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Chapter 1 Introduction

Md. Faiyazuddin (Ph.D. Thesis). Development of submicronized inhalable formulations with improved aerosolization performance (2012). 1

Formulation development encompasses an array of processes in which an active

pharmaceutical ingredient (API) is incorporated into a drug product. While biological

activity is a prerequisite for a successful dosage form, it is not the sole determinant.

Factors such as stability, processibility, delivery, and availability to the target organ

contribute to an efficacious pharmaceutical system. Optimization of these factors is a

key development mission, and the final product is often a compromise between

pharmaceutical and practical considerations. Development of pharmaceuticals for

inhalation is a particular challenge, as it involves the preparation of a formulation and

the selection of a device for aerosol dispersion. The lungs have lower buffering

capacity than other delivery sites (like GIT or blood), which limits the range of

excipients that could enhance delivery outcomes. An additional variable, unique to

pulmonary delivery, is the patient, both in terms of inhalation mode and respiratory-

tract anatomy and physiology (Timsina et al., 1994). Treating respiratory diseases

with inhalers requires delivering sufficient drug to the lungs to bring about a

therapeutic response. For optimal efficacy, drug administration must be reliable,

reproducible, and convenient. This goal can only be achieved by a combination of

improved formulation, proper dosing and inhaler design strategies (Smyth et al.,

2005). The following discussion outlines the development of submicronized inhalable

formulations with improved aerosolization performance to achieve the delivery goals.

Formulation development and characterization strategies and processing methods will

be discussed, with emphasis on their effect on stability, manufacturing feasibility,

delivery, and bioavailability.

Here first we describe bronchial asthma as a pulmonary illness, which need to be

treated by the development of submicronized (SM) inhalable formulations (i.e.,

nanoparticles; NP’s). A thorough knowledge of lungs anatomy & physiology is

important to study drug target sites and fate and effects of nanomedicines (mucociliary

movement, extrapulmonary translocation and macrophage engulfing etc.), therefore a

detail of lungs and deposition sites are also represented here. The need of a hyphenated

analytical instrument for bioesmitations at nanolevel is always in demand to facilitate

the high level translational research; therefore advances related in this field are also

mentioned.



1.1. BRONCHIAL ASTHMA (BA)

1.1.1. Global status of BA

Asthma is defined by the Global Initiative for Asthma as "a chronic inflammatory

disorder of the airways in which many cells and cellular elements play a role. The

chronic inflammation is associated with airway hyper-responsiveness that leads to

recurrent episodes of wheezing, breathlessness, chest tightness and coughing

particularly at night or in the early morning. These episodes are usually associated

with widespread, but variable airflow obstruction within the lung that is often

reversible either spontaneously or with treatment

(http://www.ginasthma.org/pdf/GINA_Report_2010.pdf14). It is thought to be

caused by a combination of genetic and environmental factors (Martinez et al., 2007).

The bronchial narrowing is usually either totally or at least partially reversible with

treatments. Bronchial tubes that are chronically inflamed may become overly sensitive

to allergens (specific triggers) or irritants (nonspecific triggers). The airways may

become "twitchy" and remain in a state of heightened sensitivity. This is called

bronchial hyper-reactivity (BHR). It is likely that there is a spectrum of bronchial

hyper-reactivity in all individuals. The narrowing that occurs in asthma is caused by

three major factors: inflammation, bronchospasm and hyperreactivity (Moore et al.,

2010). Symptoms can be prevented by avoiding triggers, such as allergens and

irritants, and by inhaling corticosteroids (Fanta et al., 2009). Leukotriene antagonists

are less effective than corticosteroids and thus less preferred (Lemanske et al., 2010).

Inflammation, or swelling, is a normal response of the body to injury or infection. The

blood flow increases to the affected site and cells rush in and ward off the offending

problem. The healing process has begun. Usually, when the healing is complete, the

inflammation subsides. Sometimes, the healing process causes scarring. The central

issue in asthma, however, is that the inflammation does not resolve completely on its

own. In the short term, this results in recurrent "attacks" of asthma. In the long term, it

may lead to permanent thickening of the bronchial walls, called airway ‘remodeling’.

If this occurs, the narrowing of the bronchial tubes may become irreversible and

poorly responsive to medications. When this fixed obstruction to airflow develops,

asthma is then classified in the group of lung conditions known as chronic obstructive



pulmonary disease (COPD). Therefore, the goals of asthma treatment are: (i) in the

short term, to control airway inflammation in order to reduce the reactivity of the

airways; and (ii) in the long term, to prevent airway remodeling (Fig. 1.1).

Fig. 1.1. The asthma cycle

1.1.2. Pathophysiology and pathogenesis of BA

One of the main pathogenetic factors is an allergic inflammation of the bronchial

mucosa. For instance, leukotrienes that are formed during an IgE-mediated immune

response exert a chemotactic effect on inflammatory cells. As the inflammation

develops, bronchi become hypersensitive to spasmogenic stimuli. Thus, stimuli other

than the original antigen(s) can act as triggers (A); e.g., breathing of cold air is an

important trigger in exercise-induced asthma (Fig. 1.2). Cyclooxygenase inhibitors

exemplify drugs acting as asthma triggers. Although there may be multiple triggers for

an inflammatory response (such as mast cell secretion), there is general agreement that

a lymphocyte-directed eosinophilic bronchitis is a hallmark of asthma. The

lymphocytes that participate in asthma pathology are biased toward the T-helper type

2 (Th2) phenotype, leading to increases in production of interleukin 4 (IL-4), IL-5, and

IL-13. The IL-4 from Th2 cells (and basophils) provides help for IgE synthesis in B

cells (http://www.cdc.gov/niosh/topics/asthma). The IL-5 provides support for

eosinophil survival. The innate or adapted immune response triggers the production of

additional cytokines and chemokines, resulting in trafficking of blood-borne cells (i.e.,

eosinophils, basophils, neutrophils, and lymphocytes) into airway tissues and these

cells further generate a variety of autocoids and cytokines. The inflammatory cascade

also leads to activation of resident cells within the airways that, in turn, can produce

panoply of cytokines, growth factors, chemokines, and autacoids (Mason et al., 2005).



Fig. 1.2. Pathophysiology of BA.

1.1.3. Triggers for BA

Triggers fall into two categories: allergens (specific) and non-allergens

(nonspecific/mostly irritants; Fig. 1.3 & 1.4) (Bouzigon et al., 1985).

i) Allergens: Seasonal pollens, dust, mites, molds, pets and insect parts; foods (Fish,

egg, peanuts, nuts, cow's milk, and soy); additives (sulfites); work-related agents

(Latex, epoxides, and formaldehyde).

Fig. 1.3. Factors limiting airflow in BA.



ii) Irritants: Respiratory infections (viral colds); drugs (Aspirin, other NSAIDs and

beta blockers); tobacco smoke; outdoor factors (Smog, weather changes, and diesel

fumes); indoor factors (Paint, detergents, deodorants, chemicals, and perfumes); night-

time; GERD (Gastro-esophageal reflux disorder); exercise (Under cold dry conditions;

work-related factors (Chemicals, dusts, gases, and metals); emotional factors

(laughing, crying, yelling, and distress); hormonal factors (Premenstrual

syndrome).The pharmacotherapy of asthma centers on controlling the disease with

drugs that inhibit airway inflammation. Other drugs that relax bronchial smooth

muscle are used for more immediate and direct relief of the symptoms of asthma. The

precise aetiology of asthma remains uncertain, but genetic and environmental factors

such as viruses, allergen exposure, early use of antibiotics, and the numbers of siblings

have all been implicated in its inception and development (Bouzigon et al., 1985).

1.1.4. BA pharmacotherapy (Antiasthmatic)

The pharmacological therapy of BA employs drugs aimed at reducing airway

inflammation (i.e., antiinflammatory agents) and drugs aimed more directly at

decreasing bronchospasm (i.e., bronchodilators). To these ends, six classes of

therapeutic agents are presently indicated for asthma treatment: b adrenergic receptor

agonists, glucocorticoids, leukotriene inhibitors, chromones, methylxanthines, and

inhibitors of immunoglobulin E (IgE). Each of these classes is discussed below.

i) Short acting β2-Adrenergic receptor agonists (SABA): These drugs are used for

acute inhalation treatment of bronchospasm. Drugs in this class include albuterol or

salbutamol (PROVENTIL, VENTOLIN), levalbuterol, the (R)-enantiomer of albuterol

(XOPENEX), metaproterenol (ALUPENT), terbutaline (BRETHAIRE), and pirbuterol

(MAXAIR). Terbutaline (BRETHINE, BRICANYL), albuterol, and metaproterenol also

are available in oral dosage form. Each of the inhaled drugs has an onset of action

within 1 to 5 minutes and produces bronchodilation that lasts for about 2 to 6 hours.

