In this paper a novel stimuli responsive hydrogel material, methacrylated sodium alginate beta-cyclodextrin (Alg-MA-β-CD), was used in combination with a microfluidic device to create microspheres. Currently there is no reliable method for fabricating homogeneous stimuli-responsive microspheres, in-house microfluidic devices are not reliable in manufacture quality or long-term use. Alginate hydrogels have many attractive characteristics for bioengineering applications and are commonly used to mimic the features and properties of the extracellular matrix (ECM). Human mesenchymal stem cells (hMSCs) are of top interest to tissue engineers. hMSCs are widely available and can be harvested and cultured directly out of human bone marrow. hMSCs have the ability to differentiate into osteoblasts, adipocytes, chondrocytes, muscle cells, and stromal fibroblasts depending on mechanical signals transmitted through surrounding ECM. The biomechanical properties of alginate based stimuli-responsive hydrogels can be tuned to match those of different types of tissues. When trying to transport and control the differentiation of hMSCs into generating new tissues or regenerating damaged tissues, it is highly beneficial to encapsulate the cells inside a microsphere made from these hydrogels. The proposed research objectives are: 1) To optimize fabrication techniques and create functional microfluidic devices; 2) Analyze the effects of flow parameters on microsphere production; and 3) Encapsulate viable hMSCs inside multi-stimuli responsive alginate microspheres using the fabricated microfluidic devices (MFDs). In this study, photolithography microfabrication methods were used to create flow-focusing style MFDs. The hydrogel materials were characterized via rheological methods. Syringe pumps controlled flow rates of fluids through the devices. Active droplets formation was monitored through a camera attached to an inverted microscope, where images were analyzed. Microsphere production was analyzed optically and characterized. Alg-MA-β-CD polymer solutions containing hMSCs were encapsulated, and a live/dead florescence assay was preformed to verify cell viability. Using a modified fabrication process it was possible to manufacture Alg-MA-β-CD microspheres and encapsulate and maintain viable hMSCs inside.

Orientador: Francisco Benedito Teixeira Pessine / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Química / Made available in DSpace on 2018-08-24T11:11:00Z (GMT). No. of bitstreams: 1 Barboza_AnaClaudiaRuedaNery_D.pdf: 6657696 bytes, checksum: 97ec0acb030267fe87c6983093726afb (MD5) Previous issue date: 2013 / Resumo: O objetivo foi deste trabalho foi obter, caracterizar e avaliar suspensões aquosas de micropartículas poliméricas bioadesivas para liberação sustentada do antisséptico digluconato de clorexidina (CHG), trabalhando somente em meio aquoso e com substâncias reconhecidamente biocompatíveis e de baixa toxicidade, para potencial aplicação na cavidade bucal. As micropartículas foram obtidas por complexação dos polieletrólitos quitosana e alginato de sódio, com auxílio de íons cálcio, e da clorexidina. O método de obtenção desenvolvido permitiu obter suspensões com distribuição de diâmetros de partículas adequada e boa estabilidade. Através de delineamento experimental fatorial, avaliou-se o efeito dos principais componentes da suspensão sobre o diâmetro médio das micropartículas e sobre a incorporação de CHG. A liberação de CHG em meio simulador da cavidade oral (saliva artificial) ocorreu de forma gradual e por longos períodos, indicando existência de interação entre o ativo, de natureza catiônica, e as terminações aniônicas do biopolímero alginato. A cinética de liberação de sistemas onde tais interações ocorrem é complexa e sua compreensão envolve diversos fenômenos físico-químicos, que se procurou identificar e discutir. Realizou-se, de forma simplificada, a modelagem da cinética de liberação através do modelo matemático semiempírico de Peppas-Korsmeyer, o que também indicou a combinação de diferentes fenômenos influenciadores da liberação. Testes