Este trabajo fue apoyado por la Fundación Princess Margaret Hospital Nacional de Ciencia e Ingeniería de Investigación de Canadá, el Instituto Canadiense para la Investigación de la Salud, la Fundación Canadiense de la Innovación y el Joey y Toby Tanenbaum / silla de la bola de Brasil en el cáncer de próstata Research, una subvención de la Fundación de Investigación de SUNY y soporte el arranque desde la Universidad de Buffalo.

1. Síntesis de pirofeofórbido-lípido <ol><li> Combinar 200 pirofeofórbido mmol (preparado a partir de Spirulina Pacifica algas como se describe anteriormente;... Zheng et al, Bioconj Chem., 2002, 13 -392) 4, 98,7 mg 16:00 lysophophatidylcholine (1-palmitoil-2-hidroxi-sn-glicero -3-fosfocolina, Avanti Polar Lipids # 855675), 76,3 mg de EDC, 48,7 mg de DMAP (4 - (dimetilamino) piridina) en 5 ml de cloroformo de amileno estabilizado para una proporción de 1:1:2:2 Pyro: Lípidos: EDC : DMPA. </li><li> Se agita la mezcla de reacción a temperatura ambiente bajo argón durante 24 h. Evaporar el cloroformo bajo presión reducida utilizando un evaporador rotatorio con calentamiento a 40 ° C. La mezcla de isómeros-piro piro lípidos (adjuntas en la posición sn-1 o sn-2) se produce. </li><li> Resuspender-piro lípidos mezcla de isómeros en 1 mM de CaCl2, 50 mM Tris (pH 8), 0,5% de Triton X-100 y 10% de metanol a la concentración de 5 mg / ml. Añadir fosfolipasa A2 (PLA2) de miel veneno de abeja en el/ Ml de concentración 0,1 mg e incubar la solución a 37 ° C durante 24 horas para digerir sn-1 piro-lípido y para producir la posición sn-2-piro lípidos isómero puro. </li><li> Añadir 2 volúmenes adicionales de cloroformo y 1,25 volúmenes de metanol, extraer, y luego eliminar el disolvente mediante evaporación rotatoria a presión reducida a 40 º C. Resuspender el producto de escisión en 1% de MeOH en DCM y se purifica sobre una columna de sílice diol pequeño para el isoméricamente pura piro-lípido. </li></ol> 2. Preparación de Porphysomes <ol><li> Disolver pirofeofórbido-lípido, diestearoil-sn-glicero-3-fosfoetanolamina-N-metoxi (polietilenglicol) (PEG-2000-PE) y colesterol en cloroformo separado, y registrar las concentraciones respectivamente. </li><li> Preparar porphysome película de lípido en un tubo de ensayo de borosilicato de 12x75 mm con un peso total de lípidos de 5 mg / tubo mediante la combinación de 65% molar de porfirina-lípido, 5% molar de PEG-2000-PE y 30% molar de colesterol. </li><li> Secar las películas lipídicas en una corriente de nitrogas generador y deje que se secan adicionalmente a vacío durante 1 hora. Almacenar las películas lipídicas a -20 ° C en atmósfera de argón hasta que la hidratación y extrusión futuro. </li><li> Añadir 1 ml de solución salina tamponada de fosfato (PBS, NaCl 150 mM, fosfato 10 mM, pH 7,4) a cada tubo de película de lípidos. Congelar el tubo de ensayo en nitrógeno líquido hasta que esté completamente congelado, y luego descongelar el tubo de 65 ° C baño de agua hasta que la muestra se ha derretido. Vortex el tubo para un total de 30 seg (3x en 10 segundos cada uno, con 5 segundos de re-calentamiento en 65 ° C baño de agua entre cada agitación). </li><li> Repita la congelación-descongelación para 5 ciclos o más sobre sea necesario hasta que no hay grandes agregados pueden ser observados en la solución hidratada. Deje la suspensión porphysome en hielo cuando haya terminado. </li><li> Montar la extrusora de alta presión (10 ml) con dos filtros de policarbonato apilados de 100 nm instalados pulg Conecte la extrusora thermobarrel a un circulador termostatizado para 65 ° C baño de agua circulante, y conecte el sistema extrusor a un tanque de nitrógeno de alta presiónAsegúrese de suministro. </li><li> Transferir la suspensión porphysome a la cámara de la extrusora con una pipeta de transferencia de vidrio de 9 pulgadas. Ajuste la presión girando el dial en el regulador del tanque de nitrógeno hasta que se observe una lectura de entre 200 y 300 psig como presión inicial de extrusión. Abrir la válvula del regulador de la presión gradualmente hasta que se alcanza la tasa de flujo suficiente porphysome o se alcanza un máximo de 800 psig (5440 kPa) de presión. </li><li> Después de la primera pasada de extrusión es completa, cerrar la válvula de control de presión y liberar la presión de la extrusora mediante la apertura de la válvula de alivio de presión en la parte superior del extrusor. Vuelva a llenar la extrusora con la solución porphysome recién extrusionados, y repetir la extrusión de al menos 10 veces para asegurar la solución extruido final es homogénea. </li><li> Determinar la concentración de la solución de porphysome final por la medición de la absorción de una muestra diluida en metanol por UV-Vis. Calcular la concentración usando la siguiente ecuación ingenioh el coeficiente de extinción de 97000 M -1 cm -1 a 410 nm para pirofeoforbida lípidos. </li></ol><img alt="Ecuación 1" src="/files/ftp_upload/50536/50536eq1.jpg" /><ol start="10"><li> Medir la distribución de tamaño final de porphysomes utilizando un Malvern Zetasizer ZS90 por dispersión de luz dinámica. Diluir solución