Mitochondrial-targeting Mn3O4/UIO-TPP nanozyme scavenge ROS to restore mitochondrial function for osteoarthritis therapy

Abstract Excessive reactive oxygen species (ROS)-induced mitochondrial damage has impact on osteoarthritis (OA). Nanozyme mimics as natural enzyme alternatives to scavenge excessive ROS has offered a promising strategy for OA therapy. Herein, we reported a novel mitochondrial-targeting Mn3O4/UIO-TPP nanozyme using metal-organic frameworks with loaded Mn3O4 as the enzyme-like active core combining mitochondria-targeting triphenylphosphine (TPP) groups to serve as ROS scavengers for therapy of OA. With sequential catalysis of superoxide dismutase-like, catalase (CAT)-like, and hydroxyl radical (·OH) scavenging potentials, the nanozyme can target mitochondria by crossing subcellular barriers to effectively eliminate ROS to restore mitochondrial function and inhibit inflammation and chondrocyte apoptosis. It also has favorable biocompatibility and biosafety. Based on anterior cruciate ligament transection-induced OA joint models, this mitochondrial-targeting nanozyme effectively mitigated the inflammatory response with the Pelletier score reduction of 49.9% after 8-week therapy. This study offers a prospective approach to the design of nanomedicines for ROS-related diseases.


Introduction
Osteoarthritis (OA), characterized by pain, joint inflammation, articular cartilage defects and limited joint movement, was one of the prevailing joint disorders across the world, threatening public health [1][2][3].Increasing evidence showed that mitochondrial dysfunction caused by reactive oxygen species (ROS)-induced mitochondrial DNA (mtDNA) damage contributes to the progression of OA, involved in PI3K-Akt, Caspase and matrix metalloproteinases (MMPs) signaling pathways in chondrocytes, as well as chondrocyte apoptosis [4][5][6][7][8][9].Antioxidants for scavenging ROS such as superoxide dismutase (SOD), Vitamins, Coenzyme Q10, Polyphenol and Polysaccharides, have been reported to reduce oxidative stress and improve the histological score of the cartilages, were regarded as a prospective approach for scavenging ROS and further therapy of OA.However, most ROS scavengers lack specific targeting and have weak ROS scavenging efficiency with short retention time in vivo [10,11], leading to unfavorable therapeutic effects.
Recently, nanozymes like noble metals, metal oxides, carbides etc. with the abilities to scavenge ROS have been extensively used in inflammatory diseases [12][13][14][15], with superior stability and ease of availability in comparison to natural enzymes.Comparatively, nanozymes have better enzyme degradation resistance in vivo [16], which could stay longer in tissues and improve the therapeutic efficacy, reducing repeated administration and the risk of infection during treatment [17].Previous researches have confirmed that nanoscale Mn 3 O 4 could present multi-enzyme-like behavior, including SOD-like, catalase (CAT)like and ÁOH scavenging activities in the medication of ROSrelated diseases, including inflammatory bowel diseases, acute kidney injury, cardiovascular and cerebrovascular diseases, and other tissue inflammation [18][19][20][21].Due to its mixed oxidation states and high stability in redox reaction, Mn 3 O 4 nanoparticles (NPs) could achieve high stability and show superiority in eliminating ROS and showed better therapeutic efficacy than clinical medicals as Mugesh et al. [22] and Wei et al. [19] reported.However, Mn 3 O 4 NPs were easy to agglomerate and sediment, leading to loss of catalytic properties and increase of cytotoxicity [23,24], discounting their therapeutic effect on ROS-related diseases.
By using carriers such as metal-organic frameworks (MOFs), transition metal carbide (MXene), and carbon dots, were proven effective to improve catalytic activity of nanozymes due to the electronic interaction between nanozymes and carriers [25].In favor of anchoring functional molecules, MOFs were a kind of newly porous crystalline material with facile synthesis, intrinsic biodegradability, tunable pore cavity and large surface area [26].These characteristics made MOFs potentially suitable for limiting the movement and promoting the interaction of NPs, ensuring the smooth diffusion of reactants by uniform dispersion [27,28].As a uniform-shaped MOFs with the highest coordination number of organic ligands and metal clusters, dense unit arrangement and strong Zr-O bond, the UIO66 series have superior hydrothermal, mechanical and chemical stability [29][30][31].This excellent stability benefits the drug bioavailability in vivo.It has been reported that UIO66 carriers for Pt, Au and Ag NPs have greatly improved the bioactivity for tumor treatment and wound healing [24,32,33].Thus, the introduction of UIO66 series MOFs may increase the catalysis and decrease the cytotoxicity of Mn 3 O 4 NPs by preventing the rapid release of Mn ions [19,34].
In our study, a multifunctional Mn 3 O 4 /UIO-TPP nanozyme (Figure 1) was developed by grafting Mn 3 O 4 NPs with UIO66 series MOFs using a hydrothermal method followed by facile conjugation with mitochondria-targeting triphenylphosphine (TPP) groups, in an attempt to scavenge mitochondrion-derived ROS to restore mitochondrial damage for OA therapy.The modified MOFs facilitate subcellular targeting at the lesion site through rapid localization of diseased cells and precisely modulation of organelle function [35], with an efficient mitochondria ROS scavenging efficiency to enhance therapeutic effect.This study may offer a promising strategy for effective mitochondrialtargeting therapy of ROS-related diseases.

