Iron oxide nanoparticles are highly researched for their use in biomedical applications such as drug delivery, diagnosis, and therapy. The inherent biodegradable and biocompatible nanoparticle properties make them highly advantageous in nanomedicine. The magnetic properties of iron oxide nanoparticles make them promising candidates for magnetic fluid hyperthermia applications. Designing an efficient iron oxide nanoparticle for hyperthermia requires synthetic, surface functionalization, stability, and biological investigations. This research focused on the following three areas: optimizing synthesis conditions for maximum radiofrequency induced magnetic hyperthermia, designing a simple and modifiable surface functionalization method for specific or broad biological stability, and in vitro and in vivo testing of surface functionalized iron oxide nanoparticles in delivering effective hyperthermia or radiotherapy. The benzyl alcohol modified seed growth method of synthesizing iron oxide nanoparticles using iron acetylacetonate as an iron precursor was investigated to identify significant nanoparticle properties that effect radiofrequency induced magnetic hyperthermia. Investigation of this synthesis under atmospheric conditions revealed a combination of thermal decomposition and oxidation-reduction mechanisms that can produce nanoparticles with larger crystallite sizes and decreased size distributions. Nanoparticles were easily surface functionalized with (3-Glycidyloxypropyl)trimethoxysilane (GLYMO) without the need for organic-aqueous phase transfer methods. The epoxy ring on GLYMO facilitated post-modifications via a base catalyzed epoxy ring opening to obtain nanoparticles with different terminal groups. Glycine, serine, γ-aminobutryic acid (ABA), (S)-(-)-4-amino-2-hydroxybutyric acid (SAHBA), ethylenediamine, and tetraethylenepentamine were successful in modifying GLYMO coated-iron oxide nanoparticles to provide colloidal and varying biological stability while also allowing for further conjugation of chemotherapeutics or radiotherapeutics. The colloidal stability of cationic and anionic nanoparticles in several biologically relevant media was studied to address claims of increased cellular uptake for cationic nanoparticles. The surface functionalized iron oxide nanoparticles were investigated to determine effects on cellular uptake and viability. In vitro tests were used to confirm the ability of iron oxide nanoparticles to provide effective hyperthermia treatment. S-2-(4-Aminobenzyl)-1,4,7,10-tetraazacyclododecane tetraacetic acid (DOTA) was coupled to SAHBA and carboxymethylated polyvinyl alcohol surface functionalized iron oxide nanoparticles and radiolabeled with 177Lu. The capability of radiolabeled iron oxide nanoparticles for delivering radiation therapy to a U87MG murine orthotopic xenograft model of glioblastoma was initially investigated.

Essa tese de doutoramento utiliza a auto-organização dos filmes finos layer-by-layer (LbL), auto-organização por alcanotióis mistas (SAMmix) e monocamada de Langmuir no desenvolvimento de dispositivos e novas metodologias para aplicações em nanomedicina. Foram desenvolvidos e aplicados biossensores utilizando as técnicas de LbL e SAM. Dentre os biossensores construídos está o sensor para óxido nítrico (NO•), que é de grande importância no sistema fisiológico. O sensor foi construído por meio da modificação de ultramicroeletrodos de fibra de carbono pela técnica LbL. A caracterização do sensor foi realizada por voltametrias e espectroscopias de impedância eletroquímica. Os resultados revelaram que a difusão de NO• é dependente do número de bicamadas empregadas e da disposição das moléculas no filme. O sensor com arquitetura CF-(PAMAM/NiTsPc), fibra de carbono (CF), ftalocianina de níquel tetrasulfonada (NiTsPc) e dendrímero poliamidoamina (PAMAM), apresentou o melhor sinal analítico. Além disso, foi analisada a detecção de NO• com interferentes como nitrito, nitrato, peróxido de hidrogênio, ácido ascórbico, dopamina, epinefrina e a norepinefrina. Os resultados mostraram alta seletividade devido à utilização do dendrímero PAMAM. O segundo biossensor utilizou a enzima acetilcolinesterase imobilizada em monocamadas auto-organizadas mistas (SAMmix) de alcanotióis. A detecção eletroquímica mostrou-se altamente sensível, uma vez que não há o uso do glutaraldeído como agente reticulante. Com essa plataforma foi possível desenvolver um biossensor de acetilcolina estável e robusto, sendo calculado o valor de Km app = 0,46x10-3 mol L-1, limite de detecção LD=3,32x10-10 mol L-1 e limite de quantificação LQ=1,11x10-9 mol L-1, valores inferiores aos encontrados