Polymeric monodispersed nanocarriers with controlled shape and size have been fabricated in the literature primarily using top down processes such as imprint lithography. In this dissertation, the geometric and material property limits of imprint based techniques have been studied. The resulting insight has led to the creation of new processes that significantly extend the limits of imprint processes in several ways: (i) Ability to print nanocarriers with ultra-soft biomaterials (<1MPa modulus); (ii) Sub-50nm diameter cylindrical particles with >3:1 aspect ratio with >5x enhanced wafer yield; (iii) Creation of reentrant barrel shapes that have the potential to be valuable in cellular uptake, such shapes being significant as they lead to fundamental demolding challenges in prior imprint processes; and (iv) Multi-layer nanocarriers which can potentially provide sophisticated functionality such as tailored release kinetics of one or more drugs. By understanding the requirements of bio-functional nanocarriers and related manufacturing constraints, a previously explored Bio Jet and Flash Imprint Lithography (Bio J-FIL) process was refined to perform successful imprints and improve the nanocarrier fabrication scalability. Next, two new fabrication processes have been developed. The first process is called Decoupled Functional Imprint Lithography (D-FIL) which allows fabrication of ultra-soft bio-functional materials (modulus of <1 MPa), challenging sizes (sub-50nm diameter cylinders with aspect ratio > 3:1), and reentrant barrel shapes. The second decoupled process, Dual Removable Layer Lithography (DRLL), has been developed to specifically create multi-layered cylindrical nanocarriers. Nanocarriers fabricated with D-FIL and DRLL process have been shown to chemically bind with an imaging agent, and model anti-cancer drugs. Drug (siRNA) retention (>90% over 9 days) and stimuli triggered release studies were performed on sub-100nm cylindrical PEGDA nanocarriers. It was found that these nanocarriers show accelerated triggered drug release when exposed to a hydrolase, Cathepsin B. While the exact mechanisms causing the triggered release are not fully understood, a few possible explanations are provided based on the experiments reported. Finally, the D-FIL, the DRLL, and the refined Bio J-FIL processes have been successfully demonstrated at the prototype scale as well as at the pilot scale in collaboration with an industrial partner, Molecular Imprints Inc. / text

O estudo sobre a utilizaÃÃo de copolÃmeros na dissoluÃÃo de fÃrmacos pouco solÃveis em Ãgua vem sendo cada vez mais praticado, devido, principalmente, à baixa toxicidade desses carreadores e da sua alta eficiÃncia no processo de dissoluÃÃo. Com o uso de copolÃmeros surfactantes, que formam micelas em soluÃÃo, à possÃvel aumentar a solubilidade e controlar o tempo e/ou atuar na liberaÃÃo de fÃrmacos hidrofÃbicos no organismo. Os copolÃmeros triblocos anfifÃlicos denominados EmPnEm (PluronicÂ), P123 (E21P69E21) e F127 (E98P69E98), onde E representa o monÃmero Ãxido de etileno [â(OCH2CH2) â], P, o monÃmero Ãxido de propileno [âOCH2CH(CH3)â], e os subscritos m e n denotam o comprimento do bloco, estÃo sendo alvos desta pesquisa devido as suas propriedades de formaÃÃo de gel a temperatura ambiente (termorresponsidade), possibilitando a aplicaÃÃo subcutÃnea, e sua capacidade de solubilizaÃÃo de ativos lipofÃlicos no nÃcleo micelar de polioxipropileno (Pn). Misturas binÃrias de Pluronics de hidrofobicidades semelhantes, em sua grande maioria, apresentam capacidade de solubilizaÃÃo maior que para os Pluronics sozinhos. Assim, visando potencializar a solubilizaÃÃo dos fÃrmacos griseofulvina, fÃrmaco modelo utilizado em vÃrios trabalhos, e mangiferina, ativo antioxidante, e promover a maior biodisponibilidade desses fÃrmacos no organismo, sistemas de micelas mistas compostas por F127 e P123 serà o foco dessa pesquisa, com finalidade de unir suas propriedades geleificantes e de biocompatibilidade para aplicaÃÃo subcutÃnea / T he use of copolymers in the dissolution of poorly soluble drugs in water is being increasingly practiced, principally, due to the low toxicity of these carriers and their high ef ficiency in the dissolution process. Using copolymer surfactants, which form micelles in solution, can enhance the solubility and control the time and act