A inexistência de um agente terapêutico único satisfatório para o tratamento do melanoma metastático e a potencialidade do quimioterápico 5FU (5-fluorouracil) motivou esta pesquisa, a qual teve por objetivo o desenvolvimento tecnológico de sistemas carreadores micro-/ e nanoestruturados contendo 5FU a fim de aumentar sua eficácia terapêutica e reduzir a toxicidade por meio da administração pulmonar. Duas formulações pulverulentas foram desenvolvidas com polímeros naturais, sulfato de condroitina e hidroxipropil-metil-celulose, denominadas 5FU-MS e 5FU-NS, utilizando as técnicas de aspersão e atomização vibracional piezoelétrica, respectivamente. Ambas as formulações foram avaliadas quanto às características físicas e químicas, perfil toxicológico in vivo (C. elegans e em ratos Wistar), e penetração e biodisponibilidade no tecido pulmonar pela quantificação da fração livre de fármaco por microdiálise pulmonar. A análise físico-química revelou a obtenção de partículas micrométricas para 5FU-MS e submicrométricas para 5FU-NS, com diâmetros médios de partícula de 2,546 ± 0,07 m e 0,652 ± 0,03 m, e fração respirável (FR%) de 55,12 ± 2,98 e 76,84 ± 0,07, respectivamente. Ambas demonstraram características e propriedades adequadas para administração pulmonar, com capacidade de deposição nas porções média e profunda. A toxicidade das formulações avaliada em C. elegans considerou o percentual de morte, desenvolvimento, DL50 e produção de ROS para os nematodos sob tratamento agudo e crônico. Os resultados evidenciaram redução significativa da toxicidade proporcionada pela redução da taxa de morte e maior desenvolvimento dos grupos tratados com as formulações 5FU-MS e 5FU-NS em comparação ao fármaco livre, sugerindo perfis de segurança satisfatórios para administração. Além disso, 5FU-MS revelou-se um agente pró-oxidante, representando um diferencial promissor deste sistema, podendo alcançar maior sensibilização das células tumorais com menores doses. A toxicidade pulmonar aguda foi avaliada pela análise de LDH e proteínas totais no fluido de lavagem bronco-alveolar (BALF) após a administração combinada das formulações 5FU-MS e 5FU-NS para administração como um sistema terapêutico único (5FU-MS/NS), e análise de dano tecidual pulmonar em ratos. Os resultados da análise bioquímica e histológica indicaram o baixo potencial de indução de lesão tecidual a partir da administração pulmonar combinada das formulações, em relação ao fármaco livre. A análise do perfil farmacocinético por microdiálise pulmonar evidenciou o êxito no desenvolvimento dos sistemas carreadores, tornando possível duplicar o t1/2 do 5FU e aumentar significativamente a biodisponibilidade no tecido pulmonar. Os resultados obtidos indicam a eficiência das formulações 5FU-MS e 5FU-NS em alcançar os benefícios terapêuticos do fármaco 5FU com menores doses e maiores intervalos de administração. Este trabalho de tese apresenta uma abordagem promissora na terapia de neoplasias com recorrência de metástase pulmonar. / The absence of a single therapeutic agent suitable for the treatment of metastatic melanoma and the potential of 5FU chemotherapy (5-fluorouracil) motivated this study, which aimed the development of carrier systems based on micro-/ and nanostructures containing 5FU to increase the therapeutic efficacy and reduce toxicity of this drug by pulmonary administration. Two different formulations of dry powders were developed with natural polymers, chondroitin sulfate and hydroxypropyl-methyl-cellulose, denomined 5FU-MS and 5FU-NS, using the spray-drying and vibrational piezoelectric atomization techniques, respectively. Both formulations were evaluated in terms of physico-chemical characteristics, in vivo toxicological behaviors (C. elegans and in Wistar rats), bioavailability and penetration in the lung tissue by quantifying of drug free fraction by lung microdialysis. The physicochemical analysis showed that were obtained as micrometric (5FU-MS) and submicron particles (5FU-NS), with average diameters of particle 2.546 ± 0.07 m and 0.652 ± 0.03 m, and respirable fraction (FR%) of 55.12 ± 2.98 and 76.84 ± 0.07, respectively. Both showed suitable characteristics and properties for pulmonary delivery, with deposition capacity in the middle and deep lung portions. The toxicity of the formulations evaluated in C. elegans considered the death rate, body development, DL50 and production of ROS to nematodes under acute and chronic treatment. The results showed significant reduction of toxicity, reducing the death rate and greater development of the groups treated with 5FU-MS and 5FU-NS formulations compared to the free drug, suggesting satisfactory safety profile for administration. In addition, 5FU-MS proved to be a pro-oxidant agent, representing a promising differential of this system which can achieve greater sensitization of tumoral cells with lower doses. Acute pulmonary toxicity was evaluated by analyzing LDH, and total protein in the bronchoalveolar lavage fluid (BALF) after combined administration of 5FU-MS formulations and 5FU-NS for administration as a single therapeutic system (5FU-MS/NS) and analysis of lung tissue damage in rats. The results of biochemical and histological analysis indicated the low potential to induce tissue damage from the pulmonary administration of combined formulations, compared to free drug. Analysis of the pharmacokinetic profile for pulmonary microdialysis showed the successful development of carrier systems, making it possible to double the t1/2 of 5FU and significantly increase bioavailability in lung tissue. The results indicate the effectiveness of the formulations 5FU-MS and 5FU-NS in achieving the therapeutic benefits of the drug 5FU at lower doses and higher dosing intervals. This thesis work presents a promising approach to cancer therapy with lung metastasis recurrence.

