Impact of the antibiotic-cargo from MSNs on Gram-positive and Gram-negative bacterial biofilms.

Mesoporous silica nanoparticles (MSNs) are promising drug nanocarriers for infection treatment. Many investigations have focused on evaluating the capacity of MSNs to encapsulate antibiotics and release them in a controlled fashion. However, little attention has been paid to determine the antibiotic doses released from these nanosystems that are effective against biofilm during the entire release time. Herein, we report a systematic and quantitative study of the direct effect of the antibiotic-cargo released from MSNs on Gram-positive and Gram-negative bacterial biofilms. Levofloxacin (LVX), gentamicin (GM) and rifampin (RIF) were separately loaded into pure-silica and amino-modified MSNs. This accounts for the versatility of these nanosystems since they were able to load and release different antibiotic molecules of diverse chemical nature. Biological activity curves of the released antibiotic were determined for both bacterial strains, which allowed to calculate the active doses that are effective against bacterial biofilms. Furthermore, in vitro biocompatibility assays on osteoblast-like cells were carried out at different periods of times. Albeit a slight decrease in cell viability was observed at the very initial stage, due to the initial burst antibiotic release, the biocompatibility of these nanosystems is evidenced since a recovery of cell viability was achieved after 72 h of assay. Biological activity curves for GM released from MSNs exhibited sustained patterns and antibiotic doses in the 2-6 µg/mL range up to 100 h, which were not enough to eradicate biofilm. In the case of LVX and RIF first-order kinetics featuring an initial burst effect followed by a sustained release above the MIC up to 96 h were observed. Such doses reduced by 99.9% bacterial biofilm and remained active up to 72 h with no emergence of bacterial resistance. This pioneering research opens up promising expectations in the design of personalized MSNs-based nanotherapies to treat chronic bone infection.

Hypoxia mimetic activity of VCE-004.8, a cannabidiol quinone derivative: implications for multiple sclerosis therapy.

BACKGROUND: Multiple sclerosis (MS) is characterized by a combination of inflammatory and neurodegenerative processes variously dominant in different stages of the disease. Thus, immunosuppression is the goal standard for the inflammatory stage, and novel remyelination therapies are pursued to restore lost function. Cannabinoids such as 9Δ-THC and CBD are multi-target compounds already introduced in the clinical practice for multiple sclerosis (MS). Semisynthetic cannabinoids are designed to improve bioactivities and druggability of their natural precursors. VCE-004.8, an aminoquinone derivative of cannabidiol (CBD), is a dual PPARγ and CB2 agonist with potent anti-inflammatory activity. Activation of the hypoxia-inducible factor (HIF) can have a beneficial role in MS by modulating the immune response and favoring neuroprotection and axonal regeneration. METHODS: We investigated the effects of VCE-004.8 on the HIF pathway in different cell types. The effect of VCE-004.8 on macrophage polarization and arginase 1 expression was analyzed in RAW264.7 and BV2 cells. COX-2 expression and PGE2 synthesis induced by lipopolysaccharide (LPS) was studied in primary microglia cultures. The efficacy of VCE-004.8 in vivo was evaluated in two murine models of MS such as experimental autoimmune encephalomyelitis (EAE) and Theiler's virus-induced encephalopathy (TMEV). RESULTS: Herein, we provide evidence that VCE-004.8 stabilizes HIF-1α and HIF-2α and activates the HIF pathway in human microvascular endothelial cells, oligodendrocytes, and microglia cells. The stabilization of HIF-1α is produced by the inhibition of the prolyl-4-hydrolase activity of PHD1 and PDH2. VCE-004.8 upregulates the expression of HIF-dependent genes such as erythropoietin and VEGFA, induces angiogenesis, and enhances migration of oligodendrocytes. Moreover, VCE-004.8 blunts IL-17-induced M1 polarization, inhibits LPS-induced COX-2 expression and PGE2 synthesis, and induces expression of arginase 1 in macrophages and microglia. In vivo experiments showed efficacy of VCE-004.8 in EAE and TMEV. Histopathological analysis revealed that VCE-004.8 treatments prevented demyelination, axonal damage, and immune cells infiltration. In addition, VCE-004.8 downregulated the expression of several genes closely associated with MS physiopathology, including those underlying the production of chemokines, cytokines, and adhesion molecules. CONCLUSIONS: This study provides new significant insights about the potential role of VCE-004.8 for MS treatment by ameliorating neuroinflammation and demyelination.

