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1.
Biochim Biophys Acta ; 1840(3): 1171-80, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24361607

RESUMO

BACKGROUND: Amorphous silica particles with the primary dimensions of a few tens of nm, have been widely applied as additives in various fields including medicine and food. Especially, they have been widely applied in powders for making tablets and to coat tablets. However, their behavior and biological effects in the gastrointestinal tracts associated with oral administration remains unknown. METHODS: Amorphous silica particles with diameters of 50, 100, and 200nm were incubated in the fasted-state and fed-state simulated gastric and intestinal fluids. The sizes, intracellular transport into Caco-2 cells (model cells for intestinal absorption), the Caco-2 monolayer membrane permeability, and the cytotoxicity against Caco-2 cells were then evaluated for the silica particles. RESULTS: Silica particles agglomerated in fed-state simultaneous intestinal fluids. The agglomeration and increased particles size inhibited the particles' absorption into the Caco-2 cells or particles' transport through the Caco-2 cells. The in vitro cytotoxicity of silica particles was not observed when the average size was larger than 100nm, independent of the fluid and the concentration. CONCLUSION: Our study indicated the effect of diet on the agglomeration of silica particles. The sizes of silica particles affected the particles' absorption into or transport through the Caco-2 cells, and cytotoxicity in vitro, depending on the various biological fluids. GENERAL SIGNIFICANCE: The findings obtained from our study may offer valuable information to evaluate the behavior of silica particles in the gastrointestinal tracts or safety of medicines or foods containing these materials as additives.


Assuntos
Absorção Intestinal , Dióxido de Silício/toxicidade , Líquidos Corporais , Células CACO-2 , Sobrevivência Celular/efeitos dos fármacos , Humanos , Tamanho da Partícula , Dióxido de Silício/química , Dióxido de Silício/farmacocinética
2.
Mol Pharm ; 11(2): 560-7, 2014 Feb 03.
Artigo em Inglês | MEDLINE | ID: mdl-24383572

RESUMO

We have previously reported the intracellular trafficking mechanism of liposomal phospholipids. In the present study, we investigated the effects of liposomal phospholipids on the intracellular trafficking of doxorubicin (DXR). In DXR-encapsulated liposomes, polyethylene glycol (PEG)-modified phospholipids have been widely used as one of the liposomal lipids. First, we investigated the intracellular trafficking mechanism of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy-PEG2000] (PEG2000-DSPE), and demonstrated that the intracellular trafficking pathways of phospholipids changed by PEG modification. Then, we evaluated the effects of liposomal DXR on the intracellular trafficking of liposomal phospholipids. Under the phosphatidylinositol transfer protein (PITP)-suppressing condition by siRNA treatment, the intracellular amounts of DSPC derived from DXR-encapsulated liposomes were larger than that from nonencapsulated liposomes. Moreover, following the effects of liposomal phospholipids on the intracellular amounts of DXR, the intracellular amounts of DXR were increased under the PITP-suppressing condition in DXR-encapsulated liposomes. We showed that intracellular DXR was associated with the complex of PITP and DSPC, and the extracellular efflux of DXR was enhanced by complex formation with PITP and DSPC.


Assuntos
Doxorrubicina/metabolismo , Sistemas de Liberação de Medicamentos , Lipossomos/química , Modelos Biológicos , Fosfolipídeos/química , Western Blotting , Cápsulas/química , Células HeLa , Humanos , Microscopia Confocal
3.
Hepatology ; 56(1): 259-69, 2012 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-22271390

