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Made available in DSpace on 2021-06-25T10:49:19Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-03-20 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Prostate cancer is one of the most frequent neoplasms, associated with high mortality. Some factors may intensify its aggressiveness, for example, the overexpression of the epidermal growth factor receptor (EGFR) in some subtypes of prostate tumors. In this context, the inhibition of EGFR helps to fight neoplasia, with the use of the chimeric anti-EGFR monoclonal antibody, cetuximab (CTX). For cancer treatment in the metastatic stage, chemotherapy using docetaxel (DTX) is widely employed, however the drug is very liposoluble, hampering its bioavailability. In this sense, liquid crystalline systems have great potential for the delivery of DTX. Particularly, liquid crystalline nanodispersions can be used in parenteral routes, with many advantages, including the possibility of prolonged drug release, improved pharmacokinetics, allowing passive tumor accumulation by the EPR effect. Thus, the objective of this work was to develop a liquid crystalline nanodispersion (LCN) based on ethoxylated cetyl alcohol 20 and propoxylated 5 as surfactant (Procetyl®), oleic acid, DSPE-PEG-MAL (anchor for CTX binding) and soy phosphatidylcholine as oily phase and 1.5 % poloxamer dispersion in PBS buffer as an aqueous phase. The formulation was loaded with DTX and covalently functionalized with CTX to evaluate its cytotoxic potential against prostate cancer cells. The results suggested that the obtained system has a predominantly hexagonal crystalline phase, was able to be nanodispersed with low polydispersity, and presented negative zeta potential. CTX did not have its structure compromised after thiolation and was successfully covalently linked to LCN. In a prostate cancer cells, PC3, LCNs functionalized with CTX underwent greater cell uptake, resulting in greater cytotoxicity, compared to free DTX and non-functionalized DTX-loaded LCNs. Therefore, the present study reports for the first time an EGFR-targeted liquid crystal nanodispersion for selective to deliver DTX to prostate cells School of Pharmaceutical Sciences of Araraquara São Paulo State University UNESP Department of Drugs and Medicines Federal University of Ceará College of Pharmacy Dentistry and Nursing Department of Pharmacy University of International Integration of the Afro-Brazilian Lusophony Institute of Health Sciences São Carlos Institute of Chemistry University of São Paulo, São Carlos School of Pharmaceutical Sciences of Ribeirão Preto University of São Paulo Ribeirão Preto School of Pharmaceutical Sciences of Araraquara São Paulo State University UNESP Department of Drugs and Medicines CNPq: # 465687/2014-8 FAPESP: #2014/50928-2

Made available in DSpace on 2021-06-25T11:06:52Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-02-20 Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Prostate cancer (PC) is the second type of cancer, causing the greatest number of deaths among the male population in the world. An attractive approach to treat PC involves the superficial modification of liposomes with specific ligands to overexpressed receptors on the surfaces of tumor cells, such as transferrin receptors (TFR's). To our knowledge, this is the first time that transferrin-functionalized liposomes for delivery of DTX to prostate cancer cells were investigated. In this study, we developed and characterized docetaxel- loaded liposomes functionalized with transferrin (LIP-DTX-TF) and evaluated their effect on PC cell lines. The results showed that the systems had a nanometric size and the presence of DTX contributed to the reduction of the vesicle size and liposomes were stable. The encapsulation efficiency of DTX was approximately 69 and 37 % for liposomes with and without functionalization, respectively. The functionalization efficiency using TF was 31 %. The functionalization process did not affect the integrity of TF. DTX encapsulated in the liposomes showed slow and sustained release for LIP-DTX and LIP-DTX-TF (51.70 % and 31.97 %, respectively). The in vitro cytotoxicity assay in PC-3 and PNT2 cell lines suggest that LIP-DTX-TF was more toxic to PC-3 than the commercial formulation. Confocal microscopy proved that functionalized liposomes were successfully internalized in PC-3 cells. Furthermore, the interaction of these systems with the TFR's overexpressed on the cell surface was further corroborated using Quartz Crystal Microbalance with Dissipation technology (QCM-D). Therefore, although additional studies in three-dimensional cell models and in vivo xenograft studies are warranted, it can be concluded that the systems developed here are promising candidates for the delivery of docetaxel to prostate cancer cells. School of Pharmaceutical Sciences São Paulo State University (UNESP) College of Pharmacy Dentistry and Nursing Federal University of Ceara (UFC) School of Pharmaceutical Science of Ribeirão Preto University of São Paulo (USP) São Carlos Institute of Chemistry University of São Paulo (USP) School of Pharmacy and Pharmaceutical Sciences Trinity Biomedical Sciences Institute Trinity College Dublin the University of Dublin School of Biochemistry and Immunology Trinity Biomedical Sciences Institute Trinity College Dublin the University of Dublin School of Medicine Trinity Biomedical Sciences Institute Trinity College Dublin the University of Dublin School of Pharmaceutical Sciences São Paulo State University (UNESP) CNPq: # 465687/2014-8 FAPESP: #2014/50928-2

