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Purpose: The global pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the lingering threat to public health has fueled the search for effective therapeutics to treat SARS-CoV-2. This study aimed to develop lipid nanoparticle (LNP) inhibitors of SARS-CoV-2 entry to reduce viral infection in the nose and upper airway. Methods: Two types of LNP formulations were prepared following a microfluidic mixing method. The LNP-Trap consisted of DOPC, DSPC, cholesterol, and DSPE-PEG-COOH modified with various spike protein binding ligands, including ACE2 peptide, recombinant human ACE2 (rhACE2) or monoclonal antibody to spike protein (mAb). The LNP-Trim consisted of ionizing cationic DLin-MC3-DMA, DSPC, cholesterol, and DMG-PEG lipids encapsulating siACE2 or siTMPRSS2. Both formulations were assayed for biocompatibility and cell uptake in airway epithelial cells (Calu-3). Functional assessment of activity was performed using SARS-CoV-2 spike protein binding assays (LNP-Trap), host receptor knockdown (LNP-Trim), and SARS-CoV-2 pseudovirus neutralization assay (LNP-Trap and LNP-Trim). Localization and tissue distribution of fluorescently labeled LNP formulations were assessed in mice following intranasal administration. Results: Both LNP formulations were biocompatible based on cell impedance and MTT cytotoxicity studies in Calu-3 cells at concentrations as high as 1 mg/mL. LNP-Trap formulations were able to bind spike protein and inhibit pseudovirus infection by 90% in Calu-3 cells. LNP-Trim formulations reduced ACE2 and TMPRSS2 at the mRNA (70% reduction) and protein level (50% reduction). The suppression of host targets in Calu-3 cells treated with LNP-Trim resulted in over 90% inhibition of pseudovirus infection. In vivo studies demonstrated substantial retention of LNP-Trap and LNP-Trim in the nasal cavity following nasal administration with minimal systemic exposure. Conclusion: Both LNP-Trap and LNP-Trim formulations were able to safely and effectively inhibit SARS-CoV-2 pseudoviral infection in airway epithelial cells. These studies provide proof-of-principle for a localized treatment approach for SARS-CoV-2 in the upper airway.
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COVID-19 , Lipossomos , Nanopartículas , Glicoproteína da Espícula de Coronavírus , Animais , Humanos , Camundongos , Glicoproteína da Espícula de Coronavírus/metabolismo , Glicoproteína da Espícula de Coronavírus/farmacologia , SARS-CoV-2 , Enzima de Conversão de Angiotensina 2/metabolismo , Enzima de Conversão de Angiotensina 2/farmacologia , ColesterolRESUMO
Monoclonal antibodies (mAbs) possess favorable pharmacokinetic properties, high binding specificity and affinity, and minimal off-target effects, making them promising therapeutic agents for central nervous system (CNS) disorders. However, their development as effective therapeutic and diagnostic agents for brain disorders is hindered by their limited ability to efficiently penetrate the blood-brain barrier (BBB). Therefore, it is crucial to develop efficient delivery methods that enhance the penetration of antibodies into the brain. Previous studies have demonstrated the potential of cadherin-derived peptides (i.e., ADTC5, HAVN1 peptides) as BBB modulators (BBBMs) to increase paracellular porosities for penetration of molecules across the BBB. Here, we test the effectiveness of the leading BBBM peptide, HAVN1 (Cyclo(1,6)SHAVSS), in enhancing the permeation of various monoclonal antibodies through the BBB using both in vitro and in vivo systems. In vitro, HAVN1 has been shown to increase the permeability of fluorescently labeled macromolecules, such as a 70 kDa dextran, 50 kDa Fab1, and 150 kDa mAb1, by 4- to 9-fold in a three-dimensional blood-brain barrier (3D-BBB) microfluidics model using a human BBB endothelial cell line (i.e., hCMEC/D3). HAVN1 was selective in modulating the BBB endothelial cell, compared to the pulmonary vascular endothelial (PVE) cell barrier. Co-administration of HAVN1 significantly improved brain depositions of mAb1, mAb2, and Fab1 in C57BL/6 mice after 15 min in the systemic circulation. Furthermore, HAVN1 still significantly enhanced brain deposition of mAb2 when it was administered 24 h after the administration of the mAb. Lastly, we observed that multiple doses of HAVN1 may have a cumulative effect on the brain deposition of mAb2 within a 24-h period. These findings offer promising insights into optimizing HAVN1 and mAb dosing regimens to control or modulate mAb brain deposition for achieving desired mAb dose in the brain to provide its therapeutic effects.
