Enhancement of polymethacrylate-mediated gene delivery by Penetratin.
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.
Christiaens
Bart
B
Department of Biochemistry, Ghent University, 9000 Ghent, Belgium. bartchrist@hotmail.com
Dubruel
Peter
P
Grooten
Johan
J
Goethals
Marc
M
Vandekerckhove
Joël
J
Schacht
Etienne
E
Rosseneu
Maryvonne
M
D000818:Animals; D019556:COS Cells; D002352:Carrier Proteins / Q000494:pharmacology*; D057846:Cell-Penetrating Peptides; D004247:DNA / Q000008:administration & dosage*; D018014:Gene Transfer Techniques; D015316:Genetic Therapy; D011109:Polymethacrylic Acids / Q000008:administration & dosage*; D014162:Transfection
D002352:Carrier Proteins; D057846:Cell-Penetrating Peptides; D011109:Polymethacrylic Acids; C030613:polymethacrylic acid; D004247:DNA; C414409:penetratin