porphysome en PBS y se realizan tres mediciones con 15 carreras cada uno para los resultados promediados. </li><li> Determinar la extinción de la fluorescencia de porphysome mediante la comparación de la emisión de fluorescencia de porphysome se inactivó (1 mM en PBS) y porphysome insaciable (1 mM en 1% de Triton X-100) utilizando espectrofluorómetro. Tome los espectros antes y después de que el detergente (Tirton X-100) se añade, y normalizar la señal de lectura a la fluorescencia máxima. Establezca la excitación de ser 410 nm, y la emisión de longitudes de onda para ser 600-800 nm. </li><li> Mantenga solución porphysomes bajo argón a 4 ° C hasta su uso en el futuro, y protegerlo de la luz por el aluminio faceite. </li></ol> 3. Preparación de los animales modelo de xenoinjerto <ol><li> Cultura de las células KB en RPMI-1640 (con 10% de SFB), y mantener las células en 5% de CO 2 y la atmósfera humedecida a 37 ° C. </li><li> Células Harvest KB utilizando la técnica de cultivo celular estándar. Mantener la suspensión de células (20000 células / ml) en tampón fosfato salino (PBS) en hielo durante el almacenamiento a corto plazo hasta el momento de la inyección. </li><li> Preparar una KB xenoinjertos en ratones desnudos. Los ratones desnudos son adquiridos de Charles River Laboratories cuando son 20 g (Categoría # Crl: NU (NCR)-Foxn1 nu, de 6-7 semanas de edad), teniendo en cuenta la dieta estándar y ropa de cama (Harlan Laboratorios; Dieta: irradiado LM-485, # 7912; Ropa de cama: camas Mazorca de maíz, ¼ &quot;, # 7097). Esterilizar todas las herramientas de manejo de animales (jeringas, pinzas, y la almohadilla animales) antes de los experimentos. Anestesie ratones con 2% de isoflurano v / v en el gas portador de oxígeno 100%. Todos los animales protocolos sean aprobados por la institución (Universidad Health Network en este caso) cuidado de los animales y el uso comité. </li><li> Inyectar las células tumorales suspendidas en PBS por vía subcutánea al flanco derecho de ratones. Los ratones son listo para ser utilizado después de 10 días cuando el tumor alcanza un diámetro de 4-5 mm y espesor de 2-3 mm. </li></ol> 4. In vivo la terapia fototérmica <ol><li> Inyectar porphysomes (que contienen 750 nmol piro a 200 l de volumen) por vía intravenosa a través de la vena de la cola. Llevar a cabo la irradiación láser de 24 horas después de la inyección de porphysomes. </li><li> En esta sección, llevar a cabo todos los procedimientos en una sala de seguridad del láser adjunto. Use gafas de seguridad láser durante la irradiación láser. </li><li> Configure el equipo de irradiación láser. Mantenga la fibra de láser y un difusor por una abrazadera en un soporte. Ajuste la potencia del láser a un nivel bajo y encienda el láser. Ajuste la altura de la fibra láser para lograr un área de irradiación de 9 mm de diámetro (DPSS Laser, LaserGlow Technologies, Toronto, Canadá), calibrar la potencia del láser a 750 mW (1,18 W / cm 2) utilizando apower metros y luego se apaga el láser para preparar a los animales. </li><li> Se anestesia a los animales con isoflurano al 2% v / v en el gas portador de oxígeno 100% a través de cono de la nariz sin barrido evidente de isoflurano un peligro de riesgo para la salud humana). Confirme que el animal está anestesiado suficientemente profundo mediante la realización de una pizca dedo del pie con unas pinzas o los dedos a las patas traseras de los animales. Los animales están listos para la irradiación de láser cuando no responden a este procedimiento. </li><li> Controlar la temperatura del tumor mediante una cámara infrarroja térmica (Mikroshot, LumaSense Technologies). Coloque la cámara cerca del tumor, se centran bien y tomar una imagen pre PTT, así como una imagen de luz blanca regular antes de comenzar la irradiación de PTT. </li><li> Encienda el láser de nuevo, y aumentar la potencia de luz de 750 mW. Coloque el tumor en el centro del rayo láser para hacer el tumor totalmente cubierto, y empezar a cronometrar el irradiación. Tomar la imagen de la temperatura del tumor cada 5 s durante un tratamiento con láser un minuto. Apague el láser a 1 min. Stop el suministro de gas de 2% de isoflurano, espere hasta que se recupere de la anestesia de los animales y lo devolvió a la jaula. </li><li> Mantenga ratones a normotermia sala de animales bajo las 12:12 h oscuridad ciclo y la luz de acuerdo a los animales protocolos aprobados por la Universidad Health Network. Inyectar buprenorfina (0,05 mg / kg, por vía subcutánea dos veces al día durante una semana después de la irradiación con láser para reducir el dolor causado por la cicatriz. Cada dos días, mida el diámetro del tumor usando una pinza y tomar una foto. Calcular el volumen de los tumores utilizando la siguiente ecuación: V = π / 6 · a · b 2, donde a es el diámetro de largo, y b es el corto. </li><li> Sacrificio de los animales poniéndolos en CO cámara 2 cuando el diámetro largo del tumor llega a 10 mm, ya que es el punto final definido en nuestro laboratorio. Realice dislocación cervical después del tratamiento CO2. Trazar la curva de supervivencia. </li></ol>

