Orientador: Edvaldo Sabadini, Fred Yukio Fujiwara / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Química / Made available in DSpace on 2018-08-19T09:00:39Z (GMT). No. of bitstreams: 1 Egidio_FernandadoCarmo_D.pdf: 2263055 bytes, checksum: 940b47bd50de20f4597683b091bd3faf (MD5) Previous issue date: 2011 / Resumo: O modelo de dois sítios, utilizado nos estudos de relaxação do solvente em soluções de carboidratos, pode ser transposto de forma satisfatória para soluções de polímeros hidrofílicos. Polímeros que possuem hidrogênios lábeis, isto é, que possuem hidrogênios disponíveis para troca química com os hidrogênios da água, causam aumento da taxa de relaxação spin-spin do solvente, R2, em relação ao valor obtido para a água pura O efeito torna-se mais intenso na medida em que a concentração dos polímeros aumenta. Polímeros que não possuem hidrogênios lábeis não alteram significativamente o R2 do solvente, pois o mecanismo de troca química não é estabelecido. A relaxação do solvente pode ser usada para estudar o processo de transição sol-gel em sistemas poliméricos. Conforme o gel e formado, as moléculas de água começam a ser confinadas e a taxa de relaxação do solvente aumenta significativamente. A estrutura de hidrogéis supramoleculares formados entre PEG e a-CD, a qual possui uma cavidade hidrofóbica em relação ao seu exterior hidrofílico, dependem das concentrações de seus constituintes e da massa molar do polímero. Quanto maior a massa molar do polímero, menor o numero de pontos de reticulação e mais frágil será o hidrogel. A partir da entrada de 2 moléculas de a-CD em uma cadeia de PEG linear 1.470 Da, a complexação torna-se mais intensa, levando ao recobrimento de toda a cadeia polimérica. A cinética de gelação entre a-CD e PEG, estudada por medidas do tempo de relaxação spin-spin (T2 = 1/R2) do solvente, fornece um perfil de curva que pode explicar o processo de complexação e formação do gel como um todo. A liberação e o confinamento de moléculas de água são refletidos no tempo de relaxação spin-spin destas. Ao liberar moléculas de água, observa-se uma diminuição nos valores de R2 da água até um ponto mínimo, a partir do qual os valores começam a aumentar devido ao confinamento destas moléculas / Abstract: The two sites model used in the studies of solvent relaxation in carbohydrate solutions was also applied for hydrophilic polymer solutions. Polymers with labile hydrogen atoms, meaning they have hydrogen available for chemical exchange with the hydrogen atoms of water, increase the solvent spin-spin relaxation rate, R2, relative to the pure water. The effect becomes more intense as the polymer concentration increases. Polymers without labile hydrogen do not affect significantly R2 of the solvent, because the chemical exchange mechanism is not established in this case. The solvent relaxation can be used to study the process of sol-gel transition in polymer systems. When the gel is formed, the water molecules become confined and the solvent relaxation rate increases significantly. The structure of supramolecular hydrogels formed from PEG and a-CD, which has a hydrophobic cavity and exterior hydrophilic, depends on the concentration of the constituents and the molar mass of the polymer. The higher is the molar mass of the polymer, the fewer is the crosslinking points and more the fragile is hydrogel. Since the inclusion of two a-CD molecules in a chain of linear PEG 1470 Da, the complexation becomes more intense, leading to the full inclusion complex. The kinetics of hydrogel formation between a-CD and PEG was studied by relaxation time measurements of the spin-spin (T2 = 1/R2) of the solvent, provides a curve, whose shape can used to explain the process of complexation and the gelation process at all. The release and confinement of water molecules of the gel are affect spin-spin relaxation the time of the water molecules. The releasing of the water molecules, results in the decreasing of R2 up to a minimum value, and beyond this point the values increases again, due to confinement of these molecules / Doutorado / Físico-Química / Doutor em Ciências

