Adenosine and guanosine-based oligonucleotides-loaded PLGA nanoparticles attenuates progression of surgically induced osteoarthritis

Adenosine and guanosine-based oligonucleotides-loaded PLGA nanoparticles attenuates progression of surgically induced osteoarthritis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Adenosine and guanosine-based oligonucleotides-loaded PLGA nanoparticles attenuates progression of surgically induced osteoarthritis Yoonhee Kim, Jin Han, Ji Young Park, Seungwoo Han This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7079137/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 15 Dec, 2025 Read the published version in Drug Delivery and Translational Research → Version 1 posted 5 You are reading this latest preprint version Abstract Osteoarthritis (OA) is a chronic degenerative joint disease that lacks effective therapies to halt its progression. While endogenous purinergic signaling—particularly via adenosine—shows promise for reducing inflammation, it is limited by short half-life and off-target effects. To address these limitations, we developed an optimal anti-inflammatory adenosine-guanosine-based oligonucleotide encapsulated in poly(lactic-co-glycolic) acid (PLGA)-based nanoparticles (NanoOligo) to enhance in vivo stability and investigated its impact on surgically induced OA models and the underlying mechanisms responsible for its anabolic effects. A large oligonucleotide library (>1,000 unique 20-mer sequences) was screened in RAW264.7 macrophages under LPS-induced inflammation to identify the most potent candidate, which was then encapsulated into PLGA nanoparticles using a microfluidic system. NanoOligo significantly protected against cartilage degeneration and alleviated pain behaviors in the rat ACLT+pMx model following intra-articular administration. In IL-1β–treated chondrocytes, it markedly suppressed inflammatory cytokines (TNFα, IL-6) and catabolic proteases (MMP-3, MMP-13, ADAMTS5). Mechanistically, NanoOligo's anti-catabolic effects were dependent on A1R and A2AR, leading to activation of the PKA–CREB axis and suppression of p38 MAPK signaling, which in turn reduced oxidative stress and cellular senescence via upregulation of the Sirt1–Nrf2–HO-1 antioxidant pathway. In conclusion, NanoOligo exerted protective effects in surgically induced OA models, which were mediated by A1R and A2AR, along with their downstream PKA–CREB axis and Sirt1–Nrf2–HO-1 antioxidant pathway. These findings highlight purinergic signaling as a potential therapeutic target for OA treatment. Osteoarthritis Purinergic signaling Adenosine Guanosine PLGA nanoparticles Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 HIGHLIGHTS A potent anti-inflammatory adenosine–guanosine-based oligonucleotide was identified by screening a large library of >1,000 sequences. Encapsulation within PLGA nanoparticles (NanoOligo) improved in vivo stability and delivery to chondrocytes. NanoOligo significantly reduced inflammatory and catabolic mediators while protecting cartilage from degeneration in a rat ACLT+pMx model as well as mice DMM model. Its therapeutic efficacy was mediated through A1R- and A2AR-dependent PKA–CREB activation and upregulation of the Sirt1–Nrf2–HO-1 antioxidant pathway. 1. INTRODUCTION Osteoarthritis (OA) is the most prevalent degenerative joint disorder, leading to significant disability, yet there is no treatment modality that slows its progression ( 1 ). Until now, the main treatment target for OA has been limited to pain control ( 2 ). NSAIDs are used as first line treatment for pain control, and in cases of persistent pain that NSAIDs cannot alleviate, intra-articular steroid injection is recommended ( 2 ). However, the cardiovascular and gastrointestinal risks of NSAIDs are well-documented, and the effects of steroid injections are only temporary ( 3 , 4 ). Moreover, recent studies have revealed that repetitive steroid injections may even hasten the joint space narrowing of knee, casting doubt on their safety ( 4 , 5 ). Given these concerns, the pressing need for safer pain management options is evident, but the development of alternatives to NSAIDs and steroid injections remains a challenge. The human body has multiple endogenous mechanisms to regulate and resolve inflammation in response to tissue damage or infection ( 6 ). These include specialized pro-resolving lipid mediators (e.g., resolvins and lipoxins), anti-inflammatory cytokines (e.g., IL-10 and TGF-β), and purinergic signaling pathways ( 6 , 7 ). Among these various mechanisms, the adenosine system has gained particular attention for its potent anti-inflammatory and tissue-regenerative capabilities ( 7 ). A line of evidence suggests that purine nucleosides such as adenosine and guanosine can exert strong anti-inflammatory and analgesic effects by activating adenosine receptors (ARs), particularly the A2A subtype, on various immune and joint-resident cells ( 7 ). During inflammatory or degenerative processes, tissue injury causes the extracellular release of ATP, DNA and RNA, and they are subsequently metabolized to adenosine by the ectonucleotidases CD39 and CD73 expressed on activated macrophages ( 8 ). The adenosine thus generated binds to its receptors to increase intracellular cAMP, exerting anti-inflammatory effects and promoting tissue repair ( 8 ). In OA models, adenosine has been shown to modulate the release of pro-inflammatory cytokines, suppress synovial inflammation, and reduce pain signaling ( 9 ). Meanwhile, although guanosine is less extensively studied, it also appears to have immunoregulatory effects through A1R and A2AR and may synergize with adenosine through related purinergic pathways ( 10 , 11 ). These observations highlight the therapeutic potential of purinergic signaling—particularly the adenosine system—for alleviating inflammation and pain in OA. Despite its therapeutic potential, adenosine-based therapy faces limitations in clinical application due to its short half-life and the risk of cardiovascular complications. Systemic administration of adenosine results in rapid clearance—its plasma half-life is only a matter of seconds—significantly narrowing the therapeutic window ( 12 ). Moreover, systemic administration of adenosine is known to slow atrioventricular (AV) nodal conduction, potentially causing transient heart block ( 13 ). These challenges underscore the need for a localized, sustained-release drug delivery strategy that maintains effective concentrations of adenosine within the target joint space while minimizing systemic risks. To address these challenges, poly(lactic-co-glycolic) acid (PLGA) nanoparticles have emerged as an attractive drug delivery vehicle ( 14 ). PLGA is biocompatible, biodegradable, and can be formulated to release its cargo over extended periods ( 14 , 15 ). By encapsulating nucleoside-based oligonucleotides in PLGA, it becomes possible to protect them from rapid degradation, extend their half-lives, and tailor their release kinetics within the joint ( 15 ). Intra-articular injection of these nanoparticles concentrates the therapeutic agent where it is needed most, potentially enhancing efficacy while minimizing systemic exposure ( 16 ). This approach may overcome the limitations associated with adenosine’s short half-life and cardiac side effects, offering a more targeted and sustained option for OA management ( 16 ). In this study, we explored the therapeutic potential of adenosine- and guanosine-based oligonucleotides delivered via PLGA nanoparticles in an experimental model of OA. We hypothesized that this localized, controlled-release strategy would reduce synovial inflammation, alleviate pain, and ultimately slow disease progression more effectively and safely than traditional treatments. To identify the most potent oligonucleotide, we screened over 1,000 different 20-mer sequences for their ability to suppress lipopolysaccharide (LPS)-induced nitric oxide (NO) production. We then encapsulated the most effective candidate within PLGA to achieve targeted, sustained release within the affected joint. The therapeutic efficacy of this PLGA–oligonucleotide complex was examined in an in vivo surgical OA model. We further characterized the mechanism of action by identifying the relevant purinergic receptors and elucidating the molecular events responsible for its anti-inflammatory and analgesic effects. Our findings provide evidence of the purinergic system’s potential as a promising, side-effect-free strategy for mitigating inflammation and pain in OA therapy. 2. MATERIALS and METHODS 2.1. Measurement of nitric oxide RAW264.7 cells were seeded in 96-well plates at a density of 1 × 10 6 cells per well. Each well was treated with a unique oligonucleotide from a library of more than 1,000 distinct oligonucleotides, alongside lipopolysaccharide (LPS) at a concentration of 1 µg/mL. The anti-inflammatory effect of each oligonucleotide was evaluated by measuring the reduction in nitric oxide (NO) production compared to LPS treated group using the nitrate/nitrite colorimetric assay (Cayman Chemical), which quantifies NO concentrations in the culture supernatant. 2.1.1. Microfluidics-based PLGA nanoparticles production The oligonucleotide with the most potent inhibitory effect on NO production in RAW264.7 cells was encapsulated in PLGA nanoparticles using the NanoAssemblr® Ignite instrument (Precision Nanosystems Inc., Canada) with a microfluidics-based approach. The aqueous phase was prepared by dissolving Tocopherol polyethylene glycol succinate (TPGS) at 0.3% w/v in pure water, followed by the addition of the oligonucleotide at a concentration optimized for encapsulation. The organic phase was prepared by dissolving PLGA at 1% w/v in ethyl acetate. Self-assembly of PLGA nanoparticles was achieved by mixing the organic and aqueous phases in a staggered herringbone chaotic structured microfluidic cartridge (Precision Nanosystems Inc., Canada). The total flow rate was fixed at 12 mL/min, with the aqueous-to-organic flow rate ratio (FRR) maintained at 3:1 by volume. After synthesis, the nanoparticles were filtered through a 0.22 µm syringe filter to remove aggregates and ensure uniform particle size. Subsequently, the nanoparticles were dialyzed against 2 liters of DPBS at room temperature for 1 hour with gentle stirring at 100 rpm using a dialysis membrane with a molecular weight cut-off (MWCO) of 12–14 kDa (Sigma-Aldrich, St. Louis, MO) to remove residual organic solvent. The purified PLGA nanoparticles were stored at 4°C for subsequent experiments. 2.1.2. Characterization of PLGA nanoparticles The synthesized PLGA nanoparticles were characterized using transmission electron microscopy (TEM), the Nano Particle Tracking Analysis, and a zeta-potential analyzer. The morphology and size of the nanoparticles were analyzed using TEM. Silicon wafers were cleaned by sonication in 100% ethanol for 8 minutes and rinsed with nanopure water. A 20 µL aliquot of the nanoparticle suspension was drop-cast onto the silicon wafers and left to evaporate overnight in a laminar flow hood at room temperature. The samples were imaged using a FEI Magellan 400 microscope equipped with an insertable concentric backscatter detector at a landing energy of 1 kV and a stage bias of 4 kV. Particle size and morphology were analyzed using ImageJ (NIH) software to calculate Feret’s diameter and circularity. Particle size and concentration were measured using the Nano Particle Tracking Analysis System (Nanosight NS 300, Malvern Panalytical). Nanoparticles were diluted with distilled water to an optimal concentration, and measurements were conducted at room temperature. Video data were analyzed with Nanosight software to determine particle size distribution and particle concentration. The zeta potential of the nanoparticles was measured to assess their surface charge and stability using a zeta-potential and particle size analyzer (ELSZ-2000ZS, Otsuka Electronics). Samples were diluted in 0.1× PBS to minimize the effects of high ionic strength, and measurements were performed at 25°C. Each sample was analyzed in triplicate, and the average zeta potential values were recorded. 2.2. Ethics and isolation of primary chondrocytes This study was conducted in compliance with the guidelines established by the National Research Council (US) Committee for the Care and Use of Laboratory Animals and was approved by the Institutional Review Board of the Kyungpook National University School of Medicine (Daegu, Korea under the approval number KNU 2023 − 0103. All procedures were performed to minimize animal discomfort and in accordance with institutional ethical standards. Primary chondrocytes were isolated from the articular cartilage of the femurs and tibias of 5-d-old C57BL/6J mice. The harvested cartilage was minced into small fragments and initially incubated with 0.25% trypsin-EDTA at 37°C for 15 minutes with gentle agitation to remove connective tissue. Subsequently, the tissue fragments were digested in 0.2% collagenase type II prepared in DMEM at 37°C for 4 hours with continuous gentle shaking to release chondrocytes. After digestion, the resulting cell suspension was filtered through a 40-µm cell strainer to remove undigested debris and washed twice with PBS. The isolated cells were resuspended in complete culture medium consisting of a 3:2 mixture of F12 and DMEM supplemented with 0.25% L-glutamine and 0.25% penicillin/streptomycin. The chondrocytes were seeded in 6-well plates at a density of 1 × 10^5 cells per well and incubated at 37°C in a humidified atmosphere with 5% CO2. Only passage 0 chondrocytes were used in the experiments to reduce the dedifferentiation effect of chondrocytes. 