Silver-doped phosphate coacervates to inhibit pathogenic bacteria associated with wound infections: an in vitro study

Abstract

There is a great demand from patients requiring skin repair, as a result of poorly healed acute wounds or chronic wounds. These patients are at high risk of constant inflammation that often leads to life-threatening infections. Therefore, there is an urgent need for new materials that could rapidly stimulate the healing process and simultaneously prevent infections. Phosphate-based coacervates (PC) have been the subject of increased interest due to their great potential in tissue regeneration and as controlled delivery systems. Being bioresorbable, they dissolve over time and simultaneously release therapeutic species in a continuous manner. Of particular interest is the controlled release of metallic antibacterial ions (e.g. Ag⁺), a promising alternative to conventional treatments based on antibiotics, often associated with antibacterial resistance (AMR). This study investigates a series of PC gels containing a range of concentrations of the antibacterial ion Ag⁺ (0.1, 0.3 and 0.75 mol%). Dissolution tests have demonstrated controlled release of Ag⁺ over time, resulting in a significant bacterial reduction (up to 7 log), against both non-AMR and AMR strains of both Gram-positive and Gram-negative bacteria (Staphylococcus aureus, Enterococcus faecalis, Escherichia coli and Pseudomonas aeruginosa). Dissolution tests have also shown controlled release of phosphates, Ca²⁺, Na⁺ and Ag⁺ with most of the release occurring in the first 24 h. Biocompatibility studies, assessed using dissolution products in contact with human keratinocyte cells (HaCaT) and bacterial strains, have shown a significant increase in cell viability (p ≤ 0.001) when gels are dissolved in cell medium compared to the control. These results suggest that gel-like silver doped PCs are promising multifunctional materials for smart wound dressings, being capable of simultaneously inhibit pathogenic bacteria and maintain good cell viability.

Figure 1

Schematic illustration of the synthesis of the phosphate coacervate gels and main studies performed in the present work.

Figure 2

Ion release of (A) phosphorus, (B) calcium, (C) sodium and (D) silver in deionized water over 7 days for all PC gels. Error bars indicate the mean ± standard deviation (n = 3).

Figure 3

pH analysis of all dissolved in deionised water (A) and cell medium (B) over 7 days. Error bars represent the SD ± mean of two biological replicates. PBS was used as a control for this experiment.

Figure 4

FTIR spectra of the undoped (PC) and silver doped (PC_Ag) gels.

Figure 5

Antibacterial activity of PC gels against non-AMR and AMR bacterial strains of S. aureus, E. faecalis, E. coli and P. aeruginosa. Bacterial reduction is expressed as the mean of CFU/mL ± standard deviation (error bars). Statistical analysis was performed using two-way ANOVA (**p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001). Asterisks illustrate the degree of statistical difference of the samples when compared to the control.

Figure 6

MTT analysis of HaCaT cells infected with bacteria in the presence of coacervate gels dissolution products in deionised water and cell medium. Error bars indicate the mean ± standard deviation. Statistical analysis was performed using one-way ANOVA (*p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001). Asterisks illustrate the degree of statistical difference when compared to the challenging sample.

Figure 7

Infection of HaCaT cells with bacterial strains after 24 h incubation with coacervate dissolution products in water and cell medium. A culture of HaCaT cells was used as a control (uninfected). Error bars indicate the mean ± standard deviation (n = 3).