Nebulizers effectiveness on pulmonary delivery of alpha-1 antitrypsin

Abstract

The nebulization of alpha-1 antitrypsin (AAT) for its administration to the lung could be an interesting alternative to parenteral infusion for patients suffering from AAT genetic deficiency (AATD). In the case of protein therapeutics, the effect of the nebulization mode and rate on protein conformation and activity must be carefully considered. In this paper two types of nebulizers, i.e., a jet and a mesh vibrating system, were used to nebulize a commercial preparation of AAT for infusion and compared. The aerosolization performance, in terms of mass distribution, respirable fraction, and drug delivery efficiency, as well as the activity and aggregation state of AAT upon in vitro nebulization were investigated. The two nebulizers demonstrated equivalent aerosolization performances, but the mesh nebulizer provided a higher efficiency in the delivery of the dose. The activity of the protein was acceptably preserved by both nebulizers and no aggregation or changes in its conformation were identified. This suggests that nebulization of AAT represents a suitable administration strategy ready to be translated to the clinical practice for delivering the protein directly to the lungs in AATD patients, either as a support therapy to parenteral administration or for subjects with a precocious diagnosis, to prevent the onset of pulmonary symptoms.
Graphical Abstract

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Drug Delivery and Translational Research
https://doi.org/10.1007/s13346-023-01346-3
ORIGINAL ARTICLE
Nebulizers effectiveness onpulmonary delivery ofalpha‑1 antitrypsin
AnnalisaBianchera1,2 · VivianaVilardo1· RobertaGiaccari1· AnnalisaMichielon1 · GianlucaBazzoli1 ·
FrancescaButtini1,2 · MarinaAiello3 · AlfredoChetta3· StefanoBruno1,2 · RuggeroBettini1,2
Accepted: 3 April 2023
© The Author(s) 2023
Abstract
The nebulization of alpha-1 antitrypsin (AAT) for its administration to the lung could be an interesting alternative to paren-
teral infusion for patients suffering from AAT genetic deficiency (AATD). In the case of protein therapeutics, the effect of
the nebulization mode and rate on protein conformation and activity must be carefully considered. In this paper two types
of nebulizers, i.e., a jet and a mesh vibrating system, were used to nebulize a commercial preparation of AAT for infusion
and compared. The aerosolization performance, in terms of mass distribution, respirable fraction, and drug delivery effi-
ciency, as well as the activity and aggregation state of AAT upon invitro nebulization were investigated. The two nebulizers
demonstrated equivalent aerosolization performances, but the mesh nebulizer provided a higher efficiency in the delivery
of the dose. The activity of the protein was acceptably preserved by both nebulizers and no aggregation or changes in its
conformation were identified. This suggests that nebulization of AAT represents a suitable administration strategy ready
to be translated to the clinical practice for delivering the protein directly to the lungs in AATD patients, either as a support
therapy to parenteral administration or for subjects with a precocious diagnosis, to prevent the onset of pulmonary symptoms.
Keywords Alpha-1 antitrypsin· Jet nebulizer· Vibrating mesh nebulizer· Protein drug delivery
Introduction
Alpha-1 antitrypsin deficiency (AATD) is a genetic disorder
that predisposes affected individuals to liver and lung dis-
ease. AATD originates from mutations of the SERPINA-1
gene, which encodes alpha-1 antitrypsin (AAT), a glyco-
sylated serpin mostly produced in the liver and one of the
main components of plasma [1]. The liver manifestations of
AATD mainly result from AAT misfolding, leading to the
formation of intracellular aggregates. In contrast, the lung-
related disease is associated with hypofunctional or non-
functional AAT variants that insufficiently inhibit neutrophil
elastase (NE), a serine protease that acts as an intracellular
and extracellular microbicidal agent. A higher NE activity
results in the proteolysis of pulmonary elastin, and, in the
long term, in pulmonary emphysema and bronchiectasis [1].