2.2.1. MTT assay The effect of the synthesized PLGA nanoparticles on cell viability was assessed using the MTT assay. Primary chondrocytes were seeded in 96-well plates at a density of 1 × 10^5 cells per well and allowed to adhere overnight. After treatment with IL-1β (1 ng/mL) and varying concentrations of the nanoparticles (0, 2, 20, 200, and 2000 nM) for 24 hours, 20 µL of MTT solution (5 mg/mL in PBS) was added to each well and incubated at 37°C for 4 hours. Following incubation, the culture medium was carefully removed, and the formazan crystals formed in viable cells were dissolved by adding 100 µL of dimethyl sulfoxide (DMSO) to each well. The absorbance was measured at 570 nm using a microplate reader, and cell viability was expressed as a percentage relative to untreated control cells. 2.2.2. Surgical OA induction in rat Male Lister Hooded (Crl:LIS) SPF rats (10 weeks old) were used to induce a surgical OA model. Rats were housed under standard conditions, three per cage, and acclimatized for two weeks prior to surgery. All procedures complied with institutional ethical guidelines for animal care and use. The anterior cruciate ligament transection with partial meniscectomy (ACLT + pMx) was performed under isoflurane anesthesia by transecting the anterior cruciate ligament and excising approximately 50% of the medial meniscus to destabilize the knee joint. Postoperative analgesia was provided with subcutaneous meloxicam (0.5 mg/kg). Rats were randomly divided into groups (seven per group) and received intra-articular injections of PBS, Lorecivivint (3 µg), and NanoOligo (0.2 µg or 1 µg) on days 7, 21, 35 and 49. Sham-operated controls received PBS injections without ACLT + pMx. Pain and functional impairment were evaluated weekly by measuring the weight bearing index. A dual-channel weight-bearing device was used to record the weight distribution between the affected (right) and unaffected (left) hind limbs. The weight bearing index was calculated as the percentage of body weight supported by the affected limb relative to the total weight borne by both hind limbs, with reduced weight bearing indicating OA-related pain. On day 56 post-surgery, all rats were euthanized under isoflurane anesthesia via transthoracic cardiac puncture. Knee joint tissues were collected for histological and micro-CT imaging analyses. 2.2.3. Safranin-O staining and microCT imaging For histological evaluation, harvested knee joints were fixed in 10% formalin for 48 hours, and decalcified in 10% ethylenediaminetetraacetic acid (EDTA) solution for 3 weeks at room temperature with regular solution changes. The samples were then embedded in paraffin, sectioned at a thickness of 5 µm, and mounted on slides. The sections were stained with Safranin-O and fast green to assess proteoglycan content in the articular cartilage. Histological images were captured using a light microscope, and the severity of cartilage degradation was graded using the Osteoarthritis Research Society International (OARSI) scoring system, which includes stage and total score evaluations ( 17 ). For structural assessment of osteophytes and subchondral bone, the knee joints were scanned using a high-resolution micro-computed tomography (microCT) system. Specimens were scanned at a resolution of 9 µm per voxel, with a voltage of 50 kV and current of 200 µA. The acquired images were reconstructed and analyzed using dedicated software to measure osteophyte volume and subchondral bone changes. Quantification of osteophyte volume (mm³) was performed, and representative 3D reconstructions of the knee joints were generated. 2.2.4. RNA isolation and quantitative PCR (qPCR) Primary chondrocytes were cultured and treated with IL-1β (1 ng/mL) and/or NanoOligo (12.5 nM) for the indicated time points. Total RNA was isolated from the cells using TRIzol reagent (Invitrogen, USA) according to the manufacturer’s instructions. RNA concentration and purity were assessed using a NanoDrop spectrophotometer (Thermo Scientific). cDNA was synthesized using a reverse transcription kit (Thermo Fisher Scientific) with 1 µg of total RNA as the template. Quantitative PCR (qPCR) was performed using SYBR Green PCR Master Mix (Applied Biosystems) on a StepOnePlus Real-Time PCR System (Applied Biosystems). The expression levels of Mmp3, Mmp13, Adamts5, Tnf-α, Il-6, Vegf, and ARs were measured, with GAPDH used as the internal control for normalization. Relative mRNA expression levels were calculated using the ΔΔCt method and expressed as fold changes compared to the control group. Primer sequences for each gene were designed using Primer-BLAST and verified for specificity. 2.2.5. Western blot analysis Primary chondrocytes were lysed in RIPA buffer containing a protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific). Protein concentration was determined using a bicinchoninic acid (BCA) protein assay kit (Pierce). Equal amounts of protein (20 µg) were separated on 10–12% SDS-PAGE gels and transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore). Membranes were blocked in 5% non-fat dry milk prepared in Tris-buffered saline with 0.1% Tween-20 (TBST) for 1 hour at room temperature. The membranes were incubated overnight at 4°C with primary antibodies specific to MMP3 (Abcam, Cambridge, UK, #ab52915), MMP13 (#ab51072), ADAMTS-5 (#ab41037), IL-1β (Cell Signaling Technology, Beverly, MA, #CS2022), TNF-α (#CS3707), IL-6 (#ab6672), IL-10 (#ab33471), p-PKA (#CS5661), PKA (#CS4782), p-CREB (#CS9198), CREB (#CS9197), p-AMPKα (#CS2531), AMPKα (#CS2532), p-p38 (#CS4631), p38 (#CS9212), p-ERK1/2 (#CS9101), ERK1/2 (BD #610123), p-IkB (#CS9246), IkB (#CS4814), p-p65 (#CS3033), p65 (#CS8242), p-FoxO3 (#CS9466), FoxO3a (#CS2497), Sirt1 (#CS9475), Nrf2 (#CS12721), HO-1 (#CS70081) and β-actin (Sigma-Aldrich, Burlington, MA, #A1978). After washing with TBST, membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 hour at room temperature. Protein bands were detected using an enhanced chemiluminescence (ECL) detection kit (Amersham) and visualized with an imaging system (Bio-Rad). Quantitative densitometric analysis was performed using ImageJ software, and protein expression was normalized to β-actin. Western blot experiments were performed in biological triplicates (three independent experiments) to ensure reproducibility. Quantified protein expression levels normalized to internal control were represented as mean ± SD in the corresponding graphs. 2.2.6. Reactive oxygen species (ROS) detection using DHE and MitoSOX staining Primary chondrocytes were seeded on coverslips in 24-well plates and treated with IL-1β (1 ng/mL) in the presence or absence of NanoOligo (6.25, 12.5, or 25 nM) or Lorecivivint (3 µg/mL) as a positive control. After 24 hours of treatment, cells were incubated with dihydroethidium (DHE; 5 µM, Thermo Fisher Scientific) or MitoSOX Red (5 µM, Thermo Fisher Scientific) for 30 minutes at 37°C in the dark. For DHE staining, fluorescence images were captured using a fluorescence microscope, and the percentage of DHE-positive cells was quantified. For MitoSOX staining, cells were washed with PBS after incubation, and fluorescence images were captured to determine the percentage of MitoSOX-positive cells. 2.2.7. Immunofluorescent 8-oxo-dG staining To detect oxidative DNA damage, cells were fixed with 4% paraformaldehyde for 15 minutes, permeabilized with 0.1% Triton X-100, and incubated with an anti-8-oxo-dG antibody (1:200, Santa Cruz Biotechnology, #sc-66036) overnight at 4°C. Cells were then incubated with a fluorophore-conjugated secondary antibody (1:500, Jackson ImmunoResearch Laboratories, West Grove, PA) for 1 hour at room temperature. Fluorescence was visualized using a fluorescence microscope, and the percentage of 8-oxo-dG-positive cells was calculated. 2.2.8. Senescence detection using β-galactosidase staining To evaluate cellular senescence, β-galactosidase staining was performed using a senescence-associated β-galactosidase staining kit (Cell Signaling Technology). After treatment with IL-1β (1 ng/mL) in the presence or absence of NanoOligo or Lorecivivint for 48 hours, cells were washed with PBS and fixed with 4% paraformaldehyde for 15 minutes at room temperature. The cells were then incubated with β-galactosidase staining solution at 37°C for 12–16 hours in a CO₂-free environment. Stained cells were imaged under a bright-field microscope, and the β-gal-positive area was quantified using ImageJ software. 2.3. Statistical Analysis All data are presented as means ± standard deviations (SDs). Statistical comparisons between two groups were performed using the Mann–Whitney U test, a nonparametric test suitable for small sample sizes that cannot be assumed to follow a normal distribution. For comparisons involving multiple groups, one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test was used. P values ≤ 0.05 were considered statistically significant. All statistical analyses were performed using Prism software version 8.0 (GraphPad Software, La Jolla, CA). 3. RESULTS 3.1. Identification and characterization of a potent anti-inflammatory oligonucleotide encapsulated in PLGA nanoparticles To identify the most potent anti-inflammatory oligonucleotide sequence, approximately 1,000 distinct oligonucleotides were each administered to RAW 264.7 macrophage cell-lines in the presence of 1 µg/mL LPS, and nitric oxide (NO) production was quantified. Several candidate oligonucleotides markedly suppressed LPS-induced NO release, and the top-performing sequences were selected for subsequent nanoparticle encapsulation studies. After further validation, the following sequence was ultimately chosen for the experiment: AGG-GAG-GGA-GGG-AGG-GAT. PLGA nanoparticles encapsulating the leading oligonucleotide candidate were characterized for morphology, size distribution, surface charge, and cytotoxicity. TEM revealed that the nanoparticles had a generally spherical shape with diameters ranging from approximately 100 to 300 nm (Figure 1B). This result was confirmed by nanoparticle tracking analysis (NTA), which showed a primary size peak at approximately 121 nm (Figure 1C). Zeta potential measurements yielded a mean value of −42 mV (Figure 1D), confirming the nanoparticles had a negatively charged surface. Such negative surface charge is commonly associated with reduced particle aggregation and enhanced colloidal stability in physiological media. To assess biocompatibility and potential cytotoxicity under inflammatory conditions, RAW 264.7 cells were treated with various concentrations of PLGA-encapsulated oligonucleotides (NanoOligo) in the presence of 1 ng/mL IL-1β and subjected to MTT assays. NanoOligo exhibited no significant cytotoxicity at concentrations below 200 nM, while viability declined markedly at 2,000 nM. Under IL‑1β‑induced inflammatory stress, 2 nM and 20 nM NanoOligo restored viability, whereas the 2,000 nM dose caused a statistically significant reduction relative to the 2 nM group (Figure 1E). These findings suggest that NanoOligo possesses cytoprotective properties under inflammatory conditions with minimal cytotoxicity. 3.2. Therapeutic efficacy of NanoOligo in an ACLT-pMx-induced rat model of OA To evaluate the therapeutic efficacy of NanoOligo in surgically induced OA model, rats underwent ACLT-pMx surgery and were treated with intra-articular injections of different doses of NanoOligo (0.2 μg or 1 μg) or Lorecivivint as a positive control on days 7, 21, 35, and 49 post-surgery. Functional impact and safety were assessed by monitoring the weight-bearing index and body weight weekly. Improvements in the weight-bearing index were evident in NanoOligo-treated groups, similar to those in Lorecivivint group, suggesting analgesic and functional benefits. Furthermore, NanoOligo treatment did not significantly affect body weight, indicating an acceptable safety profile (Figure 2A). Safranin‑O staining revealed more enhanced proteoglycan retention and reduced cartilage erosion in the NanoOligo‑treated groups relative to PBS‑treated controls (Figure 2B, upper panels). Correspondingly, microCT images demonstrated reduced osteophyte formation and improved subchondral bone integrity in NanoOligo‑treated rats compared to PBS controls (Figure 2B, lower panels). Quantitative histological assessments confirmed these observations, with significant reductions in OARSI grading, staging, and total scores, indicating robust cartilage protection by NanoOligo (Figure 2C). Additionally, microCT-based analysis showed a marked decrease in osteophyte volume and modest improvements in articular cartilage thickness in 1 μg NanoOligo-treated group (Figure 2D). To further confirm these findings, we evaluated the therapeutic potential of NanoOligo in a mouse destabilization of the medial meniscus (DMM) model, administering intra-articular injections at 1, 3, 5 and 7 weeks post-surgery. Consistent with our rat OA findings, NanoOligo-treated mice showed significantly lower total OARSI scores, reduced subchondral bone thickness, and smaller osteophyte sizes compared to controls (Supplementary Figure 1A, B). 