title: Stealth monoolein-based nanocarriers for delivery of siRNA to cancer cells.
abstract: While the delivery of small interfering RNAs (siRNAs) is an attractive strategy to treat several clinical conditions, 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 dioctadecyldimethylammonium 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 2h, by fluorescence Single Particle Tracking microscopy, PEG-coated lipoplexes were found to have better colloidal stability as they could maintain a relatively stable size. In addition, 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 colloidal stability in human serum, DODAB:MO (2:1) siRNA-lipoplexes coated with PEG-Cer are considered promising nanocarriers for further in vivo validation.
  
  
  CONCLUSIONS
  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 vectors. 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.
authors.affiliation: CBMA (Centre of Molecular and Environmental Biology), Department of Biology, University of Minho, Campus of Gualtar, 4710-057 Braga, Portugal; Centre of Physics, Department of Physics, University of Minho, Campus of Gualtar, 4710-057 Braga, Portugal.
authors.forename: Ana C N
authors.initials: AC
authors.lastname: Oliveira
authors.affiliation: Laboratory of General Biochemistry and Physical Pharmacy, Faculty of Pharmacy, Ghent University, Harelbekestraat 72, 9000 Ghent, Belgium.
authors.forename: Koen
authors.initials: K
authors.lastname: Raemdonck
authors.affiliation: Laboratory of General Biochemistry and Physical Pharmacy, Faculty of Pharmacy, Ghent University, Harelbekestraat 72, 9000 Ghent, Belgium; Center for Nano- and Biophotonics, Ghent University, Harelbekestraat 72, 9000 Ghent, Belgium.
authors.forename: Thomas
authors.initials: T
authors.lastname: Martens
authors.affiliation: Laboratory of General Biochemistry and Physical Pharmacy, Faculty of Pharmacy, Ghent University, Harelbekestraat 72, 9000 Ghent, Belgium.
authors.forename: Koen
authors.initials: K
authors.lastname: Rombouts
authors.affiliation: Immunology, Biomedical Research Center (CINBIO) and Institute of Biomedical Research of Vigo (IBIV), University of Vigo, Campus Lagoas Marcosende, 36310 Vigo, Pontevedra, Spain.
authors.forename: Rosana
authors.initials: R
authors.lastname: Simón-Vázquez
authors.affiliation: Centre of Biological Engineering (CEB), University of Minho, Campus of Gualtar, 4710-057 Braga, Portugal.
authors.forename: Cláudia
authors.initials: C
authors.lastname: Botelho
authors.affiliation: CBMA (Centre of Molecular and Environmental Biology), Department of Biology, University of Minho, Campus of Gualtar, 4710-057 Braga, Portugal; Centre of Physics, Department of Physics, University of Minho, Campus of Gualtar, 4710-057 Braga, Portugal.
authors.forename: Ivo
authors.initials: I
authors.lastname: Lopes
authors.affiliation: Centre of Physics, Department of Physics, University of Minho, Campus of Gualtar, 4710-057 Braga, Portugal.
authors.forename: Marlene
authors.initials: M
authors.lastname: Lúcio
authors.affiliation: Immunology, Biomedical Research Center (CINBIO) and Institute of Biomedical Research of Vigo (IBIV), University of Vigo, Campus Lagoas Marcosende, 36310 Vigo, Pontevedra, Spain.
authors.forename: África
authors.initials: Á
authors.lastname: González-Fernández
authors.affiliation: Centre of Physics, Department of Physics, University of Minho, Campus of Gualtar, 4710-057 Braga, Portugal.
authors.forename: M Elisabete C D
authors.initials: ME
authors.lastname: Real Oliveira
authors.affiliation: CBMA (Centre of Molecular and Environmental Biology), Department of Biology, University of Minho, Campus of Gualtar, 4710-057 Braga, Portugal. Electronic address: agomes@bio.uminho.pt.
authors.forename: Andreia C
authors.initials: AC
authors.lastname: Gomes
authors.affiliation: Laboratory of General Biochemistry and Physical Pharmacy, Faculty of Pharmacy, Ghent University, Harelbekestraat 72, 9000 Ghent, Belgium; Center for Nano- and Biophotonics, Ghent University, Harelbekestraat 72, 9000 Ghent, Belgium.
authors.forename: Kevin
authors.initials: K
authors.lastname: Braeckmans
mesh_terms: D001798:Blood Proteins / Q000378:metabolism; D045744:Cell Line, Tumor; D004337:Drug Carriers / Q000737:chemistry*; D020868:Gene Silencing; D005989:Glycerides / Q000737:chemistry*; D006801:Humans; D008081:Liposomes / Q000648:ultrastructure; D053758:Nanoparticles / Q000737:chemistry* / Q000648:ultrastructure; D009369:Neoplasms / Q000378:metabolism*; D010316:Particle Size; D011092:Polyethylene Glycols / Q000737:chemistry; D034741:RNA, Small Interfering / Q000378:metabolism*
chemical_list: D001798:Blood Proteins; D004337:Drug Carriers; D005989:Glycerides; D008081:Liposomes; D034741:RNA, Small Interfering; D011092:Polyethylene Glycols; C005953:monoolein
keywords: FFS; Monoolein; PEG; fSPT; siRNA delivery