Otorguen ayuda ha incluido NHLBI HL080532, NHLBI HL073449, CNRR RR16453, y una subvención nacional de la AHA en la concesión de ayuda (a RVS). Un especial agradecimiento se extiende al diseño de cursos a distancia y Consultoría (DCDC) del grupo, dcdcgroup.org, por su ayuda en la producción de video y el Departamento de Educación de EE.UU. Donación No. P336C050047 que fundó la DCDC.

1. Microburbujas preparación de la pasta <ol><li> En 10 ml de PBS mezclar 200 mg 1,2-dipalmitoil-sn-glicero-3-fosfatidilcolina y 50 mg de 1,2-dipalmitoil-sn-glicero-3-fosfatidiletanolamina con g de glucosa 1. </li><li> Calentar la mezcla en baño de agua hirviendo 20-30 minutos, una pipeta de mezcla cada 5 minutos. </li><li> La solución se puede almacenar a 4 ° C durante un máximo de 6 meses. </li></ol> 2. Preparación de microburbujas <ol><li> Tome un 250 l de la solución preparada de microburbujas y se incuba a 40 ° C durante 15 minutos. </li><li> La solución de microburbujas pre-calentado se transfiere entonces a un microtubo de 1,5 ml que contiene 50 l de glicerol. </li><li> 2.1 mg de ADN purificado de plásmido que codifica para una construcción de expresión de los genes de interés (en este ejemplo purificada por Qiagen endotoxinas MegaPrep kit, Qiagen, Germantown, MD, con una concentración óptima de 4mg/ml). 2.4) tampón fosfato salino se añade a un volumen final de 500 microlitros. Endotoxinas maxipreps Qiagen se utilizan, así como de PBS estéril con el fin de garantizar la esterilidad. </li><li> El aire en el microtubo luego se reemplaza con gas octafluoropropano. </li><li> El microtubo A continuación se agita vigorosamente en un amalgamador dental durante 20 segundos. </li><li> El subnatant de ADN que contienen residuales y de amortiguación que no se haya unido a las microburbujas es cuidadosamente removida y la capa de microburbujas se lavaron tres veces con PBS estéril para extraer el ADN no las tienen, y se coloca en hielo entre cada ciclo de lavado. Por lo general alcanzar una eficacia vinculante de 30-40%. Todos los reactivos son estériles y la atención se hace para minimizar la contaminación. </li><li> El plásmido ADN-vinculados microburbujas se colocan en hielo durante hasta dos horas hasta su uso. </li><li> El subnatant retirado del microtubo tras la mezcla y la PBS se lava, se puede utilizar para determinar la concentración de ADN no consolidadas, y también la cantidad límite basado en la concentración inicial conocida, mediante la medición de la densidad óptica de esta solución en una longitud de onda de 260 nm utilizando un espectrofotómetro. </li></ol> 3. Equipo de calibración <ol><li> Antes del primer uso, un transductor de 1 MHz cavitación debe ser calibrado para asegurar índice mecánico y de repetición del pulso. Un sumergible de 1 MHz, transductor de 13mm, fuera de foco está conectado a una función de 20 MHz / generador de forma de onda arbitraria a través de un amplificador de potencia. </li><li> El transductor se coloca en un recipiente de plástico lleno de agua, dirigida directamente a un hidrófono, que ha sido conectado a un osciloscopio de 500 MHz a través de un amplificador de carga. </li><li> Forma de onda, frecuencia, amplitud, ciclo de explosión, y amplificación de potencia pueden ser modificadas para obtener el ciclo de trabajo adecuado y el índice de mecánica óptima a cavitar las microburbujas. Para este experimento, se ha calibrado el sistema a un índice mecánico equivalente a aproximadamente, de 1,3 a 1 MHz. </li></ol> 4. Entrega de microburbujas y UTMD <ol><li> Antes de la entrega de microburbujas y UTMD, los ratones C57BL / 6 fueron anestesiados con ketamina y xilazina 100mg/kg 5mg/kg a través de una inyección IP. </li><li> La entrega de microburbujas se administra por vía intravenosa a través de una inyección directa en el ventrículo izquierdo del corazón a través de cateterismo o vena de la cola. Para la inyección cardíaca directa, un volumen de hasta 100 l de ADN del plásmido cargado solución de microburbujas se inyecta a través de una aguja de calibre 30 insertada en el espacio intercostal 4 ª anterior bajo visualización ultrasónica en el ventrículo izquierdo del corazón. </li><li> El bolo de solución de microburbujas resultantes de la inyección interventricular izquierda se visualiza usando Visual Sonics 38 MHz de alta frecuencia de un transductor de ultrasonido colocado en una posición fija en el tórax del ratón en una vista de eje largo con VisualSonics &#39;Vevo 2100 Sistema de Imagen. Todas las jeringas y las agujas son estériles, asegurar el ambiente más estéril posible que estas inyecciones. </li><li> Inmediatamente después de la inyección, la destrucción de microburbujas se lleva a cabo de ~ 5 minutos con un segundo, de menor tamaño de baja frecuencia 