Enhancement of polymethacrylate-mediated gene delivery by Penetratin

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Abstract

Polymethacrylates are vinyl-based polymers that are used for DNA transfection. Cationic polymethacrylates efficiently condense DNA by forming inter-polyelectrolyte complexes. Their use for DNA transfection is, however, limited due to their low ability to interact with membranes. In order to increase their transfection efficiency, we combined polymethacrylates with Penetratin, a 16-residue water-soluble peptide that internalises into cells through membrane translocation. DNA condensation was assessed using physicochemical methods, while transfection efficiency and cellular internalisation were studied using Cos-1 cells. Agarose gel electrophoresis retardation, ethidium bromide exclusion tests and dynamic light scattering measurements showed that the stability of the polymethacrylate–DNA complexes is not affected by addition of Penetratin. Transfection efficiency of polymethacrylate–DNA complexes into Cos-1 cells increased by addition of Penetratin and was higher than that of polyethylenimine (PEI)–DNA complexes and comparable to Lipofectamine™. Confocal microscopy and flow cytometry indicated that Penetratin mainly enhances endolysosomal escape polymethacrylate–DNA complexes and increases their cellular uptake. Since the cellular toxicity of polymethacrylate–DNA–Penetratin complexes remains low, especially compared to PEI, this transfection system opens new perspectives for gene therapy.

Introduction

Gene therapy consists in the delivery of polynucleotides into cells, in order to alter the expression of a given protein, thus resulting into a therapeutic benefit. The success of this strategy depends upon the efficiency of cellular delivery and expression of the gene. Spontaneous cellular uptake of polynucleotides is hampered by their negative charge, large hydrodynamic volume and degradation by nucleases (Bally et al., 1999, Garnett, 1999). In order to overcome these problems, polynucleotides have to be combined with a transfection vector.

Although viruses are at present the most efficient vectors, their use is restricted due to limitations in the DNA size, immunogenicity and putative long-term risks (Schmidt-Wolf and Schmidt-Wolf, 2003). Consequently, non-viral synthetic vectors have been developed, including cationic liposomes (Behr, 1994, Felgner et al., 1987) and polymeric gene delivery vectors (De Smedt et al., 2000, Garnett, 1999), with polyethylenimine (PEI) as one of the most successful polymers (Boussif et al., 1995). DNA is condensed and protected against degradation by formation of inter-polyelectrolyte complexes (IPEC) with partially protonated PEI (Godbey et al., 2000). The relatively high transfection efficiency of PEI-based complexes is explained by the ‘proton sponge’ theory (Behr, 1997). The positively charged complexes are taken up by cells via endocytosis (Godbey et al., 1999a, Godbey and Mikos, 2001a, Rémy-Kristensen et al., 2001) and are further protonated at low endosomal pH. The resulting influx of protons and chloride counter-ions creates an osmotic effect and subsequent endosomal swelling. Ultimately, PEI–DNA complexes are released in the cytosol. Besides polymer interactions with the plasma membrane, endosomal escape, thus represents a critical step in cellular transfection (Pouton and Seymour, 2001).

As the efficiency of PEI transfection is limited by its significant cellular toxicity (Dubruel et al., 2003, Godbey et al., 2001b), we synthesised a series of non-toxic polymethacrylates containing functional side groups with varying pKa, that efficiently condense DNA (Dubruel et al., 2000, Dubruel et al., 2002). In spite of their buffering capacity in the endosomal pH-range (pH 5.5–7.4), the transfection efficiency of these polymers remained lower than that of PEI (Dubruel et al., 2003). Confocal microscopy experiments showed that cellular internalisation and endosomal release of polymethacrylate–DNA complexes remains slow compared to PEI (Dubruel et al., 2004). As polymethacrylates are less haemolytic and cytotoxic than PEI, these data suggest that, besides endosomal buffering, membrane interactions probably modulate the efficiency of polymethacrylate-mediated transfections.

