Concept Of Nanotechnology And Nanoscale Biology Essay

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The concept of nanotechnology is wholly defined as 'Research and technology development at the atomic, molecular or macromolecular levels, in the length scale of approximately 1 - 100 nanometer range, to provide a fundamental understanding of phenomena and materials at the nanoscale and to create and use structures, devices and systems that have novel properties and functions because of their small and/or intermediate size' by the National Science Foundation, NSET, 2000 [1]. The root of its discovery is uncertain; however early documentations suggest Richard Feynman (1959) was of the first to envisage the idea of manipulating and synthesising matter on a nanometre scale i.e. the synthetic assembly of atoms. Feynman's legendary speech 'There's Plenty of Room at the Bottom' has inspired scientists since. The fundamental concepts outlined by Feynman were not branded until 1974 when a Japanese professor, Norio Taniguchi, first introduced the concept as 'Nano-Technology'.

The now rapidly advancing field of nanotechology is currently under extensive research for drug delivery, particularly in cancer therapy. The nanosize (figure 1) permits access into cells as well as cellular compartments. Nanoparticles (NPs) used for medical purposes generally observe a size definition of ≤100 nm size, although ˃100nm particles may be used when sufficient drug loading is required. Attractive features of NPs as drug delivery devices include: a large surface to mass ratio compared with other particles and an ability to interact, adsorb and contain other compounds including biologically active agents [2]. The appearance of the first commercial nanoparticle drug product, Abraxane (paclitaxel contained in albumin particles) for the treatment of breast cancer, was in early 2005. Current advancements continue to promise further breakthroughs in the development of drugs for cancer and for the delivery of drugs across the blood brain barrier.

The pharmaceutical industry is still inexperienced with this novel technology and is yet to recognise the full potential of NPs. More recently potential hazards and destructive aspects associated with nanosystems have been uncovered. Jong and Borm have reviewed the toxicological hazards of NPs in a detailed article [2]. The toxicity generally depends on the composition of the NP; however, engineered particles which are airborne may present hazardous consequences by inhalation. Extensive safety testing and evaluations of the implications of NP systems are required.

The nano unit is difficult to fully appreciate without contextualising its size relative to its superior units. The nanometre in perspective is demonstrated in figure 1 below.

Figure 1- Just how small is the nanoscale? Size comparison of a flea & a human hair to a molecular structure in the nanometre range. Adapted from reference [3]

1.2. Advancing drug delivery

Another area of current research is that of alternative, non-invasive routes of systemic delivery of drugs that cannot be delivered orally, or have undesirable pharmacokinetic profiles/ pharmaceutical properties or other disadvantageous properties as oral formulations [4]. The search for new routes has lead to discoveries of the potential exploitation of pulmonary, transdermal (TD), sublingual (SL) and intranasal (IN) routes. Of the most promising routes the successful development of a number of drugs has been formulated for TD, SL and IN use. One example is the intranasal delivery of triptans (e.g. Imitrex® manufactured by GSK) which provides faster relief of migraines in comparison to its oral equivalent. In contrast, there are currently no marketed products for systemic delivery via the pulmonary route. The short market life of Exubera® (inhaled insulin) [5] has also raised concerns into the reservations of long term use of the pulmonary route.

It is believed that the pulmonary route has many potential benefits as a portal of entry for systemic drugs. Generally, there is a higher permeability to small molecules in comparison to the gastrointestinal mucosa [6]. Evidence also suggests that the lungs display high bioavailabilities and rapid absorption of small lipophilic compounds, indicating that there is high permeability and low metabolising activity [6] [7] [8].

So why is there no drug preparation currently available for systemic pulmonary delivery? It is appropriate to deduct that this area of research is very complex. Some of factors that have to be considered in the development of an inhalation drug include: accurate, reproducible drug dosing, lower airway deposition (and hence correct aerosol characteristics), dissolving and partitioning of drug through the epithelial layer and release into the systemic circulation [4].

Incorporating nanotechnology to create a suitable nanosystem for systemic drug delivery through the lungs may allow targeted delivery and sustained release of drug. Research suggests that dry powder aerosols formulated with NPs offer stable and easy to use drug devices which hold vast potential for systemic delivery as well as local delivery for diseases such as tuberculosis [9]. However, prior to this there is still a great deal that is unknown in terms of the fate of particles within and beyond the lungs. It is necessary to continue committed research to fully exploring the potentials of pulmonary drug delivery and its long term influences.

