Rational design of polyarginine nanocapsules intended to help peptides overcoming intestinal barriers

The aim of this work was to rationally design and characterize nanocapsules (NCs) composed of an oily core and a polyarginine (PARG) shell, intended for oral peptide delivery. The cationic polyaminoacid, PARG, and the oily core components were selected based on their penetration enhancing properties. Insulin was adopted as a model peptide to assess the performance of the NCs. After screening numerous formulation variables, including different oils and surfactants, we defined a composition consisting of oleic acid, sodium deoxycholate (SDC) and Span 80. This selected NCs composition, produced by the solvent displacement technique, exhibited the following key features: (i) an average size of 180 nm and a low polydispersity (0.1), (ii) a high insulin association efficacy (80–90% AE), (iii) a good colloidal stability upon incubation in simulated intestinal fluids (SIF, FaSSIF-V2, FeSSIF-V2), and (iv) the capacity to control the release of the associated insulin for > 4 h. Furthermore, using the Caco-2 model cell line, PARG nanocapsules were able to interact with the enterocytes, and reversibly modify the TEER of the monolayer. Both cell adhesion and membrane permeabilization could account for the pronounced transport of the NCs-associated insulin (3.54%). This improved interaction was also visualized by confocal fluorescent microscopy following oral administration of PARG nanocapsulesto mice. Finally, in vivo efficacy studies performed in normoglycemic rats showed a significant decrease in their plasma glucose levels after treatment. In conclusion, here we disclose key formulation elements for making possible the oral administration of peptidesThis work was supported by the European TRANS-INT Consortium, which received funding from the European Union's Seventh Framework Programme for research, technological development and demonstration under grant agreement No. 281035. Z. Niu also would like to thank the Chinese Scholarship Council for his scholarshipS

Epigenetic alterations in creatine transporter deficiency: a new marker for dodecyl creatine ester therapeutic efficacy monitoring

Creatine transporter deficiency (CTD) is an X-linked disease caused by mutations in the Slc6a8 gene. The impaired creatine uptake in the brain leads to developmental delays with intellectual disability. We hypothesized that deficient creatine uptake in CTD cerebral cells impact methylation balance leading to alterations of genes and proteins expression by epigenetic mechanism. In this study, we determined the status of nucleic acid methylation in both Slc6a8 knockout mouse model and brain organoids derived from CTD patients’ cells. We also investigated the effect of dodecyl creatine ester (DCE), a promising prodrug that increases brain creatine content in the mouse model of CTD. The level of nucleic acid methylation was significantly reduced compared to healthy controls in both in vivo and in vitro CTD models. This hypo-methylation tended to be regulated by DCE treatment in vivo. These results suggest that increased brain creatine after DCE treatment restores normal levels of DNA methylation, unveiling the potential of using DNA methylation as a marker to monitor the drug efficacy

Dodecyl creatine ester improves cognitive function and identifies key protein drivers including KIF1A and PLCB1 in a mouse model of creatine transporter deficiency

Creatine transporter deficiency (CTD), a leading cause of intellectual disability is a result of the mutation in the gene encoding the creatine transporter SLC6A8, which prevents creatine uptake into the brain, causing mental retardation, expressive speech and language delay, autistic-like behavior and epilepsy. Preclinical in vitro and in vivo data indicate that dodecyl creatine ester (DCE) which increases the creatine brain content, might be a therapeutic option for CTD patients. To gain a better understanding of the pathophysiology and DCE treatment efficacy in CTD, this study focuses on the identification of biomarkers related to cognitive improvement in a Slc6a8 knockout mouse model (Slc6a8−/y) engineered to mimic the clinical features of CTD patients which have low brain creatine content. Shotgun proteomics analysis of 4,035 proteins in four different brain regions; the cerebellum, cortex, hippocampus (associated with cognitive functions) and brain stem, and muscle as a control, was performed in 24 mice. Comparison of the protein abundance in the four brain regions between DCE-treated intranasally Slc6a8−/y mice and wild type and DCE-treated Slc6a8−/y and vehicle group identified 14 biomarkers, shedding light on the mechanism of action of DCE. Integrative bioinformatics and statistical modeling identified key proteins in CTD, including KIF1A and PLCB1. The abundance of these proteins in the four brain regions was significantly correlated with both the object recognition and the Y-maze tests. Our findings suggest a major role for PLCB1, KIF1A, and associated molecules in the pathogenesis of CTD

