RESUMEN
Copper chelation is the most commonly used therapeutic strategy nowadays to treat Wilson's disease, a genetic disorder primarily inducing a pathological accumulation of Cu in the liver. The mechanism of action of Chel2, a liver-targeting Cu(i) chelator known to promote intracellular Cu chelation, was studied in hepatic cells that reconstitute polarized epithelia with functional bile canaliculi, reminiscent of the excretion pathway in the liver. The interplay between Chel2 and Cu localization in these cells was demonstrated through confocal microscopy using a fluorescent derivative and nano X-ray fluorescence. The Cu(i) bound chelator was found in vesicles potentially excreted in the canaliculi. Moreover, injection of Chel2 either intravenously or subcutaneously to a murine model of Wilson's disease increased excretion of Cu in the faeces, confirming in vivo biliary excretion. Therefore, Chel2 turns out to be a possible means to collect and excrete hepatic Cu in the faeces, hence restoring the physiological pathway.
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Cobre/metabolismo , Degeneración Hepatolenticular/metabolismo , Animales , Ceruloplasmina/metabolismo , Modelos Animales de Enfermedad , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Ratones , Microscopía Confocal , Espectrometría por Rayos XRESUMEN
An automatic method is established for layer-by-layer (LbL) assembly of biomimetic coatings in cell culture microplates using a commercial liquid-handling robot. Highly homogeneous thin films are formed at the bottom of each microwell. The LbL film-coated microplates are compatible with common cellular assays, using microplate readers and automated microscopes. Cellular adhesion is screened on crosslinked and peptide-functionalized LbL films and stem cell differentiation in response to increasing doses of bone morphogenetic proteins (2, 4, 7, 9). This method paves the way for future applications of LbL films in cell-based assays for regenerative medicine and high-throughput drug screening.
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Biomimética , Diferenciación CelularRESUMEN
Silver nanowires (AgNW) are attractive materials that are anticipated to be incorporated into numerous consumer products such as textiles, touchscreen display, and medical devices that could be in direct contact with skin. There are very few studies on the cellular toxicity of AgNW and no studies that have specifically evaluated the potential toxicity from dermal exposure. To address this question, we investigated the dermal toxicity after acute exposure of polymer-coated AgNW with two sizes using two models, human primary keratinocytes and human reconstructed epidermis. In keratinocytes, AgNW are rapidly and massively internalized inside cells leading to dose-dependent cytotoxicity that was not due to Ag⺠release. Analysing our data with different dose metrics, we propose that the number of NW is the most appropriate dose-metric for studies of AgNW toxicity. In reconstructed epidermis, the results of a standard in vitro skin irritation assay classified AgNW as non-irritant to skin and we found no evidence of penetration into the deeper layer of the epidermis. The findings show that healthy and intact epidermis provides an effective barrier for AgNW, although the study does not address potential transport through follicles or injured skin. The combined cell and tissue model approach used here is likely to provide an important methodology for assessing the risks for skin exposure to AgNW from consumer products.
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Insulin is known to form amyloid aggregates when agitated in a hydrophobic container. Amyloid aggregation is routinely measured by the fluorescence of the conformational dye thioflavin T, which, when incorporated into amyloid fibers, fluoresces at 480â¯nm. The kinetics of amyloid aggregation in general is characterized by an initial lag-phase, during which aggregative nuclei form on the hydrophobic surface. These nuclei then lead to the formation of fibrils presenting a rapid growth during the elongation phase. Here we describe a novel mechanism of insulin amyloid aggregation which is surprisingly devoid of a lag-time for nucleation. The excitation of thioflavin T by visible light at 440â¯nm induces the aggregation of thioflavin T-positive insulin fibrils on hydrophobic surfaces in the presence of strong agitation and at physiological pH. This process is material surface-induced and depends on the fact that surface-adsorbed insulin can bind thioflavin T. Light-induced insulin aggregation kinetics is thioflavin T-mediated and is based on an energy transfer from visible light to the protein via thioflavin T. It relies on a constant supply of thioflavin T and insulin from the solution to the aggregate. The growth rate increases with the irradiance and with the concentration of thioflavin T. The supply of insulin seems to be the limiting factor of aggregate growth. This light-induced aggregation process allows the formation of local surface-bound aggregation patterns.
