Search

Antimicrobial fatty acids derived from natural sources and renewable feedstocks are promising surface-active substances with a wide range of applications. Their ability to target bacterial membrane in multiple mechanisms offers a promising antimicrobial approach for combating bacterial infections and preventing the development of drug-resistant strains, and it provides a sustainable strategy that aligns with growing environmental awareness compared to their synthetic counterparts. However, the interaction and destabilization of bacterial cell membranes by these amphiphilic compounds are not yet fully understood. Here, we investigated the concentration-dependent and time-dependent membrane interaction between long-chain unsaturated fatty acids-linolenic acid (LNA, C18:3), linoleic (LLA, C18:2), and oleic acid (OA, C18:1)-and the supported lipid bilayers (SLBs) using quartz crystal microbalance-dissipation (QCM-D) and fluorescence microscopy. We first determined the critical micelle concentration (CMC) of each compound using a fluorescence spectrophotometer and monitored the membrane interaction in real time following fatty acid treatment, whereby all micellar fatty acids elicited membrane-active behavior primarily above their respective CMC values. Specifically, LNA and LLA, which have higher degrees of unsaturation and CMC values of 160 µM and 60 µM, respectively, caused significant changes in the membrane with net |Δf| shifts of 23.2 ± 0.8 Hz and 21.4 ± 0.6 Hz and ΔD shifts of 5.2 ± 0.5 × 10-6 and 7.4 ± 0.5 × 10-6. On the other hand, OA, with the lowest unsaturation degree and CMC value of 20 µM, produced relatively less membrane change with a net |Δf| shift of 14.6 ± 2.2 Hz and ΔD shift of 8.8 ± 0.2 × 10-6. Both LNA and LLA required higher concentrations than OA to initiate membrane remodeling as their CMC values increased with the degree of unsaturation. Upon incubating with fluorescence-labeled model membranes, the fatty acids induced tubular morphological changes at concentrations above CMC. Taken together, our findings highlight the critical role of self-aggregation properties and the degree of unsaturated bonds in unsaturated long-chain fatty acids upon modulating membrane destabilization, suggesting potential applications in developing sustainable and effective antimicrobial strategies. Ministry of Education (MOE) Published version This research was supported by the Minister of Education (MOE) in Singapore under grants RG111/20 and RG34/22. This research was also supported by the Procter & Gamble Company (P&G) under grants APG2013/129.

Cell-membrane-mimicking supported lipid bilayers (SLBs) provide an ultrathin, self-assembled layer that forms on solid supports and can exhibit antifouling, signaling, and transport properties among various possible functions. While recent material innovations have increased the number of practically useful SLB fabrication methods, typical SLB platforms only work in aqueous environments and are prone to fluidity loss and lipid-bilayer collapse upon air exposure, which limits industrial applicability. To address this issue, herein, we developed sucrose-bicelle complex system to fabricate air-stable SLBs that were laterally mobile upon rehydration. SLBs were fabricated from bicelles in the presence of up to 40 wt% sucrose, which was verified by quartz crystal microbalance-dissipation (QCM-D) and fluorescence recovery after photobleaching (FRAP) experiments. The sucrose fraction in the system was an important factor; while 40 wt% sucrose induced lipid aggregation and defects on SLBs after the dehydration-rehydration process, 20 wt% sucrose yielded SLBs that exhibited fully recovered lateral mobility after these processes. Taken together, these findings demonstrate that sucrose-bicelle complex system can facilitate one-step fabrication of air-stable SLBs that can be useful for a wide range of biointerfacial science applications. Agency for Science, Technology and Research (A*STAR) Ministry of Education (MOE) Published version This work was supported by the Ministry of Education (MOE) in Singapore under Grant AcRF TIER1-2020-T1-002-032 (RG111/20). This work was also sup‑ ported by the China-Singapore International Joint Research Institute (CSIJRI). HT is supported by a SINGA graduate scholarship from the A*STAR Graduate Academy, Singapore.

Additional file 1: Table S1. Shear viscosity of sucrose solutions without/with bicelles, and bicelle sizes in sucrose���bicelle mixtures containing various concentrations of sucrose. Figure S1. QCM-D measurements of bicelle adsorption onto silica surfaces in various concentrations of sucrose (A���F, 0���50 wt% sucrose). The baseline of ��f and ��D shifts were recorded in Tris buffer containing 150 mM NaCl, and then measurements were made in the following solutions: (1) sucrose solution in Tris buffer; (2) sucrose���bicelle mixture for SLB formation; (3) sucrose solution in Tris buffer for washing step; (4) Tris buffer for final baseline to check the quality of SLB formation. Figure S2. Air stability of SLBs after 1 month of dehydration. (A) FRAP results and recovery curves for mobility characterization of SLBs made from 20 wt% sucrose and rehydrated after 1 month of dehydration. (B) Corresponding results for SLBs made from 40 wt% sucrose. All scale bars are 20 ��m.

