Este trabajo fue financiado por donaciones a LKM-K de los Institutos Nacionales de Salud / Instituto Nacional del Cáncer (R01CA129822 y R01CA140995). Dr. Medina-Kauwe gracias C. Rey, M. M-Kauwe y D. Revetto de apoyo.

1. Entrega Sistémica En vivo <ol><li> Mezclar suficiente Herdox con solución salina estéril para equiparar 0,2 ml de una mg / kg de dosis de Herdox por inyección de 0.004 para un 6-8 semanas ratón cojinete tumores de xenoinjertos subcutáneos de flanco bilaterales nu / nu viejos. </li><li> Dibujar suavemente la mezcla Herdox en una jeringa de insulina 3/10 cc con una aguja 29G, evitando las burbujas. </li><li> La anestesia se indujo mediante una breve exposición isoflurano en una cámara de inducción equipado con un sistema de barrido de gas (velocidades de flujo de oxígeno: 0,5-1 L / min, la concentración de isoflurano: 3-4% (o inferior). </li><li> Inyectar la mezcla entera en la vena de la cola de un ratón anestesiado (0,2 ml por inyección). Inyección IV también se puede realizar en un restringido, ratón sin anestesiar. </li><li> Repetir las inyecciones en el mismo ratón durante seis días más secuenciales, una vez por día. </li></ol> 2. Fluorescencia de imágenes en vivo La acumulación de Herdox fluorescencia en los tumores puedeser detectable por el último día de la inyección (Día 7) utilizando un generador de imágenes multimodo. Los siguientes procedimientos implican el uso de un sistema de macro-iluminación y detección personalizada (Figura 1) 8. <ol><li> Encienda el Multimodo En vivo Imager óptico. </li><li> Seleccione un filtro de paso de banda de emisión (590 nm ± 30 nm) adecuado para la detección de fluorescencia de la doxorubicina. </li><li> Encienda el láser de Argón Kriptón y coloque un filtro de paso de banda de excitación (488 nm ± 10 nm) en el camino óptico del láser. </li><li> Encienda el sistema de anestesia y luego se coloca un ratón en la cámara anestesiante (caudales de oxígeno: 0,5 a 1 L / min, la concentración de isoflurano: 3-4% (o menos) equipada con un sistema de recogida de gases. </li><li> Transferir el ratón desde la cámara de anestesiar a la cámara de imágenes del Multimodo En vivo Imager óptico cuando el ratón está anestesiado. </li><li> Coloque una ojiva sobre la nariz del ratón y abrir el flujo de administrar anestesia continuasia durante la adquisición de la imagen (caudales de oxígeno: 0,5-1 L / min, la concentración de isoflurano: 2-3% (o inferior). </li><li> Adquirir imágenes de fluorescencia utilizando un tiempo de exposición de 5 a 15 segundos. </li><li> Realizar el análisis y tratamiento de imágenes incluida la corrección de fondo o el ajuste de contraste. </li></ol> 3. Fluorescencia de imágenes ex vivo Herdox fluorescencia se pueden obtener imágenes de los tumores y los órganos específicos (incluyendo el hígado, riñón, bazo, corazón y músculo esquelético) cosechadas de los ratones sacrificados a las 24 horas después de la inyección final (Día 7) de Herdox. <ol><li> Encienda el Multimodo En vivo Imager óptico. </li><li> Seleccione un filtro de paso de banda de emisión (580 nm ± 20 nm) para la detección de fluorescencia doxorrubicina. </li><li> Encienda el láser de Argón Kriptón y coloque un filtro de paso de banda de excitación (488 nm ± 10 nm) en el camino óptico del láser. </li><li> Coloque los tumores y órganos específicos dispuestos en una placa de Petri en la cámara de imágenes of En el Imager óptica multimodo vivo. </li><li> Adquirir imágenes de fluorescencia de los tejidos utilizando un tiempo de exposición de 5 a 15 segundos. Un ejemplo de adquisición de imágenes de fluorescencia inicial se muestra en la Figura 2a. Repita el mismo mediante un placa Petri vacía, lo que servirá de fondo (Figura 2b). </li><li> Realizar el análisis y tratamiento de imágenes incluida la corrección de fondo o el ajuste de contraste. Un ejemplo de una imagen corregida se muestra en la Figura 2c, que resulta de la resta de