This work was supported by the Sheikh Zayed Institute for Pediatric Surgical Innovation (RAC Awards #30000174 and 30001489).

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The authors have nothing to disclose.

Este artículo ha presentado los métodos para la síntesis de una nueva clase de agentes formadores de imágenes multimodales, moleculares basados ​​en nanopartículas azul de Prusia biofuncionalizadas. Las técnicas de imagen molecular incorporados en las nanopartículas son imágenes de fluorescencia y resonancia magnética molecular, debido a sus características complementarias. Las nanopartículas azul de Prusia biofuncionalizadas tienen un diseño de núcleo-corteza. Los pasos clave en la síntesis de estas n...

La imagen molecular es la visualización no invasiva y precisa de los procesos biológicos a nivel celular, subcelular, y los niveles moleculares 1. La imagen molecular permite un espécimen a permanecer en su microambiente nativo mientras que sus vías y mecanismos endógenos son evaluados en tiempo real. Típicamente, la imagen molecular implica la administración de un agente de imagen exógeno en forma de una pequeña molécula, macromolécula, o nanopartículas para visualizar, objetivo, y trazar procesos fisiológicos pertinentes se está estudiando 2. Las diversas modalidades de imagen que se han explorado en imagen molecular incluyen MRI, CT, PET, SPECT, ultrasonido, photoacoustics, espectroscopia Raman, la bioluminiscencia, la fluorescencia y microscopía intravital 3. Multimodal de imágenes es la combinación de dos o más modalidades de imagen donde la combinación mejora la capacidad de visualizar y caracterizar diversos procesos y eventos 4 biológicos. Multimodal de imágenes explota las ventajas de las técnicas de formación de imágenes individuales, mientras que la compensación de sus limitaciones individuales 3. En este artículo se presenta el protocolo para la síntesis de nanopartículas azul de Prusia biofuncionalizadas (PB PN) - una nueva clase de agentes de imágenes multimodales, molecular. Los PN PB se utilizan para obtener imágenes de fluorescencia y resonancia magnética molecular. PB es un pigmento que consiste de la alternancia de hierro (II) y hierro (III) átomos en una red cúbica centrada en las caras (Figura 1). La celosía PB se compone de ligandos cianuro lineales en una Fe II - CN - vinculación Fe III que incorpora cationes para equilibrar las cargas dentro de su red tridimensional 5. La capacidad de PB incorporar cationes en su celosía se explota mediante la carga por separado gadolinio y iones de manganeso en los PN PB para el contraste de resonancia magnética. La razón fundamental para la consecución de un diseño de nanopartículas para el contraste RM es debidolas ventajas de este diseño ofrece en relación con los agentes de contraste MRI actuales. La gran mayoría de agentes de contraste MRI aprobados por la FDA de Estados Unidos son quelatos de gadolinio que son paramagnéticos en la naturaleza y proporcionan contraste positivo por el mecanismo de relajación 6,7,8-spin celosía. En comparación con un solo gadolinio-quelato que proporciona baja intensidad de señal por sí mismo, la incorporación de múltiples iones de gadolinio dentro de la red PB de las nanopartículas proporciona una mayor intensidad de la señal (contraste positivo) 3,9. Además, la presencia de múltiples iones de gadolinio dentro de la red PB aumenta la densidad de espín general y la magnitud de paramagnetismo de las nanopartículas, que perturba el campo magnético local en su vecindad, generando de ese modo contraste negativo por el mecanismo de relajación espín-espín. Así, las nanopartículas que contienen gadolinio funcionar tanto como T 1 (positivo) y T 2 agentes de contraste (negativo) 10,11. En un subgrupo de pacientes con insuficiencia renal, la administración de agentes de contraste con gadolinio se ha relacionado con el desarrollo de fibrosis sistémica nefrogénica 8,12, 13. Esta observación ha llevado a investigaciones sobre el uso de iones paramagnéticos alternativos como agentes de contraste para MRI. Por lo tanto, el diseño versátil de las nanopartículas está adaptado para incorporar iones de manganeso dentro de la red PB. Similar a gadolinio quelatos, manganeso quelatos también son paramagnéticos y se utilizan normalmente para proporcionar una intensidad de señal positiva en la RM 7,14. Al igual que con gadolinio PB PN, las PB PN contienen manganeso también funcionan como T 1 (positivo) y T 2 agentes de contraste (negativo). Para incorporar capacidades de formación de imágenes de fluorescencia, la nanopartícula &quot;núcleos&quot; se recubren con una cáscara &quot;biofuncional&quot; que consiste en la avidina marcada con fluorescencia-glicoproteína (Figura 1). Avidina no sólo permite imágenes de fluorescencia, sino que también sirve como una plataforma de acoplamiento para ligandos biotinilados que se dirigen a las células y tejidos específicos. La unión de avidina-biotina es una de las más fuertes enlaces conocidos, no covalentes caracterizadas por extremadamente fuerte afinidad de unión entre avidina y biotina 15. La unión de ligandos biotinilados a la avidina-revestido PB PN confiere posibilidades de marketing moleculares a los PN PB. La motivación para la búsqueda de imágenes de fluorescencia y MR usando PB PN se debe a que estas técnicas de imagen poseen características complementarias. Imágenes de fluorescencia es una de las técnicas de imagen molecular óptica más utilizado, y permite la visualización simultánea de múltiples objetos a altas sensibilidades 1,16,17. Imágenes de fluorescencia es una modalidad segura, no invasiva, pero se asocia con bajas profundidades de penetración y resoluciones espaciales 1,3,16. Por otro lado, MRI genera un alto temporald resolución espacial de forma no invasiva y sin necesidad de radiación ionizante 1,3,16. Sin embargo MRI sufre de baja sensibilidad. Por lo tanto, imágenes de fluorescencia y la RM fueron seleccionadas como las técnicas de imagen molecular, debido a sus características complementarias de penetración de profundidad, sensibilidad y resolución espacial. En este artículo se presenta el protocolo para la síntesis y biofuncionalización de los PN PB, PB PN (GdPB), y que contiene gadolinio-PB PN (MnPB) 10,11 que contiene manganeso. Los siguientes métodos se describen: 1) medición del tamaño, la carga y la estabilidad temporal de las nanopartículas, 2) Evaluación de la citotoxicidad de las nanopartículas, 3) de medición de relaxividades MRI, y 4) la utilización de las nanopartículas para la fluorescencia y la RM molecular de una población de células diana in vitro. Estos resultados demuestran el potencial de los NPs para uso como agentes de formación de imágenes multimodales, moleculares in vivo.

