The formulation of the N-Acetylglucosamine as nanoparticles increases its anti-inflammatory activities: an in vitro study

Nanomedicine can represent a new strategy to treat several types of diseases such as those with inflammatory aetiology. Through this strategy, it is possible to obtain nanoparticles with controlled shape, size, and eventually surface charge. Moreover, the use of molecules in nanoform may allow more effective delivery into the diseased cells and tissues, reducing toxicity and side effects of the used compounds. The aim of the present manuscript was the evaluation of the effects of N-acetylglucosamine in nanoform (GlcNAc NP) in an in vitro model of osteoarthritis (OA). Human primary chondrocytes were treated with Tumor Necrosis Factor (TNF)-α to simulate a low-grade inflammation and then treated with both GlcNAc and GlcNAc NP, in order to find the lowest concentrations able to counteract the inflammatory state of the cells and ensure a chondroprotective action. The findings showed that GlcNAc NP was able to decrease the pro-inflammatory mediators, IL-6 and IL-8, which are among the main effectors of inflammation; moreover, the nanoparticles downregulated the production of metalloprotease enzymes. GlcNAc NP was effective at a very low concentration compared to GlcNAc in its native form. Furthermore, GlcNAc NP stimulated an increase in collagen type II synthesis. In conclusion, the GlcNAc in nanoform showed better performance than GlcNAc, at concentrations lower than those reached in the joints after oral administration to patients of 1.5 g/die of glucosamine

Repositioned natural compounds and nanoformulations: a promising combination to counteract cell damage and inflammation in respiratory viral infections

Respiratory viral diseases are among the most important causes of disability, morbidity, and death worldwide. Due to the limited efficacy or side effects of many current therapies and the increase in antiviral-resistant viral strains, the need to find new compounds to counteract these infections is growing. Since the development of new drugs is a time-consuming and expensive process, numerous studies have focused on the reuse of commercially available compounds, such as natural molecules with therapeutic properties. This phenomenon is generally called drug repurposing or repositioning and represents a valid emerging strategy in the drug discovery field. Unfortunately, the use of natural compounds in therapy has some limitations, due to their poor kinetic performance and consequently reduced therapeutic effect. The advent of nanotechnology in biomedicine has allowed this limitation to be overcome, showing that natural compounds in nanoform may represent a promising strategy against respiratory viral infections. In this narrative review, the beneficial effects of some promising natural molecules, curcumin, resveratrol, quercetin, and vitamin C, which have been already studied both in native form and in nanoform, against respiratory viral infections are presented and discussed. The review focuses on the ability of these natural compounds, analyzed in in vitro and in vivo studies, to counteract inflammation and cellular damage induced by viral infection and provide scientific evidence of the benefits of nanoformulations in increasing the therapeutic potential of these molecules

Biochemical and computational studies of the interaction between a glucosamine derivative, NAPA, and the IKKα kinase

The glucosamine derivative 2-(N-Acetyl)-L-phenylalanylamido-2-deoxy-β-D-glucose (NAPA), was shown to inhibit the kinase activity of IKKα, one of the two catalytic subunits of IKK complex, decreasing the inflammatory status in osteoarthritis chondrocytes. In the present work we have investigated the inhibition mechanism of IKKα by NAPA by combining computational simulations, in vitro assays and Mass Spectrometry (MS) technique. The kinase in vitro assay was conducted using a recombinant IKKα and IKKtide, a 20 amino acid peptide substrate derived from IkBα kinase protein and containing the serine residues Ser32 and Ser36. Phosphorylated peptide production was measured by Ultra Performance Liquid Chromatography coupled with Mass Spectrometry (UPLC-MS), and the atomic interaction between IKKα and NAPA has been studied by molecular docking and Molecular Dynamics (MD) approaches. Here we report that NAPA was able to inhibit the IKKα kinase activity with an IC50 of 0.5 mM, to decrease the Km value from 0.337 mM to 0.402 mM and the Vmax from 0.0257 mM·min-1 to 0.0076 mM·min-1. The computational analyses indicate the region between the KD, ULD and SDD domains of IKKα as the optimal binding site explored by NAPA. Biochemical data indicate that there is a non-significant difference between Km and Ki whereas there is a statistically significant difference between the two Vmax values. This evidence, combined with computational results, consistently indicates that the inhibition is non-competitive, and that the NAPA binding site is different than that of ATP or IKKtide