The feasibility of hot melt extrusion (HME) was explored for development of amorphous solid dispersion systems. Controlled release formulations were developed using a cellulose based derivative, AffinisolTMHPMC 100cP and 4M grades. BCS class II drugs ibuprofen and posaconazole were selected due to their difference in glass transition temperature and lipophilicity. This study focused on investigation of the impact the material attributes and process parameters on the critical quality attributes in preparation of amorphous solid dispersions using hot melt extrusion. The critical quality attributes were sub divided into three main attributes of material, process and product. Rheology of ibuprofen-Affinisol 100cP from melt phase to extrudate phase was tracked. A partial factorial design was carried out to investigate the critical parameters affecting HME. For optimisation of 40%IBU-Affinisol 100cP blends, a feed rate of 0.6kg/hr, screw speed of 500rpm and screw configuration with two mixing elements were found to be optimum for single phase extrudates. ATR-FTIR spectroscopy was found to be an indirect technique of choice in predicting the maximum ibuprofen drug load within extrudates. Prediction was based on the prepared extrudates without charging them to stability conditions. An alternative strategy of incorporation of di-carboxylic acids to increase the dissolution of posaconazole-Affinisol 4M blends was investigated. Succinic acid and L- malic acid incorporation was found to increase the dissolution of posaconazole. Although, the extrudates crystallised out quicker than the naïve posaconazole-Affinisol 4M, but free posaconazole formed eutectic and co-crystal with succinic and L-malic acid within extrudates. This lead to an increase in dissolution of the extrudates compared to day 0.

This thesis reports an investigation of hydroxypropylmethylcellulose (HPMC) as a new solid-surface room-temperature phosphorescence (SSRTP) sample matrix. The high background phosphorescence originating from filter paper substrates can interfere with the detection and quantitation of trace-level analytes. High-purity grades of HPMC were investigated as SSRTP substrates in an attempt to overcome this limitation. When compared directly to filter paper, HPMC allows the spectroscopist to achieve greater sensitivity, lower limits of detection (LOD), and lower limits of quantitation (LOQ) for certain phosphor/heavy-atom combinations since SSRTP signal intensities are stronger. For example, the determination of the analytical figures of merit for a naphthalene/sodium iodide/HPMC system resulted in a calibration sensitivity of 2.79, LOD of 4 ppm (3 ng), and LOQ of 14 ppm (11 ng). Corresponding investigations of a naphthalene/sodium iodide/filter paper system produced a calibration sensitivity of 0.326, LOD of 33 ppm (26 ng), and LOQ of 109 ppm (86 ng). Extended purging with dry-nitrogen gas yields improved sensitivities, lower LOD's, and lower LOQ's in HPMC matrices when LOD and LOQ are calculated according to the IUPAC guidelines.To test the universality of HPMC, qualitative SSRTP spectra were obtained for a wide variety of probe phosphors offering different molecular sizes, shapes, and chemical functionalities. Suitable spectra were obtained for the following model polycyclic aromatic hydrocarbons (PAHs): naphthalene, p-aminobenzoic acid, acenaphthene, phenanthrene, 2-naphthoic acid, 2-naphthol, salicylic acid, and triphenylene.Filter paper and HPMC substrates are inherently anisotropic, non-heterogeneous media. Since this deficiency cannot be addressed experimentally, a robust statistical method is examined for the detection of questionable SSRTP data points and the deletion of outlying observations. If discordant observations are discarded, relative standard deviations are typically reduced to less than 10% for most SSRTP data sets. Robust techniques for outlier identification are superior to traditional methods since they operate at a high level of efficiency and are immune to masking effects.The process of selecting a suitable sample support material often involves considerable trial-and-error on the part of the analyst. A mathematical model based on Hansen's cohesion parameter theory is developed to predict favorable phosphor-substrate attraction and interactions. The results of investigations using naphthalene as a probe phosphor and sodium iodide as an external heavy-atom enhancer support the cohesion parameter model.This document includes a thorough description of the fundamental principles of phosphorimetry and provides a detailed analysis of the theoretical and practical concerns associated with performing SSRTP. In order to better understand the properties of both filter paper and HPMC, a chapter is devoted to the discussion of the cellulose biopolymer. Experimental results and interpretations are presented and suggestions for future investigations are provided. Together, these results provide a framework that will support additional advancements in the field of solid-surface room-temperature phosphorescence spectroscopy. / Ph. D.

