Le travail de recherche mené au cours de cette thèse a été effectué dans le cadre d’une collaboration industrielle et a porté sur l’étude des caractéristiques physico-chimiques d’une matrice lipidique à libération prolongée. L’originalité de cette dernière tient à sa forme galénique, des microsphères calibrées d’environ 400 µm de diamètre, fabriquées par procédé de prilling puis conditionnées en sachets.Les caractérisations entreprises au cours de cette thèse ont été menées progressivement en partant de l’étude du comportement thermique individuel des constituants (excipients lipidiques et principe actif), puis de leurs mélanges pour définir les conditions d’existence et les caractéristiques structurales des phases solides qu’ils engendrent, notamment lors du prilling. Plusieurs diagrammes d’état partiels ont été construits pour permettre de cerner l’organisation de la matrice formant le produit fini et disposer de leviers fiables pour corriger d’éventuelles déviations du procédé de fabrication. Cette analyse était également indispensable pour décrire précisément le processus de libération du principe actif et en déterminer les paramètres cinétiques. / The research conducted in this thesis, during an industrial collaboration, is focused on the study of physicochemical characteristics of a sustained release lipid matrix. The originality of this matrix is its dosage form, microspheres of 400 microns in diameter, manufactured by a prilling process and packaged in stick-packs.The characterizations conducted during this thesis have been carried out gradually starting from the study of the thermal behavior of individual components (lipidic excipients and drug) and their mixtures to define the phase formation conditions and the structural characteristics of the solid phases generated, especially during the prilling process. Partial phase diagrams were constructed to understand the final product matrix organization and to identify actions to correct any deviations in the process. This analysis was also needed to accurately describe the drug release mechanism and to determine the kinetic parameters.

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.

La thérapie cellulaire et les médicaments de thérapie innovante sont des solutions prometteuses pour la régénération des tissus ou organes présentant des défauts fonctionnels ou organiques. Avant le stade de l'insuffisance cardiaque terminale (stade IV NYHA) suite à un infarctus du myocarde, l'implantation d'un patch de fibrine cellularisé avec des progéniteurs myocardiques sur le site de nécrose de l'infarctus, est l'une des perspectives qui permettrait de régénérer un muscle cardiaque fonctionnel et apparait comme étant une alternative nouvelle avec notamment un essai clinique de phase I en cours (ESCORT : NCT02057900). Cependant, cette thérapie innovante présente de réelles contraintes, parmi lesquelles, un protocole nécessitant, i) une utilisation pour la production de cellules progénitrices myocardiques CD15+, de DMSO, de sérum foetal bovin, de trypsine porcine, de fibroblastes murins pouvant être la source d'une contamination chimique ou microbiologique, ii) une caractérisation importante des cellules produites, pour déterminer leur viabilité, leur pureté, leur état de différenciation, iii) d'implanter le patch de fibrine cellularisé dans un délai limité avant l'obtention des résultats de stérilité et d'endotoxines, iv) d'inciser le péricarde et de former une poche, geste chirurgical très invasif, afin d'implanter le patch cellularisé. Avec l'objectif de limiter ces contraintes et de renforcer la sécurisation pharmaceutique de ce médicament de thérapie innovante, les différents axes de ce travail ont porté sur i) l'ajout, juste avant l'implantation, d'une étape de cryoconservation des cellules dans un milieu sans sérum et sans DMSO, mais avec des agents cryoprotectants de qualité pharmaceutique. L'avantage apporté par la cryoconservation étant de rendre possible une production par lot, et la réalisation des contrôles sans contrainte de temps avant l'implantation, ii) la vectorisation des cellules par une encapsulation dans des microsphères formant une suspension injectable et permettant une implantation directement au travers du péricarde et immédiatement après la décongélation, iii) l'utilisation de polymères bioadhésifs afin de maintenir les microsphères au