The authors acknowledge financial support from Houston Methodist Research Institute, the National Natural Science Foundation of China (Nos., 21231007 and 21121061), the Ministry of Education of China (Nos., 20100171110013 and 313058), the National Basic Research Program of China (973 Program No. 2014CB845604), and the Fundamental Research Funds for the Central Universities.

1. PCPS preparación de partículas <ol><li> Oxidar partículas de silicio poroso no funcionalizado en una solución al 30% de peróxido de hidrógeno a 95 ° C durante 2 hr. Aminar las partículas oxidadas en 2% (3- aminopropil) trietoxisilano solución en alcohol isopropílico durante 2 días a 65 ° C con agitación suave. </li><li> Centrifugar la solución durante 30 min a 18.800 xg y se lavan las partículas dos veces en alcohol isopropílico y tres veces en etanol, usando sonicación breve a suspender el sedimento. Deja las partículas en solución de etanol al realizar el paso 1.3 y 1.4. </li><li> Añadir un volumen conocido de suspensión de partículas (por ejemplo, 10 l) a 10 ml de diluyente isotone y contar las partículas con un analizador de recuento de partículas para determinar la concentración de partículas en la solución madre. </li><li> Activar el grupo ácido de L-arginina (0,1 nmol) con N - (3-dimetilaminopropil) - etilcarbodiimida hidrocloruro de N &#39;(EDC, 0,1 nmol) / N </em&gt; Hidroxisuccinimida (NHS, 0,1 nmol) en 20 ml de etanol durante 2 horas a RT con agitación suave. </li><li> Tratar brevemente con ultrasonidos la solución de partículas de stock y añadir 1 mil millones de partículas a la solución de L-arginina y dejar la reacción durante 18 horas a RT con agitación suave. </li><li> Activar el primer grupo de ácido aspártico de N - (terc-butoxicarbonil) -L-aspártico (Boc-Asp-OH, 1 nmol) con EDC (0,1 nmol) / NHS (0,1 nmol) en 20 ml de etanol durante 4 horas a 4 ° C con agitación suave. </li><li> Disolver 50 mg de polietilenimina en 10 ml de etanol y añadir la solución a la mezcla de Boc-Asp-OH. Deje que la reacción proceder durante 24 horas a temperatura ambiente con agitación suave. </li><li> Activar el segundo grupo de ácido aspártico de la solución de Boc-Asp-OH / PEI con EDC (0,1 nmol) / NHS (0,1 nmol) a 4 ° C durante 6 horas con agitación suave. </li><li> Para obtener partículas PCPS, añadir la solución de partículas desde el paso 1.5 en la solución de Boc-Asp-OH / PEI desde el paso 1.8. Permitir que la reacción continúe durante 18 horas a TA con suave st<html> irring. </li><li> Centrifugar la solución durante 30 min a 18.800 xg y se lava la solución de partículas tres veces con etanol, usando sonicación breve a suspender el sedimento. </li></ol> 2. PCPS Caracterización de partículas <ol><li> Medir el tamaño de las partículas utilizando un microscopio electrónico de barrido (SEM). <ol><li> Coloque una gota de suspensión de partículas (10.000 partículas / l en etanol) en una sílice limpia SEM muestra talón y dejar secar a temperatura ambiente al vacío. </li><li> Mida imágenes SEM a las 8 kV con una distancia de trabajo de 3-5 mm utilizando un detector en el objetivo. </li></ol></li><li> Medir el potencial zeta de las partículas utilizando un sistema analizador de partículas. <ol><li> Mezclar 10 l de suspensión de partículas (10.000 partículas / l en etanol) con 1 ml de 10 mM de tampón de fosfato (pH 7,4). </li><li> Cargar la muestra en células capilares plegadas y medir el potencial zeta de acuerdo con las instrucciones del fabricante. </li></ol></li></ol>le &quot;&gt; 3. Carga de siRNA en PCPS Partículas <ol><li> Secar las partículas PCPS (de partículas PCPS etapa de preparación 1.9) a vacío O / N. </li><li> Añadir siRNA (4 g) en agua libre de nucleasa (20 l) para las partículas PCPS secos y tratar brevemente con ultrasonidos. Utilice la siguiente partícula de relaciones siRNA: 2 × 10 5 partículas / 0,2 mg siRNA, 4 × 10 5 partículas / 0,2 mg siRNA, 6 × 10 5 partículas / 0,2 mg siRNA, 8 × 10 5 partículas / 0,2 mg siRNA, 10 × 10 5 partículas / 0,2 mg siRNA y 12 × 10 5 partículas / 0,2 mg siRNA. </li><li> Incubar durante 3 horas a 4 ° C en un agitador (1000 rpm) para permitir siRNA de unión a las partículas. </li></ol> 4. Optimización de la relación de partículas siRNA / PCPS <ol><li> Agregar colorante de carga de ADN a 20 l de las partículas siRNA PCPS / control con diferentes partículas a las relaciones de siRNA (ver carga de siRNA en partículas PCPS). </li><li> Cargue el samples en un gel de agarosa al 2% que contenía tinción en gel de ADN. </li><li> Realizar la electroforesis a un voltaje constante de 120 V durante 20 min en tampón. </li><li> Analizar el gel con adquisición de imágenes y el software de análisis. </li></ol> 5. Liberación de siRNA de PCPS Partículas <ol><li> Mezclar 20 l de las partículas siRNA PCPS / control con diferentes partículas a las relaciones de siRNA (ver paso 3) en dodecilsulfato de sodio (SDS, 2%) y dejar reposar durante 1 hora a temperatura ambiente. </li><li> Añadir colorante de carga de ADN de las muestras. </li><li> Cargar las muestras en un gel de agarosa al 2% que contenía la mancha gel de ADN. </li><li> Realizar la electroforesis a un voltaje constante de 120 V durante 20 min en tampón de electroforesis de ADN usando un equipo y una fuente de alimentación. </li><li> Analizar el gel con adquisición de imágenes y el software de análisis. </li></ol> 6. Microscopía Confocal de PCPS Partículas <ol><li> Añadir 5 l de partículas siRNA PCPS / fluorescentes de control (10 × 10 5 partículas / 0.2 g siRNA / 20 l) a una hoja de cubierta de vidrio. </li><li> Visualizar capas de partículas por microscopía confocal. </li></ol> 7. Caracterización de Arg-PEI / control de siRNA nanopartículas <ol><li> Para degradar el material de silicio y formar Arg-PEI nanopartículas / siRNA control, añadir partículas PCPS / siRNA (10 × 10 6 partículas PCPS / 2 g siRNA) a 100 l de solución salina tamponada con fosfato y agitar (1000 rpm) a 37 ° C durante 2 días. </li><li> Centrifugar la muestra durante 30 min a 18.800 xg y se recoge el sobrenadante. </li><li> Medir el tamaño de las nanopartículas Arg-PEI / siRNA formada con dispersión de luz dinámica (DLS). <ol><li> Mezclar 10 l del sobrenadante con 1 ml 10 mM de tampón de fosfato (pH 7,4) y ponerlo en una cubeta de plástico. </li><li> Medir el tamaño de las partículas utilizando un sistema analizador de partículas de acuerdo con las instrucciones del fabricante. </li></ol></li><li> Determinar el tamaño y la morfología de la formada Arg / PEI-siRNAnanopartículas con microscopía de fuerza atómica. <ol><li> Tomar 10 l del sobrenadante y el lugar en una oblea de silicio. </li><li> Visualice las partículas con microscopía de fuerza atómica (AFM). </li></ol></li></ol> 8. Cultivo Celular <ol><li> Cultura MDA-MB-231 células de cáncer de mama humano en medio de cultivo celular suplementado con suero bovino fetal al 10% y 1% de penicilina-estreptomicina a 5% de CO 2, 95% de humedad y 37 ° C. </li></ol> 9. Microscopía confocal de células vivas con PCPS Partículas <ol><li> Células de la placa en la cultura 2-así se desliza a una densidad de siembra de 3 x 10 5 células / pocillo durante 24 hr. </li><li> Añadir partículas PCPS cargados de control de siRNA fluorescente (10 × 10 5 partículas / 0,2 mg de partículas de relación siRNA, 50 nM siRNA) a las células. </li><li> Grabar una película de las células con un microscopio confocal (suministrado con una cámara, 5% de CO2, 95% de humedad y 37 ° C) durante 12 horas después de la expo de partículasSeguro. </li></ol> 10. Microscopía confocal de células fijadas con PCPS Partículas <ol><li> Células de la placa en la cultura 2-así se desliza a una densidad de siembra de 3 x 10 5 células / pocillo durante 24 hr. </li><li> Añadir partículas PCPS cargados de control de siRNA fluorescente (10 × 10 5 partículas / 0,2 mg de partículas de relación siRNA, 50 nM siRNA) a las células y se incuba durante 1 día, 7 días y 10 días. </li><li> Lavar las células dos veces con tampón fosfato salino y luego fijar con solución de paraformaldehído al 4% durante 10 min. </li><li> Lavar las células con solución salina tamponada con fosfato. </li><li> Permeabilizar las células con 0,1% de octil fenol etoxilado durante 10 minutos y luego lavar tres veces con tampón fosfato salino. </li><li> Bloquear las células con albúmina de suero bovino (10 mg / ml) en salina tamponada con fosfato durante 10 min a TA con agitación suave. </li><li> Para visualizar actina filamentosa, incubar las células con forma fluorescente phalloidin etiquetado (1 mu l / 40 lsolución de bloqueo) durante 20 min a TA con agitación suave y después lavar en tampón fosfato salino. </li><li> Retire las diapositivas de la trama y añadir antifade reactivo con 4 &#39;, 6-diamidino-2-fenilindol (DAPI) para visualizar el núcleo. </li><li> Añadir una cubierta de vidrio en la parte superior y tomar imágenes de las células con microscopía confocal. </li></ol> 11. citometría de flujo de células con PCPS / fluorescentes control siRNA Partículas <ol><li> Células de la placa en una placa de 6 pocillos a una densidad de siembra de 3 x 10 5 células / pocillo durante 24 horas. </li><li> Añadir partículas PCPS cargados con siRNA de control fluorescente (10 × 10 5 partículas / 0,2 g de partículas de relación de siRNA, 50 nM siRNA) a las células y se incuba durante 24 hr. </li><li> Lavar las células con solución salina tamponada con fosfato y raspar con un raspador de células. </li><li> Almacenar las células en solución salina tamponada con fosfato con suero bovino fetal al 2% antes del análisis. Use las células no tratadas como control negativo. </li><li> Realizar cy flujotometry. </li></ol> 12. La viabilidad celular de células con PCPS Partículas y PCPS / control de siRNA Partículas <ol><li> Células de la placa en una placa de 96 pocillos a una densidad celular de 3 × 10 3 células / pocillo durante 24 horas. </li><li> Tratar las células con partículas PCPS (1,5 × 10 5 / pocillo y 6 × 10 5 / pocillo) o partículas siRNA PCPS / control (10 × 10 5 partículas / 0,2 mg de partículas de relación siRNA, 10 nM y 100 nM siRNA) para 48 hr y 72 hr. Use las células no tratadas y las células tratadas con tampón fosfato salino (mismo volumen que las partículas añadidas) como controles. Analice cada muestra por triplicado. </li><li> Realizar un ensayo de proliferación celular de acuerdo con las instrucciones del fabricante. </li><li> Representar datos como la media ± desviación estándar. </li></ol> 13. Western Blot de células con PCPS / ATM mutados siRNA Partículas <ol><li> Células de la placa en una placa de 6 pocillos a una densidad celular de 2 × 10 5 células / well durante 24 horas. </li><li> Se incuban las células con siRNA PCPS / control (50 nM) partículas o PCPS / ATM siRNA (50 nM) partículas de 72 h. Use las células no tratadas como control. </li><li> Lisar las células usando un reactivo de extracción de proteínas complementado con un cóctel inhibidor de la proteasa. </li><li> Centrifugar los lisados ​​de células durante 10 min a 14.000 xg y recuperar el sobrenadante. </li><li> Determinar la concentración de proteína con un ensayo de cuantificación de proteínas de acuerdo con las instrucciones del fabricante. </li><li> Añadir tampón de carga de muestra (con 5 l 2-mercaptoetanol / ml de tampón) a las muestras y calentarlos durante 6 min a 99 ° C. </li><li> Cargar las muestras de proteínas (20 g / l) en un gel de SDS-poliacrilamida al 12% en tampón y realizar la electroforesis en gel de poliacrilamida (1 h, 120 V) utilizando un equipo de electroforesis y una fuente de alimentación. </li><li> Transferir el gel en tampón de transferencia (con 20% de metanol) a una membrana de nitrocelulosa (1 h, 100 V) utilizando un equipo de electroforesis yuna fuente de alimentación. </li><li> Bloquear la membrana con leche en polvo al 5% durante 1 hora. </li><li> Incubar la membrana con el anticuerpo primario ATM (de conejo) en solución de bloqueo (del paso 13.9) a una dilución 1: 1.000 O / N. </li><li> Se lava la membrana con solución salina tamponada de fosfato que contiene monolaurato de sorbitán polietilenglicol 0,1% y luego se incuba con el anticuerpo secundario (anti-conejo) en solución de bloqueo (del paso 13.9) a una dilución 1: 2500 durante 1 hora. </li><li> Se lava la membrana con solución salina tamponada de fosfato que contiene monolaurato de sorbitán polietilenglicol 0,1% y detectar las bandas de proteína con reactivo de detección de transferencia Western utilizando adquisición de imágenes y el software de análisis. </li><li> Para el control de carga, lavar la membrana y repita los pasos 13,9-13,12 usando un anticuerpo primario-β actina (de ratón, dilución 1: 10.000) y un anticuerpo secundario (anti-ratón 1: 4000 dilución). </li></ol>

