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Transfection of immortalized keratinocytes by low toxic poly(2-(dimethylamino)ethyl methacrylate)-based polymers
Nancy Van Overstraeten-Schlögel1, Yong Ho-Shim, Virginie Tevel
1a Research Unit of Cell Biology, University of Namur, B-5000 Namur, Belgium. nancy.vanoverstraeten@fundp.ac.be.
Abstract:
Skin carcinoma are among the most spread diagnosed tumours in the world. In this study, we investigated the transfection of immortalized keratinocytes, used as an in vitro model for skin carcinoma, using antisense technology and poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA)-based polymers, with original architecture and functionalities. We tested PDMAEMA polymers with different structures: linear, with two (DEA-PDMAEMA) or three (TEA-PDMAEMA) arms. The cytotoxicity of these polymers was assessed over a wide range of apparent M n (from 7600 to 64 600). At a N/P ratio of 7.38, cytotoxicity increases with the M n. Keratinocytes were transfected with a fluorescent oligonucleotide and then analyzed by flow cytometry. For the three architectures tested, the percentage of transfected cells and abundance of internalized oligonucleotide were closely related to the M n of the polymer. Confocal microscopy and FACS analyses showed a wide spread fine granular distribution of the oligonucleotide up to 3 days post-transfection. Then, we assessed the silencing efficiency of the polymers, targeting GFP in GFP expressing keratinocytes. The maximal silencing effect (±40%) was obtained using a DEA-PDMAEMA polymer (M n = 30 300). These results suggest that PDMAEMA-based polymers can be efficiently used to transfect immortalized keratinocytes and, thus, open new perspectives in the therapy of skin carcinoma.
Insights
Researchers explored poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA) polymers for skin carcinoma therapy. These polymers efficiently transfected immortalized keratinocytes, showing promise for future treatments.
Area of Science:
- Biomaterials Science
- Nanotechnology
- Dermatology
- Oncology
Background:
- Skin carcinoma is a prevalent global health concern.
- Effective in vitro models are crucial for developing novel cancer therapies.
- Antisense technology offers a potential therapeutic strategy for cancer.
Purpose of the Study:
- To evaluate the efficacy of poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA) polymers for transfecting immortalized keratinocytes.
- To investigate the influence of PDMAEMA polymer architecture and molecular weight on transfection efficiency and cytotoxicity.
- To assess the potential of PDMAEMA polymers in gene silencing for skin carcinoma therapy.
Main Methods:
- Synthesis and characterization of linear, two-arm (DEA-PDMAEMA), and three-arm (TEA-PDMAEMA) PDMAEMA polymers.
- Cytotoxicity assessment of PDMAEMA polymers across a range of molecular weights (M n).
- Transfection of immortalized keratinocytes with fluorescent oligonucleotides, followed by flow cytometry, confocal microscopy, and FACS analysis.
- Evaluation of gene silencing efficiency targeting GFP in GFP-expressing keratinocytes.
Main Results:
- PDMAEMA polymer cytotoxicity increased with molecular weight at a N/P ratio of 7.38.
- Transfection efficiency and oligonucleotide uptake were dependent on polymer molecular weight and architecture.
- Oligonucleotides exhibited widespread granular distribution within cells for up to 3 days post-transfection.
- A maximum of approximately 40% gene silencing was achieved using a DEA-PDMAEMA polymer (M n = 30,300).
Conclusions:
- PDMAEMA-based polymers are effective for transfecting immortalized keratinocytes.
- Polymer architecture and molecular weight significantly impact transfection outcomes.
- These findings suggest promising applications for PDMAEMA polymers in skin carcinoma therapy.
