Development of carbon monoxide-releasing molecule-2-loaded lipid nanoparticles for enhanced anti-inflammatory activity via sustained release of carbon monoxide

Abstract

The aim of this study was to enhance the anti-inflammatory effects of carbon monoxide (CO) via sustained CO release from carbon monoxide-releasing molecule-2-loaded lipid nanoparticles (CORM-2-NPs). CORM-2-NPs were prepared by hot high pressure homogenization method using trilaurin as a solid lipid core and Tween 20 (polyoxyethylene sorbitan monolaurate), Span 20 (sorbitan monolaurate), and Myrj S40 as surfactant mixture. CORM-2-NPs were prepared by optimizing different weight ratios of different lipid and surfactants. CORM-2-NPs were characterized in terms of particle size, zeta potential as surface charge, and polydispersity index. The incorporation efficiency of CORM-2 into CORM-2-NPs was determined by quantifying ruthenium using inductively coupled plasma atomic emission spectrophotometry (ICP-AES). The CO release profiles from CORM-2-NPs were assessed by myoglobin assay and compared with that from CORM-2 solutions of same CORM-2 concentration. The shape, size and morphology of blank and CORM-2-loaded lipid nanoparticles were confirmed by field-emission transmission electron microscope, with and without phosphotungstic acid staining. Non-cytotoxicity of CORM-2-NPs was confirmed by evaluating in vitro cell viability of RAW 264.7 macrophages by MTT assay. In vitro anti-inflammatory effects of CORM-2-NPs were evaluated by nitric oxide (NO) assay in lipopolysaccharide stimulated RAW 264.7 macrophages. In vivo anti-inflammatory activity of CORM-2-NPs was investigated and compared with CORM-2 solution and indomethacin as reference drug in carrageenan-induced rat paw edema model by measuring the edema volumes using digital plethysmometer. Histopathological evaluation of rat paw tissues was conducted to confirm enhanced anti-inflammatory effects of CORM-2-NPs, after 6 h of carrageenan-injection in rat paw edema model. Spherical CORM-2-NPs were successfully prepared with mean diameter of around 100 nm demonstrating narrow particle size distribution. Both blank and CORM-2-NPs showed a negative zeta potential value around -20 mV. The incorporation efficiency of CORM-2-NPs was calculated as 87.7% as determined by ICP-AES. The formation of uniform spherical particles in nanometer range was observed by TEM images. The sustained CO release from CORM-2-NPs was achieved and the CO release half-life of CORM-2 was increased up to 10 times as compared with CORM-2 solution. CORM-2-NPs showed non-cytotoxicity and improved in vitro anti-inflammatory effects by inhibition of nitric oxide production in RAW 264.7 macrophages. Edema volume in rat paws was significantly reduced after treatment with CORM-2-NPs. Moreover, histopathological evaluation confirmed enhanced in vivo anti-inflammatory effects of CORM-2-NPs. Taken together, CORM-2-NPs have a great potential for CO therapeutics against inflammation via sustained release of CO.