Research reported in this publication was supported by The Johns Hopkins Applied Physics Laboratory&#8217;s Research and Exploratory Development Department, Office of Technology Transfer, and Stuart S. Janney Fellowship Program, in addition to the National Heart, Lung, and Blood Institute of the National Institutes of Health under Award Number R21HL127355.

1. Tramitación de GLC <ol><li> Síntesis de GLC Nota: Utilice el método PIT 1,17,20 para preparar los sub-20 GLC nm 1. <ol><li> Utilice una técnica aséptica, Bioreagents o reactivos de grado de cultivo de células, y los materiales estériles (es decir, pipetas, espátulas, viales, etc.). </li><li> Utilice un gabinete de bioseguridad para la síntesis de GLC (Figura 2). </li><li> Combinar 0,6 mg de tinte fluorescente (Rojo Nilo (NIR), la concentración NiR es de aproximadamente 0,3 mg / ml) y 0,10 g de Heneicosane (lípido, 5% p / p concentración de lípidos) en un vial (15 ml), co-funde a 90 ° C, y revuelva. </li><li> Añadir 0,11 g de polioxietileno (10) oleil éter (tensioactivo, 5,5% p / p concentración de tensioactivo). Co-fundir la mezcla resultante a 90 ° C y remover. </li><li> Añadir 1,79 g de agua estéril a la mezcla, el calor a 90 ° C, y revuelva hasta que se forme una nanoemulsión transparente. Nota: Bajo continua agitación y enfriamiento controlado, SLSe crean Ns. Estas condiciones causan una inversión de la emulsión de agua en aceite a una emulsión aceite-en-agua. Moléculas lipofílicas como partición NiR a las gotas de lípidos. </li><li> Preparar una nanoemulsión en paralelo sin añadir el colorante fluorescente, con el fin de servir como una muestra de control. Combine 0,10 g de Heneicosane y 0,11 g de polioxietileno (10) oleiléter en un vial (15 ml) co-fusión a 90 ° C, y revuelva. <ol><li> Añadir 1,79 g de agua estéril a la mezcla, el calor a 90 ° C, y revuelva hasta que se forme una nanoemulsión transparente. </li></ol></li><li> Además esterilizar cada nanoemulsión usando un filtro estéril de 0,2 micras. </li></ol><img alt="Figura 2" src="/files/ftp_upload/53102/53102fig2.jpg" /> Figura 2. Esquema de la síntesis de GLC. <a href="https://www.jove.com/files/ftp_upload/53102/53102fig2large.jpg" target="_blank">Haga clic aquí para ver una versión más grande de esta figura.</a> </li><li> Tamaño de partícula y Polydispersity <ol><li> Utilice dispersión dinámica de luz (DLS) para medir el tamaño de las partículas y la polidispersidad de los GLC 1 utilizando una cubeta de vidrio y un instrumento diseñado para medir el tamaño de las partículas. </li><li> Antes de las mediciones de las muestras, medir los tamaños de partícula de dos estándares conocidos siguiendo el protocolo del fabricante para la preparación estándares y medición. </li><li> Llenar la cubeta de vidrio con 1,5 ml de solución estándar y medir la solución estándar con las mismas condiciones de medición como las muestras. Nota: Paso anterior se repitió para cada norma. </li><li> Medir las muestras como se ha preparado. Para cada uno, utilice los siguientes parámetros experimentales: una duración de 100 segundos, el índice de refracción del agua (1.33), la viscosidad del agua a 20 ° C (1.002 mPa · s). Nota: Esta técnica utiliza desplazada en frecuencia de luz para medir el tamaño de las nanopartículas. </li><li> Reporte el diámetro medio de partícula y la polidispersidad de partidistribución de tamaño de culo. </li></ol></li><li> Punto de fusión y calor latente de fusión <ol><li> Utilice un calorímetro diferencial de barrido (DSC) equipado con un muestreador automático y la fuente de enfriamiento nitrógeno líquido para investigar el comportamiento térmico de SLNs 1. </li><li> Pipetear los SLNs como preparadas en un molde de aluminio 40 l con una masa de aproximadamente 25 mg y sellar herméticamente el recipiente usando una prensa rizador universal a minimizar la pérdida de humedad durante el análisis DSC. </li><li> Mida cada nanoemulsión de 5 a 80 ° C. </li><li> Informar el punto de fusión y el calor latente de fusión del gráfico de DSC, donde el valle representa el punto de fusión y la integral de la área bajo la curva en el gráfico de DSC dividido por la cantidad de material representa el calor latente de fusión. </li></ol></li></ol> 2. GLC Interacción con fibroblastos y las células dendríticas <ol><li> Cultura de Primaria fibroblastos humanos <ol><li> Cultura fi humana primariafibroblastos de acuerdo a las instrucciones del fabricante en medio líquido para el cultivo de fibroblastos dérmicos humanos (medio 106), complementado con el kit suplemento de bajo crecimiento en suero (LSGS). Mantener las células en una atmósfera húmeda a 37 ° C, 5% de CO 2 y la subcultura al llegar a 80% de confluencia. </li><li> Por tanto MTT y análisis de imágenes, las células de semillas en una placa de 96 pocillos, a una densidad de 2 x 10 4 células por pocillo, y permitir que se equilibre a 37 ° CO / N antes de la exposición SLN. </li><li> En el tiempo cero, diluir GC en medio completo como se indica (Figura 4), ​​con respecto a la concentración de lípidos, y añadir 10 l por pocillo a cada replicar bien en la placa de 96 pocillos. </li><li> Mantener las células a 37 ° C, 5% de CO 2, durante el período de incubación. </li></ol></li><li> MTT Ensayo de toxicidad <ol><li> Después de 24 h de incubación con GLC, determinar la viabilidad celular utilizando el ensayo MTT, según el protocolo del fabricante. &lt;/ li&gt; <li> Lavar las células una vez y añadir medio fresco a cada pocillo (100 l / pocillo). Matar a los pocillos de control por incubación en una solución de metanol 70% durante 10 min antes de reemplazar con medio fresco. </li><li> Añadir reactivo MTT (10 l por pocillo) a cada pocillo de la microplaca y se incuba O / N a 37 ° C. </li><li> Después de O / N incubación, disolver los cristales de formazina intracelulares con la solución de detergente proporcionado (100 l por pocillo) de acuerdo con el protocolo del fabricante. </li><li> Después de 3 h de incubación a RT, obtener valores de absorbancia a una longitud de onda de medición de 570 nm utilizando un lector de microplacas. Reactivo MTT utilizado debe ser de menos de 1% (w / v) de 3- (4,5-dimetiltiazol-2) -2, bromuro de 5-difenil-tetrazolio: Nota. </li></ol></li><li> Microscopía de Fluorescencia <ol><li> En los puntos de tiempo específicos después de la dosificación de los fibroblastos con SLN, lavar los fibroblastos dos veces con solución salina estéril tamponada con fosfato (PBS) y fijar durante 10 minutos con 70% de metanol frío. </li><li> Después de 10 min, Sustituir el metanol con PBS y capturar imágenes utilizando un microscopio invertido, usando un paso de banda (BP) 690/50 nm conjunto de filtros. </li></ol></li><li> Cultivo de células de médula ósea de ratón dendríticas <ol><li> Inducir a las células derivadas de la médula ósea de ratón dendríticas (BMDC) anteriormente descritos 23,24. Brevemente, las suspensiones de placa de una sola célula de ratones C57BL / 10 de la médula ósea en 100 mm placas de Petri en 2 x 10 6 / placa con factor estimulante de colonias de granulocitos y macrófagos murino recombinante (GM-CSF, 300 U / ml) y murina recombinante IL-4 (células B factor estimulante, 200 U / ml). Cambie los medios de comunicación cada 3 días. </li><li> El día 7, añadir SLNs cargados-NIR a los platos a una concentración final de 0,5 y 5,0 mg / ml de lípidos y mantener las células en un 37 ° C, 5% de CO 2 incubadora para la longitud deseada de la exposición. </li><li> Recoger las células de los platos aspirando todos los medios de comunicación en la placa, recogiéndola en un tubo de 50 ml, seguido de un lavado a fondo de la placa (para desalojar ad vagamenteHerent células) con 5 ml de tampón fosfato salino (PBS) conteniendo 4 mM de ácido etilendiaminotetraacético (EDTA). Recoger la suspensión celular obtenida en el mismo tubo 50 ml. </li></ol></li><li> Evaluación del GLC Incorporación mediante citometría de flujo <ol><li> Determinar la concentración de la suspensión celular obtenida en el punto 2.4.3 y distribuir 3x10 5 células en un 12 mm x 75 mm de fondo redondo de tubo de FACS. </li><li> Lavar las células mediante la adición de 1 ml de PBS y luego centrifugar los tubos durante 5 min a 400 xg, a una temperatura de 4 ° C. </li><li> Descartar el sobrenadante y resuspender el pellet con 100 l de PBS / EDTA 2 mM / 0,1% de albúmina de suero bovino (BSA) (tampón de tinción) que contienen anticuerpo monoclonal anti-CD11c-FITC (0,25 g / ml final). Incubar durante 30 min en hielo en la oscuridad. </li><li> Lavar las células con 1 ml de PBS y centrifugar los tubos durante 5 min a 400 xg, a una temperatura de 4 ° C. </li><li> Descartar el sobrenadante y resuspender cadasedimentar con 400 l de tampón de tinción. Las células están ahora listos para la adquisición de las muestras con un citómetro de flujo (posiblemente equipado con láseres azul y rojo). </li><li> El uso de citometría de flujo de software de análisis, evaluar el nivel de incorporación de GLC por DC por gating en la población de células CD11c + (FITC positivo) y la comparación de la intensidad de GLC-asociados fluorescencia (medido en los canales PerCP-Cy5.5 o APC). </li></ol></li></ol>

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The authors have nothing to disclose.