Un traumatisme de la moelle épinière (TME) est une condition dévastatrice entraînant la perte permanente de fonctions neuronales. L’objectif de cette thèse est de formuler de microsupports pharmacologiquement actif (MPAs) avec une surface de fibronectine (FN), libérant le« brain-derived neurotrophic factor » (BDNF) de façon controlée. Nous voulons combiner ce système avec des cellules souches mésenchymateuses (CSMs) pour la réparation de TME. Le BDNF nanoprécipité a été encapsulé dans les FN-MPAs et le profil de libération in vitro a été évaluée. Elle a montré une libération biphasique et prolongée de BDNF bioactifs. Nous avons combinés des cellules souches humaines mésenchymateuse issues de la moelle osseuse adulte (cellules MIAMI) et FN-MPAs avec un hydrogel non-toxique silanisés-hydroxypropylméthylcellulose (Si-HPMC). Nous avons démontré que les FN-MPAs et le Si-HPMC augmentait l'expression de marqueurs neuraux/neuronaux de cellules MIAMI après 1 semaine. En outre, l'environnement 3D (hydrogel ou FN-MPAs) a augmenté le sécrétome thérapeutique de cellules MIAMI. Pour avoir un système facile à appliquer en clinique, nous avons choisi d’utiliser les cellules souches de la papille apicale (SCAP) et FN-MPAs libérant ou non du BDNF pour la thérapie du TME. Plus de 90 % du SCAP complexée avec FN-MPAs (libérant ou pas BDNF) demeurent viables pendant 7 jours et il y a augmentation de l'expression des gènes neuronaux/oligodendrogliaux in vitro. La récupération de la fonction locomotrice a été significativement améliorée après la transplantation du SCAP complexée avec FN-MPAs-BDNF avec une coordination cohérente du membre postérieur après 28 jours de traitement. / Traumatic spinal cord injury (SCI) is a devastating condition resulting in permanent loss of neural functions. The objective of this thesis is to develop pharmacologically active microcarriers (PAMs) with a fibronectin (FN) surface that deliver biologically active brain derived neurotrophic factor (BDNF) in a controlled manner. We want to combine this system with adult mesenchymal stem cells (MSCs) for SCI repair. The nanoprecipitated BDNF was encapsulated in FN-PAMs and the in vitro release profile was evaluated. It showed a prolonged, bi-phasic, release of bioactive BDNF, without burst effect. We combined human marrow-isolated adult multilineage-inducible (MIAMI) stem cells and FN-PAMs with an injectable non-toxic silanized-hydroxypropylmethylcellulose (Si-HPMC) hydrogel. We demonstrated that FN-PAMs and the Si-HPMC hydrogel increased the expression of neural/neuronal differentiation markers of MIAMI cells after 1 week. Moreover, the 3D environment (FN-PAMs or hydrogel) enhanced the therapeutic MIAMI cell secretome. To have a clinically translatable system, we chose to use stem cells of the apical papilla (SCAP) and FNPAMs releasing or not BDNF for SCI therapy. More than 90% of SCAP complexed with FN-PAMs (releasing or not BDNF) remained viable for 7 days and an increased neuronal-oligodendroglial gene expression in vitro. The recovery of locomotor function was significantly improved after transplantation of SCAP complexed with FN-PAMs-BDNF with frequent to consistent forelimb-hindlimb coordination after 28 days of treatment.

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Submitted by Rosangela Silva (rosangela.silva3@unioeste.br) on 2017-08-30T19:45:54Z No. of bitstreams: 4 Débora Sgorla.pdf: 1749128 bytes, checksum: e687399f0ee2e0ef0bf64d945ededc36 (MD5) ANEXO A – Artigo publicado - Development and characterization of crosslinked hyaluronic acid polymeric films for use in c~1.pdf: 2607800 bytes, checksum: f08233b805a21000ec3bd1134155499e (MD5) ANEXO B – Artigo publicado - Exploitation of lipid-polymeric matrices at nanoscale for drug delivery applications.pdf: 1272400 bytes, checksum: 817f7d9360bbc82b4da1596089608c18 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2017-08-30T19:45:54Z (GMT). No. of bitstreams: 4 Débora Sgorla.pdf: 1749128 bytes, checksum: e687399f0ee2e0ef0bf64d945ededc36 (MD5) ANEXO A – Artigo publicado - Development and characterization of crosslinked hyaluronic acid polymeric films for use in c~1.pdf: 2607800 bytes, checksum: f08233b805a21000ec3bd1134155499e (MD5) ANEXO B – Artigo publicado - Exploitation of lipid-polymeric matrices at nanoscale for drug delivery applications.pdf: 1272400 bytes, checksum: 817f7d9360bbc82b4da1596089608c18 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2017-02-03 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Introduction: Progress in the release of pharmacotherapeutic agents and improved quality of life for patients depends on the development of novel and suitable drug delivery systems because a number of conventional pharmaceutical forms can trigger multiple side effects as well as inconvenient administrations, which ultimately lead to poor treatment adhesions or inefficient treatments. Objectives: To develop new materials for application in modified drug release systems, whose potential uses are for coating solid oral dosage forms and insulin encapsulation for oral administration. Methodology: Coating films: Initially, hyaluronic acid was crosslinked with trisodium trimetaphosphate in aqueous alkaline media. Afterwards, the films were produced by evaporation method by incorporation of the unmodified and crosslinked biopolymer into the ethylcellulose dispersion in different proportions. The obtained films were characterized by morphology by scanning electron microscopy, robustness to water vapor permeability and hydration capacity in physiological simulation fluids. In addition, safety and biocompatibility were evaluated against Caco-2 and HT29-MTX intestinal cells. Lipid-polymeric nanoparticles: They were produced from the association of ethylpalmitate and HPMC-AS, through the modified solvent emulsification-evaporation technique by sonication. Subsequently, the nanoparticles were characterized by size, polydispersity index, zeta potential and encapsulation efficiency, besides morphology by scanning electron microscopy, X-ray diffraction and thermal analysis. It was also evaluated the in vitro release profile, as well as insulin uptake in a triple co-culture model, and safety and biocompatibility against Caco-2 and HT29-MTX intestinal cells. Results: Coating films: The permeability to water vapor was influenced by the increase of hyaluronic acid content in the final formulation. When immersed in gastric simulation fluid, the films presented lower swelling compared to greater hydration in intestinal simulation fluid. Simultaneously, in intestinal simulation fluid, they presented mass loss, revealing the ability