Aims Novel low toxicity formulations using biomaterial (i.e. gelatin) for triggered release and controlled manner of formulated therapeutic agent for treatment of immuno-inflammatory disease on the skin were studied in the PhD project. It is a challenging concept because of difficulties in targeting and controlling for the releases that is tailored to disease severity or lesional inflammation extent. Background Psoriasis is a complicated disease with multi-factorial pathogenesis. Potent anti-psoriatic drugs are available but for managing the symptoms of the disease. Due to the toxicity of the therapeutic agents, different strategies have been suggested to avoid severe side effects from long term or high dose usage. Psoriasis is an optimal representative for this investigation in terms of the toxicities of recognized drugs, unpredictable or relapsed nature of the disease or even life threatening developments if generalised symptoms develop as they can in some types. Method Using the rheometry in temperature sweep mode, a series of concentrations of pure gelatin and gelatin mixture were developed. In addition, using tryptic enzyme, their action was studied rheologically. A Petri dish observational method was used to investigate the permeability of formulations chosen on the basis of the rheometric performance. Then, combining the Copley diffusion cell kit and UV/VIS spectrophotometer, the release of the model drug was investigated in porous artificial membranes and porcine skin for one or more of the formulations. The preliminary part using porous artificial membranes was to investigate the amount of the release of tartrazine from a candidate gel into the circulation system. In this part, alternatives were considered for dealing with gelatin or gelatin/carbomer swelling by using mechanical stress approach or changing to octanol solvent. For the latter a dye, rhodamine, which would partition into octanol had to be substituted for tartrazine (which has iv negligible organic solubility). In the final part, using skin membrane, the amount of the release tartrazine to the skin was measured because in this, skin staining, rather than partition was needed. Results Promising results were observed in each stage. The rheological investigation on the developed gelatin/water system and gelatin/carbomer intimate system in absence and presence of tryptic enzyme showed that a responsive but convenient formulation was possible and was independent of the presence of tartrazine. Analysis of these resulting rheological profiles suggested a prediction for the best gelatin/carbomer formulations to select for the permeability tests. The latter used Petri dishes to compare differential diffusion of these candidates showed the carbomer was able to stop three-dimensional spreading of the dye through the pure gelatin or its residue (after enzyme action). The drug release studies using artificial porous membranes for preliminary work showed significant differential release between enzyme free and enzyme treated versions of the 20% gelatin/0.9% carbomer formulation. The final success was the in vitro skin experiment in which the result was obtained for the pure gelatin and shown to deliver very substantially more to areas with applied enzyme s a simulated lesion.

<p>Validerat; 20130918 (global_studentproject_submitter)</p>

In this work, the synthesis of biopolymer-based hydrogel networks with defined architecture is presented. In order to obtain materials with defined properties, the chemoselective copper-catalyzed azide-alkyne cycloaddition (or Click Chemistry) was used for the synthesis of gelatin-based hydrogels. Alkyne-functionalized gelatin was reacted with four different diazide crosslinkers above its sol-gel transition to suppress the formation of triple helices. By variation of the crosslinking density and the crosslinker flexibility, the swelling (Q: 150-470 vol.-%;) and the Young’s and shear moduli (E: 50 kPa - 635 kPa, G’: 0.1 kPa - 16 kPa) could be tuned in the kPa range. In order to understand the network structure, a method based on the labelling of free functional groups within the hydrogel was developed. Gelatin-based hydrogels were incubated with alkyne-functionalized fluorescein to detect the free azide groups, resulting from the formation of dangling chains. Gelatin hydrogels were also incubated with azido-functionalized fluorescein to check the presence of alkyne groups available for the attachment of bioactive molecules. By using confocal laser scanning microscopy and fluorescence spectroscopy, the amount of crosslinking, grafting and free alkyne groups could be determined. Dangling chains were observed in samples prepared by using an excess of crosslinker and also when using equimolar amounts of alkyne:azide. In the latter case the amount of dangling chains was affected by the crosslinker structure. Specifically, 0.1% of dangling chains were found using 4,4’-diazido-2,2’-stilbene-disulfonic acid as cosslinker, 0.06% with 1,8-diazidooctane, 0.05% with 1,12-diazidododecane and 0.022 % with PEG-diazide. This observation could be explained considering the structure of the crosslinkers. During network formation, the movements of the gelatin chains are restricted due to the formation of covalent netpoints. A further crosslinking will be possible