Development of a solid-supported light-triggered nitric oxide donor

Abstract

Nitric Oxide (NO) photocleavable donors are useful tools for interrogating nitric oxide signalling and have potential use in photopharmacological applications. There is currently intensive research into newer methods to improve NO release and kinetic profiles. Herein, we report the design and synthesis of a solid-supported photocleavable NO donor synthesized by ligating an N-nitroso photocleavable nitric oxide derivative to a TentaGel^® polymer resin bead. Illumination with 365 nm light released nitric oxide that could be tracked via a turn-on fluorescence response (λ_ex = 450 nm, λ_em = 545 nm) and measured using the Griess assay and diaminorhodamine derivatives. These beads were further shown to be compatible with living A549 cells and had the ability to deliver greater concentrations of nitric oxide to cells proximal to a bead versus cells at more distal locations within the same well.

Keywords: cellular delivery; nitric oxide; photocleavable donors; solid-phase chemistry.

Figure 1:

(A) Fluorescence image before photo-irradiation. (B) Fluorescence image after photoirradiation for 5 min using 367 nm DAPI filter cube with 20% of current intensity. (C) Changes in fluorescence intensity with respect to the duration of photo-irradiation. Bars are means ± standard deviation from two images corresponding to two independent experiments. Raw images of the replicate experiment are available in the SI (Figure S2).

Figure 2:

(A) Calibration curve using sodium nitrite to quantify NO release. The inset shows the color change of the respective nitrite concentrations obtained with the added Griess reagent. Error bars are ± SD with n = 3 technical replicates. (B) NO release from 2 mg the solid-supported light-triggered NO donor 6 after 30 seconds of photo-irradiation. (C) Schematic representation of the experiment used to generate the data in (B).

Figure 3:

Fluorescence emission spectra for 5 μM DAR-4M AM with 5 mg of solid-supported photolytic NO donor 6 suspended in solution after 10 min photoirradiation with UV light at 365 nm with a power density of 3.54 mW cm⁻². The blank contains 5 μM DAR-4M AM. All experiments were performed in phosphate buffer, pH 7.4. λ_ex = 560 nm, λ_em = 580 nm.

Figure 4:

Representative fluorescent images of A549 cells loaded with 10 μM DAR-4M AM and the solid-supported photolytic NO donor beads 6. (A) Fluorescence image of the beads 6 before photo-irradiation imaged using a 10x objective. (B) Fluorescence image of the beads 6 after photo-irradiation imaged using a 10x objective. (C) Fluorescence image after photo-irradiation in cells proximal to the irradiated bead imaged using a 20x objective. (D) Fluorescence image after photo-irradiation for cells distal from the irradiated bead imaged using a 20x objective. (E) Corrected Total Cell Fluorescence (CTCF) for proximal and distal cells to the irradiated bead. Bars are means ± standard deviation from n = 13 cells across 2 independent experiments (distal cells) or n =22 cells across three independent experiments (proximal cells).

Scheme 1:

Synthetic pathway of the solid-supported light-triggered NO donor.

Scheme 2:

NO release and fluorophore production by photoirradiation of the solid-supported photoactivatable NO donors.