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Glioblastoma multiforme (GBM) and other central nervous system (CNS) cancers have poor long-term prognosis, and there is a significant need for improved treatments. GBM initiation and progression are mediated, in part, by microRNA (miRNA), which are endogenous posttranscriptional gene regulators. Misregulation of miRNAs is a potential target for therapeutic intervention in GBM. In this work, a micelle-like nanoparticle delivery system based upon the block copolymer poly(ethylene glycol-b-lactide-b-arginine) was designed with and without a reducible linkage between the lactide and RNA-binding peptide, R15, to assess the ability of the micelle-like particles to disassemble. Using confocal live cell imaging, intracellular dissociation was pronounced for the reducible micelleplexes. This dissociation was also supported by higher efficiency in a dual luciferase assay specific for the miRNA of interest, miR-21. Notably, micelleplexes were found to have significantly better stability and higher anti-miRNA activity in cerebrospinal fluid than in human plasma, suggesting an advantage for applying micelleplexes to CNS diseases and in vivo CNS therapeutics. The reducible delivery system was determined to be a promising delivery platform for the treatment of CNS diseases with miRNA therapy.
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Antineoplásicos/farmacología , Líquido Cefalorraquídeo/metabolismo , MicroARNs/metabolismo , Arginina/administración & dosificación , Neoplasias Encefálicas/tratamiento farmacológico , Sistemas de Liberación de Medicamentos/métodos , Glioblastoma/tratamiento farmacológico , Humanos , Micelas , Nanopartículas/administración & dosificación , Poliésteres/administración & dosificación , Polietilenglicoles/administración & dosificación , Polímeros/administración & dosificaciónRESUMEN
PURPOSE: To prolong the release of a heparan sulfate binding peptide, G2-C, using a commercially available contact lens as a delivery vehicle and to demonstrate the ability of the released peptide to block herpes simplex virus-1 (HSV-1) infection using in vitro, ex vivo, and in vivo models of corneal HSV-1 infection. METHODS: Commercially available contact lenses were immersed in peptide solution for 5 days prior to determining the release of the peptide at various time points. Cytotoxicity of the released samples was determined by MTT and cell cycle analysis, and the functional activity of the released samples were assessed by viral entry, and viral spread assay using human corneal epithelial cells (HCE). The ability to suppress infection in human and pig cornea ex vivo and mouse in vivo models were also assessed. RESULTS: Peptide G2-C was released through the contact lens. Following release for 3 days, the peptide showed significant activity by inhibiting HSV-1 viral entry and spread in HCE cells. Significant suppression of infection was also observed in the ex vivo and in vivo experiments involving corneas. CONCLUSIONS: Extended release of an anti-HS peptide through a commercially available contact lens can generate significant anti-HSV-1 activity and provides a new and effective way to control corneal herpes.
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Lentes de Contacto , Córnea/virología , Infecciones Virales del Ojo/tratamiento farmacológico , Heparitina Sulfato/farmacología , Herpesvirus Humano 1 , Queratitis Herpética/tratamiento farmacológico , Animales , Córnea/metabolismo , Preparaciones de Acción Retardada , Modelos Animales de Enfermedad , Infecciones Virales del Ojo/metabolismo , Infecciones Virales del Ojo/virología , Femenino , Humanos , Queratitis Herpética/metabolismo , Queratitis Herpética/virología , Masculino , Ratones , Ratones Endogámicos BALB C , PorcinosRESUMEN
Macrophages exhibit phenotypic diversity permitting wide-ranging roles in maintaining physiologic homeostasis. Hyaluronic acid, a major glycosaminoglycan of the extracellular matrix, has been shown to have differential signaling based on its molecular weight. With this in mind, the main objective of this study was to elucidate the role of hyaluronic acid molecular weight on macrophage activation and reprogramming. Changes in macrophage activation were assessed by activation state selective marker measurement, specifically quantitative real time polymerase chain reaction, and cytokine enzyme-linked immunoassays, after macrophage treatment with differing molecular weights of hyaluronic acid under four conditions: the resting state, concurrent with classical activation, and following inflammation involving either classically or alternatively activated macrophages. Regardless of initial polarization state, low molecular weight hyaluronic acid induced a classically activated-like state, confirmed by up-regulation of pro-inflammatory genes, including nos2, tnf, il12b, and cd80, and enhanced secretion of nitric oxide and TNF-α. High molecular weight hyaluronic acid promoted an alternatively activated-like state, confirmed by up regulation of pro-resolving gene transcription, including arg1, il10, and mrc1, and enhanced arginase activity. Overall, our observations suggest that macrophages undergo phenotypic changes dependent on molecular weight of hyaluronan that correspond to either (1) pro-inflammatory response for low molecular weight HA or (2) pro-resolving response for high molecular weight HA. These observations bring significant further understanding of the influence of extracellular matrix polymers, hyaluronic acid in particular, on regulating the inflammatory response of macrophages. This knowledge can be used to guide the design of HA-containing biomaterials to better utilize the natural response to HAs.