preliminares de eficácia antimicrobiana indicaram que a clorexidina do sistema de liberação sustentada manteve sua eficácia sobre os microrganismos padrão avaliados em comparação com a clorexidina livre / Abstract: The objective of this project was to obtain, characterize and evaluate aqueous suspensions of bioadhesive polymeric microparticles for sustained release of the antiseptic chlorhexidine digluconate (CHG), working in aqueous media and with substances recognized as biocompatible and of low toxicity, for potential application to the oral cavity. Microparticles were obtained by complexation of the polyelectrolytes chitosan and sodium alginate, with pre-gelation by calcium ions, and CHG. The method of obtention developed resulted in suspensions with adequate particle size distribution and good stability. Through factorial experimental design, the effect of main suspension components on the median particle diameter and CHG incorporation was evaluated. Chlorhexidine release in oral cavity simulating media (artificial saliva) occurred gradually and for extended periods, indicating the presence of interactions between the cationic active substance, and the anionic moieties of the alginate biopolymer. The release kinetics where those interactions occur is somewhat complex and its understanding involves various physicochemical phenomena that we tried to identify and discuss. Simplified modeling of the release kinetics through the Peppas-Korsmeyer semi-empirical mathematical model was done, also indicating the combination of various release-influencing phenomena. Preliminary microbiological tests indicated that sustained-release CHG kept its efficacy against standard microorganisms evaluated in comparison to free CHG / Doutorado / Físico-Química / Doutora em Ciências

The use of Reverse osmosis (RO) membranes have been significantly increasing in water desalination, and the main operational obstacle in RO desalination plants is membrane fouling. Among other solutes, dissolved biopolymers, such as polysaccharides can lead to severe membrane fouling especially with the addition of calcium ions because of the complexation formation between the surface of membrane and foulants materials. However, this complexation can also take place in the feed bulk, resulting in foulants aggregates formation. Although there are some physical techniques that can maintain the membrane performance without reducing its lifetime, only chemical cleanings are still commonly used in RO plants. In this study, a novel cleaning method is proposed to restore the membrane performance by removing the deposited foulants without reducing the membrane lifetime. The cleaning method is based on using water saturated with dissolved CO2 gas, and its principle is based on producing spontaneous CO2 bubbles due to local pressure difference leading to nucleation of bubbles throughout the membrane surface, especially at nucleation sites, which improve the cleaning efficiency. Alginic acid sodium salt was used as a model of polysaccharides foulants in presence of different concentrations of NaCl and calcium ions aiming to enhance membrane fouling, and then CO2 cleaning solution efficiency, in terms flux recovery (FR), was tested under different operating conditions and compared to other cleaning methods. Average FR of 20%±3, 25%±3 and 80%±3 for MilliQ water, a cleaning solution at pH4, and CO2 solution at 6 bar, 0.17 m/s, and 23 ̊C ±0.2 for 6 minutes were obtained, respectively. The efficiency of this novel cleaning method was also compared to direct osmosis overnight, and the average flux was comparable (about 60%±3), though that the cleaning time was significantly different. Various calcium concentrations (0-10 mM) were added in the alginate solution to study the fouling behavior in terms of the potential for bulk complexation to form cake alginate layer on the membrane surface rather than a gel layer, and the role of CO2 bubbles nucleation to remove foulants was investigated. This cleaning method can be considered as an alternative more environmentally friendly technique in RO application.

