Nanochitin-nanolignin complexes to deliver bioactive molecules for skin regeneration

Introduction Chitin and lignin, by-products of fishery and plant biomass, can be reused and converted to high value materials for biomedical and cosmetic applications, which are bio- and eco-compatible. On the nanoscale, chitin and lignin crystalline structure and purity can be controlled, resulting inhigh antibacterial, anti-inflammatory, cicatrizing and anti-aging effectiveness (1).Combining electropositive chitin nanofibrlis (CN) and electronegative nanolignin (NL) gives rise to microcapsules able to entrap both hydrophilic and lipophilic molecules. The aim of this study was to test the efficacy CN-NL complexes in vitro with human keratinocytes (HaCat cells) and human mesenchymal stromal cells (hMSCs) for the delivery of glycyrrhetinic acid (GA), as a biomolecule with antibacterial and anti-inflammatory activity. Materials and Methods HaCat cells were cultured in presence of CNs, NL, CN-NL, 0.2% GA and CN-NL/0.2%GA and MTT assay was performed at 48 h to define the optimal concentration of each component. Therefore, the anti-inflammatory and immune responses of HaCat cells were evaluated by assaying the expression of pro-inflammatory cytokines IL-1 alfa, IL-1 beta, IL-6, IL-8 and TNF-alfa, anti-inflammatory cytokine TGF-beta, and antimicrobial peptide human beta defensin-2 (HBD-2) by RT-PCR at 6 h and 24 h. HMSCs isolated from the bone marrow were cultured with the abovementioned components for 1 week. AlamarBlue was used to monitor hMSC viability at higher and lower concentrations. After 1 week, hMSCs were osteoinduced for 1 week and mineralization was detected by von Kossa staining. Results and Discussion By using HaCat cells, non-toxic concentrations were identified for each component. Interestingly, the presence of GA, both alone and within the CN-NL complexes, increased 1.5 time the usable concentration. Our data indicated that GA markedly down-regulated the expression of proinflammatory cytokines IL-1 alfa, IL-1 beta, IL-6, IL-8 and TNF-alfa and up-regulated the expression of HBD-2 in HaCat cells.HMSCs were viable, with no appreciable differences using 2× concentration of CNs, NL, CN-NL, GA and CN-NL/GA. Their use did not modify osteo-differentiation capability of hMSCs. Conclusion These findings demonstrate that CN-NL/GA complexes are cytocompatible and have an anti- inflammatory activity on human keratinocytes.