Development of multifunctional mannan nanogel

Tese de doutoramento em Biomedical EngineeringSelf-assembled nanogels made of hydrophobized mannan or pullulan were obtained using a versatile, simple, reproducible and low-cost method. In a first reaction pullulan or mannan were modified with hydroxyethyl methacrylate or vinyl methacrylate, further modified in the second reaction with 1-hexadecanethiol. The resultant amphiphilic material self-assembles in water via the hydrophobic interaction among alkyl chains, originating the nanogel. Structural features, size, shape, surface charge and stability of the nanogels were studied using hydrogen nuclear magnetic resonance, cryo-field emission scanning electron microscopy and dynamic light scattering. Above the critical micellar concentration (cmc), evaluated by fluorescence spectroscopy with Nile red and pyrene, spherical polydisperse nanogels reveal long-term colloidal stability in aqueous medium up to six months, with a nearly neutral negative surface charge and mean hydrodynamic diameter in the nanoscale range, depending on the polymer degree of substitution. Nanogel based on vinyl methacrylated mannan was selected for further characterization among others because its synthesis is much easier, cheaper and less time consuming, its cmc and size are smaller, it is less polydisperse, and more stable at pH 3–8, in salt or urea solutions being consequently more suitable for biological applications. Proteins (bovine serum albumin or ovalbumin) and hydrophobic drugs (curcumin) are spontaneously incorporated in the mannan nanogel, being stabilized by the hydrophobic domains randomly distributed within the nanogel, opening the possibility for the development of applications as potential delivery systems for therapeutic molecules. No cytotoxicity is detected up to about 0.4 mg/mL of mannan nanogel in mouse embryo fibroblast cell line 3T3 and mouse bone marrow-derived macrophages (BMDM) using cell proliferation, lactate dehydrogenase and Live/Dead assays. Comet assay, under the tested conditions, reveals no DNA damage in fibroblasts, which seems to occur in the case of BMDM. The internalization kinetics, uptake mechanisms and intracellular trafficking pathways of mannan nanogel in mouse BMDM was assessed by flow cytometry and confocal laser scanning microscopy, using fluorescently conjugated nanogel. A time-, concentration- and energy-dependent uptake profile of the mannan nanogel is observed. Inhibition analysis unraveled mannose receptor-mediated phagocytosis and clathrin-mediated endocytosis to be involved in nanogel uptake. The mannan nanogel is also visualized in the cytosol suggesting that a fraction was able to escape from the endolysosomal system. The protein corona formed in human plasma around mannan nanogel was characterized by mass spectrometry after size exclusion chromatography or centrifugation followed by sodium dodecyl sulphate polyacrylamide gel electrophoresis. It consists of a very specific set of proteins, apolipoproteins B-100, A-I and E and human serum albumin, slowly formed following a dynamic protein exchange process. The mannan nanogel does not affect blood coagulation, does not induce complement activation and retards the fibril formation of both Alzheimer’s disease-associated amyloid β peptide and haemodialysis-associated amyloidosis β2 microglobulin, as was assessed by fluorometric thrombin generation assay, Western blot, and continuous thioflavin T fluorescence assay, respectively. Mannan nanogel has potential immunological adjuvant activity, as evaluated on the specific immune response to ovalbumin in intradermally immunized BALB/c mice. Elicited ovalbumin-specific antibodies were predominantly of IgG1 subclass indicating a T helper 2-type bias. Physicochemical characteristics, loading ability of biological agents, cytocompatibility and uptake of mannan nanogel by mouse BMDM, biosafety and biocompatibility studied at molecular level, and adjuvant activity are pronounced hints of the potential applicability of this nanosystem for macrophages targeted delivery of vaccines or drugs, acting as promising nanomedicines, always with the key goal of preventing and/or treating diseasesNanogéis poliméricos auto-organizados foram obtidos a partir de manano e pululano hidrofobicamente modificados por um método versátil, simples, reprodutível e económico. Numa primeira reação, manano ou pululano foram enxertados