Oxygen vacancy-engineered cerium oxide mediated by copper-platinum exhibit enhanced SOD/CAT-mimicking activities to regulate the microenvironment for osteoarthritis therapy | Journal of Nanobiotechnology

Methods for synthesis and characterization of nanozymes

Materials

Cerium nitrate hexahydrate (CeN₃O₉·6H₂O, 99.5%) was purchased from Macklin Company in China. Hydrazine hydrate (N₂H₄·H₂O, AR) and copper sulfate (CuSO₄·5H₂O, AR) were purchased from Chengdu Jinshan Chemical Reagent Co. Anhydrous ethanol was purchased from China National Pharmaceutical Chemical Reagent Co. Platinum tetrachloride (PtCl₄) and ethylene glycol (C₂H₄O₂, > 99%) were purchased from Macklin Company in China. All chemicals were used directly without further purification.

Synthesis of CeO₂

The nanoparticles were synthesized based on previous research [28,29,30]. Ce(NO₃)₃·6H₂O (4.6 mmol/L) was dissolved in 2 mL of deionized water, followed by the addition of acetic acid (2 mL) and ethylene glycol (52 mL). The solution was transferred to a Teflon liner, maintained at 180 °C for 200 min, centrifuged and dried to obtain the product, and calcined in air at 400 °C for 4 h to obtain CeO₂ nanospheres.

Synthesis of PtCuO_X/CeO_2-X

CuSO₄ (50 mg) and CeO₂ (100 mg) were dissolved in deionized water (4 mL), respectively, added to 80 mL of anhydrous ethanol, and hydrazine hydrate was added dropwise and reacted for 2 h to give the intermediate product, CuO_X/CeO_2-X. Similarly, PtCl₄ (20 mg) and CuO_X/CeO_2-X (100 mg) were dissolved in deionized water (4 mL) in the same manner as above. PtCuO_X/CeO_2-X nanospheres were obtained.

Characterization methods

Transmission electron microscopy (TEM) and energy dispersive spectroscopy (EDS) (FEI Talos f200s, FEI, USA) were used to determine the morphology, size, and composition of the nanospheres. Dynamic light scattering (DLS) (Zetasizer Nano ZS ZEN3600, Malvern, UK) was used for hydrodynamic diameters of nanospheres, and X-ray photoelectron spectroscopy (XPS) (Thermo Fisher ESCALAB 250Xi, USA) was used for structure–activity relationships of nanospheres. An X-ray diffractometer (Rigaku, Ultima IV, Japan) was used for the crystallinity of nanospheres. An inductively coupled plasma mass spectrometer (Thermo iCAP 6300 Duo, USA) was used for the determination of elemental content, and the JEOL JES FA200 ESR spectrometer was used for the evaluation of free radical scavenging capacity. Fourier transform infrared spectrometer (IRAffinity-1S, Shimadzu, Japan) was used for infrared absorption.

DFT computational details

All DFT calculations were carried out using the Vienna ab initio simulation package (VASP) [31]. The Perdew-Burke-Ernzerhof (PBE) [32] functional was used to treat the exchange–correlation interactions. The plane wave basis set with a kinetic energy cutoff of 400 eV and the energy convergence criterion of 10^–4 eV was used for structure relaxation. All surface calculations used a (1 × 1 × 1) Monkhorst–Pack k-point sampling. H₂O, H₂O_2, and O₂ were calculated in boxes of 15Å × 15Å × 15Å, with the gamma point only.

The adsorption energy (E_ads) is calculated as follows:

E_{(adsorbates + catalysts)}, E_adsorbates, and E_catalysts are the energy of the whole system, adsorbates, and catalysts, respectively.

Photothermal performance testing

Photothermal heating curve

CeO₂ and PtCuO_X/CeO_2-X solutions (1 mL, 50 μg/mL) were placed in Eppendorf tubes, respectively. The samples were irradiated with an 808 nm laser (RAL808T1, Quartz Laser, China) for 15 min. Temperature changes were recorded and thermographic photographs were taken.

Photothermal conversion efficiency of PtCuO_X/CeO_2-X

PtCuO_X/CeO_2-X solution (1 mL, 50 μg/mL) was irradiated with an 808 nm laser for 900 s. The laser was then turned off and allowed to cool naturally. The photothermal conversion efficiency (η) of the PtCuO_X/CeO_2-X nanospheres was then calculated using Eqs. (1)-(4).

