RESUMO
In the present study, innovative doxorubicin-loaded nanoparticles (NPs) made of a photosensitive poly(o-nitrobenzyl acrylate) (PNBA) hydrophobic matrix and an hydrophilic dextran (Dex) shell were first formulated by the emulsion-solvent evaporation process. Doxorubicin (DOX), a very well-known anticancer drug, was herein chosen as the model. DOX-loaded NPs were successfully produced by covering the hydrophobic PNBA core with Dex chains either physically adsorbed or covalently linked by changing process parameters as the presence of a catalyst (CuBr or CuSO4/ascorbic acid). It was then proved that the neutralization of DOX optimized drug loading. DOX loading and release were independent of the coverage mechanism if the catalyst used to covalently link the shell to the core was correctly chosen. Second, the kinetics of DOX release were investigated by simple diffusion or light irradiation of the NPs. Experiments showed that less than 20% of DOX was released by simple diffusion after 48 h in PBS or DMEM media when 45% of DOX released after only 30 s of light irradiation of the NPs. Finally, the impact of the phototriggered DOX release on cell viability was investigated on various cell lines [Caco-2, HepG2, HCT-116, and HT-29 cells as well as murine macrophages (RAW 264.7)]. Cellular mortality was evaluated to be dependent on the cell lines tested. Our approach provided an improved DOX release toward the human liver cancer cell line, and a high internalization of the PNBA-based NPs into HepG2 cells was observed using fluorescence microscopy.
Assuntos
Antineoplásicos/farmacologia , Materiais Biocompatíveis/farmacologia , Dextranos/farmacologia , Doxorrubicina/farmacologia , Nitrobenzenos/farmacologia , Fármacos Fotossensibilizantes/farmacologia , Polímeros/farmacologia , Animais , Antineoplásicos/química , Materiais Biocompatíveis/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Dextranos/química , Doxorrubicina/química , Portadores de Fármacos/química , Portadores de Fármacos/farmacologia , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Teste de Materiais , Camundongos , Nanopartículas/química , Nitrobenzenos/química , Tamanho da Partícula , Fármacos Fotossensibilizantes/química , Polímeros/química , Células RAW 264.7RESUMO
The mechanism of how plant-derived nanovesicles are uptaken by cells remains unknown. In this study, the garlic-derived nanovesicles (GDVs) were isolated and digested with trypsin to remove all surface proteins. Digested GDVs showed less uptake compared to undigested GDVs, confirming that the surface proteins played a role in the endocytosis. On the cell side (HepG2), interestingly, blocking the CD98 receptors significantly reduced the uptake of GDVs. During the cellular internalization of GDVs, we observed that some surface proteins of GDVs were co-localized with CD98. A total lysate of the GDV surface showed a high presence of a mannose-specific binding protein, II lectin. Blocking GDV II lectin (using mannose preincubation) highly reduced the GDV internalization, which supports that direct interaction between II lectin and CD98 plays an important role in internalization. The GDVs also exhibited in vitro anti-inflammatory effect by downregulating proinflammatory factors on the HepG2 cells. This work contributes to understanding a part of the GDV internalization process and the cellular anti-inflammatory effects of garlic.
RESUMO
Colitis-associated cancer (CAC) is a subtype of colon cancer that is driven by chronic inflammation and is prevalent in chronic ulcerative colitis patients. The development of CAC is associated with the inflammation-dysplasia-carcinoma pathway which is significantly different than adenoma-carcinoma pathway of sporadic colon cancer (CRC). Matrix Metalloproteinase 9 (MMP9) is a zinc-dependent endopeptidase against extracellular matrix (ECM) proteins expressed in the gastrointestinal tract during inflammation. We have previously shown that MMP9 plays a tumor suppressor role in CAC via "MMP9-Notch1-ARF-p53 axis" pathway. The aim of this study is to determine the role of MMP9 in maintaining genomic stability in CAC. Homozygous transgenic mice with constitutive-expression of MMP9 in the colonic epithelium (TgM9) with their wild-type littermates (WT) and stably transfected HCT116 cells with/without MMP9 were used for in vivo and in vitro experiments, respectively. As 'proof of concept' model, nanoparticles (NPs) loaded with MMP9 siRNA were used to examine the effect of MMP9 silencing in the colonic epithelium. In CAC, colonic epithelium of TgM9 mice exhibited lower amounts of reactive oxygen species (ROS), less DNA damage, and increased expression of mismatch repair genes compared to WTs. Our study showed that MMP9 expression correlates with the reduced ROS levels, decreased DNA damage, and upregulated mismatch repair pathway. This suggests that MMP9 expression is a natural biological way to suppress CAC by limiting ROS accumulation and DNA damage in the colon. Therefore, MMP9 inhibition could be deleterious for CAC patient.
