Design and optimization of ROS-reactive nanocarriers for controlled release of dexamethasone and real-time inhibition of mast cell activation

Upon activation, mast cells release many mediators, including reactive oxygen species (ROS) and tumor necrosis factor (TNF) orchestrating allergic inflammation. Nanocarriers (NCs) that can detect mast cell activation and respond by releasing anti-inflammatory compounds in real-time, would and effective therapy. ROS-responsive NCs were synthesized employing the ROS-sensitive thioketal (TK) as a linker between the amphiphilic polyethylene glycol-polylactic acid (PEG-PLA) block copolymer and dexamethasone (Dex). The resultant Dex conjugated polymeric system (PTD) readily assembled into NCs in an aqueous solution. Mouse bone marrow-derived mast cells (BMMC) were treated with the NCs, stimulated with calcium ionophore A23187 (1 microM) and generation of tumor necrosis factor (TNF) was measured by ELISA. ROS production was measured by using dichlorodihydrofluorescein diacetate (DCFH-DA). Degranulation was measured using the beta-hexosaminidase (b-hex) assay. A23187-stimulated BMMC released 540 +/- 9.8 pg/ml of TNF and Dex treatment (250 nM) decreased TNF release by 59.3 +/- 0.33 % compared to untreated control (p>0.05; n=3). BMMC treated with PTD and stimulated with A23187 to induce ROS, released significantly less TNF. Respective release of TNF in cells incubated with 1 mg/ml, 0.5 mg/ml and 0.1 mg/ml PTD was 190, 175 and 165 pg/ml, significantly lower than cells treated with 250 nM Dex (310 +/- 4.5 pg/ml). Neither Dex nor the NCs modified degranulation. We have designed and synthesized a proof-of-concept, stimulus-responsive, and on-demand Dex delivery system which inhibits TNF production by activated mast cells. Current work is on-going to further refine this system.