Immunosuppressive Cytokine‐Tethered Hydrogel for Treating Rheumatoid Arthritis

Abstract

Rheumatoid arthritis (RA) is a chronic autoimmune disorder characterized by persistent inflammation and progressive joint destruction. Current therapies primarily rely on general immune suppression, often leading to suboptimal outcomes and significant side effects. To address these challenges, an interleukin‐4 (IL‐4)‐tethered, hyaluronic acid‐based hydrogel is developed that withstands repeated mechanical stress in the joint space while fostering an immunomodulatory environment. The immunosuppressive hydrogel promotes anti‐inflammatory M2 macrophage polarization, creating an anti‐inflammatory microenvironment while preserving cartilage and mitigating joint damage. The mechanical properties of the hydrogel are carefully optimized for injectability and intra‐articular application. Its effectiveness is evaluated in a collagen‐induced arthritis (CIA) mouse model, demonstrating potential as a localized therapeutic approach. It is believed that this IL‐4‐tethered hydrogel platform will provide a foundation for the combinatorial treatment of RA and various inflammatory diseases.

An injectable hydrogel is developed by chemically tethering IL‐4, an immunosuppressive cytokine, to a hyaluronic acid‐based backbone. The hydrogel reduces joint friction and exerts immunomodulatory effects. In a rheumatoid arthritis (RA) mouse model, it reduced inflammation, preserved cartilage, and promoted anti‐inflammatory immune responses. This platform may be extended to treat other autoimmune and inflammatory diseases beyond RA.

1. Introduction

Rheumatoid arthritis (RA) is a chronic autoimmune disorder characterized by persistent inflammation of the synovial joints, leading to progressive joint destruction, pain, and functional disability.^{[ 1} , ^{2 ]} Although genetic factors are recognized as a primary cause of RA, environmental factors such as exposure to dust and microorganisms may also contribute to disease onset.^{[ 2} , ³ , ^{4 ]} However, the precise pathophysiology of RA remains largely unknown, making its treatment challenging. The biological complexity of RA, involving a diverse array of immune cells, such as macrophages and T cells, and cytokines produced by immune cells including tumor necrosis factor (TNF), has made the development of anti‐inflammatory therapies for RA particularly difficult.^{[ 2} , ⁵ , ^{6 ]} Among these immune cells, macrophages play a pivotal role in driving the inflammatory cascade. Specifically, pro‐inflammatory M1 macrophages, upon activation, secrete cytokines such as IL‐6 and TNF that amplify inflammation and contribute to disease progression. As a result, modulating macrophage behavior is essential to mitigating the symptoms of RA and breaking the cycle of chronic inflammation.

Current RA treatments, including small‐molecule drugs like methotrexate (MTX), nonsteroidal anti‐inflammatory drugs (NSAIDs), corticosteroids, and biologic therapies, primarily focus on general immune suppression or inflammation reduction.^{[ 7 ]} However, these treatments often fail to achieve long‐term remission and are associated with significant side effects. In recent years, nanoparticle‐based drug delivery systems have emerged as promising alternatives for the targeted delivery of therapeutics to inflamed joints.^{[ 8} , ⁹ , ¹⁰ , ¹¹ , ¹² , ^{13 ]} Despite their potential, these systems face limitations due to the unique biology of the synovium, where RA symptoms are most evident. Specifically, nanoparticle‐based systems often suffer from poor retention in the synovial tissue, resulting in suboptimal therapeutic efficacy.^{[ 14 ]} This highlights the urgent need for alternative biomaterial‐based approaches that address the complex pathophysiology of RA.

Hydrogels, a versatile class of biomaterials, have gained significant attention as drug‐delivery platforms in various medical fields, including cardiology, oncology, immunology, wound healing, and pain management.^{[ 15} , ¹⁶ , ¹⁷ , ¹⁸ , ¹⁹ , ²⁰ , ^{21 ]} Due to their polymeric network structure and high water content, hydrogels exhibit excellent biocompatibility and are suitable for encapsulating hydrophilic drugs.^{[ 15 ]} Additionally, their physicochemical properties, such as stiffness, porosity, and surface functionality, can be easily modified through techniques like click chemistry, allowing for customizable drug release profiles and mechanical properties. While a variety of polymers can be used to formulate hydrogels, hyaluronic acid (HA) stands out as a naturally occurring biopolymer with a well‐established safety profile, making it particularly suitable for clinical applications.^{[ 22} , ^{23 ]} HA is an anionic, hygroscopic polysaccharide composed of repeating units of D‐glucuronic acid and N‐acetyl‐D‐glucosamine.^{[ 24 ]} Its biocompatibility, biodegradability, and ease of chemical modification have contributed to the development of numerous HA‐based hydrogels, many of which are approved by the U.S. Food and Drug Administration (FDA) for viscosupplementation. These hydrogels are designed to mimic the viscoelastic properties of cartilage, thereby providing natural joint lubrication and shock absorption.^{[ 14} , ^{25 ]} Despite the potential of hydrogels for such clinical applications, previous studies on intra‐articular (IA) hydrogel injections for RA treatment have largely overlooked optimizing their mechanical properties for use in the joint space, instead focusing on conventional applications like sustained drug release. Consequently, while these studies represent significant research advancements, their clinical applicability remains limited due to inadequate mechanical performance, such as poor durability under joint stress or suboptimal lubrication properties. This underscores the critical need for hydrogel platforms that are not only biologically effective but also mechanically suited for RA management in clinical settings.^{[ 26 ]}

