Amyotrophic lateral sclerosis (ALS) is a devastating disease. Currently, there is no cure for this disease, and effective treatment strategies are greatly needed. Calpain activation plays a major role in the motor neuron degeneration that causes ALS. Therefore, therapeutic strategies can inhibit calpain activity in the central nervous system (CNS) have great clinical potential. The calpain inhibitors AK295 and MDL-28170 have been demonstrated to be neuroprotective in animal models of neurological injury, and should have great potential to treat ALS; however delivery problems have hindered their clinical success. Therefore, development of a new strategy that can locally deliver the calpain inhibitors to the central nervous system could significantly improve the treatment of ALS. The objectives of my thesis research were (1) to develop high molecular weight polyketals that provide sustained release properties for hydrophobic molecules, (2) to formulate calpain inhibitor-encapsulated polyketal microparticles which have a release half life of one month in vitro, (3) and to evaluate the performance of polyketal microparticles for delivering calpain inhibitors to the spinal cord in vivo. In completing these specific aims, we have developed biodegradable polymeric microparticles for the delivery of calpain inhibitors, AK295 and MDL-28170 to treat ALS. The results of calpain assays showed that both AK-PKMs and MDL-PKMs maintained most of their inhibitory activities even after the robust emulsion process. The in vitro release profile of MDL-28170 in MDL-PKMs showed that 50 % of the drug was released in the first 30 days. Experiments using dye-encapsulated microparticles showed that polyketal microparticles (1-2 ìm) are not easily cleared in the neutral physiological environment and can have potential to continuously release drug from the injection sites in the spinal cord. The efficacy of calpain inhibitor-encapsulated PKMs were studied by evaluation the behavior and survival of SOD1G93A rats, a genetic rat model for ALS. We observed the trend toward improvements in grip strength and rotarod performance in the first two months from the AK-PKMs treated group, however, further improvements are needed to enhance their in vivo efficacy.

Existing methods of treating glioma are not effective for eradicating the disease. Therefore, new and innovative methods of treatment alone or in combination with existing therapies are necessary. Delivery of therapeutic agents through delivery carriers such as liposomes diminishes the harmful effects of the agent in healthy tissues and allows increased accumulation in the tumor. In addition, targeted chemotherapy using liposomes provides the opportunity for further increase in drug accumulation in tumor. However, the current targeting strategies suffer accelerated plasma clearance and are not advantageous in improving efficacy. The search for new tumor targets, novel ligands, new strategies for targeting, and particle stabilization will advance our ability to improve delivery at the tumor level while decreasing toxicity to normal tissues. The global objective of this thesis was to improve the status of current liposomal therapy to achieve higher efficacy in tumors. Here, we show a novel mechanism to increase targeting to tumor while uncompromising on the long circulation of stealth liposomes. Long circulation is essential for passive accumulation of the nanocarriers due to EPR effect, in order to see benefits of targeting. Using phage display technique, a variety of tumor specific peptides were identified for use as targeting moieties. One potential advantage of the approach proposed here is the rapid identification of patient tumor specific peptide that evades the RES. This could lead to the development of a nanocarrier system with high avidity and selectivity for tumors. Therefore, tumor accumulation of the targeted formulations will be higher than that of non‐targeted liposomes due to increased drug retention at the tumor site and uncompromised blood residence time.In addition, it has been shown that the distribution of nanocarriers, spatially within the tumor, is limited that might further hinder the distribution of the encapsulated drug, thereby limiting efficacy. It is necessary to release the drug from within the nanocarrier to promote increased efficacy. Here, we were able to address the problem of drug diffusion within the tumor interstitium using a combination therapy employing a remotely triggered thermosensitive liposomal chemotherapeutic. We fabricated a thermosensitive liposomal nanocarrier that maintained its stability at physiological temperature to minimize toxicity to healthy cells. We, then, showed a remote triggering mechanism mediated by gold nanorods heated via NIR can help in achieving precise control over the desired site for drug release. These strategies enabled increased drug availability at the tumor site and contributed to tumor retardation. Additionally, we show that the synergistic therapy employing gold nanorods and thermosensitive liposomes may have great potential to be translated to the clinic.

