**2. Practical issues in the pulmonary delivery**

#### **2.1 Physiological features of the lungs**

The lung resembles an inverted tree, where the trachea or trunk subdivides into two main bronchi and these latter successively branch into more and more narrow and short bronchioles. In total, the trachea undergoes 23 bifurcations before it reaches the alveolar sacs. The first 16 generations compose the conducting region where air is filtered, warmed, humidified and conducted to the respiratory region. Gas exchange between airspaces and blood capillaries occurs in the respiratory region, which includes the respiratory bronchioles, the alveolar ducts and the alveolar sacs.

Two different epithelia line the conducting and respiratory regions. A pseudo stratified columnar epithelium lines the proximal conducting airways and is composed of ciliated columnar cells, goblet or mucus secreting cells and basal or progenitor cells (Parkes, 1994). It is progressively replaced by a simple cuboidal cell layer in the more distal airways and by a very thin epithelial lining in the alveoli. Squamous type I pneumocytes cover 95% of the alveolar surface, owing to their large apical surface and thinness (0.05 μm). Cuboidal type II pneumocytes produce the lung surfactant and are progenitor for type I cells. Type II pneumocytes are located in the corners of the alveoli. The surface area of the alveolar epithelium reaches 100 m2, which is enormous as compared to the 0.25m2 surface area of the airways (Crapo et al.,1982; Mercer et al., 1994).

Mucociliary clearance is one of the most important defense mechanisms to eliminate dust and microorganisms in the lungs (Van der Schans, 2007).

The mucus is produced by goblet cells and sub-mucosa glands. It covers the entire airway surface and its thickness ranges from 5 μm to 55 μm (Clunes & Boucher, 2007; Lai et al., 2009). It consists of an upper gel phase made of 95% water, 2% mucin, a highly glycosylated and entangled polymer, as well as salts, proteins and lipids (Bansil & Turner, 2006). A periciliary liquid layer underlies the mucus gel and its low viscosity allows effective cilia beating. The mucus is transported by the coordinated beating of the cilia and by expiratory airflow towards the oropharynx at an average flow rate of 5 mm per minute. Mucus, cells and debris coming from the nasal cavities and from the lung meet in the pharynx, are mixed with saliva and are swallowed.

Pulmonary surfactant is responsible for biophysical stabilizing activities and innate defense mechanisms. It lines the alveolar epithelial surfaces and overflows into the conductive airways so that the surfactant film is continuous between alveoli and central airways (Bernhard et al., 2004). Pulmonary surfactant is composed of 80% phospholipids (half of which being dipalmitoylphosphatidylcholine), 5–10% neutral lipids (mainly cholesterol), 5– 6% specific surfactant proteins and 3–4% non-specific proteins (Perez-Gil, 2008).

The phospholipids are mainly responsible for forming the surface active film at the respiratory air–liquid interface. In water, phospholipids self-organize in the form of bilayers.

the bloodstream following delivery to the central lung regions in humans, with a dosedependent concentrations in the serum, suggesting that large therapeutic molecules can be delivered to humans via the lungs using the FcRn-mediated transport pathway (Dumont et

The lung resembles an inverted tree, where the trachea or trunk subdivides into two main bronchi and these latter successively branch into more and more narrow and short bronchioles. In total, the trachea undergoes 23 bifurcations before it reaches the alveolar sacs. The first 16 generations compose the conducting region where air is filtered, warmed, humidified and conducted to the respiratory region. Gas exchange between airspaces and blood capillaries occurs in the respiratory region, which includes the respiratory

Two different epithelia line the conducting and respiratory regions. A pseudo stratified columnar epithelium lines the proximal conducting airways and is composed of ciliated columnar cells, goblet or mucus secreting cells and basal or progenitor cells (Parkes, 1994). It is progressively replaced by a simple cuboidal cell layer in the more distal airways and by a very thin epithelial lining in the alveoli. Squamous type I pneumocytes cover 95% of the alveolar surface, owing to their large apical surface and thinness (0.05 μm). Cuboidal type II pneumocytes produce the lung surfactant and are progenitor for type I cells. Type II pneumocytes are located in the corners of the alveoli. The surface area of the alveolar epithelium reaches 100 m2, which is enormous as compared to the 0.25m2 surface area of the

Mucociliary clearance is one of the most important defense mechanisms to eliminate dust

The mucus is produced by goblet cells and sub-mucosa glands. It covers the entire airway surface and its thickness ranges from 5 μm to 55 μm (Clunes & Boucher, 2007; Lai et al., 2009). It consists of an upper gel phase made of 95% water, 2% mucin, a highly glycosylated and entangled polymer, as well as salts, proteins and lipids (Bansil & Turner, 2006). A periciliary liquid layer underlies the mucus gel and its low viscosity allows effective cilia beating. The mucus is transported by the coordinated beating of the cilia and by expiratory airflow towards the oropharynx at an average flow rate of 5 mm per minute. Mucus, cells and debris coming from the nasal cavities and from the lung meet in the pharynx, are mixed

Pulmonary surfactant is responsible for biophysical stabilizing activities and innate defense mechanisms. It lines the alveolar epithelial surfaces and overflows into the conductive airways so that the surfactant film is continuous between alveoli and central airways (Bernhard et al., 2004). Pulmonary surfactant is composed of 80% phospholipids (half of which being dipalmitoylphosphatidylcholine), 5–10% neutral lipids (mainly cholesterol), 5–

The phospholipids are mainly responsible for forming the surface active film at the respiratory air–liquid interface. In water, phospholipids self-organize in the form of bilayers.

6% specific surfactant proteins and 3–4% non-specific proteins (Perez-Gil, 2008).

al., 2005).

**2. Practical issues in the pulmonary delivery** 

bronchioles, the alveolar ducts and the alveolar sacs.

airways (Crapo et al.,1982; Mercer et al., 1994).

with saliva and are swallowed.

and microorganisms in the lungs (Van der Schans, 2007).

**2.1 Physiological features of the lungs** 

Bilayers are also the structural form in which surfactant is assembled and stored by pneumocytes in lamellar bodies. At the air–liquid interface, phospholipids form oriented monolayers, with the hydrophilic headgroups oriented towards the aqueous phase and the hydrophobic acyl chains pointing towards the air. The higher the concentration of phopholipid molecules at the interface, the lower the surface tension, the lower the energy required to enlarge the alveolar surface during inspiration.

