RESUMEN
Probing the dynamics and quantifying the activities of intracellular protein kinases that coordinate cell growth and division and constitute biomarkers and pharmacological targets in hyperproliferative and pathological disorders remain a challenging task. Here engineering and characterization of a nanobiosensor of the mitotic kinase CDK1, through multifunctionalization of carbon nanotubes with a CDK1-specific fluorescent peptide reporter, are described. This original reporter of CDK1 activity combines the sensitivity of a fluorescent biosensor with the unique physico-chemical and biological properties of nanotubes for multifunctionalization and efficient intracellular penetration. The functional versatility of this nanobiosensor enables implementation to quantify CDK1 activity in a sensitive and dose-dependent fashion in complex biological environments in vitro, to monitor endogenous kinase in living cells and directly within tumor xenografts in mice by fluorescence imaging, thanks to a ratiometric quantification strategy accounting for response relative to concentration in space and in time.
Asunto(s)
Proteína Quinasa CDC2 , Nanotubos de Carbono , Neoplasias Experimentales/enzimología , Animales , Proteína Quinasa CDC2/metabolismo , Línea Celular Tumoral , Humanos , Ratones , FosforilaciónRESUMEN
Crystals of Boc-γ(4)(R)Val-Val-OH undergo a reversible first-order single crystal to single crystal phase transition at Tc ≈ 205 K from the orthorhombic space group P22121 (Z' = 1) to the monoclinic space group P21 (Z' = 2) with a hysteresis of â¼2.1 K. The low-temperature monoclinic form is best described as a nonmerohedral twin with â¼50% contributions from its two components. The thermal behavior of the dipeptide crystals was characterized by differential scanning calorimetry experiments. Visual changes in birefringence of the sample during heating and cooling cycles on a hot-stage microscope with polarized light supported the phase transition. Variable-temperature unit cell check measurements from 300 to 100 K showed discontinuity in the volume and cell parameters near the transition temperature, supporting the first-order behavior. A detailed comparison of the room-temperature orthorhombic form with the low-temperature (100 K) monoclinic form revealed that the strong hydrogen-bonding motif is retained in both crystal systems, whereas the non-covalent interactions involving side chains of the dipeptide differ significantly, leading to a small change in molecular conformation in the monoclinic form as well as a small reorientation of the molecules along the ac plane. A rigid-body thermal motion analysis (translation, libration, screw; correlation of translation and libration) was performed to study the crystal entropy. The reversible nature of the phase transition is probably the result of an interplay between enthalpy and entropy: the low-temperature monoclinic form is enthalpically favored, whereas the room-temperature orthorhombic form is entropically favored.
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Dipéptidos/química , Modelos Químicos , Rastreo Diferencial de Calorimetría , Frío , Cristalografía por Rayos X , Entropía , Enlace de Hidrógeno , Conformación Molecular , Movimiento (Física) , Transición de Fase , Termodinámica , Temperatura de TransiciónRESUMEN
Peptide-based hydrogels are considered of special importance due to their biocompatibility and biodegradability. They have a wide range of applications in the biomedical field, such as drug delivery, tissue engineering, wound healing, cell culture media, and biosensing. Nevertheless, peptide-based hydrogels composed of natural α-amino acids are limited for in vivo applications because of the possible degradation by proteolytic enzymes. To circumvent this issue, the incorporation of extra methylene groups within the peptide sequence and the protection of the terminal amino group can increase the enzymatic stability. In this context, we investigated the self-assembly capacity of aromatic dipeptides (Boc-α-diphenylalanine and Boc-α-dityrosine) and their ß- and γ-homologues and developed stable hydrogels. Surprisingly, only the Boc-diphenylalanine analogues were able to self-assemble and form hydrogels. A model drug, l-ascorbic acid, and oxidized carbon nanotubes (CNTs) or graphene oxide were then incorporated into the hydrogels. Under near-infrared light irradiation, the photothermal effect of the carbon nanomaterials induced the destabilization of the gel structure, which caused the release of a high amount of drug, thus providing opportunities for photocontrolled on-demand drug release.
RESUMEN
Peptides constituted of backbone homologated α-amino acids combined with carbon materials offer interesting possibilities in the modulation of cellular functions. In this work, we have prepared diphenylalanine ß- and γ-peptides and conjugated them to carbon nanotubes (CNTs). These hybrids were able to self-assemble into fibrillar dendritic structures enabling the growth of primary hippocampal cells and the modulation of their neuronal functions. In particular, following the deposition of the different nanomaterials on glass substrates, we have evaluated their effects on circuit function and geometry. The geometrical restrictions due to CNT nucleated nodes allowed growth of neuronal networks with control over network geometry, and exploring its functional impact. In diverse applications from basic neuroscience, the presence of CNT nodes may be exploited in brain interfaces able to convey highly localized electrical stimuli.
