3.
by other routes.The use of drug delivery systems for the treatment of pulmonary diseases is
increasing because of its potential for localized topical therapy in the lungs.In addition,this
route makes it possible to deposit large concentrations at disease sites,to reduce the amount
of drugs administered to patients (20–10% of the amount administered by the oral route),
to increase the local activity of drugs released at such sites, and to avoid the metabolization
of drugs due to a hepatic first-pass effect.1
Recent medical advances have established that small-airway disease is a significant
component in obstructive airway disease.2 It has also been demonstrated3 that emphysema
classically involves the terminal bronchioles, but, increasingly, there is recognition that
asthma—and in particular chronic persistent asthma—also involves the small airways. For
these reasons and in order to improve the pulmonary targeting of a potentially useful therapy,
numerous scientific contributions have been focused on the construction of suitable dosage
forms to specifically target the small airways and to increase the local bioavailability of drugs
combined with carrier systems.
It was necessary to construct such carrier systems because of the limitations of chronic
oral administration with respect to systemic side effects, including hepatic dysfunction,
skeletal malformations, hyperlipidemia, and hypercalcemia.4 At present, the clinical results
obtained with particular carrier systems suggest that some of these may offer a practical al-
ternative to systemic oral administration for chemoprevention trials or the treatment of lung
diseases.This method may substantially increase the therapeutic index of targeted compounds
by reducing the systemic complications associated with long-term administration.
Although the lungs are rich in enzymes, they also contain several protease inhibitors.
Therefore,there is some evidence that exogenous proteins may be protected from proteolytic
degradation by these inhibitors.These characteristics also make the airways a useful route of
drug administration in the inhaled or aerosol form.The mechanisms of delivery to the lungs
are perhaps more complex than for other routes. The drug fraction that reaches the lungs
depends on numerous factors,such as the amount and rate of inhaled air,the respiratory pause,
and the particle size and characteristics (homogeneity, shape, electric charges, density, and
hydrophobicity).In spite of such complex mechanisms,pulmonary delivery of a variety of drugs
such as bronchodilators and steroids has enjoyed great success.Fortunately,the advantages of
this route have been recognized, and research in the field has progressed steadily.5
The pulmonary route was long used only to treat local diseases.Recently,the use of this
route to administer drugs systemically has been the subject of intensive research studies. At
the present time, the delivery of DNAse, proteins, and peptides such as insulin, calcitonin,
PCHS: phosphatidylcholine of hydrogenated soya
PCS: phosphatidylcholine of soya
PEI: polyethylenimine
PFC: perfluorocarbon
PGLA: poly(glycolic-co-lactic acid)
PLA: poly(lactic acid)
PLAL-lys: poly(acide lactique-co-lysine)
PLV: partial liquid ventilation
PMDI: pressurized metered-dose inhaler
PTH: paratyroid hormone
PTX: paclitaxel
RB: rhodamine B
RDS: respiratory distress syndrome
RF: respirable fraction
R-PGLA: rifampicin-PGLA microspheres
SC: salmon calcitonin
SLN: solid lipid nanoparticles
SUV: small unilamellar vesicles
T½: half time of elimination
TAP: triamcinolone acetonide phosphate
TCA: triamcinolone acetonide
TNF-α: tumor necrosis factor alpha
UL: unilamellar
VEE: Venezuelan equine encephalomyelitis
4.
. . .
α-interferon, and genetic material in general is of particular interest. In order to improve
bioavailability and to optimize the release of drugs targeted to specific sites into the lungs,
several strategies have been suggested. Among these are advances in the fields of aerosol
therapy,aerosol generators,and drug delivery systems.The latter systems include liposomes,
NanoCrystals technology, polymers, nano- and microparticles, dispersed systems, salt,
and precipitates.
In spite of the development of multidose inhalers containing dry powder and portable
spray dryers,the pressurized metered-dose inhaler (pMDI) remains by far the most popular
system for inhalation therapy.pMDIs have benefited from considerable technical advances,
following the recent progressive switch from chlorofluorocarbon (CFC) to hydrofluoroalkane
(HFA) propellants.The latter have all the qualities required for pharmaceutical use (chemi-
cally stable, no toxicological effects, etc.). (Incidentally, the FDA has recently published its
intention with regard to CFC phase-out in the Federal Register.) However, because CFCs
and HFAs do not have the same physicochemical characteristics (vapor pressures, densi-
ties, solubilities), the development of new pMDIs with HFAs as propellants can require
complex reformulation, the use of new packaging materials, and the introduction of new
production processes.
This article reviews these issues and the adapted dosage forms that have been tried in
order to assess the benefits of regional drug delivery and the ability to achieve this. In this
article, the term carrier must be understood as a solid, liquid, or gaseous excipient making
it possible to target a drug and, in some specific circumstances, to modulate the absorption
kinetics and pharmacokinetics of drugs.
II. DESIGN CONSIDERATIONS
II.A. Regional Histological Differences in Respiratory Tract
The human lung is an attractive route for systemic drug administration5 in view of its enor-
mous adsorptive surface area (140 m2) and thin (0.1–0.2µm) absorption mucosal membrane
in the distal lung.6 Approximately 90% of the absorptive area of the lung is attributed to
the alveolar epithelium, which primarily consists of type I pneumocytes. Because pulmo-
nary drug administration is directly related to respiratory structure and function and to the
administration routes of the drug formulation being introduced into the lung, a summary
of the basics of the lung and of drug entrance mechanisms follows.
1. The Respiratory System
In functional terms, the respiratory system consists of three major regions: the oropharynx,
the nasopharynx, and the tracheobronchial pulmonary region. The conducting airway is
composed of the nasal cavity and associated sinuses and the nasopharynx,oropharynx,larynx,
trachea,bronchi,and bronchioles,including the first 16 generations of the airways of Weibel’s
tracheobronchial tree.The conducting airway is responsible for the filtration,humidification
and warming of inspired air.The respiratory region is composed of bronchioles,alveolar ducts,
and alveolar sacs,including generation 17–23 of Weibel’s tracheobronchial tree (Fıg.1).The
5.
respiratory gases circulate from air to blood and vice versa through 140 m2 of internal surface
area of the tissue compartment.This gas-exchange tissue is called the pulmonary parenchyma.
It consists of 130,000 lobules, each with a diameter of about 3.5 mm and containing ap-
proximately 2200 alveoli.The terminal bronchioles branch into approximately 14 respiratory
bronchioles, each of which then branches into the alveolar ducts (Fıg. 2).The ducts carry 3
or 4 spherical atria that lead to the alveolar sacs supplying 15–20 alveoli. Additional alveoli
are located directly on the walls of the alveolar ducts and are responsible for approximately
35% of total gas exchange. It has been estimated that there are 300 million alveoli in an
adult human lung. The diameter of an alveolus ranges from 250 to 290 µm, its volume is
estimated to be 1.05 × 10-5 mL, and its air–tissue interface to be 27 × 10–4 cm2. For these
calculations,it is assumed that the lung has a total volume of 4.8 L and a respiratory volume
of 3.15 L and that the air–tissue alveolar interface is 81 m2.
FIGURE 1. Tree structure of the lung. (Reprinted from Washington N, Washington C, Wilson CG.
Pulmonary drug delivery. In: Physiological Pharmaceutics Barriers to Drug Absorption, 2000, p.224,
with kind permission of Taylor & Francis Book Ltd., London, UK.)
6.
. . .
2. Barriers
PulmonarySurfactant. The elastic fibers of the lung and the wall tension of the alveoli could
cause the lungs to collapse if this were not counterbalanced by the presence of the pulmonary
surfactant system.This covers the alveolar surface to a thickness of 10–20 nm and is constantly
renewed from below.The surfactant is composed of 90% in weight of phospholipids,including
40–80% in weight of dipalmitoyl phosphatidylcholine (DPPC).The other main ingredients
are phosphatidylcholines, phophatidylglycerols, other anionic lipids, and cholesterol.7 The
other fraction (10% in weight) is composed of 4 specific proteins—the hydrophiles SP-A
and SP-C and the hydrophobes SP-B and SP-D.8 Enzymes,lipids,or detergents can destroy
this surfactant. If the pulmonary surfactant is removed quickly by pulmonary irrigation, no
damage occurs because it is quickly replaced (half-life: ∼30 hours). The surfactant is only
produced at the time of birth,which is why premature babies suffer from respiratory distress
syndrome (RDS). In this case, replacement surfactants are administered to substitute for
the missing natural surfactant.9-11
Epithelial Surface Fluid. A thin fluid layer called the mucus blanket, 5 µm in depth, covers
the walls of the respiratory tract.This barrier serves to trap foreign particles for subsequent
removal and prevents dehydration of the surface epithelium by unsaturated air during inspira-
FIGURE 2. Structure and perfusion of the alveoli. (Reprinted from Washington N, Washington C, Wilson
CG. Pulmonary drug delivery. In: Physiological Pharmaceutics Barriers to Drug Absorption, 2000:225,
with kind permission of Taylor & Francis Book Ltd., London.)
7.
tion. Hypersecretion of mucus is a result of cholinergic or α-adrenergic antagonists, which
act directly on the secreting cells of the submucosal glands. Peripheral granules, in which
mucus is stored, release a constant discharge and form a reservoir that will be secreted after
exposure to an irritating stimulus. A state of disease can modify the distribution of the cell
goblets and the composition of the fluids of the respiratory tracts.
Epithelium.12 The upper respiratory tract is made up of pseudostratified,ciliated,columnar
epithelium in cells with goblet cells.The bronchi, but not the bronchioles, have mucous and
serous glands present.However,the bronchioles possess goblet cells and smooth muscle cells
capable of narrowing the airway.The epithelium of the terminal bronchioles consists mainly
of ciliated, cuboidal cells and a small number of Clara cells (Fıg. 3). Each ciliated epithelial
FIGURE 3. Typical lung epithelia in the different pulmonary regions and thickness of the surface fluid. (a)
The bronchial epithelium (Ø 3–5 mm) showing the pseudostratified nature of the columnar epithelium,
principally comprising ciliated cells 6 µm (c), interspersed with goblet cells (g) and basal cells (b). (b)
The bronchiolar epithelium (Ø 0.5–1 mm) showing the cuboidal nature of the epithelium, principally
comprising ciliated cells (c), and interspersed with Clara cells (cl). (c) The alveolar epithelium showing the
squamous nature of the epithelium, comprising the extremely thin (Ø 5 µm) type I cell (I), which accounts
for approximately 95% of the epithelial surface, and the cuboidal (Ø 10–15 µm) type II cell (II).
8.
