Este trabajo fue apoyado por el NIH / NIBIB subvenciones R00 EB009105 (para NFS) y EB011317 P30 (para NFS), un NIH / NIBIB formación de subvención T32 EB007509 (a AMW), un Case Western Reserve University Interdisciplinario Alliance Investment Grant (para NFS), y Un Caso Comprehensive Cancer Center subvención P30 CA043703 (para NFS). Agradecemos a los investigadores de laboratorio Steinmetz pregrado de estudiantes para sus prácticas en el apoyo: Nadia Ayat, Kevin Chen, Sourav (Sid) Dey, Alice Yang, Sam Alexander, Craig D&#39;Cruz, Hern Stephen Randolph Lauren, Brian lo tanto, Pablo y Chariou .

1. VNP (CPMV, BMV, PVX y TMV) Propagación <ol><li> Poner la cámara de planta de interior controla a 15 horas del día (100% de luz, 25 º C, 65% de humedad) y 9 h de la noche (0% de luz, 22 ° C, 60% de humedad). </li><li> Se inoculan las plantas de acuerdo a la escala de tiempo en la Tabla 1. </li></ol><table border="1"><tbody><tr><td> CPMV </td><td> PVX, TMV, y BMV </td></tr><tr><td rowspan="2"> Día 0: Planta 3 caupí semillas / maceta. </td><td> Día 0: Plant ~ 30 N. benthamiana semillas / maceta. Fertilizar una vez por semana con un fertilizante cucharada 1/5 l de agua. </td></tr><tr><td> Día 14: Re-pot N. benthamiana en 1 planta / maceta. </td></tr><tr><td> Día 10: Infect deja las hojas primarias con CPMV (5 μg/50 l / hoja) por inoculación mecánica con una fina capa de carburo de silicio. </td><td> Día 28: Infect tres a five deja con PVX, TMV, o BMV (5 μg/50 l / hoja) por inoculación mecánica con una fina capa de carburo de silicio. </td></tr><tr><td> Día 20: Hojas de la cosecha y almacenar a -80 ° C. </td><td> Día 42: las hojas de la cosecha y almacenar a -80 ° C. </td></tr></tbody></table> Tabla 1. Cronograma para el cultivo, infectando, y cosechar las hojas. Nota: sólo propagación CPMV se demuestra como un ejemplo. 2. VNP (CPMV, BMV, PVX y TMV) Purificación Nota: Todos los pasos se llevan a cabo en hielo o a 4 º C. <ol><li> Homogeneizar 100 g de hojas congeladas en un mezclador estándar utilizando 2 volúmenes de tampón frío (véase la Tabla 2). Filtrar a través de 2-3 capas de estopilla. </li><li> Para PVX, ajustar el pH a 6,5 ​​utilizando 1 M de HCl. Añadir 0,2% (w / v) de ácido ascórbico y 0,2% (w / v) de sodio sulfiTE. </li><li> Centrifugar homogeneizado crudo planta a 5.500 xg durante 20 min. Recoger el sobrenadante. </li><li> Para BMV, capa de 25 ml de sobrenadante sobre 5 ml de 10% (w / v) de solución de sacarosa. Se centrifuga a 9.000 xg durante 3 horas y se resuspende el pellet en solución de CsCl 38,5% (w / v). Mezclar por agitación durante 5 horas, y luego continúe con el paso 2.12. </li><li> Extraer material de la planta mediante la adición de 0,7 volúmenes de 1:1 (v / v) de cloroformo :1-butanol. Revuelva la mezcla durante 30-60 min. </li><li> Centrifugar la solución a 5.500 xg durante 20 min. Recoger la fase acuosa superior. </li><li> Añadir NaCl a 0,2 M y 8% (w / v) PEG (MW 8.000). Para TMV, también añadir 1% (v / v) de Triton X-100. Se agita durante al menos 1 h, después se deja reposar durante al menos 1 h. </li><li> Se centrifuga la solución a 15.000 xg durante 15 min. Resuspender el sedimento en 10 ml de tampón. Para PVX, añadir 0,1% β-mercaptoetanol y urea a 0,5 M. </li><li> Centrifugar a 8.000 xg durante 30 min y recoger el líquido sobrenadante. </li><li> Ultracentrífuga sobrenadante a 160.000 xg durante 3 hr. Resuspender el sedimento en 5 ml de tampón overnight. </li><li> Preparar un gradiente de sacarosa al 10-40% usando volúmenes iguales de 10%, 20%, 30% y 40% de sacarosa en tampón (el más pesado primero). Permitir que el gradiente de equilibrar durante la noche a temperatura ambiente. </li><li> Ultracentrífuga precipitado resuspendido sobre gradiente de sacarosa a 100.000 xg durante 2 horas (24 horas para BMV). </li><li> Recoger banda de dispersión de luz y diálisis frente a tampón. </li><li> Caracterizar los VNPs (abajo) y se almacena a 4 º C. Para almacenamiento a largo plazo, almacenar a -80 ° C. </li></ol><table border="1"><tbody><tr><td> CPMV y TMV </td><td> 0,1 M de tampón de fosfato de potasio (pH 7,0) 38,5 mM KH 2 PO 4 61,5 mM K 2 HPO 4 </td></tr><tr><td> PVX </td><td> 0,5 M de tampón borato (pH 7,8) 0,5 M de ácido bórico Ajustar el pH con NaOH </td></tr><tr><td> BMV </td><td> SAMA tampón (pH 4,5) 250 mM de acetato de sodio 10 mM MgCl 2 2 mM β-mercaptoetanol (añadir nuevo) </td></tr></tbody></table> Tabla 2. Buffers y sus recetas para cada VNP. Nota: sólo propagación CPMV se demuestra como un ejemplo. 3. VNP (CPMV, BMV, PVX y TMV) Caracterización <ol><li> Espectroscopía UV / visible para determinar la concentración de VNPs. <ol><li> Medir la absorbancia de 2 l de muestra utilizando un espectrofotómetro NanoDrop. </li><li> Determinar la concentración de partículas y colorantes utilizando la ley de Beer-Lambert (A εcl =, donde A es la absorbancia, ε es el coeficiente de extinción, c es la concentración, y l es la longitud del camino). La longitud del camino es de 0,1 cm para el NanoDrop. Los coeficientes de extinción VNP-específicos son: CPMV: 8,1 cm -1 mg -1 ml (a 260 nm) PVX: 2,97 cm -1 mg -1 ml (a 260 nm) TMV: 3,0 cm -1 mg -1 ml (a 260 nm) BMV: 5,15 cm -1 mg -1 ml (a 260 nm) </li></ol></li><li> Analizar las partículas por exclusión de tamaño rápido cromatografía líquida de proteínas (FPLC). <ol><li> El uso de un Superose 6 columna de exclusión por tamaño y el Explorador de ÄKTA, carga de 50-100 g de VNPs en 200 l de 0,1 M tampón fosfato de potasio (pH 7,0). </li><li> Establecer detectores a 260 nm (ácido nucleico), 280 nm (proteína), y la longitud de onda de excitación de los colorantes unidos. </li><li> Ejecute a una velocidad de flujo de 0,5 ml / min durante 72 min. </li><li> El perfil de elución y A260: A280 nm indica si la preparación VNP es puro y si las partículas están intactos y montados. El siguiente A260: 280 proporciones indican una preparación pura VNP: CPMV: 1,8 ± 0,1 PVX: 1,2 ± 0,1 TMV:1,1 ± 0,1 BMV: 1,7 ± ​​0,1 </li></ol></li><li> Realizar desnaturalización (pre-cast NuPAGE) Bis-Tris poliacrilamida 4-12% electroforesis en gel de gradiente para analizar la pureza de la preparación y la conjugación a proteínas de recubrimiento individuales. <ol><li> Añadir 3 ml de tampón de muestra LDS 4x a 10 g de las partículas en 9 l de tampón de fosfato de potasio. Añadir un adicional de 1 l de tampón de muestra LDS 4x y 3 l de β-mercaptoetanol a BMV para reducir el elevado número de enlaces disulfuro. </li><li> Incubar en bloque de calor durante 5 minutos a 100 ° C. </li><li> Cargar muestras en un gel de SDS. </li><li> Ejecutar las muestras a 200 V durante 1 h en 1x MOPS funcionamiento de amortiguación. </li><li> Documentar el gel bajo luz UV para visualizar las proteínas fluorescentes abrigo. </li><li> Para no fluorescente de proteínas, tinción con azul de Coomassie (0,25% (w / v) azul brillante de Coomassie R-250, 30% (v / v) de metanol, 10% (v / v) de ácido acético) durante 1 hr. </li><li> Destain con 30% de metanol, 10% de ácido acético durante la noche. Change la solución si se requiere. </li><li> Documentar el gel con luz blanca. </li></ol></li><li> Analizar la integridad de las partículas por microscopía electrónica de transmisión (TEM). <ol><li> Diluir las muestras a 0.1-1 mg / ml en 20 l de agua DI. </li><li> Coloque 20 gotas l de las muestras en Parafilm. </li><li> Cubra gotas con una rejilla TEM y deje reposar durante 2 min. Wick fuera de la solución en exceso en la rejilla con papel de filtro. </li><li> Lave la red mediante la colocación de una gota de agua DI luego mecha seca. </li><li> Mancha de red mediante la colocación de una gota en 20 l de 2% (w / v) de acetato de uranilo durante 2 min. Wick fuera el exceso de tinción con papel de filtro. </li><li> Lavar rejilla una vez más en agua. </li><li> Observe red con un microscopio electrónico de transmisión. </li></ol></li></ol> 4. Conjugación química de VNPs con PEG y fluoróforos, purificación y caracterización <ol><li> Para los cálculos para las reacciones siguientes, la masa molar de los VNPs son: CPMV: 5,6x 10 6 g / mol PVX: 35 x 10 6 g / mol TMV: 41 x 10 6 g / mol BMV: 4,6 x 10 6 g / mol </li><li> Conjugar tintes y PEG a las lisinas superficiales de CPMV y PVX utilizando un solo paso N-hidroxi succinimida reacción de acoplamiento: Añadir 2.500 equivalentes molares (todos los excesos molares se refieren a un