RESUMEN
Pancreatic cancer is one of the deadliest cancers worldwide, mainly due to late diagnosis. Therefore, there is an urgent need for novel diagnostic approaches to identify the disease as early as possible. We have developed a diagnostic assay for pancreatic cancer based on the detection of naturally occurring tumor associated autoantibodies against Mucin-1 (MUC1) using engineered glycopeptides on nanoparticle probes. We used a structure-guided approach to develop unnatural glycopeptides as model antigens for tumor-associated MUC1. We designed a collection of 13 glycopeptides to bind either SM3 or 5E5, two monoclonal antibodies with distinct epitopes known to recognize tumor associated MUC1. Glycopeptide binding to SM3 or 5E5 was confirmed by surface plasmon resonance and rationalized by molecular dynamics simulations. These model antigens were conjugated to gold nanoparticles and used in a dot-blot assay to detect autoantibodies in serum samples from pancreatic cancer patients and healthy volunteers. Nanoparticle probes with glycopeptides displaying the SM3 epitope did not have diagnostic potential. Instead, nanoparticle probes displaying glycopeptides with high affinity for 5E5 could discriminate between cancer patients and healthy controls. Remarkably, the best-discriminating probes show significantly better true and false positive rates than the current clinical biomarkers CA19-9 and carcinoembryonic antigen (CEA).
RESUMEN
Mucin-1 (MUC1) glycopeptides are exceptional candidates for potential cancer vaccines. However, their autoantigenic nature often results in a weak immune response. To overcome this drawback, we carefully engineered synthetic antigens with precise chemical modifications. To be effective and stimulate an anti-MUC1 response, artificial antigens must mimic the conformational dynamics of natural antigens in solution and have an equivalent or higher binding affinity to anti-MUC1 antibodies than their natural counterparts. As a proof of concept, we have developed a glycopeptide that contains noncanonical amino acid (2S,3R)-3-hydroxynorvaline. The unnatural antigen fulfills these two properties and effectively mimics the threonine-derived antigen. On the one hand, conformational analysis in water shows that this surrogate explores a landscape similar to that of the natural variant. On the other hand, the presence of an additional methylene group in the side chain of this analog compared to the threonine residue enhances a CH/π interaction in the antigen/antibody complex. Despite an enthalpy-entropy balance, this synthetic glycopeptide has a binding affinity slightly higher than that of its natural counterpart. When conjugated with gold nanoparticles, the vaccine candidate stimulates the formation of specific anti-MUC1 IgG antibodies in mice and shows efficacy comparable to that of the natural derivative. The antibodies also exhibit cross-reactivity to selectively target, for example, human breast cancer cells. This investigation relied on numerous analytical (e.g., NMR spectroscopy and X-ray crystallography) and biophysical techniques and molecular dynamics simulations to characterize the antigen-antibody interactions. This workflow streamlines the synthetic process, saves time, and reduces the need for extensive, animal-intensive immunization procedures. These advances underscore the promise of structure-based rational design in the advance of cancer vaccine development.
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The molecular basis of antibody 5E5, which recognizes the entire GalNAc unit as a primary epitope is disclosed. The antibody's contacts with the peptide are mostly limited to two residues, allowing it to show some degree of promiscuity. These findings open the door to the chemical design of peptide-mimetics for developing efficient anti-cancer vaccines and diagnostic tools.