only in the case of crosslinker that are flexible and long enough to reach another chain. The method used to obtain defined gelatin-based hydrogels enabled also the synthesis of hyaluronic acid-based hydrogels with tailorable properties. Alkyne-functionalized hyaluronic acid was crosslinked with three different linkers having two terminal azide functionalities. By variation of the crosslinking density and crosslinker type, hydrogels with elastic moduli in the range of 0.5-3 kPa have been prepared. The variation of the crosslinking density and crosslinker type had furthermore an influence also on the hydrolytic and enzymatic degradation of gelatin-based hydrogels. Hydrogels with a low crosslinker amount experienced a faster decrease in mass loss and elastic modulus compared to hydrogels with higher crosslinker content. Moreover, the structure of the crosslinker had a strong influence on the enzymatic degradation. Hydrogels containing a crosslinker with a rigid structure were much more resistant to enzymatic degradation than hydrogels containing a flexible crosslinker. During hydrolytic degradation, the hydrogel became softer while maintaining the same outer dimensions. These observations are in agreement with a bulk degradation mechanism, while the decrease in size of the hydrogels during enzymatic degradation suggested a surface erosion mechanism. Because of the use of small amount of crosslinker (0.002 mol.% 0.02 mol.%) the networks synthesized can still be defined as biopolymer-based hydrogels. However, they contain a small percentage of synthetic residues. Alternatively, a possible method to obtain biopolymer-based telechelics, which could be used as crosslinkers, was investigated. Gelatin-based fragments with defined molecular weight were obtained by controlled degradation of gelatin with hydroxylamine, due to its specific action on asparaginyl-glycine bonds. The reaction of gelatin with hydroxylamine resulted in fragments with molecular weights of 15, 25, 37, and 50 kDa (determined by SDS-PAGE) independently of the reaction time and conditions. Each of these fragments could be potentially used for the synthesis of hydrogels in which all components are biopolymer-based materials. / In dieser Arbeit wird die Synthese Biopolymer-basierter Hydrogelnetzwerke mit definierter Architektur beschrieben. Um Materialien mit definierten und einstellbaren Eigenschaften zu erhalten, wurde die chemoselektive Kupferkatalysierte Azid-Alkin-Cycloadditionsreaktion (auch als Click-Chemie bezeichnet) für die Synthese Gelatine-basierter Netzwerke eingesetzt. Alkin-funktionalisierte Gelatine wurde mit vier verschiedenen Diazid-Quervernetzern oberhalb der Gel-Sol-Übergangstemperatur umgesetzt, um die Formierung tripelhelikaler Bereiche durch Gelatineketten zu unterdrücken. Durch Variation der Menge an Quervernetzer (und damit der Netzdichte) sowie der Länge und Flexibilität der Quervernetzer konnten u.a. die Quellung (Q: 150-470 vol.-%) sowie der Young’s - und Schermodul im kPa Bereich eingestellt werden (E: 50 kPa - 635 kPa, G’: 0.1 kPa - 16 kPa). Um die Netzwerkarchitektur zu verstehen, wurde eine Methode basierend auf dem Labeln unreagierter Azid- und Alkingruppen im Hydrogel entwickelt. Die Gelatine-basierten Hydrogele wurden mit Alkin-funktionalisiertem Fluorescein umgesetzt, um freie Azidgruppen zu detektieren, die bei einem Grafting entstehen. Darüber hinaus wurden die Hydrogele mit Azid-funktionalisiertem Fluorescein reagiert, um die Menge an freien Alkingruppen zu bestimmen, die zudem potentiell für die Anbindung bioaktiver Moleküle geeignet sind. Quervernetzung, Grafting, und die Anzahl freier Alkingruppen konnten dann mit Hilfe der konfokalen Laser Scanning Mikroskopie und der Fluoreszenzmikroskopie qualitativ und quantitativ nachgewiesen werden. Gegraftete Ketten wurden in Systemen nachgewiesen, die mit einem Überschuss an Quervernetzer hergestellt wurden, entstanden aber auch beim Einsatz äquimolarer Mengen Alkin- und Azidgruppen. Im letzteren Fall wurde in Abhängigkeit von der Struktur des Diazids unterschiedliche Anteile gegrafteter Ketten festgestellt. 0.1 mol-% von gegrafteten Ketten wurden für 4,4’-Diazido-2,2’-stilbendisulfonsäure gefunden, 0.06 mol-% für 1,8-Diazidooktan, 0.05 mol% für 1,12-diazidododecan und 0.022 mol-% für PEG-Diazid. Diese Beobachtung kann durch die unterschiedliche Flexibilität der Vernetzer erklärt werden. Während der Netzwerkbildung werden die Bewegungen der Gelatineketten eingeschränkt, so dass kovalente Netzpunkte nur erhalten werden können, wenn der Vernetzer lang und flexibel genug ist, um eine andere Alkingruppe zu erreichen. Die Strategie zur Synthese von Biopolymer-basierten Hydrogelen mit einstellbaren Eigenschaften wurde von Gelatine- auf Hyaluronsäure-basierte Gele übertragen. Alkin-funktionalisierte Hyaluronäure wurde mit drei verschiedenen Diaziden quervernetzt, wobei Menge, Länge, und Flexibilität des Quervernetzers variiert wurden. In dieser Weise wurden sehr weiche Hydrogele mit E-Moduli im Bereich von 0.5-3 kPa hergestellt. Die Variation der Vernetzungsdichte und des Vernetzertyps beeinflusste weiterhin den hydrolytischen und enzymatischen Abbau der Hydrogele. Hydrogele mit einem geringerem Anteil an Quervernetzer wurden schneller abgebaut als solche mit einem höheren Quervernetzeranteil. Darüber