3.3. Adenosine receptor-dependent anti-catabolic effects of NanoOligo in primary chondrocytes To investigate whether NanoOligo exerts anti-catabolic effects in primary chondrocytes, we first quantified the expression of matrix-degrading enzymes and inflammatory mediators following IL-1β treatment. Co-treatment with NanoOligo (12.5 nM) significantly suppressed the IL-1β-induced upregulation of Mmp3, Mmp13, Tnfα, and IL-6 , although this effect was less pronounced than that of dexamethasone. NanoOligo did not affect the transcription of Adamts5 or Vegf (Figure 3A). To confirm these observations at the protein level, we then evaluated MMP3, MMP13, and ADAMTS-5 expression at 12, 24, and 48 hours. Consistent with the gene expression data, NanoOligo markedly reduced MMP3 and MMP13 production. Notably, ADAMTS-5 protein levels were also diminished, even though its transcript level remained relatively unchanged. Furthermore, the secretion of pro-inflammatory cytokines such as IL-1β, TNFα, and IL-6 was substantially decreased, while IL-10 was increased, suggesting that NanoOligo shifts the chondrocyte environment toward anti-inflammatory state (Figure 3B). Given the established role of ARs in purinergic signaling pathway, we then investigated whether the anti-catabolic effects of NanoOligo were mediated through these receptors. Under IL-1β-induced catabolic conditions, primary chondrocytes showed significantly increased expression of A1R, A2AR, and A2BR (Figure 3C). Pharmacological inhibition of A1R with DPCPX or A2AR with ZM241385 largely abrogated the NanoOligo-induced suppression of MMP13 and ADAMTS-5, indicating that NanoOligo exerts its robust anti-catabolic effects via pathways involving A1R and A2AR. In addition, inhibition of A3R with MRS1754 further decreased MMP13 protein levels, suggesting that A3R-related signaling is involved in MMP13 production (Figure 3D). 3.4. NanoOligo activates PKA–CREB and suppresses p38 MAPK signaling under IL-1β-induced catabolic condition We then assessed how NanoOligo influences the time-course activation of cAMP-related and catabolic signaling in primary chondrocytes exposed to IL-1β–treated conditions. Treatment with NanoOligo led to a significant increase in p‑PKA and p‑CREB levels, suggesting enhanced activation of the PKA–CREB axis, which is generally associated with anabolic responses. In contrast, phosphorylation of p38 MAPK was similarly activated at 5-minute following IL-1β treatment, but thereafter, it showed a more rapid decline in the NanoOligo-treated group compared to the control group. However, the phosphorylation levels of ERK, AMPK, IκB, p65, and FOXO3a were not substantially altered by NanoOligo (Figure 4). These findings indicate that anabolic effects of NanoOligo are primarily mediated through the PKA–CREB axis and the inhibition of p38 MAPK. 3.5. NanoOligo mitigates IL‑1β–induced oxidative stress and senescence in primary chondrocytes via the Sirt1/Nrf2 axis and HO‑1 antioxidant system To further elucidate the mechanism underlying chondroprotective effects of NanoOligo, we investigated changes in ROS production and cellular senescence under IL-1β-induced catabolic conditions. Primary chondrocytes treated with IL-1β exhibited significant increases in total ROS, mitochondrial ROS, and oxidative DNA damage, as determined by DHE, MitoSOX, and 8-oxo-dG staining, respectively. Notably, NanoOligo treatment significantly reduced the proportions of DHE+, MitoSOX+, and 8-oxo-dG+ cells in a dose-dependent manner compared to the IL-1β-treated vehicle group, with reductions comparable to those observed with Lorecivivint as a positive control. Furthermore, β-galactosidase staining revealed that NanoOligo at 12.5 and 25 nM significantly diminished the accumulation of senescent chondrocytes under these conditions (Figure 5A). Based on the reduction in oxidative stress, we analysed the molecular mechanisms regulating the endogenous oxidative stress control system. NanoOligo treatment not only dose-dependently reduced levels of MMP13 and ADAMTS5 but also markedly enhanced the expression of critical antioxidant regulators, such as Sirt1, Nrf2, and HO-1. Notably, the increases in band intensity were most evident at 12.5 nM rather than at the higher dose of 25 nM, achieving levels comparable to those observed with Lorecivivint (Figure 5 B, C). 4. DISCUSSION Accumulating evidence supports that the endogenous purinergic system modulates inflammation and facilitates tissue repair through the activation of ARs ( 7 , 9 ). In this study, we evaluated the therapeutic potential of adenosine- and guanosine-based oligonucleotides encapsulated in PLGA nanoparticles to reduce pain, and slow OA progression in surgically induced OA model. Screening over 1,000 oligonucleotides identified a potent candidate, which was encapsulated in PLGA nanoparticles. Intra-articular injections of NanoOligo in a surgery-induced OA rat model preserved cartilage integrity and improved weight bearing compared to controls. Mechanistically, NanoOligo activated A1R and A2AR, stimulated the PKA–CREB signaling pathway, and inhibited the p38 MAPK pathway. Moreover, NanoOligo reduced oxidative stress and cellular senescence through activation of the Sirt1–Nrf2–HO-1 antioxidant system. Adenosine and guanosine, the primary components of NanoOligo, exhibit distinct anti-inflammatory profiles characterized by differences in receptor affinity, degradation kinetics, and signaling mechanisms. Adenosine strongly interacts with A1R, A2AR, A2BR, and A3R, particularly elevating intracellular cAMP levels through the A2AR and A2BR, leading to significant anti-inflammatory effects ( 18 , 19 ). However, its rapid enzymatic degradation by adenosine deaminase (ADA) limits its therapeutic duration ( 12 ). In contrast, guanosine can potentiate adenosine-induced cAMP signaling ( 20 ). Recent study revealed that guanosine’s neuroprotective actions depend on A2AR expression but do not involve direct binding to A1R, A2AR, or the A1R–A2AR heteromer. Instead, guanosine allosterically modulates A2AR signaling in cells co-expressing A1R–A2AR ( 21 ). This heteromer-dependent modulation allows guanosine to involve A2AR-driven responses, including cAMP accumulation and its downstream signaling pathways, without acting as a conventional agonist or antagonist ( 21 ). Moreover, guanosine is resistant to ADA-mediated degradation, affording it a longer biological half-life compared to adenosine ( 22 ). Beyond receptor heteromer modulation, guanosine also exerts anti-inflammatory actions through alternative pathways involving glutamate receptors and potassium channels, which contribute to reduced oxidative stress and suppression of pro-inflammatory cytokine production ( 11 , 23 – 25 ). Taken together, the protective activity of guanosine stems from both AR-dependent and AR-independent processes, affording it durable anti-inflammatory benefits. Hence, combining adenosine and guanosine in NanoOligo harnesses both the rapid effects of AR signaling and the longer-lasting AR-independent benefits, making NanoOligo a compelling candidate for the management of inflammatory and degenerative conditions such as OA. Our data revealed that inhibiting A1R and A2AR with its antagonist partially restored NanoOligo-mediated suppression of MMP13 and Adamts5, confirming that NanoOligo acts through both A1R and A2AR (Fig. 4 D). ARs were initially classified as either A1 or A2 based on whether they decrease or increase cAMP, respectively ( 26 ). While the anti-inflammatory function of the A2AR is well-established, the contribution of the A1R to NanoOligo’s anti-inflammatory activity appears less convincing. Typically, activation of the A1R lowers cAMP levels through Gi-mediated inhibition of adenylyl cyclase, subsequently activating catabolic kinase pathways such as PKC, PI3 kinase, and MAP kinases ( 27 ). However, our findings suggest that NanoOligo may serve as a partial agonist at A1R, thereby shifting A1R signaling from a predominantly catabolic to a more anabolic or protective role. This is consistent with earlier work showing that A1R partial agonists can modestly increase cAMP—likely through low-affinity Gs engagement—while still producing significant anti-inflammatory outcomes ( 28 ). Such partial agonists, while weaker than full agonists, can nonetheless elevate cAMP enough to support anti-inflammatory outcomes. Furthermore, A1R itself appears to exert additional cartilage-protective effects independent of cAMP modulation, as A1R deletion in ADA-deficient mice heightened inflammation and tissue damage, with increases in Th2 cytokines, chemokines, and matrix metalloproteinases ( 29 ). Collectively, these results indicate that NanoOligo's anti-inflammatory effects arise from coordinated modulation of both A1R and A2AR, promoting balanced inflammatory signaling and tissue homeostasis. NanoOligo robustly activated the PKA–CREB pathway under IL-1β-stimulated conditions, consistent with its proposed mechanism of action via ARs (Fig. 4 ). Notably, NanoOligo treatment suppressed the persistence of p38 MAPK signaling, despite previous reports indicating direct phosphorylation of p38 MAPK by PKA ( 30 ). A closer look at the signaling kinetics revealed p38 MAPK phosphorylation levels were similar in control and NanoOligo-treated cells at 5 minutes post-IL-1β stimulation; however, a substantial reduction was evident at later time points in NanoOligo-treated chondrocytes (Fig. 4 ). This finding suggests that NanoOligo does not directly inhibit the initial IL-1β-driven activation of p38 MAPK but rather rapidly terminates its activity, likely through indirect mechanisms involving reduced ROS generation. NanoOligo-mediated activation of the PKA–CREB pathway may enhance antioxidant responses via the Sirt1–Nrf2–HO-1 axis, ultimately attenuating ROS-dependent p38 MAPK signaling ( 31 , 32 ). Collectively, these findings emphasize the intricate interplay between anabolic signaling and redox homeostasis, offering valuable insights into potential therapeutic approaches for inflammation and cartilage degeneration in OA. Interestingly, while NanoOligo treatment reduced MMP13 protein levels in a dose-dependent manner, Sirt1 and Nrf2 levels peaked at 12.5 nM, and HO-1 increased at 6.25 nM and 12.5 nM but declined again at 25 nM (Fig. 5 ). Such biphasic response may result from multiple factors. High concentrations of PLGA nanoparticles may induce cellular toxicity by interacting with cell membranes or intracellular components, leading to increased ROS production and subsequent oxidative stress ( 33 , 34 ). Once internalized via endosomal-lysosomal pathways, a portion of these nanoparticles may escape the lysosomes, causing aberrant interactions of lysosomal enzymes with other cellular components and further contributing to cellular damage ( 35 , 36 ). Prolonged or excessive exposure at higher nanoparticle concentrations may overwhelm antioxidant defenses, resulting in functional fatigue and reduced expression of protective proteins. Consistent with this notion, the MTT assay demonstrated that NanoOligo concentrations up to 200 nM did not affect cell viability in the absence of IL-1β stimulation, whereas under IL-1β-induced catabolic conditions, viability improved only at lower concentrations up to 20 nM but declined at 200 nM (Fig. 1 E). Moreover, the saturation of ARs at higher NanoOligo concentrations may further limit receptor activation and downstream signaling ( 37 ). Once receptors become fully occupied, additional NanoOligo molecules may fail to enhance intracellular responses, and instead trigger receptor desensitization, internalization, or other negative feedback processes ( 38 , 39 ). Such receptor-level events can substantially reduce therapeutic efficacy and may explain the observed biphasic responses ( 39 ). Overall, these observations highlight the importance of optimizing the therapeutic window, recognizing that both insufficient and excessively high doses may diminish or even reverse the intended benefits—an essential consideration for clinical translation. 5. CONCLUSION In conclusion, adenosine- and guanosine-based oligonucleotides exert their anti-inflammatory effects by activating A1R and A2AR. This activation not only inhibits catabolic signaling via the PKA–CREB pathway but also modulates oxidative stress through the Sirt1–Nrf2–HO-1 system, which may contribute to the attenuation of OA-related catabolic progression. Our data suggests the potential of the endogenous purinergic system as a novel therapeutic strategy for OA. Declarations Ethics approval and consent to participate The authors state that they have obtained the appropriate institutional review board approval or have followed the principles outlined in the Declaration of Helsinki for all human or animal experimental investigations. The study was approved by the Animal Care Committee of Kyungpook National University (Approval No. KNU-2018-62/54). Consent for publication: Not applicable Availability of data and materials All data generated or analyzed during this study are included in this published article and its supplementary data files Competing interests The authors declare that they have no conflicts of interest with the contents of this article. Funding This research was supported by a National Research Foundation of Korea (NRF) grant funded by the Korean government (Ministry of Education) (grant number RS-2023-00274788 and RS-2024-00411212). Authors' contributions Yoonhee Kim : Writing – review & editing, Writing – original draft, Project administration, Methodology, Formal analysis, Data curation, Conceptualization. Jin Han : Writing – review & editing, Resources, Methodology, Formal analysis. Ji Young Park : Supervision, Resources, Project administration, Conceptualization. Seungwoo Han : Writing – review & editing, Writing – original draft, Supervision, Resources, Project administration, Funding acquisition, Conceptualization. Acknowledgements The authors gratefully acknowledge Dr. Donghwi Park for his insightful advice on PLGA nanoparticle formulation and for his critical review of the study design and manuscript. We also thank Ms. Yujung Kim for her expert assistance with the microfluidic fabrication and optimization of NanoOligo‑loaded PLGA nanoparticles, which was essential for the success of this study. Data Availability Statement (Required) The datasets used and/or analyzed during the current study available from the corresponding author on reasonable request. References Tang S, Zhang C, Oo WM, Fu K, Risberg MA, Bierma-Zeinstra SM, et al. Osteoarthritis. Nat Rev Dis Primers. 