Cryptophane-xenon complexes for 129Xe MRI applications

Abstract

The use of magnetic resonance imaging (MRI) and spectroscopy (MRS) in the clinical setting enables the acquisition of valuable anatomical information in a rapid, non-invasive fashion. However, MRI applications for identifying disease-related biomarkers are limited due to low sensitivity at clinical magnetic field strengths. The development of hyperpolarized (hp) ¹²⁹Xe MRI/MRS techniques as complements to traditional ¹H-based imaging has been a burgeoning area of research over the past two decades. Pioneering experiments have shown that hp ¹²⁹Xe can be encapsulated within host molecules to generate ultrasensitive biosensors. In particular, xenon has high affinity for cryptophanes, which are small organic cages that can be functionalized with affinity tags, fluorophores, solubilizing groups, and other moieties to identify biomedically relevant analytes. Cryptophane sensors designed for proteins, metal ions, nucleic acids, pH, and temperature have achieved nanomolar-to-femtomolar limits of detection via a combination of ¹²⁹Xe hyperpolarization and chemical exchange saturation transfer (CEST) techniques. This review aims to summarize the development of cryptophane biosensors for ¹²⁹Xe MRI applications, while highlighting innovative biosensor designs and the consequent enhancements in detection sensitivity, which will be invaluable in expanding the scope of ¹²⁹Xe MRI.

Fig. 1. Chemical structure and ¹²⁹Xe NMR chemical shifts of cryptophanes with varying alkoxy linker length in 1,1,2,2-tetrachloroethane-d₂ (1–4) and of a cryptophane functionalized with six [(η5-C₅Me₅)Ru^II]⁺ moieties (5) in D₂O.

Fig. 2. Top: Chemical structure of a biosensor comprised of cryptophane-A, a solubilizing peptide and a tether linking the construct to biotin. Bottom: ¹²⁹Xe NMR spectrum showing the ¹²⁹Xe(aq) signal at ca. 193 ppm and the bound ¹²⁹Xe signals at ca. 70 ppm. The peak corresponding to ¹²⁹Xe encapsulated by this biosensor shifts from ca. 70.2 to 72.5 ppm upon addition of avidin to solution. Reproduced with permission from ref. . Copyright 2001 National Academy of Sciences.

Fig. 3. Top: Mechanism of the hyper-CEST NMR experiment. Hp ¹²⁹Xe atoms (green) bind to the interior of the cryptophane cage, and are depolarized (orange) by a selective rf pulse. The exchange of bound ¹²⁹Xe out of the cage causes the depolarization of bulk ¹²⁹Xe nuclei. This scheme was modified with permission from ref. . Copyright 2014 Wiley-VCH. Bottom: Representative ¹²⁹Xe hyper-CEST NMR z-spectrum of a cryptophane host. The individual peaks represent the intensity of the ¹²⁹Xe signal as a function of the frequency at which rf pulses are applied and the black trace represents the collective z-spectrum.

Fig. 4. Top: Chemical structure of water-soluble EALA-cryptophane (WEC). Bottom: Hyper-CEST ¹²⁹Xe NMR of 5–10 μM WEC with 1 × 10⁷ HeLa cells per mL at pH 7.5 (a and b) and pH 5.5 (c and d). Reproduced with permission from ref. . Copyright 2015 American Chemical Society.

Fig. 5. Top: Scheme of biosensor for H₂S, activatable via both fluorescence and ¹²⁹Xe MRI modalities. Bottom: ¹²⁹Xe MRI images of an NMR tube containing 100 μM biosensor (A) and 100 μM biosensor with 10 equiv. HS⁻ (B). Reproduced with permission from ref. . Copyright 2017 Wiley-VCH.

Fig. 6. Top: Chemical structure of fluorescein-conjugated cryptophane (CrA-FAM). Bottom: ¹²⁹Xe hyper-CEST MRI with underlying ¹H MRI images of mouse fibroblasts labeled with CrA-FAM (15 μM intracellular concentration) and control cells, encapsulated in alginate beads. Laser scanning microscopy images of each cell population are shown, with CrA-FAM accumulated in cells in green and dead cells stained with ethidium homodimer III (EthD-III) in red. Reproduced with permission from ref. . Copyright 2014 Wiley-VCH.

Fig. 7. (a) Scheme of bacteriophage–cryptophane biosensor assembly. The capsid interior of bacteriophage MS2 was modified with cryptophane-A conjugated to maleimide and five glutamic acids to increase solubility (CryA-Mal, green circle) at ca. 110 N87C positions. The capsid exterior was modified with 54 units of the TD05.1 DNA aptamer for targeting mIgM markers on lymphoma cells. (b) Chemical structure of CryA-Mal. (c) ¹²⁹Xe hyper-CEST NMR z-spectra of the biosensor (167 nM in capsids) incubated with 2 × 10⁷ Ramos (mIgM⁺) or Jurkat (mIgM⁻) cells. (d) ¹²⁹Xe hyper-CEST MRI superimposed on ¹H MR image of tubes containing cell media only, and the two cell lines incubated with biosensor. Reproduced with permission from ref. . Copyright 2016 American Chemical Society.