The authors wish to acknowledge funding from the European Regional Development Fund &#8211; FNUSA-ICRC (no. CZ.1.05/ 1.1.00/ 02.0123), the American Heart Association Scientist Development Grant (AHA #06-35185N), and the National Institutes of Health (NIH #T32HL007111).

1. Síntesis de la magnetita de Gel <ol><li> Lave toda la cristalería usando ácido clorhídrico concentrado seguido de agua desionizada seguido por el alcohol etílico. Deje secar O / N, preferiblemente en un horno de secado. ¡CUIDADO! ácido clorhídrico es perjudicial - llevar equipo de protección personal y el trabajo en una campana de humos; alcohol etílico es perjudicial -, usar equipo de protección personal. </li><li> Utilice una botella Dreschel para de-gas 500 ml de H2O desionizada burbujeando suavemente gas N2 durante 30 minutos. </li><li> Puesta en marcha del aparato de síntesis de magnetita dentro de una campana de humos química. <ol><li> Coloque un frasco ml de tres bocas de fondo redondo de 500 dentro de un calentador isomantle y asegure la boca central utilizando una pinza y el soporte. </li><li> Instalar un septum de goma en uno de los cuellos laterales del matraz de fondo redondo y un condensador de reflujo con un septo de caucho en el cuello lado restante. Continuamente correr agua fría a través del condensador de reflujo. </li><li> Punción ªe septo de caucho del matraz de fondo redondo con una aguja conectada a una línea de gas N 2 y perforar tabique de caucho del condensador de reflujo con una aguja conectada a una línea de gas corriendo a un burbujeador (es decir, matraz con agua) para visualizar de salida de gas. </li><li> Instale una paleta de hoja en el cuello central del matraz de fondo redondo a través de un adaptador de paddle. Coloque el eje de la paleta de cuchilla para un agitador de cabeza montado en un soporte. </li></ol></li><li> Purgar el matraz de fondo redondo con gas N2 y dejar N2 gas que fluye a una velocidad baja, pero detectable. </li><li> Retire el condensador de reflujo desde el matraz de fondo redondo y añadir 1,000 g de hierro (III) cloruro, 0,6125 g de hierro (II) tetrahidrato de cloruro, y 50 ml de de-gaseados H 2 O. ¡CUIDADO! hierro (III) y cloruro de hierro (II) tetrahidrato de cloruro son perjudiciales -, usar equipo de protección personal. </li><li> Vuelva a colocar el condensador de reflujo y se agita a 1000 rpm mientras se calienta a 50° C. La agitación en estas condiciones produce 10 nanopartículas de magnetita nm de diámetro. </li><li> Una vez en 50 ° C, añadir 10 ml de solución de hidróxido de amonio al 28% mediante la inyección a través del septo de caucho en el matraz de fondo redondo mientras que todavía se agitaba. ¡CUIDADO! hidróxido de amonio es perjudicial -, usar equipo de protección personal. NOTA: La solución de hidróxido de amonio se utiliza para precipitar la magnetita y la solución debe volverse negro. </li><li> Quitar el tapón de caucho y N 2 línea de gas desde el matraz de fondo redondo y se calienta a 90 ° C a hervir el gas amoniaco mientras que todavía se agitaba. NOTA: Es opcional para mantener el flujo de N 2 en el matraz de fondo redondo por punción de tabique de caucho del condensador de reflujo, sin embargo, la oxidación de la magnetita a maghemita es insignificante durante este paso. </li><li> Una vez en 90 ° C, añadir 1 ml de ácido oleico a la matraz de fondo redondo mientras que todavía se agitaba. El ácido oleico se utiliza para recubrir la magnetita nanoparticles para formar gel de magnetita. ¡CUIDADO! ácido oleico es perjudicial -, usar equipo de protección personal. </li><li> Vuelva a colocar el tapón de goma y N 2 línea de gas en el matraz de fondo redondo y retire el condensador de reflujo. </li><li> Apagar el fuego y revuelva a 500 rpm durante 2 horas. </li><li> Retire el matraz de fondo redondo desde el calentador isomantle y decantar el líquido sobrante durante el uso de un imán fuerte sostenido contra la parte inferior del matraz para retener el gel de magnetita. ¡CUIDADO! manejar el imán fuerte con extremo cuidado para evitar daños o lesiones. </li><li> Permita gel de magnetita a O-aire seco / N (opcional). </li></ol> 2. Purificación de la magnetita de Gel <ol><li> Añadir 40 ml de hexano en el matraz de fondo redondo para disolver el gel de magnetita ¡CUIDADO! hexano es perjudicial -, usar equipo de protección personal y el trabajo en una campana de humos. </li><li> Use un embudo de separación con 40 ml de de-gaseados H2O para eliminar H 2 O residual frosoy la solución magnetita. <ol><li> Vierta lentamente la solución de magnetita en el H 2 O en el embudo de decantación y agitar suavemente el líquido de dos fases durante 5 minutos. </li><li> Escurra y deseche la fracción acuosa inferior. </li><li> Poco a poco agregue 40 ml de de-gaseados H2O para el embudo de separación de tal manera que se instala debajo de la solución de magnetita y agitar suavemente y drene como antes. </li><li> Repetir para lavar por tercera vez. </li></ol></li><li> Transfiera