European Journal of Pharmacology 533 (2006) 341 – 350

www.elsevier.com/locate/ejphar

Review
Nanomedicine for respiratory diseases
Ulrich Pison a,⁎, Tobias Welte b , Michael Giersig c , David A. Groneberg b,d
a
Department of Anesthesiology and Intensive Care Medicine, Charité Universitätsmedizin Berlin, D-13353 Berlin, Germany
b
Department of Respiratory Medicine, Hannover School of Medicine, Carl-Neuberg-Str. 1, 30625 Hannover, Germany
c
Center of Advanced European Studies and Research (Stiftung caesar), Ludwig-Erhard-Allee 2, D-53175 Bonn, Germany
d
Otto-Heubner-Centre, Pneumology and Immunology, Charité Universitätsmedizin Berlin, D-13353 Berlin, Germany
Accepted 13 December 2005
Available online 24 January 2006

Abstract

Nanotechnology provides new materials in the nanometer range with many potential applications in clinical medicine and research. Due to
their unique size-dependent properties nanomaterial such as nanoparticles offer the possibility to develop both new therapeutic and diagnostic
tools. Thus, applied nanotechnology to medical problems – nanomedicine – can offer new concepts that are reviewed.
The ability to incorporate drugs into nanosystems displays a new paradigm in pharmacotherapy that could be used for cell-targeted drug
delivery. Nontargeted nanosystems such as nanocarriers that are coated with polymers or albumin and solid lipid particles have been used as
transporter in vivo. However, nowadays drugs can be coupled to nanocarriers that are specific for cells and/or organs. Thus, drugs that are either
trapped within the carriers or deposited in subsurface oil layers could be specifically delivered to organs, tumors and cells. These strategies can be
used to concentrate drugs in selected target tissues thus minimizing systemic side effects and toxicity. In addition to these therapeutic options,
nanoparticle-based “molecular” imaging displays a field in which this new technology has set the stage for an evolutionary leap in diagnostic
imaging. Based on the recent progress in nanobiotechnology there is potential for nanoparticles and -systems to become useful tools as therapeutic
and diagnostic tools in the near future.
© 2006 Elsevier B.V. All rights reserved.

Keywords: Nanotechnology; Nanomaterial; Nanoparticle; Nanocarrier; Quantum dot; Respiratory medicine

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 342
2. Applied nanotechnology to medical problems — nanomedicine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 342
2.1. Nanodiagnostics and molecular imaging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 342
2.2. Targeted drug delivery and controlled release . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 343
2.3. Regenerative medicine. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 345
3. Respiratory diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 346
3.1. Obstructive lung diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 346
3.2. Genetic disorders affecting the airways . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 347
3.3. Infectious diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 348
3.3.1. Pulmonary tuberculosis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 348
3.3.2. Vaccination strategies. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 348
3.4. Cancer therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 349
4. Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 349

⁎ Corresponding author. Charité — Universitätsmedizin Berlin, Klinik für Anästhesiologie und operative Intensivmedizin, Standort Westend-KlinikumSpandauer
Damm 130, D-14050 Berlin, Germany. Tel.: +49 30 450 559024; fax: +49 30 450 559924.
E-mail address: [email protected] (U. Pison).

0014-2999/$ - see front matter © 2006 Elsevier B.V. All rights reserved.
doi:10.1016/j.ejphar.2005.12.068
342 U. Pison et al. / European Journal of Pharmacology 533 (2006) 341–350

Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 349
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 349