exceso molar de por VNP) de Alexa Fluor 647 éster de succinimidilo y 4.500 equivalentes de NHS- PEG (PM 5.000) disuelto en DMSO a CPMV en 0,1 M tampón fosfato de potasio. Cuando se trabaja con PVX, añadir un exceso molar de 10.000 NHS-dye y PEG-NHS. Ajustar los volúmenes de tampón y DMSO tales que la concentración final de CPMV y PVX es 2 mg / ml y el contenido de DMSO es 10% del volumen total de reacción. Incubar la mezcla de reacción durante la noche a temperatura ambiente protegido de la luz. CPMV y PVX tiene 300 y 1.270 lisinas direccionables, respectivamente. (El lector puede consultar las siguientes referencias para lecturas adicionales sobremodificación química de CPMV y PVX: 13-15). </li><li> Colorantes conjugado y PEG a tirosinas del TMV mediante un acoplamiento de diazonio seguido por el cobre (I)-catalizada azida-alquino cicloadición. <ol><li> Preparar sal de diazonio (alquino) mediante la mezcla de 400 l de 0,3 M de monohidrato de ácido p-toluenosulfónico, 25 l de nitrito de sodio 3,0 M, y 75 l de 0,68 M destilada 3-etinilanilina disolvió en acetonitrilo a 4 º C durante 1 hr. </li><li> Añadir 3,3 ml de tampón borato, pH 8,8, que contiene 100 mM NaCl a 1,25 ml de TMV (20 mg / ml de solución madre). </li><li> Reaccionar el TMV con 450 l de la sal de diazonio (alquino) de solución en un baño de hielo durante 3 horas para añadir una ligadura alquino manejar a TMV por acoplamiento de diazonio. La solución se convertirá en un color marrón claro. TMV tiene 2.140 tirosinas disponibles para la conjugación. </li><li> Se purifica el producto final utilizando un colchón de sacarosa como se describe en el paso 4,4. </li><li> Adjuntar azida-funcional Alexa Fluor 647 y PEG-azida (PM 5.000) using de cobre (I)-catalizada azida-alquino cicloadición (CuAAC). Añadir 2 equivalentes de tinte y PEG-azida por proteína de la cubierta y se incuban con 1 mM CuSO 4, 2 mM AMG, y ascorbato de sodio 2 mM a temperatura ambiente durante 15 min. Ajustar el volumen del tampón de forma que la concentración final de reacción de TMV es 2 mg / ml. (El lector puede consultar las siguientes referencias para la lectura adicional sobre la modificación química de TMV: 16,17). </li></ol></li><li> Conjugar colorantes a lisinas y PEG a cisteínas de cisteína BMV mutante (cBMV): <ol><li> Añadir 2.000 equivalentes molares de Oregon Green 488 succinimidil éster disueltos en DMSO a cBMV en 0,1 M de tampón TNKM (50 mM Tris base, 50 mM NaCl, 10 mM KCl, 5 mM MgCl 2, pH 7,4). Ajustar los volúmenes de tampón y DMSO tales que la concentración final de BMV es 1 mg / ml y el contenido de DMSO es 10% del volumen total de reacción. Incubar la mezcla de reacción durante la noche a 4 º C protegida de la luz. </li><li> Purificar las partículas mediante centrifiltros fugados como se describe en el paso 4.4. </li><li> Añadir 2.000 exceso molar de PEG-maleimida (PM 2.000) usando las mismas condiciones de reacción como antes y se incuba la mezcla de reacción durante 2 horas a 4 ° C. cBMV tiene 180 lisinas reactivas y cisteína. (El lector puede consultar las siguientes referencias para la lectura adicional sobre la modificación química de la BMV, 18). </li></ol></li><li> Purificación: Pasar la solución a través de un colchón de sacarosa al 40% (w / v) a 160.000 xg durante 2,5 hr. Volver a disolver el precipitado en tampón. Alternativamente, se dializa frente a tampón apropiada, usando 10 kDa de corte filtros spin. </li><li> Caracterización: VNPs PEGilados y marcado fluorescentemente-se analizaron usando los métodos descritos anteriormente: UV / visible de espectroscopia, la electroforesis en gel de SDS, FPLC, y TEM (no se muestra, sin embargo, se refieren a las figuras 6 y 7). </li></ol> 5. Tumor de Metas e imágenes utilizando un modelo de xenoinjertos de ratón<ol><li> Cultura HT-29 células humanas de cáncer de colon en medio RPMI suplementado con FBS al 5%, 1% de penicilina-estreptomicina, y 1% de L-glutamina, a 37 ° C en 5% de CO 2 utilizando 175 cm 2 frascos de cultivo celular. </li><li> Lavar las células dos veces con PBS estéril y la cosecha mediante la incubación con 5 ml de tripsina-EDTA a 37 º C durante 5 min. Inactivar la tripsina con 5 ml de medio RPMI. Recoger las células por centrifugación a 500 xg durante 5 min. a 4 º C y se resuspende en medio fresco RPMI a 5x10 6 células/50 medio l (determinar recuento total de células usando azul de tripano y un hemocitómetro a). Mezclar con un volumen igual de matrigel antes de la inyección (mantener todas las soluciones y reactivos estériles). </li><li> Adquirir seis semanas de edad NCR nu / nu ratones y los mantienen en una dieta libre de alfalfa durante 2 semanas. [Nota:. Todos los procedimientos con animales debe ser aprobado por el IACUC] Inducir xenoinjertos de tumores mediante inyección subcutánea de 5x10 6 células/100 l / tumor en los flancos (2 tumores / ratón) usando un manómetro 18 1/2 estérilaguja. Monitorear los animales regularmente. Medir el tamaño del tumor usando calibres y permitir que los tumores crezcan a un volumen promedio de 20 mm 3 (dentro de los próximos 12 días). Asignar los ratones en dos grupos al azar: PBS y VNP (n = 3 animales / grupo / hora punta). Uso de un 1 ml jeringa de insulina de calibre 28, administrar por la vena de la cola de inyección intravenosa 100 l de PBS estéril o 10 mg / kg formulación VNP. </li></ol> Nota: los experimentos de cultivo de tejidos y estudios con animales vivos que no se ha demostrado. Demostración práctica se limitará a procesamiento de tejidos y de adquisición de datos. Para una referencia sobre la HT-29 modelo de xenoinjerto de tumor, se remite al lector a la ref. 19 Tres técnicas se utilizan para evaluar el tumor homing de VNPs: <ol start="4"><li> Imágenes de fluorescencia mediante Maestro Imaging System: ratones sacrificio en diferentes puntos de tiempo (2, 24, y 72 h) usando CO 2gas. Disecar los animales y especiales todos los órganos principales (cerebro, corazón, pulmones, el bazo, los riñones y el hígado), junto con los tumores en los flancos, colocar los tejidos en parafilm, y analizar con el instrumento de formación de imágenes de fluorescencia utilizando excitación amarillo y filtros de emisión (800 ms exposición) para detectar la presencia de señales fluorescentes en los tejidos (derivadas de A647 etiqueta conjugarse a los VNPs). Guarde las imágenes y analizar la intensidad de fluorescencia utilizando ImageJ software 1.44o ( <a href="http://imagej.nih.gov/ij" target="_blank">http://imagej.nih.gov/ij</a> ). Comparar el patrón de absorción de los VNPs en los tumores con otros tejidos importantes con el tiempo. </li><li> Después de la imagen, recortar cada tejido por la mitad e insertar un medio en compuesto OCT para el análisis de la crio-seccionamiento y confocal. Recoge la otra mitad en predosificados crio-viales y congelar inmediatamente utilizando N2 líquido. Almacenar a -80 ° C hasta que esté listo para su posterior procesamiento. </li><li> Fluorescencia de cuantificación: Retejidos de la médula pesos. Descongelar los tejidos congelados a temperatura ambiente y colocarlos en tubos separados Falcon de 50 ml que contienen 1 ml de PBS. El uso de un homogeneizador de tejidos de mano, homogeneizar los tejidos para 2-3 min en PBS a continuación, transferir el homogenado a tubos de microcentrífuga. Se centrifuga el homogeneizado durante 10 min a 13.000 xg para eliminar la no-tejido homogeneizado. </li><li> Pipetear 100 l del sobrenadante de los tejidos de cada grupo (formulaciones PBS y VNP / puntos de tiempo) en una placa de UV 384 y negro. Evaluar la intensidad de fluorescencia (Ex / Em longitudes de onda de 600/665), utilizando un lector de placas. Normalizar los valores obtenidos fluorescentes por los pesos de los tejidos. </li><li> Inmunohistoquímica: Preparar la crio-micrótomo secciones (10 micras) y se almacena a -20 ° C. Mancha de las secciones de tejido para núcleos de las células (DAPI) y marcador de células endoteliales (marcado con FITC anti-ratón CD31 de anticuerpos). Llevar a cabo el análisis por microscopía confocal para mapear la localización intra vascular y tumoral de marcado fluorescentemente VNPs. </li></ol> Nota: Este procedimiento no se ha demostrado, los datos representativos se muestran en la Figura 8. Para una referencia sobre la inmunohistoquímica y los métodos de tinción descritos, se remite al lector a la ref. 19