. . .
cell has around 20 cilia with an average length of 6 µm and a diameter of 0.3 µm.Clara cells,
which are secretory cells, become prevalent in respiratory bronchioles. In the alveolar ducts
and alveoli, the epithelium is flatter at 0.1–0.5 µm thick. The alveoli are packed narrowly
and do not have partitioning walls; the adjacent alveoli are separated by an alveolar septum
with communication between alveoli via alveolar pores.The alveolar surface is covered with
a lipoprotein film, which is the pulmonary surfactant. The alveolar surface is mainly com-
posed of a single layer of squamous epithelial cells—type I alveolar cells—approximately 5
µm thick.Type II cells, cuboidal in shape, 10–15 µm thick, and situated at the junction of
septa,are responsible for the production of alveolar lining fluid and the regeneration of type
I cells during repair following cell damage from viruses or chemical agents.
The alveolar-capillary membrane,which separates blood from alveolar gases,is composed
of a continuous epithelium, 0.1–0.5 µm thick (Fıg. 4). The maximum absorption occurs in
FIGURE 4. Alveolar–capillary membrane.
9.
the area where the interstitium is the finest (80 nm) because the pulmonary surfactant is
also thin in this area (15 nm).The thickness of the air–blood barrier ranges from 0.2 to 10
µm.The most efficient gas exchange takes place when the air–blood barrier is less than 0.4
µm in thickness.
Interstitium. The lung interstitium is the extracellular and extravascular space between cells
in tissue.In order for a molecule to be absorbed from the airspaces to the blood,it must pass
through the interstitium. Within the interstitium are fibroblasts, tough connective fibers
(i.e., collagen fibers and basement membrane), and interstitial fluid, which slowly diffuses
and percolates through the tissue.
Vascular endothelium. The endothelium is the final barrier to a molecule being absorbed
from the airspace into the blood. Endothelial cells form capillaries that lie under Type I
cells in the alveoli (Fıg. 4).The basic alveolar structure is the septum, which is composed of
capillaries sandwiched between two epithelial monolayers.13
II.B. Controlling the Site of Aerosol Deposition in the Respiratory Tract
1. Factors Affecting Disposition of Particles
Deposition of aerosol particles in the bronchial tree is dependent on the granulometry of the
particles and the anatomy of the respiratory tract. Aerosols used in therapy are composed
of droplets or particles with different sizes and geometries. Generally, four parameters can
be used to characterize the granulometry of an aerosol:
1. Mass median diameter (MMD) corresponding to the diameter of the particles for which
50% w/w of particles have a lower diameter and 50% w/w have a higher diameter.
2. Percentage in weight of particles with a geometrical diameter of less than 5 µm.
3. Geometric standard deviation (GSD) corresponding to the ratio of the diameters of
particles from aerosols corresponding to 84% and 50% on the cumulative distribution
curve of the weights of particles.The use of a geometric standard deviation to describe
the particle size distribution requires that particle sizes are log-normally distributed.
If, as is frequently the case, particles are not log-normally distributed, the geometrical
standard deviation is meaningless and a misleading representation of the distribution.
Heterogeneous aerosols have, by definition, a GSD of greater than or equal to 1.22.14
4. Mass median aerodynamic diameter (MMAD), which makes it possible to define the
granulometry of aerosol particles by taking into account their geometrical diameter,
shape, and density: MMAD = MMD × Density½
2. Mechanisms of Particle Deposition in the Airways
There are three main particle deposition mechanisms in the lung: inertial impaction,
sedimentation, and Brownian diffusion. The deposit of particles administered by aerosol
10.
. . .
in specific areas of the respiratory tract depends on the deposition mechanism versus the
particle diameter.15
1. Inertial impaction is the most significant mechanism for the deposition of aerosol par-
ticles with an MMAD of more than 5 µm.It occurs in the upper respiratory tracts when
the velocity and mass of the particles involve an impact on the airway.It is supported by
changes in direction of inspired air and when the respiratory tracts are partially blocked.
Hyperventilation can influence impaction.
2. Sedimentation occurs in the peripheral airways and concerns small particles from an
aerosol with an MMAD ranging from 1 to 5 µm. Sedimentation is a phenomenon
resulting from the action of gravitational forces on the particles. It is proportional to
the square of the particle size (Stokes law) and is thus less significant for small particles.
This kind of deposition is independent of particle motion. Sedimentation is influenced
by breath holding, which can improve deposition.
3. Brownian diffusion is a significant mechanism for particles with an MMAD of less
than or equal to approximately 0.5 µm.The particles move by random bombardments
of gas molecules and run up against the respiratory walls. Generally, 80% of particles
with an MMAD of less than or equal to 0.5 µm are eliminated during exhalation.
The behavior of the aerosolized particles in the body is summarized in Fıgure 5.
Inhalation of
particles
Losses of particles in
atmosphere and in device
Deposit into
mouth or nose
Deposit by impact and
sedimentation in
lower respiratory tract
Deposit into alveolar area
• Specific activity
• Systemic activity
• Crossing into gastrointestinal
tract
• Specific activity by diffusion of
drug into alveolar liquids
• Systemic activity by diffusion
into capillaries of bloodstream
• Activity on walls of capillaries
by carrying through alveolo-
capillary membrane
FIGURE 5. Behavior of aerosolized particles into the body.
11.
3. Influence of Particle Size
Big particles (>10 µm) come into contact with the upper respiratory tract and are quickly
eliminated by mucociliary clearance. Particles with a diameter of 0.5–5 µm settle according
to various mechanisms. The optimum diameter for pulmonary penetration was studied on
monodispersed aerosols and is around 2–3 µm.16 Smaller particles can be exhaled before
they are deposited; holding the breath prevents this. Extremely small particles (<0.1 µm)
appear to settle effectively by means of Brownian diffusion but are difficult to produce (Fıg.
6). Often the particle size does not remain constant once it reaches the respiratory tract.
Volatile aerosols become smaller with evaporation, and hygroscopic aerosols grow bigger
with moisture from the respiratory tract. In addition, it has not yet been proven that the
retention of inhaled particles depends on their geometric diameter.17
4. Lung Permeability
The alveolar epithelium and the capillary endothelium have a very high permeability to
water, to most gases, and to lipophilic substances. However, there is an effective barrier for
many hydrophilic substances of large molecular size and for ionic species.The alveolar type
I cells have tight junctions, limiting the penetration to molecules with a radius of less than
0.6 nm. Endothelial junctions are larger, with gaps of around 4–6 nm. Normal alveolar
epithelium is almost completely impermeable to proteins and small solutes. Microvascular
endothelium, with its larger intercellular gaps, is far more permeable to all molecular sizes,
allowing proteins to flow into the systemic circulation. Pulmonary permeability increases
in smokers and in states of pulmonary disease.
Soluble macromolecules can be absorbed from the lung by passing either through the
FIGURE 6. Dependance of deposition of particulates on particle size. (Reprinted from Washington N,
Washington C, Wilson CG. Pulmonary drug delivery. In: Physiological Pharmaceutics Barriers to Drug
Absorption 2000:224, with kind permission of Taylor & Francis Book Ltd., London, UK.)
12.
. . .
cells (absorptive transcytosis) or between the cells (paracellular transport).18 It has been
postulated that molecules larger than ~40 kDa may be absorbed by transcytosis and then
enter blood either via transcytosis in the capillary or post capillary venules; molecules smaller
than ~40 kDa may directly enter the blood, primarily via the tight junctions of both the
Type I cell and the capillary.
II.C. Clearance of Inhaled Particles from the Respiratory Tract
Particles deposited and not transported across the epithelium of the respiratory tract are
cleared by either mucociliary clearance or a combination of mucociliary and alveolar clear-
ance mechanisms.
1. Mucociliary Clearance
The respiratory tract possesses series of defences against inhaled materials because of its
constant exposure to the outside environment.The lung has an efficient self-cleaning mecha-
nism known as the mucociliary escalator, in addition to other mechanisms such as coughing
and alveolar clearance.The mucus gel layer (5 µm thick) floats above the sol layer, which is
approximately 7 µm thick. The cilia extend through this layer so that the tip of the villus
protrudes into the gel. The coordinated movement of the cilia propels the mucus blanket
and deposited foreign materials at a rate of 2–5 cm.min–1 outwards towards the pharynx,
where they are swallowed. It has been estimated that 1 liter of mucus is cleared every 24
hours. Mucociliary clearance is influenced by various factors: physiological, environmental
(S2, CO2, tobacco, etc.) and diseases (asthma, cystic fibrosis, etc.).19
2. Alveolar Clearance
Particles deposited in the terminal airway units can be removed either by a nonabsorptive
or an absorptive process.20 The nonabsorptive process involves the transport of particles
from the alveoli to the ciliated region, where they are removed by the mucociliary clearance
mechanism present in the conducting airway.
The absorptive process may involve either direct penetration into the epithelial cells or
uptake and clearance by alveolar, interstitial, intravascular, and airway macrophages. In ad-
dition to their role in cleaning particles,macrophages also play an important part in inflam-
matory processes through the release of chemotactic factors to attract polymorphonuclear
neutrophils from the pulmonary vascular bed to the area. Alveolar macrophages, 15–50 µm
in diameter, lie in contact with the surfactant lining the alveoli. Foreign particles adhere
to macrophages through either electrostatic interaction or interaction with receptors for
some macromolecules, such as immunoglobulins. Following adhesion, macrophages ingest
the particles by interiorization of vacuoles, surface cavitation, or pseudopod formation.The
uptake of particles by macrophages is size dependent. Particles with a diameter of 6 µm are
phagocytosed to a much smaller extent than those with a diameter of 3 µm.Moreover,particles
with a diameter of less than 0.26 µm are minimally taken up by macrophages.The nature of
the coating material also influences the rate of phagocytosis by alveolar macrophages.21,22
13.
III. PULMONARY DRUG CARRIERS
III.A. Liposomes
Liposomes are the lung drug delivery systems that have been the subject of most studies.
Indeed,they are prepared from pulmonary surfactant endogenous phospholipids and are thus
biocompatible, biodegradable, and relatively nontoxic.23 Liposomes consist of one or more
phospholipid bilayers enclosing an aqueous phase.They can be classified as large multilamellar
vesicles (MLVs),small multilamellar vesicles (SMLVs),small unilamellar vesicles (SUVs),or
large unilamellar vesicles (LUVs), depending on their size and the number of lipid bilayers.
Liposomes are produced in a broad range of sizes and can incorporate both hydrophilic and
lipophilic drugs. A variety of drugs have been incorporated into liposomes to improve their
delivery through the airways.The advantages of drug encapsulation in liposomes are numer-
ous,with enhanced drug uptake,increased drug clearance,and reduced drug toxicity among
the most significant.The systemic toxicity of a toxic drug is markedly reduced without effect
on its efficacy once it has been incorporated into liposomes. Moreover, the composition of
liposome lipids can be carefully selected to control drug release and pulmonary retention of
the encapsulated drug.24,25 Liposomes have been studied as drug carriers for 30 years, and
some have been tested in animals and humans.26 Cytotoxic agents, anti-asthmatic drugs,
antimicrobial and antiviral drugs, and antioxidant agents with systemic actions have been
included in liposomes.27 Aerosols of liposomes containing drugs have been studied for the
treatment of bacterial,fungal,and viral infections,and as vaccines and immunomodulators.28.
