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1.
Nat Commun ; 12(1): 658, 2021 01 28.
Artigo em Inglês | MEDLINE | ID: mdl-33510169

RESUMO

A microneedle array is an attractive option for a minimally invasive means to break through the skin barrier for efficient transdermal drug delivery. Here, we report the applications of solid polymer-based ion-conductive porous microneedles (PMN) containing interconnected micropores for improving iontophoresis, which is a technique of enhancing transdermal molecular transport by a direct current through the skin. The PMN modified with a charged hydrogel brings three innovative advantages in iontophoresis at once: (1) lowering the transdermal resistance by low-invasive puncture of the highly resistive stratum corneum, (2) transporting of larger molecules through the interconnected micropores, and (3) generating electroosmotic flow (EOF). In particular, the PMN-generated EOF greatly enhances the transdermal molecular penetration or extraction, similarly to the flow induced by external pressure. The enhanced efficiencies of the EOF-assisted delivery of a model drug (dextran) and of the extraction of glucose are demonstrated using a pig skin sample. Furthermore, the powering of the PMN-based transdermal EOF system by a built-in enzymatic biobattery (fructose / O2 battery) is also demonstrated as a possible totally organic iontophoresis patch.


Assuntos
Sistemas de Liberação de Medicamentos/instrumentação , Sistemas de Liberação de Medicamentos/métodos , Epiderme/metabolismo , Pele/metabolismo , Administração Cutânea , Animais , Dextranos/administração & dosagem , Dextranos/metabolismo , Dextranos/farmacocinética , Eletro-Osmose , Fluoresceína-5-Isotiocianato/administração & dosagem , Fluoresceína-5-Isotiocianato/análogos & derivados , Fluoresceína-5-Isotiocianato/metabolismo , Fluoresceína-5-Isotiocianato/farmacocinética , Glucose/administração & dosagem , Glucose/metabolismo , Glucose/farmacocinética , Humanos , Iontoforese/instrumentação , Iontoforese/métodos , Masculino , Microinjeções , Agulhas , Padrões Moleculares Associados a Patógenos/administração & dosagem , Padrões Moleculares Associados a Patógenos/metabolismo , Padrões Moleculares Associados a Patógenos/farmacocinética , Porosidade , Suínos
2.
Int J Nanomedicine ; 15: 8401-8409, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33149583

RESUMO

Aim: The uptake pathway of liposomes into cells is mainly via endocytosis or membrane fusion; however, the relationship between the uptake pathway and the intracellular pharmacokinetics of the liposome components remains unclear. This study aimed at revealing the relationship by using cationic liposomes having similar physical properties and different uptake pathways. Materials and Methods: We prepared cationic liposomes composed of amino acid-type lipids, K3C14 and K3C16, which have different uptake pathways by a hydration method, and fluorescently modified them by encapsulating FITC-dextran and surface conjugation with Alexa Fluor® 488 (AF488). Then, we investigated their intracellular distribution in HeLa cells over time. Results: The liposomes had similar physical properties and did not cause significant cell mortality after treatment for 180 min. The delivery rate and efficiency of encapsulated FITC-dextran with the fusogenic K3C16 liposomes were 3 and 1.6 times higher, respectively, than with the endocytic K3C14 liposomes. FITC-dextran molecules delivered with K3C16 liposomes were observed throughout the cytosolic space after 10 min, while those delivered with K3C14 liposomes were mainly observed as foci and took 60 min to diffuse into the cytosolic space. K3C14 lipids modified with AF488 were distributed mostly in the cytosolic space. In contrast, fluorescently labeled K3C16 lipids were colocalized with the plasma membrane of 50% of the HeLa cells after 10 min and were gradually internalized intracellularly. Conclusion: Fusogenic K3C16 liposomes internalized into HeLa cells faster than endocytic K3C14 liposomes, and their components differently distributed in the cells.


Assuntos
Endocitose , Espaço Intracelular/metabolismo , Lipídeos/química , Lipossomos/química , Cátions , Morte Celular , Dextranos/metabolismo , Composição de Medicamentos , Sistemas de Liberação de Medicamentos , Fluoresceína-5-Isotiocianato/análogos & derivados , Fluoresceína-5-Isotiocianato/metabolismo , Células HeLa , Humanos
3.
Life Sci ; 254: 117780, 2020 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-32407844

RESUMO

AIMS: In vivo studies suggest a positive influence of fresh frozen plasma (FFP) on endothelial properties and vascular barrier function, leading to improved outcomes in animal sepsis models as well as in major abdominal surgery. However, those effects are incompletely described. It was our aim to evaluate in vitro effects of FFP on endothelial key functions and to identify underlying mechanisms. MATERIALS AND METHODS: Human pulmonary microvascular endothelial cells (HPMECs) were prestimulated with LPS, followed by incubation with FFP. Permeability for FITC-dextran was assessed, and intercellular gap formation was visualized. NF-κB nuclear translocation and expression of pro-inflammatory, pro-adhesion, and leakage-related genes were evaluated, and monocyte adhesion to ECs was assessed. Intracellular cAMP levels as well as phosphorylation of functional proteins were analyzed. In patients undergoing major abdominal surgery, Syndecan-1 serum levels were assessed prior to and following FFP transfusion. KEY FINDINGS: Post-incubation of HPMVECs with FFP increased intracellular cAMP levels that had been decreased by preceding LPS stimulation. On one hand, this reduced endotoxin-mediated upregulation of IL-8, ICAM-1, VCAM-1, VEGF, and ANG-2. Impaired phosphorylation of functional proteins was restored, and intercellular cohesion and barrier function were rescued. On the other hand, NF-κB nuclear translocation as well as monocyte adhesion was markedly increased by the combination of LPS and FFP. Syndecan-1 serum levels were lower in surgery patients that were transfused with FFP compared to those that were not. SIGNIFICANCE: Our data provide evidence for a differential modulation of crucial endothelial properties by FFP, potentially mediated by elevation of intracellular cAMP levels.