Supported lipid membranes are versatile biomimetic coatings for the chemical functionalization of inorganic surfaces. Developing simple and effective fabrication strategies to form supported lipid membranes with micropatterned geometries is a long-standing challenge. Herein, we demonstrate how the combination of chemical lift-off lithography (CLL) and easily prepared lipid bicelle nanostructures can yield micropatterned, supported lipid membranes on gold surfaces with high pattern resolution, conformal character, and biofunctionality. Using CLL, we functionalized gold surfaces with patterned arrays of hydrophilic and hydrophobic self-assembled monolayers (SAMs). Time-lapse fluorescence microscopy imaging revealed that lipid bicelles adsorbed preferentially onto the hydrophilic SAM regions, while there was negligible lipid adsorption onto the hydrophobic SAM regions. Functional receptors could be embedded within the lipid bicelles, which facilitated selective detection of receptor-ligand binding interactions in a model streptavidin-biotin system. Quartz crystal microbalance-dissipation measurements further identified that lipid bicelles adsorb irreversibly and remain intact on top of the hydrophilic SAM regions. Taken together, our findings indicate that lipid bicelles are useful lipid nanostructures for reproducibly assembling micropatterned, supported lipid membranes with precise pattern fidelity.

Plasmonic nanostructures have a wide range of applications, including chemical and biological sensing. However, the development of techniques to fabricate submicrometer-sized plasmonic structures over large scales remains challenging. We demonstrate a high-throughput, cost-effective approach to fabricate Au nanoribbons via chemical lift-off lithography (CLL). Commercial HD-DVDs were used as large-area templates for CLL. Transparent glass slides were coated with Au/Ti films and functionalized with self-assembled alkanethiolate monolayers. Monolayers were patterned with lines via CLL. The lifted-off, exposed regions of underlying Au were selectively etched into large-area grating-like patterns (200 nm line width; 400 nm pitch; 60 nm height). After removal of the remaining monolayers, a thin In2O3 layer was deposited and the resulting gratings were used as plasmonic sensors. Distinct features in the extinction spectra varied in their responses to refractive index changes in the solution environment with a maximum bulk sensitivity of ∼510 nm/refractive index unit. Sensitivity to local refractive index changes in the near-field was also achieved, as evidenced by real-time tracking of lipid vesicle or protein adsorption. These findings show how CLL provides a simple and economical means to pattern large-area plasmonic nanostructures for applications in optoelectronics and sensing.

Generation of human organoids from induced pluripotent stem cells (iPSCs) offers exciting possibilities for developmental biology, disease modelling and cell therapy. Significant advances towards those goals have been hampered by dependence on animal derived matrices (e.g. Matrigel), immortalized cell lines and resultant structures that are difficult to control or scale. To address these challenges, we aimed to develop a fully defined liver organoid platform using inverted colloid crystal (ICC) whose 3-dimensional mechanical properties could be engineered to recapitulate the extracellular niche sensed by hepatic progenitors during human development. iPSC derived hepatic progenitors (IH) formed organoids most optimally in ICC scaffolds constructed with 140 μm diameter pores coated with type I collagen in a two-step process mimicking liver bud formation. The resultant organoids were closer to adult tissue, compared to 2D and 3D controls, with respect to morphology, gene expression, protein secretion, drug metabolism and viral infection and could integrate, vascularise and function following implantation into livers of immune-deficient mice. Preliminary interrogation of the underpinning mechanisms highlighted the importance of TGFβ and hedgehog signalling pathways. The combination of functional relevance with tuneable mechanical properties leads us to propose this bioengineered platform to be ideally suited for a range of future mechanistic and clinical organoid related applications. NRF (Natl Research Foundation, S’pore) Published version

Generation of human organoids from induced pluripotent stem cells (iPSCs) offers exciting possibilities for developmental biology, disease modelling and cell therapy. Significant advances towards those goals have been hampered by dependence on animal derived matrices (e.g. Matrigel), immortalized cell lines and resultant structures that are difficult to control or scale. To address these challenges, we aimed to develop a fully defined liver organoid platform using inverted colloid crystal (ICC) whose 3-dimensional mechanical properties could be engineered to recapitulate the extracellular niche sensed by hepatic progenitors during human development. iPSC derived hepatic progenitors (IH) formed organoids most optimally in ICC scaffolds constructed with 140 μm diameter pores coated with type I collagen in a two-step process mimicking liver bud formation. The resultant organoids were closer to adult tissue, compared to 2D and 3D controls, with respect to morphology, gene expression, protein secretion, drug metabolism and viral infection and could integrate, vascularise and function following implantation into livers of immune-deficient mice. Preliminary interrogation of the underpinning mechanisms highlighted the importance of TGFβ and hedgehog signalling pathways. The combination of functional relevance with tuneable mechanical properties leads us to propose this bioengineered platform to be ideally suited for a range of future mechanistic and clinical organoid related applications.