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G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Icosahedral+virus+particles+as+addressable+nanoscale+building+blocks.">Icosahedral virus particles as addressable nanoscale building blocks.</a> Angew. Chem. Int. Ed. 41, 459-462 (2002).</li><li>Bruckman, M. A., Kaur, G., Lee, L. A., Xie, F., Sepulveda, J., Breitenkamp, R., Zhang, X., Joralemon, M., Russell, T. P., Emrick, T., Wang, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surface+modification+of+tobacco+mosaic+virus+with+"click"+chemistry.">Surface modification of tobacco mosaic virus with "click" chemistry.</a> Chembiochem. 9, 519-523 (2008).</li><li>Schlick, T. L., Ding, Z., Kovacs, E. W., Francis, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dual-surface+modification+of+the+tobacco+mosaic+virus.">Dual-surface modification of the tobacco mosaic virus.</a> J. Am. Chem. Soc. 127, 3718-3723 (2005).</li><li>Yildiz, I., Tsvetkova, I., Wen, A. M., Shukla, S., Masarapu, M. H., Dragnea, B., Steinmetz, N. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+of+Brome+mosaic+virus+for+biomedical+applications.">Engineering of Brome mosaic virus for biomedical applications.</a> RSC Advances. , (2012).</li><li>Brunel, F. M., Lewis, J. D., Destito, G., Steinmetz, N. F., Manchester, M., Stuhlmann, H., Dawson, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrazone+ligation+strategy+to+assemble+multifunctional+viral+nanoparticles+for+cell+imaging+and+tumor+targeting.">Hydrazone ligation strategy to assemble multifunctional viral nanoparticles for cell imaging and tumor targeting.</a> Nano Lett. 10, 1093-1097 (2010).</li><li>Shukla, S., Ablack, A., Wen, A., Lee, K., Lewis, J., Steinmetz, N. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+tumor+homing+and+tissue+penetration+of+the+filamentous+plant+viral+nanoparticle+Potato+virus+X.">Increased tumor homing and tissue penetration of the filamentous plant viral nanoparticle Potato virus X.</a> Molecular Pharmaceutics. , (2012).</li><li>Chatterji, A., Ochoa, W., Shamieh, L., Salakian, S. P., Wong, S. M., Clinton, G., Ghosh, P., Lin, T., Johnson, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+conjugation+of+heterologous+proteins+on+the+surface+of+Cowpea+mosaic+virus.">Chemical conjugation of heterologous proteins on the surface of Cowpea mosaic virus.</a> Bioconjug. Chem. 15, 807-813 (2004).</li></ol>

