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.

Depuis plusieurs années, la vectorisation de molécules est considérée comme la stratégie la plus prometteuse pour améliorer la pénétration cutanée des principes actifs et pour cibler et contrôler leur libération, augmentant ainsi l'efficacité thérapeutique des traitements tout en limitant leurs effets secondaires. Les nanoparticules polymériques ont fait l'objet de nombreuses études car elles possèdent une très grande stabilité et une capacité supérieure aux autres vecteurs à libérer les principes actifs de façon prolongée. Récemment, le laboratoire de Pharmacie Galénique de Besançon a montré, in vivo, que les nanoparticules polymériques de diamètre inférieure ou égale à 100 nm pénétraient spécifiquement dans la peau inflammée de la souris, alors qu'aucune pénétration n'était observée dans la peau saine. Le premier objectif de cette thèse était de confirmer ces hypothèses dans la peau inflammée d'un autre animal, le porc. Les résultats de notre étude in vivo confirment l'absence de pénétration des nanoparticules polymériques dans la peau saine du porc et montrent une pénétration taille dépendante dans la peau inflammée. Le second objectif poursuivi était de vérifier ces résultats chez l'Homme. Pour cela, une preuve de concept a été mise en place dans le service de Dermatologie du CHRU de Besançon. Les premiers résultats de cette étude clinique semblent confirmer l'absence de pénétration des nanoparticules polymérique de I 00 nm dans la peau des volontaires sains et dans la peau non lésée des patients souffrant de dermatite atopique. A l'inverse, une forte pénétration des nanoparticules est observée au niveau des plaques d'eczéma des patients. Si les résultats de l'étude clinique se confirment, nous prévoyons d'encapsuler un anti­inflammatoire ou un immunosuppresseur pour vérifier l'intérêt thérapeutique de ces vecteurs en médecine humaine dans le traitement de la dermatite atopique. Parallèlement, une évaluation pourrait être réalisée en médecine vétérinaire pour le traitement de cette pathologie fréquente chez le chien, et dont le traitement actuel repose sur l'administration per os de corticoïdes à l'origine de nombreux effets secondaires. / For several years now, nanocarriers have been considered as the most promising strategy to improve skin penetration of active ingredients. Moreover, these carriers are more efficient at targeting and controlling drug release into the skin, which leads to increased treatment efficiency and reduced side effects. Polymeric nanoparticles have been the object of an increasing number of studies due to their good physicochemical stability and prolonged release of active ingredients which is superior to any other carriers. Recently, the Laboratory of Pharmaceutical Engineering at Besançon proved in vivo, that polymeric nanoparticles with a diameter smaller or equivalent to I 00 nm specifically penetrated in inflamed skin of mice whilst no penetration was observed in healthy skin. The first aim ofthis thesis was to confirm this hypothesis on another animal's inflamed skin, in instance the pig. Our results confirm the poor penetration of polymeric nanoparticles in healthy skin ofpigs and show various degree ofpenetration depending on the size of the nanoparticles into the inflamed skin area. The second objective ofthis work was to evaluate the skin penetration of our polymeric nanoparticles in humans. A proof of concept has been developed in the Department of Dermatology at Besancon University Hospital. The first results ofthis clinical trial tend to confim1 the greater penetration ofour carrier, specifically in inflamed skin. In fact, no penetration of polymeric nanoparticles with a size close to 100 nm was observed in healthy skin ofvolunteers or in the non-inflamed skin of patients suffering from atopic dennatitis. Conversely, a high penetration ofthese carriers was observed in the skin lesions of patients with atopic dermatitis. If the results ofthis clinical trial are confirmed, we plan to load an anti-inflammatory or an immunosuppressive drug into the nanoparticles to evaluate the therapeutic value ofthese nanocarriers in human medicine in the treatment ofatopic dermatitis. Meanwhile, a similar study may be undertaken in veterinary medicine for the treatment of atopic dermatitis in dogs which is a common disease whose current treatment is based on the oral administration of corticosteroids and cause many undesirable side effects.