The coating of solid-binding peptides (SBPs) on inorganic material surfaces holds significant potential for improved surface functionalization at nano-bio interfaces. In most related studies, the goal has been to engineer peptides with selective and high binding affinity for a target material. The role of the material substrate itself in modulating the adsorption behavior of a peptide molecule remains less explored and there are few studies that compare the interaction of one peptide with different inorganic substrates. Herein, using a combination of two experimental techniques, we investigated the adsorption of a 16 amino acid-long random coil peptide to various inorganic substrates – gold, silicon oxide, titanium oxide and aluminum oxide. Quartz crystal microbalance-dissipation (QCM-D) experiments were performed in order to measure the peptide binding affinity for inorganic solid supports at the ensemble average level, and atomic force microscopy (AFM) experiments were conducted in order to determine the adhesion force of a single peptide molecule. A positive trend was observed between the total mass uptake of attached peptide and the single-molecule adhesion force on each substrate. Peptide affinity for gold was appreciably greater than for the oxide substrates. Collectively, the results obtained in this study offer insight into the ways in which inorganic materials can differentially influence and modulate the adhesion of SBPs.

The multienzyme catalytic phosphorylation of phosphatidylinositol (PI) in a supported lipid membrane platform is demonstrated for the first time. One-step treatment with PI 4-kinase IIIβ (PI4Kβ) yielded PI 4-phosphate (PI4P), while a multistep enzymatic cascade of PI4Kβ followed by PIP 5-kinase produced PI-4,5-bisphosphate (PI(4,5)P2 or PIP2). By employing quartz crystal microbalance with dissipation monitoring, we were able to track membrane association of kinase enzymes for the first time as well as detect PI4P and PI(4,5)P2 generation based on subsequent antibody binding to the supported lipid bilayers. Pharmacologic inhibition of PI4Kβ by a small molecule inhibitor was also quantitatively assessed, yielding an EC50 value that agrees well with conventional biochemical readout. Taken together, the development of a PI-containing supported membrane platform coupled with surface-sensitive measurement techniques for kinase studies opens the door to exploring the rich biochemistry and pharmacological targeting of membrane-associated phosphoinositides.

Currently, the clinical utility of taxane-based drug formulations in castration-resistant prostate cancer (CRPC) is severely limited by acquired chemotherapy resistance, dose-limiting toxicities, and nonresponders. Therefore, approaches to improve taxane-based chemotherapy are desperately required. In this review, we highlight the strategies that aim to overcome these limitations, such as bypassing therapy resistance, targeted drug delivery, and adequate prediction of therapy response. The involvement of the apoptotic pathway, ABC transporters, the glucocorticoid receptor (GR) axis, androgen receptor (AR) splicing, epithelial plasticity, and cancer stem cells in mediating taxane-resistance are outlined. Furthermore, passive and active targeted nanomedicinal drug delivery strategies and the use of circulating tumor cells in predicting docetaxel responses are discussed. Finally, recent advances towards clinical translation of these approaches in CRPC are reviewed.

The quartz crystal microbalance sensor has a resonant frequency 𝑓 and a quality factor 𝑄 which can be used to probe the properties of nanometer thick film loads. A recent review by Arnau (2008) has discussed many of the considerations necessary to accurately probe for these properties. To avoid needless duplication but to still provide an adequate background for the new user, we briefly outline the basic measurement methodologies and analytical techniques that were covered in the review. Details will be provided on some specific perspectives of the authors. For example, the special precautions necessary when dealing with soft films (polymeric and biological) under liquid are overviewed. To illustrate applications of the QCM technique, simple bilayer and vesicle behaviors are discussed, along with the structural transformation resulting from protein adsorption onto an intact vesicle adlayer. The amphipathic 𝛼 -helical (AH) peptide interaction is given as a particular example. Lastly, we summarize a top-down approach to functionalize a surface with a cell membrane and to study its interaction with proteins.

