Chitosan has been extensively studied as a genetic drug delivery platform. However, its efficiency is limited by the strength of DNA and RNA binding. Expecting a reduced binding strength of cargo with chitosan, we proposed including heparin as a competing polyanion in the polyplexes. We developed chitosan–heparin nanoparticles by a one-step process for the local delivery of oligonucleotides. The size of the polyplexes was dependent on the mass ratio of polycation to polyanion. The mechanism of oligonucleotide release was pH-dependent and associated with polyplex swelling and collapse of the polysaccharide network. Inclusion of heparin enhanced the oligonucleotide release from the chitosan-based polyplexes. Furthermore, heparin reduced the toxicity of polyplexes in the cultured cells. The cell uptake of chitosan–heparin polyplexes was equal to that of chitosan polyplexes, but heparin increased the transfection efficiency of the polyplexes two-fold. The application of chitosan–heparin small interfering RNA (siRNA) targeted to vascular endothelial growth factor (VEGF) silencing of ARPE-19 cells was 25% higher. Overall, chitosan–heparin polyplexes showed a significant improvement of gene release inside the cells, transfection, and gene silencing efficiency in vitro, suggesting that this fundamental strategy can further improve the transfection efficiency with application of non-viral vectors.

Preclinical studies frequently lack predictive value for human conditions. Human cell-based disease models that reflect patient heterogeneity may reduce the high failure rates of preclinical research. Herein, we investigated the impact of primary cell age and body region on skin homeostasis, epidermal differentiation, and drug uptake. Fibroblasts derived from the breast skin of female 20- to 30-year-olds or 60- to 70-year-olds and fibroblasts from juvenile foreskin (<10 years old) were compared in cell monolayers and in reconstructed human skin (RHS). RHS containing aged fibroblasts differed from its juvenile and adult counterparts, especially in terms of the dermal extracellular matrix composition and interleukin-6 levels. The site from which the fibroblasts were derived appeared to alter fibroblast-keratinocyte crosstalk by affecting, among other things, the levels of granulocyte-macrophage colony-stimulating factor. Consequently, the epidermal expression of filaggrin and e-cadherin was increased in RHS containing breast skin fibroblasts, as were lipid levels in the stratum corneum. In conclusion, the region of the body from which fibroblasts are derived appears to affect the epidermal differentiation of RHS, while the age of the fibroblast donors determines the expression of proteins involved in wound healing. Emulating patient heterogeneity in preclinical studies might improve the treatment of age-related skin conditions.

UV light catalyzes the ozone formation from air pollutants, like nitrogen oxides. Since ozone reacts with cutaneous sebum lipids to peroxides and, thus, promotes inflammation, tumorigenesis, and aging, even broad-spectrum sunscreens cannot properly protect skin. Meanwhile, xanthophylls, like fucoxanthin, proved their antioxidant and cytoprotective functions, but the safety of their topical application in human cell-based models remains unknown. Aiming for a more detailed insight into the cutaneous fucoxanthin toxicity, we assessed the tissue viability according to OECD test guideline no. 439 as well as changes in inflammation (IL-1α, IL-6, IL-8), homeostasis (EGFR, HSPB1) and metabolism (NAT1). First, we proved the suitability of our 24-well-based reconstructed human skin for irritation testing. Next, we dissolved 0.5% fucoxanthin either in alkyl benzoate or in ethanol and applied both solutions onto the tissue surface. None of the solutions decreased RHS viability below 50%. In contrast, fucoxanthin ameliorated the detrimental effects of ethanol and reduced the gene expression of pro-inflammatory interleukins 6 and 8, while increasing NAT1 gene expression. In conclusion, we developed an organ-on-a-chip compatible RHS, being suitable for skin irritation testing beyond tissue viability assessment. Fucoxanthin proved to be non-irritant in RHS and already showed first skin protective effects following topical application.