hinaus konnte gezeigt werden, dass Hydrogele mit Quervernetzern mit einer rigiden Struktur deutlich langsamer degradierten als Hydrogele mit flexibleren Quervernetzern. Während des hydrolytischen Abbau wurden die Materialien weicher, behielten aber ihre Form bei, was mit einem Bulk-Abbau-Modell übereinstimmt. Während des enzymatischen Abbaus hingegen änderten sich die Materialeigenschaften kaum, jedoch wurden die Proben kleiner. Diese Beobachtung stimmt mit einem Oberflächenabbaumechanismus überein. Da in allen vorgestellten Systemen nur eine kleine Menge synthetischer Vernetzer eingesetzt wurde (0.002 – 0.02 mol%), können die Materialien noch als Biopolymer-basierte Materialien klassifiziert werden. Jedoch enthalten die Materialien synthetische Abschnitte. In Zukunft könnte es interessant sein, einen Zugang zu Materialien zu haben, die ausschließlich aus Biopolymeren aufgebaut sind. Daher wurde der Zugang zu Biopolymer basierten Telechelen untersucht, die potentiell als Vernetzer dienen können. Dazu wurden durch die kontrollierte Spaltung von Gelatine mit Hydroxylamin Gelatinefragmente mit definiertem Molekulargewicht hergestellt. Hydroxalamin reagiert unter Spaltung mit der Amidbindung zwischen Asparagin und Glycin, wobei Aspartylhydroxamate und Aminoendgruppen entstehen. Die Reaktion von Gelatine mit Hydroxylamin ergab Fragmente mit Molekulargewichten von 15, 25, 37, und 50 kDa (bestimmt mit SDS-PAGE), und die Formierung dieser Fragmente war unabhängig von den weiteren Reaktionsbedingungen und der Reaktionszeit. Jedes dieser Fragmente kann potentiell für die Synthese von Hydrogelen eingesetzt werden, die ausschließlich aus Biopolymeren bestehen.

Heidelberg, Univ., Diss., 2007.

Orientadores: Marlene Rita de Queiroz, Neide Botrel Gonçalves / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia Agrícola / Made available in DSpace on 2018-08-13T02:53:54Z (GMT). No. of bitstreams: 1 Resende_JosaneMaria_D.pdf: 2330107 bytes, checksum: c1c0974d159b8ea7a1d43f517f3af29b (MD5) Previous issue date: 2007 / Resumo: A água de coco verde é extremamente perecível após a colheita dos frutos e sua qualidade está diretamente relacionada às condições do ambiente de armazenagem. Portanto, são necessárias tecnologias que permitam a conservação do coco verde por um período superior a trinta dias para que o fruto possa ser exportado e comercializado mantendo a água em condições apropriadas para o consumo ao natural. O presente trabalho teve o objetivo de desenvolver revestimentos biodegradáveis para prolongar a conservação do coco verde, variedade Anão Verde, propiciando maior tempo para a sua comercialização e consumo da água, sem alterar suas qualidades sensoriais e nutritivas. Cocos foram revestidos com biofilmes a base de quitosana, gelatina e carboximetilcelulose (CMC), nas seguintes formulações: quitosana (B1); gelatina + CMC (B2); quitosana + gelatina (B3); CMC (B4); gelatina (B5) e controle [água] (B6). Depois os frutos foram armazenados a 12 ± 2 ºC, com UR de 80%, por até 40 dias. Amostras foram retiradas a cada 10 dias para as análises físicas, fisíco-químicas, químicas, bioquímicas, microbiológicas e fisiológicas. Para a análise sensorial do fruto e da água foram retiradas amostras no início e aos 26, 33 e 40 dias de armazenamento. As características físicas, físico-químicas e químicas foram mais influenciadas pelo tempo de armazenamento do que pelos tratamentos de biofilmes. O tempo de armazenamento causou redução do volume e aumento da turbidez da água, redução do pH, dos teores de sólidos solúveis, de glicose, frutose e vitamina C, aumento da acidez total titulável, dos minerais, dos compostos fenólicos, da atividade da peroxidase e fenilalanina amônia-liase, mas não teve efeito sobre os teores de sacarose, de Na e Mn. Os biofilmes e o tempo de armazenamento influenciaram a acidez total titulável, teores de glicose e frutose, teores de N e de compostos fenólicos e a atividade da polifenoloxidase, porém as águas dos tratament os (B1), (B3) e (B5) apresentaram poucas alterações destas variáveis no decorrer do armazenamento. A presença de coliformes a 45 ºC e 35 ºC, aeróbios mesófilos, fungos filamentosos e leveduras na água foi menor nos frutos revestidos com (B1) e (B3). A contaminação foi maior na água dos frutos revestidos com (B5) e (B6). No entanto os índices de contaminação da água de todos os tratamentos foram considerados aceitáveis para o consumo. As análises fisiológicas mostraram diferenças emtre os biofilmes, sendo a concentração de O2 maior nas microcâmaras contendo frutos revestidos com quitosana e menor nas do controle. A concentração de CO2 foi maior nas microcâmaras contendo frutos do controle. As concentrações de CO2 e de O2 diminuíram nas microcâmaras ao final do armazenamento. A concentração de etileno foi muito pequena, abaixo do limite de detecção. A aceitação dos frutos diminuiu com o tempo de armazenamento, assim como a intenção de compra e expectativa em relação à água. Frutos revestidos com (B1), (B3) e (B5) tiveram maior aceitação ao final do tempo de armazenamento. A aceitação e a intenção de compra da água também diminuíram com o período de armazenamento. Frutos revestidos com gelatina não apresentaram boa aceitação da água. A água dos frutos revestidos com quitosana manteve a aceitação de sabor