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Barreira Hematoencefálica , Microfluídica , Camundongos , Animais , Humanos , Barreira Hematoencefálica/metabolismo , Camundongos Endogâmicos C57BL , Encéfalo/metabolismo , Peptídeos/metabolismo , Modelos Animais , Anticorpos Monoclonais/metabolismoRESUMO
Spermidine/spermine N1-acetyltransferase 1 (SAT1) responsible for cell polyamine catabolism is overexpressed in glioblastoma multiforme (GB). Its role in tumor survival and promoting resistance towards radiation therapy has made it an interesting target for therapy. In this study, we prepared a lipid nanoparticle-based siRNA delivery system (LNP-siSAT1) to selectively knockdown (KD) SAT1 enzyme in a human glioblastoma cell line. The LNP-siSAT1 containing ionizable DODAP lipid was prepared following a microfluidics mixing method and the resulting nanoparticles had a hydrodynamic size of around 80 nm and a neutral surface charge. The LNP-siSAT1 effectively knocked down the SAT1 expression in U251, LN229, and 42MGBA GB cells, and other brain-relevant endothelial (hCMEC/D3), astrocyte (HA) and macrophage (ANA-1) cells at the mRNA and protein levels. SAT1 KD in U251 cells resulted in a 40% loss in cell viability. Furthermore, SAT1 KD in U251, LN229 and 42MGBA cells sensitized them towards radiation and chemotherapy treatments. In contrast, despite similar SAT1 KD in other brain-relevant cells no significant effect on cytotoxic response, either alone or in combination, was observed. A major roadblock for brain therapeutics is their ability to cross the highly restrictive blood-brain barrier (BBB) presented by the brain microcapillary endothelial cells. Here, we used the BBB circumventing approach to enhance the delivery of LNP-siSAT1 across a BBB cell culture model. A cadherin binding peptide (ADTC5) was used to transiently open the BBB tight junctions to promote paracellular diffusion of LNP-siSAT1. These results suggest LNP-siSAT1 may provide a safe and effective method for reducing SAT1 and sensitizing GB cells to radiation and chemotherapeutic agents.
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Aberrant insulin-like growth factor 1 (IGF-1) signaling has been proposed as a contributing factor to the development of neurodegenerative disorders including diabetic neuropathy, and delivery of exogenous IGF-1 has been explored as a treatment for Alzheimer's disease and amyotrophic lateral sclerosis. However, the role of autocrine/paracrine IGF-1 in neuroprotection has not been well established. We therefore used in vitro cell culture systems and animal models of diabetic neuropathy to characterize endogenous IGF-1 in sensory neurons and determine the factors regulating IGF-1 expression and/or affecting neuronal health. Single-cell RNA sequencing (scRNA-Seq) and in situ hybridization analyses revealed high expression of endogenous IGF-1 in non-peptidergic neurons and satellite glial cells (SGCs) of dorsal root ganglia (DRG). Brain cortex and DRG had higher IGF-1 gene expression than sciatic nerve. Bidirectional transport of IGF-1 along sensory nerves was observed. Despite no difference in IGF-1 receptor levels, IGF-1 gene expression was significantly (P < 0.05) reduced in liver and DRG from streptozotocin (STZ)-induced type 1 diabetic rats, Zucker diabetic fatty (ZDF) rats, mice on a high-fat/ high-sugar diet and db/db type 2 diabetic mice. Hyperglycemia suppressed IGF-1 gene expression in cultured DRG neurons and this was reversed by exogenous IGF-1 or the aldose reductase inhibitor sorbinil. Transcription factors, such as NFAT1 and CEBPß, were also less enriched at the IGF-1 promoter in DRG from diabetic rats vs control rats. CEBPß overexpression promoted neurite outgrowth and mitochondrial respiration, both of which were blunted by knocking down or blocking IGF-1. Suppression of endogenous IGF-1 in diabetes may contribute to neuropathy and its upregulation at the transcriptional level by CEBPß can be a promising therapeutic approach.