1.0 MHz transductor sostiene directamente sobre el órgano deseado, apuntando a la destrucción de esta región. En este ejemplo, el ultrasonido se administró en el hígado con una frecuencia de repetición de pulso de 1,0 MHz, con un índice mecánico equivalente a aproximadamente 1.3 a 1.5, y el período de repetición del pulso de 100 ms por cada 20 ciclos. Por otra parte, el pulso puede ser cerrada en el ECG del ratón (no se muestra en este experimento) con una ráfaga de 3 fotogramas de la ecografía, cada 4-6 ciclos cardíacos. Se obtiene una mayor eficiencia de la transfección con los protocolos que permiten que el lecho capilar para volver a llenar de burbujas entre los estallidos de los ultrasonidos. </li></ol> 5. Envío Método alternativo Hemos elegido para poner de relieve la inyección interventricular debido a la complejidad del procedimiento, pero en muchos casos, como la infusión prolongada de microburbujas, una vena de la cola eninyección es el método preferido. Para el método de la cola de la vena de la entrega de microburbujas, el ratón se anestesia la misma manera. Una jeringa que contiene el plásmido ADN-vinculados microburbujas está conectado a un catéter de la vena la aguja de calibre 27 / cola. El catéter de vena de la cola se inserta en el tercio distal de cualquiera de las venas lateral derecha o la izquierda que a lo largo de la cola del ratón. La jeringa que contiene las microburbujas se coloca en una bomba de infusión que administra automáticamente un volumen uniforme preestablecido de solución en un periodo de tiempo preestablecido. Por lo general infundir 200-300μl a una tasa de 3ml/hour. El uso de animales Todos los animales fueron manejados de acuerdo con la práctica correcta de los animales según lo definido por las autoridades nacionales y / o los organismos locales de protección de los animales, y todos los animales de trabajo fue aprobado por la comisión competente (Universidad de Hawaii Cuidado de Animales institucional y el empleo, el número de autorización 07-100 -3). Anestesia adecuada (ketamina / zylazine) fue utilizado y los analgésicos (bupivicaína y buprenorfina) estaban disponibles, aunque no es necesario. 6. Los resultados representativos: La efectividad de la entrega de ADN mediada por plásmidos UTMD se puede evaluar a través de una variedad de métodos, dependiendo de los genes codificados en la construcción, tales como, pero no limitado a, la luciferasa de imágenes in vivo, B-gal tinción ex vivo, y / o inmunohistología. En particular, en imágenes de bioluminiscencia in vivo permite monitorear la presencia y duración de la expresión génica en ratones serie transfectadas con un plásmido que codifica un gen bioluminiscente (luciferasa). El Xenogen En Vivo Imaging System (IVIS) (Caliper Life Sciences, Hopkinton, MA) se utiliza para obtener imágenes de bioluminiscencia. Las imágenes se suelen considerar el conjunto de los ratones del primer día después de la transfección mediada UTMD y se repite cada tres o cuatro días hasta que la expresión del gen bioluminiscente ya no es perceptible a simple vista a través del sistema (Figura 1). Para preparar los ratones para obtener imágenes de bioluminiscencia, los primeros ratones una inyección IP de la luciferasa de la sonda D-luciferina (Caliper Life Sciences) y luego son anestesiados o 3 minutos más tarde. Biodistribución del sustrato D-luciferina se dejó transcurrir durante unos 10 minutos antes de que el animal se coloca en la cámara de IVIS de imágenes y un escáner de cuerpo completo la imagen tomada. Durante la adquisición, los fotones emitidos por la luciferasa de luciérnaga / D-luciferina reacción fotoquímica se miden. La Figura 1 también ilustra similares imágenes IVIS bioluminiscente del hígado después de UTMD, y la Figura 2 es una de epifluorescencia (100X) la imagen del hígado transfectadas utilizando un anticuerpo primario anti-luciferasa (Sigma-Aldrich) y AlexFluor-568 anticuerpo secundario conjugado (Invitrogen) . Es claro ver que la transfección mediada por el hígado UTMD ha afectado no sólo a las células endoteliales, pero el hepatocyes así como. <img src="/files/ftp_upload/2963/2963fig1.jpg" alt="Figura 1" /> Figura 1 Xenogen / IVIS imagen cardiaca de los ratones tratados UTMD (A) del ratón control negativo:.. + Plásmido PBS seguido cardiaca dirigido UTMD, (B) Tratamiento del ratón: + plásmido microburbujas seguido cardiaca dirigido UTMD, y (C) El tratamiento del ratón : microburbujas plásmido + seguido por el hígado dirigido UTMD. <img src="/files/ftp_upload/2963/2963fig2.jpg" alt="Figura 2" /> Figura 2. Inmunohistoquímica de hígado UTMD (anti-luciferasa en rojo). (A) plásmido sin UTMD, y (B) con el plásmido UTMD. Imagen confocal (100X), los núcleos son de color azul manchado DAPI.

