Este estudo avaliou a influência da fototerapia a laser (FTL) na proliferação e diferenciação de células-tronco da polpa dentária humana (DPSCs; do inglês, Dental Pulp Stem Cells ) encapsuladas em carreador injetável e termoresponsivo (PL; Pluronic® F-127, Sigma-Aldrich, MO, EUA) com incorporação de proteína morfogenética óssea 4 recombinante humana (rhBMP4) (sistema PL/rhBMP4). O biomaterial foi caracterizado de acordo com seus perfis de embebição e dissolução, liberação de rhBMP4 e sua estrutura morfológica. DPSCs foram isoladas, caracterizadas e encapsuladas em PL para confirmar sua viabilidade e seu potencial de diferenciação (adipo e osteogênico) em comparação com células-tronco mesenquimais de medula óssea (BMMSCs; do inglês, Bone Marrow Mesenchymal Stem Cells). Quando encapsuladas no sistema PL/rhBMP4, DPSCs foram irradiadas com duas densidades de energia diferentes utilizando laser de diodo de fosfeto de índio-gálio-alumínio (InGaAlP), modos contínuo, pontual e em contato [660 nm, 0,028 cm2, 20 mW, 0,71 W/cm2, 3 J/cm2 (4 s) ou 5 J/cm2 (7 s)]. Os ensaios de PKH26 (do inglês, Red Fluorescent Cell Linker), CFU-F (do inglês, Coloning Forming Units - Fibroblastic), e MTT (do inglês, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide)) foram utilizados para avaliar adesão/proliferação, diferenças na capacidade formadora de colônias e viabilidade das DPSCs (neste último caso sob estresse nutricional), respectivamente. Finalmente, a diferenciação odonto/osteogênica foi analisada por qRT-PCR e confirmada por ensaio de vermelho de alizarina. O biomaterial embebeu e dissolveu rapidamente; densa rede tubular e reticular com poros interconectados foi observada. DPSCs e BMMSCs apresentaram alta viabilidade celular quando encapsuladas em PL. Ambas as linhagens celulares tiveram êxito em se diferenciar em tecidos adiposo e ósseo. De acordo com o PKH26, DPSCs puderam aderir e proliferar no sistema PL/rhBMP4. DPSCs irradiadas encapsuladas tanto em PL como em PL/rhBMP4 formaram mais CFU-F que os controles não irradiados. Sob estresse nutricional, DPSCs semeadas no PL e irradiadas com 5 J/cm2 exibiram maior taxa de viabilidade celular em relação aos grupos não irradiados e irradiados com 3 J/cm2. Na presença de rhBMP4, os grupos irradiados tanto com 3 J/cm2 quanto com 5 J/cm2 apresentaram deposição mineral precoce quando comparados aos grupos não irradiados. Ainda, após 21 dias de diferenciação odonto/osteogênica, DPSCs irradiadas produziram maior quantidade de nódulos mineralizados. A irradiação com 5 J/cm2 levou ao aumento significativo da expressão de genes envolvidos na diferenciação odonto/osteogênica, como colágeno tipo I (COL1A1), osteocalcina (OCN), proteína da matriz dentinária 1 (DMP1), sialofosfoproteina dentinária (DSPP) e proteína heat shock 27 kDa (HSPB1). A associação entre rhBMP4 e FTL promove proliferação e diferenciação odonto/osteogênica de DPSCs acelerando e aumentando notavelmente a formação de tecido mineralizado, em especial quando a densidade de energia de 5 J/cm2 é aplicada. / This study evaluated the influence of laser phototherapy (LPT) on dental pulp stem cells (DPSCs) proliferation and differentiation upon encapsulation in an injectable and thermo-responsive cell carrier (PL; Pluronic® F-127, Sigma-Aldrich, MO, USA) loaded with human recombinant bone morphogenetic protein 4 (rhBMP4)(PL/rhBMP4 system). The biomaterial was characterized according to its swelling and dissolution profiles, release of rhBMP4 and morphological structure. DPSCs were isolated, characterized and encapsulated in PL to confirm their viability and multilineage differentiation potential (adipo and osteogenic) in comparison to bone marrow mesenchymal stem cells (BMMSCs). When encapsulated in the PL/rhBMP4 system, DPSCs were irradiated with two different energy densities using a continuous-wave indium-gallium-aluminum-phosphide (InGaAlP) diode laser [660 nm, 0.028 cm2, 20 mW, 0.71 W/cm2, 3 J/cm2 (4 s) or 5 J/cm2 (7 s)] in punctual and contact modes. The PKH26 (Red Fluorescent Cell Linker), the CFU-F (Coloning Forming Units - Fibroblastic), and the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide] assays were used to assess differences in cell adhesion/proliferation, colony forming units formation ability, and