la figura 2b de la Figura 2a. </li></ol> 4. In situ Imagen confocal de Tumores En la imagen confocal in situ permite la detección y el análisis de la acumulación tumor Herdox a nivel celular. <ol><li> Encienda una SPE microscopio confocal Leica. </li><li> Seleccione 488 luz láser nm para la excitación de la doxorrubicina y la emisión de longitudes de onda (560-620 nm) para la doxorrubicinadetección de fluorescencia. </li><li> Seleccionar un objetivo de 40X o 63X y aceite de inmersión gota en la lente del objetivo. </li><li> Extraer los tumores de los ratones recién sacrificados que hayan recibido previamente tratamientos Herdox y se burlan como se describe en el procedimiento 2. </li><li> Tumores Colocar en una placa de Petri en hielo para evitar la degradación del tejido y, a continuación, transferir los tumores a una cámara de T Delta de imagen confocal. </li><li> Adquirir confocal de imágenes de los tumores a profundidades focales secuenciales (tamaño de paso: 1 m, grueso: 20 micras). Un ejemplo de imágenes secuencialmente-adquiridos a lo largo del eje z se muestra en la Figura 3, panel izquierdo. </li><li> Realizar máxima intensidad z-proyección de las imágenes. Una proyección de intensidad máxima de z-apiladas imágenes se muestra en la Figura 3, panel derecho. </li><li> Cálculo de las intensidades medias de fluorescencia de la intensidad máxima Z -. Imágenes de proyección media de intensidades de fluorescencia de las imágenes sobre el campo de vista global fueron calculard utilizando ImageJ. </li></ol> 5. Spectral Imaging radiométrico y Análisis Imágenes espectrales y análisis radiométrico permite la discriminación entre Dox fluorescencia y autofluorescencia. <ol><li> De alimentación en un microscopio confocal de barrido de fluorescencia láser. </li><li> Adquirir 15 imágenes de los tumores Herdox tratadas y sin tratar a una profundidad especificada en el rango espectral de 510 a 650 nm, con un paso de 10 nm y excitación a 488 nm de luz usando un microscopio confocal Leica SPE. </li><li> Prepare una solución 100 mM de doxorrubicina. </li><li> Realice imagen espectral de la solución de doxorubicina 100 mM para obtener la firma espectral puro Dox fluorescencia (rango espectral: 510 a 650 nm, un tamaño de paso: 10 nm). Los resultados típicos de adquisición de la imagen de formación de imágenes espectrales y espectro de fluorescencia resultante representan como un gráfico se muestra en la Figura 4. </li><li> Adquirir la firma espectral autofluorescencia desde una imagen cube (rango espectral: 510 a 650 nm, un tamaño de paso: 10 nm) obtenida por imágenes espectrales de los tumores no tratados. Los resultados típicos de adquisición de la imagen de formación de imágenes espectrales y espectro de fluorescencia resultante representan como un gráfico se muestra en la Figura 5. </li><li> Generar cuatro firmas espectrales de referencia (autofluorescencia puro, 0.1.doxorubin 0.9. Autofluorescencia, 0.2. Doxorrubicina 0.8. Autofluorescencia, 0.3.doxorubin 0.7. Autofluorescencia) utilizando el programa que desarrollamos 9. Una curva típica que muestra cuatro firmas espectrales de referencia se muestra en la Figura 6. </li><li> Realizar la clasificación espectral de las imágenes tal como se define por las firmas espectrales de referencia a través de medida de la distancia euclidiana utilizando el programa que previamente desarrollado 9. </li><li> Realizar desmezcla espectral lineal de las imágenes mediante el uso de un programa de desmezcla espectral (plug-in en ImageJ) hemos desarrollado 7, 10, para la comparación con la imagen espectral y radiométrica análisis. Un ejemplo de separar fluorescencia Herdox de autofluorescencia por desmezcla espectral lineal se muestra en la Figura 7. </li></ol>

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El autor, Daniel Farkas, es Presidente de espectral Imagen Molecular. El resto de autores no tienen intereses en conflicto.

