Martin W. Brechbiel

Macromolecules, Dendrimers, and Nanomaterials in Magnetic Resonance Imaging: The Interplay between Size, Function, and Pharmacokinetics

Magnetism in medicine has had a long and interesting history. In the 10 th century A.D., Egyptian physician and philosopher Avicenna prescribed a grain of magnetite dissolved in milk for the accidental swallowing of rust reasoning that magnetite would render the poisonous iron inert by attracting it and accelerating its excretion through the intestine.1 A thousand years later on July 3, 1977, “Indomitable”, the little machine that could, labored for five hours to produce one image, an event that used magnetism to change the landscape of modern medicine. 2 Looking at the homemade superconducting magnet constructed from 30 miles of niobiumtitanium wire that now resides in its rightful place at the Smithsonian Institution, it is incredible to comprehend how in a mere 30 years magnetic resonance imaging (MRI) has gone from its crude, almost ugly, human scan to where physicians can now regularly order MRIs off their menu of diagnostic tools because of its exquisite anatomical resolution, routinely down to 0.5 to 1 mm. When the field was first reviewed in this journal in 1987, 3 only 39 papers were found in Medline with keywords “gado-“ and “MRI”. 4 Today, this same search on PubMed pulls out over 250,000 records, of which a significant component has been development of MR contrast agents. The human body is essentially a super-sized water bottle, with about two-thirds of its weight consisting of water. Waters hydrogen atoms are able to act as microscopic compass needles that stand “at attention” when placed in a strong magnetic field. When submitted to pulses of radio waves, their magnetic alignment is disrupted and the differences in how they relax to the previous state are used to generate images. Contrast agents can act to catalyze the process of the return to the ground relaxed state. Now commonplace in the clinic, paramagnetic or superparamagnetic metal ions are administered in 40–50% of the 7–10 million MR examinations per year. 5 These image-enhancing contrast agents add significant morphological and functional information to unenhanced MR images, allowing for enhanced tissue contrast, characterization of lesions, and evaluation of perfusion and flow-related abnormalities. In this review, we will introduce small molecule agents, but focus primarily on macromolecular MR contrast agents, particularly those containing gadolinium (Gd 3+ ) that are assembled or based in part on these same small molecules. A brief discussion on iron oxide and manganese (Mn 2+ ) agents is also provided.

MRI of tumor angiogenesis

Angiogenesis has long been established as a key element in the pathophysiology of tumor growth and metastasis. Increasingly, new molecularly targeted antiangiogenic drugs are being developed in the fight against cancer. These drugs bring with them a need for an accurate means of diagnosing tumor angiogenesis and monitoring response to treatment. Imaging techniques can offer this in a noninvasive way, while also providing functional information about the tumor. Among the many clinical imaging techniques available, MRI alone can provide relatively good spatial resolution and specificity, without ionizing radiation and with limited side effects. Arterial spin labeling (ASL) and blood oxygenation level‐dependent (BOLD) imaging techniques can be employed to confer indirect measures of angiogenesis, such as blood flow and blood volume, without the need for external contrast agents. Dynamic contrast‐enhanced (DCE)‐MRI is rapidly emerging as a standard method for directly measuring angiogenesis during angiogenesis‐inhibitor drug trials. As macromolecular MR contrast agents become available, they will inevitably be utilized in the assessment of tumor perfusion and vessel permeability. Meanwhile, technological advances have made imaging at a molecular level a possibility. They have brought the potential to directly target MR contrast agents to markers of angiogenesis, such as the αvβ3 integrin. Before this is used clinically, however, substantial gains in sensitivity brought about by improved coils, pulse sequences, and contrast agents will be needed. Herein we discuss the techniques currently available for MRI of angiogenesis, along with their respective advantages and disadvantages, and what the future holds for this evolving field of imaging. J. Magn. Reson. Imaging 2007.

Targeting dendrimer-chelates to tumors and tumor cells expressing the high-affinity folate receptor.

