Dados do Instituto Nacional do Câncer revelam 14,1 milhões de casos novos de câncer em 2012 em todo o mundo, e a estimativa para 2014/2015 no Brasil aponta para mais 500 mil novos casos. Assim, pesquisas voltadas à produção de nanopartículas e hidrogéis, constituídos de polímeros naturais ou sintéticos, são importantes para o desenvolvimento de sistemas destinados à liberação controlada de fármacos antitumorais. Nesse sentido, este estudo visou produzir derivados hidrofílicos de quitosanas, N,O-metoxi(polietilenoglicol)-g-quitosana, destinados ao encapsulamento e à liberação de fármacos antitumorais. As características estruturais e físico-químicas dos polímeros foram avaliadas por FTIR, RMN 1H, solubilidade em função do pH, viscosimetria, DRX e TGA. As quitosanas de partida, com diferentes graus médios de desacetilação e massas molares médias viscosimétricas, QD1x ( = 64,0 ± 1,1 % e = 495,6 ± 6,3.103 g mol-1), QD2x ( = 75,8 ± 0,8 % e = 346,8 ± 5,8.103 g mol-1) e QD3x ( = 92,3 ± 0,9 % e = 501,9 ± 5,9.103 g mol-1), produzidas pela aplicação do processo DAIUS à β-quitina, e quitosana comercial, Q ( = 94,7 ± 0,7 % e = 82,9 ± 2,0.103 g mol-1), foram avaliadas quanto aos teores de metais pesados por ICP OES e os resultados comparados aos limites permitidos pela ANVISA e Farmacopeia norte-americana. Teores significativos de metais foram detectados nas quitosanas QD1x, QD2x e QD3x (Ti e Al) e na quitosana comercial (Ni). O emprego de α-metoxipoli(etilenoglicol)-ω-carboxi possibilitou a síntese dos derivados PgQD1x, PgQD2x, PgQD3x e PgQ, que apresentaram graus médios de substituição semelhantes ( ≈ 40%). Os derivados foram hidrossolúveis em amplo intervalo de pH (1 – 11) e apresentaram viscosidades intrínsecas significativamente inferiores quando comparados às quitosanas. Foi observado que a estabilidade térmica e a cristalinidade das quitosanas são inferiores às de beta-quitina, enquanto os derivados exibiram maior cristalinidade e são termicamente mais estáveis quando comparados às quitosanas. Em geral, o aumento da razão molar –NH3+/TPP empregada na preparação das nano e microsuspensões de polímero/TPP pelo método de gelificação iônica resultou no aumento da densidade de cargas positivas nas superfícies das partículas. Entretanto, a ocorrência de numerosas cadeias de mPEG nos polímeros N,O-mPEG-g-quitosana levou à predominância das cadeias laterais hidrofílicas nas superfícies das partículas, blindando as cargas positivas, favorecendo a hidratação das partículas e o aumento dos diâmetros hidrodinâmicos médios. A influência das viscosidades intrínsecas dos polímeros e das concentrações poliméricas foi avaliada através do parâmetro de recobrimento (c[η]). Essa avaliação revelou que os diâmetros hidrodinâmicos médios das partículas são diretamente afetados por c[η] nos casos dos sistemas quitosana/TPP, enquanto as viscosidades intrínsecas dos derivados N,O-mPEG-g-quitosana são determinantes nos sistemas N,O-mPEG-g-quitosana/TPP. O estudo do comportamento reológico dos hidrogéis de N,O-mPEG-g-quitosana revelou que G’ > G’’ nos casos de PgQD3x, PgQD1x e PgQ, o que é característico de gel rígido, enquanto que no caso de PgQD2x foi observado G’’ > G’, indicando a predominância do comportamento viscoso sobre o elástico. O ponto de crossover foi observado acima de 38,2 ºC, indicando a formação de hidrogel estruturado termosensível. Assim, pesquisas futuras poderão investigar a influência das características estruturais e físico-químicas destes carreadores poliméricos, visando melhor eficiência