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

<ol>
	<li>Massoud, T. F., Gambhir, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+imaging+in+living+subjects:+seeing+fundamental+biological+processes+in+a+new+light.">Molecular imaging in living subjects: seeing fundamental biological processes in a new light.</a> Genes Dev. 17, 545-580 (2003).</li><li>Mankoff, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Definition+of+Molecular+Imaging.">A Definition of Molecular Imaging.</a> J Nucl Med. 48 (6), 18N-21N (2007).</li><li>James, M. L., Gambhir, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Molecular+Imaging+Primer:+Modalities,+Imaging+Agents,+and+Applications.">A Molecular Imaging Primer: Modalities, Imaging Agents, and Applications.</a> Phys Rev. 92, 897-965 (2012).</li><li>Cao, W., Chen, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multimodality+Molecular+Imaging+of+Tumor+Angiogenesis.">Multimodality Molecular Imaging of Tumor Angiogenesis.</a> J Nucl Med. 49 (2), 113S-129S (2008).</li><li>Heinrich, J. L., Berseth, P. A., Long, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+Prussian+Blue+analogues:+synthesis+and+structure+of+cubic+Cr4Co4(CN)12+and+Co8(CN)12+clusters.">Molecular Prussian Blue analogues: synthesis and structure of cubic Cr4Co4(CN)12 and Co8(CN)12 clusters.</a> Chem Commun. 11, 1231-1232 (1998).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Molecular Imaging and Contrast Agent Database (MICAD). , (2004).</li><li>Zhu, D., Liu, F., Ma, L., Liu, D., Wang, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanoparticle-Based+Systems+for+T1-Weighted+Magnetic+Resonance+Imaging+Contrast+Agents.">Nanoparticle-Based Systems for T1-Weighted Magnetic Resonance Imaging Contrast Agents.</a> Int J Mol Sci. 14 (5), 10607-1010 (2013).</li><li>Bartolini, M. E., 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=An+investigation+of+the+toxicity+of+gadolinium+based+MRI+contrast+agents+using+neutron+activation+analysis.">An investigation of the toxicity of gadolinium based MRI contrast agents using neutron activation analysis.</a> Magn. Reson. Imaging. 21 (5), 541-544 (2003).</li><li>Adel, 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=Histological+validation+of+iron-oxide+and+gadolinium+based+MRI+contrast+agents+in+experimental+atherosclerosis:+The+do's+and+don't's.">Histological validation of iron-oxide and gadolinium based MRI contrast agents in experimental atherosclerosis: The do's and don't's.</a> Atherosclerosis. 225 (2), 274-280 (2012).</li><li>Dumont, M. F., Yadavilli, S., Sze, R. W., Nazarian, J., Fernandes, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Manganese-containing+Prussian+blue+nanoparticles+for+imaging+of+pediatric+brain+tumors.">Manganese-containing Prussian blue nanoparticles for imaging of pediatric brain tumors.</a> Int J Nanomedicine. 9, 2581-2595 (2014).</li><li>Dumont, M. F., 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=Biofunctionalized+gadolinium-containing+prussian+blue+nanoparticles+as+multimodal+molecular+imaging+agents.">Biofunctionalized gadolinium-containing prussian blue nanoparticles as multimodal molecular imaging agents.</a> Bioconjug Chem. 25 (1), 129-137 (2014).</li><li>Yang, L., 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=Nephrogenic+Systemic+Fibrosis+and+Class+Labeling+of+Gadolinium-based+Contrast+Agents+by+the+Food+and+Drug+Administration.">Nephrogenic Systemic Fibrosis and Class Labeling of Gadolinium-based Contrast Agents by the Food and Drug Administration.</a> Radiology. 265 (1), 248-253 (2012).</li><li>Mendonca-Dias, M. H., Gaggelli, E., Lauterbur, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Paramagnetic+contrast+agents+in+nuclear+magnetic+resonance+medical+imaging.">Paramagnetic contrast agents in nuclear magnetic resonance medical imaging.</a> Sem in Nuc Med. 13 (4), 364-376 (1983).</li><li>Izrailev, S., Stepaniants, S., Balsera, M., Oono, Y., Schulten, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+Dynamics+Study+of+Unbinding+of+the+Avidin-Biotin+Complex.">Molecular Dynamics Study of Unbinding of the Avidin-Biotin Complex.</a> Biophys. 72 (4), 1568-1581 (1997).</li><li>Chen, Z. Y., 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=Advance+of+Molecular+Imaging+Technology+and+Targeted+Imaging+Agent+in+Imaging+and+Therapy.">Advance of Molecular Imaging Technology and Targeted Imaging Agent in Imaging and Therapy.</a> Biomed Res Int. 2014, 1-12 (2014).</li><li>Weissleder, R., Pittet, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+in+the+era+of+molecular+oncology.">Imaging in the era of molecular oncology.</a> Nature. 452 (7187), 580-589 (2008).</li><li>Jaiswal, J. K., Mattoussi, H., Mauro, J. M., Simon, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+multiple+color+imaging+of+live+cells+using+quantum+dot+bioconjugates.">Long-term multiple color imaging of live cells using quantum dot bioconjugates.</a> Nature Biotech. 21 (1), 47-51 (2002).</li><li>Mangrum, W., Christianson, K., Duncan, S., Hoang, P., Song, A., Merkle, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Duke Review of MRI Principles. 304, 304 (2012).</li><li>Shokouhimehr, M., Soehnlen, E. S., Hao, J., Griswold, M., Flask, C., Fan, X., Basilion, J. P., Basu, S., Huang, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dual+purpose+prussian+blue+nanoparticles+for+cellular+imaging+and+drug+delivery:+a+new+generation+of+T1-weighted+MRI+contrast+and+small+molecule+delivery+agents.">Dual purpose prussian blue nanoparticles for cellular imaging and drug delivery: a new generation of T1-weighted MRI contrast and small molecule delivery agents.</a> J. Mater. Chem. 20, 5251-5259 (2010).</li><li>Hoffman, H. A., Chakrabarti, L., Dumont, M. F., Sandler, A. D., Fernandes, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prussian+blue+nanoparticles+for+laser-induced+photothermal+therapy+of+tumors.">Prussian blue nanoparticles for laser-induced photothermal therapy of tumors.</a> RSC Adv. 4 (56), 29729-29734 (2014).</li></ol>