We will describe the new generation of liposomes, along with the influence of formulation
on stability (phospholipids, size, functionality) and new in vitro (bioadhesion) and in vivo
(biodistribution) studies on liposomes incorporating drugs.
1. Description
The effect of liposomes composed of hydrogenated soybean phosphatidylcholine (HSPC)
and soybean phosphatidylcholine (SPC),containing carboxyfluorescein (CF),was studied in
the mouse after prolonged inhalation.29 Pulmonary histology,along with phagocyte function,
size,and composition of the alveolar macrophages (AM),were investigated.No anomaly was
detected.AM digested the liposomes and released the CF into the phagolysosomal vacuoles.
This study showed that inhaled liposomes encapsulating an active agent can be delivered to
the lungs and, in particular, to the alveolar macrophages.
Physiological solutions of Evans blue and dry powder of liposomes composed of dipal-
mitoyl phosphatidylcholine (DPPC) and dipalmitoyl phosphatidylethanolamine (DPPE)
marked with fluorescein isocyanate were administered in aerosol form to pigs.30 After nebu-
lization, the size of the particles for both solutions was around 1.20 µm, with the size of
the liposomes initially being around 3 µm.The distribution of Evans blue is uniform in the
various pulmonary zones and is proportional to the weights of the lungs and of the animal.
Fluorescence is distributed more in the intermediary and peripheral zones of the lung.This
distribution is dependent on deposition of the liposomes and alveolar liposome-macrophage
interactions, with AM being fluorescent. These results suggest that aerosol administration
of liposomes enables local deposition in the respiratory tract and interacts with the alveolar
macrophages.
14.
. . .
2. Immunosuppressants
The immunosuppressant agent cyclosporine A (CsA) was effectively incorporated into
liposomes composed of egg yolk phosphatidylcholine (EYPC) with a molar ratio of 1:12
CsA/EYPC.31 The association percentage was high (95%).The generation of small aerosol
particles of CsA liposomes had no effect on CsA biological activity because CsA liposomes
were as effective as CsA resuspended in its normal carrier,Cremophor EL,in the inhibition
of anti-CD3 antibody stimulation of mouse spleen cell, as measured by the incorporation
of [3H] thymidine. CsA liposome particles have a mass median aerodynamic diameter of
2 µm, which permits distribution of the drug throughout the respiratory tract. Liposomes
containing CsA were given by aerosol for 15 minutes to mice, and the CsA concentration
in the lungs was found to be equivalent to that of a single daily i.v. injection 16 times more
concentrated (Fıg. 7). CsA liposomes can be produced and aerosolized in order to achieve
pulmonary concentrations with enough immunosuppressant activity to be effective in the
treatment of lung diseases.
Waldrep et al.32 proposed an optimum liposome formulation for nebulization contain-
ing glucocorticoids or immunosuppressant, using dilauroylphosphatidylcholine (DLPC)
alone instead of dipalmitoylphosphatidylcholine (DPPC), dimyristoylphosphatidylcholine
(DMPC), or egg yolk phosphatidylcholine (EYPC).
Liposomes of DLPC containing concentrated amounts of CsA and budesonide (Bud)
FIGURE 7. Comparison of CsA concentrations in blood and lung tissue after 4 days of small-particle
aerosol or intravenous administration of CsA-containing liposomes. Liposomes were composed of 2
mg of CsA/ml and 15 mg of phosphatidylcholine/mL. Three mice (26 g) were used at each time point.
Drug was administered by aerosol for 2 h twice daily, giving a dose of 1.8 mg of CsA/kg ([25 µg of
CsA/L of aerosol × 0.026 L/min {min vol} × 240 min × 0.3 {retention factor}]/0.026 kg), or for 15 min
once daily, giving a dose of 0.11 mg of CsA/kg. Intravenous administration was a single daily injection
of 0.1 mL of CsA liposomes in the tail vein, giving a dose of 1.8 mg of CsA/kg. CsA tissue concentra-
tions were determined by HPLC. (Reprinted from Gilbert et al. Characterization and administration of
cyclosporine liposomes as a small-particle aerosol. Transplantation 1993; 56(4):976, Fig. 1, with kind
permission from Lippincott Williams & Wilkins.)
15.
have been formulated and nebulized.33 Formulations 40 times more concentrated than com-
mercial ones and used by nebulization of Bud suspensions could both reduce nebulization
time and improve patient compliance.The optimum DLPC/CsA and DLPC/Bud propor-
tions are 1:7.5 and 1:15, respectively. With these, liposomes of 1–3 µm diameter could be
formulated, and after nebulization their sizes were reduced (270–560 nm).
After the inhalation of DLPC/CsA nebulized liposomes, their biodistribution was
studied in mice.34 In this study,on a per-gram-tissue basis,the lung contained approximately
18 times more CsA than the liver, and 104 times more CsA than the blood, demonstrating
the effective pulmonary targeting of the CsA/DLPC liposome aerosol.The in vitro immu-
nosuppressant effect of CsA isolated from pulmonary tissue,following delivery of nebulized
DLPC/CsA liposomes, was maintained. Inhibition (99%) of [3H]TdR by antigen-specific
stimulation reduction was revealed, along with inhibition (95%) of mitogen sensitivity.This
DLPC/CsA formulation is promising and could be used to treat chronic asthma and al-
lergies.
Liposome vectors and CsA dissociation were studied in mice following pulmonary
delivery.35 A stable radioactive complex of 99mTc-liposomes DLPC/CsA was delivered by
intratracheal (i.t.) instillation. The 99mTc-liposomes DLPC vector was retained 17 times
longer than the half-life of CsA in a normal lung and 7.5 times longer than in an inflamed
lung (Table 1).
Studies on dogs were carried out, selectively observing the immunosuppressant effect
on the lung of the aerosolized form of CsA, with the aim of seeing whether this system is
suitable for pulmonary transplants,which are compromised by chronic and acute rejection.36
The lungs absorb the nebulized CsA liposomes faster than the other organs do with weaker
concentrations of CsA. In this model, the retention of the CsA delivered by the liposomes
in the lungs was around 120 minutes.
3. Glucocorticoids
Liposomes composed of 1,2-distearoyl phosphatidylcholine (DSPC) and 1,2-distearoyl
phosphatidylglycerol (DSPG) were prepared in order to incorporate triamcinolone aceton-
TABLE 1. Half-Lives in Normal and Inflamed Lungsa
Components T1/2 α
CsA - normal lungs 17.0 ± 3.8 min
CsA - inflamed lungs 17.6 ± 7.3 min
liposomes DLPC - normal lungs 4.8 ± 0.1 h
liposomes DLPC - inflamed lungs 2.2 ± 0.9 h
HSA - normal lungs 4.2 ± 2.4 h
HSA - inflamed lungs 2.0 ± 0.3 h
a 99mTc-cyclosporine A (CsA), 99mTc-liposomes composed of DPPC, and 99mTc-
human serum albumin (HSA) (Arppe et al., 1998).
16.
. . .
ide phosphate (TAP).37 The glucocorticoid was in its hydrophilic form so that the liposome
membrane acts as a barrier and permits slow delivery.A liposome incorporating a lipophilic
glucocorticoid quickly slackens under unbalanced conditions (dilution,administration).These
liposomes are stable for 24 hours in contact with physiological fluid.Seventy-five percent of
TAP remains encapsulated, the initial encapsulation rate being 7–8.5%. Administration of
TAP solution and TAP-liposomes (207 ± 16 nm) i.t. and i.v. was compared in rats.The i.t.
administration ofTAP-liposomes enables prolonged occupation of glucocorticoid receptors,
compared with i.v. administration or with treatment with a TAP solution. Its cumulative
effect was 1.6 times higher in the lungs than in the liver.
Liposomes of EYPC–cholesterol (CHOL) incorporating dexamethasone palmitate
(DEXP),in a molar proportion of 4:3:0.3,were studied.38 Encapsulation of the DEXP was
effective (70%) in comparison with its nonesterified form (<2%).The biological activity of
DEXP was evaluated on blood mononuclear cells over a 24-hour period,measuring its anti-
lymphocyte proliferation properties and its inhibition of interferon-γ production (Table 2).
The DEXP incorporated in the liposomes kept its biological activity. Nebulization studies
in animals should confirm whether this vector is promising in drug delivery to the lungs.
DPPC liposomes containing dexamethasone (DEX) in a molar proportion of 9:1
were prepared and instilled by the i.t. route in rats.39 Encapsulation was effective (35%),
and the size of the liposome-entrapped dexamethasone (L-DEX) was approximately 231
± 32 nm. The pulmonary and blood retention levels of [3H]DEX radioactive compound
were, respectively, 50% and 1% for L-DEX and 26% and 5% for the free DEX 1.5 hours
after instillation. Its effects on reduction of white blood cell levels in peripheral blood and
of adrenocorticotropic hormone (ACTH) levels in the plasma were studied. L-DEX has a
prolonged action (>72 h) on reduction of white blood cells,whereas free DEX has no more
effect after 24 hours.Plasma ACTH levels are less significantly reduced with L-DEX (60%
in 1 h, 25% in 72 h) than with free DEX (80% in 1 h, 50% in 72 h). This study showed
that the retention of dexamethasone delivered directly into the lungs in liposomal form
was significantly prolonged (prolonged anti-inflammatory action) and that the side effects
were reduced.
Following these encouraging results, Suntres et al.40 examined the prophylactic effect
of L-DEX in an animal pulmonary model damaged by lipopolysaccharides (LPS).40 The
LPS stimulate the phagocytes to generate metabolites, which play a significant role in lung
pathogenesis.Rats were pre-treated by the i.t.route with L-DEX,DEX,or a saline solution,
then treated by the i.v.route with LPS.Measurements of the activity of various markers were
taken in: pulmonary cells (endothelial capillary cell markers,such as angiotensin-converting
TABLE 2. Inhibition (%) of Concavalin A Stimulating Proliferation of Lymphocytes and
Production of Interferon γ ( INF-γ )a
Inhibition %
concavaline A-stimulating Free DEXP Liposome-DEXP
Lymphocytes proliferation 94 94
INF-γ production 96 96
a Induced by 10–6 M of free dexamethasone palmitate (DEXP) or by DEXP loaded liposomes composed of EPC-
Cholesterol. (Benameur et al., 1995.)
17.
enzyme [ACE] and type-II alveolar epithelial cell markers, such as alkaline phosphatase
[AKP]), inflammatory response markers (myeloperoxidase [MPO] and elastase activity,
chloramine concentration) and pro-inflammatory mediators (concentration of A2 phospholi-
pase,leukotriene eicosanoid B4,and thromboxane B2 in plasma and histamine in the lungs).
