Background

Antibiotics contain diverse functional groups—such as imidazole, amino, hydroxyl, phenyl, and pyridine moieties—that can effectively displace chloride ions in hemin and serve as axial ligands to the Fe–N₄ planar center, forming a pentacoordinated structure. These units can further self-assemble into nanoaggregates through various non-covalent interactions, including hydrogen bonding, salt bridges, π–π stacking, and cation-π interactions, ultimately leading to the co-assembly of antibiotic-integrated nanozymes.

Material and Methods

Materials:Hemin,Cefalexin(CFX),Gentamicin(GM),Rapamycin(RAPA), Ceftriaxone Sodium(CRO),Fluconazole（FCZ）,sodium hydroxide (NaOH),dimethyl sulfoxide (DMSO)

Procedure

Hemin and gentamicin(or or alternatively, CFX,GM,RAPA,CRO,FCZ,etc.) were dissolved in a specified molar ratio under continuous stirring. The resulting mixture was then subjected to a drying process to obtain the final antibiotic nanozyme product.

Molecular docking and molecular dynamics simulation

Download the corresponding small molecules from the PubChem website. If a 3D structure is available, directly download the 3D structure in the SDF file format. If no 3D structure is available, download the 2D molecular structure in SDF format, then use OpenBabel to generate the 3D structure. Afterward, perform energy minimization of the molecular conformation using the UFF (Universal Force Field) with 1500 iterations.

Zeta potential measurements

A uniformly dispersed aqueous solution of the antibiotic nanozyme (approximately 20 μg/mL) was transferred to a measurement cell for simultaneous analysis of hydrodynamic diameter and zeta potential using a nanoparticle analyzer.

Scanning Electron Microscopy (SEM) Characterization of Antibiotic Nanozyme

The antibiotic nanozyme was prepared at an appropriate concentration, dispersed by sonication at 100 W for 10 min, and then drop-cast onto a smooth silicon wafer attached to a sample stub. After drying in an oven, the sample was sputter-coated with a gold layer for 30 s using an ion sputterer before surface morphology characterization by scanning electron microscopy (SEM).

Transmission Electron Microscopy (TEM) Characterization of Antibiotic Nanozyme

A 10 μL aliquot of the uniformly dispersed antibiotic nanozyme aqueous solution was carefully pipetted onto a copper grid support film. The sample was allowed to settle at room temperature for 20 minutes, after which excess liquid was gently removed from the edge of the support film using absorbent filter paper. The grid was subsequently air-dried at room temperature and examined under a 120 kV transmission electron microscope for morphological observation. High-resolution transmission electron microscopy (HRTEM) was employed for dark-field imaging, and energy-dispersive X-ray spectroscopy (EDS) was utilized for elemental mapping analysis.

Ultraviolet-Visible (UV-Vis) Full-Wavelength Scan Characterization of Antibiotic Nanozyme

The antibiotic nanozyme was prepared at an optimal concentration and dispersed by sonication at 100 W for 10 minutes. For comparative analysis, solutions of free antibiotic and hemin chloride at equivalent concentrations were prepared in parallel. The full-wavelength ultraviolet-visible (UV-Vis) absorption spectra of all three samples were recorded using a spectrophotometer.

Peroxidase-like activity of antibiotic nanozymes (including Gentamicin-，Rapamycin-, Ceftriaxone- and Fluconazole-based nanozymes) was assessed using 3,3′,5,5′-tetramethylbenzidine (TMB) as the substrate. The detailed procedure was as follows:

i. A 20 mg/mL TMB stock solution was prepared in dimethyl sulfoxide (DMSO) and stored protected from light.

ii. A 0.2 M acetate buffer (pH 4.5) was prepared.

iii. Antibiotic nanozyme solutions were prepared at 100 μg/mL in ultrapure water, followed by sonication at 100 W for 10 min. Control solutions of hemin chloride and the corresponding antibiotics at the same concentration were also prepared.

iv. The reaction was carried out in a 96-well plate with a total volume of 100 μL per well, containing 10 μL of gentamicin nanozyme or control component, 1 μL of TMB solution, 0.2 μL of 10 M H₂O₂, and 88.8 μL of 0.2 M acetate buffer (pH 4.5).

v. The absorbance at 652 nm was measured every 30 s for 10 min using a multifunctional microplate reader to monitor peroxidase-like activity.