When given in oral dosage forms, the duration of action is somewhat longer (oral

terbutaline, for example, has a duration of action of 4 to 8 hours). They are the

preferred treatment for rapid symptomatic relief of dyspnea associated with asthmatic

broncho-constriction (Stevenson & Szczeklik, 1985).



Fig. 1.4. Airway inflammation by triggers.

ii) Long acting β2-Adrenergic receptor agonists (LABA): Salmeterol xinafoate

(SEREVENT) and formoterol (FORADIL) are long-lasting adrenergic agents with very

high selectivity for the β2-receptor subtype. Inhalation of salmeterol provides

persistent bronchodilation lasting over 12 hours. LABA relax airway smooth muscle

and cause bronchodilation by the same mechanisms as short-duration agonists.

iii) Inhaled Glucocorticoids (IGC’s): ICG’s are used prophylactically to control

asthma rather than acutely to reverse asthma symptoms. Although GC’s are very

effective in controlling asthma; treatment with systemic GC’s comes at the cost of

considerable adverse effects. A major advance in BA therapy was the development of

inhaled ICG that targeted the drug directly to the relevant site of inflammation. These

formulations greatly enhance the therapeutic index of the drugs, substantially

diminishing the number and degree of side effects without sacrificing clinical utility.

There are currently five GC available for inhalation therapy: beclomethasone

(BECLOVENT, VANCERIL), triamcinolone (AZMACORT), flunisolide (AEROBID),

budesonide (PULMICORT), and fluticasone (FLOVENT). While they differ markedly

in their affinities for the GC receptor, with fluticasone and budesonide having much

higher affinities than beclomethasone, they are all effective in controlling asthma at

the appropriate doses.



iv) Leukotriene receptor antagonists (LTA’s): Zafirlukast (ACCOLATE) and

montelukast (SINGULAIR) are LT-receptor antagonists. Zileuton (ZYFLO) is an

inhibitor of 5-lipoxygenase, which catalyzes the formation of leukotrienes from

arachidonic acid.

v) Anti-IgE therapy: Omalizumab (XOLAIR) is the first "biological drug" approved

for the treatment of asthma. Omalizumab is a recombinant humanized monoclonal

antibody targeted against IgE. IgE bound to omalizumab cannot bind to IgE receptors

on mast cells and basophils, thereby preventing the allergic reaction at a very early

step in the process.

vi) Cromolyn sodium and Nedocromil sodium: The main use of cromolyn (INTAL)

and nedocromil (TILADE) is to prevent asthmatic attacks in individuals with mild to

moderate bronchial asthma. These agents are ineffective in treating ongoing broncho-

constriction. When inhaled several times daily, cromolyn inhibits both the immediate

and the late asthmatic responses to antigenic challenge or to exercise. Cromolyn was

found to inhibit antigen-induced bronchospasm as well as the release of histamine and

other autacoids from sensitized mast cells. Cromolyn has been used in the United

States for the treatment of asthma since 1973. Cromolyn has emerged as one of the

first-line agents in the treatment of mild to moderate asthma.

vii) Methylxanthines (MX’s): Theophylline & theobromine, a methylxanthine, is

among the least expensive drugs used to treat asthma, and consequently, it remains a

commonly used drug for this indication in many countries. Theophylline has proven

efficacy as a bronchodilator in asthma and formerly was considered first-line therapy.

The formation of complex double salts (Caffeine and sodium benzoate) or true salts

[Choline theophyllinate (oxtriphylline)] enhances aqueous solubility. These salts or

complexes dissociate in aqueous solution to yield the parent methylxanthines and

should not be confused with covalently modified derivatives such as dyphylline [1,3-

dimethyl-7-(2, 3-dihydroxypropyl)xanthine].

viii) Anticholinergics (ACh’s): There is a long history of the use of ACh’s in the

treatment of asthma. Renewed interest in ACh’s paralleled the realization that

parasympathetic pathways are important in bronchospasm in some asthmatics and the

availability of ipratropium bromide (ATROVENT), a quaternary muscarinic receptor

antagonist that has better pharmacological properties than prior drugs. Combined



treatment with ipratropium and β2 adrenergic agonists results in slightly greater and

more prolonged bronchodilation than with either agent alone in baseline asthma. In

acute bronchoconstriction, the combination of a β2 adrenergic agonist and ipratropium

is more effective than either agent alone and more effective than simply giving more

β2 adrenergic agonist. A metered-dose inhaler containing a mixture of ipratropium and

albuterol (COMBIVENT); and a metered-dose inhaler containing a mixture of

ipratropium and fenoterol (DUOVENT, BERODUAL) are also available.

1.1.5. Delivery of antiasthmatic drugs

Pulmonary delivery of antiasthmatic’s (AAs) is one of the interesting strategies of

drug administration for effective local therapy (BA, COPD or cystic fibrosis) and also

for systemic administration of drugs (eg. protiens and peptides). Inhaled drug delivery

systems can be divided into three principal categories: pressurized metered-dose

inhalers (pMDIs), dry powder inhalers (DPIs), and nebulizers (NBs), each class with

its unique strengths and weaknesses. This classification is based on the physical states

of dispersed-phase and continuous medium, and within each class further

differentiation is based on metering, means of dispersion, or design. Nebulizers are

distinctly different from both pMDIs and DPIs, in that the drug is dissolved or

suspended in a polar liquid, usually water. Nebulizers are used mostly in hospital and

ambulatory care settings and are not typically used for chronic-disease management

because they are larger and less convenient, and the aerosol is delivered continuously

over an extended period of time. pMDIs and DPIs are bolus drug delivery devices that

contain solid drug, suspended or dissolved in a nonpolar volatile propellant or in a dry

powder mix (DPI) that is fluidized when the patient inhales. The development of DPIs

has been motivated by the desire for alternatives to pMDIs, to reduce emission of

ozone-depleting and greenhouse gases (chlorofluorocarbons and hydrofluoroalkanes,

respectively) that are used as propellants, and to facilitate the delivery of

macromolecules and products of biotechnology. The most widely used type is a

pressurized metered dose inhaler’s (MDI’s) which uses a propellant to expel droplets

containing the pharmaceutical product to the respiratory tract. These devices are

disadvantageous on environmental grounds as they often use CFC propellants, and on

clinical grounds related to the inhalation characteristic of the devices. Concurrently,



DPIs proved successful in addressing other device and formulation-related

shortcomings of the pMDI. Table 1.1 summarizes the main advantages and

disadvantages of the DPI (versus the pMDI) (Chan H.K., 2006; Jenne & Tashkin,

1993). DPIs are easier to use, more stable and efficient systems. Because a pMDI is

pressurized, it emits the dose at high velocity, which makes premature deposition in

the oropharynx more likely (Cipolla & Johansson, 2008).

Table 1.1. DPIs versus MDIs

Advantage Disadvantage

Environmental sustainability, propellant-

free design

Deposition efficiency dependent on

patient’s inspiratory airflow

Little or no patient coordination required Potential for dose uniformity problems

Formulation stability Development and manufacture more

complex/expensive

This is a particular problem because 99% of the droplets generated are recycled back

into the reservoir to be nebulized during the next dosing. Furthermore, the droplets

produced by nebulizers are rather heterogeneous, which results in very poor drug

delivery to the lower respiratory tract. However, proteins and peptides are susceptible

to denaturation when they come into contact with these propellants or with the large

air–liquid interfaces that are constantly being generated during aerosolization. Due to

the aforesaid contrivances, DPI’s has now emerged as the best alternative of MDI’s

and other PDD devices.