D. Tech. Pharmaceutical Sciences.

The prevention and treatment of cancer and infectious diseases requires vaccines that can mediate cytotoxic T lymphocyte-based immunity. A promising strategy is protein subunit vaccines composed of purified protein antigens and immunostimulatory adjuvants, such as Toll-like receptor (TLR) agonists. In this research, we developed two new biodegradable polymeric delivery vehicles for protein antigens and TLR agonists, as model vaccine delivery systems. This work was guided by the central hypothesis that an effective vaccine delivery system would have stimulus-responsive degradation and release, biodegradability into excretable non-acidic degradation products, and the ability to incorporate various TLR-inducing adjuvants. The first vaccine delivery system is a cross-linked polyion complex micelle which efficiently encapsulates proteins, DNA, and RNA. The micelle-based delivery system consists of a block copolymer of poly(ethylene glycol) (PEG) and poly(L-lysine), cross-linked by dithiopyridyl side groups to provide transport stability and intracellular release. The second delivery system consists of solid biodegradable microparticles encapsulating proteins, nucleic acids, and hydrophobic compounds. The microparticles are composed of pH-sensitive polyketals, which are a new family of hydrophobic, linear polymers containing backbone ketal linkages. Polyketals are synthesized via a new polymerization method based on the acetal exchange reaction and degrade into non-acidic, excretable degradation products. In addition, the technique of hydrophobic ion pairing was utilized to enhance the encapsulation of ovalbumin, DNA, and RNA in polyketal microparticles via a single emulsion method. Using in vitro and in vivo immunological models, we demonstrated that the micelle- and polyketal-based vaccine delivery systems enhanced the cross-priming of cytotoxic T lymphocytes. The model vaccines were composed of ovalbumin antigen and various TLR-inducing adjuvants including CpG-DNA, monophosphoryl lipid A, and dsRNA. The results demonstrate that the cross-linked micelles and polyketal microparticles have considerable potential as delivery systems for protein-based vaccines.

Made available in DSpace on 2014-12-17T14:07:18Z (GMT). No. of bitstreams: 1 AnaCMC_DISSERT.pdf: 3568281 bytes, checksum: 78b0d72ff4bc1b9774bb0c806de32258 (MD5) Previous issue date: 2013-11-11 / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico / With the advances in medicine, life expectancy of the world population has grown considerably in recent decades. Studies have been performed in order to maintain the quality of life through the development of new drugs and new surgical procedures. Biomaterials is an example of the researches to improve quality of life, and its use goes from the reconstruction of tissues and organs affected by diseases or other types of failure, to use in drug delivery system able to prolong the drug in the body and increase its bioavailability. Biopolymers are a class of biomaterials widely targeted by researchers since they have ideal properties for biomedical applications, such as high biocompatibility and biodegradability. Poly (lactic acid) (PLA) is a biopolymer used as a biomaterial and its monomer, lactic acid, is eliminated by the Krebs Cycle (citric acid cycle). It is possible to synthesize PLA through various synthesis routes, however, the direct polycondensation is cheaper due the use of few steps of polymerization. In this work we used experimental design (DOE) to produce PLAs with different molecular weight from the direct polycondensation of lactic acid, with characteristics suitable for use in drug delivery system (DDS). Through the experimental design it was noted that the time of esterification, in the direct polycondensation, is the most important stage to obtain a higher molecular weight. The Fourier Transform Infrared (FTIR) spectrograms obtained were equivalent to the PLAs available in the literature. Results of Differential Scanning Calorimetry (DSC) showed that all PLAs produced are semicrystalline with glass transition temperatures (Tgs) ranging between 36 - 48 ?C, and melting temperatures (Tm) ranging from 117 to 130 ?C. The PLAs molecular weight characterized from Size Exclusion Chromatography (SEC), varied from 1000 to 11,000 g/mol. PLAs obtained showed a fibrous morphology characterized by Scanning Electron Microscopy (SEM) / Com os avan?os na medicina, a expectativa de vida da popula??o mundial vem crescendo consideravelmente nas ?ltimas d?cadas. Pesquisas