<ol>
	<li>Bekeredjian, R., Chen, S., Frenkel, P. A., Grayburn, P. A., Shohet, R. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrasound-targeted+microbubble+destruction+can+repeatedly+direct+highly+specific+plasmid+expression+to+the+heart.">Ultrasound-targeted microbubble destruction can repeatedly direct highly specific plasmid expression to the heart.</a> Circulation. 108, 1022-1026 (2003).</li><li>Bekeredjian, R., Katus, H. A., Kuecherer, H. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Therapeutic+use+of+ultrasound+targeted+microbubble+destruction:+a+review+of+non-cardiac+applications.">Therapeutic use of ultrasound targeted microbubble destruction: a review of non-cardiac applications.</a> Ultraschall Med. 27, 134-140 (2006).</li><li>Chen, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regeneration+of+pancreatic+islets+in+vivo+by+ultrasound-targeted+gene+therapy.">Regeneration of pancreatic islets in vivo by ultrasound-targeted gene therapy.</a> Gene Ther. 17, 1411-1420 (2010).</li><li>Miao, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrasound+enhances+gene+delivery+of+human+factor+IX+plasmid.">Ultrasound enhances gene delivery of human factor IX plasmid.</a> Hum Gene Ther. 16, 893-905 (2005).</li><li>Shimoda, M., Chen, S., Noguchi, H., Matsumoto, S., Grayburn, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+non-viral+gene+delivery+of+human+vascular+endothelial+growth+factor+improves+revascularisation+and+restoration+of+euglycaemia+after+human+islet+transplantation+into+mouse+liver.">In vivo non-viral gene delivery of human vascular endothelial growth factor improves revascularisation and restoration of euglycaemia after human islet transplantation into mouse liver.</a> Diabetologia. 53, 1669-1679 (2010).</li><li>Shohet, R. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Echocardiographic+destruction+of+albumin+microbubbles+directs+gene+delivery+to+the+myocardium.">Echocardiographic destruction of albumin microbubbles directs gene delivery to the myocardium.</a> Circulation. 101, 2554-2556 (2000).</li><li>Sirsi, S., Borden, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbubble+Compositions,+Properties+and+Biomedical+Applications.">Microbubble Compositions, Properties and Biomedical Applications.</a> Bubble Sci Eng Technol. 1, 3-17 (2009).</li><li>Li, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrasound-targeted+microbubble+destruction+enhances+AAV-mediated+gene+transfection+in+human+RPE+cells+in+vitro+and+rat+retina+in+vivo.">Ultrasound-targeted microbubble destruction enhances AAV-mediated gene transfection in human RPE cells in vitro and rat retina in vivo.</a> Gene Ther. 16, 1146-1153 (2009).</li><li>Lindner, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbubbles+in+medical+imaging:+current+applications+and+future+directions.">Microbubbles in medical imaging: current applications and future directions.</a> Nat Rev Drug Discov. 3, 527-532 (2004).</li><li>Newman, C. M., Bettinger, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+therapy+progress+and+prospects:+ultrasound+for+gene+transfer.">Gene therapy progress and prospects: ultrasound for gene transfer.</a> Gene Ther. 14, 465-475 (2007).</li><li>Vancraeynest, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myocardial+injury+induced+by+ultrasound-targeted+microbubble+destruction:+evidence+for+the+contribution+of+myocardial+ischemia.">Myocardial injury induced by ultrasound-targeted microbubble destruction: evidence for the contribution of myocardial ischemia.</a> Ultrasound Med Biol. 35, 672-679 (2009).</li></ol>

