Introduction:: Bacterial-derived cellulose (BC) has been studied as a promising material for wound dressing applications due to its biocompatibility, water holding capacity, liquid/gas permeability, and handleability properties [1]. Although BC has been studied as a dressing material for cutaneous wounds, to date, antibacterial properties have not been effectively incorporated into the cellulose material [2]. Tethered antimicrobial peptides (AMP’s) may overcome the limitations associated with conventional antibiotics and antiseptic agents, such as bacterial resistance. Bifunctional synthetically synthesized peptides can be created using carbohydrate-binding peptides (CBP) to immobilize AMPs (e.g., KR12) to the surface of BC to aid in the prevention of wound infection while also altering the inflammatory environment.
Materials and Methods:: CBP-KR12 peptide was designed and synthesized to functionalize the BC surface with antimicrobial capabilities. The following synthetic peptide was purchased: KR-12 (KRIVQRIKDFLR) conjugated to a short CBP peptide sequence (WHWTYYW) via a flexible linker (GSGSGGS). Fluorescein (5,6-FAM) was added for spectrophotometric detection and quantification.
The BC binding capabilities of the short CBP-KR12 were assessed. Peptide solutions were prepared at 20 µM in tris-buffered saline (TBS). BC samples were placed in a 96-well plate and incubated with 200 µL of short CBP-KR12 on an orbital shaker for 48 h. Peptide binding was quantified through fluorescence analysis of the incubation supernatant at 495 nm supernatant. Reduction in supernatant fluorescence compared to peptide control wells (with no BC added) correlates directly to peptide binding to BC.
For viability analysis, HaCaT cells were seeded and incubated for 24 h. Functionalized and unfunctionalized BC were then placed in cell seeded wells. Cells were also cultured on tissue culture plastic (TCP) with no BC added as a positive control. Three days after adding BC samples, a resazurin metabolic assay was performed to quantify cell metabolism.
Surface antibacterial activity was assessed against Escherichia coli. Functionalized and unfunctionalized BC was incubated in 200 µL of E. coli at 1X108 CFU/mL for 1 h in Mueller Hinton broth (MHB). Samples were rinsed in sterile MHB to remove unattached bacteria and incubated with 200 µL fresh MHB at 37 ºC for 12 h. Bacterial regrowth was determined through comparing OD600 readings on media incubated with functionalized and unfunctionalized BC.
Results, Conclusions, and Discussions:: Spectrophotometric analysis of short CBP-KR12’s binding potential indicated ~ 40% binding efficiency to BC samples (Figure 1A), indicating that the short CBP retains its binding potential regardless of the addition of KR12. This was further supported by the increase in BC surface fluorescence (Figure 1B), which was observed to be stable over a 7-day period (data not shown). Surface functionalized BC and untreated BC exhibited no statistical difference in cellular viability compared to the TCP controls (Figure 1C). Tethering the peptide to the BC surface may reduce the overall cytotoxicity of the peptide as untethered short CBP-KR12 exhibits high cytotoxicity at 5-10 µM (data not shown). The AMP tethered to the BC surface retains its anti-bacterial activity as indicated by the bacterial regrowth assay. BC functionalized with short CBP-KR12 exhibited statistically significant reduction in E. coli growth of 25% as compared to unfunctionalized BC (Figure 1D).
Overall, synthetically synthesized short CBP-KR12 retains the cellulose binding capabilities of the short CBP, and the antibacterial capabilities of KR12. Tethering the AMP provides a promising method for creating an antibacterial functionalized BC surface with reduced mammalian cell cytotoxicity. Ongoing research includes assessing the retained anti-inflammatory activity of tethered KR12 through macrophage activation analysis and endotoxin binding activity potential.
Acknowledgements (Optional): : This work was partially funded by an NSF CAREER Award to JMC.
References (Optional): : [1] Ullah H. Cellulose, 2016, 23, 2291-2314. [2] Zeng R. Cell Tissue Res, 2018, 374, 2017-232.