Assuntos
Colite/metabolismo , Neoplasias do Colo/metabolismo , Dano ao DNA , Metaloproteinase 9 da Matriz/fisiologia , Espécies Reativas de Oxigênio , Animais , Diferenciação Celular , Linhagem Celular Tumoral , Proliferação de Células , Feminino , Inativação Gênica , Células HCT116 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Receptores Notch/metabolismoRESUMO
CD98 is a multifunctional glycoprotein that is involved in various biological processes such as amino acid transport, cell adhesion, diffusion, adhesion, and proliferation. The role of CD98 in liver disease has not thoroughly been examined and is limited reports in the literature. Among these reports, direct association for CD98 in nonalcoholic fatty liver disease (NAFLD) and hepatocellular carcinoma (HCC) have been reported. Our lab has reported that targeting CD98 in high fat diet mice reduced steatosis and inflammation in NAFLD. Other reports associate CD98 in HCC due in part to the role of CD98 in activating integrin signaling. Herein, we present CD98 staining on liver biopsies from NAFLD, chronic active hepatitis, cirrhosis, and 3 stages of HCC to demonstrate the upregulation of CD98 expression throughout liver disease progression. In addition, we analyze current literature to elucidate roles and potential roles of CD98 with each stage of liver disease.
Assuntos
Proteína-1 Reguladora de Fusão/metabolismo , Hepatopatias/etiologia , Hepatopatias/metabolismo , Animais , Humanos , Hepatopatias/patologia , CamundongosRESUMO
Non-alcoholic fatty liver disease (NAFLD) is characterized by excessive lipid hepatic accumulation. Here, we investigated whether a reduction of CD98 expression mediated by CD98 siRNA-loaded nanoparticles (NPs) could attenuate liver disease markers in a mouse model of NAFLD. NPs were generated using a double emulsion/solvent evaporation technique. Mice fed a high fat diet for 8 weeks to induce fatty liver were treated with vein tail injections of CD98 siRNA-loaded NPs. In vitro, HepG2 treated with CD98 siRNA-loaded NPs showed significant downregulation of CD98 leading to a significant decrease of major pro-inflammatory cytokines and markers. In vivo, CD98 siRNA-loaded NPs strongly decreased all markers of NAFLD, including the blood levels of ALT and lipids accumulation, fibrosis evidence and pro-inflammatory cytokines. In conclusion, our results indicate that CD98 appears to function as a key actor/inducer in NAFLD, and that our NPs approach may offer a new targeted therapeutic for this disease.
Assuntos
Proteína-1 Reguladora de Fusão/genética , Inativação Gênica , Hepatopatia Gordurosa não Alcoólica/terapia , RNA Interferente Pequeno/genética , Animais , Biomarcadores/sangue , Dieta Hiperlipídica , Modelos Animais de Doenças , Regulação para Baixo , Feminino , Humanos , Camundongos , Nanopartículas/químicaRESUMO
Colitis associated cancer (CAC) is chronic inflammation driven colon cancer, prevalent among individuals with Inflammatory Bowel Disease. Matrix-metalloproteinase (MMP9) is one of the essential regulators of extra cellular matrix components. We have shown that MMP9 is protective in CAC contrary to its inflammatory role in acute-colitis. Aim of our study is to identify the mechanism of the protective role of epithelial derived-MMP9 in CAC. We used homozygous transgenic mice constitutively-expressing MMP9 in colonic-epithelium (TgM9) and wild-type (WT) littermates for in vivo experiments. Stably-transfected HCT116 with/without MMP9, and mouse embryonic-fibroblasts (WT and MMP9-/-, MEFs) were used for in vitro experiments. TgM9 mice exhibited less tumor burden, increased apoptosis, and increased expressions of active-Notch1, p53, p21WAF1/Cip1, caspase-3 and cyclin E in CAC compared to WTs. These results were supported by MEFs data. HCT116-cells overexpressing MMP9 indicated decreased cell proliferation, S-phase cell-cycle arrest and less DNA damage compared to vector. MMP9-/- mice showed attenuation of MMP9 was directly associated with p19ARF. Our study identifies the tumor suppressor role of epithelial derived-MMP9 in CAC via novel mechanistic pathway "MMP9-Notch1-ARF-p53 axis" regulating apoptosis, cell-cycle arrest and DNA damage implying, that MMP9 expression might be a natural/biological way to suppress colonic ulceration due to chronic inflammation.
