Name: biofunctionalized prussian blue nanoparticles for multimodal molecular imaging applications
Uploaded: 2015-04-28T15:00:00.000+00:00
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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).

<ol>
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The authors have nothing to disclose.

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 Prussian blue nanoparticles (PB NPs), gadolinium-containing Prussian blue nanoparticles (GdPB), or manganese-containing Prussian blue nanoparticles (MnPB), 2) biofunctionalization of the nanoparticles using fluorescent avidin by electrostatic self-assembly, and 3) attachment of the biotinylated ligands (antibodies) to the nanoparticles using robust avidin-biotin interactions. Both fluorescent avidin and the biotinylated ligands constitute the biofunctional shell of the nanoparticles.
The one-pot synthesis yields PB NPs, GdPB, or MnPB that function both as T1 and T2 contrast agents for MRI. The amounts of the paramagnetic ions (gadolinium or manganese) loaded into the nanoparticle core can be altered (increased or decreased) by varying the amounts of the paramagnetic ion-containing salts (gadolinium (III) nitrate and manganese (II) chloride) in the one-pot synthesis (Step 1.2). This results in altered (increased or decreased) MRI signal intensities. However, these modifications to the synthesis scheme may result in unstable, aggregated nanoparticles. To mitigate concerns associated with aggregation, the one-pot synthesis may be modified to incorporate size-controlling capping agents such as citrate during synthesis20.
Biofunctionalization of the nanoparticle cores is achieved by electrostatic self-assembly with fluorescent avidin, which enables fluorescence imaging. Electrostatic self-assembly requires opposite charges on the surface of the nanoparticles and the coating polymer (in this article, fluorescent avidin). To alter the surface functionality of the nanoparticles, avidin may be replaced by positively-charged polymers (e.g. polylysine or an amine-group containing polyethylene glycol) during the synthesis. However the relative proportions of the nanoparticles and coating polymer will have to be optimized to maintain nanoparticle size and stability and to prevent aggregation.
Addition of the biotinylated antibodies onto the avidin coated nanoparticles confers molecular targeting capabilities to the PB-based nanoparticles. This step is based on the robust interaction between avidin and biotin (equilibrium dissociation constant, Kd ~10-15). As with previous steps, the relative proportions of the avidin-coated nanoparticles and biotinylated ligands need to be optimized so as to prevent the biotinylated ligand from simultaneously binding two avidin coated nanoparticles resulting in nanoparticle aggregation. For molecular targeting, the antibodies may be replaced by other targeting ligands such as antibody fragments (Fab), single-chain variable fragment (scFv), peptides, or aptamers.
The key advantages of this method for synthesizing biofunctionalized nanoparticles as multimodal, molecular imaging agents are the: 1) facile one-pot (1-step) synthesis of the nanoparticle cores, and 2) sequential contacting steps (electrostatic self-assembly and avidin-biotin interactions) for coating the nanoparticle core with a biofunctional shell. Other advantages of the nanoparticles include the fact that Prussian blue (sold as Radiogardase) is already FDA-approved for human use and that the nanoparticles that results from the one-pot synthesis scheme can be used as an MRI contrast agent both in T1 (positive) and T2 (negative)-weighted sequences, which is not easily achieved using other contrast agents or nanoparticle platforms without complicated synthesis schemes or specialized chemicals for synthesis of the contrast agents. A limitation of the technique is that the synthesis can lead to polydisperse nanoparticles with aggregates if the relative proportions of the reactants in both nanoparticle core synthesis and biofunctional shell coating steps are not rigorously optimized for the reactants used in those particular steps. For example, the relative proportions for coating the nanoparticles cores with avidin cannot be extended to coating the nanoparticles with polylysine without prior optimization studies.
After mastering the technique for synthesizing biofunctionalized Prussian blue nanoparticles described here, this versatile design can be modified for molecular imaging studies in vivo. This will require PEGylation of the nanoparticles for lower immunogenicity and longer circulation times in vivo. Similar to the design described here, the PB NPs can be biofunctionalized with antibodies prior to in vivo administration. Other studies include the use of the biofunctionalized PB NPs for theranostic (simultaneous therapy + diagnostic) applications in vivo. Studies investigating the use of Prussian blue nanoparticles for photothermal therapy (based on their absorbance characteristics at near infrared wavelengths) are currently underway21.

