Name: synthesis of 68ga core-doped iron oxide nanoparticles for dual positron emission tomography /(t1)magnetic resonance imaging
Uploaded: 2018-11-20T13:00:00
Description: Watch this Scientific Journal Video about Synthesis of 68Ga Core-doped Iron Oxide Nanoparticles for Dual Positron Emission Tomography /T1Magnetic Resonance Imaging at JoVE.com

This study was supported by a grant from the Spanish Ministry for Economy and Competitiveness (MEyC) (grant number: SAF2016-79593-P) and from the Carlos III Health Research Institute (grant number: DTS16/00059). The CNIC is supported by the Ministerio de Ciencia, Innovaci&#243;n y Universidades) and the Pro CNIC Foundation and is a Severo Ochoa Centre of Excellence (MEIC award SEV-2015-0505).

1. Reagent Preparation
<ol>
	<li>0.05 M HCl
	<ol>
		<li>Prepare 0.05 M HCl by adding 208 &#181;L of 37% HCl to 50 mL of distilled water.</li>
	</ol>
	</li>
	<li>High-performance liquid chromatography eluent
	<ol>
		<li>Prepare high-performance liquid chromatography (HPLC) eluent by dissolving 6.9 g of sodium dihydrogen phosphate monohydrate, 7.1 g of disodium hydrogen phosphate, 8.7 g of sodium chloride, and 0.7 g of sodium azide in 1 L of water. Mix well and check the pH. Pass the elu...

<ol>
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(24), 3511-3524 (2009).</li><li>Lee, S., 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=Dual-modality+probes+for+in+vivo+molecular+imaging.">Dual-modality probes for in vivo molecular imaging.</a> Molecular Imaging. 8 (2), 87-100 (2009).</li><li>Louie, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multimodality+Imaging+Probes:+Design+and+Challenges.">Multimodality Imaging Probes: Design and Challenges.</a> Chemical Reviews. 110 (5), 3146-3195 (2010).</li><li>Judenhofer, M. 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K., Allard, M., Fernandez, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gallium-68:+chemistry+and+radiolabeled+peptides+exploring+different+oncogenic+pathways.">Gallium-68: chemistry and radiolabeled peptides exploring different oncogenic pathways.</a> Cancer Biotherapy &amp; Radiopharmaceuticals. 28 (2), 85-97 (2013).</li><li>Moon, S. -. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+complementary+PET/MR+dual-modal+imaging+probe+for+targeting+prostate-specific+membrane+antigen+(PSMA).">Development of a complementary PET/MR dual-modal imaging probe for targeting prostate-specific membrane antigen (PSMA).</a> Nanomedicine: Nanotechnology, Biology and Medicine. 12 (4), 871-879 (2016).</li><li>Kim, S. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hybrid+PET/MR+imaging+of+tumors+using+an+oleanolic+acid-conjugated+nanoparticle.">Hybrid PET/MR imaging of tumors using an oleanolic acid-conjugated nanoparticle.</a> Biomaterials. 34 (33), 8114-8121 (2013).</li><li>Yang, B. 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=Development+of+a+multimodal+imaging+probe+by+encapsulating+iron+oxide+nanoparticles+with+functionalized+amphiphiles+for+lymph+node+imaging.">Development of a multimodal imaging probe by encapsulating iron oxide nanoparticles with functionalized amphiphiles for lymph node imaging.</a> Nanomedicine. 10 (12), 1899-1910 (2015).</li><li>Comes Franchini, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biocompatible+nanocomposite+for+PET/MRI+hybrid+imaging.">Biocompatible nanocomposite for PET/MRI hybrid imaging.</a> International Journal of Nanomedicine. 7, 6021 (2012).