ROS-Scavenging Bamboo-Derived Carbon Dot-Methotrexate Nanocomposite Ameliorates Rheumatoid Arthritis through Dual Therapeutic Mechanisms

Abstract

Rheumatoid arthritis (RA) is a common chronic immune-mediated inflammatory disease. Current treatment strategies for RA are limited and often associated with significant adverse effects. Methotrexate (MTX), a first-line therapeutic agent for RA, demonstrates efficacy; however, its adverse effects remain a concern, particularly its tendency to increase reactive oxygen species (ROS) levels and exacerbate oxidative stress. In this study, we developed an MTX/CD complex by combining MTX with bamboo leaf-derived carbon dots (CDs). Experimental results showed that the MTX/CD complex effectively reduced ROS generation and significantly mitigated oxidative stress, while retaining the favorable therapeutic effect of MTX against RA. Moreover, the composite more effectively suppressed the abnormal proliferation of rheumatoid arthritis fibroblast-like synoviocytes (RA-FLS), thereby alleviating joint symptoms in RA patients and offering a novel therapeutic strategy for RA management.

1. Introduction

RA is a systemic autoimmune disease primarily characterized by chronic erosive arthritis, with a peak onset age between 45 and 60 years and a significantly higher prevalence in females than in males. The global incidence of RA ranges from 0.5 to 1%, while its prevalence in mainland China is approximately 0.42%, corresponding to an estimated patient population of over 5 million. − Beyond causing joint damage and disability, RA may also lead to comorbidities such as interstitial lung disease and cardiovascular disorders, imposing substantial a burden on patients and their families. Current therapeutic strategies for RA primarily include nonsteroidal anti-inflammatory drugs (NSAIDs), glucocorticoids, biologic agents, and targeted small-molecule drugs. −

As a first-line therapeutic agent for RA, MTX acts through multiple mechanisms, among which increased ROS generation represents a key pathway. , Specifically, MTX inhibits dihydrofolate reductase, disrupting purine and pyrimidine metabolism and leading to intracellular adenosine accumulation. The accumulated adenosine binds to A2A receptors, activates NADPH oxidase, and promotes ROS production. − At appropriate concentrations, ROS can induce synovial cell apoptosis and suppress synovial hyperplasia. Meanwhile, as signaling molecules, ROS downregulate the expression of pro-inflammatory cytokines such as TNF-α and IL-1β, thereby alleviating inflammatory responses. , Additionally, ROS exert anti-inflammatory effects by modulating signaling pathways including NF-κB, which contributes to the inhibition of immune cell activation. , However, excessive ROS can induce oxidative stress, underscoring the importance of maintaining MTX within a strict therapeutic window.

Bamboo leaf-derived carbon dots (CDs), a nanomaterial obtained from bamboo leaves, possess multiple functional properties such as antioxidant, antibacterial, and anti-inflammatory activities. Their unique structural and surface functional groups allow efficient scavenging of reactive oxygen species (ROS), including superoxide anions (O₂ ^–), hydrogen peroxide (H₂O₂), and hydroxyl radicals (OH^·). Through redox reactions mediated by abundant surface-active groups (e.g., phenolic hydroxyl and carboxyl groups), bamboo leaf-derived CDs convert ROS into harmless water or oxygen, thereby alleviating oxidative stress-related cellular damage. Moreover, they enhance cellular antioxidant capacity by modulating the activity of intracellular antioxidant enzymes such as superoxide dismutase (SOD) and catalase (CAT), which helps maintain ROS homeostasis.

Based on this rationale, we developed a novel MTX/CD complex by integrating MTX with bamboo leaf-derived carbon dots (CDs). Preliminary experiments and literature evidence indicate that the complex effectively reduces ROS accumulation in the joints while retaining the therapeutic efficacy of MTX, thereby ameliorating oxidative stress damage. This strategy not only shows potential for mitigating MTX-related adverse effects but may also offer a safer and more effective treatment approach for the management of RA (Figure ).

1.

Schematic illustration of the preparation of the injectable MTX/CD nanocomplex and its dual therapeutic mechanisms in rheumatoid arthritis (RA) treatment.