1.1.6. Lungs targeting strategies

1.1.6.1. Aspects of dry powder inhalers (DPI’s) design

DPIs are devices through which a dry powder formulation of an active drug is

delivered for local or systemic effect via the pulmonary route. DPIs have a number of

advantages over other methods of pulmonary drug delivery, for example, direct

delivery of drug into the deep lungs utilizing the patient’s respiration and are

increasingly being explored as a mechanism for the delivery of systemic drugs.

Successful delivery of drugs into the deep lungs depends on the integration between



powder formulations and the device performance (Peart & Clarke, 2001). Licensing

and marketing approval requires that current DPIs demonstrate in vitro performance

and in vivo efficacy and reliability. However, questions remain about the ability to

interchange DPIs and the effects of different clinical states and patient characteristics.

Dry powders for inhalation are formulated either as loose agglomerates of micronized

drug particles with aerodynamic particle sizes of less than 5 μm or as carrier-based

interactive mixtures with micronized drug particles adhered onto the surface of large

lactose carriers (Hersey JA, 1975). For topical respiratory drug delivery, a particle

size of 2–5 μm yields optimal benefit, whereas for systemic effects particle size of less

than 2 μm is needed for drug deposition in the small peripheral airways. Particles

greater than 5 µm may also result in systemic effects due to impaction in the throat

(i.e., oropharyngeal delivery) and oral absorption (Newman & Clarke, 1983). The

powder formulation is aerosolized through a DPI device, where the drug particles are

separated from the carrier (from drug–carrier mixtures) or deagglomerates drug

particles, and the dose is delivered into the patient’s deep lungs. In these systems,

particle size and flow property, formulation, drug–carrier adhesion, respiratory flow

rate and design of DPI devices extensively influence the performance (Hickey &

Concessio, 1997).

1.1.6.2. Inhaler design and the goals of asthma management

The inhaler device is very important in successful development of DPI products.

Presently, over 20 DPI devices are available on the market and more than 25 are in

development, but no device meets all of the requirements of an ideal DPI device.

Although several patents for dry powder inhalation are known from the beginning of

the 20th century (Bisgaard H, 1996), the first portable inhaler on the market (Fisons

Spinhaler) was not introduced until 1970. Early dry powder inhalers were all single-

dose devices with hard gelatin capsules as dose containers, like the Glaxo Rotahaler

and the ISF inhaler. A new dimension to dry powder inhalation was given by the

multi-dose Turbuhaler, which challenged many companies to copy this concept.

Examples of such devices include the Turbohaler (Astra Zeneca, Wilmington, DE,

USA), Diskhaler (GlaxoSmithKline, Research Triangle Park, NC, USA), Diskus

(known as the Accruable in some countries, for example the UK; GlaxoSmithKline),

Rotahaler (Glaxo-SmithKline) and Aerolizer (Novartis Pharma, Basel, Switzerland),



among others. These devices differ not only in their forms of particle generation and

delivery, but also with regard to design differences such as discrete or reservoir drug

containment, the number of doses and the presence of a dose counter. Compared with

discrete types, the performance of reservoir devices is susceptible to environmental

humidity and moisture. Additionally, dose-to-dose variations are greater. Furthermore,

dose emission for some can be dependent on inhalation flow rates. For an ideal DPI a

number of characteristics are important for device reliability, clinical efficacy and

patient acceptance. These include:

i) A device which is simple to use, convenient to carry, contains multiple doses,

protects the drug from moisture and has a indicator (audiovisual) of doses

remaining (Hickey & Crowder, 2007)

ii) Dose delivery which is accurate and uniform over a wide range of inspiratory

flow rates (Chrystyn H, 2006)

iii) Consistent dose delivery throughout the life of the inhaler and consistency of dose

when compared to other similar inhalers (Newman & Busse, 2002)

iv) Optimal particle size of drug for deep lung delivery (Clark AR, 1995)

v) Suitability for a wide range of drugs and doses (Newman et al., 2004)

vi) Minimum adhesion between drug formulation and devices (Byron PR, 2004)

vii) Product stability in the device (Byron PR, 2004; Newman et al., 2004)

viii) Cost-effectiveness (Bisgaard H, 1996), and

ix) Feedback mechanism to inform the patient of dose administration (Newman et

al., 2004).

A descriptive list of current DPI devices with delivery mechanism has been presented

in Table 1.2 and available inhalers are shown in Fig 1.5. A properly designed inhaler

containing an appropriate formulation can increase adherence and achieve the desired

therapeutic effects, and may also allow a reduction in dosing frequency. The

photographs of some currently available devices are presented in Fig. 1.5. A properly

designed inhaler containing an appropriate formulation can increase adherence and

achieve the desired therapeutic effects, and may also allow a reduction in dosing

frequency.



Table 1.2. Current DPI devices available in the market

Device DPI type Company Delivery Drugs

First generation: breath actuated single unit dose

Spinhaler Single dose Aventis Capsule SC

Rotahaler Single dose GlaxoSmithKline Capsule SS, BDP, SS +BDP

Inhalator Single dose Boeh. Ingeheim Capsule Fenoterol

Cyclohaler Single dose Pharmachemie Capsule SS, BDP, IB, BUD

Handihaler Single dose Boeh. Ingeheim Capsule Tiotropium

Aerolizer Single dose Novartis Capsule Fomoterol

FlowCaps Single dose Hovione Capsule NA

TwinCaps Single dose Hovione Capsule Anti-neuraminidase

Second generation DPIs: breath actuated multi-unit, multiple dose

Turbohaler Multi-dose Astra Zeneca Reservoir SS, TS, BUD

Diskhaler Multi-dose GlaxoSmithKline Blister SX, BDP, FP

Diskus Multi-dose GlaxoSmithKline Strip SS, SX, FP, SX+ FP

Aerohaler Multi-dose Boeh. Ingeheim - IB

Easyhaler Multi-dose Orion Pharma Reservoir SS, BDP

Ultrahaler Multi-dose Aventis Reservoir -

Pulvinal Multi-dose Chiesi Reservoir SS, BDP

Novolizer Multi-dose ASTA Reservoir BUD

MAGhaler Multi-dose Boeh. Ingeheim Reservoir SS

Taifun Multi-dose LAB Pharma Reservoir SS

Eclipse Multi-dose Aventis Capsule Na chromo-glycate

Clickhaler Multi-dose Innoveta Biomed Reservoir SS, BDP

Twisthaler Multi-dose Schering-Plough Reservoir MF

Third generation DPIs: active device

Exubera Single dose Pfizer Blister Insulin

Airmax Multi-dose Norton Health Reservoir Fm, BUD

(MF: mometasone furoate, SS: salbutamol sulphate, SX: salmeterol xinafoate, FP: fluticasone propionate, BUD: budesonide, TS: terbutaline sulphate, F: fenoterol, formoterol, IB: ipratopium bromide, Fm: formoterol, Ti: triotpium, SC: sodium cromoglycate, BDP: beclomethasone dipropinate, EFD: eformoterol fumarate).



1.1.6.3. Principles of inhaler design

The principles of DPI design is summarized and represented by Fig 1.5. Most DPIs

contain micronized drug blended with larger carrier particles, which prevents

aggregation and helps flow. These carrier particles play an important role in

pulmonary delivery of drug products. The dispersion of a dry powder aerosol is

conducted from a static powder bed. To generate the aerosol, the particles have to be

moved. Movement can be brought about by several mechanisms.

Fig. 1.5. Currently available DPI devices.

Passive inhalers employ the patient’s inspiratory flow. When the patient activates the

DPI and inhales, airflow through the device creates shear and turbulence; air is

introduced into the powder bed and the static powder blend is fluidized and enters the

patient’s airways. There, the drug particles separate from the carrier particles and are

carried deep into the lungs, while the larger carrier particles impact in the oropharynx

and are cleared. Thus, deposition into the lungs is determined by the patient’s variable



inspiratory airflow (Newman et al., 2004; Dunbar C, 2002). Inadequate drug/carrier

separation is one of the main explanations for the low deposition efficiency

encountered with DPIs (Zeng et al., 2000). Dose uniformity is a challenge in the

performance of DPIs. This is a greater concern with powders than with liquids because

of the size and discrete nature of the particulates. Various dispersion mechanisms have

been adopted for DPIs (Dunbar et al., 1998). Moreover, it has been suggested that if

shear and turbulence could be standardized by using a dispersion mechanism that is

independent of the patient’s breath, high delivery efficiency and reproducibility might

be achieved. Thus, an active inhaler might provide formulation-independent delivery

(Newman et al., 2004).

