In recent years, advances in computer architecture and lipid force field parameters have made Molecular Dynamics (MD) a powerful tool for gaining atomistic resolution of biological membranes on timescales that other tools simply cannot explore. With many key biological processes involving membranes occurring on the nanosecond timescale, MD allows us to probe the dynamics and energetics of these interactions in molecular detail. Specifically, we can observe the interactions taking place as a peptide or protein comes into contact with a lipid bilayer, and how this may shape or alter the bilayer either locally (changes in headgroup orientation, lipid fluidity) or in bulk (lipid demixing, membrane curvature). The resolution achieved through atomistic MD can be directly compared with other tools such as NMR and EPR to gain a full perspective of how these biological systems behave over different timescales. As my background is in computational physics, this thesis not only looks into broadening our understanding of various interactions with biological membranes, but also into the development of construction and analytical software to assist in my research and benefit others in the field. One aspect of biological membranes that could vastly benefit from MD simulations is that of antimicrobial peptides (AMPs). These peptides primarily target and destroy microbes by permeabilising the cell membrane through a variety of proposed mechanisms, where each mechanism relies on the AMP to adopt specific conformations upon contact with bacterial membranes. In this thesis, I present an investigation into the interactions between a synthetic AMP and an inhibitor peptide designed to regulate antimicrobial activity through the formation of a coiled coil structure, which restricts the AMP from adopting new conformations. Simulations captured the spontaneous formation of coiled coils between these peptides, and specific residues in their sequences were identified that promote unfolding. This knowledge may lead to better design of coiled coil forming peptides. Another aspect of biological membranes that can be explored with MD is the interactions between model bacterial membranes and amphipathic helices, such as the MinD membrane targeting sequence (MinD-MTS). This 11-residue helix is responsible for anchoring the MinD protein to the inner membrane of Bacillus subtilis and plays a crucial role in bacterial cell division. MinD is known to exhibit sensitivity to transmembrane potentials (TMVs), whereby its localisation and binding affinity to bacterial membranes are disrupted upon removal of the TMV. Simulations revealed rapid insertions of MinD-MTS peptides into the headgroup region of a model bacterial membrane. Analytical software was constructed to measure the membrane properties of the lipids surrounding inserted MinDMTS peptides, which revealed splayed lipid tails and suggests the MinD-MTS may be capable of inducing membrane curvature. Additional simulations were conducted to investigate the influence of a TMV on model bacterial membranes, where software was constructed to measure changes in membrane properties. An analysis of these simulations suggests that a TMV is capable of lowering the transition temperature of a model bacterial membrane by a few degrees, yielding increased fluidity in the lipids and increased perturbations on the membrane surface. Finally, another aspect of biological membranes that can be explored through MD is that of electroporation. This induction of transient water pores in cell membrane provides an exciting aspect for drug delivery applications into cells, whereby electric fields are applied to cells to increase the uptake of therapeutic drugs. Simulations of membranes with high voltage TMVs were conducted that sought to investigate the implications of electroporation across a variety of bilayer compositions at different temperatures. Software was constructed to measure changes in membrane and system properties, which revealed that pore formation occurred at the same threshold voltage for different bilayer compositions in the fluid phase (~1.9 V) and a higher voltage for DPPC bilayers in the gel phase (~2.4 V). The TMV was found to be highly dependent on the area per lipid (APL), implying that bilayers with bulkier lipids or those transitioning from gel to fluid will experience smaller TMVs and fewer pore formations. These simulations also revealed lipid flip-flopping through pores, where charged lipids tended to translocate in the direction of the electric field to produce an asymmetrically charged bilayer. Finally, simulations utilising charged peptides with membranes yielded electroporation effects, whereby the charged peptides generate an identical TMV to those produced by an ion imbalance of equal magnitude. This suggests that charged peptides, such as AMPs, may be capable of permeabilising cell membranes through electroporation mechanisms.

