RESUMO
PURPOSE: This work investigated the endocytic pathways taken by poly(isobutylcyanoacrylate) (PIBCA) nanoparticles differing in their surface composition and architecture, assuming that this might determine their efficiency of intracellular drug delivery. METHODS: Nanoparticles (A0, A25, A100, R0, R25 ) were prepared by anionic or redox radical emulsion polymerization using mixtures of dextran and fucoidan (0, 25, 100 % in fucoidan). Cell uptake was evaluated by incubating J774A.1 macrophages with nanoparticles. Endocytic pathways were studied by incubating cells with endocytic pathway inhibitors (chlorpromazine, genistein, cytochalasin D, methyl-ß-cyclodextrin and nocodazole) and nanoparticle uptake was evaluated by flow cytometry and confocal microscopy. RESULTS: The fucoidan-coated PIBCA nanoparticles A25 were internalized 3-fold more efficiently than R25 due to the different architecture of the fucoidan chains presented on the surface. Different fucoidan density and architecture led to different internalization pathway preferred by the cells. Large A100 nanoparticles with surface was covered with fucoidan chains in a loop and train configuration were internalized the most efficiently, 47-fold compared with A0, and 3-fold compared with R0 and R25 through non-endocytic energy-independent pathways and reached the cell cytoplasm. CONCLUSION: Internalization pathways of PIBCA nanoparticles by J774A.1 macrophages could be determined by nanoparticle fucoidan surface composition and architecture. In turn, this influenced the extent of internalization and localization of accumulated nanoparticles within cells. The results are of interest for rationalizing the design of nanoparticles for potential cytoplamic drug delivery by controlling the nature of the nanoparticle surface.
Assuntos
Nanopartículas , Sistemas de Liberação de Medicamentos , Emulsões , PolissacarídeosRESUMO
The aim of this study was to determine whether encapsulation of ß-lapachone (ß-lap) into liposomes interferes with its in vitro antimicrobial activity against meticillin-resistant Staphylococcus aureus (MRSA) and Cryptococcus neoformans clinical strains. Liposomes (ß-lap:lipo or ß-lap:HPß-CD-lipo) were prepared using the hydration of thin lipid film method followed by sonication. The in vitro antimicrobial activities of ß-lap-loaded liposomes against MRSA and C. neoformans were evaluated using the microdilution method according to the Clinical and Laboratory Standards Institute (CLSI). The liposomes presented a mean particle size ranging from 88.7±1.5nm to 112.4±1.9nm with a polydispersity index ranging from 0.255 to 0.340, zeta potential from -0.26±0.01mV to +0.25±0.05mV and drug encapsulation efficiency from 97.4±0.3% to 98.9±0.4%. ß-Lap and ß-lap:HPß-CD had minimum inhibitory concentrations (MICs) ranging from 2mg/L to 4mg/L, whereas the MICs of ß-lap-lipo or ß-lap:HPß-CD-lipo ranged from 4mg/L to 16mg/L for the MRSA strains tested. ß-Lap and ß-lap:HPß-CD were able to inhibit fungal growth [MIC=2-8mg/L and minimum fungicidal concentration (MFC)=4-8mg/L]. However, ß-lap-lipo and ß-lap:HPß-CD-lipo were more efficient, with MICs and MFCs of <4mg/L. These findings suggest that the liposomal formulations tested do not interfere significantly with ß-lap antibacterial activity against MRSA and improve its antifungal properties against C. neoformans.
