No description available.

No description available.

No description available.

Small extracellular vesicles (small EVs) are endogenous membrane-enclosed nanocarriers released from essentially all cells. They have been shown to carry proteins, lipids, nucleic acids to transmit biological signals throughout the body, including to the brain. Some evidence has suggested that small EVs can cross the blood-brain barrier (BBB), moving from the peripheral circulation to the central nervous system (CNS). The BBB is a dynamic barrier that regulates molecular trafficking between the peripheral circulation and the CNS. As a result, small EVs have attracted attention for their potential as a novel delivery platform for nucleic acid-based therapeutics across the BBB. Silencing RNAs (siRNAs) are a potent drug class but using “naked” siRNA is not feasible due to their short half-life, vulnerability to degradation and low penetration in cells. Despite the excitement for the development of small EV-based therapeutics, their clinical development is hampered by the lack of reliable methods for packing therapeutics into them. Reshke et al. has shown that cells can be genetically engineered to produce customizable small EVs packaged with siRNA against any protein by integrating the siRNA sequence into the pre- miR-451 structure. Mounting evidence has established that in a misfolded state, α-synuclein becomes insoluble and phosphorylated to form intracellular inclusions in neurons (known as Lewy bodies) which leads to Parkinson’s disease (PD) pathogenesis. Given that increased α-synuclein expression causes familial and idiopathic PD, decreasing its synthesis by using siRNA is an attractive therapeutic strategy. Here, we genetically engineered cells to produce small EVs packaged with siRNA against α-synuclein integrated in the pre-miR451 backbone, tested their ability to cross an in vitro BBB, and deliver its cargo to silence endogenous α-synuclein in neuron- like cells. The therapeutic potential of α-synuclein siRNA delivery by these small EVs was demonstrated by the strong mRNA (60-70%) and protein knockdown (43%) of α-synuclein in neuron-like cells. We also demonstrated that approximately at 4% and 2%, respectively of small EVs-derived from human brain endothelial cells (hCMEC/D3) and human embryonic kidney (HEK293T) were transported cross the in vitro BBB model. Interestingly, we observed that small EVs-derived from HEK293T deliver their cargo to induced brain endothelial cells (iBECs) (~74% α-synuclein mRNA reduction) but their rate of transport across BBB was lower and did not reduce α-synuclein mRNA expression in neuron-like cells, seeded on the far side of the BBB. Small EVs- derived from hCMEC/D3 reduced α-synuclein mRNA (40%) in neuron-like cells across the BBB model. This finding suggests that small EVs derived from different cell sources can undergo different intracellular trafficking routes, providing various opportunities to influence the efficiency of delivery and fate of intracellular cargo. Using small EVs-derived from hCMEC/D3, two different routes of administration, a single bolus intravenous (IV) or intra-carotid (ICD) injection, showed small EVs largely accumulated in the liver, spleen, small intestines and kidneys; and only a small amount of small EVs were detected in the brain. These results indicate that human brain endothelial cells may serve as a promising cell source for CNS treatments based on small EVs.

Amphiphilic block copolymers are unique macro-molecules capable of self-assembling into bilayers analogous to naturally occurring lipid membranes. When combined with lipids, these copolymers form hybrid membranes with unique and sometimes unpredictable properties, including increased chemical and mechanical stability. These synthetically enhanced biological structures represent a versatile platform suitable for a wide range of applications, from advanced biosensing devices to drug delivery systems. The realization of these advancements necessitates a deep understanding of material properties, including the ability to predict and control interfacial behaviors. It has been shown that in the case of pure lipid membranes, interfacial behaviors are dominated by electrostatic forces. The following work will demonstrate that, electrostatic forces also represent a major driving force behind hybrid vesicle adhesion events, such as the formation of supported bilayers or interactions with biological tissues. These electrostatic forces can be manipulated to a limited degree by adjusting suspension buffer pH which primarily modulates the substrate zeta potential. Protonation of silanol groups, in the case of silicate surfaces at low pH, results in slightly positive surface zeta potential. Unfortunately, hybrid vesicles containing BdxEOy polymers exhibit a slight negative zeta potential independent of buffer pH conditions. Therefore, pH mediation can only result in supported bilayer formation in limited cases and may be insufficiently robust for many demands of application. Furthermore, the zeta potential of hybrid vesicles is surprisingly difficult to predict and control, likely due to screening and steric effects of the PEO block. This investigation provides a model to tune and control the zeta potential of such vesicles, independent of other tunable properties. This technique, in combination with pH mediation, proves to be especially effective in controlling vesicle-substrate interaction. Furthermore, translating this understanding to interactions with tissues, could facilitate more targeted drug delivery, potentially avoiding sensitive tissues, thus reducing off-target effects. In summary, this work deepens our understanding of the complex relationship between surface-potential, pH conditions, and vesicle behavior, paving the way for novel applications in bio-sensing, drug delivery, and nanotechnology.

No description available.

This thesis presents the experimental work performed on the spectrin protein. The aim of the work was to study the direct interactions of spectrin, the cytoskeleton of RBCs, with membrane lipid to determine its effects on the mechanical properties of the lipid bilayer. Motivation for this work came from a lack of unanimity in the field of spectrin, and the hypothesized potential of the protein to perforate giant unilamellar vesicles. The work aimed to investigate and determine how spectrin-lipid interactions influence membrane mesoscopic morphology and biophysics in ways that could ultimately be important to cellular function. For this purpose, a protocol was implemented to take into account the different aspects of the binding. Direct visualisation of the spectrin-lipid interaction and distribution was achieved using confocal fluorescence microscopy. Changes in the mechanical properties of the membrane were investigated using the micropipette aspiration technique. Finally the thermodynamics of the interaction were considered with isothermal titration calorimetry experiments. This allowed evaluation of the protein-lipid interaction in a complete and coherent manner. Experiments were also performed on another elastic protein, alpha-elastin, for comparison. In addition to its similarities with spectrin (both possess hydrophobic domains and entropy elasticity), elastin is auto-fluorescent which makes it an attractive model protein. Elastin was also used as a sample model to implement new techniques using nonlinear optics microscopy.

