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

The Effects of Exercise Training and High Triglyceride Diet in an Estrogen Depleted Rat Model

Cardiovascular morbidity and mortality of premenopausal women are significantly lower compared to men of similar age. However, this protective effect evidently decreases after the onset of menopause. We hypothesized that physical exercise could be a potential therapeutic strategy to improve inflammatory processes and cardiovascular antioxidant homeostasis, which can be affected by the loss of estrogen and the adverse environmental factors, such as overnutrition. Ovariectomized (OVX, n= 40) and sham-operated (SO, n= 40) female Wistar rats were randomized to exercising (R) and non-exercising (NR) groups. Feeding parameters were chosen to make a standard chow (CTRL) or a high triglyceride diet (HT) for 12 weeks. Aortic and cardiac heme oxygenase (HO) activity and HO-1 concentrations significantly decreased in all of the NR OVX and SO HT groups. However, the 12-week physical exercise was found to improve HO-1 values. Plasma IL-6 concentrations were higher in the NR OVX animals and rats fed HT diet compared to SO CTRL rats. TNF-α concentrations were significantly higher in the NR OVX groups. 12 weeks of exercise significantly reduced the concentrations of both TNF-α and IL-6 compared to the NR counterparts. The activity of myeloperoxidase enzyme (MPO) was significantly increased as a result of OVX and HT diet, however voluntary wheel-running exercise restored the elevated values. Our results show that estrogen deficiency and HT diet caused a significant decrease in the activity and concentration of HO enzyme, as well as the concentrations of TNF-α, IL-6, and the activity of MPO. However, 12 weeks of voluntary wheel-running exercise is a potential non-pharmacological therapy to ameliorate these disturbances, which determine the life expectancy of postmenopausal women

Novel features of the rat model of inflammatory bowel disease based on 2,4,6-trinitrobenzenesulfonic acidinduced acute colitis

The 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced acute inflammatory bowel disease (IBD) model in the rat is discussed, focusing on the details of the TNBS instillation and highlighting the advantages and limitations of this model. For determination of the time-dependent action of 50% ethanol and different doses of TNBS, male Wistar rats were treated with 50% ethanol or 10 mg or 30 mg of TNBS dissolved in 50% ethanol. The TNBS-induced inflammation peaked 48-72 h after installation and the colitis caused by 30 mg of TNBS was more severe than that caused by 10 mg of TNBS. To test the effectiveness of sulfasalazine (SASP), male rats were treated with 10 mg of TNBS or with 10 mg of TNBS and SASP, and 72 h later the extent of mucosal damage was determined. Orally administered 50 mg/kg/day SASP proved to reduce the TNBS-induced colonic inflammation in rats significantly. The TNBS-induced colitis model facilitates a better understanding of the immunopathological mechanisms of IBD. Optimization of the dose of TNBS and oral SASP as positive control in TNBS-induced colitis in rats furnishes an appropriate test system for new anti-IBD drugs

HOGYAN AKNÁZHATÓK KI A VÉR-AGY GÁT TRANSZPORTRENDSZEREI A GYÓGYSZEREK IDEGRENDSZERBE VALÓ CÉLZOTT BEJUTTATÁSÁRA?

