Therapeutic Efficacy of Gold Nanoparticles in Ulcerative Colitis Management via NF-κB Pathway Inhibition

INTRODUCTION
Long-term uncontrolled colonic ulcerative colitis (UC) can lead to bloody diarrhoea, abdominal pain, weight loss, and colorectal cancer [1]. Current studies imply a complicated aetiology involving genetic susceptibility, dysregulated gut microbiota dysbiosis, dysregulated mucosal immunity and environmental stresses [2,3]. The incidence of disease has been on the rise globally, especially in the newly industrialised countries, making it a public health problem [4].
The current therapeutic arsenal includes 5-aminosalicylic acid derivatives, corticosteroids, immunomodulators and anti-TNF antibodies. These drugs induce remission in a large proportion of patients but their chronic usage is generally limited by substantial adverse effects, loss of response and high cost [5,6]. Much research is driven by the desire for safer and more selective treatments.
Nanocarriers are preferentially taken up by inflamed mucosa due to leaky epithelium and absorption of immune cells, making nanoparticle-based delivery a feasible method for intestinal inflammation [7]. Gold nanoparticles (AuNPs) are widely used metallic nanomaterials because of their tunable size, low intrinsic toxicity, easy surface functionalisation and anti-inflammatory and antioxidant capabilities [8,9].
The nuclear factor kappa B (NF-κB) signalling cascade is an important regulator of inflammatory gene transcription and is substantially active in colonic mucosa during UC [10]. Thus, targeting this route is a logical mechanistic strategy. The present investigation was designed to synthesise and characterise AuNPs and evaluate their therapeutic efficacy on DSS caused colitis in mice with a focus on NF-κB pathway modification.
Several previous studies have explored the therapeutic potential of metallic and polymeric nanoparticles in experimental colitis. Nanocrystalline silver was shown to attenuate mucosal damage and suppress TNF-α expression [23], while curcumin-loaded PLGA nanoparticles enhanced local anti-inflammatory activity through improved mucosal residence [24]. Green-synthesized selenium nanoparticles inhibited the NF-κB pathway and reduced oxidative stress in DSS-induced colitis [27], and pH-responsive polymeric nanoparticles enabled site-specific drug release at inflamed colonic regions [22]. With regard to gold nanoparticles (AuNPs) specifically, promising anti-inflammatory effects have been reported in collagen-induced arthritis [21] and in murine colitis treated with biosynthesized AuNPs [16].
Nevertheless, most prior work has relied either on green-synthesized AuNPs in which the contribution of the plant extract cannot be readily separated from that of the gold core or on combination therapies, leaving direct mechanistic evidence linking AuNPs to upstream inhibition of the canonical NF-κB pathway in ulcerative colitis relatively limited. The present study was designed to address this gap using citrate-stabilized AuNPs in a well-validated DSS model.

MATERIALS AND METHODS
Chemicals and reagents
Sigma-Aldrich (St. Louis, MO, USA) supplied hydrogen tetrachloroaurate(III) trihydrate (HAuCl₄·3H₂O, ≥99.9%) and trisodium citrate dihydrate. MP Biomedicals provided DSS (MW 36,000–50,000 Da). ELISA kits for TNF-α, IL-1β, and IL-6 were purchased from R&D Systems. Cell Signalling Technology provided primary antibodies for phospho-NF-κB p65 (Ser536), total p65, IκBα, and β-actin. The remaining compounds were analytical and utilised unpurified. Deionised water (18.2 MΩ·cm) was utilised throughout.

Synthesis of gold nanoparticles
A significantly modified Turkevich approach produced AuNPs [11]. To summarise, 50mL of 1 mM HAuCl₄ aqueous solution was heated to boiling with vigorous magnetic stirring. A 5mL 1% (w/v) trisodium citrate solution was injected quickly. The liquid boiled for 15 min, turning from pale yellow to dark blue to stable ruby red, indicating nanoparticle production. After cooling naturally to room temperature, the suspension was centrifuged at 12,000rpm for 30min., washed twice with deionised water, and redispersed in PBS for use. The suspension was gravimetrically concentrated and kept at 4°C until use.