UTMD representa un nuevo enfoque para la entrega de genes. Como una plataforma tecnológica que se puede combinar con cualquiera de las muchas posibles estrategias de terapia génica, para ofrecer una gran variedad de moléculas bioactivas en un alto grado de especificidad de tejido que se desea. La principal limitación biológica de la técnica es la baja eficiencia de transfección. Otra consideración importante es la accesibilidad de los órganos diana de la ecografía, que puede ser notablemente disminuido por intermedio de hueso o aire. La técnica requiere la optimización de la técnica basada en el tejido diana 11, así como un conocimiento básico de la acústica, para limitar el daño tisular 11 y aumentar la eficiencia. Sin embargo, proporciona un método económico, rápido y reproducible para evaluar el efecto de la expresión específica de tejido transgénico, y puede ofrecer un tratamiento eficaz para los trastornos donde los bajos niveles de expresión del transgén puede ser útil.

<table border="1"><tbody><tr><th> Nombre del reactivo / equipo </th><th> Empresa </th><th> Número de catálogo </th><th> Comentarios </th></tr><tr><td> 1,2-dipalmitoil-sn-glicero-3-fosfatidilcolina </td><td> Sigma-Aldrich </td><td> P-5911 </td><td> componente de la cáscara de lípidos de microburbujas </td></tr><tr><td> 1,2-dipalmitoil-sn-glicero-3-fosfatidiletanolamina </td><td> Sigma-Aldrich </td><td> P-3275 </td><td> componente de la cáscara de lípidos de microburbujas </td></tr><tr><td> glucosa </td><td> Sigma-Aldrich </td><td> G5400 </td><td> cree que estabiliza las microburbujas </td></tr><tr><td> tampón fosfato salino </td><td> Sigma-Aldrich </td><td> P5368 </td><td></td></tr><tr><td> glicerol </td><td> Sigma-Aldrich </td><td> G5516 </td><td> cree que para evitar que las microburbujas de coalescencia </td></tr><tr><td> Octafluoropropano gas </td><td> Airgas </td><td> N / A </td><td> gas inerte utilizado en aplicaciones clínicas </td></tr><tr><td> VialMix amalgamador dental </td><td> Bristol-Myers Squibb </td><td> N / A </td><td></td></tr><tr><td> 1 MHz, transductor de 13mm, fuera de foco </td><td> Olimpo </td><td> A303S-SU </td><td></td></tr><tr><td> 20 MHz Función / Generador de forma de onda arbitraria </td><td> Agilent </td><td> 33220A </td><td></td></tr><tr><td> Amplificador de Potencia </td><td> Krohn-Hite Co. </td><td> Modelo 7500 </td><td></td></tr><tr><td> Hidrófono </td><td> Bruel y Kjaer </td><td> Tipo 1803 </td><td></td></tr><tr><td> Amplificador de carga </td><td> Bruel y Kjaer </td><td> Tipo 2634 </td><td></td></tr><tr><td> 500 MHz Osciloscopio </td><td> LeCroy </td><td> 9354L </td><td></td></tr><tr><td> VisualSonics &#39;Vevo 2100 del sistema de imágenes con el transductor de 34 MHz </td><td> VisualSonics </td><td> 2100 </td><td></td></tr><tr><td> 27G de una pulgada de catéteres vena de la cola </td><td> VisualSonics </td><td> N / A </td><td></td></tr><tr><td> Genie Además bomba de infusión </td><td> Kent Científico </td><td> GENIE </td><td></td></tr></tbody></table>

