Active Pharmaceutical Ingredients with poor solubility have presented significant difficulties in drug product design and development including slow and ineffective absorption leading to inadequate and variable bioavailability. Therefore it has become increasingly desirable to overcome the low aqueous solubility of drug candidates and develop more novel and innovative formulation approaches to increase the dissolution rate of the poorly soluble drugs. This work focuses on the formulation of novel amorphous ibuprofen-polymer nanoconjugates based on the polymer-drug complexation in order to improve its physical and dissolution characteristics without the use of toxic organic solvents. Plain and ibuprofen-loaded binary and ternary nanoconjugates were prepared using four modified co-precipitation techniques including melt solubilization; alkaline solubilization; surfactant solubilization and hydrotropic complexation techniques. A remarkably high loading capacity was achieved ranging from 89.05 to 99.49% across the four techniques and polymer-polymer ratio of 50:50 was found to be most efficient. All the four techniques reduced the size of ibuprofen (2.87 μm) significantly in the presence of 2.0 x10-3 mM of Diethylaminoethyl Dextran (DEAE-Dextran) in the order melt solubilization (203.25 nm) > alkaline solubilization (185.68 nm) > surfactant (Tween 80) solubilization (122.17 nm) > hydrotropic complexation (77.92 nm). 5.0 x 10-4 mM of chitosan also reduced the size of ibuprofen from 2872.12 to 10.70 nm (268-fold reduction). The FTIR spectroscopic analysis revealed electrostatic, hydrophobic and hydrogen bonding interaction between solubilized ibuprofen and the cationic polymers (DEAE-Dextran and chitosan) to form a new product (an amide). Polymer-polymer complexation also occurred between DEAE-Dextran and gellan as well as chitosan and gellan to a different extent depending on the mixing ratios. 1H and 13C NMR analysis confirmed the conjugation between ibuprofen and each of the cationic polymers as well as the formation of a new amide product. DSC thermal analysis showed that the nanoconjugates exhibited new broad and diffuse peaks confirming that they did exist in amorphous state as multiple complexes. The TGA thermograms of the binary nanoconjugates exhibited one step degradation profile compared with the physical mixture which exhibited two steps. However the ternary nanoconjugates exhibited two steps degradation profile confirming the formation of multiple complexes. Marked enhancement of drug release was achieved by the four techniques compared with the ibuprofen control. All the DG (DEAE-Dextran - Gellan) complexes exhibited a higher release profile than ibuprofen control. Fickian and non-Fickian anomalous mechanisms were deduced for the drug release of ibuprofen from the binary conjugates. The ternary nanoconjugates exhibited non-Fickian (anomalous) diffusion, Fickian diffusion and Super Case II transport release mechanisms. The ternary nanoconjugate hydrogels exhibited complete release (100%) within 48 h. The lowest concentration of DEAE-Dextran, Gellan - Ibuprofen - DEAE-Dextran (GIbDD) 2:0.125, increased the release of ibuprofen by 13.4% however higher concentrations of DEAE-Dextran decreased the release profile steadily. It was concluded that DEAE-Dextran has potentials in the formulation of modified (extended) release of ibuprofen. The most prominent mechanism of release of ibuprofen from the nanoconjugate hydrogel was Super Case II transport. SEM and AFM micrographs of the drug loaded composite pharmaceutical films exhibited concentric spheres with two and three layers for the binary and ternary films respectively. This supports the evidence of internalization of ibuprofen by the polyelectrolyte complex. The FTIR and DSC results confirmed electrostatic and hydrophobic interactions between ibuprofen and DEAE-Dextran as well as between gellan and DEAE-Dextran. Thermal analysis revealed that plain bilayer films were thermally more stable than composite films. The addition of ibuprofen significantly increased (p < 0.05, n = 4) the swelling ratio of the films compared with films without the drug. The drug loaded bilayer films exhibited Fickian diffusion mechanism while the dominating mechanism for composite films was anomalous (Non-Fickian) transport. From the foregoing, it was evident that ibuprofen-polymer nanoconjugate present a novel tool for the delivery of ibuprofen with potential application for transdermal delivery.