na literatura, ressaltando a eficiencia da nova plataforma. Seguindo a mesma idéia de auto-organização, foram realizados estudos de nanotoxicidade utilizando modelos de membrana a partir de filmes de Langmuir. O principal objetivo foi elucidar a ação dos nanotubos de carbono (SWCNT), PAMAM e do nanocomplexo entre os dois materiais (SWCNT-PAMAM) nas membranas celulares, a nível molecular, usando um sistema modelo de membrana. A penetração de SWCNT e dos nanocomplexos em monocamadas lipídicas foi estudada utilizando microscopia de ângulo de Brewster (BAM) simultaneamente com cinética de absorção e pressão de superfície. Os resultados confirmaram a interação entre os nanomateriais e a membrana, indicando que a presença dos nanomateriais afeta o empacotamento dos lipídios. Foram realizados ainda estudos de citotoxicidade dos mesmos nanomateriais em sistemas celulares in vitro. Os resultados de citometria, proliferação celular, morfologia e inibição de adesão apresentaram-se evidenciaram que a combinação entre SWCNT e PAMAM, proporciona um maior índice de toxicidade em relação ao SWCNT, um comportamento diferente do que relatado nos componentes individuais. A toxicidade de nanocomplexos de SWCNT-PAMAM e de seus componentes individuais podem estar fortemente ligados ao tipo de material e como estes estão disponíveis no meio de cultura. Os estudos contidos nessa tese mostram a versatilidade dos filmes finos em sistemas auto-organizados e biomiméticos, e podem ser relevantes para o avanço de pesquisas sobre interação de nanomateriais e biossistemas. / In this thesis we employed the concept of self-organization, including the layer-by-layer (LbL) technique, alkanethiols self-assembled monolayers (SAMmix) and Langmuir monolayers, to develop new methods for materials and devices manipulation for application in nanomedicine. Two different types of biosensors were developed. The first one was based on the LbL technique to detect nitric oxide (NO•), which is of great importance in the medicine. The second biosensor was based on SAM monolayers supporting acetylcholinesterase for pesticide monitoring. The NO• was constructed by modified carbon fiber (CF) assembled with nickel phtalocyanine tetrasulfonade (NiTsPc) and polyamidoamine dendrimer (PAMAM) in the form of ultramicroelectrodes (UMEs) by the LbL technique. The sensor was characterized using differential pulse voltammetry and electrochemical impedance spectroscopy. The results showed that NO• diffusion is dependent on the number of bilayers employed and the arrangement of molecules in the film. The sensor architecture with CF-(PAMAM/NiTsPc) presented the best analytical signal. In addition, we analyzed the detection of interfering with NO• as nitrite, nitrate, hydrogen peroxide, ascorbic acid, dopamine, epinephrine and norepinephrine. The results showed high selectivity due to the use of PAMAM dendrimer as selective layer. The second biosensor used the enzyme acetylcholinesterase immobilized on SAMmix. The electrochemical detection of carbaryl was highly sensitive, since there is no use of glutaraldehyde as crosslinking agent. Using acetylcholine as a probe, Kmapp value was determined at 0.46x10-3 mol L-1, with detection limit of 3.32x10-10 mol L-1 and quantification limit of 1.11x10-9 mol L-1, values lower than those found in the literature, highlighting the efficiency of the new platform. Langmuir films made of lipids were employed as cell membrane models, in order to investigate the interactions between single-wall carbon nanotubes (SWCNT), PAMAM and their nanocomplex (SWCNT-PAMAM) at the molecular level. The interation of SWCNT and nanocomplexes in lipid monolayers was studies using Brewster angle microscopy (BAM) in conjunction with absorption kinetics and surface pressure. The results confirmed the interaction between nanomaterials and the membrane, indicating that the presence of nanomaterials affects the packing of the lipids. Cytotoxicity studies were also employed to investigate the interaction of nanomaterials in in vitro cell systems. The results of flow cytometry, cell proliferation, morphology and inhibition of adhesion revealed the toxicological aspects of the materials, demonstrating a higher toxicity to the nanocomplex, compared to SWCNT, differently of the individual components. The toxicity of SWCNT nanocomplex and its individual components can be related to the type of material and how these materials are available in the culture medium. The studies in this thesis show the versatility of self-assembly thin films on biomimetic systems and may be relevant to the advance of research on the interaction of nanomaterials and biosystems.

