**2. Fundamentals of BBB**

## **2.1 Cellular properties of Blood Brain Barrier**

BBB consisted of a monolayer of brain micro vascular endothelial cells (BMVEC) joined together by much tighter junctions than peripheral vessels and formed a cellular membrane which known as the main physical barrier of BBB (Abbott, 2005; Cardoso et.al., 2010). The main characteristics of this cellular membrane are, uniform thickness, no fenestrae, low pinocytotic activity, continues basement membrane and negative surface charge. In addition to the BMVECs, the neurovascular unit consisted of the capillary basement membrane, pericytes, astrocytes and microglia. The BMVECs are surrounded by a basement membrane which composed of structural proteins (collagen and elastin), specialized proteins (fibronectin and laminin) and proteoglycans. This structural specificity gives the basement membrane a cell establishment role. Pericytes are cellular constituents of microvessels

Blood Brain Barrier Permeation 5

suggested for tight junctions. Below the tight junctions, actin filaments (including cadherins and catenins) linked together and form a belt of adherence junctions. In addition to the contribution in the barrier function some other events such as adhesion of BMVECs to each other, the contact inhibition during vascular growth, the initiation of cell polarity and the regulation of paracellular permeability have been suggested for adherence junctions. A dynamic interaction between tight junctions and adherence junctions through signalling pathways regulate the permeability of BBB. These signalling routes mainly involve protein kinases, members of mitogen – activated protein kinases, endothelial nitric oxide synthase and G-proteins. Dynamic interactions between these pathways control the opening and closing of the paracellular route for fluids, proteins and cells to move across the endothelial cells through two main types of signal transduction procedures (e.g. signals from cell interior to tight junctions to guide their assembly and regulate their permeability, signals transmitted from tight junctions to cell interior to modulate gene expression, proliferation and differentiation). The molecular mechanisms of these interactions can be found in the literature (Ballab et al., 2004; Abbott et al., 2006). In addition to the proteins with enzymatic activities, there are other specific proteins (drug efflux transporters, multi drug resistance proteins, organic anion transporting polypeptides) work as BBB transporters which are responsible for rapid efflux of xenobiotics from the CNS (Losscher & Potschka, 2005) and

The combined effect of the special cellular and molecular properties of central nervous system result in the specific barrier functions of BBB which is important for preventing CNS from harmful xenobiotics. Because of these properties drug delivery to the CNS is among the most challenging drug development areas. In order to develop successful drug candidates for CNS disorders drug uptake mechanisms should be studied. In the next

delivery of the essential nutrients and transmitters to the brain.

section, these mechanisms are briefly reviewed.

Fig. 2. Tight junctions and adherent junctions.

including capillaries and post capillary venules that covered about 22-32% of the capillaries and shared the same basement membrane. Pericytes are responsible for a wide variety of structural and non-structural tasks in BBB. In summary they synthesis some of structural and signalling proteins and they are involved in the BMVECs proliferation, migration and differentiation. More details and references about pericytes role in BBB can be found in the literature (Cardoso et al., 2010). Fine lamellae closely opposed to the outer surface of the capillary endothelium and respective basement membrane formed by astrocytes end feet. Like pericytes, astrocytes involve in various functional and structural properties of neurovascular unit.

Microglia is immunocompetent cells of the brain that continuously survey local micro environment with highly motile extensions and change the phenotype in response to the homeostatic disturbance of the CNS (Prinz & Mildner, 2011). The interactions of brain micro vascular endothelial cells with basement membrane, neighbouring glial cells (microglia and astrocytes), neurons and perivascular pericytes leads to specific brain micro vascular biology. Presence of matrix adhesion receptors and signalling proteins form an extensive and complex matrix which is essential for maintenance of the BBB (Cardoso et al., 2010). Figure 1 shows a schematic illustration of neurovascular unit and BBB cellular components.

Fig. 1. Schematic illustration of the neurovascular unit and BBB cellular components adopted from (Cardoso et al., 2010).

#### **2.2 Molecular properties of BBB**

The BMVECs assembly are regulated by molecular constituents of tight junctions, adherence junctions and signalling pathways. Tight junctions are highly dynamic structures which are responsible for the barrier properties of BBB. Apical region of the endothelial cells sealed together by tight junctions and paracellular permeability of BMVECs are limited by them. Structurally tight junctions formed by interaction of integral transmembrane proteins with neighbouring plasma membrane. Among these proteins junction adhesion molecules, claudins and occludins (inter membrane) which bind to the cytoplasmic proteins (e.g. zonula occludens, cinguline, …) are well studied and their role in tight junctions and BBB have been evaluated (Figure 2). Beyond the main role in physical restriction of BBB, other functions such as control of gene expression, cell proliferation and differentiation have been

including capillaries and post capillary venules that covered about 22-32% of the capillaries and shared the same basement membrane. Pericytes are responsible for a wide variety of structural and non-structural tasks in BBB. In summary they synthesis some of structural and signalling proteins and they are involved in the BMVECs proliferation, migration and differentiation. More details and references about pericytes role in BBB can be found in the literature (Cardoso et al., 2010). Fine lamellae closely opposed to the outer surface of the capillary endothelium and respective basement membrane formed by astrocytes end feet. Like pericytes, astrocytes involve in various functional and structural properties of

