Previous studies have shown that pullulan films are able to stabilize enzymes and other labile molecules from thermal and oxidative degradation. Solutions made with commercially available pullulan are extremely viscous and difficult to process limiting the ability to use low-cost printing systems, such as inkjet printers, to format pullulan-containing. In this work, we show that pullulan can be made printable by decreasing its chain length by acid hydrolysis. The acid hydrolysis reaction was modelled using statistical software; the molecular weight of pullulan decreased with increasing reaction time, temperature and acid concentration. Interactions between time and temperature, and temperature and acid concentration were determined to be significant to the reaction as well. The mechanical properties and oxygen permeability of films made from pullulan with different molecular weights were also measured. The films were found to have similar tensile properties and oxygen permeabilities to each other and to those obtained using native pullulan. Using a thermally unstable enzyme (acetylcholinesterase) and an easily oxidizable small molecule (indoxyl acetate) as test materials, it was found that these films have the same ability to stabilize the enzyme and to serve as an oxygen barrier, as the films made with native pullulan. It was also found that pullulan is inkjet printable as long as the molecular weight is 56 kDa. Poor jetting and clogging of the printhead was observed when pullulan with a molecular weight higher than this threshold was used. Microarray printing was also demonstrated by a printing acetylcholinesterase/pullulan in nano-sized volumes using a Dimatix inkjet printer and showing activity of the enzyme after printing and storage at ambient conditions. Proof of concept of microarray printing opens up the potential for future applications of pullulan in other high throughput applications. / Thesis / Master of Applied Science (MASc)

The diploma thesis deals with the study of biotechnologically significant microorganisms, using the Raman spectroscopy. Content of the theoretical part is brief characteristic of Raman spectroscopy as a method, its use in practice and also use as a tool for monitoring of biotechnologically processes. Thesis was further focus on the biotechnologically significant microorganism Aureobasidium pullulans, its use in biotechnology and also for over-produced substances and in particular poly-L-maleic acid and pullulan. The content of the experimental part was study of selected strains A. pullulans, specifically stains as DSMZ, CCM F148 and CCM 8182, using Raman spectroscopy on the various types of culture media. Subject of practical part research was too production of extracellular polymers, acid poly-L-apple and pullulan, by selected strains A. pullulans. Objective of my thesis was described and determinate, spectra of individual strains as well as extracellular products, mainly pullulan, and then choose suitable production medium and optimal production strain A. pullulans. During experimental work was found, that optimal production strain was DSMZ strain culture on the mineral medium with the addition of yeast autolysate, which was optimal medium type. The content of the pullulan produced was for gravimetric determination, 6,3g/L, which also confirmed the results of the HPLC method. It was experimentally found, that Raman spectroscopy isn´t suitable method for quantification of extracellular products, but is appropriate and was used for PCA analysis of individual strains.

Interactions between polysaccharide derivatives and rod-like cellulose nanocrystals were studied by light scattering. Two replicates of cellulose nanocrystals (DOE-2-12A and DOE-2-12B) were probed with polarized and depolarized dynamic light scattering. X-ray photoelectron spectroscopy experiments showed sulfate groups on cellulose nanocrystals. Decay rates from polarized dynamic light scattering experiments exhibited a significantly smaller angular dependence for both samples. However, DOE-2-12B showed a smaller angular dependence than DOE-2-12A. Lengths and diameters of DOE-2-12A and DOE-2-12B obtained by Broersma's formula were 229 " 19 and 19 " 7 nm and 240 " 18 and 22 " 6 nm, respectively. The resultant length and diameter of DOE-2-12B were comparable to those for cellulose whiskers obtained from cotton. Adsorption of pullulan 4-chlorocinnamate (P4CC03) onto cellulose nanocrystals (DOE-2-12B) was also studied by UV-Vis spectroscopy, zeta-potential measurements, and polarized and depolarized dynamic light scattering. UV-Vis spectroscopy of the P4CC03/water binary system and in situ light scattering showed UV crosslinking of pullulan 4-chlorocinnamate occurred in binary and ternary systems but led to different aggregation behavior in the two ternary systems: PreX where P4CC03 crosslinking occurred prior to the addition of cellulose nanocrystals and Rxn where cellulose nanocrystals were present during UV exposure. These studies