in the release of hydrophobic drugs in the body. The amphiphilic triblock copolymers known E m P n E m (Pl uronicÂ), P123 (E 21 P 67 E 21 ) and F127 (E 98 P 67 E 98 ), where E is the ethylene oxide monomer [ - (OCH 2 CH 2 ) - ] and P, propylene oxide monomer [ - OCH 2 CH(CH 3 ) - ], and the subscripts, n and m , denote the block length, being targets of this research due to their gelling p roperties at room temperature, enabling the application subcutaneo us, and its ability to solubilis e the active lipophilic in the micellar core. Binary mixtures of Pluronics of similar hydrophobicities, mos tly, show greater solubilis ation capacity for Plur onics alone. Thus, i n order to enhance the solubilis ation of griseofulvin drugs, drug model used in several works, and mangiferin, active antioxidant, and promote greater bioavailability of these drugs in the body systems of mixed micelles composed of F12 7 and P123 will be the focus of this research, in order to unite their gelling properties and biocompatibility for subcutaneous administration

Biologics such as DNA and protein have immense biomedical applications, especially in diagnosis and therapy. However, many barriers hinder these applications, including biologics transport and liability in biological systems. Therefore, biocompatible and stable nanocarriers with high Biologics loading efficiency can provide a platform for advances in biologics applications. Metal-organic frameworks (MOFs) have gained significant interest within the biomedical field, mainly because of their building block versatility, porosity, stability, and chemical and biological functionality. Currently, increasing research is dedicated to improving MOFs biocompatibility, stability, and functionality for drug delivery. Using biomolecules as organic linkers could improve biocompatibility, physiological condition stability, and biological functionality. The main goal of this dissertation is to investigate the applicability of biomolecule-based Metal-organic frameworks (BioMOFs) as nanocarriers to achieve cellular delivery of active biologics. Herein, we analyzed adenine and saccharate metal−organic frameworks (BioMOF) in terms of biocompatibility, loading capability, protection, and cellular delivery of biologics. Our findings suggest that the usage of biomolecules as an organic linker generates BioMOFs with reduced cytotoxicity compared with the widely used MOFs such as Zinc Imidazole framework-8 (ZIF-8). In addition, the base-pairing functionality of coordinated adenine of KAUST-BioMOFs (KBMs) is preserved and can be used to load ssDNA. Both KAUST-BioMOFs (KBMs) and Zinc adenineated framework (ZAF) load, protect, and deliver functional ssDNA to cells. In addition, we showed the possibility of in situ encapsulation of active lysozyme in zinc saccharate (Zn-Sac) with modified synthesis procedures.

O presente estudo teve como principal objetivo aprimorar os efeitos antitumorais de uma nova estratégia imunoterapêutica para controle de tumores induzidos pelo vírus do papiloma humano tipo 16 (HPV-16). Para tal finalidade foi utilizado um novo conceito de imunoterapia ativa, baseada em uma vacina de DNA recombinante, desenvolvida no Laboratório de Desenvolvimento de Vacinas da USP (pgDE7h) e diferentes abordagens experimentais delineadas com a finalidade de aumentar a eficácia terapêutica do tratamento. Desta forma, o presente trabalho focou no desenvolvimento de novos sistemas de entrega para a vacina de DNA empregando três nanocarregadores distintos, de natureza lipídica, proteica ou peptídica, e a avaliação da eficácia terapêutica desses frente a tumores pré-estabelecidos. O presente trabalho também avaliou a combinação da imunoterapia, baseada no vetor pgDE7h, com a quimioterapia, empregando o composto cisplatina, de forma a permitir o tratamento de tumores em estágios mais avançados de crescimento. Os resultados demonstram que a associação do plasmídeo vacinal com vesículas peptídicas mostrou-se capaz de ativar células dendríticas murinas in vitro e promover aumento na sobrevivência de animais frente ao modelo de tumores subcutâneos, sem induzir toxicidade. Quando combinada ao tratamento com cisplatina, a vacina, principalmente associada à eletroporação, demonstrou um efeito sinérgico, promovendo regressão total de tumores, aumento expressivo na ativação de linfócitos T