cell viability of DPSCs (in this case under nutritional stress), respectively. Then, alizarin red and qRT-PCR analyzes were used to evaluate odonto/osteogenic differentiation. The biomaterial swelled and dissolved rapidly; dense tubular and reticular network morphology with well-interconnected pores was observed. DPSCs and BMMSCs presented high cell viability when encapsulated in PL. Both cell lineages successfully differentiated into bone or adipose tissues. According to PKH26, DPSCs were able to adhere and proliferate in the PL/rhBMP4 system. Irradiated DPSCs encapsulated in either PL or PL/rhBMP4 system formed more CFU-F than non-irradiated controls. Under nutritional stress, DPSCs encapsulated in the hydrogels with no rhBMP4 and irradiated at 5 J/cm2 exhibited higher cell viability than the other groups. In the presence of rhBMP4, the groups irradiated both at 3 and 5 J/cm2 energy densities displayed earlier mineral deposition than the non-irradiated groups. Moreover, after 21 days of odonto/osteogenic differentiation, irradiated DPSCs produced greater nodule formation than the control groups. At the energy density of 5 J/cm2, there were significant upregulation of genes involved in odonto/osteoblast differentiation, such as type I collagen (COL1A1), osteocalcin (OCN), dentin matrix protein 1 (DMP1), dentin sialophosphoprotein (DSPP) and heat shock protein 27 kDa (HSPB1). The association between rhBMP4 and LPT promotes cell proliferation and odonto/osteogenic differentiation of DPSCs accelerating and increasing the formation of mineralized tissue, in particular when the energy density of 5 J/cm2 is applied.

Made available in DSpace on 2014-12-17T14:16:37Z (GMT). No. of bitstreams: 1 EdileneGO_DISSERT.pdf: 1950686 bytes, checksum: 88f16924cd116b850ade306c274f71ac (MD5) Previous issue date: 2014-12-20 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior / New drug delivery systems have been used to increase chemotherapy efficacy due the possible drug resistance of cancer cells. Poly (lactic acid) (PLA) microparticles are able to reduce toxicity and prolong methotrexate (MTX) release. In addition, the use of PLA/poloxamer polymer blends can improve drug release due to changes in the interaction of particles with biological surfaces. The aim of this study was developing spray dried biodegradable MTX-loaded microparticles and evaluate PLA interactions with different kinds of Pluronic? (PLUF127 and PLUF68) in order to modulate drug release. The variables included different drug:polymer (1:10, 1:4.5, 1:3) and polymer:copolymer ratios (25:75, 50:50, 75:25). The precision and accuracy of spray drying method was confirmed assessing drug loading into particles (75.0- 101.3%). The MTX/PLA microparticles showed spherical shape with an apparently smooth surface, which was dependent on the PLU ratio used into blends particles. XRD and thermal analysis demonstrated that the drug was homogeneously dispersed into polymer matrix, whereas the miscibility among components was dependent on the used polymer:copolymer ratio. No new drug- polymer bond was identified by FTIR analysis. The in vitro performance of MTX-loaded PLA microparticles demonstrated an extended-release profile fitted using Korsmeyer- Peppas kinetic model. The PLU accelerated drug release rate possible due PLU leached in the matrix. Nevertheless, drug release studies carried out in cell culture demonstrated the ability of PLU modulating drug release from blend microparticles. This effect was confirmed by cytotoxicity observed according to the amount of drug released as a function of time. Thus, studied PLU was able to improve the performance of spray dried MTX-loaded PLA microparticles, which can be successfully used as carries for modulated drug delivery with potential in vivo application / Novos sistemas de libera??o de f?rmacos v?m sendo utilizados para aumentar a efic?cia de quimioter?picos devido ? poss?vel resist?ncia de c?lulas cancer?genas. As micropart?culas de poli (?cido l?ctico) (PLA) constituem uma alternativa para diminuir a toxicidade e prolongar a libera??o do metotrexato (MTX). Al?m disso, o uso de blendas polim?ricas PLA-polox?meros pode melhorar o perfil de libera??o do f?rmaco devido a mudan?as nas intera??es das part?culas