Regulation of gene expression using small interfering RNA (siRNA) is a promising strategy for research and treatment of numerous diseases. However, siRNA cannot easily cross the cell membrane due to its inherent instability, large molecular weight and anionic nature. For this reason, a carrier that protects, delivers and unloads siRNA is required for successful gene silencing. The goal of this research was to develop a potential siRNA delivery system for in vitro and in vivo applications using cationic polymers, chitosan and polyethylenimine (PEI), poly(ethylene glycol) (PEG), mannose, and poly(D,L-lactic-co-glycolic acid) (PLGA). Furthermore, the delivery system was constructed in two different ways to explore the effect of mannose location in the structure. In the first approach, mannose and PEG were directly conjugated to the chitosan/PEI backbone, while mannose was connected to the chitosan/PEI backbone through PEG spacer in the second approach. First, the ability of modified chitosan polymers to complex and deliver siRNA for gene silencing was investigated. Despite the modified chitosan polymers successfully formed nanoplexes with siRNA, entered target cells and reduced cytotoxicity of unmodified chitosan, they showed limited gene silencing efficiency. For this reason, modified PEIs were examined to improve in vitro gene knockdown. The modified PEI polymers also complexed with siRNA and facilitated endocytosis of the nanoplexes. In addition, the modifications reduced inherent cytotoxicity of unmodified PEI without compromising the gene silencing efficiency on both mRNA and protein levels. Interestingly, we found that complexation of siRNA with PEI-PEG-mannose resulted in higher cell uptake and gene silencing than complexes made with mannose-PEI-PEG. Finally, the effect of sustained release of the mannosylated pegylated PEI/siRNA nanoplexes on gene silencing was tested by encapsulating the nanoplexes within PLGA microparticles. The modified PEIs enhanced the entrapment efficiency of siRNA into the particles and resulted in reduced initial burst followed by sustained release. Incorporating the modified PEIs increased cellular uptake of siRNA, whereas it did not enhance in vitro gene knockdown efficiency due to the sustained release properties. The modified PEIs reduced the in vitro cytotoxicity and in vivo hepatotoxicity of the PLGA microparticles. In addition, encapsulating the nanoplexes into PLGA microparticles further reduced the cytotoxicity of PEI. Throughout the study, the second structure was proven more efficacious than the first structure in cellular uptake, gene silencing, siRNA encapsulation, and sustained release. We have developed novel polymeric siRNA delivery systems that enhance delivery efficiency and cellular uptake of siRNA. They have great potential for utility as a long-acting siRNA delivery system in biomedical research.

L'administration de protéines de façon locale et prolongée à partir de microsphères biodégradables de PLGA présente un réel intérêt thérapeutique (diminution du nombre d'injections, potentialisation de l'activité, diminution des effets secondaires), plus particulièrement, dans le domaine de l'ingénierie tissulaire pour l'apport de facteurs de croissance. Cependant, la faible stabilité des protéines dans ces systèmes limite leur développement. Dans ce travail, des stratégies ont été mises en place pour préserver l'intégrité de protéines à la fois lors des étapes d'encapsulation et de libération. Dans une étude préliminaire, des protéines modèles et thérapeutiques ont été précipitées afin d'éviter leur dénaturation pendant leur future encapsulation. Des particules submicroniques ont ainsi été formées par ajout d'un non solvant et d'un sel à une solution aqueuse de protéine. Après optimisation des rendements de précipitation, ces particules de protéines ont été microencapsulées par un procédé de simple émulsion (s/o/w). Aucune perte d'activité n'a été observée et ce, sans stabilisant. Ensuite, pour pallier au ralentissement de la libération après le premier jour d'incubation des microsphères, l'interaction protéine-polymère a été modélisée et des composés capables de limiter l'adsorption du lysozyme sur le PLGA ont été sélectionnés, notamment le poloxamer 188. La précipitation du lysozyme avec du poloxamer 188 avant encapsulation a ainsi conduit à une libération continue de protéine active pendant trois semaines sans effet burst à partir de microsphères de PLGA et pendant plus de six semaines avec un copolymère tribloc de PLGA-PEG-PLGA.