1. Introduction rescent biological labels (for example to trace a biological

molecule) which use both the small particle size and tunable
Respiratory diseases are increasing pathologic entities in the energy levels. Recent advances in chemistry have resulted in the
ageing population. The high expectations for better quality of preparation of monolayer-protected, high-quality, monodispersed,
life and the changing lifestyle of our society call for improved, crystalline quantum dots as small as 2 nm in diameter, which
more efficient and affordable health care. Although our under- can be conveniently treated and processed as a typical chemical
standing of the functioning of the human body at the molecular reagent.
and nanometer scale has improved tremendously, our diagnostic Artificial nanostructures, such as nanoparticles and nanode-
and therapeutic options for the effective treatment of severe and vices, being of the same size as biological entities, can readily
chronic diseases have increased only slowly over the past. interact with biomolecules on both the cell surface and within the
Diseases like cancer, diabetes, lung and cardiovascular prob- cell (see Fig. 1). Nanomedical developments range from quan-
lems, inflammatory and infectious diseases and neurological tum dots for molecular diagnostic and imaging (Akerman et al.,
disorders are serious challenges to be dealt with. Multidisci- 2002) to therapy using nanocarrier and integrated medical nano-
plinary approaches that bring together material and medical systems (Moghimi et al., 2005), which may perform complex
scientists will speed up the development of new diagnostic and repair actions at the cellular level inside the body in the future
therapeutic solutions. In this regard, applied nanotechnology to (Aldhous, 2001).
medical problems – nanomedicine – can offer new concepts For the purpose of this review, the concepts in nanomedicine
that are partially summarized in this review. We will also will be described using three interrelated themes: nanodiagnostics
extract published data regarding nanomedicine for respiratory associated with molecular imaging (Gambhir, 2002; Weissleder
diseases. and Mahmood, 2001), targeted drug delivery including controlled
release (Torchilin, 2005), and regenerative medicine (Nakashima
2. Applied nanotechnology to medical problems — et al., 2004).
nanomedicine
2.1. Nanodiagnostics and molecular imaging
Material at the nanometer scale exploits novel physical, chem-
ical and biological properties. Two principal factors cause the In nanodiagnostics, the ultimate goal is to identify disease at
properties of nanomaterials to differ significantly from other ma- the earliest stage possible, ideally at the molecular level. To
terials: increased relative surface area, and quantum effects. These achieve this goal, research and development activities in nano-
factors can change or enhance properties such as reactivity, medicine need to find new “smart” probes that signal pathologies
strength and electrical characteristics. As a particle decreases in at the level of a single cell, whether in vitro or in vivo (Czernin
size, a greater proportion of atoms are found at the surface com- and Phelps, 2002; Dayton and Ferrara, 2002; Hogemann and
pared to those inside. For example, a particle of size 30 nm has 5% Basilion, 2002; Kim, 2003; Mahmood and Josephson, 2005).
of its atoms on its surface, at 10 nm 20% of its atoms, and at 3 nm Nanotechnology offers the possibility for probes that are small
50% of its atoms. Thus nanoparticles have a much greater surface enough to cross physiological barriers, and to take different
area per unit mass compared with larger particles. As growth and measurements in parallel or to integrate several analytical steps
catalytic chemical reactions occur at surfaces, this means that a from sample preparation to detection into a single miniaturized
given mass of material in nanoparticulate form will be much more device. Such a device could contain enough hard wired in-
reactive than the same mass of material made up of larger par- telligence and robustness to be used by the patient and deliver a
ticles. These can affect the optical, electrical and magnetic be- multitude of data to the practitioner. Furthermore, the use of
havior of materials, particularly as the structure or particle size nanoelectronics will improve the sensitivity of sensors based on
approaches the smaller end of the nanoscale. Materials that exploit already established methods. Improvements of microscopic and
these effects include quantum dots, and quantum well lasers for spectroscopic techniques towards ultra-high spatial resolution,
optoelectronics. If semiconductor particles are made small enough, molecular resolution and ultra-high sensitivity will provide a
quantum effects come into play, which limit the energies at better understanding of the cell's complex “machinery” in
which electrons and holes (the absence of an electron) can exist basic research. The resulting progress should pave the way to
in the particles. As energy is related to wavelength (or color), this more innovative and powerful in vivo diagnostic tools. In
means that the optical properties of the particle can be finely general terms nanotechnology will have great impact on the
tuned depending on its size. Thus, particles can be made to emit methodologies available for both disease and drug discovery
or absorb specific wavelengths (colors) of light, merely by and consequently impact on the scope and throughput of phar-
controlling their size. Recently, quantum dots have found ap- maceutical developments (LaVan et al., 2002; Rudin and
plications in composites, solar cells (Gratzel cells) and fluo- Weissleder, 2003).
 U. Pison et al. / European Journal of Pharmacology 533 (2006) 341–350 343

Fig. 1. Nanotechnology could produce structures that have the same size than biological systems. Artificial nanostructures are given at the top, biological
nanostructures at the bottom of the figure. Nanostructured material from left to right: gold nanoparticles, 37 nm diameter, and nanorods 200 nm length; carbon
nanotubes, 200 nm diameter, with incorporated Ni particle of 8 nm; carbon nanotubes scaffolds. Biological structures from left to right: DNA; pulmonary surfactant
vesicles and liposomes; Gram negative bacteria transfected with gold nanoparticles via carbon nanotubes; fibroblast growing on scaffold made of carbon nanotubes.

