Zwitterionic-based cyclic brush polymer nanomicelles with improved lubrication and antioxidation properties

Abstract

Osteoarthritis (OA) is a degenerative bone and joint disease characterized by cartilage degradation and an inflammatory environment. Consequently, strategies aimed at remodeling the damaged joint microenvironment by concurrently enhancing lubrication and alleviating inflammation are essential for improving therapeutic efficacy. Herein, we designed multifunctional cyclic brush polymer (CP) nanomicelles composed of surface-grafted zwitterionic poly(sulfobetaine methacrylate) (PSBMA) brushes and a hydrophobic core of PHEMA. Benefiting from the unique cyclic brush topology and hydrated lubrication properties of PSBMA, the CPs exhibited effective lubrication. Tribological and wear tests showcased the CPs significantly reduced the coefficient of friction and surface wear under shear forces. Furthermore, the CPs served as nanocarriers for encapsulating the anti-inflammatory drug resveratrol (RSV) through hydrophobic interactions, serving as drug-loaded nanomicelles CP@RSV. In vitro studies indicated that CP@RSV exhibited excellent cytocompatibility, effectively eliminated reactive oxygen species (ROS) in cells and reversed mitochondrial dysfunction, thereby modulating the oxidative stress microenvironment. In conclusion, CP@RSV integrates enhanced lubrication with antioxidation properties, representing a promising strategy for the treatment of OA.

---

Jing Xie

Jing Xie obtained her bachelor's and master's degrees from Jilin University in China with Professor Bai Yang and Professor Junhu Zhang, conducting research on intelligent polymers, then she obtained her doctorate degree from Max Planck Institute for Polymer Research in Germany, supervised by Professor Hans-Juergen Butt and Dr Kaloian Koynov, conducting research on diffusion in confined environments using fluorescence correlation spectroscopy. In July 2016, she joined the Department of Biomedical Engineering, College of Polymer Science and Engineering, Sichuan University and was promoted to associate professor in 2020. Her work is mainly engaged in research on medical polymer materials, focusing on the preparation of intelligent responsive polymer materials for the treatment of bone diseases.

---

1. Introduction

Osteoarthritis (OA) is a prevalent chronic joint inflammatory condition, impacting 300 million individuals globally, with its incidence increasing due to the challenges of global aging as well as contributing factors such as mechanical injury, obesity and genetics.^1–4 OA is primarily characterized by localized inflammation and irreversible alterations in the articular cartilage.⁵ As the disease advances, the reduction in lubricating properties of joint fluids results in heightened inter-articular friction, amplifying the inflammatory conditions and inflicting greater harm on cartilage tissue.^6,7 Consequently, lubrication dysfunction plays a pivotal role in the pathogenesis of OA.^8–10 In clinical practice, the current methods of treating OA include surgery, physiotherapy and medication. Among them, surgical interventions are associated with great risks and high costs, which brings a significant financial burden to patients.^5,11 Physiotherapy provides only short-term relief from OA symptoms.^12,13 In addition, prolonged use of drugs like nonsteroidal anti-inflammatory drugs (NSAIDs) may lead to gastrointestinal ulcers, bleeding and heightened cardiovascular risk.^14,15 At the same time, local intra-articular injections offer a reduced risk of systemic side effects and enhanced drug utilization compared to traditional oral or intravenous routes.^16,17 However, free drugs injected into the joint cavity are quickly cleared by the synovial capillaries and lymphatic vessels.^18,19 To address this issue, researchers have devised diverse injectable drug delivery systems, including liposomes, polymeric micelles, and hydrogels, to improve drug retention and efficacy.^14,20,21 For instance, Zhang et al.²² developed ROS-responsive polythioketone thermoplastic polyurethane (PTKU) loaded with the anti-inflammatory drug dexamethasone (DEX) to create polyurethane nanoparticles (PTKU@DEX). This synergistically combated elevated ROS levels in OA by the degradation of the PTKU backbone to scavenge ROS and release DEX for an anti-inflammatory effect. Zhou et al.²³ developed a novel injectable hydrogel, comprising hydrazide-grafted hyaluronic acid (HA-ADH) and aldehyde-modified dextran (Dex-ALH) bonded through a Schiff base reaction (HDH), and incorporated PEG-PTK-PEG micelles loaded with dexamethasone acetate (DA) to form the multifunctional platform HDH@PDM. This platform utilized ROS-responsive thioketone bonding (TK) for drug release, demonstrating exceptional ROS scavenging abilities. The present drug delivery systems primarily target inflammation in OA treatment, with a relatively straightforward approach. However, this approach often neglects the critical issue of lubrication failure, which can lead to irreversible and progressive damage to articular cartilage, further exacerbating the progression of OA. Hence, designing a simple and multifunctional drug delivery platform that offers both anti-inflammatory and lubrication enhancement capabilities is crucial for effective treatment of OA.

In recent years, the self-assembly of amphiphilic block copolymers in solution has been the subject of extensive research.²⁴ The size and morphology of the self-assembled nanostructures can be adjusted by varying the molecular topology (e.g., linear, cyclic, rod, or bottle-brush), sequence, composition and concentration.²⁵ Among these, cyclic brush copolymers have garnered significant interest due to their unique endless chain topology, comprising a cyclic core and grafted radiating polymer brushes.^26,27 These copolymers exhibit better thermal stability and lower hydrodynamic volume compared to their linear counterparts, facilitating the creation of more stable unimolecular micelles.²⁸ It has been reported that polymers with a molecular brush topology act as lubricants for cartilage, with cyclic brush copolymers offering superior spatial stability and reduced friction in shear due to the absence of chain ends.²⁹ They also allow for drug delivery through physical encapsulation or chemical coupling, making them widely used as nanocarriers.²⁷ Zwitterionic polymers are polymers containing oppositely charged groups in their repeating units. These polymers exhibit similar structures and functions to phosphatidylcholine groups on cartilage surfaces.^30–34 For example, poly(sulfobetaine methacrylate) (PSBMA) possesses high polarity and strong hydrophilicity through ionic solvation. Consequently, they display excellent lubrication properties by attracting water molecules to form a hydrated layer, reducing friction through hydration lubrication.^12,35 Therefore, by combining zwitterionic polymers with cyclic copolymers to create zwitterionic cyclic brush polymers it is possible to minimize friction between frictional pairs. Previously, we reported a pH-responsive cyclic brush dual-drug delivery platform with lubricating properties, utilizing zwitterionic SBMA and N,N-dimethylaminoethyl methacrylate (DMAEMA) as the brush body. Of these, SBMA contributes to lubricating properties, while DMAEMA improves drug utilization.³⁶ Despite ongoing copolymerization of zwitterionic with other monomers, monopolymerisation of zwitterionic monomers on cyclic polymers remains unexplored. Hence, the development of a drug delivery platform capable of addressing both inflammation and lubrication disorders is an urgent objective in current OA treatment.

