Rozprawy doktorskie na temat „CHITOSAN-SILVER NANOPARTICLE”

Neste trabalho de mestrado foram desenvolvidos nanobiocompósitos contendo quitosana (QS) e nanopartículas de prata (AgNPs) para aplicação em matrizes poliméricas com propriedades bactericidas. O trabalho foi conduzido em 4 etapas, sendo: i) a primeira referente ao estudo e escolha do melhor modo de estabilização dos colóides de prata em solução, sendo escolhido o modo de estabilização estérica com a quitosana (QS); ii) a segunda parte está relacionada com um estudo detalhado da interação entre a QS e as nanopartículas de prata (AgNPs) além da otimização da relação QS:AgNPs no nanocompósito para se obter maior ação bactericida; iii) foi também proposta uma rota de síntese na qual não se utiliza-se o Boro Hidreto de Sódio (NaBH4) como redutor, composto altamente reativo, sendo utilizados o citrato de sódio e QS conjuntamente como redutores; iv) inserção do nanocompósito QS:AgNPs em uma matriz de polivinil álcool (PVA). Foram utilizadas as técnicas de espectroscopia UV-vis e FT IR, DLS, Potencial Zeta, MET, DR-X, ensaios microbiológicos de MIC, OD595 e teste de halo de inibição, TGA, DSC e ensaios mecânicos. Concluímos que o uso da QS como agente estabilizante em comparação ao PVA é a mais indicada, devido ao maior número de grupos funcionais interagindo com as nanopartículas de prata. O poder de ação bactericida do nanocompósito QS:AgNPs pode ser aumentado numa certa relação entre ambos, a saber 4:1 em massa. A síntese utilizando citrato de sódio e QS como redutores mostrou a possibilidade da obtenção de nanopartículas de prata pequenas, com tamanho de 2 - 5 nm com estrutura esférica ou maiores. com tamanho de 300 nm, apresentando estruturas dendríticas, dependendo apenas do tempo de reação e concentração de citrato de sódio. A última etapa revelou a possibilidade da inserção do nanocompósito no polímero PVA sem perda significativa das características térmicas e mecânicas do polímero.
The work reported here was aimed at developing chitosan/AgNPs based nanobiocomposits for bactericidal applications. The studies were divided into four main steps, viz.,: i) optimization of the silver colloids stabilition process, in which the use of chitosan resulted in the best stability, ii) a detailed investigation on the interactions between chitosan and AgNPs, as well as the optimization of the chitosan :AgNPs ratio to promote the best bactericidal effect, iii) development of a new synthetic route without using NaBH4, in a search for an environmentally-friendly route, and iv) incorporation of the chitosan:AgNps nanobiocomposites in a PVA matrix for application as smart food packaging. The nanobiocomposites were characterizaed via UV-vis and FT IR spectroscopies, DLS, Zeta potential, TEM and DR-X. Biological essays had also been carried out, as well as tensilestress and thermo analyses (DSC and TGA). The best bactericidal effect was observed for a nanobiocompostie comprising chitosan:AgNPs at a ratio of 4:1 (wt/wt). The synthetic route employing sodium citrate as reducing agent resulted in AgNPs with average diameters of 2 5 nm, as well as bigger nanoparticles with diameters of ca. 300 nm, depending on the reaction time and citrate concentration. The incorporation of the chitosan:AgNPs composites in the PVA matrix resulted in the formation of a bactericidal composite with good mechanical and thermal properties, suitable for applications as smart food packing.

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CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior
FAPEMIG - Fundação de Amparo à Pesquisa do Estado de Minas Gerais
A prata é conhecida por sua atividade antibacteriana e tem se mostrado eficiente como alternativa no contexto da resistência bacteriana a drogas, sobretudo na forma de nanopartículas (AgNPs). Nossos objetivos foram a avaliação da susceptibilidade de bactérias representativas contra AgNPs e pesquisa de efeito sinérgico ou antagônico quando associadas a quitosana (QIT) e antimicrobianos de uso terapêutico humano. Foram utilizadas oito linhagens bacterianas de referência, representativas de: Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus, S. epidermides e Enterococcus faecalis. O perfil de susceptibilidade foi determinado através do método de microdiluição em caldo, de acordo com as recomendações do CLSI. A concentração inibitória mínima (CIM90) das AgNPs para as amostras testadas foi de 64μg/mL, enquanto que para a combinação AgNPs-QIT foi de 16μg/mL. Considerando-se atividade antimicrobiana somente da QIT, a CIM90 foi de 16 μg/mL. Entre os antimicrobianos testados (meropenem, amicacina, gentamicina, levofloxacina, rifampicina, sulfametoxazol-trimetropim, tetraciclina, e oxacilina e vancomicina, estes apenas para Gram positivos), apesar de a CIM para cada linhagem bacteriana estar de acordo com os valores determinados pelo CLSI observou-se diminuição significativa para todas as drogas testadas quando combinadas com AgNPs-QIT. De modo geral, para todas as espécies bacterianas avaliadas, a associação de AgNPs com QIT ou drogas antimicrobianas apresentaram grande potencial inibitório que pode estar relacionado a estabilidade das AgNPs associadas ao polímero ou a interação positiva das nanopartículas com os antimicrobianos. O ensaio de Checkerboard foi realizado para estabelecer os efeitos sinérgicos ou antagônicos em cada combinação. A possibilidade da interação das AgNPs e da QIT com antimicrobianos já existentes é altamente relevante e reforça as recomendações da literatura acerca da reformulação de uso de antimicrobianos tradicionais, além da pesquisa de novas drogas e estratégias para sobrepujar o crescente fenômeno da resistência bacteriana aos antimicrobianos.
Silver is known for its antibacterial activity and has been shown to be effective as a potential alternative in the context of bacterial resistance to drugs, particularly in the form of nanoparticles (AgNPs). Our objectives were to evaluate the susceptibility of bacteria against AgNPs and representative survey of synergistic or antagonistic when combined with chitosan (QIT) and antimicrobials for human therapeutic use. A total of eight reference strains were evaluated of Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus, S. epidermidis and Enterococcus faecalis. The susceptibility patterns were determined by broth microdilution method, according to the CLSI guidelines. The minimum inhibitory concentration (MIC90) of AgNPS to the samples tested was 64μg/mL, while for the combination AgNPs-QIT was 16μg/mL. Considering only antimicrobial activity of QIT, the MIC90 was 16 mg / mL. Among the antimicrobials (meropenem, amikacin, gentamicin, levofloxacin, rifampicin, sulfamethoxazole- trimethoprim, tetracycline, and vancomycin and oxacillin, these only to Gram positive), while the MIC for each bacterial strain is in accordance to the reference CLSI, significant decrease was observed for all tested drugs when combined with AgNPs-QIT. In general, association of AgNPs with QIT or with antimicrobial drugs showed higher inhibitory potential, which may be related to the stability of the polymer associated AgNPs or positive interaction of nanoparticles with antibiotics. The Checkerboard assay was then performed to establish antagonistic or synergistic effects in each combination. The possibility of interaction of AgNPs and QIT with existing antimicrobials is highly relevant and reinforces the recommendations of the literature on the reformulation of traditional antimicrobial use, as well as research into new drugs and strategies to overcome the growing phenomenon of bacterial resistance to antimicrobials.