1.1.6.4. Physicochemical aspects of drug aerosol

The character of particulate systems is central to the performance of DPIs. Powders

present unique design challenges. Powders are 2-phase gas-solid systems. When

powders are static, they behave as solids; when they flow, they resemble liquids, easily

assuming the shape of the containing vessel. When a powder is dispersed in air, as is

the case after actuation of a DPI, in many ways it conforms to its carrier gas (unlike

gases or vapors, pharmaceutical powders are non-equilibrium systems).

i) Crystallinity and Polymorphism: Crystalline systems are defined by the

intermolecular spacing (ie, bond lengths and bond angles) of the unit cell, which can

be determined by x-ray diffraction. There are 7 crystal classes, which yield 14 distinct

lattice structures. The arrangement of molecules into crystals is governed by non-

covalent interactions, including hydrogen bonding, van der Waals forces,�π-π

stacking, and electrostatic interactions (Moulton & Zaworotko, 2001). However,

different polymorphs are at different energy states and thus have different properties,

including stability, solubility, and even bioavailability (Moulton & Zaworotko,

2001). The crystal habit is important because particle shape affects aerodynamic

behavior and, thus, lung deposition. Crystallization and crystal habit are influenced by

various factors, including identity of solvent (Garekani et al., 2001; Stoica et al.,

2004), impurities present during crystallization, and processing variables such as

temperature, pH, solution volume, and viscosity (Rodriguez-Hornedo et al., 2002).

Controlling crystallization is at the heart of “particle engineering,” which is a term that



is used with increasing frequency in the pharmaceutical and chemical literature.

Control over the crystallization process could yield particles with precisely engineered

morphology; co-crystallization (inclusion of functional impurities into the crystal)

could then become a formulation strategy, resulting in “supramolecular

pharmaceutics.”

ii) Moisture Content and Hygroscopicity: Hygroscopicity is the intrinsic tendency

of a material to take on moisture from its surroundings. Moisture uptake and loss due

to changes in relative humidity can result in local dissolution and recrystallization,

leading to irreversible aggregation through solid bridge formation, which can

adversely affect aerosol generation and lung deposition (Braun et al., 1996).

Hygroscopic growth involves the uptake of moisture, which will reach equilibrium in

droplets as a function of the water activity of the solution formed and the surrounding

atmosphere of water vapor; the Kelvin-Gibbs equation describes the phenomenon

involved (Hickey et al., 1990). As aerosol particles enter the lungs, they experience a

high-humidity environment (99.5% relative humidity at 37°C). Although they may not

reach equilibrium during transit, susceptible aerosol particles may be subject to

hygroscopic growth, which increases particle dimensions and affects lung deposition

(Hickey et al., 1993). Hygroscopic growth can be prevented by coating the drug

particles with hydrophobic films (Hickey et al., 1990).

iii) Particle Size: Particle size is the single most important design variable of a DPI

formulation. Methods for determining particle size and distribution use various

geometric features or physicochemical properties (Hickey et al., 1990). Among these,

aerodynamic diameter is the most relevant to lung delivery and ultimately to

therapeutic effect. There is substantial literature from the fields of industrial hygiene,

environmental and occupational medicine, and pharmaceutical sciences that link

aerodynamic size and size distribution to the probability of deposition in specific lung

sites. Several techniques are available for determining particle size distributions. The

aerosol sizing techniques can be classified as: inertial methods, light-scattering

methods, or imaging methods.

iv) Aerodynamic Diameter and Dynamic Shape Factor: Aerodynamic diameter is

the most appropriate measure of aerosol particle size, because it relates to particle

dynamic behavior and describes the main mechanisms of aerosol deposition; both



gravitational settling and inertial impaction depend on aerodynamic diameter. To reach

the peripheral airways, where drug is most efficiently absorbed, particles need to be in

the 1–5 µm aerodynamic diameter range (Bates et al., 1966). Particles larger than 5

µm usually deposit in the oral cavity or pharynx, from which they are easily cleared. In

contrast, particles smaller than 0.5 µm may not deposit at all, since they move by

Brownian motion and settle very slowly. Moreover, they are inefficient, as a 0.5- µm

sphere delivers only 0.1% of the mass that a 5 µm sphere carries into the lungs.

v) Fine Particle Fraction: FPF is the total mass of the particles that are in the fine-

particle range (Hickey et al., 1993). The fine-particle component of aerosols is usually

defined as the percentage of particles that are smaller than 5 µm aerodynamic

diameter, or, in the case of certain particle-sizing instruments, a cut-off diameter that is

close to 5 µm. Quite often this may be in the 6–7 µm range. The danger of adopting

these values as definitive measures of equivalency is associated with the effect of

particle size on deposition (Martonen et al., 1992).

vi) Surface Area and Morphology: Particle separation is the most important

performance characteristic for effective aerosol generation. To separate particles,

specific forces of interaction must be overcome.

There are 4 major forces of interaction between particles: mechanical interlocking due

to surface asperities, capillary forces from the presence of water, electrostatics arising

from the insulating nature of the material, and van der Waals forces from the

fundamental electromagnetic nature of matter. The mechanical interlocking is due to

surface features or roughness is a prominent mechanism preventing particle dispersion.

Temperature and humidity cycling, or poor drying may also result in solid bridging,

through crystallization/recrystallization phenomena at the particle surfaces (Crowder

TM, 2003). The origins of the electrostatic charge are atmospheric ionization,

chemical composition, contact with charged objects, and triboelectric charging from

motion. The strong forces (mechanical, capillary, and electrostatic) act in a

background of weak electromagnetic van der Waals forces, which relate to the

influence of point charges at a distance and can be derived from the Lennard Jones

potential (Hickey et al., 1994). Low density, high-porosity particles achieve the goal

of reducing van der Waals forces (Edwards et al., 1997).



1.2. IMPACT OF NANOSIZING ON EFFECTIVE LUNGS DEPOSITION

The different methods to produce inhalable therapeutic nanoparticles are summarized

in Fig. 1.6. In general, the processes could be grouped into two categories, top-down

and bottom-up, depending on the starting materials (Bailey & Berkland, 2009). The

top-down methods start with solid particles that are substantially larger than the

targeted nanoparticle. These large particles are mechanically broken down into smaller

sizes. Milling methods, such as wet and jet milling (Patravale et al., 2004), and

homogenization methods, such as rotor stator or high pressure homogenization

(Patravale et al., 2004; Keck & Müller, 2006), are examples of top-down processes.

Wet milling and high pressure homogenization (HPH) are the most mature top-down

processes used to produce drug nanoparticles (Eerdenbrugh et al., 2008). Both

processes are scalable, reliable, and are capable of producing nanoparticles with a

narrow size distribution (Patravale et al., 2004). However, due to their mechanical

nature, these processes are usually very time consuming (up to a few days), require

significant energy (Wiedmann et al., 1997; Ostrander et al., 1999; Ali et al., 2009),

could potentially increase the amorphous region (Chow et al., 2007; Rasenack et al.,

2004), and may introduce contamination from the milling media or the

homogenization chamber (Muller et al., 2001; Chow et al., 2007).

The bottom-up methods, on the other hand, form the particles from the molecular

level, and therefore have better control of particle properties (e.g. size, morphology,

and crystallinity) than top-down processes (Chow et al., 2007). The bottom-up

processes produce nanoparticles by crystallization and solvent removal. In general, the

bottom-up methods are more sophisticated and complex than the top-down methods.

They can be further categorized into solvent evaporation methods and antisolvent

methods, depending on the method used to induce crystallization and remove solvent.

In a solvent evaporation process, particles are formed while solvent is removed with

physical means such as heat, freezing, or vacuum drying. In an antisolvent process,

particles are produced by mixing a solution with an antisolvent, causing reduction of

dissolution power. Spray drying is the most commonly used and extensively studied

solvent evaporation method (Vehring et al., 2008; Weers et al., 2007). Solvent in an

atomized droplet is thermally removed inside the spray drying chamber. This is a one-

step process to produce dry powder and allows control of particle properties such as



morphology, density, and surface composition (Weers et al., 2007). However, the

technique has a few disadvantages, including possible degradation of heat sensitive

drugs and inefficient yields (Rogers et al., 2002; Foster et al., 1995).

