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Journal of Pharmacy
Review
And Pharmacology

Engineered PLGA nano- and micro-carriers for pulmonary

delivery: challenges and promises
Francesca Ungaro, Ivana d’ Angelo, Agnese Miro, Maria I. La Rotonda and Fabiana Quaglia
Department of Pharmaceutical and Toxicological Chemistry, University of Naples Federico II, Via D. Montesano, Naples, Italy

Keywords
microparticle; nanoparticle;
poly(lactic-co-glycolic acid); pulmonary delivery
Correspondence
Francesca Ungaro, Department of
Pharmaceutical and Toxicological Chemistry,
University of Naples Federico II, Via D.
Montesano 49, 80131 Naples, Italy.
E-mail: ungaro@unina.it
Received October 15, 2011
Accepted January 23, 2012
doi: 10.1111/j.2042-7158.2012.01486.x
Abstract
Objectives The aim of this review is to summarize the current state-of-the-art in
poly(lactic-co-glycolic acid) (PLGA) carriers for inhalation. It presents the rational
of use, the potential and the recent advances in developing PLGA microparticles and
nanoparticles for pulmonary delivery. The most promising particle engineering
strategies are discussed, highlighting the advantages along with the major challenges
for researchers working in this field.
Key findings Biodegradable polymer carriers, such as PLGA particles, may permit
effective protection and long-term delivery of the inhaled drug and, when
adequately engineered, its efficient transport to the target. The carrier can be
designed for inhalation on the basis of several strategies through the adequate com-
bination of available particle technologies and excipients. In so doing, the properties
of PLGA particles can be finely tuned at micro-size and nano-size level to fulfill spe-
cific therapeutic needs. This means not only to realize optimal in vitro/in vivo lung
deposition of the formulation, which is still crucial, but also to control the fate of the
drug in the lung after particle landing.
Summary Although many challenges still exist, PLGA carriers may be highly ben-
eficial and present a new scenario for patients suffering from chronic lung diseases
and for pharmaceutical companies working to develop novel inhaled products.

Introduction
Drug inhalation has a long and rich history that can be traced
back to the writings of many ancient civilizations, already
employing the pulmonary route of administration to deliver
natural remedies either locally or to the body through the
lung. If in the past inhaling ‘vapours’ (Egyptians in 1500) or
smoking cigarettes (Potter’s cigarettes in 1802) was sufficient,
with the rapidly growing popularity and sophistication of
therapies, there is an increasing demand for tailor-made
inhalable drug formulations able to yield the best therapeutic
outcomes via the most efficient delivery to the lungs.[1]
It is well established that key features of the formulation
(e.g., aerodynamic size, flow and aerosolization properties)
will affect the likelihood of it being deposited in the desired
region of the lung.[2] In the case of inhaled drugs required
for the treatment of lung diseases, such as asthma, chronic
obstructive pulmonary disease (COPD) and cystic fibrosis
(CF), an effective ‘local’ delivery (i.e. centrally rather than at
the periphery) of the drug is necessary.[3,4] Strategies for tar-
geting drugs to the alveoli have become increasingly impor-
tant, as many inhaled drugs are developed either to be
absorbed systemically (e.g. peptides and proteins)[5] or
to exert their pharmacological effect in this region of
the lung (e.g. antitubercular drugs directed to alveolar
macrophages).[6]
Although particle ‘landing’ at the site of interest remains
crucial in determining the therapeutic efficacy of inhaled
therapeutics, lung retention and the ability to overcome
extracellular and cellular lung barriers is just as significant.[7]
Mucociliary clearance, designed by evolution to eliminate
inhaled and possibly noxious material from the airways, may
considerably limit the residence time of the deposited formu-
lation and, subsequently, the amount of drug reaching the
target. Analogously, the nature and extent of the interactions
of the drug with lung lining fluid (LLF), airway macrophages
and lung epithelial cells will inevitably affect drug perma-
nence in situ and duration of the effect.
To overcome these drawbacks, controlled and targeted
delivery of drugs to the lungs by means of polymeric drug

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Royal Pharmaceutical Society 2012 Journal of Pharmacy and Pharmacology, 64, pp. 1217–1235 1217
PLGA carriers for inhalation Francesca Ungaro et al.

30 potential in the inhalation field. After a brief description of

Research papers (No. identified in PubMed)

the rationale behind the choice of polymer carriers for drug

inhalation, it describes the recent advances in developing
25
PLGA microparticles and nanoparticles for pulmonary deliv-
ery. The most promising particle engineering strategies are
20 discussed, highlighting the advantages along with the major
challenges for researchers working in this field.
15
Why a polymer carrier for inhalation?
10 Nowadays, drugs are administered via the pulmonary route
for two main purposes: (i) local therapies; (ii) systemic
5 absorption. It is currently believed that drug delivery into the
human lung represents the best way of treating pulmonary
diseases.[2] Inhalation allows direct delivery of the drug to the
0
site of action, with fewer systemic effects than oral therapy.
97
98
99
01
02
03
04
05
06
07
08
09
10
11 Meanwhile, the lungs may be used as a portal of entry to the
19
19
19
20
20
20
20
20
20
20
20
20
20
20

Year
Figure 1 Research studies on poly(lactic-co-glycolic acid) carriers for
pulmonary delivery identified in PubMed databases from 1997 to 2011.
delivery systems (DDS) is under investigation.[1,8] As a matter
of fact, DDS may offer substantial advantages over conven-
tional dosage forms for inhalation, including long-term
maintenance of drug concentrations within a desired thera-
peutic range, reduced doses, reduced dosing frequency, the
potential for limited side effects and, last but not least,
improved patient adherence to the therapy. This would repre-
sent a real benefit for those patients suffering from chronic
lung diseases.
Despite encouraging premises, the development of safe
and effective polymeric DDS for inhalation faces many chal-
lenges, among which the choice of the right polymer excipi-
ent[9] and its adequate engineering in the form of inhalable
particles are primary.[10] In the 1990s, Edwards and colleagues
developed a new type of inhalation aerosol based on
poly(lactic-co-glycolic acid) (PLGA), characterized by par-
ticles of small mass density and large size, which permitted
efficient and prolonged delivery of insulin into the systemic
circulation.[11] This was the first attempt to apply PLGA-based
carriers to lung delivery. After almost 10 years, we are experi-
encing the revival of the research interest in biodegradable
particles for drug inhalation (Figure 1). Nonetheless, it is
apparent from the current ‘manuscript boom’ that it is
growing and presents a future challenge.
Excellent reviews have recently summarized the new
concept of carrier-mediated lung targeting,[12,13] and the most
interesting techniques to engineer particles for inhala-
tion.[10,14,15] Nonetheless, these works only mention biode-
gradable PLGA particles among a huge range of technological
strategies. This review focuses on PLGA carriers and their
body, allowing systemic delivery of drugs via the airway sur-
faces into the bloodstream.[5] This is an attractive prospect
owing to the large surface area of the alveolar region, thick-
ness of the epithelial barrier, extensive vascularization,
relatively low proteolytic activity in the alveolar space and
absence of first-pass metabolism.
As can be seen in Table 1, locally acting drugs for inhalation
are mainly bronchodilators, corticosteroids and antibiotics,
which can reverse constriction of bronchial airways and
control airway inflammation/infection. Inhaled products
administered for their systemic effects mainly include fast-
onset analgesics, peptides and proteins, which would other-
wise need to be given by injection. In both cases, to yield the
best therapeutic outcomes, the drug has to effectively deposit
along the airways, remain in situ as long as possible, overcome
extracellular and cellular airway barriers and, when needed,
reach intracellular targets. This is the case of the emerging oli-
gonucleotide therapies for chronic respiratory diseases, able
to reach cell targets underlying disease pathogenesis.[18]
Indeed, formulating biopharmaceuticals for inhalation pre-
sents new challenges to scientists. The structural complexity
of proteins and nucleic acids compared with conventional
drugs demands an efficient drug-delivery system that allows
the intact macromolecule to gain access to the target site at
the right time and for proper duration.[38]
Lung deposition results from the complex interplay of a
range of factors, which relate to the size of the particles, the
inhalation device, the mode of inhalation or breathing
pattern and even to the lung anatomy of the individual.[2,39,40]
In particular, the aerodynamic particle size, defined as the
diameter of a sphere of unit density characterized by the same
settling velocity in air as the particle aerosol being measured,
will critically influence the deposition of the inhaled formu-
lation in the upper airways, rather than the conducting and
the alveolated airways of the lungs.[2,40,41] It is well recognized
that particles below 5 mm can be distributed deep into the

