Tunable Bio-inorganic Interfaces for Intracellular Access

Download or read book Tunable Bio-inorganic Interfaces for Intracellular Access written by Benjamin David Almquist and published by Stanford University. This book was released on 2011 with total page 101 pages. Available in PDF, EPUB and Kindle. Book excerpt: Electrophysiological tools and biologic delivery systems generally rely on non-optimal methods for gaining access through cellular membranes. Electrophysiological techniques that provide intracellular access, such as patch clamping, result in membrane holes and cell death in a matter of hours, while the delivery of bioactive materials are hampered by low bioavailability following passage through the endosomal pathways. In each case, the lipid bilayer backbone of the cellular membrane presents a formidable barrier to intracellular access. As biological gatekeepers, cell membranes not only physically define everything from whole organisms to individual organelles, they also prevent unobstructed flow of molecules between the inner and outer regions of the membrane. This occurs since the hydrophobic lipid acyl tails form a narrow hydrophobic layer a few nanometers thick, which is highly unfavorable for the passage of most hydrophilic molecules. It is this region that is one of the greatest obstacles to the dream of biotechnology seamlessly and non-destructively integrating synthetic components with biological systems. This thesis contributes to the understanding of how to rationally design devices that interact specifically with this hydrophobic region. In turn, this work begins to establish design guidelines for creating non-destructive, membrane-penetrating bio-inorganic interfaces. The beginning chapters focus on the development of the "stealth" probe platform. In nature, there exist specialized transmembrane proteins capable of incorporating into lipid bilayers by replicating the lipid hydrophilic-hydrophobic-hydrophilic structure. The stealth probe design mimics this structure by creating 2-10nm hydrophobic bands on otherwise hydrophilic structures. However, since current lithographic methods do not possess the necessary resolution, a new fabrication technique using a combination of top-down fabrication with bottom-up self-assembly methods was developed. This approach uses an evaporated chrome-gold-chrome stack and focused ion beam (FIB) milling, where the exposed edge of the embedded gold layer can be specifically functionalized with a hydrophobic thiol-mediated self-assembled monolayer. Chapter 3 explores the propensity for insertion and specific interaction of the stealth probe hydrophobic band with the hydrophobic lipid bilayer core. In order to gain quantitative insight into the interaction behavior, atomic force microscopy was used in conjunction with a new, stacked lipid bilayer testing platform. By using stacks of 100's to 1000's of lipid bilayers, substrate-probe interaction artifacts can be removed while simultaneously allowing precise determination of probe location within a lipid bilayer. It was found that completely hydrophilic probes reside in the hydrophilic hydration region between bilayers, whereas hydrophobically functionalized stealth probes preferred to reside in the bilayer core. This behavior was found to be independent of hydrophobic functionalization, with butanethiol and dodecanethiol both displaying preferential localization. The subsequent chapters explore how the molecular structure of the hydrophobic band and the band thickness affect membrane-probe interface stability. The lipid stack platform provides an easy method of force-clamp testing, which enabled quantitative extrapolation of the unstressed interface strength. A series of tests with various length alkanethiols found that the crystallinity of the molecules in the hydrophobic band is the dominant factor influencing interfacial stability. Surprisingly, hydrophobicity was found to be a secondary factor, although necessary to drive spontaneous membrane integration. Molecular length was also found to play a role in determining the ultimate interfacial strength, with short chain molecules similar in length to amino acid side chains promoting the most stable interfaces. The thickness of the hydrophobic band was found to regulate the interface structure. Bands with thicknesses comparable to that of the host lipid bilayer core likely promote a fused interface geometry, similar in structure to that of transmembrane protein-lipid bilayer interfaces. Thicker bands began to transition to a 'T-junction' interface that is characterized by a lower interface stability. Interestingly, the behavior of 10nm bands were indistinguishable from completely hydrophobic probes, reinforcing the importance of nanoscale patterning for stable membrane integration. Chapter 6 builds on the results of the previous chapters by exploring how various stealth probe geometries influence adhesion behavior. In agreement with force clamp testing, short disordered monolayers displayed strong integration into the bilayer core, while crystalline monolayers displayed extremely weak integration. Preliminary adhesion testing results with human red blood cells demonstrate that the stealth probe geometry holds promise for in vitro and in vivo platforms, expanding the results of this work from simply a biophysical test system to a real world example. Finally, the behavior of two hydrophobic bands either commensurately spaced with the hydrophobic core spacing in the bilayer stack, or incommensurately spaced in order to force one band to reside in the hydrophilic hydration layer, is explored. It was found that the commensurately spaced bands display superior strength to single band tips, which is attributed to the necessity to simultaneously rupture both membrane-hydrophobic band interfaces. Conversely, the incommensurately spaced probes display a significant destabilization of the interface. This is thought to be due to the forced residence of one hydrophobic band in a hydrophilic hydration layer. This result is intriguing for biologic delivery systems, as the nuclear double membrane presents a unique barrier geometry, and a double band system may provide a facile means for penetration.