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><tbody><tr><td>Potassium hexacyanoferrate (II) trihydrate (K&lt;sub&gt;4&lt;/sub&gt;Fe(CN)&lt;sub&gt;6&lt;/sub&gt;&amp;middot;3H&lt;sub&gt;2&lt;/sub&gt;O)</td><td>Sigma-Aldrich</td><td>P9387</td><td></td></tr><tr><td>Manganese (II) chloride tetrahydrate (MnCl&lt;sub&gt;2&lt;/sub&gt;&amp;middot;4H&lt;sub&gt;2&lt;/sub&gt;O)</td><td>Sigma-Aldrich</td><td>221279</td><td></td></tr><tr><td>Gadolinium (III) nitrate hexahydrate (Gd(NO&lt;sub&gt;3&lt;/sub&gt;)&lt;sub&gt;3&lt;/sub&gt;&amp;middot;6H&lt;sub&gt;2&lt;/sub&gt;O)</td><td>Sigma-Aldrich</td><td>211591</td><td></td></tr><tr><td>Iron (III) chloride hexahydrate (FeCl&lt;sub&gt;3&lt;/sub&gt;&amp;middot;6H&lt;sub&gt;2&lt;/sub&gt;O)</td><td>Sigma-Aldrich</td><td>236489</td><td></td></tr><tr><td>Sodium chloride (NaCl)</td><td>Sigma-Aldrich</td><td>S9888</td><td></td></tr><tr><td>Anti-NG2 Chondroitin Sulfate Proteoglycan, Biotin Conjugate Antibody</td><td>Millipore</td><td>AB5320</td><td></td></tr><tr><td>Biotinylated Anti-Human Eotaxin-3</td><td>Peprotech</td><td>500-P156GBT</td><td></td></tr><tr><td>Neuro-2a Cell Line</td><td>ATCC</td><td>CCL-131</td><td></td></tr><tr><td>BSG D10 Cell Line</td><td>Lab stock</td><td>---</td><td></td></tr><tr><td>OE21 Cell Line</td><td>Sigma-Aldrich</td><td>96062201</td><td></td></tr><tr><td>SUDIPG1 Neurospheres</td><td>Lab stock</td><td>---</td><td></td></tr><tr><td>Eol-1 Cell Line</td><td>Sigma-Aldrich</td><td>94042252</td><td></td></tr><tr><td>Poly(L-lysine) hydrobromide</td><td>Sigma-Aldrich</td><td>P1399</td><td></td></tr><tr><td>Formaldehyde</td><td>Sigma-Aldrich</td><td>F8775</td><td></td></tr><tr><td>Bovine serum albumin</td><td>Sigma-Aldrich</td><td>A2153</td><td></td></tr><tr><td>Aminoactinomycin D</td><td>Sigma-Aldrich</td><td>A9400</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich</td><td>X100</td><td></td></tr><tr><td>CellTrace Calcein Red-Orange, AM</td><td>Life Technologies</td><td>C34851</td><td></td></tr><tr><td>Avidin-Alexa Fluor 488</td><td>Life Technologies</td><td>A21370</td><td></td></tr><tr><td>Centrifuge</td><td>Eppendorf</td><td>5424</td><td></td></tr><tr><td>Peristaltic Pump</td><td>Instech</td><td>P270</td><td></td></tr><tr><td>Zetasizer Nano ZS</td><td>Malvern</td><td>ZEN3600</td><td></td></tr><tr><td>Sonicator</td><td>QSonica</td><td>Q125</td><td></td></tr><tr><td>Hot Plate/Magnetic Stirrer</td><td>VWR</td><td>97042-642</td><td></td></tr><tr><td>Ultra Clean Aluminum Foil</td><td>VWR</td><td>89107-732</td><td></td></tr><tr><td>Vortex Mixer</td><td>VWR</td><td>58816-121</td><td></td></tr><tr><td>1.7 ml conical microcentrifuge tubes</td><td>VWR</td><td>87003-295</td><td></td></tr><tr><td>15 ml conical centrifuge tubes</td><td>VWR</td><td>21008-918</td><td></td></tr><tr><td>Tube holders</td><td>VWR</td><td>82024-342</td><td></td></tr><tr><td>Disposable plastic cuvettes</td><td>VWR</td><td>7000-590 (/586)</td><td></td></tr><tr><td>Zetasizer capillary cell</td><td>VWR</td><td>DTS1070</td><td></td></tr><tr><td>Centrifugal Filters, 0.2 micrometer spin column</td><td>VWR</td><td>82031-356</td><td></td></tr><tr><td>96-well cell culture tray</td><td>VWR</td><td>29442-056</td><td></td></tr><tr><td>Trypsin EDTA 0.25% solution 1x</td><td>JR Scientific</td><td>82702</td><td></td></tr><tr><td>Cell Culture Grade PBS (1x)</td><td>Life Technologies</td><td>10010023</td><td></td></tr><tr><td>XTT Cell Proliferation Assay Kit</td><td>Trevigen</td><td>4891-025-K</td><td></td></tr><tr><td>T75 Flask</td><td>89092-700</td><td>VWR</td><td></td></tr><tr><td>Dulbecco&#039;s Modified Eagle&#039;s Medium</td><td>Biowhitaker</td><td>12-604Q</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Life Technologies</td><td>10437-010</td><td></td></tr><tr><td>Pen-Strep 1x</td><td>Life Technologies</td><td>15070063</td><td></td></tr><tr><td>Fluoview FV1200 Confocal Laser Scanning Microscope</td><td>Olympus</td><td>FV1200</td><td></td></tr><tr><td>Chambered Microscope Slides</td><td>Thermo Scientific</td><td>154534</td><td></td></tr><tr><td>Micro Cover Glasses, Square, No. 1.5</td><td>VWR</td><td>48366-227</td><td></td></tr><tr><td>Microscope Slides</td><td>VWR</td><td>16004-368</td><td></td></tr><tr><td>RPMI</td><td>Sigma-Aldrich</td><td>R8758&amp;nbsp;</td><td></td></tr><tr><td>Agarose</td><td>Sigma-Aldrich</td><td>A9539&amp;nbsp;</td><td></td></tr><tr><td>FACSCalibur Flow Cytometer</td><td>BD Biosciences</td><td></td><td></td></tr><tr><td>3 T Clinical MRI Magnet</td><td>GE Healthcare</td><td></td><td></td></tr><tr><td>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 particles based on their surface charges. The synthesized nanoparticles exhibit adequate temporal stability over a period of 5 days as indicated by their consistent sizes (hydrodynamic diameters; Figure 2C).