Molecular delivery using carriers for biological applications has been a subject of interest for the past many years, as some molecules suffer from solubility (reduced bioavailability) and stability (pH & enzyme-sensitive molecules like proteins) issues, resulting in denaturation, inactivation & loss of function. This necessitates a robust delivery vehicle, capable encapsulating such molecules, protecting them till the site of release, followed by controlled tunable release as to avoid low or excess levels, keeping the molecular concentration in the effective therapeutic window, especially in case of drug delivery. Various nano and microparticles of different sizes and surface chemistry have been used for such delivery through different modes of administration with oral being most favoured, due to ease of dosage, along with high patient compatibility. Most of these particles are synthetic in nature, involving multiple processing steps and suffer from issues like largescale synthesis, non-uniformity, particle stability (under gastric conditions etc), biocompatibility & biodegradability to name some pressing issues. Few natural and bioinspired carriers, which have relatively much lesser safety concerns, have also been explored for this purpose successfully, which pushes us to look at other such naturally available resources. Plant pollen and spores, a natural product with uniform size, physicochemical characteristics proves to be invaluable in this aspect, offering molecular loading and protection for oral delivery applications, with promising research done till date. Pollen and spores have been shown to be promising prospects for encapsulating molecules due to their double layered wall, comprising of a cellulosic intine and a exine, composed of a supreme polymer called sporopollenin. Processed pollen namely, sporopollenin exine capsules (SECs), where pollen/spores are subject chemical treatment to remove sporoplasmic contents and the intine layer, were favoured due to lower allergenicity and more loading space, with most of the work focused on singlecompartmental non-saccate pollen (Lycopodium, Sunflower etc.). Here we show that multi-compartmental pine pollen is also an effective vehicle to encapsulate and deliver molecules of interest. It has been consumed as a super- food with health-benefits such as enhanced immune and endocrine function (excellent source of testosterone), lowering of cholesterol levels along with anti-inflammatory, anti-arthritic, and anti-tumor activity and has not been explored for molecular encapsulation till date. Pine SECs however have beenproduced by sequential organic solvent, acid/base and enzymatic processing, with no process optimization nor morphological characterisation done, followed by loading of a few molecules. Here we looked at pine SECs got through acid-processing of defatted pine pollen intially, with various processing conditions like acids, concentrations and duration were explored, with the structural integrity of the capsules looked into for every processing condition with defatted pine as the source material. Given the rapid rise in protein therapeutics, SEC production was followed by protein (BSA) loading, where it was found to load thrice as much as defatted pollen of the same mass, with fluorescently tagged proteins (FITC-BSA) used to analyse the spatial loading. As the SECs suffered from structural issues, natural and defatted pollen( which involves relatively less processing) was explored more in detail in the next study, where natural pine pollen was ether-defatted. Natural pine pollen was process optimized for BSA encapsulation regarding the loading method (passive or vacuum-assisted loading), loading duration and protein concentration and then comparitive protein (BSA) loading of natural with defatted pine pollen was done to quantify protein loading with the degree of defatting. Defatted pine loaded better here due to increased pore size as measured by nitrogen adsorption/desorption isotherms. Controlled release was shown using BSA-loaded defatted pollen as the carrier, with natural polymers as binders (Xanthan Gum) or using coatings (Sodium Alginate) with tableted formulations, with simulated gastric (pH 1.2) and intestinal fluids (pH 7), where the protein was protected in the gastric phase and released gradually in the intestinal phase. FITC-BSA loading showed that the protein loaded mainly into the air-sacs with minimal central cavity loading with vacuum loading for both natural and defatted pollen, which is different from SECs spatial loading pattern, which loaded all over the particle. Finally, natural pine pollen was explored for loading of other potential molecules like dyes and drugs apart from proteins (BSA & IgG) also, through simple passive loading and vacuum–assisted loading, where the large proteins were found to load exclusively in the air-sacs with vacuum loading and minimally into the central cavity with passive loading. The dyes and drugs however loaded into the central cavity alone irrespective of the loading method. This was followed by dual molecular loading, where the dyes and drug were loaded into the central cavity through passive loading, followed by vacuum loading of BSA, which resulted in compartmentalization (distinct molecular loading pattern into air-sacs and central cavity) of molecules. The protein structure of BSA was checked into pre-loading and post-release and was found to be conserved. All this show that pine pollen is capable of encapsulating, preserving and controlled-release of proteins and other molecules, opening up myriad applications in fields like drug delivery, molecular preservation, nutraceutical delivery etc. Doctor of Philosophy (IGS)

The complement system serves as the frontline of host defense for the human body against foreign entities, and is triggered via one or more of several signaling cascades that consist of interactions between soluble and cell surface-bound complement proteins. In this thesis, the objective is to explore how lipid membranes influence the initiation, amplification, termination and regulation of complement activation, utilizing supported lipid bilayer platforms as a model experimental system. The overall hypothesis of this work is that the supported lipid bilayer platform provides a useful model system to recapitulate key protein-lipid and protein-protein interactions involved in complement activation at lipid membrane interfaces, thereby establishing an experimental framework to decipher key trigger factors (e.g., membrane surface charge) as well as evaluate candidate inhibitors acting against complement convertases and membrane attack complex assemblies. Within this scope, four different experimental studies were conducted, leading to new insights into the mechanisms by which lipid membranes influence complement activation. In the first study, the influence of membrane surface change on the adsorption properties of different pattern recognition proteins involved in initiation of complement activation was investigated. In the second study, the effects of covalent and noncovalent tethering strategies on the self-assembly of the alternative pathway C3 convertase was monitored. It was identified that the C3b immobilization scheme is a critical factor governing convertase assembly, further enabling successful evaluation of a clinically relevant complement inhibitor, compstatin. In the third study, the assembly of the complement-terminating membrane attack complex was monitored onto supported lipid bilayer platforms, with particular attention to the influence of membrane surface charge. It was determined that the membrane attack complex assembly preferentially forms on negatively charged lipid membranes, indicating the importance of membrane electrostatics. In the fourth study, the inhibitory action of vitronectin and clusterin against the membrane attack complex assembly was evaluated, offering insight into the different mechanisms by which these two regulatory proteins inhibit complement activation. In summary, the findings presented in this thesis significantly contribute to the fundamental understanding of complement activation occurring at lipid membrane interfaces, and offer a new platform to characterize the mechanisms of action for a wide range of complement inhibitors including small molecules, antibodies, and peptides. Doctor of Philosophy (MSE)