IL‐4, an anti‐inflammatory cytokine, is a promising therapeutic agent for modulating immune responses in inflammatory diseases.^{[ 27 ]} It promotes immunosuppressive M2 macrophage polarization, supports a type 2 immune response, and enhances tissue repair and immune homeostasis. IL‐4 has shown therapeutic potential in various inflammatory disease models, including arthritis, autoimmune demyelinating diseases, and psoriasis.^{[ 28} , ²⁹ , ^{30 ]} However, its clinical application is limited by its short half‐life, rapid systemic clearance, and potential toxicity at high doses.^{[ 31 ]} Therefore, the development of localized delivery systems of IL‐4 is essential to maximize its therapeutic benefits while minimizing systemic side effects.

In this study, we report the design and development of an IL‐4‐tethered hyaluronic acid‐based hydrogel for the treatment of RA. By optimizing key physical parameters such as stiffness and viscoelasticity, we synthesized a hydrogel that is mechanically suitable for clinical application to the synovium, where tissues experience repeated mechanical stress and strain.^{[ 32 ]} The hydrogel can therefore be applied to the joint space to partially reduce friction in the inflamed synovium and mitigate joint damage. Traditional hydrogels with stiffness analogous to cartilage biolubricants often have large pore sizes, which can lead to a burst release of therapeutic agents, thereby limiting sustained drug release. To overcome this challenge, we tethered IL‐4 directly to the polymeric network of a hydrogel. This approach allows the hydrogel to function as both a joint lubricant and an immunomodulatory platform that generates an anti‐inflammatory microenvironment (Figure 1 ). Overall, this study presents a dual‐functional IL‐4‐tethered hydrogel that addresses both mechanical and immunological aspects of RA pathophysiology. Our work contributes to the growing field of hydrogel‐based therapies for RA management and offers a promising platform for future translational applications.

Figure 1.

Schematic illustration of an injectable, bio‐lubricating, and immunosuppressive IL‐4‐tethered hydrogel designed for treating rheumatoid arthritis. This hydrogel induces M2 macrophage polarization, fostering an anti‐inflammatory environment that helps protect cartilage and reduce joint damage.

2. Results and Discussion

2.1. Synthesis and Characterization of IL‐4‐Conjugated Hydrogel

To synthesize an IL‐4‐tethered hyaluronic acid (HA)‐based hydrogel, a hydrogel precursor, specifically vinyl sulfonated HA (HA‐VS), was first prepared. Vinyl sulfone, a popular electron‐deficient functional group (Michael donor), efficiently participates in thiol‐Michael addition reactions under physiological pH and temperature in aqueous conditions.^{[ 33} , ³⁴ , ^{35 ]} In detail, the hydroxyl groups of HA were deprotonated under alkaline conditions to yield alkoxide ions (OH‐). Divinyl sulfone (DVS) was then added in excess (6x molar excess relative to hydroxyl groups), and the reaction time was varied to control the degree of modification (DM) of hydroxyl groups by vinyl sulfone (‐VS) groups. Note that DM is defined as the number of vinyl sulfone groups divided by the number of disaccharides repeating units.^{[ 33 ]} Consistent with previous literature, the rate of VS conjugation followed first‐order kinetics, and this was confirmed by ¹H NMR by comparing the integral signals at δ = 6.9 and δ = 2 (acetyl group of the disaccharide) while keeping other parameters such as pH and DVS molar excess constant (Figure S1, Supporting Information).^{[ 16} , ^{33 ]} Using this approach, we synthesized HA‐VS with a DM of 25%, as a higher VS density would reduce the solubility of HA‐VS, while a lower DM could lead to a prolonged gelation time, which may be unsuitable for clinical applications.^{[ 16} , ³³ , ^{36 ]} Subsequently, gelation was triggered by mixing HA‐VS with polyethylene glycol (PEG) dithiol at physiological temperature and pH at the desired gelation concentration (Figure 2a).

Figure 2.

