Single ¹⁹F Probe for Simultaneous Detection of Multiple Metal Ions Using miCEST MRI

Abstract

The local presence and concentration of metal ions in biological systems has been extensively studied ex vivo using fluorescent dyes. However, the detection of multiple metal ions in vivo remains a major challenge. We present a magnetic resonance imaging (MRI)-based method for noninvasive detection of specific ions that may be coexisting, using the tetrafluorinated derivative of the BAPTA (TF-BAPTA) chelate as a ¹⁹F chelate analogue of existing optical dyes. Taking advantage of the difference in the ion-specific ¹⁹F nuclear magnetic resonance (NMR) chemical shift offset (Δω) values between the ion-bound and free TF-BAPTA, we exploited the dynamic exchange between ion-bound and free TF-BAPTA to obtain MRI contrast with multi-ion chemical exchange saturation transfer (miCEST). We demonstrate that TF-BAPTA as a prototype single ¹⁹F probe can be used to separately visualize mixed Zn²⁺ and Fe²⁺ ions in a specific and simultaneous fashion, without interference from potential competitive ions.

A major challenge in the biomedical sciences is to monitor, characterize, quantify, and understand the multiplexity of biological events in vivo. Advanced imaging methodologies are being developed to visualize multiple biological changes simultaneously within the same anatomical frame. One strategy is the use of multimodal imaging approaches, where more than one imaging methodology is used to obtain information from multiple targets.¹⁻⁶ However, the complexity of coregistering the obtained information into an accurate spatial representation calls for probing multiple targets using a single imaging approach.

Metal ions play a pivotal role in nearly all biological processes, and deviation from normal levels is often associated with disease onset and progression.⁷ Today, our knowledge of the role of metal ions in biology is mostly based on the use of optical dyes,⁸ originally developed by Roger Tsien.^9,10 Although optical dyes have made an enormous contribution to an understanding of the role of metal ions in biological systems, the optical signal from fluorescent dyes limits their applications to in vitro studies or monitoring of surface phenomena in vivo using superficially injected dyes.¹¹ To overcome these limitations, magnetic resonance imaging (MRI) has been explored as a whole-body, noninvasive imaging technique to sense changes in metal ion levels in vivo.¹²⁻¹⁴ However, currently available probes are designed to alter the T₁ and T₂ proton relaxation rates upon binding to the metal ion of interest,¹⁵⁻¹⁷ where interpreting images and quantifying local metal levels may be difficult as this approach is not specific: changes in T₁ or T₂ may result from other sources, while the background contrast without the presence of metals is often unknown. The specific chemical shifts (Δω) of nuclear magnetic resonance (NMR)-detectable nuclei (e.g., ¹⁹F or ¹²⁹Xe) in a synthetic probe upon metal ion binding provide ultimate specificity with regard to the ion of interest.¹⁸⁻²¹ Unfortunately, NMR spectroscopy-based approaches do not provide spatial information on the location of the investigated ion and rely on the identification and integration of a specific NMR peak that may fall below a detectable signal to noise ratio (SNR).

Chemical exchange saturation transfer (CEST) imaging²²⁻²⁶ is an MRI contrast mechanism that enables the detection of low concentration solutes via the transfer of their magnetization to the bulk (high concentration) nuclei, from which the MRI signal is derived. Using ¹H as the bulk nucleus, CEST MRI has been used in a wide range of applications,²²⁻²⁶ including simultaneous imaging of different probes, based on their different Δω values.^27,28 In our previously suggested approach, which we termed ion CEST (iCEST),²⁹ a combination of ¹⁹F MRI and CEST was used to spatially monitor Ca²⁺ with high specificity, capitalizing on the dynamic exchange between the ion-bound and free ¹⁹F chelate, and the shift in the Δω of ¹⁹F upon ion binding. By using 5,5′,6,6′-tetrafluoro-BAPTA (TF-BAPTA) as the ¹⁹F iCEST probe (Figure 1a), we demonstrate here that Zn²⁺ and Fe²⁺ ions can be detected specifically and simultaneously.

Figure 1.

(a) Chemical structure of TF-BAPTA showing the ¹⁹F atom substituents on the 5 (purple) and 6 (green) positions. (b) ¹⁹F NMR spectrum (470 MHz) of 5 mM TF-BAPTA (20 mM Hepes buffer, pH = 7.2) in the presence of 0.5 mM Zn²⁺ or Fe²⁺.