com superf?cies biol?gicas. O objetivo do estudo foi desenvolver micropart?culas biodegrad?veis de MTX produzidas por spray drying e avaliar intera??es PLA-Pluronic? (PLA-PLU) para modular a libera??o do f?rmaco, utilizando diferentes tipos de Pluronic? (PLUF127 e PLUF68). As vari?veis de composi??o inclu?ram raz?es f?rmaco:pol?mero (1:10; 1:4,5; 1:3) e pol?mero:copol?mero (25:75, 50:50, 75:25). A reprodutibilidade e a efic?cia do m?todo de produ??o foram confirmadas pela alta efici?ncia de incorpora??o dos sistemas (75,0-101,3%). As micropart?culas de MTX/PLA apresentaram-se esf?ricas com superf?cie aparentemente lisa. Este formato mostrou-se dependente da raz?o pol?mero:copol?mero nas part?culas contendo blendas. A an?lise t?rmica e a difra??o de raios-X sugerem que h? dispers?o do f?rmaco por toda a matriz, enquanto que a miscibilidade entre os componentes foi dependente da raz?o pol?mero:copol?mero. Nenhuma liga??o qu?mica entre o f?rmaco e o pol?mero foi identificada pela an?lise de FTIR. As micropart?culas de PLA contendo MTX apresentaram perfil de libera??o prolongada com um prevalente modelo cin?tico de Korsmeyer-Peppas. O PLU acelerou a taxa de libera??o do f?rmaco devido a sua poss?vel sa?da da matriz polim?rica. Por outro lado, estudos de libera??o do f?rmaco realizados em cultura de c?lulas demonstraram que o PLU modula a taxa de MTX liberado a partir de micropart?culas contendo blendas. Este efeito foi confirmado pela citotoxicidade dos sistemas estudados, de acordo com a quantidade de f?rmaco liberado em fun??o do tempo. Portanto, o uso de PLU foi capaz de melhorar o perfil de libera??o de micropart?culas de PLA contendo MTX, o qual pode ser utilizado como carreador para modular a libera??o do f?rmaco com potencial aplica??o in vivo

Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Rutin is a flavonoid widely reported in the literature for its antioxidant properties, anti-inflammatory, vasoprotective, antithrombogen, among others. However, its low solubility in aqueous media reduces its bioavailability orally and, therefore, transdermal administration proves to be promising. Thus, the present work aimed to obtain, characterize and evaluate an activity and antioxidant activity of a gel. PLO (Pluronic Lecithin Organogel) containing rutin for transdermal administration.The analytical methodology for rutin quantification was developed and validated by high performance liquid chromatography (HPLC). Initially it was evaluated as an influence of the concentration of Polaxamer 407, obtained by the extrusion method with the aid of syringes, in front of the centrifugation test. The formulation that remained stable after the test was characterized by partical size determination, in vitro release study, in vitro skin penetration study, in vitro skin adhesion study. Also assessed for stability for 60 days at varying temperatures and at predetermined times, physico-chemical characteristics such as pH, density, viscosity and spreadability, as well as organoleptic characteristics, were evaluated. The antioxidant activity of the formulation was evaluated by the TRAP and TAR methods, comparing it with a positive control, Trolox. The results demonstrate that a formulation with higher concentration of Polaxamer is more stable and that is why it was characterized. The partical size were perfect for dermal administration. The formulation demonstrated controlled release of the drug after 24 hours, being able to permeate as deeper layers of the skin and to be absorbed into the systemic circulation, in addition to good adhesion to the skin surface. During the accelerated stability study, as formulations stored at low temperature, they underwent small variations in density, viscosity and spreadability relative to those stored at room temperature, while the pH remained stable throughout and favorable for application in skin. However, as observed variations were not sufficient to cause visual signs of instability. As for the antioxidant activity, a formulation showed greater activity in relation to the Trolox control and the free rutin, but it was not able to sustain an activity for a longer time, presenting a lower TAR value than Trolox. Thus, a chosen formulation has been shown capable of promoting a permeation