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PURPOSE: To (a) evaluate whether the lysine-rich protein (LRP) magnetic resonance (MR) imaging reporter gene can be engineered into G47Δ, a herpes simplex-derived oncolytic virus that is currently being tested in clinical trials, without disrupting its therapeutic effectiveness and (b) establish the ability of chemical exchange saturation transfer (CEST) MR imaging to demonstrate G47Δ-LRP. MATERIALS AND METHODS: The institutional subcommittee for research animal care approved all in vivo procedures. Oncolytic herpes simplex virus G47Δ, which carried the LRP gene, was constructed and tested for its capacity to replicate in cancer cells and express LRP in vitro. The LRP gene was detected through CEST imaging of lysates derived from cells infected with G47Δ-LRP or the control G47Δ-empty virus. G47Δ-LRP was then tested for its therapeutic effectiveness and detection with CEST MR imaging in vivo. Images of rat gliomas were acquired before and 8-10 hours after injection of G47Δ-LRP (n = 7) or G47Δ-empty virus (n = 6). Group comparisons were analyzed with a paired t test. RESULTS: No significant differences were observed in viral replication or therapeutic effectiveness between G47Δ-LRP and G47Δ-empty virus. An increase in CEST image contrast was observed in cell lysates (mean ± standard deviation, 0.52% ± 0.06; P = .01) and in tumors (1.1% ± 0.3, P = .02) after infection with G47Δ-LRP but not G47Δ-empty viruses. No histopathologic differences were observed between tumors infected with G47Δ-LRP and G47Δ-empty virus. CONCLUSION: This study has demonstrated the ability of CEST MR imaging to show G47Δ-LRP at acute stages of viral infection. The introduction of the LRP transgene had no effect on the viral replication or therapeutic effectiveness. This can aid in development of the LRP gene as a reporter for the real-time detection of viral spread. Online supplemental material is available for this article.
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Genes Reporteros , Lisina , Imagen por Resonancia Magnética , Viroterapia Oncolítica/métodos , Animales , Células Cultivadas , Masculino , Ratas , Ratas Endogámicas F344 , SimplexvirusRESUMEN
Although potent, proteins often require chemical modification for therapeutic use. Immunogenicity, difficult synthesis, and scale-up of these modifications are all engineering obstacles that stand in the way of expanding the use of these therapeutics. Melittin, a peptide derived from bee venom, has been shown to modulate inflammation. Although potentially therapeutic, the native peptide causes cell lysis and toxicity significantly hindering therapeutic application. Based upon the knowledge of the pore formation mechanism, we examined the toxicity and therapeutic effect of a melittin fusion protein with glutathione-S-transferase. The fusion of melittin and glutathione S-transferase results in diminished toxicity of the peptide and retained anti-inflammatory properties at doses that exceed toxic concentration of native melittin. Our results suggest that fusion proteins, particularly those of glutathione-S-transferase, may be facile modifications to control protein activity.
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Antiinflamatorios/farmacología , Glutatión Transferasa/farmacología , Meliteno/farmacología , Proteínas Recombinantes/farmacología , Animales , Venenos de Abeja/farmacología , Células Cultivadas , Inflamación/tratamiento farmacológico , Macrófagos/efectos de los fármacos , Ratones , Péptidos/farmacologíaRESUMEN
MicroRNAs (miRNAs) are a class of gene regulators originating from non-coding endogenous RNAs. Altered expression, both up- and down-regulation, of miRNAs plays important roles in many human diseases. Correcting miRNA dysregulation by either inhibiting or restoring miRNA function may provide therapeutic benefit. However, efficient, nontoxic miRNA delivery systems are in need. Cell penetrating peptides (CPPs) have been widely exploited for protein, DNA, and RNA delivery. Few have examined CPP transfection efficiency with single stranded anti-miRNA. The R8 peptide condensed both siRNA and anti-miRNA. Greater than 50% of cells had anti-miRNA/R8 complexes associated and in these cells 68% of anti-miRNA escapes the endosome/lysosome. Single-stranded antisense miR-21 inhibitor (anti-miR-21) administered using the R8 peptide elicited efficient downstream gene upregulation. Glioblastoma cell migration was inhibited by 25% compared to the negative control group. To our knowledge, this is the first demonstration of miRNA modulation with anti-miR-21/R8 complexes, which has laid the groundwork for further exploring octaarginine as intracellular anti-miRNAs carrier.