2025;11(1):10. Bannuru RR, Osani MC, Vaysbrot EE, Arden NK, Bennell K, Bierma-Zeinstra SMA, et al. OARSI guidelines for the non-surgical management of knee, hip, and polyarticular osteoarthritis. Osteoarthritis Cartilage. 2019;27(11):1578-89. Kearney PM, Baigent C, Godwin J, Halls H, Emberson JR, Patrono C. Do selective cyclo-oxygenase-2 inhibitors and traditional non-steroidal anti-inflammatory drugs increase the risk of atherothrombosis? Meta-analysis of randomised trials. BMJ. 2006;332(7553):1302-8. McAlindon TE, LaValley MP, Harvey WF, Price LL, Driban JB, Zhang M, et al. Effect of Intra-articular Triamcinolone vs Saline on Knee Cartilage Volume and Pain in Patients With Knee Osteoarthritis: A Randomized Clinical Trial. JAMA. 2017;317(19):1967-75. Zeng C, Lane NE, Hunter DJ, Wei J, Choi HK, McAlindon TE, et al. Intra-articular corticosteroids and the risk of knee osteoarthritis progression: results from the Osteoarthritis Initiative. Osteoarthritis Cartilage. 2019;27(6):855-62. Basil MC, Levy BD. Specialized pro-resolving mediators: endogenous regulators of infection and inflammation. Nat Rev Immunol. 2016;16(1):51-67. Antonioli L, Pacher P, Hasko G. Adenosine and inflammation: it's time to (re)solve the problem. Trends Pharmacol Sci. 2022;43(1):43-55. Deaglio S, Dwyer KM, Gao W, Friedman D, Usheva A, Erat A, et al. Adenosine generation catalyzed by CD39 and CD73 expressed on regulatory T cells mediates immune suppression. J Exp Med. 2007;204(6):1257-65. Corciulo C, Lendhey M, Wilder T, Schoen H, Cornelissen AS, Chang G, et al. Endogenous adenosine maintains cartilage homeostasis and exogenous adenosine inhibits osteoarthritis progression. Nat Commun. 2017;8:15019. Massari CM, Constantino LC, Tasca CI. Adenosine A(1) and A(2A) receptors are involved on guanosine protective effects against oxidative burst and mitochondrial dysfunction induced by 6-OHDA in striatal slices. Purinergic Signal. 2021;17(2):247-54. Bellaver B, Souza DG, Bobermin LD, Goncalves CA, Souza DO, Quincozes-Santos A. Guanosine inhibits LPS-induced pro-inflammatory response and oxidative stress in hippocampal astrocytes through the heme oxygenase-1 pathway. Purinergic Signal. 2015;11(4):571-80. Moser GH, Schrader J, Deussen A. Turnover of adenosine in plasma of human and dog blood. Am J Physiol. 1989;256(4 Pt 1):C799-806. Dennis DM, Raatikainen MJ, Martens JR, Belardinelli L. Modulation of atrioventricular nodal function by metabolic and allosteric regulators of endogenous adenosine in guinea pig heart. Circulation. 1996;94(10):2551-9. Zhang K, Tang X, Zhang J, Lu W, Lin X, Zhang Y, et al. PEG-PLGA copolymers: their structure and structure-influenced drug delivery applications. J Control Release. 2014;183:77-86. Hernandez-Giottonini KY, Rodriguez-Cordova RJ, Gutierrez-Valenzuela CA, Penunuri-Miranda O, Zavala-Rivera P, Guerrero-German P, et al. 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Effects of guanine nucleotides on adenosine and glutamate modulation of cAMP levels in optic tectum slices from chicks. Neurochem Int. 1999;34(3):213-20. Lanznaster D, Massari CM, Markova V, Simkova T, Duroux R, Jacobson KA, et al. Adenosine A(1)-A(2A) Receptor-Receptor Interaction: Contribution to Guanosine-Mediated Effects. Cells. 2019;8(12). Shin DH, Choi KS, Cho BS, Song S, Moon DC, Hong JT, et al. Pharmacokinetics of guanosine in rats following intravenous or intramuscular administration of a 1:1 mixture of guanosine and acriflavine, a potential antitumor agent. Arch Pharm Res. 2008;31(10):1347-53. Baron BM, Dudley MW, McCarty DR, Miller FP, Reynolds IJ, Schmidt CJ. Guanine nucleotides are competitive inhibitors of N-methyl-D-aspartate at its receptor site both in vitro and in vivo. J Pharmacol Exp Ther. 1989;250(1):162-9. Molz S, Dal-Cim T, Tasca CI. Guanosine-5'-monophosphate induces cell death in rat hippocampal slices via ionotropic glutamate receptors activation and glutamate uptake inhibition. Neurochem Int. 2009;55(7):703-9. Benfenati V, Caprini M, Nobile M, Rapisarda C, Ferroni S. Guanosine promotes the up-regulation of inward rectifier potassium current mediated by Kir4.1 in cultured rat cortical astrocytes. J Neurochem. 2006;98(2):430-45. Proll MA, Clark RB, Butcher RW. A1 and A2 adenosine receptors regulate adenylate cyclase in cultured human lung fibroblasts. Mol Cell Endocrinol. 1986;44(3):211-7. Jacobson KA, Gao ZG. Adenosine receptors as therapeutic targets. Nat Rev Drug Discov. 2006;5(3):247-64. Bazil CW, Minneman KP. An investigation of the low intrinsic activity of adenosine and its analogs at low affinity (A2) adenosine receptors in rat cerebral cortex. J Neurochem. 1986;47(2):547-53. Sun CX, Young HW, Molina JG, Volmer JB, Schnermann J, Blackburn MR. A protective role for the A1 adenosine receptor in adenosine-dependent pulmonary injury. J Clin Invest. 2005;115(1):35-43. Chio CC, Chang YH, Hsu YW, Chi KH, Lin WW. PKA-dependent activation of PKC, p38 MAPK and IKK in macrophage: implication in the induction of inducible nitric oxide synthase and interleukin-6 by dibutyryl cAMP. Cell Signal. 2004;16(5):565-75. Debattisti V, Gerencser AA, Saotome M, Das S, Hajnoczky G. ROS Control Mitochondrial Motility through p38 and the Motor Adaptor Miro/Trak. Cell Rep. 2017;21(6):1667-80. Jia YT, Wei W, Ma B, Xu Y, Liu WJ, Wang Y, et al. Activation of p38 MAPK by reactive oxygen species is essential in a rat model of stress-induced gastric mucosal injury. J Immunol. 2007;179(11):7808-19. Grabowski N, Hillaireau H, Vergnaud J, Tsapis N, Pallardy M, Kerdine-Romer S, et al. Surface coating mediates the toxicity of polymeric nanoparticles towards human-like macrophages. Int J Pharm. 2015;482(1-2):75-83. Platel A, Carpentier R, Becart E, Mordacq G, Betbeder D, Nesslany F. Influence of the surface charge of PLGA nanoparticles on their in vitro genotoxicity, cytotoxicity, ROS production and endocytosis. J Appl Toxicol. 2016;36(3):434-44. Panyam J, Zhou WZ, Prabha S, Sahoo SK, Labhasetwar V. Rapid endo-lysosomal escape of poly(DL-lactide-co-glycolide) nanoparticles: implications for drug and gene delivery. FASEB J. 2002;16(10):1217-26. Feng Y, Fu H, Zhang X, Liu S, Wei X. Lysosome toxicities induced by nanoparticle exposure and related mechanisms. Ecotoxicol Environ Saf. 2024;286:117215. Attie AD, Raines RT. Analysis of Receptor-Ligand Interactions. J Chem Educ. 1995;72(2):119-24. Kobayashi H, Azuma R, Yasunaga T. Expression of excess receptors and negative feedback control of signal pathways are required for rapid activation and prompt cessation of signal transduction. Cell Commun Signal. 2009;7:3. Klaasse EC, Ijzerman AP, de Grip WJ, Beukers MW. Internalization and desensitization of adenosine receptors. Purinergic Signal. 2008;4(1):21-37. Supplementary Files Graphicalabstract.png SupplementarydataFinal.docx Cite Share Download PDF Status: Published Journal Publication published 15 Dec, 2025 Read the published version in Drug Delivery and Translational Research → Version 1 posted Editorial decision: Major Revisions Needed 12 Oct, 2025 Reviewers agreed at journal 27 Aug, 2025 Reviewers invited by journal 24 Aug, 2025 Editor assigned by journal 19 Aug, 2025 First submitted to journal 17 Aug, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7079137","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":504895177,"identity":"dad93b94-0e3c-42cd-92a2-d87af7053786","order_by":0,"name":"Yoonhee Kim","email":"","orcid":"","institution":"Kyungpook National University","correspondingAuthor":false,"prefix":"","firstName":"Yoonhee","middleName":"","lastName":"Kim","suffix":""},{"id":504895178,"identity":"d2215a63-fdd9-45c1-98ee-3ecffbe7b3a1","order_by":1,"name":"Jin Han","email":"","orcid":"","institution":"Case Western Reserve University","correspondingAuthor":false,"prefix":"","firstName":"Jin","middleName":"","lastName":"Han","suffix":""},{"id":504895179,"identity":"3d6b2e01-8a6a-43ef-a5b5-977c363ba742","order_by":2,"name":"Ji Young Park","email":"","orcid":"","institution":"Kyungpook National University","correspondingAuthor":false,"prefix":"","firstName":"Ji","middleName":"Young","lastName":"Park","suffix":""},{"id":504895180,"identity":"61d32c79-87d2-499c-af46-22ab90846cc0","order_by":3,"name":"Seungwoo Han","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAz0lEQVRIiWNgGAWjYBACCWYGhg+M/2rkQJwDD4jUwjiDge2YMVhLAlFaGMBamBMbQDyitEi2Mx9s+MDDlj4/7PBDoC12croNBLRIM7MlNs6QkMndeDvNAKgl2djsAAEtcsw85o95DNhyN85OAGk5kLiNsBb+j81/EpjTDWenfyBOizQzD2MzwwHmBHnpHCJtkWxmM2zsbThmuEE6p+BAggERfpE4f/hhw8+GGnn52embP3yosJMjqAUODMAqDYhVDgLyDaSoHgWjYBSMghEFAIt+Q5GAu69gAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0002-1614-7635","institution":"Kyungpook National University","correspondingAuthor":true,"prefix":"","firstName":"Seungwoo","middleName":"","lastName":"Han","suffix":""}],"badges":[],"createdAt":"2025-07-09 03:00:34","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7079137/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7079137/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s13346-025-02020-6","type":"published","date":"2025-12-15T15:57:34+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":90343752,"identity":"e4a32910-3699-4859-8537-7df275306105","added_by":"auto","created_at":"2025-09-01 15:48:18","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":249768,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eScreening of anti-inflammatory oligonucleotides and characterization of PLGA nanoparticle-encapsulated oligonucleotides (NanoOligo).\u003c/strong\u003e\u003cbr\u003e\n(A) To find the oligonucleotide sequence with the most potent anti-inflammatory effect, RAW 264.7 cells were treated individually with approximately 1,000 different oligonucleotides alongside LPS (1 μg/mL), and NO production was quantified using a colorimetric assay. (B) Transmission electron microscopy image of PLGA nanoparticles encapsulating the oligonucleotide. The sizes of indicated PLGA nanoparticles were measured and indicated. (C) Particle size distribution of the PLGA nanoparticles was measured using Nanoparticle tracking analysis. The average size of LNPs was 121 nm. (D) Zeta potential of the PLGA nanoparticles was analyzed and the average was -42 mV. (E) The effect of PLGA nanoparticles on cell viability was assessed using an MTT assay following treatment with various concentrations of LNPs in the presence of IL-1β (1 ng/ml). *p\u0026lt;0.05, **p\u0026lt;0.01. Mann–Whitney U test.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7079137/v1/0af9e815e05fd2682a5e5f70.png"},{"id":90343754,"identity":"1059ee6c-ac95-4e66-bf5e-16189ef80791","added_by":"auto","created_at":"2025-09-01 15:48:18","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":496368,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAssessment of therapeutic efficacy and toxicity of NanoOligo in an ACLT-pMx-induced rat OA model.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) OA was induced in 10-week-old rats using ACLT-pMx surgery. Pain was assessed by measuring the weight-bearing index, and body weight was monitored weekly to evaluate potential drug toxicity. NanoOligo (0.2 μg and 1 μg) and Lorecivivint (3 μg), as a positive control for therapeutic efficacy, were administered via intra-articular injections on days 7, 21, 35, and 49 post-surgery. N=7 per group. (B) Articular cartilage degeneration was evaluated by Safranin-O staining (N=4 per group), and structural changes, including osteophyte formation and subchondral bone integrity, were analyzed by microCT imaging (N=3 per group). (C) Histological analysis included OARSI grading, staging, and total OARSI score quantification to assess the severity of cartilage damage. (D) MicroCT analysis quantified osteophyte volume and articular cartilage thickness. *p\u0026lt;0.05, **p\u0026lt;0.01, ***p\u0026lt;0.001 compared to PBS-treated control rat. Mann–Whitney U test.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7079137/v1/f7c9a67d35dd8f3a6be12d9b.png"},{"id":90343756,"identity":"80e82bb8-867e-45dc-96fa-c9a37c250921","added_by":"auto","created_at":"2025-09-01 15:48:18","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":228769,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAnti-catabolic effects of NanoOligo are dependent on A1R and A2AR in primary chondrocytes.\u003c/strong\u003e\u003cbr\u003e\n(A) Primary chondrocytes were treated with PBS, dexamethasone (1μM), or NanoOligo (12.5 nM) in the presence of IL-1β (1 ng/mL) for 6 hours, and the gene expression of \u003cem\u003eMMP3, MMP13, Adamts5, Tnfα, IL-6,\u003c/em\u003e and \u003cem\u003eVegf\u003c/em\u003e was assessed by qPCR. *p\u0026lt;0.05 compared to PBS control by Mann–Whitney U test.(B) Primary chondrocytes were treated with or without NanoOligo (12.5 nM) in the presence of IL-1β for 12, 24, and 48 hours. Protein expression of MMP3, MMP13, and Adamts5 was evaluated in total cell lysate, and cytokine levels of IL-1β, Tnfα, IL-6, and IL-10 were also measured in the supernatant. (C) The gene expression of ARs in primary chondrocytes was assessed after 6 hours of treatment with or without IL-1β. *p\u0026lt;0.05 by Mann–Whitney U test. (D) To investigate whether the anabolic effect of NanoOligo is mediated through ARs, primary chondrocytes were treated with AR inhibitors together with NanoOligo. The changes in MMP13 expression induced by IL-1β were quantified and represented in the graph. *p\u0026lt;0.05 compared to NanoOligo treated group by Mann–Whitney U test.