This research centers on the fabrication, characterization, and engineering of inverse opal scaffolds, a novel class of three-dimensional (3D) porous scaffolds made of biocompatible and biodegradable polymers, for applications in tissue engineering and regenerative medicine. The unique features of an inverse opal scaffold include a highly ordered array of pores, uniform and finely tunable pore sizes, high interconnectivity, and great reproducibility. The first part of this work focuses on the fabrication and functionalization of inverse opal scaffolds based on poly(D,L-lactic-co-glycolic acid) (PLGA), a biodegradable material approved by the U.S. Food and Drug Administration (FDA). The advantages of the PLGA inverse opal scaffolds are also demonstrated by comparing with their counterparts with spherical but non-uniform pores and poor interconnectivity. The second part of this work shows two examples where the PLGA inverse opal scaffolds were successfully used as a well-defined system to investigate the effect of pore size of a 3D porous scaffold on the behavior of cell and tissue growth. Specifically, I have demonstrated that i) the differentiation of progenitor cells in vitro was dependent on the pore size of PLGA-based scaffolds and the behavior of the cells was determined by the size of individual pores where the cells resided in, and ii) the neovascularization process in vivo could be directly manipulated by controlling a combination of pore and window sizes when they were applied to a mouse model. The last part of this work deals with the novel application of photoacoustic microscopy (PAM), a volumetric imaging modality recently developed, to tissue engineering and regenerative medicine, in the context of non-invasive imaging and quantification of cells and tissues grown in PLGA inverse opal scaffolds, both in vitro and in vivo. Furthermore, the capability of PAM to monitor and quantitatively analyze the degradation of the scaffolds themselves was also demonstrated.

Implantable sensors have been extensively investigated to facilitate diagnosis or to provide a means to generated closed loop control of therapy by yielding in vivo measurements of physical and chemical signals. Biodegradable sensors which degrade gradually after they are no longer functionally needed exhibit great potential in acute or shorter-term medical diagnostic and sensing applications due to the advantages of (a) exclusion of the need to a secondary surgery for sensor removal, and (b) reduction of the risk of long-term infection. The objective of this research is to design and characterize microfabricated RF wireless pressure sensors that are made of completely biodegradable materials and degrade at time-controlled manner (in the order of years and months). This was achieved by means of investigation of appropriate biodegradable materials and development of appropriate fabrication processes for these non-standard (Microelectromechanical systems) MEMS materials. Four subareas of research are performed: (1) Design of sensors that operate wirelessly and are made of biodegradable materials. The structure of the wireless sensor consists a very compact and relatively simple design of passive LC resonant circuits embedded in a polymer dielectric package. To design the sensor with a particular resonant frequency range, an electromagnetic model of the sensor and a mechanical model for circular plate are developed. The geometry of the sensor is established based on the analytical and finite element simulations results. (2) Investigation of the biodegradable materials in the application of implantable biodegradable wireless sensors to achieve controllable degradation lifetimes. Commercially available and FDA approved biodegradable polymers poly(L-lactic acid) (PLLA) and a "shell-core" structure of poly(lactic-co-glycolic acid) (PLGA) and polyvinyl alcohol (PVA) are utilized as the dielectric package for slow and rapid degradation sensors, respectively. Biodegradable metallic zinc and zinc/iron couples are chosen as conductor materials. The degradation behavior of Zn and Zn/Fe-couple are investigated in vitro. (3) Development of novel fabrication processes. The process exploit the advantages of MEMS technology in fabricating miniaturized devices, while protecting vulnerable biodegradable materials from the strong and/or hazardous chemicals that are commonly used in conventional MEMS fabrication process. These new processes enable the fabrication of biocompatible and biodegradable 3-D devices with embedded, near-hermetic cavities. (4) Testing the pressure response functionality and studying the degradation behavior of the wireless biodegradable pressure sensors. Both PLLA-based and PLGA/PVA-based sensors are characterized in vitro by being immersed in 0.9% saline for prolonged time. All the sensors exhibit three stages of behavior in vitro: equilibration, functional lifetime, and performance degradation. During the functional lifetime, most sensors exhibit fully stable functionality. The PLLA-based sensors show no significant weight loss within 8 month and are expected to fully degrade after 2 years, while the PLGA/PVA-based sensors can degrade completely within 26 days.

