Characterization of a fluorescent 1,8-naphthalimide-functionalized PAMAM dendrimer and its Cu(ii) complexes as cytotoxic drugs: EPR and biological studies in myeloid tumor cells.

The activity and interacting ability of a polyamidoamine (PAMAM) dendrimer modified with 4-N-methylpiperazine-1,8-naphthalimide units (termed D) and complexed by Cu(ii) ions, towards healthy and cancer cells were studied. Comparative electron paramagnetic resonance (EPR) studies of the Cu(ii)-D complex are presented: coordination mode, chemical structure, flexibility and stability of these complexes, in the absence and presence of myeloid cancer cells and peripheral blood mononuclear cells (PBMC). The interactions of Cu(ii) ions in the biological media at different equilibrium times were studied, highlighting different stability and interacting conditions with the cells. Furthermore, flow cytometry and confocal analysis, trace the peculiar properties of the dendrimers in PBMC and U937 cells. Indeed, a new probe (Fly) was used as a potential fluorescent tool for biological imaging of Cu(ii). The study highlights that dendrimer and, mainly, the Cu(ii) metallodendrimer are cytotoxic agents for the cells, specifically for U937 tumor cells, inducing mitochondrial dysfunction, ROS increase and lysosome involvement. The metallodendrimer shows antitumor selectivity, fewer affecting healthy PBMC, inducing a massive apoptotic cell death on U937 cells, in line with the high stability of this complex, as verified by EPR studies. The results underline the potentiality of this metallodendrimer to be used as anticancer drug.

Extracellular Vesicles from Campylobacter jejuni CDT-Treated Caco-2 Cells Inhibit Proliferation of Tumour Intestinal Caco-2 Cells and Myeloid U937 Cells: Detailing the Global Cell Response for Potential Application in Anti-Tumour Strategies.

Cytolethal distending toxin (CDT) is produced by a range of Gram-negative pathogenic bacteria such as Campylobacter jejuni. CDT represents an important virulence factor that is a heterotrimeric complex composed of CdtA, CdtB, and CdtC. CdtA and CdtC constitute regulatory subunits whilst CdtB acts as the catalytic subunit exhibiting phosphatase and DNase activities, resulting in cell cycle arrest and cell death. Extracellular vesicle (EV) secretion is an evolutionarily conserved process that is present throughout all kingdoms. Mammalian EVs play important roles in regular cell-to-cell communications but can also spread pathogen- and host-derived molecules during infections to alter immune responses. Here, we demonstrate that CDT targets the endo-lysosomal compartment, partially evading lysosomal degradation and exploiting unconventional secretion (EV release), which is largely involved in bacterial infections. CDT-like effects are transferred by Caco-2 cells to uninfected heterologous U937 and homologous Caco-2 cells. The journey of EVs derived from CDT-treated Caco-2 cells is associated with both intestinal and myeloid tumour cells. EV release represents the primary route of CDT dissemination, revealing an active toxin as part of the cargo. We demonstrated that bacterial toxins could represent suitable tools in cancer therapy, highlighting both the benefits and limitations. The global cell response involves a moderate induction of apoptosis and autophagic features may play a protective role against toxin-induced cell death. EVs from CDT-treated Caco-2 cells represent reliable CDT carriers, potentially suitable in colorectal cancer treatments. Our data present a potential bacterial-related biotherapeutic supporting a multidrug anticancer protocol.

Fluorescent Silica Nanoparticles Targeting Mitochondria: Trafficking in Myeloid Cells and Application as Doxorubicin Delivery System in Breast Cancer Cells.

Fluorescent silica nanoparticles (SiNPs) appear to be a promising imaging platform, showing a specific subcellular localization. In the present study, we first investigated their preferential mitochondrial targeting in myeloid cells, by flow cytometry, confocal microscopy and TEM on both cells and isolated mitochondria, to acquire knowledge in imaging combined with therapeutic applications. Then, we conjugated SiNPs to one of the most used anticancer drugs, doxorubicin (DOX). As an anticancer agent, DOX has high efficacy but also an elevated systemic toxicity, causing multiple side effects. Nanostructures are usually employed to increase the drug circulation time and accumulation in target tissues, reducing undesired cytotoxicity. We tested these functionalized SiNPs (DOX-NPs) on breast cancer cell line MCF-7. We evaluated DOX-NP cytotoxicity, the effect on the cell cycle and on the expression of CD44 antigen, a molecule involved in adhesion and in tumor invasion, comparing DOX-NP to free DOX and stand-alone SiNPs. We found a specific ability to release a minor amount of CD44+ extracellular vesicles (EVs), from both CD81 negative and CD81 positive pools. Modulating the levels of CD44 at the cell surface in cancer cells is thus of great importance for disrupting the signaling pathways that favor tumor progression.