2. Methods and Materials

2.1. Materials

Fresh bamboo leaves: Collected from Jinzhou Medical University campus (Liaoning Province), washed with deionized water and stored at low temperature; Methotrexate (MTX, ≥98% purity): MedChemExpress (HY-14519); High-glucose DMEM medium: Gibco (11965092); Fetal Bovine Serum (FBS): Gibco (A5256701); Bovine Type II Collagen: Shanghai Yuanye Bio-Technology (S12008); Complete Freund’s Adjuvant (CFA, 1 mg/mL inactivated Mycobacterium tuberculosis): MedChemExpress (HY-153808); DCFH-DA fluorescent probe: Beyotime Biotechnology (S0033S); Rat TNF-α, IL-6, and IL-1β ELISA kits: Feien Biological (ER1393, ER0042, ER1094); CCK-8 cell proliferation kit: apexbio (k1018); Antibodies: anti-IKKβ (Proteintech, 15649–1-AP), antiphospho-IKKβ (CST, 2697), anti-IκBα (Proteintech, 10268–1-AP), antiphospho-IκBα (CST, 2859), anti-COX-2 (Proteintech, 27308–1-AP), anti-β-Actin (Proteintech, 20536–1-AP); HRP-conjugated secondary antibodies (Proteintech, SA00001–1 and SA00001–2).

2.2. Preparation of Bamboo Leaf Carbon Dots and MTX-Bamboo Leaf Carbon Dot Complexes

Fresh bamboo leaves were rinsed with deionized water, dried in an oven at 60 °C to constant weight, and ground into powder. Precisely 50.00 g of the powder was placed in a corundum crucible and calcined in a tube muffle furnace under programmed heating (5 °C/min to 400 °C, held for 2 h). The calcined product was ball-milled to <50 μm, mixed with ultrapure water (1:20, w/v), and refluxed at 90 °C for 2 h. The suspension was filtered through a 0.22 μm membrane, purified sequentially using 3000 Da ultrafiltration tubes (12,000 × g, 30 min) and Sephadex G-25 chromatography, then freeze-dried to obtain bamboo leaf-derived CDs (Figure A). The lyophilized CDs were ultrasonically dispersed (300 W, 30 min) in 0.01 M PBS (pH 7.4) to prepare a 1 mg/mL stock solution. MTX was dissolved in PBS to prepare a 140 μM (0.06 mg/mL) stock solution. The CD solution (1 mL) and MTX solution (0.1 mL) were mixed at 10:1 ratio, magnetically stirred (600 rpm, 25 °C) in darkness for 10 h. Unbound drugs were removed using 3000 Da ultrafiltration tubes (12,000 × g, 30 min). The resulting MTX/CD complexes were aliquoted and stored at −80 °C.

2.

Characterization of bamboo leaf-derived carbon dots (CDs) and the MTX/CD complex. (A) TEM image of bare bamboo leaf-derived carbon dots. (B) TEM image of the MTX-loaded CD complex (MTX/CD). (C) FTIR spectra of bare CDs, free MTX, and the MTX/CD complex. (D) UV–vis absorption spectra of bare CDs, free MTX, and the MTX/CD complex. (E) XPS survey spectrum of the MTX/CD complex. (F–H) High-resolution XPS spectra of C 1s, N 1s, and O 1s in the MTX/CD complex. (I) MTX loading efficiency of CDs at varying MTX concentrations. (J) Cumulative MTX release profile from the MTX/CD complex under different pH conditions (pH 5.0 and 7.4) over 72 h.

2.3. Characterization

The morphology and size distribution of the carbon dots and complexes were characterized by high-resolution transmission electron microscopy (HR-TEM, 200 kV accelerating voltage, 0.19 nm point resolution). Selected-area electron diffraction (SAED) was employed for crystallinity analysis. UV–vis absorption spectra were recorded using a double-beam spectrophotometer with a scanning range of 200–800 nm, a resolution of 1 nm, and a 1 cm quartz cuvette, using the corresponding solvent as the reference. Fourier-transform infrared (FT-IR) spectra were collected via the KBr pellet method over the wavenumber range of 400–4000 cm^–1, with 32 accumulated scans and a spectral resolution of 4 cm^–1. X-ray photoelectron spectroscopy (XPS) analysis was conducted using monochromated Al Kα radiation (1486.6 eV), and the binding energies were calibrated against the C 1s peak at 284.8 eV. Fluorescence properties were evaluated using a fluorescence spectrophotometer.

2.4. MTX Loading and Releasing

MTX loading efficiency of CDs was determined using a dialysis-based separation method. A fixed concentration of purified bamboo CDs suspended in PBS (pH 7.4) was mixed with increasing concentrations of MTX dissolved in DMSO. The mixtures were stirred in the dark for 15 h to facilitate complex formation via noncovalent interactions. Subsequently, 3 mL of each MTX/CD solution was loaded into dialysis membranes (MWCO: 500–1000 Da) and dialyzed against 100 mL of PBS under continuous stirring for 24 h. This process allowed unbound MTX to diffuse into the dialysate while retaining the MTX/CD complexes within the membrane. The concentration of free MTX in the dialysate was quantified by measuring absorbance at 372 nm using UV–vis spectroscopy, calibrated against an MTX standard curve. Loading efficiency was calculated as

$$\mathrm{Loading}\mathrm{Efficiency}(\backslash \%)=\left(\frac{\mathrm{Total}\mathrm{mass}\mathrm{of}\mathrm{initial}\mathrm{MTX}-\mathrm{Mass}\mathrm{of}\mathrm{free}\mathrm{MTX}}{\mathrm{Total}\mathrm{mass}\mathrm{of}\mathrm{initial}\mathrm{MTX}}\right)\times 100$$