When co-incubated with cells, the nanoparticles (PB NPs, GdPB, and MnPB) exhibit negligible cytotoxicity to the cells below certain threshold concentrations (Figure 3). Cytotoxicity studies of PB NPs on Neuro2a cells indicate negligible cytotoxicity when co-incubated with Neuro2a at concentrations lower than 0.67 &#215; 10-6 mg/cell (Figure 3A). Cytotoxicity studies conducted by co-incubation of GdPB with EoL-1 and OE21 cells indicate negligible cytotoxicity of GdPB on both cell types at concentrations lower than 0.25 &#215; 10-6 mg/cell (Figure 3B). Similar, cytotoxicity studies indicate negligible cytotoxicity of MnPB when co-incubated with BSG D10 at concentrations lower than 0.25 &#215; 10-6 mg/cell (Figure 3C). The additional cytotoxicity of MnPB and GdPB can be attributed to the presence of additional ions (Mn2+ for MnPB and Gd3+ for GdPB) within the nanoparticle core.
MRI relaxivity studies were conducted using phantoms comprised of varying concentrations of PB NPs, GdPB, and MnPB. The studies indicate the utility of the nanoparticles as MRI contrast agents in both T1W and T2W sequences (Figure 4). This is demonstrated by the increased hyperintensities (positive contrast) in T1-weighted sequences and increased hypointensities (negative contrast) in T2-weighted sequences with increasing concentrations of both GdPB (Figure 4A) and MnPB (Figure 4B). Based on the relaxivity measurements, MnPB is a moderate T1 agent and a strong T2 agent while GdPB is a strong T1 agent and a moderate T2 agent (Figure 4C). The measured relaxivities of GdPB and MnPB compare favorably with those of clinically approved contrast agents10, 11.
The biofunctionalized PB NPs are able to fluorescently label a population of targeted cells in vitro (Figure 5). When biofunctionalized with both fluorescent avidin (A488) and biotinylated anti-human eotaxin-3 antibody (Eot3), GdPB can fluorescently target a population of EoL-1 cells (Figure 5B). Control GdPB nanoparticles without Eot3 exhibit negligible binding (Figure 5A) Similarly, when GdPB is biofunctionalized with both fluorescent avidin (A488) and biotinylated anti-neuronal glial antigen-2 (ANG2), the nanoparticles can fluorescently target populations of BSG D10 and SUDIPG1 neurospheres (Figures 5D and F), while control nanoparticles without the targeting antibody are unable to fluorescently label the cells (Figures 5C and E). Thus, efficient targeting and fluorescent labeling require the presence of both fluorescent avidin and the biotinylated targeting ligand.
The capability of the biofunctionalized nanoparticles to fluorescently label a population of cells, as quantified by flow cytometry, confirms the need for both fluorescent avidin and biotinylated-targeting ligand for effective fluorescent labeling (Figure 6). BSG D10 cells contacted with MnPB containing both A488 and ANG2 (MnPB-A488-ANG2) exhibit increased fluorescence (Figure 6A) and percentage of fluorescently-labeled cells (Figure 6B) when compared to control fluorescent nanoparticles with a control antibody (MnPB-A488-AbC) and without an antibody (MnPB-A488). The biofunctionalized nanoparticles are able to fluorescently target a specific sub-population of cells within a cell mixture (Figure 6C). This is demonstrated as fixed amounts of EoL-1-targeting, fluorescent GdPB (GdPB-A488-Eot3) are contacted with targeted cells (EoL-1) and control cells (OE21). Fluorescence increases as proportions of EoL-1 are increased (increased Alexa Fluor 488 signal intensity; Figure 6C) within the mixture. This indicates the specificity of the nanoparticles for this sub-population of cells within the cell mixture.
The biofunctionalized PB NPs increase MRI contrast in a population of targeted cells (Figure 7). When BSG D10 cells are contacted with equivalent concentrations of experimental (MnPB-A488-ANG2) and control (MnPB-A488-AbC and MnPB-A488) nanoparticles, phantoms exhibit increased hyperintensity for cells contacted with MnPB-A488-ANG2 compared to controls in T1W sequences, and increased hypointensity for cells contacted with MnPB-A488-ANG2 compared to controls in T2W sequences (Figure 7A). Image analysis of the ROIs within the phantom confirm this trend where cells contacted with experimental particles exhibit significantly increased intensity when compared to controls in T1W sequences and significantly decreased intensity relative to controls in T2W sequences (Figure 7B).