2. The human airway

2.1. Structure of the airway

The respiratory system (figure 2) is intricately designed to supply oxygen from the atmosphere to the alveoli and simultaneously remove carbon dioxide. On any one day, a person may inhale from 10,000 to 20,000 litres of air albeit with unwanted foreign particles. Inhalation is initiated as air enters the nasal/oral cavity, it travels to the pharynx, larynx ('voice box') into the trachea, down the branching bronchi, which successively decrease into bronchioles and finally into the terminal airspaces; the alveolar sacs. The many millions of alveoli present in the lungs present the air to a massive surface area in this gas-exchanging region for exchange to occur. In contrast, the barrier separating the air and blood is very small (~0.2 µm thickness).

Figure 2 - Structure of the respiratory system. Adapted from reference [10]

The vast yet delicate design of the respiratory tree contributes to the efficient delivery of oxygen and concurrent filtration of foreign particles. Macro particles are filtered by hairs and mucous found in the nostrils. Finer particles, dust, pollen and smoke often reach the nasal cavities and are trapped by mucous. The trachea contains ciliated and mucous secreting epithelial cells, which act to seize further foreign material such as dust. The dust adheres to sticky mucous and cilia help to propel it back up to the trachea where it is either swallowed or expectorated.

2.2 The function of the airway

2.2.1 Gaseous exchange

In the adult lung there are approximately 300 to 600 million alveoli which have the capacity to expand to 250-300μm [10] creating a huge surface area. Air flow is reduced rapidly within the respiratory tree causing air to move into the alveoli by diffusion. Each alveolus is closely associated with a network of capillaries, containing deoxygenated blood from the pulmonary artery. The alveolar epithelial wall and capillary endothelial wall is very thin allowing a rapid exchange of gases by passive diffusion along a concentration gradient. Carbon dioxide moves into the alveolus as the concentration is considerably lower in the alveolus than in the blood whilst oxygen moves out of the alveolus and into the circulation. The continuous flow of blood through the capillaries prevents saturation with oxygen.

3.3 The fate of an inhaled particle: a matter of size

The fate of a particle inspired into the lungs depends on many factors. Once the particle has overcome nasal barriers and protection from the mouth and oropharynx it moves at random through the gas. Its transport through the respiratory tree is influenced by factors such as its size, density, shape, charge, and surface properties [10]. Mechanical and electrical forces acting on the particles make their route into the lungs different from that of gases. Electrical forces act only on charged particles and since pharmaceutical particles tend not to be heavily charged, discussion of mechanical forces are most relevant. The principal mechanical forces are gravity, inertia and 'impulse transfer' from collisions with gaseous molecules [10].

The architecture of the lung sub serves to filter out particles of varying size at varying regions of the respiratory tract. Decreasing the particle size increases the distance it can travel [11]. An ultrafine particle (less than 0.1µm) moves exclusively by diffusion. Diffusional particle transport is time dependent and therefore ultrafine particles are deposited in regions of maximum residence time i.e. the gas exchange region where the residence time of the inhaled air is longer. [10]

Particles ranging from 0.1-1 µm are carried by gravitational as well as diffusional transport and larger particles are transported by inertial transport [11]. Inertia, in terms of a particle, refers to its constant movement at its current speed and direction until a force causes a change in its speed/direction. This cause may result in impaction or sedimentation (particles sinking under opposing forces of gravity) at respiratory surfaces. Inertial transport is speed dependent; therefore particles are deposited at the site of maximum particle speed i.e. the upper airways. The diagram (figure 3) below graphically represents the deposition of particles in the lung according to size.

http://pats.atsjournals.org/content/vol1/issue4/images/large/200409-046JAf2.jpeg

Figure 3 - Predicted patterns of deposition. Adapted from reference [11]

