Chapter 1

Pulmonary drug delivery

Pulmonary drug delivery
1.1 Introduction
The respiratory tract is an attractive route for the administration of therapeutics and genomics.
The past decade has been marked by intensive research efforts on pulmonary drug delivery
(PDD) for local and systemic drug and gene delivery including diagnostic agents because it has
several advantages over other routes of administration (1, 2). Various pharmaceuticals,
biopharmaceuticals, anesthetics, smoke or steam have been successfully inhaled for medical
purposes for centuries (3). Over the years inhalation therapy has established itself as a valuable
tool in the local therapy of pulmonary diseases such as asthma, chronic obstructive pulmonary
disease (COPD) (3), cystic fibrosis (CF), and pulmonary hypertension (4). The local application
of therapeutic agents to the respiratory system has several advantages over other routes of
administration, i.e., a large surface area available for absorption, high to blood flow, highly
vascularized tissue, rapid absorption, avoidance of first pass effect, avoidance of the effects of
gastric stasis and pH, smaller doses required than by the oral route to achieve equivalent
therapeutic effects useful for local treatment and systemic distribution (5, 6). Site-specific
delivery facilitates a reduction of the therapeutic dose to be administered, and thus, decreases the
associated adverse effects (6). In addition, inhalation represents a non-invasive alternative for
systemic delivery of biopharmaceuticals, which are labile to gastric acid, and thus, improves
patient compliance (7). The efficiency of inhalation therapy depends on the delivery system, the
devices used and the fate of the delivered medication in the respiratory tract. Once the
therapeutic agent has been deposited in the lung, elimination is instantly initiated, decreasing the
initial high local concentrations of the therapeutic agent in lung tissue (7, 8). Because the
concentration of the drug can decrease quickly, it often requires multiple daily inhalations which
can cause difficulty for the patient’s compliance (9).
Inhalation therapy involves the deposition of the drug in the lungs via aerosol generating devices
such as pressurized metered-dose inhalers (pMDI), nebulizers, and dry powder inhalers (DPI)
(7). The ‘conventional’ inhalation therapy does not allow targeted delivery to specific lung cells
and the deposition of the therapeutic agents in different lung areas is often poorly controlled (8).
To overcome these shortcomings, more sophisticated pulmonary delivery systems are desirable
which include potential carrier systems, and which use micro-sized and nano-sized vehicles.
These delivery systems have attracted considerable attention because of their controlled release

1
and targeting properties (9-15). These systems are mainly classified into immediate release (e.g.,
lactose-drug mixtures for DPI application) and controlled release systems (liposomes, micelles,
nanoparticles and microparticles). The modulation of the various physicochemical properties of
these systems has been explored to overcome the clearance mechanisms of the lungs and provide
prolonged residence times of the therapeutic agent within the respiratory tract (8, 10, 12) .
1.2 Anatomy and Physiology of Human Respiratory Tract
For the development of new PDD systems, one should have detailed knowledge of lung anatomy
and physiology. The respiratory tract is mainly divided into two regions i.e., the upper airway
and the lower airway with the line of division being the junction of the larynx and trachea (16).
The upper airway acts as an air transport system and consists of the nose, mouth, pharynx and
larynx. The lower respiratory tract consists of the tracheobronchial, gas-conducting airways and
the gas exchanging acini. The lower airway is divided into three zones: conducting, transitional,
and respiratory zones. The conducting zone is responsible for the bulk movement of air and
blood. In the central airways, air flow is rapid and turbulent and no gas exchange occurs. The
transitional zone has a limited role in gas exchange (16, 17) . The respiratory zone mainly
comprises of respiratory bronchioles and alveoli, where the actual gas exchange takes place (17).
Figure 1.1 shows a schematic representation of the lung. The bronchial tree trunk begins with the
trachea, which bifurcates to form the main bronchi: the left and right primary bronchi. Each
primary bronchus divides into still smaller secondary bronchi. The secondary bronchi branch
into many tertiary bronchi that further branch several times, ultimately giving rise to tiny
bronchioles that sub-divide many times, finally forming terminal bronchioles and respiratory
bronchioles. Each respiratory bronchiole sub-divides into several alveolar ducts that end in
clusters of small thin-walled air sacs called alveoli, which open into a chamber called the
alveolar sac (18) .
Two physical changes occur while moving from the trachea to the alveolar sacs in the airways
that are important in influencing airway function. Firstly, the airway diameter decreases with the
respiratory branches, e.g., a tracheal diameter (d) is 1.8 cm compared to an alveolar diameter of
0.04 cm. This permits adequate penetration of air to the lower airways for a given expansion of
the lungs. Secondly, the surface area of the airways increases with each generation, to the extent
that the total lung area at the level of the human alveolus is in the order of 140 m² (18). Alveoli
are the terminal air spaces of the respiratory system and are the actual site of gas exchange

2
between the air and the blood. About 100 million alveoli are found in each lung (19). Each
alveolus is a thin-walled polyhedral chamber of approximately 0.2 mm in diameter. Gases taken
up by inhalation need to cross alveolar epithelium, the capillary endothelium and their basement
membranes to reach blood; the distance makes up around 500 nm length (18). So, the alveolus,
providing increased surface area, becomes principal site of gas exchange in the airways (20). The
human lung consists of five lobules and ten broncho-pulmonary segments. The luminal surface
of the airways is lined by ciliated cells, which are the most common and numerous cell types.

Figure 1.1 Internal structure and organization of lungs (21).

Mucus cells are intermingled among the ciliated cells. The walls of the conducting airways are
coated by an adhesive, viscoelastic mucus layer (thickness: 5–55 μm) which is secreted by the
mucus cells. The major components of mucus are glycoproteins and water (22). This mucus
fulfils important functions, i.e., the protection of the respiratory epithelium from dehydration, the
water in the mucus promotes saturation of inhaled air, mucus contains antibacterial proteins and

3
peptides, such as defensins and lysozyme that inhibit microbial colonization of the airways, and
mucus is also involved in airway protection from inhaled xenobiotics or chemicals. Clearance of
mucus from the lung is driven by the motion of the ciliated cells ‘mucociliary escalator’, which
generates a mucus flow rate of ~5 mm/min. Thus, the mucus blanket is replaced every 20
minutes in healthy subjects (22). The mucociliary escalator serves as an important protective
mechanism for removing small inhaled particles from the lungs. The composition, thickness,
viscosity and clearance of the mucus is often altered in patients suffering from airway diseases
such as asthma, COPD and CF (7) .
The alveolar epithelium is composed of Type I and Type II alveolar cells and occasional brush
cells. The Type I pneumocytes are thin cells, cover most of the surface of the alveoli (95% of the
surface area) and the Type II pneumocytes are cuboidal secretory cells are interspersed among
the Type I cells (23) . The alveolar space is coated by a complex surfactant lining that reduces
surface tension to minimize the work of breathing and prevents collapse of the alveoli during
expiration (21). The majority of insoluble particles deposited in the upper airways are eliminated
by mucociliary clearance (24) . The most prominent defense mechanism of the respiratory region
is macrophage clearance. The particles deposited in the deeper lung will be taken up by alveolar
macrophages, which slowly migrate out of the lung, either following the broncho-tracheal
escalator or the lymphatic system (8). The blood supply to the lung is provided by a pulmonary
circulation and a systemic circulation. A drug delivered to the lower airways can enter the
systemic circulation by absorption into the alveolar capillaries of the pulmonary vascular bed
(21, 23).
1.3 Deposition mechanisms of inhaled particles
There are five mechanisms by which particles deposit in the respiratory tract namely, impaction,
sedimentation, brownion motion, interception and electrostatic precipitation. Impaction is the
inertial deposition of a particle onto an airway surface and occurs at bifurcation of airway where
flow velocities are at higher and sudden change in direction of flow take place, generating
considerable inertial forces. Higher velocity, high rate of breathing, and particles > 5 μ size and
high density increases impaction based deposition (25). Gravitational sedimentation is an
important mechanism for deposition of particles over 0.5 µm and below 5 µm in size in the small
conducting airways where the air velocity is low. Deposition due to gravity is increased by large
particle size and by longer residence times, and decreases with increasing breathing rate (25).

4
Impact of surrounding air on particles < 0.5 µm size cause a random motion in particles. Such
Brownian motion can cause particle deposition by diffusion in small airways and alveoli where
air flow is low in contrast to upper respiratory airways. Interception is usually significant only
for fibers and aggregates. Even though, center of mass of such particles remain to be in flow
stream, contact of such particles with airway wall cause deposition of such particles (25). Some
freshly generated particles can be electrically charged during the mechanical generation of
aerosols and may exhibit enhanced deposition due to charge induced deposition, though at a low
contribution to other mechanisms (26). In fact, the deposition patterns of inhaled particles may
be expressed as functions of three classes of variables: ventilatory parameters, respiratory tract
morphologies and aerosol characteristics (e.g., particle size, shape, and density). The efficiencies
of the different deposition mechanisms of inertial impaction, sedimentation and diffusion can, in
turn, be formulated in terms of these variables.
1.4 Barriers in Pulmonary Delivery
The past 30 years have been marked by intensive research efforts on PDD for local and systemic
therapy including diagnostic agents (5). The success of a PDD using aerosolized medications
depends on its ability to deliver sufficient concentration of drug to the appropriate site of action
in the lungs while exerting minimal side effects (27). Drugs for lung diseases and drugs that
undergo extensive first-pass metabolism or gastrointestinal degradation are ideal candidates for
pulmonary delivery. The lower incidence of side effects is often observed due to localized drug
deposition and reduced systemic and generalized exposure to other tissues. Despite such great
advantages of PDD, delivering therapeutic agents to lungs is a challenging task for the
formulation scientist because of the barriers of the pulmonary region and finding a suitable
delivery device.
The lungs are in direct contact with the atmosphere and thus, different lines of defense systems
exist to protect the deep parts of the lungs from exposure to particles present in the inhaled air (7)
(24). Several mechanisms are involved in the removal of particles from the upper respiratory
tract and thus, they reduce further deposition in the lower airways. The deposition of aerosol
particles in the lungs involves three mechanisms: impaction, sedimentation and diffusion (5).
Deposition in the respiratory tract is affected by the particle size, the patient’s inhalation
parameters e.g. flow rate, ventilation volume, end-inspiratory breath holding, and the aerosol
delivery system. The most crucial formulation variable for PDD is the mass median aerodynamic