site d'implantation. Dans un premier temps, ce travail a permis d'identifier l'alginate de sodium de faible viscosité à 1,2% comme polymère pour réaliser l'encapsulation à l'aide d'une buse vibrante de 120 µm de diamètre. La nature et la concentration d'agents cryoprotectants ont également été définies. Les agents cryoprotectants ont été sélectionnés parmi les oses (glucose, saccharose, tréhalose), les polyols (glycérol, mannitol, sorbitol) et l'urée, à une concentration permettant d'atteindre une osmolarité totale de 500 mOsm/L pour abaisser le point de congélation de l'eau. Enfin le chitosane de faible viscosité à 0,5% a été utilisé comme polymère bioadhésif de surface pour maintenir les propriétés mécaniques et la forme des microsphères après la congélation. Dans un second temps, une évaluation biologique a permis de mesurer l'impact des étapes du procédé d'encapsulation et de cryoconservation, sur des cellules souches mésenchymateuses humaines utilisées comme modèle. Il a ainsi été possible d'optimiser le protocole ce qui a eu pour effet d'augmenter la viabilité, évaluée après encapsulation et congélation par une analyse en cytométrie de flux avec le 7AAD, de moins de 5% à environ 35%. / Cell therapy and advanced therapy medicinal products are promising solutions for the regeneration of tissues or organs with functional or organic defects. Before the terminal heart failure stage (stage IV NYHA) following a myocardial infarction, the implantation of a cellularized fibrin patch with myocardial progenitors at the location of the infarct necrosis, is one of the perspectives that would allow a functional heart muscle to regenerate and appears to be a new alternative, in particular, with an ongoing Phase I clinical trial (ESCORT : NCT02057900). However, this innovative therapy presents real constraints, among which, a protocol requiring, i) the use for the production of CD15+ myocardial progenitor cells of, DMSO, bovine fetal serum, porcine trypsin, and murine fibroblasts which may be the source of chemical or microbiological contamination, ii) an important characterization of the produced cells, to determine their viability, purity, and state of differentiation, iii) to implant the cellularized fibrin patch within a limited time frame before getting the results of sterility and endotoxins, iv) to incise the pericardium and to form a pouch, a very invasive surgical gesture, in order to implant the cellularized patch inside. With the objective of limiting these constraints and strengthening the pharmaceutical safety of this innovative therapy medication, different axes of this work have focused on i) the addition, just before the implantation, of a step of cryopreservation of the cells in a medium without serum and without DMSO, but with pharmaceutical-grade cryoprotectants. The advantages of cryopreservation is to allow production in batches, and controls to be carried out without time constraints before the implantation, ii) the vectorization of the cells by encapsulation in microspheres forming an injectable suspension and allowing direct implantation through the pericardium immediately after thawing, iii) the use of bioadhesive polymers to maintain the microspheres at the location of the implantation. This study initially enabled to identify a low-viscosity sodium alginate at 1.2% as a polymer being used for the encapsulation with the use of a vibrating nozzle which diameter is of 120 µm. The nature and the concentration of the cryoprotectants have also been defined. The cryoprotectants were selected from oses (glucose, sucrose, trehalose), polyols (glycerol, mannitol, sorbitol) and urea, at a concentration which achieves a total osmolarity of 500 mOsm/L in order to lower the freezing point of water. Finally, low viscosity chitosan at 0.5% was used as a bioadhesive polymer at the surface of the microspheres to maintain their shapes and mechanical properties after freezing. In a second step, a biological evaluation allowed to measure the impact of the encapsulation and the cryopreservation processes, on human mesenchymal stem cells used as a model. It was thus possible to optimize the protocol, which in return increased the viability ; evaluation made after encapsulation and freezing by a flow cytometry analysis with 7AAD ; from less than 5% to about 35%.

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.