to prevent premature drug release at gastric pH, yet vulnerable to release into the intestinal environment. In addition to these results, the physico-chemical characterization suggested thermal stability of the films and physical interaction between the constituents of the formulation. Finally, cytotoxicity tests demonstrated viii that both membranes and individual materials were safe for intestinal cells when incubated for 4 h. Lipid-polymer nanoparticles: The suggested methodology yielded nanoparticles with satisfactory mean size, 297.57nm ± 29.99, PDI of 0.247 ± 0.03 and zeta potential of -19.13 ± 5.88. In addition, high encapsulation efficiency was achieved, around 83.92 ± 4.32% and DSC showed an improvement in the thermal stability of the formulation compared to individual materials. This is demonstrated by endothermic peaks of degradation that decreased in intensity and moved to higher temperatures. DRX results showed alteration of the crystalline state to amorphous, inferring the drug incorporation. The cumulative release demonstrated that only 9.0% of the encapsulated insulin was released after 2 h, reaching approximately 14% after 6h. These results altered the permeability of insulin through in vitro intestinal model. Regarding the biocompatibility with Caco-2 and HT29-MTX cells, lipid-polymeric nanoparticles did not show toxicity up to 4 hours. Conclusions: The results suggest that hyaluronic acid based films may prevent premature drug release under hostile conditions of the stomach but control the release in the more distal portions of the gastrointestinal tract when applied as coating material in solid oral dosage forms. Furthermore, they were safe to intestinal mucosa. Regarding the lipid-polymeric nanoparticles, evidences show that they can protect insulin from the hostile conditions found in the TGI, also guaranteeing the safety of the intestinal mucosa depending on its concentration. However, a better release profile and consequently better insulin uptake can be achieved by optimizing the proposed formulation. / Introdução: O progresso na liberação de ativos farmacoterapêuticos e uma melhor qualidade de vida aos pacientes depende do desenvolvimento de novos e adequados sistemas carreadores de fármacos, visto que diversas formas farmacêuticas convencionais podem desencadear múltiplos efeitos colaterais bem como administrações inconvenientes, que acabam por conduzir a fracas adesões de tratamento ou tratamentos ineficientes. Objetivos: Desenvolver novos materiais candidatos à aplicação em sistemas para liberação modificada de fármacos, cujos empregos potenciais estão voltados ao revestimento de formas farmacêuticas sólidas orais e encapsulação de insulina para administração oral. Metodologia: Filmes de revestimento: Inicialmente o ácido hialurônico foi reticulado com trimetafosfato trissódico em meio aquoso alcalino, posteriormente, os filmes foram produzidos através do método de evaporação, por incorporação do biopolímero reticulado e não modificado à dispersão de etilcelulose, em diferentes proporções. As películas obtidas foram caracterizadas em relação à morfologia por microscopia eletrônica de varredura, robustez à permeabilidade ao vapor d’água e capacidade de hidratação em fluidos de simulação fisiológicos. Além disso, a segurança e biocompatibilidade foram avaliadas contra células intestinais Caco-2 e HT29-MTX. Nanopartículas lipídico-poliméricas: Foram produzidas a partir da associação de etilpalmitato e HPMC-AS, por meio da técnica emulsificação-evaporação do solvente modificado, através de sonicação. Posteriormente, as nanopartículas foram caracterizadas em relação ao tamanho, índice de polidispersão, potencial zeta e eficiência de encapsulação, além de morfologia por microscopia eletrônica de varredura, difratometria de raios-X e análise térmica. Também avaliou-se o perfil de liberação in vitro, bem como a captação da insulina em modelo de co-cultura tripla e, segurança e biocompatibilidade contra células intestinais Caco-2 e HT29-MTX. Resultados: Filmes de revestimento: A permeabilidade ao vapor d’água foi influenciada pelo aumento do conteúdo de ácido hialurônico na formulação final. Quando imersos em fluido de simulação gástrico, os filmes apresentaram menor intumescimento comparado com uma maior hidratação em fluido de simulação intestinal. Simultaneamente, em fluido de simulação intestinal, apresentaram perda vi de massa, revelando a habilidade de prevenir a liberação prematura do fármaco em pH gástrico, todavia vulnerável a liberação em meio intestinal. Aliado a estes resultados, a caracterização físico-química sugeriu estabilidade térmica das películas e interação física entre os constituintes da formulação. Por fim, os testes de citotoxicidade demonstraram que tanto as membranas quanto os componentes individuais das formulações, quando incubadas durante 4 h, foram seguras para as células intestinais. Nanopartículas lipídico-poliméricas: A metodologia sugerida produziu nanopartículas com tamanho médio satisfatório, 297,57nm ± 29,99, PDI de 0,247 ± 0,03 e potencial zeta de -19,13 ± 5,88. Além disso, alcançou-se alta eficiência de encapsulação, em torno de 83,92 ± 4,32% e o DSC mostrou um aumento da estabilidade térmica das formulações em relação aos materiais puros, demonstrado pelos picos endotérmicos de desidratação, que diminuíram de intensidade e deslocaram-se para temperaturas superiores. Os resultados do DRX demonstraram alteração do estado cristalino para amorfo, inferindo a incorporação do fármaco. A liberação cumulativa evidenciou que apenas 9% da insulina encapsulada foi liberada após 2 h, alcançando aproximadamente 14% após 6 h, alterando, desta maneira, os resultados de permeabilidade da insulina através de modelo intestinal in vitro. Em relação à biocompatibilidade com células Caco-2 e HT29-MTX, as nanopartículas lipídico-poliméricas demonstraram ausência de citotoxicidade após 4 h. Conclusões: Os resultados sugerem que os filmes baseados em ácido hialurónico, quando aplicados como material de revestimento de formas farmacêuticas sólidas orais, poderão prevenir a liberação prematura de fármacos nas condições hostis do estômago, mas controlar a liberação nas porções mais distais do trato gastrointestinal, garantindo a segurança da mucosa intestinal. Já em relação às nanopartículas lipídico-poliméricas, evidências demonstraram que as mesmas poderão proteger a insulina das condições hostis encontradas no TGI, garantindo ainda a segurança da mucosa intestinal, todavia um melhor perfil de liberação, e consequentemente, uma melhor captação insulina podem ser alcançados por otimização da formulação proposta.

