京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21993号 / 医博第4507号 / 新制||医||1037(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 長船 健二, 教授 竹内 理, 教授 横出 正之 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22001号 / 医博第4515号 / 新制||医||1038(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 井上 治久, 教授 林 康紀, 教授 高橋 淳 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Phospholipids can regenerate oxidized tocopherols and help delay lipid oxidation. The impact of emulsifier type, tocopherol homologue and phospholipid head group on tocopherol-phospholipid interaction was investigated in this study. Three µmol tocopherol/kg emulsion and 15.0µmol/kg emulsion of PE or PS were dissolved in oil and emulsions were prepared. Tween 20 or bovine serum albumin(BSA) was used as emulsifier and the continuous phase contained 10mM imidazole/acetate buffer at pH 7. Lipid hydroperoxides and hexanal were measured as lipid oxidation products and the lag phase was determined. With Tween 20 as the emulsifier, α and δ-tocopherol had a hexanal lag phase of 2 and 4 days respectively. PE and PS both extended the lag phase to 7 and 10 days respectively in presence of δ-tocopherol. Whereas, PS extended the lag phase to 6 days and PE could not exhibit any synergism with α-tocopherol. With BSA as the emulsifier, α and δ-tocopherol had a lag phase of 4 days. PE and PS extended the lag phase to 11 days and 10 days respectively in presence of δ-tocopherol and to 7 and 8 days respectively in presence of α-tocopherol. PE and PS both exhibited synergism with mixed tocopherol and the extent of synergism was in less than δ-tocopherol but more than α-tocopherol. Phospholipids could potentially be used with tocopherols to improve the oxidative stability of emulsions. PE was more effective with BSA whereas PS was equally effective with both emulsifiers.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第23410号 / 医博第4755号 / 新制||医||1052(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 村川 泰裕, 教授 長尾 美紀, 教授 柳田 素子 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Lipid oxidation is one of the major challenges faced by the food industry as it contributes to the loss of nutritional quality and loss of flavor in food products. Studies have shown that naturally occurring phospholipids like phosphatidylserine (PS) and phosphatidylethanolamine (PE) can regenerate oxidized tocopherols and help delay the lipid oxidation in bulk oils and oil-in-water emulsions. Since consumers desire simpler and cleaner labels, without chemically synthesized antioxidants, this research is of great interest. The combination of PS and PE with tocopherols has already been studied. However, PS was a better antioxidant in combination with tocopherols in the oil-in-water emulsion system whereas PE was a better antioxidant in combination with tocopherols in bulk oils. But obtaining pure phospholipids is an expensive deal, therefore, this study uses the more economical alternative, high phosphatidylserine (PS) lecithin in combination with mixed tocopherols in soybean oil-in-water system. PS (30 µmol/kg emulsion) along with mixed tocopherols (3 µmol/kg emulsion) were dissolved in oil and emulsions stabilized by Tween20 were prepared. To determine the most effective concentration of mixed tocopherols, 0.5, 1.0, and 3 µmole of tocopherols/kg emulsion were used at pH of 3 and 7. Tocopherol with a concentration of 3 µmole/kg emulsion was found to be the most effective at pH 3. Tocopherols showed an extended lag phase at lower pH. The synergistic activities of authentic PS and high PS lecithin were compared in combination with tocopherol under similar conditions. They both had an almost similar lag phase. This combination was then tested for different pH of 3 and 7 and different salt concentrations of (0.5, 1, and 1.5 wt% of the emulsion) at pH 7 to determine the effects of external factors on the synergistic antioxidant combination. It was observed that the combination had extended antioxidant ability at lower pH of 3 whereas salt had no effect on the combination. The results showed that high PS lecithin forms a synergistic combination with mixed tocopherols to increase the lag phase in oil-in-water emulsions and can be used as a clean label antioxidant for oil-in-water emulsions.

京都大学 / 0048 / 新制・課程博士 / 博士(工学) / 甲第22763号 / 工博第4762号 / 新制||工||1745(附属図書館) / 京都大学大学院工学研究科都市環境工学専攻 / (主査)教授 高岡 昌輝, 教授 清水 芳久, 准教授 大下 和徹 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DFAM

No description available.