The sporoderms of pollen and plant spores provide an ideal source of robust monodisperse capsules for microencapsulation applications. Plant-based sporoderms are nature’s solution to protecting sensitive genetic material from harsh environmental conditions and offer a promising natural solution to the large-scale manufacture of microcapsules. The outer sporoderm layer (exine) of pollen and spores typically comprises a biopolymer, sporopollenin, with remarkable physicochemical stability, and the inner sporoderm layer (intine) typically comprises a combination of common cellulosic materials. It is important to note that the sporopollenin and cellulosic layers comprising the sporoderm are free from allergenic compounds commonly associated with pollen. Through standard wet chemistry processing methods, allergenic proteins and other contaminants may be completely removed. There currently exist a wide range of chemical processing protocols for extracting sporopollenin sporoderm microcapsules (S-SMCs), as well as various techniques for the loading of compounds within S-SMCs. However, there is still a need for a systematic analysis of the efficacy of S-SMC extraction steps, and key factors influencing the loading and release of compounds within S-SMCs. Therefore, in this thesis, detailed studies were undertaken of the most commonly utilized S-SMC extraction protocol, the applicability of this protocol to genetically distant sources of S-SMCs was explored, and the dynamics of compound loading and release from S-SMCs were elucidated. Standard S-SMC extraction steps involve, defatting of surface lipidic compounds, base-hydrolysis of proteinaceous cytoplasmic contents, acid-hydrolysis of cellulosic materials, and washing for removal of processing residues, under varying experimental conditions. Through these studies, it was shown that base-hydrolysis is redundant and may actually damage the integrity of the sporoderm from some plant species. It is now clear that defatting and acid-hydrolysis alone are adequate to isolate S-SMCs. Acid-hydrolysis temperatures can be reduced from 170°C to 70°C, and processing times can be reduced from 7 days to 5 - 30 hours. Additionally, it was observed that excessive duration acidolysis, based on existing standard protocols, destabilizes the S-SMC microstructure and causes capsule fragmentation. With regards to mechanisms of S-SMC loading and release, it was identified that air bubble collapse drives S-SMC loading, and that methods of assisted loading are important to ensure the full loading of all S-SMC cavities. Through optimization of macromolecule loading it was possible to elucidate practical loading efficiencies and distributions, as well as to estimate theoretical maximum loading efficiencies. In vitro release studies resulted in both qualitative and quantitative analysis of release dynamics. Further studies with enteric coencapsulation allowed for the development of an S-SMC system for targeted intestinal delivery with tunable controlled release. Overall, these studies provide important insights into the ongoing exploration of these promising natural microcapsules and their potential for utilization in a wide range of industrial applications. Doctor of Philosophy (MSE)

Development of functionalized surfaces that can interface with cells is essential for many biotechnological applications ranging from routine cell culture to advanced applications such as cell-based diagnostics, tissue engineering, medical implants and biosensor development. Supported lipid bilayer (SLB), is a versatile synthetic platform which mimics the fundamental properties of native biological membranes and enables studying membrane-associated cellular processes in sufficiently simplified and more controlled conditions. SLBs composed of zwitterionic lipids are resistant to nonspecific adsorption of cells and proteins. However, they can be functionalized with specific cell recognition motifs by introducing lipids with chemically selective headgroups into the bilayer for direct covalent or non-covalent coupling of biomolecules. The primary aim of this thesis work has been to explore and advance the application of SLBs as cell-surface mimetic substrates for cell interaction studies, with the special focus on functionalization of SLBs with the extracellular matrix (ECM) components. The ECM is a tissue-specific network of extracellular bio-active molecules that provides support and anchorage for cells and plays the key role in numerous cellular processes including cell growth and differentiation as well as intercellular communications. The behaviour of cells in a tissue is regulated by both chemical as well as mechanical signals arising from their microenvironment. First, we developed an experimental platform using SLB functionalized with ECM proteins for mimicking a range of substrate rigidities by tuning the bilayer viscosity. We used a recently developed method of SLB formation called solvent-assisted lipid bilayer (SALB) formation to fabricate bilayers containing various fractions of a functional lipid with a reactive headgroup to attach two major ECM components; collagen type I (Col I) and fibronectin (FN) covalently to the bilayer surface. Using, quartz crystal microbalance with dissipation (QCM-D), we demonstrated that the density of the proteins can be controlled by the fraction of reactive lipids included in the lipid precursor solution used in the SALB method. The ECM-SLB platform, supported the cell attachment, growth, and function. Importantly, fluorescent recovery after photobleaching (FRAP) analysis showed that bilayer retained its fluidity after protein conjugation and cell adhesion. The fluid nature of the bilayer led to the lateral displacement of anchored Col and FN and their accumulation underneath the cell in response to the traction forces exerted by the cells during the cell spreading process. We could control the displacement by increasing the viscosity of the membrane via incorporation of a high fraction of cholesterol (Chol) (up to 40%), a possibility offered by the SALB method. Second, we investigate the biological activity of Col I and FN which are covalently attached to the SLB and compared with those of nonspecifically adsorbed to SiO2 surface. The characterization of protein adsorption by QCM-D revealed that Col I and FN that attached to SLB have higher structural flexibility than those adsorbed onto SiO2, and their activity was higher with respect to Col I-FN interaction and cell adhesion efficiency. Finally, in attempt to develop a substrate which more closely mimics the native cell-surface environment, we functionalized the SLB with decellularized extracellular matrix (dECM) components. The dECM was obtained through a combination of chemical and enzymatic treatments of mouse adipose tissue and was solubilized under acidic conditions. We demonstrated that dECM functionalized bilayers sustain cell growth and maintain their function. The strategy and results presented in this thesis work stress the potential of ECM-SLB platform for studying cell-matrix interactions as well as studying cellular processes that occur at the membrane interface. Doctor of Philosophy (MSE)

