Name: Preparation, Purification, and Characterization of Lanthanide Complexes for Use as Contrast Agents for Magnetic Resonance Imaging (Video) | JoVE Journal
Uploaded: 2011-07-21T13:00:00.000+00:00
Duration: 13 min 21 s
Description: Learn to synthesize, purify, and characterize lanthanide complexes for use as MRI contrast agents in biological imaging. This method enables tracking of macromolecules through relaxivity measurements.

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5 minutes

We gratefully acknowledge startup funds from Wayne State University (MJA), a grant from the American Foundation for Aging Research (SMV), and a Pathway to Independence Career Transition Award (R00EB007129) from the National Institute of Biomedical Imaging and Bioengineering of the National Institutes of Health (MJA).

1. Metalation using LnCl3 salts
<ol>
	<li>Dissolve the ligand in water to produce a solution of 30-265 mM. The ligand 2-(4-isothiocyanatobenzyl)-diethylenetriamine pentaacetic acid 
	(p-SCN-Bn-DTPA) was used in this video at a concentration of 73 mM.</li>
	<li>Adjust the pH of the solution of ligand to between 5.5 and 7.0 by adding 1 M NH4OH. In this video, 0.2 mL of the 1 M NH4OH solution was used.</li>
	<li>Dissolve 1-2 equivalents of LnCl3 in water to produce a solution with a concentration of 5-1000 mM. In this video, EuCl3 
	and GdCl3 were used in concentrations of 111 mM. An excess of metal is often used to drive the metalation to completion and 
	consequently simplify purification.</li>
	<li>Add the solution of LnCl3 to the solution of ligand while stirring.</li>
	<li>After the addition of LnCl3, adjust the pH of the resulting reaction mixture to between 5.5 and 7.0 by adding 0.2 M NH4OH. A 
	total of 0.5 mL of the 0.2 M NH4OH solution was used in this video. If your ligand contains acid-sensitive functional groups, adjust 
	the pH multiple times during this step. CAUTION - If the solution becomes too basic, any base sensitive functional groups, like isothiocyanate, 
	will be rendered unusable for conjugation.</li>
	<li>Monitor the reaction via pH measurements. The reaction is complete when the pH remains constant.</li>
</ol>
2. Raising pH workup (not included in this video, but good for ligands without base-sensitive functional groups)
<ol>
	<li>Add concentrated NH4OH to the reaction mixture to adjust the pH to ≥11. This step will precipitate any uncomplexed 
	metal as the insoluble hydroxide.</li>
	<li>Filter the supernatant through a 0.2 μm filter. If the reaction mixture clogs the filter, centrifuging and decanting prior to 
	filtering is recommended.</li>
	<li>If dialysis will not be performed, remove solvent under reduced pressure (rotary evaporation or freeze drying is recommended).</li>
	<li>Steps 2.1-2.3 can be repeated if free lanthanide remains.</li>
</ol>
3. Dialysis workup
<ol>
	<li>Cut the dialysis tubing to an appropriate length (follow the manufacturer’s guidelines) to hold the sample volume while leaving extra length (approximately 10% of the sample volume). In this video, a 100-500 dalton molecular weight cut-off (MWCO) membrane was used, but larger MWCO tubing can be used as appropriate if conjugation is performed prior to metalation. Also, dialysis cassettes can be used as an alternative to dialysis tubing if desired.</li>
	<li>If appropriate based on the manufacturer’s guidelines, soak the cut dialysis tubing in water for 15 min at ambient temperature.</li>
	<li>Fill a dialysis reservoir (a 1 L beaker was used in this video) with water (dialysate). The dialysate volume should be approximately 100x that of the sample.</li>
	<li>Fold one end of the tubing twice and secure the folded portion of the tubing with a dialysis closure clamp. Wrap the end of the closure with a rubber band to ensure that it remains closed during dialysis.</li>
	<li>Filter the reaction mixture through a 0.2 μm filter, and load the filtrate into 
	the open end of the tubing being careful not to tear the tubing. Be sure to leave enough head space to close the tubing.</li>
