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. Sintesi di magnetite Gel <ol><li> Lavare la vetreria con acido cloridrico concentrato seguita da acqua deionizzata seguita da alcool etilico. Lasciare asciugare O / N, preferibilmente in un forno di essiccazione. ATTENZIONE! acido cloridrico è dannoso - indossare dispositivi di protezione individuale e lavorare in una cappa aspirante; alcol etilico è dannoso - indossare dispositivi di protezione individuale. </li><li> Utilizzare una bottiglia di Drechsel di de-gas 500 ml di H 2 O deionizzata facendo gorgogliare delicatamente N 2 gas per 30 minuti. </li><li> Set-up l&#39;apparato di sintesi di magnetite all&#39;interno di una cappa. <ol><li> Posizionare un pallone ml a tre colli a fondo tondo da 500 all&#39;interno di un riscaldatore cuffia riscaldante e fissare il collo centro utilizzando una pinza e stand. </li><li> Installare un setto di gomma in uno dei colli laterali del pallone a fondo rotondo e condensatore a riflusso con un setto di gomma nel collo lato rimanente. Continuamente scorrere acqua fredda attraverso il refrigerante a ricadere. </li><li> Puntura °e setto di gomma del pallone a fondo tondo con un ago collegato ad una linea del gas N 2 e forare il setto di gomma del condensatore a riflusso con un ago collegato ad una linea di gas in esecuzione ad un gorgogliatore (cioè, matraccio con acqua) per visualizzare efflusso del gas. </li><li> Installare una pala lama nel centro collo del pallone a fondo tondo tramite un adattatore pagaia. Attaccare albero della pala lama a un agitatore montato su un supporto. </li></ol></li><li> Spurgare il pallone a fondo tondo con N 2 gas e lasciare N 2 gas che scorre ad una velocità bassa ma rilevabile. </li><li> Rimuovere il condensatore a riflusso del pallone a fondo tondo ed aggiungere 1.000 g di ferro (III) cloruro, 0,6125 g di ferro (II) cloruro tetraidrato, e 50 ml di degassati H 2 O. ATTENZIONE! di ferro (III) cloruro e ferro (II) cloruro tetraidrato sono nocivi - indossare dispositivi di protezione individuale. </li><li> Sostituire il condensatore a ricadere e mescolare a 1.000 giri, mentre il riscaldamento a 50° C. Mescolando in queste condizioni produce 10 nm nanoparticelle di magnetite diameter. </li><li> Una volta a 50 ° C, aggiungere 10 ml di soluzione di idrossido di ammonio 28% iniettando attraverso il tappo di gomma nel pallone a fondo tondo pur agitazione. ATTENZIONE! idrossido di ammonio è dannoso - indossare dispositivi di protezione individuale. NOTA: La soluzione di idrossido di ammonio è utilizzato per precipitare magnetite e la soluzione dovrebbe diventare nero. </li><li> Rimuovere il setto di gomma e N 2 linea gas dal pallone a fondo tondo e calore a 90 ° C per evaporare l&#39;ammoniaca gassosa mentre ancora agitazione. NOTA: È facoltativa per mantenere il flusso di N 2 nel pallone a fondo tondo perforando setto di gomma della condensatore a riflusso, tuttavia, l&#39;ossidazione di magnetite per maghemite è trascurabile durante questa fase. </li><li> Una volta a 90 ° C, aggiungere 1 ml di acido oleico nel pallone a fondo tondo pur agitazione. L&#39;acido oleico è utilizzato per rivestire la magnetite nananoparticelle di per formare gel magnetite. ATTENZIONE! acido oleico è dannoso - indossare dispositivi di protezione individuale. </li><li> Sostituire il setto di gomma e N 2 linea del gas sul pallone a fondo tondo e rimuovere il refrigerante a ricadere. </li><li> Spegnere il fuoco e mescolate a 500 rpm per 2 ore. </li><li> Rimuovere il pallone a fondo tondo dal riscaldatore cuffia riscaldante e decantare il liquido in eccesso mentre usando un magnete forte tenuto contro il fondo del pallone di trattenere il gel magnetite. ATTENZIONE! gestire il forte magnete con estrema cura per evitare danni o lesioni. </li><li> Lasciare che il gel di magnetite asciugare O / N (facoltativo). </li></ol> 2. Purificazione di magnetite Gel <ol><li> Aggiungere 40 ml di esano nel pallone a fondo tondo di sciogliere il gel magnetite ATTENZIONE! esano è dannoso - indossare dispositivi di protezione individuale e lavorare in una cappa aspirante. </li><li> Utilizzare un imbuto separatore con 40 ml di de-gasati H 2 O per rimuovere residui di H 2 O from la soluzione magnetite. <ol><li> Versare lentamente la soluzione di magnetite sul H 2 O nel imbuto separatore e miscelare delicatamente il liquido in due fasi per 5 minuti. </li><li> Scolare fuori e scartare la frazione acquoso inferiore. </li><li> Aggiungere lentamente 40 ml di degassati H 2 O a imbuto separatore tale che si deposita sotto la soluzione magnetite e miscelare delicatamente e scaricare come prima. </li><li> Ripetere per lavare per la terza volta. </li></ol></li><li> Trasferire la soluzione di magnetite di una beuta, aggiungere un paio di spatole per un valore di solfato di sodio anidro e agitare per