Nanotechnology-based solutions have gained burgeoning attention in medical research, as compared with conventional therapeutic modalities, they offer advantages in efficacy, safety, and scalability. Researchers have been developing fluidic systems for nanoformulations over recent decades. Despite promising results, the clinical potential of the current nanosystems is still limited by insufficient cargo (drug and gene) loading, low production, high toxicity, low colloidal stability, unsatisfied bioavailability, and batch-to-batch variation. Flash-based self-assembly is a recently developed technology that can manufacture nanoformulations in facile, consistent, reproducible, and scalable manners. Due to the turbulent and dynamic flow generated in the mixing chamber, biomaterials self-assemble into uniform nanoparticles (NPs) through precipitation or complexation. We modified and manufactured a number of flash-based systems and evaluated their dynamic mixing profiles through simulation and empirical testing for polyplex formation and nanoparticle coating, as the dynamic fluidic control is the key for biomaterial complexation and nanoparticle coating, which provides better nanoparticle colloidal stability. In Chapter 2, we formulated polyplexes and lipid-coated NPs with controllable size and enhanced colloidal stability by exploiting the dynamic mixing of the flash-based system. Bio-inspired nanosystems with engineered functions have been advancing the field of nanomedicine. Incorporating bio-inspired components can provide nanosystems with productive ways of interacting with their surroundings by diminishing nonspecific interactions or enhancing specific targeting. Membranes from different cell types, and even organisms, can be employed and merged to meet specific goals. We derived cell membranes from distinctive mammalian cell lines to improve nanosystems with smart biological interactions, such as preserving neo-antigens or enhancing specific targeting. Another potent property of utilizing cell membranes is that they provide NPs with colloidal stability. Recent studies have reported the use of cell membrane coating onto NPs in drug delivery, imaging, phototherapies, and detoxification. The derived components from the original cell source bestow the NPs with their inherent functionality without additional complicated modulation. Cell membrane coating is a top-down technique that directly derives and harnesses the natural components, evading the technical and procedural challenges in bottom-up fabrication. However, current membrane coating techniques have problems of batch-to-batch variation and low production yield, which limits its potential for clinical translation. Taking advantage of flash-based self-assembly, we standardized and scaled up the cell membrane-coating process, which is difficult to achieve in bulk mixing approaches. The optimization of cell membrane coating was explored using various simulations. The time and cost for experimental design and optimization were reduced considerably. Cell membranes derived from tumor cells contain a rich source of tumor antigens. With the potential of cell membrane coating using flash-based self-assembly, we applied the produced cell-membrane-coated mesoporous silica nanoparticles (MSN) as a biomimetic nanovaccine for cancer immunotherapy in Chapter 3. Oral delivery of drugs and genes is a relatively convenient, patient-friendly, and safe approach. Targeted and controlled oral delivery of active pharmaceutical ingredients (API) of biomimetic nanocarriers offers significant advantages in efficacy and safety compared to conventional modalities. Besides mammalian cells, the unique functionalities of other prokaryotic and eukaryotic cell types, such as bacterium and yeasts, were exploited for macromolecule delivery. Baker’s yeast, a common yeast strain closely associated with food preparation, contains valuable polysaccharides that were reported to specifically bind to the dectin-1 receptors on the specialized intestinal epithelial cells and monocytes. Exploiting the yeast’s cell wall is a biomimetic strategy when designing an oral carrier for targeted oral drug and gene delivery. We demonstrated that the specific recognition between the microfold cells (M-cells) of the small intestine and the polysaccharides on the yeast cell wall enhances the transport of yeast-based formulations across the gut epithelium and into the lymphatic tissues in chapter 4. Utilizing the micron-sized yeast capsule or decorating a nanoparticle surface with processed yeast cell wall fragments, therapeutics were efficiently delivered to the target site through the oral path. The yeast-based formulations are biomimetic systems for targeted oral delivery of therapeutics. Taken together, the goal of this thesis is to close the gap between laboratory research and clinical translation by exploring the versatility and robustness of the developed flash technology, exploiting flash-based self-assembly for scalable production of the lipid and cell-membrane-coated nanosystems, and developing a relatively safe yeast-based drug and gene delivery platform.

