Mária DELI
scientific adviser

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Zsófia HOYK research associate
Szilvia VESZELKA senior research associate
Fruzsina WALTER research associate
Alexandra BOCSIK junior research associate
András HARAZIN junior research associate
Mária MÉSZÁROS Ph.D. student
Ilona GRÓF Ph.D. student
Ana Raquel SANTA MARIA Marie Curie research fellow, Ph.D. student
Gergő PORKOLÁB Szent-Györgyi student

BIOLOGICAL BARRIERS AND DRUG DELIVERY

Background

Outer and inner barriers protect the organisms from damaging agents and create homeostasis for physiological functions. Barriers include the epithelium of the intestinal and respiratory systems and the endothelium lining the inner surface of blood vessels. The tight intercellular junctions linking cells in epithelial and endothelial layers and the active efflux transporters in their cell membranes keeping out xenobiotics are the most important elements of the protective function of barriers. These barriers however also impede drug penetration, both from the intestines to blood and from blood to the central nervous system and thereby the effective treatment of several diseases.

Improving drug delivery across barriers

Our goal is to investigate novel methods to enhance drug penetration across barriers. There are three basic strategies studied in our experiments.


1. Modification of drugs: delivery with nanoparticles

Methods to increase the cationic or lipophilic properties of molecules are known and used to improve their pharmacokinetics for a long time. Nanotechnology provides new opportunities to improve drug delivery. In cooperation with the Department of Pharmaceutical Technology of the University of Szeged nanonized forms of the non-steroid antiinflammatory drug meloxicam were investigated (Fig. 1). Better dissolution and quicker penetration of meloxicam across epithelial cell layers were observed for meloxicam nanoparticles obtained by co-griding with excipients (Kürti et al, 2013).



Figure 1: Scanning electron microscopic image of meloxicam nanoparticles obtained by co-griding with excipients.


An ongoing project supported by OTKA (grant no. PD101206) focuses on targeted delivery of molecules across barriers by nanoparticles functionalized with ligands of SLC carriers.


2. Modification of barriers: opening intercellular tight junctions

The tight junctions between the cells forming barriers limit the paracellular flux of cells and molecules. Temporary and reversible opening of tight junctions is one of the ways to improve drug delivery (Deli 2009). The effect of short chain alkylglycerols was examined on the paracellular pathway using an in vitro model of the blood-brain barrier with our partners from the University of Göttingen (Hülper et al., 2013). Alkylglycerols could quickly and reversibly decrease the tightness of endothelial barriers (Fig. 2). Another approach to open the paracellular route in epithelial and endothelial cell layers is the use of peptides acting on tight junction proteins. The effect of tesmilifene, a chemopotentiating agent on barrier properties of endothelial cells is also investigated.



Figure 2: Effect of alkylglycerols (2-O-HG, 1-O-PG) and mannitol on immunostaining for claudin-5 junctional protein in cultured brain endothelial cells. Asterisks show holes formed between endothelial cells. Treatments cause fragmentation, loss of junctional immunostaining and cytoplasmic redistribution. The effect is temporary, 30 min after removal of alkylglycerols, but not mannitol, junctional staining is restored. Bar: 25 µm. (Hülper et al., 2013)



3. Circumvention of barriers – alternative routes

Two aspects of the nasal pathway for drug delivery were investigated. On one hand we demonstrated that a hyaluronic acid containing nasal formulation could significantly improve the penetration of both a test molecule (Horvát et al. 2009) and a biologically active peptide from the nasal cavity to different brain regions (Sipos et al. 2010) and thus circumvent the blood-brain barrier. On the other hand the nasal route as an alternative pathway was also used for the efficient delivery of meloxicam to the systemic circulation (Kürti és mtsai, 2013).


