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This paper reports on biofunctionalisation of a poly(lactic acid) (PLA) film by surface activation through cold plasma treatment followed by coating with a chitosan-gelatin xerogel. The UV cross-linking of the xerogel precursor was simultaneously performed with the fixation onto the PLA support. This has a strong effect on surface properties, in terms of wettability, surface free energy, morphology and micromechanical features. The hydrophilic - hydrophobic character of the surface, determined by contact angle measurements, was tuned along the process, passing from moderate hydrophobic PLA to enhanced hydrophilic plasma activated surface, which favors coating adhesion, then to moderate hydrophobic chitosan-gelatin coating. The coating has a Lewis amphoteric surface, with a porous xerogel-like morphology, as revealed by scanning electron microscopy images. By riboflavin mediated UV cross-linking the chitosan-gelatin coating becomes high adhesive and with a more pronounced plasticity, as shown by AFM force-distance spectroscopy. Thus prepared surface-coated PLA supports were successfully tested for growth of dermal fibroblasts, which are known for their induction potential of chondrogenic cells, which is very important in cartilage tissue engineering.
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Quitosana , Fibroblastos , Gelatina , Poliésteres , Quitosana/química , Gelatina/química , Poliésteres/química , Fibroblastos/efeitos dos fármacos , Fibroblastos/citologia , Humanos , Propriedades de Superfície , Géis/química , Raios Ultravioleta , Gases em Plasma/química , Interações Hidrofóbicas e Hidrofílicas , Materiais Revestidos Biocompatíveis/química , Reagentes de Ligações Cruzadas/química , MolhabilidadeRESUMO
Atherosclerosis (ATH) is a systemic disease characterized by a chronic inflammatory process and lipid deposition in the arterial walls. The chronic inflammation within ATH lesions results, at least in part, from the failed resolution of inflammation. This process is controlled actively by specialized pro-resolving lipid mediators (SPMs), namely lipoxins, resolvins, protectins, and maresins. Herein, biomimetic nanocarriers are produced comprising a cocktail of SPMs-loaded lipid nanoemulsions (LN) covered with macrophage membranes (Bio-LN/SPMs). Bio-LN/SPMs retain on their surface the macrophage receptors involved in cellular interactions and the "marker of self" CD47, which impede their recognition and uptake by other macrophages. The binding of Bio-LN/SPMs to the surface of endothelial cells (EC) and smooth muscle cells (SMC) is facilitated by the receptors on the macrophage membranes and partly by SPMs receptors. In addition, Bio-LN/SPMs prove functional by reducing monocyte adhesion and transmigration to/through activated EC and by stimulating macrophage phagocytic activity. After intravenous administration, Bio-LN/SPMs accumulate in the aorta of ApoE-deficient mice at the level of atherosclerotic lesions. Also, the safety assessment testing reveals no side effects or immunotoxicity of Bio-LN/SPMs. Thus, the newly developed Bio-LN/SPMs represent a reliable targeted nanomedicine for the resolution of inflammation in atherosclerosis.
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Aterosclerose , Biomimética , Animais , Camundongos , Células Endoteliais/metabolismo , Inflamação/tratamento farmacológico , Aterosclerose/patologia , Lipídeos , Mediadores da Inflamação/metabolismoRESUMO
Composite hydrogels containing apatite-like particles can act as scaffolds for osteoblast proliferation, with applications in bone tissue engineering. In this respect, porous biocompatible hydrogels were obtained from chitosan, oxidized pullulan, and PVA in different ratios. The stability of the hydrogels was ensured both by covalent bonds between aldehyde groups of oxidized pullulan and free amino groups of chitosan, and by physical bonds formed during freeze-thaw cycles and lyophilization. The deposition of calcium phosphates was performed by alternate soaking of the porous hydrogels into solutions with calcium and phosphate ions, assuring a basic pH required for hydroxyapatite formation. The mineralized hydrogels were characterized using FTIR spectroscopy, scanning electron microscopy, X-ray diffraction, and thermogravimetric analysis, showing that inorganic particles containing between 80 and 92% hydroxyapatite were deposited in a high amount on the pore walls of the polymeric matrix. The composition of the organic matrix influenced the crystallization of calcium phosphates and the mechanical properties of the composite hydrogels. In vitro biological tests showed that mineralized hydrogels support the proliferation of MG-63 osteoblast-like cells to a greater extent compared to pristine hydrogels.
