RESUMEN
Fluorophobic-driven assemblies of gold nanomaterials were stabilized into water-dispersible fluorous supraparticles by the film-forming protein hydrophobinâ II. The strategy makes use of fluorous nanomaterials of different dimensions to engineer size and inner functionalization of the resulting confined space. The inner fluorous compartments allow efficient encapsulation and transport of high loadings of partially fluorinated drug molecules in water.
Asunto(s)
Benzoxazinas/química , Celecoxib/química , Flúor/química , Leflunamida/química , Alquinos , Ciclopropanos , Oro/química , Halogenación , Sustancias Macromoleculares/química , Nanopartículas del Metal/química , Tamaño de la Partícula , Agua/químicaRESUMEN
Extracellular Vesicles (EVs) - cell secreted vesicles that carry rich molecular information of the parental cell and constitute an important mode of intercellular communication - are becoming a primary topic in translational medicine. EVs (that comprise exosomes and microvesicles/microparticles) have a size ranging from 40 nm to 1 µm and share several physicochemical proprieties, including size, density, surface charge, and light interaction, with other nano-objects present in body fluids, such as single and aggregated proteins. This makes separation, titration, and characterization of EVs challenging and time-consuming. Here we present a cost-effective and fast colorimetric assay for probing by eye protein contaminants and determine the concentration of EV preparations, which exploits the synergy between colloidal gold nanoplasmonics, nanoparticle-protein corona, and nanoparticle-membrane interaction. The assay hits a limit of detection of protein contaminants of 5 ng/µL and has a dynamic range of EV concentration ranging from 35 fM to 35 pM, which matches the typical range of EV concentration in body fluids. This work provides the first example of the exploitation of the nanoparticle-protein corona in analytical chemistry.
Asunto(s)
Colorimetría , Exosomas/química , Oro/química , Nanopartículas del Metal/química , Nanotecnología , Proteínas/análisis , Colorimetría/economía , Ojo/química , Humanos , Nanotecnología/economíaRESUMEN
Nanoparticles (NP), when exposed to biological fluids, are coated by specific proteins that form the so-called protein corona. While some adsorbing proteins exchange with the surroundings on a short time scale, described as a "dynamic" corona, others with higher affinity and long-lived interaction with the NP surface form a "hard" corona (HC), which is believed to mediate NP interaction with cellular machineries. In-depth NP protein corona characterization is therefore a necessary step in understanding the relationship between surface layer structure and biological outcomes. In the present work, we evaluate the protein composition and stability over time and we systematically challenge the formed complexes with surfactants. Each challenge is characterized through different physicochemical measurements (dynamic light scattering, ζ-potential, and differential centrifugal sedimentation) alongside proteomic evaluation in titration type experiments (surfactant titration). 100 nm silicon oxide (Si) and 100 nm carboxylated polystyrene (PS-COOH) NPs cloaked by human plasma HC were titrated with 3-[(3-Cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS, zwitterionic), Triton X-100 (nonionic), sodium dodecyl sulfate (SDS, anionic), and dodecyltrimethylammonium bromide (DTAB, cationic) surfactants. Composition and density of HC together with size and ζ-potential of NP-HC complexes were tracked at each step after surfactant titration. Results on Si NP-HC complexes showed that SDS removes most of the HC, while DTAB induces NP agglomeration. Analogous results were obtained for PS NP-HC complexes. Interestingly, CHAPS and Triton X-100, thanks to similar surface binding preferences, enable selective extraction of apolipoprotein AI (ApoAI) from Si NP hard coronas, leaving unaltered the dispersion physicochemical properties. These findings indicate that surfactant titration can enable the study of NP-HC stability through surfactant variation and also selective separation of certain proteins from the HC. This approach thus has an immediate analytical value as well as potential applications in HC engineering.
Asunto(s)
Nanopartículas/química , Proteínas/química , Tensoactivos/química , Electroforesis en Gel Bidimensional , HumanosRESUMEN
Unbalanced levels of caveolin-3 (Cav3) are involved in muscular disorders. In the present study we show that differentiation of immortalized myoblasts is affected by either lack or overexpression of Cav3. Nevertheless, depletion of Cav3 induced by delivery of the dominant-negative Cav3 (P104L) form elicited a more severe phenotype, characterized by the simultaneous attenuation of the Akt and p38 signalling networks, leading to an immature cell and molecular signature. Accordingly, differentiation of myoblasts harbouring Cav3 (P104L) was improved by countering the reduced Akt and p38 signalling network via administration of IGF-1 or trichostatin A. Furthermore, loss of Cav3 correlated with a deregulation of the TGF-ß-induced Smad2 and Erk1/2 pathways, confirming that Cav3 controls TGF-ß signalling at the plasma membrane. Overall, these data suggest that loss of Cav3, primarily causing attenuation of both Akt and p38 pathways, contributes to impair myoblast fusion.