	<li>Fold the remaining open end of the tubing twice, secure with a closure, and wrap the closure with a rubber band as in step 3.4.</li>
	<li>Attach a glass vial containing air to the clamp on one end of the dialysis tubing using a rubber band. Attach a vial containing sand to the other clamp. These vials ensure that the tubing stays immersed in the dialysate.</li>
	<li>Place the full tubing in the dialysis reservoir that contains dialysate.</li>
	<li>Stir the dialysate using a magnetic stir plate at a slow speed (no vortexing) at ambient temperature.</li>
	<li>Change the dialysate 3x over the course of a day (in this video, the dialysate was changed at 2.5, 6.5, and 11.5 h), and then allow dialysis to continue overnight (for a total of 20-28 h of dialysis).</li>
	<li>Remove the dialysis tubing from the dialysate and carefully open one closure to remove the sample. Wash the dialysis tubing 3x with water and combine the washings with the sample.</li>
	<li>Remove the water under reduced pressure. Freeze drying is used in this video.</li>
</ol>
4. Assessment of the presence of free metal
<ol>
	<li>Dissolve the metal complex in acetate buffer (buffer preparation: Dissolve 1.4 mL of acetic acid in 400 mL water, adjust the pH to 5.8 with 
	1 M NH4OH, and add water to produce a total volume of 500 mL) and add the xylenol orange indicator (16 μM in pH 5.8 buffer). In this 
	video, 0.3 mg of complex was dissolved in 0.3 mL of buffer and 3 mL of indicator solution was added.</li>
	<li>Detect the presence of free metal via observation of a color change of the indicator from yellow to violet.</li>
	<li>If desired, the amount of free metal can be quantified by creating a calibration curve1. Alternatively, the dye arsenazo III can 
	be used instead of xylenol orange2. If free metal remains, the sample should be further purified using dialysis, a desalting column, or high-performance liquid chromatography (HPLC) prior to characterization.</li>
</ol>
5. Determination of water-coordination number (q)
<ol>
	<li>Prepare a solution of the EuIII-containing complex (~1 mM) in H2O and another solution of the same concentration in 
	D2O. Prior to analysis, the D2O solution must be evaporated and dissolved in D2O three times to remove residual 
	H2O.</li>
	<li>Add the water solution to a clean cuvette, and place the cuvette into a spectrofluorometer.</li>
	<li>Perform excitation and emission scans to determine the maxima for each (~395 nm and ~595 nm, respectively).</li>
	<li>Perform a phosphorescence time-decay experiment using the following parameters: excitation and emission wavelengths determined from 
	step 5.3, excitation and emission slit widths (5 nm), flash count (100), initial delay (0.01 ms), maximum delay (13 ms), and delay 
	increment (0.1 ms). These conditions are appropriate for most complexes, but the maximum delay and increment values can be increased 
	or decreased for species with extremely long or extremely short decay times.</li>
	<li>Repeat step 5.4 with the D2O solution prepared in step 5.1.</li>
	<li>From the luminescence-decay data obtained in 5.4 and 5.5, plot the natural log of intensity versus time. The slope of these lines are 
	the decay rates (τ-1) (Figure 2). In this video, Microsoft Excel 2007 was used to generate the natural log plots from the raw data. Use the decay rates in the equation developed by Horrocks and coworkers (eq 1)3. If your ligand contains OH or NH groups coordinated to the metal, then the equation must be modified before use3.</li>
</ol>
eq 1:   <img src="/files/ftp_upload/2844/2844eq1.jpg" alt="Static equilibrium formula q calculation; equation for τ in H2O, D2O." data-altupdatedat="1788235205000" data-alt="Equation 1">
6. Relaxivity measurements
<ol>
	<li>Select the desired application mode on the relaxation time analyzer: T1 (longitudinal relaxation time) or T2 (transverse relaxation time).</li>