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Runx2 is a key transcription factor involved in valvular interstitial cells (VIC) osteodifferentiation, a process actively entwined with the calcific aortic valve disease (CAVD). We hypothesize that a strategy intended to silence Runx2 could be a valuable novel therapeutic option for CAVD. To this intent, we aimed at (i) developing targeted nanoparticles for efficient delivery of short hairpin (sh)RNA sequences specific for Runx2 to the aortic valve employing a relevant mouse model for CAVD and (ii) investigate their therapeutic potential in osteoblast-differentiated VIC (oVIC) cultivated into a 3D scaffold. Since collagen IV was used as a target, a peptide that binds specifically to collagen IV (Cp) was conjugated to the surface of lipopolyplexes encapsulating shRNA-Runx2 (Cp-LPP/shRunx2). The results showed that Cp-LPP/shRunx2 were (i) cytocompatible; (ii) efficiently taken up by 3D-cultured oVIC; (iii) diminished the osteodifferentiation of human VIC (cultured in a 3D hydrogel-derived from native aortic root) by reducing osteogenic molecules expression, alkaline phosphatase activity, and calcium concentration; and (iv) were recruited in aortic valve leaflets in a murine model of atherosclerosis. Taken together, these data recommend Cp-LPP/shRunx2 as a novel targeted nanotherapy to block the progression of CAVD, with a good perspective to be introduced in practical use.
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The improved drug delivery systems (DDS) are needed for the targeted delivery of their therapeutic cargo (biologically active protein/peptide molecules, nucleic acids, vaccines, etc.) to diseased cells. Thus, we aimed to develop magnetite nanoparticles (Fe3O4), stabilized with polyethylene glycol (PEG) and decorated (surface-functionalized) with folic acid (FA) (Fe3O4@PEG@FA) to ensure targeted internalization in cells expressing the folic acid receptors (FR). The Fe3O4@PEG@FA nanoparticles were synthesized by co-precipitation in a one-pot methodology. Curcumin (Curc), a polyphenol with anti-tumoral activity, was loaded on the nanoparticles, and FA-targeted (Fe3O4@PEG@FA@Curc) and non-targeted (Fe3O4@PEG@Curc) systems were obtained. The internalization of Fe3O4@PEG@FA@Curc and Fe3O4@PEG@Curc nanoparticles was determined in two tumor cell lines, the FR-positive MCF-7 human breast carcinoma cell line and A549 human lung adenocarcinoma cell line, expressing a low level of FR. The results showed that MCF-7 cells internalize FA-functionalized nanoparticles to a greater extent than non-targeted ones and also than A549 cells. The competitive studies performed in the presence of FA in excess suggested that internalization is an FR-dependent process. The increased internalization of Fe3O4@PEG@FA@Curc nanoparticles in MCF-7 cells is correlated with increased cytotoxicity in this cell line compared to A549 cells. In conclusion, the FA-functionalized magnetic systems can ensure a better internalization of the nanoparticles and can be used to deliver various therapeutic agents, both in cancer treatment and also in the treatment of other inflammation-associated diseases such as rheumatoid arthritis, systemic lupus erythematosus, osteoarthritis, Crohn's disease or atherosclerosis.
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Curcumina , Nanopartículas de Magnetita , Nanopartículas , Linhagem Celular Tumoral , Curcumina/farmacologia , Portadores de Fármacos/química , Sistemas de Liberação de Medicamentos/métodos , Ácido Fólico/química , Humanos , Nanopartículas de Magnetita/química , Nanopartículas/química , Polietilenoglicóis/químicaRESUMO
A wide variety of metal-based compounds have been obtained and studied for their antitumor activity since the intensely used cytostatic drugs (e.g., cisplatin) failed to accomplish their expected pharmacological properties. Thus, we aimed to develop a new vanadium-based drug and assess its antitumor properties using the human hepatocarcinoma (HepG2) cell line. The compound was synthesized from vanadyl sulfate, DL-valine, and o-vanillin and was spectrally and structurally characterized (UV-Vis, IR, CD, and single-crystal/powder-XRD). Compound stability in biological media, cell uptake, and the interaction with albumin were assessed. The mechanisms of its antitumor activity were determined compared to cisplatin by performing cytotoxicity, oxidative and mitochondrial status, DNA fragmentation, ß-Tubulin synthesis investigation, and cell cycle studies. Herein, we developed a macrocyclic tetranuclear oxidovanadium(V) compound, [(VVO)(L)(CH3O)]4, having coordinated four Schiff base (H2L) ligands, 3-methoxysalicylidenvaline. We showed that [(VVO)(L)(CH3O)]4: (i) has pH-dependent stability in biological media, (ii) binds to albumin in a dose-dependent manner, (iii) is taken up by cells in a time-dependent way, (iv) has a higher capacity to induce cell death compared to cisplatin (IC50 = 6 µM vs. 10 µM), by altering the oxidative and mitochondrial status in HepG2 cells. Unlike cisplatin, which blocks the cell cycle in the S-phase, the new vanadium-based compound arrests it in S and G2/M-phase, whereas no differences in the induction of DNA fragmentation and reduction of ß-Tubulin synthesis between the two were determined. Thus, the [(VVO)(L)(CH3O)]4 antitumor mechanism involved corroboration between the generation of oxidative species, mitochondrial dysfunction, degradation of DNA, cell cycle arrest in the S and G2/M-phase, and ß-Tubulin synthesis reduction. Our studies demonstrate the potent antitumor activity of [(VVO)(L)(CH3O)]4 and propose it as an attractive candidate for anticancer therapy.