Synthesis and characterization of HA‐based hydrogel. a) Schematic representation of the Michael addition reaction between vinylsulfonated hyaluronic acid (HA‐VS) and thiolated polyethylene glycol (PEG‐dithiol), leading to the formation of an injectable hydrogel. b) The average storage modulus of hydrogels with various Cg. c) Frequency sweep tests performed on hydrogels with different gelation concentrations (Cg). d) The G’ and G” values of the hydrogel with a Cg of 2.5% (w/v) and a degree of modification (DM) of 25%. Data are means ± s.e.m [n = 3; ^* P < 0.05, ^** P < 0.01, ^*** P < 0.001, and ^**** P < 0.0001, paired two‐tailed Student's t‐test].

Given that cartilage undergoes repeated mechanical stress over time, controlling the material properties of the hydrogel is essential for developing a long‐lasting treatment platform for joint space applications, similar to viscosupplements.^{[ 32 ]} Hydrogels with inappropriate material properties may fail to provide sufficient friction‐mitigating effects in affected joints. It has been reported that conventional intra‐articular biolubricants exhibit storage moduli (G∲) of ≈10² Pa.^{[ 37 ]} Thus, we screened hydrogels with similar storage moduli by varying the hydrogel polymer concentration (HA‐VS) while maintaining a DM of 25% and a crosslinking density of 20% with PEG‐dithiol. Three different hydrogel concentrations (2.5%, 3.75%, and 5% (w/v)) were tested, and the hydrogel formed at 2.5% (w/v) exhibited an average storage modulus of 233 Pa, comparable to commercial biolubricants (Figure 2b,c). A frequency sweep test performed on this hydrogel confirmed successful hydrogel formation, with G' values higher than G∳ values across all tested frequencies (Figure 2d). These findings are consistent with the mechanical range of HA‐based viscosupplements currently used in clinical practice, supporting the translational potential of the platform.

Our next goal was to conjugate interleukin‐4 (IL‐4), an immunosuppressive cytokine known to induce inflammatory M1 macrophages to an anti‐inflammatory M2 phenotype through STAT6 activation. STAT6 is a critical transcription factor that, upon activation by IL‐4, drives the expression of genes associated with anti‐inflammatory and tissue‐repairing functions in macrophages. This shift in macrophage phenotype has been shown to reduce inflammation and promote tissue regeneration by producing immunosuppressive cytokines such as IL‐10 and TGF‐β.^{[ 27 ]} To achieve this, IL‐4 was thiolated using Traut's reagent (2‐iminothiolane), which introduces thiol groups to primary amines of the protein and then conjugated to HA‐VS to yield IL‐4‐tethered HA (IL‐4‐HA) (Figure 3a). Note that the reaction yield between thiolated IL‐4 and HA‐VS was ≈45%, as determined by subtractive analysis using ELISA, following a previously established protocol.^{[ 5 ]} Subsequently, we investigated the impact of IL‐4 conjugation on the storage modulus of the hydrogel and observed no significant changes before and after IL‐4 conjugation (Figure 3b). To ensure the clinical applicability of the hydrogel, it is critical to confirm its injectability, where it remains liquid during injection and solidifies at the target tissue. The gelation time of the optimized IL‐4‐tethered hydrogel, at a gelation concentration of 2.5% (w/v), was ≈2 min, excluding a 1‐min equilibration time. This rapid gelation time falls within the clinically acceptable range for injectable hydrogels, meeting the practical requirement for in situ gelation during administration (Figure 3c). The ability of the hydrogel to solidify within this time frame ensures seamless delivery through a syringe and minimizes the risk of premature gelation, a key consideration for therapeutic applications in joint tissues.^{[ 38 ]} Furthermore, the yield strain of the hydrogel was determined to be ≈100% through a strain sweep test conducted from 0.01% to 1000% strain at an angular frequency of 10 rad s⁻¹ (Figure 3d). Given that the shear strain exerted in synovial joints is reported to be ≈10–15%, this yield strain suggests the suitability of the hydrogel for joint applications.^{[ 39} , ^{40 ]} To assess the resilience of the hydrogel to repeated mechanical stress, we performed 10 consecutive strain sweep tests on the same hydrogel sample from 0.01% to 100% strain. No significant changes in storage modulus were observed, indicating the resistance of the hydrogel to repeated strain (Figure 3e). Additionally, the interconnectivity of the hydrogel network was evaluated using a water‐wicking assay, a well‐established protocol for measuring bulk hydrogel interconnectivity.^{[ 41 ]} As expected, the hydrogel formed at 2.5% (w/v) exhibited the highest interconnectivity at ≈52% (Figure 3f). SEM imaging further confirmed the mesh‐like structure of the hydrogel with micro‐sized pores interspersed throughout the polymer network, which may facilitate macrophage infiltration and interaction with the IL‐4‐tethered hydrogel network (Figure 3g). However, it should be noted that SEM images may not accurately reflect the native hydrogel structure due to the dehydration and sample preparation steps involved, which can introduce artifacts.^{[ 42 ]} To further corroborate the porosity and interconnectivity of the IL‐4‐tethered hydrogel, RAW 264.7 cells were seeded on top of the pre‐formed hydrogel and cultured for 48 h. To quantify cell infiltration, the bulk hydrogel was then enzymatically dissociated using hyaluronidase to recover the infiltrated cells. Based on this analysis, 39.3 ± 2.2% (mean ± SEM, n = 4) of the surface‐seeded cells infiltrated into the hydrogel matrix. These observations partially support the porous and interconnected nature of the hydrogel, which facilitates cell infiltration and promotes cell‐hydrogel interactions, essential for potential therapeutic applications in joint tissues.

