16Positively charged ethylcelulose nanoparticles have been obtained from 17 alkylamidoammonium/Span 80 based nano-emulsion templates. Oil-in-water 18 polymeric nano-emulsions form in a broad range of oil-to-surfactant ratios and water 19 contents above 75 wt% by a low-energy method at 25ºC. Nano-emulsions with a 20 water content of 90 wt% showed droplet sizes typically below 300 nm and high 21 positive zeta potential values (~55 mV). If oleylamine is added to the system, smaller 22 droplet sizes and higher zeta potential values (~66 mV) are obtained, but the stability 23 of the nano-emulsions decreases. Although these nano-emulsions are destabilized 24 by creaming, the period of stability is large enough to allow nanoparticle preparation 25 by solvent evaporation. Polymeric nanoparticles obtained show a globular core-shell-26 like morphology, with mean diameters of around 250 nm. The surface charge of the 27 nanoparticles is similar to that of the nano-emulsion template and remains positive 28 after 24 hours dialysis, suggesting slow desorption kinetics of the 29 alkylamidoammonium from the nanoparticle surface. These results indicate that the 30 proposed nano-emulsion approach is a good strategy for the preparation of positively 31 charged nanoparticles from nonionic ethylcellulose polymers. 32 33 KEYWORDS 35 Cationic nano-emulsion; ethylcellulose nanoparticles; cationic nanoparticles; low-36 energy emulsifiaction 37 38 surface. This has been shown with polar molecules, such as PEG derivatives [10], 59 polysorbate 80 [11] or chitosan [12], which are usually added in a late step, after 60 nanoparticle preparation as an alternative to covalent linking. 61 62 A well-known method for nanoparticle preparation is nano-emulsion templating. The 63 preparation of nano-emulsions by low-energy methods is convenient because it 64 allows achieving smaller and more homogeneous droplet sizes than high energy 65 emulsification methods. Although the preparation of O/W nano-emulsions with 66 nonionic surfactants by low-energy emulsification methods has widely been studied alkylamidoammonium derivatives are a class of cationic surfactants which in spite of 95 their frequent use in the home and personal-care industry have not been explored yet, 96to our best knowledge, for biomedical applications. In contrast to their amidoamine 97 counterparts, they have the advantage of being permanently positively charged, 98 regardless of the pH of the dispersing medium, due to the quaternized ammonium 99 end group. In this paper we propose the preparation of positively charged 100 nanoparticles from ethylcellulose nano-emulsion templates formed in a mixed 101 cationic/nonionic surfactant system. The purpose of this paper is to explore the 102 4 feasibility of endowing positive surface charge to ethylcellulose nanoparticles 103 prepared by a low-energy emulsification-solvent evaporation method, by using a 104 cationic alkylamidoammonium surfactant to modulate the surface charge of the 105 nanoparticles, and assess its permanence when exposed to...

Oil-in-water nano-emulsions have been obtained in the HEPES 20 mM buffer solution / [Alkylamidoammonium:Kolliphor EL=1:1] / [6 weight % ethylcellulose in ethyl acetate] system over a wide oil-to-surfactant (O/S) range and above 35 weight% aqueous component at 25ºC. The nano-emulsion with an O/S ratio of 70/30 and 95 weight % aqueous component was used for nanoparticles preparation. These nanoparticles (mean diameter around 90 nm and zeta potential of +22 mV) were non-toxic to HeLa cells up to a concentration of 3 mM of cationic species. Successful complexation with an antisense phosphorothioate oligonucleotide targeting Renilla luciferase mRNA was achieved at cationic/anionic charge ratios above 16, as confirmed by zeta potential measurements and an electrophoretic mobility shift assay, provided that no Fetal Bovine Serum is present in the cell culture medium. Importantly, Renilla luciferase gene inhibition shows an optimum efficiency (40%) for the cationic/anionic ratio 28, which makes these complexes promising for "in vitro" cell transfection.

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