ABSTRACT
We report the design and structural characterization of cationic diblock copolymer vesicles loaded with plasmid DNA based on a single emulsion technique. For this purpose, a DNA solution was emulsified in an organic solvent and stabilized by an amphiphilic diblock copolymer. The neutral block forms an interfacial brush, whereas the cationic attachment complexes with DNA. A subsequent change of the quality of the organic solvent results in the collapse of the brush and the formation of a capsule. The capsules are subsequently dispersed in aqueous medium to form vesicles and stabilized with an osmotic agent in the external phase. Inside the vesicles, the plasmid is compacted in a liquid-crystalline fashion as shown by the appearance of birefringent textures under crossed polarizers and the increase in fluorescence intensity of labeled DNA. The compaction efficiency and the size distribution of the vesicles were determined by light and electron microscopy, and the integrity of the DNA after encapsulation and subsequent release was confirmed by gel electrophoresis. We demonstrate reverse transfection of in vitro cultured HeLa cancer cells growing on plasmid-copolymer vesicles deposited on a glass substrate.
Subject(s)
DNA/administration & dosage , Gene Transfer Techniques , Plasmids/administration & dosage , Polyvinyls/chemistry , Cations , Cell Membrane Permeability , DNA/chemistry , DNA/genetics , Drug Stability , Emulsions , Green Fluorescent Proteins/genetics , HeLa Cells , Humans , Microscopy, Electron, Scanning , Microscopy, Phase-Contrast , Microscopy, Polarization , Plasmids/chemistry , Plasmids/genetics , TransfectionABSTRACT
The structure of spherical micelles of the diblock poly(styrene-block-acrylic acid) [PS-b-PA] copolymer in water was investigated up to concentrations where the polyelectrolyte coronal layers have to shrink and/or interpenetrate in order to accommodate the micelles in the increasingly crowded volume. We obtained the partial structure factors pertaining to the core and corona density correlations with small angle neutron scattering and contrast matching in the water. The counterion structure factor was obtained with small angle x-ray scattering (SAXS) with a synchrotron radiation source. Furthermore, we have measured the flow curves and dynamic visco-elastic moduli. The functionality of the micelles is fixed with a 9 nm diameter PS core and a corona formed by around 100 PA arms. As shown by the SAXS intensities, the counterions are distributed in the coronal layer with the same density profile as the corona forming segments. Irrespective ionic strength and micelle charge, the corona shrinks with increasing packing fraction. At high charge and minimal screening conditions, the polyelectrolyte chains remain almost fully stretched and they interdigitate once the volume fraction exceeds the critical value 0.53+/-0.02. Interpenetration of the polyelectrolyte brushes also controls the fluid rheology: The viscosity increases by three orders of magnitude and the parallel frequency scaling behavior of the dynamic moduli suggests the formation of a physical gel. In excess salt, the coronal layers are less extended and they do not interpenetrate in the present concentration range.
ABSTRACT
Encapsulation of dsDNA fragments (contour length 54 nm) by the cationic diblock copolymer poly(butadiene-b-N-methyl-4-vinyl pyridinium) [PBd-b-P4VPQ] has been studied with phase contrast, polarized light, and fluorescence microscopies, as well as scanning electron microscopy. Encapsulation was achieved with a single emulsion technique. For this purpose, an aqueous DNA solution is emulsified in an organic solvent (toluene) and stabilized by the amphiphilic diblock copolymer. The PBd block forms an interfacial brush, whereas the cationic P4VPQ block complexes with DNA. A subsequent change of the quality of the organic solvent results in a collapse of the PBd brush and the formation of a capsule. Inside the capsules, the DNA is compacted as shown by the appearance of birefringent textures under crossed polarizers and the increase in fluorescence intensity of labeled DNA. The capsules can also be dispersed in an aqueous medium to form vesicles, provided they are stabilized with an osmotic agent [poly(ethylene glycol)] in the external phase. It is shown that the DNA is released from the vesicles once the osmotic pressure drops below 10(5) N/m(2) or if the ionic strength of the supporting medium exceeds 0.1 M. The method has also proven to be efficient to encapsulate pUC18 plasmid in submicrometer-sized vesicles, and the general applicability of the method has been demonstrated by the preparation of the charge inverse system: cationic poly(ethylene imine) encapsulated by the anionic diblock poly(styrene-b-acrylic acid).
ABSTRACT
The structure of spherical micelles of the diblock copolymer poly(styrene-block-acrylic acid) in water was investigated with small angle neutron scattering and contrast matching. We have monitored intermicelle correlation and the extension of the polyelectrolyte chains in the coronal layer through the overlap concentration. Irrespective of ionic strength, the corona shrinks with increasing packing fraction. Furthermore, at high charge and minimal screening conditions, the corona layers interpenetrate once the volume fraction exceeds the critical value 0.53+/-0.02.