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Movimiento Celular/efectos de los fármacos , Péptidos de Penetración Celular/farmacología , Glioblastoma/metabolismo , MicroARNs/antagonistas & inhibidores , ARN sin Sentido/farmacología , ARN Neoplásico/antagonistas & inhibidores , Línea Celular Tumoral , Péptidos de Penetración Celular/química , Glioblastoma/genética , Glioblastoma/patología , Humanos , MicroARNs/genética , MicroARNs/metabolismo , ARN sin Sentido/química , ARN sin Sentido/genética , ARN Neoplásico/genética , ARN Neoplásico/metabolismo , Transfección/métodosRESUMEN
The stem cell differentiation paradigm is based on the progression of cells through generations of daughter cells that eventually become restricted and committed to one lineage resulting in fully differentiated cells. Herein, we report on the differentiation of adult human mesenchymal stem cells (hMSCs) towards adipogenic and osteogenic lineages using established protocols. Lineage specific geneswere evaluated by quantitative real-time PCR relative to two reference genes. The expression of osteoblast-associated genes (alkaline phosphatase, osteopontin, and osteocalcin)was detected in hMSCs that underwent adipogenesis. When normalized, the expression of adipocyte marker genes (adiponectin, fatty acid binding protein P4, and leptin) increasedin a time-dependent manner during adipogenic induction. Adiponectin and leptin were also detected in osteoblast-induced cells. Lipid vacuoles that represent the adipocyte phenotype were only present in the adipogenic induction group. Conforming to the heterogeneous nature of hMSCs and the known plasticity between osteogenic and adipogenic lineages, these data indicatea marker overlap between MSC-derived adipocytes and osteoblasts. Weproposea careful consideration of experimental conditions such as investigated timepoints, selected housekeeping genesand the evidence indicating lack of differentiation into other lineageswhen evaluating hMSC differentiation.
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Cell lytic peptides are a class of drugs that can be used to selectively kill invading organisms or diseased cells. Several of these peptides have been identified as potential therapeutics. Herein, we report a novel process for purifying recombinant melittin, a cell lytic peptide that inserts into the membranes of cells causing cell lysis, from Escherichia coli. The process involves surfactant and low pH to solubilize melittin fusion proteins from the insoluble fraction of bacterial lysates. We are able to significantly improve purity of the final product and confirm the activity of the peptide. The process yields recombinant melittin that is effective when used to treat U-87 MG glioma cells and inhibits growth of the gram-positive pathogenic bacterium Streptococcus pyogenes. We demonstrate a method of repeated extraction of the insoluble protein fraction with mild detergent at a low pH that is able to generate a yield of pure, soluble melittin of â¼ 0.5-1 mg/L of E. coli culture.
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Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica , Meliteno/aislamiento & purificación , Proteínas Recombinantes/aislamiento & purificación , Antibacterianos/aislamiento & purificación , Antineoplásicos/aislamiento & purificación , Línea Celular Tumoral , Clonación Molecular , Escherichia coli/metabolismo , Humanos , Meliteno/genética , Proteínas Recombinantes/genética , Streptococcus pyogenes/efectos de los fármacosRESUMEN
Hydrogels are finding increased clinical utility as advances continue to exploit their favorable material properties. Hydrogels can be adapted for many applications, including surface coatings and drug delivery. Anti-infectious surfaces and delivery systems that actively destroy invading organisms are alternative ways to exploit the favorable material properties offered by hydrogels. Sterilization techniques are commonly employed to ensure the materials are non-infectious upon placement, but sterilization is not absolute and infections are still expected. Natural, anti-bacterial proteins have been discovered which have the potential to act as anti-infectious agents; however, the proteins are toxic and need localized release to have therapeutic efficacy without toxicity. In these studies, we explore the use of the glutathione s-transferase (GST) to anchor the bactericidal peptide, melittin, to the surface of poly(ethylene glycol) diacrylate (PEGDA) hydrogel microspheres. We show that therapeutic levels of protein can be anchored to the surface of the microspheres using the GST anchor. We compared the therapeutic efficacy of recombinant melittin released from PEGDA microspheres to melittin. We found that, when released by an activating enzyme, thrombin, recombinant melittin efficiently inhibits growth of the pathogenic bacterium Streptococcus pyogenes as effectively as melittin created by solid phase peptide synthesis. We conclude that a GST protein anchor can be used to immobilize functional protein to PEGDA microspheres and the protein will remain immobilized under physiological conditions until the protein is enzymatically released.
