RESUMEN
Aberrant activation of the Hedgehog (Hh) signalling pathway has been associated with the development and progression of pancreatic cancer. For this reason, blockade of Hh pathway by inhibitors targeting the G protein-coupled receptor Smoothened (SMO) has been considered as a therapeutic target for the treatment of this cancer. In our previous work, we obtained a new SMO ligand based on a purine scaffold (compound I), which showed interesting antitumor activity in several cancer cell lines. In this work, we report the design and synthesis of 17 new purine derivatives, some of which showed high cytotoxic effect on Mia-PaCa-2 (Hh-dependent pancreatic cancer cell lines) and low toxicity on non-neoplastic HEK-293 cells compared with gemcitabine, such as 8f, 8g and 8h (IC50 = 4.56, 4.11 and 3.08 µM, respectively). Two of these purines also showed their ability to bind to SMO through NanoBRET assays (pKi = 5.17 for 8f and 5.01 for 8h), with higher affinities to compound I (pKi = 1.51). In addition, docking studies provided insight the purine substitution pattern is related to the affinity on SMO. Finally, studies of Hh inhibition for selected purines, using a transcriptional functional assay based on luciferase activity in NIH3T3 Shh-Light II cells, demonstrated that 8g reduced GLI activity with a IC50 = 6.4 µM as well as diminished the expression of Hh target genes in two specific Hh-dependent cell models, Med1 cells and Ptch1-/- mouse embryonic fibroblasts. Therefore, our results provide a platform for the design of SMO ligands that could be potential selective cytotoxic agents for the treatment of pancreatic cancer.
Asunto(s)
Antineoplásicos , Neoplasias Pancreáticas , Purinas , Receptor Smoothened , Humanos , Receptor Smoothened/antagonistas & inhibidores , Receptor Smoothened/metabolismo , Purinas/química , Purinas/farmacología , Purinas/síntesis química , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/patología , Neoplasias Pancreáticas/metabolismo , Ligandos , Antineoplásicos/farmacología , Antineoplásicos/química , Antineoplásicos/síntesis química , Animales , Ratones , Relación Estructura-Actividad , Ensayos de Selección de Medicamentos Antitumorales , Estructura Molecular , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Células HEK293 , Línea Celular Tumoral , Células 3T3 NIH , Simulación del Acoplamiento Molecular , Proteínas Hedgehog/metabolismo , Proteínas Hedgehog/antagonistas & inhibidoresRESUMEN
BACKGROUND: Helicobacter pylori (Hp) infects the stomach of 50% of the world's population. Importantly, chronic infection by this bacterium correlates with the appearance of several extra-gastric pathologies, including neurodegenerative diseases. In such conditions, brain astrocytes become reactive and neurotoxic. However, it is still unclear whether this highly prevalent bacterium or the nanosized outer membrane vesicles (OMVs) they produce, can reach the brain, thus affecting neurons/astrocytes. Here, we evaluated the effects of Hp OMVs on astrocytes and neurons in vivo and in vitro. METHODS: Purified OMVs were characterized by mass spectrometry (MS/MS). Labeled OMVs were administered orally or injected into the mouse tail vein to study OMV-brain distribution. By immunofluorescence of tissue samples, we evaluated: GFAP (astrocytes), ßIII tubulin (neurons), and urease (OMVs). The in vitro effect of OMVs in astrocytes was assessed by monitoring NF-κB activation, expression of reactivity markers, cytokines in astrocyte-conditioned medium (ACM), and neuronal cell viability. RESULTS: Urease and GroEL were prominent proteins in OMVs. Urease (OMVs) was present in the mouse brain and its detection coincided with astrocyte reactivity and neuronal damage. In vitro, OMVs induced astrocyte reactivity by increasing the intermediate filament proteins GFAP and vimentin, the plasma membrane αVß3 integrin, and the hemichannel connexin 43. OMVs also produced neurotoxic factors and promoted the release of IFNγ in a manner dependent on the activation of the transcription factor NF-κB. Surface antigens on reactive astrocytes, as well as secreted factors in response to OMVs, were shown to inhibit neurite outgrowth and damage neurons. CONCLUSIONS: OMVs administered orally or injected into the mouse bloodstream reach the brain, altering astrocyte function and promoting neuronal damage in vivo. The effects of OMVs on astrocytes were confirmed in vitro and shown to be NF-κB-dependent. These findings suggest that Hp could trigger systemic effects by releasing nanosized vesicles that cross epithelial barriers and access the CNS, thus altering brain cells.
