Barriers that Inhaled Particles Encounter

Abstract

Inhalable particulate drug carriers—nano- and micro-particles, liposomes, and micelles—should be designed to promote drug deposition in the lung and engineered to exhibit the desired drug release property. To deposit at the desired site of action, inhaled particles must evade various lines of lung defense, including mucociliary clearance, entrapment by mucus layer, and phagocytosis by alveolar macrophages. Various physiological, mechanical, and chemical barriers of the respiratory system reduce particle residence time in the lungs, prevent particle deposition in the deep lung, remove drug-filled particles from the lung, and thus diminish the therapeutic efficacy of inhaled drugs. To develop inhalable drug carriers with efficient deposition properties and optimal retention in the lungs, particle engineers should have a thorough understanding of the barriers that particles confront and appreciate the lung defenses that remove the particles from the respiratory system. Thus, this section summarizes the mechanical, chemical, and immunological barriers of the lungs that inhaled particles must overcome and discusses the influence of these barriers on the fate of inhaled particles.

Introduction

The respiratory system and its components act as the first-line defense against airborne pollutants, microorganisms, and particles that deposit at the air-blood interface in the lung.¹ The respiratory system has evolved with a series of complex defense mechanisms to keep the respiratory tract free from foreign particles and protect the physiological environment from external harmful agents. These protective mechanisms may be considered lung barriers and can be classified into three groups: 1) mechanical barriers such as air filtration through nostrils, mucociliary clearance, coughing and sneezing; 2) chemical barriers such as actions of surfactants, antioxidants and enzymes in the local lung environment; and, 3) immunological barriers that involve activities of various inflammatory cells in recognizing and removing deposited inhaled particles and offending agents.² However, these protective mechanisms are unable to differentiate between inadvertently inhaled injurious external agents and a deliberately administered therapeutic particulate system. To develop inhalable drug carriers with superior deposition efficiency that can escape lung clearance, we should first delve into the lungs’ defensive and clearance processes. Thus, we have explained below different aspects of the respiratory anatomy and physiology that impede efficient deposition of particles in the lungs and control their fate after deposition.

Anatomical and Physiological Barriers

Similarly to an inverted tree, the respiratory apparatus is emanated from a trunk-like cylindrical trachea that bifurcates into bronchi and subsequently branches several times to form progressively narrower terminal bronchioles; further bifurcations of the terminal bronchioles yield alveolar sacs and alveoli that create a large surface area with rich blood supply for gas exchange. The human respiratory system, from the trachea to the alveoli, bifurcates about 22-23 times and exhibits a gradually thinning epithelial layer;³ this layer, in tandem with mucus and ciliated cells, forms a formidable anatomical-physiological barrier, called the mucociliary escalator, against particles that deposit on the upper respiratory tract.⁴ The alveolar region in the human lungs is continuously guarded by patrolling alveolar macrophages that engulf and digest particles and remove them from the airways.

The key physiological barriers that drug particles encounter vary depending on the routes and the sites of deposition.⁵ Ideally, inhalable particulate carriers should deposit at a specific location in the lungs; this desired location varies based on the absorption mechanism of the drug. As the structure of the lung tissue and the geometry of the airways continuously change from one airway generation to the next, the fate of nanoparticles varies based on their site of deposition in the lungs. The major physical parameter that determines the site of deposition for inhaled particles is the aerodynamic behavior described by mass median aerodynamic diameter (MMAD), a function of particle shape, density and distribution of sizes.^6,7 Particles with MMAD larger than 5 µm accumulate in the conducting zone of the respiratory system due to inertial impaction; whereas particles with MMAD smaller than 5 µm may remain airborne for a long time. Smaller particles, upon passing through various airway generations with variable air velocities, deposit in the peripheral lung.⁸ For reproducible and efficient deposition, and enhanced retention, inhaled particles should have an MMAD of 1-5 µm. Particles larger than 5 µm deposit in the mouth, oropharynx and trachea, and those smaller than 1 µm are exhaled during breathing.⁹

For efficient drug deposition and absorption from the respiratory tract, particles must also overcome the components of the host defense system that function as barriers. As shown in Figure 1, after landing on the epithelium in the lungs, depending on the site of deposition, inhaled drug particles may either be removed from the lungs or dissolved in the lung lining fluid.¹¹ The alveolar epithelium, with large surface area and rich blood supply, is the most favorable site of deposition for small molecules that are rapidly absorbed either via cell-cell tight junctions, by passive diffusion or by means of transporters.² But, high molecular weight drugs, such as large proteins and immunoglobulins, may exhibit enhanced absorption in the upper airways through receptor-mediated transcytosis.¹² Dissolved drug may undergo absorption and enter the systemic circulation, or it may be metabolized by the local enzymes. Various protective mechanisms, including coughing, mucociliary clearance, translocation to other sites, and uptake by alveolar macrophages and phagocytes, can eliminate deposited drug particles. But particle cleansing capabilities of these processes vary significantly depending on the site of particle deposition.^13,14

FIG. 1.

