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High-resolution pH imaging with ratiometric CEST and BIRDS using dual paramagnetic DOTA–tetraglycinate agents
Jelena Mihailovic1,2, Yuegao Huang1, John Walsh1, Daniel Coman1, Sara Samuel3, and Fahmeed Hyder1,3
1(1)Magnetic Resonance Research Center (MRRC), Yale University, New Haven, CT, United States, 2(2)Department of Diagnostic Radiology, Yale University, New Haven, CT, United States, 3Core Center for Quantitative Neuroscience with Magnetic Resonance (QNMR), Yale University, New Haven, CT, United States

Synopsis

Chemical Exchange Saturation Transfer (CEST) and Biosensor Imaging of Redundant Deviation in Shifts (BIRDS) biosensing methods differ respectively by detecting exchangeable and non-exchangeable protons on the agent. Given that CEST and BIRDS properties observed from the same paramagnetic agent are complimentary, we describe a novel approach for high-resolution pH imaging using dual agents of europium and thulium complexed with DOTA-tetraglycinate. In vitro results test the hypothesis that ratiometric paraCEST attributes are conserved when temperature from paraBIRDS is detected simultaneously, enabling absolute pH imaging. In vivo results in glioblastoma demonstrate feasibility of this dual paraCEST-paraBIRDS biosensing method for high-resolution pH imaging.

Introduction

MRI contrast can be improved by enhancing relaxation with paramagnetic agents(1,2).Despite great success of the gadolinium-based-agents in clinical MRI, the generated contrast is always “on” when the agent is present, and thus the approach lacks sensitivity and specificity. A novel class of responsive agents become available with chemical exchange saturation transfer (CEST) MRI(3,4), where exchange of water protons and hydroxyl/amine/amide protons are intrinsically pH and temperature dependent. The development of many paramagnetic CEST (paraCEST) agents has proceeded with the aim of pushing the chemical shift of exchangeable protons further away from the bulk water proton signal, thereby reducing direct saturation of water protons. Today many lanthanide-based paraCEST agents are used for molecular imaging(5,6). Although ratiometric paraCEST has shown some promise for molecular imaging, CEST imaging with multiple agents can also be improved accounting for the ambient conditions (e.g., temperature) to tease out the fact that any CEST contrast is intrinsically pH dependent. In addition to the exchangeable protons of the paraCEST agents, the nonexchangeable proton resonances emanating from the same paramagnetic agent are also utilized by Biosensor Imaging of Redundant Deviation in Shifts (BIRDS)(7,8)for biosensing which is a 3D chemical shift imaging (CSI) method. Various paramagnetic BIRDS (paraBIRDS) agents have been applied to map temperature and/or pH in vivo(9,10). In the present work, we hypothesize that pH imaging using ratiometric paraCEST can be calibrated by temperature mapping from paraBIRDS, where both signals are emanating from the same two agents in the same voxels.

Methods

A series of phantoms of TmDOTA-(gly)4- and EuDOTA-(gly)4- and their mixture (Macrocyclics, Dallas, TX) were prepared using 10 mM phosphate buffer at various pH values (6.4-7.9) with 10% D2O for BIRDS and CEST experiments. All NMR characterization experiments were acquired with an 11.7 T vertical magnet (Bruker, Billerica, MA). BIRDS properties for individual and the mixture samples were recorded by acquiring proton spectra at various temperatures (27-40 °C). CEST experiments were collected with 4s continuous wave saturation pulse (16.7µT) over a range of frequencies (± 100 ppm). The same phantoms were used for BIRDS and CEST imaging on a 11.7T horizontal bore spectrometer using a volume Birdcage coil (4 cm), field of view 32 × 32 mm. CSI experiments were acquired using 25 x 25 encoding steps, TR=10 ms. A 600 µs Gaussian pulse was used for excitation. CEST images were acquired using a spin-echo imaging sequence with image matrix of 64 × 64. The temperature was controlled by circulating hot air in the magnet bore, monitored by thermocouple. The same experimental setup described above was used for in vivo study. All animal experimental procedures on rats were approved by the Institutional Animal Care and Use Committee (IACUC). Fischer 344 rats received an implantation of RG2 glioma cells. The tail vein was used for injection of TmDOTA-(gly)4- and EuDOTA-(gly)4- mixture (1mmol/kg/hour). A water-heating blanket was used to control and maintain the body temperature.

Results

Proton NMR spectra of TmDOTA-(gly)4- and EuDOTA-(gly)4-, and their equivalent mixture show characteristic hyperfine shifts for nonexchangeable proton resonances (Figure 1A-C). As a result, proton resonances Tm-H2 and Tm-H6 in TmDOTA-(gly)4- and Eu-H4 in EuDOTA-(gly)4- are chose for BIRDS characterization (Figure 1D-F). The amide protons in TmDOTA-(gly)4- resonating at -52.5 ppm, while the bound water protons in EuDOTA-(gly)4- resonating at 51.5 ppm at 35 °C are indicated by CEST spectra (Figure 2). To determine the pH, BIRDS was used to read the temperature and the ratio of the CEST intensities between TmDOTA-(gly)4- and EuDOTA-(gly)4- traced pH dependency. Temperature maps from TmDOTA-(gly)4- and EuDOTA-(gly)4- were used to generate the average temperature maps of the mixture (Figure 3A) and CEST maps were used to create the ratiometric CEST maps in the mixture at different temperatures (Figure 3B). 3D surface plot (Figure 4) for pH determination was then fitted as function of temperature (T) and paraCEST ratio (R): pH=a+bR+cT+dR2+eT2+fRT+gR3+hT3+iRT2+jR2T In vivo experiment for rat’s brain tumor is shown on Figure 5A-C. Temperature mapping with BIRDS show an average temperature of 35.6±0.8°C inside the tumor, while ratiometric paraCEST imaging readout show a ratio of 2.0±0.2. The calculated pH map from ratiometric paraCEST calibrated with BIRDS indicate an average pH value of 7.0±0.7 which reflects the physiological microenvironment of solid tumors characterized by extracellular acidity.

