RESUMEN
Development of bioinspired nanomotors showing effective propulsion and cargo delivery capabilities has attracted much attention in the last few years due to their potential use in biomedical applications. However, implementation of this technology in realistic settings is still a barely explored field. Herein, we report the design and application of a multifunctional gated Janus platinum-mesoporous silica nanomotor constituted of a propelling element (platinum nanodendrites) and a drug-loaded nanocontainer (mesoporous silica nanoparticle) capped with ficin enzyme modified with ß-cyclodextrins (ß-CD). The engineered nanomotor is designed to effectively disrupt bacterial biofilms via H2O2-induced self-propelled motion, ficin hydrolysis of the extracellular polymeric matrix (EPS) of the biofilm, and controlled pH-triggered cargo (vancomycin) delivery. The effective synergic antimicrobial activity of the nanomotor is demonstrated in the elimination of Staphylococcus aureus biofilms. The nanomotor achieves 82% of EPS biomass disruption and a 96% reduction in cell viability, which contrasts with a remarkably lower reduction in biofilm elimination when the components of the nanomotors are used separately at the same concentrations. Such a large reduction in biofilm biomass in S. aureus has never been achieved previously by any conventional therapy. The strategy proposed suggests that engineered nanomotors have great potential for the elimination of biofilms.
RESUMEN
Nanoparticle-cell-nanoparticle communication by stigmergy was demonstrated using two capped nanodevices. The first community of nanoparticles (i.e.S(RA)IFN) is loaded with 9-cis-retinoic acid and capped with interferon-γ, whereas the second community of nanoparticles (i.e.S(sulf)PIC) is loaded with sulforhodamine B and capped with poly(I:C). The uptake of S(RA)IFN by SK-BR-3 breast cancer cells enhanced the expression of TLR3 receptor facilitating the subsequent uptake of S(sulf)PIC and cell killing.
Asunto(s)
Antineoplásicos/metabolismo , Comunicación Celular/efectos de los fármacos , Inductores de Interferón/metabolismo , Nanopartículas/química , Poli I-C/metabolismo , Alitretinoína/química , Antineoplásicos/química , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Inductores de Interferón/química , Interferón gamma/efectos de los fármacos , Nanopartículas/metabolismo , Poli I-C/química , Rodaminas/química , Receptor Toll-Like 3/genéticaRESUMEN
Apoptotic signaling pathways are altered in numerous pathologies such as cancer. In this scenario, caspase-9/PP2Acα interaction constitutes a key target with pharmacological interest to re-establish apoptosis in tumor cells. Very recently, a short peptide (C9h) known to disrupt caspase-9/PP2Acα interaction with subsequent apoptosis induction was described. Here, we prepared two sets of mesoporous silica nanoparticles loaded with safraninâ O (S2) or with C9h peptide (S4) and functionalized with ϵ-polylysine as capping unit. Aqueous suspensions of both nanoparticles showed negligible cargo release whereas in the presence of pronase, a marked delivery of safraninâ O or C9h was observed. Confocal microscopy studies carried out with HeLa cells indicated that both materials were internalized and were able to release their entrapped cargos. Besides, a marked decrease in HeLa cell viability (ca. 50 %) was observed when treated with C9h-loaded S4 nanoparticles. Moreover, S4 provides peptide protection from degradation additionally allowing for a dose reduction to observe an apoptotic effect when compared with C9h alone or in combination with a cell-penetrating peptide (i.e., Mut3DPT-C9h). Flow cytometry studies, by means of Annexin V-FITC staining, showed the activation of apoptotic pathways in HeLa as a consequence of S4 internalization, release of C9h peptide and disruption of caspase-9/PP2Acα interaction.
Asunto(s)
Nanopartículas/química , Péptidos/química , Polilisina/química , Dióxido de Silicio/química , Secuencia de Aminoácidos , Apoptosis/efectos de los fármacos , Caspasa 9/química , Caspasa 9/metabolismo , Dicroismo Circular , Portadores de Fármacos/síntesis química , Portadores de Fármacos/química , Células HeLa , Humanos , Microscopía Confocal , Péptidos/toxicidad , Fenazinas/química , Fenazinas/toxicidad , Porosidad , Proteína Fosfatasa 2/química , Proteína Fosfatasa 2/metabolismoRESUMEN
The possibility of achieving sophisticated actions in complex biological environments using gated nanoparticles is an exciting prospect with much potential. We herein describe new gated mesoporous silica nanoparticles (MSN) loaded with an anticoagulant drug and capped with a peptide containing a thrombin-specific cleavage site. When the coagulation cascade was triggered, active thrombin degraded the capping peptidic sequence and induced the release of anticoagulant drugs to delay the clotting process. The thrombin-dependent response was assessed and a significant increase in coagulation time in plasma from 2.6 min to 5 min was found. This work broadens the application of gated silica nanoparticles and demonstrates their ability to act as controllers in a complex scenario such as hemostasis.