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Envelhecimento/metabolismo , Axônios/patologia , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patologia , Metabolismo Energético , Fator de Crescimento Insulin-Like I/metabolismo , Células Receptoras Sensoriais/metabolismo , Animais , Anticorpos Neutralizantes/farmacologia , Axônios/efeitos dos fármacos , Axônios/metabolismo , Sequência de Bases , Proteína beta Intensificadora de Ligação a CCAAT/genética , Respiração Celular/efeitos dos fármacos , Células Cultivadas , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/patologia , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/patologia , Metabolismo Energético/efeitos dos fármacos , Gânglios Espinais/efeitos dos fármacos , Gânglios Espinais/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Glicólise/efeitos dos fármacos , Células HEK293 , Humanos , Fator de Crescimento Insulin-Like I/genética , Fígado/metabolismo , Masculino , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Fatores de Transcrição NFATC/metabolismo , Crescimento Neuronal/efeitos dos fármacos , Polímeros/metabolismo , Regiões Promotoras Genéticas/genética , Transporte Proteico/efeitos dos fármacos , Ratos Sprague-Dawley , Células Receptoras Sensoriais/patologia , Transdução de Sinais/efeitos dos fármacosRESUMO
AIM: The assessment of tumor response to therapy is of critical importance as it permits for a prospective end point evaluation and provides a guide to clinicians for making future treatment decisions. However, current practices in early evaluation of chemotherapy are insufficient. Amantadine is a substrate for SSAT-1. The present pilot study tests the hypothesis that SSAT-1 activity within the tumor, as measured by plasma acetylamantadine concentrations, can be used to monitor patient response to therapy. RESULTS: In cases with evidence of disease response, there was a reduction in the plasma acetylamantadine concentration at 4 h by approximately 32%. There was a mean increase of approximately 34% at the 4 h collection in the nonresponders. CONCLUSION: Although large-scale studies are required these findings suggest that the amantadine test could allow for determination of the efficacy of therapeutic interventions earlier, providing an effective test to assess response to treatment and for better management of patients.
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Local drug delivery approaches for treating brain tumors not only diminish the toxicity of systemic chemotherapy, but also circumvent the blood-brain barrier (BBB) which restricts the passage of most chemotherapeutics to the brain. Recently, salinomycin has attracted much attention as a potential chemotherapeutic agent in a variety of cancers. In this study, poly (ethylene oxide)/poly (propylene oxide)/poly (ethylene oxide) (PEO-PPO-PEO, Pluronic F127) and poly (dl-lactide-co-glycolide-b-ethylene glycol-b-dl-lactide-co-glycolide) (PLGA-PEG-PLGA), the two most common thermosensitive copolymers, were utilized as local delivery systems for salinomycin in the treatment of glioblastoma. The Pluronic and PLGA-PEG-PLGA hydrogels released 100% and 36% of the encapsulated salinomycin over a one-week period, respectively. While both hydrogels were found to be effective at inhibiting glioblastoma cell proliferation, inducing apoptosis and generating intracellular reactive oxygen species, the Pluronic formulation showed better biocompatibility, a superior drug release profile and an ability to further enhance the cytotoxicity of salinomycin, compared to the PLGA-PEG-PLGA hydrogel formulation. Animal studies in subcutaneous U251 xenograftednudemice also revealed that Pluronic + salinomycin hydrogel reduced tumor growth compared to free salinomycin- and PBS-treated mice by 4-fold and 6-fold, respectively within 12 days. Therefore, it is envisaged that salinomycin-loaded Pluronic can be utilized as an injectable thermosensitive hydrogel platform for local treatment of glioblastoma, providing a sustained release of salinomycin at the tumor site and potentially bypassing the BBB for drug delivery to the brain.
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Glioblastoma , Hidrogéis , Animais , Glioblastoma/tratamento farmacológico , Camundongos , Polietilenoglicóis , Piranos , TemperaturaRESUMO
Chronic exposure to ethanol is associated with enhanced leakiness in the brain microvessel endothelial cells that form the blood-brain barrier (BBB). As previous studies suggested Wnt/ß-catenin signaling could improve the BBB phenotype of brain endothelial cells, we examined the extent to which Wnt signaling is altered following ethanol exposure, using both a cell culture model of the BBB and mice exposed to ethanol, and the ability of Wnt activation to reverse the permeability effects of ethanol. The human brain endothelial cells, hCMEC/D3, were exposed to ethanol (17-200 mM) for various periods of time (0-96 hr) and Wnt signaling, as well as expression of downstream genes influencing BBB integrity in the cell monolayers were monitored. Determination of Wnt signaling in both brain homogenates and brain microvessels from mice exposed to ethanol was also performed. The effects of ethanol on the permeability of the hCMEC/D3 monolayers were examined using both small molecular weight (sodium fluorescein) and large molecular weight (IRdye 800CW PEG) fluorescent markers. Exposure of hCMEC/D3 to ethanol (50 mM) caused a down-regulation of Wnt/ß-catenin signaling, a reduction of tight junction protein expression and up-regulation of plasmalemma vesicle associated protein (PLVAP). A similar reduction in Wnt/ß-catenin activity in both cortical brain homogenates and isolated cortical cerebral microvessels were observed in mice. Other areas such as cerebellum and striatum displayed as much as 3-6 fold increases in Dkk-1, an endogenous Wnt inhibitor. Ethanol exposure caused significant changes in both sodium fluorescein and IRdye 800CW PEG permeability (2-fold compared to control). The ethanol-induced increases in permeability were attenuated by treatment with known Wnt activators (i.e. LiCl or Wnt3a). Additional screens of CNS active agents with possible Wnt activity indicated fluoxetine could also prevent the permeability effects of ethanol. These studies suggest that ethanol-induced changes in brain microvessel permeability can be reversed through activation of Wnt signaling.