Discussion

In this study, TmDOTA-(gly)4- and EuDOTA-(gly)4- were used as a model system of multivalent agents to demonstrate ratiometric imaging with paraCEST that is calibrated from temperature readout with paraBIRDS, enabling the pH-dependence of CEST contrast to be extrapolated for quantitative pH values. BIRDS properties of multivalent agents are not perturbed, whereasthe CEST properties are altered. Moreover, the temperature sensitivities in BIRDS can be enhanced in the mixture of two agents because of the increased redundancy of reporting. We show that the ratiometric CEST is temperature dependent, which can be calibrated with BIRDS, where the temperatures measured with BIRDS could be combined with the ratiometric CEST for accurate temperature-independent pH reporting. As we have shown in vivo, pH readouts vary with temperature.

Conclusions

High spatial resolution pH maps in rat brain tumors can be obtained using ratiometric paraCEST, with temperature calibrated by paraBIRDS. This paraCEST/paraBIRDS-based temperature-independent pH mapping technique can be used for longitudinal monitoring of therapeutic response in tumors, where temperatures might be different between pathological and non-pathological tissue.

Acknowledgements


References

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5. Evbuomwan, O. M, et al. CEST and PARACEST Agents for Molecular Imaging. In The Chemistry of Molecular Imaging 2014. pp. 225-243.

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7. Coman, D., H.K. Trubel, and F. Hyder, Brain temperature by Biosensor Imaging of Redundant Deviation in Shifts (BIRDS): comparison between TmDOTP5- and TmDOTMA. NMR Biomed, 2010. 23(3): p. 277-85.

8. Coman, D., et al., Brain temperature and pH measured by (1)H chemical shift imaging of a thulium agent. NMR Biomed, 2009. 22(2): p. 229-39.

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Figures

Chemical structures, 1H spectra and BIRDS characterization of the paramagnetic agents (A) TmDOTA-(gly)4- and (B) EuDOTA-(gly)4-. Exchangeable protons are highlighted. 1H spectra for (E) TmDOTA-(Gly)4-, (D) EuDOTA-(Gly)4-, and (C) the mixture of TmDOTA-(Gly)4- and EuDOTA-(Gly)4- at 11.7 T, 35 °C, and pH 7.4. The proton resonances for BIRDS characterization are labeled and temperature and pH dependencies of proton chemical shifts are shown on the right-hand side of the corresponding spectra. The 3D surfaces represent the fits of chemical shifts as function of temperature and pH.

Dependence of CEST Z-spectra on pH at 35 °C (A-C) and on temperature at pH 7.4 (D-F) for TmDOTA-(Gly)4-(A and D), EuDOTA-(Gly)4-(B and E) and the mixture of TmDOTA-(Gly)4- and EuDOTA-(Gly)4- (C and F). The CEST effect for TmDOTA-(gly)4- show increasing as the pH increases, while for EuDOTA-(gly)4- the pH effect is negligible. For the mixture (C) the pH effect is similar to that observed in single agent. Increasing the temperature has, in all samples, peaks are shifted toward the water resonance, the TmDOTA-(Gly)4- CEST intensities increase and the EuDOTA-(Gly)4- intensities decrease.

Temperature (A) and CEST ratiometric mapping (B) for mixtures containing 10 mM TmDOTA-(Gly)4- and 10 mM EuDOTA-(Gly)4- at different pHs (from left to right tubes 6.43, 6.86, 7.13, 7.42, 7.74) and different temperatures.

3D surface plot in mixture samples showing that the pH can be obtained from the temperature measured with BIRDS and CEST ratio measured with paraCEST. The CEST ratio was obtained from the CEST peaks of amide protons in TmDOTA-(gly)4- and bound water in EuDOTA-(gly)4-. The temperatures were determined with BIRDS from the chemical shift differences between protons H2 and H6 in TmDOTA-(gly)4-, and H4 in EuDOTA-(gly)4-.

In vivo pH imaging with ratiometric paraCEST calibrated using BIRDS. The tumor boundary is indicated in black. Temperature mapping with BIRDS show an average temperature of 35.6±1.0°C inside the tumor (A). Ratiometric paraCEST imaging readout show a ratio of 2.0±0.2 (B). The calculated pH map from ratiometric paraCEST calibrated with BIRDS indicate an average pH value of 7.0±0.8 (C).

Proc. Intl. Soc. Mag. Reson. Med. 28 (2020)
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