Triple-synergistic 2D material-based dual-delivery antibiotic platform

The study addresses the limited aqueous stability and suboptimal bioactivity of graphene oxide (GO) in antimicrobial applications. GO has appealing physicochemical properties, including high surface area and abundant edge carboxyl groups, and has been explored in biomedical uses such as photothermal therapy, bioassays, drug delivery, and as antibacterial agents. However, low solubility/dispersion stability in water restricts its efficacy. Silver nanoparticles (Ag NPs) are potent antimicrobials, with smaller particles offering stronger effects but prone to aggregation. Sulfadiazine (SD) and silver sulfadiazine (SSD) are clinically used broad-spectrum antibiotics, notably in burn care. The research hypothesis is that PEGylating carboxylated GO to improve aqueous stability and using it as a vector to co-load Ag NPs and SD will create a dual-delivery, 2D hybrid antibacterial system (HAS) with synergistically enhanced antimicrobial activity. The proposed mechanism involves a triple synergy of bacterial capping by GO sheets, physical puncture of bacterial membranes, and chemical growth inhibition by Ag/SD.

Materials: Graphite flakes; reagents including H2SO4, NaNO3, KMnO4, H2O2, AgNO3, HCl, NaOH, DCC, DMAP, DMF, DMSO, sodium chloroacetate (ClCH2COONa), PEG-OH (Mn ~800), SD, SSD; dialysis bags (MWCO 3.5 kDa); MTT, propidium iodide (PI), DAPI; deionized water.

Synthesis of GO-COOH: GO was prepared by a modified Hummers method. For carboxylation, GO suspension (1 mg/mL) was sonicated 30 min; ClCH2COONa (1.2 g in 40 mL) was mixed with GO (1 mg/mL, 20 mL) under ultrasonication 30 min. NaOH solution (reported as 10 mol/mL, 40 mL) was added under stirring for 3 h. The mixture was neutralized with 1 M HCl; precipitate was washed with water and alcohol by centrifugation to remove unreacted ClCH2COONa.

Synthesis of GO-PEG: GO-COOH (1 mg/mL, 20 mL in DMF) was mixed with PEG-OH (5 mg) under stirring 10 min; DCC (10 mg) and DMAP (2 mg) were added and reacted 3 h at 60 °C to form ester bonds. Excess PEG and DMF were removed by dialysis (MWCO 3.5 kDa) against water for 3 days.

Deposition of Ag NPs (GO-PEG/Ag): GO solution (0.25 mg/mL, 28 mL) was mixed with AgNO3 (5 mM, 5 mL) under ultrasonication 30 min. NaOH (1 M, 10 mL) was added as deoxygenating/reducing agent under stirring. The mixture was irradiated in a microwave oven (2.45 GHz, 200 W) for controlled times (e.g., 1–2 min). After cooling, products were collected by centrifugation and washed with water and alcohol to neutral pH.

Loading SD to form HAS (GO-PEG/Ag-SD): SD (30 mg/mL, 100 µL in DMSO) was added dropwise into GO-PEG/Ag dispersion (0.25 mg/mL, 10 mL) under ultrasonication. DMSO was removed by dialysis. The dispersion was centrifuged (1000 rpm, 10 min) to remove free SD, then freeze-dried.

Characterization: SEM (JEOL JSM-7001F); HRTEM (JEOL JEM-2100); XRD (Cu Kα, λ=0.15406 nm); UV–vis (200–800 nm; SD peak at 310 nm); FT-IR (3800–600 cm−1, 4 cm−1 resolution). Antibacterial activity assessed with a microplate reader via MTT; fluorescence microscopy for live/dead imaging.

Antibacterial testing: Models: Escherichia coli (Gram−) and Staphylococcus aureus (Gram+). LB broth used for bacterial activation (shaker 170 rpm, 37 °C, 12 h). Working inoculum: 10^5 CFU/mL.

Microdilution (MIC determination): In 96-well plates, 100 µL 0.9% saline added to wells; materials at 100 µg/mL added to first well, 50 µg/mL to second, then twofold serial dilutions; 10^5 CFU/mL bacteria added to each test well. Controls: LB only and saline with bacteria. Incubate at 37 °C; measure OD; add MTT (20 µL/well), read OD after 4 h. Material absorbance subtracted; triplicates averaged. Viable counts assessed by plating on LB-agar and incubating at 37 °C for 12 h.