O processo de micrencapsulação de proteínas em microesferas (MS) de PLGA [poli (ácido lactico-co-glicolico)] é fácil de fazer e é uma ferramenta útil para melhorar tanto uma formulação quanto para aumentar a atividade imunológica de vacinas de novas gerações. A MS-PLGA têm caráter adjuvante porque é um sistema particulado e, além disto, controla a liberação do antígeno. O escopo desta tese foi o de dar uma nova roupagem para um antígeno vacinal tradicional e muito bem estudado- o toxoide diftérico (Dtxd). Estudaram-se a produção de MS de tamanho desejado; os mecanismos que controlam danos nas proteínas durante o processo de micrencapsulação; a produção de microesferas com características de liberações em tempos distintos e ensaios biológicos. O tamanho de MS é um determinante fundamental para controlar a velocidade de liberação de um soluto. Para se produzir MS com tamanhos controlados usou-se um desenho fatorial experimental com três fatores distintos e três pontos centrais, para se determinar a influência das variáveis (concentração de poli álcool vinílico; velocidade de agitação e relação fase dispersa/fase contínua) na determinação do tamanho das MS. Foram obtidas MS esféricas e lisas de 4- 15 µm de diâmetro. Estes resultados abrem a possibilidade de se formular PLGA-MS com tamanhos planejados através de um mínimo de experimentos. O mecanismo de danos conformacionais nas proteínas nas várias fases do processo de produção de PLGA-MS é ainda uma questão em aberto. Usaram-se várias técnicas biofísicas (HPLC, espectroscopias no uv, fluorescência e CD) além de ELISA para se testar a interferência dos sais da série de Hofmeister sobre a solubilidade e estabilidade da proteína durante a emulsificação e do contacto com a interface água/cloreto de metileno (primeira etapa do processo de preparação de MS). Estudaram-se também a influência de oligômeros de PLGA e SDS sobre a estrutura da proteína no meio de liberação (etapa de liberação do soluto). A emulsificação de Dtxd na presença de Mg2+ induziu agregação protéica, com exposição de resíduos hidrofóbicos para o meio; variações no ângulo diédrico do S-S proteico e perda de identidade imunológica. Esta agregação foi quase abolida pelo caotrópico SCN- (toxicidade = 30 g/ homem adulto de 70 kg). A conformação \"nativa\" do Dtxd e sua atividade biológica foram protegidas pelo KSCN. Os oligômeros de PLGA e o SDS induziram uma conformação de Dtxd nova. A adição de KSCN na fase aquosa aumentou a eficiência de encapsulação de Dtxd pela PLGA-MS em 20 %. Esta foi a solução mais simples quando comparada com aquelas descritas na literatura. Produziram-se seis formulações diferentes (diferentes massas molares e carboximetilações do PLGA) com pelo menos três cinéticas de liberações distintas. Imunizaram-se camundongos com 5 µg de Dtxd encapsulado em MS-PLGA usando-se dois polímeros de 12 kDa (-COOH livre ou metilado) e um outro de 63 kDa (metilado). O padrão de resposta e a maturidade imunológicas foram medidos por titulações de IgG1 e IgG2a. Mantiveram-se os mesmos padrões de resposta humoral (desejável). Menores quantidades de antígenos foram necessárias para se obter os mesmos benefícios gerados pela vacina tradicional de Dtxd. Aumentaram-se a produção e a seletividade de anticorpos através de duas manipulações simples: a formulação e o tempo da aplicação da dose de reforço. Estes resultados colocam estas formulações na área de vacinas de sucesso uma vez que também foram obtidas memórias imunológicas. / The protein microencapsulation within microspheres (MS) of PLGA (Poly-lactide-co-glycolide) is easy to do and, it is a useful tool to enhance formulation and immunologic performances for new generation vaccines. MS-PLGA has adjuvant character because it is a particulate system and can control the antigen release. The question addressed in this thesis was to give this new dress for the traditional and well studied vaccine antigen - the diphtheria toxoid (Dtxd). The steps of MS control size production; mechanism to control protein damages; MS production with different polymers and biological assay were addressed here. MS size is a primary determinant of solute release velocity. A full factorial experimental design 23 with triplicate at the central point was used to determine the influence of variables (polyvinyl alcohol concentration, stirring velocity and the relationship between dispersed /continuous phase) on MS size. Uniformly spherical and smooth microspheres (4 - 15 µm of diameter) were obtained. These results open the possibility of formulating PLGA microspheres with custom sizes performing a minimum of experiments as required for specific applications. It stills an open question to detail the conformational mechanism of protein damages during the various steps of the PLGA microencapsulation process. Various techniques (HPLC gel filtration, ELISA, Fluorescence, UV and Circular dichroism spectroscopies) were tested on the interference of the Hofmeister ion series over protein solubility and stability during the emulsification and contact with the interface water/CH2Cl2 interface (First step on MS preparation). The interference of SDS and PLGA olygomers over protein structure in