CD8 E7-específicos, indução de resposta de memória efetora e memória residente e controle de populações imunossupressoras de forma sistêmica e no microambiente tumoral. Em resumo, a pesquisa ampliou os conhecimentos sobre a ação da imunoterapia ativa contra tumores induzidos por HPV e abriu perspectivas importantes para sua utilização em condições clínicas. / The present study aimed to improve the antitumor effects of a new immunotherapeutic strategy for the control of tumors induced by human papillomavirus type 16 (HPV-16). For this purpose, a new concept of active immunotherapy based on a recombinant DNA vaccine developed at the Laboratory of Vaccine Development at USP (pgDE7h) was used, and different experimental approaches were designed to increase the therapeutic efficacy of the treatment. Thus, the present work focused on the development of new delivery systems for the DNA vaccine using three distinct nanocarregadores, of a lipid, protein or peptidic nature, and the evaluation of the therapeutic efficacy of these in front of pre-established tumors. The present study also evaluated the combination of pgDE7h-based immunotherapy with chemotherapy using the cisplatin compound to allow the treatment of tumors in more advanced stages of growth. The results demonstrate that the association of the peptide-vesicle vaccine plasmid was shown to activate murine dendritic cells in vitro and promote increased survival of animals against the subcutaneous tumor model without inducing toxicity. When combined with cisplatin treatment, the vaccine, mainly associated with electroporation, demonstrated a synergistic effect, promoting total tumor regression, expressive increase in the activation of CD8 E7-specific T lymphocytes, induction of effector and resident memory response and control of immunosuppressive populations in a systemic way and in the tumor microenvironment. In summary, the research expanded knowledge about the action of active immunotherapy against HPV-induced tumors and opened important perspectives for its use under clinical conditions.

Nanoscale drug delivery systems have a great impact in current medical field. These carriers have the potential to improve the efficacy and reduce the toxicity of various medicinal products. A broad variety of different lipid based carriers had been developed and used as delivery systems in the past decades. This dissertation focused on the development of solid lipid nanoparticles (SLN) as delivery systems for a chemotherapeutic agent, docetaxel, and the use of liposomes as a carrier for recombinant protein vaccines. Docetaxel is a potent anticancer drug. However, there continues to be a need for alternative docetaxel delivery systems to improve its efficacy. Docetaxel nanoparticles comprised of lecithin as the main component were engineered using two methods, the emulsion precursor method and the solvent emulsification/evaporation method. Docetaxel in nanoparticles were more effective in killing tumor cells in culture than docetaxel solution. The intravenously injected docetaxel-nanoparticles increased the accumulation of docetaxel in tumors in mice. When administered by intravenous injection or oral routes, docetaxel-nanoparticles showed antitumor activity in tumor-bearing mice. The lecithin-based nanoparticles have the potential to be a novel biocompatible and efficacious delivery system for docetaxel. Liposomes, a well-known lipid based carrier, have been investigated extensively as a vaccine delivery system. The adjuvant activities of liposomes with different net surface charges (neutral, positive, or negative) were evaluated when simply admixed with protein antigens. Immunization study in mice after subcutaneously injection of different net charged liposomes showed different antibody responses, depending on the protein antigens. Antigens (OVA, PA) admixed with the negatively charged liposomes prepared with phospholipid, DOPA, induced a strong and functional antibody response comparable to the positively charged liposomes prepared with DOTAP lipid. The negatively charged DOPA liposomes admixed with OVA also induced OVA-specific CD8��� cytotoxic T lymphocyte responses and significantly delayed the growth of OVA-expressing B16-OVA melanoma in a mouse model. The adjuvant activity of the negatively charged liposomes may be related to the liposome's ability (i) to upregulate the expression of molecules related to the activation and maturation of antigen-presenting cells and (ii) to slightly facilitate the uptake of the antigens by antigen-presenting cells. Simply admixing certain negatively charged liposomes with certain protein antigens of interest may represent a novel platform for vaccine development. / Graduation date: 2012 / Access restricted to the OSU Community at author's request from Sept. 6, 2011 - Sept. 6, 2012

