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Application of a Stimuli‐Responsive Polymer to the Development of Novel MRI Probes

Magnetic resonance imaging is a medical diagnostic tool that is attracting considerable attention as a molecular-imaging technique; this is because MRI is a noninvasive imaging technique that provides information about the internal structure of living organisms. Recently, various chemically modified MRI contrast agents have been developed in order to study in vivo phenomena. These contrast agents, termed “MRI probes”, have various functions and include probes that detect enzyme activities, variations in pH, the ionic concentration, and so on. In order to change the probes’ relaxivities by external stimuli, probes were designed to accommodate a certain number of bound water molecules per gadolinium ion. Other probes involved the attachment of macromolecules, such as proteins, to change the molecular mobility of the probe. However, the rational development of such MRI probes is at an early stage, and the strategies for developing MRI probes are limited. In order to design novel MRI probes, we focused our attention on stimuli-responsive polymers, which can change their conformation in response to external stimuli such as chemicals, temperature, and pH. Stimuli-responsive polymers that exhibit large conformational changes have been developed for applications in drug-delivery systems, functional polymeric matrices, and artificial tissues. To the best of our knowledge, there has been no report regarding the application of stimuli-responsive polymers to the development of MRI probes. In this study, a novel MRI probe was developed by employing a strategy based on conformational changes in stimuli-responsive polymers. We demonstrated that this principle of probe design is useful in detecting pH changes. Additionally, we investigated in detail the stimuli-responsive mechanism of the probe. pH-responsive polymers were chosen for the development of the probe due to their high potential in clinical applicability for monitoring diseases such as kidney disorder and cancer. The relaxivities of MRI probes are influenced by their rotational motions; therefore, if a pH-responsive conformational change in the probe induces rotational mobility of the Gd complexes attached to the probe, pH variations would lead to alterations in the probe relaxivity. We designed a pH-responsive MRI probe, P-Gd, by attaching clinically used Gd complexes and dansyl chromophores to a pH-responsive polymer, n-octylamine-modified poly(sodium maleate-alt-ethyl vinyl ether) (poly(SM-EVE); Figure 1). At a low pH, n-octylamine-modified poly(SM-EVE) takes on a compact globule conformation because of the intramolecular hydrophobic interactions among the n-octyl groups. In contrast, at a high pH, the polymer conformation expands because of the electrostatic repulsion among the deprotonated carboxy groups. Therefore, it is assumed that the molecular rotation of the side chains of the polymer is restricted at low pH, and that the side chains in the expanded conformation rotate more rapidly at a high pH. In order to elucidate the pH-dependent changes in the polymer shape, we synthesized an aminoethyl-modified Gd(DO3A) complex (Gd(AEDO3A)) and dansyl ethylenediamine, as previously described by Li et al. and Shea et al. , respectively. Platform polymer poly(SM-EVE) anhydride was synthesized and then modified with n-octylamine according to the scheme reported by Hu et al. After attaching the Gd complexes and the dansyl chromophores to the n-octylamine-modified polymers, the residual carboxylic anhydride groups in the polymer were hydrolyzed with 1 m aqueous NaOH. The polymers were dialyzed and lyophilized to yield P-Gd (Scheme S1 in the Supporting Information). Constituents of octyl groups and Gd were estimated by using elemental analysis and ICP-MS. The approximate molecular weight of the platform polymer was determined by GPC after it was hydrolyzed by the alkaline aqueous solution. Thereafter, in order to demonstrate the possibility of using P-Gd as a pH-responsive MRI probe, the relaxivities of P-Gd at pH 4–10 were measured. The longditudinal and transverse relaxivities (r1 and r2, respectively) increased as the pH decreased and were maximal at pH 4, while the relaxivities of Gd(AEDO3A) did not increase (Figure 2). This result is consistent with the expected pH-responsive behavior of n-octylaminemodified poly(SM-EVE). It is assumed that the limited rotational motion of the side chains caused the increase in relaxivities at a low pH. By replacing the carboxy groups of the polymer side chains with functional groups whose pKa is near to physiological pH, such as sulfonamide, P-Gd could allow pathological tissues in which the extracellular pH is acidic to be imaged. The mechanism of variation in relaxation time was investigated as follows. In order to verify the conformational change in P-Gd, its fluorescence spectra were measured. The dansyl group is known to be an environmentally sensitive chromophore. The fluorescence emission spectra of the dansyl group exhibit a blue shift and their intensity increases as the group’s surroundings become hydrophobic. Therefore, we can obtain information about the conformation of P-Gd from the emission spectra of the dansyl groups attached to the polymer side chains. As the solution pH decreased, the emission spectra of P-Gd exhibited a shift toward shorter wavelengths with an increase in the fluorescence intensity, while the emission spectra of N-acetyl-N’-dansyl ethylenediamine, which is a monomer [a] S. Okada, Dr. S. Mizukami, Prof. K. Kikuchi Graduate School of Engineering, Osaka University 2–1 Yamadaoka, Suita City, Osaka 565-0871 (Japan) Fax: (+ 81) 6-6879-7875 E-mail : kkikuchi@mls.eng.osaka-u.ac.jp Supporting information for this article is available on the WWW under http ://dx.doi.org/10.1002/cbic.200900775.