The current clinical treatment for bone deficiencies in clinics includes autografts, allografts and bone graft substitutes. All these treatments, however, still have various limitations. Therefore, in this project, we aim to design, synthesise and characterise a new series of novel polymer networks (PNs) to promote bone formation and offer a new therapeutic solution. An innovative polymerisation technique was used to synthesise the novel polymer-based materials. Various lactone monomers were applied with layered double hydroxides (LDHs) as the initiator for the polymerisation. Copolymerisation was used to control the final product degradation rate, microstructure and biocompatibility. SEM and XRD were used to confirm the formation of the 3D polymer microstructure, PNs indicated interconnected fibrous microstructure (thickness of the fibre ranged from 50-150 nm). Up to 90% polymer yield was achieved using thermogravimetric analysis. Both 1H and 13C nuclear magnetic resonance (NMR) were used to understand the effects of monomer combinations in the polymerisation. The effects of the materials on osteosarcoma cell line Saos-2 were investigated. Representative material compositions and their determined mass concentrations were applied using osteogenic culture medium with Saos-2. The PNs have indicated enhanced osteogenesis using varied assays. A poly(lactide-co-caprolactone) PN (monomer mass ratio 1:1) at 6.25 mg/ml, in particular, resulted in enhanced alkaline phosphate activity not only in growth medium but also in osteogenic medium. Moreover, it indicated the highest bone nodule production in area percentage compared to the other PN samples using Saos-2. Mg-substituted calcified extracellular matrix was formed by the use of PN materials. Therefore, this novel PN could have the possibility to be used as an acellular scaffold or a sprinkle-on powder local to defect site to enhance bone formation in vivo. In addition, this novel technology could be used as a scaffold for tissue engineering.

Na presente pesquisa, produziu-se amostras de ferro puro poroso como biomaterial degradável visando a aplicação em stents, pelo processo de moldagem por injeção de pós metálicos (MPI). Os efeitos da fração volumétrica de ferro puro na mistura de injeção e da temperatura de sinterização na porosidade, microestrutura, propriedades mecânicas, propriedades de superfície, de degradação in vitro e de biocompatibilidade, foram investigados. Os resultados obtidos foram comparados com o ferro puro fabricado por fusão e com o aço inoxidável AISI 316-L. Encontrou-se que o grau de porosidade remanescente nas amostras sinterizadas foi o principal fator influenciando as propriedades mecânicas e de superfície, influenciando indiretamente os demais resultados. O ferro puro produzido por MPI exibiu valores de limite de escoamento entre 59 e 114 MPa e limite de resistência máximo de 210 MPa, com alongamento entre 10 e 50 %. A alta ductilidade é uma propriedade especialmente requerida em materiais para potencial aplicação em stents. Suas taxas de degradação em solução de Hank foram superiores às do ferro puro fabricado por fusão. O material fabricado com mistura de injeção contendo fração de ferro de 66 % (acima da fração crítica) mostrou o maior alongamento e boa taxa de degradação, um resultado interessante, pois segundo a literatura, valores acima da fração volumétrica crítica não são amplamente explorados. Os testes de biocompatibilidade mostraram excelente hemocompatibilidade do ferro puro fabricado por MPI com as células do sangue. Todas as condições testadas mostraram um nível de citotoxicidade abaixo do recomendado pela norma vigente, mas este dependendo da concentração de íons de ferro empregada e do grau de porosidade. Entre todas as condições de ensaio investigadas, as amostras contendo fração volumétrica de ferro de 62 % inicialmente na mistura de injeção e sinterizadas a 1120 oC, apresentaram a melhor combinação de propriedades para aplicação em stents. Concluiu-se que a MPI é um método tecnicamente viável como rota de produção de tubos de parede fina precursores para fabricação de stents biodegradáveis. / In the present research, an attempt was made to produce porous pure iron, as a metallic degradable biomaterial potentially for stent application, via the MIM route. The effects of iron powder loading and sintering temperature on the porosity, microstructure, mechanical properties, surface properties and in vitro degradation behavior of MIM iron were investigated. The results obtained were compared to those of cast iron. It was found that the amount of porosity remained in the as-sintered specimens had a major effect on their surface and mechanical properties. The MIM pure iron showed yield strength values between 59 and 114 MPa and maximum tensile strength of 210 MPa, with elongation values between 10 and 50 %. A high ductility is a specially required property of stent materials. Its degradation rates in Hank’s solution were superior to the degradation rate of cast iron. The material made from the feedstock containing 66 % of iron powder, above the critical powder loading, showed the highest elongation and a good in vitro degradation rate. This result is interesting, once according to the literature, powder loadings above the critical value are not well explored. The biocompatibility tests showed excellent hemocompatibility of MIM pure iron with blood cells. All conditions tested showed toxicity level below the values determined by current standards, but depending of Fe ions concentration and porosity level. Between all the conditions tested in the present investigation, the 62 % powder loading sample, sintered at 1120 oC, showed the best combination of properties for stent application. In conclusion, MIM is a promising method to be developed as a new route to produce thin-wall tubes for biodegradable stents.