Barrier dysfunction in diseases: barrier protection as a therapeutical target

An important area of research in barrier studies is the investigation of damage and dysfunction of biological barriers, including the blood-brain barrier in different diseases like diabetes mellitus, acute pancreatitis or Alzheimer’s disease. Our aim is to reveal the effect of pathogenic factors on barrier function and to identify protective molecules. We demonstrated, that amyloid peptides contributing to the pathomechanism of Alzheimer’s disease damaged not only neurons and glial cells, but brain microvessel endothelial cells and pericytes, too. This effect could be ameliorated by the polyunsaturated fatty acid DHA (Veszelka et al., 2013). In one of our projects protective molecules are screened and tested against cellular damage induced by methylglyoxal, which plays a role in metabolic syndrome and related cardiovascular complications, and Alzheimer’s disease. The role of cellular interactions between glia, endothelial cells and pericytes, and cytokines is examined in epilepsy using in vivo and in vitro approaches in our bilateral project supported by the Hungarian Academy of Sciences and CINVESTAV, Mexico.


Comprehensive and real-time measurement of cytotoxicity

The first step in the study of cellular actions of pathogenic factors, active agents or pharmaceutical excipients is the determination of their toxic doses or safety profiles. In addition to standard colorimetric tests measuring metabolic activity (MTT assay) or membrane damage in cells (LDH release) in our projects cellular viability is also determined by a real-time impedance based method (RTCA-SP, ACEA Biosciences). We could demonstrate with this novel technique that acute administration of Cremophor EL and RH40 in clinically relevant doses did not damage epithelial only endothelial cells, while prolonged treatment damaged both types of barriers, explaining the side effects of Cremophor containing drugs (Kiss et al., 2013). These observations were confirmed by morphological experiments (Fig. 3).



Figure 3: The effect of 24-h treatment with Cremophors on the immunostaining of tight junction membrane protein claudin-4 in Caco-2 human intestinal epithelial cells. Asterix, holes formed between cells, fragmentation or loss of junctional immunostaining; arrows, changes in claudin immunostaining of cell membranes. Blue color, cell nuclei of living cells; red color, dead cells; green color, claudin immunostaining.; CrEL, Cremophor EL; CrRH, Cremophor RH40; TX, Triton X-100. bar = 10 µm. (Kiss et al., 2013)



New cell culture based models for the complex investigation of barriers

Cell culture based models of the nasal, lung and intestinal epithelium and brain microvessel endothelium are used as tools for our studies on drug delivery and disease modelling. Not only cell monolayers are used, but double and triple co-cultures have been created. We established for the first time a new model of the blood-brain barrier using primary cultures of brain endothelial cells, glia and pericytes to study physiology, pathology and pharmacology of the blood-brain barrier and to estimate brain penetration of drug candidates (Nakagawa et al., 2009; patent: WO/2007/072953). A comparative analysis of endothelial blood-brain barrier models and epithelial surrogate models to estimate brain penetration of drugs was also performed with our partners from Gedeon Richter (Hellinger et al., 2012). In a joint research project with the team of András Dér from the Institute of Biophysics a new integrated microfluidic chip model is being developed which could be a big improvement as compared to the present static models of barriers.


Development and exploitation of the results

New models for the complex study of barriers are not only important for basic research but can be applied by pharmaceutical research and development and therefore be of interest for the drug industry. Research on novel ways to improve drug delivery and targeting, and protection of barriers in pathologies may contribute to better therapy of diseases.

Selected publications

Nakagawa S, Deli MA, Kawaguchi H, Shimizudani T, Shimono T, Kittel A, Tanaka K, Niwa M. (2009) A new blood-brain barrier model using primary rat brain endothelial cells, pericytes and astrocytes. NEUROCHEMISTRY INTERNATIONAL 54:(3-4) pp. 253-263.

Deli MA. (2009) Potential use of tight junction modulators to reversibly open membranous barriers and improve drug delivery BIOCHIMICA ET BIOPHYSICA ACTA-BIOMEMBRANES 1788:(4) pp. 892-910.