Παρά τις προόδους των κλασικών θεραπευτικών στρατηγικών για τον καρκίνο, η μεγάλη πλειοψηφία των ασθενών υποτροπιάζει και καταλήγει. Η ανάγκη για την αντιμετώπιση της νόσου με εναλλακτικό τρόπο οδήγησε στην ανάπτυξη ανοσοθεραπευτικών μεθόδων. Η ιδέα της ανοσοθεραπείας του καρκίνου έγινε γνωστή στα τέλη του δέκατου ένατου αιώνα, όταν ο William Coley χρησιμοποίησε ζωντανά στελέχη του πυογενούς βακτηρίου Streptococcus erysipelas με σκοπό τη δημιουργία γενικευμένης ανοσολογικής απάντησης, μέρος της οποίας να κατευθυνθεί ενάντια σε όγκους σαρκώματος. Οι σποραδικές θετικές αποκρίσεις που παρατήρησε οφείλονταν κατά πάσα πιθανότητα σε ενίσχυση της ανοσολογικής απάντησης από τις φλεγμονώδεις αντιδράσεις που προκάλεσαν τα βακτήρια. Για να επαχθεί όμως ειδική ανοσολογική απάντηση ενάντια σε όγκους απαιτείται να χαρακτηριστούν στα καρκινικά κύτταρα συγκεκριμένα αντιγόνα, ώστε να δύναται το ανοσολογικό σύστημα να τα αναγνωρίσει ως στόχους. Συνεπώς, το πρώτο βήμα στην προσπάθεια για ανοσοθεραπεία του καρκίνου είναι η απομόνωση αντιγόνων που εκφράζουν τα καρκινικά κύτταρα, τα οποία κατά προτίμηση να μην εκφράζονται από τους φυσιολογικούς ιστούς ώστε να αποφευχθεί η αυτοάνοση απάντηση. Η ταυτοποίηση ογκοειδικών αντιγόνων, τα οποία αναγνωρίζονται από τα Τ λεμφοκύτταρα, έδωσε ιδιαίτερη ώθηση στην ανάπτυξη της κατευθυνόμενης από τα Τ κύτταρα ανοσολογικής απάντησης, στο επίπεδο τόσο της έρευνας της ανοσολογίας του καρκίνου, όσο και της κλινικής ανοσοθεραπευτικής εφαρμογής και έθεσε τις βάσεις για τη χρησιμοποίηση πεπτιδικών εμβολίων στην ανοσοθεραπεία του καρκίνου. Από την πληθώρα των γνωστών καρκινικών αντιγόνων, έχουν ταυτοποιηθεί κατά κύριο λόγο επίτοποι ικανοί να συνδεθούν με μόρια του μείζονος συμπλέγματος ιστοσυμβατότητας (MHC) τάξης Ι και συνεπώς να επάγουν την ενεργοποίηση των CD8+ T κυττάρων, δεδομένου ότι οι περισσότεροι όγκοι είναι θετικοί ως προς τα μόρια MHC τάξης Ι, αλλά αρνητικοί ως προς τα μόρια MHC τάξης ΙΙ. Επιπρόσθετα, τα CD8+ Τ κύτταρα μπορούν να καταστρέφουν τα καρκινικά κύτταρα απευθείας, μέσω της αναγνώρισης του συμπλόκου MHC τάξης Ι-πεπτιδίου που εκφράζεται στην επιφάνεια του όγκου. Τα τελευταία χρόνια, δεδομένης της αναγνώρισης του κεντρικού ρόλου των CD4+ Τ λεμφοκυττάρων στην έναρξη, οργάνωση και διατήρηση της ανοσολογικής απάντησης, έχουν αναγνωριστεί και αρκετοί επίτοποι που αναγνωρίζονται από μόρια MHC τάξης ΙΙ. Πρόσφατες κλινικές μελέτες και προκλινικά μοντέλα έδειξαν ότι ο εμβολιασμός με επιτόπους που δύνανται να συνδεθούν με μόρια MHC τάξης ΙΙ, οι οποίοι εμπεριέχουν αλληλουχίες σύνδεσης για τα μόρια MHC τάξης Ι, είναι αποτελεσματικοί στην ταυτόχρονη ανάπτυξη βοηθητικών και κυτταροτοξικών Τ λεμφοκυττάρων με μακρά διάρκεια ζωής in vivo. Από τα γνωστά καρκινικά αντιγόνα, η πρωτεΐνη HER-2/neu παρουσιάζει το πλεονέκτημα της υπερέκφρασης σε ποικίλους τύπους καρκίνου, ενώ οι ασθενείς των οποίων όγκοι την υπερεκφράζουν παρουσιάζουν προϋπάρχουσα ανοσία ενάντια σε πεπτίδια αυτής. Η αυξημένη έκφρασή της στα καρκινικά κύτταρα και το γεγονός ότι πρόκειται για διαμεμβρανική πρωτεΐνη την καθιστούν στόχο για ανοσοθεραπευτικές προσεγγίσεις που περιλαμβάνουν τόσο κυτταρική όσο και χυμική ανοσία. Κλινικές έρευνες με χρήση πεπτιδίων της HER-2/neu έχουν δείξει την πρόκληση ανοσολογικής απάντησης στην πλειονότητα των ασθενών. Παρόλα αυτά, οι μεταστατικοί τύποι καρκίνου που υπερεκφράζουν τη συγκεκριμένη πρωτεΐνη παραμένουν μη θεραπεύσιμοι. Συνεπώς, υπάρχει άμεση ανάγκη για νέες θεραπευτικές προσεγγίσεις και στο σημείο αυτό η διερεύνηση των πιο ανοσογονικών τμημάτων της αλληλουχίας της πρωτεΐνης HER-2/neu, καθώς και της αντίδρασης των ασθενών σε αυτά, αποτελούν στόχο για ειδικές νέες αντικαρκινικές θεραπείες. O εγκλεισμός του αντιγόνου σε μικροσφαίρες πολυ-γαλακτικού-γλυκολικού οξέος (PLGA) έχει δειχθεί ότι επάγει ισχυρή και παρατεταμένη ανοσοαπόκριση. Μέχρι σήμερα, δεν φαίνεται να έχει αναφερθεί μελέτη στην οποία να αναλύεται η επίδραση των χαρακτηριστικών του PLGA συμπολυμερούς και του σχήματος ανοσοποίησης στον τύπο της λαμβανόμενης ανοσοαπόκρισης μετά την χορήγηση PLGA μικροσφαιρών του αντιγόνου in vivo. Στην παρούσα μελέτη διερευνήθηκε ο τύπος της ανοσοαπόκρισης που λαμβάνεται in