The authors would like to acknowledge the technical assistance of Alfred Gunasekaran in electron microscopy studies at the Institute of Micromanufacturing at Louisiana Tech University, and Dr. Jim McNamara for assistance with additional microscopy studies. The work described was supported in part by Louisiana board of Regents PKSFI Contract No. LEQSF (2007-12)-ENH-PKSFI-PRS-04 and the James E. Wyche III Endowed Professorship from Louisiana Tech University (to M.D.).

1. Planificación de Experimentos <ol><li> Determinar el volumen de nanocompuestos de cobre necesarios para la síntesis. Sobre esa base, elegir un número de frascos pequeños volumen (25 cm2), o frascos más grandes, como se indica a continuación en la preparación de materiales. </li><li> Para esta síntesis, utilizar una incubadora a 37 ° C con 5% de CO 2 y al menos 40% de humedad. Asegúrese de que una incubadora tal está disponible y que no será perturbado en repetidas ocasiones durante el periodo de síntesis (aproximadamente 24 hr). PRECAUCIÓN: apertura repetida y el cierre de la incubadora sin duda causar fluctuaciones de temperatura que pueden resultar en la síntesis alterada de las estructuras de nanocompuestos. </li></ol> 2. Preparación de los materiales <ol><li> Preparar todos los materiales frescos antes del inicio de un experimento, mediante la adición de materiales sólidos a los disolventes justo antes de la síntesis es comenzar. Mantener las soluciones stock de los materiales de partida de cistina y cobre en líquido para tiempos largos bntes no se recomienda el experimento y puede conducir a resultados variables. Una vez abierto desde el proveedor, mantener los materiales secos envolviendo la parte superior del recipiente con Parafilm de partida. Nota: El siguiente protocolo se utiliza como un ejemplo para la reacción en un 25 cm 2 frasco de cultivo celular usando 7 l de cistina, 6643 l de agua estéril, y 350 l de CNP. </li><li> Preparar una solución de 2 mg / ml de las nanopartículas de cobre pesando al menos 2 mg de CNP. Use guantes desechables durante este paso para evitar el posible contacto con la piel del CNP. Coloque las nanopartículas en una estéril 16 ml vial vaso vacío. <ol><li> Para el vial que contiene CNP, añadir agua desionizada estéril en el volumen adecuado para hacer una solución de 2 mg / ml y agitar la solución durante 20 segundos para proporcionar una dispersión de las nanopartículas antes del comienzo de síntesis (se recomienda al menos 1 ml de volumen total). No llene el camino vial más de la mitad con agua ya que esto inhibirá la mezcla por agitación. CNP will asiente rápidamente a la parte inferior del vial y aparecerá de color oscuro (gris a negro). </li><li> Sonicar la solución CNP por 17 minutos a temperatura ambiente para proporcionar la dispersión máxima de CNP antes del inicio de la síntesis. Revise periódicamente para asegurarse de que los CNP se mezclaba debido al tratamiento con ultrasonidos. Después de una sonicación éxito, CNP permanecen suspendidas en solución durante al menos 30 min y la solución será de color oscuro. </li></ol></li><li> Pesar masa suficiente de cistina para hacer una solución / ml 72,9 mg para la síntesis. Desde cistina no es directamente soluble en agua, coloque la cistina pesaron en un recipiente de pesada antiestático. <ol><li> Para el recipiente que contiene cistina pesaje, añadir un volumen suficiente de estéril, 1 M NaOH, de modo que la cistina se disuelve completamente. Por ejemplo, se disuelven 7,29 mg de cistina completamente en 100 l de 1 M NaOH, para hacer una solución 72,9 mg / ml. </li><li> Para mantener condiciones estériles, llevar a cabo este paso en un tejido flujo campana de cultivo estéril. PRECAUCIÓN: NaOHa 1 M de concentración es cáustico, así que use guantes desechables durante este paso para evitar el contacto de NaOH concentrado con la piel </li></ol></li><li> Trabajando en una campana de cultivo de tejido estéril, añadir 7 l de cistina con 6643 l de agua estéril al matraz estéril primera síntesis, y dejar incubar durante 30 min en la incubadora a 37 ° C con la tapa de matraz ventilada (suelto) para proporcionar eficaz de mezcla. Resuspender la solución de 2 mg / ml CNP por agitación durante 30 segundos, ya que los CNP se han asentado después de la etapa de sonicación. <ol><li> Añadir suficiente solución CNP en el matraz de síntesis (utilizando una técnica estéril) para mantener las relaciones de los componentes siguientes: 1 combinar partes cistina, 50 partes CNP, y 949 partes de agua estéril en un matraz de cultivo de 2 células 25 cm para iniciar la síntesis. Por ejemplo, para un volumen de síntesis 7 ml, combinar 7 l de solución de cistina Stock, 350 l de CNP, y 6.643 l de agua estéril. Vuelva a colocar la tapa en el frasco y apriete de modo que sea sege. </li><li> Después de combinar todos los componentes para la síntesis, mezclar suavemente en el matraz por agitación 4-5 veces. Colocar el frasco en la incubadora de CO 2 y ventilar el frasco aflojando el tapón de modo que no habrá intercambio de gases en y fuera del matraz durante la síntesis. </li></ol></li><li> Permita que la síntesis se ejecute en la incubadora durante aproximadamente 24 horas. Durante la síntesis, se puede observar, con microscopía y por el ojo, la formación de materiales compuestos altamente lineales. Nota: El proceso de formación de las estructuras puede ocurrir de repente en el sentido de que las estructuras son inicialmente difícil de detectar, a continuación, apariencia procede rápidamente a una densidad creciente. Por lo tanto, la formación puede ocurrir antes de las 24 horas. El proceso también puede ser observado por el ojo una vez que las estructuras se hacen más grandes y aumenta su densidad. Mientras que la generación de las estructuras se puede observar con el tiempo bajo el microscopio y por el ojo en puntos de tiempo posteriores, interrumpiendo continuamente las condiciones de síntesis y la temperatura dará lugar a pobreresultados de la síntesis. </li><li> Terminar síntesis de biocomposites por tapado herméticamente el matraz de síntesis y el almacenamiento de la embarcación en un refrigerador (4 ° C). Estructuras, una vez generados, se mantienen estables en esta forma por lo menos durante un año. Etiquetar el matraz con condiciones de síntesis, incluyendo componentes utilizados, fecha de la síntesis, y tiempo de incubación de la síntesis antes de la terminación. </li></ol> 3. Síntesis Uso de sulfato de cobre <ol><li> Llevar a cabo la síntesis de