Asunto(s)
Caveolina 3/genética , Mioblastos/citología , Mutación Puntual , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Animales , Caveolina 3/metabolismo , Diferenciación Celular/efectos de los fármacos , Fusión Celular , Línea Celular , Regulación de la Expresión Génica , Ácidos Hidroxámicos/farmacología , Factor I del Crecimiento Similar a la Insulina/farmacología , Ratones , Mioblastos/efectos de los fármacos , Mioblastos/metabolismo , Inhibidores de la Síntesis de la Proteína/farmacología , Ratas , Transducción de Señal , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
Vascular endothelial growth factor receptor-2 (VEGFR2) is an endothelial cell receptor that plays a pivotal role in physiologic and pathologic angiogenesis and is a therapeutic target for angiogenesis-dependent diseases, including cancer. By leveraging on a dedicated nanomechanical biosensor, we investigated the nanoscale mechanical phenomena intertwined with VEGFR2 surface recognition by its prototypic ligand VEGF-A and its noncanonical ligand gremlin. We found that the two ligands bind the immobilized extracellular domain of VEGFR2 (sVEGFR2) with comparable binding affinity. Nevertheless, they interact with sVEGFR2 with different binding kinetics and drive different in-plane piconewton intermolecular forces, suggesting that the binding of VEGF-A or gremlin induces different conformational changes in sVEGFR2. These behaviors can be effectively described in terms of a different "nanomechanical affinity" of the two ligands for sVEGFR2, about 16-fold higher for VEGF-A with respect to gremlin. Such nanomechanical differences affect the biological activity driven by the two angiogenic factors in endothelial cells, as evidenced by a more rapid VEGFR2 clustering and a more potent mitogenic response triggered by VEGF-A in respect to gremlin. Together, these data point to surface intermolecular interactions on cell membrane between activated receptors as a key modulator of the intracellular signaling cascade.
Asunto(s)
Fenómenos Mecánicos , Nanotecnología/métodos , Neovascularización Fisiológica , Resonancia por Plasmón de Superficie/métodos , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo , Citocinas , Transferencia Resonante de Energía de Fluorescencia , Humanos , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Ligandos , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Here, we demonstrate that introduction of halogen atoms at the tyrosine 10â phenol ring of the DSGYEV sequence derived from the flexible amyloid-ß N-terminus, promotes its self-assembly in the solid state. In particular, we report the crystal structures of two halogen-modified sequences, which we found to be stabilized in the solid state by halogen-mediated interactions. The structural study is corroborated by Non-Covalent Interaction (NCI) analysis. Our results prove that selective halogenation of an amino acid enhances the supramolecular organization of otherwise unstructured biologically-relevant sequences. This method may develop as a general strategy for stabilizing highly polymorphic peptide regions.
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Péptidos beta-Amiloides/química , Bromo/química , Tirosina/química , Secuencia de Aminoácidos , Aminoácidos/química , Cristalización , Halogenación , Enlace de Hidrógeno , Conformación Molecular , Oxidación-ReducciónRESUMEN
Polyplexes (PX) are soft materials, obtained by blending polycations and nucleic acids, designed for gene delivery applications. While much is known about the transfection properties of PX, their protein corona, the biomolecules interacting with colloids once in a biological environment, represents an underlooked parameter in gene transfection. In this study, linear and branched polyethylenimines (lPEI and bPEI), the golden standard among non-viral vectors, were selected and used throughout the work: their physicochemical properties and protein corona when complexed to DNA were studied and linked to the toxicity and transfection efficiency arisen upon their delivery to cells. Interestingly, lPEIDNA and bPEIDNA complexes were characterized by similar physicochemical features, but different biological behavior. In fact, the biological milieu where cells and PX interact greatly influences their size, stability and transfection abilities. Using PX as a soft material model system, we spotlighted structure-activity relationships and methodologies that can help interpret their biological behavior and guide future studies in the field.