Bioinspired extracellular vesicle-coated silica nanoparticles as selective delivery systems.

In recent years, there has been a breakthrough in the integration of artificial nanoplatforms with natural biomaterials for the development of more efficient drug delivery systems. The formulation of bioinspired nanosystems, combining the benefits of synthetic nanoparticles with the natural features of biological materials, provides an efficient strategy to improve nanoparticle circulation time, biocompatibility and specificity toward targeted tissues. Among others biological materials, extracellular vesicles (EVs), membranous structures secreted by many types of cells composed by a protein rich lipid bilayer, have shown a great potential as drug delivery systems themselves and in combination with artificial nanoparticles. The reason for such interest relays on their natural properties, such as overcoming several biological barriers or migration towards specific tissues. Here, we propose the use of mesoporous silica nanoparticles (MSNs) as efficient and versatile nanocarriers in combination with tumor derived extracellular vesicles (EVs) for the development of selective drug delivery systems. The hybrid nanosystems demonstrated selective cellular internalization in parent cells, indicating that the EV targeting capabilities were efficiently transferred to MSNs by the developed coating strategy. As a result, EVs-coated MSNs provided an enhanced and selective intracellular accumulation of doxorubicin and a specific cytotoxic activity against targeted cancer cells, revealing these hybrid nanosystems as promising candidates for the development of targeted treatments.

Biomimetic camouflaged nanoparticles with selective cellular internalization and migration competences.

In the last few years, nanotechnology has revolutionized the potential treatment of different diseases. However, the use of nanoparticles for drug delivery might be limited by their immune clearance, poor biocompatibility and systemic immunotoxicity. Hypotheses for overcoming rejection from the body and increasing their biocompatibility include coating nanoparticles with cell membranes. Additionally, source cell-specific targeting has been reported when coating nanoparticles with tumor cells membranes. Here we show that coating mesoporous silica nanoparticles with membranes derived from preosteoblastic cells could be employed to develop potential treatments of certain bone diseases. These nanoparticles were selected because of their well-established drug delivery features. On the other hand MC3T3-E1 cells were selected because of their systemic migration capabilities towards bone defects. The coating process was here optimized ensuring their drug loading and delivery features. More importantly, our results demonstrated how camouflaged nanocarriers presented cellular selectivity and migration capability towards the preosteoblastic source cells, which might constitute the inspiration for future bone disease treatments. STATEMENT OF SIGNIFICANCE: This work presents a new nanoparticle formulation for drug delivery able to selectively target certain cells. This approach is based on Mesoporous Silica Nanoparticles coated with cell membranes to overcome the potential rejection from the body and increase their biocompatibility prolonging their circulation time. We have employed membranes derived from preosteoblastic cells for the potential treatment of certain bone diseases. Those cells have shown systemic migration capabilities towards bone defects. The coating process was optimized and their appropriate drug loading and releasing abilities were confirmed. The important novelty of this work is that the camouflaged nanocarriers presented cellular selectivity and migration capability towards the preosteoblastic source cells, which might constitute the inspiration for future bone disease treatments.

Bacteria-Assisted Transport of Nanomaterials to Improve Drug Delivery in Cancer Therapy.

Currently, the design of nanomaterials for the treatment of different pathologies is presenting a major impact on biomedical research. Thanks to this, nanoparticles represent a successful strategy for the delivery of high amounts of drugs for the treatment of cancer. Different nanosystems have been designed to combat this pathology. However, the poor penetration of these nanomaterials into the tumor tissue prevents the drug from entering the inner regions of the tumor. Some bacterial strains have self-propulsion and guiding capacity thanks to their flagella. They also have a preference to accumulate in certain tumor regions due to the presence of different chemo-attractants factors. Bioconjugation reactions allow the binding of nanoparticles in living systems, such as cells or bacteria, in a simple way. Therefore, bacteria are being used as a transport vehicle for nanoparticles, facilitating their penetration and the subsequent release of the drug inside the tumor. This review would summarize the literature on the anchoring methods of diverse nanosystems in bacteria and, interestingly, their advantages and possible applications in cancer therapy.