auto-ensamblaje mediante la sustitución de los CNP con sal de sulfato de cobre. Utilizando una técnica estéril, disolver al menos 2 mg de sulfato de cobre en un volumen suficiente de agua desionizada estéril para hacer una solución de 2 mg / ml. Los cristales de sulfato de cobre fácilmente entran en solución a esta concentración, pero vórtice el vial si es necesario, e inspeccionar por ojo para asegurar que todos los cristales se disuelven. </li><li> Después de la preparación del sulfato de cobre, llevar a cabo la síntesis como se describe anteriormente, pero sustituyendo los CNP con el sulfato de cobre. Nota: nanocompuestos autoensambladas utilizando sulfato de cobre como material de partida se encontraron a ser mucho más coherente en forma final que para estructuras sintetizados a partir de los CNP. </li><li> Terminar la síntesis de biocomposites sulfato de cobre como para materiales compuestos CNP (paso 2.6) y almacenarlos a largo plazo a 4 ° C. </li></ol> 4. Caracterización y Manejo de Biocomposites post-síntesis <ol><li> Caracterizar biocomposites derivados de CNP y de sulfato de cobre por la luz blanca de la microscopía 9 y por microscopía electrónica 9. <ol><li> Para la caracterización y la inspección de biocomposites post-síntesis por la luz de microscopía blanco, usar un microscopio invertido como compuestos se depositan en la superficie inferior del frasco dentro de unos pocos minutos de la puesta del matraz plana, y luego se pueden poner en el foco. Utilice el ajuste de campo claro en el microscopio para maximizar el contraste entre biocomposites y el medio líquido. Compuestos derivados de CNP y la policíapor tanto el sulfato se aparecerá claro a opaco en color, pero los agregados CNP sin reaccionar aparecerá muy oscuro en color. <ol><li> Utilice una cámara digital conectada al microscopio para capturar imágenes de los materiales compuestos. Se observó un intervalo de longitudes para las estructuras individuales. </li></ol></li><li> Para la caracterización y la inspección de biocomposites post-síntesis y después de almacenamiento a 4 ° C, permiten frascos para llegar a la temperatura ambiente durante al menos 15 minutos como frascos formarán condensación inicialmente después de la retirada de la nevera, que oscurecer eficaz centrarse en el ejercicio de formación de imágenes de microscopía. Después de permitir el equilibrado a TA, limpie las superficies superior e inferior del matraz con una toalla de papel limpia para maximizar la calidad de imagen de microscopía. </li><li> Cuando se trabaja con imágenes o materiales compuestos que se han almacenado a largo plazo, vórtice del frasco durante 30 segundos para disociar grupos de compuestos que se forman mientras que en el refrigerador. Después vórtex, inspeccione las estructuras con un micrófono invertidaroscope para asegurar que los agregados han disociado, y repetir vórtex según sea necesario. </li><li> Utilice la luz blanca microscopía invertida para evaluar la eficacia de la síntesis para un experimento dado usando CNP. Por ejemplo, documentar la presencia o ausencia de los CNP sin reaccionar en matraces de síntesis utilizados para biocomposites derivados de CNP de frascos con diferentes parámetros tales como el tiempo de síntesis. Nota: Individual CNP son demasiado pequeños para observar con un microscopio de luz, pero que no ha reaccionado CNP agregados aparecerá como redondo y objetos oscuros, en contraste con los CNP-materiales compuestos han sintetizado con éxito que tendrá una alta relación de aspecto, forma lineal, y tendrá una gama de diferentes longitudes. Evitar realizar síntesis para demasiado largo de un período de tiempo antes de la terminación, ya que esto dará lugar a muy ramificadas estructuras tipo &quot;de erizo&quot;, que son difíciles de dispersar en estructuras individuales, una vez formados. </li><li> Utilice invertido microscopio de luz blanca para evaluar la eficaciade la síntesis para un experimento dado usando sulfato de cobre. Desde el sulfato de cobre va completamente en solución usando este protocolo, la solución aparecerá menos oscuro que la solución de síntesis utilizando CNP. Documentar el tamaño y la extensión de los materiales compuestos de sulfato de cobre mediante la comparación de frascos con diferentes condiciones de síntesis tales como el tiempo de síntesis antes de la terminación. Nota: Los compuestos sintetizados con éxito se mostrará una gama de diferentes longitudes. Evitar realizar síntesis para demasiado largo de un período de tiempo antes de la terminación, ya que esto dará lugar a agregados altamente ramificadas de compuestos, algunos de los cuales serán &quot;erizo-like&quot; en la estructura, y que son difíciles de dispersar en estructuras individuales, una vez formados . </li></ol></li><li> Concentrar biocomposites post-síntesis, las soluciones de centrífuga de composites en un tubo de centrífuga. Añadir 6 ml de cualquiera de las estructuras derivadas-CNP o estructuras de sulfato de cobre-derivado a un tubo de centrífuga de 15 ml. Centrifugar durante 10 minutosa 500 xg a temperatura ambiente para formar un pellet. Para volúmenes más pequeños, añadir 500 l de estructuras en solución a 0,6 ml tubos de tamaño. Centrifugar a 2000 xg a temperatura ambiente durante al menos 10 min para formar un pellet. <ol><li> Después de centrifugar durante un tiempo suficiente (al menos 10 min para microfuges), guardar el precipitado observable en la parte inferior del tubo donde las estructuras se concentran eliminando cuidadosamente el líquido sobrenadante por encima del sedimento. Estructuras biocompuesto derivados de sulfato de cobre aparecen de color azul y estructuras derivadas de los CNP son más oscuros (gris a negro). </li><li> Añadir más compuestos a este tubo y repetir el proceso en el mismo tubo para concentrar las estructuras si se desea. Para dispersar a los gránulos concentrados, añadir el volumen deseado de solución al tubo, y agitar durante 10 a 30 segundos. </li></ol></li><li> Estructuras sonicado una vez formado, para mover el tamaño de la población media (longitudes) de las estructuras a valores más bajos. Coloque las estructuras en agua desionizada estéril y sonicado durante al least 10 min. Utilizando este proceso, con el tiempo, las estructuras se fragmentan y más pequeño en longitud media (véase la figura 6 del texto). Documentar los cambios en los tamaños de compuestos con diferentes tiempos de sonicación utilizando un microscopio de luz blanca invertida y cámara digital. </li></ol>