Dox fluorescencia puede ser detectable in vivo utilizando el generador de imágenes multimodo cuando los tumores son subcutánea. Sin embargo, la dosis terapéuticamente eficaz de Herdox (0,004 mg / kg) está por debajo del umbral de detección después de una sola dosis. En contraste, después de 7 inyecciones diarias (1 vez al día durante 7 días), la acumulación del tumor y la retención de la partícula es suficiente para permitir la visualización de la fluorescencia Dox. Es ...

Tumor-targeting de la quimioterapia tiene el potencial de eliminar las células cancerosas en dosis más baja en comparación a las drogas no orientados, debido a que más de la terapia administrada se puede acumular en su destino previsto, en lugar de distribuir al tejido no tumoral. A medida que la última situación sería diluir la eficacia de la droga y por lo tanto requieren dosis más altas para ser eficaz, tumor-alcance tiene tanto ventajas terapéuticas y de seguridad más de un tratamiento no específico estándar. Orientación de la quimioterapia mediante encapsulación en nanopartículas auto-ensambladas permite que el fármaco permanezca sin modificar químicamente en contraste con fármacos que se unen covalentemente a moléculas de direccionamiento. Como tal vinculación tiene el potencial de alterar la actividad de ambos el medicamento y la molécula diana; montaje no covalente permite que la potencia del fármaco a ser retenido. Hemos demostrado anteriormente que la novela de tres componentes, complejos auto-ensamblado, Herdox, se dirige a los tumores HER2 + <em&gt; In vivo y provoca la ablación de tumores de crecimiento sin afectar al tejido normal, incluyendo el corazón 1. Herdox se forma a través de interacciones no covalentes entre la proteína de unión al receptor celular-penetración, HerPBK10, y el agente quimioterapéutico, doxorubicina (Dox), a través de un pequeño conector de ácido nucleico. HerPBK10 se une el factor de crecimiento epidérmico humano (HER) y desencadena la endocitosis mediada por el receptor 2-4, mientras que la penetración de membrana endosomal se lleva a cabo a través de la incorporación de derivados de adenovirus-pentón base de proteína de la cápside 4-6. Un dominio cargado positivamente en la proteína de unión de ácidos nucleicos permite a los 4, 5, a través del cual Dox ADN-intercalado puede ser transportado para la administración dirigida. Electrofílica, intercalación, y posiblemente interacciones oligomerización de proteínas facilitan autoensamblaje en redondo 10-20 nm partículas que son estables en la sangre y en el almacenamiento prolongado a temperaturas diferentes 1. Orientación preferencial a HER2 + células tumorales se ve facilitada por la afinidad de ligando de HER2 mejorada cuando se eleva. Nuestros estudios previos han demostrado que la administración sistémica de los rendimientos Herdox acumulación preferencial en los tumores de más de tejido no tumoral y en comparación con untargeted Dox 1, y la penetración en las células tumorales in vivo 7. Hemos observado que las liberaciones Herdox Dox después de la entrada de células tumorales, lo que permite la acumulación de Dox en el núcleo 1. Tumor-acumulación parece que se correlaciona con el nivel del receptor, como tumores relativamente bajos que expresan HER2 se acumulan menos Herdox comparación con aquellos con niveles comparativamente más altos HER2 1. Por otra parte, la concentración eficaz la muerte celular presenta una correlación inversa con HER2 visualización en líneas de células tumorales que expresan HER2 superficie celular diferentes niveles 1. Herdox presenta una ventaja terapéutica y la seguridad sobre Dox no directo, como el asesinato del tumor se produce en más de la dosis 10 veces menor en comparación con el untardrogas geted y produce ningún efecto adverso detectable en el corazón (detectado por ecocardiografía y tinción histológica) o tejido del hígado (detectado por tinción de TUNEL), en contraste con Dox no directo 1. A pesar de su derivación de una proteína de la cápside viral, HerPBK10 no exhibe inmunogenicidad detectable a niveles terapéuticos 2. Mientras que los anticuerpos pre-existentes a adenovirus conjunto pueden reconocer HerPBK10, no son capaces de impedir la unión de células 2. El volumen del tumor medido con el tiempo es un método estándar para evaluar la eficacia terapéutica de terapias dirigidas, y se ha empleado para evaluar la eficacia terapéutica de Herdox. Como complemento de este enfoque con in vivo y ex vivo de imágenes de fluorescencia de intensidad nos ha permitido evaluar mejor la eficiencia del objetivo 7. Hemos integrado específicamente en situ imagen confocal de los tumores extirpados con el análisis espectral de Dox fluorescencia para verificar que no Herdoxt sólo acumula en tumores in vivo, pero penetró en las células tumorales y Dox entregado en el citoplasma y el núcleo 7. El análisis espectral, además, nos ha permitido distinguir la fluorescencia Dox de autofluorescencia 7. Aquí se demuestra en mayor detalle nuestro enfoque para evaluar Herdox in vivo después de la administración sistémica, y lo más importante, para la evaluación de orientación a través de métodos y análisis de imágenes multimodo.