the liberation media was also studied (solute liberation step). The Dtxd emulsification in the presence of Mg2+ was followed by protein aggregation, with exposition of hydrophobic residues and changes on the dihedral S-S protein angle and loses on immunological identity. This aggregation is 95% avoided by the chaotropic and little toxic salt KSCN (30g/ adult human of 70 kg). All the \"native\" Dtxd conformation and biological properties were maintained by KSCN. MS with different liberation kinetics profile and different erosion characteristics were obtained by using six different polymers. The SDS and PLGA olygomers exerted a generation of new Dtxd molecular organization. The KSCN increased Dtxd encapsulation within PLGA-MS in more than 20 %. This was the simplest solution used to solve protein aggregation compared with others solutions used in the literature. The six different formulations produced (differing in molar mass and carboxymethylation) produced, at least, three different Dtxd liberation profiles. Mice were primed with 5 µg of Dtxd microencapsulated within MS prepared with 12 kDa (ended carboxymethylated or free PLGA) and with 63 kDa (methylated) PLGA. The response patterns and the immune maturity were measured by IgG1 and IgG2a titrations. The humoral pattern was maintained, but fewer antigens were needed to obtain the same traditional Dtxd vaccine benefits. The simple change on Dtxd-PLGA formulation and timing of the booster enhanced both, antibody production and selectivity. An immunological memory was also obtained, putting so, these formulations in the field of successful vaccine.

Developing nanoparticulate delivery systems that will allow easy movement and localisation of a drug to the target tissue and provide more controlled release of the drug in vivo is a challenge for researchers in nanomedicine. The aim of this study was to evaluate the biodistribution of two nano-delivery systems namely, poly(D,L-lactide-co-glycolide) (PLGA) nanoparticles containing samarium-153 oxide ([153Sm]Sm2O3) as radiotracer and solid lipid nanoparticles (SLNs) containing technetium-99m-methylene diphosphonate (99mTc-MDP), after oral and intravenous administration to rats to prove that orally administered nanoparticles indeed alter the biodistribution of a drug as compared to the drug on its own. Stable samarium-152 oxide ([152Sm]Sm2O3) was encapsulated in polymeric PLGA nanoparticles. These were then activated in a nuclear reactor to produce radioactive [153Sm]Sm2O3 loaded-PLGA nanoparticles. Both the stable nanoparticles as well as the fully decayed activated nanoparticles, were characterized for size, Zeta potential and morphology using dynamic light scattering and scanning electron microscopy (SEM) or transmission electron microscopy (TEM), respectively. SLNs were a form of delivery system which was used to encapsulate the radiotracer, 99mTc-MDP. 99mTc-MDP SLNs were characterized before and after encapsulation for size and Zeta potential. Both nanoparticle compounds were orally and intravenously (IV) administered to rats in order to trace their uptake and biodistribution through imaging and ex vivo biodistribution studies. The PLGA nanoparticles containing [153Sm]Sm2O3 were spherical in morphology and smaller than 500 nm, therefore meeting the objective of producing radiolabelled nanoparticles smaller than 500 nm. Various parameters were optimized to obtain an average particle size ranging between 250 and 300 nm, with an average polydispersity index (PDI) ≤ 0.3 after spray drying. The particles had a Zeta potential ranging between 5 and 20 mV. The Sm2O3-PLGA nanoparticles had an average size of 281 ± 6.3 nm and a PDI average of 0.22. The orally administered [153Sm]Sm2O3-PLGA nanoparticles were deposited in various organs which includes bone with a total of 0.3% of the Injected Dose (ID) per gram vs the control of [153Sm]Sm2O3which showed no uptake in any organs except the GI-tract. The IV injected [153Sm]Sm2O3-PLGA nanoparticles exhibit the highest localisation of nanoparticles in the spleen (8.63%ID/g) and liver (3.07%ID/g). The 99mTc-MDP-labelled SLN were spherical and smaller than 500 nm. Optimization of the MDP-loaded SLN emulsions yielded a slightly higher PDI of ≥0.5 and a size range between 150 and 450 nm. The Zeta potential was between -30 and -2 mV. The MDP-loaded SLN had an average size of 256 ± 5.27 and an average PDI of 0.245.The orally administered 99mTc-MDP SLN had the highest localisation of nanoparticles in the kidneys (8.50%ID/g) and stomach (8.04%ID/g) while the control, 99mTc-MDP had no uptake in any organs except the GI-tract. The IV injected 99mTc-MDP SLN also exhibited a high localisation of particles in the kidneys (3.87%ID/g) followed by bone (2.66%ID/g). Both the IV and oral 99mTc-MDP SLN reported significantly low deposition values in the heart, liver and spleen. Based on the imaging and the biodistribution studies, it can be concluded that there was a significant transfer of the orally administrated radiolabelled nanoparticles from the stomach to other organs vs the controls. Furthermore, this biodistribution of the nano carriers warrants surface modification and optimisation of the nanoparticles to avoid higher particle localisation in the stomach. / MSc (Pharmaceutics), North-West University, Potchefstroom Campus, 2014

