Open Access Dissertation
Date of Award
Olivia M. Merkel
This dissertation focuses on the ability of polyethyleneimine-graft-polycaprolactone-block-poly(ethylene glycol) (PEI-g-PCL-b-PEG-Fol) folate decorated tri-block copolymers ability to deliver a targeted dose of siRNA. The micelleplexes that are formed upon electrostatic interaction with siRNA are used to deliver siRNA in a targeted manner to ovarian cancer cells that over-express Folate Receptor-α (FRα). Each conjugate showed suitable sizes below 200 nm with full siRNA condensation ability. Furthermore, flow cytometry and western blot analysis demonstrated that the best FRα targeted polymer was able to effectively deliver siRNA which resulted in protein knockdown of Toll-like receptor 4 (TLR4). Consequently, TLR4 knock down within SKOV-3 cells re-sensitized them toward paclitaxel (PTX) treatment, and apoptotic events increased. This study demonstrates that PEI-g-PCL-b-PEG-Fol conjugates are a reliable siRNA delivery system and can mediate therapeutic TLR4 knockdown within ovarian cancer cells.
Subsequently, folate receptor binding studies were performed using Atomic Force Microscopy (AFM) in order to assess the binding force and probability between folic acid decorated micelleplexes and free folic acid. AFM cantilevers were decorated with active FR and our studies demonstrate that our micelleplexes have a stronger binding force and binding probability than free folic acid. Both results show that the folate decorated micelleplexes out-compete for the binding of folic acid due to their multivalent nature and therefore stronger binding avidity. Uptake studies with low concentrations of folic acid only show a slight inhibition of folic acid micelleplexes uptake, while not affecting the non-targeted micelleplexes. However, increasing concentrations of folic acid seen within literature, inhibited the uptake of targeted and non-targeted micelleplexes. These data suggest that excess folic does not blocking the micelleplexes from binding to the receptor, but perhaps affecting all nanoparticle uptake due to a cellular event, a physical destabilization of the cationic condensation of the nucleic acids, or causing an aggregation of the micelles therefore affecting its uptake.
In vivo studies were performed with an orthotopic SKOV-3/luc xenograft model in order to assess the conjugate’s effectiveness at targeted siRNA delivery and knockdown capabilities. A Bruker In-Vivo Xtreme imaging system was utilized to monitor tumor growth and luciferase knockdown. While Indium-111 labeled siRNA was designed to monitor siRNA whole body distribution, tumor targeting, and pharmacokinetic parameters by SPECT/CT and gamma scintillation counting. Upon I.P. injection, both the targeted and non-targeted siRNA containing micelleplexes showed 5-6% tumor uptake. However, when compared to scramble siRNA, the targeted micelleplexes was able to achieve a 62% luciferase knockdown. Overall, this platform for in vivo siRNA delivery with amphiphilic tri-block copolymers provides a promising option for gene knockdown for ovarian cancers.
Jones, Steven, "Folate Receptor Alpha Targeted Delivery And Characterization Of Polyethyleneimine-Graft-Polycaprolactone-Block-Poly(ethylene Glycol) Containing Sirna Micelleplexes" (2017). Wayne State University Dissertations. 1817.