vivo μετά την χορήγηση πεπτιδίων της HER-2/neu (πρότυπα αντιγόνα) συνδεμένων σε πολυ-γαλακτικού οξέος (PLA) και PLGA μικροσφαίρες. Τα πρότυπα αντιγόνα ήταν δύο: * το πεπτίδιο GSPYVSRLLGICLTSTVQLVQL, που αντιστοιχεί στην περιοχή 778-799 της ογκοπρωτεΐνης HER-2/neu. Η πεπτιδική αυτή ακολουθία περιλαμβάνει τον κυτταροτοξικό επίτοπο CLTSTVQLV (789-797) σε συνδυασμό με τον T βοηθητικό (Th) επίτοπο GSPYVSRLLGICL (778-790) της συγκεκριμένης ογκοπρωτεΐνης. * καθώς και το πεπτίδιο CLTSTVQLV (789-797), δηλαδή μόνο ο κυτταροτοξικός (CTL) επίτοπος. Ως πειραματόζωα στην συγκεκριμένη περίπτωση χρησιμοποιήθηκαν HHD διαγονιδιακοί μύες, οι οποίοι εκφράζουν ανθρώπινα HLA-A2.1 μόρια ιστοσυμβατότητας, δεδομένου ότι η ακολουθία του πεπτιδίου που έχει επιλεγεί προέρχεται από την ανθρώπινη HER-2/neu. Η μετατροπή της ανοσοαπόκρισης Th2 τύπου, εναντίον διαλυτών αντιγόνων που εκφράζονται σε καρκινικούς όγκους, σε Τh1 τύπο ανοσοαπόκρισης είναι σημαντική στην ανοσοθεραπεία του καρκίνου. Η δημιουργία αντιγονο-ειδικών CD8+ κυτταροτοξικών λεμφοκυττάρων, σε συνέργεια με τα αντίστοιχα βοηθητικά Τ (CD4+) λεμφοκύτταρα, πιστεύεται ότι θα οδηγήσουν στην απόρριψη του όγκου ή στην επιβράδυνση της ανάπτυξης αυτού. Η ταυτοποίηση του τύπου της ανοσοαπόκρισης έγινε με την ανάπτυξη ανοσοαναλυτικών τεχνικών για την μέτρηση των ολικών ειδικών ανοσοσφαιρινών IgG, των ισοτύπων αυτών (IgG1 και IgG2a). Επίσης προσδιορίσθηκε ο τύπος της ανοσοαπόκρισης σε κυτταρικό επίπεδο με την ανάπτυξη τεχνικών μέτρησης της ικανότητας του πολλαπλασιασμού των λεμφοκυττάρων και με μέτρηση των κυτοκινών, κυρίως σε υπερκείμενα καλλιεργειών λεμφοκυττάρων, αλλά και στο αίμα. Για την χορήγηση χρησιμοποιήθηκαν μικροσφαίρες PLA και PLGA με φορτωμένο το αντιγόνο με δύο διαφορετικούς τρόπους (προσροφημένο ή απλά αναμεμιγμένο). Η in vivo χορήγηση του πεπτιδικού αντιγόνου που απλά και μόνο αναμίχθηκε με PLA μικροσφαίρες προκάλεσε μια ισχυρή ανοσολογική απόκριση που ήταν συγκρίσιμη με αυτήν που προκλήθηκε από το συνδυασμό του αντιγόνου με πλήρες ανοσοενισχυτικό του Freund (CFA). Επιπλέον, μετά από ανάλυση του προφίλ των κυτοκινών που εκκρίνονται από τα Τ λεμφοκύτταρα των ανοσοποιημένων μυών, αποδείχθηκε ότι ο συνδυασμός του αντιγόνου πεπτιδίων με τις PLA μικροσφαίρες προκάλεσε μια ισχυρή Th1 ανοσολογική απόκριση στο αντιγόνο. Ο χρόνος της επώασης πεπτιδίων με τις μικροσφαίρες πριν από τη χορήγηση δεν είχε επιπτώσεις στην ανοσολογική απόκριση, γεγονός που απλοποιεί περαιτέρω την παραγωγή σε ευρεία κλίμακα αυτού του τύπου εμβολίων. Τα αποτελέσματα που ελήφθησαν από αυτή τη μελέτη δικαιολογούν την περαιτέρω διερεύνηση σε in vivo πειραματικά μοντέλα καρκίνου της δυνατότητας επαγωγής ισχυρής κυτταρικής ανοσοαπόκρισης έναντι των καρκινικών κυττάρων που υπερεκφράζουν την HER-2/neu πρωτεΐνη με απλή ανάμιξη κατάλληλων πεπτιδικών αντιγόνων της HER-2/neu με PLA μικροσφαίρες. / Despite the progress of classic therapeutic strategies developed concerning cancer the greatest number of patients deteriorates and eventually dies. The need to confront this disease in an alternative way has led to the development of new immunotherapeutic methods. The novel idea of cancer immunotherapy was born in the 19th century when William Coley used live live species of bacteria Streptococcus erysipelas in order to induce an overall immune response targeted in part against sarcoma tumors. Occasional positive immune responses that were observed were possibly due to the enhancement of the immune response from the inflammatory reactions caused by the bacteria. In order to induce a special immune response against tumors it is necessary for some specific antigens to be identified at cancer cells. So the first step in the effort to induce immunotherapy is the isolation of antigens expressed by cancer cells that are preferably not expressed at healthy tissues, to prevent autoimmune response. The identification of tumor-specific antigens that are identified by T cells gave a great boost to the development of T-cell-mediated specific immune response, both in research for tumor immunology as in its clinical appliance. That led to the beginning of peptide use in vaccines in cancer immunotherapy. From