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

Summary

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.

Abstract

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.

Protocol

1. Metalation using LnCl₃ salts

1. 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.
2. 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.
3. 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 m....

Discussion

Given the increasing number of publications that include lanthanide-based contrast agents^4-14, 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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
EuCl3∙6H2O	Sigma-Aldrich	203254-5G
p-SCN-Bn-DTPA	Macrocyclics	B-305
ammonium hydroxide	EMD Millipore	AX1303-3
Spectra/Por Biotech Cellulose Ester (CE) Dialysis Membrane - 500 D MWCO	Fisher Scientific	68-671-24
Millipore IC Millex-LG Filter Units	Fisher Scientific	SLLG C13 NL
xylenol orange tetrasodium salt	Alfa Aesar	41379
acetic acid	Fluka	49199
D2O	Cambridge Isotope Laboratories	DLM-4-25
water purifier	ELGA	Purelab Ultra
high performance liquid chromatography and mass spectrometry	Shimadzu Corporation	LCMS-2010EV
relaxation time analyzer	Bruker Corporation	mq60 minispec
UV-vis spectrophotometer	Fisher Scientific	20-624-00092
freeze dryer	Fisher Scientific	10-030-133
pH meter	Hanna Instruments	HI 221
spectrofluorometer	Horiba Instruments Inc	Fluoromax-4
Molecular Weight Calculator version 6.46 by Matthew Monr–, downloaded October 17, 2009	http://ncrr.pnl.gov/software/	Molecular Weight Calculator