<p>Predmet doktorske disertacije je razvoj aktivnog jestivog omotača na bazi hitozana sa dodatkom etarskih ulja začinskog bilja i pčelinjeg voska, kao i njegova aplikacija u cilju produženja održivosti suve fermentisane Petrovačke kobasice.<br />U cilju realizacije programa teze, formirane su tri osnovne grupe filmova: hitozan-etarsko ulje origana, hitozan-etarsko ulje kima i hitozan-etarsko ulje kima-pčelinji vosak. Paralelno sa pripremom filmova, proizvedeni su i odgovarajući kolagen-hitozan laminirani omotači. U pripremi filmova i omotača su primenjena dva vlažna postupka. Filmovi su dobijeni razlivanjem, a laminirani omotači premazivanjem filmogene emulzije preko kolagenog omotača. Tokom razvijanja, ispitivan je uticaj različitih parametara na svojstva dobijenih filmova: viskozitet i koncentracija hitozana, dodatak polietilenoksida kao kopolimera u film, dodatak različite koncentracije etarskog ulja, uslovi me&scaron;anja i temperature, kao i dodatak različite koncentracije pčelinjeg voska.<br />Dobijeni filmovi i omotači su okarakterisani ispitivanjem osobina značajnih za primenu materijala, a to su fizičko-mehanička (debljina, zatezna jačina i izduženje pri kidanju), strukturna (ATR-FTIR spektrometrija i elektron skenirajuća mikroskopija), fizičko-hemijska (sadržaj vlage, bubrenje, rastvaranje), barijerna (propustljivost gasova i brzina propustljivosti vodene pare) svojstva i biolo&scaron;ka aktivnost (antioksidatino i antimikrobno delovanje). Na osnovu detaljne karakterizacije, odabrani su filmovi i omotači optimalnih svojstava za primenu u za&scaron;titi Petrovačke kobasice tokom skladi&scaron;tenja.<br />Ispitano je dejstvo premaza na očuvanje kobasice sa i bez dodate komercijalne i autohtone starter kulture. Efekat primene premaza izučavan je ispitivanjima parametara kvaliteta Petrovačke kobasice i to, praćenjem promene sadržaja vlage, pH vrednosti, boje, oksidacije lipida, kao i senzorne ocene mirisa i ukusa ove kobasice tokom skladi&scaron;tenja.<br />Na osnovu prikazanih rezultata, može se zaključiti da je sintetisan aktivan jestivi premaz na bazi hitozana optimizovanih barijernih, fizičko-hemijskih, antioksidativnih i antimikrobnih svojstava. Sintetisani premaz ima manju brzinu propustljivosti vodene pare, uz očuvanu nisku propustljivost za gasove, smanjenu osetljivost na delovanje vlage i izraženiju antimikrobnu i antioksidativnu aktivnost. Upkos optimizovanim barijernim svojstvima za vodenu paru, hitozanski premaz sa dodatkom etarskih ulja i/ili pčelinjeg voska nije imao efekta u očuvanju sadržaja vlage suve fermentisane kobasice tokom skladi&scaron;tenja. Ispitivani premazi su doprineli očuvanju boje preseka kobasice, ali ovaj efekat nije bio potpuno izražen kod svih tipova kobasice i svih premaza. Sva tri parametra za praćenje oksidativnih promena u kobasici ukazuju na to da je nano&scaron;enjem hitozanskog premaza sa etarskim uljem origana ili kima i pčelinjim voskom, kobasica efikasno za&scaron;tićena od nepoželjnih oksidativnih promena. Između eksperimenata se može povući paralela, da hitozanski premaz pokazuje za&scaron;titni efekat prema senzornom profilu mirisa i ukusa, do blizu 5 meseci skladi&scaron;tenja. Na ovom nivou razvoja premaza, on se može preporučiti kao pomoćan ambalažni materijal.</p> / <p>The subject of the doctoral dissertation is development of active edible coating based on chitosan with added herb essential oils and beeswax, as well as its application for dry fermented sausage shelf life extension.<br />In order to realize the program of investigation, three basic groups of films were produced: chitosan-essential oil of oregano, chitosan-essential oil of caraway and chitosan-essential oil of caraway-beeswax. Simultaneously with the production of films, collagen-chitosan laminated casings were produced. In the production of films and laminated casings, two wet methods were applied. Films were produced by casting method, while laminated casings were produced by coating collagen casing with filmogenic emulsion. During production, the influence of different parameters on properties of produced films and casings was investigated: viscosity and concentration of chitosan, addition of poly (ethylene oxide) as copolymer in the films, addition of different concentration of essential oils, mixing and temperature conditions, as well as addition of different concentration of beeswax.<br />Produced films and laminated casings were characterized by the investigation of properties that are important for material application, namely physical-mechanical (thickness, tensile strength and elongation at break), structural (ATR-FTIR spectrometry and scanning electron microscopy), physical-chemical (water content, swelling, solubility), barrier (gas permeability and water vapor transmission rate) properties and biological activity (antioxidative and antimicrobial activity). Based on detail characterization, films and casings with optimal properties were selected for application on Petrovac sausage with and without the addition af commercial or indigenous starter culture for preservation during storage.<br />The effect of coating application was analyzed based on the quality parameters of Petrovac sausage, namely: water content, pH value, color, lipid oxidation and sensory profile of taste and odor of the sausage during storage.