O design de materiais na escala nanométrica está levando a sistemas com novas propriedades e aplicações as mais diversas, como em sistemas de diagnóstico e de tratamento inteligentes e sustentáveis. Melhorar a eficiência dos tratamentos de doenças através do desenvolvimento de fármacos mais eficientes e com menos efeitos colaterais, e agentes de contraste e de diagnóstico mais específicos e sensíveis para monitoramento preventivo precoce, é um dos principais objetivos da Nanomedicina. Todavia, a química de superfície necessária para realizar tais reações de funcionalização/conjugação de moléculas ainda está longe de ser adequadamente controlada, particularmente considerando-se a complexidade das biomoléculas e a estabilidade coloidal. Assim, nesta tese foram desenvolvidos processos de conjugação de nanopartículas de óxido de ferro (SPIONs) com um ou mais agentes co-funcionalizantes, gerando partículas mono, bi e multifuncionalizadas dispersáveis em meio aquoso. Os esforços foram concentrados no desenvolvimento de sistemas de diagnóstico e de entrega de fármacos baseados em nanopartículas, cujas propriedades precisam ser ajustadas pela conjugação de biomoléculas e espécies bioativas em sua superfície, num verdadeiro trabalho de engenharia a nível nanométrico/molecular. De fato, nanopartículas modificadas com moléculas co-funcionalizantes estabilizantes (glicerol-fosfato, glicose-fosfato, fosforiletanolamina, dopamina e tiron), agentes de vetorização que direcionam o nanoconjugado a células-alvo tumorais (ácido fólico e biotina), bem como com fármacos como metotrexato e ibuprofeno foram preparadas, e o efeito das mesmas sobre a eficiência de incorporação por células tumorais (HeLa e MCF-7) estudada. Os estudos de atividade biológica in vitro foram realizados em parceria com o Laboratório de Processos Fotoinduzidos e Interfaces (LPFI-IQUSP). Os resultadosobtidos confirmaram a possibilidade de se controlar a atividade biológica das nanopartículas por meio dos agentes funcionalizantes, abrindo perspectivas interessantes para o desenvolvimento de nanoagentes multifuncionais para teranóstica, conjugados com agentes de vetorização específicos (particularmente anticorpos e aptâmeros), além de agentes de contraste (radiofármacos, fluoróforos, contraste para IRM, etc.) e moléculas terapêuticas (antitumorais, anti-inflamatórios, dentre outros). Entretanto, diversos são os problemas associados aos processos químicos envolvendo a produção e funcionalização desses nanomateriais por processos convencionais em batelada, que tendem a ser demorados e apresentam dificuldade de controle dos parâmetros de reação e baixa reprodutibilidade, dificultando o escalonamento produtivo e a comercialização dos eventuais produtos. Uma estratégia promissora é o uso de reatores microfluídicos com projeto de canais adequado, além de atuadores e sensores que, juntos garantam excelente controle de processos e baixo consumo de energia e de reagentes. Assim, também foram desenvolvidos reatores microfluídicos para produção e funcionalização de nanopartículas de ouro, de forma a tornar os processos químicos programáveis, mais eficientes, controláveis e econômicos, em parceria com o Laboratório de Micromanufatura do Instituto de Pesquisas Tecnológicas (LMI-BIONANO/IPT). Essa parte do desenvolvimento foi realizando empregando a tecnologia de microfabricação em Low Temperature Co-fired Ceramics (LTCC), uma tecnologia versátil que possibilita a produção de dispositivos de diferentes geometrias em materiais cerâmicos de baixa reatividade e de baixo custo. Esses dispositivos podem tornar os processos de produção de nanopartículas multifuncionais dispersáveis suficientemente simples, versáteis e reprodutíveis para atender aos altos padrões de qualidade exigidos para produtos voltados para aplicações biomédicas / Materials design at nanoscale is leading us to intelligent systems with new properties and applications, such as more efficient diagnostic and treatment systems. Improving the treatment of diseases by the development of more specific and efficient drugs, displaying fewer or no side effects, conjugated with sensitive contrast/diagnostic agents for early preventive monitoring and treatment is one of the main goals of the Nanomedicine. However, the knowledge on surface chemistry required to perform such molecular functionalization/conjugation reactions still is far from being adequately controlled, particularly considering the complexity of biomolecules and reaching colloidal stability. Thus, in this thesis, processes of conjugation of iron oxide nanoparticles (SPIONs) with one or more co-functionalizing agents have been developed so as to generate mono, bi and multi-particles dispersible in aqueous medium. Efforts were specifically focused on the development of drug delivery and diagnostic systems based on nanoparticles whose properties must be adjusted by the conjugation of biomolecules and bioactive species on their surface, in a truly nano/molecular scale engineering work. In fact, nanoparticles modified with stabilizing co-functionalizing molecules (glycerolphosphate, glucose-phosphate, phosphorylethanolamine, dopamine and tiron), targeting agents (folic acid and biotin) to guide itself and concentrate in specific tumor cells, as well as with drugs such as methotrexate and ibuprofen were prepared, and their effect on the efficiency of uptake by tumor cells (HeLa and MCF-7) studied. In vitro biological activity studies were performed in collaboration with the Laboratory of Photo Induced Processes and Interfaces (LPFI-IQUSP). The results confirmed the possibility of controlling the biological activity of nanoparticles by anchoring suitable functionalizing agents in an additive way, opening interesting new perspectives for the development ofmultifunctional theranostics nanoagents, conjugated with specific vectorization agents (particularly antibodies and aptamers), as well as diagnostic (radiopharmaceuticals, fluorophores, MRI contrast, etc.) and therapeutic agents (antitumor, anti-inflammatory, among others). However, there are several problems associated with the production and functionalization of these nanomaterials by conventional batch processes, which tend to be time consuming and difficult to control, as confirmed by their low reproducibility, making it difficult to produce and commercialize the eventual products. A promising strategy is the use of microfluidic reactors with suitable channel designs, as well as actuators and sensors that, together, ensure excellent process control and low energy and reagent consumption. Thus, microfluidic reactors were also developed for the production and functionalization of gold nanoparticles in order to make chemical processes more predictable, efficient, controllable and economical, in partnership with the Micromanufacturing Laboratory of the Institute of Technological Research (LMI-BIONANO/IPT). This part of the development was accomplished by employing the Low Temperature Co-fired Ceramics (LTCC) microfabrication technology, a versatile technology that enables the production of devices of different geometries in ceramic materials of low reactivity and of low cost. These devices can make the production processes of dispersible multifunctional nanoparticles simple, versatile and reproducible enough to meet the high standards of quality required for products for biomedical applications

Nanopartículas de ouro têm mostrado enorme potencial de aplicação em modalidades diagnósticas e terapêuticas fotoativadas. Em especial, nanoestruturas de ouro anisotrópicas ramificadas apresentam excelente desempenho atuando tanto como contrastes de imagens fotoacústicas, quanto como agentes ativos para terapias fototérmicas de câncer. Apesar dos avanços nas suas rotas de síntese, o desenvolvimento dessas nanoestruturas de forma simples e reprodutível ainda é desafiador. O presente trabalho visou o desenvolvimento de nanopartículas de ouro anisotrópicas ramificadas, ou nanoflores, que sejam fotoativas no infravermelho-próximo para a terapia e diagnóstico de câncer. Em particular, buscou-se o desenvolvimento de uma síntese simples para sua obtenção, assim como a verificação de sua atuação como agente de contraste fotoacústico e como agente ativo para hipertermia de tumores. Para tanto, desenvolveu-se uma síntese in situ que permitiu a obtenção de nanoflores monodispersas com tamanho e propriedades ópticas controláveis. Através da variação de aspectos da síntese, como a temperatura e a concentração de ouro, foi possível sintonizar a atividade óptica das partículas entre 590 e 960 nm. Sua formação foi confirmada por microscopia eletrônica de varredura, espalhamento de luz dinâmico e espectroscopia UV-visível. As partículas apresentaram boa estabilidade de suas características físico-químicas por dois meses e meio. Ainda, as nanoflores se mostraram estáveis, também, quando suspensas em meio de cultura, sob irradiação de lasers, e quando mantidas a temperatura corpórea por longos intervalos. Sua resposta fotoacústica foi caracterizada, apresentando sinais significativos e permitindo a obtenção de imagens claras de sua localização, mesmo em baixas concentrações. Testes realizados em cultura de células mostraram que as nanoflores foram eficazes na hipertermia de uma linhagem de hepatocarcinoma de rato (HTC), ao mesmo tempo que não apresentaram sinais de toxicidade a uma linhagem de fibroblastos de camundongos (FC3H). Esses resultados revelam uma possibilidade simples de fabricação de nanoestruturas de ouro anisotrópicas ramificadas, que podem servir como uma plataforma promissora para o diagnóstico e terapia do câncer. / Gold nanoparticles have shown enormous potential of application in photodiagnostic and in phototherapeutic procedures. Notably, branched anisotropic gold nanostructures present distinguished performance acting as contrast agents of photoacoustic images and as active agents for photothermal therapies for cancer. Despite advances in their synthesis routes, the growth of these nanostructures in a simple and reproducible way is still challenging. The present study was aimed at developing branched anisotropic gold nanoparticles, coined nanoflowers, that are photoactive in the near-infrared for therapy and diagnosis of cancer. In particular, we sought to develop a simple synthesis route, as well as to verify its application for both, as photoacoustic contrast agents and as active agents for tumor hyperthermia. An in situ synthesis was developed which allowed the development of monodisperse nanoflowers with controllable size and optical properties. Through variations of certain aspects of this procedure, such as temperature and gold ions concentration, it was possible to tune the optical activity of the particles between 590 and 960 nm. The nanostructure morphology was confirmed by scanning electron microscopy, dynamic light scattering and UV-visible spectroscopy. The particles exhibited consistent physicochemical characteristics and good stability for two and a half months. Furthermore, the nanoflowers were also stable when suspended in cell culture medium, under laser irradiation and when maintained at body temperature for long intervals. Its photoacoustic response was characterized, presenting significant responses and generating clear images of its location, even at low concentrations. In vitro tests revealed that these nanoflowers were effective therapeutic agents for photothermal therapy of a rat hepatocarcinoma (HTC) lineage, while showing no signs of toxicity to mouse fibroblast (FC3H) cell line. These results reveal a simple procedure of synthesizing branched anisotropic gold nanostructures, which can serve as a promising platform for cancer diagnosis and therapy.

