Synthesis and characterization of CSL@HMSNs-Cs
The preparation protocol for CSL@HMSNs-CS is shown in Scheme 1. Solid silica nanoparticles (sSiO2) were first synthesized as a core via a modified Stöber method(19) and a mesoporous silica shell structure was coated with TEOS using the surfactant CTAB as a stabilizer. The core was selectively etched out by Na2CO3 aqueous solution at a temperature of 80℃ to form a hollow structure. The CTAB was removed by hydrochloric acid-ethanol solution and the HMSNs were collected. The diameter of the hollow core depended on the size of sSiO2, which depended on the ratio of reagents and reaction time of the sSiO2 synthesis process(19). Then CSL was loaded in the HMSNs by free diffusion. Finally, GPTMS, which contains both epoxide ring reactive to amino groups of chitosan and also hydrolyzable methoxysilyl groups, reactive to hydroxyl groups of HMSNs, was chosen as a crosslinking agent to anchor Cs onto HMSNs.
The morphologies of HMSNs and CSL@HMSNs-CS were observed by transmission electron microscopy (TEM). In Figure 1, the images show a solid sphere (Figure 1a) and a homogeneous spherical shape (size: ~260 nm) with uniform large hollow cavities(size: ~170 nm) (Figure 1b). After loaded with CSL and modification of Cs, the shell was thicker and obscured and CSL was observed in the cavity (Figure 1c).
The dynamic light scattering (DLS) particle size distribution results in Figure 2a, b showed a slight increase after coating with Cs(average 260.76 nm to 290.17 nm).
The N2 adsorption–desorption isotherm curves further manifest a classical Langmuir type IV isotherm with a type H2 hysteresis loop, indicating the existence of mesoporous structures of HMSNs (Figure 2c). The mesopores structure was obscured after surface modification and showed a significant pore-coating effect by Cs.
The BET pore diameter of the HMSNs was determined to be approximately 2.4 nm (Figure 2d) by nitrogen adsorption investigation, while the pore volume(Vpore) and specific surface area(SBET) were 0.7668 cm3/g and 1006.8 m2/g, respectively. The Vpore and SBET of CSL@HMSN were drastically decreased to 0.2476 cm3/g and 235.13 m2/g after CSL-loading. After modification with Cs, the Vpore and SBET of CSL@HMSNs-CS were further decreased to 0.1205 cm3/g and 52.816 m2/g. All these changes were results of the obstruction of pore with Cs.
The zeta potentials of the HMSNs were measured to be -32.8±1.3 mV, suggesting that HMSNs has great dispersity. The zeta potentials of CSL@HMSNs were -9.5±0.7 mV while the CSL@HMSNs-CS had positively charged with zeta potentials of 19.9±0.7 mV possibly due to the positively charged Cs modification.
Low-angle X-ray diffraction (XRD) patterns measurement showed that HMSNs exhibits a diffraction peak at 2.1° (2θ) and a weak, broad shoulder peak at 3.9°(Figure 3a), indicating the existence of wormhole mesostructures(20). CSL@HMSNs exhibit a same diffraction peak but lower intensity. CSL@HMSNs-CS exhibit no diffraction peak due to the surface modification of Cs.
Fourier transform infrared (FT-IR) spectra(Figure 3b) showed four characteristic absorption peaks of HMSNs at the peaks at 1226 cm-1 and 1050 cm-1 corresponding to the asymmetric stretching vibrations of Si-O-Si; 793 cm-1 and 958 cm-1 corresponding to the symmetric stretching vibration of Si-O-Si and the stretching vibration of Si-OH, respectively. As a result of loading CSL in the cavity, a new peak was detected at 1702 cm-1 corresponding to the vibrations of C=O. The characteristic absorption peak of Cs at 1650 and 1380 cm-1 indicated the successful coating of the CS layer(18).
All of the above results indicate that the CSL was successfully capped in the cavity with Cs. Functional modification could provide protection for the loaded drug until the nanoparticles were delivered to the target.
To evaluate the time stability, CHC was tested with Fourier transform infrared (FT-IR) spectra three months after the nanocomplex was synthesized. The result showed that the main functional group, including Si-O-Si, Si-OH, and characteristic absorption peak of Cs and CSL were not changed during the time (Supplement, Figure S2).
The temperature stability of CHC was measured by Thermogravimetric Analysis (TGA) and differential scanning calorimetry (DSC) (Supplement, Figure S3). The TGA curve showed two decompositions stage of CHC. The first decomposition was observed within 50-150 °C, with about 10 % weight loss by the evaporation of water. The second decomposition appeared within 200-450 °C with about 25 % weight loss by decomposition of organic ingredients including CSL and Cs.