of the rutin by transdermal route in a controlled manner, as well as being stable at an ambient temperature and having more significant antioxidant activity than the free rutin, and is therefore promising to administration of rutin by this route. / A rutina é um flavonoide bastante estudado devido principalmente as suas propriedades antioxidantes, anti-inflamatória, vasoprotetora e antitrombogência. No entanto, sua baixa solubilidade em meio aquoso reduz sua biodisponibilidade por via oral e, portanto, a administração por via transdérmica pode ser uma alternativa promissora. Dessa forma, o presente trabalho teve por objetivo obter, caracterizar e avaliar a estabilidade e a atividade antioxidante de um gel contendo Pluronic Lecithin Organogel (PLO) contendo rutina, para administração pela via transdérmica. A metodologia analítica para quantificação de rutina foi desenvolvida e validada por cromatografia líquida de alta eficiência (CLAE). Inicialmente foi avaliado qual a influência da concentração do Polaxamer 407, obtidos pelo método de extrusão com auxílio de seringas, frente ao teste de centrifugação. A formulação que permaneceu estável após o teste foi caracterizada através da determinação do tamanho de partícula, estudo de liberação, penetração cutânea e adesão à pele, todos in vitro. Foi também avaliada a estabilidade do produto durante 60 dias em variadas temperaturas em tempos pré-determinados, além das análises físico-químicas, como pH, densidade, viscosidade e espalhabilidade, bem como características organolépticas. A atividade antioxidante da formulação foi determinada pelos métodos TRAP e TAR, em comparação com um controle positivo, o Trolox. Os resultados demonstraram que a formulação contendo maior concentração de Polaxamer é mais estável e por isso esta foi caracterizada. O tamanho de partícula encontrado foi 4,33 μm e o sistema se mostrou homogêneo, ideal para administração cutânea. A formulação demonstrou liberação controlada do fármaco após 24 horas, sendo capaz de permear as camadas mais profundas da pele e ser absorvida para circulação sistêmica, além de boa adesão à superfície da pele. Durante o estudo de estabilidade acelerada, as formulações armazenadas em baixa e alta temperatura, sofreram pequenas variações na densidade, na viscosidade e na espalhabilidade, em relação àquelas armazenadas a temperatura ambiente, enquanto que o pH se manteve estável durante todo o tempo e favorável à sua aplicação na pele. Além disso, as variações observadas não foram suficientes para provocar alterações visuais de instabilidade. Quanto à atividade antioxidante, a formulação demonstrou maior atividade em relação ao controle Trolox e à rutina livre, porém de forma não duradoura, apresentando valor de TAR menor que o Trolox. Sendo assim, a formulação escolhida, demonstrou-se capaz de promover a permeação da rutina por via transdérmica de forma controlada, bem como estabilidade a temperatura ambiente e com atividade antioxidante significativa, sendo considerada então promissora a administração da rutina por esta via. / São Cristóvão, SE

Conselho Nacional de Desenvolvimento Científico e Tecnológico / In this work, gold nanoparticles (AuNPs) and nanoclusters (AuNCLs) were prepared by the reduction in diluted solution method, using the amphiphilic copolymer Pluronic F127(PF127) and thiolated Pluronic F127 (PF127-thiol), respectively. The copolymer was functionalized by sterification reaction using thioglycolic acid (TGA). The investigated parameters of synthesis were concentration of the PF127 solution, temperature, as well as, the UV irradiation. The nanosystems were characterized by UV/Vis absorption spectroscopy and transmission electron microscopy (TEM). Further, the system potentialities were investigated by a catalized reaction and incorporation of a model molecule (baicalein). Morphologies, sizes, polydispersity, amount of reduced ion as welll as kinetics parameters were depedent on the parameters of synthesis. The yeld of the functionalization reaction was 90%, calculated by 1H NMR. Using the PF127-thiol, the surface plasmonic ressonance band (SPR) was undetected and a strong absorption in the UV region observed, which indicated that nanoparticles smaller than 3 nm were obtained (AuNCLs). Catalitic