RESUMO
The aim of this study was the encapsulation of trans-dehydrocrotonin (t-DCTN) and its inclusion complexes with hydropropyl-beta-cyclodextrin (HP-beta-CD) in liposomes to improve t-DCTN antitumor activity. The in vitro kinetic profiles of t-DCTN-loaded liposomes (LD) and t-DCTN:HP-beta-CD-loaded liposomes (LC) were evaluated using the dialysis technique. The antitumor activity of LD and LC were investigated against Sarcoma 180 in Swiss mice. Histopathological and hematological analyses were carried out. The amounts of t-DCTN and t-DCTN:HP-beta-CD inclusion complex encapsulated in liposomes were equivalent to 1 mg of t-DCTN. The encapsulation efficiencies of LD and LC were 95.0 +/- 3.8% and 91.1 +/- 5.6%, respectively. In relation to kinetics, the drug release profiles of t-DCTN are in substantial agreement with the Fickian model. The treatment of animals with LD and LC produced tumor inhibitions of 79.4 +/- 9.6% and 63.5 +/- 5.5%, respectively. The liposomal encapsulation of t-DCTN by entrapment in the phospholipid bilayer increased at twice the antitumor activity. Moreover, the liposomal formulations reduced the hepatotoxicity effect of the drug and no significant hematological toxicity was observed in the treated animals. However, the counting of platelets was slightly decreased. Thus, the results show that the development of liposomal formulations containing t-DCTN or t-DCTN:HP-beta-CD is an important advance for enabling this drug to be use in therapy.
Assuntos
Antineoplásicos/administração & dosagem , Antineoplásicos/farmacologia , Diterpenos Clerodânicos/administração & dosagem , Diterpenos Clerodânicos/farmacologia , 2-Hidroxipropil-beta-Ciclodextrina , Animais , Antineoplásicos/química , Química Farmacêutica , Diterpenos Clerodânicos/química , Diterpenos Clerodânicos/uso terapêutico , Cinética , Lipossomos , Fígado/efeitos dos fármacos , Fígado/patologia , Masculino , Camundongos , Tamanho da Partícula , Análise de Regressão , Sarcoma/sangue , Sarcoma/tratamento farmacológico , Sarcoma/patologia , Eletricidade Estática , beta-Ciclodextrinas/químicaRESUMO
The antischistosomal activity of the sulfated polysaccharide α-D-glucan (Glu.SO4) extracted from Ramalina celastri was evaluated after encapsulation into liposomes (Glu.SO4-LIPO) in Schistosoma mansoni-infected mice. The effect of treatment with Glu.SO4 and Glu.SO4-LIPO (10 mg/kg) on egg elimination, worm burden and hepatic granuloma formation was assessed using female albino Swiss mice, 35-40 days of age, weighing 25 ± 2 g, infected with 150 cercariae/animal (Biomphalaria glabrata, BH strain). Four groups (N = 10) were studied, two controls (empty liposomes and NaCl) and two treated groups (Glu.SO4-LIPO and Glu.SO4) using a single dose. Parasitological analysis revealed that Glu.SO4-LIPO was as efficient as Glu.SO4 in reducing egg elimination and worm burden. Treatment with free Glu.SO4 and Glu.SO4-LIPO induced a statistically significant reduction in the number of granulomas (62 and 63 percent, respectively). Lectin histochemistry showed that wheat germ agglutinin intensely stained the egg-granuloma system in all treated groups. On the other hand, peanut agglutinin stained cells in the control groups, but not in the treated groups. The present results suggest a correlation between the decreasing number of hepatic egg-granulomas and the glycosylation profile of the egg-granuloma system in animals treated with free Glu.SO4 or Glu.SO4-LIPO.