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The aim of this work was the development of nanostructured formulations containing M. alternifolia essential oil. Polymeric nanocapsules and nanoemulsions (10 mg/mL) were prepared by the interfacial deposition of preformed polymer method and spontaneous emulsification, respectively. The formulations were evaluated in relation to their physicochemical characteristics (particle size, polydispersity index, zeta potential, pH and oil content). Formulations presented oil content of about 96%, nanometric mean size (160-220 nm), polydispersity index below 0.25 and negative zeta potential. The pH values were 6.43 ± 0.37 and 5.98 ± 0.00 for nanoemulsions and nanocapsules, respectively. The inclusion of oil in nanocapsules showed higher protection against volatilization. The analysis of mean size and polydispersity index of the formulations presented no significant alteration during 60 days of storage time (at 25 °C, protected from light). The in vitro antifungal activity of the formulations was evaluated through the study of the inhibition growth profile of Candida albicans yeast. In addition, the antifungal activity against Trichophyton rubrum was studied using two different in vitro models of dermatophyte nail infection. The effect of nanostructures on the growth curve of C. albicans showed inhibition and control of its growing. Nail infection models demonstrated the ability of the formulations in reducing the T. rubrum growth, and the inclusion of oil in nanocapsules was more efficient. The development of hydrogels containing the nanostructured systems were carried out. The semisolid formulations showed adequate properties for topical administration. The topical antiedematogenic effect was evaluated by ear thickness measurement before and 24 h after UVB radiation. Hydrogels containing the colloidal systems showed effect, unlike to hydrogel containing the free oil. The in vivo healing effect of hydrogels was evaluated by regression of the wound in the rat skin. Hydrogel containing the nanocapsules presented higher activity in comparison to the positive control (hydrogel containing allantoin). Analyses of the oxidative stress showed an increase of glutathione, vitamin C and catalase levels after the treatment with the hydrogel containing the nanocapsules prepared with melaleuca essential oil. / O principal objetivo deste trabalho foi o desenvolvimento de formulações nanoestruturadas contendo o óleo essencial de Melaleuca alternifolia. Suspensões de nanocápsulas poliméricas e nanoemulsões (10 mg/mL) foram preparadas pelos métodos de deposição interfacial de polímero pré-formado e emulsificação espontânea, respectivamente, e avaliadas em relação as suas características físico-químicas (tamanho de partícula, índice de polidispersão, potencial zeta, pH e determinação do conteúdo de óleo). As formulações apresentaram rendimento em óleo de, aproximadamente, 96%, diâmetro médio nanométrico (160-200 nm), índice de polidispersão inferior a 0,25 e potencial zeta negativo. Os valores de pH foram de 6,43 ± 0,37 e 5,98 ± 0,00 para nanoemulsões e suspensões de nanocápsulas, respectivamente. A nanoencapsulação do óleo apresentou uma maior proteção deste frente à volatilização. As análises de tamanho médio de partícula e índice de polidispersão não apresentaram alterações durante 60 dias de armazenamento, a uma temperatura de 25 °C, ao abrigo da luz. A avaliação da atividade antifúngica das formulações foi realizada, através do estudo do perfil de inibição do crescimento da levedura de Candida albicans. Adicionalmente, a atividade antifúngica também foi testada frente ao Trichophyton rubrum, utilizando dois diferentes modelos de infecção ungueal. O efeito das nanoestruturas na curva de crescimento da levedura demonstrou inibição e controle do crescimento. Nos modelos de infecção de unha foi possível evidenciar a habilidade das formulações em reduzir o crescimento do T. rubrum, obtendo-se resultados promissores para as nanocápsulas. O estudo de desenvolvimento de hidrogéis contendo os sistemas nanoestruturados propostos neste trabalho foi, também, realizado. As formulações semissólidas desenvolvidas apresentaram propriedades adequadas para aplicação tópica. Ainda, o efeito antiedematogênico tópico foi avaliado pela medida da espessura da orelha de camundongos antes a após 24 horas de exposição à radiação. Os hidrogéis contendo os sistemas coloidais desenvolvidos demonstraram atividade, ao passo que o gel contendo o óleo livre não apresentou efeito. Adicionalmente, o efeito cicatrizante in vivo dos hidrogéis foi avaliado através da regressão da área da ferida na pele de ratos. No modelo de cicatrização, o hidrogel contendo as nanocápsulas apresentou atividade superior ao controle positivo (hidrogel contendo alantoína). Análises de marcadores de estresse oxidativo demonstraram aumento nos níveis de glutationa, vitamina C e catalase após o tratamento com o hidrogel contendo as nanocápsulas preparadas com o óleo essencial de melaleuca.

