Efeito do uso combinado de inibidor de histona desacetilase e terapia fotodinâmica sobre o crescimento de Cryptococcus spp. in vitro

Dissertação (mestrado)—Universidade de Brasília, Instituto de Ciências Biológicas, Pós-Graduação em Nanociência e Nanobiotecnologia, 2019.Cryptococcus neoformans é um fungo patogênico oportunista encontrado mundialmente e é capaz de causar infecções potencialmente fatais, especialmente em pessoas com imunodeficiência. Limitações para o tratamento da infecção por C. neoformans envolvem a seleção de isolados resitentes aos antifúngicos convencionais, o tempo prolongado de tratamento e efeitos colaterais como nefrotoxicidade. Abordagens alternativas como a terapia fotodinâmica (TFD) e a intervenção epigenética poderiam contornar esses problemas. A TFD combina um agente fotossensibilizante (FS) não tóxico com luz vermelha e oxigênio molecular. A nanoemulsão de cloreto de ftalocianina de alumínio (NE-AlFtCl), desenvolvida por nosso grupo de pesquisa, demonstrou atividade inibitória sobre várias linhagens de células de câncer humano. Inibidores de histona desacetilase (HDACis), como o butirato de sódio (NaBut) e a tricostatina A (TSA), são frequentemente utilizados em tratamentos combinados com fármacos antineoplásicos visando a ação sinérgica e a redução de efeitos colaterais. O presente estudo teve como objetivo avaliar o efeito da TFD mediada por NE-AlFtCl, combinada a NaBut ou a TSA, sobre o crescimento de C. neoformans linhagens H99 e T1 (resistente a fluconazol) e de C. gattii NIH198. As leveduras foram cultivadas na presença de NaBut ou TSA, expostas a diferentes concentrações de NE-AlFtCl, lavadas e semeadas em placas de 96 poços para aplicação de luz no comprimento de onda de 660 nm. A incorporação do agente fotossensibilizante foi avaliada após lise celular com dimetilsulfóxido (DMSO), detecção da fluorescência no lisado por espectrofluorímetro e por microscopia confocal. A proliferação celular em resposta a diferentes tratamentos foi avaliada pela densidade óptica a 600 nm. O efeito da TFD sobre a viabilidade celular foi avaliado por meio do teste colorimétrico de brometo de 3(4,5 dimetiltiazol-2il)-2,5-difeniltetrazólio (MTT) e por diluição em série, seguida de semeadura em YPD sólido. O tempo de 30 min de incubação demonstrou-se suficiente para a incorporação máxima de NE-AlFtCl pelas leveduras. A microscopia confocal revelou que, em C. neoformans H99 e C. gattii NIH198, o AlFtCl se apresentou difuso pelo citoplasma. A linhagem T1 de C. neoformans apresentou menor incorporação do FS, que se concentrou na periferia celular. A TFD levou a redução da proliferação celular e essa redução foi acentuada pelo pré-tratamento das leveduras com NaBut ou TSA. A TFD demonstrou ainda citotoxicidade, dependente da concentração de FS, sobre as leveduras tratadas. O ensaio de diluição seriada demonstrou que o pré-tratamento com HDACi intensificou o efeito citotóxico da TDF. A linhagem T1 de C. neoformans demonstrou-se a mais resistente à ação de TFD em combinação com HDACi, enquanto C. gattii NIH 198 se mostrou mais sensível. Os resultados apresentados indicam a eficácia da TFD mediada por NE-AlFtCl, em combinação com o tratamento com HDACi, sobre o controle da proliferação de C. neoformans e C. gattii in vitro, podendo vir a representar uma proposta terapêutica alternativa para o tratamento de infecções fúngicas.Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES); Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq); Instituto Nacional de Ciência e Tecnologia (INCT) e Fundação de Apoio à Pesquisa do Distrito Federal (FAP/DF).Cryptococcus neoformans is an opportunistic pathogenic fungus found worldwide and is capable of causing life-threatening infections, especially in people with immunodeficiency. Limitations for the treatment of C. neoformans infection involve selection of isolates resistant to conventional antifungals, prolonged treatment time and side effects such as nephrotoxicity. Alternative approaches such as photodynamic therapy (PDT) and epigenetic intervention could circumvent these problems. PDT combines a non-toxic photosensitizing agent (PS) with red light and molecular oxygen. Aluminum phthalocyanine chloride (NE-AlPcCl) nanoemulsion, developed by our research group, demonstrated inhibitory activity on various human cancer cell lines. Histone deacetylase inhibitors (HDACis), such as sodium butyrate (NaBut) and trichostatin A (TSA), are often used in combination treatments with antineoplastic drugs for synergistic action and reduction of side effects. The aim of the present study was to evaluate the effect of NE-AlPcCl-mediated PDT, combined with NaBut or TSA, on the growth of C. neoformans strain H99 and T1 (fluconazole resistant) and C. gattii NIH198. Yeasts were cultured in the presence of NaBut or TSA, exposed to different concentrations of NE-AlPcCl, washed and seeded in 96-well plates for application of light at 660 nm wavelength. The incorporation of the photosensitizing agent was evaluated after cell lysis with dimethylsulfoxide (DMSO), detection of fluorescence in the lysate by spectrofluorimeter and by confocal microscopy. Cell proliferation in response to different treatments was evaluated by optical density at 600 nm. The effect of PDT on cell viability was evaluated by the colorimetric test of 3 (4,5-dimethylthiazol-2yl) -2,5-diphenyltetrazolium bromide (MTT) and by serial dilution, followed by seeding in solid YPD. The time of 30 min of incubation was demonstrated sufficient for the maximum incorporation of NE-AlPcCl by the yeasts. Confocal microscopy revealed that, in C. neoformans H99 and C. gattii NIH198, AlPcCl diffuse through the cytoplasm. The T1 line of C. neoformans presented a lower incorporation of PS, which was concentrated in the cellular periphery. PDT to reduced cell proliferation and this reduction was accentuated by yeast pretreatment with NaBut or TSA. PDT also demonstrated PS-dependent cytotoxicity on treated yeasts. The serial dilution assay demonstrated that pretreatment with HDACi enhanced the cytotoxic effect of PDT. The T1 line of C. neoformans was shown to be more resistant to the action of PDT in combination with HDACi, whereas C. gattii NIH198 was more sensitive. The results presented indicate the efficacy of NE-AlPcCl-mediated PDT, in combination with HDACi treatment, on the control of C. neoformans and C. gattii proliferation in vitro, and may represent an alternative therapeutic proposal for the treatment of fungal infections