In this study, we formulated zwitterionic cyclic brush polymer drug-loaded nanomicelles exhibiting dual functionality (Scheme 1), representing the first instance of polymerizing zwitterionic monomers directly onto cyclic polymers. Initially, we designed and synthesized the zwitterionic cyclic brush polymer cb-P(HEMA-g-PSBMA)₃₀ (CP) using the atom transfer radical polymerization (ATRP) method. The unique structure of the cyclic polymer, along with the PSBMA polymer brushes, imparted outstanding lubricating properties. Tribological assessments revealed that the CP effectively reduced the coefficient of friction (COF) and minimized surface wear. Subsequently, the drug resveratrol (RSV) was successfully encapsulated within the nanomicelles CP@RSV via hydrophobic interactions. RSV is a drug with vasoprotective, anti-inflammatory, anti-apoptotic, and anti-tumor properties, mediated through the modulation of multiple signaling pathways.³⁷ For instance, RSV inhibits the production of ROS by downregulating the expression and activity of NADPH oxidase, thereby reducing oxidative stress and exerting antioxidant effects.^38,39 Furthermore, RSV is considered an inhibitor of the key inflammatory transcription factor nuclear factor-κB (NF-κB), which suppresses the expression of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS), ultimately modulating inflammation and the immune response to infection.⁴⁰ Nevertheless, RSV faces challenges such as poor water solubility, in vivo stability and rapid metabolism, thereby limiting its clinical utility.^39,41 Therefore, encapsulating RSV within nanocarriers can effectively address these issues. In vitro free radical scavenging assays confirmed the antioxidant activity of CP@RSV, indicating its potential to effectively inhibit excess free radicals generated by inflammation. Furthermore, in vitro cellular studies demonstrated the excellent biocompatibility of CP@RSV, showing its efficacy in alleviating oxidative stress and mitigating mitochondrial damage. Therefore, the injectable nanomicelles of CP@RSV with good biocompatibility, lubrication and antioxidation properties prepared in this study have considerable potential in OA therapy and are expected to provide a new idea for intelligent drug delivery systems.

Scheme 1 Schematic illustration of the formation and mechanism of zwitterionic-based cyclic brush polymer nanomicelles with hydration lubrication and antioxidation properties.

2. Experimental

2.1. Materials

Sodium azide (NaN₃) was purchased from Shanghai Sanyou (Shanghai, China). 2-Hydroxyethyl methacrylate (HEMA, 99%, J&K) was purified through a basic Al₂O₃ column prior to use to remove the inhibitor. Bipyridine (bpy, 98%) was purchased from TCI. N,N,N′,N′′,N′′-pentamethyldiethylenetriamine (PMDETA, 99%), resveratrol (RSV, ≥99%), 2-bromoisobutyryl bromide (BIBB, ≥98%), [2-(methacryloyloxy)ethyl]dimethyl-(3-sulfopropyl) (SBMA, ≥97%) and copper(I) bromide (CuBr, >99.999%) were purchased from Aladdin. N,N-Dimethyl formamide (DMF, 99.5%), methyl alcohol (MT, 99.5%) and isopropyl alcohol (IPA, 99.7%) were purchased from Tianjin Bodi Co., Ltd (Tianjin, China). Anhydrous diethyl ether was purchased from Xilongkexue Co., Ltd (Sichuan, China). 1,1-Diphenyl-2-picrylhydrazyl (DPPH), dimethyl sulfoxide (DMSO, 99%) and 2,2-trifluoroethyl alcohol (TFEA, 99.8%) were purchased from Aladdin. The total antioxidant capacity assay kit with ABTS method (T-AOC Assay Kit) was purchased from Beyotime Biotechnology (Shanghai, China). The hydroxyl free radical assay kit was provided by Nanjing Jiancheng Biotechnology (Nanjing, China).

2-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI), TRITC Phalloidin, 2′,7′-dichlorofluorescin diacetate (DCFH-DA), lipopolysaccharides (LPS), propidium iodide (PI) staining solution, JC-1 mitochondrial membrane potential assay kit, hydrogen peroxide (H₂O₂) Content Assay Kit and fluorescein diacetate (FDA) were purchased from Solarbio Science & Technology Co., Ltd (Beijing, China). Penicillin–streptomycin solution, Dulbecco's modified Eagle's medium (DMEM), trypsin and fetal bovine serum (FBS) were bought from Gibco (USA). Phosphate buffered saline (PBS) and all consumables for cell research were purchased from Baoxin Biotechnology Co., Ltd. Cell Counting Kit-8 (CCK-8) was bought from Biosharp Biotechnology Co., Ltd (Anhui, China).

2.2. Preparation of nanomicelles

2.2.1. Preparation of zwitterionic cyclic brush polymers cb-P(HEMA-g-PSBMA)₃₀ (CPs). The polymer c-P(HEMA-Br)₃₀ was synthesized using methods similar to those described in the literature.³⁶ Briefly, l-P(HEMA)₃₀-Br was synthesized via atom transfer radical polymerization (ATRP) using propargyl 2-bromoisobutyrate as the initiator and a molar ratio of 30/1/1/1/2 for the monomer (HEMA), initiator, catalyst (CuBr), and ligand (bpy). The subsequent substitution reaction with NaN₃ produced l-P(HEMA)₃₀-N₃, which was then subjected to an intrachain click cyclization reaction to obtain c-P(HEMA)₃₀. Finally, c-P(HEMA-Br)₃₀ was obtained as the macromolecular initiator through a substitution reaction with BIBB. cb-P(HEMA-g-PSBMA)₃₀ (CP) was prepared by ATRP using c-P(HEMA-Br)₃₀ as the macroinitiator and SBMA as the monomer. For example, to prepare a polymer with a monomer concentration of 0.5 mg mmol⁻¹, c-P(HEMA-Br)₃₀ (4 mg), SBMA (235.57 mg), and bpy (4.39 mg) were dissolved in TFEA (1.687 mL) as the solvent. Solutions of the different materials were rapidly transferred to polymerization tubes and after three freeze–pump–thaw cycles, CuBr (2.02 mg) was quickly added in the fourth frozen state under nitrogen protection. After two additional cycles, the tubes were placed in an oil bath at 60 °C for three hours. Once the reaction was complete, the tube was opened and exposed to air for quenching. The product was rapidly added dropwise to 20 mL of ice diethyl ether and subsequently subjected to centrifugation at 6000 rpm. The precipitate was dissolved by adding a small quantity of deionized water, and then transferred to a dialysis bag (MWCO:1KDa) and dialyzed for 48 h. Ultimately, the white solid product CP was obtained by freeze-drying.

2.2.2. Preparation of drug-loaded nanomicelles CP@RSV. RSV (8 mg) and CP (50 mg) were individually dissolved in DMSO (1 mL) and deionized water (5 mL), respectively. The drug loading process was conducted by stirring in the dark for 12 h. To eliminate the unloaded drug and excess DMSO, the mixed solution was moved into a dialysis bag (MWCO: 1 kDa) and dialyzed for 48 h. Ultimately, the solution was freeze-dried to yield a white solid, designated as CP@RSV.

2.3. Materials characterization

2.3.1. Structural characterization of the CP. The polymer's average molecular weight (M_w) and polydispersity (PDI) were assessed using gel permeation chromatography (GPC, HLC-8320, Tosoh corporation, Japan). The ¹H NMR spectra of the CP and the products of the previous steps were recorded with a nuclear magnetic resonance spectrometer (NMR, AV III HD 400 MHz, Bruker, Germany) using DMSO-d6 and D₂O as the solvents. Fourier Transform Infrared Spectroscopy (FT-IR) spectra of the polymers were recorded in transmission mode using a Nicolet iS50 FT-IR spectrometer (Thermo Fisher, USA).

2.3.2. Characterization of the nanomicelle CP. The CP was dispersed in deionized water at a concentration of 0.5 mg mL⁻¹. The solution was sonicated for 10 minutes to promote self-assembly, and the resulting micellar solutions before and after drug loading were obtained. The average hydrodynamic size and zeta potential of the CP were evaluated utilizing the Zeta sizer (BeNano 180 zeta, Dandong Bettersize, China). Each sample was measured three times to ensure accuracy. The morphology of the CPs was examined by TEM on a JEM-2100 at an accelerating voltage of 200 keV. The observation samples were produced by adding the CP solution dropwise onto a carbon-coated copper lattice and staining with 2% (w/w) phosphotungstic acid as a negative stain. Finally, the morphology of the CP was observed after air-drying of the samples.