Specific surfactant proteins include SP-A, SP-B, SP-C and SP-D. SP-A and SP-D are hydrophilic while SP-B and SP-C are hydrophobic. SP-A is able to bind multiple ligands, including sugars, Ca2+ and phospholipids. This property allows SP-A to bind to the surface of pathogens, contributing to their elimination from the airways. Recognition of SP-A by specific receptors on alveolar macrophages stimulates phagocytosis of the pathogens. SP-B is strictly required for the biogenesis of pulmonary surfactant and its packing into lamellar bodies. Both, SP-B and SP-C promote rapid transfer of phospholipids from bilayers stores into air– liquid interfaces.

Luminal airway and alveolar macrophages are at the forefront of lung defence and their primary role is to participate in innate immune responses, that is, chemotaxis, phagocytosis, and microbial killing (Geiser, 2010). They also downregulate adaptive immune responses and protect the lung from T-cell-mediated inflammation (Holt et al., 2008). Macrophages are tightly applied on the surface of respiratory epithelia. They are immersed in the lung lining fluid beneath the surfactant film.

Although they occupy only 1% of the alveolar surface, they are capable to clean particles from the entire alveolar surface due to amoeboid movements (Geiser, 2010). In contrast to surface macrophages, interstitial macrophages are primarily involved in adaptive immunity by interfacing with lymphocytes via antigen presentation and production of cytokines (Geiser, 2010).

The lung presents a lower level of metabolism than the gastrointestinal tract and liver. Yet, various peptidases are distributed on the surface of different cell types in the lung, including bronchial and alveolar epithelial cells, submucosal glands, smooth muscles, endothelial cells, connective tissue. Proteases are largely present in lysosomes (Buhling et al., 2004). Proteases that degrade the extracellular matrix are secreted by different structural cells or are membrane bound (Stamenkovic, 2003).

Proteases play an essential role in cell and tissue growth, differentiation, repair, remodelling, cell migration and peptide-mediated inflammation (van der Velden & Hulsmann, 1999). Proteases can also be released in the airspaces by activated macrophages and neutrophils in case of inflammatory reactions in the respiratory tract (Buhling et al., 2006; Tetley, 2002). Blood supply to the lungs is divided among the pulmonary and systemic circulations (Altiere & Thompson, 1996). The pulmonary circulation consists of the pulmonary artery that leaves the right heart, branches into a dense pulmonary capillary bed that surrounds the alveoli and finally coalesces into the pulmonary vein that drains into the left heart. One hundred percent of the cardiac output flows through the pulmonary circulation. Its principal functions are gas exchange with air in the alveoli and nutrients supply to terminal respiratory units. The lungs receive a second blood supply via the systemic circulation, commonly referred to as the bronchial circulation. The bronchial circulation originates from the aorta and provides oxygenated blood and nutrients to all

Fig. 2. Deposition of nanocarriers in human respiratory tract as a function of size

(Geiser et al., 2008; Furuyama et al., 2009).

Fig. 3. Pathways involved in nanocarriers absorption

**2.2.2 Clearance mechanisms** 

Data from several of these studies have been included in this review as the pulmonary fate of atmospheric ultrafines has likely similarities with the pulmonary fate of nanomedicines

Various elimination pathways for nanoparticles exist in the lungs, including coughing, dissolution, mucociliarly escalator, translocation from the airways to other sites, phagocytosis by macrophages and neuronal uptake (Figure 3); but the quantitative relationship among these pathways has not yet been established (Muhlfeld et al., 2008).

structures of the tracheobronchial tree. Lymphatic vessels exist in close proximity of major blood vessels and of the airways (El-Chemaly et al., 2009). The lungs have unique physiological features and provide many conditions that favour the absorption of peptides and proteins.

#### **2.2 Barriers to the pulmonary delivery of active substances**

#### **2.2.1 Deposition of nanocarriers through the respiratory tract**

As pointed out in the section on lung physiology, the structure of the lung tissue largely varies according to airway generation and the fate of nanomedicines will similarly vary depending on the structures on which they deposit. The site of deposition of an inhaled formulation within the respiratory tract depends on the aerodynamic diameter of the aerosol particles. The aerodynamic diameter of a particle, daer, is equivalent to the diameter of a unit density (ρ0) sphere that has the same terminal velocity in still air as the particle:

$$d\_{air} = d\sqrt{\frac{\rho}{\rho\_0 X}}\tag{1}$$

where d is the geometric diameter of the particle, ρ is the particle density and χ is the particle dynamic shape factor denoting deviation of shape from sphericity (Hinds, 1999).

Filtering of large particles (daer >5 μm) occurs in upper airways (mouth, trachea and main bronchi) by inertial impaction. One to 5 μm daer particles deposit by gravitational settling in the central and distal tract. Particles with daer <1 μm remain suspended in the air and are mostly exhaled. Ultrafine particles (<100 nm) can largely deposit in the respiratory tract by random Brownian motion: particles <100 nm reach the alveolar region while particles <10 nm already deposit in the tracheo-bronchial region due to their high diffusion coefficients (Heyder at al., 1986).

Drug delivery inhalers, that include nebulizers, metered-dose inhalers and dry powder inhalers, generate particles with a daer in the micron-size range for deposition in the tracheobronchial tree (3–10 μm) in order to treat the airways (e.g., β2 mimetics) or in the alveolar region (1–3 μm) for systemic drug absorption (e.g., insulin) (Figure 2).

