Cationic Polymer Intercalation into the Lipid Membrane Enables Intact Polyplex DNA Escape from Endosomes for Gene Delivery.
Developing improved cationic polymer-DNA polyplexes for gene delivery requires improved understanding of DNA transport from endosomes into the nucleus. Using a FRET-capable oligonucleotide molecular beacon (OMB), we monitored the transport of intact DNA to cell organelles. We observed that for effective (jetPEI) and ineffective (G5 PAMAM) vectors, the fraction of cells displaying intact OMB in the cytosol (jetPEI ≫ G5 PAMAM) quantitatively predicted the fraction expressing transgene (jetPEI ≫ G5 PAMAM). Intact OMB delivered with PAMAM and confined to endosomes could be released to the cytosol by the subsequent addition of L-PEI, with a corresponding 10-fold increase in transgene expression. These results suggest that future vector development should optimize vectors for intercalation into, and destabilization of, the endosomal membrane. Finally, the study highlights a two-step strategy in which the pDNA is loaded in cells using one vector and endosomal release is mediated by a second agent.
Departments of †Biomedical Engineering and ‡Chemistry and Programs in §Applied Physics and ⊥Macromolecular Science and Engineering, University of Michigan , Ann Arbor, Michigan 48109, United States.
Sriram
S
Vaidyanathan
Departments of †Biomedical Engineering and ‡Chemistry and Programs in §Applied Physics and ⊥Macromolecular Science and Engineering, University of Michigan , Ann Arbor, Michigan 48109, United States.
Junjie
J
Chen
Departments of †Biomedical Engineering and ‡Chemistry and Programs in §Applied Physics and ⊥Macromolecular Science and Engineering, University of Michigan , Ann Arbor, Michigan 48109, United States.
Bradford G
BG
Orr
Departments of †Biomedical Engineering and ‡Chemistry and Programs in §Applied Physics and ⊥Macromolecular Science and Engineering, University of Michigan , Ann Arbor, Michigan 48109, United States.
Mark M
MM
Banaszak Holl
D002412:Cations / Q000378:metabolism*; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D004247:DNA / Q000378:metabolism*; D011992:Endosomes / Q000378:metabolism*; D018014:Gene Transfer Techniques; D015316:Genetic Therapy / Q000379:methods; D005822:Genetic Vectors / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D007364:Intercalating Agents / Q000378:metabolism; D008563:Membrane Lipids / Q000378:metabolism*; D010957:Plasmids / Q000378:metabolism; D011108:Polymers / Q000378:metabolism*; D014162:Transfection / Q000379:methods; D019076:Transgenes / Q000502:physiology
D002412:Cations; D007364:Intercalating Agents; D008563:Membrane Lipids; D011108:Polymers; D004247:DNA
FRET molecular beacon; gene delivery; gene therapy; linear poly(ethylenimine); membrane intercalation; molecular beacon; polyplex-cell membrane interactions; polyplexes