An in situ gelling liquid crystalline system based on monoglycerides and polyethylenimine for local delivery of siRNAs Lívia Neves Borgheti-Cardoso a , Lívia Vieira Depieri a , Sander A.A. Kooijmans b , Henrique Diniz a , Ricardo Alexandre Junqueira Calzzani c , Fabiana Testa Moura de Carvalho Vicentini a , Roy van der Meel b , Márcia Carvalho de Abreu Fantini d , Mamie Mizusaki Iyomasa e , Raymond M. Schiffelers b , Maria Vitória Lopes Badra Bentley a,⇑ a Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, Av. do Café, s/n, 14040–903 Ribeirão Preto, SP, Brazil b Laboratory of Clinical Chemistry and Haematology, University Medical Center Utrecht, Utrecht, The Netherlands c Universidad de La Frontera, Avenida Francisco Salazar 01145, Temuco, Chile d Instituto de Física, Universidade de São Paulo, São Paulo, SP, Brazil e Faculdade de Odontologia de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP, Brazil article info Article history: Received 17 December 2014 Received in revised form 16 April 2015 Accepted 20 April 2015 Available online 25 April 2015 Keywords: Self-assembly Non-viral gene delivery siRNA Local release Liquid crystal abstract The development of delivery systems able to complex and release siRNA into the cytosol is essential for therapeutic use of siRNA. Among the delivery systems, local delivery has advantages over systemic administration. In this study, we developed and characterized non-viral carriers to deliver siRNA locally, based on polyethylenimine (PEI) as gene carrier, and a self-assembling drug delivery system that forms a gel in situ. Liquid crystalline formulations composed of monoglycerides (MO), PEI, propylene glycol (PG) and 0.1 M Tris buffer pH 6.5 were developed and characterized by polarized light microscopy, Small Angle X-ray Scattering (SAXS), for their ability to form inverted type liquid crystalline phases (LC 2 ) in con- tact with excess water, water absorption capacity, ability to complex with siRNA and siRNA release. In addition, gel formation in vivo was determined by subcutaneous injection of the formulations in mice. In water excess, precursor fluid formulations rapidly transformed into a viscous liquid crystalline phase. The presence of PEI influences the liquid crystalline structure of the LC 2 formed and was crucial for com- plexing siRNA. The siRNA was released from the crystalline phase complexed with PEI. The release rate was dependent on the rate of water uptake. The formulation containing MO/PEI/PG/Tris buffer at 7.85:0.65:76.5:15 (w/w/w/w) complexed with 10 lM of siRNA, characterized as a mixture of cubic phase (diamond-type) and inverted hexagonal phase (after contact with excess water), showed sustained release for 7 days in vitro. In mice, in situ gel formation occurred after subcutaneous injection of the for- mulations, and the gels were degraded in 30 days. Initially a mild inflammatory process occurred in the tissue surrounding the gel; but after 14 days the tissue appeared normal. Taken together, this work demonstrates the rational development of an in situ gelling formulation for local release of siRNA. Ó 2015 Elsevier B.V. All rights reserved. 1. Introduction RNA interference (RNAi) has attracted much attention as a new approach for the treatment of many genetic and nongenetic human diseases since 1998, when Fire, Mello and colleagues (Fire et al., 1998) discovered that double-stranded RNAs could silence target gene expression in the nematode worm Caenorhabditis elegans. However, the clinical applicability of RNAi is limited due to various challenges related to the delivery of short interfering RNA (siRNA) molecules, which specifically induce the degradation of comple- mentary messenger RNA in the cytosol. The development of a clin- ically useful, safe and effective carrier system is required (Guzman- Villanueva et al., 2012; Peer and Lieberman, 2011). The essential design criteria for non-viral siRNA carriers include neutralization of the negatively charged phosphate backbone of nucleic acids to avoid charge repulsion by the anionic cell membrane, an appropri- ate size for cellular internalization, protection of the nucleic acids from nuclease degradation and promotion of endosomal escape (Wong et al., 2007). http://dx.doi.org/10.1016/j.ejps.2015.04.017 0928-0987/Ó 2015 Elsevier B.V. All rights reserved. ⇑ Corresponding author. E-mail address: vbentley@usp.br (M.V.L.B. Bentley). European Journal of Pharmaceutical Sciences 74 (2015) 103–117 Contents lists available at ScienceDirect European Journal of Pharmaceutical Sciences journal homepage: www.elsevier.com/locate/ejps