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7079137/v1/f260fce185bb2a70a39ab67c.png"},{"id":90344820,"identity":"bfdd8a54-4f0d-4219-a4f2-d476fcdae2e6","added_by":"auto","created_at":"2025-09-01 16:04:19","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":274381,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNanoOligo activates the PKA-CREB signaling pathway and inhibits the activation of p38 MAPK in the catabolic condition induced by IL-1β.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Primary chondrocytes were serum-starved overnight and then stimulated with IL‑1β (10 ng/mL), with or without NanoOligo (12.5 nM). Whole‑cell lysates were harvested at the indicated time points and subjected to Western blot analysis to detect total and phosphorylated forms of PKA, CREB, AMPK, p38, ERK1/2, IκB, p65, and FOXO3a. (B) Densitometric analysis of phosphorylated protein levels, normalized to the corresponding total protein, was performed using ImageJ. Data are presented as fold changes relative to the 0 min control. *p\u0026lt;0.05 compared to IL‑1β-treated group by Mann–Whitney U test.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7079137/v1/1e48780731d397ac85ac4711.png"},{"id":90343758,"identity":"793cbaec-87bf-484a-9ab8-c0444923ba6f","added_by":"auto","created_at":"2025-09-01 15:48:18","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":595762,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNanoOligo mitigates IL-1β–induced oxidative stress and senescence via Sirt1, Nrf2, and HO-1.\u003c/strong\u003e\u003cbr\u003e\n(A) Primary chondrocytes were treated with IL-1β (1 ng/mL) in the presence of NanoOligo or Lorecivivint at the indicated concentrations for 24 hours. Intracellular ROS, oxidative DNA damage, and mitochondrial ROS levels were monitored using DHE, 8-oxo-dG, and MitoSOX staining, respectively, at 24 hours, while cellular senescence was assessed by β-galactosidase staining at 48 hours. (B) After 24 hours of IL-1β treatment with or without NanoOligo or Lorecivivint, total protein lysates were harvested and subjected to Western blot analyses of MMP13, ADAMTS5, Sirt1, Nrf2, and HO-1. (C) Densitometric quantification of the Western blots was performed using ImageJ, normalized to β-actin, and presented as fold changes relative to untreated control cells. *p\u0026lt;0.05 compared to IL‑1β-treated vehicle group by Mann–Whitney U test.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7079137/v1/0b81bf114ea7feef2a876235.png"},{"id":98814047,"identity":"f28d4179-d86f-4b7a-9cc4-fbc9e46fdd01","added_by":"auto","created_at":"2025-12-22 16:10:10","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2865873,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7079137/v1/2ebca4d1-7ea0-4d58-83a5-8ca6178fa5d3.pdf"},{"id":90344728,"identity":"cf8d4441-aefa-437f-b939-b43eeb65bfe7","added_by":"auto","created_at":"2025-09-01 15:56:18","extension":"png","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":134219,"visible":true,"origin":"","legend":"","description":"","filename":"Graphicalabstract.png","url":"https://assets-eu.researchsquare.com/files/rs-7079137/v1/851bea9ab15004ee4792c20d.png"},{"id":90343772,"identity":"7e232a97-351c-4c16-b91c-b94379603181","added_by":"auto","created_at":"2025-09-01 15:48:19","extension":"docx","order_by":9,"title":"","display":"","copyAsset":false,"role":"supplement","size":11000919,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementarydataFinal.docx","url":"https://assets-eu.researchsquare.com/files/rs-7079137/v1/58e2c4eb6748c929ce28889a.docx"}],"financialInterests":"","formattedTitle":"Adenosine and guanosine-based oligonucleotides-loaded PLGA nanoparticles attenuates progression of surgically induced osteoarthritis","fulltext":[{"header":"HIGHLIGHTS","content":"\u003cul\u003e\n \u003cli\u003eA potent anti-inflammatory adenosine\u0026ndash;guanosine-based oligonucleotide was identified by screening a large library of \u0026gt;1,000 sequences.\u003c/li\u003e\n \u003cli\u003eEncapsulation within PLGA nanoparticles (NanoOligo) improved in vivo stability and delivery to chondrocytes.\u003c/li\u003e\n \u003cli\u003eNanoOligo significantly reduced inflammatory and catabolic mediators while protecting cartilage from degeneration in a rat ACLT+pMx model as well as mice DMM model.\u003c/li\u003e\n \u003cli\u003eIts therapeutic efficacy was mediated through A1R- and A2AR-dependent PKA\u0026ndash;CREB activation and upregulation of the Sirt1\u0026ndash;Nrf2\u0026ndash;HO-1 antioxidant pathway.\u003c/li\u003e\n\u003c/ul\u003e"},{"header":"1. INTRODUCTION","content":"\u003cp\u003eOsteoarthritis (OA) is the most prevalent degenerative joint disorder, leading to significant disability, yet there is no treatment modality that slows its progression (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). Until now, the main treatment target for OA has been limited to pain control (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). NSAIDs are used as first line treatment for pain control, and in cases of persistent pain that NSAIDs cannot alleviate, intra-articular steroid injection is recommended (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). However, the cardiovascular and gastrointestinal risks of NSAIDs are well-documented, and the effects of steroid injections are only temporary (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). Moreover, recent studies have revealed that repetitive steroid injections may even hasten the joint space narrowing of knee, casting doubt on their safety (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). Given these concerns, the pressing need for safer pain management options is evident, but the development of alternatives to NSAIDs and steroid injections remains a challenge.\u003c/p\u003e\u003cp\u003eThe human body has multiple endogenous mechanisms to regulate and resolve inflammation in response to tissue damage or infection (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). These include specialized pro-resolving lipid mediators (e.g., resolvins and lipoxins), anti-inflammatory cytokines (e.g., IL-10 and TGF-β), and purinergic signaling pathways (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). Among these various mechanisms, the adenosine system has gained particular attention for its potent anti-inflammatory and tissue-regenerative capabilities (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). A line of evidence suggests that purine nucleosides such as adenosine and guanosine can exert strong anti-inflammatory and analgesic effects by activating adenosine receptors (ARs), particularly the A2A subtype, on various immune and joint-resident cells (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). During inflammatory or degenerative processes, tissue injury causes the extracellular release of ATP, DNA and RNA, and they are subsequently metabolized to adenosine by the ectonucleotidases CD39 and CD73 expressed on activated macrophages (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). The adenosine thus generated binds to its receptors to increase intracellular cAMP, exerting anti-inflammatory effects and promoting tissue repair (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). In OA models, adenosine has been shown to modulate the release of pro-inflammatory cytokines, suppress synovial inflammation, and reduce pain signaling (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). Meanwhile, although guanosine is less extensively studied, it also appears to have immunoregulatory effects through A1R and A2AR and may synergize with adenosine through related purinergic pathways (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). These observations highlight the therapeutic potential of purinergic signaling\u0026mdash;particularly the adenosine system\u0026mdash;for alleviating inflammation and pain in OA.\u003c/p\u003e\u003cp\u003eDespite its therapeutic potential, adenosine-based therapy faces limitations in clinical application due to its short half-life and the risk of cardiovascular complications. Systemic administration of adenosine results in rapid clearance\u0026mdash;its plasma half-life is only a matter of seconds\u0026mdash;significantly narrowing the therapeutic window (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). Moreover, systemic administration of adenosine is known to slow atrioventricular (AV) nodal conduction, potentially causing transient heart block (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). These challenges underscore the need for a localized, sustained-release drug delivery strategy that maintains effective concentrations of adenosine within the target joint space while minimizing systemic risks. To address these challenges, poly(lactic-co-glycolic) acid (PLGA) nanoparticles have emerged as an attractive drug delivery vehicle (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). PLGA is biocompatible, biodegradable, and can be formulated to release its cargo over extended periods (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). By encapsulating nucleoside-based oligonucleotides in PLGA, it becomes possible to protect them from rapid degradation, extend their half-lives, and tailor their release kinetics within the joint (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). Intra-articular injection of these nanoparticles concentrates the therapeutic agent where it is needed most, potentially enhancing efficacy while minimizing systemic exposure (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). This approach may overcome the limitations associated with adenosine\u0026rsquo;s short half-life and cardiac side effects, offering a more targeted and sustained option for OA management (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIn this study, we explored the therapeutic potential of adenosine- and guanosine-based oligonucleotides delivered via PLGA nanoparticles in an experimental model of OA. We hypothesized that this localized, controlled-release strategy would reduce synovial inflammation, alleviate pain, and ultimately slow disease progression more effectively and safely than traditional treatments. To identify the most potent oligonucleotide, we screened over 1,000 different 20-mer sequences for their ability to suppress lipopolysaccharide (LPS)-induced nitric oxide (NO) production. We then encapsulated the most effective candidate within PLGA to achieve targeted, sustained release within the affected joint. The therapeutic efficacy of this PLGA\u0026ndash;oligonucleotide complex was examined in an in vivo surgical OA model. We further characterized the mechanism of action by identifying the relevant purinergic receptors and elucidating the molecular events responsible for its anti-inflammatory and analgesic effects. Our findings provide evidence of the purinergic system\u0026rsquo;s potential as a promising, side-effect-free strategy for mitigating inflammation and pain in OA therapy.\u003c/p\u003e"},{"header":"2. MATERIALS and METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1. Measurement of nitric oxide\u003c/h2\u003e\u003cp\u003eRAW264.7 cells were seeded in 96-well plates at a density of 1 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e cells per well. Each well was treated with a unique oligonucleotide from a library of more than 1,000 distinct oligonucleotides, alongside lipopolysaccharide (LPS) at a concentration of 1 \u0026micro;g/mL. The anti-inflammatory effect of each oligonucleotide was evaluated by measuring the reduction in nitric oxide (NO) production compared to LPS treated group using the nitrate/nitrite colorimetric assay (Cayman Chemical), which quantifies NO concentrations in the culture supernatant.\u003c/p\u003e\u003cdiv id=\"Sec4\" class=\"Section3\"\u003e\u003ch2\u003e2.1.1. Microfluidics-based PLGA nanoparticles production\u003c/h2\u003e\u003cp\u003eThe oligonucleotide with the most potent inhibitory effect on NO production in RAW264.7 cells was encapsulated in PLGA nanoparticles using the NanoAssemblr\u0026reg; Ignite instrument (Precision Nanosystems Inc., Canada) with a microfluidics-based approach. The aqueous phase was prepared by dissolving Tocopherol polyethylene glycol succinate (TPGS) at 0.3% w/v in pure water, followed by the addition of the oligonucleotide at a concentration optimized for encapsulation. The organic phase was prepared by dissolving PLGA at 1% w/v in ethyl acetate. Self-assembly of PLGA nanoparticles was achieved by mixing the organic and aqueous phases in a staggered herringbone chaotic structured microfluidic cartridge (Precision Nanosystems Inc., Canada). The total flow rate was fixed at 12 mL/min, with the aqueous-to-organic flow rate ratio (FRR) maintained at 3:1 by volume. After synthesis, the nanoparticles were filtered through a 0.22 \u0026micro;m syringe filter to remove aggregates and ensure uniform particle size. Subsequently, the nanoparticles were dialyzed against 2 liters of DPBS at room temperature for 1 hour with gentle stirring at 100 rpm using a dialysis membrane with a molecular weight cut-off (MWCO) of 12\u0026ndash;14 kDa (Sigma-Aldrich, St. Louis, MO) to remove residual organic solvent. The purified PLGA nanoparticles were stored at 4\u0026deg;C for subsequent experiments.