The DSC curve of CHC was obtained at a heating rate of 30℃/min ranged from room temperature to 800℃ under the air atmosphere. No characteristic melting peak of CSL was found in the curve, indicating the CSL in the cavity was in an amorphous state.
Evaluation of CSL loading capacity, solubility and pH-responsive release in different buffer solutions (pH=6.0, 6.8, 7.7)
Hollow spheres could load large amounts of drugs in the cavity. The difference between the amount of CSL initially employed and the content in the supernatant after stirring was defined as the loading content. The loading content of CSL in CSL@HMSNs was determined to be 28.2% although due to inevitable leakage during the process of surface modification, the content decreased to 24.3% in CSL@HMSNs-CS. The pores of HMSNs are much larger than the size of the drug molecules and provide sufficient space for drug free diffusion of the drug into and out of the carrier. The result of CSL solubility (Figure 4a) showed that crystalline CSL had poor solubility of approximately 12.96% in pH=7.4 PBS. Loading CSL with HMSNs could largely improve the solubility of CSL to 73.99%.
Approximately 40% or more of new chemical entities are poorly soluble in water, hindering their clinical application(21). Celastrol is a red acicular crystal with poor water solubility. Disruption of the highly ordered crystalline structure is the rate determining step, which requires high energy(22). Improvements in drug dissolution can be obtained via the conversion of poorly dissolved crystalline drugs from the crystalline phase to the amorphous form when drugs are loaded into the HMSNs(23).
Targeted controlled release property has been considered a significant characteristic of an expected carrier. The pH-responsive release pattern was conducted in three different pH buffer solutions(pH=6.0, 6.8, 7.7) simulating physiological (pH=7.7) and different degrees of osteoarthritis conditions (pH=6.0, 6.8). In noncoated CSL@HMSNs, a rapid burst release was observed from 0 to 30 min, and then the curve reaches a plateau state (Figure 4a). Approximately 70% of CSL was released within 60 min from CSL@HMSNs with no significant difference at pH 6.0, 6.8 or 7.7(71.2%, 73.7%, 73.9%). In contrast, in CSL@HMSNs-CS burst release was not found after stirring for 150 min at pH 7.7 and only 21.7% of CSL was released (Figure 4c), indicating the great stability of nanoparticles in physiological environments. The cumulative release amount of CSL from CSL@HMSNs-CS was higher at pH 6.0 (68.9%) than at pH 6.8 (63.5%) and pH 7.7 and the cumulative release curve in acidic environment was similar to that of uncoated nanoparticles.
Degradation of the extracellular matrix (ECM) is a key step in the pathological process of OA. During the process, the pH of the synovial fluid of osteoarthritic joints is acidic because of the inflammatory reaction. The insufficient oxygen supply and increased metabolic activity switch toward metabolism towards glycolysis, leading to the accumulation of lactate(24). The presence of NH2 groups on chains of CS provides the possibility for functional modification to HMSNs. Restricted to alkaline environments, the Cs forms a gel-like structure that remains insoluble, forming a protection layer to reduce drug leakage. Osteoarthritis can provide acidic environments to protonate free NH2 groups and improve the water solubility of Cs. The CS layer swells, leading to the exposure of pore entrances and the release of drugs was triggered(18). Medical Cs are intra-articular injected for clinical application, given their protective action on osteoarthritis by preventing type II collagen degradation(25). In the knee joint, Cs could be degraded by lysozymes, which physiologically exist in human cartilage and the main degradation product is glucosamine, preventing type II collagen from degradation clinically(26, 27). The pH-responsive property and high biodegradability of Cs make it an ideal gatekeeper for knee intra-articular injection. There were no pathological changes in the present study compared with HMSNs with HMSNs-Cs, which may be because the concentration of Cs was not high enough for clinical treatment.