<ol>
	<li>De Fougerolles, A., Vornlocher, H. P., Maraganore, J., Lieberman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interfering+with+disease:+a+progress+report+on+siRNA-based+therapeutics.">Interfering with disease: a progress report on siRNA-based therapeutics.</a> Nat Rev Drug Discov. 6 (6), 443-453 (2007).</li><li>Rolland, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+medicines:+the+end+of+the+beginning.">Gene medicines: the end of the beginning.</a> Adv Drug Deliv Rev. 57 (5), 669-673 (2005).</li><li>Oh, Y. K., Park, T. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=siRNA+delivery+systems+for+cancer+treatment.">siRNA delivery systems for cancer treatment.</a> Adv Drug Deliv Rev. 61 (10), 850-862 (2009).</li><li>Mintzer, M. A., Simanek, E. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonviral+vectors+for+gene+delivery.">Nonviral vectors for gene delivery.</a> Chem Rev. 109 (2), 259-302 (2009).</li><li>Whitehead, K. A., Langer, R., Anderson, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Knocking+down+barriers:+advances+in+siRNA+delivery.">Knocking down barriers: advances in siRNA delivery.</a> Nat Rev Drug Discov. 8 (2), 129-138 (2009).</li><li>Decuzzi, P., 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=Size+and+shape+effects+in+the+biodistribution+of+intravascularly+injected+particles.">Size and shape effects in the biodistribution of intravascularly injected particles.</a> J Control Release. 141 (3), 320-327 (2010).</li><li>Decuzzi, P., Ferrari, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+maps+for+nanoparticles+targeting+the+diseased+microvasculature.">Design maps for nanoparticles targeting the diseased microvasculature.</a> Biomaterials. 29 (3), 377-384 (2008).</li><li>Martinez, J. O., Evangelopoulos, M., Chiappini, C., Liu, X., Ferrari, M., Tasciotti, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Degradation+and+biocompatibility+of+multistage+nanovectors+in+physiological+systems.">Degradation and biocompatibility of multistage nanovectors in physiological systems.</a> J Biomed Mater Res A. 102 (10), 3540-3549 (2014).</li><li>Shen, H., 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=Enhancing+chemotherapy+response+with+sustained+EphA2+silencing+using+multistage+vector+delivery.">Enhancing chemotherapy response with sustained EphA2 silencing using multistage vector delivery.</a> Clin Cancer Res. 19 (7), 1806-1815 (2013).</li><li>Xu, R., 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=Multistage+vectored+siRNA+targeting+ataxia-telangiectasia+mutated+for+breast+cancer+therapy.">Multistage vectored siRNA targeting ataxia-telangiectasia mutated for breast cancer therapy.</a> Small. 9 (9-10), 1799-1808 .</li><li>Shen, 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=High+capacity+nanoporous+silicon+carrier+for+systemic+delivery+of+gene+silencing+therapeutics.">High capacity nanoporous silicon carrier for systemic delivery of gene silencing therapeutics.</a> ACS Nano. 7 (11), 9867-9880 (2013).</li><li>Zhang, 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=Polycation-functionalized+nanoporous+silicon+particles+for+gene+silencing+on+breast+cancer+cells.">Polycation-functionalized nanoporous silicon particles for gene silencing on breast cancer cells.</a> Biomaterials. 35 (1), 423-431 (2014).</li><li>Ozpolat, B., Sood, A. K., Lopez-Berestein, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanomedicine+based+approaches+for+the+delivery+of+siRNA+in+cancer.">Nanomedicine based approaches for the delivery of siRNA in cancer.</a> J Intern Med. 267 (1), 44-53 (2010).</li><li>Philipp, A., Dehshahri, A., Wagner, A., E, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simple+modifications+of+branched+PEI+lead+to+highly+efficient+siRNA+carriers+with+low+toxicity.">Simple modifications of branched PEI lead to highly efficient siRNA carriers with low toxicity.</a> Bioconjug Chem. 19 (7), 1448-1455 (2008).</li><li>Hunter, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+hurdles+in+polyfectin+design+and+mechanistic+background+to+polycation+induced+cytotoxicity.">Molecular hurdles in polyfectin design and mechanistic background to polycation induced cytotoxicity.</a> Adv Drug Deliv Rev. 58 (14), 1523-1531 (2006).</li><li>Andrews, P. M., Bates, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dose-dependent+movement+of+cationic+molecules+across+the+glomerular+wall.">Dose-dependent movement of cationic molecules across the glomerular wall.</a> Anat Rec. 212 (3), 223-231 (1985).</li><li>Ananta, J. S., 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=Geometrical+confinement+of+gadolinium-based+contrast+agents+in+nanoporous+particles+enhances+T1+contrast.">Geometrical confinement of gadolinium-based contrast agents in nanoporous particles enhances T1 contrast.</a> Nat Nanotechnol. 5 (11), 815-821 (2010).</li></ol>

