ABSTRACT
Comblike polyethylenimines with varying degrees of polymerization of both the main and side chains as well as different grafting densities were evaluated as gene delivery vectors. They were able to condense linear and plasmid DNA into nanosized polyplex particles with dimensions and surface potentials in the 130-330 nm and -30 to +15 mV ranges, respectively, depending on the amine/phosphate (N/P) ratio. The polyplexes remained stable in aqueous and buffer solutions from several hours up to several days. The moderate colloidal stability was also manifested in a relatively broad size distribution (PDI typically above 0.2) and structural polymorphism observed by transmission electron microscopy. Both the neat polymers and polyplexes displayed low cytotoxicity in WISH cells as the relative cell viability was more than 60%. Experiments with lysosomal fluorescence staining revealed that the internalization pathways and, in turn, transfection efficiency of the polyplex nanoparticles depended on the polymer chain topology. The vector systems based on the polymers of denser structure can be considered to be promising systems for gene transfection in eukaryotic cells.
Subject(s)
Endocytosis , Polyethyleneimine/chemistry , Polyethyleneimine/toxicity , Transfection , Flow Cytometry , Microscopy, Electron, Transmission , SolubilityABSTRACT
We report on novel gene delivery vector systems based on hybrid polymer-magnetic micelles. The hybrid micelles were prepared by codissolution of hydrophobically surface modified iron oxide and amphiphilic polystyrene-b-poly(quaternized 2-vinylpyridine) block copolymer (PS-b-P2QVP) in organic solvent. After extensive dialysis against water, micelles with positively charged hydrophilic corona of PQVP and hydrophobic PS core were prepared, in which magnetic nanoparticles were randomly distributed. The hybrid micelles were used to form complexes with linear (salmon sperm, 2000 bp, corresponding to M(w) of 1.32 × 10(6) Da) and plasmid (pEGFP-N1, 4730 bp, corresponding to M(w) of 3.12 × 10(6) Da) DNA. The resulting magnetopolyplexes of phosphate:amine (P/N) ratios in the 0.05-20 range were characterized by light scattering, ζ-potential measurements, and transmission electron microscopy as well as cytotoxicity and gel retardation assays. The investigated systems displayed a narrow size distribution, particle dimensions below 360 nm, whereas their ζ-potential values varied from positive to negative depending of the P/N ratio. The resulting vector nanosystems exhibited low toxicity. They were able to introduce pEGFP-N1 molecules into the cells. The application of a magnetic field markedly boosted the transgene expression efficiency of the magnetopolyplexes, which was even superior to those of commercial transfectants such as Lipofectamine and dendritic polyethylenimine.
Subject(s)
Gene Expression , Genetic Vectors , Magnetic Fields , Nanoparticles/chemistry , Polystyrenes/chemistry , Polyvinyls/chemistry , Transfection/methods , Animals , Cell Line , DNA/chemistry , Green Fluorescent Proteins/biosynthesis , Humans , Materials Testing , Micelles , SalmonABSTRACT
Six high molar mass polyglycidol samples were obtained by fractionation of polyglycidol synthesized by means of cationic polymerization of ethoxyethyl glycidyl ether followed by cleavage of the protective groups. The fractions covering the molar mass range from 0.1 to 2.4 x 10(6) were studied by dynamic and static light scattering. The weight-average molar masses (Mw), second virial coefficients (A2), radii of gyration (Rg), diffusion coefficients (D0), hydrodynamic radii (Rh), and dynamic virial coefficients (kDphi) were determined for the single coil in dilute aqueous solution at 25 degrees C, and scaling equations were established. It was found that polyglycidol in water does not exhibit the expected asymptotic good solvent behavior. The scaling exponents for A2, D0, and Rh are even closer to those for polymer coils in marginal solvents than to the expected ones in the excluded-volume region. The values of the interpenetration parameter, psi, and kDphi are far from reaching limiting values even for the fractions of the highest molar masses. The scaling exponent for Rg as well as the Rg/Rh ratio, which was found to increase with increasing molar mass, imply elongated coil conformation in the high molar mass region.