Barrière hémato-encéphalique humaine et endothéline 1 (régulation des transporteurs ABC et des cytokines inflammatoires)

Le système nerveux central est un organe complexe et privilégié, protégé, entre autres, par la barrière hémato-encéphalique (BHE), sur laquelle se trouvent des transporteurs d efflux telle que la P-glycoprotéine (Pgp). La BHE est impliquée dans la neuro-inflammation et les maladies cérébrales associées, et la littérature laisse penser que l endothéline 1 (ET-1) y joue un rôle important. En utilisant un modèle in vitro autologue de BHE humaine adulte et un modèle in vitro de BHE humaine fœtale (co-culture de cellules gliales et de cellules endothéliales microvasculaires cérébrales), nous avons étudié l effet de l ET-1 sur la Pgp. Une différence, en termes de modulation de l activité de la Pgp, a été observée entre la BHE fœtale et la BHE adulte suite à l exposition à l ET-1. Nous avons également évalué la modulation induite par l ET-1 sur la sécrétion de la MCP-1 (Monocyte chemoattractant protein 1) et de l IL-8. Les résultats montrent que l ET-1 diminue l activité de la Pgp et induit la sécrétion de MCP-1 et d IL-8.The central nervous system is a complex and privileged organ protected by, among others, the blood-brain barrier (BBB). On this barrier are localised efflux pumps such as P-glycoprotein. The BBB is involved in neuro-inflammation associated to many cerebral diseases. The endothelin 1 (ET-1) peptide seems to play a key role in the process. We have used an in vitro human autologous adult BBB model and an in vitro human foetal BBB model (co-culture of glial cells and brain microvascular endothelial cells) to evaluate ET-1 s influence on the P-glycoprotein. A difference was shown between the two models concerning Pgp s activity modulation by ET-1. We have also been looking at ET-1 induced Monocyte chemoattractant protein 1 and interleukin 8 secretions. The results show that ET-1 reduces Pgp s activity and induces MCP-1 and IL-8 secretion.CHATENAY M.-PARIS 11-BU Pharma. (920192101) / SudocSudocFranceF

Interactions des nanoparticules avec les barrières physiologiques

Du fait de la présence de croissante des nanoparticules dans notre environnement, les expositions et leurs effets toxiques deviennent un sujet de préoccupation. Les barrières physiologiques ont un rôle de protection de l'organisme ou d'organes particuliers. L'étude des interactions entre nanoparticules et ces barrières permet donc de comprendre leur absorption et leur distribution. Il semblerait que ces protections ne représentent pas un obstacle infaillible au passage des nanoparticules. Ces barrières exercent également des fonctions vitales qui peuvent être altérées par l'exposition aux nanoparticules. Les mécanismes d'actions toxiques souvent évoqués dans la littérature sont l'induction de stress oxydant, d'une inflammation et cytotoxicité. Les données disponibles sont encore insuffisantes pour évaluer le risque lié à l'exposition aux nanoparticules et les connaissances sur la toxicité aiguë et chroniques nécessitent d'être approfondies.Due to the presence of an increasingly large number of nanoparticles in our environment, exposure to them and their toxic effects become a cause of concern. Physiological barriers play a role in protecting the body or specific organs. The study of the interactions between nanoparticles and these barriers can therefore help the understanding of their biodistribution. It seems that these protections are not an effective obstacle against the passage of nanoparticles. These barriers also perform vital functions which may be affected by biological changes due to interference between nanoparticles and cells. In literature, the most frequently quoted toxic mechanisms of action are the induction of oxidative stress, inflammation and cytotoxicity. Available data are still insufficient to assess the risk link to nanoparticles exposure. Additional information, including in vivo studies is needed to increase knowledge about acute and chronic toxicity.RENNES1-BU Santé (352382103) / SudocLYON1-BU Santé (693882101) / SudocSudocFranceF

In vitro evidence of dysregulation of blood–brain barrier function after acute and repeated/long-term exposure to TiO2 nanoparticles