L-DEX was more effective than DEX and protected the pulmonary cells from the LPS.The
ACE and AKP activities were reduced by only 5% and 18%,respectively,while DEX reduces
them by 20 and 28%, respectively. DEX inhibited the increase in inflammatory mediator
activities. L-DEX was 15% more effective in the reduction of MPO (55%) and elastase
(68%) than DEX and 20% more effective in the reduction of chloramine (50%).The three
pro-inflammatory mediators studied are also inhibited by L-DEX and DEX: phospholipase
A2 (62 vs. 45%), leukotriene eicosanoid B4 (76 vs. 64%), and thromboxane B2 (76 vs. 64%)
in plasma.Suntres et al.40 also highlighted that pretreatment with saline solutions and blank
liposomes does not inhibit the effects induced by treatment with LPS.
4. Corticosteroids
The tolerance and safety of DLPC liposomal aerosols containing beclomethasone dipropio-
nate (BDP) were studied in 10 healthy volunteers.34 According to pulmonary function and
blood tests, exposure to aerosols containing amounts of BDP equivalent to or double those
managed by metered dose inhaler (MDI) and dry powder inhaler (DPI) for the treatment
of asthma was well tolerated.
The pulmonary distribution and clearance of DLPC-BDP liposomes and DPPC-BDP
liposomes were compared in 11 healthy volunteers.41 DLPC formed liposomes suitable for
atomization.33 Because DPPC is the major component of pulmonary surfactant and is used
for respiratory distress syndrome (RDS) therapy,9 this should also be investigated. DLPC
and DPPC liposomes had sizes of 3.5 µm and 5.0 µm,respectively,before atomizing and 0.8
µm and 0.9 µm, respectively, after atomizing.The total outputs of the nebulized liposomes
were 11.4 µg with DLPC liposomes and 3.1 µg with DPPC liposomes. This difference
could be due to phase transition temperatures (DLPC –2°C, DPPC +41°C). DPPC could
produce more rigid liposomes, which would find it difficult to pass through the openings of
the atomizer. Clearance of 99mTc-liposomes complexes was relatively slow: 24 hours after
inhalation,79% of the radioactivity originally deposited was detected using DLPC and 83%
using DPPC. Both formulations were suitable for the encapsulation of drugs because they
offered a delivery tolerated by the lower respiratory tracts. However, atomization was more
effective with the DLPC liposomes.
Liposomes containing BDP were prepared in different manners in order to improve
their stability.42 After preparation,the liposomes were freeze-dried and then rehydrated just
before atomizing. Of the series of lipids (DLPC, DMPC, DPPC, HSPC), DLPC, used
previously, was shown to be the most effective for the encapsulation of BDP, although the
encapsulation rate remains low (MLVs: 3.69 ± 0.10% m/m and SMLVs: 2.03 ± 0.08%).
Despite being increased in size after freeze-drying and rehydration, DLPC liposomes were
the smallest liposomes produced: 10.30 ± 1.35 µm and 3.87 ± 0.20 µm, before and after
atomizing,respectively.Atomization made it possible to reduce their size by breaking up any
aggregates.The best atomizing output is obtained with DLPC (78.3%),whereas the DPPC
liposomes have incorporated 25% of BDP.The RF of the DLPC liposomes was 75%,which
was 10% higher than that of the other lipids.
18.
. . .
5. Antibiotics
EYPC-CHOL liposomes encapsulating radio-marked gentamicin were instilled by the
i.t. route in rabbits.43 Gentamicin concentrations in the lungs, kidneys, and plasma were
compared according to their administration in solution or liposomes.With the latter dosage
form,the lungs contain up to 5 times more gentamicin than with the free form and,24 hours
postadministration, the gentamicin continued to diffuse. Concentrations in the kidney and
plasma were markedly lower with gentamicin liposomes than with gentamicin in solution.In
this study,the gentamicin was present in bronchoalveolar rinsings,but it was not determined
whether intact liposomes were introduced into the cells or if they were phagocytosed.In any
case,the administration of gentamicin liposomes into the lungs reduced the drug’s systemic
toxicity and provided a reservoir to slow release.
Different liposomal formulations loaded with tobramycin were studied in vitro to es-
tablish the release kinetics of tobramycin and were administered by the i.t. route in mice.24.
In vitro kinetics studies determining the quantity of tobramycin released at 37°C showed
that the best formulation contained mainly DPPC and provided gradual and sustained drug
release for at least 48 hours,especially with the formulation containing a negatively charged
lipid (DMPG) compared with a noncharged lipid (DMPC). However, both formulations
had similar patterns of about 50% tobramycin retention-release after 36 hours.Administra-
tion of tobramycin encapsulated in DPPC/DMPG (10:1) liposomes made it possible to
detect reduced quantities of tobramycin in the kidneys in comparison with the quantities
detected in the lungs.
6. Analgesics
A mixture of liposomes composed of phospholipon/CHOL encapsulating fentanyl and
free fentanyl was administered in aerosol form in healthy volunteers.44 The mean plasma
fentanyl concentration (Cfen) was significantly greater for i.v. administration than for the
aerosol mixture of free and liposome-encapsulated-fentanyl (4.67 ± 1.87 vs 1.15 ± 0.36
ng.ml–1). However, Cfen at 8 and 24 hours after aerosol administration were, respectively,
1.5 and 2 times greater than with the i.v. route.The peak absorption rate, time to peak ab-
sorption and bioavailability after inhalation were, respectively, 7.02 (± 2.34) µg min–1, 16
(± 8) min, and 12 (± 11)%. This fentanyl-liposomes formulation provides both a fast and
prolonged analgesic effect compared with i.v.administration,which can provide satisfactory
postoperative pain relief.
7. Antioxidant Agents
Radio-labeled liposomes containing DPPC–α-tocopherol in a ratio of 7:3 were administered
by the i.t. route to rats.45 No radioactivity was detected in their blood or organs other than
the lungs,for 72 hours after treatment.The α-tocopherol concentration was 16 times higher
in the lungs after this time. In vitro studies showed that pulmonary tissue, first treated by
the liposomal formulation and then incubated with Fe3+-adenosine diphosphate (ADP) pre-
oxidant, was protected from lipid peroxidation. The liposomes–α-tocopherol formulation
had a prophylactic effect against oxidant agents causing pulmonary damage.
19.
The effectiveness of the same liposomes–α-tocopherol formulation instilled by the i.t.
route in paraquat-poisoned rats was studied.46 Paraquat,a herbicide that causes serious respi-
ratory damage,led to a reduction in enzymatic activity,in particular of angiotensin-converting
enzyme and alkaline phosphatase enzyme,indicating damage to endothelial cells and type-II
alveolar cells, respectively. Paraquat reduced concentrations of the antioxidant glutathione
and supported lipid peroxidation. Administration of liposomes–α-tocopherol resulted in a
reduction of the effects of paraquat; the enzymatic activities increased, in particular, 24 and
48 hours posttreatment,along with GSH concentrations,without,however,reaching normal
levels. A significant reduction in lipid peroxidation was observed.These results suggest that
α-tocopherol,formulated in the form of liposomes and administered directly into the lungs,
may be a potential agent for the treatment of paraquat poisoning.
8. Peptides/Proteins
a. Peptides
A formulation of liposomes was optimized to permit the encapsulation and aerosol delivery
of a cationic peptide CM3, recognized for its in vitro anti-microbial and anti-endotoxin
activities.47 Cationic peptides have already been encapsulated in liposomes to induce an
anticancer response as part of the therapeutic development of anticancer vaccines.The most
effective formulation was based on liposomes made up of DMPC/DMPG (3:1), with a
size of 262 nm, with 96% of the liposomes between 190–342 nm and 4% in the range of
13–1700 nm.The size distribution of the aerosolized preparation was 2.84 ± 0.1 µm,enabling
70% CM3 encapsulation, effective atomization (50%), and a total output of 28%. Using a
mathematical model of pulmonary deposition, it was shown that the minimum inhibitory
levels (2–4 µg.mL–1) of CM3 can be reached over most of the tracheobronchial region in
the adult model and can be exceeded throughout the same region in both pediatric model
subjects using a valved jet nebulizer with a 2.5 mL volume fill.
b. Interferon
Goldbach et al.48 incorporated and nebulized interferon-γ (INF-γ) entrapped in muramyl
tripeptide-containing liposomes.48 The encapsulation efficiency was between 30 and 40%.
A microtoxicity assay was developed to measure the tumoricidal activity of murine alveolar
macrophages.Aerosolized INF-γ and liposomal immunomodulator enhanced the antitumor
properties of AM found in mice 24 hours postinhalation.
Kanaoka et al.49 showed that the presence of empty liposomes can also stabilize nebulized
INF-γ.49 INF-γ nebulized alone is unstable, with these two cysteines producing intra- and
intermolecular bonds then involving polymerization and aggregation.This method has the
advantage of avoiding the incorporation of INF-γ in the liposomes as well as separating
free INF-γ and liposomes.The liposome size remained identical before and after atomizing.
Because they are unilamellar (UL) vesicles, these liposomes were too small and too rigid to
be deformed. The size of the nebulized droplets was identical with or without liposomes.
Therefore, liposomes do not interfere in the delivery of INF-γ. It was calculated that ap-
proximately 100 liposomes were combined with a molecule of INF-γ.The most stable for-
20.
. . .
mulation was achieved when the hydrophobic interactions between the acryl chain of the
lipid and INF-γ were the strongest. Hydrogenated soybean phosphatidylcholine (HSPC),
distearoyl--α-phosphatidylcholine (DSPC), and distearoyl--α-phosphatidylglycerol
(DSPG) provided stability in the following formulation: HSPC/DSPG 10:1 and DSPC/
DPPG 10:1. Fınally, INF-γ can be nebulized thanks to the liposomes, which absorb INF-γ
on their surface (Table 3).
The prophylactic effect of an INF-γ and synthetic double-stranded polyriboinosinic-
polyribocytidylic acid (poly IC) stabilized with poly--lysine:carboxymethylcellulose (LC)
(poly[ICLC]) encapsulated in a liposomal formulation was highlighted in mice infected by
a lethal amount (10 LD50) of the influenza virus.50 The immunomodulator-liposomes were
administered intranasally, but direct lung administration is feasible.
c. Interleukin
Human serum albumin and interleukin 2 (IL-2)–loaded DMPC liposomes, as well as
free IL-2, were nebulized in dogs51 in order to compare the immunological activation
of various IL-2 formulations. A toxicity assessment revealed no side effects for either
treatment. The bronchoalveolar lavage (BAL) leukocyte cell count increased significantly
after inhalation of IL-2–liposomes versus inhalation of free IL-2. A greater proportion
of lymphocytes and eosinophils was observed after IL-2–liposomes treatment. Nontoxic
activation of pulmonary immune effectors for treating cancer in the lung may be possible
using IL-2–liposomes.