de incorporação e liberação in vitro de fármacos antitumorais. / Data from National Institute of Cancer show an increase of 14.1 million new cases of cancer over the world in 2012, and the estimate for 2014/2015 in Brazil points to over 500,00 new cases. Thus, research focused on the production of nanoparticles and hydrogels, consisting by natural or synthetic polymers, are important for the development of systems for controlled release of antitumoral drugs. Therefore, this study aimed to produce systems based on hydrophilic derivative of chitosan, N,O-methoxy(polyethyleneglycol)-g-chitosan, for the encapsulation and release of antitumor drugs. The structural and physicochemical characteristics of the chitosan and the polymers were analyzed by FTIR, 1H NMR, solubility as function of pH, viscosimetry, XRD and TGA. The parent chitosan with different average degrees of deacetylation and viscosimetric average molecular weight, QD1x ( = 64,0 ± 1,1 % e = 495,6 ± 6,3.103 g mol-1), QD2x ( = 75,8 ± 0,8 % e = 346,8 ± 5,8.103 g mol-1) e QD3x ( = 92,3 ± 0,9 % e = 501,9 ± 5,9.103 g mol-1), produced applying the USAD to β-chitin, and the comercial chitosan, Q ( = 94,7 ± 0,7 % e = 82,9 ± 2,0.103 g mol-1) were analyzed for heavy metal content by ICP OES and the results compared to the limits allowed by ANVISA and US Pharmacopoeial. Significant levels of metals were detected in chitosan QD1x, QD2x and QD3x (Ti and Al) and commercial chitosan (Ni). The use of α-methoxypoly(ethyleneglycol)-ω-carboxy enabled the synthesis of derivatives PgQD1x, PgQD2x, PgQD3x and PgQ, which had similar average degree of substitution ( ≈ 40%). The derivatives were soluble in a broad pH range (1-11) and presented significantly lower intrinsic viscosities as compared to chitosan. It was observed that the thermal stability and crystallinity of chitosan are inferior to β-chitin, while the derivatives exhibit higher crystallinity and are more thermally stable as compared to chitosan. In general, increasing molar ratio of –NH3+/TPP in the preparation of nano- and microsuspensions of polymer/TPP by ionic gelation method, resulted in an increased positive charge density on the surfaces of the particles. However, the occurrence of a great number of chains of mPEG along the N,O-mPEG-g-chitosan chains led to the predominance of hydrophilic side chains on the surfaces of the particles, shielding the positive charges, favoring the hydration of the particles and the increasing the average hydrodynamic diameter. The influence of the intrinsic viscosities of the polymers and polymer concentrations were evaluated by the overlapping parameter (c[η]). This assessment revealed that the average hydrodynamic diameter of the particles are directly affected by c[η] in the case of chitosan/TPP systems, while the intrinsic viscosities of the derivates N,O-mPEG-g-chitosan are essential for N,O-mPEG-g-chitosan/TPP systems. The study of the rheological behavior of the hydrogels of N,O-mPEG-g-chitosan revealed that G’ > G’’ in the case of PgQD3x, PgQD1x and PGQ which is characteristic of structured gel, whereas in the case of PgQD2x it was observed G’’ > G’ indicating the predominance of the viscous behavior over the elastic. The crossover point was observed above 38.2 °C, indicating the formation of structured thermosensitive hydrogel. Thus, future research will investigate the influence of structural and physicochemical characteristics of polymeric carriers, aiming a better incorporation and release in vitro efficiency of antitumor drugs.