Devido ao crescente interesse em polímeros biodegradáveis, muitos estudos têm sido realizados a fim de se obter polímeros biodegradáveis com melhores propriedades mecânicas e de processamento. O poli(3-hidroxibutirato) (PHB) tem sido apresentado como um substituto para polímeros não-biodegradáveis em aplicações comerciais. Porém, sua elevada cristalinidade, instabilidade térmica e alto custo de produção são problemas que dificultam sua comercialização. Uma alternativa para modificá-lo e melhorar suas propriedades mecânicas é através de reações de transesterificação com poli(?-caprolactona) (PCL), um poliéster sintético, também biodegradável e com alto potencial para uso como biomaterial. Neste trabalho, realizou-se a extrusão reativa de blendas de PHB e PCL e o copolímero obtido foi caracterizado por ensaios de solubilidade, calorimetria exploratória diferencial (DSC), espectroscopia no infravermelho por transformada de Fourier (FTIR), espectrometria de ressonância magnética nuclear de hidrogênio (RNM-1H) e de carbono (RNM-13C). Os resultados indicaram que houve modificação do PHB resultando no copolímero PHB-co-PCL, que apresentou menor cristalinidade que os homopolímeros de partida. Com o PHB-co-PCL, foram feitas medidas de viscosidade e ensaios de eletrofiação, variando-se os parâmetros do processo e da solução. As análises por microscopia eletrônica de varredura (MEV) apontaram formação de fibras lisas e uniformes com diâmetros médios entre 900 a 1200 nm. Análise de viabilidade celular confirmou que o material não é citotóxico, favorecendo sua aplicabilidade em mantas porosas na engenharia de tecidos. / Due to the increasing interest in biodegradable polymers, many studies have been conducted in order to obtain biodegradable polymers with improved mechanical and processing properties. The poly(3-hydroxybutyrate) (PHB) has been proposed as an ideal substitute for non-biodegradable polymers in commercial applications. However, its high crystallinity, thermal instability and high production costs are problems that have limited its commercialization. An alternative to modify it and improve its mechanical properties is through transesterification reactions with poly(?-caprolactone) (PCL), a synthetic polyester, also biodegradable and with high potential for use as a biomaterial. In this work, reactive extrusion of PHB/PCL blend was carried out, and the copolymer was characterized by solubility tests, differential scanning calorimetry (DSC), Fourier transform infrared spectroscopy (FTIR), hydrogen (1H-NMR) and carbon (13C-NMR) nuclear magnetic resonance spectroscopies. The results indicated that PHB was modified, resulting in the copolymer PHB-co-PCL, showing lower crystallinity than the homopolymers. Viscosity measurements of PHB-co-PCL solutions were performed and electrospinning tests were carried out under different conditions. The scanning electron microscopy analysis (SEM) indicated the formation of smooth and uniform fibers, with average diameter between 900-1200 nm. Cell viability analysis confirmed that the material is not cytotoxic, favoring its applicability for the production of porous fiber mats in tissue engineering.

