ENCAPSULATION EFFICIENCY AND DYNAMIC MICROCOMPARTMENTATION WITHIN GIANT LIPID VESICLES

Open Access
Author:
Dominak, Lisa M
Graduate Program:
Chemistry
Degree:
Doctor of Philosophy
Document Type:
Dissertation
Date of Defense:
July 09, 2009
Committee Members:
  • Christine Dolan Keating, Dissertation Advisor
  • Christine Dolan Keating, Committee Chair
  • Philip C. Bevilacqua, Committee Member
  • Anne M Andrews, Committee Member
  • Peter J Butler, Committee Member
Keywords:
  • protein microcompartmentation
  • synthetic cells
  • macromolecular crowding
  • encapsulation efficiency
  • lipid vesicles
  • biomimetic chemistry
Abstract:
Giant vesicles are often used as model membrane systems and as vessels in which to mimic biochemical reactions. The similarity in size and membrane curvature of giant vesicles (GVs) to intact, biological cells, and their ability to be visualized and manipulated under an optical microscope make them an attractive choice for mimicking the environment for cellular biochemistry. Our laboratory typically encapsulates aqueous two-phase systems (ATPS) within GVs to more closely mimic the cytoplasm – these models provide a way to test hypotheses of intracellular organization, namely the consequences of macromolecular crowding and phase separation on biochemical reactions within the cell cytoplasm. While previous members of our laboratory have shown dynamic control over the interior of these ATPS-containing GVs via slight changes in temperature or osmotic pressure – the overall goal of this thesis is to take dynamic control to a new level, demonstrating control over the localization of a protein within the GV interior via an external change in pH – which will be demonstrated in Chapter 5. While data confirming the overall goal of this thesis will be explained in Chapter 5, Chapters 2 – 4 answer very fundamental questions about the encapsulation of macromolecules within GVs. The data presented in these chapters pave the way with information important for the overarching objective, as well as to others using GVs as model bioreactors. Chapter 2 describes the encapsulation behaviors of dilute (<1 weight%) polymer and free dye solutions within GVs as a function of solute size, as well as reporting the effects of temperature, membrane composition, vesicle size, and incubation time on encapsulation efficiency in individual GVs. Chapter 3 describes how macromolecular crowding increases the magnitude and homogeneity of encapsulation for all polymeric solutes, while Chapter 4 describes the same for biomolecules, such as proteins and nucleic acids. Chapter 4 also describes the use of macromolecular crowding to improve bulk encapsulation efficiency within a batch of submicron vesicles, which is important for their use in industrial settings such as vehicles for drug delivery.