RESUMO

UNLABELLED: Hepatitis is often associated with the overexpression of various adhesion molecules. In particular, intracellular adhesion molecule-1 (ICAM-1), which is expressed on hepatic endothelial cells (HECs) in the early stage of inflammation, is involved in serious illnesses. Therefore, ICAM-1 suppression in HECs enables the suppression of inflammatory responses. Here, we developed an ICAM-1 small interfering RNA (siRNA) transfer method using ultrasound (US)-responsive and mannose-modified liposome/ICAM-1 siRNA complexes (Man-PEG(2000) bubble lipoplexes [Man-PEG(2000) BLs]), and achieved efficient HEC-selective ICAM-1 siRNA delivery in combination with US exposure. Moreover, the sufficient ICAM-1 suppression effects were obtained via this ICAM-1 siRNA transfer in vitro and in vivo, and potent anti-inflammatory effects were observed in various types of inflammation, such as lipopolysaccharide, dimethylnitrosamine, carbon tetrachloride, and ischemia/reperfusion-induced inflammatory mouse models. CONCLUSION: HEC-selective and efficient ICAM-1 siRNA delivery using Man-PEG(2000) BLs and US exposure enables suppression of various types of acute hepatic inflammation. This novel siRNA delivery method may offer a valuable system for medical treatment where the targeted cells are HECs.


Assuntos
Terapia Genética/métodos , Hepatite/terapia , Molécula 1 de Adesão Intercelular/efeitos dos fármacos , Molécula 1 de Adesão Intercelular/genética , RNA Interferente Pequeno/farmacologia , Terapia por Ultrassom/métodos , Doença Aguda , Análise de Variância , Animais , Células Cultivadas , Modelos Animais de Doenças , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Hepatite/patologia , Hepatócitos/citologia , Hepatócitos/metabolismo , Técnicas In Vitro , Lipossomos/química , Lipossomos/farmacologia , Masculino , Manose/química , Manose/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , RNA Interferente Pequeno/genética , Distribuição Aleatória , Valores de Referência , Transfecção/métodos
4.
Biol Pharm Bull ; 36(5): 698-702, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23649328

RESUMO

There are many potential barriers to the effective delivery of small-molecule drugs to solid tumors. Most small-molecule chemotherapeutic drugs have a large volume of distribution upon intravenous administration, which is often associated with a narrow therapeutic index due to their high level of toxicity in healthy tissues. Nanoparticle-based therapeutics for tumor targeting have emerged as one of the promising approaches to overcome the lack of tissue specificity of conventional chemotherapeutic drugs. Various different concepts have been envisioned for nanoparticle-mediated drug targeting. Among them, the passive drug targeting strategy has been the most widely investigated, and numerous preclinical studies have provided insights into the validity of the strategy. This review article briefly introduces our recent findings related to the passive drug targeting strategy including its application in anti-angiogenic therapy, along with considerations to be taken into account and implications for the rational design of a passive drug targeting strategy.


Assuntos
Antineoplásicos/administração & dosagem , Sistemas de Liberação de Medicamentos , Nanopartículas/administração & dosagem , Neoplasias/tratamento farmacológico , Animais , Resistência a Múltiplos Medicamentos , Resistencia a Medicamentos Antineoplásicos , Humanos , Neoplasias/metabolismo , Permeabilidade/efeitos dos fármacos
5.
Mol Pharm ; 8(2): 543-54, 2011 Apr 04.
Artigo em Inglês | MEDLINE | ID: mdl-21250746

RESUMO

DNA vaccination has attracted much attention as a promising therapy for the prevention of metastasis and relapse of malignant tumors, especially highly metastatic tumors such as melanoma. However, it is difficult to achieve a potent cancer vaccine effect by DNA vaccination, since the number of dendritic cells, which are the major targeted cells of DNA vaccination, is very few. Here, we developed a DNA vaccination for metastatic and relapsed melanoma by ultrasound (US)-responsive and antigen presenting cell (APC)-selective gene carriers reported previously, named Man-PEG2000 bubble lipoplexes. Following immunization using US exposure and Man-PEG(2000) bubble lipoplexes constructed with pUb-M, which expresses ubiquitylated melanoma-specific antigens (gp100 and TRP-2), the secretion of Th1 cytokines (IFN-γ and TNF-α) and the activities of cytotoxic T lymphocytes (CTLs) were specifically enhanced in the presence of B16BL6 melanoma antigens. Moreover, we succeeded in obtaining potent and sustained DNA vaccine effects against solid and metastatic tumor derived from B16BL6 melanoma specifically. The findings obtained from this study suggest that the gene transfection method using Man-PEG2000 bubble lipoplexes and US exposure could be suitable for DNA vaccination aimed at the prevention of metastatic and relapsed cancer.