International audienceThe effects of titanium dioxide nanoparticles (TiO2 NPs) on blood–brain barrier (BBB) function are unknown. Here, we report such evidence of adverse effects after in vitro exposure of a rat primary cell-based BBB model to NPs. BBB integrity was studied by measuring the flux of sucrose through the monolayer. P-glycoprotein (P-gp) activity was assessed by measuring the passage of vinblastine. Transcription profiles of P-gp and other ABC transporters as well as of cytokines were investigated by real-time PCR. Electron microscopy and particle-induced X-ray emission measurements were performed. We compared several exposure modalities, from early to chronic, mimicking a brain-to-blood transport or a systemic contamination. In the first case, BBB integrity was preserved, but P-gp activity of endothelial cells (BECs) was reduced. In the second case, BBB integrity and P-gp function were impaired from 5 μg/mL for 24 h and expression of tight junction proteins and efflux transporters was modulated. An inflammatory response had repercussions on ABC transporter expression of glial cells. We demonstrate that NPs accumulated in BECs and crossed the cell monolayer. These findings suggest that there is an immunoregulatory loop between inflammatory components, BECs and glial cells in the dysfunction of the BBB during exposure to TiO2 NPs

Regulation of P-glycoprotein by miR-27a-3p at the Brain Endothelial Barrier

Multi-drug resistance P-glycoprotein (P-gp/MDR1) is one of the most clinically relevant ABC transporters, highly enriched at the blood-brain barrier (BBB) with a broad substrate spectrum including therapeutic drugs and metabolic waste products. Altered P-gp transport function has been implicated in multi-drug resistance and in the pathogenesis and progression of neurological diseases. Recent studies have shown that P-gp expression is modulated by micro-RNAs in peripheral organs. Particularly, miR-27a-3p has been shown to play a critical role in the regulation of P-gp in multi-drug resistant cancer cells. In brain disorders, altered levels of miR-27a-3p were reported in several diseases associated with alterations in P-gp expression at the BBB. However, effect of altered miR-27a-3p expression on P-gp expression at the BBB remains to be determined. In this study, we investigated the role of miR-27a-3p in the regulation of P-gp expression and activity at the brain endothelium. Levels of miR-27a-3p were modulated by mimic and inhibitor transfection in an in-vitro model of human brain endothelial hCMEC/D3 cells. Effect of miR-27a-3p modulation on P-gp expression and activity was examined and the underlying regulatory mechanisms explored. Our results showed that transfection of hCMEC/D3 cells with miR-27a-3p mimic induces expression and activity of P-gp while miR-27a-3p inhibition exerted opposite effects. Mechanistic studies revealed that miR-27a-3p regulates P-gp by mediating Glycogen Synthase Kinase 3 Beta (GSK3ß) inhibition and activating Wnt/ß-catenin signaling. These findings shed light on miR-27a-3p/GSK3ß/ß-catenin as a novel axis that could be exploited to modulate P-gp efflux activity at the brain endothelium and help improving CNS diseases treatment or brain protection

Silencing miR-202-3p increases MMP-1 and promotes a brain invasive phenotype in metastatic breast cancer cells.

BackgroundBrain metastasis (BM) is a major cause of morbidity and mortality in breast cancer (BC) and its molecular mechanism remains poorly understood. Transmigration of metastatic cells through the brain endothelium is an essential step in BM. Metalloproteinase-1 (MMP-1) overexpression plays a key role in promoting trans-endothelial migration by degrading the inter-endothelial junctions and disrupting the endothelial integrity. However, little is known about the molecular mechanisms that induce MMP-1 in metastatic cells granting them a brain invasive phenotype. MiR-202-3p is downregulated in brain metastases compared to primary breast tumors and directly targets MMP-1. Here, we unraveled a critical role of miR-202-3p loss in MMP-1 upregulation promoting transmigration of metastatic cells through the brain endothelium.MethodsA variant of the MDA-MB-231 human BC cell line (MDA-MB-231-BrM2) selected for its propensity to form brain metastases was found to express high levels of MMP-1 and low levels of miR-202-3p compared to the parental cells. Using a gain-and-loss of function approach, we modulated levels of miR-202-3p and examined the resultant effect on MMP-1 expression. Effect of miR-202-3p modulation on integrity of the brain endothelium and the transmigrative ability of BC cells were also examined.ResultsLoss of miR-202-3p in breast cancer cells enhanced their transmigration through the brain endothelium by upregulating MMP-1 and disrupting the inter-endothelial junctions (claudin-5, ZO-1 and ß-catenin). Restoring miR-202-3p exerted a metastasis-suppressive effect and preserved the endothelial barrier integrity.ConclusionsOur study identified a critical regulatory role of miR-202-3p in brain metastasis and shed light on miR-202-3p/MMP-1 axis as a novel prognostic and therapeutic target that can be exploited to predict and prevent brain metastasis in breast cancer patients