Lipid membranes are semipermeable, allowing for water transport across the membrane but rejecting polar solutes and large molecules. Differences in solute concentrations across a lipid membrane lead to differences in the osmotic pressure Δπ causing water to flow towards regions of higher osmotic pressure (higher solute concentration) at speeds of v=-LpΔπ , where Lp is the hydraulic conductivity of the membrane. When a lipid vesicle is placed in a concentration gradient, it is predicted to move to regions of lower concentration due to osmotic flows across the membrane. John Anderson labeled this motion "osmophoresis'' and proposed a theory that predicts the vesicle velocity as U = -½𝛼LpRTG, where 𝛼 is the vesicle radius, RT is the thermal energy, and G=∇C is the imposed concentration gradient. The first experimental demonstration of osmophoresis reported a 1 μm/s migration velocity of DMPC vesicles in a 10 mM/mm sucrose gradient created between two dialysis tubes. However, Anderson's theory predicts that osmophoretic velocity should be four order of magnitude smaller than the observed velocity. A central goal of this Dissertation is to resolve this discrepancy either by validating Anderson's theory or by identifying relevant physics it may lack. Our central conclusion is that Anderson's theory is likely correct and experimental reports of faster osmophoresis can be attributed to convective flows---particularly, density driven flows caused by the solute gradients. To quantify osmophoresis, we need a steady concentration gradient and micron sized lipid vesicles which are predicted to move faster than smaller vesicles and can be observed by optical microscopy. In Chapter 1, I review methods for generating concentration gradients, including glass chambers for chemotaxis assays and microfluidic devices for sustained operation. In Chapter 2, I review the methods used in this Dissertation for producing giant lipid vesicles, which include film rehydration and emulsion templating techinques. Chapter 3 describes a quantitative investigation of the convective flows induced by solute gradients within microfluidic gradient generators. Solute gradients drive fluid motions due to combinations of gravitational body forces and diffusioosmotic surface forces. I quantify and model how these flows depend on solute type, concentration gradient, device height, and solution viscosity. I describe how undesired convective flows can be mitigated by adding thickening agents to increase the solution viscosity or by density matching between high and low concentration solutions. Chapter 4 describes experiments to measure the osmophoresis of lipid vesicles in osmotic gradients while controlling for convective flows. I use density matched sugar solutions to create 315 mM/mm gradients within a commercial gradient generator (Dunn chamber) while limiting convective flows to less than 20 nm/s. I quantify the motions of lipid vesicles and tracer particles by optical microscopy and observe that both move in a common direction at speeds of 0-10 nm/s. According to Anderson's theory, the expected osmophoretic velocity of lipid vesicles in the solute gradient we applied is 4 nm/s assuming a membrane permeability is 100 μm/s. By contrast, the previously reported motion of DMPC vesicles at speeds of 1 um/s in a 10 mM/mm gradient could not be reproduced and was likely caused by inadequate control over gradient-induced convective flows. One strategy to enhance vesicle motion in osmotic gradients is to increase the membrane permeability---for example, by addition of water channel proteins like aquaporins. The pursuit of giant vesicles with high permeability creates a challenge for measuring the membrane permeability owing to rapid vesicle swelling/shrinking in response to osmotic shocks. Chapter 5, I demonstrate how to use photo-initiated polymerization to create a light-induced osmotic shock to vesicles. Vesicle swelling rate in response to this osmotic shock can infer the membrane permeability when the reaction is fast enough. Chapter 6, I conclude how to set up a steady solute gradient with minimal convective flows to study the slow osmophoresis and highlight some specific future directions for osmophoresis, such as enhancing osmophoretic motion by increasing membrane permeability and designing a cell sorting microfluidic device to isolate living cells based on their size and permeability.

This document only includes an excerpt of the corresponding thesis or dissertation. To request a digital scan of the full text, please contact the Ruth Lilly Medical Library's Interlibrary Loan Department (rlmlill@iu.edu).