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Antibacterianos/administración & dosificación , Portadores de Fármacos/química , Glutatión Transferasa/química , Hidrogel de Polietilenoglicol-Dimetacrilato/química , Meliteno/administración & dosificación , Polietilenglicoles/química , Antibacterianos/farmacología , Portadores de Fármacos/metabolismo , Glutatión Transferasa/metabolismo , Humanos , Hidrogel de Polietilenoglicol-Dimetacrilato/metabolismo , Meliteno/farmacología , Microesferas , Modelos Moleculares , Polietilenglicoles/metabolismo , Proteolisis , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/farmacología , Infecciones Estreptocócicas/tratamiento farmacológico , Streptococcus pyogenes/efectos de los fármacos , Trombina/metabolismoRESUMEN
Oncolytic herpes simplex virus (oHSV) can potentially spread throughout the tumor, reach isolated infiltrating cells, kill them, and deliver anticancer agents. However, the host responds to oHSV by inducing intratumoral infiltration of macrophages that can engulf the virus, limiting the potential of this therapeutic strategy. Hypervascularity is a pathognomonic feature of glioblastoma (GBM) and is a promising therapeutic target. Antiangiogenic treatments have multiple benefits, including the capacity to increase oHSV efficacy by suppressing macrophage extravasation and infiltration into the tumor. Angiostatin is an antiangiogenic polypeptide, and interleukin-12 (IL-12) is an immunostimulatory cytokine with strong antiangiogenic effects. Clinical use of each has been limited by delivery issues and systemic toxicity. We tested a combination treatment strategy using oHSVs expressing angiostatin (G47Δ-mAngio) and IL-12 (G47Δ-mIL12) in two orthotopic human GBM models. Intratumoral injection of G47Δ-mAngio and G47Δ-mIL12 in mice bearing intracranial U87 or tumors derived from glioblastoma stem cells significantly prolonged survival compared to each armed oHSV alone. This was associated with increased antiangiogenesis and virus spread and decreased macrophages. These data support the paradigm of using oHSV expressing different antiangiogenic agents and show for the first time that oHSVs expressing angiostatin and IL-12 can improve efficacy in human GBM models.
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Inhibidores de la Angiogénesis/farmacología , Angiostatinas/farmacología , Neoplasias Encefálicas/tratamiento farmacológico , Glioblastoma/tratamiento farmacológico , Interleucina-12/farmacología , Virus Oncolíticos/genética , Simplexvirus/genética , Angiostatinas/genética , Animales , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/virología , Línea Celular Tumoral , Modelos Animales de Enfermedad , Femenino , Glioblastoma/metabolismo , Glioblastoma/virología , Humanos , Inyecciones Intralesiones , Interleucina-12/genética , Ratones , Ratones Desnudos , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND: Many branches of biomedical research find use for pure recombinant proteins for direct application or to study other molecules and pathways. Glutathione affinity purification is commonly used to isolate and purify glutathione S-transferase (GST)-tagged fusion proteins from total cellular proteins in lysates. Although GST affinity materials are commercially available as glutathione immobilized on beaded agarose resins, few simple options for in-house production of those systems exist. Herein, we describe a novel method for the purification of GST-tagged recombinant proteins. RESULTS: Glutathione was conjugated to low molecular weight poly(ethylene glycol) diacrylate (PEGDA) via thiol-ene "click" chemistry. With our in-house prepared PEGDA:glutathione (PEGDA:GSH) homogenates, we were able to purify a glutathione S-transferase (GST) green fluorescent protein (GFP) fusion protein (GST-GFP) from the soluble fraction of E. coli lysate. Further, microspheres were formed from the PEGDA:GSH hydrogels and improved protein binding to a level comparable to purchased GSH-agarose beads. CONCLUSIONS: GSH containing polymers might find use as in-house methods of protein purification. They exhibited similar ability to purify GST tagged proteins as purchased GSH agarose beads.
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Cromatografía de Afinidad/métodos , Glutatión Transferasa/metabolismo , Proteínas Fluorescentes Verdes/aislamiento & purificación , Cromatografía de Afinidad/instrumentación , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Glutatión/química , Glutatión Transferasa/genética , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Hidrogeles/química , Unión Proteica , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
This patent application claims an interesting and novel combination of passive accumulation of drug nanocrystals within diseased tissue, in combination with active uptake of the nanocrystals by diseased cells. The patent application further claims the hybrid nanocrystals combining imaging or stabilizing molecules as inclusions in the crystal matrix. There is a focus on cancer chemotherapy and imaging, but the initial claims are not disease specific. In this patent evaluation, the novelty and utility of this application is examined, while the state of the art in nanocrystal formulations and formulation is discussed.
