Systemic delivery of siRNA by aminated poly(α)glutamate for the treatment of solid tumors Dina Polyak a,1 , Adva Krivitsky a,1 , Anna Scomparin a,1 , Shay Eliyahu a , Hagar Kalinski b , Sharon Avkin-Nachum b , Ronit Satchi-Fainaro a, ⁎ a Department of Physiology and Pharmacology, Sackler Faculty of Medicine, Room 607, Tel Aviv University, Tel Aviv 69978, Israel b QBI Enterprises, Ltd., Ness-Ziona 70400, Israel abstract article info Article history: Received 28 April 2016 Accepted 24 June 2016 Available online 26 June 2016 Small interfering RNA (siRNA) can silence the expression of a targeted gene in a process known as RNA interfer- ence (RNAi). As a consequence, RNAi has immense potential as a novel therapeutic approach in cancer targeted therapy. However, successful application of siRNA for therapeutic purposes is challenging due to its rapid renal clearance, degradation by RNases in the bloodstream, poor cellular penetration, immunogenicity and aggregation in the blood. In addition, the few oligonucleotide-based nanomedicines that reached clinical trials either go to the liver following systemic administration or are applied topically. Treatment of solid tumors requires selective dis- tribution of siRNA to the target tissue, hence there is an unmet medical need for an efficacious and safe nano- sized delivery system for their clinical use. To overcome these hurdles, we have designed, synthesized and physico-chemically characterized a novel nanocarrier based on aminated poly(α)glutamate (PGAamine). This cathepsin B-biodegradable polymer interacts electrostatically with the siRNA to form a nano-sized polyplex sta- ble in plasma. Treatment with PGAamine-Rac1 siRNA polyplex (siRac1-polyplex) caused specific gene silencing by 80% in HeLa and SKOV-3 human ovarian adenocarcinoma cells as opposed to PGAamine-control non-targeting siRNA polyplex (siCtrl-polyplex) leading to inhibition of cell migration and wound healing abilities. A stepwise dose escalation was performed in order to determine the in vivo maximum tolerated dose (MTD). This was followed by intraperitoneal administration of siRac1-polyplex to mCherry-labeled ovarian adenocarcinoma- bearing mice leading to preferred tumor accumulation of siRac1 (8-fold) which resulted in 38% Rac1 knockdown. Furthermore, the polyplex was administered intravenously to lung carcinoma-bearing mice in which it caused 33% Rac1 knockdown. These promising results led to efficacy studies administering systemic treatment with an anticancer siRNA, siPlk1-polyplex, which inhibited tumor growth by 73% and 87% compared with siCtrl- polyplex or saline-treated mice, respectively, leading to prolonged overall survival. These findings represent the first time that a polyaminated poly(α)glutamate polymer is used for an efficacious and safe tumor delivery of RNAi following systemic administration. © 2017 Elsevier B.V. All rights reserved. Keywords: Polyplexes siRNA systemic delivery Cationic polymer Anticancer therapy Aminated poly(α)glutamate 1. Introduction RNA-guided regulation of gene expression, also known as RNA inter- ference (RNAi), holds immense potential to become a novel therapeutic approach in various diseases, among them cancer [1]. During the RNAi process, post-transcriptional gene downregulation occurs due to the presence of short sequences of double-stranded RNA, thus making this process sequence-dependent and specific to the gene of interest. Vari- ous attempts were initiated to use siRNAs as therapeutic agents but many hurdles have restricted their translation into the clinic [2,3]. These include rapid renal clearance, degradation by endogenous RN- ases, poor cellular penetration, immunogenicity, short half-life and ag- gregation. A non-viral delivery system can assist overcoming these limitations [4,5]. Such system can be a polycation nanocarrier that elec- trostatically interacts with the RNAi molecules, forming polyplexes. The polyelectrolyte complexes are self-assembled through interactions be- tween the cationic polymer and the negatively-charged siRNA. The nano-sized carriers can protect the siRNA from degradation by RNases and avert recognition by the immune system in the bloodstream [6], prolong their circulation time and enable extravasation-dependent ac- cumulation of the polyplex in the tumor due to the enhanced perme- ability and retention (EPR) effect [7,8]. This phenomenon is attributed to the leaky vessels and the poor lymphatic drainage at the tumor site allowing preferential accumulation of macromolecules and lipids. Once in the target site, positively-charged nanocarriers can facilitate cel- lular uptake of siRNA and safely deliver them to the cytoplasm. Journal of Controlled Release 257 (2017) 132–143 ⁎ Corresponding author at: Department of Physiology and Pharmacology, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv 69978, Israel. E-mail address: ronitsf@post.tau.ac.il (R. Satchi-Fainaro). 1 These authors contributed equally to this manuscript. http://dx.doi.org/10.1016/j.jconrel.2016.06.034 0168-3659/© 2017 Elsevier B.V. All rights reserved. Contents lists available at ScienceDirect Journal of Controlled Release journal homepage: www.elsevier.com/locate/jconrel