Abstract Osteoarthritis (OA) is the most common chronic inflammatory joint disease. Improving the joint inflammatory microenvironment is expected to promote early intervention and delay the progression of OA. However, effective strategies for inhibiting OA-related joint inflammation are still lacking. Lithospermic acid (LA), a polycyclic phenol carboxylic acid extracted from salvia miltiorrhiza, has strong anti-inflammatory and antioxidant effects. However, its role in the treatment of OA and the underlying mechanisms are unclear. To improve the bioavailability of LA, an LA synergistic protects etched zeolitic imidazolate framework (ZIF)-8 nanoparticles (LA@ZIF-8) was designed and developed for targeted delivery to modulate the inflammatory microenvironment in OA. This study confirmed that LA@ZIF-8 inhibits the pro-inflammatory phenotype of RAW264.7 macrophages through the NF-ĸB signaling pathway, effectively alleviates mitochondrial dysfunction, and delays articular cartilage degeneration caused by the joint inflammatory microenvironment mediated by synoval macrophages. In summary, LA@ZIF-8 delays the progression of OA by inhibiting synovial macrophage-mediated inflammatory responses, highlighting its clinical application potential. Keywords: Osteoarthritis, Synovitis, Lithospermic acid, Drug delivery, NF-κB pathway Graphical abstract [41]Image 1 [42]Open in a new tab 1. Introduction Osteoarthritis (OA) is a low-grade inflammatory disease affecting the entire joint. The key pathological changes include articular cartilage injury, subchondral osteosclerosis, and synovitis [[43]1,[44]2]. There is growing evidence that inflammatory responses in the synovium play a key role in the development and progression of OA, leading to joint swelling and pain. OA, the most common joint disease, places a significant economic burden on individuals and society [[45]3]. Current treatments for OA mainly involve local injections of glucocorticoids and oral non-steroidal anti-inflammatory drugs (NSAIDs), which only improve symptoms, such as joint pain, but do not delay the progression of the disease [[46]4]. Therefore, there is a critical necessity to develop effective therapeutic drugs that can delay OA progression. The joint microenvironment is crucial for the maintenance of cartilage homeostasis. Synovitis and cartilage degeneration are the two key pathological features of OA, with synovitis typically preceding cartilage degeneration [[47]5]. OA synovitis primarily manifests as the differentiation of synovial macrophages into the M1 pro-inflammatory phenotype, which expresses various inflammatory mediators and induces oxidative stress. Mechanistic studies have found that M1 pro-inflammatory phenotypes is regulated by the mechanistic target of rapamycin, nuclear factor-kappa B (NF-ĸB), and PI3K/Akt signaling pathways, among others [[48]6]. The activation of OA synovial macrophages produces various pro-inflammatory cytokines, such as interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF). These cytokines interact with chondrocytes through a paracrine pathway, causing chondrocyte inflammation and matrix degradation, leading to chondrocyte apoptosis and, by this means, exacerbating OA [[49]7,[50]8]. Furthermore, components such as chondrocyte extracellular matrix (ECM) degradation products continue to stimulate macrophage activation and exacerbate synovial inflammation, leading to cartilage damage [[51]9]. Therefore, early inhibition of synovial inflammation and reduction of M1 polarization in synovial macrophages are suitable intervention targets for the treatment of OA. Inflammation induces oxidative stress, leading to mitochondrial damage and reactive oxygen species (ROS) accumulation [[52][10], [53][11], [54][12]]. Simultaneously, dysfunctional mitochondria release various mitochondrial components and metabolites into the cytoplasm, further driving the inflammatory response and promoting the secretion of IL-1β, IL-18, and other inflammatory factors [[55]13,[56]14]. Mitochondria are dynamic organelles that constantly fuse or divide, a process known as mitochondrial dynamics [[57]15]. The process of fusion affects mitochondrial function, allowing mitochondria to form a network that favors oxidative phosphorylation (OXPHOS) and helps the cell resist the inflammatory environment [[58][16], [59][17], [60][18]]. Mitochondria that divide excessively into rings and fragments tend to produce high levels of ROS [[61]19]. Thus, it is speculated that mitochondrial dysfunction in OA synovial macrophages causes more macrophages to undergo M1 polarization by inducing inflammation. An increasing number of studies have confirmed that Chinese herbal medicines are effective in treating OA. Lithospermic acid (LA) is a polycyclic phenolic carboxylic acid derived from S. miltiorrhiza. It has strong anti-inflammatory [[62]20], antioxidant [[63]21], and antiviral effects [[64]22], and has demonstrated therapeutic effects in alleviating nephrotic syndrome [[65]23], mitigating acute myocardial ischemic injury [[66]24], and improving liver fibrosis [[67]25]. Studies have shown that LA effectively inhibits lipopolysaccharide (LPS)-induced macrophage inflammation [[68]20]. However, The therapeutic efficacy of the traditional Chinese medicine monomer LA in vivo is limited due to its poor stability and difficulty in crossing the cell membrane barrier for absorption. Nano-drug delivery systems offer significant advantages over traditional treatment methods, including higher drug loading rates, greatly improved drug availability, and enhanced therapeutic effects [[69]26]. Zeolitic imidazolate framework (ZIF)-8 is widely used as the basic framework for nanomedical drug delivery systems due to its easy preparation, good biocompatibility, and high stability [[70]27]. Phenolic nanotechnology is an emerging field in biomedical applications, providing a simple and versatile method to assemble nanostructures, with unique properties and high flexibility. Natural polyphenolic compounds, such as tannic acid, are rich in phenolic hydroxyl, hydroxyl, benzene rings, and double bonds, and exhibit excellent chelating ability to metal ions [[71]28]. Metal phenol complexes with unique properties and functions can be constructed by forming stable coordination bonds with metal ions. Phenolic acids are important adsorbents of heavy metal ions, and the organic complexes formed by their complexation with metal ions can effectively reduce their biological toxicity. When polyphenols and ZIF-8 are mixed in an aqueous solution, the polyphenols releases free protons, these free protons can be used to etch the structure of ZIF-8 [[72]29]. In addition, the attached phenolic acid or polyphenolic acid molecules block the pores of the metal-organic framework (MOF) due to their large molecular size, thus protecting the outer part of the MOF from further etching. The metal-phenolic networks (MPNs) produced by polyphenol-etched ZIF-8 have nanoscale shells, contain multiple functional elements, and have reduced transmembrane resistance and biotoxicity [[73]30]. We therefore hypothesised that LA can improve the joint inflammatory microenvironment by inhibiting macrophage inflammation, thereby treating OA cartilage injury. The LA@ZIF-8 nanoparticle was designed using ZIF-8 as a template and LA as an etching agent. We aimed to evaluate its biosafety in vivo and in vitro, screen and compare the inhibitory effect of the LA monomer and nanoparticles at appropriate concentrations on macrophage inflammation, and evaluate its effect on improving the OA inflammatory microenvironment and cartilage injury through co-culture with chondrocytes and intra-articular injection into the knee joint of OA mice. Transcriptome sequencing revealed the underlying mechanism of LA targeting macrophage inflammation treatment, offering valuable insights for the clinical development of innovative OA therapies and novel drug delivery systems. Continued research and development in this area has the potential to significantly advance the field of phenolic nanotechnology as well as cell therapy, providing new solutions for the treatment of various inflammatory diseases and conditions. 2. Methods 2.1. Reagent and instrument LA was purchased from MedChemExpress (USA). LPS was purchased from Solarbio (Beijing, China). Dulbecco's Modified Eagle Medium (DMEM) (21068028) and DMEM-F12(11320082) were purchased from Thermo Fisher Scientific Inc(USA). Primary antibodies against TNF-α (17590-1-AP), IL-1β (16806-1-AP), iNOS (22226-1-AP), COX-2 (66351-1-PBS), Gapdh (10494-1-AP), Col2a1(28459-1-AP), Mmp13 (18165-1-AP), Mfn1 (13798-1-AP), Opa1 (27733-1-AP), Fis1 (10956-1-AP), Drp1 (12957-1-AP) were purchased from Proteintech (Wuhan, China), Sox9 (A19710) were purchased from Abclone (Wuhan, China), and phospho-P65 (3033S), P65 (8242S), IKKβ (8943), Phospho-IKKβ (2697) were purchased from Cell Signaling Technology(USA). Cell media, penicillin/streptomycin (P/S), 0.25 % trypsin-EDTA, and fetal bovine serum (FBS) were purchased from Gibco (Grand Island, USA). Primary antibodies Anti-ROS Assay Kit (S0033S), Enhanced Mitochondrial Membrane Assay Potential Kit (C2003S), Mito-Tracker Green (C1048), Fluorine 488-labeled Goat Anti-Rabbit IgG (A0423), Goat Anti-Lamouse (A0423), fluorine 555-labeled Donkey Anti-Rabbit IgG (A0453), fluorine 555-labeled Donkey Anti-Mouse IgG (A0460), Protease and phosphatase inhibitor cocktail (P1045) for General Purpose purchased from Beyotime Biotechnology (Shanghai, China). Insulin-transferrin-selenium (ITS) was from Gibco (51500056). TRIZol™ Plus RNA Purification Kit (12183555CN) was purchased from Thermo Fisher Scientific (USA). Hieff® qPCR SYBR Green Master Mix was purchased from Yeasen Biotechnology (Shanghai, China). Deionized water used in all experiments was prepared using a Milli-Q water purification system (Millipore, Boston, MA, USA). 