Blood-brain- barrier co-culture models to study nanoparticle penetration : focus on co-culture systems

The blood-brain barrier, as a physical, active transport and metabolic barrier represents the main obstacle in the treatment of central nervous system diseases. The field of nanoparticle delivery systems is rapidly developing and nanocarriers seem to be promising for drug delivery or targeting to the brain. For testing the toxicity, uptake and transcellular transport of nanoparticles culture models of the blood-brain barrier are widely used, including immortalized brain endothelial cell lines, primary brain endothelial cells in static or dynamic culture conditions, and in co-culture systems with glial cells and/or pericytes. This mini-review gives a brief summary of blood-brain barrier co-culture models that were used for testing nanocarriers, the types of different nanoparticle systems that were examined on blood-brain barrier models, and the advantages, limitations and suitability of the blood-brain barrier models for nanoparticle penetration studies

CELL CULTURE AND IN VIVO STUDY OF MICROVESICLES FOR DRUG DELIVERY ACROSS BARRIERS

Efficient drug delivery across biological barriers, like the intestinal and blood-brain barriers is a central problem in pharmaceutical treatment of disorders [1]. Most pharmaceutical drug candidates, hydrophilic molecules, biopharmaceuticals, and efflux transporter ligands have a low permeability across barriers. To solve this unmet therapeutical need colloidal drug delivery systems utilizing physiological transporters of the barriers hold a great promise. The aim of our study was to test nanosized, biocompatible and biodegradable vesicles which can encorporate both hydrophilic and hydrophobic drug cargos and present on their surfaces ligands for solute carrier (SLC) proteins. Glucose analogues and amino acids were used to achieve increased specificity and efficacy for drug delivery across barriers. Bilayered microvesicles of non-ionic surfactants, niosomes are able to encapsulate solutes and serve as potential drug carriers. Niosomes with an average hydrodynamical size of 200 nm were prepared containing different ligands and their combinbations, and Evans blue-albumin as a model molecule. Human Caco-2 intestinal epithelial and D3 brain endothelial cells, a model of the blood-brain barrier [2], were used for toxicity measurements by colorimetric methods and real-time cell microelectric sensing, permeability experiments and morphological examinations. The presence of glucose and amino acid ligands on microvesicles increased the uptake of Evans blue-albumin to the cells and its penetration across the cell layers. A kinetic in vivo study in nude mice by eXplore Optix, a near infrared fluorescence time-domain optical imaging demonstrated the elevated accumulation of Evans blue-albumin in the brain after the intravenous injection of glucose analogue and amino acid labeled niosomes. These results indicate that microvesicles labeled with SLC transporter ligands can be used for targeting hydrophilic biomolecules across barriers

Nanovesicles for drug delivery across blood-brain barrier: a cell culture study

Efficient drug delivery across biological barriers is a central problem in pharmaceutical treatment of diseases. Most pharmaceutical drug candidates including hydrophilic molecules, biopharmaceuticals, and efflux transporter ligands have a low permeability across barriers. To solve this unmet therapeutical need colloidal drug delivery systems targeting physiological transporters of barriers hold a great promise. Nanosized, biocompatible and biodegradable vesicles containing Evans blue-albumin as a model molecule were prepared and characterized by our partners at the University of Szeged. The aim of our study was to test the cellular toxicity and penetration across barriers of nanovesicles loaded with albumin and containing ligands for solute carrier proteins. Primary rat and human hCMEC/D3 brain endothelial and Caco-2 human intestinal epithelial cells were used as in vitro model sytems of the blood-brain and intestinal barriers, respectively. The cellular toxicity of the nanoparticles was measured by real-time cell microelectric sensing (RTCA-SP, ACEA Biosciences) and MTT assay. The results of the MTT assay and impedance measurement for vesicles without targeting molecule correlated well. The uptake of targeted and loaded nanovesicles interfered with the colorimetric MTT assay in brain endothelial cells because of Evans blue, therefore kinetical data from impedance measurements were more informative on the cellular toxicity of these nanoparticles. The non-toxic doses determined by the cell viability tests proved to be optimal for further studies. The presence of glucose analogue in nanovesicles increased the uptake of the model molecule to cultured brain endothelial cells indicating that ligands for solute carrier proteins can be used for targeting brain endothelial cells

NANOPARTICLES: TOXICITY AND PENETRATION ACROSS BIOLOGICAL BARRIERS

Nanoparticles provide new opportunities for drug delivery and human therapy. To fulfill the therapeutical potential of nanoparticles two major aspects, toxicity and penetration across barriers of the body need to be studied. Different ex vivo and in vitro cell culture based models of the skin, nasal, lung, intestinal and blood-brain barriers have been established in our laboratory that can be used for both purposes. Three different types of nanoparticles were tested on the different models. Amorphous nanoparticles from the antiinflammatory drug meloxicam were obtained by by co-grinding with polyvinylpyrrolidone. Nanosized bilayered vesicles of non-ionic surfactants bearing glucose and amino acid ligands were prepared to specifically target solute carriers on the blood-brain barrier [1]. Poly(ferrocenyl silane) redox responsive polymer nanocarriers were also studied [2]. Several methods were applied parallelly to measure the toxicity of nanoparticles. In addition to colorimetric tests like MTT dye reduction assay, release of the cytoplasmic enzyme lactate dehydrogenase cellular events were also monitored in real time. By measuring impedance across microelectrodes covered with cells quantitative information on cell viability and intercellular adherence indicating paracellular permeability could be obtained. Co-culture models of the barriers prepared from primary cultures or human cell lines [3] served for permeability experiments to test the penetration of nanocarriers across cell layers. In the case of the blood-brain barrier a kinetic in vivo study in mice was also performed by near infrared fluorescence time-domain optical imaging. The results indicate that (i) toxicity measurements are very important to obtain the optimal dose of nanoparticles on living cells, (ii) nanonization of drugs can improve drug dissolution, absorption and pharmacokinetics, (iii) targeting of microvesicles increases their penetration across barriers

