Cytocompatibility, Antimicrobial and Antioxidant Activity of a Mucoadhesive Biopolymeric Hydrogel Embedding Selenium Nanoparticles Phytosynthesized by Sea Buckthorn Leaf Extract

Abstract

Phytosynthesized selenium nanoparticles (SeNPs) are less toxic than the inorganic salts of selenium and show high antioxidant and antibacterial activity. Chitosan prevents microbial biofilm formation and can also determine microbial biofilm dispersal. Never-dried bacterial nanocellulose (NDBNC) is an efficient carrier of bioactive compounds and a flexible nanofibrillar hydrophilic biopolymer. This study aimed to develop a selenium-enriched hydrogel nanoformulation (Se-HNF) based on NDBNC from kombucha fermentation and fungal chitosan with embedded biogenic SeNPs phytosynthesized by an aqueous extract of sea buckthorn leaves (SbLEx)-SeNPsSb-in order to both disperse gingival dysbiotic biofilm and prevent its development. We determined the total phenolic content and antioxidant activity of SbLEx. Liquid chromatography-mass spectrometry (LC-MS) and high-performance liquid chromatography (HPLC) were used for the identification of polyphenols from SbLEx. SeNPsSb were characterized by transmission electron microscopy-energy-dispersive X-ray spectroscopy (TEM-EDX), dynamic light scattering (DLS), zeta potential, Fourier transform infrared spectroscopy (FTIR) and X-ray diffraction (XRD) in small- and wide-angle X-ray scattering (SAXS and WAXS). The hydrogel nanoformulation with embedded SeNPsSb was characterized by SEM, FTIR, XRD, rheology, mucin binding efficiency, contact angle and interfacial tension measurements. We also assessed the in vitro biocompatibility, antioxidant activity and antimicrobial and antibiofilm potential of SeNPsSb and Se-HNF. TEM, DLS and SAXS evidenced polydisperse SeNPsSb, whereas FTIR highlighted a heterogeneous biocorona with various biocompounds. The contact angle on the polar surface was smaller (52.82 ± 1.23°) than that obtained on the non-polar surface (73.85 ± 0.39°). The interfacial tension was 97.6 ± 0.47 mN/m. The mucin binding efficiency of Se-HNF decreased as the amount of hydrogel decreased, and the SEM analysis showed a relatively compact structure upon mucin contact. FTIR and XRD analyses of Se-HNF evidenced an interaction between BNC and CS through characteristic peak shifting, and the rheological measurements highlighted a pseudoplastic behavior, 0.186 N adhesion force and 0.386 adhesion energy. The results showed a high degree of cytocompatibility and the significant antioxidant and antimicrobial efficiency of SeNPsSb and Se-HNF.

Keywords: bacterial nanocellulose; fungal chitosan; gingival dysbiotic biofilm; phytosynthesized selenium nanoparticles; sea buckthorn leaf extract.

Figure 1

ATR-FTIR spectra and XRD analyses of the selenium nanoparticles (SeNPsSb): (a,b) ATR-FTIR spectra of SeNPsSb compared with: (a) Na₂SeO₃, ascorbic acid and sea buckthorn leaves aqueous extract (SbLEx); (b) thiamine, catechin, BSA and palmitic acid; (c) XRD-WAXS of SeNPsSb compared with the extract SbLEx, Na₂SeO₃ and ascorbic acid; (d) XRD-SAXS of SeNPsSb presented as a convoluted signal between three different analyses, with SAXS1, SAXS2 and SAXS3 performed between the 2θ angles 0–0.8°, 0–1.2° and 0–1.5°, respectively; (e) particle size distribution of SeNPsSb determined by XRD-SAXS.

Figure 2

TEM-EDX analysis of the selenium nanoparticles (SeNPsSb): (a) TEM analysis; (b) EDX analysis.

Figure 3

Antioxidant activity of the selenium nanoparticles (SeNPsSb) at different concentrations (±error bars, α < 0.05, n = 3; different letters mean statistically significant differences between the samples); DPPH: 2,2-diphenyl-1-picrylhydrazyl, FRAP: ferric reducing antioxidant power, CUPRAC: cupric reducing antioxidant activity.

Figure 4

Cytocompatibility and in vitro antioxidant activity of SeNPsSb, HNF and Se-HNF: (a) CCK-8 assay for SeNPsSb; C− (untreated cells, negative cytotoxicity control), C+ (cells treated with 7.5% dimethyl sulfoxide (DMSO), positive cytotoxicity control); (b) quantifying total ROS after H₂DCFDA labeling of HGF-1 cells treated with SeNPsSb; C− (untreated cells, negative control), C+ (cells treated with 37 µM H₂O₂-ROS inducer, positive control); (c) CCK-8 assay for Se-HNF; C− (untreated cells, negative cytotoxicity control), C+ (cells treated with 7.5% dimethyl sulfoxide (DMSO), positive cytotoxicity control), HNF—5% water-soluble chitosan in 0.4% never-dried bacterial nanocellulose; 0.5 Se-HNF—HNF with 0.5 µg/mL SeNPsSb; 2.5 Se-HNF—HNF with 2.5 µg/mL SeNPsSb; (d) quantifying total ROS after H₂DCFDA labeling of HGF-1 cells treated with Se-HNF; C− (untreated cells, negative control), C+ (cells treated with 37 µM H₂O₂-ROS inducer, positive control), HNF—5% water-soluble chitosan in 0.4% never-dried bacterial nanocellulose; 0.5 Se-HNF—HNF with 0.5 µg/mL SeNPsSb; 2.5 Se-HNF—HNF with 2.5 µg/mL SeNPsSb (±error bars, α < 0.05, n = 3; different letters mean statistically significant differences between the samples).