Developing nanoparticulate delivery systems that will allow easy movement and localisation of a drug to the target tissue and provide more controlled release of the drug in vivo is a challenge for researchers in nanomedicine. The aim of this study was to evaluate the biodistribution of two nano-delivery systems namely, poly(D,L-lactide-co-glycolide) (PLGA) nanoparticles containing samarium-153 oxide ([153Sm]Sm2O3) as radiotracer and solid lipid nanoparticles (SLNs) containing technetium-99m-methylene diphosphonate (99mTc-MDP), after oral and intravenous administration to rats to prove that orally administered nanoparticles indeed alter the biodistribution of a drug as compared to the drug on its own. Stable samarium-152 oxide ([152Sm]Sm2O3) was encapsulated in polymeric PLGA nanoparticles. These were then activated in a nuclear reactor to produce radioactive [153Sm]Sm2O3 loaded-PLGA nanoparticles. Both the stable nanoparticles as well as the fully decayed activated nanoparticles, were characterized for size, Zeta potential and morphology using dynamic light scattering and scanning electron microscopy (SEM) or transmission electron microscopy (TEM), respectively. SLNs were a form of delivery system which was used to encapsulate the radiotracer, 99mTc-MDP. 99mTc-MDP SLNs were characterized before and after encapsulation for size and Zeta potential. Both nanoparticle compounds were orally and intravenously (IV) administered to rats in order to trace their uptake and biodistribution through imaging and ex vivo biodistribution studies. The PLGA nanoparticles containing [153Sm]Sm2O3 were spherical in morphology and smaller than 500 nm, therefore meeting the objective of producing radiolabelled nanoparticles smaller than 500 nm. Various parameters were optimized to obtain an average particle size ranging between 250 and 300 nm, with an average polydispersity index (PDI) ≤ 0.3 after spray drying. The particles had a Zeta potential ranging between 5 and 20 mV. The Sm2O3-PLGA nanoparticles had an average size of 281 ± 6.3 nm and a PDI average of 0.22. The orally administered [153Sm]Sm2O3-PLGA nanoparticles were deposited in various organs which includes bone with a total of 0.3% of the Injected Dose (ID) per gram vs the control of [153Sm]Sm2O3which showed no uptake in any organs except the GI-tract. The IV injected [153Sm]Sm2O3-PLGA nanoparticles exhibit the highest localisation of nanoparticles in the spleen (8.63%ID/g) and liver (3.07%ID/g). The 99mTc-MDP-labelled SLN were spherical and smaller than 500 nm. Optimization of the MDP-loaded SLN emulsions yielded a slightly higher PDI of ≥0.5 and a size range between 150 and 450 nm. The Zeta potential was between -30 and -2 mV. The MDP-loaded SLN had an average size of 256 ± 5.27 and an average PDI of 0.245.The orally administered 99mTc-MDP SLN had the highest localisation of nanoparticles in the kidneys (8.50%ID/g) and stomach (8.04%ID/g) while the control, 99mTc-MDP had no uptake in any organs except the GI-tract. The IV injected 99mTc-MDP SLN also exhibited a high localisation of particles in the kidneys (3.87%ID/g) followed by bone (2.66%ID/g). Both the IV and oral 99mTc-MDP SLN reported significantly low deposition values in the heart, liver and spleen. Based on the imaging and the biodistribution studies, it can be concluded that there was a significant transfer of the orally administrated radiolabelled nanoparticles from the stomach to other organs vs the controls. Furthermore, this biodistribution of the nano carriers warrants surface modification and optimisation of the nanoparticles to avoid higher particle localisation in the stomach. / MSc (Pharmaceutics), North-West University, Potchefstroom Campus, 2014

Cancerous solid tumors can migrate and lead to metastatic bone disease. Drugs prescribed to reduce bone resorption from metastasis, such as zoledronic acid and the RANKL-inhibitory antibody Denosumab, cause side effects such as osteonecrosis of the jaw when delivered systemically. This project used two biocompatible materials, acrylic bone cement (PMMA) and poly(lactic-co-glycolic acid) (PLGA), to incorporate and sustain release of anti-resorptive agents. Results showed similar mechanical properties for acrylic bone cements loaded up to 6.6% drug by weight. Results showed sustained zoledronic acid release for 8 weeks from both systems, with PMMA releasing up to 22% of loaded drug and PLGA films releasing over 95%. The antibody release rate was lower, with the majority of antibody still inside the PLGA films after 8 weeks. In vitro bioactivity remained above 50% for zoledronic acid eluted from both materials at early, middle, and late time points. This study sheds light on the behavior of these biocompatible polymers at high drug weight percent loadings compared to previous studies. PLGA demonstrated superior release kinetics but inferior bioactivity of eluted drug. By incorporating anti-resorptive drugs into locally implantable materials, this work could lead to a treatment offering improved quality of life for cancer patients.