The drug development process is notoriously long and expensive. During preclinical studies, inaccurate prediction of pharmacokinetic properties such as the ability of a drug candidate to passively permeate cell plasma membranes contributes to the high failure rate of drug candidates during clinical trials. Passive drug permeability is currently predicted using in vitro techniques such as parallel artificial membrane permeability assays, or PAMPA. In PAMPA, drug transport is predicted between aqueous compartments via a synthetic filter filled with a phospholipid solution in an organic solvent. The lack of translatability of preclinical predictions to humans can be attributed, in part, to lack of biological similarly between models used for permeability prediction and cell plasma membranes in vivo. Here, I demonstrate a new method for pharmacokinetic prediction, built by using droplet interface bilayers (DIBs) as human-mimetic artificial cell membranes. DIBs are bilayer sections created at the interface of two aqueous droplets. In the literature, DIBs have been used as artificial cell plasma membranes to study, for example, electrophysiological properties, protein insertion, water permeability, and molecular transport. DIBs can be formed between droplets of differing composition such that one droplet can be used as a donor compartment and the other as an acceptor compartment for the quantification of molecular transport across the artificial cell membrane. DIBs have previously been used to measure the passive permeability of numerous fluorophores as well as the drugs caffeine and doxorubicin. However, the extent to which DIBs have been tuned to mimic human cell plasma membranes and transport across them is limited. I present here the use of microfluidic platforms for bespoke DIB formation, where variables such as temperature, bilayer composition, and droplet contents are customized to create biomimetic cells-on-a-chip. These artificial cells are then used to measure molecular transport with the aim of predicting permeability. In Chapter 2, I investigate the effectiveness of literature methods for the modification of polydimethylsiloxane (PDMS) microfluidic device channels for aqueous droplet formation and storage. While numerous techniques have been presented as mitigation strategies for common challenges in droplet microfluidics, it is not clear from the literature if any of these methods would be effective or necessary for the formation and analysis of DIBs. With the aim of facilitating aqueous droplet formation, I tested the effect of PDMS silanization using trichloro(1H,1H,2H,2H-perfluorooctyl)silane (PFOS) on surface hydrophobicity and oleophobicity. To assess their effect on reducing the rate of aqueous droplet evaporation, I tested surface treatment of PDMS with Teflon AF or Aquapel. I also tested modifications to the device fabrication process by bonding a glass coverslip to the surface of the device and soaking the device in water overnight. To quantify changes in PDMS surface chemistry, I performed contact angle measurements, aqueous droplet formation experiments, and measurements of droplet size during on-chip storage. I determined that baking PDMS microfluidic devices at 65 C overnight produced channel surfaces which allowed for aqueous droplet formation and storage. In Chapter 3 I present a systematic study on the role of temperature in DIB formation using naturally derived phospholipids. The use of increased temperature to form DIBs using total lipid extracts has previously been demonstrated, but has never before been investigated systematically using naturally derived phospholipids and bespoke formulations thereof. I hypothesized that, in order to form complete phospholipid monolayers and DIBs, the microfluidic device must be held at the phase transition temperature of the phospholipids. Using a custom-built heating platform, I tested DIB formation over a range of temperatures to determine conditions which allowed DIB formation rather than droplet coalescence. I show that temperature is a key parameter for DIB formation using naturally derived phospholipids in a microfluidic device. In Chapter 4, I demonstrate the use of DIBs as a new type of pharmacokinetic compartment model for intestinal absorption. Using three-droplet networks, the components of which were designed to mimic the intestinal space, the enterocyte cytosol, and the blood, I measured fluorescein permeability across intestine-mimetic DIBs. The model was able to predict the transport of fluorescein more accurately than the current state-of-the-art technique, PAMPA. Chapter 5 describes the development of complex DIB models for pharmacologically relevant membranes as well as an investigation into novel methods of drug transport detection on-chip. I created a new DIB model for the small intestine, incorporating more components of the enterocyte plasma membrane such as cholesterol. Measurement of calcein permeability served as a control experiment, as calcein does not cross cell plasma membranes. Measurement of fluorescein permeability yielded a significantly shorter permeation half-life than was determined in Chapter 4, indicating an