2.3.3. Tribological analysis. Tribological tests of CP solutions were performed at room temperature using a multifunctional friction and wear tester (UMT-3, Bruker, USA) in reciprocating mode with a ball-and-disc friction pair. Si₃N₄ ceramic balls with a diameter of 6.35 mm were used for the upper friction pair, while polytetrafluoroethylene (PTFE) blocks were used for the lower friction pair. Specifically, the polymer solution (1 mL) was applied to the contact area of the upper and lower friction pair, and deionized water (H₂O) was used as the control group. The sliding speed, sliding amplitude, and test time were set at 3 mm s⁻¹, 3 mm, and 8 min, respectively. The coefficient of friction-time curves of the polymer solutions were recorded by varying the concentration (0.0625–5.0 mg mL⁻¹), frequency (1–3 Hz) and load (1–3 N). The lubricating properties of the polymer solutions were evaluated based on these curves.

The surface wear of the PTFE block after the friction test was examined utilizing a confocal laser scanning microscope (CLSM, LSM700, Zeiss, Germany). 3D wear strength was measured with ImageJ software. In addition, the morphology of the wear marks was observed and analyzed using a white light interferometer (WLI, Chotest Superview W1, Chotest Technology Inc., China).

2.3.4. In vitro drug loading and drug release. The freeze-dried CP@RSV were re-dispersed in deionized water and their absorbance at 304 nm was measured. The concentration of RSV in nanomicelles can be determined from the standard curve. The drug loading content (DLC) and encapsulation efficiency (EE) were determined using the following formulas:

where W_drug is the weight of the actual loaded RSV, W_micelle is the weight of the micelles, and W_{initial loaded drug} is the initial weight of RSV loaded in the micelles.

The drug release profiles of CP@RSV were determined as follows. First, 1 mL of CP@RSV solution was moved into a dialysis bag (MWCO:1kDa). The dialysis bag containing the solution was then placed in a 100 mL centrifuge tube containing 20 mL PBS of pH 5.5 and pH 7.4. Finally, the tube was placed in a shaker at 37 °C with an agitation speed of 120 rpm. At designated time points (0.5, 1, 2, 4, 6, and 12 hours), 2 mL of sample was withdrawn and replaced with an equivalent volume of fresh PBS. The cumulative drug release from the nanoparticles was determined based on the RSV standard curve, and time-dependent drug release profiles were obtained.

The particle size and zeta potential of the sample during drug release were measured as described above. At the indicated time points, the sample solution was taken from the dialysis bag and analyzed by the Zeta sizer.

2.3.5. Characterization of drug-loaded nanomicelles CP@RSV. The particle size, morphology, Zeta potential and tribological experiments of the CP@RSV nanomicelles were evaluated using methods similar to those employed for the CP.

2.3.6. Injectability tests. The CP and CP@RSV were dispersed in deionized water at a concentration of 0.125 mg mL⁻¹. The resulting solutions, along with H₂O, were subsequently loaded into 1 mL syringes for the injection test. The injection force of the materials was measured using a texture analyzer (TA. Xtc-20, Shanghai Baosheng Industrial Development Co., Ltd.) in compression mode, with an injection rate of 0.5 mm s⁻¹. The average force measured between 10 and 30 seconds was used as the injection force. Three replicates were performed for each sample.

2.3.7. In vitro antioxidant evaluation.
2.3.7.1. H₂O₂ scavenging assay. Each sample solution (0.125 mg mL⁻¹) was incubated with H₂O₂ solution (1 mM) at 37 °C for 4 hours. The residual concentration of H₂O₂ was then determined using the Hydrogen Peroxide Content Assay Kit. Absorption spectra were recorded using a UV-visible spectrophotometer, and the absorbance at 415 nm was measured using a microplate reader. The elimination of the H₂O₂ radical was calculated using the following formula:
where A_0 sample denotes the absorbance of the sample alone, and A_sample and A_control represent the absorbance the application solution with and without the sample, respectively.
2.3.7.2. Hydroxyl radical (˙OH) scavenging assay. The different material solutions were mixed with the application solution following the protocol of the hydroxyl free radical assay kit. After incubation at room temperature for 20 minutes, the absorption spectra and absorbance values at 550 nm were measured using a UV-visible spectrophotometer and a microplate reader, respectively. The elimination of the ˙OH radical was calculated using the following formula:
where A_0 sample denotes the absorbance of the sample alone, and A_sample and A_control represent the absorbance the application solution with and without samples, respectively.
2.3.7.3. DPPH scavenging assay. 1 mL of each material solution was mixed with 1 mL of DPPH solution (0.1 mM) at room temperature and incubated in the dark for 30 minutes. The absorption spectra were then recorded with a UV-visible spectrophotometer, and the absorbance was measured at 517 nm by a microplate reader. The elimination of the DPPH radical was calculated using the following formula:
where A_0 sample represents the absorbance of the sample solution alone, A_sample represents the absorbance of the sample mixed with DPPH, and A_control represents the absorbance of the DPPH.
2.3.7.4. Evaluation of total antioxidant capacity. The total antioxidant capacity of CP@RSV was assessed utilizing the T-AOC Assay Kit with the ABTS method. The working solution of ABTS (900 μL) was prepared according to the kit's instructions, and then mixed with the sample solution (100 μL). The absorbance at 734 nm was determined after the sample was incubated at room temperature for 10 minutes. The antioxidant capacity of the samples was calculated by incubating 100 μL of different concentrations of the standard solution with 900 μL of the working solution for 10 minutes to generate a standard curve.

2.4. Cellular experiment

2.4.1. Cell viability study. First, chondrocytes and RAW 264.7 cells were cultured in DMEM medium containing 1% antibiotics and 10% FBS. The two types of cells were then inoculated into well plates at a density of 3000 cells per well and cultured for 24 hours. Different concentrations of RSV, CP and CP@RSV solutions were prepared using complete culture medium as the solvent. 100 μL of each solution were added to the wells to co-cultivate with the cells for 1, 3, and 5 days. Afterward, the solvent was removed, and 100 μL of CCK-8 reagent (volume ratio of CCK-8 to complete medium 1 : 10) was added under dark conditions, followed by co-cultivation for an additional 3 hours. The optical density (OD) at 450 nm was ultimately recorded utilizing an enzyme labeler (ST-360, KHB, Shanghai). The formula for calculating cell viability is as follows:
where OD_{Experimental group} indicates the absorbance value of cell wells under the co-culture of different experimental groups. OD_{Experimental blank} indicates the absorbance value of the experimental materials (without cells) after reaction with CCK-8 reagent. OD_{Control group} represents the absorbance value of only the cells. OD_{Control blank} represents the absorbance value of the culture medium (without cells) after reaction with CCK-8 reagent.

2.4.2. Live/dead staining. Chondrocytes were inoculated into 48-well plates at a density of 6000 cells per well and cultured at 37 °C with 5% CO₂ for 24 hours until the cells were fully adhered to the plate. After that, 200 μL of RSV, CP and CP@RSV medium solutions were added and co-cultured for 1, 3, and 5 days. Staining working solutions were prepared with 50 μg mL⁻¹ of FDA for detecting live cells (green color) and 10 μg mL⁻¹ of PI for detecting dead cells (red color). After adding 200 μl of the working solution to each well, the staining was performed in the dark for 15 minutes. Subsequently, the cells underwent five washes with PBS and were promptly observed under a fluorescence microscope (Olympus IX71, Japan).