Assuntos
Células Endoteliais/fisiologia , Endotélio Vascular/metabolismo , Plasma/fisiologia , Idoso , Adesão Celular/fisiologia , Permeabilidade da Membrana Celular/fisiologia , Células Cultivadas , AMP Cíclico/metabolismo , Dextranos/metabolismo , Fluoresceína-5-Isotiocianato/análogos & derivados , Fluoresceína-5-Isotiocianato/metabolismo , Junções Comunicantes/fisiologia , Humanos , Lipopolissacarídeos , Pessoa de Meia-Idade , Monócitos/fisiologia , NF-kappa B/metabolismo , Fosforilação , Sindecana-1/sangue
4.
Int J Food Microbiol ; 327: 108652, 2020 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-32442778

RESUMO

Food-grade waste and side streams should be strictly kept in food use in order to achieve sustainable food systems. At present, the baking industry creates food-grade waste as excess and deformed products that are mainly utilized for non-food uses, such as bioethanol production. The purpose of this study was therefore to explore the potential of waste wheat bread recycling for fresh wheat bread production. Waste bread recycling was assessed without further processing or after tailored fermentation with lactic acid bacteria producing either dextran or ß-glucan exopolysaccharides. When non-treated waste bread slurry was added to new bread dough, bread quality (specific volume and softness) decreased with increasing content of waste bread addition. In situ EPS-production (dextran and microbial ß-glucan) significantly increased waste bread slurry viscosity and yielded residual fructose or glucose that could effectively replace the sugar added for yeast leavening. Furthermore, fermentation acidified waste bread matrix, thus improving the hygienic safety of the process. Bread containing dextran synthesized in situ by Weissella confusa A16 showed good technological quality. The produced dextran compensated the adverse effect of recycled bread on new bread quality attributes by 12% increase in bread specific volume and 37% decrease in crumb hardness. In this study, a positive technological outcome of the bread containing microbial ß-glucan was not detected. The waste bread fermented by W. confusa A16 containing dextran appears to enable safe bread recycling with low acidity and minimal quality loss.


Assuntos
Pão/microbiologia , Pão/normas , Fermentação , Indústria Alimentícia , Resíduos Industriais , Reciclagem/métodos , Triticum/microbiologia , Dextranos/metabolismo , Ácido Láctico/metabolismo , Weissella/metabolismo , Leveduras/metabolismo , beta-Glucanas/metabolismo
5.
Biochem Biophys Res Commun ; 526(4): 1077-1084, 2020 06 11.
Artigo em Inglês | MEDLINE | ID: mdl-32312522

RESUMO

Bilobalide, one of the key bioactive components of Ginkgo biloba leaves, exerts prominent neuroprotective properties in central nervous system (CNS) disease. However, the effect of bilobalide on blood-brain barrier (BBB) permeability remains unknown. In this study, we investigated the effect of bilobalide on BBB permeability and its potential mechanism involved. Both the in vitro and in vivo results showed that significant enhancement of BBB permeability was found following bilobalide treatment, evidenced by the reduced transendothelial electrical resistance (TEER), the increased fluorescein sodium (Na-F) penetration rate in vitro and the leakage of FITC-dextran in vivo. Transmission electron microscope (TEM) images demonstrated that bilobalide modulated BBB permeability by changing the ultrastructure of tight junctions (TJs). In addition, actin-binding proteins ezrin, radixin and moesin (ERM) and Myosin light chain (MLC) phosphorylation was observed following bilobalide treatment. Moreover, the effect of bilobalide on TEER reduction and ERM/MLC phosphorylation was counteracted by adenosine A1 receptor (A1R) siRNA. The current findings suggested that bilobalide might reversibly modulate BBB permeability by the alteration of TJs ultrastructure through A1R-mediated phosphorylation of actin-binding proteins.