5
 diameter (MMAD) of the particles. Figure 1.2 shows the effects of particle size on the deposition
efficiency of particles in respiratory system. Large particles with a MMAD of more than 5 μm
experience impaction in the oropharynx and upper conducting airways because of their high
momentum, while particles with an MMAD between 1 and 5 μm sediment in the deeper airways
and bronchioles. Small particles with an MMAD below 0.5 μm obey the principle of Brownian
diffusion, remain suspended in air and are exhaled during normal breathing (Table 1.1) (27).
Apart from the size, the deposition of aerosol particles also depends on density, hygroscopicity,
and the shape of the aerosolized particles. The anatomy of the airways and the breathing pattern
also determines impaction, sedimentation and diffusion of particles in the airflow. It is
commonly accepted that the optimal particle size (1-5 μm) is essential for the effective delivery
of particles to lungs, as particles smaller than 1 μm can possibly be exhaled without being
deposited (28). However, some recent investigations showed that nanoscale particles are also
effectively deposited in the alveolar region because of increased diffusional mobility, especially
in individuals suffering from asthma and increasingly during physical exercise (29).
Table 1.1: Deposition fate of inhaled particles in lungs

Sr. Aerodynamic Fate of inhaled particle in lungs Phagocytosis

No. diameter (µm)
1 <1 µm Particle remains suspended in Less liable to phagocytosis
lungs
2 1-5 µm Deposition of particle in the deep Highly liable to
lungs phagocytosis
3 >5 µm Particles deposited in upper Less liable to phagocytosis
respiratory tract

6
Figure 1.2 Deposition efficiency of particle in the respiratory system as function of the particle
size (28) .

The respiratory mucus covers the conducting airways and captures foreign matter inhaled with
each breath. The non-absorptive process involves transport of particles to the ciliated region
following clearance by the mucociliary escalator. In normal airways, the respiratory cilia
transport mucus at a rate of 2.5-5 mm/min towards the oropharynx where it is either swallowed
or expectorated (30). The mucus acts as a physical barrier, as increased viscosity of mucus
reduces drug penetration and its diffusion. Upon deposition in the lung, the particles are wetted
by mucus and subsequently transported toward the oesophagus by ciliated cells. The mucociliary
clearance of mucus-trapped foreign substances is an important pulmonary defense mechanism
against inhaled pathogens and particles and as well it acts as a barrier to gene transfer vectors
(31). The alveolar epithelium is not covered by mucus but a thin layer of alveolar fluid is
secreted on the surface of the alveoli epithelium. Alveolar fluid is composed of phospholipids
and lung surfactant excreted from Type II pneumocytes. The surface activity of the surfactant is
mainly provided by the phospholipids, surfactant proteins B and C, which also lower the surface
tension, whereas surfactant proteins A and D can opsonize foreign matter in the lungs (32). The
alveolar macrophages located in the alveoli can rapidly engulf the foreign particles by
phagocytosis as a defense mechanism. Every single alveolus is covered by 12-14 macrophages,
which gives approximately 19,000 alveolar macrophages per microlitre of bronchoalveolar
7
lavage fluid (BALF) (33). Crystalline powders of pharmaceutical application which exhibit
specific gravity approaching 1 and which are in the range of respirable aerodynamic diameter
show rapid macrophage uptake and clearance. Such uptake has been shown to be dependent on
the geometric diameter of inhaled particles, while lung deposition depends the aerodynamic
diameter of particles (34).
These anatomical and physiological barriers of the lungs play an important role in pulmonary
delivery of therapeutics and various approaches have been utilized to overcome these limitations.
Particulate nanocarriers can be used to improve the therapeutic index, reduce metabolism,
prolong half-life or reduce toxicity, increase bioavailability and site specific targeting resulting in
a reduction in the biological dosage frequency and improved patient compliance.
1.5 Delivery devices
Delivery device for pulmonary delivery should produce aerosolized particles ideally in the range
of 0.5-5 μ size with reproducible dosing and should maintain physical and chemical stability of
drug formulation. Additionally, the system should be simple, easy to use, cheap and portable
(35). Inhaled drug delivery devices can be divided into three principal categories: nebulizers,
pressurized metered-dose inhalers (pMDIs) and dry powder inhalers (DPIs), each class with its
unique strengths and weaknesses.

1.5.1 Nebulizers
Nebulizers have been used in inhalation therapy since the early 19th century. Marketed
respiratory solutions are generally composed of drug dissolved in aqueous, isotonic solvent
systems that may contain preservatives to reduce microbial growth. There are two traditional
devices: air-jet and ultrasonic nebulizers. For a typical jet nebulizer (Figure 1.3), compressed air
passes through a narrow hole and entrains the drug solution from one or more capillaries mainly
by momentum transfer. Large droplets impact on baffles and gets refined to the size required,
while droplets with smaller size run in a streamline flow of air bypassing the impact on baffles
(36). Approximately 50-60% of the particles produced are in the respirable range.

8
Figure 1.3: Schematic presentation of a jet nebulizer (36)

Alternatively, ultrasonic nebulizers use a high frequency vibrating plate to provide the energy
required to aerosolize the liquid. The frequency of the vibrating piezoelectric crystal determines
the droplet size for a given solution. Approximately 70% of the particles produced present sizes
of between 1 and 5 µm. However, heat generated from frictional forces induced by movement of
the transducing crystal may be harmful to thermolabile formulations. Some of the most
commonly available nebulizers on the market are: Ventolin® (Salbutamol, ß2-mimetic
bronchodilator), Bricanyl® (Terbutaline, ß2-mimetic bronchodilator), Atrovent® (Ipratropium,
anticholinergic bronchodilator), Pulmozyme® (Dornase alpha, mucolytic) and Tobi®
(Tobramycin, antibiotic).

But nebulization has many well-documented disadvantages, including extended administration

time, high cost, low efficiency, poor reproducibility and great variability, risk of bacterial
contamination and constant cleaning requirements, and sometimes the need for bulky
compressors or gas cylinders (37). Moreover, in some cases, the presence of preservatives such
as sodium metabisulfite, benzalkonium chloride and ethylene diamine tetraacetic acid (EDTA)
has caused coughing and bronchoconstriction (38). One of the principal problems of nebulizers is
that the device includes a large “dead volume” of solution. A large fraction of the amount (up to
50%) can thus remain trapped in the apparatus. Moreover, the aerosolized drug is generated
continuously, leading to drug waste (38). With a continuously working compressor (continuous
droplet generation), part of the aerosol cloud may be wasted into the environment through the
vent when the patient stops or interrupts inhalation or does not inhale fast enough. The amount

9
inspired is equivalent, more or less, to half of the delivered amount. Of this inhaled amount, it is
still necessary to remove a fraction of particles that are not in the “respirable range”. In
conclusion, the pulmonary fractions obtained using a nebulizer may vary from 2-10% of the
nominal dose. For example, 2.5 ml of Pulmozyme at 1 mg/ml is delivered by a jet nebulizer with
a delivery efficiency of 10% (39).

1.5.2 Pressurized Metered-Dose Inhalers

Traditional asthma therapy has primarily used the pMDI. Since the 1950s, they have been the
backbone of inhalation therapy (40). In a pMDI, a propellant pressurized to a liquid state is
holding the drug either in a suspended or dissolved form. Metered release of the fluid through a
valve causes expansion and evaporation of propellant leaving the drug in the form of a high
velocity aerosol. Rapid release of the propellant into the valve stem along with the actuator
seating forms an expansion chamber where propellant starts to boil (Figure 1.4). The liquefied
propellant serves both as a source of energy for expelling the formulation from the valve in the
form of rapidly evaporating droplets and as a dispersion medium for the drug and other
excipients.

Figure 1.4: Schematic representation of a typical pressurized metered-dose inhaler (41)

Physicochemical properties of the formulation determine the particle size and size distribution of
an MDI aerosol. For example, aerosol size for suspension formulations may be reduced if the
formulation has a high vapour pressure, small particle size and low drug concentration. For
instance, studies have shown that the aerosol size for suspension formulations may be reduced if
the formulation has a high vapor pressure, a small drug particle size, or a low drug concentration
(42). A surfactant such as sorbitan trioleate, oleic acid, and lecithins, at levels between 0.1% and

10
2.0% w/w, are typically added to aid the suspension or solubility of drug and to lubricate the
metering mechanism (41). Flavors and suspended sweeteners may be present to combat the
unpleasant taste. To enhance chemical stability, antioxidants (ascorbic acid) or chelating agents
(EDTA) may be added to formulations (38) .