4. Amid the inside and out; the epithelial boundary

4.1. Epithelial cells

The epithelium is essentially a barrier placed between two separate environments. Epithelial cells are found on all mucosal surfaces including the respiratory (figure 4), gastrointestinal and genitourinary tracts, separating the inside from the outside, as well as on the body's external surface, organs, the lining of body cavities, tubes and ducts. Structurally, the epithelial barrier is organised as a tightly connected continuous layer of cells characterised by distinct apical and basolateral surfaces with specific structural modifications. The functions of epithelial cells include absorption and secretion, in addition to other specialised functions (ciliary, sensory and contractility). However, the ultimate function of the epithelial layer is to serve as a powerful barrier for protection. [12] [13]

= Tight junctions

Basal membrane

Apical membrane

Figure 4- Shows the diversity of epithelial cells present in various regions of the respiratory surface and their size characterisations and comparison of a typical aerosol particle. Adapted from reference [14]

The morphology of epithelium varies depending upon its site and is often reflected in its functional requirements. It can be generally characterised into simple (single layers) and stratified (more than two layers).

4.1.1 Alveolar epithelium

The airways can be split into proximal conducting airways (towards the beginning) and distal conducting airways (the distant end). The epithelium lining the proximal airways is pseudo-stratified columnar and consists of three major cell types: basal cells, ciliated cells and goblet (secretor) cells. The internal nasal region is lined by epithelium that is similar to the proximal conducting airways in terms of function and morphology. Goblet cells secrete mucus which coats the epithelium and acts as a trap to provide protection from the constant contact with noxious external environmental agents. Mucus also contains natural antibiotics (definsins) and lysosymes which help to destroy bacteria [10] [13].

A fourth cell, the Clara cell, is a secretory cell, which becomes increasingly dominant as the airway diameter decreases. It is thought to be a progenitor cell for the goblet cell as well as for type II alveolar cells and the ciliated cell [15]. The density of ciliated cells and goblet cells begins to decrease towards the periphery; ciliated cells are only present up to the bronchioli whereas few goblet cells are found in the bronchioli.

The distal air spaces of the lung are lined with a continuous epithelium consisting of two major types of alveolar epithelial cells, type I and type II pneumocytes, joined by tight junctions. This alveolar layer offers a strong barrier against diffusion of small molecules compared to a more permeable, endothelial barrier lining the lung vasculature [16] [17]. Tight junctions (see figure 4) are also an important barrier, preventing transport between interacting cells. Tight junctions are sites between cells that are closely fused together. It is thought that they may be tighter in alveoli compared to the rest of the airway tight junctions [18]. The alveolar epithelium is covered by two layers which constitutes the epithelial lining fluid (EPL). Firstly, a surfactant layer and a second layer containing a number of substances including apoprotein, IgG, albumin, phospholipids and carbohydrates.

Type I epithelial cells are large squamous cells which are extremely thin and make up more than 98% of the alveolar surface [19].Type II cells are smaller, cuboidal cells that are present in a higher number (60%) [20] compared to Type I cells yet only constitute less than 2% of the alveolar surface [19]. Type II cells have many functions including synthesis and reuptake of surfactant, regeneration of epithelium following injury to the alveoli surface and transportation of water [21].

5. Transport pathways

5.1 Small molecules and solutes etc.

The epithelial lining the airways are selectively permeable to molecules fated to be absorbed into or released out of the body. There are two routes small molecules may utilise to transport themselves; the paracellular and transcellular routes (figure 5).

Paracellular transport is the selective, passive movement of small molecules between cells through intracellular tight junctions depending on their size and charge. The flow across the cell is linear to the solute molecular size and becomes non-linear when the hydrated molecular radius is increased.

Apical membrane

Basal membrane

nrd1067-f2.gif

Figure 5 - Transport pathways across the intestinal epithelium: (a) transcellular transport (b) paracellular transport (c) transcytosis: receptor independent/receptor mediated endocytosis (RME). Adapted from reference [22]

Passive transport via the transcellular route involves transport of small molecules such as glucose and ions (e.g. Na+) as well as water through the epithelial cell traversing through the apical and basal membranes. Water channels have been found for transportation of water [23], its movement is driven by an osmotic gradient to remove excess water from distal air spaces of the lung. Water is also transported from capillaries into the airspace, this is believed to be in order to replace evaporative losses of water and maintain hydration [23].