la solución de magnetita a un matraz Erlenmeyer, añadir unas espátulas valor de sulfato de sodio anhidro, y agitar para eliminar cualquier H 2 O residual que queda de la solución de magnetita. </li><li> Filtrar la solución a través de papel de filtro de magnetita 1 m en un embudo de filtro para eliminar el sulfato de sodio y H 2 O. residual NOTA: Se recomienda la asistencia de vacío. </li><li> Transferir la solución de magnetita a un matraz de evaporación de 50 ml y el uso de un evaporador rotatorio para evaporar el hexano durante2 h con las siguientes condiciones: Velocidad de rotación moderada, el vacío aplicado, evaporando matraz en un baño de agua 50 ° C, y 24 ° C de circulación de agua a través del condensador. NOTA: De forma opcional, almacenar el gel magnetita antes de recubrir con PLGA. </li></ol> 3. Recubrimiento de nanopartículas de magnetita con PLGA Shell <ol><li> Disolver 3,60 g de PLGA (75/25 mezcla) en 240 ml de acetato de etilo para crear un (m / v) solución de 1,5%. PRECAUCIÓN: acetato de etilo es perjudicial -, usar equipo de protección personal y el trabajo en una campana de humos. </li><li> Disolver 25,00 g de Pluronic F-127 en 500 ml de de-gaseados H 2 O usando un agitador magnético para crear un (m / v) solución de 5,0%. NOTA: Pluronic F-127 es un copolímero de bloques anfifílico no iónico que actúa como un agente tensioactivo biocompatible. Esto ayuda a estabilizar la emulsión aceite-en-agua en la etapa 3.3.2. </li><li> Utilizando una microespátula, recoger el gel de magnetita en seis alícuotas 0,040 g dentro de viales de vidrio ponderados. Perform el siguiente proceso de recubrimiento y lavado para cada alícuota. NOTA: Las alícuotas son necesarias para asegurar una manipulación eficiente y decantación magnética, que maximizará pureza y el rendimiento y reducir al mínimo la degradación antes de la liofilización en el paso 4. <ol><li> Añadir un 0,040 g alícuota de gel de magnetita y 40 ml de la solución de PLGA a un vaso de precipitados de plástico y se somete a ultrasonidos en un limpiador ultrasónico durante 10 minutos. </li><li> Añadir 80 ml de la solución de Pluronic al vaso de precipitados de plástico y emulsionar inmediatamente con un mezclador de laboratorio en la posición más alta durante 7 minutos para formar el revestimiento de PLGA en las nanopartículas de magnetita como una emulsión de aceite-en-agua. </li><li> Inmediatamente diluir la solución SPION en 1 litro de H2O desionizada y sonicar durante 1 h en una campana de extracción química para evaporar el acetato de etilo. </li><li> Coloque un imán fuerte junto a la solución SPION y revuelva suavemente para recoger SPIONs parduscos en el imán. NOTA: Puede ser necesario agitar de forma intermitente durante varias horass antes de que la solución se vuelve blanquecina que indica que la mayoría de los SPIONs se han recogido. </li><li> Decantar la solución acuosa al tiempo que conserva los SPIONs en el vaso de precipitados con el imán. </li><li> Lavar las SPIONs tres veces como sigue. <ol><li> Suspender los SPIONs en 1 litro de H2O desionizada </li><li> Sonicar la solución SPION durante 20 minutos. </li><li> Coloque un imán fuerte junto a la solución SPION y revuelva suavemente para recoger SPIONs parduscos en el imán. Puede ser necesario agitar intermitentemente durante varias horas antes de que la solución se vuelve clara que indica que la mayoría de los SPIONs han sido recogidos. </li><li> Decantar la solución acuosa al tiempo que conserva los SPIONs en el vaso de precipitados con el imán. </li></ol></li></ol></li><li> Recoge los SPIONs sintetizados a partir de cada una de las seis partes alícuotas de gel de magnetita en un único vial de vidrio ponderada como una suspensión acuosa. Opcionalmente decantar el exceso de agua magnéticamente según sea necesario. </li></ol> 4. CongelaciónSECADO de SPIONs <ol><li> Congelar la solución SPION. </li><li> Liofilizar la solución SPION O / N en un liofilizador. </li><li> Pesar los SPIONs liofilizados. SPIONs liofilizados pueden almacenarse a -20 ° C hasta su uso para el marcaje celular. NOTA: El almacenamiento a -20 ° C dramáticamente reduce la cinética de degradación y aumenta la vida útil. </li></ol> 5. Etiquetado de células con SPIONs <ol><li> Suspender una alícuota de SPIONs en solución salina tamponada con fosfato (PBS) a una concentración de 40 mg / ml y sonicar durante 30 minutos. </li><li> Añadir la solución a un matraz SPION casi confluente de células a una concentración de 5 l / ml de medio de cultivo celular. Asegurar una distribución uniforme moviendo suavemente el matraz. </li><li> Se incuban las células durante 16 horas a 37 ° C. </li><li> Aspirar suavemente el medio de cultivo y lavar las células dos veces con PBS. </li><li> Recoger las células marcadas magnéticamente y usar para los experimentos. </li><li> Solución SPION no utilizado se puede almacenar a 4 ° C y debe ser nosotrosed dentro de unos meses. Sonicar durante 30 minutos antes de cada uso. </li></ol>