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Barreira Hematoencefálica/efeitos dos fármacos , Barreira Hematoencefálica/metabolismo , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Etanol/toxicidade , Via de Sinalização Wnt/fisiologia , Animais , Permeabilidade Capilar/efeitos dos fármacos , Permeabilidade Capilar/fisiologia , Humanos , Masculino , Camundongos Endogâmicos C57BLRESUMO
Although doxorubicin (DOX) is an effective anti-cancer drug with cytotoxicity in a variety of different tumors, its effectiveness in treating glioblastoma multiforme (GBM) is constrained by insufficient penetration across the blood-brain barrier (BBB). In this study, biocompatible magnetic iron oxide nanoparticles (IONPs) stabilized with trimethoxysilylpropyl-ethylenediamine triacetic acid (EDT) were developed as a carrier of DOX for GBM chemotherapy. The DOX-loaded EDT-IONPs (DOX-EDT-IONPs) released DOX within 4 days with the capability of an accelerated release in acidic microenvironments. The DOX-loaded EDT-IONPs (DOX-EDT-IONPs) demonstrated an efficient uptake in mouse brain-derived microvessel endothelial, bEnd.3, Madin-Darby canine kidney transfected with multi-drug resistant protein 1 (MDCK-MDR1), and human U251 GBM cells. The DOX-EDT-IONPs could augment DOX's uptake in U251 cells by 2.8-fold and significantly inhibited U251 cell proliferation. Moreover, the DOX-EDT-IONPs were found to be effective in apoptotic-induced GBM cell death (over 90%) within 48 h of treatment. Gene expression studies revealed a significant downregulation of TOP II and Ku70, crucial enzymes for DNA repair and replication, as well as MiR-155 oncogene, concomitant with an upregulation of caspase 3 and tumor suppressors i.e., p53, MEG3 and GAS5, in U251 cells upon treatment with DOX-EDT-IONPs. An in vitro MDCK-MDR1-GBM co-culture model was used to assess the BBB permeability and anti-tumor activity of the DOX-EDT-IONPs and DOX treatments. While DOX-EDT-IONP showed improved permeability of DOX across MDCK-MDR1 monolayers compared to DOX alone, cytotoxicity in U251 cells was similar in both treatment groups. Using a cadherin binding peptide (ADTC5) to transiently open tight junctions, in combination with an external magnetic field, significantly enhanced both DOX-EDT-IONP permeability and cytotoxicity in the MDCK-MDR1-GBM co-culture model. Therefore, the combination of magnetic enhanced convective diffusion and the cadherin binding peptide for transiently opening the BBB tight junctions are expected to enhance the efficacy of GBM chemotherapy using the DOX-EDT-IONPs. In general, the developed approach enables the chemotherapeutic to overcome both BBB and multidrug resistance (MDR) glioma cells while providing site-specific magnetic targeting.
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Antineoplásicos/farmacologia , Neoplasias Encefálicas/tratamento farmacológico , Doxorrubicina/farmacologia , Portadores de Fármacos , Glioblastoma/tratamento farmacológico , Nanopartículas Magnéticas de Óxido de Ferro/química , Animais , Apoptose , Materiais Biocompatíveis/química , Barreira Hematoencefálica , Linhagem Celular Tumoral , Cães , Resistência a Múltiplos Medicamentos , Resistencia a Medicamentos Antineoplásicos , Humanos , Células Madin Darby de Rim Canino , Camundongos , Permeabilidade , Espécies Reativas de OxigênioRESUMO
Salinomycin is an antibiotic introduced recently as a new and effective anticancer drug. In this study, magnetic iron oxide nanoparticles (IONPs) were utilized as a drug carrier for salinomycin for potential use in glioblastoma (GBM) chemotherapy. The biocompatible polyethylenimine (PEI)-polyethylene glycol (PEG)-IONPs (PEI-PEG-IONPs) exhibited an efficient uptake in both mouse brain-derived microvessel endothelial (bEnd.3) and human U251 GBM cell lines. The salinomycin (Sali)-loaded PEI-PEG-IONPs (Sali-PEI-PEG-IONPs) released salinomycin over 4 days, with an initial release of 44% ± 3% that increased to 66% ± 5% in acidic pH. The Sali-IONPs inhibited U251 cell proliferation and decreased their viability (by approximately 70% within 48 h), and the nanoparticles were found to be effective in reactive oxygen species-mediated GBM cell death. Gene studies revealed significant activation of caspases in U251 cells upon treatment with Sali-IONPs. Furthermore, the upregulation of tumor suppressors (i.e., p53, Rbl2, Gas5) was observed, while TopII, Ku70, CyclinD1, and Wnt1 were concomitantly downregulated. When examined in an in vitro blood-brain barrier (BBB)-GBM co-culture model, Sali-IONPs had limited penetration (1.0% ± 0.08%) through the bEnd.3 monolayer and resulted in 60% viability of U251 cells. However, hyperosmotic disruption coupled with an applied external magnetic field significantly enhanced the permeability of Sali-IONPs across bEnd.3 monolayers (3.2% ± 0.1%) and reduced the viability of U251 cells to 38%. These findings suggest that Sali-IONPs combined with penetration enhancers, such as hyperosmotic mannitol and external magnetic fields, can potentially provide effective and site-specific magnetic targeting for GBM chemotherapy.