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Authors have nothing to disclose.

Mientras que la evaluación de potenciales efectos tóxicos de los nanomateriales incluyendo CNP, se observó que en el largo plazo, CNP fueron transformados de una distribución de partículas más dispersa inicialmente a una forma más grande, agregada (Figura 2). En algunos casos, estas formaciones altamente agregados que se produjeron en la placa de cultivo de células, en condiciones biológicas, formaron proyecciones altamente lineales del agregado central, que recuerda cobre descrito anteriorment...

Copper is a highly reactive metal that in the biological world is essential in some enzyme functions 1,2, but in higher concentrations is potently toxic including in the nanoparticulate form 3,4. Concern over copper toxicity has become more relevant as CNPs and other copper-based nanomaterials are utilized, due to the increased surface area/mass for nanostructures. Thus, even a small mass of copper, in nanoparticle form, could cause local toxicity due to its ability to penetrate the cell and be broken down into reactive forms. Some biological species can complex with and chelate metal ions, and even incorporate them into biological structures as has been described in marine mussels 5. In studying the potential toxic effects of nanomaterials 4, it was discovered that over time, and under biological conditions used for typical cell culturing (37 &#176;C and 5% CO2), stable copper biocomposites could be formed with a high-aspect ratio (linear) structure.
By a process of elimination, the initial discovery of these linear biocomposites, which occurred in complete cell culture media, was simplified to a defined protocol of essential elements needed for the biocomposites to self-assemble. Self-assembly of two types of highly linear biocomposites was discovered to be possible with two starting metal components: 1) CNPs and 2) copper sulfate, with the common biological component being cystine. Although more complex, so called &#8220;urchin&#8221; and &#8220;nanoflower&#8221; type copper-containing structures with nanoscale and microscale features have been previously reported, these were produced under non-biological conditions, such as temperatures of 100 &#176;C or greater 6-8. To our knowledge, synthesis of individual, linear copper-containing nanostructures that are scalable in liquid phase under biological conditions has not been previously described. 
One of the starting materials utilized for synthesis of nanocomposites, namely CNPs, has been reported previously to be very toxic to cells 4. It has recently been reported that after the nanocomposites are formed, these structures are less toxic on a per mass basis than the starting NPs 9. Thus, the synthesis described here may be derived from a biological and biochemical reaction that has utility in stabilizing reactive copper species, both in the sense of transforming the NP form into larger structures and in producing composites less toxic to cells. 
In contrast to many other nanomaterial forms which are known to aggregate or clump upon interaction with biological liquid media 10,11, once formed, the highly linear composites described here avoid aggregation, possibly due to a redistribution of charge which has been previously reported 9. As detailed in the current work, this avoidance of aggregation is convenient for the purposes of working with the structures once formed for at least 3 reasons: 1) composite structures once formed may be concentrated using centrifugation and then easily dispersed again using vortex mixing; 2) formed structures can be decreased in average size by sonication for different periods of time; and 3) the formed linear structures may provide an additional tool for avoiding the recently described &#8220;coffee ring effect&#8221; 12 and thus provide a dopant for creating more evenly distributed coatings of materials, especially those containing spherical particulates.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Mini Vortexer</td>
			<td>VWR (https://us.vwr.com)</td>
			<td>58816-121</td>
			<td></td>
		</tr>
		<tr>
			<td>CO2 Incubator Model # 2425-2</td>
			<td>VWR (https://us.vwr.com)</td>
			<td>Contact vendor</td>
			<td>Current model calalog # 98000-360</td>
		</tr>
		<tr>
			<td>Eppendorf Centrifuge (Refrigerated Microcentrifuge)</td>
			<td>Labnet (http://labnetinternational.com/)</td>
			<td>C2500-R</td>
			<td>Model Prism R</td>
		</tr>
		<tr>
			<td>Cell Culture Centrifuge Model Z323K</td>
			<td>Labnet (http://labnetinternational.com/)</td>
			<td>Contact vendor</td>
			<td>Current model Z206A catalog # C0206-A</td>
		</tr>
		<tr>
			<td>Sonicator (Ultrasonic Cleaner)</td>
			<td>Branson Ultrasonics Corporation (http://www.bransonic.com/)</td>
			<td>1510R-MTH</td>
			<td></td>
		</tr>
		<tr>
			<td>Balance</td>
			<td>Sartorius (http://dataweigh.com)</td>
			<td></td>
			<td>Model CP225D similar model CPA225D</td>
		</tr>
		<tr>
			<td>Olympus IX51 Inverted Light Microscope</td>
			<td>Olympus (http://olympusamerica.com</td>
			<td>Contact vendor</td>
			<td></td>
		</tr>
		<tr>
			<td>Olympus DP71 microscope digital camera</td>
			<td>Olympus (http://olympusamerica.com</td>
			<td>Contact vendor</td>
			<td></td>
		</tr>
		<tr>
			<td>external power supply unit- white light for Olympus microscope</td>
			<td>Olympus (http://olympusamerica.com</td>
			<td>TH4-100</td>
			<td></td>
		</tr>
		<tr>
			<td>10x, 20, and 40x microscope objectives</td>
			<td>Olympus (http://olympusamerica.com</td>
			<td>Contact vendor</td>
			<td></td>
		</tr>
		<tr>
			<td>Scanning Electron Microscope</td>
			<td>Hitachi (http://hitachi-hitec.com/global/em/sem/sem_index.html)</td>
			<td>model S-4800</td>
			<td></td>
		</tr>
		<tr>
			<td>Transmission Electron Microscope</td>
			<td>Zeiss (http://zeiss.com/microscopy/en_de/products.html)</td>
			<td>model Libra 120</td>
			<td></td>
		</tr>
		<tr>
			<td>Table Top Work Station Unidirectional Flow Clean Bench</td>
			<td>Envirco (http://envirco-hvac.com)</td>
			<td>model PNG62675</td>
			<td>Used for sterile cell culture technique</td>
		</tr>
	</tbody>
</table>

