Redoxable heteronanocrystals functioning magnetic relaxation switch for activatable T₁ and T₂ dual-mode magnetic resonance imaging

Abstract

T₁/T₂ dual-mode magnetic resonance (MR) contrast agents (DMCAs) have gained much attention because of their ability to improve accuracy by providing two pieces of complementary information with one instrument. However, most of these agents are “always ON” systems that emit MR contrast regardless of their interaction with target cells or biomarkers, which may result in poor target-to-background ratios. Herein, we introduce a rationally designed magnetic relaxation switch (MGRS) for an activatable T₁/T₂ dual MR imaging system. Redox-responsive heteronanocrystals, consisting of a superparamagnetic Fe₃O₄ core and a paramagnetic Mn₃O₄ shell, are synthesized through seed-mediated growth and subsequently surface-modified with polysorbate 80. The Mn₃O₄ shell acts as both a protector of Fe₃O₄ in aqueous environments to attenuate T₂ relaxation and as a redoxable switch that can be activated in intracellular reducing environments by glutathione. This simultaneously generates large amounts of magnetically decoupled Mn²⁺ ions and allows Fe₃O₄ to interact with the water protons. This smart nanoplatform shows an appropriate hydrodynamic size for the EPR effect (10–100 nm) and demonstrates biocompatibility. Efficient transitions of OFF/ON dual contrast effects are observed by in vitro imaging and MR relaxivity measurements. The ability to use these materials as DMCAs is demonstrated via effective passive tumor targeting for T₁- and T₂-weighted MR imaging in tumor-bearing mice.

Introduction

Magnetic resonance (MR) imaging has been widely applied in a variety of clinical diagnostic fields as a powerful noninvasive technique [1], [2]. MR imaging is particularly appealing because it can provide anatomical images of tissues and organs with excellent spatial resolution [3], [4]. The diagnostic capabilities of MR imaging can be greatly improved by introducing exogenous medical media, such as T₁ or T₂ contrast agents, for enhanced target tissue contrast on T₁-or T₂-weighted imaging sequences, respectively [5], [6]. T₁ contrast agents, represented by clinically-available gadolinium (Gd³⁺) chelates, are paramagnetic materials, which shorten the longitudinal relaxation time (T₁) of nearby water protons [7], [8]. However, despite their ability to generate a positive (bright) contrast with high signal intensity, the use of T₁ contrast agents is compromised by their lack of sensitivity and intrinsic low MR relaxivity [9]. Alternatively, T₂ contrast agents commonly consist of superparamagnetic nanoparticles, such as superparamagnetic iron oxide nanoparticles (SPION), and can be magnetically saturated by the typical magnetic field strengths in MRI scanners [2], [10]. These agents work by shortening the traverse relaxation time (T₂) of water protons in their vicinity [11], [12]. While they have high detection sensitivity for lesions, their magnetic susceptibility to artifacts and inherent negative (dark) contrast effects can induce low signal-to-noise ratios, which limits their application [13], [14]. Consequentially, MR contrast agents that integrate both T₁ and T₂ contrast capabilities are highly desirable for advanced MR imaging techniques [14], [15]. These dual-mode agents are able to overcome the limitations of individual T₁ and T₂ imaging modalities by improving the accuracy. This is done by validating the reconstruction and visualizing the data that is simultaneously provided by two complementary sources of information within a single instrument [15], [16].

Recent investigations into T₁/T₂ dual-mode contrast agents (DMCAs) have evolved to combine T₁ and T₂ contrast agents into a single nanoprobe [13], [16], [17], [18], [19]. In this case, severe proton relaxation interference between the two different contrast agents (magnetic coupling), which attenuates the T₁ contrast effect by the strong local magnetic field of the superparamagnetic T₂ material, is inevitable when they are in close proximity to one another [15], [16]. Several designs have demonstrated that inserting distance modulators between the T₁ and T₂ materials, such as silica shells [13], [15] or inorganic linkers [16], can reduce the undesirable magnetic coupling effect. For those nanoplatforms, however, fastidious processing of the nanoparticles and well-defined DMCA design are required to avoid quenching the T₁ and T₂ contrast effects. Furthermore, most of the contrast agents are “always ON” systems, exerting the MR contrast effect regardless of their proximity or interaction with target cells or environmental markers in biology; this can result in poor target-to-background ratios [20], [21].

The development of inventive imaging strategies based on stimuli-responsive smart nanosystems with high T₁ and T₂ relaxivity would be an ultimate solution and enable accurate imaging of biological targets. In particular, the redox potential variation induced by concentration changes of reducing agents across different intra/extracellular regions has been one of the most extensively employed biological triggers for stimuli-responsive imaging nanoparticles [22], [23]. Glutathione (GSH) is an abundant reducing agent in cytoplasm (1∼15 mM), which plays a central role in cell growth and function [24], [25]; the fact that cancer cells have higher GSH concentrations than corresponding normal cells enables redox-responsive nanoplatforms to be promising cancer diagnostic probes [24], [26]. Therefore, the introduction of a redox-responsive moiety that can cage T₁ and T₂ contrast agents in a magnetically coupled state and initially shield them from water molecules would greatly improve the MR signal transition in stimuli-responsive DMCA systems for cancer diagnosis.

In this study, we developed an activatable T₁/T₂ dual-mode imaging probe with a magnetic relaxation switch (MGRS) that exhibits high sensitivity and effective silencing/activation of the MR contrast effect in target conditions. This was accomplished by constructing heteronanocrystals that consist of a superparamagnetic core and a redox-responsive paramagnetic shell. For a proof of concept, we designed a Mn₃O₄-coated Fe₃O₄ hybrid nanocrystal that is encapsulated within amphiphilic polyethylene glycol (PEG) derivatives; we refer to these crystals as redox-responsive activatable nanostarshells (RANS). They can be activated under an intracellular reducing environment for ultrasensitive bimodal MR imaging in vivo. To date, various Mn-based materials, such as Mn chelates [27], MnO nanoparticles [28], and hybrid nanomaterials [11], [29], have been applied for MR imaging with good biocompatibility. However, the application of a redoxable Mn₃O₄ layer as the MGRS of a DMCA has not been reported. The schematic illustration in Fig. 1 depicts the design of RANS and its operation mechanism. The Fe₃O₄ core of RANS is initially shielded from the aqueous environment by the Mn₃O₄ shell. The Mn center is also restricted within the Mn₃O₄ structure, resulting in low accessibility to water and magnetic coupling with the superparamagnetic core [30]; the T₁ and T₂ contrast effects are quenched (“OFF” state). After accumulation of RANS on the tumor tissue, which is caused by the enhanced permeation and retention (EPR) effect, and subsequent internalization into the tumor cells, the Mn₃O₄ shells are dissolved into Mn²⁺ ions by a redox reaction (“redox-mediated peeling”) in the presence of abundant GSH in the cytoplasm. Numerous high-spin Mn²⁺ ions and exposed Fe₃O₄ cores can individually serve as MR contrast agents; these can be collectively activated to generate a dramatic enhancement in the T₁ and T₂ signal contrast (“ON” state). The heteronanocrystals were synthesized by sequential seed-mediated growth using an iron precursor and a manganese precursor through a thermal decomposition method. Using an emulsion and solvent evaporation method, these nanocrystals were subsequently transferred into an aqueous solution using polysorbate 80 (P80) as a biocompatible capping molecule. The transition efficacy of the OFF/ON dual contrast effects and the potential usefulness of RANS as a DMCA were confirmed through in vitro and in vivo experiments.