The authors declare that they have no competing financial interests.

Este protocolo describe un método para la entrega exitosa y transfección de ARNsi en las células. En particular, la entrega de siRNA se logra mediante el uso de una plataforma multifuncional que consiste de partículas pSi policatión-funcionalizado. El uso de la terapia de ARNip tiene un gran potencial, por ejemplo, el tratamiento del cáncer, ya que varios oncogenes pueden ser dirigidos con alta especificidad. Por lo tanto, existe una demanda para desarrollar vehículos de administración de ARNsi, que pue...

Los pequeños RNAs de interferencia (siRNAs) son moléculas de ARN de doble cadena que pueden suprimir la expresión de genes. En los últimos años, siRNAs se han desarrollado como una nueva generación de biofármacos que muestran potencial terapéutico para uso futuro en aplicaciones clínicas 1-5. Sin embargo, la implementación exitosa de la terapia siRNA sigue siendo un reto considerable, debido a la degradación por nucleasas, mala absorción intracelular, la baja eficiencia de transfección y liberación ineficiente desde el endosoma / lisosoma 5. Muchos de estos obstáculos pueden ser superados por el desarrollo de plataformas de distribución, que puede entregar de forma segura y eficiente siRNA al tejido enfermo. En comparación con los portadores virales, las plataformas no virales proporcionan varias ventajas, como la seguridad, bajo costo y facilidad de adaptación. En particular, las nanopartículas catiónicos, tales como polímeros y lípidos, han demostrado su utilidad para la entrega siRNA 3. Anteriormente, hemos desarrollado un dr discoidalug sistema de entrega, denominado el vector de múltiples etapas (MSV). Esta plataforma se basa en etapas secuenciales, en los que un vehículo se libera de otro. La primera etapa del vehículo es una micropartícula biodegradable hecha de silicio poroso (psi), mientras que los segundos vehículos etapa son nanopartículas cargadas con fármacos o agentes de contraste 6,7. Las nanopartículas, que están incrustados en el material de la ISP, se liberan gradualmente a medida que se degrada el Si 8. Una ventaja de usar partículas de Si es que las características de morfología y de la superficie pueden adaptarse fácilmente para lograr la biodistribución óptima y liberación del fármaco. Recientemente, el uso exitoso de la plataforma MSV para la entrega de liposomas siRNA a tejido tumoral se muestra en un modelo de ratón de ovario y cáncer de mama 9, 10. En este trabajo, hemos fabricado un sistema de entrega universal para siRNA basado en los principios de la plataforma MSV. La eficacia de este sistema de entrega previamente nos ha demostradoing diferentes siRNA moléculas 11. El sistema es un portador de silicio poroso policatión-funcionalizado (PCPS), que consiste de la ISP injertado con polietilenimina (PEI) y arginina (Arg). PEI puede ayudar en la formación de interacciones electrostáticas con siRNA, mientras que Arg y la ISP pueden servir para reducir la toxicidad de PEI, como anteriormente demonstarted 11. Además, la presencia de PEI puede ayudar en la absorción intracelular y el escape endosomal, mientras que las micropartículas pSi permiten la protección siRNA y de liberación sostenida. Las partículas pSi degradan gradualmente en condiciones fisiológicas, lo que resulta en la formación de nanopartículas Arg-PEI / siRNA (Figura 1), que tienen una morfología distinta y una distribución de tamaño estrecha 11. Para obtener más información con respecto a la estabilidad del sistema PCPS / siRNA, por favor referirse al estudio de Shen et al. 11. Esta plataforma PCPS difiere del MSV convencional, ya que las nanopartículas segunda etapa no se prese inicialmentent en el portador, pero se forman con el tiempo como el portador de la primera etapa se degrada 11, 12. La eficiencia de carga siRNA eficiencia, la citotoxicidad y silenciamiento de los genes del sistema de PCPS se ha evaluado in vitro. La eficacia de transfección se midió usando siRNA contra de la ataxia telangiectasia mutada (ATM) oncogén, que está implicado en la reparación del ADN 10. Anteriormente, la supresión de la ATM se ha demostrado que disminuye el crecimiento del tumor en un modelo de cáncer de mama 10.