Synthesis of Mn 3 O 4 NPs
Mn 3 O 4 NPs synthesizing was referred from the method in previous work [36] with slightly modified.According to a routine procedure, 613 mg Mn(CH 3 COO) 2 Á4H 2 O was added into 30 ml ethanol and heated up to 120 C (5 C/min) for 24 h in an autoclave after fully dissolved under ultrasound.Once naturally cooled to ambient temperature, the Mn 3 O 4 NPs were subsequently obtained by centrifugation and dried after 12 h in a 60 C vacuum chamber.

Synthesis of UIO66-COOH
The method for synthesizing precursor UIO66 was referred from previous work [37] with slightly modified.According to a routine procedure, 160 mg ZrCl 4 and 114 mg BDC were added into a degassed solution (40 ml DMF and 1.2 ml HAc) and heated up to 120 C (5 C/min) for 24 h in an autoclave after fully dissolved under ultrasound.Once naturally cooled to ambient temperature, centrifugated to collect and soaked the precipitates into methanol for three days, then dried after 12 h in a 60 C vacuum chamber.
The obtained UIO66 was carboxylated by a post-synthesis exchange method referred from previous work [38,39] with slightly modified.Typically, 105 mg of 1,2,4-benzene tricarboxylic acid was dissolved it in 2 ml of 4% KOH solution, then the solution pH was adjusted to 7 6 0.2 with 1 M HCl with an additional 28 mg UIO66 and 0.5 ml DMF.The obtained solution was next heated up to 85 C (5 C/min) for 24 h in an autoclave after ultrasonic dispersion for 30 min.Once naturally cooling to ambient temperature, centrifugated to collect and soaked the precipitates into methanol for three days, then dried after 12 h in a 60 C vacuum chamber.

Synthesis of Mn 3 O 4 /UIO nanozymes
Typically, 32 mg Mn(CH 3 COO) 2 Á4H 2 O and 100 mg UIO66-COOH were added into 20 ml ethanol.The obtained solution was next ultrasonic dispersion for 15 min, and then heated up to 120 C (5 C/min) for 24 h in an autoclave.Once naturally cooled to ambient temperature, centrifugated to collect and washed the precipitates with methanol three times, then dried after 12 h in a 60 C vacuum chamber.
For TPP conjugation, 20 mg Mn 3 O 4 /UIO3 nanozyme was ultrasonically dispersed in 20 ml ethanol in a flask.The obtained solution was next stirred at ambient temperature for 20 min with an additional 9.59 mg EDC and 4.95 mg NHS.Then added 20 mg TPP-NH 2 into the solution and stirred for another 24 h at ambient temperature.Once centrifugated to collect and washed with methanol three times, the product was dried after 12 h in a 60 C vacuum chamber and named Mn 3 O 4 /UIO-TPP.

In vitro enzyme-like activity
The SOD-like activity was measured, which was the superoxide (ÁO À 2 ) scavenging ability of Mn 3 O 4 /UIO nanozymes, by using a SOD activity assay kit (Sigma-Aldrich, USA).Typically, 20 ll sample solution (0, 5, 10, 20, 50, 100 lg/ml) was incubated for 20 min at 37 C after being mixed with 200 ll WST-8/enzyme working solution mix, and was recorded via a microplate reader (Thermo Fisher, USA) at the absorbance of 450 nm based on the manufacturer's guidelines.
The CAT-like activity was measured, which was the H 2 O 2 scavenging ability of Mn 3 O 4 /UIO nanozymes, by using a CAT activity assay kit (Beyotime, China).Typically, 10 ll 250 mM H 2 O 2 and 40 ll sample solution were incubated for 5 min at 25 C after being mixed with a working solution mix, then took 10 ll of this reacted solution and added into a chromogenic working solution, and was recorded after incubated for 15 min at 25 C via a microplate reader at the absorbance of 520 nm based on the manufacturer's guidelines.
The ÁOH scavenging ability of Mn 3 O 4 /UIO nanozymes was measured by using a ÁOH detection kit (Solarbio, China).Typically, 15 ll sample solution was added into 140 ll working solution mix and incubated for 20 min at 37 C, then took 100 ll supernatant and recorded via a microplate reader at the absorbance of 536 nm based on the manufacturer's guidelines.

Extraction and culture of chondrocytes
The joint tissues of male Sprague-Dawley (SD) rats within three days of birth were used for harvesting chondrocytes.The harvested chondrocytes were cultured in fresh Dulbecco modified eagle medium (DMEM, Gibco, USA) with an additional 10% (V/V) fetal bovine serum (Sijiqing, China) and 1% penicillin/streptomycin (Solarbio).Refreshed the cultured medium every 72 h, and the chondrocytes were sub-cultured or collected when reaching 75-85% density for further the further use of experiments.

Live and dead staining assay
Chondrocytes in six-well plates with a density of 2 Â 10 5 cells/ well were cultured overnight.Then, 20 lg/ml of UIO66-COOH and Mn 3 O 4 /UIOs were, respectively, added into the medium.The cells were stained with Calcein-AM/PI (Sigma-Aldrich, USA) based on the manufacturer's guidelines when the 24 h incubation finished, and were washed with PBS buffer three times before being captured via a fluorescence microscope (Olympus BX53, Japan).Besides, the survival of chondrocytes under oxidative stress was also evaluated.Six-well plates with a density of 2 Â 10 5 cells/well of chondrocytes were incubated with 200 lM H 2 O 2 for 24 h to establish the cell model with oxidative stress damage.Then, 20 lg/ml of UIO66-COOH, Mn 3 O 4 /UIO3 and Mn 3 O 4 /UIO-TPP were, Regenerative Biomaterials, 2023, Vol. 10, rbad078 | 3 respectively, added into the medium.The cells were stained with Calcein-AM/PI when the 24 h incubation finished and were washed with PBS buffer three times before being captured.