The antifungal activity

i. C. albicans stored at -80°C was thawed, inoculated into 10 mL SDB liquid medium and incubated overnight in an air incubator at 37 °C with oscillations. The following day, the bacterial suspension was transferred to fresh medium and cultured for an additional 6-7 h at 37°C to activate the fungus,ensuring it reached the logarithmic growth phase.

ii. the initial inoculum concentration of the C. albicans suspension was approximately 10⁵-10⁶ CFU mL⁻¹, and the medium volume was 10 mL. To each well, 100 µL of C. albicans suspension and 100 µL of FCZ (Hemin) solution (or RAPA (Hemin), CRO (Hemin), or other components) at different concentrations were added to 800 µL of SDB medium and mixed thoroughly. As controls, the corresponding resistant antibiotics (FCZ, ITZ, or 5-FC) were added in the same volume (100 µL) along with 100 µL of bacterial solution and 800 µL of SDB medium as the negative control. The blank control consisted of 100 µL of bacterial solution and 900 µL of SDB medium. All groups were cultured at 37°C with shaking in an incubator for 24 h. After incubation, 200 µL of the suspension from each group was transferred to a microplate, and the absorbance was measured at 600 nm using a microplate reader.

The antibacterial activity

The antibacterial activity of GM (Hemin) against E.coli(BNCC358264) and MRSA (ATCC43300) was determined using the same experimental method as for C. albicans.

i. The E. coli and MRSA strains, stored at -80°C, were thawed and inoculated into 5 mL of LB liquid medium, followed by incubation with shaking at 37°C overnight. The next day, the bacterial solution was transferred to fresh LB medium at a 1:100 dilution and incubated at 37°C for approximately 2 h to activate the bacteria and allow them to enter the logarithmic growth phase, with a bacterial concentration of approximately 8×10⁸ CFU mL-1.

ii. 100 µL of E. coli or MRSA suspension and 100 µL of GM (Hemin) solution (or other components) were added to 800 µL of LB liquid medium. The bacterial cultures were then incubated at 37°C for 24 h with continuous shaking. The absorbance at 600 nm of each group was measured using a multifunctional microplate reader to assess the antibacterial effect.

TEM for MRSA

i. An overnight culture of MRSA was subcultured and activated for 4 hours until it reached the mid-logarithmic phase (approximately 1 × 10⁹ CFU/mL).

ii. One-milliliter aliquots of the bacterial suspension were centrifuged in separate tubes, and the supernatant was discarded. The bacterial pellets were resuspended in 1 mg/mL solutions of either the gentamicin nanozyme or its individual components, followed by incubation at 37°C for 12 hours.

iii. After incubation, the bacteria were washed twice with phosphate-buffered saline (PBS). The pellets were then fixed with 1 mL of 2.5% glutaraldehyde and stored at 4°C for a minimum of 2 hours.

iv. The fixed bacterial samples were centrifuged and washed three times with ddH₂O. Subsequently, they were subjected to sequential dehydration in a graded ethanol series (50%, 70%, 85%, 95%, and 100%). After critical point drying, the specimens were sputter-coated and imaged using a scanning electron microscope to examine the surface morphology.

v. For TEM observation, the fixed samples were embedded in resin and ultrathin-sectioned. The sections were then placed on copper grids and observed under a transmission electron microscope to assess the internal structure and morphology.

SEM for C. albicans

The bacterial suspension cultured overnight was transferred and activated for 6h. A 1 mL aliquot of the fungal suspension was taken from each centrifuge tube, centrifuged, and the supernatant was discarded. The drug-resistant working suspension was then treated with FCZ (Hemin) (at a final concentration of 1 mg mL-1), while the drug-resistant bacteria treated with FCZ and Hemin were used as the control group. The samples were fixed with 4% paraformaldehyde, followed by scanning electron microscopy (SEM) imaging.

Confocal Laser Scanning Microscopy (CLSM) for Biofilm Eradication Assessment

i. Overnight bacterial culture was subcultured and activated for 4 h to approximately 1×10⁹ CFU/mL for subsequent use.

ii. A 24-well plate was prepared with cell climbing slides, and 1 mL of bacterial suspension was added to each well. The plate was incubated at 37°C for 24 h to allow biofilm formation.

The medium was carefully aspirated, and 1 mL of 100 μg/mL gentamicin nanozyme or control solutions was gently added along the well wall. The plate was incubated at 37°C for 3 h.

iv. After treatment, the liquid was removed, and biofilms were stained with 0.5 μM SYTO 9 solution at 37°C in the dark for 30 min. The slides were then washed with PBS and carefully retrieved.

v. Each slide was mounted inverted on a clean glass slide with 5 μL of anti-fade mounting medium and imaged using confocal laser scanning microscopy.