<table border="0" cellpadding="0" cellspacing="0" width="1812">
	<tbody>
		<tr height="55">
			<td height="55" style="height:55px;width:605px;">Name of the Material/Equipment</td>
			<td style="width:421px;">Company/Institution</td>
			<td style="width:133px;">Catalog Number</td>
			<td style="width:653px;">Comments/Description</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:605px;">Polyethylenimine (PEI), branched</td>
			<td style="width:421px;">Sigma-Aldrich</td>
			<td style="width:133px;">408727</td>
			<td style="width:653px;">Average molecular weight ~25,000 Da</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:605px;">L-Arginine</td>
			<td style="width:421px;">Sigma-Aldrich</td>
			<td style="width:133px;">A5006</td>
			<td style="width:653px;">Reagent grade, &#8805;98%</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:605px;">Boc-Asp-OH</td>
			<td style="width:421px;">Sigma-Aldrich</td>
			<td style="width:133px;">408-468</td>
			<td style="width:653px;">99%</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:605px;">(3-&#8203;Aminopropyl)&#8203;triethoxysilane</td>
			<td style="width:421px;">Sigma-Aldrich</td>
			<td style="width:133px;">440140</td>
			<td style="width:653px;">99%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:605px;">Hydrogen peroxide solution</td>
			<td style="width:421px;">Sigma-Aldrich</td>
			<td style="width:133px;">216763</td>
			<td style="width:653px;">30&#160;wt. % in H2O</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:605px;">Sulfuric acid</td>
			<td style="width:421px;">Sigma-Aldrich</td>
			<td style="width:133px;">339741</td>
			<td style="width:653px;">100.00%</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:605px;">Isopropyl alcohol</td>
			<td style="width:421px;">Sigma-Aldrich</td>
			<td style="width:133px;">W292907</td>
			<td style="width:653px;">&#8805;99.7%</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:605px;">Ethanol</td>
			<td style="width:421px;">Sigma-Aldrich</td>
			<td style="width:133px;">459844</td>
			<td>&#8805;99.5%&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">N-(3-Dimethylaminopropyl)-N&#8242;-ethylcarbodiimide hydrochloride (EDC)</td>
			<td>Sigma-Aldrich</td>
			<td>03449</td>
			<td>&#8805;99%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Albumin from bovine serum</td>
			<td>Sigma-Aldrich</td>
			<td>A7030-10G</td>
			<td>Blocking agent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:605px;">Ataxia-telangiectasia mutated siRNA&#160;</td>
			<td style="width:421px;">Sigma-Aldrich</td>
			<td style="width:133px;"></td>
			<td style="width:653px;">Designed in-house</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:605px;">Tris Acetate-EDTA buffer</td>
			<td style="width:421px;">Sigma-Aldrich</td>
			<td style="width:133px;">T9650</td>
			<td style="width:653px;">For DNA agarose gel electrophoresis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:605px;">Penicillin-Streptomycin</td>
			<td style="width:421px;">Sigma-Aldrich</td>
			<td>P4333</td>
			<td style="width:653px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:605px;">Fetal Bovine Serum</td>
			<td style="width:421px;">Sigma-Aldrich</td>
			<td>F2442</td>
			<td style="width:653px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:605px;">TWEEN 20</td>
			<td style="width:421px;">Sigma-Aldrich</td>
			<td>P1379</td>
			<td style="width:653px;">Polyethylene glycol sorbitan monolaurate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:605px;">2-Mercaptoethanol</td>
			<td style="width:421px;">Sigma Aldrich</td>
			<td>M6250</td>
			<td style="width:653px;">For Western blot</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:605px;">Sodium dodecyl sulfate</td>
			<td style="width:421px;">Sigma-Aldrich</td>
			<td>L3771</td>
			<td style="width:653px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:605px;">Sodium phosphate monobasic</td>
			<td style="width:421px;">Sigma-Aldrich</td>
			<td>71496</td>
			<td style="width:653px;">For making phosphate buffer&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:605px;">Sodium phosphate dibasic</td>
			<td style="width:421px;">Sigma-Aldrich</td>
			<td>71640</td>
			<td style="width:653px;">For making phosphate buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-Mouse IgG</td>
			<td style="width:421px;">Sigma-Aldrich</td>
			<td style="width:133px;">A4416</td>
			<td style="width:653px;">Secondary antibody (anti-mouse) for Western blot</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:605px;">N-Hydroxysuccinimide (NHS)</td>
			<td style="width:421px;">Sigma-Aldrich</td>
			<td style="width:133px;">130672</td>
			<td style="width:653px;">98%</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:605px;">CELLSTAR 96W Microplate Tissue Culture Treated Clear w/ Lid</td>
			<td style="width:421px;">Greiner Bio-One</td>
			<td style="width:133px;">655182</td>
			<td style="width:653px;">96-well plate</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:605px;">10X Tris-Glycine Liquid</td>
			<td style="width:421px;">Li-Cor&#160;</td>
			<td style="width:133px;">928-40010</td>
			<td style="width:653px;">Transfer buffer for Western blot</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:605px;">Paraformaldehyde solution 4% in PBS&#160;</td>
			<td style="width:421px;">Santa Cruz</td>
			<td>Sc-281692</td>
			<td style="width:653px;">Fixation of cells</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">CellTiter 96 AQueous Non-Radioactive Cell Proliferation Assay (MTS)</td>
			<td style="width:421px;">Promega</td>
			<td style="width:133px;">G5421</td>
			<td style="width:653px;">Proliferation assay</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:605px;">Phosphate buffered saline</td>
			<td style="width:421px;">Fisher Scientific</td>
			<td style="width:133px;">BP399-500</td>
			<td style="width:653px;">10 X Solution</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:605px;">Corning cellgro Dulbecco&#39;s Modification of Eagle&#39;s (Mod.)</td>
			<td style="width:421px;">Fisher Scientific&#160;</td>
			<td style="width:133px;">MT-15-017-CM</td>
			<td style="width:653px;">Cell culture media, 1X solution</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:605px;">Triton X-100</td>
			<td style="width:421px;">Fisher Scientific</td>
			<td>AC21568-2500</td>
			<td style="width:653px;">Octyl phenol ethoxylate, permeabilization agent</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:605px;">Cover glass</td>
			<td style="width:421px;">Fisher Scientific</td>
			<td>12-530C&#160;</td>
			<td style="width:653px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:605px;">Methanol</td>
			<td style="width:421px;">Fisher Scientific&#160;</td>
			<td>A412-1</td>
			<td style="width:653px;">For Western blot transfer buffer</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:605px;">Plastic Cuvettes</td>
			<td style="width:421px;">Fisher Scientific&#160;</td>
			<td style="width:133px;">14-377-010&#160;</td>
			<td style="width:653px;">For size measurements using Zetasizer Nano ZS</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:605px;">Molecular BioProducts RNase AWAY Surface Decontaminant</td>
			<td style="width:421px;">Fisher Scientific</td>
			<td style="width:133px;">14-754-34</td>
			<td style="width:653px;">Spray for removing RNAse contamination</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:605px;">Agarose</td>
			<td style="width:421px;">Fisher Scientific</td>
			<td style="width:133px;">BP165-25</td>
			<td style="width:653px;">Low melting point, for running RNA samples</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ProLong Gold Antifade Reagent with DAPI&#160;</td>
			<td style="width:421px;">Invitrogen</td>
			<td>P36935</td>
			<td style="width:653px;">Antifade reagent with DAPI, nucelus detection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:605px;">Alexa Fluor 488 Phalloidin</td>