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Francesca Ungaro et al. PLGA carriers for inhalation

Table 1 Drugs marketed or undergoing preclinical/clinical studies by the pulmonary route

Current
Therapeutic development
application Drug class Drug Inhalation device* status**
[16,17]
Local delivery Asthma Short-acting b2 agonist Salbutamol (albuterol) Nebulizer, pMDI, DPI Marketed
Fenoterol pMDI
Pirbuterol Nebulizer, pMDI
Long-acting b2 agonist[16,17] Salmeterol pMDI, DPI Marketed
Formeterol DPI
Anticholinergic Ipratropium bromide Nebulizer, pMDI Marketed
compounds[16,17] Tiotropium bromide pMDI, DPI
Inhaled corticosteroids[16,17] Beclomethasone dipropionate Nebulizer, pMDI Marketed
Budesonide Nebulizer, DPI
Fluticasone propionate pMDI, DPI
COPD
Antisense EPI-2010 Nebulizer Terminated
oligonucleotides [18,19] AIR-645 Phase II
PXSTPI-1100 Preclinical
ATL-1102 Preclinical
CpG oligonucleotides [18,19] AVE-7279 Nebulizer Terminated
QAX-935 (IMO-2134) Phase I
siRNA[18,19] Excellair Nebulizer Phase II
Cystic fibrosis Antibiotics[20–23] Tobramycin Nebulizer, DPI Marketed
Aztreonam lisine Nebulizer Marketed
Colistimethate sodium Nebulizer Pilot trials
DPI Preclinical
Liposomal ciprofloxacin (ARD-3100) Nebulizer Phase II
Levofloxacin (Aeroquin, Nebulizer Phase III
formerly MP-376)
Mucolytics/mucous Dornase alfa Nebulizer Marketed
mobilizers[20,24] Lancovutide (Moli1901) Phase II
Antiproteases[25–27] a1-antitrypsin (AAT) Nebulizer Phase II
Respiratory Distress Pulmonary surfactant[28] Phospholipids/surfactant proteins Endo-tracheal tube Marketed
Syndrome
Systemic delivery Analgesia Opioids[29,30] Fentanyl (Staccato) Nebulizer Phase I
Liposomal fentanyl (AeroLEF) Phase II
Diabetes Peptides[31–37] Insulin (Affrezza) DPI Phase II
Glucagon-like peptide (MKC253) Phase I
Osteoporosis Calcitonin DPI Preclinical
Parathyroid hormone

*DPI, dry powder inhaler; pMDI, pressurized Metered Dose Inhaler. **More details on clinical studies can be found at http://clinicaltrials.gov/.

smaller airways and this penetration correlates well with a
good clinical response to local treatment. Differently, a par-
ticle fraction with an aerodynamic diameter in the 1–2 mm
range is probably the most efficient for deposition into the
capillary-rich alveolar airspaces, the target for the systemic
delivery of drugs. Finally, submicron particles can be exhaled,
if they are not aggregated and/or if insufficient time is avail-
able for their migration to the lung walls.
A number of inhalation devices are available on the market
to effectively deliver inhaled drugs to the lungs, such as nebu-
lizers, pressurized metered dose inhalers (pMDIs) and dry
powder inhalers (DPIs).[42] Since they have superior handling,
metering and reliability as compared with nebulizers, pMDIs
are the most commonly used inhalers to treat asthma and
COPD.[43] Nonetheless, pMDIs require the use of liquid drug
formulations (mainly suspensions) in polluting propel-
lants (chlorofluorocarbon and hydrofluoroalkane are gases
causing ozone depletion and greenhouse effects) and delivery
efficiency strongly depends on the actuation-breath coordi-
nation of the user.[44] DPIs, on the other hand, are breath-
actuated and do not cause any coordination problem and,
thus, have become accepted increasingly by both patients and
formulators.[45,46] In particular, DPIs are currently regarded as
the device of choice for macromolecule delivery to the lungs,
due to their potential to overcome solubility, bioavailability
and stability issues encountered by nebulizers and pMDIs.[14]
From a technological point of view, the handling, process-
ing and inhalation of excipient-free dried drug particles
are challenging. Novel strategies have been developed and
investigated for drug inhalation via DPIs, including

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Royal Pharmaceutical Society 2012 Journal of Pharmacy and Pharmacology, 64, pp. 1217–1235 1219
PLGA carriers for inhalation
Table 2 Main advantages of polymer carriers in pulmonary delivery
Microcarriers Protection of the entrapped drug
Sustained drug release
Reduction of daily drug doses (i.e. side effects)
Combined therapy
Dry formulation
Macrophage escape (>10 mm)
Nanocarriers Protection of the entrapped drug
Sustained drug release
Reduction of daily drug doses (i.e. side effects)
Combined therapy
Efficient transport through lung lining fluid
Cell targeting (e.g. macrophages)
Mucosal vaccination
microparticles.[8,9] Advanced products include Pulmo-
Sphere™ particles, engineered tobramycin-containing
hollow porous microparticles made of dipalmitoyl-
phosphatidylcholine (DPPC), the principal component of
endogenous lung surfactants.[23] Technosphere™ Insulin
(MannKind Corp., Valencia, USA), based on the intermo-
lecular self-assembly of fumaryl diketopiperazine (FDKP),
forming a three-dimensional highly porous sphere, is also
facing the market.[31,33] Nonetheless, these systems may suffer
from the short duration of their therapeutic effect (i.e. fre-
quent administrations). In the same way, lipid-based vesicles
(e.g. liposomes, lipoplexes, solid lipid nanoparticles), the
most extensively investigated carriers for controlled delivery
to the lung, do not allow sustained drug release and are
mainly delivered by nebulization.[8,9]
In this scenario, biodegradable polymer carriers have
become an increasingly attractive option for inhalation
therapies (Table 2).[12,15,47] Although calling into question
safety issues, which are far to be fully settled, the use of
polymer carriers may allow the reduction of daily drug doses,
and this could contribute to better control of local chronic
infections while diminishing the rate of drug appearance in
the bloodstream (i.e. nonspecific distribution to non-target
tissues) and subsequent side effects. The carrier can also offer
the potential to combine different drugs[48] or a drug with
helper excipients (e.g. absorption promoters, mucolytic
agents).[49] This can be very important in the case of complex
therapeutic regimens, such as those currently required to
treat or, better, to eradicate chronic pulmonary diseases. Fur-
thermore, polymer particles can play a crucial role in improv-
ing the therapeutic index of macromolecular drugs by: (i)
increasing the stability of the drug both ‘in the bottle’ and
in vivo; (ii) increasing the amount of drug that reaches the site
of action; (iii) prolonging the residence time of the drug
in situ (i.e. sustained pharmacological effect).[50,51]
Another important issue that can be potentially addressed
by polymer carrier-based systems intended for inhalation is
overcoming extracellular and cellular barriers imposed by the
lungs (Figure 2).[7]
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Francesca Ungaro et al.
Although controversial, literature data highlight that LLFs,
especially lung mucus, along with resident macrophages, rep-
resent a critical barrier to carrier-based therapies. By virtue of
its peculiar size and surface properties, a polymer carrier may
allow easy diffusion of a drug through the LLF,[52,53] its prefer-
ential uptake from lung epithelial cells or improved escape
from locally circulating macrophages.[11,54] It has been dem-
onstrated that the potential for access of particles to airway
mucus can be tuned at size level.[53] While large nanoparticles
with a mean size around 560 nm were almost completely
blocked by a 220 mm-thick CF sputum layer, small nanopar-
ticles were retarded only by a factor of 1.3 as compared with
buffer.[53] It is worth noting that particle size plays a crucial
role also in determining its preferential uptake by resident
macrophages, which can rapidly engulf the foreign particles
as a defence mechanism. A size of 1–2 mm for inhaled par-
ticles is ideal for macrophages to phagocytose (cell diameters
~15–22 mm), with smaller (below 200 nm) size being taken
up less efficiently.[5] In contrast, large particles with geometric
diameters of 10–20 mm, able to penetrate deep into lungs and
avoid macrophage engulfment, are considered ideal for sys-
temic delivery of protein drugs.[11] Nonetheless, the reduction
of the particle size to a nanometric range has been suggested
to further enhance the potential of biodegradable polymer
carriers for mucosal delivery of vaccines.[55]
Beside particle dimensions, the role played by the surface
chemistry of the carrier in its interactions with, and adhe-
sion to, lung cellular and extracellular barriers should also be
considered.[52] It has now become clear that slight differences
in surface properties may have significant implications in the
cellular uptake and the interactions of the carrier with the
biological environment. Particular attention must be paid to
imparting a charge or adhesiveness to the particle. When
modified on the surface with carboxyl groups, particles as
small as 59 nm may be completely immobilized by human
mucus.[56] On the other hand, large (200–500 nm) non-
adhesive nanoparticles achieved by covalent modification of
polystyrene particles with polyethylenglycol (PEG) have
been demonstrated to rapidly penetrate human mucus.[57]
More recently, hydrophilic poloxamer-coated biodegradable
nanoparticles, displaying a negative charge, have been dem-
onstrated to more easily diffuse across the mucus barrier
leading to a higher intracellular accumulation as compared
with positively charged nanoparticles modified with chito-
san.[58] Thus, a thorough understanding of carrier interac-
tions with LLFs is a condition sine qua non to achieve
particles engineered to cross mucus barriers and, thus, for
lung delivery.
Assuming the carrier successfully overcomes extracellular
barriers, the drug has still to interact with the target cells of
the respiratory tract to be effective. The interaction of the
carrier with cells is critical in many applications such as
mucosal vaccination or novel oligodeoxynucleotide (ODN)-
© 2012 The Authors. JPP © 2012
Francesca Ungaro et al. PLGA carriers for inhalation