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Wnt/ß-catenin signaling is important for blood-brain barrier (BBB) development and is implicated in BBB breakdown under various pathophysiological conditions. In the present study, a comprehensive characterization of the relevant genes, transport and permeability processes influenced by both the autocrine and external activation of Wnt signaling in human brain endothelial cells was examined using hCMEC/D3 culture model. The hCMEC/D3 expressed a full complement of Wnt ligands and receptors. Preventing Wnt ligand release from hCMEC/D3 produced minimal changes in brain endothelial function, while inhibition of intrinsic/autocrine Wnt/ß-catenin activity through blocking ß-catenin binding to Wnt transcription factor caused more modest changes. In contrast, activation of Wnt signaling using exogenous Wnt ligand (Wnt3a) or LiCl (GSK3 inhibitor) improved the BBB phenotypes of the hCMEC/D3 culture model, resulting in reduced paracellular permeability, and increased P-glycoprotein (P-gp) and breast cancer resistance associated protein (BCRP) efflux transporter activity. Further, Wnt3a reduced plasmalemma vesicle associated protein (PLVAP) and vesicular transport activity in hCMEC/D3. Our data suggest that this in vitro model of the BBB has a more robust response to exogenous activation of Wnt/ß-catenin signaling compared to autocrine activation, suggesting that BBB regulation may be more dependent on external activation of Wnt signaling within the brain microvasculature.
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Barreira Hematoencefálica/metabolismo , Células Endoteliais/metabolismo , Via de Sinalização Wnt , Comunicação Autócrina/efeitos dos fármacos , Barreira Hematoencefálica/efeitos dos fármacos , Células Cultivadas , Endocitose/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Ligantes , Cloreto de Lítio/farmacologia , Proteínas de Membrana/metabolismo , Oxazóis/farmacologia , Fenótipo , Receptores de Superfície Celular/metabolismo , Via de Sinalização Wnt/efeitos dos fármacos , Via de Sinalização Wnt/genéticaRESUMO
The blood-brain barrier (BBB) poses a major obstacle by preventing potential therapeutic agents from reaching their intended brain targets at sufficient concentrations. While transient disruption of the BBB has been used to enhance chemotherapeutic efficacy in treating brain tumors, limitations in terms of magnitude and duration of BBB disruption exist. In the present study, the preliminary safety and efficacy profile of HAV6, a peptide that binds to the external domains of cadherin, to transiently open the BBB and improve the delivery of a therapeutic agent, was evaluated in a murine brain tumor model. Transient opening of the BBB in response to HAV6 peptide administration was quantitatively characterized using both a gadolinium magnetic resonance imaging (MRI) contrast agent and adenanthin (Ade), the intended therapeutic agent. The effects of HAV6 peptide on BBB integrity and the efficacy of concurrent administration of HAV6 peptide and the small molecule inhibitor, Ade, in the growth and progression of an orthotopic medulloblastoma mouse model using human D425 tumor cells was examined. Systemic administration of HAV6 peptide caused transient, reversible disruption of BBB in mice. Increases in BBB permeability produced by HAV6 were rapid in onset and observed in all regions of the brain examined. Concurrent administration of HAV6 peptide with Ade, a BBB impermeable inhibitor of Peroxiredoxin-1, caused reduced tumor growth and increased survival in mice bearing medulloblastoma. The rapid onset and transient nature of the BBB modulation produced with the HAV6 peptide along with its uniform disruption and biocompatibility is well-suited for CNS drug delivery applications, especially in the treatment of brain tumors.