Assuntos
Animais , Feminino , Masculino , Camundongos , Anti-Helmínticos/farmacologia , Glucanos/farmacologia , Líquens/química , Extratos Vegetais/farmacologia , Schistosoma mansoni/efeitos dos fármacos , Esquistossomose mansoni/tratamento farmacológico , Anti-Helmínticos/administração & dosagem , Fezes/parasitologia , Glucanos/administração & dosagem , Glucanos/isolamento & purificação , Imuno-Histoquímica , Intestinos/parasitologia , Intestinos/patologia , Lipossomos , Fígado/parasitologia , Fígado/patologia , Extratos Vegetais/administração & dosagemRESUMO
The antischistosomal activity of the sulfated polysaccharide α-D-glucan (Glu.SO(4)) extracted from Ramalina celastri was evaluated after encapsulation into liposomes (Glu.SO(4)-LIPO) in Schistosoma mansoni-infected mice. The effect of treatment with Glu.SO(4) and Glu.SO(4)-LIPO (10 mg/kg) on egg elimination, worm burden and hepatic granuloma formation was assessed using female albino Swiss mice, 35-40 days of age, weighing 25 ± 2 g, infected with 150 cercariae/animal (Biomphalaria glabrata, BH strain). Four groups (N = 10) were studied, two controls (empty liposomes and NaCl) and two treated groups (Glu.SO(4)-LIPO and Glu.SO(4)) using a single dose. Parasitological analysis revealed that Glu.SO(4)-LIPO was as efficient as Glu.SO(4) in reducing egg elimination and worm burden. Treatment with free Glu.SO(4) and Glu.SO(4)-LIPO induced a statistically significant reduction in the number of granulomas (62 and 63%, respectively). Lectin histochemistry showed that wheat germ agglutinin intensely stained the egg-granuloma system in all treated groups. On the other hand, peanut agglutinin stained cells in the control groups, but not in the treated groups. The present results suggest a correlation between the decreasing number of hepatic egg-granulomas and the glycosylation profile of the egg-granuloma system in animals treated with free Glu.SO(4) or Glu.SO(4)-LIPO.
Assuntos
Anti-Helmínticos/farmacologia , Glucanos/farmacologia , Líquens/química , Extratos Vegetais/farmacologia , Schistosoma mansoni/efeitos dos fármacos , Esquistossomose mansoni/tratamento farmacológico , Animais , Anti-Helmínticos/administração & dosagem , Fezes/parasitologia , Feminino , Glucanos/administração & dosagem , Glucanos/isolamento & purificação , Imuno-Histoquímica , Intestinos/parasitologia , Intestinos/patologia , Lipossomos , Fígado/parasitologia , Fígado/patologia , Masculino , Camundongos , Extratos Vegetais/administração & dosagemRESUMO
The aim was to synthesize and characterize fucoidan-coated poly(isobutylcyanoacrylate) nanoparticles. The nanoparticles were prepared by anionic emulsion polymerization (AEP) and by redox radical emulsion polymerization (RREP) of isobutylcyanoacrylate using fucoidan as a new coating material. The nanoparticles were characterized, and their cytotoxicity was evaluated in vitro on J774 macrophage and NIH-3T3 fibroblast cell lines. Cellular uptake of labeled nanoparticles was investigated by confocal fluorescence microscopy. Results showed that both methods were suitable to prepare stable formulations of fucoidan-coated PIBCA nanoparticles. Stable dispersions of nanoparticles were obtained by AEP with up to 100% fucoidan as coating material. By the RREP method, stable suspensions of nanoparticles were obtained with only up to 25% fucoidan in a blend of polysaccharide composed of dextran and fucoidan. The zeta potential of fucoidan-coated nanoparticles was decreased depending on the percentage of fucoidan. It reached the value of -44 mV for nanoparticles prepared by AEP with 100% of fucoidan. Nanoparticles made by AEP appeared more than four times more cytotoxic (IC(50) below 2 µg/mL) on macrophages J774 than nanoparticles made by RREP (IC(50) above 9 µg/mL). In contrast, no significant difference in cytotoxicity was highlighted by incubation of the nanoparticles with a fibroblast cell line. On fibroblasts, both types of nanoparticles showed similar cytotoxicity. Confocal fluorescence microscopy observations revealed that all types of nanoparticles were taken up by both cell lines. The distribution of the fluorescence in the cells varied greatly with the type of nanoparticles.
