Des nanoparticules d’or fonctionnelles ont été synthétisées avec pour objectifs l’élaboration de nanomatériaux pour applications biomédicales et propriétés physiques originales. L’étude de la fonctionnalisation a conduit à utiliser le chimie “click” entre des nanoparticules d’or portant des ligands thiolates azoturés et des alcynes terminaux en utilisant un catalyseur au cuivre (I) renforcé par un ligand polyazoté, ce qui a permis d’introduire des fonctions très variées. En particulier le récepteur folate a été greffé de cette façon pour la synthèse de nouveaux vecteurs du docétaxel, un puissant agent anti-cancéreux dont l’étude est menée en collaboration. Des nanoparticules d’or fonctionnelles ont aussi servi de support pour le greffage de complexes du fer (II) à transition de spin, ce qui a permis de réaliser l’étude en 2D par différentes techniques physiques en collaboration. Enfin un nouveau ligand azoté a été mis au point pour la synthèse de nanoparticules d’or originales. / The thesis concerns functionalizations and applications of gold nanoparticles (AuNPs). In the aspects of functionalization of AuNPs, we concentrated on efficiently functionalized AuNPs by “Click” chemistry. In the aspects of applications, the PEG capped AuNPs was prepared to encapsulate vitamins, which has a potential use in hydrophobic part of human body; the folate functionalized AuNPs was used for docetaxel delivery for cancer therapy; the novel synthesis of triazole stabilized AuNPs used for biosensors; and the citrate capped AuNPs introduced into the silica thin films to check the SERS effect and spin crossover of iron complexes.

Rendre possible l'administration orale du docétaxel (Dtx), un puissant agent anticancéreux administré par voie intraveineuse, représente un défi important en cancérologie. Disposer de formulations administrables par voie orale, moins toxiques et mieux tolérées, représenterait une avancée majeure au plan clinique. Toutefois, plusieurs études ont montré que la très faible biodisponibilité du Dtx par voie orale résulte simultanément de : (i) sa faible solubilité aqueuse, (ii) son faible passage transépithélial au niveau intestinal, (iii) son efflux par les pompes d’efflux (P-gp) et son métabolisme par le cytochrome P450. Nous avons conçu une formulation capable de répondre simultanément à ces différents problèmes. Ainsi, nous avons tout d’abord fait appel aux cyclodextrines (CDs) pour augmenter la solubilité apparente du Dtx. La complexation du Dtx avec la méthyl-β-CD a permis d’augmenter la solubilité apparente du Dtx d’environ 5000 fois. Ce complexe a ensuite été associé à des nanoparticules(NPs) polymères composées d’un coeur de poly(cyanoacrylate d’alkyle) et recouvert en surface de chitosane thiolé afin de leur conférer des propriétés mucoadhésives et de diminuer localement le métabolisme. Ces NPs ont montré in vitro et ex vivo leur capacité à arriver intactes au niveau de l’intestin, d’y adhérer et de libérer le Dtx de manière contrôlée dans le temps, et finalement d’améliorer son absorption intestinale. Une évaluation de la toxicité de cette formulation vis-à-vis de la muqueuse intestinale suggère que l’encapsulation du Dtx dans les NPs assure une certaine protection de la muqueuse. Au final, la formulation orale proposée offre en perspective la possibilité de moduler la dose administrée, donc d’ajuster finement la posologie et finalement d’offrir au corps médical et aux patients les bénéfices d’une thérapie personnalisée. / Docetaxel (Dtx) is an anticancer drug widely used in therapy. However, severe allergic reactions and peripheral neurotoxicity are caused by the intravenous administration of the commercial formulation Taxotere®, requiring thus the oral administration of dexamethasone and antihistamine before infusion. In this context, there is an urgent need to design new orally administered Dtx formulations to reduce these side effects and improve the patient’s qualityof life. Dtx belongs to the Class IV of the Biopharmaceutical Classification System, which comprises substances with both low solubility in aqueous fluids and low apparent intestinal permeability. This represents a major drawback when foreseeing oral delivery. Moreover, Dtx has been shown to be substrate of biological transporters and/or metabolized in the intestinal barrier. We designed a formulation able to overcome these different problems. First of all, we solved the low solubility problem by using cyclodextrin (CDs). The complexation of Dtx with the Methyl-ß-CD allowed increasing the apparent solubility of the Dtx about 5000 times. This complex was then associated to polymeric core-shell nanoparticles (NPs) based on poly(isobutyl cyanoacrylate) coated with thiolated chitosan. Among the characteristics of this system, mucoadhesion properties are the most important for an oral administration. The presence of the positively charged chitosan chains, and the thiol groups at the surface allow NPs to adhere to the mucus layer. In vitro and ex vivo experiments showed that these NPs were able to ensure a time-controlled release of Dtx and to improve its absorption at the intestinal level. An evaluation of the local intestinal toxicity of this formulation suggests that the encapsulation of Dtx into polymeric NPs had a protective effect allowing a preservation of the mucosa integrity. The further step will be to confirm by in vivo studies if this kind of nanoparticles is able to enhance the bioavailability of Dtx allowing to display an anti-tumor activity.