introduction to the ultrasound targeted microbubble destruction technique

En las moléculas UTMD, bioactivos, tales como carga negativa vectores de ADN plásmido que codifica un gen de interés, se añaden a las capas de lípidos catiónicos de agentes de contraste de microburbujas 7-9. En ratones estas microburbujas llevar por vectores se puede administrar por vía intravenosa o directamente en el ventrículo izquierdo del corazón. En animales de mayor tamaño también pueden ser inyectados a través de un catéter intracoronario. La posterior entrega de la circulación a un órgano blanco se produce por la cavitación acústica a una frecuencia de resonancia de las microburbujas. Parece probable que la energía mecánica generada por los resultados de la destrucción de microburbujas en la formación de poros transitorios en o entre las células endoteliales de la microvasculatura de la región en estudio 10. Como resultado de este efecto sonoporación, la eficiencia de transfección en ya través de las células endoteliales se ha mejorado, y la codificación de los transgenes-vectores se depositan en el tejido circundante. ADN plásmido en el torrente circulatorio se degrada rápidamente por nucleasas en la sangre, lo que reduce aún más la probabilidad de parto para que no se sonicaron tejidos y da lugar a muy específica de órgano blanco de la transfección.

Watch this Scientific Journal Video about Introduction to the Ultrasound Targeted Microbubble Destruction Technique at JoVE.com

Introduction to the Ultrasound Targeted Microbubble Destruction Technique

Ultrasonido dirigido destrucción de microburbujas (UTMD) se puede utilizar para dirigir el sitio de entrega específicos de moléculas bioactivas, incluyendo genes terapéuticos, para dirigirse a los órganos accesibles a la ecografía, como el corazón y el hígado<sup&gt; 1-6</sup&gt;.

Introducción a la técnica de ultrasonido enfocado destrucción de microburbujas

In UTMD, bioactive molecules, such as negatively charged plasmid DNA vectors encoding a gene of interest, are added to the cationic shells of lipid ...

introduction-to-ultrasound-targeted-microbubble-destruction

Nanyang Technological University

Research

JoVE Journal

Bioengineering

15.5K vistas.  University of Hawaii. Ultrasonido dirigido destrucción de microburbujas (UTMD) se puede utilizar para dirigir el sitio de entrega específicos de moléculas bioactivas, incluyendo genes terapéuticos, para dirigirse a los órganos accesibles a la ecografía, como el corazón y el hígado 1-6.

Video: Introduction to the Ultrasound Targeted Microbubble Destruction Technique

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

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic valve leaflets are composed of interstitial cells suspended within an extracellular matrix (ECM) and are lined with an endothelial cell monolayer. The valve withstands a harsh, dynamic environment and is constantly exposed to shear, flexion, tension, and compression. Research has shown calcific lesions in diseased valves occur in areas of high mechanical stress as a result of endothelial disruption or interstitial matrix damage1-3. Hence, it is not surprising that epidemiological studies have shown high blood pressure to be a leading risk factor in the onset of aortic valve disease4. 
The only treatment option currently available for valve disease is surgical replacement of the diseased valve with a bioprosthetic or mechanical valve5. Improved understanding of valve biology in response to physical stresses would help elucidate the mechanisms of valve pathogenesis. In turn, this could help in the development of non-invasive therapies such as pharmaceutical intervention or prevention. Several bioreactors have been previously developed to study the mechanobiology of native or engineered heart valves6-9. Pulsatile bioreactors have also been developed to study a range of tissues including cartilage10, bone11 and bladder12. The aim of this work was to develop a cyclic pressure system that could be used to elucidate the biological response of aortic valve leaflets to increased pressure loads. 
The system consisted of an acrylic chamber in which to place samples and produce cyclic pressure, viton diaphragm solenoid valves to control the timing of the pressure cycle, and a computer to control electrical devices. The pressure was monitored using a pressure transducer, and the signal was conditioned using a load cell conditioner. A LabVIEW program regulated the pressure using an analog device to pump compressed air into the system at the appropriate rate. The system mimicked the dynamic transvalvular pressure levels associated with the aortic valve; a saw tooth wave produced a gradual increase in pressure, typical of the transvalvular pressure gradient that is present across the valve during diastole, followed by a sharp pressure drop depicting valve opening in systole. The LabVIEW program allowed users to control the magnitude and frequency of cyclic pressure. The system was able to subject tissue samples to physiological and pathological pressure conditions. This device can be used to increase our understanding of how heart valves respond to changes in the local mechanical environment.

