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Purpose: To study the stability of polymer-plasmid complexes (polyplexes) both as an aqueous dispersion and in their lyophilized form., Methods: The characteristics of the polyplexes (size, charge and transfection potential) were monitored at different temperatures. Moreover, we studied possible changes in the secondary and tertiary structure of the plasmid by agarose gel electrophoresis and by CD spectroscopy to gain insight into the mechanism of polyplex degradation., Results: The polyplexes preserved almost their full transfection potential after aging in an aqueous solution of 20 mM Hepes (pH 7.4) containing 10% sucrose at 4 and 20 degrees C for 10 months. On the other hand, the polyplexes aged at 40 degrees C were rather unstable and lost their transfection capability with a half-life of around 2 months. During storage, conformational changes in the secondary and tertiary structure of DNA were observed. When naked plasmid DNA was aged at 40 degrees C as an aqueous solution and complexed with polymer just before the transfection experiment, a slower drop in its transfection capability was observed. The freeze-dried polyplexes using sucrose as lyoprotectant almost fully retained their transfection efficiency, even when aged at 40 degrees C for 10 months., Conclusions: This study provides information about polyplex stability in aqueous dispersions on storage and demonstrates that freeze-drying is an excellent method to ensure long term stability.

In this paper the effect of the topology of plasmid DNA (supercoiled, open-circular and linear) on its binding characteristics with the polymeric transfectant poly((2-dimethylamino)ethyl methacrylate) was studied. The formed polyplexes were also evaluated for their transfection properties in vitro in two different cell lines. Anion-exchange chromatography was used for the separation of supercoiled and open-circular plasmid from a plasmid stock solution. Linear plasmids were prepared by endonucleases that cleaved the plasmid either in the promoter region or in a region not specific for expression (ampicillin resistance region). Plasmid DNA was also heat-denatured for 6 h at 70 degrees C, resulting in DNA mainly in the open-circular and oligomeric forms. The transfection of two different cell lines was dependent on the topology of the DNA in the order supercoiled>open-circular approximately heat-denatured>linear DNA prepared by cleaving in the nonspecific region>linear DNA prepared by cleaving in the promoter region. No differences in the size of the complexes or in the quenching of the DNA-intercalating fluorophore acridine orange were found as function of the topology. However, circular dichroism spectroscopy revealed differences between the topological plasmid species, both in the free form and in the presence of excess of cationic polymer.

The aim of this study was to gain insight into the formulation parameters affecting the size of poly((2-dimethylamino)ethyl methacrylate)-plasmid complexes (polyplexes). Experimental designs were applied to screen and optimize several variables, which may influence the complex size. In a screening design, it was demonstrated that at a fixed concentration of plasmid (40 micrograms/ml) after incubation with polymer, the size of the resulting polyplexes was highly dependent on the polymer/plasmid ratio as well as on the pH, viscosity (i.e. sucrose concentration) and ionic strength of the aqueous solution. However, the temperature, PEG 600 (up to 5% (v/v)) and Tween 80 (up to 0.2%) had a marginal effect on the size of the polyplexes. In an optimization design, the effect of the pH, polymer/plasmid ratio and Tween on the size of the polymer/plasmid complexes prepared at relatively high concentration of plasmid (50-200 micrograms/ml) was evaluated. Based on the results of the optimization design, a mathematical model was derived, which describes the relationship between the size of the polyplexes and the different formulation parameters. This model shows that even at high plasmid concentration (200 micrograms/ml), small sized polyplexes were formed at low pH and ionic strength, especially when the solution contains 20% (w/v) sucrose. This concentrated polyplex dispersion (polymer/plasmid ratio > 3/1 (w/w), 200 micrograms plasmid/ml) can be diluted down to 5 micrograms/ml plasmid without significant changes in particle size and transfection potential. At lower ratios, a growth in particle size was observed upon dilution of the complexes, which might also explain the low transfection efficiency of these polyplexes in vitro.

Purpose: The aim of this study was to gain insight into the relation between the physical characteristics of particles formed by a plasmid and a synthetic cationic polymer (poly(2-dimethylamino)ethyl methacrylate, PDMAEMA) and their transfection efficiency., Methods: The PDMAEMA-plasmid particles were characterized by dynamic light scattering (size) and electrophoretic mobility measurements (charge). The transfection efficiency was evaluated in cell culture (COS-7 cells) using a pCMV-lacZ plasmid coding for beta-galactosidase as a reporter gene., Results: It was shown that the optimal transfection efficiency was found at a PDMAEMA-plasmid ratio of 3 (w/w), yielding stable and rather homogeneous particles (diameter 0.15 micron) with a narrow size distribution and a slightly positive charge. Particles prepared at lower weight ratios, showed a reduced transfection efficiency and were unstable in time as demonstrated by DLS measurements. Like other cationic polymers, PDMAEMA is slightly cytotoxic. This activity was partially masked by complexing the polymer with DNA. Interestingly, the transfection efficiency of the particles was not affected by the presence of serum proteins., Conclusions: PDMAEMA is an interesting vector for the design of in vivo and ex vivo gene transfection systems.