A Nitronaphthalimide Probe for Fluorescence Imaging of Hypoxia in Cancer Cells

Abstract

The bioreductive enzymes typically upregulated in hypoxic tumor cells can be targeted for developing diagnostic and drug delivery applications. In this study, a new fluorescent probe 4-(6-nitro-1,3-dioxo-1H-benzo[de]isoquinolin-2(3H)-yl)benzaldehyde (NIB) based on a nitronaphthalimide skeleton that could respond to nitroreductase (NTR) overexpressed in hypoxic tumors is designed and its application in imaging tumor hypoxia is demonstrated. The docking studies revealed favourable interactions of NIB with the binding pocket of NTR-Escherichia coli. NIB, which is synthesized through a simple and single step imidation of 4-nitro-1,8-naphthalic anhydride displayed excellent reducible capacity under hypoxic conditions as evidenced from cyclic voltammetry investigations. The fluorescence measurements confirmed the formation of identical products (NIB-red) during chemical as well as NTR-aided enzymatic reduction in the presence of NADH. The potential fluorescence imaging of hypoxia based on NTR-mediated reduction of NIB is confirmed using in-vitro cell culture experiments using human breast cancer (MCF-7) cells, which displayed a significant change in the fluorescence colour and intensity at low NIB concentration within a short incubation period in hypoxic conditions.

Keywords: Docking; Fluorescence; Hypoxia; Molecular probe; Nitronaphthalimide; Nitroreductase.

Scheme 1.

Synthetic route for NIB and NIB-red

Fig. 1

(A) 2D view of molecular interactions of NIB with amino acid residues of NTR: hydrogen bond interactions are shown in purple arrow, the amino acid residues that form hydrophobic contacts are presented in green and π- π stacking with green arrows. (B) Overlay of NIB in the active binding pocket of 3X21. (C) Proposed reduction of NIB in presence of NTR to NIB-red

Fig. 2

Cyclic voltammograms recorded for 1 mmol of NIB alone and NIB in presence of 0.5 mM NADH solution in 0.1 M TBAFB₄ (DMSO) at (A) normal oxygen environment (normoxic) and (B) nitrogen environment (hypoxic)

Fig. 3

(A) Time-dependent variation in the fluorescence emission of 1 μM NIB in PBS (pH = 7.4) with 1% DMSO in the presence of 20 μg/mL NTR and 500 μM NADH (λ_exc = 410 nm) and (B) Fluorescence intensity of 1 μM NIB-red vs. the reaction time on treatment with different concentrations of NTR (0 to 20 µg/mL) in presence of 500 µM NADH (λ_exc = 433 nm)

Fig. 4

Fluorescence images of MCF-7 cells under normoxic (20% O₂) and hypoxic (1% O₂) conditions after 2 h of NIB incubation, imaged for bright and blue/green fluorescence (blue: λ_exc = 370−410 nm; λ_em = 429–462 nm; green: λ_exc = 473–491 nm; λ_em = 502–561 nm). Scale bar: 100 µM

Fig. 5

(A) Percentage conversion of fluorescence intensities and (B) relative fluorescence intensity from blue (λ_em = 478 nm) to green (λ_em = 539 nm) wavelength in hypoxic conditions with respect to control as mentioned in materials and methods. The raw intensities of the images were calculated using Image J software and percentage of conversion was calculated by normalising the intensities with respect to control. The values are average of two independent experiments