Although perfusion-based MR methods such as DCE have proven effective in diagnosing tumors as small as 1–2 cm and conventional MR methods are still being improved for more accurate cancer detection and prognosis,28,29
the sensitivity of these methods for detecting malignancy in its earliest stages, before gross anatomical changes have taken place, is lacking. In addition, conventional MRI methods frequently lack specificity for typing cancers for the purpose of differential diagnosis. Thus a concerted effort has been made to develop MR CAs that have the ability to detect even subtle changes in the intra and extracellular milieu during oncogenesis. The transformation of benign cells into malignancy is characterized by a concomitant array of biochemical changes in the microenvironment, i.e., remodeling of the extracellular matrix, changes to the vascular endothelium, activation of inflammatory pathways and upregulation of cell surface proteins and enzymes involved in chemotaxis and adhesion. These changes provide ample potential targets for MR CA development.
Cells expressing a large number of binding sites accessible to targeting molecules can be potentially imaged using the systemic delivery of antibody- or non-antibody ligands either by injecting paramagnetic targeted agents directly into systemic circulation, or after pre-targeting the cancer-associated targeted sites with affinity molecules and injecting paramagnetic CA after the clearance of non-bound affinity molecules. In both cases, the imaging of cancer is improved by linking paramagnetic metals to large molecules, which have the inherent problem of nonspecific uptake and slow removal from circulation. Although the uptake by the cells of the reticuloendothelial system is a key characteristic in the utility of SPIOs as diagnostic agents, endocytosis/phagocytosis by professional phagocytes also removes nanoparticles from the circulation, thus preventing them from reaching specific cellular targets. Fortunately, the level of SPIO opsonization in blood, and thus the macrophage uptake and blood half-life of these agents, can in most cases be altered by changing the coating layer of the nanoparticles. It has been reported that low hydrophobicity of the surface and/or negative surface charge can hinder rapid phagocytosis and clearance from the circulation after intravenous injections.
The greatest limiting factor in the development of cell or receptor-targeted MR CAs is sensitivity. Even in the case of receptors, which are greatly overexpressed in cancerous cells, the ability to link sufficient quantities of CA to the target to produce a detectable contrast change in the MR image is limited. Early attempts to detect Gd3+
chelates linked to monoclonal antibodies (mAb) targeted against cell surface receptors were unsuccessful due to the limited number of Gd3+
ions per antibody 30,31,32
. It is estimated that local concentrations of Gd3+
must be in the range of 10 to 500 uM to produce detectable contrast enhancements 33,6
, which translates into a substantial number (greater than 10) of Gd3+
ions per mAb even if the antigen (i.e., a cell surface receptor) is highly expressed in the tissue.
In some cases, the problem of sensitivity can be addressed by designing CAs that are taken up via passive or receptor-mediated transport into the cells of interest, thus providing an avenue for accumulation of the probe. Continued development of carrier molecules for paramagnetic agents such as micelles, liposomes, micro-emulsions, apoferritin, and lipoproteins further increase the potential for MRI of cancer targets that exist in low concentration in tumors and pre-malignant cells.
Another approach that has been used to deliver higher concentrations of Gd3+
-chelates to tumors is the use of dendrimers. Dendrimers are synthetic molecules that consist of a core surrounded by concentric shells (termed generations) made up of covalently linked branched chemical moieties. Moieties in the outer shell can serve as attachment points for functional groups, substrates, or CAs. In most cases, the linking of chelated Gd3+
to dendrimeric carriers increases relaxivity of the metal, resulting in greater signal enhancement in the MR image (reviewed in 34
). Aptamers (DNA or RNA oligonucleotides) also have been used as cancer targeting agents for conjugation to IOs and are well suited for this purpose due to their low molecular weights, lack of immunogenicity, and availability. Hwang do et al.35
developed a multimodality imaging probe (for concurrent fluorescence, radionuclide and MRI) based on a cobalt-ferrite nanoparticle bound to the cancer-specific aptamer, AS1411. Twenty-four hours after intravenous administration to nude mice bearing C6 tumors, T2-weighted MR images could be clearly visualized as black spots, whereas no change in signal was seen for control injected animals.
Although Gd-linked dendrimers or IO nanoparticles can localize greater amounts of CA to a tissue, alternative strategies have been sought to increase the target to background/contrast ratio produced by these agents. ‘Activatable’, or ‘amplifiable’ (MRamp) agents rely on the activity of endogenous or exogenous enzymes or other biomolecules to chemically modify the CA after it has bound its target, thereby causing the CAs to be retained in the tissue of interest (i.e., a tumor).
Our laboratory has demonstrated a method whereby a low molecular weight MRamp agent, di- 5-hydroxytryptamide of DTPA(Gd) (5-HT) is caused to oligomerize and accumulate at the site of xenografted A431 tumors (human squamous cell carcinoma) after the 5HT substrate is oxidized by exogenous horseradish peroxidase (HRP). 36,37
HRP (and the catalyst glucose oxidase) is selectively brought to the site of the tumor by linking it to the tumor-specific monoclonal antibody (anti-L6 mAb) that binds the Lewis Y antigen overexpressed in many carcinomas (). Using this technique, we observed a stronger regional enhancement of tumors over a longer time span (a 160% increase at approx. at 55 min post contrast) in experimental animals than in control ones. In this case, the relaxivity changes were due to the activation of bis-5HT- DTPAGd by peroxidases and accumulation at the site of antibody binding as a result of oligomerization and binding to tyrosine-containing proteins. Enzymatic MR CA signal amplification has shown great promise for detecting malignancies in animal models of cancer, especially in instances where only a small number of malignant cells are present, or when the cancer-specific target (such as a receptor) exists at a low concentration.
Figure 2 A) Schema of mAb conjugate synthesis with a formation of stable bisatomatic hydrazone bond using HydraLinK. B) HRP/GO-targeted coupled catalysis in the presence of glucose. Di-5HT-DTPA(Gd) is used as a reducer of oxidized HRP. Top: Plot of signal intensity (more ...)
A different approach that has been used to amplify the signal generated by extremely low concentration molecular targets is paramagnetic chemical exchange saturation technique (PARACEST) 38
. In this technique, protons in direct contact with the target CA molecule are saturated (made ‘MR silent’) by a selective radiofrequency pulse. The ensuing transient loss of signal is then transferred to nearby protons because of inhomogeneities created in the surrounding environment.