Novel strategy of senescence elimination via toxicity-exempted kinome perturbations by nanoliposome-based thermosensitive hydrogel for osteoarthritis therapy

Cellular senescence and the senescence-associated secretory phenotype (SASP) have been implicated in osteoarthritis (OA). This study aims to determine whether multi-kinase inhibitor YKL-05-099 (Y099) has potential in senescence elimination and OA therapy and whether delivering Y099 by nanoliposmal hydrogel improves the performance of the kinase inhibitor. Y099 inhibited IL-1β-induced inflammation and catabolism and promoted anabolism of chondrocytes. To attenuate the inhibition of cell viability, nanoliposomal Y099-loaded thermosensitive hydrogel (Y099-Lip-Gel) was developed for sustained release and toxicity exemption. Notably, Y099-Lip-Gel exhibited a pronounced effect on promoting anabolism and suppressing catabolism and inflammation without causing the inhibition of chondrocyte viability. Moreover, Y099-Lip-Gel remarkably increased the master regulator of chondrocyte phenotype Sox9 expression. After four intra-articular injections of Y099-Lip-Gel in the OA murine model, the histological lesions of cartilage were attenuated by Y099-Lip-Gel with subchondral bone loss and osteoclast formation inhibited. Transcriptomic analysis and experimental validations revealed that Y099-Lip-Gel suppressed cellular senescence by inhibiting the expression of senescence inducers and SASP factors. Furthermore, the phosphoproteomic analysis showed that Y099-Lip-Gel exerted a significant influence on kinome phosphorylation, inhibiting the MAPK and NF-κB signaling activations. The protective effects of Y099-Lip-Gel were also validated in cultured human OA cartilage explants. In conclusion, nanoliposomal Y099-loaded thermosensitive hydrogel has considerable potential in OA therapy. Nanoliposome-based hydrogel system has strength in reducing kinase inhibition-induced cytotoxicity, enhancing cellular tolerance to kinome perturbation, and improving the performance of protein kinase inhibitors. Senescence elimination via toxicity-exempted kinome perturbations achieved by advanced nanotechnology is a promising strategy for OA.


Introduction
Osteoarthritis (OA) is a common age-related degenerative joint disease, characterized by progressive articular cartilage degradation, synovial inflammation, osteophyte formation, subchondral bone lesions, and abnormal angiogenesis, which causes chronic pain, swelling, stiffness, and motion restriction [1,2]. Due to the high incidence, the absence of effective therapy for early OA, and the high cost of joint replacement for advanced OA, OA has become a worldwide challenge. Recent research advances in the mechanisms of OA have promoted the understanding of this disease and indicated some novel therapies based on these findings, which may be promising for OA therapy. Cellular senescence has been implied in a wide range of age-related diseases, including OA [3,4]. As a hallmark of aging, accumulated cellular senescence is a permanent state of cell-cycle arrest accompanied by the release of a group of pro-inflammatory cytokines, chemokines, matrix metalloproteases (MMPs), and growth factors with autocrine, paracrine, and endocrine activities, which is a senescence-related secretome termed Junlai Wan, Zhiyi He, and Yingchao Zhao contributed equally to this work. senescence-associated secretory phenotype (SASP) [5]. Cellular senescence is caused by oxidative stress, DNA damage, and dysfunctional oncogenes, contributing to inflammation, the decline of the tissue regenerative potential and function, and tumorigenesis [6]. Due to the intrinsic relationship between cellular senescence and aging and aging-related inflammation, targeting senescence is a promising novel strategy for OA therapy.
Kinome is a collection that comprises 538 kinases playing critical functions by catalyzing protein phosphorylation and regulating intracellular signaling events [7]. Due to the critical roles of protein kinases in the mechanisms of diseases, such as inflammation [8] and aging [9], small molecule kinase inhibitors have been introduced into research and approved for clinical applications [10]. Multiple kinases and related signaling pathways, such as the mitogen-activated protein kinase (MAPK) pathway and NF-κB pathway, have also been implied in reactive oxygen species (ROS) or mechanical stress-induced cellular senescence and the SASP productions in OA [11,12]. Although the experimental findings have demonstrated the potential of targeting kinases in OA [13,14], kinase-based targeting strategies have not been fully established in OA therapy due to the challenges in improving target selectivity and attenuating toxicity associated with off-target effects [8]. YKL-05-099 (Y099) is a novel multi-kinase kinase inhibitor that increases bone formation without increasing bone resorption by dual-targeting salt-inducible kinases and CSF1R in the experimental osteoporosis model [15,16]. Since subchondral bone is an essential element supporting cartilage and the subchondral bone loss has been characterized as an early OA change [17], Y099 may be promising in targeting the subchondral bone to prevent OA progress. Furthermore, Y099 has been shown to inhibit tyrosine kinases, which have been identified as promising drug targets for OA [16,18]. However, the cytotoxicity of Y099 has also been suggested since it can be used as a chemotherapy agent in cancer therapy [19]. Thus, the effects and potential of Y099 in OA therapy remain unclear.