Cellular barriers Extracellular barriers

Trachea

Mucus

Luminal mucus layer

Bronchi Periciliary layer

Bronchioles Alveolar lining fluid

Pulmonary surfactant
Alveoli
Aqueous subphase

Figure 2 Schematic representation of cellular and extra-cellular barriers imposed by the lung to inhaled drugs. The trachea and bronchi epithelia are
made up of basal, ciliated, brush and goblet cells (~60 mm-height). The same cells are present in bronchiolar epithelium but are not as tall (~10 mm). In the
alveolar region, type-I and secretor type-II cells build up the epithelium. The conducting airways are lined with airway surface liquid, a mucus gel-aqueous
sol complex composed by the periciliary and the luminal mucus layers, which get thinner from the level of the trachea (up to 100 mm) to the bronchioles
(~3 mm). The alveoli are lined with alveolar subphase fluid and pulmonary surfactant, which is composed of approximately 80–85% phospholipids,
5–10%proteins and 5–10% other lipids. All the lung surfaces are supervised by resident mononuclear phagocytes (i.e. airway macrophages). They
are highly represented in the alveolar lining fluid (~80% of the bronchoalveolar lavage cellular components) and so are often referred to as alveolar
macrophages.[5,30,31]

based therapies. These applications require a firm control
over particle–cell interactions, which are mainly dictated by
size and surface properties of the delivery system. The induc-
tion of humoral immune responses at the mucosal lymphoid
tissue (MALT) of the respiratory tract requires the transport
of particulate antigens by M-cells and their delivery to the
sub-mucosa, where dendritic cells and macrophages process
and present the antigen to naïve T-cells in the adjacent
mucosal lymph nodes. Similarly, at the alveoli, particulate
antigens are taken up and processed by circulating antigen-
presenting cells. Thus, when designing new inhalable
vaccines, surface-modified polymer carriers, especially nano-
particles, may represent a paramount tool not only to protect
the protein-based antigen, but also to tune its interaction
with immune cells.[59,60] The challenges of nucleic acid deliv-
ery via the pulmonary route are possibly greater, since the
drug has to cross the cellular membrane and gain access into
the cytoplasm/nuclei, where the final targets are located.[19,61]
Therefore, the main function of the carrier, along with mac-
romolecule protection against enzymatic attack, is to facili-
tate ODN cellular uptake, promote its endosomal escape and
release it at the final intracellular target. This can be achieved
by biodegradable polymer carriers.[52,62–64]
Poly(lactic-co-glycolic acid) carriers
in drug delivery and their potential
for inhalation
Among synthetic biodegradable polymers, thermoplastic ali-
phatic poly(ester)s, PLGAs, have generated tremendous
research interest due to their excellent biocompatibility as
well as the possibility of tailoring their biodegradability by
varying composition (lactide/glycolide ratio), molecular
weight and chemical structure (i.e. capped and uncapped
end-groups).[51,65] PLGAs characterized by very different in
vivo life-times, ranging from three weeks to over a year, are
available and approved for human use. Drug encapsulation
within PLGA copolymers is regarded as a powerful means to
achieve its sustained release for long time-frames and, in the
case of labile drugs, effectively protect the molecule from in
vivo degradation occurring at the administration site. Thus,
injectable PLGA microparticles (< 250 mm – ideally
< 125 mm) with different formulation design have been
developed for the delivery of small drugs as well as protein
therapeutics and nucleic acids,[50,51] resulting in several mar-
keted products. On the other hand, PLGA nanoparticles
(< 1 mm) represent a well-established tool to achieve

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Table 3 Examples of poly(lactic-co-glycolic acid) (PLGA)-based carriers tested for pulmonary delivery

Type Drug Formulation Key in vitro/in vivo findings

[67,68]
PLGA Conventional Insulin Microparticles/ Prolonged hypoglycaemic effect in vivo
microparticles microparticles mannitol blends Minimal signs of lung toxicity
Interleukin-2[69,70] Microparticles/ Preservation of in vitro biological activity (T-cells)
mannitol blends
Large porous particles Anthocyanin[71] Dry powders Prolonged inhibition of A549 cells death induced by
cigarette smoke
Persistence in mouse lung (20 days)
No signs of lung inflammation (10 days)
Deslorelin[72] Dry powders High drug concentrations in plasma up to 7 days
Heparin[73] Dry powders Increased plasma half-life of the drug
No cytotoxicity (Calu-3)
Insulin[49] Dry powders Preservation of protein structural integrity
In vivo alveolar deposition
Hypoglycaemic activity at low doses in diabetic rats
Prolonged hypoglycaemic effect in diabetic rats
Prostaglandin E1[74,75] Dry powders Increased stability in rat lung homogenates
Increased plasma half-life of the drug
No cytotoxicity (Calu-3)
No in vivo acute toxicity
Hollow porous particles Palmytil-acylated- Dry powders In vivo deposition in the central lung
exendin-4[76] Prolonged hypoglycaemic effect in diabetic rats
No signs of lung inflammation (2 weeks)
PLGA Nanoparticle dispersions Combined Dispersion for Sustained plasma therapeutic drug levels
nanoparticles antitubercular nebulizer Increased plasma half-life
therapy [77] Enhanced drug bioavailability
Hepatitis B vaccine[78] Dispersion for Enhanced mucosal and humoral responses to the vaccine
nebulizer Efficient uptake of nanoparticles by rat alveolar
macrophages
No cytotoxicity (Calu-3)
Insulin[79] Dispersion for Prolonged hypoglycaemic effect in diabetic
nebulizer
Trojan particles (porous Hepatitis B surface Dry powders High mucosal immune response in the lungs
nanoparticle-aggregate antigen[80]
particles) Rifampicin[81] Dry powders Sustained plasma therapeutic drug levels
Drug persistence in lung tissue and cells
Nano-embedded siRNA[64] Dry powders Preservation of siRNA structural integrity
microparticles Efficient in vitro gene silencing
No cytotoxicity (H1299 cells)
TAS-103 Dry powders Enhanced cytotoxicity against A549 cells
anticancer drug[82] High local drug concentration
Tobramycin[83] Dry powders In vitro antimicrobial activity against P. aeruginosa
Nanoparticle composition affects in vivo deposition

