Este trabajo fue financiado por el NIH (U54CA143837), los del MD Anderson Cancer Center de Subvenciones de apoyo de los NIH (CA016672), la Fundación V (SAC), y una beca de investigación sin restricciones de la Fundación de Investigación Kanzius (SAC, Erie, PA). Agradecemos a Kristine Ceniza del Departamento de Oncología Quirúrgica, Centro de Cáncer MD Anderson, por la asistencia administrativa.

Una visión general de ensayo completo se representa en la Figura 1. Más detalles se representan en los pasos 1-3 a continuación. 1. Evaluar Calefacción RF de NP Coloides: AuNPs como ejemplo <ol><li> En general, para que se investigó cada muestra NP, primero Lavar la muestra varias veces a través de un filtro de centrifugación con agua desionizada (DI) para eliminar los iones y contaminantes de fondo. Todos los iones y contaminantes se han eliminado de la suspensión AuNP cuando el líquido que se lavan a cabo tiene velocidades de calentamiento de RF similares (HR) como agua DI. Este proceso de purificación también permite mayores concentraciones de PN a ser obtenidos. Vale la pena señalar que, aunque el uso de AuNPs en este ejemplo, los principios fundamentales se pueden aplicar a otros materiales NP. </li><li> Como un ejemplo, purificar una botella de 500 ml de AuNPs disponibles comercialmente de 5 nm de diámetro y, a continuación someterlos a un campo de RF de 13,56 MHz de eléctrica-Field fuerza 90 kV / m. <ol><li> Tome ~ 125 ml de la solución madre AuNP y dividido entre seis 50 kDa tubos de filtración centrífuga. Se centrifuga a 3.000 rpm. durante 2 min 5 seg. Borrar filtros de amortiguamiento y recarga filtrados con más solución de reserva. Repita hasta que se ha filtrado a los 500 ml. </li><li> Reemplazar el tampón se filtró con un volumen similar de agua DI y repetir aproximadamente 8 veces (o hasta que los HRs RF tampón filtrados son equivalentes a agua DI). Nota, análisis UV-Vis también se puede utilizar para controlar los picos de absorción de contaminantes. Una vez que los contaminantes de amortiguamiento se han eliminado totalmente, pipeta aproximadamente 0,5 ml de agua DI en cada filtro y resuspender las AuNPs por pipeteo repetido. Esto debería eliminar por completo los AuNPs del filtro y permitir la completa resuspensión. Combine todos los seis suspensiones en un tubo de 15 ml Eppendorf. </li><li> Una vez que los AuNPs se han purificado y concentrado, analizar la muestra mediante ICP-OES y / o ICP-MS, UV-vis y el potencial Zeta de datos sobre concentratioN y NP estabilidad, respectivamente. SEM y / o análisis de TEM también se puede utilizar para obtener datos morfológicos. La preparación detallada de la muestra para estas técnicas se puede encontrar en la literatura 4. </li></ol></li><li> Usando el sistema de Kanzius de RF descrito en estudios previos 8, o derivaciones de este sistema, colocar un 1,3 ml cilíndrica cubeta de cuarzo de modo que el campo eléctrico de RF en el aire (sin muestra presente) sería ~ 90 kV / m en el interior de la cubeta. Para una muestra estándar de solución salina (0,9% NaCl)-el campo eléctrico se reduciría a ~ 1,1 kV / m. Estas son las condiciones aproximadas utilizados para permitir que se hagan comparaciones entre los diferentes sistemas. <ol><li> Pipetear 1,3 ml de una muestra de 1.000 mg / l de coloide AUNP purificada en la cubeta de cuarzo y introducir este en el campo de RF. Esto se puede hacer mediante el uso de un soporte de muestras de teflón a la medida. Exponer a la muestra de campo de RF para un período de 120 segundos o hasta que la muestra alcanza 70 ° C para evitar la formación de arco eléctrico o de ebullición rápida. Californiapture los datos de imagen térmica (así como las áreas de control) usando una cámara IR y el software asociado. Repita este procedimiento en tres ocasiones. </li><li> Se filtra la muestra a través de otro filtro de centrífuga de 50 kDa a extraer los AuNPs de la memoria intermedia de agua DI. Volver a exponer el búfer para el campo de RF, de nuevo tres veces. La diferencia en jonrones entre el coloide AuNP y el búfer de fondo de agua DI determina la HR por los propios AuNPs. Esperar a obtener jonrones de ~ 0.3 ° C / seg y 0,05 º C / s para dar un HR depende AuNP de ~ 0,25 ° C / seg. Resuspender las AuNPs hayan quedado en el filtro en 1,3 ml de agua para los experimentos in vitro / in vivo. </li></ol></li></ol> 2. La hipertermia inducida RF-Nanopartículas asistida: Estudios in vitro <ol><li> Estos estudios in vitro se pueden aplicar a cualquier tipo de cáncer tipo de células que forman monocapas 2D. En este experimento utilizar carcinoma hepatocelular humano derivado células Hep 3B. <ol><li> Placa ~ 50.000 cells en los tres delanteros pocillos de una placa de 12 pocillos con 1 ml de medio de crecimiento. Repita este 6 veces (usar tres placas para estudios de NP y tres placas como controles). Incubar a 37,5 ° C durante 24 horas antes de la introducción de los PN. Esterilizar NPs primero usando Bioseguridad Gabinete de exposición de luz UV durante 5 min. </li><li> En cada pocillo introducir 0,1 ml de una solución de 1,000 mg / L AuNP y dejar actuar durante otras 24 horas. Añadir 0,1 ml de agua en cada pocillo de las tres placas de células de control y también dejar durante 24 