Therapeutic proteins can be delivered to the lungs by any medical inhaler. Yet, medical inhalers are not designed to produce ultrafine particles. Ultrafines require enormous energy for their creation, that is, for the atomization of the solution into nano-sized liquid droplets or for the complete de-agglomeration of nanosized dry powder particles. Therefore, drug formulations based on nanoparticles are most often delivered to the respiratory tract by nebulization of colloidal suspensions (Dailey et al., 2003). Developments of the preparation of dry powder microparticles as nanoparticles carriers for pulmonary drug delivery were also reported quite recently (Tsapis et al., 2002). It should be noted, ultrafines are generated in abundance in our environment by the most significant pollution sources, which are those related to combustion processes (Morawska et al., 2005). Epidemiologic studies have provided evidence that an increase in atmospheric ultrafines is associated with adverse pulmonary and cardiovascular effects in susceptible parts of the population. Therefore, significant research has focused on the fate of inhaled ultrafines in the body and ultrafines have been frequently produced in laboratories using spark generators (Geiser et al., 2008).

structures of the tracheobronchial tree. Lymphatic vessels exist in close proximity of major blood vessels and of the airways (El-Chemaly et al., 2009). The lungs have unique physiological features and provide many conditions that favour the absorption of peptides

As pointed out in the section on lung physiology, the structure of the lung tissue largely varies according to airway generation and the fate of nanomedicines will similarly vary depending on the structures on which they deposit. The site of deposition of an inhaled formulation within the respiratory tract depends on the aerodynamic diameter of the aerosol particles. The aerodynamic diameter of a particle, daer, is equivalent to the diameter of a unit density (ρ0) sphere that has the same terminal velocity in still air as the particle:

where d is the geometric diameter of the particle, ρ is the particle density and χ is the particle dynamic shape factor denoting deviation of shape from sphericity (Hinds, 1999).

*d d aer*

*X*

0 

Filtering of large particles (daer >5 μm) occurs in upper airways (mouth, trachea and main bronchi) by inertial impaction. One to 5 μm daer particles deposit by gravitational settling in the central and distal tract. Particles with daer <1 μm remain suspended in the air and are mostly exhaled. Ultrafine particles (<100 nm) can largely deposit in the respiratory tract by random Brownian motion: particles <100 nm reach the alveolar region while particles <10 nm already deposit in the tracheo-bronchial region due to their high diffusion coefficients

Drug delivery inhalers, that include nebulizers, metered-dose inhalers and dry powder inhalers, generate particles with a daer in the micron-size range for deposition in the tracheobronchial tree (3–10 μm) in order to treat the airways (e.g., β2 mimetics) or in the alveolar

Therapeutic proteins can be delivered to the lungs by any medical inhaler. Yet, medical inhalers are not designed to produce ultrafine particles. Ultrafines require enormous energy for their creation, that is, for the atomization of the solution into nano-sized liquid droplets or for the complete de-agglomeration of nanosized dry powder particles. Therefore, drug formulations based on nanoparticles are most often delivered to the respiratory tract by nebulization of colloidal suspensions (Dailey et al., 2003). Developments of the preparation of dry powder microparticles as nanoparticles carriers for pulmonary drug delivery were also reported quite recently (Tsapis et al., 2002). It should be noted, ultrafines are generated in abundance in our environment by the most significant pollution sources, which are those related to combustion processes (Morawska et al., 2005). Epidemiologic studies have provided evidence that an increase in atmospheric ultrafines is associated with adverse pulmonary and cardiovascular effects in susceptible parts of the population. Therefore, significant research has focused on the fate of inhaled ultrafines in the body and ultrafines have been frequently produced in laboratories using spark generators (Geiser et al., 2008).

region (1–3 μm) for systemic drug absorption (e.g., insulin) (Figure 2).

**2.2 Barriers to the pulmonary delivery of active substances 2.2.1 Deposition of nanocarriers through the respiratory tract** 

and proteins.

(Heyder at al., 1986).

Fig. 2. Deposition of nanocarriers in human respiratory tract as a function of size

Data from several of these studies have been included in this review as the pulmonary fate of atmospheric ultrafines has likely similarities with the pulmonary fate of nanomedicines (Geiser et al., 2008; Furuyama et al., 2009).

#### **2.2.2 Clearance mechanisms**

(1)

Various elimination pathways for nanoparticles exist in the lungs, including coughing, dissolution, mucociliarly escalator, translocation from the airways to other sites, phagocytosis by macrophages and neuronal uptake (Figure 3); but the quantitative relationship among these pathways has not yet been established (Muhlfeld et al., 2008).

Fig. 3. Pathways involved in nanocarriers absorption

with respect to healthy individuals. Actually, MCC and dissolution occur simultaneously and their relative importance should depend on the elimination rate from each of these contributions. While insoluble particles of 6 μm are practically all cleared from the bronchial airways by MCC in 24 h, smaller particles are retained for a longer period showing almost an inverse relationship between the 24 h airway retention and the geometric particle size. Nanoparticles with enhanced mobility may partition through the mucus into the periciliary spaces, where they can be taken up by the airway macrophages and bronchial epithelial

Alveolar macrophages are responsible for clearance of nanoparticles deposited in the alveolar region, in which MCC is absent. In response to the deposited nanoparticles, alveolar macrophages will migrate to the particles and phagocytize them via chemotaxis involving opsonisation. Macrophage uptake is believed to complete within 6–12 h after deposition of the particles in the alveoli (Oberdörster, 2007). nce internalized in the macrophages, the particles will be either disintegrated (e.g. by enzymes in lysosomes) or accumulated in the lymphatic system (Schmid et al., 2009) draining both airways and alveoli and finally terminating in the mediastinal and hilar lymph nodes (Geiser & Kreyling, 2010). A minor fraction of the particle-carrying macrophages will migrate to the ciliated airways where they are removed by MCC (Schmid et al., 2009). However, with a retention half-time of up to 700 days in humans (Oberdörster, 2007), clearance of solid particles by alveolar macrophages is a relatively slow mechanism. Phagocytosis of particles below 100 nm is not effective (Oberdörster, 2007), most probably because of a less effective recognition (~20%) of nanoparticles by the macrophages (Schmid et al., 2009). The reduced recognition is possibly due to more scattered and diluted chemotactic signals as a result of i) higher number concentration of nanoparticles (compared with micron-sized particles at the same dose) and ii) fewer opsonin molecules available per particle. Conversely, since nanoparticles are more readily taken up by epithelial cells, they become less available to be phagocytized by macrophages (Madl & Pinkerton, 2009). Macrophages are also present in the ciliated airway but their role in nanoparticle

This process involves transcytosis of the particles into the epithelial cells and/or across the epithelia of the respiratory tract into the interstitium and then to blood and lymph. As described earlier, translocation to the lymphatic system can be facilitated by macrophage uptake. The transport may be protein-mediated, requiring binding of certain proteins on the nanoparticle surface for recognition by the receptors (Schmid et al., 2009). The transport may also be receptor-mediated transcytosis via caveolae (Oberdörster, 2007), which have a diameter of 50–100 nm. Surface coating of the particles by albumin and lecithin may facilitate cellular uptake by pneumocytes and transcytosis across capillaries (Yang et al., 2008). Once internalized, nanoparticles can bind to mitochondria and even DNA in the

When translocated to the systemic circlulation, nanoparticles could cause unwanted effects on the blood (e.g. accumulation in platelets) and other organs in the body (Oberdörster, 2007). Some biological effects may include inflammation, oxidative stress, cytotoxicity,

fibrosis, and immunologic responses ( Madl & Pinkerton, 2009; Unfried et al., 2007).

cells, causing a reduction of MCC (Schmid et al., 2009).

clearance is probably less important compared with MCC.