Deli MA, Veszelka S, Csiszar B, Toth A, Kittel A, Csete M, Sipos A, Szalai A, Fulop L, Penke B, Ábrahám CS, Niwa M. (2010) Protection of the Blood-Brain Barrier by Pentosan Against Amyloid-beta-Induced Toxicity JOURNAL OF ALZHEIMER'S DISEASE 22:(3) pp. 777-794.

Veszelka S, Tóth AE, Walter FR, Datki Z, Mózes E, Fülöp L, Bozsó Z, Hellinger É, Vastag M, Orsolits B, Környei Z, Penke B, Deli MA. (2013) Docosahexaenoic acid reduces amyloid β-induced toxicity in cells of the neurovascular unit. JOURNAL OF ALZHEIMER'S DISEASE 36:(3) pp. 487-501.

Hülper P, Veszelka S, Walter FR, Wolburg H, Fallier-Becker P, Piontek J, Blasig IE, Lakomek M, Kugler W, Deli MA. (2013) Acute effects of short-chain alkylglycerols on blood-brain barrier properties of cultured brain endothelial cells BRITISH JOURNAL OF PHARMACOLOGY 169:(7) pp. 1561-1573.

Tóth AE, Walter FR, Bocsik A, Sántha P, Veszelka S, Nagy L, Puskás LG, Couraud PO, Takata F, Dohgu S, Kataoka Y, Deli MA. (2014) Edaravone protects against methylglyoxal-induced barrier damage in human brain endothelial cells. PLOS ONE 9:(7) Paper e100152. 14 p.

Kiss L, Hellinger É, Pilbat A-M, Kittel Á, Török Z, Füredi A, Szakács G, Veszelka S, Sipos P, Ózsvári B, Puskás LG, Vastag M, Szabó-Révész P , Deli MA. (2014) Sucrose esters increase drug penetration, but do not inhibit P-glycoprotein in Caco-2 intestinal epithelial cells. JOURNAL OF PHARMACEUTICAL SCIENCES 03:(10) pp. 3107-3119.

Walter FR, Veszelka S, Pásztói M, Péterfi ZA, Tóth A, Rákhely G, Cervenak L, Ábrahám CS, Deli MA. (2015) Tesmilifene modifies brain endothelial functions and opens the blood-brain/blood-glioma barrier. JOURNAL OF NEUROCHEMISTRY 134:(6) pp. 1040-1054.

Sántha P, Veszelka S, Hoyk Z, Mészáros M, Walter FR, Tóth AE, Kiss L, Kincses A, Oláh Z, Seprényi G, Rákhely G, Dér A, Pákáski M, Kálmán J, Kittel Á, Deli MA. (2016) Restraint stress-induced morphological changes at the blood-brain barrier in adult rats. FRONTIERS IN MOLECULAR NEUROSCIENCE 8: Paper 88. 15 p.

Bocsik A, Walter FR, Gyebrovszki A, Fülöp L, Blasig IE, Dabrowski S, Ötvös F, Tóth A, Rákhely G, Veszelka S, Vastag M, Szabó-Révész P, Deli MA. (2016) Reversible opening of intercellular junctions of intestinal epithelial and brain endothelial cells with tight junction modulator peptides. JOURNAL OF PHARMACEUTICAL SCIENCES 105:(2) pp. 754-765.

Helms HC, Abbott NJ, Burek M, Cecchelli R, Couraud P-O, Deli MA, Förster C, Galla HJ, Romero IA, Shusta EV, Stebbins MJ, Vandenhaute E, Weksler B, Brodin B. (2016) In vitro models of the blood-brain barrier: An overview of commonly used brain endothelial cell culture models and guidelines for their use. JOURNAL OF CEREBRAL BLOOD FLOW AND METABOLISM 36:(5) pp. 862-890.

Walter FR, Valkai S, Kincses A, Petneházi A, Czeller T, Veszelka S, Ormos P, Deli MA, Dér A. (2016) A versatile lab-on-a-chip tool for modeling biological barriers. SENSORS AND ACTUATORS B-CHEMICAL 222: pp. 1209-1219.