Lysosomal escape and mitochondrial-targeting
Chondrocytes in confocal dishes with a density of 1 Â 10 5 cells/ dish were cultured overnight.Medium with additional FITClabeled Mn 3 O 4 /UIO-TPP and FITC-labeled Mn 3 O 4 /UIO3 nanozymes (20 lg/ml) were, respectively, replaced.After 3, 6 and 12 h incubation, the medium was refreshed and the chondrocytes were stained in the dark with LysoTracker Red DND-99 (50 nM, 1 ml, Thermo Fisher) or MitoTracker Red CMXRos working solution (100 nM, 1 ml, Thermo Fisher) at 37 C for 60 min.The chondrocytes were next fixed with 4% paraformaldehyde (4% PFA, Biosharp, China) for 15 min and stained the chondrocyte nuclei with DAPI (Beyotime).The chondrocytes were captured via a confocal microscopy (Leica, Germany) and the correlated Pearson's R value was analyzed by Fiji software.

Subcellular superoxide staining assay
The subcellular superoxide levels were detected by using a MitoSOX fluorescence probe (Yeason, China).Briefly, the treated chondrocytes (200 lM H 2 O 2 , 24 h) in six-well plates, with a density of 2 Â 10 5 cells/well, have been co-cultured with 20 lg/ml of UIO66-COOH, Mn 3 O 4 /UIO3 and Mn 3 O 4 /UIO-TPP for 24 h.Once washed with PBS buffer three times after discarding the medium, the cells were stained in the dark with 5 lM MitoSOX solution at 37 C for 10 min and then stained the chondrocyte nuclei with Hoechst 33342 (Solarbio).The chondrocytes were washed with PBS buffer three times before being captured via a fluorescence microscope and the mean fluorescence intensity (MFI) quantitative results were analyzed by Image J software.

Intracellular mitochondrial membrane potential (DWm) assay
Intracellular DWm was measured by using a Mitochondrial membrane potential assay kit (Beyotime).Briefly, the treated chondrocytes (200 lM H 2 O 2 , 24 h) in 24-well plates, with a density of 4 Â 10 4 cells/well, have been co-cultured with 20 lg/ml of UIO66-COOH, Mn 3 O 4 /UIO3 and Mn 3 O 4 /UIO-TPP for 24 h.And were stained in the dark with 10 lM TMRE working solution at 37 C for 30 min, and then stained the chondrocyte nuclei with Hoechst 33342.The chondrocytes were washed with PBS buffer three times before being captured via a fluorescence microscope and the MFI quantitative results were analyzed by Image J software.

Intracellular mitochondrial calcium level assay
Intracellular mitochondrial calcium level was measured by using Rhod2/AM fluorescent probe (Yeason, China).Briefly, the treated chondrocytes (200 lM H 2 O 2 , 24 h) in 24-well plates, with a density of 4 Â 10 4 cells/well, have been co-cultured with 20 lg/ml of UIO66-COOH, Mn 3 O 4 /UIO3 and Mn 3 O 4 /UIO-TPP for 24 h.And were stained in the dark with 10 lM Rhod2/AM working solution (containing 0.02% Pluronic F-127) at 37 C for 30 min, and then incubated with fresh DMEM for another 30 min.The chondrocytes were washed with PBS buffer three times before being captured via a fluorescence microscope and the MFI quantitative results were analyzed by Image J software.

Intracellular ATP level assay
Intracellular ATP level was measured by using an ATP assay kit (Beyotime) based on the manufacturer's guidelines.Briefly, the treated chondrocytes (200 lM H 2 O 2 , 24 h) in six-well plates, with a density of 2 Â 10 5 cells/well, have been co-cultured with 20 lg/ml of UIO66-COOH, Mn 3 O 4 /UIO3 and Mn 3 O 4 /UIO-TPP for 24 h.Once washed with PBS buffer three times after discarding the medium, the cells were collected and lysed in RIPA buffer.A volume of 20 ll supernatant was next isolated by centrifugation and was added to 100 ll ATP detection working solution in a 96-well plate, respectively, and the results were obtained via a fluorometer (BioTek, USA).

Intracellular mtDNA evaluation
The intracellular mtDNA of the treated chondrocytes was collected using a Universal DNA extraction kit (Magen, China) based on the manufacturer's guidelines.The mtDNA (mitochondrially encoded Cytochrome C Oxidase II, MT-CO2) copy number was further calculated by quantitative real-time PCR (qRT-PCR) proceeded following the steps of 95 C for 10 min, 95 C for 15 s and 60 C for 60 s via a Detection System (Roche, Switzerland), and ACTB (b-actin) was selected as the reference gene.The primer sequences were listed in Supplementary Table S4.

