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1.
Ars pharm ; 62(1): 40-51, ene.-mar. 2021. tab, graf
Artigo em Inglês | IBECS-Express | IBECS | ID: ibc-ET6-1767

RESUMO

INTRODUCTION: Several natural products exhibit promising antineoplastic activity against bladder cancer cells, including allyl isothiocyanate (AITC). However, the AITC irritates the mucous membranes and induces eczematous or vesicular skin reactions. Thus, pharmaceutical formulations are necessary to overcome these problems. The aim was to develop micellar solutions containing AITC and investigate their antitumoral activity in bladder carcinoma cell lines. METHOD: The micellar solutions were prepared by cold dispersion method. Subsequently, we evaluated cytotoxicity, cell proliferation, cell cycle kinetics and long-term effects of micelles in bladder cancer cells. RESULTS: Cytotoxicity and cell proliferation assays showed there was an increase in AITC activity when it was encapsulated in micelles. We also observed cell cycle arrest in the S phase after treatment with AITC-micelles. Furthermore, the formulation was able to maintain the long-term effects of free AITC. CONCLUSIONS: The micellar solutions developed can become an interesting approach for administration of AITC in the treatment of bladder cancer


INTRODUCCIÓN: Varios productos naturales exhiben actividad antineoplásica prometedora contra las células can¬cerosas de vejiga, incluido el isotiocianato de alilo (AITC). Sin embargo, el AITC irrita las membranas mucosas e induce reacciones cutáneas vesiculares o eccematosas. Por tanto, las formulaciones farmacéuticas son necesarias para superar estos problemas. El objetivo era desarrollar soluciones micelares que contengan AITC e investigar su actividad antitumoral en líneas celulares de carcinoma de vejiga. MÉTODO: Las soluciones micelares se prepararon mediante el método de dispersión en frío. Posteriormente, eval¬uamos la citotoxicidad, la proliferación celular, la cinética del ciclo celular y los efectos a largo plazo de las micelas en las células del cáncer de vejiga. RESULTADOS: Los ensayos de citotoxicidad y proliferación celular mostraron que hubo un aumento en la actividad de AITC cuando se encapsuló en micelas. También observamos la detención del ciclo celular en la fase S después del tratamiento con micelas AITC. Además, la formulación pudo mantener los efectos a largo plazo del AITC libre. CONCLUSIONES: Las soluciones micelares desarrolladas pueden convertirse en un enfoque interesante para la ad¬ministración de AITC en el tratamiento del cáncer de vejiga

2.
Braz. J. Pharm. Sci. (Online) ; 56: e18411, 2020. tab, graf
Artigo em Inglês | LILACS-Express | LILACS | ID: biblio-1132049

RESUMO

Antimicrobial and antitumor activities of resveratrol, a compound found mainly in grapes, have already been demonstrated. However, its low bioavailability is a limiting factor for therapeutic application. Polymeric micelles can be an approach to solve this problem since they can encapsulate hydrophobic substances. We developed and characterized micellar formulations containing resveratrol and evaluated their cytotoxic and antimicrobial effects. The formulations were prepared by the cold dispersion method with different concentrations of F127 (5 or 10% w/w) and resveratrol (500 or 5000 µM). The formulations were characterized according to size, polydispersity index, pH, encapsulation rate and in vitro release. Cytotoxic effect was evaluated on a bladder cancer cell line and antimicrobial effect was evaluated on E. coli, S. aureus and C. albicans. One of the formulations (10% w/w of F127 and 5000 µM of resveratrol) was a monodispersed solution with high encapsulation rate, thus it was chosen for the cytotoxicity and antimicrobial assays. MS- 10+RES-3 was able to preserve the antimicrobial and cytotoxic activity of resveratrol. This is the first study that evaluated antimicrobial potential and cytotoxicity of micelles containing resveratrol on bladder cancer cells and the results showed that micellar nanostructures could ensure the maintenance of the biological activity of resveratrol.

3.
Biomed Pharmacother ; 109: 779-787, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30551531

RESUMO

New therapeutic strategies against leishmaniasis are desirable, since the treatment against disease presents problems, such as the toxicity, high cost and/or parasite resistance. As consequence, new antileishmanial compounds are necessary to be identified, as presenting high activity against Leishmania parasites, but low toxicity in mammalian hosts. Flau-A is a naphthoquinone derivative recently showed to presents an in vitro effective action against Leishmania amazonensis and L. infantum species. In the present work, the in vivo efficacy of Flau-A, which was incorporated into a Poloxamer 407-based micelle system, was evaluated in a murine model against L. amazonensis infection. Amphotericin B (AmB) and Ambisome® were used as controls. The animals were infected and later treated with the compounds. Thirty days after the treatment, parasitological and immunological parameters were evaluated. Results showed that AmB, Ambisome®, Flau-A or Flau-A/M-treated animals presented significantly lower average lesion diameter and parasite burden in tissue and organs evaluated, when compared to the control (saline and micelle) groups. Flau-A or Flau-A/M-treated mice were those presenting the most significant reductions in the parasite burden, when compared to the others. These animals developed also a more polarized antileishmanial Th1 immune response, which was based on significantly higher levels of IFN-γ, IL-12, TNF-α, GM-CSF, and parasite-specific IgG2a isotype; associated with low levels of IL-4, IL-10, and IgG1 antibody. The absence of toxicity was found in these animals, although mice receiving AmB have showed high levels of renal and hepatic damage markers. In conclusion, results suggested that the Flau-A/M compound may be considered as a possible therapeutic target to be evaluated against human leishmaniasis.