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section3\"\u003e\u003ch2\u003e2.1.2. Characterization of PLGA nanoparticles\u003c/h2\u003e\u003cp\u003eThe synthesized PLGA nanoparticles were characterized using transmission electron microscopy (TEM), the Nano Particle Tracking Analysis, and a zeta-potential analyzer. The morphology and size of the nanoparticles were analyzed using TEM. Silicon wafers were cleaned by sonication in 100% ethanol for 8 minutes and rinsed with nanopure water. A 20 \u0026micro;L aliquot of the nanoparticle suspension was drop-cast onto the silicon wafers and left to evaporate overnight in a laminar flow hood at room temperature. The samples were imaged using a FEI Magellan 400 microscope equipped with an insertable concentric backscatter detector at a landing energy of 1 kV and a stage bias of 4 kV. Particle size and morphology were analyzed using ImageJ (NIH) software to calculate Feret\u0026rsquo;s diameter and circularity.\u003c/p\u003e\u003cp\u003eParticle size and concentration were measured using the Nano Particle Tracking Analysis System (Nanosight NS 300, Malvern Panalytical). Nanoparticles were diluted with distilled water to an optimal concentration, and measurements were conducted at room temperature. Video data were analyzed with Nanosight software to determine particle size distribution and particle concentration. The zeta potential of the nanoparticles was measured to assess their surface charge and stability using a zeta-potential and particle size analyzer (ELSZ-2000ZS, Otsuka Electronics). Samples were diluted in 0.1\u0026times; PBS to minimize the effects of high ionic strength, and measurements were performed at 25\u0026deg;C. Each sample was analyzed in triplicate, and the average zeta potential values were recorded.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.2. Ethics and isolation of primary chondrocytes\u003c/h2\u003e\u003cp\u003e This study was conducted in compliance with the guidelines established by the National Research Council (US) Committee for the Care and Use of Laboratory Animals and was approved by the Institutional Review Board of the Kyungpook National University School of Medicine (Daegu, Korea under the approval number KNU 2023\u0026thinsp;\u0026minus;\u0026thinsp;0103. All procedures were performed to minimize animal discomfort and in accordance with institutional ethical standards.\u003c/p\u003e\u003cp\u003ePrimary chondrocytes were isolated from the articular cartilage of the femurs and tibias of 5-d-old C57BL/6J mice. The harvested cartilage was minced into small fragments and initially incubated with 0.25% trypsin-EDTA at 37\u0026deg;C for 15 minutes with gentle agitation to remove connective tissue. Subsequently, the tissue fragments were digested in 0.2% collagenase type II prepared in DMEM at 37\u0026deg;C for 4 hours with continuous gentle shaking to release chondrocytes. After digestion, the resulting cell suspension was filtered through a 40-\u0026micro;m cell strainer to remove undigested debris and washed twice with PBS. The isolated cells were resuspended in complete culture medium consisting of a 3:2 mixture of F12 and DMEM supplemented with 0.25% L-glutamine and 0.25% penicillin/streptomycin. The chondrocytes were seeded in 6-well plates at a density of 1 \u0026times; 10^5 cells per well and incubated at 37\u0026deg;C in a humidified atmosphere with 5% CO2. Only passage 0 chondrocytes were used in the experiments to reduce the dedifferentiation effect of chondrocytes.\u003c/p\u003e\u003cdiv id=\"Sec7\" class=\"Section3\"\u003e\u003ch2\u003e2.2.1. MTT assay\u003c/h2\u003e\u003cp\u003eThe effect of the synthesized PLGA nanoparticles on cell viability was assessed using the MTT assay. Primary chondrocytes were seeded in 96-well plates at a density of 1 \u0026times; 10^5 cells per well and allowed to adhere overnight. After treatment with IL-1β (1 ng/mL) and varying concentrations of the nanoparticles (0, 2, 20, 200, and 2000 nM) for 24 hours, 20 \u0026micro;L of MTT solution (5 mg/mL in PBS) was added to each well and incubated at 37\u0026deg;C for 4 hours. Following incubation, the culture medium was carefully removed, and the formazan crystals formed in viable cells were dissolved by adding 100 \u0026micro;L of dimethyl sulfoxide (DMSO) to each well. The absorbance was measured at 570 nm using a microplate reader, and cell viability was expressed as a percentage relative to untreated control cells.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section3\"\u003e\u003ch2\u003e2.2.2. Surgical OA induction in rat\u003c/h2\u003e\u003cp\u003eMale Lister Hooded (Crl:LIS) SPF rats (10 weeks old) were used to induce a surgical OA model. Rats were housed under standard conditions, three per cage, and acclimatized for two weeks prior to surgery. All procedures complied with institutional ethical guidelines for animal care and use. The anterior cruciate ligament transection with partial meniscectomy (ACLT\u0026thinsp;+\u0026thinsp;pMx) was performed under isoflurane anesthesia by transecting the anterior cruciate ligament and excising approximately 50% of the medial meniscus to destabilize the knee joint. Postoperative analgesia was provided with subcutaneous meloxicam (0.5 mg/kg). Rats were randomly divided into groups (seven per group) and received intra-articular injections of PBS, Lorecivivint (3 \u0026micro;g), and NanoOligo (0.2 \u0026micro;g or 1 \u0026micro;g) on days 7, 21, 35 and 49. Sham-operated controls received PBS injections without ACLT\u0026thinsp;+\u0026thinsp;pMx. Pain and functional impairment were evaluated weekly by measuring the weight bearing index. A dual-channel weight-bearing device was used to record the weight distribution between the affected (right) and unaffected (left) hind limbs. The weight bearing index was calculated as the percentage of body weight supported by the affected limb relative to the total weight borne by both hind limbs, with reduced weight bearing indicating OA-related pain. On day 56 post-surgery, all rats were euthanized under isoflurane anesthesia via transthoracic cardiac puncture. Knee joint tissues were collected for histological and micro-CT imaging analyses.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section3\"\u003e\u003ch2\u003e2.2.3. Safranin-O staining and microCT imaging\u003c/h2\u003e\u003cp\u003eFor histological evaluation, harvested knee joints were fixed in 10% formalin for 48 hours, and decalcified in 10% ethylenediaminetetraacetic acid (EDTA) solution for 3 weeks at room temperature with regular solution changes. The samples were then embedded in paraffin, sectioned at a thickness of 5 \u0026micro;m, and mounted on slides. The sections were stained with Safranin-O and fast green to assess proteoglycan content in the articular cartilage. Histological images were captured using a light microscope, and the severity of cartilage degradation was graded using the Osteoarthritis Research Society International (OARSI) scoring system, which includes stage and total score evaluations (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eFor structural assessment of osteophytes and subchondral bone, the knee joints were scanned using a high-resolution micro-computed tomography (microCT) system. Specimens were scanned at a resolution of 9 \u0026micro;m per voxel, with a voltage of 50 kV and current of 200 \u0026micro;A. The acquired images were reconstructed and analyzed using dedicated software to measure osteophyte volume and subchondral bone changes. Quantification of osteophyte volume (mm\u0026sup3;) was performed, and representative 3D reconstructions of the knee joints were generated.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section3\"\u003e\u003ch2\u003e2.2.4. RNA isolation and quantitative PCR (qPCR)\u003c/h2\u003e\u003cp\u003ePrimary chondrocytes were cultured and treated with IL-1β (1 ng/mL) and/or NanoOligo (12.5 nM) for the indicated time points. Total RNA was isolated from the cells using TRIzol reagent (Invitrogen, USA) according to the manufacturer\u0026rsquo;s instructions. RNA concentration and purity were assessed using a NanoDrop spectrophotometer (Thermo Scientific). cDNA was synthesized using a reverse transcription kit (Thermo Fisher Scientific) with 1 \u0026micro;g of total RNA as the template. Quantitative PCR (qPCR) was performed using SYBR Green PCR Master Mix (Applied Biosystems) on a StepOnePlus Real-Time PCR System (Applied Biosystems). The expression levels of Mmp3, Mmp13, Adamts5, Tnf-α, Il-6, Vegf, and ARs were measured, with GAPDH used as the internal control for normalization. Relative mRNA expression levels were calculated using the ΔΔCt method and expressed as fold changes compared to the control group. Primer sequences for each gene were designed using Primer-BLAST and verified for specificity.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section3\"\u003e\u003ch2\u003e2.2.5. Western blot analysis\u003c/h2\u003e\u003cp\u003ePrimary chondrocytes were lysed in RIPA buffer containing a protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific). Protein concentration was determined using a bicinchoninic acid (BCA) protein assay kit (Pierce). Equal amounts of protein (20 \u0026micro;g) were separated on 10\u0026ndash;12% SDS-PAGE gels and transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore). Membranes were blocked in 5% non-fat dry milk prepared in Tris-buffered saline with 0.1% Tween-20 (TBST) for 1 hour at room temperature. The membranes were incubated overnight at 4\u0026deg;C with primary antibodies specific to MMP3 (Abcam, Cambridge, UK, #ab52915), MMP13 (#ab51072), ADAMTS-5 (#ab41037), IL-1β (Cell Signaling Technology, Beverly, MA, #CS2022), TNF-α (#CS3707), IL-6 (#ab6672), IL-10 (#ab33471), p-PKA (#CS5661), PKA (#CS4782), p-CREB (#CS9198), CREB (#CS9197), p-AMPKα (#CS2531), AMPKα (#CS2532), p-p38 (#CS4631), p38 (#CS9212), p-ERK1/2 (#CS9101), ERK1/2 (BD #610123), p-IkB (#CS9246), IkB (#CS4814), p-p65 (#CS3033), p65 (#CS8242), p-FoxO3 (#CS9466), FoxO3a (#CS2497), Sirt1 (#CS9475), Nrf2 (#CS12721), HO-1 (#CS70081) and β-actin (Sigma-Aldrich, Burlington, MA, #A1978). After washing with TBST, membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 hour at room temperature. Protein bands were detected using an enhanced chemiluminescence (ECL) detection kit (Amersham) and visualized with an imaging system (Bio-Rad). Quantitative densitometric analysis was performed using ImageJ software, and protein expression was normalized to β-actin. Western blot experiments were performed in biological triplicates (three independent experiments) to ensure reproducibility. Quantified protein expression levels normalized to internal control were represented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD in the corresponding graphs.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section3\"\u003e\u003ch2\u003e2.2.6. Reactive oxygen species (ROS) detection using DHE and MitoSOX staining\u003c/h2\u003e\u003cp\u003ePrimary chondrocytes were seeded on coverslips in 24-well plates and treated with IL-1β (1 ng/mL) in the presence or absence of NanoOligo (6.25, 12.5, or 25 nM) or Lorecivivint (3 \u0026micro;g/mL) as a positive control. After 24 hours of treatment, cells were incubated with dihydroethidium (DHE; 5 \u0026micro;M, Thermo Fisher Scientific) or MitoSOX Red (5 \u0026micro;M, Thermo Fisher Scientific) for 30 minutes at 37\u0026deg;C in the dark. For DHE staining, fluorescence images were captured using a fluorescence microscope, and the percentage of DHE-positive cells was quantified. For MitoSOX staining, cells were washed with PBS after incubation, and fluorescence images were captured to determine the percentage of MitoSOX-positive cells.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section3\"\u003e\u003ch2\u003e2.2.7. Immunofluorescent 8-oxo-dG staining\u003c/h2\u003e\u003cp\u003eTo detect oxidative DNA damage, cells were fixed with 4% paraformaldehyde for 15 minutes, permeabilized with 0.1% Triton X-100, and incubated with an anti-8-oxo-dG antibody (1:200, Santa Cruz Biotechnology, #sc-66036) overnight at 4\u0026deg;C. Cells were then incubated with a fluorophore-conjugated secondary antibody (1:500, Jackson ImmunoResearch Laboratories, West Grove, PA) for 1 hour at room temperature. Fluorescence was visualized using a fluorescence microscope, and the percentage of 8-oxo-dG-positive cells was calculated.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section3\"\u003e\u003ch2\u003e2.2.8. Senescence detection using β-galactosidase staining\u003c/h2\u003e\u003cp\u003eTo evaluate cellular senescence, β-galactosidase staining was performed using a senescence-associated β-galactosidase staining kit (Cell Signaling Technology). After treatment with IL-1β (1 ng/mL) in the presence or absence of NanoOligo or Lorecivivint for 48 hours, cells were washed with PBS and fixed with 4% paraformaldehyde for 15 minutes at room temperature. The cells were then incubated with β-galactosidase staining solution at 37\u0026deg;C for 12\u0026ndash;16 hours in a CO₂-free environment. Stained cells were imaged under a bright-field microscope, and the β-gal-positive area was quantified using ImageJ software.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003e2.3. Statistical Analysis\u003c/h2\u003e\u003cp\u003eAll data are presented as means\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviations (SDs). Statistical comparisons between two groups were performed using the Mann\u0026ndash;Whitney U test, a nonparametric test suitable for small sample sizes that cannot be assumed to follow a normal distribution. For comparisons involving multiple groups, one-way analysis of variance (ANOVA) followed by Tukey\u0026rsquo;s post hoc test was used. P values\u0026thinsp;\u0026le;\u0026thinsp;0.05 were considered statistically significant. All statistical analyses were performed using Prism software version 8.0 (GraphPad Software, La Jolla, CA).