Autonomous Treatment of Bacterial Infections in Vivo Using Antimicrobial Micro- and Nanomotors

Abstract

The increasing resistance of bacteria to existing antibiotics constitutes a major public health threat globally. Most current antibiotic treatments are hindered by poor delivery to the infection site, leading to undesired off-target effects and drug resistance development and spread. Here, we describe micro- and nanomotors that effectively and autonomously deliver antibiotic payloads to the target area. The active motion and antimicrobial activity of the silica-based robots are driven by catalysis of the enzyme urease and antimicrobial peptides, respectively. These antimicrobial motors show micromolar bactericidal activity in vitro against different Gram-positive and Gram-negative pathogenic bacterial strains and act by rapidly depolarizing their membrane. Finally, they demonstrated autonomous anti-infective efficacy in vivo in a clinically relevant abscess infection mouse model. In summary, our motors combine navigation, catalytic conversion, and bactericidal capacity to deliver antimicrobial payloads to specific infection sites. This technology represents a much-needed tool to direct therapeutics to their target to help combat drug-resistant infections.

Keywords: antimicrobial peptides; autonomous treatment; bacterial infection; nanomotors; nanoparticles; self-propulsion.

Figure 1

Bioactive micro- and nanomotors coated with antimicrobial peptides for the autonomous treatment of infections. Schematic of the AMP-coating process of the urease micro- and nanomotors and their autonomous propulsion to target pathogenic infections both in vitro and in vivo. Briefly, AMP–urease motors will encounter and hydrolyze urea (yellow spheres) in solution. The hydrolysis reaction will propel the motors, and when exposed to bacterial membranes, the AMPs onto their surface will act as antimicrobials and lyse bacterial cells in controlled (e.g., in vitro assays) and complex (e.g., infected wound) biological environments.

Figure 2

Effect of antimicrobial peptides on the active motion of bioactive micromotors. (a) SEM micrograph of the hollow silica microcapsules. Inset: TEM micrograph of the hollow silica microparticles. (b) Average speed of urease micromotors for different concentrations of urea. Inset: Representative 15 s trajectories for different concentrations of urea. (c) Average speed and zeta potential of urease micromotors for different concentrations of LL-37 peptide used to functionalize the silica surface. (d) Average speed and zeta potential of urease micromotors for different concentrations of K7-Pol peptide used to functionalize the silica surface. All results are shown as the mean ± standard error of the mean.

Figure 3

Effect of antimicrobial peptides on the active motion of bioactive nanomotors. (a) SEM micrograph of the mesoporous silica nanoparticles. Inset: TEM micrograph of the mesoporous silica nanoparticles. (b) Diffusion coefficient of urease nanomotors for different concentrations of urea. (c) Diffusion coefficient and zeta potential of urease nanomotors for different concentrations of LL-37 peptide used to functionalize the silica surface. (d) Diffusion coefficient and zeta potential of urease nanomotors for different concentrations of K7-Pol peptide used to functionalize the silica surface. All results are shown as the mean ± standard error of the mean.

Figure 4

Antimicrobial activity of bioactive micro- and nanomotors functionalized with antimicrobial peptides. (a) Schematic depicting experimental design of in vitro biological activity assays. Briefly, 10⁵ bacterial cells and peptides, the antibiotic polymyxin B, or urease micro- and nanomotors (0–500 μg mL^–1) were added to a 96-well plate and incubated at 37 °C. One day after the exposure, the solution in each well was measured in a microplate reader (600 nm) to check inhibition of bacteria compared to the untreated controls. (b) Heat map of the antimicrobial activity of each system against five bacterial strains: A. baumannii AB177, E. coli ATCC11775, K. pneumoniae ATCC13883, P. aeruginosa PAO1, and S. aureus ATCC12600. Assays were performed in three independent replicates, and heat map OD₆₀₀ values are the arithmetic mean of the replicates in each condition. This figure was created with BioRender.com.

Figure 5

Mechanism of action of the antimicrobial motors. (a) Bioactive micro- and nanomotors cause the depolarization of bacterial membranes at their MIC concentration against (b) A. baumannii AB177 and (c) K. pneumoniae ATCC13883. Briefly, micro- and nanomotors functionalized with LL-37 and K7-Pol, respectively, enabled the higher depolarization of K. pneumoniae cells than the nonfunctionalized motors. When A. baumannii cells were exposed to them both, functionalized and nonfunctionalized motors presented depolarizing effect. The potent permeabilizer antimicrobial polymyxin B was used as a negative control for depolarization. Assays were performed in three independent replicates. This figure was created with BioRender.com.

Figure 6

Anti-infective activity of the antimicrobial motors in vivo. (a) Mice had their dorsal region shaved, scratched (1-cm-long wound), and infected with A. baumannii AB177. After 1 h, functionalized and nonfunctionalized micro- and nanomotors or peptides were added to the infection site. Mice were euthanized, the tissue from the infection site was harvested, and the bacterial cells of treated and untreated samples were counted by plating. (b) Schematic representation of the wound site infected and the addition of urea before the treatment with (i) micro- and nanomotors or (ii) peptides free is solution. Antimicrobial micro- and nanomotors self-propelled, driven by urea, through a distance of 1 cm to enable the autonomous treatment of the target infected area. On the other hand, peptides by themselves exhibited antimicrobial activity only within the area they were administered and did not clear the infection at a distance. Briefly, after the infection was established, urea was spread over the entire length of the wound (1 cm). Next, the micro- and nanomotors coated with peptides and the peptides alone were inoculated to one of the extremities of the infected wound. (c) Four days postinfection, 1 cm² of the infected area was excised and the ability of the micro- and nanomotors to travel throughout the wound alone and when functionalized with peptides was assessed. (d) Treatment with peptides alone decreased bacterial counts only in the extremity where they were administered (light yellow background), as revealed by similar bacterial counts detected in areas at a distance from the administration site (dark yellow background) and those of untreated control groups. (e) Mouse weight was monitored throughout the experiments, serving as a proxy to assess the toxicity of both micro- and nanomotors and (f) peptides in solution. None of the treatment groups led to toxicity in mice. Eight animals were used per group. This figure was created with BioRender.com.