the plethora of already known cancer antigens, epitopes have been identified as capable of forming complex with MHC (Major Histocompatibility Complex) class I molecules, which consequently induce the activation of CD8+ T cells, given that most tumors are positive for the MHC class I molecules, but negative to MHC class II molecules. Moreover, CD8+ T cells can kill cancer cells directly, through identification of the MHC class I–peptide complex that is expressed on the tumor surface. Recently many epitopes that are recognized by MHC class II molecules have been identified, since it is well known that the CD4+ T cells play an important role in the initiation, organization and maintenance of the immune response. Recent clinical studies and preclinical models have shown that immunization with epitopes that are eminent to form a complex with MHC class II molecules, which comprise amino acid sequences that can connect with MHC class I molecules, are effective in the simultaneous induction of helper and cytotoxic long life T-cell in vivo. Among all known cancer antigens, the HER-2/neu protein demonstrates the advantage of being overexpressed in various types of cancer, while patients whose tumors overexpress the protein exhibit preexisting immunity against its peptides. HER-2/neu is a transmembrane protein that is overexpressed in cancer cells and therefore the perfect target for immunotherapy concerning both cellular and humoral immunity. Clinical studies using HER-2/neu peptides have shown induction of immune response in the majority of patients. However, metastatic tumors overexpressing HER-2/neu protein still remain incurable. As a result, there is ample need for respective new therapeutic strategies and at this point more potent immunogenic sequences of the protein are under investigation, as is the response of patients to those sequences, in hope of creating more specific anticancer therapies. Encapsulation of antigen into poly (lactic-co-glycolic) acid (PLGA) microspheres has proven to induce potent and long lasting immune response. Up to date, there is no study analyzing the influence of PLGA polymer characteristics or the immunization scheme, regarding the type of the immune response following the administration of PLGA antigen microspheres in vivo. In the current study, the type of the immune response after in vivo administration of HER-2/neu peptide adsorbed on poly-lactic acid (PLA) and PLGA microspheres is investigated. The model antigens used were the following two: • GSPYVSRLLGICLTSTVQLVQL peptide corresponds to the 779-799 amino acid sequence of the HER-2/neu protein. This amino acid sequence contains the cytotoxic epitope CLTSTVQLV (789-797) in combination with the Th epitope GSPYVSRLLGICL (778-790) of the HER-2/neu protein. • CLTSTVQLV (789-797) peptide, which corresponds to merely the cytotoxic epitope. HHD transgenic mice expressing human HLA-A2.1 histocompatibility molecules were used as subjects, given the fact that the amino acid sequence chosen has derived from the human HER-2/neu protein. Converting the preexisting Th2 type of immune response, against soluble antigens expressed in tumors, to the Th1 type is extremely important in curing cancer. The production of antigen-specific CD8+ cytotoxic lymphocytes with the relevant helper T (CD4+) lymphocytes is believed to trigger the rejection of the tumor or the delay of its development. The type of the immune response was identified with immuno-analytic techniques developed for measuring the total amount of IgG immunoglobulins, and their isotypes (IgG1 and IgG2a). Moreover the type of the immune response has been determined at cellular level using proliferation assay and cytokine measurement assay, usually at cell culture supernatants but also in blood samples. For the peptide administration, PLA and PLGA microspheres were used. The antigen was administered in two different ways, either absorbed or adsorbed (just mixed). The in vivo administration of the peptide antigen just admixed with PLA microspheres induced potent immune response, comparable to that caused by the antigen administration using complete Freund’s