Premium
Purification of polyethylenimine polyplexes highlights the role of free polycations in gene transfer
Author(s) -
Boeckle Sabine,
von Gersdorff Katharina,
van der Piepen Silke,
Culmsee Carsten,
Wagner Ernst,
Ogris Manfred
Publication year - 2004
Publication title -
the journal of gene medicine
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.689
H-Index - 91
eISSN - 1521-2254
pISSN - 1099-498X
DOI - 10.1002/jgm.598
Subject(s) - polyethylenimine , transfection , gene delivery , in vitro , chemistry , in vivo , dna , microbiology and biotechnology , biophysics , biochemistry , biology , gene
Background Nonviral vectors based on polyethylenimine (PEI) usually contain an excess of PEI that is not complexed to DNA. Since unbound PEI contributes to cellular and systemic toxicity, purification of polyplexes from unbound PEI is desirable. Methods Size exclusion chromatography (SEC) was used to purify PEI polyplexes of free PEI. Transfection properties of purified polyplexes and the effect of free PEI on gene delivery were studied in vitro and in vivo after systemic application into mice. Results SEC did not change the size and zeta‐potential of polyplexes. Independent of the amount of PEI used for complex formation, purified PEI polyplexes had the same final PEI nitrogen/DNA phosphate ratio of 2.5. Notably, purified PEI polyplexes demonstrated low cellular and systemic toxicity. High transfection efficiency was achieved with purified polyplexes at high DNA concentrations (8–15 µg/ml). At low DNA concentrations (2–4 µg/ml) gene transfer with purified particles was less efficient than with polyplexes containing free PEI both in vitro and in vivo . Mechanistic studies showed that free PEI partly blocked cellular association of DNA complexes but was essential for the following intracellular gene delivery. Adding free PEI to cells treated with purified particles with a delay of up to 4 h resulted in significantly enhanced transfection efficiency compared with non‐purified particles or purified particles without free PEI. Conclusions This study presents an efficient method to remove free PEI from PEI polyplexes by SEC. Our results from transfection experiments demonstrate that free PEI substantially contributes to efficient gene expression but also mediates toxic effects in a dose‐dependent manner. Purified polyplexes without free PEI have to be applied at increased concentrations to achieve high transfection levels, but exhibit a greatly improved toxicity profile. Copyright © 2004 John Wiley & Sons, Ltd.