Characterization of AuNPs
Optical absorption was recorded between 300 and 800 nm using a UV–Vis spectrophotometer (Shimadzu UV-1800). FTIR spectra of dried samples were obtained on a Bruker Tensor 27 instrument over 400–4000 cm⁻¹ using KBr pellets. The crystalline structure was determined by XRD (Rigaku MiniFlex, Cu Kα, λ = 1.5406 Å) at 2θ values from 20° to 80°. Particle morphology was examined by FESEM (TESCAN MIRA3) after mounting on conductive carbon tape and gold sputter coating. Hydrodynamic diameter and zeta potential were measured using a Malvern Zetasizer Nano ZS instrument.

Animal model and experimental design
Male BALB/c mice (8–10 weeks old, 22–25 g) were obtained from the institutional animal house and kept under standard conditions (22 ± 2 °C, 12-h light/dark cycle) with free access to standard rodent chow and water. All experimental procedures were approved by the institutional animal ethics committee in accordance with national guidelines for the care of laboratory animals.
After 7 days of acclimatization, animals were randomly assigned to four groups (n = 8 per group): Group I (Control) received normal drinking water only; Group II (DSS) received 3% (w/v) DSS in drinking water for 7 days, followed by 7 days of normal water; Group III (DSS + AuNPs) received DSS as above plus AuNPs (5 mg/kg/day, oral gavage) starting from day 1 for a total of 14 days; and Group IV (DSS + 5-ASA) served as a positive-control group receiving DSS plus the standard reference drug 5-aminosalicylic acid (50 mg/kg/day) over the same period. Body weight, stool consistency, and presence of blood in faeces were recorded daily and used to calculate the disease activity index (DAI) according to the standard scoring system [12].

Sample collection
At day 15, animals were slaughtered under mild anaesthesia. Excise the colon from caecum to anus, measure its length with a ruler, and rinse in cold PBS. The distal segments were either fixed in 10% buffered formalin for histology or snap-frozen in liquid nitrogen for biochemical and molecular studies at -80°C.

Histopathological analysis
Formalin-fixed colons were paraffin-embedded, sectioned at 5µm, and stained with haematoxylin and eosin. A pathologist blinded to the treatment graded sections using a light microscope for mucosal injury, crypt loss, and inflammatory cell infiltration on an 0-4 scale. Higher numbers indicated greater damage.

Myeloperoxidase activity assay
Hexadecyltrimethylammonium bromide buffer homogenised colon tissue, freeze-thawed three times, and centrifuged. At 460nm, o-dianisidine and H2O2 were used to measure myeloperoxidase (MPO) activity in units per gram of wet tissue [17].

Western blot analysis
Total protein was extracted using RIPA buffer with protease and phosphatase inhibitor cocktails and the concentrations were measured using BCA. The equivalent amount of 40 µg was resolved on 10% SDS-PAGE and transferred onto PVDF membranes, blocked in 5% non-fat milk. Membranes were incubated overnight at 4°C with primary antibodies (1:1000) against phospho-NF-кB p65, total p65, IкBα and β-actin. Enhanced chemiluminescence was seen on membranes incubated with HRP-conjugated secondary antibody following washing. Quantification of band intensities with Image-J.

Cytokine measurement
TNF-α, IL-1β, and IL-6 levels in colonic homogenates were measured using commercial ELISA kits according to the manufacturers’ instructions. Absorbance was read at 450nm and concentrations were expressed as pg per mg of total protein.

Statistical analysis
Data are presented as mean ± standard deviation (SD). Differences among groups were analysed by one-way ANOVA followed by Tukey’s post-hoc test using GraphPad Prism v9. P values below 0.05 were considered statistically significant.