\u003c/p\u003e\u003c/div\u003e"},{"header":"3. RESULTS","content":"\u003cp\u003e\u003cstrong\u003e3.1. Identification and characterization of a potent anti-inflammatory oligonucleotide encapsulated in PLGA nanoparticles\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo identify the most potent anti-inflammatory oligonucleotide sequence, approximately 1,000 distinct oligonucleotides were each administered to RAW 264.7 macrophage cell-lines in the presence of 1 \u0026micro;g/mL LPS, and nitric oxide (NO) production was quantified. Several candidate oligonucleotides markedly suppressed LPS-induced NO release, and the top-performing sequences were selected for subsequent nanoparticle encapsulation studies. After further validation, the following sequence was ultimately chosen for the experiment: AGG-GAG-GGA-GGG-AGG-GAT.\u003c/p\u003e\n\u003cp\u003ePLGA nanoparticles encapsulating the leading oligonucleotide candidate were characterized for morphology, size distribution, surface charge, and cytotoxicity. TEM revealed that the nanoparticles had a generally spherical shape with diameters ranging from approximately 100 to 300 nm (Figure 1B). This result was confirmed by nanoparticle tracking analysis (NTA), which showed a primary size peak at approximately 121 nm (Figure 1C). Zeta potential measurements yielded a mean value of \u0026minus;42 mV (Figure 1D), confirming the nanoparticles had a negatively charged surface. Such negative surface charge is commonly associated with reduced particle aggregation and enhanced colloidal stability in physiological media. To assess biocompatibility and potential cytotoxicity under inflammatory conditions, RAW 264.7 cells were treated with various concentrations of PLGA-encapsulated oligonucleotides (NanoOligo) in the presence of 1 ng/mL IL-1\u0026beta; and subjected to MTT assays. NanoOligo exhibited no significant cytotoxicity at concentrations below 200 nM, while viability declined markedly at 2,000 nM. Under IL‑1\u0026beta;‑induced inflammatory stress, 2 nM and 20 nM NanoOligo restored viability, whereas the 2,000 nM dose caused a statistically significant reduction relative to the 2 nM group (Figure 1E). These findings suggest that NanoOligo possesses cytoprotective properties under inflammatory conditions with minimal cytotoxicity.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.2. Therapeutic efficacy of NanoOligo in an ACLT-pMx-induced rat model of OA\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo evaluate the therapeutic efficacy of NanoOligo in surgically induced OA model, rats underwent ACLT-pMx surgery and were treated with intra-articular injections of different doses of NanoOligo (0.2 \u0026mu;g or 1 \u0026mu;g) or Lorecivivint as a positive control on days 7, 21, 35, and 49 post-surgery. Functional impact and safety were assessed by monitoring the weight-bearing index and body weight weekly. Improvements in the weight-bearing index were evident in NanoOligo-treated groups, similar to those in Lorecivivint group, suggesting analgesic and functional benefits. Furthermore, NanoOligo treatment did not significantly affect body weight, indicating an acceptable safety profile (Figure 2A). Safranin‑O staining revealed more enhanced proteoglycan retention and reduced cartilage erosion in the NanoOligo‑treated groups relative to PBS‑treated controls (Figure 2B, upper panels). Correspondingly, microCT images demonstrated reduced osteophyte formation and improved subchondral bone integrity in NanoOligo‑treated rats compared to PBS controls (Figure 2B, lower panels). Quantitative histological assessments confirmed these observations, with significant reductions in OARSI grading, staging, and total scores, indicating robust cartilage protection by NanoOligo (Figure 2C). Additionally, microCT-based analysis showed a marked decrease in osteophyte volume and modest improvements in articular cartilage thickness in 1 \u0026mu;g NanoOligo-treated group (Figure 2D). To further confirm these findings, we evaluated the therapeutic potential of NanoOligo in a mouse destabilization of the medial meniscus (DMM) model, administering intra-articular injections at 1, 3, 5 and 7 weeks post-surgery. Consistent with our rat OA findings, NanoOligo-treated mice showed significantly lower total OARSI scores, reduced subchondral bone thickness, and smaller osteophyte sizes compared to controls (Supplementary Figure 1A, B).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.3. Adenosine receptor-dependent anti-catabolic effects of NanoOligo in primary chondrocytes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate whether NanoOligo exerts anti-catabolic effects in primary chondrocytes, we first quantified the expression of matrix-degrading enzymes and inflammatory mediators following IL-1\u0026beta; treatment. Co-treatment with NanoOligo (12.5 nM) significantly suppressed the IL-1\u0026beta;-induced upregulation of \u003cem\u003eMmp3, Mmp13, Tnf\u0026alpha;,\u003c/em\u003e and \u003cem\u003eIL-6\u003c/em\u003e, although this effect was less pronounced than that of dexamethasone. NanoOligo did not affect the transcription of \u003cem\u003eAdamts5\u003c/em\u003e or \u003cem\u003eVegf\u003c/em\u003e (Figure 3A). To confirm these observations at the protein level, we then evaluated MMP3, MMP13, and ADAMTS-5 expression at 12, 24, and 48 hours. Consistent with the gene expression data, NanoOligo markedly reduced MMP3 and MMP13 production. Notably, ADAMTS-5 protein levels were also diminished, even though its transcript level remained relatively unchanged. Furthermore, the secretion of pro-inflammatory cytokines such as IL-1\u0026beta;, TNF\u0026alpha;, and IL-6 was substantially decreased, while IL-10 was increased, suggesting that NanoOligo shifts the chondrocyte environment toward anti-inflammatory state (Figure 3B).\u003c/p\u003e\n\u003cp\u003eGiven the established role of ARs in purinergic signaling pathway, we then investigated whether the anti-catabolic effects of NanoOligo were mediated through these receptors. Under IL-1\u0026beta;-induced catabolic conditions, primary chondrocytes showed significantly increased expression of A1R, A2AR, and A2BR (Figure 3C). Pharmacological inhibition of A1R with DPCPX or A2AR with ZM241385 largely abrogated the NanoOligo-induced suppression of MMP13 and ADAMTS-5, indicating that NanoOligo exerts its robust anti-catabolic effects via pathways involving A1R and A2AR. In addition, inhibition of A3R with MRS1754 further decreased MMP13 protein levels, suggesting that A3R-related signaling is involved in MMP13 production (Figure 3D).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.4. NanoOligo activates PKA\u0026ndash;CREB and suppresses p38 MAPK signaling under IL-1\u0026beta;-induced catabolic condition\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe then assessed how NanoOligo influences the time-course activation of cAMP-related and catabolic signaling in primary chondrocytes exposed to IL-1\u0026beta;\u0026ndash;treated conditions. Treatment with NanoOligo led to a significant increase in p‑PKA and p‑CREB levels, suggesting enhanced activation of the PKA\u0026ndash;CREB axis, which is generally associated with anabolic responses. In contrast, phosphorylation of p38 MAPK was similarly activated at 5-minute following IL-1\u0026beta; treatment, but thereafter, it showed a more rapid decline in the NanoOligo-treated group compared to the control group. However, the phosphorylation levels of ERK, AMPK, I\u0026kappa;B, p65, and FOXO3a were not substantially altered by NanoOligo (Figure 4). These findings indicate that anabolic effects of NanoOligo are primarily mediated through the PKA\u0026ndash;CREB axis and the inhibition of p38 MAPK.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.5. NanoOligo mitigates IL‑1\u0026beta;\u0026ndash;induced oxidative stress and senescence in primary chondrocytes via the Sirt1/Nrf2 axis and HO‑1 antioxidant system\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo further elucidate the mechanism underlying chondroprotective effects of NanoOligo, we investigated changes in ROS production and cellular senescence under IL-1\u0026beta;-induced catabolic conditions. Primary chondrocytes treated with IL-1\u0026beta; exhibited significant increases in total ROS, mitochondrial ROS, and oxidative DNA damage, as determined by DHE, MitoSOX, and 8-oxo-dG staining, respectively. Notably, NanoOligo treatment significantly reduced the proportions of DHE+, MitoSOX+, and 8-oxo-dG+ cells in a dose-dependent manner compared to the IL-1\u0026beta;-treated vehicle group, with reductions comparable to those observed with Lorecivivint as a positive control. Furthermore, \u0026beta;-galactosidase staining revealed that NanoOligo at 12.5 and 25 nM significantly diminished the accumulation of senescent chondrocytes under these conditions (Figure 5A).\u003c/p\u003e\n\u003cp\u003eBased on the reduction in oxidative stress, we analysed the molecular mechanisms regulating the endogenous oxidative stress control system. NanoOligo treatment not only dose-dependently reduced levels of MMP13 and ADAMTS5 but also markedly enhanced the expression of critical antioxidant regulators, such as Sirt1, Nrf2, and HO-1. Notably, the increases in band intensity were most evident at 12.5 nM rather than at the higher dose of 25 nM, achieving levels comparable to those observed with Lorecivivint (Figure 5 B, C).\u003c/p\u003e"},{"header":"4. DISCUSSION","content":"\u003cp\u003eAccumulating evidence supports that the endogenous purinergic system modulates inflammation and facilitates tissue repair through the activation of ARs (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). In this study, we evaluated the therapeutic potential of adenosine- and guanosine-based oligonucleotides encapsulated in PLGA nanoparticles to reduce pain, and slow OA progression in surgically induced OA model. Screening over 1,000 oligonucleotides identified a potent candidate, which was encapsulated in PLGA nanoparticles. Intra-articular injections of NanoOligo in a surgery-induced OA rat model preserved cartilage integrity and improved weight bearing compared to controls. Mechanistically, NanoOligo activated A1R and A2AR, stimulated the PKA\u0026ndash;CREB signaling pathway, and inhibited the p38 MAPK pathway. Moreover, NanoOligo reduced oxidative stress and cellular senescence through activation of the Sirt1\u0026ndash;Nrf2\u0026ndash;HO-1 antioxidant system.\u003c/p\u003e\u003cp\u003eAdenosine and guanosine, the primary components of NanoOligo, exhibit distinct anti-inflammatory profiles characterized by differences in receptor affinity, degradation kinetics, and signaling mechanisms. Adenosine strongly interacts with A1R, A2AR, A2BR, and A3R, particularly elevating intracellular cAMP levels through the A2AR and A2BR, leading to significant anti-inflammatory effects (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). However, its rapid enzymatic degradation by adenosine deaminase (ADA) limits its therapeutic duration (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). In contrast, guanosine can potentiate adenosine-induced cAMP signaling (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). Recent study revealed that guanosine\u0026rsquo;s neuroprotective actions depend on A2AR expression but do not involve direct binding to A1R, A2AR, or the A1R\u0026ndash;A2AR heteromer. Instead, guanosine allosterically modulates A2AR signaling in cells co-expressing A1R\u0026ndash;A2AR (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). This heteromer-dependent modulation allows guanosine to involve A2AR-driven responses, including cAMP accumulation and its downstream signaling pathways, without acting as a conventional agonist or antagonist (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). Moreover, guanosine is resistant to ADA-mediated degradation, affording it a longer biological half-life compared to adenosine (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e). Beyond receptor heteromer modulation, guanosine also exerts anti-inflammatory actions through alternative pathways involving glutamate receptors and potassium channels, which contribute to reduced oxidative stress and suppression of pro-inflammatory cytokine production (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan additionalcitationids=\"CR24\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). Taken together, the protective activity of guanosine stems from both AR-dependent and AR-independent processes, affording it durable anti-inflammatory benefits. Hence, combining adenosine and guanosine in NanoOligo harnesses both the rapid effects of AR signaling and the longer-lasting AR-independent benefits, making NanoOligo a compelling candidate for the management of inflammatory and degenerative conditions such as OA.