até 40 dias de armazenamento. Entre os biofilmes testados, o de quitosana apresentou o melhor desempenho, contribuindo para a manutenção das características nutricionais e sensoriais da água de coco e proporcionando maior aceitação do consumidor e melhor aparência do fruto por 40 dias de armazenamento, o que permite indicar este revestimento para fins de exportação / Abstract: The green coconut water is extremely perishable after harvest and its quality is directly related to storage ambient conditions. Therefore, technologies to allow the preservation of green coconut by a larger period than thirty days are need so that the fruit can be exported and commercialized maintaining the water in adequate conditions for in natura consumption. The present study had the objective of developing biodegradable coatings to prolong the conservation of green coconut, Anão Verde variety, to obtain larger time for its commercialization and consumption of water, without changing its sensorial and nutritional qualities. Coconuts were covered with bio-films made from chitosan, gelatin and carboxymethylcellulose (CMC), using the follow formulations: chitosan (B1); gelatin + CMC (B2); chitosan + gelatin (B3); CMC (B4); gelatin (B5) and control [water] (B6). Then the fruits were stored for up to 40 days at 12° ± 2°C and RH of 80%. Samples were removed every 10 days for physical, physicochemical, chemical, biochemical, microbiological and physiological analyses. For the fruit and water sensorial analysis, samples were removed in the beginning and at 26, 33 and 40 days of storage. The physical, physico-chemical and chemical characteristics were more affected by storage time than by the treatments with the bio-films. The storage time promoted the decrease of water volume and the increase of water turbidity, reduction of pH and soluble solid contents, glucose, frutose and vitamin C, increase of the total titratable acidity, minerals contents, phenolic compounds contents and the activity of peroxidase and phenylalanine ammonia-lyase but had no effect on the sucrose content and on some of minerals, such as Na and Mn. The bio-films and storage time affected the total titratable acidity, glucose, fructose contents, N and phenolic compounds contents and the polyphenoloxidase activity, although the water from the fruits coated with (B1), (B3) and (B5) showed little alteration respect to these variables during the storage time. The presence of coliforms at 45 ºC and 35 ºC, mesophilic aerobics, filamentous fungi and yeasts in the water was lower on the fruits coated with (B1) and (B3). Contamination was highest in the water of fruits coated with (B5) and (B6). However, all the water contamination indexes were considered to be within the acceptable levels for consumption. The physiological analyses showed differences among bio-films: the concentration of O2 was larger inside the microchambers containing fruits coated with chitosan and smaller on that containing fruits of the control. The concentration of CO2 was larger inside microchamber containing fruits of the control. The concentrations of CO2 and O2 inside microchambers decreased at the end of storage time. The ethylene concentration was very small, below the detection limit. The acceptance of the fruits decreased with storage time, as did the purchasing intention and expectation respect to the water. Fruits coated with (B1), (B3) and (B5) showed the greater acceptance at the end of the storage time. The acceptance and the purchasing intention of the water also decreased with storage period. Fruits coated with bio-films of gelatin did not present good acceptance of water. The water of the fruits coated with chitosan maintained the flavor acceptance up to 40 storage days. Among the bio-films tested, the chitosan presented the best performance, contributing to maintain the nutritional and sensorial characteristics of the coconut water and providing larger consumer acceptance and better appearance of the fruit for 40 days of storage, indicating this coconut coating for the exportation ends / Doutorado / Tecnologia Pós-Colheita / Doutor em Engenharia Agrícola

Diplomová práca sa zaoberá novou kvalitatívnou analýzou kolagénových vrstiev pomocou cirkulárneho dichroizmu v pevnej fáze. Teoretická časť opisuje kolagén a jeho sekundárnu štruktúru, spektroskopické metódy cirkulárneho dichroizmu (CD) a zoslabenej úplnej reflektancie (ATR). Experimentálna časť sa zaoberá prípravou homogénnych a transparentných kolagénových filmov a optimalizáciou objemu, koncentrácie a sušenia. Následne sa zmerala a vyhodnotila kalibračná krivka s rôznymi pomermi kolagénu a želatíny. Získaná rovnica lineárnej regresie sa použila na stanovenie obsahu kolagénu v modifikovaných vzorkách. Výsledky hodnotených modifikovaných vzoriek boli porovnané s meraniami ATR spektroskopie. Merania ATR ukázali vyšší obsah kolagénu ako CD merania. Vzorky s najnižším a najvyšším obsahom kolagénu sú pre obe metódy rovnaké. Dokazuje to, že môžme porovnávať vzorky s rôznym obsahom kolagénu navzájom, ale nemôžeme určiť presný obsah kolagénu pripojením týchto dvoch metód. Optimalizovaná metóda CD sa môže použiť na prípravu a meranie presného obsahu kolagénu vo vzorkách, najmä v rámci výroby kolagénu, aby sa dosiahla konštantná kvalita kolagénu v každom kroku výroby.