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Helicobacter pylori , Ratones , Animales , Helicobacter pylori/metabolismo , Astrocitos , Ureasa/metabolismo , Ureasa/farmacología , FN-kappa B/metabolismo , Factor B del Complemento/metabolismo , Factor B del Complemento/farmacología , Modelos Animales de Enfermedad , Espectrometría de Masas en Tándem , NeuronasRESUMEN
The Smoothened (SMO) receptor is the most druggable target in the Hedgehog (HH) pathway for anticancer compounds. However, SMO antagonists such as vismodegib rapidly develop drug resistance. In this study, new SMO antagonists having the versatile purine ring as a scaffold were designed, synthesised, and biologically tested to provide an insight to their mechanism of action. Compound 4s was the most active and the best inhibitor of cell growth and selectively cytotoxic to cancer cells. 4s induced cell cycle arrest, apoptosis, a reduction in colony formation and downregulation of PTCH and GLI1 expression. BODIPY-cyclopamine displacement assays confirmed 4s is a SMO antagonist. In vivo, 4s strongly inhibited tumour relapse and metastasis of melanoma cells in mice. In vitro, 4s was more efficient than vismodegib to induce apoptosis in human cancer cells and that might be attributed to its dual ability to function as a SMO antagonist and apoptosis inducer.
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Antineoplásicos/farmacología , Neoplasias/tratamiento farmacológico , Purinas/farmacología , Receptor Smoothened/antagonistas & inhibidores , Animales , Antineoplásicos/química , Antineoplásicos/uso terapéutico , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Células HT29 , Proteínas Hedgehog/metabolismo , Humanos , Ratones Endogámicos C57BL , Neoplasias/metabolismo , Purinas/química , Purinas/uso terapéutico , Transducción de Señal/efectos de los fármacos , Receptor Smoothened/metabolismoRESUMEN
BACKGROUND: Extracellular vesicles (EVs) have shown great potential for targeted therapy, as they have a natural ability to pass through biological barriers and, depending on their origin, can preferentially accumulate at defined sites, including tumors. Analyzing the potential of EVs to target specific cells remains challenging, considering the unspecific binding of lipophilic tracers to other proteins, the limitations of fluorescence for deep tissue imaging and the effect of external labeling strategies on their natural tropism. In this work, we determined the cell-type specific tropism of B16F10-EVs towards cancer cell and metastatic tumors by using fluorescence analysis and quantitative gold labeling measurements. Surface functionalization of plasmonic gold nanoparticles was used to promote indirect labeling of EVs without affecting size distribution, polydispersity, surface charge, protein markers, cell uptake or in vivo biodistribution. Double-labeled EVs with gold and fluorescent dyes were injected into animals developing metastatic lung nodules and analyzed by fluorescence/computer tomography imaging, quantitative neutron activation analysis and gold-enhanced optical microscopy. RESULTS: We determined that B16F10 cells preferentially take up their own EVs, when compared with colon adenocarcinoma, macrophage and kidney cell-derived EVs. In addition, we were able to detect the preferential accumulation of B16F10 EVs in small metastatic tumors located in lungs when compared with the rest of the organs, as well as their precise distribution between tumor vessels, alveolus and tumor nodules by histological analysis. Finally, we observed that tumor EVs can be used as effective vectors to increase gold nanoparticle delivery towards metastatic nodules. CONCLUSIONS: Our findings provide a valuable tool to study the distribution and interaction of EVs in mice and a novel strategy to improve the targeting of gold nanoparticles to cancer cells and metastatic nodules by using the natural properties of malignant EVs.