Figure 3.

Synthesis and characterization of IL‐4‐tethered hydrogel. a) Schematic representation of the synthesis of IL‐4‐tethered hydrogel. b) The storage modulus of the optimal hydrogel formulation (Cg = 2.5% and DM = 25%) before and after IL‐4 conjugation. c) The gelation time of the optimal hydrogel tethered with IL‐4. d) Amplitude sweep test performed on the optimal hydrogel formulation (Cg = 2.5% and DM = 25%). e) Consecutive amplitude sweep tests were performed on the same hydrogel, indicating no significant fatigue upon exerting repeated strains. f) Interconnectivity of hydrogels formed with different Cg. g) An SEM image of the IL‐4‐tethered hydrogel synthesized (Scale bar: 50 µm). Data are means ± s.e.m [n = 3; ^* P < 0.05, ^** P < 0.01, ^*** P < 0.001, and ^**** P < 0.0001, paired two‐tailed Student's t‐test].

2.2. Macrophage Repolarization Capability of IL‐4‐Tethered Hydrogel

Prior to investigating the repolarization ability of the IL‐4‐tethered hydrogel on inflammatory M1 macrophages in vitro, the optimal concentration of IL‐4 required to induce M1 macrophage repolarization toward an immunosuppressive M2 phenotype was first determined using free IL‐4. To achieve this, bone marrow‐derived macrophages (BMDMs) were harvested from the tibia and femur of DBA/1 mice. Since the primary objective was to facilitate M2 polarization from M1 macrophages, BMDMs were polarized into the M1 phenotype by treatment with IFN‐γ for 24 h, following a previously established protocol with slight modifications (Figure 4a).^{[ 43 ]} Upon exposure of IFN‐γ‐polarized M1 BMDMs to increasing concentrations of IL‐4 (1 to 100 ng mL⁻¹), differential expression of M1 and M2 phenotypic markers was observed via flow cytometry (Figure S2, Supporting Information). Specifically, a reduction in M1‐associated markers, CD86 and iNOS, was evident, plateauing at 1 and 20 ng mL⁻¹, respectively, with significant reductions compared to untreated M1 macrophages. Concurrently, an upregulation of M2‐associated markers, CD206 and Arginase‐1, was observed, commencing at 10 and 10 ng mL⁻¹, respectively, with significantly higher expression compared to M1 macrophages. These findings established 20 ng mL⁻¹ of IL‐4 as the concentration yielding the maximal reduction in M1 markers (CD86 and iNOS) and the most pronounced increase in M2 markers (CD206 and Arginase‐1). These results align with previous studies, further supporting the role of IL‐4 in reprogramming M1 macrophages into the M2 phenotype to create an immunosuppressive microenvironment conducive to treating autoimmune disorders.^{[ 31} , ⁴⁴ , ⁴⁵ , ^{46 ]} Note that although we additionally tested a combination of IL‐4 and IL‐13, the combination did not show a significantly enhanced effect compared to IL‐4 alone at 20 ng mL⁻¹ (Figure S3, Supporting Information).

Figure 4.

Re‐polarization capability of IL‐4‐tethered hydrogel on inflammatory M1 macrophages in‐vitro. a) An experimental timeline. b) Phenotypic changes induced on M1 macrophages by the IL‐4‐tethered hydrogel (M1 marker: CD86 and iNOS, M2 marker: CD206 and Arginase‐1). c) Various cytokines produced by the hydrogel‐experienced macrophages (Pro‐inflammatory cytokine: TNF‐α and IL‐1β, Anti‐inflammatory cytokine: IL‐10 and TGF‐β). Data are means ± s.e.m [n = 3; ^* P < 0.05, ^** P < 0.01, ^*** P < 0.001, and ^**** P < 0.0001, paired two‐tailed Student's t‐test].