<table border="1">
<tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments (optional)</th
 ></tr>
 <tr>
 <td >Fluorescence laser scanning confocal microscope</td>
 <td >Leica</td>
 <td >SPE</td>
 <td></td>
 </tr>
 <tr>
 <td>In Vivo Optical Imager</td>
 <td>Spectral Molecular Imaging</td>
 <td>Multimode In Vivo Optical Imager</td>
 <td></td>
 </tr>
 <tr>
 <td >Doxorubicin-HCl</td>
 <td >Sigma-Aldrich</td>
 <td >D4035</td>
 <td></td>
 </tr>
 <tr>
 <td >Nude (NU/NU) mouse, female, 6-8 week</td>
 <td >Charles River</td>
 <td >Strain code 088</td>
 <td></td>
 </tr>
 <tr>
 <td >MDA-MB-435 human HER2+ tumor cells</td>
 <td >NCI-Frederick Cancer DCTD Tumor/Cell Line Repository</td>
 <td >0507292</td>
 <td></td>
 </tr>
 <tr>
 <td >3/10 cc insulin syringe U-100 with 29G x 1/2" Ultra-FineIV permanently attached needle</td>
 <td >BD</td>
 <td >309301</td>
 <td></td>
 </tr>
 <tr>
 <td >Delta T chamber </td>
 <td >Bioptechs </td>
 <td >04200417B</td>
 <td></td>
 </tr>
 </tbody>
</table>

analysis of targeted viral protein nanoparticles delivered to her2+ tumors

La nanopartícula HER2 + del tumor-específica, Herdox, exhibe acumulación tumoral-preferencial y la ablación de tumores de crecimiento en un modelo animal de cáncer de HER2 +. Herdox está formado por no covalente de auto-ensamblaje de una proteína de penetración celular específica del tumor con el agente de quimioterapia, doxorubicina, a través de un pequeño conector de ácido nucleico. Una combinación de electrofílico, intercalación y las interacciones oligomerización facilitar autoensamblaje en redondo 10-20 nm partículas. Herdox muestra una estabilidad en la sangre, así como en el almacenamiento prolongado a temperaturas diferentes. El suministro sistémico de Herdox en ratones portadores de tumores da como resultado la muerte de las células del tumor, sin efectos adversos detectables en el tejido no tumoral, incluyendo el corazón y el hígado (que se someten a daño marcado por la doxorubicina no dirigidos). HER2 elevación facilita orientación a las células que expresan el receptor del factor de crecimiento epidérmico humano, por lo tanto, los tumores se presentan niveles elevados de HER2 presentan una mayor acumulación de Herdox en comparación con las células de expresióncantar niveles más bajos, tanto in vitro como in vivo. La intensidad de fluorescencia de imagen combinada con el análisis y confocal espectral situ nos ha permitido verificar en el tumor vivo focalización y la penetración de células tumorales de Herdox después de la administración sistémica. A continuación te detallamos los métodos de evaluación de tumor de la orientación a través de imágenes multimodo después de la administración sistémica.