generation of scalable, metallic high-aspect ratio nanocomposites in a biological liquid medium

El objetivo de este protocolo es describir la síntesis de dos nuevos biocomposites con estructuras de elevada relación de aspecto. Los biocomposites consisten en cobre y cistina, ya sea con nanopartículas de cobre (CNP) o sulfato de cobre contribuye el componente metálico. La síntesis se llevó a cabo en un líquido en condiciones biológicas (37 ° C) y la forma composites autoensambladas después de 24 hr. Una vez formados, estos compuestos son muy estables tanto en medios líquidos y en una forma seca. Los materiales compuestos escala de la nano- a micro gama de longitud, y desde unos pocos micrómetros a 25 nm de diámetro. Emisión de campo microscopía electrónica de barrido con espectroscopia de rayos X de energía dispersiva (EDX) demostró que el azufre estaba presente en las estructuras lineales derivados de NP, mientras que estaba ausente de material de partida CNP, confirmando así la cistina como la fuente de azufre en los nanocompuestos finales . Durante la síntesis de estos nano y micro-compuestos lineales, una amplia gama de longitudes de structures se forma en el recipiente de síntesis. La sonicación de la mezcla líquida después de la síntesis se demostró para ayudar a controlar el tamaño promedio de las estructuras por la disminución de la longitud media con un aumento del tiempo de sonicación. Dado que las estructuras formadas son muy estables, no se aglomeran, y están formados en fase líquida, centrifugación también puede ser usada para ayudar en la concentración y la segregación de los materiales compuestos formados.

La figura 1 muestra un diagrama de flujo esquemático de las etapas de síntesis para formar los biocomposites lineales descritos en este trabajo. CNP o sulfato de cobre como materiales de partida se combinan con agua estéril para formar una solución 2 mg / ml, esta solución se mezcla y se sometió a ultrasonidos para proporcionar una mezcla homogénea, y esta solución de cobre se mezcla a continuación en la siguiente relación para la síntesis de: 949 partes estéril agua: 50 partes de la mezcla ...

Watch this Scientific Journal Video about Generation of Scalable, Metallic High-Aspect Ratio Nanocomposites in a Biological Liquid Medium at JoVE.com

Generation of Scalable, Metallic High-Aspect Ratio Nanocomposites in a Biological Liquid Medium

Aquí se presenta un protocolo para sintetizar nuevos, biocomposites elevada relación de aspecto en condiciones biológicas y en medios líquidos. Los biocomposites escala de nanómetros a micrómetros de diámetro y longitud, respectivamente. Nanopartículas de cobre (CNP) y sulfato de cobre en combinación con cistina son los componentes clave para la síntesis.

