The lanthanides commonly written as Ln are the elements from lanthanum to lutetium and usually exist as trivalent ions, Ln3+. They have the general formula [Xe]4fn where n = 0-14. One of the main properties of the lanthanides is their similarity to calcium; they have a very similar ionic radius but a higher charge. Therefore, Ln3+ ions have a high affinity for biological Ca2+ sites and can act either as a Ca2+ inhibitor or probe. [1]
The 4f orbitals on the lanthanides are shielded by the filled 5p66s2 sub-shells, resulting in unique spectroscopic properties, including characteristic narrow-line emissions in the visible or near-infrared regions. Most lanthanide ions are luminescent, but some produce more prominent emissions than others depending on the ease with which their excited state(s) can be populated and the minimisation of non-radiative deactivation paths. Luminescent probes containing Eu(III) and Tb(III) ions have been popular previously due to the large energy gap between their excited and ground states. [2] The lanthanide ions can be used in medical applications such as imaging, selective labelling and sensing of specific biomarkers. In this article, new developments and applications of the lanthanides in medical imaging and for the diagnosis of disease are reviewed.
Paramagnetic effects provide extra structural information in NMR in the form of residual dipolar couplings (RDCs) and pseudocontact chemical shifts (PCSs). Therefore the introduction of artificial paramagnetic metals into proteins is an area of great interest where lanthanide chemistry can play a key role.
Liu et al. [3] have developed a new lanthanide-based probe, CLaNP-7, which improves on many properties of earlier probes. Previously, paramagnetic probes based on cyclen have been presented which attach to a target protein via two disulphide bridges, helping to reduce the probe's mobility relative to the protein. CLaNP-5, the most recently developed probe, [4] contains two chelating pyridine-N-oxide arms which force the probe to exist as one single diastereomer, giving a single peak in NMR data and having a single magnetic susceptibility tensor. However, the charge of this probe (+3) affects the electrostatic potential of the surface to which it binds and could affect molecular interactions with ligands and proteins.
The new probe, CLaNP-7, has p-nitrophenol arms instead of the pyridine-N-oxide arms in CLaNP-5, providing more space for a ninth coordination site and giving the overall complex a net charge of either +1 or zero, depending on whether H2O or OH occupy this ninth site. The p-nitrophenol groups also have a yellow colour associated with them, allowing for easy detection and purification of the tagged proteins.
It was found that pH dependence of the CLaNP-7 probe could be introduced by placing the lanthanide ion near a histidine residue on the protein.
It was suggested that the induced pH dependence was due to hydrogen bonding between the imidazole ring on the histidine and the H2O or OH in the ninth coordination site (Figure 2). This dependence allows two sets of distance restraints to be obtained from the same probe, simply by altering the pH.
This new probe provides great potential for the structural identification of macromolecules within the body, allowing two sets of information to be obtained from just one probe at a single position on the protein.
Another area of great demand is for a sensitive, low cost method to monitor acetylsalicylate and its metabolites within the body. Aspirin (acetylsalicylic acid or ASA) is one of the most widely used therapeutic substances and so the understanding of its pharmacodynamics and pharmacokinetics is vital. Salicylates are also used as markers to assess free radical damage in vivo due to OH radicals.
One of the major metabolites of ASA in the body is salicylic acid (SA), further metabolised to salicyluric acid (SU), which is eliminated in the urine with a high elimination rate constant. It is therefore possible to use the excretion of SU as an indicator to measure SA concentrations in vivo in a non-invasive way. Unusual concentrations of SU in urine have also been linked to several diseases and conditions, giving the proposed method potential in diagnosis.
Using the binary complex [Tb(DO2A)]+, Esplin et al. [5] have presented the first lanthanide / macrocycle receptor used to detect SU in urine. Terbium has a large energy gap between its lowest energy excited state and its ground state, allowing for an intense emission in the visible region. The SU analyte coordinates to form a ternary [Tb(DO2A)(SU)]- complex which is strongly luminescent, increasing the intensity of this emission.
Figure 2: The structure of the probe CLaNP-7 and its interactions with the imizadole ring of a nearby histidine
Through experiment, Esplin et al. found that a sample dilution factor of 1:350 produced a reproducible, linear correlation between the SU concentration and the emission intensity, which was independent of the donor.
This method allows sample preparation and analysis to be conducted in as little as five minutes. There is potential for optimisation of the Tb receptor site by adding functional groups to improve the limit of detection (LOD) by at least one order of magnitude (from 1.8 mg L-1 SU in urine), which would make the detection more sensitive than any other reported technique.