La Figura 1 muestra el prototipo de imágenes in vivo óptica, que fue construido para el propósito de la adquisición de imágenes bajo múltiples modalidades, incluyendo la intensidad de fluorescencia, espectral, curso de la vida, 2-fotón confocal, intra-vitales, y en imágenes de bioluminiscencia. Además, la alta cámara sensible enfriada y líneas de láser de alta potencia incorporado en este sistema los rendimientos de imágenes de fluorescencia de mayor contraste en comparaci...

Watch this Scientific Journal Video about Analysis of Targeted Viral Protein Nanoparticles Delivered to HER2+ Tumors at JoVE.com

Analysis of Targeted Viral Protein Nanoparticles Delivered to HER2+ Tumors

Este artículo detalla los procedimientos para el análisis de imágenes ópticas de las nanopartículas de tumor específico, Herdox. En particular, se describe aquí el uso detallada del dispositivo de formación de imágenes multimodo para la detección de tumor de la orientación y la evaluación de la penetración del tumor.

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. ...

analysis-targeted-viral-protein-nanoparticles-delivered-to-her2

Research

JoVE Journal

Bioengineering

10.9K vistas.  University of Southern California. Este artículo detalla los procedimientos para el análisis de imágenes ópticas de las nanopartículas de tumor específico, Herdox. En particular, se describe aquí el uso detallada del dispositivo de formación de imágenes multimodo para la detección de tumor de la orientación y la evaluación de la penetración del tumor.

Video: Analysis of Targeted Viral Protein Nanoparticles Delivered to HER2+ Tumors

Introduction to the Ultrasound Targeted Microbubble Destruction Technique

In UTMD, bioactive molecules, such as negatively charged plasmid DNA vectors encoding a gene of interest, are added to the cationic shells of lipid microbubble contrast agents7-9. In mice these vector-carrying microbubbles can be administered intravenously or directly to the left ventricle of the heart. In larger animals they can also be infused through an intracoronary catheter. The subsequent delivery from the circulation to a target organ occurs by acoustic cavitation at a resonant frequency of the microbubbles. It seems likely that the mechanical energy generated by the microbubble destruction results in transient pore formation in or between the endothelial cells of the microvasculature of the targeted region10. As a result of this sonoporation effect, the transfection efficiency into and across the endothelial cells is enhanced, and transgene-encoding vectors are deposited into the surrounding tissue. Plasmid DNA remaining in the circulation is rapidly degraded by nucleases in the blood, which further reduces the likelihood of delivery to non-sonicated tissues and leads to highly specific target-organ transfection.

Ultrasound Targeted Microbubble Destruction (UTMD) can be used to direct site-specific delivery of bioactive molecules, including therapeutic genes, to target organs accessible to ultrasound, such as the heart and liver1-6.

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 ...

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 ...