Assuntos
Bilobalídeos/farmacologia , Barreira Hematoencefálica/metabolismo , Proteínas dos Microfilamentos/metabolismo , Receptor A1 de Adenosina/metabolismo , Animais , Barreira Hematoencefálica/efeitos dos fármacos , Dextranos/metabolismo , Fluoresceína-5-Isotiocianato/análogos & derivados , Fluoresceína-5-Isotiocianato/metabolismo , Humanos , Masculino , Camundongos , Peso Molecular , Permeabilidade/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Proteínas de Junções Íntimas/metabolismo
6.
Nat Commun ; 11(1): 1809, 2020 04 14.
Artigo em Inglês | MEDLINE | ID: mdl-32286269

RESUMO

Small interfering RNAs (siRNAs) are a new class of promising therapeutic molecules that can be used for sequence-specific downregulation of disease-causing genes. However, endosomal entrapment of siRNA is a key hurdle for most delivery strategies, limiting the therapeutic effect. Here, we use live-cell microscopy and cytosolic galectin-9 as a sensor of membrane damage, to probe fundamental properties of endosomal escape of cholesterol-conjugated siRNA induced by endosome-disrupting compounds. We demonstrate efficient release of ligand-conjugated siRNA from vesicles damaged by small molecules, enhancing target knockdown up to ∼47-fold in tumor cells. Still, mismatch between siRNA-containing and drug-targeted endolysosomal compartments limits siRNA activity improvement. We also show widespread endosomal damage in macroscopic tumor spheroids after small molecule treatment, substantially improving siRNA delivery and knockdown throughout the spheroid. We believe the strategy to characterize endosomal escape presented here will be widely applicable, facilitating efforts to improve delivery of siRNA and other nucleic acid-based therapeutics.


Assuntos
Endossomos/metabolismo , Imageamento Tridimensional , RNA Interferente Pequeno/metabolismo , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Colesterol/metabolismo , Citosol/efeitos dos fármacos , Citosol/metabolismo , Dextranos/metabolismo , Endossomos/efeitos dos fármacos , Galectinas/metabolismo , Técnicas de Silenciamento de Genes , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Células MCF-7 , Bibliotecas de Moléculas Pequenas/farmacologia , Esferoides Celulares/metabolismo , Esferoides Celulares/patologia
7.
Biol Pharm Bull ; 43(2): 319-324, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32009118

RESUMO

We examined the influence of liver disease on the absorption from the liver surface of fluorescein isothiocyanate (FITC)-dextran 10 (FD-10, MW: 11000) and several marker compounds with different molecular weights. The purpose of this study was to determine the feasibility of liver surface application of macromolecular compounds in the disease state. We used male Wistar rats treated with carbon tetrachloride (CCl4) or D-galactosamine (GAL). FD-10 and other marker compounds were applied to the liver surface using a cylindrical diffusion cell in liver-intoxicated rats. The blood, bile, urine, and the remaining solution in the diffusion cell were collected for assay. FD-10 was absorbed by first-order kinetics from the liver surface in the liver-intoxicated rat models. The calculated rate constant ka values in the normal, CCl4 and GAL groups were 0.000965, 0.00125 and 0.00104 min-1, respectively. Increased absorption of FITC-dextrans in the liver-intoxicated rats was observed. In both CCl4 and GAL groups, an inverse relationship was observed between the molecular weight and ka from the rat liver surface of the marker compounds. The limits of the molecular weight absorbed from the liver surface were extrapolated to be 71200, 135000, and 105000 in the normal, CCl4, and GAL groups, respectively. In conclusion, increased absorbability from the rat liver surface indicates that liver surface application for liver targeting of macromolecules in the diseased state is indeed feasible. Therefore, our findings can support further research on liver surface application of drugs under liver disease.


Assuntos
Tetracloreto de Carbono/toxicidade , Dextranos/metabolismo , Fluoresceína-5-Isotiocianato/análogos & derivados , Galactosamina/toxicidade , Fígado/efeitos dos fármacos , Fígado/metabolismo , Animais , Dextranos/sangue , Fluoresceína-5-Isotiocianato/metabolismo , Masculino , Ratos , Ratos Wistar
8.
Biochem Biophys Res Commun ; 523(3): 651-657, 2020 03 12.
Artigo em Inglês | MEDLINE | ID: mdl-31948759

RESUMO

Non-digestible oligosaccharides have wide food industrial applications as dietary fibers and prebiotics. The aim of this study is to realize the effective biosynthesis of isomalto-oligosaccharides (IMOs) and reduce the production of by-product dextran. In the presence of acceptors improved the dextransucrase reaction shifting to oligosaccharides formation but a number of by-products dextran appeared. Maltose acceptor performed stronger inhibition behaviors in dextran synthesis than lactose and glucose acceptor due to its higher efficiencies. Acceptors had no influence on the structure of by-product dextran which mainly composed of α-(1,6)-glycosidic linkages and low α-(1,3)-glycosidic branch. In addition, the Mw and contents of IMOs and oligodextrans synthesized by dual-enzyme were hard to control. Addition of maltose acceptor in the dual-enzyme reaction, the adequate dextranase preferentially degraded dextran than the acceptor products to yield the IMOs. Results indicated that the combined use of the dual-enzyme and the maltose acceptor is a simple and effective method to promote the high-quality of functional IMOs.