The transport of macromolecular systems is likely to be excluded from the paracellular pathway due to size restrictions. However, macromolecules can be facilitated through the epithelial cell via active transcellular transport used to transport larger molecules such as proteins across the epithelial cell

5.2 Macromolecular transport

Endocytosis and exocytosis are active transcellular mechanisms. Endocytosis is the invagination of the epithelial cell surface membrane around macromolecular material, from the external cellular environment, pocketed inwardly and its subsequent migration towards the cells interior. The opposite i.e. the discharge of material across the cell and release of its contents on the opposite side into the external environment is exocytosis. Endocytosis can be classified in terms of the type of cargo that is being carried as well as the nature of the protein vesicle coat. Endocytic pathways can be categorised into clathrin dependent (5.2.1) and clathrin independent pathways (5.2.2).

Transcytosis is essentially the coupling of endocytosis and exocytosis in order to carry macromolecular goods across cells. It is a sophisticated process which maintains the composition of both the external and internal cellular environments via membrane bound vesicles representing the vehicle for transfer of the goods. Transcytosis can occur in both apical to basal and basal to apical directions. It is mostly recognised as a dominant mode of transport in sheets of polarised epithelial cells, although it is widely adopted by most if not all cells of the body, trafficking a range of molecular goods throughout the body. The ligands involved macromolecular transports across the alveolar epithelial cell layer are outlined in section 5.3.

Transcytosis can generally be categorised into receptor mediated (see section 5.2.1) and receptor in dependent. The latter includes fluid phase (see section 5.2.1) and adsorptive uptake into cells. Adsorptive uptake involves electrostatic interaction between receptor and ligand.

5.2.1 Clathrin dependent

The clathrin coat was first identified in 1975 [24]. Today it is the most characterised vesicular coat in terms of its biochemical composition and role in internalisation and transcytosis. The structural form of clathrin comprises of six molecules three of which are heavy-chain and three light chains. This is known as a triskelion, which assembles to form a closed confinement with prominent pentagonal and hexagonal surfaces (figure 38).

As well as clathrin, the coated pits also contain various adaptor proteins. These proteins help to assemble clathrin coats and concentrate cargo receptors in pits from the golgi apparatus and cell membrane [25], physically connecting cargo receptors to the clathrin coat. Adaptors are part of a diverse collection of proteins that recognise receptors with varying types of cargo. The adaptor proteins (APs) comprise a family with AP1, AP2, AP3 and AP4 proteins which are closely related. The structural components of these proteins have presented key information on the functions. AP2 is linked to RME in a complex sequence [26]. It is intimately associated with the coat assembly as clathrin assembles onto the adaptors constituting the external layer of the coat. This stabilises the curve in the cellular membrane which develops into a pit. The internalisation of receptors present on cellular membranes alongside their respective ligands, adaptors, regulatory and accessory molecules and clathrin occurs. This is known as receptor mediated endocytosis (RME); there are various membrane proteins constantly being internalised via RME.

Figure 8 - A diagrammatic summary of the events involved in endocytosis via clathrin coated pits. Adapted from figure [26]

The clathrin coated pit has a very short life, invaginating within a minute after being assembled and subsequently forming a clathrin coated vesicle. The vesicle transiently sheds its coat and fuses with early endosomes. Fluid from the external cellular environment is contained within the pits and also invaginates ensuring that dissolved substances are internalised. This is known as fluid phase endocytosis.

Clathrin coated pits exist in both alveolar type I and type II cells. Whilst the presence of clathrin independent caveolin pathways (5.2.2) was thought to be only in type I cells and endothelial cells, recent evidence suggests otherwise [27].

5.2.2 Clathrin independent

There are a number of clathrin independent pathways; the best-studied is the caveolar pathway. Caveolae are flask-shaped invaginations (figure 9) present on the plasma membrane that form vesicles of around 50-100nm in size which are 'smoother' than the clathrin coated pits. These are present in type I pneumocytes in high abundance.

Structurally, caveolae is composed of cholesterol, sphingolipids, and various lipid-modified and transmembrane proteins [28]. Areas of the cell membrane differ in rigidity depending on the various concentrations of cholesterol, sphinolipids and phospholipids. Increasingly rigid regions are known as 'lipid rafts' (figure 9), these areas are biochemically distinct to the rest of the membrane however they are indistinctive from caveolae, which are similarly concentrated in cholesterol and sphingolipids [49]. Caveolae can be recognised from the formation of cave like invaginations. Caveolins are a family of proteins that are responsible for this difference between caveolae and lipid rafts. The family members include Caveolin-1,-2, and -3 [30] [31]. The abundance of each type of caveolin varied from cell type, only smooth muscle cells express all three proteins [31].