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The cellular internalization of drug carriers occurs via different endocytic pathways that ultimately involve the endosomes and the lysosomes, organelles where the pH value drops to 6.0 and 5.0, respectively. We aimed to design and characterize pH/temperature-responsive carriers for the effective delivery of the anti-tumoral drug doxorubicin. To this purpose, poly(N-isopropylacrylamide-co-vinylimidazole) was synthesized as an attractive pH/temperature-sensitive copolymer. Microspheres made of this copolymer, loaded with doxorubicin (MS-DXR), disintegrate in monodisperse nanospheres (NS-DXR) under conditions similar to that found in the bloodstream (pH = 7.4, temperature of 36 °C) releasing a small amount of payload. However, in environments that simulate the endosomal and lysosomal conditions, nanospheres solubilize, releasing the entire amount of drug. We followed the NS-DXR internalization using two cancer cell lines, hepatic carcinoma HepG2 cells and lung adenocarcinoma A549 cells. The data showed that NS-DXR are internalized to a greater extent by HepG2 cells than A549 cells, and this correlated with increased cytotoxicity induced by NS-DXR in HepG2 cells compared with A549 cells. Moreover, NS-DXR particles do not cause hemolysis and erythrocytes aggregation. Administered in vivo, NS-DXR localized in the liver and kidneys of mice, and the loading of DXR into NS resulted in the reduced renal clearance of DXR. In conclusion, the newly developed poly(N-isopropylacrylamide-co-vinyl imidazole) particles are biocompatible and may be introduced as carriers for doxorubicin to hepatic tumors.
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Calcific aortic valve disease (CAVD) is a progressive inflammatory disorder characterized by extracellular matrix remodeling and valvular interstitial cells (VIC) osteodifferentiation leading to valve leaflets calcification and impairment movement. Runx2, the master transcription factor involved in VIC osteodifferentiation, modulates the expression of other osteogenic molecules. Previously, we have demonstrated that the osteoblastic phenotypic shift of cultured VIC is impeded by Runx2 silencing using fullerene (C60)-polyethyleneimine (PEI)/short hairpin (sh)RNA-Runx2 (shRunx2) polyplexes. Since the use of polyplexes for in vivo delivery is limited by their instability in the plasma and the non-specific tissue interactions, we designed and obtained targeted, lipid-enveloped polyplexes (lipopolyplexes) suitable for (1) systemic administration and (2) targeted delivery of shRunx2 to osteoblast-differentiated VIC (oVIC). Vascular cell adhesion molecule (VCAM)-1 expressed on the surface of oVIC was used as a target, and a peptide with high affinity for VCAM-1 was coupled to the surface of lipopolyplexes encapsulating C60-PEI/shRunx2 (V-LPP/shRunx2). We report here that V-LPP/shRunx2 lipopolyplexes are cyto- and hemo-compatible and specifically taken up by oVIC. These lipopolyplexes are functional as they downregulate the Runx2 gene and protein expression, and their uptake leads to a significant decrease in the expression of osteogenic molecules (OSP, BSP, BMP-2). These results identify V-LPP/shRunx2 as a new, appropriately directed vehicle that could be instrumental in developing novel strategies for blocking the progression of CAVD using a targeted nanomedicine approach.