In this project an overview of how synthetic lipid nanoparticles and exosomes can be used for targeted drug delivery is compiled. The goal is to identify aspects that can be in favor for targeted drug delivery and the development of products at Cytiva. The most important fields for Cytiva to understand is the methods and the challenges of cell culturing for production of exosomes, productions of lipid nanoparticles, purification of exosomes, analysis of both exosomes and lipid nanoparticles, and how exosomes and lipid nanoparticles are used as tools for drug delivery. To understand these aspects a description focusing on structural components, specific delivery and cargo loading is also included in the report. Many different components and methods have been found in the different fields mentioned, and the ones that we believe are the most relevant for Cytiva are presented and discussed in the report. We conclude that both exosomes and lipid nanoparticle are suitable options as drug delivery vehicles, especially for their ability to be modified for targeted delivery, encapsulate therapeutic compounds and cross biological barriers. Exosomes are also biostable and possess low immunogenicity. For production the methods identified with highest potential are Hollow-Fiber Bioreactor for cell culturing in production of exosomes and Microemulsion and High-Pressure Homogenization for lipid nanoparticles. Purification is required for exosomes and the most prominent method is Size-Exclusion Chromatography, because of its scalability. After production and purification it is important to be able to detect the vesicles and the most developed and used methods are Nanoparticle Tracking Analysis and Flow Cytometry, beacuse they can use labeling techniques and single vesicle analysis.

Understanding the cellular membrane interaction with membrane active biomolecules and antimicrobial agents provides an insight in their working mechanism. Here, we studied the effect of antimicrobial agents; a recently developed peptidomimetics E107-3 and colistin as well as the N-terminal helix H0, of Endophilin A1 on the lipid bilayer. It is important to discern the interaction mechanism of antimicrobial peptides with lipid membranes in battling multidrug resistant bacterial pathogens. We study the modification of structural and mechanical properties with a recently reported peptidomimetic on lipid bilayer. The compound referred to as E107-3 is synthesized based on the acylated reduced amide scaffold and has been shown to exhibit good antimicrobial potency. This compound increases lipid bilayer permeability as indicated by our vesicle leakage essay. Micropipette aspiration experiment shows that exposure of GUV to the compound causes the protrusion length Lp to spontaneously increase and then decrease, followed by GUV rupture. Solution atomic force microscopy (AFM) is used to visualize lipid bilayer structural modulation within a nanoscopic regime. This compound induces nanoscopic heterogeneous structures rather than pore like structures as produced by melittin. Finally, we use AFM-based force spectroscopy to study the impact of the compound on lipid bilayer’s mechanical properties. With the incremental addition of this compound, we found the bilayer puncture force decreases moderately and a 39% decrease of the bilayer area compressibility modulus KA. To explain our experimental data, we propose a membrane interaction model encompassing disruption of lipid chain packing and extraction of lipid molecules. The later action mode is supported by our observation of a double-bilayer structure in the presence of fusogenic calcium ions. Polyanionic Lipopolysaccharides LPS are important in regulating the permeability of outer membrane (OM) of gram-negative bacteria. To initiate the bactericidal activity of polymyxins, it is essential to impair the LPS-enriched OM. Here, we study the mechanism of membrane permeability caused by colistin (Polymyxin E) of LPS/phospholipid bilayers. Our vesicle leakage experiment showed that colistin binding enhanced bilayer permeability; the maximum increase in the bilayer permeability was positively correlated with the LPS fraction. Addition of magnesium ions abolished the effect of LPS in enhancing bilayer permeabilization. Solution atomic force microscopy (AFM) measurements on planar lipid bilayers shows the formation of nano- and macro clusters which protruded from the bilayer by ~2nm. Moreover, increasing the fraction of LPS or colistin enhances the formation of clusters but inhibits by magnesium ions addition. To explain our experimental data, we proposed a lipid-clustering model where colistin binds to LPS to form large-scale complexes segregated from zwitterionic phospholipids. The discontinuity (and thickness mismatch) at the edge of LPS-colistin clusters will create a passage that allows solutes to permeate through. The proposed model is consistent with all data obtained from our leakage and AFM experiments. Our results of LPS-dependent membrane restructuring provided useful insights into the mechanism that could be used by polymyxins in impairing the permeability barrier of the OM of Gram-negative bacteria. Also, we studied the effect of helix H0 of a membrane modification inducing protein endophilin, on planar bilayer. We obtained transmembrane defects on the bilayer when scanned.with AFM.

Cholesterol is a substance of a steroidal nature that has a number of functions in the human body. One of them is also an irreplaceable role in the proper functioning of the skin barrier. Cholesterol is an integral part of the lipid matrix, together with ceramides and free fatty acids in an equimolar ratio (1: 1: 1), and 5% cholesterol sulfate, which fills the intercellular space between stratum corneum cells and is responsible for the barrier properties of the skin. Cholesterol is therefore necessary for epidermal homeostasis, but its role in SC permeability is unknown. The aim of this work was to study the influence of cholesterol concentration on the permeability and microstructure of model lipid membranes of the skin barrier. Eight sets of model membranes with decreasing cholesterol concentrations (100%, 80%, 70%, 60%, 40%, 20%, 0%) and cholesterol alone were studied for which permeability and microstructure were monitored.The study of permeability of membranes was carried out in the Franz diffusion cells by measuring four permeation parameters: water loss, electrical impedance measurement, and the cumulative amount of two model drugs (theophylline and indomethacin). The microstructure of these model membranes was verified by X-ray powder diffraction. X-ray powder diffraction measurements...