The current therapeutic approach to AATD treatment
consists of weekly slow intravenous administrations of
AAT purified from human plasma to restore the protective
plasma level of about 60mg/dL [2]. Infusions are costly
and require ambulatorial treatment and healthcare person-
nel. Moreover, despite the lungs being the organs mainly
affected by AATD, only 3% of intravenously administered
AAT is reported to reach the pulmonary epithelium [3].
Therefore, the local administration of AAT has been con-
sidered a potential alternative, if an appropriate amount of
active AAT reaches the lower respiratory tract in the active
form. This idea is not new in the field of AAT therapy: in
the past, AAT has been administered by nebulization with jet
nebulizers to healthy volunteers [4], cystic fibrosis patients
[5], and AATD patients [6].
The products for nebulization currently on the market for
chronic diseases are commonly prescribed with a recom-
mended nebulizer since the inhaled dose can vary consider-
ably depending on the nebulization system. In fact, inhala-
tion medicines are true drug/device combination products. In
the case of protein therapeutics, the effect of the nebulization
* Ruggero Bettini
ruggero.bettini@unipr.it
1 Food andDrug Department, University ofParma, Parco
Area Delle Scienze 27/a, Parma, Italy
2 Interdepartmental Center Biopharmanet-Tec, University
ofParma, Parco Area Delle Scienze Building 33, Parma,
Italy
3 Department ofMedicine andSurgery, University ofParma,
Via Gramsci 14, Parma, Italy

Drug Delivery and Translational Research
1 3
mode and rate on protein conformation and activity should
also be considered. Indeed, a protein therapeutic could dena-
ture—and therefore lose its activity—due to the interaction
with the air–liquid interface, oxidation, thermal degradation,
and mechanical stress [7]. These issues were addressed for
AAT by Flament etal. [8, 9], who focused on the effects of
formulation excipients and technological parameters on the
nebulization by jet and ultrasonic nebulization systems. In
these works, the addition of surfactants improved the respir-
able fraction of AAT, but the type of nebulizer and operating
conditions played a major role. The advent of vibrating mesh
nebulizers in the early 2000s—which eliminated the prob-
lem of solution recirculation—opened new possibilities for
the efficient nebulization of drugs, particularly biopharma-
ceuticals. However, during nebulization, heating of the drug
solution occurs, potentially affecting thermolabile proteins.
Wild-type AAT, in particular, was reported to denature at a
temperature of about 48°C [10]. Moreover, protein aggrega-
tion by interfacial degradation was reported with these types
of nebulizers [7, 11].
Research works and some clinical trials have described
the administration of AAT to patients by nebulization to
exploit its anti-inflammatory properties. Most of them
involved CF patients [12–15]. All results agreed on the
safety of this approach and provided evidence of its effi-
cacy in reducing lung inflammation. On the other hand,
when considering the inhibition of elastase activity in AAT
patients [16–18], a high peripheral deposition of AAT was
reported, but the clinical parameters did not provide con-
clusive evidence of elastase inhibition and improvement
in pulmonary function, as reviewed elsewhere [19]. In our
opinion, these results suffer from a limited consideration of
the biological activity and aggregation of the protein upon
nebulization, since the sole estimation of the dose of protein
reaching the deep lungs is not sufficient to guarantee the
desired therapeutic effect, particularly in consideration that
all nebulization systems expose protein solutions to poten-
tially damaging conditions.
To address these issues, in this paper we investigated
not only the drug delivery efficiency but also the activity
and aggregation state of AAT upon invitro nebulization
with two types of nebulizers, i.e., a jet and a mesh vibrat-
ing system as crucial element for the efficient translation of
liquid formulations into effective therapeutic medicines for
nebulization.
Materials andmethods
Materials
Prolastin® powder for infusion, containing 1000mg of
human plasma-purified AAT (Grifols, Barcelona, Spain,
Batch G45BE00351) was divided into aliquots and dissolved
in ultrapure water produced by an Arium® purification sys-
tem (Sartorius, Goettingen, Germany). A 25mM potas-
sium phosphate buffer solution was prepared by dissolv-
ing 2.336g of K2HPO4 and 1.577g of KH2PO4, both from
A.C.E.F. (Fiorenzuola, Italy), in 1 L of ultrapure water. The
Bradford assay kit was from Bio-Rad Laboratories (Milan,
Italy). Porcine elastase was from Sigma-Aldrich (St. Louis,
Missouri, USA). All other materials were from Sigma-
Aldrich, unless otherwise stated.