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The use of dietary supplements as an alternative treatment for joint-related pathologies such as osteoarthritis (OA) is increasing. However, there is little scientific evidence to support the intended use. The aim of this study was to evaluate the anti-inflammatory effects of creatine- and amino acid-based supplements in primary cultured canine chondrocytes (CnCs) as an in-vitro model of OA and compare the effects to more commonly used agents, such as the non-steroidal anti-inflammatory drug (NSAID), carprofen, and the joint supplement, glucosamine (GS). CnCs were stimulated with interleukin-1ß (IL-1ß) and the subsequent release of prostaglandin E2 (PGE2) and tumor necrosis factor alpha (TNFα) was measured using an enzyme-linked immunosorbent assay (ELISA). Changes in oxylipins were also assessed using high-performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS). All compounds examined were able to significantly reduce the release of PGE2 and TNFα and were associated with reductions in cyclooxygenase-2 (COX-2) expression and nuclear factor-kappaB (NF-κB) phosphorylation. The creatine- and amino acids-based supplements also altered the profile of oxylipins produced. All compounds examined were less effective at reducing the release of PGE2 than carprofen. Carprofen significantly increased release of TNFα from CnCs, however, while the other agents reduced TNFα release. This study suggests that creatine- and amino acid-based supplements may have a beneficial role in preventing inflammation within the joint and that further studies are warranted.
L'utilisation de suppléments alimentaires à titre de traitement alternatif pour les pathologies associées aux articulations telle que l'arthrose (OA) est en augmentation. Toutefois, il y a peu d'évidences scientifiques qui supportent l'utilisation proposée. L'objectif de la présente étude était d'évaluer les effets anti-inflammatoires de suppléments à base de créatine et d'acides aminés sur des cultures primaires de chondrocytes canins (CnCs) utilisés comme modèle in vitro d'OA et de comparer les effets à des agents plus communément utilisés, tel que l'agent anti-inflammatoire non-stéroïdien (AINS) carprofen, et le supplément articulaire, glucosamine (GS). Les CnCs furent stimulés avec de l'interleukine-1ß (IL-1ß) et la libération subséquente de prostaglandine E2 (PGE2) et le facteur nécrosant de tumeur alpha (TNFα) fut mesurée par épreuve immuno-enzymatique (ELISA). Les changements dans les oxylipines furent également mesurés par chromatographie en phase liquide à haute performance/spectrométrie de masse tandem (HPLC/MS/MS). Tous les composés examinés étaient en mesure de réduire significativement la libération de PGE2 et de TNFα et étaient associés avec des réductions d'expression de cyclooxygénase-2 (COX-2) et de phosphorylation du facteur nucléaire kappaB (NF-κB). Les suppléments à base de créatine et d'acides aminés ont également altéré le profil des oxylipines produits. Tous les composés examinés étaient moins efficaces que le carprofen pour réduire la libération de PGE2. Le carprofen augmentait significativement la libération de TNFα par les CnCs, alors que les autres agents la réduisaient. La présente étude suggère que les suppléments à base de créatine et d'acides aminés pourraient avoir un rôle bénéfique dans la prévention de l'inflammation dans l'articulation et que des études supplémentaires sont requises.(Traduit par Docteur Serge Messier).
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Condrócitos/efeitos dos fármacos , Suplementos Nutricionais , Doenças do Cão/tratamento farmacológico , Inflamação/veterinária , Osteoartrite/veterinária , Animais , Anti-Inflamatórios não Esteroides/farmacologia , Carbazóis/farmacologia , Sobrevivência Celular , Células Cultivadas , Condrócitos/metabolismo , Citocinas/genética , Citocinas/metabolismo , Cães , Regulação da Expressão Gênica/efeitos dos fármacos , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Osteoartrite/tratamento farmacológicoRESUMO
Salinomycin is an antibiotic that has recently been introduced as a novel and effective anti-cancer drug. In this study, PLGA nanofibers (NFs) containing salinomycin (Sali) were fabricated by electrospinning for the first time. The biodegradable PLGA NFs had stability for approximately 30 days and exhibited a sustained release of the drug for at least a 2-week period. Cytotoxicity of the NFs + Sali was evaluated on human glioblastoma U-251 cells and more than 50% of the treated cells showed apoptosis in 48 h. Moreover, NFs + Sali was effective to induce intracellular reactive oxygen species (ROS) leading to cell apoptosis. Gene expression studies also revealed the capability of the NFs + Sali to upregulate tumor suppressor Rbl1 and Rbl2 as well as Caspase 3 while decreasing Wnt signaling pathway. In general, the results indicated anti-tumor activity of the Sali-loaded NFs suggesting their potential applications as implantable drug delivery systems in the brain upon surgical resection of the tumor.
