Multiple hurdles, such as drug solubility, stability, and physical barriers in the body, hinder bioavailability of many promising therapeutics. Polymeric nanocarriers can encapsulate the therapeutics to protect non-target areas from side effects but also protect the drug from premature degradation for increased circulation and bioavailability. To capitalize on these advantages, the polymer nanoparticle must be properly engineered for increased control in size distribution, therapeutic encapsulation, colloidal stability, and release kinetics. However, each application requires a specific set of characteristics and properties. Being able to tailor these by manipulation of different design parameters is essential to optimize nanoparticles for the application of interest. This study of nanoparticle fabrication and characterization takes us a step closer to building effective delivery systems tailored for specific treatments. Poly(ethylene oxide)-b-poly(D,L-lactic acid) (PEO-b-PDLLA) based nanoparticles were produced to range from 100-200 nm in size. They were fluorescently labeled with a hydrophobic dye 6-13 bis(triisopropylsilylethynyl) pentacene (TIPS pentacene) at an optimal loading of 0.5 wt% with respect to the core. Surfaces were successfully coated with streptavidin to be readily functionalized with various biotinylated compounds such as PD-L1 antibodies or A488 fluorophore. Using the same PEO-b-PDLLA, iron oxide and a conjugated polymer poly(2- methoxy-5-(2-ethylhexyloxy)-1,4-phenylenevinylene) (MEH-PPV) were co-encapsulated to form fluorescently labeled magnetic particles. Using poly(lactic-co-glycolic acid), CRISPR-Cas9 plasmids were encapsulated at 1.6 wt% and most of the payload released within the first 24 hours. The incorporated plasmids were intact enough to have mammalian macrophages successfully express the bacterial protein Cas9. Using similar PLGA based particles, the surface was functionalized with streptavidin and bound to the surface of bacteria as an active carrier for increased penetration of solid tumors averaging ~23 particles per bacterium. PEO-b-PLGA based particles were used in conjunction with a hydrophobic salt former to encapsulate a peptide designed to reduce platelet binding to cancer cells and mitigate extravasation. The peptide encapsulated was increased from < 2 wt% without salt former to 8.5 wt% with the used of hexadecyl phosphonic acid. Although the applications across these projects can be broad, the fundamentals and important design parameters considered contribute to the overarching field of effective carriers for drug delivery. / Ph. D. / There are many reasons why many promising pharmaceutical formulations never make it through regulation and onto market, including low solubility of the drug, low absorbance by the body, and harmful side effects, to name a few. Using polymer drug carriers, these difficulties can be overcome by holding the drug in a more soluble carrier, releasing it on a certain timeline or to a specific location to increase absorbance and decrease side effects. When designing a carrier, the requirements for the product are dependent on the application and the disease of interest. This work looks at the material types and conditions during particle formation to see how it affects the final product to better define and understand how these parameters change the performance. This work shows that the carrier size can be manipulated depending on how much of one material is used versus the other, they can be labeled to fluoresce so they can be tracked during cell and animal studies, and they can be coated with targeting compounds on the surface to increase the specificity of the carrier to localize to a target location of interest. Different particles containing DNA for gene editing, peptides for cancer therapies, and magnetic iron oxides to increase transport across difficult cell barriers have all be fabricated and characterized. The lessons learned through these projects will help guide future work to more effective and efficient delivery of pharmaceuticals to the body.

