RESUMO
Engineered peptides capable of binding to silica have been used to provide contrast in chemical force microscopy and tested for their capacity to selectively capture silica nanoparticles (NPs). Gold coated atomic force microscopy (AFM) microcantilevers with integrated tips and colloidal probes were functionalized with engineered peptides through a thiol group of a terminal cysteine which was linked via a glycine trimer to a 12-mer binding sequence. The functionalized probes demonstrated a significantly increased binding force on silicon oxide areas of a gold-patterned silicon wafer, whereas plain gold probes, and those functionalized with a random permutation of the silica binding peptide motif or an all-histidine sequence displayed similar adhesion forces to gold and silicon oxide. As the functionalized probes also allowed contact mode imaging subsequently to the adhesion mapping, also the associated friction contrast was measured and found to be similar to the adhesion contrast. Furthermore, the adsorption of silica NPs onto planar gold surfaces functionalized in the same manner was observed to be selective. Notably, the surface coverage with silica NPs was found to decrease with increasing pH, implying the importance of electrostatic interactions between the peptide and the NPs. Finally, the adsorption of silica NPs was monitored via the decrease in fundamental resonance frequency of an AFM microcantilever functionalized with silica binding peptides.
Assuntos
Microscopia de Força Atômica/métodos , Nanopartículas/metabolismo , Proteínas Recombinantes/metabolismo , Ouro/metabolismo , Nanopartículas/química , Óxidos/metabolismo , Ligação Proteica , Compostos de Silício/metabolismoRESUMO
Direct polymerization of an oxaliplatin analogue was used to reproducibly generate amphiphiles in one pot, which consistently and spontaneously self-assemble into well-defined nanoparticles (NPs). Despite inefficient drug leakage in cell-free assays, the NPs were observed to be as cytotoxic as free oxaliplatin in cell culture experiments. We investigated this phenomenon by super-resolution fluorescence structured illumination microscopy (SIM) and nanoscale secondary ion mass spectrometry (NanoSIMS). In combination, these techniques revealed NPs are taken up via endocytic pathways before intracellular release of their cytotoxic cargo. As with other drug-carrying nanomaterials, these systems have potential as cellular delivery vehicles. However, high-resolution methods to track nanocarriers and their cargo at the micro- and nanoscale have been underutilized in general, limiting our understanding of their interactions with cells and tissues. We contend this type of combined optical and isotopic imaging strategy represents a powerful and potentially generalizable methodology for cellular tracking of nanocarriers and their cargo.
Assuntos
Antineoplásicos/química , Complexos de Coordenação/química , Portadores de Fármacos/química , Nanopartículas/química , Imagem Óptica/métodos , Compostos Organoplatínicos/química , Piridinas/química , Células A549 , Antineoplásicos/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Complexos de Coordenação/farmacologia , Liberação Controlada de Fármacos , Endocitose , Fluorescência , Células HeLa , Humanos , Compostos Organoplatínicos/farmacologia , Tamanho da Partícula , Polímeros/química , Piridinas/farmacologia , Propriedades de SuperfícieRESUMO
The encapsulation efficiency of high-Tg polynorbornene micelles was probed with a hydrophobic dye 2,6-diiodoboron-dipyrromethene (BODIPY). Changes in the visible absorption spectra of aggregated versus monomeric dye molecules provided a probe for assessing encapsulation. Polynorbornene micelles are found to be capable of loading up to one BODIPY dye per ten polymers. As the hydrophilic block size increased in the polymeric amphiphiles, more of the dye was incorporated within the micelles. This result is consistent with the dye associating with the polymer backbone in the shell of the micelles. The encapsulation rate varied significantly with temperature, and a slight dependence on micellar morphology was also noted. Additionally, we report a 740 µs triplet lifetime for the encapsulated BODIPY dye. The lifetime is the longest ever recorded for a BODIPY triplet excited state at room temperature and is attributed to hindered triplet-triplet annihilation in the high-viscosity micellar shell.
RESUMO
Adherent cells respond to a wide range of substrate cues, including chemistry, topography, hydrophobicity, and surface energy. The cell-substrate interface is therefore an important design parameter in regenerative medicine and tissue engineering applications, where substrate cues are used to influence cell behavior. Thin films comprising 4.5 nm (average diameter) gold nanoparticles coated with a mixture of two alkanethiols can confer hemispherical topography and specific chemistry to bulk substrates. The behavior of murine embryonic stem cells (ESCs) on the thin films can then be compared with their behavior on self-assembled monolayers of the same alkanethiols on vapor-deposited gold, which lack the topographical features. Cells cultured both with and without differentiation inhibitors are characterized by immunofluorescence for Oct4 and qPCR for Fgf5, Foxa2, Nanog, Pou5f1, and Sox2. Nanoscale chemistry and topography are found to influence stem cell differentiation, particularly the early differentiation markers, Fgf5 and Foxa2. Nanoscale topography also affects Oct4 localization, whereas the chemical composition of the substrate does not have an effect. It is demonstrated for the first time that ESCs can sense topographical features established by 4.5 nm particles, and these findings suggest that nanoscale chemistry and topography can act synergistically to influence stem cell differentiation. This study furthers the understanding of the effects of these substrate properties, improving our ability to design materials to control stem cell fate.
Assuntos
Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/efeitos dos fármacos , Nanopartículas Metálicas/química , Animais , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Marcadores Genéticos/genética , Ouro/química , Ouro/farmacologia , Fator 3-beta Nuclear de Hepatócito/genética , Fator 3-beta Nuclear de Hepatócito/metabolismo , Camundongos , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Reação em Cadeia da PolimeraseRESUMO
The accurate characterization of submicrometer and nanometer sized particles presents a major challenge in the diverse applications envisaged for them including cosmetics, biosensors, renewable energy, and electronics. Size is one of the principal parameters for classifying particles and understanding their behavior, with other particle characteristics usually only quantifiable when size is accounted for. We present a comparative study of emerging and established techniques to size submicrometer particles, evaluating their sizing precision and relative resolution, and demonstrating the variety of physical principles upon which they are based, with the aim of developing a framework in which they can be compared. We used in-house synthesized Stöber silica particles between 100 and 400 nm in diameter as reference materials for this study. The emerging techniques of scanning ion occlusion sensing (SIOS), differential centrifugal sedimentation (DCS), and nanoparticle tracking analysis (NTA) were compared to the established techniques of transmission electron microscopy (TEM), scanning mobility particle sizing (SMPS), and dynamic light scattering (DLS). The size distributions were described using the mode, arithmetic mean, and standard deviation. Uncertainties associated with the six techniques were evaluated, including the statistical uncertainties in the mean sizes measured by the single-particle counting techniques. Q-Q plots were used to analyze the shapes of the size distributions. Through the use of complementary techniques for particle sizing, a more complete characterization of the particles was achieved, with additional information on their density and porosity attained.