Quantitative physical characterization of lipid-polycation-DNA lipopolyplexes.
Quantitative assays for the characterization of multi-component lipopolyplexes and their individual constituents are crucial for determining the consistency of formulation protocols which are ultimately reflected in biological activity. Lipid-polycation-DNA formulations consisting of lipids, polycations and DNA are of interest because they have been demonstrated to be efficient gene-delivery vehicles when administered systemically. We have developed a panel of analytical techniques to characterize these lipopolyplexes. Complexes were measured for size by dynamic light scattering and surface-charge characteristics by zeta potential. Interaction between DNA and the polycation, protamine sulphate, was determined using a PicoGreen dye-exclusion technique. Total DNA in the lipopolyplex was assayed through decomplexation of the formulation by addition of heparin sulphate and subsequent DNA quantification by PicoGreen reagent. Protamine sulphate in the lipopolyplex was determined using a novel Amido Black-staining protocol which is linearly sensitive in a range of 0.25-3 microg of protein. Lipids were quantified by HPLC after extraction in chloroform/methanol (2:1). In this method elution is conducted over 40 min, with 1,2-dioleoyl-3-trimethylammonium-propane and cholesterol being resolved by greater than 10 min. Such assays are essential for product characterization and release tests, as well as development of a better understanding of the correlation between physical structure and biological function.
Tsai
Jeannette T
JT
Targeted Genetics Corporation, 1100 Olive Way, Suite 100, Seattle, WA 98101, U.S.A. paulr@targen.com
Furstoss
Kevin J
KJ
Michnick
Timothy
T
Sloane
David L
DL
Paul
Ralph W
RW
D000580:Amido Black; D000818:Animals; D016466:CHO Cells / Q000378:metabolism; D002784:Cholesterol / Q000032:analysis* / Q000235:genetics; D002851:Chromatography, High Pressure Liquid; D006224:Cricetinae; D004247:DNA / Q000032:analysis / Q000235:genetics; D005229:Fatty Acids, Monounsaturated / Q000032:analysis* / Q000378:metabolism; D005456:Fluorescent Dyes; D008055:Lipids / Q000032:analysis*; D008081:Liposomes / Q000032:analysis* / Q000378:metabolism; D009930:Organic Chemicals; D010957:Plasmids / Q000032:analysis* / Q000235:genetics; D011073:Polyamines / Q000032:analysis; D000071228:Polyelectrolytes; D011479:Protamines / Q000737:chemistry; D000644:Quaternary Ammonium Compounds / Q000032:analysis* / Q000378:metabolism; D012680:Sensitivity and Specificity; D014162:Transfection
D000580:Amido Black; D002784:Cholesterol; D004247:DNA; D005229:Fatty Acids, Monounsaturated; D005456:Fluorescent Dyes; D008055:Lipids; D008081:Liposomes; D009930:Organic Chemicals; D011073:Polyamines; D000071228:Polyelectrolytes; D011479:Protamines; D000644:Quaternary Ammonium Compounds; C099476:PicoGreen; C070046:1,2-dioleoyloxy-3-(trimethylammonium)propane