horas. </li><li> Después de haber pasado 24 horas, aspirar el medio celular y lavar con PBS para eliminar cualquier AuNPs unidos a la superficie. Sustituya el medio celular. Las células están listas para la exposición RF. </li></ol></li><li> Coloque cada paquete celular de 12 pocillos dentro del campo de RF. Espere hasta que las células se hayan enfriado a 31 ° C. Encienda el generador de RF y exponer de 3,5 min. La temperatura final de los medios de comunicación celular será ~ 37 ° C. Apague el campo de RF. Retire las células y colocarlas en una incubadora durante 24 horas antes del análisis. <ol> &lt;li&gt; Eliminar las células de la incubadora y medio celular aspirado. Añadir 1,6 ml de medio de células a cada pocillo así en 0,4 ml de reactivo MTT. Se incuban las células durante 4 horas. Medios aspirar y renovar con 2 ml de dimetilsulfóxido (DMSO). Coloca las placas de células sobre un rockero banco y dejar actuar durante 10 minutos para permitir que el DMSO para solubilizar los reactivos MTT. Por último, la pipeta 100 l de cada pocillo en una placa de 96 pocillos y ópticamente leer el pozo a 570 nm usando un lector de placas tales como el lector de placas SPECTROstar Nano. </li></ol></li></ol> 3. La hipertermia inducida RF-nanopartículas asistida: estudios in vivo <ol><li> Estos estudios in vivo se pueden aplicar a cualquier tipo de cáncer que se forma tumores sólidos en un modelo murino ortotópico o ectópico. Este experimento utiliza células de cáncer de hígado Hep 3B en un BALB-C modelo de ratón desnudo del tumor ectópico. </li><li> Nota: todos los experimentos in vivo se ejecutan en cumplimiento de todas las directrices, reglamentos y las agencias reguladoras. Además, el protocolo que se está demostrado se llevó a cabo bajo la dirección y aprobación de la Universidad de Texas MD Anderson Cancer Centros de Cuidado de Animales y el empleo Comisión Institucional (IACUC). <ol><li> Crecer un número apropiado de células (~ 100 K) en un matraz de cultivo de tejidos con el medio de crecimiento apropiado. Incubar en una incubadora a 37 ° C con 5% de CO 2 durante la duración del cultivo celular. </li><li> Tratar las células con tripsina (para separar del matraz) y producir una solución de 2 millones de células por cada 25 l. Añadir una cantidad igual de Matrigel (en hielo) y mezclar a fondo para preparar la solución de la inyección final. Inyectar esta solución en la posición deseada en el lomo del ratón y esperar una cantidad adecuada de tiempo para que los tumores crecen hasta el tamaño deseado (2-4 semanas para la mayoría de las células). Antes de la exposición a RF, BALB-C ratones desnudos deben tener tumores ectópicos sólidos 0,5-1 cm de diámetro. </li></ol></li><li><ol><li> Preparar a los ratones a utilizar (en este caso BALB-C ratones Desnudos) por anaesthetizing ellos con una solución de ketamina y xilazina por inyección IP. Mientras que los ratones están cayendo dormido, mantenerlos en una cámara de temperatura controlada a 37 º C. Se necesitarán 20 ratones en total, todos los tumores tamaño similar rodamientos. Diez ratones se utilizarán en conjunción con y sin AuNPs (este último es sólo inyecciones de PBS), mientras que los 10 ratones restantes se dividirán entre los controles de RF-expuesta sin exposición de RF y: todos sin inyecciones AUNP. </li><li> Una vez adecuadamente anestesiados, inyectar los AuNPs directamente en el tumor usando una jeringa de 1 cc con una aguja de calibre 27. La solución AUNP debe ser a una concentración de Au de 200 mg / L en 0,1 ml de PBS. Después de la inyección, utilice un hisopo de cirugía para absorber la sangre y limpiar el lugar de inyección con un algodón con alcohol. </li><li> A continuación, monte el ratón para ser tratado en la cabeza de recepción del generador de RF. El ratón debe colocarse de manera que el tumor es más cercano a la cabeza de la transmisión. Proteja las áreas que no deben ser tratados, así como la sensibilidadtiva áreas tales como los ojos, los oídos y dedos de los pies, con la cinta de cobre. Asegúrese de que la cinta de cobre está en contacto con el avión a tierra apropiadamente de modo que no se produzca la acumulación de carga. Además, el área de exposición tiene que tener un espacio de al menos 1 cm mayor que el tamaño del sitio de tratamiento deseado. </li><li> Cámara térmica Posición IR para que el tumor y el área de tratamiento son visibles. Encienda RF durante 5 min. Registre la curva de temperatura resultante. Si la terapia alcanza temperaturas superiores a 42 ° C de tratamiento para inmediatamente. </li></ol></li><li><ol><li> Después de la exposición a RF de los ratones a recuperarse de la anestesia en una cámara caliente hasta que estén conscientes. </li><li> Repita el experimento con los mismos ratones 48 horas más tarde (pasos 3.3.1 - 3.4.1). </li><li> Después del experimento, la eutanasia a los ratones de acuerdo con los protocolos y procedimientos institucionales. Anotar el peso del tumor. Para el análisis histológico fijan los tumores utilizando formol e incrustarlos en parafina. Secciones tumorales suelen ser manchadas ingenioh hematoxilina y eosina y metas pertinentes para medir la eficacia terapéutica, por ejemplo, Ki-67, exfoliados caspasa-3, etc. </li></ol></li></ol>