RATIONALE AND OBJECTIVES The authors developed a new method for delivering contrast agents to tumors and tumor cells. Gadolinium complexes of folate-conjugated dendrimer-chelates increased the longitudinal relaxation rate of tumor cells expressing the high-affinity folate receptor, hFR. The coupling of folate to polymeric chelates, composed of a dendrimer backbone, targets these chelates to endogenous folate binding proteins. These proteins exist in both the serum of patients with cancer and on the cell surface of many human cancers of epithelial origin. METHODS The authors attached folic acid to a generation four ammonia core polyamidoamine dendrimer. The folate-dendrimer was reacted with 2-(4-isothiocyanatobenzyl)-6-methyl-diethylenetriaminepentaacetic acid to form the polymeric chelate f-PAMAM-TU-DTPA. For fluorescent studies, the generation four dendrimer was reacted with fluorescein-5-isothiocyanate and carboxytetramethylrhodamine succinimidyl ester, followed by capping the remaining amines with succinic anhydride. RESULTS The study results show that cells accumulate the folate-conjugated dendrimer in a receptor specific manner. Tumor cells expressing the high-affinity folate receptor showed a 650% increase in the mean fluorescence. This increase occurred with a rapid rise to 325%, followed by a slow increase to 650%. It required both the expression of the hFR and the coupling of folic acid to the dendrimer. Excess free folic acid inhibited the binding of the folate conjugated polymer. Fluorescent microscopic study showed that the folate-conjugated dendrimer binds to the cell surface and is accumulated within the cells. Treatment of tumor cells that express the hFR with gadolinium complexes of the folate-conjugated polymeric chelate increases the longitudinal relaxation rate by 110%. This increase was inhibited by an excess of free folic acid. CONCLUSIONS These data demonstrate that folate-conjugated magnetic resonance imaging contrast agents represent a promising new approach to tumor targeting.

Specific targeting of folate-dendrimer MRI contrast agents to the high affinity folate receptor expressed in ovarian tumor xenografts.

The need to develop target-specific MRI contrast agents to aid in disease characterization remains highly essential. In this study, we present a generation four polyamidoamine (PAMAM) folate-dendrimer that specifically targets the high affinity folate receptor (hFR) overexpressed on more than 80% of ovarian tumors. In vitro, mouse erythroleukemia cells expressing the hFR bind the radiolabeled folate-dendrimer chelate resulting in over 2700% increase in binding compared with untreated cells. The binding was inhibited by free folic acid to levels observed on folate-receptor-negative cells. In vivo, ovarian tumor xenografts resulted in a 33% contrast enhancement, following the folate-dendrimer chelate administration, that was significantly different compared with results obtained with a non-specific, extracellular fluid space agent, Gd-HP-DO3A. In addition, this contrast enhancement was absent in saline-treated animals, folate-receptor-negative tumors, and was inhibited by free folic acid. Results suggest that a macromolecular, dendrimeric MRI agent with high molecular relaxivities (1646 mM−1 s−1) can be used in specifically targeting the hFR on tumor cells and ovarian tumors.

A phase I clinical trial with monoclonal antibody ch806 targeting transitional state and mutant epidermal growth factor receptors

An array of cell-surface antigens expressed by human cancers have been identified as targets for antibody-based therapies. The great majority of these antibodies do not have specificity for cancer but recognize antigens expressed on a range of normal cell types (differentiation antigens). Over the past two decades, our group has analyzed thousands of mouse monoclonal antibodies for cancer specificity and identified a battery of antibodies with limited representation on normal human cells. The most tumor-specific of these antibodies is 806, an antibody that detects a unique epitope on the epidermal growth factor receptor (EGFR) that is exposed only on overexpressed, mutant, or ligand-activated forms of the receptor in cancer. In vitro immunohistochemical specificity analysis shows little or no detectable 806 reactivity with normal tissues, even those with high levels of wild-type (wt)EGFR expression. Preclinical studies have demonstrated that 806 specifically targets a subset of EGFR expressed on tumor cells, and has significant anti-tumor effects on human tumor xenografts, primarily through abrogation of signaling pathways. The present clinical study was designed to examine the in vivo specificity of a chimeric form of mAb 806 (ch806) in a tumor targeting/biodistribution/pharmacokinetic analysis in patients with diverse tumor types. ch806 showed excellent targeting of tumor sites in all patients, no evidence of normal tissue uptake, and no significant toxicity. These in vitro and in vivo characteristics of ch806 distinguish it from all other antibodies targeting EGFR.

Dendrimer-based macromolecular MRI contrast agents: characteristics and application.

Numerous macromolecular MRI contrast agents prepared employing relatively simple chemistry may be readily available that can provide sufficient enhancement for multiple applications. These agents operate using a approximately 100-fold lower concentration of gadolinium ions in comparison to the necessary concentration of iodine employed in CT imaging. Herein, we describe some of the general potential directions of macromolecular MRI contrast agents using our recently reported families of dendrimer-based agents as examples. Changes in molecular size altered the route of excretion. Smaller-sized contrast agents less than 60 kDa molecular weight were excreted through the kidney resulting in these agents being potentially suitable as functional renal contrast agents. Hydrophilic and larger-sized contrast agents were found better suited for use as blood pool contrast agents. Hydrophobic variants formed with polypropylenimine diaminobutane dendrimer cores created liver contrast agents. Larger hydrophilic agents are useful for lymphatic imaging. Finally, contrast agents conjugated with either monoclonal antibodies or with avidin are able to function as tumor-specific contrast agents, which also might be employed as therapeutic drugs for either gadolinium neutron capture therapy or in conjunction with radioimmunotherapy.