Figure 9 - Representation of a caveolae 'flask' and a lipid raft. Adapted from reference 32

The caveolin proteins have numerous properties which enable them to selectively localise within and induce invagination of caveolae. Cav-1 was demonstrated to have a high binding affinity for sphingolipids and cholesterol [33] [34]. Caveolins may also oligomerize forming units containing 14-16 proteins and subsequently form oligomer-oligomer interactions resulting in large complexes [32]. The above properties are thought to provide a suitable environment for the formation of invaginations in the sub 100 nm range. Researchers therefore considered caveolae for vehicles for the intracellular movement of macromolecules. It has since been revealed that cells use this function of caveolae to selectively take in molecules ranging in size (folates to albumin). Further research has now shown that Caveolae invaginations migrate from the apical to the basal membrane as a route of transcytosis. It is interesting to note that viruses as well as bacteria also exploit this pathway [32].

5.3 Ligands

Biological ligands have been found to utilise transcytotic pathways in type I and type II alveolar epithelial cells. Immunoglobulin G and albumin are ligands which have been found to be transported across alveolar epithelium intact and are of the most studied proteins that cross the epithelial barrier

Figure 10 - reference Adapted from ?

5.3.1 IgG and its FcRn receptor

Immunoglobulin G (IgG) is the major type of antibody, found in all body fluids. It is the smallest but most prominent class of antibody (75% to 80%) in the body [35]. IgG antibodies are important in fighting bacterial and viral infections. In the lung, they have been found in bronchoalveolar lavage (BAL) fluid, which is fluid used to assess biochemical and inflammatory changes in and effects of therapy on the interstitial lung tissue, it is obtained by washout of the alveolar compartment of the lung.

It was initially discovered that IgGs are intestinally absorbed by suckling newborn mice from the mothers breast milk providing passive immunity [36]. However, the size of these proteins is too large to cross the epithelium and so the absorption of IgG could not be explained by passive diffusion, transport through intercellular tight junctions or fluid phase endocytosis. Binding to the FcRn receptor present on the neonatal intestinal brush border allowed IgG molecules to 'hitch a ride' across the epithelium [36] [37]. This complementary trafficking receptor of IgG is a MHC class I-related Fc-receptor.

Figure 11- Diagram showing the FcRn present on the bruch border in the intestinal epithelium of rodents, its binding with IgG and subsequent transcytosis. Adapted from [38]

Blumberg then found that the FcRn receptor is also present in adult mice which has lead to further research which suggests that it is present in various anatomical sites in the adult human [39][40][41][42]. Transport studies via the FcRn receptor have been carried out using rat epithelial monolayers. It was found that IgG is transported in the apical to basal direction as well as in the basal to apical direction [43] although the former is far more superior. In a study by Kim et al [44] investigating unidirectional fluxes of rat IgG labelled with biotin (biot-rIgG), showed saturation of FcRn receptors when the concentration of rIgG was raised. The flux of biot-rIgG from apical to basal decreased drastically when competing with excess unlabelled rat Fc in the apical fluid. This suggests that the IgG receptor specifically interacts with the Fc region of IgG and no influence by other parts of IgG (Fab or F(ab')2) was demonstrated [44].

A study by Gerburg M. Spiekermann et al [45] confirmed the above findings. The characteristics of FcRn binding of IgG and its transport across mucosal epithelial cells in human and mouse lung in vivo were investigated. A fusion protein was constructed using: an Fc fragment of murine IgG and erythropoietin (Epo), which is a hormone responsible for stimulating red blood cell production. This fusion protein (Epo-Fc) was utilised as a tracer to monitor transport. Its function with and without the presence of a competing ligand (IgG) was examined. In the presence of competition the activity of Epo-Fc was completely extinguished. This provides further evidence that transport of Epo-Fc depends on receptor mediated transcytosis probably via the FcRn receptor and the Fc portion of IgG is responsible for receptor binding.