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Antineoplásicos/química , Vesículas Extracelulares/química , Oro/química , Neoplasias Pulmonares/diagnóstico por imagen , Neoplasias Pulmonares/metabolismo , Melanoma/química , Nanopartículas del Metal/química , Adenocarcinoma/diagnóstico por imagen , Adenocarcinoma/terapia , Animales , Antineoplásicos/farmacología , Línea Celular Tumoral , Permeabilidad de la Membrana Celular , Neoplasias del Colon/diagnóstico por imagen , Neoplasias del Colon/terapia , Colorantes Fluorescentes/química , Humanos , Pulmón/metabolismo , Melanoma Experimental/diagnóstico por imagen , Melanoma Experimental/terapia , Ratones , Ratones Endogámicos C57BL , Imagen Óptica , Propiedades de Superficie , Distribución TisularRESUMEN
BACKGROUND: Stable and non-toxic fluorescent markers are gaining attention in molecular diagnostics as powerful tools for enabling long and reliable biological studies. Such markers should not only have a long half-life under several assay conditions showing no photo bleaching or blinking but also, they must allow for their conjugation or functionalization as a crucial step for numerous applications such as cellular tracking, biomarker detection and drug delivery. RESULTS: We report the functionalization of stable fluorescent markers based on nanodiamonds (NDs) with a bifunctional peptide. This peptide is made of a cell penetrating peptide and a six amino acids long ß-sheet breaker peptide that is able to recognize amyloid ß (Aß) aggregates, a biomarker for the Alzheimer disease. Our results indicate that functionalized NDs (fNDs) are not cytotoxic and can be internalized by the cells. The fNDs allow ultrasensitive detection (at picomolar concentrations of NDs) of in vitro amyloid fibrils and amyloid aggregates in AD mice brains. CONCLUSIONS: The fluorescence of functionalized NDs is more stable than that of fluorescent markers commonly used to stain Aß aggregates such as Thioflavin T. These results pave the way for performing ultrasensitive and reliable detection of Aß aggregates involved in the pathogenesis of the Alzheimer disease.
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Enfermedad de Alzheimer/diagnóstico , Amiloide/análisis , Colorantes Fluorescentes/química , Nanodiamantes/química , Amiloide/metabolismo , Péptidos beta-Amiloides/análisis , Péptidos beta-Amiloides/metabolismo , Animales , Benzotiazoles/química , Benzotiazoles/toxicidad , Biomarcadores/análisis , Línea Celular , Supervivencia Celular/efectos de los fármacos , Péptidos de Penetración Celular/química , Colorantes Fluorescentes/toxicidad , Humanos , Ratones Transgénicos , Nanodiamantes/toxicidad , Agregado de ProteínasRESUMEN
Here we report the incorporation of gold nanostructures (nanospheres or nanorods, functionalized with carboxylate-end PEG) and curcumin oil-in-water (O/W) nanoemulsions (CurNem) into alginate microgels using the dripping technique. While gold nanostructures are promising nanomaterials for photothermal therapy applications, CurNem possess important pharmacological activities as reported here. In this sense, we evaluated the effect of CurNem on cell viability of both cancerous and non-cancerous cell lines (AGS and HEK293T, respectively), demonstrating preferential toxicity in cancer cells and safety for the non-cancerous cells. After incorporating gold nanostructures and CurNem together into the microgels, microstructures with diameters of 220 and 540 µm were obtained. When stimulating microgels with a laser, the plasmon effect promoted a significant rise in the temperature of the medium; the temperature increase was higher for those containing gold nanorods (11â»12 °C) than nanospheres (1â»2 °C). Interestingly, the incorporation of both nanosystems in the microgels maintains the photothermal properties of the gold nanostructures unmodified and retains with high efficiency the curcumin nanocarriers. We conclude that these results will be of interest to design hydrogel formulations with therapeutic applications.
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Portadores de Fármacos/química , Oro/química , Nanosferas/química , Nanotubos/química , Antineoplásicos/administración & dosificación , Antineoplásicos/química , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Curcumina/administración & dosificación , Curcumina/química , Liberación de Fármacos , Emulsiones , Geles , Células HEK293 , Humanos , Rayos Láser , Tamaño de la Partícula , Fotoquimioterapia/métodos , Polietilenglicoles/química , Solubilidad , Propiedades de SuperficieRESUMEN
Gold nanoparticles (AuNPs) have been extensively used in biological applications because of their biocompatibility, size, and ease of characterization, as well as an extensive knowledge of their surface chemistry. These features make AuNPs readily exploitable for biomedical applications, including drug delivery and novel diagnostic and therapeutic approaches. In a previous work, we studied ex vivo distribution of the conjugate C(AuNP)-LPFFD for its potential uses in the treatment of Alzheimer's disease. In this study, we covalently labeled the conjugate with [(18)F]-fluorobenzoate to study the in vivo distribution of the AuNP by positron emission tomography (PET). After intravenous administration in rat, the highest concentration of the radiolabeled conjugate was found in the bladder and urine with a lower proportion in the intestine, demonstrating progressive accumulation compatible with biliary excretion of the conjugate. The conjugate also accumulated in the liver and spleen. PET imaging allowed us to study the in vivo biodistribution of the AuNPs in a noninvasive and sensitive way using a reduced number of animals. Our results show that AuNPs can be covalently and radioactively labeled for PET biodistribution studies.