Submitted by DIEGO FARIA COLA null (coladf@gmail.com) on 2016-03-02T22:46:11Z No. of bitstreams: 1 dissertacao.pdf: 1500429 bytes, checksum: 0915c0a782a07bc716f94ac8d6e25db8 (MD5) / Rejected by Ana Paula Grisoto (grisotoana@reitoria.unesp.br), reason: Solicitamos que realize uma nova submissão seguindo as orientações abaixo: Por favor verifique a numeração do arquivo submetido. O número de páginas está inconsistente com o sumário. A versão submetida é considerada a versão final da dissertação/tese, portanto não poderá ocorrer qualquer alteração após a aprovação. Caso a troca do arquivo no Repositório seja imprescindível, o aluno deverá entrar em contato com a Seção Técnica de Pós-Graduação de sua unidade e verificar quais os procedimentos necessários devem ser realizados para a substituição. Agradecemos a compreensão. on 2016-03-04T13:53:10Z (GMT) / Submitted by DIEGO FARIA COLA null (coladf@gmail.com) on 2016-03-04T14:12:33Z No. of bitstreams: 1 dissertacao.pdf: 1500361 bytes, checksum: bff7d0721d385321109c2e58dfa5697a (MD5) / Approved for entry into archive by Ana Paula Grisoto (grisotoana@reitoria.unesp.br) on 2016-03-04T14:43:05Z (GMT) No. of bitstreams: 1 cola_df_me_soro.pdf: 1500361 bytes, checksum: bff7d0721d385321109c2e58dfa5697a (MD5) / Made available in DSpace on 2016-03-04T14:43:05Z (GMT). No. of bitstreams: 1 cola_df_me_soro.pdf: 1500361 bytes, checksum: bff7d0721d385321109c2e58dfa5697a (MD5) Previous issue date: 2016-02-26 / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / O estudo de características e aplicações de estruturas em escala nanométrica passou a ter um grande interesse de pesquisadores das áreas médicas (humana e animal), ambiental e agrícola. Entre essas estruturas, destacam-se os nanocarreadores, que permitiram a melhoraria da biodisponibilidade de muitos compostos bioativos e a diminuição de possíveis efeitos toxicológicos. Além disso, evitar a ocorrência de altos prejuízos na agropecuária com o desenvolvimento de novos nanocarreadores voltados ao combate de endo e ectoparasitas causadores de grandes prejuízos aos pecuaristas. Fármacos como a Ivermectina e o Metopreno são utilizados no combate de ectoparasitas, no entanto, estes compostos possuem problemas relacionados à biodisponibilidade. Este trabalho teve como objetivo preparar e caracterizar sistemas carreadores lipídicos, como as Nanopartículas Lipídicas Sólidas e os Carreadores Lipídicos Nanoestruturados a fim de melhorar e produzir alternativas para o uso destes compostos visando aplicações em veterinária. Na primeira etapa do trabalho foram preparadas e caracterizadas as nanopartículas lipídicas sólidas e os carreadores lipídicos nanoestruturados contendo os fármacos ivermectina e metopreno. As propriedades físico-químicas das suspensões coloidais, como, diâmetro, polidispersão, potencial zeta, pH e eficiência de encapsulação foram avaliadas em função do tempo. A eficiência de encapsulação alcançada foi acima de 99% para ambos os fármacos, sendo que as suspensões coloidais apresentaram estabilidade durante o período investigado. Os resultados do ensaio de citotoxicidade e genotoxicidade demonstram que as nanopartículas testadas para a encapsulação possibilitaram um aumento na viabilidade celular das células testadas, onde as nanopartículas lipídicas sólidas apresentaram menor toxicidade do que os carreadores lipídicos nanoestruturados. As nanopartículas lipídicas sólidas apresentaram tamanho médio de 285,6 ± 15,8 nm e os carreadores lipídicos nanoestruturados de 257,5 ± 7,4 nm. Todas os carreadores apresentaram morfologia esférica, sem formação de agregados e sem alterações morfológicas após a encapsulação. Portanto, os resultados deste trabalho demostraram que as nanopartículas apresentaram boas características coloidais e a presença ou ausência de drogas não provocam alterações nas características das nanopartículas, possibilitando