Assuntos
Dextranase/metabolismo , Glucosiltransferases/metabolismo , Leuconostoc mesenteroides/enzimologia , Maltose/metabolismo , Oligossacarídeos/metabolismo , Dextranos/química , Dextranos/metabolismo , Hidrólise , Leuconostoc mesenteroides/química , Leuconostoc mesenteroides/metabolismo , Oligossacarídeos/química , Especificidade por Substrato
9.
Biochem Biophys Res Commun ; 523(3): 573-579, 2020 03 12.
Artigo em Inglês | MEDLINE | ID: mdl-31932036

RESUMO

The applications of dextran depend not only on the molecular weight but also on the types and number of branches. In this study, dextran generated from Leuconostoc mesenteroides (L.M.CICC-20724) was characterized by fourier-transform infrared spectrum and nuclear magnetic resonance spectroscopy. Our analyses showed that dextran was a polysaccharide composed of d-glucose units with predominantly α(1 â†’ 6) linkages in the main chain and few α(1 â†’ 3) linkages in the branch. Periodate oxidation, a classic chemical method, was usually combined with Smith degradation and gas chromatography to analyze glycosidic linkages in polysaccharide quantitatively. In this study, we calculated the exact straight-chain/branched-chain ratio in the dextran using periodate oxidation only. The ratios obtained by periodate oxidation only were compared to the ratios obtained by nuclear magnetic resonance. The results showed that the ratios of the two groups were nearly equal, and the average relative error between the two groups was 0.83%. This method was evaluated and found to be accurate and stable. This technique provided a convenient and straightforward chemical method for the quantitative analysis of the straight-chains and branched-chains in polysaccharides which had a similar structure. The ratios during the enzymatic synthesis process of dextran were analyzed by this method and were found to be stable with a high level of approximately 95% on average.


Assuntos
Dextranos/química , Leuconostoc mesenteroides/química , Biocatálise , Configuração de Carboidratos , Dextranos/metabolismo , Leuconostoc mesenteroides/metabolismo , Oxirredução , Ácido Periódico/química , Espectroscopia de Prótons por Ressonância Magnética , Espectroscopia de Infravermelho com Transformada de Fourier
10.
Carbohydr Polym ; 232: 115802, 2020 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-31952601

RESUMO

A series of biocompatible and non- toxic polysaccharide molecules have been successfully fabricated and explored their potential application for scavenging the carbonyl species in vitro. These macromolecules were dextrans with different hydrazide substitution ratios determined by TNBS assay, NMR and FTIR characterization. The colorimetric assay had demonstrated that these macromolecules could effectively scavenge acrolein, oxidized bovine serum albumin (BSA) in buffer solutions as well as carbonyl proteins from serum. The scavengers could achieve twice more scavenging effects for modified dextrans with high molecular weight (Mw = 100,000) than those of low ones (Mw = 40,000) with the same substitution ratio. Protein gel electrophoresis confirmed that the formation of the complex between carbonyls and modified dextrans resulted in appearance of slower bands. It also revealed that such macromolecules could protect cultured cells against the toxicity of acrolein or its derivatives. The proposed macromolecules indicated a very promising capability as scavengers for oxidative stress plus its derivatives without side effects.


Assuntos
Dextranos/metabolismo , Depuradores de Radicais Livres/metabolismo , Hidrazinas/metabolismo , Soroalbumina Bovina/metabolismo , Animais , Bovinos , Dextranos/química , Depuradores de Radicais Livres/química , Hidrazinas/química , Estrutura Molecular , Carbonilação Proteica , Soroalbumina Bovina/química
11.
J Dairy Sci ; 103(2): 1141-1150, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31785876

RESUMO

Food protein allergies are a major global concern. Hydrolysis of food proteins reduces their allergenicity, but another novel approach is the covalent attachment of polysaccharides to proteins via the Maillard reaction (i.e., glycation), which blocks some IgE binding epitopes on the protein allergen. We wanted to examine whether enzymatic hydrolysis, combined with glycation, could further reduce IgE binding for people with a cow milk protein allergy. Whey protein isolate (WPI) was hydrolyzed by immobilized trypsin and chymotrypsin to degree of hydrolysis (DH) values of 17 to 27%. Immobilized enzymes were used to avoid heat-treating the hydrolysate (to inactivate the enzymes, because heating could also affect the IgE binding ability of the protein). The resultant whey protein isolate hydrolysates (WPIH) were then glycated with 10-kDa dextran (DX) in aqueous solutions held at 62°C for 24 h. We analyzed the molar mass (MW) of WPIH samples and their corresponding glycates (WPIH-DX) using size-exclusion chromatography with multi-angle laser light scattering. We obtained blood sera from 8 patients who had been diagnosed with a cow milk protein allergy, and we used a composite serum for IgE binding analysis. The average MW values of samples WPIH-1 to WPIH-3 decreased from 11.15, 9.46, and 7.57 kDa with increasing DH values of 18.7, 22.5, and 27.1%. Glycation significantly reduced the high bitterness of the WPIH samples, as assessed by a trained sensory panel. The WPIH-DX glycates had significantly reduced WPI-specific IgE binding capacity compared to WPI or unglycated WPIH; we found an almost 99% reduction in IgE binding for the WPIH-DX glycate made from WPIH with a DH value of 27.1%. Hydrolysis of WPI followed by glycation with DX via the Maillard reaction significantly decreased the allergenicity of whey proteins.