[EN] In the present Doctoral Thesis, different strategies to improve functional properties of starch films for food packaging applications were analysed: study of the effect of amylose:amylopectin ratio, blend with other polymers poly(vinyl alcohol) (PVA), and incorporation of different fillers (rice bran and cellulose nanocrystals-CNCs) and antimicrobial agents (neem oil-N, oregano essential oil-O and silver nanoparticles-AgNPs). Likewise, a biodegradation study of the films as affected by antimicrobials was carried out. Among different starches with distinct amylose:amylopectin ratio, pea starch was selected with higher values of this ratio. The high content of amylose gave rise to stiffer and more resistant to fracture films, with lower oxygen permeability, which decreased during storage. Pea starch-PVA (S-PVA) blend films represented a good strategy to improve film properties without a notable increase in cost. The PVA addition led to films which were less water soluble and not as sensitive to water sorption, more extensible and resistant than neat starch films, while they maintained the high oxygen barrier and provided stability to the matrix during ageing. In this sense, S-PVA ratios near to 1:1 are recommendable. When rice bran with the smallest particle size was used as filler, it improved the elastic modulus of the films, but reduced the film stretchability and barrier properties, due to the enhancement of the water binding capacity and the introduction of discontinuities in the matrix. Incorporation of CNCs into S-PVA films, as a reinforcing material, enhanced phase separation of polymers, did not imply changes in water vapour barrier of the films but improved the film mechanical performance: they became stiffer and more stretchable, while crystallization of PVA was partially inhibited. Silver loaded S-PVA films exhibited antimicrobial activity against the fungi (Aspergillus niger and Penicillium expansum) and bacteria (Listeria innocua and Escherichia coli) genera, depending on the silver concentration. Silver nanoparticles provoked notable changes in the film colour and transparency but no relevant changes in the other physical properties. Silver was completely delivered to aqueous simulants within the firsts 60 minutes of contact, but the film release capacity drastically decreased in the non-polar simulant, where the overall migration limit for food contact packaging materials (60 mg/Kg simulant) was accomplished. So, the use of the developed films as food packaging materials should be restricted to fat-rich foodstuffs. The incorporation of oregano essential oil (O), as antimicrobial agents, into the S-PVA matrix gave rise to active films against bacteria and fungi, whereas films containing neem oil (N) were not effective. Higher O concentration in the films was required to observe antifungal effect with respect to the antibacterial activity. Oils did not notably affect water sensitivity and water barrier properties of the films, but at high ratio, oils implied weaker film networks, affecting their mechanical performance. Blend of starch with PVA significantly improved PVA biodegradation and disintegration behaviour. Incorporation of silver species to S-PVA films slowed down their biodegradation kinetics while increased their degradation ratio. However, no significant effect of O was observed on the biodegradability indices despite the antimicrobial activity detected for this oil. N even promoted biodegradation of S-PVA films. Finally, a food application of biopolymer coatings was studied, using chitosan and essential oils (oregano or rosemary) to prevent weight loss and fungal development of semi-hard goat's milk cheeses. Coating were highly effective at reducing fungal growth and cheese weight loss while slightly reduced lipolytic and proteolytic activities in the cheese. Sensory evaluation revealed that the cheeses double coated with chitosan-oregano oil were the best evaluated in terms of aroma and flavour.
[ES] En la Tesis Doctoral, se han analizado diferentes estrategias para mejorar las propiedades funcionales de películas de almidón para aplicaciones de envasado de alimentos: el estudio del ratio amilosa:amilopectina, mezclas con otros polímeros (alcohol de polivinilo-PVA), y la incorporación de diferentes refuerzos (salvado de arroz y nanocristales de celulose-CNCs) y agentes antimicrobianos (aceite de neem-N, aceite esencial de orégano-O y nanopartículas de plata-AgNPs). También se realizó un estudio de biodegradación de las películas observando el efecto de los antimicrobianos. Entre los diferentes almidones con distinto ratio amilosa:amilopectina, se seleccionó el almidón de guisante con altos valores de amilosa. El alto contenido en amilosa dio lugar a películas más rígidas y resistentes a la fractura, con baja permeabilidad al oxígeno, la cual disminuyó durante el almacenamiento. Las películas de mezcla de almidón-PVA (S-PVA) representaron una buena estrategia para mejorar las propiedades de las películas sin incrementar notablemente el precio. La adición de PVA dio lugar a películas menos solubles en agua, menos sensibles a su absorción y más extensible y resistentes que las de almidón puro, mientras mantuvieron alta barrera al oxígeno y dieron estabilidad a la matriz durante el envejecimiento. Son recomendables los ratios de S-PVA cercanos a 1:1. El salvado de arroz con menor tamaño de partícula, mejoró el modulo elástico de las películas, pero su extensibilidad y propiedades barrera empeoraron debido a la mejor capacidad de retención de agua y a las discontinuidades introducidas. La incorporación de CNCs en las películas de S-PVA incrementó la separación de fases de los polímeros, sin cambios en la permeabilidad al vapor de agua, y mejoró las prestaciones mecánicas: películas más rígidas y extensibles, mientras que inhibió parcialmente la cristalización del PVA. Las películas de S-PVA con partículas de plata exhibieron actividad antimicrobiana contra dos hongos y dos bacterias, dependiendo de la concentración de plata. Las AgNPs provocaron cambios en el color de las películas así como en su transparencia. La plata fue completamente liberada en los primeros 60 minutos en contacto con simulantes acuosos, sin embargo la liberación disminuyó en simulantes no polares, donde se cumple el límite de migración global. Por lo tanto, el uso de las películas desarrolladas para envasado de alimentos debe ser restringido a productos alimenticios ricos en grasas. La incorporación de O en la matriz de S-PVA dio lugar a películas activas contra bacterias y hongos, mientras que las películas con N no fueron efectivas. Se necesitaron concentraciones más altas de O en las películas para observar un efecto antifúngico respecto a la actividad bactericida. Los aceites no afectaron notablemente la sensibilidad ni las propiedades barreras al agua de las películas, aunque la mayor proporción de aceite dio lugar a películas con una red más débil, afectando sus prestaciones mecánicas. La mezcla de S con PVA mejoró significativamente el comportamiento de biodegradación y desintegración de las películas. La incorporación de AgNPs a películas de S-PVA disminuyó su cinética de biodegradación mientras aumentó su ratio de degradación. La adición de O no presentó efecto significativo en el índice de biodegradación a pesar de la actividad antimicrobiana detectada. El N incluso mejoró la biodegradación de las películas. Finalmente, se realizó una aplicación de recubrimientos basados en biopolímeros, usando quitosano-CH y aceite esencial de orégano o romero para evitar la pérdida de peso y el desarrollo de hongos en quesos de cabra semicurados. Los recubrimientos fueron efectivos en la reducción del crecimiento fúngico y la pérdida de peso de los quesos, mientras que la actividad lipolítica y proteolítica ligeramente disminuyó. El análisis sensorial reveló q
[CAT] S'han analitzat diferents estratègies per a millorar les propietats funcionals de pel·lícules de midó per a aplicacions d'envasat d'aliments: l'estudi de la ràtio amilosa:amilopectina, mescles amb altres polímers (alcohol polivinílic-PVA), i la incorporació de diferents reforços (segó d'arròs i nanocristalls de celulosa-CNCs) i agents antimicrobians (oli de neem-N, oli essencial d'orenga-O i nanopartícules de plata). També es va fer un estudi de biodegradació de les pel·lícules per observar l'efecte dels antimicrobians. Entre els diferents midons amb distint ràtio amilosa:amilopectina, es va seleccionar el midó de pèsol amb alts valors d'amilosa. L'alt contingut en amilosa va donar lloc a pel·lícules més rígides i resistents a la fractura, amb baixa permeabilitat a l'oxigen, la qual va disminuir durant l'emmagatzemament. Les pel·lícules de mescla de S-PVA van representar una bona estratègia per a millorar les propietats de les pel·lícules sense incrementar notablement el preu. L'addició de PVA va donar lloc a pel·lícules menys solubles en aigua, menys sensibles a la seua absorció i més extensible i resistents que les de midó pur. A més van mantenir l'alta barrera a l'oxigen i van donar estabilitat a la matriu durant l'envelliment. Son recomanables les ràtios de S- PVA pròxims a 1:1. El segó d'arròs amb menor tamany de partícula, va millorar el mòdul elàstic de les pel·lícules, però la seua extensibilitat i propietats barrera van empitjorar. La incorporació de CNCs en les pel·lícules de S-PVA, va incrementar la separació de fases dels polímers, sense implicar canvis en la permeabilitat al vapor d'aigua, però millorant les prestacions mecàniques: pel·lícules més rígides i extensibles, al mateix temps que es va inhibir parcialment la cristal·lització del PVA. Les pel·lícules de S-PVA amb partícules de plata van exhibir activitat antimicrobiana front a dos de fongs i dos bacteris, depenent de la concentració de plata. Les AgNPs van provocar canvis en el color de les pel·lícules així com en la seua transparència. La plata va ser completament alliberada en els primers 60 minuts en contacte amb simulants aquosos. La capacitat d'alliberament va disminuir en simulants no polars, on es complix el límit de migració global. Per tant, l'ús de les pel·lícules desenvolupades per a l'envasat d'aliments ha de ser restringit a productes alimentaris rics en greixos. La incorporació d'O en la matriu de S-PVA va donar lloc a pel·lícules actives front a bacteris i fongs. Pel contrari les pel·lícules amb N no van ser efectives. Van ser necessàries concentracions més elevades d'O en les pel·lícules per a observar un efecte antifúngic respecte a l'activitat bactericida. Els olis no van afectar la sensibilitat a l'aigua ni les propietats barrera a l'aigua de les pel·lícules, però la major proporció d'oli va donar lloc a pel·lícules amb una xarxa més dèbil, afectant les seues prestacions mecàniques. La mescla de S amb PVA va millorar significativament el comportament de biodegradació i desintegració. La incorporació de partícules de plata a pel·lícules de S-PVA va disminuir la seua cinètica de biodegradació mentre que va augmentar la seua ràtio de degradació. No obstant això, l'addició d'O no va presentar un efecte significatiu en els índexs de biodegradació a pesar de l'activitat antimicrobiana detectada. L'N fins i tot va millorar la biodegradació de les pel·lícules de S-PVA. Finalment, es va realitzar una aplicació de recobriments basats en biopolímers, fent ús de quistosà-CH i olis essencials de orenga i romer per evitar la pèrdua de pes i el desenvolupament de fongs en formatges de cabra semi-curats. Els recobriments van ser altament efectius en la reducció del creixement fúngic i la pèrdua de pes dels formatges. L'activitat lipolítica i proteolítica en els formatges va disminuir suaument. L'anàlisi sensorial va revelar que els form
Cano Embuena, AI. (2015). Different strategies to improve the functionality of biodegradable films based on starch and other polymers [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/55383
TESIS
Premiado

