Entrega de fotossensibilizadores mediada por nanofolhas de óxido de grafeno para terapias fotodinâmica e fototérmica combinadas no tratamento de carcinoma mamário in vitro e in vivo

Tese (doutorado)—Universidade de Brasília, Departamento de Biologia Celular, Instituto de Ciências Biológicas, Programa de Pós-Graduação em Biologia Molecular, 2017.As fototerapias têm se mostrado como uma abordagem promissora frente às terapias convencionais para o tratamento do câncer. O presente estudo teve como objetivo produzir e avaliar a eficácia de ação da plataforma de nanofolhas de óxido de grafeno carboxilado associadas ao azul de metileno (NanoGO-AM) no uso das terapias fotodinâmica (TFD) e fototérmica (TFT) combinadas contra modelo de carcinoma mamário murino ortotópico singênico. In vitro, NanoGO-AM apresentou produção de espécies reativas de oxigênio (EROs) após irradiação com luz de LED de 660 nm, e aumento de temperatura de 35,6 ° C seguido de irradiação com luz de laser. Os ensaios in vivo demonstraram um efeito aditivo obtido por NanoGO-AM com uso das TFD/TFT combinadas, que promoveram a ablação completa do tumor em 5/5 camundongos portadores de tumores de células 4T1-Luciferase. Até 30 dias após o último tratamento, não houve recidiva do tumor comparado aos grupos de TFD ou TFT apenas, que apresentaram bioluminescência tumoral 63 vezes maior que o grupo de tratamento para TFD/TFT combinadas. Estudos histológicos confirmaram que as terapias combinadas foram capazes de prevenir o crescimento tumoral e as metástases no fígado, pulmão e baço. Todos os dados sugerem o potencial de NanoGO-AM no tratamento de câncer de mama e prevenção de metástases. Na segunda parte do estudo, utilizou-se o cloreto de ftalocianina de alumínio (AlClFt), que é um fotossensibilizador hidrofóbico com alto rendimento quântico de oxigênio singleto. A combinação de nanofolhas de óxido de grafeno com AlClFt resultou em um dispositivo no qual o fotossensibilizador e o agente fototérmico estão em uma única nanoestrutura com potencial para uso em terapias combinadas. As nanofolhas de óxido de grafeno (NanoGO) foram produzidas a partir do método de Hummers tradicional e AlClFt foi adsorvido a sua superfície. Sob irradiação para TFD, NanoGO-AlClFt apresentou considerável produção de ERO em ambiente biológico. Células normais e tumorais (humanas e murinas), como MCF-7 (carcinoma mamário humano), MCF-10A (células normais de mama humana), 4T1-Luciferase (carcinoma mamário murino bioluminescente) e NIH/3T3 (célula de fibroblasto de embrião de camundongo) foram tratadas com NanoGO-AlClFt e irradiadas para TFD, TFT ou TFD/TFT combinadas. Houve uma redução significativa na viabilidade das células tumorais de até 90\%, mostrando o potencial da plataforma NanoGO-AlClFt em produzir danos celulares irreversíveis em células de carcinoma de mamário humano e murino. Os ensaios in vivo com camudongos portadores de tumor de células 4T1-Luciferase demonstraram a eficiência do NanoGO-AlClFt na mediação da ablação parcial do tumor e na prevenção da progressão tumoral com o uso da TFD/TFT combinadas.Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES); Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq); Financiadora de Estudos e Projetos (Finep); Fundação de Apoio a Pesquisa do Distrito Federal (FAP-DF) e Institutos Nacionais de Ciência e Tecnologia (INCT).Phototherapies have been shown as a promising alternative to the conventional therapies for cancer treatment. The present study aimed to prepare and use the nanographene oxide carboxylated-methylene blue platform (NanoGO-MB) to promote tumor ablation using combined photodynamic (PDT) and photothermal (PTT) therapies against a syngeneic orthotopic murine breast cancer model. In vitro, NanoGO-MB presented reactive oxygen species (ROS) production after LED light irradiation, and a temperature increase of approximately 40 °C followed by laser light irradiation. In vivo assays demonstrated an additive effect obtained by NanoGO-MB with the combined PDT/PTT therapies, which promoted complete tumor ablation in 5/5 4T1- Luciferase tumor-bearing mice. Up to 30 days after the last treatment, there was no tumor regrowth compared with PDT or PTT only groups, which displayed tumoral bioluminescence 63-fold higher than the combined PDT/PTT treatment group. Histological studies confirmed that the combined therapies were able to prevent tumor growth and liver, lung, and spleen metastasis. All data suggest the potential of NanoGO-MB in the treatment of breast cancer and metastasis prevention. In the second part of the study, it was used aluminum phthalocyanine chloride (AlPc), which is a hydrophobic photosensitizer with a high singlet oxygen species quantum yield. The combination of nanographene oxide and AlPc resulted in a device which the photosensitizer and the photothermal agent are in a single nanostructure with potential for use in the combined therapies. Graphene oxide nanosheets (NanoGO) were produced from a traditional Hummers method and AlPc was adsorbed on its surface. NanoGO-AlPc upon irradiation for PDT therapy displayed considerable ROS production in a biological environment. Normal and tumor cells (human and murine), as MCF-7 (human breast carcinoma cells), MCF-10A (human breast normal cells), 4T1-Luc (murine mammary tumor bioluminescent cells) and NIH/3T3 (murine embryo fibroblast cell) were treated with NanoGO-AlPc and irradiated for single PDT or single PTT and combined PDT/PTT therapies. There was a significant decrease in tumor cells viability of up to 90% showing the potential of platform NanoGO-AlPc in produce irreversible cell damage in human and murine breast carcinoma cells. In vivo assays with 4T1-Luc tumor-bearing mice demonstrated the efficiency of NanoGO-AlPc in mediating partial tumor ablation and preventing tumor progression with PDT and PTT combined therapies

