Pentadecanoic Acid-Releasing PDMS: Towards a New Material to Prevent S. epidermidis Biofilm Formation

Abstract

Microbial biofilm formation on medical devices paves the way for device-associated infections. Staphylococcus epidermidis is one of the most common strains involved in such infections as it is able to colonize numerous devices, such as intravenous catheters, prosthetic joints, and heart valves. We previously reported the antibiofilm activity against S. epidermidis of pentadecanoic acid (PDA) deposited by drop-casting on the silicon-based polymer poly(dimethyl)siloxane (PDMS). This material exerted an antibiofilm activity by releasing PDA; however, a toxic effect on bacterial cells was observed, which could potentially favor the emergence of resistant strains. To develop a PDA-functionalized material for medical use and overcome the problem of toxicity, we produced PDA-doped PDMS by either spray-coating or PDA incorporation during PDMS polymerization. Furthermore, we created a strategy to assess the kinetics of PDA release using ADIFAB, a very sensitive free fatty acids fluorescent probe. Spray-coating resulted in the most promising strategy as the concentration of released PDA was in the range 0.8-1.5 μM over 21 days, ensuring long-term effectiveness of the antibiofilm molecule. Moreover, the new coated material resulted biocompatible when tested on immortalized human keratinocytes. Our results indicate that PDA spray-coated PDMS is a promising material for the production of medical devices endowed with antibiofilm activity.

Keywords: ABIFAB; PDMS; S. epidermidis; antibiofilm; pentadecanoic acid; surface coating.

Figure 1

Effect of PDA on PDMS hydrophobicity. Pictures of water droplets and water contact angle measurements of uncoated PDMS, PDA-incorporated PDMS, and PDMS spray-coated with PDA. Each data represents the mean ± SD of three independent measurements.

Figure 2

Evaluation of biofilm formed in convertible flow cells on PDMS surfaces. (A) CLSM analysis of S. epidermidis RP62A biofilms formed on uncoated PDMS (NC), PDA-incorporated PDMS and PDA spray-coated PDMS. Bi-dimensional and three-dimensional biofilm structures were obtained using the LIVE/DEAD^® Biofilm Viability Kit. (B) COMSTAT quantitative analysis of biomass (µm³/µm²), average thickness (µm), and roughness coefficient (Ra*) of biofilms formed on uncoated PDMS (NC), PDA-incorporated PDMS and PDMS spray-coated with PDA. Differences in mean values were compared to the uncoated PDMS (NC) values and considered significant when p < 0.05 (** p < 0.01, *** p < 0.001) according to the Student’s t-test.

Figure 3

Analysis of PDA release from PDA spray-coated PDMS using the fluorescent probe ADIFAB. (A) ADIFAB fluorescence emission spectra of PDA spray-coated PDMS in agitation over time: ADIFAB in fresh PBS, time = 0 (black line), in PBS buffer after 1 h (red line), 3 h (green line), 8 h (yellow line), 24 h (blue line) and 48 h (pink line). (B) PDA concentration in the PBS samples. (C) Average rates of PDA release.

Figure 4

Long-term efficacy of the PDA spray-coated PDMS. (A) CLSM analysis of S. epidermidis RP62A biofilms formed in convertible flow cells on uncoated PDMS (NC) (left panel), PDA spray-coated PDMS freshly coated with pentadecanoic acid (central panel) and PDA spray-coated PDMS exposed to a constant PBS buffer flow for 21 days (right panel). Three-dimensional biofilm structures were obtained using the LIVE/DEAD^® Biofilm Viability Kit. (B) COMSTAT quantitative analysis of biomass (µm³/µm²), average thickness (µm), and roughness coefficient (Ra*) of biofilm on uncoated PDMS (NC), PDA spray-coated PDMS freshly coated with PDA and PDA spray-coated PDMS exposed to a constant PBS buffer flow for 21 days. Differences in mean values were compared to the uncoated PDMS (NC) values and considered significant when p < 0.05 (** p < 0.01) according to the Student’s t-test.

Figure 5

Release capability and biocompatibility of PDA spray-coated PDMS. (A) Rate of PDA release over time. The instantaneous rate of PDA release at 1, 3, 6, 24 h, 7 days, and 21 days of cell-flow experiments is reported. (B) Effect of PDMS on cell viability. Immortalized human keratinocytes were incubated in the presence of uncoated PDMS (dark grey bar), and PDA spray-coated PDMS for 24 or 48 h (light grey and white bars, respectively). Cell survival was expressed as the percentage of viable cells in the presence of different PDMS-based materials under test compared with control cells (i.e., untreated cells, black bar). Values are given as means ± SD (n ≥ 3). Differences in mean values were compared to the uncoated PDMS (NC) values and considered significant when p < 0.05 according to the Student’s t-test. The data were not significantly different compared to the control.