Skin
carcinomas are among the most commonly diagnosed
tumors in the world. In this study, we investigated the transfection of immortalized keratinocytes, used as an in vitro model for skin
carcinoma, using the antisense technology and
poly(2-(dimethylamino)ethyl methacrylate) (
PDMAEMA)-based copolymers. In order to improve the transfection efficiency of the classic
PDMAEMA polymers, copolymers were synthesized including a poly(N-
morpholino)
ethylmethacrylate) (PMEMA) moiety for an improved
proton-sponge effect, intended to favour the release of the
oligonucleotide from the acidic endosome. These copolymers were synthesized either statistically (with alternating
PDMAEMA and PMEMA fragments) or in blocks (one
PDMAEMA block followed by one PMEMA block). MTT assays were performed using the
PDMAEMA-PMEMA copolymers and revealed no significant cytotoxicity of these
polymers at an N/P ratio of 7.3. Using fluorescent
oligonucleotides and analyzing transfection efficiency by flow cytometry, we noticed no significant differences between the two kinds of copolymers. However copolymers with a higher
DMAEMA content and a higher Mn were also those displaying the highest vectorization efficiency. Confocal microscopy showed that these copolymers induced a fine granular distribution of the transfected
antisense oligonucleotides inside the cells. We also assessed the functionality of the transfected
antisense oligonucleotide by transfecting immortalized GFP expressing keratinocytes with a GFP
antisense oligonucleotide using these copolymers. A significant silencing was achieved with a
PDMAEMA-PMEMA in block copolymer (Mn=41,000, 89 %
PDMAEMA). Together, these results suggest that
PDMAEMA-PMEMA copolymers combining low toxicity, vectorization and
proton sponge properties, can be efficiently used to transfect immortalized keratinocytes and so open new perspectives in the
therapy of skin
carcinomas as well as of other
skin diseases of genetic or immunological origin.