Stepwise Nitric Oxide Release and Antibacterial Activity of a Nitric Oxide Photodonor Hosted within Cyclodextrin Branched Polymer Nanocarriers

Abstract

A hydrophobic nitric oxide (NO) photodonor integrating both nitroso and nitro functionalities within its chromophoric skeleton has been synthesized. Excitation of this compound with blue light triggers the release of two NO molecules from the nitroso and the nitro functionalities via a stepwise mechanism. Encapsulation of the NO photodonor within biocompatible neutral, cationic, and anionic β-cyclodextrin branched polymers as suitable carriers leads to supramolecular nanoassemblies, which exhibit the same nature of the photochemical processes but NO photorelease performances enhanced by about 1 order of magnitude when compared with the free guest. Antibacterial tests carried out with methicillin-resistant Staphylococcus aureus and Acinetobacter baumannii demonstrate an effective antibacterial activity exclusively under light activation and point out a differentiated role of the polymeric nanocarriers in determining the outcome of the antibacterial photodynamic action.

Scheme 1. Molecular Structure of NOPD1 and the Poly-βCDs Used as Host Nanocarriers; Inset: Stepwise Mechanism for the NO Photorelease

Scheme 2

Reagents and conditions: a) octylamine, K₂CO₃, CH₃CN, r.t., 24 h; b) NOPD2, NaNO₂, THF/CH₃COOH 2:1, 12 h, 0 °C → r.t.

Figure 1

Absorption spectra of NOPD1 (a) and NOPD2 (b). PBS (10 mM; pH 7.4):MeOH 1:1 v/v; T = 25 °C.

Figure 2

Absorption spectral changes observed upon exposure of an air-equilibrated solution of NOPD1 (45 μM) at λ_exc = 420 nm at different irradiation times from 0 to 40 min (A) and from 40 to 100 min (B). The arrows indicate the course of the spectral profile with illumination time (C). HPLC traces related to solutions of NOPD1 (a), the authentic photoproduct NOPD2 used as a reference (b), and NOPD1 after 30 min irradiation (c). PBS (10 mM; pH 7.4):MeOH 1:1 v/v; T = 25 °C.

Figure 3

Normalized absorption spectra of NOPD1 (A) and NOPD2 (B) in the presence of aqueous dispersions (4 mg mL^–1) of N-poly-βCD (a), C-poly-βCD (b), and A-poly-βCD (c). The insets show representative hydrodynamic diameters in the case of nanoassemblies NOPD1/N-poly-βCD (A) and NOPD2/N-poly-βCD (B). PBS (10 mM; pH 7.4); T = 25 °C.

Figure 4

Absorption spectral changes observed upon exposure of an air-equilibrated aqueous suspension of NOPD1/C-poly-βCD at λ_exc = 420 nm at different irradiation times from 0 to 5 min (A) and from 5 to 31 min (B). The arrows indicate the course of the spectral profile with the illumination time. (C) Evolution of the absorbance at 400 nm upon irradiation at λ_exc = 420 nm of air-equilibrated aqueous suspensions of NOPD1/N-poly-βCD (■), NOPD1/C-poly-βCD (●), and NOPD1/A-poly-βCD (▲). NOPD1/N-poly-βCD ([NOPD1] = 12 μM); NOPD1/C-poly-βCD ([NOPD1] = 10 μM); NOPD1/A-poly-βCD ([NOPD1] = 7 μM); [N-poly-βCD] = [C-poly-βCD] = [A-poly-βCD] = 4 mg mL^–1. PBS (10 mM; pH 7.4); T = 25 °C.

Figure 5

NO release profiles observed upon alternate ON/OFF cycles of irradiation at λ_exc = 405 nm for aqueous suspensions of (A) NOPD1/N-poly-βCD (a), NOPD1/C-poly-βCD (b), and NOPD1/A-poly-βCD (c) and (B) NOPD2/N-poly-βCD (a), NOPD2/C-poly-βCD (b), and NOPD2/A-poly-βCD (c). [NOPD1] = [NOPD2] = 8 μM; [N-poly-βCD] = [C-poly-βCD] = [A-poly-βCD] = 4 mg mL^–1. PBS (10 mM; pH 7.4); T = 25 °C.

Figure 6

Planktonic assay for (A) MRSA and (B) A. baumannii (1 × 10⁸ CFU mL^–1) incubated with the free NOPD1 (1% DMSO) and its nanoassemblies with poly-βCD, suspended in PBS (10 mM; pH 7.4) and either kept in the dark or irradiated with blue light at λ_exc = 420 nm (ca. 96 mW cm^–2). Untreated bacteria were used as a control. Statistical analyses via One Way Anova and Tukey tests indicate a statistically significant difference (*p < 0.01). [NOPD1] = 10 μM; [N-poly-βCD] = [C-poly-βCD] = [A-poly-βCD] = 4 mg mL^–1.