	<li>Prepare a series of samples that contain different concentrations of GdIII-containing complex in an aqueous solvent. In this video, water was used as the solvent and solutions of 10.0, 5.00, 2.50, 1.25, 0.625, and 0 mM were prepared. Other aqueous solvents or buffers could be used, but it is important to use the solvent as the blank. The final volume of the sample is specific to the instrument that is being used.</li>
	<li>Place a sample in the instrument and let it sit for 5 min to equilibrate to the temperature of the instrument (37 °C in this video).</li>
	<li>Determine the relaxation time (in units of s) by adjusting the parameters of the software to obtain a smooth exponential curve for T1 or T2 (representative curves for T1 and T2 are shown in Figure 3).</li>
	<li>Repeat steps 6.3 and 6.4 for all samples including the blank.</li>
	<li>Calculate the inverse of the measured T1 or T2 values in units of s-1.</li>
	<li>Plot the T1-1 or T2-1 values versus GdIII concentration (in units of mM). Because of the hygroscopic nature of GdIII-containing complexes, confirm the concentration of GdIII using atomic absorption spectrophotometry or inductively coupled plasma mass spectrometry. Fit the plot with a straight line. A representative plot is shown in Figure 4.</li>
	<li>The slope of the fitted line is the relaxivity (r1 or r2 for T1 and T2, respectively) and has units of mM-1s-1.</li>
</ol>
7. Representative Results
Representative data for the steps in this protocol have been included in the Tables and Figures section. In addition to 
the water-coordination number and relaxivity characterization described in the protocol, it is important to characterize final products 
using standard chemical techniques. The identity of the compound can be obtained using mass spectrometry, and representative mass spectra 
showing the diagnostic isotope patterns for GdIII- and EuIII-containing complexes are shown in Figure 5. Furthermore, for non-GdIIIlanthanide-containing complexes, NMR spectroscopy can be used for identification of the product. To characterize the purity of the complex, HPLC or elemental analysis or both can be used.
<img src="/files/ftp_upload/2844/2844fig1.jpg" alt="Chemical synthesis process diagram, LnCl3 complex formation and purification, showing vacuum removal." data-altupdatedat="1788235205000" data-alt="Figure 1"> Figure 1. General scheme for metalation and purification: Scheme depicting the general procedure for metalation and reasons for choosing different purification routes.
<img src="/files/ftp_upload/2844/2844fig2.jpg" alt="Exponential decay graph, ln intensity vs. time, linear fit, R²=0.99, data analysis result." data-altupdatedat="1788235205000" data-alt="Figure 2"> Figure 2. Luminescence-intensity plot: Representative plot of the natural log of intensity versus time from section 5. The slopes of the lines generated from similar curves acquired for water and D2O solutions are used with eq 1 to characterize the water-coordination number of EuIII-containing complexes.
<img src="/files/ftp_upload/2844/2844fig3.jpg" alt="Recovery rate vs. time graphs; dynamic response analysis; data fitting diagrams." data-altupdatedat="1788235205000" data-alt="Figure 3"> Figure 3. Relaxation decay time curves: Representative data for (left) T1 and (right) T2 acquisition. Deviations from these curve shapes would produce unreliable data.
<img src="/files/ftp_upload/2844/2844fig4.jpg" alt="Linear relationship graph; concentration vs. inverse T1, equation: y=4.33x-0.0129, data fitting." data-altupdatedat="1788235205000" data-alt="Figure 4"> Figure 4. Relaxivity determination: A representative plot of 1/T1 versus the concentration of GdIII. The slope of the fitted line is relaxivity and has units of mM-1s-1.
<img src="/files/ftp_upload/2844/2844fig5.jpg" alt="Mass spectrometry graph showing m/z vs. relative intensity; peaks indicate ion distribution analysis." data-altupdatedat="1788235205000" data-alt="Figure 5"> Figure 5. Mass spectra: Representative mass spectra showing the diagnostic isotope patterns for (left) GdIII-containing complexes and (right) EuIII-containing complexes. The black Gaussian peaks represent the theoretical isotope distribution and the red lines are the actual data.