</li><li>Karageorgou, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gallium-68+Labeled+Iron+Oxide+Nanoparticles+Coated+with+2,3-Dicarboxypropane-1,1-diphosphonic+Acid+as+a+Potential+PET/MR+Imaging+Agent:+A+Proof-of-Concept+Study.">Gallium-68 Labeled Iron Oxide Nanoparticles Coated with 2,3-Dicarboxypropane-1,1-diphosphonic Acid as a Potential PET/MR Imaging Agent: A Proof-of-Concept Study.</a> Contrast Media &amp; Molecular Imaging. 2017, 1-13 (2017).</li><li>Madru, R., 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=(68)Ga-labeled+superparamagnetic+iron+oxide+nanoparticles+(SPIONs)+for+multi-modality+PET/MR/Cherenkov+luminescence+imaging+of+sentinel+lymph+nodes.">(68)Ga-labeled superparamagnetic iron oxide nanoparticles (SPIONs) for multi-modality PET/MR/Cherenkov luminescence imaging of sentinel lymph nodes.</a> American Journal of Nuclear Medicine and Molecular Imaging. 4 (1), 60-69 (2013).</li><li>Lahooti, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PEGylated+superparamagnetic+iron+oxide+nanoparticles+labeled+with+68Ga+as+a+PET/MRI+contrast+agent:+a+biodistribution+study.">PEGylated superparamagnetic iron oxide nanoparticles labeled with 68Ga as a PET/MRI contrast agent: a biodistribution study.</a> Journal of Radioanalytical and Nuclear Chemistry. 311 (1), 769-774 (2017).</li><li>Lee, H. -. 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=PET/MRI+dual-modality+tumor+imaging+using+arginine-glycine-aspartic+(RGD)-conjugated+radiolabeled+iron+oxide+nanoparticles.">PET/MRI dual-modality tumor imaging using arginine-glycine-aspartic (RGD)-conjugated radiolabeled iron oxide nanoparticles.</a> Journal of Nuclear Medicine. 49 (8), 1371-1379 (2008).</li><li>Patel, D., 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=The+cell+labeling+efficacy,+cytotoxicity+and+relaxivity+of+copper-activated+MRI/PET+imaging+contrast+agents.">The cell labeling efficacy, cytotoxicity and relaxivity of copper-activated MRI/PET imaging contrast agents.</a> Biomaterials. 32 (4), 1167-1176 (2011).</li><li>Choi, J., 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=A+Hybrid+Nanoparticle+Probe+for+Dual-Modality+Positron+Emission+Tomography+and+Magnetic+Resonance+Imaging.">A Hybrid Nanoparticle Probe for Dual-Modality Positron Emission Tomography and Magnetic Resonance Imaging.</a> Angewandte Chemie International Edition. 47 (33), 6259-6262 (2008).</li><li>Wong, R. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+size-controlled+synthesis+of+dextran-coated,+64Cu-doped+iron+oxide+nanoparticles.">Rapid size-controlled synthesis of dextran-coated, 64Cu-doped iron oxide nanoparticles.</a> ACS Nano. 6 (4), 3461-3467 (2012).</li><li>Osborne, E. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+microwave-assisted+synthesis+of+dextran-coated+iron+oxide+nanoparticles+for+magnetic+resonance+imaging.">Rapid microwave-assisted synthesis of dextran-coated iron oxide nanoparticles for magnetic resonance imaging.</a> Nanotechnology. 23 (21), 215602 (2012).</li><li>Pellico, J., 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=Fast+synthesis+and+bioconjugation+of+68+Ga+core-doped+extremely+small+iron+oxide+nanoparticles+for+PET/MR+imaging.">Fast synthesis and bioconjugation of 68 Ga core-doped extremely small iron oxide nanoparticles for PET/MR imaging.</a> Contrast Media &amp; Molecular Imaging. 11 (3), 203-210 (2016).</li><li>Pellico, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+imaging+of+lung+inflammation+with+neutrophil-specific+68Ga+nano-radiotracer.">In vivo imaging of lung inflammation with neutrophil-specific 68Ga nano-radiotracer.</a> Scientific Reports. 7 (1), 13242 (2017).</li></ol>