<table border="0" cellpadding="0" cellspacing="0" width="854">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:455px;">Name of Material/ Equipment</td>
			<td style="width:115px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Potassium hexacyanoferrate (II) trihydrate (K4Fe(CN)6 &#183; 3H2O)</td>
			<td>Sigma-Aldrich</td>
			<td>P9387</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Manganese (II) chloride tetrahydrate (MnCl2 &#183; 4H2O)</td>
			<td>Sigma-Aldrich</td>
			<td>221279</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gadolinium (III) nitrate hexahydrate (Gd(NO3)3 &#183; 6H2O)</td>
			<td>Sigma-Aldrich</td>
			<td>211591</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Iron (III) chloride hexahydrate (FeCl3 &#183; 6H2O)</td>
			<td>Sigma-Aldrich</td>
			<td>236489</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium chloride (NaCl)</td>
			<td>Sigma-Aldrich</td>
			<td>S9888</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-NG2 Chondroitin Sulfate Proteoglycan, Biotin Conjugate Antibody</td>
			<td>Millipore</td>
			<td>AB5320</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Biotinylated Anti-Human Eotaxin-3</td>
			<td>Peprotech</td>
			<td>500-P156GBT</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Neuro-2a Cell Line</td>
			<td>ATCC</td>
			<td>CCL-131</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BSG D10 Cell Line</td>
			<td>Lab stock</td>
			<td>---</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">OE21 Cell Line</td>
			<td>Sigma-Aldrich</td>
			<td>96062201</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SUDIPG1 Neurospheres</td>
			<td>Lab stock</td>
			<td>---</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Eol-1 Cell Line</td>
			<td>Sigma-Aldrich</td>
			<td>94042252</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Poly(L-lysine) hydrobromide</td>
			<td>Sigma-Aldrich</td>
			<td>P1399</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Formaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>F8775</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bovine serum albumin</td>
			<td>Sigma-Aldrich</td>
			<td>A2153</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Aminoactinomycin D</td>
			<td>Sigma-Aldrich</td>
			<td>A9400</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>X100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CellTrace Calcein Red-Orange, AM</td>
			<td>Life Technologies</td>
			<td>C34851</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Avidin-Alexa Fluor 488</td>
			<td>Life Technologies</td>
			<td>A21370</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Centrifuge</td>
			<td>Eppendorf</td>
			<td>5424</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Peristaltic Pump</td>
			<td>Instech</td>
			<td>P270</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Zetasizer Nano ZS</td>
			<td>Malvern</td>
			<td>ZEN3600</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sonicator</td>
			<td>QSonica</td>
			<td>Q125</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hot Plate/Magnetic Stirrer</td>
			<td>VWR</td>
			<td>97042-642</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ultra Clean Aluminum Foil</td>
			<td>VWR</td>
			<td>89107-732</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vortex Mixer</td>
			<td>VWR</td>
			<td>58816-121</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1.7 mL conical microcentrifuge tubes</td>
			<td>VWR</td>
			<td>87003-295</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">15 mL conical centrifuge tubes</td>
			<td>VWR</td>
			<td>21008-918</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tube holders</td>
			<td>VWR</td>
			<td>82024-342</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Disposable plastic cuvettes</td>
			<td>VWR</td>
			<td>7000-590 (/586)</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Zetasizer capillary cell</td>
			<td>VWR</td>
			<td>DTS1070</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Centrifugal Filters, 0.2 micrometer spin column</td>
			<td>VWR</td>
			<td>82031-356</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">96-well cell culture tray</td>
			<td>VWR</td>
			<td>29442-056</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trypsin EDTA 0.25% solution 1X</td>
			<td>JR Scientific</td>
			<td>82702</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cell Culture Grade PBS (1X)</td>
			<td>Life Technologies</td>
			<td>10010023</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">XTT Cell Proliferation Assay Kit</td>
			<td>Trevigen</td>
			<td>4891-025-K</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">T75 Flask</td>
			<td>89092-700</td>
			<td>VWR</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dulbecco&#39;s Modified Eagle&#39;s Medium</td>
			<td>Biowhitaker</td>
			<td>12-604Q</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fetal Bovine Serum</td>
			<td>Life Technologies</td>
			<td>10437-010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pen-Strep 1X</td>
			<td>Life Technologies</td>
			<td>15070063</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fluoview FV1200 Confocal Laser Scanning Microscope</td>
			<td>Olympus</td>
			<td>FV1200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Chambered Microscope Slides</td>
			<td>Thermo Scientific</td>
			<td>154534</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Micro Cover Glasses, Square, No. 1.5</td>
			<td>VWR</td>
			<td>48366-227</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microscope Slides</td>
			<td>VWR</td>
			<td>16004-368</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RPMI</td>
			<td>Sigma-Aldrich</td>
			<td>R8758&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Agarose</td>
			<td>Sigma-Aldrich</td>
			<td>A9539&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FACSCalibur Flow Cytometer</td>
			<td>BD Biosciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">3 T Clinical MRI Magnet</td>
			<td>GE Healthcare</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">100 mL round-bottom flask</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

