A Study of Cobalt (III) Oxide Nanoparticle Delivery of siRNA Molecules Directed Against Signaling Intermediates of the P2Y2 Receptor

Author

Rachel Blair Stroud, Missouri State UniversityFollow

Date of Graduation

Summer 2022

Degree

Master of Science in Cell and Molecular Biology

Department

Biomedical Sciences

Committee Chair

Richard Garrad

Abstract

G protein-coupled receptors are evolutionarily ubiquitous sensors of extracellular signals, propagating intracellular signal cascades through heterotrimeric G proteins. P2Y₂ receptors are GPCRs which are activated by extracellular nucleotides to mediate signaling cascades via Gαq coupling. Many GPCRs are subject to a common mechanism for signal termination involving phosphorylation of the C-terminal tail followed by β-arrestin binding and subsequent endocytic internalization of the complex. This effect has been described for the P2Y₂ R in the 1321N1 astrocytoma cell line, and UTP-induced activation and desensitization profiles have been previously defined. There is need to develop molecular vehicles for safe and effective delivery of nucleic acids such as siRNA, a therapeutic target largely unrealized. Cobalt (III) Oxide nanoparticles, chosen due to preliminary success, were used to deliver β-arrestin1 siRNA to 1321N1 cells. MTT assay shows Co₃O₄NP are not toxic in this cell line. No baseline for β-arrestin1 expression could be established during qRT-PCR, as such neither LipofectamineTM nor Co₃O₄NP delivery of the siRNA conferred measurable knockdown. Calcium assays reveal no significant differences between desensitization proficiency with or without siRNA, though there is visible grouping between treatments. Similarly, unexpected wild type dose-response desensitization trends complicated calcium assay analysis, such that previously reported behavior could not be replicated. Overall, no significant trends were observed, and further trials will be required, largely due to the difficult nature of working with these inorganic nanoparticles.

Keywords

: P2Y2 receptor, nucleotide signaling, β-arrestin, cobalt (III) oxide nanoparticles (Co3O4NP), siRNA, nucleic acid delivery, qRT-PCR, calcium assay, G protein-coupled receptors (GPCR)

Subject Categories

Cell Biology | Molecular Biology | Nanotechnology | Nucleic Acids, Nucleotides, and Nucleosides

Copyright

© Rachel Blair Stroud

Recommended Citation

Stroud, Rachel Blair, "A Study of Cobalt (III) Oxide Nanoparticle Delivery of siRNA Molecules Directed Against Signaling Intermediates of the P2Y2 Receptor" (2022). MSU Graduate Theses. 3789.
https://bearworks.missouristate.edu/theses/3789