Oligomerization of Sulfolobus solfataricus

The recombinant amidase from the hyperthermophylic archaeon Sulfolobus solfataricus (SSAM) a signature amidase, was cloned, purified and characterized. The enzyme is active on a large number of aliphatic and aromatic amides over the temperature range 60–95 °C and at pH values between 4.0 and 9.5, with an optimum at pH 5.0. The recombinant enzyme is in the form of a dimer of about 110 kD that reversibly associates into an octamer in a pH-dependent reaction. The pH dependence of the state of association was studied using gel permeation chromatography, analytical ultracentrifugation and dynamic light scattering techniques

Nanostructured Poly-l-lactide and Polyglycerol Adipate Carriers for the Encapsulation of Usnic Acid: A Promising Approach for Hepatoprotection

The present study investigates the utilization of nanoparticles based on poly-l-lactide (PLLA) and polyglycerol adipate (PGA), alone and blended, for the encapsulation of usnic acid (UA), a potent natural compound with various therapeutic properties including antimicrobial and anticancer activities. The development of these carriers offers an innovative approach to overcome the challenges associated with usnic acid’s limited aqueous solubility, bioavailability, and hepatotoxicity. The nanosystems were characterized according to their physicochemical properties (among others, size, zeta potential, thermal properties), apparent aqueous solubility, and in vitro cytotoxicity. Interestingly, the nanocarrier obtained with the PLLA-PGA 50/50 weight ratio blend showed both the lowest size and the highest UA apparent solubility as well as the ability to decrease UA cytotoxicity towards human hepatocytes (HepG2 cells). This research opens new avenues for the effective utilization of these highly degradable and biocompatible PLLA-PGA blends as nanocarriers for reducing the cytotoxicity of usnic acid

Nutraceuticals and the Network of Obesity Modulators

Obesity is considered an increasingly widespread disease in the world population, regardless of age and gender. Genetic but also lifestyle-dependent causes have been identified. Nutrition and physical exercise play an important role, especially in non-genetic obesity. In a three-compartment model, the body is divided into fat mass, fat-free mass and water, and obesity can be considered a condition in which the percentage of total fat mass is in excess. People with a high BMI index or overweight use self-medications, such as food supplements or teas, with the aim to prevent or treat their problem. Unfortunately, there are several obesity modulators that act both on the pathways that promote adipogenesis and those that inhibit lipolysis. Moreover, these pathways involve different tissues and organs, so it is very difficult to identify anti-obesity substances. A network of factors and cells contributes to the accumulation of fat in completely different body districts. The identification of natural anti-obesity agents should consider this network, which we would like to call “obesosome”. The nutrigenomic, nutrigenetic and epigenetic contribute to making the identification of active compounds very difficult. This narrative review aims to highlight nutraceuticals that, in vitro or in vivo, showed an anti-obesity activity or were found to be useful in the control of dysfunctions which are secondary to obesity. The results suggest that it is not possible to use a single compound to treat obesity, but that the studies have to be addressed towards the identification of mixtures of nutraceuticals

A Review of the Effect of a Nanostructured Thin Film Formed by Titanium Carbide and Titanium Oxides Clustered around Carbon in Graphitic Form on Osseointegration

Improving the biocompatibility of implants is an extremely important step towards improving their quality. In this review, we recount the technological and biological process for coating implants with thin films enriched in titanium carbide (TiC), which provide improved cell growth and osseointegration. At first, we discuss the use of a Pulsed Laser Ablation Deposition, which produced films with a good biocompatibility, cellular stimulation and osseointegration. We then describe how Ion Plating Plasma Assisted technology could be used to produce a nanostructured layer composed by graphitic carbon, whose biocompatibility is enhanced by titanium oxides and titanium carbide. In both cases, the nanostructured coating was compact and strongly bound to the bulk titanium, thus particularly useful to protect implants from the harsh oxidizing environment of biological tissues. The morphology and chemistry of the nanostructured coating were particularly desirable for osteoblasts, resulting in improved proliferation and differentiation. The cellular adhesion to the TiC-coated substrates was much stronger than to uncoated surfaces, and the number of philopodia and lamellipodia developed by the cells grown on the TiC-coated samples was higher. Finally, tests performed on rabbits confirmed in vivo that the osseointegration process of the TiC-coated implants is more efficient than that of uncoated titanium implants