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Radioisótopos de Flúor/farmacocinética , Oro/química , Nanopartículas del Metal , Péptidos/farmacocinética , Animales , Cromatografía Líquida de Alta Presión , Evaluación Preclínica de Medicamentos , Masculino , Microscopía Electrónica de Transmisión , Péptidos/química , Tomografía de Emisión de Positrones , Ratas , Ratas Sprague-Dawley , Espectrofotometría Ultravioleta , Resonancia por Plasmón de Superficie , Distribución TisularRESUMEN
Gold nanoparticles (AuNP) capped with biocompatible layers have functional optical, chemical, and biological properties as theranostic agents in biomedicine. The ferritin protein containing in situ synthesized AuNPs has been successfully used as an effective and completely biocompatible nanocarrier for AuNPs in human cell lines and animal experiments in vivo. Ferritin can be uptaken by different cell types through receptor-mediated endocytosis. Despite these advantages, few efforts have been made to evaluate the toxicity and cellular internalization of AuNP-containing ferritin nanocages. In this work, we study the potential of human heavy-chain (H) and light-chain (L) ferritin homopolymers as nanoreactors to synthesize AuNPs and their cytotoxicity and cellular uptake in different cell lines. The results show very low toxicity of ferritin-encapsulated AuNPs on different human cell lines and demonstrate that efficient cellular ferritin uptake depends on the specific H or L protein chains forming the ferritin protein cage and the presence or absence of metallic cargo. Cargo-devoid apoferritin is poorly internalized in all cell lines, and the highest ferritin uptake was achieved with AuNP-loaded H-ferritin homopolymers in transferrin-receptor-rich cell lines, showing more than seven times more uptake than apoferritin.
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In advanced stages of cancer disease, caveolin-1 (CAV1) expression increases and correlates with increased migratory and invasive capacity of the respective tumor cells. Previous findings from our laboratory revealed that specific ECM-integrin interactions and tyrosine-14 phosphorylation of CAV1 are required for CAV1-enhanced melanoma cell migration, invasion and metastasis in vivo. In this context, CAV1 phosphorylation on tyrosine-14 mediated by non-receptor Src-family tyrosine kinases seems to be important; however, the effect of Src-family kinase inhibitors on CAV1-enhanced metastasis in vivo has not been studied. Here, we evaluated the effect of CAV1 and c-Abl overexpression, as well as the use of the Src-family kinase inhibitors, PP2 and dasatinib (more specific for Src/Abl) in lung metastasis of B16F10 melanoma cells. Overexpression of CAV1 and c-Abl enhanced CAV1 phosphorylation and the metastatic potential of the B16F10 murine melanoma cells. Alternatively, treatment with PP2 or dasatinib for 2â¯h reduced CAV1 tyrosine-14 phosphorylation and levels recovered fully within 12â¯h of removing the inhibitors. Nonetheless, pre-treatment of cells with these inhibitors for 2â¯h sufficed to prevent migration, invasion and trans-endothelial migration in vitro. Importantly, the transient decrease in CAV1 phosphorylation by these kinase inhibitors prevented early steps of CAV1-enhanced lung metastasis by B16F10 melanoma cells injected into the tail vein of mice. In conclusion, this study underscores the relevance of CAV1 tyrosine-14 phosphorylation by Src-family kinases during the first steps of the metastatic sequence promoted by CAV1. These findings open up potential options for treatment of metastatic tumors in patients in which Src-family kinase activation and CAV1 overexpression favor dissemination of cancer cells to secondary sites.