Assuntos
Vacinas Anticâncer/administração & dosagem , Neoplasias Pulmonares/prevenção & controle , Manose/química , Melanoma Experimental/prevenção & controle , Antígenos Específicos de Melanoma/genética , Ultrassom , Vacinas de DNA/administração & dosagem , Adenocarcinoma/imunologia , Adenocarcinoma/patologia , Adenocarcinoma/prevenção & controle , Animais , Células Apresentadoras de Antígenos/imunologia , Apoptose , Western Blotting , Neoplasias do Colo/imunologia , Neoplasias do Colo/patologia , Neoplasias do Colo/prevenção & controle , Citocinas/metabolismo , Células Dendríticas , Ensaio de Imunoadsorção Enzimática , Feminino , Citometria de Fluxo , Técnicas de Transferência de Genes , Terapia Genética , Humanos , Lipossomos , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/secundário , Linfoma/imunologia , Linfoma/patologia , Linfoma/prevenção & controle , Masculino , Melanoma Experimental/imunologia , Melanoma Experimental/patologia , Antígenos Específicos de Melanoma/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Polietilenoglicóis/química , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Linfócitos T Citotóxicos/imunologia , Ubiquitinação , Vacinação , Vacinas de DNA/genética , Vacinas de DNA/imunologia
6.
Int J Pharm ; 359(1-2): 234-40, 2008 Jul 09.
Artigo em Inglês | MEDLINE | ID: mdl-18448289

RESUMO

To elucidate the determinants of the in vivo anti-tumor efficacy of polyethylene glycol (PEG)-modified liposomal doxorubicin (DOX), we examined its anti-tumor effect against three different tumor cell lines (Lewis lung cancer (LLC), Colon-26 (C26) and B16BL6 melanoma (B16)) in vitro and in vivo. In vitro, LLC was the most sensitive tumor to DOX and liposomal DOX based on the MTT assay. However, the strongest in vivo anti-tumor effect was observed in the C26 tumor-bearing mice. The in vivo accumulation of radiolabelled PEG liposome in the C26 tumor after intravenous injection was significantly larger than in other tumors. The extent of vascularity assessed by immunohistochemical staining of CD31 was not directly related with the tumor accumulation of PEG liposome. On the other hand, Evans blue extravasation and secretion of VEGF in C26 tumors were higher than in LLC tumors, clearly demonstrating that the vasculature permeability was higher within C26 tumors. These results indicated that the vascular permeability within the tumor substantially affects the tumor accumulation of PEG liposome and may be one of the important determinants in the in vivo anti-tumor efficacy of PEG liposomal DOX.


Assuntos
Antibióticos Antineoplásicos/administração & dosagem , Permeabilidade Capilar/efeitos dos fármacos , Doxorrubicina/administração & dosagem , Neoplasias Experimentais/tratamento farmacológico , Animais , Linhagem Celular Tumoral , Portadores de Fármacos/química , Ensaios de Seleção de Medicamentos Antitumorais , Injeções Intravenosas , Lipossomos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Polietilenoglicóis/química
7.
Carbohydr Res ; 405: 78-86, 2015 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-25500195

RESUMO

Novel sugar-conjugated cholesterols, ß-Gal-, α-Man-, ß-Man-, α-Fuc-, and ß-Man-6P-S-ß-Ala-Chol, were synthesized and incorporated into liposomes. In vitro experiments using the glyco-coated liposomes showed that the glyco-coated liposomes are efficiently taken up by cells expressing carbohydrate-binding receptors selectively. Glyco-coated liposomes are promising candidates for drug delivery vehicles.