O Histoplasma capsulatum é um fungo dimórfico responsável por infecções pulmonares graves caracterizadas por reação granulomatosa. Sua incidência vem aumentando nos últimos anos devido principalmente às alterações imunológicas relacionadas ao comprometimento da imunidade celular. Anteriormente, nosso grupo de pesquisa demonstrou o envolvimento dos leucotrienos (LTs) nos mecanismos de defesa do hospedeiro durante a histoplasmose. Além disso, demonstrou que antígenos livres de células (CFAgs, do inglês cell-free antigens), derivados de H. capsulatum, quando empregados na imunização de animais, conferem proteção eficiente aos mesmos e controle da infecção, uma vez que ativam a imunidade celular, e aumento da produção de LTs nos pulmões dos animais imunizados. Assim, nosso grupo desenvolveu microesferas (MS) biodegradáveis, constituídas de ésteres derivados dos ácidos láctico e glicólico (PLGA), contendo LTB4. Estas MS foram avidamente fagocitadas por macrófagos in vitro e aumentaram o recrutamento de leucócitos para os pulmões, quando administradas intratraquealmente. Diante do papel dos LTs na histoplasmose e dos potenciais terapêutico e profilático dos CFAgs, o objetivo deste estudo foi avaliar as atividades biológicas in vitro e in vivo de MS contendo LTB4 e/ou CFAgs. Assim, foram desenvolvidas MS (PLGA) contendo LTB4 e/ou CFAgs através do processo de simples ou dupla emulsão seguido pela extração do solvente. Este método permitiu uma eficiente encapsulação tanto do mediador lipídico quanto dos antígenos protéicos e um perfil de liberação sustentada ao longo dos dias avaliados. O potencial zeta e a morfologia das MS não foram alterados com o processo de microencapsulação; da mesma forma, a integridade dos CFAgs não foi interferida. Para estudos in vitro, empregamos macrófagos diferenciados de medula óssea murina (BMDM). O tamanho adequado das MS contribuiu para sua eficiente fagocitose pelos BMDM e as MS contendo LTB4 e/ou CFAgs modularam a produção de TNF-, IL-1, IL-6 e IL-12, quimiocinas (KC, MCP-1 e RANTES), e nitrito, sendo que as MS-CFAgs mostraram-se mais potentes. O estímulo com as diferentes MS induziu discreto aumento na expressão de CD86 na superfície de BMDM. Neste contexto, verificamos o envolvimento do fator de transcrição NF-B durante a ativação de BMDM induzida pelas MS. Apesar da eficiente ativação dos BMDM induzida pelas MS, não foi possível evidenciar uma resposta imune celular em animais imunizados com as MS-LTB4+CFAgs ou MS-CFAgs. Portanto, futuros experimentos deverão ser realizados a fim de investigar o potencial profilático das MS contendo LTB4 e/ou CFAgs na histoplasmose. / Histoplasma capsulatum is a dimorphic pathogenic fungus that causes a pulmonary disease characterized by chronic granulomatous reaction. In the last years, the incidence of histoplasmosis has increased, mainly as a result of the immunological alterations involved with deficiency of the cellular immunity. Previously, our research group demonstrated the involvement of leukotrienes (LTs) on host defense mechanisms during the histoplasmosis. Cell-free antigens (CFAgs) derived from H. capsulatum, when employed for animals´ immunization, can confer efficient protection and control of the infection, since they activate the cellular immunity. Furthermore, the protection of CFAgs-immunized mice was associated with increased LTB4 generation in the lungs. Based on these results, our group developed biodegradable microspheres (MS) based on PLGA containing LTB4. We showed that these MS were phagocytosed by macrophages in vitro and increased the leukocyte recruitment into the lungs, when