Assessment of Lipid Peroxidation by Malondialdehyde (MDA) Assay

i. An overnight culture of MRSA was subcultured and activated for 4 hours until it reached the mid-logarithmic phase (approximately 1 × 10⁹ CFU/mL).

ii. The bacterial suspension was centrifuged at 5000 rpm for 3 minutes. The resulting pellet was then washed and resuspended in an equal volume of sterile ddH₂O.

iii. In 1.5 mL sterile microcentrifuge tubes, 900 μL of the bacterial suspension was mixed with 100 μL of either the gentamicin nanozyme or its individual component solutions (final concentration: 1 mg/mL). The mixtures were incubated at 37°C for 1 hour.

iv. After treatment, the bacteria were collected by centrifugation, and the supernatant was discarded. The pellet was washed three times with ice-cold PBS. Subsequently, 400 μL of lysis buffer was added to the pellet. The cells were lysed by sonication on ice using an ultrasonic cell disruptor at 120 W power for a total of 20 minutes (with a cycle of 3 seconds pulse-on and 2 seconds pulse-off).

v. Following sonication, the lysates were centrifuged at 10,822 rpm for 10 minutes at 4°C. The resulting supernatant was carefully collected for the subsequent MDA assay.

Protein Leakage Detection

i.The overnight bacterial culture was subcultured and activated for 4 hours until it reached an optical density corresponding to approximately 1 × 10⁹ CFU/mL.

ii.One milliliter of the bacterial suspension was aliquoted into centrifuge tubes. After centrifugation and removal of the supernatant, the bacterial pellets were resuspended in 1 mg/mL gentamicin nanozyme or its individual component solutions. The tubes were then incubated at 37°C for 12 hours.

iii. Following incubation, the bacteria were centrifuged. The supernatant was collected, and its protein content was quantified using a bicinchoninic acid (BCA) protein assay kit.

Effect of Gentamicin Nanozyme on Hydrogen Sulfide (H₂S) Production in MRSA

i. An overnight culture of MRSA was subcultured and activated for 4 hours until reaching the mid-logarithmic phase (approximately 1 × 10⁹ CFU/mL).

ii. The bacterial suspension was centrifuged at 5000 rpm for 3 minutes. The resulting pellet was washed and resuspended in an equal volume of sterile ddH₂O.

iii. In 1.5 mL sterile microcentrifuge tubes, 900 μL of the prepared bacterial suspension was mixed with 100 μL of either the gentamicin nanozyme solution or its individual component solutions (final concentration: 1 mg/mL). The mixtures were then incubated at 37°C for 3 hours.

iv. Following incubation, 25 μM of the H₂S-sensitive fluorescent probe WSP-5 was added to each tube, followed by an additional 30 minutes of incubation at 37°C in the dark.

v. The bacterial cells were collected by centrifugation, washed, and resuspended to remove any unbound probe. Subsequently, 200 μL aliquots of each suspension were transferred to a black 96-well microplate. The fluorescence intensity was measured using a multimode microplate reader with an excitation wavelength of 502 nm and an emission wavelength of 525 nm

Ferroptosis Measurement

i. A single colony of C. albicans ATCC 10231 was selected and inoculated onto SDB liquid medium for culture. The cultures were incubated overnight at 37°C. The next day, the fungal suspension was diluted into SDB liquid medium at 1:100 and cultured for about 6 h for activation, reaching a concentration of about 1 × 10⁶-10⁷ CFU mL-1.

ii. In 700 μL SDB liquid medium system, 100 µL of the ferroptosis inducer Ferroheme(0.5mM), 100 μL of FCZ (Hemin) (CRO (Hemin), GM (Hemin), FCZ, CRO, GM) at different concentrations ,and 100 μL of activated fungal suspension were added. Meanwhile, the same system without the ferroptosis inducer was set up as the control group, a blank control group was prepared by adding 900 µL of SDB liquid medium and 100 µL of the activated fungal suspension., All groups were incubated at 37°C .The antifungal effect of the ferroptosis on antibiotics and antibiotic nanozymes was evaluated by measuring the OD at 600 nm and through plate coating.The antifungal effect of Ferrostatin-1, a ferroptosis inhibitor, on antibiotics and antibiotic nanozymes was assessed using the same experimental protocol.