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	<li>Gannon, C. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Carbon+nanotube-enhanced+thermal+destruction+of+cancer+cells+in+a+noninvasive+radiofrequency+field.">Carbon nanotube-enhanced thermal destruction of cancer cells in a noninvasive radiofrequency field.</a> Cancer. 110, 2654 (2007).</li><li>Curley, S. A., Cherukuri, P., Briggs, K., Patra, C. R., Upton, M., Dolson, E., Mukherjee, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noninvasive+radiofrequency+field-induced+hyperthermic+cytotoxicity+in+human+cancer+cells+using+cetuximab-targeted+gold+nanoparticles.">Noninvasive radiofrequency field-induced hyperthermic cytotoxicity in human cancer cells using cetuximab-targeted gold nanoparticles.</a> J. Exp. Ther. Oncol. 7, 313 (2008).</li><li>Gannon, C. J., Patra, C. R., Bhattacharya, R., Mukherjee, P., Curley, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracellular+gold+nanoparticles+enhance+non-invasive+radiofrequency+thermal+destruction+of+human+gastrointestinal+cancer+cells.">Intracellular gold nanoparticles enhance non-invasive radiofrequency thermal destruction of human gastrointestinal cancer cells.</a> Journal of Nanobiotechnology. 6, 2 (2008).</li><li>Raoof, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stability+of+antibody-conjugated+gold+nanoparticles+in+the+endolysosomal+nanoenvironment:+implications+for+noninvasive+radiofrequency-based+cancer+therapy.">Stability of antibody-conjugated gold nanoparticles in the endolysosomal nanoenvironment: implications for noninvasive radiofrequency-based cancer therapy.</a> Nanomedicine. 8, 1096 (2012).</li><li>Glazer, E. S., Massey, K. L., Zhu, C., Curley, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pancreatic+carcinoma+cells+are+susceptible+to+noninvasive+radio+frequency+fields+after+treatment+with+targeted+gold+nanoparticles.">Pancreatic carcinoma cells are susceptible to noninvasive radio frequency fields after treatment with targeted gold nanoparticles.</a> Surgery. 148, 319 (2010).</li><li>Glazer, E. S., Curley, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Radiofrequency+field-induced+thermal+cytotoxicity+in+cancer+cells+treated+with+fluorescent+nanoparticles.">Radiofrequency field-induced thermal cytotoxicity in cancer cells treated with fluorescent nanoparticles.</a> Cancer. 116, 3285 (2010).</li><li>Glazer, E. S., Curley, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-invasive+radiofrequency+ablation+of+malignancies+mediated+by+quantum+dots,+gold+nanoparticles+and+carbon+nanotubes.">Non-invasive radiofrequency ablation of malignancies mediated by quantum dots, gold nanoparticles and carbon nanotubes.</a> Therapeutic Delivery. 2, 1325 (2011).</li><li>Corr, S. J., Raoof, M., Mackeyev, Y., Phounsavath, S., Cheney, M. A., Cisneros, B. T., Shur, M., Gozin, M., McNally, P. J., Wilson, L. J., Curley, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Citrate-Capped+Gold+Nanoparticle+Electrophoretic+Heat+Production+in+Response+to+a+Time-Varying+Radiofrequency+Electric-Field.">Citrate-Capped Gold Nanoparticle Electrophoretic Heat Production in Response to a Time-Varying Radiofrequency Electric-Field.</a> J. Phys. Chem. C. 116, 24380 (2012).</li><li>Kruse, D. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Radio-Frequency+Coupling+Network+for+Heating+of+Citrate-Coated+Gold+Nanoparticles+for+Cancer+Therapy:+Design+and+Analysis.">A Radio-Frequency Coupling Network for Heating of Citrate-Coated Gold Nanoparticles for Cancer Therapy: Design and Analysis.</a> IEEE Transactions on Biomedical Engineering. 58, 10 (2011).</li><li>Li, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Negligible+absorption+of+radiofrequency+radiation+by+colloidal+gold+nanoparticles.">Negligible absorption of radiofrequency radiation by colloidal gold nanoparticles.</a> J. Colloid Interf. Sci. 358, 47 (2011).</li><li>Liu, X., Chen, H. J., Chen, X., Parini, C., Wen, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low+frequency+heating+of+gold+nanoparticle+dispersions+for+non-invasive+thermal+therapies.">Low frequency heating of gold nanoparticle dispersions for non-invasive thermal therapies.</a> Nanoscale. , (2012).</li><li>Sassaroli, E., Li, K. C. P., O'Neill, B. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Radio+frequency+absorption+in+gold+nanoparticle+suspensions:+a+phenomenological+study.">Radio frequency absorption in gold nanoparticle suspensions: a phenomenological study.</a> J. Phys. D App. Phys. 45, 075303 (2012).</li><li>Worle-Knirsch, J. M., Pulskamp, K., Krug, H. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oops+they+did+it+again!+Carbon+nanotubes+hoax+scientists+in+viability+assays.">Oops they did it again! Carbon nanotubes hoax scientists in viability assays.</a> Nano Lett. 6, 1261 (2006).</li></ol>

No tenemos nada que revelar.

Estos protocolos permiten que el experimentador para analizar completamente la medida en que los nanomateriales (en este caso AuNPs) puede aumentar la hipertermia inducida por RF para el tratamiento del cáncer. El primer protocolo se ocupa específicamente de analizar la producción de calor a partir de muestras AUNP altamente concentrados y purificados. Aunque otros grupos han informado de la producción de calor principalmente a partir de los tampones que los AuNPs se suspenden en y no los propios 9-11 AuN...

