Destruction of Opportunistic Pathogens via Polymer Nanoparticle-Mediated Release of Plant-Based Antimicrobial Payloads

Abstract

The synthesis of antimicrobial thymol/carvacrol-loaded polythioether nanoparticles (NPs) via a one-pot, solvent-free miniemulsion thiol-ene photopolymerization process is reported. The active antimicrobial agents, thymol and carvacrol, are employed as "solvents" for the thiol-ene monomer phase in the miniemulsion to enable facile high capacity loading (≈50% w/w), excellent encapsulation efficiencies (>95%), and elimination of all postpolymerization purification processes. The NPs serve as high capacity reservoirs for slow-release and delivery of thymol/carvacrol-combination payloads that exhibit inhibitory and bactericidal activity (>99.9% kill efficiency at 24 h) against gram-positive and gram-negative bacteria, including both saprophytic (Bacillus subtilis ATCC 6633 and Escherichia coli ATCC 25922) and pathogenic species (E. coli ATCC 43895, Staphylococcus aureus RN6390, and Burkholderia cenocepacia K56-2). This report is among the first to demonstrate antimicrobial efficacy of essential oil-loaded nanoparticles against B. cenocepacia - an innately resistant opportunistic pathogen commonly associated with debilitating respiratory infections in cystic fibrosis. Although a model platform, these results point to promising pathways to particle-based delivery of plant-derived extracts for a range of antimicrobial applications, including active packaging materials, topical antiseptics, and innovative therapeutics.

Keywords: antimicrobial; carvacrol; miniemulsion polymerization; polymer nanoparticles; thymol.

Figure 1

a) The thiol-ene reaction involves alternating chain transfer and propagation b) with various multifunctional monomers used to generate polythioether nanoparticles via thiol-alkene photopolymerization in miniemulsion. c) Representative TEM of thymol/carvacrol-loaded nanoparticles.

Figure 2

a) Amounts of essential oils extracted from the supernatant after pelleting NPs by ultracentrifugation. b) Calculated release profiles for both C_NPs and TC_NPs over 24 h.

Figure 3

Well diffusion assay identifies treatments with antimicrobial activity. Bacteria were incubated with a) Hitenol BC-20 (70 × 10⁻³ M), b) Control_NPs, c) C_NPs, and d) TC_NPs at 10¹³ NPs mL⁻¹. Zones of inhibition (ZOI, mm) are reported below each image.

Figure 4

Percent viability of various bacteria upon treatment with TC_NPs. The 0 μg mL⁻¹ EO {10¹¹} was treated with Control_NPs. Inset images show the corresponding spot tests for the presence of live bacteria.

Figure 5

Effect of TC_NPs on the viability of a) B. subtilis ATCC 6633 and b) E. coli ATCC 25922, as monitored by confocal laser scanning microscopy. Representative images of control cultures (top row), and cultures treated with 10¹¹ TC_NPs (middle and bottom row) at the indicated time points. The green signal (SYTO 9) indicates viable live cells, whereas red signal (propidium iodide) indicates damaged or dead cells. Scale bars = 10 μm.

Figure 6

Evaluation of antimicrobial activity for 10¹¹ TC_NPs mL–1 on the viability of (●) E. coli ATCC 25922, (■) S. aureus RN6390, (▲) B. subtilis ATCC 6633, (▼) E. coli ATCC 43895 (serotype O157:H7), and (◆) B. cenocepacia K56-2 via a) a kinetic terminal dilution and b) percentage of bacteria killed and over 48 h. *Dashed line represents the limit of quantitation.

Figure 7

a) TEM of E. coli ATCC 25922 and b) B. subtilis ATCC 6633 control cultures and cultures that were challenged with 10¹¹ TC_NPs for various times. c) High resolution SEM of the control culture of B. subtilis ATCC 6633 and the culture treated for 24 h with 10¹¹ TC_NPs.