A new non-viral DNA delivery vector: the terplex system.
A new DNA delivery vector (the terplex system) based on a balanced hydrophobicity and net surface charge between stearyl-poly(L-lysine), low density lipoprotein (LDL), and genetic material (i.e. plasmid DNA or antisense oligonucleotide) was developed. The pSV-beta-gal plasmid in terplex system showed a 2-5-fold increase in beta-galactosidase expression on murine smooth muscle cells (A7R5) compared to Lipofectin. Delivery of unmodified c-myb antisense oligonucleotide to A7R5 cells was also facilitated significantly by the terplex system, requiring as little as 5.4 nM of antisense oligonucleotide to achieve a 50% antiproliferative effect. Similar antiproliferative effect was observed when the c-myb antisense/terplex formulation was tested on CCD-32 Lu human lung fibroblasts. Characterization of the physical properties of the terplex system was performed using various techniques. Plasmid DNA was condensed by addition of stearyl-PLL and LDL, resulting in the terplex system of about 100 nm in diameter as shown by atomic force microscopy. A strong hydrophobic interaction between stearyl-poly(L-lysine) and LDL was registered by 1H-NMR spectrometry, showing a significant decrease in the epsilon-methylene signal of poly(L-lysine) backbone when stearyl-poly(L-lysine) was mixed with LDL; however, this phenomenon was not observed with unmodified poly(L-lysine). Agarose gel electrophoresis revealed that electrophoretic mobility of the terplex system decreased with increasing amounts of stearyl-poly(L-lysine), indicating that the surface charge of the terplex system became more positive by addition of stearyl-poly(L-lysine). Zeta-potential measurement showed that the terplex system exerted a slightly positive charge (+2 mV) at a 1:1:1 weight ratio of plasmid DNA:LDL:stearyl-poly(L-lysine). The obtained results will be utilized in the design of more efficient and safer DNA delivery vectors for in vivo gene therapy.
Kim
J S
JS
Department of Pharmaceutics and Pharmaceutical Chemistry, Center for Controlled Chemical Delivery, University of Utah, Salt Lake City 84112, USA.
Kim
B I
BI
Maruyama
A
A
Akaike
T
T
Kim
S W
SW
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D004247:DNA / Q000008:administration & dosage*; D004587:Electrophoresis, Agar Gel; D005822:Genetic Vectors; D006801:Humans; D008077:Lipoproteins, LDL / Q000008:administration & dosage* / Q000737:chemistry; D051379:Mice; D018625:Microscopy, Atomic Force; D016376:Oligonucleotides, Antisense / Q000008:administration & dosage* / Q000737:chemistry; D010957:Plasmids; D011107:Polylysine / Q000008:administration & dosage* / Q000737:chemistry; D011518:Proto-Oncogene Proteins / Q000235:genetics; D020598:Proto-Oncogene Proteins c-myb; D055672:Static Electricity; D013228:Stearates / Q000008:administration & dosage* / Q000737:chemistry; D015534:Trans-Activators / Q000235:genetics
D008077:Lipoproteins, LDL; D016376:Oligonucleotides, Antisense; D011518:Proto-Oncogene Proteins; D020598:Proto-Oncogene Proteins c-myb; D013228:Stearates; D015534:Trans-Activators; C115579:stearyl-poly(lysine); D011107:Polylysine; D004247:DNA