High-throughput RNA sequencing transcriptome analysis of ABC-DLBCL reveals several tumor evasion strategies.

Activated B-cell (ABC) lymphoma, a distinct molecular entity within diffuse large B-cell lymphoma (DLBCL), remains highly incurable, showing a worse response to standard immunochemotherapy. The discouraging results obtained in several clinical trials using proteasome inhibitors, tyrosine kinase inhibitors, or immunomodulators, lead to an intense search for new, potentially druggable biomarkers in DLBCL. In this study, we designed an experimental strategy for DLBCL to discover high- and low-abundance RNA-seq-derived transcripts involved in the oncogenic phenotype in patients diagnosed with ABC-DLBCL. Based on the results of a comparative analysis, 79 DE genes and two enriched gene sets related to metabolism and immunity were selected. Genes related to drug resistance, anti-inflammatory response, and tumor-cell dissemination were found to be up-regulated, while tumor suppressor genes were down-regulated. Then, we searched for the perturbagens most suitable for gene expression profiling (GEP) by iLINCS-CMap. Herein, we present a novel experimental approach that connects the omics signature of DLBCL with potential drugs for more accurate treatments.

A novel CDC25A/DYRK2 regulatory switch modulates cell cycle and survival.

The cell division cycle 25A (CDC25A) phosphatase is a key regulator of cell cycle progression that acts on the phosphorylation status of Cyclin-Cyclin-dependent kinase complexes, with an emergent role in the DNA damage response and cell survival control. The regulation of CDC25A activity and its protein level is essential to control the cell cycle and maintain genomic integrity. Here we describe a novel ubiquitin/proteasome-mediated pathway negatively regulating CDC25A stability, dependent on its phosphorylation by the serine/threonine kinase DYRK2. DYRK2 phosphorylates CDC25A on at least 7 residues, resulting in its degradation independent of the known CDC25A E3 ubiquitin ligases. CDC25A in turn is able to control the phosphorylation of DYRK2 at several residues outside from its activation loop, thus affecting DYRK2 localization and activity. An inverse correlation between DYRK2 and CDC25A protein amounts was observed during cell cycle progression and in response to DNA damage, with CDC25A accumulation responding to the manipulation of DYRK2 levels or activity in either physiological scenario. Functional data show that the pro-survival activity of CDC25A and the pro-apoptotic activity of DYRK2 could be partly explained by the mutual regulation between both proteins. Moreover, DYRK2 modulation of CDC25A expression and/or activity contributes to the DYRK2 role in cell cycle regulation. Altogether, we provide evidence suggesting that DYRK2 and CDC25A mutually control their activity and stability by a feedback regulatory loop, with a relevant effect on the genotoxic stress pathway, apoptosis, and cell cycle regulation.

A versatile multicomponent mesoporous silica nanosystem with dual antimicrobial and osteogenic effects.