Assuntos
Colite/patologia , Neoplasias do Colo/patologia , Mucosa Intestinal/enzimologia , Metaloproteinase 9 da Matriz/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Animais , Apoptose , Caspase 3/metabolismo , Proliferação de Células , Colite/induzido quimicamente , Colite/complicações , Colo/imunologia , Colo/metabolismo , Colo/patologia , Neoplasias do Colo/etiologia , Ciclina E/metabolismo , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Dano ao DNA , Sulfato de Dextrana/toxicidade , Fibroblastos , Células HCT116 , Humanos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Metaloproteinase 9 da Matriz/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neoplasias Experimentais/etiologia , Neoplasias Experimentais/patologia , Receptor Notch1/metabolismo , Pontos de Checagem da Fase S do Ciclo Celular , Transdução de Sinais , Transfecção , Carga Tumoral , Proteína Supressora de Tumor p53/metabolismo , Proteínas Supressoras de Tumor/genéticaRESUMO
Glial cell line-derived neurotrophic factor (GDNF) protects against high-fat diet (HFD)-induced hepatic steatosis in mice, however, the mechanisms involved are not known. In this study we investigated the effects of GDNF overexpression and nanoparticle delivery of GDNF in mice on hepatic steatosis and fibrosis and the expression of genes involved in the regulation of hepatic lipid uptake and de novo lipogenesis. Transgenic overexpression of GDNF in liver and other metabolically active tissues was protective against HFD-induced hepatic steatosis. Mice overexpressing GDNF had significantly reduced P62/sequestosome 1 protein levels suggestive of accelerated autophagic clearance. They also had significantly reduced peroxisome proliferator-activated receptor-γ (PPAR-γ) and CD36 gene expression and protein levels, and lower expression of mRNA coding for enzymes involved in de novo lipogenesis. GDNF-loaded nanoparticles were protective against short-term HFD-induced hepatic steatosis and attenuated liver fibrosis in mice with long-standing HFD-induced hepatic steatosis. They also suppressed the liver expression of steatosis-associated genes. In vitro, GDNF suppressed triglyceride accumulation in Hep G2 cells through enhanced p38 mitogen-activated protein kinase-dependent signaling and inhibition of PPAR-γ gene promoter activity. These results show that GDNF acts directly in the liver to protect against HFD-induced cellular stress and that GDNF may have a role in the treatment of nonalcoholic fatty liver disease.
Assuntos
Dieta Hiperlipídica , Fígado Gorduroso/metabolismo , Fígado Gorduroso/prevenção & controle , Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Fígado/metabolismo , PPAR gama/metabolismo , Animais , Antígenos CD36/genética , Antígenos CD36/metabolismo , Fígado Gorduroso/etiologia , Fígado Gorduroso/patologia , Fator Neurotrófico Derivado de Linhagem de Célula Glial/administração & dosagem , Fator Neurotrófico Derivado de Linhagem de Célula Glial/genética , Fator Neurotrófico Derivado de Linhagem de Célula Glial/uso terapêutico , Células Hep G2 , Humanos , Fígado/patologia , Camundongos , Camundongos Transgênicos , Nanopartículas/administração & dosagem , Nanopartículas/uso terapêutico , PPAR gama/genética , Transdução de Sinais/fisiologia , Triglicerídeos/metabolismoRESUMO
Inflammatory bowel diseases (IBDs) are chronic and progressive inflammatory disorders of the gastrointestinal tract. In IBD, protein serological biomarkers could be relevant tools for assessing disease activity, performing early-stage diagnosis and managing the treatment. Using the interleukin-10 knockout (IL-10(-/-)) mouse, a model that develops a time-dependent IBD-like disorder that predominates in the colon; we performed longitudinal studies of circulating protein biomarkers in IBD. Circulating protein profiles in serum samples collected from 30-, 93-, to 135-day-old IL-10(-/-) mice were investigated using two-dimensional differential gel electrophoresis and MALDI-TOF/TOF tandem mass spectrometry. A total of 15 different proteins were identified and confirmed by ELISA and Western blot to be differentially accumulated in serum samples from mid- to late-stage IL-10(-/-) mice compared to early non-inflamed IL-10(-/-) mice. The use of another model of colitis and an extra-intestinal inflammation model validated this biomarker panel and demonstrated that comprised some global inflammatory markers, some intestinal inflammation-specific markers and some chronic intestinal inflammation markers. Statistical analyses using misclassification error rate charts validated the use of these identified proteins as powerful biomarkers of colitis. Unlike standard biomarker screening studies, our analyses identified a panel of proteins that allowed the definition of protein signatures that reflect colitis status. BIOLOGICAL SIGNIFICANCE: Crohn's disease (CD) and ulcerative colitis (UC) are the most common inflammatory bowel diseases (IBDs) occurring in humans. The major current diagnosis tool is colonoscopy, which is invasive and could lead to false diagnosis. The emergence of serological biomarkers enables the use of new diagnosis tools such as protein signatures for IBD diagnosis/management. Using 2D-DIGE coupled to mass spectrometry, our longitudinal study in a mouse model of colitis identified a signature of protein biomarkers for specific stages of disease.