Fig. 1.6. Particle formation processes.

Spray freezing into liquid overcame these disadvantages. Instead of evaporating the

solvent, dry particles are produced by spraying atomized droplets into a cryogenic

liquid and subsequently lyophilizing the frozen droplets. Accordingly, thermal

decomposition is avoided due to the cryogenic operating conditions and the yield can

be as high as 95% (Barron et al., 2003). A series of cryogenic methods, such as ultra

rapid freezing, have since been developed. The rapid expansion of supercritical

solutions is a unique process that produces particles by expanding a supercritical

solution through a nozzle. This process produces small particles with a narrow

distribution and little to no residual solvent. Other solvent evaporation methods, such

as evaporative precipitation into aqueous solution and microemulsion methods, have

also been demonstrated to produce inhaled drug nanoparticles (Chen et al., 2002;

Dickinson et al., 2001). Antisolvent processes vary depending on the antisolvent

(supercritical fluids or liquids) and the crystallization apparatus used. Liquid

antisolvent processes use a different means of process intensification, such as high

gravity (Zhao et al., 2010) and sonication (Dhumal et al., 2009), to achieve small

particle size. High-speed impinging crystallization is another novel method of process



intensification, which was recently developed to generate nanoparticles (Panagiotou

et al., 2008) but has not been used to generate inhaled drug particles to date. High

production yields from 80% to approximately 100% have been reported with most of

the nanoparticle formation methods (Reddy et al., 2004; Kesisoglou et al., 2007;

Deng et al., 2008; Chan et al., 2003; Chougule et al., 2007).

1.2.1. Nanosizing techniques

1.2.1.1. Top-down process

i) Wet milling: Wet milling processes have been successfully used to produce a few

FDA approved drugs (Eerdenbrugh et al., 2008). The milling media, usually made of

glass (Chiang et al., 2009), zirconium oxide (Yang et al., 2010) or highly cross-

linked polystyrene resin (Patravale et al., 2004) and slurry containing raw drug

particles and stabilizer are circulated through a milling chamber. A rotating milling

shaft produces both high shear rate and impaction between the milling media and drug

particles. The energy generated from the high shear and impaction disintegrates the

raw material into nanosized particles, generally in the size range of 100–400 nm range

(Eerdenbrugh et al., 2008). NanoCrystal™ is an example of a wet milling process

(Burgess et al., 2005). The wet milling process can be scaled up from a 100 mg lab

scale to a 500 kg commercial scale without introducing significant changes in particle

size distribution and batch-to-batch variation (Burgess et al., 2005).

ii) High pressure homogenization (HPH): Similar to the wet milling process, HPH is

scalable and has been commercially used for several FDA approved drugs. A slurry

feeding stream, usually composed of drug coarse particle and stabilizer, is pressurized

with an intensifier pump to 100–2000 bars. The high pressure stream then passes

through a relief valve where cavitation, high shear force, and collision between the

particles are induced by a sudden release of pressure (Bhavna et al., 2009). The HPH

process has also been used to produce nanosized bulking agents, including α-lactose

and sucrose nanoparticles which have been shown to improve aerosol performance of

low dose pMDI formulations (James et al., 2008). The homogenization pressure used

in the reported studies varied from 690 bar (10,000 psi) to 1724 bar (25,000 psi)

(James et al., 2008). Compared to the wet milling process, HPH generally requires

shorter processing time, from less than 30 min to a few hours (Bhavna et al., 2008).



1.2.1.2. Bottom-up process

1.2.1.2.1. Solvent evaporation methods

i) Spray drying (SD): In these methods, solid or hollow/dimpled/wrinkled particles

are formed, depending on the Peclet number (Pe) and the final particle size is

determined by the initial droplet size and the concentration of the starting solution

(Okuyama & Lenggoro, 2003; Thybo et al., 2008). The typical size of spray dried

pharmaceutical particles ranges from 0.5 to 50 μm. Although the spray drying of NP’s

in production is severely limited, the process is widely used in the post-processing

steps to generate dry nanoparticles (Yang et al., 2008).

ii) Cryogenic solvent evaporation (CSE): A series of techniques, including spray

freezing into liquid (SFL), have been proposed to evaporate solvents with cryogenic

fluids and freeze drying at a wide range of cooling rates (102–106 K/s). To prevent

particle growth during the cooling process, the cooling rate is maintained at a

sufficiently higher rate than those uses in the conventional lyophilization (~1 K/s)

(Engstrom et al., 2008). These cryogenic methods generally produce amorphous

particles (Yang et al., 2008). The SFL process atomizes the feeding stream below the

surface of the cryogen where additional impinging between the feed stream and

cryogenic liquid enhances the atomization. The evaporated cryogen around the

droplets provides a sufficient insulating effect to prevent particle growth. Other

cryogenic techniques, namely ultra rapid freezing (URF) (Yang et al., 2008) or thin

film freezing (TFF), have also been developed (Engstrom et al., 2009). URF utilizes a

cooling rate between those used for SFL and lyophilization, i.e. 102–104 K/s. In this

process, a drug solution and stabilizer is dripped onto a frozen hard surface whose

temperature is maintained by dry ice. Upon impacting with the frozen surface, the

droplet is frozen into a disk, which is then lyophilized to remove solvent (Overhoff et

al., 2007).

iii) Evaporative precipitation into aqueous solution (EPAS): EPAS is similar to

SFL except that heated water is used instead of the cryogen. An organic solution of

drug is pre-heated through a coil, injected under the surface of a heated aqueous

solution with added surfactant(s) to stabilize the particles. Intensive atomization occurs

below the liquid surface that produces a large interface between the organic and



aqueous solutions, causing rapid evaporation of the organic solvent and precipitation

of particles. The use of higher temperatures was shown to produce smaller particles

due to the faster evaporation rate and mass transfer.

iv) Nanoemulsion (NE): NE method involves trapping the drug solution in the nano-

domain and then the solvent can be either vacuum evaporated or frozen and freeze

dried to generate either a nanosuspension or dry nanoparticles, respectively [Dickinson

et al., 2001].

v) Condensation aerosol generation (CAG): Condensation aerosol generation

produces nanoparticles by condensing vaporized liquid or solid. In one application, a

drug solution is fed through a heated capillary with a precisely controlled energy input

(Gupta et al., 2003). The solution is vaporized in the capillary and exits the capillary

as a jet of hot vapor. The resulting particles are subsequently cooled and condensed by

ambient air to form particulates suitable for inhalation (Li et al., 2005; Hong et al.,

2002). The MMAD of the generated aerosol varied from 390 to 420 nm with a narrow

standard deviation (2–4 nm). A novel process was developed to generate nanoparticles

by quickly evaporating a thin layer of drug substrate and cooling it into nanoparticles

(Simis et al., 2008).

vi) Rapid expansion of supercritical solutions (RESS): RESS, the oldest

supercritical particle engineering technique, utilizes the tunable dissolution power of a

supercritical fluid (e.g. supercritical CO2) (Pasquali et al., 2008). A pre-heated

supercritical drug solution is rapidly expanded through a nozzle, resulting in a sudden

drop in density and the dissolution power, causing the solute to precipitate. The

instantaneous generation of homogeneous supersaturation allows production of small

particles with a narrow size distribution (Martín et al., 2008). Unfortunately, this

method is only applicable for drugs that are highly soluble in the supercritical fluid.