2.2. Fabrication of ZIF-8 and LA@ZIF-8 nanoparticles The development of nanometric ZIF-8 and LA@ZIF-8 particles was carried out in a mixed solvent environment with H[2]O and methanol. Before synthesis, 150 mg (mg) of zinc nitrate hexahydrate (Zn(NO[3]) [2]·6H[2]O) were dissolved in 5 mL (mL) of deionized water, while 165 mg of 2-methylimidazole were uniformly dispersed in 5 mL of methanol, Ultrasonic assisted dissolution. Lithospermic acid was carefully dissolved in methanol to obtain a drug concentration of 60 mg per milliliter (60 mg/mL). At the beginning of the synthesis, the Zn(NO[3]) [2]·6H[2]O solution (5 mL) was stirred vigorously at a speed of 500 revolutions per minute (rpm). Next, the 2-methylimidazole solution (5 mL) was carefully poured into the Zn(NO[3]) [2] · 6H[2]O solution with vigorous stirring, and then 4 mL of methanol was added. After a 2-h reaction period at room temperature, ZIF-8 nanoparticles were collected by centrifugation, during which they underwent three thorough washes with methanol to remove undigested reagents. Mirroring the procedure for ZIF-8, 5 mL of 2-methylimidazole solution were gradually incorporated into Zn (NO[3]) [2]·6H[2]O stock solution (5 mL), then join 2 mL lithospermic acid solution, stirred at 500 rpm/min, then 4 mL of methanol was added. The stirred mixture was then allowed to react for 20 min at room temperature. The nanoscopic LA@ZIF-8 particles were then isolated by centrifugation at 12,000 rpm for 10 min, subjected to three exhaustive methanol washes to remove the unpackaged lithospermic acid. After vacuum lyophilization at −80 °C, nanoscale ZIF-8 and LA@ZIF-8 particles were prepared for further characterization efforts. Preparation of ZIF-8 nanoparticles supported with fluorescein GFP or Rhodamine B (RhoB): 50 μg of GFP or Rhodamine B (RhoB) is added to 2-methylimidazole solution, and the subsequent synthesis steps are described above to synthesize nanoparticles with GFP or RhoB fluorescence. 2.3. Comprehensive characterization of LA@ZIF-8 nanoparticles The characterization of ZIF-8 and LA@ZIF-8 nanoparticles was conducted through TEM (JEOL, Tokyo, Japan). The structure of the ZIF-8 and LA@ZIF-8 were monitored using X-ray diffractometer (XRD, Bruker D8 Focus Powder XRD) analysis. X-ray photoelectron spectroscopy (XPS) was conducted with an X-ray photoelectron spectroscope (ESCALAB 250 XI, Thermo Fisher Scientific, Waltham, MA, USA).To further investigate its physical properties, dynamic light scattering (DLS) technology from Malvern, UK was used to accurately quantify its zeta potential and particle size distribution. 2.4. Drug loading and release After a careful rinsing procedure with methyl alcohol to remove any unencapsulated lithospermic acid, LA@ZIF-8 nanoparticles were successfully synthesized. A certain amount of dry LA@ZIF-8 nanoparticles is taken and denoted as m[0]. Subsequently, the nanoparticles are completely dissolved using a dilute nitric acid solution, then centrifuged and filtered, where the residue represents the extracted LA, LA was freeze-dried and weighed as m[L].The loading ratio (LR) = m[L]/m[0]∗100 %. The lithospermic acid release profile was studied under acidic (pH 6.0) and neutral (pH 7.2) conditions. Initially, 1 mL of LA@ZIF-8 nanoparticles was encapsulated in a dialysis membrane (MWCO, 3500 Da). This membrane was immersed in 30 mL of phosphate buffered saline (PBS), supplemented with 5 % polysorbate 80, at the specified pH levels. The system was shaken on a vibrating table at a constant speed of 100 rpm and maintained at a constant temperature of 37 °C. At predetermined time intervals (0, 0.5, 1, 2, 4, 6, 8, 12, 24h), PBS was removed for analysis and refreshed with fresh PBS. The concentrations of lithospermic acid released in the PBS samples were accurately determined by UV–visible spectroscopy at 370 nm. 2.5. Cell culture and treatment RAW264.7 cells were grown in DMEM medium supplemented with 10 % fetal bovine serum and 1 % penicillin-streptomycin only. RAW264.7 cells are induced into M1-type macrophages after 12 h of induction with 100 ng/mL LPS. Cells grown in fresh medium were used as a control. The ATDC5 cell line was cultured in DMEM/F12 medium supplemented with 5 % (v/v) fetal bovine serum (FBS) and 1 % penicillin-streptomycin solution at 37 °C in a humidified incubator maintained at 5 % CO[2]. To stimulate ATDC5 cell differentiation, 1 % (v/v) insulin-transferrin-selenium (ITS) supplement was used. After an incubation period of 96 h, a sophisticated pattern of cell proliferation, indicative of differentiation, materialized. 2.6. Macrophages conditioned medium (CM) collection RAW264.7 cells were seeded at a density of 1 × 10^6 cells/well in a 6-well plate and then incubated with 100 ng/mL LPS plus 50 μg/mL LA, 50 μg/mL ZIF-8 or 50 μg/mL LA@ZIF-8 for 12 h, cells cultured in fresh medium were used as control. After that, the supernatant of each group was centrifuged at 1000 g for 5 min and stored at −80 °C for further experiments. CM was diluted with serum-free DMEM-F12 medium to a final concentration of 50 % and added to Petri dishes for chondrocyte culture [[74]31]. 2.7. In vitro cytotoxicity Cell cytotoxicity was assessed using the Cell Counting Kit-8 (CCK-8). Briefly, Raw264.7 cells were cultured in 96-well plates at a density of 1 × 10^4 cells/well, followed by LA treatments (0, 12.5, 25, 50, 100, 200 or 400 μg/mL),ZIF-8 treatments (0, 12.5, 25, 50, 100, 200 or 400 μg/mL) or LA@ZIF-8 treatments (0, 12.5, 25, 50, 100, 200 or 400 μg/mL) for 24 h. Each plate was then filled with 100 μl of 10 % CCK-8 solution and incubated at 37 °C for 120 min. The absorbance of the wells was measured at 450 nm using a microplate reader (Leica Microsystems, Wetzlar, Germany). All results are received through three independent experiments. 