Formulation Optimisation: Results and Discussion

An important goal for non-viral gene therapy is the development of stable delivery systems that can be injected into the blood stream and will deliver DNA to the appropriate tissues as well as being easy to characterise thereby gaining agency approval (Feigner, 1997). As noted by many authors it has proven difficult to obtain homogeneous synthetic gene delivery complexes with size distribution suitable for systemic injection due to instability in aqueous suspension and under physiological environments and therefore metastable preparations of plasmid complexes are used in the majority of studies (Anchordoquy, 1999a; Hong et a l , 1997). For example the binding of albumin may be enough to cause aggregation of delivery complexes in the blood stream by reducing their zeta potential and hence reducing the charge repulsion between the particles (Pouton and Seymour, 1998). However with many authors holding different views the ideal size remains undetermined. Much of the problem stems from the lack of correlation between the optimal formulation for in vitro and in vivo investigations and the differing requirements for the method of administration in vivo (Gorman et al. 1997). For instance studies have shown that in cell culture large complexes are more effective transfection agents than small complexes (Bally et a l,

1999). This is supported by in vitro studies, whereby the large complexes are thought to have higher tendency than small complexes to sediment, thereby facilitating cell

Chapter 4._____________________________________Formulation Optimisation: Results and Discussion

contact, especially in cultures of adherent cells. McLean et al. (1997) obtained data that showed that although clustering of lipopolyplexes occurred in the bloodstream, the aggregates were generally smaller than erythrocytes and often less than 1pm in diameter. In practice, however, even delivery of 50-100nm particles to the liver is proving to be elusive and poorly reproducible after i.v. administration (Pouton and Seymour, 1998). Authors such as Wasan and colleagues and Bally et at. report small particles size of less than 200nm to be a requirement for effective systemic bio­ distribution as larger aggregate structures are eliminated rapidly through uptake into the recticuloendothelial system (RES) (Bally et a l , 1999; Barratt, 1999; Hope et a/., 1998; Murphy et a l, 2000; Selby et a l, 2000; Verbann et a l , 2001; Wasan et a l,

1999). Experiments using size fractionated complexes have also shown that the highest levels of gene expression in all tissues were produced by complexes 200-450nm range (Smyth Templeton and Lasic, 1999). The prevailing view is that smaller and more well defined particle sizes will be more appropriate to overcome barriers to diffusion found

in vivo, especially for systemic i.v. administration (Murphy et a l , 2000).

The development of efficient non-viral gene delivery vehicles is reportedly hampered by an insufficient understanding of their physical characteristics. A better characterisation of complexes could lead to improved delivery methods and improve the marketability of gene therapies thereby reducing their dosage levels and making them more effective (Anchordoquy, 1999b). The study presented here highlights some of the problems associated with achieving the ideal small well-defined DNA complexes whilst producing viable products in terms of DNA dose and buffer composition. The experiments were carried out with either commercially available calf thymus DNA and poly-L-lysine or plasmid DNA and poly-L-lysine. Calf thymus DNA is a short DNA molecule that has been used here and by others (Deng and Bloomfield, 1999; Geall et a l, 1999; Shapiro et a l, 1969) to serve as an inexpensive and easily available reference for studying the behaviour of plasmid DNA during formulation.

The physical characteristics and biological activity (transfection rate for example) of given preparation has previously been shown to be dependent on a number of factors. The size and zeta potential of condensates such as polyplexes have been shown to be

Chapter 4._____________________________________Formulation Optimisation: Results and Discussion

dependent on the polymer/DNA ratio and is therefore an important factor in determining transfection efficiency (Verbaan et at., 2001). According to Jeong and Park (2002) the diameter of complex particles gradually decreases by increasing the charge ratio. Additionally, transfection efficiency is said to increase with increasing charge ratio that could therefore be explained by the structural compactness of the complexes thereby supporting the theory that small particles are optimum (Jeong and Park, 2002). For the purposes of this investigation previous work in laboratories at UCL has shown that a charge ratio of +2.0 was optimum for the preparation of small stable DNA/PLL complexes and has been used throughout work detailed here. Anchordoquy (1999a) showed that physical characteristics can depend on the precise mixing protocol, media composition and time allowed for complex formation. Murphy and colleagues reported that aggregation of DNA complexes normally occurs when attempting to prepare systems containing condensed DNA especially at concentrations and in physiologically compatible buffer systems that are appropriate for in vivo

administration. The effect of such factors on the aggregative characteristics of DNA/PLL complexes was assessed here on the basis o f particle size.

Figure 4.1 represents a typical sample of ctDNA/PLL complexes prepared at a charge ratio of +2.0 with a DNA concentration of 25jxgml‘^ in 20mM HEPES, pH 7.2 buffer solution. The complexes were prepared using the twin syringe pump with a tubing assembly with a 0.8mm internal diameter (ID). These are relatively mild conditions for complex preparation and do not represent the conditions that are likely to be experienced in the environment of the human body. Under these conditions it is possible to produce a very evenly distributed population with a mean diameter of less than lOOnm. It has previously been reported by M olas and colleagues (2002) that in certain conditions the process of DNA condensation, facilitated by electrostatic neutralisation could result in particles of DNA/PLL of approximately 20nm in diameter, significantly smaller than those generated here. These results demonstrate the ability to produce small, stable and well defined complexes. However in the remainder o f experiments detailed in this section a number o f formulation factors were varied resulting in a change to the normal size distribution and mean size represented in Figure 4.1.

Chapter 4. Formulation Optimisation: Results and Discussion