Synthesis and biological characterization of a new fluorescent probe for vesicular trafficking based on polyazamacrocycle derivative.

The fluorescent probes represent an important tool in the biological study, in fact characterization of cellular structures and organelles are an important tool-target for understanding the mechanisms regulating most biological processes. Recently, a series of polyamino-macrocycles based on 1,4,7,10-tetraazacyclododecane was synthesized, bearing one or two NBD units (AJ2NBD·4HCl) useful as sensors for metal cations and halides able to target and to detect apolar environment, as lipid membranes. In this paper, we firstly illustrate the chemical synthesis of the AJ2NBD probe, its electronic absorption spectra and its behavior regarding pH of the environment. Lack of any cellular toxicity and an efficient labelling on fresh, living cells was demonstrated, allowing the use of AJ2NBD in biological studies. In particular, this green fluorescent probe may represent a potential dye for the compartments involved in the endosomal/autophagic pathway. This research's field should benefit from the use of AJ2NBD as a vesicular tracer, however, to ensure the precise nature of vesicles/vacuoles traced by this new probe, other more specific tests are needed.

Rapamycin Re-Directs Lysosome Network, Stimulates ER-Remodeling, Involving Membrane CD317 and Affecting Exocytosis, in Campylobacter Jejuni-Lysate-Infected U937 Cells.

The Gram-negative Campylobacter jejuni is a major cause of foodborne gastroenteritis in humans worldwide. The cytotoxic effects of Campylobacter have been mainly ascribed to the actions of the cytolethal distending toxin (CDT): it is mandatory to put in evidence risk factors for sequela development, such as reactive arthritis (ReA) and Guillain-Barré syndrome (GBS). Several researches are directed to managing symptom severity and the possible onset of sequelae. We found for the first time that rapamycin (RM) is able to largely inhibit the action of C. jejuni lysate CDT in U937 cells, and to partially avoid the activation of specific sub-lethal effects. In fact, we observed that the ability of this drug to redirect lysosomal compartment, stimulate ER-remodeling (highlighted by ER-lysosome and ER-mitochondria contacts), protect mitochondria network, and downregulate CD317/tetherin, is an important component of membrane microdomains. In particular, lysosomes are involved in the process of the reduction of intoxication, until the final step of lysosome exocytosis. Our results indicate that rapamycin confers protection against C. jejuni bacterial lysate insults to myeloid cells.

Curcumin-1,2,3-Triazole Conjugation for Targeting the Cancer Apoptosis Machinery.

The burden of neoplastic diseases is widely recognized as a severe cause of mortality. The clinical inadequacy of most anticancer therapeutics urgently prompted intense drug discovery efforts toward the identification of new chemical entities endowed with a potent and safe antitumor profile. In this scenario, targeting cancer cells apoptosis machinery has emerged as a relevant strategy, useful for tackling the emergence of drug resistance. On this basis, a small library of naturally inspired hybrid molecules was obtained by combining, through a click chemistry approach, "privileged" synthons such as curcumin scaffold and 1,2,3-triazole building block. Compound 1, bearing a para-fluoro phenyl moiety, showed low-micromolar potency against T acute lymphoblastic leukemia cell growth. More in-depth biologic studies demonstrated, for this analog, cell death-inducing properties associated with its capability to simultaneously activate both the receptor and the mitochondrial apoptosis cascades. This peculiar behavior offers promises for achieving an expanded anticancer effect, namely intense cytotoxic response coupled with reduced predisposition of chemoresistance insurgence. Altogether, this study allowed the identification of compound 1 as a lead compound worth to be progressed as an anticancer drug candidate.

Melatonin Modulates Cell Cycle Dynamics and Promotes Hippocampal Cell Proliferation After Ischemic Injury in Neonatal Rats.