Nebulizers andnebulization procedure
Two commercially available inhalation systems were tested
for the invitro evaluation of nebulization of AAT: a breath-
enhanced jet nebulizer system, TurboBOY®, equipped
with a PARI LC Sprint® ampoule, and an electronic active
vibrating membrane nebulizer, the eFlow® rapid appara-
tus, both from PARI GmbH (Munich, Germany). Adequate
amounts of freeze-dried powder were weighed and dis-
solved under gentle stirring in ultrapure water to a final
protein concentration of 25mg/mL (2.5% w/v). Six mL
of protein solution (i.e., 150mg of AAT) were loaded in
ampoules for nebulization both for the delivered dose and
aerodynamic profile tests.
Determination ofdelivered dose anddrug
delivery rate
The delivered dose (DD), representing the overall mass of
protein emitted, the drug delivery rate (DDR), representing
the mass emitted per minute, and the time needed to aero-
solize the entire dose loaded in the ampoule, were deter-
mined following the specifications in the European Phar-
macopoeia 11th ed. [20]. A breathing simulator (sine pump
Model SRV500CC, VCS Srl, Parma, Italy) was set to mimic
an adult breathing pattern of 15 breaths/min, with a tidal
volume of 500mL per inspiration. The inhalation:expiration
ratio was 1:1. For both nebulizers, the mouthpiece of each
ampoule was connected to the sine pump by a specific
rubber adaptor and through a filter holder (PARI Pharma
GmbH, Munich, Germany) equipped with a filter (type A/E
glass diameter 76mm, Pall Corporation, New York, US)
to entrap the nebulized aerosol. For DDR, the nebulizer
was run for 1min; thereafter, the filter, coded as F1, was
removed, and substituted with a new filter (F2), then the
nebulizer was activated and run until the end of the nebu-
lization. Filter F2 was kept for a maximum of 5min, then
substituted with a new filter (F3) to prevent breakage and
overload. For the jet nebulizer, the end of nebulization was
set 1min after hearing sputtering; for the mesh nebulizer, the
end of nebulization was determined by its automatic stop.

Drug Delivery and Translational Research
1 3
To estimate the amount of AAT deposited on the filters,
each of them was transferred into a glass crystallizer and
washed in ice-cold 25mM phosphate buffer for 5min to
recover the nebulized protein. Thereafter, the solution was
filtered through a cellulose acetate membrane with a pore
size of 0.45μm, transferred in a 10-mL flask, brought to
volume with cold phosphate buffer, and stored on ice until
the concentration of AAT was determined.
The DDR was estimated by quantifying the mass of AAT
collected by F1, while DD was determined as the sum of the
mass collected by all filters. The efficiency of the proce-
dure for the protein recovery from filters was estimated by
quantifying with Bradford assay (see below) the amount of
AAT collected from a filter imbibed with 0.5mL of AAT
solution (25mg/mL) and recollected in 10mL of cold phos-
phate buffer.
Each test was carried out in triplicate.