<div>Fresh produce has been the leading source of foodborne illness outbreaks in the US, surpassing typical pathogen carriers such as meat, dairy, and seafood. Among the fresh produce popular to the consumers, cantaloupe and sprouts are mostly susceptible to pathogen contaminations and outbreaks. However, it has been a challenge to address the key factor in the contamination - the biofilms formed by pathogens are highly resistant to conventional washing and cleaning procedures. For cantaloupe, the net-like and porous surface forms a barrier for washing. For sprouts, the fragile texture of seedlings prevents aggressive cleaning operation and biofilm removal.</div><div><br></div><div>In this study, innovative interventions were developed to improve microbial safety of fresh produce, using cantaloupe and alfalfa sprouts as models. For cantaloupe, abrasive brushing was designed to remove pathogen biofilm from cantaloupe. Our research found pathogens could form biofilm at cantaloupe rind surface as the residence time of pathogens increased. Biofilm formed on cantaloupe rind was imaged by cryo-scanning electron microscopy (cryo-SEM), and its resistance to sodium hypochlorite and lauroyl arginate ethyl (LAE) was confirmed. Furthermore, abrasive brushing with peroxyacetic acid (PAA) could effectively remove biofilm formed at cantaloupe rind. The efficacy of this novel cleaning technique was highly desirable, which could achieve 3 log reduction in pathogen population. Mechanism of abrasive brushing to remove biofilm at cantaloupe rind surface was also proposed. Conceivably, brushing with diatomaceous earth (DE) and PAA could be an innovative and cost-effective method to remove pathogen biofilm from cantaloupe rind.</div><div><br></div><div>For alfalfa sprouts, since most of the outbreaks are linked to the sprouting seeds, seed disinfection treatments are considered to be the most effective method to improve microbial safety of sprouts. In this study, a newly developed alginate-based, antimicrobial seed coating treatment was evaluated for its efficacy to reduce foodborne pathogens from alfalfa seeds and sprouts. The calcium alginate coating in the presence of 2.5% lactic acid (CA-LA coating) reduced foodborne pathogens inoculated on alfalfa seeds to an undetectable level on day 1 during 28 day-seed storage, while chlorine (20,000 ppm) or lactic acid (2.5%) treatment took longer time to reach the same level. With sprouts, CA-LA coating resulted in > 2.5 log reduction for pathogen cells. In contrast, log reduction was < 0.6 for either chlorine (20,000 ppm) or lactic acid (2.5%) treatment. In general, this study indicated the effect of calcium alginate coating on reducing bacterial load of alfalfa seeds and sprouts, however, the germination rate of treated seeds was compromised due to the addition of lactic acid in the seed coating. Further study is needed to select antimicrobial compounds with minimum impact on germination rate of seeds.</div><div><br></div>

The diploma thesis if focused on the study of bioactive hydrogél and nanofiber wound dressings composed of natural biopolymers, which were functionalized by active compounds in the form of analgesic, antibiotics and enzymes. Hydrogél wound dressings were constituted from alginate and chitosan and nanofibers were created from polyhydroxybutyrate. The following 7 active compounds were selected to be added to the wound dressings: ampicillin, streptomycin, ibuprofen, papain, bromelain, collagenase and trypsin. In the theoretical part the structure of the skin and types of wound injuries were described. This part also talks about types of wound dressing and their applications, as well as treatment of skin wounds using enzymes and compounds with analgesic and antimicrobial properties. In addition, this section describes safety assays, in particular cytotoxicity assays on human cells. At the beginning of the experimental part, the process of preparation of hydrogél wound dressing was optimised. Subsequently, the dressings were enriched with active compounds and the rate of gradual releasing of the substances into model environment was monitored. In the case of enzymes, their proteolytic activity was also tested after their incorporation to the wound dressings. Furthermore, the prepared bioactive wound dressings were analyzed for possible cytotoxic effect on human keratinocytes. Finally, the wound dressing with combined content of active substances was created and also characterized for the rate of substance release, proteolytic activity and cytotoxicity. Antimicrobial activity of this wound dressings, against two selected strains of microorganisms: Escherichia coli and Staphylococcus epidermidis, was also evaluated.

The diploma thesis is focused on the characterisation and isolation of naturally occurred active compounds, especially on lipophilic compounds. The next part of the thesis deals with enzymes and their encapsulation into alginate particles to suggest new enzymatic dietary supplement for children with optimal nutrient composition. The theoretical part is focused on the issue of child nutrition, accordingly the necessary nutrients that children need to grow without any health difficulties. It is also focused on the availability of enzymatic food supplements for children. Finally, the theoretical part deals with the lack of chosen natural substances and their effects in connection with cancer. In the experimental part, four types of oils obtained from seeds were characterized. The oils were obtained by two methods: cold pressing with a Yoda kitchen press and extraction in an organic solvent with Soxtherm. Seeds were linseed, sesame, pumpkin, and cumin. With these oils were characterized natural active substances, polyphenols, flavonoids, antioxidants, carotenoids, chlorophylls, and fatty acids. Liposomes were also prepared from selected extracts, in which an effect on intestinal tumour cells were observed. Furthermore, some enzymes were selected, and they were encapsulated into alginate particles with average size 450 µm. In these particles was specified encapsulation efficiency as well as the proteolytic activity after testing in digest juices. The last part of this thesis was focused on the optimization of a complete enzymatic food supplement with addition of alginate particles as enzyme carriers. Samples of these supplements were finally subjected to a sensory analysis.

