Halochromic Bacterial Cellulose/Anthocyanins Hybrid Polymer Film with Wound-Healing Potential

Abstract

Polymer-based dressings deriving from natural biomaterials have advantages such as nontoxicity, biocompatibility, and mechanical stability, which are essential for efficient wound healing and microbial infection diagnostics. Here, we designed a prototype of an intelligent hydrogel dressing on the base of bacterial cellulose (BC) for monitoring wound microbial infection due to the uploaded natural pH dye-sensor, anthocyanins (ANC) of elderberry fruit (Sambucus nigra L.). The highest sensor responses to bacterial metabolites for ANC immobilized to BC were observed at pH 5.0 and 6.0. The detection limit of the sensor signals was 3.45 A.U., as it was evaluated with a smartphone-installed application. The FTIR spectral analysis of the hybrid BC/ANC hydrogel films has proved the presence of anthocyanins within the BC matrix. Hybrid films differed from the control ones by thicker microfibrils and larger pores, as detected with scanning electron microscopy. Halochromic BC/ANC films exhibited antimicrobial activities mainly against gram-positive bacteria and yeast. They showed no cytotoxicity for the in vitro human cell lines and mouse fibroblasts within a selected range of anthocyanin concentrations released from the BC/ANC film/dressing prototype. Compared to the control, the in vitro healing test showed overgrowth of primary mouse fibroblasts after applying 0.024-2.4 µg/mL ANC.

Keywords: anthocyanins; bacterial cellulose; hybrid polymer; microbial infection; natural pH-sensor; wound healing.

Figure 1

A pH-dependent color line of the elderberry fruit extract (pH 4–12) (A), bacterial cellulose (BC) hydrogels filled in the anthocyanin extract (AE) at pH from 4.0 to 12.0 (B), the anthocyanin UV-VIS spectra at pH 4–12 in the range λ 400–700 nm (C), dynamics of the cumulative liquid release through the BC/AE and BC (control) films into a model leather (D); the absorbance of anthocyanin extract by a hydrogel film (E), and the anthocyanin extract release from the hydrogel into a distilled water (F).

Figure 2

Smartphone-based sensor system based on bacterial cellulose (BC) hydrogel membranes and natural anthocyanins of black elderberry (Sambucus nigra L.) design. (A,B) changes in the color of the BC hydrogel disks with natural anthocyanins immobilized in their structure at the time of incubation on the Bacillus subtilis (A) and Pseudomonas aeruginosa (B) lawns and on pH of the 100 mM phosphate-citrate buffer used in immobilization procedure. Incubation time: 0 min (a), 20 min (b), 40 min (c), 60 min (d), 120 min (e), 180 min (f). (C,D) dependence of the value of the sensor responses (intensities of red (a) and navy blue (b) staining) of the smartphone sensor based on BC hydrogel membranes with the immobilized natural anthocyanins on the time of incubation on the lawn of B. subtilis (C,D) and P. aeruginosa (E,F). The 100 mM phosphate-citrate buffer solution pH 5.0 was used in the immobilization procedure. (G)—the dependence of the value of differential sensor responses (dark violet color intensity) of the BC hydrogel-based sensor membranes with natural anthocyanins on pH of the 100 mM phosphate-citrate buffer used in the immobilization procedure. The sensor membranes were incubated on lawns of B. subtilis (grey bars) and P. aeruginosa (white bars) for 30 min (G), the differential sensor response was calculated as the difference in the intensity of blue staining of the sensor BC hydrogel-based membranes before the contact with bacteria and after 30 min incubation on the bacterial lawns.

Figure 3

Antimicrobial activity of hydrogels with the elderberry anthocyanin extract (AE) and undecylenic acid (UA). (A) Staphylococcus aureus; (B) Bacillus subtilis; (C) Candida albicans; (D) Escherichia coli lawns, where the cellulose-based hydrogel discs impregnated with AE (on the left) and +UA added (on the right) placed and incubated 16 h. As controls, pure hydrogel (on the top) and hydrogel saturated with UA without AE (on the bottom).

Figure 4

Characterization of bacterial cellulose (BC) films loaded with anthocyanin extract (AE) with Fourier-transform infrared spectroscopy (FTIR) spectral analysis (A) and scanning microscopy analysis (B). (Aa), the FTIR spectra of BC/AE. (A(b,c)), spectra fragments demonstrating AE’s most pronounced influence on BC. 1, 2, 3—spectra for AE, BC, BC/AE, accordingly. (Ba), an SEM image of the purified BC’s nanostructure; (Bb), an SEM micrograph of the BC loaded with AE. Scale bar: 1 μm. The mean width for BC microfibrils is 113 ± 28 nm; the mean width for BC/AE microfibrils is 172 ± 58 nm. Mean values are significantly different (p < 0.05).

Figure 5

Effects of elderberry anthocyanin extract on the viability of mesenchymal stem cells from the umbilical cord (A), primary mouse fibroblast cells primary mouse fibroblast cells primary mouse fibroblast cells (B), and human embryonic kidney cells (C). The MTT assay determined the cytotoxicity data, as mentioned in Materials and Methods. The error bars represent the standard deviation of the mean (SD). Statistically significant changes in treatment groups compared with control are indicated by asterisks (* p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001).

Figure 6

In vitro fibroblast migration assay results. (A) microscopic images showing a scratched area at the beginning of the experiment (0 h) and after 24 and 48 h of treatment; (B) scratched areas calculated between scratch edges before and after the treatment in different periods. The scratch and MTT assay determined were performed, as mentioned in Materials and Methods. The error bars represent the standard deviation of the mean (SD). Statistically significant changes in scratched areas compared with control are indicated by asterisks (* p < 0.05; *** p < 0.001; **** p < 0.0001).