**2.2.2.4 Translocation into cells, blood and lymph** 

nucleus (Muhlfeld et al., 2008; Oberdörster, 2007).

**2.2.2.3 Macrophage uptake** 

When a nanoparticle has landed on the airways, it first encounters the surfactant on the top of the airway lining fluid. The surfactant will enhance particle wetting thus helping it to sink into the fluid, passing first through the gel phase and then to the sol phase. Compared with sulphur colloidal particles (220 nm), human serum albumin molecules (HSA, 66 kDa) were cleared 3 times more slowly from the bronchi of dogs. This difference was attributed to the possibility that sulphur nanoparticles resided on the gel phase (i.e. the top layer of the periciliary fluid) whereas HSA dissolved and partitioned into the sol (i.e. bottom layer) which may be transported less effectively by mucociliary clearance (MCC). If extrapolated to inhaled drugs, the more soluble ones will behave like HSA and should be hence less susceptible to MCC (Edsbacker et al., 2008), but more likely absorbed through the epithelium. For nanoparticle agglomerates, it is likely that the particles will first reside on the gel phase. Depending on the aqueous dispersibility and solubility in the airway fluid, the agglomerates may remain in the gel phase behaving like the microsized particles, or they may then disperse into nanoparticles followed by dissolution and absorption. Nanoparticles delivered within a liquid droplet (e.g. from a nebulizer) might be different from dry particles, as the droplet liquid may interact with the gel layer making the nanoparticles easier to wet and partition into the sol layer, i.e. potentially more readily to escape the MCC (Zhang et al., 2011).

#### **2.2.2.1 Dissolution**

Dissolution depends on the site of deposition, which determines the volume of airway fluids available for dissolution and, hence, whether the dissolution occurs in sink or non-sink conditions, as well as on the solubility and dose of the drugs. Freely water soluble drugs include organic salts (e.g., salbutamol sulphate, terbutaline sulphate and disodium cromoglycate) and polar compounds (e.g. mannitol). These drugs will dissolve readily in the airway fluid followed by absorption or elimination by the mucociliarly escalator. Sparingly soluble drugs include the inhaled corticosteroids, which have aqueous solubility ranging from 140 to below 0.1 μg/mL (Edsbacker et al., 2008). Once dissolved, the drug molecules are diluted in the airway fluid where they can bind to proteins, opsonins, or other constituents and be metabolized and/or absorbed into the blood and lymph ( Schmid et al., 2010).

Absorption of the drugs depends on the site such as alveolar or conducting airways (which affects the barrier thickness and surface area) and the drug molecule itself (which impacts on passive diffusion and active uptake by the epithelium. It must be pointed out that absorption of most drugs from the lungs is rapid: as an example, it has been reported that following inhalation of formoterol fumarate (4.5 nmol/L) and budesonide (136 pmol/L, the peak plasma concentrations occurred at 20 and 10 min, respectively.

#### **2.2.2.2 Mucociliary clearance (MCC)**

MCC operates in the ciliated airways where the movement of the cilia transports the mucus carrying the drug nanoparticles or dissolved drug (not yet absorbed) on the epithelial surface towards the pharynx/larynx. The drug-containing mucus will then be swallowed to the GI tract. The average transport velocity in the human trachea has been estimated at 3–10 mm/min, but the value varies widely among subjects. Using well-developed techniques of depositing radiolabelled sulfur colloids in the central airways, Daviskas and her colleagues reported a MCC rate remarkably reduced in patients with bronchiectasis, asthma, and CF,

When a nanoparticle has landed on the airways, it first encounters the surfactant on the top of the airway lining fluid. The surfactant will enhance particle wetting thus helping it to sink into the fluid, passing first through the gel phase and then to the sol phase. Compared with sulphur colloidal particles (220 nm), human serum albumin molecules (HSA, 66 kDa) were cleared 3 times more slowly from the bronchi of dogs. This difference was attributed to the possibility that sulphur nanoparticles resided on the gel phase (i.e. the top layer of the periciliary fluid) whereas HSA dissolved and partitioned into the sol (i.e. bottom layer) which may be transported less effectively by mucociliary clearance (MCC). If extrapolated to inhaled drugs, the more soluble ones will behave like HSA and should be hence less susceptible to MCC (Edsbacker et al., 2008), but more likely absorbed through the epithelium. For nanoparticle agglomerates, it is likely that the particles will first reside on the gel phase. Depending on the aqueous dispersibility and solubility in the airway fluid, the agglomerates may remain in the gel phase behaving like the microsized particles, or they may then disperse into nanoparticles followed by dissolution and absorption. Nanoparticles delivered within a liquid droplet (e.g. from a nebulizer) might be different from dry particles, as the droplet liquid may interact with the gel layer making the nanoparticles easier to wet and partition into the sol layer, i.e. potentially more readily to escape the MCC

Dissolution depends on the site of deposition, which determines the volume of airway fluids available for dissolution and, hence, whether the dissolution occurs in sink or non-sink conditions, as well as on the solubility and dose of the drugs. Freely water soluble drugs include organic salts (e.g., salbutamol sulphate, terbutaline sulphate and disodium cromoglycate) and polar compounds (e.g. mannitol). These drugs will dissolve readily in the airway fluid followed by absorption or elimination by the mucociliarly escalator. Sparingly soluble drugs include the inhaled corticosteroids, which have aqueous solubility ranging from 140 to below 0.1 μg/mL (Edsbacker et al., 2008). Once dissolved, the drug molecules are diluted in the airway fluid where they can bind to proteins, opsonins, or other constituents and be metabolized and/or absorbed into the blood and lymph ( Schmid et al.,