The essential components of an MDI are the container, the metering valve, and the actuator.
Usual valve volumes range from 25-100 µl, which deliver a drug dose of about 50 µg to 5 mg.
The most commonly available MDIs on the market are: Flixotide® (Fluticasone, corticosteroid),
Atrovent® (Ipratropium bromide, anticholinergic bronchodilator), Ventolin® (Salbutamol, ß2-
mimetic bronchodilator) and Combivent® (Ipratropium bromide + Salbutamol, anticholinergic +
ß2-mimetic bronchodilator). Chlorofluorocarbon (CFC)-based MDIs usually contain a mix of
liquefied low boiling-poing propellant (CFC 12- dichlorodifluoromethane) and a high boiling-
point propellant (CFC 11- trichlorofluoromethane or CFC 114 – dichlorotetrafluoromethane).
However, the success of CFC propellant-driven MDIs has been overshadowed by their
contribution to ozone depletion in the upper atmosphere and the concomitant health effects (43).
In fact, the international community agreed to phase out CFC propellants in 2000. So nowadays,
the main emphasis in MDI developments is on the introduction of non-CFC propellants. But the
non-CFC propellant hydrofluoroalkanes (HFA 134a - 1, 1, 1, 2-tetrafluoroethane and HFA 227 -
heptafluoropropane) exhibit very different physicochemical properties and extremely low solvent
properties. This however is advantageous in preventing the dissolution of small drug particles but
may also be disadvantageous This feature is beneficial in preventing the dissolution of small
drug particles, but it is also disadvantageous in that the commonly used surface-active agents are
almost totally insoluble and not able to provide any physical stabilization of drug particles in
suspension. A number of approaches have been undertaken to overcome the problems of drug
particles instability in HFAs, addition of cosolvents, requirement to develop new specific surface
active agents, modification of surface properties of particles to reduce interfacial tension, and
production of particles compatible to HFA (44). However, MDIs have several other
disadvantages. There may also be chances of “cold-Freon effect” which may cause inhalation
problems to patients due to cold propellant spray on the back of the throat (45). Moreover,
because an MDI is pressurized, it emits the dose at high velocity, which makes premature
deposition in the oropharynx more likely (46). Thus MDIs require careful coordination of
actuation and inhalation. Only a small fraction of the drug (10-20%) escaping the inhaler

11
penetrates the patient’s lungs due to a combination of high particle exit velocity and poor
coordination between actuation and inhalation. The deposition of aerosolized drugs in the mouth
and the oropharyngeal regions varies considerably according to the application technique, but
losses using the pressurized devices are routinely greater than 70% and can exceed 90%. Particle
losses that occur proximally to the lung are a long-documented problem that continues to
compromise the effectiveness of current aerosol therapy protocols.

Spacer devices and reservoirs were developed to allow the deceleration of the aerosol cloud
before reaching the throat and to make perfect coordination between actuation and inhalation
slightly less important (Figure 1.5). Nevertheless, adding a chamber to an MDI makes it less
portable, and many chambers can develop static electrical charges on the inner walls and thereby
reduce the lung delivery (47). Despite these enhancements, incorrect use of MDIs is still a
prevalent problem (48). About 75% of patients made at least one error when using an MDI (49).

Figure 1.5: Representation of the use of a spacer device with an MDI (50)

The introduction of “breath-actuated” MDI devices has provided a convenient and portable
means to achieve coordination between actuation and inhalation. Breath-actuated inhalers have
been developed that sense patient’s inhalation and automatically fire the inhaler. Examples are
the Autohaler® (3M Pharmaceuticals, U.S.A) or Easybreathe® (Norton Healthcare, U.K.) (51).

1.5.3 Dry Powder Inhalers

As a result of the problems encountered with MDIs, the design and use of alternative inhalers
that do not use propellants were developed. These devices combine powder technology with

12
device design in order to disperse dry particles as an aerosol in the patient’s inspiratory airflow
(52).

All DPIs have four basic features: a dose-metering mechanism, an aerosolization mechanism, a
deaggregation mechanism, and an adaptor to direct the aerosol into the mouth. DPIs are subject
to strict pharmaceutical and manufacturing standards by regulatory bodies, the most challenging
of which is the demonstration of device reliability in terms of delivered dose uniformity and
delivered dose deposition (53). Indeed, comparative in-vitro data for a generic product versus the
innovator product must be provided on the complete individual stage particle size distribution
profile using a multistage impactor/impinger with various impaction stages, such as the NGI
(53).

For DPIs, the dose received by the patient is dependent on four interrelated factors (54)

1. The properties of the drug formulation, particularly powder flow, particle size and drug carrier
interaction

2. The performance of the inhaler device, including aerosol generation and delivery

3. Correct inhalation technique for deposition in the lungs

4. The inspiratory flow rate

Therefore, a balance between the design of an inhaler device, drug formulation, and the
inspiratory flow rate of patient is required (55). DPIs are a widely-accepted inhaled delivery
dosage form, particularly in Europe where they are currently used by an estimated 40% of
patients to treat asthma and chronic obstructive pulmonary disease. Their use will continue to
grow (54). Presently, over 20 DPI devices are available on the market (Table 1.2) and more than
25 are in development (Table 1.3). Today there are essentially two types of DPIs: those in which
the drug is packaged in individual doses (capsules) (Single dose or Unit-dose devices) and those
that contain multiple doses in a foil-foil blister or a reservoir of drug from which the doses are
metered out (Multi-unit and Multi-dose devices).

13
Table 1.2: DPI devices currently available on the market (56)

Device DPI type Company Delivery Drug(s)

method
Breath-actuated single unit dose
Spinhaler Single Aventis Capsule Sodium cromoglycate
dose
Rotahaler Single GlaxoSmithKline Capsule Salbutamol sulphate
dose Beclomethasone dipropionate
Inhalator Single Boeringher-Ingelheim Capsule Fenoterol
dose
Handihale Single Boeringher-Ingelheim Capsule Tiotropium
r dose
Aerolizer Single Novartis Capsule Formoterol
dose
FlowCaps Single Hovione Capsule Salbutamol Sulphate
dose
TwinCaps Single Hovione Capsule Zanamivir
dose
Breath-actuated multiple dose
Turbuhale Multi- Astra Zeneca Reservoir Salbutamol sulphate
r dose Terbutaline sulphate
Budesonide
Diskhaler Multi- GlaxoSmithKline Blister Salmeterol xinafoate
dose Beclomethasone dipropionate
Fluticasone propionate
Zanamivir
Diskus Multi- GlaxoSmithKline Strip pack Salbutamol sulphate
dose Salmeterol xinafoate
Fluticasone propionate
Aerohaler Multi- Boeringher-Ingelheim Reservoir Ipratopium bromide
dose

14
Easyhaler Multi- Orion Pharma Reservoir Salbutamol sulphate
dose Beclomethasone dipropionate
Ultrahaler Multi- Aventis Reservoir Ciclesonide
dose
Pulvinal Multi- Chiesi Reservoir Salbutamol sulphate
dose Beclomethasone dipropionate
Novolizer Multi- ASTA Reservoir Budesonide
dose
MAGhaler Multi- Boeringher-Ingelheim Reservoir Salbutamol sulphate
dose
Taifun Multi- LAB Pharma Reservoir Salbutamol sulphate
dose
Eclipse Multi- Aventis Capsule Sodium cromoglycate
dose
Clickhaler Multi- Innoveta Biomed Reservoir Salbutamol sulphate
dose Beclomethasone dipropionate
Twisthaler Multi- Schering-Plough Reservoir Mometasone furoate
dose
Active device
Airmax Multi- Norton Healthcare Reservoir Formoterol
dose Budesonide
Inhance Single Pfizer Blister Insulin
dose

Table 1.3: DPIs approved or in development stage (56)

Device DPI type Company Delivery method Drug(s)

Aspirair Multi-dose Vectura Powder/Active Apomorphine
hydrochloride
Actispire Single dose Britania Powder/Active Pumactant
JAGO Multi-unit SkyPharma Reservoir Salbutamol sulphate

15
Airmax Multi-unit Norton Healthcare Reservoir Formoterol,
Budesonide
AIR Single dose Alkermes Capsule Placebo powders
MicroDose Multi-unit MicroDose/ 3M Powder/Electronic Insulin
Cyclovent Multi-unit Pharmachemie Reservoir Morphine
Conix One Single dose Cambridge Consultant Foil seal Vaccines
Microhaler Single dose Harris Pharmaceutical Capsule Sodium cromoglycate
Spiros Multi-unit Dura Blister/Active Albuterol sulphate
Miat-Haler Multi-unit MiatSpA Reservoir Formoterol,
Fluticasone
propionate,
Budesonid
Acu-Breath Multi-unit Respirics Powder Fluticasone
propionate
Swinhaler Multi-unit Otsuka Powder Budesonide
Pharmaceutical
Certihaler Multi-unit Novartis Powder Formoterol
1.5.4 Unit-dose devices
The Spinhaler® (Aventis) was the first dry powder device, described in 1971 (57) which works
on the mechanism to pierce the capsule. The caps fits into an impeller. The impeller rotates as
the patient breaths through the device (Figure 1.6) during which powder deaggregates and
relative motion imparted due to impeller rotation. This low-resistance device has presented low
in vitro fine particle fractions (FPF) (% < 5 µm) of 4-12% (58).

A similar DPI, the Rotahaler® (GlaxoSmithKline) breaks the capsules into two, the body part
falling inside the device and the cap being retained in the entry port. As the patient inhales, the
portion of the capsule containing the dug experiences erratic motion in the airstream, causing
dislodged particles to be entrained and subsequently inhaled. Turbulence due to grid upstream of
the mouthpiece deaggregates particles. A FPF of 26% has been reported for this low resistance
device (58).