Asunto(s)
Caveolina 1/metabolismo , Dasatinib/farmacología , Neoplasias Pulmonares/secundario , Melanoma Experimental/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Pirimidinas/farmacología , Neoplasias Cutáneas/metabolismo , Familia-src Quinasas/antagonistas & inhibidores , Animales , Caveolina 1/genética , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Dasatinib/uso terapéutico , Femenino , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/patología , Masculino , Melanoma Experimental/patología , Ratones , Ratones Endogámicos C57BL , Neuropéptidos/metabolismo , Fosforilación/efectos de los fármacos , Inhibidores de Proteínas Quinasas/uso terapéutico , Pirimidinas/uso terapéutico , Neoplasias Cutáneas/patología , Transfección , Tirosina/metabolismo , Proteína de Unión al GTP rac1/metabolismoRESUMEN
BACKGROUND: Collagen, the most abundant protein in the animal kingdom, represents a promising biomaterial for regenerative medicine applications due to its structural diversity and self-assembling complexity. Despite collagen's widely known structural and functional features, the thermodynamics behind its fibrillogenic self-assembling process is still to be fully understood. In this work we report on a series of spectroscopic, mechanical, morphological and thermodynamic characterizations of high purity type I collagen (with a D-pattern of 65 nm) extracted from Wistar Hannover rat tail. Our herein reported results can be of help to elucidate differences in self-assembly states of proteins using ITC to improve the design of energy responsive and dynamic materials for applications in tissue engineering and regenerative medicine. METHODS: Herein we report the systematic study on the self-assembling fibrillogenesis mechanism of type I collagen, we provide morphological and thermodynamic evidence associated to different self-assembly events using ITC titrations. We provide thorough characterization of the effect of pH, effect of salts and protein conformation on self-assembled collagen samples via several complementary biophysical techniques, including circular dichroism (CD), Fourier Transform infrared spectroscopy (FTIR), differential scanning calorimetry (DSC), atomic force microscopy (AFM), scanning electron microscopy (SEM), dynamic mechanical thermal analysis (DMTA) and thermogravimetric analysis (TGA). RESULTS: Emphasis was made on the use of isothermal titration calorimetry (ITC) for the thermodynamic monitoring of fibrillogenesis stages of the protein. An overall self-assembly enthalpy value of 3.27 ± 0.85 J/mol was found. Different stages of the self-assembly mechanism were identified, initial stages take place at pH values lower than the protein isoelectric point (pI), however, higher energy release events were recorded at collagen's pI. Denatured collagen employed as a control exhibited higher energy absorption at its pI, suggesting different energy exchange mechanisms as a consequence of different aggregation routes.
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The inclusion compound (IC) of cyclodextrin (CD) containing the antitumor drug Methotrexate (MTX) as a guest molecule was obtained to increase the solubility of MTX and decrease its inherent toxic effects in nonspecific cells. The IC was conjugated with gold nanoparticles (AuNPs), obtained by a chemical method, creating a ternary intelligent delivery system for MTX molecules, based on the plasmonic properties of the AuNPs. Irradiation of the ternary system, with a laser wavelength tunable with the corresponding surface plasmon of AuNPs, causes local energy dissipation, producing the controlled release of the guest from CD cavities. Finally, cell viability was evaluated using MTS assays for ß-CD/MTX and AuNPs + ß-CD/MTX samples, with and without irradiation, against HeLa tumor cells. The irradiated sample of the ternary system AuNPs + ß-CD/MTX produced a diminution in cell viability attributed to the photothermal release of MTX.
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Low effectiveness and resistance to treatments are commonplace in disorders of the central nervous system (CNS). These issues concern mainly the blood-brain barrier (BBB), which preserves homeostasis in the brain and protects this organ from toxic molecules and biohazards by regulating transport through it. BBB shuttles-short peptides able to cross the BBB-are being developed to help therapeutics to cross this barrier. BBB shuttles can be discovered by massive exploration of chemical diversity (e.g. computational means, phage display) or rational design (e.g. derivatives from a known peptide/protein able to cross). Here we present the selection of a peptide shuttle (HAI) from several candidates and the subsequent in-depth in vitro and in vivo study of this molecule. In order to explore the chemical diversity of HAI and enhance its biostability, and thereby its bioactivity, we explored two new protease-resistant versions of HAI (i.e. the retro-D-version, and a version that was N-methylated at the most sensitive sites to enzymatic cleavage). Our results show that, while both versions of HAI are resistant to proteases, the retro-D-approach preserved better transport properties.