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Nanotubos de Carbono , Red Nerviosa , Neuronas , Ingeniería de Tejidos/instrumentación , Ingeniería de Tejidos/métodos , Animales , Materiales Biocompatibles , Hipocampo , RatasRESUMEN
Molecular gels formed by the self-assembly of low-molecular-weight gelators have received increasing interest because of their potential applications in drug delivery. In particular, the ability of peptides and amino acids to spontaneously self-assemble into three-dimensional fibrous network has been exploited in the development of hydrogels. In this context, we have investigated the capacity of binary mixtures of aromatic amino acid derivatives to form hydrogels. Carbon nanomaterials, namely oxidized carbon nanotubes or graphene oxide, were incorporated in the two most stable hydrogels, formed by Fmoc-Tyr-OH/Fmoc-Tyr(Bzl)-OH and Fmoc-Phe-OH/Fmoc-Tyr(Bzl)-OH, respectively. The structural and physical properties of these gels were assessed using microscopic techniques and rheology. Circular dichroism and molecular dynamics simulations demonstrated that the hydrogel formation was mainly driven by aromatic interactions. Finally, a model hydrophilic drug (l-ascorbic acid) was loaded into the hybrid hydrogels at a high concentration. Under near-infrared light irradiation, a high amount of drug was released triggered by the heat generated by the carbon nanomaterials, thus offering interesting perspectives for controlled drug delivery.
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Sistemas de Liberación de Medicamentos , Hidrogeles/química , Nanotubos de Carbono/química , Péptidos/química , Aminoácidos/química , Dicroismo Circular , Liberación de Fármacos/efectos de la radiación , Grafito/química , Humanos , Hidrogeles/efectos de la radiación , Concentración de Iones de Hidrógeno , Rayos Infrarrojos , Simulación de Dinámica Molecular , Nanoestructuras/química , ReologíaRESUMEN
Carbon nanotubes (CNTs) are a unique tool in nanotechnology owing to their exceptional properties that offer a variety of opportunities for applications in different fields. Nevertheless, their low dispersibility in organic solvents and in aqueous media hampers their development. The functionalization of their surface allows overcoming this issue, while exploiting and tuning their properties. Thanks to their high specific surface area, multi-functionalization strategies give the possibility to conjugate several copies of different molecules to endow the nanotubes with multiple functionalities. In this context, this review wishes to focus on the preparation of multimodal CNTs designed by covalent multi-functionalization. More specifically, we describe the different approaches that have been developed to prepare multi-functionalized CNTs through double and triple covalent functionalization of the nanotube framework. We also emphasize the strategies used to control the derivatization of multi-functionalized CNTs with molecules of interest mainly via sequential or simultaneous methodologies.
RESUMEN
The integration of carbon nanotubes (CNTs) into organized nanostructures is of great interest for applications in materials science and biomedicine. In this work we studied the self-assembly of ß and γ homologues of diphenylalanine peptides under different solvent and pH conditions. We aimed to investigate the role of peptide backbone in tuning the formation of different types of nanostructures alone or in combination with carbon nanotubes. In spite of having the same side chain, ß and γ peptides formed distinctively different nanofibers, a clear indication of the role played by the backbone homologation on the self-assembly. The variation of the pH allowed to transform the nanofibers into spherical structures. Moreover, the co-assembly of ß and γ peptides with carbon nanotubes covalently functionalized with the same peptide generated unique dendritic assemblies. This comparative study on self-assembly using diphenylalanine backbone homologues and of the co-assembly with CNT covalent conjugates is the first example exploring the capacity of ß and γ peptides to adopt precise nanostructures, particularly in combination with carbon nanotubes. The dendritic organization obtained by mixing carbon nanotubes and peptides might find interesting applications in tissue engineering and neuronal interfacing.
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Materiales Biocompatibles/química , Materiales Biocompatibles/síntesis química , Nanotubos de Carbono/química , Nanotubos de Carbono/ultraestructura , Fenilalanina/análogos & derivados , Dipéptidos , Nanotecnología/métodos , Fenilalanina/químicaRESUMEN
Unconstrained γ(4) amino acid residues derived by homologation of proteinogenic amino acids facilitate helical folding in hybrid (αγ)n sequences. The C12 helical conformation for the decapeptide, Boc-[Leu-γ(4)(R)Val]5-OMe, is established in crystals by X-ray diffraction. A regular C12 helix is demonstrated by NMR studies of the 18 residue peptide, Boc-[Leu-γ(4)(R)Val]9-OMe, and a designed 16 residue (αγ)n peptide, incorporating variable side chains. Unconstrained (αγ)n peptides show an unexpectedly high propensity for helical folding in long polypeptide sequences.
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Aminoácidos/química , Péptidos/química , Cristalografía por Rayos X , Modelos Moleculares , Conformación Molecular , Estructura Molecular , Estructura Secundaria de ProteínaRESUMEN
Monosubstituted γ(4)-residues (γ(4)Leu, γ(4)Ile, and γ(4)Val) form helices even in short homooligomeric sequences. C14 helix formation is established by X-ray diffraction in homooligomeric (γ)n tetra-, hexa- and decapeptide sequences demonstrating the high propensity of γ residues, with proteinogenic side chains, to adopt locally folded conformations.