			<td style="width:421px;">Invitrogen</td>
			<td style="width:133px;">A12379</td>
			<td style="width:653px;">Dissolve 300 units in 1.5 ml methanol, detection of filamentous actin</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:605px;">SYBR Safe DNA Gel Stain</td>
			<td style="width:421px;">Invitrogen</td>
			<td style="width:133px;">S33102</td>
			<td style="width:653px;">Visualization of RNA</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:605px;">Negative Control siRNA</td>
			<td style="width:421px;">Qiagen</td>
			<td style="width:133px;">1022076</td>
			<td style="width:653px;">Control siRNA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:605px;">AllStars Neg. siRNA AF 555</td>
			<td style="width:421px;">Qiagen</td>
			<td style="width:133px;">1027294</td>
			<td style="width:653px;">Fluorescent control siRNA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:605px;">Cell scraper</td>
			<td style="width:421px;">Celltreat</td>
			<td>229310</td>
			<td style="width:653px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BioLite Multidishes and Microwell Plates</td>
			<td style="width:421px;">Thermo Scientific</td>
			<td>130184</td>
			<td style="width:653px;">6-well plate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pierce LDS Sample Loading Buffer (4X)&#160;</td>
			<td style="width:421px;">Thermo Scientific</td>
			<td>84788</td>
			<td style="width:653px;">Sample loading buffer for Western blot</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:605px;">Pierce BCS Protein Assay Kit</td>
			<td style="width:421px;">Thermo Scientific</td>
			<td style="width:133px;">23227</td>
			<td style="width:653px;">Protein quantification assay</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Halt Protease Inhibitor Single-Use Cocktails (100X)</td>
			<td style="width:421px;">Thermo Scientific</td>
			<td style="width:133px;">78430</td>
			<td style="width:653px;">Protease inhibitor cocktail, use at 1X</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">M-PER Mammalian Protein Extraction Reagent</td>
			<td style="width:421px;">Thermo Scientific&#160;</td>
			<td style="width:133px;">78501</td>
			<td style="width:653px;">Protein extraction reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:605px;">Sorvall Legend Micro 21R</td>
			<td>Thermo Scientific</td>
			<td>75002440</td>
			<td>Centrifuge</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:605px;">Beta Actin Antibody&#160;</td>
			<td>Thermo Scientific&#160;</td>
			<td>MA1-91399</td>
			<td>&#946;-actin primary antibody (from mouse) for western blor</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">6X TriTrack DNA Loading Dye</td>
			<td style="width:421px;">Thermo Scientific</td>
			<td>R1161</td>
			<td style="width:653px;">DNA loading dye</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nuclease-Free Water&#160;</td>
			<td style="width:421px;">Life Technologies</td>
			<td>AM9938</td>
			<td style="width:653px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Non-Fat Dry Milk</td>
			<td style="width:421px;">Lab Scientific</td>
			<td>M0841</td>
			<td style="width:653px;">For Western blot</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2-well BD Falcon culture slides&#160;</td>
			<td style="width:421px;">BD Biosciences</td>
			<td>354102</td>
			<td style="width:653px;">2-well culture slides</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Amersham ECL Western blot detection reagent.&#160;</td>
			<td style="width:421px;">GE Healthcare Life Sciences</td>
			<td style="width:133px;">RPN2106</td>
			<td style="width:653px;">Western blot detection reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BA Membranes</td>
			<td style="width:421px;">GE Healthcare Life Sciences</td>
			<td>10402096</td>
			<td style="width:653px;">Nitrocellulose membrane for Wester blot</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ATM (D2E2) Rabbit mAb</td>
			<td style="width:421px;">Cell Signaling</td>
			<td>2873S</td>
			<td style="width:653px;">ATM primary antibody (from rabbit) for Western blot</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-rabbit IgG, HRP-linked Antibody</td>
			<td style="width:421px;">Cell Signaling&#160;</td>
			<td>7074</td>
			<td style="width:653px;">Secondary antibody (anti-rabbit) for Western blot&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:605px;">Folded capillary cells</td>
			<td style="width:421px;">Malvern&#160;</td>
			<td style="width:133px;">DTS 1061</td>
			<td style="width:653px;">For zeta potentail measurements using Zetasizer Nano ZS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:605px;">MDA-MB-231 cell line</td>
			<td style="width:421px;">ATCC</td>
			<td style="width:133px;">HTB-26</td>
			<td style="width:653px;">Mammary Gland/Breast</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:605px;">12% Mini-PROTEAN TGX Gel</td>
			<td>Bio-rad</td>
			<td>456-1043&#160;</td>
			<td style="width:653px;">For Western blot</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Biorad PowerPac HC</td>
			<td>Bio-rad</td>
			<td>164-5052</td>
			<td>Power supply for electrophoresis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">10x Tris/Glycine/SDS Buffer</td>
			<td>Bio-rad</td>
			<td>161-0732</td>
			<td>Running buffer for Western blot</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Wide Mini-Sub Cell GT Cell</td>
			<td>Bio-rad</td>
			<td>170-4405</td>
			<td>Electrophoresis equipment for DNA agarose gel&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mini-PROTEAN Tetra cell</td>
			<td>Bio-rad</td>
			<td>165-8000</td>
			<td>Electrophoresis equipment for Western blot</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ChemiDoc XRS+ System with Image Lab Software</td>
			<td>Bio-rad</td>
			<td>170-8265</td>
			<td>Image acquisition and analysis software for gels and blots</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">4&#34; (10cm) dia., 5x7mm diced Silicon Wafer</td>
			<td>Ted Pella&#160;</td>
			<td>16007</td>
			<td>Silicon waferfor scanning electron microscopy and atomic force microscopy</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Thermomixer R</td>
			<td>Eppendorf</td>
			<td>22670107</td>
			<td>Shaker&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Isoton II diluent</td>
			<td>Beckman Coulter</td>
			<td>8546719</td>
			<td>Isoton diluent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Multisizer 4 Coulter Counter</td>
			<td>Beckman Coulter</td>
			<td>A63076</td>
			<td>Particle counting analyzer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:605px;">Non-functionalized porous silicon particles&#160;</td>
			<td style="width:421px;">Microelectronics Research Center, University of Texas at Austin</td>
			<td style="width:133px;"></td>
			<td style="width:653px;">Dicoidal shape. 2.6 &#956;m (diameter) x 0.7 &#956;m (hight), provided in isopropyl alcohol</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:605px;">Zetasizer Nano ZS</td>
			<td style="width:421px;">Malvern</td>
			<td style="width:133px;"></td>
			<td style="width:653px;">Particle analyzer system for size and zeta potential&#160;</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:605px;">Scanning Electron Microscope</td>
			<td style="width:421px;">FEI</td>
			<td style="width:133px;"></td>
			<td style="width:653px;">Particle size and shape&#160;</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:605px;">Atomic Force Microscope</td>
			<td style="width:421px;">Bruker</td>
			<td style="width:133px;"></td>
			<td style="width:653px;">Particle size and shape</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:605px;">Fluo ViewTM 1000 Confocal Microscope</td>
			<td style="width:421px;">Olympus</td>
			<td style="width:133px;"></td>
			<td style="width:653px;">Visualization of fixed and live cells</td>
		</tr>
	</tbody>
</table>