Fluorinated systems are defined by unique properties that offer advantages in drug delivery, material synthesis and industrial applications. In comparison to their hydrocarbon counterparts, the design of fluorinated solutes for tailored applications is limited by the inability to predict the effect of fluorination on phase behavior. This work examines and interprets the influence of fluorination on the phase behavior of fluorinated solutes and surfactants, with emphasis on their impact on vesicle bilayers. Thermodynamic partitioning of functionalized series of fluorinated and hydrocarbon nicotinate prodrugs fashioned to promote solubility in a fluorocarbon solvent (perfluorooctyl bromide; PFOB) is measured. Predictive approaches are also employed to describe partitioning of these nicotinates between immiscible phases relevant to drug delivery. The findings reveal no strong correlation of the partitioning trends with biological markers of cytotoxicity and prodrug uptake for PFOB mediated delivery. However, partitioning in model membranes (liposomes), which, increases with the hydrophobicity of the perhydrocarbon nicotinates, suggests incorporation in a cellular matrix is chain length dependent. The impact of incorporating fluorinated surfactants in catanionic vesicles, which form spontaneously in dilute aqueous solutions and serve as potential substitutes to conventional meta-stable liposome-based vesicles, is studied. Much larger isotropic vesicle regions are observed in the phase map of the partially fluorinated catanionic surfactant pair, cetylpyridinium bromide/ sodium perfluorooctanoate (CPB/SPFO) than in fully fluorinated HFDPC (1,1,2,2,-tetrahydroperfluorododecyl pyridinium chloride )/SPFO. Fluorescence probing of the vesicle bilayers suggest more fluid bilayers in CPB/SPFO than in HFDPC/SPFO due to better chain packing in the fully fluorinated bilayer. However, the vesicle region is expanded in more asymmetric fluorinated bilayers of HFDPC/SPFH (sodium perfluorohexanoate). The increased chain asymmetry in HFDPC/SPFH results in reduced packing density and more fluid bilayers than in HFDPC/SPFO. The robustness of CPB/SPFO and HFDPC/SPFO vesicles is demonstrated in the synthesis of silica hollow spheres by templating and the retention of encapsulated solutes. Higher colloidal stability of the silica spheres is achieved in HFDPC/SPFO relative to CPB/SPFO due to the barrier effect of the fluorinated bilayer. Similarly, higher solute retention in HFDPC/SPFO is observed. The modulation of phase behavior with fluorination offers opportunities in tunable applications of fluorinated bilayers.

<p>Enterotoxigenic Escherichia coli (ETEC), the leading cause of traveler's diarrhea and childhood mortality due to diarrhea in the developing world, has been shown to secrete heat-labile enterotoxin (LT) in association with outer membrane vesicles. However, studies on the effect of LT have been performed using soluble LT, which is not its physiologically relevant presentation context. The effect of LT associated with vesicles and its trafficking within human intestinal epithelial cells were compared with soluble LT. Cytokine responses and trafficking of standardized samples of soluble LT and vesicle-associated LT were evaluated in polarized intestinal epithelial cells. Using real-time PCR, immunoblotting, and ELISAs, we found that compared to soluble LT, vesicle-bound LT showed delayed kinetics in the activation of LT. Vesicles containing LT or not also produced cytokines through different signaling pathways than soluble LT. We found that this difference in signaling was due to different trafficking within the cell. Interestingly, not all LT associated with vesicles is active within cells. Vesicle-associated LT must bind to the host receptor GM1 in lipid rafts to be active within cells. This suggests that although vesicles can deliver large amounts of LT to a cell, much of the LT would be inactive and not produce a physiological response. To test this hypothesis, we attempted to develop animal models for ETEC-induced diarrhea. Although the models were largely unsuccessful, the mouse model appears promising for determining the physiological response of a host to LT as fluid accumulation was observed in response to vesicles containing LT. The results in this thesis provide further understanding of the mechanism of LT-induced diarrhea and emphasize the importance of study toxins in their natural context.</p> / Dissertation

Mitochondria are dynamic; they alter their shape through fission, fusion and budding of vesicles. Mitochondrial vesicles serve as a quality control mechanism enabling these organelles to rid themselves of damaged lipids and proteins. Dysregulation in mitochondrial dynamics and quality control have been linked to Parkinson’s Disease, making the identification of molecules requisite for these processes a priority. We identified the endocytic protein, Sorting nexin 9 (Snx9) through a genome wide siRNA screen for genes which substantially alter mitochondrial morphology and therefore are important for its maintenance. In this work, the role of Snx9 in mitochondrial morphology is examined. Ultrastructural imaging of mitochondria within cells silenced for Snx9 revealed unbudded vesicles along a hyperfused mitochondrial reticulum suggesting a role for Snx9 in the release of these vesicles. The vesicular profiles contained concentric membranous whorls enriched for neutral lipids. Localization studies suggest the Parkinson’s disease genes, Parkin and Vps35 localize to the unbudded profiles.