PVM/MA-shelled selol nanocapsules promote cell cycle arrest in A549 lung adenocarcinoma cells

Background: Selol is an oily mixture of selenitetriacylglycerides that was obtained as a semi-synthetic compound containing selenite. Selol is effective against cancerous cells and less toxic to normal cells compared with inorganic forms of selenite. However, Selol’s hydrophobicity hinders its administration in vivo. Therefore, the present study aimed to produce a formulation of Selol nanocapsules (SPN) and to test its effectiveness against pulmonary adenocarcinoma cells (A549). Results: Nanocapsules were produced through an interfacial nanoprecipitation method. The polymer shell was composed of poly(methyl vinyl ether-co-maleic anhydride) (PVM/MA) copolymer. The obtained nanocapsules were monodisperse and stable. Both free Selol (S) and SPN reduced the viability of A549 cells, whereas S induced a greater reduction in non-tumor cell viability than SPN. The suppressor effect of SPN was primarily associated to the G2/M arrest of the cell cycle, as was corroborated by the down-regulations of the CCNB1 and CDC25C genes. Apoptosis and necrosis were induced by Selol in a discrete percentage of A549 cells. SPN also increased the production of reactive oxygen species, leading to oxidative cellular damage and to the overexpression of the GPX1, CYP1A1, BAX and BCL2 genes. Conclusions: This study presents a stable formulation of PVM/MA-shelled Selol nanocapsules and provides the first demonstration that Selol promotes G2/M arrest in cancerous cells

Citotoxidade induzida por nanoemulsões de selol em células de adenocarcinoma de pulmão humano