targeted drug or gene delivery, with special emphasis on
cancer treatment.[66]
In the attempt to discover novel strategies for the pro-
longed release of drugs in the lung, PLGA nanoparticles and
microparticles have been studied also as carriers for inhala-
tion. As summarized in Table 3, studies with PLGA micropar-
ticles for inhalation have been carried out mostly with the
intention of developing inhalable formulations of proteins
and other macromolecules.[49,67–70,72–74,84] Currently, protein
encapsulation within PLGA copolymers is considered a
very useful strategy to entrap and deliver the native macro-
molecule in a sustained manner.[50,51,85]
Among the techniques developed for the production
of PLGA microparticles, solvent evaporation/extraction
methods are the most widely employed for the microencap-
sulation of therapeutic macromolecules.[51] The quality of the
final product is related to the preservation of protein integrity
along manufacturing and in vivo application.[86,87] Excellent
reviews have summarized all the possible causes of protein
degradation during the microencapsulation process high-
lighting the strategies efficient in preventing the phenom-
enon.[88,89] As a function of the critical steps affecting the
quality of the final product, it is useful to single out a class of
excipients and select, on the basis of the literature reports,

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Francesca Ungaro et al.
those more suitable to the specific design.[65] For inhalable
microparticles, the microencapsulation technique and the
selected excipients should also contribute to the correct aero-
solization properties of the particles. The technological
paradox is that respirable particles have to be small for depo-
sition, but also large enough to allow metering during manu-
facturing and delivery of the dosage form.
Thus, microparticle porosity, which is generally considered
a microsphere defect to be overcome,[90] may oddly become a
desired feature for inhalation.[91,92] Conversely, conventional
microparticles may require the use of a inert carrier (e.g.
mannitol) to become respirable.[69,70]
Beside macromolecule inhalation, the potential of PLGA
particles as carriers for lung delivery of antibiotics has been
also deeply investigated (Table 3).[77,81,93–97] Here, the primary
goal is to increase the local concentration of the antibiotic
within the macrophages, the host cells for some bacteria, such
as Mycobacterium tuberculosis. Although some successful
attempts have been made to deliver antibiotics to the lungs
via PLGA microparticles, nanoparticles seem to better fit
the purpose. When encapsulated into PLGA nanoparticles,
the elimination half-life and mean residence time of the
entrapped antibiotics are significantly prolonged as com-
pared with the orally-administered parent drugs, resulting in
an enhanced drug bioavailability.[77]
Despite the advantage of reduced doses and dosing fre-
quency, the first-developed PLGA nanoparticle-based ‘liquid’
formulations pose several challenges to practical implementa-
tion. Long delivery times, low delivery efficiencies, stability
of sustained-release formulations in aqueous solution and
access to clean water in developing countries are only a few
of the challenges highlighted by researchers in this field.
Novel formulation approaches for antibiotic delivery have
involved the creation of micron-scale dry powders based on
PLGA nanoparticles.[81,83,97] In fact, dry powders based on self-
assembled rifampicin-loaded PLGA nanoparticles (‘porous
nanoparticle-aggregate particles’ or PNAPs) were devel-
oped.[81] Alternatively, mannitol microparticles containing
rifampicin-loaded PLGA nanoparticles for inhalation therapy
of tuberculosis were prepared in one step using a four-fluid
nozzle spray drier.[97] These technologies have demonstrated a
great potential also in the development of dry powders for
protein delivery, with particular regard to mucosal vaccina-
tion,[80,98] and short interference RNA (siRNA) delivery.[64]
Although current literature data suggest the potential of
PLGA particles for prolonged drug delivery in the lung, their
use for inhalation is still embryonic and presents some short-
comings. In particular, a huge amount of work has still to be
produced on clearance mechanisms and persistence in vivo
that may considerably limit the benefit of sustained-release
inhalation therapy. The persistence of PLGA particles in vivo
has been only indirectly demonstrated by some authors, who
showed the presence of the released drug in the lung for up to
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Royal Pharmaceutical Society 2012 Journal of Pharmacy and Pharmacology, 64, pp. 1217–1235
PLGA carriers for inhalation
20 days.[71] If this feature is desirable to prolong the therapeu-
tic effect, its influence on the safety of respirable PLGA
particles has not been elucidated yet. Anyway, in vitro cyto-
toxicity studies have shown that native PLGA has no manifest
toxicity against healthy lung macrophages,[99] CF bronchial
cells[100] or lung carcinoma cell lines.[64] Furthermore, the pro-
inflammatory potential of PLGA nanoparticles has been
recently investigated in vivo and results suggest that biode-
gradable nanoparticles are safer than non-biodegradable
polystyrene particles[101] and do not induce lung tissue dam-
age,[102] also up to 10 days.[71] Nonetheless, chronic toxicologi-
cal data on PLGA particles are still poor and demand for
further studies.
As polyesters, PLGAs undergo hydrolysis in vivo, forming
biologically compatible moieties that should be cleared from
the lung. With respect to biodegradation, among all commer-
cially available PLGA types, those characterized by a rapid in
vitro degradation are undoubtedly preferred for inhalation.
The creation of synthetic PLGA derivatives more hydrophilic
than native PLGAs has been attempted and novel poly(vinyl
alcohol) (PVA)-based branched polyesters with PLGA side
chains, characterized by faster degradation rates compared
with linear PLGA of similar molecular weight, have been
employed for pulmonary delivery [62,103,104].
Although few experimental studies have been performed
so far to fully explore the potential and drawbacks of PLGA in
pulmonary delivery, respirable dry powders based on PLGA
particles appear an enticing technological approach for inha-
lation. However, depositing drug doses reliably into the lungs
through the newest DPIs is straightforward in principle, but
challenging in reality. The peculiar properties of the drug par-
ticles, such as size, shape, density and surface properties, will
affect handling, processing, and inhalation of the formula-
tion and, therefore, its likelihood of being deposited in the
desired region of the lung [10,105]. In the following, a critical
account of the most promising production and formulation
techniques employed to engineer PLGA microparticles and
nanoparticles for inhalation is provided, highlighting the
goals already achieved and those to be gained.
Engineering poly(lactic-co-glycolic
acid) microparticles for inhalation
Respirable PLGA microparticles have been generated,
making use of several techniques, that feature partly compet-
ing, partly complementary characteristics. Great efforts have
been made to improve lung deposition of the particle and
overcoming intracellular and extracellular barriers imposed
by lungs. Besides the PLGA polymeric platform, excipients
are sometimes needed to optimize the final particle proper-
ties as a function of the preparation technique. The desirable
product characteristics firstly include the correct aerosoliza-
tion properties (i.e. low mass mean aerodynamic diameter
1223
PLGA carriers for inhalation
Conventional Large porous Hollow porous
microparticles particles microparticles
Figure 3 Typical architectures of respirable poly(lactic-co-glycolic acid)
microparticles.
(MMAD), high fine particle fraction (FPF) and emitted
dose). To this end, PLGA particles have been engineered in
very different ways, taking into account that the particles
should also comply with the requirements of drug stability
and prolonged release. (Figure 3)
Among the parameters that can be adjusted to optimize the
efficiency of dry powders for inhalation, particle mass density
and size have recently drawn researchers’ attention to limit
loss of drug owing to PLGA particle aggregation in the inhaler
and macrophage-mediated clearance of the particles from the
lungs.[5,10] The idea is that conventional PLGA particles
achieved by micronization (i.e. 1–3 mm) are both prone to
aggregation and can be rapidly phagocytosed in the deep lung
by alveolar macrophages. On the contrary, large porous par-
ticles (LPPs) with low mass density (< 0.4 g/cm3) and high
geometric diameter (> 5 mm) can be used to enhance both
particle aerosolization behaviour and residence time in the
lung.[106] Thus, working on size, shape and density of the par-
ticles, the first PLGA-based LPPs for insulin delivery were
achieved.[11] More recently, great efforts have been made to
achieve ‘regularly shaped’ porous particles by operating selec-
tively on size and density, thus achieving large porous ‘micro-
spheres’. In so doing, PLGA-based LPPs have been engineered
to sustain deslorelin delivery via the deep lungs,[72] allow effi-
cient pulmonary adsorption of low-molecular-weight hep-
arin[73] and increase insulin systemic availability.[49]
Large porous particles
From a technological standpoint, PLGA-based porous micro-
spheres for inhalation have been typically obtained by the
double emulsion (wi/o/we) technique in the presence of a
porogen (Table 4).
When microspheres are prepared by the wi/o/we technique,
the presence of osmotically active drugs and/or excipients in
the internal aqueous phase (wi), as well as their concentra-
tion, generates an osmotic pressure gradient which can
strongly affect particle porosity (Figure 4a).[65] Thus, LPPs
can be achieved adding osmogens in the internal aqueous
phase of the double emulsion, thus causing water influx from
the external to the internal aqueous phase during solvent
evaporation (i.e. particle hardening). Under the influence of
an osmotic gradient, the organic phase of the double emul-
sion acts as a semi-permeable membrane allowing the
1224
Francesca Ungaro et al.
passage of water across the organic phase. In particular, high
osmotic pressures in wi generate an influx of water from the
external aqueous phase (we) to wi. The occurrence of this
process during particle hardening results in the formation of
a typically porous structure.
In this sense, to control PLGA particle porosity and, conse-
quently, flow and aerosolization properties of the developed
dry powders, hydroxypropyl-b-cyclodextrin (HPbCD) has
been recently tested as aid-excipient in insulin-loaded PLGA
microparticles intended for pulmonary delivery.[49,91] The use
of HPbCD in LPP production is very intriguing not only in
the light of cyclodextrin’s osmotic properties but also for its
potential to enhance macromolecule adsorption through
respiratory epithelium. Several formulations, differing in
HPbCD and insulin loadings, were produced by the wi/o/we
technique and their properties compared.[91] Insulin and,
when added, HPbCD were solubilized in wi. The technologi-
cal results show that the combination of appropriate amounts
of insulin and HPbCD plays a crucial role in achieving PLGA/
HPbCD/insulin LPPs with the desired bulk and aerodynamic
properties (i.e. a highly porous structure, a very low density
(0.1 g/ml) and a theoretical MMAD (MMADt) lower than
10 mm).[91] The good aerosolization behaviour of the devel-
oped LPPs was confirmed by in vitro aerosolization tests,
showing that optimized PLGA/HPbCD/insulin particles had
an experimental MMAD (MMADexp) ranging from 4.01 to
7.00 and an FPF estimated to be 26.9–89.6% at the different
airflow rates tested (i.e. 30–90 l/min).[49] Confocal micro-
scopy studies, performed after in vivo administration of
labelled PLGA/HPbCD/insulin LPPs to the rat lung by means
of a low-scale DPI, suggest that particles reach the alveoli and
remain in situ after delivery. Finally, the therapeutic potential
of optimized PLGA/HPbCD/insulin LPPs was confirmed by
dose–response studies performed in both normoglycaemic
and streptozotocin-induced diabetic rats.[49] In vivo data show
that PLGA/HPbCD/insulin LPPs are able to reach the alveoli
and release insulin, which is absorbed in its bioactive form.
While insulin solutions administered via pulmonary route
are unable to cause a significant hypoglycaemic effect, insulin
delivered through PLGA/HPbCD/insulin LPPs at the same
doses (0.5–4.0 IU/kg) significantly reduces blood glucose
level as a function of the administered dose in both animal
models. The developed LPPs, tested in hyperglycaemic rats in
evident pathological conditions, exert a very significant and
longer hypoglycaemic effect even at insulin doses as low as
0.5 IU/kg (about 0.5 mg of PLGA/HPbCD/insulin LPP per
rat) as compared with an insulin solution. The duration of
the effect was consistent with previous results achieved by
Edwards and colleagues,[11] who showed falling glucose levels
for the first 10 h after inhalation of PLGA/insulin LPPs, fol-
lowed by relatively constant low glucose levels for the remain-
der of the 96-h period. It is worth noting that these effects
were observed for PLGA/insulin LPPs at much higher insulin
© 2012 The Authors. JPP © 2012
Royal Pharmaceutical Society 2012 Journal of Pharmacy and Pharmacology, 64, pp. 1217–1235
Francesca Ungaro et al. PLGA carriers for inhalation