biofunctionalized prussian blue nanoparticles for multimodal molecular imaging applications

Multimodal, la imagen molecular permite la visualización de los procesos biológicos a resoluciones celulares, subcelulares, ya nivel molecular utilizando múltiples técnicas de imagen complementarias. Estos agentes de formación de imágenes facilitan la evaluación en tiempo real de las vías y mecanismos in vivo, que mejoran tanto la eficacia diagnóstica y terapéutica. En este artículo se presenta el protocolo para la síntesis de nanopartículas azul de Prusia biofuncionalizadas (PB PN) - una nueva clase de agentes para su uso en aplicaciones de imágenes multimodales, molecular. Las técnicas de imagen incorporados en el nanopartículas, imágenes de fluorescencia y la resonancia magnética (MRI), tienen características complementarias. Los PN PB poseen un diseño de núcleo-corteza donde gadolinio y iones de manganeso incorporados dentro de los espacios intersticiales de la red PB generan contraste MRI, tanto en T1 y T2 y secuencias. Los PN PB están recubiertas con avidina fluorescente usando electrostática auto comoblea, que permite imágenes de fluorescencia. Las nanopartículas recubiertas con avidina se modifican con ligandos biotinilados que confieren posibilidades de marketing moleculares a las nanopartículas. La estabilidad y la toxicidad de las nanopartículas se miden, así como sus capacidades de relajación de MRI. Las capacidades de imágenes multimodales, moleculares de estas biofuncionalizadas PN PB luego se demostraron utilizándolos para imágenes de fluorescencia y resonancia magnética molecular in vitro.