No hay conflictos de interés declarado.

Este protocolo proporciona un método para la modificación química de VNPs y sus aplicaciones para la formación de imágenes de tumores in vivo. Las técnicas de formación de imágenes de fluorescencia de animales, la cuantificación de fluorescencia, y la inmunohistoquímica presentados aquí son útiles para el estudio de biodistribución y la evaluación de tumor homing. Estas técnicas proporcionan información valiosa con respecto al acceso de las nanopartículas en el tumor por el efecto EPR....

<table border="1">
	<tbody>
		<tr>
			<td>Material Name</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Comments (optional)</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>VNP production</td>
		</tr>
		<tr>
			<td>Indoor plant chamber</td>
			<td>Percival Scientific</td>
			<td>E-41L2</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>V. unguiculata seeds (California black-eye no. 5)</td>
			<td>Burpee</td>
			<td>51771A</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>N. benthamiana seeds</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>N. benthamiana seeds were a gift from Salk Institute. Seeds are produced through plant propagation.</td>
		</tr>
		<tr>
			<td>Carborundum</td>
			<td>Fisher</td>
			<td>C192-500</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Pro-mix BX potting soil</td>
			<td>Premier Horticulture</td>
			<td>713400</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Jack's Professional 20-10-20 Peat-Lite Fertilizer</td>
			<td>JR Peters</td>
			<td>77860</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>50.2 Ti rotor</td>
			<td>Beckman</td>
			<td>337901</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>SW 32 Ti rotor</td>
			<td>Beckman</td>
			<td>369694</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Optima L-90K ultracentrifuge</td>
			<td>Beckman</td>
			<td>365672</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>SLA-3000 rotor</td>
			<td>Thermo Scientific</td>
			<td>07149</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>SS-34 rotor</td>
			<td>Thermo Scientific</td>
			<td>28020</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sorvall RC-6 Plus centrifuge</td>
			<td>Thermo Scientific</td>
			<td>46910</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Polypropylene bottle</td>
			<td>Beckman</td>
			<td>355607</td>
			<td>For SLA-3000 rotor</td>
		</tr>
		<tr>
			<td>Polycarbonate bottle</td>
			<td>Beckman</td>
			<td>357002</td>
			<td>For SS-34 rotor</td>
		</tr>
		<tr>
			<td>Ultra-Clear tube</td>
			<td>Beckman</td>
			<td>344058</td>
			<td>For sucrose gradient and SW 32 Ti rotor</td>
		</tr>
		<tr>
			<td>Polycarbonate bottle</td>
			<td>Beckman</td>
			<td>355618</td>
			<td>For pelleting and 50.2 Ti rotor</td>
		</tr>
		<tr>
			<td>NanoDrop spectrophotometer</td>
			<td>Thermo Scientific</td>
			<td>NanoDrop2000c</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>PowerEase 500 pre-cast gel system</td>
			<td>Invitrogen</td>
			<td>EI8675EU</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Superose 6 10/300 GL (24 ml) size-exclusion column</td>
			<td>GE Healthcare</td>
			<td>17-5172-01</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&Auml;KTA Explorer 100 Chromatograph</td>
			<td>GE Healthcare</td>
			<td>28-4062-66</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Allegra X-12R</td>
			<td>Beckman</td>
			<td>392302</td>
			<td>Benchtop centrifuge</td>
		</tr>
		<tr>
			<td>Cryostat</td>
			<td>Leica</td>
			<td>CM1850</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Maestro 2</td>
			<td>Caliper Life Sciences</td>
			<td>&nbsp;</td>
			<td>In vivo imaging system</td>
		</tr>
		<tr>
			<td>Tissue-Tearor</td>
			<td>Biospec Products</td>
			<td>985370-395</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Microplate reader</td>
			<td>Tecan</td>
			<td>Infinite-200</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Transmission electron microscope</td>
			<td>ZEISS</td>
			<td>Libra 200FE</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>FluoView laser scanning confocal microscope</td>
			<td>Olympus</td>
			<td>FV1000</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Chemicals and Reagents</td>
		</tr>
		<tr>
			<td>3-ethynylaniline</td>
			<td>Sigma Aldrich</td>
			<td>498289-5G</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>384 well black plate</td>
			<td>BD Biosciences</td>
			<td>353285</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>4-12% Bis-Tris NuPAGE SDS gel</td>
			<td>Invitrogen</td>
			<td>NP0321BOX</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>4X LDS sample buffer</td>
			<td>Invitrogen</td>
			<td>NP0008</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Acetic Acid</td>
			<td>Fisher</td>
			<td>A385-500</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Acetonitrile</td>
			<td>Sigma Aldrich</td>
			<td>271004-1L</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Alexa Fluor 647 azide</td>
			<td>Invitrogen</td>
			<td>A10277</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Alexa Fluor 647 carboxylic acid, succinimidyl ester</td>
			<td>Invitrogen</td>
			<td>A20006</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Amicon Ultra-0.5 ml Centrifugal Filters</td>
			<td>Millipore</td>
			<td>UFC501096</td>
			<td>10 kDa cut-off</td>
		</tr>
		<tr>
			<td>Aminoguanidine hydrochloride</td>
			<td>Acros Organics</td>
			<td>36891-0250</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Boric acid</td>
			<td>Fisher</td>
			<td>A74-500</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Coomassie Brilliant Blue R-250</td>
			<td>Fisher</td>
			<td>BP101-25</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>CsCl</td>
			<td>Acros Organics</td>
			<td>42285-1000</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>MP Biomedicals</td>
			<td>157574</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide</td>
			<td>Fisher</td>
			<td>BP231-100</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Filter paper</td>
			<td>Fisher</td>
			<td>09-801K</td>
			<td>P5 grade</td>
		</tr>
		<tr>
			<td>FITC anti-mouse CD31</td>
			<td>BioLegend</td>
			<td>102406</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Goat serum</td>
			<td>Invitrogen</td>
			<td>16210-064</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Fisher</td>
			<td>BP366-500</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>L-ascorbic acid sodium salt</td>
			<td>Acros Organics</td>
			<td>35268-0050</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Fisher</td>
			<td>A412P-4</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Fisher</td>
			<td>BP214-500</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Microscope slides</td>
			<td>Fisher</td>
			<td>12-544-3</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Microscope cover glass</td>
			<td>VWR</td>
			<td>48366-277</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>MOPS buffer</td>
			<td>Invitrogen</td>
			<td>NP0001</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>mPEG-mal</td>
			<td>Nanocs</td>
			<td>PG1-ML-2k</td>
			<td>MW 2000</td>
		</tr>
		<tr>
			<td>mPEG-N3</td>
			<td>Nanocs</td>
			<td>PG1-AZ-5k</td>
			<td>MW 5000</td>
		</tr>
		<tr>
			<td>mPEG-NHS</td>
			<td>Nanocs</td>
			<td>PG1-SC-5k</td>
			<td>MW 5000</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Fisher</td>
			<td>BP358-212</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Oregon Green 488 succinimidyl ester *6-isomer*</td>
			<td>Invitrogen</td>
			<td>O-6149</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>p-toluenesulfonic acid monohydrate</td>
			<td>Acros Organics</td>
			<td>13902-0050</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Permount</td>
			<td>Fisher</td>
			<td>SP15-100</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Potassium phosphate dibasic</td>
			<td>Fisher</td>
			<td>BP363-1</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Potassium phosphate monobasic</td>
			<td>Fisher</td>
			<td>BP362-1</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sodium acetate</td>
			<td>Fisher</td>
			<td>BP333-500</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sodium nitrite</td>
			<td>Acros Organics</td>
			<td>42435-0050</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sodium sulfite</td>
			<td>Amresco</td>
			<td>0628-500G</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Fisher</td>
			<td>S6-500</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>TEM grid</td>
			<td>Ted Pella</td>
			<td>FCF-400Cu</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Tris base</td>
			<td>Fisher</td>
			<td>BP152-500</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>EMD Chemicals</td>
			<td>TX1568-1</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&beta;-mercaptoethanol</td>
			<td>Fisher</td>
			<td>O3446I-100</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Tissue Culture</td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Invitrogen</td>
			<td>12483-020</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Hemocytometer</td>
			<td>Fisher</td>
			<td>0267110</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>HT-29 cells</td>
			<td>ATCC</td>
			<td>HTB-38</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Invitrogen</td>
			<td>25030-080</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Cellgro</td>
			<td>21-040-CV</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Penicillin-streptomycin</td>
			<td>Invitrogen</td>
			<td>10378-016</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>RPMI-1640</td>
			<td>Invitrogen</td>
			<td>31800-089</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Tissue culture flasks</td>
			<td>Corning</td>
			<td>431080</td>
			<td>175 cm2</td>
		</tr>
		<tr>
			<td>Trypan Blue</td>
			<td>Thermo Scientific</td>
			<td>SV30084.01</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Trypsin, 0.05% (1X) with EDTA 4Na, liquid</td>
			<td>Invitrogen</td>
			<td>25300-054</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Animal Studies</td>
		</tr>
		<tr>
			<td>18% Protein Rodent Diet</td>
			<td>Harlan Teklad</td>
			<td>Teklad Global 2018S</td>
			<td>Alfalfa free diet</td>
		</tr>
		<tr>
			<td>Insulin syringe</td>
			<td>BD Biosciences</td>
			<td>329410</td>
			<td>28 gauge</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Baxter</td>
			<td>AHN3637</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Matrigel Matrix basement membrane</td>
			<td>BD Biosciences</td>
			<td>356234</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>NCR nu/nu mice</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>CWRU School 
			of Medicine Athymic Animal and Xenograft Core Facility</td>
		</tr>
		<tr>
			<td>Sterile syringe</td>
			<td>BD Biosciences</td>
			<td>305196</td>
			<td>18 1/2 gauge</td>
		</tr>
		<tr>
			<td>Tissue-Tek CRYO-OCT Compound</td>
			<td>Andwin Scientific</td>
			<td>4583</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