The fate of inhaled particles in the respiratory apparatus (modified from¹⁰).

Mechanical Barriers

Particles encounter three major mechanical barriers imposed by airway geometry, local environment, lung epithelium and mucociliary clearance, which are elaborated below:

Airway geometry

Starting from the trachea, the structure of the airways changes at each bifurcation and the airway lumen diameter decreases with successive generations. To appreciate the influence of airway geometry on particle deposition, it is important to first understand the role of deposition mechanisms that dictate the fate of airborne particles according to their aerodynamic behavior.⁸ Larger particles usually deposit on the upper airways by inertial impaction because they are unable to change their trajectory as the airflow direction changes from one generation of airways to another due to their mass and velocity. Particle deposition by sedimentation takes place in the small airways and peripheral lung where the air velocity is relatively slower and residence time of the tidal air is longer.⁹ Sedimentation, a time-dependent mechanism, causes particles, which still remain in the air stream and have not been removed by impaction in the upper airways, to settle slowly on the lungs’ luminal surfaces. Particles deposit by diffusion, the third mechanism, in the terminal airways and alveolar region where smaller particles (∼0.1 µm) exhibit time-dependent deposition through Brownian motion.⁴ The variable structure of the airways creates turbulence in the inspired air, which may also influence particle deposition in the lungs. For efficient deep lung deposition, particulate drug carriers should remain airborne when travelling through continuous branching and tapering of the airways and should not deposit on upper airway surfaces prematurely by impaction. The extent of particle deposition by impaction depends directly on the particle size, velocity of the inhaled air, and the bifurcation curves but changes inversely based on the airway diameter.¹⁵ The influence of these factors decreases in the peripheral lung and alveolar regions; the deposition in the peripheral airways occurs via sedimentation and diffusion that are influenced by the physicochemical characteristics of the particles.

Airway microenvironment

The microenvironment of the respiratory apparatus, such as the relative humidity of the lungs (99.5%), may influence the deposition of inhaled particles. The humidity-induced changes in the particle may reduce particle deposition efficiency and change distribution patterns in the lungs.

Under the influence of high relative humidity in the airways, aerosol droplets with different tonicity may experience either increase or decrease in their particle size and thus the mechanism of particle deposition (Figure 2). Depending on the tonicity of aerosol droplets, the size may change 2- to 5-fold at equilibrium compared with the initially inhaled size.^16,17

FIG. 2.

Influence of high relative humidity in airways on the size of saline droplets administered via the pulmonary route. Depending on the saline tonicity, droplet size increases or decreases due to hygroscopic growth or shrinkage in the humid environment of the respiratory tract (modified from¹⁶).

Epithelium

The tightly arranged epithelial cells in the airway serve as a strong barrier against the entry of foreign materials into the systemic circulation. The ciliated epithelium participates in removal of the mucus and thus the extraneous particles and infectious agents entrapped in the mucus. The epithelium also secretes surfactant lipids that prevent airway collapse and control inflammatory responses.¹⁸ The cellular composition of the airway epithelial layer varies depending on airway generations (Figure 3).

FIG. 3.

The scheme of the epithelial layer at different sites in the lungs (modified from¹⁹).

The tracheobronchial airways are composed of ciliated pseudostratified columnar cells embedded with goblet, brush, Clara, and basal cells. The epithelium of the terminal bronchioles contains cuboidal or columnar cells interspersed with ciliated and Clara cells. The alveolar level has simple squamous alveolar epithelial Type I and II cells. In addition to the structural cells, the respiratory epithelium contains various levels of resident and/or inflammatory/migratory cells, including alveolar macrophages, dendritic cells, lymphocytes, leukocytes and mast cells; the total population of migratory cells varies depending on the lung’s pathophysiology.²⁰ Type I cells with large cytoplasm form much of the single layered epithelium of the alveolar region that allows an easy pass to drug absorption from drug particles that reach the alveolar site.²¹ Thus, the microanatomical features of the conducting and respiratory epithelium, such as differences in the tightness of cell junctions, influence drug absorption from the particles depending on their site of deposition.