Intracellular ROS level evaluation
The intracellular ROS level was measured by using a 2 0 ,7 0 -dichlorofluorescin diacetate fluorescent probe (DCFH, Solarbio) Briefly, the treated chondrocytes (200 lM H 2 O 2 , 24 h) in six-well plates, with a density of 2 Â 10 5 cells/well, have been co-cultured with 20 lg/ml of UIO66-COOH, Mn 3 O 4 /UIO3 and Mn 3 O 4 /UIO-TPP for 24 h.Once washed with PBS buffer three times after discarding the medium, the cells were stained in the dark with 10 lM DCFH working solution at 37 C for 30 min.The chondrocytes were washed with PBS buffer three times before being captured via a fluorescence microscope and the MFI quantitative results were analyzed by Image J software.

qRT-PCR analysis
The total RNA of the chondrocytes in experiments was obtained by using a Total RNA extraction kit (Magen, China) based on the manufacturer's guidelines.After the obtained RNA was reverse transcript, the qRT-PCR was proceeded following the steps of 95 C for 10 min, 95 C for 15 s and 60 C for 60 s via a Detection System (Roche, Switzerland).The IL6, MMP13, COX2 and MMP3 gene expression levels were analyzed, and glyceraldehyde-3phosphate dehydrogenase (GAPDH) was selected as the reference gene.The primary sequences were listed in Supplementary Table S4.

Immunofluorescence staining
The IL6 and MMP13 protein expression levels were further analyzed by using immunofluorescence staining.Briefly, the treated chondrocytes (200 lM H 2 O 2 , 24 h) in six-well plates, with a density of 2 Â 10 5 cells/well, have been co-cultured with 20 lg/ml of UIO66-COOH, Mn 3 O 4 /UIO3 and Mn 3 O 4 /UIO-TPP for 24 h.Once the chondrocytes were fixed with 4% PFA for 20 min, 3% H 2 O 2 was used for another 15 min before being blocked with goat serum working solution (ZS-bio, China) to eliminate nonspecific staining.Primary antibodies of IL-6 and MMP-13 (1:200, Bioss, China) were used to incubate with the chondrocytes overnight in a cool condition (4 C).Subsequently, an FITC-coupled anti-rabbit secondary antibody (1:100, Boster, China) was next used in the dark for 1 h, and the chondrocyte nuclei were stained with DAPI.The chondrocytes were further washed with PBS buffer three times before being captured via a fluorescence microscope and the MFI quantitative results were analyzed by Image J software.Besides, the evaluation of the DNA damage proceeded with the same treatments as described above, and the DNA damage of cells was measured based on the manufacturer's guidelines by using a DNA damage assay kit by c-H2AX immunofluorescence (Beyotime).The chondrocytes were further washed with PBS buffer three times before being captured via a fluorescence microscope and the MFI quantitative results were analyzed by Image J software.

In vivo experiments
The animal experiment was sanctioned by the Animal Ethics Committee of Guangxi Medical University (No. 202106011).Male SD rats (140-180 g) were acquired from Guangxi Medical University Laboratory Animal Center, and the experimental OA animal models were next constructed by performing an anterior cruciate ligament transection (ACLT) surgery on these rats.After the surgery, the rats were randomly categorized into five groups: (i) Sham group: Normal rats have only incised the skin and joint capsule without any further process; (ii) OA group: OA rats with PBS buffer injection at the dosage of 100 ll, two times a week; (iii) UIO66-COOH group: OA rats with UIO66-COOH injection at the dosage of 100 ll (20 lg/ml), two times a week; (iv) Mn 3 O 4 /UIO3 group: OA rats with Mn 3 O 4 /UIO3 injection at the dosage of 100 ll (20 lg/ml), two times a week; (v) Mn 3 O 4 /UIO-TPP: OA rats with Mn 3 O 4 /UIO-TPP injection at the dosage of 100 ll (20 lg/ml), two times a week.An overdose of pentobarbital sodium (Nembutal) was carried out for the euthanasia of rats at 4 and 8 weeks, and the knee joints and other major organs of these rats were harvested for further experiments, the heart, liver, spleen, lung and kidney were included.
The collected knee joints of rats were partially captured and scored based on Pelletier's macroscopic scores by three individuals, and the others were fixed for 2 days by using 4% PFA and decalcified for 6 weeks via an Ultrasonic Decalcifying Unit (USE 33, Germany) at pH ¼ 7.2 with 0.5 M EDTA (Macklin).After that, 3 lm thick sections of each group were acquired after the decalcified knee joints were embedded and sliced.These sections were soon stained by using hematoxylin-eosin (HE, Solarbio) and safranin O-fast green (S-G, Solarbio), then captured via an optical microscope (Olympus BX53, Japan).The captured photographs were later scored based on the Mankin scores by three individuals.Meanwhile, the collected organs of these rats were also fixed for 2 days by using 4% PFA, then 3 lm thick organ sections of each group were acquired after the organs were embedded and sliced.And in vivo cytotoxicity was further investigated based on these organs' sections by histological analysis.
To further investigated the inflammatory factors (IL-6 and MMP-13) expression levels in joint tissues.The synovial fluid harvested from the treated rats' joints was also detected by using an enzyme-linked immunosorbent assay (ELISA, Meimian, China) based on the manufacturer's guidelines.The results were obtained via a microplate reader at the absorbance of 450 nm.
To further investigate the retention time of Mn 3 O 4 /UIO-TPP nanozyme in the articular cavity of SD rats.Cy5.5-labeled Mn 3 O 4 / UIO-TPP nanozyme was firstly dispersed in PBS buffer to form a solution at the concentration of 20 lg/ml and was next injected 100 ll into the knee joints of rats.The fluorescence signals and fluorescence intensity quantification were recorded via in vivo imaging system (AniView 100, BLT, China) at various time points (0, 3, 6, 12, 24, 48, 72, 96 h).Different ex vivo organs were also harvested after 96 h, and then recorded and quantified fluorescence intensity via the same equipment.