Assuntos
Antiprotozoários/uso terapêutico , Leishmania/efeitos dos fármacos , Leishmaniose/tratamento farmacológico , Micelas , Naftoquinonas/uso terapêutico , Poloxâmero/uso terapêutico , Animais , Antiprotozoários/química , Antiprotozoários/farmacocinética , Excipientes/química , Excipientes/farmacocinética , Excipientes/uso terapêutico , Feminino , Leishmania/metabolismo , Leishmaniose/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Naftoquinonas/química , Naftoquinonas/farmacocinética , Poloxâmero/química , Poloxâmero/farmacocinética , Resultado do Tratamento
4.
Parasitol Int ; 68(1): 63-72, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30339837

RESUMO

Clioquinol (5-chloro-7-iodoquinolin-8-ol or ICHQ) was recently showed to presents an in vitro effective antileishmanial action, causing changes in membrane permeability, mitochondrial functionality, and parasite morphology. In the present study, ICHQ was incorporated into a Poloxamer 407-based polymeric micelles system (ICHQ/M), and its antileishmanial activity was in vivo evaluated in L. amazonensis-infected BALB/c mice. Amphotericin B (AmpB) and its liposomal formulation (Ambisome®) were used as controls. Parasitological and immunological evaluations were performed 30 days after the treatment. Results indicated more significant reductions in the average lesion diameter and parasite burden in ICHQ or ICHQ/M-treated mice, which were associated with the development of a polarized Th1 immune response, based on production of high levels of IFN-γ, IL-12, TNF-α, GM-CSF, and antileishmanial IgG2a antibody. Control groups´ mice produced high levels of IL-4, IL-10, and IgG1 isotype antibody. No organic toxicity was found by using ICHQ or ICHQ/M to treat the animals, although those receiving AmpB and Ambisome® have presented higher levels of renal and hepatic damage markers. In conclusion, results suggested that the ICHQ/M composition can be considered as an antileishmanial candidate to be tested against human leishmaniasis.


Assuntos
Antiprotozoários/imunologia , Antiprotozoários/uso terapêutico , Clioquinol/imunologia , Clioquinol/uso terapêutico , Leishmania mexicana/efeitos dos fármacos , Leishmaniose Visceral/tratamento farmacológico , Poloxâmero/administração & dosagem , Anfotericina B/administração & dosagem , Anfotericina B/uso terapêutico , Anfotericina B/toxicidade , Animais , Anticorpos Antiprotozoários/sangue , Antígenos de Protozoários/administração & dosagem , Antígenos de Protozoários/imunologia , Antígenos de Protozoários/uso terapêutico , Antiprotozoários/administração & dosagem , Antiprotozoários/toxicidade , Clioquinol/administração & dosagem , Citocinas/biossíntese , Citocinas/imunologia , Sistemas de Liberação de Medicamentos/métodos , Humanos , Imunoglobulina G/sangue , Interferon gama/biossíntese , Interferon gama/imunologia , Leishmania mexicana/crescimento & desenvolvimento , Leishmaniose Visceral/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Micelas , Carga Parasitária , Poloxâmero/química , Células Th1
5.
Sci Rep ; 8(1): 15310, 2018 10 17.
Artigo em Inglês | MEDLINE | ID: mdl-30333575

RESUMO

This study aimed to investigate the potential of an oral formulation (QV formulation) containing Quercetin and a Dipeptidyl Peptidase-4 Inhibitor (DPP-4 inhibitor), Vildagliptin, in improving metabolic homeostasis in type 1 diabetes model. Female albino Fischer rats were divided into four groups: untreated control animals (C), untreated diabetic animals (D), diabetic animals treated with QV formulation (DQV), and diabetic animals treated with insulin (DI). Diabetes was induced by injection of alloxan (135 mg kg body mass)-1 and confirmed by glycemic test. After the 30-day treatment period, biochemical parameters were analyzed in the pancreas, liver, and serum. Histopathological changes in pancreatic tissue were examined by Hematoxyline & Eosin staining and the insulin content in the islet measured by immunohistochemistry with anti-insulin antibody. The glycogen content in the hepatocytes was quantified by Periodic Schiff Acid staining. The QV formulation reduced the glycemia, preserved the pancreatic architecture, increased insulin levels, furthermore ameliorated lipid profile and to promote higher survival rate of animals. Together, our data suggest that the QV formulation treatment was able to normalize metabolic homeostasis in type 1 diabetic rats.