Iron oxide nanoparticles are a well-established contrast agent for T2-weighted MRI. However, due to the drawbacks of this type of contrast for the diagnosis of certain pathologies, T1-weighted or bright contrast is many times preferred. The nanoparticles presented here not only overcome these limitations by offering positive contrast in MRI but also offer a signal in a functional imaging technique, such as PET, via 68Ga incorporation in their core. Microwave technology enhances t...

The combination of imaging techniques for medical purposes has triggered the quest for different methods to synthesize multimodal probes1,2,3. Due to the sensitivity of positron emission tomography (PET) scanners and the spatial resolution of MRI, PET/MRI combinations seem to be one of the most attractive possibilities, providing anatomical and functional information at the same time4. In MRI, T2-weighted sequences can be used, darkening the tissues in which they accumulate. T1-weighted sequences may also be used, producing the brightening of the specific accumulation location5. Among them, positive contrast is often the most adequate option, as negative contrast makes it much harder to differentiate signal from endogenous hypointense areas, including those often presented by organs such as the lungs6. Traditionally, Gd-based molecular probes have been employed to obtain positive contrast. However, Gd-based contrast agents present a major drawback, namely their toxicity, which is critical in patients with renal problems7,8,9. This has motivated research in the synthesis of biocompatible materials for their use as T1 contrast agents. An interesting approach is the use of iron oxide nanoparticles (IONPs), with an extremely small core size, that provide positive contrast10. Due to this extremely small core (~2 nm), most of the Fe3+ ions are on the surface, with 5 unpaired electrons each. This increases longitudinal relaxation time (r1) values and yields much lower transversal/longitudinal (r2/r1) ratios compared to traditional IONPs, producing the desired positive contrast11.
To combine IONPs with a positron emitter for PET, there are two key issues to take into account: radioisotope election and nanoparticle radiolabeling. Regarding the first issue, 68Ga is an alluring choice. It has a relatively short half-life (67.8 min). Its half-life is suitable for peptide labeling since it matches common peptide biodistribution times. Moreover, 68Ga is produced in a generator, enabling the synthesis in bench modules and avoiding the need for a cyclotron nearby12,13,14. In order to radiolabel the nanoparticle, surface-labeling radioisotope incorporation is the prevalent strategy. This can be done using a ligand that chelates 68Ga or taking advantage of the affinity of the radiometal toward the surface of the nanoparticle. Most examples in the literature concerning IONPs use a chelator. There are examples of the use of heterocyclic ligands such as 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)15, 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA)16,17, and 1,4,7-triazacyclononane,1-glutaric acid-4,7-acetic acid (NODAGA)18, and the use of 2,3-dicarboxypropane-1,1-diphosphonic acid (DPD), a tetradentate ligand 19. Madru et al.20 developed a chelator-free strategy in 2014 to label IONPs using a chelator-free method used by another group posteriorly21.
However, major drawbacks of this approach include a high risk of in vivo transmetalation, low radiolabeling yields, and lengthy protocols unsuitable for short-lived isotopes22,23,24. For this reason, Wong et al.25 developed the first example of core-doped nanoparticles, managing to incorporate 64Cu in the core of the IONPs in a 5-min synthesis using microwave technology.
Here, we describe a rapid and efficient procedure to incorporate the radionuclide into the core of the nanoparticle, eluding many of the drawbacks presented by traditional methods. For this purpose, we propose the use of a microwave-driven synthesis (MWS), which reduces reaction times considerably, increases yields, and enhances reproducibility, critically important parameters in IONP synthesis. The refined performance of MWS is due to dielectric heating: rapid sample heating as molecular dipoles try to align with the alternating electric field, being polar solvents and reagents more efficient for this type of synthesis. In addition, the use of citric acid as a surfactant, together with microwave technology, results in extremely small nanoparticles, producing a dual T1-weighted MRI/PET26 signal, herein denoted as 68Ga Core-doped iron oxide nanoparticles (68Ga-C-IONP).
The protocol combines the use of microwave technology, 68GaCl3 as positron emitter, iron chloride, sodium citrate, and hydrazine hydrate, resulting in dual T1-weighted MRI/PET nanoparticulate material in hardly 20 min. Moreover, it yields consistent results over a range of 68Ga activities (37 MBq, 111 MBq, 370 MBq, and 1110 MBq) with no significant effects on the main physicochemical properties of the nanoparticles. The reproducibility of the method using high 68Ga activities extends the field of possible applications, including large animal models or human studies. In addition, there is a single purification step included in the method. In the process, any excess of free gallium, iron chloride, sodium citrate, and hydrazine hydrate are removed by gel filtration. Total free isotope elimination and the purity of the sample ensure no toxicity and enhance imaging resolution. In the past, we have already demonstrated the usefulness of this approach in targeted molecular imaging27,28.

<table>
	<tbody>
		<tr>
			<td>Iron (III) chloride hexahydrate</td>
			<td>POCH</td>
			<td>2317294</td>
			<td></td>
		</tr>
		<tr>
			<td>Citric acid, trisodium salt dihydrate 99%</td>
			<td>Acros organics</td>
			<td>227130010</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrazine hydrate</td>
			<td>Aldrich</td>
			<td>225819</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrochloric acid 37%</td>
			<td>Fisher Scientific</td>
			<td>10000180</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium dihydrogen phosphate monohydrate</td>
			<td>Aldrich</td>
			<td>S9638</td>
			<td></td>
		</tr>
		<tr>
			<td>Disodium phosphate dibasic</td>
			<td>Aldrich</td>
			<td>S7907</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Aldrich</td>
			<td>746398</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Azide</td>
			<td>Aldrich</td>
			<td>S2002</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium dihydrogen phosphate anhydrous</td>
			<td>POCH</td>
			<td>799200119</td>
			<td></td>
		</tr>
		<tr>
			<td>68Ga Chloride</td>
			<td>&#160;ITG Isotope Technologies Garching GmbH, Germany</td>
			<td>68Ge/68Ga generator system</td>
			<td></td>
		</tr>
		<tr>
			<td>Microwave</td>
			<td>Anton Paar</td>
			<td>Monowave 300</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Hettich</td>
			<td>Universal 320</td>
			<td></td>
		</tr>
		<tr>
			<td>Size Exclusion columns</td>
			<td>GE Healthcare</td>
			<td>PD-10</td>
			<td></td>
		</tr>
	</tbody>
</table>

synthesis of 68ga core-doped iron oxide nanoparticles for dual positron emission tomography /(t1)magnetic resonance imaging

Here, we describe a microwave synthesis to obtain iron oxide nanoparticles core-doped with 68Ga. Microwave technology enables fast and reproducible synthetic procedures. In this case, starting from FeCl3 and citrate trisodium salt, iron oxide nanoparticles coated with citric acid are obtained in 10 min in the microwave. These nanoparticles present a small core size of 4.2 &#177; 1.1 nm and a hydrodynamic size of 7.5 &#177; 2.1 nm. Moreover, they have a high longitudinal relaxivity (r1) value of 11.9 mM-1&#183;s-1 and a modest transversal relaxivity value (r2) of 22.9 mM-1&#183;s-1, which results in a low r2/r1 ratio of 1.9. These values enable positive contrast generation in magnetic resonance imaging (MRI) instead of negative contrast, commonly used with iron oxide nanoparticles. In addition, if a 68GaCl3 elution from a 68Ge/68Ga generator is added to the starting materials, a nano-radiotracer doped with 68Ga is obtained. The product is obtained with a high radiolabeling yield (&#62; 90%), regardless of the initial activity used. Furthermore, a single purification step renders the nano-radiomaterial ready to be used in vivo.