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Glioblastoma/metabolismo , Nanofibras/química , Copolímero de Ácido Poliláctico e Ácido Poliglicólico/química , Piranos/química , Piranos/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Humanos , Microscopia Eletrônica de Varredura , Espécies Reativas de Oxigênio/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Creatine is an ergogenic compound used by athletes to enhance performance. Supplementation with creatine monohydrate (CM) has been suggested for musculoskeletal and neurological disorders. Until now, little is known about its pharmacokinetic profile. Our objective was to determine the oral bioavailability of CM and the influence of dose on oral absorption. Rats were dosed orally with low dose (10 mg/kg) or high dose (70 mg/kg) 13C-labeled CM. Blood samples were removed at various time points. Muscle and brain tissue were collected at the conclusion of the study. Plasma and tissue levels of 13C-labeled creatine were determined using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Physiologically based pharmacokinetic (PBPK) models of CM were built using GastroPlus™. These models were used to predict the plasma concentration-time profiles of creatine hydrochloride (CHCL), which has improved aqueous solubility compared to CM. Absolute oral bioavailability for low dose CM was 53% while high dose CM was only 16%. The simulated Cmax of 70 mg/kg CHCL was around 35 µg/mL compared to 14 µg/mL for CM with a predicted oral bioavailability of 66% with CHCL compared to 17% with CM. Our results suggest that the oral bioavailability of CM is less than complete and subject to dose and that further examination of improved dosage formulations of creatine is warranted.
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A facile one-pot method for synthesizing amine-functionalized nonspherical Fe3O4 nanoparticles in gram-scale quantities is presented using just a single source of iron (iron(II) chloride) and an amine (triethylamine). The amine not only transforms iron salt to Fe3O4, but also directs the morphology of the nanoparticles along with the temperature of the reaction and functionalizes them, making the synthesis very economical. By modifying the surface further, these nanoparticles promise to offer useful biomedical applications. For example, after biocide coating, the particles are found to be 100% effective in deactivating methicillin-resistant Staphylococcus aureus (MRSA) bacteria in 2 h. Cellular-uptake studies using biocompatible EDTA-Na3 (N-(trimethoxysilyl-propyl)ethylenediaminetriacetate, trisodium salt)-coated nanoparticles in human glioblastoma U-251 cells show that the majority of the particles are internalized by the cells in the presence of a small dc-magnetic field, making these particles a potential candidate as drug carriers for magnetic field-targeted delivery and hyperthermia.
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Aminas/química , Materiais Biomiméticos/química , Óxido Ferroso-Férrico/química , Pesquisa Biomédica , Tamanho da Partícula , Propriedades de SuperfícieRESUMO
There is an urgent need to develop new and alternative methods to deliver functional biomolecules to the brain for diagnosis and treatment of brain diseases. The goal of this study was to evaluate the activity of blood-brain barrier (BBB) modulators (i.e., HAV and ADT peptides) to deliver functional biomolecules (i.e., galbumin, IRdye800cw-cLABL, and cIBR7) to the brains of mice and rats. HAV6, cHAVc3, and ADTC5 peptides but not HAV4 peptide significantly enhanced the brain delivery of 65 kDa galbumin compared to control in Balb/c mice as quantified by magnetic resonance imaging (MRI). Ten-minute pretreatment with ADTC5 peptide still significantly increased brain delivery of galbumin; however, no enhancement was observed after 10-min pretreatment with HAV6. There was no enhancement of galbumin deposition following 40-min pretreatment with ADTC5 or HAV6, suggesting a short duration of the BBB opening for large molecules. ADTC5 peptide also improved the brain delivery of IRdye800cw-cLABL peptide about 3.5-fold compared to control in Balb/c mice as detected by near infrared fluorescence (NIRF). The BBB modulator activity of ADTC5 to deliver cIBR7 peptide was also evaluated in vivo using Sprague-Dawley rats. The amount of cIBR7 in the brain was detected by LC-MS/MS. ADTC5 peptide enhanced the delivery of cIBR7 peptide into rat brain about 4-fold compared to control and the intact cIBR7 can be efficiently extracted and detected in rat brain. In conclusion, HAV and ADT peptides enhance the brain delivery of functional peptides (e.g., cLABL and cIBR7) and protein (e.g., 65 kDa galbumin) in two animal models, and the duration of the BBB opening for a large molecule (e.g., galbumin) was short.
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OBJECTIVES: In conventional in-vitro blood-brain barrier (BBB) models, primary and immortalized brain microvessel endothelial cell (BMEC) lines are often cultured in a monolayer or indirect coculture or triculture configurations with astrocytes or pericytes, for screening permeation of therapeutic or potentially neurotoxic compounds. In each of these cases, the physiological relevancy associated with the direct contact between the BMECs, pericytes and astrocytes that form the BBB and resulting synergistic interactions are lost. We look to overcome this limitation with a direct contact coculture model. METHODS: We established and optimized a direct interaction coculture system where primary human astrocytes are cultured on the apical surface of a Transwell® filter support and then human cerebral microvessel endothelial cells (hCMEC/D3) seeded directly on the astrocyte lawn. KEY FINDINGS: The studies suggest the direct coculture model may provide a more restrictive and physiologically relevant model through a significant reduction in paracellular transport of model compounds in comparison with monoculture and indirect coculture. In comparison with existing methods, the indirect coculture and monoculture models utilized may limit cell-cell signaling between human astrocytes and BMECs that are possible with direct configurations. CONCLUSIONS: Paracellular permeability reductions with the direct coculture system may enhance therapeutic agent and potential neurotoxicant screening for BBB permeability better than the currently available monoculture and indirect coculture in-vitro models.
