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We have developed a versatile delivery platform comprising a novel composite of two biomaterials with proven track records: apatite and poly(lactic-co-glycolic acid) (PLGA). These composites have been tested in the delivery of a model protein, bovine serum albumin (BSA), as well as a growth factor, bone morphogenetic protein-2 (BMP-2), which is a potent inducer of bone formation. The controlled release strategy is based on the use of a polymer with acidic degradation products to control the dissolution of a basic inorganic component, resulting in protein release. The release profile can be modified systematically by changing variables that affect polymer degradation and/or apatite dissolution, such as polymer molecular weight, polymer composition, apatite loading, and apatite particle size. We have found that an increase in polymer molecular weight and polymer hydrophobicity led to slower polymer degradation, and in turn, slower apatite dissolution and protein release. Protein release was enhanced by reducing apatite particle size and by lowering the apatite content in the composites. We anticipate that this delivery platform can be extended to the controlled release of other therapeutic proteins and chemicals. / Singapore-MIT Alliance (SMA)

Spinal cord injury (SCI) leads to axonal damage and limits the ability of the brain to communicate with the rest of the body. Several bioengineered approaches have been developed for the recovery of SCI. Among these techniques, degradable guidance tubes have shown promising results. However, design of nerve guide tubes requires several design considerations and has been a significant challenge. To assess the efficacy of a prototypical implanted nerve guide tubes, it is essential to perform continuous monitoring. In this respect, magnetic resonance imaging (MRI) is one of the most reliable imaging techniques as it offers the ability to achieve extraordinary high temporal and spatial resolution in addition to its non-invasive features. In spite of the excellent image quality of non-enhanced MRI various types of contrast agents have been developed to further enhance the contrast and allow improved visualization. The MRI contrast agents principally work by shortening the T1 or T2 relaxation times of protons located nearby. The presented study was intended to evaluate the in vitro degradation of the nerve guide tubes made of poly (lactic-co-glycolic acid) (PLGA). PLGA tubes incorporated with different concentrations of superparamagnetic iron oxide (SPIO) were scanned by MRI 3T on weekly basis during the degradation period. Spin-echo (SE) sequence with various echo times (TEs) ranged from 13.3 to 314.4 msec was applied. T2 mapping was computed using in-house algorithm developed in Matlab. Least square fit was used to find the slope of the decay curve by plotting log intensity on the y-axis and echo time on the x-axis. The average T2 values were calculated. Mass loss and water uptake of the degrading tubes were also measured weekly. Moreover, the micro-structural changes of the tubes were investigated using the scanning electron microscope (SEM). The MRI results showed that the concentration of SPIO affects the signal intensity of the T2 weighted images reducing the T2 relaxation time value. Accordingly, a linear correlation between SPIO concentration and T2 relaxation time was found. At the beginning of degradation, the SPIO nanoparticles were trapped within the polymeric network. Therefore, water penetration was the predominant factor affecting the T2 relaxation times. At week 5, a significant mass loss was observed. From this stage onwards, the trapped SPIO were released from the polymeric network increasing T2 relaxation time dramatically. According to SEM images, the size of the pores in PLGA guide tubes was increased with the degradation. Approaching the end of degradation, shrinkage of the tubes was observed and the degraded nerve guide tubes were shown to be collapsed. Similar shape variation was observed in T2 weighted MR images. In summary, this study provided an approach to non-invasive monitoring of degradation behavior of nerve guide tubes using contrast enhancement. The developed technique is of great importance since it opened an insight to non-invasive monitoring of tissue engineered scaffolds for in vivo studies.