O adenocarcinoma ocorre em 90% dos casos de tumores malignos dos ovários e apresenta-se bilateralmente em 30 a 50% das pacientes. Devido à falta de sintomas iniciais da doença e à baixa especificidade dos marcadores tumorais existentes, quando detectado, o câncer encontra-se em estadios III e IV da doença, os quais indicam disseminação na cavidade peritoneal. O paclitaxel (PCX) é o fármaco de primeira escolha para o tratamento do câncer de ovário, entretanto a sua baixa solubilidade aquosa reduz sua biodisponibilidade. Dessa forma, no medicamento comercial, Taxol®, o PCX está solubilizado em uma mistura de tensoativos tóxicos. O desenvolvimento de nanocarreadores de fármacos tem sido investigado para promover a redução dos efeitos tóxicos e aumentar a segurança e a eficiência terapêutica com PCX. A funcionalização dos nanocarreadores é uma das estratégias utilizadas para aumentar a seletividade às células tumorais que superexpressam receptores de folato. O objetivo do presente trabalho foi desenvolver e caracterizar nanopartículas poliméricas contendo paclitaxel, funcionalizadas e não funcionalizadas com ácido fólico, visando a otimização do tratamento do câncer de ovário. Os sistemas foram obtidos com sucesso, utilizando técnica de nanoprecipitação. Os resultados obtidos mostraram que as formulações NPPCX e NPPCX-AF apresentaram distribuição de tamanhos de partículas de 140 e 154,6 nm, respectivamente, e índices de polidispersão menores que 0,1, com alta eficiência de encapsulação do PCX. Os resultados obtidos foram adequados para a via de administração endovenosa e promoção do direcionamento ativo no ambiente tumoral. Os resultados in vitro em testes de citotoxicidade em linhagens celulares SKOV-3 e OVCAR-3 demonstraram que as nanopartículas contendo PCX (NPPCX e NPPCX-AF) foram capazes de disponibilizar o PCX e reduzir a viabilidade celular. Os ensaios de citometria de fluxo e de microscopia confocal demonstraram a capacidade de ambas as nanopartículas apresentaram um uptake celular tempo dependente, mostrando a capacidade dos nanocarreadores serem internalizados. Além disso, comparando as duas linhagens celulares, a SKOV-3 apresentou maior uptake por apresentar maior número de receptores de folato. Dessa forma, as análises in vitro sugerem que os nanocarreadores, NPPCX e NPPCX-AF, apresentam potencial para a otimização da terapia do câncer de ovário. No entanto, estudos in vivo são necessários para a obtenção de resultados adicionais relativos à eficiência e à segurança para o tratamento de câncer de ovário. / Adenocarcinoma occurs in 90% of cases of malignant ovarian cancer and is present bilaterally in 30 to 50% of patients. Due to lack of initial symptoms and the low specificity of the existing tumor markers, the cancer is detected when it\'s in stages III and IV, which indicate spread into the peritoneal cavity. Paclitaxel (PCX) is the drug of first choice for ovarian cancer treatment, but it has low aqueous solubility, which reduces its bioavailability. Thus, in the commercial drug, Taxol®, PCX is solubilized in a mixture of toxic surfactants. The development of drug nanocarriers has been investigated to promote the reduction of toxic effects and increase the safety and therapeutic efficacy of PCX. Functionalization of nanocarriers is one of the strategies used to increase selectivity to tumor cells that overexpress folate receptors. The aim of the present work was the development and characterization of folate-modified nanoparticles (NPPCX-AF) and unmodified nanoparticles (NPPCX) and evaluation of in vitro efficacy of developed systems using adenocarcinoma cell lines. The systems were successfully obtained using nanoprecipitation technique. The results showed that the NPPCX and NPPCX-AF formulations had a particle size distribution of 140 and 154.6 nm, respectively, and polydispersity indexes smaller than 0.1, with high PCX encapsulation efficiency. The results obtained were suitable for the intravenous administration route and promotion of active targeting in the tumor microenvironment. The in vitro cellular cytotoxicity assays of SKOV-3 and OVCAR-3 cell lines demonstrated that NPPCX and NPPCX-AF were able to release PCX and reduce cell viability. The flow cytometry assays demonstrated that both nanoparticles presented a time dependent cellular uptake, showing the ability of nanocarriers to be uptake. In addition, comparing both cell lines, SKOV-3 showed a higher uptake due to its greater amount of folate receptors. Thus, in vitro results suggested that the nanocarriers, NPPCX and NPPCX-AF, present a distinguish potential for ovarian cancer therapy optimization. In vivo studies are needed to confirm the in vitro results and provide additional data regarding safety and efficacy of ovarian cancer treatment.

