Development and Characterization of Azithromycin-Loaded Microemulsions: A Promising Tool for the Treatment of Bacterial Skin Infections

In recent years, the treatment of bacterial skin infections has been considered a major healthcare issue due to the growing emergence of antibiotic-resistant strains of Staphylococcus aureus. The incorporation of antibiotics in appropriate nanosystems could represent a promising strategy, able to overcome several drawbacks of the topical treatment of infections, including poor drug retention within the skin. The present work aims to develop microemulsions containing azithromycin (AZT), a broad-spectrum macrolide antibiotic. Firstly, AZT solubility in various oils, surfactants and co-surfactants was assessed to select the main components. Subsequently, microemulsions composed of vitamin E acetate, Labrasol (R) and Transcutol (R) P were prepared and characterized for their pH, viscosity, droplet size, zeta potential and ability to release the drug and to promote its retention inside porcine skin. Antimicrobial activity against S. aureus methicillin-resistant strains (MRSA) and the biocompatibility of microemulsions were evaluated. Microemulsions showed an acceptable pH and were characterized by different droplet sizes and viscosities depending on their composition. Interestingly, they provided a prolonged release of AZT and promoted its accumulation inside the skin. Finally, microemulsions retained AZT efficacy on MRSA and were not cytotoxic. Hence, the developed AZT-loaded microemulsions could be considered as useful nanocarriers for the treatment of antibiotic-resistant infections of the skin

Assessment and imaging of intracellular magnesium in saos-2 osteosarcoma cells and its role in proliferation

Magnesium is an essential nutrient involved in many important processes in living organ-isms, including protein synthesis, cellular energy production and storage, cell growth and nucleic acid synthesis. In this study, we analysed the effect of magnesium deficiency on the proliferation of SaOS-2 osteosarcoma cells. When quiescent magnesium-starved cells were induced to proliferate by serum addition, the magnesium content was 2–3 times lower in cells maintained in a medium without magnesium compared with cells growing in the presence of the ion. Magnesium depletion inhibited cell cycle progression and caused the inhibition of cell proliferation, which was associated with mTOR hypophosphorylation at Serine 2448. In order to map the intracellular magnesium distribution, an analytical approach using synchrotron-based X-ray techniques was applied. When cell growth was stimulated, magnesium was mainly localized near the plasma membrane in cells maintained in a medium without magnesium. In non-proliferating cells growing in the presence of the ion, high concentration areas inside the cell were observed. These results support the role of magnesium in the control of cell proliferation, suggesting that mTOR may represent an important target for the antiproliferative effect of magnesium. Selective control of magnesium availability could be a useful strategy for inhibiting osteosarcoma cell growth

Analysis of intracellular magnesium and mineral depositions during osteogenic commitment of 3d cultured saos2 cells

In this study, we explore the behaviour of intracellular magnesium during bone phenotype modulation in a 3D cell model built to mimic osteogenesis. In addition, we measured the amount of magnesium in the mineral depositions generated during osteogenic induction. A two-fold increase of intracellular magnesium content was found, both at three and seven days from the induction of differentiation. By X-ray microscopy, we characterized the morphology and chemical composition of the mineral depositions secreted by 3D cultured differentiated cells finding a marked co-localization of Mg with P at seven days of differentiation. This is the first experimental evidence on the presence of Mg in the mineral depositions generated during biomineralization, suggesting that Mg incorporation occurs during the bone forming process. In conclusion, this study on the one hand attests to an evident involvement of Mg in the process of cell differentiation, and, on the other hand, indicates that its multifaceted role needs further investigation

Magnesium Deprivation Potentiates Human Mesenchymal Stem Cell Transcriptional Remodeling

Magnesium plays a pivotal role in energy metabolism and in the control of cell growth. While magnesium deprivation clearly shapes the behavior of normal and neoplastic cells, little is known on the role of this element in cell differentiation. Here we show that magnesium deficiency increases the transcription of multipotency markers and tissue-specific transcription factors in human adipose-derived mesenchymal stem cells exposed to a mixture of natural molecules, i.e., hyaluronic, butyric and retinoid acids, which tunes differentiation. We also demonstrate that magnesium deficiency accelerates the osteogenic differentiation of human bone marrow-derived mesenchymal stem cells. We argue that magnesium deprivation generates a stressful condition that modulates stem cell plasticity and differentiation potential. These studies indicate that it is possible to remodel transcription in mesenchymal stem cells by lowering extracellular magnesium without the need for genetic manipulation, thus offering new hints for regenerative medicine applications

X-ray fluorescence microscopy of light elements in cells: self-absorption correction by integration of compositional and morphological measurements