Absorption of the drugs depends on the site such as alveolar or conducting airways (which affects the barrier thickness and surface area) and the drug molecule itself (which impacts on passive diffusion and active uptake by the epithelium. It must be pointed out that absorption of most drugs from the lungs is rapid: as an example, it has been reported that following inhalation of formoterol fumarate (4.5 nmol/L) and budesonide (136 pmol/L, the

MCC operates in the ciliated airways where the movement of the cilia transports the mucus carrying the drug nanoparticles or dissolved drug (not yet absorbed) on the epithelial surface towards the pharynx/larynx. The drug-containing mucus will then be swallowed to the GI tract. The average transport velocity in the human trachea has been estimated at 3–10 mm/min, but the value varies widely among subjects. Using well-developed techniques of depositing radiolabelled sulfur colloids in the central airways, Daviskas and her colleagues reported a MCC rate remarkably reduced in patients with bronchiectasis, asthma, and CF,

peak plasma concentrations occurred at 20 and 10 min, respectively.

**2.2.2.2 Mucociliary clearance (MCC)** 

(Zhang et al., 2011). **2.2.2.1 Dissolution** 

2010).

with respect to healthy individuals. Actually, MCC and dissolution occur simultaneously and their relative importance should depend on the elimination rate from each of these contributions. While insoluble particles of 6 μm are practically all cleared from the bronchial airways by MCC in 24 h, smaller particles are retained for a longer period showing almost an inverse relationship between the 24 h airway retention and the geometric particle size. Nanoparticles with enhanced mobility may partition through the mucus into the periciliary spaces, where they can be taken up by the airway macrophages and bronchial epithelial cells, causing a reduction of MCC (Schmid et al., 2009).

#### **2.2.2.3 Macrophage uptake**

Alveolar macrophages are responsible for clearance of nanoparticles deposited in the alveolar region, in which MCC is absent. In response to the deposited nanoparticles, alveolar macrophages will migrate to the particles and phagocytize them via chemotaxis involving opsonisation. Macrophage uptake is believed to complete within 6–12 h after deposition of the particles in the alveoli (Oberdörster, 2007). nce internalized in the macrophages, the particles will be either disintegrated (e.g. by enzymes in lysosomes) or accumulated in the lymphatic system (Schmid et al., 2009) draining both airways and alveoli and finally terminating in the mediastinal and hilar lymph nodes (Geiser & Kreyling, 2010). A minor fraction of the particle-carrying macrophages will migrate to the ciliated airways where they are removed by MCC (Schmid et al., 2009). However, with a retention half-time of up to 700 days in humans (Oberdörster, 2007), clearance of solid particles by alveolar macrophages is a relatively slow mechanism. Phagocytosis of particles below 100 nm is not effective (Oberdörster, 2007), most probably because of a less effective recognition (~20%) of nanoparticles by the macrophages (Schmid et al., 2009). The reduced recognition is possibly due to more scattered and diluted chemotactic signals as a result of i) higher number concentration of nanoparticles (compared with micron-sized particles at the same dose) and ii) fewer opsonin molecules available per particle. Conversely, since nanoparticles are more readily taken up by epithelial cells, they become less available to be phagocytized by macrophages (Madl & Pinkerton, 2009). Macrophages are also present in the ciliated airway but their role in nanoparticle clearance is probably less important compared with MCC.

#### **2.2.2.4 Translocation into cells, blood and lymph**

This process involves transcytosis of the particles into the epithelial cells and/or across the epithelia of the respiratory tract into the interstitium and then to blood and lymph. As described earlier, translocation to the lymphatic system can be facilitated by macrophage uptake. The transport may be protein-mediated, requiring binding of certain proteins on the nanoparticle surface for recognition by the receptors (Schmid et al., 2009). The transport may also be receptor-mediated transcytosis via caveolae (Oberdörster, 2007), which have a diameter of 50–100 nm. Surface coating of the particles by albumin and lecithin may facilitate cellular uptake by pneumocytes and transcytosis across capillaries (Yang et al., 2008). Once internalized, nanoparticles can bind to mitochondria and even DNA in the nucleus (Muhlfeld et al., 2008; Oberdörster, 2007).

When translocated to the systemic circlulation, nanoparticles could cause unwanted effects on the blood (e.g. accumulation in platelets) and other organs in the body (Oberdörster, 2007). Some biological effects may include inflammation, oxidative stress, cytotoxicity, fibrosis, and immunologic responses ( Madl & Pinkerton, 2009; Unfried et al., 2007).

respiratory system will determine the pharmacodynamic response. For instance, the rapid uptake of particles by alveolar macrophages can be a way of targeting anti-tuberculosis drugs to this type of cells (Nimje et al., 2009). Conversely, macrophages uptake represents a clearance pathway for drugs acting on other cells within the lungs (e.g., β2 mimetics).

Nowadays, biopharmaceuticals and conventional drugs are frequently engineered or incorporated in carriers in order to direct their fate in preferential pathways (Schmidt, 2009; Veronese & Pasut, 2005). Nano-size drug carriers can incorporate various therapeutics (e.g.,poorly water soluble drugs) and present several advantages for drug delivery to the lung including controlled release, protection from metabolism and degradation, decreased drug toxicity and targeting capabilities. Moreover, the successful integration of novel drugs with devices capable of delivering defined doses to the respiratory tract has resulted in a proven track record for inhalation as a route of administration that limits systemic exposure and provides localized topical delivery. Thus, a number of orally inhaled products have been successfully developed over the last 50 years, providing symptomatic relief to millions

There are numerous types of nanoparticle systems now being explored for drug delivery to

The types of nanoparticle used at present in research for cancer therapeutic applications include polymeric nanoparticles, protein nanoparticles, ceramic nanoparticles, viral

Liposomes are the most extensively investigated system for controlled drug delivery to the lungs (Mansour et al., 2009). A few liposome-encapsulated antibiotics have been delivered to the lungs in phase II clinical trials. These include amikacin (Weers et al., 2009) and ciprofloxacin (Bruinenberg et al., 2010) Multiple treatment cycles with ARIKACE™ (liposomal amikacin for inhalation) showed sustained improvement in lung function with significant reduction in bacterial density in CF patients who have chronic Pseudomonas

A nanoscale liposomal formulation of amikacin has been shown to slowly release the drug in rat lungs and to penetrate Pseudomonas biofilms and CF sputum in vitro (Meers et al., 2008).

of patients with asthma and chronic obstructive pulmonary disease (COPD).

lungs, especially in cancer treatment (Haley & Frenkel, 2008).

nanoparticles and metallic nanoparticles (Balak et al., 2010).

lung infections (Okusanya et al., 2009).