viral nanoparticles for in vivo tumor imaging

El uso de nanomateriales tiene el potencial de revolucionar la medicina y ciencia de los materiales. Actualmente, un número de diferentes nanopartículas están siendo investigados para aplicaciones en la formación de imágenes y terapia. Nanopartículas virales (VNPs) derivados de plantas pueden ser considerados como auto-ensambladas bionanomaterials con tamaños definidos y formas. Los virus de plantas bajo investigación en el laboratorio de Steinmetz incluyen partículas icosaédricas formadas por virus del mosaico del caupí (CPMV) y el virus del mosaico de Brome (BMV), ambos de los cuales son 30 nm de diámetro. También estamos desarrollando estructuras en forma de varilla y filamentosas derivados de los virus de las plantas siguientes: virus del mosaico del tabaco (TMV), que forma varillas rígidas con unas dimensiones de 300 nm por 18 nm, y Potato virus X (PVX), que forman partículas filamentosas 515 nm de longitud y 13 nm de ancho (se remite al lector a las refs. 1 y 2 para obtener más información sobre VNPs). <p class="jove_content"&gt; Desde el punto de un científico de materiales de vista, VNPs son bloques de construcción atractivos por varias razones: las partículas son monodispersas, se puede producir con facilidad a gran escala en planta, son excepcionalmente estables, y biocompatible. También, VNPs son &quot;programables&quot; unidades, que pueden ser específicamente diseñados mediante modificación genética o métodos químicos bioconjugación 3. La estructura de VNPs se conoce a resolución atómica, y modificaciones pueden llevarse a cabo con precisión espacial a nivel atómico 4, un nivel de control que no se puede lograr utilizando nanomateriales sintéticos con estado actual de la técnica de las tecnologías. En este trabajo se describe la propagación de CPMV, PVX, TMV, y la BMV en Vigna ungiuculata y plantas de Nicotiana benthamiana. Protocolos de extracción y purificación para cada VNP se dan. Métodos para la caracterización de purificado y VNPs químicamente marcados se describen. En este estudio, nos centramos en chetiquetado emical de VNPs con fluoróforos (por ejemplo, Alexa Fluor 647) y polietilenglicol (PEG). Los colorantes de facilitar el seguimiento y la detección de los VNPs 5-10, y PEG reduce la inmunogenicidad de las nanopartículas proteináceos al tiempo que mejora su farmacocinética 8,11. Demostramos tumor homing de VNPs PEGilados utilizando un modelo de ratón xenograft tumor. Una combinación de imágenes de fluorescencia de tejidos ex vivo mediante Maestro del sistema de imágenes, la cuantificación de fluorescencia en tejidos homogeneizados, y la microscopía confocal se utiliza para estudiar la biodistribución. VNPs se eliminan a través del sistema reticuloendotelial (RES); tumor homing se logra pasivamente a través del aumento de la permeabilidad y retención (EPR) efecto 12. La nanotecnología VNP es un potente plug-and-play de tecnología para el tratamiento de la imagen y sitios de la enfermedad in vivo. Seguimos desarrollando VNPs para llevar cargas de drogas y restos de formación de imágenes clínicamente relevantes, así como ligandos específicos de tejido aorientar receptores moleculares sobreexpresa en el cáncer y la enfermedad cardiovascular.