RESULTS AND DISCUSSION
Synthesis and characterization of AuNPs
A clear colour change from pale yellow to wine-red within minutes after citrate addition confirmed gold ion reduction to AuNPs. UV–Vis. spectra exhibited a prominent surface plasmon resonance peak around 520 nm, indicating tiny spherical AuNPs [13]. The tight band revealed a homogeneous size distribution with no shoulder indicating significant aggregation.
Fig. 1 shows FTIR, XRD, FESEM, and particle-size distribution together to show the synthesised particles’ nanoscale characterisation. FTIR analysis revealed successful surface capping by citrate ions, with bands near 3400 cm⁻¹ (hydroxyl group O–H stretching), 1620 cm⁻¹, and 1390 cm⁻¹ (asymmetric and symmetric C=O stretches of citrate carboxylate) [14]. Face-centered cubic gold (JCPDS no. 04-0784) had characteristic reflections at 38.2°, 44.4°, 64.6°, and 77.5° in the (111), (200), (220), and (311) planes. This orientation favours crystallographic development due to the strong (111) reflection. FESEM imaging showed spherical, well-distributed nanoparticles with an average diameter of 20 ± 4 nm and minimal aggregation. The DLS analysis showed a hydrodynamic size of around 32 nm and a zeta potential of −34 mV, indicating excellent electrostatic stabilisation from surface citrate [15].

Effect of AuNPs on disease activity index and body weight
On day 4, DSS mice began losing weight and by day 7, had lost 18% of their body mass and had blood-tinged faeces. The treated group (DSS + AuNPs) lost weight more slowly and recovered from day 9 onwards, resulting in a ~6% weight reduction compared to the control group. The AuNP-treated group had significantly lower DAI scores (weight loss, stool consistency, and rectal bleeding) than the DSS group from day 6 onwards (P < 0.05). Fig. 2 shows DAI and daily body weight curves.
AuNPs have been shown to reduce intestinal inflammation in models of intestinal damage [16,28]. Importantly, the 5-ASA-treated reference group improved similarly, though less so, demonstrating that AuNPs at 5 mg/kg can meet or outperform a conventional aminosalicylate in this acute scenario. Before determining chronic safety, prolonged follow-up is needed.

Macroscopic and histopathological changes in the colon
Muscle thickening and shortness of the organ are caused by chronic inflammation, hence colon shortening is a useful predictor of severity in mouse models of colitis. The colon length was reduced in the DSS treated group compared to controls (mean length 5.0±0.45 cm vs. 8.0±0.30 cm), however, AuNP treatment was able to significantly reverse this effect with a mean length of 7.2±0.40 cm. Fig. 3 shows macroscopic schematics and quantitative comparisons.
Macroscopic observations were confirmed by microscopic investigation. Sections from control mice had intact mucosal architecture, well-preserved crypts, and little inflammation. In contrast, the DSS group had significant mucosal ulcers, crypt loss, submucosal oedema, and intense polymorphonuclear and mononuclear cell infiltration. AuNP therapy restored mucosal continuity and crypt integrity, but infiltration persisted (Fig. 4). Histopathological scores decreased from 3.6±0.4 in the DSS group to 1.5±0.3 in the therapy group (P<0.01), indicating protection.

Reduced neutrophil infiltration
Most neutrophils store MPO in azurophilic granules, and its tissue activity is utilised to measure neutrophil infiltration in inflamed tissues [17]. Compared to controls, MPO activity in the DSS group increased nearly four-fold (12.4 ± 2.1 vs. 3.2 ± 0.7 U/g tissue). AuNP therapy reduced MPO activity by 60% (5.0 ± 1.1 U/g), aligning with histologically found lower inflammatory infiltration. Table 1 summarises clinical and biochemical characteristics for all groups.

Suppression of NF-κB activation
During UC, the NF-κB pathway is crucial for transcription of pro-inflammatory cytokines, chemokines, and adhesion molecules. Western blotting assessed pathway activation. Fig. 5 shows that the DSS group had significantly higher levels of phospho-NF-κB p65 (Ser536) and lower levels of IκBα, indicating canonical pathway activation. AuNP therapy significantly reduced p65 phosphorylation by 65% and restored IκBα protein levels, indicating active suppression of IKK-mediated signalling [18,19]. Total p65 levels did not differ significantly between groups, showing that activation rather than expression caused the effect.
Previous investigations have shown that metallic nanoparticles can reduce inflammation by modulating NF-κB in several disease situations [20,21]. Reactive-oxygen-species scavenging by AuNPs against upstream regulators like IKK subunits’ thiol groups may indirectly decrease the cascade and lessen redox-sensitive activation. Detailed mechanistic dissection requires silencing and direct binding experiments within cells.

