Orientador: David Mendez Soares / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Fisica Gleb Wataghin / Made available in DSpace on 2018-08-15T00:08:17Z (GMT). No. of bitstreams: 1 Gomes_WyllersonEvaristo1983-_M.pdf: 20027750 bytes, checksum: ac9756c2723f3eb80a562d792355af61 (MD5) Previous issue date: 2010 / Resumo: As propriedades dinâmicas e estruturais de filmes de surfatantes adsorvidos em superfícies são de interesse fundamental e aplicado. Investigamos a formação de estruturas auto-organizadas de surfatantes sobre superfícies de substrato sólido de ouro. Estudamos sua dinâmica de formação e estabilidade. As estruturas foram feitas em ambiente aquoso, sob condições físico-químicas controladas. Tais estruturas são potenciais candidatas a modelos in vitro de membrana biológica (sistema biomimético) / Abstract: The structural and dynamical properties of surfactant films are both of fundamental and applied interest. To understand the formation mechanism of these structures we have studied the formation of surfactant self-assembled aggregates on gold surfaces. Their dynamic and stability were investigated. All experiments were performed in aqueous media, under specific physical and chemical conditions. These structures are potential candidates of in vitro models for biological membranes (bio-mimetic systems) / Mestrado / Físico-Química / Mestre em Física

O presente trabalho tem como objetivo principal a eletrofiação de nanofibras poliméricas de poliacrilonitrila (PAN) e polifluoreto de vinilideno (PVDF), incorporadas com negro de fumo (NF) e ftalocianina de cobre (CuPc), visando aplicações em dispositivos sensores. Inicialmente foram preparadas soluções de PAN puro a 6 % em peso e PVDF puro a 20% em peso e foram misturadas a essas soluções partículas de negro de fumo e ftalocianina de cobre, obtendo soluções de PAN/NF, PVDF/NF, PAN/CuPc e PVDF/CuPc. Foi determinada a viscosidade absoluta das soluções. Realizou-se a eletrofiação para obtenção de nanofibras que foram caracterizadas segundo o diâmetro e morfologia, usando microscópio óptico e microscópio eletrônico de varredura. Para avaliar as interações polímero-polímero, polímero-partícula foram analisadas por espectroscopia FITR e Raman. Com as fibras de PAN/NF foi analisada a resistência e condutância elétrica das membranas usando um picoamperímetro digital, visando aplicação como filtro eletrostático. Foi construído canal na lâmina de silício usando um feixe de laser visando a deposição de fibras dentro do canal usando a técnica de focagem eletrodinâmica com tensão aplicada em máscaras de cobre. Foi usada a técnica da microbalança de cristal de quartzo para determinar a variação de massa adsorvida por membranas de PAN/CuPc e PVDF/CuPc por meio da medida da variação de frequência usando um frequencímetro digital, onde se observou que essas membranas são promissoras para atuar como sensores de vapor de amônia. / The main objective of this work is the incorporation of different particles in order to electrospun polymeric nanofibers of polyacrylonitrile (PAN) and polyvinylidene fluoride (PVDF), aiming at applications in sensor devices. Initially, solutions of PAN pure 6 wt% and PVDF pure 20 wt% were prepared and these solutions were mixed with carbon black (NF) particles and copper phthalocyanine (CuPc), obtaining solutions of PAN/NF, PVDF/NF, PAN/CuPc and PVDF/CuPc. The absolute viscosity of the solutions was determined. The electrospinning was performed to obtain nanofibers that were characterized according to the diameter and morphology, using optical microscope and scanning electron microscopy. To evaluate the polymer-polymer and polymer-particle interactions, FITR and Raman spectroscopy were performed. The resistance and conductance of the membranes electrospun from PAN/NF solution were analyzed using a digital picoammeter, and an increase in the resistance was measured. This result shows that the membrane is suitable to be applied as electrostatic filter. A channel was constructed on the silicon wafer using a laser beam for the deposition of fibers inside the channel using the electrodynamic focusing technique. The quartz crystal microbalance technique was used to determine the applicability of the membranes as sensor layer. The results of PAN/CuPc and PVDF/CuPc membranes suggests that these membranes are promising to act such as ammonia vapor sensors.