<img alt="Figure 1" src="/files/ftp_upload/52621/52621fig1.jpg" /> 
Figure 1: The core-shell design of the PB NPs. The core consists of a PB lattice, which is comprised of linear cyanide ligands in a FeII&#8211; CN &#8211; FeIII linkage. These linkages enable PB NPs to incorporate cations within its three-dimensional network as a means of balancing charges5. This cation-binding ability of Prussian blue is utilized to load gadolinium and manganese ions within the lattice, which provides MRI contrast. The core is coated with a biofunctional shell comprised of fluorescent avidin to enable fluorescence imaging and biotinylated ligands to enable molecular targeting.
<img alt="Figure 2" src="/files/ftp_upload/52621/52621fig2.jpg" /> 
Figure 2: Size, charge, and stability of the PB NPs. (A) Size distributions of PB (blue), GdPB (red) and MnPB (black) nanoparticles measured by DLS. (B) Zeta potentials of PB NPs, GdPB, and MnPB. (C) Temporal stability of PB (blue), GdPB (red) and MnPB (black) nanoparticles in water (solid) and DMEM (dotted) for five days after their synthesis, measured by DLS.
<img alt="Figure 3" src="/files/ftp_upload/52621/52621fig3.jpg" /> 
Figure 3: Cytotoxicity of the PB NPs. Cytotoxicity studies using various concentrations of (A) PB NPs added to a fixed number of Neuro2a cells (B) GdPB added to a fixed amount of EoL-1 and OE-21 cells, respectively and (C) MnPB added to a fixed number of BSG D10 cells. Cell survival rate was calculated at 24 and 48 hr. Reproduced with permission from Ref 11, copyright 2014 American Chemical Society; Ref 10 with permission from Dove Press Ltd; and Ref 21 with permission from The Royal Society of Chemistry.
<img alt="Figure 4" src="/files/ftp_upload/52621/52621fig4.jpg" /> 
Figure 4: MR images and relaxivities of GdPB and MnPB at 3 T. Hyperintensity in T1-weighted sequences and hypointensity in T2-weighted sequences of (A) GdPB and (B) MnPB as a function of concentration of the nanoparticles. (C) Tabulation of the relaxitivies of PB NPs, GdPB, and MnPB measured at 3 T. Reproduced with permission from Ref 11, copyright 2014 American Chemical Society.
<img alt="Figure 5" src="/files/ftp_upload/52621/52621fig5.jpg" /> 
Figure 5: Fluorescent labeling of targeted cells using the biofunctionalized PB NPs, as measured by laser scanning confocal microscopy. Images of EoL-1 cells treated with (A) control (GdPB-A488) and (B) experimental (GdPB-A488-Eot3) nanoparticles. Images of BSG neurospheres treated with (C) control (MnPB-A488) and (D) experimental (MnPB-A488-ANG2) nanoparticles. Images of SUDIPG1 neurospheres treated with (E) control (MnPB-A488) and (F) experimental (MnPB-A488-ANG2) nanoparticles. Reproduced with permission from Ref 11, copyright 2014 American Chemical Society. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52621/52621fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" src="/files/ftp_upload/52621/52621fig6.jpg" /> 
Figure 6: Fluorescent labeling of targeted cells using the biofunctionalized PB NPs, as measured by flow cytometry. (A) Flow cytometry histograms of BSG D10 cells treated with experimental (MnPB-A488-ANG2) and control (MnPB-A488-AbC and MnPB-A488) nanoparticles. (B) Percentage of BSG D10 cells from panel (A) that are fluorescent (% of Alexa-Fluor positive) upon treatment with experimental (MnPB-A488-ANG2) and control (MnPB-A488-AbC and MnPB-A488) nanoparticles, ** p &#60;0.05. (C) Flow cytometry scatter plots of a cell mixture containing varying proportions of EoL-1 (targeted cells) and OE21 (control cells) targeted by the nanoparticles (GdPB-A488-Eot3). Reproduced with permission from Ref 11, copyright 2014 American Chemical Society and from Ref 10 with permission from Dove Press Ltd. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52621/52621fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" src="/files/ftp_upload/52621/52621fig7.jpg" /> 
Figure 7: Increasing MRI contrast in targeted cells using the biofunctionalized PB NPs. (A) T1-weighted and T2-weighted contrast enhancement in phantoms comprised of a fixed number of BSG D10 cells treated with experimental (MnPB-A488-ANG2) and control (MnPB-A488-AbC and MnPB-A488) nanoparticles. (B) Normalized signal intensity for BSG D10 cells treated with experimental (MnPB-A488-ANG2) and control (MnPB-A488-AbC and MnPB-A488) nanoparticles, ** p &#60;0.05. Reproduced from Ref 10 with permission from Dove Press Ltd.