A leishmaniose é uma doença tropical negligenciada que afeta milhares de pessoas podendo até levar a óbito em sua forma visceral. Durante o seu ciclo de vida, o parasita passa por diversas mudanças ambientais como mudança de temperatura e pH, principalmente quando da transfecção do inseto vetor para o hospedeiro mamífero. Tais mudanças geram estresse celular que pode levar proteínas ao enovelamento incorreto assim como a processos agregativos, sendo necessários sistemas de controle de qualidade proteico para manter a homeostase celular, do qual fazem parte as chaperonas moleculares. Chaperonas como a Hsp100, ajudam a manter a homeostase celular e a adaptação desempenhando um papel importante para protozoários como a Leishmania braziliensis, causador da leishmaniose. A Hsp100 tem papel desagregase, atuando com outras chaperonas moleculares para a extração de polipeptídios de agregados proteicos possibilitando seu desenovelamento e posterior reenovelamento, evitando seu efeito tóxico sobre a célula. A Hsp100 parece ser essencial para esses microrganismos, no entanto não há muito dados disponíveis para Hsp100 em Leishmania sp. e Plasmodium sp. Neste trabalho está descrito o protocolo para expressão e purificação da Hsp100 recombinante de L. braziliensis (rLbHsp100), assim como sua caracterização estrutural e funcional inicial in vitro. A proteína foi analisada por espectropolarimetria de dicroísmo circular, apresentando estrutura típica de proteínas ricas em hélices α, a fluorescência estática de triptofano demonstrou que a proteína possui estrutura terciária local com seus triptofanos parcialmente expostos ao solvente. Por cromatografia de exclusão molecular analítica, observou-se que a LbHsp100 se comporta como um oligômero cujo estado é influenciado tanto pela concentração proteica como pela presença de nucleotídeos adenosina. Análises por ultracentrifugação analítica evidenciaram que a rLbHsp100 em solução apresenta um equilíbrio de diversas espécies havendo deslocamento para um hexâmero de maneira concentração dependente. Análises de SAXS confirmaram a estrutura hexamérica e proporcionaram a obtenção de um modelo ab initio da proteína. Através de microscopia eletrônica de transmissão pode-se observar a forma toróide e a dispersividade do sistema. Por fim, atestou-se que a proteína foi obtida funcional com fraca atividade ATPásica, apresentando também interações com nucleotídeos adenosina (ATP e ADP) assim como com a suramina. Leishmaniasis is a neglected tropical disease that affects thousands of people and may even lead to death in its visceral form. During its life cycle, the parasite undergoes several environmental changes such as temperature and pH changes, especially when transfecting from the vector insect into the mammalian host. Such changes generate a cellular stress that can lead to misfolding as well as to aggregative processes, therefore a protein quality control system is necessary to maintain cell homeostasis, which includes molecular chaperones. Chaperones such as Hsp100 can help maintain cellular homeostasis and adaptation playing an important role for protozoa such as Leishmania braziliensis, which causes leishmaniasis. The Hsp100 has a disaggregase action, acting with other proteins of the chaperone system to extract polypeptides from protein aggregates, allowing their unfolding and subsequent refolding, avoiding their toxic effect on the cell. Hsp100 appears to be essential for these microorganisms, however there is not much data available for Hsp100 in Leishmania sp. and Plasmodium sp. This work describes the protocol for expression and purification of the recombinant Hsp100 of Leishmania braziliensis (rLbHsp100), as well as its initial in vitro characterization. The protein was analyzed by circular dichroism spectropolarimetry, presenting a typical structure of ?-helix rich protein as well as a concentration-dependent structure gain, static fluorescence of tryptophan demonstrated that the protein has local tertiary structure with its tryptophans partially exposed to the solvent. Analytical size exclusion chromatography showed that LbHsp100 behaves as an oligomer whose state is influenced by both the concentration and the presence of adenosine nucleotides. Analysis by analytical ultracentrifugation has shown that the rLbHsp100 in solution exhibits an equilibrium of several species shifting towards a hexamer in a concentration dependent manner. SAXS analyzes confirm the hexameric structure and had provide an ab initio model for the protein. Transmission electron microscopy shows the toroidal form and dispersivity of the system. Finally, the obtained protein had showed catalytic function, and also interacted with adenosine nucleotides (ATP and ADP) as well as suramine.