Nanostructured lipid carrier mediates effective delivery of methotrexate to induce apoptosis of rheumatoid arthritis via NF-kB and FOXO1 Neeraj K. Garg a, 1 , Rajeev K. Tyagi b,c, 1 , Bhupinder Singh a,d , Gajanand Sharma a , Pradip Nirbhavane a , Varun Kushwah e , Sanyog Jain e , Om Prakash Katare a, * a Drug Delivery Research Group, University Institute of Pharmaceutical Sciences, UGC Centre of Advanced Studies, Panjab University, Chandigarh 160014, India b Department of Periodontics, College of Dental Medicine Georgia Regents University, 1120, 15th Street, Augusta, GA 30912, USA c Institute of Science, Nirma University, Sarkhej-Gandhinagar Highway, Ahmedabad, 382 481 Gujarat, India d UGC-Centre of Excellence in Applications of Nanomaterials, Nanoparticles & Nanocomposites (Biomedical Sciences), Panjab University, Chandigarh 160 014, India e Centre for Pharmaceutical Nanotechnology, Department of Pharmaceutics, National Institute of Pharmaceutical Education and Research (NIPER), Sector 67, SAS Nagar (Mohali), Punjab 160062, India A R T I C L E I N F O Article history: Received 26 October 2015 Received in revised form 24 November 2015 Accepted 21 December 2015 Available online 6 January 2016 Keywords: Nanostructured lipid carriers (NLCs) Methotrexate Rheumatoid Arthritis Apoptosis NF-kB-IkB FOXO1 RT-PCR a-Terpineol Phospholipids S-100 Gelucire 50/13 Transcutol P Transdermal delivery A B S T R A C T Present study was designed to develop novel nano-structured lipid carriers (NLCs) formulated by lipid mixture and chemical permeation enhancer-based hydrogel for an effective transdermal delivery of methotrexate (MTX). The prepared NLCs were optimized with different preparative variables such as particle size <200 nm, poly-dispersity index (PDI) <0.2, and entrapment efficiency 85%. The drug incorporated into NLCs-gel base showed excellent spread ability without any grittiness during rheological behavior and texture profile analysis. The in vitro release showed biphasic release pattern with initial fast release of drug (>50%) in 8 h followed by sustained release (up to 85%) by the end of 48th h. NLCs showed greater uptake in human hyper-proliferative keratinocyte cell line (HaCaT). NLCs showed increased expression of inflammatory mediators as well asapoptosis in U937 monocytic cells. The greater expression of pro-apoptotic gene Bim regulated by NF-kB-IkB and FOXO1 is supported by fold regulations calculated for various apoptotic and pro-inflammatory biomarkers carried out by RT-PCR. The immunocytochemistry to detect IL-6 expression and immunofluorescence assay suggested that induced apoptosis occurs in experimentally induced in vitro arthritis model treated with NLCs-MTX. We saw reduced inflammation and triggered apoptosis through NF-kB & FOXO1 pathways induced by MTX loaded NLCs in rheumatoid arthritic cells. In addition, formulated NLCs exhibit better skin permeation with higher permeation flux & enhancement ratio as shown by confocal laser scanning microscopy (CLSM). Moreover, histopathological examinations of skin are suggestive of safety potential of NLCs. ã 2015 Elsevier B.V. All rights reserved. 1. Introduction Methotrexate (MTX) is a folate anti-metabolite widely used for anti-neoplastic activity, and known for anti-inflammatory effect. MTX has been used for treating psoriasis, cancer and rheumatoid arthritis (RA) (Amarji et al., 2015; Jain et al., 2015a; Paulus, 1990). Among various disease-modifying anti-rheumatic drugs (DMARDs), oral administration of MTX has been commonly used for treating RA (Paulus, 1990; Weinblatt et al., 1991). MTX has reportedly shown to slowdown the rate of joint destruction by inhibiting pro-inflammatory cytokines such as interferon (IFN-g), tumor necrosis factor (TNF-a), interleukin (IL)-1b , IL-6, IL-15, and IL-18 (Choy and Panayi, 2001; Cutolo et al., 2001; Feldmann et al., 1996; Zwerina et al., 2005), and joint-destructive enzymes viz. inducible nitric oxide synthase (iNOS), NOS 2 and cyclooxygenase-2 (COX-2) during chronic inflammation in joints (Choy and Panayi, 2001; Feldmann et al., 1996; Lee et al., 2012; Zwerina et al., 2005). Although MTX tablets and injections have been licensed to the market, long term usage results into adverse effects including mucosal ulceration, stomatitis, bone marrow suppression, loss of * Correspondence author at: Division of Pharmaceutics, University Institute of Pharmaceutical Sciences, UGC-Centre of Advanced Study, Panjab University, Chandigarh 160 014, India. Fax: +91 172 2543101. E-mail address: drkatare@yahoo.com (O.P. Katare). 1 Co- first author (contributed equally to this work). http://dx.doi.org/10.1016/j.ijpharm.2015.12.061 0378-5173/ ã 2015 Elsevier B.V. All rights reserved. International Journal of Pharmaceutics 499 (2016) 301–320 Contents lists available at ScienceDirect International Journal of Pharmaceutics journal homepage: www.elsev ier.com/locate /ijpharm