En este estudio, la síntesis de GLC y su aplicabilidad para aplicaciones dirigidas intracelulares fueron exploradas. Estas nanopartículas biocompatibles se han mostrado prometedores como vehículos de administración para múltiples aplicaciones, incluyendo la administración de fármacos, el silenciamiento de genes y tecnologías de vacunas 25-30. Ultra-pequeños GLC se sintetizaron utilizando un proceso fácil, y sus interacciones con las células cutáneas primarias y células inmunes primarias fueron ex...

Vehículos de administración basados ​​en nanopartículas han mostrado una gran promesa para aplicaciones dirigidas intracelulares, proporcionando un mecanismo para alterar específicamente la señalización celular y la expresión génica. Estos vehículos pueden ser cargados con fármacos, proteínas y ácidos nucleicos diseñados para afectar las respuestas celulares y lograr un efecto deseado en los tejidos diana. Muchos tipos de nanovehículos se han explorado para el beneficio terapéutico y de diagnóstico incluyendo lípidos, polímeros, silicio y materiales magnéticos. Estos sistemas son atractivos debido a su potencial para la entrega localizada de fármacos, aumento de la concentración terapéutica en tejidos diana, y la reducción de la toxicidad sistémica. Nanopartículas lipídicas sólidas (GLC) son un ejemplo bien estudiado de un sistema de liberación de nanopartículas que ha surgido como un vehículo de suministro de fármaco prometedor en los últimos años. SLNs se pueden formular fácilmente para múltiples aplicaciones, incluyendo la detección de bio-1, cosméticos 2, y tentrega herapeutic 3-7. Su utilidad se deriva del hecho de que se componen enteramente de, lípidos no tóxicos reabsorbibles, resulta en una mayor biocompatibilidad. Durante la síntesis, fármacos lipófilos se pueden incorporar en vehículos SLN, lo que aumenta la solubilidad del fármaco y la idoneidad para la administración parenteral. Vehículos SLN también ayudan a estabilizar la terapéutica encapsulados, reduciendo su degradación y la limpieza, y la maximización de la acción terapéutica. Estos vehículos son particularmente bien adaptado para acción prolongada, preparaciones de liberación controlada debido a su estabilidad en 3,4,8,9 la temperatura corporal. Es importante destacar que la encapsulación de fármacos en nanopartículas lipídicas altera los perfiles farmacocinéticos intrínsecas de las moléculas de fármaco. Esto proporciona una ventaja potencial al permitir la liberación controlada de fármacos con un índice terapéutico estrecho. La velocidad de liberación de la terapéutica incorporado SLN-puede ser sintonizado basa en la tasa de degradación de lípidos o de la tasa de difusión del fármaco en elmatriz lipídica. SLNs son a menudo diseñados para acumularse en tejidos diana específicos. Por ejemplo, su tamaño (típicamente mayor que 10 nm) potencia la retención en la circulación, donde la vasculatura tumoral con fugas de tejido facilita la deposición. Además, la vía de administración de partículas se ha demostrado que altera la biodistribución con el potencial para apuntar estructuras fisiológicas específicas, tales como los ganglios linfáticos 10,11. Tras la deposición en los tejidos diana, el logro de las interacciones celulares apropiadas y eventual internalización de nanopartículas es un reto debido a la capacidad de las membranas celulares para controlar selectivamente el flujo de iones y moléculas dentro y fuera de la celda 12. Para facilitar la absorción celular, es posible modificar nanovehículos con ligandos específicos que incluyen péptidos, moléculas pequeñas, y anticuerpos monoclonales 13,14. Varios mecanismos que incluyen tanto la penetración pasiva y transporte activo de nanopartículasa través de la membrana celular se han descrito previamente 3,12,15. En general, se ha demostrado que las interacciones célula-nanopartículas están influenciados por las propiedades fisicoquímicas de las nanopartículas incluyendo el tamaño, forma, carga superficial y la química de superficie, además de los parámetros específicos de células tales como el tipo celular o fase del ciclo celular 12. Una investigación anterior demostró la síntesis de sub-10 GLC nm de tópicos 16 y biomarcadores aplicaciones de detección de 1 utilizando el método de la temperatura de inversión de fases (PIT) 17. Este es un método de síntesis suave donde la composición 2 se mantiene constante mientras que la temperatura se cambia gradualmente. Agitación continua de la solución calentada, mientras se enfría a los resultados de RT en una nanoemulsión. Este proceso resulta en la síntesis de GLC con tamaño de partícula menor 1 que se informó anteriormente utilizando diversos métodos para la síntesis de lípidos nanoparticles 17-22. La escala de tamaño resultante, a menos de 20 nm, proporciona una ventaja para aplicaciones de focalización intracelulares debido a una mayor área de superficie y el potencial para las interacciones celulares mejoradas. A esquemática de GLC, diseñado para ofrecer un colorante fluorescente o terapéutico, se muestra en la Figura 1. Los SLNs constan de un lípido interior (por ejemplo, alcano lineal) permitiendo la incorporación de compuestos lipófilos (por ejemplo, tintes o agentes terapéuticos) y un exterior tensioactivo (tensioactivo no iónico, por ejemplo, lineal) rodeada de agua. En este estudio, SLNs se cargaron con un colorante fluorescente y se usan como un modelo para investigar las interacciones célula-partícula. Fibroblastos dérmicos humanos primarios y células dendríticas de ratones fueron expuestos a teñir-cargadas SLN lo largo del tiempo con el fin de caracterizar las interacciones con respecto a la toxicidad y la captación de partículas. A 3- (4,5-dimetiltiazol-2-il) -2,5-diphenylphenyltetrazolium (MTT) ensayo fue utilized el fin de establecer los niveles de dosificación apropiados. La microscopía de fluorescencia y citometría de flujo fueron dos métodos empleados para examinar la captación de partículas in vitro. <img alt="Figura 1" src="/files/ftp_upload/53102/53102fig1.jpg" /> Figura 1. Esquema de GLC que muestra los componentes principales. <a href="https://www.jove.com/files/ftp_upload/53102/53102fig1large.jpg" target="_blank">Haga clic aquí para ver una versión más grande de esta figura.</a>