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Barrera Hematoencefálica/metabolismo , Péptidos de Penetración Celular/síntesis química , Péptidos de Penetración Celular/farmacocinética , Receptores de Transferrina/análisis , Animales , Péptidos de Penetración Celular/química , Sistemas de Liberación de Medicamentos , Diseño de Fármacos , Estabilidad de Medicamentos , Humanos , Péptido Hidrolasas/metabolismo , Permeabilidad , RatasRESUMEN
BACKGROUND: Numerous studies have proposed the use of fluorescent semiconductor nanoparticles or quantum dots (QDs) as novel tools to label cells and tumors. However, QD applications are limited by their toxicity in biological systems and little is known about whether QDs affect the capacity of cancer cells to metastasize. Previously, we described the "biomimetic" synthesis of CdTe-QDs (QDs-glutathione [GSH]) with increased biocompatibility and the potential utility in labeling cells. PURPOSE: In order to determine the feasibility of using QDs-GSH as a tool for tracking tumor cells during early metastasis, we characterized here for the first time, the in vitro and in vivo effects of the incorporation of green or red biomimetic QDs-GSH into B16F10 cells, a syngeneic mouse melanoma line for metastasis assays in C57BL/6 mice. METHODS: B16F10 cells were labeled with green or red biomimetic QDs-GSH in the presence or absence of n-acetylcysteine. Then, migration, invasion and proliferation of labeled B16F10 were evaluated in vitro. Finally, the B16F10 cells labeled with red QDs-GSH were used to monitor in vivo lung metastasis at early time points (5 minutes to 24 hours) or after 21 days in C57BL/6 mice. RESULTS: We developed a methodology that allows obtaining QDs-GSH-labeled B16F10 cells (nearly 100% viable labeled cells), which remained viable for at least 5 days and migrated similarly to control cells. However, proliferation, invasion, and the capacity to form metastatic nodules in the lungs were severely attenuated. Fluorescence imaging revealed that distribution/accumulation of QDs-GSH-labeled B16F10 cells could be tracked following injection into C57BL/6 mice (syngeneic preclinical metastasis model) and that these cells preferentially accumulated in the perialveolar area in lungs as early as 5 minutes post-injection. CONCLUSION: The methodology described here represents a useful alternative for monitoring initial events during tumor cell metastasis.
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Materiales Biomiméticos/química , Diagnóstico por Imagen , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/secundario , Melanoma Experimental/patología , Puntos Cuánticos/química , Coloración y Etiquetado , Acetilcisteína/farmacología , Animales , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Glutatión/química , Humanos , Hidrodinámica , Ratones Endogámicos C57BL , Especies Reactivas de Oxígeno/metabolismo , Electricidad Estática , Distribución Tisular/efectos de los fármacosRESUMEN
Curcumin is widely considered beneficial to human health, but insolubility and instability greatly hamper reproducible exploitation of the advantageous traits. Here we report on the development, characterization and evaluation of a curcumin-loaded nanoemulsion (CUR-NEM) that is highly effective in preventing post-surgery tumor reincidence and metastasis. The method of fabrication utilized safe excipients and generated particles of 200 nm (PDI ≤ 0.2) with negative zeta potential (-30 mV) and a high yield of curcumin (95%), which can be converted by lyophilization to a dry powder. In vitro assays showed that CUR-NEM is safe in non-cancerous human cells (HEK-293T) and preferentially cytotoxic in gastric (AGS), colon (HT29-ATCC, HT29-US), breast (MDA-MB-231) and melanoma (B16F10) cells. In addition, in melanoma cells the nanoformulation increases intracellular curcumin accumulation and reactive oxygen species (ROS) formation, while preventing cell-migration and invasion. In vivo studies in C57BL/6 mice demonstrated that a single dose, applied topically to the wounded area after surgical excision of primary tumors formed upon subcutaneous injection of syngeneic B16F10 cells, was sufficient to completely prevent reincident tumor growth and spontaneous lung metastasis, while in untreated animals 70% reincidence and metastasis were observed. In vivo experiments also showed that the fluorescence signal due to curcumin was maintained at least 15 days after topical application of CUR-NEM, while when administered in DMSO the curcumin signal disappeared within 4 days. Importantly, the administration of a dose 22 times larger than that applied topically to animals after tumor surgery did not alter biochemical parameters. Due to the safety and efficacy of the formulation, we envisage it as ideal for topical application in cancer patients following surgery, to prevent tumor reincidence and metastasis. In addition, other routes of administration/protocols could also be proposed to treat/prevent malignant tumors in patients.