v?m sendo realizadas visando manter a qualidade de vida da popula??o atrav?s do desenvolvimento de novas drogas e novos procedimentos cir?rgicos. Os biomateriais s?o estudados atualmente, e sua utiliza??o vai desde a reconstru??o de tecidos e ?rg?os afetados por patologias ou outros tipos de falha, ? utiliza??o em sistema de libera??o de f?rmacos capazes de prolongar o tempo do medicamento no organismo e aumentar a sua biodisponibilidade. Os biopol?meros consistem em uma classe de biomateriais muito visada pelos pesquisadores, uma vez que apresentam propriedades ideais para tal aplica??o, como alta biocompatibilidade e biodegradabilidade. O Poli(?cido l?ctico) (PLA) ? um biopol?mero usado como biomaterial, e seu mon?mero, o ?cido l?ctico, ? eliminado pelo ciclo do ?cido c?trico (ciclo de Krebs). ? poss?vel sintetizar o PLA atrav?s de v?rias rotas de s?ntese, no entanto, a policondensa??o direta ? a rota mais barata devido ao uso de poucas etapas de polimeriza??o. Neste trabalho foi utilizada a ferramenta de planejamento fatorial para produzir PLAs com diferentes massas molares, a partir da policondensa??o direta do ?cido l?ctico, com caracter?sticas adequadas para a utiliza??o em sistema de libera??o de f?rmacos (SLFs). Por meio do planejamento observou-se que o tempo de esterifica??o ? o est?gio mais importante na obten??o de uma maior massa molar na rota de policondensa??o. Os Infravermelhos por Transformada de Fourier (FTIR) obtidos apresentaram espectrogramas equivalentes ao de PLAs dispon?veis na literatura. Resultados de Calorimetria Explorat?ria Diferencial (DSC) mostraram que todos os PLAs produzidos s?o semicristalinos, com temperaturas de transi??o v?treas (Tgs) variando entre 36 48?C e temperaturas de fus?o (Tm) variando entre 117 130?C. As massas molares, caracterizadas a partir de Cromatografia por Exclus?o de Tamanho (SEC), variaram entre 1000 11000 g/mol. Os PLAs obtidos apresentaram uma morfologia fibrosa caracterizada por meio de Microscopia Eletr?nica de Varredura (MEV)

L’objectif de cette thèse a été d’optimiser la réponse immunitaire anti-tumorale induite par les cellules dendritiques (DC) et les cellules iNKTs, en réponse à la prise en charge du KRN7000 (a-galactosyl-céramide) par la molécule CD1d située sur les DCs. Le premier axe de travail visait à synthétiser de nouveaux analogues du KRN7000, en fonctionnalisant la position C6 du sucre et en greffant un groupement phényl sur l’une des chaînes grasses. Les études in vitro ont montré que les modifications apportées par rapport au KRN7000 n’ont pas altéré la prise en charge des molécules obtenues par les DCs. Dans tous les cas, une sécrétion de cytokines a pu être observée. Des études complémentaires visant à décrire le profil cytokinique in vivo sont en cours. Le second axe a consisté en la mise au point d’une stratégie de vectorisation du KRN7000 afin de favoriser sa présentation aux DCs, en l’associant à des molécules d’intérêt comme un peptide spécifique d’une tumeur, une molécule de ciblage des DCs ou des ligands des TLRs. Dans les conditions utilisées, le phénomène d’anergie induit classiquement par l’administration répétée du KRN7000 n’a pas pu être levé. Cependant, nous avons montré d’une part que le KRN7000 vectorisé dans les liposomes est toujours pris en charge par les cellules dendritiques, et d’autre part qu’une réponse immunitaire se traduisant par la production de cytokines par les cellules iNKTs est induite. / The aim of this project was to optimize the anti-tumor immune response induced by dendritic and iNKTs cells, in response to KRN7000 (a-galactosyl-ceramide), which interacts with CD1d molecule situated on DCs. At first we synthesized new analogues of KRN7000, by functionalizing the C6 position of the carbohydrate moiety and by grafting a phenyl group on one of the fatty chain. In vitro studies indicated that chemical modifications of KRN7000 did not alter its interaction with CD1d. In all cases, cytokine secretion was observed. Further studies are in progress to describe the in vivo cytokine profile. In a second step, we developed liposomal constructs incorporating KRN7000 to optimize its presentation to DCs. Some constructions containing KRN7000 were able to associate a peptide, a targetting molecule of DCs or TLR ligands. Even if the anergy phenomenon induced by repeated administrations of KRN7000 could not be regulated by the use of liposomes, we have shown that encapsulated KRN7000 is still supported by DCs and that an immune response resulting in cytokines secretion by iNKT cells is induced.

