Characterisation of plasmid DNA conjugates as a basis for their processing

Abstract

Plasmid DNA complexes have been used by several research groups for gene therapy applications and have given promising results during preliminary clinical trials. However, their eventual adoption for the treatment of a wide range of genetic diseases requires considerable progress with regards to carrier formulation and processing. Amongst the key parameters that are known to be important and need further elucidation are the size and surface charge of the conjugates and the binding ef®ciency of the carrier to the DNA.The present study has focused on two synthetic carriers with different formulation and charge characteristics, both of which have been recommended in the literature as having the potential to deliver genes to target cells. These were a cationic lipid (DC-chol/DOPE) and a polylysinecondensed anionic liposome (OA/DOPE/Chol). Conjugate size, zeta potential, stability and binding ef®ciency were measured for conjugates of DNA plasmids of molecular weights between 6.9 kb and 29 kb. Plasmids were condensed by polylysine of two different molecular weights with or without cationic lipids.

The formation of plasmid DNA loaded pharmaceutical powders using supercritical fluid technology

“…For example, nonpolar buffer salts which are soluble in the ternary supercritical mixture may be carried out of the drying chamber together with the water and the organic solvent and hence may provide both stabilization and decreased formulation load. Alternatively, DNA complexation may stabilize against depurination (Wolfert and Seymor, 1996;Adami et al, 1998;Tsai et al, 1999). The SEDS process provides the flexibility to optimize product stability based on the understanding gained in this study.…”

Quantitative physical characterization of lipid–polycation–DNA lipopolyplexes

“…As previously observed, polycation/DNA or lipid/DNA particles tend to aggregate at high concentrations [25,27], at charge ratios near neutrality [27] and at high ionic strengths [27,28]. To avoid aggregation and reduce variability in size, we mixed the protamine/DNA using a syringe pump and worked at a ratio away from the isoelectric point.…”

Preparation of small unilamellar vesicles (SUV) and biophysical characterization of their complexes with poly‐l‐lysine‐condensed plasmid DNA

“…pDNA (pSVβ) was produced in Escherichia coli DH5α, extracted from the host cells and purified using the method described previously by Levy et al [19]. pDNA was condensed with PLL (Sigma–Aldrich) of molecular mass 29 kDa at a charge ratio of 2 by the method of Tsai et al [20] using an infuse/withdraw syringe pump (PDH 2000; Harvard Apparatus, Holliston, MA, U.S.A.) equipped with a T mixing head. The PDL complexes were formed by mixing PLL‐ DNA and liposomes vesicles (SUV) at the desired charge ratio using the same mixing apparatus.…”

“…Zeta potential and particle‐size measurements were made by laser doppler spectrometry using the Malvern Zetasizer 3000 (Malvern Instruments, Malvern, Worcs., U.K.). For zeta‐size measurements we used the method of Tsai et al [20]. Samples were injected into the in situ cuvette using 5 ml disposable syringes.…”

“…Any DNA released was separated from the system by extraction with phenol/chloroform/3‐methylbutan‐1‐ol (Amersham Pharmacia Biotech, Rainham, Essex, U.K.), followed by ethanol precipitation and resuspension in 1 ml of TE buffer (10 mM Tris/HCl/1 mM EDTA, pH 8). DNA released was assayed using Pico Green (Molecular Probes Inc.) [20]. The percentages of free and encapsulated PLL‐DNA were calculated in terms of the total DNA present in the complex, which was maintained constant at 2 μg in all of the experiments.…”