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Insulina/química , Luz , Agregado de Proteínas/efectos de la radiación , Tiazoles/química , Benzotiazoles , Interacciones Hidrofóbicas e Hidrofílicas , Microscopía Confocal , Microscopía Electrónica de Rastreo , Poliestirenos/química , Unión Proteica , Propiedades de SuperficieRESUMEN
Surface coatings delivering BMP are a promising approach to render biomaterials osteoinductive. In contrast to soluble BMPs which can interact with their receptors at the dorsal side of the cell, BMPs presented as an insoluble cue physically bound to a biomimetic matrix, called here matrix-bound (bBMP-2), are presented to cells by their ventral side. To date, BMP-2 internalization and signaling studies in cell biology have always been performed by adding soluble (sBMP-2) to cells adhered on cell culture plates or glass slides, which will be considered here as a "reference" condition. However, whether and how matrix-bound BMP-2 can be internalized by cells and its relation to canonical (SMAD) and non-canonical signaling (ALP) remain open questions. In this study, we investigated the uptake and processing of BMP-2 by C2C12 myoblasts. This BMP-2 was presented either embedded in polyelectrolyte multilayer films (matrix-bound presentation) or as soluble form. Using fluorescently labeled BMP-2, we showed that the amount of matrix-bound BMP-2 internalized is dependent on the level of crosslinking of the polyelectrolyte films. Cav-1-mediated internalization is related to both SMAD and ALP signaling, while clathrin-mediated is only related to ALP signaling. BMP-2 internalization was independent of the presentation mode (sBMP-2 versus bBMP-2) for low crosslinked films (soft, EDC10) in striking contrast with high crosslinked (stiff, EDC70) films where internalization was much lower and slower for bBMP-2. As anticipated, internalization of sBMP-2 barely depended on the underlying matrix. Taken together, these results indicate that BMP-2 internalization can be tuned by the underlying matrix and activates downstream BMP-2 signaling, which is key for the effective formation of bone tissue. STATEMENT OF SIGNIFICANCE: The presentation of growth factors from material surfaces currently presents significant challenges in academic research, clinics and industry. Being able to deliver efficiently these growth factors by a biomaterial will open new perspectives for regenerative medicine. However, to date, very little is known about how matrix-bound growth factors are delivered to cells, especially whether they are internalized and how they are signaling to drive key differentiation events. These initial steps are crucial as they will guide the subsequent processes leading to tissue regeneration. In this work, we investigate the uptake and processing by cells of BMP-2 ligands embedded in polyelectrolyte multilayer films in comparison to soluble BMP-2. We show that BMP-2 responsive cells can internalize matrix-bound BMP-2 and that internalization is dependent on the cross-linking level of the polyelectrolyte films. In addition, we show that internalization is mediated by both clathrin- and caveolin-dependent pathways. While inhibiting clathrin-dependent endocytosis affects only non-canonical signaling, blocking caveolin-1-dependent endocytosis reduces both canonical and non-canonical BMP signaling. The signaling pathways found for matrix-bound BMP-2 are similar to those found for soluble BMP-2. These results highlight that BMP-2 presented by a biomaterial at the ventral side of the cell can trigger major endocytic and associated signaling pathways leading to bone regeneration.