Assuntos
Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 1/metabolismo , Inibidores da Dipeptidil Peptidase IV/farmacologia , Homeostase/efeitos dos fármacos , Quercetina/farmacologia , Vildagliptina/farmacologia , Administração Oral , Animais , Glicemia/metabolismo , Diabetes Mellitus Experimental/sangue , Diabetes Mellitus Tipo 1/sangue , Inibidores da Dipeptidil Peptidase IV/administração & dosagem , Quimioterapia Combinada , Feminino , Insulina/metabolismo , Lipídeos/sangue , Quercetina/administração & dosagem , Ratos Endogâmicos F344 , Fatores de Tempo , Vildagliptina/administração & dosagem
6.
Exp Parasitol ; 186: 24-35, 2018 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-29448040

RESUMO

Amphotericin B (Amp) has been well-successfully used to treat against Leishmania infection, although high toxicity has been found in patients. In the present study, Amp was administered in Leishmania infantum-infected BALB/c mice by three distinct delivery systems aiming to compare their efficacy against challenge infection, as well as their side effects in a murine visceral leishmaniasis (VL) model. This product was administered in a Poloxamer P407 (Pluronic® F127)-based polymeric micelle system (Amp/M), in the Ambisome® formulation (Lip-Amp) or in a free format (free Amp). Glucantime® (Gluc) was used as a comparative drug. Aiming to evaluate different endpoints of the treatments, the efficacy of the compounds was investigated one and 15-days after the therapeutic regimens, determining the parasite load by a limiting dilution assay and a quantitative PCR (qPCR) technique, as well as evaluating the immune response generated in the infected and treated animals. In the results, Amp/M or Lip-Amp-treated mice presented the best outcomes, since significant parasite load reductions were found in the evaluated organs, as well as a parasite-specific Th1 immune response was observed in the animals. In addition, no hepatic or renal damage was found in these mice. On the other hand, free Amp or Gluc induced toxicity in the animals, which was associated with a low Th1 immune response. Comparatively, Amp/M was the most effective drug in our experimental model, and results showed that the Amp-carrying system could be considered as a future alternative in studies against VL.


Assuntos
Anfotericina B/administração & dosagem , Antiprotozoários/administração & dosagem , Sistemas de Liberação de Medicamentos/normas , Leishmaniose Visceral/tratamento farmacológico , Anfotericina B/toxicidade , Animais , Antiprotozoários/toxicidade , Citocinas/metabolismo , Modelos Animais de Doenças , Feminino , Rim/efeitos dos fármacos , Leishmania infantum/efeitos dos fármacos , Fígado/efeitos dos fármacos , Meglumina/administração & dosagem , Antimoniato de Meglumina , Camundongos , Camundongos Endogâmicos BALB C , Micelas , Nitritos/metabolismo , Compostos Organometálicos/administração & dosagem , Organismos Livres de Patógenos Específicos , Baço/citologia , Baço/imunologia
7.
Parasitol Res ; 115(11): 4083-4095, 2016 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-27365053

RESUMO

The current treatment of leishmaniasis has been hampered due to the high toxicity of the available drugs and long duration protocols, which often lead to its abandonment. In the present study, a poloxamer 407-based delivery system was developed, and a molecule, 8-hydroxyquinoline (8-HQN), was incorporated with it, leading to an 8-HQN/micelle (8-HQN/M) composition. Assays were performed to evaluate the in vitro antileishmanial activity of 8-HQN/M against Leishmania amazonensis stationary promastigotes. The cytotoxicity in murine macrophages and in human red cells, as well as the efficacy of the treatment in macrophages infected by parasites, was also assessed. This product was also evaluated for the treatment of murine tegumentary leishmaniasis, using L. amazonensis-infected BALB/c mice. To evaluate the in vivo efficacy of the treatment, the average lesion diameter (area) in the infected tissue, as well as the parasite load at the site of infection (skin), spleen, liver and draining lymph nodes were examined. Non-incorporated micelle (B-8-HQN/M) and the free molecule (8-HQN) were used as controls, besides animals that received only saline. The parasite burden was evaluated by limiting dilution and quantitative real-time PCR (qPCR) techniques, and immunological parameters associated with the treatments were also investigated. In the results, the 8-HQN/M group, when compared to the others, presented more significant reductions in the average lesion diameter and in the parasite burden in the skin and all evaluated organs. These animals also showed significantly higher levels of parasite-specific IFN-γ, IL-12, and GM-CSF, associated with low levels of IL-4 and IL-10, when compared to the saline, 8-HQN/M, and B-8-HQN groups. A predominant IL-12-driven IFN-γ production, against parasite proteins, mainly produced by CD4+ T cells, was observed in the treated animals, post-infection. In conclusion, 8-HQN/M was highly effective in treating L. amazonensis-infected BALB/c mice and can be considered alone, or combined with other drugs, as an alternative treatment for tegumentary leishmaniasis. Graphical Abstract Therapeutic scheme and immunological and parasitological parameters developed in the present study.