The pH-dependent release profile of MTX from the MTX/CD complex was evaluated under simulated physiological conditions (pH 5.0 and 7.4). Briefly, 3 mL of the MTX/CD complex (equivalent to 0.28 mg/mL MTX) was dialyzed (MWCO: 500–1000 Da) against 100 mL of PBS at pH 5.0 or 7.4, with constant stirring (100 rpm) at 37 °C. Aliquots of dialysate (1 mL) were collected at predetermined intervals (0, 2, 4, 8, 12, 24, 48, and 72 h) and replaced with fresh PBS. The released MTX concentration was quantified by UV–vis absorbance at 372 nm. Cumulative release (%) was calculated as

$$\mathrm{Release}\mathrm{Efficiency}(\backslash \%)=\frac{\mathrm{Released}\mathrm{MTX}\mathrm{at}\mathrm{time}t}{\mathrm{Total}\mathrm{Loaded}\mathrm{MTX}}\times 100$$

Triplicate experiments were performed for statistical validation.

2.5. Animals

SPF-grade female SD rats (230 ± 20 g, 6–8 weeks old) were obtained from the Laboratory Animal Center of Jinzhou Medical University (License No. SCXK (Liao) 2024–0003) and housed in a barrier facility (22 ± 1 °C, 50 ± 5% humidity, 12 h light/dark cycle). All experimental procedures were approved by the Animal Ethics Committee of Jinzhou Medical University and conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Efforts were made to minimize animal suffering, including the use of appropriate anesthesia during surgical procedures and humane end points for euthanasia following AVMA guidelines.

2.6. Cell Culture

RA-FLS were obtained from Wuhan Procell Life Technology Co., Ltd. (Cat. CP-H248). Cultivation was performed in high-glucose DMEM supplemented with 10% FBS, under standard conditions of 37 °C and 5% CO₂ in a humidified incubator.

2.7. CIA Model Establishment

Bovine type II collagen (5 mg) was accurately weighed and dissolved in 2.5 mL of 0.1 mol/L glacial acetic acid solution (pH 3.2) under continuous agitation at 4 °C for 12 h. The resulting solution was emulsified with an equal volume of Complete Freund’s Adjuvant (CFA) using a dual-syringe emulsification method to prepare a stable oil-in-water emulsion (1 mg/mL). SPF female SD rats were subjected to a primary immunization via subcutaneous injection of 0.1 mL emulsion into the left hind paw pad, followed by a booster immunization with an equivalent volume of emulsion administered subcutaneously at the tail base 14 days later. After successful model establishment, the rats were randomly divided into four groups (n = 8): the model and control group received daily oral gavage of saline (10 mL/kg); the MTX group was treated with MTX solution (0.5 mg/kg); and the MTX/CD group received the MTX/CD nanocomplex containing an equivalent MTX dose (0.5 mg/kg). Joint swelling indices were recorded following 28 consecutive days of treatment.

2.8. HE Staining

Fixed joint specimens were decalcified in 8% hydrochloric acid decalcification solution for 14 days, followed by dehydration, clearing, and paraffin embedding. Serial coronal sections (5 μm thickness) were prepared and subjected to HE staining: sections were stained with hematoxylin for 5 min, differentiated in 1% acid ethanol for 30 s, and counterstained with eosin for 2 min. Whole-slide images were acquired using a digital pathological scanning system, and synovial pathology was evaluated according to the Krenn synovitis scoring system to assess synovial hyperplasia, inflammatory cell infiltration, and pannus formation. Concurrently, heart, liver, spleen, lung, and kidney tissues were fixed with 4% paraformaldehyde, paraffin-embedded, and sectioned at 4 μm thickness for HE staining.

2.9. CCK-8 Assay

To validate the cytotoxic effects of bamboo leaf-derived CDs on FLS, a CCK-8 assay was performed. RA-FLS cells from passages 3–5 were seeded in 96-well plates at a density of 1 × 10⁴ cells/well. After adherence, cells were treated with complete medium containing gradient concentrations of CDs (2.5–160 μM) for 24 h. Subsequently, 10 μL of CCK-8 reagent was added to each well, followed by 1-h incubation at 37 °C. Absorbance at 450 nm was measured using a microplate reader, and cell viability (%) was calculated as (OD_{experimental group}/OD_{control group}) × 100.