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Proteína Morfogenética Ósea 2/metabolismo , Endocitosis , Matriz Extracelular/metabolismo , Transducción de Señal , Proteínas Smad/metabolismo , Fosfatasa Alcalina/metabolismo , Animales , Fenómenos Biomecánicos , Receptores de Proteínas Morfogenéticas Óseas/metabolismo , Caveolina 1/metabolismo , Línea Celular , Clatrina/metabolismo , Reactivos de Enlaces Cruzados/química , Dinaminas/metabolismo , Ácido Hialurónico/química , Concentración de Iones de Hidrógeno , Ratones , Mioblastos/metabolismo , Fosforilación , Polilisina/química , Unión Proteica , SolubilidadRESUMEN
UNLABELLED: The increasing demand for bone repair solutions calls for the development of efficacious bone scaffolds. Biphasic calcium phosphate (BCP) scaffolds with both macropores and micropores (MP) have improved healing compared to those with macropores and no micropores (NMP), but the role of micropores is unclear. Here, we evaluate capillarity induced by micropores as a mechanism that can affect bone growth in vivo. Three groups of cylindrical scaffolds were implanted in pig mandibles for three weeks: MP were implanted either dry (MP-Dry), or after submersion in phosphate buffered saline, which fills pores with fluid and therefore suppresses micropore-induced capillarity (MP-Wet); NMP were implanted dry. The amount and distribution of bone in the scaffolds were quantified using micro-computed tomography. MP-Dry had a more homogeneous bone distribution than MP-Wet, although the average bone volume fraction, BVFâ¾, was not significantly different for these two groups (0.45±0.03 and 0.37±0.03, respectively). There was no significant difference in the radial bone distribution of NMP and MP-Wet, but the BVFâ¾, of NMP was significantly lower among the three groups (0.25±0.02). These results suggest that micropore-induced capillarity enhances bone regeneration by improving the homogeneity of bone distribution in BCP scaffolds. The explicit design and use of capillarity in bone scaffolds may lead to more effective treatments of large and complex bone defects. STATEMENT OF SIGNIFICANCE: The increasing demand for bone repair calls for more efficacious bone scaffolds and calcium phosphate-based materials are considered suitable for this application. Macropores (>100µm) are necessary for bone ingrowth and vascularization. However, studies have shown that microporosity (<20µm) also enhances growth, but there is no consensus on the controlling mechanisms. In previous in vitro work, we suggested that micropore-induced capillarity had the potential to enhance bone growth in vivo. This work illustrates the positive effects of capillarity on bone regeneration in vivo; it demonstrates that micropore-induced capillarity significantly enhances the bone distribution in the scaffold. The results will impact the design of scaffolds to better exploit capillarity and improve treatments for large and load-bearing bone defects.
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Huesos/efectos de los fármacos , Fosfatos de Calcio/farmacología , Acción Capilar , Andamios del Tejido/química , Animales , Regeneración Ósea/efectos de los fármacos , Huesos/irrigación sanguínea , Huesos/diagnóstico por imagen , Tamaño de los Órganos , Porosidad , Sus scrofa , Microtomografía por Rayos XRESUMEN
Controlling the different steps of cell differentiation in vitro using bioactive surfaces may be useful in view of future cell therapies. Substrates presenting peptides, which are minimal fragments of extracellular matrix (ECM) proteins may be used for this purpose. In this work, we used polyelectrolyte multilayer films presenting two peptides derived from different muscle ECM proteins to target syndecan or/and integrin receptors. We showed that the presence of laminin-derived peptide to target syndecan-1 promotes lamellipodia formation, increases migration speed, directionality, and cell proliferation but impaired myotube formation. The cellular effects of L2synd are under the control of Rac1 and Cdc42 activities and involved ß1 integrin in contrast to RGD-containing peptide, which enabled adhesion via ß3 integrins and muscle cell differentiation. Our results show that peptides grafted onto multilayered films can guide the proliferation/differentiation balance and reveal crosstalk between different adhesion receptors.
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The fabrication of hyaluronic acid (HA) nanogels using a thiol-ene reaction has been demonstrated. HA was modified with pentenoate groups and then cross-linked with poly(ethylene glycol)-bis(thiol) by exposure to UV light. The cross-linking density and thereby the rigidity of the obtained gels were precisely controlled by the degree of substitution of pentenoate-modified HA. Their swelling properties also depended on cross-linking density. To produce hydrogels at the nanoscale, hyaluronic acid precursors were solely confined inside liposomes before cross-linking and purified after cross-linking. The size of the resulting nanogels followed their swelling properties and was also affected by their cross-linking density. Such bionanogels with tunable mechanical and swelling properties have potential in drug delivery.