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Nile Red (NiR)</td>
			<td>Sigma</td>
			<td>19123</td>
			<td>BioReagent, suitable for fluorescence, &#8805;98.0%</td>
		</tr>
		<tr>
			<td>Heneicosane</td>
			<td>Aldrich</td>
			<td>286052</td>
			<td>98%</td>
		</tr>
		<tr>
			<td>Brij O10</td>
			<td>Sigma</td>
			<td>P6136</td>
			<td>Brij 97, C18-1E10, Polyoxyethylene (10) oleyl ether</td>
		</tr>
		<tr>
			<td>Water</td>
			<td>Sigma</td>
			<td>W3500</td>
			<td>Sterile-filtered, BioReagent, suitable for cell culture</td>
		</tr>
		<tr>
			<td>Syringe Filter 0.2 &#181;m Supor Membrane Low Protein Binding</td>
			<td>Life Sciences</td>
			<td>PN4612</td>
			<td>Non-Pyrogenic</td>
		</tr>
		<tr>
			<td>Nanotrac Ultra&#160;</td>
			<td>Microtrac</td>
			<td>serial number U1985IS</td>
			<td>Instrument</td>
		</tr>
		<tr>
			<td>Differential Scanning Calorimeter</td>
			<td>Mettlet-Toledo</td>
			<td>----</td>
			<td>Instument</td>
		</tr>
		<tr>
			<td>Primary human fibroblasts&#160;</td>
			<td>Life Technologies</td>
			<td>C-004-5C</td>
			<td>Neonatal (HDFn)</td>
		</tr>
		<tr>
			<td>Medium 106</td>
			<td>Life Technologies</td>
			<td>M-106-500</td>
			<td>A sterile, liquid medium for the culture of human dermal fibroblasts.</td>
		</tr>
		<tr>
			<td>Low Serum Growth Supplement Kit (LSGS Kit)</td>
			<td>Life Technologies</td>
			<td>S-003-K</td>
			<td>All the components of complete LSGS</td>
		</tr>
		<tr>
			<td>MTT Cell Proliferation Assay Kit</td>
			<td>Trevigen</td>
			<td>4890-025-K</td>
			<td>Sensitive kit for the measurement of cell proliferation based upon the reduction of the tetrazolium salt, 3,[4,5-dimethylthiazol-2- yl]-2,5-diphenyl-tetrazolium bromide (MTT)</td>
		</tr>
		<tr>
			<td>Safire2 microplate reader</td>
			<td>Tecan</td>
			<td>----</td>
			<td>Instrument</td>
		</tr>
		<tr>
			<td>Phosphate buffered saline&#160;</td>
			<td>Sigma</td>
			<td>P5493</td>
			<td>For molecular biology</td>
		</tr>
		<tr>
			<td>Recombinant murine GM-CSF&#160;</td>
			<td>R&#38;D Systems</td>
			<td>415</td>
			<td>&#62;97%, by SDS-PAGE under reducing conditions and visualized by silver stain.</td>
		</tr>
		<tr>
			<td>Recombinant murine IL-4&#160;</td>
			<td>R&#38;D Systems</td>
			<td>404</td>
			<td>&#62;97%, by SDS-PAGE under reducing conditions and visualized by silver stain.</td>
		</tr>
	</tbody>
</table>

solid lipid nanoparticles (slns) for intracellular targeting applications

Nanoparticle-based delivery vehicles have shown great promise for intracellular targeting applications, providing a mechanism to specifically alter cellular signaling and gene expression. In a previous investigation, the synthesis of ultra-small solid lipid nanoparticles (SLNs) for topical drug delivery and biomarker detection applications was demonstrated. SLNs are a well-studied example of a nanoparticle delivery system that has emerged as a promising drug delivery vehicle. In this study, SLNs were loaded with a fluorescent dye and used as a model to investigate particle-cell interactions. The phase inversion temperature (PIT) method was used for the synthesis of ultra-small populations of biocompatible nanoparticles. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylphenyltetrazolium bromide (MTT) assay was utilized in order to establish appropriate dosing levels prior to the nanoparticle-cell interaction studies. Furthermore, primary human dermal fibroblasts and mouse dendritic cells were exposed to dye-loaded SLN over time and the interactions with respect to toxicity and particle uptake were characterized using fluorescence microscopy and flow cytometry. This study demonstrated that ultra-small SLNs, as a nanoparticle delivery system, are suitable for intracellular targeting of different cell types.