Doctor of Philosophy / Department of Chemistry / Santosh Aryal / Nanotechnology offers exciting options for the site-selective delivery of chemotherapeutics and diagnostic agents using nanoparticles. Varieties of organic and inorganic nanomaterials have been explored extensively as a delivery system either in the form of drug carriers or imaging agents. Successful stories include the clinical translation of anticancer nanomedicines such as PEGylated liposomal doxorubicin (DOXIL®), albumin-bound paclitaxel (Abraxane®), and polymeric micelle loaded paclitaxel (Genexol®), which are currently used in the clinic as one of the first lines for cancer chemotherapies. These conventional nanomedicines rely on passive-drug targeting taking advantage of leaky tumor vasculature, called the Enhanced Permeability and Retention (EPR) effect. However, delivering biologically active components selectively to the diseased cell, for example, cancer, is highly challenging due to the biological barriers in the body including blood pool cells/proteins, heterogeneous microenvironment, and intracellular degradation. Therefore, the goal of this dissertation is to develop nanoplatforms that can deliver the agents of interest in targeted fashion to cancer while bypassing or collaborating with the biological barriers. The design consideration of these nanoplatforms centralizes on using simple chemical reactions and cell biology to engineer nanoparticles. The presented nanoparticles were extensively studied and evaluated for their biological functions using in vitro and in vivo models. These nanoconstructs described herein address current limitations of conventional nanomedicine such as (1) the lack of understanding of the interaction of nanoparticle and biological system, and (2) the lack of an effective targeting strategy to deliver drugs to the cancer cell in the tumors. The significant findings of each system will be highlighted and discussed throughout this dissertation. Results obtained highlight key findings such as NP intracellular fate, maximized tumor accumulation, and unique pharmacokinetics could open the avenues for systemic investigations for personalized medicine and lay the foundation for nanomedicine design to accelerate clinical translation.

The need for chemoselective bond formation within complex biological systems has driven much research in chemical biology and chemical medicine and has allowed control over the structure and biological properties of a range of chemical entities. Reactions that are highly biocompatible, selective and occur at low concentration are classified as being bioorthogonal. Although bioorthogonal reactions have been successfully applied to bioconjugation and imaging in living systems, only a few examples exist of bioorthogonal reactions being utilised for the activation of prodrugs. The tetrazine mediated inverse electron demand Diels-Alder reaction is characterized by excellent reaction rates and high biocompatibility in both in vitro and in vivo applications. To date, this chemistry has found only limited application in prodrug activation or drug release strategies. Herein, a series of tetrazine-trigger systems are reported in which an active drug is liberated from its inactive form upon triggering with tetrazine. It is shown that the release of encapsulated and conjugated drugs from polymeric nanoparticles can be triggered by tetrazines providing an on-demand release within biological systems. In a totally new approach that fully complies with the principle of bioorthogonality by avoiding the generation of any by-products, tetrazine was utilised as a prodrug scaffold leading to symbiotic and traceless dyadic prodrug activation. The simultaneous formation of two active drugs (here the anticancer drug camptothecin and a known micro RNA inhibitor) was confirmed and validated within a biological environment. The use of tetrazines as a trigger to activate or release an active drug will open new directions in the field of chemical biology/medicine.