Diagnostic concepts and improvements based on nanotech- forming implants. Such new drug carriers, drug conjugates and
nology with the perspective of molecular imaging are not per se drug-nanosystems can be engineered to control degradation, react
beneficial. If such concepts will be successful in the future, it to stimuli and be site-specific (Fig. 2). A successful drug carrier
will ultimately change our self-conception including the defi- system needs to demonstrate optimal drug loading and release
nition of health and disease. properties, long shelf-life and low toxicity. Colloidal systems,
such as micellar solutions, vesicle and liquid crystal dispersions,
2.2. Targeted drug delivery and controlled release as well as nanoparticle dispersions consisting of small particles of
10–400 nm diameter show great promise as carriers in drug de-
In addition to the potential improvements in the diagnostic livery systems. Already new nanoparticle formulations make use
field, nanotechnology offers advantages that allow a more tar- of the fact that an enlarged surface/volume ratio results in en-
geted drug delivery and a more controllable release of a thera- hanced activity. Nanoparticles are also useful as drug carriers for
peutic compound. The aim of targeted drug delivery and a the effective transport of poorly soluble therapeutics. When a
controlled release is to better manage drug pharmacokinetics, drug is suitably encapsulated, in nanoparticulate form, it can be
pharmacodynamics, non-specific toxicity, immunogenicity and delivered to the appropriate site, released in a controlled way and
biorecognition of systems in the quest for improved efficacy. protected from undergoing premature degradation. This results
Two aspects drive the progress of nanostructured drug devel- in higher efficacy and dramatically minimizes undesirable side
opment: drug formulation and route of administration. New drug effects. Such nanoparticulate delivery systems can be used to
formulation that takes advantage from material science and more effectively treat cancer and a wide range of other diseases,
nanotechnology give rise for new micro and nanoparticles, micro which call for drugs of high potency. Drug-delivering microchip
and nanocapsules, liposomes and micelles, dendrimers, liquid technology, resulting from the convergence of controlled release
crystals, hydrogels, molecularly imprinted polymers, conjuga- and fabrication technologies evolved for the electronics industry,
tions of biological molecules and synthetic polymers, and in situ is also benefiting from the application of nanotechnology.
344 U. Pison et al. / European Journal of Pharmacology 533 (2006) 341–350

Fig. 2. Drug carriers, drug conjugates and drug-nanosystems can be engineered to control degradation, react to stimuli and be site-specific.

Further miniaturization and the ability to store and release structural biology to provide new strategies and opportunities in
chemicals on demand offer new treatment options in the fight the design of novel drug delivery systems adapted to today's
against disease. A future vision is that nanoparticles will carry demands, has not been fully embraced. However, it is critical for
therapeutic payloads or genetic content into diseased cells, all nanoparticulates that drug safety is considered in parallel with
minimizing side effects as the nanoparticles will only become efficacy (Table 1).
active upon reaching their ultimate destination. They may even The route of administration is as important as the drug itself
check for overdosage before becoming active, thus preventing for a therapeutic success. Nano-based approaches to drug
drug-related poisoning. In the past 3 decades, the number and delivery are focused on crossing a particular physical barrier,
variety of controlled release systems for drug delivery applica- such as the blood–brain barrier; or on finding alternative and
tions has increased dramatically. Many utilize polymers that acceptable routes for the delivery of a new generation of
have particular physical or chemical characteristics, such as protein-based drugs other than via the gastro-intestinal tract,
biodegradability, biocompatibility or responsiveness to pH or where degradation can occur. Material science and nanotech-
temperature changes and could be designed with various nology are thus the basis of innovative delivery techniques that
functions (Fig. 3). In spite of many successful examples, the offer great potential benefits to patients and new markets to
notion of combining polymer science with concepts from pharmaceutical and drug delivery companies.

Fig. 3. Nanocarrier could be designed with various functions. The inorganic iron core provide MRI imaging features, diblock copolymers could be selected to give a
hydrophilic shell and a polypeptide layer. The former could sense and react according to the environment (pH, temperature, ion content etc.); the latter provide binding
features for drugs.
 U. Pison et al. / European Journal of Pharmacology 533 (2006) 341–350 345