property of the AuNPs were investigated by reduction of p-nitrophenol, in which the AuNPs synthesised by PF127 were more efficent than the systems obtained by citrate reduction. The encapsulation efficiency of the baicalein were around 90%. In conclusion, the synthesis of AuNPs by PF127 is not only adequate to modulate sizes and morphology but also to provide environmental advantage in comparison to some tradicional methods. In addition, these are promissing systems to biologic applications because of the biocompatibility of PF127. / Neste trabalho, nanopartículas e nanoclusters de ouro (AuNPs e AuNCLs, respectivamente) foram obtidos pelo método de redução em solução diluída, empregando o polímero anfifílico Pluronic F 127 não funcionalizado (PF127) e funcionalizado (PF 127-Tiol) como agente redutor/estabilizador, respectivamente. A funcionalização do polímero foi realizada por reação de esterificação com ácido tioglicólico (TGA), inserindo-se grupos tióis terminais. A concentração de PF127 e temperatura de obtenção foram parâmetros variados nos processos, além da incidência ou não de radiação UV. A caracterização dos nanosistemas foi realizada por espectroscopia de absorção no UV-Vis e por microscopia eletrônica de transmissão (MET). Os sistemas foram testados quanto à atividade catalítica, na redução do p-nitrofenol em borohidreto de sódio e na incorporação de uma molécula modelo, a baicaleína. As diferentes condições de síntese influíram sobre a morfologia, tamanho, quantidade, polidispersão e cinética de formação das nanopartículas. O rendimento da reação de funcionalização, estimado por 1H RMN, foi de 90%. O produto obtido (PF127-Tiol) foi empregado na redução do Au3+ e, nanopartículas menores que 3 nm foram obtidas, nesse caso, AuNCLs, evidenciados pelo desaparecimento da banda SPR e surgimento de uma forte absorção no UV, além da observação por MET. Os sistemas a partir do PF127 foram investigados quanto a aplicação em catálise, na qual as AuNPs preparados utilizando Pluronic foi mais eficiente do que as obtidas por redução por citrato. Na investigação da incorporação de uma molécula modelo, as eficiências de encapsulamento foram superiores a 90%. Dessa forma, esses sistemas mostraram-se promissores para uma diversa gama de aplicações de nanopartículas ou clusters de ouro, tais como na catálise de reações químicas e encapsulamamento de bioativos. A síntese por PF127 mostrou-se uma rota ambientalmente amigável e capaz de modulação nos tamanhos e formas das AuNPs,.Os sistemas ainda são promissores para uso em sistemas biológicos, tendo em vista a biocompatibilidade do PF127.

Este estudo avaliou a influência da fototerapia a laser (FTL) na proliferação e diferenciação de células-tronco da polpa dentária humana (DPSCs; do inglês, Dental Pulp Stem Cells ) encapsuladas em carreador injetável e termoresponsivo (PL; Pluronic® F-127, Sigma-Aldrich, MO, EUA) com incorporação de proteína morfogenética óssea 4 recombinante humana (rhBMP4) (sistema PL/rhBMP4). O biomaterial foi caracterizado de acordo com seus perfis de embebição e dissolução, liberação de rhBMP4 e sua estrutura morfológica. DPSCs foram isoladas, caracterizadas e encapsuladas em PL para confirmar sua viabilidade e seu potencial de diferenciação (adipo e osteogênico) em comparação com células-tronco mesenquimais de medula óssea (BMMSCs; do inglês, Bone Marrow Mesenchymal Stem Cells). Quando encapsuladas no sistema PL/rhBMP4, DPSCs foram irradiadas com duas densidades de energia diferentes utilizando laser de diodo de fosfeto de índio-gálio-alumínio (InGaAlP), modos contínuo, pontual e em contato [660 nm, 0,028 cm2, 20 mW, 0,71 W/cm2, 3 J/cm2 (4 s) ou 5 J/cm2 (7 s)]. Os ensaios de PKH26 (do inglês, Red Fluorescent Cell Linker), CFU-F (do inglês, Coloning Forming Units - Fibroblastic), e MTT (do inglês, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide)) foram utilizados para avaliar adesão/proliferação, diferenças na capacidade formadora de colônias e viabilidade das DPSCs (neste último caso sob estresse nutricional), respectivamente. Finalmente, a diferenciação odonto/osteogênica foi analisada por qRT-PCR e confirmada por ensaio de vermelho de alizarina. O biomaterial embebeu e dissolveu rapidamente; densa rede tubular e reticular