Nanotubos de Carbono (Carbon Nanotubes - CNTs) são nanoestruturas de carbono na forma de tubos cilíndricos que apresentam excelentes propriedades ópticas, elétricas, térmicas e mecânicas. A imobilização dos CNTs em conjunto com polieletrólitos condutores e naturais na forma de filmes ultrafinos, utilizando a técnica automontagem, apresenta uma nova alternativa para o desenvolvimento de nanocompósitos aplicados em dispositivos sensores e biossensores. Neste trabalho de mestrado foram construídos e estudados filmes automontados contendo CNTs funcionalizados com grupos ácidos carboxílicos e imobilizados com os polieletrólitos polianilina (PANI) e quitosana (QUIT). Também foram fabricados para comparação filmes de PANI e poliestireno sulfonado (PSS). O crescimento dos filmes de PANI/CNT e PANI/PSS revelou um crescimento linear com o número de bicamadas, conforme mostraram as medidas de espectroscopia UV-Vis e voltametria cíclica (CV), enquanto que para o filme de QUIT/CNT, as medidas de microbalança de cristal de quartzo (QCM) mostraram um crescimento exponencial. Análises de espectroscopias Raman e infravermelho com transformada de Fourier (FTIR) indicaram alterações nas bandas características dos filmes, comparadas com seus materiais precursores, indicando interação entre os componentes nas multicamadas dos filmes. Análises morfológicas obtidas por AFM mostraram um aumento da rugosidade com o aumento do número de bicamadas. O mapeamento Raman revelou que os filmes contendo CNTs apresentaram maior heterogeneidade química na superfície do que aos filmes somente com polieletrólitos. Foi observada uma queda na resistividade dos filmes, com o aumento do número de bicamadas, sendo esta, mais acentuada com a presença dos CNTs. Os eletrodos contendo os filmes automontados apresentaram alta estabilidade eletroquímica, a presença dos CNTs aumentou a intensidade das correntes de pico e tornou os sistemas mais reversíveis. Os eletrodos que se mostraram mais sensíveis à detecção de peróxido de hidrogênio foram selecionados para imobilização da enzima Glicose Oxidase (GOx). Foram investigados os seguintes sistemas biossensores: (PANI/CNT)7, (PANI/PSS)1 e (QUIT/CNT)5 apresentando na mesma ordem: valores de sensibilidades de 190 nA/(mmol/L), 36 nA/(mmol/L) e 220 nA/(mmol/L); Limite de detecção de 2,2 µmol/L, 67,5 µmol/L e 8,5 µmol/L, e valores deconstante de Michealis-Menten(\'K IND.M\'POT.APP\' ) de 2,2 µmol/L, 67,5 µmol/L e 8,5 µmol/L. Os resultados indicam que a utilização dos CNTs é bastante promissora para fabricação de dispositivos biossensores para aplicação em Nanomedicina, e além disso, a escolha dos componentes para formação dos nanocompósitos exerce grande influência no desempenho do dispositivo. / Carbon Nanotubes (CNTs) are cylindrical carbon nanostructures exhibiting excellent electrical, thermal, optical and mechanical properties. The immobilization of CNTs in nanostructured thin films in conjunction with polymers using the Layer-by-Layer (LbL) technique provides a new alternative for development of nanocomposites to be used as sensors and biosensors. In this study we report the fabrication of LbL films containing CNTs functionalized with carboxylic acid immobilized in conjunction with polyaniline (PANI) or chitosan (QUIT). Films comprising PANI and sulfonated polystyrene (PSS) were also produced for comparison. Film growth was monitored by UV-Vis spectroscopy, cyclic voltammetry (CV) and quartz crystal microbalance (QCM) and the results showed a linear increase with the number of bilayers in PANI films, and an exponential growth for QUIT/CNT films. FTIR and Raman analyses