This diploma thesis deals with encapsulation of natural extracts. In the theoretical part the methods of encapsulation, materials for particle preparation, as well as application of encapsulation techniques in food industry were described. In experimental part selected natural extracts of propolis, green barley and probiotics were characterized. There substances were encapsulated into alginate and chitosan. In the total of 25 types of prepared particles long-term stability in some model physiological conditions as well as in four different model foods was evaluated. Additionally, stability of selected particles in several real milk-based products was followed too. The stability of particles was determined spectroptohometrically. In natural extract a content of polyphenols, proteins, chlorophylls, as well as total antioxidant activity were analysed. To analysis of probiotics optical and fluorescence microscopy were used. In propolis and green barley antimicrobial activity was tested too. Moreover, in the sample of propolis also cytotoxic assay was applied. Agar-chitosan was chosen as the best shell material for propolis due to its optimal stability in model physiological conditions as well as model foods. Liposomes were evaluated as unstable and were not recommended for further application. As the suitable shell material for powdered green barley starch-alginate (rate 1:4) and agar-chitosan were proposed, while the second one showed better stability for released proteins. Agar-chitosan shell material was usable for fresh green barley too. For probiotics encapsulation alginate or alginate-starch were chosen because of their porosity and possibility of nutrients diffusion. In real foods the best results were reached with application of probiotic particles into milk. Coencapsulation of powdered barley and probiotics did not confirm inhibition of culture growth. Neither the antimicrobial effect of propolis and barley nor the cytotoxic effect of propolis were confirmed.

>Magister Scientiae - MSc / Human Immunodeficiency Virus (HIV), which is the cause of Acquired Immunodefiency Syndrome (AIDS) is a major global public health issue affecting over 37 million people worldwide and is responsible for claiming over 32 million lives since the discovery of the disease in 1981. Through effective diagnosis, treatment and prevention HIV is a manageable disease. Today, advanced antiretrovirals, known as HAART, serve as effective, first-line drug regimens, consisting of a variety of viral inhibitors, and have successfully helped viral suppression. However, issues arise with antiretrovirals due to patient non-adherence and the development of drug resistant mutations. Coupled with dormant HIV reservoirs, viral extinction is attenuated. It is therefore essential that effective alternative treatments are investigated. The exploration of nanomedicine for targeted drug delivery has shown an ability to prolong the drug circulation time, target drugs to specific sites in the body, and enhance drug effectiveness. A previous study demonstrated a novel therapeutic strategy that was based on a mutant version of the caspase-3 enzyme that can induce apoptosis in HIV infected cells. This therapeutic strategy has the potential to wipe out reservoirs of HIV infection. However, the therapeutic strategy lacked selectivity because the delivery mechanism was based on protein transduction technology which will result in the nonselective delivery of the drug. In this study, preliminary work towards the development of a targeted nanoparticle delivery system for this mutant caspase-3 enzyme is described. The study describes the synthesis of green fluorescent protein loaded alginate/chitosan nanoparticles that were functionalized with a DNA aptamer intended to target the nanoparticles to CD4 expressing cells, that are also targeted by HIV. The THP-1 cell line was used due to the ability of the cells to express CD4 receptors on the cell surface. The nanoparticles were synthesized through ionotropic gelation. The size, polydispersity, zeta potential and morphology were investigated by Dynamic Light Scattering and Scanning Electron Microscopy, respectively. The strongly negative zeta potential studies revealed stability of the nanoparticles in suspension and Scanning Electron Microscopy results showed an indicative collapse of the polymer network for the empty nanoparticles (i.e. nanoparticles not loaded with GFP), whereas solid, cuboid nanoparticles were shown for the GFP-loaded nanoparticles. Image-based fluorescence cytometry demonstrated that the GFP-loaded nanoparticles bind to the THP-1 cells that express the CD4 receptor. The results obtained are indicative of a potential drug delivery system for HIV treatment however, adjustments would need to be made to the current study to further develop this nanocarrier.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第20440号 / 農博第2225号 / 新制||農||1049(附属図書館) / 学位論文||H29||N5061(農学部図書室) / 京都大学大学院農学研究科応用生命科学専攻 / (主査)教授 植田 充美, 教授 小川 順, 教授 渡邊 隆司 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