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Curcumina/química , Emulsiones/química , Neoplasias/patología , Células A549 , Adenocarcinoma/patología , Animales , Línea Celular Tumoral , Movimiento Celular , Supervivencia Celular , Portadores de Fármacos/química , Células HEK293 , Humanos , Neoplasias Pulmonares/patología , Melanoma Experimental , Ratones , Ratones Endogámicos C57BL , Nanopartículas/química , Metástasis de la Neoplasia , Especies Reactivas de Oxígeno/metabolismo , Solventes/químicaRESUMEN
AIM: To track early events during lung metastasis, we labeled cells expressing (B16F10CAV1) or lacking CAV1 (B16F10mock) with gold nanoparticles conjugated to the peptide TAT (AuNPs-PEG-TAT). METHODS: B16F10 expressing or lacking CAV1 were labeled with AuNPs-PEG-TAT. The physicochemical properties and cytotoxicity of these nanoparticles, as well as their effects on migration and invasiveness of B16F10 cells in vitro were evaluated. Ex vivo lung distribution of the labeled cells after tail vein injection into C57BL/6 mice was examined. RESULTS: AuNPs-PEG-TAT did not affect B16F10 viability, migration and invasiveness. The metastatic and tumorigenic capability of the labeled B16F10 was also not modified in comparison to unlabeled B16F10 cells. CAV1 expression favored the retention of B16F10 cells in the lungs of mice 2 h post injection, suggesting CAV1 promoted adherence to endothelial cells and transendothelial migration. CONCLUSIONS: We developed a protocol to label B16F10 cells with AuNPs-PEG-TAT that permits subsequent tracking of cells in mice. CAV1 overexpression was found to increase retention and transendothelial migration of B16F10 cells in the lung.
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Caveolina 1/genética , Rastreo Celular , Melanoma Experimental/diagnóstico por imagen , Nanopartículas del Metal/administración & dosificación , Animales , Caveolina 1/química , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Endoteliales/metabolismo , Regulación Neoplásica de la Expresión Génica , Oro/química , Humanos , Melanoma Experimental/genética , Melanoma Experimental/patología , Nanopartículas del Metal/química , Ratones , Metástasis de la NeoplasiaRESUMEN
With its capacity for anaerobic methane oxidation and denitrification, the bacterium Methylomirabilis oxyfera plays an important role in natural ecosystems. Its unique physiology can be exploited for more sustainable wastewater treatment technologies. However, operational stability of full-scale bioreactors can experience setbacks due to, for example, bacteriophage blooms. By shaping microbial communities through mortality, horizontal gene transfer, and metabolic reprogramming, bacteriophages are important players in most ecosystems. Here, we analyzed an infected Methylomirabilis sp. bioreactor enrichment culture using (advanced) electron microscopy, viral metagenomics and bioinformatics. Electron micrographs revealed four different viral morphotypes, one of which was observed to infect Methylomirabilis cells. The infected cells contained densely packed ~55 nm icosahedral bacteriophage particles with a putative internal membrane. Various stages of virion assembly were observed. Moreover, during the bacteriophage replication, the host cytoplasmic membrane appeared extremely patchy, which suggests that the bacteriophages may use host bacterial lipids to build their own putative internal membrane. The viral metagenome contained 1.87 million base pairs of assembled viral sequences, from which five putative complete viral genomes were assembled and manually annotated. Using bioinformatics analyses, we could not identify which viral genome belonged to the Methylomirabilis- infecting bacteriophage, in part because the obtained viral genome sequences were novel and unique to this reactor system. Taken together these results show that new bacteriophages can be detected in anaerobic cultivation systems and that the effect of bacteriophages on the microbial community in these systems is a topic for further study.
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An exciting and emerging field in nanomedicine involves the use of gold nanoparticles (AuNPs) in the preclinical development of new strategies for the treatment and diagnosis of brain-related diseases such as neurodegeneration and cerebral tumors. The treatment of many brain-related disorders with AuNPs, which possess useful physical properties, is limited by the blood-brain barrier (BBB). The BBB highly regulates the substances that can permeate into the brain. Peptides and proteins may represent promising tools to improve the delivery of AuNPs to the central nervous system (CNS). In this review, we summarize the potential applications of AuNPs to CNS disorders, discuss different strategies based on the use of peptides or proteins to improve the delivery of AuNPs to the brain, and examine the intranasal administration route, which bypasses the BBB. We also analyze the potential neurotoxicity of AuNPs and the perspectives and new challenges concerning the use of peptides and proteins to enhance the delivery of AuNPs to the brain. The majority of the work described in this review is in a preclinical stage of experimentation, or in select cases, in clinical trials in humans. We note that the use of AuNPs still requires substantial study before being translated into human applications. However, for further clinical research, the issues related to the potential use of AuNPs must be analyzed.
