Mangiferin is a natural glucosyl xanthone with antioxidant and anti-inflammatory activity, making it suitable for protection against cutaneous diseases. In this study ethosomes and transethosomes were designed as topical delivery systems for mangiferin. A preformulation study was conducted using different surfactants in association with phosphatidylcholine. Vesicle dimensional distribution was monitored by photon correlation spectroscopy, while antioxidant capacity and cytotoxicity were respectively assessed by free radical scavenging analysis and MTT on HaCaT keratinocytes. Selected nanosystems were further investigated by cryogenic transmission electron microscopy, while mangiferin entrapment capacity was evaluated by ultracentrifugation and HPLC. The diffusion kinetics of mangiferin from ethosomes and transethosomes evaluated by Franz cell was faster in the case of transethosomes. The suitability of mangiferin-containing nanovesicles in the treatment of skin disorders related to pollutants was investigated, evaluating, in vitro, the antioxidant and anti-inflammatory effect of ethosomes and transethosomes on human keratinocytes exposed to cigarette smoke as an oxidative and inflammatory challenger. The ability to induce an antioxidant response (HO-1) and anti-inflammatory status (IL-6 and NF-kB) was determined by RT-PCR and immunofluorescence. The data demonstrated the effectiveness of mangiferin loaded in nanosystems to protect cells from damage. Finally, to gain insight into the keratinocytes’ uptake of ethosome and transethosome, transmission electron microscopy analyses were conducted, showing that both nanosystems were able to pass intact within the cells.

In order to design valid protocols for drug release via nanocarriers, it is essential to know the mechanisms of cell internalization, the interactions with organelles, and the intracellular permanence and degradation of nanoparticles (NPs) as well as the possible cell alteration or damage induced. In the present study, the intracellular fate of liposomes, polymeric NPs and mesoporous silica NPs (MSN) has been investigated in an in vitro cell system by fluorescence and transmission electron microscopy. The tested nanocarriers proved to be characterized by specific interactions with the cell: liposomes enter the cells probably by fusion with the plasma membrane and undergo rapid cytoplasmic degradation; polymeric NPs are internalized by endocytosis, occur in the cytoplasm both enclosed in endosomes and free in the cytosol, and then undergo massive degradation by lysosome action; MSN are internalized by both endocytosis and phagocytosis, and persist in the cytoplasm enclosed in vacuoles. No one of the tested nanocarriers was found to enter the nucleus. The exposure to the different nanocarriers did not increase cell death; only liposomes induced a reduction of cell population after long incubation times, probably due to cell overloading. No subcellular damage was observed to be induced by polymeric NPs and MSN, whereas transmission electron microscopy revealed cytoplasm alterations in liposome-treated cells. This important information on the structural and functional relationships between nanocarriers designed for drug delivery and cultured cells further proves the crucial role of microscopy techniques in nanotechnology.

In this pilot study, ethosomes and transethosomes were investigated as potential delivery systems for cholecalciferol (vitamin D3), whose deficiency has been correlated to many disorders such as dermatological diseases, systemic infections, cancer and sarcopenia. A formulative study on the influence of pharmaceutically acceptable ionic and non-ionic surfactants allowed the preparation of different transethosomes. In vitro cytotoxicity was evaluated in different cell types representative of epithelial, connective and muscle tissue. Then, the selected nanocarriers were further investigated at light and transmission electron microscopy to evaluate their uptake and intracellular fate. Both ethosomes and transethosomes proven to have physicochemical properties optimal for transdermal penetration and efficient vitamin D3 loading; moreover, nanocarriers were easily internalized by all cell types, although they followed distinct intracellular fates: ethosomes persisted for long times inside the cytoplasm, without inducing subcellular alteration, while transethosomes underwent rapid degradation giving rise to an intracellular accumulation of lipids. These basic results provide a solid scientific background to in vivo investigations aimed at exploring the efficacy of vitamin D3 transdermal administration in different experimental and pathological conditions.

Ozone therapy is a modestly invasive procedure based on the regeneration capabilities of low ozone concentrations and used in medicine as an alternative/adjuvant treatment for different diseases. However, the cellular mechanisms accounting for the positive effects of mild ozonization are still largely unexplored. To this aim, in the present study the effects of low ozone concentrations (1 to 20 µg O3/mL O2) on structural and functional cell features have been investigated in vitro by using morphological, morphometrical, cytochemical and immunocytochemical techniques at bright field, fluorescence and transmission electron microscopy. Cells exposed to pure O2 or air served as controls. The results demonstrated that the effects of ozoneadministration are dependent on gas concentration, and the cytoskeletal organization, mitochondrial activity and nuclear transcription may be differently affected. This suggests that, to ensure effective and permanent metabolic cell activation, ozone treatments should take into account the cytological and cytokinetic features of the different tissues.Â

In clinical practice, administration of low ozone (O₃) dosages is a complementary therapy for many diseases, due to the capability of O₃ to elicit an antioxidant response through the Nuclear Factor Erythroid 2-Related Factor 2 (Nrf2)-dependent pathway. Nrf2 is also involved in the adipogenic differentiation of mesenchymal stem cells, and low O₃ concentrations have been shown to stimulate lipid accumulation in human adipose-derived adult stem cells in vitro. Thus, O₃ treatment is a promising procedure to improve the survival of explanted adipose tissue, whose reabsorption after fat grafting is a major problem in regenerative medicine. In this context, we carried out a pilot study to explore the potential of mild O₃ treatment in preserving explanted murine adipose tissue in vitro. Scanning and transmission electron microscopy, Western blot, real-time polymerase chain reaction and nuclear magnetic resonance spectroscopy were used. Exposure to low O₃ concentrations down in the degradation of the explanted adipose tissue and induced a concomitant increase in the protein abundance of Nrf2 and in the expression of its target gene Hmox1. These findings provide a promising background for further studies aimed at the clinical application of O₃ as an adjuvant treatment to improve fat engraftment.

Ozone is a strong oxidant, highly unstable atmospheric gas. Its medical use at low concentrations has been progressively increasing as an alternative/adjuvant treatment for several diseases. In this study, we investigated the effects of mild ozonisation on human adipose-derived adult stem (hADAS) cells i.e., mesenchymal stem cells occurring in the stromal-vascular fraction of the fat tissue and involved in the tissue regeneration processes. hADAS cells were induced to differentiate into the adipoblastic lineage, and the effect of low ozone concentrations on the adipogenic process was studied by combining histochemical, morphometric and ultrastructural analyses. Our results demonstrate that ozone treatment promotes lipid accumulation in hADAS without inducing deleterious effects, thus paving the way to future studies aimed at elucidating the effect of mild ozonisation on adipose tissue for tissue regeneration and engineering.