Avaliação das propriedades anti-inflamatórias de Nanocápsulas com Núcleo Lipídico (LNC) carregadas com metotrexato

Abstract

Methotrexate is an anti-rheumatic drug that modifies the course of the disease, being the first anti-rheumatic drug for most patients with rheumatoid arthritis (RA), however some patients do not respond to treatment with MTX, these patients have low expression of CD39 in regulatory T cells. Macrophages are immune cells that play a central role in the inflammatory response of RA, but these cells are resistant to apoptosis. The Lipid Nucleus (LNC) nanocapsules, is a new model of nanocapsules created by a research group from the Universidade Federal do Rio Grande do Sul, these nanocapsules have greater stability in the body fluid and are easily phagocytosed by macrophages. LNCs loaded with MTX (MTX-LNC), were used to evaluate the apoptotic and immunomodulatory potential of MTX-LNC in biological models of arthritis (in vivo) and macrophages (in vitro). Lewis rats were induced to arthritis by intradermal injection of Freund's complete adjuvant, the results showed that the MTX-LNC group showed better results when compared with MTX, both in reducing the volume of the paw and in the evaluation of the joint components: joint space (0.0880.012, 0.050.002; p=0.3291), synovial membrane (22.612.291, 29.350.9004; p=0.0211), cartilage (joint: 0.040.0063, 0.0530.0037; p=0.0031, calcified: 0.0350.0058, 0.0540.005; p=0.0436) and chondrocytes (articular: 614.3225.0, 108987.72; p=0.0081, calcified: 495.3180.6, 954.576.16; p=0.0041). The formulations were also tested on human (THP-1) and mouse (J774) macrophages (in vitro) and the calculated IC50%, THP-1: MTX 17.365nM and MTX-LNC 194.2nM 48 hours and J774: MTX 17.188nM (r2=0.47) and 175.2nM MTX-LNC (r2=0.98) in 24 hours. MTX-LNC induces more iROS and membrane with lower mitochondrial potential (m) for both cells compared to MTX (P<0.0001). When assessing the DNA damage index by the comet assay, both formulations (MTX / MTX-LNC) showed a significant increase. MTX-LNC induces more activation of caspase-8 (p = 0.002), 3 (p <0.0001) and Annexin V + (p<0.0001) than MTX. The analysis of gene expression related to apoptosis, showed that MTX-LNC positively regulates the TNFRSF25 gene (84,29) purchased the MTX formulation, extrinsic pathway genes (FASLG, TRADD, CRADD) and pro-apoptotic genes (BAD, BCL2L10) are also positively regulated, suggesting activation of the extrinsic apoptosis pathway. The new MTX-LNC formulation is promising and more efficient than traditional MTX, both in vivo tests as a modifier of the disease course in an experimental model, and in vitro tests, improving the apoptotic effects of this drug in THP1 and J774 macrophages by improving the activation of apoptosis effector caspases increasing DNA damage and upregulating pro-apoptotic genes via extrinsic pathway.