Promoting neural cell proliferation may represent an important strategy for enhancing brain repair after developmental brain injury. The present study aimed to assess the effects of melatonin on cell proliferation after an ischemic injury in the developing hippocampus, focusing on cell cycle dynamics. After in vivo neonatal hypoxia-ischemia (HI), hippocampal cell cycle dynamics were assessed by flow cytometry, together with histological evaluation of dentate gyrus cellularity and proliferation. Melatonin significantly increased the number of proliferating cells in the G2/M phase as well as the proliferating cell nuclear antigen (PCNA) and doublecortin (DCX) labeling reduced by HI. In vivo BrdU labeling revealed a higher BrdU-positivity in the dentate gyrus of ischemic rats treated with melatonin, an effect followed by increased cellularity and preserved hippocampal tissue integrity. These results indicate that the protective effect of melatonin after ischemic injury in neonatal rats may rely on the modulation of cell cycle dynamics of newborn hippocampal cells and increased cell proliferation.

Efficient and highly reproducible production of red blood cell-derived extracellular vesicle mimetics for the loading and delivery of RNA molecules.

Extracellular vesicles (EVs) are promising natural nanocarriers for the delivery of therapeutic agents. As with any other kind of cell, red blood cells (RBCs) produce a limited number of EVs under physiological and pathological conditions. Thus, RBC-derived extracellular vesicles (RBCEVs) have been recently suggested as next-generation delivery systems for therapeutic purposes. In this paper, we show that thanks to their unique biological and physicochemical features, RBCs can be efficiently pre-loaded with several kinds of molecules and further used to generate RBCEVs. A physical vesiculation method, based on "soft extrusion", was developed, producing an extremely high yield of cargo-loaded RBCEV mimetics. The RBCEVs population has been deeply characterized according to the new guidelines MISEV2023, showing great homogeneity in terms of size, biological features, membrane architecture and cargo. In vitro preliminary results demonstrated that RBCEVs are abundantly internalized by cells and exert peculiar biological effects. Indeed, efficient loading and delivery of miR-210 by RBCEVs to HUVEC has been proven, as well as the inhibition of a known mRNA target. Of note, the bench-scale process can be scaled-up and translated into clinics. In conclusion, this investigation could open the way to a new biomimetic platform for RNA-based therapies and/or other therapeutic cargoes useful in several diseases.

Flow cytometric analysis of hepatopancreatic cells from Armadillidium vulgare highlights terrestrial isopods as efficient environmental bioindicators in ex vivo settings.

Several studies have reported the high bioindication capacity of Isopoda (Crustacea, Oniscidea), which is related to their important ability to accumulate contaminants, usefulness in soil ecotoxicology and bioindication activities. Any change in the isopod population, diversity and life cycle can indicate relevant pollution levels. The analysis of target tissues, such as the hepatopancreas, is another emerging approach (from a cytologic/histological level) to detect contaminant accumulation from different sources. In this study, tissue disaggregation procedures were optimised in the hepatopancreas, and flow cytometry (FC) was applied to detect cell viability and several cell functions. After disaggregation, two hepatopancreatic cell types, small (S) and big (B), were still recognisable: they differed in morphology and behaviour. The analyses were conducted for the first time on isopods from sites under different conditions of ecological disturbance through cytometric re-interpretation of ecological-environmental parameters. Significant differences in cell functional parameters were found, highlighting that isopod hepatopancreatic cells can be efficiently analysed by FC and represent standardisable, early biological indicators, tracing environmental-induced stress through cytologic/histologic analyses.

Extracellular Vesicles as New Players in Drug Delivery: A Focus on Red Blood Cells-Derived EVs.