Assuntos
Proteínas Sanguíneas/metabolismo , Colite Ulcerativa/sangue , Doença de Crohn/sangue , Animais , Biomarcadores/sangue , Modelos Animais de Doenças , Feminino , Camundongos , Camundongos KnockoutRESUMO
Clustered, regularly interspaced, short palindromic repeats-CRISPR associated (CRISPR-Cas) systems defend bacteria against foreign nucleic acids, such as during bacteriophage infection and transformation, processes which cause envelope stress. It is unclear if these machineries enhance membrane integrity to combat this stress. Here, we show that the Cas9-dependent CRISPR-Cas system of the intracellular bacterial pathogen Francisella novicida is involved in enhancing envelope integrity through the regulation of a bacterial lipoprotein. This action ultimately provides increased resistance to numerous membrane stressors, including antibiotics. We further find that this previously unappreciated function of Cas9 is critical during infection, as it promotes evasion of the host innate immune absent in melanoma 2/apoptosis associated speck-like protein containing a CARD (AIM2/ASC) inflammasome. Interestingly, the attenuation of the cas9 mutant is complemented only in mice lacking both the AIM2/ASC inflammasome and the bacterial lipoprotein sensor Toll-like receptor 2, but not in single knockout mice, demonstrating that Cas9 is essential for evasion of both pathways. These data represent a paradigm shift in our understanding of the function of CRISPR-Cas systems as regulators of bacterial physiology and provide a framework with which to investigate the roles of these systems in myriad bacteria, including pathogens and commensals.
Assuntos
Proteínas de Bactérias/imunologia , Farmacorresistência Bacteriana/imunologia , Francisella/imunologia , Infecções por Bactérias Gram-Negativas/imunologia , Evasão da Resposta Imune/imunologia , Inflamassomos/imunologia , Lipoproteínas/imunologia , Animais , Membrana Celular/genética , Membrana Celular/imunologia , Farmacorresistência Bacteriana/genética , Francisella/genética , Infecções por Bactérias Gram-Negativas/genética , Evasão da Resposta Imune/genética , Inflamassomos/genética , Sequências Repetidas Invertidas/imunologia , Lipoproteínas/genética , Camundongos , Camundongos KnockoutRESUMO
Patients suffering from inflammatory bowel disease (IBD) are currently treated by systemic drugs that can have significant side effects. Thus, it would be highly desirable to target TNFα siRNA (a therapeutic molecule) to the inflamed tissue. Here, we demonstrate that TNFα siRNA can be efficiently loaded into nanoparticles (NPs) made of poly (lactic acid) poly (ethylene glycol) block copolymer (PLA-PEG), and that grafting of the Fab' portion of the F4/80 Ab (Fab'-bearing) onto the NP surface via maleimide/thiol group-mediated covalent bonding improves the macrophage (MP)-targeting kinetics of the NPs to RAW264.7 cells in vitro. Direct binding was shown between MPs and the Fab'-bearing NPs. Next, we orally administered hydrogel (chitosan/alginate)-encapsulated Fab'-bearing TNFα-siRNA-loaded NPs to 3% dextran sodium sulfate (DSS)-treated mice and investigated the therapeutic effect on colitis. In vivo, the release of TNFα-siRNA-loaded NPs into the mouse colon attenuated colitis more efficiently when the NPs were covered with Fab'-bearing, compared to uncovered NPs. All DSS-induced parameters of colonic inflammation (e.g., weight loss, myeloperoxidase activity, and Iκbα accumulation) were more attenuated Fab'-bearing NPs loaded with TNFα siRNA than without the Fab'-bearing. Grafting the Fab'-bearing onto the NPs improved the kinetics of endocytosis as well as the MP-targeting ability, as indicated by flow cytometry. Collectively, our results show that Fab'-bearing PLA-PEG NPs are powerful and efficient nanosized tools for delivering siRNAs into colonic macrophages.
Assuntos
Colite/tratamento farmacológico , Fragmentos Fab das Imunoglobulinas/administração & dosagem , Nanopartículas/administração & dosagem , RNA Interferente Pequeno/administração & dosagem , Fator de Necrose Tumoral alfa/genética , Animais , Anticorpos/imunologia , Antígenos de Diferenciação/imunologia , Linhagem Celular , Colite/induzido quimicamente , Colite/metabolismo , Colite/patologia , Colo/efeitos dos fármacos , Colo/metabolismo , Colo/patologia , Sulfato de Dextrana , Fragmentos Fab das Imunoglobulinas/química , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Nanopartículas/química , Polietilenoglicóis/química , RNA Interferente Pequeno/químicaRESUMO
BACKGROUND: Krüppel-like factor 4 (KLF4) is a zinc finger transcription factor expressed in the differentiated epithelial cells lining of the intestine. Under physiological conditions, KLF4 inhibits cell proliferation. Conversely, KLF4 mediates proinflammatory signaling in macrophages and its overexpression in the esophageal epithelium activates cytokines, leading to inflammation-mediated esophageal squamous cell cancer formation in mice. Here, we tested whether KLF4 has a proinflammatory activity in experimental colitis in mice. METHODS: Villin-Cre;Klf4 mice with intestine-specific Klf4 deletion (Klf4) and control mice with floxed Klf4 gene (Klf4) were treated or not with 3% dextran sodium sulfate (DSS) for 7 days to induce colitis. Additionally, WT mice were administered or not, nanoparticles loaded with scrambled or Klf4-siRNA, and concomitantly given DSS. RESULTS: Compared with DSS-treated Klf4 mice, DSS-treated Klf4 mice were significantly less sensitive to DSS-induced colitis. DSS treatment of Klf4 mice induced Klf4 expression in the crypt zone of the colonic epithelium. DSS-treated Klf4 mice had increased proliferation relative to DSS-treated control mice. DSS treatment induced NF-κB signaling pathway in Klf4 mice colon but not Klf4 mice. Additionally, WT mice given DSS and nanoparticle/Klf4-siRNA were less sensitive to colitis and had reduced Klf4 expression and while maintaining the proliferative response in the colonic epithelium. CONCLUSIONS: Our results indicate that Klf4 is an important mediator of DSS-induced colonic inflammation by modulating NF-κB signaling pathway and could be involved in the pathogenesis and/or propagation of inflammatory bowel disease. Thus, Klf4 may represent a novel therapeutic target in inflammatory bowel disease.