Table 1 pulmonary delivery of protein drugs is diminished by proteases

Potential applications of nanotechnology in respiratory medicine and macrophages in the lung, which reduce their overall bio-
Therapy Diagnostics Research availability, and by the barrier between capillary blood and
Drug delivery and Molecular Probing of DNA structure alveolar air.
controlled release imaging Targeting mechanisms can make advantage of both, drug
Gene delivery MRI contrast Detection of proteins formulation and route of administration, and can be either pas-
enhancement
sive or active. An example of passive targeting is the preferential
Regenerative medicine Bio detection of Pharmacokinetic studies
and tissue pathogens accumulation of chemotherapeutic agents in solid tumors as a
engineering result of the differences in the vascularization of the tumor tissue
Tumor destruction Fluorescent Separation and purification of compared with healthy tissue. Active targeting involves the
biological labels biological molecules and cells chemical ‘decorating’ of the surface of drug carriers with mole-
cules enabling them to be selectively attached to diseased cells
(Fig. 2). The controlled release of drugs is also important for
The peroral route is the most frequently used drug delivery therapeutic success. Controlled release can be sustained or pul-
route due to patient acceptability and convenience. However, an satile. Sustained (or continuous) release of a drug involves
increasing number of new effective therapeutics are protein and polymers that release the drug at a controlled rate, by diffusion
peptide-based, but they do not easily cross mucosal surfaces and out of the polymer or by degradation of the polymer over time.
biological membranes, and do not resist degradation mechan- Pulsatile release is often preferred, as it closely mimics the way
isms that exist in the gastrointestinal tract (e.g. acid-induced by which the body naturally produces hormones such as in-
hydrolysis in the stomach, enzymatic degradation throughout sulin. It is achieved by using drug-carrying polymers that
the gastrointestinal tract, bacterial fermentation in the colon). respond to specific stimuli (e.g. exposure to light, changes in
Nanocarrier and capsulation may protect against degradation pH or temperature).
through the gastrointestinal tract and may allow crossing mu- The long-term objective of drug delivery systems is the
cosal surfaces. Thus the peroral route may be used in the future ability to target selected cells and/or receptors within the body.
for administration of sensitive compounds if appropriate pro- At present, the development of new drug delivery techniques is
tection could be added to drugs through nanoconstructs. driven by the need on the one hand to more effectively target
Transdermal drug delivery avoids problems such as gastro- drugs to the site of disease, to increase patient acceptability and
intestinal irritation, metabolism, variations in delivery rates and reduce healthcare costs; and on the other hand, to identify novel
interference due to the presence of food. It is also suitable for ways to deliver new classes of pharmaceuticals that cannot be
unconscious patients. The technique is generally non-invasive, effectively delivered by conventional means. Nanotechnology is
well accepted by patients and can be used to provide local critical in reaching these goals.
delivery over several days. Limitations include slow penetration
rates, lack of dosage flexibility and/or precision, and a restriction 2.3. Regenerative medicine
to relatively low dosage drugs.
Parenteral routes of drug delivery using, e.g. intravenous, Besides molecular diagnostics and targeted drug delivery
intramuscular or subcutaneous administration are very important including their controlled release material science with nanotech-
but more invasive compared to peroral and transdermal route of nology will enable regenerative medicine (Aldhous, 2001; Zaret,
drug delivery. But nanoscaled drug carriers have the potential for 2004). The focus of regenerative medicine is to work with the
improving the delivery of drugs through nasal and sublingual body's own repair mechanisms to prevent and treat disabling
routes, both of which avoid first-pass metabolism. It has been chronic diseases such as allergies, obstructive lung disease, dia-
possible to deliver peptides and vaccines systemically using the betes, osteoarthritis, and degenerative disorders of the cardiovas-
nasal route through the association of active drug macromole- cular and central nervous system and to help victims of disabling
cules with nanoparticles (Vila et al., 2004). In addition, there is injuries. A cellular and molecular basis has been established for
the possibility of improving the ocular bioavailability of drugs if the development of innovative disease-modifying therapies for in
administered in a colloidal drug carrier. situ tissue regeneration and repair, requiring only minimally in-
Another important route of drug delivery is through the lungs. vasive surgery. Rather than targeting the symptoms or attempting
Pulmonary delivery using metered dose inhaler systems for to delay the progress of these diseases, future therapies will be
aerosols, powders or solutions may contain nanostructures such designed to rectify chronic conditions using the body's own
as liposomes, micelles, nanoparticles and dendrimers. Aerosol healing mechanisms. To name some examples: facilitating the
products for pulmonary delivery comprise more than 30% of the regeneration of healthy cartilage in an osteoathritic joint, re-
global drug delivery market. Research into lung delivery is driven establishing a physiological release profile in diabetic pancre-
by the potential for successful protein and peptide drug delivery atic islets, or promoting self-repair mechanisms in areas of the
by this route and by the promise of an effective delivery mecha- central nervous system and of the heart (Petit-Zeman, 2001).
nism for gene therapy (e.g. in the treatment of cystic fibrosis). Nanotechnology can play a pivotal role in the development of
Pulmonary drug delivery offers local targeting for the treatment of cost-effective therapies for in situ tissue regeneration. This
respiratory diseases and increasingly appears to be a viable option involves not only a deeper understanding of the basic biology
for the delivery of drugs systemically. However, the success of of tissue regeneration, but also identifying effective ways to
346 U. Pison et al. / European Journal of Pharmacology 533 (2006) 341–350