No description available.

Η βλέννα του σαλιγκαριού έχει χρησιμοποιηθεί στην ιατρική από την αρχαιότητα από την εποχή του Ιπποκράτη, ο οποίος είχε προτείνει τη χρήση της βλέννας για την καταπολέμηση του πόνου, εγκαυμάτων και άλλων τραυμάτων. Πρόσφατη επιστημονική μελέτες του εκκρίματος του σαλιγκαριού Helix aspersa επιβεβαίωσαν ότι η βλέννα περιέχει ένα ασυνήθιστο συνδυασμό φυσικών προϊόντων με ευεργετικές και θεραπευτικές ιδιότητες στο ανθρώπινο δέρμα. Η βλέννα του σαλιγκαριού παρουσιάζει σημαντικό οικονομικό - εμπορικό ενδιαφέρον, ως προς την αξιοποίηση των χημικών συστατικών της όπως η αλλαντοΐνη και το γλυκολικό οξύ που χρησιμοποιούνται ευρύτατα ως δραστικά συστατικά κατά την παρασκευή φαρμακευτικών και καλλυντικών προϊόντων. Η αλλαντοΐνη είναι μία ετεροκυκλική οργανική ένωση που παράγεται από την κατάλυση των πουρινών από το ένζυμο καταλάση. Παρουσιάζει μια μακρά ιστορία όσον αφορά στη χρήση της σε μια ποικιλία από τοπικά φάρμακα και καλλυντικά προϊόντα για την περιποίηση του δέρματος. Το γλυκολικό οξύ είναι το μικρότερο α-υδροξύ - οξύ (AHA). Χάρη στην εξαιρετική ικανότητά του να διαπερνάει το δέρμα, βρίσκει εφαρμογές σε διάφορα προϊόντα φροντίδας του δέρματος, για τη βελτίωση της εμφάνισης και της υφής του δέρματος μειώνοντας ρυτίδες, ουλές ακμής, υπέρχρωση και άλλα και σε άλλες δερματικές παθήσεις. Στην παρούσα εργασία έγινε προσπάθεια ανάπτυξης μεθοδολογιών για τον ποιοτικό και ποσοτικό προσδιορισμό της αλλαντοΐνης και του γλυκολικού οξέος. Με χρωματογραφία υψηλής απόδοσης (HPLC-DAD) σε στήλη Synergi RP (Phenomenex ®) επετεύχθη πολύ καλός διαχωρισμός εντός 6 min. Οι τελικές συνθήκες διαχωρισμού (ισοκρατική έκλουση με εκλούτη ρυθμιστικό διάλυμα 10 mM KH2PO4 pH 2.7 και η ροή ρυθμίστηκε στο 0.7 mL/min και στους 30 C) και προκατεργασίας δείγματος (αραίωση της βλέννας στο ρυθμιστικό διάλυμα, θέρμανση στους 60 C για 20 λεπτά και εκχύλιση με εξάνιο) επιλέχθηκαν μετά από μελέτη διαφόρων παραμέτρων που αφορούν στη σύσταση του εκχυλιστικού μέσου και της κινητής φάσης. Η μέθοδος επικυρώθηκε ως προς τα αναλυτικά της χαρακτηριστικά (γραμμικότητα, επαναληψιμότητα, ακρίβεια και σταθερότητα). Ανάλυση της βλέννας του σαλιγκαριού Helix aspersa έδειξε ότι περιέχει 48.610 ± 0.002, 47.590 ± 0.001, 52.820 ± 0.001 mg/L αλλαντοΐνης και 3753.01 ± 0.01, 3170.51 ± 0.02, 1979.23 ± 0.02 mg/L γλυκολικού οξέος για τις τρεις παρτίδες που αναλύσαμε, αντίστοιχα. Η προτεινόμενη μεθοδολογία χρησιμοποιήθηκε για την ποσοτική ανάλυση τριών καλλυντικών κρεμών (Elicina της εταιρίας Cosméticos Elicina Ltda, Santiago, Χιλή, Labcconte της εταιρίας Cosméticos Concepción Ltda, Χιλή και μια κρέμα από την εταιρία Helix ir στην Κρήτη) στις οποίες η βλέννα του σαλιγκαριού αναγράφεται να περιέχεται σε υψηλό ποσοστό (85%). Τα αποτελέσματα μας δείχνουν