Assuntos
Leishmania mexicana/efeitos dos fármacos , Leishmaniose Cutânea/tratamento farmacológico , Oxiquinolina/uso terapêutico , Animais , Citocinas/metabolismo , Modelos Animais de Doenças , Eritrócitos/parasitologia , Feminino , Humanos , Leishmaniose Cutânea/parasitologia , Fígado/parasitologia , Macrófagos/parasitologia , Camundongos , Camundongos Endogâmicos BALB C , Micelas , Oxiquinolina/administração & dosagem , Carga Parasitária , Polímeros , Baço/parasitologia , Linfócitos T/imunologia
8.
Parasitol Int ; 65(6 Pt A): 728-736, 2016 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-27425599

RESUMO

New therapeutics are urgently needed to treat visceral leishmaniasis (VL). Due to the fact that drug discovery is a long and expensive process, the development of delivery systems to carry old and toxic drugs could be considered, as well as the evaluation of new molecules that have already shown to present biological activity. In this context, the present study evaluated the in vitro and in vivo antileishmanial activity of an 8-hydroxyquinoline (8-HQN)-containing polymeric micelle (8-HQN/M) system against Leishmania infantum, the main causative agent of VL in the Americas. The experimental strategy used was based on the evaluation of the parasite load by a limiting-dilution technique in the spleen, liver, bone marrow and draining lymph nodes of the infected and treated animals, as well as by a quantitative PCR (qPCR) technique to also assess the splenic parasite load. The immune response developed was evaluated by the production of IFN-γ, IL-4, IL-10, IL-12 and GM-CSF cytokines, as well as by antileishmanial nitrite dosage and antibodies production. Hepatic and renal enzymes were also investigated to verify cellular injury as a result of treatments toxicity. In the results, 8-HQN/M-treated mice, when compared to the other groups: saline, free amphotericin B (AmpB, as a drug control), 8-HQN and B-8-HQN/M (as a micelle control) showed more significant reductions in their parasite burden in all evaluated organs. These animals also showed an antileishmanial Th1 immunity, which was represented by high levels of IFN-γ, IL-12, GM-CSF and nitrite, associated with a low production of IL-4 and IL-10 and anti-Leishmania IgG1 isotype antibodies. In addition, any hepatic or renal damage was found in these treated animals. In conclusion, 8-HQN/M was effective in treating L. infantum-infected BALB/c mice, and can be considered alone, or combined with other drugs, as an alternative treatment for VL.


Assuntos
Antiparasitários/uso terapêutico , Portadores de Fármacos/uso terapêutico , Leishmania infantum/efeitos dos fármacos , Leishmaniose Visceral/tratamento farmacológico , Oxiquinolina/uso terapêutico , Anfotericina B/uso terapêutico , Animais , Anticorpos Antiprotozoários/sangue , Feminino , Fator Estimulador de Colônias de Granulócitos e Macrófagos/biossíntese , Imunoglobulina G/sangue , Interferon gama/biossíntese , Interleucina-10/biossíntese , Interleucina-12/biossíntese , Interleucina-4/biossíntese , Leishmania infantum/imunologia , Leishmaniose Visceral/parasitologia , Camundongos , Camundongos Endogâmicos BALB C , Micelas , Carga Parasitária
9.
Exp Parasitol ; 169: 34-42, 2016 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-27427166

RESUMO

In the present study, a Poloxamer 407-based amphotericin B (AmpB)-containing polymeric micelles system (AmpB/M) was employed in the treatment of Leishmania amazonensis-infected BALB/c mice. Initially, the in vitro antileishmanial activity (IC50 value) of AmpB/M and B-AmpB/M (empty micelles) against stationary promastigotes and amastigotes-like forms of the parasites was determined, and results were of 1.83 ± 0.4 and 22.1 ± 0.7 µM, respectively, for the promastigotes, and of 2.27 ± 0.5 and 33.98 ± 2.6 µM, respectively, for the amastigotes-like. The cytotoxic concentration (CC50) values of these products were also evaluated, and we found the results of 119.5 ± 9.6 and 134.7 ± 10.3 µM, respectively. With these values, the selectivity index (SI) was calculated and results were of 65.3 and 5.4, respectively, for the promastigotes, and of 59.3 and 3.96, respectively, for the amastigotes-like of the parasites. Free AmpB showed IC50 values of 1.2 ± 0.3 and 2.5 ± 0.5 µM for the promastigotes and amastigotes-like, respectively, whereas the CC50 value was of 9.5 ± 0.4 µM. The SI values of this drug were of 7.9 and 3.8, respectively, for the promastigote and amastigote-like stages of the parasites. After, animals were infected and received saline or were treated subcutaneously with free AmpB, AmpB/M or B-AmpB/M. In the results, free AmpB-treated and infected mice showed reductions in their body weight, which were associated with hepatic and renal damage; however, no organic alteration was observed in the AmpB/M-treated animals. In addition, these animals showed significant reductions in their lesion average size and in the parasite burden in all evaluated infected tissue and organs, when compared to the other groups; as well as significantly higher levels of antileishmanial IFN-γ, IL-12, GM-CSF and nitrite, which were associated with low production of IL-4, IL-10 and IgG1 isotype antibodies. In conclusion, this AmpB/M system could be considered as an alternative for future studies in the treatment of tegumentary leishmaniasis.