1.2.1.2.2. Antisolvent methods

Mixing a solution with an antisolvent generates supersaturation that subsequently

induces nucleation and simultaneous growth by condensation and coagulation (Dalvi

et al., 2009). The drug must be soluble in the solvent but practically not soluble in the

antisolvent. The solvent and antisolvent must also be miscible at the operating

conditions. In this process, mixing is a critical factor for controlling the final particle



size and size distribution (Yang et al., 2005; Tung et al., 2009; Matteucci et al.,

2006). When supersaturation is sufficiently high and mixing is uniform, fast nucleation

consumes most of the solute and arrests crystal growth, enabling great control of

particle size and distribution (Matteucci et al., 2006). The effect of mixing is

expressed as the Damkoehler Number (Da), which is the ratio between the mixing time

(τmix) to the precipitation time (τprecipitation). Da<1 (uniform mixing) is required to form

small particles with controlled size distribution (Matteucci et al., 2006).

i) Effect of surfactants: Absorbed surfactants on the precipitating drug surface

stabilize the particle by steric and electrostatic stabilization. This increases particle

growth by condensation and coagulation, and effectively increases precipitation time

and decreases Da (Matteucci et al., 2006). Therefore, higher surfactant concentrations

were required to generate smaller particles, i.e. the higher the surfactant concentration,

the smaller the particle size, until the size reduction reached a plateau (Da<1). In

maintaining small particle size, the surfactant in the solvent phase has more effects

than antisolvent phase. This is due to elimination of mass transfer within the

antisolvent phase and diffusion through the solvent/antisolvent interface.

ii) Effect of process intensification: Process intensification, such as rotating packed

bed (Chen et al., 2004), reduces mixing time τmix, yielding lower Da and enabling

good control of size and distribution. High-gravity controlled precipitation (HGCP)

processes utilize a rotating packed bed (RPB) to intensify micromixing and mass

transfer (Hu et al., 2008). The HGCP process provides good control of particle size

and size distribution. A variety of SCF technologies, such as gas antisolvent (GAS),

supercritical fluid antisolvent (SAS), and solution enhanced dispersion by supercritical

fluids (SEDS) (Okamoto et al., 2008; Reverchon et al., 2006), also utilizes process

intensification.

1.2.2. Aerosol characterization

Aerosolized nanoparticle formulations are characterized by a variety of in-vitro, in-

vivo, and ex-vivo methods. Table 1.3 lists several important techniques that have been

commonly used.

1.2.2.1. Dissolution rate: It is used to predict particle behavior in-vivo, including

absorption, therapeutic activities, metabolism, and elimination of the drugs. A few



Pharmacopoeia standard dissolution tests (paddle, basket, and flow-through) have been

developed and are used for characterizing solid oral dosage drugs. The applicability of

these methods on nanoparticles is affected by the compromised wetting behavior as

the particle size decreases (Heng et al., 2008), and the flow-through method was

found the most appropriate for measuring nanoparticle dissolution rate.

Table 1.3. In vitro/ in vivo characterization

Type Method Measurement

In-vitro

Dissolution test Dissolution rate

Inertial impaction Aerodynamic particle size distribution

Delivered dose assay Dose delivered and dose uniformity

Laser diffraction Particle size and size distribution

Laser doppler velocimetry Aerosol velocity

In-vivo

Scintigraphy Visualization and quantification of aerosol

deposition in respiratory tract

Pharmacokinetics (HPLC;

LC-MS; UHPLC/MS) PK parameters

Phamacodynamics Biochemical and physiological effects

Ex-vivo Isolated perfused lung Drug transport and disposition effects

However, there is no standard method for characterizing inhaled drug nanoparticles.

For these drugs, solubility and kinetics of drug dissolution in the lung fluid are critical

for predicting drug behavior in the lungs (Son et al., 2009). This test is generally

carried out by adding the drug particles into a dissolution medium under agitation. The

solution is then sampled at a pre-defined time interval to measure the extent of

dissolution. Since there are no standard methods developed to date, various media

[distilled water (Dickinson et al., 2001), phosphate buffer (Tam et al., 2008),

simulated lung fluid (SLF) and modified SLF (Sinswat et al., 2008)], apparatus flow-

through cell (Davies et al., 2003), standard USP dissolution apparatus (Sinswat et al.,

2008) and a stirring vessel (Dickinson et al., 2001), test conditions (sinking (Sinswat

et al., 2008), supersaturated condition (Tam et al., 2008), and particle introduction

into the medium (adding the particles directly (Yang et al., 2008a) or in aerosolized



for (Tam et al., 2008), have been utilized to characterize the dissolution rate of

inhaled drugs.

1.2.2.2. Inertial impaction: It is a standard in-vitro technique used to predict aerosol

deposition in the lungs. The impactor measures aerodynamic particle size distribution

(APSD) (USP, 2009; Hickey et al., 2004) by passing the aerosol through a nozzle.

The impaction efficiency can be estimated using Stokes number, which is defined as

the ratio of the stopping distance to the orifice diameter (Merkus et al., 2008; USP,

2009). The Anderson Cascade Impactor (ACI) is the pharmacopeia method and the

most commonly used instrument of inertial impactor.

1.2.2.3. Delivered dose assay: It is used to quantitatively determine the delivered dose

and dose uniformity using a sampling apparatus (USP, 2009). The apparatus is

connected to an aerosol delivery system through a mouthpiece adapter and a vacuum

pump is connected at the other end to provide the desired air flow. The aerosol

released into the sampling apparatus is captured onto a filter that is then assayed to

quantify the delivered dose.

1.2.2.4. Gamma scintigraphy: It is a non-invasive imaging technique that enables

visualization of drug deposition in respiratory track and lungs, predicts in-vivo

efficacy data (Newman et al., 1998 & 1999) and estimates mucociliary clearance

(Snell et al., 1999). The drug formulation is radio labelled with gamma ray-emitting

radiotracer, such as 99mTc, prior to inhalation. Radiolabelling could be achieved by

mixing the particles with a solution of a radiotracer. An appropriate in-vitro validation

is required to ensure distribution of the radiotracer across particle size range and not

affecting particle size distribution. After inhalation of the radiolabelled drug, a gamma

camera is used to visualize and quantify the drug deposition in the respiratory track

and lungs (Newman et al., 2003). A few 3D methods, such as single photon emission

computed tomography (SPECT), have also been explored and developed to overcome

limitations of the 2D method in differentiating the overlaid anatomic structures, which

is particularly useful to establish in- vivo and in vitro correlations (Eberl et al., 2006;

Chan et al., 2006).

1.2.2.5. Pharmacokinetics: PK studies investigate adsorption, distribution,

metabolism, and excretion (ADME) of drugs using either animal or human models.

Lung deposition and plasma concentration data are collected from test subjects to



extract critical PK parameters, including drug absorption parameters such as the peak

drug concentration (Cmax), the time Cmax occurs (Tmax), the absorption rate constant

(ka) to determine the absorption rate, the area under curve (AUC) to estimate

bioavailability and half life of the drug (T0.5). Small rodents, such as mice and rats, are

commonly used in animal PK studies, where the drug is directly administered through

intranasal or intratracheal routes; or using passive inhalation (Sakagami et al., 2006;

Cryan et al., 2007).

1.2.2.6. Ex vivo study: It is used to study lung specific PK without interference of

systemic (Sakagami et al., 2006). The test utilizes isolated perfused lung models,

while maintaining its tissue functionality and architecture. Since the lungs are isolated

from the body, possible complications that may occur during in-vivo tests are avoided

Cryan et al., 2007). The shortcoming of ex-vivo methods include complex

experimental set up, lack of tracheo-bronchial circulation and the short period for

viable use of the perfused lungs (Cryan et al., 2007).

1.3. FATE AND EFFECTS OF SUBMICRONIZED PARTICLES IN LUNGS

The actual fate and effect of small particles can’t be observed without having a detail

of lungs anatomy and physiology concerned.

1.3.1. Lungs physiology

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 (http://www.ecompendium.be2011). 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 (Fig. 1.7) (Parkes et al., 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 0.25 m2 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 et al., 2007; Lai et al., 2009).

Fig. 1.7. Pulmonary tissue distribution.

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 et al., 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 min-1.



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; DPPC), 5–10% neutral lipids (mainly cholesterol), 5–

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

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. 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 head groups oriented towards the

aqueous phase and the hydrophobic acyl chains pointing towards the air. The higher

the concentration of phospholipid 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 et al., 2010). They also down regulate

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 et al.,



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 et al., 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 et al., 2003). Proteases

play an essential role in cell and tissue growth, differentiation 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 et al., 1994). It is progressively replaced by a simple cuboidal cell layer in

themore 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 CP, 2004). 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

et al., 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 et al., 2006). 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 I, 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 TD, 2002). Blood supply to the lungs is

divided among the pulmonary and systemic circulations (Altiere et al., 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 % 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 structures of the trachea-bronchial tree.

Lymphatic vessels exist in close proximity of major blood vessels and of the airways

(El-Chemaly et al., 2009).

