New cationized LDL-DNA complexes: their targeted delivery to fibroblasts in culture.
Low density lipoproteins (LDL) have been cationized using the water-soluble carbodiimide, N-ethyl-N'-(3-trimethylpropylammonium) carbodiimide iodide at a reagent: lipoprotein mole ratio of 10 000:1. This was shown to increase the innate DNA-binding capacity of LDL 10-fold. [125I]-labeled carbodiimide-modified LDL ([125I])-labeled ECDI-LDL) appeared to recognize the LDL receptor on normal human skin fibroblasts, although some nonspecific binding also was detected. To demonstrate the large ionic component in the lipoprotein-DNA interactions, epsilon -NH2 amino groups on the apolipoprotein B-100 (apoB-100) component of LDL were acetylated with acetic anhydride. A nitrocellulose filter-binding assay revealed that acetylated LDL bound approximately 25% of the [3H]-labeled pBR322 plasmid DNA bound by native LDL under the same conditions. ECDI-LDL-[3H]-labeled plasmid DNA complexes were considerably more stable to NaCl challenge than complexes formed between [3H]-labeled plasmid DNA and native LDL. Thus, the half dissociation of ECDI-LDL containing complexes was achieved at 0.28 M NaCl, whereas for LDL-plasmid DNA complexes this was reached at 0.18 M NaCl. Displacement studies with native LDL studies showed that ECDI-LDL-[3H]-labeled plasmid DNA complexes retained the ability to recognize the LDL receptor on normal skin fibroblasts. Finally, ECDI-LDL complexes with pSV2CAT expression plasmid were shown to transfect CV-1 fibroblasts, a cell line known to specifically recognize apoB-liposome conjugates.
Khan
Zainub
Z
Biochemistry, School of Life and Environmental Sciences, University of Durban-Westville, Durban, South Africa.
Hawtrey
Arthur O
AO
Ariatti
Mario
M
D000818:Animals; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D004247:DNA / Q000008:administration & dosage* / Q000737:chemistry*; D004337:Drug Carriers / Q000737:chemistry*; D005022:Ethyldimethylaminopropyl Carbodiimide / Q000031:analogs & derivatives / Q000737:chemistry; D005347:Fibroblasts / Q000378:metabolism*; D018014:Gene Transfer Techniques; D006801:Humans; D007456:Iodine Isotopes; D008077:Lipoproteins, LDL / Q000737:chemistry*; D010957:Plasmids; D011973:Receptors, LDL / Q000378:metabolism; D012867:Skin / Q000166:cytology / Q000378:metabolism; D012965:Sodium Chloride / Q000737:chemistry
D004337:Drug Carriers; D007456:Iodine Isotopes; D008077:Lipoproteins, LDL; D011973:Receptors, LDL; D012965:Sodium Chloride; C041829:1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide methiodide; D004247:DNA; D005022:Ethyldimethylaminopropyl Carbodiimide