Table 4 Preparation techniques employed to develop porous poly(lactic-co-glycolic acid) (PLGA) microparticles for inhalation

Preparation technique Porosigen agent Delivered drug

[72]
Supercritical fluids (CO2SC) Cyclodextrins Deslorelin
Precision particle Oils[107] Ciprofloxacin
Fabrication (PPF)*
Single emulsion (o/w) Poloxamer (Pluronic F68 and F127) [84,108] Human Growth Hormone
Palmytil-acylated-exendin-4
Double emulsion (wi/o/we) Cyclodextrins[49,76,91,109] Insulin
Bovine Serum Albumin
Palmytil-acylated-exendin-4
Sodium chloride[76] Palmytil-acylated-exendin-4
Poly(ethylenimine)[73,74] LMW Heparin
Prostaglandin E1
Ammonium bicarbonate[92,100,110,111] Budesonide
Decoy oligonucleotide
Doxorubicin
Lysozyme
Rhodamine-dextran
Poly(vinyl alcohol)[112] Hepatitis B surface antigen
None[113]** Capreomycin sulfate

*Patented technology that uses acoustic excitation and/or flow-limited field-injection electrostatic spraying to fabricate uniformly sized particles. **Par-
ticle porosity has been achieved by rapid evaporation of the organic solvent.

(a)
wi
Osmogen o we

Osmogen
Water we
wi o
influx

Osmogen

(b)
wi/o/we

w1

o
w2

Figure 4 Production of large porous particles by the double-emulsion technique. (a) Generation of an osmotic pressure gradient between wi and we.
(b) Addition of an effervescent salt (e.g. ammonium bicarbonate) in wi.

doses (about 9 mg of LPPs containing 20% of insulin by
weight were administered per rat) as compared with PLGA/
HPbCD/insulin LPPs.
HPbCD has been recently employed to produce also
albumin-coated hollow porous PLGA particles for pulmo-
nary delivery of palmityl-acylated exendin-4 (Pal-Ex4), a
potent glucagon-like peptide-1 agonist with great potential
for the treatment of diabetes.[76] The contemporary addition
of HPbCD and sodium chloride within wi, along with
controlled solvent evaporation, allowed the achievement of