com poros interconectados foi observada. DPSCs e BMMSCs apresentaram alta viabilidade celular quando encapsuladas em PL. Ambas as linhagens celulares tiveram êxito em se diferenciar em tecidos adiposo e ósseo. De acordo com o PKH26, DPSCs puderam aderir e proliferar no sistema PL/rhBMP4. DPSCs irradiadas encapsuladas tanto em PL como em PL/rhBMP4 formaram mais CFU-F que os controles não irradiados. Sob estresse nutricional, DPSCs semeadas no PL e irradiadas com 5 J/cm2 exibiram maior taxa de viabilidade celular em relação aos grupos não irradiados e irradiados com 3 J/cm2. Na presença de rhBMP4, os grupos irradiados tanto com 3 J/cm2 quanto com 5 J/cm2 apresentaram deposição mineral precoce quando comparados aos grupos não irradiados. Ainda, após 21 dias de diferenciação odonto/osteogênica, DPSCs irradiadas produziram maior quantidade de nódulos mineralizados. A irradiação com 5 J/cm2 levou ao aumento significativo da expressão de genes envolvidos na diferenciação odonto/osteogênica, como colágeno tipo I (COL1A1), osteocalcina (OCN), proteína da matriz dentinária 1 (DMP1), sialofosfoproteina dentinária (DSPP) e proteína heat shock 27 kDa (HSPB1). A associação entre rhBMP4 e FTL promove proliferação e diferenciação odonto/osteogênica de DPSCs acelerando e aumentando notavelmente a formação de tecido mineralizado, em especial quando a densidade de energia de 5 J/cm2 é aplicada. / This study evaluated the influence of laser phototherapy (LPT) on dental pulp stem cells (DPSCs) proliferation and differentiation upon encapsulation in an injectable and thermo-responsive cell carrier (PL; Pluronic® F-127, Sigma-Aldrich, MO, USA) loaded with human recombinant bone morphogenetic protein 4 (rhBMP4)(PL/rhBMP4 system). The biomaterial was characterized according to its swelling and dissolution profiles, release of rhBMP4 and morphological structure. DPSCs were isolated, characterized and encapsulated in PL to confirm their viability and multilineage differentiation potential (adipo and osteogenic) in comparison to bone marrow mesenchymal stem cells (BMMSCs). When encapsulated in the PL/rhBMP4 system, DPSCs were irradiated with two different energy densities using a continuous-wave indium-gallium-aluminum-phosphide (InGaAlP) diode laser [660 nm, 0.028 cm2, 20 mW, 0.71 W/cm2, 3 J/cm2 (4 s) or 5 J/cm2 (7 s)] in punctual and contact modes. The PKH26 (Red Fluorescent Cell Linker), the CFU-F (Coloning Forming Units - Fibroblastic), and the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide] assays were used to assess differences in cell adhesion/proliferation, colony forming units formation ability, and cell viability of DPSCs (in this case under nutritional stress), respectively. Then, alizarin red and qRT-PCR analyzes were used to evaluate odonto/osteogenic differentiation. The biomaterial swelled and dissolved rapidly; dense tubular and reticular network morphology with well-interconnected pores was observed. DPSCs and BMMSCs presented high cell viability when encapsulated in PL. Both cell lineages successfully differentiated into bone or adipose tissues. According to PKH26, DPSCs were able to adhere and proliferate in the PL/rhBMP4 system. Irradiated DPSCs encapsulated in either PL or PL/rhBMP4 system formed more CFU-F than non-irradiated controls. Under nutritional stress, DPSCs encapsulated in the hydrogels with no rhBMP4 and irradiated at 5 J/cm2 exhibited higher cell viability than the other groups. In the presence of rhBMP4, the groups irradiated both at 3 and 5 J/cm2 energy densities displayed earlier mineral deposition than the non-irradiated groups. Moreover, after 21 days of odonto/osteogenic differentiation, irradiated DPSCs produced greater nodule formation than the control groups. At the energy density of 5 J/cm2, there were significant upregulation of genes involved in odonto/osteoblast differentiation, such as type I collagen (COL1A1), osteocalcin (OCN), dentin matrix protein 1 (DMP1), dentin sialophosphoprotein (DSPP) and heat shock protein 27 kDa (HSPB1). The association between rhBMP4 and LPT promotes cell proliferation and odonto/osteogenic differentiation of DPSCs accelerating and increasing the formation of mineralized tissue, in particular when the energy density of 5 J/cm2 is applied.

