Ta-NH2 NPs synthesis and characterization.
Pristine Ta NPs with high density have poor colloidal stability in common aqueous [24]. To improve the colloidal stability of Ta NPs and increase their retention in negatively charged articular cartilage extracellular matrix (ECM) [25], the surface of Ta NPs was modified with amino group. Ta-NH2 NPs were synthesized by a simple and efficient silane-coupling approach, which was widely used for modification of the surface of inorganic NPs [26]. In detail, the commercial raw Ta NPs were dispersed in ethanol and then centrifugated to separate large-sized NPs. Amino groups were coated on the surface of Ta NPs by refluxing reaction with (3-Aminopropyl) trimethoxysilane in ethanol. From scanning electron microscopy (SEM) (Fig. 2A and B) images, Ta-NH2 NPs exhibited uniform monodispersed compared with Ta NPs. Meanwhile, in consistent with previous report [24], as shown in the TEM image (supplementary Fig. 1A, the Ta NPs and Ta-NH2 NPs were observed with irregular elliptical morphology. HR-TEM elemental mapping images (supplementary Fig. 1B) indicated that Ta, O, and N elements were uniformly distributed in Ta-NH2 NPs. Whereas only elemental Ta and O emerged in Ta NPs, verifying the successful modification of amino group on Ta NPs surface. As shown in Fig. 2C, the hydrodynamic sizes of Ta NPs and Ta-NH2 NPs determined by dynamic light scattering (DLS) were 116.73 ± 0.31 and 265.10 ± 9.45 nm with a narrow PDI (0.18 ± 0.01 vs. 0.33 ± 0.05), respectively. Meanwhile, the zeta potential changed from negative (Ta NPs, -36.63 ± 1.31 mV) to positive charge (Ta-NH2 NPs, 32.27 ± 0.51 mV) (Fig. 2D), which could be explained by the modification of Ta NPs with amino group ligands spreading out in solutions. As shown in the XRD patterns (Fig. 2E), Ta-NH2 NPs maintained all the characteristic peaks of the Ta NPs at high angles of 30° − 80°, which indicated that the modification of amino group on Ta NPs surface did not decrease the crystalline purity of Ta NPs. In addition, from FT-IR spectra (Fig. 2F), the Ta-NH2 NPs appeared new characteristic bands around 3300 cm− 1 of -NH2, confirming that amino group had been successfully bonded on the surface of Ta NPs. In addition, the chemical valence of Ta2O5 and metallic Ta could be detected at 28 eV (Ta4f7/2) and 26 eV (Ta4f5/2) from the XPS spectra (Fig. 2G), which could be attributed to partial oxidation on the surface of Ta NPs and turned into a more stable form [27]. The oxygenic groups on metal surface were beneficial to the reaction of silane with surface oxygenic groups of Ta NPs [28]. Meanwhile, the chemical valence of Si and N elements also could be observed at 100 eV (Si2p) and 404 eV (N1s) from the XPS spectra (supplementary Fig. 2), respectively, further confirming the successful modification of (3-Aminopropyl) trimethoxysilane on Ta NPs surface. As shown in Fig. 2H, Ta-NH2 NPs exhibited stable dispersion in H2O for 5 days. Furthermore, no obvious hydrodynamic particle size change was observed in Ta-NH2 NPs after incubation in H2O for 5 days, indicating a good colloidal stability (Fig. 2I). Taken together, our data demonstrated that amino groups had been successfully decorated on the surface of Ta NPs with improved colloidal stability.
H2 O2 scavenging activity of Ta-NH2 NPs.