In vitro cell viability and cytotoxicity assay
The nanoparticle cell viability assay showed no significant cytotoxicity at relatively low concentration (50, 100, 200 μg/mL) of HMSNs and HMSN-Cs after incubation for 3 h and 24 h. There was no significance among each groups (Supplement, Figure S4). After 24 h incubation, when the concentration of HMSNs and HMSN-Cs increased to 400 μg/mL, obvious cell cytotoxicity was observed (Figure 5a). These results suggested that HMSNs and HMSNs-Cs showed great biocompatibility. In Figure 5b, the administration of CSL@HMSNs-Cs increases the cell viability of chondrocytes after stimulation by IL -1β in pH 6.0 culture medium compared with pH 6.8 and pH 7.7, indicating the pH-responsive treatment of CSL@HMSNs-CS. In figure 5c, the data of concentration-dependent therapeutic effects showed that at low concentration(4 μg/mL, approximately 1 μg/mL CSL after calculated by the loading capacity of CSL@HMSNs-CS) of CSL@HMSNs-CS, there is no difference compared with OA groups. At the concentration of 40 μg/mL, the cell viability was increased but no significant difference compared with 400 μg/mL group.
A cell viability assay was applied to examine the therapeutic effect of CSL and CSL@HMSNs-CS (Figure 5d). After stimulation with IL-1β to simulate the OA condition, compared with the control group without nanoparticle intervention, the cell viability was decreased. Treatment with simple CSL slightly increased the cell viability, whereas notable growth was observed after treatment with CSL@HMSNs-CS compared with OA condition. The data of the HMSNs and HMSNs-Cs group showed no significant difference with the OA condition.
All these results demonstrated the pH-responsive property of CSL@HMSNs-CS and that cell viability could be reversed by treatment with CSL and CSL@HMSNs-CS. The data of the in vitro cell viability assay demonstrate an improvement in CSL@HMSNs-CS compared with simple CSL, indicating that the HMSN loading and the Cs coating provide an extraordinary bioavailability. In the laboratory, to prepare drugs with poor solubility, solvent dimethyl sulfoxide (DMSO) was often chosen as a virtual ‘universal solvent’. But the cell permeabilizing effects of DMSO may influence the results. HMSNs are excellent candidates for loading traditional medicine facing the issue of their poor solubility. The ability to improve bioavailability has been demonstrated in many drugs with poor solubilities such as resveratrol and albendazole(28, 29). The high volume of pores provides a large contact area to the vehicle solution, leading to better drug loading and release. The high loading capacity of HMSNs could reduce potential cytotoxicity of CSL owing to the rapid release of the drug.
Enzyme-linked immunosorbent assay (ELISA)
The expression levels of inflammatory factors and MMP-3, MMP-13 in chondrocyte supernatant were evaluated via ELISA(Figure 6). The expression levels of IL-1β, IL-6, TNF-α, MMP-3, and MMP-13 dramatically increased in the OA, HMSNs, HMSNs-Cs groups, with no significant differences among the groups. In the simple CSL group, the expression level was slightly decreased, and a further decrease was observed in the CSL@HMSNs-CS group, revealing the good anti-inflammatory effect of nanoparticle medicine.
The increase in inflammatory factors probably due to the activation of pro-inflammatory enzymes involved in the processing of inflammatory factor precursor synthesis. The present study showed that Inflammatory factors, which are part of a vicious cycle in OA progression, could be inhibited by CSL@HMSNs-CS. Inhibition of IL-1β and TNF-α can block the OA occurrence and the development of the early and late course of OA, as well as the infiltration of immune cells and the destruction of cartilage structural integrity.
Matrix metalloproteinases(MMPs) are a broad family of secreted or transmembrane zinc-dependent endoproteinases that play a significant role in the degradation of ECM(30). Among them, MMP-3 and MMP-13 are powerful collagenolytic enzymes that show proteolytic activity on type II collagen, which is the most abundant protein component of cartilage and maintain morphology and function of cartilage(31). Stimulation by inflammatory reactions could induce the secretion of these enzymes from cartilage and synovial cells in the early stages of OA. Overexpression of proteolytic enzymes triggered an increase in the breakdown products from the ECM, leading to the phagocytosis by the synovial cells. Such positive-feedback regulation may amplify the development of OA(32). Using an integrated systems pharmacology method, it was predicted that the MMPs family is the direct target of CSL in rheumatoid arthritis, partially involved in the therapeutic effects in rheumatoid arthritis(33). The results of the present study indicated that CSL@HMSNs-Cs provide an ideal treatment by downregulating the expression of MMP-3 and MMP-13, which could be responsible for the anti-inflammatory effect previously mentioned.
Evaluation of pain behavior
Von Frey filaments are the gold standard way of evaluating sensory thresholds. Mechanical sensitivity is expressed by the paw withdrawal threshold(PWT) upon pricking the hind limb with von Frey filaments. The up-down method was used to estimate and modify thresholds(34, 35). According to the results of the pain behavior test(Figure 7), the PWT decreased in the first week after MIA injection. In the second week, drug intervention was conducted via intra-articular injection. After that, an increase in PWT was observed in both the CSL and CSL@HMSNs-CS groups. A significant difference between the CSL and CSL@HMSNs-CS groups was demonstrated after the fourth week, showing the effect of inhibiting central sensitization.
