Influence of DNA condensation state on transfection efficiency in DNA/polymer complexes: An AFM and DLS comparative study

Abstract

Atomic force microscopy (AFM) is used to describe the formation process of polymer/DNA complexes. Two main objectives of this research are presented. The first one is to apply AFM as an effective tool to analyse DNA molecules and different polycation/DNA complexes in order to evaluate their degree of condensation (size and shape). The other one is to search for a relationship between the condensation state of DNA and its transfection efficiency. In this study, linear methacrylate based polymers and globular SuperFect polymers are used in order to induce DNA condensation. Ternary complexes, composed of methacrylate based polymers and polyethylene glycol (PEG)-based copolymers, are also investigated. AFM allows us to confirm good condensation conditions and relate them (or not) to transfection efficiencies. These AFM results (obtained after drying in air) are compared with measurements deduced from Dynamic Light Scattering (DLS) experiments performed in water. This comparison allowed us to identify the structural modifications resulting from deposition on the mica surface.

Introduction

The development of an efficient gene delivery vehicle is a major challenge for gene therapy. The DNA condensation process has thus drawn a large interest in biology the last decade. Two types of vectors of nucleic acids exist: viral agents (like recombinant retroviruses and adenoviruses (Ledley, 1996, Wilson, 1995)) and synthetic vectors (as cationic lipids and polymers). However, viral agents yet suffer from a limited success in delivering genes, partially due to the immune and toxic response they induce, but also because of biosafety problems (Miller, 2003, Maguire-Zeiss and Federoff, 2004, Feldman, 2003). To avoid these problems, non-viral carriers are presently investigated in many laboratories. For example, DNA condensation is achieved with proteins like protamine (Allen et al., 1997) or liposomes and lipid intermediates (Gao and Huang, 1996). Promising results are also obtained with polycationic polymers (van de Wetering et al., 1997, van de Wetering et al., 1998), such as poly-l-lysine (Choi et al., 1999, Toncheva et al., 1998) or protonated amino-functionalised polymethacrylate (Pirotton et al., 2004). Such polymer/DNA complexes are formed through electrostatic interactions between the negatively charged phosphate groups of DNA and protonated amino groups of the polymer.

Although polycationic polymers have some advantages over viral vectors, their efficiency as a transfecting agent is still limited (Itaka et al., 2003). Indeed, several biophysical requirements on such polyelectrolyte particles are encountered. Apart from the polymer characteristics (charge density, molecular weight), which have been previously discussed (Pirotton et al., 2004), we wondered if the physico-chemical properties of the formed polymer/DNA complexes influence the transfection efficiency process. Indeed, a physical view of the condensates (including their dimensions) related to a biological view (including transfection efficiency) could facilitate improvement in this gene delivery technology (Dunlap et al., 1997). In this context, atomic force microscopy (AFM) has proven to be an excellent tool having the ability to image soft biological structures like cells, bacteria or proteins (Radmacher et al., 1992, Razatos et al., 1998, Reich et al., 2001). DNA molecules, such as plasmids in particular, have been intensively studied and detailed structures of individual molecules have yet been revealed (Hansma et al., 1992, Yang et al., 1992, Lyubchenko et al., 1993, Argaman et al., 1997). Moreover, AFM measurements have the advantage on DLS experiments that they can be performed after deposition on any surface and have an intrinsic better resolution.

Polycationic polymethacrylate derived chains are sufficient to condense DNA in order to produce binary complexes capable of transfecting cells in culture. However, these chains alone do not protect the particles from the environment. The interactions between the serum proteins and the complexes often impair the transfection in vitro. In vivo, these interactions lead to the rapid elimination of the complexes by phagocytosing macrophages (Howard et al., 2000). These problems can be avoided by the introduction of hydrophilic sequences of poly(ethylene glycol) (PEG) at the surface of the complexes. Previous results indeed highlighted that cell transfection with binary complexes is feasible exclusively in the absence of serum and that, on the contrary, the PEGylated complexes allowed transfection even in the presence of serum. The presence of the hydrophilic sequences of PEG at the complex surface also improved the hemocompatibility properties of the complexes (Pirotton et al., 2004). These kinds of complexes are also analysed by AFM.

Finally, globular dendrimeric polymers are also used to condense DNA. Dendrimers represent a class of polymers that exhibit a molecular architecture characterized by regular dendritic branching with a radial symmetry (Frechet, 1994). Their structural difference compared to linear polycationic polymers induces different condensed structures, which are also observed and discussed in detail.

More precisely, in this research, linear methacrylate based polycationic polymers (PDMAEMA), PEGylated linear methacrylate based polycationic polymers and globular SuperFect dendrimers are used in order to induce DNA condensation. AFM data obtained on PDMAEMA/DNA binary complexes are exposed and discussed as a function of the polymer/DNA weight ratio and transfection efficiency. Characteristics of ternary complexes are then compared to the binary complex properties. Measurements obtained on these two kinds of complexes are thus compared to Dynamic Light Scattering data, obtained in wet conditions. The globular SuperFect/DNA structures are finally exposed. In order to appreciate the architecture of polymer/DNA complexes, an image of uncondensed dsDNA has been provided for comparison.