Charles University in Prague, Faculty of Pharmacy in Hradec Králové Department of: Pharmaceutical Technology Consultants: PharmDr. Ondřej Holas, Ph.D. Mgr. Jana Kubačková Student: Adélka Semrádová Title of thesis: Acidoresistant polymeric nanoparticles: preparation and assessment Use of oral drug delivery nanosystems has a great potential in therapy of inflammatory bowel disease, which includes Crohn's disease and ulcerative colitis. Nanosized delivery systems are more efficiently accumulated at the inflammatory site, targeting specifically macrophages to resolve inflammation locally and reduce systemic adverse effects. The aim of this research was to prepare pharmaceutical formulations based on polymeric nanoparticles. Three types of poly(lactide-co-glycolide) - two linear and one branched polymer - together with the acidoresistant polymer cellulose-acetate phthalate (CAF) in various ratios were used to prepare nanoparticles by nanoprecipitation method. Rhodamine B was used as model active substance. The effect of acidoresistant component content on size and zeta potential of the nanoparticles was evaluated. Furthermore, dissolution tests were performed at both acidic and physiological pH. It was found that CAF doesn't have any significant effect on size of the particles and their stability....

L’embolisation thérapeutique est devenu le traitement de choix pour l’hémorragie, les malformations artériovéneuses ou certains types de cancer. Parmi différents agents d’embolisation,les microsphères non dégradables (Embozene®, Bead BlockTM,…) sont les plus utilisées. Leur forme bien sphérique et leur taille calibrée permettent un meilleur ciblage dans les vaisseaux et une bonne qualité de l’occlusion. Dans certains cas cliniques, l’embolisation temporaire, envisageable avec l’utilisation des microsphères résorbables peut être bénéfique pour les patients. Le but du travail réalisé au cours de cette thèse a été le développement de microsphères résorbables satisfaisant les différents critères pour être employées comme matériaux d’embolisation (taille calibrée,biocompatibles, élastique pour être injectée au travers des cathéters mais suffisamment rigide pour résister à la pression sanguine). Dans cet objectif, nous avons développé une méthode de synthèse de microsphères constituées d’hydrogels hydrolysables par polymérisation en suspension. Une large gamme de microsphères ont été synthétisées en modulant la nature du réticulant et/ou la composition des milieux de polymérisation. Les expériences in vitro ont démontré que les microsphères obtenues sont satisfaisantes pour permettre leur injection au travers des cathéters. La dégradation rapide des ponts de réticulation a été confirmée à travers la diminution du module élastique G’ et du pH du surnageant, accompagnée d’une augmentation du taux de gonflement.Malgré une dégradation partielle des microsphères (due à une réaction secondaire formant des liaisons de réticulation non dégradables), le temps de l’hydrolyse a répondu parfaitement au cahier de charges (entre 7 et 49 jours). Des études complémentaires pour optimiser la réaction de polymérisation vont permettre le développement de microsphères totalement dégradables. / Therapeutic embolization is nowadays a first line treatment for haemorrhage, arteriovenous malformation or tumors. Among different embolization agents, non degradable microspheres(Embozene®, Bead BlockTM,…) are the most employed thanks to their well calibrated spherical shape which allows good occlusion. In some cases including treatment of uterine fibroids or chemo-sensitive tumors, it may be interesting to achieve a temporary embolization to avoid definitive destruction of the tissue. Temporary embolization would be possible using biodegradable microspheres. The aim of our work was to develop degradable microspheres having all requiredcharacteristics to be used as embolization material (well calibrated in size, biocompatible, rigide enough to resist blood pressure but elastic enough to remain intact during injection through catheter). To this purpose, we have developed hydrolysable hydrogel based microspheres by suspension polymerization. A wide range of microspheres was synthesized by varying the type of crosslinker and composition of the polymerization medium. In vitro test showed that the microspheres have suitable characteristics to pass through catheter. Degradation studies revealed a rapid diminution of G’ modulus and the pH of the supernatants, accompanied by an increase of swelling ratio due to the hydrolysis of the crosslinkings. Although microspheres were not totally degradable as expected (since a side reaction had created non degradable crosslinking during the polymerisation), characterisations showed promising results that the degradation did occur within a suitable time scale requirements for temporal embolization.

