Calcium–siRNA nanocomplexes: What reversibility is all about Emil Ruvinov a, ⁎, Olga Kryukov a , Efrat Forti a , Efrat Korin a , Matan Goldstein a , Smadar Cohen a,b,c a Avram and Stella Goldstein-Goren Department of Biotechnology Engineering, Ben-Gurion University of the Negev, Beer-Sheva 8410501, Israel b Regenerative Medicine and Stem Cell (RMSC) Research Center, Ben-Gurion University of the Negev, Beer-Sheva 8410501, Israel c The Ilse Katz Institute for Nanoscale Science and Technology, Ben-Gurion University of the Negev, Beer-Sheva 8410501, Israel abstract article info Article history: Received 25 November 2014 Received in revised form 14 February 2015 Accepted 17 February 2015 Available online 19 February 2015 Keywords: Calcium complex Drug delivery Gene silencing RNA interference siRNA delivery Transfection Gene silencing using small interfering RNA (siRNA) relies on the critical need for a safe and effective carrier, capable of strong but reversible complexation, siRNA protection, cellular uptake, and cytoplasmatic unloading of its cargo. We hypothesized that a delivery platform based on the eletrostatic interactions of siRNA with calcium ions in solution would fulfill these needs, ultimately leading to effective gene silencing. Physical characterization of the calcium–siRNA complexes, using high resolution microscopy and dynamic light scattering (DLS), showed the formation of stable nanosized complexes ~80 nm in diameter, bearing mild (~ -7 mV) negative surface charge. The complexes were extremely stable in the presence of serum proteins or high concentrations of heparin; they maintained their nanosized features in suspension for days; and effectively protected the siRNA from enzymatic degradation. The Ca–siRNA complexes were disintegrated in the presence of Ca-chelating ion exchange resin, thus proving their reversibility. Excellent cytocompatibility of calcium–siRNA complexes was achieved using physiological calcium ion concentrations. The calcium–siRNA complexes successfully induced a very high (~80%) level of gene silencing in several cell types, at both mRNA and protein levels, associated with efficient cellular uptake. Collectively, our results show that the developed delivery platform based on reversible calcium–siRNA interactions offers a simple and versatile method for enhancing the therapeutic efficiency of siRNA. © 2015 Elsevier B.V. All rights reserved. 1. Introduction Small interfering RNA (siRNA) represents a promising type of thera- peutics and a powerful research tool, which exploits a natural post- transcriptional mechanism of RNA interference (RNAi), leading to effec- tive and highly specific silencing of target genes [1,2]. However, the efficiency of siRNA delivery into the cell cytoplasm is very low, as is the resultant gene silencing, both in vitro and in vivo. Obstacles for successful siRNA application include its strong negative charge, fast elimination and degradation rates, and inadequate cellular entry and trafficking [3–5]. Thus, various non-viral carriers (lipids, polymers and many others) have been investigated, aimed at protecting siRNA while facilitating its intracellular delivery [2,6]. In designing such siRNA car- riers several important criteria are considered, such as biocompatibility, stability, effective cellular uptake, effective endosomal escape, and, ultimately, successful unloading of the siRNA cargo in the cytoplasm, where the RNAi machinery is deployed [3,5,6]. Complexation of siRNA, to be mediated by the specific carrier, repre- sents a critical step required for siRNA protection from degradation, charge masking, and subsequent cellular entry. One of the first carriers applied for siRNA complexation was the synthetic cationic polymer, polyethylenimine (PEI), originally used for plasmid DNA condensation for subsequent transfection, exemplifying the initial belief that cationic polymers would be excellent candidates for siRNA delivery [7–9]. Despite strong PEI–siRNA polyplex formation, PEI was found to be less effective in siRNA-mediated gene silencing than in DNA plasmid trans- fection, and was also associated with increased toxicity, both aspects demanding time-consuming chemical modifications, or even revisiting its potential use as a delivery vehicle for siRNA [9–14]. Unloading and release of siRNA from its carrier inside the cytoplasm is the final and essential step required for the silencing action of the delivered siRNA molecule. This step is critically dependent on the exis- tence of sufficient, but readily reversible interactions between the siRNA and its complexing agent. With that in mind, we designed a novel siRNA delivery platform based on the well-known electrostatic interactions of siRNA with calcium ions, but, however, using a completely different preparation strategy, compared to generally used approaches. Calcium formulations, particularly calcium phosphate (CaP)-based, are being ex- tensively used for DNA transfection. However, CaP use as a nucleic acid carrier suffers from several critical drawbacks, such as uncontrollable growth of CaP crystals in solution, low reproducibility, absolute require- ment for immediate application after preparation, possible impairment of calcium homeostasis, and cell death [15–19]. Moreover, silencing Journal of Controlled Release 203 (2015) 150–160 ⁎ Corresponding author at: Avram and Stella Goldstein-Goren Department of Biotechnology Engineering, Ben-Gurion University of the Negev, Beer-Sheva, POB 653, 8410501, Israel. E-mail address: ruvinove@bgu.ac.il (E. Ruvinov). http://dx.doi.org/10.1016/j.jconrel.2015.02.029 0168-3659/© 2015 Elsevier B.V. All rights reserved. Contents lists available at ScienceDirect Journal of Controlled Release journal homepage: www.elsevier.com/locate/jconrel