Controlled Delivery of Nitric Oxide Using Stable and Stimuli-Responsive S-nitrosothiols
Nitric oxide (NO) is a short-lived, endogenously produced gas which plays multiple roles in mammalian physiology. A broad range of NO donors has emerged as potential therapeutics in different pathological processes including cardiovascular and respiratory disorders, wound healing, the immune response to infection and cancer. However, limited NO payloads, too rapid NO release, and the lack of organ or tissue specificity have limited the clinical utility of currently available NO donors. Herein, three main strategies were used to make stable and stimuli-responsive NO donors using S-nitrosothiols (SNTs). First, we have developed photoactive nanoparticles (NPs) by encapsulation of a hydrophobic and stable SNT, tert-dodecane s-nitrosothiol (tDodSNO), into polystyrene maleic acid (SMA) to make nano-sized SMA-tDodSNO particles. Encapsulation of tDodSNO physically protects it from metabolic breakdown as well as trans-nitrosation reactions with proteins and thiols, thereby inhibits unspecific NO release. In the absence of photo-irradiation, NPs had a half-life (t1/2) of approximately 104 h, while photoactivation (cold light with the intensity of 2700 W/m2) decreased this to just 3.5 min. Second, a stable and tuneable NO-releasing compound was synthesized which contains hydrophobic and sterically hindered SNT as well as a carboxylate group far from the SNT group. The compound showed sustained NO release in the dark (t1/2= 300 h) while upon photoactivation is degraded within a few mins (t1/2= 5.5 min). Third, by combining these two approaches and attachment of the synthesized SNT to SMA to produce a novel NO-releasing polymer (SMASNO). The unactivated polymer has a NO release half-life of approximately 392 h, and photoactivation decreased this to 23 min. Photoactivation of SMA-tDodSNO resulted in an 18-fold shift in the EC50 of rat’s aortic ring vasorelaxation and also induced localized hyperpermeability in rats mesenteric bed with microscopic analysis indicating that the SMA-tDodSNO increased tissue uptake through a combination of photoactivation induced vasodilation and extravasation. SMA-tDodSNO showed significant cytotoxicity in 4T1 cell line and when combined with doxorubicin (Dox) it synergistically arrested cell proliferation and induced apoptosis. Due to NO release, it enhanced the endocytosis of a Dox-loaded NP (SMA-Dox) and increased the permeability of the endosomal membrane, hence facilitated the escape of the NPs, and reduced Dox efflux from the cells. Similarly, photoactivation of SMASNO shifted the EC50 of the aortic ring to 12-fold lower concentrations. The polymer could self-assemble to form containing the hydrophobic fluorescent dye Nile Red (NR) as a traceable drug analog to form NRNPs. The NR release from the particles was increased in either the presence of high levels of glutathione or in response to photoactivation, indicating that the NP could release its cargo in response to specific stimuli. When NRNPs were perfused into a rat mesenteric bed, then a region of the bed irradiated by cold light was significantly higher than other parts of the bed. In conclusion, stable and stimuli-responsive NO-releasing compounds were made using hydrophobic and bulky SNTs covalently attached or self-assembled with hydrophilic moieties. We report that the stimuli-responsive NO-releasing NPs can be used to overcome some of the barriers in front of efficient chemotherapy, as they can enhance blood supply, vessel permeability in tumor tissues, endocytosis and endosomal escape of NPs, and inhibit drug resistance.
Advisor: Giles, Gregory; Gamble, Allan; Greish, Khaled
Degree Name: Doctor of Philosophy
Degree Discipline: Pharmacology and toxicology
Publisher: University of Otago
Keywords: Nitric Oxide; S-nitrosothiols; Stimuli-Responsive; Controlled Delivery
Research Type: Thesis