16
Figure 1.6: Schematic presentation of the Spinhaler (59)

The Handihaler® (Boehringer Ingelheim) delivers drug contained in a capsule through rumbling
motion after piercing pins opens the capsule. The particles are dispersed through turbulence due
to plastic grid during inhalation. This device is more complicated as it requires at least 7 distinct
steps to deliver the dose. For some patients, two inhalations are required to completely empty the
capsule and achieve the therapeutic dose (54)

Unit-dose devices are considered as not patient-friendly and not easy to use because there are
several manoeuvres to accomplish before inhalation, such as taking the capsule from the
package, loading it and piercing it within the device. Furthermore, there have been recent reports
of patients ingesting the capsule instead of placing it in the device and inhaling the contents (60).
However some studies show that adherence to correct inhaler use depends on the importance
given to appropriate training prior to product use and device education by health-care providers
(61).

1.5.5 Multi-dose devices

Multi-dose DPIs have been developed, either as multi-unit dose or as multi-dose reservoir
devices. Inhalator M® (Boehringer Ingelheim) has a rotating drum magazine for the storage of
six capsules. Capsule pierced at both ends stays stagnant while the high pressure drop across the
capsule causes the emptying of the of the contents and shear stress and collision occurring during
the process causes deagrregation (58).

The Diskhaler® (GlaxoSmithKline) makes use of individual doses packed on disc type blister
packs (Figure 1.7). Piercing and subsequent flow of air through packaging depression containing
the dose leads to dispersion of powder. The aerosol stream is mixed with a bypass flow entering

17
through two holes in the mouthpiece that, together with a grid, gives rise to turbulence that
promotes deagglomeration.

Figure 1.7: Schematic presentation of the Diskhaler (62)

The Diskus® (GlaxoSmithKline) is quite similar except that it contains a foil strip with 60 single
dose blisters (Figure 1.8). FPF have been reported to be approximately 23-30% for these two low
resistance devices (58).

Figure 1.8: Schematic presentation of the Diskus (62)

Turbuhaler® (AstraZeneca) is another sophisticated multi-dose reservoir system that contains

200 doses of small pellets of micronized drug. The drug deagrregation takes place during
metering and inhalation. One dose can be dispensed into the dosing chamber by a simple back-
and-forth twisting action on the base of the reservoir (Figure 1.9).

18
Figure 1.9: Schematic presentation of Turbuhaler (62)

The advantages of the reservoir systems are their relative ease and low cost of manufacture and
the ease of including a large number of doses within the device (63). Nevertheless, reproducible
dose metering remains the most difficult challenge in device design. Indeed, the variability of
dose emissions from DPIs, and in particular from the reservoir system, at the recommended flow
rate has been found to be relatively high, with a total relative standard deviation of more than
15% about the average emitted dose for the Pulmicort Turbuhaler (64). Furthermore, powders
contained in reservoirs may be more susceptible to deterioration through entrance of moisture,
and the use of a desiccant is recommended. For these reasons, pre-metered doses from multi-unit
dose systems are more consistent than doses delivered from the reservoir devices, as they are
individually sealed and protected from the environment until the point of use by the patient. It is
also vital to ensure that no accidental or additional dose is inhaled. This has led to the
incorporation of dose counters on new reservoir devices.

1.6 Importance of the inspiratory airflow

DPIs require little or no coordination between the actuation and inhalation due to their activation
through inhalation by patient, they often result in better lung delivery than that achieved with
comparable MDIs. Since DPIs are typically formulated as one-phase, solid-particle blends, they
are also preferred from the standpoints of stability and processing (65). Moreover, DPI are a
propellant-free and thus environmentally friendly. The effectiveness of DPIs is depend on the
age, gender, disease, and the breathing cycle of the device user. One of the most important
disadvantages of DPIs includes requirement of moderate inspiratory effort to draw the
formulation from the device, and some patients are not capable of such effort. Low-resistance
passive DPIs are less dependent on flow rate than high-resistance devices. Devices with higher
19
resistance need a higher inspiratory force from patients to achieve the desired air flow. This
could be difficult for patients with severe asthma and for children and infants. Thus, a balance
between resistance and turbulence is essential to achieve the desired therapeutic effect from DPI
formulations. Additionally, it has been demonstrated that drug deposition deep in the lung from
DPI formulations is determined not only by the peak flow rate but also by the flow increase rate.
It was reported that a high peak flow rate did not necessarily guarantee a high aerosol deposition
if the initial flow increase rate was insufficiently high (66). Most of DPIs are breath-activated,
depend on inhalation for aerosol generation, several power-assisted devices i.e. pneumatic,
impact force, and vibratory (67) have been developed or are currently under development. These
“active” inhalers are not subject to the same limitations as passive inhalers and have a different
advantage/disadvantage profile. It has been advised 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.

1.7 Future research and new developments

Since the manufacture of the first DPI Spinhaler®, device technology has continued to grow and
a lot of devices are now currently available on the market. However, no devices have shown
notable efficiency in delivering drugs from the formulation. Additionally, the concept of powder
interaction with the device on powder dispersion has generally been poorly understood.
Recently, computational fluid dynamics has enhanced understanding of the impact of inhaler
design on powder dispersion and deposition, and has demonstrated that small variations in device
design can produce significant variations in performance (68). Nowadays, ways to improve the
efficiency of drug delivery from DPIs are developed by changing formulation technology, drug
and carrier particle engineering and designing new devices. Indeed, the design of a device needs
to be coordinated with drug formulations (i.e., powder in capsules, disks, bulk powders or
agglomerates), so that the drugs are aerosolized during inhalation and deliver a dose to the lungs
that achieves maximum therapeutic benefits. It is often the case that the drug formulation and
inhaler device need to be optimized together to ensure reliable and effective drug delivery.
Therefore, the inhaler-drug combination is generally considered as a single medication whose in
vitro performance and in vivo efficacy must be demonstrated. So, in the design of a new DPI,
consideration must be given to optimizing the formulation of the powder containing the drug
substance to ensure a chemically stable and consistent dose, and to optimizing the design of the

20
metering system within the inhaler itself to produce a convenient device that is comfortable and
easy to use for the patient.

1.7.1 Formulation of dry powder inhalers

In order to obtain an effective delivery of drugs to the lungs, various ways of formulations were
developed. Often, the drug is mixed with carrier particles or encapsulated in liposomes,
nanoparticles and microparticles. Large porous particles were also created for DPI
administration. The particle size distribution affects the deposition of drug in the respiratory
tract. However, the smaller the particles, the stronger the cohesive forces, resulting in increased
adhesion between the particles and subsequently poor flow properties (69). So, one way to
improve the flow properties of a drug is through the addition of excipients. Excipients are used to
enhance the physical or chemical stability of the active pharmaceutical ingredient, its mechanical
properties, and/or its pharmaceutical properties, such as dissolution and permeation. In DPI
formulations, excipients function first and foremost as carrier particles. Usually, no more than a
few milligrams of drug need to be delivered (e.g. corticosteroids for asthma therapy), and
excipients provide bulk, which improves handling, dispensing, and metering of the drug.

1.7.1.1 Nature of excipients

The lungs have a large surface area and thin membranes. Unlike the gastrointestinal tract, the
lungs have limited buffering capacity. Many compounds that could enhance drug delivery
outcomes also have the potential to irritate or injure the lungs (70). Thus, the choice of potential
excipients is limited to compounds that are endogenous to the lung and can easily be metabolized
or cleared. Currently, lactose is the most commonly used excipient in marketed DPIs. The
reasons for this are as much historical as they are physicochemical/pharmaceutical in nature:
lactose has long been used as an excipient in oral dosage forms before being deployed in DPIs. It
has an established safety and stability profile, different manufacturing processes with tight
controls over purity and physical properties, and is easily available at different grades and is
inexpensive. Moreover, lactose is highly crystalline, less hygroscopic than other sugars and has
the smooth surfaces and satisfactory flow properties desirable for a DPI carrier particle (71).

Other sugars, such as glucose, mannitol and trehalose (55, 71-73) have been shown to be
feasible alternatives to lactose, and it is expected that these sugars will eventually find their way
into approved products. An additional benefit that may be gained from the use of a sugar carrier

21
is the taste/sensation on inhaling, which can assure the patient that a dose has been taken (63).
Phospholipids and cholesterol have also been used in experimental liposomal formulations (74)
or as solid lipidic carriers or fillers (75). The availability of the active drug depends afterwards
on the redispersion of the particles in the inspired air, as a function of the cohesive forces
between drug particles and the adhesive forces between drug and carrier particles. The larger
carrier particles deposit on the oropharynx, while the fine drug particles partly reach the deep
lung. These adhesive forces must be carefully considered, as inadequate separation of drug and
carrier is the main reason for deposition problems. So, as excipients can make up over 99% of
the product by weight, their choice is a crucial determinant of overall DPI performance. Despite
the apparent lack of choices, the excipient must be carefully selected, as its physicochemical
properties, such as size and morphology, profoundly affect the performance of the formulation
(76). In general, the morphology and roughness of carrier particles are not uniform, containing
regions that exhibit different roughness parameters. Clearly, these variations in physicochemical
properties in the surface of a carrier material may lead to differences in apparent adhesion
properties of drug particles. Furthermore, during the dynamic process of mixing, the adherence
of drug particles to the more adhesive areas of the carrier surface is likely to occur (77). Indeed,
it has been proposed that the surfaces of larger particles consisted of distinct regions containing
so-called “active sites”. It was further suggested that when the amount of fine carrier particles in
the mixture is below the saturation limit of the large carrier particles’ adhesive potential, the fine
particles will preferentially bind to these active sites. When these active sites have been
completely occupied by fine particles, a binary carrier system will then exist, i.e., carrier with
strongly bound fine particles, and carrier with weakly bound fine particles or free fine particles
(78).

