Chitosan microparticles loaded with essential oils inhibit duo-biofilms of Candida albicans and Streptococcus mutans

Oral candidiasis is a common fungal infection that affects the oral mucosa, and happens when Candida albicans interacts with bacteria in the oral microbiota, such as Streptococcus mutans, causing severe early childhood caries. C. albicans and S. mutans mixed biofilms are challenging to treat with conventional antimicrobial therapies, thus, new anti-infective drugs are required. Objective: This study aimed to test a drug delivery system based on chitosan microparticles loaded with geranium and lemongrass essential oils to inhibit C. albicans and S. mutans mixed biofilms. Methodology: Chitosan microparticles loaded with essential oils (CM-EOs) were obtained by spray-drying. Susceptibility of planktonic were performed according CLSI at 4 to 2,048 µg/mL. Mixed biofilms were incubated at 37ºC for 48 h and exposed to CM-EOs at 256 to 4,096 µg/mL. The antimicrobial effect was evaluated using the MTT assay, with biofilm architectural changes analyzed by scanning electron microscopy. RAW 264.7 cell was used to evaluate compound cytotoxicity. Results: CM-EOs had better planktonic activity against C. albicans than S. mutans. All samples reduced the metabolic activity of mixed C. albicans and S. mutans biofilms, with encapsulated oils showing better activity than raw chitosan or oils. The microparticles reduced the biofilm on the slides. The essential oils showed cytotoxic effects against RAW 264.7 cells, but encapsulation into chitosan microparticles decreased their toxicity. Conclusion: This study demonstrates that chitosan loaded with essential oils may provide an alternative method for treating diseases caused by C. albicans and S. mutans mixed biofilm, such as dental caries

Chitosan microparticles loaded with essential oils inhibit duo-biofilms of Candida albicans and Streptococcus mutans

Abstract Oral candidiasis is a common fungal infection that affects the oral mucosa, and happens when Candida albicans interacts with bacteria in the oral microbiota, such as Streptococcus mutans, causing severe early childhood caries. C. albicans and S. mutans mixed biofilms are challenging to treat with conventional antimicrobial therapies, thus, new anti-infective drugs are required. Objective This study aimed to test a drug delivery system based on chitosan microparticles loaded with geranium and lemongrass essential oils to inhibit C. albicans and S. mutans mixed biofilms. Methodology Chitosan microparticles loaded with essential oils (CM-EOs) were obtained by spray-drying. Susceptibility of planktonic were performed according CLSI at 4 to 2,048 µg/mL. Mixed biofilms were incubated at 37ºC for 48 h and exposed to CM-EOs at 256 to 4,096 µg/mL. The antimicrobial effect was evaluated using the MTT assay, with biofilm architectural changes analyzed by scanning electron microscopy. RAW 264.7 cell was used to evaluate compound cytotoxicity. Results CM-EOs had better planktonic activity against C. albicans than S. mutans. All samples reduced the metabolic activity of mixed C. albicans and S. mutans biofilms, with encapsulated oils showing better activity than raw chitosan or oils. The microparticles reduced the biofilm on the slides. The essential oils showed cytotoxic effects against RAW 264.7 cells, but encapsulation into chitosan microparticles decreased their toxicity. Conclusion This study demonstrates that chitosan loaded with essential oils may provide an alternative method for treating diseases caused by C. albicans and S. mutans mixed biofilm, such as dental caries

Evaluation of citoprotector effect of amifostine on the doxorubicin-induced acute cardiotoxicity.