Assuntos
Anfotericina B/administração & dosagem , Antiprotozoários/administração & dosagem , Excipientes , Leishmania mexicana/efeitos dos fármacos , Leishmaniose Cutânea/tratamento farmacológico , Poloxâmero , Anfotericina B/farmacologia , Anfotericina B/uso terapêutico , Anfotericina B/toxicidade , Animais , Antiprotozoários/farmacologia , Antiprotozoários/uso terapêutico , Antiprotozoários/toxicidade , Sobrevivência Celular/efeitos dos fármacos , Citocinas/biossíntese , Relação Dose-Resposta a Droga , Feminino , Imunoglobulina G/biossíntese , Concentração Inibidora 50 , Leishmania mexicana/crescimento & desenvolvimento , Leishmania mexicana/imunologia , Leishmaniose Cutânea/imunologia , Leishmaniose Cutânea/parasitologia , Leishmaniose Cutânea/patologia , Fígado/parasitologia , Linfonodos/parasitologia , Macrófagos Peritoneais/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Micelas , Polímeros , Ratos , Baço/citologia , Baço/imunologia , Baço/parasitologia
10.
Int J Pharm ; 430(1-2): 359-65, 2012 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-22525078

RESUMO

We recently demonstrated that agitation during lipoplex formation (vorLTsiR) improves the gene knockdown effect of siRNA because the resultant decrease in lipoplex size leads to an enhanced uptake by cells. In furthering this line of research, the present study was focused on the interaction of siRNA to cationic liposomes during lipoplex preparation. A fluorescence resonance energy transfer (FRET) study indicated that the application of agitation in the presence of siRNA effectively reorganized positively charged lipids (DC-6-14 and DOPE) in an order that effectively promoted further electrostatic interaction between the negatively charged phosphate backbone of siRNA and the positively charged lipids in the cationic liposome membrane. A circular dichroism (CD) study indicated that the agitation did not bring about a change in the A-form helix of siRNA, therefore the interactions between the lateral anionic groups of siRNA - responsible for the characteristic bands of the A-form helix - and cationic liposomes were effectively promoted. Factorial design coupled with response surface methodology was used to statistically analyze the influence of vortex speed and time and siRNA dose on the in vitro gene knockdown effects of siRNA-lipoplex that were spontaneously formulated (spoLTsiR) along with that formulated under agitation (vorLTsiR). The analysis indicated that vortex speed plays the most important role in enhancing the gene knockdown effect of siRNA among the three variables, although all three are important. It was concluded that the high energy transmitted by applying agitation during lipoplex formation harmonized the interaction of siRNA to positively charged lipids (DC-6-14 and DOPE) in cationic liposomes, resulting in a superior gene knockdown efficacy of vorLTsiR compared to spoLTsiR. Our study suggests that the preparation procedure is one of the critical factors in producing the enhanced gene knockdown effect of siRNA.


Assuntos
Etanolaminas/química , Técnicas de Silenciamento de Genes , Miristatos/química , Fosfatidiletanolaminas/química , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Transfecção/métodos , Cátions , Dicroísmo Circular , Análise Fatorial , Transferência Ressonante de Energia de Fluorescência , Células HeLa , Humanos , Lipossomos , Luciferases/biossíntese , Luciferases/genética , Movimento (Física) , Conformação de Ácido Nucleico , RNA Interferente Pequeno/química , Fatores de Tempo
11.
Int J Pharm ; 410(1-2): 153-60, 2011 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-21392562

RESUMO

The successful delivery of therapeutic siRNA to the designated target cells and their availability at the intracellular site of action are crucial requirements for successful RNAi therapy. In the present study, we focused on the siRNA-lipoplex preparation procedure and its effect on the gene-knockdown efficiency of siRNA in vitro. Agitation (vortex-mixing) during siRNA-lipoplex (vor-LTsiR) preparation and its effect on the gene-knockdown efficiency of stably expressed cell GFP was investigated, and their efficiency was compared with that of spontaneously formed lipoplex (spo-LTsiR). A dramatic difference in size between lipoplexes was observed at the N/P ratio of 7.62 (siRNA dose of 30 nM), even though both lipoplexes were positively charged. With the siRNA dose of 30 nM, vor-LTsiR accomplished a 50% gene-knockdown, while spo-LTsiR managed a similar knockdown effect at the 120 nM level, suggesting that the preparation procedure remarkably affects the gene-knockdown efficacy of siRNA. The uptake of vor-LTsiR was mainly via clathrin-mediated endocytosis, whereas that of spo-LTsiR was via membrane fusion. In addition, by inhibiting clathrin-mediated endocytosis, the gene-knockdown efficiency was significantly lowered. The size of the lipoplex, promoted by the preparation procedure, is likely to define the entry pathway, resulting in an increased amount of siRNA internalized in cells and an enhanced gene-knockdown efficacy. The results of the present study definitively show that a proper siRNA-lipoplex preparation procedure makes a significant contribution to the efficiency of cellular uptake, and thereby, to the gene-knockdown efficiency of siRNA.