Edaravone protects against methylglyoxal-induced barrier damage in human brain endothelial cells

BACKGROUND: Elevated level of reactive carbonyl species, such as methylglyoxal, triggers carbonyl stress and activates a series of inflammatory responses leading to accelerated vascular damage. Edaravone is the active substance of a Japanese medicine, which aids neurological recovery following acute brain ischemia and subsequent cerebral infarction. Our aim was to test whether edaravone can exert a protective effect on the barrier properties of human brain endothelial cells (hCMEC/D3 cell line) treated with methylglyoxal. METHODOLOGY: Cell viability was monitored in real-time by impedance-based cell electronic sensing. The barrier function of the monolayer was characterized by measurement of resistance and flux of permeability markers, and visualized by immunohistochemistry for claudin-5 and β-catenin. Cell morphology was also examined by holographic phase imaging. PRINCIPAL FINDINGS: Methylglyoxal exerted a time- and dose-dependent toxicity on cultured human brain endothelial cells: a concentration of 600 µM resulted in about 50% toxicity, significantly reduced the integrity and increased the permeability of the barrier. The cell morphology also changed dramatically: the area of cells decreased, their optical height significantly increased. Edaravone (3 mM) provided a complete protection against the toxic effect of methylglyoxal. Co-administration of edaravone restored cell viability, barrier integrity and functions of brain endothelial cells. Similar protection was obtained by the well-known antiglycating molecule, aminoguanidine, our reference compound. CONCLUSION: These results indicate for the first time that edaravone is protective in carbonyl stress induced barrier damage. Our data may contribute to the development of compounds to treat brain endothelial dysfunction in carbonyl stress related diseases

Blood-brain carrier co-culture models to study nanoparticle penetration : focus on co-culture systems

The blood-brain barrier, as a physical, active transport and metabolic barrier represents the main obstacle in the treatment of central nervous system diseases. The field of nanoparticle delivery systems is rapidly developing and nanocarriers seem to be promising for drug delivery or targeting to the brain. For testing the toxicity, uptake and transcellular transport of nanoparticles culture models of the blood-brain barrier are widely used, including immortalized brain endothelial cell lines, primary brain endothelial cells in static or dynamic culture conditions, and in co-culture systems with glial cells and/or pericytes. This mini-review gives a brief summary of blood-brain barrier co-culture models that were used for testing nanocarriers, the types of different nanoparticle systems that were examined on blood-brain barrier models, and the advantages, limitations and suitability of the blood-brain barrier models for nanoparticle penetration studies

The Absence of N-Acetyl-D-glucosamine Causes Attenuation of Virulence of Candida albicans upon Interaction with Vaginal Epithelial Cells In Vitro.

To better understand the molecular events underlying vulvovaginal candidiasis, we established an in vitro system. Immortalized vaginal epithelial cells were infected with live, yeast form C. albicans and C. albicans cultured in the same medium without vaginal epithelial cells were used as control. In both cases a yeast to hyphae transition was robustly induced. Whole transcriptome sequencing was used to identify specific gene expression changes in C. albicans. Numerous genes leading to a yeast to hyphae transition and hyphae specific genes were upregulated in the control hyphae and the hyphae in response to vaginal epithelial cells. Strikingly, the GlcNAc pathway was exclusively triggered by vaginal epithelial cells. Functional analysis in our in vitro system revealed that the GlcNAc biosynthesis is involved in the adherence to, and the ability to kill, vaginal epithelial cells in vitro, thus indicating the key role for this pathway in the virulence of C. albicans upon vulvovaginal candidiasis

Simvastatin, edaravone and dexamethasone protect against kainate-induced brain endothelial cell damage

Excitotoxicity is a central pathological pathway in many neurological diseases with blood-brain barrier (BBB) dysfunction. Kainate, an exogenous excitotoxin, induces epilepsy and BBB damage in animal models, but the direct effect of kainate on brain endothelial cells has not been studied in detail. Our aim was to examine the direct effects of kainate on cultured cells of the BBB and to test three anti-inflammatory and antioxidant drugs used in clinical practice, simvastatin, edaravone and dexamethasone, to protect against kainate-induced changes.Primary rat brain endothelial cell, pericyte and astroglia cultures were used to study cell viability by impedance measurement. BBB permeability was measured on a model made from the co-culture of the three cell types. The production of nitrogen monoxide and reactive oxygen species was followed by fluorescent probes. The mRNA expression of kainate receptors and nitric oxide synthases were studied by PCR.Kainate damaged brain endothelial cells and made the immunostaining of junctional proteins claudin-5 and zonula occludens-1 discontinuous at the cell border indicating the opening of the barrier. The permeability of the BBB model for marker molecules fluorescein and albumin and the production of nitric oxide in brain endothelial cells were increased by kainate. Simvastatin, edaravone and dexamethasone protected against the reduced cell viability, increased permeability and the morphological changes in cellular junctions caused by kainate. Dexamethasone attenuated the elevated nitric oxide production and decreased the inducible nitric oxide synthase (NOS2/iNOS) mRNA expression increased by kainate treatment.Kainate directly damaged cultured brain endothelial cells. Simvastatin, edaravone and dexamethasone protected the BBB model against kainate-induced changes. Our results confirmed the potential clinical usefulness of these drugs to attenuate BBB damage