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ADN/química , Técnicas de Transferencia de Gen , Polietileneimina/química , Corona de Proteínas/química , Supervivencia Celular/genética , ADN/genética , Electroforesis en Gel de Poliacrilamida , Células HeLa , Humanos , Corona de Proteínas/metabolismo , Electricidad EstáticaRESUMEN
One of the main hurdles in nanomedicine is the low stability of drug-nanocarrier complexes as well as the drug delivery efficiency in the region-of-interest. Here, we describe the use of the film-forming protein hydrophobin HFBII to organize dodecanethiol-protected gold nanoparticles (NPs) into well-defined supraparticles (SPs). The obtained SPs are exceptionally stable in vivo and efficiently encapsulate hydrophobic drug molecules. The HFBII film prevents massive release of the encapsulated drug, which, instead, is activated by selective SP disassembly triggered intracellularly by glutathione reduction of the protein film. As a consequence, the therapeutic efficiency of an encapsulated anticancer drug is highly enhanced (2 orders of magnitude decrease in IC50). Biodistribution and pharmacokinetics studies demonstrate the high stability of the loaded SPs in the bloodstream and the selective release of the payloads once taken up in the tissues. Overall, our results provide a rationale for the development of bioreducible and multifunctional nanomedicines.
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Antineoplásicos Fitogénicos/administración & dosificación , Proteínas Fúngicas/química , Oro/química , Hypocrea/química , Nanopartículas del Metal/química , Paclitaxel/administración & dosificación , Animales , Antineoplásicos Fitogénicos/farmacocinética , Línea Celular Tumoral , Liberación de Fármacos , Femenino , Humanos , Ratones Endogámicos BALB C , Paclitaxel/farmacocinéticaRESUMEN
Nanomedicine aims to find novel solutions for urgent biomedical needs. Despite this, one of the most challenging hurdles that nanomedicine faces is to successfully target therapeutic nanoparticles to cells of interest in vivo. As for any biomaterials, once in vivo, nanoparticles can interact with plasma biomolecules, forming new entities for which the name protein coronas (PCs) have been coined. The PC can influence the in vivo biological fate of a nanoparticle. Thus for guaranteeing the desired function of an engineered nanomaterial in vivo, it is crucial to dissect its PC in terms of formation and evolution within the body. In this contribution we will review the 'good' and 'bad' sides of the PC, starting from the scientific aspects to the technological applications.
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Vías de Administración de Medicamentos , Nanomedicina , Proteínas/químicaRESUMEN
The fibroblast growth factor (FGF)/FGF receptor (FGFR) system plays a crucial role in cancer by affecting tumor growth, angiogenesis, drug resistance, and escape from anti-angiogenic anti-vascular endothelial growth factor therapy. The soluble pattern recognition receptor long-pentraxin 3 (PTX3) acts as a multi-FGF antagonist. Here we demonstrate that human PTX3 overexpression in transgenic mice driven by the Tie2 promoter inhibits tumor growth, angiogenesis, and metastasis in heterotopic, orthotopic, and autochthonous FGF-dependent tumor models. Using pharmacophore modeling of the interaction of a minimal PTX3-derived FGF-binding pentapeptide with FGF2, we identified a small-molecule chemical (NSC12) that acts as an extracellular FGF trap with significant implications in cancer therapy.
Asunto(s)
Proteína C-Reactiva/genética , Factores de Crecimiento de Fibroblastos/genética , Regulación Neoplásica de la Expresión Génica , Neoplasias/genética , Componente Amiloide P Sérico/genética , Animales , Western Blotting , Proteína C-Reactiva/metabolismo , Ciclo Celular/efectos de los fármacos , Ciclo Celular/genética , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Células Cultivadas , Femenino , Factores de Crecimiento de Fibroblastos/metabolismo , Factores de Crecimiento de Fibroblastos/farmacología , Humanos , Estimación de Kaplan-Meier , Masculino , Ratones Endogámicos C57BL , Ratones Desnudos , Ratones Transgénicos , Estructura Molecular , Neoplasias/metabolismo , Neoplasias/terapia , Neovascularización Patológica/genética , Neovascularización Patológica/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Componente Amiloide P Sérico/metabolismo , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacología , Carga Tumoral/efectos de los fármacos , Carga Tumoral/genética , Ensayos Antitumor por Modelo de Xenoinjerto/métodosRESUMEN
Freestanding lipid bilayers were challenged with 15 nm Au nanospheres either coated by a citrate layer or passivated by a protein corona. The effect of Au nanospheres on the bilayer morphology, permeability and fluidity presents strong differences or similarities, depending on the observation length scale, from the colloidal to the molecular domains. These findings suggest that the interaction between nanoparticles and lipid membranes should be conveniently treated as a multiscale phenomenon.