Gelatin is increasingly becoming an important raw material in many different fields. The pretreatment stage of the production of gelatin from pork skin plays an important role, it has big effect on the quality of obtained products. A survey on 5 types of acid HCl, H2SO4, H3PO4, CH3COOH, Citric was conducted. It showed that the influence of these types of acid on the pretreatment process was different. Acetic acid made the best result with the protein after extraction reaching 54.88 mg/ml, the product bloom reached 223. The most appropriate proportion of acetic acid was 3%. A survey on the effect of soaking time on the capacity of extraction was conducted. The result showed that the 16-hour extraction brought the best result reaching 56.16 mg/ml, the product bloom reached 245. The most suitable proportion of soaking pork skin:acid was 1:2. The most appropriate temperature of acid immersion was 15°C, at which the protein after extraction reached 68.39 mg/ml, the product bloom reached 299, the viscosity reached 23 mPas. / Gelatin ngày càng trở thành nguồn nguyên liệu quan trọng trong nhiều lĩnh vực khác nhau. Giai đoạn tiền xử lý của việc sản xuất gelatin từ bì lợn đóng vai trò quan trọng và có ảnh hưởng lớn tới chất lượng sản phẩm thu được. Khảo sát 5 loại axit: HCl, H2SO4, H3PO4, CH3COOH, Citric, thấy rằng ảnh hưởng của các loại axit này trong quá trình tiền xử lý là khác nhau. Axit axetic cho kết quả tốt nhất với hàm lượng protein sau trích ly đạt 54,88 mg/ml, độ bloom sản phẩm đạt 223. Nồng độ thích hợp nhất của axit axetic là 3%. Khảo sát ảnh hưởng của thời gian ngâm đến khả năng trích ly thấy rằng 16 giờ cho kết quả tốt nhất đạt 56,16 mg/ml, độ bloom sản phẩm đạt 245. Tỷ lệ ngâm bì lợn: axit thích hợp nhất là 1:2. Nhiệt độ ngâm axit thích hợp nhất là 15oC, tại đó hàm lượng protein sau trích ly đạt 68,39 mg/ml, độ bloom sản phẩm đạt 299, độ nhớt đạt 23 mPs.

Depuis une dizaine d'année, le rôle des biofilms dans la résistance aux antibiotiques et à la réponse immunitaire a été mis en évidence dans les infections chroniques. Les plaies chroniques possèdent ces mêmes particularités de résistance aux divers traitements. La formation de biofilm dans les plaies induit une protection des bactéries qui participe potentiellement à la chronicité et est responsable de l'inefficacité des soins. Le but de ma thèse est de concevoir un biogel actif contre les biofilms pouvant être présents dans les plaies chroniques.Nous avons développé un gel de gélatine innovant contenant divers agents anti-biofilms et permettant l'éradication des bactéries pathogènes. Ces gels sont des solides mous composés d'une phase liquide emprisonnée dans un réseau de biopolymères. Biocompatibles et biorésorbables, ils présentent des avantages pour une utilisation en tant que biomatériaux pour la santé. Il est possible d'y inclure des molécules qui vont ensuite diffuser vers le milieu extérieur. Pour mieux appréhender cette cinétique de diffusion, nous avons étudié le relargage de molécules modèles de différents poids moléculaires et de différentes charges.La stratégie anti-biofilm adoptée consiste à prévenir la colonisation bactérienne, déstabiliser le biofilm et éradiquer les bactéries pathogènes de la plaie (Diminution de la biomasse d'au moins 5 logarithmes). Pour développer ce système, 3 types de molécules ont été combinées : un antiseptique commercial, utilisé dans le cadre des soins des plaies chroniques, un chélateur d'ions actifs contre les MMPs surexprimées dans les plaies et une protéase capable de dégrader la matrice du biofilm. Cette étude a été réalisée sur des biofilms mono-espèce de P. aeruginosa et S. aureus, bactéries pathogènes fréquemment retrouvées dans les plaies chroniques. La combinaison des principes actifs a été testée en solution, en contact direct avec le biofilm. Puis, les agents ont été encapsulés dans un biogel. Nous avons étudié à la fois les propriétés viscoélastiques de ces gels mais aussi leur efficacité contre un biofilm, comparé au traitement non encapsulé. Lors de ce travail, nous avons élaboré un gel manipulable capable d'éradiquer un biofilm mono espèce ou multi espèces, constitué de souches de laboratoire ou de souches cliniques issues de plaie chronique. / Over the past 10 years, researchers have highlighted the role of biofilms on resistance to immune response and antibiotics treatments of chronic infections.Chronic wounds share these characteristics as they are resistant to care treatments. The possible biofilm formation in wound can protect bacteria participating to the chronicity and the resistance to wound care treatment. The