ότι τα καλλυντικά σκευάσματα περιέχουν 0.110 ± 0.003, 0.260 ± 0.010 , 0.370 ± 0.001 mg/L αλλαντοΐνης και 4.520 ± 0.010, 5.350 ± 0.010, 5.690 ± 0.001 mg/L γλυκολικού οξέος, αντίστοιχα. / The snail mucus has been used in medicine since the time of Hippocrates, who proposed the use of mucus to combat pain, burns and other wounds. Recent scientific studies of the snail secretion Helix Aspersa confirmed that the snail contains an unusual combination of natural products which are beneficial for human skin. The last time the mucus of the snail has important economic - commercial interest in the use of chemical components such as allantoin and glycolic acid are widely used as active ingredients in the manufacture of pharmaceutical and cosmetic products. Allantoin is a heterocyclic organic compound produced by the breakdown of purines by the enzyme catalase. It has a long history in the use of a variety of topical medications and cosmetics for skin care. The glycolic acid is the smallest alpha-hydroxy acid (AHA). Thanks to its excellent ability to penetrate the skin, it finds applications in various skin care products, for improving the appearance and texture of skin by reducing wrinkles, acne scars, hyperpigmentation and also can improve many other skin disorders. In this study we developed analytical methodology for qualitative and quantitative determination of allantoin and glycolic acid. Application of highperformance chromatography (HPLC-DAD) on a Synergi RP (Phenomenex ®) column has provided a very good separation within 6 min. The final separation (isocratic elution with buffer 10 mM KH2PO4 pH 2.7 as eluent and flow rate 0.7 mL/min at 30 °C) and sample pretreatment (by dilution the mucus in the buffer, heated at 60 °C for 20 min and extracted with hexane) were selected after a study of various parameters relating to the extraction medium and the mobile phase. The method was validated in terms of analytical characteristics (linearity, repeatability, accuracy and stability). Analysis of the snail secretions Helix aspersa showed that it contains 48.610 ± 0.002, 47.590 ± 0.001, 52.820 ± 0.001 mg/L allantoin and 3753.01 ± 0.01, 3170.51 ± 0.02, 1979.23 ± 0.02 mg/L glycolic acid for three batches that we analyzed, respectively. The methodology was used for quantitative analysis of three cosmetic creams (Elicina from the company: Cosméticos Elicina Ltda, Santiago, Chile, Labcconte from the comany: Cosméticos Concepción Ltda, Chile and a cream from the company: Helix ir in Crete) where the snail mucus is deemed to be about 85%. Our results show that cosmetic products contain 0.110 ± 0.003, 0.260 ± 0.010 , 0.370 ± 0.001 mg/L allantoin and 4.520 ± 0.010, 5.350 ± 0.010, 5.690 ± 0.001 mg/L glycolic acid, respectively.