<br />Based on presented results, it can be concluded that active edible coating based on chitosan was synthesized, with optimized barrier, physical-chemical, antioxidant and antibacterial properties. Synthesized coating has lower water transmission rate with preserved low gas permeability, lowered sensibility to humidity and more pronounced antimicrobial and antioxidant activity. Despite optimized water vapor barrier properties, chitosan coating with addition of essential oils and/or beeswax did not show the effect in preserving the water content in the sausage during storage. Investigated coatings contributed to the preservation of core color of the sausage, but this effect was not fully expressed in all types of sausages and coatings. All three parameters for investigation of oxidative changes in the sausage showed that chitosan coating with essential oils of oregano and caraway and beeswax efficiently preserved sausage from unwanted oxidative changes. Mutual conclusion from the experiments is that chitosan coating shows preservative effect on the sensory profile of taste and odor till approximately 5 months of storage. On this level of coating development, it can be recommended as auxiliary packaging material.</p>

L'exportation des mangues est limitée par le mûrissement rapide et la prolifération microbienne sur les fruits. Cette thèse propose une nouvelle approche sûre et saine utilisant des molécules d'origine naturelle pour améliorer la conservation post-récolte des mangues et ainsi participer à la préservation de la santé des consommateurs et à une amélioration des potentialités du commerce international de certains pays exportateurs. Ces travaux ont montré que l'immobilisation du système lactoperoxydase dans le film de chitosane appliqué sur l'épiderme des mangues pouvait maintenir la qualité microbiologique et physicochimique des fruits traités. Le couplage Chitosane-Système lactoperoxydase a prolongé la durée de conservation des mangues durant plus de deux semaines sans altérer leurs qualités organoleptiques.Ce travail a permis également de mettre en évidence la synergie entre le système lactoperoxydase et la concentration de chitosane. Un enrobage optimum de 1% de chitosane a permis de fixer le système enzymatique et de maintenir les mangues en bon état sanitaire. La présence d'iode dans le système lactoperoxydase n'agit pas de manière significative sur la conservation des mangues. / The mango export is limited by the rapid ripening and microbial growth on the fruit. This thesis proposes a new approach to safe and healthy using natural molecules to improve post-harvest conservation of mango and thus help preserve the health of consumers and improved the potential of international trade in certain exporting countries. This work shown that the immobilization of the lactoperoxidase in the chitosan film and applied as coating of mangoes could maintain the microbiological and physicochemical quality of fruits. Chitosan-coupling lactoperoxidase system extended the shelf life of mangoes for over two weeks without affecting their organoleptic quality.This work also helped to highlight the synergy between the lactoperoxidase and the concentration of chitosan. An optimum coating made from 1% chitosan allowed to fix the enzyme system and to maintain the mangoes in a good sanitary condition. The presence of iodine in the lactoperoxidase does not act significantly on the conservation of mangoes.

O objetivo deste estudo clínico foi avaliar a influência da quitosana na biomodificação da dentina após a remoção seletiva das lesões de cárie com o laser de Er:YAG em dentes decíduos, quanto à redução da quantidade de Streptococus mutans e Lactobacillus ssp, e o comportamento clínico das restaurações realizadas. A amostra foi composta por 52 crianças que apresentaram lesão de cárie ativa atingindo dentina, localizada na superfície oclusal em molar decíduo. Para a análise microbiológica (n=10), os dentes foram distribuídos aleatoriamente em 4 grupos de acordo com a remoção seletiva das lesões de cárie: laser de Er:YAG (250mJ/4Hz) ou contra-ângulo (método convencional) e o tratamento da superfície da dentina: sem biomodificação e com biomodificação (gel de quitosana a 2,5%). Foram realizadas coletas da dentina infectada, dentina afetada e após a biomodificação com gel de quitosana a 2,5%. As unidades formadoras de colônias (UFC/mg) foram contadas e os dados foram analisados por meio do teste Shapiro-Wilk e teste de Mann-Whitney para o laser de Er:YAG após aplicação da quitosana. Após a remoção seletiva da lesão de cárie com o laser de Er:YAG houve redução significativa na quantidade de S. mutans (p=0,0068). Para Lactobacillus ssp não foi observada diferença significante entre os métodos de remoção (p=0,089). Após a biomodificação com gel de quitosana a 2,5%, houve redução significativa na quantidade de S. mutans, para ambos os métodos de remoção (p=0,0524). A aplicação de quitosana promoveu redução da quantidade de Lactobacillus ssp, após remoção seletiva pelo método convencional, mas não com laser de Er:YAG (p=0,000487). Para a análise clínica os dentes foram restaurados com adesivo (Single Bond Universal/3M) e resina composta (Z350 Filtek/3M) e avaliados nos períodos de 7 dias (baseline), 6 meses e 1 ano, utilizando os critérios USPHS modificados. Foram realizadas imagens fotográficas para auxiliar na análise clínica das restaurações. Para análise da adaptação marginal da réplica da restauração em microscopia eletrônica, os dentes foram moldados com silicona de adição. Os dados obtidos na análise clínica foram submetidos aos testes não-paramétrico de Kruskal Wallis e complementar de Dun (p< 0.05). Na análise clínica observou-se diferença significativa para a remoção seletiva da lesão da cárie utilizando o laser de Er:YAG com relação aos critérios descoloração e adaptação marginal (p<0,05). Concluiu-se que o laser de