<ol>
	<li>Lovell, J. F., 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=Porphysome+nanovesicles+generated+by+porphyrin+bilayers+for+use+as+multimodal+biophotonic+contrast+agents.">Porphysome nanovesicles generated by porphyrin bilayers for use as multimodal biophotonic contrast agents.</a> Nat. Mater. 10, 324-332 (2011).</li><li>Lovell, J. F., 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=Enzymatic+regioselection+for+the+synthesis+and+biodegradation+of+porphysome+nanovesicles.">Enzymatic regioselection for the synthesis and biodegradation of porphysome nanovesicles.</a> Angew Chem Int Ed Engl. 51, 2429-2433 (2012).</li><li>Liu, T. W., MacDonald, T. D., Shi, J., Wilson, B. C., Zheng, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intrinsically+Copper-64-Labeled+Organic+Nanoparticles+as+Radiotracers.">Intrinsically Copper-64-Labeled Organic Nanoparticles as Radiotracers.</a> Angewandte Chemie. , (2012).</li><li>Zheng, G., 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=Low-density+lipoprotein+reconstituted+by+pyropheophorbide+cholesteryl+oleate+as+target-specific+photosensitizer.">Low-density lipoprotein reconstituted by pyropheophorbide cholesteryl oleate as target-specific photosensitizer.</a> Bioconjug Chem. 13, 392-396 (2002).</li><li>Chen, B., Pogue, B. W., Hasan, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liposomal+delivery+of+photosensitising+agents.">Liposomal delivery of photosensitising agents.</a> Expert Opin Drug Deliv. 2, 477-487 (2005).</li><li>Jin, C. S., Zheng, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liposomal+nanostructures+for+photosensitizer+delivery.">Liposomal nanostructures for photosensitizer delivery.</a> Lasers Surg Med. 43, 734-748 (2011).</li><li>Olson, F., Hunt, C. A., Szoka, F. C., Vail, W. J., Papahadjopoulos, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+liposomes+of+defined+size+distribution+by+extrusion+through+polycarbonate+membranes.">Preparation of liposomes of defined size distribution by extrusion through polycarbonate membranes.</a> Biochim Biophys Acta. 557, 9-23 (1979).</li><li>Berger, N., Sachse, A., Bender, J., Schubert, R., Brandl, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Filter+extrusion+of+liposomes+using+different+devices:+comparison+of+liposome+size,+encapsulation+efficiency,+and+process+characteristics.">Filter extrusion of liposomes using different devices: comparison of liposome size, encapsulation efficiency, and process characteristics.</a> Int J Pharm. 223, 55-68 (2001).</li><li>Jesorka, A., Orwar, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liposomes:+technologies+and+analytical+applications.">Liposomes: technologies and analytical applications.</a> Annu Rev Anal Chem (Palo Alto Calif). 1, 801-832 (2008).</li><li>Lapinski, M. M., Castro-Forero, A., Greiner, A. J., Ofoli, R. Y., Blanchard, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+liposomes+formed+by+sonication+and+extrusion:+rotational+and+translational+diffusion+of+an+embedded+chromophore.">Comparison of liposomes formed by sonication and extrusion: rotational and translational diffusion of an embedded chromophore.</a> Langmuir. 23, 11677-11683 (2007).</li><li>Liu, D., Huang, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Size+Homogeneity+of+a+Liposome+Preparation+is+Crucial+for+Liposome+Biodistribution+In+Vivo.">Size Homogeneity of a Liposome Preparation is Crucial for Liposome Biodistribution In Vivo.</a> Journal of Liposome Research. 2, 57-66 (1992).</li><li>Zhang, Y., Lovell, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Porphyrins+as+Theranostic+Agents+from+Prehistoric+to+Modern+Times.">Porphyrins as Theranostic Agents from Prehistoric to Modern Times.</a> Theranostics. 2, 905-915 (2012).</li></ol>

No tenemos nada que revelar.

En el desarrollo de tecnologías de administración de fármacos, las nanopartículas multifuncionales son actualmente en amplias investigaciones para un seguimiento preciso del vehículo de suministro de fármaco mientras se mantiene una carga útil elevada de fármaco. Liposomas de porfirina-cargada se han desarrollado para mejorar las propiedades farmacocinéticas y la entrega más eficiente que la administración directa de porfirina, sino que existen obstáculos, incluyendo la cantidad de carga restringido de porfi...