Cette étude décrit le développement de systèmes nanostructurés muco-adhésives, qui comprennent les nanoparticules décorées avec du chitosane et les films contenant des nanoparticules, visant la libération buccale de la curcumine. Deux différents systèmes de nanoparticules ont été développés: les nanoparticules de polycaprolactone (PCL) et les nanoparticules xyloglucane-bloc-polycaprolactone (XGO-b-PCL). Les nanoparticules de PCL ont été préparées par la technique de nanoprécipitation, alors que les nanoparticules XGO-b-PCL ont été préparées par la méthode du co-solvant. Les deux systèmes colloïdaux ont montré une forme sphérique et distribution monodisperse de particules. La décoration des nanoparticules avec du chitosane a été confirmée en mesurant la taille des particules et le potentiel zêta. Les études de la diffusion dynamique de la lumière (DLS) montrent un rayon hydrodynamique autour de 100 nm pour les nanoparticules de PCL et 50 nm pour les nanoparticules de XGO-b-PCL. Les suspensions de nanoparticules chargées en curcumine présentent des valeurs d'efficacité d'encapsulation proche de 100 %, et un taux moyen de principe actif autour de 450 μg/mL et 180 μg/mL pour les nanoparticules de PCL et XGO-b-PCL, respectivement. Les systèmes de nanoparticules ont aussi été caractérisées par la technique d'analyse du suivi des nanoparticules (NTA) et microscopie électronique à transmission (MET). Les propriétés mucoadhésives des nanoparticules ont été évaluées en mesurant les interactions de ces systèmes avec la glycoprotéine mucine (mucine sous-maxillaire bovine, BSM) par diffusion dynamique de la lumière (DLS), microbalance à cristal de quartz avec la mesure de dissipation d'énergie (QCM-D) et résonance plasmonique de surface (SPR). Toutes les nanoparticules décorées avec du chitosane ont démontré une excellente capacité d'interaction avec la mucine via de forces électrostatiques formées entre les groupes amines du polysaccharide et les groupes chargés négativement de la glycoprotéine, indiquant leur potentiel comme vecteurs mucoadhésives de médicaments. Les nanoparticules PCL ont aussi été caractérisées en termes de perméabilité et de rétention de curcumine à travers la muqueuse oesophagienne de porc. La rétention de concentrations de curcumine dans la muqueuse indique la possibilité d'obtention des effets locaux sur la surface muqueuse. Les études in vitro ont montré que les nanoparticules de PCL et XGO-b-PCL contenant la curcumine ont présenté une activité cytotoxique plus petite par rapport à la curcumine libre. Les nanoparticules de PCL contenant de la curcumine ont aussi démontré une activité antimicrobienne contre Candida albicans, ce qui suggère leur potentiel utilisation dans le traitement des infections fungiques. Les films mucoadhésifs contenant des nanoparticules ont été préparés par la technique de « casting », après l'incorporation des nanoparticules de PCL dans les solutions de chitosane plastifié. Les films obtenus en utilisant du chitosane de moyenne et haute masse molaire sont homogènes et flexibles. Les nanoparticules de PCL chargées de curcumine étaient uniformément distribuées sur les surfaces des films, comme le montrent les images de microscopie à force atomique (AFM) et de microscopie électronique à balayage de haute résolution (MEB-FEG). L'analyse par MEB-FEG des sections transversales des films a démontré la présence de nanoparticules à l'intérieur des films. En plus, les films ont montré un taux important d'hydratation en milieu salivaire et une libération contrôlée-prolongée de la curcumine. Par conséquent, les