Des revêtements polymères basés sur la physisorption de copolymères en peigne, dérivés du (Poly-L-Lysine)-g-poly(N Isopropylacrylamide) noté PLL-g-PNIPAM, ont été développés afin de contrôler réversiblement l'adhésion cellulaire. La préparation de ces revêtements repose sur l'adsorption spontanée de la poly(lysine) sur la plupart des substrats anioniques usuels en biologie, aisément mis en oeuvre par le non-spécialiste : une simple immersion du substrat dans une solution de PLL-g-PNIPAM donne une monocouche résistante au rinçage et dense en chaines poly(N-Isopropylacrylamide) thermosensibles.L'adsorption de copolymères en peigne dérivés de la PLL et portant des chaînes latérales de natures variées permet d'ajuster à souhait les propriétés du revêtement. Notre attention s'est portée particulièrement sur l'étude de couchesthermosensibles poly(N-Isopropylacrylamide-coligand), où le ligand est soit un groupe biotine, soit un peptide d'adhésion.Une voie de synthèse générique de dérivés PLL-g- PNIPAM est décrite. Elle se fonde sur la polymérisation RAFT du N-acryloxysuccinimide et la post-modification des unités de répétition puis des extrémités de chaînes. Des mesures de la température de transition soluble/insoluble en solution et des caractérisations par AFM et QCM-d de l'extension des chaînes en surface ont permis de démontrer que la transition au sein des revêtements s'opère à une température proche de celle en solution. La stabilité en termes de composition et d'épaisseur des revêtements suite à des cycles de transitions thermiques a été vérifiée. Une étude de l'adsorption de particules décorées par des protéines comme l'avidine a permis de montrer la capacité des couches adsorbée à exposer/masquer la biotine, et à réguler la formation de liaisons spécifiques par simple changement de température. Pour s'assurer de la possibilité d'ajustement du contraste d'accessibilité de ligands, cette étude a été complétée par des mesures sur des couches mixtes contenant des mélanges de chaînes fonctionnelles ou non fonctionnalisées. Enfin, en utilisant un peptide d'adhésion (RGD) comme ligand, il a été montré que l'interaction avec la surface de cellules HeLA peut être réversiblement contrôlée via une variation de température de quelques degrés, moyennant d'optimiser la densité en peptides et la présence de chaînes répulsives en surface.Un control local par stimulation lumineuse a été envisagé. Des poly(N-Isopropylacrylamide) contenant des azobenzenes furent synthétisés mais ne montrent qu'une faible réponse lumineuse. / Polymer coatings based on the physorption of comb-like polymers, (Poly-L-Lysine)-g-poly(NIsopropylacrylamide)derivatives (PLL-g-PNIPAM), have been developed to reversibly modulate specific cell adhesion on demand.This technique of coating relies on the spontaneous adsorption of PLL on typical anionic substrates used in biology: a simple bath application of substrates in solution of PLLg-PNIPAM affords a stable polymer adlayer with dense and thermoresponsive poly(NIsopropylacrylamide) brushes. Adsorption of comb-like polymers with different strands enables to easily modulate properties of the coatings. We focused on thermoresponsive poly(N-Isopropylacrylamide-co-ligand) brusheswhere ligand is Biotin or a peptide of adhesion.A versatile synthesis of PLL-g-PNIPAM is presented. This synthesis is based on RAFT polymerization of N-acryloxysuccinimide and post-modifications of both the backbone and the polymer ends. Detection of the transition soluble/insoluble in solution and AFM/QCM-d studies of PNIPAM adlayers demonstrate that the critical temperature in adlayers are close to the one in solution. Stability in terms of composition and thickness has been checked after temperature cycles. The study of the adsorption of Avidin-coated particles suggests that these adlayers can expose/mask Biotin, and so regulate specific interaction by simple change in temperature. To optimize the thermal contrast of ligand accessibility, this study includes measurements on mixed adlayers with functionalized/unfunctionalized brushes. Finally, specific adhesion of HeLa cells can be thermally modulated on adlayers presenting repellent brushes and with controlled densities of adhesive peptides (RGD).A local control has been considered on such coatings by using light stimulus. In this context, azobenzene-containing Poly(NIsopropylacrylamide) were synthesized but they shown only a weak light-response.