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Estenose da Valva Aórtica , Calcinose , Valva Aórtica/metabolismo , Valva Aórtica/patologia , Estenose da Valva Aórtica/metabolismo , Calcinose/metabolismo , Células Cultivadas , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Humanos , Osteoblastos/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Molécula 1 de Adesão de Célula Vascular/metabolismoRESUMO
The receptor for advanced glycation end products (RAGE) plays a central role in the chronic inflammatory process associated with atherosclerosis development. We aimed to develop lipoplexes carrying RAGE-short hairpin (sh) RNA, targeted to the adhesion molecule P-selectin, selectively expressed on the surface of activated endothelium (Psel-lipo/shRAGE) to down-regulate RAGE expression as a therapeutic strategy for atherosclerosis. In vitro, Psel-lipo/shRAGE lipoplexes were efficiently taken up by activated endothelial cells (EC), decreased the expression of RAGE protein, and proved to be functional by reducing the monocyte adhesion to activated EC. In ApoE-deficient mice, the targeted lipoplexes accumulated specifically and efficiently transfected the aorta. The repeated administration of Psel-lipo/shRAGE lipoplexes, twice per week for one month: i) reduced the expression of RAGE protein in the aorta by decreasing the expression of NF-kB and TNF-α; ii) diminished the plasma levels of TNF-α, IL6, IL-1ß, and MCP-1; iii) inhibited the atherosclerotic plaque development and iv) had no significant adverse effects. In conclusion, the newly developed Psel-lipo/shRAGE lipoplexes reduce the inflammatory processes associated with RAGE signaling and the progression of atherosclerosis in ApoE-deficient mice. Downregulation of RAGE employing these lipoplexes may represent a promising new targeted therapy to block atherosclerosis progression.
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Aterosclerose , Células Endoteliais , Animais , Aterosclerose/genética , Aterosclerose/terapia , Inflamação/terapia , Camundongos , Camundongos Knockout , Selectina-P , RNA Interferente Pequeno , Receptor para Produtos Finais de Glicação AvançadaRESUMO
Hepatic cancer is one of the most widespread maladies worldwide that requires urgent therapies and thus reliable means for testing anti-cancer drugs. The switch from two-dimensional (2D) to three-dimensional (3D) cell cultures produced an improvement in the in vitro outcomes for testing anti-cancer drugs. We aimed to develop a novel hyaluronic acid (HA)-based 3D cell model of human hepatocellular carcinoma (HepG2 cells) for drug testing and to assess comparatively in 3D vs. 2D, the cytotoxicity and the apoptotic response to the anti-tumor agent, cisplatin. The 3D model was developed by seeding HepG2 cells in a HA/poly(methylvinylether-alt-maleic acid) (HA3P50)-based scaffold. Compared to 2D, the cells grown in the HA3P50 scaffold proliferate into larger-cellular aggregates that exhibit liver-like functions by controlling the release of hepatocyte-specific biomarkers (albumin, urea, bile acids, transaminases) and the synthesis of cytochrome-P450 (CYP)7A1 enzyme. Also, growing the cells in the scaffold sensitize the hepatocytes to the anti-tumor effect of cisplatin, by a mechanism involving the activation of ERK/p38α-MAPK and dysregulation of NF-kB/STAT3/Bcl-2 pathways. In conclusion, the newly developed HA-based 3D model is suitable for chemotherapeutic drug testing on hepatocellular carcinoma. Moreover, the system can be adapted and employed as experimental platform functioning as a proper tissue/tumor surrogate.