© 2012 The Authors. JPP © 2012

Royal Pharmaceutical Society 2012 Journal of Pharmacy and Pharmacology, 64, pp. 1217–1235 1225
PLGA carriers for inhalation
hollow LPPs. Plain particles were further processed with
human serum albumin (HSA), which was conjugated with
the carboxylate groups of poly(ethylene-alt-maleic anhy-
dride), to achieve HSA-coated hollow LPPs based on PLGA.
The developed Pal-Ex4 loaded particles demonstrated great
potential for inhalation, in terms of drug loading, release rate,
in vitro/in vivo aerosolization efficiency and duration of the
hypoglycemic effects in vivo. On the basis of the results, the
authors ascribe several benefits to HSA coating: prevention of
particle aggregation, improvement of LPP aerosolization
properties and, last but not least, potential for tight binding
of the fatty-acid conjugated peptide, so as to control its release
in vivo.[76]
Despite encouraging results, severe limitation of the
osmogen-based technological approach is represented by the
poor control of drug encapsulation efficiency, ascribable to
mass exchanges and consequent drug loss occurring between
the two phases during particle hardening. This phenomenon,
as well as rapid drug release due to the macroporous structure
of the system, can be particularly dramatic in the case of
highly hydrophilic macromolecules, such as nucleic acids.[114]
Establishment of interactions of the drug with helper excipi-
ents may limit macromolecule loss. Along this line, an alter-
native osmotic agent that has been tested to produce LPPs is
polyethylenimine (PEI), a hydrophilic polycation that forms
complexes by electrostatic interactions with different anionic
macromolecules (e.g. nucleic acids, heparin).[115] A
concentration-dependent increase in particle porosity was
experienced after PEI addition in wi during preparation of
PLGA microparticles for the delivery of oligonucle-
otides.[114,116] The use of PEI as core-modifying agent has been
re-examined to achieve respirable PLGA LPPs of low-
molecular-weight heparin[73] and, more recently, prostaglan-
din E1 (PGE1).[74] Despite particle porosity, LPPs showed high
entrapment efficiency, ascribed to drug–polycation inter-
actions. Furthermore, in both cases, a remarkable extension
of the plasma half-life of the drug (from 6 h to 24 h) was
observed.
An alternative formulation strategy to achieve PLGA-
based LPPs for the prolonged release of hydrophilic macro-
molecules relies on the use of an effervescent agent, namely
ammonium bicarbonate, which decomposes into ammonia
and carbon dioxide during emulsification, forming a porous
matrix as the gas products escape (i.e. gas-foamed LPPs)[110]
(Figure 4b). Since pore formation depends on effervescence
rather than on diffusional mass exchanges between aqueous
phases, this technique allows efficient encapsulation of mac-
romolecules in highly porous PLGA particles.[92,100,110]
The new process was firstly applied by Yang et al. for the
encapsulation of two model molecules, lysozyme and doxo-
rubicin hydrochloride.[110] The addition of an appropriate
amount of ammonium bicarbonate was essential to achieve a
homogeneous population of highly porous particles with
1226 Royal Pharmaceutical Society 2012 Journal of Pharmacy and Pharmacology, 64, pp. 1217–1235
Francesca Ungaro et al.
optimal aerodynamic properties. Although the authors suc-
ceeded in improving drug encapsulation efficiency, poor
control over release properties was achieved without further
modification of formulation conditions. To further engineer
the system, lipid-engineered gas-foamed LPPs were recently
developed in our laboratory.[92] To control LPP release prop-
erties, two lipid aid excipients were tested. Our first choice
was DPPC, the major component of human lung surfactant,
which is gaining increasing research interest in the develop-
ment of respirable dry powders for a number of reasons,
including its biocompatibility.[9] As an alternative lipid
excipient, we investigated the potential of 1,2-dioleoyl-3-
trimethylammonium-propane (DOTAP), a cationic lipid
extensively studied as transfection agent for nucleic acids.[117]
The effect of the presence of DPPC or DOTAP upon the prop-
erties of gas-foamed LPPs containing a model hydrophilic
macromolecule, rhodamine B isothiocyanate–dextran
(Rhod-dex), was assessed. We found that in the case of hydro-
philic macromolecules unable to interact with PLGA end-
groups, such as Rhod-dex, excipient addition was essential
to increase the amount of drug entrapped within LPPs, being
as high as 80% only for DPPC-engineered or DOTAP-
engineered gas-foamed LPPs. The Rhod-dex release profile
from LPPs was also strongly affected by excipient addition in
the initial formulation, with lipid-engineered LPPs allowing
for a more prolonged release of Rhod-dex as compared with
excipient-free LPPs. Conceiving the developed LPPs for drug
inhalation, DPPC-engineered and DOTAP-engineered LPPs
displayed optimal FPF and MMADexp. In vivo deposition
studies performed after intratracheal administration of LPPs
in rats confirmed the ability of the developed dry powders
to deposit along bronchi and bronchioles. On the basis of
encouraging technological results, we are currently develop-
ing respirable DPPC-engineered gas-foamed LPPs for pro-
longed pulmonary delivery of a decoy oligodeoxynucleotide
to nuclear factor-kB (NF-kB) (dec-ODN) for the treatment
of chronic lung inflammation (e.g. CF).[100] Dec-ODN LPPs
represent the first attempt to delivery ODN-based therapeu-
tics to the lung in the form of dry powders. Dec-ODN was
effectively encapsulated (~80%) within biodegradable par-
ticles with controlled porosity, confirming that the use of
lipid excipients and gas foaming may result in efficient
microencapsulation of a highly hydrophilic macromolecule
(i.e. dec-ODN) within highly porous PLGA particles. Fur-
thermore, a sustained release of dec-ODN from LPPs was
achieved. Particle aserosol performance, as evaluated by
multi-stage liquid impinger, demonstrated that the devel-
oped dec-ODN LPPs have very good flow properties, with
more than 95% of the capsule content being emitted during
aerosolization. The MMADexp of < 6 mm, along with findings
of previous studies, strongly supports a preferential depo-
sition of dec-ODN LPPs in the distal airways.[63] In vitro
pharmacological studies performed in CF human bronchial
© 2012 The Authors. JPP © 2012
Francesca Ungaro et al.
o/w
o
w w
Figure 5 Production of large porous particles by the single-emulsion technique. Interconnected pores are generated by addition of extractable poro-
gens in the organic phase.
epithelial IB3-1 cells showed that lipopolysaccharide (LPS)
challenge caused an increase of NF-kB/DNA binding activity,
which was significantly inhibited by DPPC-engineered
dec-ODN LPPs at 24 and 72 h.[100] This inhibitory effect on
NF-kB/DNA binding activity was correlated with decreased
interleukin (IL)-6 and IL-8 secretion and mRNA levels.In con-
trast, naked dec-ODN exhibited these effects only at 24 h.
These results were supported by an enhanced structural stabil-
ity of dec-ODN in cultured cells due to entrapment in LPPs.[63]
PLGA-based LPPs have been also achieved by the single
emulsion technique (Table 1)[84,108]. Porous biodegradable
PLGA microparticles with interconnected pores have been
fabricated using poloxamers as extractable porogens
(Figure 5). Pores are generated by the time difference between
PLGA hardening and the extraction of the hydrophilic surfac-
tants from the organic phase using water. Nonetheless, the
drug should be loaded by adsorption onto LPPs. Thus, it is
very important to favour drug interactions with PLGA. This
is the case of the fatty acid-conjugated peptide Pal-Ex4, the
palmitic acid moieties of which were demonstrated to
improve Ex4 interactions with the hydrophobic PLGA and,
consequently, to extend its in vitro release (completed after
five days).[76] In turn, an extended therapeutic duration in vivo
was observed, though ascribable not only to sustained drug
delivery but also to the increased plasma half-life of Pal-Ex4
due to the induction of albumin binding, typical of the con-
jugated peptide itself.
Novel technologies, such as the supercritical fluid process
and the patented precision particle fabrication (PPF), have
been also tested for the achievement of PLGA-based LPPs
(Table 1). Supercritical CO2 processing was applied to
prepare deslorelin-loaded LPPs with the aim of reducing the
residual solvent content of the particles, retain protein integ-
rity and sustain its release in vivo.[72] The developed particles
effectively sustained the systemic delivery of deslorelin via
© 2012 The Authors. JPP © 2012
Royal Pharmaceutical Society 2012 Journal of Pharmacy and Pharmacology, 64, pp. 1217–1235
PLGA carriers for inhalation
o
the deep lungs for up to seven days as compared with plain
deslorelin (plasma concentrations sustained up to three days)
and conventional deslorelin PLGA particles (plasma concen-
trations 2-fold lower at day 7). More recently, the patented
PPF technology was used to create monodisperse PLGA-
based LPPs for pulmonary delivery of nano-precipitated
ciprofloxacin (NanoCipro dry powders) using oils as extract-
able porogens.[107] Notably, particle morphology strictly
depended upon the nature and concentration of the extract-
able oil – canola oil led to PLGA particles with a porous web-
like internal structure and with silicon oil hollow porous
particles were achieved. The main limitation of the technique
was the low ciprofloxacin encapsulation efficiency.
Despite the peculiar limitations, all the advanced particle-
engineering techniques, along with the traditional methods of
microencapsulation, have contributed to the increased possi-
bility of formulating LPPs for pulmonary delivery. Notably,
PLGA particles have been effectively engineered into LPPs
with aerosol and release properties suitable for the prolonged
delivery of drugs deep in the lungs. The challenge for research-
ers working in this field is that none of these techniques can
really provide the solution to all types of molecules.
Engineering poly(lactic-co-glycolic
acid) nanoparticles for inhalation
In the current era of nanotechnology,biodegradable nanopar-
ticles are gaining momentum as carriers for the inhalation
of drugs.[12,118] Indeed, nanoparticle delivery to the lungs is an
attractive concept because it can cause retention of the carrier
in the lungs (particles of a few hundred nanometers represent a
tenacious resident of the lungs) accompanied by a prolonged
drug release,meaning improved lung bioavailability over con-
ventional PLGA microparticles. Furthermore, nanoparticles
represent a promising tool to more simply penetrate airway
1227
PLGA carriers for inhalation Francesca Ungaro et al.