Targeted delivery aids in minimizing most of the drug-originated systemic toxic effects as well as improving the pharmacokinetic properties of anticancer therapeutics. Tumor targeting using hyaluronic acid (HA) as the targeting ligand has attracted a great deal of interest among a host of strategies developed to target the overexpressed tumor specific receptors. HA is an endogenous molecule that possesses a lot of biological functions in the human body. The role of HA synthases, HA degrading enzymes and the interaction of HA with its primary receptor CD44 in tumor metastasis and angiogenesis is really complex and controversial to date. However, overexpression of CD44receptors on tumor surface has been well studied, which have been utilized to direct tumor targeted drugs. Most of the HA based targeting systems were HA drug conjugates and surface modified colloidal carriers which required covalent modification. The lack of accurate structural characterization of these systems resulted in modification of HA binding sites that could affect the efficient cellular uptake. LbL technique is a simple and facile method to incorporate several materials into polyelectrolyte assemblies for drug delivery applications. HA being a negatively charged polysaccharide can be easily incorporated into such systems without any covalent modification. Although HA based polyelectrolyte multilayer films and microcapsules have been reported in combination with polycations like PAH, PLL and chitosan, their application as targeted drug delivery systems have not yet been explored. Herein, two LbL architectures with HA as the terminal layer have been investigated as targeted drug carriers, which can recognize overexpressed CD44 receptors in metastatic breast cancer cells. In the first part of the thesis, a novel polyelectrolyte nanocapsule system composed of biopolymers HA and protamine sulphate (PR) as the wall components was prepared and characterized. These pH and enzyme responsive nanocapsules were then utilized for efficient loading and release of anticancer drug doxorubicin (dox). Higher drug release was observed in simulated intracellular conditions like acidic pH and presence of hyaluronidase enzyme as compared to physiological pH. In the second part of the thesis, dox incorporated bovine serum albumin (BSA) nanoparticles modified with HA-Poly(l-Lysine) multilayers were developed and characterized. The drug release pattern of the dox loaded BSA nanoparticles was found to depend on the presence of a protease enzyme trypsin than pH variations. Both of these drug delivery systems were then evaluated for their cell targeting efficiency and cytotoxicity in CD44+ positive metastatic breast cancer cell line MDA MB 231. The final layer HA facilitated targeted delivery of these drug carriers via CD44 receptor mediated endocytosis. The enhanced cellular uptake followed by sustained delivery of dox by virtue of slow intracellular enzymatic degradation of the drug carriers resulted in their improved cytotoxicity as compared to free dox. Further in vitro biodistribution and tumor suppression efficiency of both the systems were studied in breast cancer xenograft models using BALB/c nude mice. Enhance accumulation of dox in the tumor tissue and significant tumor reduction were observed when treated with encapsulated dox using the HA based nanocarriers as opposed to free dox.