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, ...

biofunctionalized-prussian-blue-nanoparticles-for-multimodal

Universidad San Sebastian

Research

JoVE Journal

Bioengineering

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

Video: Biofunctionalized Prussian Blue Nanoparticles for Multimodal Molecular Imaging Applications

Registered Bioimaging of Nanomaterials for Diagnostic and Therapeutic Monitoring 

Nanomedications can be carried by blood borne monocyte-macrophages into the reticuloendothelial system (RES; spleen, liver, lymph nodes) and to end organs. The latter include the lung, RES, and brain and are operative during human immunodeficiency virus type one (HIV-1) infection. Macrophage entry into tissues is notable in areas of active HIV-1 replication and sites of inflammation. In order to assess the potential of macrophages as nanocarriers, superparamagnetic iron-oxide and/or drug laden particles coated with surfactants were parenterally injected into HIV-1 encephalitic mice. This was done to quantitatively assess particle and drug biodistribution. Magnetic resonance imaging (MRI) test results were validated by histological coregistration and enhanced image processing. End organ disease as typified by altered brain histology were assessed by MRI. The demonstration of robust migration of nanoformulations into areas of focal encephalitis provides '"proof of concept" for the use of advanced bioimaging techniques to monitor macrophage migration. Importantly, histopathological aberrations in brain correlate with bioimaging parameters making the general utility of MRI in studies of cell distribution in disease feasible. We posit that using such methods can provide a real time index of disease burden and therapeutic efficacy with translational potential to humans.

Registered Bioimaging of Nanomaterials for Diagnostic and Therapeutic Monitoring

Bioimaging methods used to assess cell biodistribution of nanoparticles are applicable for therapeutic and diagnostic monitoring of nanoformulated compounds. The methods described herein are sensitive and specific when assessed by histological coregistration. The methodologies provide a translational pathway from rodent to human applications.

Nanomedications can be carried by blood borne monocyte-macrophages into the reticuloendothelial system (RES; spleen, liver, lymph nodes) and to end ...

Synthesis of Immunotargeted Magneto-plasmonic Nanoclusters

Magnetic and plasmonic properties combined in a single nanoparticle provide a synergy that is advantageous in a number of biomedical applications including contrast enhancement in novel magnetomotive imaging modalities, simultaneous capture&#160;and detection of circulating tumor cells (CTCs),&#160;and multimodal molecular imaging combined with photothermal therapy of cancer cells. These applications have stimulated significant interest in development of protocols for synthesis of magneto-plasmonic nanoparticles with optical absorbance in the near-infrared (NIR) region and a strong magnetic moment. Here, we present a novel protocol for synthesis of such hybrid nanoparticles that is based on an oil-in-water microemulsion method. The unique feature of the protocol described herein is synthesis of magneto-plasmonic nanoparticles of various sizes from primary blocks which also have magneto-plasmonic characteristics. This approach yields nanoparticles with a high density of magnetic and plasmonic functionalities which are uniformly distributed throughout the nanoparticle volume. The hybrid nanoparticles can be easily functionalized by attaching antibodies through the Fc moiety leaving the Fab portion that is responsible for antigen binding available for targeting.