Generación de Scalable, metálico de alta Aspect Ratio nanocompuestos en una Biológica Medio Líquido

The goal of this protocol is to describe the synthesis of two novel biocomposites with high-aspect ratio structures. The biocomposites consist of copper ...

generation-scalable-metallic-high-aspect-ratio-nanocomposites

Research

JoVE Journal

Bioengineering

9.0K vistas.  Centenary College of Louisiana.  Aquí se presenta un protocolo para sintetizar nuevos, biocomposites elevada relación de aspecto en condiciones biológicas y en medios líquidos. Los biocomposites escala de nanómetros a micrómetros de diámetro y longitud, respectivamente. Nanopartículas de cobre (CNP) y sulfato de cobre en combinación con cistina son los componentes clave para la síntesis. 

Video: Generation of Scalable, Metallic High-Aspect Ratio Nanocomposites in a Biological Liquid Medium

Cellular Lipid Extraction for Targeted Stable Isotope Dilution Liquid Chromatography-Mass Spectrometry Analysis

The metabolism of fatty acids, such as arachidonic acid (AA) and linoleic acid (LA), results in the formation of oxidized bioactive lipids, including numerous stereoisomers1,2. These metabolites can be formed from free or esterified fatty acids. Many of these oxidized metabolites have biological activity and have been implicated in various diseases including cardiovascular and neurodegenerative diseases, asthma, and cancer3-7. Oxidized bioactive lipids can be formed enzymatically or by reactive oxygen species (ROS). Enzymes that metabolize fatty acids include cyclooxygenase (COX), lipoxygenase (LO), and cytochromes P450 (CYPs)1,8. Enzymatic metabolism results in enantioselective formation whereas ROS oxidation results in the racemic formation of products.
While this protocol focuses primarily on the analysis of AA- and some LA-derived bioactive metabolites; it could be easily applied to metabolites of other fatty acids. Bioactive lipids are extracted from cell lysate or media using liquid-liquid (l-l) extraction. At the beginning of the l-l extraction process, stable isotope internal standards are added to account for errors during sample preparation. Stable isotope dilution (SID) also accounts for any differences, such as ion suppression, that metabolites may experience during the mass spectrometry (MS) analysis9. After the extraction, derivatization with an electron capture (EC) reagent, pentafluorylbenzyl bromide (PFB) is employed to increase detection sensitivity10,11. Multiple reaction monitoring (MRM) is used to increase the selectivity of the MS analysis. Before MS analysis, lipids are separated using chiral normal phase high performance liquid chromatography (HPLC). The HPLC conditions are optimized to separate the enantiomers and various stereoisomers of the monitored lipids12. This specific LC-MS method monitors prostaglandins (PGs), isoprostanes (isoPs), hydroxyeicosatetraenoic acids (HETEs), hydroxyoctadecadienoic acids (HODEs), oxoeicosatetraenoic acids (oxoETEs) and oxooctadecadienoic acids (oxoODEs); however, the HPLC and MS parameters can be optimized to include any fatty acid metabolites13.
Most of the currently available bioanalytical methods do not take into account the separate quantification of enantiomers. This is extremely important when trying to deduce whether or not the metabolites were formed enzymatically or by ROS. Additionally, the ratios of the enantiomers may provide evidence for a specific enzymatic pathway of formation. The use of SID allows for accurate quantification of metabolites and accounts for any sample loss during preparation as well as the differences experienced during ionization. Using the PFB electron capture reagent increases the sensitivity of detection by two orders of magnitude over conventional APCI methods. Overall, this method, SID-LC-EC-atmospheric pressure chemical ionization APCI-MRM/MS, is one of the most sensitive, selective, and accurate methods of quantification for bioactive lipids.

This protocol will demonstrate the extraction and analysis of free and esterified bioactive fatty acids from cells. Fatty acids are accurately quantified using stable isotope dilution, chiral liquid chromatography, electron capture atmospheric chemical ionization multiple reaction monitoring mass spectrometry (SID-LC-ECAPCI-MRM/MS).

Extracción de los lípidos celulares orientadas por dilución de isótopos estables análisis de espectrometría de cromatografía de líquidos-Mass

The metabolism of fatty acids, such as arachidonic acid (AA) and linoleic acid (LA), results in the formation of oxidized bioactive lipids, including ...

Investigación

Bioingeniería

Environmentally-controlled Microtensile Testing of Mechanically-adaptive Polymer Nanocomposites for ex vivo Characterization

Implantable microdevices are gaining significant attention for several biomedical applications1-4. Such devices have been made from a range of materials, each offering its own advantages and shortcomings5,6. Most prominently, due to the microscale device dimensions, a high modulus is required to facilitate implantation into living tissue. Conversely, the stiffness of the device should match the surrounding tissue to minimize induced local strain7-9. Therefore, we recently developed a new class of bio-inspired materials to meet these requirements by responding to environmental stimuli with a change in mechanical properties10-14. Specifically, our poly(vinyl acetate)-based nanocomposite (PVAc-NC) displays a reduction in stiffness when exposed to water and elevated temperatures (e.g. body temperature). Unfortunately, few methods exist to quantify the stiffness of materials in vivo15, and mechanical testing outside of the physiological environment often requires large samples inappropriate for implantation. Further, stimuli-responsive materials may quickly recover their initial stiffness after explantation. Therefore, we have developed a method by which the mechanical properties of implanted microsamples can be measured ex vivo, with simulated physiological conditions maintained using moisture and temperature control13,16,17. 
To this end, a custom microtensile tester was designed to accommodate microscale samples13,17 with widely-varying Young's moduli (range of 10 MPa to 5 GPa). As our interests are in the application of PVAc-NC as a biologically-adaptable neural probe substrate, a tool capable of mechanical characterization of samples at the microscale was necessary. This tool was adapted to provide humidity and temperature control, which minimized sample drying and cooling17. As a result, the mechanical characteristics of the explanted sample closely reflect those of the sample just prior to explantation.
The overall goal of this method is to quantitatively assess the in vivo mechanical properties, specifically the Young's modulus, of stimuli-responsive, mechanically-adaptive polymer-based materials. This is accomplished by first establishing the environmental conditions that will minimize a change in sample mechanical properties after explantation without contributing to a reduction in stiffness independent of that resulting from implantation. Samples are then prepared for implantation, handling, and testing (Figure 1A). Each sample is implanted into the cerebral cortex of rats, which is represented here as an explanted rat brain, for a specified duration (Figure 1B). At this point, the sample is explanted and immediately loaded into the microtensile tester, and then subjected to tensile testing (Figure 1C). Subsequent data analysis provides insight into the mechanical behavior of these innovative materials in the environment of the cerebral cortex.

