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

In vitro antiviral and anti-inflammatory activities of N-Acetylglucosamine: development of an alternative and safe approach to fight viral respiratory infections

Viral respiratory tract infections (RTIs) are responsible for significant morbidity and mortality worldwide. A prominent feature of severe respiratory infections, such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, is the cytokine release syndrome. Therefore, there is an urgent need to develop different approaches both against viral replication and against the consequent inflammation. N-acetylglucosamine (GlcNAc), a glucosamine (GlcN) derivative, has been developed as an immunomodulatory and anti-inflammatory inexpensive and non-toxic drug for non-communicable disease treatment and/or prevention. Recent studies have suggested that GlcN, due to its anti-inflammatory activity, could be potentially useful for the control of respiratory virus infections. Our present study aimed to evaluate in two different immortalized cell lines whether GlcNAc could inhibit or reduce both viral infectivity and the inflammatory response to viral infection. Two different viruses, frequent cause of upper and lower respiratory tract infections, were used: the H1N1 Influenza A virus (IAV) (as model of enveloped RNA virus) and the Human adenovirus type 2 (Adv) (as model of naked DNA virus). Two forms of GlcNAc have been considered, bulk GlcNAc and GlcNAc in nanoform to overcome the possible pharmacokinetic limitations of GlcNAc. Our study suggests that GlcNAc restricts IAV replication but not Adv infection, whereas nano-GlcNAc inhibits both viruses. Moreover, GlcNAc and mainly its nanoformulation were able to reduce the pro-inflammatory cytokine secretion stimulated by viral infection. The correlation between inflammatory and infection inhibition is discussed

Topoisomerase II-Mediated DNA Damage Is Differently Repaired during the Cell Cycle by Non-Homologous End Joining and Homologous Recombination

Topoisomerase II (Top2) is a nuclear enzyme involved in several metabolic processes of DNA. Chemotherapy agents that poison Top2 are known to induce persistent protein-mediated DNA double strand breaks (DSB). In this report, by using knock down experiments, we demonstrated that Top2α was largely responsible for the induction of γH2AX and cytotoxicity by the Top2 poisons idarubicin and etoposide in normal human cells. As DSB resulting from Top2 poisons-mediated damage may be repaired by non-homologous end joining (NHEJ) or homologous recombination (HR), we aimed to analyze both DNA repair pathways. We found that DNA-PKcs was rapidly activated in human cells, as evidenced by autophosphorylation at serine 2056, following Top2-mediated DNA damage. The chemical inhibition of DNA-PKcs by wortmannin and vanillin resulted in an increased accumulation of DNA DSB, as evaluated by the comet assay. This was supported by a hypersensitive phenotype to Top2 poisons of Ku80- and DNA-PKcs- defective Chinese hamster cell lines. We also showed that Rad51 protein levels, Rad51 foci formation and sister chromatid exchanges were increased in human cells following Top2-mediated DNA damage. In support, BRCA2- and Rad51C- defective Chinese hamster cells displayed hypersensitivity to Top2 poisons. The analysis by immunofluorescence of the DNA DSB repair response in synchronized human cell cultures revealed activation of DNA-PKcs throughout the cell cycle and Rad51 foci formation in S and late S/G2 cells. Additionally, we found an increase of DNA-PKcs-mediated residual repair events, but not Rad51 residual foci, into micronucleated and apoptotic cells. Therefore, we conclude that in human cells both NHEJ and HR are required, with cell cycle stage specificity, for the repair of Top2-mediated reversible DNA damage. Moreover, NHEJ-mediated residual repair events are more frequently associated to irreversibly damaged cells

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

The nutraceuticals as modern key to achieve erythrocyte oxidative stress fighting in osteoarthritis