Pain is the major symptom of OA and is involved in both peripheral nociceptive stimuli and central sensitization. The acidic environment in OA could activate the osteoclasts, leading to the attenuation of the subchondral plate. The explosion of subchondral nerves because of the destruction of osteochondral integrity induces a continuous peripheral stimulation(36). In addition, sensory neurons are sensitive to H+, so nociceptive sensory neurons could be directly excited by H+-gated currents in the acidosis extracellular environment(37). The acute inflammatory response caused by the injection of MIA would be resolved by week 1 but could give rise to the sensitization of peripheral receptors. Increased stimulation input from peripheral nociceptors enhances the excitability of dorsal horn neurons at the dorsal horn, the so-called central sensitization, leading to the mechanical hypersensitivity (38). CSL could relieve knee OA pain by decreasing cytokines expression, inhibiting inflammatory infiltration and reducing peripheral stimulation, and interrupting the process of pain formation.
MRI and Safranin O Fast Green staining of the knee joint
MRI images of the knee joints(Figure 8a) revealed that articular surface erosion (marked by arrow) and prominent joint effusion (marked by star) were present in the OA group. The images showed no obvious effect on osteoarthritis in the simple CSL group, whereas a great improvement in articular surface erosion and joint effusion was observed in the CSL@HMSNs-CS group, indicating extraordinary therapeutic efficacy. The safranin O fast green staining result showed the same pathological changes as observed by MRI(Figure 8b). Comparing with the saline group, the OA group showed obvious vast cartilage loss, disorganized chondrocytes and structural destruction. Injection of simple CSL could improve the changes, but the cartilage layer is thinner and shows fissures. A dramatic improvement in pathological changes, such as smooth cartilage surface, undulating tide line and cartilage thickness was observed in the CSL@HMSNs-CS group.
Intra-articular injection of monosodium iodoacetate is a commonly used method to build an OA animal model to imitate human OA cartilage and bone pathology(39). At 2 weeks post-injection, a high dosage of MIA(2 mg) could simulate the pathological characteristics of subchondral bone and subchondral trabecular bone, such as erosion of subchondral trabecular bone, destruction of articular cartilage, and exposure of subchondral bone, similar to end-stage human OA joints(40). Subchondral bone stiffening and the destruction of articular cartilage play a critical role in the progression of OA. The dynamic balance between osteoclasts and osteoblasts leads to disorganized cartilage and bone structure. The image of the OA group showed severe loss of joint cartilage and remodeling of subchondral bone. CSL, forming an insoluble precipitate, can lower joint lubrication and induce the secretion of inflammatory factors in cartilage. According to the MRI results, a profoundly reduced knee swelling and improvement in synovial inflammation and cartilage integrity were demonstrated in the CSL@HMSNs-CS group, suggesting a protective effect on the structure of cartilage and subchondral bone.
Protein expression of NF-κB signalling pathway
The protein expression levels of phosphorylated p65, phosphorylated IKKβ and phosphorylated IκBα were demonstrated in Figure 9. Compared with the control group, the expression levels of phosphorylated p-65, phosphorylated IKKβ and phosphorylated IκBα were dramatically up-regulated in OA, HMSNs, HMSNs-Cs group. Simple CSL could downregulate the expression level but is of limited effectiveness. In CSL@HMSNs-CS group, the protein expression level was significantly downregulated suggesting the notable effect in inhibiting the expression level of the NF-κB signalling pathway in chondrocytes.
Celastrol has various of anti-inflammatory cellular targets and can significantly reduce the upregulated expression of MMPs family and protect chondrocytes against the IL-1β-induced inflammatory response and apoptosis(41). NF-κB signalling pathway is a typical signalling pathway involved in the development of OA pathobiology. Cartilage degradation can be induced by NF-κB transcription factors to enhance the secretion of various degradative enzymes such as MMP and A disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) which play a critical role in the degradation of ECM structural protein (42, 43). CSL attenuates NF-κB translocation to the nuclear, and pretreatment with CSL reduces the matrix degradation induced by IL-1β(44). IκBα phosphorylation by the IKK complex can be inhibited by celastrol in different cell types, which is a key step in NF-κB activation, consistent with the present study(45).