The electrochemical quartz crystal microbalance (EQCM) has been proven an effective mean of monitoring up to nano-scale mass changes related to electrode potential variations at its surface. The principles of operation are based on the converse piezoelectric response of quartz crystals to mass variations on the crystal surface. In this work, principles and operations of the EQCM and piezo-electrodes are discussed. A conductive oxide, ruthenium oxide (RuO2) is a promising material to be used as a diffusion barrier for metal interconnects. Characterization of copper underpotential deposition (UPD) on ruthenium and RuO2 electrodes by means of electrochemical methods and other spectroscopic methods is presented. Copper electrodeposition in platinum and ruthenium substrates is investigated at pH values higher than zero. In pH=5 solutions, the rise in local pH caused by the reduction of oxygen leads to the formation of a precipitate, characterized as posnjakite or basic copper sulfate by means of X-ray electron spectroscopy and X-ray diffraction. The mechanism of formation is studied by means of the EQCM, presenting this technique as a powerful in-situ sensing device.

Healthcare associated infections (HAIs) cost the US healthcare system over $45 billion to treat and cause millions of deaths annually. A large subset of HAIs are associated with medical devices that are meant to improve and save lives. Infected devices are treated using traditional antibiotics, contributing to development of antibiotic resistance. Antibiotic resistance is expected to cost $100 trillion and kill more people a year than cancer by 2050; thus, new alternative antimicrobials for the treatment of device-associated HAIs are critically needed. Antimicrobial peptides (AMPs), such as 26 amino-acid (aa), marine-derived Chrysophsin-1 (CHY1), are poised to reduce HAIs due to their broad antimicrobial activity and unique mechanisms of action that do not promote bacterial resistance. AMPs are short (12-50aa), positively charged (+2-+9) proteins found in the innate immune systems of many different species. Their high separation of hydrophilic and hydrophobic residues leads to many unique mechanisms derived from many unique secondary and tertiary structures that are not yet well understood. Despite the discovery of over 2000 natural AMPs and many more synthetically designed AMPs, none have been successfully commercialized for healthcare applications due to challenges surrounding cytotoxicity, short in vivo half-life (degradation), high costs of production and effectiveness in physiological environments (such as those with high-salt). Several strategies have been investigated to overcome these challenges, for example, truncation of cytotoxic sequences or D-amino acid substitution to improve AMP toxicity and stability; however, many of these strategies can reduce antimicrobial effectiveness. A unique strategy of increasing stability, reducing cytotoxicity, and maintaining antimicrobial activity that is relevant for medical devices is the covalent tethering (binding) of AMPs via a flexible tethering molecule to the surface. However, the effect of tethering parameters on resulting AMP mechanisms and activity is still widely debated. AMP activity can vary widely by utilizing different tethering strategies, which include additional variables such as: (1) peptide choice and properties (such as native mechanism, concentration, charge, and structure), (2) tether choice and properties (such as chemical composition, length, charge, surface density, and flexibility), and (3) testing conditions (such as temperature, solvent composition and substrate type). Some studies suggest that AMP performance may be tether-dependent, for example some AMPs require longer tethers while others do not and some need a flexible tether. Thus, models for predicting successful tethering strategies for different AMP properties, which currently do not exist, must be developed. Further, complicated and often destructive techniques, such as XPS and SEM, are typically implemented to study the relationship of all these parameters vs. antimicrobial activity, which are labor-intensive and limited in scope. Predictive models guiding tether strategy need to be constructed, but also new techniques to study tethering be developed. If these technical milestones are achieved they can serve as a predicate for commercial implementation of a host of new therapies targeted at reducing device-associated HAIs. The overall goal of this thesis was to study the relationship between antimicrobial activity of tethered C-CHY1 examining both spacer length and peptide surface density and the development of a feasible clinical business case for tethered AMPs. To achieve this goal, a traditional entrepreneurial approach was taken in which a minimally-viable product was first designed