porous silicon microparticles for delivery of sirna therapeutics

Small interfering RNA (siRNA) can be used to suppress gene expression, thereby providing a new avenue for the treatment of various diseases. However, the successful implementation of siRNA therapy requires the use of delivery platforms that can overcome the major challenges of siRNA delivery, such as enzymatic degradation, low intracellular uptake and lysosomal entrapment. Here, a protocol for the preparation and use of a biocompatible and effective siRNA delivery system is presented. This platform consists of polyethylenimine (PEI) and arginine (Arg)-grafted porous silicon microparticles, which can be loaded with siRNA by performing a simple mixing step. The silicon particles are gradually degraded over time, thereby triggering the formation of Arg-PEI/siRNA nanoparticles. This delivery vehicle provides a means for protecting and internalizing siRNA, without causing cytotoxicity. The major steps of polycation functionalization, particle characterization, and siRNA loading are outlined in detail. In addition, the procedures for determining particle uptake, cytotoxicity, and transfection efficacy are also described.

Este protocolo describe el uso de un sistema de suministro no virales para la transfección segura y eficiente siRNA. Los resultados de SEM revelan que las partículas de los PCP son de forma cilíndrica y tener un diámetro de 2,6 m (Figura 2A). Las partículas están cargadas positivamente con un potencial zeta de aproximadamente 8,21 (Figura 2B), permitiendo de este modo unión electrostática con nucleótidos cargadas negativamente. Confocal de imágenes de diferentes capas de las p...

Watch this Scientific Journal Video about Porous Silicon Microparticles for Delivery of siRNA Therapeutics at JoVE.com

Porous Silicon Microparticles for Delivery of siRNA Therapeutics

Entrega sigue siendo el principal desafío para la aplicación terapéutica de pequeños ARN de interferencia (siRNA). Este protocolo implica el uso de una plataforma de entrega siRNA multifuncional y biocompatible, que consiste en arginina y polietilenimina injertada micropartículas de silicio poroso.

Silicio Poroso micropartículas para la entrega de siRNA Terapéutica

Small interfering RNA (siRNA) can be used to suppress gene expression, thereby providing a new avenue for the treatment of various diseases. However, the ...

porous-silicon-microparticles-for-delivery-of-sirna-therapeutics

Research

JoVE Journal

Bioengineering

10.8K vistas.  Houston Methodist Research Institute.  Entrega sigue siendo el principal desafío para la aplicación terapéutica de pequeños ARN de interferencia (siRNA). Este protocolo implica el uso de una plataforma de entrega siRNA multifuncional y biocompatible, que consiste en arginina y polietilenimina injertada micropartículas de silicio poroso. 

Video: Porous Silicon Microparticles for Delivery of siRNA Therapeutics

Soft Lithographic Functionalization and Patterning Oxide-free Silicon and Germanium

The development of hybrid electronic devices relies in large part on the integration of (bio)organic materials and inorganic semiconductors through a stable interface that permits efficient electron transport and protects underlying substrates from oxidative degradation. Group IV semiconductors can be effectively protected with highly-ordered self-assembled monolayers (SAMs) composed of simple alkyl chains that act as impervious barriers to both organic and aqueous solutions. Simple alkyl SAMs, however, are inert and not amenable to traditional patterning techniques. The motivation for immobilizing organic molecular systems on semiconductors is to impart new functionality to the surface that can provide optical, electronic, and mechanical function, as well as chemical and biological activity. 
Microcontact printing (&mu;CP) is a soft-lithographic technique for patterning SAMs on myriad surfaces.1-9 Despite its simplicity and versatility, the approach has been largely limited to noble metal surfaces and has not been well developed for pattern transfer to technologically important substrates such as oxide-free silicon and germanium. Furthermore, because this technique relies on the ink diffusion to transfer pattern from the elastomer to substrate, the resolution of such traditional printing is essentially limited to near 1 &mu;m.10-16
In contrast to traditional printing, inkless &mu;CP patterning relies on a specific reaction between a surface-immobilized substrate and a stamp-bound catalyst. Because the technique does not rely on diffusive SAM formation, it significantly expands the diversity of patternable surfaces. In addition, the inkless technique obviates the feature size limitations imposed by molecular diffusion, facilitating replication of very small (&lt;200 nm) features.17-23 However, up till now, inkless &mu;CP has been mainly used for patterning relatively disordered molecular systems, which do not protect underlying surfaces from degradation.
Here, we report a simple, reliable high-throughput method for patterning passivated silicon and germanium with reactive organic monolayers and demonstrate selective functionalization of the patterned substrates with both small molecules and proteins. The technique utilizes a preformed NHS-reactive bilayered system on oxide-free silicon and germanium. The NHS moiety is hydrolyzed in a pattern-specific manner with a sulfonic acid-modified acrylate stamp to produce chemically distinct patterns of NHS-activated and free carboxylic acids. A significant limitation to the resolution of many &mu;CP techniques is the use of PDMS material which lacks the mechanical rigidity necessary for high fidelity transfer. To alleviate this limitation we utilized a polyurethane acrylate polymer, a relatively rigid material that can be easily functionalized with different organic moieties. Our patterning approach completely protects both silicon and germanium from chemical oxidation, provides precise control over the shape and size of the patterned features, and gives ready access to chemically discriminated patterns that can be further functionalized with both organic and biological molecules. The approach is general and applicable to other technologically-relevant surfaces.

Here we describe a simple method for patterning oxide-free silicon and germanium with reactive organic monolayers and demonstrate functionalization of the patterned substrates with small molecules and proteins. The approach completely protects surfaces from chemical oxidation, provides precise control over feature morphology, and provides ready access to chemically discriminated patterns.

Funcionalización suaves litográfica y Patrones libre de óxido de silicio y el germanio

The development of hybrid electronic devices relies in large part on the integration of (bio)organic materials and inorganic semiconductors through a ...

Investigación

Bioingeniería

Self-reporting Scaffolds for 3-Dimensional Cell Culture

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting 3D cellular construct can often be more relevant to studying the molecular events and cell-cell interactions than similar experiments studied in 2D. To create effective 3D cultures with high cell viability throughout the scaffold the culture conditions such as oxygen and pH need to be carefully controlled as gradients in analyte concentration can exist throughout the 3D construct. Here we describe the methods of preparing biocompatible pH responsive sol-gel nanosensors and their incorporation into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds along with their subsequent preparation for the culture of mammalian cells. The pH responsive scaffolds can be used as tools to determine microenvironmental pH within a 3D cellular construct. Furthermore, we detail the delivery of pH responsive nanosensors to the intracellular environment of mammalian cells whose growth was supported by electrospun PLGA scaffolds. The cytoplasmic location of the pH responsive nanosensors can be utilized to monitor intracellular pH (pHi) during ongoing experimentation.

Biocompatible pH responsive sol-gel nanosensors can be incorporated into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds. The produced self-reporting scaffolds can be used for in&#160;situ monitoring of microenvironmental conditions whilst culturing cells upon the scaffold. This is beneficial as the 3D cellular construct can be monitored in real-time without disturbing the experiment.

Los andamios de autoinforme para el 3-Dimensional Cultivo Celular

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting ...