Er:YAG reduziu a quantidade de S. mutans e Lactobacillus ssp e a biomodificação com gel de quitosana a 2,5% foi efetiva para potencializar o efeito antimicrobiano, e que o comportamento clínico das restaurações não foi influenciado pelo método de remoção da lesão de cárie e biomodificação da dentina afetada / The objective of this clinical study was to evaluate the effect of Er: YAG laser for the removal of caries lesions in primary teeth, followed by the biomodification of dentin affected with 2.5% chitosan gel, reducing the amount of Streptococcus mutans and Lactobacillus ssp, and the clinical behavior of the performed restorations. The sample consisted of 52 children who presented lesions of active caries reaching dentin, located on the occlusal surface in a deciduous molar. For the microbiological analysis (n = 10), the teeth were randomly distributed in 4 groups according to the selective removal of caries lesions: Er: YAG laser (250mJ / 4Hz) or contra-angle (conventional method) and treatment of the dentin surface: without biomodification and with biomodification (chitosan gel). Dentin tissue was collected in three periods: before selective removal of the caries lesion, after selective removal of the caries lesion and after the dentin biomodification with chitosan gel. The colony forming units (CFU/mg) were counted and the data were analyzed by means of the Shapiro-Wilk test and Mann-Whitney test for the Er: YAG laser after application of chitosan. After the selective removal of the caries lesion with Er:YAG laser there was a significant reduction in the amount of S. mutans (p = 0.0068). For Lactobacillus ssp, no significant difference was observed between the removal methods (p = 0.089). After biomodification with 2.5% chitosan gel, there was a significant reduction in the amount of S. mutans, for both removal methods (p=0.0524). The application of chitosan promoted reduction of the amount of Lactobacillus ssp after selective removal by the conventional method, but not with Er: YAG laser (p = 0.000487). For the clinical analysis, the teeth were restored with adhesive (Single Bond Universal / 3M) and composite resin (Z350 Filtek / 3M) and evaluated in 7-day periods (baseline), 6 months and 1 year using modified USPHS criteria. Photographic images were taken to aid in the clinical analysis of the restorations. For analysis of the marginal adaptation of the replica of the restoration in electron microscopy, the teeth were molded with addition silicone. The data obtained in the clinical analysis were submitted to nonparametric Kruskal Wallis tests and Dun\'s complement (p<0.05). In the clinical analysis, a significant difference was observed for the selective removal of the caries lesion using the Er: YAG laser in relation to the descoloration and marginal adaptation criteria (p<0.05). It was concluded that the Er: YAG laser reduced the amount of S. mutans and Lactobacillus ssp and the biomodification with chitosan was effective to potentiate the antimicrobial effect, and that the clinical behavior of the restorations was not influenced by the method of removing the lesion from caries and biomodification of the affected dentin

Quitosana é um biopolímero natural obtido a partir da desacetilação de quitina. Embora a quitosana já seja utilizada na Odontologia, seu papel sobre o substrato dentinário de dentes decíduos não está bem elucidado. Portanto, o objetivo do presente estudo foi avaliar o papel da incorporação do gel de quitosana a 2,5 % na dentina de dentes decíduos afetada por lesão de cárie. Dentes decíduos extraídos foram coletados e submetidos à indução de lesão de cárie artificial. Após teste de microdureza inicial (n=28), os dentes foram estratificados para receber gel de quitosana a 2,5%. A superfície dentinária hígida (n=3), dentina desmineralizada (n=3) e dentina biomodificada com gel de quitosana a 2,5% (n=3) foram submetidas à Espectroscopia de Energia Dispersiva de Raio-X (EDS) e Microscopia Eletrônica de Varredura (MEV). O teste de microdureza também foi realizado após a indução de lesão de cárie artificial e após a biomodificação da dentina. Os dados foram avaliados usando o teste paramétrico one-way ANOVA para medidas repetidas. A análise dos dados para EDS foi efetuada por meio de teste não paramétrico de Kruskal-Wallis seguido pelo ajuste dos valores de significância pela correção de Bonferroni para múltiplos testes, bem como por meio de estatística descritiva dos dados obtidos através da fórmula: Variável de estudo controle/ controle × 100. A biomodificação da dentina não alterou a microdureza da superfície dentinária (p=0,339). A porcentagem atômica de cálcio revelou diferenças estatisticamente significantes antre a dentina hígida e biomodificada com quitosana (p<0.022), assim como a porcentagem atômica de fósforo que se mostrou superior no grupo que sofreu a biomodificação. A MEV revelou um expressivo número de túbulos dentinários obliterados, porém com maior diâmetro. As imagens topográficas revelaram, ainda, uma superfície lisa e regular após a biomodificação. Embora a aplicação do gel de quitosana a 2,5% na dentina parcialmente desmineralizada em dentes decíduos não foi capaz de aumentar o valor de microdureza, a biomodificação gerou uma superfície dentinária apropriada para procedimentos restauradores adesivos / Chitosan is a natural biopolymer obtained from chitin deacetylation. Although chitosan is already used in dentistry, this role on the primary teeth dentin substrate is not well elucidated. So, the aim to this study was to evaluate the role of the 2.5% chitosan gel incorporation in primary