The Neuromuscular Disorder Mediated by Extracellular Vesicles in Amyotrophic Lateral Sclerosis.

Amyotrophic lateral sclerosis (ALS) represents a neurodegenerative disorder characterized by the progressive loss of both upper and lower motor neurons, resulting in muscular atrophy and eventual paralysis. While much research has concentrated on investigating the impact of major mutations associated with ALS on motor neurons and central nervous system (CNS) cells, recent studies have unveiled that ALS pathogenesis extends beyond CNS imbalances, encompassing dysregulation in other tissues such as skeletal muscle. Evidence from animal models and patients supports this broader perspective. Skeletal muscle, once considered solely as an effector organ, is now recognized as possessing significant secretory activity capable of influencing motor neuron survival. However, the precise cellular and molecular mechanisms underlying the detrimental effects observed in muscle and its associated structures in ALS remain poorly understood. Additionally, emerging data suggest that extracellular vesicles (EVs) may play a role in the establishment and function of the neuromuscular junction (NMJ) under both physiological and pathological conditions and in wasting and regeneration of skeletal muscles, particularly in neurodegenerative diseases like ALS. This review aims to explore the key findings about skeletal muscle involvement in ALS, shedding light on the potential underlying mechanisms and contributions of EVs and their possible application for the design of biosensors.

Looking to the Future of the Role of Macrophages and Extracellular Vesicles in Neuroinflammation in ALS.

Neuroinflammation is a common pathological feature of amyotrophic lateral sclerosis (ALS). Although scientific evidence to date does not allow defining neuroinflammation as an ALS trigger, its role in exacerbating motor neuron (MNs) degeneration and disease progression is attracting research interest. Activated CNS (Central Nervous System) glial cells, proinflammatory peripheral and infiltrated T lymphocytes and monocytes/macrophages, as well as the immunoreactive molecules they release, represent the active players for the role of immune dysregulation enhancing neuroinflammation. The crosstalk between the peripheral and CNS immune cells significantly correlates with the survival of ALS patients since the modification of peripheral macrophages can downregulate inflammation at the periphery along the nerves and in the CNS. As putative vehicles for misfolded protein and inflammatory mediators between cells, extracellular vesicles (EVs) have also drawn particular attention in the field of ALS. Both CNS and peripheral immune cells release EVs, which are able to modulate the behavior of neighboring recipient cells; unfortunately, the mechanisms involved in EVs-mediated communication in neuroinflammation remain unclear. This review aims to synthesize the current literature regarding EV-mediated cell-to-cell communication in the brain under ALS, with a particular point of view on the role of peripheral macrophages in responding to inflammation to understand the biological process and exploit it for ALS management.

Molecular Characterization of Temozolomide-Treated and Non Temozolomide-Treated Glioblastoma Cells Released Extracellular Vesicles and Their Role in the Macrophage Response.

Extracellular vesicles (EVs) are widely investigated in glioblastoma multiforme (GBM) for their involvement in regulating GBM pathobiology as well as for their use as potential biomarkers. EVs, through cell-to-cell communication, can deliver proteins, nucleic acids, and lipids that are able to reprogram tumor-associated macrophages (TAMs). This research is aimed to concentrate, characterize, and identify molecular markers of EVs subtypes released by temozolomide (TMZ)-treated and non TMZ-treated four diverse GBM cells. Morphology, size distribution, and quantity of small (sEVs) and large (lEVs) vesicles were analyzed by cryo-TEM. Quality and quantity of EVs surface markers were evaluated, having been obtained by Western blotting. GBM cells shed a large amount of EVs, showing a cell line dependent molecular profile A comparative analysis distinguished sEVs and lEVs released by temozolomide (TMZ)-treated and non TMZ-treated GBM cells on the basis of quantity, size and markers expression. Finally, the GBM-derived sEVs and lEVs, irrespective of TMZ treatment, when challenged with macrophages, modulated cell activation toward a tendentially M2b-like phenotype.

Moderate Static Magnetic Field (6 mT)-Induced Lipid Rafts Rearrangement Increases Silver NPs Uptake in Human Lymphocytes.

