Kinetic Stabilization of Interfaces in Biologically Inspired Phase Systems

Open Access
Author:
Dewey, Daniel Carroll
Graduate Program:
Chemistry
Degree:
Doctor of Philosophy
Document Type:
Dissertation
Date of Defense:
April 29, 2015
Committee Members:
  • Christine Dolan Keating, Dissertation Advisor
  • Philip C. Bevilacqua, Committee Member
  • Scott A Showalter, Committee Member
  • Prof Peter Butler, Committee Member
Keywords:
  • ATPS
  • aqueous biphasic systems
  • biomimetic
  • bioreactor
  • vesicle
  • emuslion
Abstract:
Systems created to mimic living systems can be used to provide better control for understanding biological processes, and to apply knowledge gained outside of living systems. This work focuses on the stabilization of interfaces in biomimetic media formed from aqueous polymeric phases and amphiphiles. Polymeric phase systems are used to mimic the aqueous, crowded environment of the cytoplasm, and can also provide non-membranous phases used as compartments within living cells. Amphiphilic molecules can create membranous compartments where the membrane is the interface. Artificial vesicles assembled from amphiphiles are used to mimic the cell membrane and other biological membranes. Biological mimics composed of aqueous phases and vesicles are attractive to origin-of-life studies because of their self-assembly from dilute solution, and their underlying roles in contemporary life. Artificial creation of biological compartments and reaction conditions can be used to create microscale bioreactors that may be applied to research or production. Chapter 2 analyzes an RNA enzyme bioreactor formed from stabilization of a poly(ethylene glycol) (PEG) 8 kDa and dextran 10 kDa aqueous two-phase system. The bioreactors are formed in phase droplets that are stabilized by lipid vesicles. The emulsion is characterized in terms of droplet size, stabilization mechanism, and interfacial diffusion. Droplet size was found to correlate with nearly complete partitioning of vesicles to the interface, where they were observed to stabilize droplets by electrostatic forces. Nucleic acid probe molecules were found to diffuse across the stabilized interface. An RNA enzyme is partitioned to the interior of the bioreactor droplet, a substrate strand diffuses into the droplet, the substrate cleaves, and substrate products leave the dextran-rich phase bioreactor through partitioning. Poly(acrylamide) gel electrophoresis and confocal microscopy were used to monitor the reaction. In Chapter 3, the forces that bring particles to the interface in aqueous phase systems are investigated in terms of depletion forces and interfacial tension. An oil-water interface was created and the water phase was crowded with poly(acrylic acid) (PAA) 2.1 kDa. Silica particles in the aqueous solution changed in their adsorption behavior depending on PAA concentration, despite minimal change in interfacial tension. In a PEG and dextran ATPS, total polymer concentration is raised and particle adsorption is observed as well as the interfacial tension. Caboxylated latex particles were not observed to adsorb to the interface until a greater interfacial tension was present than predicted from standard interfacial tension models. Particles that are adsorbed to the interface still sterically stabilize with behavior expected from oil-water emulsions, including bridging. Chapter 4 expands the electrostatic stabilization observed in Chapter 2. Vesicles without PEGylated surface chemistry are partitioned in a PEG and dextran ATPS as well as polyelectrolytes. Large unilamellar vesicles (LUVs) are found to partition strongly to the dextran-rich phase, even at low ionic strength. Dextran sulfate and diethylaminoethyl dextran were partitioned to create interfacial potentials in a similar matter to the LUVs. Interfacial electrostatic potentials caused by asymmetric ion partitioning are observed to cause steric inhibition of coalescence. Changes in salt concentration led to changes in partitioning and ionic screening changes that altered phase droplet stabilization. The interplay between coacervate phase system interfaces and amphiphilic membrane formation is investigated in Chapter 5 in relation to abiogenesis. Oleic acid vesicles were created and homogenized. Temperature-dependent vesicle formation was observed at ionic strength values relevant to that of an early ocean. PAA 2.1 kDa and poly(allyl ammine hydrochloride) (PAH) 58 kDa coacervates were developed that varied in surface charge depending on polyelectolyte ratio. Interaction between vesicles and coacervates depended on coacervate charge and on phosopholipid incorporation in vesicle membranes. Dioleoylphosphatidylcholine as an additive increases membrane stability and resulted in reduced LUV adsorption. This dissertation describes work controlling amphiphilic membrane adsorption at interfaces in all-aqueous media. A bioreactor is developed from a stabilization mechanism assembled and investigated within the work, and other bioreactors and stabilized phase morphologies mimicking those found within cells may also be developed. Conceptual versions of these applications are proposed and outlined as future directions.