com hidroxietil metacrilato ou vinil metacrilato, que por sua vez foram substituídos numa segunda reação com 1- hexadecanetiol. O material anfifílico resultante auto-organiza-se em água através da associação das cadeias alquílicas hidrofóbicas, originando o nanogel. As características estruturais, o tamanho, a forma, a carga de superfície e a estabilidade dos nanogéis foram estudados por espectroscopia de ressonância magnética nuclear 1H, microscopia crio-eletrónica de varrimento e dispersão dinâmica de luz. Acima da concentração micelar critica (cmc), avaliada por espectroscopia de fluorescência usando o vermelho de Nilo e o pireno, os nanogéis esféricos polidispersos revelam longa estabilidade coloidal em meios aquosos até seis meses, com carga de superfície negativa praticamente neutra, e diâmetro médio num intervalo nanométrico, cujo valor depende do grau de substituição do polímero. Dos nanogéis produzidos, o nanogel baseado em manano enxertado com vinil metacrilato foi selecionado para uma caracterização mais aprofundada porque a sua síntese é mais fácil, económica e rápida, a cmc e o tamanho são menores, é menos polidisperso e mais estável no intervalo de pH 3–8 bem como na presença de sal e ureia, sendo consequentemente mais apropriado para aplicações biológicas. Proteínas (albumina sérica bovina ou ovalbumina) e drogas hidrofóbicas (curcumina) são incorporadas espontaneamente no nanogel de manano, sendo estabilizados nos domínios hidrofóbicos aleatoriamente distribuídos no interior do nanogel, abrindo perspetivas para o desenvolvimento de aplicações em sistemas de libertação de moléculas terapêuticas. Nenhuma citotoxicidade é detetada com o nanogel de manano até 0.4 mg/mL na linha celular de fibroblastos de embrião de ratinho 3T3 e nos macrófagos derivados da medula óssea de ratinho usando ensaios de proliferação celular, lactato desidrogenase e “Live/Dead”. O ensaio cometa nas condições testadas, não revela dano no ADN dos fibroblastos, que possivelmente ocorre, no entanto, no caso dos macrófagos. A cinética de internalização, os mecanismos de internalização e as vias de tráfego intracelular do nanogel de manano nos macrófagos derivados da medula óssea de ratinho foram avaliados por citometria de fluxo e microscopia de confocal de varrimento laser, usando o nanogel conjugado com um fluorocromo. O perfil de internalização do nanogel é dependente do tempo, da concentração e de energia. A análise com inibidores revelou que a fagocitose mediada pelo receptor da manose e a endocitose mediada por clatrina estão envolvidos na internalização do nanogel. O nanogel de manano é também visualizado no citosol sugerindo que uma fração é capaz de escapar do sistema endolisossomal. A corona de proteínas formada no plasma humano em redor do nanogel de manano foi caracterizada por espectrometria de massa, após cromatografia de exclusão por tamanho ou centrifugação, seguidas de eletroforese em gel de poliacrilamida na presença de dodecil sulfato de sódio. A corona consiste num conjunto específico de proteínas, apoliproteínas B-100, A-I e E e albumina sérica humana, que se forma após um lento e dinâmico processo de troca de proteínas. O nanogel de manano não afeta a coagulação do sangue, não induz ativação do complemento e retarda a formação de fibras do péptido β amiloide associado à doença de Alzheimer e de β2 microglobulina na amiloidose associada à hemodiálise, como foi avaliado por teste fluorimétrico de geração de trombina, Western blot e análise continua da fluorescência de tioflavina T, respectivamente. O nanogel de manano tem potencial atividade adjuvante, avaliada na resposta imune específica para ovalbumina em ratinhos BALB/c imunizados por via intradérmica. Os anticorpos específicos para ovalbumina induzidos foram predominantemente da subclasse IgG1, o que indica uma propensão para induzir uma resposta mediada por células “T helper” tipo 2. As características físico-químicas, a capacidade de incorporação de agentes biológicos, a citocompatibilidade e a internalização do nanogel de manano por macrófagos derivados da medula óssea de ratinho, a biossegurança e biocompatibilidade estudadas a nível molecular, e a atividade adjuvante deixam entrever a potencial aplicação deste nanosistema na libertação direcionada a macrófagos, quer de vacinas quer de fármacos, atuando como promissores nanomedicamentos, sempre com o objetivo chave de prevenir e/ou tratar doenças