Polymers have deep roots as drug delivery tools, and are widely used in clinical to private settings. Currently, however, numerous traditional therapies exist which may be improved through use of polymeric biomaterials. Through our work with infectious and oxidative stress disease prevention and treatment, we aimed to develop application driven, enhanced therapies utilizing new classes of polymers synthesized in-house. Applying biodegradable poly(β-amino ester) (PBAE) polymers, covalent-addition of bioactive substrates to these PBAEs avoided certain pitfalls of free-loaded and non-degradable drug delivery systems. Further, through variation of polymer ingredients and conditions, we were able to tune degradation rates, release profiles, cellular toxicity, and material morphology. Using these fundamentals of covalent drug-addition into biodegradable polymers, we addressed two problems that exist with the treatment of patients with high-risk wound-sites, namely non-biodegradability that require second-surgeries, and free-loaded antibiotic systems where partially degraded materials fall below the minimum inhibitory concentration, allowing biofilm proliferation. Our in situ polymerizable, covalently-bound vancomycin hydrogel provided active antibiotic degradation products and drug release which closely followed the degradation rate over tunable periods. With applications of antioxidant delivery, we continued with this concept of covalent drug addition and modified a PBAE, utilizing a disulfide moiety to mimic redox processes which glutathione/glutathione disulfide performs. This material was found to not only be hydrolytically biodegradable, but tunable in reducibility through cleavage of the disulfide crosslinker, forming antioxidant groups of bound-thiols, similar to drugs currently used in radioprotective therapies. The differential cellular viability of degradation products containing disulfide or antioxidant thiol forms was profound, and the antioxidant form significantly aided cellular resistance to a superoxide attack, similar to that of a radiation injury. Pathophysiological oxidation in the form of radiation injury or oxidative stress based diseases are often region specific to the body and thus require specific targeting, and nanomaterials are widely researched to perform this. Utilizing a tertiary-amine base-catalyst, we were able to synthesize a high drug content (20-26 wt%) version of the disulfide PBAE previously unattainable. The reduced version of this material created a linear-chain polymer capable of single-emulsion nanoparticle formulation for use with intravenous antioxidant delivery applications instead of local.

L’objectif de cette thèse était d'étudier les propriétés physico-chimiques de réseaux interpénétrés formés par auto-assemblage de copolymères à blocs amphiphiles stimulables. La première partie du travail a consisté à élaborer des hydrogels interpénétrés IPSAN (InterPenetrated Self-Assembled Network) à partir d’un simple mélange de copolymères triblocs associatifs. Ainsi, l’IPSAN correspond à une combinaison des deux réseaux polymères. Le premier réseau est formé par un copolymère tribloc à base de poly(oxyde d'éthylène) (tPOE) porteur de blocs hydrophobes polymérisables sous UV. Le deuxième réseau est constitué d’un copolyélectrolyte tribloc pH sensible à base de poly(acide acrylique) (TH50).L’influence des concentrations en copolymères et du pH sur la structure et les propriétés mécaniques des hydrogels IPSAN a été systématiquement étudiée. Dans une deuxième partie, nous nous sommes intéressés à l'influence de la formation d'un réseau transitoire sur la séparation de phases dans des mélanges de tPOE et de polymères linéaires en solution aqueuse. Cette étude a mis en évidence une augmentation de l'incompatibilité entre les deux polymères induite par l’association du copolymère tribloc. Ce système a permis l'élaboration d'hydrogels macroporeux photo-réticulables.Enfin, la diffusion de polymères linéaires et de particules solides micrométriques dans des réseaux de copolymères pH-sensibles a été étudiée par des techniques de recouvrement de fluorescence après photobleaching (FRAP) et de diffusion dynamique de la lumière. La formation du réseau transitoire ralentit le mouvement de traceurs linéaires de petite taille de la même manière que dans le cas d'hydrogels covalents. Le contrôle de la structure et de la dynamique du réseau par le pH est un levier puissant pour contrôler la diffusion dans ces hydrogels. / The objective of this thesis was to investigate the physical chemical properties of interpenetrating networks formed by self-association of responsive amphiphilic block copolymers. The