Assuntos
Carboidratos/química , Colesterol/química , Lipossomos/química , Lipossomos/síntese química , Animais , Transporte Biológico , Técnicas de Química Sintética , Células Hep G2 , Humanos , Lipossomos/metabolismo , Melanoma Experimental , Camundongos
8.
J Drug Target ; 22(8): 732-8, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-24830302

RESUMO

Development of an efficient small interfering RNA (siRNA) delivery method using non-viral carriers is necessary to determine potent therapeutic effects of RNA interference. Inflammatory responses induced by siRNA interaction with Toll-like receptors and retinoic-acid-inducible gene I protein/melanoma differentiation-associated gene 5 (RIG-I/MDA-5) are obstacles to the application of siRNAs in clinically. Here, we evaluated the effects on inflammatory responses by our siRNA delivery method using bubble lipoplexes with ultrasound (US) exposure in cultured macrophages. The effective gene suppression effects were obtained under low-toxic conditions in this siRNA transfer method. The interferon (IFN)-α after siRNA transfer using lipoplexes/bubble lipoplexes with US exposure was not detected. However, low levels of type I IFN mRNA production were induced through interaction of siRNA and cytoplasmic RIG-I/MDA-5, but not Toll-like receptors. Our findings indicate that it is possible to develop a safe and efficient siRNA delivery technique using mannosylated bubble lipoplexes and US exposure.


Assuntos
Inflamação/metabolismo , Macrófagos Peritoneais/efeitos dos fármacos , Manose/química , Animais , Células Cultivadas , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , Feminino , Regulação da Expressão Gênica , Helicase IFIH1 Induzida por Interferon , Interferon-alfa/genética , Interferon-alfa/metabolismo , Lipossomos/química , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos ICR , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno , Receptor de Interferon alfa e beta/genética , Receptor de Interferon alfa e beta/metabolismo , Receptores de Superfície Celular , Transdução de Sinais , Receptor 3 Toll-Like/genética , Receptor 3 Toll-Like/metabolismo , Transfecção/métodos , Ultrassom
9.
Int J Pharm ; 475(1-2): 401-7, 2014 Nov 20.
Artigo em Inglês | MEDLINE | ID: mdl-25218184

RESUMO

A method involving the use of doxorubicin-loaded polyethylene-glycol-modified liposomes and transfection using mannose-modified bubble lipoplexes in combination with ultrasound irradiation may be a promising approach to cancer treatment; it could not only suppress early-stage tumor growth but also enhance transfection efficacy in antigen-presenting cells, thus enhancing the therapeutic potential of a DNA vaccine. However, to date only limited research has been carried out regarding this combination DNA vaccination method for use in cancer therapy. In this study, we examined the anti-tumor effect of DNA vaccination using an ultrasound-responsive mannose-modified gene carrier combined with doxorubicin-encapsulated polyethylene-glycol-modified liposomes. Doxorubicin-encapsulated PEGylated liposomes activated transcriptional factors, such as nuclear factor-κB and AP-1 in the spleen; subsequently pUb-M, ubiquitylated melanoma-related antigen encoding plasmid DNA expression in splenic cells was significantly enhanced. Moreover, effective cytotoxic T-lymphocyte activities were stimulated by DNA vaccination combined with the administration of doxorubicin-encapsulated polyethylene-glycol-modified liposomes. Furthermore, potent DNA vaccine effects against established solid tumor and metastatic tumor derived from B16BL6 melanoma were observed. These results suggest that the combined use of DNA vaccination with doxorubicin-encapsulated polyethylene-glycol-modified liposomes could be an effective method for the treatment of melanoma using immunotherapy.


Assuntos
Antineoplásicos/farmacologia , Doxorrubicina/análogos & derivados , Melanoma/tratamento farmacológico , Linfócitos T Citotóxicos/imunologia , Terapia por Ultrassom/métodos , Vacinas de DNA/farmacologia , Animais , Células Apresentadoras de Antígenos/imunologia , Linhagem Celular Tumoral , Doxorrubicina/farmacologia , Manose/química , Melanoma/imunologia , Camundongos , NF-kappa B/biossíntese , Plasmídeos , Polietilenoglicóis/farmacologia , RNA Mensageiro , Fator de Transcrição AP-1/biossíntese , Ativação Transcricional/imunologia , Transfecção
10.
J Pharm Sci ; 103(8): 2464-9, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24985750