(a)

Nanoparticle on Nano-embedded
Trojan particles
coarse inert carrier microparticles

Figure 6 Inhalable poly(lactic-co-glycolic acid) nanoparticles.

Nanoparticle-based dry powders for inhalation can be achieved by:
adsorption on coarse inert carriers, nanoparticle self-assembling (Trojan
particles or porous nanoparticle-aggregate particles) or embedding
nanoparticles into an inert microparticle.

barriers, which can be better overcome at nanosize level.[52]

Finally, carriers of nanometric size offer potential for drug
×10000 2 μm 2.00 kV 5 mm
targeting to specific lung tissue and cell populations.[119]
From a technological point of view, the development of (b)
effective PLGA nanocarriers for inhalation first requires
adequate engineering of the particles at the nanosize level. On
this matter, PLGA nanoparticles with different features
(size, morphology, zeta-potential) may be achieved by con-
trolling the parameters specific to the production method
employed.[120,121] Since the potential of nanoparticles to inter-
act with lung tissue strongly depends on nanoparticle size and
surface charge,[52] this is an important aspect to be taken into
account for excipient selection and formulation design.
Nonetheless, another great challenge of using nanoparticles
for inhalation is the low inertia of dry powders with a mean
size lower than 1 mm, which are generally exhaled upon inha-
lation.[5,12] Thus, the most widely employed nanoparticle-
×30000 1 μm 2.00 kV 5 mm
based inhalation approach relies on the nebulization of
nanoparticulate aqueous dispersions.
Figure 7 Scanning electron micrographs of spray dried rifampicin
Different strategies to engineer the system at the microsize porous nanoparticle-aggregate particles (PNAPs) containing 80% nano-
level and achieve ‘micrometric’ nanoparticle-based dry particles by weight (PNAP80). A magnification of the surface of the PNAP
powders have recently been explored (Figure 6).[98,122] Among indicates a shell of aggregated nanoparticles with intact structure (scale
them, self-assembly of nanoparticles or their embedding bars represent: 2 mm (a), 1 mm (b)).[81]
within an inert ‘microcarrier’ have been extensively investi-
gated and, probably, represent the most promising techno-
rophage targeting) could be combined with the ease of flow,
logical approach.
processing and aerosolization potential of LPPs.
Formation of the large porous nanoparticle aggregates
The ‘Trojan’ approach
occurs via a spray-drying process that ensures the drying time
Since nanoparticle drying to develop dry powders for inhala- of the sprayed droplet (i.e. time required for a droplet to dry)
tion is very difficult to achieve, given that nanoparticles is sufficiently shorter than the characteristic time for redi-
aggregate excessively in the dry state, large porous carriers stribution of nanoparticles by diffusion within the drying
have been used to deliver nanoparticles deep in the lung (i.e. droplet (i.e. time required for nanoparticles to diffuse from
Trojan particles) in the form of dry powders.[123] As in Vir- the edge of the droplet to its centre). The proof of principle of
gilio’s epic poem, which tells of the Greek army entering and using Trojan particles for inhalation was given on polystyrene
destroying the city of Troy staying hidden in a horse, PNAPs nanoparticles. This approach has been found useful for
have been developed to act as carrier particles that release achieving hybrid DPPC and hyaluronic acid large porous
nanoparticles once delivered into the body, thereby acting as a carriers for PLGA nanoparticles releasing dexamethasone
‘Trojan’ delivery systems for nanoparticles (Figure 7).[80,81,123] acetate.[124] Scanning electron microscopy and confocal laser
In so doing, all the advantages of nanoparticles for inhalation scanning microscopy analysis showed that dexamethasone-
(e.g. long-term residence in situ, prolonged drug release, mac- loaded Trojan particles are spherical, hollow and possess an

