Cerenkov Probes to detect Redox Status

Objectives The objective is to develop a robust method to utilize the emission of Cerenkov photons in conjunction with functional fluorinated chromophores to determine redox state in tissue. Resazurin (RA) is a commonly used viability dye and redox sensor. When reduced in cells, RA is converted into resorufin (RAred), a highly fluorescent molecule. Here we describe fluorination, characterization and application of the resulting molecule in vitro, in cells and in mouse models.

Methods RA was fluorinated using electrophilic fluorination under acidic conditions. The resulting mixture of fluorinated derivatives was purified using preparative HPLC; 5 fractions were identified and collected. Mass spectrometry, UV-vis spectroscopy, NMR (1H and 19F) and pKa studies were run to fully characterize the compounds. To determine the reduction capability of MFRA and DFRA, chemical reduction of the molecule was performed using sodium dithionite. DU145 cell studies determined the feasibility of the compounds to be reduced in cellulo. Preliminary tumor bearing mouse studies were run to determine the delivery of the probe to the tumor area, tracking the probe with both aPET and optical imaging.

Results Mass spectrometry analysis confirmed the synthesis of mono and difluorinated compounds. 1H-NMR and 19F-NMR analysis confirmed the presence of two isomers of monoflurinated resazurin (MFRA) in one of the fractions and an isomer of difluororesazurin (DFRA) in each of two fractions. The pKa results followed a trend of higher acidity with increased degree of fluorination. Analysis of the Cerenkov emission using an IVIS Spectrum determined that both MFRA (with the addition of FDG as photon source) and 18F-MFRA were able to be reduced chemically. The reduced compound is highly fluorescent resulting in a longer wavelength emission due to Cerenkov Radiation Energy Transfer (CRET). The maximum ratio of emission between MFRA and MFRAred was 3.5 at 620 nm. The difference in photonic emissions between 620-660 nm suggests that these are the best filters to use for in cellulo and/or in vivo application. A similar study with DFRA showed comparable results between the 19F and the 18F-labeled compounds. Cell studies demonstrated that DU145 prostate cancer cells are able to reduce both MFRA and DFRA readily. Preliminary mouse studies show a maximum accumulation of 0.25% injected dose in the tumor. Excretion is primarily renal and to a lesser extent hepatic within a couple of hours.

Conclusions Successful fluorination of the resazurin molecule was achieved. Chemical and cellular reduction led to increased fluorescence capability of MFRAred and DFRAred leading to a bathochromic shift of Cerenkov emission due to CRET. Cellular activity of the fluorinated derivatives is an indication of a possible in vivo application. The detection of redox stress is a potential tool to non-invasively detect several medically relevant scenarios including antioxidant studies, inflammation and potentially neurodegenerative diseases. The eventual impact of this technique lies in the ability translate it into the clinic coupled with PET for a dual imaging modality.