IntroduÃÃo: A Doxorubicina (DOX), antineoplÃsico antracÃclico, à largamente utilizada no tratamento dos mais diversos tipos de tumores sÃlidos e neoplasias hematolÃgicas. A Cardiotoxicidade provocada pelo uso de antibiÃticos antracÃclicos tem sido observada hà algumas dÃcadas como fator agravante e limitante do uso terapÃutico da DOX. Apesar do conhecimento de inÃmeros fatores que podem mediar a induÃÃo da cardiotoxicidade pela DOX, os mecanismos fisiopatolÃgicos continuam nÃo esclarecidos. Objetivo: Avaliar o efeito da amifostina e glutationa na cardiotoxicidade aguda induzida por doxorubicina e estudar a morfologia do tecido cardÃaco de camundongos tratados com doxorubicina por microscopia de forÃa atÃmica. Material e MÃtodos: Camundongos C57BL/6 fÃmeas (n=8) foram tratados com doxorubicina (25mg/Kg i.p.) ou salina (0,2mL i.p.) e sacrificados 96 horas apÃs tratamento. Outro grupo experimental foi tratado com amifostina (AMF 25, 50 e 100 mg/Kg s.c.), glutationa (GLT 125, 250 e 500mg/Kg s.c.) ou salina 30 mim antes da injeÃÃo de doxorubicina, no caso da glutationa a administraÃÃo foi diÃria atà o dia do sacrifÃcio. Os parÃmetros analisados foram: eletrocardiograma, Ãndices cardÃaco e esplÃnico, dosagem de grupos sulfidrilas nÃo protÃicos, dosagem de CK e CK-MB, parÃmetros histolÃgicos, dosagem por ELISA de TNF-&#61537;, IL-1&#61538;&#61484; expressÃo por imunohistoquÃmica de TNF-&#61537;, IL-1&#61538;, iNOS, apoptose e anÃlise por microscopia de forÃa atÃmica. Resultados: O tratamento com AMF nas doses de 50 e 100mg/Kg e GLT 250 e 500 mg/Kg foi capaz de aumentar a percentagem de sobrevivÃncia dos animais que foram submetidos a cardiotoxicidade aguda induzida por DOX (25 mg/Kg) quando comparados com o grupo injetado somente com DOX. A AMF e GLT tambÃm foram capazes de prevenir, em comparaÃÃo ao grupo DOX (p<0,05), as alteraÃÃes nos valores eletrocardiogrÃficos, (aumento do QRS e QTc e diminuiÃÃo da amplitude de R), as alteraÃÃes nos Ãndices cardÃacos e esplÃnicos, a elevaÃÃo dos nÃveis sÃricos das enzimas CK e CK-MB, a reduÃÃo dos nÃveis de grupos sulfidrilas nÃo protÃicos no tecido cardÃaco e as alteraÃÃes histolÃgicas (degeneraÃÃo hidrÃpica e vacuolizaÃÃo, focos de hialinizaÃÃo de fibras cardÃacas, picnose e necrose) induzidas pela DOX (25mg/Kg). A DOX induziu aumento da marcaÃÃo imunohistoquÃmica para cÃlulas apoptÃticas e expressÃo de iNOS e diminuiu a expressÃo de TNF-&#61537;. A AMF foi capaz de prevenir estas alteraÃÃes, sendo esta prevenÃÃo apenas discreta para a expressÃo de TNF-&#61537;. A microscopia de forÃa atÃmica revelou alteraÃÃes morfolÃgicas nÃo vistas pela microscopia Ãptica e mostrou ser uma ferramenta valiosa na avaliaÃÃo de efeitos de drogas. ConclusÃo: Nossos resultados sugerem o efeito citoprotetor da amifostina pelo aumento da atividade da glutationa peroxidase no tecido cardÃaco e que esta se mostra tÃo eficiente quanto a droga de referencia dexrazoxane. A utilizaÃÃo da microscopia atÃmica introduz uma ferramenta de anÃlise comparativa em escala nanomÃtrica, tornando possÃvel observar a destruiÃÃo membranar cardÃaco condizente com dano oxidativo.Introduction: Doxorubicin (DOX) is an antineoplasic anthracyclic agent used on the treatment of several solid tumors and hematological cancers. DOX-induced cardiotoxicity has been studied for decades as a limiting factor on the anticancer therapy with this drug. Despite the current knowledge concerning the mechanisms of DOX-induced cardotoxicity, its pathophysiology is still not clear. Purpose: To evaluate of the amifostine and glutathione citoprotective effect on the DOX-induced acute cardiotoxicity and to study the morphology of cardiac tissue through the use of atomic force microscopy as a tool. Materials and Methods: C57BL/6 female mice were treated with doxorubicin (25mg/Kg i.p.) or saline (0,2mL i.p.) and