Asunto(s)
Anticuerpos Monoclonales/química , Antineoplásicos/química , Vacunas contra el Cáncer/química , Lectinas/química , Mucina-1/química , Anticuerpos Monoclonales/farmacología , Antineoplásicos/farmacología , Vacunas contra el Cáncer/farmacología , Ensayos de Selección de Medicamentos Antitumorales , Glicopéptidos/química , Glicosilación , Humanos , Enlace de Hidrógeno , Lectinas/farmacología , Simulación de Dinámica Molecular , Fragmentos de Péptidos/química , Conformación Proteica , Relación Estructura-ActividadRESUMEN
N-methyl-d-aspartate receptors (NMDARs) are ionotropic glutamate receptors controlling fundamental physiological processes in the central nervous system, such as learning and memory. Excessive activation of NMDARs causes excitotoxicity and results in neurodegeneration, which is observed in a number of pathological conditions. Because of their dichotomous role, therapeutic targeting of NMDAR is difficult. However, several lines of evidence suggest that excitotoxicity is predominantly linked to extrasynaptically located NMDARs. Here, we report on a nanoparticle-based strategy to inhibit extrasynaptic NMDARs exclusively and subtype selectively, while allowing synaptic NMDARs activity. We designed gold nanoparticles (AuNPs) carrying conopeptide derivatives conjugated on their poly(ethylene glycol) coating as allosteric NMDAR inhibitors and show that these nanoparticles antagonize exclusively extrasynaptic NMDAR-mediated currents in cultured hippocampal neurons. Additionally, we show that conopeptide-functionalized AuNPs are neuroprotective in an in vitro model of excitotoxicity. By using AuNPs carrying different allosteric inhibitors with distinct NMDAR subtype selectivity such as peptide conantokin-G or peptide conantokin-R, we suggest activation of extrasynaptic GluN2B-containing diheteromeric NMDARs as the main cause of excitotoxicity.
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Nanopartículas del Metal , Receptores de N-Metil-D-Aspartato , Oro , Hipocampo , Neuronas/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Sinapsis/metabolismoRESUMEN
Gold nanomaterials hold great potential for biomedical applications. While this field is evolving rapidly, little attention has been paid to precise nanoparticle design and functionalization. Here, we show that when using proteins as targeting moieties, it is fundamental to immobilize them directionally to preserve their biological activity. Using full-length leptin as a case study, we have developed two alternative conjugation strategies for protein immobilization based on either a site-selective or a nonselective derivatization approach. We show that only nanoparticles with leptin immobilized site-selectively fully retain the ability to interact with the cognate leptin receptor. These results demonstrate the importance of a specified molecular design when preparing nanoparticles labeled with proteins.
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Oro/química , Proteínas Inmovilizadas/química , Leptina/química , Nanopartículas del Metal/química , Humanos , Leptina/metabolismo , Células MCF-7 , Receptores de Leptina/metabolismoRESUMEN
GalNAc-glycopeptides derived from mucin MUC1 are an important class of tumor-associated antigens. α- O-glycosylation forces the peptide to adopt an extended conformation in solution, which is far from the structure observed in complexes with a model anti-MUC1 antibody. Herein, we propose a new strategy for designing potent antigen mimics based on modulating peptide/carbohydrate interactions by means of O â S/Se replacement at the glycosidic linkage. These minimal chemical modifications bring about two key structural changes to the glycopeptide. They increase the carbohydrate-peptide distance and change the orientation and dynamics of the glycosidic linkage. As a result, the peptide acquires a preorganized and optimal structure suited for antibody binding. Accordingly, these new glycopeptides display improved binding toward a representative anti-MUC1 antibody relative to the native antigens. To prove the potential of these glycopeptides as tumor-associated MUC1 antigen mimics, the derivative bearing the S-glycosidic linkage was conjugated to gold nanoparticles and tested as an immunogenic formulation in mice without any adjuvant, which resulted in a significant humoral immune response. Importantly, the mice antisera recognize cancer cells in biopsies of breast cancer patients with high selectivity. This finding demonstrates that the antibodies elicited against the mimetic antigen indeed recognize the naturally occurring antigen in its physiological context. Clinically, the exploitation of tumor-associated antigen mimics may contribute to the development of cancer vaccines and to the improvement of cancer diagnosis based on anti-MUC1 antibodies. The methodology presented here is of general interest for applications because it may be extended to modulate the affinity of biologically relevant glycopeptides toward their receptors.