initiate and control the regenerative process. This ‘nanobiomi- asthma, airway inflammation is characterized in most cases by
metic’ strategy depends on three basic elements: intelligent an increased number of activated T-lymphocytes, particularly
biomaterials, bioactive signalling molecules, and cells (Correa- CD4+ Th2 cells, and sometimes eosinophils and mast cells. The
Duarte et al., 2004). By ‘tailoring’ resorbable polymers at the most notable difference of chronic severe asthma compared
molecular level for specific cellular responses, nanotechnology with mild to moderate asthma is an increased number of
can assist in the development of biomimetic, intelligent bio- neutrophils. In stable COPD, airway inflammation is character-
materials. These biomaterials are designed to react positively to ized by an increased number of T-lymphocytes, particularly CD8+
changes in the immediate environment, stimulating specific T cells, macrophages and neutrophils. With the progression of the
regenerative events at the molecular level, directing cell pro- disease severity macrophage and neutrophil numbers increase.
liferation, cell differentiation, and extracellular matrix produc- Although there may be a partial overlap between asthma and
tion and organization. The sequential signaling of bioactive COPD in some patients, the differences in functional, structural
molecules, which triggers regenerative events at the cellular and pharmacological features clearly demonstrate the consensus
level, is necessary for the fabrication and repair of tissues. that asthma and COPD are different diseases along all their stages
Nano-assisted technologies should enable the sequential deliv- of severity. Since the major pathway of administering anti asthma
ery of proteins, peptides and genes to mimic nature's signaling and anti COPD drug is the aerosolic route, major advantages may
cascade. As a result, bioactive materials are produced, which be drawn out of nanocarrier systems.
release signaling molecules at controlled rates that in turn For bronchial asthma, experimental studies have already
activate the cells in contact with the stimuli. been conducted to assess the use of such nanosystems. Nano-
Finally, a major focus of ongoing and future efforts in re- particle technology was applied to discover a potent nanopar-
generative medicine will be to effectively exploit the enormous ticle P-selectin antagonist with strong anti-inflammatory effects
self-repair potential that has been observed in adult stem cells in a murine model allergic asthma (John et al., 2003). The
(Nakashima et al., 2004; Pomerantz and Blau, 2004; Reya et al., background of the study was to assess the role of P-selectin for
2001). In this regard, nano-assisted technologies will aid in the development and progression of peribronchial inflammation
achieving three main objectives — to identify signaling systems, in allergic airway disease. Since selective P-selectin inhibitors
in order to leverage the self-healing potential of endogenous may lead to an attenuation of the ongoing inflammatory pro-
adult stem cells; to develop efficient targeting systems for stem cesses present in allergic bronchial asthma, a panel of novel
cell therapies, and to monitor single cells after transplantation. P-selectin inhibitors were synthesized using polyvalent poly-
Of huge impact would also be the ability to implant cell-free, mer nanoparticles (John et al., 2003). First, a construct that
intelligent bioactive materials that would effectively provide binds efficiently to P-selectin was generated by assembling a
signaling to stimulate the self-healing potential of the patient's particle with the ligands acting as mimetics of the binding
own stem cells. elements that mediate the adhesion of P-selectin to its ligand P-
selectin glycoprotein ligand-1 (PSGL-1). Then, an in vitro
3. Respiratory diseases assay was used to evaluate the different inhibitors by
examining the interactions between P-selectin-coated capillary
Applied nanotechnology to medical problems – nanomedi- tubes and circulating cells. It was shown that they preferentially
cine – provides new concepts for diagnoses and therapies that bind to selectins expressed on activated endothelial cells (John
could be described using the three interrelated themes molecular et al., 2003). After these in vitro experiments, in vivo studies
imaging, targeted drug delivery including controlled release, were conducted using a murine model of allergic asthma and a
and regenerative medicine. Because nanocarrier systems may significant reduction of allergen-induced peribronchial inflam-
be administered to the airways easily, a number of respiratory mation airway and airway hyperreactivity present. (John et al.,
diseases may be approached using nanoparticles: obstructive 2003) indicating the validity of the new compounds.
lung diseases, genetic disorders affecting the airways, infectious A further study used chitosan/interferon (IFN)-γ pDNA nano-
diseases including tuberculosis, and cancer. particles. These particles were established to analyze the quantity
of in situ IFN-γ production. The reason for this approach is that
3.1. Obstructive lung diseases adenovirus-mediated IFN-γ gene transfer reduces airway hyper-
responsiveness in mice but is limited so far by the frequency of
The obstructive airway diseases chronic obstructive pulmo- gene delivery required. The nanocarriers were given to oval-
nary disease (COPD) and bronchial asthma represent one of the bumin-sensitized mice to assess their efficacy to modulate oval-
major global causes of disability and death. In this respect, bumin-induced inflammation and airway hyperresponsiveness
COPD is estimated to become the third most common cause of (Kumar et al., 2003). It was shown that the mice treated intranasal
death by 2020. The structural and pathophysiologic findings in with 25 μg of chitosan-IFN-γ nanoparticles (i.n.) had a lower
both diseases appear to be easily differentiated in the extremes airway hyperresponsiveness to methacholine challenge. They
of clinical presentation. However, a significant overlap may also had less histopathological signs of airway inflammation.
exist in individual patients regarding features such as airway The nanoparticles also led to an increased production of IFN-γ
wall thickening on computer tomography or reversibility and while Th2-cytokines, interleukin (IL)-4, IL-5, and ovalbumin-
airway hyperresponsiveness in lung function tests. Airway specific serum IgE were reduced in comparison to the control
inflammation differs between the two diseases. In bronchial group (Kumar et al., 2003). It was also demonstrated that the
 U. Pison et al. / European Journal of Pharmacology 533 (2006) 341–350 347