DMPC liposomes containing IL-2 were administered by aerosol in several immuno-
deficient patients.52 The rate of encapsulation,or at least of association,was very high (98.8%),
and the average diameter of these liposomes was around 1.1 µm. Patient compliance, safety,
toxicity,and the immune effects of IL-2 liposomes were studied in individuals with primary
immune deficiency and,subsequently,a larger cohort of patients with hepatitis C.According
to the authors of this study,a biological activity of aerosol IL-2 liposomes has been observed
in viral disease (hepatitis C), and additional studies on aerosol Il-2 liposomes in individuals
with hepatitis C and HIV are planned.
TABLE 3. Liposome Formulations Having Adsorbed INF-γ at Their Surface, To Have
Efficient Nebulization of INF-γa
Liposome
composition
Size of liposomes
(nm, average ± SD)
Size of aerosols
(µm, average ± SD)
% of recovery
remaining
% of recovery
aerosolized
None 3.06 ± 1.99 3.1 ± 0.7 0.4 ± 0.2
HSPC/DSPG (10/1) 45.0 ± 24 4.88 ± 2.84 27.2 ± 4.7 25.7 ± 12.6
DSPC/DPPG (10/1) 28.5 ± 19 — 29.8 ± 2.6 43.1 ± 16.6
EPC/DSPG (10/1) 43.7 ± 23 3.79 ± 2.29 16.2 ± 13.0 15.8 ± 2.6
EPC 40.8 ± 24 4.99 ±3.06 3.7± 1.0 1.2 ± 0.4
a Kanaoka et al., 1999
21.
d. Insulin
DPPC–CHOL (7:2) liposomes encapsulating insulin of various oligomerization degrees
were instilled by the i.t. route in rats.53 These studies revealed that only the initial response
(10 min) of encapsulated hexameric insulin is slower than that of dimeric insulin,suggesting
a slower permeability through the pulmonary epithelium.However,the hypoglycemic effect
was identical for both encapsulated oligomers, as it was for the physical mixture of insulin
and blank liposomes. Prolonged absorption of insulin is not due to encapsulation but to the
liposome surface connection and probably to an interaction between the exogenous DPPC
and pulmonary surfactant.
The absorption of insulin was studied in the presence of DPPC phospholipids or
pulmonary rinsing fluid and compared with a dispersion of insulin and blank liposomes.54
Compared to a free insulin dispersion, the presence of liposomes supported the absorption
of insulin by type-II alveolar cells.Glucose levels decreased more quickly and more intensely
in the presence of a physical mixture of insulin–DPPC than in the presence of the insulin–
liposomes dispersion. When the pulmonary rinsing fluid was added to these mixtures, the
hypoglycemic effect was reinforced, especially for the insulin-liposomes dispersion, which
remained less effective than the insulin–DPPC dispersion.In conclusion,the bonds between
the insulin and phospholipids were promoted in the case of the DPPC dispersion compared
to the liposomes, in which the DPPC molecules were sterically restricted.
9. Gene Therapy
The administration of liposomes complexed to deoxyribonucleic acid (DNA) in the form of
a plasmid—termed lipofection—has been demonstrated as a promising gene delivery strategy
in vivo. Plasmid–cationic liposome complexes composed of pCMV4α1-AT and lipofectin
(Fıg. 8) were delivered by repeated aerosol or i.v. administration in rabbits.55 Gene transfer
to the lungs after either i.v. or aerosol administration was similar. This was demonstrated
by the presence of human α1-antitrypsin (Hα1-AT) proteins in the airway epithelial cells.
A weaker protein signal was detected in the kidney and liver in rabbits receiving aerosol
administration. No reverse effect was found on lung compliance or lung resistance, along
with no toxicity.
The delivery of cationic liposomes complexed to plasmid DNA by small particle aero-
sol was investigated.56 It was found that DNA–liposome complexes were damaged to a
significant degree during nebulization, such that the activity of the transfected gene was
FIGURE 8. pCMV4α1-AT plasmid. A: promoter sequence of major immediate early gene from cy-
tomegalovirus. B: translation enhancer. C: human α1-antitrypsin (hα1-AT) cDNA. D: 3’ untranslated
sequence from human growth hormone gene. (Reprinted from Canonico et al. No lung toxicity after
repeated aerosol or intravenous delivery of plasmid–cationic liposomes complexes. J Appl Physiol
1994; 77:416, Fig. 1, with kind permission of The American Physiology Society.)
22.
. . .
diminished.A more stable DNA–cationic liposome complex is desirable for aerosol delivery,
as well as a suitable flow rate and reservoir volume—all factors that influence the stability of
complexes.Complexes with liposomes composed of N-(2-hydroxyethyl)-N,N-dimethyl-2,3-
bis(tetradecytoxy)-1-propanaminium bromide/dioleoylphosphatidylethanolamine (DMRIE/
DOPE) permitted a longer period of active particle delivery.The particle size range was 1–2
µm.The aerosol output was consistent from 0 to 5 minutes. From these experiments, it was
concluded that the aerosol delivery of DNA–cationic liposome complexes to the lungs is
possible for the purposes of gene therapy to the lung.
Cationic liposomes, composed of 1,2-dimyristoyl-Sn-glycero-3-ethylphosphatidyl-
choline (EDMPC)/CHOL (1:1) were used to complex DNA encoding the human cystic
fıbrosis transmembrane regulator conductance gene (hCFTR).57 These DNA–liposome
complexes were nebulized in monkeys by aerosol. Measurements were made to determine
DNA delivery and RNAm transcription by the expression of proteins. No signs of toxicity
were detected. Proteins were widely distributed in the pulmonary tract and were located on
the apical level of the pulmonary epithelial cells, which is the drug application site.
The effects of the cationic DNA–liposome formulation on both transfection efficiency
and stability during nebulization were assessed.58 The effects of nebulization on the size of the
particles and on their morphology were also examined.The cationic lipid bis-guanidinium-
tren-cholesterol (BGTC) in combination with the neutral colipid dioleoylphosphatidyl-
ethanolamine (DOPE) was found to have a degree of stability suitable for effective gene
delivery by the aerosol route.These studies are promising with respect to clinical applications
for aerosol gene delivery.
10. Anticancer Agents
DLPC liposomes containing anticancer agent 9-nitrocamptothecin (9NC) were nebulized
in mice for the treatment of different types of human cancers: i.e., xenografts implanted by
the subcutaneous route and osteosarcomas and melanomas by the intravenous route, with
all three producing pulmonary metastases.59 Once nebulized, the particles have a diameter
of 300 nm. In all cases, cancer growth was inhibited (Fıg. 9).The amount of effective 9NC
contained in the liposomes is 10–50 times lower than that used by other routes of adminis-
tration.The greater therapeutic effectiveness is a result of rapid absorption in the respiratory
tract and, more specifically, in the pulmonary tissues, and penetration into the organ and
tumor sites.Moreover,the lactone form of camptothecin is preserved in the liposomes during
pulmonary deposition, even in the presence of albumin. In fact, albumin combines with the
camptothecin carboxyl form, involving an almost total loss of 9NC anticancer activity. No
toxicity was detected, even if the 9NC was present in the kidneys, liver, or spleen.
Other studies were investigated with 9NC-liposomes (L9NC),by atomizing them into
mice with pulmonary metastases caused by B16 melanoma or human osteosarcoma.60 In
both cases, the administration of L-9NC in aerosol form led to a reduction in pulmonary
weight and the number and size of metastases (Table 4).Treatment with L-9NC appeared
to be effective against pulmonary tumors.
Koshkina et al.61 showed in mice that 5% CO2-enriched air enhanced the pulmonary
delivery of two anticancer agents, paclitaxel (PTX) and camptothecin (CPT), contained
in DPPC nebulized liposomes.61 With the addition of 5% CO2, the size of the nebulized
liposomes increased significantly, from 340 ± 11 nm to 490 ± 7 nm for CPT-liposomes (L-
23.
FIGURE 9. Treatment of human breast cancer (CLO) xenografts in nude mice with 9-NC liposomes
aerosol. Aerosol was administered to mice in a sealed plastic cage for 15 min daily, 5 times weekly
for 31 days. The dose of 9-NC was 8.1/lg/kg per day (᭺ untreated, n = 5;. ᭹ 9-NC liposomes, n =
6). (Reprinted from Knight et al. Anticancer activity of 9-nitrocamptothecin liposome aerosol in mice.
Transactions of The American Clinical and Climatological Association 2000, 111, Fig, 5, p.139, with
kind permission of The American Clinical and Climatological Association.)
CPT) and from 130 ± 18 nm to 230 ± 17 nm for PTX-liposomes (L-PTX).CPT distribution
after 30 minutes of administration was 3.5 times higher with the 5% CO2-enriched air than
with normal air, increasing from ~134 ± 123 ng to ~ 476 ± 216 ng CTP/g of tissue. CPT
distribution in other organs also increased with the addition of 5% CO2,twofold in the liver
and eightfold in the brain. The pulmonary pharmacokinetic profile of CPT was similar in
both cases,whereas it was higher for PTX with 5% CO2-enriched air (Fıg.10).These results
show that when liposomes are nebulized with 5% CO2-enriched air,the pulmonary delivery
of encapsulated drugs is enhanced.
The therapeutic effect of liposomes containing paclitaxel (PTX-liposomes) was stud-
ied in mice with metastases, inoculated with pulmonary renal cell carcinoma.62 Aerosol
treatment with PTX-liposomes was more efficient than with i.v. administration (Fıg. 11).
TABLE 4. Effect of 9-Nitrocamptothecine Loaded Liposomes (L-9NC) Treatment by
Aerosol on Lung Melanoma Metastasesa
Mice
Lung weight
(mg) Tumor number
Size of biggest
tumor (mm)
% of biggest
tumor
Nontreated 311 ± 111 85 ± 47 2.2 ± 0.8 50 ± 0
L-9NC treated 177 ± 17 32 ± 10 0.6 ± 0.2 22 ± 7
a Koshikina et al., 2000
24.
. . .
FIGURE 11. Pulmonary phamacokinetics of PTX-DLPC administered by aerosol (᭺) or i.v. (᭹). Mice
inhaled the drug for 30 min; starting time, 0 (total deposited dose, 5 mg of PTX/kg). Bolus i.v. injection
with 5 mg of PTX/kg was given into tail vein at time 0 (Reprinted from Koshkina et al. Paclitaxel lipo-
some aerosol treatment induces inhibition of pulmonary metastases in murine renal carcinoma model.
Clin Cancer Res 2001; 7:3260, Fig 1, with kind permission of Cancer Research.)
FIGURE 10. Tissue distribution of CPT after a 30-min exposure to liposome aerosol generated with
normal air (solid gray) or with 5% CO2-enriched air (hatched). At the end of treatment (30 min) organs
from 3 mice per group were resected and the drug content determined by HPLC. Mean values and SD
were calculated. P-values for 5% CO2-renriched air compared to normal air were 0.02, 0.13, 0.04, 0.04,
0.03, 0.01 for lungs, liver, spleen, kidney, blood and brain, respectively (Student’s t test, two-tailed).