O presente trabalho apresenta a síntese e a caracterização de derivados de quitosana, bem como o preparo e caracterização de filmes de nanocompósitos à base de quitosana comercial (ou seus derivados), argila (MMT) e nanopartículas de prata (NPs-Ag), obtidas via Fotoquímica. Para tanto, foram preparados, a partir da quitosana comercial (QC), os derivados: quitosana desacetilada (Q30des), quitosana purificada (QP), quitosanas parcialmente despolimerizas (QD30, QD21 e QD5), quitosanas hidrofílicas (QD21-40DEAE e QD5-49DEAE) e quitosanas anfifílicas (QD21-40DEAE-6DD, QD21-40DEAE-18DD, QD5-49DEAE-6DD e QD5-49DEAE-17DD). O grau médio de desacetilação das QC, QP e Q30des e de substituição por grupos DEAE e dodecila foram determinados por Espectroscopia de Ressonância Magnética Nuclear de Hidrogênio (RMN de 1H). Ademais, os biopolímeros foram caracterizados por Espectroscopia no Infravermelho (FTIR-ATR), Viscosimetria, Análise Termogravimétrica e Microscopia Eletrônica de Varredura (MEV). Em seguida, foi estudada a síntese de nanopartículas de prata, sob radiação UV, em filmes de nanocompósitos de quitosana comercial ou seus derivados e argila. Em um primeiro momento, estudou-se a formação das NPs-Ag em filmes de QC com diferentes formulações e posteriormente em filmes de derivados de quitosana contendo 10% de MMT (m/m). A técnica de Difração de Raios-X (DRX) foi utilizada para a determinação do espaçamento interlamelar da argila montmorilonita pura e nos compósitos preparados. A síntese das NPs-Ag foi acompanhada por Espectrofotometria de Absorção Molecular no UV-vis, e monitorada após um ano de sua formação, sendo suas características morfológicas, bem como a dispersão da argila nos nanocompósitos examinados por Microscopia Eletrônica de Transmissão (MET). Por fim, a atividade antimicrobiana dos filmes de nanocompósitos foi avaliada pelo método de Disco de Difusão contra as bactérias Escherichia coli e Bacillus subtilis.
The present work presents the synthesis and characterization of chitosan derivatives, as well as the preparation and characterization of nanocomposite films based on commercial chitosan (or its derivatives), clay (MMT) and silver nanoparticles (NPs-Ag) obtained by photochemical method. Therefore, were prepared from commercial chitosan (QC): deacetylated chitosan (Q30des); purified chitosan (QP); partially depolymerized chitosans (QD30, QD21 and QD5); hydrophilic chitosans (QD21-40DEAE and QD5-QD5) and amphiphilic chitosans (QD21-40DEAE-6DD, QD21-40DEAE-18DD, QD5-49DEAE-6DD and QD5-49DEAE-17DD). The deacetylation degrees of QC, QP and Q30des were determined by Nuclear Magnetic Resonance Spectroscopy (1H-NMR). This technique also used to determine the degrees of substitution by DEAE and dodecyl groups. In addition, the biopolymers were characterized by Infrared Spectroscopy (FTIR-ATR), Viscosimetry, Thermogravimetry and Scanning Electron Microscopy. Moreover, the NPs-Ag synthesis under UV radiation was studied on nanocomposite films of commercial chitosan or its derivatives and clay. At first, the Ag-NPs formation was studied on QC films with different formulations and secondarily, on films of chitosan derivatives containing 10 wt % of MMT. The X-Ray Diffraction (XRD) was used to determine the interlamellar spacing of pure montmorillonite clay and in the nanocomposites prepared. The synthesis of the NP-Ag was accompanied by UV-vis Spectroscopy. Its morphological characteristics, as well as the clay dispersion in the nanocomposites were examined by Electron Transmission Electron Microscopy (TEM). Finally, the antimicrobial activities of materials were investigated by the disk diffusion method against the bacteria Escherichia coli e Bacillus subtilis.