2.8. Intracellular uptake of nanoparticles Endocytosis was monitored using LA@ZIF-8 for fluorescence localization. LA@ZIF-8 was incubated with RAW264.7 cells in a 6-well plate. After a 12 h incubation, the cells were washed with PBS and then incubated with DAPI. Finally, the slides were examined under a confocal laser scanning fluorescence microscope (CLSM). 2.9. Therapeutic effect on M1 polarization of RAW264.7 As described before, LPS has been used to simulate synovitis in the development of osteoarthritis. RAW264.7 cells were divided into four groups: 1) control group: RAW264.7 treated with fresh medium only; 2) LPS group: RAW264.7 cells induced in macrophages M1-type modeled after 12 h induction with 100 ng/mLLPS; 3)ZIF-8 group: RAW264.7 was incubated with 100 ng/mL LPS plus 50 μg/mL ZIF-8 for 12 h. 4) LA group: RAW264.7 was incubated with 100 ng/mL LPS plus 50 μg/mL LA for 12 h; 5) LA@ZIF-8 group: RAW264.7 was incubated with 100 ng/mL LPS plus 50 μg/mL LA@ZIF-8 for 12 h. 2.10. ROS detection After 12 h of culture in 6-well plates, ROS levels in different groups of RAW264.7 cells were detected using the fluorescence probe DCFH-DA. The cells were observed under a fluorescence microscope (DM4000 B; Leica, Germany). 2.11. Western blotting RAW264.7 cells or ATDC5 chondrocytes were seeded in 6-well plates to collect total protein. RAW264.7 cells were stimulated with LPS (100 ng/mL) in the presence or absence of LA (50 μg/mL), ZIF-8 (50 μg/mL) or LA@ZIF-8 (50 μg/mL) treatment for 12 h. CM was co-cultured with ATDC5 chondrocytes for 24 h. RAW264.7 cells or ATDC5 chondrocytes were lysed with radioimmunoprecipitation lysis buffer (RIPA) containing a cocktail of protease and phosphatase inhibitors. Quantification of lysates was evaluated with a BCA (bicinchoninic acid) test kit before its denaturation process at 99 °C for a duration of 10 min. The samples were transferred to nitrocellulose filter membranes (Millipore, USA). For the detection of specific proteins, target antibodies are used during the incubation process. Proteins were visualized and documented using BIO-RAD's ChemiDOC Western blot imaging system. Antibodies used for western blotting were TNF-α, IL-1β, iNOS, COX-2, GAPDH, Col2a1, Mmp13, Phospho-NF-κB, NF-κB, IKKβ, p-IKKβ, Sox9, Mfn, Opa, Fis1, Drp1. GAPDH was used as an internal control. 2.12. Quantitative real–time PCR RAW264.7 or ATDC5 cells were plated in 6-well plates to collect total RNA. After treatment in each group, the TRIzol™ Plus RNA purification kit was used to lyse RAW264.7 or ATDC5 cells according to the manufacturer's instructions. Complementary cDNA was synthesized by PrimeScript™ RT Master Mix and real-time PCR analyzes were performed in QuantStudio 7 (Thermo) using Hieff® qPCR SYBR Green Master Mix. The cDNA was predenatured at 95 °C for 2 min and denatured at 95 °C for 10 s and annealing/stretching at 60 °C for 30s. The primers used were the following: β-actin, forward, 5′-TATGCTTCCCTCACGCCATCC-3'; reverse, 5′-GTCACGCACGATTTCCCTCTCTCAG-3'; IL-1β, forward, 5′-TCCGAGCAGCACATCAACAAGAG-3'; reverse, 5′-AGGTTCCACGGGAAAGACACAGG-3'; COX-2, forward, 5′-TTCAACACACTCTATCACTGGC-3′, reverse, 5′-AGAAGCGTTTGCGGTACTCAT-3'; iNOS, forward, 5′-GTTCTCAGCCCAACAATACAAGA3′, reverse, 5′-GTGGACGGTCGATGTCAC3'; Sox9, forward, 5′-GAGCCGGATCTGAAGAGGGA-3'; reverse, 5′-GCTTGACGTGTGGCTTGTTC-3'; Mmp9, forward, 5′-CGCCACCACAGCCAACTATGACA-3′ reverse, 5′-CTGCTTGCCCAGGAAGACGAAGA-3'.Tnf-α, forward, 5′ GACGTGGAACTGGCAGAAGAG-3′ reverse, 5′-TTGGTGGTTTTGTGAGTGTGAG-3'; Adamts5, forward, 5′-CAACAGGAGGATCATCGCAGATACAG-3'; reverse, 5′-CCAAGGTCACCATCATTACACCAAGT-3'; Mmp13, forward, 5′-TGGAGTAATCGCATTGTGAGAGTC-3′, reverse, 5′-CCAGCCACGCATAGTCATATAGATAC -3'. Expression levels of specific RNAs were normalized to β-actin. 2.13. Immunofluorescence of RAW264.7 and ADTC5 in vitro LPS-treated RAW264.7 cells were co-cultured with LA (50 μg/mL),ZIF-8 (50 μg/mL)or LA@ZIF-8 (50 μg/mL)in 6-well plates for 12 h. CM was co-cultured with ATDC5 chondrocytes for 24 h. Then, the cells were washed three times with PBS and fixed with a 4 % paraformaldehyde solution. After incubating with 0.5 % Triton X-100 in PBS for 15 min, the cells were blocked with 5 % goat serum for 1 h. Cells were then treated with primary antibodies against Tnf-α, iNOS, Col2a1 and Mmp13 applied at 4 °C overnight. Secondary antibodies were incubated for 60 min at room temperature with Alexa Fluor 555 label or Alexa Fluor 488 label. Images were examined by fluorescence microscopy after 1 min of DAPI labeling. 2.14. Alcian blue staining of ATDC5 in vitro Alcian blue staining of ATDC5 cells in 6-well plates was performed to visualize cartilage degeneration. ATDC5 cells were cultured in 6-well plates overnight. Following a 24-h treatment with CM, the cells were stained with Alcian blue according to the manufacturer's recommended protocol. Finally, images were analyzed using a light microscope. 