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Materiais Biomiméticos/química , Carcinoma Hepatocelular/metabolismo , Cisplatino/farmacologia , Ácido Hialurônico/química , Neoplasias Hepáticas/metabolismo , Biomarcadores Tumorais/metabolismo , Carcinoma Hepatocelular/tratamento farmacológico , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Colesterol 7-alfa-Hidroxilase/metabolismo , Cisplatino/química , Ensaios de Seleção de Medicamentos Antitumorais , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células Hep G2 , Humanos , Neoplasias Hepáticas/tratamento farmacológico , Alicerces TeciduaisRESUMO
Naringenin, an anti-inflammatory citrus flavonoid, is restrained from large-scale use by its reduced water solubility and bioavailability. To overcome these limitations, naringenin was loaded into lipid nanoemulsions directed towards vascular cell adhesion molecule (VCAM)-1, exposed by activated endothelium, and delivered intravenously in a murine model of lipopolysaccharide (LPS)-induced inflammation. To follow the in vivo bio-distribution, naringenin-loaded nanoemulsions were labeled with near-infrared probe Indocyanine Green (ICG). Based on ICG fluorescence, a VCAM-1-dependent retention of nanoemulsions was detected in the heart and aorta, while ultra-high-performance liquid chromatography (UHPLC) measurements showed a target-selective accumulation of naringenin in the heart and lungs. Correlated, fluorescence and UHPLC data indicated a mixed behavior of the VCAM-1 directed nanoparticles, which were driven not only by the targeting moiety but also by passive retention. The treatment with naringenin-loaded nanoemulsions reduced the mRNA levels of some inflammatory mediators in organs harvested from mice with acute inflammation, indicative of their anti-inflammatory potential. The data support a novel theranostic nanoplatform for inflammation, the naringenin/ICG-loaded nanoparticles that either by passive accumulation or effective targeting of the activated endothelium can be employed for imaging inflamed vascular areas and efficient delivery of the encapsulated therapeutic agent.
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Diabetes reduces the number and induces dysfunction in circulating endothelial progenitor cells (EPCs) by mechanisms that are still uncovered. This study aims to evaluate the number, viability, phenotype, and function of EPCs in dyslipidemic mice with early diabetes mellitus and EPC infiltration in the aortic valve in order to identify possible therapeutic targets in diabetes-associated cardiovascular disease. A streptozotocin-induced diabetic apolipoprotein E knock-out (ApoE-/-) mouse model was used to identify the early and progressive changes, at 4 or 7 days on atherogenic diet after the last streptozotocin or citrate buffer injection. Blood and aortic valves from diabetic or nondiabetic ApoE-/- animals were collected.EPCs were identified as CD34 and vascular endothelial growth factor receptor 2 positive monocytes, and the expression levels of α4ß1, αVß3, αVß5, ß1, αLß2, α5 integrins, and C-X-C chemokine receptor type 4 chemokine receptor on EPC surface were assessed by flow cytometry. The number of CD34 positive cells in the aortic valve, previously found to be recruited progenitor cells, was measured by fluorescence microscopy. Our results show that aortic valves from mice fed 7 days with atherogenic diet presented a significantly higher number of CD34 positive cells compared with mice fed only 4 days with the same diet, and diabetes reversed this finding. We also show a reduction of circulatory EPC numbers in diabetic mice caused by cell senescence and lower mobilization. Dyslipidemia induced EPC death through apoptosis regardless of the presence of diabetes, as shown by the higher percent of propidium iodide positive cells and higher cleaved caspase-3 levels. EPCs from diabetic mice expressed α4ß1 and αVß3 integrins at a lower level, while the rest of the integrins tested were unaffected by diabetes or diet. In conclusion, reduced EPC number and expression of α4ß1 and αVß3 integrins on EPCs at 4 and 7 days after diabetes induction in atherosclerosis-prone mice have resulted in lower recruitment of EPCs in the aortic valve.
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Diabetes Mellitus Experimental/metabolismo , Dislipidemias/fisiopatologia , Células Progenitoras Endoteliais/metabolismo , Integrina alfa4beta1/metabolismo , Integrina alfaVbeta3/metabolismo , Células-Tronco/metabolismo , Estreptozocina/uso terapêutico , Animais , Valvopatia Aórtica , Células Cultivadas , Masculino , Camundongos , Camundongos KnockoutRESUMO
Calcific aortic valve disease (CAVD) is a progressive disorder that increases in prevalence with age. An important role in aortic valve calcification is played by valvular interstitial cells (VIC), that with age or in pathological conditions acquire an osteoblast-like phenotype that advances the disease. Therefore, pharmacological interventions aiming to stop or reverse the osteoblastic transition of VIC may represent a therapeutic option for CAVD. In this study, we aimed at developing a nanotherapeutic strategy able to prevent the phenotypic switch of human aortic VIC into osteoblast-like cells. We hypothesize that nanocarriers designed for silencing the Runt-related transcription factor 2 (Runx2) will stop the progress or reverse the osteodifferentiation of human VIC, induced by high glucose concentrations and pro-osteogenic factors. We report here the potential of fullerene (C60)-polyethyleneimine (PEI)/short hairpin (sh)RNA-Runx2 nano-polyplexes to efficiently down-regulate Runx2 mRNA and protein expression leading subsequently to a significant reduction in the expression of osteogenic proteins (i.e. ALP, BSP, OSP and BMP4) in osteoblast-committed VIC. The data suggest that the silencing of Runx2 could represent a novel strategy to impede the osteoblastic phenotypic shift of VIC and the ensuing progress of CAVD.