RESUMO

Angiogenesis, the sprouting of capillaries from preexisting ones, is essential for the sustained growth of solid tumors. In this study, we used SU5416, a hydrophobic molecule with potent tyrosine kinase inhibitor of type 2 receptor for vascular endothelial growth factor (VEGF), as PEGylated emulsion (SU5416-PE), and evaluated the antitumor potency of this formulation in Lewis lung cancer (LLC), Colon-26 (C26), and B16BL6 melanoma (B16) tumor-bearing mice. Intravenous injection of SU5416-PE into tumor-bearing mice significantly suppressed the growth of C26 and B16 tumors, but had no effect on the growth of LLC tumors. MTT assay revealed that SU5416 inhibited the proliferation of human umbilical vein endothelial cells in a concentration-dependent manner but did not show such an inhibitory effect on all types of tumor cells examined, demonstrating the specificity of SU5416 for endothelial cells. Considering that VEGF levels within C26 and B16 tumors were found to be about 10-fold and 20-fold higher than that in LLC tumors, respectively, it was suggested that SU5416-PE would inhibit angiogenesis in certain types of tumor tissue such as C26 and B16 where VEGF plays a major role for promoting angiogenesis, leading to the suppression of in vivo tumor growth.


Assuntos
Inibidores da Angiogênese/uso terapêutico , Antineoplásicos/uso terapêutico , Emulsões/química , Indóis/uso terapêutico , Neoplasias/tratamento farmacológico , Veículos Farmacêuticos/química , Polietilenoglicóis/química , Pirróis/uso terapêutico , Inibidores da Angiogênese/administração & dosagem , Inibidores da Angiogênese/química , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/química , Linhagem Celular , Linhagem Celular Tumoral , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/patologia , Humanos , Indóis/administração & dosagem , Indóis/química , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Neoplasias/patologia , Neovascularização Patológica/tratamento farmacológico , Neovascularização Patológica/patologia , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/uso terapêutico , Pirróis/administração & dosagem , Pirróis/química , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores
11.
Biomaterials ; 35(5): 1347-58, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24290812

RESUMO

Block copolymer micelles have shown promise for the intracellular delivery of chemotherapeutic agents, proteins, and nucleic acids. Understanding the mechanism of their intracellular trafficking and fate, including the extracellular efflux of the polymers, will help improve their efficacy and minimize their safety risks. In this Leading Opinion paper, we discuss the molecular mechanism of block copolymer micelle trafficking, from intracellular uptake to extracellular efflux, on the basis of studies with HeLa cells. By using FRET (fluorescence resonance energy transfer) with confocal microscopy, we found that, following their intracellular transport via endocytosis, the micelles dissociated into their polymeric components in late endosomes and/or lysosomes. Furthermore, we confirmed that the intrinsic proteins NPC1 and ORP2 are involved in the intermembrane transfer of polymers from the endosome to the plasma membrane via the ER (endoplasmic reticulum) by using knockdown experiments with siRNAs. After the polymers were transported to the plasma membrane with the aid of ORP2, they were extruded into the cell medium via ABC transporter, ABCB1. Experiments with ABCB1-expressing vesicles indicated that the polymer itself, and not the fluorescent compounds, was recognized by the transporter. These findings, and the analysis of related mechanisms, provide valuable information that should help minimize the potential risks associated with the intracellular accumulation of block copolymer micelles and to improve their therapeutic efficacy.


Assuntos
Micelas , Polímeros/metabolismo , Subfamília B de Transportador de Cassetes de Ligação de ATP , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Sequência de Bases , Transporte Biológico , Endocitose , Endossomos/metabolismo , Transferência Ressonante de Energia de Fluorescência , Células HeLa , Humanos , Lisossomos/metabolismo , Microscopia Confocal , RNA Interferente Pequeno
12.
Yakugaku Zasshi ; 132(11): 1273-9, 2012.
Artigo em Japonês | MEDLINE | ID: mdl-23123719

