Hydrodynamically Directed Superbundling of Peptide Amphiphiles

Kolberg-Edelbrock, Jack

doi:https://doi.org/10.21985/n2-9r8h-qx50

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Hydrodynamically Directed Superbundling of Peptide Amphiphiles

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The extracellular matrix (ECM) is a masterwork of biology, and its development was a key part of the transformation from monocellular to multicellular life. With an ECM, cells acquired the ability to cooperatively build a dynamic support network that facilitated their movement, specialization, and communication. This ECM is a hierarchical structure, built from individual proteins, polysaccharides, and inorganic crystals such that single cells can contribute to the macroscopic whole. The fundamentally bottom-up design of ECM has inspired material in applications ranging from non-fouling naval hulls to cortical implants. This work examines the ability of peptide amphiphiles (PAs) to be hierarchically structured into biomaterials by hydrodynamically shaping PA nanofibers into micron-scale, highly aligned “superbundle” (SB) gels.Briefly, chapter one addresses the multidisciplinary nature of this topic by introducing concepts from the fields of tissue engineering, supramolecular chemistry, and fluid dynamics. Chapter two covers the inspiration for this project, as well as the processes developed to transform continuous, liquid phase supramolecular materials into discrete, hyper-aligned, micron-scale gels. Chapter three examines the conditions under which PA SBs can be created by evaluating a variety of processing parameters, as well as identifying material properties necessary for PAs to successfully form SBs. In chapter four, preliminary applications of PA SBs to biological systems are reported, addressing both their biocompatibility and effect on resident cells. The final chapter summarizes the findings reported in chapters two, three, and four and suggests opportunities for future research on PA SBs. Chapter one begins by introducing historical developments in tissue engineering and identifies the primary challenges faced by tissue engineers today. It subsequently reviews the structure of natural tissues with a focus on the ECM. This review of natural tissue is followed by a discussion of commonly used biomaterials and their lifecycle. It then focuses in on supramolecular biomaterials, providing a detailed chemical and biological review of peptide amphiphiles (PAs), the biomaterial studied in this thesis. Chapter one concludes with an introduction to flow-focusing microfluidics for biomaterials fabrication. Chapter two details the development of a flow-focusing microfluidic system capable of processing annealed PA nanofibers into the hyper-aligned, micron-scale gels called superbundles (SBs). Fundamental concepts introduced in this chapter include the interactions present between PA nanofibers, manners by which PA gelation occurs, and the motivation for creating the described microfluidic system. This introduction is followed by a summary of the engineering process used to create a system capable of extruding SBs. The discussion focuses on the development of an appropriate microfluidic junction geometry, the effects of intrinsic fluid properties on microfluidic device behavior, and the eventual conditions that led to the first SBs. These engineering details are followed by a theoretical exploration of the PA gelation process. This analysis is based on gelation studies performed by Olav Smidsrød. After this theoretical examination of the interfacial processes occurring within the microfluidic device, chapter two proposes and argues for one possible explanation for the hyper-aligned morphology observed in SBs. This explanation focuses on the ability of laminar flow conditions to align high-aspect-ratio particles in the direction of fluid flow and posits that this flow alignment allows PA nanofibers to undergo entropy-driven condensation out of the surrounding solvent. This proposed mechanism is based on the principles put forth by the Asakura-Oosawa Theory (AOT) and successor theories. Chapter two closes with characterization experiments of the initial, “exploratory phase” SBs. The observed sizes of SBs are explained through the production of a mathematical model, as well as the hypothesis that SBs break down along their mechanically weak short axis during the post-extrusion deceleration process. The mutual necessity of PA nanofibers and a gelator to SB formation is demonstrated by the inability to form SBs in the absence of either PA nanofibers or divalent cations. Finally, the primary role of inter-nanofiber interactions in SB formation is supported by a spectroscopic interrogation of SB’s internal β-sheet structure which demonstrates persistence of the β-sheet structure after SB formation. Chapter three explores