Orientador: : Leonardo Fernandes Fraceto / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-23T08:25:53Z (GMT). No. of bitstreams: 1 Pereira_AndersondoEspiritoSanto_M.pdf: 3153536 bytes, checksum: 2427b9dc08e349cd1eadf5c8a001ceb9 (MD5) Previous issue date: 2013 / Resumo: A "Revolução Verde" ocorreu na década de 60 e previa o aumento da produção agrícola devido ao crescimento populacional, onde foi intensificado o estudo de novas tecnologias para o aumento da produção de alimentos e rentabilidade dos agricultores. Entre as várias tecnologias, destacou-se a utilização de defensivos agrícolas no controle, prevenção e na eliminação de doenças que interferem na produtividade agrícola. O uso de nanopartículas (NPs) para o carreamento de compostos bioativos para liberação sustentada aumenta o tempo de ação e estabilidade química do ativo no meio, bem como a disponibilidade para ação junto ao organismo alvo. Na agricultura, o uso de NPs visa reduzir a concentração efetiva do ativo a ser utilizado, reduzir de aplicações, reduzir a toxicidade, diminuir a periculosidade e os riscos de contaminação ambiental. O presente trabalho propôs o desenvolvimento de NPs poliméricas de poli-épsilon caprolactona (PCL) como sis-temas carreadores para o herbicida atrazina (ATZ) bem como a avaliação das características físico-químicas destes sistemas, a atividade herbicida e a genotoxicidade das formulações preparadas. As nanocápsulas (NCs/ATZ) e as nanoesferas (NEs/ATZ) contendo ATZ apresentaram diâmetro médio de 483,1 ± 10,4 nm e 408,5 ± 2,5 nm, respectivamente. A ATZ apresentou uma eficiência de encapsulação acima de 90% para as formulações de NE e NC e foram observadas alterações no perfil de liberação da ATZ em comparação com o herbicida ATZ. A estabilidade coloidal e físico-química das formulações foi mantida por um período de 90 dias. O uso de NPs aumentou a retenção da ATZ em ensaios com coluna de solo, sendo que a atividade herbicida se mostrou mais eficaz quando comparada ao ativo ATZ apenas e ao de uma formulação comercial (Gesaprin). A investigação da genotoxicidade das formulações, utilizando o ensaio de aberração cromossômica Allium cepa, mostrou que a encapsulação da ATZ reduziu os efeitos sobre o número de aberrações cromossômicas quando comparadas ao ativo ATZ e à formulação comercial. As formulações de NPs contendo ATZ preparadas neste trabalho apresentam grande potencial para aplicação na agricultura, uma vez que estas podem ter ação herbicida utilizando menor concentração de ativo, reduzem a mobilidade da ATZ no solo e diminuem os efeitos genotóxicos, tornando-se mais seguras ao meio ambiente e reduzindo os riscos de contaminação / Abstract: The "Green Revolution" occurred during the decade of 60 and it aimed towards the rapid increase on agriculture production due to population growth, when was intensified the research and development to increase agriculture production and profitability. Among several technologies, herbicides and pesticides have emerged to control, prevent and destroy diseases that interfere in the agricultural productivity. The use of nanoparticles (NPs), as drug delivery system loads to modified drug release profile, increase time of action and increased chemical stability is wells is increase in bioavailability. In agriculture the use of NPs can reduces the amount of chemical used and the number of applications with decrease in toxicity, minimizing the risks of an environmental contamination. This study aims to develop NPs pre-pared with poly-épsilon-caprolactone (PCL) as a carrier system for the herbicide atrazine (ATZ). The formulations were characterized and the herbicide activity and genotoxicity were investigated. The nanocapsules (NCs/ATZ) and nanospheres (NE/ATZ) containing ATZ showed a size average diameter of 483.1±10.4 nm and 408.5±2.5 nm respectively. The ATZ presented encapsulation efficiency over 90% on formulations of NC and NE. The release profiles of the ATZ encapsulated in NPs were changed in relation to the ATZ herbicide only. The colloidal stability over 90 days showed that the formulations were stable. The use of NPs increased the retention of ATZ on soil column and showing that the herbicide was more active when compared to ATZ or a commercial formulation (Gesaprin). The genotoxicity evaluation showed that the encapsulation of ATZ reduced the toxic effects on the number of chromosomal aberration when compared to active ATZ and commercial formulation. NPs formulation containing ATZ prepared in this study presented a great potential for application in agriculture, since these formulations have the same herbicide activity (using lower concentration of active compound), reduce the ATZ soil mobility and also decrease the genotoxicity effects of ATZ, and in this way, reducing the risks of environmental contamination / Mestrado / Bioquimica / Mestre em Biologia Funcional e Molecular