The authors have nothing to disclose.

Cet article a présenté les méthodes pour la synthèse d&#39;une nouvelle classe de multimodales, agents d&#39;imagerie moléculaire à base de nanoparticules bleu de Prusse biofunctionalized. Les modalités d&#39;imagerie moléculaire incorporés dans les nanoparticules sont l&#39;imagerie de fluorescence et l&#39;IRM moléculaire, en raison de leurs caractéristiques complémentaires. Les nanoparticules bleu de Prusse biofunctionalized ont un design noyau-enveloppe. Les principales étapes de la synthèse de ces nan...

L&#39;imagerie moléculaire est la visualisation non invasive et ciblée des processus biologiques au niveau cellulaire, subcellulaire et une échelle moléculaire. L&#39;imagerie moléculaire permet un spécimen de rester dans son microenvironnement native tandis que ses voies et mécanismes endogènes sont évalués en temps réel. Typiquement, l&#39;imagerie moléculaire implique l&#39;administration d&#39;un agent d&#39;imagerie exogène sous la forme d&#39;une petite molécule, macromolécule, ou nanoparticules de visualiser, cible, et de tracer les processus physiologiques pertinents étudié deux. Les différentes modalités d&#39;imagerie qui ont été explorées en imagerie moléculaire comprennent l&#39;IRM, CT, PET, SPECT, ultrasons, photoacoustique, la spectroscopie Raman, la bioluminescence, fluorescence et microscopie intravitale 3. Imagerie multimodale est la combinaison de deux ou plusieurs modalités d&#39;imagerie où la combinaison améliore la capacité de visualiser et de caractériser les différents processus et des événements biologiques 4. Multimodal imagerie exploite les forces des techniques d&#39;imagerie individuels, tout en compensant leurs limitations individuelles 3. Cet article présente le protocole pour la synthèse de nanoparticules bleu de Prusse biofunctionalized (PB PN) - une nouvelle classe d&#39;agents d&#39;imagerie multimodaux, moléculaires. Les IP PB sont utilisés pour l&#39;imagerie de fluorescence et l&#39;IRM moléculaire. PB est un pigment constitué d&#39;une alternance de fer (II) et de fer (III) des atomes dans un réseau cubique à faces centrées (figure 1). Le treillis est composé de PB de ligands cyanure linéaires dans un Fe II - CN - liaison Fe III qui incorpore des cations pour équilibrer les charges au sein de son réseau 5 tridimensionnel. La capacité des PB à intégrer cations dans son réseau est exploité en chargeant séparément gadolinium et ions manganèse dans les IP PB pour le contraste IRM. La justification de la poursuite d&#39;une conception de nanoparticules pour le contraste IRM est à cause deles avantages de cette conception offre par rapport aux agents de contraste d&#39;IRM actuels. La grande majorité des agents de contraste IRM approuvés par la FDA des États-Unis sont chélates de gadolinium paramagnétique qui sont dans la nature et fournissent contraste positif par le mécanisme de relaxation 6,7,8 de spin-réseau. Par rapport à un simple-chelate de gadolinium qui fournit une faible intensité de signal lui-même, l&#39;incorporation de plusieurs ions gadolinium dans le réseau PB des nanoparticules qui assure une meilleure intensité de signal (contraste positif) 3,9. En outre, la présence d&#39;ions de gadolinium multiples dans le réseau de PB augmente la densité de spins global et l&#39;ampleur du paramagnétisme des nanoparticules, ce qui perturbe le champ magnétique local