increase in permeability with the more complex, biomimetic phospholipid formulation. I also developed sex-specific models for intestinal absorption to investigate the effect of sex-based membrane differences on permeability. This relationship has never before been explored in the literature. In comparison to the initial intestinal phospholipid formulation, the sex-specific formulations contained acyl chain tail groups which have been found in different ratios in male and female cells. A significantly longer half-life for fluorescein permeability was found in female intestine-mimetic DIBs, mirroring the slower drug absorption observed in female patients. I also used DIBs to model blood-brain barrier permeability. I demonstrate this application using two different brain lipid extracts, polar and total brain lipids. Polar brain lipids have previously been used in PAMPA to predict blood-brain barrier permeability, but have been found to overpredict the permeation of charged molecules in comparison to custom lipid formulations which mimic the composition of human brain endothelial cells. Permeability measurements in DIBs formed using polar brain lipids gave results which agree with PAMPA, as DIBs formed using polar brain lipids were permeable to fluorescein, but those formed using total brain lipids were not. Blood-brain barrier-mimetic DIBs formed using either lipid extract are impermeable to calcein and FITC-dextrans (both 40 and 500 kDa). I also show the formation of the first DIBs to be created using a total lipid extract from human cells as well as their impermeability to calcein. The extract tested was prepared from testicular Sertoli cells, which exhibit properties similar to the blood-brain barrier, but future work will focus on extracts prepared from human brain endothelial cells. Finally, I explore new options for the on-chip detection of the transport of nonfluorescent molecules. To move away from reliance on fluorescent molecules for permeability measurements, I selected three fluorogenic molecular recognition agents (fluorescamine, Chromeo P540, and DimerDye 4) whose fluorescence signal is activated by amine groups. None of the tested methods proved to be viable in DIBs, potentially due to slow permeation, low quantum yield, and side reactions with phospholipids. Overall, I demonstrate here the microfluidic formation and application of several novel types of biomimetic DIBs to permeability prediction. My work shows that DIBs can be used to predict permeability and mirror effects observed in vivo. Future work will focus on the development of new methods for the detection of drug transport and the application of the pharmacokinetic compartment models presented to predicting drug permeability. Further work using total lipid extracts prepared from human cells will also be vital to enhancing the use of biomimetic DIBs as pharmacokinetic permeability prediction tools. As their biological similarity and capacity to accurately predict transport increase, so will the potential of DIBs to improve the accuracy and translatablity of preclinical drug development. / Graduate / 2023-08-09

Modern drug development is an astronomically expensive and time consuming undertaking. Because of this, studying the pharmacokinetic properties of drugs in vitro has become an integral step early in the process of drug development, with the goal of preventing costly failures late in the process, and dangerous side effects. Artificial phospholipid bilayers known as droplet interface bilayers (DIBs) have the potential to be used for these pharmacokinetics assays, combining the low cost of cell-free assays with the ability to more closely mimic structures found in life than current cell-free in vitro techniques. Combined with the reproducibility, ease of use, and low reagent consumption found with microfluidic methods, disruptive new low cost techniques for assessing pharmacokinetics in drug development may be possible using DIBs as an artificial cell membrane model. In this work, I establish the potential of DIBs to be used as a pharmacokinetics modelling platform, and advance the use of microfluidic methods for carrying out pharmacokinetics assays in drug discovery. I first developed a new microfluidic platform for the formation of DIBs, which sought to solve some of the shortcomings of current microfluidic methods for DIB formation (Chapter 2). This device is the first that can be used to form DIB networks from dissimilar droplets in parallel, without use of active controls, and with droplet contact gentle enough to enable use of biomimetic lipid mixtures. I examine for the first time the behaviour of phospholipids on microfluidic devices, and characterise the interaction that they have with a common material used to construct microfluidic devices (Chapter 3). Not only has this interaction never been studied before, but my unexpected findings indicate a new area requiring further study in order to advance the adoption of DIBs on microfluidic devices. In collaboration with my colleague Jaime Korner, I use my newly developed microfluidic platform to carry out an on-chip permeation assay for the first time using biomimetic lipid