68Ga-C-IONP were synthesized by combining FeCl3, 68GaCl3, citric acid, water, and hydrazine hydrate. This mixture was introduced into the microwave for 10 min at 120 &#176;C and 240 W under controlled pressure. Once the sample had cooled down to room temperature, the nanoparticles were purified by gel filtration to eliminate unreacted species (FeCl3, citrate, hydrazine hydrate) and free 68Ga (Figure 1</...

Watch this Scientific Journal Video about Synthesis of 68Ga Core-doped Iron Oxide Nanoparticles for Dual Positron Emission Tomography /T1Magnetic Resonance Imaging at JoVE.com

Synthesis of 68Ga Core-doped Iron Oxide Nanoparticles for Dual Positron Emission Tomography /T1Magnetic Resonance Imaging

Here, we present a protocol to obtain 68Ga core-doped iron oxide nanoparticles via fast microwave-driven synthesis. The methodology renders PET/(T1)MRI nanoparticles with radiolabeling efficiencies higher than 90% and radiochemical purity of 99% in a 20-min synthesis.

Synthesis of 68Ga Core-doped Iron Oxide Nanoparticles for Dual Positron Emission Tomography /(T1)Magnetic Resonance Imaging

Here, we describe a microwave synthesis to obtain iron oxide nanoparticles core-doped with 68Ga. Microwave technology enables fast and reproducible ...

This method provides some fascinating synthesis of gallium-68 core-doped nanoparticles for hybrid PET/MR molecular imaging. The main advantage of this technique is that thanks to the use of microwave technology, the synthesis is fast and more importantly, completely reproducible. First, dissolve 75 milligrams of iron chloride hexahydrate and 80 milligrams of citric acid trisodium salt dihydrate in nine milliliters of water.Transfer the mixture to a microwave-adapted flask. Next, load a dynamic protocol in the microwave. Set the temperature to 120 degrees Celsius, the time to 10 minutes, the pressure to 250 psi, and the power to 240 watts.Add one milliliter of hydrazine hydrate to the reaction mixture. Then start the microwave protocol. In the meantime, rinse a gel filtration desalting column with 20 milliliters of distilled water.Once the protocol is finished, and the flask cooled to room temperature, pipette 2.5 milliliters of the final mixture onto the column and discard the flowthrough. Following this, add three milliliters of distilled water to the column and collect the nanoparticles in a plastic tube. Add 75 milligrams of iron chloride hexahydrate and 80 milligrams of citric acid trisodium salt dihydrate into a vial.Elute the gallium-68 generator using the recommended volume and concentration of hydrochloric acid according to the vendor. After the hydrochloric acid injection in the self-shielded generator, four milliliters of gallium-68 chloride is obtained, ready to use without further processing. Add four milliliters of gallium-68 chloride to the microwave-adapted flask.Then pipette five milliliters of distilled water into the flask and mix well. Now load a dynamic protocol in the microwave. Set the temperature to 120 degrees Celsius, the time to 10 minutes, the pressure to 250 psi, and the power to 240 watts.Add one milliliter of hydrazine hydrate to the reaction mixture. Then start the microwave protocol. In the meantime, rinse a gel filtration desalting column with 20 milliliters of distilled water.Once the protocol is finished and the flask cooled to room temperature, pipette 2.5 milliliters of the final mixture onto the column and discard the flowthrough. Then add three milliliters of distilled water to the column and collect the nanoparticles in a glass vial. To measure the hydrodynamic size of the gallium-68 nanoparticles, pipette 60 microliters of the sample into a cuvette and perform three dynamic light scattering measurements per sample.To assess the colloidal stability of the gallium-68 nanoparticles, incubate 500 microliters of sample in different buffers at 37 degrees Celsius for different times, ranging from zero to 24 hours. At the selected times, transfer 60 microliter aliquots to cuvettes and measure their hydrodynamic size. To obtain a gel filtration radiochromatogram, fractionate the elution from the size exclusion column into 500 microliter aliquots during the gel filtration purification step.Then measure the radioactivity present in each aliquot using an activimeter. To determine the radiochemical stability, incubate the gallium-68 nanoparticles in mouse serum for 30 minutes at 37 degrees Celsius. After incubation, purify the nanoparticles by ultrafiltration.Then measure the radioactivity present in the nanoparticles and filtrate. Hydrodynamic size data for the gallium-68 nanoparticles revealed a narrow size distribution and mean hydrodynamic size of 7.9 nanometers. Measurements of five different syntheses proved method reproducibility.The hydrodynamic size of gallium-68 nanoparticles incubated in different media from zero to 24 hours showed no significant changes, meaning the sample is stable in different buffers and serums. Because of the fast heating achieved using microwave technology, nanoparticles present ultra small core sizes of about four nanometers. Electron microscopy images revealed homogeneous core sizes and the absence of aggregation.The gallium-68 nanoparticle gel filtration chromatogram shows a main radioactivity peak corresponding to the nanoparticles and a reduced peak corresponding to free gallium-68. The radiolabeling yield was 92%which translates into a specific activity relative to 7.1 gigabecquerel per millimole of iron. An excellent longitudinal value of 11.9 and a modest relaxation value of 22.9 were obtained for five gallium-68 nanoparticle syntheses, yielding an average ratio of 1.9, meaning gallium-68 nanoparticles are ideal for T1-weighted MRI.MR phantom images at different gallium-68 nanoparticle concentrations show an increase in the iron concentration and positive contrast. An increasing iron concentration implies an increasing gallium-68 concentration, and the PET signal is increasingly intense. The use of microwave technology allows for the reproducible and rapid synthesis of iron oxide nanoparticles for multi-modal imaging.Following this procedure, we have produced a tracer that can be used for targeted molecular imaging with PET, T1 MRI, or hybrid approaches. After its development, this technique pave the way for researchers to explore the use of hybrid molecular imaging in fields such as oncology and cardiovascular diseases. Don't forget that working with radioactive compounds can be extremely hazardous, and radioprotection precautions should always be taken while performing this procedure.