Environmentally-controlled Microtensile Testing of Mechanically-adaptive Polymer Nanocomposites for ex vivo Characterization

A method is discussed by which the in vivo mechanical behavior of stimuli-responsive materials is monitored as a function of time. Samples are tested ex vivo using a microtensile tester with environmental controls to simulate the physiological environment. This work further promotes understanding the in vivo behavior of our material.

Prueba Microtracción ambientalmente controlado mecánicamente de adaptación nanocompuestos poliméricos para<em&gt; Ex vivo</em&gt; Caracterización

Implantable microdevices are gaining significant attention for several biomedical applications1-4. Such devices have been made from a range of materials, ...

In situ TEM of Biological Assemblies in Liquid

Researchers regularly use Transmission Electron Microscopes (TEMs) to examine biological entities and to assess new materials. Here, we describe an additional application for these instruments- viewing viral assemblies in a liquid environment. This exciting and novel method of visualizing biological structures utilizes a recently developed microfluidic-based specimen holder. Our video article demonstrates how to assemble and use a microfluidic holder to image liquid specimens within a TEM. In particular, we use simian rotavirus double-layered particles (DLPs) as our model system. We also describe steps to coat the surface of the liquid chamber with affinity biofilms that tether DLPs to the viewing window. This permits us to image assemblies in a manner that is suitable for 3D structure determination. Thus, we present a first glimpse of subviral particles in a native liquid environment.

In situ TEM of Biological Assemblies in Liquid

Here we describe a procedure to image viral complexes in liquid at nanometer resolution using a transmission electron microscope.

In situ TEM de Ensamblajes Biológicos en Líquido

Researchers regularly use Transmission Electron Microscopes (TEMs) to examine biological entities and to assess new materials. Here, we describe an ...

Synthesis of Keratin-based Nanofiber for Biomedical Engineering

Electrospinning, due to its versatility and potential for applications in various fields, is being frequently used to fabricate nanofibers. Production of these porous nanofibers is of great interest due to their unique physiochemical properties. Here we elaborate on the fabrication of keratin containing poly (&#949;-caprolactone) (PCL) nanofibers (i.e., PCL/keratin composite fiber). Water soluble keratin was first extracted from human hair and mixed with PCL in different ratios. The blended solution of PCL/keratin was transformed into nanofibrous membranes using a laboratory designed electrospinning set up. Fiber morphology and mechanical properties of the obtained nanofiber were observed and measured using scanning electron microscopy and tensile tester. Furthermore, degradability and chemical properties of the nanofiber were studied by FTIR. SEM images showed uniform surface morphology for PCL/keratin fibers of different compositions. These PCL/keratin fibers also showed excellent mechanical properties such as Young&#39;s modulus and failure point. Fibroblast cells were able to attach and proliferate thus proving good cell viability. Based on the characteristics discussed above, we can strongly argue that the blended nanofibers of natural and synthetic polymers can represent an excellent development of composite materials that can be used for different biomedical applications.

Electrospun nanofibers have a high surface area to weight ratio, excellent mechanical integrity, and support cell growth and proliferation. These nanofibers have a wide range of biomedical applications. Here we fabricate keratin/ PCL nanofibers, using the electrospinning technique, and characterize the fibers for possible applications in tissue engineering.

Síntesis de nanofibras a base de Queratina de Ingeniería Biomédica

Electrospinning, due to its versatility and potential for applications in various fields, is being frequently used to fabricate nanofibers. Production of ...

Alternating Magnetic Field-Responsive Hybrid Gelatin Microgels for Controlled Drug Release

Magnetically-responsive nano/micro-engineered biomaterials that enable a tightly controlled, on-demand drug delivery have been developed as new types of smart soft devices for biomedical applications. Although a number of magnetically-responsive drug delivery systems have demonstrated efficacies through either in vitro proof of concept studies or in vivo preclinical applications, their use in clinical settings is still limited by their insufficient biocompatibility or biodegradability. Additionally, many of the existing platforms rely on sophisticated techniques for their fabrications. We recently demonstrated the fabrication of biodegradable, gelatin-based thermo-responsive microgel by physically entrapping poly(N-isopropylacrylamide-co-acrylamide) chains as a minor component within a three-dimensional gelatin network. In this study, we present a facile method to fabricate a biodegradable drug release platform that enables a magneto-thermally triggered drug release. This was achieved by incorporating superparamagnetic iron oxide nanoparticles and thermo-responsive polymers within gelatin-based colloidal microgels, in conjunction with an alternating magnetic field application system.