\u003c/p\u003e\u003cp\u003eOur data revealed that inhibiting A1R and A2AR with its antagonist partially restored NanoOligo-mediated suppression of MMP13 and Adamts5, confirming that NanoOligo acts through both A1R and A2AR (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). ARs were initially classified as either A1 or A2 based on whether they decrease or increase cAMP, respectively (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). While the anti-inflammatory function of the A2AR is well-established, the contribution of the A1R to NanoOligo\u0026rsquo;s anti-inflammatory activity appears less convincing. Typically, activation of the A1R lowers cAMP levels through Gi-mediated inhibition of adenylyl cyclase, subsequently activating catabolic kinase pathways such as PKC, PI3 kinase, and MAP kinases (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e). However, our findings suggest that NanoOligo may serve as a partial agonist at A1R, thereby shifting A1R signaling from a predominantly catabolic to a more anabolic or protective role. This is consistent with earlier work showing that A1R partial agonists can modestly increase cAMP\u0026mdash;likely through low-affinity Gs engagement\u0026mdash;while still producing significant anti-inflammatory outcomes (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). Such partial agonists, while weaker than full agonists, can nonetheless elevate cAMP enough to support anti-inflammatory outcomes. Furthermore, A1R itself appears to exert additional cartilage-protective effects independent of cAMP modulation, as A1R deletion in ADA-deficient mice heightened inflammation and tissue damage, with increases in Th2 cytokines, chemokines, and matrix metalloproteinases (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). Collectively, these results indicate that NanoOligo's anti-inflammatory effects arise from coordinated modulation of both A1R and A2AR, promoting balanced inflammatory signaling and tissue homeostasis.\u003c/p\u003e\u003cp\u003eNanoOligo robustly activated the PKA\u0026ndash;CREB pathway under IL-1β-stimulated conditions, consistent with its proposed mechanism of action via ARs (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Notably, NanoOligo treatment suppressed the persistence of p38 MAPK signaling, despite previous reports indicating direct phosphorylation of p38 MAPK by PKA (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e). A closer look at the signaling kinetics revealed p38 MAPK phosphorylation levels were similar in control and NanoOligo-treated cells at 5 minutes post-IL-1β stimulation; however, a substantial reduction was evident at later time points in NanoOligo-treated chondrocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). This finding suggests that NanoOligo does not directly inhibit the initial IL-1β-driven activation of p38 MAPK but rather rapidly terminates its activity, likely through indirect mechanisms involving reduced ROS generation. NanoOligo-mediated activation of the PKA\u0026ndash;CREB pathway may enhance antioxidant responses via the Sirt1\u0026ndash;Nrf2\u0026ndash;HO-1 axis, ultimately attenuating ROS-dependent p38 MAPK signaling (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e). Collectively, these findings emphasize the intricate interplay between anabolic signaling and redox homeostasis, offering valuable insights into potential therapeutic approaches for inflammation and cartilage degeneration in OA.\u003c/p\u003e\u003cp\u003eInterestingly, while NanoOligo treatment reduced MMP13 protein levels in a dose-dependent manner, Sirt1 and Nrf2 levels peaked at 12.5 nM, and HO-1 increased at 6.25 nM and 12.5 nM but declined again at 25 nM (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Such biphasic response may result from multiple factors. High concentrations of PLGA nanoparticles may induce cellular toxicity by interacting with cell membranes or intracellular components, leading to increased ROS production and subsequent oxidative stress (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e). Once internalized via endosomal-lysosomal pathways, a portion of these nanoparticles may escape the lysosomes, causing aberrant interactions of lysosomal enzymes with other cellular components and further contributing to cellular damage (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e). Prolonged or excessive exposure at higher nanoparticle concentrations may overwhelm antioxidant defenses, resulting in functional fatigue and reduced expression of protective proteins. Consistent with this notion, the MTT assay demonstrated that NanoOligo concentrations up to 200 nM did not affect cell viability in the absence of IL-1β stimulation, whereas under IL-1β-induced catabolic conditions, viability improved only at lower concentrations up to 20 nM but declined at 200 nM (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). Moreover, the saturation of ARs at higher NanoOligo concentrations may further limit receptor activation and downstream signaling (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e). Once receptors become fully occupied, additional NanoOligo molecules may fail to enhance intracellular responses, and instead trigger receptor desensitization, internalization, or other negative feedback processes (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e). Such receptor-level events can substantially reduce therapeutic efficacy and may explain the observed biphasic responses (\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e). Overall, these observations highlight the importance of optimizing the therapeutic window, recognizing that both insufficient and excessively high doses may diminish or even reverse the intended benefits\u0026mdash;an essential consideration for clinical translation.\u003c/p\u003e"},{"header":"5. CONCLUSION","content":"\u003cp\u003eIn conclusion, adenosine- and guanosine-based oligonucleotides exert their anti-inflammatory effects by activating A1R and A2AR. This activation not only inhibits catabolic signaling via the PKA\u0026ndash;CREB pathway but also modulates oxidative stress through the Sirt1\u0026ndash;Nrf2\u0026ndash;HO-1 system, which may contribute to the attenuation of OA-related catabolic progression. Our data suggests the potential of the endogenous purinergic system as a novel therapeutic strategy for OA.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors state that they have obtained the appropriate institutional review board approval or have followed the principles outlined in the Declaration of Helsinki for all human or animal experimental investigations. The study was approved by the Animal Care Committee of Kyungpook National University (Approval No. KNU-2018-62/54).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication:\u0026nbsp;\u003c/strong\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analyzed during this study are included in this published article and its supplementary data files\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no conflicts of interest with the contents of this article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was supported by a National Research Foundation of Korea (NRF) grant funded by the Korean government (Ministry of Education) (grant number RS-2023-00274788 and RS-2024-00411212).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eYoonhee Kim\u003c/strong\u003e: Writing \u0026ndash; review \u0026amp; editing, Writing \u0026ndash; original draft, Project administration, Methodology, Formal analysis, Data curation, Conceptualization. \u003cstrong\u003eJin Han\u003c/strong\u003e: Writing \u0026ndash; review \u0026amp; editing, Resources, Methodology, Formal analysis. \u003cstrong\u003eJi Young Park\u003c/strong\u003e: Supervision, Resources, Project administration, Conceptualization.\u0026nbsp;\u003cstrong\u003eSeungwoo Han\u003c/strong\u003e: Writing \u0026ndash; review \u0026amp; editing, Writing \u0026ndash; original draft, Supervision, Resources, Project administration, Funding acquisition, Conceptualization.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors gratefully acknowledge Dr. Donghwi Park for his insightful advice on PLGA nanoparticle formulation and for his critical review of the study design and manuscript. We also thank Ms. Yujung Kim for her expert assistance with the microfluidic fabrication and optimization of NanoOligo‑loaded PLGA nanoparticles, which was essential for the success of this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement (Required)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analyzed during the current study available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eTang S, Zhang C, Oo WM, Fu K, Risberg MA, Bierma-Zeinstra SM, et al. Osteoarthritis. Nat Rev Dis Primers. 2025;11(1):10.\u003c/li\u003e\n\u003cli\u003eBannuru RR, Osani MC, Vaysbrot EE, Arden NK, Bennell K, Bierma-Zeinstra SMA, et al. OARSI guidelines for the non-surgical management of knee, hip, and polyarticular osteoarthritis. 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Pharmacokinetics of guanosine in rats following intravenous or intramuscular administration of a 1:1 mixture of guanosine and acriflavine, a potential antitumor agent. Arch Pharm Res. 2008;31(10):1347-53.\u003c/li\u003e\n\u003cli\u003eBaron BM, Dudley MW, McCarty DR, Miller FP, Reynolds IJ, Schmidt CJ. Guanine nucleotides are competitive inhibitors of N-methyl-D-aspartate at its receptor site both in vitro and in vivo. J Pharmacol Exp Ther. 1989;250(1):162-9.\u003c/li\u003e\n\u003cli\u003eMolz S, Dal-Cim T, Tasca CI. Guanosine-5\u0026apos;-monophosphate induces cell death in rat hippocampal slices via ionotropic glutamate receptors activation and glutamate uptake inhibition. Neurochem Int. 2009;55(7):703-9.\u003c/li\u003e\n\u003cli\u003eBenfenati V, Caprini M, Nobile M, Rapisarda C, Ferroni S. Guanosine promotes the up-regulation of inward rectifier potassium current mediated by Kir4.1 in cultured rat cortical astrocytes. 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Cell Commun Signal. 2009;7:3.\u003c/li\u003e\n\u003cli\u003eKlaasse EC, Ijzerman AP, de Grip WJ, Beukers MW. Internalization and desensitization of adenosine receptors. Purinergic Signal. 2008;4(1):21-37.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"drug-delivery-and-translational-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ddtr","sideBox":"Learn more about [Drug Delivery and Translational Research](https://www.springer.com/journal/13346)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/ddtr/default.aspx","title":"Drug Delivery and Translational Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Osteoarthritis, Purinergic signaling, Adenosine, Guanosine, PLGA nanoparticles","lastPublishedDoi":"10.21203/rs.3.rs-7079137/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7079137/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eOsteoarthritis (OA) is a chronic degenerative joint disease that lacks effective therapies to halt its progression. While endogenous purinergic signaling—particularly via adenosine—shows promise for reducing inflammation, it is limited by short half-life and off-target effects. To address these limitations, we developed an optimal anti-inflammatory adenosine-guanosine-based oligonucleotide encapsulated in poly(lactic-co-glycolic) acid (PLGA)-based nanoparticles (NanoOligo) to enhance in vivo stability and investigated its impact on surgically induced OA models and the underlying mechanisms responsible for its anabolic effects. A large oligonucleotide library (\u0026gt;1,000 unique 20-mer sequences) was screened in RAW264.7 macrophages under LPS-induced inflammation to identify the most potent candidate, which was then encapsulated into PLGA nanoparticles using a microfluidic system. NanoOligo significantly protected against cartilage degeneration and alleviated pain behaviors in the rat ACLT+pMx model following intra-articular administration. In IL-1β–treated chondrocytes, it markedly suppressed inflammatory cytokines (TNFα, IL-6) and catabolic proteases (MMP-3, MMP-13, ADAMTS5). Mechanistically, NanoOligo's anti-catabolic effects were dependent on A1R and A2AR, leading to activation of the PKA–CREB axis and suppression of p38 MAPK signaling, which in turn reduced oxidative stress and cellular senescence via upregulation of the Sirt1–Nrf2–HO-1 antioxidant pathway. In conclusion, NanoOligo exerted protective effects in surgically induced OA models, which were mediated by A1R and A2AR, along with their downstream PKA–CREB axis and Sirt1–Nrf2–HO-1 antioxidant pathway. These findings highlight purinergic signaling as a potential therapeutic target for OA treatment.\u003c/p\u003e","manuscriptTitle":"Adenosine and guanosine-based oligonucleotides-loaded PLGA nanoparticles attenuates progression of surgically induced osteoarthritis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-01 15:48:13","doi":"10.21203/rs.3.rs-7079137/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major Revisions Needed","date":"2025-10-12T08:23:45+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2025-08-27T12:31:39+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-08-24T20:43:43+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-08-20T00:02:19+00:00","index":"","fulltext":""},{"type":"submitted","content":"Drug Delivery and Translational Research","date":"2025-08-17T22:41:29+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"drug-delivery-and-translational-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ddtr","sideBox":"Learn more about [Drug Delivery and Translational Research](https://www.springer.com/journal/13346)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/ddtr/default.aspx","title":"Drug Delivery and Translational Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"cd1e6e49-5789-40e3-aa47-1ba0c71371eb","owner":[],"postedDate":"September 1st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-12-22T16:04:08+00:00","versionOfRecord":{"articleIdentity":"rs-7079137","link":"https://doi.org/10.1007/s13346-025-02020-6","journal":{"identity":"drug-delivery-and-translational-research","isVorOnly":false,"title":"Drug Delivery and Translational Research"},"publishedOn":"2025-12-15 15:57:34","publishedOnDateReadable":"December 15th, 2025"},"versionCreatedAt":"2025-09-01 15:48:13","video":"","vorDoi":"10.1007/s13346-025-02020-6","vorDoiUrl":"https://doi.org/10.1007/s13346-025-02020-6","workflowStages":[]},"version":"v1","identity":"rs-7079137","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7079137","identity":"rs-7079137","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}