Wagner et al. previously used influenza virus hemagglutinine HA-2 derived peptides to enhance the transfection efficiency of polylysine–DNA complexes (Wagner, 1999). In the current work, polymethacrylates were combined with Penetratin, in an attempt to increase the transfection efficiency on Cos-1 cell cultures. Penetratin is a non-toxic 16-residue peptide derived from the third helix of the DNA-binding domain of the Drosophila Antennapedia homeoprotein (Derossi et al., 1994). Penetratin belongs to the family of water-soluble cell-penetrating peptides (CPP) with low lytic activity, which has been used as vectors for cellular internalisation of hydrophilic biomolecules (Derossi et al., 1998, Lindgren et al., 2000, Stephens and Pepperkok, 2001). The mechanism for cellular internalisation of Penetratin has not been unravelled yet. Penetratin interacts with negatively charged phospholipid vesicles and translocates through phospholipid bilayers with minimal perturbation of the bilayer integrity (Binder and Lindblom, 2003, Drin et al., 2001, Drin et al., 2003, Christiaens et al., 2002, Christiaens et al., 2004, Persson et al., 2004, Terrone et al., 2003). The energy- and receptor-independent cellular internalisation of this non-toxic peptide is attributed to interactions with the plasma membrane, followed by membrane translocation (Prochiantz, 1996). At 37 °C, endosomal escape further contributes to the efficient uptake of Penetratin (Fischer et al., 2004). The tryptophan residues and the positively charged residues seem to be crucial for both the interactions with lipid bilayers and for the cellular uptake of the peptide (Christiaens et al., 2002, Christiaens et al., 2004, Derossi et al., 1994, Derossi et al., 1996). Penetratin has been successfully used for (phospho-)peptides and peptide nucleic acid (PNA) internalisation, but DNA internalisation is restricted to 55-mer oligonucleotides (Braun et al., 2002, Dunican and Doherty, 2001, Pooga et al., 1998, Prochiantz, 1998).

In order to optimise the use of polymethacrylates for ex vivo gene transfer (Garnett, 1999), we carried out a systematic physicochemical study of the influence of Penetratin upon DNA condensation by polymethacrylates and measured the cellular uptake and transfection efficiency of polymethacrylate–DNA complexes combined with Penetratin on Cos-1 cell cultures.

Section snippets

Materials

Branched PEI (Mw = 25 kDa) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) were from Aldrich. 4-Chloro-7-nitro-1,2,3-benzoxadiazole (NBD-Cl), ethidium bromide, linear calf thymus DNA (15–23 kb), boric acid, agarose (electrophoresis grade), N-(2-hydroxyethyl)piperazine-N′-2-ethane-sulfonic acid (HEPES) and bromophenol blue were from Sigma. Ethylenediamine tetra-acetic acid (EDTA) was from Fluka. Glycerol was from Vel. LysoTracker® Red DND-99 and Oregon Green® 488-X

Polymethacrylate and Penetratin synthesis

The chemical structures of the polymethacrylates, containing either primary (AEMA) and tertiary amine groups (DMAEMA), or a combination of those groups with imidazole side groups (HYMIMMA), are shown in Fig. 1A and B. The chemical composition, molecular weight, polydispersity and mass per charge of the polymers and of polyethylenimine are summarized in Table 1. The sequence of the Penetratin peptide is given in Fig. 1C.

Effect of Penetratin addition on the polymethacrylates–DNA interaction

The Penetratin interaction with free DNA was first investigated in water.

Discussion

In this study, we compared the efficiency of methacrylate-based copolymers, containing positively charged primary (AEMA) and tertiary amine (DMAEMA) groups and uncharged imidazole (HYMIMMA) groups, for DNA transfection, in the absence and presence of Penetratin.

Transfection vectors are aimed at compensating the properties that hamper spontaneous DNA uptake by cells, i.e. its high negative charge, large hydrodynamic volume and susceptibility towards nuclease degradation (Bally et al., 1999,

Conclusion

The present study shows that a combination of DNA condensating polymethacrylates and the membrane-translocating Penetratin peptide yields an efficient transfection vector. Penetratin increases cellular uptake and endolysosomal escape of polymethacrylate–DNA complexes, without affecting the DNA condensation properties of these polymers. As a consequence, transfection levels higher than PEI and comparable to Lipofectamine™ can be achieved. These data clearly demonstrate that, besides DNA

Acknowledgements

The authors wish to thank Jan Tavernier for the use of the flow cytometer; Vera Goosens for technical assistance during the confocal microscopy experiments; and Berlinda Vanloo, Alain Prochiantz and Alain Joliot for fruitful discussions. Bart Christiaens was supported by a grant from the ‘Institute for the Promotion of Innovation through Science and Technology in Flanders’ (IWT-Vlaanderen). Etienne Schacht and Peter Dubruel acknowledge the financial support of the Belgian Government, Belgian

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