Porphysomes son novedosos nanovesículas multifuncionales que hemos desarrollado recientemente, que son capaces de imagen multimodal y la terapia 1. Se forman a partir bicapas de porfirina auto-ensambladas y contienen una gran densidad de porfirina (más de 83, 000/porphysome de partículas), que genera gran coeficiente de extinción y los resultados en la fluorescencia única de auto-extinción dependiente de la estructura. Porphysomes tienen buenas in vivo farmacocinéticos y de biodistribución propiedades: que exhiben una vida media de 12 h de sangre después de la administración sistemática, y de forma pasiva se acumulan en los tumores de xenoinjertos con 7,5% ID / g a las 24 h después de la inyección 2. Su singular estructura y las propiedades físico-químicas hacen porphysome un buen candidato para la imagen multimodal y la terapia guiada por imágenes. En primer lugar, que contiene porfirina, porphysomes son intrínsecamente adecuados para imágenes de fluorescencia de los tumores de la acumulación del tumor 1.Además, cada uno de porfirina tiene un sitio estable para radioisótopos quelantes, por lo tanto, porphysomes pueden ser fácilmente etiquetados con radioisótopos tales como 64Cu para formación de imágenes de PET 3. Además, la energía de la luz absorbida se disipa térmicamente bajo exposición de irradiación láser cuando porphysome estructura está intacta, por lo porphysomes también exhiben formación de imagen fotoacústica único y capacidades de PTT. Se ha demostrado que 24 horas después de la inyección intravenosa de porphysomes, la irradiación con láser del tumor porphysome-acumulada indujo un aumento rápido de la temperatura y fuerte ablación del tumor fototérmica. Esto demostró que porphysomes son potenciadores fototérmicos eficientes, con un coeficiente de extinción tan alto como las nanopartículas de oro (AuNPs) 1. Por otra parte, en comparación con otros agentes fototérmicos inorgánicos, incluyendo AuNPs, porphysomes muestran una ventaja excepcional en la bioseguridad debido a su naturaleza orgánica. Porphysomes son enzimáticamente biodegradable e inducen toxicit aguda mínimay en ratones con dosis intravenosas tan altas como 1000 mg / kg 1. Además, de forma similar a los liposomas, la gran núcleo acuoso de porphysomes podría ser pasiva o activamente cargado con agentes terapéuticos o de formación de imágenes. Las propiedades ópticas y biocompatibilidad de porphysomes demuestran el potencial multimodal de nanopartículas orgánicas para imagen biofotónica y la terapia. En este trabajo, presentamos el método de síntesis de los conjugados pirofeoforbida lípidos, la fabricación y el método de caracterización de porphysomes por extrusión a alta presión. In vivo PTT en ratones se lleva a cabo también para demostrar la eficiencia de porphysome habilitados PTT en el tumor tratamiento usando un modelo de tumor de xenoinjerto subcutáneo.

<table border="1">
	<tbody>
		<tr>
			<td>Name of Equipment</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments (optional)</td>
		</tr>
		<tr>
			<td>Rotary evaporator</td>
			<td>Thermo-Savant</td>
			<td>SPD131DDA</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Votex</td>
			<td>Scientific Industries</td>
			<td>SI-0236</td>
			<td>Model number: G560</td>
		</tr>
		<tr>
			<td>High pressure extruder</td>
			<td>LIPEX, Northern Lipids Inc.</td>
			<td>T.001</td>
			<td>10 ml Thermobarrel Extruder</td>
		</tr>
		<tr>
			<td>Heated bath circulators (Thermostatted circulator)</td>
			<td>Thermo SCIENTIFIC</td>
			<td>SC100-S5P</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Polycarbonate filters</td>
			<td>Avanti Polar Lipids</td>
			<td>610005</td>
			<td>Pore size 100 nm, and membrane diameter 19 mm</td>
		</tr>
		<tr>
			<td>Zetasizer</td>
			<td>Malvern Instruments</td>
			<td>ZS90</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>UV/Visible Spectrophototmeter</td>
			<td>Varian Australia</td>
			<td>Cary 50 Bio UV/ Visible Spectrophotometer</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Spectrofluorometer</td>
			<td>HORIBA Scientific</td>
			<td>FluoroMax-4</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Transmission Electron Microscopy (TEM)</td>
			<td>Hitachi</td>
			<td>H-7000</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Cell culture incubator</td>
			<td>SANYO</td>
			<td>MCO-18AIC</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Powermeter</td>
			<td>Thorlabs</td>
			<td>PM100D</td>
			<td>with sensor S142C</td>
		</tr>
		<tr>
			<td>671nm Laser</td>
			<td>LaserGlow Technologies</td>
			<td>LRS-0671_PFN-02000-05</td>
			<td>S/N:10097270</td>
		</tr>
		<tr>
			<td>Infrared Thermometer</td>
			<td>Mikroshot, LUMASENSE Technologies</td>
			<td>9102409</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Laser protective eye goggles</td>
			<td>LaserGlow Technologies</td>
			<td>AGF6602XX</td>
			<td>Optical Density: 1.5+ at 630-700 nm</td>
		</tr>
	</tbody>
</table>

one minute, sub-one-watt photothermal tumor ablation using porphysomes, intrinsic multifunctional nanovesicles

Recientemente hemos desarrollado porphysomes como nanovesículas intrínsecamente multifuncionales. Un fotosensibilizador, pirofeoforbida α, se conjugó con un fosfolípido y luego auto-ensamblado de vesículas esféricas en liposomas similares. Debido a la altísima densidad de porfirina en la bicapa lipídica-porfirina, porphysomes generan grandes coeficientes de extinción, dependiente de la estructura de la fluorescencia de auto-extinción, y una excelente eficacia fototérmica. En nuestra formulación, porphysomes se sintetizaron usando extrusión de alta presión, y muestran un tamaño medio de partícula de alrededor de 120 nm. Inyección Veinticuatro horas después de la intravenosa de porphysomes, la temperatura local del tumor aumentó de 30 ° C a 62 ° C rápidamente tras la exposición durante un minuto de 750 mW (1,18 W / cm 2), 671 nm irradiación con láser. Tras la ablación térmica completa del tumor, escaras formado y curado en 2 semanas, mientras que en los grupos de control de los tumores continuaron creciendo y todos llegaron a la linea definidapunto d dentro de 3 semanas. Estos datos muestran cómo porphysomes se pueden utilizar como terapia fototérmica potentes agentes (PTT).