Preparation and characterization of Y099-loaded nanoliposomes
Y099-loaded nanoliposomes were prepared using the thin film evaporation method. Briefly, 1 mg of Y099, 1 mg of cholesterol (Sigma, USA), and 8 mg of phospholipids (Avanti, USA) were dissolved in a round-bottom flask containing chloroform. The solution was then rotated and evaporated to form a lipid film. Then, the thin film was dissolved in deionized water under ultrasonic conditions in a water bath and extruded through polycarbonate membranes with pore sizes of 400, 200, and 100 nm to obtain Y099loaded nanoliposomes, which were lyophilized for later use.
To prepare Cy5-labeled Y099-loaded nanoliposomes, Cy5 dye (Thermo Fisher Scientific, USA) was additionally added at a mass fraction of 0.5% using the same method described above. After Y099-loaded nanoliposomes were diluted 1:100 with pure water, the particle size of the nanoliposomes was determined using a Malvern Mastersizer 3000 (Malvern Panalytical, UK). The morphologies of nanoliposomes were investigated using transmission electron microscopy (TEM) (JEOL 2010, Japan).

Preparation and characterization of nanoliposome-loaded thermosensitive hydrogel
The thermosensitive hydrogel with a mass fraction of 25% and a phase transition temperature of ~ 35 ℃ was prepared using PLGA1500-PEG1200-PLGA1500 polymers (Sun-Lipo NanoTech, China) dissolved in pure water by stirring at 4 ℃. Next, a certain amount of lyophilized Y099 nanoliposomes (w/w = 3%, drug concentration = ~ 3 mg/mL) was added to the gel and dispersed uniformly using lowtemperature vortexing to produce a temperature-sensitive and sustained-release water-based hydrogel loaded with Y099 nanoliposomes (Y099-Lip-Gel). Additionally, the hydrogels containing 3% Y099 nanoliposomes or not were lyophilized, placed onto the adhesive tape, vacuum-dried, and sputter-coated with gold before observing the distribution of drug nanoliposomes in the gel under a scanning electron microscope (SEM) (Gemini 300, Zeiss, German). Y099-Lip-Gel was diluted with different volumes of pure water to prepare the solutions with concentrations of 10%, 13%, 16%, 19%, 22%, and 25%. The solutions were heated from 20 to 65 ℃ in a constant temperature water bath. The precipitation-gel-solution phase transition temperature values of Y099-Lip-Gel were measured, and a phase transition diagram was plotted. The modulus-temperature relationship and the modulus-time relationship (37 ℃) of Y099-Lip-Gel (25 wt%) were subsequently measured to obtain gelation temperature and gelation time (37 ℃). The thermal stability of Y099-Lip-Gel was analyzed by a thermogravimetric analyzer TGA 550 (TA Instruments, USA). The samples were heated to 800 °C at the rate of 5 °C/min. In vitro release of drugs of Y099-Lip-Gel was detected by high-performance liquid chromatography (HPLC). Ten-microliter PBS, Cy5-labeled nanoliposomes, hydrogel, or hydrogel loaded with Cy5-labeled nanoliposomes were administrated by intra-articular injections to check the joint retention using Bruker MI fluorescence imaging system with the excitation at 650 nm and the emission at 666 nm.

Cytotoxicity assays
Cytotoxicity of Y099 or Y099-Lip-Gel was assessed using the Cell Counting Kit-8 (CCK8) kit (#96992, Sigma, USA) as previously described [32]. Chondrocytes were seeded at a density of 5000-10,000 cells/well and cultured in 96-well plates for 24 h, followed by Y099 treatment for 24 h or Y099-Lip-Gel treatment for 24 h or 72 h. After the incubation with the CCK8 reagent, the absorbance value in the wavelength of 450 nm was detected using a microplate reader (BioTek, USA).

Chondrocytes isolation, culture, and drug treatment
Chondrocytes were obtained from the knee joints of 3-dayold C57BL/6 mice by sequential enzymatic digestion as previously described [33].

Micromass culture
Micromass cultures were performed as previously described [34]. Briefly, passage 1st chondrocytes were plated at a density of 2.5 × 10 5 cells/10 μL drop on 24-well plates. The chondrocytes were treated with 5 ng/mL IL-1β and with or without Y099-Lip-Gel (Y099: 5 μM) for 72 h. Extracellular matrix proteoglycan contents were identified by Alcian blue staining.

RNA extraction, cDNA synthesis, and quantitative real-time PCR
Trizol reagent (#15596026, Invitrogen, USA) was used to extract RNA according to the manufacturer's instructions. Total RNA (1 μg) was used to synthesize cDNA using the HiScript II 1st Strand cDNA Synthesis Kit (#R211-01, Vazyme, China). mRNA levels of each gene of interest were normalized to Gapdh mRNA in the same sample, and the relative expression of the genes of interest was determined using the formula of Livak and Schmittgen [35]. The primers used for quantitative real-time PCR (qPCR) are listed in Table S1.