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Acenocumarol/química , Anticoagulantes/química , Nanopartículas/química , Dióxido de Silicio/química , Trombina/química , Acenocumarol/farmacología , Animales , Anticoagulantes/farmacología , Coagulación Sanguínea/efectos de los fármacos , Ácidos Pentanoicos/química , Conejos , Tiempo de Coagulación de la Sangre TotalRESUMEN
We describe herein a Toll-like receptor 3 (TLR3) targeting delivery system based on mesoporous silica nanoparticles capped with the synthetic double stranded RNA polyinosinic-polycytidylic acid (poly(I:C)) for controlled cargo delivery in SK-BR-3 breast carcinoma cells. Our results show that poly(I:C)-conjugated nanoparticles efficiently targeted breast cancer cells due to dsRNA-TLR3 interaction. Such interaction also triggered apoptotic pathways in SK-BR-3, significantly decreasing cells viability. Poly(I:C) cytotoxic effect in breast carcinoma cells was enhanced by loading nanoparticles' mesopores with the anthracyclinic antibiotic doxorubicin, a commonly used chemotherapeutic agent.
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Antineoplásicos/química , Antineoplásicos/farmacología , Neoplasias de la Mama/química , Neoplasias de la Mama/tratamiento farmacológico , Doxorrubicina/química , Nanopartículas/química , Poli I-C/química , Poli I-C/farmacología , ARN Bicatenario/química , Dióxido de Silicio/química , Línea Celular Tumoral , Femenino , Humanos , Inmunidad Innata , ARN Bicatenario/farmacologíaRESUMEN
Core-shell nanoparticles comprised of Fe3O4 cores and a mesoporous silica shell with an average expanded pore size of 6.07 nm and coated with a poly(N-isopropylacrylamide) (PNIPAM) layer (CS-MSNs-EP-PNIPAM) were prepared and characterized. The nanoparticles was loaded with (Ru(bipy)3(2+)) dye or an antibacterial enzyme, lysozyme, to obtain CS-MSNs-EP-PNIPAM-Ru(bipy)3(2+) and CS-MSNs-EP-PNIPAM-Lys, respectively. The lysozyme loading was determined to be 160 mg/g of nanoparticle. It was seen that Ru(bipy)3(2+) and lysozyme release was minimal at a room temperature of 25 °C while at physiological temperature (37 °C), abrupt release was observed. The applicability of the CS-MSNs-EP-PNIPAM-Lys was further tested with two Gram-positive bacteria samples, Bacillus cereus and Micrococcus luteus. At physiological temperature, the nanoparticles were shown to reduce bacterial growth, indicating a successful release of lysozyme from the nanoparticles. This nanoparticle system shows potential as a nanocarrier for the loading of similarly sized proteins or other species as a drug delivery platform.
Asunto(s)
Resinas Acrílicas/química , Antibacterianos/química , Compuestos Ferrosos/química , Muramidasa/química , Nanopartículas/química , Dióxido de Silicio/química , Antibacterianos/administración & dosificación , Bacillus cereus/efectos de los fármacos , Micrococcus luteus/efectos de los fármacos , Muramidasa/administración & dosificación , Porosidad , TemperaturaRESUMEN
In recent years, mesoporous silica nanoparticles (MSNs) have been used as effective supports for the development of controlled-release nanodevices that are able to act as multifunctional delivery platforms for the encapsulation of therapeutic agents, enhancing their bioavailability and overcoming common issues such as poor water solubility and poor stability of some drugs. In particular, redox-responsive delivery systems have attracted the attention of scientists because of the intracellular reductive environment related to a high concentration of glutathione (GSH). In this context, we describe herein the development of a GSH-responsive delivery system based on poly(ethylene glycol)- (PEG-) capped MSNs that are able to deliver safranin O and doxorubicin in a controlled manner. The results showed that the PEG-capped systems designed in this work can be maintained closed at low GSH concentrations, yet the cargo can be delivered when the concentration of GSH is increased. Moreover, the efficacy of the PEG-capped system in delivering the cytotoxic agent doxorubicin in cells was also demonstrated.