administrated via intratracheal. Because the role of leukotrienes in the histoplasmosis and therapeutic and profilatic effects of CFAgs, the aim of this study was evaluate the in vitro and in vivo biological activities of MS containing LTB4 and/or CFAgs. Then, MS (PLGA) containing LTB4 and/or CFAgs were developed through simple or double emulsion/extraction process. This method allowed an efficient encapsulation of the lipid mediator and CFAgs, and a sustained release profile during the evaluated days. Zeta potential and morphology of MS were not altered with the microencapsulation process; CFAgs integrity was not interfered. For in vitro studies, we employed bone marrow-derived macrophages (BMDM). The appropriate size of MS contributed for efficient uptake by BMDM. MS containing LTB4 and/or CFAgs modulated the TNF-, IL-1, IL-6 and IL-12, chemokines (KC, MCP-1 and RANTES), and nitrite production by BMDM, since the MS-CFAgs showed potent immunostimulant effect. Moreover, the stimulus with different MS provoked a discreet increase in the CD86 cell expression. Also we verified an involvement of the transcription factor NF-B during the BMDM activation induced by MS. Even though the in vitro biological activities on BMDM, it was not possible to evidence a cellular immune response in immunized mice with the MS-LTB4+CFAgs or MS-CFAgs. Therefore, future experiments should be conducted in order to investigate the profilatic potential of MS containing LTB4 and/or CFAgs in the histoplasmosis.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / O objetivo desse trabalho foi desenvolver filmes biodegradÃveis à base de amido e goma de cajueiro, acrescidos ou nÃo de montmorilonita. Em uma primeira etapa, foi feito um delineamento experimental composto central a partir de misturas entre amido e goma de cajueiro (1:1, m:m) adicionadas de diferentes concentraÃÃes da argila montmorilonita (0 a 15% - MMT) e tempos de ultrassom (0 a 5 min), onde foi realizado um estudo das variaÃÃes de desempenho de filmes produzidos por deposiÃÃo (casting), em funÃÃo das variÃveis. Os filmes foram avaliados em termos de propriedades Ãticas, mecÃnicas, de barreira e tÃrmicas e analisados por Microscopia eletrÃnica de varredura (MEV) e Ãngulo de contato. Em uma segunda etapa do trabalho, as formulaÃÃes dos filmes amido e goma de cajueiro (AGC) e os de amido, goma de cajueiro e montmorilonita AGC-MMT foram testadas como revestimentos para amÃndoas de castanhas de caju. As amÃndoas revestidas com AGC e AGC-MMT, assim como um controle (amÃndoas sem revestimento), foram acondicionadas em sacos plÃsticos de polietileno de baixa densidade, estocadas em temperatura e umidade ambiente e tiveram sua estabilidade avaliada. Os testes de estabilidade realizados foram baseados em medidas de absorÃÃo de umidade, atividade de Ãgua e do grau de oxidaÃÃo das amÃndoas (determinado por Ãndice de perÃxido e Ãndice de acidez). O incremento de MMT interferiu nas propriedades Ãticas dos filmes, aumentando sua opacidade. A permeabilidade ao vapor de Ãgua dos filmes produzidos foi significativamente (p < 0,05) reduzida pela MMT. A resistÃncia à traÃÃo e o mÃdulo elÃstico apresentaram valores mÃximos em uma faixa intermediÃria de concentraÃÃo de MMT. O tempo de ultrassom favoreceu a dispersÃo da argila em concentraÃÃes baixas de MMT, porÃm em concentraÃÃes maiores favorece a formaÃÃo de aglomerados, o que tambÃm à visualizado nas micrografias obtidas por MEV. Com o aumento do tempo de ultrassom, a resistÃncia à traÃÃo e o mÃdulo tenderam a aumentar. As curvas de DSC do amido, goma de cajueiro e do filme produzido sem MMT