adjuvant (CFA). Moreover, upon the analysis of the cytokine profile secreted from T lymphocytes of immunized mice, the PLA admixed peptide proved to induce a specific and potent Th1 immune response. The incubation time of the peptide with PLA microspheres had no implications to the immune response, therefore further simplifying future mass production of such vaccine types. The results extracted by this study justify further investigation of the in vivo experimental cancer models for inducing potent cellular immune response against cancer cells that overexpress the HER-2/neu protein by simply mixing the appropriate HER-2/neu peptide antigens with PLA microspheres.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Foram sintetizadas microesferas poliméricas com propriedades magnéticas e morfologia casca-núcleo por meio da técnica de polimerização em suspensão em duas etapas. O núcleo foi constituído por poli(estireno-co-divinilbenzeno) e magnetita modificada com ácido oleico. Foi avaliada a influência da velocidade de agitação e da concentração de iniciador sobre as características das microesferas utilizadas como núcleo (morfologia, tamanho de partículas, propriedades magnéticas e estabilidade térmica). A casca foi constituída por poli(estireno-co-divinilbenzeno) sem material magnético. Foi avaliado o método de adição da emulsão dos monômeros formadores da casca e o tempo de inchamento dos núcleos na emulsão. As microesferas casca-núcleo foram caracterizadas quanto ao seu aspecto morfológico e à estabilidade térmica. Os núcleos não apresentaram ciclos de histerese, estando assim próximos de um material com propriedades superparamagnéticas. O copolímero sintetizado com a maior velocidade de agitação e a menor concentração de iniciador foi o que apresentou o maior teor de ferro incorporado (3,317 %), a maior magnetização de saturação (2,99 emu/g) e o menor diâmetro médio de partículas (81 m). As microesferas casca-núcleo apresentaram apenas um estágio de degradação e as suas Tmáx foram menores do que a do núcleo. O mapa composicional de ferro confirmou a presença de magnetita na superfície das microesferas casca-núcleo / Polymeric microspheres with magnetic properties and core-shell morphology were synthesized by the technique of suspension polymerization in two stages. The core was constituted by poly(styrene-co-divinylbenzene) and magnetite coated with oleic acid. The influence of stirring rate and initiator concentration on microspheres characteristics (morphology, particle size, magnetic properties and thermal stability) was studied. The shell was constituted by poly(styrene-co-divinylbenzene) without magnetic material. It was also evaluated the methods of the monomers emulsion addition and the coress swelling time in the emulsion.. The microspheres were characterized by morphologic aspect and thermal stability. The cores did not presented hysteresis cycles, thus proving the superparamagnetic properties. Copolymer synthesized with the higher stirring rate and lower initiator concentration presented the higher incorporated iron content (3.317 %), higher saturation magnetization (2.99 emu/g) and the lower average particle diameter (81 m). Core-shell microspheres presented only one stage of degradation and the Tmáx were smaller than temperature of the cores. Iron compositional map confirmed the presence of magnetite on the surface of core-shell microspheres

Nesta dissertação, microesferas poliméricas com propriedades magnéticas à base de estireno e divinilbenzeno foram sintetizadas por polimerização em suspensão. O material utilizado para conferir as propriedades magnéticas foi magnetita sintetizada em laboratório. Foram estudados os efeitos da modificação da magnetita com ácido oleico, da velocidade de agitação, do teor de iniciador e de teor de agente de suspensão sobre as características das partículas poliméricas obtidas. As microesferas foram caracterizadas quanto ao seu aspecto morfológico, à estabilidade térmica, ao tamanho e à distribuição do tamanho de partículas e quanto à suas propriedades magnéticas. Foram obtidos copolímeros com morfologia esférica e tamanho