formulations and bespoke compartments modelled after the human intestine. We demonstrate that this on-chip assay has predictive accuracy greater than that of a current widely used cell-free technique (Chapter 4). Finally, I demonstrate that a DIB based microfluidic platform enables, and is critical for, characterising the effect of structural features such as membrane asymmetry on drug permeation. With this, I find measurable, previously unknown effects of membrane asymmetry on the absorption of the chemotherapy drug doxorubicin, highlighting a possible contributing factor to chemoresistance in some cancers (Chapter 5). I find, and demonstrate throughout the body of this work that microfluidic methods and DIBs can not only provide alternatives to current cell-free in vitro pharmacokinetics assays, but that they can exceed the performance of existing assays, and be used for entirely new ways of examining pharmacokinetics. Through building bespoke artificial cell membranes from the ground up, I hope to demonstrate herein the great potential of these powerful new cell-free methods. / Graduate / 2022-09-12

Lipid molecules are amphiphilic in nature consisting of a hydrophilic head group and hydrophobic hydrocarbon tails. The lipid bilayer consists of two layers of lipid molecules arranged with their hydrophobic tails facing each other and their hydrophilic head groups solvated by water. Lipid bilayers with hydrophilic polymer chains grafted onto the head groups have applications in various fields, such as stabilization of liposomes designed for targeted drug delivery, synthesis of supported bilayers for biomaterial applications, surface modification of implanted medical devices to prevent biological fouling and design of in vitro biosensors. The focus of this thesis lies in understanding the effects of polymer grafting on the thermodynamics and mechanical properties of lipid bilayers. Dissipative particle dynamics (DPD) has evolved as a promising method to study complex soft matter systems. The basic DPD algorithm, and its implementation are discussed in Chapter 2 of this thesis. It is important to achieve a tensionless state while studying phase transitions and deducing the mechanical properties of the bilayer. We proposed a modification of the Andersen barostat which can be incorporated in a DPD simulation to achieve the tensionless state as well as carry out simulations at a prescribed tension. In Chapter 3 of this thesis the effect of polymer grafting on single tailed lipid bilayers is studied. Simulations are carried out by varying the grafting fraction, Gf, defined as the ratio of the number of polymer molecules to the number of lipid molecules. At lowGf, the bilayer shows a sharp transition from the gel (Lβ) to the liquid crystalline (Lα) phase. This main melting transition temperature is lowered as Gf is increased. Corresponding to this, an increase in the area per head group is also observed. Above a critical value of Gf the interdigitated, LβI phase is observed prior to the main transition for the longer lipid tails. The analysis for two tailed lipids as a function of polymer chain length is extensively studied in Chapter 5. For the case of two tailed lipids, an intermediate interdigitated phase was not observed and the decrease in the melting temperature is more pronounced as the length of the polymer chain is increased. The scaling for fractional change in the area per head group, as well as the decrease in transition temperature as a function of polymer grafting are in good agreement with mean field theory predictions. The bending modulus (k) and area stretch modulus (kA) are essential for determining the shape and the mechanical stability of biological cells or lipid based vesicles. In simulations, the bending modulus k is evaluated from the Fourier transform of the out-of-plane fluctuations of the bilayer mid-plane. In Chapter 4 of this thesis, we illustrate that a surface representation based on Delanuay triangulation provides a robust parameter free representation of the bilayer surface. By evaluating the bending modulus for single tail lipids of different tail lengths, the continuum scaling relation d2 is verified. To our knowledge this is the first systematic investigation and verification of this scaling relationship using computer simulations. Using the continuum relation, =kAd2/ we find that α depends weakly on the tail lengths of the bilayer. Nevertheless we illustrate that a value of α=130 can be used to reliably estimate the bending modulus from the area stretch modulus for polymer free bilayers. Using our method, we are also able to capture the low q scalings and obtain the bending modulus of the gel (Lβ) phase. Grafted polymer was found to increase the value of the bending modulus for single tail lipids. Although the presence of polymer directly increases the area per head group, the suppressed height fluctuations dominate and the bending modulus increases for the single tail lipids. For two tail lipids a small decrease in the bending modulus was observed at low grafting fractions and short polymer chains. For large polymer lengths the bending modulus was found to increase monotonically.

