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Drug loaded dendritic core-multishell (CMS) nanocarriers are of especial interest for the treatment of skin diseases, owing to their striking dermal delivery efficiencies following topical applications. CMS nanocarriers are composed of a polyglycerol core, connected by amide-bonds to an inner alkyl shell and an outer methoxy poly(ethylene glycol) shell. Since topically applied nanocarriers are subjected to biodegradation, the application of conventional amide-based CMS nanocarriers (10-A-18-350) has been limited by the potential production of toxic polyglycerol amines. To circumvent this issue, three tailored ester-based CMS nanocarriers (10-E-12-350, 10-E-15-350, 10-E-18-350) of varying inner alkyl chain length were synthesized and comprehensively characterized in terms of particle size, drug loading, biodegradation and dermal drug delivery efficiency. Dexamethasone (DXM), a potent drug widely used for the treatment of inflammatory skin diseases, was chosen as a therapeutically relevant test compound for the present study. Ester-and amide-based CMS nanocarriers delivered DXM more efficiently into human skin than a commercially available DXM cream. Subsequent in vitro and in vivo toxicity studies identified CMS (10-E-15-350) as the most biocompatible carrier system. The anti-inflammatory potency of DXM-loaded CMS (10-E-15-350) nanocarriers was assessed in TNF alpha supplemented skin models, where a significant reduction of the pro-inflammatory cytokine IL-8 was seen, with markedly greater efficacy than commercial DXM cream. In summary, we report the rational design and characterization of tailored, biodegradable, ester-based CMS nanocarriers, and their subsequent stepwise screening for biocompatibility, dermal delivery efficiency and therapeutic efficacy in a top-down approach yielding the best carrier system for topical applications. (C) 2016 Elsevier B.V. All rights reserved.
Engineered nanogels are of high value for a targeted and controlled transport of compounds due to the ability to change their chemical properties by external stimuli. As it has been indicated that nanogels possess a high ability to penetrate the stratum corneum, it cannot be excluded that nanogels interact with dermal dendritic cells, especially in diseased skin. In this study the potential crosstalk of the thermore-sponsive nanogels (tNGs) with the dendritic cells of the skin was investigated with the aim to determine the immunotoxicological properties of the nanogels. The investigated tNGs were made of dendritic polyglycerol (dPG) and poly(glycidyl methyl ether-co-ethyl glycidyl ether) (p(GME-co-EGE)), as polymer conferring thermoresponsive properties. Although the tNGs were taken up, they displayed neither cytotoxic and genotoxic effects nor any induction of reactive oxygen species in the tested cells. Interestingly, specific uptake mechanisms of the tNGs by the dendritic cells were depending on the nanogels cloud point temperature (Tcp), which determines the phase transition of the nanoparticle. The study points to caveolae-mediated endocytosis as being the major tNGs uptake mechanism at 37 degrees C, which is above the Tcp of the tNGs. Remarkably, an additional uptake mechanism, beside caveolae-mediated endocytosis, was observed at 29 degrees C, which is the Tcp of the tNGs. At this temperature, which is characterized by two different states of the tNGs, macropinocytosis was involved as well. In summary, our study highlights the impact of thermoresponsivity on the cellular uptake mechanisms which has to be taken into account if the tNGs are used as a drug delivery system.
Background/Aims: Preterm birth (PTB) and low birth weight (LBW) significantly influence mortality and morbidity of the offspring in early life and also have long-term consequences in later life. A better understanding of the molecular mechanisms of preterm birth could provide new insights regarding putative preventive strategies. Metabolomics provides a powerful analytic tool to readout complex interactions between genetics, environment and health and may serve to identify relevant biomarkers. In this study, the association between 163 targeted maternal blood metabolites and gestational age was investigated in order to find candidate biomarkers for PTB. Methods: Five hundred twenty-three women were included into this observational study. Maternal blood was obtained before delivery. The concentration of 163 maternal serum metabolites was measured by flow injection tandem mass spectrometry. To find putative biomarkers for preterm birth, a three-step analysis was designed: bivariate correlation analysis followed by multivariable regression analysis and a comparison of mean values among gestational age groups. Results: Bivariate correlation analysis showed that 2 acylcarnitines (C16:2, C2), 1 amino acids (xLeu), 8 diacyl-PCs (PCaaC36:4, PCaaC38:4, PCaaC38:5, PCaaC38:6, PCaaC40:4, PCaaC40:5, PCaaC40:6, PCaaC42:4), and 1 Acylalkyl-PCs (PCaeC40:5) were inversely correlated with gestational age. Multivariable regression analysis confounded for PTB history, maternal body mass index (BMI) before pregnancy, systolic blood pressure at the third trimester, and maternal body weight at the third trimester, showed that the diacyl-PC PCaaC38:6 was the only metabolite inversely correlated with gestational age. Conclusions: Maternal blood concentrations of PCaaC38:6 are independently associated with gestational age. (C) 2016 The Author(s) Published by S. Karger AG, Basel