(Reprinted from Koshkina et al. Improved respiratory delivery of the anticancer drugs, camptothecin
and paclitaxel, with 5% CO2-enriched air: pharmacokinetic studies. Cancer Chemother Pharmacol
2001; 47:453, Fig. 1, with kind permission of Springer-Verlag.)
25.
The weight of the lungs and the number of visible tumors decreased by ~26% and ~ 32%,
respectively, compared with the untreated mice.Their life expectancy also increased, by ~10
days. This study reveals the potential therapeutic application of aerosols for the treatment
of pulmonary cancer.
11. Bioadhesive Liposomes
Bioadhesive drug delivery systems were introduced in order to prolong and intensify the con-
tact between controlled delivery forms and the mucous apical pole,inducing active transport
processes.63 Contact with the mucus of the epithelium is called muco-adhesion, and direct
contact with the cellular membrane is called cyto-adhesion. Lectins are nonimmunological
glycoproteins that have the capacity to recognize and bind to glycoproteins exposed at the
epithelial cell surface.
Liposomes functionalized with lecithins appeared to be capable of improving their bind-
ing to human alveolar cells (A549 and primary cells).64 In this study, the unfunctionalized
liposome formulation was optimized by measuring the loss of carboxyfluorescein (CF) loaded
in the liposomes during atomization.Liposomes composed of DPPC–CHOL (50–50% mol)
were more stable during atomization (8% CF loss) than DPPC liposomes (15–20% CF loss),
even in the presence of pulmonary surfactant. Lehr et al.63 reported that the atomization
of DPPC–CHOL liposomes with lecithin functionalization did not significantly influence
their physical stability.The cell-binding capacity of functionalized liposomes is much higher
than that of unfunctionalized liposomes, even after atomization (Fıg. 12).
Immunoliposomes—liposomes carrying specific antibodies—can target cells carrying a
specific antigen. Margalit65 reported that they have been used to target pulmonary tumors
in vitro and in vivo.
12. Dry Powder Liposomes
An optimum formulation of dehydrated liposomes depends on several factors: the liposome
composition, the presence of cholesterol (CHOL), the incorporation of a cryoprotective
sugar, the preparation method, and the nature and proportion of the incorporated drug. An
optimum liposome formulation corresponds to an optimum size, lamellarity (unilamellar
[UL] or multilamellar [ML]), has a maximum drug incorporation efficiency and oxidation
index. An optimum dry powder formulation is characterized by its repose angle, its com-
pressibility index, and its dispersible and respirable fractions.
In the past, several formulations of liposome dry powder inhaler (DPI) have been de-
veloped. Among these, a formulation of liposome DPI containing anti-asthmatic ketotifen
fumarate (KF),was optimized.25 Liposomes formed by two successive hydrations before and
after sonification (1 and 2 hours, respectively) and with a molar composition KF/(EYPC-
CHOL) (1:15) demonstrate a maximum encapsulation rate.In this case,sucrose is revealed
to be the best system cryoprotector, with a mass lipid/sugar ratio of 1:12 and a maximum
concentration of 500 mM. When lactose monohydrate (Sorbolac-400) was added before
freeze-drying, 97.92 ± 0.54% KF retention was achieved. The oxidation of liposome lipids
is not inhibited by the presence of nitrogen or antioxidant agents, with the oxidation index
increasing from 0.427 ± 0.01 to 1.510 ± 0.01 (Table 5).Fınally,the respirable fraction of this
26.
. . .
formulation (21.59 ± 1.53%) was comparable with a commercial control (26.49 ± 1.52%).
The KF-liposome DPI was successfully prepared according to the respirable fraction to be
delivered to the central and peripheral pulmonary tract. Obviously, the choice of the cryo-
protector is dependent on the chemical structure of the drug. For example, as a reducing
sugar, the sucrose would be entirely unsuitable for protein or peptide delivery.
Table 5. Formulation of Dry Powder Inhaler (DPI) Liposomesa
Formulations KF : EYPC : CHOL Size (µm)
% of
encapsulated KF Oxidation index
KF[1] 1 : 15 : 0 1.56 ± 0.26 86 1.510 ± 0.01
KF[2] 1 : 10 : 5 1.70± 0.12 70 1.425 ± 0.01
KF[3] 1 : 7.5 : 7.5 2.05 ± 0.10 64 1.328 ± 0.01
a Liposomes are composed of egg yolk phosphatidylcholine (EYPC) and cholesterol (CHOL), which permit the
highest ketotifen fumarate (KF) incorporation rate, with an oxidation index that is still high (Joshi and Misra25).
FIGURE 12. Interaction of lectin-functionalized liposomes with alveolar epithelial cells. Cell association
of 200 µg wheat germ agglutinin (WGA)-liposomes with A549 cells. WGA liposomes = WGA-functional-
ized liposomes; blank liposomes = DPPC:cholesterol liposomes; WGA liposomes + free WGA = WGA
liposomes and 20-fold free WGA; inhibitory sugar = 20 µl of 20.0 mM diacetylchitobiose; LS = alveoafact
(lung surfactant). Results represent the average and standard deviation of at least 3 determinations
from 2 different passage numbers for A549 cells. (Reprinted from Abu-Dahab et al. Lectin-functional-
ized liposomes for pulmonary drug delivery: effect of nebulization on stability and bioadhesion. Eur J
Pharm Sci 2001; 14:43, Fig. 6b, with kind permission of Elsevier Science.)
27.
III.B. Polymeric Microspheres and Nanospheres
1. Microspheres
The term microparticles includes microspheres (uniform spheres), microcapsules (with a
core and an outer layer of polymer), and irregularly-shaped particles.66 Microparticles are
composed of biodegradable polymers, which may be natural or synthetic. They have been
widely used as vectors of drugs via different administration routes.These particles have the
characteristics required to target and support drug delivery. They are prepared in a wide
range of sizes, from 1 to 999 µm, which is a decisive factor for delivery of drugs in vivo. A
number of lipophilic and hydrophilic molecules are able to be encapsulated or incorporated
in the microspheres. In comparison with liposomes, microspheres are physicochemically
more stable in vivo and in vitro and would thus allow slower release and a more prolonged
action of the encapsulated drugs.The pulmonary administration of aerosolized microspheres
may therefore provide an opportunity for the prolonged delivery of a systemically active
agent, with the drug protected from enzymatic hydrolysis. Microspheres have already been
prepared from various polymers: albumin,poly(glycolic-co-lactic acid) (PGLA),poly(lactic
acid) (PLA), poly(butylcyanoacrylate) (PBC), etc.
Microspheres can be produced to meet certain morphological requirements,such as size,
shape, and porosity, by varying the process parameters. Microspheres are less susceptible to
the effects of hygroscopic growth within the airways.67 Furthermore,Sakagami et al.66 sug-
gested enhancing pulmonary absorption by delaying mucociliary clearance through the use
of hydroxypropylcellulose microspheres, because the highly viscous hydroxypropylcellulose
demonstrates mucoadhesive properties.Because cellulose derivatives are not metabolized and
the lung is not a conduit like the GI tract, the accumulation of such drug delivery devices
can be prejudicial. For site-specific delivery, Steiner et al.66 developed microspheres formed
from a material (diketopiperazine) releasing the drug at a specific pH.
a. Albumin Microspheres
Albumin microspheres may be a suitable carrier for airway delivery because of their biocom-
patibility and biodegradability.Albumin microspheres encapsulating an anti-silicotic agent,
tetrandine,were studied as carriers for pulmonary drug delivery.69 The entrapment efficiency
was approximately 40% and the mean diameter of the microspheres was 4.41 µm, which is
suitable for inhalation.The respirable fraction (RF) was assessed in vitro with a twin-stage
liquid impinger: more than 11% of the delivered drug was collected in the lower stage, and
this fraction is believed to reach the lower airway.These types of albumin microspheres have
potential for the targeting and controlled release of an anti-silicotic drug within the lung.
Albumin microspheres loaded with ciprofloxacin (CIPRO), a quinolone used to treat
various microbial diseases, were investigated for their drug release in vitro as a potential dry
powder to inhale.70 The CIPRO-loaded albumin microspheres were smaller than 5 µm,a size
suitable for DPIs.Drug entrapment depended on the drug/material ratio and was around 50%
for CIPRO/albumin (1:1 w/w).The in vitro drug release profile from the microspheres was
dependent on the thermal treatment of the microspheres.With the best thermal treatment,
within 0.5 hours the burst effect indicates that 10 ~ 20% CIPRO has been released from
the microspheres, and within 12 hours 70 ~ 90% CIPRO is released. The CIPRO release
28.
. . .
rate fell as the albumin ratio increased. In conclusion, sustained-release microspheres were
suitable for dry powder inhaled pulmonary drug delivery systems.
b. Target or Avoid Alveolar Macrophages?
Targeting drugs to alveolar macrophages has the distinct advantage of delivering high concen-
trations of drug to a cell that plays a central role in the progression of disease (tuberculosis)
and in immune responses.
The microspheres can target alveolar macrophages (AMs) without eliciting a pulmonary
inflammatory response in vitro.22 In fact, a cell culture of AM, in the presence of micro-
spheres composed of PLA, produces negligible quantities of oxidants and tumor necrosis
factor alpha (TNF-α) inflammatory cytokines. Interactions between PLA microspheres,
marked by rhodamine 6G,which is a fluorescent agent,and AM are concentration-dependent
(~30% interactions with a concentration of 50,000 particles /mL). Endocytosis of the mi-
crospheres was revealed in the presence of certain endocytosis inhibitors—lysosomotropic
agents, NH4Cl, and chloroquine—reducing AM–particle interaction by around 50%. This
study demonstrated that microspheres can enter alveolar macrophages without activating
them, thus enabling possible drug delivery to target macrophages, for example, in the case
of tuberculosis.
Wang et al.71 showed that the coencapsulation of an immunomodulator (monophos-
phoryl lipid A [MPLA]) in PLGA microspheres makes it possible to increase the rate of
phagocytosis (Fıg. 13). In the case of other diseases, alveolar macrophages must be avoided
FIGURE 13. Effect of coencapsulated MPLA on phagocytosis of PLGA microspheres containing plasmid
DNA. J774A-1 cells were incubated with PLGA microspheres (6000 g/mole) containing MPLA (᭜) or
no MPLA () for 0.75, 1.5, 3, 6, 12 and 24 h. Free microspheres were removed by PBS washing, cells
were fixed, and the number of microspheres per cell was counted by phase contrast microscopy. Error
bars indicate S.D. (n = 3). (Reprinted from Wang et al. Encapsulation of plasmid DNA in biodegradable
poly(D,L-lactic-co-glycolic acid) microspheres as a novel approach for immunogene delivery. J Control
Release 1999; 57:16, Fig. 9b, with kind permission of Elsevier Science.)
29.
in order to prevent phagocytosis clearance and thus to enhance the alveolar half-life and
bioavailability of the drug.