micrométrico, com propriedades magnéticas comparáveis a materiais superparamagnéticos. O tamanho de partícula foi afetado principalmente pela velocidade de agitação, enquanto que a distribuição deste tamanho foi fortemente influenciada pela alteração dos teores de iniciador e agente de suspensão. A magnetização de saturação para as microesferas ficou próxima dos valores encontrados na literatura, comparando-se resinas com o mesmo teor de material magnético. A modificação com ácido oleico foi considerada importante para a incorporação do material magnético / In this dissertation, polymeric microspheres with magnetic properties based on styrene and divinylbenzene were synthesized by suspension polymerization technique. In order to obtain magnetic properties, magnetite was synthesized in laboratory. The effects of magnetite modification with oleic acid, stirring velocity, concentration of initiator and concentration of stabilizer on the particles properties were studied. The magnetic microspheres were characterized according to morphology, thermal stability, particle size and its distribution and magnetic properties. It was verified that the microspheres presented spheric shape, micrometric size and magnetic properties comparable to superparamagnetic materials. Particle size was mainly affected by stirring velocity, while its dispersion was strongly affected by concentration of initiator and stabilizer. The saturation magnetization for the microshpheres was similar to the values reported in the literature, taking in account resins with the same content of magnetic material. The modification with oleic acid was considered important to the incorporation of the magnetic material

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Foram sintetizadas microesferas poliméricas com propriedades magnéticas e morfologia casca-núcleo por meio da técnica de polimerização em suspensão em duas etapas. O núcleo foi constituído por poli(estireno-co-divinilbenzeno) e magnetita modificada com ácido oleico. Foi avaliada a influência da velocidade de agitação e da concentração de iniciador sobre as características das microesferas utilizadas como núcleo (morfologia, tamanho de partículas, propriedades magnéticas e estabilidade térmica). A casca foi constituída por poli(estireno-co-divinilbenzeno) sem material magnético. Foi avaliado o método de adição da emulsão dos monômeros formadores da casca e o tempo de inchamento dos núcleos na emulsão. As microesferas casca-núcleo foram caracterizadas quanto ao seu aspecto morfológico e à estabilidade térmica. Os núcleos não apresentaram ciclos de histerese, estando assim próximos de um material com propriedades superparamagnéticas. O copolímero sintetizado com a maior velocidade de agitação e a menor concentração de iniciador foi o que apresentou o maior teor de ferro incorporado (3,317 %), a maior magnetização de saturação (2,99 emu/g) e o menor diâmetro médio de partículas (81 m). As microesferas casca-núcleo apresentaram apenas um estágio de degradação e as suas Tmáx foram menores do que a do núcleo. O mapa composicional de ferro confirmou a presença de magnetita na superfície das microesferas casca-núcleo / Polymeric microspheres with magnetic properties and core-shell morphology were synthesized by the technique of suspension polymerization in two stages. The core was constituted by poly(styrene-co-divinylbenzene) and magnetite coated with oleic acid. The influence of stirring rate and initiator concentration on microspheres characteristics (morphology, particle size, magnetic properties and thermal stability) was studied. The shell was constituted by poly(styrene-co-divinylbenzene) without magnetic material. It was also evaluated the methods of the monomers emulsion addition and the coress swelling time in the emulsion.. The microspheres were characterized by morphologic aspect and thermal stability. The cores did not presented hysteresis cycles, thus proving the superparamagnetic properties. Copolymer synthesized with the higher stirring rate and lower initiator concentration presented the higher incorporated iron content (3.317 %), higher saturation magnetization (2.99 emu/g) and the lower average particle diameter (81 m). Core-shell microspheres presented only one stage of degradation and the Tmáx were smaller than temperature of the cores. Iron compositional map confirmed the presence of magnetite on the surface of core-shell microspheres