Here, we describe a protocol for synthesis of magneto-plasmonic nanoparticles with a strong magnetic moment and a strong near-infrared (NIR) absorbance. The protocol also includes antibody conjugation to the nanoparticles through the Fc moiety for various biomedical applications which require molecular specific targeting.

Magnetic and plasmonic properties combined in a single nanoparticle provide a synergy that is advantageous in a number of biomedical applications ...

Chemistry

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.

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

Iron Oxide Nanoparticles for Image-Guided Therapy Delivery in Murine Glioblastoma

Nanoparticle Delivery of an Oligonucleotide Payload in a Glioblastoma Multiforme Animal Model

Glioblastoma multiforme (GBM) is the most common and aggressive form of primary brain malignancy for which there is no cure. The blood-brain barrier is a significant hurdle in the delivery of therapies to GBM. Reported here is an image-guided, iron oxide-based therapeutic delivery nano platform capable of bypassing this physiological barrier by virtue of size and accumulating in the tumor region, delivering its payload. This 25 nm nano platform consists of crosslinked dextran-coated iron oxide nanoparticles labeled with Cy5.5 fluorescent dye and containing antisense oligonucleotide as a payload. The magnetic iron oxide core enables tracking of the nanoparticles through in vivo magnetic resonance imaging, while Cy5.5 dye allows tracking by optical imaging. This report details the monitoring of the accumulation of this nanoparticle platform (termed MN-anti-miR10b) in orthotopically implanted glioblastoma tumors following intravenous injection. In addition, it provides insight into the in vivo delivery of RNA oligonucleotides, a problem that has hampered the translation of RNA therapeutics into the clinic.

Author Spotlight: Innovative Cancer Therapies with Iron Oxide Nanoparticles for Glioblastoma Treatment

The blood-brain barrier is a significant hurdle in the delivery of therapies for glioblastoma, a disease for which there is no cure. Here, we report an in vivo image-guided iron oxide therapeutic nano platform that can bypass this physiological barrier by virtue of size and accumulate in the tumor.

Glioblastoma multiforme (GBM) is the most common and aggressive form of primary brain malignancy for which there is no cure. The blood-brain barrier is a ...

Cancer Research

Hydrothermal Synthesis of Persistent Luminescent Nanoparticles

Synthesis of Persistent Luminescent Nanoparticles for Rewritable Displays and Illumination Applications

Persistent luminescent nanoparticles (PLNPs) possess the capabilities to maintain extended longevity and robust emission even after the excitation has ceased. PLNPs have been widely used across various domains, including information displays, data encryption, biological imaging, and artistic decoration with sustained and vivid luminosity, providing boundless possibilities for a variety of innovative technology and artistic projects. This protocol focuses on an experimental procedure for the hydrothermal synthesis of PLNPs. The successful synthesis of enduring luminescent nanomaterials with Mn2+ or Cr3+ serving as a luminescent center in Zn2GeO4: Mn (ZGO: Mn) or ZnGa2O4: Cr highlights the universality of this synthetic method. On the other hand, the optical properties of ZGO: Mn can be changed by adjusting the pH of precursor solutions, demonstrating the tunability of the protocol. When charged with ultraviolet (UV) at a wavelength of 365 nm for 3 min and then stopped, PLNPs exhibit the remarkable capacity to generate afterglow efficiently and consistently, which makes them ideal for making two-dimensional rewritable displays and three-dimensional transparent, luminous artworks. This protocol outlined in this paper provides a feasible method for the synthesis of persistent luminescent nanoparticles for further illumination and imaging applications, opening up novel prospects for the fields of science and art.

Author Spotlight: Advancing Bioimaging and Therapy with Functional Nanomaterials

A protocol is presented for the synthesis of persistent luminescent nanomaterials (PLNPs) and their potential applications in rewritable displays and artistic processing utilizing the afterglow effect under ultraviolet light (365 nm) irradiation.

Persistent luminescent nanoparticles (PLNPs) possess the capabilities to maintain extended longevity and robust emission even after the excitation has ...