rimuovere ogni residuo H 2 O residua dalla soluzione di magnetite. </li><li> Filtrare la soluzione attraverso carta da filtro magnetite 1 micron in un imbuto filtro per rimuovere il solfato di sodio e residua H 2 O. NOTA: è consigliabile farsi accompagnare vuoto. </li><li> Trasferire la soluzione magnetite ad un pallone di evaporazione 50 ml e utilizzare un evaporatore rotante per evaporare l&#39;esano per2 h con le seguenti condizioni: velocità di rotazione moderate, vuoto applicato, evaporando pallone in un bagno a 50 ° C di acqua, e 24 ° C l&#39;acqua che circola attraverso il condensatore. NOTA: In alternativa, conservare il gel di magnetite prima del rivestimento con PLGA. </li></ol> 3. Rivestimento di magnetite di nanoparticelle con PLGA Shell <ol><li> Sciogliere 3,60 g di PLGA (75/25 blend) in 240 ml di acetato di etile per creare un (m / v) di 1,5%. ATTENZIONE: acetato di etile è dannoso - indossare dispositivi di protezione individuale e lavorare in una cappa aspirante. </li><li> Sciogliere 25.00 g di Pluronic F-127 in 500 ml di degassati H 2 O con un agitatore magnetico per creare un (m / v) 5,0%. NOTA: Pluronic F-127 è un copolimero a blocchi anfifilo non ionico che agisce come un tensioattivo biocompatibile. Aiuta a stabilizzare l&#39;emulsione olio-in-acqua nel passo 3.3.2. </li><li> Utilizzando un microspatula, raccogliere il gel di magnetite in sei 0,040 g aliquote all&#39;interno fiale di vetro ponderati. Perform il seguente processo di rivestimento e lavaggio per ciascuna aliquota. NOTA: Le aliquote sono necessari per garantire una gestione efficiente e decantazione magnetico, che consentirà di massimizzare purezza e resa, riducendo al minimo la degradazione prima di liofilizzazione al punto 4. <ol><li> Aggiungere a 0,040 g di gel un&#39;aliquota magnetite e 40 ml della soluzione di PLGA in un beaker di plastica e sonicare in ultrasuoni per 10 minuti. </li><li> Aggiungere 80 ml della soluzione Pluronic nel becher di plastica ed emulsionare immediatamente con un miscelatore da laboratorio al valore massimo per 7 minuti per formare il rivestimento PLGA sulle nanoparticelle di magnetite come una emulsione olio-in-acqua. </li><li> Diluire immediatamente la soluzione Spion in 1 L di H 2 O deionizzata e ultrasuoni per 1 h in una cappa per evaporare l&#39;acetato di etile. </li><li> Posizionare un forte magnete accanto alla soluzione Spion e mescolare delicatamente per raccogliere SPIONs brunastre al magnete. NOTA: potrebbe essere necessario mescolare ad intermittenza per diverse ores prima che la soluzione diventa biancastra indicando che la maggior parte dei SPIONs sono stati raccolti. </li><li> Decantare la soluzione acquosa, pur mantenendo le SPIONs nel bicchiere con il magnete. </li><li> Lavare i SPIONs tre volte di seguito. <ol><li> Sospendere le SPIONs in 1 L di deionizzata H 2 O. </li><li> Sonicare la soluzione Spion per 20 minuti. </li><li> Posizionare un forte magnete accanto alla soluzione Spion e mescolare delicatamente per raccogliere SPIONs brunastre al magnete. Potrebbe essere necessario agitare intermittenza per diverse ore prima la soluzione diventa chiara indicando che la maggior parte dei SPIONs sono stati raccolti. </li><li> Decantare la soluzione acquosa, pur mantenendo le SPIONs nel bicchiere con il magnete. </li></ol></li></ol></li><li> Raccogliere le SPIONs sintetizzati da ciascuna delle sei aliquote gel magnetite in una singola fiala di vetro ponderata in sospensione acquosa. Opzionalmente decantare l&#39;acqua in eccesso magneticamente, se necessario. </li></ol> 4. Fermo-Essiccazione di SPIONs <ol><li> Congelare la soluzione spion. </li><li> Liofilizzare la soluzione Spion O / N in un liofilizzatore. </li><li> Pesare i SPIONs liofilizzati. SPIONs liofilizzati possono essere conservati a -20 ° C fino al momento per l&#39;etichettatura delle cellule. NOTA: Conservare a -20 ° C riduce drammaticamente cinetica di degradazione e aumenta la durata di conservazione. </li></ol> 5. Etichettatura di Celle con SPIONs <ol><li> Sospendere una aliquota di SPIONs in tampone fosfato salino (PBS) ad una concentrazione di 40 mg / ml e con ultrasuoni per 30 minuti. </li><li> Aggiungere la soluzione in un pallone Spion quasi confluenti di cellule ad una concentrazione di 5 microlitri / ml di mezzo di coltura cellulare. Garantire una distribuzione uniforme agitandola lentamente il pallone. </li><li> Incubare le cellule per 16 ore a 37 ° C. </li><li> Aspirare delicatamente terreno di coltura e lavare le cellule due volte con PBS. </li><li> Raccogliere le cellule magneticamente marcate e utilizzare per gli esperimenti. </li><li> Soluzione spion non utilizzata può essere conservato a 4 ° C e dovrebbe essere noicato nel giro di pochi mesi. Con ultrasuoni per 30 minuti prima di ogni utilizzo. </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.