Agar diffusion: MH agar inoculated with 10^5 CFU/mL; paper disks (impregnated with 10 µL of 1 mg/mL suspension; 10 mg/disk nominal) loaded with HAS, HAS without SD (HAS SD−), and SSD placed on agar. Incubate 25 h at 37 °C; measure inhibition zone diameters.

Live/dead fluorescence assay: Bacteria at 10^5 CFU/mL incubated with HAS (50 µg/mL) for 1 h at 37 °C (shaking). Cells stained with PI (1 µg/mL, 15 min) and counterstained with DAPI (5 µg/mL, 5 min) in dark; imaged by fluorescence microscopy.

Stability assessment: Visual observation of GO, GO-COOH, and GO-PEG in water over prolonged storage (reported up to 96 days).

• PEGylation markedly enhanced aqueous stability: GO-PEG remained stably dispersed in water without precipitation over extended storage (tested up to 96 days), outperforming GO and GO-COOH.
• Successful carboxylation and PEG conjugation confirmed by FT-IR: increased COOH-related peak at ~1720 cm−1 after carboxylation and new ester carbonyl signal at 1735 cm−1 after PEGylation.
• Ag nanoparticle deposition and crystallinity verified: TEM/HRTEM showed highly monodispersed Ag NPs embedded on GO-PEG with lattice spacing of 0.236 nm (Ag (111)). XRD showed fcc Ag reflections at 2θ ≈ 38.2°, 44.2°, 64.5°, 77.3° alongside GO (001) at 10.8°.
• Ag NP size tunable by microwave time at 200 W: ~8 nm after 1 min and ~50 nm after 2 min, without agglomeration.
• SD loading onto GO-PEG/Ag confirmed by FT-IR with new bands at 3424, 3356, 2874, 2136, 1654, 1156, and 1097 cm−1; UV–vis used to quantify loading (standard curve referenced).
• Antibacterial efficacy: The hybrid antibacterial system (GO-PEG/Ag-SD, HAS) exhibited antibacterial activity enhanced by over 3-fold compared to the SD-free system (HAS SD−), against E. coli and S. aureus. The enhanced performance is attributed to triple synergy: bacterial capping by GO sheets, physical puncture, and chemical inhibition by Ag and SD.

Functionalizing GO with PEG increased hydrophilicity and colloidal stability, addressing a key barrier to biomedical deployment of GO-based antimicrobials. Using GO-PEG as a high-surface-area vector enabled stable anchoring of Ag NPs and co-loading of sulfadiazine, providing a dual-delivery platform. Microwave-assisted synthesis afforded rapid, green control over Ag NP size, with smaller (~8 nm) particles expected to provide higher antimicrobial activity due to increased surface area and ion release while remaining well-dispersed on GO-PEG. Spectroscopic and diffraction analyses confirmed each construction step: carboxylation, PEGylation, Ag deposition, and SD loading. Biologically, the platform leverages triple synergy: (1) GO sheet interactions can wrap or cap bacteria, enhancing contact; (2) sharp edges and embedded nanoparticles can physically disrupt membranes (puncture); and (3) chemical inhibition arises from Ag+ release and SD’s antibacterial action. Together these mechanisms yielded substantially greater killing than the SD-free composite, across both Gram-negative and Gram-positive models. Improved dispersion stability likely increases bioavailability and contact with cells, further boosting efficacy. The approach broadens the design and application space for 2D carbon-based antimicrobials, with tunable nanoparticle size offering a lever to balance potency and safety.

This work presents a PEGylated graphene oxide-based dual-delivery antibacterial platform (HAS) that co-loads silver nanoparticles and sulfadiazine. The synthesis integrates carboxylation, covalent PEGylation, rapid microwave-assisted Ag NP deposition, and post-loading of SD. The resulting 2D hybrid shows markedly improved aqueous stability and a triple-synergistic antibacterial mechanism (capping, puncture, inhibition), achieving over threefold enhancement in antibacterial activity versus the SD-free counterpart. Structural and spectroscopic characterization validates successful fabrication, and Ag NP size is tunable via microwave time. The platform highlights a generalizable strategy for stabilizing and functionalizing 2D carbon materials for biomedical use. Future work could quantify MICs and inhibition zones across broader pathogens, optimize Ag size and SD loading for efficacy/safety balance, assess cytotoxicity and hemocompatibility, evaluate performance in biofilm models, and conduct in vivo infection and wound-healing studies.