Western blot analysis
Protein extraction and western blot analysis were performed as the previous study described [36]. Briefly, total protein was extracted using radio-immunoprecipitation assay buffer according to the manufacturer's instructions. The total protein (10 μg) was resolved by sodium dodecyl-sulfate polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane. After being blocked with 5% bovine serum albumin, the membrane was incubated with the primary antibodies at 4 °C overnight and was rinsed thrice with tris-buffered saline with 0.1% Tween 20 for 15 min. The membrane was incubated with secondary antibodies for 1 h at room temperature and washed thrice with tris-buffered saline with 0.1% Tween 20 for 15 min. Protein blots were developed using a Western ECL substrate kit (#32106, Thermo Fisher Scientific, USA) and a Bio-Rad scanner (BioRad, USA). The intensity of bands was quantified by digital image analysis software (Bio-Rad, USA). Western blot analyses were performed in triplicate.
The sequencing data were processed as previously described [38]. Gene expression levels were indicated by FPKM (fragment per kilobase of transcript per million mapped reads). Principal component analysis (PCA) was used to determine the overall difference in transcriptome between each group. Differentially expressed genes (DEGs) were identified by the criteria of FDR < 0.05 using the R package DESeq2 (v1.38.2) [39]. The perturbation amplitude of gene expression induced by the treatments was defined by expression variance calculated by the absolute value of the difference in normalized gene expression (FPKM) between the two groups to identify the robustly regulated genes. To identify the commonly prioritized genes in the ranked list of up-or down-regulated genes induced by IL-1β and in the ranked list of the down-and up-regulated genes by Y099-Lip-Gel, which were ranked in descending order of expression variance values, normalized rescue score was defined using the R package RobustRankAggreg (v1.1) [40]. By comparing the perturbation amplitude induced by IL-1β or Y099-Lip-Gel, Type-1 and Type-2 rescued genes were defined. Type-1 genes were defined as the completely rescued ones, whose perturbation amplitudes induced by Y099-Lip-Gel were greater than the ones induced by IL-1β. Type-2 genes were the partially rescued ones whose perturbation amplitudes induced by Y099-Lip-Gel were smaller than the ones induced by IL-1β. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses and gene set enrichment analysis (GSEA) were performed using the R package clusterProfiler (v3.11) [41]. Senescence-related gene sets were sourced from CellAge (Build 2) [42]. For GSEA, FDR < 0.25 was considered significant.

Phosphoproteomic analysis
Chondrocytes were prepared and treated as they were done in the RNA-seq study. Two biological replicates of each group were prepared for phosphoproteomic analysis. After the drug treatment, chondrocyte pallets were collected. Subsequent protein sample preparation and liquid chromatography-tandem mass spectrometry (LC-MS/MS) were performed at Jingjie PTM Biolab Co., Ltd. (China). Briefly, the protein of chondrocyte pallets was extracted, and the protein samples underwent trypsin digestion to generate peptide mixtures which were further labeled with tandem mass tag (TMT) (ThermoFisher Scientific, USA) according to the manufacturer's instructions. The TMT-labeled peptides were fractionated by high pH reverse-phase HPLC using Agilent 300 Extend C18 column (5 μm particles, 4.6 mm ID, 250 mm length). Then, the phosphopeptides of each fraction were enriched with immobilized metal affinity chromatography (IMAC)-TiQ2 and analyzed using LC-MS/MS. Data were processed using the MaxQuant search engine (v.1.5.2.8) and analyzed using bioinformatics methods [43,44]. Phosphosites with a ratio of estimated phosphorylation levels > 1.3 or < 1/1.3 and a coefficient of variation (CV) < 0.1 were considered significant [45]. Kinome tree was visualized using Coral [46]. KEGG enrichment analysis and GSEA of the significant phosphosites were performed using the R package clusterProfiler (v3.11) [41]. For GSEA, FDR < 0.25 was considered significant.

Animal study
All the protocols of animal study were approved by the Experimental Animal Ethics Committee of Tongji Medical College, Huazhong University of Science and Technology (Wuhan, China) (IACUC: 2737). Sixty 8-week-old male C57BL/6 J mice were purchased from Wuhan Beiente Biology Science and Technology Co., Ltd. and fed in a specificpathogen-free animal environment. The OA model was established by the surgical destabilization of the medial meniscus (DMM) after inhalation-induced anesthesia with isoflurane. The sham animals were subjected to the surgical incision of the joint cavity and excision of the anterior fat pad. The mice were randomly and evenly assigned into five groups, including (1) sham; (2) DMM; (3) DMM treated with the hydrogel loaded with drug-free nanoliposomes (Lip-Gel) (DMM-0 mg/mL); (4) DMM treated with Y099-Lip-Gel loaded with 1.5 mg/mL Y099 (DMM-1.5 mg/ mL); and (5) DMM treated with Y099-Lip-Gel loaded with 3.0 mg/mL Y099 (DMM-3.0 mg/mL). One-week postsurgery, the mice were treated with the first intra-articular injections of 10 μL drugs and subsequently treated every 2 weeks for 6 weeks. The mice of the sham or DMM group received 10 μL saline solution. The mice were sacrificed, and the right knee joints were collected for analysis after all the injections were finished. The major organs, including the heart, liver, spleen, kidney, and testis, were fixed by 4% PFA and subjected to hematoxylin and eosin (H&E) staining following standard protocol.