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Encéfalo/metabolismo , Portadores de Fármacos , Oro , Nanopartículas del Metal , Nanomedicina/métodos , Péptidos , Encefalopatías , Portadores de Fármacos/química , Portadores de Fármacos/farmacocinética , Portadores de Fármacos/uso terapéutico , Portadores de Fármacos/toxicidad , Oro/química , Oro/farmacocinética , Oro/uso terapéutico , Oro/toxicidad , Humanos , Nanopartículas del Metal/química , Nanopartículas del Metal/uso terapéutico , Nanopartículas del Metal/toxicidad , Péptidos/química , Péptidos/farmacocinética , Péptidos/uso terapéutico , Péptidos/toxicidad , Proteínas/química , Proteínas/farmacocinética , Proteínas/uso terapéutico , Proteínas/toxicidadRESUMEN
In this article we review the flow chemistry methodologies for the controlled synthesis of different kind of nano and microparticles for biomedical applications. Injection mechanism has emerged as new alternative for the synthesis of nanoparticles due to this strategy allows achieving superior levels of control of self-assemblies, leading to higher-ordered structures and rapid chemical reactions. Self-assembly events are strongly dependent on factors such as the local concentration of reagents, the mixing rates, and the shear forces, which can be finely tuned, as an example, in a microfluidic device. Injection methods have also proved to be optimal to elaborate microsystems comprising polymer solutions. Concretely, extrusion based methods can provide controlled fluid transport, rapid chemical reactions, and cost-saving advantages over conventional reactors. We provide an update of synthesis of nano and microparticles such as core/shell, Janus, nanocrystals, liposomes, and biopolymeric microgels through flow chemistry, its potential bioapplications and future challenges in this field are discussed.
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Investigación Biomédica/métodos , Microesferas , Nanopartículas/química , Investigación Biomédica/instrumentaciónRESUMEN
Gold nanorods used in therapy and diagnosis must be nontoxic and stable in biological media and should be specific for the target. The complete combination of these three factors has hindered the use of gold nanorods as carriers in biological and biomedical applications. In this study, we produced a conjugate of gold nanorods with the peptide CLPFFD that recognizes toxic ß-amyloid aggregates present in Alzheimer's disease, demonstrates colloidal stability, maintains plasmonic properties, and shows no effects on cell viability in the SH-SY5Y cell line. Furthermore, the irradiation of ß-amyloid in the presence of the conjugate with near-infrared region irradiation energy reduces the amyloidogenic process reducing also its cytotoxicity. The nanorods were synthesized following the seed-mediated method in cetyltrimethylammonium bromide (CTAB) and were conjugated with the N-terminal cysteine peptide, CLPFFD. The conjugate was exhaustively characterized using different techniques (Absorption spectroscopy, X-ray photoelectron spectroscopy, electron energy loss spectroscopy, and zeta potential). The effects on cell viability and cell penetration by transmission electron microscopy of the conjugate were evaluated. The chemisorption of the peptide on the surface of gold nanorods increases their stability and reduces their effects on cell viability.
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Oro/química , Nanotubos , Péptidos/química , Supervivencia Celular , Microscopía Electrónica de Transmisión de Rastreo , Espectroscopía de Fotoelectrones , Espectroscopía Infrarroja CortaRESUMEN
The treatment of Alzheimer's disease and many other brain-related disorders is limited because of the presence of the blood-brain barrier, which highly regulate the crossing of drugs. Metal nanoparticles have unique features that could contribute to the development of new therapies for these diseases. Nanoparticles have the capacity to carry several molecules of a drug; furthermore, their unique physico-chemical properties allow, for example, photothermal therapy to produce molecular surgery to destroy tumor cells and toxic structures. Recently, we demonstrated that gold nanoparticles conjugated to the peptide CLPFFD are useful to destroy the toxic aggregates of ß-amyloid, similar to the ones found in the brains of patients with Alzheimer's disease. However, nanoparticles, like many other compounds, have null or very low capacity to cross the blood-brain barrier. In order to devise a strategy to improve drug delivery to the brain, here we introduced the peptide sequence THRPPMWSPVWP into the gold nanoparticle-CLPFFD conjugate. This peptide sequence interacts with the transferrin receptor present in the microvascular endothelial cells of the blood-brain barrier, thus causing an increase in the permeability of the conjugate in brain, as shown by experiments in vitro and in vivo. Our results are highly relevant for the therapeutic applications of gold nanoparticles for molecular surgery in the treatment of neurodegenerative diseases such as Alzheimer's disease.