first part of the work was to develop IPSAN hydrogels(InterPenetrated Self-Assembled Network) simply by mixing two triblock copolymers. Thus, the IPSAN corresponds to a combination of the two polymer networks. The first network is formed by a triblock copolymer based on poly(ethylene oxide) (tPEO) bearing UV-cross-linkable hydrophobic blocks. The second network consists of a pH-sensitive triblock polyelectrolyte based on poly(acrylic acid) (TH50). The influence of the concentration of the copolymers and of the pH on the structure and the mechanical properties of the IPSAN has been systematically studied. In the second part we investigated the influence ofthe formation of a transient network on the phase separation in mixtures of tPEO and linear polymers in aqueous solution. This study revealed an increase of the incompatibility between the two polymers induced by the association of the triblock copolymer. This system enabled the elaboration of photo-cross-linked macroporous hydrogels. Finally, the diffusion of linear polymers and solid particles in pH-sensitive networks was studied by fluorescence recovery after photobleaching (FRAP)and dynamic light scattering. The formation of a transient network restricts the movement of small linear tracers in the same way as a covalent hydrogel would. The control of the structure and dynamics of the network by the pH is a powerful tool to control the diffusion in this hydrogels.

Nous développons une nouvelle approche des actuateurs microfluidiques à base d’hydrogels stimulables. Contrairement aux approches précédentes, les hydrogels sont synthétisés et greffés à la paroi inférieure du microcanal avant la fermeture du système. Nous utilisons une nouvelle stratégie de synthèse pour les films d’hydrogels stimulables par chimie click thiol-ène. Les films sont obtenus par dépôt, sur des surfaces fonctionnalisées avec des thiols, d’un mélange de chaînes polymères préformées et d’un réticulant dithiol. Le greffage et la réticulation s’obtiennent simultanément par une activation thermique ou par irradiation UV. Des films et des micro-motifs sont obtenus sur une large gamme d’épaisseur allant de la centaine de nanomètres à plusieurs microns. Nous intégrons les motifs d’hydrogels stimulables dans les microcanaux pour réaliser des actionneurs. Nous étudions des actionneurs thermosensibles réalisés avec des hydrogels de poly(N-isopropylacrylamide). Avec la température, les motifs d’hydrogels gonflent ou dégonflent en absorbant/expulsant l’eau de manière réversible. L’effet est rapide (inférieur à la seconde), la transition abrupte (quelques degrés autour de la LCST à 32°C) et l’amplitude de déformation est importante (gonflement de 400%). Les micro-vannes réalisées avec cette nouvelle approche présentent de très bonnes performances et une grande durabilité. Nous avons aussi réalisé de nouveaux actionneurs reconfigurables fonctionnant comme des pièges microfluidiques. Ces actionneurs microfluidiques innovants offrent de nombreuses perspectives de par la facilité de leur mise en œuvre, leurs performances et l’intégration sur des micro-puces à haute densité. / We develop a new method to build microactuators using stimuli-responsive hydrogels. The hydrogel is synthesized with covalent attachment to the microchannel bottom walls prior to closing the microsystem, contrarily to previous approaches. We use a new stimuli-responsive hydrogel films synthesis pathway. This synthesis is based on thiol-ene click chemistry. The formation of films is achieved by adding bifunctional thiol molecules as cross-linkers to ene-functionalized preformed polymers on thiol-modified surfaces. The cross-linking and grafting are simultaneously performed either by thermal activation or UV-irradiation. Hydrogel films and micro-patterns are easily obtained in a wide range of thickness from hundred nanometers to several microns. We show that these responsive hydrogels patterns can be integrated into microfluidics channels to build microactuators. We focus on thermo-sensitive actuators made from poly(N-isopropylacrylamide). Under temperature, hydrogel patterns reversibly swell and collapse by absorbing/expulsing water. The phase transition is rapid (lower than 1 second), abrupt (a few degrees around the LCST at 32°C) and the deformation amplitude is high (400% swelling). Microvalves obtained by this approach exhibit high performances and durability. Moreover, we develop new reconfigurable actuators functioning as microfluidic traps. These new-concept microfluidic actuators offer wide possibilities because of their ease of fabrication, their performances and their ability to be integrated into high density.