Cellular Lipid Extraction for Targeted Stable Isotope Dilution Liquid Chromatography-Mass Spectrometry Analysis

The metabolism of fatty acids, such as arachidonic acid (AA) and linoleic acid (LA), results in the formation of oxidized bioactive lipids, including numerous stereoisomers1,2. These metabolites can be formed from free or esterified fatty acids. Many of these oxidized metabolites have biological activity and have been implicated in various diseases including cardiovascular and neurodegenerative diseases, asthma, and cancer3-7. Oxidized bioactive lipids can be formed enzymatically or by reactive oxygen species (ROS). Enzymes that metabolize fatty acids include cyclooxygenase (COX), lipoxygenase (LO), and cytochromes P450 (CYPs)1,8. Enzymatic metabolism results in enantioselective formation whereas ROS oxidation results in the racemic formation of products.
While this protocol focuses primarily on the analysis of AA- and some LA-derived bioactive metabolites; it could be easily applied to metabolites of other fatty acids. Bioactive lipids are extracted from cell lysate or media using liquid-liquid (l-l) extraction. At the beginning of the l-l extraction process, stable isotope internal standards are added to account for errors during sample preparation. Stable isotope dilution (SID) also accounts for any differences, such as ion suppression, that metabolites may experience during the mass spectrometry (MS) analysis9. After the extraction, derivatization with an electron capture (EC) reagent, pentafluorylbenzyl bromide (PFB) is employed to increase detection sensitivity10,11. Multiple reaction monitoring (MRM) is used to increase the selectivity of the MS analysis. Before MS analysis, lipids are separated using chiral normal phase high performance liquid chromatography (HPLC). The HPLC conditions are optimized to separate the enantiomers and various stereoisomers of the monitored lipids12. This specific LC-MS method monitors prostaglandins (PGs), isoprostanes (isoPs), hydroxyeicosatetraenoic acids (HETEs), hydroxyoctadecadienoic acids (HODEs), oxoeicosatetraenoic acids (oxoETEs) and oxooctadecadienoic acids (oxoODEs); however, the HPLC and MS parameters can be optimized to include any fatty acid metabolites13.
Most of the currently available bioanalytical methods do not take into account the separate quantification of enantiomers. This is extremely important when trying to deduce whether or not the metabolites were formed enzymatically or by ROS. Additionally, the ratios of the enantiomers may provide evidence for a specific enzymatic pathway of formation. The use of SID allows for accurate quantification of metabolites and accounts for any sample loss during preparation as well as the differences experienced during ionization. Using the PFB electron capture reagent increases the sensitivity of detection by two orders of magnitude over conventional APCI methods. Overall, this method, SID-LC-EC-atmospheric pressure chemical ionization APCI-MRM/MS, is one of the most sensitive, selective, and accurate methods of quantification for bioactive lipids.

This protocol will demonstrate the extraction and analysis of free and esterified bioactive fatty acids from cells. Fatty acids are accurately quantified using stable isotope dilution, chiral liquid chromatography, electron capture atmospheric chemical ionization multiple reaction monitoring mass spectrometry (SID-LC-ECAPCI-MRM/MS).

Extracción de los lípidos celulares orientadas por dilución de isótopos estables análisis de espectrometría de cromatografía de líquidos-Mass

The metabolism of fatty acids, such as arachidonic acid (AA) and linoleic acid (LA), results in the formation of oxidized bioactive lipids, including ...

Therapeutic Gene Delivery and Transfection in Human Pancreatic Cancer Cells using Epidermal Growth Factor Receptor-targeted Gelatin Nanoparticles