El método PIT fue utilizado para sintetizar los GLC y la temperatura de inversión de fase se determinó utilizando un baño de agua. Las muestras se calienta lentamente y suavemente agitado hasta que la solución parecía clara. La temperatura de inversión de fase para los GLC hechas usando lípidos heneicosane es de 45 ° C. La Tabla 1 resume el tamaño de partícula, polidispersidad, punto de fusión y el calor latente de fusión de los GLC. Los SLNs sintetizados usando las condiciones de procesami...

Watch this Scientific Journal Video about Solid Lipid Nanoparticles SLNs for Intracellular Targeting Applications at JoVE.com

Solid Lipid Nanoparticles SLNs for Intracellular Targeting Applications

In this study, a method for synthesizing ultra-small populations of biocompatible nanoparticles was described, as well as several in vitro methods by which to assess their cellular interactions.

Sólido lípidos nanopartículas (GLC) Aplicaciones para intracelular de focalización

Nanoparticle-based delivery vehicles have shown great promise for intracellular targeting applications, providing a mechanism to specifically alter ...

solid-lipid-nanoparticles-slns-for-intracellular-targeting

Research

JoVE Journal

Bioengineering

Solid Lipid Nanoparticles (SLNs) for Intracellular Targeting Applications

16.9K vistas.  The Johns Hopkins Applied Physics Laboratory. In this study, a method for synthesizing ultra-small populations of biocompatible nanoparticles was described, as well as several in vitro methods by which to assess their cellular interactions. 

Video: Solid Lipid Nanoparticles SLNs for Intracellular Targeting Applications

Viral Nanoparticles for In vivo Tumor Imaging

The use of nanomaterials has the potential to revolutionize materials science and medicine. Currently, a number of different nanoparticles are being investigated for applications in imaging and therapy. Viral nanoparticles (VNPs) derived from plants can be regarded as self-assembled bionanomaterials with defined sizes and shapes. Plant viruses under investigation in the Steinmetz lab include icosahedral particles formed by Cowpea mosaic virus (CPMV) and Brome mosaic virus (BMV), both of which are 30 nm in diameter. We are also developing rod-shaped and filamentous structures derived from the following plant viruses: Tobacco mosaic virus (TMV), which forms rigid rods with dimensions of 300 nm by 18 nm, and Potato virus X (PVX), which form filamentous particles 515 nm in length and 13 nm in width (the reader is referred to refs. 1 and 2 for further information on VNPs).
From a materials scientist's point of view, VNPs are attractive building blocks for several reasons: the particles are monodisperse, can be produced with ease on large scale in planta, are exceptionally stable, and biocompatible. Also, VNPs are "programmable" units, which can be specifically engineered using genetic modification or chemical bioconjugation methods 3. The structure of VNPs is known to atomic resolution, and modifications can be carried out with spatial precision at the atomic level4, a level of control that cannot be achieved using synthetic nanomaterials with current state-of-the-art technologies.
In this paper, we describe the propagation of CPMV, PVX, TMV, and BMV in Vigna ungiuculata and Nicotiana benthamiana plants. Extraction and purification protocols for each VNP are given. Methods for characterization of purified and chemically-labeled VNPs are described. In this study, we focus on chemical labeling of VNPs with fluorophores (e.g. Alexa Fluor 647) and polyethylene glycol (PEG). The dyes facilitate tracking and detection of the VNPs 5-10, and PEG reduces immunogenicity of the proteinaceous nanoparticles while enhancing their pharmacokinetics 8,11. We demonstrate tumor homing of PEGylated VNPs using a mouse xenograft tumor model. A combination of fluorescence imaging of tissues ex vivo using Maestro Imaging System, fluorescence quantification in homogenized tissues, and confocal microscopy is used to study biodistribution. VNPs are cleared via the reticuloendothelial system (RES); tumor homing is achieved passively via the enhanced permeability and retention (EPR) effect12. The VNP nanotechnology is a powerful plug-and-play technology to image and treat sites of disease in vivo. We are further developing VNPs to carry drug cargos and clinically-relevant imaging moieties, as well as tissue-specific ligands to target molecular receptors overexpressed in cancer and cardiovascular disease.

Viral Nanoparticles for In vivo Tumor Imaging

Plant viral nanoparticles (VNPs) are promising platforms for applications in biomedicine. Here, we describe the procedures for plant VNP propagation, purification, characterization, and bioconjugation. Finally, we show the application of VNPs for tumor homing and imaging using a mouse xenograft model and fluorescence imaging.

Las nanopartículas virales para<em&gt; En vivo</em&gt; Imágenes de tumores

The use of nanomaterials has the potential to revolutionize materials science and medicine. Currently, a number of different nanoparticles are being ...