2.4.3. Morphological staining. Chondrocytes were cultured as previously described. The cells were co-cultured with RSV, CP and CP@RSV medium solutions in 48-well plates for 1, 3, and 5 days, and then removing the medium solutions. Afterward, the cells were treated with 4% paraformaldehyde solution to fix them before being permeabilized with 0.5% Triton X-100 in the dark. Next, rhodamine-labeled phalloidin was added to stain the cells for 30 minutes, and DAPI (blue) was applied to stain the nuclei for 10 minutes. Finally, the cells were subjected to five washes with PBS and were immediately placed under a fluorescence microscope for observation.

2.4.4. Cellular ROS scavenging activity. Intracellular ROS generation was evaluated by employing DCFH-DA as the ROS probe. RAW 264.7 cells and chondrocytes were inoculated into well plates at a density of 6000 cells per well. After a 24-hour incubation period, they were exposed to 1 μg mL⁻¹ LPS for another 24 hours. Subsequently, they were treated with DMEM, RSV, CP and CP@RSV for 24 hours, respectively. Cells that were not stimulated with LPS were used as controls. After that, 200 μL of DCFH-DA solutions were dispensed into each well for a 30-minute incubation in a darkened environment. Finally, the cells underwent three washes with PBS and were observed. The fluorescence intensity was then quantified utilizing ImageJ software.

2.4.5. Mitochondrial membrane potential staining. The mitochondrial function of chondrocytes and RAW 264.7 cells were examined using a mitochondrial membrane potential assay kit containing JC-1. Both cell types were seeded into well plates and incubated for 24 hours. Except for the control group, all four groups were stimulated with 200 μL of H₂O₂ (100 μM) for 24 h to mimic oxidative stress, followed by incubation with DMEM, RSV, CP, and CP@RSV for 24 h, respectively. Afterwards, 200 μL of JC-1 staining solutions were included into each well. After that, these cells were incubated for 20 minutes, followed by two washes with JC-1 staining buffer, preparing them for observation. The fluorescence intensity was quantified utilizing ImageJ software.

3. Results and discussion

3.1. Syntheses and characterization of the CPs

The synthetic route for the zwitterionic cyclic brush polymer (cb-P(HEMA-g-PSBMA)₃₀, CP) is shown in Fig. 1A and Fig. S1 (ESI†). Initially, the l-P(HEMA)₃₀-Br was synthesized by ATRP, and its degree of polymerization (DP) was ascertained to be ∼30 by comparing the ratio of the integrated intensity of peak b assigned to the methylene protons adjacent to the ester bonds to that of the peak a attributed to the proton of the triple bond in the ¹H NMR spectrum (Fig. S2, ESI†). Subsequently, c-P(HEMA)₃₀ was synthesized through intrachain click cyclization of the linear precursor l-P(HEMA)₃₀-N₃. The success of cyclization was verified through FT-IR and GPC analysis to confirm the structure of c-P(HEMA)₃₀. As shown in the FT-IR plot in Fig. 1B, azide groups (∼2120 cm⁻¹) were absent after the click reaction. The GPC curves (Fig. 1C) show that the c-P(HEMA)₃₀ polymer exhibits longer retention times (lower molecular weights) with respect to the linear precursor, and that these polymers all exhibited single peaks and narrowly distributed MWs, indicating the achievement of controlled polymerization. Furthermore, a comparison of the ¹H NMR spectra of l-P(HEMA)₃₀-N₃ and c-P(HEMA)₃₀ (Fig. S3, ESI†) reveals the disappearance of the ethylene proton signal at 4.65 ppm, further validating the success of the cyclization reaction. After that, the macromolecular initiator c-P(HEMA-Br)₃₀ was prepared by reacting c-P(HEMA)₃₀ with BIBB. The disappearance of the hydroxyl peak at 4.8 ppm in the ¹H NMR spectrum (Fig. S4, ESI†) confirmed the successful synthesis of the macromolecular initiator. Finally, to prepare cyclic brush polymers designed to enhance lubrication, the zwitterionic cyclic brush polymer CP was synthesized using c-P(HEMA-Br)₃₀ as the macroinitiator and zwitterionic SBMA as the monomer. The 1H NMR spectra of c-P(HEMA-Br)₃₀ and polymer CP are presented in Fig. 1D, which distinctly show the proton signals of SBMA at 4.45, 3.77, 3.19, 2.94, and 2.23 ppm. The MW, DP, yield and PDI of the CPs with different monomer concentrations are shown in Fig. 1E. Moreover, the GPC images of the CPs (Fig. S5, ESI† and Table S1 (ESI†)) indicate that CP2 exhibited a high yield, a single-peak distribution in GPC analysis, and more importantly, the highest DLC and EE, thus CP2 was selected for subsequent tribological experiments.

Fig. 1 (A) Synthesis route and schematic diagram of the zwitterionic cyclic brush polymer (CP). (B) FTIR spectra of l-P(HEMA)₃₀-N₃ and c-P(HEMA)₃₀. (C) GPC curves of l-P(HEMA)₃₀-Br, l-P(HEMA)₃₀-N₃ and c-P(HEMA)₃₀. (D) ¹H NMR spectra of c-P(HEMA-Br)₃₀ and the CP. (E) M_w, DP, yield and PDI of the CPs at varying monomer concentrations.

3.2. Lubrication and wear properties

Improving lubrication to reduce joint friction is the key to treating OA.⁸ To assess the friction-reducing properties of the CPs, tribological experiments were conducted using a multifunctional friction and wear tester in cyclic reciprocating mode, as depicted in Fig. 2A. Si₃N₄ ceramic balls and PTFE were used for the upper and lower friction subsets, respectively. Firstly, we measured the COF – time plots of CP solutions with different concentrations at 1 Hz with a 3 N load (Fig. 2B). The graph shows that the COF values remained fairly stable during sliding, with only minor fluctuations. Notably, the friction time curves for various concentrations of CP solutions were lower than that of H₂O, which is precisely due to the hydration lubrication of zwitterionic brushes. The positively charged –N⁺(CH₃)₂– and the negatively charged –SO₃⁻ functional groups in PSBMA can combine with large numbers of water molecules through ionic dipole interactions, generating a dense hydration layer around the charges. This hydration layer can tolerate high pressures without shifting position, while still transitioning quickly to a relaxed state, thus exhibiting a fluidic behaviour under shear, effectively reducing friction at the joint interface.^42,43 In addition, the COF value of 0.125 mg mL⁻¹ was lower than that of other concentrations, thus it was utilized for the subsequent experiments. The lubrication performance of CP under varying loading forces and reciprocating frequencies were investigated. The histograms of the average COF values of H₂O and CP at 1 Hz, 1 N; 1 Hz, 3 N; and 3 Hz, 3 N are shown in Fig. 2C–E. The lubrication effect of the CPs significantly improved, yielding lower COF values compared to H₂O under different test conditions. Specifically, increasing the applied normal load at the same reciprocating frequency resulted in a decrease in COF. Increasing the load from 1 N to 3 N at 1 Hz, the COF of CP reduced from 0.035 to 0.026, while the COF of H₂O decreased from 0.042 to 0.036, likely due to boundary lubrication effects. The increase in reciprocation frequency from 3 Hz to 1 Hz had a lesser impact on COF at the same normal load of 3 N. The above data indicate that the zwitterionic cyclic brush polymer CP can maintain a stable and excellent lubrication effect under shear.