2.15. Mitochondrial membrane potential (JC-1) analysis After culture for 24 h with CM in 6-well plates, ATDC5 chondrocytes were washed once with PBS, 1 mL of cell culture medium and 1 mL of JC-1 staining medium were added and cultivated at 37 °C for 20 min. Remove the supernatant and wash three times with JC-1 staining buffer. By adding 2 mL of cell medium, the cells were observed under a confocal laser microscope [[75]32]. 2.16. Mitochondrial morphology analysis After 24 h of culturing ATDC5 chondrocytes with CM in 6-well plates, mitochondrial morphology was assessed using the aforementioned methodologies and reagents. Then, Mito-Tracker Green (1:5000) and Mito-Tracker Red CMXRos (1:1000) solution were introduced and incubated at 37 °C for 30 min. The MitoTracker Red CMXRos solution was then discarded and fresh cell culture medium prewarmed to 37 °C was added. The cells were then examined using the laser confocal microscope (TCS SP8; Leica, Germany). 2.17. Transmission electron microscopy (TEM) analysis ATDC5 chondrocyte cells after treatment in 6-well plates were examined using TEM. Adherent cell blocks, obtained by centrifugation, were subjected to fixation and dehydration procedures. Specifically, the sample was initially fixed with 2.5 % glutaraldehyde for a duration of 2.5 h, followed by three washes in phosphate buffered saline (PBS) (0.1 M, pH 7.0), each lasting 3 min and fixed in 1 % tetroxidedosmium for 2 h. After that, the cells are washed three times. Cells were subjected to a continuous dehydration process using a graded ethanol series, followed by two cycles of dehydration in a 1:1 mixture of ethanol and acetone, and finally in pure acetone. Each time, the cells were infiltrated with acetone: resin 3:1, 1:1, 1:1, 1:1, 1:1 and 1:1 for 1 h. Cells were impregnated with resin overnight and embedded in fresh resin for 3 h. After an additional polymerization at 37 °C for 8 h and at 65 °C for 48 h, The resin segments were sliced into ultrathin slices (70–100 nm). Each ultrathin tissue section was precisely placed on a copper grid with carbon film and then stained with uranyl acetate at 4 °C for 7 min. After staining, slices were carefully cleared, followed by a second lead citrate stain at 25 °C for 3 min, then observed and images captured (Talos L120C, FEI; Thermo Fisher Scientific). 2.18. Establishment of anterior cruciate ligament transection (ACLT) in mice 24 male C57BL/6 mice, 8 weeks old, were obtained from Shanghai SIPPR BK Laboratory Animals Ltd. (Shanghai, China) for the study. Mice were maintained under a 12 h light/dark cycle with ad libitum access to food and water. All animals used in this study received ethical approval and were cared for in accordance with the institutional guidelines established by the Experimental Animal Ethics Committee of Shanghai Jiao Tong University School of Medicine (XHEC-F-2025-003). Osteoarthritis (OA) models were created by the ACLT method, divided into the sham operation group, the ACLT group,the ACLT + ZIF-8 group, the ACLT + LA group and the ACLT + LA@ZIF-8 group. Five days after surgery, intra-articular injections of LA, ZIF-8 or LA@ZIF-8 were administered for subsequent histological analysis. Specifically, mice in ACLT group were injected intra-articularly with PBS, and mice in ACLT + ZIF-8 group and ACLT + LA@ZIF-8 were injected intra-articularly with 100 μL of 50 μg/mL ZIF-8 or LA@ZIF-8 NPs, and mice in LA group were injected intra-articularly with 100 μL of 50 μg/mL LA every 3 days for 4 weeks. 2.19. In vivo imaging system (IVIS) imaging The residence time and biodistribution behaviors of LA@ZIF-8 were observed using a noninvasive IVIS (AniView 100, BLT, China) by the administration of NPs into OA mice intra-articularly. After 72 h, specific tissues, such as the heart, spleen, lung, liver, and kidney, were obtained for IVIS. 2.20. Histological and immunofluorescence analyses and OARSI scoring The knee joints of mice with osteoarthritis (OA) were collected and then fixed in 4 % paraformaldehyde (PFA). The samples were decalcified with 10 % EDTA before being embedded in paraffin. Safranin O-fast green and hematoxylin and eosin (H&E) staining were used to assess changes in cartilage microstructure after LA, ZIF-8 or LA@ZIF-8 treatment in 4-μm knee sections. As mentioned, the Mankin score and the International Osteoarthritis Research Society score (OARSI) were calculated. Immunohistochemical (IHC) staining was performed with antibodies against iNOS, p-P65, Col2a1, Aggrecan, Sox9. Immunofluorescence staining was performed with an antibody against iNOS, Drp1. The percentage of positively stained cells or positive field in synovium and articular cartilage was determined by Image J software. In addition, to evaluate the systemic side effect of nanoparticles, the main organs of a subgroup of mice, especially the heart, spleen, lungs. liver and kidney, were removed. To confirm the therapeutic effect roles of LA@ZIF-8 in vivo. 2.21. Statistical analysis SPSS 20.0 software was used for this statistical analysis. All quantitative data sets are presented as mean ± standard deviation (SD). Differences between groups were analyzed by one-way analysis of variance (ANOVA), followed by Tukey's post hoc test. Statistical significance is considered ∗ p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. 