1.3.2. Influence of size in regional lung deposition

Particles are deposited in the respiratory tract when they are removed in a definitive

fashion from the flow streamline generated by the breathing maneuver. The

physicochemical properties of inhaled aerosols that can determine deposition are: size,

size distribution, shape, charge, density and hygroscopicity (Pilcer et al., 2010). The

site of deposition of an inhaled formulation within the respiratory tract depends on the

aerodynamic diameter of the aerosol particles. In the field of aerosol medicine, particle

size is a formulation design variable that can be engineered accordingly, aiming the

development of pulmonary drug delivery systems (Fig 1.8) (Chow et al., 2007). From

the mechanisms of deposition, particle diameter is the primary factor determining

pulmonary deposition of aerosols in the various regions of the respiratory tract.



Ultimately, inspiratory flow rate also plays an important role in the particle deposition

following pulmonary administration (Dolovich et al., 2009).

Fig. 1.8. Particle size of nanomedicine used for lungs.

Generally, there are five different mechanisms by which particle deposition can occur

in the lungs: inertial impaction, sedimentation, diffusion, interception and electrostatic

precipitation. The two latter mechanisms are related, respectively, to particle shape

(e.g. elongated particles) and electrostatic charges; and have been reviewed in detail

elsewhere (Gonda et al., 2004). The mechanisms of deposition directly (or inversely)

related to particle size are presented in Fig. 1.8.

1.3.2.1. Inertial impaction: It occurs when airborne particles possess enough

momentum to keep its trajectory despite changes in direction of the air stream,

consequently colliding with the walls of the respiratory tract.

The chances of deposition by impact are increased when the particles are more likely

to travel longer distances, S, which is based on the particle mobility (velocity per unit

force), B, mass, m, and velocity, v, according to Eq. (1) (Gonda et al., 2004);

S = B ·m· v …(1)

The dimensionless Stokes’ number, Stk, more specifically describes the probability of

particle deposition in the airways via impaction.



The higher the Stokes’ number, the more readily particles will be deposited by inertial

impaction, according to Eq. (2):

Stk = ρp· d2 · V/18 ·η· R …(2)

(Where ρp is the particle density, d is the particle diameter, V is the air velocity, η is

the air viscosity and R is the airway radius).

Therefore, considering the bifurcated architecture of the lungs, large particles

travelling through the airways at high airflow velocity are more likely to impact in the

proximal portion of the respiratory tract (upper airways) (Zeng et al., 2001).

Fig. 1.9. Diagram represents the smaller particles depositing in the lower airways

(d: particle diameter; Stk: Stokes number; ρp: particle density; V: air velocity; η: air viscosity; R: airway radius; Vts: terminal settling velocity; ρa: air density; g:

gravitational acceleration; Dif: diffusion coefficient; k: Boltzmann’s constant; T: absolute temperature; dae: aerodynamic diameter; ρ0: unity density).

1.3.2.2. Sedimentation: It is a time-dependent process in which particles settle due to

the influence of gravity. Hence, breathing maneuvers in which more time is allowed

for the particles to sediment (e.g. breath-holding) may increase lung deposition (Zeng

et al., 2001). The Stokes’ Law assumes that the relative velocity between the surface

of the particle and the airstream is null. Considering unit density spheres of 1–40 μm,



Stokes’ law can be used to predict the terminal settling velocity, Vts, according to Eq.

(3):

Vts = (ρp − ρa) · d2 · g/ 18 · η …(3)

(Where ρa is the density of air (ρp > ρa) and g is the gravitational acceleration (Gonda

et al., 2004). However, for particles smaller than 10 μm, a slip correction factor (Cc)

derived by Cunningham should be applied to Stokes’ law, as described in Eq. (4)

(Crowder et al., 2002);

Cc = 1 + Kn · [A1 + A2 . exp (− A3/Kn)]…(4)

(Kn is the Knudsen Number, and A1, A2 and A3 are constants). The size-dependence of

this equation is related to the balance between the downward force exerted by the

particle and the resistant force for which Stokes’ law is valid. With increased air flow,

the stream becomes turbulent and the deposition by impaction increases (Zeng et al.,

2001). Therefore, this equation assumes laminar flow within the airways, as defined by

the Reynolds number, Re (Eq. 5):

Re = ρa · V · d/η …(5)

Interestingly, the effect of gravity on particle sedimentation has recently been

evaluated by the National Aeronautics and Space Administration (NASA). Inhalation

of lunar dust is a concern for potential toxicological effects to future explorers of the

moon (Darquenne et al., 2008). In this study, the researchers found that, although the

deposition of fine particles is greater on earth, peripheral deposition was improved at

low gravity for those particles that are actually deposited in the lunar environment.

1.3.2.3. Diffusion: Diffusion occurs when particles are sufficiently small to undergo a

random motion due to molecular bombardment. This process, also known as Brownian

motion, is correlated to particle size, according to Stokes–Einstein equation {Eq. (6)}

(Gonda et al., 2004);

Dif = k · T/3π · η · d…(6)

(Where Dif is the diffusion coefficient, k is the Boltzmann’s constant and T is the

absolute temperature).



Different from impaction and sedimentation mechanisms, diffusional deposition is

therefore inversely related to particle size.

1.3.3. Aerodynamics in aerosolization performance

The aerodynamic diameter, dae is, by definition, the diameter of a sphere with unit

density (ρ=1), having the same terminal settling velocity in still air as the particle in

consideration (de Boer et al., 2002). Considering the particle characteristics, this

independent variable can therefore correlate the effect of geometric diameter and

particle density, as described in Eq. (7);

dae = d. √ρ/ ρ0.χ …(7)

Where‘d’ is the geometric diameter of the particle, ρ is the particle density, ρ0 is the

unit density and χ is the particle dynamic shape factor denoting deviation of shape

from sphericity (Hinds et al., 1999). The relationship between the geometric diameter

and the particle density for aerodynamic diameter is illustrated by a study of large

porous particles for pulmonary delivery (Edwards et al., 1997). In vitro aerodynamic

size determination of inhalation products can be performed by cascade impactors,

including Andersen Cascade Impactors, Multi-Stage Liquid Impinger and Next

Generation Cascade Impactors (de Boer et al., 2002). This characterization is essential

to evaluate the particle deposition in the lungs. The mass median aerodynamic

diameter (MMAD) is the cut off particle size in which 50% of the mass of the aerosol

is smaller and the other 50% is larger than the referred parameter (Jaafar-Maalej et

al., 2009). MMAD is a measure of central tendency while geometric standard

deviation (GSD) indicates the magnitude of dispersity from the MMAD value (Pilcer

et al., 2008). In order to be deposited in the deeper lung, it is essential that the drug

particles have an aerodynamic diameter between 0.5 and 5 mm (Tsapis et al., 2002).

Smaller particles will be exhaled while larger particles will impact in the mouth or

throat due to their inertia. Because of their low particle mass, such fine powders are

very cohesive. Cohesivity emerges because of inter-particle interactions which impede

flowability. Interactions mainly arise through Vander Waals and electrostatic forces.

During the inhalation process of pharmaceutical products, a specific device is

positioned in the mouth of the patients and the drug particles are aerosolized. These

particles travel throughout the airways with a number of factors determining its



deposition in the respiratory tract, which can be anywhere from the oral cavity to the

alveoli. Filtering of large particles (daer5>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 et al., 1986). Drug delivery

inhalers include nebulizers, metered-dose inhalers and dry powder inhalers. These

inhalers generate particles with a daer in the micron-size range for deposition in the

tracheo-bronchial tree (3–10 μm) in order to treat the airways (β2 mimetics) or in the

alveolar region (1–3 μm) for systemic drug absorption (insulin). Recent development

has also been done on the preparation of dry powder microparticles as nanoparticles

carriers for pulmonary drug delivery (Tsapis et al., 2002). Regional lung deposition of

micronized aerosols is more frequently reported, the investigation of nanometer

aerosols deposition in specific regions of the lungs is not available in the literature.

Considering the late advances in nanotechnology, the regional dosimetry of

nanoaerosols has been identified as a specific gap by the National Institute for

Occupational Safety and Health (NIOSH, 2005). The most common one found in

literatures related to development of formulations for pulmonary delivery is that

particles in the range of 1–5 μm are deposited in the deep lungs; while those larger

than 10 µm are generally deposited in the oropharyngeal region and the particles

smaller than 0.5 μm are exhaled (Haughney et al., 2010; Usmani et al., 2009).