Osteoarthritis (OA), the most common joint disease, shows an increasing prevalence in the aging population in industrialized countries. OA is characterized by low-grade chronic inflammation, which causes degeneration of all joint tissues, such as articular cartilage, subchondral bone, and synovial membrane, leading to pain and loss of functionality. Erythrocytes, the most abundant blood cells, have as their primary function oxygen transport, which induces reactive oxygen species (ROS) production. For this reason, the erythrocytes have several mechanisms to counteract ROS injuries, which cause damage to lipids and proteins of the cell membrane. Oxidative stress and inflammation are highly correlated and are both causes of joint disorders. In the synovial fluid and blood of osteoarthritis patients, erythrocyte antioxidant enzyme expression is decreased. To date, OA is a non-curable disease, treated mainly with non-steroidal anti-inflammatory drugs and corticosteroids for a prolonged period of time, which cause several side effects; thus, the search for natural remedies with anti-inflammatory and antioxidant activities is always ongoing. In this review, we analyze several manuscripts describing the effect of traditional remedies, such as Harpagophytum procumbens, Curcumin longa, and Boswellia serrata extracts, in the treatments of OA for their anti-inflammatory, analgesic, and antioxidant activity. The effects of such remedies have been studied both in in vitro and in vivo models, considering both joint cells and erythrocytes

NAPA, a glucosamine derivative: biochemical analysis of interaction with IKKα Kinase

The IKB kinase (IKK) complex, involved in several cellular pathways, comprises two catalytic subunits, IKKα and IKKβ, and a regulatory subunit, NEMO. Globally, the organization of the two kinases is similar, even if significant differences can be observed. IKKβ, associated with NEMO, activates the canonical pathway of NF-κB, while IKKα mainly activates the non-canonical pathway. NAPA, 2-(N-Acetyl)-L-phenylalanylamido-2-deoxy-b-D-glucose, is a glucosamine derivative synthetized and tested in vitro in our lab on human primary chondrocytes isolated from cartilage of patients with Osteoarthritis (OA) the most common inflammatory joints disease. The NF-κB kinases have a prominent role in the activation of inflammatory processes in OA such as the stimulation of several molecules, matrix metalloproteinases (MMPs) and transcriptional factors. Considering that the IKKα kinase activity resulted inhibited by NAPA, as shown in our previous work, the aim of this study is to explore the interaction between NAPA and IKKα through in vitro kinase assay. The IKKα enzymatic activity was studied performing an in vitro assay using a recombinant IKKα kinase and a synthetic peptide, IKKtide, containing the two serine residues. Interestingly, in relation of these reaction conditions, IKKα was able to phosphorylate the IKKtide preferable on one serine only, as pIKKtide was largely exceeding the ppIKKtide amount revealed by UPLC/MS. To evaluate the inhibitory effect of NAPA on the IKKtide phosphorylation, different concentrations of NAPA were incubated with IKKα, ATP and IKKtide, as substrate. The NAPA IC50 was found to be 0.5 ± 0.086 mM. that could seem high, anyway, it has to be considered that a strong inhibition of IKKα could result in a detrimental effect for cells and could display side effects in humans. To verify if NAPA inhibited IKKα interacting with the active site of the kinase, the assay was performed using different amount of ATP, but all these concentrations were unable to revert the inhibition of NAPA, suggesting that NAPA did not interact with the ATP binding site of IKKα. To confirm this hypothesis, a kinetic assay was performed using a fixed amount of enzyme and increasing concentrations of substrate, IKKtide. The same experiment was conducted in presence of different concentrations of NAPA. The difference between Km and Ki obtained resulted not statistically significant, in contrast, the difference between the Vmax values obtained in absence or in the presence of NAPA strongly suggested that the inhibition could be non-competitive. It can be concluded that this inhibition of IKKα kinase activity makes NAPA a very appealing molecule considering that phosphorylation has a central role in biological regulation of intracellular pathways. Our findings demonstrated that NAPA does not bind to the ATP binding site and made this molecule extremely interesting and particularly suitable for long-term treatments, such as those required for OA

