Stabilized integrin-targeting ternary LPD (lipopolyplex) vectors for gene delivery designed to disassemble within the target cell.
Recent research in the field of nonviral gene delivery vectors has focused on preparing nanoparticles that are stabilized by the incorporation of a PEG coating and where one of the vector components is also cleavable. Here,we describe the synthesis, formulation, transfection properties, and biophysical studies of a PEG-stabilized ternary lipopolyplex vector in which, for the first time, both the lipid and peptide components are designed to be cleaved once the vector has been internalized. A series of cationic lipids, bearing short tri- or hexaethylene glycol groups, attached to the headgroup via an ester linkage, has been prepared. Trifunctional peptides have also been prepared, consisting of a Lys(16) sequence at the N-terminus (to bind and condense plasmid DNA); a spacer group (containing a sequence recognized and cleaved by endosomal enzymes) and an optional PEG4 amino acid; and an integrin-targeting cyclic peptide sequence (allowing the resulting nanoparticle to be internalized via receptor-mediated endocytosis). Differing combinations of these lipids and peptides have been formulated with DOPE and with plasmid DNA, and complex stability, transfection, and cleavage studies carried out. It was shown that optimal transfection activities in a range of cell types and complex stabilities were achieved with lipids bearing short cleavable triethylene glycol moieties, whereas the incorporation of PEG4 amino acids into the cleavable peptides had little effect. We have synthesized appropriate fluorescently labeled components and have studied the uptake of the vector, endosomal escape, peptide cleavage, and plasmid transport to the nucleus in breast cancer cells using confocal microscopy. We have also studied the morphology of these compact, stabilized vectors using cryo-EM.
Mustapa
M Firouz Mohd
MF
Department of Chemistry, University College London, Christopher Ingold Laboratories, 20 Gordon Street, London, UK.
Grosse
Stephanie M
SM
Kudsiova
Laila
L
Elbs
Martin
M
Raiber
Eun-Ang
EA
Wong
John B
JB
Brain
Anthony P R
AP
Armer
Hannah E J
HE
Warley
Alice
A
Keppler
Melanie
M
Ng
Tony
T
Lawrence
M Jayne
MJ
Hart
Stephen L
SL
Hailes
Helen C
HC
Tabor
Alethea B
AB
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D045744:Cell Line, Tumor; D002470:Cell Survival; D020285:Cryoelectron Microscopy; D004247:DNA / Q000008:administration & dosage*; D011992:Endosomes / Q000378:metabolism; D006801:Humans; D016023:Integrins / Q000378:metabolism*; D050356:Lipid Metabolism; D008055:Lipids / Q000138:chemical synthesis / Q000737:chemistry*; D051379:Mice; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism; D010957:Plasmids / Q000008:administration & dosage; D011092:Polyethylene Glycols / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism; D014162:Transfection
D016023:Integrins; D008055:Lipids; D010455:Peptides; D011092:Polyethylene Glycols; D004247:DNA