Statistical analysis
Statistical analysis of all the data obtained from the experiments was proceeded using SPSS 26.0 statistical software with at least three repeated experiments and reported as means 6 standard deviations.The comparison between the two groups was proceeded by using one-way ANOVA with the least significant difference analysis.P < 0.05 was regarded statistically significant, and * P<0.05, ** P<0.01, *** P<0.001 and **** P<0.0001.

Synthesis and characterization of the Mn 3 O 4 /UIO nanozymes
The Mn 3 O 4 /UIO nanozymes were synthesized based on the precursor UIO66-COOH loaded with different contents of Mn 3 O 4 .In brief, the UIO66-COOH NPs were synthesized and then redispersed in ethanol at 120 C with different amounts of Mn(CH 3 COO) 2 Á4H 2 O (4, 16 and 32 mg, respectively) to form Mn 3 O 4 /UIOs (named Mn 3 O 4 /UIO1, Mn 3 O 4 /UIO2 and Mn 3 O 4 /UIO3) by electrostatic interaction [41].TEM images (Figure 2A; Supplementary Figure S1) showed that compared with UIO66 and UIO66-COOH, Mn 3 O 4 /UIOs preserved its 3D octahedral structure and uniform size after Mn 3 O 4 loading at the condition of Mn(CH 3 COO) 2 Á4H 2 O as an Mn 2þ source.An appropriate loading capacity was achieved while further increasing the quantity of Mn(CH 3 COO) 2 Á4H 2 O to 64 mg resulting in unbound particles surrounding the Mn 3 O 4 /UIO (Supplementary Figure S2).DLS studies confirmed that compared with UIO66-COOH, the hydrodynamic diameters of Mn 3 O 4 /UIOs (Supplementary Figure S3) were slightly increased according to the amount of Mn 2þ .And the same trend was also shown for zeta potential with the order of Mn 3 O 4 / UIO1 < Mn 3 O 4 /UIO2 < Mn 3 O 4 /UIO3 (Supplementary Figure S4).Notably, the hydrodynamic diameters of the three Mn 3 O 4 /UIO nanozymes were far less than bare Mn 3 O 4 NPs, indicating the favorable dispersibility in aqueous solution.EDX mapping results of Mn 3 O 4 /UIO further proved the existence of the Mn element in the NPs (Figure 2B and C).
XRD spectra (Figure 2D) of the synthesized UIO66-COOH illustrated iconic diffraction peaks at 2h angles of 7.56 , 8.70 , 25.90 , 30.88 , 43.48 , 50.50 and 56.82 , almost consistent with standard UIO66 sample (CCDC No. 837796, Supplementary Figure S5A), indicating that the carboxylated process did not change its crystal structures.However, no iconic diffraction peaks of Mn 3 O 4 (JCPDS24-0734, Supplementary Figure S5B) were detected in the patterns of Mn 3 O 4 /UIOs, probably due to that Mn 3 O 4 crystals loaded on the UIO66-COOH were ultrasmall and hard to detect.Peaks at 1716.6 and 1734.2/cm of FTIR spectra (Figure 2E) and those at 1615/cm of Raman spectra (Supplementary Figure S6) verified free carboxylic acid and its dimer in both Mn 3 O 4 /UIOs and UIO66-COOH [42][43][44].Yet, characteristics of FTIR or Raman spectra of Mn 3 O 4 were hardly found in Mn 3 O 4 /UIOs because of weak signals from ultrasmall Mn 3 O 4 crystals (Figure 2E; Supplementary Figures S6 and S7).
XPS results (Supplementary Figure S8A) further proved the apparent Mn element in the Mn 3 O 4 /UIOs.And the fine spectra of Mn 2p (Supplementary Figure S8B) showed the peaks of Mn 2p 3/2 and Mn 2p 1/2 at 641.1 and at 653.1 eV, respectively, indicating the Mn element was mainly presented as Mn 3 O 4 in these samples.And the ratio of Mn 2þ and Mn 3þ ions in the samples was $1:2 according to the area ratio, which was consistent with the theoretical value reported previously [45][46][47].Meanwhile, TGA results (Supplementary Figure S9) showed that compared with Regenerative Biomaterials, 2023, Vol. 10, rbad078 | 5 UIO66-COOH, Mn 3 O 4 /UIOs differ in the remaining mass percentage at 800 C, which was caused by the different content of Mn 3 O 4 loading.And similar to the work previously reported [48], this increase of Mn 3 O 4 loadings occupied the pore structure of MOFs also leading to a diminution in their BET surface area and pore volume (Supplementary Table S2).Further ICP-OES study (Supplementary Table S3) demonstrated that the mass fraction of Mn increased with the trend of Mn 3 O 4 /UIO1 < Mn 3 O 4 / UIO2 < Mn 3 O 4 /UIO3, were 0.80%, 3.39% and 5.16%, respectively.
The above all demonstrated the successful synthesis of Mn 3 O 4 /UIOs with varying levels of Mn 3 O 4 loading.The loading of Mn 3 O 4 did not significantly alter the crystal and molecular  Based on the above results, it demonstrated a sequential catalysis behavior of Mn3O4/UIOs for ROS scavenging, especially for Mn 3 O 4 /UIO3 due to its highest absolute content of Mn 3 O 4 , potentiating the application for OA therapy.

