3.1. Morphology and Structure Characterizations of Cu-EGCG Nanosheets
The Cu-EGCG nanosheets were designed and synthesized by the oxidative coupling assembly of polyphenol with strong chelating ability (EGCG) and Cu2+ (Scheme 1), and the copper ions coordinated with EGCG self-assembled into metal-phenolic nanocomplexes. TEM and SEM were conducted to observe the nanosheets morphology of Cu-EGCG (Fig. 1A-B). TEM and SEM analysis revealed that the NPs generated were ultrathin and flaky. DLS analysis was further characterized the diameter of nanosheets, and the result showed that nanosheets had a diameter of 218 nm (Fig. 1C), which was consistent with TEM and SEM results. EDS element energy spectrum display that there were C, N, O and Cu elements in Cu-EGCG nanosheets (Fig. 1D). In addition, TEM-mapping analysis also show that C, N, O and Cu elements were distributed on Cu-EGCG nanosheets. The XRD analysis of Cu-EGCG nanosheets showed that a unique crystalline structure was observed in 2θ = 30o ~ 40o, different with other Cu-based materials like Cu, Cu2O, CuO, and Cu (OH)2 (Fig. 1E), possibly due to the coordination of EGCG changed the crystal structure of Cu2+. The coordination between Cu2+ and EGCG was further detected by XPS spectra (Fig. 1F-G) and Fourier transform infrared spectra (FTIR) (Fig. 1H). The spectra of Cu 2p showed the main peaks of Cu 2p3/2 at 932.5 eV and Cu 2p1/2 at 952.6 eV, with the shake-up satellite peaks at 943.25 eV and 962.5 eV respectively (Fig. 1G). The successful synthesis of Cu2+ and EGCG was confirmed by FTIR. Compared with single EGCG, the phenolic hydroxyl stretching vibration peak at 3360 cm− 1 shifted to a relative high wavenumber after the EGCG coordination with copper ions, possibly attributed to the partial disruption of hydrogen bonds in EGCG (Fig. 1H)[25, 32]. In addition, the deformation vibration peak at 1450 cm− 1 and the C-O-H stretching vibration peak at 1150 cm− 1 of the phenolic hydroxyl in Cu-EGCG became weaker than the EGCG without coordinating, which indicated that copper ions were coupled to EGCG by the phenolic hydroxyl [33–35]. Moreover, the thermal stability of EGCG also greatly improved after the coordination between EGCG and Cu2+ (Fig. 1I), which was vital to the complicated vivo environment.
The sustained release behavior of Cu2+ was important for the utilization of copper ions[28, 31, 36]. We detected the release profile of copper ions, with the in vitro release behavior presented in Fig. 1J. Whether Cu-EGCG nanosheets was incubated in PBS, copper ions were continuously released within 60 h. About 75% copper ions was released among the 10 h, and the same amount was continuously released for the following time. Moreover, by combining the copper release and CCK-8 assay results (Fig. 3A), the toxicity of copper ions is reduced due to the sustained release of copper ions.
3.2. Multienzyme-like antioxidative activity of Cu-EGCG Nanosheets.
During the process of inflammatory response, the ROS, including H2O2, hydroxyl radicals (•OH) and super-oxide anions (O2• –) are the majority injury factors[37–39]. Therefore, it is critical to eliminate excess ROS for protecting tissues from the inflammatory injury effectively. The H2O2, •OH, and O2• – were used to estimate the ROS scavenging ability of Cu-EGCG nanosheets in vitro. The DPPH result showed that Cu-EGCG could significantly scavenge DPPH free radicals and was dose-dependent, which showed that Cu-EGCG had a good total antioxidant capacity. And the free radical scavenging ability was 60% with 100 µg/mL Cu-EGCG was (Fig. 2A). The catalase-like activity of Cu-EGCG was confirmed. As shown in Fig. 2B, the O2 generated from the decomposition of H2O2 was observed in Cu-EGCG treatment group, while the EGCG group showed no generation of O2. Furthermore, the dynamically generated O2 was found by dissolved oxygen meter, The decomposition of H2O2 into H2O and O2 was dependent on the concentration of both substrate and catalyst. Compared with the blank control, the oxygen production of Cu-EGCG group was greatly enhanced, and high concentration of Cu-EGCG could decompose hydrogen peroxide rapidly (Fig. 2C). When the concentration of Cu-EGCG was 100 µg/mL and 200 µg/mL, the resulting oxygen concentration is up to 24 mg/L after 10 min treatment. However, when its concentration is 12.5 µg/mL, the oxygen production concentration is only 9 mg/L. These results indicate that Cu-EGCG has strong decomposition ability of hydrogen peroxide, which is consistent with the observed results. When Cu-EGCG is used to treat different concentrations of H2O2, the higher the concentration of H2O2, the more oxygen is produced (Fig. 2D), indicating that Cu-EGCG could effectively remove H2O2, and the decomposition rates increased in a dose-dependent manner of both Cu-EGCG nanosheets (Fig. 2C) and H2O2 (Fig. 2D). These results revealed the intrinsic catalase-like activity of Cu-EGCG nanosheets. We further investigated the ROS scavenging ability of Cu-EGCG nanosheets by monitoring individual group reactions. As shown in Fig. 2E, the •OH after reacting with different concentrations of EGCG and Cu-EGCG nanosheets with 1 h, both EGCG and Cu-EGCG nanosheets exhibited •OH scavenging capacity, while the •OH elimination efficiency of Cu-EGCG nanosheets were better than EGCG under the condition of 80 µg/mL, demonstrating its high •OH scavenging capacity. Figures 2F-2I exhibit the •OH and O2•– scavenging capacity by using the electron paramagnetic resonance (EPR). With the increasing of Cu-EGCG concentrations, the ESR signals from the BMPO/•OH and DEPMPO/•OOH spin adducts decrease obviously, indicating the increasing scavenging efficiency toward •OH and O2•– (Fig. 2G&2I). Together, these results revealed that Cu-EGCG nanosheets was efficient in ROS scavenging ability.