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Biological membranes form the structural boundaries and compartments of cells, owing to their robustness and impermeability facilitated by phospholipid bilayers. The strength of biological membranes is intricately linked to the behavior of membrane pores, whose formation and expansion can lead to membrane rupture. However, processes essential for drug delivery, gene editing via genetic material transfer, and antimicrobial peptide action necessitate controlled membrane disruption for efficient cellular entry. Likewise, fundamental phenomena such as exocytosis, including neurotransmitter release between neurons and hormone secretion for physiological responses, rely on membrane breach to release cargo beyond cell confines. Exocytosis involves the fusion of cargo-contained vesicle membranes with the cell's plasma membrane, resulting in the release of cargo into the extracellular milieu. Post-release, these fused vesicles may either integrate with the plasma membrane, remain stationary, enlarge, or depart the release site through fusion pore closure, which, in turn, can modulate exocytosis rate through site availability. However, the precise mechanism of membrane rupture remains elusive. Similarly, the pathway of membrane fusion facilitated by SNARE proteins, pivotal in cellular fusion machinery, remains a subject of debate. Additionally, the mechanisms governing exocytosis remain incompletely understood. To address these inquiries, we employ ultra-coarse-grained molecular dynamics simulations which can explore these phenomena in physiological timescale. These simulations explore membrane rupture mechanisms via pore formation and expansion under varying membrane tension. Furthermore, the research addresses how SNARE proteins drive membrane fusion. In addition, we also rigorously analyze confocal microscopy data from Ling-Gang Wu's research group and develop a quantitative model to elucidate exocytosis rate regulation. Furthermore, the research verifies the robustness of a mathematical model outlining Ca2+-mediated membrane fusion and establishes that hemifusion diaphragms (HDs), where only the outer leaflets of membranes fuse, act as hubs in the Ca2+-mediated fusion network. This finding casts new light on the role of membranes in SNARE-mediated fusion. In the extra study, we analyzed fission yeast contractile ring behavior based on z-stack confocal microscopy data from Mohan Balasubramanian's research group, offering insights into the mechanism behind a critical step in cytokinesis. Chapter one examines membrane pore energetics and bilayer rupture times through highly coarse-grained simulations operating at submillisecond time scales. No metastable states are detected during pore formation. At lower tensions, small hydrophobic pores mature into large hydrophilic pores that ultimately rupture from reversible hydrophilic pores, aligning with classical tension-dependent rupture times. At higher tensions, membranes rupture directly from small hydrophobic pores, with rupture times exhibiting exponential tension dependence. Upon reaching a minimum hydrophobic pore size, a critical tension threshold prompts immediate rupture. This analysis corroborates established experimental findings but reveals that the high-tension exponential regime is not related to long-lived pre-pore defects but rather to the instability of hydrophilic pores beyond a critical tension, leading to significant changes in pore dynamics and rupture kinetics. Chapter two describes utilizing ultra-coarse-grained simulations to dissect the core requirements of membrane fusion and unravel the intricacies of SNARE-mediated fusion. Remarkably, simulations conducted on a millisecond timescale expose the inefficiency of fusion through simple body forces pushing vesicles together. Successful inter-vesicle fusion hinges on the rod-like structure of fusogens, ensuring their sufficient length for effective fusion and subsequent clearance from the fusion site via entropic forces. Simulations featuring rod-shaped fusogens and SNARE proteins demonstrate the fusion of 50-nanometer vesicles in submilliseconds, propelled by entropic forces that direct a predictable fusion pathway. The entropic force hypothesis of SNARE-mediated membrane fusion garners strong support from these findings, emphasizing the necessity of the rod-like configuration of the SNARE complexes for entropic force generation and fusion. Chapter three focuses on the spatiotemporal dynamics of dense-core vesicle exocytosis events in chromaffin cells, deducing a novel mechanism for exocytosis regulation based on the availability of release sites. Repeated fusion supports membrane reservoir comprising incompletely merged or closed vesicles, occupying release sites and dampening exocytosis frequency. Mathematical modeling suggests reservoir formation relies on locally reduced membrane tension, eliminating the driving force for vesicle merging. Endocytosis facilitates the clearance of unmerged vesicles from the reservoir, ultimately restoring release site availability for subsequent exocytosis events. Chapter four introduces a mathematical model pinpointing the hemifusion diaphragm (HD) as the decision nexus dictating the outcomes of pathways and the fate of final products during multivalent cation-mediated membrane interactions. Transient formation of a high-tension hemifusion interface between membrane-enclosed compartments underscores the model's prediction of fusion, dead-end hemifusion, or vesicle lysis. This comprehensive framework offers predictive insights into interactions mediated by cationic fusogens within membrane-enclosed compartments. Chapter five offers a unique exploration of writhing contractile rings in fission yeast cell ghosts, resulting from controlled digestion of the cell wall and subsequent membrane permeabilization. This innovative approach unveils the intricate dynamics of contractile rings under exceptional circumstances. Writhing of rings is attributed to the detachment of sections from the weakened membrane, followed by their coiling due to apparent twisting torques at anchoring points. Iterative rotations give rise to multiple coils within the rings.

