The influence of physico‐chemical and process conditions on the physical stability of plasmid DNA complexes using response surface methodology

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.

Gene delivery to human bone marrow mesenchymal stem cells by microporation

“…The first reported study regarding this topic aimed to demonstrate the utility of central composite design in modeling responses such as vector size, zeta potential and transfection efficiency of several non-viral gene delivery vectors (Birchall et al, 2001). Similarly other authors have verified the effect of several parameters and their interaction on physical stability of polyplexes (Gazori et al, 2009;Mount et al, 2003;Zhong et al, 2007). Regarding electroporation, and to our knowledge there is no reported study aiming at multivariate optimising procedure for in vitro gene delivery to human MSC, mainly considering cell recoveries or yield of transfection as output values.…”

Structural aspects of histone H1–DNA complexes and their relation to transfection efficiency

“…A new quality in the mechanistic investigations of the colloidal properties of polycation–DNA vectors has been introduced by Lee et al [24,25], Sarkar et al [26] and Mount et al [27]. These authors have impressively demonstrated the aggregation kinetics of polycation–DNA complexes, mainly polylysine–DNA or liposome/polylysine–DNA complexes.…”

Stable gene transfection mediated by polysulfobetaine/PDMAEMA diblock copolymer in salted medium

“…As the concentration of NaCl increases, the binding affinity of routine cationic polymers (PEI, chitosan, PLL) for DNA decreases and a salt induced aggregation may occur. This kind of aggregation is a consequence of the decrease in the repulsive electrical double layer (Debye-Huckel length) around the already-formed cationic complexes particles and occurs rapidly after mixing of the DNA solution and the cationic agent [20,[35][36][37]. Even a very little amount of NaCl solution added into the PDMAEMA 100 /DNA complex can induce rapid increase in absorbance of solution as shown in Fig.…”