La absorción de energía de RF por el tejido biológico (debido a su permitividad eléctrica inherente) da lugar a temperaturas elevadas de tejido como una función del tiempo, lo que finalmente conduce a la muerte celular por hipertermia. Se planteó la hipótesis de que la hipertermia cáncer se puede optimizar mediante el uso de nanomateriales específicas que internalizan dentro de la célula del cáncer y actúan como transductores de RF-térmicas, dejando las células sanas, normales vecinas intacta. Varios informes han demostrado ya que una variedad de NPS puede actuar como fuentes de calor RF eficaz que ayuda en la necrosis cáncer de 1-4. En estos aspectos, NP de oro (AuNPs) 3-5, los nanotubos de carbono 1, y los puntos cuánticos 6, 7 han mostrado características interesantes cuando se utiliza en tanto in vitro como in vivo en experimentos de RF. Aunque todavía se está debatiendo la naturaleza exacta del mecanismo de calentamiento de estos PN cuando se expone a un campo de RF, una serie deexperimentos fundamentales utilizando AuNPs ha dado gran importancia tanto en el tamaño de NP y estados de agregación. Se demostró que sólo AuNPs con diámetros &lt;10 nm se calentarán cuando se expone a un campo de RF 8. Además, este mecanismo de calentamiento se atenúa significativamente cuando se agregan los AuNPs. Esta condición agregación también fue validado dentro de los modelos in vitro que dio importancia a la optimización AuNP estabilidad coloidal dentro de los compartimentos intracelulares endolysomal para la terapia de RF eficaz 4. Sin embargo, las técnicas y los principios experimentales utilizados para recoger y evaluar estos datos pueden ser problemáticos, especialmente en el caso de la validación de perfiles de calor de RF de coloides NP. Varios informes han demostrado que el calentamiento Joule del fondo suspensión iónica que los PN se suspenden en puede ser la fuente principal de la producción de calor de RF y no los propios 9-12 PN. Aunque nuestro reciente documento de 8 ha validado tl uso de las interacciones de RF en la generación de calor de AuNPs de diámetro inferior a 10 nm, nuestro objetivo es describir estos protocolos con más detalle en este artículo. También demuestran los protocolos y técnicas necesarios para evaluar la eficacia de AuNPs como agentes térmicos de hipertermia en tanto in vitro como en experimentos in vivo para modelos de cáncer de hígado. Aunque nos centramos principalmente en coloides simples de AuNPs citrato-capsulado, las mismas técnicas se pueden aplicar a otros híbridos AUNP tales como anticuerpos y complejos de quimioterapia-conjugado. Al adherirse a estos principios el experimentalista con suerte debe ser capaz de evaluar rápidamente el potencial para cualquier nanomaterial para ser un agente de hipertermia térmica inducida por RF eficaz.

<table border="1">
	<tbody>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Reagent/Material</td>
		</tr>
		<tr>
			<td>500 ml gold nanoparticles (5 nm)</td>
			<td>Ted Pella, INC</td>
			<td>15702-5</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Amicon Ultra-4/-15 Centrifugal Filter Units (50 kDa)</td>
			<td>Millipore</td>
			<td>UFC805024/UFC910096</td>
			<td>(4 ml and 15 ml volumes)</td>
		</tr>
		<tr>
			<td>MEM X1 Cell Culture Media</td>
			<td>Cellgro</td>
			<td>10-101-CV</td>
			<td>(add extra nutrients as necessary)</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Sigma</td>
			<td>F4135-500 ml</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Copper Tape</td>
			<td>Ted Pella</td>
			<td>16072</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>Kanzius RF System (13.56 MHZ)</td>
			<td>ThermMed, LLC, Inc. (Erie, PA, USA)</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>IR Camera</td>
			<td>FLIR SC 6000, FLIR Systems, Inc. (Boston, MA, USA)</td>
			<td>Contact FLIR</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>1.3 ml Quartz Cuvette</td>
			<td>ThermMed, LLC, Inc. (Erie, PA, USA)</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Teflon Sample holder with Rotary Stage</td>
			<td>ThermMed, LLC, Inc. (Erie, PA, USA)</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>SPECTROstar Nano Microplate reader</td>
			<td>BGM Labtech</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>UV-Vis spectrometer</td>
			<td>Applied Nanofluorescence, Houston, TX)</td>
			<td>NS1 NanoSpectralyzer</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>ICP-OES</td>
			<td>PerkinElmer</td>
			<td>Optima 4300 DV</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Zetasizer</td>
			<td>Malvern</td>
			<td>Zen 3600 Zetasizer</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

protocols for assessing radiofrequency interactions with gold nanoparticles and biological systems for non-invasive hyperthermia cancer therapy

Terapias de cáncer que son menos tóxicos y invasiva que sus contrapartes existentes son altamente deseables. El uso de la RF-campos eléctricos que penetran profundamente en el cuerpo, provocando una toxicidad mínima, en la actualidad se está estudiando como un medio viable para la terapia del cáncer no invasivo. Se prevé que las interacciones de la energía de RF con nanopartículas internalizados (NPS) pueden liberar calor que puede causar un sobrecalentamiento (hipertermia) de la célula, en última instancia, que termina en la necrosis celular. En el caso de los sistemas no biológicos, se presentan los protocolos detallados en relación con cuantificación del calor liberado por los coloides NP altamente concentradas. Para los sistemas biológicos, en el caso de los experimentos in vitro, se describen las técnicas y las condiciones que deben cumplirse a fin de exponer con eficacia las células cancerosas a la energía de RF sin artefactos de calentamiento del material a granel que oscurecen considerablemente los datos. Por último, damos una metodología detallada fo in vivo en modelos de ratones con tumores de cáncer hepático ectópicos.

1. Evaluación de calentamiento por RF de coloides NP: AuNPs como un ejemplo. Después de seguir el apartado 1.1 - 1.2.3 esperar tener una solución altamente concentrada, estable y purificada de 5 nm y 10 nm de diámetro AuNPs. De los 500 ml AS-comprado solución madre, esperar para obtener al menos 4 ml de solución a una concentración de 1000 mg / L. La diferencia en los CR entre los AuNPs y la solución tampón de agua DI fondo a esta concentración debe ser ~ 0,25 ° C / s y 0,1 º C / s ...