aim of this thesis is to conceive a biogel against biofilms in chronic wounds.We have developed an innovative gelatin gel containing various anti-biofilm agents able to eradicate pathogenic bacteria biofilm. This gel is soft matter composed of a liquid phase entrapped in a biopolymer network. It is biocompatible and bioresorbable; these properties are necessary for a biomaterial. The gel has the capacity to deliver molecules with controlled release. We have studied the release of model molecules with different charges and weights.The anti-biofilm strategy consisted in preventing bacterial colonization, disrupting the biofilm and eradicating pathogen bacteria in wound (a decrease biomass of at least 5 log reductions). The system developed consisted in the combination of three different types of molecules: a commercial antiseptic usually applied in chronic wound care, an ion chelator active against MMPs which are over-expressed in chronic wounds and a protease which can disrupt the matrix of the biofilm.The study has been carried out on mono-species biofilms synthesized in vitro, with P. aeruginosa and S. aureus, two pathogenic bacteria frequently encountered in chronic wounds. The combination of the active agents was tested in solution or in directly contact with the biofilm. Then molecules were entrapped in the biogel. The viscoelastic properties of the gel were studied and the efficacy of the entrapped treatment compared to that of the solution. A handeable and efficient biomaterial has been elaborated during this study. It is able to eradicate mono – and multi- species biofilms from both laboratory and clinical bacterial strains.

This work presents the development of entropy-elastic gelatin based networks in the form of films or scaffolds. The materials have good prospects for biomedical applications, especially in the context of bone regeneration. Entropy-elastic gelatin based hydrogel films with varying crosslinking densities were prepared with tailored mechanical properties. Gelatin was covalently crosslinked above its sol gel transition, which suppressed the gelatin chain helicity. Hexamethylene diisocyanate (HDI) or ethyl ester lysine diisocyanate (LDI) were applied as chemical crosslinkers, and the reaction was conducted either in dimethyl sulfoxide (DMSO) or water. Amorphous films were prepared as measured by Wide Angle X-ray Scattering (WAXS), with tailorable degrees of swelling (Q: 300-800 vol. %) and wet state Young’s modulus (E: 70 740 kPa). Model reactions showed that the crosslinking reaction resulted in a combination of direct crosslinks (3-13 mol.-%), grafting (5-40 mol.-%), and blending of oligoureas (16-67 mol.-%). The knowledge gained with this bulk material was transferred to the integrated process of foaming and crosslinking to obtain porous 3-D gelatin-based scaffolds. For this purpose, a gelatin solution was foamed in the presence of a surfactant, Saponin, and the resulting foam was fixed by chemical crosslinking with a diisocyanate. The amorphous crosslinked scaffolds were synthesized with varied gelatin and HDI concentrations, and analyzed in the dry state by micro computed tomography (µCT, porosity: 65±11–73±14 vol.-%), and scanning electron microscopy (SEM, pore size: 117±28–166±32 µm). Subsequently, the work focused on the characterization of the gelatin scaffolds in conditions relevant to biomedical applications. Scaffolds showed high water uptake (H: 630-1680 wt.-%) with minimal changes in outer dimension. Since a decreased scaffold pore size (115±47–130±49 µm) was revealed using confocal laser scanning microscopy (CLSM) upon wetting, the form stability could be explained. Shape recoverability was observed after removal of stress when compressing wet scaffolds, while dry scaffolds maintained the compressed shape. This was explained by a reduction of the glass transition temperature upon equilibration with water (dynamic mechanical analysis at varied temperature (DMTA)). The composition dependent compression moduli (Ec: 10 50 kPa) were comparable to the bulk micromechanical Young’s moduli, which were measured by atomic force microscopy (AFM). The hydrolytic degradation profile could be adjusted, and a controlled decrease of mechanical properties was observed. Partially-degraded scaffolds displayed an increase of pore size. This was likely due to the pore wall disintegration during degradation, which caused the pores to merge. The scaffold cytotoxicity and immunologic responses were analyzed. The porous scaffolds enabled proliferation of human dermal fibroblasts within the implants (up to 90 µm