Name: biofunctionalized prussian blue nanoparticles for multimodal molecular imaging applications
Uploaded: 2015-04-28T15:00:00
Description: Watch this Scientific Journal Video about Biofunctionalized Prussian Blue Nanoparticles for Multimodal Molecular Imaging Applications at JoVE.com

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

1. Synthesis of PB NPs, GdPB, and MnPB
Synthesis of the nanoparticles (PB NPs, GdPB, or MnPB) is achieved using a one-pot synthesis scheme by performing the steps detailed below:
<ol>
	<li>Prepare solution 'A' containing 5 ml of 5 mM potassium hexacyanoferrate (II) in deionized (DI) water. Depending on the type of nanoparticle being synthesized &#8212; PB NPs, GdPB, or MnPB, prepare solution 'B' as follows:
	<ol>
		<li>For PB NPs: prepare 10 ml of a solution containing 2.5 m...

<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>

This article has presented the methods for the synthesis of a novel class of multimodal, molecular imaging agents based on biofunctionalized Prussian blue nanoparticles. The molecular imaging modalities incorporated into the nanoparticles are fluorescence imaging and molecular MRI, due to their complementary features. The biofunctionalized Prussian blue nanoparticles have a core-shell design. The key steps in the synthesis of these nanoparticles are the: 1) one-pot synthesis which yields the cores that are comprised of P...