In this manuscript, we propose a simple and versatile methodology to design nanosystems based on biocompatible and multicomponent mesoporous silica nanoparticles (MSNs) for infection management. This strategy relies on the combination of antibiotic molecules and antimicrobial metal ions into the same nanosystem, affording a significant improvement of the antibiofilm effect compared to that of nanosystems carrying only one of these agents. The multicomponent nanosystem is based on MSNs externally functionalized with a polyamine dendrimer (MSN-G3) that favors internalization inside the bacteria and allows the complexation of multiactive metal ions (MSN-G3-Mn+). Importantly, the selection of both the antibiotic and the cation may be done depending on clinical needs. Herein, levofloxacin and Zn2+ ion, chosen owing to both its antimicrobial and osteogenic capability, have been incorporated. This dual biological role of Zn2+ could have and adjuvant effect thought destroying the biofilm in combination with the antibiotic as well as aid to the repair and regeneration of lost bone tissue associated to osteolysis during infection process. The versatility of the nanosystem has been demonstrated incorporating Ag+ ions in a reference nanosystem. In vitro antimicrobial assays in planktonic and biofilm state show a high antimicrobial efficacy due to the combined action of levofloxacin and Zn2+, achieving an antimicrobial efficacy above 99% compared to the MSNs containing only one of the microbicide agents. In vitro cell cultures with MC3T3-E1 preosteoblasts reveal the osteogenic capability of the nanosystem, showing a positive effect on osteoblastic differentiation while preserving the cell viability. STATEMENT OF SIGNIFICANCE: A simple and versatile methodology to design biocompatible and multicomponent MSNs based nanosystems for infection management is proposed. These nanosystems, containing two antimicrobial agents, levofloxacin and Zn2+, have been synthetized by external functionalization of MSNs with a polycationic dendrimer (MSNs-G3), which favours its internalization inside the bacteria and lead the complexation with metal ions through the amines of the dendrimer. The nanosystems offer a notable improvement of the antibiofilm effect (above 99%) than both components separately as well as osteogenic capability with positive effect on the osteoblastic differentiation and preserved cell viability.

Betulinic Acid Hydroxamate is Neuroprotective and Induces Protein Phosphatase 2A-Dependent HIF-1α Stabilization and Post-transcriptional Dephosphorylation of Prolyl Hydrolase 2.

Huntington's disease (HD) is a neurodegenerative disorder characterized by unwanted choreatic movements, behavioral and psychiatric disturbances, and dementia. The activation of the hypoxic response pathway through the pharmacological inhibition of hypoxia-inducing factor (HIF) prolyl-hydroxylases (PHDs) is a promising approach for neurodegenerative diseases, including HD. Herein, we have studied the mechanism of action of the compound Betulinic acid hydroxamate (BAH), a hypoximimetic derivative of betulinic acid, and its efficacy against striatal neurodegeneration using complementary approaches. Firstly, we showed the molecular mechanisms through which BAH modifies the activity of the PHD2 prolyl hydroxylase, thus directly affecting HIF-1α stability. BAH treatment reduces PHD2 phosphorylation on Ser-125 residue, responsible for the control of its hydrolase activity. HIF activation by BAH is inhibited by okadaic acid and LB-100 indicating that a protein phosphatase 2A (PP2A) is implicated in the mechanism of action of BAH. Furthermore, in striatal cells bearing a mutated form of the huntingtin protein, BAH stabilized HIF-1α protein, induced Vegf and Bnip3 gene expression and protected against mitochondrial toxin-induced cytotoxicity. Pharmacokinetic analyses showed that BAH has a good brain penetrability and experiments performed in a mouse model of striatal neurodegeneration induced by 3-nitropropionic acid showed that BAH improved the clinical symptoms. In addition, BAH also prevented neuronal loss, decreased reactive astrogliosis and microglial activation, inhibited the upregulation of proinflammatory markers, and improved antioxidant defenses in the brain. Taken together, our results show BAH's ability to activate the PP2A/PHD2/HIF pathway, which may have important implications in the treatment of HD and perhaps other neurodegenerative diseases.

Nanoparticles Coated with Cell Membranes for Biomedical Applications.

Nanoparticles designed for diagnosing and treating different diseases have impacted the scientific research in biomedicine, and are expected to revolutionize the clinic in the near future through a new area called nanomedicine. In the last few years, a new approach in this field has emerged: the use of cell membranes for coating nanoparticles in an attempt to mimic the ability of cells to interface and interact with physiological environments. Although such functions have been replicated through synthetic techniques, many research groups are now employing naturally derived cell membranes to coat different types of nanoparticles in an attempt to improve their performance for a wide range of applications. This review summarizes the literature on nanoparticles coated with cell membranes and, more importantly, aims at inspiring and encouraging new developments to this technology in the biomedical area.

Effect of N-acyl-dopamines on beta cell differentiation and wound healing in diabetic mice.