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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+corona+significantly+reduces+active+targeting+yield.">Protein corona significantly reduces active targeting yield.</a> Chem Commun (Camb). 49 (25), 2557-2559 (2013).</li><li>Salvati, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transferrin-functionalized+nanoparticles+lose+their+targeting+capabilities+when+a+biomolecule+corona+adsorbs+on+the+surface.">Transferrin-functionalized nanoparticles lose their targeting capabilities when a biomolecule corona adsorbs on the surface.</a> Nat Nanotechnol. 8 (2), 137-143 (2013).</li><li>Landazuri, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetic+targeting+of+human+mesenchymal+stem+cells+with+internalized+superparamagnetic+iron+oxide+nanoparticles.">Magnetic targeting of human mesenchymal stem cells with internalized superparamagnetic iron oxide nanoparticles.</a> Small. 9 (23), 4017-4026 (2013).</li><li>Sun, J. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+labeling+of+endothelial+progenitor+cells+isolated+from+peripheral+blood+with+superparamagnetic+iron+oxide+nanoparticles.">In vitro labeling of endothelial progenitor cells isolated from peripheral blood with superparamagnetic iron oxide nanoparticles.</a> Mol Med Rep. 6 (2), 282-286 (2012).</li><li>Zhang, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+viability+of+transplanted+beta+cells+labeled+with+a+novel+contrast+agent+-+polyvinylpyrrolidone-coated+superparamagnetic+iron+oxide+nanoparticles+by+magnetic+resonance+imaging.">Detection of viability of transplanted beta cells labeled with a novel contrast agent - polyvinylpyrrolidone-coated superparamagnetic iron oxide nanoparticles by magnetic resonance imaging.</a> Contrast Media Mol Imaging. 7 (1), 35-44 (2012).</li><li>Song, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Labeling+efficacy+of+superparamagnetic+iron+oxide+nanoparticles+to+human+neural+stem+cells:+comparison+of+ferumoxides,+monocrystalline+iron+oxide,+cross-linked+iron+oxide+(CLIO)-NH2+and+tat-CLIO.">Labeling efficacy of superparamagnetic iron oxide nanoparticles to human neural stem cells: comparison of ferumoxides, monocrystalline iron oxide, cross-linked iron oxide (CLIO)-NH2 and tat-CLIO.</a> Korean J Radiol. 8 (5), 365-371 (2007).</li></ol>

The authors declare that they have no competing financial interests.

Como con cualquier protocolo de síntesis de nanopartículas, la pureza de los productos químicos reactivos es crítico para la consecución de SPIONs de alta calidad que tendrán efectos citotóxicos mínimos. Por tanto, es importante adquirir reactivos muy puros incluyendo el ácido oleico (≥99%), hierro (II) tetrahidrato de cloruro de (≥99.99%), (III) cloruro de hierro (≥99.99%), acetato de etilo (grado HPLC, ≥99.9% ), hexano (grado HPLC, ≥97.0%), hidróxido de amonio (≥99.99%), y sulfato de sodio (≥99...