Made available in DSpace on 2016-12-08T15:56:18Z (GMT). No. of bitstreams: 1 KATIUSCA WESSLER MIRANDA.pdf: 4397696 bytes, checksum: da27bc7c1adc7efa3e74ecfcf87feb0e (MD5) Previous issue date: 2015-08-07 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / This paper studied ring opening polymerizations L-lactide (LLA) and ε-caprolactone (ε-CL) by bulk polymerization and solution polymerization, in order to evaluate the influence of initiator concentration, reaction time and reaction temperature on the yield and molecular weight of both polymers. Tin octoate and methanol was employed as initiators. It was observed that LLA and ε-CL speed ring opening polymerization, increases with temperature and that the monomer/polymer conversion immediately occurs in the first hours of synthesis when 2% of initiator is used. The molar mass, determined by capillary viscometry, was approximately 7x103 g/mol, for both polymers. It was also studied the copolymerization of LLA and ε-CL by solution polymerization employing different solvents. Weight percentages of LLA/ε-CL equal to 100/0, 95/5, 90/10, 85/15 and 80/20 were studied. The toluene was the only solvent that enabled the production of polymers and copolymers at 120 ° C. Initiator concentration equal to 0.015% and reaction time equal to 24 hours, were assessed, generating polymers with average molar mass (Mw) around 2x104 g / mol, determined by GPC. It was observed by proton and carbon 13 nuclear magnetic resonance (13 C-NMR and 1H-NMR) that reactions conducted with ε-CL concentrations lower than 15% do not yield copolymer, only PLLA. Bulk polymerization it was also studied and the main difference compared to solution polymerization is associated with the molar mass of the copolymer P(LLA-co-εCL)80/20. This product had a higher molar mass when synthesized by the first technique. After the study of copolymerization, the functionalization of nanocrystalline cellulose particles (CNC) with LLA and ε-CL, by polymerization in solution, was studied. Three compositions were analyzed: (i) PLLA-CNC, (II) P(LLA-co-εCL)85/15-CNC and (III) P(LLA-co εCL)80/20-CNC; the reactions were conducted at 120 ° C for 24 hours. It was possible to functionalize CNC particles in situ, using 80/20 LLA/ε-CL systems, with tin octoate (0.015 wt%) as catalyst and toluene as solvent (composition III). The functionalization was confirmed by Fourier transform infrared spectroscopy (FTIR). The compatibility of the P(LLA-co-εCL)80/20-CNC particle increased in toluene, confirming the reduction of hydrophilicity of these particles. Unlike occurred with the compositions (I) and (II), the composition (III) only allowed the production CNC functionalized particles. PLLA, PCL and / or P (LLA-co-εCL) were not produced. This fact indicates that the concentration of ε-CL and the presence of cellulose hydroxyl groups decreased the reactivity between the monomers. / Neste trabalho foram estudadas as reações de abertura de anel dos monômeros L-lactídeo (LLA) e ε-caprolactona (ε-CL), pela técnica de polimerização em massa com o intuito de avaliar a influência da concentração de iniciador, do tempo e da temperatura de reação sobre o rendimento reacional e a massa molar de ambos os polímeros. O iniciador empregado neste estudo foi o octoato de estanho e como co-iniciador, metanol foi empregado. Foi observado que a velocidade de reação de obtenção do poli(L-ácido láctico) (PLLA) e da poli(ε-caprolactona) (PCL) aumenta em função da temperatura e que com 2% de iniciador a conversão de monômero em polímero ocorre logo nas primeiras horas de síntese. A massa molar determinada por viscosimetria capilar foi de aproximadamente 7x103 g/mol para ambos os polímeros. Também foi estudada a obtenção de copolímeros de LLA e ε-CL por reações de polimerização em solução empregando diferentes solventes. As porcentagens mássicas de LLA/ε-CL empregadas foram 100/0, 95/5, 90/10, 85/15 e 80/20. O tolueno foi o único solvente que possibilitou a produção de polímeros e copolímeros a 120 °C. Para estas sínteses a concentração de iniciador foi igual a 0,015% e o tempo de reação foi de 24 horas, gerando polímeros com massa molar ponderal média (Mw) ao redor de 2x104 g/mol, determinadas por GPC. Foi observado por ressonância magnética nuclear de hidrogênio e de carbono 13 (RMN1H e RMN13C) que as reações conduzidas com concentrações de ε-CL inferiores à 15% não rendem copolímero, apenas PLLA é produzido. A obtenção de copolímeros pela técnica de polimerização em massa também foi estudada e a principal diferença em relação a polimerização em solução está associada à massa molar do copolímero P(LLA-co-εCL)80/20, sendo que o produto da polimerização em massa apresentou massa molar mais elevada que o obtido pela polimerização em solução. Após o estudo da obtenção dos copolímeros, foi estudada a funcionalização de partículas de celulose nanocristalina (CNC) com os monômeros de LLA e ε-CL pela técnica de polimerização em solução. Foram analisadas três composições: (I) PLLA-CNC, (II) P(LLA-co-εCL)85/15-CNC e (III) P(LLA-co-εCL)80/20-CNC; as reações foram conduzidas a 120°C durante 24 horas. Foi possível funcionalizar partículas de CNC in situ, empregando 80% de LLA, 20% de ε-CL, 0,015% de octoato de estanho e tolueno como solvente (composição III). A funcionalização foi confirmada por análises de espectroscopia de infravermelho por transformada de Fourier (FTIR). Testes de dispersão do P(LLA-co-εCL)80/20-CNC em tolueno confirmaram a diminuição da hidrofilicidade destas partículas. Ao contrário do ocorrido com as composições (I) e (II), a composição (III) permitiu somente a produção partículas de CNC funcionalizadas. PLLA, PCL e/ou P(LLA-co-εCL) não foram produzidos. Este fato indica que o aumento da concentração de ε-CL e a presença de grupos hidroxilas de celulose diminuíram a reatividade entre os monômeros.