Microcomputed tomography analysis
After the fixation in 4% paraformaldehyde, microcomputed tomography (μCT) analysis of the joint was conducted using Scanco vivaCT 40 (Scanco Medical, Switzerland) with the following parameters of calcified tissue visualization: voxel size of 10 µm resolution, voltage of 100 kV, current of 98 µA, and integration time of 300 ms. The 3D reconstructed images and quantitative analysis data were generated by 3D built-in software. The parameters of the tibial subchondral bone, including trabecular bone volume (BV)/tissue volume (TV) fraction (BV/TV), trabecular thickness (Tb. Th), trabecular spacing (Tb. Sp), and trabecular number (Tb. N), were measured as previously described [47]. The surface roughness analysis was performed in the medial femoral condyle using ImageJ software v1.53 k (National Institutes of Health, USA).

Histomorphometric and immunohistochemistry analyses
The knee joints were decalcified in 10% ethylenediaminetetraacetic acid (EDTA) for 30 days after the fixation in 4% paraformaldehyde at 4 °C, subsequentially embedded in paraffin, and sliced into 5-μm sections. Safranin O (Saf-O)/fast green staining was performed for histological assessment of cartilage to observe chondrocyte morphology and arrangement, articular cartilage structure, and extracellular matrix abundance. The OA Research Society International (OARSI) scoring system (0 to 6) and modified Mankin scoring system (0 to 16) were used to semi-quantitatively analyze cartilage lesions [48,49]. Saf-O abundance reflecting proteoglycan content was scored (0 to 12) according to the previous studies [47,50]. The mean value of three individual scores assessed by three blinded researchers was calculated for one slide, and the mean score of randomly chosen ten slides was calculated to represent one sample. A higher score indicates more advanced histological lesions of OA. Tartrate-resistant acid phosphatase (TRAP) staining was performed to analyze the osteoclast number in the femoral condyle as previously described [51]. Immunohistochemistry (IHC) analyses of the joints and human cartilage explants were performed as previously described [52].

Statistical analysis
All data analyses were conducted using GraphPad Prism software v7.0 (GraphPad, USA). Comparisons between the two groups were performed by unpaired t-test. Comparisons among multiple groups were performed by one-way analysis of variance (ANOVA) followed by Tukey's test. A p value < 0.05 was recognized as significant. All the experiments were repeated three times, and independently triplicate data were displayed as means and standard deviations.

Y099 notably suppresses inflammation and catabolism and promotes anabolism in IL-1β-treated chondrocytes while causing viability inhibition
The cytotoxicity of Y099 was first checked by the CCK8 method ( Fig. 1A and B). After 24-h treatment, the cell viability of chondrocytes was notably suppressed by Y099 (0.5-5 μM) in a dose-dependent manner (~ 20% decreased at 1.0 μM; ~ 50% at 5.0 μM) (Fig. 1B). To assess whether Y099 affects the anabolism and catabolism of IL-1β-treated chondrocytes, the expression of cartilage anabolic indicators aggrecan and Type-II collagen and catabolic indicator MMP-13 was detected by IF analyses (Fig. 1C). IL-1β (5 ng/ mL) suppressed the abundance of aggrecan and Type-II collagen and induced the expression of MMP-13, whereas Y099 (5.0 μM) rescued the effects of IL-1β by increasing aggrecan and Type-II collagen and inhibiting MMP-13 after 24-h treatment. These findings were validated by western blot (Fig. 1D and E). The western blot showed that MMP-3, a catabolic indicator like MMP-13, was more sensitive to  In line with the changes of aggrecan and Type-II collagen, SOX9, the master regulator of chondrocyte phenotypes, was also moderately increased by the Y099 treatment.
Furthermore, the expression of inflammation indicators COX and iNOS were notably enhanced by IL-1β, whereas Y099 showed a pronounced inhibition in COX and iNOS even at a low concentration (0.5 μM) (Fig. 1D and E). These findings demonstrated that Y099 has a remarkable suppressive effect