nanosystems inhibited the epithelial inflammation within 6 h of The major component of airway mucus is made up of large,
delivery which was paralleled by an induction of apoptosis of oligomeric, gel-forming mucin glycoproteins with molar masses
goblet cells. On the molecular level, the treatment with nano- ranging between 10 and 40 million Da (Davies et al., 1996; Gupta
carriers involved signal transducer and activator of transcription and Jentoft, 1992; Thornton et al., 1990; Thornton et al., 1991). A
4 (STAT4) signaling because STAT4-deficient mice did exhibit number of different mucin proteins which are products of different
reduced airway hyperresponsiveness and inflammation (Kumar genes have been identified in the respiratory tract so far. The
et al., 2003). Together, these studies using chitosan/interferon mucins are produced primarily by 2 different airway cell types:
(IFN)-γ pDNA nanoparticles demonstrated that nanoparticles goblet cells and glandular cells. While MUC2 and MUC5AC
can be coupled to bioactive molecules that target inflammatory expression has been localised to cells of the surface epithelium
events in allergic airway inflammation and that the intranasal (Chung et al., 2004; Davies et al., 1996; Groneberg et al., 2002; Li
administration of these nanocarriers can effectively reduce esta- et al., 1997; Wickstrom et al., 1998) (Chung et al., 2004), MUC5B
blished allergen-induced experimental asthma. and MUC7 mucins are expressed primarily in glandular cells
A further study reported the use of nanoparticle technology (Groneberg et al., 2003; Sharma et al., 1998; Wickstrom et al.,
in the discovery of a potent nanoparticle P-selectin antagonist 1998) but can also be induced in epithelial cells under states of
with anti-inflammatory effects in allergic airway disease (John inflammation (Groneberg et al., 2002). Out of the different res-
et al., 2003). The study background the role of P-selectin as a piratory mucins, MUC5AC and MUC5B have been identified as
crucial factor in the development and progression of peribron- major gel-forming mucins whereas MUC2 contibutes only to a
chial inflammation in allergic asthma. Since selective inhibitors lesser extent to the matrix (Davies et al., 1996).
of P-selectin may attenuate inflammatory processes associated In view of the high levels of CFTR expression in glandular
with allergic airway disease, novel P-selectin inhibitors were cells which play an important role in the composition of mucus
generated by the use of polyvalent polymer nanoparticles (John hydration and their attributed pathophysiological importance in
et al., 2003). A construct binding efficiently to P-selectin was the progression of cystic fibrosis (Engelhardt et al., 1993), nu-
generated by assembling a particle that presented an array of merous studies have focussed on the identification of differences
groups, with the ligands acting as mimetics of the binding between cystic fibrosis and normal airway secretions (Boat and
elements that mediate the adhesion of P-selectin to its ligand Cheng, 1980; Roussel et al., 1975; Zhang et al., 1995). Alter-
PSGL-1. Using an in vitro assay, the inhibitors were evaluated ations such as increased sulfation and fucosylation and de-
by examining the interactions between P-selectin-coated capil- creased sialytion of secreted mucins have been demonstrated.
lary tubes and circulating cells and it was found that they pre- Biochemical studies also indicated a higher heterogeneity of
ferentially bind to selectins expressed on activated endothelial cystic fibrosis mucin as compared to the normal mucus com-
cells (John et al., 2003). Further in vivo experiments in a murine position (Chace et al., 1985). However, a molecular treatment
model of asthma were then conducted that demonstrated a sig- option targeting mucin gene expression has not been established
nificant reduction of allergen-induced peribronchial inflamma- so far and only one study has led to promising results using a
tion airway and airway hyperreactivity (John et al., 2003). myristoylated, alanine-rich C-kinase substrate (MARCKS)
In contrast to these two studies focusing on the use of nano- protein (Singer et al., 2004). Since not only mucus hypersecre-
carriers in bronchial asthma, no studies have so far been con- tion but also the deficient channel protein CFTR may be targeted
ducted to assess the potential use of nanosystems for the treatment by nanosystems, cystic fibrosis seems to be an ideal candidate
of COPD. However, with new compounds being developed cur- for the therapeutic use of such systems.
rently, experiments using this new technology in COPD will be In a recent study, a gelatine and DNA nanoparticle coacervate
conducted soon using animal models of COPD (Chung et al., containing chloroquine and calcium has been developed as a
2004). gene delivery vehicle. In this vehicle the cell ligand transferring
is covalently bound to gelatine (Truong-Le et al., 1999). The
3.2. Genetic disorders affecting the airways coacervation conditions which resulted in the formation of
distinct nanoparticles were studied and it was demonstrated that
Cystic fibrosis, a frequent inherited, autosomal recessive dis- the nanosystems formed within a narrow range of DNA con-
order, is caused by a dysfunction of the epithelial chloride channel centrations and achieved incorporation of more than 98% of the
CFTR (cystic fibrosis transmembrane regulator) (Rosenstein and DNA in the reaction (Truong-Le et al., 1999). It was further
Zeitlin, 1998). So far, more than 500 mutations of the CFTR gene studied whether the crosslinking of gelatine to stabilize the
are known that are associated with cystic fibrosis (Stern, 1997). particles affects electrophoretic mobility of the DNA but no
Apart from gastrointestinal manifestations such as pancreatic in- effect was found. The DNA in the nanosystems was also shown
sufficiency, the major cause of morbidity results from airway dis- to be partially resistant to digestion with concentrations of
ease (Rosenstein and Zeitlin, 1998). The hypersecretory-induced DNase I that result in extensive degradation of free DNA but is
airway changes in cystic fibrosis are characterized by submucosal completely degraded by high concentrations of DNase. An op-
gland and goblet cell hyper- and metaplasia, leading to mucus timum cell transfection by nanosphere DNA required the pre-
overproduction and distortion of the muco-ciliary clearance. As a sence of calcium and nanospheres containing transferrin. The
result, airway plugging by mucus leads to chronic inflammatory biological integrity of the nanosphere DNA was demonstrated
changes and bacterial colonization (Groneberg et al., 2002; with a model system utilizing DNA encoding the cystic fibrosis
Ramsey, 1996). transport regulator (CFTR). In transfection studies of cultured
348 U. Pison et al. / European Journal of Pharmacology 533 (2006) 341–350