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Anticuerpos Monoclonales/inmunología , Antígenos de Neoplasias/inmunología , Neoplasias de la Mama/microbiología , Carbohidratos/inmunología , Glicopéptidos/inmunología , Oxígeno/inmunología , Animales , Anticuerpos Monoclonales/química , Neoplasias de la Mama/patología , Neoplasias de la Mama/terapia , Carbohidratos/química , Diseño de Fármacos , Femenino , Glicopéptidos/química , Glicósidos/química , Glicósidos/inmunología , Glicosilación , Humanos , Neoplasias Mamarias Experimentales/inmunología , Neoplasias Mamarias Experimentales/patología , Neoplasias Mamarias Experimentales/terapia , Ratones , Ratones Endogámicos BALB C , Estructura Molecular , Oxígeno/química , Selenio/química , Selenio/inmunología , Azufre/química , Azufre/inmunologíaRESUMEN
Effective and selective targeting of the αV ß3 integrin subtype is of high relevance in cancer research for the development of therapeutic systems with improved efficacy and of diagnostic imaging probes. We report here a new class of highly selective, αV ß3 -targeted gold nanoparticles (AuNPs), which carry cyclic 4-aminoproline-RGD semipeptides (cAmpRGD) as the targeting moiety immobilized at low surface density on the poly(ethylene glycol) (PEG)-based nanoparticle coating. We show that these nanoparticles are potent inhibitors of the integrin-mediated melanoma tumor cell adhesion to vitronectin and are selectively internalized via receptor-mediated endocytosis. Furthermore, we have developed bifunctional cAmpRGD-functionalized AuNPs by conjugation of a fluorophore (FAM or TAMRA) to a separate set of reactive groups on the PEG-based coating. These bifunctional AuNPs not only recapitulate the binding properties of cAmpRGD-AuNPs but also can be visualized via confocal laser microscopy, allowing direct observation of nanoparticle internalization. The peculiar molecular design of these nanoparticles and their precisely defined architecture at the molecular level accounts for their selective integrin binding with very low nonspecific background.
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miRNAs are key regulators of gene expression, and alterations in their expression levels correlate with the onset and progression of cancer. Although miRNAs have been proposed as biomarkers for cancer diagnosis, their application in routine clinical praxis is yet to come. Current quantification strategies have limitation, and there is a great interest in developing innovative ones. Since a few years, nanotechnology-based approaches for miRNA quantification are emerging at fast pace but there is urgent need to go beyond the proof-of-concept stage. Nanotechnology will have a strong impact on cancer diagnosis through miRNA detection only if it is demonstrated that the newly developed approaches are indeed working on 'real-world' samples under standardized conditions.
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Biomarcadores de Tumor/análisis , MicroARNs/análisis , Nanotecnología/métodos , Neoplasias/diagnóstico , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Humanos , MicroARNs/metabolismo , Nanopartículas/química , Neoplasias/patología , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
Printed polymer electronics has held for long the promise of revolutionizing technology by delivering distributed, flexible, lightweight and cost-effective applications for wearables, healthcare, diagnostic, automation and portable devices. While impressive progresses have been registered in terms of organic semiconductors mobility, field-effect transistors (FETs), the basic building block of any circuit, are still showing limited speed of operation, thus limiting their real applicability. So far, attempts with organic FETs to achieve the tens of MHz regime, a threshold for many applications comprising the driving of high resolution displays, have relied on the adoption of sophisticated lithographic techniques and/or complex architectures, undermining the whole concept. In this work we demonstrate polymer FETs which can operate up to 20 MHz and are fabricated by means only of scalable printing techniques and direct-writing methods with a completely mask-less procedure. This is achieved by combining a fs-laser process for the sintering of high resolution metal electrodes, thus easily achieving micron-scale channels with reduced parasitism down to 0.19 pF mm-1, and a large area coating technique of a high mobility polymer semiconductor, according to a simple and scalable process flow.