This presence of active sites has obvious implications for DPI drug delivery since retention of
drug particles on these relatively high-energy sites during processing and aerosolization would
result in a decrease in apparent respirable drug fraction, as suggested by Staniforth (78).
Furthermore, it is suggested that the active sites present on the surface of the carrier will have a
specific energy distribution with a critical, average adhesion point below which particles - drug
or lactose could be removed. Current methods for overcoming such issues include ”filling” the
potential active sites by increasing the fine particle content present on the carrier surface or
pacifying the effects of active sites by the addition of so-called “force control agents” such as

22
magnesium stearate (79, 80). This additional or ternary component can be added to occupy and
presaturate higher-energy binding sites on the carrier before the drug is added, consequently
increasing the release and the respirable fraction of the active drug.

1.7.1.2 Blending

After drug and excipient(s) have individually been brought to their desired forms, they are
combined in the blending process. The flow properties of the components of the powder blend
will play an important role in the efficiency of blending and, ultimately, in aerosol dispersion.
Blended formulations consist of small drug particles that are mixed with large excipient carrier
particles (50-200 µm). It is a critical step in the manufacture of a DPI product and is in fact
subject to substantial optimization work during development. When mixing powders with
different properties, particle sizes, and ratios, as is the case with DPI formulations, inadequate
mixing can cause poor dose uniformity. In many cases, inadequate mixing cannot be overcome
simply by increasing the mixing time. Mixer selection, rotation speed, capacity, and fill level are
all subject to optimization, as they can all affect the blend homogeneity (81). Different powders
may have different mixing requirements, depending on the interaction forces present between the
various particles (78). For low concentration (drug-carrier ratio) blends, geometric dilutions are
necessary, using multiple pre-blending steps. Various blending options are available: low-energy
tumbling blending, tumbling blending with sieving to break up agglomerates of micronized drug
and aid distribution with the powder mass, and high energy blending, with paddles, impeller
blades and redistributing powder within the blending vessel (82). An interactive mixture of the
two components is prepared by blending until the mixture can remain intact during the filling
process (to produce an accurate metered dose) and then freely separate into its primary
components during inhalation. 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. Inadequate drug/carrier separation is one of the main explanations for the low deposition
efficiency encountered with DPIs (83).

Though there are tremendous advantages of the pulmonary drug delivery, pulmonary
administration of these therapeutics is limited by the defense mechanisms of the lung. The
mucociliary escalator, coughing, and alveolar clearance are the 3 major physical ways of

23
removing deposited particles. In the conducting airways deposited particles are rapidly cleared
by the mucociliary clearance into the pharynx. In the terminal airways (alveoli), absorptive or
non-absorptive processes remove deposited particles. The absorptive process may involve either
direct penetration into the epithelial cells or uptake and clearance by the alveolar macrophages.
The non-absorptive process involves transport of particles to the ciliated region (conducting
airways) followed by clearance by the mucociliary escalator (84). To overcome these challenges
encountered during pulmonary delivery, particulate drug delivery system is another option for
pulmonary administration.

1.8 Nanocarriers for pulmonary delivery

These systems can be broadly classified into immediate release [e. g. lactose-drug mixtures for
dry powder inhaler (DPI) application] and controlled release systems (such as liposomes,
micelles, nano- and microparticles based on polymers). Particulate nanocarriers such as
liposomes, polymerosomes, micelles, microparticles and nanoparticles can be used to improve
the therapeutic index of new or established drugs by modifying drug absorption, reducing
metabolism, prolonging biological half-life or reducing toxicity, increase bioavailability, better
drug targeting and delivery. Drug distribution is then controlled primarily by properties of the
carrier and no longer by physicochemical characteristics of the drug substance only.
Nanocarriers could provide the advantage of sustained release in the lung tissue and thus the
systemic circulation, resulting in a reduction in dosage frequency and improved patient
compliance (85). These nanocarriers are removed due to the binding of plasma complement,
immunoglobulins, and other proteins (opsonization), leading to uptake by macrophages
(absorptive process of pulmonary clearance). This clearance can be markedly delayed by the
grafting of synthetic hydrophilic polymers such as poly (ethylene glycol) (PEG) onto the surface
of the vehicles. PEG forms a hydrated shell hindering protein interaction with drug vehicles or
drugs themselves (e.g., PEG-coated insulin), thereby greatly reducing opsonization and uptake
by macrophages. Such “stealth” DDS have prolonged pharmacokinetics and lesser side effects of
activation of host defense (immune response, cytokine release, complement activation) (86).

1.8.1 Nanoparticle for pulmonary drug formulations

Drug nanoparticle formulations are usually created in one of two ways. Particles may be
precipitated out of solution (bottom-up), or they are milled from larger particles (top-down) (18).

24
In both mechanisms, the total surface area increases, which increases the free energy of the
particles. The system compensates for this increase in free energy by dissolving crystalline nuclei
and precipitating onto other particles in a process known as Ostwald Ripening (19), or by
agglomerating smaller particles. Generating stable nanoparticle colloids typically necessitates the
use of surfactants, which decrease the surface tension at the particle surface and thereby help to
reduce the increase in free energy (18). This decreases the degree of Ostwald Ripening and
agglomeration within the system. Many chemical processing technologies have been used to
produce nanostructured materials suitable for pulmonary delivery. Some processes that are
currently under investigation involve wet milling (18), supercritical fluid extraction (21), spray
drying (22), electrospray (86), high-pressure homogenization and recrystallization via solvent
displacement (87). Wet milling is a process that utilizes either ceramic or metallic milling media
to grind a suspension of insoluble drug and surfactant. Wet milling has also been used to
formulate budesonide for nebulized delivery to the lungs (18). Spray drying is a process that
forces fluid through a nozzle, producing a mist that is dried to produce a fine powder. The
technique employs a variety of different types of nozzles, some of which use ultrasound or air-jet
shear to nebulize drug suspensions. Supercritical fluid extraction is a technique that is currently
being developed for use in nanoparticle drug formulations. It uses supercritical fluid to extract a
solvent from a drug emulsion or solution, leaving behind a suspension of drug particles (21).
These processes are advantageous because they generally offer better scalability, and are
therefore industrially relevant. Unfortunately, some processes (such as spray drying) often utilize
co-solvents to improve drying and/or large amounts of excipient to stabilize the drug and to
maintain powder properties. In addition to chemical processing technologies, multiple recent
studies have examined different polymeric nanoparticle fabrication methods as applied to
pulmonary drug formulations (88, 89). These techniques generally involve polyelectrolyte
complex formation, double emulsion/solvent evaporation techniques, or emulsion polymerization
techniques. Polyelectrolyte complexes use oppositely charged polymers to entrap drugs into a
polymeric matrix nanoparticle, which then releases the drug either through polymer degradation
or drug diffusion. Double emulsion/solvent evaporation techniques involve dissolving the drug
and polymer in an organic solvent, which is then emulsified in an aqueous solution. The organic
solvent diffuses out of the polymer phase and into the aqueous phase, and is then evaporated,
leaving behind drug-loaded polymeric nanoparticles. Emulsion polymerization is similar to

25
emulsion/solvent evaporation except that monomer is emulsified into droplets and then
polymerization is initiated. Liposomal formulations are typically produced by extruding or
homogenizing a suspension of dissolved, hydrated lipids. (90). This suspension can then be
delivered via nebulization, freeze-dried, or incorporated into larger particles.

1.8.2 Liposomal Nanocarrier Formulations

Liposomal nanoparticles have been investigated as potential drug carriers to the lung. Lot of
lipids used for liposome preparation are already present in lungs making liposomal carriers better
tolerated systems (90). Moreover, diverse groups of amphiphilic molecules available for
constructing liposomes provides required variability in physichochemical parameters like
particles size etc. and this in turn makes them more attractive carriers for drug delivery (91). A
possible concern is that lipids themselves modify the surface tension of the lungs and have
themselves been used as therapeutics (34). In general, liposomes are interesting potential drug
delivery vehicles because of their stability in suspension. For instance, upon dilution or changes
in ionic strength that may be encountered during administration, liposomes will theoretically
remain intact. This is because liposomal formulations are not thermodynamically equilibrated
systems, but are kinetically trapped systems that are unable to respond to thermodynamic
perturbations (91). This property might make them suitable for formulations that require long-
term storage as liquid suspensions, such as those used in nebulization. Liposomes have also been
investigated as potential targeting agents for alveolar macro-phages, partially because
macrophages are associated with lung surfactant metabolism (92, 93). Studies have also shown
that mannosylation of liposomes and particle size influence the uptake of liposomes by
macrophages (92, 93). The studies demonstrate that liposomal uptake increases as particle size
increases over a range of 100 - 2,000 nm and as the degree of mannosylation increases on the
surface of the particles (92, 93). This is likely due to the fact that mannose receptors are
exclusively expressed on the surface of alveolar macrophages (93).