<ol><li>Barge, A., Cravotto, G., Gianolio, E., Fedeli, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((How+to+determine+free+Gd+and+free+ligand+in+solution+of+Gd+chelates.+A+technical+note%5BTitle%5D)+AND+%22Contrast+Med.+Mol.+Imaging%22%5BJournal%5D)">How to determine free Gd and free ligand in solution of Gd chelates. A technical note.</a> Contrast Med. Mol. Imaging. 1, 184-188 (2006).</li>
<li>Nagaraja, T. N., Croxen, R. L., Panda, S., Knight, R. A., Keenan, K. A., Brown, S. L., Fenstermacher, J. D., Ewing, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Application+of+arsenazo+III+in+the+preparation+and+characterization+of+an+albumin-linked%2C+gadolinium-based+macromolecular+magnetic+resonance+contrast+agent%5BTitle%5D)+AND+%22J.+Neurosci.+Methods%22%5BJournal%5D)">Application of arsenazo III in the preparation and characterization of an albumin-linked, gadolinium-based macromolecular magnetic resonance contrast agent.</a> J. Neurosci. Methods. 157, 238-245 (2006).</li>
<li>Supkowski, R. M., Horrocks, W. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((On+the+determination+of+the+number+of+water+molecules%2C+q%2C+coordinated+to+europium%28III%29+ions+in+solution+from+luminescence+decay+lifetimes%5BTitle%5D)+AND+%22Inorg.+Chim.+Acta%22%5BJournal%5D)">On the determination of the number of water molecules, q, coordinated to europium(III) ions in solution from luminescence decay lifetimes.</a> Inorg. Chim. Acta. 340, 44-48 (2002).</li>
<li>Menjoge, A. R., Kannan, R. M., Tomalia, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dendrimer-based+drug+and+imaging+conjugates%3A+design+considerations+for+nanomedical+applications%5BTitle%5D)+AND+%22Drug+Discovery+Today%22%5BJournal%5D)">Dendrimer-based drug and imaging conjugates: design considerations for nanomedical applications.</a> Drug Discovery Today. 15, 171-185 (2010).</li>
<li>Que, E. L., Chang, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Responsive+magnetic+resonance+imaging+contrast+agents+as+chemical+sensors+for+metals+in+biology+and+medicine%5BTitle%5D)+AND+%22Chem.+Soc.+Rev%22%5BJournal%5D)">Responsive magnetic resonance imaging contrast agents as chemical sensors for metals in biology and medicine.</a> Chem. Soc. Rev. 39, 51-60 (2010).</li>
<li>Uppal, R., Caravan, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Targeted+probes+for+cardiovascular+MR+imaging%5BTitle%5D)+AND+%22Future+Med.+Chem%22%5BJournal%5D)">Targeted probes for cardiovascular MR imaging.</a> Future Med. Chem. 2, 451-470 (2010).</li>
<li>Major, J. L., Meade, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Bioresponsive%2C+cell-penetrating%2C+and+multimeric+MR+contrast+agents%5BTitle%5D)+AND+%22Acc.+Chem.+Res%22%5BJournal%5D)">Bioresponsive, cell-penetrating, and multimeric MR contrast agents.</a> Acc. Chem. Res. 42, 893-903 (2009).</li>
<li>Datta, A., Raymond, K. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Gd-hydroxypyridinone+%28HOPO%29-based+high-relaxivity+magnetic+resonance+imaging+%28MRI%29+contrast+agents%5BTitle%5D)+AND+%22Acc.+Chem.+Res%22%5BJournal%5D)">Gd-hydroxypyridinone (HOPO)-based high-relaxivity magnetic resonance imaging (MRI) contrast agents.</a> Acc. Chem. Res. 42, 938-947 (2009).</li>
<li>León-Rodríguez, L. M. D., Lubag, A. J. M., Malloy, C. R., Martinez, G. V., Gillies, R. J., Sherry, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Responsive+MRI+agents+for+sensing+metabolism+in+vivo%5BTitle%5D)+AND+%22Acc.+Chem.+Res%22%5BJournal%5D)">Responsive MRI agents for sensing metabolism in vivo.</a> Acc. Chem. Res. 42, 948-957 (2009).</li>