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We report the preparation of gold nanoparticle (AuNP)-based vaccine candidates against the tumor-associated form of the mucin-1 (MUC1) glycoprotein. Chimeric peptides, consisting of a glycopeptide sequence derived from MUC1 and the T-cell epitope P30 sequence were immobilized on PEGylated AuNPs and the ability to induce selective antibodies in vivo was investigated. After immunization, mice showed significant MHC-II mediated immune responses and their antisera recognized human MCF-7 breast cancer cells. Nanoparticles designed according to this report may become key players in the development of anticancer vaccines.
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Vacunas contra el Cáncer/inmunología , Epítopos de Linfocito T/inmunología , Glicopéptidos/inmunología , Oro/química , Nanopartículas del Metal/química , Mucina-1/inmunología , Secuencia de Aminoácidos , Animales , Vacunas contra el Cáncer/química , Epítopos de Linfocito T/química , Genes MHC Clase II , Glicopéptidos/química , Humanos , Inmunización , Células MCF-7 , Ratones , Datos de Secuencia Molecular , Mucina-1/química , Neoplasias/inmunología , Neoplasias/prevención & controlRESUMEN
MicroRNAs (miRNAs) are important regulators of gene expression, and many pathological conditions, including cancer, are characterized by altered miRNA expression levels. Therefore, accurate and sensitive quantification of miRNAs may result in correct disease diagnosis establishing these small noncoding RNA transcripts as valuable biomarkers. Aiming at overcoming some limitations of conventional quantification strategies, nanotechnology is currently providing numerous significant alternatives to miRNA sensing. In this review an up-to-date account of nanotechnology-based strategies for miRNA detection and quantification is given. The topics covered are: nanoparticle-based approaches in solution, sensing based on nanostructured surfaces, combined nanoparticle/surface sensing approaches, and single-molecule approaches.
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MicroARNs/análisis , Nanotecnología/métodos , Animales , Humanos , MicroARNs/genética , Análisis de Matrices Tisulares/métodosRESUMEN
DNA-gold nanoparticle probes are implemented in a simple strategy for direct microRNA (miRNA) quantification. Fluorescently labeled DNA-probe strands are immobilized on PEGylated gold nanoparticles (AuNPs). In the presence of target miRNA, DNA-RNA heteroduplexes are formed and become substrate for the endonuclease DSN (duplex-specific nuclease). Enzymatic hydrolysis of the DNA strands yields a fluorescence signal due to diffusion of the fluorophores away from the gold surface. We show that the molecular design of our DNA-AuNP probes, with the DNA strands immobilized on top of the PEG-based passivation layer, results in nearly unaltered enzymatic activity toward immobilized heteroduplexes compared to substrates free in solution. The assay, developed in a real-time format, allows absolute quantification of as little as 0.2 fmol of miR-203. We also show the application of the assay for direct quantification of cancer-related miR-203 and miR-21 in samples of extracted total RNA from cell cultures. The possibility of direct and absolute quantification may significantly advance the use of microRNAs as biomarkers in the clinical praxis.
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Técnicas de Química Analítica/métodos , ADN/química , Oro/química , Nanopartículas del Metal/química , MicroARNs/análisis , Bioensayo , ADN/metabolismo , Fluorescencia , Límite de Detección , MicroARNs/química , Sondas Moleculares/químicaRESUMEN
Point mutations in the Kirsten rat sarcoma viral oncogene homologue (KRAS) gene are being increasingly recognized as important diagnostic and prognostic markers in cancer. In this work, we describe a rapid and low-cost method for the naked-eye detection of cancer-related point mutations in KRAS based on gold nanoparticles. This simple colorimetric assay is sensitive (limit of detection in the low picomolar range), instrument-free, and employs nonstringent room temperature conditions due to a combination of DNA-conjugated gold nanoparticles, a probe design which exploits cooperative hybridization for increased binding affinity, and signal enhancement on the surface of magnetic beads. Additionally, the scheme is suitable for point-of-care applications, as it combines naked-eye detection, small sample volumes, and isothermal (PCR-free) amplification.
