Physical properties and in vitro transfection efficiency of gene delivery vectors based on complexes of DNA with synthetic polycations
Introduction
Gene therapy is a rapidly progressing technology devized for the treatment of a variety of diseases [1]. Since naked DNA is successfully used only for local delivery, but is not suitable for systemic delivery into distant targets in vivo due to its degradation by serum nucleases, delivery systems have to be used. There are some requirements that an efficient gene delivery system has to fulfill: (a) biocompatibility and non-immunogenicity, (b) good stability in the bloodstream, (c) protection of the DNA from degradation during transport, (d) avoidance of uptake by components of the reticuloendothelial system (RES), (e) a size small enough for effective extravasation, (f) tissue and cell specificity, (g) effective endosomal escape after entering the target cell and avoidance of degradation by lysosomal enzymes and (h) transportation of the DNA to the nucleus. Three basic types of vectors for gene delivery are being intensively studied: viral vectors, cationic liposomes and lipids, and polycation/DNA complexes—each having its advantages and disadvantages [2], [3]. Viruses are considered to be the most efficient gene delivery vectors. Their use in clinical treatment, however, is associated with the risk of an immune response against viral particles and of random integration mediated by viruses, or their recombination with wild-type viruses [4].
Our research is focused on the development of non-viral polycation/DNA complexes prepared by self-assembly of polyions in solution. The advantage of using synthetic polycations for self-assembly with DNA is based on an easy, cheap and reproducible synthesis of such material. The possibility of optimization of the structure of the polycations by selecting respective monomers or optimum composition of copolymers, of changes in molecular weight or the density of positive charge along the polymer chain as well as of modification of the polycation/DNA complexes with biologically active molecules are some of the most important factors predetermining synthetic polymers for successful use in the preparation of gene delivery vectors. Further improvement of membrane transport of the non-viral gene delivery vectors based on polycation/DNA complexes can be achieved by the introduction of targeting moieties and fusogenic groups into complex structures [5], [6], [7]. It was shown that the self-assembly of DNA with polycations results in DNA protection from enzymatic degradation [5]. The chemical structure and molecular weight of the polycations significantly affect the stability of polycation/DNA complexes in inorganic salts [8], [9], their dissociation in the presence of strong polymer acids [10], [11] and in vitro transfection activities of the complexes [12], [13].
In this study we have systematically examined biophysical properties of complexes formed by self-assembly of DNA with synthetic linear polycations bearing primary and tertiary amino or quaternary ammonium groups (Scheme 1), with the aim of improving selected properties of polycation/DNA vectors. Size, shape, density and selected physicochemical properties of the complexes were characterized by atomic force microscopy as well as by static and dynamic light scattering methods. The ability of the PEC vectors to protect DNA from release and degradation during transport on the one hand, and to release DNA in the cytoplasm or nucleus on the other, was investigated in models by testing the dissociation of cooperative binding of various PECs in NaCl solutions and buffers in correlation with the results of in vitro cell transfection experiments.
Section snippets
Chemicals
2-(Dimethylamino)ethyl methacrylate (DMAEM), methacryloyl chloride (MACl) (freshly distilled), triethylamine (TEA), ethylenediamine (EDA), 2-(dimethylamino)ethylamine (DMAEA), di-tert-butyl dicarbonate, trifluoroacetic acid, methyl chloride, acetonitrile, dimethylformamide (DMF), 2,2′-azobisisobutyronitrile (AIBN) and ethidium bromide were from Fluka A.G., Prague, Czech Republic. 2,4,6-Trinitrobenzene-1-sulfonic acid (TNBSA) was purchased from Serva, Heidelberg, Germany. Calf thymus DNA (sodium
Characterization of the dissociation of partially ionized polycations
The dependence of the apparent dissociation constant (pKapp) of PLL, PAEMA, PDMAEMA and PDMAEM on the degree of its ionization (α) in water is shown in Fig. 1. The lowest values of pKapp were found for PDMAEM, which can be assigned to cyclic conformations of ionized and non-ionized tertiary aminoalkyl esters [16]. In the region of low values of α (pH>7) the basicity increases in the order: PDMAEMA<PAEMA<PLL.
Formation of the complexes investigated by fluorescence assay
N/P ratios characterizing complete DNA condensation, N/Pcc, obtained by the ethidium
Conclusions
Physicochemical characteristics, stability in aqueous solutions and in vitro transfection activity of polycation/DNA complexes, for use as gene delivery vectors, can be controlled by proper choice of the polycation structure and the conditions for their self-assembly with DNA. An appropriate choice of conditions for polycation/DNA complex preparation results in condensed, small and stable particles fulfilling requirements for extravasation of the vectors from circulation.
In vitro transfection
Acknowledgements
This work was supported by the Grant Agency of the Czech Republic (grant No. 307/96/K226), the European Union Program (QLK6-2000-00280) and the Grant Agency of the Academy of Sciences of the Czech Republic (A1050101). The authors thank Dr Pientka for his kind help with obtaining AFM images, Dr Wyatt for the kind loan of a multiangle light scattering detector DAWN DSP-F (Wyatt Technology Corp., Santa Barbara USA), Mrs Oupicka for technical assistance and Dr R. Dales for careful reading of the
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