Fig. 2 (A) Schematic of the tribological testing method and lubricating mechanism of the CP. (B) CP concentrations of 0.0625–5.0 mg mL⁻¹ for COF-time curves. Histograms of COF for H₂O and CP solutions with a concentration of 0.125 mg mL⁻¹ at (C) 1 Hz 1N, (D) 1 Hz 3N and (E) 3 Hz 3N. (F) 2D and 3D images of surface wear and distance-wear intensity curves for the blank, H₂O and CP groups after friction tests at 3 Hz with a 3 N load. (G) WLI morphology and cross-sectional profiles of PTFE surfaces and H₂O and CP wear marks at 3 Hz with a 3 N load.

To further illustrate the lubrication effect of CP, we employed CLSM to analyze the wear morphology of PTFE surfaces after tribological experiments. Fig. 2F presents two-dimensional (2D) and ImageJ processed three-dimensional (3D) images of the PTFE surface wear obtained for friction between CP and H₂O as lubricants at 3 Hz with 3 N load, and a blank group without friction was used as a control. Moreover, the quantitative data demonstrate the wear intensity versus the distance along the green line in the middle of the 2D image. The results indicate that the wear surface topography was pronounced in the H₂O and CP groups compared to the blank group, but the CP group displayed the shallowest wear. Notably, the wear intensity-distance curves and 3D images demonstrated a significantly lower wear intensity in CP than in H₂O. Additionally, Fig. S6 and S7 (ESI†) depict the degree of surface wear under 1 Hz, 1 N and 1 Hz, 3 N conditions, which present the same results. Additionally, WLI was employed to observe the three-dimensional morphology of the PTFE surface and the cross-sectional profile of the wear marks (Fig. 2G). The unabraded PTFE surface displayed only post-processing marks with no visible wear. In contrast, both the CP and water groups exhibited more pronounced wear marks, although the depth of these marks in the CP group was significantly smaller than that observed in the H₂O group. This finding provides a clear demonstration that the CP effectively reduces friction and minimizes wear. In conclusion, our findings demonstrate that the CP exhibits stable lubrication properties that significantly reduce interfacial friction and surface wear. Therefore, it shows a promising approach as a lubricant.

3.3. Characterization and antioxidant properties of CP@RSV

Zwitterionic cyclic brush polymers can form stable drug-loaded nanomicelles by self-assembly due to their amphiphilic structure.^24,44 CP can physically encapsulate the RSV via hydrophobic interactions, without altering the drug's properties, resulting in the formation of CP@RSV, as schematically illustrated in Fig. 3A. The DLC and EE of the polymers at varying monomer concentrations, calculated from the RSV standard curve (Fig. S8, ESI†), are detailed in Table S1 (ESI†). Notably, compared to other polymers, CP2 exhibited the highest DLC of 5.72% and an EE of 35.75%. This is likely due to the hydrophilic branched brushes of medium chain length (M_w), which provide enhanced encapsulation space and optimal hydrophilic–hydrophobic interactions, thereby maximizing the drug-loading capacity of the micelles.^26,45 Therefore, we selected CP2 (referred to as CP) as the model polymer. TEM observations revealed that both CP and CP@RSV displayed a uniform and regular spherical morphology, with a significant increase in the size of the nanomicelle following drug loading (Fig. 3B and C). The particle sizes of CP and CP@RSV were further measured by DLS and found to be 125.8 nm and 228.1 nm, respectively (Fig. 3D), indicating the successful preparation of CP@RSV. It is noteworthy that the particle size measured by DLS was notably larger than those observed by TEM. This variation can be attributed to the fact that DLS assesses the hydrodynamic size of nanoparticles in an aqueous phase, while TEM reflects the size of the nanoparticles in a dehydrated and dried state. The hydrodynamic diameter change of CP over time was further detected by dynamic light scattering (DLS).^46,47 Furthermore, the Zeta potential data showed that the CP@RSV had a higher potential compared to the CP, which confirms the successful loading of RSV.

Fig. 3 (A) Schematic diagram of the drug-loaded nanomicelles CP@RSV preparation. TEM images of CP (B) and CP@RSV (C) at a polymer concentration of 0.25 mg mL⁻¹. (D) Hydrodynamic diameters and Zeta potentials of CP and CP@RSV in water measured by DLS. (E) Cumulative release of RSV in PBS solution at pH 7.4 and pH 5.5. Changes in (F) particle sizes and (G) Zeta potentials over time during drug release. (H) Injection force of CP and CP@RSV at a concentration of 0.125 mg mL⁻¹. UV-vis absorption spectra of (I) H₂O₂, (K) ˙OH and (M) DPPH radicals after incubation with RSV and CP@RSV. Scavenging capacity of (J) H₂O₂, (L) ˙OH and (N) DPPH. (O) Total antioxidant capacity.

The cumulative release profile of RSV is shown in Fig. 3E. The data show that the drug was released rapidly during the first four hours, with a cumulative release of 70.8% at pH 7.4 and 82% at pH 5.5. After this initial burst, the release rate slowed down. Notably, the release rate of RSV was faster at pH 5.5 than at pH 7.4. These results suggest that a greater amount of drug was released at pH 5.5, which can be attributed to a pH-responsive phenomenon.⁴⁸ At lower pH, the zwitterionic groups of the polymer experience an increase in the charge density of their cationic groups (–N⁺(CH₃)₂–) and a decrease in the deprotonation of their anionic groups (–SO₃⁻). This charge reversal disrupts the electrostatic equilibrium within the micelles, leading to micelle swelling and enhanced drug release. This is further supported by the larger particle size observed at pH 5.5, as shown in Fig. S9A (ESI†). The zeta potential of CP and CP@RSV in PBS at pH 7.4 and pH 5.5 was analyzed using DLS (Fig. S9B, ESI†). The results demonstrated that the zeta potentials at pH 7.4 and pH 5.5 were notably higher than those in deionized water. This phenomenon may be attributed to the cations present in the PBS buffer solution, which interact with the negatively charged nanomicelles, effectively shielding the negative charges on the nanomicelles’ surfaces and thereby resulting in a significant increase in the Zeta potential. The change of particle size during drug release was further analyzed (Fig. 3F). The data show that the particle size gradually decreased over time, with a slower rate of change observed after the fourth hour. This suggests that a substantial amount of drug was released within the first four hours, causing a rapid decrease in particle size, followed by stabilization. Additionally, the change in zeta potential during drug release was also examined (Fig. 3G). The results indicated that the zeta potential increased rapidly within the first four hours and then stabilized. Notably, the changes in both particle size and zeta potential closely mirrored the drug release profile. The observed differences in particle size and zeta potential of CP@RSV following drug release, compared to CP nanomicelles, can be attributed to the incomplete release of the drug. A portion of the drug remains encapsulated within the nanomicelles. In addition, the injectability of the CP@RSV was evaluated. As shown in Fig. 3H, the injection forces of CP and CP@RSV were measured to be 0.377 N and 0.388 N, respectively, which were not significantly different from that of H₂O (0.368 N). All the injection force curves were smooth (Fig. S10, ESI†). These minimal differences indicate that drug loading had a negligible impact on the injectability of the nanocarrier system. Tribological experiments were subsequently conducted on CP@RSV. The data presented in Table S2 (ESI†) indicate that the COF values for CP@RSV were similar to those of the CP group, with both being significantly lower than those observed for the H₂O group. Wear data shown in Fig. S11 (ESI†) reveal that the wear intensity and wear depth of CP@RSV are considerably lower than those of the H₂O group under 1 Hz 1 N, 1 Hz 3 N and 3 Hz 3 N conditions. These results suggest that CP@RSV exhibits a sustained and effective lubrication performance.