Dissertação (mestrado)-Universidade de Brasília, Instituto de Ciências Biol ógicas, Departamento de Biologia Celular, Programa de Pós-Graduação em Biologia Molecular, 2013.As neoplasias de pulmão constituem uma das principais causas de morte por câncer no mundo. São classificadas em dois principais grupos histológicos: carcinoma de pulmão de células pequenas (CPCP) e carcinoma de pulmão de células não-pequenas (CPCNP), sendo o último o principal causador de carcinomas e foco deste trabalho. O tratamento de CPCNP apresenta baixa eficiência e segurança e, deste modo, novos potenciais quimioterápicos estão sendo estudados para melhorar os protocolos terapêuticos. Neste contexto, o selol, um selenitotriacilglicerídeo derivado do óleo da semente de girassol, contendo selênio a nível de oxidção IV, tem mostrado propriedades antioxidantes e anticarcinogênicas, rápida absorção pelo organismo e menor toxicidade cumulativa, quando comparado aos compostos selênicos clássicos. Entretanto, sua alta hidrofobicidade faz necessária a associação dessa droga a um sistema de entrega de drogas. Esse sistema pode ser desenhado para se acumular, passiva ou ativamente no tecido tumoral e interagir fortemente com as células cancerígenas. Isto pode ser alcançado pelo carreamento do selol dentro de nanoestruturas e pelo controle de suas propriedades de superfície, como pela cobertura das nanoestruturas com o copolímero PVM/MA, que possui como principais características a atoxicidade e alta bioadesividade. O presente estudo teve por objetivo o desenvolvimento de nanoemulsões (NEs) de selol, cobertas ou não com o copolímero PVM/MA, a fim de avaliar sua eficácia como sitemas de entrega de drogas e investigar a citotoxicidade dessas nanoemulsões frente células de adenocarcinoma de pulmão humano (linhagem A549) in vitro. As nanoemulsões de selol foram estabilizadas pelo tensoativo Cremophor ELP R e cobertas ou não com diferentes quantidade do copolímero PVM/MA. As NEs resultantes foram caracterizadas para diâmetro hidrodinâmico, potencial zeta e estabilidade por espalhamento de luz dinâmica. A morfologia das gotículas foi determinada por microscopia eletrônica de transmissão e varredura. A viabilidade das células foi analisada por ensaio colorimétrico (MTT). Os parâmetros de morte celular e fragmentação de DNA foram avaliados por citometria de fluxo. As alterações morfológicas foram analisadas em microscopia de contraste de fase. As NEs de selol, cobertas ou não com o copolímero PVM/MA, mostraram-se nanossistemas de entrega de drogas estáveis pelo período de até 150 dias após o desenvolvimento das nanogotículas, armazenadas a 25_C e 4_C. As nanogotículas foram citotóxicas e promoveram morte em células A549, apresentando, contudo, menor toxicidade frente as células normais. Todas essas características evidenciam o potencial das NEs de selol de serem utilizadas como agentes quimioterápicos, sozinhas ou em associação com outras drogas, no tratatmento do adenocarcinoma de pulmão humano. _______________________________________________________________________________________ ABSTRACTLung cancers are the leading causes of cancer death worldwide. They are classified into two main histological groups: small cell lung cancer and non-small cell lung cancer (NSCLC). NSCLC is the most prevalent and this study focused on its adenocarcinoma subtype. The treatment of NSCLC presents low efficiency and safety and, thus, new potential chemotherapics are being studied in order to improve therapeutical protocols. In this context, selol, a selenium (IV)-containing compound has shown to be an efficient anticancer drug and to be safer than classical selenium compounds. However, its high hydrophobicity makes it necessary to load this drug in a drug delivery system. This drug delivery system may also be designed to accumulate, passively and/or actively, in the tumor tissue and to strongly interact with cancer cells; this can be achieved by loading selol into nanostructures and by controlling its surface properties. Therefore, the aim of the present study was to develop selol nanoemulsions (NEs) as drug delivery systems and test their anticancer efficiency against human pulmonary adenocarcinoma cells (A549 lineage) in vitro. Selol nanodroplets were stabilized by the surfactant Cremophor ELP R and covered or not with different amounts of the bioadhesive copolymer PVM/MA The resulting NEs were characterized by size, charge, and stability in dynamic light scattering (DLS) and morphology in transmission (TEM) and scanning electron microscopy. The cells viability was measured by the colorimetric assay (MTT). Parameters of cell death and DNA fragmentation were assessed by ow cytometry and the morphological changes were analyzed in phase contrast microscopy. The selol NEs, uncovered or covered with PVM/MA copolymer, were drug delivery nanosystems stable for up to 150 days after the development of droplet, stored at 4 C and 25 C. The nanodroplets were cytotoxic and promoted death in A549 cells, showing, however, lower toxicity against normal cells. All these features demonstrate the potential of the selol NEs be used as chemotherapeutic agents, alone or in combination with other drugs, in the treatment of human pulmonary adenocarcinoma

Nanographene oxide-methylene blue as phototherapies platform for breast tumor ablation and metastasis prevention in a syngeneic orthotopic murine model

Abstract Background In the photodynamic therapy (PDT), the photosensitizer absorbs light and transfers the energy of the excited state to the oxygen in the cell environment producing reactive oxygen species (ROS), that in its turn, may cause cell damage. In the photothermal therapy (PTT), light also is responsible for activating the photothermal agent, which converts the absorbed energy in heat. Graphene oxide is a carbon-based material that presents photothermal activity. Its physical properties allow the association with the photosensitizer methylene blue and consequently the production of ROS when submitted to light irradiation. Therefore, the association between nanographene oxide and methylene blue could represent a strategy to enhance therapeutic actions. In this work, we report the nanographene oxide-methylene blue platform (NanoGO-MB) used to promote tumor ablation in combination with photodynamic and photothermal therapies against a syngeneic orthotopic murine breast cancer model. Results In vitro, NanoGO-MB presented 50% of the reactive oxygen species production compared to the free MB after LED light irradiation, and a temperature increase of ~ 40 °C followed by laser irradiation. On cells, the ROS production by the nanoplatform displayed higher values in tumor than normal cells. In vivo assays demonstrated a synergistic effect obtained by the combined PDT/PTT therapies using NanoGO-MB, which promoted complete tumor ablation in 5/5 animals. Up to 30 days after the last treatment, there was no tumor regrowth compared with only PDT or PTT groups, which displayed tumoral bioluminescence 63-fold higher than the combined treatment group. Histological studies confirmed that the combined therapies were able to prevent tumor regrowth and liver, lung and spleen metastasis. In addition, low systemic toxicity was observed in pathologic examinations of liver, spleen, lungs, and kidneys. Conclusions The treatment with combined PDT/PTT therapies using NanoGO-MB induced more toxicity on breast carcinoma cells than on normal cells. In vivo, the combined therapies promoted complete tumor ablation and metastasis prevention while only PDT or PTT were unable to stop tumor development. The results show the potential of NanoGO-MB in combination with the phototherapies in the treatment of the breast cancer and metastasis prevention