One of the most relevant drawbacks in medicine is the ability of drugs and/or imaging agents to reach cells. Nanotechnology opened new horizons in drug delivery, and silver nanoparticles (AgNPs) represent a promising delivery vehicle for their adjustable size and shape, high-density surface ligand attachment, etc. AgNPs cellular uptake involves different endocytosis mechanisms, including lipid raft-mediated endocytosis. Since static magnetic fields (SMFs) exposure induces plasma membrane perturbation, including the rearrangement of lipid rafts, we investigated whether SMF could increase the amount of AgNPs able to pass the peripheral blood lymphocytes (PBLs) plasma membrane. To this purpose, the effect of 6-mT SMF exposure on the redistribution of two main lipid raft components (i.e., disialoganglioside GD3, cholesterol) and on AgNPs uptake efficiency was investigated. Results showed that 6 mT SMF: (i) induces a time-dependent GD3 and cholesterol redistribution in plasma membrane lipid rafts and modulates gene expression of ATP-binding cassette transporter A1 (ABCA1), (ii) increases reactive oxygen species (ROS) production and lipid peroxidation, (iii) does not induce cell death and (iv) induces lipid rafts rearrangement, that, in turn, favors the uptake of AgNPs. Thus, it derives that SMF exposure could be exploited to enhance the internalization of NPs-loaded therapeutic or diagnostic molecules.

Intracellular Transport of Silver and Gold Nanoparticles and Biological Responses: An Update.

Medicine, food, and cosmetics represent the new promising applications for silver (Ag) and gold (Au) nanoparticles (NPs). AgNPs are most commonly used in food and cosmetics; conversely, the main applications of gold NPs (AuNPs) are in the medical field. Thus, in view of the risk of accidentally or non-intended uptake of NPs deriving from the use of cosmetics, drugs, and food, the study of NPs⁻cell interactions represents a key question that puzzles researchers in both the nanomedicine and nanotoxicology fields. The response of cells starts when the NPs bind to the cell surface or when they are internalized. The amount and modality of their uptake depend on many and diverse parameters, such as NPs and cell types. Here, we discuss the state of the art of the knowledge and the uncertainties regarding the biological consequences of AgNPs and AuNPs, focusing on NPs cell uptake, location, and translocation. Finally, a section will be dedicated to the most currently available methods for qualitative and quantitative analysis of intracellular transport of metal NPs.

Recent Advances in Photodynamic Therapy: Metal-Based Nanoparticles as Tools to Improve Cancer Therapy.

Cancer remains a significant global health challenge, with traditional therapies like surgery, chemotherapy, and radiation often accompanied by systemic toxicity and damage to healthy tissues. Despite progress in treatment, these approaches have limitations such as non-specific targeting, systemic toxicity, and resistance development in cancer cells. In recent years, nanotechnology has emerged as a revolutionary frontier in cancer therapy, offering potential solutions to these challenges. Nanoparticles, due to their unique physical and chemical properties, can carry therapeutic payloads, navigate biological barriers, and selectively target cancer cells. Metal-based nanoparticles, in particular, offer unique properties suitable for various therapeutic applications. Recent advancements have focused on the integration of metal-based nanoparticles to enhance the efficacy and precision of photodynamic therapy. Integrating nanotechnology into cancer therapy represents a paradigm shift, enabling the development of strategies with enhanced specificity and reduced off-target effects. This review aims to provide a comprehensive understanding of the pivotal role of metal-based nanoparticles in photodynamic therapy. We explore the mechanisms, biocompatibility, and applications of metal-based nanoparticles in photodynamic therapy, highlighting the challenges and the limitations in their use, as well as the combining of metal-based nanoparticles/photodynamic therapy with other strategies as a synergistic therapeutic approach for cancer treatment.

Extracellular Vesicles from NSC-34 MN-like Cells Transfected with Mutant SOD1 Modulate Inflammatory Status of Raw 264.7 Macrophages.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease targeting the brain and spinal cord. Non-neuronal cells, including macrophages, may contribute to the disruption of motor neurons (MNs), neuromuscular junction dismantling and clinical signs of ALS. Understanding the modality and the effect of MNs-macrophage communication is pivotal. Here, we focus on extracellular vesicle (EVS)-mediated communication and, in particular, we analyze the response of macrophages. NSC-34 cells transfected with mutant SOD1 (G93A, A4V, G85R, G37R) and differentiated towards MN-like cells, and Raw 264.7 macrophages are the cellular models of the study. mSOD1 NSC-34 cells release a high number of vesicles, both large-lEVs (300 nm diameter) and small-sEVs (90 nm diameter), containing inflammation-modulating molecules, and are efficiently taken up by macrophages. RT-PCR analysis of inflammation mediators demonstrated that the conditioned medium of mSOD1 NSC-34 cells polarizes Raw 264.7 macrophages towards both pro-inflammatory and anti-inflammatory phenotypes. sEVs act on macrophages in a time-dependent manner: an anti-inflammatory response mediated by TGFß firstly starts (12 h); successively, the response shifts towards a pro-inflammation IL-1ß-mediated (48 h). The response of macrophages is strictly dependent on the SOD1 mutation type. The results suggest that EVs impact physiological and behavioral macrophage processes and are of potential relevance to MN degeneration.