Abnormal elevations of ROS and reactive nitrogen species (RNS) in synovial fluid and chondrocytes play a crucial role in the development of OA and are involved in the entire inflammatory process. Mechanistic studies have confirmed that excessive ROS and RNS negatively affect chondrocyte function, leading to extracellular matrix degradation, mitochondrial damage, and apoptosis, which ultimately contribute to chronic inflammation and cartilage destruction.^49–51 Therefore, the development of a biomedical material with free radical scavenging capabilities is highly advantageous for OA treatment. As is well known, RSV is a natural polyphenol with phenolic hydroxyl groups that can provide antioxidant properties.³⁷ To assess whether RSV encapsulated within CP retains its antioxidant capacity, we conducted assays to evaluate its ability to scavenge ROS and RNS, including H₂O₂, ˙OH, and DPPH radicals. The UV-Vis absorption spectra (Fig. 3I and K) demonstrated the greatest reduction in the characteristic peaks of H₂O₂ and ˙OH following CP@RSV addition, confirming its excellent ROS scavenging ability. Quantitative data from Fig. 3J and L further show that CP@RSV exhibited the strongest scavenging activity against these three radicals, with scavenging rates of 31.9% and 52.1%, respectively. These results indicate that CP@RSV outperforms RSV in free radical scavenging, which can be attributed to the encapsulation of RSV in the CP. The CPs with a unique cyclic topology and zwitterionic brush structure can significantly enhance the utilization of RSV by overcoming the limitations of RSV's short half-life and poor stability in free form. Furthermore, the ability of CP@RSV to scavenge RNS was evaluated through DPPH radical scavenging assays. Fig. 3M illustrates a decrease in absorbance at 517 nm, indicating the elimination of DPPH radicals by RSV and CP@RSV. Quantitative data (Fig. 3N) revealed that CP@RSV exhibited a 62.2% scavenging rate for DPPH radicals, demonstrating its effective RNS scavenging capacity. Finally, the total antioxidant capacity of CP@RSV was calculated from a standard curve (Fig. S12, ESI†) using the total antioxidant capacity kit. Fig. 3O showed that CP@RSV had the highest total antioxidant capacity. In summary, CP@RSV significantly enhances antioxidant activity, which can effectively inhibit inflammation to produce overexpressed free radicals ROS and RNS. Thus, CP@RSV can serve as an effective scavenger of reactive oxygen species.

3.4 Cytocompatibility evaluation

To assess the feasibility of CP@RSV in the treatment of OA, it is necessary to conduct cytocompatibility tests. Firstly, the effects of RSV, CP and CP@RSV on the viability of chondrocytes and Raw264.7 cells were evaluated at various concentrations using the CCK-8 assay. As illustrated in Fig. 4A, at the highest concentration of 500 μg mL⁻¹, chondrocyte viability remained above 80% in all groups. Notably, while the RSV group exhibited a slightly lower cell survival rate compared to the other groups, the CP@RSV group maintained a viability of approximately 100%. This finding suggests that loading RSV into the cyclic brush polymer CP considerably decreases drug toxicity and enhances drug bioavailability. Similarly, the viability of Raw264.7 cells was also found to be high across all groups, with no notable difference between CP@RSV and the control group (Fig. 4B). The findings suggest that CP@RSV does not adversely affect the viability of either chondrocytes or Raw264.7 cells and exhibits minimal cytotoxicity.

Fig. 4 Cell viability of chondrocytes (A) and Raw264.7 cells (B) at different concentrations of RSV, CP and CP@RSV determined by CCK-8. (C) Cell proliferation of different materials co-cultured with chondrocytes for 1, 3 and 5 days. (D) Fluorescence pictures of chondrocyte live/dead staining. (E) Fluorescence pictures of morphological staining of chondrocytes.

To further explore the cytocompatibility of the materials, cell proliferation was evaluated using the CCK-8 assay, live–dead cell staining and morphological staining. Fig. 4C shows the OD values measured at 450 nm after co-culturing different materials with chondrocytes for 1, 3, and 5 days. The OD values increased steadily from day 1 to day 5, with minimal differences between groups. Live–dead staining (Fig. 4D) revealed a significant increase in the number of chondrocytes across all groups, with fluorescence images predominantly showing green fluorescence (live cells) and very little red fluorescence (dead cells), confirming normal proliferative behavior. Additionally, the effects of the various groups on chondrocyte growth morphology were assessed using DAPI and Phalloidin staining, which highlighted the cytoskeleton (red fluorescence) and nuclei (blue fluorescence). The fluorescence images in Fig. 4E demonstrate that chondrocytes from all groups exhibited clearly defined nuclei and intact cytoskeletons, with cells evenly distributed and neatly arranged in a normal spindle shape, indicating healthy growth. In summary, the results of these cell experiments strongly indicate that CP@RSV possesses excellent biocompatibility, thus highlighting its potential for further applications.

3.5. Intracellular ROS scavenging evaluation

The inflammatory environment often leads to the induction of oxidative stress, resulting in the generation of a high level of ROS.⁵¹ The ROS fluorescent indicator DCFH-DA was used to evaluate the ROS-scavenging ability of CP@RSV. Briefly, DCFH-DA is easily absorbed by cells and is hydrolyzed by intracellular esterases into DCFH. Upon interaction with intracellular ROS, DCFH is oxidized to green fluorescent DCF. Firstly, chondrocytes and Raw264.7 cells were stimulated with LPS, a potent activator commonly used to stimulate cells. As illustrated in Fig. 5A and B, after 24 hours, Raw264.7 cells and chondrocytes exhibited strong green fluorescence compared to the control group, indicating a significant elevation of intracellular ROS levels. 3D surface plots showed that the LPS group had the widest distribution of fluorescence and the highest intensity. However, RSV treatment significantly attenuated the LPS-induced elevation of ROS levels. Moreover, CP@RSV showed the best scavenging efficiency, which resulted in long-term drug stability and enhanced antioxidant efficacy due to the unique topology of the ring-brush polymer. The 3D surface plots and fluorescence quantifications showed that, compared to the CP and RSV groups, CP@RSV had the least fluorescence distribution and weakest fluorescence intensity. Thus, CP@RSV effectively scavenged excessive ROS in Raw264.7 cells and chondrocytes, with the ROS levels in the cells showing no notable difference compared to the control group. Quantitative fluorescence assessments (Fig. 5C and D) indicated the fluorescence intensities of Raw264.7 cells and chondrocytes were 12.2 and 15.6 after LPS treatment, respectively, while they reduced to 2.5 and 1.0 following co-culture with CP@RSV, respectively. Our data indicates that CP@RSV has excellent antioxidant activity and significantly inhibits the expression of intracellular ROS. Therefore, it shows great potential for use in reducing oxidative stress, inhibiting the development of inflammation, and treating inflammation related diseases.

Fig. 5 DCFH-DA staining to observe ROS production in Raw264.7 cells (A) and chondrocytes (B). Quantification of ROS fluorescence in Raw264.7 cells (C) and chondrocytes (D) after different treatments (n = 3, *p < 0.05, **p < 0.01 and ***p < 0.001 indicate a significant difference in comparison to the control group, ^☆p < 0.05, ^☆☆p < 0.01 and ^☆☆☆p < 0.001 demonstrate statistical differences between the other experimental groups, ns p > 0.05 suggests no significant difference).