apresentaram picos endotÃrmicos similares em torno de 110ÂC, que podem estar relacionados à superposiÃÃo de eventos como vaporizaÃÃo da Ãgua e fusÃo da fase cristalina dos componentes. A medida do Ãngulo de contato comprovou que os filmes produzidos sÃo altamente hidrofÃlicos. Os testes de estabilidade realizados mostram que os revestimentos AGC e AGC-MMT conferem menor acidez e Ãndice de perÃxidos quando comparados a um controle podendo ser uma alternativa para aumento da vida de prateleira das amÃndoas de castanhas de caju, servindo como embalagem primÃria. / The purpose of this study was to develop biodegradable films based on starch and cashew gum, added or not with montmorillonite. At a first stage, a central composite experimental design was carried out from mixtures of starch and cashew gum (1:1, m: m) added with different concentrations of montmorillonite (0-15% - MMT) and submitted to variable ultrasound times (0-5 min), In a study of variations in the performance of films produced by casting. The films were evaluated in terms of optical properties, mechanical, barrier and thermal. As well as scanning electron microscopy (SEM) and contact angle. In a second stage of the work, the formulations of the films based on starch and cashew tree gum (SCG) and starch cashew tree gum and montmorillonite (SCG-MMT) were tested as coatings for cashew kernels. The kernels coated with SCG and SCG-MMT, as a control (uncoated kernels) were placed in plastic bags of low density polyethylene, stored at room temperature and relative humidity and their stability were evaluated. Stability tests were based on measurements of moisture absorption, water activity and the degree of oxidation of almonds (determined by peroxide value and acid value). The increase of MMT concentration interfered with the optical properties of the films, increasing its opacity. The water vapor permeability of films was significantly (p <0.05) reduced by the MMT. The tensile strength and elastic modulus showed a peak in an intermediate range of concentration of MMT. The time of ultrasound favors the dispersion of clay in low concentrations of MMT, but at higher concentrations et led to formation of agglomerates, which also appeared in SEM micrographs. With the increase of ultrasound times tensile strength and modulus tended to increase. DSC curves of starch, cashew gum and the film produced without MMT the endothermic peaks had similar MMT about 110  C, that may be related superposition events such as the vaporization of water and melting of the crystalline phase of the components. The contact angle measurement showed that the films are highly hydrophilic. Stability tests performed show that the coatings AG and AG-MMT give less acidity and peroxide value when compared to a control, may be an alternative of the use to increase the shelf life of almonds cashew nuts serving as primary packaging.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / Este trabalho busca o desenvolvimento de filmes biodegradÃveis a partir de gelatina obtida de resÃduos de tilÃpia (Oreochromis niloticus) e galactomanana extraÃda da Algaroba (Prosopis juliflora). Foi utilizado um delineamento experimental do tipo centrÃide simplex para avaliar o efeito das proporÃÃes entre os dois componentes â gelatina (GE) e galactomanana (GA) - sobre as propriedades dos filmes formados. Os resultados dos filmes obtidos a partir do delineamento de misturas indicaram que o filme de GE e GA nas mesmas proporÃÃes apresentaram os melhores resultados de permeabilidade ao vapor de Ãgua e ensaios mecÃnicos, sendo posteriormente esse filme, submetido à