<img alt="Figure 1" src="/files/ftp_upload/4352/4352fig1.jpg" /> 
 Figure 1. Plant virus-infected plants. Vigna unguiculata plants infected with CPMV (A). Nicotiana benthamiana plants infected with PVX (B), TMV (C), and BMV (D). The pictures were taken about 10 days post infection by mechanical inoculation.
<img alt="Figure 2" fo:content-width="3in" fo:src="/files/ftp_upload/4352...

Watch this Scientific Journal Video about Viral Nanoparticles for In vivo Tumor Imaging at JoVE.com

Viral Nanoparticles for In vivo Tumor Imaging

Nanopartículas de plantas virales (VNPs) son prometedoras plataformas para aplicaciones en biomedicina. A continuación, se describen los procedimientos para la propagación de plantas VNP, purificación, caracterización y bioconjugación. Finalmente, mostramos la aplicación de VNPs para la recalada del tumor y la formación de imágenes mediante un modelo de xenotrasplante de ratón y de imágenes de fluorescencia.

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

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

viral-nanoparticles-for-in-vivo-tumor-imaging

Research

JoVE Journal

Bioengineering

Viral Nanoparticles for In vivo Tumor Imaging

17.1K vistas.  Case Western Reserve University. Nanopartículas de plantas virales (VNPs) son prometedoras plataformas para aplicaciones en biomedicina. A continuación, se describen los procedimientos para la propagación de plantas VNP, purificación, caracterización y bioconjugación. Finalmente, mostramos la aplicación de VNPs para la recalada del tumor y la formación de imágenes mediante un modelo de xenotrasplante de ratón y de imágenes de fluorescencia.

Video: Viral Nanoparticles for In vivo Tumor Imaging

Simple Microfluidic Devices for in vivo Imaging of C. elegans, Drosophila and Zebrafish

Micro fabricated fluidic devices provide an accessible micro-environment for in vivo studies on small organisms. Simple fabrication processes are available for microfluidic devices using soft lithography techniques 1-3. Microfluidic devices have been used for sub-cellular imaging 4,5, in vivo laser microsurgery 2,6 and cellular imaging 4,7. In vivo imaging requires immobilization of organisms. This has been achieved using suction 5,8, tapered channels 6,7,9, deformable membranes 2-4,10, suction with additional cooling 5, anesthetic gas 11, temperature sensitive gels 12, cyanoacrylate glue 13 and anesthetics such as levamisole 14,15. Commonly used anesthetics influence synaptic transmission 16,17 and are known to have detrimental effects on sub-cellular neuronal transport 4. In this study we demonstrate a membrane based poly-dimethyl-siloxane (PDMS) device that allows anesthetic free immobilization of intact genetic model organisms such as Caenorhabditis elegans (C. elegans), Drosophila larvae and zebrafish larvae. These model organisms are suitable for in vivo studies in microfluidic devices because of their small diameters and optically transparent or translucent bodies. Body diameters range from ~10 &mu;m to ~800 &mu;m for early larval stages of C. elegans and zebrafish larvae and require microfluidic devices of different sizes to achieve complete immobilization for high resolution time-lapse imaging. These organisms are immobilized using pressure applied by compressed nitrogen gas through a liquid column and imaged using an inverted microscope. Animals released from the trap return to normal locomotion within 10 min.
We demonstrate four applications of time-lapse imaging in C. elegans namely, imaging mitochondrial transport in neurons, pre-synaptic vesicle transport in a transport-defective mutant, glutamate receptor transport and Q neuroblast cell division. Data obtained from such movies show that microfluidic immobilization is a useful and accurate means of acquiring in vivo data of cellular and sub-cellular events when compared to anesthetized animals (Figure 1J and 3C-F 4).
Device dimensions were altered to allow time-lapse imaging of different stages of C. elegans, first instar Drosophila larvae and zebrafish larvae. Transport of vesicles marked with synaptotagmin tagged with GFP (syt.eGFP) in sensory neurons shows directed motion of synaptic vesicle markers expressed in cholinergic sensory neurons in intact first instar Drosophila larvae. A similar device has been used to carry out time-lapse imaging of heartbeat in ~30 hr post fertilization (hpf) zebrafish larvae. These data show that the simple devices we have developed can be applied to a variety of model systems to study several cell biological and developmental phenomena in vivo.

Simple Microfluidic Devices for in vivo Imaging of C. elegans, Drosophila and Zebrafish

A simple microfluidic device has been developed to perform anesthetic free in vivo imaging of C. elegans, intact Drosophila larvae and zebrafish larvae. The device utilizes a deformable PDMS membrane to immobilize these model organisms in order to perform time lapse imaging of numerous processes such as heart beat, cell division and sub-cellular neuronal transport. We demonstrate the use of this device and show examples of different types of data collected from different model systems.

Simples dispositivos de microfluidos para<em&gt; In vivo</em&gt; Imágenes de<em&gt; C. elegans</em&gt;,<em&gt; Drosophila</em&gt; Y pez cebra

Micro fabricated fluidic devices provide an accessible micro-environment for in vivo studies on small organisms. Simple fabrication processes are ...

Investigación

Bioingeniería

Analysis of Targeted Viral Protein Nanoparticles Delivered to HER2+ Tumors

The HER2+ tumor-targeted nanoparticle, HerDox, exhibits tumor-preferential accumulation and tumor-growth ablation in an animal model of HER2+ cancer. HerDox is formed by non-covalent self-assembly of a tumor targeted cell penetration protein with the chemotherapy agent, doxorubicin, via a small nucleic acid linker. A combination of electrophilic, intercalation, and oligomerization interactions facilitate self-assembly into round 10-20 nm particles. HerDox exhibits stability in blood as well as in extended storage at different temperatures. Systemic delivery of HerDox in tumor-bearing mice results in tumor-cell death with no detectable adverse effects to non-tumor tissue, including the heart and liver (which undergo marked damage by untargeted doxorubicin). HER2 elevation facilitates targeting to cells expressing the human epidermal growth factor receptor, hence tumors displaying elevated HER2 levels exhibit greater accumulation of HerDox compared to cells expressing lower levels, both in vitro and in vivo. Fluorescence intensity imaging combined with in situ confocal and spectral analysis has allowed us to verify in vivo tumor targeting and tumor cell penetration of HerDox after systemic delivery. Here we detail our methods for assessing tumor targeting via multimode imaging after systemic delivery.