3.6. Intracellular mitochondrial function evaluation

The excessive generation of ROS during the progression of OA significantly impacts cellular function in various aspects. While mitochondria are the primary organelles for ROS generation in cells, mitochondrial dysfunction is closely associated with oxidative stress. Among the various indicators of mitochondrial health, mitochondrial membrane potential (MMP) is closely associated with mitochondrial function.^52–54 To evaluate the effects of CP@RSV on the mitochondrial function of chondrocytes and Raw 264.7 cells, the JC-1 MMP fluorescence probe was employed. Under conditions of intact MMP and normal mitochondrial function, JC-1 accumulates in the mitochondrial matrix, forming polymers that emit red fluorescence. Conversely, when the MMP is compromised, JC-1 exists as a monomer and emits green fluorescence.⁵⁵ As shown in Fig. 6A, the control group exhibited strong red fluorescence and weak green fluorescence, indicative of normal mitochondrial function in Raw264.7 cells. In contrast, after 24 hours of H₂O₂ treatment, the red fluorescence significantly decreased while the green fluorescence increased markedly, indicating that H₂O₂ disrupted the MMP of Raw264.7 cells, leading to the dissociation of JC-1 aggregates and the emergence of JC-1 monomers that emit strong green fluorescence. Quantitative analysis of fluorescence (Fig. 6C) revealed that the fluorescence ratio of JC-1 aggregates to JC-1 monomers (A/M ratio) was significantly reduced by H₂O₂ compared to the control group. Notably, CP@RSV exhibited the least green fluorescence signals and the highest A/M ratio compared to the RSV and CP groups, indicating that CP@RSV effectively inhibited H₂O₂-induced damage to Raw264.7 cells mitochondria. Similarly, chondrocytes stimulated by H₂O₂ displayed increased green fluorescence and a significant drop in A/M ratio, reflecting substantial mitochondrial dysfunction leading to mitochondrial membrane damage (Fig. 6B and D). Conversely, the A/M ratio in CP@RSV showed a significant increase compared to the other H₂O₂-treated groups, suggesting a substantial restoration of the mitochondrial damage induced by H₂O₂. In summary, even though the inflammatory processes create an anoxic microenvironment that leads to mitochondrial damage and eventual apoptosis in cells, CP@RSV demonstrates the ability to effectively inhibit oxidative stress, reverse mitochondrial damage, and facilitate the recovery of MMP essential for maintaining normal cellular activities.

Fig. 6 JC-1 staining to observe the mitochondrial membrane potential in Raw264.7 cells (A) and chondrocytes (B). Quantification of the fluorescence ratio between JC-1 aggregates and JC-1 monomers in Raw264.7 cells (C) and chondrocytes (D) (n = 3, *p < 0.05, **p < 0.01 and ***p < 0.001 imply significant differences in comparison with the H₂O₂ group, ns p > 0.05 suggests no significant difference).

4. Conclusion

Lubrication failure and inflammation play a pivotal role in the pathogenesis of OA. Therefore, a combined therapeutic strategy that synergizes lubrication and anti-inflammatory effects is highly desirable. In this work, zwitterionic cyclic brush polymer nanomicelles CP@RSV were designed and synthesized with lubrication enhancement and effective RSV loading. The cyclic brush polymer CP, consisting of SBMA as the brush body, was prepared by intrachain click cyclization and ATRP. The synergistic effects of the hydration effect of PSBMA and the ring-brush-like topology of CP contributed to its favorable lubrication properties. Tribological and wear tests demonstrated that CP exhibited lower COF values under varying loads and frequencies, leading to a notable reduction in surface wear. In addition, the hydrophobic inner cavity of CP enabled effective encapsulation and release of RSV. In vitro assessments confirmed the biocompatibility of CP@RSV, with no adverse impact on cell morphology and growth on chondrocytes and Raw264.7 cells. Additionally, CP@RSV exhibited antioxidant properties, effectively scavenging intracellular ROS and improving mitochondrial function to support normal cellular processes on chondrocytes and Raw264.7 cells. In conclusion, CP@RSV not only minimizes interfacial friction but also enables efficient drug encapsulation, making it a promising nanomicelle for OA therapy.

Author contributions

Huizhu Wang: conceptualization, methodology, data curation, validation, writing – original draft, software, formal analysis, investigation, resources, and data curation. Miao Zhang: conceptualization, methodology, data curation, resources, investigation and software. Ying Chen: resources, methodology, and software. Shaopei Ding: investigation, resources and software. Meng Qin: data curation. Tong Wu: supervision and resources. Jianshu Li: supervision, project administration and funding acquisition. Jing Xie: supervision, conceptualization, writing – review & editing and funding acquisition.

Conflicts of interest

The authors declare no competing interests.

Data availability

All data are provided in the article and its ESI.†

Acknowledgements

This work was supported by the National Natural Science Foundation of China (No. U22A20158), Sichuan Science and Technology Program (No. 2025ZNSFSC0338), the Science Research Foundation of Aier Eye Hospital Group (Grant No. AGK2307D05), XiangJiang Philanthropy Foundation (Grant NO. KY24020), Chengdu Science and Technology Program (2024-YF05-00530-SN) and the State Key Laboratory of Advanced Polymer Materials (Grant No. sklpme 2023-2-16).