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Sistemas de Liberación de Medicamentos , Ácido Hialurónico/química , Nanopartículas/químicaRESUMEN
Copper oxide nanoparticles (CuO-NP) were studied for their toxicity and mechanism of action on hepatocytes (HepG2), in relation to Cu homeostasis disruption. Indeed, hepatocytes, in the liver, are responsible for the whole body Cu balance and should be a major line of defence in the case of exposure to CuO-NP. We investigated the early responses to sub-toxic doses of CuO-NP and compared them to equivalent doses of Cu added as salt to see if there is a specific nano-effect related to Cu homeostasis in hepatocytes. The expression of the genes encoding the Cu-ATPase ATP7B, metallothionein 1X, heme oxygenase 1, heat shock protein 70, superoxide dismutase 1, glutamate cysteine ligase modifier subunit, metal responsive element-binding transcription factor 1 and zinc transporter 1 was analyzed by qRT-PCR. These genes are known to be involved in response to Cu, Zn and/or oxidative stresses. Except for MTF1, ATP7B and SOD1, we clearly observed an up regulation of these genes expression in CuO-NP treated cells, as compared to CuCl2. In addition, ATP7B trafficking from the Golgi network to the bile canaliculus membrane was observed in WIF-B9 cells, showing a need for Cu detoxification. This shows an increase in the intracellular Cu concentration, probably due to Cu release from endosomal CuO-NP solubilisation. Our data show that CuO-NP enter hepatic cells, most probably by endocytosis, bypassing the cellular defence mechanism against Cu, thus acting as a Trojan horse. Altogether, this study suggests that sub-toxic CuO-NP treatments induce successively a Cu overload, a Cu-Zn exchange on metallothioneins and MTF1 regulation on both Cu and Zn homeostasis.
Asunto(s)
Cobre/química , Regulación de la Expresión Génica/efectos de los fármacos , Hepatocitos/efectos de los fármacos , Homeostasis/efectos de los fármacos , Nanopartículas del Metal/química , Adenosina Trifosfatasas/metabolismo , Proteínas de Transporte de Catión/metabolismo , Supervivencia Celular , ATPasas Transportadoras de Cobre , Aparato de Golgi/efectos de los fármacos , Células Hep G2 , Humanos , Espectrometría de Masas , Nanopartículas del Metal/toxicidad , Microscopía Electrónica de Rastreo , Microscopía Fluorescente , Nanotecnología , Estrés Oxidativo , Reacción en Cadena de la Polimerasa , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
We present the preparation of nanogels made of hyaluronic acid (HA) with a well-controlled structure. To this end, HA precursors with polymerizable methacrylate groups (HA-MA) were confined within water-in-oil nanoemulsion droplets as nanoreactors and further photopolymerized under UV. Particular attention was paid to the preparation of a stable nanoemulsion template with a homogeneous droplet size. Upon UV irradiation of the emulsion containing HA-MA, crosslinked HA-MA particles with a well-defined size were obtained. Moreover, by varying the photopolymerization conditions, i.e. the number of received photons, we could control the conversion rate of the polymerization, as proved by 1H-NMR. Nanogels with controlled cross-linking densities were thus obtained. Not only could their crosslinking densities be controlled by the number of incident photons, but also by the degree of methacrylation (DM) of HA-MA derivatives. In addition, the swelling properties of the nanogels depended on external factors, showing their pH and ionic strength responsiveness. We show that these structures were highly biocompatible, stable under storage and enzymatically biodegradable, which opens the route for their application as drug delivery systems. Finally, insulin was loaded in the nanogels and its pH-dependent release was demonstrated. This versatile method of nanogel preparation, which can be applied to every type of hydrophilic precursor, offers a potential synthetic route to design other types of fully biocompatible drug delivery systems.