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

A cyclic pressure bioreactor capable of subjecting heart valve tissue to physiological and pathological pressure conditions has been designed. A LabVIEW program allows users to control pressure magnitude, amplitude and frequency. This device can be used to study the mechanobiology of heart valve tissue or isolated cells.

Diseño de un biorreactor de presión cíclica de la Ex Vivo Estudio de las válvulas aórtica del corazón

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic ...

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. ...

Wideband Optical Detector of Ultrasound for Medical Imaging Applications

Optical sensors of ultrasound are a promising alternative to piezoelectric techniques, as has been recently demonstrated in the field of optoacoustic imaging. In medical applications, one of the major limitations of optical sensing technology is its susceptibility to environmental conditions, e.g. changes in pressure and temperature, which may saturate the detection. Additionally, the clinical environment often imposes stringent limits on the size and robustness of the sensor. In this work, the combination of pulse interferometry and fiber-based optical sensing is demonstrated for ultrasound detection. Pulse interferometry enables robust performance of the readout system in the presence of rapid variations in the environmental conditions, whereas the use of all-fiber technology leads to a mechanically flexible sensing element compatible with highly demanding medical applications such as intravascular imaging. In order to achieve a short sensor length, a pi-phase-shifted fiber Bragg grating is used, which acts as a resonator trapping light over an effective length of 350 &#181;m. To enable high bandwidth, the sensor is used for sideway detection of ultrasound, which is highly beneficial in circumferential imaging geometries such as intravascular imaging. An optoacoustic imaging setup is used to determine the response of the sensor for acoustic point sources at different positions.

Optical detection of ultrasound is impractical in many imaging scenarios because it often requires stable environmental conditions. We demonstrate an optical technique for ultrasound sensing in volatile environments with miniaturization and sensitivity levels appropriate for optoacoustic imaging in restrictive scenarios, e.g. intravascular applications.

Banda ancha Detector óptico de ultrasonido para aplicaciones de imágenes médicas

Optical sensors of ultrasound are a promising alternative to piezoelectric techniques, as has been recently demonstrated in the field of optoacoustic ...

Manufacture of Concentrated, Lipid-based Oxygen Microbubble Emulsions by High Shear Homogenization and Serial Concentration

Gas-filled microbubbles have been developed as ultrasound contrast and drug delivery agents. Microbubbles can be produced by processing surfactants using sonication, mechanical agitation, microfluidic devices, or homogenization. Recently, lipid-based oxygen microbubbles (LOMs) have been designed to deliver oxygen intravenously during medical emergencies, reversing life-threatening hypoxemia, and preventing subsequent organ injury, cardiac arrest, and death. We present methods for scaled-up production of highly oxygenated microbubbles using a closed-loop high-shear homogenizer. The process can produce 2 L of concentrated LOMs (90% by volume) in 90 min. Resulting bubbles have a mean diameter of ~2 &#956;m, and a rheologic profile consistent with that of blood when diluted to 60 volume %. This technique produces LOMs in high capacity and with high oxygen purity, suggesting that this technique may be useful for translational research labs.

We describe methods for the manufacture of large volumes of lipid-based oxygen microbubbles (LOMs) designed for intravenous oxygen delivery using high-shear homogenization and serial concentration.

Fabricación de concentrado, de oxígeno a base de lípidos microburbujas Emulsiones por High Shear Homogeneización y Concentración de serie

Gas-filled microbubbles have been developed as ultrasound contrast and drug delivery agents. Microbubbles can be produced by processing surfactants using ...