H2O2, the representative ROS [29] was selected to investigate the ROS scavenging activity of Ta-NH2 NPs in vitro. As shown in Fig. 3A, 1 mM H2O2 was reacted with different concentration of Ta-NH2 NPs at 37 ℃ for 1 h. Ta-NH2 NPs exhibited ROS scavenging activity in a concentration-dependent manner. Approximately 35% of the H2O2 was decomposed by 50 µg/mL Ta-NH2 NPs, and almost 40% H2O2 could be scavenging in the concentration 100 µg/mL with excellent pH and temperature stabilities (Fig. 3B and C). Compared with the working concentrations of reported metal nanoparticles, recently reported ultrasmall copper oxide [13] and manganese dioxide nanoparticles [30] might manifest better catalytic efficiency than Ta-NH2 NPs. However, these metal nanomaterials might be ionized by body fluid [15] to generate free metal ions, which further led to element imbalance or even metal poisoning [16, 17]. In addition, compared with inert metal such as cerium and gold nanoparticles [31], Ta-NH2 NPs showed stronger H2O2 decomposition. Compared with traditional antioxidants, the stability analysis indicated that the H2O2 scavenging of Ta-NH2 NPs was not influenced by pH or temperature [32]. It is well known that the activities of ROS scavenging in traditional biological enzymes [33] were affected by the microenvironment of joint cavity, and this drawback might affect the therapeutic outcome due to the fluctuation in physical and chemical properties of osteoarthritic joints.
The biocompatibility of Ta-NH2 NPs in vitro.
The biocompatibility of Ta-based materials has been widely validated in orthopedic implants [18]. In the present study, our in vitro biocompatibility analysis suggested that Ta-NH2 NPs also shared the same characteristics in safety. The CCK-8 assay results showed no significant cytotoxicity at test concentration after 24 h and 48 h co-culture (Fig. 3D). But slightly decreased chondrocyte viability was noticed when concentration reached 200 µg/mL. Chondrocytes after cocultured with Ta-NH2 NPs did not manifest obvious morphology change. In addition, Western blot (Fig. 3E and G) results showed that no significant changes in protein levels of COL-II, SOX9, ACAN, MMP13, RUNX2, and ADAMTS5 post 24h treatment. Furthermore, crystal violet staining, alcian blue staining, and immunofluorescent staining of COL-II and ACAN results indicated Ta-NPs treatment did not affect the deposition of cartilaginous ECM(Fig. 3I).
Ta-NH2 NPs protects viability and hyaline-like phenotype in chondrocyte under oxidative stress in vitro.
During the progression of OA, iNOS from OA-affected cartilage may contribute to the inflammation and pathogenesis of cartilage destruction [7]. Chondrocytes isolated from unhealthy OA cartilage showed over expression of iNOS mainly in the superficial zone [34]. Expression of iNOS could reflect the degree of oxidative stress [7].To investigate the inhibitory effect of Ta-NH2 NPs on iNOS and ROS production in chondrocyte under oxidative stress, we pre-treated cells with Ta-NH2 NPs (100 µg/mL) or catalase (CAT, 100 µg/mL) for 1 h. Cells were then challenged with H2O2 for 24 h (400 µM). 2′,7′-Dichlorofluorescin diacetate (DCFH-DA) staining indicated significant increase in intra-cellular ROS level in H2O2 treated group (Fig. 4A), which was inhibited by either Ta-NH2 NPs or CAT. In agreement with previous studies, our data showed significant increase in iNOS expression post H2O2 challenge. While pre-treatment with Ta-NH2 NPs, but not CAT reversed the iNOS level. In addition, live and dead staining data suggested, Ta-NH2 NPs or CAT pre-treatment successfully protected chondrocyte viability via inhibiting intra-cellular ROS production. These data indicated that although Ta-NH2 NPs and CAT showed similar protective effect under oxidative stress, different mechanism might be involved. In the aspect of phenotypic alternation, our immunofluorescence staining and Western blot results suggested that H2O2 significantly decreased the hyaline-like phenotype in chondrocytes, while increased fibrotic (COL-I) and hypertrophic (COL-X, RUNX-2, and MMP-13) markers [35, 36]. It is worth mentioning that fibrosis and hypertrophy are important pathological change in osteoarthritic cartilage [7, 8]. Previous studies have revealed that reduction of oxidative stress attenuated fibrosis and hypertrophy indexes effectively in tissue-organ level, including liver [37], kidney [38], and heart [39]. Similar to CAT, Ta-NH2 NPs inhibited the increment in protein levels of COL-I, RUNX-2, and MMP-13 (Fig. 4C), and maintained the hyaline-like phenotype (ACAN and COL-II) in H2O2 treated chondrocytes. Compared with CAT group, it is worth noticing that Ta-NH2 NPs group showed more significant increase in ACAN and COL-II, while decrease in ADAMTS-5 protein. The observation that Ta-NH2 NPs was more capable of restoring the balance between catabolism and anabolism might be attributed to the inhibitory ability of Ta-NH2 NPs in iNOS expression. Previous study suggested that NO promoted degradation of ECM by enhancing the activity of matrix metalloproteinase (MMPs) that subsequently led to joint destruction [40].