Die Wundheilung stellt einen physiologischen Vorgang zur Regeneration zerstörten Gewebes dar. Der reibungslose Ablauf der Heilung wird durch ein komplexes Zusammenspiel von Zellen und extrazellulären Komponenten gewährleistet. Durch vielfältige Faktoren kann dieser fein abgestimmte Prozess zum Erliegen kommen, so dass eine chronische Wundheilungsstörung resultiert. Trotz zahlreicher Behandlungsmöglichkeiten chronischer Wunden bleibt jedoch die erfolgreiche Heilung oftmals aus. Die Anwendung von biodegradierbaren Materialen (Biomaterialien) in der Wundheilung wurde in den vergangenen Jahren intensiv erforscht und teilweise erfolgreich am Patienten eingesetzt. An dem Ziel, ein Biomaterial herzustellen, welches alle funktionellen und strukturellen Fähigkeiten gesunder menschlicher Haut mit sich bringt, wird jedoch nach wie vor gearbeitet. In verschiedenen Studien konnte die gute Verträglichkeit und Biodegradierbarkeit des Biopolymers PLGA, bestehend aus Lactat und Glycolsäure, bereits gezeigt werden. In der vorliegenden Dissertation wurden dreidimensionale (3D)-Gerüste (Scaffolds) aus PLGA hinsichtlich ihrer Wechselwirkungen mit humanen dermalen Fibroblasten (Fb) untersucht. Dermale Fb leisten einen entscheidenden Beitrag zur erfolgreichen Wundheilung, da sie unter der Einwirkung diverser Wachstumsfaktoren zur Migration ins Wundgebiet sowie zur Neusynthese und Reorganisation extrazellulärer Matrix (ECM) befähigt sind. In den Untersuchungen wurden grundlegende Kenntnisse zum Verhalten der Zellen bezüglich Proliferation sowie Synthese nativer ECM in den PLGA-Scaffolds gewonnen und das Migrationsverhalten der Fb in das Biomaterial untersucht. Dabei zeigte sich, dass dermale Fb in den PLGA-Scaffolds nicht nur proliferieren sondern auch einen ausgeprägten Matrixstoffwechsel aufweisen. Sie sind in der Lage, Kollagen und Hyaluronsäure, wichtige Bestandteile der ECM, abzulagern. Zudem konnte mit Hilfe der Arbeit der Einfluss von Wachstumsfaktoren sowohl auf relevante ECM-Komponenten im 3D-Kultursystem als auch auf das Migrationsverhalten der Fb in das Biomaterial verdeutlicht werden. Aus den Ergebnissen geht hervor, dass 3D-PLGA-Scaffolds geeignete Substrate zur Kultivierung dermaler Fb darstellen. Die zukünftig geplante Weiterentwicklung und Funktionalisierung dieses Biopolymers für den Einsatz in der Wundheilung scheint somit viel versprechend.

A variety of synthetic bone graft materials such as the polymer poly(lactic-co-glycolic acid) (PLGA) have been investigated as alternatives to current tissue based bone graft materials. In this study, efforts have been made to improve the tissue-PLGA interface by immobilizing bone morphogenetic protein-2 (BMP-2) in an oriented manner on scaffolds using covalently immobilized heparin. The results demonstrated a four-fold increase in covalently immobilized heparin compared to non-specific heparin attachment. Furthermore, the scaffolds with covalently attached heparin retained approximately three-fold more BMP-2 than did either scaffolds with no heparin attached or scaffolds with non-specific heparin attachment. The activity of scaffolds with BMP-2 immobilized in various manners was examined using an alkaline phosphatase assay on C3H10T1/2-seeded scaffolds. These results indicated approximately twice the amount of activity with scaffolds that had BMP-2 immobilized with covalently attached heparin than on scaffolds with adsorption of BMP-2 and a three-fold increase in activity when compared to scaffolds that had non-specific heparin attachment as the mechanism for BMP-2 immobilization. These results demonstrated that PLGA with covalently linked heparin has potential to immobilize BMP-2 in a specific orientation that is favorable for cell-receptor binding, leading to the more efficient use of the bone-growth factor.

Spinal cord injury (SCI) leads to axonal damage and limits the ability of the brain to communicate with the rest of the body. Several bioengineered approaches have been developed for the recovery of SCI. Among these techniques, degradable guidance tubes have shown promising results. However, design of nerve guide tubes requires several design considerations and has been a significant challenge. To assess the efficacy of a prototypical implanted nerve guide tubes, it is essential to perform continuous monitoring. In this respect, magnetic resonance imaging (MRI) is one of the most reliable imaging techniques as it offers the ability to achieve extraordinary high temporal and spatial resolution in addition to its non-invasive features. In spite of the excellent image quality of non-enhanced MRI various types of contrast agents have been developed to further enhance the contrast and allow improved visualization. The MRI contrast agents principally work by shortening the T1 or T2 relaxation times of protons located nearby. The presented study was intended to evaluate the in vitro degradation of the nerve guide tubes made of poly (lactic-co-glycolic acid) (PLGA). PLGA tubes incorporated with different concentrations of superparamagnetic iron oxide (SPIO) were scanned by MRI 3T on weekly basis during the degradation period. Spin-echo (SE) sequence with various echo times (TEs) ranged from 13.3 to 314.4 msec was applied. T2 mapping was computed using in-house algorithm developed in Matlab. Least square fit was used to find the slope of the decay curve by plotting log intensity on the y-axis and echo time on the x-axis. The average T2 values were calculated. Mass loss and water uptake of the degrading tubes were also measured weekly. Moreover, the micro-structural changes of the tubes were investigated using the scanning electron microscope (SEM). The MRI results showed that the concentration of SPIO affects the signal intensity of the T2 weighted images reducing the T2 relaxation time value. Accordingly, a linear correlation between SPIO concentration and T2 relaxation time was found. At the beginning of degradation, the SPIO nanoparticles were trapped within the polymeric network. Therefore, water penetration was the predominant factor affecting the T2 relaxation times. At week 5, a significant mass loss was observed. From this stage onwards, the trapped SPIO were released from the polymeric network increasing T2 relaxation time dramatically. According to SEM images, the size of the pores in PLGA guide tubes was increased with the degradation. Approaching the end of degradation, shrinkage of the tubes was observed and the degraded nerve guide tubes were shown to be collapsed. Similar shape variation was observed in T2 weighted MR images. In summary, this study provided an approach to non-invasive monitoring of degradation behavior of nerve guide tubes using contrast enhancement. The developed technique is of great importance since it opened an insight to non-invasive monitoring of tissue engineered scaffolds for in vivo studies.