Determination ofmass distribution andrespirable
fraction ofAAT bynextgeneration impactor
The mass distribution of AAT emitted by the two nebu-
lizers was analyzed by Next Generation Impactor (NGI,
Copley Scientific, Nottingham, UK). The apparatus was
pre-cooled at 4°C to prevent droplet evaporation and deter-
minations were performed within 5min of the removal of
the impactor from the refrigerator. A continuous aspiration
flow of 15 L/min was obtained by connecting the apparatus
to a pump (VP1000, Erweka, Italy) governed by a Criti-
cal Flow Controller (TPK, Copley Scientific, Nottingham,
UK). Each ampoule filled with the appropriate amount of
protein solution was connected to the induction port of the
apparatus by a rubber adaptor. The nebulizer was activated
for 4min, as prescribed in European Pharmacopeia 11th
edition [20], or for 2, 6, or 9min, or until exhaustion of
the solution in the ampoule, to assess the consistency of
nebulization in time. The induction port, each stage, and
the filter of the Micro Orifice Collector were rinsed with
ice-cold phosphate buffer and each sample was collected
and brought to volume in 20-mL volumetric flasks. The
concentration of protein in each sample was quantified by
Bradford protein assay [21] and the resulting amount (sum
of the quantity recovered in each stage) used to calculate
the emitted dose. The cumulative undersize mass percent-
age of AAT found in each stage was used to build a mass
distribution plot with respect to the cut-off diameters of
each stage: in particular, the median mass aerodynamic
diameter (MMAD) and the geometric standard deviation
(GSD) were calculated from the plot of cumulative under-
size percentage of the collected drug (in probit scale) with
respect to the log cut-off values of each stage according
to European Pharmacopoeia specifications (2.9.18, 11th
ed.) [20], built with Microsoft Excel for Mac (ver. 16.57).
This plot also allowed the determination of the fine particle
fraction (FPF%), corresponding to the percentage mass of
protein emitted in droplets having an aerodynamic diameter
lower than 5μm. This value was also used to estimate fine
particle dose, namely the milligrams of proteins expected
to reach the lower respiratory airways, as a product between
the delivered dose, derived from DD/DDR experiments,
and FPF%. Each test was carried out in triplicate.
Protein quantification
The amount of AAT in each sample was estimated by
UV–visible absorption spectroscopy at 280nm using a
Cary4000 spectrophotometer (Agilent Technologies, Santa
Clara, USA), by Bradford assay (Bio-rad Laboratories,
Milano, Italy) [21] or by 12% SDS electrophoresis (SDS-
PAGE) [22] followed by densitometric analysis of the AAT
band using a Bio-rad system. The Bradford assay was per-
formed with a Spark 10M microplate reader (Tecan, Manne-
dorf, Switzerland) as per manufacturer’s instructions. Each
measurement was carried out in triplicate.
Determination ofAAT activity
The samples deriving from DD/DDR tests or NGI tests were
snap-frozen in liquid nitrogen and then stored at −80°C to
prevent activity loss over time [23]. They were then thawed
immediately before the activity assays were performed. AAT
activity was assayed for each sample and normalized to its
quantity, as determined by densitometric analysis of SDS-
PAGE electropherograms. Briefly, AAT was incubated at
5nM concentration, as determined by SDS-PAGE, with
10nM porcine elastase in a solution containing 0.1 M
HEPES, 0.5M NaCl, and 0.05% Triton, pH 7.4 at 37°C
for 45min. The residual elastase activity was determined
by following the hydrolysis of N-succinyl-Ala-Ala-Ala-
p-nitroanilide to p-nitroaniline at 410nm in a Cary 4000
UV–Vis spectrophotometer. The fractional AAT residual
activity (A) was calculated from the initial velocity of the
elastase assay in the absence (Vctrl) and presence (VI) of
AAT, according to Eq.1:
Protein precipitation was assessed by SDS-PAGE as pre-
viously described [23]. All the measurements were carried
out in replicate.
Evaluation ofprotein alteration uponnebulization
Protein aggregation and structural integrity upon nebuli-
zation were evaluated by dynamic light scattering (DLS),
(1)
A
=1−(2
V
ctr l
−V
I
V
ctr l
)

Drug Delivery and Translational Research
1 3
size exclusion chromatography (SEC), and circular dichro-
ism (CD). To prevent any protein alteration ascribable to
its recovery from filters, as in DD/DDR experiments, these
tests were performed on samples collected by nebulizing the
solutions in a Twin Stage Impinger, according to European
Pharmacopoeia specifications (2.9.18, 11th ed.) [20] using a
continuous aspiration flow of 60 L/min. Briefly, 7 or 30mL
of 25mM phosphate buffer were introduced in the upper
and lower impingement chambers. The nebulizers were run
until sputtering (for jet nebulizer) or after the automatic stop
of the device (for mesh nebulizer). Each sample from the
upper impingement chamber was collected and brought to
a final volume of 10mL, while each sample from the lower
impingement chamber was collected and brought to a final
volume of 50mL with potassium phosphate buffer. The
solutions were immediately tested by dynamic light scatter-
ing, size exclusion chromatography, and circular dichroism.