Pirofeofórbido se conjuga con el fosfolípido como un monómero de lípidos estable (Figura 1a) y el conjugados auto-ensamblan para formar porphysomes por el método de extrusión de la membrana usando una extrusora de alta presión. Por lo general es difícil de extruir suspensión porphysome lípidos en los 3 primeros ciclos de extrusión con una tasa de flujo relativamente lento. Como se repiten más extrusiones, la tasa de flujo de la solución extruido aumenta gradualmente, y la presión se puede ...

Watch this Scientific Journal Video about One Minute, Sub-One-Watt Photothermal Tumor Ablation Using Porphysomes, Intrinsic Multifunctional Nanovesicles at JoVE.com

One Minute, Sub-One-Watt Photothermal Tumor Ablation Using Porphysomes, Intrinsic Multifunctional Nanovesicles

Desarrollamos nuevos nanovesículas multifuncionales intrínsecas llamados porphysomes, que tienen la estructura dependiente de fluorescencia propiedades fototérmicos únicas de auto-extinción y, por lo que funciona agentes de terapia fototérmica como potentes. Formulamos porphysomes por extrusión a alta presión y se investigó su eficacia de la terapia fototérmica en un modelo de tumor de xenotrasplante.

Un Minuto, Sub-Uno-Watt fototérmica Ablación Usando Porphysomes, Intrinsic Multifuncional nanovesículas

We recently developed porphysomes as intrinsically multifunctional nanovesicles. A photosensitizer, pyropheophorbide &alpha;, was conjugated to a ...

one-minute-sub-one-watt-photothermal-tumor-ablation-using-porphysomes

Research

JoVE Journal

Bioengineering

10.7K vistas.  University of Toronto.  Desarrollamos nuevos nanovesículas multifuncionales intrínsecas llamados porphysomes, que tienen la estructura dependiente de fluorescencia propiedades fototérmicos únicas de auto-extinción y, por lo que funciona agentes de terapia fototérmica como potentes. Formulamos porphysomes por extrusión a alta presión y se investigó su eficacia de la terapia fototérmica en un modelo de tumor de xenotrasplante. 

Video: One Minute, Sub-One-Watt Photothermal Tumor Ablation Using Porphysomes, Intrinsic Multifunctional Nanovesicles

Contrast Ultrasound Targeted Treatment of Gliomas in Mice via Drug-Bearing Nanoparticle Delivery and Microvascular Ablation

We are developing minimally-invasive contrast agent microbubble based therapeutic approaches in which the permeabilization and/or ablation of the microvasculature are controlled by varying ultrasound pulsing parameters.  Specifically, we are testing whether such approaches may be used to treat malignant brain tumors through drug delivery and microvascular ablation.  Preliminary studies have been performed to determine whether targeted drug-bearing nanoparticle delivery can be facilitated by the ultrasound mediated destruction of "composite" delivery agents comprised of 100nm poly(lactide-co-glycolide) (PLAGA) nanoparticles that are adhered to albumin shelled microbubbles. We denote these agents as microbubble-nanoparticle composite agents (MNCAs). When targeted to subcutaneous C6 gliomas with ultrasound, we observed an immediate 4.6-fold increase in nanoparticle delivery in MNCA treated tumors over tumors treated with microbubbles co-administered with nanoparticles and a 8.5 fold increase over non-treated tumors. Furthermore, in many cancer applications, we believe it may be desirable to perform targeted drug delivery in conjunction with ablation of the tumor microcirculation, which will lead to tumor hypoxia and apoptosis. To this end, we have tested the efficacy of non-theramal cavitation-induced microvascular ablation, showing that this approach elicits tumor perfusion reduction, apoptosis, significant growth inhibition, and necrosis. Taken together, these results indicate that our ultrasound-targeted approach has the potential to increase therapeutic efficiency by creating tumor necrosis through microvascular ablation and/or simultaneously enhancing the drug payload in gliomas.

Insonation of microbubbles is a promising strategy for tumor ablation at reduced time-averaged acoustic powers, as well as for the targeted delivery of therapeutics. The purpose of the present study is to develop low duty cycle ultrasound pulsing strategies and nanocarriers to maximize non-thermal microvascular ablation and payload delivery to subcutaneous C6 gliomas.

Ecografía de contraste dirigidos tratamiento de los gliomas en ratones a través de la entrega de nanopartículas de drogas y la ablación Teniendo microvascular

We are developing minimally-invasive contrast agent microbubble based therapeutic approaches in which the permeabilization and/or ablation of the ...