Poly[lactic co-glycolic] acid (PLGA) is a biocompatible polymer commonly used in the field of tissue engineering, but its mechanical properties tend to be less than ideal for most orthopedic applications. Five PLGA composites, reinforced with 0 to 1% nonfunctionalized single-walled carbon nanotubes, were prepared and tested for tensile strength. In order to achieve consistent nanotube dispersions, sodium dodecyl sulfate was incorporated as a surfactant. The polymer scaffold fabrication methods were successful at creating suitable samples for tensile testing. After the tests were performed, scanning electron microscope images were taken to examine the fractured edges and determine the cause of failure. Analysis of fractured surfaces indicated good nanotube dispersions in all composite samples, and an increase in tensile strength, with respect to the control (0.532 MPa), was found for composites at the 0.07% nanotube and 0.09% nanotube concentrations (0.570 MPa and 0.643 MPa respectively). Total length at failure decreased as carbon nanotube concentration increased. This experiment showed a promising trend toward increasing the mechanical properties of PLGA/carbon nanotube composites and represented a prospective foundation for future research.

Made available in DSpace on 2014-06-11T19:24:11Z (GMT). No. of bitstreams: 0 Previous issue date: 2006-08-04Bitstream added on 2014-06-13T19:48:18Z : No. of bitstreams: 1 oliveira_lc_me_arafcf.pdf: 1008043 bytes, checksum: 986417155d3a5f5fe5fe070125ff8109 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Neste trabalho estudou-se a encapsulação do cloridrato de ciprofloxacino (CIPRO) em lipossomas revestidos de ácido poli-láctico-co-glicólico (PLGA) ou o copolímero termosensível Pluronic® F127 (PLU). A eficiência de encapsulação foi obtida a partir das frações contendo lipossomas carregados de CIPRO separadas por cromatografia de exclusão em gel de Sephadex G-50, e mostrou-se melhor para as amostras contendo PLGA e maior concentração de CIPRO (5 mg/mL). A determinação do diâmetro médio dos lipossomas foi realizada por espalhamento dinâmico de luz (Light Scattering) e demonstrou redução no tamanho das estruturas quando PLGA ou PLU estavam presentes nas preparações. A incorporação de CIPRO aos lipossomas provocou aumento do tamanho das estruturas quando comparadas com as preparações isentas de fármaco. Verificou-se que o aumento na concentração de fármaco provocou a diminuição do diâmetro médio dos lipossomas. Experimentos de liberação in vitro mostraram que a liberação do CIPRO a partir dos lipossomas foi mais lenta em relação ao CIPRO não encapsulado. A liberação do CIPRO a partir de lipossomas revestidos com PLGA mostrou que a liberação foi mais lenta em relação aos lipossomas não revestidos. Os resultados demonstram que as preparações de CIPRO em lipossomas revestidos com PLGA ou PLU podem representar sistemas de liberação de fármacos antibióticos com grande potencial de utilização. O estudo de biodisponibilidade ocular demonstrou que lipossomas revestidos com PLGA e PLU mantiveram a MIC90 de CIPRO para os principais patógenos oculares por mais tempo que o CIPRO em solução no humor aquoso, quando administrados por via subconjuntival. Estes resultados demonstram que a associação de PLGA e PLU com lipossomas pode ser utilizada como um eficiente sistema de liberação ocular de fármacos. / In the present work we studied the encapsulation of ciprofloxacin hydrochloride (CIPRO) in liposomes coated either by the poly-lactic-co-glycolic acid (PLGA) or thermosensitive copolymer Pluronic® F127 (PLU). Unilamellar liposomes containing 40 or 50 mM of hydrogenated soy phosphatidylcholine (FSH) were prepared by reverse phase evaporation (REV) method, followed by sonication. Encapsulation efficiency was obtained using fractions containing liposomes loaded with CIPRO, separated by exclusion chromatography on Sephadex G-50 gel. Best encapsulation efficiency was obtained with samples containing PLGA with CIPRO at a higher concentration (5 mg/mL). Liposome medium diameter was determined by dynamic light scattering, and showed a size reduction when either PLGA or PLU was present in the preparations. The incorporation of CIPRO into the liposomes caused a size increasement of the structures when compared to preparations lacking the drug. Nevertheless, increasing the drug concentration caused a decrease of the liposome medium diameter. Experiments of in vitro release showed that the liberation of CIPRO from the liposomes was slower when compared to not encapsulated CIPRO. The release of CIPRO from liposomes coated by PLGA showed that the liberation was slower when compared to non-coated liposomes. The results show that liposome preparations containing CIPRO and covered either by PLGA or PLU represent antibiotic drug delivery systems with great possibilities. The bioavailability study shows that liposomes covered by both PLGA and PLU maintained the MIC90 of CIPRO against the main ocular pathogens for longer time than CIPRO solution in aqueous humor, when subconjunctivally injected. These results demonstrate that the PLGA and PLU association with liposomes can be used as an efficient ocular drug delivery system.