showed P4CC03 adsorbed onto cellulose nanocrystals and UV induced crosslinking occurred at the surface of the cellulose nanocrystals. Zeta-potential measurements also showed that P4CC03 adsorbed onto cellulose nanocrystals. Finally, adsorption of 2-hydroxypropyltrimethylammonium xylans (HPMAXs) of degree of molar substitution MS = 0.34 onto rod-like cellulose nanocrystals (DOE-2-12Bs) were probed with zeta-potential measurements and polarized and depolarized dynamic light scattering. Zeta-potential changes of HPMAX/water, HPMAX/DOE-2-12B/water, and DOE-2-12B/water systems showed HPMAX adsorption onto DOE-2-12Bs. Intensity autocorrelation functions from Hv and Vv mode exhibited partial heterodyning. Decay time distributions of the binary and ternary systems showed that aggregates existed in the binary system but disappeared in the ternary system. These observations revealed that HPMAX adsorbed onto a fraction of the cellulose nanocrystals in the ternary system at low concentrations of HPMAX. Decreasing translational and rotational diffusion coefficients with increasing HPMAX concentration indicated HPMAX adsorption onto cellulose nanocrystals. A significant HPMAX concentration dependence of the ratio of rotational diffusion coefficient to translational diffusion coefficient showed strong adsorptive interactions between HPMAX and DOE-2-12B. These studies showed there were interactions between polysaccharides and cellulose nanocrystals even in very dilute solutions. Also, it was shown that probe diffusion studies with rod-like cellulose nanocrystals is a promising strategy for probing complicated polymer solutions. / Ph. D.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20672号 / 医博第4282号 / 新制||医||1024(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 妹尾 浩, 教授 野田 亮, 教授 岩田 想 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Pullulan is an exopolysaccharide secreted extracellularly by the black yeast-like fungi Aureobasidium pullulans. Due to an alpha-(1-->6) linked maltotriose repeat unit, which interferes with hydrogen bonding and crystallization, pullulan is completely water soluble unlike cellulose. It has also been tested and shown to possess non-toxic, biodegradable, non-mutagenic and non-carcinogenic properties. Chemical modification of polysaccharides to increased hydrophobicity and increase functionality has shown great promise in drug delivery systems. Particularly in amorphous solid dispersion (ASD) formulations, hydrophobicity increases miscibility with hydrophobic, crystalline drugs and carboxy functionality provides stabilization with drug moieties and well as pH specific release. Successful synthesis of cellulose w-carboxyalkanoates have been reported and showed great promise as ASD polymers based on their ability to retard the recrystallization of the HIV drug ritonavir and antibacterial clarithromycin. However, these cellulose derivatives have limitations due to their limited water solubility. Natural pullulan is water-soluble and modification with w-carboxyalkanoate groups would provide a unique set of derivatives with increased solubility therefore stronger polymer-drug interactions in solution. We have successfully prepared novel pullulan w-carboxyalkanoates, which exhibit good solubility in polar aprotic and polar protic solvents. All derivatives exhibit high thermal stability and most recorded high glass transition temperatures. Due to unknown impact of their three dimensional structure on miscibility and stabilization of drug against crystallization, each of these polymers possesses great potential for use in various drug delivery applications. / Master of Science

Ideally, every person in the world should have access to a safe and clean water supply; if not all sources of water are clean and safe, at the very least, an effective method to detect water contamination should be readily available. An effective detection method should not only be sensitive, rapid, robust, and affordable, but, ideally, it should also be equipment-free and easy to transport and deliver to the end-users. The main goal of this project is to develop a variety of bits and pieces of bioassay systems, with a particular focus on paper-based bioactive devices in order to provide portable and ready-to-use biosensors which can be useable by anyone anywhere around the world without requiring formal training. According to the World Health Organization (WHO), 76,000 people each year die in India alone because of pesticide poisoning. Long term exposure to organophosphate pesticides is known to have adverse effects on neurological function and can lead to Alzheimer's Disease, Attention Deficit Hyperactivity Disorder (ADHD), and reduced Intelligence Quotient (IQ). The likelihood of long term exposure to pesticides is heightened in developing countries, so a reliable and inexpensive pesticide sensor is a much-needed device in the developing world. To address this need, this project reports on the development of a fully-automated bioactive