2.10. Colony Formation Assay

RA-FLS cells were seeded in 6-well plates at a density of 500 cells/well and treated with CDs (10 μM), MTX (0.5 μM), or MTX/CD nanocomplex (equivalent MTX concentration). Drug-containing medium was replaced every 3 days. After 14 days, cells were fixed with 4% paraformaldehyde for 20 min and stained with 1% crystal violet (dissolved in 20% methanol) for 15 min. Colonies containing >50 cells were counted, and the clonogenic rate (%) was calculated as (colony number/initial seeded cell number) × 100.

2.11. Transwell Assay

RA-FLS cell suspension (1 × 10⁴ cells/well in serum-free medium) was seeded into the upper chambers of 8 μm pore-size Transwell plates. The lower chambers were loaded with chemotactic medium containing 20% FBS. After 48 h of treatment, nonmigrated cells in the upper chambers were removed with cotton swabs, followed by fixation with 4% paraformaldehyde and staining with 0.1% crystal violet. Membrane-penetrating cells were quantified by counting five randomly selected fields under an inverted microscope at 200× magnification.

2.12. Scratch Assay

RA-FLS cells (5 × 10⁵ cells/well) were seeded in 6-well plates and allowed to form confluent monolayers. A linear scratch wound was created using a 200 μL sterile pipet tip. After PBS washing to remove detached cells, the medium was replaced with 1% FBS-containing medium. Cells were then treated with CDs (10 μM), MTX (0.5 μM), and their complex, respectively. Scratch regions were captured at 0, 24, and 48 h under an inverted phase-contrast microscope (10× objective). The healing rate was calculated using ImageJ software with the formula: (1 – scratch area/initial area) × 100%.

2.13. ROS Detection

RA-FLS cells were seeded in confocal dishes at a density of 5 × 10⁴ cells/dish. After 24 h of treatment, cells were loaded with 10 μM DCFH-DA probe and incubated at 37 °C in the dark for 30 min. Following three PBS washes, fluorescence images were immediately acquired using a laser scanning confocal microscope (excitation wavelength: 488 nm, emission wavelength: 525 nm). Fluorescence intensity was quantified as arbitrary units per cell (a.u./cell) via ImageJ software.

2.14. Inflammatory Cytokine Detection

Blood samples were collected via caudal vein puncture and allowed to clot for 30 min. Serum was obtained by centrifugation at 3000 × g for 15 min. Rat-specific ELISA kits were processed according to the manufacturer’s protocols. Optical densities were measured using a microplate reader at dual wavelengths (primary wavelength: 450 nm; reference wavelength: 630 nm). Cytokine concentrations were determined via standard curves and expressed in picograms per milliliter (pg/mL).

2.15. Antioxidant Enzyme Activity assay

Antioxidant enzyme activities were quantitatively analyzed using commercial kits. Superoxide dismutase (SOD) activity was determined by the WST-8 method: Cell lysates from treated RA-FLS were mixed with reaction working solution according to the kit instructions, followed by incubation at 37 °C in the dark for 30 min before measuring absorbance at 450 nm. One unit of SOD activity was defined as the amount of enzyme required to inhibit 50% of superoxide anion radical generation, with results expressed as U/mg protein. Catalase (CAT) activity was assessed through hydrogen peroxide degradation kinetics: Cell lysates reacted with substrate (65 μM H₂O₂) in PBS for 1 min, after which ammonium molybdate was added to terminate the reaction. The decrease in absorbance at 405 nm was measured, and activity was calculated as μmol of H₂O₂ decomposed per min per mg protein (μmol/min/mg pro). Total protein concentration was determined by BCA assay to normalize enzyme activity data.

2.16. Western Blot

Western blot analysis was performed to detect key proteins in the NF-κB signaling pathway. Total protein was extracted from RA-FLS cells of each group using RIPA lysis buffer. After separation by 10% SDS-PAGE, 30 μg protein samples were transferred to PVDF membranes and blocked with 5% skim milk at room temperature for 1 h. Membranes were then incubated overnight at 4 °C with primary antibodies: anti-IKKβ, antiphospho-IKKβ, antiphospho-IκBα, antitotal IκBα, and anti-COX-2. Following TBST washes, membranes were incubated with HRP-conjugated secondary antibodies at room temperature for 1 h. Protein bands were visualized using ECL chemiluminescence reagent, and band intensities were quantified with ImageJ software. β-Actin served as the loading control for normalization.

2.17. Statistical Analysis

Data are expressed as mean ± standard deviation (mean ± SD). Statistical analyses were performed using SPSS 19.0 software, with one-way analysis of variance (ANOVA) and Tukey’s multiple comparison tests conducted through GraphPad Prism 9.5. Statistical significance thresholds were defined as * p < 0.05 (significant), ** p < 0.01 (highly significant), and *** p < 0.001 (extremely significant).