3. Results 3.1. LA@ZIF-8 synthesis and characterisation LA etching ZIF-8 synthesis process and mechanism diagram ([76]Fig. 1A),LA, as a polyphenolic compound featuring phenolic hydroxyl groups that act as chelating sites, can coordinate with zinc ions on the outside of ZIF-8 particles. This coordination facilitates the release of free H+ ions, which attach to LA molecules and subsequently infiltrate into ZIF-8, achieving gradual internal etching. Due to the strong metal-phenol interaction, a large amount of LA in the solution tends to adhere to the surface of ZIF-8, stabilising the overall crystal form of ZIF-8 and promoting the transformation of the surface of ZIF-8 from hydrophobic to hydrophilic. TEM images clearly show that the ZIF-8 particles are ZIF-8 NPs has a uniform, monodisperse hexagonal morphology and a smooth surface, with a size of about 200–300 nm. After LA modification, the surface of ZIF-8 particles became rough, and small voids were created within each ZIF-8 particle and a network structure appeared, confirming that protons were dissociated from LA and etched inward through the hydrophilic surface of ZIF-8 ([77]Fig. 1B). The distribution of O elements is more dense than that of ZIF-8 particles, possibly due to the large amount of phenol hydroxyl group contained in LA.Compared to the element distribution map of ZIF-8 particles, there are notable areas where elemental distribution appears absent ([78]Fig. 1C). The XRD characterization verifies the structure of LA-modified ZIF-8, as evidenced by characteristic diffraction signals that match those of pristine ZIF-8 crystals, demonstrating the preservation of framework topology during LA incorporation([79]Fig. 1D). The XPS survey spectrum of the LA@ZIF-8 revealed the presence of O, C, N, and Zn([80]Fig. 1E). The XPS data of C 1s spectrum of the LA@ZIF-8 showed typical types of carbon bonds: C-N, C=O, C-C and C=C respectively([81]Fig. 1F). N 1s indicated typical types of carbon bonds: C-N and C=N ([82]Fig. 1G). Zn 2p indicated typical types of bonds: Zn2p 2/3 and Zn2p 1/2([83]Fig. 1H). After LA etching, the zeta potential of ZIF-8 decreased from 19 to −7 mV, possibly due to the large number of phenolic hydroxyl groups on the surface of ZIF-8, further confirming the successful preparation ([84]Fig. 1I). LA@ZIF-8 has a slightly larger diameter compared to ZIF-8, possibly due to the coordination of LA on the surface of ZIF-8 with zinc ions, forming a metal-phenol network ([85]Fig. 1J). The particle size of LA@ZIF-8 varies slightly between 291 and 317 nm, indicating excellent stability over 7 days ([86]Fig. 1K). In order to investigate the pH response behavior of ZIF-8 NPs, in vitro release studies of LA were conducted in PBS with different pH values (pH6.0, pH 7.2), Under pH 7.2 conditions, the drug release rate of LA was only 19.8 % at 12 h and 37.2 % at 24 h. In contrast, under pH 6.0 conditions, the cumulative drug release rate was significantly accelerated, reaching 81.2 % at 12 h and 96.2 % at 24 h (Fig. 1L). These findings indicate that LA@ZIF-8 exhibits a responsive behavior in mildly acidic environments, thereby enabling controlled release of LA. These results suggest that LA@ZIF-8 responds to mildly acidic environments and allows for controlled release of LA. Fig. 1. [87]Fig. 1 [88]Open in a new tab Synthesis and characterization of LA@ZIF-8. A) LA etching ZIF8 synthesis process and mechanism diagram. B) TEM images of ZIF-8 and LA@ZIF-8. C) Mapping of ZIF-8 and LA@ZIF-8. D) XRD images of ZIF-8 and LA@ZIF-8. E) XPS of ZIF-8 and LA@ZIF-8 for full survey scan. F) XPS analyses of C 1s in LA@ZIF-8. G) XPS analyses of N 1s in LA@ZIF-8. H) XPS analyses of Zn2p in LA@ZIF-8. I) Zetal potential of ZIF-8 and LA@ZIF-8. J) DLS of ZIF-8 and LA@ZIF-8. K) The stability analysis of LA@ZIF-8. L) Cumulative in vitro drug release of LA@ZIF-8 in PBS (pH 6.0 and 7.2). The data are presented as the mean ± SD. n = 3. 3.2. Cytotoxicity and cellular uptake of LA@ZIF-8 To determine the safe concentrations of LA and LA@ZIF-8 acting on RAW264.7 cells, we conducted a co-incubation experiment with ZIF-8, LA or LA@ZIF-8 for 24 h ([89]Fig. 2A). The effect of ZIF-8,LA and LA@ZIF-8 on RAW264.7 cells viability was measured using the CCK-8 assay. When the concentration of ZIF-8 reaches 200 μg/mL, it will produce obvious toxic effect on raw264.7 cells, indicating that ZIF-8 has high biocompatibility ([90]Fig. 2B). The results indicated that LA had no significant cytotoxic effect on RAW264.7 cells in the concentration range of 0–50 μg/mL. However, the cells viability decreased significantly when the LA concentration exceeded 50 μg/mL ([91]Fig. 2C). The obvious toxicity of LA@ZIF-8 was observed only when the concentration of LA@ZIF-8 reached 100 μg/mL., ([92]Fig. 2D). Consequently, a concentration of 50 μg/mL was selected for subsequent experiments on LA and LA@ZIF-8. GFP was encapsulated in ZIF-8 and LA@ZIF-8 and co-incubated with RAW264.7 cells for 24 h. Confocal laser microscopy revealed green fluorescence localised around the nucleus, confirming cellular uptake of ZIF-8 and LA@ZIF-8 ([93]Fig. 2E). Fig. 2. [94]Fig. 2 [95]Open in a new tab Evaluate the impact of LA@ZIF-8 on M0 macrophages. A) Schematic diagram to explore the safe concentration of ZIF-8, LA and LA@ZIF-8. B-D) CCK8 assay of macrophages treated with various concentrations of ZIF-8, LA and LA@ZIF-8 for 24h. E) Fluorescence images of RAW264.7 cells endocytosis with ZIF8 and LA@ZIF-8 for 24 h. Scale bars are 25 μm. The data are presented as the mean ± SD. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, n = 3. 3.3. LA@ZIF-8 suppressed LPS-induced M1 polarization of RAW264.7 macrophages RAW264.7 cells were treated with 100 ng/mL LPS to induce M1 macrophages, establishing a model of arthritic synovitis cells, followed by treatment with LA or LA@ZIF-8. Therapeutic effects were evaluated based on the expression of inflammation-related genes or proteins ([96]Fig. 3A). The inflammatory factors produced by M1 macrophages can cause mitochondrial dysfunction, leading to oxidative stress and ROS accumulation. The macrophages in each group were labeled with DCFH-DA to evaluate the effects of LA and LA@ZIF-8 on ROS clearance by M1 macrophages. LPS-treated M1 macrophages produced a substantial amount of ROS, and the green fluorescence intensity was significantly reduced following LA and LA@ZIF-8 treatments. LA@ZIF-8, on the other hand, showed a stronger ability to clear ROS (p < 0.0001) (Fig. 3B and C). RAW264.7 macrophages induced by LPS were treated with 50 μg/mL LA, 50 μg/mL ZIF-8 or 50 μg/mL LA@ZIF-8, and the mRNA expression levels of M1 macrophage markers TNF-α, IL-1β, iNOS, and COX-2 were measured in each group. The expression of inflammation-related genes in RAW264.7 macrophages was significantly downregulated following treatment with LA or LA@ZIF-8 ([97]Fig. 3D). Further analysis of inflammation-related protein expression in macrophages revealed significant upregulation of TNF-α, iNOS, COX-2, and IL-1β in RAW264.7 cells in the LPS group. Treatment with LA or LA@ZIF-8 downregulated these protein levels, with LA@ZIF-8 showing stronger anti-inflammatory effects ([98]Fig. 3E and F). M1-type macrophage markers, TNF-α and iNOS, were labeled by immunofluorescence staining. Following treatment in the LA and LA@ZIF-8 groups, the level of M1 macrophage markers, TNF-α, and iNOS, were significantly decreased compared to those in the LPS group, demonstrating strong anti-inflammatory effects. The efficacy of LA was enhanced in the LA@ZIF-8 group ([99]Fig. 3G and H). In conclusion, LA@ZIF-8 inhibited the LPS-induced pro-inflammatory phenotype of RAW264.7 macrophages and effectively reduced ROS accumulation caused by inflammation. Fig. 3. [100]Fig. 3 [101]Open in a new tab Effect of LA@ZIF-8 on anti-inflammatory in LPS-activated RAW264.7 cells. A) Schematic diagram of explore the effect of ZIF-8, LA and LA@ZIF-8 on M1 macrophage inflammation. B) Representative immunofluorescence images of LPS-stimulated RAW264,7 cells stained with ROS fluorescent probe DCFH-DA. Scale bars are 50 μm. C) Quantitative analysis of ROS level. D) Expression of M1 macrophage markers COX-2, Tnf-α, iNOS and IL-1β were examined by qPCR. E) Western blot showing the expression of Tnf-α, iNOS, COX-2 and IL-1β in macrophages after various treatments. F) Quantification of expression levels of Tnf-α, iNOS, COX-2 and IL-1β protein. G) Representative images of immunostaining for M1 marker (TNF-α and iNOS) in RAW264.7 cells in the different groups. Scale bars are 50 μm. H) Quantification of the fluorescence intensity in different groups. The data are presented as the mean ± SD. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, n = 3. 3.4. LA@ZIF-8 salvage inflammation-induced mitochondrial damage in RAW264.7 macrophages To further verify the therapeutic effects of LA, ZIF-8 and LA@ZIF-8 on mitochondrial dysfunction in LPS-induced M1-polarized macrophages, RAW264.7 cells were treated with 100 ng/mL LPS. The mitochondrial membrane potential, mitochondrial morphology, and related protein expression levels were compared before and after LA or LA@ZIF-8 treatment in all groups ([102]Fig. 4A). Fluorescence staining revealed that the mitochondrial membrane potential of macrophages was significantly reduced after LPS stimulation, indicating that LPS induces mitochondrial dysfunction in M1 macrophages. After treatment with LA, ZIF-8 or LA@ZIF-8, the red fluorescence of macrophage mitochondrial polymers significantly increased, while the green fluorescence of mitochondrial monomers significantly decreased ([103]Fig. 4B and C). Mitochondria are highly dynamic, with their form and function regulated through fusion and division. Excessive mitochondrial division leads to fragmentation and mitochondrial network damage. Mitochondrial probes were used to label the macrophage mitochondria in each group, and LA@ZIF-8 effectively reversed LPS-induced mitochondrial division in macrophages ([104]Fig. 4D and E). The morphology of mitochondria was observed using TEM, revealing oval or oblong cross-sections with regular ridge arrangements in normal mitochondria. Following LPS stimulation, the mitochondrial matrix swelled, and