Assuntos
Colite/prevenção & controle , Sulfato de Dextrana/toxicidade , Inflamação/prevenção & controle , Mucosa Intestinal/metabolismo , Fatores de Transcrição Kruppel-Like/fisiologia , Proteínas dos Microfilamentos/fisiologia , NF-kappa B/metabolismo , Animais , Western Blotting , Adesão Celular , Proliferação de Células , Células Cultivadas , Colite/induzido quimicamente , Colite/patologia , Modelos Animais de Doenças , Feminino , Imunofluorescência , Técnicas Imunoenzimáticas , Inflamação/induzido quimicamente , Inflamação/patologia , Integrases/metabolismo , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/patologia , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/antagonistas & inibidores , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Macrófagos/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , NF-kappa B/genética , Nanopartículas/administração & dosagem , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de SinaisRESUMO
Treatment strategies for inflammatory bowel disease have been constrained by limited therapeutic efficacy and serious adverse effects owing to a lack of receptor for targeted drug delivery to the inflamed colon. Upon inflammation, CD98 expression is highly elevated in colonic epithelial cells and infiltrating immune cells. To investigate whether CD98 can be used as a colitis-targeted delivery receptor, we constructed CD98 Fab'-bearing quantum dots (QDs)-loaded nanoparticles (Fab'-NPs). The resultant Fab'-NPs had desired particle size (~458 nm) with a narrow size distribution and zeta-potential (approximately +19 mV), low cytotoxicity, and excellent fluorescence properties. Electron microscopy images provided direct evidence for the well-dispersed distribution of QDs within spherical Fab'-NPs. Cellular uptake experiments demonstrated that Fab'-NPs were efficiently internalized into Colon-26 and RAW 264.7 cells through the CD98-mediated endocytosis pathway, and showed that the targeting effect of CD98 Fab' markedly increased their cellular uptake efficiency compared with control pegylated QDs-loaded NPs (PEG-NPs). Furthermore, ex vivo studies showed much more effective accumulation of Fab'-NPs in colitis tissue than that of PEG-NPs. These findings suggest that because of inflammation-dependent over-expression of CD98, active colitis-targeted delivery can be accomplished using NPs decorated with CD98 antibody.
RESUMO
BACKGROUND & AIMS: Nanoparticles have been explored as carriers of small interfering RNAs (siRNAs) and might be developed to treat patients with inflammatory bowel disease (IBD). Overexpression of CD98 on the surface of colonic epithelial cells and macrophages promotes the development and progression of IBD. We developed an orally delivered hydrogel that releases nanoparticles with single-chain CD98 antibodies on their surface (scCD98 functionalized) and loaded with CD98 siRNA (siCD98). We tested the ability of the nanoparticles to reduce levels of CD98 in the colons of mice with colitis. METHODS: scCD98-functionalized siCD98-loaded nanoparticles were fabricated using a complex coacervation technique. We investigated the cellular uptake and lysosome escape profiles of the nanoparticles in Colon-26 cells and RAW 264.7 macrophages using fluorescence microscopy. Colitis was induced by transfer of CD4(+)CD45RB(high) T cells to Rag(-/-) mice or administration of dextran sodium sulfate to C57BL/6 mice. Mice were then given hydrogel (chitosan and alginate) containing scCD98-functionalized nanoparticles loaded with siCD98 or scrambled siRNA (control) via gavage. RESULTS: The scCD98-functionalized nanoparticles were approximately 200 nm in size and had high affinity for CD98-overexpressing cells. The scCD98-functionalized siCD98-loaded nanoparticles significantly reduced levels of CD98 in Colon-26 cells and RAW 264.7 macrophages, along with production of inflammatory cytokines (tumor necrosis factor α, interleukin-6, and interleukin-12). In mice with colitis, administration of the scCD98-functionalized siCD98-loaded nanoparticles reduced colon expression of CD98. Importantly, the severity of colitis was also reduced compared with controls (based on loss of body weight, myeloperoxidase activity, inflammatory cytokine production, and histological analysis). Approximately 24.1% of colonic macrophages (CD11b(+)CD11c(-)F4/80(+)) in the mice had taken up fluorescently labeled siRNA-loaded nanoparticles within 12 hours of administration. CONCLUSIONS: Nanoparticles containing surface CD98 antibody and loaded with siCD98 reduce expression of this protein by colonic epithelial cells and macrophages, and oral administration decreases the severity of colitis in mice. This nanoparticle in hydrogel (chitosan/alginate) formulation might be developed to treat patients with IBD.