paper-based sensor for the detection of organophosphate pesticides. In the proposed biosensor, two innovations were implemented to achieve a full-automated format for the pesticide sensor: (I) First is a PUMP ON A PAPER (Jahanshahi-Anbuhi et al., LOC, 2012) that increases the flow rate of fluids within paper-based microfluidic analytical devices and sequentially brings two separate liquid streams to the enzyme test zone on the paper sensor, and (II) the second innovation is a PIPETTE ON A PAPER (Jahanshahi-Anbuhi et al., LOC, 2014) that involved the creation of a pullulan (a natural non-ionic polysaccharide) temporary bridge-system to transfer a known amount of solution to the sensing zone that, gives the enzyme zone a chance to dry and accept the substrate solution from the slow channel after a fixed period of time. This proposed format results in a simplified assay that detects the presence of pesticides automatically without any further manipulation from the user. However, the shelf life of this assay kit is challenging due to instability of both enzyme (AChE) and substrate (IDA) at room temperature. AChE loses its enzymatic activity when stored at room temperature and IDA becomes oxidized quickly. This problem is not unique to these two bio reagents, however; almost all bioassays which use bio-reagents (such as enzymes and small-molecular substrates) are unstable to varying degrees and require special shipping and storage. The instability of these molecules can arise from either thermal denaturation or chemical modification, such as oxidation or hydrolysis. Because of these issues, they often have to be shipped on dry ice with special packaging, which is costly. The cost of maintaining a cold chain for distributing bio-reagents accounts for up to 80% of the cost. Aside from the cost, these reagents also have to be stored in bulk in refrigerators or freezers to minimize the loss of activity, but they must be thawed and aliquoted for their intended tests. Repeated freezing and thawing can result in a significant loss of activity, which often leads to less reliable test results. These issues make running such assays in resource-limited settings a significant challenge. There is, therefore, an urgent need for an assay system with stable reagents that is easy to use, simple to read, inexpensive, and that includes a method for the long-term stabilization of enzymes and other unstable reagents in pre-measured quantities. To overcome to all these issues, pullulan is utilized for the development of pill-based-biosensors. Pullulan dissolves quickly in aqueous solutions and shows very high oxygen barrier properties in its film form. Considering the unique properties of pullulan, it is hypothesized that pullulan may be suitable for producing assay pills with encapsulated enzymes or other unstable molecules and may provide a simplified platform for carrying out bioassays in resource-limited settings. The application of these pill-based-biosensors is shown via the entrapment of AChE and IDA for the creation of an assay kit that can detect organophosphate pesticides (Jahanshahi-Anbuhi et al., Angew. Chem., 2014). Moreover, this thesis reports on the stabilization of highly unstable firefly luciferase for the detection of microorganisms and, more particularly, ATP. Through the use of pullulan, this thesis demonstrates that both the enzyme and the substrate can be protected, immobilized, and stabilized at room temperature, instead of the existing storage methods, which require temperatures <-20˚C. This innovation allows for a more convenient method of shipping the bioassay kits around the world without any extra care. Furthermore, pullulan-based films are utilized for the development of a method for controlled multidirectional flow within paper-based biosensors. This method provides the possibility of trapping labile and volatile reagents and stabilizing them by forming thin films with pullulan. The trapped reagents within pullulan films can be strategically stacked and assembled on a paper strip in different directions. Furthermore, should the need arise, these reagents can be released and delivered sequentially or simultaneously in both vertical and lateral directions through the paper. The application of this method is shown for: (I) creation of "ready-to-use" assay kit for the detection of Escherichia coli (E. Coli). This assay kit has the step of cell lysing and proceeds automatically to the step in which enzymes react. The second application (II) shows the trapping of Simon’s reagents, which is widely used for methamphetamine detection. Overall, these unique fabrication techniques can be widely used for the preparation of highly stable, ready-to-use, and user-friendly biosensors. We are currently working on the detection of other contaminants such as heavy metals, and we are starting on vaccine stabilization and delivery, which would have a tremendous impact for society. / Dissertation / Doctor of Engineering (DEng)

No description available.