and business case analyzed, followed by studies to better optimize and understand the underlying structure-mechanism relationships. CHY1 with a C-terminus cysteine to allow for surface-binding (C-CHY1) was tethered onto a silicon dioxide surface via a flexible poly(ethylene glycol) (PEG) tether, and then both surface binding behavior and antimicrobial success of C-CHY1 were examined as a function of tether properties and reaction conditions. For these studies, quartz-crystal microbalance with dissipation (QCM-D) was the primary technique, a real-time, non-destructive flow method that was then coupled with downstream characterization techniques: fluorescent microscopy and contact angle measurements. In parallel a deep dive into domestic and international business models for commercializing AMP technologies. Specifically, tether length and surface density effects on C-CHY1 mechanisms were studied, followed by the effect of temperature, type of microbe, and salt concentration on the antimicrobial mechanisms of tethered C-CHY1. QCM-D was used to measure binding of C-CHY1 via three different length tethers, PEG molecular weight (MW) 866, 2000 and 7500, followed by microscopy to measure antimicrobial effectiveness against two model microbes Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus). Modeling of QCM-D data allowed for surface density and thickness to be calculated and related to C-CHY1 antimicrobial activity. PEG 7500 allowed proper C-CHY1 orientation and mobility, allowing for its native pore-forming mechanism and highest activity while PEG 866 tethers led to denser grafting and an effective, yet non-native ion displacement mechanism. The QCM-D was used to characterize the effect of salt concentration and temperature reaction conditions on the grafting density of C-CHY1 tethered via PEG 866 and PEG 7500, which was then related to antimicrobial activity. For PEG MW 866, neither temperature nor salt concentration increases significantly altered the grafting density of C-CHY1 while for PEG 7500 increasing temperature allowed for significantly increased grafting density. C-CHY1 density had no significant effect on antimicrobial activity against either microbe. Temperature of bacterial incubation did demonstrate microbe-specific changes in C-CHY1 antimicrobial activity. These results demonstrated that small changes in reaction conditions can drastically change membrane selectivity of C-CHY1. An in-depth investigation of the effects of bacterial membrane composition and temperature on soluble C-CHY1 mechanism was implemented to better understand the molecular membrane- and temperature-dependent selectivity and structure-function of C-CHY1. Supported lipid bilayers (SLBs) formed in QCM-D can be used as model membranes to elucidate AMP action mechanisms against membranes of different compositions. Two and three component SLBs representative of Gram-negative phosphatidylethanolamine (PE) and phosphatidyglycerol acid (PG) with and without charged lipopolysaccharide, LPS and Gram-positive bacteria phosphatidylcholine (PC) and PG with and without charged lipoteichoic acid, (LTA) were formed at both 23°C and 37°C. C-CHY1 at 5 µM was exposed to the different membranes and mechanistic surface action was studied. The membranes formed highly different baseline responses in QCM-D, indicative of vastly different membrane structures, thicknesses and deposition behaviors on SiO2, warranting future studies. Further, significant effects of LTA incorporation were observed in both peptide interaction and deposition. There were measurable effects of temperature on membrane formation as well as peptide interaction kinetics and even mode of interaction. Lastly, business models for the commercialization of novel medical device technologies such as surface-tethered C-CHY1 were investigated. While this technology has the potential to solve many unmet needs, there must a commercialization plan implemented in order to have an impact. There is a clear disconnect between technology development in academia and technology commercialization in industry that must be connected. Development of an entrepreneurial mindset at the graduate school level, can help bridge the gap. A thorough investigation of domestic and international business models for commercializing AMP technologies was carried out and distilled in the form of the Business Model Canvas developed by Alexander Osterwalder that can be used as a roadmap for commercialization efforts. Using the QCM-D a relationship between both spacer length and peptide surface density and the antimicrobial activity of tethered C-CHY1 was determined. A business plan was developed in order to increase the impact of this and other AMP based work. This work provides a roadmap for future researchers to quickly develop and commercial novel AMP based coating technology.