Assuntos
Colite/prevenção & controle , Colo/metabolismo , Proteína-1 Reguladora de Fusão/genética , Proteína-1 Reguladora de Fusão/imunologia , Terapia Genética/métodos , Nanomedicina/métodos , Nanopartículas , Interferência de RNA , RNA Interferente Pequeno/administração & dosagem , Anticorpos de Cadeia Única/administração & dosagem , Administração Oral , Alginatos/química , Animais , Linhagem Celular , Quitosana/química , Colite/induzido quimicamente , Colite/genética , Colite/imunologia , Colite/metabolismo , Colite/patologia , Colo/imunologia , Colo/patologia , Sulfato de Dextrana , Modelos Animais de Doenças , Ácido Glucurônico/química , Ácidos Hexurônicos/química , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Hidrogéis , Interleucina-12/metabolismo , Interleucina-6/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , RNA Interferente Pequeno/metabolismo , Índice de Gravidade de Doença , Fatores de Tempo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Intestinal CD98 expression plays a crucial role in controlling homeostatic and innate immune responses in the gut. Modulation of CD98 expression in intestinal cells therefore represents a promising therapeutic strategy for the treatment and prevention of inflammatory intestinal diseases, such as inflammatory bowel disease. Here, the advantages of nanoparticles (NPs) are used, including their ability to easily pass through physiological barriers and evade phagocytosis, high loading concentration, rapid kinetics of mixing and resistance to degradation. Using physical chemistry characterizations techniques, CD98 siRNA/polyethyleneimine (PEI)-loaded NPs was characterized (diameter of ~480 nm and a zeta potential of -5.26 mV). Interestingly, CD98 siRNA can be electrostatically complexed by PEI and thus protected from RNase. In addition, CD98 siRNA/PEI-loaded NPs are nontoxic and biocompatible with intestinal cells. Oral administration of CD98/PEI-loaded NPs encapsulated in a hydrogel reduced CD98 expression in mouse colonic tissues and decreased dextran sodium sulfate-induced colitis in a mouse model. Finally, flow cytometry showed that CD98 was effectively downregulated in the intestinal epithelial cells and intestinal macrophages of treated mice. Finally, the results collectively demonstrated the therapeutic effect of "hierarchical nano-micro particles" with colon-homing capabilities and the ability to directly release "molecularly specific" CD98 siRNA in colonic cells, thereby decreasing colitis.
Assuntos
Proteína-1 Reguladora de Fusão/química , Inflamação/metabolismo , Mucosa Intestinal/metabolismo , Nanopartículas/química , Polietilenoimina/química , RNA Interferente Pequeno/química , Animais , Linhagem Celular , Sobrevivência Celular , Colo/efeitos dos fármacos , Colo/metabolismo , Colo/patologia , Células Dendríticas/metabolismo , Sulfato de Dextrana/efeitos adversos , Proteína-1 Reguladora de Fusão/genética , Técnicas de Transferência de Genes , Inflamação/induzido quimicamente , Inflamação/genética , Inflamação/patologia , Doenças Inflamatórias Intestinais/genética , Doenças Inflamatórias Intestinais/metabolismo , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/patologia , Intestinos/efeitos dos fármacos , Intestinos/patologia , Macrófagos/metabolismo , Camundongos , Nanopartículas/administração & dosagem , Nanopartículas/ultraestrutura , RNA Interferente Pequeno/genéticaRESUMO
BACKGROUND: Dextran sodium sulfate (DSS) is commonly used in mouse studies to induce a very reproducible colitis that effectively mimics the clinical and histological features of human inflammatory bowel disease (IBD) patients, especially ulcerative colitis. However, the mechanisms of action of DSS remain poorly understood, and observations by our laboratory and other groups indicate that DSS contamination of colonic tissues from DSS-treated mice potently inhibits the quantitative reverse-transcription polymerase chain reaction (qRT-PCR) amplification of mRNA. RESULTS: A prior study used poly-A-mediated mRNA purification to remove DSS from RNA extracts, but we herein report a second efficient and cost-effective approach to counteract this inhibition, using lithium chloride precipitation to entirely remove DSS from RNAs. We also explored how DSS interferes with qRT-PCR process, and we report for the first time that DSS can alter the binding of reverse transcriptase to previously primed RNA and specifically inhibits the enzymatic activities of reverse transcriptase and Taq polymerase in vitro. This likely explains why DSS-treated colonic RNA is not suitable to qRT-PCR amplification without a previous purification step. CONCLUSION: In summary, we provide a simple method to remove DSS from colonic RNAs, and we demonstrate for the first time that DSS can inhibit the activities of both polymerase and reverse transcriptase. In order to reliably analyze gene expression in the colonic mucosa of DSS-treated mice, the efficiency rate of qRT-PCR must be the same between all the different experimental groups, including the water-treated control group, suggesting that whatever the duration and the percentage of the DSS treatment, RNAs must be purified.