RESUMO

In the post-genome era, the analysis of disease-related genes has rapidly advanced, and the medical application of the information obtained from gene analysis is being put into practice. In particular, the development of a novel system to transfect the gene of interest selectively and efficiently into targeted cells is essential for the gene therapy of refractory diseases, in vivo functional analysis of genes, and establishment of animal models for diseases. However, a suitable carrier for selective gene delivery to targeted cells remains to be developed. The sonoporation method using microbubbles and ultrasound (US) exposure is one of the most promising approaches for effective gene transfection. However, it is difficult to transfect the therapeutic gene into the targeted organs/cells selectively by the conventional sonoporation method. Recently, our group has developed mannose-modified and US-responsive carriers/nucleic acid complexes (Man-PEG(2000) bubble lipoplexes), and succeeded in obtaining the enhanced and selective gene expression in mannose receptor-expressing cells by combination with US exposure. In this review, I described our gene transfection methods using Man-PEG(2000) bubble lipoplexes and external US exposure. Additionally, I also reviewed the enhancing mechanism of gene expression focusing on the intracellular transporting processes, in vivo distribution, and the activation of transcriptional factors. I believe that these findings help in the development of an effective gene transfection system using US-exposing system.


Assuntos
Técnicas de Transferência de Genes , Terapia Genética/métodos , Lipossomos , Transfecção/métodos , Ultrassom , Animais , Expressão Gênica , Humanos , Lectinas Tipo C , Lipossomos/química , Manose , Receptor de Manose , Lectinas de Ligação a Manose , Microbolhas , Ácidos Nucleicos , Polietilenoglicóis , Receptores de Superfície Celular , Fatores de Transcrição/metabolismo
13.
Biomaterials ; 33(32): 8131-41, 2012 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-22858002

RESUMO

Liposomes are widely used as drug delivery vehicles to transfer chemotherapeutic agents, proteins, and nucleic acids into target cells. To improve therapeutic effects and reduce unexpected toxic side-effects, it is necessary to understand the mechanism of liposomal uptake into cells, and the intracellular fate of internalized liposomes. The intracellular fate of synthesized components used in the construction of liposomes remains unclear. In the work presented here, we investigated the trafficking processes from intracellular uptake to extracellular efflux using conventional liposomes constructed with phospholipids (DOPC) and cholesterols (Chol). Following intracellular transport of liposomes via endocytosis, DOPC was localized in the endoplasmic reticulum (ER) and Golgi apparatus after escape from the endosome/lysosome, whereas Chol was only localized in the ER. Moreover, proteins involved in the intracellular trafficking of liposomal components were identified. Additionally, we showed that DOPC was partly effluxed via ABCG1, while Chol was partly effluxed via ABCA1 or ABCB1; suggesting that each liposomal component examined in this study was effluxed through different transporters. Our findings offer valuable information regarding targeted delivery to specific intracellular organelles, and how to possibly avoid unexpected toxic effects following multiple applications of liposome formulations.


Assuntos
Colesterol/farmacocinética , Lipossomos/farmacocinética , Fosfatidilcolinas/farmacocinética , Linhagem Celular , Colesterol/química , Colesterol/metabolismo , Retículo Endoplasmático/metabolismo , Endossomos/metabolismo , Complexo de Golgi/metabolismo , Humanos , Lipossomos/química , Lipossomos/metabolismo , Lisossomos/metabolismo , Fosfatidilcolinas/química , Fosfatidilcolinas/metabolismo
14.
Biomaterials ; 32(20): 4659-69, 2011 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-21481454

RESUMO

The development of gene transfection methods enhancing the level of gene expression under simple and low-toxic condition is required for gene therapy in clinical. Our group has developed the ultrasound (US)-mediated gene transfection method using Man-PEG(2000) bubble lipoplexes, which are US-responsive and mannose-modified gene carriers, and succeeded in obtaining the enhanced gene expression in mannose receptor-expressing cells selectively by the gene transfer using Man-PEG(2000) bubble lipoplexes with US exposure in vitro and in vivo. Here, we investigated pDNA transferring mechanism followed by US exposure to unmodified and Man-PEG(2000) bubble lipoplexes, in particular, focused on US exposure timing. Following investigation of intracellular transferring characteristics, a large amount of pDNA was transferred into the cytoplasm followed by US-mediated destruction of bubble lipoplexes in the gene transfer using both bubble lipoplexes with US exposure. Moreover, the effective gene expression was obtained without TNF-α production when US was exposed until 5 min after the addition of bubble lipoplexes. These findings suggest that the gene transfer using unmodified and Man-PEG(2000) bubble lipoplexes with US exposure enables to transfer pDNA into the cytoplasm, and optimized US exposure timing is important to achieve the high level of gene expression and the low level of pro-inflammatory cytokine production.