Watch this Scientific Journal Video about Protocols for Assessing Radiofrequency Interactions with Gold Nanoparticles and Biological Systems for Non-invasive Hyperthermia Cancer Therapy at JoVE.com

Protocols for Assessing Radiofrequency Interactions with Gold Nanoparticles and Biological Systems for Non-invasive Hyperthermia Cancer Therapy | Medicine | Video

Protocols for Assessing Radiofrequency Interactions with Gold Nanoparticles and Biological Systems for Non-invasive Hyperthermia Cancer Therapy

Se describen los protocolos que se utilizan para investigar las interacciones de 13.56 MHz de radiofrecuencia (RF) de electricidad-los campos con coloides de nanopartículas de oro en ambos sistemas no biológicos y biológicos (in vitro / in vivo). Estas interacciones están siendo investigados para su aplicación en la terapia del cáncer.

Protocolos para la Evaluación de radiofrecuencia Interacciones con nanopartículas de oro y Sistemas Biológicos para la terapia del cáncer de hipertermia no invasiva

Cancer therapies  which are less toxic and invasive than their existing counterparts are highly  desirable. The use of RF electric-fields that penetrate ...

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10.7K vistas.  University of Texas M.D. Anderson Cancer Center.  Se describen los protocolos que se utilizan para investigar las interacciones de 13.56 MHz de radiofrecuencia (RF) de electricidad-los campos con coloides de nanopartículas de oro en ambos sistemas no biológicos y biológicos (in vitro / in vivo). Estas interacciones están siendo investigados para su aplicación en la terapia del cáncer. 

Video: Protocols for Assessing Radiofrequency Interactions with Gold Nanoparticles and Biological Systems for Non-invasive Hyperthermia Cancer Therapy

Staining Protocols for Human Pancreatic Islets

Estimates of islet area and numbers and endocrine cell composition in the adult human pancreas vary from several hundred thousand to several million and beta mass ranges from 500 to 1500 mg 1-3. With this known heterogeneity, a standard processing and staining procedure was developed so that pancreatic regions were clearly defined and islets characterized using rigorous histopathology and immunolocalization examinations. 
Standardized procedures for processing human pancreas recovered from organ donors are described in part 1 of this series. The pancreas is processed into 3 main regions (head, body, tail) followed by transverse sections. Transverse sections from the pancreas head are further divided, as indicated based on size, and numbered alphabetically to denote subsections. This standardization allows for a complete cross sectional analysis of the head region including the uncinate region which contains islets composed primarily of pancreatic polypeptide cells to the tail region.
The current report comprises part 2 of this series and describes the procedures used for serial sectioning and histopathological characterization of the pancreatic paraffin sections with an emphasis on islet endocrine cells, replication, and T-cell infiltrates. Pathology of pancreatic sections is intended to characterize both exocrine, ductular, and endocrine components. The exocrine compartment is evaluated for the presence of pancreatitis (active or chronic), atrophy, fibrosis, and fat, as well as the duct system, particularly in relationship to the presence of pancreatic intraductal neoplasia4. Islets are evaluated for morphology, size, and density, endocrine cells, inflammation, fibrosis, amyloid, and the presence of replicating or apoptotic cells using H&amp;E and IHC stains. 
The final component described in part 2 is the provision of the stained slides as digitized whole slide images. The digitized slides are organized by case and pancreas region in an online pathology database creating a virtual biobank. Access to this online collection is currently provided to over 200 clinicians and scientists involved in type 1 diabetes research. The online database provides a means for rapid and complete data sharing and for investigators to select blocks for paraffin or frozen serial sections.

This video demonstrates procedures for characterization of human pancreatic islets using hematoxylin and eosin (H&E) and immunohistochemistry (IHC). Pancreatic sections from head, body, and tail regions are stained by both H&amp;E and IHC to determine islet endocrine composition (insulin, glucagon, and pancreatic polypeptide), cell replication (Ki67), and inflammatory infiltrates (H&E, CD3). The uncinate region is localized using IHC for pancreatic polypeptide.

Protocolos de tinción de islotes pancreáticos humanos

Estimates of islet area and numbers and endocrine cell composition in the adult human pancreas vary from several hundred thousand to several million and ...

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Medicina

Cerenkov Luminescence Imaging (CLI) for Cancer Therapy Monitoring

In molecular imaging, positron emission tomography (PET) and optical imaging (OI) are two of the most important and thus most widely used modalities1-3. PET is characterized by its excellent sensitivity and quantification ability while OI is notable for non-radiation, relative low cost, short scanning time, high throughput, and wide availability to basic researchers. However, both modalities have their shortcomings as well. PET suffers from poor spatial resolution and high cost, while OI is mostly limited to preclinical applications because of its limited tissue penetration along with prominent scattering optical signals through the thickness of living tissues.
 Recently a bridge between PET and OI has emerged with the discovery of Cerenkov Luminescence Imaging (CLI)4-6. CLI is a new imaging modality that harnesses Cerenkov Radiation (CR) to image radionuclides with OI instruments. Russian Nobel laureate Alekseyevich Cerenkov and his colleagues originally discovered CR in 1934. It is a form of electromagnetic radiation emitted when a charged particle travels at a superluminal speed in a dielectric medium7,8. The charged particle, whether positron or electron, perturbs the electromagnetic field of the medium by displacing the electrons in its atoms. After passing of the disruption photons are emitted as the displaced electrons return to the ground state. For instance, one 18F decay was estimated to produce an average of 3 photons in water5. 
 Since its emergence, CLI has been investigated for its use in a variety of preclinical applications including in vivo tumor imaging, reporter gene imaging, radiotracer development, multimodality imaging, among others4,5,9,10,11. The most important reason why CLI has enjoyed much success so far is that this new technology takes advantage of the low cost and wide availability of OI to image radionuclides, which used to be imaged only by more expensive and less available nuclear imaging modalities such as PET.
 Here, we present the method of using CLI to monitor cancer drug therapy. Our group has recently investigated this new application and validated its feasibility by a proof-of-concept study12. We demonstrated that CLI and PET exhibited excellent correlations across different tumor xenografts and imaging probes. This is consistent with the overarching principle of CR that CLI essentially visualizes the same radionuclides as PET. We selected Bevacizumab (Avastin; Genentech/Roche) as our therapeutic agent because it is a well-known angiogenesis inhibitor13,14. Maturation of this technology in the near future can be envisioned to have a significant impact on preclinical drug development, screening, as well as therapy monitoring of patients receiving treatments.