caries-affected dentin teeth. Extracted primary teeth were collected and submitted to artificial caries induction. Teeth were stratified to receive 2.5% chitosan gel after dentin microhardness initial test (n= 28). Healthy dentin (n=3), demineralized dentin (n=3) and biomodified dentin with 2.5% chitosan gel (n=3) were submitted to Energy Dispersive X-ray Spectroscopy (EDS) and Scanning Electron Microscopy (SEM). Microhardness Test was performed too after artificial caries induction and after dentin biomodification. Data were evaluated using one-way ANOVA repeated measures parametric test. Data analysis for EDS was performed using non-parametric Kruskal-Wallis test followed by adjustment of significance values by Bonferroni correction for multiple tests, as well as by means of descriptive statistics of the data obtained using the formula: Study variable control/control × 100. Dentin biomodification did not alter the subsurface microhardness of dentin (p=0,339). The calcium atomic percentage showed statistically significant differences between healthy and biomodificated dentin (p<0.022) and too presented superior phosphorus atomic percentage. SEM revealed expressive number of dentinal tubules obliterated, but a larger diameter. Topographic images revealed a smooth and regular surface in biomodified dentin. Although 2.5% chitosan gel application on partially demineralized dentin in primary teeth was not able to increase microhardness, the biomodification generated an appropriate dentin surface for adhesive restorative procedures

O objetivo do presente estudo foi avaliar o efeito da biomodificação com carbodiimida e quitosana fosforilada na dentina afetada por cárie. A amostra foi composta por 75 espécimes de dentina bovina submetidos à indução de lesão de cárie pelo método de ciclagem do pH (8h em solução desmineralizante e 16h em solução remineralizante) durante 14 dias. Foi realizada a remoção seletiva da lesão de cárie com broca em baixa rotação. Os espécimes foram divididos de acordo com a biomodificação da dentina: sem biomodificação (controle), carbodiimida (EDC) e quitosana fosforilada (P-chi). Os espécimes foram condicionados com ácido fosfórico 35%, e tratados com o agente biomodificador correspondente. Os espécimes foram restaurados com adesivo (Single Bond Universal - 3M ESPE) e resina composta (Z250 - 3M ESPE). Trinta espécimes foram submetidos à análise de microdureza da subsuperfície dentinária em quatro momentos: dentina hígida, dentina afetada por cárie, após a remoção seletiva da lesão de cárie (dentina residual) e, após a restauração dos espécimes. Os 36 espécimes restantes foram submetidos a ensaio de resistência de união à microtração (&mu;TBS) realizado após 24 h e 6 meses de armazenamento em água e microscopia eletrônica de varredura (MEV). Os dados foram analisados por meio de testes paramétricos e não paramétricos (&alpha;=0,05). A biomodificação da dentina residual afetada por cárie com EDC e P-chi não alterou a microdureza da subsuperfície dentinária (p=0,141). Na análise de &mu;TBS, não foram encontradas diferenças significativas entre os grupos 24 h e após 6 meses de armazenamento. Ao longo do tempo, o grupo controle (p<0,001) teve redução da &mu;TBS, o grupo EDC (p=0,099) foi capaz de manter a &mu;TBS e o grupo P-chi (p=0,005) promoveu aumento da &mu;TBS. A análise de MEV mostrou que os biomodificadores preservaram a interface adesiva após 6 meses de armazenamento. A biomodificação com carbodiimida e quitosana fosforilada não influenciou a microdureza da subsuperfície da dentina afetada por cárie, porém foi capaz de inibir a degradação da interface adesiva, mantendo a resistência de união da resina composta à dentina afetada após 6 meses de armazenamento em água / The aim of this study was to evaluate in vitro the effect of biomodification with carbodiimide and phosphorylated chitosan on caries affected dentin. The sample was composed by 75 bovine incisors, submitted to caries induction by pH cycling (8h in demineralizing solution and 16h in remineralizing solution) for 14 days. The selective caries removal was performed with bur at low-speed handpiece. Specimens were divided according to the dentin biomodification: no biomodification (control), carbodiimide (EDC) and phosphorylated chitosan (P-chi). All specimens were conditioned with 35% phosphoric acid and then treated with the correspondent biomodificator. Specimens were restored with an adhesive system (Single Bond Universal - 3M ESPE) and composite resin (Z250 - 3M ESPE). Thirty specimens were submitted to subsurface microhardness test in four periods of time: on sound dentin, on caries-affected dentin, after the selective removal of caries (residual caries-affected dentin) and after the restoration. The remaining 36 specimens were submitted to microtensile bond strength test (&mu;TBS) after 24 h and 6 months of water storage and to scanning electron microscopy analysis (SEM). Data were statistically analyzed by parametric and non-parametric tests (&alpha;=0.05). The biomodification of residual caries-affected dentin with EDC and P-chi did not change the subsurface microhardness (p=0.141). In the &mu;TBS test, there were no significant difference between groups at 24 h and 6 months of water storage. Over time, the control group (p<0.001) had a decreased on the &mu;TBS, the EDC group (p=0.099) was capable of maintained &mu;TBS and the P-chi group (p=0.005) increased in &mu;TBS. SEM analysis showed that biomodification preserved the adhesive interface after 6 months of water storage. The biomodification with carbodiimide and phosphorylated chitosan did not influence the subsurface microhardness of caries-affected dentin, however, it was capable of inhibit degradation of the adhesive interface, maintaining the microtensile bond strength of the composite resin to caries affected dentin after 6 months of water storage