The expression of FcRn receptor differs in terms of sites across species. In rats and cows FcRn expression is predominantly in the bronchiolar and alveolar regions of epithelium, while primates have a higher expression of FcRn in the upper airway epithelium. [46, 47, 48, 49]. A study by Dumont et al [50] has accomplishedly demonstrated the systemic delivery of Epo-Fc (Fc from human IgG) through the upper airways in a phase I clinical study performed on healthy male volunteers. However, focus is still concentrated on the lower airways following in vitro evidence of IgG transport and access to a larger surface area.

Evidence presented in studies such as those described above [44] [45] has caused speculation amongst scientists to further exploit the FcRn receptor. Coupling biological agents with Fc fragments could be used to exploit FcRn mediated transport for systemic drug delivery.

5.3.2 Albumin and its gp60 receptor

Human serum albumin (HSA), the most abundant protein to be found in human blood plasma is also present in BAL fluid at a concentration of ~8-10% of that in blood [51]. There is evidence to suggest that it is transported through endothelial cells via transcytotic mechanisms mediated through a specific albumin binding receptor known as the gp60 receptor [52] [53]. Albumin absorption (although relatively slow) across alveolar epithelium models in vitro suggests this receptor may also be present on alveolar epithelial cells [54] [55]. The mechanism of albumin transport is still largely unknown.

Studies using radiolabelled bovine serum albumin ([3H]- methylated BSA) across rat alveolar epithelial monolayers show that the flux of BSA from the apical to basal membrane was greater than from the basal to the apical direction. Labelling BSA with methyl groups showed no advantage over labelling with FITC as both resulted in similar rates of flux of intact albumin [55] [56].

John et al identified the gp60 receptor in rat type II alveolar cells in vitro using the anti-gp60 antibody: cy3 [57]. They found that cross-linking gp60 with antibodies stimulated endocytosis in type II alveolar epithelial cells. This phenomenon was previously described relative to the gp60 receptor in endothelial cells which enhances trancystosis by stimulating Src protein tyrosine kinase [28] [59]. This activation is dependent on temperature as well as duration of cross-linking. Cross-linking for a shorter period of time significantly increased endocytosis of fluorescently labelled albumin, however cross-linking for over one hour lead to damage of gp60 and consequently no albumin uptake [60].

The review by Kim et al [56] suggests that passive diffusion and non-specific adsorptive endocytosis of albumin is unlikely. The latter is thought to be true since the net negative charge owing to cationic molecules on cellular membranes would prevent adsorption of albumin since it is also negatively charged at physiological pH. From the presentation of their findings it may be assumed that albumin is largely taken up by epithelial cells through transcytotic mechanisms.

However, some studies advocate that the main route of albumin clearance in the alveoli is through paracellular mechanisms. Wangensteen et al [61] studied the transport of riboflavin conjugated to albumin vs. albumin alone. They found that [3H]riboflavin-albumin was cleared faster than the [3H]albumin tracer by twofold after instilling into the lungs of anesthetized rats. This suggests that the transport of both of these may be different. Another experiment by them also measured the affects of transcytosis inhibitors (nocodazole and monensin) in isolated perfused rabbit lungs on the permeability-surface area product (PS) in riboflavin-albumin and albumin transport. The PS product of sucrose was used as a marker for paracellular transport. Both the product for sucrose and albumin transport was unaffected by endocytosis inhibitors. Other studies also support the idea of paracellular transport of albumin [62]. Further studies are required to establish the dominant transport pathways of albumin and alveolar epithelial cells.

5.3.3 Folate and its folate receptor

Folates are a group of compounds also known as vitamin B9 that are an important constituent of the human diet. Folic acid is the non-physiological, synthetic, oxidized form of the vitamin. Tetrahydrofolate (THF) and dihydrofolate, in their biologically active form, act as carriers/donors of methyl groups and function as important cofactors in the synthesis of purines and pyrimidines [63]. The total body content of folate is estimated to be 38-96 mg [63].

Folate carriers are expressed in the epithelium of various organs. At physiological pH, folates are hydrophilic and anionic in nature which prevents their diffusion in a passive manner. They are transported by various carriers including the reduced folate carrier (RFC). Here, the folate receptor (FR) is of interest in terms of its presence on the apical surface of type I and type II alveolar epithelial cells [7]. The glycosyl phosphatidylinositol (GPI)-anchored glycopoly-peptide FR binds to 5-methyltetrahydrofolate form as well as folic acid [1]. The affinities of different folate compounds for the folate receptor varies from a 2-fold to 100-fold affinity, although folic acid consistently binds with a high affinity [64] [65].