An overview on immunogenic cell death in cancer biology and therapy

Immunogenic cell death (ICD) is a modality of regulated cell death that is sufficient to promote an adaptive immune response against antigens of the dying cell in an immunocompetent host. An important characteristic of ICD is the release and exposure of damage-associated molecular patterns, which are potent endogenous immune adjuvants. As the induction of ICD can be achieved with conventional cytotoxic agents, it represents a potential approach for the immunotherapy of cancer. Here, different aspects of ICD in cancer biology and treatment are reviewed

Oral delivery of fish oil in oil-in-water nanoemulsion : development, colloidal stability and modulatory effect on in vivo inflammatory induction in mice

To improve the oral absorption of fish oil and test its anti-inflammatory effect, a fish oil nanoemulsion was developed using cis-4,7,10,13,16,19-docosahexaenoic fatty acid as a biomarker for oral administration. The colloidal stability tests of the fish oil nanoemulsion showed an average size of 155.44 nm ± 6.46 (4 °C); 163.04 nm ± 9.97 (25 °C) and polydispersity index 0.22 ± 0.02 (4 °C), 0.21 ± 0.02 (25 °C), indicating systems with low polydispersity and stable droplets. The fish oil nanoemulsion did not alter the cell viability of the RAW 264.7 macrophages and, at a concentration of 0.024 mg/mL, was kinetically incorporated into the cells after 18 h of contact. The nanoemulsion was maintained in the gastrointestinal region for a significantly shorter period of time (p ≤ 0.05) compared to the intake of fish oil in free form. Inflammatory tests demonstrated that nanoemulsion and fish oil showed less (p ≤ 0.05) neutrophil infiltration after 24h of sepsis induction and there was a significant reduction (p ≤ 0.05) in the volume of paw edema in female adult Balb/c mice who received the nanoemulsion diet compared to the other experimental groups (control, formalin, fish oil and sunflower oil). These results indicate that the fish oil nanoemulsion was significantly effective in the dietary conditions tested here, presenting satisfactory responses in the modulation of inflammatory disorders, demonstrating interesting and beneficial nutraceutical effects

Database Survey of Anti-Inflammatory Plants in South America: A Review

Inflammation is a complex event linked to tissue damage whether by bacteria, physical trauma, chemical, heat or any other phenomenon. This physiological response is coordinated largely by a variety of chemical mediators that are released from the epithelium, the immunocytes and nerves of the lamina propria. However, if the factor that triggers the inflammation persists, the inflammation can become relentless, leading to an intensification of the lesion. The present work is a literature survey of plant extracts from the South American continent that have been reported to show anti-inflammatory activity. This review refers to 63 bacterial families of which the following stood out: Asteraceae, Fabaceae, Euphorbiaceae, Apocynaceae and Celastraceae, with their countries, parts used, types of extract used, model bioassays, organisms tested and their activity

Recent Advances in Antimicrobial Nano-Drug Delivery Systems

Infectious diseases are among the major health issues of the 21st century. The substantial use of antibiotics over the years has contributed to the dissemination of multidrug resistant bacteria. According to a recent report by the World Health Organization, antibacterial (ATB) drug resistance has been one of the biggest challenges, as well as the development of effective long-term ATBs. Since pathogens quickly adapt and evolve through several strategies, regular ATBs usually may result in temporary or noneffective treatments. Therefore, the demand for new therapies methods, such as nano-drug delivery systems (NDDS), has aroused huge interest due to its potentialities to improve the drug bioavailability and targeting efficiency, including liposomes, nanoemulsions, solid lipid nanoparticles, polymeric nanoparticles, metal nanoparticles, and others. Given the relevance of this subject, this review aims to summarize the progress of recent research in antibacterial therapeutic drugs supported by nanobiotechnological tools

Fish Oil Nanoemulsion Supplementation Attenuates Bleomycin-Induced Pulmonary Fibrosis BALB/c Mice

Diets rich in omega-3 or -6 fatty acids will produce different profiles for cell membranes phospholipid constitutions. Omegas 3 and 6 are part of the diet and can modulate the inflammatory profile. We evaluated the effects of the oral absorption of fish oil, when associated with a lipid nanoemulsion in an experimental pulmonary inflammatory model. Pulmonary fibrosis is a disease associated with excessive extracellular matrix deposition. We determined to investigate the morphophysiological mechanisms in mice that were pretreated after induction with bleomycin (BLM). The pretreatment was for 21 days with saline solution, sunflower oil (SO), fish oil (FO), and fish oil nanoemulsion (NEW3). The animals received a daily dose of 50 mg/Kg of docosahexaenoic acid DHA and 10 mg/Kg eicosapentaenoic (EPA) (100 mg/Kg), represented by a daily dose of 40 µL of NEW3. The blank group was treated with the same amount daily (40 µL) during the 21 days of pretreatment. The animals were treated with SO and FO, 100 mg/Kg (containing 58 mg/Kg of polyunsaturated fats/higher% linoleic acid) and 100 mg/Kg (50 mg/Kg of DHA and 10 mg/Kg EPA), respectively. A single dose of 5 mg/mL (50 μL) bleomycin sulfate, by the intratracheal surgical method in BALB/cAnNTac (BALB/c). NEW3 significantly reduced fibrotic progression, which can be evidenced by the protection from loss of body mass, increase in respiratory incursions per minute, decreased spacing of alveolar septa, decreased severity of fibrosis, and changes in the respiratory system. NEW3 attenuated the inflammatory changes developed in the experimental model of pulmonary fibrosis, while group SO showed a significant increase in inflammatory changes. This concluded that the presented results demonstrated that is possible to positively modulate the immune and inflamamtory response to an external agressor, by changing the nutitional intake of specific fatty acids, such as omega-3 placed in fish oil. Moreover, these benefits can be improved by the nanoencapsulation of fish oil in lipid nanoemulsions