Targeted delivery and capture of cells to specific sites within the body is desirable for a variety of biomedical applications. Delivery of neural stem cells to the brain by MRI-guided focused ultrasound has been proposed as a possible treatment option for neurodegenerative disease, traumatic brain injury, and stroke1. Mesenchymal stem cells are being studied for their ability to deliver anti-cancer drugs to tumors due to their natural tumor-tropic properties2,3. Cardiac stem cells have been delivered to the heart as a possible treatment for myocardial infarction4,5. Vascular stents have been developed with CD34 antibodies to capture circulating progenitor cells6. While promising, these cell targeting approaches present drawbacks including lack of cell specificity, inconsistent cell retention, and off-target cell delivery.
The overall goal of the current method is to enable magnetically directed targeting of cells for a variety of cell delivery and sorting applications. Magnetic targeting allows for controlled delivery of specific cells to a specific target site with minimal off-target effects7. The magnetic fields can be generated by implanted or external devices to safely direct the movement of magnetically-labeled cells within the body8. Numerous research efforts have focused on magnetically directed targeting of stem cells to injured tissues such as the heart9-14, retina15, lung16, skin17, spinal cord18,19, bone20, liver21, and muscle22,23 in order to improve regeneration outcomes.
Magnetic targeting of cells has also been studied extensively as a means to endothelialize implantable cardiovascular devices. A uniform and complete endothelium provides a barrier between the device and circulating blood elements to mitigate thrombosis and inflammation. Endothelial cells can be delivered to the device either prior to implantation or via the vascular system following implantation. In both cases, magnetic fields are used to capture cells to the surface of the device and retain the cells when subjected to the shear stress generated by circulating blood. Magnetic vascular stents24-27 and vascular grafts28 have both been fabricated and tested for this purpose.
Magnetic cell targeting requires a strategy for labeling cells with magnetic carrier particles. These particles can be bound to the surface of cells via antibodies or ligand/receptor pairs or they can be endocytosed into the cells. Superparamagnetic iron oxide nanoparticles (SPION) are biodegradable, biocompatible, and readily endocytosed by a variety of cell types29. These particles effectively render a cell responsive to magnetic fields and are naturally degraded over time. SPIONs provide a straightforward and safe means of magnetically labeling cells in culture for a variety of magnetic targeting and sorting applications. A method for synthesizing SPIONs with a magnetite (Fe3O4) core and poly(lactic-co-glycolic acid) (PLGA) shell is provided. In addition, a method for labeling cells in culture with SPIONs is provided.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Ammonium Hydroxide solution, 28% NH3 in H2O, &#8805;99.99% trace metal basis</td>
			<td>Sigma-Aldrich</td>
			<td>338818-100ML&#160;</td>
			<td>Harmful reagent - wear personal protective equipment</td>
		</tr>
		<tr>
			<td>Dreschel bottle, 500 mL</td>
			<td>Ace Glass</td>
			<td>5516-16</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethyl Acetate, CHROMASOLVR Plus, for HPLC, 99.9%&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>650528-1L</td>
			<td>Harmful reagent - wear personal protective equipment &#38; work in fume hood</td>
		</tr>
		<tr>
			<td>Ethyl alcohol</td>
			<td>Sigma-Aldrich</td>
			<td>E7023</td>
			<td>Harmful reagent - wear personal protective equipment</td>
		</tr>
		<tr>
			<td>Filter paper, 3 cm dia, grade 1</td>
			<td>Fisher</td>
			<td>09-805P</td>
			<td>For use with glass filter funnel</td>
		</tr>
		<tr>
			<td>Glass beakers, 1 L</td>
			<td>Fisher</td>
			<td>FB-101-1000</td>
			<td>For washing SPIONs</td>
		</tr>
		<tr>
			<td>Glass filter funnel, vacuum hose adapter, fits 24/40, 30 mL</td>
			<td>Fisher</td>
			<td>K954100-0344&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass vial caps</td>
			<td>Fisher</td>
			<td>03-391-46</td>
			<td>For use with glass vials</td>
		</tr>
		<tr>
			<td>Glass vials, 2 mL</td>
			<td>Fisher</td>
			<td>03-391-44</td>
			<td>For collecting magnetite gel &#38; SPIONs</td>
		</tr>
		<tr>
			<td>Hexane, CHROMASOLVR, for HPLC, &#8805;97.0% (GC)</td>
			<td>Sigma-Aldrich</td>
			<td>34859-1L&#160;</td>
			<td>Harmful reagent - wear personal protective equipment &#38; work in fume hood</td>
		</tr>
		<tr>
			<td>Hydrochloric acid</td>
			<td>Sigma-Aldrich</td>
			<td>H1758</td>
			<td>Harmful reagent - wear personal protective equipment &#38; work in fume hood</td>
		</tr>
		<tr>
			<td>Iron(II) chloride tetrahydrate, &#8805;99.99% trace metals basis&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>380024-5G</td>
			<td>Harmful reagent - wear personal protective equipment</td>
		</tr>
		<tr>
			<td>Iron(III) chloride anhydrous, powder, &#8805;99.99% trace metals basis</td>
			<td>Sigma-Aldrich</td>
			<td>451649-1G</td>
			<td>Harmful reagent - wear personal protective equipment</td>
		</tr>
		<tr>
			<td>Isomantle heater, 500 mL</td>
			<td>Voight Global</td>
			<td>EM0500/CEX1</td>
			<td></td>
		</tr>
		<tr>
			<td>Laboratory mixer</td>
			<td>Silverson</td>
			<td>L5M-A</td>
			<td></td>
		</tr>
		<tr>
			<td>Lyophilizer</td>
			<td>Labconco</td>
			<td>7670520</td>
			<td></td>
		</tr>
		<tr>
			<td>Microspatulas</td>
			<td>Fisher</td>
			<td>21-401-25A</td>
			<td>For transfering magnetite gel</td>
		</tr>
		<tr>
			<td>NdFeB magnet, 1 in x 1 in x 1 in</td>
			<td>Amazing Magnets</td>
			<td>C1000H-M</td>
			<td>Very strong magnet, handle with care</td>
		</tr>
		<tr>
			<td>Oleic acid, &#8805;99% (GC)</td>
			<td>Sigma-Aldrich</td>
			<td>O1008-5G&#160;</td>
			<td>Store in freezer; Harmful reagent - wear personal protective equipment</td>
		</tr>
		<tr>
			<td>Overhead stirrer</td>
			<td>IKA</td>
			<td>2572201</td>
			<td></td>
		</tr>
		<tr>
			<td>Overhead stirrer clamp</td>
			<td>IKA</td>
			<td>2664000</td>
			<td>For use with overhead stirrer</td>
		</tr>
		<tr>
			<td>Overhead stirrer H-stand</td>
			<td>IKA</td>
			<td>1412000</td>
			<td>For use with overhead stirrer</td>
		</tr>
		<tr>
			<td>Phosphate buffered saline</td>
			<td>Life Technologies</td>
			<td>10010-023</td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic beakers, 250 mL</td>
			<td>Fisher</td>
			<td>02-591-28</td>
			<td></td>
		</tr>
		<tr>
			<td>PLGA PURASORB PDLG (75/25 blend)</td>
			<td>Purac</td>
			<td>PDLG 7502</td>
			<td>PDLG 7502A may be used as well; Store in freezer</td>
		</tr>
		<tr>
			<td>Pluronic F-127 powder, BioReagent, suitable for cell culture</td>
			<td>Sigma-Aldrich</td>
			<td>P2443-250G&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>PTFE expandable blade paddle, 8 mm dia</td>
			<td>SciQuip</td>
			<td>SP4018</td>
			<td></td>
		</tr>
		<tr>
			<td>PTFE vessel adapter, fits 24/40, 8 mm dia paddle</td>
			<td>Monmouth Scientific</td>
			<td>PTFE Vessel Adaptor A480</td>
			<td>For use with PTFE expandable blade paddle</td>
		</tr>
		<tr>
			<td>Recirculating chiller</td>
			<td>Clarkson</td>
			<td>696613</td>
			<td>For use with rotoevaporator</td>
		</tr>
		<tr>
			<td>Reflux condenser, fits 24/40, 250 mm</td>
			<td>Ace Glass</td>
			<td>5997-133</td>
			<td></td>
		</tr>
		<tr>
			<td>Rotoevaporator</td>
			<td>Clarkson</td>
			<td>216949</td>
			<td></td>
		</tr>
		<tr>
			<td>Round bottom flask, 50 mL, 24/40 joint</td>
			<td>Sigma-Aldrich</td>
			<td>Z414484</td>
			<td>For use with rotoevaporator</td>
		</tr>
		<tr>
			<td>Rubber septa, fits 24/40</td>
			<td>Ace Glass</td>
			<td>9096-56</td>
			<td></td>
		</tr>
		<tr>
			<td>Separatory funnel with stopper, 250 mL</td>
			<td>Fisher</td>
			<td>10-438E</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium sulfate ACS reagent, &#8805;99.0%, anhydrous, granular</td>
			<td>Sigma-Aldrich</td>
			<td>239313-500G&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Three neck round bottom flask, angled, 24/40 joints, 500 mL</td>
			<td>Ace Glass</td>
			<td>6948-16</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrasonic cleaner perforated pan</td>
			<td>Fisher</td>
			<td>15-335-20A</td>
			<td>For use with ultrasonic cleaner</td>
		</tr>
		<tr>
			<td>Ultrasonic cleaner, 2.8 L</td>
			<td>Fisher</td>
			<td>15-335-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Vacuum controller</td>
			<td>Clarkson</td>
			<td>216639</td>
			<td>For use with rotoevaporator (optional)</td>
		</tr>
		<tr>
			<td>Vacuum pump</td>
			<td>Clarkson</td>
			<td>219959</td>
			<td>For use with rotoevaporator</td>
		</tr>
	</tbody>
</table>