Healthcare associated infections (HAIs) cost the US healthcare system over $45 billion to treat and cause millions of deaths annually. A large subset of HAIs are associated with medical devices that are meant to improve and save lives. Infected devices are treated using traditional antibiotics, contributing to development of antibiotic resistance. Antibiotic resistance is expected to cost $100 trillion and kill more people a year than cancer by 2050; thus, new alternative antimicrobials for the treatment of device-associated HAIs are critically needed. Antimicrobial peptides (AMPs), such as 26 amino-acid (aa), marine-derived Chrysophsin-1 (CHY1), are poised to reduce HAIs due to their broad antimicrobial activity and unique mechanisms of action that do not promote bacterial resistance. AMPs are short (12-50aa), positively charged (+2-+9) proteins found in the innate immune systems of many different species. Their high separation of hydrophilic and hydrophobic residues leads to many unique mechanisms derived from many unique secondary and tertiary structures that are not yet well understood. Despite the discovery of over 2000 natural AMPs and many more synthetically designed AMPs, none have been successfully commercialized for healthcare applications due to challenges surrounding cytotoxicity, short in vivo half-life (degradation), high costs of production and effectiveness in physiological environments (such as those with high-salt). Several strategies have been investigated to overcome these challenges, for example, truncation of cytotoxic sequences or D-amino acid substitution to improve AMP toxicity and stability; however, many of these strategies can reduce antimicrobial effectiveness. A unique strategy of increasing stability, reducing cytotoxicity, and maintaining antimicrobial activity that is relevant for medical devices is the covalent tethering (binding) of AMPs via a flexible tethering molecule to the surface. However, the effect of tethering parameters on resulting AMP mechanisms and activity is still widely debated. AMP activity can vary widely by utilizing different tethering strategies, which include additional variables such as: (1) peptide choice and properties (such as native mechanism, concentration, charge, and structure), (2) tether choice and properties (such as chemical composition, length, charge, surface density, and flexibility), and (3) testing conditions (such as temperature, solvent composition and substrate type). Some studies suggest that AMP performance may be tether-dependent, for example some AMPs require longer tethers while others do not and some need a flexible tether. Thus, models for predicting successful tethering strategies for different AMP properties, which currently do not exist, must be developed. Further, complicated and often destructive techniques, such as XPS and SEM, are typically implemented to study the relationship of all these parameters vs. antimicrobial activity, which are labor-intensive and limited in scope. Predictive models guiding tether strategy need to be constructed, but also new techniques to study tethering be developed. If these technical milestones are achieved they can serve as a predicate for commercial implementation of a host of new therapies targeted at reducing device-associated HAIs. The overall goal of this thesis was to study the relationship between antimicrobial activity of tethered C-CHY1 examining both spacer length and peptide surface density and the development of a feasible clinical business case for tethered AMPs. To achieve this goal, a traditional entrepreneurial approach was taken in which a minimally-viable product was first designed and business case analyzed, followed by studies to better optimize and understand the underlying structure-mechanism relationships. CHY1 with a C-terminus cysteine to allow for surface-binding (C-CHY1) was tethered onto a silicon dioxide surface via a flexible poly(ethylene glycol) (PEG) tether, and then both surface binding behavior and antimicrobial success of C-CHY1 were examined as a function of tether properties and reaction conditions. For these studies, quartz-crystal microbalance with dissipation (QCM-D) was the primary technique, a real-time, non-destructive flow method that was then coupled with downstream characterization techniques: fluorescent microscopy and contact angle measurements. In parallel a deep dive into domestic and international business models for commercializing AMP technologies. Specifically, tether length and surface density effects on C-CHY1 mechanisms were studied, followed by the effect of temperature, type of microbe, and salt concentration on the antimicrobial mechanisms of tethered C-CHY1. QCM-D was used to measure binding of C-CHY1 via three different length tethers, PEG molecular weight (MW) 866, 2000 and 7500, followed by microscopy to measure antimicrobial effectiveness against two model microbes Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus). Modeling of QCM-D data allowed for surface density and thickness to be calculated and related to C-CHY1 antimicrobial activity. PEG 7500 allowed proper C-CHY1 orientation and mobility, allowing for its native pore-forming mechanism and highest activity while PEG 866 tethers led to denser grafting and an effective, yet non-native ion displacement mechanism. The QCM-D was used to characterize the effect of salt concentration and temperature reaction conditions on the grafting density of C-CHY1 tethered via PEG 866 and PEG 7500, which was then related to antimicrobial activity. For PEG MW 866, neither temperature nor salt concentration increases significantly altered the grafting density of C-CHY1 while for PEG 7500 increasing temperature allowed for significantly increased grafting density. C-CHY1 density had no significant effect on antimicrobial activity against either microbe. Temperature of bacterial incubation did demonstrate microbe-specific changes in C-CHY1 antimicrobial activity. These results demonstrated that small changes in reaction conditions can drastically change membrane selectivity of C-CHY1. An in-depth investigation of the effects of bacterial membrane composition and temperature on soluble C-CHY1 mechanism was implemented to better understand the molecular membrane- and temperature-dependent selectivity and structure-function of C-CHY1. Supported lipid bilayers (SLBs) formed in QCM-D can be used as model membranes to elucidate AMP action mechanisms against membranes of different compositions. Two and three component SLBs representative of Gram-negative phosphatidylethanolamine (PE) and phosphatidyglycerol acid (PG) with and without charged lipopolysaccharide, LPS and Gram-positive bacteria phosphatidylcholine (PC) and PG with and without charged lipoteichoic acid, (LTA) were formed at both 23°C and 37°C. C-CHY1 at 5 µM was exposed to the different membranes and mechanistic surface action was studied. The membranes formed highly different baseline responses in QCM-D, indicative of vastly different membrane structures, thicknesses and deposition behaviors on SiO2, warranting future studies. Further, significant effects of LTA incorporation were observed in both peptide interaction and deposition. There were measurable effects of temperature on membrane formation as well as peptide interaction kinetics and even mode of interaction. Lastly, business models for the commercialization of novel medical device technologies such as surface-tethered C-CHY1 were investigated. While this technology has the potential to solve many unmet needs, there must a commercialization plan implemented in order to have an impact. There is a clear disconnect between technology development in academia and technology commercialization in industry that must be connected. Development of an entrepreneurial mindset at the graduate school level, can help bridge the gap. A thorough investigation of domestic and international business models for commercializing AMP technologies was carried out and distilled in the form of the Business Model Canvas developed by Alexander Osterwalder that can be used as a roadmap for commercialization efforts. Using the QCM-D a relationship between both spacer length and peptide surface density and the antimicrobial activity of tethered C-CHY1 was determined. A business plan was developed in order to increase the impact of this and other AMP based work. This work provides a roadmap for future researchers to quickly develop and commercial novel AMP based coating technology.