Multidrug resistance phenotype of tumor cells describes resistance to wide range of structurally unrelated anticancer agents and is a serious limitation to effective chemotherapy. It is a multifactor yet not fully elucidated phenomenon by the involvement of diverse cellular pathways. Aim of this study was to investigate the resistance mechanisms developed against docetaxel and doxorubicin that are widely used in the treatment of breast cancer in model cell line MCF-7. Resistant sublines were developed by application of drugs in dose increments and effect of docetaxel and doxorubicin on drug applied cells were investigated by cell viability assays. Expression analysis of P-gp, MRP1, BCRP, Bcl-2, Bax and &amp / #946 / -tubulin isotypes were performed by RT-PCR, qPCR, Western blot and immunocytochemistry. Genome-wide expression analysis was also performed by cDNA microarray. According to cell viability assays, drug applied cells developed varying degree of resistance to docetaxel and doxorubicin. Gene expression analysis demonstrated that de novo expression of P-gp contributed significantly to drug resistance. Expression levels of class II, III and V &amp / #946 / -tubulin isotypes increased in docetaxel resistant sublines. According to microarray analysis, a variety of genes showed significantly altered expression levels particularly drug metabolizing and detoxification enzymes (i.e. increased GPX1 and GSTP1 with decreased POR), survival proteins (e.g. decreased TRAIL together with increased decoy receptors and CD40), extracellular matrix components (e.g. increased integrin signaling), growth factors and cytokines (e.g. EGFR1, FGFR1, CTGF, IL6, IL8 and IL18 overexpression), epithelial-mesenchymal transition proteins (i.e. increased vimentin and N-cadherin with decreased E-cadherin and occludin) and microtubule dynamics related proteins (e.g. increased MAP1B and decreased MAP7). Development of cross-resistance and combined drug effects on resistant sublines were also studied. Results demonstrated that docetaxel and doxorubicin resistant cells developed cross-resistance to paclitaxel, vincristine, ATRA, tamoxifen and irradiation. Finally, modulatory effects of verapamil and promethazine in combined drug applications were investigated and verapamil and promethazine were shown to decrease MDR1 expression level thus reverse the MDR. They also showed synergic and additive effects in combined docetaxel and doxorubicin applications. Identification of resistance mechanisms may personalize chemotherapy potentially increasing efficacy of chemotherapy and life quality of patients.

A recent clinical trial for locally advanced breast cancer patients treated with epirubicin and docetaxel prior to surgery reported significant dose-dependent reductions in tumour RNA integrity values which correlated with pathological complete response. The purpose of the present study was to assess whether similar chemotherapy-dependent alterations in RNA integrity could occur in vitro and to assess its relationship, if any, to apoptosis. Treatment of wildtype A2780 ovarian carcinoma cells with taxanes resulted in dose- and time-dependent RNA degradation, identified as several unique bands on electropherograms having mobilities lower than the 28S and 18S rRNAs. We refer to this chemotherapy-dependent generation of aberrant RNA bands on electropherograms as “RNA disruption”. RNA disruption was found to be temporally associated with the induction of apoptosis, as determined by the appearance of a sub G1 peak of DNA content, positive annexin-V staining, and both PARP-1 and caspase-3 cleavage. Treatment of cells with a caspase-3 inhibitor resulted in a significant reduction in rRNA disruption, suggesting the involvement of caspase-3 or related caspases in RNA disruption. In contrast, docetaxel-dependent rRNA disruption was absent when docetaxel was administered to docetaxel-resistant A2780DXL cells, indicating that changes in RNA integrity may possibly differentiate between responsive and non-responsive tumours in cancer patients.

No description available.

No description available.