Orientador: Leonardo Fernandes Fraceto / Resumo: A demanda pelo uso de produtos para uma agricultura sustentável com um menor impacto no ambiente tem sido cada vez maior. Sendo assim, a utilização do controle biológico é uma alternativa para diminuir o uso dos agrotóxicos e os consequentes riscos. O fungo Trichoderma harzianum é um exemplo de controle biológico eficaz contra o patógeno Sclerotinia sclerotiorum (mofo branco), o qual atinge diversas culturas e traz perdas na produção que podem chegar até 100 %. Porém, a utilização desse fungo de controle biológico pode encontrar alguns problemas como, por exemplo, estresse do fungo por fatores bióticos e abióticos. Uma possível solução para esse tipo de problema é a microenpcaulação. Desta maneira, este trabalho teve por objetivo desenvolver micropartículas de alginato de cálcio e de quitosana como sistema carreador de fungos, caracterizar o sistema de micropartículas através de métodos físico-químicos, avaliação molecular da microbiota do solo, antagonismo contra o fitopatógeno S. sclerotiorum e o efeito das micropartículas em plantas. O tamanho médio das micropartículas de alginato de cálcio e quitosana foi de 2000 μm e 2500 μm, respectivamente. A microscopia eletrônica de varredura confirmou a morfologia esférica das micropartículas após o processo de desidratação. O ensaio de fotoestabilidade revelou uma maior proteção do fungo Trichoderma harzianum quando encapsulado nas micropartículas de alginato de cálcio. Os ensaios de Calorimetria Diferencial de Varredura (DSC) e Es... (Resumo completo, clicar acesso eletrônico abaixo) / Doutor

Les nucléotides naturels et les analogues nucléotidiques présentent des activités pharmacologiques importantes : par exemple, le nucléotide adénosine triphosphate (ATP) présente un intérêt pour le traitement de l'ischémie ou de plaques d'athérosclérose. L'utilisation clinique de ces molécules est cependant limitée en raison de la présence d'un groupe triphosphate, qui est sujet à l'hydrolyse in vivo, et responsable de la forte hydrophilie des molécules, ce qui limite fortement leur capture par les cellules cibles et l'accès à leurs cibles pharmacologiques intracellulaires. Pour surmonter ces limitations et permettre l'administration de nucléotides et d’analogues nucléotidiques, l'utilisation de systèmes de drug delivery comme les nanoparticules pourrait assurer la protection et l'administration ciblée des molécules actives. Cependant, les nanoparticules conçues pour l’administration intraveineuse ne sont pas toujours adaptées au traitement de certaines maladies chroniques. C’est pour cela qu’un implant sous-cutané avec des caractéristiques de libération prolongée peut représenter une alternative valable, tout en étant peu invasif et capable d’atteindre les tissus lymphatiques, cible importante de plusieurs thérapies.Le premier chapitre de cette thèse porte sur la formulation de nanoparticules pour encapsuler l’ATP ou la zidovudine triphosphate (AZT-TP), grâce à la présence du chitosane (CS). Ces nanoparticules sont formées par interactions ioniques entre les charges positives du chitosane et les charges négatives des groupes triphosphates de l’ATP ou de l’AZT-TP. Dans ce travail, les nanoparticules sont caractérisées et leur délivrance cellulaire de l’ATP et de l’AZT-TP est démontrée sur une lignée cellulaire de macrophages. Dans un deuxième temps, la stabilité de ces systèmes a été améliorée afin d'obtenir un meilleur comportement en conditions physiologiques. Cette amélioration de la stabilité a été obtenue par la complexation du fer(III) au chitosane (CS-Fe). Cette stratégie a été appliquée aux nanoparticules de tripolyphosphate (TPP) et d’ATP. Les nanoparticules ont été ensuite testées sur deux lignées de cellules macrophagiques, montrant une internalisation améliorée de l’ATP par rapport aux nanoparticules précédentes. Enfin, les nanoparticules à base de CS-Fe et ATP ont été dispersées dans une solution de PLGA, dans le but de mettre au point un implant à formation in situ. Une fois en contact avec les fluides physiologiques, la suspension prend la forme d’un dépôt solide. Des études de libération in vitro montrent la capacité des systèmes de retenir les nanoparticules à l’intérieur de la matrice et de les libérer de façon progressive pendant 5 jours. Après administration sous-cutanée chez la souris, les implants de PLGA contenant les nanoparticules ont retenu l’ATP au lieu de l’injection jusqu’à 50 heures, comparé à quelques heures pour l’ATP libre et les nanoparticules libres, montrant ainsi leur pertinence comme systèmes pour la libération prolongée de nucléotides. / Natural nucleotides and nucleotide analogs display important pharmacological activities: for example the nucleotide adenosine triphosphate (ATP) could be an interesting molecule for the treatment of ischemia or atherosclerotic plaques. The clinical use of these molecules is however limited due to the presence of a triphosphate group, which is prone to hydrolysis in vivo, and responsible for the high hydrophilicity of the molecules, thereby strongly limiting their uptake by targeted cells and access to their intracellular pharmacological targets. To overcome these limitations and enable the administration of nucleotides and nucleotide analogs, the use of drug delivery systems such as nanoparticles may