Inkjet printing (jetting) technology, due to its high speed of operation and accuracy, is utilised in Additive Manufacturing (AM) of three dimensional parts. Commercially available AM processes that use jetting technology include three dimensional printing (3DP by Z-Corporation), Polyjet (by Objet), Multi Jet Modelling (MJM by 3D Systems) and three dimensional printing by Solidscape. Apart from 3D Printing by Z-corporation, all the other jetting based processes require a support material to successfully build a part. The support material provides a base to facilitate the removal of the part from the build platform and it helps manufacturing of cavities, holes and overhanging features. These support materials present challenges in terms of their removability and reusability. This research is therefore, aimed towards finding a support material composition that can be used with jetting based AM processes. The support material should be easily removable either by melting or by dissolution and also, if possible, it should be reusable. AM processes often process materials with poor mechanical properties and therefore, the parts produced by these processes have limited functionality. In an attempt to obtain complex shaped, functional parts made of nylon (i.e. Polyamide 6), a new jetting based AM process is under research at Loughborough University. The process uses two different mixtures of caprolactam (i.e. the monomer used to produce polyamide). These mixtures are to be jetted using inkjet heads and subsequently polymerised into polyamide 6. Therefore, another aim of this research was to consider the support material s suitability for jetting of caprolactam. Two different polymers were researched which included Pluronic F-127 and methylcellulose (MC). Both these polymers are known for gel formation upon heating in aqueous solutions. Due to the inhibition of polymerisation of polyamide 6 by the presence of water, non-aqueous solvents such as ethylene glycol, propylene glycol and butylene glycol were studied. Since both F-127 and MC in the glycols mentioned above had not been studied before, all the compositions prepared and investigated in this report were novel. F-127 did not show gel formation in propylene and butylene glycol but formed a gel in ethylene glycol at a concentration of 25% (w/w) F-127. MC, on the other hand, showed gel formation upon cooling in all the three glycols at concentrations as low as 5% for ethylene glycol and 1% for both propylene and butylene glycol. These compositions were characterized using experimental techniques such as Fourier Transform Infrared (FTIR) spectroscopy, hot stage microscopy, differential scanning calorimetry (DSC) and X-ray diffraction (XRD). A mechanism of gelation for both F-127 and MC in glycols is presented based on the results of these characterisation techniques. Viscosity and surface tension measurements along with the texture analysis of selected compositions were also performed to evaluate their suitability for jetting. All these compositions, due to their water solubility and/or low melting temperatures (i.e. near 500C) present the advantage of ease of removal. Removal by melting at low temperatures can also provide reusability of these support materials and thus advantages such as reduction in build cost and environmental effect can be achieved.

Among the great breakthroughs in nanoscience and nanotechnology is the emergence of synthetic polymers that demonstrate biological activity and thus can be exploited for biomedical applications, extending from agents in therapeutics to drug delivery and tissue engineering. A key factor in the fabrication of such polymeric materials is the ability to tune and control their properties. To this end, an insight into the mode of interactions with biological systems is imperative. Computer simulations have proved to be a valuable tool that can compliment experiments and provide -otherwise inaccessible- information. In the context of this thesis, different aspects of the polymeric biological activity were investigated by studying two polymeric materials suitable for different types of applications, aiming to clarify yet undisclosed mechanisms that govern the polymers' behaviour either in solution or in conjunction with model lipid membranes. The first part of the thesis is dedicated to a nonionic amphiphilic copolymer known as Pluronic L64 that is considered as a candidate for the design of novel hybrid polymer-lipid vesicles that will act as carriers for drugs or genes. The hybrid bilayers are subjected to mechanical stress and their properties are compared to those of pure lipid bilayers. The simulations showed that the hybrid membranes can sustain increased surface tension prior to rupture, are stiffer, thicker and the polymers can induce higher lipid tail packing and also reduce the lipid mobility, rendering the membranes more ordered and less fluid. At high values of lateral pressure, which leads to pore formation, the copolymer chains decelerate the pore growth. The examination of the defect formation mechanism reveals that the hydrophilic PEO segment plays the most vital role. The same systems were also observed in varying temperatures and the impact of the inserted polymers on the phase behaviour was investigated. The data suggested that the polymers change the nature of the phase transition from a discontinuous to a continuous one. The hybrid membranes transform between the ordered and the disordered phase in a continuous manner and not at a critical melting temperature. Interestingly, the effect of polymers is different at the low and high temperature regions, as proved by the analysis of the mechanical, structural and dynamic membrane properties. The second part is focused on the study of polyhexamethylene biguanide (PHMB), a biguanide-based polyelectrolyte, that possesses remarkable biocidal properties. Even though PHMB's activity is known, the specific mode of action against bacterial membranes is still puzzling. Our work revealed that the polyelectrolyte assumes a counterintuitive behaviour in aqueous solution tending to self-organise into ordered compact structures, despite the repulsive electrostatic interactions of its positively charged segments. The formed nano-objects are thermodynamically stable, as was confirmed by free energy calculations and could be linked to PHMB's antibacterial mechanism. These findings pave the way for further computational and experimental exploration of these fascinating and promising materials that could lead to the design of novel smart biologically active nanoparticles.