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Diabetes contributes directly to the development of cardiovascular aortic valve disease. There is currently no drug therapy available for a dysfunctional valve and this urges the need for additional research to identify distinctive mechanisms of cardiovascular aortic valve disease evolution. The aim of this study was to evaluate changes of valvular aortic lesions induced in a hyperlipemic ApoE-/- mouse model by early type 1 diabetes onset (at 4 and 7 days after streptozotocin induction). The haemodynamic valve parameters were evaluated by echography and blood samples and aortic valves were collected. Plasma parameters were measured, and inflammatory, remodelling and osteogenic markers were evaluated in the aortic valves. Next, correlations between all parameters were determined. The results showed early aortic valve dysfunction detected by echography after 1 week of diabetes; lesions were found in the aortic root. Moreover, increased expression of cell adhesion molecules, extracellular matrix remodelling and osteogenic markers were detected in hyperlipemic ApoE-/- diabetic mice. Significant correlations were found between tissue valve biomarkers and plasmatic and haemodynamic parameters. Our study may help to understand the mechanisms of aortic valve disease in the diabetic milieu in order to discover and validate new biomarkers of cardiovascular aortic valve disease in diabetes and reveal new possible targets for nanobiotherapies.
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Valva Aórtica , Aterosclerose/complicações , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Tipo 1/complicações , Doenças das Valvas Cardíacas/etiologia , Animais , Valva Aórtica/metabolismo , Valva Aórtica/patologia , Valva Aórtica/fisiopatologia , Aterosclerose/metabolismo , Aterosclerose/patologia , Glicemia/metabolismo , Moléculas de Adesão Celular/metabolismo , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patologia , Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 1/patologia , Matriz Extracelular/metabolismo , Matriz Extracelular/patologia , Hemoglobinas Glicadas/metabolismo , Doenças das Valvas Cardíacas/metabolismo , Doenças das Valvas Cardíacas/patologia , Doenças das Valvas Cardíacas/fisiopatologia , Hemodinâmica , Mediadores da Inflamação/metabolismo , Lipídeos/sangue , Masculino , Camundongos Knockout para ApoE , Osteogênese , Fatores de TempoRESUMO
Citrus flavonoids have well-documented protective effects on cardiovascular system, but the poor water solubility and reduced bioavailability restrict their therapeutic use. We aimed to overcome these limitations and encapsulated naringenin and hesperetin into lipid nanoemulsions (LNs), targeted to vascular cell adhesion molecule-1 (VCAM-1), which is expressed on activated endothelial cells (ECs). LNs were characterized by a hydrodynamic size of ~200 nm, negative zeta potential, an encapsulation efficiency of flavonoids higher than 80%, good in vitro stability and steady release of the cargo. The LNs were neither cytotoxic to human ECs line EA.hy926, nor provoked in vitro lysis of murine erithrocytes. Then, we tested whether these nanoformulations reduce tumor necrosis factor-alpha (TNF-α) induced EC-activation. We found that flavonoid-loaded LNs, either non-targeted or targeted to the endothelium, were taken up by the EA.hy926 cells in a dose-dependent manner, but dependent on TNF-α only in the case of endothelium-targeted LNs. Moreover, these nanoparticles inhibited both the adhesion and transmigration of THP-1 monocytes on/through activated ECs, by mechanisms involving a reduced expression of the pro-inflammatory chemokine monocyte chemotactic protein 1 (MCP-1) and diminished nuclear translocation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB).
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The potential human health risks following the exposure to inorganic nanoparticles (NPs) is a very important issue for their application in leather finishing industry. The aim of our study was to investigate the cytotoxic effect of silver (Ag)/titanium dioxide (TiO2) NPs on human cells. Photocatalytic NPs were prepared by electrochemical deposition of Ag on the surface of TiO2 and nitrogen (N)-TiO2 NPs and, subsequently, physico-chemical characterized. Then, a set of experiments have been performed to study the cytotoxicity and cell death mechanisms involved, the changes in cell morphology and the production of ROS induced in human keratinocytes (HaCaT) and human lung epithelial cells (A549) by exposure to NPs. Moreover, the changes in major signaling pathways and the inflammatory response induced by Ag/N-TiO2 NPs in A549 cells were investigated. The data showed that cell death by late apoptosis/necrosis is induced in cells as function of the dose and the type of NPs and is characterized by morphological changes and cytoskeletal disorganization and an increase in reactive oxygen species (ROS) production. The exposure of A549 cells to Ag/N-TiO2 NPs determine the activation of ERK1/2 MAP-kinase pathway and the release of pro-inflammatory mediators CXCL1, GM-CSF and MIF, known to be involved in the recruitment of circulating neutrophils and monocytes.