3. Results

3.1. Characterization of Bamboo Leaf-Derived Carbon Dots and MTX/CD Complex

The structural and functional properties of CDs and their MTX complexes were systematically characterized. Transmission electron microscopy (TEM) revealed that bare CDs formed monodisperse quasi-spherical nanoparticles with an average diameter of 3 nm (Figure A), while MTX conjugation increased the particle size to approximately 4 nm (Figure B), indicating successful drug loading. Fourier-transform infrared (FTIR) spectroscopy further elucidated the interaction mechanisms: bare CDs exhibited characteristic peaks at 3426.69 cm^–1 (O–H stretching) and 1604.91 cm^–1 (CC vibrations), whereas MTX showed distinct bands at 1645 cm^–1 (amide I) and 1205 cm^–1 (C–N stretching). Critically, the MTX/CD complexes displayed shifted peaks at 1658 cm^–1 (hydrogen-bonded CO) and 1543 cm^–1 (−NH₂ bending), confirming noncovalent interactions between the carboxyl groups of the CDs and the amine groups of MTX (Figure C).

UV–vis absorption spectra demonstrated the π–π* transition of bare CDs at 280 nm and the pteridine ring absorption of MTX at 304 nm. The MTX/CD complex exhibited a redshift to 292 nm with a shoulder at 350 nm (Figure D), suggesting ground-state complex formation through electron delocalization. X-ray photoelectron spectroscopy (XPS) quantified elemental changes: nitrogen content increased from 7.90% in CDs to 10.2% in the complexes due to the pteridine nitrogen from MTX, while oxygen decreased from 27.15 to 24.8%, consistent with hydrogen bonding (Figure E–H). High-resolution N 1s spectra revealed new pyridinic N (399.8 eV) and protonated −NH₃ ⁺ (401.5 eV) peaks, further verifying conjugation.

In summary, bamboo leaf-derived CDs integrate ultrasmall size, high-density functionalization, and distinctive optoelectronic properties, establishing a robust material foundation for their application as multifunctional nanocarriers.

3.2. Efficiency of MTX Loading and Releasing

Bamboo-derived carbon dots demonstrated high loading capacity for MTX across a range of concentrations. At lower MTX concentrations (0.009–0.12 mM), loading efficiencies reached approximately 92%. Even at higher MTX concentrations (up to 0.47 mM), the efficiency remained notably high at 85.9%, corresponding to a loading content of 0.37 mg of MTX per mg of CDs. The gradual decrease in efficiency observed with increasing MTX concentrations suggested saturation of available binding sites on the CD surface. (Figure I). This exceptional performance is attributed to electrostatic interactions and hydrogen bonding between hydrophilic functional groups (−COOH, −OH, −NH₂) on the bamboo-derived CDs and complementary groups in the MTX molecule. The robust loading efficiency confirms the suitability of bamboo CDs as effective nanocarriers for MTX delivery.

The MTX/CD complex demonstrated distinct pH-dependent release kinetics when evaluated under simulated physiological conditions (pH 5.0 and 7.4) using dialysis (MWCO: 500–1000 Da). At pH 5.0 (mimicking the acidic synovial microenvironment of inflamed joints), a burst release of 42.1 ± 3.2% occurred within 8 h, followed by sustained release reaching 69.3 ± 2.1% at 72 h. In contrast, release at physiological pH (7.4) was significantly attenuated, achieving only 46.7 ± 1.9% after 72 h (Figure J). This biphasic profilecharacterized by rapid initial dissociation of surface-adsorbed MTX (0–8 h) and gradual diffusion from the CD matrix (8–72 h)was attributed to protonation of carboxyl groups on the CDs under acidic conditions, which weakened MTX-CD electrostatic/hydrogen-bonding interactions and accelerated drug liberation.

3.3. In Vivo Therapeutic Efficacy and Safety of the MTX/CD Complex

A 28-day pharmacological intervention revealed distinct therapeutic outcomes among the experimental groups. The MTX/CD complex group showed significantly enhanced efficacy compared to MTX monotherapy, as evidenced by standardized paw circumference measurements that indicated an 18.13% greater reduction in inflammation (**p < 0.01). Notably, the CDs-only group (administered at a carbon dot dosage equivalent to that in the MTX/CD complexes) exhibited negligible anti-inflammatory activity, with only a 6.2% reduction in joint swelling (p > 0.05 vs model group). This finding highlights the limited standalone therapeutic value of carbon dots, despite their known ROS-scavenging capability (Figure A,D).

3.

In vivo therapeutic efficacy and biocompatibility of the MTX/CD complex in a CIA rat model. (A) Representative images of paw swelling in different treatment groups. (B) H&E-stained sections of rat ankle joints. (C) H&E-stained sections of major organs (heart, liver, spleen, lung, kidney). (D) Quantification of paw swelling. (E) Quantitative synovitis score based on H&E-stained joint sections. (F) Serum biochemical analysis of liver function markers across treatment groups.