<li>Castelli, D. D., Gianolio, E., Crich, S. G., Terreno, E., Aime, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Metal+containing+nanosized+systems+for+MR-molecular+imaging+applications%5BTitle%5D)+AND+%22Coord.+Chem.+Rev%22%5BJournal%5D)">Metal containing nanosized systems for MR-molecular imaging applications.</a> Coord. Chem. Rev. 252, 2424-2443 (2008).</li>
<li>Caravan, P., Ellison, J. J., McMurry, T. J., Lauffer, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Gadolinium%28III%29+chelates+as+MRI+contrast+agents%3A+structure%2C+dynamics%2C+and+applications%5BTitle%5D)+AND+%22Chem.+Rev%22%5BJournal%5D)">Gadolinium(III) chelates as MRI contrast agents: structure, dynamics, and applications.</a> Chem. Rev. 99, 2293-2352 (1999).</li>
<li>Lauffer, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Paramagnetic+metal+complexes+as+water+proton+relaxation+agents+for+NMR+imaging%3A+theory+and+design%5BTitle%5D)+AND+%22Chem.+Rev%22%5BJournal%5D)">Paramagnetic metal complexes as water proton relaxation agents for NMR imaging: theory and design.</a> Chem. Rev. 87, 901-927 (1987).</li>
<li>Yoo, B., Pagel, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+overview+of+responsive+MRI+contrast+agents+for+molecular+imaging%5BTitle%5D)+AND+%22Front.+Biosci%22%5BJournal%5D)">An overview of responsive MRI contrast agents for molecular imaging.</a> Front. Biosci. 13, 1733-1752 (2008).</li>
<li>Pandya, S., Yu, J., Parker, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Engineering+emissive+europium+and+terbium+complexes+for+molecular+imaging+and+sensing%5BTitle%5D)+AND+%22Dalton+Trans%22%5BJournal%5D)">Engineering emissive europium and terbium complexes for molecular imaging and sensing.</a> Dalton Trans. 23, 2757-2766 (2006).</li>
<li>Nwe, K., Xu, H., Regino, C. A. S., Bernardo, M., Ileva, L., Riffle, L., Wong, K. J., Brechbiel, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+new+approach+in+the+preparation+of+dendrimer-based+bifunctional+diethylenetriaminepentaacetic+acid+MR+contrast+agent+derivatives%5BTitle%5D)+AND+%22Bioconjugate+Chem%22%5BJournal%5D)">A new approach in the preparation of dendrimer-based bifunctional diethylenetriaminepentaacetic acid MR contrast agent derivatives.</a> Bioconjugate Chem. 20, 1412-1418 (2009).</li>
<li>Nwe, K., Bernardo, M., Regino, C. A. S., Williams, M., Brechbiel, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Comparison+of+MRI+properties+between+derivatized+DTPA+and+DOTA+gadolinium-dendrimer+conjugates%5BTitle%5D)+AND+%22Bioorg.+Med.+Chem%22%5BJournal%5D)">Comparison of MRI properties between derivatized DTPA and DOTA gadolinium-dendrimer conjugates.</a> Bioorg. Med. Chem. 18, 5925-5931 (2010).</li>
<li>Caravan, P., Das, B., Deng, Q., Dumas, S., Jacques, V., Koerner, S. K., Kolodziej, A., Looby, R. J., Sun, W. -C., Zhang, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+lysine+walk+to+high+relaxivity+collagen-targeted+MRI+contrast+agents%5BTitle%5D)+AND+%22Chem.+Commun%22%5BJournal%5D)">A lysine walk to high relaxivity collagen-targeted MRI contrast agents.</a> Chem. Commun. , 430-432 (2009).</li>
<li>León-Rodríguez, L. M. D., Kovacs, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+synthesis+and+chelation+chemistry+of+DOTA-peptide+conjugates%5BTitle%5D)+AND+%22Bioconjugate+Chem%22%5BJournal%5D)">The synthesis and chelation chemistry of DOTA-peptide conjugates.</a> Bioconjugate Chem. 19, 391-402 (2008).</li>
<li>Boswell, C. A., Eck, P. K., Regino, C. A. S., Bernardo, M., Wong, K. J., Milenic, D. E., Choyke, P. L., Brechbiel, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Synthesis%2C+characterization%2C+and+biological+evaluation+of+integrin+%CE%B1V%CE%B23-targeted+PAMAM+dendrimers%5BTitle%5D)+AND+%22Mol.+Pharm%22%5BJournal%5D)">Synthesis, characterization, and biological evaluation of integrin αVβ3-targeted PAMAM dendrimers.</a> Mol. Pharm. 5, 527-539 (2008).</li>
</ol>

No conflicts of interest declared.