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Antineoplásicos/administración & dosificación , Sistemas de Liberación de Medicamentos , Nanopartículas , Animales , Antineoplásicos/farmacocinética , Diseño de Fármacos , Humanos , Nanotecnología/métodos , Neoplasias/tratamiento farmacológico , Neoplasias/patología , Patentes como AsuntoRESUMEN
Bevacizumab (BEV) is an antiangiogenic drug approved for glioblastoma (GBM) treatment. However, it does not increase survival and is associated with glioma invasion. Angiostatin is an antiangiogenic polypeptide that also inhibits migration of cancer cells, but is difficult to deliver. Oncolytic viruses (OV) can potentially spread throughout the tumor, reach isolated infiltrating cells, kill them and deliver anticancer agents to uninfected cells. We have tested a combination treatment of BEV plus an OV expressing angiostatin (G47Δ-mAngio) in mice-bearing human GBM. Using a vascular intracranial human glioma model (U87) in athymic mice, we performed histopathological analysis of tumors treated with G47Δ-mAngio or BEV alone or in combination, followed tumor response by magnetic resonance imaging (MRI), and assessed animal survival. Our results indicate that injection of G47Δ-mAngio during BEV treatment allows increased virus spread, tumor lysis, and angiostatin-mediated inhibition of vascular endothelial growth factor (VEGF) expression and of BEV-induced invasion markers (matrix metalloproteinases-2 (MMP2), MMP9, and collagen). This leads to increased survival and antiangiogenesis and decreased invasive phenotypes. We show for the first time the possibility of improving the antiangiogenic effect of BEV while decreasing the tumor invasive-like phenotype induced by this drug, and demonstrate the therapeutic advantage of combining systemic and local antiangiogenic treatments with viral oncolytic therapy.
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Inhibidores de la Angiogénesis/genética , Inhibidores de la Angiogénesis/uso terapéutico , Angiostatinas/genética , Anticuerpos Monoclonales Humanizados/uso terapéutico , Glioma/terapia , Herpesvirus Humano 1/genética , Virus Oncolíticos/genética , Inhibidores de la Angiogénesis/administración & dosificación , Angiostatinas/metabolismo , Animales , Anticuerpos Monoclonales Humanizados/administración & dosificación , Antineoplásicos/administración & dosificación , Bevacizumab , Línea Celular Tumoral , Chlorocebus aethiops , Femenino , Terapia Genética , Vectores Genéticos/administración & dosificación , Glioma/genética , Glioma/mortalidad , Glioma/patología , Herpesvirus Humano 1/metabolismo , Humanos , Inyecciones , Ratones , Ratones Desnudos , Viroterapia Oncolítica , Virus Oncolíticos/metabolismo , Resultado del Tratamiento , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/metabolismo , Células Vero , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
PURPOSE: Inflammation occurs routinely when managing gliomas and is not easily distinguishable from tumor regrowth by current MRI methods. The lack of noninvasive technologies that monitor inflammation prevents us to understand whether it is beneficial or detrimental for the patient, and current therapies do not take this host response in consideration. We aim to establish whether a gadolinium (Gd)-based agent targeting the inflammatory enzyme myeloperoxidase (MPO) can selectively detect intra- and peritumoral inflammation as well as glioma response to treatment by MRI. METHODS: We carried out serial Gd-bis-5-HT-DTPA (MPO-Gd) MRI before and after treating rodent gliomas with different doses of oncolytic virus (OV) and analyzed animal survival. The imaging results were compared with histopathologic and molecular analyses of the tumors for macrophage/microglia infiltration, virus persistence, and MPO levels. RESULTS: Elevated MPO activity was observed by MRI inside the tumor and in the peritumoral cerebrum at day 1 post-OV injection, which corresponded with activation/infiltration of myeloid cells inhibiting OV intratumoral persistence. MPO activity decreased, whereas tumor size increased, as the virus and the immune cells were cleared (days 1-7 post-OV injection). A 10-fold increase in viral dose temporally decreased tumor size, but augmented MPO activity, thus preventing extension of viral intratumoral persistence. CONCLUSIONS: MPO-Gd MRI can distinguish enhancement patterns that reflect treatment-induced spatiotemporal changes of intratumoral and intracerebral inflammation from those indicating tumor and peritumoral edema. This technology improves the posttreatment diagnosis of gliomas and will increase our understanding of the role of inflammation in cancer therapy.