Figure 5

Effect of SeNPsSb, HNF and Se-HNF treatment on the HGF-1 cell morphology: (a) untreated cells (negative cytotoxicity control); (b) cells treated with 7.5% DMSO (positive cytotoxicity control); (c) 2.5 µg/mL SeNPsSb; (d) 25 µg/mL HNF; (e) 1000 µg/mL HNF; (f) 25 µg/mL 2.5 Se-HNF; (g) 1000 µg/mL 2.5 Se-HNF; HNF—5% water-soluble chitosan in 0.4% never-dried bacterial nanocellulose; 2.5 Se-HNF—HNF with 2.5 µg/mL SeNPsSb (green fluorescence indicates the labeled actin cytoskeleton and blue fluorescence indicates the stained nuclei).

Figure 6

Antimicrobial activity of SeNPsSb at 24 h after treatment: (a) inhibition of B. cereus growth; (b) inhibition of E. faecalis growth; (c) inhibition of S. aureus growth; (d) inhibition of C. albicans growth (±error bars, α < 0.05, n = 3; different letters mean statistically significant differences between the samples).

Figure 7

Quantitative screening of the antibacterial activity of hydrogel formulations: (a) B. cereus growth inhibition 24 h after hydrogel treatment; (b) E. faecalis growth inhibition 24 h after hydrogel treatment; (c) S. aureus growth inhibition 24 h after hydrogel treatment; (d) C. albicans growth inhibition 24 h after hydrogel treatment; (±error bars, α < 0.05, n = 3; different letters mean statistically significant differences between the samples; each microbial density was analyzed separately for statistical significance investigation); HNF—5% water-soluble chitosan in 0.4% never-dried bacterial nanocellulose; 0.5 Se-HNF—HNF with 0.5 µg/mL SeNPsSb; 2.5 Se-HNF—HNF with 2.5 µg/mL SeNPsSb.

Figure 8

Mucin binding efficiency and SEM analysis of 2.5 Se-HNF: (a) periodic acid–Schiff (PAS) (±standard deviation, α < 0.5, n = 3, **—σ between 0.01 and 0.001); (b–d) SEM analyses of: (b) 2.5 Se-HNF; (c) mucin (Mu); (d) 2.5 Se-HNF-Mu; 2.5 Se-HNF—5% water-soluble chitosan in 0.4% never-dried bacterial nanocellulose enriched with 2.5 µg/mL SeNPsSb; Mu—3.5% aqueous mucin suspension; 2.5 Se-HNF-Mu—2.5 Se-HNF mixed with the 3.5% aqueous mucin suspension at the ratio 1:1 (v/v).

Figure 9

ATR-FTIR spectra and XRD structure analysis of the 2.5 Se-HNF hydrogel and its interaction with mucin: (a,b) ATR-FTIR spectra of 2.5 Se-HNF compared with: (a) chitosan (CS) and bacterial nanocellulose (BNC); (b) mucin (Mu) and 2.5 Se-HNF-Mu system; (c) 2.5 Se-HNF diffractogram compared with the diffractograms of bacterial nanocellulose (BNC) and chitosan (CS); (d) Se-HNF diffractogram compared with the diffractograms of mucin (Mu) and the Se-HNF-Mu system; 2.5 Se-HNF—5% water-soluble chitosan in 0.4% never-dried bacterial nanocellulose enriched with 2.5 µg/mL SeNPsSb; Mu—3.5% aqueous mucin suspension; 2.5 Se-HNF-Mu—2.5 Se-HNF mixed with the 3.5% aqueous mucin suspension at the ratio 1:1 (v/v).

Figure 10

Rheological properties determined: (a) in oscillatory mode with hysteresis for the hydrogel 2.5 Se-HNF; (b) in oscillatory mode with hysteresis for Mu; (c) in oscillatory mode with hysteresis for 2.5 Se-HNF-Mu; (d) in hysteresis shear mode for hydrogel 2.5 Se-HNF; (e) in hysteresis shear mode for Mu; (f) in hysteresis shear mode for hydrogel with mucin 2.5 Se-HNF-Mu; (g) in axial mode for the hydrogel 2.5 Se-HNF; (h) in axial mode for Mu; (i) in axial mode for hydrogel with mucin 2.5 Se-HNF-Mu; 2.5 Se-HNF—5% water-soluble chitosan in 0.4% never-dried bacterial nanocellulose enriched with 2.5 µg/mL SeNPsSb; Mu—3.5% aqueous mucin suspension; 2.5 Se-HNF-Mu—2.5 Se-HNF mixed with the 3.5% aqueous mucin suspension at the ratio 1:1 (v/v).