Cerenkov Luminescence Imaging CLI for Cancer Therapy Monitoring

Use of Cerenkov Luminescence Imaging (CLI) for monitoring preclinical cancer treatment is described here. This method takes advantage of Cerenkov Radiation (CR) and optical imaging (OI) to visualize radiolabeled probes and thus provides an alternative to PET in preclinical therapeutic monitoring and drug screening.

Cerenkov Luminiscencia Imaging (CLI) para la Vigilancia del Cáncer Terapia

In  molecular imaging, positron emission tomography (PET) and optical imaging (OI)  are two of the most important and thus most widely used modalities1-3. ...

Non-invasive Optical Measurement of Cerebral Metabolism and Hemodynamics in Infants

Perinatal brain injury remains a significant cause of infant mortality and morbidity, but there is not yet an effective bedside tool that can accurately screen for brain injury, monitor injury evolution, or assess response to therapy. The energy used by neurons is derived largely from tissue oxidative metabolism, and neural hyperactivity and cell death are reflected by corresponding changes in cerebral oxygen metabolism (CMRO2). Thus, measures of CMRO2 are reflective of neuronal viability and provide critical diagnostic information, making CMRO2 an ideal target for bedside measurement of brain health.
Brain-imaging techniques such as positron emission tomography (PET) and single-photon emission computed tomography (SPECT) yield measures of cerebral glucose and oxygen metabolism, but these techniques require the administration of radionucleotides, so they are used in only the most acute cases.
Continuous-wave near-infrared spectroscopy (CWNIRS) provides non-invasive and non-ionizing radiation measures of hemoglobin oxygen saturation (SO2) as a surrogate for cerebral oxygen consumption. However, SO2 is less than ideal as a surrogate for cerebral oxygen metabolism as it is influenced by both oxygen delivery and consumption. Furthermore, measurements of SO2 are not sensitive enough to detect brain injury hours after the insult 1,2, because oxygen consumption and delivery reach equilibrium after acute transients 3. We investigated the possibility of using more sophisticated NIRS optical methods to quantify cerebral oxygen metabolism at the bedside in healthy and brain-injured newborns. More specifically, we combined the frequency-domain NIRS (FDNIRS) measure of SO2 with the diffuse correlation spectroscopy (DCS) measure of blood flow index (CBFi) to yield an index of CMRO2 (CMRO2i) 4,5.
With the combined FDNIRS/DCS system we are able to quantify cerebral metabolism and hemodynamics. This represents an improvement over CWNIRS for detecting brain health, brain development, and response to therapy in neonates. Moreover, this method adheres to all neonatal intensive care unit (NICU) policies on infection control and institutional policies on laser safety. Future work will seek to integrate the two instruments to reduce acquisition time at the bedside and to implement real-time feedback on data quality to reduce the rate of data rejection.

We combined frequency-domain near-infrared spectroscopy measures of cerebral hemoglobin oxygenation with diffuse correlation spectroscopy measures of cerebral blood flow index to estimate an index of oxygen metabolism. We tested the utility of this measure as a bedside screening tool to evaluate the health and development of the newborn brain.

No invasiva de medición óptica del metabolismo cerebral en lactantes y Hemodinámica

Perinatal brain injury remains a significant cause of infant mortality  and morbidity, but there is not yet an effective bedside tool that can  accurately ...

Non-invasive Assessment of Microvascular and Endothelial Function

The authors have utilized capillaroscopy and forearm blood flow techniques to investigate the role of microvascular dysfunction in pathogenesis of cardiovascular disease. Capillaroscopy is a non-invasive, relatively inexpensive methodology for directly visualizing the microcirculation. Percent capillary recruitment is assessed by dividing the increase in capillary density induced by postocclusive reactive hyperemia (postocclusive reactive hyperemia capillary density minus baseline capillary density), by the maximal capillary density (observed during passive venous occlusion). Percent perfused capillaries represents the proportion of all capillaries present that are perfused (functionally active), and is calculated by dividing postocclusive reactive hyperemia capillary density by the maximal capillary density. Both percent capillary recruitment and percent perfused capillaries reflect the number of functional capillaries. The forearm blood flow (FBF) technique provides accepted non-invasive measures of endothelial function: The ratio FBFmax/FBFbase is computed as an estimate of vasodilation, by dividing the mean of the four FBFmax values by the mean of the four FBFbase values. Forearm vascular resistance at maximal vasodilation (FVRmax) is calculated as the mean arterial pressure (MAP) divided by FBFmax. Both the capillaroscopy and forearm techniques are readily acceptable to patients and can be learned quickly.
The microvascular and endothelial function measures obtained using the methodologies described in this paper may have future utility in clinical patient cardiovascular risk-reduction strategies. As we have published reports demonstrating that microvascular and endothelial dysfunction are found in initial stages of hypertension including prehypertension, microvascular and endothelial function measures may eventually aid in early identification, risk-stratification and prevention of end-stage vascular pathology, with its potentially fatal consequences.