Involvement of Sphingosine 1-Phosphate in Palmitate-Induced Non-Alcoholic Fatty Liver Disease
(2016)
Background/Aims: Ectopic lipid accumulation in hepatocytes has been identified as a risk factor for the progression of liver fibrosis and is strongly associated with obesity. In particular, the saturated fatty acid palmitate is involved in initiation of liver fibrosis via formation of secondary metabolites by hepatocytes that in turn activate hepatic stellate cells (HSCs) in a paracrine manner Methods: a-smooth muscle actin-expression (alpha-SMA) as a marker of liver fibrosis was investigated via western blot analysis and immunofluorescence microscopy in HSCs (LX-2). Sphingolipid metabolism and the generation of the bioactive secondary metabolite sphingosine I-phosphate (SIP) in response to palmitate were analyzed by LC-MS/MS in hepatocytes (HepG2). To identify the molecular mechanism involved in the progression of liver fibrosis real-time PCR analysis and pharmacological modulation of SIP receptors were performed. Results: Palmitate oversupply increased intra- and extracellular SIP-concentrations in hepatocytes. Conditioned medium from HepG2 cells initiated fibrosis by enhancing alpha-SMA-expression in LX-2 in a S1P-dependent manner In accordance, fibrotic response in the presence of SIP was also observed in HSCs. Pharmacological inhibition of SIP receptors demonstrated that S1P(3) is the crucial receptor subtype involved in this process. Conclusion: SIP is synthesized in hepatocytes in response to palmitate and released into the extracellular environment leading to an activation of HSCs via the S1P(3) receptor (C) 2016 The Author(s) Published by S. Karger AG, Basel
CD4(+) Foxp3(+) regulatory T cells (Tregs) depend on CD28 signaling for their survival and function, a receptor that has been previously shown to activate the acid sphingomyelinase (Asm)/ceramide system. In this article, we show that the basal and CD28-induced Asm activity is higher in Tregs than in conventional CD4(+) T cells (Tconvs) of wild-type (wt) mice. In Asm-deficient (Smpd1(-/-); Asm(-/-)) mice, as compared with wt mice, the frequency of Tregs among CD4(+) T cells, turnover of the effector molecule CTLA-4, and their suppressive activity in vitro were increased. The biological significance of these findings was confirmed in our Treg-sensitive mouse model of measles virus (MV) CNS infection, in which we observed more infected neurons and less MV-specific CD8(+) T cells in brains of Asm(-/-) mice compared with wt mice. In addition to genetic deficiency, treatment of wt mice with the Asm inhibitor amitriptyline recapitulated the phenotype of Asm-deficient mice because it also increased the frequency of Tregs among CD4(+) T cells. Reduced absolute cell numbers of Tconvs after inhibitor treatment in vivo and extensive in vitro experiments revealed that Tregs are more resistant toward Asm inhibitor-induced cell death than Tconvs. Mechanistically, IL-2 was capable of providing crucial survival signals to the Tregs upon inhibitor treatment in vitro, shifting the Treg/Tconv ratio to the Treg side. Thus, our data indicate that Asm-inhibiting drugs should be further evaluated for the therapy of inflammatory and autoimmune disorders.
Background: Gestational diabetes mellitus (GDM) is associated with adverse pregnancy outcomes. It is known that GDM is associated with an altered placental function and changes in placental gene regulation. More recent studies demonstrated an involvement of epigenetic mechanisms. So far, the focus regarding placental epigenetic changes in GDM was set on gene-specific DNA methylation analyses. Studies that robustly investigated placental global DNA methylation are lacking. However, several studies showed that tissue-specific alterations in global DNA methylation are independently associated with type 2 diabetes. Thus, the aim of this study was to characterize global placental DNA methylation by robustly measuring placental DNA 5-methylcytosine (5mC) content and to examine whether differences in placental global DNA methylation are associated with GDM. Methods: Global DNA methylation was quantified by the current gold standard method, LC-MS/MS. In total, 1030 placental samples were analyzed in this single-center birth cohort study. Results: Mothers with GDM displayed a significantly increased global placental DNA methylation (3.22 +/- 0.63 vs. 3.00 +/- 0.46 %; p = 0.013; +/- SD). Bivariate logistic regression showed a highly significant positive correlation between global placental DNA methylation and the presence of GDM (p = 0.0009). Quintile stratification according to placental DNA 5mC levels revealed that the frequency of GDM was evenly distributed in quintiles 1-4 (2.9-5.3 %), whereas the frequency in the fifth quintile was significantly higher (10.7 %; p = 0.003). Bivariate logistic models adjusted for maternal age, BMI, ethnicity, recurrent miscarriages, and familiar diabetes predisposition clearly demonstrated an independent association between global placental DNA hypermethylation and GDM. Furthermore, an ANCOVA model considering known predictors of DNA methylation substantiated an independent association between GDM and placental DNA methylation. Conclusions: This is the first study that employed a robust quantitative assessment of placental global DNA methylation in over a thousand placental samples. The study provides large scale evidence that placental global DNA hypermethylation is associated with GDM, independent of established risk factors.