Reduction of pro-inflammatory cytokines
Lowering NF-κB activation is likely to minimise downstream cytokine production. By ELISA, DSS therapy elevated TNF-α, IL-1β, and IL-6 by 4–6 fold in colonic tissue compared to controls. AuNP therapy reduced each cytokine to 35–45% of DSS levels (Fig. 6 and Table 2). These pleiotropic cytokines cause UC mucosal injury and are therapeutic targets [26]. The cytokine response reported here is remarkably similar to that seen in studies using anti-TNF or 5-ASA based therapy, but with the practical benefit that AuNPs can in principle be functionalised for targeted delivery to inflamed mucosa [22].

Mechanistic interpretation
Taken together, the biochemical, molecular, and histological data converge on a coherent mechanistic picture, schematized in Fig. 7. AuNPs interrupt the NF-κB cascade upstream by reducing IκBα phosphorylation and degradation, which in turn prevents nuclear translocation of the p65/p50 dimer and subsequent transcription of inflammatory mediators. The reduction in MPO activity and neutrophil infiltration is most likely a downstream consequence of decreased expression of NF-κB-dependent chemokines and adhesion molecules. In addition, the well-documented antioxidant action of AuNPs may contribute by lowering the oxidative burden on colonocytes, thereby protecting the mucosal barrier and reducing further activation of innate immunity [29].

Comparison with previous nanoparticle studies
Comparing our findings with earlier reports, the magnitude of NF-κB inhibition obtained here with citrate-stabilised AuNPs at 5 mg/kg is comparable to that reported for selenium nanoparticles at higher doses [27], and similar to the protective effects seen with curcumin-loaded polymeric nanoparticles [24]. Compared with green-synthesized AuNPs—where the contribution of phytochemicals from plant extracts is difficult to disentangle from that of the gold core itself [30]—our chemically prepared particles allowed a more direct attribution of the observed effects to the AuNPs themselves, while preserving comparable efficacy. This relative methodological clarity is, in our view, a useful contribution of the present work.

Limitations of the current study
Several limitations of the present study should be acknowledged. The DSS model, although widely used and well validated, mimics acute rather than chronic UC and therefore does not capture the full complexity of relapsing-remitting disease in patients [25]. The 14-day treatment was also too short to allow assessment of long-term safety, including potential gold accumulation in the liver, spleen, or kidneys, which is a known concern for metallic nanomaterials. In addition, while NF-κB suppression was clearly demonstrated, other cooperating pathways such as MAPK, JAK/STAT, and the NLRP3 inflammasome were not examined here and certainly warrant future investigation. The route of administration—oral gavage of free AuNPs in PBS—is also not optimized in terms of mucosal targeting; future work could combine AuNPs with pH-responsive carriers or targeting ligands to improve site-specific delivery and potentially reduce the effective dose further.

CONCLUSION
Citrate-stabilized gold nanoparticles produced by a simple chemical reduction process and thoroughly characterised by FTIR, XRD and FESEM showed a clear protective effect against DSS-induced ulcerative colitis in BALB/c mice. Administration orally of 5 mg/kg/day decreased disease activity, maintained colon length, lowered histopathological scores and reduced MPO activity. NF-κB p65 phosphorylation was inhibited, IκBα expression was restored, and colonic levels of TNF-α, IL-1β and IL-6 were lowered at the molecular level, confirming NF-κB pathway suppression as a primary mechanism of action. Further study is needed to clarify long-term safety, biodistribution and potential synergy with current UC medications, but these data support the argument for AuNPs as a promising candidate for nanomedicine-based management of inflammatory bowel disease. Future work should also investigate approaches for surface functionalisation to further optimise mucosal targeting and evaluate efficacy in models of chronic and relapsing colitis that better emulate the actual illness.

CONFLICT OF INTEREST
The authors declare that there is no conflict of interests regarding the publication of this manuscript.