This article details the procedures for optical imaging analysis of the tumor-targeted nanoparticle, HerDox. In particular, detailed use of the multimode imaging device for detecting tumor targeting and assessing tumor penetration is described here.

Análisis de la proteína viral dirigida Nanopartículas Entregado a tumores HER2 +

The HER2+ tumor-targeted nanoparticle, HerDox, exhibits tumor-preferential accumulation and tumor-growth ablation in an animal model of HER2+ cancer. ...

PLGA Nanoparticles Formed by Single- or Double-emulsion with Vitamin E-TPGS

Poly(lactic-co-glycolic acid) (PLGA) is a biocompatible member of the aliphatic polyester family of biodegradable polymers. PLGA has long been a popular choice for drug delivery applications, particularly since it is already FDA-approved for use in humans in the form of resorbable sutures. Hydrophobic and hydrophilic drugs are encapsulated in PLGA particles via single- or double-emulsion. Briefly, the drug is dissolved with polymer or emulsified with polymer in an organic phase that is then emulsified with the aqueous phase. After the solvent has evaporated, particles are washed and collected via centrifugation for lyophilization and long term storage. PLGA degrades slowly via hydrolysis in aqueous environments, and encapsulated agents are released over a period of weeks to months. Although PLGA is a material that possesses many advantages for drug delivery, reproducible formation of nanoparticles can be challenging; considerable variability is introduced by the use of different equipment, reagents batch, and precise method of emulsification. Here, we describe in great detail the formation and characterization of microparticles and nanoparticles formed by single- or double-emulsion using the emulsifying agent vitamin E-TPGS. Particle morphology and size are determined with scanning electron microscopy (SEM). We provide representative SEM images for nanoparticles produced with varying emulsifier concentration, as well as examples of imaging artifacts and failed emulsifications. This protocol can be readily adapted to use alternative emulsifiers (e.g. poly(vinyl alcohol), PVA) or solvents (e.g.&#160;dichloromethane, DCM).

We describe the production and characterization of nanoparticles and microparticles composed of poly(lactic-co-glycolic acid) using vitamin E-TPGS as an emulsifier. By varying formulation parameters such as the concentration of emulsifier, it is possible to produce nanoparticles with mean diameters ranging from 220 nm to&#160;1.98 &#181;m.

Poly(lactic-co-glycolic acid) (PLGA) is a biocompatible member of the aliphatic polyester family of biodegradable polymers. PLGA has long been a popular ...

Imaging Denatured Collagen Strands In vivo and Ex vivo via Photo-triggered Hybridization of Caged Collagen Mimetic Peptides

Collagen is a major structural component of the extracellular matrix that supports tissue formation and maintenance. Although collagen remodeling is an integral part of normal tissue renewal, excessive amount of remodeling activity is involved in tumors, arthritis, and many other pathological conditions. During collagen remodeling, the triple helical structure of collagen molecules is disrupted by proteases in the extracellular environment. In addition, collagens present in many histological tissue samples are partially denatured by the fixation and preservation processes. Therefore, these denatured collagen strands can serve as effective targets for biological imaging. We previously developed a caged collagen mimetic peptide (CMP) that can be photo-triggered to hybridize with denatured collagen strands by forming triple helical structure, which is unique to collagens. The overall goals of this procedure are i)&#160;to image denatured collagen strands resulting from normal remodeling activities in vivo, and ii) to visualize collagens in ex vivo tissue sections using the photo-triggered caged CMPs. To achieve effective hybridization and successful in vivo and ex vivo imaging, fluorescently labeled caged CMPs are either photo-activated immediately before intravenous injection, or are directly activated on tissue sections. Normal skeletal collagen remolding in nude mice and collagens in prefixed mouse cornea tissue sections are imaged in this procedure. The imaging method based on the CMP-collagen hybridization technology presented here could lead to deeper understanding of the tissue remodeling process, as well as allow development of new diagnostics for diseases associated with high collagen remodeling activity.

Imaging Denatured Collagen Strands In vivo and Ex vivo via Photo-triggered Hybridization of Caged Collagen Mimetic Peptides

This procedure demonstrates in vivo near IR fluorescence imaging of collagen remodeling activities in mice as well as ex vivo staining of collagens in tissue sections using caged collagen mimetic peptides that can be photo-triggered to hybridize with denatured collagen strands.

Imaging desnaturalizadas colágeno Strands<em&gt; En vivo</em&gt; Y<em&gt; Ex vivo</em&gt; A través de Photo-desencadenada Hibridación de Caged colágeno Mimetic Péptidos

Collagen is a major structural component of the extracellular matrix that supports tissue formation and maintenance. Although collagen remodeling is an ...

Harmonic Nanoparticles for Regenerative Research

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

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

Armónica Nanopartículas para Regenerativa de Investigación

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

Biofunctionalized Prussian Blue Nanoparticles for Multimodal Molecular Imaging Applications

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

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

Biofuncionalizadas prusiana nanopartículas azul para Multimodal Molecular Aplicaciones de imágenes

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

Microwave-driven Synthesis of Iron Oxide Nanoparticles for Fast Detection of Atherosclerosis

A fast and reproducible microwave-driven protocol has been developed for the synthesis of neridronate-functionalized nanoparticles. Starting from the synthesis of hydrophobic nanoparticles, our method is based on an adaptation from thermal decomposition method to microwave driven synthesis. The new methodology produces a decrease in the reaction times in comparison with traditional procedures. Moreover, the use of the microwave technology increases the reproducibility of the reactions, something important from the point of view of clinical applications. The novelty of this iron oxide nanoparticle is the attachment of Neridronate. The use of this molecule leads a bisphosphonate moiety towards the outside of the nanoparticle that provides Ca2+ binding properties in vitro and selective accumulation in vivo in the atheroma plaque. The protocol allows the synthesis and plaque detection in about 3 hr since the initial synthesis from organic precursors. Their accumulation in the atherosclerotic area in less than 1 hr provides a contrast agent particularly suitable for clinical applications.

Microwave technology enables extremely fast synthesis of iron oxide nanoparticles for atherosclerosis plaque characterization. The use of an aminobisphosphonate in the external side of the nanoparticle provides a fast accumulation in the atherosclerotic area.

Síntesis microondas impulsada de nanopartículas de óxido de hierro para la detección rápida de la aterosclerosis

A fast and reproducible microwave-driven protocol has been developed for the synthesis of neridronate-functionalized nanoparticles. Starting from the ...

