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Overview of Experimental Results

Antibiotic Nanozyme

Antibiotic Nanozymes Coassembled by Antibiotics and Hemin

Molecular docking and molecular dynamics simulation

Computational analysis revealed that the primary amine of gentamicin forms a metal-coordination bond (2.3 Å) with the central iron atom of hemin, while its methylamine coordinates at a bond length of 2.7 Å. Furthermore, a single gentamicin molecule was shown to simultaneously interact with two hemin molecules through dual metal-coordination bonds.

Fig.1 MD simulation of GM (Hemin) co-assembly based on the unit obtained
Fig.1 MD simulation of GM (Hemin) co-assembly based on the unit obtained

MD Simulation of GM (Hemin) Co-assembly

Based on molecular dynamics simulations performed on the gentamicin nanozyme as a representative model, we demonstrate the theoretical feasibility of forming the antibiotic nanozyme structure.

Fig.2
Fig.2 MD simulation of GM (Hemin) co-assembly based on the unit obtained

Peroxidase(POD)-like Activity Assay

We systematically evaluated the POD activity of nanozymes formed by the conjugation of various antibiotics with hemin. The results demonstrated markedly enhanced POD activity across all tested nanozymes, validating the broad applicability and efficacy of this assembly strategy for multiple antibiotic classes.

Fig.2 POD-like activity of Levofloxacin HCl(LH) (Hemin) (a)、Fluconazole (FCZ) (Hemin) (b)、pyrithione (Hemin) (c)、Rapamycin(RAPA) (Hemin) (d)
Fig.3 POD-like activity of Levofloxacin HCl(LH) (Hemin) (a)、Fluconazole (FCZ) (Hemin) (b)、pyrithione (Hemin) (c)、Rapamycin(RAPA) (Hemin) (d)

Theoretical Insight into the Bioinspired Activity

To better elucidate the molecular catalytic mechanism, we simulated the POD-like activitiy of H2O2 molecules oxidizing TMB on FCZ(Hemin), and the corresponding proposed reaction pathways were investigated using density functional theory (DFT) calculations as shown in Figure 4.

Therefore, the following equation serve as plausible mechanisms for the POD-like activity:

H2O2 + 2TMB → 2H2O + 2oxTMB (1)

The whole POD-like catalytic cycle (eq (1)) consists of seven steps as described in Figure 4a, and two TMB molecules can be oxidized by a H2O2 molecule converting to two oxTMB molecules on nanozymes.

Fig.4 The corresponding proposed reaction pathways of FCZ(Hemin)

In Vitro Antimicrobial Activity

Antibacterial efficacy of the synthesized nanozymes was evaluated to determine whether the assembly strategy enhances bactericidal activity and broad-spectrum applicability. All antibiotic–hemin co-assemblies exhibited significantly stronger antimicrobial effects compared to their individual components.

Fig.3 Anti-C. albicans activity of FCZ (Hemin)、CRO(Hemin)、RAPA (Hemin)
Fig.5 Anti-C. albicans activity of FCZ (Hemin)、CRO(Hemin)、RAPA (Hemin)

Affinity Landscape of Antibiotic-Hemin Interactions

What’s more,using benzene ring-containing antibiotics as an example, we initially predicted and ranked the affinity between various antibiotics and chlorinated heme. Benzylpenicillin (BPN) exhibited the strongest affinity, while polymyxin B (PB) showed the weakest. When comparing the assembled products of these two antibiotics with chlorinated heme, PB failed to form a Soret band, indicating its inability to establish axial coordination and a catalytic active center. As a result, PB (Hemin) does not enhance the bactericidal activity of PB.

Fig.4 (a)Binding energy sequencing heat map of antibiotic affinity with Hemin;Comparison of antibacterial activity of benzathine penicillin (b) and polymyxin B (c) against MRSA after assembly with hemin (d,e).
Fig.6 (a)Binding energy sequencing heat map of antibiotic affinity with Hemin;Comparison of antibacterial activity of benzathine penicillin (b) and polymyxin B (c) against MRSA after assembly with hemin (d,e).

