Development of Proteoliposome- and Nanowell-based Platforms to Assess P-glycoprotein Transport Activity

Open Access
Author:
Park, Soohyun
Graduate Program:
Bioengineering
Degree:
Doctor of Philosophy
Document Type:
Dissertation
Date of Defense:
October 03, 2016
Committee Members:
  • Sheereen Majd, Dissertation Advisor
  • Sheereen Majd, Committee Chair
  • William O Hancock, Committee Member
  • Peter J Butler, Committee Member
  • Manish Kumar, Outside Member
  • William O Hancock, Committee Chair
Keywords:
  • Giant liposome
  • P-glycoprotein
  • Multidrug resistance
  • Pore-spanning bilayer
Abstract:
This thesis presents two platforms developed to assess transport activities of multidrug resistance (MDR) efflux pump, called P-glycoprotein (Pgp). Pgp is a transmembrane protein that is believed to be responsible for MDR of cells against broad range of small molecules. This nonspecific transport activity limits the efficacy of a number of orally induced drugs, as Pgp is expressed on the membranes of various cell types including apical surface of abdomen, epithelial cells of blood-brain barrier, and especially cancer cells. Food and Drug Administration stresses criticality of Pgp in the drug development phase by reinforcing drug screening against Pgp in the approval process. Therefore, effective and efficient techniques to screen the intercations between candidate drugs and Pgp are in high demand. To this end, we developed two assay platforms employing model membrane systems: 1) giant proteoliposome-based assay and 2) a platform with pore-spanning bilayer over an array of nanosized wells. Giant proteoliposomes bearing Pgp with size and composition similar to that of cell plasma membrane are excellent biomimetic system to exclusively monitor the interaction between Pgp and its substrates. In this thesis, transport activity of Pgp across the giant proteoliposome membrane was assessed by translocation of Pgp fluorescent substrate, rhodamine123 (Rho123). We monitored Rho123 transport into the giant proteoliposome lumen upon activation by ATP. The results showed that Rho123 transport rate with ATP concentration followed Michaelis-Menton kinetics, and complete inhibition was achieved with 40 µM verapamil, a Pgp competitive inhibitor. For the nanowell-based Pgp transport activity, an array of nanowells with 200 nm diameter and 800 nm depth was fabricated on a quartz substrate. Giant proteoliposome ruptured on the substrate surface, forming pore-spanning bilayer. Using this platform, we monitored Rho123 transport into the nanowells by active Pgp. In conclusion, giant-proteoliposome and nanowell-based platforms were developed to assess the transport activity of Pgp. This thesis presents the detailed records of optimization processes and assay results and analysis from the two platforms. Finally, we anticipate that these techniques will provide outstanding platforms to assess interactions between Pgp and drug candidates under development in near future.