o desenvolvimento de sistemas carreadores para estas drogas visando aplicações veterinárias. / The study characteristics and structures of applications in nanoscale have a great interest of researchers in medical areas (human and animal), environment and agriculture. Among these, the nanocarriers stand out, that allowed improve the bioavailability of many bioactive compounds and the reduction of potential toxicological effects, and prevent the occurrence of high losses in agriculture with the development of new nanocarriers aimed at fighting and endo ectoparasites causing great harm to farmers. Drugs such as Ivermectin and Methoprene are used to combat ectoparasites, however, these compounds possess bioavailability problems related. This study aimed to prepare and characterize lipid carrier systems, such as Solid Lipid Nanoparticles and Nanostructured Lipid carriers to improve and produce alternatives to the use of these compounds targeting applications in veterinary. In the first stage of the work we have been prepared and characterized the solid lipid nanoparticles and nanostructured lipid carriers containing the drug ivermectin or methoprene. The physico-chemical properties of colloidal suspensions such as, diameter, polydispersity, Zeta potential, pH and encapsulation efficiency were measured as a function of time. The encapsulation efficiency was achieved above 99% for both drugs, and colloidal suspensions were stable during the period investigated. The results of the cytotoxicity and genotoxicity test demonstrate that the tested nanoparticles for encapsulating allowed an increase in cell viability of test cells, where the solid lipid nanoparticles had lower toxicity than the nanostructured lipid carriers. The solid lipid nanoparticles have an average size of 285,6 ± 15,8 nm and nanostructured lipid carriers 257,5 ± 7,4 nm. All the carriers had spherical morphology without forming aggregates and without morphological changes after encapsulation. Therefore, the results of this study demonstrated that the colloidal nanoparticles showed good characteristics and the presence or absence of drug do not cause changes in the characteristics of the nanoparticles, allowing the development of carrier systems for drug targeting these veterinary applications. / FAPESP: 2013/24788-6

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / No presente trabalho, nanopartÃculas de M0,5Zn0,5Fe2O4 (M= Ni ou Mn) foram preparadas por sÃntese hidrotÃrmica sob condiÃÃes brandas sem qualquer procedimento de calcinaÃÃo. Amostras de composiÃÃo MFe2O4 (M = Zn, Ni ou Mn) tambÃm foram sintetizadas para fins de comparaÃÃo. As propriedades estruturais e magnÃticas das amostras foram investigadas por DifraÃÃo de Raios-X (DRX), Espectroscopia na RegiÃo do Infravermelho com Transformada de Fourier (FTIR), Espectroscopia Raman, Espectroscopia MÃssbauer, MagnetÃmetro de Amostra Vibrante (VSM) e Microscopia EletrÃnica de TransmissÃo (TEM). As anÃlises de DRX exibiram picos caracterÃsticos da fase de espinÃlio em todas as amostras sintetizadas. O tamanho mÃdio de partÃcula foi obtido por DRX, VSM e TEM e apresentou valor em torno de 10 nm para o M0,5Zn0,5Fe2O4. As imagens de TEM exibiram nanopartÃculas de morfologia cÃbica. Os parÃmetros magnÃticos observados por MÃssbauer e VSM mostraram comportamento superparamagnÃtico para todas as amostras contendo Zn, alÃm de altos valores de magnetizaÃÃo de saturaÃÃo (~55 meu/g) para a amostra Ni0,5Zn0,5Fe2O4. Devido a isto, este nÃcleo magnÃtico foi escolhido para a formulaÃÃo do nanocarreador. Desta forma, este foi inicialmente modificado com Ãcido olÃico. As anÃlises de TG e FTIR evidenciaram a presenÃa de molÃculas de oleato na superfÃcie da nanopartÃcula. AlÃm disso, o coeficiente de revestimento do processo realizado foi 3,7 oleato / nm2. Um estudo de adsorÃÃo da piplartina na nanopartÃcula modificada (NiZn-AO) foi realizado e observou-se relaÃÃo (NiZn-AO : Piplartina) Ãtima de 1 : 2 (m/m). ApÃs o processo adsortivo, o sistema contendo NiZn-AO e piplartina foi revestido com matriz polimÃrica constituÃda de P123 e F127 para conferir biocompatibilidade ao sistema, e formar o nanocarreador. Testes preliminares de hipertermia foram realizados na nanopartÃcula e observou-se que um campo de 126 Oe conduz a geraÃÃo de calor para alcanÃar uma temperatura de 42ÂC, dentro da faixa de hipertermia