Bhavane et al. developed a novel type of drug delivery vehicle composed of liposomal
nanocarriers covalently linked by enzymatically labile spacers (90). The liposomes were between
80 and 195 nm in size and loaded with ciprofloxacin with 90% efficiency. Agglomeration was
induced using dimethyl 3,3-dithiobispropionimidate 2HCl (DTBP), which is a homobifunctional
imidoester capable of reacting with primary amines and also contains thiol-cleavable disulfide

26
bonds, making it enzymatically labile (90). Agglomerated liposomes differ from
unagglomerated liposomes in their release characteristics, the former being more slow releasing
than latter, as well as agglomerated particles may give burst release. Upon nebulization, the
agglomerated particles were reported to retain the encapsulated drug and nebulized droplets had
aerodynamic diameters between 1 and 5 µm, putting them within the respirable range (90).
Liposomal formulations have also been investigated in potential dry powder formulations.
Chougule et al. loaded nano-sized liposomes with tacrolimus, an immunosuppressant often used
to prevent rejection of allotropic lung transplants. The liposomal suspension with lactose,
sucrose, or trehalose and L-leucine was spray dried to produce particles with an aerodynamic
diameter of approximately 2.2 µm that demonstrated good aerosolization properties when
administered from a DPI (94)

1.8.3 Biodegradable microspheres

Biodegradable microspheres produced from natural and synthetic polymers (e.g. poly (lactic-co-
glycolic) acid polymer (PLGA), Poly Lactic acid (PLA)) have been extensively investigated as
drug carriers for administration via a number of different routes in order to ensure targeting and
sustained drug release. PLGA microspheres have been used for controlled release of a wide
range of drugs including peptides and proteins, and their potential use in pulmonary delivery has
been explored, principally for anti-asthmatic drugs (95). A number of microspheres have proved
to be non-toxic, biodegradable and non-immunogenic following systemic injection. Duddu et al
produced micron-size hollow-porous microspheres (PulmoSpheres®) using a two-step process
(96). First, a fluorocarbon-in-water emulsion is prepared by high pressure homogenization, using
a saturated phosphatidylcholine as the surfactant. The emulsion is then combined with a second
aqueous solution containing the active agent and other wall-forming material (e.g., co-
surfactants, sugars and salts). The second step involves spray drying the aqueous dispersion.
Mean geometric and aerodynamic diameters of spray dried particles werre about 5 and 7 µm,
respectively, and the bulk density is about 0.4 g/cm³. Morphologically, the particle walls have a
sponge-like appearance with pores (96). Particle size, morphology and density can be controlled
through the selection of the blowing agent type and its concentration. High respirable fractions
are obtained in vitro as a result of a significant decrease in interparticle attractive forces and
improved aerodynamic properties due to the hollow porous design. One clinical evaluation on

27
healthy volunteers of dry powder tobramycin, using lipid-based Pulmosphere technology, has
already been made, giving a mean whole lung deposition of 34 ± 6 % (37, 96) .

1.8.4 Large porous particles

In general, therapeutic dry powder aerosols are made with drug particle mass densities of
approximately 1 ± 0.5 g/cm³ and mean geometric diameters of less than 5 µm to avoid deposition
in the oropharyngeal cavity and in the DPI devices. Now, a new type of inhalation aerosol
characterized by particles of mass density significantly lower than 1 (0.1- 0.5 g/cm³) has been
recently proposed, so that an aerodynamic diameter in the respirable size range can be achieved
with a geometric particle size greater than 10 µm (97). These large porous particles represent an
important step in pulmonary delivery for future applications (34). The increased aerosolization
efficiency lowers the probability of deposition losses before particle entry in the intrapulmonary
airways, thereby increasing the systemic bioavailability of an inhaled drug. This is due to a lower
tendency for powder aggregation as a result of a reduced fractional surface area of particle-
particle contact in the dry powder. Moreover, these large porous particles can escape the natural
clearance mechanisms of the lungs, such as the mucociliary escalator and phagocytosis by
alveolar macrophages, since very large particles may escape that kind of clearance, giving
enough time for the drug to be effectively delivered (98). Thin-walled large porous particles have
also been prepared by macroscale aggregation of nanoparticles via a spray-drying process with
an additional control over physical characteristics by adding other components to the spray-dried
solutions such as sugars, lipids, polymers and proteins. Nanoparticulate drug delivery systems
are converted to large porous microsystems to combine the release and delivery potential of
former with flow, processing and aerosolization potentials of latter (99).

1.9 Toxicity considerations of nanocarrier formulations

A major concern with nanoparticle therapeutics is the unforeseen negative health impact that
nanoparticles may have. Studies in rodents have shown that intratracheally administered carbon
nanoparticles accelerate vascular thrombosis (100). Additionally, it has also been demonstrated
that inhaled iridium particles may migrate from the lung to the systemic circulation, which may
have detrimental vascular effects (101). Inhaled carbon nanoparticles show accumulation in
brain, however their toxicity needs to be determined (102). Conversely, a recent study has shown
that metallic nanoparticles are no less cytotoxic than metallic microparticles, suggesting that the

28
small size of nanoparticles may not be the most important factor affecting their toxicity (103).
This notwithstanding, the toxicity of nanoparticles is a legitimate concern and should be
thoroughly investigated. In addition to the possible inherent toxic effects of nanoparticles, some
materials used to formulate nanoparticles may have toxic effects and therefore may not be viable
for developing therapeutic products. For example, the toxicity of polycyanoacrylates has been
demonstrated by Brzoska et al (104). They prepared poly (butyl) cyanoacrylate and poly
(hexyl)cyanoacrylate nanoparticles. They determined that both types of nanoparticles caused an
increase in lactate dehydrogenase (LDH) activity in human pulmonary epithelial cells. The
degree of toxicity was greater for the poly (butyl) cyanoacrylate and independent of the stabilizer
used, which stands to reason because shorter chain polycyanoacrylates have been associated with
higher cytotoxicity. The degree of toxicity also increased with increasing nanoparticle
concentration, most likely due to the subsequent increase in polycyanoacrylate concentration.
Polyethyleneimine (PEI) has also demonstrated cytotoxicity in lung cells (105). Bivas-Benita et
al. also reported increasing cytotoxicity of PEI-DNA complexes with increasing ratio of PEI to
DNA (106). Despite this, Dailey et al. have shown that PLGA nanoparticles induce less
inflammation than polystyrene particles of similar size when delivered to the lungs (88). Based
on this observation, nanoparticle toxicity in the lungs may be more dependent on material choice
than particle size. Therefore, it is essential to investigate the toxicity of these particles and
alternatives or methods can be investigated to mitigate pulmonary toxicity of these system.

Nanocarrier drug formulations offer many advantages over traditional aerosol powders and
liquid pulmonary dose formulations. The bioavailability of poorly water-soluble drugs can be
greatly enhanced by the large surface area of drug nanocarrier formulations. Additionally,
nanoparticles can be formulated in such a way to offer enhanced control over the morphology of
dry powder drug formulations and the ability to produce structures with both a low-density
microstructure for delivery to the deep lung and nanostructure for enhanced dissolution and
bioavailability. The literature suggests many different formulation approaches for drugs that use
a variety of excipients to fabricate nanocarrier or nanocarrier complexes suitable for pulmonary
delivery. Many chemical processing techniques such as lyophilization, supercritical fluid
extraction and spray drying have been successfully used for therapeutic nanoparticle processing.
Additionally, a range of formulation techniques are available, for example, emulsion
polymerization, double emulsion/solvent evaporation, polyelectrolyte complexation,

29
nanoprecipitation and liposomal loading that suits different requirements. These techniques offer
the ability to produce nanocarriers with a high degree of control over particle size, however,
residual solvents, presence of cytotoxic components, drug loading problems as well as scale up
issues need to be addressed to have commercial applicability of such formulations. With the
perfection of pulmonary nanocarrier drug formulations, the lungs may become a preferred route
of drug delivery for many more local and systemic therapeutic interventions.

1.10 References
1. Groneberg DA, Eynott PR, Doring F, Dinh QT, Oates T, Barnes PJ, et al. Distribution and
function of the peptide transporter PEPT2 in normal and cystic fibrosis human lung.
Thorax. 2002;57(1):55-60.
2. Groneberg DA, Nickolaus M, Springer J, Doring F, Daniel H, Fischer A. Localization of
the peptide transporter PEPT2 in the lung: implications for pulmonary oligopeptide uptake.
The American journal of pathology. 2001;158(2):707-14.
3. Hiller FC. In: Hickey AJ, editor. Pharmaceutical Inhalation Aerosol Technology. New
York: Marcel Dekker Inc.; 1992. p. 385.
4. Olschewski H, Simonneau G, Galie N, Higenbottam T, Naeije R, Rubin LJ, et al. Inhaled
iloprost for severe pulmonary hypertension. The New England journal of medicine.
2002;347(5):322-9.
5. Courrier HM, Butz N, Vandamme TF. Pulmonary drug delivery systems: recent
developments and prospects. Critical reviews in therapeutic drug carrier systems.
2002;19(4-5):425-98.
6. Groneberg DA, Witt C, Wagner U, Chung KF, Fischer A. Fundamentals of pulmonary
drug delivery. Respiratory medicine. 2003;97(4):382-7.
7. Patton JS, Byron PR. Inhaling medicines: delivering drugs to the body through the lungs.
Nature reviews Drug discovery. 2007;6(1):67-74.
8. Geiser M, Kreyling WG. Deposition and biokinetics of inhaled nanoparticles. Particle and
fibre toxicology. 2010;7:2.
9. Gessler T, Seeger W, Schmehl T. Inhaled prostanoids in the therapy of pulmonary
hypertension. Journal of aerosol medicine and pulmonary drug delivery. 2008;21(1):1-12.
10. Kurmi BD, Kayat J, Gajbhiye V, Tekade RK, Jain NK. Micro- and nanocarrier-mediated
lung targeting. Expert opinion on drug delivery. 2010;7(7):781-94.