The study of materials composed from the combination of chitosan and silver nanoparticles has attracted the interest of researchers from different areas due to large variety of applications of these systems, such as the development of electro-optical devices and the disease treatment like cancer. Despite the existence of several works devoted to the characterization of the biological activity of these materials, many physical properties have not been explored so far. In this work, we study thermo-optical, interfacial and morphological properties of fluids and lms based on chitosan, by considering the effects associated with the addition of silver nanoparticles. Using a large variety of experimental techniques, it is investigated how the concentration of silver nanoparticles modifies the heat diffusion, fluid adhesion and photodegradation process in chitosan samples. From the time-resolved z-scan measurements in solution of chitosan and silver nanoparticles, it is studied how the heat transport induced by the visible radiation is modifed by the insertion of the metallic nanoparticles. Our results show that thermo-optical coefficient and thermal diffusivity are sensible to the variation of colloids concentration. With respect to morphological properties of chitosan film doped with silver nanoparticles, it is observed that the introduction of guest particles tends to modify the superficial structure of the films, with the formation large clusters of nanoparticles. In relation to interfacial properties associated with wetting phenomenom, it is observed a non-monotonic behavior of the contact angle of castor oil droplets as the silver nanoparticles concentration is increased. The parameters related to wetting phenomenon are computed from the analysis of time-evolution of the droplet spreading, by using a molecular-kinetic theory. Further, the effects of silver concentration on the photodegradation of chitosan films are examined, when the films are exposed to the electromagnetic radiation in the visible and ultraviolet regions. The present results reveal that the photodegradation process of the films depends on the radiation wavelength and the used excitation regime.
O estudo de materiais formados pela associação da quitosana a nanopartículas de prata tem atraído a atenção de pesquisadores de diferentes áreas devido à grande variedade de aplicações destes sistemas, tais como a fabricação de dispositivos eletro-ópticos e o tratamento de doenças como câncer. Apesar da existência de inúmeros trabalhos voltados à caracterização da atividade biológica destes materiais, diversas propriedades físicas ainda não foram devidamente caracterizadas. Neste trabalho, estudaremos as propriedades termo-ópticas, interfaciais e morfológicas de fluidos e filmes baseados em quitosana, considerando os efeitos associados à adição de nanopartículas de prata. Usando uma grande variedade de técnicas experimentais, é investigado como a concentração de nanopartículas de prata altera os processos de difusão de calor, de adesão à fluidos e de fotodegradação nas amostras de quitosana. A partir de medidas de varredura Z resolvida no tempo em soluções de quitosana e nanopartículas de prata, é averiguado como o transporte de calor induzido pela radiação visível incidente é modificado pela inserção da nanopartículas metálicas. Nossos resultado mostram que o coeficiente termo-óptico e a difusidade térmica são sensíveis à variação da concentração de nanopartículas de prata. No que diz respeito às propriedades morfológicas de filmes de quitosana dopada com nanopartículas de prata, é observado que a introdução das partículas hóspedes tende a transformar a estrutura super cial dos lmes, com a formação de grandes aglomerados de nanopartículas. Com relação as propriedades de interfaciais associadas ao fenômeno de molhagem, é observado um comportamento não-monotônico do ângulo de contato de gotículas de óleo de mamona, à medida que a concentração de nanopartículas aumenta. Os parâmetros associados ao fenômeno de molhagem são calculados a partir da análise da evolução temporal do espalhamento das gotas, usando a teoria cinética-molecular. Além disso, os efeitos da concentração de nanopartículas sobre a fotodegradação dos filmes de quitosana dopada são verificados, quando submetidos à radiação eletromagnéticas nas regiões do visível e ultravioleta. Os presentes resultados revelam que o processo de fotodegradação dos filmes depende do comprimento de onda e do regime de excitação utilizado.

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A utilização de produtos naturais conjuntamente com nanopartículas de prata tem atraído interesse de pesquisadores, tanto do ponto de vista tecnológico quanto cientifico devido à ação desses produtos no controle de doenças e pragas. O objetivo desse trabalho foi testar produtos naturais (associados ou não a nanoprata) com ação antimicrobiana sobre Bipolaris oryzae, in vitro, e no tratamento de sementes de arroz. Para o estudo dos compostos contendo quitina e própolis adicionadas opcionalmente nanoprata foram utilizados sementes de arroz na cultivar IRGA 424 inoculadas com um isolado de Bipolaris oryzae. Os compostos utilizados são naturais, têm propriedades antiadesivas e atividade antimicrobiana contra microrganismos, possuem propriedades biocompatíveis e não fitotóxicas aos vegetais em geral. O processo de sínteses dos compostos contendo própolis, quitina e nanoprata, foi realizado por meio da aplicação de sonicação para a dispersão e mistura entre as substâncias. Foram testados os produtos no desenvolvimento e crescimento do fungo Bipolaris oryzae em placas de petri e em sementes de arroz infectadas. Os tratamentos foram: quitina, própolis, prata, quitina+própolis, quitina+própolis+nanoprata, quitina+prata, própolis+nanoprata e testemunha (T - sementes sem tratamentos), totalizando neste estudo 10 tratamentos. Logo a eficiência dos produtos e suas soluções foram testados por meio do blotter test (teste de sanidade de sementes), porcentagem de inibição e diâmetro de crescimento do fungo, germinação e comprimento de plântulas das sementes tratadas com os produtos. Os resultados demonstram que os oligômeros de quitosana e os compósitos de quitosana com extrato de própolis e nanopartículas de prata controlam o crescimento do patógeno. A importância deste achado reside no fato de que é um passo em direção ao objetivo de diminuir o uso de fungicidas químicos. A partir de testes de germinação poderá concluir-se que os melhores resultados foi o tratamento com quitosano/prata compósito NPs, com uma taxa de germinação de 91%. Os novos materiais poliméricos são considerados como muito promissores para o controle de Bipolaris oryzae no arroz, tornando-se assim alternativas ecológicas aos fungicidas químicos.
The use of natural products in conjunction with silver nanoparticles has attracted interest of many researches, from both scientific and technological point of view due to their action for disease and pests control. The objective was to test natural products (with or without nanosilver) with antimicrobial action against Bipolaris oryzae, in vitro, and in the treatment of rice seeds. To study the compounds containing chitin and propolis optionally added nanosilver rice seeds were used in IRGA inoculated with an isolated 424 Bipolaris oryzae. The compounds used are natural, have anti-adhesive properties and antimicrobial activity against microorganisms, have biocompatible properties and not phytotoxic to plants in general. The process of synthesis of compounds containing propolis, chitin and nanosilver was performed by applying the sonication for the dispersion and mixing of the substances. Products were tested on the development and growth of Bipolaris oryzae fungus in petri dishes and infected rice seeds. The treatments were: chitin, propolis, silver, chitin + propolis, chitin + Propolis + nanosilver, chitin + silver, propolis + nanosilver and control (T - treatments seeded), totaling 10 treatments in this study. The results were conclusive in showing that chitosan oligomers and the composites of chitosan with propolis extract and silver nanoparticles led to a remarkable reduction in growth of the pathogen. The importance of this finding lies in the fact that it is a step towards the goal of decreasing the use of chemical fungicides in plant pathology. From germination tests it could be concluded that the best results was the treatment with chitosan/silver NPs composite, with a germination rate of 91%. The novel polymeric materials can thus be deemed as very promising for the control of the Bipolaris oryzae of rice and pave the way towards eco-friendly alternatives to chemical fungicides.