moderada. Portanto, o nanocarreador formulado apresenta potencial para aplicaÃÃes biomÃdicas. / In this work, nanoparticles of the M0,5Zn0,5Fe2O4 (M = Ni ou Mn) have been prepared by hydrothermal synthesis in mild conditions without any calcinations process. MFe2O4 (M = Zn, Ni or Mn) nanoparticles were also prepared for comparison. The structural and magnetic properties of the ferrites were investigated by X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), Raman spectroscopy, MÃssbauer spectroscopy, vibrating sample magnetometer (VSM) and Transmission electron microscopy (TEM). XRD analysis showed peaks of the spinel phase for all samples. The average particle size was obtained by XRD, TEM and VSM and values around 10 nm were found for M0,5Zn0,5Fe2O4. TEM images showed particles of cubic morphology. The magnetic parameters observed by MÃssbauer and VSM shown superparamagnetic behavior for the samples containing Zn and high saturation magnetization values (~55 emu/g) for Ni0,5Zn0,5Fe2O4. Due to this, it was chosen for formulating the nanocarrier. Thus, it was first modified with oleic acid. The TG and FTIR analysis revealed the presence of oleate molecules on the surface of the nanoparticle. Furthermore, the coating coefficient of the process carried was 3.7 oleate / nm2. A study of piplartine adsorption on the modified nanoparticle (NiZn-AO) was performed and the optimum relationship (NiZn-AO: piplartine) was 1 : 2 (w / w). After the adsorptive process, the system containing NiZn-AO and piplartine was coated with polymeric matrix consisting of F127 and P123 to confer biocompatibility to the system and form the nanocarrier. Hyperthermia tests were performed in nanoparticles and it was observed that a field of 126 Oe leads to heat generation to attain a temperature of 42ÂC within the range of moderate hyperthermia. Therefore, the fabricated nanocarrier had potential for biomedical applications.

Submitted by MARCIA ROVADOSCHI (marciar@unifra.br) on 2018-08-17T19:15:30Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertacao_MichelePereiraMoreira.pdf: 1257282 bytes, checksum: ec3939cec40b071e77b87bd241002dab (MD5) / Made available in DSpace on 2018-08-17T19:15:30Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertacao_MichelePereiraMoreira.pdf: 1257282 bytes, checksum: ec3939cec40b071e77b87bd241002dab (MD5) Previous issue date: 2017-03-31 / Simvastatin is used to treat hypercholesterolemia acting by inhibition of 3-hydroxy-methylglutaryl-coenzyme A (HMG-CoA) reductase responsible for cholesterol synthesis. Studies carried out in humans, in animal models and in vitro observed neural damage reduction and increased glutamate uptake after simvastatin treatment. Glutamate is the major excitatory neurotransmitter in the central nervous system with crucial role in several physiological mechanisms, but at high levels it leads to cell death and participate to neurodegenerative diseases. Despite of pharmaceutical characteristics of simvastatin, as lipophilic drug that can cross cell membranes it has low bioavailability and non-specific biodistribution, simvastatin reach nervous system at low rates, limiting the pleiotropic effects described. Nanoemulsions may enhance solubility and increase biodistribution of lipophilic drugs, thus this technology can improve simvastatin performance in the neurodegenerative diseases treatment. The aim of study is producing simvastatin-loaded nanoemulsion, evaluate its physicochemical characteristics besides biological safety, and effect against glutamatergic toxicity. Blank nanoemulsion (without drug; BNE) and 1 mg/mL simvastatin nanoemulsion (SNE) were produced by spontaneous emulsification. Physicochemical characterization and stability were determined at three different temperatures (± 4 oC, ± 23 oC or ± 40 oC) for 15 days. Toxicity of formulations at 0.1 μg/mL; 1 μg/mL e 10 μg/mL was evaluated in Vero cell linage cultures and hippocampal slices from rat. Effect of formulations against glutamatergic excitotoxicity (10 mM glutamate) was evaluated at hippocampal slices from rat. All procedures with animals were previously approved by local Ethical Committee (CEUA no 05/2016). Formulation showed macroscopically homogeneous aspect and white color with Tyndall effect. Particle size was approximately 204 nm (BNE) and 139 (SNE). Zeta potential was -3 mV and -5 mV, for BNE and SNE respectively. Both of them appeared polydispersity index lower than 0.3, pH ± 6.5. Drug content was ± 1.01 mg/mL with encapsulation efficiency ± 98%. Formulations did not show any physicochemical alterations following 15 days at ± 23 oC or ± 40 oC, but simvastatin content was reduced. However, nanoemulsions exposed to ± 4 oC had constant drug content with increased particle size and polydispersity index. For all formulations, Zeta potential and pH were constant during 15 days. Cytotoxicity was no detected for all nanoemulsions. When hippocampal slices were incubated with glutamate with free simvastatin or SNE, 42% of cells were died, without effect of simvastatin. At the present work, nanoemulsions were produced efficiently, without cytotoxicity at parameters assayed. Additional studies should be performed to improve nanoemulsions stability. Simvastatin-loaded nanoemulsion or free did not show effect against glutamatergic damage evaluate in hippocampal slices from rat. / A sinvastatina é um fármaco prescrito para hipercolesterolemia, que atua inibindo a 3-metil-hidróxi-glutaril coenzima A (HMG-CoA) redutase, que controla a síntese do colesterol. Estudos em humanos e modelos in vitro e/ou animais indicam que a sinvastatina é capaz de reduzir o dano neural, e também aumentar a captação do glutamato. O glutamato é o principal neurotransmissor excitatório do sistema nervoso central, sendo fundamental em diferentes mecanismos fisiológicos, mas em altas concentrações pode induzir à morte celular e doenças neurodegenerativas. Apesar da sinvastatina ser um fármaco lipofílico e atravessar facilmente as barreiras celulares, a sua baixa biodisponibilidade e biodistribuição inespecífica fazem com que ela alcance o sistema nervoso em baixas concentrações, limitando os efeitos pleiotrópicos descritos. O uso de nanoemulsões pode aumentar a solubilidade e melhorar a biodistribuição de fármacos lipofílicos e, assim, pode melhorar o desempenho da sinvastatina no tratamento de doenças neurológicas. Portanto, o objetivo deste trabalho é preparar uma nanoemulsão contendo sinvastatina, avaliar suas características físico-químicas e de segurança, e avaliar o efeito contra a excitotoxicidade glutamatérgica. As nanoemulsões branca (NEB) ou contendo sinvastatina (NES) na concentração de 1 mg/mL foram preparadas por emulsificação espontânea. Foi realizada a caracterização físico-química e a estabilidade das formulações foi determinada em três temperaturas (±4 oC, ±23 oC ou ±40 oC), por 15 dias. A toxicidade das formulações foi avaliada nas concentrações de 0,1 μg/mL; 1 μg/mL e 10 μg/mL em células Vero e em fatias de hipocampo de ratos. A avaliação de efeito contra excitotoxicidade foi realizada nas fatias de hipocampo submetidas ao glutamato 10 mM. Os procedimentos descritos foram aprovados pela Comissão de Ética no Uso de Animais (CEUA) do Centro Universitário Franciscano (protocolo 05/2016). As formulações produzidas apresentaram-se homogêneas, de cor branca levemente azulada. O tamanho de partícula foi de aproximadamente 204 nm (NEB) e 139 nm (NES). O potencial zeta foi de -3 mV e -5 mV, para NEB e NES, respectivamente. Ambas apresentaram índice de polidispersão abaixo de 0,3, e o pH ± 6,5. O teor de fármaco encontrado em NES foi de ± 1,01 mg/mL com taxa de associação de ± 98%. As nanoemulsões não apresentaram alterações nas características físico-químicas após 15 dias de armazenamento, mantidas a ± 23 oC ou ± 40 oC, mas houve redução no teor de sinvastatina. Enquanto as nanoemulsões mantidas em ± 4 oC mantiveram o teor do fármaco, mas sofreram aumento no tamanho e índice de polidispersão. O potencial zeta e o pH mantiveram-se estáveis em todas as formulações desde o preparo até após 15 dias. Não houve indicativo de toxicidade por parte das nanoemulsões nos ensaios propostos. Nas fatias de hipocampo submetidas ao glutamato 10 mM e à sinvastatina livre ou em nanoemulsão, observou-se redução em até 42% da viabilidade, sem efeito da sinvastatina. Foi possível, no presente trabalho, produzir com sucesso nanoemulsões contendo sinvastatina, que não demonstraram toxicidade através dos parâmetros avaliados. Contudo estudos adicionais para melhorar a estabilidade deverão ser realizados. A sinvastatina livre ou em nanoemulsão, não foi capaz de reverter o dano provocado pelo glutamato no modelo proposto.