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Antineoplásicos/química , Portadores de Fármacos/química , Nanopartículas/química , Dióxido de Silicio/química , Antineoplásicos/farmacología , Supervivencia Celular/efectos de los fármacos , Preparaciones de Acción Retardada , Doxorrubicina/química , Doxorrubicina/farmacología , Diseño de Fármacos , Liberación de Fármacos , Glutatión/metabolismo , Células HeLa , Humanos , Espacio Intracelular/metabolismo , Oxidación-Reducción/efectos de los fármacos , Fenazinas/química , Fenazinas/farmacología , Polietilenglicoles/química , PorosidadRESUMEN
ß-Lapachone (ß-lap) is a novel anticancer agent that selectively induces cell death in human cancer cells, by activation of the NQO1 NAD(P)H dehydrogenase and radical oxygen species (ROS) generation. We characterized the gene expression profile of budding yeast cells treated with ß-lap using cDNA microarrays. Genes involved in tolerance to oxidative stress were differentially expressed in ß-lap treated cells. ß-lap treatment generated reactive oxygen species (ROS), which were efficiently blocked by dicoumarol, an inhibitor of NADH dehydrogenases. A yeast mutant in the mitochondrial NADH dehydrogenase Nde2p was found to be resistant to ß-lap treatment, despite inducing ROS production in a WT manner. Most interestingly, DNA damage responses triggered by ß-lap were abolished in the nde2Δ mutant. Amino acid biosynthesis genes were also induced in ß-lap treated cells, suggesting that ß-lap exposure somehow triggered the General Control of Nutrients (GCN) pathway. Accordingly, ß-lap treatment increased phosphorylation of eIF2α subunit in a manner dependent on the Gcn2p kinase. eIF2α phosphorylation required Gcn1p, Gcn20p and Nde2p. Gcn2p was also required for cell survival upon exposure to ß-lap and to elicit checkpoint responses. Remarkably, ß-lap treatment increased phosphorylation of eIF2α in breast tumor cells, in a manner dependent on the Nde2p ortholog AIF, and the eIF2 kinase PERK. These findings uncover a new target pathway of ß-lap in yeast and human cells and highlight a previously unknown functional connection between Nde2p, Gcn2p and DNA damage responses.
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Antineoplásicos/farmacología , Activación Enzimática/efectos de los fármacos , Naftoquinonas/farmacología , eIF-2 Quinasa/metabolismo , Antineoplásicos/metabolismo , Dicumarol/farmacología , Humanos , Immunoblotting , NAD(P)H Deshidrogenasa (Quinona)/antagonistas & inhibidores , NAD(P)H Deshidrogenasa (Quinona)/metabolismo , Naftoquinonas/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Proteínas Serina-Treonina Quinasas/metabolismo , ARN Interferente Pequeño/genética , Especies Reactivas de Oxígeno/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomycetales , Sales de Tetrazolio , Tiazoles , TranscriptomaRESUMEN
We report herein the design of a smart delivery system in which cargo delivery from capped mesoporous silica (MS) nanoparticles is controlled by an integrated enzyme-based "control unit". The system consists of Janus-type nanoparticles having opposing Au and MS faces, functionalized with a pH-responsive ß-cyclodextrin-based supramolecular nanovalve on the MS surface and two effectors, glucose oxidase and esterase, immobilized on the Au face. The nanodevice behaves as an enzymatic logical OR operator which is selectively fueled by the presence of D-glucose and ethyl butyrate.
Asunto(s)
Sistemas de Liberación de Medicamentos , Esterasas/metabolismo , Glucosa Oxidasa/metabolismo , Oro/metabolismo , Nanopartículas/química , Dióxido de Silicio/metabolismo , Enzimas Inmovilizadas/química , Enzimas Inmovilizadas/metabolismo , Esterasas/química , Glucosa Oxidasa/química , Oro/química , Concentración de Iones de Hidrógeno , Cinética , Modelos Moleculares , Estructura Molecular , Tamaño de la Partícula , Porosidad , Dióxido de Silicio/química , Propiedades de SuperficieRESUMEN
The classical role of the conserved Gcn2 kinase of yeast and mammals is to activate the translation of the transcription factors Gcn4 in yeast and activating transcription factor 4 in mammals by phosphorylating the eukaryotic translation initiation factor 2α. Gcn2 is activated by uncharged tRNAs in response to amino acid starvation and this regulatory system is important for tolerance to nutrient deprivation and other stresses and for development, differentiation, and normal function of mammalian organs. In the past few years, the classical Gcn2 pathway has been shown to modulate life span, tumor cell survival, and immune responses. In addition, Gcn2 modulates translation of novel mRNAs such as those of an unknown regulator of leucine transport and of sulfiredoxin SRX1 in yeast (activation of translation) and of inducible nitric oxide synthase, ErBb2, HIF1a, and 5'-terminal oligopyrimidine tract mRNAs in mammals (inhibition of translation). Finally, Gcn2 directly phosphorylates novel proteins such as methionyl-tRNA synthetase in mammals, and this triggers a pathway for DNA repair. These findings anticipate many expanding roles of Gcn2 in the future, with relevance for stress responses and human disease.
