Gene Interference with Morpholinos in a Gold Nanoparticle-Based Delivery Platform in Rat PC12 Cells

Area of Science:

• Neuroscience
• Molecular Biology
• Biotechnology

Background:

• Pituitary adenylate cyclase-activating polypeptide (PACAP) plays a role in neural cell differentiation.
• The PACAP receptor 1 (PAC1R) is crucial for mediating PACAP's effects.
• Developing targeted methods to study PACAP/PAC1R signaling is essential.

Purpose of the Study:

• To demonstrate the efficiency of gene knockdown of PAC1R using novel gold nanocomplexes.
• To investigate the impact of PAC1R gene knockdown on PACAP-induced neurite outgrowth in PC12 cells.
• To establish a new tool for studying PACAP's neurotropic effects.

Main Methods:

• Gold nanoparticle-cholera toxin B conjugates loaded with morpholinos (MOs) were synthesized.
• Nanocomplexes were delivered into PC12 cells via lipid raft-dependent endocytosis.
• UV light triggered the release of antisense MO to knock down PAC1R mRNA.

Main Results:

• Maximal PAC1R gene knockdown efficiency of 65% ± 12 reduction in protein level was achieved within 24 hours.
• Reduced PAC1R impaired PC12 cell responsiveness to PACAP.
• Neurite outgrowth decreased to 10% ± 8 and 11% ± 9 after PACAP exposure in knockdown cells.

Conclusions:

• Gold nanocomplexes provide an efficient method for PAC1R gene knockdown.
• PAC1R is critical for PACAP-mediated neurite outgrowth and neural differentiation in PC12 cells.
• This approach enhances understanding of PACAP's neurotropic roles.