Nanostructured TiC Layer: a suitable surface for osteointegration

In recent years, the number of patients undergoing arthroplasty surgery for joint problems, such as osteoarthritis and accidental fractures, has grown considerably. Wide research has been conducted to study the possible use of biomaterials in orthopedic surgery that would provide bone fixation or able to induce new bone tissue formation and osteointegration. Titanium is the gold standard material used for permanent implants in contact with bone, thanks to its biocompatibility, resistance to corrosion and mechanical properties. In our laboratory, nanostructured titanium-derivative surfaces have been analysed with the aim to find a surface with the best osseointegration features. Titanium carbide (TiC) layer was produced by IPPA deposition directly on glass slides, obtaining surfaces with 25% light transmittance ability. This feature allows to perform several kinds of experiments on cells, retaining the good characteristics of nanostructured titanium. We studied the adhesion, proliferation, and morphology of cells on nanostructured TiC surfaces, comparing them to both cell-culture-treated polystyrene dishes and poly-d-lysinated glass slides (poly-d-Lys). For a more reliable investigation, we chose to use human primary cells, isolated from patients undergoing arthroplasty surgery. Three different types of cells were studied, dermal fibroblasts (FBs), human osteoblasts (hOBs) and human chondrocytes (HPCs). To study the effect of the different surfaces on cell adhesion and morphology, an immunofluorescence experiment was performed evaluating the actin filament organization. Very interestingly, the cells cultivated on TiC showed an actin structure more similar to the tissue disposition. The FBs were arranged in 3D structure, showing filaments disposed on different planes. HPCs formed a particular structure with the nucleus on one side and the cytoplasm on the other side and the hOBs showed a complex network, with a larger number of contact points among cells. These differences in adhesion are also confirmed by the Atomic Force Microscopy (AFM) images, where the cells grown on TiC substrates show well-defined actin filaments which are not evident on the membrane of cells grown on polystyrene and poly-d-Lys. Moreover, the presence of these stress fibers and the overall height of the cells over the substrate indicate that the cells have a better attachment on TiC. In order to analyse the effect of TiC surface on cellular metabolism involved factors release, an ELISA assay was performed. The amount of Fibroblast Growth Factor-2 (FGF-2), Bone Morphogenetic Protein-2 (BMP-2) and Osteocalcin (OC) was measured in cell culture medium of FBs, HPCs and hOBs, respectively. FBs cultivated on TiC produced a higher amount of FGF-2 compared to polystyrene and poly-d-Lys, while HPCs cultured on poly-d-Lys and TiC produced a higher amount of BMP-2 compared to polystyrene. Osteocalcin is used as a serum marker of bone formation and as indicator of the proliferative and differentiated state of osteoblasts in cell culture. In hOBs cultivated on TiC a higher amount of OC compared to polystyrene and poly-d-Lys was obtained. Considering the involvement of FBs, HPCs and hOBs in the production of extracellular matrix components in the in vivo tissues, the effect of TiC surface on Collagen protein expression was evaluate by immunofluorescence. In order to allow cells to produce a detectable amount of collagens, cells were cultivated for seven days before analysis. Collagen type I in hFBs and hOBs, and Collagen type II in HPCs resulted increased in cells seeded on TiC compared to the other two substrates. All these results suggest how TiC is an excellent additional layer to cell culture and that can be considered as a biomaterial useful for in vivo osteointegration. [1] Lopreiato M., Mariano A. and Cocchiola R., Condensed matter, 2020, 5, 29

Different Expression and Localization of Phosphoinositide Specific Phospholipases C in Human Osteoblasts, Osteosarcoma Cell Lines, Ewing Sarcoma and Synovial Sarcoma

Background: bone hardness and strength depends on mineralization, which involves a complex process where calcium phosphate, produced by bone-forming cells, were shed around the fibrous matrix. This process is strictly regulated, and a number of signal transduction systems were interested in calcium metabolism, such as the Phosphoinositide (PI) pathway, including the Phospholipase C (PLC) enzymes. Methods: we analysed the PLC enzymes in human osteoblasts and osteosarcoma cell lines MG-63 and SaOS-2. We compared the results to the expression of PLCs in samples of patients affected with Ewing Sarcoma (ES) and Synovial Sarcoma (SS). Results: In osteoblasts, MG-63 cells and SaOS-2 significant differences were identified in the expression of PLC 4 and PLC  subfamily isoforms. Differences were also identified regarding the expression of PLCs in ES and SS. Most ES and SS did not express PLCB1, which was expressed in most osteoblasts, MG-63 and SaOS-2 cells. Conversely, PLCB2, unexpressed in the cell lines, was expressed in some ES and SS. However, PLCH1 was expressed in SaOS-2 and inconstantly expressed in osteoblasts, while it was expressed in ES and unexpressed in SS. The most relevant difference observed in ES compared to SS regarded PLC  and PLC  isoforms. Conclusion: MG-63 and SaOS-2 osteosarcoma cell lines probably do not represent the appropriate experimental model for studies about the analysis of signal transduction in osteoblasts