(A-22) Improved antibacterial activity on surfaces containing amphoteric aminated condensed tannin (tanfloc)-based polyelectrolyte multilayers

Introduction:: The adhesion and proliferation of bacteria on biomedical device surfaces can cause device failure. Bacterial biofilms can also form, which protect the bacteria from antimicrobial agents, making them difficult to eradicate. Surface chemistry and topography of scaffolds can impact bacterial activity. The layer-by-layer (LbL) assembly of polyelectrolyte multilayers (PEMs) is a promising technique for preparing antimicrobial surfaces. In this study, Tanfloc (TAN), an amphoteric and aminated condensed tannin biopolymer, was used to develop PEMs with antimicrobial properties. TAN has been shown to be cytocompatible and exhibit antibacterial characteristics [1,2]. TAN can function as either a polyanion or a polycation depending on the pH, making it a versatile candidate for use in PEMs. The goal of this research was to develop and analyze bioinspired surfaces that possess antibacterial properties through the incorporation of physical and chemical features PEMs including TAN paired with other biopolymers. To demonstrate its flexibility for the preparation of PEMs, TAN was paired with various polyanions and polycations, including heparin, hyaluronan, chondroitin sulfate, polyethylene imine, and trimethyl chitosan (TMC). Both positively and negatively charged terminated layers were prepared, taking into account the surface properties of PEMs. The antimicrobial activity of the resulting PEMs was evaluated against both Gram-positive and Gram-negative bacteria.


Materials and Methods:: TMC was synthesized to be used as a polycation for the LbL fabrication of PEMs. The PEM deposition was accomplished by alternately exposing the surfaces to solutions in the following sequence: polycation, rinse, polyanion, rinse. Each polyelectrolyte deposition and rinse step were performed for 5 minutes under shaking. Polyanion-terminated and polycation-terminated PEMs were constructed using 12 and 13 layers, respectively, and the deposition process was monitored through Fourier-transform surface plasmon resonance.

Surface chemistry and topography of the PEMs were evaluated using X-ray photoelectron spectroscopy (XPS) and atomic force microscopy (AFM), respectively. The antibacterial activity of the surfaces was assessed against Gram-negative Pseudomonas aeruginosa and Gram-positive Staphylococcus aureus cultures in nutrient media over time. After 6 and 24 hours on the samples, bacteria adhesion and morphology/biofilm formation were examined using fluorescence microscopy and scanning electron microscopy (SEM) images. The experimental procedures were performed in triplicate.

Results, Conclusions, and Discussions:: The results from LBL assembly of the polyelectrolyte multilayer PEM consisting of 12 layers are shown in Figure 1. Combining TAN with various biopolymers, either as polyanion or polycation, was found to produce a stable polyelectrolyte multilayer film on the surface, as demonstrated in this study. The surface chemistry of the PEMs was analyzed using both survey and high-resolution XPS spectra (Figure 2). Figure 3 displays three-dimensional topography images of the assembled PEM surfaces, along with the root-mean-square surface roughness (Rq). One of the major obstacles in implementing biomedical devices is the occurrence of bacterial infections. In order to investigate the antibacterial activity of PEMs containing TAN as both polyanion and polycation, TAN-HEP and TAN-TMC in different terminated layers were selected. Since the mechanisms to combat Gram-positive and Gram-negative bacteria due to differences in their cell walls, it is important to investigate how the biomaterial surface interacts with both. The fluorescence microscopy images of live and dead S. aureus and P. aeruginosa are displayed in Figure 4 and Figure 5, respectively. TAN-HEP and TAN-TMC significantly reduce the adhesion of live S. aureus bacteria after 24 hours compared to the control (glass). After 24 hours, a significant portion of the bacteria attached to the PEM-coated surface are dead. TAN-HEP PEMs significantly decrease the adhesion of live P. aeruginosa bacteria after 24 hours compared to the control (polystyrene and glass) and they have almost no bacteria attached. These PEMs also demonstrate better antiadhesive properties than TAN-TMC PEMs. All surfaces dramatically reduce the bacteria adhesion surfaces compared to controls. The amount of dead bacteria adhered significantly increased on TAN-TMC with negatively charged terminated layers compared to control.

Conclusions: In this study, the LbL assembly approach is used to create PEMs from tanfloc as polycation and polyanion that have optimal surface coverage. The chemical and topographical properties demonstrate how highly desired this coating is for modifying biomedical devices. The antimicrobial studies indicate that PEMs possess bactericidal properties against both P. aeruginosa and S. aureus, suggesting their potential as effective agents in preventing bacterial infections.


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References (Optional): : [1] R.M. Sabino, G. Mondini, M.J. Kipper, A.F. Martins, K.C. Popat, Tanfloc/heparin polyelectrolyte multilayers improve osteogenic differentiation of adipose-derived stem cells on titania nanotube surfaces, Carbohydr. Polym. 251 (2021) 117079. https://doi.org/10.1016/j.carbpol.2020.117079.

[2] R.M. Sabino, K. Kauk, L.Y.C. Madruga, M.J. Kipper, A.F. Martins, K.C. Popat, Enhanced hemocompatibility and antibacterial activity on titania nanotubes with tanfloc/heparin polyelectrolyte multilayers, J. Biomed. Mater. Res. - Part A. 108 (2020) 992–1005. https://doi.org/10.1002/jbm.a.36876.