Histopathological evaluation via H&E staining further supported these results: MTX/CD treatment markedly reduced the synovial hyperplasia area to (0.46 ± 0.05) mm² (vs model group: 1.24 ± 0.11 mm²), whereas CDs-only intervention failed to suppress tissue proliferation (1.08 ± 0.09 mm²) (Figure B,E). This confirms that clearing ROS alone is insufficient to alleviate inflammation-driven joint destruction in the absence of MTX’s pharmacological action.

Although CDs-only treatment did not significantly suppress pro-inflammatory mediators, the MTX/CD complexes substantially reduced cytokine levels, linking their superior efficacy to the synergistic modulation of inflammatory signaling. Importantly, histopathological assessment of major organs (heart, liver, spleen, lungs, and kidneys) showed preserved tissue architecture in both the MTX/CD- and CDs-only treated rats, with organ indices comparable to those of healthy controlsthus verifying the safety profile of the carbon dots even in the absence of therapeutic benefit (Figure C). Serum biochemical analysis further confirmed that neither the CDs nor the MTX/CD complex induced significant liver damage (Figure F).

3.4. Cytotoxicity and Anti-Proliferative Effects

Quantitative analysis of RA-FLS cells treated with bamboo leaf-derived CDs at various concentrations showed cell viability above 90% across all dosage groups. CCK-8 assay results confirmed the negligible cytotoxicity of CDs within the 2.5–160 μM range (Figure A), demonstrating the excellent biocompatibility of the nanomaterials with FLS cells.

4.

Effects of the MTX/CD complex on RA-FLS viability and proliferation. (A) Cell viability of RA-FLS treated with various concentrations of CDs, as determined by the CCK-8 assay. (B) Representative images of colony formation in RA-FLS under different treatments. (C) Quantification of colony formation rates. (D) Transwell migration assay images. (E) Quantification of migrated cells per field. (F) Scratch wound healing assay images at 0, 24, and 48 h. (G) Quantification of the wound closure rate.

Colony formation assays further revealed that the MTX/CD complex exhibited stronger antiproliferative effects than MTX monotherapy, significantly reducing the colony formation rate (Figure B,C). This enhanced efficacy may be attributed to the nanocarrier properties of the CDs, which potentially improve intracellular drug delivery or modulate cell cycle-related signaling pathways, thereby more effectively suppressing the pathological proliferation of RA-FLS cells.

3.5. Suppression of RA-FLS Migration by the MTX/CD Complex

Transwell migration assays demonstrated the superior efficacy of the MTX/CD complex in inhibiting cell migration. The number of membrane-penetrating cells was significantly lower in the MTX/CD group (150 ± 10 cells/field) compared to the MTX-alone group (253 ± 8 cells/field), representing a substantial reduction (Figure D,E). These results indicate that the composite formulation is highly effective in suppressing the transmembrane migration of RA-FLS cells.

This enhanced inhibitory effect was further corroborated by a scratch wound healing assay. The MTX/CD complex treatment resulted in a substantially slower wound closure rate compared to the other groups (Figure F,G), confirming its potent suppressive effect on the pathological migration of RA-FLS cells.

3.6. ROS Scavenging and Oxidative Stress Modulation Mechanisms

To elucidate the therapeutic mechanism further, we quantified intracellular ROS levels. Quantitative analysis using the DCFH-DA fluorescent probe revealed distinct ROS modulation patterns across treatment groups. While CD monotherapy significantly reduced fluorescence intensity (4.30 ± 0.7 a.u.) compared to the blank control (9.38 ± 1.2 a.u.), MTX monotherapy markedly increased ROS accumulation (45.35 ± 1.4 a.u.). Crucially, the MTX/CD complex substantially attenuated this MTX-induced ROS overproduction (19.31 ± 1.9 a.u.) (Figure A,B). These findings demonstrate the potent ROS-scavenging capacity of the CDs and their ability to counteract the pro-oxidant effect of MTX.

5.

ROS scavenging and anti-inflammatory effects of the MTX/CD complex in RA-FLS. (A) Representative fluorescence images of intracellular ROS levels detected by DCFH-DA staining. (B) Quantitative analysis of ROS fluorescence intensity. (C) Real-time kinetics of superoxide generation measured by DHE fluorescence. (D) Calculated rates of ROS generation. (E–G) Serum levels of inflammatory cytokines (TNF-α, IL-6, IL-1β) measured by ELISA.