synthesis-68ga-core-doped-iron-oxide-nanoparticles-for-dual-positron

Research

JoVE Journal

Biology

Labeling hESCs and hMSCs with Iron Oxide Nanoparticles for Non-Invasive in vivo Tracking with MR Imaging

In recent years, stem cell research has led to a better understanding of developmental biology, various diseases and its potential impact on regenerative medicine. A non-invasive method to monitor the transplanted stem cells repeatedly in vivo would greatly enhance our ability to understand the mechanisms that control stem cell death and identify trophic factors and signaling pathways that improve stem cell engraftment. MR imaging has been proven to be an effective tool for the in vivo depiction of stem cells with near microscopic anatomical resolution. In order to detect stem cells with MR, the cells have to be labeled with cell specific MR contrast agents. For this purpose, iron oxide nanoparticles, such as superparamagnetic iron oxide particles (SPIO), are applied, because of their high sensitivity for cell detection and their excellent biocompatibility. SPIO particles are composed of an iron oxide core and a dextran, carboxydextran or starch coat, and function by creating local field inhomogeneities, that cause a decreased signal on T2-weighted MR images. This presentation will demonstrate techniques for labeling of stem cells with clinically applicable MR contrast agents for subsequent non-invasive in vivo tracking of the labeled cells with MR imaging.

For the evaluation of new stem cell therapies it is important to non-invasively track the injected cells in vivo. This video will show you how to label human mesenchymal and embryonic stem cells with iron oxide based contrast agents in vivo for subsequent MR imaging in vivo.

In recent years, stem cell research has led to a better understanding of developmental biology, various diseases and its potential impact on regenerative ...

In vitro Labeling of Human Embryonic Stem Cells for Magnetic Resonance Imaging

Human embryonic stem cells (hESC) have demonstrated the ability to restore the injured myocardium. Magnetic resonance imaging (MRI) has emerged as one of the predominant imaging modalities to assess the restoration of the injured myocardium. Furthermore, ex-vivo labeling agents, such as iron-oxide nanoparticles, have been employed to track and localize the transplanted stem cells. However, this method does not monitor a fundamental cellular biology property regarding the viability of transplanted cells. It has been known that manganese chloride (MnCl2) enters the cells via voltage-gated calcium (Ca2+) channels when the cells are biologically active, and accumulates intracellularly to generate T1 shortening effect. Therefore, we suggest that manganese-guided MRI can be useful to monitor cell viability after the transplantation of hESC into the myocardium.
In this video, we will show how to label hESC with MnCl2 and how those cells can be clearly seen by using MRI in vitro. At the same time, biological activity of Ca2+-channels will be modulated utilizing both Ca2+-channel agonist and antagonist to evaluate concomitant signal changes.

In this video, we are showing how to label human embryonic stem cells (hESC) with manganese chloride (MnCl2) which can enter cells via voltage-gated calcium channels when the cells are biologically active. Additionally, we show the use of MnCl2 as cellular MRI contrast agent to determine the in vitro viability of hESC.

Human embryonic stem cells (hESC) have demonstrated the ability to restore the injured myocardium. Magnetic resonance imaging (MRI) has emerged as one of ...