depth). Furthermore, indirect eluate tests were carried out with L929 cells to quantify the material cytotoxic response. Here, the effect of the sterilization method (Ethylene oxide sterilization), crosslinker, and surfactant were analyzed. Fully cytocompatible scaffolds were obtained by using LDI as crosslinker and PEO40 PPO20-PEO40 as surfactant. These investigations were accompanied by a study of the endotoxin material contamination. The formation of medical-grade materials was successfully obtained (<0.5 EU/mL) by using low-endotoxin gelatin and performing all synthetic steps in a laminar flow hood. / Diese Arbeit beschreibt die Entwicklung Entropie-elastischer Gelatine-basierter Netzwerke als Filme und Scaffolds. Mögliche Anwendungen für die entwickelten Materialien liegen im biomedizinischen Bereich, insbesondere der Knochenregeneration. Im ersten Schritt der Arbeit wurden Entropie-elastische, Gelatine-basierte Hydrogel-Filme entwickelt, deren mechanische Eigenschaften durch die Veränderung der Quervernetzungsdichte eingestellt werden konnten. Dazu wurde Gelatine in Lösung oberhalb der Gel-Sol-Übergangstemperatur kovalent quervernetzt, wodurch die Ausbildung helikaler Konformationen unterdrückt wurde. Als Quervernetzer wurden Hexamethylendiisocyanat (HDI) oder Lysindiisocyanat ethylester (LDI) verwendet, und die Reaktionen wurden in Dimethylsulfoxid (DMSO) oder Wasser durchgeführt. Weitwinkel Röntgenstreuungs Spektroskopie (WAXS) zeigte, dass die Netzwerke amorph waren. Der Quellungsgrad (Q: 300-800 vol. %) und der Elastizitätsmodul (E: 70 740 kPa) konnten dabei durch die systematische Veränderung der Quervernetzungsdichte eingestellt werden. Die Analyse der Quervernetzungsreaktion durch Modellreaktionen zeigte, dass die Stabilisierung der Hydrogele sowohl auf kovalente Quervernetzungen (3-13 mol.-%) als auch auf Grafting von (5-40 mol.-%) und Verblendung mit Oligoharnstoffen (16-67 mol.-%) zurückgeführt werden kann. Die Erkenntnisse aus dem Umgang mit dem Bulk-Material wurden dann auf einen integrierten Prozess der Verschäumung und chemischen Quervernetzung transferiert, so dass poröse, dreidimensionale Scaffolds erhalten wurden. Dafür wurde eine wässrige Gelatinelösung in Gegenwart eines Tensids, Saponin, verschäumt, und durch chemische Quervernetzung mit einem Diisocyanat zu einem Scaffold fixiert. Die Scaffolds hergestellt mit unterschiedlichen Mengen HDI und Gelatine, wurden im trockenen Zustand mittels Mikro Computertomographie (µCT, Porosität: 65±11–73±14 vol.-%) und Rasterelektronenmikroskopie (SEM, Porengröße: 117±28–166±32) charakterisiert. Anschließend wurden die Scaffolds unter Bedingungen charakterisiert, die für biomedizinische Anwendungen relevant sind. Die Scaffolds nahmen große Mengen Wasser auf (H: 630 1680 wt.-%) bei nur minimalen Änderungen der äußeren Dimensionen. Konfokale Laser Scanning Mikroskopie zeigte, dass die Wasseraufnahme zu einer verminderten Porengröße führte (115±47–130±49 µm), wodurch die Formstabilität erklärbar ist. Eine Formrückstellung der Scaffolds wurde beobachtet, wenn Scaffolds im nassen Zustand komprimiert wurden und dann entlastet wurden, während trockene Proben in der komprimierten Formen blieben (kalte Deformation). Dieses Entropie-elastische Verhalten der nassen Scaffolds konnte durch die Verminderung der Glasübergangstemperatur des Netzwerks nach Wasseraufnahme erklärt werden (DMTA). Die zusammensetzungsabhängigen Kompressionsmoduli (Ec: 10 50 kPa) waren mit den mikromechanischen Young’s moduli vergleichbar, die mittels Rasterkraftmikroskopie (AFM) gemessen wurden. Das hydrolytische Degradationsprofil konnte variiert werden, und während des Abbaus kam es nur zu kontrolliert-graduellen Änderungen der mechanischen Eigenschaften. Während der Degradation konnte ein Anstieg der mittleren Porengröße beobachtet werden, was durch das Verschmelzen von Poren durch den Abbau der Wände erklärt werden kann. Die Endotoxinbelastung und die Zytotoxizität der Scaffolds wurden untersucht. Humane Haut-Fibroblasten wuchsen auf und innerhalb der Scaffolds (bis zu einer Tiefe von 90 µm). Indirekte Eluat-Tests mit L929 Mausfibroblasten wurden genutzt, um die Zytotoxizität der Materialien, insbesondere den Einfluss des Quervernetzertyps und des Tensids, zu bestimmen. Vollständig biokompatible Materialien wurden erzielt, wenn LDI als Quervernetzer und PEO40 PPO20-PEO40 als Tensid verwendet wurden. Durch den Einsatz von Gelatine mit geringem Endotoxin-Gehalt, und die Synthese in einer Sterilarbeitsblank konnten Materialien für medizinische Anwendungen (Endotoxin-Gehalt < 0.5 EU/mL) hergestellt werden.