Assuntos
Antineoplásicos/toxicidade , Sistemas de Liberação de Medicamentos , Nanopartículas/toxicidade , Polissacarídeos/toxicidade , Adsorção , Animais , Antineoplásicos/química , Antineoplásicos/metabolismo , Linhagem Celular , Cianoacrilatos/química , Cianoacrilatos/toxicidade , Composição de Medicamentos , Avaliação Pré-Clínica de Medicamentos , Emulsões , Embucrilato , Excipientes/química , Fibroblastos/efeitos dos fármacos , Fibroblastos/fisiologia , Fluorescência , Formazans/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/fisiologia , Camundongos , Microscopia Confocal , Nanopartículas/química , Tamanho da Partícula , Phaeophyceae , Fitoterapia , Extratos Vegetais , Polimerização , Polissacarídeos/química , Polissacarídeos/metabolismo , Sais de Tetrazólio/metabolismoRESUMO
Usnic acid, a lichen metabolite, is known to exert antimitotic and antiproliferative activities against normal and malignant human cells. Many chemotherapy agents exert their activities by blocking cell cycle progression, inducing cell death through apoptosis. Microtubules, protein structure involved in the segregation of chromosomes during mitosis, serve as chemotherapeutical targets due to their key role in cellular division as well as apoptosis. The aim of this work was to investigate whether usnic acid affects the formation and/or stabilisation of microtubules by visualising microtubules and determining mitotic indices after treatment. The breast cancer cell line MCF7 and the lung cancer cell line H1299 were treated with usnic acid 29 µM for 24 hours and two positive controls: vincristine (which prevents the formation of microtubules) or taxol (which stabilizes microtubules). Treatment of MCF7 and H1299 cells with usnic acid did not result in any morphological changes in microtubules or increase in the mitotic index. These results suggest that the antineoplastic activity of usnic acid is not related to alterations in the formation and/or stabilisation of microtubules.(AU)
O ácido úsnico, um metabólito de liquens, é conhecido por sua atividade antimitótica e antiproliferativa em células humanas normais e malignas. Muitos quimioterápicos exercem suas atividades bloqueando a progressão do ciclo celular e induzindo morte celular por apoptose. Os microtúbulos, estruturas protéicas envolvidas na segregação dos cromossomos durante a mitose, servem como alvo quimioterapêutico devido ao seu importante papel tanto na divisão celular quanto nos mecanismos de morte celular por apoptose. O objetivo deste trabalho foi investigar se o ácido úsnico afeta a formação e/ou estabilização dos microtúbulos, a partir da visualização de microtúbulos e determinação de índices mitóticos após o tratamento. Células de câncer de mama MCF7 e de câncer de pulmão H1299 foram tratadas por 24 horas com 29 µM de ácido úsnico e dois controles positivos: vincristina (que impede a formação de microtúbulos) e taxol (que estabiliza microtúbulos). O tratamento das células MCF7 e H1299 com o ácido úsnico não resultou em aumento do índice mitótico. Os resultados sugerem que a atividade antineoplásica do ácido úsnico não está relacionada a alterações na formação e/ou estabilização de microtúbulos.(AU)
Assuntos
Humanos , Índice Mitótico/métodos , Líquens/metabolismo , Microtúbulos , /administração & dosagem , Neoplasias da Mama/tratamento farmacológico , Neoplasias Pulmonares/tratamento farmacológico , Antimitóticos/isolamento & purificação , Anticarcinógenos/administração & dosagemRESUMO
Usnic acid, a lichen metabolite, is known to exert antimitotic and antiproliferative activities against normal and malignant human cells. Many chemotherapy agents exert their activities by blocking cell cycle progression, inducing cell death through apoptosis. Microtubules, protein structure involved in the segregation of chromosomes during mitosis, serve as chemotherapeutical targets due to their key role in cellular division as well as apoptosis. The aim of this work was to investigate whether usnic acid affects the formation and/or stabilisation of microtubules by visualising microtubules and determining mitotic indices after treatment. The breast cancer cell line MCF7 and the lung cancer cell line H1299 were treated with usnic acid 29 µM for 24 hours and two positive controls: vincristine (which prevents the formation of microtubules) or taxol (which stabilizes microtubules). Treatment of MCF7 and H1299 cells with usnic acid did not result in any morphological changes in microtubules or increase in the mitotic index. These results suggest that the antineoplastic activity of usnic acid is not related to alterations in the formation and/or stabilisation of microtubules.