Given the increasing number of publications that include lanthanide-based contrast agents4-14, it is important that care is taken in preparing, purifying, and characterizing products to ensure reproducible and comparable results. These complexes are often considered challenging to purify and characterize relative to organic molecules due to their paramagnetic nature and the sensitivity 
of any functional groups that could be used for bioconjugation. We have described common methods for the synthesis, purificat...

<table><tbody><tr><td>EuCl&lt;sub&gt;3&lt;/sub&gt;&amp;#8729;6H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma-Aldrich</td><td>203254-5G</td><td></td></tr><tr><td>&lt;em&gt;p&lt;/em&gt;-SCN-Bn-DTPA</td><td>Macrocyclics</td><td>B-305</td><td></td></tr><tr><td>ammonium hydroxide</td><td>EMD Millipore</td><td>AX1303-3</td><td></td></tr><tr><td>Spectra/Por Biotech Cellulose Ester (CE) Dialysis Membrane - 500 D MWCO</td><td>Fisher Scientific</td><td>68-671-24</td><td></td></tr><tr><td>Millipore IC Millex-LG Filter Units</td><td>Fisher Scientific</td><td>SLLG C13 NL</td><td></td></tr><tr><td>xylenol orange tetrasodium salt</td><td>Alfa Aesar</td><td>41379</td><td></td></tr><tr><td>acetic acid</td><td>Fluka</td><td>49199</td><td></td></tr><tr><td>D&lt;sub&gt;2&lt;/sub&gt;O</td><td>Cambridge Isotope Laboratories</td><td>DLM-4-25</td><td></td></tr><tr><td>water purifier</td><td>ELGA</td><td>Purelab Ultra</td><td></td></tr><tr><td>high performance liquid chromatography and mass spectrometry</td><td>Shimadzu Corporation</td><td>LCMS-2010EV</td><td></td></tr><tr><td>relaxation time analyzer</td><td>Bruker Corporation</td><td>mq60 minispec</td><td></td></tr><tr><td>UV-vis spectrophotometer</td><td>Fisher Scientific</td><td>20-624-00092</td><td></td></tr><tr><td>freeze dryer</td><td>Fisher Scientific</td><td>10-030-133</td><td></td></tr><tr><td>pH meter</td><td>Hanna Instruments</td><td>HI 221</td><td></td></tr><tr><td>spectrofluorometer</td><td>Horiba Instruments Inc</td><td>Fluoromax-4</td><td></td></tr><tr><td>Molecular Weight Calculator version 6.46 by Matthew Monr&amp;#8211;, downloaded October 17, 2009</td><td><a href="http://ncrr.pnl.gov/software/" target="_blank"><img src="/img/new_window.png"> http://ncrr.pnl.gov/software/</a></td><td>Molecular Weight Calculator</td><td></td></tr></tbody></table>

preparation, purification, and characterization of lanthanide complexes for use as contrast agents for magnetic resonance imaging

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 ...