cell labeling and targeting with superparamagnetic iron oxide nanoparticles

La administración dirigida de las células y agentes terapéuticos se beneficiaría de una amplia gama de aplicaciones biomédicas concentrando el efecto terapéutico en el sitio diana, mientras que se minimizan los efectos deletéreos a sitios desviado. Focalización celular magnética es una técnica de suministro eficiente, seguro y sencillo. Nanopartículas de óxido de hierro superparamagnético (SPION) son biodegradables, biocompatibles, y pueden ser endocitosis en células para hacerlos sensibles a los campos magnéticos. El proceso de síntesis implica la creación de magnetita (Fe 3 O 4) Nanopartículas seguido por emulsificación de alta velocidad para formar un poli (ácido láctico-co-glicólico) revestimiento (PLGA). Los SPIONs PLGA-magnetita son de aproximadamente 120 nm de diámetro incluyendo el núcleo de magnetita diámetro aproximadamente 10 nm. Cuando se coloca en medio de cultivo, SPIONs son naturalmente endocitosis por las células y se almacenan como pequeños grupos dentro de endosomas citoplasmáticos. Estas partículas imparten masa magnética suficiente para las célulaspara permitir la orientación dentro de los campos magnéticos. Numerosos clasificación de células y aplicaciones dirigidas están habilitadas por la prestación de diversos tipos de células sensibles a los campos magnéticos. SPIONs tienen una variedad de otras aplicaciones biomédicas, así como el uso como agente de contraste de imagen médica, la administración de fármacos o gen diana, ensayos de diagnóstico, y la generación de la hipertermia local para la terapia de tumores o de soldadura de tejidos.

