Nanoemulsion-templated polylelectrolyte multifunctional nanocapsules for DNA entrapment and bioimaging.

The emerging field of bionanotechnology aims at advancing colloidal and biomedical research via introduction of multifunctional nanoparticle-based containers intended for both gene therapy and bioimaging. In the present contribution we entrapped the model genetic material (herring testes DNA) in the newly-designed non-viral vectors, i.e., multifunctional nanocapsules obtained by layer-by-layer (LbL) adsorption of DNA and oppositely charged polysaccharide-based chitosan (CHIT) on the nanoemulsion core, loaded by IR-780 indocyanine (used as the fluorescent marker) and stabilized by gemini-type ammonium salts: N,N,N',N'-tetramethyl-N,N'-di(dodecyl)-ethylenediammonium bromide, d(DDA)PBr and N,N,N',N'-tetramethyl-N,N'-di(dodecyl)-butylenediammonium d(DDA)BBr. Ternary phase diagrams of the surfactant-oil-water systems were determined by titration method. Then, the stability of the nanoemulsions obtained with IR-780 solubilized in the oleic acid (OA) or isopropyl myristate (IPM) phase was evaluated by backscattering (BS) profiles and ζ-potential measurements. In the next step, CHIT and DNA layers were subsequently deposited on the kinetically stable nanoemulsion cores. The IR-780-loaded nanocarriers covered by (DNA/CHIT)4 bilayers shown the high ζ-potential value (about +43mV provided by Doppler electrophoresis), the size <120nm and the spherical shape as analyzed by dynamic light scattering (DLS), atomic force microscopy (AFM) and scanning electron microscopy (SEM). Finally, the long-lasting nanosystems were subjected to in vitro biological studies on human cancer cell lines - doxorubicin-sensitive breast (MCF-7/WT), epithelial lung adenocarcinoma (A549) and skin melanoma (MEWO). Biological response of the cell culture was expressed as cytotoxic activity evaluated by MTT-based proliferation assay as well as bioimaging of intracellular localization of IR-780 molecules loaded in the multilayer DNA-deposited nanocontainers - provided by confocal laser scanning microscopy (CLSM) and total internal reflection fluorescence microscopy (TIRFM). Our results demonstrate that the fabricated oil-core CHIT-coated nanocapsules stabilized by both d(DDA)PBr and d(DDA)BBr surfactants are promising as multifunctional nanocarriers for DNA delivery and cancer diagnostics.