The increasing spread of antibiotic resistance among bacteria poses a major threat to the public health. There is an urge for the development of innovative formulations of existing and new antibiotics. One area of interest is in wound care, where two interesting antimicrobials are the conventional antibiotic amoxicillin and the antimicrobial peptide AP114. The objective of this work was to systemically evaluate topical gel formulations of these APIs by following a Quality by Design approach. A short excipient compatibility study was performed and the thickening agents poloxamer 407 and HPMC were chosen to be included in the following Design of Experiment (DoE) study of formulation composition and storage climate. The DoE set up was generated by the software MODDE Pro® and a short stability study of four weeks was performed, including analysis of the apparent pH, rheology stability, appearance, BCA assay, UV-Vis and FTIR spectroscopy and Franz cell diffusion. The results showed that AP114 formulations stored at 2-8˚C with poloxamer 407 should be with buffer pH 5-6 and 5-15 wt% organic phase to maximize stability, while HPMC based AP114 gels should be with buffer pH 6-8 and 10-40 wt% organic phase. Poloxamer 407 was not preferrable for amoxicillin formulations. The optimal HPMC based amoxicillin formulations included the storage temperature 25˚C, a buffer pH 7-8 and 40-60 wt% organic phase. HPMC based amoxicillin gels stored at 2-8˚C should be composed with buffer pH 6-8 and 10-40 wt% organic phase.

Abstract Biodegradable starch-glycerol based films were obtained. The influence of lipid compounds (palmitic, stearic and oleic acid), other polymers (hydroxypropylmethylcellulose and sodium caseinate) and bioactive compounds (¿-tocoferol, D-limonene and orange essential oil) on film properties (oxygen and water vapour barrier, optical, mechanical, nano- and microstructural). Furthermore the effect of storage time on films¿ properties was also considered. Fatty acids addition did not improve the water vapour ability of films except for non-stored saturated fatty acids containing films. X-ray diffraction results showed that cristallinity of films increased with storage time, thus increasing the stiffness and decreasing the gloss of films. Furthermore, crystallinity affected the water sorption capacity of films as function of relative humidity and temperature. Glass transition temperature of starch films varied with saturated fatty acids addition. However, oleic acid did not affect this parameter. The presence of fatty acids promoted the formation of V-type structures, thus indicatin the formation of amylose-lipid complexes that inhibited the developmet of other crystalline structures. The effect of the incorporation of other biopolymers to improve the functionality of starch films was also studied. Hydroxypropylmethylcellulose (HPMC) addition inhibited starch retrogradation. However, obtained films were more permeable, specially in case of oxygen. HPMC addition produced phase separation as it was observed by scanning electron microscopy. On the contrary, sodium caseinate incorporation (NaCas) allowed to obtain homogeneous films and less permeable to oxygen. Obtained films showed less mechanical resistance in comparison with pure starch films but a greater flexibility without increasing the water vapour permeability. Rearrangement of polymers chains during storage reduced the mechanical resistance, the extensibility and the gloss of composite films. Regarding the obtained results, the film including a starch:protein ratio of 50:50 was choosen as the film with the most adequate properties. Composite film (starch:Nacas ratio = 50:50) was studied as a matrix for the incorporation o active compounds (¿-tocopherol, D-limonene and orange essential oil). The effect of ¿-tocopherol addition was compared with the incorporation of oleic acid and their mixture. Lipids addition promoted phase separation between starch and NaCas due to the different interactions between each polymer and the lipids. Furthermore, oleic acid addition increased significantly the oxygen permeability whereas ¿-tocopherol greatly improved the antioxidant capacity of films without affecting the oxygen permeability. D-limonene and orange essential oil incorporation was carried out by forming rapeseed and soy nanoliposomes, which acted as carriers of bioactive components. Nanoliposomes incorporation was performed directly in starch-NaCas dispersions without any homogenization, to avoid nanoliposomes damages. Bioactive compounds addition did not confer antimicrobial capacity to the films (except for soy-orange oil nanoliposomes containing film) probably due to the high stability of nanoliposomes and the low antibacterial activity of D-limonene and orange essential oil. / Se han desarrollado y caracterizado films biodegradables a base de almidón de maíz y glicerol como plastificante, evaluando al mismo tiempo el efecto de la adición de componentes lipídicos (ácido palmítico, esteárico y oleico), otros polímeros (hidroxipropilmetilcelulosa y caseinato de sodio) y compuestos bioactivos (¿-tocoferol, aceite esencial de naranja y D-limoneno) sobre las propiedades de los films (propiedades barrera al vapor de agua y al oxígeno, ópticas, mecánicas, micro y nanoestructurales). Asimismo se evaluó la influencia del tiempo de almacenamiento en las propiedades de