Assuntos
Terapia Genética/métodos , Lipossomos/química , Manose/química , Transfecção/métodos , Ultrassom , Animais , Células Cultivadas , Feminino , Humanos , Macrófagos/citologia , Macrófagos/metabolismo , Teste de Materiais , Camundongos , Camundongos Endogâmicos C57BL , Fator de Necrose Tumoral alfa/metabolismo
15.
J Control Release ; 156(3): 355-63, 2011 Dec 20.
Artigo em Inglês | MEDLINE | ID: mdl-21756951

RESUMO

Recently, our group developed ultrasound (US)-responsive and mannose-modified gene carriers (Man-PEG(2000) bubble lipoplexes), and successfully obtained a high level of gene expression in mannose receptor-expressing cells following gene transfection using Man-PEG(2000) bubble lipoplexes and US exposure. We also reported that large amounts of plasmid DNA (pDNA) were transferred into the cytoplasm of the targeted cells in the gene transfection using this method. In the present study, we investigated the involvement of transcriptional processes on enhanced gene expression obtained by unmodified and Man-PEG(2000) bubble lipoplexes with US exposure. The transcriptional process related to activator protein-1 (AP-1) and nuclear factor-κB (NFκB) was activated by US exposure, and was founded to be involved in enhanced gene expression obtained by gene transfection using unmodified and Man-PEG(2000) bubble lipoplexes with US exposure. On the other hand, activation of AP-1 and NFκB pathways followed by US exposure was hardly involved in the inflammatory responses in the gene transfection using this method. These findings suggest that activation of AP-1 and NFκB followed by US exposure is involved in the enhanced gene expression using unmodified and Man-PEG(2000) bubble lipoplexes with US exposure, and the selection of pDNAs activated by US exposure is important in this gene transfection method.


Assuntos
DNA/administração & dosagem , Lipídeos/química , Manose/química , NF-kappa B/genética , Fator de Transcrição AP-1/genética , Transfecção , Ultrassom/métodos , Animais , Linhagem Celular , Células Cultivadas , Citocinas/imunologia , Feminino , Lipossomos/química , Camundongos , Camundongos Endogâmicos C57BL , NF-kappa B/imunologia , Plasmídeos/administração & dosagem , Polietilenoglicóis/química , Fator de Transcrição AP-1/imunologia
16.
Hum Gene Ther ; 21(1): 65-74, 2010 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-19719400

RESUMO

To achieve effective gene therapy, it is necessary to selectively and efficiently transfect therapeutic gene into targeted cells. In this study, we developed a combination method using mannosylated lipoplexes, which show selectivity to antigen-presenting cells such as macrophages and dendritic cells, and bubble liposomes (BLs), which are known to enhance transfection efficiency on exposure to ultrasound (US). In cultured mouse macrophages, known for the expression of mannose receptors, the transfection efficiency of this combination method using mannosylated lipoplexes and BLs with US was higher than that of naked pDNA or unmodified lipoplexes and BLs. In the liver and spleen, the in vivo transfection efficiency of this combination method was significantly higher than that of naked pDNA or unmodified lipoplexes and BLs with US. Transfection in hepatic nonparenchymal cells using this combination method was about 12 times higher than that in hepatic parenchymal cells. As far as splenic transfection activities were concerned, the transfection efficiency of this combination method in CD11c(+) cells was significantly higher than that in CD11c(-) cells. In conclusion, we demonstrated that the gene transfection efficiency in macrophages and dendritic cells was significantly increased by this combination method using mannosylated lipoplexes and BLs with US exposure.