Investigación

Bioingeniería

Harmonic Nanoparticles for Regenerative Research

In this visualized experiment, protocol details are provided for in vitro labeling of human embryonic stem cells (hESC) with second harmonic generation nanoparticles (HNPs). The latter are a new family of probes recently introduced for labeling biological samples for multi-photon imaging. HNPs are capable of doubling the frequency of excitation light by the nonlinear optical process of second harmonic generation with no restriction on the excitation wavelength.
Multi-photon based methodologies for hESC differentiation into cardiac clusters (maintained as long term air-liquid cultures) are presented in detail. In particular, evidence on how to maximize the intense second harmonic (SH) emission of isolated HNPs during 3D monitoring of beating cardiac tissue in 3D is shown. The analysis of the resulting images to retrieve 3D displacement patterns is also detailed.

Protocol details are provided for in vitro labeling human embryonic stem cells with second harmonic generating nanoparticles. Methodologies for hESC investigation by multi-photon microscopy and their differentiation into cardiac clusters are also presented.

Armónica Nanopartículas para Regenerativa de Investigación

In this visualized experiment, protocol details are provided for in vitro labeling of human embryonic stem cells (hESC) with second harmonic generation ...

Production and Targeting of Monovalent Quantum Dots

The multivalent nature of commercial quantum dots (QDs) and the difficulties associated with producing monovalent dots have limited their applications in biology, where clustering and the spatial organization of biomolecules is often the object of study. We describe here a protocol to produce monovalent quantum dots (mQDs) that can be accomplished in most biological research laboratories via a simple mixing of CdSe/ZnS core/shell QDs with phosphorothioate DNA (ptDNA) of defined length. After a single ptDNA strand has wrapped the QD, additional strands are excluded from the surface. Production of mQDs in this manner can be accomplished at small and large scale, with commercial reagents, and in minimal steps. These mQDs can be specifically directed to biological targets by hybridization to a complementary single stranded targeting DNA. We demonstrate the use of these mQDs as imaging probes by labeling SNAP-tagged Notch receptors on live mammalian cells, targeted by mQDs bearing a benzylguanine moiety.

We provide detailed instructions for the preparation of monovalent targeted quantum dots (mQDs) from phosphorothioate DNA of defined length. DNA wrapping occurs in high yield, and therefore, products do not require purification. We demonstrate the use of the SNAP tag to target mQDs to cell-surface receptors for live-cell imaging applications.

Producción y focalización de monovalente Quantum Dots

The multivalent nature of commercial quantum dots (QDs) and the difficulties associated with producing monovalent dots have limited their applications in ...

Biofunctionalized Prussian Blue Nanoparticles for Multimodal Molecular Imaging Applications

Multimodal, molecular imaging allows the visualization of biological processes at cellular, subcellular, and molecular-level resolutions using multiple, complementary imaging techniques. These imaging agents facilitate the real-time assessment of pathways and mechanisms in vivo, which enhance both diagnostic and therapeutic efficacy. This article presents the protocol for the synthesis of biofunctionalized Prussian blue nanoparticles (PB NPs) - a novel class of agents for use in multimodal, molecular imaging applications. The imaging modalities incorporated in the nanoparticles, fluorescence imaging and magnetic resonance imaging (MRI), have complementary features. The PB NPs possess a core-shell design where gadolinium and manganese ions incorporated within the interstitial spaces of the PB lattice generate MRI contrast, both in T1 and T2-weighted sequences. The PB NPs are coated with fluorescent avidin using electrostatic self-assembly, which enables fluorescence imaging. The avidin-coated nanoparticles are modified with biotinylated ligands that confer molecular targeting capabilities to the nanoparticles. The stability and toxicity of the nanoparticles are measured, as well as their MRI relaxivities. The multimodal, molecular imaging capabilities of these biofunctionalized PB NPs are then demonstrated by using them for fluorescence imaging and molecular MRI in vitro.

This protocol describes the synthesis of biofunctionalized Prussian blue nanoparticles and their use as multimodal, molecular imaging agents. The nanoparticles have a core-shell design where gadolinium or manganese ions within the nanoparticle core generate MRI contrast. The biofunctional shell contains fluorophores for fluorescence imaging and targeting ligands for molecular targeting.

Biofuncionalizadas prusiana nanopartículas azul para Multimodal Molecular Aplicaciones de imágenes

Multimodal, molecular imaging allows the visualization of biological processes at cellular, subcellular, and molecular-level resolutions using multiple, ...