Nanopartículas de magnetita son de aproximadamente 10 nm de diámetro, como resultado de agitación una solución acuosa de hierro (III) y cloruro de hierro (II) cloruro tetrahidrato a 50 ° C y 1000 rpm (Figura 1). Estos resultados demuestran la síntesis exitosa de nanopartículas de magnetita. Es importante para verificar el tamaño y la forma de nanopartículas de magnetita tomadas de una pequeña muestra del lote cuando se intenta la síntesis por primera vez. Microscopía electrónica de transmis...

Watch this Scientific Journal Video about Cell Labeling and Targeting with Superparamagnetic Iron Oxide Nanoparticles at JoVE.com

Cell Labeling and Targeting with Superparamagnetic Iron Oxide Nanoparticles

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 ...

cell-labeling-targeting-with-superparamagnetic-iron-oxide

Research

JoVE Journal

Bioengineering

11.9K vistas.  Mayo Clinic. 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.

Video: Cell Labeling and Targeting with Superparamagnetic Iron Oxide Nanoparticles

Production and Targeting of Monovalent Quantum Dots

The multivalent nature of commercial quantum dots (QDs) and the difficulties associated with producing monovalent dots have limited their applications in biology, where clustering and the spatial organization of biomolecules is often the object of study. We describe here a protocol to produce monovalent quantum dots (mQDs) that can be accomplished in most biological research laboratories via a simple mixing of CdSe/ZnS core/shell QDs with phosphorothioate DNA (ptDNA) of defined length. After a single ptDNA strand has wrapped the QD, additional strands are excluded from the surface. Production of mQDs in this manner can be accomplished at small and large scale, with commercial reagents, and in minimal steps. These mQDs can be specifically directed to biological targets by hybridization to a complementary single stranded targeting DNA. We demonstrate the use of these mQDs as imaging probes by labeling SNAP-tagged Notch receptors on live mammalian cells, targeted by mQDs bearing a benzylguanine moiety.

We provide detailed instructions for the preparation of monovalent targeted quantum dots (mQDs) from phosphorothioate DNA of defined length. DNA wrapping occurs in high yield, and therefore, products do not require purification. We demonstrate the use of the SNAP tag to target mQDs to cell-surface receptors for live-cell imaging applications.

Producción y focalización de monovalente Quantum Dots

The multivalent nature of commercial quantum dots (QDs) and the difficulties associated with producing monovalent dots have limited their applications in ...

Investigación

Bioingeniería

Fabrication of a Functionalized Magnetic Bacterial Nanocellulose with Iron Oxide Nanoparticles

In this study, bacterial nanocellulose (BNC) produced by the bacteria Gluconacetobacter xylinus is synthesized and impregnated in situ with iron oxide nanoparticles (IONP) (Fe3O4) to yield a magnetic bacterial nanocellulose (MBNC). The synthesis of MBNC is a precise and specifically designed multi-step process. Briefly, bacterial nanocellulose (BNC) pellicles are formed from preserved G. xylinus strain according to our experimental requirements of size and morphology. A solution of iron(III) chloride hexahydrate (FeCl3&#183;6H2O) and iron(II) chloride tetrahydrate (FeCl2&#183;4H2O) with a 2:1 molar ratio is prepared and diluted in deoxygenated high purity water. A BNC pellicle is then introduced in the vessel with the reactants. This mixture is stirred and heated at 80 &#176;C in a silicon oil bath and ammonium hydroxide (14%) is then added by dropping to precipitate the ferrous ions into the BNC mesh. This last step allows forming in situ magnetite nanoparticles (Fe3O4) inside the bacterial nanocellulose mesh to confer magnetic properties to BNC pellicle. A toxicological assay was used to evaluate the biocompatibility of the BNC-IONP pellicle. Polyethylene glycol (PEG) was used to cover the IONPs in order to improve their biocompatibility. Scanning electron microscopy (SEM) images showed that the IONP were located preferentially in the fibril interlacing spaces of the BNC matrix, but some of them were also found along the BNC ribbons. Magnetic force microscope measurements performed on the MBNC detected the presence magnetic domains with high and weak intensity magnetic field, confirming the magnetic nature of the MBNC pellicle. Young&#39;s modulus values obtained in this work are also in a reasonable agreement with those reported for several blood vessels in previous studies.