SEM/EDX analysis of stomach contents of a sea slug snacking on a polluted seafloor reveal microplastics as a component of its diet.

Understanding the impacts of microplastics on living organisms in aquatic habitats is one of the hottest research topics worldwide. Despite increased attention, investigating microplastics in underwater environments remains a problematic task, due to the ubiquitous occurrence of microplastic, its multiple modes of interactions with the biota, and to the diversity of the synthetic organic polymers composing microplastics in the field. Several studies on microplastics focused on marine invertebrates, but to date, the benthic sea slugs (Mollusca, Gastropoda, Heterobranchia) were not yet investigated. Sea slugs are known to live on the organisms on which they feed on or to snack while gliding over the sea floor, but also as users of exogenous molecules or materials not only for nutrition. Therefore, they may represent a potential biological model to explore new modes of transformation and/or management of plastic, so far considered to be a non-biodegradable polymer. In this study we analysed the stomachal content of Bursatella leachii, an aplysiid heterobranch living in the Mar Piccolo, a highly polluted coastal basin near Taranto, in the northern part of the Ionian Sea. Microplastics were found in the stomachs of all the six sampled specimens, and SEM/EDX analyses were carried out to characterize the plastic debris. The SEM images and EDX spectra gathered here should be regarded as a baseline reference database for future investigations on marine Heterobranchia and their interactions with microplastics.

Micro and Nanoplastics Identification: Classic Methods and Innovative Detection Techniques.

Micro and nanoplastics are fragments with dimensions less than a millimeter invading all terrestrial and marine environments. They have become a major global environmental issue in recent decades and, indeed, recent scientific studies have highlighted the presence of these fragments all over the world even in environments that were thought to be unspoiled. Analysis of micro/nanoplastics in isolated samples from abiotic and biotic environmental matrices has become increasingly common. Hence, the need to find valid techniques to identify these micro and nano-sized particles. In this review, we discuss the current and potential identification methods used in microplastic analyses along with their advantages and limitations. We discuss the most suitable techniques currently available, from physical to chemical ones, as well as the challenges to enhance the existing methods and develop new ones. Microscopical techniques (i.e., dissect, polarized, fluorescence, scanning electron, and atomic force microscopy) are one of the most used identification methods for micro/nanoplastics, but they have the limitation to produce incomplete results in analyses of small particles. At present, the combination with chemical analysis (i.e., spectroscopy) overcome this limit together with recently introduced alternative approaches. For example, holographic imaging in microscope configuration images microplastics directly in unfiltered water, thus discriminating microplastics from diatoms and differentiates different sizes, shapes, and plastic types. The development of new analytical instruments coupled with each other or with conventional and innovative microscopy could solve the current problems in the identification of micro/nanoplastics.

Toxicity, Bioaccumulation and Biotransformation of Glucose-Capped Silver Nanoparticles in Green Microalgae Chlorella vulgaris.

Silver nanoparticles (AgNPs) are one of the most widely used nanomaterials in consumer products. When discharged into the aquatic environment AgNPs can cause toxicity to aquatic biota, through mechanisms that are still under debate, thus rendering the nanoparticles (NPs) effects evaluation a necessary step. Different aquatic organism models, i.e., microalgae, mussels, Daphnia magna, sea urchins and Danio rerio, etc. have been largely exploited for NPs toxicity assessment. On the other hand, alternative biological microorganisms abundantly present in nature, i.e., microalgae, are nowadays exploited as a potential sink for removal of toxic substances from the environment. Indeed, the green microalgae Chlorella vulgaris is one of the most used microorganisms for waste treatment. With the aim to verify the possible involvement of C. vulgaris not only as a model microorganism of NPs toxicity but also for the protection toward NPs pollution, we used these microalgae to measure the AgNPs biotoxicity and bioaccumulation. In particular, to exclude any toxicity derived by Ag+ ions release, green chemistry-synthesised and glucose-coated AgNPs (AgNPs-G) were used. C. vulgaris actively internalised AgNPs-G whose amount increases in a time- and dose-dependent manner. The internalised NPs, found inside large vacuoles, were not released back into the medium, even after 1 week, and did not undergo biotransformation since AgNPs-G maintained their crystalline nature. Biotoxicity of AgNPs-G causes an exposure time and AgNPs-G dose-dependent growth reduction and a decrease in chlorophyll-a amount. These results confirm C. vulgaris as a bioaccumulating microalgae for possible use in environmental protection.