Microglia is immunocompetent cells of the brain that continuously survey local micro environment with highly motile extensions and change the phenotype in response to the homeostatic disturbance of the CNS (Prinz & Mildner, 2011). The interactions of brain micro vascular endothelial cells with basement membrane, neighbouring glial cells (microglia and astrocytes), neurons and perivascular pericytes leads to specific brain micro vascular biology. Presence of matrix adhesion receptors and signalling proteins form an extensive and complex matrix which is essential for maintenance of the BBB (Cardoso et al., 2010). Figure 1 shows a schematic illustration of neurovascular unit and BBB cellular components.

Fig. 1. Schematic illustration of the neurovascular unit and BBB cellular components

The BMVECs assembly are regulated by molecular constituents of tight junctions, adherence junctions and signalling pathways. Tight junctions are highly dynamic structures which are responsible for the barrier properties of BBB. Apical region of the endothelial cells sealed together by tight junctions and paracellular permeability of BMVECs are limited by them. Structurally tight junctions formed by interaction of integral transmembrane proteins with neighbouring plasma membrane. Among these proteins junction adhesion molecules, claudins and occludins (inter membrane) which bind to the cytoplasmic proteins (e.g. zonula occludens, cinguline, …) are well studied and their role in tight junctions and BBB have been evaluated (Figure 2). Beyond the main role in physical restriction of BBB, other functions such as control of gene expression, cell proliferation and differentiation have been

neurovascular unit.

adopted from (Cardoso et al., 2010).

**2.2 Molecular properties of BBB** 

suggested for tight junctions. Below the tight junctions, actin filaments (including cadherins and catenins) linked together and form a belt of adherence junctions. In addition to the contribution in the barrier function some other events such as adhesion of BMVECs to each other, the contact inhibition during vascular growth, the initiation of cell polarity and the regulation of paracellular permeability have been suggested for adherence junctions. A dynamic interaction between tight junctions and adherence junctions through signalling pathways regulate the permeability of BBB. These signalling routes mainly involve protein kinases, members of mitogen – activated protein kinases, endothelial nitric oxide synthase and G-proteins. Dynamic interactions between these pathways control the opening and closing of the paracellular route for fluids, proteins and cells to move across the endothelial cells through two main types of signal transduction procedures (e.g. signals from cell interior to tight junctions to guide their assembly and regulate their permeability, signals transmitted from tight junctions to cell interior to modulate gene expression, proliferation and differentiation). The molecular mechanisms of these interactions can be found in the literature (Ballab et al., 2004; Abbott et al., 2006). In addition to the proteins with enzymatic activities, there are other specific proteins (drug efflux transporters, multi drug resistance proteins, organic anion transporting polypeptides) work as BBB transporters which are responsible for rapid efflux of xenobiotics from the CNS (Losscher & Potschka, 2005) and delivery of the essential nutrients and transmitters to the brain.

The combined effect of the special cellular and molecular properties of central nervous system result in the specific barrier functions of BBB which is important for preventing CNS from harmful xenobiotics. Because of these properties drug delivery to the CNS is among the most challenging drug development areas. In order to develop successful drug candidates for CNS disorders drug uptake mechanisms should be studied. In the next section, these mechanisms are briefly reviewed.

Fig. 2. Tight junctions and adherent junctions.

Blood Brain Barrier Permeation 7

In this method, substances (e.g. macromolecules) are engulfed by membrane and pass through the cell by vesicles and release in the other side (Kerns & Di, 2008). Endocytosis occurs via two main methods: bulk phase endocytosis (fluid phase or pinocytosis) and mediated or facilitated endocytosis (receptor and absorptive mediated). Fluid phase endocytosis is a nonsaturable, non-competitive and non-specific method for uptake of extra

Receptor mediated endocytosis facilitates the larger essential molecules uptake selectively using specific receptors present in luminal membrane. Hormones, growth factors, enzymes

Absorptive mediated endocytosis is based on an electrostatic interaction between negatively charged plasma membrane luminal surfaces (glycocalyx which is a negatively charged proteoglycan or glycosaminoglycan) with cationic substances (e.g. peptides) and uptake it in a vesicle into the endothelial cell and release it on the other side (Figure 4)

This has lower affinity and higher capacity than receptor mediated endocytosis (Alam et al., 2010). Mechanism of vesicle formation (caveolin dependent, dynamin dependent and caveolin- dynamin independent) is not discussed in this chapter and more details could be

Fig. 3. Free and facilitated passive diffusion.

found in the literature (Lajoie et al., 2010).

cellular fluids which is temperature and energy dependent.

and plasma proteins are targets for specific receptors (Pardridge, 2007).

**3.2 Endocytosis** 

(Ueno, 2009).