Submitted by Caroline Xavier (caroline.xavier@pucrs.br) on 2017-06-26T14:56:31Z No. of bitstreams: 1 TES_RICARDO_FERNANDES_GARCIA_PARCIAL.pdf: 159331 bytes, checksum: aacf8f2e7202562d6ba8bfb886ab0c8d (MD5) / Made available in DSpace on 2017-06-26T14:56:31Z (GMT). No. of bitstreams: 1 TES_RICARDO_FERNANDES_GARCIA_PARCIAL.pdf: 159331 bytes, checksum: aacf8f2e7202562d6ba8bfb886ab0c8d (MD5) Previous issue date: 2017-03-08 / More and more studies are focusing on the combination of matrices that have osteoconductive characteristics with osteoinductive proteins. These proteins can stimulate the differentiation of mesenchymal and osteoprogenitor cells into osteoblasts and thereby increase the migration of cells related to bone formation within the defect site. The main materials used for these purposes are biodegradable polymers, such as PLA (poly lactic acid) and PLGA (poly lactic glycolic acid). Several drugs are used to be released in these systems, such as antibiotics, contraceptives and proteins, including human growth hormone (GH). RhGH in bone tissue promotes the increased deposition of proteins by chondrocytes and osteoblasts, an increase in the number of mitoses and the conversion of chondrocytes into osteoblasts. Thus, it is necessary to evaluate this biomaterial (PLGA \ rhGH) as a controlled release device. Thus achieving the presence of rhGH in the site of bone healing. For this study was used as animal model, wistar rats. The study was carried out in accordance with Law No. 11,794 of October 8, 2008, as well as following the Brazilian Directive of Practice for the Care and Use of Animals for Scientific and Educational Purposes - DBPA of CONCEA. Project approved by CEUA of PUCRS under number 15/00461. Thirty adult wistar rats were used, in which they were submitted to bone defects with Carbide spherical drill number 704, the defect corresponded to the diameter of the drill. The rats were submitted to the same treatment but suffered euthanasia at different times (07, 15 and 20 days). The femurs of the rats were radiographed in the range of 7, 15, and 20 days to gauge the optical density. Slides were then produced for histological analysis, with cuts of approximately 5 ?m thick and stained with hematoxylin / eosin (HE). Three more representative cuts of each blade were chosen. The systemic repercussion of rhgh was assessed through IGF-1 levels through blood collection that was performed before each euthanasia, in the control group and in the experimental group. The blood collected was centrifuged (2500 revolutions per minute for 10 min) and serum obtained stored in a freezer at -20 ? C for further determination of IGF-1 plasma levels using the method based on an enzymatically amplified assay of the " Sandwich, "held at the Senhor dos Passos laboratory in Porto Alegre, RS. For statistical analysis, the Tukey test was applied, 5% significance level and ANOVA variance analysis. There were no statistically significant differences in bone mineral densities, however numerically the PLGA\RhGH grafts always had a greater amount of shades of gray. In conclusion, defects with PLGA\RhGH grafts showed a higher optical density. A higher mineral density compared to the autogenous graft In the histological analyzes there was also no statistically significant difference between the autogenous and PLGA\RhGH grafts. However, by numerical analysis we observed a better and equal performance between the PLGA\RhGH graft and autogenous graft. Defects with PLGA\RhGH grafting probably had faster healing and maturation than