A associação lipídio/citocromo c é importante e deve ser estudada, pois repercute na atividade peroxidática da proteína abordada e pode contribuir para o processo apoptótico, ou morte programada da célula, e também desempenha um papel significativo na cadeia respiratória. A natureza e a especificidade da interação do citocromo c com bicamadas lipídicas têm sido bastante investigadas ultimamente, mas informações detalhadas e precisas sobre tais assuntos ainda não existem. É aceito que ocorre primeiramente uma interação eletrostática entre a proteína citocromo c e as membranas fosfolipídicas. Em seguida, há uma interação hidrofóbica. Entretanto, ainda não é bem compreendido o papel da cadeia fosfolipídica. A associação do citocromo c com membranas lipídicas induz mudanças no estado de spin do átomo de ferro. A interação entre as vesículas carregadas e o citocromo c induz mudanças estruturais na proteína, as quais são refletidas no seu centro ativo, ou grupo heme. As mudanças do campo cristalino no sítio do ferro hemínico de forte para fraco são acompanhadas por mudanças do estado de spin de baixo para alto, respectivamente. Neste trabalho, estuda-se sistematicamente a natureza da interação entre o citocromo c e a cadeia fosfolipídica. As mudanças estruturais no grupo heme foram correlacionadas com a natureza do lipídio, ou seja, com a carga da cabeça e com o tamanho e o tipo da cadeia fosfolipídica. Foram utilizados treze lipídios diferentes, naturais e sintetizados, com cabeças polares negativas e neutras e com cadeias carbônicas saturadas e insaturadas de diferentes comprimentos. Para tal investigação, utilizamos as técnicas: Ressonância Paramagnética Eletrônica (RPE) Onda Contínua (CW) e Pulsada (PW) e Dicroísmo Circular Magnético (MCD). As técnicas enunciadas avaliam as mudanças de estado de spin e a simetria do citocromo c nos seus estados férrico e ferroso. A interação lipoprotéica lipídio/citocromo c foi avaliada com lipídios diferentes, inclusive com o lipossomo PCPECL, que mimetiza a membrana interna da mitocôndria nos eucariontes. A partir dos resultados experimentais, sugerimos um modelo para esse tipo de associação. / This association lipid/cytochrome c is interesting to study in order to understand the peroxidase activity of this protein, that plays an important role in the respiratory chain and in the apoptosis process or the programmed cell death. The nature and specificity of the interaction of cytochrome c with lipid bilayers have been major goals in recent studies, but detailed information on that issue is not yet widely available. In this regard, it is generally accepted that the electrostatic interaction is an important factor in the association of cytochrome c with phospholipid membranes, followed by a hydrophobic interaction. However, the role played by the phospholipid chain is not well understood. The association of cytochrome c with negative membranes induces a change in the heme iron spin state. The interaction between the charged vesicles and cytochrome c leads to structural changes in the active central or heme group. The changing of the crystalline field of the heme iron from strong to weak is accompanied by spin states changes from low to high spin, respectively. These facts concerned us to investigate more systematically the nature of the interaction between cytochrome c and the phospholipid chains. The lipid-induced effects in the heme iron crystalline field are correlated to the nature of the charged head group and to the size and type of the phospholipid chain. Thirteen different lipids, nature and synthetic, were used, with negative and neutra1 polar head group and saturated and unsaturated acyl chains with different length. This work investigates the change of heme iron spin state and symmetry of ferric cytochrome c using Continuous Wave (CW) and pulsed (PW) Electron Paramagnetic Resonance (EPR) and Magnetic Circular Dichroism (MCD) techniques. These techniques analyze the spin state change and the symmetry of the iron cytochrome c in its ferric and ferrous states. The effect of the different lipids were analyzed, including PCPECL membrane that mimetics the inner mitocondrial membrane in eukaryotes.