Orientador: Eliana Aparecida de Rezende Duek / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia Mecanica / Made available in DSpace on 2018-08-14T20:10:35Z (GMT). No. of bitstreams: 1 Silva_KatiaFernanda_M.pdf: 16700696 bytes, checksum: 568261b128b361204100ac55aba7640b (MD5) Previous issue date: 2008 / Resumo: A Ciência dos Biomateriais é uma área de conhecimento multidisciplinar e está relacionada a implantes cirúrgicos e dispositivos médicos e odontológicos. Dentre as atuais aplicações dos biomateriais, a utilização de polímeros biorreabsorvíveis como arcabouço (scaffolds) para cultura de células tem se destacado como alternativa para tratamento de lesões e perda de tecidos. O objetivo deste trabalho foi obter e caracterizar arcabouços da blenda de poli(e-caprolactona) (PCL) e poli(D,L-ácido lático-co-ácido glicólico) (PLGA) nas diferentes composições (70/30, 50/50 e 30/70), preparados pelo método de fusão com adição e lixiviação de citrato de sódio. Os resultados obtidos através das análises de MEV evidenciaram que as amostras da blenda de PCL/PLGA 30/70 possuem tamanhos e quantidades de poros mais homogêneos quando comparadas às suas análogas de PCL/PLGA nas composições 50/50 e 70/30. Os materiais foram utilizados como arcabouço para cultura de osteoblastos e o estudo in vivo foi realizado pelo implante em tíbia de ratos. A cultura de células nas blendas de PCL/PLGA 70/30 e 30/70 mostraram um padrão de adesão e proliferação satisfatório, com intensa atividade celular comparado com a blenda na composição 50/50. No estudo in vivo as amostras de PCL/PLGA 30/70 com cultura apresentaram um crescimento celular mais intenso quando comparada com as blendas nas composições 70/30 com cultura, e nas diferentes composições sem cultura de células. Os resultados mostraram que a técnica utilizada no preparo das amostras é útil na confecção de arcabouços altamente porosos. Em função das análises in vitro e in vivo as blendas de PCL/PLGA 30/70 foram os arcabouços mais indicados para aplicações na engenharia de tecidos ósseos. / Abstract: The use of bioreabsorbable polymers as scaffolds for the cells culture has received special attention as an alternative for the treatment of lesions and the loss of tissue. The aim of this work was to obtain and to characterize porous scaffolds of poly(e-caprolactone) (PCL) and poly(D,Llactic acid-co-glycolic acid) (PLGA), blends, in different composition (30/70, 50/50 e 70/30), prepared by melting method with addition and leaching of sodium citrate. The morphology of samples obtained by SEM showed that PCL/PLGA 30/70 blends presented size and size distribution of porous more homogeneous when compared to PCL/PLGA 50/50 and 30/70. The culture of cells in PCL/PLGA 70/30 and 30/70 showed a satisfactory adhesion and proliferation, with intense cellular activity compared to PCL/PLGA 50/50. In the in vivo study, the samples of PCL/PLGA 30/70 showed a higher cellular grown compared to the results obtained in in vivo study of 70/30 blends with cells, 70/30 and 30/70 without cells. The results showed that the proposed technique allowed the preparation of highly porous scaffolds and the in vitro and in vivo showed that PCL/PLGA 30/70 blends were the most suitable scaffolds for applications in the bone tissue engineering. / Mestrado / Materiais e Processos de Fabricação / Mestre em Engenharia Mecânica

Orientadores: Paulo Pinto Joazeiro, Eliana Aparecida de Rezende Duek / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-15T19:20:15Z (GMT). No. of bitstreams: 1 Lucchesi_Carolina_D.pdf: 5696358 bytes, checksum: 89bc8546c0a1d6b5a11d7260168dd236 (MD5) Previous issue date: 2010 / Resumo: Os polímeros biorreabsorvíveis, tais como, PHB, PCL e PLGA, têm sido estudados como dispositivo para engenharia de tecidos por serem biocompatíveis, suportarem o crescimento e diferenciação celular e os produtos de sua degradação serem atóxicos. No entanto, a escolha do biomaterial depende das necessidades exigidas para uma determinada aplicação. Os suportes para engenharia de tecidos devem se basear na construção de réplicas biológicas in vitro, como que o biomaterial se tornasse parte integrada para transplante in vivo para a recuperação de perdas ou mau funcionamento de tecidos ou órgãos, devendo subseqüentemente, atuar sem agredir o restante do organismo, isto é, sem o risco de rejeição ou complicação. Muitas estratégias têm sido desenvolvidas com o intuito de substituir tecidos ou órgãos danificados, incluindo a aplicação de suportes tridimensionais (3D), os quais devem possuir características