Quartz Crystal Microbalance (QCM) is a sensitive and effective technique to analyze mass changes at the interface between a solid material and a liquid environment. In this Master thesis, QCM was employed for evaluating the interactions between selected degradable polymers and nanodiamond particles (nDP), fibronectin and the growth factor BMP-2.   Many parameters must be adapted to allow QCM measurements involving degradable polymers. These parameters were then tailored to allow QCM measurements with PLA, poly(LLA-co-CL), poly(TMC-D-LA) and PS.   Moreover, QCM provides quantitative measurements of protein adsorption on degradable polymers. The behavior of PLA and poly(LLA-co-CL) was further evaluated and compared with respect to protein adsorption. This behavior was demonstrated to be different for the two polymers considered and to be dependent on protein concentration in solution.   Eventually, exploiting QCM it was also possible to assess the relationship between nDP and the adsorption of fibronectin and BMP-2 onto PLA and poly(LLA-co-CL).

Electrochemical quartz crystal microbalance has been employed to investigate electro-responsiveness of an ionic liquid as an additive in lubricant oils on a gold surface. Polarisation of the surface reveals changes in frequency where an increase in magnitude amplified the observed response, corresponding to a controllable alternation of the ionic liquid configuration on the surface as a function of applied potential. The frequency changes are due to different packing of the anion and cation, respectively, on the surface as their mass densities and geometries are different. Relaxation of the system was reversible to the application of a potential and it was also found to be diffusion dependent, where the ratio between the ion diffusivities could be extracted from the results. Measurement of the system relaxation reveals a potential decay of that of a discharging capacitor, with an internal resistance inducing an initial potential drop due to the resistivity of the oil medium. The discharge behaviour was also proven to show high internal reproducibility validity within experiments. This newly discovered insight in responsive differences of ion packing is of importance, not only for ionic liquid additives in tribology, but for understanding and exploiting ionic liquids in an array of electrochemical applications.

Current dissolution apparatuses require several hundred milligrams of sample per trial, measure dissolution rate indirectly via concentration sampling, and cannot maintain sink conditions throughout the duration of a test. This work describes a novel dissolution testing methodology developed using a commercial quartz crystal microbalance (QCM) system to measure dissolution rates of drugs while overcoming the limitations of current dissolution methods. The apparatus was characterized for a sample drug system of benzoic acid dissolved using a dissolution medium of deionized water at flow rates of 1000, 100, 50, and 10 &mgr;L/min. Using an analysis method that combines the responses of resonance frequency and resistance of the quartz crystal during dissolution, the dissolution rate of benzoic acid was found to be 4.029 ± 0.743, 2.026 ± 0.913, 1.565 ± 0.349, and 1.060 ± 0.103 % mass/s, for each flow rate, respectively. The QCM dissolution apparatus method can be used to measure drug dissolution directly by quantifying mass loss (rather than indirectly via concentration changes as with current methods), reduce sample sizes compared with current methods by three orders of magnitude onto the microgram scale, and maintain sink conditions throughout the duration of the test.

京都大学 / 0048 / 新制・課程博士 / 博士(工学) / 甲第19189号 / 工博第4066号 / 新制||工||1627(附属図書館) / 32181 / 京都大学大学院工学研究科合成・生物化学専攻 / (主査)教授 北川 進, 教授 濵地 格, 教授 森 泰生 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DGAM

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