The synergistic effect of ferrous protoporphyrin in promoting the anti-MRSA activity of gentamicin (GM)

Zeta Potential and Hydrodynamic Size

Zeta potential measurements indicated that the co-assembled gentamicin nanozyme exhibits uniform nanoscale particle size with a peak hydrodynamic diameter of approximately 170 nm, demonstrating monodisperse characteristics suitable for biological applications.

Fig.6 Particle size distribution (a) and Zeta potential (b) of GM (Hemin).
Fig.7 Particle size distribution (a) and Zeta potential (b) of GM (Hemin).

TEM Characterization of GM (Hemin)

TEM revealed a cross-linked spherical nanostructure for the GM (Hemin) (Fig. 8a). High-angle annular dark-field imaging (Fig. 8b) showed uniform iron distribution at 5 nm resolution, while energy-dispersive X-ray spectroscopy(EDS) (Fig.8c,d) confirmed homogeneous distribution of Fe, C, N, and O elements throughout the assembly, verifying successful incorporation of hemin-derived iron.

Fig.7 TEM images (a), High-Angle Annular Dark-Field image (b), and EDS (c: Iron; d: Carbon, Nitrogen, Oxygen, Iron) of GM(Hemin)
Fig.8 TEM images (a), High-Angle Annular Dark-Field image (b), and EDS (c: Iron; d: Carbon, Nitrogen, Oxygen, Iron) of GM(Hemin)

UV-Vis Spectral Analysis

UV-Vis spectroscopy indicated coordination between hemin and gentamicin via the porphyrin iron center. Post-assembly spectral changes included a Soret band at 415 nm and decreased Q-band intensity, suggesting iron center coordination and possible axial distortion of the porphyrin plane.

Fig.8 Full-wavelength UV-Vis absorption spectrum of GM (Hemin)
Fig.9 Full-wavelength UV-Vis absorption spectrum of GM (Hemin)

CLSM for Biofilm Eradication Assessment

Three-dimensional confocal laser scanning microscopy revealed significant reduction in bacterial density and biofilm thickness following gentamicin nanozyme treatment, providing direct visual evidence of biofilm disruption capability consistent with quantitative plate counting results.

Fig.9 The scavenging effect of GM (Hemin) on Methicillin-Resistant Staphylococcus aureus (MRSA) biofilm was observed by laser confocal imaging.
Fig.10 The scavenging effect of GM (Hemin) on Methicillin-Resistant Staphylococcus aureus (MRSA) biofilm was observed by laser confocal imaging.

TEM Analysis of MRSA

TEM images of MRSA ultrastructure showed control cells maintaining regular, turgid morphology with intact cell walls and membranes. In contrast, gentamicin nanozyme-treated bacteria exhibited severe cellular shrinkage, membrane disruption, uneven cytoplasmic density, and evident intracellular content leakage.

Fig.10 Observation of MRSA treated with GM (Hemin) by transmission electron microscope.
Fig.11 Observation of MRSA treated with GM (Hemin) by transmission electron microscope.

Protein Leakage Detection

Quantitative analysis of post-treatment bacterial suspensions showed significantly increased protein content in MRSA exposed to gentamicin nanozyme, indicating cell disruption and cytoplasmic leakage. Control groups displayed no significant differences in protein levels, confirming maintained cellular integrity.

Fig.11 Quantitative detection of protein leakage in GM (Hemin) treated MRSA.
Fig.12 Quantitative detection of protein leakage in GM (Hemin) treated MRSA.

Assay Assessment of Lipid Peroxidation

Malondialdehyde assay results demonstrated significantly increased lipid peroxidation in MRSA treated with gentamicin nanozyme, confirming that peroxidase activity induces lipid peroxidation as a crucial antibacterial mechanism.

Fig.12 Detection of lipid peroxidation via Malondialdehyde levels
Fig.13 Detection of lipid peroxidation via Malondialdehyde levels

Effects on Hydrogen Sulfide(H2S) Production

Fluorescence quantification revealed significantly reduced intracellular H₂S levels in MRSA following gentamicin nanozyme treatment, suggesting the nanozyme inhibits bacterial H₂S production and/or consumes H₂S to reverse resistance and enhance antibacterial effects. The specific mechanism requires further investigation.