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During a traumatic insult to the brain, tissue is subjected to large stresses at high rates which often surpass cellular thresholds leading to cell dysfunction or death. Cellular events that occur at the time of and immediately after an insult are poorly understood. Immediately following traumatic brain injury (TBI), the neuronal plasma membrane may become disrupted and potentiate detrimental pathways by allowing extracellular contents to gain access to the cytosol. In the current study, neuronal plasma membrane disruption was assessed in vivo following moderate unilateral controlled cortical impact in rats using a normally cell-impermeant fluorescent compound as a plasma membrane permeability marker. This fluorescent dye was injected into the cerebrospinal fluid and was allowed to diffuse into the brain. TBI caused a widespread acute disruption of neuronal membranes which was significantly different compared to uninjured brains. Affected cells were present in cortex and hippocampal regions. These findings were complemented by an in vitro model of TBI where membrane disruption was quantified and its mechanisms elucidated. Permeability marker(s) were added to neuronal cultures before the insult as indicators for increases in plasma membrane permeability. The percentage of cells containing the permeability marker was dependent on the molecular mass, as smaller molecules gained access to a higher percentage of cells than larger ones. Permeability increases were also positively correlated with the rate of insult. Membrane disruption was transient, evidenced by a robust resealing within the first minute after the insult. In addition, membrane resealing was found to be dependent on extracellular Ca2+, as chelation of the ion abolished a significant amount of resealing. We have also investigated the effects of mechanically-induced plasma membrane disruptions on neuronal network electrical activity. We have developed a multielectrode array system that allows the study of electrical activity before, during, and after a traumatic insult to neurons. Endogenous electrical activity of neuronal cultures presented a heterogeneous response following mechanical insult. Moreover, spontaneous firing dysfunction induced by injury outlasted the presence of membrane disruptions. This study provides a multi-faceted approach to elucidate the role of neuronal plasma membrane disruptions in TBI and its functional consequences.

The physiological role of the teichoic acid polymers found in Gram-positive bacterial cell walls is not known. Studies of Bacillus subtilis hybrid strains implicate a defined chromosomal region, which includes the tag-1 locus, as necessary for teichoic acid biosynthesis. A set of ten mutants carrying lesions in this region was identified from among forty-four temperature-sensitive (ts) mutants generated by nitrosoguanidine mutagenesis and bacteriophage 029 selection. This protocol gave a population enriched for ts, versus auxotrophic, mutants. For each of the ten mutants, the frequency of genetic reconstruction, or correction, of the ts phenotype indicated that it was due to change(s) in a single gene. Results of two-factor transformation crosses sorted the mutants into three complementation groups; all ten could complement tag-1. Mutants in two complementation groups were transformed to ts⁺ with cloned rodC DNA. The map order of the newly isolated ts markers was determined from the results of two factor crosses. Orientation with respect to the hisA marker was inferred from transduction experiments. The newly isolated strains were shown to be conditional rod⁻ mutants. Growth at 48°C resulted in reduced growth rates and spherically shaped cells. Additional phenotypes seen for some mutants, namely 029 phage resistance and ts spore outgrowth, appeared closely associated with the ts rod⁻ mutation. Wall phosphate content for two of the mutants, following growth at 48°C, was found to be reduced in comparison to the wild-type control. Taken together these results lend support to the argument that the tag-1 region of the chromosome, which most likely directs teichoic acid biosynthesis, is important for establishment and maintenance of the normal bacillary morphology seen for B. subtilis. The importance of other gene products to the organization of newly synthesized wall was examined using B. subtilis macrofibers. Left- and right-handed macrofibers were converted to spheroplasts and the multi-celled structures regenerated under the two sets of conditions conducive for production of the original, and inverse hand. The helix hands observed for the regenerated structures always corresponded to those expected on the basis of the parental genotype.