In vivo biodistribution and biocompatibility assessment of Ta-NH2 NPs.
Currently, drug delivery by intra-articular injection suffers from short retention in the joint cavity [10]. Small molecules are rapidly cleared from the joint cavity within hours via synovial vasculature, and macromolecules within days via synovial lymphatics [11]. In order to achieve stable pharmacokinetics, increased dosage or repetitive injection are often necessary [12]. In addition to biocompatibility, the ideal therapeutic antioxidant should also have high efficiency and long-term effect. In the present study, the positive charged NPs were designed to prolong the retention of Ta-NPs in articular cartilage. As shown in Fig. 5, after intra-articular injection of Cy5.5 labeled Ta-NH2 NPs (100 µg/mL) and CAT (100 µL), in vivo fluorescence imaging indicated gradually decreased concentration of both antioxidants at 1, 3, 7, 14 and 28 d. Whereas the Cy5.5 dye alone showed rapid decrease in fluorescence intensity after 1 h post injection (supplementary Fig. 5). Compared with CAT group (at 3 d), significantly longer joint retention (at 28 d) of Ta-NH2 NPs was noticed. In addition, further analysis in dissected knee joints confirmed that Cy5.5 labeled Ta-NH2 NPs, but not Cy5.5 labeled CAT (supplementary Fig. 5) was still detectable after 28 d post injection in femoral articular cartilage (Fig, 5B and C). Agreed with previous study, these data confirmed the positively charged Ta NPs we prepared was able to stay in the joint cavity, particularly in articular cartilage [41]. From the imaging of the dissected femoral condyle, sustained fluorescence over 28 d observational period outlined the shape of trochlear cartilage, indicated that the Ta-NPs after amino modification could be adsorbed in the surface of the articular cartilage.
To further explore the biocompatibility and potential organ toxicity of Ta-NH2 NPs, we analyzed the biodistribution in main organs. Liver, heart, spleen, lung, kidney and brain from rat post intra-articular injection of Cy5.5 labeled Ta-NH2 NPs were tested. No hemorrhage, atrophy, or necrosis was found in the analyzed organs. At the macroscopic level, Ta-NH2 NPs mainly accumulated in the liver, spleen and kidney. Among them, the fluorescence intensity in liver and kidney reached the peak at day 3 post injection and decayed by day 7. However, the fluorescence intensity in liver and kidney increased again at day 14. This observation suggested that Ta-NH2 NPs in the organ might be gradually metabolized, but required more than 28 days. To evaluate the organ toxicity of Ta-NH2 NPs, histological analysis was employed. H&E staining of main organs indicated no necrosis, congestion, or hemorrhage in the heart, liver, spleen, and lung at 1, 3, 7, 14 and 28 days (supplementary Fig. 6) after single dose intra-articular injection of Ta NPs. Moreover, no distinguishable inflammatory, lesion or tissue damage was observed in the glomerulus, tubules, collecting ducts, and urethra, illustrating the excellent biocompatibility of Ta-NH2 NPs. To further explore the functional changes of these organs, the hemogram and blood biochemistry analyses were performed. We selected AST and ALT, CRE and BUN to reflect the liver and renal functions, respectively. Compared with control group, our data showed no significant alternation in the size, number, and composition of blood cells, and in AST, ALT, BUN, and CRE levels.