Fig.13 Hydrogen Sulfide content in MRSA after treatment with GM (Hemin)
Fig.14 Hydrogen Sulfide content in MRSA after treatment with GM (Hemin)

Conclusion

GM(Hemin) effectively eradicates biofilms primarily. Subsequently, leveraging its peroxidase (POD)-like activity, GM(Hemin) generates reactive oxygen species (ROS) that enhance bacterial lipid peroxidation, disrupt membrane permeability.What’s more,it elevates intracellular ROS levels to potentiating antibiotic efficacy. Additionally, the antibiotic nanozyme may inflict physical damage on bacterial cells , leading to protein leakage. Together, these mechanisms form a multifaceted antibacterial strategy that enhances ferroptosis-like pathways to suppress bacterial survival.

The synergistic effect of ferrous protoporphyrin in promoting the antifungal activity of Fluconazole (FCZ)

SEM Characterization of FCZ(Hemin)

SEM characterization confirmed nanoscale dimensions of the co-assembled fluconazole nanozyme, with EDS mapping showing homogeneous distribution of characteristic Fe and F elements, providing direct evidence of successful hemin-fluconazole co-assembly.

Fig.14 SEM image and element energy spectrum analysis (Fe and F) of FCZ (Hemin)
Fig.15 SEM image and element energy spectrum analysis (Fe and F) of FCZ (Hemin)

Ferroptosis Measurement

The FCZ (Hemin) demonstrated significantly enhanced antibacterial efficacy in the absence of ferroptosis inhibitor, which was abolished upon inhibitor addition. These findings indicate ferric porphyrin reduction to ferrous heme acts as a ferroptosis inducer that intensifies fungal ferroptosis, thereby boosting antimicrobial efficacy.

Fig.15 Antifungal activity of FCZ(Hemin) and CRO(Hemin) against C. albicans.
Fig.16 Antifungal activity of FCZ(Hemin)(a) and CRO(Hemin)(b) against C.albicans.

SEM Analysis of C. albicans

SEM observations revealed that the control C. albicans cells exhibited a plump and intact morphology. In contrast, the cells treated with the fluconazole nanozyme displayed a collapsed and shrunken surface with evident pores.

Fig.16 SEM images of C. albicans treated with FCZ (Hemin).
Fig.17 SEM images of C. albicans treated with FCZ (Hemin).

Conclusion

FCZ(Hemin) acts as a ferroptosis inducer. This process enhances fungal ferroptosis, thereby improving the antimicrobial efficacy of fluconazole (FCZ).The free radicals generated by FCZ(Hemin) catalysis further elevate intracellular ROS levels, thereby promoting enhanced ferroptosis.

Conclusion

This study successfully synthesized a series of antibiotic nanozymes by co-assembling various antibiotics with hemin via a coordination-precipitation method. Morphological characterization revealed that the synthesized antibiotic nanozymes possessed relatively uniform particle sizes. Molecular docking simulations further confirmed the theoretical feasibility of their structures. Subsequently, using gentamicin nanozymes and fluconazole nanozymes as representative examples, their antibacterial effects were systematically investigated. Through growth curve assays, electron microscopy, and protein leakage experiments, we validated their ability to inhibit the growth and viability of planktonic bacteria, demonstrating potent antibacterial properties.Further mechanistic studies revealed that the peroxidase (POD)-like activity of the nanozymes generates reactive oxygen species (ROS), as confirmed by fluorescent staining, which contribute to bacterial damage and death. Additionally, membrane potential assays and lipid peroxidation measurements verified membrane permeabilization and damage induced by the gentamicin nanozyme. The use of a ferroptosis inhibitor confirmed that the fluconazole nanozyme enhances its antibacterial efficacy primarily through the ferroptosis pathway.

Based on these collective findings, antibiotic nanozymes effectively eradicate drug-resistant bacteria and exhibit superior performance compared to conventional antibiotics. In conclusion, antibiotic nanozymes represent a promising class of candidates that optimize antibiotic efficacy, effectively target both planktonic bacteria and biofilms, and can serve as potential alternatives to current antibiotics in bacterial treatment. Moreover, their applicability to various antibiotics and the flexibility in ligand design underscore their broad utility and generalizability.