OBJECTIVE: To determine the effect of clinically used zoledronate (ZOL) and docetaxel on breast cancer cells and bone biology under both bone remodeling stages and the rate of tumor dissemination and state of dormancy. METHODS: The effect of clinically used zoledronate (ZOL) was examined on MDA-MB-231 and MDA-BO cells in a roller tube system under bone resorption and formation conditions. Three groups; calvaria alone, calvarial co-cultured with tumor cells, and calvaria with tumor cells treated with four repeat doses of 2 µM of ZOL were cultured for 8, 14 and 20 days. The formation groups were supplemented with 150 µg/ml ascorbic acid. Cell counts were performed on trypsinized calvaria harvested at 2, 8 and 14 days. Media was changed every 2 days and the changed media was re-seeded in a 24-well for 20 days. To test the impact of chemotherapy agents on cancer-bone metastasis the effect of 10 µM of docetaxel was tested on breast cancer cells under formation and resorption conditions using the above design. Tumor burden was assessed at 8 days. RESULTS: Tumor burden: no statistically significant difference between ZOL treated and untreated groups under resorption and formation conditions in both cell lines. Exposure to docetaxel revealed that ~30% of the cells were affected by chemotherapy in formation model, while ~70% was affected in resorption model in both cell types. Dissemination model: the dissemination rate for MDA and BO cells under formation condition is significantly less than for resorption conditions. Fluorescent microscopy: MDA and BO tumor-calvaria were treated with Ki 67 antibody showed that under bone resorption conditions the cancer-bone cells colony were predominantly in proliferation stage while under formation conditions cancer cells were in dormancy. Confocal microscopy: observation confirmed the relation of the mode of cancer cell attachment to bone endosteal cell layer with the dormancy and cell proliferation states. CONCLUSIONS: Both cancer cell lines showed resistance to ZOL under formation and resorption conditions. Drug resistance to docetaxel was more evident under formation condition, where cells are dormant and not proliferating. The dissemination rate is significantly higher in resorption condition, suggesting that cells in formation are dormant with lower dissemination rate. / 2019-07-23T00:00:00Z