Assuntos
DNA Polimerase Dirigida por DNA/química , Sulfato de Dextrana/química , Cloreto de Lítio/química , RNA Mensageiro/isolamento & purificação , DNA Polimerase Dirigida por RNA/química , Inibidores da Transcriptase Reversa/química , Animais , Fracionamento Químico/métodos , Colite/induzido quimicamente , Colite/genética , Colite/patologia , Sulfato de Dextrana/farmacologia , Modelos Animais de Doenças , Humanos , Camundongos , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/normasRESUMO
The application of RNA interference (RNAi) for inï¬ammatory bowel disease (IBD) therapy has been limited by the lack of non-cytotoxic, efficient and targetable small interfering RNA (siRNA) carriers. TNF-α is the major pro-inflammatory cytokine mainly secreted by macrophages during IBD. Here, a mannosylated bioreducible cationic polymer (PPM) was synthesized and further spontaneously assembled nanoparticles (NPs) assisted by sodium triphosphate (TPP). The TPP-PPM/siRNA NPs exhibited high uniformity (polydispersity index = 0.004), a small particle size (211-275 nm), excellent bioreducibility, and enhanced cellular uptake. Additionally, the generated NPs had negative cytotoxicity compared to control NPs fabricated by branched polyethylenimine (bPEI, 25 kDa) or Oligofectamine (OF) and siRNA. In vitro gene silencing experiments revealed that TPP-PPM/TNF-α siRNA NPs with a weight ratio of 40:1 showed the most efficient inhibition of the expression and secretion of TNF-α (approximately 69.9%, which was comparable to the 71.4% obtained using OF/siRNA NPs), and its RNAi efficiency was highly inhibited in the presence of mannose (20 mm). Finally, TPP-PPM/siRNA NPs showed potential therapeutic effects on colitis tissues, remarkably reducing TNF-α level. Collectively, these results suggest that non-toxic TPP-PPM/siRNA NPs can be exploited as efficient, macrophage-targeted carriers for IBD therapy.
Assuntos
Doenças Inflamatórias Intestinais/terapia , Macrófagos/metabolismo , Manose/metabolismo , Nanopartículas/química , Interferência de RNA , Fator de Necrose Tumoral alfa/metabolismo , Animais , Células CACO-2 , Morte Celular/efeitos dos fármacos , Eletroforese em Gel de Ágar , Endocitose/efeitos dos fármacos , Humanos , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Espectroscopia de Ressonância Magnética , Masculino , Camundongos , Nanopartículas/ultraestrutura , Tamanho da Partícula , Polímeros/síntese química , Polímeros/química , Polímeros/farmacologia , Polifosfatos/química , Polifosfatos/farmacologia , Interferência de RNA/efeitos dos fármacos , RNA Interferente Pequeno/metabolismo , TitulometriaRESUMO
This review describes the state of the art in nanoparticle and nanodevice applications for medical diagnosis and disease treatment. Nanodevices, such as cantilevers, have been integrated into high-sensitivity disease marker diagnostic detectors and devices, are stable over long periods of time, and display reliable performance properties. Nanotechnology strategies have been applied to therapeutic purposes as well. For example, nanoparticle-based delivery systems have been developed to protect drugs from degradation, thereby reducing the required dose and dose frequency, improving patient comfort and convenience during treatment, and reducing treatment expenses. The main objectives for integrating nanotechnologies into diagnostic and therapeutic applications in the context of intestinal diseases are reviewed.
