(A-11) Layer-by-Layer Functionalized Gauze for Synthetic Anti-Biofilm Peptide Releasing Wound Dressings

Introduction::
Microbial biofilms on wounds lead to longer hospital stays, painful mechanical debridement, and higher mortality. Biofilms are dense communities of surface associated cells that secrete an extracellular matrix (ECM) comprised of polysaccharides, DNA, and proteins. Some of these proteins form functional amyloid structures stabilizing the ECM; thus, amyloid fibrils are a potential anti-biofilm target. Before amyloid fibril formation, soluble protein monomers undergo a conformational change, causing aggregation into toxic, soluble oligomeric intermediates [1]. These oligomeric transient intermediates share a novel secondary backbone structure called an “α-sheet” [1]. Identification of these α-sheets has led to the development of synthetic peptides with a stable, monomeric α-sheet structure [2, 3]. These de novo α-sheet peptides inhibit amyloid formation and reduce biofilm growth in several bacterial species [4]. α-sheet inhibitor peptides are a novel approach to destabilize the biofilm ECM and prevent biofilm formation in wounds.




Alginate (ALG) and chitosan (CH) are widely used in wound dressings due to their adhesive and antimicrobial activity. We have developed a layer-by-layer (LbL) functionalized gauze with alternating layers of ALG and CH that is loaded with α-sheet peptides for controlled release at the wound site to prevent biofilm formation and increase antibiotic susceptibility.




Materials and Methods::
For the LbL gauze functionalization (Fig. 1A), a dip coating method was adapted from Gomes, et al [5]. Briefly, cotton gauze was negatively charged by immersion in a TEMPO solution for 30 minutes, followed by rinsing with DI water. The gauze was then alternately immersed in ALG and CH solutions for 5 minutes each. Between each polyelectrolyte exposure, the samples were rinsed with DI water. The charged gauze was functionalized with four layers (gauze/CH/ALG/CH/ALG) and then immersed in a solution of α-sheet peptides for 24 hours. Peptide absorption was measured over time with a Nanodrop™ instrument. Gauze substrates were then rinsed with DI water containing calcium for ALG gel formation. A final CH layer was added and the gauze was dried for 24 hours. Peptide release was measured in PBS using a standard Bradford protein assay. Gauze surface properties were characterized by attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy, SEM, and x-ray photoelectron spectroscopy (XPS). Antibacterial tests were conducted in vitro using a broth culture method where gauze samples were added to a 24 well plate with 1 mL of standardized bacteria (Escherichia coli, UTI89 strain). After 42 hours, antibiotics were added to some wells. After 48 hours, gauze samples were rinsed and added to PBS, sonicated to remove adherent bacteria, and biofilm formation was quantified by serial dilutions and colony forming units (CFU).




Results, Conclusions, and Discussions::
ATR-FTIR spectra (Fig. 1B) for functionalized gauze showed that the TEMPO solution created a charged surface (peak at 1,600 nm) and that the polyelectrolyte layers were successfully deposited on the cotton surface (peak changes at 3,300 nm). ATR-FTIR also showed successful peptide incorporation with characteristic peptide peaks (1,558 and 1,637 nm). SEM images of plain gauze and LbL functionalized gauze showed morphology and texture changes to the gauze indicating successful deposition of ALG and CH layers and stability after washing (Fig. 1C, D). Next, α-sheet peptide release kinetics from the LbL gauze showed a burst peptide release of 90 µg of peptide over the first 6 hours followed by sustained release up to 48 hours for a total release of 140 µg of peptide from a 1 cm² functionalized gauze (Fig. 1E). This represents ~70% of absorbed peptide release over 48 hours. For the in vitro broth culture assay, the LbL+peptide gauze showed an 81% and 96% reduction in CFUs as compared to the plain gauze for the non-antibiotic and antibiotic (+Gentamicin) conditions, respectively (p< 0.001). Comparison of the ratio of CFUs with antibiotics to the control plain gauze without antibiotics showed a statistically significant reduction for the CT5+peptide gauze (p=0.0004) (Fig. 1F).




A biocompatible, LbL functionalized gauze loaded with synthetic α-sheet peptides presents a novel way to combat wound biofilm formation. ATR-FTIR, XPS, and SEM analysis demonstrated successful LbL deposition on charged cotton gauze. Immersion in a calcium solution caused ALG gel formation containing the α-sheet peptides and facilitated sustained peptide release over 72 hours. Administration of peptides in solution caused a dose-dependent reduction in biofilm formation. A broth culture assay showed that LbL+peptide gauze significantly reduced biofilm formation on the gauze. Increased antibiotic susceptibility was also observed. Current work is focused on repeating the antibiotic tests with Staphylococcus aureus and performing cytocompatibility studies.




Acknowledgements (Optional): :
Research is supported in part by funding of the author by a TL1 fellowship from the Institution of Translational Health Sciences at the University of Washington.




References (Optional): :
[1] Daggett VD. Acc Chem Res. 2006; 39:594-602. [2] Hopping G. eLife. 2014; 3:e01681. [3] Kellock J. JMB. 2016; 428:2317-2328. [4] Bleem A. NPJ Biofilms Microbes. 2017.3:16. [5] Gomes AP. Carbohydr Polyms. 2015; 127:451-461.