The fundamental structure of all biological membranes is the lipid bilayer. At- tributed to the multifaceted features of lipids and its dynamical interaction with other membrane-integrated molecules, the lipid bilayer is involved in a variety of physiological phenomena such as transmembrane transportation, cellular signalling transduction, energy storage, etc. Due to the nanoscale but high complexity of the lipid bilayer system, experimental investigation into many important processes at the molecular level is still challenging. Molecular dynamics (MD) simulation has been emerging as a powerful tool to study the lipid membrane at the nanoscale. Utilizing atomistic MD, we have quantitatively investigated the effect of lamellar and nonlamellar lipid composition changes on a series of important bilayer properties, and how membranes behave when exposed to a high-pressure environment. A series of membrane properties such as lateral pressure and dipole potential pro les are quanti ed. Results suggest the hypothesis that compositional changes, involving both lipid heads and tails, modulate crucial mechanical and electrical features of the lipid bilayer, so that a range of biological phenomena, such as the permeation through the membrane and conformational equilibria of membrane proteins, may be regulated. Furthermore, water also plays an essential role in the biomembrane system. To balance accuracy and efficiency in simulations, a coarse-grained ELBA water model was developed. Here, the ELBA water model is stress tested in terms of temperature- and pressure-related properties, as well as hydrating properties. Results show that the accuracy of the ELBA model is almost as good as conventional atomistic water models, while the computational efficiency is increased substantially.

Micro/nano-patterning of supported lipid bilayers (SLBs) has shown considerable potential for addressing fundamental biophysical questions about cell membrane behavior and the creation of a new generation of biosensors. Herein are presented several novel lithographic methods for the size-controlled patterning of SLBs from the microscale to the nanoscale. Using these methods, chemically distinct types of phospholipid bilayers and/or Escherichia Coli (E. Coli) membranes can be spatially addressed on a single microchip. These arrays can, in turn, be employed in the studies of multivalent ligand-receptor interactions, enzyme kinetics, SLBs size limitation, and membrane-associated species separation. The investigations performed in the Laboratory for Biological Surface Science include the following projects. Chapters II and III describe the creation of lab-on-a-chip based platforms by patterning SLBs in microfluidic devices, which were employed in high throughput binding assays for multivalent ligand-receptor interactions between cholera toxin B subunits (CTB) and ganglioside GM1. The studies on the effect of ligand density for multivalent CTB-GM1 interactions revealed that the CTB-GM1 binding weakened with increasing GM1 density. Such a result can be explained by the clustering of GM1 on the supported phospholipid membranes, which in turn inhibits the binding of CTB. Chapter IV characterizes the enzymatic activity of phosphatase tethered to SLBs in a microfluidic device. Higher turnover rate and catalytic efficiency were observed at low enzyme surface densities, ascribing to the low steric crowding hindrance and high enzyme fluidity, as well as the resulting improvement of substrate accessibility and affinity of enzyme catalytic sites. Chapter V presents sub-100 nm patterning of supported biomembranes by atomic force microscopy (AFM) based nanoshaving lithography. Stable SLBs formed by this method have a lower size limit of ~ 55 nm in width. This size limit stems from a balance between a favorable bilayer adhesion energy and an unfavorable bilayer edge energy. Finally, chapter VI demonstrates the electrophoretic separation of membrane-associated fluorophores in polymer-cushioned lipid bilayers. This electrophoretic method was applied to the separation of membrane proteins in E. Coli ghost membranes.

In the last few decades, an increasing number of physicists specialized in soft matter, including polymers, have turned their attention to biologically relevant materials. The properties of various molecules and fibres, such as DNA, RNA, proteins, and filaments of all sorts, are studied to better understand their behaviours and functions. Self-assembled biological membranes, or lipid bilayers, are also the focus of much attention as many life processes depend on these. Small lipid bilayers vesicles dubbed liposomes are also frequently used in the pharmaceutical and cosmetic industries. In this thesis, work is presented on both the elastic properties of polymers and the response of lipid bilayer vesicles to extrusion in narrow-channels. These two areas of research may seem disconnected but they both concern deformed soft materials. The thesis contains four articles: the first presenting a fundamental study of the entropic elasticity of circular chains; the second, a simple universal description of the effect of sequence on the elasticity of linear polymers such as DNA; the third, a model of the symmetric thermophoretic stretch of a nano-confined polymer; the fourth, a model that predicts the final sizes of vesicles obtained by pressure extrusion. These articles are preceded by an extensive introduction that covers all of the essential concepts and theories necessary to understand the work that has been done.