The article is divided into several sections, focusing on extracellular vesicles' (EVs) nature, features, commonly employed methodologies and strategies for their isolation/preparation, and their characterization/visualization. This work aims to give an overview of advances in EVs' extensive nanomedical-drug delivery applications. Furthermore, considerations for EVs translation to clinical application are summarized here, before focusing the review on a special kind of extracellular vesicles, the ones derived from red blood cells (RBCEVs). Generally, employing EVs as drug carriers means managing entities with advantageous properties over synthetic vehicles or nanoparticles. Besides the fact that certain EVs also reveal intrinsic therapeutic characteristics, in regenerative medicine, EVs nanosize, lipidomic and proteomic profiles enable them to pass biologic barriers and display cell/tissue tropisms; indeed, EVs engineering can further optimize their organ targeting. In the second part of the review, we focus our attention on RBCEVs. First, we describe the biogenesis and composition of those naturally produced by red blood cells (RBCs) under physiological and pathological conditions. Afterwards, we discuss the current procedures to isolate and/or produce RBCEVs in the lab and to load a specific cargo for therapeutic exploitation. Finally, we disclose the most recent applications of RBCEVs at the in vitro and preclinical research level and their potential industrial exploitation. In conclusion, RBCEVs can be, in the near future, a very promising and versatile platform for several clinical applications and pharmaceutical exploitations.

Clozapine suppresses NADPH oxidase activation, counteracts cytosolic H2O2, and triggers early onset mitochondrial dysfunction during adipogenesis of human liposarcoma SW872 cells.

Long-term treatment of schizophrenia with clozapine (CLZ), an atypical antipsychotic drug, is associated with an increased incidence of metabolic disorders mediated by poorly understood mechanisms. We herein report that CLZ, while slowing down the morphological changes and lipid accumulation occurring during SW872 cell adipogenesis, also causes an early (day 3) inhibition of the expression/nuclear translocation of CAAT/enhancer-binding protein ß and peroxisome proliferator-activated receptor γ. Under the same conditions, CLZ blunts NADPH oxidase-derived reactive oxygen species (ROS) by a dual mechanism involving enzyme inhibition and ROS scavenging. These effects were accompanied by hampered activation of the nuclear factor (erythroid-derived2)-like 2 (Nrf2)-dependent antioxidant responses compared to controls, and by an aggravated formation of mitochondrial superoxide. CLZ failed to exert ROS scavenging activities in the mitochondrial compartment but appeared to actively scavenge cytosolic H2O2 derived from mitochondrial superoxide. The early formation of mitochondrial ROS promoted by CLZ was also associated with signs of mitochondrial dysfunction. Some of the above findings were recapitulated using mouse embryonic fibroblasts. We conclude that the NADPH oxidase inhibitory and cytosolic ROS scavenging activities of CLZ slow down SW872 cell adipogenesis and suppress their Nrf2 activation, an event apparently connected with increased mitochondrial ROS formation, which is associated with insulin resistance and metabolic syndrome. Thus, the cellular events characterised herein may help to shed light on the more detailed molecular mechanisms explaining some of the adverse metabolic effects of CLZ.

Automated-Mechanical Procedure Compared to Gentle Enzymatic Tissue Dissociation in Cell Function Studies.

The first step to obtain a cellular suspension from tissues is the disaggregation procedure. The cell suspension method has to provide a representative sample of the different cellular subpopulations and to maximize the number of viable functional cells. Here, we analyzed specific cell functions in cell suspensions from several rat tissues obtained by two different methods, automated-mechanical and enzymatic disaggregation. Flow cytometric, confocal, and ultrastructural (TEM) analyses were applied to the spleen, testis, liver and other tissues. Samples were treated by an enzymatic trypsin solution or processed by the Medimachine II (MMII). The automated-mechanical and enzymatic disaggregation procedures have shown to work similarly in some tissues, which displayed comparable amounts of apoptotic/necrotic cells. However, cells obtained by the enzyme-free Medimachine II protocols show a better preservation lysosome and mitochondria labeling, whereas the enzymatic gentle dissociation appears to constantly induce a lower amount of intracellular ROS; nevertheless, lightly increased ROS can be recognized as a complimentary signal to promote cell survival. Therefore, MMII represents a simple, fast, and standardized method for tissue processing, which allows to minimize bias arising from the operator's ability. Our study points out technical issues to be adopted for specific organs and tissues to obtain functional cells.

A microfluidic approach to fabricate sucrose decorated liposomes with increased uptake in breast cancer cells.