More than 32,000 patients are diagnosed with pancreatic cancer in the United States per year and the disease is associated with very high mortality 1. Urgent need exists to develop novel clinically-translatable therapeutic strategies that can improve on the dismal survival statistics of pancreatic cancer patients. Although gene therapy in cancer has shown a tremendous promise, the major challenge is in the development of safe and effective delivery system, which can lead to sustained transgene expression. 
Gelatin is one of the most versatile natural biopolymer, widely used in food and pharmaceutical products. Previous studies from our laboratory have shown that type B gelatin could physical encapsulate DNA, which preserved the supercoiled structure of the plasmid and improved transfection efficiency upon intracellular delivery. By thiolation of gelatin, the sulfhydryl groups could be introduced into the polymer and would form disulfide bond within nanoparticles, which stabilizes the whole complex and once disulfide bond is broken due to the presence of glutathione in cytosol, payload would be released 2-5. Poly(ethylene glycol) (PEG)-modified GENS, when administered into the systemic circulation, provides long-circulation times and preferentially targets to the tumor mass due to the hyper-permeability of the neovasculature by the enhanced permeability and retention effect 6. Studies have shown over-expression of the epidermal growth factor receptor (EGFR) on Panc-1 human pancreatic adenocarcinoma cells 7. In order to actively target pancreatic cancer cell line, EGFR specific peptide was conjugated on the particle surface through a PEG spacer.8
Most anti-tumor gene therapies are focused on administration of the tumor suppressor genes, such as wild-type p53 (wt-p53), to restore the pro-apoptotic function in the cells 9. The p53 mechanism functions as a critical signaling pathway in cell growth, which regulates apoptosis, cell cycle arrest, metabolism and other processes 10. In pancreatic cancer, most cells have mutations in p53 protein, causing the loss of apoptotic activity. With the introduction of wt-p53, the apoptosis could be repaired and further triggers cell death in cancer cells 11. 
Based on the above rationale, we have designed EGFR targeting peptide-modified thiolated gelatin nanoparticles for wt-p53 gene delivery and evaluated delivery efficiency and transfection in Panc-1 cells.

Type B gelatin-based engineered nanovectors system (GENS) was developed for systemic gene delivery and transfection in the treatment of pancreatic cancer. By modification with epidermal growth factor receptor (EGFR) specific peptide on the surface of nanparticles, they could target on EGFR receptor and release plasmid under reducing environment, such as high intracellular glutathione concentrations.

Entrega y transfección de genes en células humanas de cáncer de páncreas con factor de crecimiento epidérmico receptor dirigidos a base de gelatina Nanovectors Ingeniería

More than 32,000 patients are diagnosed with pancreatic cancer in the United States per year and the disease is associated with very high mortality 1. ...

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 ...

Evaluation of Polymeric Gene Delivery Nanoparticles by Nanoparticle Tracking Analysis and High-throughput Flow Cytometry

Non-viral gene delivery using polymeric nanoparticles has emerged as an attractive approach for gene therapy to treat genetic diseases1 and as a technology for regenerative medicine2. Unlike viruses, which have significant safety issues, polymeric nanoparticles can be designed to be non-toxic, non-immunogenic, non-mutagenic, easier to synthesize, chemically versatile, capable of carrying larger nucleic acid cargo and biodegradable and/or environmentally responsive. Cationic polymers self-assemble with negatively charged DNA via electrostatic interaction to form complexes on the order of 100 nm that are commonly termed polymeric nanoparticles. Examples of biomaterials used to form nanoscale polycationic gene delivery nanoparticles include polylysine, polyphosphoesters, poly(amidoamines)s and polyethylenimine (PEI), which is a non-degradable off-the-shelf cationic polymer commonly used for nucleic acid delivery1,3 . Poly(beta-amino ester)s (PBAEs) are a newer class of cationic polymers4 that are hydrolytically degradable5,6 and have been shown to be effective at gene delivery to hard-to-transfect cell types such as human retinal endothelial cells (HRECs)7, mouse mammary epithelial cells8, human brain cancer cells9 and macrovascular (human umbilical vein, HUVECs) endothelial cells10.
A new protocol to characterize polymeric nanoparticles utilizing nanoparticle tracking analysis (NTA) is described. In this approach, both the particle size distribution and the distribution of the number of plasmids per particle are obtained11. In addition, a high-throughput 96-well plate transfection assay for rapid screening of the transfection efficacy of polymeric nanoparticles is presented. In this protocol, poly(beta-amino ester)s (PBAEs) are used as model polymers and human retinal endothelial cells (HRECs) are used as model human cells. This protocol can be easily adapted to evaluate any polymeric nanoparticle and any cell type of interest in a multi-well plate format.

A protocol for nanoparticle tracking analysis (NTA) and high-throughput flow cytometry to evaluate polymeric gene delivery nanoparticles is described. NTA is utilized to characterize the nanoparticle particle size distribution and the plasmid per particle distribution. High-throughput flow cytometry enables quantitative transfection efficacy evaluation for a library of gene delivery biomaterials.