Cell Labeling and Targeting with Superparamagnetic Iron Oxide Nanoparticles

Targeted delivery of cells and therapeutic agents would benefit a wide range of biomedical applications by concentrating the therapeutic effect at the target site while minimizing deleterious effects to off-target sites. Magnetic cell targeting is an efficient, safe, and straightforward delivery technique. Superparamagnetic iron oxide nanoparticles (SPION) are biodegradable, biocompatible, and can be endocytosed into cells to render them responsive to magnetic fields. The synthesis process involves creating magnetite (Fe3O4) nanoparticles followed by high-speed emulsification to form a poly(lactic-co-glycolic acid) (PLGA) coating. The PLGA-magnetite SPIONs are approximately 120 nm in diameter including the approximately 10 nm diameter magnetite core. When placed in culture medium, SPIONs are naturally endocytosed by cells and stored as small clusters within cytoplasmic endosomes. These particles impart sufficient magnetic mass to the cells to allow for targeting within magnetic fields. Numerous cell sorting and targeting applications are enabled by rendering various cell types responsive to magnetic fields. SPIONs have a variety of other biomedical applications as well including use as a medical imaging contrast agent, targeted drug or gene delivery, diagnostic assays, and generation of local hyperthermia for tumor therapy or tissue soldering.

Targeted cell delivery is useful in a variety of biomedical applications. The goal of this protocol is to use superparamagnetic iron oxide nanoparticles (SPION) to label cells and thereby enable magnetic cell targeting approaches for a high degree of control over cell delivery and localization.

Etiquetado Celular y Orientación con superparamagnético nanopartículas de óxido de hierro

Targeted delivery of cells and therapeutic agents would benefit a wide range of biomedical applications by concentrating the therapeutic effect at the ...

Fabricating Superhydrophobic Polymeric Materials for Biomedical Applications

Superhydrophobic materials, with surfaces possessing permanent or metastable non-wetted states, are of interest for a number of biomedical and industrial applications. Here we describe how electrospinning or electrospraying a polymer mixture containing a biodegradable, biocompatible aliphatic polyester (e.g., polycaprolactone and poly(lactide-co-glycolide)), as the major component, doped with a hydrophobic copolymer composed of the polyester and a stearate-modified poly(glycerol carbonate) affords a superhydrophobic biomaterial. The fabrication techniques of electrospinning or electrospraying provide the enhanced surface roughness and porosity on and within the fibers or the particles, respectively. The use of a low surface energy copolymer dopant that blends with the polyester and can be stably electrospun or electrosprayed affords these superhydrophobic materials. Important parameters such as fiber size, copolymer dopant composition and/or concentration, and their effects on wettability are discussed. This combination of polymer chemistry and process engineering affords a versatile approach to develop application-specific materials using scalable techniques, which are likely generalizable to a wider class of polymers for a variety of applications.

Two- and three-dimensional superhydrophobic polymeric materials are prepared by electrospinning or electrospraying biodegradable polymers blended with a lower surface energy polymer of similar composition.

Fabricación superhidrófobas materiales poliméricos para aplicaciones biomédicas

Superhydrophobic materials, with surfaces possessing permanent or metastable non-wetted states, are of interest for a number of biomedical and industrial ...

Manufacture and Drug Delivery Applications of Silk Nanoparticles

Silk is a promising biopolymer for biomedical and pharmaceutical applications due to its outstanding mechanical properties, biocompatibility and biodegradability, as well its ability to protect and subsequently release its payload in response to a trigger. While silk can be formulated into various material formats, silk nanoparticles are emerging as promising drug delivery systems. Therefore, this article covers the procedures for reverse engineering silk cocoons to yield a regenerated silk solution that can be used to generate stable silk nanoparticles. These nanoparticles are subsequently characterized, drug loaded and explored as a potential anticancer drug delivery system. Briefly, silk cocoons are reverse engineered first by degumming the cocoons, followed by silk dissolution and clean up, to yield an aqueous silk solution. Next, the regenerated silk solution is subjected to nanoprecipitation to yield silk nanoparticles &#8211; a simple but powerful method that generates uniform nanoparticles. The silk nanoparticles are characterized according to their size, zeta potential, morphology and stability in aqueous media, as well as their ability to entrap a chemotherapeutic payload and kill human breast cancer cells. Overall, the described methodology yields uniform silk nanoparticles that can be readily explored for a myriad of applications, including their use as a potential nanomedicine.

Nanoparticles are emerging as promising drug delivery systems for a broad range of indications. Here, we describe a simple yet powerful method to manufacture silk nanoparticles using reverse engineered Bombyx mori silk. These silk nanoparticles can be readily loaded with a therapeutic payload and subsequently explored for drug delivery applications.

Fabricación y entrega de medicamentos Aplicaciones de seda nanopartículas

Silk is a promising biopolymer for biomedical and pharmaceutical applications due to its outstanding mechanical properties, biocompatibility and ...

Antimicrobial Characterization of Advanced Materials for Bioengineering Applications

The development of new advanced materials with enhanced properties is becoming more and more important in a wide range of bioengineering applications. Thus, many novel biomaterials are being designed to mimic specific environments required for biomedical applications such as tissue engineering and controlled drug delivery. The development of materials with improved properties for the immobilization of cells or enzymes is also a current research topic in bioprocess engineering. However, one of the most desirable properties of a material in these applications is the antimicrobial capacity to avoid any undesirable infections. For this, we present easy-to-follow protocols for the antimicrobial characterization of materials based on (i) the agar disk diffusion test (diffusion method) and (ii) the ISO 22196:2007 norm to measure the antimicrobial activity on material surfaces (contact method). This protocol must be performed using Gram-positive and Gram-negative bacteria and yeast to cover a broad range of microorganisms. As an example, 4 materials with different chemical natures are tested following this protocol against Staphylococcus aureus, Escherichia coli, and Candida albicans.The results of these tests exhibit non-antimicrobial activity for the first material and increasing antibacterial activity against Gram-positive and Gram-negative bacteria for the other 3 materials. However, none of the 4 materials are able to inhibit the growth of Candida albicans.