Assuntos
Dextranos/metabolismo , Hipersensibilidade Alimentar/imunologia , Imunoglobulina E/sangue , Hipersensibilidade a Leite/imunologia , Proteínas do Soro do Leite/metabolismo , Alérgenos/imunologia , Alérgenos/metabolismo , Animais , Bovinos , Criança , Quimotripsina/metabolismo , Epitopos/metabolismo , Feminino , Hipersensibilidade Alimentar/sangue , Glicosilação , Humanos , Hidrólise , Reação de Maillard , Hidrolisados de Proteína/metabolismo , Tripsina/metabolismo , Proteínas do Soro do Leite/imunologia
12.
Int J Pharm ; 576: 118987, 2020 Feb 25.
Artigo em Inglês | MEDLINE | ID: mdl-31870961

RESUMO

A novel Liposome Aggregate Platform (LAP) system for prolonged retention of drugs in the posterior eye segment after intravitreal injection (IVT) was developed and evaluated. Calcein, FITC-dextran-4000 (FD4) and Flurbiprofen (FLB), were encapsulated in negatively charged liposomes, and mixed with protamine to produce the LAP. The lipid/protamine ratio was fixed, in order to have a convenient aggregation rate permitting IVT injection and also a sustained release of liposome-entrapped molecules (in vitro) from LAP. In vitro release studies confirmed the potential of LAP system consisted of HPC/DPPG/Chol liposomes and protamine (at 1:1 w/w to lipid), to delay calcein, FD4 and FLB release, compared to free liposomes. In vivo studies demonstrated increased vitreous retention of liposomes and LAP for all molecules, compared to the corresponding solutions; however the retention of FD4 is similar for non-aggregated liposomes and LAP, and calcein retention is only slightly increased by LAP compared to liposomes. The later result may be connected with the visible ocular inflammation caused by both dyes; interestingly inflammation was moderately reduced when dyes were entrapped in liposomes and even more when in LAP. No visible inflammation was demonstrated for FLB, and the LAP system significantly increased the retention of FLB in the ocular tissues (compared to non-aggregated liposomes). Taking into account the capability of the novel LAP system to decrease inflammatory reactions towards calcein and FD4, and prolong the retention of FLB in ocular tissues, it is concluded that such systems, after further optimization, may be considered as promising effective and safe approaches for treatment of posterior segment ocular pathologies.


Assuntos
Anti-Inflamatórios não Esteroides/administração & dosagem , Flurbiprofeno/administração & dosagem , Lipídeos/química , Lipossomos , Segmento Posterior do Olho/metabolismo , Protaminas/química , Animais , Anti-Inflamatórios não Esteroides/química , Anti-Inflamatórios não Esteroides/farmacocinética , Preparações de Ação Retardada , Dextranos/administração & dosagem , Dextranos/química , Dextranos/metabolismo , Composição de Medicamentos , Liberação Controlada de Fármacos , Fluoresceína-5-Isotiocianato/administração & dosagem , Fluoresceína-5-Isotiocianato/análogos & derivados , Fluoresceína-5-Isotiocianato/química , Fluoresceína-5-Isotiocianato/metabolismo , Fluoresceínas/administração & dosagem , Fluoresceínas/química , Fluoresceínas/metabolismo , Flurbiprofeno/química , Flurbiprofeno/farmacocinética , Injeções Intravítreas , Modelos Biológicos , Coelhos , Distribuição Tecidual
13.
Theranostics ; 9(24): 7447-7457, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31695779

RESUMO

Human serum albumin (HSA) is the most abundant plasma protein. The main reason for using HSA as a versatile tool for drug delivery is based on its ability to accumulate in tumors. However, the mechanism of albumin accumulation in tumors is not yet clear. Many researchers using HSA as a drug-carrier have focused on the passive tumor targeting by enhanced permeability and retention (EPR) effect, while other investigators proposed that albumin binding proteins mediate albumin accumulation in tumors. We investigated whether HSA accumulation in tumors is mediated by the EPR effect or by secreted protein acidic and rich in cysteine (SPARC), which is known to be an albumin-binding protein. Methods: To investigate the role of SPARC on HSA accumulation in tumors, we compared HSA uptake in U87MG glioblastoma cells with different SPARC expression. U87MG cells generally express high levels of SPARC and were, therefore, used as SPARC-rich cells. SPARC-less U87MG (U87MG-shSPARC) cells were established by viral-shSPARC transduction. We detected cellular uptake of fluorescence-labeled HSA by confocal microscopy in U87MG and U87MG-shSPARC cells. To demonstrate the mechanism of HSA accumulation in tumors, we injected FNR648-labeled HSA and FITC-labeled dextran in U87MG and U87MG-shSPARC tumor-bearing mice and observed their micro-distribution in tumor tissues. Results: HSA was internalized in cells by binding with SPARC in vitro. HSA accumulation in U87MG glioma was associated with SPARC expression in vivo. FITC-dextran was distributed in U87MG tumors in the vicinity of blood vessels. The distribution of HSA, on the other hand, was observed in the regions remote from blood vessels of U87MG tumor tissues but not in U87MG-shSPARC tumor tissues. Conclusion: Our results demonstrate that the tumor-distribution of HSA is affected not only by the EPR-effect but also by SPARC expression. SPARC enhances HSA accumulation in U87MG glioma and mediates active targeting of HSA in tumors.


