Fe8-Based MRI Contrast Agents Targeted to Breast and Ovarian Cancer Cells
Parga Rivera, Kenia A.
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Magnetic resonance imaging (MRI) contrast agents (CA’s) are of great interest for cancer imaging as a complementary tool in cancer diagnosis. Tumor-targeting MRI-CA conjugates based on an [Fe8O4(pz)12Cl4] paramagnetic cluster were synthesized to generate a breast/ovarian cancer tumor-targeting CA. Fe8 clusters were chosen for their stability, and because biochemical mechanisms for the efficient elimination of iron exist. Conjugates of Fe8 and hyaluronic acid (HA) work by targeting CD44, a proteoglycan which is over expressed on most human breast and ovarian cancer cell lines as well as on tumor specimens, and is a major receptor for HA. The ultimate goal of this project was to study the properties of an ovarian cancer tumor-targeting CA. Several Fe8-phenol complexes were prepared and characterized, including Fe8-Tyr. Taking a cue from the demonstrated affinity of iron for phenolate ligands, the synthesis and characterization of new Fe(III)-Tyr-HA conjugates was subsequently developed, with Tyramine-substituted Hyaluronic Acid (Tyr-HA). The conjugates produced are soluble in water. Tyr- HA5 and Tyr-HA16 refer to the tyramine functionalized 5 KDa and 16 KDa HA, respectively. The synthetic work of the conjugates encompasses three steps, (1) coupling of 4-hydroxyphenethylamine (Tyr) to the targeting group -- hyaluronic acid (HA) to form Tyr-HA (Tyr-HA5 and Tyr-HA16) conjugates -- (2) synthesis of the Fe8 complex, and (3) coupling of the Fe8 complex, by the substitution of a chloride terminal ligand, to the Tyr-HA conjugate to form the targeted Fe8- Tyr-HA5 and Fe8-Tyr-HA16 CA’s. The characterization of the conjugates was carried out by EDS, TEM, IR, and Mössbauer spectroscopies, and TGA. The data generated led to the following general conjugate formula for Fe8-Tyr-HA5: (Fecubane)4(Feouter-Tyr-HA)1.6(Feouter-O-Feouter)2.4 Experimental studies in aqueous solution, such as pH, Dynamic Light Scattering and Zeta Potential, demonstrate the conjugate’s stable behavior in aqueous solution, with an average hydrodynamic diameter of 145 ± 0.4 nm and 407.4 ± 0.5 nm, and a zeta potential of -28 ± 2 mV and -30 ± 3 for Fe8- Tyr-HA5 and Fe8-Tyr-HA16, respectively. The relaxivities of the Fe8-Tyr-HA conjugates were determined by their T1 and T2 values in water. For Fe8-Tyr-HA5, r1 = 0.11 mM-1s-1 and r2 = 26.20 mM-1s-1 at 7.2 T, while r1 = 0.11 mM-1s-1 and r2 = 52.18 mM-1s-1 at 11.9 T. For Fe8-Tyr-HA16, r1 = 0.39 mM-1s-1 and r2 = 0.10 mM-1s-1 at 7.2 T, while r1 = 0.47 mM-1s-1 and r2 = 0.23 mM-1s-1 at 11.9 T. The relaxivity values were compared to the relaxivities of similar complexes in the current literature. Preliminary in vivo experiments conducted using nude mice have shown that the lead Fe8-Tyr-HA5 conjugate is physiologically compatible and non-toxic in the dose ranges administered. Clear visibility of CA following an intratumoral (direct) injection was observed; clear negative contrast in circulating blood was evident, with attenuation persisting for more than 10 minutes following injection. Moreover, with an intravenous administration heterogeneous signal change within the tumor was evident by 15 minutes after injection and was increasingly conspicuous with time over this window of observations.