estruturais e mecânicas para guiar a proliferação e espalhamento de células in vitro e in vivo. Os suportes, feitos de materiais sintéticos ou naturais, servem como substitutos para a matriz extracelular (MEC) nativa. Ênfase especial é dada as técnicas com controle computadorizado, como a fabricação sólida com forma livre (SFF), conhecida como prototipagem rápida (RP), a qual permite preparar suportes 3D com geometrias complexas, tanto externamente como internamente, além de ser uma técnica rápida e de baixo custo. Além disso, grande parte dos polímeros possuem superfície hidrofóbica, característica inadequada para a maior parte dos diferentes tipos celulares, o que dificulta aplicação na engenharia de tecidos. Uma alternativa a este problema é o tratamento da superfície por plasma. Este tratamento induz modificação restrita ao topo da superfície, conferindo carater hidrofílico à superfície, dependendo do gás utilizado. Neste estudo, arcabouços de polímeros biorreabsorvíveis PCL, PLGA e PHB foram preparados por diferentes técnicas, casting e sinterização seletiva a laser, avaliando-se o comportamento de células osteoblásticas diferenciadas sobre os biomateriais poliméricos tridimensionais. Inicialmente o trabalho foi desenvolvido com os polímeros PCL e PLGA preparando-se blendas poliméricas, as quais demonstraram melhorar as características gerais dos polímeros, quando utilizados como dispositivos para tecido ósseo, como as propriedades mecânicas. Com o intuito de aprimorar o design tridimensional do material, optou-se pela realização da técnica de sinterização seletiva a laser. No entanto, a técnica exige uma grande quantidade de material e devido ao alto do custo do PCL e PLGA, este foi substituído pelo polímero PHB, o qual é produzido pela indústria nacional Biocycle possuindo um baixo custo e ainda ser biocompatível. Os dados são apresentados em capítulos independentes. Arcabouços porosos de PCL, PLGA e suas blendas foram preparados pela técnica de evaporação do solvente, onde sais de citrato de sódio com granulometria de 180-250 µm, foram adicionados a solução para a promoção dos poros, sendo posteriormente lavados do arcabouço. Células osteoblásticas provenientes de calota craniana de ratos Wistar foram semeadas sobre os arcabouços, sendo avaliado o comportamento de citoxicidade do material e o comportamento de adesão e morfologia celular através de ensaios bioquímicos e MEV. Os arcabouços de PHB foram obtidos por uma das técnicas de RP, a sinterização seletiva a laser (SSL), sendo sua superfície modificada por plasma pelos gases oxigênio e nitrogênio. Células osteoblásticas provenientes de calota craniana de coelhos foram semeadas sobre os arcabouços realizando-se o estudo in vitro, através de análises bioquímicas pela técnica do MTT, para viabilidade e adesão celular, quantificação de colágeno por Sírius Red e Microscopia Eletrônica de Varredura (MEV). Para o estudo in vivo, após o cultivo celular sobre os arcabouços, defeitos ósseos foram provocados em coelhos e os arcabouços contendo as células foram então implantados, avaliando-se a interação PHB/osteoblasto/tecido, através da análise histológica. Todos os arcabouços estudados, PCL, PLGA e suas blendas, assim como o PHB, não apresentaram índices de citotoxicidade, permitiram às células a capacidade de adesão, proliferação e síntese de matriz, mantendo seu fenótipo osteoblástico. As amostras de PHB tratadas por plasma de Nitrogênio mostrou melhorar a capacidade de adesão celular. Os arcabouços de PHB contendo células mostraram-se os mais adequados para o preenchimento de defeitos ósseos, melhorando o processo de regeneração apresentando uma boa osteointegração. A sinterização seletiva a laser apresentou-se uma excelente técnica para a obtenção de PHB 3D para a Engenharia de Tecidos. / Abstract: The bioresorbable polymers as, PHB, PCL and PLGA have been studied as device for Tissue Engineering for their biocompatibility and to support the cell growth and differentiation and their degradation products are nontoxics. However, the choice of the biomaterial depends on the needs demanded for a certain application. The scaffolds for tissue engineering have to be designed to mimetize the biological conditions in vitro to became part integrated for transplant for the recovery of tissue or organs lost or without function, and subsequently, to work in a cordial way with the remaining of the organism without the rejection risk or complication. A lot of strategies have been developed with to substitute damaged tissues or organs, and it has