autogenous grafts. / Cada vez mais estudos est?o focando a combina??o de matrizes que possuam caracter?sticas osteocondutora com prote?nas osteoindutivas. Essas prote?nas podem estimular a diferencia??o de c?lulas mesenquimais e osteoprogenitoras em osteoblastos e assim aumentar a migra??o de c?lulas relacionadas ? forma??o ?ssea dentro do s?tio do defeito. Os principais materiais utilizados para esses objetivos s?o os pol?meros biodegrad?veis, como o PLA (poli ?cido l?tico) e o PLGA (poli ?cido glic?lico l?tico). Diversas drogas s?o utilizadas para serem liberadas nesses sistemas, como antibi?ticos, anticoncepcionais e prote?nas, incluindo o horm?nio do crescimento humano recombinante (rhGH). O rhGH no tecido ?sseo, promove a deposi??o aumentada de prote?nas pelos condr?citos e osteoblastos, aumento do n?mero de mitoses e a convers?o de condr?citos em osteoblastos. Desta forma se faz necess?rio a avalia??o desse biomaterial (PLGA\rhGH) como dispositivo de libera??o controlada. Conseguindo com isso a presen?a do rhGH intimamente no local da cicatriza??o ?ssea. Para este estudo foi utilizado como modelo animal,ratos wistar. O estudo foi todo realizado de acordo com a Lei N0 11.794, de 8 de outubro de 2008 bem como seguindo a diretriz Brasileira de Pr?tica para o Cuidado e a Utiliza??o de Animais para Fins Cient?ficos e Did?ticos ? DBPA do CONCEA. Projeto aprovado pelo CEUA da PUCRS sob n?mero 15/00461. Foram utilizados trinta ratos wistar adultos, nos quais foram submetidos a defeitos ?sseos nos f?mures com broca, os defeitos tinham todos mais ou menos 05mm de di?metro. Os ratos foram submetidos ao mesmo tratamento, por?m divididos em tr?s grupos de acordo com o tempo das eutan?sia :Grupo 01 eutan?sia em 07dias , Grupo 02 eutan?sia em 15 dias e Grupo 03 eutan?sia em 20 dias. Os f?mures dos ratos foram radiografados no intervalo entre 07, 15, e 20 dias para aferir a densidade ?ptica. Em seguida foram produzidas l?minas para an?lises histol?gicas, com cortes de aproximadamente 5?m de espessura e corados com hematoxilina/eosina (HE). Foram escolhidas tr?s cortes mais representativos de cada l?mina. A repercuss?o sist?mica do rhGH foi avaliada atrav?s dos n?veis de IGFI atrav?s da coleta de sangue que foi realizada antes de cada eutan?sia, no grupo-controle e no grupo experimental. O sangue coletado foi centrifugado (2500 rota??es por minuto por 10 min) e o soro obtido armazenado em freezer a -20?C para posterior determina??o dos n?veis plasm?ticos de IGFI usando o m?todo que se baseia em um ensaio enzimaticamente amplificado do tipo ?sandu?che?, realizado no laborat?rio Senhor dos Passos em Porto Alegre,RS. Para an?lise estat?stica foi aplicado o Teste Tukey, n?vel de 5% de signific?ncia e an?lise de vari?ncia ANOVA. N?o houve diferen?a estatisticamente significante em rela??o as densidades minerais ?sseas, por?m numericamente os enxertos com PLGA\rhGH tiveram sempre uma quantidade maior de tons de cinza. Conclui-se que os defeitos com enxerto de PLGA\rhGH apresentaram uma maior densidade ?ptica e uma maior densidade mineral em compara??o ao enxerto aut?geno. Nas an?lises histol?gicas tamb?m n?o houve diferen?a estatisticamente significante entre os enxertos aut?geno e de PLGA\rhGH. Por?m pelas analises num?ricas observamos um desempenho melhor e/ou igual entre o enxerto de PLGA\rhGH e enxerto aut?geno. Os defeitos com enxerto de PLGA\rhGH, de ocordo com a metodologia empregada, densidade mineral ?ptica e pelas l?minas histol?gicas, observamos uma cicatriza??o e matura??o mais r?pida do que os enxerto aut?genos.