Artificial tissue expansion is required to generate new skin prior to reconstructive surgery, in order to compensate for a deficit of healthy tissue. Hydrogel tissue expanders, which expand anisotropically, show great promise in overcoming clinical limitations in the field, thus allowing the technique to be used in a wider range of surgeries. These devices consist of pellets of dry poly(methyl methacrylate-co-vinylpyrrolidone), compressed into discs through a hot compression moulding process. However, a number of significant problems still exist in these devices, and this thesis aims to address these issues. To date, there has been a lack of investigation of the factors governing the behaviour of anisotropic swelling. For this reason, a range of different compression ratios have been investigated, with particular focus on the relationship between the material flow during compression and the swelling behaviour of the resulting device. It was found that samples of the same initial size expand to the same reference swelling dimensions, regardless of compression ratio. During hot pressing, the material flow was found to be governed by slip-stick behaviour at the interface between the hot press and the device, affecting the properties and swelling behaviour of the devices. Based on these findings, devices were developed which could expand from a disc into a non-prismatic shape (dome or wedge). Such devices could reduce complication rates and allow the growth of new tissue with anisotropic resting tension. The devices were tested in a small in vivo trial, where it was shown that there were no adverse effects on the tissue produced, and that the shape of the expander (dome) was retained. As devices are being produced for medical use, understanding the effect of sterilization by γ-irradiation is essential, but to date this has been overlooked in the literature. It was found that γ-irradiation caused an increase in cross-linking in the P(MMA-co-NVP). Whilst this produced little change in swelling behaviour for isotropic devices, in the case of anisotropic devices it caused a change in the shape of expansion, reducing the area of new skin which could be generated by the device. It was found that by reducing the concentration of impurities (residual molecules from the polymer synthesis) the impact of γ-irradiation could be greatly reduced. Finally, controlling the rate of expansion is essential in order to avoid clinical complications. In order to control the rate of expansion, particularly during the initial period of swelling, semi-permeable PDMS coatings were applied to the compressed devices. Coatings of thickness greater than 0.375mm were found to effectively control the rate of swelling, for both cylindrical and non-prismatic shapes. As the coating thickness increased, the maximum swelling size decreased. However, it has been shown that change in height (the parameter which governs the area of skin produced) is affected less than the change in mass or diameter.

In der vorliegenden Arbeit wurde eine neue Klasse von implantierbaren biochemischen Sensoren bezüglich ihrer Sensitivität im physiologisch relevanten pH- (pH 7,4) sowie Glukose-Konzentrationsbereich (2 - 20 mM) entwickelt und untersucht. Die Glukose-sensitiven Hydrogele basieren auf der Bindung von 5-fach-Zuckern an Boronsäuregruppen, die in einem Acrylamid-basierten Hydrogel mit N,N′-Methylenbisacrylamid (BIS) als Vernetzter (AAm/APB/BIS, 80/20/0,75 mol%) verankert sind. Weiterhin konnten pH-sensitive Hydrogele auf Basis von 2-(Dimethylamino)ethyl Methacrylate (DMAEMA), Hydroxypropyl-methacrylat (HPMA) sowie Tetraethyleneglycol dimethacrylate (TEGDMA) als Vernetzter in unterschiedlichen Zusammensetzungen und Geometrien untersucht werden. Die verwendeten Hydrogele wurden hinsichtlich der Diffusionsprozesse sowie ihrer Quellkinetik charakterisiert, um deren Sensitivität, Selektivität, Reproduzierbarkeit und