The overall goal of this procedure is to synthesize, purify, and characterize lanthanide containing complexes that can be used for tagging biological macromolecules. This is accomplished by first mixing the metal starting material and ligand while controlling the pH of the solution. The second step of the procedure is to purify the resulting complex using dialysis.Next, the absence of free metal needs to be verified. The final step of the procedure is the characterization of the properties of the complex that are relevant to imaging. Ultimately, results can be obtained that show that the synthesized complexes will behave as contrast agents through relax activity measurements.This method can help answer key questions in the imaging field, such as whether the water coordination number of a contrast agent changes upon binding to a protein. Generally, individuals need this method will struggle because net carefully controlling the pH will result in rendering the LI and useless for bio education. Demonstrating the dialysis and activity measurements will be Buda and Sashi.Graduate students from my laboratory To begin methation using lanthanide salts first dissolve one equivalent of ligand in water to produce a 30 to 265 millimolar solution. The ligand Paraiso Benzyl DTPA is used here at a concentration of 73 Millimolar. Next, adjust the pH of the ligand solution to between 5.5 and seven by adding one molar ammonium hydroxide.In this video, 0.2 milliliters of the one molar ammonium hydroxide solution is used now dissolve one to two equivalents of lanthanide chloride in water to produce a solution with a concentration of five to 1000 millimolar. Either euro bium chloride or gadolinium chloride can be used in concentrations of 111 millimolar. Often an excess of metal is used to drive the ventilation to completion and consequently simplify purification.Next, add each solution of lathan salts to the prepared solution of ligand while stirring. Now adjust the pH of the resulting reaction mixtures to between 5.5 and seven by adding 0.2 molar ammonium hydroxide. Here, a total of 0.5 milliliters of the 0.2 molar ammonium hydroxide solution is used to adjust the solution.If the ligand being used contains acid sensitive functional groups, adjust the pH multiple times during this step. Be sure to use caution when adjusting the pH because if the solution becomes too basic, any base sensitive functional groups like Isothiocyanate will be rendered unusable. For conjugation, closely monitor the reaction via pH measurements.The reaction is complete when the pH remains constant For ligands without any base sensitive functional groups, A workup that involves raising the pH may also be useful. To begin dialysis workup, determine an appropriate length of dialysis tubing to hold the sample volume plus extra length to hold an additional 10%of the sample volume. Then follow manufacturer guidelines to cut the tubing to this length.In this video, a 100 to 500 Dalton molecular weight cutoff membrane was used, but larger molecular weight cutoff tubing can be used as appropriate if conjugation is performed prior to methation, if appropriate, based on the manufacturer's guidelines, soak the cut dialysis tubing, and water for 15 minutes at ambient temperature. Next, fill a dialysis reservoir with water, which will serve as the dialysate. A one liter beaker is used here.The dialysate volume should be approximately 100 times that of the sample. Now fold one end of the tubing twice and secure the folded portion with the dialysis closure clamp. Wrap the end of the closure with a rubber band to ensure that it remains closed during dialysis.Filter the previously prepared reaction mixture through a 0.2 micron filter. Then load the filtrate into the open end of the tubing. Being careful not to tear the tubing.Be sure to leave enough head space to close the tubing. Fold the remaining open end of the tubing twice, then secure it with a closure and wrap the closure with a rubber band. Next, attach a glass vial containing air to the clamp on one end of the dialysis tubing using another rubber band.Then attach a vial containing sand to the other clamp. These vials ensure that the tubing stays immersed in the dialysate. Now, place the full tubing in the dialysis reservoir that contains dialysate.Stir the dialysate using a magnetic stir plate at a slow speed at ambient temperature. Be sure the stir speed remains slow and the solution does not.Vortex. Change the dialate three times over the course of a day.In this video, the dialate is changed at 2.5, 6.5, and 11.5 hours. Allow dialysis to continue overnight for total of 20 to 28 hours. Once the dialysis is complete, remove the tubing from the dialate and carefully open one closure to remove the sample.Wash the dialysis tubing three times with water and combine the washings with the sample. Finally, remove the water from the sample under reduced pressure here. This is accomplished by freeze drying.The sample is frozen and then placed on a freeze drying apparatus. To assess the presence