RESUMEN
Quercetin is a natural compound that has shown several biological activities. However, it displays poor water solubility and, therefore, low bioavailability. In this study, oil-in-water nanosized emulsions were obtained by the hot solvent diffusion method, using castor oil as oily phase and poly(ethylene glycol) (660)-12-hydroxystearate (PEG 660-stearate) and lecithin as surfactants. The effect of the PEG 660-stearate concentration on the droplet size of the nanosized emulsions and on the ability of these systems to load quercetin was investigated. Dynamic light scattering (DLS), transmission electron microscopy (TEM), cryo-TEM, and small-angle X-ray scattering (SAXS) were used to characterize the systems. We have demonstrated that a critical concentration of PEG 660-stearate (2.5 wt%) was needed to obtain colloidal dispersions displaying microemulsion characteristics. This colloidal dispersion, that was not optically birefringent, was constituted by a monodisperse population of 20 nm-large droplets, and exhibited excellent stability. Besides, this system was able to solubilize five times more quercetin than nanoemulsions prepared using 0.25 wt% PEG 660-stearate. SAXS results suggest that the spherical droplets have a core-shell structure. With regard to the hot solvent diffusion method, both diffusion of the solvent towards the aqueous phase and increase of the temperature above the phase inversion temperature (PIT) of PEG 660-stearate appeared to be required for obtaining clear and isotropic colloidal dispersions.
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Emulsiones/química , Nanopartículas/química , Polietilenglicoles/química , Quercetina/química , Estearatos/química , Tensoactivos/química , Aceite de Ricino , Lecitinas , Luz , Microscopía Electrónica de Transmisión , Tamaño de la Partícula , Quercetina/análisis , Dispersión de Radiación , Dispersión del Ángulo Pequeño , Solubilidad , Solventes , Difracción de Rayos XRESUMEN
This paper discusses the thermoresponsive nanoparticles obtained by self-assemblies of nonlinear oligosaccharide-based diblock copolymer systems. These diblock copolymers were synthesized by Cu(I)-catalyzed 1,3-dipolar azide/alkyne cycloaddition ("click" reaction) of propargyl-functionalized ß-cyclodextrin (ßCyD) and xyloglucooligosaccharide (XGO) with poly(N-isopropylacrylamide) (PNIPAM) having a terminal azido group prepared by atom transfer radical polymerization (ATRP). Elastic and quasi-elastic light scattering analysis of the dibock copolymers in H(2)O indicated that thermodynamic phase transitions of the PNIPAM blocks at their cloud points (T(cp)s ≈ 34 °C), around lower critical solution temperatures (LCSTs), triggered their self-assemblies into the nanoparticles. These nanoparticles had narrow size distributions and small interphases (i.e., sharp boundaries). The mean hydrodynamic radii (R(h)s) of the ßCyD and XGO-based nanoparticles were determined to be around 150 and 250 nm upon slow heating (i.e., step-by-step heating), and 364 and 91.5 nm upon fast heating, respectively, depending on a predominance of the interchain association or the intrachain contraction. Transmission electron microscope (TEM) and field emission gun-scanning electron microscopy (FEG-SEM) images of the nanoparticles clearly showed compact spherical nanoparticles whose cores are mainly made with the PNIPAM blocks, whereas the rough shells consist in the oligosaccharidic blocks.
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Acrilamidas/química , Nanopartículas/química , Oligosacáridos/síntesis química , Polímeros/química , Temperatura , Acrilamidas/síntesis química , Resinas Acrílicas , Catálisis , Cobre/química , Ciclización , Estructura Molecular , Oligosacáridos/química , Polímeros/síntesis químicaRESUMEN
Polycaprolactone (PCL) nanoparticles decorated with a mucoadhesive polysaccharide chitosan (CS) containing curcumin were developed aiming the buccal delivery of this drug. These nanoparticles were prepared by the nanoprecipitation method using different molar masses and concentrations of chitosan and concentrations of triblock surfactant poloxamer (PEO-PPO-PEO), in order to optimize the preparation conditions. Chitosan-coated nanoparticles showed positive surface charge and a mean particle radius ranging between 114 and 125 nm, confirming the decoration of the nanoparticles with the mucoadhesive polymer, through hydrogen bonds between ether and amino groups from PEO and CS, respectively. Dynamic Light Scattering (DLS) studies at different scattering angles and concentrations have shown that the nanoparticles are monodisperse (polydispersity indices were lower than 0.3). The nanoparticle systems were also examined with Nanoparticle Tracking Analysis (NTA), and the results were in good agreement with those obtained by DLS. Colloidal systems showed mean drug content about 460 µg/mL and encapsulation efficiency higher than 99%. Finally, when coated with chitosan, these nanoparticles show a great ability to interact with mucin indicating also their suitability for mucoadhesive applications.