Orientador: Eliana Aparecida de Resende Duek / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia Mecanica / Made available in DSpace on 2018-08-09T22:07:51Z (GMT). No. of bitstreams: 1 Barauna_GrazielledosSantos_M.pdf: 1217156 bytes, checksum: ded7e97c7818fe0c3d87cb97d8c8f8aa (MD5) Previous issue date: 2007 / Mestrado / Materiais e Processos de Fabricação / Mestre em Engenharia Mecânica

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[ES] Los tumores malignos del cerebro y del sistema nervioso central representan el 21% de los tumores en niños y son la segunda causa principal de muerte por cáncer pediátrico después de la leucemia. Los gliomas de alto grado siguen siendo incurables y poseen altas tasas de mortalidad: una supervivencia a cinco años del 4,7 % para pacientes con glioblastoma multiforme (GBM) y menos de un año en pacientes con glioma pontino intrínseco difuso (DIPG). Los bajos niveles de penetración del fármaco a través de la barrera hematoencefálica y la baja tasa de supervivencia asociada resaltan en la necesidad de nuevas propuestas de tratamiento para esta necesidad clínica no cubierta. La administración intranasal ofrece un enfoque no invasivo prometedor que elude el metabolismo hepato-gastrointestinal y la barrera hematoencefálica, constituyendo una ruta directa de la nariz al cerebro. Los poliglutamatos (PGA) representan excelentes candidatos para la administración de agentes terapéuticos en el cerebro debido a su biodegradabilidad y multivalencia, lo que permite la unión covalente de fármacos y grupos directores que puedan contribuir a cruzar las diferentes barreras biológicas existentes desde la nariz hasta el cerebro. La conjugación covalente del agente terapéutico a la cadena polimérica ofrece una estabilidad prolongada en la circulación sanguínea y un mayor control sobre la liberación del fármaco en el microambiente tumoral. Esta tesis se centra en el diseño racional y desarrollo de nuevos conjugados PGA-fármaco y una plataforma intranasal segura y eficiente para la administración dirigida y la liberación del fármaco en el cerebro como tratamiento del glioma pediátrico. Se desarrolló una familia de conjugados de poliglutamatos que incorporan palbociclib, un inhibidor de CDK, utilizando diferentes espaciadores sensibles a estímulos, así como diferente carga de fármacos. Estudiamos el efecto de la carga y conjugación de un fármaco sobre la conformación en solución de PGAs lineales y en forma de estrella. Con dispersión de rayos X de ángulo pequeño y dicroísmo circular, demostramos la evolución del conjugado polipeptídico al aumentar la carga de fármaco. Además, establecimos el vínculo entre la conformación de los conjugados y su actividad biológica en células GBM y DIPG derivadas de pacientes. Nuestros hallazgos ilustran la necesidad de una comprensión profunda de las propiedades fisicoquímicas de los nanosistemas estudiados que pueden ayudar a predecir su resultado biológico. Para el cribado rápido y la validación de la formulación intranasal, establecimos con éxito una plataforma de detección ex vivo basada en células de difusión verticales de Franz con mucosa nasal de oveja. Varios sistemas basados en PGA lineal y en forma de estrella modificadas con diferentes ligandos (incluyendo el ácido docosahexaenoico, ácido hialurónico (HA), odorranalectina), así como sistemas entrecruzados del PGA en forma de estrella y la mezcla física con un HA entrecruzado (HA-CP®). A continuación, se realizó estudios de biodistribución mediante administración intranasal de los candidatos seleccionados, seguidos de la cuantificación ex vivo con la técnica IVIS y el ensayo de fluorescencia estándar después de la homogeneización de órganos y los estudios histológicos del cerebro. Los datos obtenidos mostraron claramente la presencia de las diferentes plataformas en el cerebro e, su internalización en células del bulbo olfativo y una buena difusión en el cerebro a través de diferentes áreas, llegando incluso a detectarse en el hipocampo. Los candidatos