Stealth monoolein-based nanocarriers for delivery of siRNA to cancer cells Ana C.N. Oliveira a,b , Koen Raemdonck c , Thomas Martens c,d , Koen Rombouts c , Rosana Simón-Vázquez e , Cláudia Botelho f , Ivo Lopes a,b , Marlene Lúcio b , África González-Fernández e , M. Elisabete C.D. Real Oliveira b , Andreia C. Gomes a,⇑ , Kevin Braeckmans c,d a CBMA (Centre of Molecular and Environmental Biology), Department of Biology, University of Minho, Campus of Gualtar, 4710-057 Braga, Portugal b Centre of Physics, Department of Physics, University of Minho, Campus of Gualtar, 4710-057 Braga, Portugal c Laboratory of General Biochemistry and Physical Pharmacy, Faculty of Pharmacy, Ghent University, Harelbekestraat 72, 9000 Ghent, Belgium d Center for Nano- and Biophotonics, Ghent University, Harelbekestraat 72, 9000 Ghent, Belgium e Immunology, Biomedical Research Center (CINBIO) and Institute of Biomedical Research of Vigo (IBIV), University of Vigo, Campus Lagoas Marcosende, 36310 Vigo, Pontevedra, Spain f Centre of Biological Engineering (CEB), University of Minho, Campus of Gualtar, 4710-057 Braga, Portugal article info Article history: Received 15 January 2015 Received in revised form 13 July 2015 Accepted 23 July 2015 Available online xxxx Keywords: FFS fSPT Monoolein PEG siRNA delivery abstract While the delivery of small interfering RNAs (siRNAs) is an attractive strategy to treat several clinical con- ditions, siRNA-nanocarriers’ stability after intravenous administration is still a major obstacle for the development of RNA-interference based therapies. But, although the need for stability is well recognized, the notion that strong stabilization can decrease nanocarriers’ efficiency is sometimes neglected. In this work we evaluated two stealth functionalization strategies to stabilize the previously validated dioctade- cyldimethylammonium bromide (DODAB):monoolein (MO) siRNA-lipoplexes. The nanocarriers were pre- and post-pegylated, forming vectors with different stabilities in biological fluids. The stealth nanocarriers’ behavior was tested under biological mimetic conditions, as the production of stable siRNA-lipoplexes is determinant to achieve efficient intravenous siRNA delivery to cancer cells. Upon incubation in human serum for 2 h, by fluorescence Single Particle Tracking microscopy, PEG-coated lipo- plexes were found to have better colloidal stability as they could maintain a relatively stable size. In addi- tion, using fluorescence fluctuation spectroscopy, post-pegylation also proved to avoid siRNA dissociation from the nanocarriers in human serum. Concomitantly it was found that PEG-coated lipoplexes improved cellular uptake and transfection efficiency in H1299 cells, and had the ability to silence BCR-ABL, affecting the survival of K562 cells. Based on an efficient cellular internalization, good silencing effect, good siRNA retention and good col- loidal stability in human serum, DODAB:MO (2:1) siRNA-lipoplexes coated with PEG-Cer are considered promising nanocarriers for further in vivo validation. Statement of Significance This work describes two stealth functionalization strategies for the stabilization of the previously validated dioctadecyldimethylammonium bromide (DODAB):monoolein (MO) siRNA-lipoplexes. These nanocarriers are capable of efficiently incorporating and delivering siRNA molecules to cells in order to silence genes whose expression is implicated in a pathological condition. The main objective was to functionalize these nanocarriers with a coating conferring protection to siRNA in blood without compromising its efficient delivery to cancer cells, validating the potential of DODAB:MO (2:1) siRNA-lipoplexes as therapeutic vec- tors. We show that the stealth strategy is determinant to achieve a stable and efficient nanocarrier, and that DODAB:MO mixtures have a very promising potential for systemic siRNA delivery to leukemic cells. Ó 2015 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.actbio.2015.07.032 1742-7061/Ó 2015 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved. Abbreviations: CLSM, confocal laser scanning microscopy; CML, chronic myeloid leukemia; DLS, dynamic light scattering; DODAB, dioctadecyldimethylammonium bromide; DSPE–PEG, poly(ethylene glycol) 2000 –distearoyl-phosphatidylethanolamine; FFS, fluorescence fluctuation spectroscopy; DG, free energy of Gibbs; fSPT, fluorescence Single Particle Tracking; HSA, human serum albumin; K b , binding constant; MO, 1-monooleoyl-rac-glycerol, monoolein; PEG, poly(ethylene)glycol; PEG-Cer, Poly(ethylene glycol) 2000 C(8)ceramide; PEI, polyethylenimine; f-potential, zeta potential. ⇑ Corresponding author. E-mail address: agomes@bio.uminho.pt (A.C. Gomes). Acta Biomaterialia xxx (2015) xxx–xxx Contents lists available at ScienceDirect Acta Biomaterialia journal homepage: www.elsevier.com/locate/actabiomat Please cite this article in press as: A.C.N. Oliveira et al., Stealth monoolein-based nanocarriers for delivery of siRNA to cancer cells, Acta Biomater. (2015), http://dx.doi.org/10.1016/j.actbio.2015.07.032