The concept of treating an infected wound has been addressed for centuries, long before the knowledge of microbes was known to human. A wide range of attributes such as spider web, seaweed, honey, leaches etc. has been used in the past decades. Also, nobel metals have been used to fight various pathogens of wounds. Historically, silver (Ag) has been used in the field for long but it gained ground as an antiseptic in 1960’s. A wide range of silver wound dressings has been introduced since then. A Silver based dressing functions through the release of silver ions (Ag+) from elemental silver or silver compounds incorporated into the dressing. Silver ions (Ag+) then act as an antimicrobial agent by binding to the walls and DNA of the cells, thus interfering with cell division and replication. The presence of moisture is a prerequisite for Ag+ to be released. For this reason most test methods in the field involve completely wetting of products prior to measuring antimicrobial effects. Chitosan and silver nanoparticles (AgNPs) have shown wound healing properties individually and combination of both of these two may show improvement in wound healing activity. Thus, the composite films prepared in this project were evaluated for various in vitro evaluation tests. The AgNPs films have shown a significant difference in water absorption capacity and antibacterial activity as compared to the blank films. The aim of this project work is to study and investigate properties and characteristics of chitosan-silver nanocomposite film. The chitosan-silver nanocomposite films were prepared by mixing silver nitrate (AgNO3) with distilled water and acetic acid to prepare a 100 ml solution. Different concentrations of AgNO3, viz., 0%, 2%, 3% were prepared in 100ml of aqueous chitosan solution. The different mixtures were then poured into separate petri dishes and kept aside for cooling. A thin layer of Chitosan-silver nanocompositefilm was formed and extracted from the petri dish. These composite films were then characterized by Scanning electron microscopy (SEM), X-ray diffraction (XRD), Universal testing machine (UTM), and Fourier transform infrared (FTIR) spectroscopy techniques. Further, the water absorption and antimicrobial properties of the composite films were subsequently studied and reported in the present work.

博士
義守大學
電機工程學系
103
Silver nanoparticles (AgNPs) have been widely applied in various fields including nanotechnology, biotechnology, and medical industry. In order to reduce the AgNPs potential toxicity for human body, various synthesis strategies have been developed in recent years. The stability and distribution characteristics of the synthesized AgNPs are two crucial parameters in synthetic techniques. In our study, we established two synthetic systems to fabricate AgNPs-embedded chitosan particles with different size scales. In the first system, a novel approach for the one-step synthesis of AgNPs-embedded chitosan particles was proposed by using the pump-driven syringe method. The method was planned to simultaneously obtain and stabilize AgNPs in chitosan polymer matrix in situ, and the product was called silver nanoparticles-chitosan composite particles (Ag@chitosan). The diameters of the synthesized Ag@chitosan spheres ranged from 1.7 to 2.5 mm, and those of embedded AgNPs were measured to be 15±3.3 nm. Furthermore, we utilized various instruments to analyze the characteristics of the prepared Ag@chitosan spheres including the ultraviolet-visible absorbance spectrophotometer (UV-Vis), Fourier transform infrared spectrometer (FTIR), X-ray diffractometer (XRD), energy dispersive spectroscopy (EDS), scanning electron microscope (SEM), and transmission electron microscope (TEM). The results showed that the AgNPs were homogeneously distributed over the chitosan sphere, and these synthesized spheres possessed porous structure that could be utilized in multiple fields. Then, the antifungal effect of the Ag@chitosan spheres was evaluated, and it was found that they could inhibit the growth of Cordyceps militaris (Cm) but not Antrodia cinnamomea (Ac). Ac is an important cash crop with highly economic value in Taiwan, and our products might be an option to become a beneficial additive for cultivating Ac by inhibiting other fungi and bacteria. In the second system, we developed the microfluidic chip device made by Polymethyl methacrylate (PMMA) to synthesize the Ag@chitosan spheres with micrometer scale. The diameters of the Ag@chitosan spheres synthesized by the microfluidic method ranged from 262 to 558 μm, and their sizes could be controlled by altering the concentration of chitosan, concentration of NaOH, flow rate of continuous phase, and flow rate of dispersed phase. Moreover, we utilized various instruments to analyze the characteristics of the prepared Ag@chitosan spheres including the UV-Vis spectrophotometer, FTIR spectrometer, EDS, and SEM. The results showed that the AgNPs were also homogeneously distributed and the sphere structure was also porous as expected. In order to assess the possible toxicity of Ag@chitosan for human cells, we executed the MTT assay on both NIH-3T3 and MCF-7 cell lines. The results demonstrated that there was no observable toxicity even if the concentration of Ag@chitosan up to 1000μg/mL. Furthermore, the antibacterial effect of Ag@chitosan was evaluated by using Escherichia coli (E. Coli) broths, and the results showed that E. Coli growth could be inhibited by Ag@chitosan with dose-dependent relationship. Generally speaking, we have established two novel systems to synthesize homogeneous and stable AgNPs embedded in the chitosan particles successfully, and have confirmed the product to be highly biocompatible, non-toxic, antibacterial, and antifungal. Our synthetic systems and the synthesized products will provide a novel option of the antimicrobial research, and may be utilized in medicine and other fields in the future.

碩士
國立成功大學
材料科學及工程學系碩博士班
100
Chitosan, known as sustainable materials with high metal binding activity and potential antibacterial properties is a promising natural polymer for high performance filtration. In case of “environmental concern”, creating an antibacterial material with a “green technology” and is an urgent. Through this research, low pressure plasma is used to synthesize silver nanopaticles in electrospinning chitosan nanofibers. Low pressure plasma offers way to reduce the usage of toxic chemical, time efficiency, and lower temperature processing in compare to other method such as chemical reduction, thermal, and UV- irradiation. This incorporation is expected to increase antibacterial properties of chitosan nanofibers. Chitosan nanofibers had been successfully electrospun on pvc grid from 5.4% w/v of chitosan and 0.6% PEO. Fibers diameter was 136 nm ± 18% without bead structures. AgNO3 addition up to 2% did not significantly change chitosan nanofibers diameter. Plasma treatment reduced Ag+ from AgNO3 precursor into Ag0 so that Ag nanoparticles were created. Besides, plasma also etched chitosan nanofibers so that reduction of fibers diameter is observed. UV- visible spectroscopy and XPS analysis revealed silver intensity will be increasing proportionally with plasma treatment time. XPS analysis also observed interaction between chitosan and Ag nanoparticles (N--Ag). Furthermore, TEM evaluation showed particle size average after 1.5 minutes plasma treatment was 1.5 nm in average. By addition of Ag nanoparticles, antibacterial activity of chitosan nanofibers could be enhanced.

