A rapid one-step synthesis of silver and copper coordinated chlorine functionalized fullerene nanoparticles with enhanced antibacterial activity

Abstract

Nanoparticle modification demonstrates a remarkable synergetic effect in combating bacteria, particularly resistant bacteria, enhancing their efficacy by simultaneously targeting multiple cellular pathways. This approach positions them as a potent solution against the growing challenge of antimicrobial-resistant (AMR) strains. This research presents an investigation into the synthesis, characterization, and antibacterial evaluation of silver-coordinated chloro-fullerenes nanoparticles (Ag-C₆₀-Cl) and copper-coordinated chloro-fullerenes nanoparticles (Cu-C₆₀-Cl). Utilizing an innovative, rapid one-step synthesis approach, the nanoparticles were rigorously characterized using X-ray Photoelectron Spectroscopy (XPS), Scanning Electron Microscopy-Energy Dispersive X-ray Spectrometer (SEM-EDS), High-Resolution Transmission Electron Microscopy (HR-TEM), Fourier-Transform Infrared Spectroscopy (FTIR), and Raman spectroscopy. In conjunction with the analytical techniques, a computational approach was utilized to corroborate the findings from Raman spectroscopy as well as the surface potential of these nanoparticles. Moreover, the antibacterial activities of the synthesized nanoparticles were assessed against Escherichia coli (E. coli) and Methicillin-Resistant Staphylococcus aureus (MRSA). These findings demonstrated that the synthesized Ag-C₆₀-Cl and Cu-C₆₀-Cl nanoparticles exhibited minimum inhibitory concentrations (MIC) of 3.9 μg mL^-1 and 125 μg mL^-1, respectively. Reactive oxygen species (ROS) quantification, catalase assay, and efflux pump inhibition results revealed promising broad-spectrum antibacterial effects.

Fig. 1. Schematic representation for the synthesis of the nanoparticles.

Fig. 2. Raman spectra and morphological images of C₆₀-Cl, Ag-C₆₀-Cl, and Cu-C₆₀-Cl fullerenes synthesized through the liquid–liquid interfacial method. The Raman spectra display the inherent vibrational modes of (red trace) C₆₀-Cl, (blue trace) Ag-C₆₀-Cl, and (green trace) Cu-C₆₀-Cl. Insets show the corresponding morphological images of the nanoparticles taken through a Raman microscope at 50× with a scale bar of 20 μm. All spectra were background-subtracted and normalized.

Fig. 3. (I) Graphical models illustration of relaxed geometry of six metal ions in relation to the fullerenes. (a) Configuration with six metal ions positioned inside the fullerene cage and (b) configuration with six metal ions positioned outside the fullerene cage. Theoretical Raman spectra for (II) C₆₀-Cl, (III) Ag ions outside the cage Ag-C₆₀-Cl, (IV) Ag ions inside the cage Ag-C₆₀-Cl, (V) Cu ions outside the cage Cu-C₆₀-Cl, and (VI) Cu ions inside the cage Cu-C₆₀-Cl. The spectral peak alignment is further corroborated by the matching peaks in the experimental spectra (Fig. 2).

Fig. 4. Electrostatic potential mapped onto the total electron density of fullerenes. Isometric scale bar values are shown in blue (positive) and red (negative) for (a) pristine fullerene +/− 6.488 × 10⁻² [eV], (b) fullerene modified with copper atoms exhibiting increased polarization and repulsive interactions with the carbon framework, with an isometric scale bar of +/− 4.52 × 10⁻² [eV], and (c) fullerene modified with silver atoms also showing increased polarization and repulsive interactions with the carbon framework, with an isometric scale bar of +/− 3.594 × 10⁻² [eV].

Fig. 5. (a) XPS survey spectra of C₆₀-Cl and high-resolution scans for (b) C 1s and (c) Cl 2p.

Fig. 6. (a) XPS survey spectra of Ag-C₆₀-Cl and high-resolution scans for (b) C 1s, (c) Ag 3d, and (d) Cl 2p.

Fig. 7. (a) XPS survey spectra of Cu-C₆₀-Cl and high-resolution scans for (b) C 1s, (c) Cu 2p, and (d) Cl 2p.

Fig. 8. SEM-EDS of C₆₀-Cl, Ag-C₆₀-Cl, and Cu-C₆₀-Cl fullerenes. SEM image and EDS results of (a) C₆₀-Cl, (b) Ag-C₆₀-Cl, and (c) Cu-C₆₀-Cl.

Fig. 9. FTIR spectra of C₆₀, C₆₀-Cl, Ag-C₆₀-Cl, and Cu-C₆₀-Cl fullerenes. The black FTIR spectrum represents pristine fullerenes, the red FTIR spectrum represents C₆₀-Cl, the blue represents Ag-C₆₀-Cl, and the green represents Cu-C₆₀-Cl.

Fig. 10. (a) TEM image of C₆₀ nanoparticles at 10k×; (b) corresponding SAED pattern of C₆₀; (c) TEM image of C₆₀-Cl at 10k×; (d) its corresponding SAED pattern; (e) TEM image of Ag-C₆₀-Cl at 10k×; (f) its corresponding SAED pattern; (g) TEM image of Cu-C₆₀-Cl at 10k×; (h) its corresponding SAED pattern.

Fig. 11. EtBr cartwheel assay assessing efflux pump inhibition in E. coli and MRSA. Evaluation of (a) control (without nanoparticles), (b) C₆₀-Cl fullerenes, (c) Ag-C₆₀-Cl fullerenes, and (d) Cu-C₆₀-Cl fullerenes in E. coli and MRSA.

Fig. 12. Relative fluorescence intensity indicating ROS production in E. coli and MRSA treated with C₆₀-Cl, Ag-C₆₀-Cl, and Cu-C₆₀-Cl fullerenes. (a) E. coli and (b) MRSA were treated with increasing concentrations of C₆₀-Cl, Ag-C₆₀-Cl, and Cu-C₆₀-Cl fullerenes from 0.031 MIC to 0.5× MIC, or H₂O₂ alone (control). Data is represented by three independent assays (+/−SD). Statistical significance was determined using a T-test, comparing the MIC-treated samples to the H₂O₂ control, a potent inducer of ROS. Significance is denoted as p > 0.05 (*).

Fig. 13. Change in fluorescence indicating catalase activity in E. coli and MRSA treated with C₆₀-Cl, Ag-C₆₀-Cl, and Cu-C₆₀-Cl nanoparticles. E. coli and MRSA were treated with C₆₀-Cl, Ag-C₆₀-Cl, and Cu-C₆₀-Cl fullerenes with concentrations ranging from 7.8–500 μg mL⁻¹, along with appropriate controls. Data is represented by three independent assays (+/−SD). Statistical significance was determined using a T-test. Significance is denoted as follows: p > 0.05 (*) and p ≤ 0.005 (**). Significant differences between nanoparticle treatments are indicated by brackets above the bars.

Fig. 14. SEM images for E. coli and MRSA before and after the treatment with C₆₀-Cl, Ag-C₆₀-Cl, and Cu-C₆₀-Cl nanoparticles.