Mucociliary clearance

Removal of mucus and entrapped particles from the respiratory tract, by coordinated cell signaling that produces synchronized movement of cilia in the upper airways, is a major host defense mechanism of the lung.²² Mucociliary clearance rate in the nasal cavity is around 3-25 mm/minute and is around 4-20 mm/minute in the trachea.²³ The rate decreases with the increase in the number of airway generations. With successive airway generations, the number of ciliated cells decreases, the cilia become shorter, beating frequency declines, and overall mucus secretion falls because of reduced number of secretory cells in the deep lungs.²⁴ The mucus usually forms a continuous layer in the conducting zone of the lung, but the thickness changes from ∼8 μm in the trachea to ∼2 μm in the bronchioles;²⁵ especially, in the lower airways, mucus becomes noncontinuous and appears as small upward moving patches. The serous cells of the submucosal glands and goblet cells normally secrete mucus that is composed mainly of water (95%); the other (5%) components of the mucus are glycoproteins, proteins, inorganic salts, and lipids (1%). The viscoelastic properties of the mucus are controlled by its water content.

Mucociliary clearance removes the inhaled particles deposited in the conducting zone. Ciliary movement transports the mucus-loaded particles or dissolved particles towards the oropharynx, which are then either swallowed or excreted.²⁶ The mucociliary clearance rate decreases in bronchiectasis, asthma and cystic fibrosis. This process can remove, within 24 hours, practically all of the particles larger than 6 μm that deposit in the ciliated airways. Smaller particles exhibit resistance to this process and thus remain in the lung for a longer period. Nano-sized particles may escape the process of mucociliary clearance due to their enhanced mobility and reduced level of internalization by the macrophages. In fact, particle uptake by alveolar macrophages is the major pathway for elimination in the peripheral airways because mucociliary clearance is absent in the peripheral regions of the lungs.²⁷

The site of deposition affects the residence time and the clearance rate of inhaled drug particles. The mucus layer in the airways acts as the key barrier during the process of particle deposition and entraps a significant proportion of the drug entering the lungs after inhalation; the entrapped particles are then cleared from the lung via the mucociliary clearance. Thus, for efficient deposition and extended retention, inhaled drug particulate systems should be engineered for particle charge, size, solubility, lipophilicity, and mucus penetrability.²⁸

Chemical barriers

Dissolution of the drug in the lung fluid, lung surfactants and metabolic enzymes are three major chemical barriers of the respiratory system.

Dissolution

The process of wetting and dissolution of inhaled particles deposited in the lungs is very important because the efficacy of encapsulated drug is only observed in its dissolved state. The dissolution of the inhaled particles varies depending on the site of deposition, drug solubility, dose, and volume of lung fluid.²⁹ Drug particles with high solubility in the lung fluid are either rapidly absorbed via alveolar epithelium or removed by the mucociliary escalator mechanism. Further, the dilution of the dissolved drug in the airway fluid allows drug molecules to interact with proteins, enzymes, opsonins and other components followed by metabolism and/or absorption into the systemic circulation and/or lymphatic system.

Epithelial Lining Fluid and Surfactants

The epithelial lining fluid, whose volume is very small, is distributed in the lungs in the form of a thin layer with a thickness of 5-10 μm in the upper airway. The thickness of the lining fluid gradually decreases with each successive airway generation, which becomes 0.05-0.08 μm in the alveolar region. Because of the small volume and difficulty in reaching the smaller airways, the exact composition of the epithelial lining fluid is not fully understood. Further, the airway mucosa, which is coated with a layer of phospholipids, along with mucin, lubricate and guard the epithelium against harmful foreign agents, impede adhesion of cilia with the mucus gel, and facilitate ciliary beating.³⁰ The lung surfactants, produced and secreted by the alveolar type II cells, are composed of phospholipids and surfactant-specific proteins. The thin surfactant film above the alveolar lining fluid reduces the surface tension, helps expand air-blood interface during inspiration and prevents alveolar collapse during expiration. The surfactants also defend the lung by preventing the adhesion of inhaled particles or microorganisms and thus facilitate their uptake by alveolar macrophages.³¹

Inhaled drug particles, after deposition and dissolution, may interact with the components of the fluid and surfactants lining the airways and alveolar region. Particle-fluid interactions may either increase or decrease drug solubility and thus the absorption of certain inhaled drugs.³² To enhance the deposition and retention of the drug, simulated lung surfactants have been used as delivery vehicles for pulmonary administration.^33,34 But these studies^33,34 demonstrated a complex interplay between drugs and lung surfactants, which should be taken into account for development and optimization of inhalable formulations.