Usando el esquema de síntesis en un solo recipiente, las nanopartículas de Pb NPs (diámetro medio de 78,8 nm, índice de polidispersidad (PDI) = 0,230; calculada por el instrumento de dispersión de luz dinámica), GdPB, o MnPB (diámetro de 164,2 nm, PDI = 0,102 significa) ( diámetro medio 122,4 nm, PDI = 0,124) que son monodispersas (medido por DLS) se puede sintetizar constantemente (Figura 2A). Los potenciales zeta medidos de las nanopartículas sintetizadas están a menos de -30 mV (Fig...

Watch this Scientific Journal Video about Biofunctionalized Prussian Blue Nanoparticles for Multimodal Molecular Imaging Applications at JoVE.com

Biofunctionalized Prussian Blue Nanoparticles for Multimodal Molecular Imaging Applications

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

Biofuncionalizadas prusiana nanopartículas azul para Multimodal Molecular Aplicaciones de imágenes

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

biofunctionalized-prussian-blue-nanoparticles-for-multimodal

Research

JoVE Journal

Bioengineering

10.2K vistas.  Children's National Medical Center. This protocol describes the synthesis of biofunctionalized Prussian blue nanoparticles and their use as multimodal, molecular imaging agents. The nanoparticles have a core-shell design where gadolinium or manganese ions within the nanoparticle core generate MRI contrast. The biofunctional shell contains fluorophores for fluorescence imaging and targeting ligands for molecular targeting. 

Video: Biofunctionalized Prussian Blue Nanoparticles for Multimodal Molecular Imaging Applications

Fluorescence Lifetime Imaging of Molecular Rotors in Living Cells

Diffusion is often an important rate-determining step in chemical reactions or biological processes and plays a role in a wide range of intracellular events. Viscosity is one of the key parameters affecting the diffusion of molecules and proteins, and changes in viscosity have been linked to disease and malfunction at the cellular level.1-3 While methods to measure the bulk viscosity are well developed, imaging microviscosity remains a challenge. Viscosity maps of microscopic objects, such as single cells, have until recently been hard to obtain. Mapping viscosity with fluorescence techniques is advantageous because, similar to other optical techniques, it is minimally invasive, non-destructive and can be applied to living cells and tissues.
Fluorescent molecular rotors exhibit fluorescence lifetimes and quantum yields which are a function of the viscosity of their microenvironment.4,5 Intramolecular twisting or rotation leads to non-radiative decay from the excited state back to the ground state. A viscous environment slows this rotation or twisting, restricting access to this non-radiative decay pathway. This leads to an increase in the fluorescence quantum yield and the fluorescence lifetime. Fluorescence Lifetime Imaging (FLIM) of modified hydrophobic BODIPY dyes that act as fluorescent molecular rotors show that the fluorescence lifetime of these probes is a function of the microviscosity of their environment.6-8 A logarithmic plot of the fluorescence lifetime versus the solvent viscosity yields a straight line that obeys the F&ouml;rster Hoffman equation.9 This plot also serves as a calibration graph to convert fluorescence lifetime into viscosity.
Following incubation of living cells with the modified BODIPY fluorescent molecular rotor, a punctate dye distribution is observed in the fluorescence images. The viscosity value obtained in the puncta in live cells is around 100 times higher than that of water and of cellular cytoplasm.6,7 Time-resolved fluorescence anisotropy measurements yield rotational correlation times in agreement with these large microviscosity values. Mapping the fluorescence lifetime is independent of the fluorescence intensity, and thus allows the separation of probe concentration and viscosity effects. 
In summary, we have developed a practical and versatile approach to map the microviscosity in cells based on FLIM of fluorescent molecular rotors.

Fluorescence Lifetime Imaging (FLIM) has emerged as a key technique to image the environment and interaction of specific proteins and dyes in living cells. FLIM of fluorescent molecular rotors allows mapping of viscosity in living cells.

Imágenes de fluorescencia vida de rotores moleculares en las células vivas

Diffusion is often an important rate-determining step in chemical reactions or biological processes and plays a role in a wide range of intracellular ...

Investigación

Bioingeniería

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 ...

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 ...

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 ...