sacrificed 96 hours after treatment. In another experimental setting, mice were given amifostine (AMF 25, 50 e 100 mg/Kg s.c.), glutathione (GLT 125, 250 e 500mg/Kg s.c.) or vehicle, 30 mim before the administration of DOX, except glutathione that was injected daily. Analitical parameters included: electrocardiograms, cardiac and spleen indices, non protein suphidrils groups levels, CK and CK-MB cardiac enzymes levels, histological analysis, cardiac levels of (TNF-&#61537;, IL-1&#61538;, iNOS) determined by ELISA, immunohistochemistry for TNF-&#61537;, IL-1&#61538;, iNOS, apoptosis and atomic force microscopy tissue analysis. Results: AMF (100mg/Kg) and GLT (250 e 500 mg/Kg) treatments were able of improve the survival rate of animals in spite of the injection of DOX (25 mg/Kg) in comparison to DOX-treated only group (p<0.05). A AMF e GLT were also able to prevent electrocardiographic changes (rising of QRS e QTc and reduced R amplitude), changes in the cardiac and spleen indices, the augmentation of blood levels of CK e CK-MB, reduction of non proteic suphidrils groups levels, and histological changes induced by DOX (25mg/Kg). DOX induced the augmentation of the immunostaining for apoptotic cells and iNOS what was prevented by the administration of amifostine. The atomic force microscopy reveals morphological changes on the tissue organizational structure which is not possible to be observed through optical microscopy. Conclusion: Our results suggest that the amifostine citoprotective effect on DOX-induced acute cardiotoxicity is due the rising of glutathione peroxidase activity in the cardiac tissue. The citoprotective effect of amifostine is as efficient as the reference drug dexrazoxane. The use of atomic force microscopy as a new pharmacological tool for comparative analysis in nanometric scale allow us to observe DOX-induced membrane destruction what is suggestive of oxidative stress process.IntroduÃÃo: A Doxorubicina (DOX), antineoplÃsico antracÃclico, à largamente utilizada no tratamento dos mais diversos tipos de tumores sÃlidos e neoplasias hematolÃgicas. A Cardiotoxicidade provocada pelo uso de antibiÃticos antracÃclicos tem sido observada hà algumas dÃcadas como fator agravante e limitante do uso terapÃutico da DOX. Apesar do conhecimento de inÃmeros fatores que podem mediar a induÃÃo da cardiotoxicidade pela DOX, os mecanismos fisiopatolÃgicos continuam nÃo esclarecidos. Objetivo: Avaliar o efeito da amifostina e glutationa na cardiotoxicidade aguda induzida por doxorubicina e estudar a morfologia do tecido cardÃaco de camundongos tratados com doxorubicina por microscopia de forÃa atÃmica. Material e MÃtodos: Camundongos C57BL/6 fÃmeas (n=8) foram tratados com doxorubicina (25mg/Kg i.p.) ou salina (0,2mL i.p.) e sacrificados 96 horas apÃs tratamento. Outro grupo experimental foi tratado com amifostina (AMF 25, 50 e 100 mg/Kg s.c.), glutationa (GLT 125, 250 e 500mg/Kg s.c.) ou salina 30 mim antes da injeÃÃo de doxorubicina, no caso da glutationa a administraÃÃo foi diÃria atà o dia do sacrifÃcio. Os parÃmetros analisados foram: eletrocardiograma, Ãndices cardÃaco e esplÃnico, dosagem de grupos sulfidrilas nÃo protÃicos, dosagem de CK e CK-MB, parÃmetros histolÃgicos, dosagem por ELISA de TNF-&#61537;, IL-1&#61538;&#61484; expressÃo por imunohistoquÃmica de TNF-&#61537;, IL-1&#61538;, iNOS, apoptose e anÃlise por microscopia de forÃa atÃmica. Resultados: O tratamento com AMF nas doses de 50 e 100mg/Kg e GLT 250 e 500 mg/Kg foi capaz de aumentar a percentagem de sobrevivÃncia dos animais que foram submetidos a cardiotoxicidade aguda induzida por DOX (25 mg/Kg) quando comparados com o grupo injetado somente com DOX. A AMF e GLT tambÃm foram capazes de prevenir, em comparaÃÃo ao grupo DOX (p<0,05), as alteraÃÃes nos valores eletrocardiogrÃficos, (aumento do QRS e QTc e diminuiÃÃo