Dissertação (mestrado)—Universidade de Brasília, Instituto de Ciências Biológicas, Programa de Pós-Graduação em Biologia Animal, 2018. / Submitted by Raquel Almeida (raquel.df13@gmail.com) on 2018-05-04T17:05:06Z No. of bitstreams: 1 2017_BarbaraYasminGarciaAndrade.pdf: 5450012 bytes, checksum: 8be71c305259f744cf3f0d62389cd15b (MD5) / Approved for entry into archive by Raquel Viana (raquelviana@bce.unb.br) on 2018-05-29T20:33:56Z (GMT) No. of bitstreams: 1 2017_BarbaraYasminGarciaAndrade.pdf: 5450012 bytes, checksum: 8be71c305259f744cf3f0d62389cd15b (MD5) / Made available in DSpace on 2018-05-29T20:33:56Z (GMT). No. of bitstreams: 1 2017_BarbaraYasminGarciaAndrade.pdf: 5450012 bytes, checksum: 8be71c305259f744cf3f0d62389cd15b (MD5) Previous issue date: 2018-05-29 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES). / O câncer epitelial de ovário aparece como o tipo de câncer ginecológico com maior mortalidade em todo o mundo. Atualmente, o tratamento padrão é baseado na combinação de cisplatina com um taxano, porém tem como principal obstáculo para sua eficiência o desenvolvimento de resistência aos tratamentos pelas células tumorais. O docetaxel é um importante antineoplásico hidrofóbico amplamente utilizado na clínica no tratamento de tumores como mama, ovário, próstata e outros.Entretanto, sua toxidade sistêmica é alta devido ao solvente utilizado na administração, o Tween 80, responsável por agravar os efeitos adversos e provocar reações alérgicas. A associação do docetaxel com nanopartículas lipídicas sólidas mostra-se como uma opção para diminuir os problemas relacionados à aplicação biológica do docetaxel, pois permite um maior nível de absorção pelas células tumorais, além de protegê-lo da degradação dentro do organismo antes que chegue ao alvo. Esse trabalho teve como objetivo avaliar a atividade antitumoral de duas formulações de nanopartículas lipídicas sólidas (NLS) associadas ao docetaxel (DTX) nas linhagens de carcinoma de ovário humano sensível A2780 e resistente ao docetaxel A2780-MDR#2 e A2780-MDR#3. Para obtenção das linhagens resistentes A2780-MDR#2 e A2780-MDR#3 foi feito a transdução das células A2780 com VLP contendo o plasmídeo retroviral com gene MDR (pHaMDR). As linhagens obtidas possuem morfologia alterada, com células maiores e crescimento em colônias, características diferentes da linhagem parental e apresentam sensibilidadereduzida ao DTX. Foi avaliado a ação citotóxica de três nanoformulações: (1) NLS-BRANCA, para verificação da citotoxidade do sistema; (2) NLS-DTX-1, formulação contendo 1 mg/mL de docetaxel; (3) NLS-DTX-2, formulação contendo 2 mg/mL de docetaxel. Como controle positivo foi utilizado o DTX livre. Com os resultados obtidos foi possível observar que as NLS-DTX-1 e NLS-DTX-2 possuem atividade citotóxica a partir de 10 ng/mL nos tempos de tratamento de 24, 48 e 72 horas e seu veículo, a NLS-BRANCA, não apresentou toxicidade significativa tanto na linhagem tumoral sensível (A2780) como nas resistentes à docetaxel (A2780-MDR#2 e A2780-MDR#3). Por microscopia de luz foi possível visualizar que o tratamento com as NLS-DTX alterou a morfologia das células A2780, tornando-as arrendondadas e menores, além de desorganizar o citoesqueleto das células, causando danos aos microtúbulos, fato esse observado por microscopia confocal a laser. Esses danos a morfologia e ao citoesqueleto se dá pela atividade estabilizadora de microtúbulos de docetaxel, o que promove a desorganização do citoesqueleto. Foi avaliado a capacidade clonogênica das células A2780 após 24 horas de tratamento com as NLS-DTX e foi visto que as células não foram capazes de se proliferar e formar colônias como ocorre naturalmente a essa linhagem. A atividade antimitótica de DTX presente nas formulações foi observada pelo impedimento da progressão do ciclo celular em G2/M. A interrupção do ciclo celular levou as células A2780 a morte celular. Portanto, com esse trabalho foi possível concluir que a associação de DTX com o nanossistema NLS é eficiente pois mostrou ação citotóxica em células de carcinoma de ovário sensíveis e resistentes à docetaxel, além de preservar a atividade antimitótica característica do fármaco. / The epithelial ovarian cancer is the most common cause of women deaths among gynecological cancers worldwide. Currently, the standard treatment is based on the combination of cisplatin with a taxane, however the efficacy of this combination faces a main obstacle: the development of resistance to treatments by tumor cells. Docetaxel is an important hydrophobic antineoplastic applied in clinical treatment for breast, ovary, prostate and solid tumors in general, but it shows high systemic toxicity due to the utilization of Tween 80 as solvent. The use of Tween 80 is responsible to increase the side effects and to cause allergic reactions. The association of docetaxel with solid lipid nanoparticles is shown as an option to enhance biological application of docetaxel, because it provides a better absorption by the tumor cells besides preventing the drug degradation within the organism before it arrives at the target. The aim of this study was to evaluate the antitumor activity of two solid lipid nanoparticle (NLS) formulations associated with docetaxel (DTX) in sensitive human ovary carcinoma A2780 cell line and resistant to docetaxel A2780-MDR#2 and A2780-MDR#3 strains. A2780-MDR#2 and A2780-MDR#3 strains were generated by transduction of A2780 cells with VLP containing retroviral plasmid with MDR gene (pHaMDR). Transduced cell lines showed altered morphology presenting bigger cells growing in colonies, different from the parental cells and were less sensitive to DTX. Cytotoxicity assay for three nanoformulations was performed: (1) NLS-WHITE, to evaluate cytotoxicity caused by the system; (2) NLS-DTX1, formulation with 1mg/mL of docetaxel; (3) NLS-DTX-2, formulation with 2 mg/mL of docetaxel. Free docetaxel was used as a positive control. The results demonstrated that NLS-DTX-1 and NLS-DTX-2 have cytotoxic activity beginning from 10 ng/mL concentration within 24, 48 and 72 hours of treatment and their vehicle, NLS-WHITE showed no significant toxicity both in the sensitive (A2780) and in resistant cell lines (A2780-MDR#2 e A2780-MDR#3). Also, the treatment with NLS-DTX altered the morphology of A2780 cells, making them rounded and smaller. This morphology damage is due to the stabilization of microtubules by the DTX, which promotes the cytoskeleton disorganization, observed by confocal microscopy. Clonogenic assay was performed with A2780 cell after 24h of treatment with NLSDTX and it was seen that these cells were not able to proliferate and make colonies, as naturally occurs in this cell line. The antimitotic activity of DTX present in the NLSDTX formulations was observed by the prevention of cell cycle progression in G2/M leading cells to death. Finally, it was possible to conclude that the association of DTX with the NLS nanosystem is an efficient treatment since it shows cytotoxic activity in sensitive ovarian tumor cells as well as in docetaxel resistant cells and keeps the antimitotic activity presented by free docetaxel.

