ABSTRACT
Research is progressing fast to find safe and effective methods of delivering therapeutic genes to patients afflicted with a range of genetic and acquired diseases that either do not respond at all, or respond poorly, to treatment with small-molecule drugs or protein-replacement therapy. A technical barrier that remains relates to the need for scalable operations that can consistently and reproducibly make large quantities of the therapeutic gene vectors under the current Good Manufacturing Practice ('cGMP'). The present investigation focuses on these issues and introduces a new method of assessing the engineering effects of process and material factors on the colloidal properties of plasmid-DNA delivery systems based on response surface methodology (RSM) and experimental techniques. Previously, experiments have shown that several factors can reduce the physical stability of non-viral delivery systems. Specifically, it has been demonstrated that the mean size and charge of plasmid DNA condensed by cationic agents are affected by many factors, including the pH and ionic strength of the buffer, and the method of preparation. For example, the method and intensity of mixing of the DNA with condensing and conjugating agents have been shown to be important. Using RSM to analyse new experimental data in the present paper, we report on the impact of these factors and, more crucially, the effects of interaction between the factors on the colloidal properties of the DNA-vector complexes. Specifically, for plasmid DNA condensed by poly-L-lysine, interactions between ionic strength, pH and DNA concentrations play a critical role. Whether poly-L-lysine should be used as a condensing agent in the final delivery system remains to be demonstrated. However, the use of RSM combined with the scaleable experimental approach described in this paper may be applied to other delivery systems.
Subject(s)
Colloids/chemistry , Combinatorial Chemistry Techniques/methods , Drug Delivery Systems/methods , Drug Stability , Liposomes/chemistry , Materials Testing/methods , Plasmids/chemistry , Polylysine/chemistry , Hydrogen-Ion Concentration , Particle Size , Plasmids/administration & dosage , Salts/chemistry , Statistics as TopicABSTRACT
BACKGROUND: The formulation of polycationic complexes containing plasmid DNA for optimal transfection in vitro and in vivo for DNA vaccination, gene therapy and other applications continues to be a major research goal. Here we present new data on the biophysical properties of an integrin-targeted plasmid DNA (LID) formulation. MATERIAL/METHODS: Two plasmids (D), pEGFP (4.7 kb) and pCI-luc (5.7 kb), were mixed with a synthetic a5b1 integrin-targeted peptide (I), [K]16 GACRRETAWACG, in the presence of a cationic liposome (L), Lipofectin, composed of DOTMA and DOPE to form LID complexes. The physical properties of the complexes were measured using a variety of techniques including dynamic light scattering and fluorescence methods. The in vitro gene delivery to neuroblastoma cells with LID complexes was also assessed. RESULTS: We demonstrate the effects of complex size and charge ratio on in vitro transfection of mouse (Neuro-2A) and human (IMR-32) neuroblastoma cells. We report a significant increase in the level of luciferase and green fluorescent protein expression when transfection is performed in buffers of physiological ionic strength and hypothesise that the enhancement in transfection is caused by an increase in the size of the complexes observed during mixing and maturation. CONCLUSIONS: Cell transfection is also shown to be dependent on complex size and charge ratio, with large complexes prepared at charge ratios above 4.0 demonstrating efficient transfection.