incorporaÃÃo de nanocelulose extraÃda da vagem da algaroba. A formulaÃÃo dos filmes de gelatina e galactomanana foi feita utilizando 25% de glicerol (em base seca de gelatina e galactomanana) variando a quantidade de nanocristais de celulose da fibra da algaroba nas concentraÃÃes de 2,5, 5 e 7,5% (em base seca de gelatina e galactomanana). Os filmes foram obtidos por casting e caracterizados quanto à permeabilidade ao vapor de Ãgua (PVA), propriedades mecÃnicas de resistÃncia à traÃÃo, elongaÃÃo e mÃdulo elÃstico, microscopia Ãtica (MO) e microscopia eletrÃnica de varredura (MEV), infravermelho por transformada de Fourier (FTIR), opacidade, anÃlise termogravimÃtrica (TGA) e calorimetria exploratÃria diferencial (DSC). Os nanocristais de celulose foram efetivos para reduzir a PVA quando adicionados a 5%. A adiÃÃo de nanocelulose incrementou a resistÃncia à traÃÃo e o mÃdulo a partir de 2,5%, com um pequeno decrÃscimo na concentraÃÃo de 7,5%. NÃo houve efeito significativo com a adiÃÃo de nanocelulose na elongaÃÃo dos filmes. A anÃlise de TGA mostrou que crescentes concentraÃÃes de nanocelulose aumentaram a estabilidade tÃrmica dos filmes, e a anÃlise de DSC comprovou a formaÃÃo de blenda. As imagens de microscopia permitiram observar aglomeraÃÃes de nanocristais à medida que se aumentou as concentraÃÃes. Na anÃlise de FTIR nÃo houve modificaÃÃo na estrutura dos filmes, ocorrendo apenas deslocamento ou reduÃÃo de algumas bandas. A opacidade aumentou com a incorporaÃÃo de nanocelulose, porem nÃo aumentou com a variaÃÃo das concentraÃÃes. / This work seeks developing biodegradable films from gelatin obtained from waste tilapia (Oreochromis niloticus) and galactomannan extracted from Mesquite (Prosopis juliflora). An experimental design type simplex centroid was used to assess the effect of the ratios between the two components - gelatin (GE) and galactomannan (GA) â on the properties of the formed films. The results of the films obtained from the mixture design indicated that the film containing a GE:GA of 1:1 showed the best results for water vapor permeability and mechanical tests, being this film later submitted to the incorporation of cellulose nanocrystals extracted from the leguminous of the mesquite. The formulation of the films of gelatin and galactomannan was performed using glycerol 25% (on a dry basis of gelatin and galactomannan) varying the amount of nanocellulose from mesquite fiber on concentrations of 2.5, 5 and 7, 5% (dry basis of gelatin and galactomannan). The films were obtained by casting and were characterized in terms of their water vapor permeability (WVP), mechanical properties (tensile strength, elongation and elastic modulus), optical scanning electron microscopy (SEM), optical microscopy (OM), Fourier Transform Infrared (FTIR), opacity, thermal gravimetric analysis (TGA) and differential scanning calorimetry (DSC). The nanocellulose at 5% was effective to reduce the WVP. Nanocellulose increased the tensile strength and modulus with a slight decrease at the highest concentration (7.5 %). Nanocellulose did not affect significantly the film elongation. TGA analysis showed that increasing nanocellulose concentrations increased the film thermal stability, and the DSC analysis proved the formation of blends. The microscopy images revealed the formation of clusters of nanocrystals as their concentration increased. FTIR analysis revealed no major changes in the film structure apart from offset or reduction of some bands. The opacity increased with the incorporation of nanocellulose, although it did not increase with increasing nanocellulose concentrations.