los films. La adición de ácidos grasos no mejoró notablemente la permeabilidad al vapor de agua excepto en el caso de los films con ácidos grasos saturados y solo en films no almacenados. Los resultados de difracción de rayos X mostraron que la cristalinidad aumentó con el tiempo de almacenamiento, incrementándose la rigidez, y disminuyendo el brillo de los films. Del mismo modo, la cristalinidad afectó a la capacidad de sorción de agua de los films en función de la humedad relativa y la temperatura. La temperatura de transición vítrea de los films de almidón se vio afectada por la adición de ácidos grasos saturados pero no por la adición de ácido oleico. La presencia de dichos componentes promovió la formación de estructuras cristalinas tipo V, indicando la formación de complejos entre los lípidos y las cadenas de amilosa e inhibiendo la formación de otros tipos de formas cristalinas. Se analizó también el efecto de la incorporación de otros biopolímeros en la posible mejora de la funcionalidad de los films de almidón. En las mezclas con hidroxipropilmetilcelulosa (HPMC), se inhibió la retrogradación del almidón en los films composite, pero se observó un efecto negativo en las propiedades barrera de los mismos, que fueron más permeables, principalmente al oxígeno. La adición de HPMC produjo separación de fases en los films (observada por microscopía electrónica de barrido). Por el contrario, la incorporación de caseinato de sodio (NaCas) permitió formar films homogéneos y menos permeables al oxígeno. Los films presentaron una resistencia mecánica algo menor que los films de almidón puro pero una mayor flexibilidad sin incrementar los valores de permeabilidad al vapor de agua. La reorganización de las cadenas de los polímeros con el tiempo de almacenamiento provocó la disminución de la resistencia mecánica, la deformabilidad y el brillo de los films composite. Atendiendo a los efectos observados, se eligió como formulación más adecuada el film composite formado por almidón y NaCas con un ratio de polímeros del 50:50. El film composite de almidón y NaCas (50:50) se estudió como matriz para la incorporación de compuestos bioactivos como son el ¿-tocoferol y el aceite esencial de naranja o su principal componente, el D-limoneno. El efecto de la adición de ¿-tocoferol se comparó con la influencia de la adición de ácido oleico y también con la adición de ambos compuestos. La adición de lípidos provocó una separación de fases entre el almidón y el NaCas debido a la diferente interacción entre cada polímero y los lípidos. Asimismo la adición de ácido oleico incrementó significativamente la permeabilidad al oxígeno, al contrario que el ¿-tocoferol, que además impartió a los films una elevada capacidad antioxidante. La incorporación de aceite esencial de naranja y D-limoneno se realizó utilizando nanoliposomas de lecitina de soja y lecitina de colza que encapsularon los compuestos activos. La incorporación de nanoliposomas en los films se realizó directamente en las dispersiones acuosas sin posterior homogeneización para evitar su ruptura. La adición de los compuestos bioactivos en forma de nanoliposomas no confirió capacidad antimicrobiana a los films, salvo en el caso de los nanoliposomas de lecitina de soja con aceite esencial, debido probablemente a la dificultad de los compuestos encapsulados para difundir en el film por la gran estabilidad de los liposomas y a la baja actividad antilisteria del D-limoneno y el aceite esencial de naranja. / Jiménez Marco, A. (2013). Propiedades de films de almidón de maíz. Influencia de la incorporación de lípidos, biopolímeros y compuestos bioactivos [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/28214 / TESIS / Premios Extraordinarios de tesis doctorales

<p>У оквиру ове докторске дисертације испитана је биолошка активност екстраката плодних тела и потопљених култура (мицелије и филтрата) аутохтоних врста гљива <em>Coprinus comatus</em> и<em> Coprinellus truncorum</em>. Такође, испитан је&nbsp; метаболизам фосфата мицелија обе врсте употребом нуклеарно магнетне резонантне спректроскопије (³¹Р NMR), утицај ванадијума на метаболизам фосфата као и идентификација облика ванадата присутних у ћелији мицелије (⁵¹V NMR). Утврђена је антирадикалска и антиоксидативна активност&nbsp; етанолних,метанолних и водених екстраката гљива при чему су се екстракти потопљених култура издвојили по антирадикалској, а екстракти плодних тела по антиоксидативној активности. Екстракти потопљених култура истакли су се и у погледу антибактеријске активности, где се као најпотентнији показао&nbsp; хлороформски екстракт филтрата потопљене културе<em> C. comatus</em>. Такође, етанолни екстракт филтрата потопљене културе <em>C. comatus</em> показао се као најпотентнији у анти-ацетилхолинестеразној активности у односу на&nbsp; конвенционални лек донепезил. Испитан је и утицај екстраката на вијабилност ћелијских линија HepG2 (хумане хепатома ћелије) и Rin-5F (&szlig; ћелије панкреаса пацова).</p><p>Спектрофотометријским методама одређен је укупан садржај фенола и флавоноида у већини анализираних екстраката.</p><p>LC/MS идентификацијом и квантификацијом фенолних киселина уочена је разлика између фенолних једињења присутних у плодном телу, мицелији и филтрату потопљене културе. Екстракти потопљених култура бележе већи број и већи садржај једињења. Укупан садржај протеина одређен само у воденим екстрактима, а укупан садржај угљених хидрата у полисахаридним екстрактима.