It has been previously demonstrated that different ¹⁹F-BAPTA derivatives have different Δω values in their respective ¹⁹F NMR spectra and variable K_d properties for various metal ions.¹⁹ Because of the fact that free TF-BAPTA exchanges too rapidly (k_ex ≈ 10,000 s^–1) with Ca²⁺-bound TF-BAPTA³⁰ to be useful for generating iCEST contrast, we hypothesized that it could be used for the detection of other metal ions. Figure 1b shows the ¹⁹F NMR spectrum of TF-BAPTA in the presence of either Zn²⁺ or Fe²⁺ (10:1 molar ratio). The Δω of the ¹⁹F atoms at 5- (purple) and 6-positions (green) are shifted downfield and upfield, respectively, in the presence of either of the ions, with a larger effect for the paramagnetic Fe²⁺. One of the potential drawbacks of ¹⁹F MRI using 5F-BAPTA is a possible line broadening of the bulk signal of the free ligand in vivo in live tissue,¹⁹ such as seen when high amounts of Mg²⁺ are added (Figure S1, Supporting Information (SI)). As a result, images with reduced SNR may be experienced and smaller observed Δω values may not be sufficient for selective saturation of poorly shifted nuclei without direct bulk saturation. However, as also previously demonstrated,³⁰ the fast exchange between Ca²⁺ and TF-BAPTA broadens the peak that is related to the 5-positioned ¹⁹F atom and does not affect the NMR characteristics of the 6-positioned ¹⁹F atom (Figure S2, SI). Additionally, a high Mg²⁺ concentration does not affect the NMR properties of 5F and 6F atoms of TF-BAPTA (Figure S2, SI), making the latter a suitable ¹⁹F MRI probe in a biological setup. The 6-positioned ¹⁹F atom for the signal of the bulk (in ¹⁹F-CEST experiments) is thus preferable since it does not broaden due to ion exchange. The two frequencies that are observed in the ¹⁹F NMR spectrum of TF-BAPTA require the center frequency offset (O₁) to be placed at the resonance of one of these frequencies when performing ¹⁹F MRI. Therefore, all ¹⁹F MRI experiments in this study were performed with O₁ set at the frequency of the 6-positioned ¹⁹F, while the signal from the 5-positioned ¹⁹F was suppressed using a spectrally selective excitation pulse and spoiler gradient.

Figure 2b,c shows the ¹H and ¹⁹F MR images of seven tubes containing 10 mM TF-BAPTA and 200 μM added ion, without any observable changes in ¹H or ¹⁹F MR contrast. However, the ¹⁹F iCEST images show a clear differential MR contrast between the samples containing Zn²⁺ (Figure 2d) and Fe²⁺ (Figure 2e), for a saturation pulse applied at Δω = −2.8 and −18 ppm, respectively. These Δω values were chosen from the ¹⁹F NMR spectra, using the offset values of TF-BAPTA upon the addition of Zn²⁺ or Fe²⁺, respectively (see Figure 1b). Figure 2f clearly shows that both ions can be simultaneously visualized using TF-BAPTA as a single iCEST probe. Figure 3 shows the corresponding ¹⁹F iCEST spectra for samples containing either Zn²⁺ (Figure 3a) or Fe²⁺ (Figure 3b). The dynamic ¹⁹F exchange between TF-BAPTA and [M²⁺-TF-FBAPTA] results in an iCEST effect for both ions, at Δω = −2.8 ppm for [Zn²⁺-TF-BAPTA] and at Δω = −18 ppm for [Fe²⁺-TF-BAPTA], respectively. Using Bloch simulations (Figure 3a,b), the exchange rate (k_ex) between free and bound TF-BAPTA is estimated to be ∼20 s^–1 for both ions. This k_ex is rather low, and much higher CEST contrast may be obtained for ¹⁹F chelates with higher k_ex values. Despite this slow exchange, we were still able to detect 10% CEST contrast for a 200 μM ion concentration with the sensitivity from a 10 mM signal strength. The use of ¹⁹F based CEST enables a reduction in the concentration of the ¹⁹F iCEST probe to a biological relevant molar ratio (probe: ion), a feat that is not possible with ¹H CEST, which is based on water. Additionally, ¹⁹F enables “hot spot” tracer detection without an endogenous background signal,³¹ contrary to ¹H CEST, which suffers from a large nonspecific endogenous background signal. This may further reduce the ¹⁹F probe concentration to below 10 mM, alleviating potential toxicity effects from Ca²⁺ buffering. Although TF-BAPTA did not show a significant buffering effect for intracellular Ca²⁺,³⁰ further studies are needed prior to its use in vivo. Importantly, when balanced salt solutions containing physiological levels of other ions (1.3 mM Ca²⁺, 0.9 mM Mg²⁺, 5.9 mM K⁺, and 143 mM Na⁺) and glucose (6 mM) were used, the iCEST effect from Zn²⁺ was not affected (Figure S3, SI). This is a great advantage for the use of TF-BAPTA as an iCEST probe compared to 5F-BAPTA, which exchanges much faster with other metal ions, causing broadening of the bulk signal in the ¹⁹F NMR spectrum, limiting its applications.