Aliado ao fato dos biomateriais ainda serem pouco explorados pelas indústrias alimentícias, este trabalho propôs o desenvolvimento de embalagens que sejam, além de biodegradáveis, também ativas através do uso de um agente antimicrobiano natural capaz de inibir a proliferação de fungos correntes em produtos de panificação (Penicillium commune e Eurotium amstelodami). Primeiramente, filmes biodegradáveis a base de fécula de mandioca foram elaborados pela técnica de casting, usando açúcares e glicerol como plastificantes. O aumento do conteúdo de glicerol causou diminuição da resistência máxima à tração e elevação dos valores de propriedades de barreira. Numa segunda etapa do trabalho, a introdução de nanopartículas de argila esmectita influenciou positivamente as propriedades de barreira dos filmes, devido à diminuição observada nos valores de permeabilidade ao vapor de água e coeficiente de permeabilidade ao oxigênio. Nesta fase, a variação do conteúdo de glicerol também afetou significativamente as propriedades mecânicas e de barreira dos filmes biodegradáveis. As concentrações inibitórias mínimas dos óleos essenciais de cravo e de canela contra os fungos estudados foram definidas e o óleo essencial de canela foi selecionado, para ser incorporado aos filmes biodegradáveis, em três conteúdos distintos, pois foi o composto que mostrou uma inibição mais eficiente. A atividade antimicrobiana dos filmes biodegradáveis com incorporação de óleo essencial de canela foi testada sobre os micro-organismos escolhidos através de testes de difusão em halo, cujos resultados foram suficientes para demonstrar o potencial ativo da embalagem desenvolvida. Como método alternativo de incorporação do agente antimicrobiano, gás carbônico (CO2) em estado supercrítico foi utilizado como solvente. Os resultados obtidos foram promissores, uma vez que se observou incorporação de agente antimicrobiano dentro da matriz polimérica em quantidade suficiente para inibir a proliferação dos fungos testados. / This work concerned the development of biodegradable and active packaging based on cassava starch and natural antimicrobial agents that efficiently inhibit growth of fungi commonly found in bread products (Penicillium commune and Eurotium amstelodami). Biodegradable films were prepared by the casting technique, using sugars and glycerol as plasticizers. An increase in glycerol content caused a decrease in tensile strength and an elevation on barrier properties results. In a second phase of the work, incorporation of nanoclays diminished water vapor and oxygen permeability thus improving film packaging properties. Two antimicrobial agents were tested (cinnamon and clove essential oils) and cinnamon essential oil was chosen to be incorporated in biodegradable films based on cassava starch because it was more efficient against Penicillium commune and Eurotium amstelodami. Antimicrobial activity of biodegradable films with cinnamon essential oil was evaluated by means of a halo diffusion test in which an inhibition zone around a sample of the biodegradable and active film was analysed. As alternative method of antimicrobial agent incorporation, carbon dioxide (CO2) in supercritical state was used as solvent. Promising results were obtained since the amount of antimicrobial agent incorporated inside the polymeric matrix was sufficient to inhibit proliferation of fungi.

Vaccines have become an important approach for the treatment of cancer. Cancer vaccines help the immune system to detect and eradicate tumor cells. Also, cancer vaccines are designed to stimulate an effective immune response that can create long-term immune memory to prevent tumor recurrence. This treatment approach involves the administration of a vaccine comprising or encoding an antigen and can often be combined with an adjuvant to further promote the immune response. The goal of this research was to study the effect of the polyanhydride composition of prophylactic cancer vaccine formulations on the tumor-specific immune response. To achieve this goal, three different amphiphilic polyanhydride copolymers were generated comprising different ratios of 1,6-bis-(p-carboxyphenoxy)-hexane (CPH) and 1,8-bis-(p-carboxyphenoxy)-3,6-dioxaoctane (CPTEG) or sebacic anhydride (SA) monomers. These copolymers were used to fabricate particles encapsulating a model antigen, ovalbumin (OVA), using a double emulsion solvent evaporation technique. The ability of the three different compositions of amphiphilic polyanhydride copolymers (50:50 CPTEG:CPH, 20:80 CPTEG:CPH, and 20:80 CPH:SA) encapsulating OVA to elicit immune responses was investigated. Further, the impact of soluble unmethylated oligodeoxynucleotides containing deoxycytidyl-deoxyguanosine dinucleotides (CpG ODN), an immunologic adjuvant, on the immune response to the three formulations was also studied. The immune response to cancer vaccines was measured after treatment of C57BL/6J mice with two subcutaneous injections, seven days apart, of 50 μg OVA encapsulated in particles composed of different polyanhydride copolymers with or without 25 μg CpG ODN. In vivo studies showed that 20:80 CPTEG:CPH particles encapsulating OVA significantly stimulated the highest level of CD8+ T lymphocytes, generated the highest serum titers of OVA-specific IgG antibodies, and produced longer survival in comparison to formulations involving the other polyanhydride copolymers. The results also revealed that supplementing the vaccine formulations with CpG ODN did not enhance the immunogenicity of OVA. These results accentuate the crucial role of the copolymer composition of polyanhydrides in stimulating the immune response and improving cancer vaccine efficacy.