Investigación

Bioingeniería

Optical Frequency Domain Imaging of Ex vivo Pulmonary Resection Specimens: Obtaining One to One Image to Histopathology Correlation

Lung cancer is the leading cause of cancer-related deaths1. Squamous cell and small cell cancers typically arise in association with the conducting airways, whereas adenocarcinomas are typically more peripheral in location. Lung malignancy detection early in the disease process may be difficult due to several limitations: radiological resolution, bronchoscopic limitations in evaluating tissue underlying the airway mucosa and identifying early pathologic changes, and small sample size and/or incomplete sampling in histology biopsies. High resolution imaging modalities, such as optical frequency domain imaging (OFDI), provide non-destructive, large area 3-dimensional views of tissue microstructure to depths approaching 2 mm in real time (Figure 1)2-6. OFDI has been utilized in a variety of applications, including evaluation of coronary artery atherosclerosis6,7 and esophageal intestinal metaplasia and dysplasia6,8-10.
Bronchoscopic OCT/OFDI has been demonstrated as a safe in vivo imaging tool for evaluating the pulmonary airways11-23 (Animation). OCT has been assessed in pulmonary airways16,23 and parenchyma17,22 of animal models and in vivo human airway14,15. OCT imaging of normal airway has demonstrated visualization of airway layering and alveolar attachments, and evaluation of dysplastic lesions has been found useful in distinguishing grades of dysplasia in the bronchial mucosa11,12,20,21. OFDI imaging of bronchial mucosa has been demonstrated in a short bronchial segment (0.8 cm)18. Additionally, volumetric OFDI spanning multiple airway generations in swine and human pulmonary airways in vivo has been described19. Endobronchial OCT/OFDI is typically performed using thin, flexible catheters, which are compatible with standard bronchoscopic access ports. Additionally, OCT and OFDI needle-based probes have recently been developed, which may be used to image regions of the lung beyond the airway wall or pleural surface17.
While OCT/OFDI has been utilized and demonstrated as feasible for in vivo pulmonary imaging, no studies with precisely matched one-to-one OFDI:histology have been performed. Therefore, specific imaging criteria for various pulmonary pathologies have yet to be developed. Histopathological counterparts obtained in vivo consist of only small biopsy fragments, which are difficult to correlate with large OFDI datasets. Additionally, they do not provide the comprehensive histology needed for registration with large volume OFDI. As a result, specific imaging features of pulmonary pathology cannot be developed in the in vivo setting. Precisely matched, one-to-one OFDI and histology correlation is vital to accurately evaluate features seen in OFDI against histology as a gold standard in order to derive specific image interpretation criteria for pulmonary neoplasms and other pulmonary pathologies. Once specific imaging criteria have been developed and validated ex vivo with matched one-to-one histology, the criteria may then be applied to in vivo imaging studies. Here, we present a method for precise, one to one correlation between high resolution optical imaging and histology in ex vivo lung resection specimens. Throughout this manuscript, we describe the techniques used to match OFDI images to histology. However, this method is not specific to OFDI and can be used to obtain histology-registered images for any optical imaging technique. We performed airway centered OFDI with a specialized custom built bronchoscopic 2.4 French (0.8 mm diameter) catheter. Tissue samples were marked with tissue dye, visible in both OFDI and histology. Careful orientation procedures were used to precisely correlate imaging and histological sampling locations. The techniques outlined in this manuscript were used to conduct the first demonstration of volumetric OFDI with precise correlation to tissue-based diagnosis for evaluating pulmonary pathology24. This straightforward, effective technique may be extended to other tissue types to provide precise imaging to histology correlation needed to determine fine imaging features of both normal and diseased tissues.

Optical Frequency Domain Imaging of Ex vivo Pulmonary Resection Specimens: Obtaining One to One Image to Histopathology Correlation

A method to image ex vivo pulmonary resection specimens with optical frequency domain imaging (OFDI) and obtain precise correlation to histology is described, which is essential to developing specific OFDI interpretation criteria for pulmonary pathology. This method is applicable to other tissue types and imaging techniques to obtain precise imaging to histology correlation for accurate image interpretation and assessment. Imaging criteria established with this technique would then be applicable to image assessment in future in vivo studies.

Frecuencia de imagen óptica de dominio de<em&gt; Ex vivo</em&gt; Piezas de resección pulmonar: Obtención de One to One de imagen para estudio histopatológico de correlación

Lung cancer is the leading cause of cancer-related deaths1. Squamous cell and small cell cancers typically arise in association with the conducting ...

Viral Nanoparticles for In vivo Tumor Imaging

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

Viral Nanoparticles for In vivo Tumor Imaging

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

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

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

Construction of a Preclinical Multimodality Phantom Using Tissue-mimicking Materials for Quality Assurance in Tumor Size Measurement

World Health Organization (WHO) and the Response Evaluation Criteria in Solid Tumors (RECIST) working groups advocated standardized criteria for radiologic assessment of solid tumors in response to anti-tumor drug therapy in the 1980s and 1990s, respectively. WHO criteria measure solid tumors in two-dimensions, whereas RECIST measurements use only one-dimension which is considered to be more reproducible 1, 2, 3,4,5. These criteria have been widely used as the only imaging biomarker approved by the United States Food and Drug Administration (FDA) 6. In order to measure tumor response to anti-tumor drugs on images with accuracy, therefore, a robust quality assurance (QA) procedures and corresponding QA phantom are needed.
To address this need, the authors constructed a preclinical multimodality (for ultrasound (US), computed tomography (CT) and magnetic resonance imaging (MRI)) phantom using tissue-mimicking (TM) materials based on the limited number of target lesions required by RECIST by revising a Gammex US commercial phantom 7. The Appendix in Lee et al. demonstrates the procedures of phantom fabrication 7. In this article, all protocols are introduced in a step-by-step fashion beginning with procedures for preparing the silicone molds for casting tumor-simulating test objects in the phantom, followed by preparation of TM materials for multimodality imaging, and finally construction of the preclinical multimodality QA phantom. The primary purpose of this paper is to provide the protocols to allow anyone interested in independently constructing a phantom for their own projects. QA procedures for tumor size measurement, and RECIST, WHO and volume measurement results of test objects made at multiple institutions using this QA phantom are shown in detail in Lee et al. 8.