Ansprechzeit gegenüber den physiologischen Parametern (pH, pCO2, Glukose) zu verbessern. Die aufgebauten pCO2-Sensoren zeigten vielversprechende Ansprechzeiten von wenigen Minuten. Die Glukose und pH-Sensoren wiesen im physiologischen Medium (PBS) deutlich höhere Ansprechzeiten von mehreren Stunden auf. Die Kombination von piezoresistiven Drucksensoren mit Stimuli-sensitiven Hydrogelen bietet nicht nur eine große Vielfalt bezüglich der zu detektierenden Analyten, sondern ermöglicht auch miniaturisierte und implantierbare Sensoren für die kontinuierliche Erfassung von physiologischen Parametern. So war die Verkapselung zum Schutz und zugleich zur Gewährleistung der Biokompatibilität und ohne Beeinträchtigung der Funktionalität und Flexibilität der elektronischen Bauteile das Ziel. Dazu wurden die Sensoren mit dem Polymer Parylene C eingehaust, dass zusätzlich eine Polyethylenglykolschicht enthielt. Hierfür wurden Blockcopolymere mittels Ringöffnungspolymerisation synthetisiert, die Polyaminosäuren als Linkermoleküle und PEG zur gezielten Funktionalisierung enthalten. Nach kovalenter Anbindung an die inerte Parylene C-Oberfläche zeigten sich deutlich veränderte Oberflächeneigenschaften und eine verbesserte Zellkompatibilität und Hämokompatibilität. Zudem wurde der sogenannte Tarnkappeneffekt von PEG-Ketten, die sich in der Schicht nach außen ausrichten, festgestellt. Damit wurde Adsorption von Proteinen (Fibronektin, Fibrinogen), die in Entzündungsreaktionen, der Zelladhäsion sowie der Blutgerinnung maßgebend sind, deutlich verringert. / In this work a new class of implantable biochemical sensors with a high sensitivity at physiological pH (pH 7,4) and glucose (2 – 20 mM) ranges were developed and tested. The glucose sensitive hydrogel was made of acrylamide and N,N′-methylene-bis(acrylamide) as a crosslinker (AAm/APB/BIS, 80/20/0,75 mol%). The swelling mechanism was based on the reversible interaction of sugar molecules and the boronic acid groups in the hydrogel. Also a pH sensitive hydrogel made of 2-(dimethylamino) ethyl methacrylate (DMAEMA), hydroxypropyl-methacrylat (HPMA) and the crosslinker tetraethylene glycol dimethacrylate (TEGDMA) with different molar ratios and geometries was characterized. The swelling kinetics as well as the diffusion processes of different hydrogels were studied to advance sensitivity, selectivity, reproducibility and response time with respect to physiological parameters (pH, pCO2, glucose). pCO2 sensors showed promising short response times of about 4 min whereas glucose and pH sensors displayed longer response times of several hours in phosphate-buffered saline solution. The combination of piezoresistive pressure sensors and stimuli-sensitive hydrogels enables a great diversity of detecting analytes as well as miniaturized and implantable sensors for continuous measuring of physiological parameters. However, to implant the sensors an encapsulation strategy is needed that secures the electronics as well as ensures the biocompatibility without loss of functionality and flexibility. For this, the devices were coated with the polymer parylene C and an additional layer of blockcopolymers composed of polyaminoacid (PAA) and polyethyleneglycol (PEG) blocks synthesized via ring-opening polymerization. The functionalization units are carried out by the PEG blocks whereas the PAA blocks perform as linker molecules onto the activated parylene C surface. After covalent coupling of blockcopolymers to the inert polymer the surface characteristics changed and hence the cell and blood compatibility was improved. Furthermore the stealth effect of the outwards PEG chains was utilized to reduce the adsorption of proteins like fibronectin or fibrinogen. These proteins play a major role in inflammatory processes, cell adhesion and blood coagulation. The results gave proof that the encapsulation leads to decisively reduced physiological reactions.