Assuntos
Técnicas de Silenciamento de Genes/métodos , Inativação Gênica , Lipídeos/química , RNA Interferente Pequeno/administração & dosagem , Cátions , Linhagem Celular Tumoral , Clatrina/metabolismo , Endocitose , Humanos , Lipossomos
12.
Drug Deliv Transl Res ; 1(4): 277-88, 2011 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-25788362

RESUMO

Small interfering RNAs (siRNAs) are small RNA molecules that have a potent, sequence-specific gene silencing effect and therefore show promise for therapeutic use as molecular-targeted drugs for the treatment of various genetic diseases, including cancer. The aim of the present study was to evaluate whether Argonaute2 (Ago2) is a therapeutically effective target for siRNA-based cancer therapy. Ago2 is the key protein in mammalian RNAi and is also known as the only member of the Ago family that mediates the microRNA (miRNA)-dependent cleavage of targeted mRNAs. It is assumed that these unique properties of the Ago2 protein can play a central role in the regulation of the miRNA pathway and subsequent translational inhibition of miRNA-targeted mRNAs, including cell survival and cancer progression. To assess its therapeutic effect, siRNA against Ago2 (Ago2-siRNA) was transfected into HT1080 human fibrosarcoma cells, which are malignant cancer cells. Ago2 gene silencing resulted in the inhibition of cell growth and the induction of apoptosis and G0/G1 arrest in the cell cycle. In addition, Ago2 knockdown induced morphological changes and actin stress fiber formation in the cells. The results of a microarray study showed that Ago2 suppression stimulated several crucial genes related to apoptosis, the cell cycle, immune response, cell adhesion, metabolism, etc. Repeated intratumoral injection of Ago2-siRNA/cationic liposome complex induced tumor growth suppression in an HT1080 xenograft model. These results suggest that the suppression of the Ago2 gene may be useful for the inhibition of cancer progression and that Ago2 may be a desirable target for siRNA-based cancer therapy.

13.
Methods Mol Biol ; 605: 461-72, 2010.
Artigo em Inglês | MEDLINE | ID: mdl-20072901

RESUMO

In the last two decades, cationic liposomes have been investigated as vehicles for nucleic acids [plasmid DNA (pDNA) and small interfering RNA (siRNA)] delivery in vitro and in vivo. The formation of cationic liposomes-nucleic acids complexes, termed lipoplexes, depends on a number of experimental variables. The quality of the nucleic acid and the cationic liposome as well as the selection of diluents for diluting the concentrated stocks strongly affect the resulting lipoplexes and their efficiency of gene-expression or gene-silencing effect following transfection. In addition, the molar ratio of cationic lipid nitrogen (N) to siRNA or pDNA phosphate (P) (N/P ratio) influences the final characteristics of the lipoplexes, such as size, surface zeta potential, and reproducibility, thereby reflecting their efficiency following transfection. The methods presented in this chapter could be helpful to obtain reliable and reproducible lipoplexes and experimental results.


Assuntos
DNA/administração & dosagem , Lipossomos/química , RNA Interferente Pequeno/administração & dosagem , Transfecção , Cátions/química , DNA/química , Inativação Gênica , Células HeLa , Humanos , Lipossomos/classificação , Plasmídeos/administração & dosagem , RNA Interferente Pequeno/química
14.
Pharm Res ; 25(11): 2497-504, 2008 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-18581204

RESUMO

PURPOSE: The purpose of this study is to determine if the treatment with siRNA-lipoplexes significantly influences on global gene expression in the treated cells. METHODS: We investigated global gene expression in a HT1080 cell line by a cDNA microarray. We also evaluated the effect of lipofection on global gene expression by determining the change of the expression of an exogenous gene, green fluorescence protein (GFP), and also determined treatment-related cytotoxicity. RESULTS: Treatment of the cells with either siRNA-lipoplexes or cationic liposomes altered the expression of approximately 2,500 genes. When lipoplexes containing non-specific siRNAs were used, GFP expression was enhanced. In this case the effect was independent on the dose and type of siRNA in the formulation. By contrast, when lipoplexes containing a specific siRNA against GFP was used, GFP expression was markedly diminished. These results clearly indicate that an efficient reduction of a targeted gene expression by a specific siRNA is accompanied by a significant alteration of the expression of numerous non-targeted genes. In addition, treatment-related cytotoxicity increased with siRNA- and cationic lipid-doses, but was not dependent on siRNA type. CONCLUSION: Non-specific effects of siRNA-lipoplexes may either enhance, attenuate or even fully mask the desired outcomes of siRNA-based biochemical studies and therapies.


Assuntos
Perfilação da Expressão Gênica , Lipossomos , RNA Interferente Pequeno/administração & dosagem , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Proteínas de Fluorescência Verde/genética , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , RNA Interferente Pequeno/farmacologia
15.
Int J Pharm ; 357(1-2): 199-205, 2008 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-18343610

RESUMO

In this report, we investigated the combined effect of drug liposomalization and addition of glycerol on the transdermal delivery of isosorbide 5-nitrate (ISN) in rat abdominal skin in vitro. Occlusive application of both liposomal and aqueous ISN solution, with and without addition of 5% glycerol, showed that drug liposomalization and addition of glycerol has far-reaching implications for ISN permeation and accumulation in 4 and 8 weeks old rat abdominal skin. Using 8 weeks old rat abdominal skin, the optimal concentration of glycerol to be added to liposomal ISN was found to be 5%. The ISN mean values permeated through and accumulated in stripped 8 weeks old rat abdominal skin from those formulations described above were not significant different, which might indicate the combined effect of glycerol and liposomal ISN resides solely in the stratum corneum (SC). Based on previous reports, the enhancement effect of glycerol might be due to an increase in the SC hydration, and perhaps due to subtle changes in the lipid organization caused by penetration of liposomal lipids within the SC intercellular spaces. These data might provide evidence that glycerol action on SC is useful to facilitate skin permeation and accumulation of drugs formulated in liposome.