Dynamic light scattering, DLS
The starting protein solution and the residual solutions in the
ampoules were collected and quantified. To perform DLS
analysis, protein concentration was optimized by comparing
the signals obtained from samples as such or after dilution
in 10mM NaCl aqueous solution. When no difference was
observed between the original sample and its diluted coun-
terpart, the measurement of the sample as such was consid-
ered (e.g., for samples deriving from impingement cham-
bers), since no interaction was supposed to occur between
molecules in solution; otherwise, the diluted sample was
considered, as in the case of those deriving from ampoule,
which were diluted 1:10 in 10mM NaCl aqueous solution.
Measurements were performed with a Zetasizer Nano (Mal-
vern Instruments, UK) equipped with a 633-nm laser, using
NIBS detection (173° backscatter) at 25°C. Samples were
analyzed immediately after they were obtained, without
any prolonged storage or freezing step to avoid time- and
temperature-dependent aggregation. Three measurements
were performed for each sample, and considered as valid if
the intercept of the correlation function was between 0.8 and
1. The three replicates were averaged to calculate the mean
size and polydispersity index.
Size exclusion chromatography, SEC
Chromatographic separation was achieved on a Prominence
HPLC system coupled with a UV detector and LabSolu-
tions software (Shimadzu, Kyoto, Japan) using a BioSep-
SEC-2000 column (300mm, 1.50mm, 5μM Phenomenex,
Torrance, CA, USA) in isocratic elution mode with a mobile
phase composed of 50mM K2HPO4, 300mM NaCl pH 7 and
pumped at a flow rate of 1mL/min. The injection volume
was 50 μL at an estimated protein concentration of 1mg/
mL. The detection was performed with an SPD-20A Model
UV detector (Shimadzu Kyoto, Japan) at 220nm. Samples
were centrifuged for 30min at 16,000 × g before loading.
Each chromatographic run lasted 15min. The size of the
aggregates was estimated by means of a calibration curve
built with a Column Performance Check Standard, Aque-
ous SEC 1 (Phenomenex), including myoglobin (17kDa),
ovalbumin (44kDa), IgG (150kDa), IgA (300kDa), and
bovine thyroglobulin (670kDa).
Circular dichroism, CD
CD spectra were collected using a Jasco J-1500 spectropola-
rimeter (Jasco, Tokyo, Japan) equipped with a Peltier ther-
mostatic unit set at 20°C using 0.1mm optical length quartz
cells and driven by a JASCO Spectra Manager II software.
The protein concentration was 5µM in 10mM K2HPO4
buffered at pH 7. The spectral scans were collected between
250 and 180nm, 0.5 nm data pitch, 8s DIT, bandwidth
2nm, at 50nm/min scanning speed. Each spectrum was
the result of 3 averaged accumulations. Secondary structure
estimation was performed by using the Dichroweb server
[24]. All CD spectra were corrected for buffer background.
Far‐UV CD signal changes at 220nm were monitored as a
function of increasing temperature from 20 to 90°C, with
steps of 5°C and with an equilibration time of 1s at each
temperature before recording the measurement. The thermal
transitions were analyzed with the CalFitter algorithm [25].
Statistical analysis
Data were analyzed with Microsoft Excel for Mac using a
t-test. Statistical significance was assumed at p < 0.05. If not
otherwise specified, measurements were carried out in trip-
licate and represented as mean value and standard deviation.
Results anddiscussion
Preliminary assessment ofdose andpotential
administration regimen
In this work, we investigated the use of nebulized AAT as
an alternative, or at least as a support, to parenteral infusion.