Cell viability
The cytotoxicity of Mn 3 O 4 /UIOs were detected by using a CCK-8 assay kit (Figure 4A).As shown, UIO66-COOH displayed no obvious cytotoxicity at all concentration sets.Meanwhile, Mn 3 O 4 /UIO nanozymes only showed little cytotoxicity at a concentration of 5-20 lg/ml with cell viability maintained at no <80%.Once the concentration of Mn 3 O 4 /UIOs increased upper than 50 lg/ml, the cell viability would decrease to lower than 70%.For comparison, bare Mn 3 O 4 NPs showed cytotoxic effect at the concentration of 5 lg/ml in Supplementary Figure S10.This biocompatibility difference was probably due to the released Mn ions into the cytoplasm after endocytosis of the material, because free Mn ions from ultra-small manganese oxides were reported to have cytotoxicity [34].Therefore, without apparent cytotoxicity, 20 lg/ml Mn 3 O 4 /UIO nanozymes were chosen for further experiments (Figure 4B and 4C).Meanwhile, live and dead staining of chondrocytes also confirmed this.Considering the amalgamation of catalytic proficiency and biocompatibility, a concentration of 20 lg/ml was preferred in the subsequent experiments of Mn 3 O 4 / UIO3 nanozyme.

Conjugation with mitochondria-targeting TPP group to form Mn 3 O 4 /UIO-TPP
The existence of an oxidative respiratory pathway and ion pump in mitochondria maintains its membrane potential difference of $180 mV [49], making it highly affinity for some chemical molecules by charge attraction, such as the TPP group [50].TPP group was combined with the free carboxyl group on the surface of UIO66-COOH via amidation through a classical NHS/EDC reaction system to form a mitochondrial-targeting Mn 3 O 4 /UIO-TPP nanozyme.
The FTIR spectra of the prepared Mn 3 O 4 /UIO-TPP nanozyme were compared with those of Mn 3 O 4 /UIO3 (Supplementary Figure S11), and the broad peak at the wavenumber of 2500-3200/cm, corresponded to free carboxylic acid O-H bonds, were decreased, which were caused by the consumption of carboxyl groups to form amide bonds.Correspondingly, Mn 3 O 4 /UIO-TPP showed a new absorption peak at wavenumber of 1660-1725/cm, correlated to C¼O bonds Band I in amide bonds [51].Besides, the surface charge of Mn 3 O 4 /UIO-TPP turned to 8.01 mV (Supplementary Figure S12A), with little significant changes in its hydrodynamic diameter (Supplementary Figure S12B).All above illustrated the successful fabrication of Mn 3 O 4 /UIO-TPP nanozyme.

Subcellular tracking and mitochondrial-targeting
Mitochondria were the primary generator of intracellular superoxide and the origin of cellular oxidative damage [52,53], thus the subcellular location of the nanozymes was critical for the scavenging of subcellular ROS.However, previous reported NPs as antioxidant drugs for mitochondria ROS scavenging were unrealistic in targeting effect by systemic administration [54], turned out to a weak ROS scavenging efficiency, and were easily cleared and metabolized in a short period [55], consequently resulting in limited therapeutic effect.This led to the need for repeated dosing and further increasing the toxic risk of the treatment, which also revealed improvements were still needed in the research of the relevant field.
Generally, the nanomaterials were first transported into lysosomes after cellular uptake [56], which may prevent effective targeting of NPs to mitochondria.It is imperative to break the lysosomal barriers to improve ROS scavenging efficiency [57].In our study, following the cellular uptake in chondrocytes, the nanozymes were located in lysosomes (Figure 5A).After incubation for 3 h, both FITC-labeled Mn 3 O 4 /UIO3 and Mn 3 O 4 /UIO-TPP showed strong colocalization signals primarily with lysosomes stained by LysoTracker Red (R ¼ 0.60 and R ¼ 0.50, Figure 5B).After 12 h, the colocalization coefficient with lysosomes of Mn 3 O 4 /UIO-TPP treated cells was significantly reduced, while it slightly decreased for Mn 3 O 4 /UIO3-treated cells, demonstrating the accelerated lysosomal escape effect of Mn 3 O 4 /UIO-TPP achieved by the positive surface charge after the TPP conjugation.
The capability of Mn 3 O 4 /UIO-TPP of mitochondrial-targeting (Figure 5C) was further investigated.Following labeling the mitochondria with MitoTracker Red, it was obvious to found Mn 3 O 4 / UIO-TPP showed preferential mitochondrial accumulation.After 12 h incubation, colocalization analysis (Figure 5D) illustrated the colocalization coefficient with mitochondria of Mn 3 O 4 /UIO-TPP nanozyme was R ¼ 0.52, higher than that of Mn 3 O 4 /UIO3 (R ¼ 0.19), revealing that Mn 3 O 4 /UIO-TPP nanozyme have the ability of breaking lysosomal barriers and mitochondrial-targeting.