We further employed Dihydroethidium (DHE) to monitor real-time superoxide anion generation. Kinetic analysis over 40 min showed a linear increase in fluorescence in all groups (Figure C). The MTX group exhibited the most rapid ROS generation rate (186.3 ± 7.0 RFU/min), significantly higher than the RA-FLS control (116.0 ± 7.1 RFU/min). In contrast, CDs alone markedly suppressed the rate to 65.0 ± 7.0 RFU/min. Importantly, the MTX/CD complex effectively mitigated the excessive ROS generation induced by MTX, yielding a rate (134.3 ± 10.6 RFU/min) that was significantly lower than the MTX group and comparable to the control (Figure D). This kinetic data confirms that the nanocomposite preserves MTX’s therapeutic action while neutralizing its propensity to exacerbate oxidative stress.

Complementary ELISA of inflammatory cytokines in rat serum showed that the MTX/CD complex induced marked reductions in key pro-inflammatory mediators: TNF-α (52.89 ± 4.15 pg/mL vs MTX 97.44 ± 9.51 pg/mL), IL-6 (26.97 ± 4.14 pg/mL vs 53.15 ± 6.36 pg/mL), and IL-1β (26.35 ± 4.01 pg/mL vs 48.39 ± 3.05 pg/mL) (Figure E–G). This suggests the composite formulation synergistically suppresses inflammation, potentially through ROS-mediated regulation of the NF-κB signaling pathway.

Further supporting evidence from antioxidant enzyme activity assays confirmed the restorative capacity of MTX/CD complexes against oxidative stress. Compared to the RA-FLS model group, the MTX monotherapy group exhibited a further reduction in antioxidant enzyme activity, whereas the CD monotherapy group demonstrated significantly elevated activity. The MTX/CD combination therapy group demonstrated superior efficacy in restoring antioxidant enzyme activity­(Figure A,B).

6.

Mechanisms of oxidative stress regulation and NF-κB pathway inhibition. (A) SOD activity in RA-FLS under different treatments. (B) CAT activity in RA-FLS under different treatments. (C) Western blot analysis of key proteins in the NF-κB pathway. (D–H) Densitometric quantification of protein expression levels: p-IKKβ, IKKβ, p-IκBα, IκBα, and COX-2, normalized to β-actin.

Additionally, Western blot analysis of the NF-κB signaling pathway validated its anti-inflammatory mechanism. Both the MTX monotherapy group and the MTX/CD combination therapy group showed reduced levels of P-IKKβ and P–IκBα, mitigated degradation of total IκBα protein, and significantly downregulated COX-2 expression levels compared to the RA-FLS model group and the CDs monotherapy group (Figure C–H). These collective findings demonstrate that the MTX/CD complex effectively inhibits the activation of the NF-κB signaling pathway.

4. Discussion

The comprehensive characterization of bamboo leaf-derived carbon dots (CDs) confirmed their suitability as nanocarriers, exhibiting an ultrasmall size (∼3 nm), abundant surface functional groups (hydroxyl, carboxyl, and CC bonds), and intrinsic fluorescence. These ultrasmall dimensions facilitate penetration through synovial barriers for targeted drug delivery to inflamed joints, while the surface hydroxyl and carboxyl groups enable high MTX loading efficiency via hydrogen bonding and electrostatic interactions, concurrently serving as active sites for ROS scavenging. The natural origin of these CDs minimizes biotoxicity risks, which is supported by the high RA-FLS viability (>90%) observed across a wide concentration range (2.5–160 μM). This favorable safety profile contrasts with certain synthetic nanomaterials, such as liposomes or polymers.

Our newly presented data on MTX loading and release demonstrate exceptional loading efficiency (85.9% at 0.47 mM MTX), attributable to electrostatic and hydrogen-bonding interactions between the hydrophilic groups (−COOH, −OH) on the CDs and MTX. Notably, the pH-dependent release kinetics revealed accelerated MTX liberation under acidic conditions (69.3 ± 2.1% at pH 5.0 vs 46.7 ± 1.9% at pH 7.4 over 72 h). This biphasic release profilecharacterized by an initial rapid dissociation followed by sustained diffusionaligns well with the acidic synovial milieu of inflamed joints (pH ∼ 5.0–6.5). This ensures targeted drug availability at the disease site while potentially minimizing systemic exposure.

Expanded mechanistic studies elucidated the dual therapeutic action of the MTX/CD composite. MTX mediates its antiproliferative effects primarily by inhibiting dihydrofolate reductase, thereby disrupting nucleotide metabolism and inducing apoptosis in RA-FLS. , Our colony formation and Transwell assays confirmed that the MTX/CD complex more effectively suppresses RA-FLS proliferation and migration than MTX alone, an enhancement likely due to improved intracellular delivery facilitated by the CD carriers. Complementing this, the CDs contribute through potent ROS scavenging and antioxidant restoration. New data from antioxidant enzyme assays demonstrate that CDs upregulate SOD and CAT activities in RA-FLS, effectively counteracting MTX-induced oxidative stress and synergizing with MTX’s antiarthritic effects by restoring redox homeostasis.