This paper describes in-house procedures of constructing a preclinical multimodality phantom made of tissue-mimicking (TM) materials for quality assurance (QA) of tumor size measurement in animal imaging modalities such as ultrasound (US), computed tomography (CT) and magnetic resonance imaging (MRI).

Construcción de un Phantom multimodalidad preclínicos Utilización de los materiales de tejidos que imitan para la Garantía de la Calidad en la medida del tamaño del tumor

World Health Organization (WHO) and the Response Evaluation Criteria in Solid Tumors (RECIST) working groups advocated standardized criteria for ...

Production of Large Numbers of Size-controlled Tumor Spheroids Using Microwell Plates

Tumor spheroids are increasingly recognized as an important in vitro model for the behavior of tumor cells in three dimensions. More physiologically relevant than conventional adherent-sheet cultures, they more accurately recapitulate the complexity and interactions present in real tumors. In order to harness this model to better assess tumor biology, or the efficacy of novel therapeutic agents, it is necessary to be able to generate spheroids reproducibly, in a controlled manner and in significant numbers.
The AggreWell system consists of a high-density array of pyramid-shaped microwells, into which a suspension of single cells is centrifuged. The numbers of cells clustering at the bottom of each microwell, and the number and ratio of distinct cell types involved depend only on the properties of the suspension introduced by the experimenter. Thus, we are able to generate tumor spheroids of arbitrary size and composition without needing to modify the underlying platform technology. The hundreds of microwells per square centimeter of culture surface area in turn ensure that extremely high production levels may be attained via a straightforward, nonlabor-intensive process. We therefore expect that this protocol will be broadly useful to researchers in the tumor spheroid field.

We introduce a protocol for the generation of large numbers (thousands to hundreds of thousands) of uniform size- and composition-controlled tumor spheroids, using commercially available microwell plates.

La producción de grandes cantidades de esferoides tumorales Tamaño controlados utilizando placas de micropocillos

Tumor spheroids are increasingly recognized as an important in vitro model for the behavior of tumor cells in three dimensions. More physiologically ...

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 ...

Longitudinal Measurement of Extracellular Matrix Rigidity in 3D Tumor Models Using Particle-tracking Microrheology

The mechanical microenvironment has been shown to act as a crucial regulator of tumor growth behavior and signaling, which is itself remodeled and modified as part of a set of complex, two-way mechanosensitive interactions. While the development of biologically-relevant 3D tumor models have facilitated mechanistic studies on the impact of matrix rheology on tumor growth, the inverse problem of mapping changes in the mechanical environment induced by tumors remains challenging. Here, we describe the implementation of particle-tracking microrheology (PTM) in conjunction with 3D models of pancreatic cancer as part of a robust and viable approach for longitudinally monitoring physical changes in the tumor microenvironment, in situ. The methodology described here integrates a system of preparing in vitro 3D models embedded in a model extracellular matrix (ECM) scaffold of Type I collagen with fluorescently labeled probes uniformly distributed for position- and time-dependent microrheology measurements throughout the specimen. In vitro tumors are plated and probed in parallel conditions using multiwell imaging plates. Drawing on established methods, videos of tracer probe movements are transformed via the Generalized Stokes Einstein Relation (GSER) to report the complex frequency-dependent viscoelastic shear modulus, G*(&#969;). Because this approach is imaging-based, mechanical characterization is also mapped onto large transmitted-light spatial fields to simultaneously report qualitative changes in 3D tumor size and phenotype. Representative results showing contrasting mechanical response in sub-regions associated with localized invasion-induced matrix degradation as well as system calibration, validation data are presented. Undesirable outcomes from common experimental errors and troubleshooting of these issues are also presented. The 96-well 3D culture plating format implemented in this protocol is conducive to correlation of microrheology measurements with therapeutic screening assays or molecular imaging to gain new insights into impact of treatments or biochemical stimuli on the mechanical microenvironment.

Particle-tracking microrheology can be used to non-destructively quantify and spatially map changes in extracellular matrix mechanical properties in 3D tumor models.

Medida longitudinal de la matriz extracelular Rigidez en modelos de tumores en 3D utilizando partículas de seguimiento Microrheology

The mechanical microenvironment has been shown to act as a crucial regulator of tumor growth behavior and signaling, which is itself remodeled and ...