Visualizing Angiogenesis by Multiphoton Microscopy In Vivo in Genetically Modified 3D-PLGA/nHAp Scaffold for Calvarial Critical Bone Defect Repair

The reconstruction of critically sized bone defects remains a serious clinical problem because of poor angiogenesis within tissue-engineered scaffolds during repair, which gives rise to a lack of sufficient blood supply and causes necrosis of the new tissues. Rapid vascularization is a vital prerequisite for new tissue survival and integration with existing host tissue. The de novo generation of vasculature in scaffolds is one of the most important steps in making bone regeneration more efficient, allowing repairing tissue to grow into a scaffold. To tackle this problem, the genetic modification of a biomaterial scaffold is used to accelerate angiogenesis and osteogenesis. However, visualizing and tracking in vivo blood vessel formation in real-time and in three-dimensional (3D) scaffolds or new bone tissue is still an obstacle for bone tissue engineering. Multiphoton microscopy (MPM) is a novel bio-imaging modality that can acquire volumetric data from biological structures in a high-resolution and minimally-invasive manner. The objective of this study was to visualize angiogenesis with multiphoton microscopy in vivo in a genetically modified 3D-PLGA/nHAp scaffold for calvarial critical bone defect repair. PLGA/nHAp scaffolds were functionalized for the sustained delivery of a growth factor pdgf-b gene carrying lentiviral vectors (LV-pdgfb) in order to facilitate angiogenesis and to enhance bone regeneration. In a scaffold-implanted calvarial critical bone defect mouse model, the blood vessel areas (BVAs) in PHp scaffolds were significantly higher than in PH scaffolds. Additionally, the expression of pdgf-b and angiogenesis-related genes, vWF and VEGFR2, increased correspondingly. MicroCT analysis indicated that the new bone formation in the PHp group dramatically improved compared to the other groups. To our knowledge, this is the first time multiphoton microscopy was used in bone tissue-engineering to investigate angiogenesis in a 3D bio-degradable scaffold in vivo and in real-time.

Visualizing Angiogenesis by Multiphoton Microscopy In Vivo in Genetically Modified 3D-PLGA/nHAp Scaffold for Calvarial Critical Bone Defect Repair

Here, we present a protocol to visualize blood vessel formation in vivo and in real-time in 3D scaffolds by multiphoton microscopy. Angiogenesis in genetically modified scaffolds was studied in a murine calvarial critical bone defect model. More new blood vessels were detected in the treatment group than in controls.

Visualización de angiogénesis por microscopía multifotón En Vivo en modificados genéticamente 3D-PLGA/nHAp andamio para Calvarial críticos de la reparación del defecto óseo

The reconstruction of critically sized bone defects remains a serious clinical problem because of poor angiogenesis within tissue-engineered scaffolds ...

Initial Evaluation of Antibody-conjugates Modified with Viral-derived Peptides for Increasing Cellular Accumulation and Improving Tumor Targeting

Antibody-conjugates (ACs) modified with virus-derived peptides are a potentially powerful class of tumor cell delivery agents for molecular payloads used in cancer treatment and imaging due to increased cellular accumulation over current ACs. During early AC in vitro development, fluorescence techniques and radioimmunoassays are sufficient for determining intracellular localization, accumulation efficiency, and target cell specificity. Currently, there is no consensus on standardized methods for preparing cells for evaluating AC intracellular accumulation and localization. The initial testing of ACs modified with virus-derived peptides is critical especially if several candidates have been constructed. Determining intracellular accumulation by fluorescence can be affected by background signal from ACs at the cell surface and complicate the interpretation of accumulation. For radioimmunoassays, typically treated cells are fractionated and the radioactivity in different cell compartments measured. However, cell lysis varies from cell to cell and often nuclear and cytoplasmic compartments are not adequately isolated. This can produce misleading data on payload delivery properties. The intravenous injection of radiolabeled virus-derived peptide-modified ACs in tumor bearing mice followed by radionuclide imaging is a powerful method for determining tumor targeting and payload delivery properties at the in vivo phase of development. However, this is a relatively recent advancement and few groups have evaluated virus-derived peptide-modified ACs in this manner. We describe the processing of treated cells to more accurately evaluate virus-derived peptide-modified AC accumulation when using confocal microscopy and radioimmunoassays. Specifically, a method for trypsinizing cells to remove cell surface bound ACs. We also provide a method for improving cellular fractionation. Lastly, this protocol provides an in vivo method using positron emission tomography (PET) for evaluating initial tumor targeting properties in tumor-bearing mice. We use the radioisotope 64Cu (t1/2 = 12.7 h) as an example payload in this protocol.

Viral-derived peptides coupled to antibody-conjugates (ACs) is an approach gaining momentum due to the potential of delivering molecular payloads with increased tumor cell accumulation. Utilizing common methods to evaluate peptide conjugation, AC and payload intracellular accumulation, and tumor targeting, this protocol helps researchers during the key initial development phases.

Evaluación inicial de los conjugados de anticuerpos modificados con péptidos virales derivados para aumentar la acumulación celular y mejorar Tumor Targeting

Antibody-conjugates (ACs) modified with virus-derived peptides are a potentially powerful class of tumor cell delivery agents for molecular payloads used ...

Correlative Light Electron Microscopy (CLEM) for Tracking and Imaging Viral Protein Associated Structures in Cryo-immobilized Cells

Due to its high resolution, electron microscopy (EM) is an indispensable tool for virologists. However, one of the main difficulties when analyzing virus-infected or transfected cells via EM are the low efficiencies of infection or transfection, hindering the examination of these cells. In order to overcome this difficulty, light microscopy (LM) can be performed first to allocate the subpopulation of infected or transfected cells. Thus, taking advantage of the use of fluorescent proteins (FPs) fused to viral proteins, LM is used here to record the positions of the "positive-transfected" cells, expressing a FP and growing on a support with an alphanumeric pattern. Subsequently, cells are further processed for EM via high pressure freezing (HPF), freeze substitution (FS) and resin embedding. The ultra-rapid freezing step ensures excellent membrane preservation of the selected cells that can then be analyzed at the ultrastructural level by transmission electron microscopy (TEM). Here, a step-by-step correlative light electron microscopy (CLEM) workflow is provided, describing sample preparation, imaging and correlation in detail. The experimental design can be also applied to address many cell biology questions.

Correlative Light Electron Microscopy CLEM for Tracking and Imaging Viral Protein Associated Structures in Cryo-immobilized Cells

A correlative light electron microscopy (CLEM) method is applied to image virus-induced intracellular structures via electron microscopy (EM) in cells that are previously selected by light microscopy (LM). LM and EM are combined as a hybrid imaging approach to achieve an integrated view of virus-host interactions.

Correlativos luz microscopia electrónica (CLEM) para el seguimiento y proyección de imagen proteína Viral asociada a estructuras en las células inmovilizadas de Cryo

Due to its high resolution, electron microscopy (EM) is an indispensable tool for virologists. However, one of the main difficulties when analyzing ...