La evaluación de la administración de genes nanopartículas poliméricas por nanopartículas de análisis de seguimiento y de alto rendimiento Citometría de Flujo

Non-viral gene delivery using polymeric nanoparticles has emerged as an attractive approach for gene therapy to treat genetic diseases1 and as a ...

Harmonic Nanoparticles for Regenerative Research

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

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

Armónica Nanopartículas para Regenerativa de Investigación

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

Production of Metal Nanoparticles by Pulsed Laser-ablation in Liquids: A Tool for Studying the Antibacterial Properties of Nanoparticles

The emergence of multidrug-resistant bacteria is a global clinical concern leading some to speculate about our return to a "pre-antibiotics" era of medicine. In addition to efforts to identify novel small-molecule antimicrobial drugs, there has been great interest in the use of metal nanoparticles as coatings for medical devices, wound dressings, and consumer packaging, due to their antimicrobial properties. The wide variety of methods available for nanoparticle synthesis results in a broad spectrum of chemical and physical properties which can affect antibacterial efficacy. This manuscript describes the pulsed laser-ablation in liquids (PLAL) method to create nanoparticles. This approach allows for the fine tuning of nanoparticle size, composition, and stability using post-irradiation methods as well as the addition of surfactants or volume excluders. By controlling particle size and composition, a large range of physical and chemical properties of metal nanoparticles can be explored which may contribute to their antimicrobial efficacy thereby opening new avenues for antibacterial development.

The antimicrobial properties of metals such as copper and silver have been recognized for centuries. This protocol describes pulsed laser-ablation in liquids, a method of synthesizing metal nanoparticles that provides the ability to fine tune the properties of these nanoparticles to optimize their antimicrobial effects.

Producción de nanopartículas metálicas por ablación por láser pulsado en líquidos: una herramienta para estudiar las propiedades antibacterianas de las nanopartículas

The emergence of multidrug-resistant bacteria is a global clinical concern leading some to speculate about our return to a "pre-antibiotics" era of ...

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

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

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

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

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

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

Protein Kinase C-delta Inhibitor Peptide Formulation using Gold Nanoparticles

Protein kinase C-delta inhibitor (PKC&#948;i) is a promising drug to prevent ischemia-reperfusion-induced organ injury. It is usually conjugated to a cell-penetrating peptide, TAT, for intracellular delivery. However, TAT has shown non-specific biological activities. Gold nanoparticles (GNPs) can be used as drug delivery carriers without recognized toxicity. Therefore, we have used a GNP/peptide hybrid to deliver PKC&#948;i. Two short peptides (P2: CAAAAE and P4: CAAAAW), at a 95:5 ratio, were used to modify the surface properties of GNP. GNPs conjugated with PKC&#948;i (GNP/PKCi) are stable in distilled water, 0.9% NaCl, and phosphate-buffered saline (PBS) containing bovine serum albumin or fetal bovine serum. Intravenous injection of GNP-PKCi was previously shown to prevent ischemia-reperfusion injury of the lung. This article outlines a protocol to formulate GNP/PKCi and assess the physiochemical properties of GNP/PKCi. We have used similar methods to formulate other peptide-based drugs with GNP. This article will hopefully draw more attention to this novel intracellular drug delivery technology and its applications in vivo.

We have previously used a gold nanoparticle peptide hybrid to intravenously deliver a synthetic peptide, protein kinase C-delta inhibitor, which reduced ischemia-reperfusion-induced acute lung injury. Here we show the detailed protocol of the drug formulation. Other intracellular peptides can be formulated similarly.

Proteína quinasa c-Delta inhibidor péptido formulación usando nanopartículas de oro

Protein kinase C-delta inhibitor (PKC&#948;i) is a promising drug to prevent ischemia-reperfusion-induced organ injury. It is usually conjugated to a ...