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Colorimetría/métodos , Oro/química , Nanopartículas del Metal/química , Neoplasias/genética , Mutación Puntual , Proteínas Proto-Oncogénicas/genética , Proteínas ras/genética , Secuencia de Bases , Línea Celular Tumoral , Colorimetría/economía , Análisis Costo-Beneficio , ADN Helicasas/metabolismo , Humanos , Modelos Moleculares , Conformación de Ácido Nucleico , Hibridación de Ácido Nucleico , Sondas de Oligonucleótidos/química , Sondas de Oligonucleótidos/genética , Tamaño de la Partícula , Proteínas Proto-Oncogénicas p21(ras)RESUMEN
We show that water soluble InP/ZnS core/shell QDs are a safer alternative to CdSe/ZnS QDs for biological applications, by comparing their toxicity in vitro (cell culture) and in vivo (animal model Drosophila). By choosing QDs with comparable physical and chemical properties, we find that cellular uptake and localization are practically identical for these two nanomaterials. Toxicity of CdSe/ZnS QDs appears to be related to the release of poisonous Cd(2+) ions and indeed we show that there is leaching of Cd(2+) ions from the particle core despite the two-layer ZnS shell. Since an almost identical amount of In(III) ions is observed to leach from the core of InP/ZnS QDs, their very low toxicity as revealed in this study hints at a much lower intrinsic toxicity of indium compared to cadmium.
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Compuestos de Cadmio/toxicidad , Drosophila/efectos de los fármacos , Indio/toxicidad , Fosfinas/toxicidad , Compuestos de Selenio/toxicidad , Compuestos de Zinc/toxicidad , Animales , Ensayo de Materiales , Puntos Cuánticos , Tasa de SupervivenciaRESUMEN
In the bone marrow, hematopoietic stem cells (HSCs) reside in endosteal and vascular niches. The interactions with the niches are essential for the maintenance of HSC number and properties. Although the molecular nature of these interactions is well understood, little is known about the role of physical parameters such as matrix elasticity. Osteoblasts, the major cellular component of the endosteal HSC niche, flatten during HSC mobilization. We show that this process is accompanied by osteoblast stiffening, demonstrating that not only biochemical signals but also mechanical properties of the niche are modulated. HSCs react to stiffer substrates with increased cell adhesion and migration, which could facilitate the exit of HSCs from the niche. These results indicate that matrix elasticity is an important factor in regulating the retention of HSCs in the endosteal niche and should be considered in attempts to propagate HSCs in vitro for clinical applications.
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Células de la Médula Ósea/citología , Adhesión Celular/fisiología , Movimiento Celular/fisiología , Células Madre Hematopoyéticas/citología , Células de la Médula Ósea/metabolismo , Comunicación Celular/fisiología , Diferenciación Celular/fisiología , Línea Celular , Línea Celular Tumoral , Elasticidad , Células Madre Hematopoyéticas/metabolismo , HumanosRESUMEN
We report the preparation of gold nanoparticles (AuNPs) functionalized with the peptide-toxin conantokin-G and their selective binding to N-methyl-d-aspartate (NMDA) receptors recombinantly expressed by transfected HEK 293 cells. The AuNPs are passivated with a mixed self-assembled monolayer of ω-carboxy- and ω-amino-polyethylene glycol (PEG) thiols. We compare two different passivation systems: the alkyl-PEG600 system is characterized by a C(11)-alkyl chain between the thiol group and the PEG segment, whereas the PEG3000 system lacks this alkyl-chain. We show that only the alkyl-PEG600 passivation system allows selective conjugation of cysteine-terminated peptides to the periphery of the passivation layer via a heterobifunctional linker strategy. In contrast, using the PEG3000 passivation system, peptides are immobilized both on the passivation layer and directly on the gold surface via concurrent place-exchange reaction. We therefore recommend the use of the alkyl-PEG600 system to precisely control the number of immobilized peptides on AuNPs. In fact, we show that the number of conjugated peptides per particle can be varied with good control simply by varying the composition of the self-assembled monolayer. Finally, we demonstrate that conjugation of the conantokin-G peptide to the solvent-exposed interface of the passivation layer results in maximal binding interaction between the peptide-functionalized AuNPs and the targeted NMDA receptors on the cell surface. Conantokin G-coupled AuNP may be used to spatially restrict NMDA-receptor-blockade on neuronal surfaces.