Acting during phase II metabolism, sulfotransferases (SULTs) serve detoxification by transforming a broad spectrum of compounds from pharmaceutical, nutritional, or environmental sources into more easily excretable metabolites. However, SULT activity has also been shown to promote formation of reactive metabolites that may have genotoxic effects. SULT subtype 1E1 (SULT1E1) was identified as a key player in estrogen homeostasis, which is involved in many physiological processes and the pathogenesis of breast and endometrial cancer. The development of an in silico prediction model for SULT1E1 ligands would therefore support the development of metabolically inert drugs and help to assess health risks related to hormonal imbalances. Here, we report on a novel approach to develop a model that enables prediction of substrates and inhibitors of SULT1E1. Molecular dynamics simulations were performed to investigate enzyme flexibility and sample protein conformations. Pharmacophores were developed that served as a cornerstone of the model, and machine learning techniques were applied for prediction refinement. The prediction model was used to screen the DrugBank (a database of experimental and approved drugs): 28% of the predicted hits were reported in literature as ligands of SULT1E1. From the remaining hits, a selection of nine molecules was subjected to biochemical assay validation and experimental results were in accordance with the in silico prediction of SULT1E1 inhibitors and substrates, thus affirming our prediction hypotheses.
Background & Aims: Exosomes are small membrane vesicles involved in intercellular communication. Hepatocytes are known to release exosomes, but little is known about their biological function. We sought to determine if exosomes derived from hepatocytes contribute to liver repair and regeneration after injury. Methods: Exosomes derived from primary murine hepatocytes were isolated and characterized biochemically and biophysically. Using cultures of primary hepatocytes, we tested whether hepatocyte exosomes induced proliferation of hepatocytes in vitro. Using models of ischemia/reperfusion injury and partial hepatectomy, we evaluated whether hepatocyte exosomes promote hepatocyte proliferation and liver regeneration in vivo. Results: Hepatocyte exosomes, but not exosomes from other liver cell types, induce dose-dependent hepatocyte proliferation in vitro and in vivo. Mechanistically, hepatocyte exosomes directly fuse with target hepatocytes and transfer neutral ceramidase and sphingosine kinase 2 (SK2) causing increased synthesis of sphingosine-1-phosphate (S1P) within target hepatocytes. Ablation of exosomal SK prevents the proliferative effect of exosomes. After ischemia/reperfusion injury, the number of circulating exosomes with proliferative effects increases. Conclusions: Our data shows that hepatocyte-derived exosomes deliver the synthetic machinery to form S1P in target hepatocytes resulting in cell proliferation and liver regeneration after ischemia/reperfusion injury or partial hepatectomy. These findings represent a potentially novel new contributing mechanism of liver regeneration and have important implications for new therapeutic approaches to acute and chronic liver disease. (C) 2015 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.
pH-sensitive nanoparticles which release in a controlled fashion on the skin or dissolve in the hair follicle could significantly improve treatment effectiveness and make transfollicular drug delivery a success. Dexamethasone-loaded Eudragit L 100 nanoparticles were prepared by nanoprecipitation from an organic drug-polymer solution. Their toxicity potential was assessed using isolated human fibroblasts. pH-dependent swelling and erosion kinetics of the nanoparticles were investigated by dynamic light scattering and viscosity measurements and its effect on drug release was assessed in vitro with Franz diffusion cells. Stable, 100-550 nm-sized dexamethasone-loaded Eudragit L 100 nanoparticles with drug loading capacity and entrapment efficiency as high as 83% and 85%, respectively, were obtained by using polyvinyl alcohol as a stabilizer and ethanol as organic solvent The nanoparticles showed little or no toxicity on isolated normal human fibroblasts. Dexamethasone existed in the nanoparticles as solid solution or in amorphous form. The nanoparticles underwent extensive swelling and slow drug release in media with a low buffer capacity (as low as 10 mM) and a higher pH or at a pH close to the dissolution pH of the polymer (pH 6) and a higher buffer capacity. In 40 mM buffer and above pH 6.8, the nanoparticles eroded fast or dissolved completely and thus released the drug rapidly. pH-sensitive nanoparticles which potentially release in a controlled manner on the stratum corneum but dissolve in the hair follicle could be prepared. (C) 2016 Elsevier B.V. All rights reserved.