Figure 2. ROS scavenging activities of Cu-EGCG nanosheets. (A) The DPPH scavenging capabilities with different concentrations of Cu-EGCG nanosheets. (B) bubbles were generated after adding Cu-EGCG nanosheets into the H2O2 solution. (C) Catalase-like activity of Cu-EGCG nanosheets. Increased decomposition rates of 10× 10− 3 M H2O2 into H2O and O2 with increased concentration of Cu-EGCG nanosheets. (D) Increased decomposition rates of H2O2 into H2O and O2 in the presence of 80 µg/mL Cu-EGCG nanosheets with increased concentration of H2O2. (E) EGCG and Cu-EGCG nanosheets concentration dependent •OH elimination rates using SA indicator. (F) EPR spectra of BMPO/•OH in the presence EGCG and Cu-EGCG nanosheets after reacted with •OH, respectively. (G) EPR spectra represents •OH scavenging activities with the different concentration of Cu-EGCG nanosheets. (H) EPR spectra of DEPMPO/•OOH in the presence EGCG and Cu-EGCG nanosheets after reacted with O2• –, respectively. (I) EPR spectra represents O2• – with the different concentration of Cu-EGCG nanosheets.
3.3. Cytotoxicity of NPs.
As previously reported, the toxicity of copper ions could be decreased by the sustained release[40]. In this study, the Cu-EGCG nanosheets showed the sustained release of Cu2+ within 60 h with or without H2O2 solution. The cytotoxicity of the copper release, EGCG and Cu-EGCG nanosheets on Raw264.7 macrophages was estimated by CCK-8. As shown in Fig. 3A, the EGCG and Cu-EGCG nanosheets exhibited negligible cytotoxicity to Raw264.7 at concentrations below 100 µg/mL, indicating Cu-EGCG nanosheets with good biocompatibility. In contrast, the copper release compound, CuCl2, showed notable cytotoxicity to Raw264.7 with Cu-EGCG at the same concentration of 100 µg/mL. These results confirmed that by forming Cu-EGCG nanosheets, the cytotoxicity of copper ions can decrease. In addition, nanosheets were utilized at 80 µg/mL for the further experiments. We also used Calcein AM/PI to estimate cells activity. As shown in Fig. 3B, there was little red fluorescence representing dead cells in the groups treated with EGCG and Cu-EGCG, and the Cu-EGCG group had fewer dead cells than the EGGG-treated group compared to group M1, which showed results consistent with those in Fig. 3C. These results indicate that Cu-EGCG nanosheets has good biocompatibility.
3.4. Antioxidant and anti-inflammatory activities of Cu-EGCG nanosheets
Previous studies have confirmed that oxidative stress induced by ROS is involved in the process of human aging diseases, such as OA, which may induce chondrocyte apoptosis and cartilage degeneration[41, 42]. Therefore, maintaining the balance of oxidative and anti-oxidative effects in vivo and removing excess ROS is considered a feasible solution for the treatment of OA[43]. The intracellular ROS scavenging ability of Cu-EGCG nanosheets was estimated by using LPS-induced RAW264.7 macrophages with the DCFH-DA probe [44]. LPS can induce an inflammatory response in RAW264.7 macrophages. Shown in Fig. 4A, LPS-stimulated RAW264.7 macrophages (M1 macrophages) showed an intense DCF fluorescence intensity compared to untreated cells. And the fluorescence signal was significantly reduced by the addition of free drug EGCG and Cu-EGCG nanosheets, suggesting the intracellular ROS regulatory ability of EGCG and Cu-EGCG nanosheets. However, the inhibitory effect on intracellular ROS generation in Cu-EGCG nanosheets treating group was better than free drug EGCG due to the short retention time of small molecule drugs, as shown in Fig. 4B. The fluorescence increased ≈ 16.36-fold after LPS-induce ROS production in RAW264.7 macrophages, which analyzed by FACS (Fig. 4C). Both free drug EGCG and Cu-EGCG nanosheets could scavenge the intracellular ROS, while Cu-EGCG nanosheets exhibits the strongest effect. All these results were consistent with the fluorescent microscopy.
