VÉR-AGY GÁT TRANSZPORTEREK ÉS GYÓGYSZERBEJUTTATÁS A KÖZPONTI IDEGRENDSZERBE

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. A 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

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

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

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

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

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

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

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