The first polysulfenamides were synthesized with S-N and N-S-N bonds along the backbone. We demonstrated that sulfenamides were stable in polar protic and aprotic solvents, but degraded rapidly when exposed to acidic conditions. Microparticles were fabricated from polysulfenamides with S-N bonds, their surfaces were readily functionalized, and they were internalized by cells allowing for intracellular delivery of their cargo. These microparticles were also stable at physiological pH, degraded under acidic conditions, and possessed minimal toxicity towards cells. This work demonstrated that polysulfenamides form the basis for a new set of polymers for drug delivery that greatly differ from prior work in this field. New biodegradable polymers with N-S-N bonds along the backbone were synthesized. These were the first polymers with these bonds and possessed many of the same characteristics as polymers synthesized with S-N bonds. The synthesis and characterization of comb block copolymers with arms composed of poly(lactic acid), poly(butyl acrylate), and poly(styrene-b-vinylpyridine) were described. The self-assembled morphologies in the solid state of comb tri- and tetrablock copolymers with poly(styrene) were also described. These assemblies demonstrated that well-ordered and complex morphologies were assembled from these polymers. The steric effect of substitutions on oxanorbornenes in ring opening metathesis polymerization (ROMP) was investigated. Oxanorbornenes substituted with methyls at the bridgehead positions showed limited reactivity with the Grubbs first and second generation catalysts and the Grubbs first generation methylidene catalyst.

Cancer immunotherapy has proven to be challenging as it depends on overcoming multiple mechanisms that mediate immune tolerance to self-antigens. In situ immunization is based on the concept that it is possible to break immune tolerance by inducing tumor cell death in situ in a manner that provides antigen presenting cells such as dendritic cells (DCs) with a wide selection of tumor antigens that can then be presented to the immune system and result in a therapeutic anticancer immune response. Based on recent advances in the understanding of antitumor immunity, we designed a three-step approach to in situ immunization to lymphoma: (1) Inducing immunogenic tumor cell death with the chemotherapeutic drug Doxorubicin (Dox). Dox enhances the expression of "eat-me" signals by dying tumor cells, facilitating their phagocytosis by dendritic cells (DCs). Due to the vesicant activity of Dox, microparticles (MPs) made of PLGA (a biodegradable polymer) can safely deliver Dox intratumorally and are effective vaccine adjuvants; (2) Enhancing antigen presentation and T cell activation using anti-OX40; (3) Sustaining T cell responses by checkpoint blockade using anti-CTLA-4. In vitro, Dox MPs were less cytotoxic to DCs than to B lymphoma cells, did not require internalization by the lymphoma cells, and significantly enhanced phagocytosis of tumor cells by DCs as compared to soluble Dox. In mice, this three-step therapy induced CD4- and CD8-dependent systemic immune responses that enhanced T cell infiltration into distant lymphoma tumors leading to their eradication and significantly improving survival. Our findings demonstrate that systemic antitumor immune responses can be generated locally by three-step therapy and merit further investigation of three-step therapy for immunotherapy of lymphoma patients. Furthermore, we designed another in situ immunization approach using PLGA MPs loaded with both Dox and CpG oligodeoxynucleotides (CpG). The addition of CpG was to further enhance the Dox MP design by including an agent that addresses Step Two in situ, by enhancing tumor antigen presentation by DCs. In vitro, we show that Dox/CpG MPs can kill B and T lymphoma cells and are less toxic to DCs than soluble Dox. In vivo, Dox/CpG MPs combined with anti-CTLA-4 and anti-OX40 generated systemic immune responses that suppressed injected and distant tumors in a murine B lymphoma model, leading to tumor-free mice. The combination regimen was also effective at reducing T cell lymphoma and melanoma tumor burdens. In conclusion, Dox/CpG MPs represent a versatile, efficient and safe tool for in situ immunization that could provide a promising component of immunotherapy for patients with a variety of types of cancer.