We present here a new methodology for quantitative mapping of light elements in cells, based on combination of compositional and morphological information, derived respectively by X-ray Fluorescence Microscopy (XRFM), Atomic Force Microscopy and Scanning Transmission X-ray Microscopy (STXM). Since XRFM of light elements (carbon, nitrogen, oxygen, sodium and magnesium), are strongly influenced by self-absorption, we developed an algorithm to correct for this effect, using the morphological and structural information provided by AFM and STXM. Finally, the corrected distributions have been obtained, thus allowing quantitative mapping

The EGF- induced cell proliferation and migration in HT29 is inhibited by 9-hydroxystearic acid

: Previous studies have shown that p21 (1) and histone hyperacetylation are important in basal growth inhibition by 9-HSA (2). It is known that serum-starved cells exit G1 and enter the G0 phase but can be induced to reenter the cell cycle through treatment with either serum or specific growth factors. We were therefore interested in determining whether colon cancer cells that had been withdrawn in the G0 phase of the cell cycle by treatment with 9-HSA were also capable of reentering the cell cycle after treatment with epidermal growth factor (EGF). Previous studies have shown that FAK is important in linking EGFR activation to the cellular machinery that promotes proliferation and cell migration (3). To determine wether 9-HSA treatment interferes with signal trasduction involving EGFR and FAK, we analyzed FAK expression and its phosphorylation levels, HT29 invasion index after EGF stimulation, in control and 9-HSA treated cells. Our data indicate that the treatment with 9-HSA induces a molecular phenotype characterized by a lack of growth factor responsiveness and diminished cell migration

The role of caspase-9, bax, and p53 in the apoptotic response triggered by 9-hydroxystearic acid

HDAC1 (Histone deacetylase) activity is inhibited by 9-hydroxystearic acid (9-HSA) (1,2). HDAC1 inhitors specifically induce differentiation/apoptosis of transformed cells in vitro and suppres tumor growth in vivo. 9-HSA, an endogenous lipid peroxidation product, induces apoptosis characterized by mitochondrial stress, but so far the critical elements of these apoptotic program remain poorly defined. To characterize this apoptotic program in more detail we used a human ostesarcoma cell line p53 w.t. and we studied important elements of the apoptotic response such as p53, Bax and caspase 9. We demonstrate that Bax expression level and caspase 9 activity are critical for apoptosis induced by 9-HSA and that the susceptibility to cell death in response to 9-HSA treatment is regulated by p53 acetylation level

The assessment of intracellular magnesium: Different strategies to answer different questions

The role of magnesium in cell metabolism is complex and still not completely clarified. Although magnesium has been shown to modulate many phenomena in cells, its intracellular distribution and subcellular compartmentalization have not yet elucidated in detail, mainly as a consequence of the inadequacy of analytical techniques. The method usually employed to quantify total magnesium in cells or tissue are F-AAS or more sensitive techniques as graphite furnace AAS and inductively coupled plasma mass spectroscopy (MS). Thanks to the development of new specific fluorescent dyes, several progresses have been made in the comprehension of the fundamental biological process at the cellular and sub-cellular level. Moreover, the biological function of a chemical element in cells does not only require the determination of its intracellular quantity but also the spatial distribution of its concentration. Most of Mg2+-sensitive fluorescent dyes detect only the free metal ions, precluding the possibility of identifying the total pool of Mg. This review aims at giving an overview on different techniques focusing on two approaches to quantify total Mg in a small cell population or in single cells: i) Indirect Mg detection, labelbased methods that represent the best choice to quantify the elemental concentration on a large cell population; ii) direct Mg detection (label-free), Synchrotron-based x-ray microscopy techniques that offer the possibility of achieving a detailed map of the intracellular concentration of a specific chemical element on single cell

A new imidazothiazolylmethylen-oxindole induces apoptosis and decline in intracellular free and total magnesium in HT29 colon cancer cells

Recent studies demonstrated that 2MM-67, a new 3-(5-imidazo[2,1-b]thiazolylmethylene)-2-indolinone, analog of recently published compounds is able to induce apoptotic cell death in HT-29, a colon carcinoma cell line. The cells treated with the proapoptotic agent (500 nM) showed arrest in G2/M phase and increased expression of p21 protein. Moreover it was observed a significant reduction of mitochondrial potential (Δψm), enhanced expression of Bax and, finally, a marked induction of effector caspases. The aim of this work was to assess cellular magnesium levels during 2MM-67-induced apoptosis and if they correlate to Δψm decrease and to an increase in ROS production, characteristic feature of mitochondrion-mediated apoptosis. Intracellular free and total magnesium were evaluated in flow cytometry, respectively by the commercial probe MagFluo4-AM (Kd=4.7 mM), and the new synthesized DCHQ7 (Kd=8.3 μM), while ROS production was assessed by DiClFDA and Dihydrohetidine. Our results indicate that 2MM-67 treatment in HT29 cells can result in rapid declines in intracellular free and total magnesium concomitant with the decrease in mitochondrial membrane potential and the increase of ROS production