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Quitosano/química , Curcumina/química , Portadores de Fármacos/química , Nanopartículas/química , Administración Bucal , Curcumina/administración & dosificación , Luz , Mucinas/química , Mucoproteínas/química , Tamaño de la Partícula , Polietilenglicoles/química , Glicoles de Propileno/química , Dispersión de Radiación , Propiedades de SuperficieRESUMEN
This work describes the synthesis and self-assembly of carbohydrate-clicked rod-coil amphiphilic systems. Copper-catalyzed Huisgen cycloaddition was efficiently employed to functionalize the hydrophilic extremity of PEG-b-tetra(p-phenylene) conjugates by lactose and N-acetyl-glucosamine ligands. The resulting amphiphilic systems spontaneously self-assembled into nanoparticles when dissolved in aqueous media, as evidenced by dynamic light scattering (DLS), transmission electron microscopy (TEM), and small-angle X-ray scattering (SAXS). The formation of highly monodisperse micelles having a mean diameter of 10 nm was observed for systems containing a PEG 900 core, and a decrease in the hydrophilic moiety (PEG 600) led to the formation of vesicles with a broader size distribution. The presence of carbohydrate residues on the surfaces of the micelles and their ability to establish specific interactions with wheat germ agglutinin (WGA) and peanut agglutinin (PNA) were further highlighted by light-scattering measurements, thus confirming the attractive applications of such sugar micelles in biosensor devices.
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Adhesivos , Glicoconjugados/química , Lectinas/química , Nanopartículas , Microscopía Electrónica de Rastreo , Microscopía Electrónica de Transmisión , Dispersión del Ángulo Pequeño , Difracción de Rayos XRESUMEN
This work discusses the self-assembly properties of thermoresponsive hybrid oligosaccharide-block-poly(N-isopropylacrylamide) copolymer systems: maltoheptaose-block-poly(N-isopropylacrylamide) (Mal(7)-b-PNIPAM(n)) copolymers. Those systems at different molar masses and volume fractions were synthesized using Cu(I)-catalyzed 1,3-dipolar azide/alkyne cycloaddition, so-called "click" chemistry, between an alkynyl-functionalized maltoheptaose (1) and poly(N-isopropylacrylamide) having a terminal azido group (N(3)-PNIPAM(n)) prepared by atom transfer radical polymerization (ATRP). While the cloud point (T(cp)) of the N(3)-PNIPAM(n) ranged from 36.4 to 51.5 degrees C depending on the degree of polymerization, those obtained of the diblock copolymers ranged from 39.4 to 73.9 degrees C. The self-assembly of such systems is favored due to the hydrophobicity of the PNIPAM in water above the T(cp). While the N(3)-PNIPAM(n) present polydisperse globular shape with a mean diameter of 500 nm, well-defined vesicular morphologies with an approximate diameter of 300 nm are obtained in diblock copolymer systems. These results were obtained and confirmed using static and dynamic light scattering as well as imaging techniques such as transmission electron microscope experiments.
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Acrilamidas/química , Glucanos/química , Oligosacáridos/química , Oligosacáridos/síntesis química , Polímeros/química , Temperatura , Resinas Acrílicas , Catálisis , Cobre/químicaRESUMEN
We describe here a short and efficient synthetic route for incorporating terpyridine based metal complexes at the 3'-extremity of both single and bis-oligonucleotide (bis-ODN) stretches to form novel metal complex-ODN conjugates. All single stranded mono and bis-ODN tethered metal complexes and the respective duplex ODNs were characterized by circular dichroism spectroscopy and UV-Vis melting experiments. Duplexes formed by these hybrid metal complex-DNA conjugates showed around 4-5 degrees C stabilization with respect to the unmodified duplexes. Moreover hybridization of two complementary bis-ODN tethered metal complexes at different ratios in solution gave rise to a self-assembled nanometric linear network, which was characterized by non-denaturing gel electrophoresis and TEM studies. Thus, our simple synthetic strategy would contribute to develop multimetallic 2D-DNA nanoarrays where we can place different metal complexes at regular intervals along the ODN stretches.