Master thesis deals with the use of bacteriophage as a theranostic drug nanocarrier. The term theranostics is used in recent years for systems that allow connecting of diagnostics, targeted drug delivery and monitoring of patient’s response to administered treatment in a single modality. These systems are very suitable especially with heterogeneous diseases, such as cancer. Nowadays, the treatment of cancer has often severe side effects to the patient’s body, which lowers his capability to fight the disease. Theoretical part of this work is focused on the properties of viral capsids, proteins and inorganic materials as drug nanocarriers. In practical part of this work, different methods for cultivation of bacteriophage are compared, both in liquid and solid medium and with different concentrations of the maltose, trough whose receptors bacteriophage is able to enter the host cell. Optimal was cultivation with 0.2% maltose in solid medium. Practical part is focused mainly on the use of bacteriophage as a nanocarrier for cytotoxic drug doxorubicin. Bacteriophage was able to encapsulate all applied concentrations of doxorubicin (0; 12.5; 25; 50; 100 and 200 g/ml), which was proved using fluorescent detection. Different times of encapsulation (2; 4; 8 and 12 hours) were studied. Optimal time was 2 hours. Encapsulation properties of bacteriophage were compared to apoferritin. Bacteriophage was able to encapsulate 4× higher concentrations of doxorubicin and its release during rinsing with water was 10× lower compared to apoferritin. This work concludes that bacteriophage is a very suitable platform for targeted drug delivery in theranostics.

Il a été démontré que l’acidité de l’environnement tumoral jouait un rôle dans la résistance aux chimiothérapies. L’utilisation de nanovecteurs, tels que les nanocapsules lipidiques (LNC), permet non seulement d’améliorer le temps de biodistribution de substances actives, mais aussi de cibler l’environnement tumoral tout en protégeant les actifs de cet environnement acide. L’objectif de cette thèse porte ainsi sur l’optimisation et l’évaluation de LNC furtives et pH-sensibles dans le contexte du mélanome.Dans un premier temps, ces travaux ont consisté à caractériser la vascularisation de mélanomes humain et murin. Ces études ont permis de comparer différentes tumeurs (densité, taille et structure) et de déterminer si l’usage de nanomédecines est approprié dans ce contexte.La seconde partie s’est orientée sur l’élaboration de polymères combinant furtivité et pH-sensibilité. Ces copolymères composés de N-vinylpyrrolidone (NVP)et de vinylimidazole ont été synthétisés par polymérisation RAFT et post-insérés à la surface des LNC. Ces modifications ont donné lieu à des LNC présentant des charges de surface pH-dépendantes,entrainant une augmentation de leur internalisation à pH acide dans des cellules de mélanome. Finalement, des études de biodistribution ont mis en évidence l’intérêt de la NVP dans le développement de nanovecteurs furtifs. En conclusion, les copolymères développés ont permis de prolonger le temps de circulation, mais aussi d’apporter des propriétés pH-sensibles qui pourraient améliorer l’internalisation tumorale des LNC in vivo et donc de potentialiser l’effet d’une thérapie anticancéreuse. / Tumor acidity has been shown to play a major role in resistance to chemotherapy. The use of nanomedicines, as lipid nanocapsules (LNC), allows to protect drugs from this acidic environment. They can also improve the biodistribution of therapeutics and to target the tumor environment. The aim of this thesis concerns the evaluation and characterization of stealth and pH-sensitive LNC in the context of melanoma. Firstly, these works consisted in characterizing the vascularization of human and mice melanoma. These studies allowed to compare different tumors (density, size and structure), and determine if the used of nanocarrier is suitable in the context of melanoma.The second part of this thesis described the development and the characterization of new copolymers, combining stealth and pH-sensitive properties. These copolymers, composed of Nvinylpyrrolidone(NVP) and vinylimidazole, were synthesized by RAFT polymerization and were post in sertedonto LNC surface. These modifications allowed to obtain charge reversal nanocarriers, leading to increase their melanoma cell uptake underacid pH. Finally, biodistribution of these modified nanoparticles was studied in vivo and highlighted the interest of NVP in the development of stealth nanocarriers. To conclude, the developed copolymers able to extend nanocarrier circulation time and to provide pH-responsive properties which should increase the tumor internalization of LNC invivo and potentiate the effect of anticancer drugs.