dans son voisinage, générant de ce fait contraste négatif par le mécanisme de relaxation spin-spin. Ainsi, les nanoparticules contenant du gadolinium fonctionnent à la fois comme T 1 (positif) et T deux agents (négative) de contraste 10,11. Dans un sous-ensemble de patients atteints d&#39;insuffisance rénale, l&#39;administration d&#39;agents de contraste à base de gadolinium a été liée au développement de la fibrose néphrogénique systémique 8,12, 13. Cette observation a conduit des enquêtes sur l&#39;utilisation d&#39;ions paramagnétiques alternatives comme agents de contraste pour IRM. Par conséquent, la conception souple des nanoparticules est adapté pour incorporer des ions manganèse dans le réseau de PB. Similaire à gadolinium-chélates, manganèse-chélates sont également paramagnétique et sont généralement utilisés pour fournir une intensité de signal positif en IRM 7,14. Comme contenant du gadolinium PB IP, les contenant du manganèse PB IP fonctionnent comme T 1 (positif) et T 2 agences (négative) de contraste. Pour incorporer des capacités d&#39;imagerie de fluorescence, les «noyaux» nanoparticules sont enrobées d&#39;une enveloppe &quot;biofonctionnel&quot; consistant en avidine la glycoprotéine marqué par fluorescence (Figure 1). L&#39;avidine non seulement permet l&#39;imagerie de fluorescence, mais également sert de plate-forme d&#39;accueil pour des ligands biotinylés qui ciblent les cellules et les tissus spécifiques. La liaison avidine-biotine est un des plus forts liens connus, non-covalentes caractérisés par une très forte affinité de liaison entre l&#39;avidine et de la biotine 15. La fixation des ligands biotinylés à l&#39;avidine revêtu PB IP confère des capacités de ciblage moléculaire au SNM PB. La motivation pour la poursuite de la fluorescence et IRM utilisant PB IP est parce que ces modalités d&#39;imagerie possèdent des caractéristiques complémentaires. imagerie de fluorescence est une des techniques les plus largement utilisés optiques d&#39;imagerie moléculaire, et permet la visualisation simultanée de plusieurs objets à des sensibilités élevées 1,16,17. imagerie de fluorescence est un coffre-fort, la modalité non-invasive, mais est associée à de faibles profondeurs de pénétration et des résolutions spatiales 1,3,16. D&#39;autre part, l&#39;IRM génère une haute temporelled résolution spatiale de manière non invasive et sans la nécessité d&#39;un rayonnement ionisant 1,3,16. Cependant IRM souffre d&#39;une faible sensibilité. Par conséquent imagerie de fluorescence et l&#39;IRM ont été choisis comme les techniques d&#39;imagerie moléculaire en raison de leurs caractéristiques complémentaires de pénétration en profondeur, la sensibilité et la résolution spatiale. Cet article présente le protocole pour la synthèse et biofonctionnalisation des IP PB, PB IP (PIBb), et contenant du gadolinium-PB IP (MnPB) 10,11 contenant du manganèse. Les méthodes suivantes sont décrites: 1) mesure de la taille, la charge et la stabilité temporelle des nanoparticules, 2) évaluation de la cytotoxicité des nanoparticules, 3) Mesure de la relaxivité IRM, et 4) l&#39;utilisation des nanoparticules pour la fluorescence et l&#39;imagerie par résonance magnétique moléculaire d&#39;une population de cellules cibles in vitro. Ces résultats démontrent le potentiel de PN pour l&#39;utilisation comme agents multimodal, d&#39;imagerie moléculaire in vivo.