Having identified 20 ng mL⁻¹ as the optimal concentration for IL‐4‐mediated M1 macrophage repolarization, an equivalent amount of IL‐4 was conjugated to HA‐VS to synthesize IL‐4‐tethered HA (HA‐IL‐4). Specifically, the absolute mass of IL‐4 required for conjugation was back‐calculated to match a final concentration of 20 ng mL⁻¹, based on the volume of culture medium used to stimulate an equivalent number of macrophages. The HA concentration was maintained at the gelation concentration of 2.5 w/v (%). Notably, pre‐formed hydrogels were excluded from this study as they yield insufficient cell numbers for downstream analyses and primarily facilitate surface‐level interactions, which do not accurately recapitulate in vivo conditions.^{[ 47 ]} M1 macrophages were subsequently treated with HA‐IL‐4, and its repolarization potential was compared to that of free IL‐4 at the same concentration. This experimental design ensured that the IL‐4 conjugation did not compromise its bioactivity (Figure 4b,c). Upon treatment with HA‐IL‐4, a significant upregulation of M2 markers (CD206 and Arginase‐1) and a downregulation of M1 markers (CD86 and iNOS) were observed, with statistically significant differences compared to M0 or M1 macrophages (Figure 4b). Notably, CD206 expression increased by over 2‐fold, while Arginase‐1 showed an increase of 20‐fold compared to M1 macrophages. Meanwhile, both CD86 and iNOS levels exhibited a 2‐fold reduction in response to HA‐IL‐4 treatment compared to M1 macrophages. These findings confirmed that IL‐4 retained its functionality post‐conjugation. However, the extent of phenotypic changes induced by HA‐IL‐4 was less pronounced compared to free IL‐4, likely due to the confinement of IL‐4 within the HA polymer backbone, limiting its interaction frequency with macrophages. Despite this limitation, HA‐IL‐4 effectively facilitated the repolarization of M1 macrophages into the anti‐inflammatory M2 phenotype. To further validate the functional capacity of reprogrammed M2 macrophages, the secretion profiles of pro‐inflammatory cytokines (TNF‐α and IL‐1β) and anti‐inflammatory cytokines (IL‐10 and TGF‐β) were quantified (Figure 4c). As expected, both free IL‐4 and HA‐IL‐4 significantly reduced the secretion of pro‐inflammatory cytokines TNF‐α and IL‐1β relative to M1 macrophages. Similarly, both treatments robustly enhanced the production of anti‐inflammatory cytokines IL‐10 and TGF‐β compared to M0 or M1 macrophages. These findings further corroborate the efficacy of HA‐IL‐4 in promoting an anti‐inflammatory environment. Additionally, cytocompatibility assessments demonstrated that the IL‐4‐tethered hydrogel exhibited excellent biocompatibility over a 48‐h co‐incubation period with three distinct cells, specifically RAW264.7, BMDMs, and MCT3‐E1, which represent osteoblasts (Figure S4, Supporting Information). Collectively, these results highlight the potential of HA‐IL‐4 for in vivo therapeutic applications.

2.3. Therapeutic Effects of IL‐4‐Tethered Hydrogel in an RA Mouse Model

To evaluate the therapeutic effects of IL‐4‐tethered hydrogel in a collagen‐induced arthritis (CIA) mouse model, a standard experimental schedule was employed, including primary immunization (day ‐41), boosting (day ‐20), and sample injection (day 0), followed by analysis on day 28 (Figure 5a,b). The CIA model, a widely used RA model, effectively mimics the pathophysiology of human rheumatoid arthritis, enabling the study of joint inflammation and erosion.^{[ 48 ]} Mice were randomly distributed into experimental groups (healthy control, untreated RA, hydrogel‐only, IL‐4‐only, and IL‐4‐tethered hydrogel), and all analyses, including paw volume measurement and ankle thickness assessment, were conducted via blinded testing to ensure unbiased evaluation. Note that a physically encapsulated IL‐4 group was not included in the in vivo study, as it exhibited burst release behavior in vitro (≈85% IL‐4 released within 24 h), making it likely to behave similarly to the free IL‐4 group already included as a control (Figure S5, Supporting Information). Specifically, CIA‐induced mice received intra‐articular injections of either free IL‐4 (100 ng), 20 µL of blank hydrogel, or 20 µL of IL‐4‐tethered hydrogel containing 100 ng of conjugated IL‐4 (absolute mass). Paw and ankle thickness changes (fold) were calculated relative to the baseline thickness measured on the day of primary immunization (day ‐41). The IL‐4‐tethered hydrogel demonstrated superior therapeutic efficacy compared to the untreated and control groups. By day 28, mice treated with the IL‐4‐tethered hydrogel exhibited significant reductions in paw volume and ankle thickness (Figure 5c,d). Histological analyses using Safranin O and hematoxylin and eosin (H&E) staining also revealed similar findings, with the IL‐4‐tethered hydrogel group showing enhanced cartilage preservation (as evidenced by red staining of chondrocytes), reduced multinucleated osteoclast formation, and inflammatory infiltration compared to untreated RA mice (Figure 5e‐g). These therapeutic effects were further supported by micro‐CT imaging, which revealed improved joint integrity and reduced bone erosion in treated mice relative to untreated RA controls (Figure 5h). Cytokine profiling revealed a notable increase in anti‐inflammatory markers (IL‐10 and TGF‐β) in foot tissue treated with IL‐4‐tethered hydrogel compared to untreated mice, indicating its potential to modulate the inflammatory microenvironment (Figure 5i,j). These results confirm that IL‐4‐tethered hydrogel effectively alleviates RA symptoms by creating a local immunosuppressive environment, reducing inflammation, preserving joint structure, and protecting cartilage integrity.