Fig. 4. Nanocarrier size

Surface area has been proposed as the single most important particle dose parameter for the toxicity of nanoparticles (Schmid et al., 2009). This is particularly relevant for inflammatory and oxidative stress reactions, such as surface area of a catalyst (i.e. nanoparticles), that determines the oxidative reaction rate. However, oxidative stress involves the formation of reactive oxygen species (ROS) from the particles containing reactants such as transition metals or polyaromatic hydrocarbons (which induce the expression of the CYP1A1 protein). Drug nanoparticles, which do not contain such reactants, are therefore less likely to cause oxidative stress in the lungs. Biodegradabile nanoparticles indeed showed significantly lower inflammatory response in-vitro (Sung et al., 2007). Interestingly, translocation in the reverse direction with particles re-entrained from the lung capillaries or interstitium to the luminal side of the epithelium have been shown in animal models using rabbits and rats. Such back-translocation was suggested to be macrophage-mediated.

#### **2.2.2.5 Neuronal uptake**

Translocation into afferent vagal nerves in the tracheobronchial airways has been proposed but still not well studied (Oberdorster et al., 2005). Nanoparticles deposited in the nasal cavity have been reported to be taken up by the olfactory lobe and translocated to the central nervous system (Oberdorster et al., 2005). However, such a neuronal uptake pathway is relevant only if the drug nanoparticles are inhaled nasally. Existing data from epidemiologic and toxicological studies showed longer retention of inhaled nanoparticles in the lungs, but the applicability of these findings on nanoparticles is under investigation. In theory, inhaled nanodrug particles have the potential to be retained longer in the lungs followed by cellular uptake and translocation into the systemic circulation thus causing nanotoxicity. It can be speculated that the fate of the nanoparticles in the lungs, regarding the elimination pathways, will depend on the properties of both the particle and the drug molecule. Micron-sized aggregates of nanoparticles will deposit by sedimentation in the tracheobronchial (TB) region where MCC will operate to eliminate both the dissolved and undissolved drugs. Even discrete nanoparticles can deposit by diffusion in the TB region. Drug nanoparticles deposited in the alveolar region will dissolve in the airway fluid and be absorbed. This is likely to be the case even for hydrophobic drugs with low aqueous solubility like inhaled corticosteroids due to the relatively low doses that are used. As a result of the low persistence of drug nanoparticles, dissolution and mucociliary escalator will likely be the major clearance pathways responsible for these particles before macrophage phagocytosis and other translocation pathways would start to play a significant role.

#### **3. Nanocarriers for lung delivery**

Nanomedicine can be defined as the application of nanotechnology to medicine. Nanotechnology involves the understanding and control of matter at dimensions of 1 to 100- 200 nm, where unique phenomena enable novel applications. Artificial nanostructures are of the same size as biological entities and can readily interact with biomolecules on both the cell surface and within the cell (Figure 4). Here our attention is focused on the fate of nanomedicines delivered to the lung, in particular an innovative glucocorticoid delivery system will be considered.

The understanding the fate of nanomedicines in the lungs is important because fate and therapeutic activity are closely related. Interaction of nanomedicines with cells of the

Surface area has been proposed as the single most important particle dose parameter for the toxicity of nanoparticles (Schmid et al., 2009). This is particularly relevant for inflammatory and oxidative stress reactions, such as surface area of a catalyst (i.e. nanoparticles), that determines the oxidative reaction rate. However, oxidative stress involves the formation of reactive oxygen species (ROS) from the particles containing reactants such as transition metals or polyaromatic hydrocarbons (which induce the expression of the CYP1A1 protein). Drug nanoparticles, which do not contain such reactants, are therefore less likely to cause oxidative stress in the lungs. Biodegradabile nanoparticles indeed showed significantly lower inflammatory response in-vitro (Sung et al., 2007). Interestingly, translocation in the reverse direction with particles re-entrained from the lung capillaries or interstitium to the luminal side of the epithelium have been shown in animal models using rabbits and rats.

Translocation into afferent vagal nerves in the tracheobronchial airways has been proposed but still not well studied (Oberdorster et al., 2005). Nanoparticles deposited in the nasal cavity have been reported to be taken up by the olfactory lobe and translocated to the central nervous system (Oberdorster et al., 2005). However, such a neuronal uptake pathway is relevant only if the drug nanoparticles are inhaled nasally. Existing data from epidemiologic and toxicological studies showed longer retention of inhaled nanoparticles in the lungs, but the applicability of these findings on nanoparticles is under investigation. In theory, inhaled nanodrug particles have the potential to be retained longer in the lungs followed by cellular uptake and translocation into the systemic circulation thus causing nanotoxicity. It can be speculated that the fate of the nanoparticles in the lungs, regarding the elimination pathways, will depend on the properties of both the particle and the drug molecule. Micron-sized aggregates of nanoparticles will deposit by sedimentation in the tracheobronchial (TB) region where MCC will operate to eliminate both the dissolved and undissolved drugs. Even discrete nanoparticles can deposit by diffusion in the TB region. Drug nanoparticles deposited in the alveolar region will dissolve in the airway fluid and be absorbed. This is likely to be the case even for hydrophobic drugs with low aqueous solubility like inhaled corticosteroids due to the relatively low doses that are used. As a result of the low persistence of drug nanoparticles, dissolution and mucociliary escalator will likely be the major clearance pathways responsible for these particles before macrophage phagocytosis and other translocation pathways would start to play a significant

Nanomedicine can be defined as the application of nanotechnology to medicine. Nanotechnology involves the understanding and control of matter at dimensions of 1 to 100- 200 nm, where unique phenomena enable novel applications. Artificial nanostructures are of the same size as biological entities and can readily interact with biomolecules on both the cell surface and within the cell (Figure 4). Here our attention is focused on the fate of nanomedicines delivered to the lung, in particular an innovative glucocorticoid delivery

The understanding the fate of nanomedicines in the lungs is important because fate and therapeutic activity are closely related. Interaction of nanomedicines with cells of the

Such back-translocation was suggested to be macrophage-mediated.