Inhibition of mitochondrial damage
In order to reveal the intracellular functions of Mn 3 O 4 /UIO-TPP nanozyme on mitochondria under oxidative stress conditions, H 2 O 2 was used to induce chondrocytes to a state of oxidative stress, and mitochondrial ROS levels were first detected by MitoSOX fluorescence probe.As shown in Figure 6A and D, chondrocytes (G2) emitted a strong red fluorescent signal after being treated with H 2 O 2 or H 2 O 2 þ UIO66-COOH compared with the normal group (G1), which was lowered after treatment by Mn 3 O 4 / UIO3 (G4) or Mn 3 O 4 /UIO-TPP (G5) nanozyme.Besides, quantification analysis indicated that Mn 3 O 4 /UIO-TPP nanozyme (G5) were more efficient in mitochondrial superoxide scavenging than Mn 3 O 4 /UIO3 (G4), which means that Mn 3 O 4 /UIO-TPP nanozyme have a better mitochondrial protective function.
This advantage of mitochondrial protective function was also manifested in promoting DWm recovery.The maintenance of DWm is crucial for the proper functioning of mitochondria, as it enables electron transfer through an intact electron transport chain and facilitates ATP generation, among other essential mitochondrial activities [49].However, DWm was fragile and easy to depolarize under oxidative stress conditions [58].Herein, the protective effect of Mn 3 O 4 /UIO-TPP nanozyme on oxidative stressinduced DWm depolarization was evaluated by using a tetramethylrhodamine ethyl ester perchlorate (TMRE) fluorescent probe.Normally, the TMRE probe accumulated rapidly in healthy mitochondrial and generated a red fluorescence.When cells were under oxidative stress conditions, negligible fluorescence was detected compared with the normal group (G1).However, after incubation with additional Mn 3 O 4 /UIO3 or Mn 3 O 4 /UIO-TPP nanozyme, an apparent increase in the MFI could be detected (Figure 6B).The quantitative results (Figure 6E) showed that the Mn 3 O 4 /UIO-TPP group (G5) presented the highest fluorescence intensity among the experimental groups, restored to 70.8% of the normal group (G1), indicating Mn 3 O 4 /UIO-TPP could recover the DWm levels of chondrocytes due to oxidative damage to a certain extent.
Moreover, excessive intracellular ROS would induce mitochondrial Ca 2þ overload, unbalanced calcium homeostasis, mitochondrial function failure and cell death [59].Thereby, a mitochondrial Ca 2þ -specific Rhod2/AM fluorescent probe was next used to assess the intracellular mitochondrial calcium levels.As shown in Figure 6C and F of Ca 2þ levels, the fluorescence intensity increased within chondrocytes mitochondria after being stimulated with H 2 O 2 (G2), which was particularly reversed by Mn 3 O 4 /UIO-TPP (G5) nanozyme with MFI decreased by 54.6% compared with H 2 O 2 treated group (G2).The results indicated that Mn 3 O 4 /UIO-TPP nanozyme could rebalance mitochondrial calcium homeostasis under oxidative stress conditions.And due to intracellular ATP being mainly produced by mitochondria [60], ATP levels could also be used as a measure of mitochondrial function.As results in Figure 6G, Mn 3 O 4 /UIO-TPP nanozyme sufficiently alleviated the reduction of ATP in oxidative damaged cells.Finally, mtDNA expression levels as another important criterion [61] were further performed to evaluate mitochondrial function by qRT-PCR via its copy number (Figure 6H).The increased protective effect of Mn 3 O 4 /UIO-TPP nanozyme on mtDNA was better than Mn 3 O 4 /UIO3, implying that Mn 3 O 4 /UIO-TPP nanozyme was sufficient to eliminate mitochondrial superoxide, providing efficient protection of mitochondrial functions.

Inhibition of oxidative and inflammatory in H 2 O 2 -induced chondrocytes
To estimate the antioxidant and anti-inflammatory potency of Mn 3 O 4 /UIO-TPP in H 2 O 2 -induced chondrocytes, the survival of chondrocytes was first detected by live and dead staining.As shown in Supplementary Figure S13, H 2 O 2 pro-treated chondrocytes had a cell viability of 35.1%, demonstrating oxidative stress injury.However, Mn 3 O 4 /UIO3 and Mn 3 O 4 /UIO-TPP increased the cell viability to 63.3% and 77.0%, indicating they could prevent the H 2 O 2 -induced death of cells.
Later, the intracellular ROS levels were identified by a DCFH probe.Briefly, DCFH is an ROS probe that generated a green fluorescence when oxidized by intracellular ROS.Obviously, more intense green fluorescent signals could be noticed in Figure 7A and B for the H 2 O 2 -treated group (G2) compared with the normal group (G1).However, Mn 3 O 4 /UIO3 and Mn 3 O 4 /UIO-TPP significantly weakened fluorescence, especially for the Mn 3 O 4 /UIO-TPP group (G5), with the MFI reduction of 48.2% compared to the H 2 O 2 group (G2).Moreover, the intracellular ROS levels of chondrocytes was also investigated via a flow cytometry after labeling with DCFH (Figure 7C), and the trend presented was consistent with that under the fluorescence microscope.
The anti-inflammatory ability of Mn 3 O 4 /UIO-TPP nanozyme was measured by qRT-PCR.The induction of H 2 O 2 caused a significant upregulation of IL6, MMP13, COX2 and MMP3 expression levels (Figure 7D), which were pro-inflammatory factors and high correlation with OA inflammation [62][63][64][65].Compared with the H 2 O 2 group (G2), Mn 3 O 4 /UIO-TPP efficiently downregulated the gene expression of IL6, MMP13, COX2 and MMP3, which was also confirmed by immunofluorescence staining of IL6 and MMP13 (Figure 7E and F).Mn 3 O 4 /UIO-TPP nanozyme also significantly reduced the H 2 O 2 -induced oxidative damage of cellular DNA, which was demonstrated by the detection of c-H2AX foci (Figure 6G  and H).
All the above results indicated that Mn 3 O 4 /UIO-TPP nanozyme, as a multifunctional antioxidant, was effective in scavenging cellular ROS, anti-oxidative stress and anti-inflammatory in OA.