been used the application of three-dimensional supports (3D), which should possess structural and mechanical applications to guide the cells proliferation and spread in vitro and in vivo. The scaffolds, made from synthetic or natural materials, serve as substitutes for the extracellular matrix (ECM) native. Special emphasis is given the techniques with computerized control, as the free solid form (SFF), known as rapid prototyping (RP), which allows to prepare three-dimensional supports with complex geometries, so much externally as internally, besides to be a fast technique with low cost. Besides, great part of the polymers possess hydrophobic surface, inadequate characteristic for most of the different cell types, which is not desirable for tissue engineering applications. An alternative to this problem is the surface treatment by plasma. Plasma treatment induces restricted modification to the top of the surface, improving the surface hydrophilicity, depending on the gas used. In this study, scaffolds of bioresorbable polymer PCL, PLGA e PHB were prepared by different techniques, casting and selective laser sintering, being evaluated the osteoblast cells behavior on the 3D polymer scaffolds. Previously we developed the studies preparing the polymeric blends with PCL and PLGA, which demonstrated improve the general characteristic of the material, as the mechanical properties, as devices for bone tissue. With the intention to improve the design of the scaffolds, we chose for the selective laser sintering technique. However, the technique demands a great amount of material and due to the high cost of PCL and PLGA, those weres substituted by PHB polymer, which is produced by Brazilian industry Biocycle with low cost and still to be biocompatible. For those reasons the data are presented in independent chapters. PCL, PLGA porous scaffolds and their blends were prepared those scaffolds by casting solvent, and sodium citrate with 180-250 µm were added to the solution for porous formation when the salt was washed later of the scaffolds. Osteoblast cells from rat Wistar calvaria were seemed on the scaffolds, being evaluated the behavior of cell adhesion and viability behavior, cell morphology through biochemical assays, and scanning electron microscopy. Three-dimensional PHB scaffolds were obtained by selective sintering laser (SSL), with the surface modified by nitrogen and oxygen plasma. Osteoblast cells obtained from rabbit calvaria were seemed on the scaffolds to the in vitro studies, through biochemical analyses by MTT test for cell viability and cell adhesion, collagen quantification of by Sirius Red colorimetric assay and scanning electron microscopy (SEM). For the in vivo studies, bone defects were provoked in rabbits and they were filling out with 3D PHB with osteoblast cells culture prior implant. We evaluated the PHB/osteoblast/tissue, interaction through the histological analysis. All the scaffolds studied PCL, PLGA and their blends, as well as the PHB did not showed cytotoxicity effects, allowed cells adhere, proliferated, and matrix synthesized, maintaining their osteoblastic phenotype. The PHB samples treated by nitrogen plasma have been showed to improve the cell adhesion. The PHB scaffolds with cell seeded previously demonstrated to be more suitable for filling out bone defects, improving the regeneration process showing a good osteointegration. The selective laser sintering was excellent technique to obtain PHB scaffolds for Tissue Engineering. / Doutorado / Biologia Celular / Doutor em Biologia Celular e Estrutural

Cette thèse vise à développer des nanoparticules de PLGA pour la vectorisation et à étudier l’interaction de ces nanoparticules avec des bicouches phospholipidiques imitant les membranes cellulaires. Pour la vectorisation passive, les changements physico-chimiques ont été contrôlés en incubant les NPs de PLGA (50:50) dans différentes conditions de pH tamponné à des intervalles de temps accrus. Le PLGA a montré plusieurs comportements de dégradation différant selon le pH. La formation de pores a été observée à pH élevé (conditions basiques) tout en préservant le volume des particules mais en modifiant la densité. Par opposition, à faible pH, une érosion superficielle des particules conduisant à une diminution de leur taille a été démontrée. Cette étude a été réalisée à l'aide de la DLS, l’ESEM et la spectrophotométrie. Pour la vectorisation active, les parois des capsules de PLGA (75:25) ont été modifiées par addition de phospholipides. La libération de la sonde fluorescente