Novel Therapeutic Delivery of Nanocurcumin in Central Nervous System Related Disorders.

Nutraceuticals represent complementary or alternative beneficial products to the expensive and high-tech therapeutic tools in modern medicine. Nowadays, their medical or health benefits in preventing or treating different types of diseases is widely accepted, due to fewer side effects than synthetic drugs, improved bioavailability and long half-life. Among herbal and natural compounds, curcumin is a very attractive herbal supplement considering its multipurpose properties. The potential effects of curcumin on glia cells and its therapeutic and protective properties in central nervous system (CNS)-related disorders is relevant. However, curcumin is unstable and easily degraded or metabolized into other forms posing limits to its clinical development. This is particularly important in brain pathologies determined blood brain barrier (BBB) obstacle. To enhance the stability and bioavailability of curcumin, many studies focused on the design and development of curcumin nanodelivery systems (nanoparticles, micelles, dendrimers, and diverse nanocarriers). These nanoconstructs can increase curcumin stability, solubility, in vivo uptake, bioactivity and safety. Recently, several studies have reported on a curcumin exosome-based delivery system, showing great therapeutical potential. The present work aims to review the current available data in improving bioactivity of curcumin in treatment or prevention of neurological disorders.

Sonication-Assisted Production of Fosetyl-Al Nanocrystals: Investigation of Human Toxicity and In Vitro Antibacterial Efficacy against Xylella Fastidiosa.

Recently, there is a growing demand in sustainable phytopathogens control research. Nanotechnology provides several tools such as new pesticides formulations, antibacterial nanomaterials and smart delivery systems. Metal nano-oxides and different biopolymers have been exploited in order to develop nanopesticides which can offer a targeted solution minimizing side effects on environment and human health. This work proposed a nanotechnological approach to obtain a new formulation of systemic fungicide fosetyl-Al employing ultrasonication assisted production of water dispersible nanocrystals. Moreover, chitosan was applicated as a coating agent aiming a synergistic antimicrobial effect between biopolymer and fungicide. Fosetyl-Al nanocrystals have been characterized by morphological and physical-chemical analysis. Nanotoxicological investigation was carried out on human keratinocytes cells through cells viability test and ultrastructural analysis. In vitro planktonic growth, biofilm production and agar dilution assays have been conducted on two Xylella fastidiosa subspecies. Fosetyl-Al nanocrystals resulted very stable over time and less toxic respect to conventional formulation. Finally, chitosan-based fosetyl-Al nanocrystals showed an interesting antibacterial activity against Xylella fastidiosa subsp. pauca and Xylella fastidiosa subsp. fastidiosa.

Clinical isolates of the modern Mycobacterium tuberculosis lineage 4 evade host defense in human macrophages through eluding IL-1ß-induced autophagy.

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), has infected over 1.7 billion people worldwide and causes 1.4 million deaths annually. Recently, genome sequence analysis has allowed the reconstruction of Mycobacterium tuberculosis complex (MTBC) evolution, with the identification of seven phylogeographic lineages: four referred to as evolutionarily "ancient", and three "modern". The MTBC strains belonging to "modern" lineages appear to show enhanced virulence that may have warranted improved transmission in humans over ancient lineages through molecular mechanisms that remain to be fully characterized. To evaluate the impact of MTBC genetic diversity on the innate immune response, we analyzed intracellular bacterial replication, inflammatory cytokine levels, and autophagy response in human primary macrophages infected with MTBC clinical isolates belonging to the ancient lineages 1 and 5, and the modern lineage 4. We show that, when compared to ancient lineage 1 and 5, MTBC strains belonging to modern lineage 4 show a higher rate of replication, associated to a significant production of proinflammatory cytokines (IL-1ß, IL-6, and TNF-α) and induction of a functional autophagy process. Interestingly, we found that the increased autophagic flux observed in macrophages infected with modern MTBC is due to an autocrine activity of the proinflammatory cytokine IL-1ß, since autophagosome maturation is blocked by an interleukin-1 receptor antagonist. Unexpectedly, IL-1ß-induced autophagy is not disadvantageous for the survival of modern Mtb strains, which reside within Rab5-positive phagosomal vesicles and avoid autophagosome engulfment. Altogether, these results suggest that autophagy triggered by inflammatory cytokines is compatible with a high rate of intracellular bacilli replication and may therefore contribute to the increased pathogenicity of the modern MTBC lineages.