A Microfluidic Technique to Probe Cell Deformability

Here we detail the design, fabrication, and use of a microfluidic device to evaluate the deformability of a large number of individual cells in an efficient manner. Typically, data for ~102 cells can be acquired within a 1 hr experiment. An automated image analysis program enables efficient post-experiment analysis of image data, enabling processing to be complete within a few hours. Our device geometry is unique in that cells must deform through a series of micron-scale constrictions, thereby enabling the initial deformation and time-dependent relaxation of individual cells to be assayed. The applicability of this method to human promyelocytic leukemia (HL-60) cells is demonstrated. Driving cells to deform through micron-scale constrictions using pressure-driven flow, we observe that human promyelocytic (HL-60) cells momentarily occlude the first constriction for a median time of 9.3 msec before passaging more quickly through the subsequent constrictions with a median transit time of 4.0 msec per constriction. By contrast, all-trans retinoic acid-treated (neutrophil-type) HL-60 cells occlude the first constriction for only 4.3 msec before passaging through the subsequent constrictions with a median transit time of 3.3 msec. This method can provide insight into the viscoelastic nature of cells, and ultimately reveal the molecular origins of this behavior.

We demonstrate a microfluidics-based assay to measure the timescale for cells to transit through a sequence of micron-scale constrictions.

Una técnica de microfluidos investigará Deformabilidad Cell

Here we detail the design, fabrication, and use of a microfluidic device to evaluate the deformability of a large number of individual cells in an ...

A Microfluidic Chip for ICPMS Sample Introduction

This protocol discusses the fabrication and usage of a disposable low cost microfluidic chip as sample introduction system for inductively coupled plasma mass spectrometry (ICPMS). The chip produces monodisperse aqueous sample droplets in perfluorohexane (PFH). Size and frequency of the aqueous droplets can be varied in the range of 40 to 60 &#181;m and from 90 to 7,000&#160;Hz, respectively. The droplets are ejected from the chip with a second flow of PFH and remain intact during the ejection. A custom-built desolvation system removes the PFH and transports the droplets into the ICPMS. Here, very stable signals with a narrow intensity distribution can be measured, showing the monodispersity of the droplets. We show that the introduction system can be used to quantitatively determine iron in single bovine red blood cells. In the future, the capabilities of the introduction device can easily be extended by the integration of additional microfluidic modules.

We present a discrete droplet sample introduction system for inductively coupled plasma mass spectrometry (ICPMS). It is based on a cheap and disposable microfluidic chip that generates highly monodisperse droplets in a size range of 40&#8722;60 &#181;m at frequencies from 90 to 7,000 Hz.

Un chip de microfluidos para ICPMS Introducción Muestra

This protocol discusses the fabrication and usage of a disposable low cost microfluidic chip as sample introduction system for inductively coupled plasma ...

An Experimental Protocol for Assessing the Performance of New Ultrasound Probes Based on CMUT Technology in Application to Brain Imaging

The possibility to perform an early and repeatable assessment of imaging performance is fundamental in the design and development process of new ultrasound (US) probes. Particularly, a more realistic analysis with application-specific imaging targets can be extremely valuable to assess the expected performance of US probes in their potential clinical field of application.
The experimental protocol presented in this work was purposely designed to provide an application-specific assessment procedure for newly-developed US probe prototypes based on Capacitive Micromachined Ultrasonic Transducer (CMUT) technology in relation to brain imaging.
The protocol combines the use of a bovine brain fixed in formalin as the imaging target, which ensures both realism and repeatability of the described procedures, and of neuronavigation techniques borrowed from neurosurgery. The US probe is in fact connected to a motion tracking system which acquires position data and enables the superposition of US images to reference Magnetic Resonance (MR) images of the brain. This provides a means for human experts to perform a visual qualitative assessment of the US probe imaging performance and to compare acquisitions made with different probes. Moreover, the protocol relies on the use of a complete and open research and development system for US image acquisition, i.e. the Ultrasound Advanced Open Platform (ULA-OP) scanner.
The manuscript describes in detail the instruments and procedures involved in the protocol, in particular for&#160;the calibration, image acquisition and registration of US and MR images. The obtained results prove the effectiveness of the overall protocol presented, which is entirely open (within the limits of the instrumentation involved), repeatable, and covers the entire set of acquisition and processing activities for US images.