résultats obtenus indiquent que les systèmes mucoadhésives développés, nanoparticules décorées avec du chitosane et films contenant des nanoparticules, offrent une nouvelle stratégie pour la libération buccale de la curcumine, et sont potentiellement intéressant dans des applications de traitement locaux des maladies de la cavité orale. / This study describes the development of mucoadhesive nanostructured systems, including chitosan-coated nanoparticles and films containing nanoparticles, aiming the buccal delivery of curcumin. Two different systems of nanoparticles were developed: polycaprolactone (PCL) nanoparticles and xyloglucan-block-polycaprolactone (XGO-b-PCL) nanoparticles. PCL nanoparticles were prepared by the nanoprecipitation technique, while XGO-b-PCL nanoparticles were prepared by the co-solvent method. Both colloidal systems displayed spherical shape and monodisperse distribution of particles. The decoration of nanoparticles with chitosan was confirmed by particle size and zeta potential measurements. Dynamic light scattering (DLS) studies shown hydrodynamic radius around of 100 nm for PCL nanoparticles, and 50 nm for XGO-b-PCL nanoparticles. Curcumin-loaded nanoparticle suspensions exhibited encapsulation efficiency values close to 100 %, and a mean drug content about of 450 μg/mL and 180 μg/mL for PCL and XGO-b-PCL nanoparticles, respectively. Nanoparticle systems were also characterized by nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM). The mucoadhesive properties of nanoparticles were evaluated by monitoring the interactions of these systems with glycoprotein mucin (bovine submaxillary mucin, BSM) by dynamic light scattering (DLS), quartz crystal microbalance with dissipation monitoring (QCM-D) and surface plasmon resonance (SPR). All chitosan-coated nanoparticles demonstrate excellent ability to interact with mucin though electrostatic forces formed between amino groups of polysaccharide and negatively charged groups of glycoprotein, indicating their potential as mucoadhesive drug carriers. PCL nanoparticles were also characterized in terms of curcumin permeability and retention across the porcine esophageal mucosa. Amount of curcumin retained in the mucosa highlights the possibility of application for obtaining local effects on the mucosal surface. In vitro studies demonstrated that curcumin into copolymer nanoparticles showed reduced cytotoxicity when compared to free drug. Curcumin-loaded PCL nanoparticles also exhibited antimicrobial activity against Candida albicans strains, suggesting their potential use in the treatment of fungal infections. Mucoadhesive films containing nanoparticles were prepared using the casting technique, after the incorporation of PCL nanoparticles in plasticized chitosan solutions. Films obtained using medium and high molar mass chitosan showed to be homogeneous and flexible. Curcumin-loaded PCL nanoparticles were uniformly distributed on the films surface, as evidenced by atomic force microscopy (AFM) and high-resolution scanning electron microscopy (FEG-SEM) images. Analyses of films cross sections by FEG-SEM demonstrate the presence of nanoparticles inside the films. In addition, films showed good rate of hydration in saliva medium, and in vitro prolonged-controlled delivery of curcumin. Therefore, the results obtained indicate that the developed mucoadhesive systems, chitosan-coated nanoparticles and films containing nanoparticles, offer a novel strategy to buccal curcumin delivery and have potential applications in the local treatment of oral cavity disease.