The M1 macrophages are primarily phenotype that secreting various inflammatory cytokines promotes OA progression[5, 45]. The expression levels of pro-inflammatory cytokines including TNF-α, iNOS, IL-1β and IL-6 were detected by using qRT-PCR to investigate the anti-inflammatory capability of Cu-EGCG nanosheets on LPS-induced RAW264.7 cells. In Fig. 5A, the levels of TNF-α, iNOS, IL-1β and IL-6 secreted by RAW264.7 cells obviously increased after LPS induce. Nevertheless, Cu-EGCG dramatically suppressed those expression compared with free drug EGCG, indicating the potent anti-inflammatory activity of Cu-EGCG nanosheets. Immunofluorescence staining was also conducted to evaluate the protein expressions of iNOS secreting by M1 macrophages. The fluorescence images by fluorescent microscopy in Fig. 5B&5D shows that the level of iNOS was significantly upregulated in the LPS-activated RAW264.7 macrophages, suggesting the M1 polarization. After Cu-EGCG nanosheets treatment, iNOS levels decreased remarkably than EGCG. In addition, CD206 as a surface marker of M2-type macrophages[46], M2 macrophages participate in inflammation resolution, wound healing, tumor growth, produce anti-inflammatory mediators and growth factors in vitro [47–49]. As shown in Fig. 5C&5E, the fluorescence images indicates that the level of CD206 increases obviously after EGCG and Cu-EGCG treatment, while the Cu-EGCG group showed stronger fluorescence intensification than the group treated with EGCG, all these suggest the LPS-induced RAW264.7 macrophages successfully repolarize from M1 to M2.
3.5. NPs Treatment Inhibits Chondrocyte inflammation.
To further investigate the influence of RAW264.7 in cartilage inflammation-associated cells, we cultured chondrocyte with CM. The effects of Cu-EGCG nanosheets on chondrocytes were firstly determined. As shown in Fig. 6A, when Cu-EGCG concentration was 80 µg/ml, the cell viability of chondrocytes was about 80%. This result indicates that the concentration of Cu-EGCG nanosheets treated macrophages had little effect on the viability of chondrocytes. To estimate the influence of Cu-EGCG nanosheets on the OA catabolic markers or cartilage-related genes (such as IL-6, IL-1β, TNF-α, MMP-13, MMP-3, ACAN and Col2a1) expression levels, chondrocytes pretreated with 10 µg/ml LPS were co-cultured with different concentrations of Cu-EGCG nanosheets for 24 hours, where the chondrocytes without treatment were used as a blank control group. As shown in Fig. 6B, the inflammatory markers expression was remarkably elevated after LPS treatment. As a comparison, Cu-EGCG nanosheets result in significantly decreasing regulation of all tested mRNA.
ROS levels in chondrocytes treated with various CM were detected by DCFH-DA staining (Fig. 7A). The fluorescence was stronger in M1-CM treated cells than RAW-CM cells, suggesting excessive ROS production. After M1 + EGCG-CM or M1 + Cu-EGCG-CM treatment, significant reduction in fluorescence signal due to efficient ROS scavenging. Relative quantitative measurements showed that total ROS level could be upregulated to 4.68-fold after M1-CM stimulation, but treatment with Cu-EGCG-CM eliminated excess ROS from the cells to a healthy level (Fig. 7B). These results showed that Cu-EGCG nanosheets can inhibit macrophage CM-induced chondrocyte inflammation. At molecular level, we investigated the relative expression of cartilage related mRNAs (COL2A1 and ACAN) and catabolic markers of OA (IL-1β, IL-6, MMP-13 and MMP-3) (Fig. 7C). Compared with the RAW-CM group, the expression levels of IL-1β, IL-6, MMP-13 and MMP-3 were notably increased by 26.24-fold, 24.14-fold, 3.4-fold, and 2.65- fold respectively in CM cultured chondrocyte from the M1 supernatant group. And the expression of COL2A1 and ACAN was decreased obviously. All these inflammatory factors all decreased were decrease after treatment with EGCG-CM or Cu-EGCG-CM, while Cu-EGCG-CM showed better performance in reducing the inflammatory factor upregulation and improving the expression of cartilage related maker. Secretions of MMP-13 and IL-6 plays a crucial role in OA. Immunofluorescence staining indicated that high green signal of MMP-13 and IL-6 was observed in the M1-CM group (Fig. 7D-7F). The expression of MMP-13 and IL-6 was reduced in both experimental groups, with the optimal effect of Cu-EGCG-CM. Both the results of qRT-PCR and immunofluorescence staining showed that anti-inflammatory cytokines secreted by RAW264.7 activated by Cu-EGCG with better protective effects on chondrocytes than the other two groups.