Developing targeted drug delivery systems is an urgent need to decrease the side effects and increase the drug's efficiency. Most cancer cells show an increased sugar consumption compared to healthy cells due to the deregulation of sugar transporters. Consequently, liposomes, as a biocompatible nanocarrier, could be surface decorated by sugars to enhance drug targeting into cancer cells. Our work outlines a new strategy to easily manufacture sucrose decorated liposomes using sucrose stearate, a biocompatible and biodegradable non-ionic surfactant, with a scalable microfluidic approach. Sucrose decorated liposomes were loaded with berberine hydrochloride, a well-known phytochemical compound to investigate its effects on triple-negative breast cancer cells (MDA-MB-231). Using the microfluidic manufacturing system, we prepared berberine-loaded liposomes using a mixture of phosphatidylcholine and cholesterol with and without sucrose stearate with a size up to 140 nm and narrow polydispersity. Stability was confirmed for 90 days, and the in vitro release profile was evaluated. The formulations showed acceptable in vitro biocompatibility and significantly higher anti-proliferative effect on MDA-MB-231 cell line. These results have been confirmed by an increased uptake evaluated by flow cytometry and confocal microscopy. Taken together, our findings represent an innovative, easy, and scalable approach to obtain sugar decorated liposomal formulations without any surface-chemistry reactions. They can be potentially used as an anticancer targeted drug delivery system.

Temporal correlation of morphological and biochemical changes with the recruitment of different mechanisms of reactive oxygen species formation during human SW872 cell adipogenic differentiation.

Human SW872 preadipocyte conversion to mature adipocytes is associated with time-dependent changes in differentiation markers' expression and with morphological changes accompanied by the accumulation of lipid droplets (LDs) as well as by increased mitochondriogenesis and mitochondrial membrane potential. Under identical conditions, the formation of reactive oxygen species (ROS) revealed with a general probe was significant at days 3 and 10 of differentiation and bearly detectable at day 6. NADPH oxidase (NOX)-2 activity determined with an immunocytochemical approach followed a very similar pattern. There was no evidence of mitochondrial ROS (mROS), as detected with a selective fluorescence probe, at days 3 and 6, possibly due to the triggering of the Nrf-2 antioxidant response. mROS were instead clearly detected at day 10, concomitantly with the accumulation of very large LDs, oxidation of both cardiolipin and thioredoxin 2, and decreased mitochondrial glutathione. In conclusion, the morphological and biochemical changes of differentiating SW872 cells are accompanied by the discontinuous formation of ROS derived from NOX-2, increasingly implicated in adipogenesis and adipose tissue dysfunction. In addition, mROS formation was significant only in the late phase of differentiation and was associated with mitochondrial dysfunction.

Which ones, when and why should renin-angiotensin system inhibitors work against COVID-19?

The article describes the possible pathophysiological origin of COVID-19 and the crucial role of renin-angiotensin system (RAS), providing several "converging" evidence in support of this hypothesis. SARS-CoV-2 has been shown to initially upregulate ACE2 systemic activity (early phase), which can subsequently induce compensatory responses leading to upregulation of both arms of the RAS (late phase) and consequently to critical, advanced and untreatable stages of COVID-19 disease. The main and initial actors of the process are ACE2 and ADAM17 zinc-metalloproteases, which, initially triggered by SARS-CoV-2 spike proteins, work together in increasing circulating Ang 1-7 and Ang 1-9 peptides and downstream (Mas and Angiotensin type 2 receptors) pathways with anti-inflammatory, hypotensive and antithrombotic activities. During the late phase of severe COVID-19, compensatory secretion of renin and ACE enzymes are subsequently upregulated, leading to inflammation, hypertension and thrombosis, which further sustain ACE2 and ADAM17 upregulation. Based on this hypothesis, COVID-19-phase-specific inhibition of different RAS enzymes is proposed as a pharmacological strategy against COVID-19 and vaccine-induced adverse effects. The aim is to prevent the establishment of positive feedback-loops, which can sustain hyperactivity of both arms of the RAS independently of viral trigger and, in some cases, may lead to Long-COVID syndrome.

Lock, Stock and Barrel: Role of Renin-Angiotensin-Aldosterone System in Coronavirus Disease 2019.