Figure 5.

Therapeutic effects of IL‐4‐tethered hydrogel in an RA mouse model. a) Experimental timeline: Primary immunization (day ‐41), boosting (day ‐20), sample injection (day 0), and analysis (day 28). b) Representative images of mouse paws before and after CIA induction and treatment with IL‐4‐tethered hydrogel. c) Paw and d) ankle thickness changes (fold) measured relative to baseline thickness on the day of primary immunization (day ‐41). Representative e) Safranin‐O, and f) hematoxylin and eosin (H&E), staining of joint tissues, comparing RA, healthy, hydrogel‐only, IL‐4‐only, and IL‐4‐tethered hydrogel treatment groups. g) Representative hematoxylin and eosin (H&E) stained images showing the multinucleated osteoclasts (indicated with arrows). h) Micro‐CT images illustrating bone morphology across experimental groups: untreated, healthy, hydrogel‐only, IL‐4‐only, and IL‐4‐tethered hydrogel. Cytokine analysis of i) IL‐10 and j) TGF‐β levels from foot tissue treated with different samples. Data are means ± s.e.m [n = 3; ^* P < 0.05, ^** P < 0.01, ^*** P < 0.001, and ^**** P < 0.0001, paired two‐tailed Student's t‐test].

3. Conclusion

We successfully developed an IL‐4‐tethered hydrogel platform designed for the localized treatment of RA. This innovative platform enables joint lubrication and immunomodulation by promoting M2 macrophage polarization and therefore creating an anti‐inflammatory microenvironment. The hydrogel was imparted with optimized mechanical properties, ensuring its injectability and resilience under joint stress. Its effectiveness was evaluated in vitro and, in a collagen‐induced arthritis mouse model, demonstrating promising therapeutic outcomes, including reduced inflammation, enhanced cartilage preservation, and modulation of cytokine profiles. We believe that this modular platform can be further utilized to tether other therapeutic agents beyond IL‐4, enabling the development of multifunctional hydrogels for combinatorial treatment strategies that address the complex pathophysiology of RA. Overall, this IL‐4‐tethered hydrogel system holds great promise as a localized therapeutic approach for RA and potentially other inflammatory diseases, offering a foundation for future hydrogel‐based therapies.

4. Experimental Section

Preparation of Hydrogel Precursors

Hyaluronic acid (HA)‐based hydrogel precursors, specifically, vinylsulfonated HA (HA‐VS) and thiolated HA (HA‐SH) were synthesized according to previous literature.^{[ 16} , ^{33 ]} In brief, hyaluronic acid (Lifecore Biomedical) with a molecular weight of 100 kDa was dissolved in 0.1 m NaOH at a concentration of 2% (w/v) and was reacted with 3x molar excess of divinyl sulfone (DVS, Thermo Scientific) under ambient temperature with continuous stirring. Different degrees of modification (DM) of HA hydroxyl groups were achieved by controlling the reaction time. To stop the VS conjugation reaction, 6 m of HCl was added dropwise to the reaction mixture until pH became 4. The resulting solution was then dialyzed against deionized (DI) water for 3 days using Spectra/Por^® dialysis membrane (MWCO: 3500 Da). Afterward, the solution was freeze‐dried and subjected to ¹H Nuclear Magnetic Resonance (NMR) analysis by Bruker Avance III™ HD 400 MHz NMR spectrometer. The success of HA‐VS synthesis was confirmed by comparing the integral signals at δ = 6.9 and δ = 2 (acetyl group of the disaccharide).

IL‐4 Conjugation to HA‐VS

To conjugate IL‐4 (Peprotech) to the vinylsulfone groups of HA‐VS, IL‐4 was first thiolated using Traut's reagent (Thermo Scientific) according to the manufacturer's protocol. Briefly, IL‐4 was mixed with 0.1 m borate buffer of pH 8 and a 20‐fold molar excess of Traut's reagent was added to the mixture. Afterward, the mixture was allowed to react for 1 h with constant shaking. The thiolated IL‐4 was then purified using a desalting column (Thermo Scientific) pre‐equilibrated with PBS containing 5 mm EDTA. Subsequently, the thiolated IL‐4 was reacted with HA‐VS in PBS for at least 2 h. The unreacted IL‐4 was removed by using a 30 kDa (Molecular weight cut‐off) Amicon Centrifugal filter (Merck/Millipore). Afterward, the amount of IL‐4 conjugated to HA‐VS was determined by subtractive analysis using ELISA (Invitrogen), following a previously established protocol.^{[ 5} , ^{31 ]}

IL‐4 Conjugated Hydrogel Formation

To prepare the IL‐4‐tethered hydrogel, IL‐4‐conjugated HA‐VS and poly(ethylene glycol) dithiol (MW: 2000 Da, Sigma) were separately dissolved in PBS at predetermined concentrations. The two solutions were then mixed at specific polymer concentrations and DMs to induce hydrogel formation through thiol‐ene click chemistry. The resulting hydrogels were subsequently utilized for in vitro immunological assessments and physical characterizations to further optimize their structural and functional properties.