DPPC plays a role in alveolar macrophage phagocytotic of microparticles.21 The in-
teractions of PLGA and DPPC/PLGA microspheres containing peroxidase, as a protein
model, have been evaluated on an AM cell culture by confocal microscopy. After incuba-
tion for 1 hour, the PLGA particles are located in the macrophage cytoplasm (95 ± 1.35%),
while the DPPC–PGLA particles are instead located at their surface (26.2 ± 13.9%). X-ray
photoelectron spectroscopy (XPS) results indicated that the inclusion of DPPC in the
microspheres altered the microsphere surface chemistry, with the DPPC covering a large
portion of the microsphere surface, but did not entirely mask the PLGA. The dominance
of DPPC on the microsphere surface was highly beneficial in moderating the interactions
occurring between the microspheres and phagocytic cells in the lung. DPPC reduced the
adsorption of opsonic proteins,thereby reducing microsphere phagocytosis occurring in the
alveoli, which enabled possible alveolar drug delivery (Table 6). These microspheres could
be designed to act as a controlled delivery system for small molecules, peptides, or proteins
for pulmonary administration.
Other studies were investigated to understand the inhibition of pulmonary phagocyto-
sis. In fact, respirable PGLA microspheres (2–3 µm) containing a fluorophore (rhodamine
B [RB]) were used as a model.20 RB’s loading efficiency was approximately 18%, and its
burst effect was very low, with less than 0.5% being released up to 19 hours. Two alveolar
macrophage types were used for this study: the NR8383 cell line and alveolar macrophages
(AM) freshly isolated from the lungs of rats. Seventy percent of the NR8383 population
phagocytosed a mean of 3.24 ± 0.69 microspheres per cell. The use of inhibitors (cytocha-
lasin D, Na azide) prevented phagocytosis. The phagocytosis of microspheres coated with
polaxomer 338 depended on the microspheres-per-cell ratio R. Compared to the control,
when R = 5, the phagocytosis reduction was 20% and 15% for AM and NR8383, respec-
tively; and when R ≥ 10,phagocytosis was 10–15% reduced for AM,while no reduction was
found for NR8383.The phagocytosis of microspheres coated with DPPC was significantly
lower than the control at all microsphere-per-cell ratios. Even at excess ratios, around 65%
of phagocytosis was inhibited for both cell types.
c. Importance of Encapsulated Drug Nature
El-Baseir et al.67 studied the in vitro release kinetics of nedocromil sodium (NS) (hydro-
soluble compound) and beclomethasone dipropionate (BDP) (hydrophobic compound) from
poly(-lactic acid) (PLA) microspheres. The release kinetics of NS exhibited a biphasic
TABLE 6. Effect of DPPC on Microparticle Internalization by Alveolar Macrophages
(AM)a
Particles PGLA PGLA/DPPC
Size (µm) 3.5 ± 1.72 3.3 ± 1.00
(%) of internalization in AM 65.1 ± 15.8 26.2 ± 13.9
a Evora et al.21
30.
. . .
pattern characterized by an initial and rapid release, probably of the drug located near the
surface of the microspheres, followed by a period of continuous slow release (80–100% of
drug released over an 8-day test period).The initial phase is particle-size dependent.In fact,
27% of the drug was immediately released when the particles had a diameter of 2.79 µm,
and 42–60% was released for larger particle sizes (3.52 and 4.88 µm diameter).The release
profile of NS was found to follow a square root of time-dependent mechanism as defined
by the Higuchi equation (Q = kt½), where Q is the cumulative release of the drug, k the
constant release rate, and t the time period.
BDP-loaded PLA microspheres demonstrated much higher entrapment levels and
smaller particles than the more hydrophilic NS (88% and 9% and 0.9-1.2 µm and 2.5-5 µm,
respectively).Differential scanning calorimetry (DSC) data indicated the possibility of sus-
tained release of BDP for over 6 days. BDP-loaded PLA microspheres were stable upon
immersion in phosphate-buffered saline, in contrast with NS-loaded PLA microspheres.
These results may indicate that lipophilic drug particles are not adsorbed near or on the
surface of the microsphere but that they are molecularly dispersed in the polymeric matrix,
and therefore that no initial burst effect can occur. The deposition of PLA microspheres
loaded with NS or BDP in the Andersen Cascade Impactor is presented in Table 7.
d. Corticosteroids
To prevent rapid dissolution in bronchial fluid and the fast absorption of corticoids via the
lung surface,Wichert72 encapsulated beclomethasone dipropionate (BDP) in PLA or PGLA
microparticles. Only 20% of BDP was encapsulated in the microspheres, but both particle
diameters were suitable for pulmonary delivery—namely, 2.6 ± 0.4 µm and 2.8 ± 0.7 µm
for PLA and PGLA microspheres, respectively. Microparticles with the same drug content
but different matrix polymers demonstrated marked differences in their release patterns.
PGLA (MW 15,000) released only about 20% within 8 hours, whereas PLA (MW 2000)
released nearly all the encapsulated drug (Fıg. 14). BDP release was found to be concentra-
tion dependent: a lower amount of polymer per drug molecule presented fewer barriers for
drug diffusion within the polymer matrix.The in vitro degradation of PLA microparticles in
bronchial fluid was studied in order to see whether microparticles are biodegradable within
an acceptable time span. After 1 hour of incubation with some bronchial fluid at 37°C, the
particles demonstrated an obvious deterioration of their surface characteristics, including
deep holes.This study also revealed that particles made with a lower molecular weight PLA
could be suitable for inhalatory sustained-release formulations. An evaluation of the com-
patibility and toxicity is necessary at this stage.
e. Antibiotics
Rifampicin-loaded PGLA microspheres (R-PGLA) were administered by insufflation or
nebulization to guinea pigs infected by mycobacterium tuberculosis (MTB).73 The in vitro
growth of MTB was inhibited in the presence of an appropriate dose of R-PGLA. The
R-PGLA microspheres, the sizes of which are within the respiratory range (1–5 µm), sig-
nificantly reduced the lung bacterial loads (tenfold) when compared to that of the controls
(Fıg. 15). R-PGLA–treated animals also exhibited reduced inflammation and lung damage
32.
. . .
compared to untreated controls or rifampicin-solution–treated animals. Nebulization was
more efficient in reducing the number of viable microorganisms in the lungs at equivalent
doses of R-PGLA than was insufflation. This study indicated the potential of R-PGLA
microspheres, delivered by nebulization directly to the lungs, to treat the early development
of pulmonary tuberculosis.
FIGURE 15. Number of viable bacteria (cfu/mL) in lung () and spleen (ٗ) tissues (4–5 weeks postin-
fection) following nebulization of R-PLGA microspheres (1.03–1.72 mg/kg), RIF (1.03–1.72 mg/kg)
and PLGA. Animals including control group were exposed to MTB 24 h after drug administration.
Bars represent mean :t. SD for n = 3–5. * p < 0.05 (level of significance for R-PLGA microspheres).
(Reprinted from Suarez et al. Respirable PLGA microspheres containing rifampicin for the treatment
of tuberculosis: screening in an infectious disease model. Pharm Res 2001; 18(9):1317, Fig 3, with kind
permission of Kluwer Academic Plenum Publishers.)
FIGURE 14. Effect of the matrix polymer on drug release (mean of three batches ± coefficient of
variation). Drug content is 16% in all cases (Reprinted from Wichert et al. Low molecular weight PLA:
a suitable polymer for pulmonary administred microparticles. J Microencaps 1993; 10(2):202, Fig. 3,
with kind permission of Taylor & Francis Ltd, www.tandf.co.uk/journals.)
33.
f. Proteins
Calcitonin. The pulmonary administration and in vitro degradation of gelatin microspheres
loaded with salmon calcitonin (SC) was studied by Morimoto et al.74 Gelatin microspheres
made it possible to prevent particle degradation by enzymes. The in vitro release study
(Fıg. 16) revealed that SC seems to be dependent on the gelatin microsphere load and
not on the particle size. Within 2 hours, approximately 85% of SC was released from
positively-charged gelatin microspheres, while 40% was released from negatively-charged
gelatin microspheres. These results suggested that the SC released from the microspheres
depended on the electrostatic repulsion between SC (isoelectric point [IEP] = 8.3) and
positively charged gelatin microspheres (IEP = 9). However, the initial release of SC from
negatively-charged microspheres was suppressed by the formation of a poly–ion complex.
Consequently, the electrostatic forces relationship between the incorporated proteins and
gelatin may be an important factor affecting the release rate of incorporated proteins from
gelatin microspheres.
The results for intratracheal administration of SC-loaded gelatin microspheres are
given in Fıgure 17. The hypocalcemic effect following the administration of SC in both
types of gelatin microspheres was significantly greater than that following administration
in aqueous solution (in pH 7.0 PBS).The hypocalcemic effect following the administration
FIGURE 16. Release profiles of salmon calcitonin from gelatin microspheres with different charge
(A) and different particle sizes (B) in pH 7.0 PBS at 37°C. Positively charged microspheres: 11.2 µm
(᭝). Negatively charged microspheres: 10.9 µm (᭡). Each point represents the mean ± s.e.m., n = 4.
(Reprinted from Morimoto et al. Gelatin microspheres as a pulmonary delivery system: evaluation
of salmon calcitonin absorption. J Pharm Pharmacol 2000; 52:614, Fig. 2, with kind permission of
Pharmaceutical Press.)
34.
. . .
FIGURE 17. Time course of hypocalcæmic effect in rats after pulmonary administration of salmon
calcitonin (3.0 U.kg–1) in gelatin microspheres with different charge (A) and different particle sizes (B).
Solution (᭹) ; positively charged microspheres: 3.4 µm (᭺) ; 11.2 µm (᭝) ; 22.5 µm (ٗ) ; 71.5 µm (᭛) ;
negatively charged microspheres: 10.9 µm (᭡). Each point represents the mean ± s.e.m of at least 4
animals. (Reprinted from Morimoto et al. Gelatin microspheres as a pulmonary delivery system: evalua-
tion of salmon calcitonin absorption. J Pharm Pharmacol 2000; 52:615, Fig. 3a/b, with kind permission
of Pharmaceutical Press.)
35.
of SC in positively-charged gelatin microspheres was significantly greater than that after
administration in negatively-charged gelatin microspheres. Furthermore, Fıgure 17 shows
that the administration of smaller particles produced a greater hypocalcemic effect. In fact,
small particle sizes appeared to reach the lower regions of rat lungs—the alveoli—where
the respiratory tract promotes drug absorption.The pharmacological availability of SC was
greater when given via the lungs in positively-charged gelatin microspheres (particle sizes
3.4 and 11.2 µm) than in solution (50% and 15%, respectively) and was similar to that after
intramuscular administration of an SC solution.