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Queratinócitos/efeitos dos fármacos , Nanopartículas Metálicas/toxicidade , Mucosa Respiratória/efeitos dos fármacos , Compostos de Prata/toxicidade , Titânio/toxicidade , Células A549 , Apoptose/efeitos dos fármacos , Catálise , Citocinas/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , Mediadores da Inflamação/metabolismo , Queratinócitos/metabolismo , Queratinócitos/patologia , Necrose , Estresse Oxidativo/efeitos dos fármacos , Processos Fotoquímicos , Mucosa Respiratória/metabolismo , Mucosa Respiratória/patologia , Medição de Risco , Transdução de SinaisRESUMO
: The progress in small-interfering RNA (siRNA) therapeutics depends on the development of suitable nanocarriers to perform specific and effective delivery to dysfunctional cells. In this paper, we questioned whether P-selectin, a cell adhesion molecule specifically expressed on the surface of activated endothelial cells (EC) could be employed as a target for nanotherapeutic intervention. To this purpose, we developed and characterized P-selectin targeted PEGylated cationic liposomes able to efficiently pack siRNA and to function as efficient vectors for siRNA delivery to tumour necrosis factor-α (TNF-α) activated EC. Targeted cationic liposomes were obtained by coupling a peptide with high affinity for P-selectin to a functionalized PEGylated phospholipid inserted in the liposomes' bilayer (Psel-lipo). As control, scrambled peptide coupled cationic liposomes (Scr-lipo) were used. The lipoplexes obtained by complexation of Psel-lipo with siRNA (Psel-lipo/siRNA) were taken up specifically and at a higher extent by TNF-α activated b.End3 endothelial cells as compared to non-targeted Scr-lipo/siRNA. The Psel-lipo/siRNA delivered with high efficiency siRNA into the cells. The lipoplexes were functional as demonstrated by the down-regulation of the selected gene (GAPDH). The results demonstrate an effective targeted delivery of siRNA into cultured activated endothelial cells using P-selectin directed PEGylated cationic liposomes, which subsequently knock-down the desired gene.
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BACKGROUND: Lipopolysaccharide (LPS) is widely recognized as a potent activator of monocytes/macrophages, and its effects include an altered production of key mediators, such as inflammatory cytokines and chemokines. The involvement of Gi protein in mediating LPS effects has been demonstrated in murine macrophages and various cell types of human origin. PURPOSE: The aim of the present work was to evaluate the potential of a Gi-protein inhibitor encapsulated in liposomes in reducing the inflammatory effects induced by LPS in monocytes/macrophages. MATERIALS AND METHODS: Guanosine 5'-O-(2-thiodiphosphate) (GOT), a guanosine diphosphate analog that completely inhibits G-protein activation by guanosine triphosphate and its analogs, was encapsulated into liposomes and tested for anti-inflammatory effects in LPS-activated THP1 monocytes or THP1-derived macrophages. The viability of monocytes/macrophages after incubation with different concentrations of free GOT or liposome-encapsulated GOT was assessed by MTT assay. MAPK activation and production of IL1ß, TNFα, IL6, and MCP1 were assessed in LPS-activated monocytes/macrophages in the presence or absence of free or encapsulated GOT. In addition, the effect of free or liposome-encapsulated GOT on LPS-stimulated monocyte adhesion to activated endothelium and on monocyte chemotaxis was evaluated. RESULTS: We report here that GOT-loaded liposomes inhibited activation of MAPK and blocked the production of the cytokines IL1ß, TNFα, IL6, and MCP1 induced by LPS in monocytes and macrophages. Moreover, GOT encapsulated in liposomes reduced monocyte adhesion and chemotaxis. All demonstrated events were in contrast with free GOT, which showed reduced or no effect on monocyte/macrophage activation with LPS. CONCLUSION: This study demonstrates the potential of liposomal GOT in blocking LPS proinflammatory effects in monocytes/macrophages.