New Insights on the Mechanism of Polyethylenimine Transfection and Their Implications on Gene Therapy and DNA Vaccines

28 Pages Posted: 3 May 2021

See all articles by Juan Sabín

Juan Sabín

Universidade de Santiago de Compostela - Biophysics and Interfaces Group

Manuel Alatorre-Meda

Tecnológico Nacional de México - Cátedras CONACyT

Jose Miñones

Universidade de Santiago de Compostela - Department of Physical Chemistry

Vicente Domínguez-Arca

Universidade de Santiago de Compostela - Biophysics and Interfaces Group

Gerardo Prieto

Universidade de Santiago de Compostela - Biophysics and Interfaces Group

Abstract

Polyethylenimine (PEI) has been demonstrated as an efficient DNA delivery vehicle both in vitro and in vivo. There is a consensus that PEI-DNA complexes enter the cells by endocytosis and escape from endosomes by the so-called “proton sponge” effect. However, little is known on how and where the polyplexes are de-complexed for DNA transcription and replication to occur inside the cell nucleus. To better understand this issue, we (i) tracked the cell internalization of PEI upon transfection to human epithelial cells and (ii) studied the interaction of PEI with phospholipidic layers mimicking nuclear membranes. Both the biological and physicochemical experiments provided evidence of a strong binding affinity between PEI and the lipidic bilayer. Firstly, confocal microscopy revealed that PEI alone could not penetrate the cell nucleus; instead, it arranged throughout the cytoplasm and formed a sort of aureole surrounding the nuclei periphery. Secondly, surface tension measurements, fluorescence dye leakage assays, and differential scanning calorimetry demonstrated that a combination of hydrophobic and electrostatic interactions between PEI and the phospholipidic monolayers/bilayers led to the formation of stable defects along the model membranes, allowing the intercalation of PEI through the monolayer/bilayer structure. Results are also supported by molecular dynamics simulation of the pore formation in PEI-lipidic bilayers. As discussed throughout the text, these results might shed light on a the mechanism in which the interaction between PEI and the nucleus membrane might play an active role on the DNA release: on the one hand, the PEI-membrane interaction is anticipated to facilitate the DNA disassembly from the polyplex by establishing a competition with DNA for the PEI binding and on the other hand, the forming defects are expected to serve as channels for the entrance of de-complexed DNA into the cell nucleus. A better understanding of the mechanism of transfection of cationic polymers opens paths to development of more efficiency vectors to improve gene therapy treatment and the new generation of DNA vaccines that has been used during the SARS-CoV-2 pandemic.

Suggested Citation

Sabín, Juan and Alatorre-Meda, Manuel and Miñones, Jose and Domínguez-Arca, Vicente and Prieto, Gerardo, New Insights on the Mechanism of Polyethylenimine Transfection and Their Implications on Gene Therapy and DNA Vaccines. Available at SSRN: https://ssrn.com/abstract=3838461 or http://dx.doi.org/10.2139/ssrn.3838461

Juan Sabín

Universidade de Santiago de Compostela - Biophysics and Interfaces Group ( email )

Spain

Manuel Alatorre-Meda

Tecnológico Nacional de México - Cátedras CONACyT ( email )

Durango
Mexico

Jose Miñones

Universidade de Santiago de Compostela - Department of Physical Chemistry ( email )

Complejo Docente - Campus Universitario de Lugo
Lugo, A Coruña 15704
Spain

Vicente Domínguez-Arca

Universidade de Santiago de Compostela - Biophysics and Interfaces Group ( email )

Spain

Gerardo Prieto (Contact Author)

Universidade de Santiago de Compostela - Biophysics and Interfaces Group ( email )

Spain

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