Crucially, Western blot analysis identified the inhibition of the NF-κB pathway as a central anti-inflammatory mechanism. The MTX/CD complex significantly reduced the phosphorylation of IKKβ and IκBα, suppressed IκBα degradation, and downregulated cyclooxygenase-2 (COX-2) expression. This molecular evidence explains the marked reduction in key pro-inflammatory cytokines (TNF-α, IL-6, IL-1β), as NF-κB is a master regulator of their transcription. The ROS-scavenging capacity of the CDs likely interrupts the crosstalk between ROS and the NF-κB pathway, , thereby amplifying the anti-inflammatory impact of MTX.

In the CIA rat model, the MTX/CD complex outperformed MTX monotherapy, reducing paw swelling by an additional 18.13% and synovial hyperplasia by 63% (0.46 ± 0.05 mm² vs 1.24 ± 0.11 mm²). Safety assessments revealed no histopathological damage in major organs, underscoring the composite’s improved safety profile over conventional MTX, which is often associated with hepatotoxicity. This finding aligns with in vivo safety reports of other innovative MTX carriers, such as albumin-based systems, and highlights the potential of natural nanocarriers for reducing systemic toxicity. The sustained efficacy observed over the 28-day treatment period mirrors that of biologic agents like certolizumab, but without the associated risks of immunogenicity or infection.

Despite the compelling therapeutic outcomes, a limitation of this study is the lack of direct biodistribution data for the MTX/CD complex in the CIA rat model. While the significant reduction in joint inflammation and synovial hyperplasia provides strong indirect evidence of the nanocomposite’s ability to localize to and act upon the diseased joints, detailed pharmacokinetics and tissue distribution profiles would offer deeper insights into its targeting efficiency and in vivo fate. We acknowledge that such data are crucial for the comprehensive translational development of this nanomedicine. Therefore, investigating the biodistribution of the MTX/CD complex, potentially using radiolabeling or near-infrared fluorescence imaging techniques, will be a primary objective of our subsequent pharmacokinetic studies.

Compared to other micro/nanoparticle systems, the MTX/CD nanocomposite offers distinct advantages. For instance, while selenium nanoparticles (SeNPs) possess recognized antioxidant and anti-inflammatory properties, they often require surface modification to achieve efficient drug loading and controlled release. In contrast, our bamboo-derived CDs inherently possess both high drug-loading capacity and ROS-scavenging functionality without the need for additional functionalization. Similarly, heparin-loaded silk fibroin microparticles (Hep@SFMPs) demonstrate excellent sustained release, but their fabrication involves multistep processes. Our MTX/CD system is synthesized via a simpler, one-pot assembly, enhancing reproducibility and scalability. Furthermore, unlike silica-core nanoparticles that may require complex surface engineering for biotracking, the intrinsic fluorescence and biocompatibility of bamboo CDs allow for straightforward monitoring and offer improved biosafety.

5. Conclusions

This study successfully developed a bamboo leaf-derived carbon dot-methotrexate nanocomposite (MTX/CD) that synergistically addresses key limitations in rheumatoid arthritis therapy. The nanocomposite demonstrated high MTX loading efficiency (85.9%) and pH-responsive release kinetics, achieving significantly accelerated drug liberation in acidic microenvironments (69.3% at pH 5.0 vs 46.7% at pH 7.4), enabling targeted delivery to inflamed joints. In vitro studies revealed dual therapeutic actions: MTX/CD potently suppressed RA-FLS proliferation and migration while concurrently mitigating oxidative stress through direct ROS scavenging (reducing intracellular ROS by 57.4% versus MTX alone) and restoration of antioxidant enzyme activities. Mechanistically, the complex inhibited NF-κB pathway activation, evidenced by reduced phosphorylation of IKKβ/IκBα and downregulated COX-2 expression. In CIA rat models, MTX/CD outperformed MTX monotherapy with an 18.13% greater reduction in paw swelling and 63% suppression of synovial hyperplasia, while maintaining excellent biocompatibility across major organs. This work establishes MTX/CD as a promising paradigm for enhancing RA treatment efficacy and safety, warranting future investigations into in vivo biodistribution and long-term toxicity profiles.

All data generated or analyzed during this study are included in this published article and its Supporting Information files.

X.-R.H. and F.W. have contributed equally. Y.W. and F.X. contributed to the conception and design of the study. X.-R.H. and F.W. performed the bioinformatic analysis. X.-R.H. and B.-F.Z. performed the experiment and the statistical analysis. X.-R.H. wrote the first draft of the manuscript. All authors contributed to manuscript revision, read, and approved the submitted version. Yuan Wang was responsible for article revision and confirmation of the final draft.

The authors declare no competing financial interest.