Nesta dissertação, microesferas poliméricas com propriedades magnéticas à base de estireno e divinilbenzeno foram sintetizadas por polimerização em suspensão. O material utilizado para conferir as propriedades magnéticas foi magnetita sintetizada em laboratório. Foram estudados os efeitos da modificação da magnetita com ácido oleico, da velocidade de agitação, do teor de iniciador e de teor de agente de suspensão sobre as características das partículas poliméricas obtidas. As microesferas foram caracterizadas quanto ao seu aspecto morfológico, à estabilidade térmica, ao tamanho e à distribuição do tamanho de partículas e quanto à suas propriedades magnéticas. Foram obtidos copolímeros com morfologia esférica e tamanho micrométrico, com propriedades magnéticas comparáveis a materiais superparamagnéticos. O tamanho de partícula foi afetado principalmente pela velocidade de agitação, enquanto que a distribuição deste tamanho foi fortemente influenciada pela alteração dos teores de iniciador e agente de suspensão. A magnetização de saturação para as microesferas ficou próxima dos valores encontrados na literatura, comparando-se resinas com o mesmo teor de material magnético. A modificação com ácido oleico foi considerada importante para a incorporação do material magnético / In this dissertation, polymeric microspheres with magnetic properties based on styrene and divinylbenzene were synthesized by suspension polymerization technique. In order to obtain magnetic properties, magnetite was synthesized in laboratory. The effects of magnetite modification with oleic acid, stirring velocity, concentration of initiator and concentration of stabilizer on the particles properties were studied. The magnetic microspheres were characterized according to morphology, thermal stability, particle size and its distribution and magnetic properties. It was verified that the microspheres presented spheric shape, micrometric size and magnetic properties comparable to superparamagnetic materials. Particle size was mainly affected by stirring velocity, while its dispersion was strongly affected by concentration of initiator and stabilizer. The saturation magnetization for the microshpheres was similar to the values reported in the literature, taking in account resins with the same content of magnetic material. The modification with oleic acid was considered important to the incorporation of the magnetic material

Submitted by Marco Antonio Oliveira da Silva (maosilva@ipen.br) on 2016-08-25T18:22:57Z No. of bitstreams: 0 / Made available in DSpace on 2016-08-25T18:22:57Z (GMT). No. of bitstreams: 0 / A radioterapia interna seletiva é uma alternativa para o tratamento de alguns tipos de cânceres como o carcinoma hepatocelular (CHC), ou câncer de fígado primário. Neste tratamento, microesferas de vidro ou polimérica contendo em sua estrutura radionuclídeos emissores de partículas β- são introduzidas no fígado por meio da artéria hepática e migram, preferencialmente, para regiões hipervascularizadas, que são características da presença de tecido canceroso. Neste trabalho, foram propostos o desenvolvimento de vidros fosfato contendo hólmio para produção de microesferas e sua aplicação em radioterapia interna seletiva no Brasil. O vidro desenvolvido apresentou durabilidade química adequada, densidade de 2,7(3)g/cm3, alta estabilidade térmica e as impurezas encontradas não inviabilizam o tratamento. As microesferas foram produzidas pelos métodos da chama e da queda gravitacional e foram caracterizadas por diversas técnicas em que se observaram forma, granulometria, atividade e biocompatibilidade apropriados para o tratamento pretendido. Propõe-se que as microesferas possam ser submetidas a testes in vivo. / Tese (Doutorado em Tecnologia Nuclear) / IPEN/T / Instituto de Pesquisas Energeticas e Nucleares - IPEN-CNEN/SP