Asunto(s)
Biosíntesis de Proteínas , Proteínas Serina-Treonina Quinasas/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Animales , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/genética , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Transporte Biológico , Supervivencia Celular , Factor 2 Eucariótico de Iniciación/genética , Factor 2 Eucariótico de Iniciación/metabolismo , Regulación de la Expresión Génica , Humanos , Fosforilación , Proteínas Serina-Treonina Quinasas/metabolismo , Saccharomyces cerevisiae/enzimología , Proteínas de Saccharomyces cerevisiae/metabolismo , Transducción de Señal , Estrés Fisiológico , Factor de Transcripción 4 , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Intracellular pH conditions many cellular systems, but its mechanisms of regulation and perception are mostly unknown. We have identified two yeast genes important for tolerance to intracellular acidification caused by weak permeable acids. One corresponded to LEU2 and functions by removing the dependency of the leu2 mutant host strain on uptake of extracellular leucine. Leucine transport is inhibited by intracellular acidification, and either leucine oversupplementation or overexpression of the transporter gene BAP2 improved acid growth. Another acid-tolerance gene is GCN2, encoding a protein kinase activated by uncharged tRNAs during amino acid starvation. Gcn2 phosphorylates eIF2α (eukaryotic initiation factor 2α) (Sui2) at Ser51 and this inhibits general translation, but activates that of Gcn4, a transcription factor for amino acid biosynthetic genes. Intracellular acidification activates Gcn2 probably by inhibition of aminoacyl-tRNA synthetases because we observed accumulation of uncharged tRNAleu without leucine depletion. Gcn2 is required for leucine transport and a gcn2-null mutant is sensitive to acid stress if auxotrophic for leucine. Gcn4 is required for neither leucine transport nor acid tolerance, but a S51A sui2 mutant is acid-sensitive. This suggests that Gcn2, by phosphorylating eIF2α, may activate translation of an unknown regulator of amino acid transporters different from Gcn4.
Asunto(s)
Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/enzimología , Estrés Fisiológico/fisiología , Ácido Acético , Adaptación Fisiológica , Aminoácidos/metabolismo , Aminoacil-ARNt Sintetasas/genética , Aminoacil-ARNt Sintetasas/metabolismo , Regulación Fúngica de la Expresión Génica/fisiología , Homeostasis , Concentración de Iones de Hidrógeno , Leucina/metabolismo , Mutación , Plásmidos , Proteínas Serina-Treonina Quinasas/genética , ARN de Hongos/genética , ARN de Hongos/metabolismo , ARN de Transferencia/genética , ARN de Transferencia/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
The eIF2α kinases integrate translation initiation rates with nutrient availability, thus allowing cells to adapt to nutrient scarcity. Recent evidence has uncovered new functions of these kinases in tumour cell biology, ranging from regulation of cell cycle progression, maintenance of genome stability, control of apoptosis, and cell survival under nutrient stress and hypoxia. Accordingly, active eIF2α kinases modulate the antineoplasic activity of several antitumour drugs, either by exacerbating their cytotoxic effect or by promoting chemoresistance. Understanding of eIF2α kinases molecular roles may provide mechanistic insights into how tumour cells sense and adapt to nutrient restriction, thus helping to implement more effective approaches for cancer chemotherapy.
