Concurrent Dual-Contrast Enhancement Using Fe₃O₄ Nanoparticles to Achieve a CEST Signal Controllability

Traditional T₂ magnetic resonance imaging (MRI) contrast agents have defects inherent to negative contrast agents, while chemical exchange saturation transfer (CEST) contrast agents can quantify substances at trace concentrations. After reaching a certain concentration, iron-based contrast agents can "shut down" CEST signals. The application range of T₂ contrast agents can be widened through a combination of CEST and T₂ contrast agents, which has promising application prospects. The purpose of this study is to develop a T₂ MRI negative contrast agent with a controllable size and to explore the feasibility of dual contrast enhancement by combining T₂ with CEST contrast agents. The study was carried out in vitro with HCT-116 human colon cancer cells. A GE SIGNA Pioneer 3.0 T medical MRI scanner was used to acquire CEST images with different saturation radio-frequency powers (1.25/2.5/3.75/5 μT) by 2D spin echo-echo planar imaging (SE-EPI). Magnetic resonance image compilation (MAGiC) was acquired by a multidynamic multiecho 2D fast spin-echo sequence. The feasibility of this dual-contrast enhancement method was assessed by scanning electron microscopy, transmission electron microscopy, Fourier transform infrared spectroscopy, dynamic light scattering, ζ potential analysis, inductively coupled plasma, X-ray photoelectron spectroscopy, X-ray powder diffraction, vibrating-sample magnetometry, MRI, and a Cell Counting Kit-8 assay. The association between the transverse relaxation rate r₂ and the pH of the iron-based contrast agents was analyzed by linear fitting, and the linear relationship between the CEST effect in different B₁ fields and pH was analyzed by the ratio method. Fe₃O₄ nanoparticles (NPs) with a mean particle size of 82.6 ± 22.4 nm were prepared by a classical process, and their surface was successfully modified with -OH active functional groups. They exhibited self-aggregation in an acidic environment. The CEST effect was enhanced as the B₁ field increased, and an in vitro pH map was successfully plotted using the ratio method. Fe₃O₄ NPs could stably serve as reference agents at different pH values. At a concentration of 30 μg/mL, Fe₃O₄ NPs "shut down" the CEST signals, but when the concentration of Fe₃O₄ NPs was less than 10 μg/mL, the two contrast agents coexisted. The prepared Fe₃O₄ NPs had almost no toxicity, and when their concentration rose to 200 μg/mL at pH 6.5 or 7.4, they did not reach the half-maximum inhibitory concentration (IC₅₀). Fe₃O₄ magnetic NPs with a controllable size and no toxicity were successfully synthesized. By combining Fe₃O₄ NPs with a CEST contrast agent, the two contrast agents could be imaged simultaneously; at higher concentrations, the iron-based contrast agent "shut down" the CEST signal. An in vitro pH map was successfully plotted by the ratio method. CEST signal inhibition can be used to realize the pH mapping of solid tumors and the identification of tumor active components, thus providing a new imaging method for tumor efficacy evaluation.