Gene Interference with Morpholinos in a Gold Nanoparticle-Based Delivery Platform in Rat PC12 Cells.
For the first time the efficiency of gene knockdown of the pituitary adenylate cyclase-activating polypeptide (PACAP) receptor 1 (PAC1R) is demonstrated by employing gold nanocomplexes. This gene knockdown subsequently affects the action of PACAP on neurite outgrowth in PC12 cells. These nanocomplexes comprise cholera toxin B (CTB)-gold nanoparticle conjugates loaded with double-stranded morpholinos (MOs) (photo MO and antisense MO). Nanocomplexes are introduced into cells via lipid raft-dependent endocytosis. After UV light exposure, the photolinker in the photo MO is cleaved, bisecting the photo MO and releasing the antisense MO from the conjugate. The antisense MO then binds the PAC1R mRNA and decreases gene expression. The maximal efficiency of gene knockdown is observed after 24 hours, resulting in a 65% ± 12 reduction of the protein level. This reduction in PAC1R impairs the responsiveness of cells to PACAP exposure. Following PAC1R gene knockdown, only 10% ± 8 and 11%± 9 of cells exhibit neurite outgrowth after 4-day exposure to PACAP-38 and PACAP-27, respectively. These results demonstrate an efficient PAC1R gene knockdown and noticeable difference in response to PACAP action on neural cell differentiation, adding an extra dimension to determine the involvement of PACAP and its PAC1R in the neurotropic effect to PC12 cells.
Wei
W
Deng
Melissa M J
MM
Farnham
Ewa M
EM
Goldys
Suja
S
Mohammed
Paul M
PM
Pilowsky
D000818:Animals; D001483:Base Sequence; D001692:Biological Transport; D002454:Cell Differentiation / Q000187:drug effects / Q000528:radiation effects; D002470:Cell Survival / Q000187:drug effects / Q000528:radiation effects; D002772:Cholera Toxin / Q000378:metabolism; D004337:Drug Carriers / Q000737:chemistry / Q000378:metabolism; D055785:Gene Knockdown Techniques; D006046:Gold / Q000737:chemistry* / Q000378:metabolism; D053768:Metal Nanoparticles; D008958:Models, Molecular; D060172:Morpholinos / Q000737:chemistry* / Q000235:genetics*; D020932:Nerve Growth Factor / Q000494:pharmacology; D009474:Neurons / Q000166:cytology; D009690:Nucleic Acid Conformation; D016716:PC12 Cells; D010446:Peptide Fragments / Q000494:pharmacology; D051219:Pituitary Adenylate Cyclase-Activating Polypeptide / Q000737:chemistry; D051381:Rats; D051237:Receptors, Pituitary Adenylate Cyclase-Activating Polypeptide, Type I / Q000172:deficiency* / Q000235:genetics*; D014466:Ultraviolet Rays
D004337:Drug Carriers; D060172:Morpholinos; D010446:Peptide Fragments; D051219:Pituitary Adenylate Cyclase-Activating Polypeptide; D051237:Receptors, Pituitary Adenylate Cyclase-Activating Polypeptide, Type I; D006046:Gold; D002772:Cholera Toxin; D020932:Nerve Growth Factor