O ácido úsnico, um metabólito de liquens, é conhecido por sua atividade antimitótica e antiproliferativa em células humanas normais e malignas. Muitos quimioterápicos exercem suas atividades bloqueando a progressão do ciclo celular e induzindo morte celular por apoptose. Os microtúbulos, estruturas protéicas envolvidas na segregação dos cromossomos durante a mitose, servem como alvo quimioterapêutico devido ao seu importante papel tanto na divisão celular quanto nos mecanismos de morte celular por apoptose. O objetivo deste trabalho foi investigar se o ácido úsnico afeta a formação e/ou estabilização dos microtúbulos, a partir da visualização de microtúbulos e determinação de índices mitóticos após o tratamento. Células de câncer de mama MCF7 e de câncer de pulmão H1299 foram tratadas por 24 horas com 29 µM de ácido úsnico e dois controles positivos: vincristina (que impede a formação de microtúbulos) e taxol (que estabiliza microtúbulos). O tratamento das células MCF7 e H1299 com o ácido úsnico não resultou em aumento do índice mitótico. Os resultados sugerem que a atividade antineoplásica do ácido úsnico não está relacionada a alterações na formação e/ou estabilização de microtúbulos.
Assuntos
Feminino , Humanos , Antimitóticos/farmacologia , Antineoplásicos/farmacologia , Benzofuranos/farmacologia , Microtúbulos/efeitos dos fármacos , Paclitaxel/farmacologia , Vincristina/farmacologia , Neoplasias da Mama/patologia , Linhagem Celular Tumoral/efeitos dos fármacos , Neoplasias Pulmonares/patologiaRESUMO
Usnic acid, a lichen metabolite, is known to exert antimitotic and antiproliferative activities against normal and malignant human cells. Many chemotherapy agents exert their activities by blocking cell cycle progression, inducing cell death through apoptosis. Microtubules, protein structure involved in the segregation of chromosomes during mitosis, serve as chemotherapeutical targets due to their key role in cellular division as well as apoptosis. The aim of this work was to investigate whether usnic acid affects the formation and/or stabilisation of microtubules by visualising microtubules and determining mitotic indices after treatment. The breast cancer cell line MCF7 and the lung cancer cell line H1299 were treated with usnic acid 29 microM for 24 hours and two positive controls: vincristine (which prevents the formation of microtubules) or taxol (which stabilizes microtubules). Treatment of MCF7 and H1299 cells with usnic acid did not result in any morphological changes in microtubules or increase in the mitotic index. These results suggest that the antineoplastic activity of usnic acid is not related to alterations in the formation and/or stabilisation of microtubules.
Assuntos
Antimitóticos/farmacologia , Antineoplásicos/farmacologia , Benzofuranos/farmacologia , Microtúbulos/efeitos dos fármacos , Paclitaxel/farmacologia , Vincristina/farmacologia , Neoplasias da Mama/patologia , Linhagem Celular Tumoral/efeitos dos fármacos , Feminino , Humanos , Neoplasias Pulmonares/patologiaRESUMO
BACKGROUND: Investigations in the field of pharmaceutical analysis and quality control of medicines require analytical procedures that achieve suitable performance. An analytical curve is one of the most important steps in the chemical analysis presenting a direct relationship to features such as linearity. OBJECTIVE: This study has the aim of developing a new methodology, the stationary cuvette, to derive analytical curves by spectroscopy for drug analysis. METHODOLOGY: The method consists basically of the use of a cuvette with a path length of 10 cm, containing a constant volume of solvent in which increasing amounts of a stock solution of the sample are added, droplet by droplet. After each addition, the cuvette is stirred and the absorbance is measured. This procedure was compared with the currently used methodology, which requires a labour-intensive dilution process, and possible sources of variation between them were evaluated. RESULTS: The results demonstrated that the proposed technique presented high sensitivity and similar reproducibility compared with the conventional methodology. In addition, a number of advantages were observed, such as user-friendliness, cost-effectiveness, accuracy, precision and robustness. CONCLUSION: The stationary cuvette approach may be considered to be an appropriate alternative to derive analytical curves for analysing drug content in raw materials and medicines through UV-VIS spectrophotometry.