Assessment of new biocompatible Poly(N-(morpholino)ethyl methacrylate)-based copolymers by transfection of immortalized keratinocytes

Autor:	Philippe Dubois, Nancy Van Overstraeten-Schlögel, Yong Ho Shim, Virginie Tevel, Jacques Piette, Martine Raes, Géraldine Piel
Rok vydání:	2012
Předmět:	Keratinocytes Materials science Polymers Pharmaceutical Science Biocompatible Materials Endosomes Transfection Methacrylate Flow cytometry Polymer chemistry Copolymer medicine Humans Moiety Cytotoxicity Cells Cultured chemistry.chemical_classification medicine.diagnostic_test Oligonucleotide Gene Transfer Techniques General Medicine Polymer Oligonucleotides Antisense Combinatorial chemistry Nylons chemistry Methacrylates
Zdroj:	Drug Delivery. 19:112-122
ISSN:	1521-0464 1071-7544
DOI:	10.3109/10717544.2011.649219
Popis:	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.
Databáze:	OpenAIRE
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