From Fast Fluorescence Imaging to Molecular Diffusion Law on Live Cell Membranes in a Commercial Microscope

It has become increasingly evident that the spatial distribution and the motion of membrane components like lipids and proteins are key factors in the regulation of many cellular functions. However, due to the fast dynamics and the tiny structures involved, a very high spatio-temporal resolution is required to catch the real behavior of molecules. Here we present the experimental protocol for studying the dynamics of fluorescently-labeled plasma-membrane proteins and lipids in live cells with high spatiotemporal resolution. Notably, this approach doesn&#8217;t need to track each molecule, but it calculates population behavior using all molecules in a given region of the membrane. The starting point is a fast imaging of a given region on the membrane. Afterwards, a complete spatio-temporal autocorrelation function is calculated correlating acquired images at increasing time delays, for example each 2, 3, n repetitions. It is possible to demonstrate that the width of the peak of the spatial autocorrelation function increases at increasing time delay as a function of particle movement due to diffusion. Therefore, fitting of the series of autocorrelation functions enables to extract the actual protein mean square displacement from imaging (iMSD), here presented in the form of apparent diffusivity vs average displacement. This yields a quantitative view of the average dynamics of single molecules with nanometer accuracy. By using a GFP-tagged variant of the Transferrin Receptor (TfR) and an ATTO488 labeled 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphoethanolamine (PPE) it is possible to observe the spatiotemporal regulation of protein and lipid diffusion on &#181;m-sized membrane regions in the micro-to-milli-second time range.

Spatial distribution and temporal dynamics of plasma membrane proteins and lipids is a hot topic in biology. Here this issue is addressed by a spatio-temporal image fluctuation analysis that provides conceptually the same physical quantities of single particle tracking, but it uses small molecular labels and standard microscopy setups.

De Fluorescencia Fast Imaging a la Ley de Difusión Molecular en membranas de células vivas en un microscopio Comercial

It has become increasingly evident that the spatial distribution and the motion of membrane components like lipids and proteins are key factors in the ...

Cell Labeling and Targeting with Superparamagnetic Iron Oxide Nanoparticles

Targeted delivery of cells and therapeutic agents would benefit a wide range of biomedical applications by concentrating the therapeutic effect at the target site while minimizing deleterious effects to off-target sites. Magnetic cell targeting is an efficient, safe, and straightforward delivery technique. Superparamagnetic iron oxide nanoparticles (SPION) are biodegradable, biocompatible, and can be endocytosed into cells to render them responsive to magnetic fields. The synthesis process involves creating magnetite (Fe3O4) nanoparticles followed by high-speed emulsification to form a poly(lactic-co-glycolic acid) (PLGA) coating. The PLGA-magnetite SPIONs are approximately 120 nm in diameter including the approximately 10 nm diameter magnetite core. When placed in culture medium, SPIONs are naturally endocytosed by cells and stored as small clusters within cytoplasmic endosomes. These particles impart sufficient magnetic mass to the cells to allow for targeting within magnetic fields. Numerous cell sorting and targeting applications are enabled by rendering various cell types responsive to magnetic fields. SPIONs have a variety of other biomedical applications as well including use as a medical imaging contrast agent, targeted drug or gene delivery, diagnostic assays, and generation of local hyperthermia for tumor therapy or tissue soldering.

Targeted cell delivery is useful in a variety of biomedical applications. The goal of this protocol is to use superparamagnetic iron oxide nanoparticles (SPION) to label cells and thereby enable magnetic cell targeting approaches for a high degree of control over cell delivery and localization.

Etiquetado Celular y Orientación con superparamagnético nanopartículas de óxido de hierro

Targeted delivery of cells and therapeutic agents would benefit a wide range of biomedical applications by concentrating the therapeutic effect at the ...

Solid Lipid Nanoparticles (SLNs) for Intracellular Targeting Applications

Nanoparticle-based delivery vehicles have shown great promise for intracellular targeting applications, providing a mechanism to specifically alter cellular signaling and gene expression. In a previous investigation, the synthesis of ultra-small solid lipid nanoparticles (SLNs) for topical drug delivery and biomarker detection applications was demonstrated. SLNs are a well-studied example of a nanoparticle delivery system that has emerged as a promising drug delivery vehicle. In this study, SLNs were loaded with a fluorescent dye and used as a model to investigate particle-cell interactions. The phase inversion temperature (PIT) method was used for the synthesis of ultra-small populations of biocompatible nanoparticles. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylphenyltetrazolium bromide (MTT) assay was utilized in order to establish appropriate dosing levels prior to the nanoparticle-cell interaction studies. Furthermore, primary human dermal fibroblasts and mouse dendritic cells were exposed to dye-loaded SLN over time and the interactions with respect to toxicity and particle uptake were characterized using fluorescence microscopy and flow cytometry. This study demonstrated that ultra-small SLNs, as a nanoparticle delivery system, are suitable for intracellular targeting of different cell types.

