Abstract
Cationic solid lipid nanoparticles (SLN) have been recently proposed as non-viral vectors in systemic gene therapy. The aim of this study was to evaluate the effect of the protamine amount used as the transfection promoter in SLN-mediated gene delivery. Three protamine-SLN samples (Pro25, Pro100, and Pro200) prepared by adding increasing amounts of protamine were characterized for their size, zeta potential, and protamine loading level. The samples were evaluated for pDNA complexation ability by gel-electrophoresis analysis and for cytotoxicity and transfection efficiency by using different cell lines (COS-I, HepG2, and Na1300). The size of SLN was ~230 nm and only Pro200 showed few particle aggregates. Unlike the Pro25 sample with the lowest protamine loading level, the others SLN samples (Pro100 and Pro200) exhibited a good ability in complexing pDNA. A cell-line dependent cytotoxicity lower than that of the positive control PEI (polyethilenimmine) was observed for all the SLN. Among these, only Pro100, having an intermediate amount of protamine, appeared able to promote pDNA cell transfer, especially in a neuronal cell line (Na1300). In conclusion, the amount of protamine as the transfection promoter in SLN affects not only the gene delivery ability of SLN but also their capacity to transfer genes efficiently to specific cell types.
Highlights
Gene therapy requires the introduction of foreign DNA encoding a therapeutic gene into the target cells, using viral or non-viral delivery systems
Protamine loading In all solid lipid nanoparticles (SLN) samples both the total and the free protamine content increased with the increase of the initial amount of protamine (Table 1)
The protamine fraction associated to the nanocarriers, calculated as the difference between the total and the free content, increased significantly from 4.5% to 13% w/w by increasing the initial amount of protamine in Pro25 and Pro100, respectively, and it remained constant by further protamine increase (Pro200), reaching a plateau value
Summary
Gene therapy requires the introduction of foreign DNA encoding a therapeutic gene into the target cells, using viral or non-viral delivery systems. In order to promote the transfection, cationic non-viral vectors are described to facilitate an electrostatic interaction of the nanocarrier with both the negative backbone of DNA and the negative cell surface inducing unspecific endocythosis. For this reason, cationic lipids such as Esterquat 1 (EQ1) (N,N-di(β-stearoylethyl)-N,N-dimethylammonium chloride) or DOTAP (N-(1-(2,3-dioleoyloxy) propyl)-N,N,Ntrimethylammoniumchloride) have been used in the development of cationic gene delivery devices (Olbrich et al, 2001; del Pozo-Rodriguez et al, 2007). Protamine is a cationic nuclear protein enriched in arginine, FDA approved for the parenteral administration, and widely studied in the gene delivery (Tsuchiya et al, 2006) owing to its ability to improve DNA packaging in sperm cells (Braun, 2001), to protect
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