(where “η” is the photothermal conversion coefficient, “h” is the heat transfer coefficient, “A” is the surface area of the vessel, and “h A” can be determined by a linear relationship between time and the negative logarithm of the cooling cycle (-lnθ). “T_max” is the equilibrium temperature, “T_surr” is the ambient temperature, “Qs” is the heat generation of the solvent, “I” is the irradiated laser power, “Aλ” is the absorbance of the PtCuO_X/CeO_2-X nanospheres at 808 nm, “τs” is the time, “M_D” is the mass of the solvent, “C_D” is the heat capacity of the solvent, “θ” is the cooling cycle, and “ΔT” is the temperature difference during a given period).

The calculation procedure is as follows:

△T_max = T_max-T_surr = 48.1–25 = 23.1℃

hA = M_D * C_D / τs = 4.2*1/335.2 = 0.01252.

Qs = M_D * C_D (T_PBS-T_surr) = 4.2*0.001*(28.2–25) = 0.0013.

η = (hA△T_max-Qs) /(I(1–10- Aλ)) = (0.0125*23.1–0.0013) /π*0.52*(1–10-0.47) = 0.5541.

Photothermal stability

PtCuO_X/CeO_2-X was dissolved in PBS (50 μg/mL) and irradiated with NIR light for 15 min before stopping the irradiation and allowing it to cool to room temperature before starting the next round of irradiation. This was repeated four times and the temperature changes were recorded to evaluate the photothermal stability.

Stability experiments of nanospheres

The dispersion of PtCuO_X/CeO_2-X was recorded over 7 days and in different solvents. In addition, the average particle size and the polydispersity index (PDI) of the nanospheres in different solvents were measured by dynamic light scattering.

Enzyme mimetic properties of nanospheres

H₂O₂ decomposition test

We investigated the decomposition of H₂O₂ by PtCuO_X/CeO_2-X. Briefly, H₂O₂ solution (5 μL, 10 mM) was added to PtCuO_X/CeO_2-X aqueous solution (5 mL, 50 μg/mL). The amount of oxygen released was quantified using a portable dissolved oxygen meter. The ability of the PtCuO_X/CeO_2-X to continuously decompose H₂O₂ in vitro and in vivo was evaluated by adding H₂O₂ solution at the same time intervals using water and synovial fluid from OA rats, respectively, as the reaction system and the amount of oxygen released was recorded for four consecutive times.

ESR for ·OH detection

A solution of H₂O₂ derived from hydroxyl radicals was generated by full-band xenon irradiation for 5 min and scavenged with 5-tert-butylcarbonyl-5-methyl-1-pyrroline-N-oxide (BMPO, 10 mM). The scavenging ability of PtCuO_X/CeO_2-X towards ·OH was evaluated by monitoring the changes in peak intensities compared to the control.

ESR for ·O₂
⁻ detection

In the ESR experiments, the ·O₂⁻ was generated from KO₂ using 18-crown-6 as a stabilizer, while 5-(deoxy)-5-tert-butylcarbonyl-5-methyl-1-pyrroline-N-oxide (DMPO) was used as a scavenger. Peak intensities were measured for PtCuO_X/CeO_2-X nanozymes and control materials.

·O₂
⁻, H₂O₂, ·OH, and DPPH scavenging ability

The ability of CeO₂, PtCuO_X/CeO_2-X, and PtCuO_X/CeO_2-X + NIR to scavenge ·O₂⁻ was compared using a total superoxide dismutase assay kit (Beyotime, China). In addition, to verify whether the SOD-like activity of the nanozymes was concentration-dependent, the removal of ·O₂⁻ by PtCuO_X/CeO_2-X at concentrations of 20, 50, and 100 mg/mL was compared. The absorbance at 450 nm was measured using an enzyme-labeling device (Thermo Scientific, USA), and then the free radical scavenging rate was determined.

The scavenging activities of CeO₂, PtCuO_X/CeO_2-X, and PtCuO_X/CeO_2-X + NIR against H₂O₂, ·OH, DPPH, and total ROS were compared using the catalase assay kit (Beyotime, China), the hydroxyl radical scavenging capacity assay kit (Solarbio, China), the DPPH radical scavenging capacity assay kit (Solarbio, China), and the total antioxidant capacity assay kit (Beyotime, China), respectively.

Primary chondrocyte harvest and culture

Sprague–Dawley (SD) rats were obtained from the Experimental Animal Center of Guangxi Medical University, and their articular cartilage was harvested and primary chondrocytes were extracted. The fresh cartilage tissue was minced, digested with trypsin for 30 min, centrifuged, resuspended with type II collagenase, and further digested for 4 h. Finally, cells were cultured in DMEM medium. Subsequent validation using third-generation anterior chondrocytes.