Developing non-invasive and accurate diagnostics that are easily manufactured, robust and reusable will provide monitoring of high-risk individuals in any clinical or point-of-care environment, particularly in the developing world. There is currently no rapid, low-cost and generic sensor fabrication technique capable of producing narrow-band, uniform, reversible colorimetric readouts with a high-tuneability range. This thesis aims to present a theoretical and experimental basis for the rapid fabrication, optimisation and testing of holographic sensors for the quantification of pH, organic solvents, metal cations, and glucose in solutions. The sensing mechanism was computationally modelled to optimise its optical characteristics and predict the readouts. A single pulse of a laser (6 ns, 532 nm, 350 mJ) in holographic “Denisyuk” reflection mode allowed rapid production of sensors through silver-halide chemistry, in situ particle size reduction and photopolymerisation. The fabricated sensors consisted of off-axis Bragg diffraction gratings of ordered silver nanoparticles and localised refractive index changes in poly(2-hydroxyethyl methacrylate) and polyacrylamide films. The sensors exhibited reversible Bragg peak shifts, and diffracted the spectrum of narrow-band light over the wavelength range λpeak ≈ 500-1100 nm. The application of the holographic sensors was demonstrated by sensing pH in artificial urine over the physiological range (4.5-9.0), with a sensitivity of 48 nm/pH unit between pH 5.0 and 6.0. For sensing metal cations, a porphyrin derivative was synthesised to act as the crosslinker, the light absorbing material, the component of a diffraction grating, as well as the cation chelating agent. The sensor allowed reversible quantification of Cu2+ and Fe2+ ions (50 mM - 1 M) with a response time within 50 s. Clinical trials of a glucose sensor in the urine samples of diabetic patients demonstrated that the glucose sensor has an improved performance compared to a commercial high-throughput urinalysis device. The experimental sensitivity of the glucose sensor exhibited a limit of detection of 90 µM, and permitted diagnosis of glucosuria up to 350 mM. The sensor response was achieved within 5 min and the sensor could be reused about 400 times without compromising its accuracy. Holographic sensors were also tested in flake form, and integrated with paper-iron oxide composites, dyed filter and chromatography papers, and nitrocellulose-based test strips. Finally, a generic smartphone application was developed and tested to quantify colorimetric tests for both Android and iOS operating systems. The developed sensing platform and the smartphone application have implications for the development of low-cost, reusable and equipment-free point-of-care diagnostic devices.

Tissue engineering offers a paradigm shift in the treatment of back pain. Engineered intervertebral discs could replace degenerated tissue and overcome the limitations of current treatments that disrupt the biomechanics of the spine. New materials, which exhibit sophisticated mechanical responses, are needed to provide templates for tissue regeneration. These behaviours include time-dependent deformation---facilitating shock absorption and nutrient transfer---and strong material anisotropy and tensile-compressive nonlinearities---providing flexibility in controlled directions. In this work, frameworks for the design of materials with controllable structure-property relationships are developed. The time-dependent mechanical properties of composites of agar, alginate and gelatin hydrogels are investigated. It is shown that the time-dependent responses of the composites can be tuned over a wide range. It is then demonstrated that materials mimicking the fibre-reinforced nature of natural tissues can be developed by infiltrating thick electrospun fibre networks with alginate. These fibre-reinforced hydrogels have tensile and compressive properties that can be separately altered. To better understand the mechanical behaviour of these hydrogel-based materials, improved methods for characterising poroelastic and poroviscoelastic time-dependent material properties using indentation are developed. It is shown that poroviscoelastic relaxation is the product of separate poroelastic and viscoelastic relaxation responses. The techniques developed here provide a methodology to rapidly characterise the properties of time-dependent materials and to create materials with complex structure-property relationships similar to those found in natural tissues; they present a framework for biomimetic materials design. The work in this thesis can be used to inform the design of clinically relevant tissue engineering treatments and help the quarter of a million people each year who undergo spinal surgery to reduce back pain.