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Oro/química , Nanopartículas del Metal/química , Péptidos/química , Péptidos/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Secuencia de Aminoácidos , Conotoxinas/metabolismo , Células HEK293 , Humanos , Proteínas Inmovilizadas/química , Proteínas Inmovilizadas/metabolismo , Datos de Secuencia Molecular , Polietilenglicoles/química , Unión Proteica , Compuestos de Sulfhidrilo/químicaRESUMEN
Despite tremendous progress in recent years, nanopatterning of hydrated polymeric systems such as hydrogels still represents a major challenge. Here, we employ block copolymer nanolithography to arrange gold nanoparticles on a solid template, followed by the transfer of the pattern to a polymeric hydrogel. In the next step, these nanoparticles serve as specific anchor points for active biomolecules. We demonstrate the engineering of poly(ethylene glycol) hydrogel surfaces with respect to elasticity, nanopatterning, and functionalization with biomolecules. For the first time, biomolecule arrangement on the nanometer scale and substrate stiffness can be varied independently from each other. Young's moduli, a measure of the compliance of the substrates, can be tuned over 4 orders of magnitude, including the values for all of the different tissues found in the human body. Structured hydrogels can be used to pattern any histidine-tagged protein as exemplified for his-protein A as an acceptor for immunoglobulin. When cell-adhesion-promoting peptide cRGDfK is selectively coupled to gold nanoparticles, the surfaces provide cues for cell-surface interaction and allow for the study of the modulation of cellular adhesion by the mechanical properties of the environment. Therefore, these substrates represent a unique multipurpose platform for studying receptor/ligand interactions with adhering cells, mechanotransduction, and cell-adhesion-dependent signaling.
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Polímeros/química , Adhesión Celular , Células Cultivadas , Elasticidad , Humanos , Hidrogeles/química , Microscopía Confocal , Microscopía Fluorescente , Tamaño de la PartículaRESUMEN
DNA hybrid catalysis goes organometallic: A DNA strand functionalized with diene ligands forms iridium(I) complexes that can efficiently catalyze an allylic amination in aqueous medium (see scheme). The DNA-based complexes show high stability and activity, and their secondary structure influences the stereoselectivity of the reaction.
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ADN/química , Iridio/química , Polienos/química , Aminación , Catálisis , Cinética , Oligonucleótidos/química , EstereoisomerismoRESUMEN
We present a fluorescence-based assay for the characterization of functionalized gold nanoparticles (AuNPs) capped with a self-assembled monolayer of mixed thiols derived from poly(ethylene glycol) (PEG). These water-soluble AuNPs carry primary amino groups at the solvent-exposed interface, which can be used for further conjugation of biologically active molecules. The reported assay allows quantification of the average number of functionalizable amino groups per particle (N(NH(2))) with a relative uncertainty below or equal to +/-14% (95% confidence interval), thus providing essential information for the successive derivatization of the AuNPs. Here, a fluorescently labeled derivative of peptide-neurotoxin conantokin-G was coupled to the amino groups of the particle ligand shell via a flexible linker. We quantitatively determined the average number of peptides per particle (N(pept)) and the yield of the two-step conjugation strategy. AuNPs carrying 50-70 copies of the peptide were obtained. In addition, we have gained insights into the deterioration of the self-assembled monolayer due to thiol desorption with time. Under ordinary storage conditions in solution and at room temperature, a decrease in N(NH(2)) between 48% and 75% could be observed at the end of the period of investigation (42-56 days). Slow desorption of the conjugated peptides upon storage was also observed and quantified ( approximately 25% in 14 days).