Smörjmedel är en nödvändighet för maskiner för att minska slitage och energiförbrukning. Dock är över 95% av de använda smörjmedlen i dag fossilbaserade. Smörjmedel baserade på vegetabiliska oljor finns tillgängliga men har ofta en lägre oxidativ stabilitet och sämre lågtemperaturegenskaper än deras fossilbaserade motsvarigheter. Ett annat hinder att övervinna är att ersätta vanligt använda förtjockningsmedel i halvfast smörjmedel med biobaserat alternativ vilket också visat sig vara en utmanande uppgift. Representanter för marina biomassa som mikro- och makroalger har potential att användas i framtiden som förnybar råvara källor på grund av deras kemiska sammansättningar såväl som gynnsamma odlingsförhållanden. Mikroalger har ett högt lipidinnehåll och vissa arter har ett högt innehåll av kolväten som liknar råolja. Makroalger har ett högt innehåll av polysackarider med en förtjockningsförmåga i vatten. Även om mikroalger innehåller lovande kolväten och lipider som kan användas i smörjmedelsapplikationer är det idag inte ekonomiskt möjligt att använda mikroalger för att producera produkter med lågt värde. Även makroalger är för kostsamma att kultivera för billiga produkter, För att kunna använda polysackarider som tillsatser i olja måste de vara mer hydrofoba. I denna studie oxiderades alginat först i en ringöppningsreaktion, för att senare reduceras i en Schiff-basformation och reduktion till en mer amfifil polysackarid. Resultaten visade en hög nedbrytning av polysackariden både i oxidationsreaktionen och i reduktionsreaktionen, från en startmolekylvikt av 580 000 g/mol till ~ 10 000 g/mol. Etanol kunde användas i oxidationsreaktionen för att öka den möjliga alginatkoncentrationen. Både FTIR- och 1H-NMR-resultaten indikerade en lyckad oxidation och reduktion. Framtida arbete involverar inblandning av det hydrofobt modifierade alginatet i en basolja och utvärdering av effekten av den erhållna biobaserade basoljekomponenten på oljeegenskaperna, till exempelökning av viskositeten, oxidationsstabilitet och förtjockningsbeteenden. / Lubricants are a necessity for machineries in order to reduce friction and wear. However, over 95% of the lubricants used today are fossil-based. Vegetable oil-based lubricants are available but often possess lower oxidative stability and poorer low temperature properties than their fossil-based counterparts. Vegetable oils are therefore not a perfect solution to reduce crude oil usage. Another obstacle to overcome would be a replacement of commonly used thickeners in semisolid lubricants with bio-based alternative, which has proven to be a challenging task. Marine biomass representatives such as micro-and macroalgae have the potential to be used in the future as renewable feedstock sources due to their chemical compositions as well as beneficial cultivation conditions such as usage of non-arable land and saltwater. Microalgae have a high lipid content, and in some species a high content of hydrocarbons similar to crude oil. Macroalgae have a high content of polysaccharides, some with gelling abilities. Even though microalgae contain promising hydrocarbons and lipids that can be used in lubricant applications, it is currently not economically feasible to use microalgae to produce low valueproducts. Macroalgae are also too expensive to cultivate to be used in low price products. In order to use polysaccharides as additives in oil, they need to be more amphiphilic. In this study, alginate, derived from brown seaweed, was first oxidized in a ring opening reaction to later be reduced in a Schiff base formation and reduction to introduce more hydrophobic side groups. The results revealed a severe degradation of the polysaccharide both in the oxidation and in the reduction reaction, from a starting molecular weight of 580 000 g/mol to ~ 10 000 g/mol. Ethanol was proved to be a suitable solvent in the oxidation reaction which increased the possible alginate concentration. Both FTIR and 1H-NMR results indicated a successful oxidation and reduction. Future work involves incorporation of the hydrophobically modified alginate in a base oil and evaluation of the presence of the obtained bio-based base oil component on the oil properties, for example viscosity increase, oxidation stability and thickening behaviors.