Assuntos
Osteonectina/metabolismo , Albumina Sérica Humana/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto , Animais , Neoplasias Encefálicas/metabolismo , Linhagem Celular Tumoral , Dextranos/metabolismo , Fluoresceína-5-Isotiocianato/análogos & derivados , Fluoresceína-5-Isotiocianato/metabolismo , Glioma/metabolismo , Humanos , Masculino , Camundongos Endogâmicos BALB C , Camundongos Nus , Ligação Proteica , Distribuição Tecidual
14.
Sci Rep ; 9(1): 16796, 2019 11 14.
Artigo em Inglês | MEDLINE | ID: mdl-31727909

RESUMO

Non-steroidal anti-inflammatory drugs (NSAIDs) cause damage in the small intestine in a bacteria-dependent manner. As high-fat diet (HFD) is a potent inducer of gut dysbiosis, we investigated the effects of HFD on bacterial flora in the small intestine and NSAID-induced enteropathy. 16S rRNA gene analysis revealed that the population of Bifidobacterium spp. significantly decreased by fold change of individual operational taxonomic units in the small intestine of mice fed HFD for 8 weeks. HFD increased intestinal permeability, as indicated by fluorescein isothiocyanate-dextran absorption and serum lipopolysaccharide levels, accompanied by a decrease in the protein expressions of ZO-1 and occludin and elevated mRNA expression of interleukin (IL)-17A in the small intestine. HFD-fed mice exhibited increased susceptibility to indomethacin-induced damage in the small intestine; this phenotype was observed in normal diet-fed mice that received small intestinal microbiota from HFD-fed mice. Administration of neutralizing antibodies against IL-17A to HFD-fed mice reduced intestinal permeability and prevented exacerbation of indomethacin-induced damage. Thus, HFD-induced microbial dysbiosis in small intestine caused microinflammation through the induction of IL-17A and increase in intestinal permeability, resulting in the aggravation of NSAID-induced small intestinal damage.


Assuntos
Anti-Inflamatórios não Esteroides/efeitos adversos , Dieta Hiperlipídica/efeitos adversos , Disbiose/microbiologia , Interleucina-17/genética , Intestino Delgado/microbiologia , RNA Ribossômico 16S/genética , Animais , Bifidobacterium/classificação , Bifidobacterium/efeitos dos fármacos , Bifidobacterium/isolamento & purificação , Dextranos/metabolismo , Modelos Animais de Doenças , Disbiose/sangue , Disbiose/induzido quimicamente , Fluoresceína-5-Isotiocianato/análogos & derivados , Fluoresceína-5-Isotiocianato/metabolismo , Indometacina/efeitos adversos , Intestino Delgado/efeitos dos fármacos , Intestino Delgado/imunologia , Lipopolissacarídeos/sangue , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Permeabilidade/efeitos dos fármacos , Análise de Sequência de DNA , Regulação para Cima
15.
Molecules ; 24(20)2019 Oct 17.
Artigo em Inglês | MEDLINE | ID: mdl-31627389

RESUMO

The cellular transport process of DNA is hampered by cell membrane barriers, and hence, a delivery vehicle is essential for realizing the potential benefits of gene therapy to combat a variety of genetic diseases. Virus-based vehicles are effective, although immunogenicity, toxicity and cancer formation are among the major limitations of this approach. Cationic polymers, such as polyethyleneimine are capable of condensing DNA to nanoparticles and facilitate gene delivery. Lack of biodegradation of polymeric gene delivery vehicles poses significant toxicity because of the accumulation of polymers in the tissue. Many attempts have been made to develop biodegradable polymers for gene delivery by modifying existing polymers and/or using natural biodegradable polymers. This review summarizes mechanistic aspects of gene delivery and the development of biodegradable polymers for gene delivery.


Assuntos
Quitosana/metabolismo , Técnicas de Transferência de Genes/classificação , Nanopartículas/metabolismo , Polietilenoimina/metabolismo , Polilisina/metabolismo , Animais , Transporte Biológico , Quitosana/química , Dextranos/química , Dextranos/metabolismo , Endossomos/metabolismo , Terapia Genética/métodos , Glucanos/química , Glucanos/metabolismo , Humanos , Ácido Hialurônico/química , Ácido Hialurônico/metabolismo , Hidrólise , Lisossomos/metabolismo , Nanopartículas/química , Polietilenoimina/química , Polilisina/química
16.
J Agric Food Chem ; 67(46): 12806-12815, 2019 Nov 20.
Artigo em Inglês | MEDLINE | ID: mdl-31650841

RESUMO

Lactic acid bacteria are known to produce extracellular polysaccharides such as α-glucan, levan, and inulin, which are promising for applications in food systems because of their prebiotic properties. In this work, a novel glucansucrase (GS) Gtf-DSM from Lactobacillus ingluviei DSM 14792 was recombinantly expressed and biochemically characterized as a dextransucrase capable of producing a dextran polysaccharide with four types of linkages, including 69% (α1 → 6), 24% (α1 → 3), 6% (α1 → 4), and 1% (α1 → 2). Intriguingly, the dextransucrase Gtf-DSM had a sequence identity of 99.3% with the reuteransucrase GtfO producing a reuteran with 21% (α1 → 6) and 79% (α1 → 4) linkages. Thus, the dextransucrase Gtf-DSM is a unique target for understanding the linkage specificity of GSs and the investigation of site-directed mutagenesis using Gtf-DSM and GtfO as templates is underway.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Glucosiltransferases/química , Glucosiltransferases/metabolismo , Lactobacillus/enzimologia , Proteínas de Bactérias/genética , Dextranos/química , Dextranos/metabolismo , Glucosiltransferases/genética , Glicosiltransferases/química , Glicosiltransferases/genética , Glicosiltransferases/metabolismo , Lactobacillus/química , Lactobacillus/genética , Lactobacillus/metabolismo , Mutagênese Sítio-Dirigida , Especificidade por Substrato
17.
Pharm Res ; 36(12): 172, 2019 Oct 28.
Artigo em Inglês | MEDLINE | ID: mdl-31659456