Употребом Фуријеве инфрацрвене спектроскопске методе (FTIR) детектоване су везе између угљених хидрата&nbsp; присутних у полисахаридним екстрактима, а планарном&nbsp; хроматографијом показано је да екстракти плодног тела и филтрата врсте <em>С. truncorum</em>, као и екстракт плодног тела врсте <em>C</em>. <em>comatus</em>, садрже велику&nbsp; количину D-глукозе, док екстракт мицелије <em>C. truncorum</em>, баш као и екстракти филтрата и мицелије <em>C. comatus</em>, садрже највише галактозе. Квалитативном и квантитативном елементарном анализом (ААS) утврђен је виши садржај&nbsp; калијума и гвожђа у анализираним узорцима. GC-МS идентификацијом и квантификацијом масних киселина указано је на значајно присуство линолне киселине код обе врсте.&nbsp;<br />Како за аутохтону врсту&nbsp; <em>C.truncorum </em>постоји мало података у литератури, подаци о њеном хемијском саставу могу се сматрати иновативним.<br />Компаративним прегледом биолошке активности и хемијског састава екстраката плодног тела и мицелије и филтрата (потопљених култура) указано је да су анализирани екстракти извори биоактивних супстанци са медицинским потенцијалом, а потопљене културе датих гљива представљају атрактивне кандидате за даља биотехнолошка истраживања.</p> / <p>U okviru ove doktorske disertacije ispitana je biološka aktivnost ekstrakata plodnih tela i potopljenih kultura (micelije i filtrata) autohtonih vrsta gljiva <em>Coprinus comatus</em> i<em> Coprinellus truncorum</em>. Takođe, ispitan je&nbsp; metabolizam fosfata micelija obe vrste upotrebom nuklearno magnetne rezonantne sprektroskopije (³¹R NMR), uticaj vanadijuma na metabolizam fosfata kao i identifikacija oblika vanadata prisutnih u ćeliji micelije (⁵¹V NMR). Utvrđena je antiradikalska i antioksidativna aktivnost&nbsp; etanolnih,metanolnih i vodenih ekstrakata gljiva pri čemu su se ekstrakti potopljenih kultura izdvojili po antiradikalskoj, a ekstrakti plodnih tela po antioksidativnoj aktivnosti. Ekstrakti potopljenih kultura istakli su se i u pogledu antibakterijske aktivnosti, gde se kao najpotentniji pokazao&nbsp; hloroformski ekstrakt filtrata potopljene kulture<em> C. comatus</em>. Takođe, etanolni ekstrakt filtrata potopljene kulture <em>C. comatus</em> pokazao se kao najpotentniji u anti-acetilholinesteraznoj aktivnosti u odnosu na&nbsp; konvencionalni lek donepezil. Ispitan je i uticaj ekstrakata na vijabilnost ćelijskih linija HepG2 (humane hepatoma ćelije) i Rin-5F (&szlig; ćelije pankreasa pacova).</p><p>Spektrofotometrijskim metodama određen je ukupan sadržaj fenola i flavonoida u većini analiziranih ekstrakata.</p><p>LC/MS identifikacijom i kvantifikacijom fenolnih kiselina uočena je razlika između fenolnih jedinjenja prisutnih u plodnom telu, miceliji i filtratu potopljene kulture. Ekstrakti potopljenih kultura beleže veći broj i veći sadržaj jedinjenja. Ukupan sadržaj proteina određen samo u vodenim ekstraktima, a ukupan sadržaj ugljenih hidrata u polisaharidnim ekstraktima.Upotrebom Furijeve infracrvene spektroskopske metode (FTIR) detektovane su veze između ugljenih hidrata&nbsp; prisutnih u polisaharidnim ekstraktima, a planarnom&nbsp; hromatografijom pokazano je da ekstrakti plodnog tela i filtrata vrste <em>S. truncorum</em>, kao i ekstrakt plodnog tela vrste <em>C</em>. <em>comatus</em>, sadrže veliku&nbsp; količinu D-glukoze, dok ekstrakt micelije <em>C. truncorum</em>, baš kao i ekstrakti filtrata i micelije <em>C. comatus</em>, sadrže najviše galaktoze. Kvalitativnom i kvantitativnom elementarnom analizom (AAS) utvrđen je viši sadržaj&nbsp; kalijuma i gvožđa u analiziranim uzorcima. GC-MS identifikacijom i kvantifikacijom masnih kiselina ukazano je na značajno prisustvo linolne kiseline kod obe vrste.&nbsp;<br />Kako za autohtonu vrstu&nbsp; <em>C.truncorum </em>postoji malo podataka u literaturi, podaci o njenom hemijskom sastavu mogu se smatrati inovativnim.<br />Komparativnim pregledom biološke aktivnosti i hemijskog sastava ekstrakata plodnog tela i micelije i filtrata (potopljenih kultura) ukazano je da su analizirani ekstrakti izvori bioaktivnih supstanci sa medicinskim potencijalom, a potopljene kulture datih gljiva predstavljaju atraktivne kandidate za dalja biotehnološka istraživanja.</p> / <p>The biological activity of extracts of basidiocarps (fruiting bodies)&nbsp; and submerged cultures (mycelium and filtrate) of autochthonous mushroom species&nbsp; <em>Coprinus comatus</em> and&nbsp; <em>Coprinellus truncorum&nbsp;</em> was examined. Furthermore, the metabolism of phosphate&nbsp; of mycelia&nbsp; of both types was studied using nuclear magnetic&nbsp; resonance spectros-copy ( ³¹ R NMR), the influence of vanadium on phosphate metabolism and the identification of vanadate oxidation states present in the mycelia cell ( ⁵¹ V NMR). The antiradical and antioxidant activity of methanolic, ethanolic and water fungal extracts was determined. Extracts of submerged cultures achieved the best anti- radical activity while fruit body extracts showed the best antioxidant activity. Extracts of submerged cultures also highlighted in terms of antibacterial activity, where the chloroform extract of the submerged culture&nbsp; <em>C. comatus</em>&nbsp; showed as the most potent. Also, the ethanolic extract of the submerged culture of<em>&nbsp; C. comatus</em>&nbsp; was found to be most relevant in anti-acetylcholinesterase activity&nbsp; compared with&nbsp; the conventional donepezil drug. The influence of extracts on the viability of cell lines HepG2 (human hepatocytes cells) and Rin-5F (&szlig; pancreatic cells of the rat) was also examined.