No description available.

The expanding field of tissue engineering provides a new approach to regenerative medicine for common ailments such as cardiovascular disease and type-I diabetes. Biomaterials can be administered as a delivery vehicle to introduce therapeutic cells to sites of damaged or diseased tissue. A specific class of biomaterials, termed hydrogels, is suitable for this application as they can provide a biocompatible, biodegradable scaffold that mimics the physical properties of the native soft tissue. Injectable hydrogels are increasingly being developed for biomedical applications due to their ability to be delivered in a minimally invasive manner. One potential use for such materials is in the delivery of therapeutics such as cells or growth factor-releasing particles. In this study, the first aim was to determine the interactive effects between collagen-based hydrogels and additives (cells and microspheres) for cardiac regeneration. The results demonstrated that the addition of either cells or microspheres to a collagen-based hydrogel decreased its gelation time and increased its viscosity. Increased cross-linker concentrations resulted in lower cell viability. However, this cell loss could be minimized by delivering cells with the cross-linker neutralizing agent, glycine. As a potential application of these materials, the second aim of this study was to develop a hydrogel for use as an ectopic islet transplant site. Specifically, collagen-chitosan hydrogels were synthesized and characterized, with and without laminin, and tested for their ability to support angiogenic and islet cell survival and function. Matrices synthesized with lower chitosan content (20:1 collagen:chitosan) displayed greater cell compatibility for both angiogenic cells and for islets and weaker mechanical properties, while matrices with higher chitosan content (10:1 collagen:chitosan) had the opposite effect. Laminin did not affect the physical properties of the matrices, but did improve angiogenic cell and islet survival and function. Overall the proposed collagen-based hydrogels can be tailored to meet the physical property requirements for cardiac and islet tissue engineering applications and demonstrated promising cell support capabilities.

In recent years, the degradation and subsequent loss of tissues is an issue that has affected people worldwide. Although there are treatments addressing the degradation of tissues, such treatments involve complicated and expensive procedures, where full tissue regeneration is not achieved. For these reasons, in recent years, tissue engineering has developed cutting-edge biomaterials capable of inducing effective tissue regeneration both under cellular or acellular conditions. Peptide hydrogels are versatile biomaterials composed of the basic components of life amino acids, which act as building blocks to form hierarchical structures, which subsequently go on to form well-defined scaffolds. Biomaterials have been widely used for the culture of mammalian cells, tissue engineering, regenerative medicine, drug delivery, etc. This is thanks to their capability of providing a three-dimensional architecture to cells, which mimics the natural architecture of the extracellular matrix (ECM). Peptide- based hydrogels can be easily functionalised with active biological cues, which can direct the cellular response. It has been shown that the ionic-complementary FEFEFKFK hydrogel, succeeded to support the culture of mammalian cells such as bovine chondrocytes. In this work, we used the same FEFEFKFK hydrogel to investigate the capability of this hydrogel to support the three-dimensional culture of both human osteoblasts (hOBs), and human mesenchymal stem cells (hMSCs) for bone regeneration applications. To achieve this goal, hOBs were cultured within both FEFEFKFK (non-functionalised) and RGD-FEFEFKFK (functionalised) gels. Then the suitability of the FEFEFKFK gels to induce cellular proliferation, synthesis of bone ECM and mineralisation was explored. In addition, taking advantage of the inherent plasticity of hMSCs, we also investigated the capability of the FEFEFKFK gel to foster the osteogenic differentiation of hMSCs, and subsequently to induce bone mineralisation in 3-D under osteogenic stimulation. Based on the results obtained in this work, the FEFEFKFK gel arises as a promising biomaterial for both bone and dental tissue regeneration applications.