Y099-Lip-Gel remains beneficial effects of Y099 on chondrocytes, enhancing the promotion of Sox9 expression without causing viability inhibition
To overcome the limitation of Y099 in cytotoxicity, Y099-Lip-Gel was developed for sustained release and toxicity exemption. As shown in Fig. 2A, Y099 is loaded in the lipid bilayer of nanoliposomes. The Y099-loaded nanoliposomes with a mean particle size of 145.6 nm were evenly dispersed (Fig. 2B). The PLGA1500-PEG1200-PLGA1500 polymers (25 wt%) displayed temperature-dependent solution-gel transition behavior, which exhibited solution-like behavior at 4 °C and room temperature (25 °C) and formed a gel at body temperature (37 °C) ( Fig. 2C and D). The modulustemperature cure showed that the maximum temperature of the modulus of the polymer solution (25 wt%) was 33.8 °C (Fig. 2E). The modulus-time cure demonstrated that the gelation time was 99.2 s at 37 °C (Fig. 2E), which is appreciated for performing the intra-articular injections of this gelforming polymer solution and for the retention in the joint by gelation. SEM showed that the Y099-loaded nanoliposomes were evenly dispersed in the hydrogel (Fig. 2F). Thermogravimetric analysis showed that the hydrogel promoted the thermal stability of Y099-loaded nanoliposomes (Fig. 2G). Moreover, the drug release assay showed that more than 80% of Y099 was released from the nanoliposome in 5 days, whereas the hydrogel prolonged the drug release and more than 30% drug could be retained on the 20th day (Fig. 2F).
To determine whether the sustained release of Y099 could reduce cytotoxicity, the viability of the chondrocytes treated with Y099-Lip-Gel loaded with 1.0, 2.5, 5.0, 10.0, or 100.0 μM Y099 for 72 h was assessed by the CCK8 method. As shown in Fig. 3A, nanoliposomes or hydrogel themselves show no cytotoxicity, and Y099-Lip-Gel shows good cytocompatibility at the concentration from 1.0 to 5.0 μM. Of note, Y099-Lip-Gel (5.0 μM) showed considerable strength in toxicity exemption when compared with Y099 at 5.0 μM (Fig. 3B). To investigate whether Y099-Lip-Gel remained the beneficial effects of Y099 on chondrocytes, Alcian blue staining of micromass was performed to check the matrix abundance under the treatment of Y099-Lip-Gel. As shown in Fig. 3C, IL-1β reduces the matrix abundance of micromass, whereas Y099-Lip-Gel rescues this reduction caused by IL-1β. Western blot analyses showed that Y099-Lip-Gel remained the suppressive effects on the IL-1β-induced catabolic and inflammatory indicators (Fig. 3D). Although the suppressions of COX and iNOS were attenuated due to sustained release, the effects on promoting SOX9 and inhibiting MMP-13 were enhanced (Fig. 3D). These findings of MMP-3, MMP-13, and SOX9 were further validated by qPCR, which showed that Y099 regulated the expression of these indicators at the transcriptional levels (Fig. 3E). Of note, both western blot and qPCR showed that Y099-Lip-Gel exhibited an enhanced promotive effect on Sox9 expression which is critical for cartilage homeostasis and OA development, suggesting a notable potential of Y099-Lip-Gel in OA therapy. These findings together indicated that sustained release of Y099 achieved by thermosensitive hydrogel loaded with nanoliposomes succeeded in toxicity exemption and reached a good balance on anabolism promotion and catabolism inhibition.

Y099-Lip-Gel attenuates histological cartilage lesions and subchondral bone loss in the OA murine model
To check whether hydrogel prolongs the retention of nanoliposomes in the joint, 10 μL PBS, Cy5-labeled nanoliposomes, hydrogel, or hydrogel loaded with Cy5labeled nanoliposomes were administrated by intra-articular injections (Fig. 4A). The Cy5 fluorescence was dismissed 48 h after the injection of Cy5-labeled liposomes, whereas the hydrogel preserved the fluorescence for 72 h (Fig. 4B and To evaluate the potential of Y099-Lip-Gel in OA therapy, the murine OA model was established by DMM surgery and received four injections of Y099-Lip-Gel by intra-articular administration for 2 months (Fig. 4D). H&E staining showed that Y099-Lip-Gel did not induce appreciable histological changes in the key organ after the 2-month treatment (Supplemental Fig. 1). The Saf-O/fast green staining of articular cartilage showed that DMM surgery induced typical OA-like cartilage degeneration, characterized by matrix loss, disorganized chondrocyte sequence, and fissures, whereas these changes were attenuated by Y099-Lip-Gel (Fig. 4E). The histological lesions of cartilage were further quantitatively analyzed by OARSI and modified Mankin scoring system. As shown in Fig. 4G, the DMM and DMM-0 mg/mL groups both show a considerable increase in OARSI and Mankin scores. However, both treatment groups showed lower scores than the DMM or DMM-0 mg/mL groups (Fig. 4G). Moreover, Saf-O scoring was used as a supplementary assessment of matrix loss, which showed that Y099-Lip-Gel partially rescued the elevation of the Saf-O score induced by DMM (Fig. 4G).
To further assess the anabolic effect of Y099-Lip-Gel, IHC analyses of aggrecan were performed. The results showed that Y099-Lip-Gel attenuated the loss of aggrecan induced by DMM (Fig. 4F), which is consistent with the quantitative analyses of aggrecan positive area (Fig. 4G). These findings suggested that Y099-Lip-Gel attenuated histological cartilage lesions and promoted aggrecan synthesis against traumainduced OA. Osteophyte or bone spur formation is characterized in OA patients and experimental models, causing OA-related chronic pain. The 3D images of the joints developed by μCT analysis demonstrated that the obvious osteophytes were formed on the femoral surface of the DMM group, whereas osteophyte formation was ameliorated by Y099-Lip-Gel (Fig. 5A). The surface roughness analyses of the medial femoral condyle showed that the roughness was notably increased in the DMM group, whereas Y099-Lip-Gel attenuated the increased roughness of bone surface induced by DMM (Fig. 5B). Since subchondral bone is a critical element of the osteochondral unit in maintaining cartilage homeostasis, the bone mass and microstructure of tibial subchondral bone were analyzed by μCT (Fig. 5C). The coronal section of femurs and tibias showed that subchondral bone was reduced in the DMM group compared with the sham group, whereas Y099-Lip-Gel efficiently prevented the subchondral bone loss induced by DMM (Fig. 5D). The quantitative analyses of tibias showed that Y099-Lip-Gel exhibited an efficient action in increasing Tb. Th, thus leading to an increased BT/TV (Fig. 5D). Since osteoclast is an essential regulator of subchondral bone remodeling, subchondral osteoclast numbers were analyzed by TRAP staining. As shown in Fig. 5E and Supplemental  Fig. 2, the osteoclast number is increased in the DMM group whereas decreased in the Y099-Lip-Gel groups. These findings indicated that Y099-Lip-Gel inhibited the OA-related osteophyte formation, subchondral bone loss, and osteoclast formation in the OA murine model.