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

biofunctionalized prussian blue nanoparticles for multimodal molecular imaging applications

Multimodal, l&#39;imagerie moléculaire permet la visualisation des processus biologiques à des résolutions cellulaires, subcellulaires, et au niveau moléculaire en utilisant plusieurs techniques d&#39;imagerie, complémentaires. Ces agents d&#39;imagerie facilitent l&#39;évaluation en temps réel des voies et mécanismes in vivo, qui améliorent à la fois l&#39;efficacité diagnostique et thérapeutique. Cet article présente le protocole pour la synthèse de nanoparticules bleu de Prusse biofunctionalized (PB PN) - une nouvelle classe d&#39;agents pour une utilisation dans des applications multimodales, d&#39;imagerie moléculaire. Les modalités d&#39;imagerie incorporé dans les nanoparticules, l&#39;imagerie par fluorescence et l&#39;imagerie par résonance magnétique (IRM), ont des caractéristiques complémentaires. Les IP PB possèdent une conception noyau-enveloppe où le gadolinium et des ions manganèse incorporés dans les espaces interstitiels du treillis PB génèrent contraste IRM, à la fois en T 1 et T 2 pondérées en séquences. Les IP PB sont revêtues d&#39;avidine fluorescent à l&#39;aide électrostatique comme auto-Assemblée, qui permet l&#39;imagerie de fluorescence. Les nanoparticules revêtues d&#39;avidine sont modifiés avec des ligands biotinylés qui confèrent des capacités de ciblage moléculaire aux nanoparticules. La stabilité et la toxicité des nanoparticules sont mesurées, ainsi que leurs relaxivité d&#39;IRM. Les capacités multimodales, d&#39;imagerie moléculaire de ces IP PB biofunctionalized sont ensuite démontrées par leur utilisation pour l&#39;imagerie de fluorescence et l&#39;IRM moléculaire in vitro.

En utilisant le schéma de synthèse d&#39;un pot, des nanoparticules de PB IP (diamètre moyen de 78,8 nm, indice de polydispersité (PDI) = 0,230; calculé par l&#39;instrument dynamique de diffusion de la lumière), PIBb (diamètre moyen 164,2 nm, PDI = 0,102), ou MnPB ( diamètre moyen de 122,4 nm, PDI = 0,124) qui sont monodisperse (tel que mesuré par DLS) peut être synthétisé constamment (figure 2A). Les potentiels zêta mesurées des nanoparticules synthétisées sont inférieures à -30 mV <...

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

Biofunctionalized Prussian Blue Nanoparticles for Multimodal Molecular Imaging Applications

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

Biofunctionalized bleu de Prusse nanoparticules pour l&#39;imagerie moléculaire multimodale Applications

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

biofunctionalized-prussian-blue-nanoparticles-for-multimodal

Research

JoVE Journal

Bioengineering

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

Vidéo: Biofunctionalized Prussian Blue Nanoparticles for Multimodal Molecular Imaging Applications

Fluorescence Lifetime Imaging of Molecular Rotors in Living Cells

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

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

Imagerie de vie de fluorescence des rotors moléculaires dans les cellules vivantes