碩士
明志科技大學
化學工程研究所
101
The carboxymethyl chitosan is an amphiprotic ether derivative, which contains carboxyl, hydroxyl and amine groups in the molecule, and makes it possible to offer enough chelate groups. The carboxymethyl chitosan has a higher chelation capacity than chitosan, which has potential implications for improved Ag nanoparticle formation and immobilization. By adjusting the concentration of carboxymethyl chitosan, and the reduction temperature and time of silver nanoparticles, the silver nanoparticle solution with optical properties could be synthesized. Observations through a UV-Vis absorption spectrometer discovered that the Ag nanoparticle solution had a surface plasmon absorption peak of 400 nm. Under optimal conditions, transmission electron microscopy analysis showed that the size of Ag nanoparticles was in the range from 5 to 10 nm, and the uniform dispersion. The synthesized Ag nanoparticle solution was stable; its optical properties do not change over time. When adding solutions of different metal ions (Pb2+, Ni2+, Cr3+, Co2+, Cu2+, Cd2+, Hg2+) to the silver nanoparticle solution, observations discovered that only Hg2+ions would cause the surface plasmon absorption peak of the silver nanoparticles to displace and decrease. It was possible to use these changes in optical properties for the detection of mercury ions (Hg2+).

碩士
亞東技術學院
應用科技研究所
100
The research prepared silver nanoparticles by NaBH4 chemical reduction method, and it were added to blending hydrogel by chitosan (Ch)/carboxymethyl cellulose (CMC)：(ChxCy) to prepare GCwChxCyAgz polymer solutions and membranes at last by adjusting pH and concentration of silver nanoparticles and genepin, and analyzed with UV-Vis, FTIR-ATR spectrum, DSC, mechanical properties, SEM morphology and antibacterial test. UV-Vis spectrums reveal that CMCAg3 stabilizer works on dispersibility of silver nanoparticles and better than ChAg3 stabilizer. Besides, polymer solution of low concentration of (Ch5C1)0.25Ag3 shows supreme absorbance (1.91). Ionic strength decreases with concentration of ionic stabilizer, but dispersibility of silver nanaparticles increases and obtains miniaturization. FTIR-ATR spectrums show that the highest hydrogen bond strength of 1.0wt%Ch1C1 membrane, it provides with approximate hydrogen bond sites. The sample of ChxCyAgz membrane displays absorption (1595cm-1) of COO- of CMC and NH3+ of chitosan ,and both rising substantially. Ag+ destroyed the ionic bond between COO- and NH3+ by electrostatic attraction and valence electrons of O, N. In addition, because of silver nanoparticles were added to Ch5C1 and Ch1C1 membrane, it resulting intercalation reaction with CMC and Ch sites of matrices, hydrogen bond strength descends sharply. Blend hydrogel cross-linking by genipin shows that tertiary amine (1262 cm-1 and 1735 cm-1) of six-ring diene three nitrogen amine absorption peaks and of C=O of ester group raises, would get membrans in blue. DSC thermal characterizations show that Tm and enthalpy of blend hydrogen membrane in first amd secondary heating that it’s higher than both of CMC and Ch, it means hydrogel blends well and make new interaction, and Ch1C1 membrane is the best. 0.3mM of Ag added to blend hydrogel that its sites were occupied and ionic bond attraction were destruct. Mechanical property tests suggest that (Ch1C1)0.1 membrane has the highest tensile strength (59.37 MPa) and elongation (36%). Young’s modulus ,tensile strength and elongation of 1.0wt% (Ch1C1)Ag3 membrane which is still better than others after addition of silver nanoparticles. But young’s modulus and tensile strength of 1.0wt%Ch3C1Ag2 and 1.0wt%Ch5C1Ag2 membranes enhance obviously, it’s similar to 0.5wt%Ch5C1Ag2 membrane, and it obtained the highest tensile strength (85.24MPa). Tensile strength of blend cross-linking hydrogel will increase with concentration of chitosan and cross-linking agent, but elongation is contrary. SEM observations show that irregular surface of CMC and many particles spreaded on it that it is cause of weak acid. CMC surface attracted and covered with silver nanoparticles by electrostatic attraction or chelating that it made many raised globes, otherwise cross-linking CMC surface was pocked that it is cause of bond strength shortened the distance. Antibacterial test results show that inhibition rate of Staphylococcus aureus increases with concentration of chitosan and time. In conclusion, the inhibition rate is about 96.09-100% when contacted with Staphylococcus aureus for 6 hours.

碩士
國立成功大學
材料科學及工程學系
104
Plasma-synthesized silver nanoparticles (Ag NPs) were immobilized on the surface of chitosan-based nanofibers fabricated by two kinds of dual-supplied electrospinning processes. In the first fabrication approach, the dual-supplied electrospinning was conducted by two flows that carried the chitosan and the crosslinking agent separately. The two flows were mixed right before the ejection of the electrospinning solutions. The second approach involved the use of a lab-made co-axial nozzle that constructed the chitosan nanofiber cores and the poly(ethylene oxide) (PEO) outer sheath which also carried the crosslinking agent and the silver precursor. Electrospinning processes were carefully investigated in this research project. The resulting chitosan nanofibers with an average fiber diameter as small as about 135 nm were then treated with the argon plasma bombardment. Silver precursors, the silver nitrate, within the chitosan nanofibers were converted to metallic Ag NPs, which were immobilized on the surface of these nanofibers. Water resistance confirmed these robust chitosan nanofibers, as well as the stabilities of surface-immobilized Ag NPs. Following antibacterial tests based on the water filtration apparatus were investigated. And the antibacterial performance of these electrospun chitosan nanofibers were proportionally enhanced by the population of the surface-immobilized Ag NPs.