Assuntos
Glicerol/farmacologia , Dinitrato de Isossorbida/análogos & derivados , Lipossomos/química , Vasodilatadores/administração & dosagem , Administração Cutânea , Animais , Química Farmacêutica , Portadores de Fármacos , Técnicas In Vitro , Dinitrato de Isossorbida/administração & dosagem , Dinitrato de Isossorbida/farmacocinética , Masculino , Ratos , Ratos Wistar , Absorção Cutânea , Vasodilatadores/farmacocinética
16.
Int J Pharm ; 353(1-2): 65-73, 2008 Apr 02.
Artigo em Inglês | MEDLINE | ID: mdl-18155369

RESUMO

Metronomic chemotherapy is a novel approach to the control of advanced cancer, as it appears to preferentially inhibit endothelial cell activity in the growing vasculature of tumors. Doxorubicin-containing sterically stabilized liposomes (DXR-SL) accumulate in large amounts in tumor tissue, resulting in enhanced antitumor effects of the encapsulated DXR. In the present study, it was hypothesized that metronomic chemotherapy may further augment the accumulation of DXR-SL, improving its therapeutic efficacy. This study tests the antitumor efficacy for the combination of a metronomic cyclophosphamide (CPA)-dosing schedule with sequential intravenous injections of DXR-SL in the treatment of lung metastatic B16BL6 melanoma-bearing mice. Three dosing schedules for the combination of metronomic CPA injections (s.c. 170 mg/kg every 6 days) plus either a low or a high dose of DXR-SL (i.v. 1 or 5 mg/kg every 6 days) were set: Schedule I, DXR-SL was given 3 days before the first CPA treatment; Schedule II, DXR-SL and CPA were given simultaneously; and, Schedule III, DXR-SL was given 3 days after the first CPA treatment. Lung weight and median survival time (MST) were evaluated. As expected, both the dosing schedule as well as the dose of DXR-SL improved therapeutic efficacy. Schedule I with the low DXR dose and Schedule II with the low or high DXR dose significantly increased MST, compared with regular metronomic CPA therapy. Under the dosing schedules (Schedule I with the low DXR dose and Schedule II with the high DXR), there was a strong relationship between increased MST and decreased lung weight. However, Schedule I with high DXR dose resulted in significantly lower lung weights, but did not increase MST, suggesting that chemotherapy may result in increased toxicity in some conditions. Although treatment regimens require optimization, the results of the present study may prove useful in further explorations of combining metronomic chemotherapy with liposomal anticancer drugs in the treatment of solid tumors.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Ciclofosfamida/administração & dosagem , Doxorrubicina/análogos & derivados , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/secundário , Polietilenoglicóis/administração & dosagem , Animais , Linhagem Celular Tumoral , Doxorrubicina/administração & dosagem , Esquema de Medicação , Masculino , Melanoma Experimental/patologia , Camundongos , Camundongos Endogâmicos C57BL
17.
Biol Pharm Bull ; 30(4): 751-7, 2007 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-17409515

RESUMO

Cationic liposomes (CL) are one of the most widely studied non-viral vectors for gene delivery. It is well-known that CL induces cytotoxicity following lipofection. However, little is known regarding the mechanism involved in the cytotoxicity. In this study, the in vitro cytotoxicity of CL and its complex with pDNA (lipoplex) was investigated, and a part of the mechanism of induction as well. While free pDNA did not show any cytotoxicity, pDNA increased the cytotoxicity of CL via the formation of lipoplex. In addition, the lipoplex-induced cytotoxicity increased in a lipoplex dose-dependent manner, irrespective of the type of pDNA, cell line and the absence or presence of serum. An assay showed that apoptosis was largely induced by treatment with the lipoplex (lipofection), but not with CL alone, in the tested range of concentration of CL and pDNA. Furthermore, following treatment with lipoplexes, the cells exhibited the morphological features of apoptosis and DNA fragmentation. A cDNA microarray study showed that the lipofection up-regulated 45 genes related to apoptosis, transcription regulation and immune response. These results clearly indicate that pDNA in the lipoplex increases the cytotoxicity of CL as a result of inducing apoptosis. The fundamental principle for gene therapy is to deliver gene-based therapeutics to target cells for specific gene targeting with minimal cytotoxicity. Our results suggest the possibility that cytotoxicity induced by lipofection, accompanied by gene changes, could intrinsically exacerbate, attenuate or even mask the desired effects of gene-based therapy.