Born Normalization for Fluorescence Optical Projection Tomography for Whole Heart Imaging

Optical projection tomography is a three-dimensional imaging technique that has been recently introduced as an imaging tool primarily in developmental biology and gene expression studies. The technique renders biological sample optically transparent by first dehydrating them and then placing in a mixture of benzyl alcohol and benzyl benzoate in a 2:1 ratio (BABB or Murray s Clear solution). The technique renders biological samples optically transparent by first dehydrating them in graded ethanol solutions then placing them in a mixture of benzyl alcohol and benzyl benzoate in a 2:1 ratio (BABB or Murray s Clear solution) to clear. After the clearing process the scattering contribution in the sample can be greatly reduced and made almost negligible while the absorption contribution cannot be eliminated completely. When trying to reconstruct the fluorescence distribution within the sample under investigation, this contribution affects the reconstructions and leads, inevitably, to image artifacts and quantification errors.. While absorption could be reduced further with a permanence of weeks or months in the clearing media, this will lead to progressive loss of fluorescence and to an unrealistically long sample processing time. This is true when reconstructing both exogenous contrast agents (molecular contrast agents) as well as endogenous contrast (e.g. reconstructions of genetically expressed fluorescent proteins).

We suggest a Born normalized approach for Optical Projection Tomography (BnOPT) that accounts for the absorption properties of imaged samples to obtain accurate and quantitative fluorescence tomographic reconstructions. We use the proposed algorithm to reconstruct the fluorescence molecular probe distribution within small animal organs.

Optical projection tomography is a three-dimensional imaging technique that has been recently introduced as an imaging tool primarily in developmental ...

Mesoscopic Fluorescence Tomography for In-vivo Imaging of Developing Drosophila

Visualizing developing organ formation as well as progession and treatment of disease often heavily relies on the ability to optically interrogate molecular and functional changes in intact living organisms. Most existing optical imaging methods are inadequate for imaging at dimensions that lie between the penetration limits of modern optical microscopy (0.5-1mm) and the diffusion-imposed limits of optical macroscopy (&gt;1cm) [1]. Thus, many important model organisms, e.g. insects, animal embryos or small animal extremities, remain inaccessible for in-vivo optical imaging. 
Although there is increasing interest towards the development of nanometer-resolution optical imaging methods, there have not been many successful efforts in improving the imaging penetration depth. The ability to perform in-vivo imaging beyond microscopy limits is in fact met with the difficulties associated with photon scattering present in tissues. Recent efforts to image entire embryos for example [2,3] require special chemical treatment of the specimen, to clear them from scattering, a procedure that makes them suitable only for post-mortem imaging. These methods however evidence the need for imaging larger specimens than the ones usually allowed by two-photon or confocal microscopy, especially in developmental biology and in drug discovery.
We have developed a new optical imaging technique named Mesoscopic Fluorescence Tomography [4], which appropriate for non-invasive in-vivo imaging at dimensions of 1mm-5mm. The method exchanges resolution for penetration depth, but offers unprecedented tomographic imaging performance and it has been developed to add time as a new dimension in developmental biology observations (and possibly other areas of biological research) by imparting the ability to image the evolution of fluorescence-tagged responses over time. As such it can accelerate studies of morphological or functional dependencies on gene mutations or external stimuli, and can importantly, capture the complete picture of development or tissue function by allowing longitudinal time-lapse visualization of the same, developing organism.
The technique utilizes a modified laboratory microscope and multi-projection illumination to collect data at 360-degree projections. It applies the Fermi simplification to Fokker-Plank solution of the photon transport equation, combined with geometrical optics principles in order to build a realistic inversion scheme suitable for mesoscopic range. This allows in-vivo whole-body visualization of non-transparent three-dimensional structures in samples up to several millimeters in size.
We have demonstrated the in-vivo performance of the technique by imaging three-dimensional structures of developing Drosophila tissues in-vivo and by following the morphogenesis of the wings in the opaque Drosophila pupae in real time over six consecutive hours.

Mesoscopic Fluorescence Tomography for In-vivo Imaging of Developing Drosophila

Mesoscopic fluorescence tomography operates beyond the penetration limits of tissue-sectioning fluorescence microscopy. The technique is based on multi-projection illumination and a photon transport description. We demonstrate in-vivo whole-body 3D visualization of the morphogenesis of GFP-expressing wing imaginal discs in Drosophila melanogaster.

Visualizing  developing organ formation as well as progession and treatment of disease often  heavily relies on the ability to optically interrogate ...