Medicines for inhalation are combination products whose
performance, in terms of delivered dose and fraction of the
dose reaching the targeted deep lung, is strongly affected by
the proper combination of formulation and device. As for the
suitable dose, an assessment of the potential amount of AAT
to be administered was preliminarily required. The protec-
tive threshold of AAT in the epithelial lining fluid (ELF) is
reported as 1.3–1.7μM [26], although the level in healthy
subjects is about 3μM (0.15 mg/mL—lower threshold

Drug Delivery and Translational Research
1 3
0.07mg/mL). Since the volume of ELF is estimated between
10 and 30mL [27], the mass of active protein that should
reach the deep lung is between 1.5 and 4.5mg (lower thresh-
old 0.7–2.1mg). Considering the potential loss of protein
during nebulization, and that part of the solution (about
1mL) remains in the ampoule at the end of nebulization,
we set a target dose of 150mg. This dose is almost double
the dose administered in other clinical trials (NCT01217671
and NCT04204252 [18, 28]), but this should not constitute a
safety concern since up to 500mg/day of recombinant AAT
(rAAT) were previously administered to patients without
signs of allergic reaction [14], and 100mg of hAAT were
administered twice a day for 1week, also with no adverse
effects [29]. The AAT concentration that we chose was
25mg/mL, which corresponds to that of Prolastin® upon
reconstitution in water, as per the manufacturer’s instruc-
tions. This choice would allow the use of the commercial
product off-label, without modifying the final concentration
of the salts and excipients present in the lyophilized product.
Indeed, hypertonicity could induce adverse reactions [30]
such as bronchoconstriction [31] and cough [32], thus reduc-
ing the effectiveness of the deposition in the lungs. Given a
concentration of 25mg/mL, the proposed dose of 150mg
can be reached through a single nebulization of 6mL, which
also corresponds to the maximum capacity of the ampoules
of the two nebulizers.
Evaluation oftheaerosolization performance
ofnebulizers
We measured the effect of the two nebulizers on the size
distribution of the droplets obtained from 25mg/mL AAT
solutions with 4min of nebulization (Table1). MMAD val-
ues were comparable, despite a higher GSD for the jet nebu-
lizer. In particular, MMAD for the jet nebulizer (3.9µm)
was in agreement with the producer’s specifications and
was consistent with the AAT values reported by Griese
etal. (3.5µm) [15] and Hatley etal. (3.67 ± 0.72 µm by
laser diffraction) [33], and not statistically different from
the ones reported by Brand etal. (4.4 ± 2 μm) [16]. The
apparent difference with the latter could be ascribed either
to the different type of impactor (an eight-stage cascade
impactor, instead of an NGI, which is considered more
efficient [11]) or to the different AAT concentration used
in that study (50mg/mL AAT vs. 25mg/mL), although
increased viscosity, due to higher protein concentration,
would be expected to decrease droplet size [34, 35]. The
presence of salts in Prolastin® after reconstitution should
also be taken into consideration and could be partially
responsible for the reduction of MMAD [34]. This effect
could also explain the MMAD upon nebulization with the
mesh nebulizer (3.3μm), which was lower than the one
reported in the producer’s specifications (4.1μm), as well
as the values reported by Hatley (4.4μm), though measured
by laser diffraction on salbutamol solutions [33], and by
Brand etal. (4 ± 1.6μm), with an analogous vibrating mesh
device (AKITA2 APIXNEB) on radiolabelled Prolastin®
solution at 35mg/mL [17]. Finally, the fine particle fraction
of both nebulizers was significantly higher than previously
reported by Hatley etal. (67.5 ± 1.0% for the jet nebulizer
and 59.9 ± 3.7% for the mesh nebulizer) [33] and Brand etal.
(67–70% for AKITA2 APIXNEB) [17].
The fine particle fraction produced by the mesh nebulizer
was significantly higher than that obtained with the jet nebu-
lizer (t-test, p < 0.001).
