Extracellular and intracellular factors influencing gene transfection mediated by 1,4-dihydropyridine amphiphiles.
Double-charged 1,4-dihydropyridine (1,4-DHP) amphiphiles have been shown to condense DNA and efficiently transfect it into cells in vitro [Hyvönen et al., Biochim. Biophys. Acta 1509 (2000) 451]. Alkyl chain length and buffering capacity at endosomal pH range (5.0-7.4) affected complexation and transfection activity. In this study we examined how those chemical modifications of amphiphile-DNA complexes (amphiplexes) affect their interactions with extracellular polyanions (glycosaminoglycans, albumin) and lipid bilayers, their cellular uptake and intracellular distribution. To evaluate cellular uptake, CV1-P cells were incubated with labeled DNA-amphiphile complexes and analyzed by flow cytometry. Confocal laser fluorescence microscopy was used to investigate the intracellular distribution of amphiplexes. The results showed that biophysical properties of compounds can be changed by slight structural modifications. These factors determine the intracellular kinetics and transfection efficacy of the compounds. Some extracellular glycosaminoglycans and serum interfere with 1,4-DHP-amphiphile-mediated transfection by destabilizing the amphiplexes. Neither high cellular uptake, membrane destabilizing activity nor buffering capacity alone is adequate for high transfection efficacy. The activity results from complex interplay of various factors that determine intracellular kinetics and, consequently, transfection.
Hyvönen
Zanna
Z
Department of Pharmaceutics, University of Kuopio, P.O. Box 1627, Finland. zanna.hyvonen@uku.fi
Ruponen
Marika
M
Rönkkö
Seppo
S
Suhonen
Pekka
P
Urtti
Arto
A
D000818:Animals; D002460:Cell Line / Q000378:metabolism; D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000493:pharmacokinetics; D004095:Dihydropyridines / Q000737:chemistry / Q000493:pharmacokinetics*; D005110:Extracellular Space / Q000235:genetics / Q000378:metabolism*; D000882:Haplorhini; D007424:Intracellular Fluid / Q000378:metabolism*; D008081:Liposomes; D013501:Surface-Active Agents / Q000737:chemistry / Q000493:pharmacokinetics*; D014162:Transfection / Q000379:methods*
D004095:Dihydropyridines; D008081:Liposomes; D013501:Surface-Active Agents; C038806:1,4-dihydropyridine; D004247:DNA