Real-time Monitoring of High Intensity Focused Ultrasound (HIFU) Ablation of In Vitro Canine Livers Using Harmonic Motion Imaging for Focused Ultrasound (HMIFU)

Harmonic Motion Imaging for Focused Ultrasound (HMIFU) is a technique that can perform and monitor high-intensity focused ultrasound (HIFU) ablation. An oscillatory motion is generated at the focus of a 93-element and 4.5 MHz center frequency HIFU transducer by applying a 25 Hz amplitude-modulated signal using a function generator. A 64-element and 2.5 MHz imaging transducer with 68kPa peak pressure is confocally placed at the center of the HIFU transducer to acquire the radio-frequency (RF) channel data. In this protocol, real-time monitoring of thermal ablation using HIFU with an acoustic power of 7 W on canine livers in vitro is described. HIFU treatment is applied on the tissue during 2 min and the ablated region is imaged in real-time using diverging or plane wave imaging up to 1,000 frames/second. The matrix of RF channel data is multiplied by a sparse matrix for image reconstruction. The reconstructed field of view is of 90&#176; for diverging wave and 20 mm for plane wave imaging and the data are sampled at 80 MHz. The reconstruction is performed on a Graphical Processing Unit (GPU) in order to image in real-time at a 4.5 display frame rate. 1-D normalized cross-correlation of the reconstructed RF data is used to estimate axial displacements in the focal region. The magnitude of the peak-to-peak displacement at the focal depth decreases during the thermal ablation which denotes stiffening of the tissue due to the formation of a lesion. The displacement signal-to-noise ratio (SNRd) at the focal area for plane wave was 1.4 times higher than for diverging wave showing that plane wave imaging appears to produce better displacement maps quality for HMIFU than diverging wave imaging.

Real-time Monitoring of High Intensity Focused Ultrasound HIFU Ablation of In Vitro Canine Livers Using Harmonic Motion Imaging for Focused Ultrasound HMIFU

This article describes real-time monitoring of HIFU ablation in canine liver with high frame rate ultrasound imaging using diverging and plane wave imaging. Harmonic Motion Imaging for Focused Ultrasound is used to image the decrease of acoustic radiation force induced displacement in the ablated region.

Monitoreo en tiempo real de Ultrasonido de alta intensidad (HIFU) Ablación de<em&gt; In Vitro</em&gt; Canine hígados Uso Movimiento armónico Imaging para Ultrasonido Focalizado (HMIFU)

Harmonic Motion Imaging for Focused Ultrasound (HMIFU) is a technique that can perform and monitor high-intensity focused ultrasound (HIFU) ablation. An ...

Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber

In reconstructive surgery, there is a clinical need for an alternative to the current methods of autologous reconstruction which are complex, costly and trade one defect for another. Tissue engineering holds the promise to address this increasing demand. However, most tissue engineering strategies fail to generate stable and functional tissue substitutes because of poor vascularization. This paper focuses on an in vivo tissue engineering chamber model of intrinsic vascularization where a perfused artery and a vein either as an arteriovenous loop or a flow-through pedicle configuration is directed inside a protected hollow chamber. In this chamber-based system angiogenic sprouting occurs from the arteriovenous vessels and this system attracts ischemic and inflammatory driven endogenous cell migration which gradually fills the chamber space with fibro-vascular tissue. Exogenous cell/matrix implantation at the time of chamber construction enhances cell survival and determines specificity of the engineered tissues which develop. Our studies have shown that this chamber model can successfully generate different tissues such as fat, cardiac muscle, liver and others. However, modifications and refinements are required to ensure target tissue formation is consistent and reproducible. This article describes a standardized protocol for the fabrication of two different vascularized tissue engineering chamber models in vivo.

Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber

This is a guideline for constructing in vivo vascularized tissue using a microsurgical arteriovenous loop or a flow-through pedicle configuration inside a tissue engineering chamber. The vascularized tissues generated can be employed for organ regeneration and replacement of tissue defects, as well as for drug testing and disease modeling.

Ingeniería de tejidos por intrínseca Vascularización en una<em&gt; En Vivo</em&gt; Cámara Ingeniería de Tejidos

In reconstructive surgery, there is a clinical need for an alternative to the current methods of autologous reconstruction which are complex, costly and ...

A Rapid and Chemical-free Hemoglobin Assay with Photothermal Angular Light Scattering

Photo-thermal angular light scattering (PT-AS) is a novel optical method for measuring the hemoglobin concentration ([Hb]) of blood samples. On the basis of the intrinsic photothermal response of hemoglobin molecules, the sensor enables high-sensitivity, chemical-free measurement of [Hb]. [Hb] detection capability with a limit of 0.12 g/dl over the range of 0.35 - 17.9 g/dl has been demonstrated previously. The method can be readily implemented using inexpensive consumer electronic devices such as a laser pointer and a webcam. The use of a micro-capillary tube as a blood container also enables the hemoglobin assay with a nanoliter-scale blood volume and a low operating cost. Here, detailed instructions for the PT-AS optical setup and signal processing procedures are presented. Experimental protocols and representative results for blood samples in anemic conditions ([Hb] = 5.3, 7.5, and 9.9 g/dl) are also provided, and the measurements are compared with those from a hematology analyzer. Its simplicity in implementation and operation should enable its wide adoption in clinical laboratories and resource-limited settings.

A photo-thermal angular light scattering (PT-AS) sensor enables the rapid and chemical-free hemoglobin assay of nanoliter-scale blood samples. Here, details of the PT-AS setup and a measurement protocol for the hemoglobin concentration in blood are provided. Representative results for anemic blood samples are also presented.

Un ensayo de hemoglobina rápida y libre de químicos con fototérmica angular de dispersión de luz

Photo-thermal angular light scattering (PT-AS) is a novel optical method for measuring the hemoglobin concentration ([Hb]) of blood samples. On the basis ...