the breadth and flexibility of microfluidic PA gelation. It starts by explaining the benefits and drawbacks of the considered techniques for quantifying SB morphology and defends the decision to use blinded human confocal microscopy scoring to determine the processing parameters under which SB most easily formed. This argument is followed by an explanation of the possible processing parameters that were available for exploration and explains the decision to focus on flow rate ratios (FRRs) and PA concentration. The data indicate that SB formation occurs most reliably at high FRRs and high PA concentrations. These findings are hypothesized to occur due to the combined importance of flow confinement and a percolating nanofiber network. Flow confinement is shown to support proper alignment of PA nanofibers, and a percolating nanofiber network helps SBs remain intact after extrusion. These findings are supported by electron and confocal microscopy images demonstrating the absence of aligned structures at low FRRs and the loss of cohesion present within a single SB when PA concentration falls below 0.1 weight percent (wt%), respectively. After exploring the transformation of PA nanofibers into SBs using calcium as the gelator, chapter three examines the use of alternative gelators including non-physiologic cations, polyions, and oppositely charged PA nanofibers. Regardless of the gelator used, the same principle of high PA concentration and high FRR was shown to produce the best SBs. Interestingly, these explorations suggested that high-molecular-weight gelators promoted primarily interfacial gelation, while ions promoted bulk gelation. Chapter three closes with the hypothesis that SB formation can be extended to a wide variety of PA chemistries. Chapter four explores chapter three’s hypothesis of the flexibility of SB formation and the ability to harness that flexibility to use SBs in biological applications. Specifically, it demonstrates that PAs with pendant GRGDS, IKVAV, and BMP2-binding epitopes can be successfully incorporated into SBs by following the design rules developed in chapter three, shows that SBs encapsulate and store therapeutic proteins with a variety of isoelectric points, and highlights the morphological similarities between SBs and decellularized ECM (dECM). Following these proofs-of-concept, the chapter details the wide array of experiments performed to demonstrate both biocompatibility and biofunctionality of SBs. These experiments include engineering discussions of how to take “as-extruded” SBs and form them into a macroscopic object that is suitable for cell culture, an evaluation of the differences between seeding cells onto SBs versus co-extruding cells with the PA, and a demonstration that cells prefer to interact with SBs over size-matched, microscopically-unaligned PA fragments. Briefly, as-extruded SBs are most effectively condensed into cell-culture-ready materials through either gentle packing onto a transwell membrane or absorption onto an absorbable collagen sponge (ACS). Cells were shown to survive both co-extrusion, where annealed PAs are mixed with cells prior to extrusion, and post-extrusion seeding, where a suspension of cells is deposited onto prefabricated SBs. Interestingly, the size of the co-extruded cells appeared to influence their location in the final biomaterial structure. Large cells, such as human mesenchymal stem cells (hMSCs) were located outside of SBs; small cells, such as mouse myoblasts (C2C12s) remained embedded within single SBs. The preferential interaction of multiple cell types with SBs versus size-matched, microscopically un-aligned PA “fragments” is demonstrated by morphological analysis. After these initial cellular experiments are described, chapter four delves into the use of SBs in osteogenic applications. Although conclusive data was not collected on this topic, initial results suggest that SB-like morphology may accelerate the differentiation of rat bone-marrow mesenchymal stem cells (rBMSCs) into an osteogenic lineage under both non-differentiation promoting and differentiation promoting conditions. This thesis concludes with chapter five, which connects the findings of the earlier chapters and details possible future projects that could be performed using the SB microfluidic platform, such as non-osteogenic applications, controlled macroscopic assembly of SBs, and experimental analyses of the kinetics and dynamics of SB formation. Overall, the key findings of this thesis include the morphological similarity of PA SBs to decellularized mammalian extracellular matrix, the identification of processing parameters key to SB formation, the flexibility of the PA SB fabrication process, and the interplay between processing parameters and the response of cells cultured on their surfaces.

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