Made available in DSpace on 2014-12-17T14:25:23Z (GMT). No. of bitstreams: 1 AbdoFN_TESE.pdf: 1739510 bytes, checksum: 7f520b9c4a32fd74e279d017c0bdd143 (MD5) Previous issue date: 2014-03-31 / Foam was developed as a novel vehicle for streptokinase with the purpose of increasing the contact time and area between the fibrinolytic and the target thrombus, which would lead to a greater therapeutic efficacy at lower doses, decreasing the drug s potential to cause bleeding. Fibrinolytic foams were prepared using CO2 and human albumin (at different v:v ratios), as the gas and liquid phases, respectively, and streptokinase at a low total dose (100,000 IU) was used as fibrinolytic agent conveyed in 1 mL of foam and in isotonic saline solution. The foams were characterized as foam stability and apparent viscosity. The thrombolytic effect of the streptokinase foam was determined in vitro as thrombus lysis and the results were compared to those of a fibrinolytic solution (prepared using the same dose of streptokinase) and foam without the fibrinolytic. In vitro tests were conducted using fresh clots were weighed and placed in test tubes kept at 37 ? C. All the samples were injected intrathrombus using a multiperforated catheter. The results showed that both foam stability and apparent viscosity increased with the increase in the CO2:albumin solution ratio and therefore, the ratio of 3:1 was used for the incorporation of streptokinase. The results of thrombus lysis showed that the streptokinase foam presented the highest thrombolytic activity (44.78 ? 9.97%) when compared to those of the streptokinase solution (32.07 ? 3.41%) and the foam without the drug (19.2 ? 7.19%). We conclude that fibrinolytic foam showed statistically significant results regarding the enhancement of the lytic activity of streptokinase compared to the effect of the prepared saline solution, thus it can be a promising alternative in the treatment of thrombosis. However, in vivo studies are needed in order to corroborate the results obtained in vitro / Uma espuma foi desenvolvida como novo ve?culo para a estreptoquinase com vistas a aumentar a ?rea de contato e o tempo de perman?ncia junto ao trombo, de modo a se obter maior efici?ncia terap?utica em doses menores, diminuindo suas potenciais complica??es hemorr?gicas. A espuma fibrinol?tica foi preparada com CO2, albumina humana e estreptoquinase, em dispositivo desenvolvido para tal fim, com diferentes raz?es de fases g?s/l?quido. Ensaios de estabilidade e viscosidade aparente foram realizados para caracteriza??o das espumas e a escolha da mais est?vel. A estreptoquinase em dose total reduzida (100.000 UI) foi utilizada como fibrinol?tico veiculado em 1 mL de espuma e em solu??o salina isot?nica (0,9%). A espuma sem fibrinol?tico tamb?m foi utilizada como comparativo. Testes in vitro foram realizados utilizando-se co?gulos frescos, que foram pesados e colocados em tubos de ensaio mantidos a 37?C. As espumas com e sem fibrinol?tico e a solu??o fibrinol?tica foram testadas por aplica??o intraco?gulo em doses id?nticas atrav?s de cateter multiperfurado e pistola de inje??o. Os resultados in vitro evidenciaram atrav?s da diminui??o dos pesos dos co?gulos, que a espuma trombol?tica apresentou atividade l?tica de 44,78 ? 9,97%, enquanto as mesmas doses da estreptoquinase em solu??o salina isot?nica promoveram 32,07 ?3,41% de lise dos co?gulos. Na espuma sem fibrinol?tico a redu??o do trombo foi de 19,2 ? 7,19%. Conclui-se que a espuma fibrinol?tica apresentou resultados estatisticamente significativos no tocante ? potencializa??o da atividade l?tica da estreptoquinase, quando comparado ao efeito da solu??o preparada com solu??o salina, podendo ser uma alternativa promissora nos tratamentos das tromboses. Os dados obtidos sinalizam para necessidade de estudos in vivo para comprova??o dos obtidos nos in vitro