**2.2.2.5 Neuronal uptake** 

role.

**3. Nanocarriers for lung delivery** 

system will be considered.

Fig. 4. Nanocarrier size

respiratory system will determine the pharmacodynamic response. For instance, the rapid uptake of particles by alveolar macrophages can be a way of targeting anti-tuberculosis drugs to this type of cells (Nimje et al., 2009). Conversely, macrophages uptake represents a clearance pathway for drugs acting on other cells within the lungs (e.g., β2 mimetics).

Nowadays, biopharmaceuticals and conventional drugs are frequently engineered or incorporated in carriers in order to direct their fate in preferential pathways (Schmidt, 2009; Veronese & Pasut, 2005). Nano-size drug carriers can incorporate various therapeutics (e.g.,poorly water soluble drugs) and present several advantages for drug delivery to the lung including controlled release, protection from metabolism and degradation, decreased drug toxicity and targeting capabilities. Moreover, the successful integration of novel drugs with devices capable of delivering defined doses to the respiratory tract has resulted in a proven track record for inhalation as a route of administration that limits systemic exposure and provides localized topical delivery. Thus, a number of orally inhaled products have been successfully developed over the last 50 years, providing symptomatic relief to millions of patients with asthma and chronic obstructive pulmonary disease (COPD).

There are numerous types of nanoparticle systems now being explored for drug delivery to lungs, especially in cancer treatment (Haley & Frenkel, 2008).

The types of nanoparticle used at present in research for cancer therapeutic applications include polymeric nanoparticles, protein nanoparticles, ceramic nanoparticles, viral nanoparticles and metallic nanoparticles (Balak et al., 2010).

Liposomes are the most extensively investigated system for controlled drug delivery to the lungs (Mansour et al., 2009). A few liposome-encapsulated antibiotics have been delivered to the lungs in phase II clinical trials. These include amikacin (Weers et al., 2009) and ciprofloxacin (Bruinenberg et al., 2010) Multiple treatment cycles with ARIKACE™ (liposomal amikacin for inhalation) showed sustained improvement in lung function with significant reduction in bacterial density in CF patients who have chronic Pseudomonas lung infections (Okusanya et al., 2009).

A nanoscale liposomal formulation of amikacin has been shown to slowly release the drug in rat lungs and to penetrate Pseudomonas biofilms and CF sputum in vitro (Meers et al., 2008).

including particle clearance, cellular targeting, intracellular trafficking, and drug absorption are needed to better design formulations that deliver to the ''target'' with the optimal balance of pharmacodynamic, pharmacokinetic, and safety profiles. More specifically, continued advances are needed in the development of: (1) controlled release formulations; (2) formulations with improved regional targeting within the lungs (e.g., airway versus alveoli and vice versa); (3) formulations containing active targeting moieties; (4) formulation strategies for improving the systemic bioavailability of inhaled macromolecules; (5) formulation strategies for delivering macromolecules, including siRNA and DNA, into cells; and (6 ) formulations with improved dose consistency. It is likely that such innovation will require the development of novel excipients and particle engineering strategies. Future innovation must also take into account the changing marketplace and the diverse set of customers (patient, healthcare professional, heath authorities, payers, and politicians) who must be satisfied. The pharmacoeconomics of new delivery systems will be closely scrutinized, so it is imperative that cost factors should be taken into account. Otherwise, the new technology option may overshoot the

Epidemiological studies have confirmed a positive correlation between levels of particulate pollution and increased morbidity and mortality rates among general populations (Gwinn &

The adverse health effects seem to be dominated by pulmonary symptoms. For instance, many reports have addressed that occupational exposure of inhaled rigid nanoparticles (NPs) can lead to respiratory diseases such as pneumoconiosis (pulmonary fibrosis) and

Increasing inhalation of ambient ultrafine particles has been linked with exacerbation of respiratory symptoms and mortality among COPD sufferers (Xia et al., 2009). It has also been documented that NPs can instigate oxidative stress and cellular toxicity in various

It was also reported that chronic exposure to NPs can potentially predispose humans to lung

A concentration range of NPs within the level found in ambience and in nanotechnology

The second safety aspect of deep lung deposition is the interaction of nanoparticles with the alveolar environment. The alveolar space is covered with a thin surfactant film. This film has important physiological functions e.g. to accelerate gas exchange and to lower the surface tension in the alveolar space. Compromising these functions by inhalable nanoparticles might cause life threatening consequences. Therefore, the compatibility of a delivery system with the alveolar environment must be considered (Azarmi et al., 2008).

For these reasons vesicular nanocarriers, composed of lung surfactants and/or synthetic amphiphiles, provide an efficient delivery system for the treatment of pulmonary disorders due to their biocompatibility, biodegradability and non-toxic nature (Taylor & Newton,

evolving inhalation marketplace.

Vallyathan, 2006; Stone et al., 2007).

types of cells (Huang et al., 2009).

2004).

inflammation and increase the risk of COPD.

**4. Toxicity of nanoparticles to the lung** 

bronchitis (Byrne & Baugh, 2008; Lkhasuren et al., 2007).

industries (Klaine et al., 2008) can promote mucin aggregation.

Mitsopoulos and Suntres reported that the delivery of N-acetylcysteine as a liposomal formulation improves its effectiveness in counteracting Paraquat-induced cytotoxicity (Mitsopoulos & Suntres, 2011).

Liposomal drug dry powder formulations, realized to obtain novel devices capable of delivering defined doses of drugs, represent promising tools for pulmonary drug administration, such as selective localization of drug, reduced local and systemic toxicities, increased patient compliance and high dose loading.

In liposomal dry powder formulations, drug encapsulating liposomes are homogenized, dispersed into the carrier and converted into dry powder by using freeze drying, spray drying or supercritical fluid technologies.

The most commonly used liposomes are composed of lung surfactants and synthetic lipids. Liposomal formulation have been proposed to delivery anticancer drugs, corticosteroids, immunosuppressants, antimicotic drugs, antibiotics for local pulmonary infections and CF and opioid analgesics for pain management using. Many of them have reached the stage of clinical trials for the treatment of several pulmonary diseases (Misra et al., 2009).

