The Use of Time Correlated Single Photon Counting to Optimize siRNA Encapsulation in Nanoliposomes

Open Access
Author:
Tanella, Elaine Marie
Area of Honors:
Bioengineering
Degree:
Bachelor of Science
Document Type:
Thesis
Thesis Supervisors:
  • Peter J Butler, Thesis Supervisor
  • Peter J Butler, Honors Advisor
  • Margaret June Slattery, Faculty Reader
Keywords:
  • siRNA
  • nanoliposome
  • molecular brightness
  • time correlated single photon counting
  • fluorescence correlation spectroscopy
Abstract:
Short interfering RNA (siRNA) mediates gene silencing in mammalian cells. The potential of this molecule to serve as a therapeutic agent for disease such as cancer is currently being examined. In order to be effective, siRNA must interact with the target cell’s RNA machinery found in its cytoplasm. However, siRNA, being negatively charged, cannot spontaneously enter a cell’s cytoplasm due to the nature of the phospholipid bilayer. Additionally, the stability of siRNA is questionable due to the fact that it can be easily degraded by RNAases naturally found in the human body. These problems could be solved by utilizing nanoliposomes as vehicles to transport siRNA to a targeted tumor cell area, bypassing extracellular RNAses. This project uses time-correlated single photon counting (TCSPC) to perform fluorescence correlation spectroscopy (FCS) and to determine molecular brightness (MB) values of fluorescently-tagged siRNA molecules in order to measure the encapsulation efficiency of nanoliposome-siRNA encapsulation. Using this method, the following quantitative information can be extracted from the nanoliposome-siRNA samples: diffusion coefficient (τD), hydrodynamic radii (R), and average concentration (C). TCSPC allows all of this information to be obtained by one system instead of multiple systems. We found that siRNA was able to be encapsulated into the fabricated nanoliposomes, though the quantity of siRNA in each nanoliposome varied. Additionally, we found that TCSPC was an ideal method to obtain multiple important characteristics of these siRNA-nanoliposomes simultaneously.