Y099-Lip-Gel recuses IL-1β-induced gene expression and regulates OA-related signaling pathways
To decipher the mechanism by which Y099-Lip-Gel is against OA, transcriptomic analyses by RNA-seq were performed (Fig. 6A). The PCA result indicated the overall transcriptomic similarity and differences of samples (Fig. 6B). As shown in Fig. 6B, IL-1β induced a transcriptomic shift in the PC1 axis, which was opposed to the effect of Y099-Lip-Gel on the transcriptomic shift in the PC1 axis. Compared with the Ctl-Veh group, 2319 DEGs were identified in the Ctl-IL-1β group, including 1258 up-regulated and 1061 downregulated genes (Fig. 6C). However, compared with the Ctl-IL-1β group, 2922 up-regulated and 2917 down-regulated genes were identified in the Y099-IL-1β group (Fig. 6C). Of note, Mmp3, Chil1, Saa3, Fth1, Lcn2, Spp1, Mt2, Cxcl1, and Ldha were notably up-regulated by IL-1β, whereas they were remarkably down-regulated by Y099-Lip-Gel (Fig. 6C). Indeed, ~ 69.4% of up-regulated genes (n = 805) and ~ 46.6% of down-regulated genes (n = 494) induced by IL-1β were rescued by Y099-Lip-Gel (Fig. 6D). Moreover, two types of rescued genes were further characterized by comparing the perturbation amplitudes of gene expression (Fig. 6E). The Type-1 genes (n = 889; 68.4%) were defined as completely rescued genes since the perturbation amplitudes induced by Y099-Lip-Gel were greater than the ones induced by IL-1β. The Type-2 genes (n = 410; 31.6%) were defined as partially rescued genes, which were much less than the Type-1 genes (Fig. 6E). To identify the most significantly rescued genes, a robust rank aggregation algorithm was employed to define a normalized rescued score which was to identify the common polarized gene in the comparisons between the Ctl-IL-1β and Ctl-Veh groups or between the Y099-IL-1β and Ctl-IL-1β groups and reflected the rescuing efficacy of Y099-Lip-Gel to IL-1β treatment at gene levels. According to the normalized rescued scores, the top 30 rescued genes are shown in Fig. 6F. In addition to catabolic genes such as Mmp3 and Mmp13, inflammation-related genes (e.g., Nos2, Cx3cl1, Ccl2, Cxcl3, and Cxcl10) and tissue remodeling-related genes (e.g., Spp1, Timp1, and Ogn) were also identified in the top rescued gene list. Of note, Cdkn1a, which encodes cyclin-dependent kinase inhibitor 1A (P21 Cip1 ) and is a critical regulator and indicator of cellular senescence, was remarkably induced by IL-1β but suppressed by Y099-Lip-Gel. To understand the pathways related to these recused genes, these genes were subjected to GO and KEGG enrichment analyses (Fig. 6G). The pathways related to tissue remodeling and homeostasis pathways (e.g., tissue migration, ECM organization), inflammation pathways (e.g., inflammation response, cytokine production), stress pathway (e.g., response to oxidative stress), cartilage homeostasis (e.g., cartilage development, chondrocyte differentiation), bone homeostasis (e.g., ossification, osteoblast differentiation), and protein kinase activity (e.g., regulation of protein serine/threonine kinase activity, negative regulation of phosphorylation) were identified by GO analyses (Fig. 6G). Moreover, KEGG analyses showed that the rescued genes were related to OA-related signaling pathways, such as TNF, IL-17, NF-κB, and MAPK signaling pathways. Of note, KEGG analyses also indicated that cellular senescence was significantly associated with the effect of Y099-Lip-Gel on transcriptome (Fig. 6G).