Submitted by PPG Engenharia e Tecnologia de Materiais (engenharia.pg.materiais@pucrs.br) on 2018-06-25T13:24:34Z No. of bitstreams: 1 Dissserta??o final_Lucas Weber Dias.pdf: 2263503 bytes, checksum: ea9c29b6b538c56ea795610f7bc5a808 (MD5) / Approved for entry into archive by Sheila Dias (sheila.dias@pucrs.br) on 2018-06-29T13:30:33Z (GMT) No. of bitstreams: 1 Dissserta??o final_Lucas Weber Dias.pdf: 2263503 bytes, checksum: ea9c29b6b538c56ea795610f7bc5a808 (MD5) / Made available in DSpace on 2018-06-29T13:38:08Z (GMT). No. of bitstreams: 1 Dissserta??o final_Lucas Weber Dias.pdf: 2263503 bytes, checksum: ea9c29b6b538c56ea795610f7bc5a808 (MD5) Previous issue date: 2017-08-29 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPES / The main function of the packaging is to protect the cosmetic from the external environment, avoiding undesirable changes in the product that is contained inside, and thus indispensable for its storage and transpotation. However, its disposal improperly generates a large volume of solid waste, which is directly related to the environmental impact. One of the alternatives to minimize this impact is the use of packages made from biodegradable polymeric films based on polyacrylic acid (PLA), poly (lactic acid ? co- glycolic acid) (PLGA) and polycaprolactone (PCL), blends of theses polymers and incorporation of Zeolites 13x. The thermal, mechanical optical, water vapor barrier and degradation time in saline solution of polymeric systems are evaluated for application as cosmetic packaging. The technique used for the preparation of the polymer films was that of Solvent Casting. The polymer films were characterized by the techniques of UV-Vis Spectrophotometry, Differential Scanning Calorimetry (DSC), Thermogravimetric Analysis (TGA), Scsannig Electron Microscopy (SEM), transparency, water vapor permeability, mechanical assay and absorption spectroscopy atomic. The transmittance results showed that the PCL and PLA blends showed greater blockage to the passage of radiation (190 - 1100nm) and water vapor permeability. The incorporation of PLA or PLGA to the pure PCL resulted in material with batter thermal properties, but more rigid and brittle, not modifying the degradation process until 120 days of incubation. The incorporation of 15% Zeolite 13x into the PCL/PLA blends did not significantly modify the properties evaluated. / As embalagens t?m como principal fun??o, proteger o cosm?tico do meio externo, evitando que ocorram altera??es indesej?veis no produto que est? contido no seu interior, sendo assim indispens?vel para o seu armazenamento e transporte. Por?m, o seu descarte de forma inadequada gera um grande volume de res?duos s?lidos, que est? diretamente relacionado com o impacto ambiental. Uma das alternativas para minimizar este impacto ? a utiliza??o de embalagens confeccionadas a partir de pol?meros biodegrad?veis. Desta maneira, o presente trabalho tem como objetivo a prepara??o e caracteriza??o de filmes polim?ricos biodegrad?veis baseados em Poli ? ?cido l?ctico (PLA), Poli (?cido l?ctico ? co- ?cido glic?lico) (PLGA) e Policaprolactona (PCL), blendas destes pol?meros e incorpora??o de Ze?litas 13x. S?o avaliadas as propriedades t?rmicas, mec?nicas, ?pticas, de barreira a vapor de ?gua e o tempo de degrada??o em solu??o salina dos sistemas polim?ricos, visando aplica??o como embalagem para cosm?ticos. A t?cnica utilizada para a prepara??o dos filmes polim?ricos foi a de Solvent Casting. Os filmes polim?ricos foram caracterizados pelas t?cnicas de Espectrofotometria UV-Vis, Calorimetria Explorat?ria Diferencial (DSC), An?lise Termogravim?trica (TGA), Microscopia Eletr?nica de Varredura (MEV-FEG), transpar?ncia, permeabilidade a vapor de ?gua, ensaio mec?nico e Espectroscopia de absor??o at?mica. Os resultados de transmit?ncia mostraram que a blenda de PCL e PLA apresentou maior bloqueio ? passagem de radia??o (190 nm ? 1100 nm) e ? permeabilidade de vapor de ?gua. A incorpora??o de PLA ou PLGA ao PCL puro resultou num material com melhores propriedades t?rmicas, por?m mais r?gido e quebradi?o, n?o modificando o processo de degrada??o at? 120 dias de incuba??o. A incorpora??o de 15% Ze?lita 13x ? blenda de PCL/PLA n?o modificou significativamente as propriedades avaliadas.