Solid Lipid Nanoparticles SLNs for Intracellular Targeting Applications

In this study, a method for synthesizing ultra-small populations of biocompatible nanoparticles was described, as well as several in vitro methods by which to assess their cellular interactions.

Sólido lípidos nanopartículas (GLC) Aplicaciones para intracelular de focalización

Nanoparticle-based delivery vehicles have shown great promise for intracellular targeting applications, providing a mechanism to specifically alter ...

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

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

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

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

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

Multimodal Imaging and Spectroscopy Fiber-bundle Microendoscopy Platform for Non-invasive, In Vivo Tissue Analysis

Recent fiber-bundle microendoscopy techniques enable non-invasive analysis of in vivo tissue using either imaging techniques or a combination of spectroscopy techniques. Combining imaging and spectroscopy techniques into a single optical probe may provide a more complete analysis of tissue health. In this article, two dissimilar modalities are combined, high-resolution fluorescence microendoscopy imaging and diffuse reflectance spectroscopy, into a single optical probe. High-resolution fluorescence microendoscopy imaging is a technique used to visualize apical tissue micro-architecture and, although mostly a qualitative technique, has demonstrated effective real-time differentiation between neoplastic and non-neoplastic tissue. Diffuse reflectance spectroscopy is a technique which can extract tissue physiological parameters including local hemoglobin concentration, melanin concentration, and oxygen saturation. This article describes the specifications required to construct the fiber-optic probe, how to build the instrumentation, and then demonstrates the technique on in vivo human skin. This work revealed that tissue micro-architecture, specifically apical skin keratinocytes, can be co-registered with its associated physiological parameters. The instrumentation and fiber-bundle probe presented here can be optimized as either a handheld or endoscopically-compatible device for use in a variety of organ systems. Additional clinical research is needed to test the viability of this technique for different epithelial disease states.

Multimodal Imaging and Spectroscopy Fiber-bundle Microendoscopy Platform for Non-invasive, In Vivo Tissue Analysis

The assembly and use of a multimodal microendoscope is described which can co-register superficial tissue image data with tissue physiological parameters including hemoglobin concentration, melanin concentration, and oxygen saturation. This technique can be useful for evaluating tissue structure and perfusion, and can be optimized for individual needs of the investigator.

Multimodal de imágenes y la Plataforma Microendoscopy Espectroscopia de fibra paquete para no invasiva,<em&gt; En Vivo</em&gt; Análisis de Tejidos

Recent fiber-bundle microendoscopy techniques enable non-invasive analysis of in vivo tissue using either imaging techniques or a combination of ...

Manufacture and Drug Delivery Applications of Silk Nanoparticles

Silk is a promising biopolymer for biomedical and pharmaceutical applications due to its outstanding mechanical properties, biocompatibility and biodegradability, as well its ability to protect and subsequently release its payload in response to a trigger. While silk can be formulated into various material formats, silk nanoparticles are emerging as promising drug delivery systems. Therefore, this article covers the procedures for reverse engineering silk cocoons to yield a regenerated silk solution that can be used to generate stable silk nanoparticles. These nanoparticles are subsequently characterized, drug loaded and explored as a potential anticancer drug delivery system. Briefly, silk cocoons are reverse engineered first by degumming the cocoons, followed by silk dissolution and clean up, to yield an aqueous silk solution. Next, the regenerated silk solution is subjected to nanoprecipitation to yield silk nanoparticles &#8211; a simple but powerful method that generates uniform nanoparticles. The silk nanoparticles are characterized according to their size, zeta potential, morphology and stability in aqueous media, as well as their ability to entrap a chemotherapeutic payload and kill human breast cancer cells. Overall, the described methodology yields uniform silk nanoparticles that can be readily explored for a myriad of applications, including their use as a potential nanomedicine.

Nanoparticles are emerging as promising drug delivery systems for a broad range of indications. Here, we describe a simple yet powerful method to manufacture silk nanoparticles using reverse engineered Bombyx mori silk. These silk nanoparticles can be readily loaded with a therapeutic payload and subsequently explored for drug delivery applications.