RESUMO

PURPOSE: Caco-2 monolayers are the most common model of the intestinal epithelium and are critical to the development of oral drug delivery strategies and gastrointestinal disease treatments. However, current monolayer systems are cost- and/or time-intensive, hampering progress. This study evaluates two separate methods to reduce resource input: FB Essence as a fetal bovine serum (FBS) alternative and a new, 3-day Caco-2 system deemed "thrifty, rapid intestinal monolayers" (TRIM). METHODS: Caco-2 cells were cultured with FB Essence and compared to cells in 10% FBS for proliferation and monolayer formation. TRIM were compared to commonly-used 21-day and Corning® HTS monolayer systems, as well as mouse intestines, for permeability behavior, epithelial gene expression, and tight junction arrangement. RESULTS: No amount of FB Essence maintained Caco-2 cells beyond 10 passages. In contrast, TRIM compared favorably in permeability and gene expression to intestinal tissues. Furthermore, TRIM cost $109 and required 1.3 h of time per 24-well plate, compared to $164 and 3.7 h for 21-day monolayers, and $340 plus 1.0 h for the HTS system. CONCLUSIONS: TRIM offer a new approach to generating Caco-2 monolayers that resemble the intestinal epithelium. They are anticipated to accelerate the pace of in vitro intestinal experiments while easing financial burden.


Assuntos
Mucosa Intestinal/metabolismo , Administração Oral , Animais , Células CACO-2 , Proliferação de Células , Células Cultivadas , Colágeno/química , Dextranos/metabolismo , Liberação Controlada de Fármacos , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Permeabilidade , Propriedades de Superfície , Junções Íntimas/metabolismo
18.
Parasit Vectors ; 12(1): 486, 2019 Oct 16.
Artigo em Inglês | MEDLINE | ID: mdl-31619276

RESUMO

BACKGROUND: In the animal production sector, enteritis is responsible for serious economic losses, and intestinal parasitism is a major stress factor leading to malnutrition and lowered performance and animal production efficiency. The effect of enteric parasites on the gut function of teleost fish, which represent the most ancient bony vertebrates, is far from being understood. The intestinal myxozoan parasite Enteromyxum leei dwells between gut epithelial cells and causes severe enteritis in gilthead sea bream (Sparus aurata), anorexia, cachexia, growth impairment, reduced marketability and increased mortality. METHODS: This study aimed to outline the gut failure in this fish-parasite model using a multifaceted approach and to find and validate non-lethal serum markers of gut barrier dysfunction. Intestinal integrity was studied in parasitized and non-parasitized fish by immunohistochemistry with specific markers for cellular adhesion (E-cadherin) and tight junctions (Tjp1 and Cldn3) and by functional studies of permeability (oral administration of FITC-dextran) and electrophysiology (Ussing chambers). Serum samples from parasitized and non-parasitized fish were analyzed using non-targeted metabolomics and some significantly altered metabolites were selected to be validated using commercial kits. RESULTS: The immunodetection of Tjp1 and Cldn3 was significantly lower in the intestine of parasitized fish, while no strong differences were found in E-cadherin. Parasitized fish showed a significant increase in paracellular uptake measured by FITC-dextran detection in serum. Electrophysiology showed a decrease in transepithelial resistance in infected animals, which showed a diarrheic profile. Serum metabolomics revealed 3702 ions, from which the differential expression of 20 identified compounds significantly separated control from infected groups in multivariate analyses. Of these compounds, serum inosine (decreased) and creatine (increased) were identified as relevant and validated with commercial kits. CONCLUSIONS: The results demonstrate the disruption of tight junctions and the loss of gut barrier function, a metabolomic profile of absorption dysfunction and anorexia, which further outline the pathophysiological effects of E. leei.


Assuntos
Enterite/veterinária , Doenças dos Peixes/parasitologia , Metabolômica , Myxozoa/patogenicidade , Doenças Parasitárias em Animais/parasitologia , Dourada/parasitologia , Animais , Caderinas/metabolismo , Claudina-3/metabolismo , Creatina/sangue , Dextranos/metabolismo , Modelos Animais de Doenças , Eletrofisiologia , Enterite/parasitologia , Ensaio de Imunoadsorção Enzimática , Fluoresceína-5-Isotiocianato/análogos & derivados , Fluoresceína-5-Isotiocianato/metabolismo , Imuno-Histoquímica , Inosina/sangue , Mucosa Intestinal/metabolismo , Intestinos/parasitologia , Intestinos/patologia , Doenças Parasitárias em Animais/patologia , Permeabilidade , Proteína da Zônula de Oclusão-1/metabolismo
19.
Enzyme Microb Technol ; 131: 109432, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31615671