Self-assembly of polysaccharide derivatives at liquid/solid interfaces was studied by surface plasmon resonance spectroscopy (SPR) and quartz crystal microbalance with dissipation monitoring (QCM-D). Carboxymethyl cellulose (CMC) adsorption onto cellulose surfaces from aqueous solutions was enhanced by electrolytes, especially by divalent cations. A combination of SPR and QCM-D results showed that CMC formed highly hydrated layers on cellulose surfaces (90 to 95% water by mass). Voigt-based viscoelastic modeling of the QCM-D data was consistent with the existence of highly hydrated CMC layers with relatively low shear viscosities of ~ 10-3 N·s·m-2 and elastic shear moduli of ~ 105 N·m-2. Adsorption of pullulan 3-methoxycinnamates (P3MC) and pullulan 4-chlorocinnamates (P4CC) with different degrees of cinnamate substitution (DSCinn) onto cellulose, cellulose acetate propionate (CAP), poly(L-lactic acid) (PLLA), and methyl-terminated self-assembled monolayer (SAM-CH3) surfaces was also studied by SPR and QCM-D. Hydrophobic cinnamate groups promoted the adsorption of pullulan onto all surfaces and the adsorption onto hydrophobic surfaces was significantly greater than onto hydrophilic surfaces. SPR and QCM-D results showed that P3MC and P4CC also formed highly hydrated layers (70 to 90% water by mass) with low shear viscosities and elastic shear moduli. Finally, cellulose adsorption and activity on pullulan cinnamate (PC) and cellulose blend films were studied via QCM-D and in situ atomic force microscopy (AFM). The hydrophobicity of PC surfaces was controlled by adjusting the degree of cinnamate substitution per anhydroglucose unit (DSCinn). It was found that cellulase showed weak adsorption onto low DSCinn PC surfaces, whereas cellulase adsorbed strongly onto high DSCinn PC surfaces, a clear indication of the role surface hydrophobicity played on enzyme adsorption. Moreover, cellulase catalyzed hydrolysis of cellulose/PC and cellulose/polystyrene (PS) blend surfaces was studied. The QCM-D results showed that the cellulase hydrolysis rate on cellulose in cellulose/PC blend surfaces decreased with increasing DSCinn. AFM images revealed smooth surfaces for cellulose/PC (DSCinn = 0.3) blend surfaces and laterally phase separated morphologies for cellulose/PC (DSCinn ≥ 0.7) blend surfaces. The combination of QCM-D and AFM measurements indicated that cellulase catalyzed hydrolysis was strongly affected by surface morphology. The cellulase hydrolysis activity on cellulose in cellulose/PS blend surfaces was similar with cellulose/PC blend surfaces (DSCinn ≥ 0.7). These studies showed self-assembly of macromolecules could be a promising strategy to modify material surfaces and provided further fundamental understanding of adsorption phenomena and bioactivity of macromolecules at liquid/solid interfaces. / Ph. D.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Polymeric nanocapsules have been studied extensively for drug delivery by various routes of administration. Currently, the nanoencapsulation of drugs is considered the most efficient means of ensuring controlled release, specific targeting and reduction of adverse effects. In this context, the aim of this work was to develop polymeric nanocapsules for pulmonary delivery of beclomethasone dipropionate (BD). Nanocapsules have been prepared from 2 polymers, poly(-caprolactone) (PCL) and ethyl cellulose (EC). To quantify the drug in the nanostructures, the analytical method was developed and validated. This method showed to be specific, linear, precise, accurate and robust. Nanocapsules were prepared by interfacial deposition of preformed polymers and were evaluated as to pH, particle diameter, polydispersity index, drug content, encapsulation efficiency and zeta potential. All samples showed encapsulation efficiency greater than 98%, negative zeta potential, pH value in the range of neutrality and drug contents close to their theoretical values. The size distribution was nanometric (158-270 nm) with polydispersity index lower than 0.2. The results of the photodegradation study showed that polymeric nanocapsules were able to protect BD from UVC radiation when compared to the free drug solution. In vitro release experiments were performed using the dialysis bag technique, which showed, for all formulations, a prolonged drug release mediated by anomalous transport and first order kinetics. Free drug in solution took between 24 and 36 h to reach 100% of release, whereas nanocapsules were able to control the drug release for up to 108 h, depending on the polymer employed. Nanocapsules of EC and PCL were evaluated for in vitro and in vivo toxicity and the results suggest that the proposed formulations are safe. In the final stage of the work, pullulan was proposed as stabilizer agent for PCL nanocapsules and the results obtained for the zeta potential and the drug content suggested that these formulations have become more stable. Thus, the nanocapsules developed in this work represent a promising alternative for the pulmonary delivery of BD in the treatment of asthma and other respiratory disorders. / Nanocápsulas poliméricas vem sendo estudadas extensivamente para liberação de fármacos por diversas vias de administração. Atualmente a nanoencapsulação de fármacos é considerada o meio mais eficiente de assegurar liberação controlada, direcionamento específico e redução dos efeitos adversos. Neste contexto, o