Electron Spin Resonance Micro-imaging of Live Species for Oxygen Mapping

This protocol describes an electron spin resonance (ESR) micro-imaging method for three-dimensional mapping of oxygen levels in the immediate environment of live cells with micron-scale resolution1. Oxygen is one of the most important molecules in the cycle of life. It serves as the terminal electron acceptor of oxidative phosphorylation in the mitochondria and is used in the production of reactive oxygen species. Measurements of oxygen are important for the study of mitochondrial and metabolic functions, signaling pathways, effects of various stimuli, membrane permeability, and disease differentiation. Oxygen consumption is therefore an informative marker of cellular metabolism, which is broadly applicable to various biological systems from mitochondria to cells to whole organisms. Due to its importance, many methods have been developed for the measurements of oxygen in live systems. Current attempts to provide high-resolution oxygen imaging are based mainly on optical fluorescence and phosphorescence methods that fail to provide satisfactory results as they employ probes with high photo-toxicity and low oxygen sensitivity. ESR, which measures the signal from exogenous paramagnetic probes in the sample, is known to provide very accurate measurements of oxygen concentration. In a typical case, ESR measurements map the probe's lineshape broadening and/or relaxation-time shortening that are linked directly to the local oxygen concentration. (Oxygen is paramagnetic; therefore, when colliding with the exogenous paramagnetic probe, it shortness its relaxation times.) Traditionally, these types of experiments are carried out with low resolution, millimeter-scale ESR for small animals imaging. Here we show how ESR imaging can also be carried out in the micron-scale for the examination of small live samples. ESR micro-imaging is a relatively new methodology that enables the acquisition of spatially-resolved ESR signals with a resolution approaching 1 micron at room temperature2. The main aim of this protocol-paper is to show how this new method, along with newly developed oxygen-sensitive probes, can be applied to the mapping of oxygen levels in small live samples. A spatial resolution of ~30 x 30 x 100 &mu;m is demonstrated, with near-micromolar oxygen concentration sensitivity and sub-femtomole absolute oxygen sensitivity per voxel. The use of ESR micro-imaging for oxygen mapping near cells complements the currently available techniques based on micro-electrodes or fluorescence/phosphorescence. Furthermore, with the proper paramagnetic probe, it will also be readily applicable for intracellular oxygen micro-imaging, a capability which other methods find very difficult to achieve.

This protocol describes a method for micron-scale three-dimensional imaging of oxygen concentration in the immediate environment of live cells by electron spin resonance microscopy.

This protocol describes an electron spin resonance (ESR) micro-imaging method for three-dimensional mapping of oxygen levels in the immediate environment ...

Preparation, Purification, and Characterization of Lanthanide Complexes for Use as Contrast Agents for Magnetic Resonance Imaging

Polyaminopolycarboxylate-based ligands are commonly used to chelate lanthanide ions, and the resulting complexes are 
useful as contrast agents for magnetic resonance imaging (MRI). Many commercially available ligands are especially useful because they contain 
functional groups that allow for fast, high-purity, and high-yielding conjugation to macromolecules and biomolecules via amine-reactive 
activated esters and isothiocyanate groups or thiol-reactive maleimides. While metalation of these ligands is considered common 
knowledge in the field of bioconjugation chemistry, subtle differences in metalation procedures must be taken into account when 
selecting metal starting materials. Furthermore, multiple options for purification and characterization exist, and selection of the most 
effective procedure partially depends on the selection of starting materials. These subtle differences are often neglected in published 
protocols. Here, our goal is to demonstrate common methods for metalation, purification, and characterization of lanthanide complexes 
that can be used as contrast agents for MRI (Figure 1). We expect that this publication will enable biomedical scientists to incorporate 
lanthanide complexation reactions into their repertoire of commonly used reactions by easing the selection of starting materials and 
purification methods.

We demonstrate the metalation, purification, and characterization of lanthanide complexes. The complexes described here can be conjugated to macromolecules to enable tracking of these molecules using magnetic resonance imaging.

Polyaminopolycarboxylate-based ligands are commonly used to chelate lanthanide ions, and the resulting complexes are 
useful as contrast agents for ...

Quantifying Mixing using Magnetic Resonance Imaging

Mixing is a unit operation that combines two or more components into a homogeneous mixture. This work involves mixing two viscous liquid streams using an in-line static mixer. The mixer is a split-and-recombine design that employs shear and extensional flow to increase the interfacial contact between the components. A prototype split-and-recombine (SAR) mixer was constructed by aligning a series of thin laser-cut Poly (methyl methacrylate) (PMMA) plates held in place in a PVC pipe. Mixing in this device is illustrated in the photograph in Fig. 1. Red dye was added to a portion of the test fluid and used as the minor component being mixed into the major (undyed) component. At the inlet of the mixer, the injected layer of tracer fluid is split into two layers as it flows through the mixing section. On each subsequent mixing section, the number of horizontal layers is duplicated. Ultimately, the single stream of dye is uniformly dispersed throughout the cross section of the device.
 Using a non-Newtonian test fluid of 0.2% Carbopol and a doped tracer fluid of similar composition, mixing in the unit is visualized using magnetic resonance imaging (MRI). MRI is a very powerful experimental probe of molecular chemical and physical environment as well as sample structure on the length scales from microns to centimeters. This sensitivity has resulted in broad application of these techniques to characterize physical, chemical and/or biological properties of materials ranging from humans to foods to porous media 1, 2. The equipment and conditions used here are suitable for imaging liquids containing substantial amounts of NMR mobile 1H such as ordinary water and organic liquids including oils. Traditionally MRI has utilized super conducting magnets which are not suitable for industrial environments and not portable within a laboratory (Fig. 2). Recent advances in magnet technology have permitted the construction of large volume industrially compatible magnets suitable for imaging process flows. Here, MRI provides spatially resolved component concentrations at different axial locations during the mixing process. This work documents real-time mixing of highly viscous fluids via distributive mixing with an application to personal care products.

Magnetic resonance imaging (MRI) provides a powerful tool to evaluate the effectiveness of process equipment during operation. We discuss the use of MRI to visualize mixing in a static mixer. The application is relevant to personal care products, but can be applied to a broad range of food, chemical, biomass and biological fluids.