Content: Nowadays, protein-based nanoparticle as a biodegradable, biocompatible product attracts considerable interest for new uses in specialized technical areas. Gelatin is a denatured, biodegradable, and nonimmunogenic protein obtained by controlled hydrolysis of the triple-helix structure of collagen into single-strain molecules. As an amphiphilic biopolymer, gelatin can easily assemble into different kinds of aggregates under the defined pH and temperature and the resulting gelatin nanoparticles have been developed to be applied in the food industry and biomedical fields. Herein we report a novel macromolecular microsphere composites (MMC) hydrogels with the use of prepared methacrylated gelatin nanoparticles (MA-GNP) as the cross-linker. MA-GNP have the ability of chemical crosslinking by the polymerization of C=C bonds, such that the composite hydrogels can be formed by radical polymerization of acrylamide (AAm) on the surface of MA-GNP. The smooth spherical particles with an average size of ~100 nm have been synthesized through a modified two-step desolvation method as proved by atomic force microscopy (AFM). The results of nuclear magnetic resonance and dynamic light scattering further confirm the presence of reactive groups (C=C bonds) in the particles and its narrow sizes distribution. The resulting composite hydrogels (MA-GNP/PAAm) are porous materials with tunable pore sizes and exhibit enhanced compressive resistance and elasticity as well. Increasing appropriately the dosage of MA-GNP reduces the equilibrium swelling ratio and improves thermal stability of the gels. Moreover, all the hydrogels exhibit prolonged blood-clotting time, nonhemolytic nature and strong suitability for cell proliferation, indicating the improved antithrombogenicity and excellent cyto-compatibility. It suggests that the novel MA-GNP/PAAm hydrogels have potential application as tissue engineer scaffold materials, and the MA-GNP can be a promising macromolecular microsphere cross-linker for application in biomedical materials. The present work not only exploits new strategies to fabricate MMC hydrogels but also advance the potential application of biodegradable gelatin-based nanoparticles in biomedical fields. Take-Away: 1. A well-dispersed methacrylated gelatin nanoparticle (MA-GNP) with an average size of ~100 nm is presented by a modified two-step desolvation method. 2. MA-GNP is readily introduced into the polyacrylamide (PAAm) system as a cross-linker to prepare macromolecular microsphere composites (MMC) hydrogels via a free radical polymerization reaction. 3. MA-GNP is an effective cross-linker, improving both the compressive resistance and elasticity of MMC hydrogels as well as the biocompatibility.

The content of this diploma thesis was testing the applicability of available structural analysis techniques on a appropriate model hydrogel system. The main aim was to evaluate the advantages and disadvantages of selected structural analysis techniques and the influence of the composition change of the hydrogel system on its internal structure. Semi-interpenetrated hydrogels based on agarose of different concentration were chosen as model system, this type of material was chosen for simple and repeatable preparation and also for its application potential. Electron microscopy (SEM, cryoSEM), mercury porosimetry and turbidimetry were chosen as structural analysis techniques. Experimental results could be used to improve knowledge about the influence of hydrogel composition on its structure and the comparison of chosen techniques will be used for the choice of appropriate structural analysis in the future, which will be applicable to other hydrogel systems as well.

The thesis deals with molecular dynamic simulation of the influence of water on the deformation of hydrogels. Hydrogels are model materials formed from macromolecular networks solvated with water. It was found that water can form bridges between macromolecules that take the form of temporary ionic crosslinks. These bridges affect the behavior of the network during deformation. Water bridges are water molecules that have a limited radius of motion in the space between two macromolecules. The concentration of the water bridges was regulated by a partial charge on the macromolecular chain in the organic network. Bridges are a type of interaction that is relatively strong but significantly delocalized. It is not possible to dissociate the water bridge, after dissociation it will be re-created in another place in a short time. The influence of water bridges was compared with other types of network crosslinks, especially covalent and physical bonds. Covalent crosslinks are modeled as a simple binding interaction between two macromolecules. They are undissociable and are local throughout the simulation. Physical bonds are modeled as micelles, where hydrophobic groups form the core and hydrophilic groups form the micelle shell. Physical bonds have the nature of dissociable bonds that are local. Different types of crosslinks have different effects on deformation properties. The deformation of a network containing a combination of two types of crosslinks was simulated: (i) physically-covalent, (ii) ionically-covalent, and (iii) physically-ionic networks and (iv) ternary physically-covalent-ion networks. For individual and combined networks, the behavior depending on simple networks was verified. The number of water bridges was fundamentally affected by the primary structure of the chains. When the PEG chain was replaced with hydrophobic polyoxymethylene (POM) or polyoxytrimethylene (POTM), their solvation and mechanical behavior deteriorated.