Polymeric nanogels as vaccine delivery systems

Polymeric nanogels find a relevant field of application in the formulation of a new generation of therapeutic and preventive vaccines, aiming at the fine-tuned modulation of the immune response. Intrinsic properties of polymeric nanogels, such as material chemistry, size and shape, surface charge, and hydrophobicity or hydrophilicity, may be determining factors in shaping the induced immune response. These materials can thus work as synthetic adjuvants, which can also be conjugated with immunostimulants. Polymeric nanogels protect vaccine antigens from degradation in vivo and, surface-conjugated with antibodies or specific ligands, could increase active targeting specificity. This review covers the recent published data concerning the modulation of innate and adaptive immune responses by engineered polymeric nanogels and their potential application as delivery systems in vaccination.S.A. Ferreira is the recipient of a fellowship from International Iberian Nanotechnology Laboratory (INL)

Supramolecular assembled nanogel made of mannan

Introduction: The supramolecular assembled nanogel made of mannan was synthesed and characterized with the purpose to obtain a potential pharmaceutical delivery system able to work both as a therapeutic and prophylactic vaccine adjuvant and antigen carrier. These systems are expected to perform as carriers for proteins and peptides, acting like antigens, optimizing delivery to antigen-presenting cells, by targeting their mannose receptors. Immunity might be improved conjugating this system with other immune response modifiers.info:eu-repo/semantics/publishedVersio

Printing Technologies on Flexible Substrates for Printed Electronics

Printing technologies have been demonstrated to be highly efficient and compatible with polymeric materials (both inks and substrates) enabling a new generation of flexible electronics applications. Conductive flexible polymers are a new class of materials that are prepared for a wide range of applications, such as photovoltaic solar cells, transistors molecular devices, and sensors and actuators. There are many possible printing techniques. This chapter provides an opportunity to review the most common printing techniques used at the industrial level, the most commonly used substrates and electronic materials, giving an overall vision for a better understanding and evaluation of their different features. Several technological solutions (contact/noncontact) and its critical challenges are also presented. Inkjet Printing Technology (IPT) has been receiving a great attention and therefore higher focus is given to this technology. An overview of IPT is presented to evidence its importance and potential as a key-technology on the research field for printed electronics development, as well as on large scale industrial manufacturing. A background and a review on prior work are presented along with used materials, developed applications and potential of IPT technology. The main features of the different printing technologies, advantages and main challenges are also compared

Setting rents in residential real estate: a methodological proposal using multiple criteria decision analysis

The real estate sector has been negatively affected by the recent economic recession, which has forced structural changes that impact property value and price. Recent pressures have also motivated reduced liquidity and access to credit, causing a drop in property sales and, thus, boosting the rental housing market. It is worth noting, however, that the rental housing segment is not with-out difficulties and complexity, namely in terms of legislation and rental value revaluation. In light of this reasoning, this study aims to develop a multiple criteria decision support system for calculation of residential rents. By integrating cognitive maps and the measuring attractiveness by a categorical based evaluation technique (MACBETH), we also aim to introduce simplicity and transparency in the decision making framework. The practical implications, advantages and shortfalls of our proposal are also analyzed

Is the plant Bolboschoenus maritimus an adequate biomonitor for trace metal contamination in saltmarshes? A field study from the Óbidos lagoon (Portugal)