Figure 2.

iCEST maps. (a) The orientation of the samples in the phantom containing 10 mM TF-BAPTA and 200 μM ion (pH = 7.2). (b) ¹H MRI, (c) ¹⁹F MRI, (d) iCEST (Δω = −2.8 ppm) overlaid on ¹⁹F MRI, (e) iCEST (Δω = −18 ppm) overlaid on ¹⁹F MRI, and (f) both iCEST results (Δω = −2.8 ppm, Δω = −18 ppm) overlaid on ¹⁹F MRI.

Figure 3.

¹⁹F iCEST spectra for samples containing 10 mM TF-BAPTA and 200 μM Zn²⁺ (a) and Fe²⁺ (b). Circles represent experimental signal; solid lines represent Bloch simulations (two-pool model).

The unique and different Δω value of the exchangeable moiety is one of the most exceptional characteristics of iCEST compared to other MRI sensors. This feature gives CEST sensors an artificial color designation, by which they can be tagged in a singular specific frequency, much like fluorescent dyes. For ¹H CEST, this has been exploited for “multi-color” MRI of live cells²⁸ and in vivo.²⁷ Here (Figure 4) we investigated whether Zn²⁺ and Fe²⁺ could be distinguished from each other when mixed together and with other ions. When a saturation pulse was applied at the resonance of the Zn²⁺-TF-BAPTA complex (i.e., Δω = −2.8 ppm), only the tubes that contained Zn²⁺ ions generated an observable iCEST contrast. The contrast did not change when competing ions, such as Ca²⁺, Mg²⁺, or Fe²⁺, were included in the sample solution. Similarly, when the saturation pulse was applied at Δω = −18 ppm (Δω of Fe²⁺-TF-BAPTA), only the samples that included Fe²⁺ generated iCEST contrast, without interference from the other coexisting ions Ca²⁺, Mg²⁺, or Zn²⁺. Notably, when both Zn²⁺ and Fe²⁺ ions were mixed with TF-BAPTA (center tube in Figures 4a–d), the iCEST contrast could be obtained at both Δω values of Zn²⁺-TF-BAPTA (−2.8 ppm) and Fe²⁺-TF-BAPTA (−18 ppm). The unique ability to detect two different ions using a single imaging probe (TF-BAPTA) is clearly reflected in the two distinctive peaks that were obtained in the iCEST spectra (Figure 4e). These experimental results were further supported by Bloch simulations using a three-pool model (Figure 4e).

Figure 4.

Simultaneous detection of multiple metal ions. (a) ¹⁹F MRI and the orientation of the tubes in the phantom containing 10 mM of TF-BAPTA and 200 μM of mixed ions, (b) iCEST (Δω = −2.8 ppm), and (c) iCEST (Δω = −18 ppm) overlaid on ¹⁹F MRI. (d) Merged iCEST image highlights (orange–purple scale) the shared iCEST contrast voxels shown in panels b and c. (e) ¹⁹F iCEST spectra. Circles represent experimental signal; lines represent Bloch simulations (three-pool model).

The capability of detecting ¹⁹F probes at sub millimolar concentrations,³² the high sensitivity of the ¹⁹F NMR spectrum Δω values to changes in the chemical environment,³³ together with the frequency being specific of these Δωs for certain metal ions, should inspire further development of novel responsive contrast agents for iCEST MRI. One strategy that allows a local increase of the ¹⁹F probe concentration and eliminating the need of systemic administration is to coencapsulate the imaging probe with the transplanted target cells.³⁹ For example, such an approach may be used for the detection of transplanted β cells that release Zn²⁺ upon the release of insulin.¹² By adding ¹⁹F atoms to the two 6-positions of 5F-BAPTA (which previously allowed the detection of only Ca²⁺ using iCEST^29,34) it became possible to detect both Zn²⁺ and Fe²⁺. Adding one ¹⁹F atom to the BAPTA backbone dramatically changes the binding properties of TF-BAPTA.³⁵ At the same time, the added ¹⁹F atom induces k_ex values that allow the detection of Zn²⁺ and Fe²⁺ with ¹⁹F iCEST MRI. Although other ¹H MRI probe can be used to detect Zn^2+12,36−38 with a potential higher sensitivity as compared to ¹⁹F probes, the specificity of iCEST to simultaneously detect different coexisting ions using the same sensor represents a new concept for the rational design of novel MRI probes. While BAPTA derivatives are widely used for the fluorescent detection of metal homeostasis in vitro, the possibility to probe metals in vivo noninvasively with MRI would have profound implications for the biological sciences.

Supporting Information Available

Experimental methods and figures. This material is available free of charge via the Internet at http://pubs.acs.org.

The authors declare no competing financial interest.

Funding Statement

National Institutes of Health, United States