Molecular imaging is the non-invasive and targeted visualization of biological processes at the cellular, subcellular, and molecular levels1. Molecular imaging permits a specimen to remain in its native microenvironment while its endogenous pathways and mechanisms are assessed in real-time. Typically, molecular imaging involves the administration of an exogenous imaging agent in the form of a small molecule, macromolecule, or nanoparticle to visualize, target, and trace relevant physiological processes being studied2. The various imaging modalities that have been explored in molecular imaging include MRI, CT, PET, SPECT, ultrasound, photoacoustics, Raman spectroscopy, bioluminescence, fluorescence, and intravital microscopy3. Multimodal imaging is the combination of two or more imaging modalities where the combination enhances the ability to visualize and characterize various biological processes and events4. Multimodal imaging exploits the strengths of the individual imaging techniques, while compensating for their individual limitations3.
This article presents the protocol for the synthesis of biofunctionalized Prussian blue nanoparticles (PB NPs) - a novel class of multimodal, molecular imaging agents. The PB NPs are utilized for fluorescence imaging and molecular MRI. PB is a pigment consisting of alternating iron (II) and iron (III) atoms in a face-centered cubic network (Figure 1). The PB lattice is comprised of linear cyanide ligands in a FeII- CN - FeIII linkage that incorporates cations to balance charges within its three-dimensional network5. The ability of PB to incorporate cations into its lattice is exploited by separately loading gadolinium and manganese ions into the PB NPs for MRI contrast.
The rationale for pursuing a nanoparticle design for MRI contrast is because of the advantages this design offers relative to current MRI contrast agents. The vast majority of US FDA-approved MRI contrast agents are gadolinium chelates that are paramagnetic in nature and provide positive contrast by the spin-lattice relaxation mechanism6,7,8. As compared to a single gadolinium-chelate that provides low signal intensity on its own, the incorporation of multiple gadolinium ions within the PB lattice of the nanoparticles provides enhanced signal intensity (positive contrast)3,9. Further, the presence of multiple gadolinium ions within the PB lattice increases the overall spin density and the magnitude of paramagnetism of the nanoparticles, which disturbs the local magnetic field in its vicinity, thereby generating negative contrast by the spin-spin relaxation mechanism. Thus the gadolinium-containing nanoparticles function both as T1 (positive) and T2 (negative) contrast agents10,11.
In a subset of patients with impaired renal function, the administration of gadolinium-based contrast agents has been linked to the development of nephrogenic systemic fibrosis8,12, 13. This observation has prompted investigations into the use of alternative paramagnetic ions as contrast agents for MRI. Therefore, the versatile design of the nanoparticles is adapted to incorporate manganese ions within the PB lattice. Similar to gadolinium-chelates, manganese-chelates are also paramagnetic and are typically used to provide positive signal intensity in MRI7,14. As with gadolinium-containing PB NPs, the manganese-containing PB NPs also function as T1 (positive) and T2 (negative) contrast agents.
To incorporate fluorescence imaging capabilities, the nanoparticle &#8220;cores&#8221; are coated with a &#8220;biofunctional&#8221; shell consisting of the fluorescently-labeled glycoprotein avidin (Figure 1). Avidin not only enables fluorescence imaging, but also serves as a docking platform for biotinylated ligands that target specific cells and tissue. The avidin&#8211;biotin bond is one of the strongest known, non-covalent bonds characterized by extremely strong binding affinity between avidin and biotin15. The attachment of biotinylated ligands to the avidin-coated PB NPs confers molecular targeting capabilities to the PB NPs.
The motivation for pursuing fluorescence and MR imaging using PB NPs is because these imaging modalities possess complementary features. Fluorescence imaging is one of the most widely used optical molecular imaging techniques, and allows for the simultaneous visualization of multiple objects at high sensitivities1,16,17. Fluorescence imaging is a safe, non-invasive modality but is associated with low depths of penetration and spatial resolutions1,3,16. On the other hand, MRI generates high temporal and spatial resolution non-invasively and without a need for ionizing radiation1,3,16. However MRI suffers from low sensitivity. Therefore fluorescence imaging and MRI were selected as the molecular imaging techniques due to their complementary features of depth penetration, sensitivity, and spatial resolution.
This article presents the protocol for the synthesis and biofunctionalization of the PB NPs, gadolinium-containing PB NPs (GdPB), and manganese-containing PB NPs (MnPB)10,11. The following methods are described: 1) measurement of size, charge, and temporal stability of the nanoparticles, 2) evaluation of cytotoxicity of the nanoparticles, 3) measurement of MRI relaxivities, and 4) utilization of the nanoparticles for fluorescence and molecular MR imaging of a population of targeted cells in vitro. These results demonstrate the potential of the NPs for use as multimodal, molecular imaging agents 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, molecular imaging allows the visualization of biological processes at cellular, subcellular, and molecular-level resolutions using multiple, complementary imaging techniques. These imaging agents facilitate the real-time assessment of pathways and mechanisms in vivo, which enhance both diagnostic and therapeutic efficacy. This article presents the protocol for the synthesis of biofunctionalized Prussian blue nanoparticles (PB NPs) - a novel class of agents for use in multimodal, molecular imaging applications. The imaging modalities incorporated in the nanoparticles, fluorescence imaging and magnetic resonance imaging (MRI), have complementary features. The PB NPs possess a core-shell design where gadolinium and manganese ions incorporated within the interstitial spaces of the PB lattice generate MRI contrast, both in T1 and T2-weighted sequences. The PB NPs are coated with fluorescent avidin using electrostatic self-assembly, which enables fluorescence imaging. The avidin-coated nanoparticles are modified with biotinylated ligands that confer molecular targeting capabilities to the nanoparticles. The stability and toxicity of the nanoparticles are measured, as well as their MRI relaxivities. The multimodal, molecular imaging capabilities of these biofunctionalized PB NPs are then demonstrated by using them for fluorescence imaging and molecular MRI in vitro.

Using the one-pot synthesis scheme, nanoparticles of PB NPs (mean diameter 78.8 nm, polydispersity index (PDI) = 0.230; calculated by the dynamic light scattering instrument), GdPB (mean diameter 164.2 nm, PDI = 0.102), or MnPB (mean diameter 122.4 nm, PDI = 0.124) that are monodisperse (as measured by DLS) can be consistently synthesized (Figure 2A). The measured zeta potentials of the synthesized nanoparticles are less than -30 mV (Figure 2B), indicating moderate stability of the parti...

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.

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

Research

JoVE Journal

Bioengineering

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 ...

Viral Nanoparticles for In vivo Tumor Imaging

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 ...

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 ...

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 ...

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 ...

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 ...

Solid Lipid Nanoparticles (SLNs) for Intracellular Targeting Applications

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 ...

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 ...