Metabolic Activities in the Lungs

Drug metabolizing enzymes in the lung could significantly change drug concentrations in the lung and efficacy of inhaled drugs after deposition, although the knowledge of drug-metabolizing capabilities of the lung is very limited. The major classes of drug metabolizing enzymes present in the liver are also present in the conducting and respiratory regions of the lungs, but at a significantly reduced level. The concentration of cytochrome P450 enzymes, for example, is 5-20-fold lower in the lung compared with that in the liver. Other Phase 1 enzymes in the lungs are monoamine oxidase, aldehyde dehydrogenase and flavin-containing monooxygenases. The alveolar macrophages contain a higher concentration of esterase, but esterase levels in alveolar Type I and II cells are much lower. Macromolecules, especially proteins and peptides, undergo hydrolysis by proteases, such as endopeptidase and cathepsin H, present in the lungs. Metabolic degradation varies depending on the protein involved; vasoactive intestinal polypeptide, for example, is completely degraded after pulmonary administration.³⁵ But other proteins, such as insulin, exhibit high systemic absorption because they do not undergo metabolism in the alveolar region.³⁶

Cellular and Immunological Barriers

Cells of the immune system, such as macrophages and other elements of the lymphatic system can also act as barriers against particle deposition, drug absorption, and particle clearance and can eventually influence the therapeutic efficacy of inhaled particulate systems.

Uptake of Inhaled Particles by Macrophages

The alveolar macrophages, present at the air-blood barrier, are the first-line of defense that clean inhaled pollutants and pathogens from the deep lung region where involvement of the mucociliary clearance mechanism is minimal (Figure 4). Because of the presence of macrophages at the epithelial layer, submerged in the lung lining fluid, alveolar macrophages can efficiently engulf and remove foreign particles. In response to inhaled particles, alveolar macrophages migrate toward the particle deposition site, detect particles based on shape, size and composition or by means of opsonization, and finally internalize the particles. The whole process of uptake by macrophages may take 6–12 hours after deposition of particles in the alveolar region.³⁷ Particles engulfed by the macrophages are either degraded by the lysosomal enzymes or cleared via the lymphatic system. Some of the alveolar macrophages with engulfed particles move upward to the upper ciliated airways and are subsequently removed via the mucociliary escalator system.²⁷ The physicochemical properties of the particles can influence the phagocytosis of the particles by the alveolar macrophages. Macrophages fail to detect and surround individual particles smaller than 100 nm; some solid particles remain in the lungs for years because alveolar macrophages are too slow to remove those particles from the lung.³⁷

FIG. 4.

The schematic representing the alveolar region and patrolling alveolar macrophages in the lungs.

FIG. 5.

The elimination pathways for the nanoparticles deposited in the lungs (modified from¹³).

Alveolar macrophages are dominant phagocytes in mediating inflammatory and immunological responses against foreign material deposited in the lungs.³⁸ In response to inhaled pathogenic organisms, alveolar macrophages synthesize and secrete a series of inflammatory mediators—including cytokines, chemokines, arachidonic acid metabolites, lysozymes and antimicrobial proteins—to amplify the inflammatory and phagocytic responses, and recruit activated neutrophils into the alveolar region.³⁹ Recent studies demonstrate that the composition and surface tension of the surfactant layer in the lung may change the shape of alveolar macrophages and their phagocytic activity.^40,41

Alveolar macrophages can recognize and remove micron-sized particles from the lungs, but nanoparticles are more easily internalized by epithelial cells, leaving a few for uptake by alveolar macrophages.⁴² Depending on the desired therapeutic outcome, an inhalable drug delivery system can be tuned to exhibit enhanced or reduced detection and uptake by alveolar macrophages. The ciliated airways of the upper respiratory tract also contain macrophages, but compared with the mucociliary clearance mechanism, these airway macrophages contribute minimally to the overall elimination of deposited nanoparticles from the upper respiratory tract.