Capillaroscopy is a non-invasive, relatively inexpensive methodology for directly visualizing the microcirculation. The forearm blood flow technique provides accepted non-invasive measures of endothelial function.

La evaluación no invasiva de la función endotelial microvascular y

The authors have utilized capillaroscopy and forearm blood flow techniques to investigate the role of microvascular dysfunction in pathogenesis of ...

Transplantation into the Anterior Chamber of the Eye for Longitudinal, Non-invasive In vivo Imaging with Single-cell Resolution in Real-time

Intravital imaging has emerged as an indispensable tool in biological research. In the process, many imaging techniques have been developed to study different biological processes in animals non-invasively. However, a major technical limitation in existing intravital imaging modalities is the inability to combine non-invasive, longitudinal imaging with single-cell resolution capabilities. We show here how transplantation into the anterior chamber of the eye circumvents such significant limitation offering a versatile experimental platform that enables non-invasive, longitudinal imaging with cellular resolution in vivo. We demonstrate the transplantation procedure in the mouse and provide representative results using a model with clinical relevance, namely pancreatic islet transplantation. In addition to enabling direct visualization in a variety of tissues transplanted into the anterior chamber of the eye, this approach provides a platform to screen drugs by performing long-term follow up and monitoring in target tissues. Because of its versatility, tissue/cell transplantation into the anterior chamber of the eye not only benefits transplantation therapies, it extends to other in vivo applications to study physiological and pathophysiological processes such as signal transduction and cancer or autoimmune disease development.

Transplantation into the Anterior Chamber of the Eye for Longitudinal, Non-invasive In vivo Imaging with Single-cell Resolution in Real-time

A new approach combining intraocular transplantation and confocal microscopy enables longitudinal, non-invasive real-time imaging with single-cell resolution within grafted tissues in vivo. We demonstrate how to transplant pancreatic islets into the anterior chamber of the mouse eye.

Trasplante en la cámara anterior del ojo para Longitudinal, no invasiva<em&gt; En vivo</em&gt; Imagen con una sola célula de resolución en tiempo real

Intravital imaging has emerged as an indispensable tool in biological research.  In the process, many imaging techniques have been developed to study ...

Non-invasive Imaging and Analysis of Cerebral Ischemia in Living Rats Using Positron Emission Tomography with 18F-FDG

Stroke is the third leading cause of death among Americans 65 years of age or older1. The quality of life for patients who suffer from a stroke fails to return to normal in a large majority of patients2, which is mainly due to current lack of clinical treatment for acute stroke. This necessitates understanding the physiological effects of cerebral ischemia on brain tissue over time and is a major area of active research. Towards this end, experimental progress has been made using rats as a preclinical model for stroke, particularly, using non-invasive methods such as 18F-fluorodeoxyglucose (FDG) coupled with Positron Emission Tomography (PET) imaging3,10,17. Here we present a strategy for inducing cerebral ischemia in rats by middle cerebral artery occlusion (MCAO) that mimics focal cerebral ischemia in humans, and imaging its effects over 24 hr using FDG-PET coupled with X-ray computed tomography (CT) with an Albira PET-CT instrument. A VOI template atlas was subsequently fused to the cerebral rat data to enable a unbiased analysis of the brain and its sub-regions4. In addition, a method for 3D visualization of the FDG-PET-CT time course is presented. In summary, we present a detailed protocol for initiating, quantifying, and visualizing an induced ischemic stroke event in a living Sprague-Dawley rat in three dimensions using FDG-PET.

Non-invasive Imaging and Analysis of Cerebral Ischemia in Living Rats Using Positron Emission Tomography with 18F-FDG

Brain damage resulting from cerebral ischemia may be non-invasively imaged and studied in rats using pre-clinical positron emission tomography coupled with the injectable radioactive probe, 18F-fluorodeoxyglucose. Further, the use of modern software tools that include volume of interest (VOI) brain templates dramatically increase the quantitative information gleaned from these studies.

De imagen no invasivas y Análisis de la isquemia cerebral en ratas vivas utilizando Tomografía por emisión de positrones con<sup&gt; 18</sup&gt; F-FDG

Stroke is the third leading cause of death among Americans 65 years of age or older1. The quality of life for patients who suffer from a stroke fails to ...

Murine Model for Non-invasive Imaging to Detect and Monitor Ovarian Cancer Recurrence

Epithelial ovarian cancer is the most lethal gynecologic malignancy in the United States. Although patients initially respond to the current standard of care consisting of surgical debulking and combination chemotherapy consisting of platinum and taxane compounds, almost 90% of patients recur within a few years. In these patients the development of chemoresistant disease limits the efficacy of currently available chemotherapy agents and therefore contributes to the high mortality. To discover novel therapy options that can target recurrent disease, appropriate animal models that closely mimic the clinical profile of patients with recurrent ovarian cancer are required. The challenge in monitoring intra-peritoneal (i.p.) disease limits the use of i.p. models and thus most xenografts are established subcutaneously. We have developed a sensitive optical imaging platform that allows the detection and anatomical location of i.p. tumor mass. The platform includes the use of optical reporters that extend from the visible light range to near infrared, which in combination with 2-dimensional X-ray co-registration can provide anatomical location of molecular signals. Detection is significantly improved by the use of a rotation system that drives the animal to multiple angular positions for 360 degree imaging, allowing the identification of tumors that are not visible in single orientation. This platform provides a unique model to non-invasively monitor tumor growth and evaluate the efficacy of new therapies for the prevention or treatment of recurrent ovarian cancer.

