Polymer-Based Bioorthogonal Nanocatalysts for the Treatment of Bacterial Biofilms

Abstract

Bioorthogonal catalysis offers a unique strategy to modulate biological processes through the in situ generation of therapeutic agents. However, the direct application of bioorthogonal transition metal catalysts (TMCs) in complex media poses numerous challenges due to issues of limited biocompatibility, poor water solubility, and catalyst deactivation in biological environments. We report here the creation of catalytic "polyzymes", comprised of self-assembled polymer nanoparticles engineered to encapsulate lipophilic TMCs. The incorporation of catalysts into these nanoparticle scaffolds creates water-soluble constructs that provide a protective environment for the catalyst. The potential therapeutic utility of these nanozymes was demonstrated through antimicrobial studies in which a cationic nanozyme was able to penetrate into biofilms and eradicate embedded bacteria through the bioorthogonal activation of a pro-antibiotic.

Figure 1.

a) Structures of [Fe(TPP)]Cl and polymeric nanoparticle. b) Transmission electron microscopy (TEM) imaging and dynamic light scattering measurement (DLS). Scale bar is 100 nm. c) Schematic representation of the bioorthogonal activation for imaging and therapeutics.

Figure 2.

a) Schematic representation of the bioorthogonal activation of aryl azide-protected resorufin. b) Catalytic activity of polyzyme (100 nM) was tracked by measuring changes in fluorescence (Ex. 570nm, Em. 590nm) of pro-resorufin solutions at different concentrations. The curve is fit with a non-linear exponential equation. 1 mM GSH was used as the cofactor. c) Polyzyme kinetics is shown as a function of substrate concentration; line is the regression curve corresponding to Michaelis-Menten kinetics.

Figure 3.

Polyzyme analog (200 nM) diffusion into GFP - expressing E. coli (CD-2) biofilms after incubation for 1hr in M9 media, as measured by confocal microscopy images. (a) free TPP, (b) encapsulated TPP. Scale bars are 50 μm. 1 mM GSH was used as the cofactor.

Figure 4.

Confocal microscopy images of biofilms treated with polyzyme (1 hr, 200 nM) followed by incubation with pro-dye (1 hr, 4 μM); negative controls are biofilms incubated with (a) pro-dye only and (b) free catalyst followed by incubation with pro-dye. Scale bars are 50 μm. 1 mM GSH was used as the cofactor.

Figure 5.

Schematic representation of the activation of (a) pro-Moxifloxacin and (b) pro-Ciprofloxacin. 1 mM GSH was used as the cofactor. (c)Viability of E. coli (CD-2) biofilms incubated with polyzyme (2 hrs, 200 nM) followed by treatment with prodrugs (6 hrs). (d) Viability of P. aeruginosa (ATCC-27853) biofilms incubated with polyzyme (2 hrs, 1 μM) followed by treatment with prodrugs (6 hrs). Biofilms treated with prodrug, drug and prodrug + polymers were used as controls. Each experiment was performed in triplicate. Error bars represent standard deviation.

Figure 6.

Viability of 3T3 fibroblast cells after treating with polyzyme for overnight. Each result is an average of three experiments, and the error bars designate the standard deviations.