Made available in DSpace on 2014-06-12T16:26:08Z (GMT). No. of bitstreams: 2 arquivo2100_1.pdf: 3223358 bytes, checksum: 09b24234d9d2402b923206e4ce568f13 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2008 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Uma estratégia tecnológica para superar as limitações hoje encontradas na aplicação clínica da Interleucina 2 (IL-2) para imunoterapia contra o câncer é o desenvolvimento de um sistema de liberação controlada que mantenha a estabilidade da proteína encapsulada e promova uma liberação sustentada dessa proteína, para estimular o próprio sistema imune do paciente a combater a doença neoplásica, sem causar efeitos indesejáveis. Sistemas de liberação controlada, microcápsulas ou microesferas, podem ser preparados através do uso de polímeros biodegradáveis e biocompatíveis, tais como o copolímero de ácido láctico e glicólico (PLGA) aprovado pela vigilância sanitária americana Food and Drug Administration (FDA) para tratamento clínico. Estes sistemas poliméricos apresentam como principal vantagem a possibilidade de modular características da partícula, como a eficiência da microencapsulação e o perfil de liberação da proteína. O principal desafio deste trabalho foi desenvolver microesferas biodegradáveis de PLGA contendo Interleucina-2 pelo método de emulsificação/evaporação do solvente com propriedades controladas como tamanho de partícula, eficiência de encapsulação e cinética de liberação in vitro, para posterior ensaio pré-clínico. Investigou-se o efeito de variáveis do processo como o efeito do agente emulsificante, o tipo de solvente e a velocidade de agitação, encapsulando-se albumina de soro bovina (BSA), como proteína modelo, em microesferas de PLGA. Neste estudo, foram definidas as condições de processo e formulação para a obtenção de microesferas de PLGA contendo IL-2, onde também se explorou o efeito da presença de agentes estabilizantes como polietilenoglicol e BSA na encapsulação de IL-2. Inovando no processo de produção de micropartículas poliméricas, o uso de micromisturadores desenvolvidos em LTCC (Cerâmica com baixa temperatura de sinterização) foi explorado como alternativa tecnológica ao processo convencional que envolve agitação mecânica na etapa de emulsificação. Microesferas de PLGA foram produzidas utilizando diferentes tipos de dispositivos microfluídicos e os resultados obtidos mostraram a viabilidade técnica desta inovação tecnológica, gerando microesferas contendo IL-2, com eficiência de encapsulação e tamanho de partículas similares às microesferas obtidas pelo processo convencional (eficiência de encapsulação da ordem de 80%, partículas menores que 40 μm). As microesferas de PLGA contendo IL-2 foram desenvolvidas na tentativa de se estabelecer um perfil cinético de liberação de IL-2 que possa solucionar problemas relacionados à aplicação intra-tumoral da IL-2 livre, visando o desenvolvimento de terapias biológicas para câncer avançado de cabeça e pescoço. O estudo pré-clínico das microesferas de PLGA contendo IL-2 foi realizado em dois modelos experimentais de tumor, Melanoma B16F10 e Carcinoma de Erlich. Os resultados obtidos no estudo préclínico realizado com os dois modelos tumorais coloca em evidência a importância de ajuste dos modelos experimentais utilizados no desenvolvimento de terapias biológicas de câncer usando sistemas de liberação controlada, onde a velocidade de evolução do tumor e o tempo de duração de ensaios são decisivos para o desenvolvimento da resposta biológica desejada