Multifunctional 3D-Printed Wound Dressings Containing a Combination of Synergistic Antimicrobials in the Management of MRSA Infected Topical Wounds

Abstract

Despite increased pre- and postoperative care and aseptic practices in surgical rooms, methicillin-resistant Staphylococcus aureus (MRSA) continues to colonize acute surgical wounds. MRSA is also present in chronic nonhealing wounds, such as diabetic foot and pressure ulcers. In this work, advanced antimicrobial-loaded wound dressings are 3D printed using fused deposition modeling. To achieve a high antimicrobial effect, the topical antiseptic octenidine (OCT) was incorporated into the pellets used in the feeder of the extruder prior to fused modeling. Lysostaphin (LYS), a lytic enzyme that cleaves MRSA peptidoglycan, was incorporated by supramolecular interactions on the surface of the OCT-loaded dressings to exploit the anti-MRSA synergy identified here between OCT and LYS showing a fractional inhibition concentration index (FICI) of 0.156. Minimum inhibitory concentration (MIC) and bactericidal concentration (MBC) values for the OCT were 1 and 25 μg/mL, respectively, whereas the MIC and MBC values for the LYS were 0.1 and 0.2 μg/mL, respectively. The resulting dressings completely eradicate MRSA USA 300 inocula (10⁵ CFU/mL) in 96 h. The bactericidal mechanisms exerted by these dressings were identified through molecular techniques, showing lytic effects on the cell wall peptidoglycans of treated bacteria. Additionally, OCT at 1 μg/mL was able to reduce lipopolysaccharide (100 ng/mL)-induced NO production on murine J774A.1 macrophages by more than 90% demonstrating its simultaneous anti-inflammatory action. This effect was also corroborated by the qRT-PCR analysis of several pro-inflammatory genes including IL-1β, IL-6, TNF-α, and Nos2. The combination of OCT and LYS within the dressings reveals higher in vivo therapeutic effects compared to free compounds or individual antimicrobial-loaded dressings. In vitro and in preclinical models, the use of OCT-LYS dressings effectively reduces MRSA bioburden and inflammation, promoting fast wound healing.

Keywords: 3D printing; MRSA; antimicrobial therapies; wound dressings; wound healing.

1

Analysis of antimicrobial activity and thermal stability of selected antimicrobials. (A) Antimicrobial activity assay against MRSA before and after heat treatment at 220 °C for 5 min. Results are presented as the mean ± standard deviation (SD) from three independent experiments (n = 3). (B) TGA (thermogravimetric analysis) thermograms of the antimicrobials in air.

2

(A) Images of 3D printed dressings. (B) FTIR (Fourier transform infrared spectroscopy) analysis of printed dressings and those of the free antimicrobial compounds. (C) OCT release profile in different media under sink conditions; data are presented as the mean ± SD (n = 5). (D) Contact angle data and images of water droplets on the printed materials over time (1 min). (E) Mechanical testing stress–strain graph on the 3D printed materials and their corresponding Young’s moduli.

3

Evaluation of antimicrobial activity and synergism of PLA and PLA:PEO-based dressings against MRSA. (A) Antimicrobial activity of PLA-OCT (left) and PLA:PEO-OCT (right) printed dressings (concentrations presented as total dressing weight per volume adjusted to contain all the same OCT content). (B) Durability test of the PLA:PEO-OCT dressings. A fresh MRSA inoculum was added at days 1, 3, and 5 on the same single dressing. (C) Fractional inhibitory concentration index (FICI) analysis to show the synergism identified between the OCT and LYS against MRSA. (D) Antimicrobial assay of 8 mm in diameter dressings coated with different LYS concentrations (concentrations presented as PLGA nanoparticle suspensions at 5000 and 2500 μg/mL (which corresponds with a LYS loading of 0.8 ppm and 0.4 ppm, respectively). (E) Evaluation of the LYS MBC and MIC against MRSA. (F) SEM images of dressings with particles. SEM images, first row: PLA:PEO dressings were immersed in various LYS-loaded PLGA particle concentrations (from 1250 to 5000 μg/mL). Second row: PLA:PEO-OCT dressings immersed in various LYS-loaded PLGA particle concentrations (from 1250 to 5000 μg/mL). Bar graphs show the mean ± SD (n = 3 independent experiments).

4

Evaluation of MRSA membrane depolarization and DNA degradation. (A) Cell depolarization assay. (B) Qubit assay for the evaluation of DNA degradation (ssDNA). (C) Agarose gel analysis of released DNA. Statistical significance between groups is indicated by horizontal bars, with p-values as follows: ns (nonsignificant), p < 0.05 (*), p < 0.01 (**), p < 0.001 (***), p < 0.0001 (****). Bar graphs show the mean ± SD (n = 3 independent experiments).

5

Cell viability and inflammatory response in various cell types exposed to treatments. (A) Cell viability in keratinocytes, fibroblasts, and macrophages exposed to the OCT (left) and LYS (right). (B) Cell viability in fibroblasts (left) and keratinocytes (right) when exposed to PLA-OCT and PLA:PEO-OCT dressings exudates released after 24 h. (C) Nitric oxide (NO) production after inflammatory response of LPS-induced macrophages to OCT. Statistical significance between control and all treated groups is indicated by a horizontal bar, with the p-value as follows: p < 0.0001 (****). (D) qPCR quantification of pro-inflammatory genes expression after OCT treatment (p < 0.0001 (****)). (E) Cell viability in fibroblasts exposed to PLA:PEO + 0.8 ppm LYS and PLA:PEO-OCT + LYS dressings (25 and 0.8 ppm, respectively). (F) Cell viability in keratinocytes exposed to PLA:PEO + 0.8 ppm LYS and PLA:PEO-OCT + LYS dressings (25 ppm and 0.8 ppm, respectively). (G) Cell viability in macrophages exposed to PLA:PEO + LYS 0.8 ppm and PLA:PEO-OCT + LYS dressings (25 ppm and 0.8 ppm, respectively). Bar graphs show mean ± SD (n = 3 independent experiments).

6

Semiquantitative bacterial counts and analysis from infected wounds in treated animals. (A) Diagram of the experimental procedure followed in the wound infection model used. (B) Images showing the progression of wounds in the different experimental groups. Bacterial growth was classified semiquantitatively using the streak plate method as follows: (−) no growth, (+) minor, (++) moderate, (+++) extensive, and (++++) massive growth. (C) Summary and analysis of the bacterial load obtained by the streak plate method. Data are presented as the mean ± SD (n = 8). Statistical significance between groups is indicated by horizontal bars, with p-values as follows: p < 0.05 (*), p < 0.01 (**), p < 0.001 (***), p < 0.0001 (****).

7

Histopathological evaluation of the experimental groups at 7 days after surgical intervention (PSI). Upper row: hematoxylin–eosin staining (all images taken at ×4 magnification). Lower row: Gram staining (mostly at ×60 magnification except for PLA:PEO-OCT and PLA:PEO-OCT + LYS, which were taken at ×10 magnification).