30
11. Langer R, Vacanti JP. Tissue engineering. Science (New York, NY). 1993;260(5110):920-
6.
12. Chow AH, Tong HH, Chattopadhyay P, Shekunov BY. Particle engineering for pulmonary
drug delivery. Pharmaceutical research. 2007;24(3):411-37.
13. Sung JC, Pulliam BL, Edwards DA. Nanoparticles for drug delivery to the lungs. Trends in
biotechnology. 2007;25(12):563-70.
14. Azarmi S, Roa WH, Lobenberg R. Targeted delivery of nanoparticles for the treatment of
lung diseases. Advanced drug delivery reviews. 2008;60(8):863-75.
15. Rytting E, Nguyen J, Wang X, Kissel T. Biodegradable polymeric nanocarriers for
pulmonary drug delivery. Expert opinion on drug delivery. 2008;5(6):629-39.
16. JB. West PW. Ventilation-perfusion relationships.in The Lung: Scientific Foundations. In:
R.G. Crystal JBW, P.J. Barnes and E.R. Weibal, Raven Press, editor. 1991. p. 1289.
17. Yu J, Chien YW. Pulmonary drug delivery: physiologic and mechanistic aspects. Critical
reviews in therapeutic drug carrier systems. 1997;14(4):395-453.
18. Thompson AJHaDC. In: A.J. Hickey MD, editor. Pharmaceutical Inhalation Aerosol
Technology. 2nd Edition ed. New York, NY, USA2004. p. 1.
19. Stone KC, Mercer RR, Gehr P, Stockstill B, Crapo JD. Allometric relationships of cell
numbers and size in the mammalian lung. American journal of respiratory cell and
molecular biology. 1992;6(2):235-43.
20. Crapo JD, Barry BE, Gehr P, Bachofen M, Weibel ER. Cell number and cell characteristics
of the normal human lung. The American review of respiratory disease. 1982;126(2):332-7.
21. C.A. D’Angelis JJCaRMR. In: Zimmerman BPFaJJ, editor. Pediatric Critical Care. 4th
Edition ed. Philadelphia, PA, USA2011.
22. Lai SK, Wang YY, Wirtz D, Hanes J. Micro- and macrorheology of mucus. Advanced drug
delivery reviews. 2009;61(2):86-100.
23. Thompson RJAaDC. Inhalation Aerosols: Physical and Biological Basis for Therapy. New
York, NY, USA1996. p. 83.
24. van der Schans CP. Conventional chest physical therapy for obstructive lung disease.
Respiratory care. 2007;52(9):1198-206; discussion 206-9.

31
25. Y. MTBaY. Deposition mechanics of pharmaceutical particles in human airways:. In: AJ
H, editor. Inhalation aerosols, Physical and biological basis for therapy. 94. New york,
NY, pp 3-271996.
26. Lippmann M, Schlesinger RB. Interspecies comparisons of particle deposition and
mucociliary clearance in tracheobronchial airways. Journal of toxicology and
environmental health. 1984;13(2-3):441-69.
27. Martonen TB, Katz IM. Deposition patterns of aerosolized drugs within human lungs:
effects of ventilatory parameters. Pharmaceutical research. 1993;10(6):871-8.
28. Lam JK, Liang W, Chan HK. Pulmonary delivery of therapeutic siRNA. Advanced drug
delivery reviews. 2012;64(1):1-15.
29. Daigle CC, Chalupa DC, Gibb FR, Morrow PE, Oberdorster G, Utell MJ, et al. Ultrafine
particle deposition in humans during rest and exercise. Inhalation toxicology.
2003;15(6):539-52.
30. Yeates DB, Aspin N, Levison H, Jones MT, Bryan AC. Mucociliary tracheal transport rates
in man. Journal of applied physiology. 1975;39(3):487-95.
31. P.B. McCray J, G. Wang, B. Davidson, M. Bodner, S.M. Herrmann, D.J. Jolly, inventor;
The University of Iowa Research Foundation Chiron Corp, assignee. Methods and
compositions for increasing the infectivity of gene transfer vectors. U patent US 6855549.
2005.
32. Goerke J. Pulmonary surfactant: functions and molecular composition. Biochimica et
biophysica acta. 1998;1408(2-3):79-89.
33. Sutinen S, Riska H, Backman R, Sutinen SH, Froseth B. Alveolar lavage fluid (ALF) of
normal volunteer subjects: cytologic, immunocytochemical, and biochemical reference
values. Respiratory medicine. 1995;89(2):85-92.
34. Edwards DA, Hanes J, Caponetti G, Hrkach J, Ben-Jebria A, Eskew ML, et al. Large
porous particles for pulmonary drug delivery. Science (New York, NY).
1997;276(5320):1868-71.
35. Dolovich MB, Ahrens RC, Hess DR, Anderson P, Dhand R, Rau JL, et al. Device selection
and outcomes of aerosol therapy: Evidence-based guidelines: American College of Chest
Physicians/American College of Asthma, Allergy, and Immunology. Chest.
2005;127(1):335-71.

32
36. Le Brun PP, de Boer AH, Heijerman HG, Frijlink HW. A review of the technical aspects of
drug nebulization. Pharmacy world & science : PWS. 2000;22(3):75-81.
37. Newhouse MT, Nantel NP, Chambers CB, Pratt B, Parry-Billings M. Clickhaler (a novel
dry powder inhaler) provides similar bronchodilation to pressurized metered-dose inhaler,
even at low flow rates. Chest. 1999;115(4):952-6.
38. Dalby R.N. TSL, Hickey A.J. Medical devices for the delivery of therapeutic aerosols to
the lungs. In: AJ H, editor. Inhalation aerosols, Physical and biological basis for therapy.
vol. 94. New york, NY1996. p. 441-73.
39. Cipolla D, Gonda I, Shire SJ. Characterization of aerosols of human recombinant
deoxyribonuclease I (rhDNase) generated by jet nebulizers. Pharmaceutical research.
1994;11(4):491-8.
40. Crompton G. A brief history of inhaled asthma therapy over the last fifty years. Primary
care respiratory journal. 2006;15(6):326-31.
41. Newman SP. Principles of metered-dose inhaler design. Respiratory care. 2005;50(9):1177-
90.
42. Polli GP, Grim WM, Bacher FA, Yunker MH. Influence of formulation on aerosol particle
size. Journal of pharmaceutical sciences. 1969;58(4):484-6.
43. Molina MJ, Rowland FS. Stratospheric sink for chlorofluoromethanes: chlorine atom-
catalysed destruction of ozone. Nature. 1974;249(5460):810-12.
44. Pethick C, Smith H. Bose-Einstein condensation in dilute gases: Cambridge university
press; 2002.
45. Crompton GK. Problems patients have using pressurized aerosol inhalers. European journal
of respiratory diseases Supplement. 1982;119:101-4.
46. Ganderton D. General factors influencing drug delivery to the lung. Respiratory medicine.
1997;91 Suppl A:13-6.
47. Geller DE, Pitlick WH, Nardella PA, Tracewell WG, Ramsey BW. Pharmacokinetics and
bioavailability of aerosolized tobramycin in cystic fibrosis. Chest. 2002;122(1):219-26.
48. Giraud V, Roche N. Misuse of corticosteroid metered-dose inhaler is associated with
decreased asthma stability. The European respiratory journal. 2002;19(2):246-51.

33
49. Khassawneh BY, Al-Ali MK, Alzoubi KH, Batarseh MZ, Al-Safi SA, Sharara AM, et al.
Handling of inhaler devices in actual pulmonary practice: metered-dose inhaler versus dry
powder inhalers. Respiratory care. 2008;53(3):324-8.
50. Bateman ED, Hurd SS, Barnes PJ, Bousquet J, Drazen JM, FitzGerald M, et al. Global
strategy for asthma management and prevention: GINA executive summary. The European
respiratory journal. 2008;31(1):143-78.
51. Rubin BK, Fink JB. Optimizing aerosol delivery by pressurized metered-dose inhalers.
Respiratory care. 2005;50(9):1191-200.
52. Delaby I, Ernst B, Froelich D, Muller R. Droplet deformation in immiscible polymer
blends during transient uniaxial elongational flow. Polymer Engineering & Science.
1996;36(12):1627-35.
53. Newman SP, Busse WW. Evolution of dry powder inhaler design, formulation, and
performance. Respiratory medicine. 2002;96(5):293-304.
54. Atkins PJ. Dry powder inhalers: an overview. Respiratory care. 2005;50(10):1304-12;
discussion 12.
55. Steckel H, Thies J, Müller BW. Micronizing of steroids for pulmonary delivery by
supercritical carbon dioxide. International journal of pharmaceutics. 1997;152(1):99-110.
56. Islam N, Gladki E. Dry powder inhalers (DPIs)--a review of device reliability and
innovation. International journal of pharmaceutics. 2008;360(1-2):1-11.
57. Bell JH, Hartley PS, Cox JS. Dry powder aerosols. I. A new powder inhalation device.
Journal of pharmaceutical sciences. 1971;60(10):1559-64.
58. Dunbar CA, Hickey AJ, Holzner P. Dispersion and Characterization of Pharmaceutical
DryPowder Aerosols. Kona Powder and Particle Journal. 1998;16:7-45.
59. Concessio NM, Hickey AJ. Descriptors of irregular particle morphology and powder
properties. Advanced drug delivery reviews. 1997;26(1):29-40.
60. Tezky T, Holquist C. Misadministration of capsules for inhalation. Drug Topics.
2005;4:48-9.
61. Melani AS, Zanchetta D, Barbato N, Sestini P, Cinti C, Canessa PA, et al. Inhalation
technique and variables associated with misuse of conventional metered-dose inhalers and
newer dry powder inhalers in experienced adults. Annals of allergy, asthma &
immunology. 2004;93(5):439-46.