seleccionados se han escalado y en la actualidad su evaluación biológica en un modelo in vivo está en curso. En conclusión, se ha diseñado, desarrollado y validado diferentes plataformas basadas en PGA capaces de llegar al cerebro a través de la administración intranasal, constituyendo una base prometedora para el tratamiento de múltiples trastornos relacionados con el cerebro. / [CA] Els tumors malignes del cervell i del sistema nerviós central representen el 21% dels tumors en nens i són la segona causa principal de mort per càncer pediàtric després de la leucèmia. Els gliomes d'alt grau que continuen sent incurables amb taxes de mortalitat altes: una supervivència a cinc anys del 4,7 % per a pacients amb glioblastoma multiforme (GBM) i menys d'un any en pacients amb glioma pontí intrínsec difús (DIPG). Els nivells baixos de penetració del fàrmac a través de la barrera hematoencefàlica i la baixa taxa de supervivència associada ressalten en la necessitat de noves propostes de tractament per a una necessitat clínica no satisfeta. L'administració intranasal ofereix un enfocament no invasiu prometedor que eludeix el metabolisme hepatogastrointestinal i la barrera hematoencefàlica, cosa que permet l'administració directa del nas al cervell. Els poliglutamats (PGA) representen excel·lents candidats per a l'administració al cervell a causa de la seva biodegradabilitat i multivalència, la qual cosa dóna suport a la introducció covalent de fàrmacs i grups objectiu i pot facilitar la transició del nas al cervell. La conjugació covalent de l'agent terapèutic a la cadena polimèrica ofereix una estabilitat prolongada en la circulació sanguínia i un major control sobre l'alliberament del fàrmac al microambient tumoral. Aquesta tesi se centra en el desenvolupament de nous conjugats PGA-fàrmac dissenyats racionalment i una plataforma intranasal segura i eficient per a l'administració dirigida i l'alliberament del fàrmac al cervell com a tractament del glioma pediàtric. Es va desenvolupar una família de conjugats de poliglutamat que incorporen l'inhibidor de CDK palbociclib, utilitzant diferents enllaçadors sensibles a estímuls i nivells variables de càrrega de fàrmacs. Estudiem l'efecte del fàrmac hidrofòbic conjugat sobre la conformació en solució de PGA lineals i en forma d'estrella. Amb dispersió de raigs X d'angle petit i dicroisme circular, demostrem l'evolució del sistema conjugat en augmentar la càrrega de fàrmac. A més, establim el vincle entre la conformació dels conjugats i la seva activitat biològica provada en cèl·lules GBM i DIPG derivades de pacients. Les nostres troballes van il·lustrar la necessitat d'una comprensió profunda de les propietats fisicoquímiques dels nanosistemes estudiats que poden ajudar a predir-ne el resultat biològic. Per a la detecció ràpida de la formulació intranasal basada en els portadors de PGA, vam establir amb èxit una plataforma de detecció ex vivo basada en cèl·lules de difusió verticals de Franz amb mucosa d'ovella. Diversos sistemes basats en PGA lineal i en forma d'estrella amb fraccions conjugades (inclòs l'àcid docosahexaenoic, àcid hialurònic (HA), odorranalectina), així com partícules reticulades (a través de cicloaddició azida-alquí o enllaços disulfur) i la barreja física amb un HA es van provar els polímers creuats (HA-CP®). Els estudis de biodistribució després de l'administració intranasal es van fer en candidats seleccionats, seguits de la quantificació ex vivo amb la tècnica IVIS i l'assaig de fluorescència estàndard després de l'homogeneïtzació d'òrgans i els estudis histològics del cervell. Les dades obtingudes van mostrar clarament el transport del nas al cervell, la internalització cel·lular dels conjugats al bulb olfactiu i, el que és més interessant, una bona difusió del cervell a través de diferents àrees, arribant fins i tot a l'hipocamp. Els sistemes seleccionats es van ampliar i la preparació per a l'avaluació biològica en un model in vivo està en curs. En general, vam ser capaços de dissenyar, desenvolupar i