Glucose capped silver nanoparticles induce cell cycle arrest in HeLa cells.

This study aims to determine the interaction (uptake and biological effects on cell viability and cell cycle progression) of glucose capped silver nanoparticles (AgNPs-G) on human epithelioid cervix carcinoma (HeLa) cells, in relation to amount, 2×103 or 2×104 NPs/cell, and exposure time, up to 48h. The spherical and well dispersed AgNPs (30±5nm) were obtained by using glucose as reducing agent in a green synthesis method that ensures to stabilize AgNPs avoiding cytotoxic soluble silver ions Ag+ release. HeLa cells take up abundantly and rapidly AgNPs-G resulting toxic to cells in amount and incubation time dependent manner. HeLa cells were arrested at S and G2/M phases of the cell cycle and subG1 population increased when incubated with 2×104 AgNPs-G/cell. Mitotic index decreased accordingly. The dissolution experiments demonstrated that the observed effects were due only to AgNPs-G since glucose capping prevents Ag+ release. The AgNPs-G influence on HeLa cells viability and cell cycle progression suggest that AgNPs-G, alone or in combination with chemotherapeutics, may be exploited for the development of novel antiproliferative treatment in cancer therapy. However, the possible influence of the cell cycle on cellular uptake of AgNPs-G and the mechanism of AgNPs entry in cells need further investigation.

Microscopies at the Nanoscale for Nano-Scale Drug Delivery Systems.

One of the frontier of nanoscience is undoubtedly represented by the use of nanotechnologies in the pharmaceutical research. During the last decades a big family of nanostructures that have a surface-acting action, such as NanoParticles (NPs), lipid nanocarriers and many more, have been developed to be used as Drug Delivery Systems (DDSs). However, these nanocarriers opened also new frontiers in nanometrology, requiring an accurate morphological characterization, near atomic resolution, before they are really available to clinicians to ascertain their elemental composition, to exclude the presence of contaminants introduced during the synthesis procedure and to ensure biocompatibility. Classical Transmission (TEM) and Scanning Electron Microscopy (SEM) techniques frequently have to be adapted for an accurate analysis of formulation morphology, especially in case of hydrated colloidal systems. Specific techniques such as environmental scanning microscopy and/or cryo preparation are required for their investigation. Analytical Electron Microscopy (AEM) techniques such as Electron Energy-Loss Spectroscopy (EELS) or Energy-Dispersive X-ray Spectroscopy (EDXS) are additional assets to determine the elemental composition of the systems. Here we will discuss the importance of Electron Microscopy (EM) as a reliable tool in the pharmaceutical research of the 21(st) century, focalizing our attention on advantages and limitations of different kind of NPs (in particular silver and carbon NPs, cubosomes) and vesicles (liposomes and niosomes).

Administration Dependent Antioxidant Effect of Carica papaya Seeds Water Extract.

Carica papaya is widely used in folk medicine as herbal remedy to prevent, protect against, and cure several diseases. These curative properties are based on the presence in different parts of the plant of phytochemical nutrients with antioxidant effect. Seeds are the less exploited part; thus this study is aimed at assessing the antioxidant activities of the C. papaya seeds water extract against hydrogen peroxide (H2O2) oxidative stress in human skin Detroit 550 fibroblasts. C. papaya seeds water extract is not toxic and acts as a potent free radical scavenger, providing protection to Detroit 550 fibroblasts that underwent H2O2 oxidative stress. Data show that (i) the maximum protective effect is achieved by the simultaneous administration of the extract with 1 mM H2O2; (ii) the extract in presence of an oxidative stress does not increase catalase activity and prevents the release of cytochrome C and the inner mitochondrial transmembrane potential (Δψ m ) loss; (iii) the extract is more efficient than vitamin C to hamper the oxidative damage; (iv) the purified subfractions of the seeds water extract exert the same antioxidant effect of whole extract. In conclusion, C. papaya seeds water extract is potentially useful for protection against oxidative stress.