Preparation of Silicon Nanowire Field-effect Transistor for Chemical and Biosensing Applications

Surveillance using biomarkers is critical for the early detection, rapid intervention, and reduction in the incidence of diseases. In this study, we describe the preparation of polycrystalline silicon nanowire field-effect transistors (pSNWFETs) that serve as biosensing devices for biomarker detection. A protocol for chemical and biomolecular sensing by using pSNWFETs is presented. The pSNWFET device was demonstrated to be a promising transducer for real-time, label-free, and ultra-high-sensitivity biosensing applications. The source/drain channel conductivity of a pSNWFET is sensitive to changes in the environment around its silicon nanowire (SNW) surface. Thus, by immobilizing probes on the SNW surface, the pSNWFET can be used to detect various biotargets ranging from small molecules (dopamine) to macromolecules (DNA and proteins). Immobilizing a bioprobe on the SNW surface, which is a multistep procedure, is vital for determining the specificity of the biosensor. It is essential that every step of the immobilization procedure is correctly performed. We verified surface modifications by directly observing the shift in the electric properties of the pSNWFET following each modification step. Additionally, X-ray photoelectron spectroscopy was used to examine the surface composition following each modification. Finally, we demonstrated DNA sensing on the pSNWFET. This protocol provides step-by-step procedures for verifying bioprobe immobilization and subsequent DNA biosensing application.

We describe key steps for biosensing by using polysilicon nanowire field-effect transistors, including the preparation of the device and the immobilization and confirmation of a DNA molecular probe on the nanowire surface, as well as conditions for DNA sensing.

Preparación de silicio de nanocables de transistor de efecto de campo de químicos y biosensores Aplicaciones

Surveillance using biomarkers is critical for the early detection, rapid intervention, and reduction in the incidence of diseases. In this study, we ...

Synthesis of Thermogelling Poly(N-isopropylacrylamide)-graft-chondroitin Sulfate Composites with Alginate Microparticles for Tissue Engineering

Injectable biomaterials are defined as implantable materials that can be introduced into the body as a liquid and solidify in situ. Such materials offer the clinical advantages of being implanted minimally invasively and easily forming space-filling solids in irregularly shaped defects. Injectable biomaterials have been widely investigated as scaffolds for tissue engineering. However, for the repair of certain load-bearing areas in the body, such as the intervertebral disc, scaffolds should possess adhesive properties. This will minimize the risk of dislocation during motion and ensure intimate contact with the surrounding tissue, providing adequate transmission of forces. Here, we describe the preparation and characterization of a scaffold composed of thermally sensitive poly(N-isopropylacrylamide)-graft-chondroitin sulfate (PNIPAAM-g-CS) and alginate microparticles. The PNIPAAm-g-CS copolymer forms a viscous solution in water at RT, into which alginate particles are suspended to enhance adhesion. Above the lower critical solution temperature (LCST), around 30 &#176;C, the copolymer forms a solid gel around the microparticles. We have adapted standard biomaterials characterization procedures to take into account the reversible phase transition of PNIPAAm-g-CS. Results indicate that the incorporation of 50 or 75 mg/ml&#160;alginate particles into 5% (w/v) PNIPAAm-g-CS solutions quadruple the adhesive tensile strength of PNIPAAm-gCS alone (p&#60;0.05). The incorporation of alginate microparticles also significantly increases swelling capacity of PNIPAAm-g-CS (p&#60;0.05), helping to maintain a space-filling gel within tissue defects. Finally, results of the in vitro toxicology assay kit, 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) and Live/Dead viability assay indicate that the adhesive is capable of supporting the survival and proliferation of encapsulated Human Embryonic Kidney (HEK) 293 cells over 5 days.

Synthesis of Thermogelling PolyN-isopropylacrylamide-graft-chondroitin Sulfate Composites with Alginate Microparticles for Tissue Engineering

An injectable tissue engineering scaffold composed of poly(N-isopropylacrylamide)-graft-chondroitin sulfate (PNIPAAm-g-CS)-containing alginate microparticles was prepared. The adhesive strength, swelling properties and in vitro biocompatibility are analyzed in this study. The characterization techniques developed here may be applicable to other thermogelling systems.

Síntesis de termogelificante poli (N-isopropilacrilamida) compuestos de sulfato de condroitina-injertado con alginato micropartículas de Ingeniería de Tejidos

Injectable biomaterials are defined as implantable materials that can be introduced into the body as a liquid and solidify in situ. Such materials offer ...

Delivery of Therapeutic siRNA to the CNS Using Cationic and Anionic Liposomes

Prion diseases result from the misfolding of the normal, cellular prion protein (PrPC) to an abnormal protease resistant isomer called PrPRes. The emergence of prion diseases in wildlife populations and their increasing threat to human health has led to increased efforts to find a treatment for these diseases. Recent studies have found numerous anti-prion compounds that can either inhibit the infectious PrPRes isomer or down regulate the normal cellular prion protein. However, most of these compounds do not cross the blood brain barrier to effectively inhibit PrPRes formation in brain tissue, do not specifically target neuronal PrPC, and are often too toxic to use in animal or human subjects. 
We investigated whether siRNA delivered intravascularly and targeted towards neuronal PrPC is a safer and more effective anti-prion compound. This report outlines a protocol to produce two siRNA liposomal delivery vehicles, and to package and deliver PrP siRNA to neuronal cells. The two liposomal delivery vehicles are 1) complexed-siRNA liposome formulation using cationic liposomes (LSPCs), and 2) encapsulated-siRNA liposome formulation using cationic or anionic liposomes (PALETS). For the LSPCs, negatively charged siRNA is electrostatically bound to the cationic liposome. A positively charged peptide (RVG-9r [rabies virus glycoprotein]) is added to the complex, which specifically targets the liposome-siRNA-peptide complexes (LSPCs) across the blood brain barrier (BBB) to acetylcholine expressing neurons in the central nervous system (CNS). For the PALETS (peptide addressed liposome encapsulated therapeutic siRNA), the cationic and anionic lipids were rehydrated by the PrP siRNA. This procedure results in encapsulation of the siRNA within the cationic or anionic liposomes. Again, the RVG-9r neuropeptide was bound to the liposomes to target the siRNA/liposome complexes to the CNS. Using these formulations, we have successfully delivered PrP siRNA to AchR-expressing neurons, and decreased the PrPC expression of neurons in the CNS.

The goal of this protocol is to use cationic/anionic liposomes with a neuro-targeting peptide as a CNS delivery system to enable siRNA to cross the BBB. The optimization of a delivery system for treatments, like siRNA, would allow for more treatment options for prion and other neurodegenerative diseases.

Entrega de Therapeutic siRNA al SNC Usando catiónicos y aniónicos Los liposomas

Prion diseases result from the misfolding of the normal, cellular prion protein (PrPC) to an abnormal protease resistant isomer called PrPRes.  The ...

Designing Porous Silicon Films as Carriers of Nerve Growth Factor

Despite the great potential of NGF for treating neurodegenerative diseases, its therapeutic administration represents a significant challenge as the protein does not cross the blood-brain barrier, owing to its chemical properties, and thus requires long-term delivery to the brain to have a biological effect. This work describes fabrication of nanostructured PSi films as degradable carriers of NGF for sustained delivery of this sensitive protein. The PSi carriers are specifically tailored to obtain high loading efficacy and continuous release of NGF for a period of four weeks, while preserving its biological activity. The behavior of the NGF-PSi carriers as a NGF delivery system is investigated in vitro by examining their capability to induce neuronal differentiation and outgrowth of PC12 cells and dissociated DRG neurons. Cell viability in the presence of neat and NGF-loaded PSi carriers is evaluated. The bioactivity of NGF released from the PSi carriers is compared to the conventional treatment of repetitive free NGF administrations. PC12 cell differentiation is analyzed and characterized by the measurement of three different morphological parameters of differentiated cells; (i) the number of neurites extracting from the soma (ii) the total neurites' length and (iii) the number of branching points. PC12 cells treated with the NGF-PSi carriers demonstrate a profound differentiation throughout the release period. Furthermore, DRG neuronal cells cultured with the NGF-PSi carriers show an extensive neurite initiation, similar to neurons treated with repetitive free NGF administrations. The studied tunable carriers demonstrate the long-term implants for NGF release with a therapeutic potential for neurodegenerative diseases.