Cellular uptake assay

Grafting of fluorescent groups to nanospheres

PtCuO_X/CeO_2-X nanospheres (0.5 g) were added to a mixture of 3-aminopropyltrimethoxysilane (120 μL) and 95% ethanol (6 mL) and reacted for 1 h under light protection to obtain the intermediate containing amino-functionalized nanospheres. Dissolve 0.1 g of the above product in dimethylsulfoxide (400 μL), add Cy5-NHS ester (60 μL), then add dimethylsulfoxide until the volume reaches 800 μL, add 20 μL of triethylamine, and react for 24 h away from light.

Cellular uptake

The chondrocytes (0.5 × 10⁵ cells/well) were inoculated into 6-well plates, and after 24 h of culture, Cy5-PtCuO_X/CeO_2-X nanospheres (50 μg/mL) were co-cultured with the cells, and the cells were fixed with 4% paraformaldehyde after 0.5, 6, and 12 h. The cytoskeleton was labeled with flavonoids and the nuclei were stained with DAPI. Confocal microscopy was performed to observe the uptake of nanospheres by cells.

Cell viability assay

Chondrocytes were cultured to attachment, co-cultured with the addition of nanospheres (30, 40, 50, 60, 70, and 80 μg/mL), and the cytotoxicity of nanozymes was evaluated by measuring the absorbance at 450 nm using a cell counting kit-8 (CCK-8, Biosharp, China) and a microplate reader (Molecular Devices, USA). In addition, the protective effect of nanozymes on inflammatory chondrocytes was evaluated. Briefly, chondrocytes were cultured to attachment, IL-1β was added, the culture was continued for 12 h, 50 μg/ml of nanozymes was added, and cell viability was determined after 24 h using a CCK-8 kit.

Observation of live and dead cell staining

Chondrocytes were cultured to adherence induced by the addition of IL-1β (10 ng/mL) for 12 h and then co-cultured with the addition of CeO₂, PtCuO_X/CeO_2-X, or PtCuO_X/CeO_2-X + NIR (1.0 W/cm², 5 min) for 24 h. Cells were incubated with 1 μM calcein-AM and 1 μM propidium iodide (PI) for 30 min. Dead and live cells were then observed and recorded under a fluorescence microscope.

Measurement of ROS scavenging capacity in vitro

DCFH-DA Probe

ROS scavenging ability was detected using the fluorescent probe DCFH-DA (Beyotime, China). Chondrocytes (1 × 10⁵ cells/well) were cultured until wall-adherent and induced by IL-1β for 12 h, then CeO₂, PtCuO_X/CeO_2-X, and PtCuO_X/CeO_2-X + NIR were added and cultured for 24 h, the probes were added, and the images were captured by fluorescence microscope (BD Biosciences, USA) after 30 min.

DAF-FM DA Probe

Nitric oxide (NO) levels in chondrocytes were detected using the Nitric Oxide Fluorescent Probe Assay Kit (Beyotime, China). Cells were treated in the same way as the image acquisition process described above.

DHE Probe

The level of intracellular superoxide (·O₂⁻) was detected with the fluorescent probe DHE (Beyotime, China). The chondrocytes were treated as described above. Fluorescence images of each group of chondrocytes after different treatments were captured by fluorescence microscope.

HPF probe

The intracellular hydroxyl radicals (·OH) levels were detected using the HPF fluorescent probe. Cells were treated in the same way as the image acquisition process described above.

[Ru (DPP)3]CI2 (luminescent oxygen sensor)

The chondrocytes were treated as described above. Each group received an addition of [Ru(DPP)3]Cl2 (0.01 mg/mL, 10 μL), and fluorescence imaging was performed by fluorescence microscopy.

qRT-PCR

Chondrocytes (1 × 10⁵ cells/well) were cultured to adherence, induced with IL-β for 12 h, and then co-cultured with CeO₂, PtCuO_X/CeO_2-X, and PtCuO_X/CeO_2-X + NIR for 24 h. Total RNA was extracted with TRIZOL and cell lysis buffer, and cDNA was reverse transcribed with PrimeScript according to the instructions. qRT-PCR was performed by adding SYBR premix to the cDNA of the real-time PCR system (Thermo Fisher, USA), preincubating for 1 cycle at 95 °C for 600 s, followed by 45 cycles at 95 °C for 10 s, and amplifying at 60 °C for 60 s. The 2^−ΔΔCt method was used to quantify relative mRNA expression, normalized to GAPDH. The experiment was repeated in triplicate. Primer sequences are shown in Table 1.