The cellular membrane is a complex, self-assembled biological structure that is responsible for numerous physiological functions and also serves to compartmentalize various cell organelles. There is great interest in understanding how membrane-active agents perturb cellular membranes from diverse origins, e.g., mammalian and bacterial, as well as other biological entities such as much smaller, enveloped viruses. This interest has directed countless efforts to attempt to mimic the complex nature of cellular membranes in order to create different types of model membrane platforms. In this thesis, the main objective is to examine the biophysical interactions between membrane-active agents such as amphipathic peptides, fatty acids, and monoglycerides, and model membrane platforms in order to distinguish the corresponding membrane morphological responses. The overall hypothesis is that probing membrane morphological responses induced in model lipid membrane platforms can distinguish the mechanisms of action of membrane-active peptides and monoglycerides, thereby revealing novel biophysical insights into molecular evolution and anti-infective applications. Within this scope, three main experimental studies were conducted that were aimed at developing more biologically relevant model platforms as well as investigating unexplored monoglyceride molecules of biological importance. In the first study, in order to create a defect-free supported lipid bilayer, an α-helical (AH) peptide derived from nonstructural protein (NS5A) of the hepatitis C virus, was employed to repair the still unruptured vesicles trapped in the initially formed bilayer after the vesicle fusion, revealing a correlation between vesicle size and rupture potency. For the second study, the preparation of giant unilamellar vesicles (GUVs) that include fractions of human liver microsome (HLM) extracts was conducted in order to study the interactions against AH peptide. Based on the experimental results obtained at different HLM fractions and peptide concentrations in a systematic fashion, it was identified that HLM-GUVs were more resistant to peptide-induced disruption in direct comparison with DOPC-GUVs, which are composed of wholly synthetic lipids. In the third study, the interaction between GUVs and glycerol monolaurate was explored for the first time. Depending on the aggregation state of glycerol monolaurate (monomeric versus micellar states), vesicle fission and fusion behaviors were observed in both simple and complex GUV compositions. This is the first example of a single molecule that can induce both vesicle fusion and fission, highlighting the potentially significant role of monoglycerides in evolutionary biology as a more advanced molecular structure going beyond the earliest, primitive cellular membranes composed of fatty acids. In summary, the findings in this thesis contribute to a fundamental understanding of how different classes of molecules – peptides, fatty acids, monoglycerides – induce membrane morphological responses in biologically relevant model membranes and offer insights into the roles in which these morphological responses play in therapeutic applications as well as molecular evolution. Doctor of Philosophy (MSE)

Surface and interfacial science is an essential field of study in determining the structure-property design parameters of materials. The fundamental knowledge is especially important in modern days whereby many practical applications rely on the compatibility of organic and inorganic substances. Protein adsorption is one of such areas of research that has been considered highly necessary and relevant for various applications, yet much of its details remain under vigorous scrutiny due to its complexity. Protein adsorption is the first step in the acute biological reaction to an exposed surface, in which the functionalities or properties of a material in query could possibly be altered or even contaminated depending on the biomaterials accumulated on the surface. The protein-surface interaction occurs spontaneously and almost at random but the detailed process can be broken down into step-by-step phases whereby a certain level of higher governance is embedded within the system that dictates its kinetics and trends. The major factors can be largely classified into extrinsic and intrinsic factors, which include many other subfactors that are cross-interdependent of one another. Therefore, even with highly sophisticated computerized simulations, the theoretical studies are limited without the inclusion of systematic studies emphasizing data-driven empirical analysis. With the emphasis on the empirical establishment of fundamental insights, this dissertation dives in to the interfacial science of protein-surface interactions using carefully structured experimental designs and intuition-based analyses to form fresh arguments to explain protein adsorption. While most of the experimental layouts are in reference to past studies, the methods were reinforced with modern techniques, and speculations were minimized through data verification via complimentary pairing of analytical devices. The project is presented as a three-part series starting from a broad macroscopic view of the protein adsorption in ensemble, which then gradually narrows down to the sub-molecular level of protein-surface interactions. Thorough investigations revealed that protein adsorption is indeed reliant on the environmental conditions (through control of ionic strength), but more specifically by factors that directly intervene with the conformational stability of the proteins. However, it was further revealed that the extrinsic controls were insufficient in determining the final outcome, ultimately guided by the protein’s potential entropic energy that is confined within the malleable structure. Finally, based on the insights and the unexpected and/or expected observations from the series of studies, scientific and practical solutions were discussed for relevant applications, particularly in regards to achieving an efficient and optimized surface passivation protocol. Revealing the need for a disruptive solution condition for the effective tuning of stable protein layers was especially critical considering the implications that it may have on the many relevant surface passivation applications that were considered common practice. This is only one such example, and clearly there is more room for improvement as there is much to be uncovered from the study of protein adsorption. Doctor of Philosophy