In vivo therapeutic effect of Ta-NH2 NPs in MIA induced osteoarthritic model.
As shown in Fig. 7A, the rat osteoarthritis model was induced by sodium iodoacetate MIA injection, followed by the corresponding antioxidant injection. At 8 w post injection, compared with that in the sham operation group, the micro-CT scanning indicated obvious bone defects in the patella and femoral condyle, and alternation in subchondral bone structure in vehicle group (Fig. 7B). The subchondral bone structure was significantly improved by Ta-NH2 NPs or CAT injection (P < 0.05, Fig. 7C). And compared with CAT group, Ta-NH2 NPs showed superior therapeutic effect in patellar bone tissue restoration.
In order to verify the long-term anti-oxidative stress effect of Ta-NH2 NPs post single intra-articular injection, iNOS expression was detected in articular cartilage, subchondral bone and synovium during the entire process. The expression of iNOS in articular cartilage was significantly increased, and the expression reached the peak at the 8th week after MIA injection (Fig. 7D). Antioxidant treatment decreased the expression of iNOS, particularly in Ta-NH2 NPs treated group. In addition, Ta-NH2 NPs, but not CAT showed prolonged inhibition of iNOS expression at the 8th week after MIA injection (Fig. 7D). Cartilage is the main source of NO in OA [42], and iNOS expression is more enhanced in chondrocytes compared with synovial cells from patients with OA [42]. Furthermore, in chondrocytes isolated from osteoarthritic cartilage, the over expression of iNOS was mainly concentrated in the superficial region [34], whereas chondrocytes isolated from patients without OA did not express iNOS. These observations were in lined with our data, in which we showed iNOS expression was mainly confined to articular cartilage, especially in the superficial zone. The cartilage-targeting ability of Ta-NH2 NPs might act through direct oxidation resistance in the ECM, or reduce intracellular iNOS synthesis via phagocytosis. The iNOS expression in synovium tissue was similar to cartilage, whereas no significant difference in iNOS expression in subchondral bone was noticed.
The synovium is a thin connective tissue that attaches to the joints, its inflammation is mediated by activation of mitochondrial dysfunction [6], cytokines, and metabolites in synovial cells. The inter-communication between chondrocytes and synovial cells is thought to be beneficial to joint homeostasis [43]. Once synovitis is activated, cartilage undergoes subsequent adverse changes.In addition to the immunohistochemical staining of iNOS, we further observed synovial inflammation and angiogenesis by H&E staining. It was not difficult to notice the MIA-induced synovial inflammation (Fig. 7D). In vehicle group, obvious hyperplasia and inflammatory cell infiltration were seen in synovium at 4 weeks post intra-articular injection. In addition, vessel hyperplasia was also noticed at 8 weeks. The inflammation of the synovium was alleviated after both anti-oxidative treatments. Notably, synovitis score indicated synovial inflammation in the Ta-NH2 NPs group was significantly lower than that in the CAT group at 8 weeks post injection (Fig. 7E).
The OARSI scores were shown in Fig. 8. H&E and safranin-o-fast green staining showed that the vehicle group presented obvious typical osteoarthritic features such as surface irregularity, decreased expression of glycosaminoglycans, and cartilage defects. Compared with vehicle group, both antioxidants attenuated the OARSI score in femur, and significantly decreased the OARSI score in tibia. Agreed with our previous data, Ta-NH2 NPs exhibited excellent long-term therapeutic effect as evidence by more homogeneous glycosaminoglyc and cell arrangement (P < 0.05 when compared with CAT group).