objetivo do presente trabalho foi desenvolver nanocápsulas poliméricas para a liberação pulmonar do dipropionato de beclometasona (DB). Nanocápsulas foram preparadas a partir de 2 polímeros, a poli(-caprolactona) (PCL) e a etilcelulose (EC). Para a quantificação do fármaco nas nanoestruturas, o método analítico foi desenvolvido e validado. Este mostrou ser específico, linear, preciso, exato e robusto. As nanocápsulas foram preparadas por deposição interfacial do polímero pré-formado e avaliadas quanto ao pH, diâmetro de partícula, índice de polidispersão, teor, eficiência de encapsulamento e potencial zeta. Todas as amostras apresentaram eficiência de encapsulamento maior que 98%, valor de potencial zeta negativo, valor de pH na faixa da neutralidade e teores próximos aos teóricos. A distribuição de tamanho foi nanométrica (158-270 nm) com índice de polidispersão menor que 0,2. Os resultados do estudo de fotodegradação mostraram que as nanocápsulas poliméricas foram capazes de proteger o DB da radiação UVC quando comparadas com uma solução do fármaco. Os experimentos de liberação in vitro foram realizados empregando a técnica de sacos de diálise, a qual mostrou, para todas as formulações, uma liberação prolongada do DB, mediada por transporte anômalo e cinética de primeira ordem. A solução etanólica de DB levou entre 24 e 36 h para alcançar 100% de liberação, enquanto que as nanocápsulas foram capazes de controlar a liberação do fármaco por até 108 h, dependendo do polímero empregado. Nanocápsulas de EC e PCL foram avaliadas quanto à toxicidade in vitro e in vivo e os resultados obtidos sugerem que as formulações propostas são seguras. Na etapa final do trabalho, o pullulan foi proposto como agente estabilizador de nanocápsulas de PCL e os resultados obtidos para o potencial zeta e o teor de fármaco sugerem que estas formulações tornaram-se mais estáveis. Desta forma, as nanocápsulas desenvolvidas neste trabalho representam uma alternativa promissora para a liberação pulmonar do DB no tratamento da asma e de outras desordens do trato respiratório.

Lors de cette dernière décennie, le développement de formes pharmaceutiques innovantes permettant d'améliorer l'efficacité, la sécurité et l'acceptabilité des médicaments pédiatriques est en pleine croissance. Les films orodispersibles (ODF) appartiennent à ces nouvelles formes galéniques améliorant la compliance des patients. Ils sont constitués d'une matrice de polymère hydrophile dans laquelle un ou des principe(s) actif(s) (PA) sont dissous ou dispersés. Après dépôt de l'ODF sur la langue ou dans la cavité buccale, la matrice se désagrège libérant le PA pour une action locale ou systémique. Dans cette étude, la mise au point d'ODF, par la méthode de coulée/évaporation de solvant a été explorée afin d'administrer un PA d'intérêt en pédiatrie, la tétrabénazine (TBZ). Les caractérisations physicochimiques et biopharmaceutiques des ODF ont mis en évidence une augmentation de la vitesse et du taux de dissolution de la TBZ induit par son état amorphe. Le système constitué d'un support polymère et d'un PA sous forme amorphe peut être assimilé aux dispersions solides amorphes (SD). Les études réalisées démontrent l'importance de la nature du polymère utilisé pour maintenir les propriétés initiales du système dans le temps. La formation de liaisons hydrogène entre la PA étudié et le polymère est un facteur essentiel pour assurer la stabilité des SD. De plus, l'incorporation de cyclodextrines (CD) prolonge l'état amorphe du PA en générant des liaisons hydrogène avec la TBZ et en l'entourant d'une barrière chimique. Cette association favorise la libération du PA par effet synergique améliorant la biodisponibilité. Cette forme innovante représente un intérêt majeur dans l'amélioration de l'observance dans le cadre d'un traitement pédiatrique / During the last decade, various strategies to develop innovating oral dosage forms for pediatric population were investigated in order to improve treatment efficiency, safety and acceptability. Among these new delivery systems, orodispersible films (ODF) present a great potential to enhance patient compliance. In ODF, drug is dissolved or dispersed in a hydrophilic film-forming polymer. Once the ODF is in the mouth, polymeric matrix disintegrates releasing the drug for local or systemic action. In this study, ODF, produced with the solvent casting/evaporation method, were developed to administer a drug of interest for pediatric population, the tetrabetazine (TBZ). Physicochemical and biopharmaceutic characterizations showed that ODF allowed a major improvement of TBZ dissolution profile in simulated saliva, mainly due to the amorphous state of the drug in ODF. ODF were identified as amorphous solid dispersion (SD) composed of both amorphous TBZ and polymer matrix. We demonstrated that the choice of the polymer plays an important role to maintain initial properties of the system and amorphous state stability over the time. H-bonding formation between TBZ and polymer is essential to assure the preservation of TBZ amorphous state. Moreover, the incorporation of cyclodextrins (CD), by generating H-bonding with TBZ, has extended its stability. By synergic effect, this association produces an improvement of drug release leading to promote bioavailability. As they are easy to swallow and allow enhancing treatment efficiency, ODF appear as suitable delivery forms for pediatric patients