Moreover, Morimoto et al.74 claimed that the enzyme responsible for the degradation
of SC exhibited a fourfold higher activity in the membrane fraction of lung homogenate
than in the cytosol fraction. The degradation of SC by secreted or membrane-associated
enzymes in the mucus layer of the lung would be physically prevented by the use of gelatin
microspheres.Moreover,coadministration with enzyme inhibitors could be suggested.Indeed,
inhibitors such as chymostatin, antipain, and bacitracin have the greatest inhibiting effects
on enzymes involved in SC degradation.75
In conclusion, gelatin microspheres have been shown to be a useful carrier for pulmo-
nary delivery of SC and to increase its absorption via the respiratory tract. Other proteins
or peptides (such as insulin) could also be administered via this route, but the most useful
carrier (positively- or negatively-charged microspheres) depends on the IEP of the protein
and its electrostatic interaction with the type of gelatin used.
Prospects for protein encapsulation in microspheres. Proteins, such as erythropoietin or bo-
vine serum albumin (BSA), have already been encapsulated in PGLA microspheres.76,77
However, the particle sizes were much too large (50–600 µm) to be administered by the
pulmonary tract.
DNA can be encapsulated in PLGA microspheres without compromising its structural
and functional integrity.71 Encapsulation efficiencies (EE) seemed to depend on the increased
molecular mass (MW) of the polymer (EE = 30.0% for MW = 12,500 and EE = 53.3% for
MW = 50,000).The diameter of microspheres ranged between 0.4 and 2 µm,which is within
the respirable range. Moreover, PLGA microspheres can protect plasmids from nuclease
degradation and therefore offer an effective approach for in vivo gene delivery, especially to
phagocyte cells, for inducing immunization.
g. Viruses
Venezuelan equine encephalomyelitis (VEE) was inactivated by 60Co-irradiation and microen-
capsulated in PGLA microspheres (≤10 µm) with the aim of studying the effectiveness in
inducing immune responses against aerosol challenge with VEE virus.78 Mice were primed by
s.c.or i.t.administration of microencapsulated VEE virus,followed 30 days later by a single
immunization given by the oral, i.t., or s.c. route. Mice boosted by i.t. or s.c. administration
had higher plasma IgG anti-VEE levels than orally immunized animals.The levels of IgG
and IgA antibody activity in the bronchoalveolar lavage (BAL) from mice boosted by the
i.t. route were higher than those in animals boosted by the other routes (Fıg. 18). Mucosal
immunization via the i.t. route appeared to be the most effective regimen, because 100% of
the mice resisted the virus challenge.
36.
. . .
FIGURE 18. Time course of plasma IgG anti-VEE antibody response in mice immunized by systemic
followed by mucosal route with methylene chloride processed microsphere vaccine. Groups of BALB/c
mice (5/group) were immunized by administration of 50 µg of formalin-fixed, 6OCo-inactivated TC-53
virus in microspheres by s.c. (50:50 DL-PLG; batch G320-140-00, methylene chlorjde solvent) or i.t.
(50:50 DL-PLG; batches H456-092-OQ and H456-109-00, methylene chloride solvent) routes on day
0 and boosted on day 30 by s.c., oral, or i.t. administration of 50 µg of the same microencapsulated
virus vaccine. Plasma was collected at 10-day intervals and assayed for antibody activity by ELISA.
(Reprinted from Greenway et al. Induction of protective immune responses against Venezuelan equine
encephalitis (VEE) virus aerosol challenge with microencapsulated VEE virus vaccine. Vaccine 1998;
15(13):1318, Fig. 2, with kind permission of Elsevier Science.)
h. Antigens
Recombinant F1 (rF1) and V (rV) subunit antigens were entrapped within PLA micro-
spheres and were administered by the i.t or i.m. route to mice challenged afterwards with a
virulent strain of Yersinia pestis.79 The introduction of antigenic material into the respiratory
tree triggers the production of locally produced specific antibodies in the lung,which should
improve protection against pneumonic plague infection.Microspheres had loadings of 1.2%
(w/w) rV and 5% (w/w) rF1. Following injection of 107 U Y. Pestis, the group immunized
with microspheres by the i.t.route had the highest percentage of survivors (55%),compared
with those immunized with microspheres by the i.m. route (50%), with antigen solution
administrated by the i.t. route (33%) and administrated by the i.m. route (20%). Only i.t.
instillation of microspheres induced significant quantities of anti-F1 and -V specific IgA
in bronchoalveolar lavage (Table 8). This study showed that the introduction of F1 and V
subunits into the respiratory tract may be an alternative to parenteral immunization schedules
for protecting individuals from plague.
37.
TABLE 8. Mean (± SE) Anti-F1 and V IgG Endpoint Titers in Day 82 Lung Washesa
Treatment Anti-V IgG Anti-F1 IgG Anti-V IgA Anti-F1 IgA
MS i.t. 2048 ± 627 115 ± 14 18 ± 13 18 ± 13
Sol i.t. 780 ± 397 20 ± 13 1.6 ± 0.8 <1.0
MS i.m. 972 ± 343 42 ± 11 <1.0 <1.0
Sol i.m. 275 ± 116 13 ± 6 1.0 ± 0.5 <1.0
a Generated by day 1 and 60 immunizations with microspheres (MS) coencapsulated (5 µg F1, 1 µg V) or soluble
(sol) admixed rF1 and rV subunits (5 µg F1, 1 µg V). Mice were immunized by either i.t. or i.m. routes (n = 5).
(Eyles et al., 2000).
i. Mucoadhesive Microparticles
Mucoadhesive microspheres of hydroxypropylcellulose (HPC) encapsulating beclomethasone
dipropionate (BDP) were administered as powder aerosols to healthy or asthmatic guinea
pigs.68 The pharmacokinetics and pharmacodynamics of BDP were compared for different
BDP formulations: pure crystalline BDP (cBDP), amorphous BDP incorporated in HPC
microspheres (aBDP-HPC),and crystalline BDP-loaded HPC microspheres (cBDP-HPC).
Powder aerosols were produced within a respirable size range of 1.7–2.9 µm.The pharmaco-
kinetic profiles for these three powders were dissolution modulated.It was shown that at 180
minutes postadministration,more than 95% and 85% of BDP were absorbed from the lung
following aBDP-HPC and cBDP administration, respectively; whereas 86% of BDP were
absorbed at 180 minutes following cBDP-HPC administration.A prolonged lung retention
of BDP may be beneficial in maximizing the efficacy of BDP dose delivery to the lung and
in reducing the side effects caused by its extra lung absorption.The duration of inhibition of
eosinophil infiltration into the airways of asthma-induced guinea pigs was assessed following
cBDP and cBDP-HPC administration. While cBDP (1.37 mg.kg–1) inhibited eosinophil
infiltration for only 1–6 hours, cBDP-HPC, with a lower drug dosage (0.25 mg.kg–1), was
able to maintain these inhibitory effects for 24 hours following administration. This study
showed that this HPC microsphere system has the potential to prolong the therapeutic
duration of BDP following inhalation.
j. Porosity: A Decisive Factor
Rogerson et al. 80 highlighted that the difficulty with many sustained-release inhalation
therapies is that solid (or more dense) particles will be removed by clearance mechanisms
before acting as a drug reservoir.To avoid these problems, Rogerson et al.80 and Edwards et
al.81 developed particles of small mass density (<0.4 gram per cubic cm) with relatively large
geometric diameter (>5 µm),which permitted the highly efficient delivery of inhaled thera-
peutics into the systemic circulation and prevented the phagocytosis by macrophages.The use
of relatively low-density perforated (or porous) microparticles significantly reduced attractive
forces between the particles,thereby reducing the shear forces and increasing the flowability
of the resulting powders.82 This made it possible to prevent aggregation.The microstructures
38.
. . .
allowed the fluid suspension medium to freely permeate or perfuse the particulate boundary
and,hence,to reduce or minimize density differences between the dispersion components.82
Moreover, as a consequence of their large size and low mass density, porous particles can be
aerosolized from a DPI more efficiently than can smaller nonporous particles, resulting in
higher respirable fractions of inhaled drugs.81 In conclusion, in view of these advantages,
dispersions of this invention are particularly compatible with inhalation therapies.
Large porous particles are more efficient for the pulmonary administration of potent
drugs by a dry powder inhaler than are small porous or nonporous particles.81 Porous par-
ticles (ρ < ~0.4 g.cm-3, d > 5 µm) and nonporous particles (ρ ~ 1± 0.5 g.cm-3, d < 5 µm) of
PLGA,with the same aerodynamic diameter,were prepared with incorporated testosterone
and were then tested on an in vitro lung model of the Andersen cascade impactor (ACI).
The respirable fraction for the porous system is higher than that for the nonporous system:
50 ± 10% and 20.5 ± 3.5%, respectively.The highly efficient respirable fraction for the large
porous particles can be attributed to their smaller surface-to-volume ratio,their low aggrega-
tion, and their ability to exit the DPI as single particles.The particle composition has little
influence, with the respirable fraction analogous between PLGA particles and polylactic
acid-co-lysine-graft-lysine (PLAL-lys) particles: 50 ± 10% and 57 ± 1.9%, respectively. In
vivo studies on the bioavailability and inflammatory response of particles incorporating
insulin and delivered by aerosol were conducted in rats. Only 46% of porous particles are
deposited in the trachea, compared with the deposition of 79% nonporous particles. For
large porous particles, insulin bioavailability relative to subcutaneous injection was 87.5%,
whereas the small nonporous particles yielded a relative bioavailability of 12% after inhala-
tion. Given the short systemic half-life of insulin (11 min) and the 12- to 24-hour time
scale of particle clearance from the central and upper airways, the appearance of exogenous
insulin in the bloodstream several days after inhalation appeared to indicate that large
porous particles achieve long, nonphagocytosed lifetimes in the deep lungs. These studies
also demonstrated that the phagocytosis of particles fell sharply when the particle diameter
increased beyond 3 µm.Indeed,large porous particles with a mean diameter of 20.4 µm lead
to 177% bioavailability for the subcutaneous injection of testosterone, whereas only 53% of
relative bioavailability was observed for large porous particles of 10.1 µm.
2. Nanoparticles
Nanoparticles have the same characteristics as microparticles,being composed of biodegrad-
able polymers and drug binding at the surface or in the interior of the host minicarrier,83
also providing protection against enzymatic digestion and improving drug bioavailability
via controlled release.The mean size of a nanoparticles is between 1 and 999 nm.These are
new carriers for drugs84 or diagnostic products.85 The methods of preparation,drug loading,
drug release, and surface modification methods have already been reviewed.86
Furthermore, the use of bioadhesive hydrogel polymers increases the length of time
for which the nanoparticles are in contact with the respiratory mucosa, preventing the det-
rimental action of mucociliary clearance.87 In this field, Dunn87 described a new method
allowing the inhalation delivery of large macromolecules. Following Dunn’s invention, by
using cyclodextrins, sensitive molecules can be protected during the granulations of nano-
particles production phase.
The size, structure (Fıg. 19), characteristics (nanosphere recovery, drug content, drug