We present a protocol for the antimicrobial characterization of advanced materials. Here, the antimicrobial activity on material surfaces is measured by two methods that complement each other: one is based on the agar disk diffusion test, and the other is a standard procedure based on the ISO 22196:2007 norm.

Caracterización antimicrobiana de materiales avanzados para aplicaciones de bioingeniería

The development of new advanced materials with enhanced properties is becoming more and more important in a wide range of bioengineering applications. ...

Uptake of New Lipid-coated Nanoparticles Containing Falcarindiol by Human Mesenchymal Stem Cells

Nanoparticles are the focus of an increased interest in drug delivery systems for cancer therapy. Lipid-coated nanoparticles are inspired in structure and size by low-density lipoproteins (LDLs) because cancer cells have an increased need for cholesterol to proliferate, and this has been exploited as a mechanism for delivering anticancer drugs to cancer cells. Moreover, depending on drug chemistry, encapsulating the drug can be advantageous to avoid degradation of the drug during circulation in vivo. Therefore, in this&#160;study, this design is used to fabricate lipid-coated nanoparticles of the anticancer drug falcarindiol, providing a potential new delivery system of falcarindiol in order to stabilize its chemical structure against degradation and improve its uptake by tumors. Falcarindiol nanoparticles, with a phospholipid and cholesterol monolayer encapsulating the purified drug core of the particle, were designed. The lipid monolayer coating consists of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol (Chol), and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE PEG 2000) along with the fluorescent label DiI (molar ratios of 43:50:5:2). The nanoparticles are fabricated using the rapid injection method, which is a fast and simple technique to precipitate nanoparticles by good-solvent for anti-solvent exchange. It consists of a rapid injection of an ethanol solution containing the nanoparticle components into an aqueous phase. The size of the fluorescent nanoparticles is measured using dynamic light scattering (DLS) at 74.1 &#177; 6.7 nm. The uptake of the nanoparticles is tested in human mesenchymal stem cells (hMSCs) and imaged using fluorescence and confocal microscopy. The uptake of the nanoparticles is observed in hMSCs, suggesting the potential for such a stable drug delivery system for falcarindiol.

This article describes the encapsulation of falcarindiol in lipid-coated 74 nm nanoparticles. The cellular uptake of the nanoparticles&#160;by human stem cells into lipid droplets is monitored by fluorescent and confocal imaging. Nanoparticles are fabricated by the rapid injection method of solvent shifting, and their size is measured with the dynamic light scattering technique.

Absorción de nuevas nanopartículas recubiertas de lípidos que contienen Falcarindiol por células madre mesenquimales humanas

Nanoparticles are the focus of an increased interest in drug delivery systems for cancer therapy. Lipid-coated nanoparticles are inspired in structure and ...

Applications for Open Source Microplate-Compatible Illumination Panels

Microplates are commonly used in the modern laboratory environment for a wide variety of tasks both in small-scale laboratory benchtop operations as well as large-scale high-throughput screening (HTS) campaigns. Though laboratory automation has greatly increased the utility of microplates there remain instances where automation-based instrumentation is not feasible, cost-effective or compatible with microplate formatting needs. In these cases, microplates must be manually prepared. Problematic to manual microplate manipulations is that a number of difficulties can arise pertaining to the accurate tracking of sample operations, data record keeping and quality control (QC) inspection for well artifacts or formatting errors. As microplate well densities increase (i.e., 96-well, 384-well, 1536-well) the potential for introducing errors also drastically increases.&#160; Moreover, for small bench-top laboratory operations there exists a need to improve the ease and accuracy of sample handling in a cost-effective fashion. Herein, we describe a system that acts as a semi-automated pipetting guide referred to as the Microplate Assistive Pipetting Light Emitter (M.A.P.L.E.). &#160;M.A.P.L.E. has multiple uses for supporting compound hit-picking and microplate preparation for assay development in high-throughput screening or laboratory benchtop operations, as well as QC/quality assurance (QA) diagnostic evaluation of microplate quality or visualizing well formatting errors.

Microplate Assistive Pipetting Light Emitter (M.A.P.L.E.) is a computer-driven device that systematically illuminates microtiter wells to provide guidance for the manual preparation of microplates.&#160; M.A.P.L.E. improves the accuracy of microplate preparation while automating data recordkeeping. &#160;In addition, it can assist with examining microplate quality or aid in the detection of errors.&#160; &#160;

Aplicaciones para paneles de iluminación compatibles con microplacas de código abierto

Microplates are commonly used in the modern laboratory environment for a wide variety of tasks both in small-scale laboratory benchtop operations as well ...