revealed changes in the bands of nanocomposites compared to their precursor materials, indicating the interactions between the components in the multilayers. Morphological analysis of the films obtained by atomic force microscopy (AFM) showed that the roughness of the films increased with the number of bilayers. Raman mapping showed that the presence of CNTs generated a high heterogeneity in film surface in comparison to a films formed only by polyelectrolytes. Electrical resistivity of the films decrease upon increasing the number of bilayers, especially for CNTs-containing films. Furthermore, electrodes containing LbL films exhibited high electrochemical stability, in which the presence of CNTs increased the intensity of response signal. The electrodes that exhibited best performance toward hydrogen peroxide detection were employed for immobilization of glucose oxidase (GOx) and used as glucose biosensors. The systems (PANI/CNT)7, (PANI/PSS) 1 and (QUIT/CNT)5 exhibited a sensitivity of 190 nA/(mmol/L), 36 nA/(mmol/L) and 220 nA/(mmol/L), respectively. The detection limit was estimated at 2.2 mmol/L, 67.5 mmol/L and 8,5 mmol/L, whereas the Michaelis-Menten constant ( \'K IND.M\'POT.APP\') values was found to be 2.2 mmol/L, 67.5 mmol/L and 8.5 mmol/L, respectively, to the three systems employed. The results indicated that the use of CNTs in Layer-by-layer thin films is promising for use as biosensors. Furhtermore, we showed that the choice of the polyelectrolyte is a crucial parameter to tailor specific, high performance sensors.

Drug based nanoparticle (NP) formulations have gained considerable attention over the past decade for their use in various drug delivery systems. NPs have been shown to increase bioavailability, decrease side effects of highly toxic drugs, and prolong drug release. Furthermore, polymer based, biodegradable nanodelivery has become increasing popular in the field of NP formulation because of their high degree of compatibility and low rate of toxicity. Due to their popularity, commercially available polymers such as poly lactic acid (PLA), poly glycolic acid (PGA) and polylactic-co-glycolic acid (PLGA) are commonly used in the development and design of new nano based delivery systems. Nonsteriodal anti-inflammatory drugs (NSAIDs) are commonly used for the treatment of pain and inflammation. NSAIDs such as diclofenac and celecoxib function by blocking cyclooxygenase expression and reducing prostaglandin synthesis. Unfortunately, the pharmacological actions of NSAIDs can lead to the development of several adverse side effects such as gastrointestinal ulceration and bleeding. The aim of this study was to formulate and optimize diclofenac or celecoxib entrapped polymer NPs using an emulsion-diffusion-evaporation technique. NP formulations were evaluated based on specific formula parameters such as particle size, zeta potential, morphology, and entrapment efficiency. Effects of stabilizer type, stabilizer concentration, centrifugal force, drug amount, and/or emulsifier (lecithin) on nanoparticle characterization were examined for formula optimization. Results of the formulation studies showed that NPs developed using polylactide-co-glycolide (PLGA) polymers and the stabilizer didodecyldimethylammonium bromide (DMAB) demonstrated enhanced stability, drug entrapment, and reduced particle size. These findings demonstrate an effective method for polymer NP formulation of diclofenac or celecoxib. Furthermore, the results reported herein support a novel method of drug delivery that may function to reduce known adverse effects of these pharmacotherapeutic agents.