Chapter I: This chapter briefly gives an introduction about microorganisms, their varieties, growth, reproduction etc. In particular, about bacterial function. A sincere attempt is made to review this briefly, including an account of the studies already reported in the literature. Chapter II deals with the antimicrobial activity of Ag/agar film on Escherichia coli (E. coli), Staphylococcus aureus (S. aureus), and Candida albicans (C. albicans). In particular, films were repeatedly cycled for its antimicrobial activity. The antimicrobial activity of Ag/agar film was found to be in the order, C. albicans > E. coli > S. aureus. Chapter III describes the synthesis of Ag@AgI NPs in agarose matrix. A detailed antibacterial studies including repetitive cycles are carried out on E. coli and S. aureus bacteria. EPR and TEM studies are carried out on the Ag@AgI/Agarose and the bacteria, respectively, to elucidate a possible mechanism for killing of the bacteria. The hybrid could be recycled for the antibacterial activity many times and is found to be non toxic towards human cervical cancer cell (HeLa cells). Chapter IV reports the antibacterial efficacy of silver nanoparticles (Ag NPs) deposited alternatively layer by layer (LBL) on chitosan polymer in the form of a thin film over a quartz plate and stainless steel strip against E. coli. AFM studies are carried out on the microbe to know the morphological changes affected by the hybrid film. The hybrid films on aging (3 months) are found to be as bioactive as before. Cytotoxicity experiments indicated good biocompatibility. Chapter V describes the fabrication of carbon foam porous electrode modified with reduced graphene oxide-Ag nanocomposites. The device can perform sterilization by killing pathogenic microbes with the aid of just one 1.5V battery with very little power consumption. Chapter VI Here we have studied in particular a property say the influence of surface defect in the production of ROS by ZnO NPs and in turn the bactericide activity. Secondly, a homogeneous ZnO and ZnO/Ag nanohybrid has been synthesized by employing chitosan as mediator. The synergistic antibacterial effect of ZnO/Ag nanohybrid on bacteria is found to be more effective, compared to the individual components (ZnO and Ag). A possible mechanism has been proposed for the death of bacteria by ZnO/Ag nanohybrid, based on EPR studies and TEM studies.

Chapter I: This chapter briefly gives an introduction about microorganisms, their varieties, growth, reproduction etc. In particular, about bacterial function. A sincere attempt is made to review this briefly, including an account of the studies already reported in the literature. Chapter II deals with the antimicrobial activity of Ag/agar film on Escherichia coli (E. coli), Staphylococcus aureus (S. aureus), and Candida albicans (C. albicans). In particular, films were repeatedly cycled for its antimicrobial activity. The antimicrobial activity of Ag/agar film was found to be in the order, C. albicans > E. coli > S. aureus. Chapter III describes the synthesis of Ag@AgI NPs in agarose matrix. A detailed antibacterial studies including repetitive cycles are carried out on E. coli and S. aureus bacteria. EPR and TEM studies are carried out on the Ag@AgI/Agarose and the bacteria, respectively, to elucidate a possible mechanism for killing of the bacteria. The hybrid could be recycled for the antibacterial activity many times and is found to be non toxic towards human cervical cancer cell (HeLa cells). Chapter IV reports the antibacterial efficacy of silver nanoparticles (Ag NPs) deposited alternatively layer by layer (LBL) on chitosan polymer in the form of a thin film over a quartz plate and stainless steel strip against E. coli. AFM studies are carried out on the microbe to know the morphological changes affected by the hybrid film. The hybrid films on aging (3 months) are found to be as bioactive as before. Cytotoxicity experiments indicated good biocompatibility. Chapter V describes the fabrication of carbon foam porous electrode modified with reduced graphene oxide-Ag nanocomposites. The device can perform sterilization by killing pathogenic microbes with the aid of just one 1.5V battery with very little power consumption. Chapter VI Here we have studied in particular a property say the influence of surface defect in the production of ROS by ZnO NPs and in turn the bactericide activity. Secondly, a homogeneous ZnO and ZnO/Ag nanohybrid has been synthesized by employing chitosan as mediator. The synergistic antibacterial effect of ZnO/Ag nanohybrid on bacteria is found to be more effective, compared to the individual components (ZnO and Ag). A possible mechanism has been proposed for the death of bacteria by ZnO/Ag nanohybrid, based on EPR studies and TEM studies.

Chapter–I Introduction Shock waves appear in nature whenever the different elements in a fluid approach one another with a velocity larger than the local speed of sound. Shock waves are essentially non-linear waves that propagate at supersonic speeds. Such disturbances occur in steady transonic or supersonic flows, during explosions, earthquakes, tsunamis, lightening strokes and contact surfaces in laboratory devices. Any sudden release of energy (within few μs) will invariably result in the formation of shock wave since it is one of the efficient mechanisms of energy dissipation observed in nature. The dissipation of mechanical, nuclear, chemical, and electrical energy in a limited space will result in the formation of a shock wave. However, it is possible to generate micro-shock waves in laboratory using different methods including controlled explosions. One of the unique features of shock wave propagation in any medium (solid, liquid or gases) is their ability to instantaneously enhance pressure and temperature of the medium. Shock waves have been successfully used for disintegrating kidney stones, non-invasive angiogenic therapy and osteoporosis treatment. In this study, we have generated a novel method to produce micro-shock waves using micro-explosions. Different biological applications were developed by further exploring the physical properties of shock waves. Chapter – II Bacterial transformation using micro-shock waves In bacteria, uptake of DNA occurs naturally by transformation, transduction and conjugation. The most widely used methods for artificial bacterial transformation are procedures based on CaCl2 treatment and electroporation. In this chapter, controlled micro-shock waves were harnessed to develop a unique bacterial transformation method. The conditions have been optimized for the maximum transformation efficiency in E. coli. The highest transformation efficiency achieved (1 × 10-5 transformants per cell) was at least 10 times greater than the previously reported ultrasound mediated transformation (1 × 10-6 transformants per cell). This method has also been successfully employed for the efficient and reproducible transformation of Pseudomonas aeruginosa and Salmonella Typhimurium. This novel method of transformation has been shown to be as efficient as electroporation with the added advantage of better recovery of cells, economical (40 times cheaper than commercial electroporator) and growth-phase independent transformation. Chapter – III Needle-less vaccine delivery using micro-shock waves Utilizing the instantaneous mechanical impulse generated behind the micro-shock wave during controlled explosion, a novel non-intrusive needleless vaccine delivery system has been developed. It is well established, that antigens in the epidermis are efficiently presented by resident Langerhans cells, eliciting the requisite immune response, making them a good target for vaccine delivery. Unfortunately, needle free devices for epidermal delivery have inherent problems from the perspective of patient safety and comfort. The penetration depth of less than 100 µm in the skin can elicit higher immune response without any pain. Here the efficient utilization of the device for micro-shock wave mediated vaccination was demonstrated. Salmonella enterica serovar Typhimurium vaccine strain pmrG-HM-D (DV-STM-07) was delivered using our device in the murine salmonellosis model and the effectiveness of the delivery system for vaccination was compared with other routes of vaccination. The device mediated vaccination elicits better protection as well as IgG response even in lower vaccine dose (ten-fold lesser), compare to other routes of vaccination. Chapter – IV In vitro and in vivo biofilm disruption using shock waves Many of the bacteria secrete highly hydrated framework of extracellular polymer matrix on encountering suitable substrates and get embedded within the matrix to form biofilm. Bacterial colonization in biofilm form is observed in most of the medical devices as well as during infections. Since these bacteria are protected by the polymeric matrix, antibiotic concentration of more than 1000 times of the MIC is required to treat these infections. Active research is being undertaken to develop antibacterial coated medical implants to prevent the formation of biofilm. Here, a novel strategy to treat biofilm colonization in medical devices and infectious conditions by employing shock waves was developed. Micro-shock waves assisted disintegration of Salmonella, Pseudomonas and Staphylococcus biofilm in urinary catheters was demonstrated. The biofilm treated with micro-shock waves became susceptible to antibiotics, whereas the untreated was resistant. Apart from medical devices, the study was extended to Pseudomonas lung infection model in mice. Mice exposed to shock waves responded well to ciprofloxacin while ciprofloxacin alone could not rescue the mice from infection. All the mice survived when antibiotic treatment was provided along with shock wave exposure. These results clearly demonstrate that shock waves can be used along with antibiotic treatment to tackle chronic conditions resulting from biofilm formation in medical devices as well as biological infections. Chapter – V Shock wave responsive drug delivery system for therapeutic application Different systems have been used for more efficient drug delivery as well as targeted delivery. Responsive drug delivery systems have also been developed where different stimuli (pH, temperature, ultrasound etc.) are used to trigger the drug release. In this study, a novel drug delivery system which responds to shock waves was developed. Spermidine and dextran sulfate was used to develop the microcapsules using layer by layer method. Ciprofloxacin was loaded in the capsules and we have used shock waves to release the drug. Only 10% of the drug was released in 24 h at pH 7.4, whereas 20% of the drug was released immediately after the particles were exposed to shock waves. Almost 90% of the drug release was observed when the particles were exposed to shock waves 5 times. Since shock waves can be used to induce angiogenesis and wound healing, Staphylococcus aureus skin infection model was used to show the effectiveness of the delivery system. The results show that shock wave can be used to trigger the drug release and can be used to treat the wound effectively. A brief summary of the studies that does not directly deal with the biological applications of shock waves are included in the Appendix. Different drug delivery systems were developed to check their effect in Salmonella infection as well as cancer. It was shown for the first time that silver nanoparticles interact with serum proteins and hence the antimicrobial properties are affected. In a nutshell, the potential of shock waves was harnessed to develop novel experimental tools/technologies that transcend the traditional boundaries of basic science and engineering.