Antimicrobial lipids such as fatty acids and monoglycerides are promising antibacterial agents that destabilize bacterial cell membranes, representing a new treatment approach that might overcome the challenges of antibiotic-resistant bacteria. To date, most studies on antimicrobial lipids have focused on evaluating inhibitory activity against target bacterium by indirect biological methods. While it is known that antimicrobial lipids act against bacterial cell membranes, it remains to be understood how antimicrobial lipids interact with phospholipid membranes. In this thesis, the objective is to develop a comprehensive physicochemical understanding of how fatty acids and monoglycerides interact with model phospholipid membranes, within the broader context of establishing an experimental framework based on supported lipid bilayers (SLBs) to correlate the molecular self-assembly of antimicrobial lipids with their corresponding biophysical and biological activities. The overall hypothesis of this thesis is that SLB platforms can provide a predictive model system to evaluate the potency and mechanism of action of antimicrobial lipids. To test this hypothesis, a fluid-phase, zwitterionic phospholipid SLB platform was established to investigate the membrane morphological responses induced by antimicrobial lipids, and surface-sensitive measurement techniques revealed that fatty acids promote tubule formation while monoglycerides induce membrane budding. Concentration-dependent trends in membrane-disruptive behavior were observed and correlated with the extent of inhibitory activity against Staphylococcus aureus. It was further determined that fatty acids induce tubule formation and increase membrane fluidity only at or above the corresponding critical micelle concentration (CMC) value, while monoglycerides exhibit dual-mode behavior by inducing membrane budding at or above CMC and promoting tubule formation at lower concentrations. In both cases, monoglycerides also increase membrane fluidity. By taking advantage of new SLB fabrication methods, it was also possible to study the interaction of fatty acids and monoglycerides with cholesterol-containing SLBs across different membrane phase states. Depending on the type of induced membrane morphological response, it was found that cholesterol either inhibited or promoted membrane remodeling in a manner consistent with how cholesterol affects the material properties of phospholipid/cholesterol lipid bilayers. The experimental observations were rationalized by taking into account the chemical structure and self-assembly properties of antimicrobial lipids along with how these molecular characteristics influence membrane translocation and membrane strain in phospholipid membranes. In summary, the findings presented in this thesis demonstrate how SLB platforms can provide a predictive materials science tool for studying antimicrobial lipids, and offer a broadly applicable, integrated experimental approach to characterize the potency and mechanism of action of particular antimicrobial lipids. Doctor of Philosophy

Artificial organs are vital for drug development since preclinical animal testing has a limitation in human toxicity prediction, occasionally leading to severe damages. In particular the liver which is an organ that serves the functions in drug metabolism and detoxification, would play a critical role in toxicity screening when reconstructed in vitro. However, primary hepatocytes lose their functions when seeded onto plain substrates, and maintaining the functions ex vivo has been challenging. One of the cues found in liver tissue engineering to keep hepatocyte phenotype is the structural dimensionality; three-dimensional (3D) culture systems which emulate the liver microenvironment, provide enhanced cell-cell interactions and cell-material interactions compared to those in two-dimensions, resulting in prolonged of hepatocyte functions. Therefore, in-vivo-like platforms that mimic the liver microstructure have been in high demand. The overall goal of this dissertation is to create a liver-mimicking platform that can aid in maintaining hepatic functions. Besides cells, the liver is composed of extracellular matrices (ECM) with highly-ordered, porous structure. On the other hand, fabrication of such ECM-based highly-ordered scaffold has been challenging. Problems lie in high viscosity and in slow crosslinking of the aqueous protein solutions for building complex configuration. In this thesis, the material chosen for the platform is a photocrosslinkable protein, gelatin methacryloyl (GelMA). Gelatin is a hydrolyzed form of collagen, which is the main component of the liver structure. While preserving biological advantages of collagen/gelatin, the functionalized gelatin can be crosslinked in minutes in the presence of ultraviolet light and a photoinitiator. Furthermore, aqueous solutions of GelMA are much less viscous than the parent materials. However, the synthesis method has not been optimized in a systematic manner, leaving room for improvement. The first work presented in this thesis is to revamp GelMA synthesis through finding appropriate buffer systems and reaction conditions such as pH, molarity, temperature, and time. The second part of the thesis work entails characterizing the physical properties of GelMA hydrogels with a simplified model. Rheological experiments on mechanical stiffness, swelling measurements in size and mass, and enzymatic degradation experiments were carried out for comprehensive characterization. Finally, a liver-mimicking hydrogel platform was developed, possessing highly-ordered pores and interconnections. Protein-based inverted colloidal crystal (ICC) scaffolds were fabricated with GelMA and a sacrificial polymer lattice. The use of GelMA enabled easy infiltration into the lattices at a high protein concentration and fast crosslinking. Liver model cells, Huh7.5 cells, in GelMA ICC scaffolds attached well to the surface of GelMA ICC and formed 3D cell constructs with cell multi layers during a culture period of high viability. Relative to GelMA plain surface, cells in GelMA ICC exhibited higher hepatic functions. The results demonstrate the potential of GelMA ICC to be an artificial liver platform – that can maintain hepatic functions and foresee human drug toxicity – and would be contributive to drug discovery and development. Doctor of Philosophy (IGS)

