Chitin Nanofibrils and Nanolignin as Functional Agents in Skin Regeneration

Abstract

Chitin and lignin, by-products of fishery and plant biomass, can be converted to innovative high value bio- and eco-compatible materials. On the nanoscale, high antibacterial, anti-inflammatory, cicatrizing and anti-aging activity is obtained by controlling their crystalline structure and purity. Moreover, electropositive chitin nanofibrlis (CN) can be combined with electronegative nanolignin (NL) leading to microcapsule-like systems suitable for entrapping both hydrophilic and lipophilic molecules. The aim of this study was to provide morphological, physico-chemical, thermogravimetric and biological characterization of CN, NL, and CN-NL complexes, which were also loaded with glycyrrhetinic acid (GA) as a model of a bioactive molecule. CN-NL and CN-NL/GA were thermally stable up to 114 °C and 127 °C, respectively. The compounds were administered to in vitro cultures of human keratinocytes (HaCaT cells) and human mesenchymal stromal cells (hMSCs) for potential use in skin contact applications. Cell viability, cytokine expression and effects on hMSC multipotency were studied. For each component, CN, NL, CN-NL and CN-NL/GA, non-toxic concentrations towards HaCaT cells were identified. In the keratinocyte model, the proinflammatory cytokines IL-1α, IL-1 β, IL-6, IL-8 and TNF-α that resulted were downregulated, whereas the antimicrobial peptide human β defensin-2 was upregulated by CN-LN. The hMSCs were viable, and the use of these complexes did not modify the osteo-differentiation capability of these cells. The obtained findings demonstrate that these biocomponents are cytocompatible, show anti-inflammatory activity and may serve for the delivery of biomolecules for skin care and regeneration.

Keywords: anti-inflammatory; bio-based; chitin; cosmetics; glycyrrhetinic acid; immunomodulation; keratinocytes; lignin; mesenchymal stem cells; nanomaterials; skin regeneration.

Figure 1

FE-SEM micrographs of the powders: (A) Spray dried CN; (B) Spray dried CN-NL complex; (C) Morphology of NL powder taken from the pristine NL sample; (D) Zoomed-in magnification of NL (16,000×) in back scattered modality to observe the NL nanostructure.

Figure 2

FE-SEM micrographs of (A) pure CN from deposition from diluted water suspension; (B) CN-NL complex; (C) and (D) CN-NL complex at higher magnification.

Figure 3

Infrared ATR spectra of (A): CN; (B) NL; (C) GA.

Figure 4

Infrared ATR spectra of (A) CN-NL and CN-NL/GA powders; (B) CN-NL, CN-LN/GA and GA in the region 2400-4000 cm⁻¹; (C) CN-NL, CN-LN/GA and GA in the region 1100-2000 cm^-1.

Figure 5

Thermogravimetric trends related to: (A) GA, NL and CN; (B) CN-NL and CN-NL/GA.

Figure 6

Results of MTT assay for different concentrations of CN, CN-NL, CN-NL/GA (0.2%), using HaCaT cells. The results were normalized by the viability of untreated cells as control.

Figure 7

Micrographs at the inverted optical microscope of hMSCs in culture after 24 h of treatment with: (A) CN at 10 µg/mL; (B) NL at 10 µg/mL; (C) CN-NL at 0.2 µg/mL; (D) CN-NL/GA at 0.5 µg/mL; (E) GA at 0.5 µg/mL; (F) control. Micrographs are representative of each compound administrated at the intermediate concentration in the tested ranges. Original magnification 100×, scale bar = 100 µm.

Figure 8

Bar graphs showing metabolic activity, as obtained by alamarBlue® test, performed on undifferentiated hMSCS using CN-NL, GA and CN-LN/GA complexes: (A–C) at different time points (1, 4 and 8 days in culture) and selected concentrations for each compound; (D) on day 8, comparing the three compounds within homogeneous concentration groups (* p < 0.01, ** p < 0.001, *** p < 0.0001).

Figure 9

Light micrographs of von Kossa staining performed on (A) undifferentiated (control) and (B) osteodifferentiated hMSCs after incubation with all the nanocomponents (CN, NL, GA, CN-NL, CN-NL/GA) for 15 days at the highest concentrations in the tested ranges. Original magnification 200×; scale bar = 100 µm.

Figure 10

Bar graphs showing the results of RT-PCR performed on HaCaT cells exposed to the biopolymer nanomaterials al 6 h and 24 h for different cytokines involved in inflammatory response and HBD-2 and an antimicrobial peptide. The results are normalized by the expression in untreated cells as control.