Here, we present a protocol to make a bacterial nanocellulose (BNC) magnetic for applications in damaged blood vessel reconstruction. The BNC was synthesized by G. xylinus strain. On the other hand, magnetization of the BNC was realized through in situ precipitation of Fe2+ and Fe3+ ferrous ions inside the BNC mesh.

La fabricación de una funcionalizado magnética bacteriana NANOCELULOSA con nanopartículas de óxido de hierro

In this study, bacterial nanocellulose (BNC) produced by the bacteria Gluconacetobacter xylinus is synthesized and impregnated in situ with iron oxide ...

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 ...

Identification of Metal Oxide Nanoparticles in Histological Samples by Enhanced Darkfield Microscopy and Hyperspectral Mapping

Nanomaterials are increasingly prevalent throughout industry, manufacturing, and biomedical research. The need for tools and techniques that aid in the identification, localization, and characterization of nanoscale materials in biological samples is on the rise. Currently available methods, such as electron microscopy, tend to be resource-intensive, making their use prohibitive for much of the research community. Enhanced darkfield microscopy complemented with a hyperspectral imaging system may provide a solution to this bottleneck by enabling rapid and less expensive characterization of nanoparticles in histological samples. This method allows for high-contrast nanoscale imaging as well as nanomaterial identification. For this technique, histological tissue samples are prepared as they would be for light-based microscopy. First, positive control samples are analyzed to generate the reference spectra that will enable the detection of a material of interest in the sample. Negative controls without the material of interest are also analyzed in order to improve specificity (reduce false positives). Samples can then be imaged and analyzed using methods and software for hyperspectral microscopy or matched against these reference spectra in order to provide maps of the location of materials of interest in a sample. The technique is particularly well-suited for materials with highly unique reflectance spectra, such as noble metals, but is also applicable to other materials, such as semi-metallic oxides. This technique provides information that is difficult to acquire from histological samples without the use of electron microscopy techniques, which may provide higher sensitivity and resolution, but are vastly more resource-intensive and time-consuming than light microscopy.

Enhanced darkfield microscopy and hyperspectral imaging with spectral mapping enable screening, localization, and identification of nanoscale materials in histological samples with improved speed and accuracy over traditional methods. The goal of this paper is to provide methods for darkfield imaging and hyperspectral mapping of metal oxide nanoparticles in histological samples.

Identificación de las nanopartículas de óxido de metal en muestras histológicas por Enhanced Mapeo de campo oscuro Microscopía y hiperespectrales

Nanomaterials are increasingly prevalent throughout industry, manufacturing, and biomedical research. The need for tools and techniques that aid in the ...

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 ...

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

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

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

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

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

Protocol for MicroRNA Transfer into Adult Bone Marrow-derived Hematopoietic Stem Cells to Enable Cell Engineering Combined with Magnetic Targeting

While CD133+ hematopoietic stem cells (SCs) have been proven to provide high potential in the field of regenerative medicine, their low retention rates after injection into injured tissues as well as the observed massive cell death rates lead to very restricted therapeutic effects. To overcome these limitations, we sought to establish a non-viral based protocol for suitable cell engineering prior to their administration. The modification of human CD133+ expressing SCs using microRNA (miR) loaded magnetic polyplexes was addressed with respect to uptake efficiency and safety as well as the targeting potential of the cells. Relying on our protocol, we can achieve high miR uptake rates of 80&#8211;90% while the CD133+ stem cell properties remain unaffected. Moreover, these modified cells offer the option of magnetic targeting. We describe here a safe and highly efficient procedure for the modification of CD133+ SCs. We expect this approach to provide a standard technology for optimization of therapeutic stem cell effects and for monitoring of the administered cell product via magnetic resonance imaging (MRI).

This protocol illustrates a safe and efficient procedure to modify CD133+ hematopoietic stem cells. The presented non-viral, magnetic polyplex-based approach may provide a basis for the optimization of therapeutic stem cell effects as well as for monitoring the administered cell product via magnetic resonance imaging.

Protocolo para la transferencia de microARN en médula ósea hematopoyéticas células madre adultas para permitir ingeniería celular combinado con orientación magnética

While CD133+ hematopoietic stem cells (SCs) have been proven to provide high potential in the field of regenerative medicine, their low retention rates ...