Facile, one-step synthesis of self-assembling, cationic block copolymers of poly(2-N-(dimethylaminoethyl) methacrylate) (pDMAEMA) and PEO-PPO-PEO (Pluronic®) is developed. The copolymers are obtained via free-radical polymerization of DMAEMA initiated by Pluronic-radicals generated by cerium (IV). The copolymers possess surface activity, are polycationic at pH<7.1, and self-assemble into micelle-like aggregates when neutralized. Potential applications of the novel copolymers for DNA transfection in gene therapy are discussed. / Singapore-MIT Alliance (SMA)

Poly(acrylic acid) (PAA) was grafted onto both termini of Pluronic F87 (PEO₆₇-PPO₃₉-PEO₆₇) via atom transfer radical polymerization to produce a novel muco-adhesive block copolymer PAA₈₀-b-F₈₇-b-PAA₈₀. It was observed that PAA₈₀-F₈₇-PAA₈₀ forms stable complexes with weakly basic anti-cancer drug, Doxorubicin. Thermodynamic changes due to the drug binding to the copolymer were assessed at different pH by isothermal titration calorimetry (ITC). The formation of the polymer/drug complexes was studied by turbidimetric titration and dynamic light scattering. Doxorubicin and PAA-b-F87-b-PAA block copolymer are found to interact strongly in aqueous solution via non-covalent interactions over a wide pH range. At pH>4.35, drug binding is due to electrostatic interactions. Hydrogen-bond also plays a role in the stabilization of the PAA₈₀-F₈₇-PAA₈₀/DOX complex. At pH 7.4 (α=0.8), the size and stability of polymer/drug complex depend strongly on the doxorubicin concentration. When CDOX <0.13mM, the PAA₈₀-F₈₇-PAA₈₀ copolymer forms stable inter-chain complexes with DOX (110 ~ 150 nm). When CDOX >0.13mM, as suggested by the light scattering result, the reorganization of the polymer/drug complex is believed to occur. With further addition of DOX (CDOX >0.34mM), sharp increase in the turbidity indicates the formation of large aggregates, followed by phase separation. The onset of a sharp enthalpy increase corresponds to the formation of a stoichiometric complex. / Singapore-MIT Alliance (SMA)

<p>Multifunctional drug delivery systems incorporated with stimuli-sensitive drug release, magnetic nanoparticles and magnetic resonance (MR) <em>T</em>₂ contrast agents is attracting increasing attention recently. In this thesis, works on polymer nanospheres response to temperature change, superparamagnetic iron oxide nanoparticles (SPION)/polymeric composite materials for MR imaging contrast agents are summarized.</p><p>A “shell-in-shell” polymeric structure has been constructed through a “modified double-emulsion method”. Thermosensitive inner shell is comprised of poly(<em>N</em>-isopropylacrylamide) which undergoes phase transition at body temperature. Such a feature could facilitate drug release at an elevated temperature upon administration. Furthermore, the dual-shell structure is covered by a layer of gold nanoparticles. According to the cytotoxicity tests, the biocompatibility is shown to be enhanced due to the layer of gold.</p><p>SPION have been prepared using a high temperature decomposition method. Particle growth of SPION is monitored by transmission electron microscope and synchrotron X-ray diffraction. Poly(L,L-lactide)@SPION (PLLA@SPION) composite particles have been prepared through surface-initiated ring-opening polymerization which has been developed in our lab. For biomedical applications, it is essential to transfer the particles to physiological solutions from organic solutions. Phase transfer of SPION has been carried out by utilizing small molecules. Stability at the neutral pH is of large concern for such transfer systems. A novel phase transfer agent, Pluronic F127 (PF127), a triblock copolymer has been applied and the stability of the aqueous PF127@oleic acid (OA)@SPION solution has been greatly enhanced over a broad pH range. Most interestingly, PF127@OA@SPION show remarkable efficacy as T2 contrast agents as indicated by relaxometric measurements compared with commercially available products.</p>