Drug-Free Enzyme-Based Bactericidal Nanomotors against Pathogenic Bacteria

Abstract

The low efficacy of current conventional treatments for bacterial infections increases mortality rates worldwide. To alleviate this global health problem, we propose drug-free enzyme-based nanomotors for the treatment of bacterial urinary-tract infections. We develop nanomotors consisting of mesoporous silica nanoparticles (MSNPs) that were functionalized with either urease (U-MSNPs), lysozyme (L-MSNPs), or urease and lysozyme (M-MSNPs), and use them against nonpathogenic planktonic Escherichia coli. U-MSNPs exhibited the highest bactericidal activity due to biocatalysis of urea into NaHCO₃ and NH₃, which also propels U-MSNPs. In addition, U-MSNPs in concentrations above 200 μg/mL were capable of successfully reducing 60% of the biofilm biomass of a uropathogenic E. coli strain. This study thus provides a proof-of-concept, demonstrating that enzyme-based nanomotors are capable of fighting infectious diseases. This approach could potentially be extended to other kinds of diseases by selecting appropriate biomolecules.

Keywords: E. coli; biofilms; enzymatic nanomotors; infections; nanomachines; self-propulsion.

Figure 1

Fabrication and characterization of enzyme-based mesoporous silica nanoparticles. (A) Scheme of the stepwise fabrication process to synthesize enzyme-based nanomotors. (B) Scanning electron microscopy (SEM) image of mesoporous silica nanoparticles (MSNPs). (C) TEM image of MSNPs showing the porous particle surface. (D) Surface charge of the unmodified MSNPs, the amino-modified MSNPs (NH₂-MSNPs), the urease-modified MSNPs (U-MSNPs), the lysozyme-modified MSNPs (L-MSNPs), and the urease- and lysozyme-modified MSNPs (MMSNPs) (N = 3, error bars indicate SE).

Figure 2

Evaluating the bactericidal capacity of the different enzyme-based micromotors: (A) optical density (OD₆₀₀) of nonpathogenic E. coli after 24 h in the presence of different concentrations of urease, U-MSNPs, L-MSNPs, and M-MSNPs. (B) Fluorescence images and (C) percentage of dead bacteria determined by live/dead assay after 2 h of 1 × 10⁸ CFU/mL E. coli treated with 12.5 μg/mL (minimum inhibitory concentration, MIC₅₀) for urease, U-MSNPs, L-MSNPs, and M-MSNPs. (D) E. coli counts (log 10 CFU/mL) after 2 and 4 h of treatment with 12.5 μg/mL (MIC₅₀) urease, U-MSNPs, L-MSNPs, and M-MSNPs. (E) Photographs of Petri plates at 10³ CFU dilution used to measure the efficacy of urease, U-MSNPs, L-MSNPs, and M-MSNPs against E. coli after 2 and 4 h. All experiments were carried out at [urea] = 50 mM (N = 3, error bars represent SE).

Figure 3

Bacteria imaged with SEM: Examples of (A) live E. coli MG1655; (B) dead bacteria after having been treated with U-MSNPs for 2 h in the presence of 50 mM urea. Yellow box depicts a zoom image of (B) the bacteria in the top row.

Figure 4

Motion analysis of urease-based nanomotors (U-MSNPs) in PBS, LB, and simulated urine. (A) Representative trajectories of U-MSNPs at 0 mM (top) and 100 mM urea (bottom). (B) Mean-squared displacements (MSDs) of U-MSNPs at 0 and 100 mM. (C) Effective diffusion coefficients calculated from the MSDs at different urea concentrations (N = 20, error bars show SE).

Figure 5

Evaluating the bactericidal capacity of urease-based nanomotors (U-MSNPs) against uropathogenic E. coli (CFT073): (A) optical density (OD₅₅₀) of planktonic uropathogenic E. coli for different concentrations of urease and U-MSNPs; (B) percentage of the biofilm biomass from uropathogenic E. coli remaining after treatment with U-MSNP nanomotors and excess of urease (at 5- to 10-fold, the highest U-MSNP nanomotor concentrations applied); and (C) simulated fluorescence projections and orthogonal view sections of 4-day uropathogenic E. coli biofilm before and 6 h after treatment with different concentrations of urease and U-MSNPs (scale bar = 50 μm). All experiments were carried out at [urea] = 100 mM. (N = 3, error bars represent SE).