA samples showed strong signals at ∼3517 G, where the value of g-factor is ∼2.0037 (Figure 2a). Interestingly, all the arginine-doped PDA nanoparticles displayed stronger signal intensity or higher spin values than the conventional PDA-0, as shown in Figure 2a, indicating that, indeed, there was a higher free-radical concentration within the arginine-doped samples. In particular, PDA-2 showed the strongest signal in the EPR spectra among all samples. Notably, the radical concentration within the arginine-doped PDA nanoparticles would increase first, and then decrease with a gradual increase of arginine doping content from 0% to 8.69%, based on the result from Figure 2b. Accordingly, we assumed that at low doping content, the incorporated arginine might destroy the formation of compact π-conjugation aggregates in PDA, further leading to the decreased density and increased radical concentration, while at higher arginine doping content the total radical concentration could be reduced since the number of decreased catechol group on the particle surface became a primary issue by causing an inhibition of arginine from generating or scavenging free radicals. Such observation is consistent with our observations made in the typical hydroxyl free-radical (•OH) scavenging activity results of the DPPH experiments. In the first test, we found that the intensity of all signal peaks of the hydroxy radical DMPO–OH significantly decreased after adding the PDA nanoparticles (Figure 2c), indicating that all melanin-like PDA nanoparticles demonstrated promising •OH scavenging capacities. Notably, PDA-2 showed the best hydroxyl radicals scavenging property among all the testing samples based on the observation in Figure 2b. For the DPPH testing, PDA-2 also exhibited a stronger radical scavenging effect at the same dose, compared with PDA-0, as visualized by the faster color fading of the DPPH solution from purple to yellow (Figure 2d). Further quantitative analysis showed that all the PDA samples exhibited a dose- and time-dependent free-radical scavenging properties ().43 Intriguingly, we also revealed that the radical scavenging capacity of arginine-doped PDA nanoparticles increased initially, and then decreased with increasing dose of the arginine doping. Again, the PDA-2 demonstrated the highest scavenging activity among the samples (Figure 2e). Thus, we employed these samples in further biological investigations of their antioxidant properties, both in vitro and in vivo. Figure 2 | (a) Various physical parameters of PDA-i (i = 0–3). (b) EPR spectra of PDA-i (i = 0–3). (c) EPR spectra of DMPO/•OH after incubation with PDA-i (i = 0–3). (d) Optical image of PDA derivatives and/or stable free-radical molecules in ethanol solution. (I) PDA-0, (II) PDA-2, (III) DPPH, (IV) DPPH+PDA-0, and (V) DPPH+PDA-2. DPPH solution (0.1 mM) was incubated with different PDA samples (0.021 mg/mL) for 5 min. (e) DPPH radical scavenging activities of PDA-i (i = 0–3). DPPH solution (0.1 mM) was incubated with different PDA samples (0.021 mg/mL) over a time course of 25 min as a function of scavenging activity. Download figure Download PowerPoint Next, we investigated the antioxidant performances of the selected melanin-like PDA nanoparticles (PDA-2) and the conventional PDA-0 by studying the protection of cells against oxidative stress. Prior to that, MTT proliferation assay (30–100 μg/mL particle concentration, 24 h and 48 h incubation) was carried out to demonstrate the promising biocompatibility of the PDA-2 melanin-inspired biopolymer, and the conventional PDA-0 (). To mimic ROS scavenging properties of the melanin-like PDA nanoparticles in a pathological condition, hDPSCs were exposed to hydrogen peroxide (H2O2) at 100 μM and then treated with PDA-0 or PDA-2. As expected, the ROS level of H2O2-treated hDPSCs was highly increased within 2 days, while its level diminished distinctly by cotreatment with the melanin-like PDA nanoparticles (Figure 3a and b). Notably, PDA-2 demonstrated better antioxidant effect than PDA-0 at a wide range of particle concentrations (). Furthermore, both AO/EB double-staining assay and MTT assay results (Figure 3c and d) suggested that the cells treated by H2O2 decreased in viability by up to ∼30% in 2 days, and also, lost the ability to proliferate, while both of the melanin-like PDA samples protected the cells well from oxidative damage. In particular, the cell viability increased up to ∼85% and ∼70% at 80 μg/mL and 50 μg/mL of PDA-2, respectively, and maintained the capacity to proliferate (). To further understand the antioxidant behaviors of melanin-like PDA nanoparticles, both the production of lipid oxidation and depletion of GSH in the cytoplasm of hDPSCs were quantitatively analyzed. As shown in Figure 3d and 3e, the level of MDA generated from the lipid oxidation reaction as a result of excessive ROS,44 could be suppressed by pretreatment with the melanin-like PDA nanoparticles, whereas, the c