Here, we present a protocol to design and fabricate nanostructured porous silicon (PSi) films as degradable carriers for the nerve growth factor (NGF). Neuronal differentiation and outgrowth of PC12 cells and mice dorsal root ganglion (DRG) neurons are characterized upon treatment with the NGF-loaded PSi carriers.

Diseño de películas de silicio poroso como portadores de Factor de crecimiento nervioso

Despite the great potential of NGF for treating neurodegenerative diseases, its therapeutic administration represents a significant challenge as the ...

3D Printed Porous Cellulose Nanocomposite Hydrogel Scaffolds

This work demonstrates the use of three-dimensional (3D) printing to produce porous cubic scaffolds using cellulose nanocomposite hydrogel ink, with controlled pore structure and mechanical properties. Cellulose nanocrystals (CNCs, 69.62 wt%) based hydrogel ink with matrix (sodium alginate and gelatin) was developed and 3D printed into scaffolds with uniform and gradient pore structure (110-1,100 &#181;m). The scaffolds showed compression modulus in the range of 0.20-0.45 MPa when tested in simulated in vivo conditions (in distilled water at 37 &#176;C). The pore sizes and the compression modulus of the 3D scaffolds matched with the requirements needed for cartilage regeneration applications. This work demonstrates that the consistency of the ink can be controlled by the concentration of the precursors and porosity can be controlled by the 3D printing process and both of these factors in return defines the mechanical properties of the 3D printed porous hydrogel scaffold. This process method can therefore be used to fabricate structurally and compositionally customized scaffolds according to the specific needs of patients.&#160;

The three critical steps of this protocol are i) developing the right composition and consistency of the cellulose hydrogel ink, ii) 3D printing of scaffolds into various pore structures with good shape fidelity and dimensions and iii) demonstration of the mechanical properties in simulated body conditions for cartilage regeneration.

3D impreso celulosa poroso nanocompuesto hidrogel andamios

This work demonstrates the use of three-dimensional (3D) printing to produce porous cubic scaffolds using cellulose nanocomposite hydrogel ink, with ...

Preparation of Neutrally-charged, pH-responsive Polymeric Nanoparticles for Cytosolic siRNA Delivery

The success of siRNA as a targeted molecular medicine is dependent upon its efficient cytosolic delivery to cells within the tissue of pathology. Clinical success for treating previously &#8216;undruggable&#8217; hepatic disease targets with siRNA has been achieved. However, efficient tumor siRNA delivery necessitates additional pharmacokinetic design considerations, including long circulation time, evasion of clearance organs (e.g., liver and kidneys), and tumor penetration and retention. Here, we describe the preparation and in vitro physicochemical/biological characterization of polymeric nanoparticles designed for efficient siRNA delivery, particularly to non-hepatic tissues such as tumors. The siRNA nanoparticles are prepared by electrostatic complexation of siRNA and the diblock copolymer poly(ethylene glycol-b-[2-(dimethylamino)ethyl methacrylate-co-butyl methacrylate]) (PEG-DB) to form polyion complexes (polyplexes) where siRNA is sequestered within the polyplex core and PEG forms a hydrophilic, neutrally-charged corona. Moreover, the DB block becomes membrane-lytic as vesicles of the endolysosomal pathway acidify (&#60; pH 6.8), triggering endosomal escape and cytosolic delivery of siRNA. Methods to characterize the physicochemical characteristics of siRNA nanoparticles such as size, surface charge, particle morphology, and siRNA loading are described. Bioactivity of siRNA nanoparticles is measured using luciferase as a model gene in a rapid and high-throughput gene silencing assay. Designs which pass these initial tests (such as PEG-DB-based polyplexes) are considered appropriate for translation to preclinical animal studies assessing the delivery of siRNA to tumors or other sites of pathology.

Methods to prepare and characterize the physicochemical properties and bioactivity of neutrally-charged, pH-responsive siRNA nanoparticles are presented. Criteria for successful siRNA nanomedicines such as size, morphology, surface charge, siRNA loading, and gene silencing are discussed.

La preparación de nanopartículas poliméricas con capacidad de pH, cargadas de neutralmente, para la entrega de siRNA citosólica

The success of siRNA as a targeted molecular medicine is dependent upon its efficient cytosolic delivery to cells within the tissue of pathology. Clinical ...

Silicon Nanowires and Optical Stimulation for Investigations of Intra- and Intercellular Electrical Coupling

Myofibroblasts can spontaneously internalize silicon nanowires (SiNWs), making them an attractive target for bioelectronic applications. These cell-silicon hybrids offer leadless optical modulation capabilities with minimal perturbation to normal cell behavior. The optical capabilities are obtained by the photothermal and photoelectric properties of SiNWs. These hybrids can be harvested using standard tissue culture techniques and then applied to different biological scenarios. We demonstrate here how these hybrids can be used to study the electrical coupling of cardiac cells and compare how myofibroblasts couple to one another or to cardiomyocytes. This process can be accomplished without special equipment beyond a fluorescent microscope with coupled laser line. Also shown is the use of a custom-built MATLAB routine that allows the quantification of calcium propagation within and between the different cells in the culture. Myofibroblasts are shown to have a slower electrical response than that of cardiomyocytes. Moreover, the myofibroblast intercellular propagation shows slightly slower, though comparable velocities to their intracellular velocities, suggesting passive propagation through gap junctions or nanotubes. This technique is highly adaptable and can be easily applied to other cellular arenas, for in vitro as well as in vivo or ex vivo investigations.

This protocol describes the use of silicon nanowires for intracellular optical bio-modulation of cell in a simple and easy to perform method. The technique is highly adaptable to diverse cell types and can be used for in vitro as well as in vivo applications.

Nanohilos de silicio y estimulación óptica para investigaciones de acoplamiento eléctrico intra e intercelular

Myofibroblasts can spontaneously internalize silicon nanowires (SiNWs), making them an attractive target for bioelectronic applications. These ...

Tools for Surface Treatment of Silicon Planar Intracortical Microelectrodes

Intracortical microelectrodes hold great therapeutic potential. But they are challenged with significant performance reduction after modest implantation durations. A substantial contributor to the observed decline is the damage to the neural tissue proximal to the implant and subsequent neuroinflammatory response. Efforts to improve device longevity include chemical modifications or coating applications to the device surface to improve the tissue response. Development of such surface treatments is typically completed using non-functional "dummy" probes that lack the electrical components required for the intended application. Translation to functional devices requires additional consideration given the fragility of intracortical microelectrode arrays. Handling tools greatly facilitate surface treatments to assembled devices, particularly for modifications that require long procedural times. The handling tools described here are used for surface treatments applied via gas-phase deposition and aqueous solution exposure. Characterization of the coating is performed using ellipsometry and x-ray photoelectron spectroscopy. A comparison of electrical impedance spectroscopy recordings before and after the coating procedure on functional devices confirmed device integrity following modification. The described tools can be readily adapted for alternative electrode devices and treatment methods that maintain chemical compatibility.

The present protocol describes tools for handling silicon planar intracortical microelectrodes during treatments for surface modification via gas deposition and aqueous solution reactions. The assembly of the components used to handle the devices throughout the procedure is explained in detail.

Herramientas para el tratamiento superficial de microelectrodos intracorticales planos de silicio

Intracortical microelectrodes hold great therapeutic potential. But they are challenged with significant performance reduction after modest implantation ...