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Antineoplásicos/uso terapéutico , Factor 2 Eucariótico de Iniciación/antagonistas & inhibidores , Neoplasias/tratamiento farmacológico , Factor 2 Eucariótico de Iniciación/genética , Factor 2 Eucariótico de Iniciación/metabolismo , Humanos , Neoplasias/genética , Neoplasias/metabolismoRESUMEN
Beta-lapachone is an anticancer agent that selectively induces cell death in several human cancer cells. The mechanism of beta-lapachone cytotoxicity is not yet fully understood. Here we report that beta-lapachone treatment delayed cell cycle progression at the G(1)/S transition, incremented phosphorylation of the Rad53p checkpoint kinase and decreased cell survival in the budding yeast Saccharomyces cerevisiae. Furthermore, beta-lapachone induced phosphorylation of histone H2A at serine 129. These checkpoint responses were regulated by Mec1p and Tel1p kinases. Mec1p was required for Rad53p/histone H2A phosphorylation and cell survival following beta-lapachone treatment in asynchronous cultures, but not for the G(1) delay. The tel1Delta mutation increased sensitivity to beta-lapachone in a mec1 defective strain and compromised checkpoint responses in G(1). Both Rad53p phosphorylation and G(1) delay were fully dependent on a functional Mre11p-Rad50p Xrs2p (XMR) complex, and mutants in the XMR complex were hypersensitive to beta-lapachone treatment. Finally, XRS2 and TEL1 worked epistatically regarding beta-lapachone sensitivity and Xrs2p was phosphorylated in a Tel1p-dependent manner after beta-lapachone treatment. Taken together, these findings indicate that beta-lapachone activates a Mre11p-Tel1p checkpoint pathway in budding yeast. Given the conserved nature of the Mre11p-Tel1p pathway, these results suggest that activation of the Mre11-Tel1p checkpoint could be of significance for beta-lapachone anti-tumour activity.
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Antiinfecciosos/farmacología , Endodesoxirribonucleasas/fisiología , Exodesoxirribonucleasas/fisiología , Péptidos y Proteínas de Señalización Intracelular/fisiología , Naftoquinonas/farmacología , Proteínas Serina-Treonina Quinasas/fisiología , Proteínas de Saccharomyces cerevisiae/fisiología , Saccharomyces cerevisiae/genética , Ciclo Celular , Secuencia Conservada , Endodesoxirribonucleasas/metabolismo , Exodesoxirribonucleasas/metabolismo , Fase G1 , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Cinética , Mutación , Fosforilación , Plásmidos/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Fase S , Saccharomyces cerevisiae/fisiología , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomycetales , Proteína p53 Supresora de Tumor/fisiologíaRESUMEN
The discovery of genotoxic amino acids derived from phenylglycine, and possessing halogen substituents, is described. The utility of hypervalent iodine reagents in the synthesis of this class of compounds is highlighted. The mechanism of action of the (haloaryl)glycines was studied in Saccharomyces cerevisiae.
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Daño del ADN , Glicina/química , Glicina/toxicidad , Halógenos/química , Benzamidinas/química , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Glicina/análogos & derivados , Glicina/síntesis química , Humanos , Estructura Molecular , Nitrógeno/química , Oxígeno/química , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/genética , Estereoisomerismo , Relación Estructura-ActividadRESUMEN
The immunosuppressant Tacrolimus (FK506) has increased the survival rates of organ transplantation. FK506 exerts its immunosuppressive effect by inhibition of the protein phosphatase calcineurin in activated T-cells. Unfortunately, FK506 therapy is associated with undesired non-therapeutic effects involving targets other than calcineurin. To identify these targets we have addressed FK506 cellular toxicity in budding yeast. We show that FK506 increased cell sensitivity upon osmotic challenge independently of calcineurin and the FK506-binding proteins Fpr1p, -2p, -3p, and -4p. FK506 also induced strong amino acid starvation and activation of the general control (GCN) pathway. Tryptophan prototrophy or excess tryptophan overcame FK506 toxicity, showing that tryptophan deprivation mediated this effect. Mutation of the GCN3 and -4 genes partially alleviated FK506 toxicity, suggesting that activation of the GCN pathway by FK506 was also involved in osmotic tolerance. FK506 enhanced osmotic stress-dependent Hog1p kinase phosphorylation that was not accompanied by induction of a Hog1p-dependent reporter. Interestingly, deletion of the GCN2 gene suppressed FK506-dependent Hog1p hyperphosphorylation and restored Hog1p-dependent reporter activity. Conversely, deletion of the HOG1 gene impaired FK506-dependent activation of Gcn2p kinase and translation of a GCN4-LacZ reporter, highlighting functional cross-talk between the Gcn2p and Hog1p protein kinases. Taken together, these data demonstrate that both FK506-induced amino acid starvation and activation of the GCN pathway contribute to cell sensitivity to osmotic stress and reveal a positive regulatory loop between the Hog1p and Gcn2p pathways. Given the conserved nature of Gcn2p and Hog1p pathways, this mechanism of FK506 toxicity could be relevant to the non-therapeutic effects of FK506 therapy.