Pheomelanin effect on UVB radiation-induced oxidation/nitration of L-tyrosine

Pheomelanin is a natural yellow-reddish sulfur-containing pigment derived from tyrosinase-catalyzed oxidation of tyrosine in presence of cysteine. Generally, the formation of melanin pigments is a protective response against the damaging effects of UV radiation in skin. However, pheomelanin, like other photosensitizing substances, can trigger, following exposure to UV radiation, photochemical reactions capable of modifying and damaging cellular components. The photoproperties of this natural pigment have been studied by analyzing pheomelanin effect on oxidation/nitration of tyrosine induced by UVB radiation at different pH values and in presence of iron ions. Photoproperties of pheomelanin can be modulated by various experimental conditions, ranging from the photoprotection to the triggering of potentially damaging photochemical reactions. The study of the photomodification of l-Tyrosine in the presence of the natural pigment pheomelanin has a special relevance, since this tyrosine oxidation/nitration pathway can potentially occur in vivo in tissues exposed to sunlight and play a role in the mechanisms of tissue damage induced by UV radiation

Development of polysaccharide-based nano-systems containing lactoferrin for treatment of respiratory infections

Viral respiratory infections affect people of all ages, the emergence of new viruses and the selection of drug-resistant viral strains have prompted the development of new therapeutic strategies. The advent of nanotechnology in biomedicine has opened the way to new therapies for the delivery of drugs, proteins and nucleic acids. The small size provides these systems with excellent chemical-physical and pharmacokinetic properties, making it possible for them to transit inside small capillaries and cross biological barriers [1]. Among nano-carriers, the most promising in the biomedical field are those based on polysaccharides thanks to their biodegradability, bioactivity and biocompatibility as well as the presence of many functional groups that can be used to improve drug loading. This work was aimed at developing innovative therapies for the treatment of infection by respiratory viruses, such as influenza virus and adenovirus, and of the inflammatory processes caused by them. Lactoferrin (Lf) possesses many biological functions including antimicrobial, antitumor, anti-inflammatory activities [2]. Many studies have shown protective effects of lactoferrin against common viral infections [3]. Polysaccharide-based nanocarriers were developed in order to improve the lactoferrin therapeutic index. Alginate and hyaluronic acid were used to prepare two different nano-complexes for protein delivery using the complex coacervation method. The obtained systems were characterized by DLS technique to investigate their dimensions, polydispersity and stability. Morphological analysis, performed by scanning electron microscopy (SEM), confirmed the presence of nanostructures while thermal analysis (TGA) evidenced the formation of polysaccharide-protein interactions with an improvement in thermal stability (increase in TD) of the nanocomplexes as protein concentration increased. Circular dichroism measurements displayed no conformational change of lactoferrin after sonication treatment or interaction with alginate and hyaluronic acid. By physicochemical characterization were identified the most promising complexes then subjected to cytotoxicity analysis by MTT test on two different cell lines (A549 and MDCK). Preliminary results were positive. Further tests are ongoing to evaluate the anti-inflammatory and antiviral activity of the nanocomplexes

Harpagophytum procumbens root extract mediates anti-inflammatory effects in osteoarthritis synoviocytes through CB2 activation

The endocannabinoid system is involved in the nociceptive and anti-inflammatory pathways, and a lowered expression of CB2 receptors has been associated with inflammatory conditions, such as osteoarthritis (OA). This suggests that CB2 modulators could be novel therapeutic tools to treat OA. In the present study, the involvement of Harpagophytum procumbens root extract, a common ingredient of nutraceuticals used to treat joint disorders, in CB2 modulation has been evaluated. Moreover, to clarify the effects of the pure single components, the bioactive constituent, harpagoside, and the main volatile compounds were studied alone or in a reconstituted mixture. Human fibroblast-like synoviocytes, extracted by joints of patients, who underwent a total knee replacement, were treated with an H. procumbens root extract dissolved in DMSO (HPEDMSO). The effectiveness of HPEDMSO to affect CB2 pathways was studied by analyzing the modulation of cAMP, the activation of PKA and ERK MAP kinase, and the modulation of MMP-13 production. HPEDMSO was able to inhibit the cAMP production and MAP kinase activation and to down-regulate the MMP-13 production. Pure compounds were less effective than the whole phytocomplex, thus suggesting the involvement of synergistic interactions. Present findings encourage further mechanistic studies and support the scientific basis of the use of H. procumbens in joint disorders