34
62. How to Use Your Inhaler 2008. Available from:
http://www.asthma.ca/adults/treatment/howToUse.php.
63. Sumby B, Slater A, Atkins PJ, Prime D. Review of dry powder inhalers. Advanced drug
delivery reviews. 1997;26(1):51-8.
64. Hindle M, Byron PR. Dose emissions from marketed dry powder inhalers. International
journal of pharmaceutics. 1995;116(2):169-77.
65. Ashurst II, Malton A, Prime D, Sumby B. Latest advances in the development of dry
powder inhalers. Pharmaceutical science & technology today. 2000;3(7):246-56.
66. De Boer A.H. GD, Hagedoorn P. . Inhalation characteristics and their in vitro drug
delivery. Part 2: Effect of peak flow rate (PIFR) and inspiration time of the in vitro drug
release from three different types of commercial dry powder inhaler. Int J Pharm.
1996;138:45-56.
67. Tobyn M, Staniforth JN, Morton D, Harmer Q, Newton ME. Active and intelligent inhaler
device development. International journal of pharmaceutics. 2004;277(1-2):31-7.
68. Coates MS, Fletcher DF, Chan HK, Raper JA. Effect of design on the performance of a dry
powder inhaler using computational fluid dynamics. Part 1: Grid structure and mouthpiece
length. Journal of pharmaceutical sciences. 2004;93(11):2863-76.
69. Feeley JC, York P, Sumby BS, Dicks H. Determination of surface properties and flow
characteristics of salbutamol sulphate, before and after micronisation. International journal
of pharmaceutics. 1998;172(1–2):89-96.
70. Telko MJ, Hickey AJ. Dry powder inhaler formulation. Respiratory care. 2005;50(9):1209-
27.
71. Smyth HC, Hickey A. Carriers in drug powder delivery. Am J Drug Deliv. 2005;3(2):117-
32.
72. Tee SK, Marriott C, Zeng XM, Martin GP. The use of different sugars as fine and coarse
carriers for aerosolised salbutamol sulphate. International journal of pharmaceutics.
2000;208(1–2):111-23.
73. Steckel H, Bolzen N. Alternative sugars as potential carriers for dry powder inhalations.
International journal of pharmaceutics. 2004;270(1–2):297-306.
74. Shah SP, Misra A. Development of liposomal amphotericin B dry powder inhaler
formulation. Drug delivery. 2004;11(4):247-53.

35
75. Sebti T, Amighi K. Preparation and in vitro evaluation of lipidic carriers and fillers for
inhalation. European journal of pharmaceutics and biopharmaceutics. 2006;63(1):51-8.
76. Islam N, Stewart P, Larson I, Hartley P. Effect of carrier size on the dispersion of
salmeterol xinafoate from interactive mixtures. Journal of pharmaceutical sciences.
2004;93(4):1030-8.
77. Young PM, Edge S, Traini D, Jones MD, Price R, El-Sabawi D, et al. The influence of
dose on the performance of dry powder inhalation systems. International journal of
pharmaceutics. 2005;296(1-2):26-33.
78. Staniforth JN, Rees JE, Lai FK, Hersey JA. Interparticle forces in binary and ternary
ordered powder mixes. The Journal of pharmacy and pharmacology. 1982;34(3):141-5.
79. Zeng XM, Martin GP, Marriott C, Pritchard J. The influence of carrier morphology on drug
delivery by dry powder inhalers. International journal of pharmaceutics. 2000;200(1):93-
106.
80. Ferrari F, Cocconi D, Bettini R, Giordano F, Santi P, Tobyn M, et al. The surface
roughness of lactose particles can be modulated by wet-smoothing using a high-shear
mixer. AAPS PharmSciTech. 2004;5(4):e60.
81. Alexander A, Shinbrot T, Johnson B, Muzzio FJ. V-blender segregation patterns for free-
flowing materials: effects of blender capacity and fill level. International journal of
pharmaceutics. 2004;269(1):19-28.
82. Malcolmson RJ, Embleton JK. Dry powder formulations for pulmonary delivery.
Pharmaceutical science & technology today. 1998;1(9):394-8.
83. Zeng XM, Martin GP, Marriott C, Pritchard J. The influence of carrier morphology on drug
delivery by dry powder inhalers. International journal of pharmaceutics. 2000;200(1):93-
106.
84. Roy I, Vij N. Nanodelivery in airway diseases: Challenges and therapeutic applications.
Nanomedicine: Nanotechnology, Biology and Medicine. 2010;6(2):237-44.
85. Fernandes CA, Vanbever R. Preclinical models for pulmonary drug delivery. Expert
opinion on drug delivery. 2009;6(11):1231-45.
86. Agu RU, Ugwoke MI. In vitro and in vivo testing methods for respiratory drug delivery.
Expert opinion on drug delivery. 2011;8(1):57-69.

36
87. Jacobs C, Muller RH. Production and characterization of a budesonide nanosuspension for
pulmonary administration. Pharmaceutical research. 2002;19(2):189-94.
88. Dailey LA, Jekel N, Fink L, Gessler T, Schmehl T, Wittmar M, et al. Investigation of the
proinflammatory potential of biodegradable nanoparticle drug delivery systems in the lung.
Toxicology and applied pharmacology. 2006;215(1):100-8.
89. Dailey LA, Kleemann E, Wittmar M, Gessler T, Schmehl T, Roberts C, et al. Surfactant-
free, biodegradable nanoparticles for aerosol therapy based on the branched polyesters,
DEAPA-PVAL-g-PLGA. Pharmaceutical research. 2003;20(12):2011-20.
90. Vincent JH, Johnston AM, Jones AD, Bolton RE, Addison J. Kinetics of deposition and
clearance of inhaled mineral dusts during chronic exposure. British journal of industrial
medicine. 1985;42(10):707-15.
91. Chono S, Tanino T, Seki T, Morimoto K. Influence of particle size on drug delivery to rat
alveolar macrophages following pulmonary administration of ciprofloxacin incorporated
into liposomes. Journal of drug targeting. 2006;14(8):557-66.
92. Edwards DA, Dunbar C. Bioengineering of therapeutic aerosols. Annual review of
biomedical engineering. 2002;4:93-107.
93. A.V. Annapragada PSaFM. In: A.L. Adjel and P.K. Gupta MD, editor. Inhalation Delivery
of Therapeutic Peptides and Proteins. New York, NY, USA1997. p. 27.
94. Chougule M, Padhi B, Misra A. Nano-liposomal dry powder inhaler of tacrolimus:
preparation, characterization, and pulmonary pharmacokinetics. International journal of
nanomedicine. 2007;2(4):675-88.
95. Tee SK, Marriott C, Zeng XM, Martin GP. The use of different sugars as fine and coarse
carriers for aerosolised salbutamol sulphate. International journal of pharmaceutics.
2000;208(1-2):111-23.
96. Duddu SP, Sisk SA, Walter YH, Tarara TE, Trimble KR, Clark AR, et al. Improved lung
delivery from a passive dry powder inhaler using an Engineered PulmoSphere powder.
Pharmaceutical research. 2002;19(5):689-95.
97. Musante CJ, Schroeter JD, Rosati JA, Crowder TM, Hickey AJ, Martonen TB. Factors
affecting the deposition of inhaled porous drug particles. Journal of pharmaceutical
sciences. 2002;91(7):1590-600.

37
98. Edwards DA, Ben-Jebria A, Langer R. Recent advances in pulmonary drug delivery using
large, porous inhaled particles. Journal of applied physiology (Bethesda, Md : 1985).
1998;85(2):379-85.
99. Tsapis N, Bennett D, Jackson B, Weitz DA, Edwards DA. Trojan particles: large porous
carriers of nanoparticles for drug delivery. Proceedings of the National Academy of
Sciences of the United States of America. 2002;99(19):12001-5.
100. Tetley TD. Health effects of nanomaterials. Biochemical Society transactions. 2007;35(Pt
3):527-31.
101. Semmler M, Seitz J, Erbe F, Mayer P, Heyder J, Oberdorster G, et al. Long-term clearance
kinetics of inhaled ultrafine insoluble iridium particles from the rat lung, including transient
translocation into secondary organs. Inhalation toxicology. 2004;16(6-7):453-9.
102. Oberdorster G, Sharp Z, Atudorei V, Elder A, Gelein R, Lunts A, et al. Extrapulmonary
translocation of ultrafine carbon particles following whole-body inhalation exposure of
rats. Journal of toxicology and environmental health Part A. 2002;65(20):1531-43.
103. Cha KE, Myung H. Cytotoxic effects of nanoparticles assessed in vitro and in vivo. Journal
of microbiology and biotechnology. 2007;17(9):1573-8.
104. Brzoska M, Langer K, Coester C, Loitsch S, Wagner TO, Mallinckrodt C. Incorporation of
biodegradable nanoparticles into human airway epithelium cells-in vitro study of the
suitability as a vehicle for drug or gene delivery in pulmonary diseases. Biochemical and
biophysical research communications. 2004;318(2):562-70.
105. Kleemann E, Neu M, Jekel N, Fink L, Schmehl T, Gessler T, et al. Nano-carriers for DNA
delivery to the lung based upon a TAT-derived peptide covalently coupled to PEG-PEI.
Journal of controlled release. 2005;109(1-3):299-316.
106. Bivas-Benita M, Romeijn S, Junginger HE, Borchard G. PLGA-PEI nanoparticles for gene
delivery to pulmonary epithelium. European journal of pharmaceutics and
biopharmaceutics. 2004;58(1):1-6.

38