We describe a non-invasive animal imaging platform that allows the detection, quantification, and monitoring of ovarian cancer growth and recurrence. This intra-peritoneal xenograft model mimics the clinical profile of patients with ovarian cancer.

Modelo murino de imagen no invasivas para detectar y vigilar cáncer ovárico de Recurrencia

Epithelial ovarian cancer is the most lethal gynecologic malignancy in the United States. Although patients initially respond to the current standard of ...

Non-invasive Assessment of the Efficacy of New Therapeutics for Intestinal Pathologies Using Serial Endoscopic Imaging of Live Mice

Animal models of inflammatory bowel disease (IBD) and colorectal cancer (CRC) have provided significant insight into the cell intrinsic and extrinsic mechanisms that contribute to the onset and progression of intestinal diseases. The identification of new molecules that promote these pathologies has led to a flurry of activity focused on the development of potential new therapies to inhibit their function. As a result, various pre-clinical mouse models with an intact immune system and stromal microenvironment are now heavily used. Here we describe three experimental protocols to test the efficacy of new therapeutics in pre-clinical models of (1) acute mucosal damage, (2) chronic colitis and/or colitis-associated colon cancer, and (3) sporadic colorectal cancer. We also outline procedures for serial endoscopic examination that can be used to document the therapeutic response of an individual tumor and to monitor the health of individual mice. These protocols provide complementary experimental platforms to test the effectiveness of therapeutic compounds shown to be well tolerated by mice.

We describe methods for longitudinal monitoring of the efficacy of therapeutics for the treatment of colonic pathologies in mice using a rigid endoscope. This protocol can be readily used for the characterization of the therapeutic response of an individual tumor in live mice and also for monitoring potential disease relapse.

Evaluación no invasiva de la eficacia de nuevas terapias para Intestinal Patologías recurriendo a imágenes de Endoscópica serie de ratones vivos

Animal models of inflammatory bowel disease (IBD) and colorectal cancer (CRC) have provided significant insight into the cell intrinsic and extrinsic ...

Non-invasive Assessment of Dorsiflexor Muscle Function in Mice

Assessment of skeletal muscle contractile function is an important measurement for both clinical and research purposes. Numerous conditions can negatively affect skeletal muscle. This can result in a loss of muscle mass (atrophy) and/or loss of muscle quality (reduced force per unit of muscle mass), both of which are prevalent in chronic disease, muscle-specific disease, immobilization, and aging (sarcopenia). Skeletal muscle function in animals can be evaluated by a range of different tests. All tests have limitations related to the physiological testing environment, and the selection of a specific test often depends on the nature of the experiments. Here, we describe an in vivo, non-invasive technique involving a helpful and easy assessment of force frequency-curve (FFC) in mice that can be performed on the same animal over time. This permits monitoring of disease progression and/or efficacy&#160;of a potential therapeutic treatment.

Measurement of rodent skeletal muscle contractile function is a useful tool that can be used to track disease progression as well as efficacy of therapeutic intervention. We describe here the non-invasive, in vivo assessment of the dorsiflexor muscles that can be repeated over time in the same mouse.

Evaluación no invasiva de la función muscular de los dorsiflexores en ratones

Assessment of skeletal muscle contractile function is an important measurement for both clinical and research purposes. Numerous conditions can negatively ...

Oxygenation-sensitive Cardiac MRI with Vasoactive Breathing Maneuvers for the Non-invasive Assessment of Coronary Microvascular Dysfunction

Oxygenation-sensitive cardiac magnetic resonance imaging (OS-CMR) is a diagnostic technique that uses the inherent paramagnetic properties of deoxyhemoglobin as an endogenous source of tissue contrast. Used in combination with standardized vasoactive breathing maneuvers (hyperventilation and apnea) as a potent non-pharmacologic vasomotor stimulus, OS-CMR can monitor changes in myocardial oxygenation. Quantifying such changes during the cardiac cycle and throughout vasoactive maneuvers can provide markers for coronary macro- and microvascular function and thereby circumvent the need for any extrinsic, intravenous contrast or pharmacologic stress agents.
OS-CMR uses the well-known sensitivity of T2*-weighted images to blood oxygenation. Oxygenation-sensitive images can be acquired on any cardiac MRI scanner using a modified standard clinical steady-state free precession (SSFP) cine sequence, making this technique vendor-agnostic and easily implemented. As a vasoactive breathing maneuver, we apply a 4-min breathing protocol of 120 s of free breathing, 60 s of paced hyperventilation, followed by an expiratory breath-hold of at least 30 s. The regional and global response of myocardial tissue oxygenation to this maneuver can be assessed by tracking the signal intensity change. The change over the initial 30 s of the post-hyperventilation breath-hold, referred to as the breathing-induced myocardial oxygenation reserve (B-MORE) has been studied in healthy people and various pathologies. A detailed protocol for performing oxygen-sensitive CMR scans with vasoactive maneuvers is provided.
As demonstrated in patients with microvascular dysfunction in yet incompletely understood conditions, such as inducible ischemia with no obstructive coronary artery stenosis (INOCA), heart failure with preserved ejection fraction (HFpEF), or microvascular dysfunction after heart transplantation, this approach provides unique, clinically important, and complementary information on coronary vascular function.

The assessment of microvascular function by oxygenation-sensitive cardiac magnetic resonance imaging in combination with vasoactive breathing maneuvers is unique in its ability to assess rapid dynamic changes in myocardial oxygenation in vivo and, thus, may serve as a critically important diagnostic technique for coronary vascular function.

Resonancia magnética cardíaca sensible a la oxigenación con maniobras de respiración vasoactiva para la evaluación no invasiva de la disfunción microvascular coronaria