In Vitro IL‐4 Release

Hydrogels containing either physically encapsulated IL‐4 or covalently tethered IL‐4 were prepared as described above. Each sample was incubated in phosphate‐buffered saline (PBS) at 37 °C. After 24 h, the supernatant was collected, and the IL‐4 concentration was quantified by ELISA (Thermo Scientific) according to the manufacturer's instructions. The cumulative release was calculated as the percentage of the initially loaded IL‐4 mass (M/M_o).

Cell Infiltration Study in IL‐4‐Conjugated Hydrogels

An IL‐4‐conjugated hydrogel bedding was pre‐formed in 48‐well cell culture plates by adding 100 µL of hydrogel solution per well under sterile conditions. RAW 264.7 cells were then seeded on the surface of the pre‐formed hydrogel at a density of 1×10⁵ cells/ml in 600 µL of culture medium and incubated for 48 h at 37 °C. After the incubation period, the culture medium was removed, and cells remaining on the hydrogel surface were thoroughly washed off using PBS (without calcium) containing 0.53 mM EDTA. Afterward, the bulk hydrogel was then dissociated by treating it with 2% (w/v) hyaluronidase (Sigma aldrich) for 24 h at 37 °C to recover cells that had infiltrated the hydrogel network. The harvested cells were subsequently counted using a LUNA™ automated cell counter.

Physical Characterization of Hydrogels

The rheological properties of hydrogels synthesized were measured with an Anton Paar rheometer. Parallel plates of 25 mm diameter were utilized in this study. To determine the storage (G′) and loss (G″) moduli of hydrogels synthesized, 600 µL of hydrogel were gently placed in between the parallel plates with the gap height of 1 mm. Afterward, frequency sweep tests were performed in the frequency range spanning from 0.1 to 100 rad s⁻¹ at 0.1% strain at 25 °C. The gelation time was measured by performing time sweep tests, with the temperature adjusted to 37 °C, while the yield strength was measured by performing strain sweep tests from 0.01% to 1000% strain at 10 rad s⁻¹ angular frequency.

Percent Interconnectivity

To evaluate the interconnectivity (%) of the synthesized IL‐4‐conjugated hydrogel, the hydrogel was fully swollen in PBS for 24 h. After swelling, the hydrated hydrogel was weighed, and a Kimwipe was gently pressed against the surface for 30 s to remove loosely bound water. The mass was then recorded again. The interconnected volume was determined by calculating the percentage of the mass of water removed relative to the total hydrated mass.^{[ 41 ]}

Cell Isolation and Culture

RAW 264.7 cells were maintained in DMEM containing 10% fetal bovine serum (FBS) (Hyclone) and 1% penicillin/streptomycin (Hyclone) prior to initiating differentiation. For the differentiation into osteoclasts, RAW 264.7 cells were grown in αMEM supplemented with 10% FBS, 1% penicillin/streptomycin, and 20 ng mL⁻¹ RANKL (Sigma). MC3T3‐E1 preosteoblast cells (subclone 4) were cultured in αMEM (ascorbic acid‐free, Gibco) supplemented with 10% FBS and 1% penicillin/streptomycin. To induce osteoblast differentiation, MC3T3‐E1 cells were grown in αMEM with 10% FBS, 1% penicillin/streptomycin, 150 µg mL⁻¹ ascorbic acid (Sigma), 10 mm β‐glycerophosphate (Sigma), and 10 nm dexamethasone (Sigma).

Bone marrow cells were extracted from the femurs and tibias of DBA/1 mice and cultured in complete DMEM supplemented with 20 ng mL⁻¹ murine M‐CSF (referred to as BMDM culture medium) at a density of 6×10⁶ cells per 10 mL. The medium was replaced with 10 mL of fresh BMDM culture medium on day 3. On day 5, cells were detached using Accutase cell detachment solution, reseeded at the required concentration for specific assays, and cultured for an additional day. By day 6, the cells were fully differentiated into macrophages, designated as M0. To generate M1 or M2 BMDMs, M0 macrophages were further cultured for 24 h in BMDM culture medium supplemented with 20 ng mL⁻¹ murine IFN‐γ or 20 ng mL⁻¹ murine IL‐4, respectively. All centrifugation steps were conducted at 350×g, 4 °C, for 5 min, using a solution containing at least 1% FBS.