validar diferents plataformes basades en PGA capaces d'arribar al cervell mitjançant l'administració intranasal, la qual cosa podria ser la base per al tractament de múltiples trastorns relacionats amb el cervell. / [EN] Malignant brain and central nervous system tumors account for ~21% of tumors in children and represent the second leading cause of pediatric cancer deaths after leukemia. Standard chemotherapy allows a ~95% five-year survival rate for patients with low-grade gliomas. Unfortunately, high-grade gliomas remain generally incurable and suffer from high mortality rates - a 4.7% five-year survival for glioblastoma multiform (GBM) patients and less than one year in patients with diffuse intrinsic pontine glioma (DIPG). Low levels of drug delivery through the blood-brain barrier (BBB) and the associated poor survival rate highlight the necessity for novel treatment approaches for an unmet clinical need. Intranasal administration offers a promising non-invasive approach that circumvents hepato-gastrointestinal metabolism and the BBB, thereby enabling direct nose-to-brain delivery. Polyglutamates (PGAs), represent excellent candidates for brain delivery due to their biodegradability and multivalency, which supports the covalent introduction of drugs and targeting moieties to facilitate the nose-to-brain transition. Covalent conjugation of therapeutic agents to the polymeric main chain offers prolonged stability in the blood circulation and increased control over the drug release in the tumor microenvironment if adequately designed considering endogenous triggers, including acidic pH, an increased reductive environment, or the overexpression of specific proteases. This thesis focuses on developing novel, rationally-designed PGA-drug conjugates as a safe and efficient intranasal platform for targeted delivery and drug release in the brain as a pediatric glioma treatment. We developed a family of PGA-drug conjugates incorporating the CDK inhibitor palbociclib that employed different stimuli-responsive linkers and varying drug loading levels and studied the effect of drug loading on the solution conformation of linear and star-shaped PGAs. We demonstrated system evolution upon increased drug loading with SAXS and CD. Moreover, we established a link between conjugate conformation and their biological activity, as evaluated in patient-derived GBM and DIPG cells. Our findings illustrated the necessity of a deep understanding of the physico-chemical properties of studied nanosystems that can aid in predicting their biological outcome. For the development of an intranasal formulation, we successfully established an ex vivo screening platform based on vertical Franz diffusion cells with sheep mucosa. We evaluated several systems based on linear and star-shaped PGA with conjugated moieties (including docosahexaenoic acid, hyaluronic acid, odorranalectin), crosslinked particles (via azide-alkyne cycloaddition or disulfide bonds), and physical mixtures with a hyaluronic acid cross polymer (HA-CP®). After intranasal administration with selected candidates, we performed biodistribution studies, followed by ex vivo quantification with the IVIS spectrum in vivo imaging system and fluorescent assays after organ homogenization and brain histological studies. The obtained data demonstrated nose-to-brain transportation, internalization of conjugates in the olfactory bulb and robust diffusion through different brain areas, even reaching the hippocampus. We also scaled up selected systems and preparations for biological evaluation in an in vivo model that is currently ongoing. Overall, we were able to design, develop, and validate different PGA-based platforms capable of arriving at the brain via intranasal, which might be the base for the treatment of multiple brain-related disorders. / Melnyk, T. (2024). Drug Dellivery to the Brain Using Polymer Therapeutics as an Intranasal Platform for Pediatric Glioblastoma Treatment [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/203498