ABSTRACT
Gene therapy is expected to treat various incurable diseases including viral infections, autoimmune disorders, and cancers. Cationic lipids (CL) have been used as carriers of therapeutic DNAs for gene therapy because they can form a complex with DNA and such a complex can be incorporated into cells and transport the bound DNA to cytosol. The CL/DNA complexes are called lipoplexes and categorized as a non-viral vector. Lipoplexes are often prepared by adding a neutral phospholipid dioleoylphosphatidylethanolamine (DOPE) to CL in order to enhance transfection. However, the role of DOPE is not fully understood. We synthesized a new CL having an ethylenediamine cationic head group, denoted by DA, and found that addition of DOPE to DA achieved a good efficiency, almost in the similar level of commonly used transfection reagent Lipofectamine 2000 (Invitrogen). The composition of DA:DOPE=1:1 showed the highest efficiency. This lipoplex showed structural transition when pH was changed from 7 to 4, corresponding pH lowering in late endosome, while DOPE itself showed structural transition at more basic pH around 8. The present data showed that the DOPE/DA composition determines the structural transition pH and choosing a suitable pH, i.e., a suitable composition, is essential to increase the transfection efficiency.
Subject(s)
Ethylenediamines/chemistry , Phosphatidylethanolamines/chemistry , Biophysics/methods , Cations , DNA/chemistry , Endocytosis , Endosomes/metabolism , Gene Expression Regulation , Genetic Therapy/methods , Hep G2 Cells , Humans , Hydrogen-Ion Concentration , Lipids/chemistry , Models, Chemical , Phospholipids/chemistry , Plasmids/metabolism , Scattering, Radiation , Transfection , X-RaysABSTRACT
We synthesized new calix[4]arene-based lipids, denoted by CPCaLn, bearing the choline phosphate (CP) group which is an inverse phosphoryl choline (PC) structure. Small-angle X-ray scattering and multi-angle light scattering coupled with field flow fractionation showed that these lipids form monodisperse micelles with a fixed aggregation number and diameters of 1.9 and 2.6 nm for lipids bearing C3 and C6 alkyl tails, respectively. Furthermore, when CPCaLn was mixed with the fluorescein isothiocyanate (FITC)-bearing lipids and added to cells, strong fluorescence was observed at 37 °C, but not at 4 °C, indicating that the micelles were taken up by the cells through endocytosis. Recent studies have shown that replacement of polymer-attached PC groups with CP groups markedly promotes cellular uptake, even though the surface charge is neutral. On the basis of the idea, CPCaLn micelles interacted with cells in the same way, suggesting that the micelles bearing CP groups are expected to use as carriers in the drug delivery system.
Subject(s)
Calixarenes/chemistry , Micelles , Phospholipids/chemistry , Phosphorylcholine/chemistry , Calixarenes/chemical synthesis , Cell Line , Endocytosis , Fluorescein-5-isothiocyanate/chemistry , Humans , Static ElectricityABSTRACT
The size, surface charge, and microstructure of lipoplexes comprising cationic lipids and nucleic acids are important factors for transfection efficiency. As these properties are largely determined by the cationic lipids used, a number of studies on the relationship between cationic lipids and the transfection efficiency have been reported. Among the many cationic lipids, lipids with multivalent cationic head groups are expected to be potent transfection reagents. Here, we prepared calix[4]arene-based lipids with different alkyl chain lengths from C3 to C15 and evaluated the relationship between the alkyl chain length and the transfection efficiency. C6 lipoplexes exhibited the highest transfection efficiency among all lipoplexes. The gene expression with C9 and C12 lipoplexes was slightly lower than that with C6 lipoplexes. C3 lipoplexes hardly induced gene expression, while C15 lipoplexes exhibited no complexation with plasmid DNA. Although all lipoplexes exhibited nearly identical characteristics, they exhibited different behaviours in terms of the interactions between the lipoplexes and anionic micelles comprising phosphatidylserine, a model of endosomal vehicle. After mixing with phosphatidylserine micelles, C6 lipoplexes released the bound plasmid DNA at pH 5 but not at pH 7, indicating that they can interact with the late endosomal membrane after being incorporated into cells. No plasmid DNA was released from C9 or C12 lipoplexes at either pH values. Thus, the alkyl chain length of cationic lipids is related to their interaction with the endosomal compartment and can provide a basis for the design of novel transfection reagents.