Comparing the whole-lung deposition as a function of fine particle fraction, they found

that the scattered data straddled the line of identity when particles were smaller than 3

µm. Results based on impaction studies indicate that particles smaller than 3 µm are

more likely to deposit in the deep lungs.

1.3.4. Mucociliary clearance of particles

Various elimination pathways for SMs exist in the lungs, including coughing,

dissolution, mucociliary escalator, translocation from the airways to other sites,

phagocytosis by macrophages and neuronal uptake (Fig. 1.10). Mucociliary clearance



effectively eliminates submicrotherapeutics that are unable to penetrate the mucus, that

bind to the mucin, or that freely diffuse through the mucus but are unable to cross the

airway epithelium effectively. The mucin fibers form a network with water filled pores

of diameters ranging from 20 nm to 800 nm (Olmsted et al., 2001; Yudin et al.,

1989). Particles larger than the mucus mesh pore size or particles adhering to mucin

will be cleared with the mucus gel. Particles can adhere to the mucin through various

adhesive forces, including electrostatic interactions with the carboxyl or sulfate groups

on the mucin, hydrophobic forces, polymer chain interpenetration and hydrogen

bonding. Mucoadhesion can increase the residence time of particles delivered to the

gastro-intestinal tract where mucus renewal is slower than the transit time of the

luminal content (Varum et al., 2008). Yet, in respiratory airways, if one would like to

prolong the residence time of particles, one needs to design the particles so that they

freely diffuse in the upper gel phase of mucus and reach the lower watery and slowly

cleared periciliary liquid (Lai et al., 2008).

Fig. 1.10. Potential pathways determining the fate of inhaled nanoparticles.

Polymeric nanoparticles as large as 500 nm in diameter can rapidly diffuse through

mucus as far as they are densely coated with low molecular weight poly-ethylene

glycol (Lai et al., 2007). In contrast, the transport of uncoated particles through



mucus was largely hindered with diffusion coefficients 4500–(500 nm) and 2400-fold

(200 nm) lower than that in water. Polystyrene nanoparticles are trapped in mucus

because hydrophobic polystyrene beads form polyvalent bonds with hydrophobic

domains in mucin fibers. Coating particles with PEG is hypothesized to create a

hydrophilic and neutral shell that minimizes hydrophobic adhesive interactions with

mucus. Insoluble and non-biodegradable nanoparticles have been shown to remain in

the lung for several weeks and to clear via the mucociliary escalator into the gastro-

intestinal tract (Moller et al., 2008). Nanoparticles were cleared from the lungs via the

airways into the gastro-intestinal tract and were found in feces.

1.3.5. Macrophages uptake

Alveolar macrophages are responsible for clearance of nanoparticles deposited in the

alveolar region, in which mucociliary clearance is absent. In response to the deposited

nanoparticles, alveolar macrophages will migrate to the particles and phagocytise them

via chemotaxis involving opsonisation (Fig. 1.11). Macrophage uptake is believed to

complete in 6–12 h after deposition of the particles in the alveoli (Oberdörster et al.,

2007).

Fig. 1.11. Pulmonary fate of submicron particles (mucociliary clearance; lung-surface macrophages take up a limited amount of nanoparticles and

translocation of nanoparticles across the alveolar epithelium).



Once 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 et al., 2010; Videira et al., 2002; Yang et al., 2008). A minor

fraction of the particle-carrying macrophages will migrate to the ciliated airways

where they are removed by MCC (Oberdörster et al., 2007). Phagocytosis of

particles below 100 nm is not effective (Oberdörster et al., 2007), which may be due

to less effective recognition (~20%) of nanoparticles by the macrophages (Borm et al.,

2006). The reduced recognition is 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, because nanoparticles are more readily taken up by epithelial

cells, they become lessavailable to be phagocytized by macrophages (Madl et al.,

2009). Macrophages are also present in the ciliated airway (McWilliam et al., 2000)

but their role in nanoparticle clearance is probably less important compared with

MCC. Free nanoparticles were found in broncho-alveolar lavage immediately

following inhalation, but later they were associated with alveolar macrophages.

Instead, the particles were located in large vesicles and the vesicles contained other

material like surfactant, suggesting nanoparticles uptake during phagocytosis of other

material (Geiser et al., 2008).

1.3.6. Extrapulmonary translocation

Translocation of SM’s from the lungs into the blood and into extrapulmonary organs

represents in general a minor pathway in the fate of nanomedicines. So many times,

TEM micrographs showed the presence of nanoparticles in endocytic vesicles in

alveolar epithelial cells and in phagocytotic vacuoles in alveolar macrophages,

suggesting transcytosis across the alveolar epithelium as well as migration of particle-

laden alveolar macrophages via the blood circulation to extrapulmonary organs. The

chemical composition, shape, size and surface charge of nanoparticles and also the

physicochemical properties influence the rapid nanoparticles translocation from the

alveolar spaces towards the systemic circulation (Choi et al., 2010). NP’s with

hydrodynamic diameter <34 nm and a noncationic surface charge translocate across



the alveolar epithelium into the septal interstitium, followed by translocation to the

regional draining lymph nodes where further translocation into the bloodstream could

occur. NP’s with hydrodynamic diameter <6 nm can traffic rapidly from the lungs to

lymph nodes and the bloodstream, and then be subsequently cleared by the kidneys.

Six nm is the macromolecules upper size that allows paracellular diffusion across the

alveolar epithelium in vitro (Matsukawa et al., 1997). Paracellular diffusion also

explain the enhanced and rapid translocation of NP’s <6 nm from the lungs into the

blood. NP’s (≥20 nm) have been shown to translocate across alveolar epithelial cells

through transcellular pathways (Takenaka et al., 2006; Furuyama et al., 2009). The

physicochemical properties of the NP’s largely affected transport rates, with

positively-charged particles being transported 20 to 40 times faster than negatively

charged particles and smaller particles (20 nm) being transported 3 times faster than

larger particles (120 nm) with similar net surface charge density (Yacobi et al., 2008).

1.4. ANALYTICAL METHOD FOR NANOLEVEL QUANTIFICATION

The ultra high performance liquid chromatography�electrospray ionization-tandem

mass spectrometry (UHPLC/ESI-Q-TOF-MS) is a novel chromatographic technique

utilizing high linear velocities, which is based on concept using columns with smaller

packing (1.7-1.8 µm porous particles) and operated under high pressure (up to 15,000

psi) (Ultra Performance LCTM by design, 2004). This is an extremely powerful

approach which dramatically improves peak resolution, sensitivity and speed of

analysis (Luo et al., 2010). In addition, time-of-flight mass spectrometry (Q-TOF-MS)

allows the generation of mass information with higher accuracy and precision.

UHPLC is specially designed to resist higher back-pressures, with the advantages of

fast injection cycles, low injection volumes, negligible carryover and temperature

control (4–40 ºC), which collectively contributes to speedy and sensitive analysis (Fig

1.12) (Novákov et al., 2006). Furthermore, acquity UHPLC columns contain hybrid

X-Terra sorbent, which utilizes bridged ethylsiloxane/ silica hybrid (BEH) structure,

ensures the column stability under the high pressure and wide pH range (1–12)

(Novákov et al., 2006). In addition to UHPLC, the use of orthogonal quadrupole

time-of-flight mass spectrometry (Q-TOF-MS), with low and high collision-energy



full scans acquisition simultaneously performed, offers more possibilities in screening

and identification, resulting in valuable fragmentation information (Novákov et al.,

2006; Plumb et al., 2004).

Fig. 1.12. Elements of an UHPLC–MS system: (a) Autosampler (loads the samples

onto the UHPLC); (b) HPLC; (c) ionization source (interface for LC to MS); (d) Mass spectrometer.

Consequently UHPLC/ESI-Q-TOF-MS has been emerged as a powerful hyphenated

technique for bioanalytical investigation (USFDA, 2005). However, for bulk drug

characterization HPTLC method has also been considered as a tool having economical

and proficient importance (Ahmad et al., 2009). HPTLC has advantages over other

methods because of rapidity, selectivity, economy and overall versatility in QC aspects

of drugs.

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