Assuntos
Astrócitos/citologia , Barreira Hematoencefálica/citologia , Células Endoteliais/citologia , Microvasos/citologia , Barreira Hematoencefálica/metabolismo , Circulação Cerebrovascular/fisiologia , Técnicas de Cocultura , Endotélio Vascular/citologia , Humanos , PermeabilidadeRESUMO
A room temperature reduction-hydrolysis of Fe(III) precursors such as FeCl3 or Fe(acac)3 in various lyotropic liquid crystal phases (lamellar, hexagonal columnar, or micellar) formed by a range of ionic or neutral surfactants in H2O is shown to be an effective and mild approach for the preparation of iron oxide (IO) nanomaterials with several morphologies (shapes and dimensions), such as extended thin nanosheets with lateral dimensions of several hundred nanometers as well as smaller nanoflakes and nanodiscs in the tens of nanometers size regime. We will discuss the role of the used surfactants and lyotropic liquid crystal phases as well as the shape and size differences depending upon when and how the resulting nanomaterials were isolated from the reaction mixture. The presented synthetic methodology using lyotropic liquid crystal solvents should be widely applicable to several other transition metal oxides for which the described reduction-hydrolysis reaction sequence is a suitable pathway to obtain nanoscale particles.
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Alzheimer's disease pathology includes, beside neuronal damage, reactive gliosis and reduced blood-brain barrier (BBB) integrity. Microglia are intimately associated with the BBB and upon AD pathology, pro-inflammatory responses of microglia could contribute to BBB damage. To study whether microglia can directly affect BBB integrity, the effects of amyloid beta (Aß) -stimulated primary murine microglia on co-cultured mouse brain endothelial cells (bEnd3) and murine astrocyte cultures were assessed. We also assessed whether microglial phenotype modulation via poly(ADP-ribose) polymerase-1 (PARP-1) inhibition/ablation can reverse microglial impact on these BBB forming cells. Unstimulated microglia promoted expression of tight junction proteins (TJPs), zonula ocluden-1 (ZO-1) and occludin in co-cultured endothelia cells, whereas Aß-stimulated microglia reduced endothelial expression of ZO-1 and occludin. Astrocytes co-cultured with microglia showed elevated glial fibrillary acidic protein (GFAP) expression, which was further increased if microglia had been stimulated with Aß. Aß induced microglial release of nitric oxide (NO) and tumour necrosis factor alpha (TNFα), which resulted in reduced endothelial expression of TJPs and increased paracellular permeability. Microglial PARP-1 inhibition attenuated these Aß-induced events. These findings demonstrate that PARP-1 mediated microglial responses (NO and TNFα) can directly reduce BBB integrity by promoting TJP degradation, increasing endothelial cell permeability and inducing astrogliosis. PARP-1 as a modulator of microglial phenotype can prevent microglial BBB damaging events, and thus is a potential therapeutic target.
Assuntos
Endotélio Vascular/metabolismo , Microglia/fisiologia , Poli(ADP-Ribose) Polimerase-1/fisiologia , Junções Íntimas/metabolismo , Peptídeos beta-Amiloides/farmacologia , Animais , Barreira Hematoencefálica/efeitos dos fármacos , Barreira Hematoencefálica/metabolismo , Técnicas de Cocultura , Endotélio Vascular/efeitos dos fármacos , Feminino , Masculino , Camundongos , Camundongos Knockout , Microglia/efeitos dos fármacos , Junções Íntimas/efeitos dos fármacosRESUMO
Common chemotherapy is often associated with adverse effects in normal cells and tissues. As an alternative approach, localized chemotherapy can diminish the toxicity of systemic chemotherapy while providing a sustained release of the chemotherapeutics at the target tumor site. Therefore, injectable biodegradable hydrogels as drug delivery systems for chemotherapeutics have become a matter of importance. Here, we review the application of a variety of injectable hydrogel-based drug delivery systems, including thermosensitive, pH-sensitive, photosensitive, dual-sensitive, as well as active targeting hydrogels, for the treatment of different types of cancer. Generally, injectable hydrogel-based drug delivery systems are found to be more efficacious than the conventional systemic chemotherapy in terms of cancer treatment.