Catalytic Scavenging of Plant Reactive Oxygen Species In Vivo by Anionic Cerium Oxide Nanoparticles

Reactive oxygen species (ROS) accumulation is a hallmark of plant abiotic stress response. ROS play&#160;a dual role in plants by acting as signaling molecules at low levels and damaging molecules at high levels. Accumulation of ROS in stressed plants can damage metabolites, enzymes, lipids, and DNA, causing a reduction of plant growth and yield. The ability of cerium oxide nanoparticles (nanoceria) to catalytically scavenge ROS in vivo provides a unique tool to understand and bioengineer plant abiotic stress tolerance. Here, we present a protocol to synthesize and characterize poly (acrylic) acid coated nanoceria (PNC), interface the nanoparticles with plants via leaf lamina infiltration, and monitor their distribution and ROS scavenging in vivo using confocal microscopy. Current molecular tools for manipulating ROS accumulation in plants are limited to model species and require laborious transformation methods. This protocol for in vivo ROS scavenging has the potential to be applied to wild type plants with broad leaves and leaf structure like Arabidopsis thaliana.

Catalytic Scavenging of Plant Reactive Oxygen Species In Vivo by Anionic Cerium Oxide Nanoparticles

Here, we present a protocol for the synthesis and characterization of cerium oxide nanoparticles (nanoceria) for ROS (reactive oxygen species) scavenging in vivo, nanoceria imaging in plant tissues by confocal microscopy, and in vivo monitoring of nanoceria ROS scavenging by confocal microscopy.

Depuración catalítica de planta oxígeno reactivo especie In Vivo de nanopartículas de óxido de cerio aniónicos

Reactive oxygen species (ROS) accumulation is a hallmark of plant abiotic stress response. ROS play&#160;a dual role in plants by acting as signaling ...

Screening and Identification of Small Peptides Targeting Fibroblast Growth Factor Receptor2 using a Phage Display Peptide Library

The human Fibroblast Growth Factor Receptor (FGFR) family comprises four members, namely, FGFR1, FGFR2, FGFR3, and FGFR4, that are involved in various biological activities including cell proliferation, survival, migration and differentiation. Several aberrations in the FGFR signaling pathway, owing to mutations or gene amplification events, have been identified in various types of cancers. Hence, recent research has focused on developing strategies involving therapeutic targeting of FGFRs. Current FGFR inhibitors at various stages of pre-clinical and clinical development include either small molecule inhibitors of tyrosine kinases or monoclonal antibodies, with only a few peptide- based inhibitors in the pipeline. Here, we provide a protocol using phage display technology to screen small peptides as antagonists of FGFR2. Briefly, a library of phage-displayed peptides was incubated in a plate coated with FGFR2. Subsequently, unbound phage was washed off by TBST (TBS + 0.1% [v/v] Tween-20), and bound phage was eluted with 0.2 M glycine-HCl buffer (pH 2.2). The eluted phage was further amplified and used as input for the next round of biopanning. Following three rounds of biopanning, the peptide sequences of individual phage clones were identified by DNA sequencing. Finally, the screened peptides were synthesized and analyzed for affinity and biological activity.

Herein, we present a detailed protocol for screening small peptides that bind to FGFR2 using a phage display peptide library. We further analyze the affinity of the selected peptides toward FGFR2 in vitro and its ability to suppress cell proliferation.

Detección e identificación de pequeños péptidos dirigidos al receptor de factor de crecimiento de fibroblastos2 utilizando una biblioteca de péptidos de pantalla de fago

The human Fibroblast Growth Factor Receptor (FGFR) family comprises four members, namely, FGFR1, FGFR2, FGFR3, and FGFR4, that are involved in various ...

Using Magnetometry to Monitor Cellular Incorporation and Subsequent Biodegradation of Chemically Synthetized Iron Oxide Nanoparticles

Magnetic nanoparticles, made of iron oxide, present a peculiar interest for a wide range of biomedical applications for which they are often internalized in cells and then left within. One challenge is to assess their fate in the intracellular environment with reliable and precise methodologies. Herein, we introduce the use of the vibrating sample magnetometer (VSM) to precisely quantify the integrity of magnetic nanoparticles within cells by measuring their magnetic moment. Stem cells are first labeled with two types of magnetic nanoparticles; the nanoparticles have the same core produced via a fast and efficient microwave-based nonaqueous sol gel synthesis and differ in their coating: the commonly used citric acid molecule is compared to polyacrylic acid. The formation of 3D cell-spheroids is then achieved via centrifugation and the magnetic moment of these spheroids is measured at different times with the VSM. The obtained moment is a direct fingerprint of the nanoparticles&#8217; integrity, with decreasing values indicative of a nanoparticle degradation. For both nanoparticles, the magnetic moment decreases over culture time revealing their biodegradation. A protective effect of the polyacrylic acid coating is also shown, when compared to citric acid.

Iron oxide nanoparticles are synthesized via a nonaqueous sol gel procedure and coated with anionic short molecules or polymer. The use of magnetometry for monitoring the incorporation and biotransformations of magnetic nanoparticles inside human stem cells is demonstrated using a vibrating sample magnetometer (VSM).

Uso de magnetometría para monitorear la incorporación celular y la posterior biodegradación de nanopartículas de óxido de hierro sintetizadas químicamente

Magnetic nanoparticles, made of iron oxide, present a peculiar interest for a wide range of biomedical applications for which they are often internalized ...