Brewster angle microscopy and PMIRRAS study of DNA interactions with BGTC, a cationic lipid used for gene transfer.
The lipid bis(guanidinium)-tris(2-aminoethyl)amine-cholesterol (BGTC) is a cationic cholesterol derivative bearing guanidinium polar headgroups which displays high transfection efficiency in vitro and in vivo when used alone or formulated as liposomes with the neutral colipid 1,2-di-[ cis-9-octadecenoyl]- sn-glycero-3-phosphoethanolamine (DOPE). Since transfection may be related to the structural and physicochemical properties of the self-assembled supramolecular lipid-DNA complexes, we used the Langmuir monolayer technique coupled with Brewster angle microscopy (BAM) and polarization modulation infrared reflection absorption spectroscopy (PMIRRAS) to investigate DNA-BGTC and DNA-BGTC/DOPE interactions at the air/water interface. We herein show that BGTC forms stable monolayers at the air/water interface. When DNA is injected into the subphase, it adsorbs to BGTC at 20 mN/m. Whatever the (+/-) charge ratio of the complexes used, defined as the ratio of positive charges of BGTC in the monolayer versus negative charges of DNA injected in the subphase, the DNA interacts with the cationic lipid and forms either an incomplete (no constituent in excess) or a complete (DNA in excess) monolayer of oriented double strands parallel to the lipid monolayer plan. We also show that, under a homogeneous BGTC/DOPE (3/2) monolayer at 20 mN/m, DNA adsorbs homogeneously to form an organized but incomplete layer whatever the charge ratio used (DNA in default or in excess). Compression beyond the collapse of these mixed DNA-BGTC/DOPE systems leads to the formation of dense DNA monolayers under an asymmetric lipid bilayer with a bottom layer of BGTC in contact with DNA and a top layer mainly constituted of DOPE. These results allow a better understanding of the mechanisms underlying the formation of the supramolecular BGTC-DNA complexes efficient for gene transfection.
Castano
Sabine
S
UMR 5248 CBMN, CNRS-Universite Bordeaux 1-ENITAB, IECB, 2 rue Robert Escarpit, 33607 Pessac, France. s.castano@cbmn.u-bordeaux.fr
Delord
Brigitte
B
Février
Annie
A
Lehn
Jean-Marie
JM
Lehn
Pierre
P
Desbat
Bernard
B
D000388:Air; D001703:Biophysics / Q000379:methods; D002412:Cations; D002784:Cholesterol / Q000031:analogs & derivatives* / Q000737:chemistry; D004247:DNA / Q000737:chemistry*; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D006146:Guanidines / Q000737:chemistry*; D008051:Lipid Bilayers / Q000737:chemistry; D008055:Lipids / Q000737:chemistry; D008853:Microscopy / Q000379:methods*; D011312:Pressure; D013055:Spectrophotometry, Infrared; D013499:Surface Properties; D013997:Time Factors; D014867:Water / Q000737:chemistry
C101389:3-(N(4)-(N(1),N(8)-diguanidinospermidine)carbamoyl)cholesterol; D002412:Cations; D006146:Guanidines; D008051:Lipid Bilayers; D008055:Lipids; D014867:Water; D004247:DNA; D002784:Cholesterol