Submitted by Emanuel Varela Cardoso (emanuel.varela@ufcg.edu.br) on 2018-06-13T20:55:07Z No. of bitstreams: 1 PAULA CHRISTIANNE GOMES GOUVEIA SOUTO MAIA – DISSERTAÇÃO (PPG-CEMat) 2015.pdf: 2231072 bytes, checksum: ae7116b6547ccb83d6170ccf2e265c42 (MD5) / Made available in DSpace on 2018-06-13T20:55:07Z (GMT). No. of bitstreams: 1 PAULA CHRISTIANNE GOMES GOUVEIA SOUTO MAIA – DISSERTAÇÃO (PPG-CEMat) 2015.pdf: 2231072 bytes, checksum: ae7116b6547ccb83d6170ccf2e265c42 (MD5) Previous issue date: 2015-06-19 / O estudo sobre sistema de liberação controlada de fármaco está em constante crescimento, pois visa melhorar e prolongar o controle da administração de fármacos. Insulina oral é um sonho dos pacientes e um desafio para os cientistas. Para os doentes, não é apenas o alívio da dor da aplicação de múltiplas injeções, mas também a proteção das células betas do pâncreas. A quitosana é um biomaterial considerado atóxico, não alergênica, biodegradável, biofuncional, biocompatível e as suas atividades biológicas compreendem a ação antioxidante, antimicrobiana, analgésica, aceleração da cicatrização, anti-inflamatórias além de ser muito estudada como matriz polimérica em sistemas de liberação controlada de fármacos. A gelatina por sua vez está sendo muito utilizada na área farmacêutica com a finalidade de favorecer o intumescimento do sistema e consequentemente acelerar o processo de liberação. Sendo assim, este trabalho teve como objetivo desenvolver membranas de quitosana, gelatina e insulina para uso em sistema de liberação controlada de fármacos. As membranas desenvolvidas foram caracterizadas pelas técnicas de Difração de raios-X (DRX), Espectroscopia na Região de Infravermelho com Transformada de Fourier (FTIR), Microscopia Eletrônica de Varredura (MEV) com Espectroscopia por Energia Dispersiva de raios X (EDS) e Microscopia Óptica (MO). Na técnica de DRX verificou que o que não ocorreu alteração significativa na cristalinidade das membranas. Com a técnica de FTIR verificou que a gelatina, insulina e o tripolifosfato de sódio não interferiram nos grupos funcionais de superfície da quitosana, mantendo desta forma as propriedades da mesma. No EDS foi possível detectar os elementos químicos característicos do material. Foi possível perceber, através das técnicas de MO e MEV, alteração na morfologia da membrana contendo insulina, gelatina e tripolifosfato de sódio quando comparada a de quitosana pura. Baseado nos resultados pode-se concluir que a insulina foi encapsulada pela quitosana e que a presença da gelatina influenciou no tamanho e forma dos poros das membranas e que a neutralização com NaOH diminuiu a quantidade de aglomerados nas superfícies dos arcabouços reticulados com tripolifosfato de sódio. / The study of drug controlled release system is constantly growing, it aims to improve and extend the control of drug administration. Oral insulin is a dream of patients and a challenge for scientists. For patients, it's not just pain relief applying multiple injections, but also the protection of beta cells of the pancreas. Chitosan, a biomaterial is considered non-toxic, non-allergenic, biodegradable, biofunctional, biocompatible and their biological activities include the antioxidant action, antimicrobial, analgesic, acceleration of wound healing, anti-inflammatory as well as being widely studied as polymer matrix systems controlled drug release. Gelatin turn is being widely used in the pharmaceutical field for the purpose of favoring the system swelling and consequently accelerate the release process. Thus, this study aimed to develop chitosan membranes, gelatin and insulin for use in controlled release system of drugs. The developed membranes were characterized by powder Diffraction X-ray (XRD), spectroscopy in the infrared region with a Fourier transform (FTIR) analysis, Scanning Electron Microscopy (SEM) with Energy Dispersive Spectroscopy X-ray (EDS) and Microscopy optical (MO). In XRD technique we found that what was no significant change in the crystallinity of the membranes. With FTIR technique found that gelatin, insulin and sodium tripolyphosphate did not affect the surface functional groups of chitosan, thereby maintaining the properties thereof. The EDS was possible to detect the characteristic chemical elements of the material. It was possible to see, through the techniques of OM and SEM, change in morphology of the membrane containing insulin, gelatin and sodium tripolyphosphate compared to pure chitosan. Based on the results it can be concluded that insulin was encapsulated by chitosan and the presence of the gelatin influence the size and shape of the pores of the membranes and neutralization with NaOH decreased the amount of agglomerates on the surfaces of scaffolds crosslinked with sodium tripolyphosphate.