The present work is divided into two sections. The first section deals with the synthesis of regenerable adsorbents for the removal of arsenic from contaminated waters. An adsorbent based on carboxymethylated polyethylenimine grafted agarose gels was synthesized and characterized as a regenerable synthetic ferric oxide adsorbent with high capacity for arsenate ions at pH 3.0. Similarly, four metal ion chelating adsorbents based on dipicolylamine were synthesized and characterized with respect to their Cu(II), Fe(III) and As(V) adsorption capacities. The most efficient adsorbents were Nov-PEI-DPA and Nov-TREN-DPA. Additionally, a commercial ion exchange resin was modified with permanganate to oxidize arsenite into arsenate. A complete oxidation-adsorption system was proposed in which a column packed with the oxidation resin was connected in series with an adsorbent column composed of the polyethylenimine grafted agarose gels.The second section involved work with magnetic nanoparticles. First, composite adsorbents consisting of magnetic particles encapsulated within agarose beads with and without grafted iminodiacetic acid (IDA) chelating groups were synthesized. The adsorption capacity of the adsorbents for Cu(II), Fe(III) and As(V) at different concentrations was investigated. Batch experiments were carried out to determine the Fe(III) and As(V) adsorption isotherms for the magnetic Novarose-IDA. Regenerability of the adsorbent was achieved with a pH change of the inlet solution, without affecting its magnetic or adsorption properties.Magnetic composite particles were synthesized for biomedical applications. First, magnetic nanoparticles were coated with silica and then used for gold nanoshell production. These nanoshells were functionalized with a Brij S10 derivative, containing carboxylic groups, using dodecanethiol as a bridging agent to incorporate a fluorescent biomolecule.Finally, magnetic and gold particles were encapsulated in PLGA nanoparticles. Docetaxel was loaded on these multifunctional nanoparticles and released studies were performed at 37°C. The presence of magnetite, colloidal gold and gold nanoshells in the PLGA nanoparticles was revealed by the coloration acquired by the polymeric nanoparticles. The release of drug from the polymeric nanoparticles showed a biphasic behavior with an initial burst followed by a prolonged slow release. There was no effect of the presence of magnetic or metallic particles on docetaxel release.

O presente trabalho tem como intuito avaliar e otimizar o processo de confecção e as propriedades de nanopartículas poliméricas (NPs) e de lipossomos (LPs) para o transporte de substâncias de interesse visando o tratamento e prevenção de doenças que atingem o sistema nervoso central. As NPs de PLGA com polissorbato-80 (T-80) adsorvido foram otimizadas quanto ao tipo de tensoativo, regime de sonicação, volume de solução de T-80 e tipo de agitador sobre o tamanho, polidispersão e potencial zeta das NPs; ao passo que LPs funcionalizados com anticorpo anti-transferrina também tiveram sua confecção otimizada e suas propriedades foram avaliadas para diferentes misturas de lipídeos contendo DPPC ou DOPC. Após a otimização dos nanocarreadores, ensaios celulares em células Neuro-2a e SH-SY5Y foram realizados de modo comparar a citotoxicidade e a cinética de internalização de tais sistemas. Demonstra-se que o processo de confecção dos carreadores para aplicação no sistema nervoso central requer investigação pormenorizada para melhores resultados na aplicação biológica. / This study aims at analyzing and optimizing the obtainment process and properties of polymeric nanoparticles (NPs) and liposomes (LPs) for the transport of substance of interest with the purpose of treatment and prevention of various central nervous system diseases. PLGA NPs coated with polissorbate-80 (T-80) were optimized regarding the type of surfactant, sonication regime, T-80 solution dispersion volume and size of stirrer on the size, polidispersity and zeta potencial of NPs; whilst anti-transferrin antibody functionalized LPs also had their synthesis optimized and their properties were also compared for different lipid mixtures containing DPPC or DOPC. After optimization, the nanocarriers were submitted to in vitro assays with Neuro-2a and SH-SY5Y cells in order to compare their cytotoxicity and internalization kinetics. It is demonstrated that the process to obtain nanocarriers for central nervous system applications require detailed analysis when seeking the most favorable biological results.