Y099-Lip-Gel suppresses cellular senescence by inhibiting senescence inducers and the senescence-associated secretory phenotype factors
Due to the findings of transcriptome strongly suggesting the connection between Y099-Lip-Gel and cellular senescence, the senescence-related gene sets were further investigated by bioinformatics. GSEA showed that IL-1β had the potential in promoting senescence-related genes, whereas Y099-Lip-Gel had a suppressive effect on them (Fig. 7A). Moreover, IL-1β significantly activated senescence inducers and inhibited the senescence suppressors (Fig. 7A). Although Y099-Lip-Gel showed moderate effects on senescence suppressors, it notably inhibited the expression of senescence inducers (FDR < 0.05) (Fig. 7A). Leading edge analyses showed the senescencerelated genes significantly up-regulated by IL-1β but downregulated by Y099-Lip-Gel (Fig. 7A). Of note, senescence inducer Serpine1 was the top 2 in the leading edge of IL-1β and the top 1 in the leading edge of Y099-Lip-Gel, indicating its critical role in the mechanism of Y099-Lip-Gel against OA (Fig. 7A). To validate whether Y099-Lip-Gel suppresses senescence, β-galactosidase staining was performed, which showed that Y099-Lip-Gel reduced IL-1β-induced senescence of chondrocytes (Fig. 7B). The IF of γH2AX showed that Y099-Lip-Gel decreased IL-1β-induced elevation of γH2AX abundance in nuclei, suggesting that IL-1β-induced DNA damage was rescued by Y099-Lip-Gel (Fig. 7C). The suppression of Y099-Lip-Gel on senescence indicators P21 Cip1 and P16 INK4A was further validated by western blot (Fig. 7D). Moreover, the expression of Cdkn1a and the rescued SASP factors (n = 15) were characterized (Fig. 7E) and validated by qPCR (Fig. 7F). These findings suggested that Y099-Lip-Gel suppresses senescence by inhibiting senescence inducers and the SASP factors.

Y099-Lip-Gel exhibits a therapeutic effect on human OA cartilage by promoting aggrecan and suppressing MMP-13 and senescence makers
To explore whether Y099-Lip-Gel has therapeutic effects on human OA cartilage, human OA cartilage explants were harvested from the medial femoral condyle of TKA patients and subjected to Y099-Lip-Gel treatment (Fig. 10A). The cartilage explants from the same patients were divided into two, which were maintained in the IL-1β-contained culture medium to preserve the gene expression of OA phenotypes and treated with or without Y099-Lip-Gel for 48 h, respectively (Fig. 10A). As shown in Fig. 10B, the cartilage explants are around 5 × 5 × 5 mm 3 with a complete cartilagesubchondral bone structure (~ 2 mm cartilage and ~ 3 mm subchondral bone in thickness). Safranin O-fast green staining showed OA-related histological characteristics, such as the loss of the cartilage content and chondrocytes and the wear of the cartilage surface (Fig. 10C). To check the effects of Y099-Lip-Gel on anabolism, catabolism, and senescence, the IHC analyses of aggrecan, MMP-13, and P21 Cip1 were performed, which showed that the Y099-Lip-Gel was efficient to increase the abundance of aggrecan and suppress the expression of MMP-13 and P21 Cip1 in the human OA cartilage after 48-h treatment ( Fig. 10D and E). These findings supported that Y099-Lip-Gel had a therapeutic effect on human OA cartilage by promoting aggrecan and suppressing MMP-13 and senescence-related gene expression.