A vér-agy gát anatómiai alapját képező agyi endotélsejtek genomjának 11 %-át transzporterek génjei teszik ki, ami jól jelzi a szállítófehérjék fontosságát. Az agyi hajszálerekben az Slc (solute carrier) szállítófehérje család látja el a központi idegrendszert tápanyagokkal, vitaminokkal, nyomelemekkel, metabolikus prekurzorokkal. Ezek a karrierek főképpen az agyba irányuló transzportban vesznek részt, míg a vér-agy gát efflux transzporterei a neurotranszmitterek és metabolitok szintjét szabályozzák az agyban, valamint megakadályozzák a potenciálisan toxikus anyagok, xenobiotikumok bejutását a vérből az agyba. Az efflux transzportereknek köszönhető a legtöbb központi idegrendszeri gyógyszerjelölt molekula alacsony átjutása is a vér-agy gáton. A probléma megoldására jelentős erővel folynak olyan kutatások, amelyek a hatóanyagokat a vér-agy gát szállítófehérjéinek segítségével juttatják be a központi idegrendszerbe. Megvizsgáltuk a gyógyszerek transzportja és célzott bevitele szempontjából kulcsfontosságú Slc és efflux pumpa fehérjecsaládok génexpressziós mintázatát izolált patkány agyi mikroerekben. A glükóz transzporterek közül a Glut-1 expessziós mRNS szintje volt a legmagasabb, de a Glut-3 és -5 is kifejeződött. Minden vizsgált aminosav transzporter esetében magas génexpressziós szintet mértünk, a legmagasabb a gyógyszerek bejutásában is szerepet játszó Lat-1, valamint a Cat-1 és Sn-1 szintje volt. A peptid transzporterek esetében a Pht-2 szintje szignifikánsan magasnak bizonyult, míg a Pept-1, -2 génexpressziója nem volt mérhető. A kreatint (Crt), taurint (Taut) és C-vitamint (Asct-1) szállító fehérjék is expresszálódtak agyi mikroerekben. Az efflux pumpák esetében az ABC transzporter P-glikoprotein (Abcb1), a mellrák rezisztencia fehérje (Bcrp, Abcg2), és a multidrog rezisztencia proteinek közül az Mrp-1, 4, 5 mRNS szinje volt a legmagasabb, míg az Mrp-2 nem volt mérhető. A legtöbb vizsgált vér-agy gát transzporter mRNS expressziójára jó egyezést kaptunk izolált agyi mikroerek és a vér-agy gát tenyészetes modellje között. Mivel az Slc transzporterek jelentős mértékben és egyedi mintázatban expresszálódnak a vér-agy gáton, Slc transzportfehérjék ligandjainak kombinációjával ellátott nanorészecskék felvételét teszteltük agyi endotélsejteken, és megállapítottuk, hogy a nanorészecskék felszínére kötött ligandok szignifikánsan magasabb bejutást eredményeznek, mint a jelöletlen partikulumok. Az előadásban ismertetett kutatásokat az OTKA PD105622 pályázat, valamint az MTA Bolyai János Kutatási Ösztöndíj támogatta

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

Background: Efficient drug delivery across the blood-brain barriers (BBB) is a central problem in pharmaceutical treatment of neurological 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 vesicular or solid nanoparticle drug delivery systems targeting physiological transporters of the BBB hold a great promise. Curcumin extracted from the plant turmeric possesses anti-oxidative, anti-inflammatory and neuroprotective properties and is a potential treatment for different cerebral diseases. However, the clinical application of this natural compound is hampered by its poor water solubility and absorption, rapid metabolism and systemic elimination resulting in low bioavailability. Nanosized, biocompatible and biodegradable vesicles containing Evans blue-albumin as a model molecule and a hydrophobic therapeutic biomolecule, curcumin were prepared and characterized. In addition, fluorescent solid nanoparticles were also examined. The aim of our study was to test the cellular toxicity and penetration of nanovesicles loaded with albumin or curcumin and fluorescent nanoparticles all containing ligands for transporters on culture models of the BBB. Methods: The nanovesicles and fluorescent solid nanoparticles were labelled with different ligands, biotin, a glucose analogue and glutathione. Primary rat and human hCMEC/D3 brain endothelial cells were used as in vitro model systems of the BBB. The cellular toxicity of the nanoparticles was measured by real-time cell microelectric sensing (RTCA-SP, ACEA Biosciences) and MTT assay. The permeability tests were performed on triple co-culture BBB model and hCMEC/D3 cells using Transwell inserts. Brain endothelial uptake of nanoparticles was quantified by fluorescent spectroscopy and visualized by confocal microscopy. Results: No toxicity for loaded or unloaded nanovesicles or solid nanoparticles was found by MTT assay and impedance measurements. The loading of curcumin into liposomes significantly decreased its toxicity for brain endothelial cells at high concentrations. The presence of a glucose analogue in nanovesicles increased the uptake of the model molecule to cultured brain endothelial cells. The brain endothelial uptake of both loaded nanovesicles and solid nanoparticles could be followed by confocal microscopy. Conclusion: Our data indicate that encapsulation of lipophilic or macromolecular drugs into nanovesicles may decrease cellular toxicity and increase uptake and transport at the BBB, however the type of the targeting ligand and its coupling to the nanoparticle may be crucial for efficacy