Les macrophages (Mɸ) infiltrés dans les tumeurs orchestrent les différentes étapes du développement tumoral. De par leur capacité à internaliser les nanoparticules (NPs) et leur plasticité phénotypique, ils sont impliqués dans l’efficacité thérapeutique des actifs vectorisés par un rôle de réservoir de NPs ou une modulation de leur réponse envers les cellules néoplasiques. Le calcitriol, métabolite actif de la vitamine D, possède des activités à la fois anticancéreuse et immunomodulatrice. Sa vectorisation via des NPs est une approche thérapeutique intéressante pour potentialiser ses activités tout en limitant les effets secondaires s’opposant à son utilisation clinique dans le cadre de la chimiothérapie. Une étude de formulation a permis de développer des NPs à base d’acide poly(D,L)lactique et de triglycérides (ratio 1:2) d’une taille de 200 nm et présentant une libération prolongée du calcitriol. Des études in vitro menées sur les cellules de cancer du sein MCF-7 ont permis de mettre en évidence l’avantage d’une libération prolongée du calcitriol vis-à-vis de son activité antiproliférative aboutissant à une réduction de 65% de la viabilité cellulaire après 10 jours par rapport au contrôle, non observable avec le calcitriol libre. La participation active des M? à l’activité cytotoxique du calcitriol sur les lignées cellulaires de cancer du sein MCF-7 et de leucémie MV4-11 a aussi été mise en évidence par un modèle de co-culture in vitro. En effet, les NPs de calcitriol, après internalisation par les Mɸ, provoquent une action cytotoxique prolongée contre les cellules MCF-7 en co-culture au bout de 10 jours avec seulement 20% de cellules viables vs 70% en l’absence de Mɸ / Tumor associated macrophages (Mɸ) orchestrate the different stages of tumor development. They are able to internalize nanoparticles (NPs) and are known for their phenotypic plasticity, which make them interesting targets for cancer treatment through the storage of NPs or a modulation of their activity towards the neoplastic cells. Calcitriol, the active metabolite of vitamin D, exerts both anticancer and immunomodulatory activities. Its vectorization via NPs is an interesting therapeutic approach to potentiate its activities while limiting its side effects, which hamper its current clinical use in chemotherapy. We developed poly (D, L) lactic acid and triglyceride-based NPs (1:2 ratio) measuring 200 nm and exhibiting a sustained release of calcitriol. In vitro studies, performed on breast cancer cells (MCF-7), showed the advantages of a sustained release of calcitriol regarding its antiproliferative activity with a 65%-decrease in cell viability after 10 days compared to unexposed cells, while it was unobservable for free calcitriol. The implication of Mɸ in the cytotoxic activity of calcitriol towards MCF-7 cells and MV4-11 cells (leukemia) cells has been demonstrated using an in vitro co-culture model. Calcitriol-NPs showed a sustained cytotoxic activity towards MCF-7 cells in co-cultures after 10 days, through their uptake by Mɸ, with a decrease in cell viability of 80% vs 30% in mono-cultures

O paclitaxel (PCX) é o fármaco de primeira escolha para o tratamento do câncer de ovário. Contudo, a baixa solubilidade do PCX torna necessária a adição de adjuvantes tóxicos à formulação comercial (Taxol®). Assim, o desenvolvimento de sistemas de liberação nanoestruturados têm sido estudados para promover a redução dos efeitos tóxicos e aumentar a segurança e a eficiência terapêutica do PCX. Uma das estratégias utilizadas para aumentar a seletividade dos nanocarreadores às células tumorais é a funcionalização de suas superfícies com folato, devido a superexpressão dos receptores de folato nas células tumorais. O objetivo do presente trabalho foi desenvolver e caracterizar nanopartículas de PLGA funcionalizadas (NP-PCX-AF) e não funcionalizadas (NP-PCX) para a veiculação do PCX, bem como a avaliação in vitro da eficácia dos sistemas desenvolvidos em linhagens celulares de adenocarcinoma de ovário. Os nanocarreadores foram produzidos pela técnica de nanoprecipitação e avaliados por meio das análises físico-químicas de diâmetro médio de partícula, índice de polidispersão, potencial zeta, eficiência de encapsulação, estabilidade coloidal e espectro de infravermelho. Além disso, foi avaliada a eficácia antitumoral e o uptake dos sistemas desenvolvidos por meio dos ensaios de citotoxicidade celular por resazurina e internalização por microscopia confocal e citometria de fluxo. Os resultados obtidos mostraram que as formulações funcionalizadas e não funcionalizadas apresentaram reduzidos valores