Novel graft copolymers enhance in vitro delivery of antisense oligonucleotides in the presence of serum.
Antisense technology holds tremendous potential in the research and clinical settings. However, successful delivery of antisense oligodeoxynucleotides (ODNs) to the intracellular site of action requires the passage of many barriers, including survival against extracellular serum nucleases and escape from endolysosomal degradation. Previous work has shown that the effectiveness of antisense delivery by the cationic liposome, dioleoyl-3-trimethylammonium-propane (DOTAP), is enhanced substantially by the incorporation of a pH-sensitive polymer, poly (propylacrylic acid) (PPAA), in serum-free media. To improve this system for application in serum-containing media conditions, PPAA was modified in this work by grafting onto it either poly(ethylene oxide) (PEO) or a more hydrophobic analog, poly (oxyalkylene amine), known as Jeffamine. The ternary formulation of DOTAP/ODN/PPAA-g-Jeffamine resulted in 8-fold increased uptake of fluorescently-labeled ODNs compared to DOTAP/ODN/PPAA and ~80% silencing of green fluorescent protein (GFP) expression in CHO-d1EGFP cells treated in the presence of 10% FBS-containing media. In contrast, the carrier systems that contained PPAA or PPAA-g-PEO failed to display any significant antisense activity in the presence of serum, even though all of the delivery systems displayed moderate to high levels of antisense activity in serum-free conditions. The results reveal that the carrier system with the Jeffamine graft copolymer effectively mediates specific gene silencing in the presence of serum, while the system with the PEO graft copolymer fails to do so. While the pH-dependent lytic functionality of PPAA was found to be lost upon grafting with PEO or Jeffamine, the hydrophobicity of the latter was sufficient to mediate cellular internalization and endosomal escape. Thus, the PPAA-g-Jeffamine copolymers hold substantial promise as agents for controlled therapeutic delivery of antisense oligonucleotides.
Peddada
Lavanya Y
LY
Department of Biomedical Engineering, Rutgers University, Piscataway, NJ 08854, USA.
Harris
Nicole K
NK
Devore
David I
DI
Roth
Charles M
CM
D000179:Acrylates / Q000737:chemistry* / Q000633:toxicity; D000588:Amines / Q000737:chemistry*; D000818:Animals; D001692:Biological Transport; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D011992:Endosomes / Q000378:metabolism; D005229:Fatty Acids, Monounsaturated / Q000737:chemistry*; D020868:Gene Silencing; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000235:genetics; D006461:Hemolysis / Q000187:drug effects; D006863:Hydrogen-Ion Concentration; D057927:Hydrophobic and Hydrophilic Interactions; D016376:Oligonucleotides, Antisense / Q000737:chemistry / Q000378:metabolism*; D011092:Polyethylene Glycols / Q000737:chemistry* / Q000633:toxicity; D011108:Polymers / Q000737:chemistry* / Q000633:toxicity; D000644:Quaternary Ammonium Compounds / Q000737:chemistry*; D044967:Serum / Q000378:metabolism*; D014162:Transfection
D000179:Acrylates; D000588:Amines; D005229:Fatty Acids, Monounsaturated; D016376:Oligonucleotides, Antisense; D011108:Polymers; D000644:Quaternary Ammonium Compounds; C485184:enhanced green fluorescent protein; C501628:poly(propyl acrylic acid); D049452:Green Fluorescent Proteins; D011092:Polyethylene Glycols; C070046:1,2-dioleoyloxy-3-(trimethylammonium)propane
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