of free metal in the sample, first prepare acetate buffer by dissolving 1.4 milliliters of acetic acid in 400 milliliters of water, and adjust the pH to 5.8 with one molar ammonium hydroxide, and add water to produce a total volume of 500 milliliters. Then dissolve 0.3 milligrams of each complex in 0.3 milliliters of buffer.Now prepare xol orange indicator, which should be 16 micromolar in the pH 5.8 buffer. Add three milliliters of this indicator solution to the previously dissolved metal complexes. Detect the presence of free metal via observation of a color change of the indicator from yellow to violet.If desired, the amount of free metal can be quantified by creating a calibration curve. If free metal remains, the sample should be further purified using dialysis or high performance liquid chromatography prior to characterization. Next, to determine the water coordination number for a opium sample, prepare a solution of about one millimolar of the opium containing complex in water, and then another solution of the same concentration in deuterium oxide.Prior to analysis, the deuterium oxide solution must be evaporated and dissolved in deuterium oxide three times to remove residual water, turn on the spectro fluorimeter, add the water solution to a clean vete and place the vete into the spectro fluorimeter. Now perform excitation and emissions to determine the maxima for each at approximately 395 nanometers, 595 nanometers respectively. Next, perform a phosphorescence time decay experiment using the excitation and emission wavelengths just determined and the parameters displayed here.Repeat this step with a prepared deuterium oxide solution from the luminescence decay data just obtained plot, the natural log of intensity versus time. The slope of these lines are the decay rates. In this video, Microsoft Excel 2007 was used to generate the natural log plots from the raw data.Use the decay rates in the equation developed by hors and coworkers seen here. If your ligand contains OH or NH groups coordinated to the metal, then the equation must be modified before use. To determine the relativity measurements of the sample first, select the desired application mode on the relaxation time analyzer, either T one or T two.Here, the T one setting is selected. Next, prepare a series of samples that contain different concentrations of the lanthanide containing complex in an acquiesce solvent. Here, water is used as the solvent and solutions of ten five two 0.5, 1.25, 0.625 and zero.Millimolar are prepared. Other acquiesce solvents or buffers can be used, but it is important to use the solvent as the blank. The final volume of the sample is specific to the instrument that is being used.Place a sample in the instrument and let it sit for five minutes to equilibrate to the temperature of the instrument, which is 37 degrees Celsius for the unit used in this video. Now determine the relaxation time in units of seconds by adjusting the parameters of the software to obtain a smooth exponential curve for T one or T two. Repeat this for all samples, including the blank.Now, calculate the inverse of the measured T one or T two relaxation values and plot. These values versus Lathan concentration and units of millimolar fit the plot with a straight line. The slope of the fitted line is the relax activity being R one or R two for T one and T two respectively.Here we see the general scheme for mediation and purification. This scheme depicts the general procedure for methation and reasons for choosing different purification roots. Here we see a representative luminescence decay plot in which the natural log of decay is plotted versus time the slopes of the lines generated from similar curves required for water and deuterium oxide solutions are used with equation one to determine the water coordination number of opium containing complexes.A representative example of livity measurements can be seen here with a slope of the fitted line being the sample's T one livity. In addition to the water coordination number and livity characterization described in the protocol, it is also important to characterize final products using standard chemical techniques. The identity of the compound can be obtained using mass spectrometry representative mass spectra showing the diagnostic isotope patterns for gadolinium and opium containing complexes are shown here.Following this procedure, other methods like endr spectroscopy HPLC and elemental analysis can be performed to further characterize the resulting complexes. After watching this video, you should have a good understanding of how to synthesize, purify, and characterize lanthanide containing complexes that can be used for tagging biological macromolecules.

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Preparation, Purification, and Characterization of Lanthanide Complexes for Use as Contrast Agents for Magnetic Resonance Imaging (Video) | JoVE Journal

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

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