Assuntos
DNA/metabolismo , Vetores Genéticos/toxicidade , Lipossomos/toxicidade , Plasmídeos/metabolismo , Animais , Apoptose/efeitos dos fármacos , Cátions/química , Linhagem Celular Tumoral , DNA/administração & dosagem , DNA/química , DNA/classificação , DNA/genética , Fragmentação do DNA/efeitos dos fármacos , DNA Complementar/análise , DNA Complementar/genética , Relação Dose-Resposta a Droga , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Terapia Genética/métodos , Glioma/metabolismo , Glioma/patologia , Glioma/terapia , Células HeLa , Humanos , Técnicas In Vitro , Lipossomos/administração & dosagem , Lipossomos/química , Lipossomos/metabolismo , Melanoma Experimental/metabolismo , Melanoma Experimental/patologia , Melanoma Experimental/terapia , Camundongos , Análise de Sequência com Séries de Oligonucleotídeos , Ratos , Transfecção
18.
Int J Pharm ; 333(1-2): 62-9, 2007 Mar 21.
Artigo em Inglês | MEDLINE | ID: mdl-17097247

RESUMO

Efficient delivery is a key issue in translating interference RNA technology into a feasible therapy. The efficiency of carrier systems used for this technology is commonly tested by co-transfection, i.e. simultaneous transfection with an exogenous gene and with the siRNA. Two approaches can be distinguished: (1) with the two transfectants in the same carrier complex (siRNA/pDNA/carrier) and (2) with the two transfectants in different carrier complexes (pDNA/carrier and siRNA/carrier). The process to prepare the nucleic acid(s)-carrier complexes and the transfection procedure may affect the effectiveness of the gene-silencing process. In this study, two preparation methods were compared, namely the co-preparation of an siRNA/pDNA/liposome lipoplex (Method I) and the separate preparation of an siRNA/liposome lipoplex and a pDNA/liposome lipoplex (Method II). siRNA in the lipoplex produced by Method I showed a stronger gene-silencing effect than that in the lipoplexes prepared by Method II. There was no significant difference between the two methods in the amount of siRNA delivered to cells. Cellular entry and intracellular trafficking of siRNA/pDNA/liposome lipoplex is likely to differ from those of the separate lipoplexes. When in Method II non-transcriptional pDNA was included in the complex with siRNA, the gene-silencing effect was significantly enhanced. If and to what extent the experimental design is suitable to quantify RNA interference remains to be demonstrated.


Assuntos
Lipídeos/química , Lipossomos , Plasmídeos/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Transfecção/métodos , Animais , Cátions , Linhagem Celular Tumoral , Colesterol/química , Citoplasma/metabolismo , Etanolaminas/química , Genes Reporter , Luciferases de Vaga-Lume , Luciferases de Renilla , Melanoma Experimental/genética , Melanoma Experimental/metabolismo , Camundongos , Miristatos/química , Tamanho da Partícula , Fosfatidiletanolaminas/química , RNA Interferente Pequeno/genética
19.
J Control Release ; 115(1): 94-102, 2006 Sep 28.
Artigo em Inglês | MEDLINE | ID: mdl-16919352

RESUMO

In order to investigate the effect of liposomal drugs on skin delivery, it was postulated that the process of liposomalization might lead the drug to an overpredicted solubility state which has far-reaching implications for drug skin permeation and accumulation. In this regard, conventional (CL) and flexible liposomes (FL) were prepared by the lipid film hydration method and the particles were downsized by sonication using hydrocortisone (HC) as a poorly water soluble model drug. The solutions derived from the whole CL and FL suspensions eluted on a Sephadex G-50 column (SG-50) demonstrated that most part of HC not only resides solely in the water phase but also it might exist in an improved solubility state. The results of the in vitro study using rat abdominal skin and occlusive application indicated that HC penetrated and accumulated much better solely than when associated with CL or FL. In regard to the penetration of the non-entrapped HC associated to liposomes bilayer fragments, a very small amount of phospholipids in the non-liposomal part eluted on SG-50 was found that could not justify by itself the penetration of HC associated to liposome bilayer fragments. It was proposed that all the steps of the liposomes preparation process might contribute for the increased HC solubility state, but definitively the presence of phospholipids played a crucial role on improving the HC solubility in the absence of sodium cholate. In comparison with commercially available ointments, the non-entrapped HC solution derived from the whole CL suspension eluted on SG-50 showed a higher concentration of HC accumulated and more uniformly distributed as well in the epidermis and dermis compartments. In addition, the thermodynamic activity of the non-entrapped HC solutions maintaining a driving force of the drug across the skin barrier pointed out that the level of HC solubility achieved during liposome preparation has far-reaching implication for drug skin permeation and accumulation in the experimental conditions used. The findings also indicated that the non-entrapped drug solutions obtained on the process of liposomalization could be useful on transdermal drug delivery systems, particularly for improving the permeation and accumulation capacity of poorly soluble drugs.


Assuntos
Anti-Inflamatórios/administração & dosagem , Hidrocortisona/administração & dosagem , Administração Cutânea , Animais , Anti-Inflamatórios/química , Anti-Inflamatórios/farmacocinética , Portadores de Fármacos , Hidrocortisona/química , Hidrocortisona/farmacocinética , Técnicas In Vitro , Lipossomos , Masculino , Microscopia Eletrônica de Varredura , Pomadas , Tamanho da Partícula , Fosfolipídeos/análise , Fosfolipídeos/química , Ratos , Ratos Wistar , Pele/química , Absorção Cutânea/efeitos dos fármacos , Suspensões
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