A biomimetic cell membrane bioelectronic transistor consisting of a supported phospholipid bilayer atop chemical vapor deposition (CVD) graphene or solution processed reduced graphene oxide on titanium dioxide (TiO2) has been successfully demonstrated for biosensing application. For the first time, a unique bonding interaction between titanium and carbon atom on graphene oxide has been experimentally identified by X-ray Photoelectron Spectroscopy (XPS). The covalent interaction acts as a strong surface anchorage in facilitating uniform adhesion and near to ideal surface coverage of spin coated graphene oxide on titanium bearing substrate using the in-situ solid-liquid phase exfoliation method. The liquid gated Reduced Graphene Oxide (RGO) biotransistor is sensitive to environmental changes in ionic strength of liquid environment by shift in electrical output signals at a sensitivity level of ~0.030mA per millimolar at gate bias VG=-8V and 0.035mA per millimolar at drain bias VD=1V. Incorporation of a phospholipid membrane atop the RGO transformed the sensing transistor to a bioelectronic transistor capable of sensing attached biomolecules on functionalized lipid/RGO surface acting as pseudo gate bias which modulated the electrical signals output. Supported neutral and charged lipid membranes on RGO at active source-drain region were formed by solvent assisted lipid bilayer (SALB) formation. A modulation of transistor conductance induced by gate charges were observed by the measurement of transistor drain current. Based on the 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)/RGO biotransistor platform, a functional supported lipid bilayer (SLB) was demonstrated using biotin-streptavidin binding event. The quantification of biotin composition with respect to transistor drain current modulation was monitored which showed a linear relationship between the percentage of changes in drain current versus the percentage of biotin composition in the complex SLB. For instance, a 12% change in drain current was measured for a 1wt % biotinylated DOPC lipid complex membrane upon streptavidin binding. The results illustrated the potential of using lipid bilayer as an efficient biofunctionalization tool on graphene oxide as a highly versatile and sensitive biosensing platform for a variety of receptor-ligand binding events. Doctor of Philosophy

Pollen microparticles and sporopollenin - sporoderm microcapsules (S-SMCs) provide a compelling all-natural solution for use in a wide range of oil/water emulsion-type applications. To enhance the utility of pollen microparticles and S-SMCs, surface modification was shown to be a mechanism for altering surface chemistry and thereby tuning microparticle wetting properties, emulsification characteristics, and particle/cell adhesion. This project explored the extraction and surface modification of discrete pollen particles from bee-collected pollen granules and the extraction of S-SMCs from pollen particles. Characterization of the ultraviolet-ozone (UV-O) surface modified particles (pollen and S-SMCs) by X-ray photoelectron spectroscopy (XPS) and attenuated total reflectance Fourier transform infrared (ATR-FTIR) to give insight to the UV-O modifications to sporopollenin (both native and acid extracted). This project will also be exploring the possible applications for such microparticles as functional microbeads. UV- O treatment is shown to increase the proportion of surface elemental oxygen and C=O bonds, leading to enhanced particle dispersion properties, control over Pickering emulsion characteristics, and increased particle/cell binding affinities. Looking forward, bee-collected pollen is widely available, competitively priced, and is considered regulation-free for oral consumption due to a long history of use as a food and medicine, with extensive literature purporting numerous health benefits. Beyond facilitating the utilization of pollen and S-SMCs as a functional microparticle system, a greater understanding of UV-O surface modification of sporopollenin also provides insights into the inherent UV protective properties of pollen shells, and provides insights into the use of pollen sporopollenin for exploring climate change through fossil records. Master of Engineering

The interfacial science of phospholipid assemblies on solid supports is principally governed by a set of fundamental forces that exists in all colloidal and interfacial systems. In this thesis, the objective is to investigate how the interplay of different interfacial forces contributes to the fabrication and stabilization of phospholipid assemblies, including adsorbed vesicles and supported lipid bilayers. The overall hypothesis is that the adhesion of phospholipid assemblies on solid supports can be rationalized by taking into account the fundamental material properties of the system and corresponding experimental parameters. A combination of experimental and theoretical approaches is employed, leading to new physical insights into the contributions of the different interfacial forces as well as methods to controllably fabricate phospholipid assemblies. In the first part, two studies are reported clarifying the role of two experimental parameters, osmotic pressure and vesicle lamellarity, in the fabrication of supported lipid bilayers on silicon oxide. In the second part, two additional studies investigate the factors behind the difficulty to fabricate supported lipid bilayers on titanium oxide and aluminum oxide. It is identified that the hydration force plays a governing role to influence the lipid-substrate interaction on both substrates, and it is demonstrated that bilayer formation on aluminum oxide can be achieved by solvent-assisted lipid self-assembly. In the third part, a set of three studies extends the application of localized surface plasmon resonance sensors to track vesicle adsorption and rupture, including factors such as the adsorption rate and substrateinduced deformation of adsorbed vesicles at low surface coverage. In summary, the findings presented in this thesis demonstrate the collective importance of the interfacial forces for the fabrication and stabilization of phospholipid assemblies on solid supports, and offer integrated approaches for scrutinizing the adsorption of biological macromolecules in general. Doctor of Philosophy (MSE)