the ridge density decreased. Mitochondrial damage in each group was characterized by the mitochondrial cristae volume density analysis [[105]10,[106]12]. After treatment with LA@ZIF-8, mitochondrial matrix swelling and ridge disorders were significantly reduced ([107]Fig. 4F and G). The expression levels of mitochondrial division and fusion proteins Drp1, Fis1, Opa1, and Mfn1 were analyzed. It was observed that the levels of Drp1 and Fis1 proteins associated with mitochondrial division significantly increased in M1 macrophages after LPS induction, while the levels of Opa1 and Mfn1 proteins associated with mitochondrial fusion significantly decreased ([108]Fig. 4H and I). These results indicate that LPS-induced mitochondrial division was predominant in M1 macrophages. Compared to the LPS group, the protein levels of Drp1 and Fis1 were significantly downregulated following treatment with LA or LA@ZIF-8, while the protein levels of Opa1 and Mfn1 were significantly upregulated. This suggests that LA or LA@ZIF-8 treatment had an inhibitory effect on LPS-induced mitochondrial hyperdivision in M1 macrophages. In conclusion, the treatment of mitochondrial dysfunction with ZIF-8 alone has no effective therapeutic effect. LA@ZIF-8 significantly alleviated LPS-induced mitochondrial dysfunction of RAW264.7 macrophages and markedly improved mitochondrial morphology; its efficacy was superior to that of LA alone. Fig. 4. [109]Fig. 4 [110]Open in a new tab Mitochondrial Function Recovery by LA@ZIF-8 A) Schematic diagram of mechanism by which LA@ZIF-8 reduces mitochondrial dysfunction in M1-polarized macrophages. B) Representative images of Mitochondrial membrane potential of M1 macrophages after various treatments stained with JC-1. Scale bars are 50 μm. C) Quantitative analysis of JC-1 monomer/JC-1 aggregates ratio. D) RAW264.7 cells were treated with various treatment incubation with or without LPS and stained with Mito-Tracker observe mitochondrial function. E) Quantitative analysis of mitochondrial length. F) Mitochondrial morphology observed by TEM. G) Quantitative analysis of mitochondrial cristae volume density. H) The protein expression of Mfn1, Opa1, Fis1 and Drp1 in RAW264.7 cells after various treatments. I) Quantification of expression levels of Mfn1, Opa1, Fis1 and Drp1 protein. The data are presented as the mean ± SD. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, n = 3. 3.5. LA@ZIF-8 protects ATDC5 chondrocytes by inhibiting the polarization of RAW264.7 macrophages To assess the effect of M1 macrophages on chondrocytes, the CM of treated RAW264.7 cells was incubated with ATDC5 chondrocytes, which were induced by ITS for 24 h. The synthesis and degradation function of the chondrocytes were then evaluated ([111]Fig. 5A). After 24 h of incubation, the M1+LA-CM and M1+LA@ZIF-8-CM groups experienced reversed dysregulation of chondrocyte synthesis and catabolic homeostasis caused by M1-CM, but ZIF-8 alone has no therapeutic effect ([112]Fig. 5B). Expression of the catabolism-related genes/proteins Adamts5, Mmp9, and Mmp13 in the M1-CM group was significantly reduced, while the expression of the metabolism-related proteins Col2a1 and Sox9 significantly increased ([113]Fig. 5C–E). DCFH-DA was used to label each group to evaluate the effect of different CMs on ROS production in ATDC5 chondrocytes. Results indicated that ATDC5 chondrocytes treated with M1-CM produced more ROS, and using ZIF-8 alone does not reduce ROS generation. LA and LA@ZIF-8 significantly reversed chondrocyte ROS accumulation induced by M1-CM ([114]Fig. 5F and G). Immunofluorescence analysis confirmed that Mmp13 expression significantly increased, while Col2a1 expression significantly decreased in ATDC5 chondrocytes treated with M1-CM, M1+LA-CM and M1+LA@ZIF-8-CM groups significantly reversed this phenomenon ([115]Fig. 5H and I). In summary, LA@ZIF-8 has stronger protection against macrophage inflammation in OA chondrocytes compared with LA. Fig. 5. [116]Fig. 5 [117]Open in a new tab Protective effect of LA@ZIF-8 on chondrocytes. A) Schematic diagram of the CMs was co-cultured with ATDC5 induced by ITS to verify the protective effect of LA@ZIF-8-CM on chondrocytes. B) Alcian blue staining of mucopolysaccharide (accharide) -like proteoglycan in ATDC5 chondrocytes. C) The catabolic genes(Adamts5、Mmp9、Mmp13) were identified by qPCR. D) The protein expression of synthesis and decomposition of related protein (Col2a1, Sox9 and Mmp13) in ATDC5 chondrocytes after various treatments. E) Quantification of expression levels of Col2a1, Sox9 and Mmp13 protein. F) Representative immunofluorescence images of ATDC5 chondrocytes after different treatments stained with ROS fluorescent probe DCFH-DA. Scale bars are 200 μm. G) Quantitative analysis of ROS. H) Representative images of immunostaining for Mmp13 and Col2a1 in ATDC5 chondrocytes after different treatments. I) Quantification of the fluorescence intensity in different groups. The data are presented as the mean ± SD. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, n = 3. (For interpretation of the references to colour in this figure legend, the reader is referred to