</p><p>Spectrophotometric methods determined the total con-tent of phenol and flavonoids in most of the analyzed extracts.</p><p>The LC/MS identification and quantification of phenolic acids revealed the difference between the phenolic compounds present in the fruiting body, mycelium, and the submerged culture filtrate. Extracts of submerged cultures record a greater number and higher content of compounds.</p><p>The total content of proteins determined only in water extracts&nbsp; and the total content of&nbsp; carbohydrates in poly-saccharide extracts. Using the Fourier infrared spectro-scopic method (FTIR), the links between the sugar pre-sent in the&nbsp; polysaccharide extracts were detected, and planar chromatography showed that the extracts&nbsp; of the fruiting body and the filtrate of type<em>&nbsp; C. truncorum</em>, as well as the extract of the fruiting body of the species&nbsp; <em>C. comatus</em>, contain a large amount of D-glucose, while the extract of the&nbsp;<em> C</em>. <em>truncorum</em>&nbsp; mycelia&nbsp; and&nbsp; mycelia&nbsp; of&nbsp; <em>C. comatus</em>, contain the most galactose. GC-MS identification and quantification of fatty acids indicated a significant presence of linoleic acid in both species, while qualitative and quantitative elemental analysis (AAS) has determined a higher content of potas-sium and iron in the analyzed samples. Since there is no data in the literature for the autochtho-nous species&nbsp;<em> C</em>. <em>truncorum</em>, the studies on its chemical composition can be considered advanced аs innovative. A comparative review of the biological activity and the chemical composition of the extracts of the fruiting body and&nbsp; mycelia&nbsp; and filtrates&nbsp; of&nbsp; medium of&nbsp; submerged cultures&nbsp; indicated that the extracts were analyzed by sources of bioactive substances with medical potential, and the submerged cultures of these mushrooms are attractive candidates for biotechnological research.</p> / <p>В рамках данной работы была исследованна биологическая активность экстракта плодородных тел и погружонных видов култур (мицелии и филтрата) автотоных видов грибов <em>Coprinus comatus</em> и <em>Coprinellus truncorum</em>. Также, исследованн метаболизм фосфата обеих видов&nbsp; мицелий с помощью ядерного магнитного резонанса спектроскопии (³¹Р ЯМР), влияние на содержание ванадия в метаболизме фосфата, а также идентификация формы ванадата присущего в клеток мицеллий (⁵¹V ЯМР). Установленная антирадикальная и антиоксидантная активность метанольных, этанольных и водных экстрактов гриб, причём выделяются экстракты погружённых культур по антирадикальной активности и&nbsp; экстракты плодородных тел по антиоксидантной активности.</p><p>Экстракты погружённых культур выделялись и в плане антибактериальной активности, причем,&nbsp; наиболее мощным из филтратов показался экстракт хлороформа погруженной культуры <em>C. comatus.</em> А также этанольный экстракт филтрата погружённой культуры<em> C. comatus</em> оказался найболее мощным в анти-ацетихолинестеразной активностипо сравнению с традиционным лекарством донепезилом. Было исследовано и влияние экстрактов на виябильность клеток линий&nbsp;&nbsp; HepG2 (гуманые хепатома клетки) и Rin-5F (&szlig; клетки поджелудочной железы крыс).<br />Методом спектрофотометрии определена совокупность фенола и флавоноида в большинстве проанализированных экстрактах.<br />С помощью ЛС ̸МС идентификации и квантификации фенолных кислот была замечена разница между соединениями фенола, присущих в плодородном теле, и мицелии, и филтрата погружённой культуры. Экстракты погружённых культур отражают больше количество и более высокое содержание соединений.<br />Общее содержание белков выделен только в водяных экстрактах, и общее содержание углеводов в полисахаридных экстрактах. Используя инфракрасный метод спектроскопии Фурия (ИКМСФ) были обнаружены связи между сахарами, присущими в полисахаридных экстрактах, а планарной хромотографиой было показано, что экстракты плодородного тела и филтратов вида <em>С. truncorum</em>,&nbsp; а&nbsp; также и экстракты плодородного тела вида <em>C. comatus</em> содержат большое количество D-глюкозы, в то время как экстракт мицелии <em>C. truncorum,</em> именно как и экстракт фильтрата и мицелии <em>C. comatus</em>, содержат больше всего галактозы.<br />GC-МS идентификацией и квантификацией жирных кислот показано значительное наличие линолевой кислоты у обоих видах. А качественным и квантитативным элементарным анализом установленно большее содержание калиума и железа в анализированых шаблонах.<br />Из-за того, что для автохтонного вида <em>C. truncorum</em> практически не было данных в литературе, данные о её химическом составе можно считать прогрессивным и инновационным.<br />Сравнительный анализ биологической активности и химического состава экстрактов плодородного тела и мицелии и фильтрат (погружённых культур) показаывает, что проанализированные экстракты &mdash; источники биологически активных веществ с медицинским потенциалом, и погружённые культуры данных гриб являются привлекательными кандидатами для биотехнологических исследований.</p>