3D-micro-MR angiography of mice using macromolecular MR contrast agents with polyamidoamine dendrimer core with reference to their pharmacokinetic properties.

Four novel macromolecular MRI contrast agents, all of which had the same chemical composition but different molecular weights, were prepared using generation‐3, ‐4, ‐5, and ‐6 polyamidoamine (PAMAMTM) dendrimers conjugated with a bifunctional diethylenetriaminepentaacetic acid derivative to change the blood retention, tissue perfusion, and excretion. Size‐dependent changes in the pharmacokinetics were observed in the biodistribution study. 153Gd‐labeled generation‐6 PAMAMTM‐conjugates remained in the blood significantly longer than all of the other preparations (P < 0.001). The increase in blood‐to‐organ ratio of the preparations was found to correlate with increasing molecular size (P < 0.001). Additionally, 3D‐micro MR images and angiography of mice of high quality and detail were obtained using PAMAMTM‐(1B4M‐Gd)x as a macro‐molecular MRI contrast agent with a 1.5‐T clinical MRI instrument. Numerous fine vessels of ∼200 μm diameter were visualized on subtracted 3D‐MR angiographms with G6D‐(1B4M‐Gd)192. The quality of the images was sufficient to estimate the microvasculature of cancerous tissue for anti‐angiogenesis therapy and to investigate knockout mice. Magn Reson Med 45:454–460, 2001.

Dendrimer-based nanosized MRI contrast agents.

Paramagnetic metals can induce T1 shortening by interaction with free water molecules. Two metal ions, Gadolinium and Manganese, are currently available for human use. Gadolinium-based MRI contrast agents (CAs) can operate using a approximately 100-fold lower concentration of Gadolinium ions in comparison to the necessary concentration of Iodine atoms employed in CT imaging in the tissues. Therefore, numerous macromolecular MRI CAs prepared employing relatively simple chemistry are readily available that can provide sufficient enhancement for multiple applications. Herein, we describe the synthesis, characteristics, and potential applications of dendrimer-based macromolecular MRI CAs in our recently reported libraries. This entire series of dendrimer-based macromolecular MRI CAs have a spherical shape and possess similar surface charges. Changes in molecular size altered the route of excretion. Smaller sized contrast agents, of less than 60 kD molecular weight, were excreted through the kidney resulting in these agents being potentially suitable as functional renal contrast agents. Less hydrophilic and larger sized contrast agents were found better suited for use as blood pool contrast agents. Hydrophobic variants of CAs formed with polypropylenimine diaminobutane dendrimer cores quickly accumulated in the liver and can function as liver contrast agents. Larger hydrophilic agents are also useful for lymphatic imaging. Finally, contrast agents conjugated with either monoclonal antibodies or with avidin are able to function as tumor-specific contrast agents and might also be employed as therapeutic drugs for either gadolinium neutron capture therapy or in conjunction with radioimmunotherapy.

A dendrimer-based nanosized contrast agent dual-labeled for magnetic resonance and optical fluorescence imaging to localize the sentinel lymph node in mice

To preoperatively and intraoperatively localize the sentinel lymph node (SLN), a single hybrid probe for MR and near infrared (NIR) optical imaging was synthesized and tested.

Pharmacokinetics and enhancement patterns of macromolecular MR contrast agents with various sizes of polyamidoamine dendrimer cores

Four macromolecular contrast agents are synthesized to visualize small vessels by MRI using generation‐3 (G3D), ‐4 (G4D), ‐5 (G5D), and ‐6 (G6D) polyamidoamine dendrimers conjugated to chelated gadolinium (Gd). The pharmacokinetics, enhancement patterns, and the ability of these constructs to visualize fine vessels is evaluated by dynamic MRI in relationship to their size. Gd‐G6D and ‐G5D exhibit a prolonged high vascular (ventricular) signal intensity (SI) with high ventricle‐to‐organ SI ratios. The initial high vascular SI with Gd‐G4D decreases to a value as low as that obtained with Gd‐G3D and Gd‐dimeglumine‐diethylenetriaminepentaacetic acid (Gd‐DTPA). Gd‐G5D, ‐G4D, and ‐G3D show high renal SIs, and Gd‐DTPA prominently enhances the skin. Gd‐G6D and ‐G5D present fine vasculature significantly more clearly than Gd‐G3D and ‐DTPA (P < 0.005). As the molecular size increases, the excretion of the 153Gd‐conjugates is retarded. In conclusion, Gd‐G6D and ‐G5D are retained in the blood and present fine vessels with high quality and detail, and should be adequate for visualizing small tumor vasculature. Magn Reson Med 46:1169–1173, 2001.