enable the protection and the targeted delivery of these drugs. Nanoparticles designed for intravenous injections are however not always convenient, e.g. in the case of chronic diseases. Therefore, a subcutaneous implant with sustained release features might represent a valid alternative, which is less invasive and can reach lymphatic tissues (important targets of many therapies). The first chapter of this thesis presents the formulation of nanoparticles to encapsulate ATP as well as zidovudine triphosphate (AZT-TP), thanks to the presence of chitosan (CS). These nanoparticles are formed through ionic interactions between the positive charges of chitosan and the negative charges of the triphosphate groups of ATP or AZT-TP. In this work, nanoparticles are characterized and their cellular delivery of ATP and AZT-TP inside a macrophage cell line is demonstrated. In a second time, the stability of these systems has been improved in order to obtain a better behavior in physiological conditions. This improved stability has been achieved through the complexation of chitosan to iron(III) (CS-Fe). This strategy has been applied to TPP and ATP nanoparticles. These nanoparticles have been tested on two macrophages cell lines showing an improved internalization compared to the previous ones. Finally, CS-Fe/ATP nanoparticles have been dispersed in a PLGA solution in order to develop an in situ forming implant. Once in contact with physiological fluids, the suspension turns into a solid depot. In vitro release studies show the ability of the systems to retain nanoparticles inside the matrix and to gradually release them over 5 days. After subcutaneous administration to mice, PLGA implants containing nanoparticles were able to retain ATP at the injection site for up to 50 hours, as compared to few hours of free ATP or free nanoparticles, showing therefore their relevance as sustained release systems of nucleotides.

Neste trabalho, a irradiação por feixe de elétrons foi estudada como uma ferramenta para a reticulação do tecido de pericárdio bovino (PB). A conjugação de quitosana e fibroína de seda no tecido de PB liofilizado também foi objeto de estudo deste trabalho. Para isto, amostras de PB foram liofilizadas e irradiadas em um acelerador de elétrons utilizando-se diferentes doses e taxas de dose. Essas amostras foram analisadas por calorimetria exploratória diferencial (DSC), análises termogravimétricas (TGA), espectroscopia Raman, teste de intumescimento, microscopia eletrônica de varredura (MEV), microscopia eletrônica de transmissão (MET), testes de tração e quanto sua biofuncionalidade. Após as análises foi possível concluir que a irradiação do tecido na ausência de oxigênio favorece a reticulação das fibras de colágeno, enquanto na presença de oxigênio observou-se preferencialmente a cisão das fibras de colágeno. Ambas as amostras apresentaram diminuição de suas propriedades mecânicas. Também pôde-se concluir que a irradiação tanto na ausência quanto na presença de oxigênio produz um novo biomaterial cuja adesão e proliferação de células endoteliais é favorecida ao longo do tecido. Na segunda etapa deste trabalho amostras de PB liofilizadas foram incorporadas em soluções de quitosana, fibroína de seda e suas misturas (1:3, 1:1, 3:1). Depois de modificadas, as amostras foram novamente liofilizadas e submetidas à irradiação. Estas amostras foram caracterizadas por espectroscopia Raman, e avaliadas quanto sua citotoxicidade, biofuncionalidade e potencial de calcificação. Após lavagens das amostras de biomaterial com solução aquosa (NaCl 0,9%), as mesmas não apresentaram toxicidade. O teste de biofuncionalidade mostrou que as amostras de PQSFI (todas as proporções) favoreceram a adesão e crescimento das células endoteliais. Todas as amostras induziram a calcificação, entretanto apresentaram uma relação Ca/P menor do que a da hidroxiapatita. / In this work, electron beam irradiation was studied as a crosslinker of bovine pericardium tissue (BP). The treatment of BP with chitosan and silk fibroin was also the goal of this study. Samples of BP were freeze-dried and irradiated in an electron beam accelerator using different doses and dose rates. Samples were analyzed by differential scanning calorimetry (DSC), thermogravimetric analysis (TGA), Raman spectroscopy, water uptake tests, scanning electron microscopy (SEM), transmission electron microscopy (TEM), tensile tests and evaluation as biofunctionality. After analysis, it was concluded that irradiation of the tissue in the absence of oxygen promotes crosslinking of collagen fibers. On the other hand, in the presence of oxygen the chain scission of the collagen fibers was observed, and both samples suffered a decrease in their mechanical properties. It was also concluded that irradiation, both in the absence or presence of oxygen, promotes the adhesion and proliferation of endothelial cells throughout the tissue. In the second part of this work, lyophilized BP were incorporated into chitosan, silk fibroin solutions and mixtures of chitosan/silk fibroin in the ratio of 1:3, 1:1, 3:1. Then, the samples were again lyophilized and submitted to irradiation. These samples were characterized by Raman spectroscopy, and tested for their cytotoxicity, biofunctionality, and calcification. After washing the biomaterial with aqueous solution (NaCl 0.9%), the samples did not show cytotoxicity. Biofunctionality test showed that PQSFI samples (all proportions) promoted the adhesion and growth of endothelial cells. All samples induced calcification, but they exhibited a Ca/P ratio lower than hydroxyapatite.