Orientador: Maria Palmira Daflon Gremião / Banca: Marcelo Bispo de Jesus / Banca: Heloisa Sobreiro Selistre de Araujo / Banca: Beatriz Stringhetti Ferreira Cury / Banca: Maria Palmira Daflon Gremião / Resumo: Os glioblastomas multiformes (GBMs) são os tumores mais invasivos do sistema nervoso central (SNC) e sua invasividade está relacionada com a interação entre glicoproteínas da matriz extracelular com integrinas presentes nas células tumorais. As integrinas α5β1, αvβ5, e αvβ3 são fortemente expressas em GBMs, e podem ser utilizadas como moléculas alvo na terapia e no diagnóstico por imagem dos tumores do SNC. Os inibidores de integrinas cRGDfV, cRGDfK e vicrostatin têm sido estudadas no tratamento de GBMs pois inibem as integrinas αvβ3, αvβ5 e α5β1, e, assim, exibem potentes propriedades antitumorais. Entretanto, a aplicação de proteínas terapêuticas é limitada freqüentemente pela baixa estabilidade em fluídos fisiológicos e a baixa penetração através das membranas biológicas. Para contornar estes problemas, tem˗se buscado novos sistemas de liberação de fármacos e rotas alternativas de administração, como por exemplo, a via nasal. Assim, este trabalho teve como objetivo a encapsulação dos peptídeos cRGDfV e cRGDfK em nanopartículas de quitosana, pelo método de gelificação ionotrópica, empregando o planejamento fatorial 23. Este sistema baseado em quitosana promoveu um aumento da mucoadesão e podem auxiliar o acesso dos peptideos cRGDfV e cRGDfK ao SNC pela via nasal. Outra estratégia utilizada nesse trabalho foi a utilização das nanopartículas de quitosana escolhidas no planejamento fatorial para serem modificadas com PEG por meio de interações supramoleculares e também por int... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Glioblastoma multiforme (GBMs) is the most invasive tumor from central nervous system (CNS) and its invasiveness is related to interaction between glycoproteins of extracellular matrix and integrins present in tumor cells. Integrins α5β1, αvβ5, e αvβ3 are overexpressed in GBMs and they could be used as target molecules in the treatment and diagnostic imaging of the tumors from CNS. The disintegrins cRGDfV, cRGDfK and vicrostatin have been studied for the treatment of GBMs because they inhibit the integrins αvβ3, αvβ5, and α5β1, and, therefore, they show potent anti˗tumor properties. However, the administration of therapeutic proteins is limited by their low stability in physiological fluids and low penetration in biological membranes. To overcome these problems, drug delivery systems have been developed and alternative route of administration, such as nasal administration, has been used. Thus, the aim of this study was the development of chitosan nanoparticles, employing factorial design, by the means of ionotropic gelification and the encapsulation of disintegrins cRGDfV e cRGDfK. The chitosan˗based drug delivery system increased the mucoadhesion, which could promote the blood˗brain barrier permeation of disintegrins cRGDfV and cRGDfK. Another strategy aimed in this work was the use of chitosan nanoparticles selected by factorial design to be modified with PEG by supramolecular interactions and also by covalent interactions with methoxy˗polyethylene glycol and maleimide˗polyethylene glycol˗hydroxisuccinimide. The modified nanoparticles by covalent interactions were functionalized through linkage of disintegrin cRGDfK on nanoparticles' surface in order to improve the targeting of nanoparticles to tumor environment by specific interactions with the integrins overexpressed in... (Complete abstract click electronic access below) / Doutor

INTRODUÇÃO: Sistemas nanométricos carreadores de fármacos, ao alcançarem a circulação sanguínea, se concentram em seus sítios de ação (\"drug-targeting\"), evitando tecidos saudáveis. O diâmetro médio e a polidispersidade de nanopartículas são parâmetros relevantes, pois podem influenciar no percurso pelo fluxo sanguíneo da partícula e na interação celular. A LDE, uma nanopartícula lipídica que mimetiza a lipoproteína de baixa densidade (LDL), é capaz de carrear fármacos como o quimioterápico paclitaxel para tecidos com alta taxa de proliferação celular, como por exemplo, lesões ateroscleróticas e tecidos neoplásicos. Assim, é relevante investigar a influência de diferentes tamanhos da LDE na captação celular e na eficácia terapêutica em aterosclerose experimental. OBJETIVO: Avaliar a influência de duas faixas de tamanhos da nanopartícula lipídica carreadora do quimioterápico paclitaxel (LDE-paclitaxel), na captação celular e na resposta terapêutica do tratamento da aterosclerose em coelhos submetidos à dieta rica em colesterol. MÉTODOS: A associação LDE-paclitaxel foi preparada por emulsificação por alta pressão. A separação da LDE-paclitaxel original em LDE-paclitaxel grande e LDE-paclitaxel pequena foi feita por ultracentrifugação por gradientes de densidade. Nos estudos com células endoteliais HUVEC foram avaliados citotoxicidade, internalização celular e detecção de apoptose/necrose. Para estudo em animal, foram utilizados coelhos New Zealand machos, com aterosclerose induzida por dieta, divididos em dois grupos: LDE-paclitaxel-grande (n=9) e LDE-paclitaxel-pequena (n=10). O tratamento com