OBJECTIVE: One of the major issues in current radiotherapy (RT) is the normal tissue toxicity. A smart combination of agents within the tumor would allow lowering the RT dose required while minimizing the damage to healthy tissue surrounding the tumor. We chose gold nanoparticles (GNPs) and docetaxel (DTX) as our choice of two radiosensitizing agents. They have a different mechanism of action which could lead to synergistic effect. Our first goal was to assess the variation in GNP uptake, distribution, and retention in the presence of DTX. Our second goal was to assess the therapeutic results of the triple combination, RT/GNPs/DTX. METHODS: We used HeLa and MDA-MB-231 cells for our study. Cells were incubated with GNPs (0.2nM) in the absence and presence of DTX (50nM) for 24 hrs for determination of uptake, distribution, and retention of NPs. For RT experiment, treated cells were given a 2 Gy dose of 6 MV photons using a linear accelerator. RESULTS: Concurrent treatment of DTX and GNPs resulted in over 85% retention of GNPs in tumor cells. DTX treatment also forced GNPs to be closer to the most important target, the nucleus, resulting in a significant decrease in cell survival with the triple combination of RT, GNPs, and DTX vs. RT plus DTX alone. Our experimental therapeutics results are supported by Monte Carlo simulations. CONCLUSION: The ability to not only trap GNPs at clinically feasible doses but also to retain them within the cells could lead to meaningful fractionated treatments in future combined cancer therapy. Furthermore, the suggested triple combination of RT/GNPs/DTX may allow lowering the RT dose to spare surrounding healthy tissue. ADVANCES IN KNOWLEDGE: This is the first study to show intracellular GNP transport disruption by DTX, and its advantage in radiosensitization. / Graduate / 2020-10-31

Orientador: Marlus Chorilli / Resumo: O câncer de próstata (CP) é a neoplasia maligna urológica mais comum e a segunda principal causa de mortalidade de homens associada ao câncer no mundo. O tratamento é iniciado por meio de intervenções cirúrgicas que abrangem a prostatectomia radical, tratamentos radioterápicos, terapias envolvendo privação androgênica e as imunoterapias. A quimioterapia também é bastante utilizada e recentes estudos demonstraram um controle notável do crescimento do CP utilizando docetaxel (DTX). No entanto, o DTX apresenta efeitos colaterais associados à toxicidade, tanto do fármaco como da formulação, Taxotere®. Em consequência disso, o desenvolvimento de sistemas nanotecnológicos de liberação de fármacos, como lipossomas, tem sido promissor para o tratamento do CP, em virtude das múltiplas características favoráveis desses nanosistemas, como biocompatibilidade e biodegradabilidade. Uma abordagem atrativa é a modificação superficial dos lipossomas com ligantes específicos dos receptores regulados nas superfícies de células tumorais, como os receptores de transferrina, que podem ser superexpressos em células tumorais de próstata. O atual trabalho visou desenvolver, caracterizar e avaliar a citotoxicidade in vitro de lipossomas funcionalizados com transferrina para veiculação do DTX no tratamento do CP. Os lipossomas foram obtidos por meio da técnica clássica da hidratação do filme lipídico e foram constituídos pela mistura de fosfatidilcolina de soja (SPC): colesterol (Col) : DSPE-PEG (2000)... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Prostate cancer (PC) is the most common urological malignancy and the second leading cause of cancer-related mortality in men worldwide. Treatment is initiated through surgical interventions that include radical prostatectomy, radiotherapy treatments, androgen deprivation therapies and immunotherapies. Chemotherapy is also widely used and recent studies have shown remarkable control of CP growth using docetaxel (DTX). However, DTX has side effects associated with the toxicity of both the drug and the formulation Taxotere®. As a result, the development of nanotechnological drug delivery systems, such as liposomes, has been promising for the treatment of CP, due to the multiple favorable characteristics of these nanosystems, such as biocompatibility and biodegradability. An attractive approach is the superficial modification of liposomes with receptor-specific ligands regulated on tumor cell surfaces, such as transferrin receptors, which can be overexpressed in prostate tumor cells. The present work aimed to develop, characterize and evaluate the in vitro cytotoxicity of transferrinfunctionalized liposomes for DTX delivery in the treatment of CP. The liposomes were obtained by the classical technique of hydration of the lipid film and consisted of soybean phosphatidylcholine (SPC): cholesterol (Col): DSPE-PEG (2000) -Maleimide and SPC: Col: DSPE-PEG (2000) mixture. ) -Maleimide: DTX, in the molar ratios 10: 2: 0.78 and 10: 2: 0.78: 0.52, respectively. The characterizations were... (Complete abstract click electronic access below) / Mestre