human tracheal epithelial cells (9HTEo) with nanospheres con- studies, the effects of oral administration of nanoparticle-encap-
taining the plasmid, a CFTR expression in over 50% of the cells sulated drugs to Mycobacterium tuberculosis-infected mice every
was found (Truong-Le et al., 1999). It was also demonstrated 10th day was studied. No tubercle bacilli were detected after 5 oral
that human bronchial epithelial cells (IB-3-1) defective in doses (day 50 of treatment). This finding suggests that nano-
CFTR-mediated chloride transport regained an effective trans- particle-based antituberculous therapy may be beneficial since it
port activity when transfected with nanospheres containing the may lead to a reduction in dosing frequency (Pandey et al., 2003).
CFTR transgene (Truong-Le et al., 1999). Therefore, nanosys- Next to this study on oral administration, also the aerosolic
tems might be an attractive candidate to deliver new therapeutic bioavailability of antitubercular drugs was examined (Pandey et al.,
compounds in cystic fribrosis. 2003) using a formulation of rifampicin, isoniazid and pyrazina-
mid encapsulated in poly (DL-lactide-co-glycolide) nanoparticles.
3.3. Infectious diseases These nanosystems are suitable for nebulization and are prepared
by the use of the multiple emulsion technique.
Bacterial and viral infections play a crucial role in the pro- Different drugs were examined for pharmacokinetic char-
gression of chronic lung diseases such as COPD or CF and lead acteristics and chemotherapeutic potentials after nebulization in
via a progressive destruction of lung tissue to respiratory failure a model of M. tuberculosis infected guinea pigs. It was shown
(Koch and Hoiby, 1993). Infections of the lower respiratory tract that the aerosolized particles exhibit a mass median aerodynamic
invariably occur in patients with CF and despite major ad- diameter of 1.88 ± 0.11 μm that is known to be favorable for a
vantages in antibiotic drug therapy, pneumonia remains a major bronchoalveolar deposition (Groneberg et al., 2003). A single
cause of morbidity and mortality (Rosenstein and Zeitlin, 1998). nebulization to guinea pigs was found to lead to sustained thera-
Since the local concentration of antimicrobial agents in the lung peutic levels in the lungs for up to 11 days and in the plasma for
is the most important factor for a successful eradication of 6–8 days. Nanoparticle-encapsulation also led to a significantly
bacteria, the alveolar and bronchial epithelium is a site with prolonged mean residence time and elimination half-life of the
major opportunity for drug delivery and therapy (Chung et al., different drugs when compared to the orally administered con-
2004; Ramsey, 1996). Direct delivery of antibiotics to the lower ventional drugs formulations. A comparison to the oral route of
airways by aerosol administration has potential advantages: administration resulted in an enhanced relative bioavailability for
deposition to the alveolar site of the infection can reach high the aerosolically delivered nanoparticle-encapsulated prepara-
local concentration (Honeybourne, 1994) and therefore inhaled tions (rifampicin 12.7-fold, pyrazinamide 14.7-fold, and iso-
drugs can reduce the occurrence of serious systemic adverse niazid 32.8-fold). In addition to these beneficial features, the
effects by dose reduction. Given the central role of antibiotic and absolute bioavailability in comparison to intravenous admin-
antiviral treatment in numerous respiratory diseases and the mul- istration was increased (6.5-fold for rifampicin, 13.4-fold for
titude of clinical studies concerning sytemical and topical anti- pyrazinamide, and 19.1-fold for isoniazid).
biotic therapy (Bressolle et al., 1992; Groneberg et al., 2003; Nebulization of the nanoparticle-encoated drug in the M.