Induction of Immunogenic Cell Death by Photodynamic Therapy Mediated by Aluminum-Phthalocyanine in Nanoemulsion

Photodynamic therapy (PDT) has been clinically employed to treat mainly superficial cancer, such as basal cell carcinoma. This approach can eliminate tumors by direct cytotoxicity, tumor ischemia, or by triggering an immune response against tumor cells. Among the immune-related mechanisms of PDT, the induction of immunogenic cell death (ICD) in target cells is to be cited. ICD is an apoptosis modality distinguished by the emission of damage-associated molecular patterns (DAMP). Therefore, this study aimed to analyze the immunogenicity of CT26 and 4T1 treated with PDT mediated by aluminum-phthalocyanine in nanoemulsion (PDT-AlPc-NE). Different PDT-AlPc-NE protocols with varying doses of energy and AlPc concentrations were tested. The death mechanism and the emission of DAMPs–CRT, HSP70, HSP90, HMGB1, and IL-1β–were analyzed in cells treated in vitro with PDT. Then, the immunogenicity of these cells was assessed in an in vivo vaccination-challenge model with BALB/c mice. CT26 and 4T1 cells treated in vitro with PDT mediated by AlPc IC50 and a light dose of 25 J/cm2 exhibited the hallmarks of ICD, i.e., these cells died by apoptosis and exposed DAMPs. Mice injected with these IC50 PDT-treated cells showed, in comparison to the control, increased resistance to the development of tumors in a subsequent challenge with viable cells. Mice injected with 4T1 and CT26 cells treated with higher or lower concentrations of photosensitizer and light doses exhibited a significantly lower resistance to tumor development than those injected with IC50 PDT-treated cells. The results presented in this study suggest that both the photosensitizer concentration and light dose affect the immunogenicity of the PDT-treated cells. This event can affect the therapy outcomes in vivo

Synthesis and Evaluation of New Potential Benzo[a]phenoxazinium Photosensitizers for Anticancer Photodynamic Therapy

The use of photodynamic therapy (PDT) and development of novel photosensitizers (PSs) for cancer treatment have received more and more attention nowadays. In the present work, five benzo[a]phenoxazinium derivatives have been prepared and evaluated for their in vitro anticancer photodynamic activity for the first time. They are red light absorbers and show low fluorescence quantum yield. Of these compounds, PS4 exhibited a higher quantum yield for reactive oxygen species (ROS) generation. The assays with cells in vitro showed that PS1 and PS4 were not significantly toxic in the dark, but was robustly toxic against the murine breast adenocarcinoma cells 4T1 and normal murine fibroblast cells NIH-3T3 upon photoactivation. More interestingly, PS5 was particularly selective towards 4T1 cancer cells and nearly non-phototoxic to non-cancerous NIH-3T3 cells. The results described in this report suggest that these new benzo[a]phenoxazinium derivatives are potential candidates as PSs for anticancer PDT. Further investigation of benzo[a]phenoxaziniums for anticancer PDT is warranted