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

Recherche

Bio-ingénierie

Viral Nanoparticles for In vivo Tumor Imaging

The use of nanomaterials has the potential to revolutionize materials science and medicine. Currently, a number of different nanoparticles are being investigated for applications in imaging and therapy. Viral nanoparticles (VNPs) derived from plants can be regarded as self-assembled bionanomaterials with defined sizes and shapes. Plant viruses under investigation in the Steinmetz lab include icosahedral particles formed by Cowpea mosaic virus (CPMV) and Brome mosaic virus (BMV), both of which are 30 nm in diameter. We are also developing rod-shaped and filamentous structures derived from the following plant viruses: Tobacco mosaic virus (TMV), which forms rigid rods with dimensions of 300 nm by 18 nm, and Potato virus X (PVX), which form filamentous particles 515 nm in length and 13 nm in width (the reader is referred to refs. 1 and 2 for further information on VNPs).
From a materials scientist's point of view, VNPs are attractive building blocks for several reasons: the particles are monodisperse, can be produced with ease on large scale in planta, are exceptionally stable, and biocompatible. Also, VNPs are "programmable" units, which can be specifically engineered using genetic modification or chemical bioconjugation methods 3. The structure of VNPs is known to atomic resolution, and modifications can be carried out with spatial precision at the atomic level4, a level of control that cannot be achieved using synthetic nanomaterials with current state-of-the-art technologies.
In this paper, we describe the propagation of CPMV, PVX, TMV, and BMV in Vigna ungiuculata and Nicotiana benthamiana plants. Extraction and purification protocols for each VNP are given. Methods for characterization of purified and chemically-labeled VNPs are described. In this study, we focus on chemical labeling of VNPs with fluorophores (e.g. Alexa Fluor 647) and polyethylene glycol (PEG). The dyes facilitate tracking and detection of the VNPs 5-10, and PEG reduces immunogenicity of the proteinaceous nanoparticles while enhancing their pharmacokinetics 8,11. We demonstrate tumor homing of PEGylated VNPs using a mouse xenograft tumor model. A combination of fluorescence imaging of tissues ex vivo using Maestro Imaging System, fluorescence quantification in homogenized tissues, and confocal microscopy is used to study biodistribution. VNPs are cleared via the reticuloendothelial system (RES); tumor homing is achieved passively via the enhanced permeability and retention (EPR) effect12. The VNP nanotechnology is a powerful plug-and-play technology to image and treat sites of disease in vivo. We are further developing VNPs to carry drug cargos and clinically-relevant imaging moieties, as well as tissue-specific ligands to target molecular receptors overexpressed in cancer and cardiovascular disease.

Viral Nanoparticles for In vivo Tumor Imaging

Plant viral nanoparticles (VNPs) are promising platforms for applications in biomedicine. Here, we describe the procedures for plant VNP propagation, purification, characterization, and bioconjugation. Finally, we show the application of VNPs for tumor homing and imaging using a mouse xenograft model and fluorescence imaging.

Les nanoparticules virales pour<em&gt; In vivo</em&gt; Imagerie des tumeurs

The use of nanomaterials has the potential to revolutionize materials science and medicine. Currently, a number of different nanoparticles are being ...

Wideband Optical Detector of Ultrasound for Medical Imaging Applications

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

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

Wideband détecteur optique des ultrasons pour les applications d&#39;imagerie médicale

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

Harmonic Nanoparticles for Regenerative Research

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

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

Harmonique nanoparticules régénératrice recherche

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

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

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

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

De Fluorescence Imaging rapide à moléculaire loi Diffusion sur les membranes cellulaires en direct dans un microscope commercial

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

Cell Labeling and Targeting with Superparamagnetic Iron Oxide Nanoparticles

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

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

Cellule étiquetage et le ciblage des nanoparticules d&#39;oxyde de fer Superparamagnetic

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

Solid Lipid Nanoparticles (SLNs) for Intracellular Targeting Applications

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

Solid Lipid Nanoparticles SLNs for Intracellular Targeting Applications

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

Solide Lipid nanoparticules (SLN) Applications pour le ciblage intracellulaire

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

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

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

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

Synthèse entraînée par micro-ondes de l&#39;oxyde de fer Nanoparticules pour la détection rapide de l&#39;athérosclérose

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

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

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

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

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

Multimodal Imaging and Platform microendoscopie Spectroscopy Fiber-bundle pour non-invasive,<em&gt; In Vivo</em&gt; L&#39;analyse des tissus

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

Manufacture and Drug Delivery Applications of Silk Nanoparticles

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

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