Recent advances in ratiometric fluorescence imaging of enzyme activity in vivo

Abstract

Among molecular imaging modalities that can monitor enzyme activity in vivo, optical imaging provides sensitive, molecular-level information at low-cost using safe and non-ionizing wavelengths of light. Yet, obtaining quantifiable optical signals in vivo poses significant challenges. Benchmarking using ratiometric signals can overcome dependence on dosing, illumination variability, and pharmacokinetics to provide quantitative in vivo optical data. This review highlights recent advances using fluorescent probes that are processed by enzymes to induce photophysical changes that can be monitored by ratiometric imaging. These diverse strategies include caged fluorophores that change photophysical properties upon enzymatic cleavage, as well as multi-fluorophore systems that are triggered by enzymatic cleavage to alter optical outputs in one or more fluorescent channels. The strategies discussed here have great potential for further development as well as potential broad applications for targeting diverse enzymes important for a wide range of human diseases.

Keywords: Enzyme probes; Fluorescent probes; Optical imaging.

Figure 1.

Representative strategies for ratiometric optical imaging. Schematic of enzymatic processing and resulting spectral changes by a) release of a caging group that induces photophysical changes in a single fluorophore, b) release of a photophysically interacting second fluorophore, and c) release of a caging group that unquenches a fluorophore in the presence of a second, spectrally separated fluorophore. Shaded spectra indicate change upon enzymatic reaction in the direction of the appended arrow. Unshaded spectra indicate spectra remain constant upon enzymatic reaction.

Figure 2.

Single fluorophore-based ratiometric fluorescent probes for in vivo imaging. a) Ester-capped TCFIS chromophore 1 is cleaved by BChE, resulting in 2, with a blue shifted emission wavelength. b) Ratiometric in vivo imaging of a lung cancer model with 1 (ex. 570 nm). c) NTR-InD (3) is converted to 4 with a red-shifted absorption wavelength by treatment with NTR and reduction and elimination of the nitrobenzyl group. d) Rap-N (5) is converted to red-shifted 6 upon treatment with NTR and reduction and elimination of the nitrobenzylcarbamate. e) Ratiometric in vivo imaging of a breast tumor model with 5 (ex. 808 nm).

Figure 3.

Caged single-fluorophores imaged with two-photon ratiometric imaging. a) L-glutamate caging group on naphthalene-benzothiazole chromophore 7 is cleaved by GGT, resulting in chromophore 8, with a red-shifted emission wavelength. b) Neutrophil elastase cleaves the pentafluoropropanamide group on rTP-BC3 (9) resulting in 10, with a red-shifted emission wavelength. c) Ratiometric imaging of collected tissues of an IBD mouse model, treated with 9 (2-photon ex. 800 nm).

Figure 4.

Imaging of enzyme activity with ratiometric FRET probes. a) Structure of ratiometric FRET probe AVB-620 (7) that responds to MMP-9 protease activity. b) Ratio image (I_Cy5/I_Cy7) of cervical lymph nodes following dosing with AVB-620 (11). Cyan and red arrows designate cancer negative and positive lymph nodes, respectively. c) Structure of Death-Cat-RATIO (12), a dual substrate “AND-gated” ratiometric FRET probe with response to cathepsins and caspase-3. d) Ex-vivo fluorescent images of metastases-like breast tumors after injection with Death-Cat-RATIO 24 h prior to imaging. Ratio channel is (I_Cy5/I_FRET). Dashed lines indicate the tumor boundaries and areas of background tissue.

Figure 5.

Ratiometric imaging with an “always on” and a turn-on” fluorophore. a) Ratiometric probe for GGT (9) before and after enzymatic cleavage. Ratio images of HeLa tumor mouse models after treatment with 13 (mCy-Cl channel: λ_ex = 660 nm, λ_em = 710 ± 20 nm); BODIPY channel: λ_ex = 480 nm, λ_em 520 ± 20 nm). b) Conjugated polymer (PCPDTBT) nanoparticles appended with MMP-2 cleavable peptide flanked with Cy5.5 and a quencher. Ratio images of primary gastric tumors in mice after treatment with polymer agent (Cy5.5 channel: λ_ex = 660 nm, λ_em = 690 nm; PCPDTB channel: λ_ex = 660 nm, λ_em 830 nm;). c) Ratiometric probe for MMP-9 (14) before and after enzymatic cleavage. Ratio images of LS180 tumors after treatment with 14 to observe pH mapping (ANNA channel 1: λ_ex = 465–500 nm, λ_em 500 nm; ANNA channel 2: λ_ex = 465–500 nm, λ_em 540 nm) and MMP-9 activity (ANNA channel: λ_ex = 465–500 nm, λ_em 500 nm LP; Cy5.5 channel: λ_ex = 640–675 nm, λ_em 680 nm LP).