hydrophile, la calcéine, a été contrôlée en augmentant la température. On a observé qu'avec le DOPC (0,31 mM), la vectorisation peut être déclenchée à l'aide de détergents ou d'une enzyme (PLA2). Dans le cadre de cette étude, nous avons proposé la formation d'un complexe lipide-polymère ayant lieu à l'intérieur de la matrice, ce qui le rend vulnérable aux enzymes ou détergents induisant sa libération. L'effet des NPs de PLGA sur les bicouches phospholipidiques imitant la membrane cellulaire a été réalisé à l'aide de sondes fluorescentes moléculaires (Prodan et Laurdan). L'étude a été effectuée en calculant la polarisation généralisée (GP) sous l'influence des NPs de PLGA (50:50 et 75:25). L'interaction ayant lieu s’avérait être un phénomène de surface et aucune effet des NPs sur la perméabilité des membranes modèles LUVs et SUVs n’a été souligné. La valeur de Tm des phospholipides est également maintenue lorsque l’étude est menée avec le Laurdan. Les études de GP mené avec la sonde Prodan fournissent la première méthode originale pour déterminer la Tg de PLGA dans des conditions aqueuses. C'est une méthode rapide et facile qui détermine la valeur de Tg de PLGA en temps réel et en utilisant une très petite quantité de l'échantillon. Cette interaction n'est pas affectée par la composition des membranes cellulaires imitant les bicouches. / This thesis aims at developing PLGA nanoparticles for controlled release and investigating its interaction with phospholipid bilayers mimicking cell membranes. For passive controlled release the physiochemical changes were monitored by incubating the PLGA (50:50) NPs in different buffered pH conditions at increased time intervals. PLGA exhibited dissimilar degradation behavior with pore formation for high pH (basic conditions) maintaining the volume of the particles but change in the density, while at low pH it showed surface erosion. There is decrease in the particle size upon incubating in low pH. This study was carried out using DLS, ESEM and spectrophotometry. For active release the walls of PLGA (75:25) capsules were modulated using phospholipids. The release of hydrophilic fluorescent probe Calcein was monitored with increasing the temperature. It was observed that with DOPC (0.31mM) the release can be triggered using detergents or an enzyme (PLA2). We propose the formation of a lipid-polymer complex within the polymer matrix forming plugs which are vulnerable to enzymes/detergents inducing release. The effect of PLGA NPs over the phospholipid bilayers mimicking cell membrane was carried out using molecular fluorescent probes (Prodan and Laurdan). The study was carried out by calculating the generalised polarisation (GP) under the influence of PLGA NPs (50:50 and 75:25). It is found that the interaction is a surface phenomenon and there is no influence of NPs over the permeability of model membranes LUVs and SUVs. The Tm value of the phospholipids is also maintained when studied with Laurdan. Prodan probe GP studies provide first original method to determine the Tg of PLGA in complete aqueous conditions. It is a rapid and easy method which determines the Tg value of PLGA in real time using very small quantity of the sample. This interaction is not affected by the composition of the bilayer mimicking cell membranes.

This work deals with the using and application of separation techniques for analysis of polymers degradation and polycyclic aromatic hydrocarbons. Thereby this work is separated to two special parts. In the first part, the degradation properties of synthetic biopolymers based on lactic acid, gylcolic acid and poly(ethyleneglycol) PLGA-PEG-PLGA and ITA-PLGA-PEG-PLGA-ITA (modified by itaconic acid) were studied. These copolymers (firstly their thermosensitive hydrogels) should be used for therapy of fractures in orthopedy (as adhesives). Therefore, the sol-gel and gel-sol phase diagrams were determinated for selected samples of copolymers. The samples forming gel at 37 C was used for other study. Polymer samples were depredated in phosphate buffer at 37°C. The degradation process of physical hydrogels was described by the decrease of molecular weight and the increase of concentration lactic acid and glycolic acid in phosphate buffer. The obtained results confirmed that the degradation of polymer modified by itaconic acid is faster process than no modified polymer and polymers with lower ratio PLGA/PEG degrade also faster than lower ration PLGA/PEG. The influence of pH it was also tested. The rate of degradation of polymers was follow pH 4,0