Come con qualsiasi protocollo di sintesi di nanoparticelle, la purezza dei reagenti chimici è fondamentale per ottenere SPIONs alta qualità che avranno effetti citotossici minimi. È quindi importante per l&#39;acquisto reagenti molto puri compreso l&#39;acido oleico (≥99%), ferro (II) tetraidrato Cloruro (≥99.99%), ferro (III) cloruro (≥99.99%), acetato di etile (grado HPLC, ≥99.9% ), esano (grado HPLC, ≥97.0%), idrossido di ammonio (≥99.99%) e solfato di sodio (≥99.0%). È di particolare importanza per...

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

Somministrazione mirata di cellule e agenti terapeutici a favore di un&#39;ampia gamma di applicazioni biomediche concentrando l&#39;effetto terapeutico al sito bersaglio, riducendo al minimo gli effetti deleteri a siti fuori bersaglio. Il targeting cellule magnetica è una tecnica di consegna efficiente, sicuro, e diretto. Nanoparticelle di ossido di ferro (superparamagnetic spion) sono biodegradabili, biocompatibili, e possono essere endocitosi in cellule di renderli sensibili ai campi magnetici. Il processo di sintesi prevede la creazione di magnetite (Fe 3 O 4) nanoparticelle seguita da emulsificazione ad alta velocità per formare una poli (lattico-co-glicolico) (PLGA) rivestimento. Le SPIONs PLGA-magnetite sono circa 120 nm di diametro compreso nucleo diametro magnetite circa 10 nm. Quando posto in terreno di coltura, SPIONs sono naturalmente endocitosi dalle cellule e memorizzati come piccoli cluster all&#39;interno di endosomi citoplasmatici. Queste particelle impartiscono massa magnetica sufficiente per le celluleper consentire il targeting entro campi magnetici. Numerosi separazione delle cellule e applicazioni destinate sono attivati ​​per rendere vari tipi di cellule sensibili ai campi magnetici. SPIONs hanno una varietà di altre applicazioni biomediche e compreso l&#39;uso come agente di contrasto imaging medico, farmaco o gene somministrazione mirata, saggi diagnostici, e la generazione di ipertermia locale per la terapia tumorale o saldatura del tessuto.

Nanoparticelle di magnetite sono circa 10 nm di diametro a seguito di agitazione una soluzione acquosa di ferro (III) cloruro e ferro (II) cloruro tetraidrato a 50 ° C e 1.000 rpm (Figura 1). Questi risultati dimostrano riuscita sintesi di nanoparticelle di magnetite. È importante verificare la dimensione e la forma delle nanoparticelle di magnetite prelevati da un piccolo campione di batch quando si tenta la sintesi per la prima volta. Microscopia elettronica a trasmissione (TEM) è il metodo preferi...

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.

Etichettatura delle cellule e Targeting con superparamagnetico ossido di ferro nanoparticelle

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 Views.  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.

Produzione e Targeting di monovalente Quantum Dots

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

Ricerca

Bioingegneria

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.

Realizzazione di un funzionalizzato magnetico batterica Nanocellulose con nanoparticelle di Ossido di Ferro

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.

Solid Lipid nanoparticelle (linfonodi sentinella) Applicazioni per intracellulare targeting

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.

Identificazione di ossido di metallo nanoparticelle in campioni istologici da avanzato Mapping Darkfield Microscopy e iperspettrale

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.

Forno a microonde-driven sintesi di ossido di ferro Nanoparticelle per il rilevamento rapido di aterosclerosi

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.

Valutazione iniziale di anticorpo-coniugati modificate con peptidi virali-derivati per accumulo cellulare di aumentare e migliorare il Targeting tumorale

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.

Protocollo per il trasferimento di MicroRNA in adulto cellule staminali emopoietiche derivate da midollo osseo per abilitare ingegneria cellulare combinata con il Targeting magnetico

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.

Lo Scavenging catalitica di pianta reattive dell'ossigeno specie In Vivo di nanoparticelle di ossido di cerio anionici

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.

Screening e identificazione di piccoli peptidi mirati Fibroblast Growth Factor Receptor2 utilizzando una 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 ...

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).

Utilizzo della magnetometria per monitorare l'incorporazione cellulare e la successiva biodegradazione delle nanoparticelle di ossido di ferro sintetizzate chimicamente

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