A promising application of nanocarriers to lung targeting is related to gene delivery. Gene therapy is currently being developed for a wide range of acute and chronic lung diseases, incuding CF, cancer and asthma (Griesenbach & Alton, 2009; Lam et al., 2011). Nguyen et al (2009) developed a highly efficient nanocomposite aerosol for pulmonary gene delivery, consisting of a biodegradable polymer core.

Respiratory diseases have attracted particular attention as targets of siRNA - mediated therapeutics, due to the lethality and prevalence of certain illnesses and the lung's accessibility to therapeutic agents via both local and systemic delivery routes. However, one of the major challenges to realize the RNAi therapeutic potential in lung diseases is to deliver the siRNAs to the lung tissue, in particular, to the target cells with high efficiency and high specificity (Yuan et al., 2011).

Several clinical trials have been conducted in order to assess the efficacy and safety of pulmonary DNA delivery using viral and non-viral vectors, especially in the case of CF. Yet, none of these formulations have been pursued due to low transfection efficiency, transient gene expression or immune elimination of the gene vector. Identifying the barriers to cell transfection might help to improve gene transfer efficiency of non-viral vectors (Griesenbach & Alton, 2009). An efficient and safe cationic lipid, 6-lauroxyhexyl lysinate (LHLN), was proposed to prepare cationic liposomes. *In vitro* tests showed that, compared with Lipofectamine2000, the new cationic liposome formulation using LHLN exhibited lower cytotoxicity and similar transfection efficiency in A549 and HepG2 lung cancer cells (Peng et al., 2011).

Ishitsuka et al. (2011) developed a multifunctional envelope-type nano device (MEND), in which plasmid DNA is condensed using a polycation to form a core particle that is encapsulated in a lipid envelope, modified with the IRQ peptide (IRQRRRR) to enhance transgene expression in lungs. (Ishitsuka et al., 2011).

Clinical applications of liposomes and nanoparticles for drug delivery to the respiratory tract are still in early stages. The key to future innovation may lie at the interface between biology and particle engineering. Improved understanding of biological processes

Mitsopoulos and Suntres reported that the delivery of N-acetylcysteine as a liposomal formulation improves its effectiveness in counteracting Paraquat-induced cytotoxicity

Liposomal drug dry powder formulations, realized to obtain novel devices capable of delivering defined doses of drugs, represent promising tools for pulmonary drug administration, such as selective localization of drug, reduced local and systemic toxicities,

In liposomal dry powder formulations, drug encapsulating liposomes are homogenized, dispersed into the carrier and converted into dry powder by using freeze drying, spray

The most commonly used liposomes are composed of lung surfactants and synthetic lipids. Liposomal formulation have been proposed to delivery anticancer drugs, corticosteroids, immunosuppressants, antimicotic drugs, antibiotics for local pulmonary infections and CF and opioid analgesics for pain management using. Many of them have reached the stage of

A promising application of nanocarriers to lung targeting is related to gene delivery. Gene therapy is currently being developed for a wide range of acute and chronic lung diseases, incuding CF, cancer and asthma (Griesenbach & Alton, 2009; Lam et al., 2011). Nguyen et al (2009) developed a highly efficient nanocomposite aerosol for pulmonary gene delivery,

Respiratory diseases have attracted particular attention as targets of siRNA - mediated therapeutics, due to the lethality and prevalence of certain illnesses and the lung's accessibility to therapeutic agents via both local and systemic delivery routes. However, one of the major challenges to realize the RNAi therapeutic potential in lung diseases is to deliver the siRNAs to the lung tissue, in particular, to the target cells with high efficiency

Several clinical trials have been conducted in order to assess the efficacy and safety of pulmonary DNA delivery using viral and non-viral vectors, especially in the case of CF. Yet, none of these formulations have been pursued due to low transfection efficiency, transient gene expression or immune elimination of the gene vector. Identifying the barriers to cell transfection might help to improve gene transfer efficiency of non-viral vectors (Griesenbach & Alton, 2009). An efficient and safe cationic lipid, 6-lauroxyhexyl lysinate (LHLN), was proposed to prepare cationic liposomes. *In vitro* tests showed that, compared with Lipofectamine2000, the new cationic liposome formulation using LHLN exhibited lower cytotoxicity and similar transfection efficiency in A549 and HepG2 lung cancer cells (Peng et

Ishitsuka et al. (2011) developed a multifunctional envelope-type nano device (MEND), in which plasmid DNA is condensed using a polycation to form a core particle that is encapsulated in a lipid envelope, modified with the IRQ peptide (IRQRRRR) to enhance

Clinical applications of liposomes and nanoparticles for drug delivery to the respiratory tract are still in early stages. The key to future innovation may lie at the interface between biology and particle engineering. Improved understanding of biological processes

clinical trials for the treatment of several pulmonary diseases (Misra et al., 2009).

(Mitsopoulos & Suntres, 2011).

increased patient compliance and high dose loading.

drying or supercritical fluid technologies.

consisting of a biodegradable polymer core.

and high specificity (Yuan et al., 2011).

transgene expression in lungs. (Ishitsuka et al., 2011).

al., 2011).

including particle clearance, cellular targeting, intracellular trafficking, and drug absorption are needed to better design formulations that deliver to the ''target'' with the optimal balance of pharmacodynamic, pharmacokinetic, and safety profiles. More specifically, continued advances are needed in the development of: (1) controlled release formulations; (2) formulations with improved regional targeting within the lungs (e.g., airway versus alveoli and vice versa); (3) formulations containing active targeting moieties; (4) formulation strategies for improving the systemic bioavailability of inhaled macromolecules; (5) formulation strategies for delivering macromolecules, including siRNA and DNA, into cells; and (6 ) formulations with improved dose consistency. It is likely that such innovation will require the development of novel excipients and particle engineering strategies. Future innovation must also take into account the changing marketplace and the diverse set of customers (patient, healthcare professional, heath authorities, payers, and politicians) who must be satisfied. The pharmacoeconomics of new delivery systems will be closely scrutinized, so it is imperative that cost factors should be taken into account. Otherwise, the new technology option may overshoot the evolving inhalation marketplace.