Understanding the assembly of monomeric components into specific molecular motifs is a central theme in materials and surface engineering. Motif designs, specifically using a controllable template, can yield materials with desired optical or electronic properties. The objective of this thesis is to understand the aggregate size, packing, and monomer orientation for the cationic dye, pseudoisocyanine. These organic molecules assemble into crystals in solution, on planar bilayer templates, and on the membranes of living cells. Pseudoisocyanine J-aggregates were found to form on top of the heterogeneous lipid domains in a phospholipid bilayer. This behaviour is limited to a few headgroup chemistries and lateral packing motifs, allowing one to control aggregation via a combination of these two factors. These aggregates are low-dimensional and display polymorphism. Using atomic force microscopy and visible-light spectroscopy, distinct optical characteristics can be correlated to different bilayer templated J-aggregate morphologies. The molecular packing of a similar J-aggregate crystal was resolved using both atomic force microscopy and selected area electron diffraction. The infrared absorption spectra of different polymorphs also displayed distinct differences. These separate examinations enabled a perspective that clarifies the geometry, packing, orientation, and size of templated J-aggregates. Insights into the templating of J-aggregates on the molecular scale reveals that they are sensitive reporters of membrane phase in adherent cells, and are compatible with established cell biology techniques. Lipid domains in live mammalian cells were visualized using fluorescent J-aggregates in combination with endogenous marker proteins of the endocytic process. Analysis of live cell images and additional biophysical work revealed that pseudoisocyanine J-aggregates formed on domains of the anionic lipid bis(monoacylglycerol)phosphate. Only by using J-aggregates can this lipid be shown to form well-ordered domains during endosomal maturation, leading to multivesicular body formation. These data demonstrate that a correlated optical and topographical approach is necessary to understand the structure of fluorescent molecular assemblies, and form the basis for utilizing such aggregates in a biological context.

Understanding the assembly of monomeric components into specific molecular motifs is a central theme in materials and surface engineering. Motif designs, specifically using a controllable template, can yield materials with desired optical or electronic properties. The objective of this thesis is to understand the aggregate size, packing, and monomer orientation for the cationic dye, pseudoisocyanine. These organic molecules assemble into crystals in solution, on planar bilayer templates, and on the membranes of living cells. Pseudoisocyanine J-aggregates were found to form on top of the heterogeneous lipid domains in a phospholipid bilayer. This behaviour is limited to a few headgroup chemistries and lateral packing motifs, allowing one to control aggregation via a combination of these two factors. These aggregates are low-dimensional and display polymorphism. Using atomic force microscopy and visible-light spectroscopy, distinct optical characteristics can be correlated to different bilayer templated J-aggregate morphologies. The molecular packing of a similar J-aggregate crystal was resolved using both atomic force microscopy and selected area electron diffraction. The infrared absorption spectra of different polymorphs also displayed distinct differences. These separate examinations enabled a perspective that clarifies the geometry, packing, orientation, and size of templated J-aggregates. Insights into the templating of J-aggregates on the molecular scale reveals that they are sensitive reporters of membrane phase in adherent cells, and are compatible with established cell biology techniques. Lipid domains in live mammalian cells were visualized using fluorescent J-aggregates in combination with endogenous marker proteins of the endocytic process. Analysis of live cell images and additional biophysical work revealed that pseudoisocyanine J-aggregates formed on domains of the anionic lipid bis(monoacylglycerol)phosphate. Only by using J-aggregates can this lipid be shown to form well-ordered domains during endosomal maturation, leading to multivesicular body formation. These data demonstrate that a correlated optical and topographical approach is necessary to understand the structure of fluorescent molecular assemblies, and form the basis for utilizing such aggregates in a biological context.