Assuntos
Células Dendríticas/metabolismo , Lipossomos/metabolismo , Macrófagos Peritoneais/metabolismo , Manose/metabolismo , Transfecção/métodos , Ultrassom , Animais , Células Cultivadas , Regulação da Expressão Gênica , Injeções Intravenosas , Espaço Intracelular/metabolismo , Fígado/diagnóstico por imagem , Fígado/metabolismo , Luciferases/genética , Luciferases/metabolismo , Camundongos , Plasmídeos/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Baço/diagnóstico por imagem , Fatores de Tempo , Ultrassonografia
17.
Biomaterials ; 31(30): 7813-26, 2010 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-20656348

RESUMO

Development of a gene delivery system to transfer the gene of interest selectively and efficiently into targeted cells is essential for achievement of sufficient therapeutic effects by gene therapy. Here, we succeeded in developing the gene transfection method using ultrasound (US)-responsive and mannose-modified gene carriers, named Man-PEG(2000) bubble lipoplexes. Compared with the conventional lipofection method using mannose-modified carriers, this transfection method using Man-PEG(2000) bubble lipoplexes and US exposure enabled approximately 500-800-fold higher gene expressions in the antigen presenting cells (APCs) selectively in vivo. This enhanced gene expression was contributed by the improvement of delivering efficiency of nucleic acids to the targeted organs, and by the increase of introducing efficiency of nucleic acids into the cytoplasm followed by US exposure. Moreover, high anti-tumor effects were demonstrated by applying this method to DNA vaccine therapy using ovalbumin (OVA)-expressing plasmid DNA (pDNA). This US-responsive and cell-specific gene delivery system can be widely applied to medical treatments such as vaccine therapy and anti-inflammation therapy, which its targeted cells are APCs, and our findings may help in establishing innovative methods for in-vivo gene delivery to overcome the poor introducing efficiency of carriers into cytoplasm which the major obstacle associated with gene delivery by non-viral carriers.


Assuntos
Técnicas de Transferência de Genes , Terapia Genética/métodos , Manose/química , Transfecção/métodos , Ultrassom , Vacinas de DNA/administração & dosagem , Animais , Apresentação de Antígeno , Linhagem Celular , Citocinas/metabolismo , Feminino , Expressão Gênica , Genes MHC Classe I , Humanos , Lipossomos/química , Lipossomos/metabolismo , Fígado/metabolismo , Teste de Materiais , Camundongos , Camundongos Endogâmicos C57BL , Estrutura Molecular , Polietilenoglicóis/química , Vacinas de DNA/genética , Vacinas de DNA/imunologia
18.
J Control Release ; 133(1): 4-10, 2009 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-18840484

RESUMO

We evaluated the in vivo anti-tumor effect of polyethylene glycol-modified liposomal doxorubicin (PEG liposomal DOX) in the DOX-resistant Colon-26 cancer cells (C26/DOX)-bearing mice model. IC(50) value of DOX to C26/DOX in vitro (40.0 microM) was about 250 times higher than that to control C26 (C26/control) (0.15 microM). However, in vivo anti-tumor effect of PEG liposomal DOX was similar in both C26/control- and C26/DOX-bearing mice, suggesting that the in vivo anti-tumor effect of PEG liposomal DOX was not directly reflecting the sensitivity of these tumor cells to DOX. IC(50) value (0.10 microM) of DOX to HUVEC, a model vascular endothelial cell, was similar to that of C26/control. Double immunohistochemical staining of vascular endothelial cells and apoptotic cells within the tumor tissue after intravenous administration of PEG liposomal DOX showed that the extent of co-localization of apoptotic cells with endothelial cells was significantly higher for C26/DOX tumors (60%) than C26/control ones (20%), suggesting that the apoptosis is caused preferentially for vascular endothelial cells in C26/DOX tumor. From these results, it was suggested that the cytotoxic effect of DOX on vascular endothelial cells in the tumor would be involved in the in vivo anti-tumor effect of PEG liposomal DOX in C26/DOX-bearing mice.


Assuntos
Doxorrubicina/uso terapêutico , Células Endoteliais/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Polietilenoglicóis/química , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Animais , Antibióticos Antineoplásicos/administração & dosagem , Antibióticos Antineoplásicos/química , Antibióticos Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Linhagem Celular , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Doxorrubicina/administração & dosagem , Doxorrubicina/química , Interações Medicamentosas , Resistencia a Medicamentos Antineoplásicos , Células Endoteliais/citologia , Humanos , Concentração Inibidora 50 , Lipossomos/química , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Neoplasias/metabolismo , Neoplasias/patologia , Análise de Sobrevida , Verapamil/farmacologia
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