Mixing is a unit operation that combines two or more components into a homogeneous mixture.  This work involves mixing two viscous liquid streams using an ...

Strategies for Optimization of Cryogenic Electron Tomography Data Acquisition

Cryogenic electron tomography (cryoET) is a powerful method to study the 3D structure of biological samples in a close-to-native state. Current state-of-the-art cryoET combined with subtomogram averaging analysis enables the high-resolution structural determination of macromolecular complexes that are present in multiple copies within tomographic reconstructions. Tomographic experiments usually require a vast amount of tilt series to be acquired by means of high-end transmission electron microscopes with important operational running-costs. Although the throughput and reliability of automated data acquisition routines have constantly improved over the recent years, the process of selecting regions of interest at which a tilt series will be acquired cannot be easily automated and it still relies on the user's manual input. Therefore, the set-up of a large-scale data collection session is a time-consuming procedure that can considerably reduce the remaining microscope time available for tilt series acquisition. Here, the protocol describes the recently developed implementations based on the SerialEM package and the PyEM software that significantly improve the time-efficiency of grid screening and large-scale tilt series data collection. The presented protocol illustrates how to use SerialEM scripting functionalities to fully automate grid mapping, grid square mapping, and tilt series acquisition. Furthermore, the protocol describes how to use PyEM to select additional acquisition targets in off-line mode after automated data collection is initiated. To illustrate this protocol, its application in the context of high-end data collection of Sars-Cov-2 tilt series is described. The presented pipeline is particularly suited to maximizing the time-efficiency of tomography experiments that require a careful selection of acquisition targets and at the same time a large amount of tilt series to be collected.

The increasing demand for large-scale data collection in cryogenic electron tomography requires high-throughput image acquisition routines. Described here is a protocol that implements the recent developments of advanced acquisition strategies aimed at maximizing the time-efficiency and throughput of tomographic data collection.

Cryogenic electron tomography (cryoET) is a powerful method to study the 3D structure of biological samples in a close-to-native state. Current ...

Imaging Replicative Domains in Ultrastructurally Preserved Chromatin by Electron Tomography

Principles of DNA folding in the cell nucleus and its dynamic transformations that occur during the fulfillment of basic genetic functions (transcription, replication, segregation, etc.) remain poorly understood, partially due to the lack of experimental approaches to high-resolution visualization of specific chromatin loci in structurally preserved nuclei. Here we present a protocol for the visualization of replicative domains in monolayer cell culture in situ,&#160;by combining EdU labeling of newly synthesized DNA with subsequent label detection with Ag-amplification of Nanogold particles and ChromEM staining of chromatin. This protocol allows for the high-contrast, high-efficiency pre-embedding labeling, compatible with traditional glutaraldehyde fixation that provides the best structural preservation of chromatin for room-temperature sample processing. Another advantage of pre-embedding labeling is the possibility to pre-select cells of interest for sectioning. This is especially important for the analysis of heterogeneous cell populations, as well as compatibility with electron tomography approaches to high-resolution 3D analysis of chromatin organization at sites of replication, and the analysis of post-replicative chromatin rearrangement and sister chromatid segregation in the interphase.

This protocol presents a technique for high-resolution mapping of replication sites in structurally preserved chromatin in situ that employs a combination of pre-embedding EdU-streptavidin-Nanogold labeling and ChromEMT.

Principles of DNA folding in the cell nucleus and its dynamic transformations that occur during the fulfillment of basic genetic functions (transcription, ...

Quantitating Iron Transport Across the Mouse Placenta In Vivo Using Nonradioactive Iron Isotopes

Iron is essential for maternal and fetal health during pregnancy, with approximately 1 g of iron needed in humans to sustain a healthy pregnancy. Fetal iron endowment is entirely dependent on iron transfer across the placenta, and perturbations of this transfer can lead to adverse pregnancy outcomes. In mice, measurement of iron fluxes across the placenta traditionally relied on radioactive iron isotopes, a highly sensitive but burdensome approach. Stable iron isotopes (57Fe and 58Fe) offer a nonradioactive alternative for use in human pregnancy studies.
Under physiological conditions, transferrin-bound iron is the predominant form of iron taken up by the placenta. Thus, 58Fe-transferrin was prepared and injected intravenously in pregnant dams to directly assess placental iron transport and bypass maternal intestinal iron absorption as a confounding variable. Isotopic iron was quantitated in the placenta and mouse embryonic tissues by inductively coupled plasma mass spectrometry (ICP-MS). These methods can also be employed in other animal model systems of physiology or disease to quantify in vivo iron dynamics.

Quantitating Iron Transport Across the Mouse Placenta In Vivo Using Nonradioactive Iron Isotopes

This article demonstrates how to prepare and administer transferrin-bound nonradioactive isotopic iron for studies of iron transport in mouse pregnancy. The approach for quantifying isotopic iron in fetoplacental compartments is also described.

Iron is essential for maternal and fetal health during pregnancy, with approximately 1 g of iron needed in humans to sustain a healthy pregnancy. Fetal ...