Asunto(s)
Oro/química , Membrana Dobles de Lípidos/química , Nanopartículas del Metal/química , Nanopartículas del Metal/ultraestructura , Modelos Químicos , Difusión , Ensayo de Materiales , Modelos MolecularesRESUMEN
Self-assembled monolayers of Thrombin Binding Aptamers (TBA) were prepared on gold surfaces with typical surface densities of close-packed ssDNA (4×10(12) and 8×10(12)molecules/cm(2)). CONtact Angle MOlecular REcognition (CONAMORE) in captive bubble geometry was then assessed to scan the surface work triggered by TBAs when switching from the elongated to the G-quadruplex conformation upon binding with Na(+) or K(+) cations. We found Na(+) and K(+) could induce comparable linear to G-quadruplex strokes, and resulted in values for surface work of ~-70 pN nm/molecule (~18 kBT). The strokes change the in-plane van der Waals and weak electrostatic interactions and accumulate to result in macroscopic surface work. Micro- to macroscopic translation strongly depends on the nature of the cation and TBA surface density. In particular, the K(+) stimulus triggers a macroscopic surface work of -2.2±0.2 and -5.3±0.2 mN/m for low and high packed monolayers, respectively, while Na(+) triggers up to -6.7±1.0 mN/m in the highly packed monolayer, but creates negligible work for the low packed monolayer. These results show that CONAMORE can contribute important information for the development of devices based on DNA switches, and ultimately help address some of the open challenges for DNA-based nanomachinery.
Asunto(s)
Aptámeros de Nucleótidos/química , ADN de Cadena Simple/química , Oro/química , Trombina , Humanos , Nanotecnología/métodos , Potasio/química , Sodio/química , Propiedades de SuperficieRESUMEN
Biological surface science is receiving great and renewed attention owing the rising interest in applications of nanoscience and nanotechnology to biological systems, with horizons that range from nanomedicine and biomimetic photosynthesis to the unexpected effects of nanomaterials on health and environment. Biomolecule surface transformations are among the fundamental aspects of the field that remain elusive so far and urgently need to be understood to further the field. Our recent findings indicate that surface thermodynamics can give a substantial contribution toward this challenging goal. In the first part of the article, we show that biomolecule surface transformations can be framed by a general and simple thermodynamic model. Then, we explore its effectiveness by addressing some typical cases, including ligand-receptor surface binding, protein thin film machines, nanomechanical aspects of the biomolecule-nanoparticle interface and nanomechanical biosensors.
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Factores Biológicos/química , Nanopartículas/química , Receptores de Superficie Celular/química , Termodinámica , Fenómenos Biomecánicos , Técnicas Biosensibles , Humanos , Ligandos , Modelos Químicos , Nanotecnología , Unión Proteica , Soluciones , Propiedades de SuperficieRESUMEN
Any molecular recognition reaction supported by a solid phase drives a specific change of the solid-solution interfacial tension. Sessile contact angle (CA) experiments can be readily used to track this thermodynamic parameter, prompting this well-known technique to be reinvented as an alternative, easy-access and label-free way to probe and study molecular recognition events. Here we deploy this technique, renamed for this application CONAMORE (CONtact Angle MOlecular REcognition), to study the interaction of the tumor-derived pro-angiogenic vascular endothelial growth factor-A (VEGF-A) with the extracellular domain of its receptor VEGFR2. We show that CONAMORE recognizes the high affinity binding of VEGF-A at nanomolar concentrations to surface-immobilized VEGFR2 regardless of the presence of a ten-fold excess of a non-specific interacting protein, and that it further proofs its specificity and reliability on competitive binding experiments involving neutralizing anti-VEGF-A antibodies. Finally, CONAMORE shows the outstanding capability to detect the specific interaction between VEGFR2 and low molecular weight ligands, such as Cyclo-VEGI, a VEGFR2 antagonist cyclo-peptide, that weighs about 2 kDa.