Uptake of inhaled particles by structural cells, blood and lymphatic systems

The lymphatic vasculature is largely present in the interstitial region surrounding small airways and blood vessels; the alveolar region and the air-blood interface are devoid of any lymphatic circulation.⁴³ The major functions of the pulmonary lymphatic system are to drain out any fluid and proteins that exude into the interstitial region of the lungs from the vascular compartment and prevent edema formation due to fluid accumulation in the lungs. Inhaled particles deposited in the deep lungs may translocate into the structural cells such as epithelial cells and subsequently cross the pulmonary interstitium to enter the systemic and/or lymphatic circulation. Micron-sized protein particles and microorganisms can easily translocate into the lymphatic fluid via the highly permeable epithelium. The uptake of inhaled particles by alveolar macrophages facilitates their translocation into the lymphatic circulation. Particles are then filtered through local lymph nodes before returning to the venous blood circulation. Moreover, inhaled nanoparticles that enter the general circulation, after translocation via transcytosis or other active transport mechanisms, may produce undesirable adverse effects such as platelet activation and aggregation in the blood and other inflammatory and immunologic reactions in certain organs.⁴²

Further, dendritic cells in the respiratory tract are the major antigen presenting cells in airway mucosa and lung parenchyma. These cells control immunological homeostasis in response to inhaled antigens that the lungs continuously encounter. Respiratory dendritic cells maintain the balance between tolerance and immunity against inhaled antigens by directing the appropriate responses in the respiratory system. Dendritic cells perhaps extend dendrites transepithelially into the airway lumen to grab antigen or particles directly. They prefer to internalize smaller (20 nm) particles compared with larger (1000 nm) ones.^44,45

Pathophysiological Barriers

Various pathological conditions, such as inflammation, edema and tissue injury due to exposure to harmful chemicals or insoluble particles, may alter the physiology of the airways and the lung tissue. Airway remodeling occurs due to chronic lung inflammation or recurring exposure of the mucus to external environmental substances such as allergens and pollutants; chronic structural changes may also occur at the subepithelial level.⁴⁶ The extent of the airway remodeling varies depending on the severity and duration of the underlying pathological condition and the lung function.

The severity of pathological changes influences the permeability of the air-blood barrier, deposition efficiency and clearance rate of inhaled particles. In healthy lungs, the mucus clearance rate changes based on the airway region, the number of ciliated cells, and ciliary beating rate in the particular region. The ciliary functions, and the amount and quality of mucus, may alter the mucus clearance, which is usually faster in the upper airways compared with that in the peripheral airways. An intact airway epithelial layer, undamaged ciliary structure, efficient ciliary movement, and physiologically normal amount, composition and viscosity of the mucus layer are important requirements for normal functioning of the mucociliary clearance system. Certain lung diseases may impair the mucociliary clearance system; immotile cilia syndrome and bronchiectasis, for example, almost completely destroy the ciliary movement and functions. In contrast, although the ciliary structure and functions are not adversely affected in cystic fibrosis, persistence of large amounts of viscous mucus in the airways impairs mucociliary clearance. The pathological changes in inflammatory disorders such as asthma and chronic bronchitis also reduce the mucociliary clearance rate. With airway remodeling, the increased number of submucosal glands and goblet cells secrete excessive mucus that causes obstruction of the airways. In fact, fluctuations in the airflow because of transient airway obstruction may affect deposition patterns of inhaled drug particles in the lungs.⁴⁷ Overall, these pathological conditions significantly reduce mucociliary clearance of inhaled particles deposited in the upper airways.

Infections, allergens, and cigarette smoke also cause pathophysiological changes in the airways and lung tissues. Although the influence of the underlying inflammatory conditions on the air-blood barrier permeability has been extensively studied, data regarding the changes on the permeability of alveolar epithelium are inconclusive. Thus, in airway inflammation, the permeability of alveolar epithelium may increase, remain unchanged, or decrease.^48,49 For example, repeated exposure to cigarette smoke increases epithelial permeability to water soluble hydrophilic drugs when compared with nonsmokers.^50,51

SUMMARY

• The pulmonary route offers the advantages of the large absorptive surface, reduced first-pass loss, high permeability, thin alveolar epithelium, and rich blood supply, the features that favor rapid systemic absorption of the drug.²

• The respiratory system has evolved with an array of protective mechanisms to keep the entry of invading and unwanted airborne particles at bay.

• Pathological changes in the lungs can also elevate or reduce the protective capabilities of the barriers.

• To reach the desired site and produce a therapeutic effect, inhaled particles must bypass both physiological protective barriers, including mechanical, chemical, cellular and immunological barriers, and disease-induced barriers (Figure 5).

• Thus, inhaled particles should be engineered and empowered to elude various lines of lung defense, promote deposition, and extend retention time.

Abbreviation Used

MMAD

mass median aerodynamic diameter

Author Disclosure Statement

The authors declare no conflict of interest.