Assuntos
Gastroenteropatias/diagnóstico , Gastroenteropatias/terapia , Nanomedicina , Nanoestruturas/uso terapêutico , Neoplasias Colorretais/diagnóstico , Dendrímeros/uso terapêutico , Humanos , Doenças Inflamatórias Intestinais/tratamento farmacológico , Lipossomos/uso terapêutico , Nanoconchas/uso terapêutico , Nanofios , Pontos QuânticosRESUMO
CD98 is a type II transmembrane glycoprotein whose expression increases in intestinal epithelial cells (IECs) during intestinal inflammation. Enteropathogenic Escherichia coli (EPEC) is a food-borne human pathogen that attaches to IECs and injects effector proteins directly into the host cells, thus provoking an inflammatory response. In the present study, we investigated CD98 and EPEC interactions in vitro and ex vivo and examined FVB wild-type (WT) and villin-CD98 transgenic mice overexpressing human CD98 in IECs (hCD98 Tg mice) and infected with Citrobacter rodentium as an in vivo model. In vivo studies indicated that CD98 overexpression, localized to the apical domain of colonic cells, increased the attachment of C. rodentium in mouse colons and resulted in increased expression of proinflammatory markers and decreased expression of anti-inflammatory markers. The proliferative markers Ki-67 and cyclin D1 were significantly increased in the colonic tissue of C. rodentium-infected hCD98 Tg mice compared to that of WT mice. Ex vivo studies correlate with the in vivo data. Small interfering RNA (siRNA) studies with Caco2-BBE cells showed a decrease in adherence of EPEC to Caco2 cells in which CD98 expression was knocked down. In vitro surface plasmon resonance (SPR) experiments showed direct binding between recombinant hCD98 and EPEC/C. rodentium proteins. We also demonstrated that the partial extracellular loop of hCD98 was sufficient for direct binding to EPEC/C. rodentium. These findings demonstrate the importance of the extracellular loop of CD98 in the innate host defense response to intestinal infection by attaching and effacing (A/E) pathogens.
Assuntos
Infecções por Enterobacteriaceae/imunologia , Proteína-1 Reguladora de Fusão/metabolismo , Imunidade Inata , Mucosa Intestinal/metabolismo , Animais , Células CACO-2 , Citrobacter rodentium , Colo , Infecções por Enterobacteriaceae/microbiologia , Escherichia coli Enteropatogênica , Células Epiteliais/imunologia , Células Epiteliais/metabolismo , Feminino , Proteína-1 Reguladora de Fusão/genética , Regulação da Expressão Gênica/imunologia , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Peroxidase , Reação em Cadeia da Polimerase em Tempo Real/métodos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodosRESUMO
Modification of specific Gram-negative bacterial cell envelope components, such as capsule, O-antigen and lipid A, are often essential for the successful establishment of infection. Francisella species express lipid A molecules with unique characteristics involved in circumventing host defences, which significantly contribute to their virulence. In this study, we show that NaxD, a member of the highly conserved YdjC superfamily, is a deacetylase required for an important modification of the outer membrane component lipid A in Francisella. Mass spectrometry analysis revealed that NaxD is essential for the modification of a lipid A phosphate with galactosamine in Francisella novicida, a model organism for the study of highly virulent Francisella tularensis. Significantly, enzymatic assays confirmed that this protein is necessary for deacetylation of its substrate. In addition, NaxD was involved in resistance to the antimicrobial peptide polymyxin B and critical for replication in macrophages and in vivo virulence. Importantly, this protein is also required for lipid A modification in F. tularensis as well as Bordetella bronchiseptica. Since NaxD homologues are conserved among many Gram-negative pathogens, this work has broad implications for our understanding of host subversion mechanisms of other virulent bacteria.
Assuntos
Amidoidrolases/metabolismo , Proteínas de Bactérias/metabolismo , Francisella/enzimologia , Francisella/patogenicidade , Infecções por Bactérias Gram-Negativas/microbiologia , Lipídeo A/metabolismo , Amidoidrolases/química , Amidoidrolases/genética , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Linhagem Celular , Feminino , Francisella/genética , Francisella/metabolismo , Francisella tularensis/enzimologia , Francisella tularensis/genética , Humanos , Macrófagos/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Alinhamento de Sequência , VirulênciaRESUMO
BACKGROUND: Adenosine, an endogenous purine nucleoside, is involved in several physiological functions. We have previously shown that A(2B)AR plays a pro-inflammatory role during colitis. AIMS: Our goals were to determine if A(2B)AR expression was necessary on immune cells/non-immune cells during colitis and if A(2B)AR was a suitable target for treating intestinal inflammation. METHODS: Wild-type and A(2B)AR knockout mice were utilized in bone marrow transplants to explore the importance of immune/non-immune A(2B)AR expression during the development of colitis. Additionally, a T-cell transfer model of colitis was used in Rag1 knockout or A(2B)AR/RAG1 double knockout recipients. Finally, A(2B)AR small interfering RNA nanoparticles were administered to dextran sodium sulphate-treated mice. RESULTS: Wild-type mice receiving wild-type or knockout bone marrow developed severe colitis after dextran sodium sulphate treatment, whereas colitis was significantly attenuated in knockout mice receiving wild-type or knockout bone marrow. Colitis induced in Rag1 knockout animals was attenuated in A(2B)AR/RAG1 double knockout recipients. Animals receiving nanoparticles exhibited attenuated parameters of colitis severity compared to mice receiving control nanoparticles. CONCLUSIONS: Our results suggest that A(2B)AR on non-immune cells plays an important role for the induction of colitis and targeting A(2B)AR expression during colitis may be useful for alleviating symptoms of intestinal inflammation.