RESUMO

The potential anticancer activity of arginine deiminase (ADI) via deimination of l-arginine into citrulline has been extensively verified against various arginine-auxotrophic tumors, however, the higher antigenicity, structural instability and in vivo proteolysis are the major challenges that limit this enzyme from further clinical implementation. Since, this clinically applied enzyme was derived from Mycobacterium spp, thus, searching for ADI from eukaryotic microbes "especially thermophilic fungi" could have a novel biochemical, conformational and catalytic properties. Aspergillus nidulans ADI was purified with 5.3 folds, with molecular subunit structure 48 kDa and entire molecular mass 120 kDa, ensuring its homotrimeric identity. The peptide fingerprinting analysis revealing the domain Glu95-Gly96-Gly97 as the conserved active site of A. nidulans ADI, with higher proximity to Mycobacterium ADI clade IV. In an endeavor to fortify the structural stability and anticancer activity of A. nidulans ADI, the enzyme was chemically modified with dextran. The optimal activity of Dextran-ADI conjugates was determined at 0.08:20 M ratio of ADI: Dextran, with an overall increase to ADI molecular subunit mass to ˜100 kDa. ADI was conjugated with dextran via the ε-amino groups interaction of surface lysine residues of ADI. The resistance of Dextran-ADI conjugate to proteolysis had been increased by 2.5 folds to proteinase K and trypsin, suggesting the shielding of >50% of ADI surface proteolytic recognition sites. The native and Dextran-ADI conjugates have the same optimum reaction temperature (37 °C), reaction pH and pH stability (7.0-8.0) with dependency on K+ ions as a cofactor. Dextran-ADI conjugates exhibited a higher thermal stability by ˜ 2 folds for all the tested temperatures, ensuring the acquired structural and catalytic stability upon dextran conjugation. Dextran conjugation slightly protect the reactive amino and thiols groups of surface amino acids of ADI from amino acids suicide inhibitors. The affinity of ADI was increased by 5.3 folds to free L-arginine with a dramatic reduction in citrullination of peptidylarginine residues upon dextran conjugation. The anticancer activity of ADI to breast (MCF-7), liver (HepG-2) and colon (HCT8, HT29, DLD1 and LS174 T) cancer cell lines was increased by 1.7 folds with dextran conjugation in vitro. Pharmacokinetically, the half-life time of ADI was increased by 1.7 folds upon dextran conjugation, in vivo. From the biochemical and hematological parameters, ADIs had no signs of toxicity to the experimental animals. In addition to the dramatic reduction of L-arginine in serum, citrulline level was increased by 2.5 folds upon dextran conjugation of ADI. This is first report exploring thermostable ADI from thermophilic A. nidulans with robust structural stability, catalytic efficiency and proteolytic resistance.


Assuntos
Antineoplásicos/química , Antineoplásicos/metabolismo , Aspergillus nidulans/enzimologia , Dextranos/metabolismo , Estabilidade Enzimática , Hidrolases/química , Hidrolases/metabolismo , Animais , Antineoplásicos/farmacocinética , Antineoplásicos/farmacologia , Arginina/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Citrulina/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Hidrolases/farmacocinética , Hidrolases/farmacologia , Cinética , Camundongos , Peso Molecular , Multimerização Proteica , Proteólise , Temperatura
20.
Immunobiology ; 224(6): 758-764, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31522781

RESUMO

PURPOSE: This study aimed to investigate the mechanism of PACAP38 on house dust mite (HDM)-induced asthmatic airway epithelial barrier destruction. METHODS: The HDM-induced asthma mice model and 16HBE cell model was established respectively. The enzyme linked immunosorbent assay (ELSIA), cell count and immunohistochemical assay were performed on mice in control group, HDM group and PACAP38 + HDM group.The cAMP/PKA activity, p-CREB and total CREB expression, TEER and the FITC-DX were investigated on cells in control-16HBE group, HDM-16HBE group and PACAP38 + HDM-16HBE group. RESULTS: The levels of IL-4 and IL-5 in the HDM group were significantly higher than those in the control group (P < 0.05), while the above indexes in the PACAP38 + HDM group were lower than those in the HDM group (P < 0.05). E-cadherin, ß-catenin, ZO-1 and occludin in the control group were highly immunoreactive in airway epithelial cells, whereas connexin staining was attenuated after HDM induction. The TEER level, cAMP levels and PKA activity were decreased, while FITC-DX transmittance was increased in HDM-16HBE group (P < 0.05) compared with the control-16HBE group. CONCLUSION: PACAP38 could reduce the airway inflammation, weaken the AJC protein heterotopia and activate cAMP/PKA signaling pathway in HDM-induced asthma, which indicate that PACAP38 may be an important contributor in HDM-induced asthma.


Assuntos
Alérgenos/imunologia , Antígenos de Dermatophagoides/imunologia , Asma/imunologia , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/metabolismo , Pyroglyphidae/imunologia , Mucosa Respiratória/imunologia , Animais , Asma/sangue , Asma/metabolismo , Asma/patologia , Líquido da Lavagem Broncoalveolar/citologia , Linhagem Celular , AMP Cíclico/metabolismo , Dextranos/metabolismo , Humanos , Imunoglobulina E/sangue , Interleucina-4/imunologia , Interleucina-5/imunologia , Pulmão/imunologia , Pulmão/metabolismo , Pulmão/patologia , Masculino , Proteínas de Membrana/metabolismo , Camundongos Endogâmicos BALB C , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/imunologia , Mucosa Respiratória/metabolismo , Mucosa Respiratória/patologia
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