This doctoral thesis studies the properties and behaviour of phase-separated hydrogels. These can be prepared by interaction of polyelectrolytes with oppositely charged surfactants. Negatively charged polysaccharide hyaluronan and cationic surfactant carbethopendecinium bromide (Septonex), whose properties, behaviour and utilization are described in the first two chapters of theoretical part, were selected for this role. Hyaluronan is naturally-occuring in living organisms and is known for his specific targeting to the tumour cells. Septonex is used as antiseptic and disinfectant. Experimental part of this work is focused on examination of the structure and behaviour of these types of hydrogels especially from rheological point of view. The crucial part of this study was to establish mechanical properties and their dependence on environment. This led to design further studies. Structural analysis was held using the methods of ATR-FTIR, ionic chromatography and ICP-OES helping to measure the inner content of entry materials in the gels and supernatants respectively. The last chapter deals with antimicrobial activity. The results of this study indicate a potential usage of these substances in the field of medical applications.

This master´s thesis deals with the study on mechanical and transport properties of hybrid hydrogel systems. Considering applications of hydrogels, especially in chemical industry, pharmacy, or eventually medical applications, for the study gellan and alginate-based hydrogels were selected. In order to compare individual characteristics physical and hybrid hydrogels were prepared. Gellan hydrogels were prepared in deionization aqua solution, calcium chloride dihydrate and tween 80 solution. Alginate hydrogels were prepared in calcium chloride dihydrate solution as well, and polyacrylamide with N,N´–methylenbisacrylamide. For the study of mechanical properties moisture analyser and rheology measurements were selected. Transport properties were studied using the diffusion experiments combined with UV-VIS spectroscopic detection. Concluding of this thesis is summarization of measured values, which provides comprehensive review of the problematics. It was discovered that the conveniently selected concentrations of structural components of hydrogel matrix and the additives can influence both the mechanical as well as the transport properties of studied hydrogels.

The content of this diploma thesis is study of interactions between humic acids and model probes (such as organic dyes and ions of heavy metals) by the simple diffusion experiments in diffusion cell. The main aim was to desctibe the influence of charge of different diffusion sonds (solved substances) on interactions with humic acids imobilized in model gel phase. As a appropriete model probe was chosen copper ions (as a representation of heavy metals) and organic dyes (methylene blue, rhodamine, fluorescein and chicago blue). Experimental results could be used to improve knowledge based on natural barier properties of humic acids and to inovate simple diffusion laboratory techniques for characterization of reaktivity of biopolymers in general.

Diploma thesis main goal is to obtain new pieces of knowledge about relationship between hydrogel structures and its flow and transport properties. Thesis is mainly focusing on combining pertinent biopolymers into model hydrogels based on agarose. Then perform correlation of results obtained by diffusion methods, and by rheologic measurements on macroscopic and microscopic level. Properties of hydrogels were measured by selected rheologic measurements, dynamic light scattering method, and correlative fluorescence spectroscopy. From these methods various parameters (MSD modules, values of complex viscosity) were obtained. Afterwards transport properties of prepared hydrogels were studied by observing Rhodamine 6G diffusion. Here two different approaches were used. From macroscopic perspective, simple principles of mass diffusion from dye solution to cuvettes filled with hydrogels containing individual biopolymers were used. From microscopic perspective, dye was added during the sample preparation and then the mass diffusion was investigated using FCS. Based on evaluated results it was discovered that added biopolymers don’t influence properties of carrier medium, in this case agarose hydrogels. During the study of prepared hydrogels’ reactivity and barrier properties some differences were observed. Charge of biopolymer and its charge density were discovered as main factors influencing transport of charged solutes into prepared hydrogels.