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ADN/química , Hierro/química , Nanotecnología , Análisis de Secuencia por Matrices de Oligonucleótidos , Compuestos Organometálicos/síntesis química , Rutenio/química , Dicroismo Circular , Electroforesis en Gel de Poliacrilamida , Ligandos , Microscopía Electrónica de Transmisión , Estructura Molecular , Oligonucleótidos/química , Compuestos Organometálicos/química , Tamaño de la Partícula , Estereoisomerismo , Propiedades de Superficie , Temperatura de TransiciónRESUMEN
This study describes the preparation and the characterization of poly[ N-(2-hydroxypropyl methacrylamide)] hydrogel with bulk-modified saccharidic portion of ganglioside GM 3 (Neu5Ac-alpha2,3-Gal-beta1,4-Glc). The 3'-sialyllactose is a bioactive epitope recognized by many cell surface receptors on viruses, bacteria, and human cells such as growth factor receptors. Acrylated 3'-sialyllactose was synthesized and incorporated into the macromolecular network of hydrogels by free radical cross-linking copolymerization. Fluorescence techniques coupled to confocal laser scanning microscopy was employed to characterize the binding and accessibility of the sialyl group in the polymer network by using a monoclonal antibody against GM 3 and the lectin wheat germ agglutinin. The morphology of the network was examined by scanning electron microscopy and confocal microscopy to image the gel morphology. The water content of sialyllactosyl-HPMA hydrogel compared to unmodified gel was characterized by swelling measurements and thermogravimetry. A preliminary implantation study in rat brain was performed to examine the biofunctionality of the sialyllactosyl hydrogel using an experimental model of Parkinson's disease.
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Gangliósido G(M3)/química , Hidrogeles/química , Implantes Experimentales , Oligosacáridos/química , Ácidos Polimetacrílicos/química , Animales , Anticuerpos Monoclonales/química , Axones/patología , Materiales Biocompatibles/síntesis química , Materiales Biocompatibles/química , Cuerpo Estriado/patología , Modelos Animales de Enfermedad , Dopamina/metabolismo , Fluorescencia , Hidrogeles/síntesis química , Inmunohistoquímica , Ensayo de Materiales , Microscopía Confocal , Microscopía Electrónica de Rastreo , Oligosacáridos/síntesis química , Oxidopamina , Tamaño de la Partícula , Lectinas de Plantas/química , Ácidos Polimetacrílicos/síntesis química , Ratas , Propiedades de Superficie , Temperatura , Factores de Tiempo , Aglutininas del Germen de Trigo/químicaRESUMEN
The objective of this work was to investigate the formation of hollow microcapsules composed of hyaluronic acid (HA) and poly(allylamine) (PAH) by layer-by-layer adsorption on CaCO 3 microparticles and subsequent core removal by addition of chelating agents for calcium ions. We found that the molecular weight of HA as well as the HA solution concentration used during deposition are crucial parameters influencing the multilayer structure. Whereas the effect of molecular weight of HA was mainly attributed to the porous structure of the template which allows penetration of polyelectrolytes when their size is below the maximum pore size of the template ( approximately 60 nm), that of the concentration of the HA solution was related to the intrinsic properties of the polysaccharide. Indeed, as shown by quartz crystal microbalance with dissipation monitoring as well as electron microscopy techniques, the latter leads to dense structures for concentrations from five to ten times the critical overlap concentration during adsorption. Such conditions were found to be favorable for the formation of hollow shells. Regarding conditions for core dissolution, we demonstrated the possibility to use either ethylenediaminetetraacetic acid (EDTA) or citric acid as chelating agents. However, in some cases, it was necessary to chemically cross-link the shell to maintain its integrity.