Notes and references

1. Y. Peng, Y. Wang, R. Bai, K. Shi, H. Zhou and C. Chen, Adv. Healthcare Mater., 2024, 13, 2400615.
2. D. J. Hunter and S. Bierma-Zeinstra, Lancet, 2019, 393, 1745–1759.
3. P. Yu, Y. Li, H. Sun, H. Zhang, H. Kang, P. Wang, Q. Xin, C. Ding, J. Xie and J. Li, Adv. Mater., 2023, 35, 2303299.
4. K. M. Dhanabalan, B. Padhan, A. A. Dravid, S. Agarwal, N. M. Pancheri, A. Lin, N. J. Willet, A. K. Padmanabhan and R. Agarwal, J. Mater. Chem. B, 2024, 12, 11172–11186.
5. N. K. Arden, T. A. Perry, R. R. Bannuru, O. Bruyère, C. Cooper, I. K. Haugen, M. C. Hochberg, T. E. McAlindon, A. Mobasheri and J.-Y. Reginster, Nat. Rev. Rheumatol., 2021, 17, 59–66.
6. H. Zhang, S. Yuan, B. Zheng, P. Wu, X. He, Y. Zhao, Z. Zhong, X. Zhang, J. Guan, H. Wang, L. Yang and X. Zheng, Small, 2024, 21, 2407885.
7. L. Yang, X. Zhao, X. Liao, R. Wang, Z. Fan, S. Ma and F. Zhou, J. Colloid Interface Sci., 2023, 629, 859–870.
8. R. Yan, H. Yang, Y. Liu, Y. Wang, S. Liu, R. Xie and L. Ren, ACS Appl. Mater. Interfaces, 2024, 16, 20273–20285.
9. X. Zhang, J. Wang, H. Jin, S. Wang and W. Song, J. Am. Chem. Soc., 2018, 140, 3186–3189.
10. S. Ma, L. Liu, W. Zhao, R. Li, X. Zhao, Y. Zhang, B. Yu, Y. Liu and F. Zhou, Nat. Commun., 2025, 16, 398.
11. L. Sharma, N. Engl. J. Med., 2021, 384, 51–59.
12. X. He, S. He, G. Xiang, L. Deng, H. Zhang, Y. Wang, J. Li and H. Lu, Adv. Mater., 2024, 36, 2405943.
13. P. G. Conaghan, A. D. Cook, J. A. Hamilton and P. P. Tak, Nat. Rev. Rheumatol., 2019, 15, 355–363.
14. S. Brown, S. Kumar and B. Sharma, Acta Biomater., 2019, 93, 239–257.
15. X. An, F. Zhou, G. Li, Y. Wei, B. Huang, M. Li, Q. Zhang, K. Xu, R. C. Zhao and J. Su, J. Mater. Chem. B, 2024, 12, 4148–4161.
16. G. Li, S. Liu, Y. Chen, J. Zhao, H. Xu, J. Weng, F. Yu, A. Xiong, A. Udduttula, D. Wang, P. Liu, Y. Chen and H. Zeng, Nat. Commun., 2023, 14, 3159.
17. N. Joshi, J. Yan, S. Levy, S. Bhagchandani, K. V. Slaughter, N. E. Sherman, J. Amirault, Y. Wang, L. Riegel, X. He, T. S. Rui, M. Valic, P. K. Vemula, O. R. Miranda, O. Levy, E. M. Gravallese, A. O. Aliprantis, J. Ermann and J. M. Karp, Nat. Commun., 2018, 9, 1275.
18. L. M. Mancipe Castro, A. J. García and R. E. Guldberg, J. Biomed. Mater. Res., 2021, 109, 426–436.
19. Y. Cao, X. Dong and X. Chen, Pharmaceutics, 2022, 14, 778.
20. P. Prabhu, J. Drug Delivery Sci. Technol., 2024, 97, 105801.
21. D. Pareek, Md Zeyaullah, S. Patra, O. Alagu, G. Singh, K. Wasnik, P. S. Gupta and P. Paik, J. Mater. Chem. B, 2025, 13, 3094–3113.
22. H. Zhang, H. Xiong, W. Ahmed, Y. Yao, S. Wang, C. Fan and C. Gao, Chem. Eng. J., 2021, 409, 128147.
23. T. Zhou, H. Xiong, S. Q. Wang, H. L. Zhang, W. W. Zheng, Z. R. Gou, C. Y. Fan and C. Y. Gao, Mater. Today Nano, 2022, 17, 100164.
24. Y. Mai and A. Eisenberg, Chem. Soc. Rev., 2012, 41, 5969.
25. J. Yang, R. Wang and D. Xie, Macromolecules, 2019, 52, 7042–7051.
26. Y. Wang, Z. Wu, Z. Ma, X. Tu, S. Zhao, B. Wang, L. Ma and H. Wei, Polym. Chem., 2018, 9, 2569–2573.
27. Z. Liu, Y. Huang, X. Zhang, X. Tu, M. Wang, L. Ma, B. Wang, J. He, P. Ni and H. Wei, Macromolecules, 2018, 51, 7672–7679.
28. S. Honda, T. Yamamoto and Y. Tezuka, Nat. Commun., 2013, 4, 1574.
29. B. A. Lakin, B. G. Cooper, L. Zakaria, D. J. Grasso, M. Wathier, A. M. Bendele, J. D. Freedman, B. D. Snyder and M. W. Grinstaff, ACS Biomater. Sci. Eng., 2019, 5, 3060–3067.
30. Y. Liu, Z. Ma, X. Wang, J. Liang, L. Zhao, Y. Zhang, J. Ren, S. Zhang and Y. Liu, Adv. Sci., 2024, 11, 2406027.
31. L. Li, M. Yue, Q. Peng, X. Pu and Z. Li, Adv. Funct. Mater., 2024, 34, 2309594.
32. P. Yu, X. Peng, H. Sun, Q. Xin, H. Kang, P. Wang, Y. Zhao, X. Xu, G. Zhou, J. Xie and J. Li, Mater. Horiz., 2024, 11, 5352–5365.
33. Q. Jin, Y. Deng, X. Chen and J. Ji, ACS Nano, 2019, 13, acsnano.8b07746.
34. Q. Li, C. Wen, J. Yang, X. Zhou, Y. Zhu, J. Zheng, G. Cheng, J. Bai, T. Xu, J. Ji, S. Jiang, L. Zhang and P. Zhang, Chem. Rev., 2022, 122, 17073–17154.
35. J. Hou, Y. Lin, C. Zhu, Y. Chen, R. Lin, H. Lin, D. Liu, D. Guan, B. Yu, J. Wang, H. Wu and Z. Cui, Adv. Sci., 2024, 11, 2402477.
36. M. Zhang, X. Peng, Y. Ding, X. Ke, K. Ren, Q. Xin, M. Qin, J. Xie and J. Li, Mater. Horiz., 2023, 10, 2554–2567.
37. M. R. De Oliveira, S. F. Nabavi, A. Manayi, M. Daglia, Z. Hajheydari and S. M. Nabavi, Biochim. Biophys. Acta, Gen. Subj., 2016, 1860, 727–745.
38. J. Wang, J.-S. Gao, J.-W. Chen, F. Li and J. Tian, Rheumatol. Int., 2012, 32, 1541–1548.
39. A. Y. Berman, R. A. Motechin, M. Y. Wiesenfeld and M. K. Holz, npj Precis. Oncol., 2017, 1, 35.
40. S. Sheng, X. Wang, X. Liu, X. Hu, Y. Shao, G. Wang, D. Mao, C. Li, B. Chen and X. Chen, Front. Pharmacol., 2022, 13, 829677.
41. L. Wei, Q. Pan, J. Teng, H. Zhang and N. Qin, Mater. Today Bio, 2024, 24, 100884.
42. J. Yang, Y. Han, J. Lin, Y. Zhu, F. Wang, L. Deng, H. Zhang, X. Xu and W. Cui, Small, 2020, 16, 2004519.
43. C. Sun, Y. Zhang, F. Dong, J. Zhao, P. Zhang, S. Li, Y. Gao, Y. Wang and G. Gao, Chem. Eng. J., 2024, 488, 150944.
44. N. S. Cameron, M. K. Corbierre and A. Eisenberg, Can. J. Chem., 1999, 77, 1311–1326.
45. F. Liu, D. Wang, M. Zhang, L. Ma, C.-Y. Yu and H. Wei, Acta Biomater., 2022, 144, 15–31.
46. X. Tu, C. Meng, Y. Wang, L. Ma, B. Wang, J. He, P. Ni, X. Ji, M. Liu and H. Wei, Macromol. Rapid Commun., 2018, 39, 1700744.
47. C. Chang, H. Wei, J. Feng, Z.-C. Wang, X.-J. Wu, D.-Q. Wu, S.-X. Cheng, X.-Z. Zhang and R.-X. Zhuo, Macromolecules, 2009, 42, 4838–4844.
48. S. Ding, P. Yu, X. Peng, Y. Liu, X. Xu, W. Sun, J. Xie and J. Li, Adv. Funct. Mater., 2025, 2505638.
49. S. Zhang, L. Wang, Y. Kang, J. Wu and Z. Zhang, Acta Biomater., 2023, 162, 1–19.
50. Z. Jiang, H. Wang, Z. Zhang, J. Pan and H. Yuan, J. Nanobiotechnol., 2022, 20, 419.
51. X. Huang, D. He, Z. Pan, G. Luo and J. Deng, Mater. Today Bio, 2021, 11, 100124.
52. G. Chen, L. Cui, P. Luo, J. Fang, X. Xie and C. Jiang, ACS Appl. Mater. Interfaces, 2024, 16, 34705–34719.
53. Y. Hui, J. Mao, M. Rui, Y. Huang, X. Jiang, Y. Xu, W. Wang, J. Wu, L. Zhou, K. Xi, L. Huang and L. Chen, Adv. Healthcare Mater., 2024, 13, 2402596.
54. H. Lu, J. Wei, K. Liu, Z. Li, T. Xu, D. Yang, Q. Gao, H. Xiang, G. Li and Y. Chen, ACS Nano, 2023, 17, 6131–6146.
55. M. Zhang, X. Peng, H. Xu, X. Sun, Y. Liu, Q. Li, Y. Ding, S. Ding, J. Luo, J. Xie and J. Li, Adv. Sci., 2024, 11, 2404143.

---

Footnote

† Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d5tb00704f

---