Monitoring the negative impacts of trace metals is crucial to assess the health and stability of ecosystems. In salt marshes, halophyte plants were reported as possible bioaccumulators of these elements. The aim of this work was to explore the bioaccumulation potential of Bolboschoenus maritimus as a tool for monitoring the presence of metals in coastal environments. Bolboschoenus maritimus were collected from a brackish water lagoon, and the presence of the trace metals lead, cadmium, and nickel were seasonally evaluated in distinct parts of the plants, and in water and sediment samples. Lead was the trace metal with the highest concentration detected in water and sediments of the sampling site. The highest lead concentrations in B. maritimus were recorded in the spring season. The transport index indicated an accumulation of lead in the leaves of around 70% in the spring of 2009. Cadmium in leaves in spring and summer of 2009 reached values above 5 mg Cd. kg−1. Nickel was not detected in most samples collected. Bolboschoenus maritimus was considered an adequate biomonitor for lead and cadmium, since it bioaccumulates both metals with seasonally distinct results, as the bioaccumulation factor results indicated.info:eu-repo/semantics/publishedVersio

Self assembling nanogels

Este resumo faz parte de: Book of abstracts of the Meeting of the Institute for Biotechnology and Bioengineering, 2, Braga, Portugal, 2010. A versão completa do livro de atas está disponível em: http://hdl.handle.net/1822/1096

Avaliação da sensibilidade in vitro à quinidina em isolados brasileiros de Plasmodium falciparum: análise comparativa à quinina e à cloroquina

Malária falciparum representa grave e crescente problema de saúde pública mundial, dada a resistência do parasito à maioria dos fármacos disponíveis. Em algumas áreas endêmicas, a quinidina, diastereoisômero do antimalárico quinina, vem sendo empregada em substituição a este último. Com o objetivo de avaliar o emprego da quinidina como alternativa à perda crescente de sensibilidade de cepas brasileiras de P. falciparum à quinina, como o observado na região Amazônica, realizamos ensaio comparativo entre quinidina, quinina e cloroquina. A técnica in vitro do microteste de sensibilidade foi utilizada. Todos os isolados mostraram-se altamente resistentes à cloroquina. Resistência à quinina não foi observada, embora altos valores de CMI (concentração mínima inibitória) tenham sido encontrados. Estes resultados corroboram o decréscimo de suscetibilidade de cepas brasileiras à quinina. Observou-se variação de IC50 de 0,053 a 4,577 mimol/L de sangue para a quinidina, enquanto para a quinina estimou-se IC50 de 0,053 a 8,132 mimol/L de sangue. Ademais, observou-se clareamento da parasitemia em concentrações inferiores à da quinidina quando empregada como fármaco antiarrítmico, confirmando estudo anterior por nós realizado. Resultados semelhantes foram encontrados em isolado oriundo da África.Falciparum malaria represents a serious and an increasing world public health problem due to the acquired parasite's resistance to the most available drugs. In some endemic areas, quinidine, a diastereoisomer of the antimalarial quinine, has been employed for replacing the latter. In order to evaluate the use of quinidine as an alternative to the increasing loss of quinine effectiveness in Brazilian P. falciparum strains, as has been observed in the Amazon area, we have assayed quinidine, quinine and chloroquine. The in vitro microtechnique was employed. All isolates showed to be highly resistant to chloroquine. Resistance to quinine was not noted although high MIC (minimal inhibitory concentration) values have been observed. These data corroborate the decreasing sensitivity to quinine in strains from Brazil. Quinidine showed IC50 from 0.053 to 4.577 mumol/L of blood while IC50 from 0.053 to 8.132 mumol/L of blood was estimated for quinine. Moreover, clearance of the parasitemia was observed in concentrations lower than that used for quinidine in antiarrhythmic therapy, confirming our previous data. The results were similar to African isolate