For the cell viability assays, 1×10⁴ BMDMs or RAW 264.7 cells and 5×10³ MC3T3‐E1 cells were seeded in a 96‐well plate and cultured for 24 h. The cells were then treated with IL‐4‐tethered HA. Cell viability was assessed using the Cell Counting Kit‐8 (CCK‐8, Sigma–Aldrich) assay, following the manufacturer's protocol.

Macrophage Repolarization In‐Vitro

M0, M1, M2, and BMDMs pre‐polarized to M1 BMDMs which are further treated with various concentrations of IL‐4, and IL‐4 tethered HA, were analyzed for phenotypes. The cells were detached in Accutase cell dissociation reagent (Thermo Scientific), labeled with fluorophore‐conjugated antibodies (APC‐conjugated anti‐Arginase 1 anti‐mouse monoclonal antibody (Invitrogen), eFluor 450‐conjugated anti‐mouse CD206 antibody (Invitrogen), FITC‐conjugated anti‐mouse CD86 antibody (Biolegend), and PE‐conjugated anti‐mouse iNOS antibody (Invitrogen)) and analyzed by flow cytometry. The culture supernatants were also collected after centrifugation, and the levels of TNFα, IL‐1β, IL‐10, and TGF‐ β were measured by ELISA (Thermo Scientific).

Mouse Model of Collagen‐Induced Arthritis

All animal experiments were conducted with approval from the Animal Care and Use Committees at KAIST (Approval No. KA2024‐117‐v1 and KA2021‐061). Male DBA/1 mice, aged 12 weeks, were obtained from Orient Bio (South Korea). To induce collagen‐induced arthritis (CIA), a solution of 2 mg mL⁻¹ type II collagen (Chondrex) emulsified with 4 mg mL⁻¹ complete Freund's adjuvant (CFA, Chondrex) in a 1:1 ratio was prepared and injected intradermally at the base of the tail of each DBA/1 mouse. After three weeks, a booster injection containing an emulsion of type II collagen and incomplete Freund's adjuvant (IFA, Chondrex) was administered intradermally.^{[ 48 ]}

In vivo therapy of CIA‐induced mice

CIA‐induced mice were intra‐articularly injected with free IL‐4 (100 ng), 20 µL of blank hydrogel, or 20 µL of IL‐4‐conjugated hydrogel. Healthy mice and untreated CIA‐induced mice were included as controls. Clinical scores were assessed in a blinded manner, and the thickness of each paw and ankle was measured using a caliper at three‐time points: at the time of CIA induction (healthy state), on the first day of treatment (Day 0), and at the end of the study (Day 28). On Day 28, hind paws were collected for histological analysis. Additionally, foot tissue fluids were extracted by incubating the excised paw tissues in 1 mL of RIPA lysis buffer (Sigma) supplemented with Complete Protease Inhibitor Cocktail (Sigma), followed by vigorous vortexing for 15 min at 4 °C. The supernatants were collected after centrifugation and stored at −80 °C for subsequent ELISA analysis. The concentrations of TNF‐α, IL‐1β, IL‐10, and TGF‐β (Invitrogen) in the synovial fluid were quantified using ELISA according to the manufacturer's protocol.

Histological Analysis

Hind paws were collected and fixed in 10% neutral buffered formalin (NBF) for 48 h, followed by decalcification in a 1 m EDTA solution for 2–3 weeks. The samples were embedded in paraffin, and 5 µm sections were prepared. Micro‐CT imaging (SkyScan 1173, Bruker) was used to analyze the general morphology of the bone after respective treatments (Genoss, South Korea). The tissue sections were stained with hematoxylin and eosin (H&E) and Safranin‐O following standard procedures.

Statistical Analysis

The level of significance in all statistical analyses was set at p < 0.05. Data were evaluated using either an unpaired Student's t‐test for two groups or one‐way or two‐way one‐way analysis of variance (ANOVA) for three or more groups. Analyses were performed using Prism 8.4 (GraphPad Software).

Conflict of Interest

The authors declare no conflict of interest.

Author Contributions

W.B. was responsible for the conceptualization, visualization, and original draft writing of the study. The methodology was developed by W.B. and M.J., while the investigation was conducted by W.B. and H.‐T.L. J.‐H.P. supervised the project. The review and editing of the manuscript were carried out by W.B., H.‐T.L., and J.‐H.P.

Supporting information

Supporting Information

Back W., Jeong M., Le H.‐T., Park J.‐H., Immunosuppressive Cytokine‐Tethered Hydrogel for Treating Rheumatoid Arthritis. Adv. Healthcare Mater. 2025, 14, e2501613. 10.1002/adhm.202501613

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.