Immunofluorescence of relevant inflammatory genes, chondroprotective genes, and apoptosis-related factors

Chondrocytes (1 × 10⁵ cells/well) were cultured until apposition, induced by the addition of IL-1β for 12 h, and then co-cultured with the addition of CeO₂, PtCuO_X/CeO_2-X, and PtCuO_X/CeO_2-X + NIR for 24 h. Cells were fixed with 4% paraformaldehyde solution (Biosharp, China) for 15 min, permeabilized with 3% H₂O₂ for 30 min, and blocked with goat serum for 30 min to detect non-specific antibodies. IL-6 (Affinity Biosciences, #DF6087), MMP-13(Proteintech Group,#18,165–1-AP), Col2a1(Affinity Biosciences,#AF0135), Bcl-2(Affinity Biosciences,#AF6139), Bax(Affinity Biosciences,#AF0120), Caspase-3(Affinity Biosciences,#AF6311) and β-actin(Affinity Biosciences,#AF7018) antibodies were incubated for 8–12 h, followed by FITC-conjugated anti-rabbit IgG (Boston, China) for 1 h. Finally, fluorescence images were captured with a fluorescence microscope (Olympus, Japan) and quantified using ImageJ software.

Mitochondrial membrane potential assay

JC-1 fluorescent staining

Cells were treated as described above, and after cells were incubated with JC-1 (Solarbio, China) working solution for 20 min, changes in JC-1 monomers and aggregates were recorded under a fluorescence microscope for each group, and fluorescence intensities were quantified using ImageJ software.

JC-1 flow cytometry

Cells were treated as described above. Cells in each treatment group were stained with a JC-1 fluorescent probe for 30 min, and the cells were washed three times with 4 °C PBS to precipitate. The intensity of red and green fluorescence signals were detected by flow cytometry (BD FACSCaliburTM Flow Cytometer).

Cytoplasmic Ca²⁺ concentration assay

Cell culture and treatment as described in Sect. “Immunofluorescence of relevant inflammatory genes, chondroprotective genes, and apoptosis-related factors“, and at the end of the treatment, Fluo-4 AM (Beyotime, China) solution was co-incubated with chondrocytes for 30 min, and the fluorescence intensity of the cells was observed by fluorescence microscopy and finally quantitatively analyzed by fluorescence using ImageJ.

ATP measurement

Cell culture was performed as described above, and after 24 h, the cells were treated with IL-1β for 12 h. According to the Enhanced ATP Assay Kit (Beyotime, China) instructions, the cells of each group were completely lysed, and the standard curve was plotted first, after which the ATP content of each group was analyzed and calculated using a fluorescence microplate reader.

Apoptosis detection

Chondrocytes were cultured until apposition, then induced with IL-1β for 12 h. CeO₂, PtCuO_X/CeO_2-X, and PtCuO_X/CeO_2-X + NIR were added to co-cultivate the cells for 24 h. Cells were then counted and 1.0 × 10⁵ cells were collected, apoptosis detection reagents were added, and the cells were mixed and incubated away from light for 15 min, and the ratio of apoptosis was detected using Annexin V-APC /7-AAD Apoptosis Detection Kit (KeyGEN BioTECH, China) and flow cytometry (FCM) and analyzed by FlowJo_V10 software.

Immunoblotting assay for apoptosis-related proteins and ROS/Rac-1/NF-κB pathway proteins

The expression of apoptotic proteins and inflammatory pathway-related proteins was detected by Western blotting analysis using the following protein species and antibody sources: Caspase-3(Affinity Biosciences,#AF6311), Bcl-2(Affinity Biosciences,#AF6139), Bax(Affinity Biosciences,#AF0120), Rac-1(Affinity Biosciences,#AF4200), p65(Affinity Biosciences,#AF5006), p-p65(Affinity Biosciences,#AF2006). The cell treatment was the same as above, and then the proteins were extracted, gel electrophoresis was performed, the membrane was transferred, the membrane was closed, the primary antibody was incubated for 8 h, and the secondary antibody was incubated for 1 h, and the protein bands were observed by BIO-RAD imaging system, and the intensity of the bands was analyzed by ImageJ software.

Establishment of a rat OA model

With the ethical approval of the Ethics Committee of Guangxi Medical University, the OA model was established using the anterior cruciate ligament transection (ACLT) method in randomized groups of 60 rats, body weight: 200–220 g, male SD rats, and rearing temperature: 23–25 °C. Treatments: weekly injection of drugs and NIR irradiation in the light group twice a week for 5 min each. Samples were collected in batches after 4 and 8 weeks of continuous treatment.