LDE-paclitaxel foi iniciado após 4 semanas da dieta. Aortas dos coelhos foram coletadas para análise macro e microscópica das lesões ateroscleróticas. RESULTADOS: A LDE-paclitaxel original foi caracterizada com diâmetro médio de 75nm; após a ultracentrifugação, a LDE-paclitaxel grande apresentou diâmetro médio de 83nm e a LDE-paclitaxel pequena de 40nm. Os ensaios de citotoxicidade mostraram que, após incubação por 24 horas, a LDE-paclitaxel pequena alcançou o IC50 com menor concentração que a LDE-paclitaxel grande. No ensaio de internalização, a LDE-paclitaxel pequena foi internalizada em menores concentrações e em menor tempo em comparação com as partículas da LDE-paclitaxel original ou LDE-paclitaxel grande. Nos ensaios para detecção de apoptose/necrose, LDE-paclitaxel, independentemente do tamanho, aumentou a porcentagem de células necróticas. A LDE-paclitaxel pequena também aumentou a percentagem de células apoptóticas, em comparação às outras partículas. No estudo in vivo, não houve diferença entre os tratamentos LDE-paclitaxel grande e LDEpaclitaxel pequena: a razão área de lesão/área total foi igual entre os grupos, assim como a quantificação de macrófagos e de células de músculo liso na íntima das aortas. CONCLUSÃO: O tamanho da LDE, apesar de ser um relevante parâmetro físico-químico, não influenciou no efeito antiaterosclerótico da associação LDE-paclitaxel. Portanto, em relação ao tamanho das partículas, a LDE-paclitaxel original, que possui ambas as populações, é eficienteno tratamento da aterosclerose experimental induzida por dieta rica em colesterol / INTRODUCTION: As nanometric drug carriers enter the blood circulation, they concentrate on their action sites, avoiding healthy tissue. The average diameter and polydispersity of nanoparticles are relevant parameters because they have influence on the particles course through the blood flow and on cell interaction. LDE, a lipid nanoparticle that mimics low-density lipoprotein (LDL), is capable of carrying chemotherapeutic drugs such as paclitaxel, for tissues with a high rate of cell proliferation, such as atherosclerotic lesions. Thus, it is important to investigate the influence of different sizes in the cellular uptake of LDE and therapeutic efficacy in experimental atherosclerosis. OBJECTIVE: To evaluate the influence of two size ranges of the lipid nanoparticle carrier of the chemotherapeutic drug paclitaxel (LDE-paclitaxel) in cellular uptake and therapeutic response in experimental atherosclerosis. METHODS: LDEpaclitaxel was prepared by emulsification by high energy. The separation of original-LDE-paclitaxel in large-LDE-paclitaxel particles and small-LDEpaclitaxel particles was performed by ultracentrifugation by density gradients. In studies with HUVEC endothelial cells, cytotoxicity, cell internalization and detection of apoptosis/necrosis were assessed. In in vivo study, New Zealand male rabbits, with atherosclerosis induced by cholesterol-rich diet, were divided into two groups: large-LDE-paclitaxel (n=9) and small-LDE-paclitaxel (n=10). Treatment with LDE-paclitaxel started after 4 weeks of diet. Aortas of the rabbits were collected for macroscopic and microscopic analysis of atherosclerotic lesions. RESULTS: The original-LDE-paclitaxel was characterized with an average diameter of 75nm. After ultracentrifugation, the large-LDE-paclitaxel showed average diameter of 83nm and small-LDE-paclitaxel 40nm. The cytotoxicity assay showed that, after incubation for 24 hours, small-LDE paclitaxel reached the IC50 in lower concentration than large-LDE paclitaxel. In internalization assays, small-LDE-paclitaxel was internalized in lower concentrations and shorter time as compared with the original and large particles. LDE-paclitaxel, independently of particle size, increased the percentage of necrotic cells. Small-LDE-paclitaxel was also able to increase the percentage of apoptotic cellsas compared with the original and large particles. In experimental study, there was no difference between large-LDE-paclitaxel and small-LDE-paclitaxel treatment: lesion area / total area ratio was similar between groups as well as the quantification of macrophages and smooth muscle cells of the intima of aortas.CONCLUSION: The size of the LDE, although an important physicochemical parameter, did not influence the antiatherosclerotic effect of LDE-paclitaxel. Therefore, with respect to particle size, the original-LDE-paclitaxel that has both populations is efficient to treat experimental atherosclerosis induced by a cholesterol-rich diet