<p style="text-align: justify;">Mikrokapsulacija je tehnika kojom se nestabilne, međusobno inkompatibilne i biolo&scaron;ki aktivne supstance prevode u stabilniji oblik, ili se omogućuje njihovo kontrolisano i ciljano oslobađanje. Osobine formiranih mikrokapsula, kinetika i mesto otpu&scaron;tanja inkapsuliranih materija zavise, pre svega, od njihovog omotača. Savremene tendencije razvoja prehrambenih proizvoda i proizvoda farmaceutske i kozmetičke industrije, su sve vi&scaron;e usmerene ka upotrebi prirodnih i biorazgradivih polimernih materija za formiranje omotača mikrokapsula.<br />Cilj ove disertacije je mogućnost primene hitozana, netoksičnog, biorazgradivog derivata hitina, kao materije omotača mikrokapsula sa uljnim sadržajem. S obzirom na njegovu slabu povr&scaron;insku aktivnost, istraživanja su usmerena na primenu interakcija hitozana sa suprotno naelektrisanim jonskim povr&scaron;inski aktivnim materijama (PAM) u vodenim rastvorima, kao mehanizmu za njegovo deponovanje na graničnoj povr&scaron;ini ulje/voda.<br />Primenom različitih metoda (tenziometrija, viskozimetrija, turbidimetrija, merenje elektroforetske pokretljivosti) interakcije hitozana sa natrijum-dodecil-sulfatom (SDS) i natrijum-lauriletar-sulfatom (SLES) su detaljno ispitane. Definisane su promene kako na granici faza, tako i unutar rastvora, a mehanizam formiranja kompleksa hitozan/PAM različitih osobina je u potpunosti razja&scaron;njen. Utvrđeno je da do formiranja stabilnog koacervata dolazi pri masenom odnosu hitozan:SLES 1:2 i hitozan:SDS 1:2.<br />Ispitivanje uticaja interakcije hitozan-PAM na osobine emulzionih sistema tipa ulje u vodi (veličina i raspodela veličina kapi, stabilnost) omogućilo je odabir hitozan-SLES sistema kao omotača pogodnog za dobijanje mikrokapsula uljnog sadržaja. Na osnovu ovih rezulatata kao uljna faza odabrani su trigliceridi srednje dužine ugljovodoničnih lanca (TSDL). Su&scaron;enjem emulzija primenom spray drying postupka dobijene se mikrokapsule uljnog sadržaja sa vitaminom E i ispitan je uticaj umreživača na njihove osobine. Karakterizacijom dobijenih mikrokapsula (određivanje sadržaja vlage, ispitivanje morfologije povr&scaron;ine, efiksanost inkapsulacije vitamina E, kinetika otpu&scaron;tanja vitamina E u in vitro uslovima) zaključeno je da na osobine mikrokapsula utiče vrsta i koncentracija umreživača. Mikrokapsule čiji omotač nije umrežen pokazale su najbolje karakteristike.</p> / <p>Microencapsulation is a technique that unstable, incompatible and biologically active substances converted to a more stable form, or allow their controlled and targeted release. The properties of formed microcapsules, kinetics and the place of release of encapsulated substances, primarly depend on their shell characteristic. Modern trends in the development of food products and products of the pharmaceutical and cosmetic industries are increasingly focused on the use of natural and biodegradable polymeric materials for coatings.<br />The aim of this dissertation is to investigate the possibility of using chitosan, non-toxic and biodegradable chitin derivative, as a shell material of microcapsules with oil content. Due to its low surface activity, the research is focused on the utilization of chitosan interactions with opositly charged ionic surfactants (sodium dodecyl sulfate (SDS) and sodium lauryl ether sulfate (SLES)) in aqueous solutions as mechanisms for its depositing at the oil/water interface.<br />Based on detailed investigation of interactions in the chitosan-ionic surfactant systems using different methods (tensiometry, viscometry, turbidimetry, measurement of electrophoretic mobility), changes have been defined both at the interface and within the bulk, as well as the mechanism of formation of the coacervate phase. It has been determined that at chitosan:SLES and chitosan:SDS mass ratio of 1:2 stabile coacervate were formed.<br />An investigation of the influence of interaction on the properties of oil-in-water emulsion systems (size and distribution of droplet size, stability) enabled the chitosan-SLES system to be selected as a shell suitable for obtaining microcapsules of the oil content. Also, based on these results, medium-chain triglycerides were selected as the oil phase of the emulsion.<br />Microcapsules with vitamin E were obtained by spray drying of emulsions stabilized with chitosan/SLES complex. The influence of the crosslinker on the properties of microcapsules was investigated. Characterization of obtained microcapsules (moisture content determination, investigation of the surface morphology, efficiency of the vitamin E encapsulation, release in vitro kinetics of vitamin E) showed that type and concentration of crosslinking agents had influences the properties of microcapsules. Microcapsules without crosslinking agents have the most suitable characteristics.</p>