Les nanotechnologies appliquées à la médecine, et plus particulièrement à l’oncologie, ont permis le développement d’une nouvelle classe d’entités, appelées communément nanomédicaments ou médicaments vectorisés. Ce projet de recherche a pour objectif d’encapsuler du docétaxel dans un vecteur lipidique unilamellaire furtif, puis de greffer en surface le trastuzumab afin d’en améliorer le profil pharmacocinétique, notamment en optimisant la spécificité de la phase de distribution. Les résultats obtenus montrent qu’il est possible de développer un immunoliposome furtif de 140 nm encapsulant 90 % de docétaxel avec un taux de greffage de trastuzumab de 30 %. L’approche en cytométrie de flux que nous avons développée et appliquée a permis une quantification absolue du nombre d’anticorps présents en surface. In vitro, un double screening en culture 2D et en sphéroïde 3D a démontré la supériorité antiproliférative de l’immunoliposome comparativement à tous les autres traitements, indépendamment du statut Her2 des lignées étudiées. Les études in vivo ont confirmé cette supériorité, y compris comparativement au T-DM1, l’antibody-drug conjugate de référence dans la pathologie. Les études de biodistribution ont montré que l’accumulation de notre forme vectorielle dépendait de la taille et du degré de vascularisation des tumeurs, plus que statut Her2 tumoral. En conclusion, nous avons démontré l’intérêt thérapeutique de développer des formes vectorielles dans la prise en charge du cancer du sein, comparativement aux traitements standard. Une optimisation de la phase de distribution explique la supériorité antiproliférative obtenue avec l’immunoliposome. / The application of nanotechnology in medicine, especially oncology, has allowed for the development of a new class of entities, commonly called nanomedicine or vectorized medicine.This research project aims to encapsulate docetaxel in a stealthy, unilamellar, lipidic vector, then graft trastuzumab onto its surface to improve its pharmacokinetic profile, specifically by optimizing the specificity of the distribution phase.The results show that it is possible to develop a stealthy immunoliposome of 140 nm encapsulating 90% docetaxel and a trastuzumab grafting rate of 30 %. The flow cytometry approach that we developed and applied allowed for an absolute quantification of the number of antibodies present on the surface. In vitro, a double screening in 2D culture and in 3D spheroid showed the antiproliferative superiority of the immunoliposome compared to all the other treatments, regardless of the Her2 status in the cells studied. In vivo studies have confirmed said superiority compared to T-DM1; the benchmark antibody-drug conjugate for this pathology. Biodistribution studies have shown that the accumulation of our vector depends moreover on the size and degree of tumor vascularization than its Her2 status. In conclusion, we have demonstrated the therapeutic value of developing vector forms in the management of breast cancer therapy compared to standard treatments. The optimization of the distribution phase explains the antiproliferative superiority obtained by using the immunoliposome.

Chronic Obstructive Pulmonary Disease (COPD) is a leading cause of death worldwide. Alveolar wall destruction is a significant contributor to COPD. Inflammatory macrophages are a major source of the Extracellular Matrix (ECM) proteolysis. ECM breakdown causes air to get trapped in the alveoli, obstructing airflow. One step in curing COPD may be to convert inflammatory to pro-regenerative macrophages. Recently, decellularized ECM scaffolds have shown the ability to induce a pro-regenerative phenotype. Yet these scaffolds are incapable for reaching the alveolar region of the lungs. To reach the alveolar region particles need a diameter of 1-5 μm or smaller than 300 nm. We used protein from decellularized lung tissue to create nanoparticles. By first digesting the protein in acid, we electrosprayed the solution into nanoparticles. The average size of the nanoparticles is 225 (± 67) nm, within the requirements to reach alveoli. However, another barrier exists for treating this disease. That barrier is mucus; mucus hypersecretion is another sign of COPD. The formed particles are capable of penetrating the mucus layer in COPD. After characterizing the particles, we began in vitro investigations. First, we measured cytotoxicity of the nanoparticles. In alveolar epithelial cells, adding nanoparticles to the media increased cellular proliferation. We then added the nanoparticles to isolated murine macrophages. The nanoparticles induced a pro-regenerative phenotypic shift in murine macrophages. These experiments reveal that these nanoparticles may become an effective treatment for degenerative lungs diseases, such as COPD, after further investigation.