The FR has been found in caveolae [66] and undergoes endocytic processes to gain entry into the alveolar cell. However, there is conflicting views that suggest FRs are endocytosed via clathrin coated pits [67] [68]. Furthermore, Maxfield and McGraw advised that the pathway of FR may be complicated and involve a number of sorting procedures [69].

As well as its high binding affinity to FR, folic acid has many attractive properties as a targeting ligand for internalisation of conjugated therapeutics. Folic acid is soluble in water, it has low immunogenicity and a relatively low MW (MW=441.4gmol-1), it is easily available and has a good storage stability in addition to simple conjugation chemistry [70, 71]. These properties have allowed drug delivery experts to conjugate various materials on to folic acid. In particular various anti-cancer agents have been considered since the FR is over-expressed in cancerous cells and can be exploited for targeted drug delivery.

5.3.3 Basal- apical transport: IgA

Immunoglobulin A (IgA) is also transported across the epithelial barrier; however it is transcytosed in the basal to apical direction. Its delivery across the cell has been clearly defined. Dimeric IgA binds to its complementary pIgR (polymeric immunoglobulin receptor) at the basolateral surface; binding stimulates endocytosis of the IgA-pIgR complex into clathrin coated pits. The endosome containing this complex moves away from the membrane components and is trafficked into the common endosome and to the apical recycling endosome. The vesicle containing the complex fuses with the apical plasma membrane. PIgR undergoes preteolytic cleavage by a serine protease localised in the apical membrane. Cleavage releases 'secretory' IgA from the mucosal surface. The presence of dimeric IgA in the airway is well established [72].

The function of these receptors is to contribute to mucosal defense of the upper airways by apical secretion of IgA via the pIgA receptor mediated transport. There are relatively low levels of pIgR expression in the distal airway compared to the upper airways [73] although airway goblet cells do not express pIgR. It has emerged that some pathogens have exploited the small uncleaved IgA-pIgR percentage present in the apical membrane of nasopharyngeal epithelial cells in order to enter the underlying interstitium [74].

5.3.4 Other material

Numerous studies investigating the characteristics of the alveolar epithelial layer using a variety of materials have been published. Some of these will be discussed below:

Transferrin is a glycoprotein, present in blood plasma, that is tightly bound (binding affinity of 1023 M-1 at pH 7.4 [75]) to iron which can be released for intracellular delivery of iron. Transferrin can carry two iron (Fe3+) ions and associate with transferrin receptors (TfR) on the surface of cells leading to endocytosis of transferrin. TfR is then recycled by transport of the vesicle to the cell surface membrane in order to bind to more iron. Radio-labelled transferrin, 125I-transferrin, demonstrated a high apical to basal flux in rat alveolar monolayers alongside albumin and IgG [76]. Further studies are required to establish the mechanisms of transferrin and its potentials.

Horse radish peroxide (HRP) is an enzyme derived from horse radish it is widely used as a retrograde label; it has also been used as a pinocytosis marker. In comparison to transferrin, HRP displays low symmetric permeability in the apical to basal and basal to apical membrane although its transport is intact [77]. Since its transport is mediated by pinocytosis, this demonstrates that there may be low pinocytic (fluid phase endocytosis) absorption of macromolecules across the alveolar epithelium.

Observations of endocytic movement of ferritin, dextran and other tracer substances have been indicated in type II alveolar epithelial cells [78] [79]. Pulmonary surfactant protein (SP-A) is another compound endocytosed specifically in to the apical surface of type II epithelial cells via RME [80].

Transport studies

The study of transport mechanisms in vitro requires suitable cell monolayer's that can imitate conditions of in vivo cell layers. Kim et al have described in vitro models that are useful for the study of transport across alveolar epithelial layers. Primary cultured rat monolayer's have demonstrated similar morphological traits and transport processes to that which are present in type I cells in vivo. These monolayer's also display phenotypic evolution into cells that resemble type I alveolar cells. These are of exceptional value in the study of pulmonary drug delivery.

Nanoparticles that have been utilised for study as potential therapeutic vehicles include:

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