Discussion
In the present study, the nanoliposome-based thermosensitive hydrogel system succeeded in reducing kinase inhibition-induced cytotoxicity, enhancing cellular tolerance to kinome perturbation, and improving the performance of Y099 in promoting anabolism of cartilage. The findings of this study demonstrated that nanoliposomal Y099 has a pronounced effect on promoting anabolism via notably increasing Sox9 expression and on suppressing catabolism and inflammation, without causing the inhibition of chondrocyte viability. Moreover, this study suggested that nanoliposomal Y099 can attenuate OA cartilage lesions and preserve subchondral bone by rescuing the OA-related transcriptomic perturbation, regulating kimone modifications, and inhibiting cellular senescence and the SASP productions via inhibiting the MAPK and NF-κB signaling pathways. This study demonstrates the potential of Y099 in OA therapy and proposes a promising and novel strategy of senescence elimination via toxicity-exempted kinome perturbations achieved by advanced nanotechnology for OA. Targeting senescence by nanoscale platforms has been suggested as an effective strategy for OA. Ren et al. reported that ceria nanoparticles could eliminate the senescent synoviocytes in the rat OA model [53]. The thermosensitive hydrogel loaded with hydroxytyrosol-encapsulated chitosan nanoparticles succeeded in suppressing oxidative stress and inflammation and reducing the senescent cells induced by hydrogen peroxide [54]. However, the exact mechanisms by which these nanoparticles eliminate senescence are largely unknown. RNA interference combined with nanoscale delivery platforms has also been shown to reduce chondrocyte senescence and slow the progression of OA. Zhu et al. proposed a novel injectable self-assembling peptide nanofiber hydrogel to deliver aging-related mir-29b-5p, which exhibited the capabilities of senescence elimination, anabolism promotion, catabolism inhibition, and cartilage repair [55]. Moreover, a direct strategy targeting senescence facilitated by p16INK4a siRNA-loaded PLGA nanoparticles was proven to suppress p16INK4a in fibroblast-like synoviocytes [56]. Although the senescence elimination effects of RNAbased strategies have been demonstrated, single miRNA or siRNA targeting might fail since the intracellular RNA regulation network is complicated and dynamic. Compared with the existing studies, the present study is the first to target senescence by a kinome perturbation strategy facilitated by the nanoliposome-loaded hydrogel. Many kinases and related signaling pathways have also been implied in cellular senescence and the SASP productions in OA. Kinome perturbation is a comprehensive strategy modifying a batch of senescence-related kinases, thus widely inhibiting the expression of the downstream senescence-related genes.
Current protein kinase inhibitors, which have been developed since 2001 when the first tyrosine kinase inhibitor imatinib was approved for chronic myeloid leukemia, are most for cancer therapy [57]. Very few of them, such as tofacitinib and upadacitinib, are approved for skeleton diseases [58,59]. Although the functions of various protein kinases, such as the MAPK family, have been well-characterized in OA mechanisms [18,60], no protein kinase inhibitors have been approved for OA therapy. The insufficient selectivity and off-target toxicity of kinase inhibitors, which are caused by the sequence and structural similarities shared by protein kinase families [61], hinder the development of protein kinase inhibitors. The challenges also include the toxicity facilitated by elevated lipophilicity which causes the binding to adventitious targets [62]. Rationally designed nanoplatforms have strength in sustained and controlled release of the drug, thus enhancing the bioavailability and reducing the dose-related toxicity [28,63]. Nanoliposomes are biocompatible, biodegradable, low toxic, and site-specific for both A Gene ontology analysis showing the pathways significantly related to differentially modified phosphosites induced by Y099-Lip-Gel and the kinases enriched in these pathways. The MAPK and NF-κB signalingrelated pathways are labeled in bold in the right dot plot. GeneRatio indicates the kinase number ratio in each GO item. The color and size of the dot represent the P value adjusted by Benjamini and Hochberg method (FDR) and the gene number assigned to the corresponding GO item, respectively. The kinases related to significant pathways are shown in the right heatmap and represented by official gene symbols. B Western blot analyses of the phosphorylation of MAPK and NF-κB signaling in the vehicle-induced or IL-1β-induced chondrocytes treated with or without Y099-Lip-Gel (Y099: 5 μM) for 24 h hydrophilic and hydrophobic drugs [64]. Lecithin is a type of natural phospholipid wildly used for nanoparticle synthesis and drug delivery [65]. The present study utilized lecithin nanoliposomes, which are simple to prepare and effective at reducing drug toxicity while enhancing the beneficial effects of the drug.
The existing treatments used in clinical practices have significant drawbacks, such as gastrointestinal issues caused by systemic administration of nonsteroidal anti-inflammatory drugs and the lack of clarity regarding the effectiveness of intra-articular corticosteroids or hyaluronic acid. Moreover, the low accumulation and retention of drugs in joints resulting from conventional systemic or intra-articular administrations lead to decreased drug efficacy and dose-limiting toxicities. Hydrogels, like the PLGA-PEG-PLGA thermosensitive hydrogels, are beneficial due to their biocompatibility, lack of toxicity, biodegradability, and excellent absorption ability, Representative images of immunohistochemistry analyses of the protein abundance of the anabolic indicator aggrecan, catabolic indicator MMP-13, and senescence indicators P21 Cip1 in the IL-1β (10 ng/ mL)-maintained human explants treated with or without Y099-Lip-Gel (Y099: 5 μM) for 48 h. Scar bar: 200 μm. E Quantitative analyses of immunohistochemistry. Data represent mean ± SD; N = 6/group; ** P < 0.01 by paired t-test which make them an ideal choice for intra-articular drug delivery [64,66]. The thermosensitive hydrogel in the present study prolonged the retention and release of nanoliposomal Y099, as well as promoted the stability of nanoliposomes. Thus, Y099-Lip-Gel has the potential to be utilized as a novel therapy for the treatment of OA.
Several limitations need to be considered in this study. First, although the injections could retain and work locally in the joint cavity, the delivery system in the present study lacks the targeting effects of cartilage. A composite and pleiotropic drug delivery system with high delivery efficiency and specific targeting ability is expected to further improve Y099-Lip-Gel. Secondly, the mechanical properties of Y099-Lip-Gel were not assessed in this study. More efforts are needed to develop a physical-activity-adapted and mechanic-resistant hydrogel to improve its therapeutic effects. Moreover, since the effects of Y099-Lip-Gel on the other joint elements were not assessed, whether Y099-Lip-Gel affected them remained unclear. Finally, although histological assessments of key organs were performed for the evaluation of systemic toxicity, the complete blood count and biochemical indicators of peripheral blood were absent.

Conclusions
Nanoliposomal Y099-loaded thermosensitive hydrogel rescues the OA-related transcriptome and regulates kinome modifications to suppress catabolism, promote anabolism, and eliminate cellular senescence. Nanoliposomal Y099loaded thermosensitive hydrogel is simple to prepare, effective at reducing drug toxicity, prolonging retention and release, promoting stability, and enhancing the beneficial effects of the drug; thus, it has the potential to be a novel therapeutic agent for OA. Nanoliposome-based hydrogel system has strength in reducing kinase inhibition-induced cytotoxicity, enhancing cellular tolerance to kinome perturbation, and improving the performance of protein kinase inhibitors. Senescence elimination via toxicity-exempted kinome perturbations achieved by advanced nanotechnology is a promising strategy for OA.