In vivo curative effect for therapy of OA
In order to further estimate the in vivo curative effect of OA therapy, an ACLT surgery was performed to construct the OA models on SD rats.Once the OA models were constructed, an overdose of pentobarbital sodium (Nembutal) was carried out for the euthanasia of rats at 4 and 8 weeks, and the intra-articular (IA) injected knee joints were harvested after that (Figure 8A).
Macroscopic observation (Figure 8B) for the collected knee joints showed that the femoral plateau and tibial plateau were smooth and shiny in the sham group, however, both plateaus showed severe wear and damage over time in the OA group.More importantly, after IA injection of Mn 3 O 4 /UIO-TPP nanozyme, the degree of destruction of the femoral plateau and the tibial plateau was greatly reduced in the Mn 3 O 4 /UIO3 and Mn 3 O 4 /UIO-TPP groups compared with the OA group, especially for Mn 3 O 4 /UIO-TPP group.The results of Pelletier scores (Figure 8C) showed the OA group scored 4.33 and 5.33 at 4 and 8 weeks, respectively.However, the Mn 3 O 4 /UIO-TPP group was scored as 1.33 and 2.67, which decreased by 69.3% and 49.9% compared to the OA group, respectively.Besides, in order to investigate the levels of IL6 and MMP13 expression in the joint cavity, synovial fluid was extracted and analyzed using ELISA.The results of the Mn 3 O 4 / UIO-TPP group in Figure 8D and Supplementary Figure S14 showed a significant reduction in the expression of IL6 and MMP13 in chondrocytes, illustrating its excellent antiinflammatory effect in vivo.
Furthermore, histological staining further demonstrated the therapeutic effect of Mn 3 O 4 /UIO3 and Mn 3 O 4 /UIO-TPP.The HE staining results shown in Figure 9A illustrated chondrocyte loss and collagen disruption that occurred over time on the joint surface of the OA group.S-G staining (Figure 9B) further confirmed this, in the OA group, cartilage erosion and proteoglycan loss with faint staining of safranine O were apparent on the joint surface.Importantly, these changes were well improved after IA injection of Mn 3 O 4 /UIO-TPP.The staining results of the Mn 3 O 4 /UIO-TPP group showed a smooth cartilage surface, close to the normal chondrocytes, which was further supported by Mankin scores (Supplementary Figure S15).Moreover, inflammatory factors (IL6 and MMP13) expression levels in chondrocytes were also evaluated by using immunohistochemical staining.As shown in Figure 9C and D, there was positive staining (dark brown) on the articular surface of the OA group, indicating the highly expressed IL6 and MMP13, which were greatly decreased in the Mn 3 O 4 /UIO-TPP group.
Meanwhile, in order to evaluate the retention time of Mn 3 O 4 / UIO-TPP nanozyme in vivo, Cy5.5-labeled Mn 3 O 4 /UIO-TPP was detected via an in vivo imaging system after IA injection (Supplementary Figure S16A).Since a knee joint injection of Mn 3 O 4 /UIO-TPP nanozyme was performed, a slow decrease of fluorescence intensity was detected in the first 6 h, and the fluorescence signals persevere at a high level for another 48 h (Supplementary Figure S16B).Furthermore, these fluorescence signals were also presented in other organs of SD rats (Supplementary Figure S17), especially for the kidney, spleen and liver, indicating that Mn 3 O 4 /UIO-TPP nanozyme could be cleared by hepatic and renal pathways.
Major organs were harvested after IA injection for in vivo cytotoxicity evaluation, the heart, liver, spleen, lung and kidney were included.And the histological images of these organs of rats were shown in Supplementary Figure S18.All organs of Mn 3 O 4 /UIO-TPP treated rats presented no apparent damages, similar to the sham group.
All above has demonstrated that mitochondrial-targeting Mn 3 O 4 /UIO-TPP nanozyme has a realistic therapeutic effect on OA, with favorable biosafety, outstanding stability and long retention in the joint cavity.

Conclusions
In summary, an artificially mitochondrial-targeting Mn 3 O 4 /UIO-TPP nanozyme as a potent ROS scavenger was developed by grafting Mn 3 O 4 NPs with UIO66 series MOFs and mitochondriatargeting triphenylphosphine (TPP) groups for OA therapy.Results showed that Mn 3 O 4 /UIO-TPP nanozyme possesses efficient cascade catalysis for ÁO À 2 and H 2 O 2 , presenting SOD-like, CAT-like and ÁOH scavenging activities.
By targeting mitochondria, Mn 3 O 4 /UIO-TPP nanozyme significantly normalized and rehabilitated the functions of ROSinduced damaged mitochondria and prevent chondrocyte

Figure 1 .
Figure 1.Fabrication route of Mn 3 O 4 /UIO-TPP and lowering ROS levels, reducing mtDNA/DNA damage, and anti-inflammatory for therapy of OA by mitochondrial-targeting.
3 O 4 content on nanozyme, Mn 3 O 4 / UIO nanozymes with lower Mn 3 O 4 content were also synthesized by adjusting the dosage of Mn(CH 3 COO) 2 Á4H 2 O to 4 mg or 16 mg while other procedures remain the same.Á4H 2 O from low to high.The brief reaction conditions were shown in Supplementary TableS1.