N-acyl-dopamines are endolipids with neuroprotective, antiinflammatory and immunomodulatory properties. Previously, we showed the ability of these compounds to induce HIF-1α stabilization. Hypoxia and HIF-1α play an important role in the most relevant stages of diabetic pathogenesis. This work analyzes the possible role of these molecules on beta cell differentiation, insulin production and diabetic foot ulcer. Hypoxia response pathway has been characterized in beta-cell differentiation in rat pancreatic acinar cell line and human islet-derived precursor cells. Protein and mRNA expression of key proteins in this process have been analyzed, as well as those involved in beta cells reprogramming. The effect of N-acyl-dopamines on hypoxia response pathway, beta cells reprogramming and insulin production have been studied in both cell types, as well as its role in angiogenesis models in vitro and wound closure in type 2 diabetic mice. Our results show how the hypoxia response pathway is altered during beta cells differentiation, accompanied by an induction of the transcription factor HIF-1α. We demonstrate how some N-acyl-dopamines induce beta cell differentiation and insulin production in two different cell models. In parallel, these endolipids promote angiogenesis in vitro and wound closure in type 2 diabetic mice. These results provide a biological mechanism through which some endolipids could induce beta cell differentiation. We demonstrate how N-acyl-dopamines can modulate insulin production and, in parallel, reverse HIF-1α inhibition in a wound healing model in diabetic mice. Therefore, the potential use of the pharmacological modulation of N-acyl-dopamines may have implications for diabetes prevention and treatment strategies.

Metabolomic profiling of human lung tumor tissues - nucleotide metabolism as a candidate for therapeutic interventions and biomarkers.

Although metabolomics has attracted considerable attention in the field of lung cancer (LC) detection and management, only a very limited number of works have applied it to tissues. As such, the aim of this study was the thorough analysis of metabolic profiles of relevant LC tissues, including the most important histological subtypes (adenocarcinoma and squamous cell lung carcinoma). Mass spectrometry-based metabolomics, along with genetic expression and histological analyses, were performed as part of this study, the widest to date, to identify metabolic alterations in tumors of the most relevant histological subtypes in lung. A total of 136 lung tissue samples were analyzed and 851 metabolites were identified through metabolomic analysis. Our data show the existence of a clear metabolic alteration not only between tumor vs. nonmalignant tissue in each patient, but also inherently intrinsic changes in both AC and SCC. Significant changes were observed in the most relevant biochemical pathways, and nucleotide metabolism showed an important number of metabolites with high predictive capability values. The present study provides a detailed analysis of the metabolomic changes taking place in relevant biochemical pathways of the most important histological subtypes of LC, which can be used as biomarkers and also to identify novel targets.

VCE-004.8, A Multitarget Cannabinoquinone, Attenuates Adipogenesis and Prevents Diet-Induced Obesity.

Over the past few years, the endocannabinoid system (ECs) has emerged as a crucial player for the regulation of food intake and energy metabolism, and its pharmacological manipulation represents a novel strategy for the management of metabolic diseases. The discovery that VCE-004.8, a dual PPARγ and CB2 receptor agonist, also inhibits prolyl-hydroxylases (PHDs) and activates the HIF pathway provided a rationale to investigate its effect in in vitro models of adipogenesis and in a murine model of metabolic syndrome, all processes critically regulated by these targets of VCE-004.8. In accordance with its different binding mode to PPARγ compared to rosiglitazone (RGZ), VCE-004.8 neither induced adipogenic differentiation, nor affected osteoblastogenesis. Daily administration of VCE-004.8 (20 mg/kg) to HFD mice for 3-wks induced a significant reduction in body weight gain, total fat mass, adipocyte volume and plasma triglycerides levels. VCE-004.8 could also significantly ameliorate glucose tolerance, reduce leptin levels (a marker of adiposity) and increase adiponectin and incretins (GLP-1 and GIP) levels. Remarkably, VCE-004.8 increased the FGF21 mRNA expression in white and brown adipose, as well as in a BAT cell line, qualifying cannabinoaminoquinones as a class of novel therapeutic candidates for the management of obesity and its common metabolic co-morbidities.