Potent Antibacterial Nanoparticles against Biofilm and Intracellular Bacteria

Abstract

The chronic infections related to biofilm and intracellular bacteria are always hard to be cured because of their inherent resistance to both antimicrobial agents and host defenses. Herein we develop a facile approach to overcome the above conundrum through phosphatidylcholine-decorated Au nanoparticles loaded with gentamicin (GPA NPs). The nanoparticles were characterized by scanning electron microscopy (SEM), dynamic light scattering (DLS) and ultraviolet-visible (UV-vis) absorption spectra which demonstrated that GPA NPs with a diameter of approximately 180 nm were uniform. The loading manner and release behaviors were also investigated. The generated GPA NPs maintained their antibiotic activities against planktonic bacteria, but more effective to damage established biofilms and inhibited biofilm formation of pathogens including Gram-positive and Gram-negative bacteria. In addition, GPA NPs were observed to be nontoxic to RAW 264.7 cells and readily engulfed by the macrophages, which facilitated the killing of intracellular bacteria in infected macrophages. These results suggested GPA NPs might be a promising antibacterial agent for effective treatment of chronic infections due to microbial biofilm and intracellular bacteria.

Figure 1

SEM image of PA NPs (A) and GPA NPs (B); (C) size distribution of GPA estimated using Image J (about 1000 particles were counted); (D) Hydrodynamic size and (E) surface zeta potential of bare Au NPs, PA NPs and GPA NPs measured by dynamic light scattering; (F) absorption spectrum of PA NPs, GPA NPs and gentamicin.Scale bar represented 1 μm.

Figure 2. The load (A) or release profiles (C) of gentamicin on GPA NPs in 6 mM HEPES buffer (pH 7.4) and 6 mM Tris-HCl buffer (pH 4.5).

The effect of ionic strength on the load (B) or release (D) of gentamicin on GPA NPs. NaCl was added to 6 mM HEPES buffer (pH 7.4) and 6 mM Tris-HCl buffer (pH 4.5) to obtain the ionic strengths as indicated.

Figure 3. Crystal violet assay to assess the antibiofilm activity of samples against P. aeruginosa biofilm (A,C) and L. monocytogenes (B,D).

Figure 4. Fluorescence images (A) and SEM images (B) of P. aeruginosa biofilm.

Scale bar for SEM and fluorescence images are 1 μm and 10 μm, respectively.

Figure 5. The inhibitory effects of samples on P. aeruginosa (A) and S. aureus (B) biofilm formation.

Figure 6. Fluorescence image (A,C) and brightfield image (B,D) of macrophages alone, or macrophages incubated with GPA NPs for 2 h followed by rinsing with new medium.

Scale bar represented 10 μm. (E) Cellular uptake of GPA NPs measured by ICP-MS. RAW264.7 cells were incubated in the presence of 0.116 mg/mL of GPA NPs as time indicated. Data are represented as pg Au/cell.

Figure 7. The colony-forming units of residual P. aeruginosa in infected macrophages treated with different samples.

Figure 8. Schematic of the procedure for preparing GPA NPs.