© 2012 The Authors. JPP © 2012

1228 Royal Pharmaceutical Society 2012 Journal of Pharmacy and Pharmacology, 64, pp. 1217–1235
Francesca Ungaro et al.
irregular surface ascribable to nanoparticles, likely held
together by DPPC and hyaluronic acid.
More recently, rifampicin-loaded PLGA nanoparticles have
been dispersed throughout a matrix of an inert excipient,
l-leucine, a hydrophobic amino acid demonstrated to
improve powder dispersibility and aerosolization proper-
ties.[81] Rifampicin-loaded PNAPs possessed properties suit-
able for efficient deposition in the lungs. In vitro release
showed an initial burst of rifampicin, with the remainder
available for release beyond 8 h. Analogously, systemic levels
of rifampicin were detected for 6–8 h after delivery of PNAPs
to guinea-pigs by insufflation. A prolonged permanence of
rifampicin both in bronco-alveolar lavage (BAL) and lung
tissue was observed as compared with porous particles con-
taining free rifampicin.
A recent paper described dry powders intended for vaccine
delivery were based on PNAP technology.[80] In particular,
PLGA/PEG nanoparticles composed of a PLGA core and a
PEG hydrophilic shell, containing recombinant hepatitis B
surface antigen (rHBsAg), were prepared by the double emul-
sion method and subsequently spray-dried with l-leucine.
Again, hollow and low-density particles with a rough inner
surface, ascribed to intact PLGA/PEG nanoparticles, were
achieved. Various PNAP formulations containing rHBsAg
were administered to guinea-pigs by the pulmonary route
and the immune response elicited in the systemic circulation
and in the lungs assessed. The IgG titres were measured in the
serum for 24 weeks after the initial immunization; whereas
the titres of IgA, an indicator of mucosal immunity, were
measured in BAL. Results showed that rHBsAg PNAPs have
the advantage of eliciting a high mucosal immune response in
the lungs without traditional adjuvants. Nonetheless, no for-
mulation was able to elicit a systemic response comparable
with that of a control antigen adsorbed on alum administered
via the parenteral route.
Nano-embedded microparticles
Although direct drying of nanoparticle suspensions has dem-
onstrated some success in the creation of dry powders com-
posed of agglomerated PLGA nanoparticles, this technique
requires great attention to preserve nanoparticle integrity
upon drying and may not always offer the desired control over
their flow and aerosolization properties. An alternative early-
investigated approach consists of nanocomposite particles
obtained by inclusion of drug-loaded PLGA nanoparticles
within sugar micoparticles, used as inert carriers.[83,125–128]
The nanoparticle-containing micron-sized particles, also
termed nano-embedded microparticles (NEMs), are
designed to release primary nanoparticles after reaching
deep lung, upon the dissolution of the inert carrier in LLF
(Figure 8).
© 2012 The Authors. JPP © 2012
Royal Pharmaceutical Society 2012 Journal of Pharmacy and Pharmacology, 64, pp. 1217–1235
PLGA carriers for inhalation
2~10 μm
Lung lining fluid
Lung epithelium
Figure 8 Proposed mechanism of nanoparticle release from nano-
embedded microparticles into lung lining fluid.
Among the inert excipients available for NEM production,
lactose is the first choice and the most commonly used in
marketed DPIs. It has an established safety and stability
profile, may be processed by different manufacturing tech-
niques with tight controls over purity and physical proper-
ties, is easily available at different grades and is inexpensive.[9]
It is conceived that the fine particles of lactose reaching the
lungs are rapidly absorbed and metabolized by the intestinal
epithelium and are principally excreted in urine. Further-
more, in contrast to oral administration, lactose swallowed at
the levels present in inhaled preparations (up to 25 mg) is
unlikely to present problems even in patients with lactose
intolerance.[129]
Other safe inert excipients, such as mannitol and trehalose,
have been tested as potential carriers in naoparticle-based dry
powders for inhalation and have found their way into mar-
keted products for inhalation.[9] Research interest in mannitol
is increased by its ability to act as an airway rehydrating agent,
inducing water flux into the bronchial lumen.[130] Indeed, the
first attempts to produce NEMs for inhalation have been
made with mannitol particles loaded with insulin-loaded
PLGA nanoparticles by a spray-drying fluidized bed granula-
tor.[79] This resulted in soft matrix composite granules dis-
playing aerosolization properties superior to those of freeze-
dried nanoparticles. In vivo deposition studies showed that
more than 50% of the composite granules were able to reach
rat bronchioles and alveoli. As a result, a prolonged pharma-
cological effect lasting more than 12 h was achieved as
compared with insulin solutions administered either intrave-
nously or intratracheally.
Recently, inhalable composite particles made of sugars and
PLGA have been achieved by a classical spray-drying tech-
nique.[82,83,125,126] Trehalose and lactose were tested as inert car-
riers for rifampicin-loaded PLGA nanoparticles.[125,126]
Formulation studies were useful to assess the effect of the
preparation conditions – inlet temperature, size and weight
ratio of primary NPs – on the properties of NEMs. Notably,
1229
PLGA carriers for inhalation
the authors found that the inlet temperature of the spray-
drier was crucial to allow the decomposition of NEMs into
intact nanoparticles and its adjustment was necessary when
the size of the primary nanoparticles or the sugar carrier were
changed. Contrariwise, once achieved at optimum tempera-
tures, the ratio by weight of nanoparticles did not affect the
inhalation performance of NEMs. On the basis of encourag-
ing preliminary data, PLGA nanoparticles were loaded with
an anti-cancer drug, TAS-103, and spray-dried with threal-
ose.[82] The cytotoxicity of the developed NEMs against A549
cells was higher than that of the free drug. Furthermore, when
NEMs were administered to rats by inhalation, the cytotoxic
agent reached higher concentrations in the lung than in the
plasma or those achieved after intravenous administration of
free drug.
In a recent work, we tried to shed light on the role played by
nanoparticle composition on the interactions of NEMs with
the lung environment.[83] To this purpose, we designed and
developed a pulmonary delivery system for antibiotics, such
as tobramycin, based on NEMs consisting of PLGA nanopar-
ticles embedded in an inert microcarrier made of lactose. At
nanosize level, helper hydrophilic polymers were used to
impart the desired surface, bulk and release properties to
PLGA nanoparticles prepared by a modified emulsion-
solvent diffusion technique.[83] Results showed that PVA and
chitosan are essential to optimize the size and modulate the
surface properties of tobramycin-loaded PLGA nanopar-
ticles, whereas the use of alginate allows efficient tobramycin
entrapment within nanoparticles and its release up to one
month. Optimized formulations displayed good in vitro
antimicrobial activity against Pseudomonas aeruginosa plank-
tonic cells. Nonetheless, since nanoparticles will be embed-
ded in vivo in the lung mucus, which will prevent them from
reaching the target, selected nanoparticle formulations were
subjected to further in vitro studies to assess their ability to
interact with mucus environment. The rough determination
of nanoparticle–mucin interactions by the mucin-particle
method suggest that chitosan-engineered nanoparticles have
a higher tendency to interact with mucin as compared with
PVA-engineered ones. The strength of interaction was con-
firmed by the z-potential of the mucin–nanoparticle disper-
sion, indicating a strong adsorption of the polymer onto the
surface of chitosan-engineered nanoparticles. This phenom-
enon will likely affect nanoparticle diffusion through mucus.
Fluorescence-assisted transport studies performed on a arti-
ficial mucus model,[131] showed that nanoparticle transport
through mucus is facilitated when mucin shields the nano-
particle surface charge (i.e. chitosan-engineered nanopar-
ticles). Notably, in vivo biodistribution studies of NEMs
engineered by spray-drying showed that PVA-engineered
alginate/PLGA nanoparticles reached the deep lung, while
chitosan-engineered nanoparticles were found in great
amounts in the upper airways, lining lung epithelial surfaces.
1230 Royal Pharmaceutical Society 2012 Journal of Pharmacy and Pharmacology, 64, pp. 1217–1235
Francesca Ungaro et al.
Thus, PLGA nanoparticle composition appears to play a
crucial role in determining not only the technological fea-
tures of nanoparticles but also, once processed in the form of
NEMs, their in vitro/in vivo deposition pattern.
A crucial issue pertaining to NEMs is related to the drug-
to-powder ratio, which still needs to be improved to achieve
safe and effective NEMs for in vivo inhalation of antibiot-
ics.[83] Actually, the addition of an inert ingredient in elevated
ranges decreases the amount of PLGA nanoparticles and, in
turn, the dose of active drug administered. Nonetheless, this
is not a challenge for drugs that are therapeutically active at
very low doses, such as biotech molecules (i.e. proteins rather
than oligonucleotides). On this matter, it should be under-
lined that spray-drying has been demonstrated to be an excel-
lent technique also for engineering of siRNA-loaded PLGA
nanoparticles.[64,127] Mannitol NEMs with aerosol properties
suited for deep lung delivery have been engineered without
destroying the biological activity of sensitive molecules.
In the light of current literature data it can be concluded
that PNAPs and NEMs are very promising systems for sus-
tained delivery of conventional and biotech drugs in the
lungs. In perspective, they may represent important tools for
mucosal vaccination as well as for efficient treatment of local
lung diseases, which demand nanosized carriers.
Conclusions
Over the last 20 years, drug delivery to the respiratory tract
has become of choice for the treatment of asthma, COPD and
respiratory infections. The main objective is to confine the
therapeutic agent to the airways so as to maximize drug con-
centration at the site of action and, in so doing, maximize
drug efficacy. In addition, the lung is under investigation as a
portal of entry to the body, permitting delivery of drugs, espe-
cially peptides and proteins, via the airway surfaces into the
bloodstream. Literature review shows that advanced particle
engineering techniques have contributed to the increased
possibility of formulating nano-carriers and micro-carriers
for both local and systemic pulmonary delivery of conven-
tional drugs and biotech macromolecules, such as emerging
inhaled proteins and oligonucleotides.
Although carrier-free formulations for inhalation are
somewhat advisable due to their low toxicological profile,
biodegradable and biocompatible polymer particles may
permit efficient protection and long-term delivery of the
inhaled drug. On this matter, PLGA particles for inhalation
have been produced on the basis of numerous strategies that
use a combination of available particle technologies and
excipients to develop the desired dosage form. Particle prop-
erties can be finely tuned at micro-size and nano-size level to
fulfill specific therapeutic needs. The challenge to carrier
development is that none of the technological approaches
reported in the literature is universal and it is very difficult
© 2012 The Authors. JPP © 2012
Francesca Ungaro et al. PLGA carriers for inhalation

to choose a priori the one that best fits the purpose. In vitro/in
vivo studies represent a critical step before selection of the
best formulations as candidates for use in humans. Although
lung deposition studies are still crucial, there is an impelling
need for in vitro/in vivo models to investigate what happens
after the particle has landed. Special attention must be paid to
the evaluation of the safety of PLGA in the lung. In particular,
the shortage of chronic toxicological data on engineered
PLGA particles must be balanced with studies to definitively
assess the toxicological potential of such particles in the lung,
before their in vivo use.
In the light of these considerations, nano-carriers and
micro-carriers of PLGA seem an emerging formulation strat-
egy in pulmonary delivery. When adequately engineered,
PLGA particles may provide sustained drug delivery to the
lungs, extend duration of action, reduce the therapeutic dose
and reduce the adverse effects of highly toxic drugs, thus
improving patient compliance. Although many challenges
still exist, PLGA carriers represent a real benefit for both phar-
maceutical companies working to develop novel inhaled
products and patients suffering from chronic lung diseases.
Declarations
Conflict of interest
The Author(s) declare(s) that they have no conflicts of inter-
est to disclose.
Funding
The authors wish to thank Fondazione per la Ricerca sulla
Fibrosi Cistica-Onlus – Delegazione di Torino (FFC#5/2007)
and – Delegazione del Lago di Garda con i GdS dell’Isola Berga-
masca e di Chiasso (FFC#23/2011) for supporting their
research on the development of respirable PLGA carriers for
cystic fibrosis therapy.

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