We present a facile method to fabricate a biodegradable gelatin-based drug release platform that is magneto-thermally responsive. This was achieved by incorporating superparamagnetic iron oxide nanoparticles and poly(N-isopropylacrylamide-co-acrylamide) within a spherical gelatin micro-network crosslinked by genipin, in conjunction with an alternating magnetic field application system.

Alternando microgeles de gelatina de campo híbrido-Responsive magnéticos para Controlled Drug Release

Magnetically-responsive nano/micro-engineered biomaterials that enable a tightly controlled, on-demand drug delivery have been developed as new types of ...

Preparation of Monodomain Liquid Crystal Elastomers and Liquid Crystal Elastomer Nanocomposites

LCEs are shape-responsive materials with fully reversible shape change and potential applications in medicine, tissue engineering, artificial muscles, and as soft robots. Here, we demonstrate the preparation of shape-responsive liquid crystal elastomers (LCEs) and LCE nanocomposites along with characterization of their shape-responsiveness, mechanical properties, and microstructure. Two types of LCEs &#8212; polysiloxane-based and epoxy-based &#8212; are synthesized, aligned, and characterized. Polysiloxane-based LCEs are prepared through two crosslinking steps, the second under an applied load, resulting in monodomain LCEs. Polysiloxane LCE nanocomposites are prepared through the addition of conductive carbon black nanoparticles, both throughout the bulk of the LCE and to the LCE surface. Epoxy-based LCEs are prepared through a reversible esterification reaction. Epoxy-based LCEs are aligned through the application of a uniaxial load at elevated (160 &#176;C) temperatures. Aligned LCEs and LCE nanocomposites are characterized with respect to reversible strain, mechanical stiffness, and liquid crystal ordering using a combination of imaging, two-dimensional X-ray diffraction measurements, differential scanning calorimetry, and dynamic mechanical analysis. LCEs and LCE nanocomposites can be stimulated with heat and/or electrical potential to controllably generate strains in cell culture media, and we demonstrate the application of LCEs as shape-responsive substrates for cell culture using a custom-made apparatus.

We demonstrate the preparation of siloxane-based and epoxy-based liquid crystal elastomers (LCEs) and LCE nanocomposites. The LCEs are characterized with respect to reversible strain, liquid crystal ordering, and stiffness. As a potential application, we demonstrate their use as shape-responsive substrates in a custom device for active cell culture.

Preparación de monodominio de cristal líquido elastómeros y elastómeros de cristal líquido nanocompuestos

LCEs are shape-responsive materials with fully reversible shape change and potential applications in medicine, tissue engineering, artificial muscles, and ...

Automated Robotic Liquid Handling Assembly of Modular DNA Devices

Recent advances in modular DNA assembly techniques have enabled synthetic biologists to test significantly more of the available &#34;design space&#34; represented by &#34;devices&#34; created as combinations of individual genetic components. However, manual assembly of such large numbers of devices is time-intensive, error-prone, and costly. The increasing sophistication and scale of synthetic biology research necessitates an efficient, reproducible way to accommodate large-scale, complex, and high throughput device construction.
Here, a DNA assembly protocol using the Type-IIS restriction endonuclease based Modular Cloning (MoClo) technique is automated on two liquid-handling robotic platforms. Automated liquid-handling robots require careful, often times tedious optimization of pipetting parameters for liquids of different viscosities (e.g. enzymes, DNA, water, buffers), as well as explicit programming to ensure correct aspiration and dispensing of DNA parts and reagents. This makes manual script writing for complex assemblies just as problematic as manual DNA assembly, and necessitates a software tool that can automate script generation. To this end, we have developed a web-based software tool, http://mocloassembly.com, for generating combinatorial DNA device libraries from basic DNA parts uploaded as Genbank files. We provide access to the tool, and an export file from our liquid handler software which includes optimized liquid classes, labware parameters, and deck layout. All DNA parts used are available through Addgene, and their digital maps can be accessed via the Boston University BDC ICE Registry. Together, these elements provide a foundation for other organizations to automate modular cloning experiments and similar protocols.
The automated DNA assembly workflow presented here enables the repeatable, automated, high-throughput production of DNA devices, and reduces the risk of human error arising from repetitive manual pipetting. Sequencing data show the automated DNA assembly reactions generated from this workflow are ~95% correct and require as little as 4% as much hands-on time, compared to manual reaction preparation.

Here, an automated workflow to perform modular DNA &#34;device&#34; assembly using a modular cloning DNA assembly method on liquid-handling robots is presented. The protocol uses an intuitive software tool for generating liquid handler picklists for combinatorial DNA device library generation, which we demonstrate using two liquid handling platforms.

Automatizado de montaje robotizada manipulación líquido de dispositivos modulares de la DNA

Recent advances in modular DNA assembly techniques have enabled synthetic biologists to test significantly more of the available &#34;design space&#34; ...

Synthesis of Biocompatible Liquid Crystal Elastomer Foams as Cell Scaffolds for 3D Spatial Cell Cultures

Here, we present a step-by-step preparation of a 3D, biodegradable, foam-like cell scaffold. These scaffolds were prepared by cross-linking star block co-polymers featuring cholesterol units as side-chain pendant groups, resulting in smectic-A (SmA) liquid crystal elastomers (LCEs). Foam-like scaffolds, prepared using metal templates, feature interconnected microchannels, making them suitable as 3D cell culture scaffolds. The combined properties of the regular structure of the metal foam and of the elastomer result in a 3D cell scaffold that promotes not only higher cell proliferation compared to conventional porous templated films, but also better management of mass transport (i.e., nutrients, gases, waste, etc.). The nature of the metal template allows for the easy manipulation of foam shapes (i.e., rolls or films) and for the preparation of scaffolds of different pore sizes for different cell studies while preserving the interconnected porous nature of the template. The etching process does not affect the chemistry of the elastomers, preserving their biocompatible and biodegradable nature. We show that these smectic LCEs, when grown for extensive time periods, enable the study of clinically relevant and complex tissue constructs while promoting the growth and proliferation of cells.