Hodson et al., 1981; Nolan et al., 1982), the use of nanosystems to tuberculosis infection model on every 10th day led to an absence
deliver such drugs is of particular interest and a number of studies of M. tuberculosis in the lungs of the infected animals after five
have already addressed this issue. treatment doses. By contrast, 46 daily doses of orally administered
drug were needed to obtain similar therapeutic benefits. Together
3.3.1. Pulmonary tuberculosis these studies demonstrated that an aerosolic administration of
Next to the experimental studies addressing its role for the nanoparticle-encoated antitubercular drugs can be used experi-
treatment of allergic asthma nanoparticle technology has also mentally to improve pharmacological management of pulmonary
been evaluated for its potential use in antimicrobial therapy. One tuberculosis.
of the first studies addressed the issue of nanoparticle-encap-
sulated antitubercular drugs as potential oral drug delivery sys- 3.3.2. Vaccination strategies
tems against murine tuberculosis (TB) (Pandey et al., 2003). One Nanocarrier systems may also be used to stimulate long lasting
of the major problems with long-duration TB chemotherapy is and protective immune responses to respiratory viral infections. A
patient non-compliance. A reduction in the frequency of dosing number of studies already assessed this issue experimentally, i.e.
using nanoparticle-encapsulated compounds might therefore lead by using polylactic acid nanoparticles coated with a hydrophilic
to a significant improvement in the therapy. To assess this issue, polyethyleneglycol coating and tetanus toxoid (Vila et al., 2004)
poly (DL-lactide-co-glycolide) (PLG) nanoparticle-encapsulated or chitosan-DNA nanospheres containing a cocktail of plasmid
formulations of the three frontline antitubercular drugs rifampicin, DNAs encoding respiratory syncytial virus (RSV) antigens (Kumar
isoniazid and pyrazinamide were studied in mice. The drug en- et al., 2002). In mice, a significant reduction of viral antigen load
capsulation efficiencies were reported to be 66.3% ± 5.8% for and viral titers after an acute RSV infection were found. Treatment
isoniazid, 68% ± 5.6% for pyrazinamide, and 56.9% ± 2.7% for also resulted in the induction of RSV-specific nasal IgA antibodies,
rifampicin. After single oral administrations of the nanoparticle cytotoxic T lymphocytes, IgG antibodies, and interferon-gamma
formulations the preparations were found in the circulation for production suggesting a potential use of these nanocarriers in the
over 9 days (isoniazid and pyrazinamide) and 6 days (rifampicin) development of new RSV vaccination strategies (Kumar et al.,
while the therapeutic concentrations in the tissues were main- 2002). Nanoparticles may also be used to induce immunity to
tained for 9 to 11 days (Pandey et al., 2003). In further infection parainfluenza virus infections. Proteins of the bovine parainfluenza
 U. Pison et al. / European Journal of Pharmacology 533 (2006) 341–350 349

type 3 virus (PI-3) were incorporated into the two nanoparticles ceftazidime in patients with nosocomial pneumonia: pharmacokinetics and
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Kumar, M., Behera, A.K., Lockey, R.F., Zhang, J., Bhullar, G., De La Cruz, C.P.,
The financial support of the European Fund for Regional Chen, L.C., Leong, K.W., Huang, S.K., Mohapatra, S.S., 2002. Intranasal
Development (EFRE, 2000–2006 2ü/1, “Nanocarrier”) and the gene transfer by chitosan-DNA nanospheres protects BALB/c mice against
DFG (PI 165/8 and 10, GR2014/2-1, SFB 587 B13) is gratefully acute respiratory syncytial virus infection. Hum. Gene Ther. 13, 1415–1425.
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Mohapatra, S.S., 2003. Chitosan IFN-gamma-pDNA nanoparticle (CIN)
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