Quantum Dot Nanobeads for Enhanced Sensitivity in Biomarker Detection

Fluorescent Lateral Flow Immunoassay Based on Quantum Dots Nanobeads

Quantum dots, also known as semiconductor nanocrystals, are novel fluorescent labels for biological imaging and sensing. However, quantum dot-antibody conjugates with small dimensions (~10 nm), prepared through laborious purification procedures, exhibit limited sensitivity in detecting certain trace disease markers using lateral flow immunoassay strips. Herein, we present a method for the preparation of quantum dot nanobeads (QDNB) using a one-step emulsion evaporation method. Using the as-prepared QDNB, a fluorescent lateral flow immunoassay was fabricated to detect disease biomarkers using C-reactive protein (CRP) as an example. Unlike single quantum dot nanoparticles, quantum dot nanobead-antibody conjugates are more sensitive as immunoassay labels due to signal amplification by encapsulating hundreds of quantum dots in one polymer composite nanobead. Moreover, the larger size of QDNBs facilitates easier centrifugation separation when conjugating QDNBs with antibodies. The fluorescent lateral flow immunoassay based on QDNBs was fabricated, and the CRP concentration in the sample was measured in 15 min. The test results can be qualitatively assessed under UV light illumination and quantitatively measured using a fluorescent reader within 15 min.

Author Spotlight: High-Quality Quantum Dot Nanobeads for Sensitive Fluorescent Lateral Flow Immunoassays

Here, we describe a protocol for the preparation of quantum dot nanobeads (QDNB) and the detection of disease biomarkers using QDNB-based lateral flow immunoassay strips. The test results can be qualitatively assessed under UV light illumination and quantitatively measured using a fluorescent strip reader within 15 min.

Quantum dots, also known as semiconductor nanocrystals, are novel fluorescent labels for biological imaging and sensing. However, quantum dot-antibody ...

Biochemistry

Combined Near-infrared Fluorescent Imaging and Micro-computed Tomography for Directly Visualizing Cerebral Thromboemboli

Direct thrombus imaging visualizes the root cause of thromboembolic infarction. Being able to image thrombus directly allows far better investigation of stroke than relying on indirect measurements, and will be a potent and robust vascular research tool. We use an optical imaging approach that labels thrombi with a molecular imaging thrombus marker&#160;&#8212; a Cy5.5 near-infrared fluorescent (NIRF) probe that is covalently linked to the fibrin strands of the thrombus by the fibrin-crosslinking enzymatic action of activated coagulation factor XIIIa during the process of clot maturation. A micro-computed tomography (microCT)-based approach uses thrombus-seeking gold nanoparticles (AuNPs) functionalized to target the major component of the clot: fibrin. This paper describes a detailed protocol for the combined in vivo microCT and ex vivo NIRF imaging of thromboemboli in a mouse model of embolic stroke. We show that in vivo microCT and fibrin-targeted glycol-chitosan AuNPs (fib-GC-AuNPs) can be used for visualizing both in situ thrombi and cerebral embolic thrombi. We also describe the use of in vivo microCT-based direct thrombus imaging to serially monitor the therapeutic effects of tissue plasminogen activator-mediated thrombolysis. After the last imaging session, we demonstrate by ex vivo NIRF imaging the extent and the distribution of residual thromboemboli in the brain. Finally, we describe quantitative image analyses of microCT and NIRF imaging data. The combined technique of direct thrombus imaging allows two independent methods of thrombus visualization to be compared: the area of thrombus-related fluorescent signal on ex vivo NIRF imaging vs. the volume of hyperdense microCT thrombi in vivo.

This protocol describes the application of combined near-infrared fluorescent (NIRF) imaging and micro-computed tomography (microCT) for visualizing cerebral thromboemboli. This technique allows the quantification of thrombus burden and evolution. The NIRF imaging technique visualizes fluorescently labeled thrombus in excised brain, while the microCT technique visualizes thrombus inside living animals using gold-nanoparticles.

Direct thrombus imaging visualizes the root cause of thromboembolic infarction. Being able to image thrombus directly allows far better investigation of ...

Medicine

Biofunctionalization of Magnetic Nanomaterials

Magnetic nanomaterials have received great attention in different biomedical applications. Biofunctionalizing these nanomaterials with specific targeting agents is a crucial aspect to enhance their efficacy in diagnostics and treatments while minimizing the side effects. The benefit of magnetic nanomaterials compared to non-magnetic ones is their ability to respond to magnetic fields in a contact-free manner and over large distances. This allows to guide or accumulate them, while they can also be monitored. Recently, magnetic nanowires (NWs) with unique features were developed for biomedical applications. The large magnetic moment of these NWs enables a more efficient remote control of their movement by a magnetic field. This has been utilized with great success in cancer treatment, drug delivery, cell tracing, stem cell differentiation or magnetic resonance imaging. In addition, the NW fabrication by template-assisted electrochemical deposition provides a versatile method with tight control over the NW properties. Especially iron NWs and iron-iron oxide (core-shell) NWs are suitable for biomedical applications, due to their high magnetization and low toxicity.
In this work, we provide a method to biofunctionalize iron/iron oxide NWs with specific antibodies directed against a specific cell surface marker that is overexpressed in a large number of cancer cells. Since the method utilizes the properties of the iron oxide surface, it is also applicable to superparamagnetic iron oxide nanoparticles. The NWs are first coated with 3-aminopropyl-tri-ethoxy-silane (APTES) acting as a linker, which the antibodies are covalently attached to. The APTES coating and the antibody biofunctionalization are proven by electron energy loss spectroscopy (EELS) and zeta potential measurements. In addition, the antigenicity of the antibodies on the NWs is tested by using immunoprecipitation and western blot. The specific targeting of the biofunctionalized NWs and their biocompatibility are studied by confocal microscopy and a cell viability assay.