IVIS imaging evaluation

The Cy5-labeled PtCuO_X/CeO_2-X and the free Cy5 were injected into a knee joint of the rat. Then the IVIS images were collected by IVIS Spectrum Imaging System (BLT, China) at predetermined times (excitation wavelength 675 nm, emission wavelength 680 nm). In addition, the important organs were also scanned by the IVIS system to clarify the metabolic pathway of PtCuO_X/CeO_2-X.

Thermographic analysis of rat knee joints

The right knee joints of rats were injected with PBS, CeO₂, and PtCuO_X/CeO_2-X (100 μL, 50 μg/mL), respectively. 12 h later, the right knee joints were irradiated with NIR light. Images and temperatures during NIR irradiation were recorded with an anterior NIR camera.

Gait analysis

After successful modeling of OA, the gait of rats at weeks 4 and 8 was analyzed using the Animal Visual Gait Analysis System. Gait duration and mean gait speed were assessed.

Histologic analysis

Enzyme-linked immunosorbent assay (ELISA)

We used IL-6(MEIMIAN, #MM-0190R2), MMP-13(MEIMIAN, #MM-0110R2), and Col2a1(Zeye Bio, #ZY0324ER) ELISA kits to detect the expression of related proteins in joint fluid and cell supernatants of OA rats, and IL-1β (Solarbio, #SEKR-0002), IL-17 (Solarbio, #SEKR-0007), and TGF-α (Zeye Bio, #ZY0126ER) kits to detect the expression of immune response-related factors. Add specimens according to instructions, incubate at 37 °C for 30 min, and wash 5 times with Wash Solution. Add 50 μL enzyme reagent, incubate for 30 min, wash 5 times, add dyes A and B sequentially, incubate for 10 min at 37 °C without light and add the termination solution. Measure the absorbance (OD value) of each well using an enzyme meter. Calculate the concentration from the standard curve.

Immunohistochemistry staining (IHC)

Tissue sections were routinely deparaffinized and dehydrated using a universal two-step detection kit (ZSGB-BIO, China, #PV-9000), and the antibody was diluted 1:200 and incubated overnight at 4 °C, protected from light, and then conjugated with biotinylated secondary antibody. The slides were photographed with a light microscope (OLYMPUS BX53F, Japan).

Hematoxylin–eosin (HE) and saffron-o-fast green staining

Tissue sections and major organs were stained with hematoxylin–eosin kit (HE, Solarbio, China), saffron-o-fast green staining kit (Solarbio, China), and the sections were observed under a microscope and photographed for histological analysis. In addition, Venous blood samples were collected for routine and blood biochemical analyses to evaluate the biotoxicity of the nanozymes.

Tissue reactive oxygen species assay

Tissue homogenate was prepared by adding 1 mL buffer to 50 mg of knee cartilage tissue, 190 μL supernatant was collected by centrifugation, 10 μL BBcellProbeTM O11 ROS probe (BestBio, China) was added, and the tissue was incubated at 37 °C for 30 min under light protection. The ROS level was determined using a fluorescence microplate reader (Bio-Tek Instruments, USA) with an excitation wavelength of 488 nm and an emission wavelength of 530 nm.

Nanozymes cartilage penetration capability test

100 μL of Cy5-PtCuO_X/CeO_2-X (50 μg/mL) was injected into the knee joints of OA rats. The articular cartilage (including subchondral bone) of the femoral and tibial sides of the knee joints were harvested at 24, 48, and 72 h after injection, and the knee joints were immersed in saline for 6 h under light avoidance conditions, and then cryosections were performed, stained with DAPI, and sealed, and the images were visualized and recorded under a fluorescence microscope.

Hemolysis assay

Arterial blood was collected from rats and cell suspension was prepared. Then, 900 μL of ultrapure water (positive control) and PBS containing different concentrations of PtCuO_X/CeO_2-X nanozymes were added, and the mixtures were gently shaken and allowed to stand at 37 °C for 2 h. Photographs were taken to compare hemolysis between different groups. The supernatant was transferred to a 96-well plate and the percentage of hemolysis was calculated by recording the absorbance at 540 nm using the enzyme marker.

Statistical analysis

Data were expressed as mean ± standard deviation and found to exhibit a normal/Gaussian distribution under the Shapiro–Wilk test. Analyses were performed using GraphPad Prism software (v. 9.4.1). Unpaired Student’s t-test was performed for comparisons between 2 groups, and one-way analysis of variance (ANOVA) was performed for 3 or more groups, followed by Tukey’s test. The sample size for each analysis was presented within the figure legends. * and # for P < 0.05, ** and ## for P < 0.01, *** and ### for P < 0.001, and **** and #### for P < 0.0001.