Esplin et al. have found a low cost, rapid method of detection with the possibility of automation. This could be used as an easy way of observing levels of SU within the body for the detection of certain medical conditions and for monitoring the pharmacokinetics of aspirin.
Lanthanides also have great potential for more specific disease diagnosis, with one of the most exciting and promising areas of new development involving the early diagnosis of cancer.
There are often problems with false results regarding the diagnosis of cancer, especially in the early stages. Integrin αvβ3, a membrane protein, is overexpressed in cancerous cells and different levels of overexpression have been linked to different stages of the disease, allowing for potential early stage diagnosis. Zhang et al. [6] have developed a probe to quantify expression levels of cancerous biomarkers as well as cancer cells themselves. It consists of three parts; a fluorescent dye for fluorescence imaging (FI), 2-aminoethyl-monoamide-DOTA (MMA-DOTA) for loading the europium and for quantification using ICPMS and a guiding cyclic Arg-Gly-Asp peptide which targets integrin αvβ3 specifically (Figure 3).
The binding of Eu to the complex (mmA-AMF-DOTA) does not affect the energy level of the fluorescein moiety, showing that the Eu complex can be used for both ICPMS of the targeted cancer cells and FI of the cells. Experiments show that there is no species-dependent response in ICPMS and combined with the low LOD this shows that ICPMS could be used as a highly sensitive and accurate method to quantify the Eu labelled cancer cells.
It was found that 69.2 - 309.4 amol of αvβ3 are expressed per cell of αvβ3 - positive cancer cells compared to 1.6 - 5.2 a mol on αvβ3-negative or healthy cells. The LOD of αvβ3 suggests that, depending on the type of cancer cell, the minimum required number of cells for detection is between 17 and 75. The results from counting cells using ICPMS were compared with results obtained using flow cytometry and showed a high level of agreement, suggesting that ICPMS is a reliable method for counting the cancer cells. However, above levels of 400,000 cells the ICPMS intensity of labelled cells began to deviate. This may be due to reduced surface area for labelling, due to cell aggreagation at such high concentrations. However, ICPMS was found to be much more sensitive than fluorescence confocal microscopy in the early stages of cancer when there may not be enough cancerous cells to see via microscopy.
The developed probe can also penetrate into the nucleus of certain cancer cells even with the incubation temperature controlled at 4áµ’C, providing potential for further investigation into possible drug developments. Zhang et al. have successfully found a trifunctional probe with high potential for earlier and more accurate diagnosis of cancer. Further work could also involve developing probes selective to other cell-membrane biomarkers for other disease diagnosis.
Figure 3: Structure of the trifunctional probe developed by Zhang et al.Recently, probes based on lanthanides have been introduced to target other specific analytes, such as thiols within the blood. Links have been made between low cysteine (Cys) levels and health issues and also between high homocysteine (HCys) levels and certain disorders. Therefore there is great interest in the development of selective sensors for sulfhydryl-containing amino acids. Previously Yoon et al. [7] have developed fluorescent probes for the detection of such species but, although the probes were highly selective, they consisted of short wavelength emitting fluorophores with very short lifetimes. This is a major disadvantage in biological applications.
Glutathione (GSH), the most predominant tripeptide thiol in the blood, is rapidly oxidised to its dimeric form, GSSG in response to oxidative stress within a cell. This suggests that if the ratio of GSH:GSSG is monitored, the overall health of a cell and its ability to resist oxidative damage can be measured.
McMahon and Gunnlaugsson [8] have designed a probe called 1. Tb; an octadentate macrocyclic Tb(III) cyclen conjugate containing a maleimide group which, upon 1,4-Michael addition of a thiol group to the double bond of the alkene, shows large shifts in Tb(III) centred emission. This Tb(III) emission is enhanced by up to 500% in the presence of GSH. The emission intensity increases as a function of GSH concentration within the biological pH range and is unaffected by the presence of amino acids other than Cys and HCys.
As well as monitoring the presence of Cys and HCys in the blood in order to diagnose certain disorders, the ratio of GSH:GSSG can also be monitored. GSSG does not contain the nucleophilic thiol moiety which GSH does and so 1.Tb can selectively sense GSH and thus can be used to observe the reaction kinetics of the redox process.
The new developments discussed here are just a few examples of the many advancements being made in lanthanide chemistry. The unique properties of these elements allow their use in promising new methods of disease diagnosis and even give them potential for medical treatments.