The development of new ultrasound (US) probes based on Capacitive Micromachined Ultrasonic Transducer (CMUT) technology requires an early realistic assessment of imaging capabilities. We describe a repeatable experimental protocol for US image acquisition and comparison with magnetic resonance images, using an ex vivo bovine brain as an imaging target.

Un protocolo Experimental para evaluar el desempeño de nuevas sondas de ultrasonido basado en la tecnología CMUT en aplicación a la proyección de imagen de cerebro

The possibility to perform an early and repeatable assessment of imaging performance is fundamental in the design and development process of new ...

Comprehensive Evaluation of the Effectiveness and Safety of Placenta-Targeted Drug Delivery Using Three Complementary Methods

No effective treatments currently exist for placenta-associated pregnancy complications, and developing strategies for the targeted delivery of drugs to the placenta while minimizing fetal and maternal side effects remains challenging. Targeted nanoparticle carriers provide new opportunities to treat placental disorders. We recently demonstrated that a synthetic placental chondroitin sulfate A binding peptide (plCSA-BP) could be used to guide nanoparticles to deliver drugs to the placenta. In this protocol, we describe in detail a system for assessing the efficiency of drug delivery to the placenta by plCSA-BP that employs three separate methods used in combination: in vivo imaging, high-frequency ultrasound (HFUS), and high-performance liquid chromatography (HPLC). Using in vivo imaging, plCSA-BP-guided nanoparticles were visualized in the placentas of live animals, while HFUS and HPLC demonstrated that plCSA-BP-conjugated nanoparticles efficiently and specifically delivered methotrexate to the placenta. Thus, a combination of these methods can be used as an effective tool for the targeted delivery of drugs to the placenta and development of new treatment strategies for several pregnancy complications.

We describe a system that utilizes three methods to evaluate the safety and effectiveness of placenta-targeted drug delivery: in vivo imaging to monitor nanoparticle accumulation, high-frequency ultrasound to monitor placental and fetal development, and HPLC to quantify drug delivery to tissue.

Evaluación integral de la efectividad y seguridad de la entrega de drogas orientada a Placenta utilizando tres métodos complementarios

No effective treatments currently exist for placenta-associated pregnancy complications, and developing strategies for the targeted delivery of drugs to ...

Preparing Protein Producing Synthetic Cells using Cell Free Bacterial Extracts, Liposomes and Emulsion Transfer

The bottom-up assembly approach for construction of synthetic cells is an effective tool for isolating and investigating cellular processes in a cell mimicking environment. Furthermore, the development of cell-free expression systems has demonstrated the ability to reconstitute the protein production, transcription and translation processes (DNA&#8594;RNA&#8594;protein) in a controlled manner, harnessing synthetic biology. Here we describe a protocol for preparing a cell-free expression system, including the production of a potent bacterial lysate and encapsulating this lysate inside cholesterol-rich lipid-based giant unilamellar vesicles (GUVs) (i.e., stable liposomes), to form synthetic cells. The protocol describes the methods for preparing the components of the synthetic cells including the production of active bacterial lysates, followed by a detailed step-by-step preparation of the synthetic cells based on a water-in-oil emulsion transfer method. These facilitate the production of millions of synthetic cells in a simple and affordable manner with a high versatility for producing different types of proteins. The obtained synthetic cells can be used to investigate protein/RNA production and activity in an isolated environment, in directed evolution, and also as a controlled drug delivery platform for on-demand production of therapeutic proteins inside the body.

This protocol describes the method, materials, equipment and steps for bottom-up preparation of RNA and protein producing synthetic cells. The inner aqueous compartment of the synthetic cells contained the S30 bacterial lysate encapsulated within a lipid bilayer (i.e., stable liposomes), using a water-in-oil emulsion transfer method.

Preparación de células sintéticas productoras de proteínas mediante extractos bacterianos libres de células, liposomas y transferencia de emulsión

The bottom-up assembly approach for construction of synthetic cells is an effective tool for isolating and investigating cellular processes in a cell ...