da amplitude de R), as alteraÃÃes nos Ãndices cardÃacos e esplÃnicos, a elevaÃÃo dos nÃveis sÃricos das enzimas CK e CK-MB, a reduÃÃo dos nÃveis de grupos sulfidrilas nÃo protÃicos no tecido cardÃaco e as alteraÃÃes histolÃgicas (degeneraÃÃo hidrÃpica e vacuolizaÃÃo, focos de hialinizaÃÃo de fibras cardÃacas, picnose e necrose) induzidas pela DOX (25mg/Kg). A DOX induziu aumento da marcaÃÃo imunohistoquÃmica para cÃlulas apoptÃticas e expressÃo de iNOS e diminuiu a expressÃo de TNF-&#61537;. A AMF foi capaz de prevenir estas alteraÃÃes, sendo esta prevenÃÃo apenas discreta para a expressÃo de TNF-&#61537;. A microscopia de forÃa atÃmica revelou alteraÃÃes morfolÃgicas nÃo vistas pela microscopia Ãptica e mostrou ser uma ferramenta valiosa na avaliaÃÃo de efeitos de drogas. ConclusÃo: Nossos resultados sugerem o efeito citoprotetor da amifostina pelo aumento da atividade da glutationa peroxidase no tecido cardÃaco e que esta se mostra tÃo eficiente quanto a droga de referencia dexrazoxane. A utilizaÃÃo da microscopia atÃmica introduz uma ferramenta de anÃlise comparativa em escala nanomÃtrica, tornando possÃvel observar a destruiÃÃo membranar cardÃaco condizente com dano oxidativo.Introduction: Doxorubicin (DOX) is an antineoplasic anthracyclic agent used on the treatment of several solid tumors and hematological cancers. DOX-induced cardiotoxicity has been studied for decades as a limiting factor on the anticancer therapy with this drug. Despite the current knowledge concerning the mechanisms of DOX-induced cardotoxicity, its pathophysiology is still not clear. Purpose: To evaluate of the amifostine and glutathione citoprotective effect on the DOX-induced acute cardiotoxicity and to study the morphology of cardiac tissue through the use of atomic force microscopy as a tool. Materials and Methods: C57BL/6 female mice were treated with doxorubicin (25mg/Kg i.p.) or saline (0,2mL i.p.) and sacrificed 96 hours after treatment. In another experimental setting, mice were given amifostine (AMF 25, 50 e 100 mg/Kg s.c.), glutathione (GLT 125, 250 e 500mg/Kg s.c.) or vehicle, 30 mim before the administration of DOX, except glutathione that was injected daily. Analitical parameters included: electrocardiograms, cardiac and spleen indices, non protein suphidrils groups levels, CK and CK-MB cardiac enzymes levels, histological analysis, cardiac levels of (TNF-&#61537;, IL-1&#61538;, iNOS) determined by ELISA, immunohistochemistry for TNF-&#61537;, IL-1&#61538;, iNOS, apoptosis and atomic force microscopy tissue analysis. Results: AMF (100mg/Kg) and GLT (250 e 500 mg/Kg) treatments were able of improve the survival rate of animals in spite of the injection of DOX (25 mg/Kg) in comparison to DOX-treated only group (p<0.05). A AMF e GLT were also able to prevent electrocardiographic changes (rising of QRS e QTc and reduced R amplitude), changes in the cardiac and spleen indices, the augmentation of blood levels of CK e CK-MB, reduction of non proteic suphidrils groups levels, and histological changes induced by DOX (25mg/Kg). DOX induced the augmentation of the immunostaining for apoptotic cells and iNOS what was prevented by the administration of amifostine. The atomic force microscopy reveals morphological changes on the tissue organizational structure which is not possible to be observed through optical microscopy. Conclusion: Our results suggest that the amifostine citoprotective effect on DOX-induced acute cardiotoxicity is due the rising of glutathione peroxidase activity in the cardiac tissue. The citoprotective effect of amifostine is as efficient as the reference drug dexrazoxane. The use of atomic force microscopy as a new pharmacological tool for comparative analysis in nanometric scale allow us to observe DOX-induced membrane destruction what is suggestive of oxidative stress process