In this work, we provide a protocol to biofunctionalize magnetic nanomaterials with antibodies for specific cell targeting. As examples, we utilize iron nanowires to target cancer cells.

Magnetic nanomaterials have received great attention in different biomedical applications. Biofunctionalizing these nanomaterials with specific targeting ...

Biomolecular Imaging of Cellular Uptake of Nanoparticles using Multimodal Nonlinear Optical Microscopy

Probing gold nanoparticles (AuNPs) in living systems is essential to reveal the interaction between AuNPs and biological tissues. Moreover, by integrating nonlinear optical signals such as stimulated Raman scattering (SRS), two-photon excited fluorescence (TPEF), and transient absorption (TA) into an imaging platform, it can be used to reveal biomolecular contrast of cellular structures and AuNPs in a multimodal manner. This article presents a multimodal nonlinear optical microscopy and applies it to perform chemically specific imaging of AuNPs in cancer cells. This imaging platform provides a novel approach for developing more efficient functionalized AuNPs and determining whether they are within vasculatures surrounding the tumor, pericellular, or cellular spaces.

This article presents the integration of a spectral-focusing module and a dual-output pulse laser, enabling rapid hyperspectral imaging of gold nanoparticles and cancer cells. This work aims to demonstrate the details of multimodal nonlinear optical techniques on a standard laser scanning microscope.

Probing gold nanoparticles (AuNPs) in living systems is essential to reveal the interaction between AuNPs and biological tissues. Moreover, by integrating ...

A Bright NIR-II Fluorescence Probe for Vascular and Tumor Imaging

As an emerging imaging technology, near-infrared II (NIR-II, 1000-1700 nm) fluorescence imaging has significant potential in the biomedical field, owing to its high sensitivity, deep tissue penetration, and superior imaging with spatial and temporal resolution. However, the method to facilitate the implementation of NIR-II fluorescence imaging for some urgently needed fields, such as medical science and pharmacy, has puzzled relevant researchers. This protocol describes in detail the construction and bioimaging applications of a NIR-II fluorescence molecular probe, HLY1, with a D-A-D (donor-acceptor-donor) skeleton. HLY1 showed good optical properties and biocompatibility. Furthermore, NIR-II vascular and tumor imaging in mice was performed using a NIR-II optics imaging device. Real-time high-resolution NIR-II fluorescence images were acquired to guide the detection of tumors and vascular diseases. From probe preparation to data acquisition, the imaging quality is greatly improved, and the authenticity of the NIR-II molecular probes for data recording in intravital imaging is ensured.

The present protocol describes a detailed, real-time NIR-II fluorescence imaging operation of a mouse using a NIR-II optics imaging device.

As an emerging imaging technology, near-infrared II (NIR-II, 1000-1700 nm) fluorescence imaging has significant potential in the biomedical field, owing ...

Biofunctionalized Prussian Blue Nanoparticles for Multimodal Molecular Imaging Applications

Summary

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Chapters in this video

Registered Bioimaging of Nanomaterials for Diagnostic and Therapeutic Monitoring

Synthesis of Immunotargeted Magneto-plasmonic Nanoclusters

Viral Nanoparticles for In vivo Tumor Imaging

Nanoparticle Delivery of an Oligonucleotide Payload in a Glioblastoma Multiforme Animal Model

Synthesis of Persistent Luminescent Nanoparticles for Rewritable Displays and Illumination Applications

Fluorescent Lateral Flow Immunoassay Based on Quantum Dots Nanobeads

Combined Near-infrared Fluorescent Imaging and Micro-computed Tomography for Directly Visualizing Cerebral Thromboemboli

Biofunctionalization of Magnetic Nanomaterials

Biomolecular Imaging of Cellular Uptake of Nanoparticles using Multimodal Nonlinear Optical Microscopy

A Bright NIR-II Fluorescence Probe for Vascular and Tumor Imaging