Nitric Oxide-Releasing Cyclodextrins

Abstract

A series of secondary amine-modified cyclodextrin (CD) derivatives was synthesized with diverse exterior terminal groups (i.e., hydroxyl, methyl, methoxyl, and primary amine). Subsequent reaction with nitric oxide (NO) gas under alkaline conditions yielded N-diazeniumdiolate-modified CD derivatives. Adjustable NO payloads (0.6-2.4 μmol/mg) and release half-lives (0.7-4.2 h) were achieved by regulating both the amount of secondary amine precursors and the functional groups around the NO donors. The bactericidal action of these NO-releasing cyclodextrin derivatives was evaluated against Pseudomonas aeruginosa, a Gram-negative pathogen, with antibacterial activity proving dependent on both the NO payload and exterior modification. Materials containing a high density of NO donors or primary amines exhibited the greatest ability to eradicate P. aeruginosa. Of the materials prepared, only the primary amine-terminated heptasubstituted CD derivatives exhibited toxicity against mammalian L929 mouse fibroblast cells. The NO donor-modified CD was also capable of delivering promethazine, a hydrophobic drug, thus demonstrating potential as a dual-drug-releasing therapeutic.

Figure 1.

(a) Synthetic route for CD-HEDA7/NO. (b) ¹H NMR spectra for CD-HEDA7 (top line) and CD-HEDA7/NO (bottom line). (c) UV-Vis spectra for CD-HEDA7 (solid line) and CD-HEDA7/NO (dash line).

Figure 2.

(a) Proton-initiated decomposition of N-diazeniumdiolate-modified CD derivatives. (b) Cumulative NO release given as t[NO] versus time for NO-releasing CD derivatives. Solid line represents CD-PA/NO; dash line represents CD-MA7/NO; dot line represents CD-HEDA7/NO. (c) Proposed structure for stabilization of N-diazeniumdiolate CD derivatives by neighboring cationic ammonium groups.

Figure 3.

(a–c) Confocal laser scanning microscopy (CLSM) images of P. aeruginosa exposed to 300 μg/mL CD-PA/NO for 2 h. DAF-2 green fluorescence indicates intracellular NO delivery. The appearance of PI red fluorescence is attributed to cellular membrane destruction (cell death). (a) Bright field; (b) DAF-2; (c) PI. (d–f) CLSM images of P. aeruginosa exposed to 300 μg/mL CD-EDA/NO for 2 h. (d) Bright field; (e) DAF-2; (f) PI.

Figure 4.

Cell viability (%) of L929 mouse fibroblasts following exposure to blank (buffer only), control (non-NO-releasing) and NO-releasing CD solutions at 250–2000 μg/mL for 4 h. The data represents the mean standard deviation of at least three determinations. (a) Mono-substituted CD derivatives; (b) Hepta-substituted CD derivatives.

Figure 5.

(a) Illustration of promethazine and NO co-delivery for antibacterial activity. (b) Bactericidal efficacy of PM (circle), the complex of PM and CD-DETA (triangle) and the complex of PM and CD-DETA/NO (square) against Gram-negative P. aeruginosa. PM and CD derivatives were delivered in a molar ratio of 1:1. The X-axis is the concentration of PM in different systems. (c) Cell viability (%) of L929 mouse fibroblasts following exposure to PM, the complex of PM and CD-DETA, and the complex of PM and CD-DETA/NO at the MBC_4h concentrations. Red bar was PM; green bar was the complex of PM and CD-DETA; blue bar was the complex of PM and CD-DETA/NO.

Scheme 1.

Synthesis of secondary amine- and N-diazeniumdiolate-functionalized CD derivatives. (a) Preparation of secondary amine-modified CDs; reagents and conditions: (i) TsOCl, NaOH, H₂O/CH₃CN, room temp.; (ii) Primary amine (RNH₂), 75 °C; (iii) Bromine, P(Ph)₃, DMF, 80 °C; (iv) Primary amine (RNH₂), DMF, room temp. (b) Subsequent N-diazeniumdiolate formation.