This study presents a methodology to prepare 3D, biodegradable, foam-like cell scaffolds based on biocompatible side-chain liquid crystal elastomers (LCEs). Confocal microscopy experiments show that foam-like LCEs allow for cell attachment, proliferation, and the spontaneous alignment of C2C12s myoblasts.

Síntesis de biocompatibles de cristal líquido de elastómero Espumas como andamios celulares para 3D espaciales Cultivos Celulares

Here, we present a step-by-step preparation of a 3D, biodegradable, foam-like cell scaffold. These scaffolds were prepared by cross-linking star block ...

Simultaneous Quantification of Selected Kynurenines Analyzed by Liquid Chromatography-Mass Spectrometry in Medium Collected from Cancer Cell Cultures

The kynurenine pathway and the tryptophan catabolites called kynurenines have received increased attention for their involvement in immune regulation and cancer biology. An in vitro cell culture assay is often used to learn about the contribution of different tryptophan catabolites in a disease mechanism and for testing therapeutic strategies. Cell culture medium that is rich in secreted metabolites and signaling molecules reflects the status of tryptophan metabolism and other cellular events. New protocols for the reliable quantification of multiple kynurenines in the complex cell culture medium are desired to allow for a reliable and quick analysis of multiple samples. This can be accomplished with liquid chromatography coupled with mass spectrometry. This powerful technique is employed in many clinical and research laboratories for the quantification of metabolites and can be used for measuring kynurenines.
Presented here is the use of liquid chromatography coupled with single quadrupole mass spectrometry (LC-SQ) for the simultaneous determination of four kynurenines, i.e., kynurenine, 3-hydroxykynurenine, 3-hydroxyanthranilic, and xanthurenic acid in the medium collected from in vitro cultured cancer cells. SQ detector is simple to use and less expensive compared to other mass spectrometers. In the SQ-MS analysis, multiple ions from the sample are generated and separated according to their specific mass-to-charge ratio (m/z), followed by the detection using a Single Ion Monitoring (SIM) mode.
This paper draws the attention on the advantages of the reported method and indicates some weak points. It is focused on critical elements of LC-SQ analysis including sample preparation along with chromatography and mass spectrometry analysis. The quality control, method calibration conditions and matrix effect issues are also discussed. We described a simple application of 3-nitrotyrosine as one analog standard for all target analytes. As confirmed by experiments with human ovary and breast cancer cells, the proposed LC-SQ method&#160;generates reliable results and can be further applied to other in vitro cellular models.

Described here is a protocol for the determination of four different tryptophan metabolites generated in the kynurenine pathway (kynurenine, 3-hydroxykynurenine, xanthurenic acid, 3-hydroxyanthranilic acid) in the medium collected from cancer cell cultures analyzed by liquid chromatography coupled with a single quadrupole mass spectrometry.

Cuantificación simultánea de kynureninas seleccionadas analizadas por cromatografía líquida-espectrometría de masas en medio recogido de cultivos celulares oncológicos

The kynurenine pathway and the tryptophan catabolites called kynurenines have received increased attention for their involvement in immune regulation and ...

Scalable Generation of Mature Cerebellar Organoids from Human Pluripotent Stem Cells and Characterization by Immunostaining

The cerebellum plays a critical role in the maintenance of balance and motor coordination, and a functional defect in different cerebellar neurons can trigger cerebellar dysfunction. Most of the current knowledge about disease-related neuronal phenotypes is based on postmortem tissues, which makes understanding of disease progression and development difficult. Animal models and immortalized cell lines have also been used as models for neurodegenerative disorders. However, they do not fully recapitulate human disease. Human induced pluripotent stem cells (iPSCs) have great potential for disease modeling and provide a valuable source for regenerative approaches. In recent years, the generation of cerebral organoids from patient-derived iPSCs improved the prospects for neurodegenerative disease modeling. However, protocols that produce large numbers of organoids and a high yield of mature neurons in 3D culture systems are lacking. The protocol presented is a new approach for reproducible and scalable generation of human iPSC-derived organoids under chemically-defined conditions using scalable single-use bioreactors, in which organoids acquire cerebellar identity. The generated organoids are characterized by the expression of specific markers at both mRNA and protein level. The analysis of specific groups of proteins allows the detection of different cerebellar cell populations, whose localization is important for the evaluation of organoid structure. Organoid cryosectioning and further immunostaining of organoid slices are used to evaluate the presence of specific cerebellar cell populations and their spatial organization.

This protocol describes a dynamic culture system to produce controlled size aggregates of human pluripotent stem cells and further stimulate differentiation in cerebellar organoids under chemically-defined and feeder-free conditions using a single-use bioreactor.

Generación escalable de organoides cerebelosos maduros a partir de células madre pluripotentes humanas y caracterización por inmunosuización

The cerebellum plays a critical role in the maintenance of balance and motor coordination, and a functional defect in different cerebellar neurons can ...