Since the end of 2019, the medical-scientific community has been facing a terrible pandemic caused by a new airborne viral agent known as SARS-CoV2. Already in the early stages of the pandemic, following the discovery that the virus uses the ACE2 cell receptor as a molecular target to infect the cells of our body, it was hypothesized that the renin-angiotensin-aldosterone system was involved in the pathogenesis of the disease. Since then, numerous studies have been published on the subject, but the exact role of the renin-angiotensin-aldosterone system in the pathogenesis of COVID-19 is still a matter of debate. RAAS represents an important protagonist in the pathogenesis of COVID-19, providing the virus with the receptor of entry into host cells and determining its organotropism. Furthermore, following infection, the virus is able to cause an increase in plasma ACE2 activity, compromising the normal function of the RAAS. This dysfunction could contribute to the establishment of the thrombo-inflammatory state characteristic of severe forms of COVID-19. Drugs targeting RAAS represent promising therapeutic options for COVID-19 sufferers.

Elaborated study of Cu(II) carbosilane metallodendrimers bearing substituted iminopyridine moieties as antitumor agents.

Iminopyridine-decorated carbosilane metallodendrimers have recently emerged as a promising strategy in the treatment of cancer diseases. Their unique features such as the nanometric size, the multivalent nature and the structural perfection offer an extraordinary platform to explore structure-to-property relationships. Herein, we showcase the outstanding impact on the antitumor activity of a parameter not explored before: the iminopyridine substituents in meta position. New Cu(II) carbosilane metallodendrimers, bearing methyl or methoxy substituents in the pyridine ring, were synthesized and thoroughly characterized. Electron Paramagnetic Resonance (EPR) was exploited to unveil the properties of the metallodendrimers. This study confirmed the presence of different coordination modes of the Cu(II) ion (Cu-N2O2, Cu-N4 and Cu-O4), whose ratios were determined by the structural features of the dendritic molecules. These metallodendrimers exhibited IC50 values in the low micromolar range (<6 µM) in tumor cell lines such as HeLa and MCF-7. The subsequent in vitro assays on both healthy (PBMC) and tumor (U937) myeloid cells revealed two key facts which improved the cytotoxicity and selectivity of the metallodrug: First, maximizing the Cu-N2O2 coordination mode; second, adequately selecting the pair ring-substituent/metal-counterion. The most promising candidates, G1(-CH3)Cl (8) and G1(-OCH3)NO3(17), exhibited a substantial increase in the antitumor activity in U937 tumor cells, compared to the non-substituted counterparts, probably through two different ROS-production pathways.

Uptake and Intracellular Trafficking Studies of Multiple Dye-Doped Core-Shell Silica Nanoparticles in Lymphoid and Myeloid Cells.

INTRODUCTION: Since most biologically active macromolecules are natural nanostructures, operating in the same scale of biomolecules gives the great advantage to enhance the interaction with cellular components. Noteworthy efforts in nanotechnology, particularly in biomedical and pharmaceutical fields, have propelled a high number of studies on the biological effects of nanomaterials. Moreover, the determination of specific physicochemical properties of nanomaterials is crucial for the evaluation and design of novel safe and efficient therapeutics and diagnostic tools. In this in vitro study, we report a physicochemical characterisation of fluorescent silica nanoparticles (NPs), interacting with biological models (U937 and PBMC cells), describing the specific triggered biologic response. METHODS: Flow Cytometric and Confocal analyses are the main method platforms. However TEM, NTA, DLS, and chemical procedures to synthesize NPs were employed. RESULTS: NTB700 NPs, employed in this study, are fluorescent core-shell silica nanoparticles, synthesized through a micelle-assisted method, where the fluorescence energy transfer process, known as FRET, occurs at a high efficiency rate. Using flow cytometry and confocal microscopy, we observed that NTB700 NP uptake seemed to be a rapid, concentration-, energy- and cell type-dependent process, which did not induce significant cytotoxic effects. We did not observe a preferred route of internalization, although their size and the possible aggregated state could influence their extrusion. At this level of analysis, our investigation focuses on lysosome and mitochondria pathways, highlighting that both are involved in NP co-localization. Despite the main mitochondria localization, NPs did not induce a significant increase of intracellular ROS, known inductors of apoptosis, during the time course of analyses. Finally, both lymphoid and myeloid cells are able to release NPs, essential to their biosafety. DISCUSSION: These data allow to consider NTB700 NPs a promising platform for future development of a multifunctional system, by combining imaging and localized therapeutic applications in a unique tool.