de diâmetros médios de partículas (menor que 150 nm) e índices de polidispersão reduzidos (menor que 0,2) com elevada eficiência de encapsulação do PCX, próximo a 100%, o que é adequado para a via de administração endovenosa e promoção do direcionamento ativo e passivo no ambiente tumoral. Os resultados de citotoxicidade celular in vitro em linhagens OVCAR-3 e SKOV-3 demostraram a elevada capacidade das formulações desenvolvidas em liberar o PCX em tempo hábil para ocasionar dano celular. Além disso, a formulação NPPCX- AF apresentou maior capacidade citotóxica que a formulação NP-PCX nas menores concentrações de PCX avaliadas, com redução máxima da viabilidade celular, aproximadamente 22 % para a linhagem celular SKOV-3 e 11% para OVCAR-3, utilizando a concentração de 1000 nM no tempo de 72 horas. Os ensaios de citometria de fluxo e de microscopia confocal mostraram a elevada capacidade de ambos os carreadores serem internalizados através do processo de endocitose, com valores de internalização celular superiores a 80% após 1 hora de tratamento. Estudos in vivo subsequentes serão necessários para confirmar o potencial da formulação NP-PCX-AF no tratamento do câncer de ovário / Paclitaxel (PCX) is a first-line medicinal treatment for ovarian cancer. However, the low PCX water solubility makes necessary the addition of toxic adjuvants to the commercial formulation (Taxol®). Thus, the development of nanostructured delivery systems has been studied to promote a reduction on side effects and an increase on safety and PCX therapeutic efficacy. One of strategies used to increase nanocarriers selectivity to tumor cells is the modification of their surfaces with folate due to folate receptor overexpression on tumor cells membrane. The aim of this work was the development and characterization of folate-modified PLGA nanoparticles (NP-PCXAF) and unmodified nanoparticles (NP-PCX) to PCX delivery, as well as the evaluation of in vitro efficacy of developed systems using human ovarian cancer cell lines. The nanocarriers were produced through nanoprecipitation technique and evaluated through physico-chemical parameters like particle size, polydispersity index, zeta potential, encapsulation efficiency, colloidal stability and infrared spectroscopy. Besides that, antitumoral efficacy and cellular uptake were evaluated using resazurin cytotoxicity assay, confocal microscopy and flow cytometry. The results obtained for folate-modified nanoparticles and unmodified nanoparticles showed low particle size distribution (less than 150 nm) and polydispersity index (less than 0.2) with high PCX encapsulation efficiency (about 100%) which is suitable for intravenous administration and for promoting active and passive targeting to tumor microenvironment. The in vitro cellular cytotoxicity assays of OVCAR-3 and SKOV-3 cell lines demonstrated the high ability of both formulations to release PCX and consequently to cause cellular damage. Furthermore, in lower concentrations NP-PCX-AF formulation showed higher cytotoxicity capacity than NP-PCX with higher cell viability reduction, about 22% for SKOV-3 cell line and 11% for OVCAR-3, using concentration of 1000 nM at 72 hours. Finally, flow cytometry and confocal microscopy assays indicated a high capacity of both nanocarriers to be internalized through endocytosis process. Therefore, subsequent in vivo studies are necessary to confirm the NP-PCX-AF potential for ovarian cancer treatment

This theses focuses on preparation and characterisation and comparison of two types of polymer nanoparticles using photophysical methods. The first type was polystyrene nanoparticles prepared by modified nanoprecipitation method from polymeric sulfonated nanofiber membranes with diffrent content of sulfonated groups. The second type was polymer nanoparticles with diferent lenght of hydrophobic polycaprolactone (PCL) and hydrophilic poly(ethyleneglycol) (PEG) blocks. Both types of nanoparticles conteined encapsulated photosensitizer 5,10,15,20-meso-tetraphenylporphyrin with a high quantum singlet oxygen yield. In the prepared nanoparticles with a photosensitizer, the kinetics of singlet oxygen generation was studied using time resolved spectroscopy. The relative efficiency of photooxidation was studied using external akceptor of singlet oxygen. The effect of temperature, size and matrix (type of polymer) of nanoparticles on photooxidation efficiency was also studied. In the next part of this these was studied the influence of time on the size and stability of nanoparticles and also the effect of solution pH and ionic strength on prepared nanoparticles.