Polycaprolactone Nanofibers Functionalized by Fibronectin/Gentamicin and Implanted Silver for Enhanced Antibacterial Properties, Cell Adhesion, and Proliferation

Abstract

Novel nanomaterials used for wound healing should have many beneficial properties, including high biological and antibacterial activity. Immobilization of proteins can stimulate cell migration and viability, and implanted Ag ions provide an antimicrobial effect. However, the ion implantation method, often used to introduce a bactericidal element into the surface, can lead to the degradation of vital proteins. To analyze the surface structure of nanofibers coated with a layer of plasma COOH polymer, fibronectin/gentamicin, and implanted with Ag ions, a new X-ray photoelectron spectroscopy (XPS) fitting method is used for the first time, allowing for a quantitative assessment of surface biomolecules. The results demonstrated noticeable changes in the composition of fibronectin- and gentamicin-modified nanofibers upon the introduction of Ag ions. Approximately 60% of the surface chemistry has changed, mainly due to an increase in hydrocarbon content and the introduction of up to 0.3 at.% Ag. Despite the significant degradation of fibronectin molecules, the biological activity of Ag-implanted nanofibers remained high, which is explained by the positive effect of Ag ions inducing the generation of reactive oxygen species. The PCL nanofibers with immobilized gentamicin and implanted silver ions exhibited very significant antipathogen activity to a wide range of Gram-positive and Gram-negative strains. Thus, the results of this work not only make a significant contribution to the development of new hybrid fiber materials for wound dressings but also demonstrate the capabilities of a new XPS fitting methodology for quantitative analysis of surface-related proteins and antibiotics.

Keywords: Immobilization; PCL nanofibers; XPS; fibronectin; plasma; silver; surface functionalization.

Figure 1

Schematics of nanofiber fabrication and their subsequent modification (a) and chemical structure of polymer and immobilized compounds (b).

Figure 2

SEM micrographs of PCL-ref (a), PCL-COOH (b), PCL-COOH-FBn (c), and PCL-COOH-FBn-Ag2.5 (d) samples.

Figure 3

XPS C1 spectra of PCL-ref (a), PCL-COOH (b), PCL-COOH-FBn (c), PCL-COOH-GM (d), PCL-COOH-FBn-Ag2.5 (e), and PCL-COOH-GM-Ag2.5 (f) materials.

Figure 4

XPS N1 spectra of PCL-COOH (a), PCL-COOH-FBn (b), PCL-COOH-GM (c), PCL-COOH-FBn-Ag2.5 (d), and PCL-COOH-GM-Ag2.5 (e) materials.

Figure 5

FTIR spectra of samples PCL-ref, PCL-COOH, PCL-COOH-FBN, PCL-COOH-FBN-Ag2.5, and PCL-COOH-FBN-Ag5. The insets show enlarged regions of the spectra, highlighted by dashed squares.

Figure 6

Effect of fibronectin and Ag ion implantation on cell colonization, cell morphology, and formation of mature cytoskeleton. (A). The number of cells on the surface after 24 h of cultivation was determined by counting cell nuclei stained with the DNA intercalating dye Hoechst 33342. (B). Representative fluorescence photographs of cells cultured on the studied nanofibers. Actin fibers of the cytoskeleton were stained with Phalloidin 488. (C). Average areas of fibroblast cells measured by phalloidin staining of cellular actin filaments. * p ≤ 0.1.

Figure 6

Effect of fibronectin and Ag ion implantation on cell colonization, cell morphology, and formation of mature cytoskeleton. (A). The number of cells on the surface after 24 h of cultivation was determined by counting cell nuclei stained with the DNA intercalating dye Hoechst 33342. (B). Representative fluorescence photographs of cells cultured on the studied nanofibers. Actin fibers of the cytoskeleton were stained with Phalloidin 488. (C). Average areas of fibroblast cells measured by phalloidin staining of cellular actin filaments. * p ≤ 0.1.