<p>Although various proof-of-concept studies have demonstrated the eventual potential of a multifunctional SERS-active metallic nanostructures for biological applications such as single cell analysis/measurement and drug delivery, the actual development and testing of such a system in vitro has remained challenging. One key point at which many potentially useful biomethods encounter difficulty lies in the translation of early proof-of-concept experiments in a clean, aqueous solution to complex, crowded, biologically-active environments such as the interior of living cells. The research hypotheses for this work state that multifunctional nanoconstructs can be fabricated and used effectively in conjunction with surface-enhanced Raman scattering (SERS) spectroscopy and other photonics-based methods to make intracellular measurements in and deliver treatment to single cells. The results of experimental work address the specific research aims, to 1) establish temporal and spatial parameters of nanoprobe uptake and modulation, 2) demonstrate targeting of functionalized nanoparticles to the cytoplasm and nucleus of single cells, 3) deliver to and activate drug treatment in cells using a multifunctional nanosystem, and 4) make intracellular measurements in normal and disease cells using external nanoprobes,</p><p>Raman spectroscopy and two-dimensional Raman imaging were used to identify and locate labeled silver nanoparticles in single cells using SERS detection. To study the efficiency of cellular uptake, silver nanoparticles were functionalized with three differently charged SERS/Raman labels and co-incubated with J774 mouse macrophage cell cultures for internalization via normal cellular processes. The surface charge on the nanoparticles was observed to modulate uptake efficiency, demonstrating a dual function of the surface modifications as tracking labels and as modulators of cell uptake. </p><p>To demonstrate delivery of functionalized nanoparticles to specific locations within the cell, silver nanoparticles were co-functionalized with the HIV-1 TAT (49-57) peptide for cell-penetrating and nuclear-targeting ability and p-mercaptobenzoic acid (pMBA) molecules as a surface-enhanced Raman scattering (SERS) label for tracking and imaging. Two-dimensional SERS mapping was used to track the spatial and temporal progress of nanoparticle uptake in PC-3 human prostate cells and to characterize localization at various time points, demonstrating the potential for an intracellularly-targeted multiplexed nanosystem. Silver nanoparticles co-functionalized with the TAT peptide showed greatly enhanced cellular uptake and nuclear localization as compared with the control nanoparticles lacking the targeting moiety. </p><p>The efficacy of targeted nanoparticles as a drug delivery vehicle was demonstrated with development and testing of an anti-cancer treatment in which novel scintillating nanoparticles functionalized with HIV-1 TAT (49-57) for cell-penetrating and nuclear-targeting ability were loaded with tethered psoralen molecules as cargo. The experiments were designed to investigate a nanodrug system consisting of psoralen tethered to a nuclear targeting peptide anchored to UVA-emitting, X-ray luminescent yttrium oxide nanoparticles. Absorption of the emitted UVA photons by nanoparticle-tethered psoralen has the potential to cross-link adenine and thymine residues in DNA located in the nucleus. Such cross-linking by free psoralen following activation with UVA light has previously been shown to cause apoptosis in vitro and an immunogenic response in vivo. Experimental results using the PC-3 human prostate cancer cell line demonstrate that X-ray excitation of these psoralen-functionalized Y2O3 nanoscintillators yields concentration-dependent reductions in cell number density when compared to control cultures containing psoralen-free Y2O3 nanoscintillators. </p><p>The development and demonstration of a small molecule-sensitive SERS-active fiber-optic nanoprobe suitable for intracellular bioanalysis was demonstrated using pH measurements in single living human cells. The proof-of-concept for the SERS-based fiber-optic nanoprobes was illustrated by measurements of intracellular pH in MCF-7 human breast cancer, HMEC-15/hTERT immortalized normal human mammary epithelial, and PC-3 human prostate cancer cells. Clinical relevance was demonstrated by pH measurements in patient biopsy cell samples. The results indicated that that fiber-optic nanoprobe insertion and interrogation provide a sensitive and selective means to monitor biologically relevant small molecules at the single cell level.</p> / Dissertation

Magistro baigiamajame darbe yra nagrinėjamas dabartinis nanomedicinos teisinis reglamentavimas ES ir JAV. Ši tema Lietuvoje iki šiol dar nebuvo tyrinėjimų objektu. Pirmoji darbo dalis supažindina skaitytoją su nanomedicina bei su ja susijusių sąvokų problematiką. Antroji dalis atskleidžia nanomedicinos teisinio reglamentavimo prielaidas ES, o trečioji dalis – JAV. Be to, atsižvelgiant į gautus rezultatus, yra įvertinamos galimos nanomedicinos teisinio reglamentavimo perspektyvos ateityje. Darbe plačiai analizuojami atitinkami pasirinktų tarptautinės teisės subjektų teisės aktai bei specialioji teisinė literatūra (įvairių institucijų dokumentai, teisės mokslininkų darbai) bei tam tikra mokslinė-techninė literatūra (įvairių institucijų ir organizacijų ataskaitos ir tyrimai, mokslininkų tyrimai). / The master thesis researches the current regulation of nanomedicine in the EU and the US. Until now, this topic has not yet been researched in Lithuania. In the first part of the thesis nanomedicine and the problem of nanomedicine-related definitions are introduced to the reader. The second part reveals the basis of the regulation of nanomedicine in the EU, and the third part – in the US. Furthermore, considering the achieved results, the perspective of the future regulation of nanomedicine is assessed. The thesis extensively analyses the legal acts of the chosen subjects of international law and specific legal literature (documents of various institutions, works of law scholars) as well as some scientific-technical literature (reports and researches of various institutions and organizations, scientists’ researches).