Chapter–I Introduction Shock waves appear in nature whenever the different elements in a fluid approach one another with a velocity larger than the local speed of sound. Shock waves are essentially non-linear waves that propagate at supersonic speeds. Such disturbances occur in steady transonic or supersonic flows, during explosions, earthquakes, tsunamis, lightening strokes and contact surfaces in laboratory devices. Any sudden release of energy (within few μs) will invariably result in the formation of shock wave since it is one of the efficient mechanisms of energy dissipation observed in nature. The dissipation of mechanical, nuclear, chemical, and electrical energy in a limited space will result in the formation of a shock wave. However, it is possible to generate micro-shock waves in laboratory using different methods including controlled explosions. One of the unique features of shock wave propagation in any medium (solid, liquid or gases) is their ability to instantaneously enhance pressure and temperature of the medium. Shock waves have been successfully used for disintegrating kidney stones, non-invasive angiogenic therapy and osteoporosis treatment. In this study, we have generated a novel method to produce micro-shock waves using micro-explosions. Different biological applications were developed by further exploring the physical properties of shock waves. Chapter – II Bacterial transformation using micro-shock waves In bacteria, uptake of DNA occurs naturally by transformation, transduction and conjugation. The most widely used methods for artificial bacterial transformation are procedures based on CaCl2 treatment and electroporation. In this chapter, controlled micro-shock waves were harnessed to develop a unique bacterial transformation method. The conditions have been optimized for the maximum transformation efficiency in E. coli. The highest transformation efficiency achieved (1 × 10-5 transformants per cell) was at least 10 times greater than the previously reported ultrasound mediated transformation (1 × 10-6 transformants per cell). This method has also been successfully employed for the efficient and reproducible transformation of Pseudomonas aeruginosa and Salmonella Typhimurium. This novel method of transformation has been shown to be as efficient as electroporation with the added advantage of better recovery of cells, economical (40 times cheaper than commercial electroporator) and growth-phase independent transformation. Chapter – III Needle-less vaccine delivery using micro-shock waves Utilizing the instantaneous mechanical impulse generated behind the micro-shock wave during controlled explosion, a novel non-intrusive needleless vaccine delivery system has been developed. It is well established, that antigens in the epidermis are efficiently presented by resident Langerhans cells, eliciting the requisite immune response, making them a good target for vaccine delivery. Unfortunately, needle free devices for epidermal delivery have inherent problems from the perspective of patient safety and comfort. The penetration depth of less than 100 µm in the skin can elicit higher immune response without any pain. Here the efficient utilization of the device for micro-shock wave mediated vaccination was demonstrated. Salmonella enterica serovar Typhimurium vaccine strain pmrG-HM-D (DV-STM-07) was delivered using our device in the murine salmonellosis model and the effectiveness of the delivery system for vaccination was compared with other routes of vaccination. The device mediated vaccination elicits better protection as well as IgG response even in lower vaccine dose (ten-fold lesser), compare to other routes of vaccination. Chapter – IV In vitro and in vivo biofilm disruption using shock waves Many of the bacteria secrete highly hydrated framework of extracellular polymer matrix on encountering suitable substrates and get embedded within the matrix to form biofilm. Bacterial colonization in biofilm form is observed in most of the medical devices as well as during infections. Since these bacteria are protected by the polymeric matrix, antibiotic concentration of more than 1000 times of the MIC is required to treat these infections. Active research is being undertaken to develop antibacterial coated medical implants to prevent the formation of biofilm. Here, a novel strategy to treat biofilm colonization in medical devices and infectious conditions by employing shock waves was developed. Micro-shock waves assisted disintegration of Salmonella, Pseudomonas and Staphylococcus biofilm in urinary catheters was demonstrated. The biofilm treated with micro-shock waves became susceptible to antibiotics, whereas the untreated was resistant. Apart from medical devices, the study was extended to Pseudomonas lung infection model in mice. Mice exposed to shock waves responded well to ciprofloxacin while ciprofloxacin alone could not rescue the mice from infection. All the mice survived when antibiotic treatment was provided along with shock wave exposure. These results clearly demonstrate that shock waves can be used along with antibiotic treatment to tackle chronic conditions resulting from biofilm formation in medical devices as well as biological infections. Chapter – V Shock wave responsive drug delivery system for therapeutic application Different systems have been used for more efficient drug delivery as well as targeted delivery. Responsive drug delivery systems have also been developed where different stimuli (pH, temperature, ultrasound etc.) are used to trigger the drug release. In this study, a novel drug delivery system which responds to shock waves was developed. Spermidine and dextran sulfate was used to develop the microcapsules using layer by layer method. Ciprofloxacin was loaded in the capsules and we have used shock waves to release the drug. Only 10% of the drug was released in 24 h at pH 7.4, whereas 20% of the drug was released immediately after the particles were exposed to shock waves. Almost 90% of the drug release was observed when the particles were exposed to shock waves 5 times. Since shock waves can be used to induce angiogenesis and wound healing, Staphylococcus aureus skin infection model was used to show the effectiveness of the delivery system. The results show that shock wave can be used to trigger the drug release and can be used to treat the wound effectively. A brief summary of the studies that does not directly deal with the biological applications of shock waves are included in the Appendix. Different drug delivery systems were developed to check their effect in Salmonella infection as well as cancer. It was shown for the first time that silver nanoparticles interact with serum proteins and hence the antimicrobial properties are affected. In a nutshell, the potential of shock waves was harnessed to develop novel experimental tools/technologies that transcend the traditional boundaries of basic science and engineering.