This difference was due to the fact that most of the drop-
lets emitted by the mesh nebulizer accumulated in stages
3–6 of the NGI having a cut-off between 5.39 and 1.36μm,
at 15 L/min (Fig.1), and this could be ascribed to the posi-
tive effect exerted by the mesh membrane that contrib-
uted to the production of smaller droplets. This result is
in good agreement with data reported by Brand etal. with
AKITA2 APIXNEB [17]. An analogous difference was
observed between the same two nebulizers when 160mg
colistimethate sodium were aerosolized (71.9% mesh vs.
59.7% jet) [36].
By prolonging the nebulization time, both nebulizers
showed a linear relationship between emitted fraction as well
as in fine particle fraction, and nebulization time (Fig.2).
The slope of the obtained regression lines was similar for the
two nebulizers. Thus, no reduction in the efficiency of nebu-
lization was observed, differently from what was reported
by Steckel and Eskandar for a jet nebulization system analo-
gous to the one used in these tests [37]: in that work, the
decrease of efficiency was attributed to the increase of the
concentration of the solution in the ampoule stemming from
solvent evaporation. This aspect was investigated in the pre-
sent work by sampling the undiluted nebulized product col-
lected in pre-cooled 50-mL test tubes: samples were centri-
fuged before quantitation to remove any insoluble aggregate
deriving from protein denaturation. No visible aggregates
were detected, nor variation of the original concentration of
protein solution, as assessed by UV absorbance, for any of
the samples collected from the two nebulizers.
Nebulization time did not affect droplet size distribution,
as no shift occurred in MMAD, for both types of nebulizers.
Table 1 Mass median aerodynamic diameter, geometric standard
deviation, and fine particle fraction obtained from the two nebulizers
evaluated after 4min by NGI (mean ± standard deviation; n = 3)
Jet nebulizer Mesh nebulizer
MMAD (μm) 3.9 ± 0.1 3.3 ± 0.3
GSD (μm) 2.5 ± 0.5 1.7 ± 0.1
Fine particle fraction (%) 71.8 ± 2.4 78.2 ± 1.5

Drug Delivery and Translational Research
1 3
However, the breadth of the distribution was smaller when
using the mesh nebulizer, as reflected by the lower values
of GSD (Table2).
Determination ofDDR andDD
To assess DDR and DD, ampoules were filled with 6mL of
AAT solution, and nebulization was performed by connect-
ing the nebulizer to a sine pump (Table3). Particular atten-
tion was paid to protein recovery from the collection filters
to avoid denaturing conditions. This is a critical step, as pre-
viously reported [11]. Therefore, filters were not subjected
to sonication to extract the protein, a cold phosphate buffer
was used as a diluent, and all samples were centrifuged and
processed in the shortest time possible. In these conditions,
the efficiency of mass recovery was estimated at 98 ± 7%.
By combining the value of the respirable fraction obtained
by NGI with the DD, the fine particle dose was estimated
at 25.1mg for the jet nebulizer and 39.9mg for the mesh
nebulizer, respectively. The DDR, as assessed according to
pharmacopeial specifications, was comparable between the
two nebulizers, but the total drug delivered was significantly
higher when using the mesh nebulizer (p = 0.033 by t-test).
Therefore, the latter was more efficient than the jet nebulizer
in delivering the solution, also considering that the time to
complete nebulization was lower and that about 1mL of
the loaded solution was left over in the ampoule due to the
automatic stop of the mesh device.
Fig. 1 Mass distribution of
AAT obtained after nebuliza-
tion of the AAT solution in the
NGI with the jet nebulizer and
the mesh nebulizer. The bars
represent the standard deviation
(n = 3)
Fig. 2 Emitted fraction (left) and respirable fraction (right) of AAT
after nebulization of the solution with the jet nebulizer (empty
square) and the mesh nebulizer (solid triangle) in NGI. The bars rep-
resent the standard deviation (n = 3). The lines are the graphical rep-
resentation of the linear regression of the experimental data. Emitted
fraction jet nebulizer y = 3.1429x + 0.381 (R2 = 0.9727) dotted line;
mesh nebulizer y = 3.0351x + 2.5272 (R2 = 0.9662) dashed line; res-
pirable fraction jet nebulizer y = 2.6202x + 0.7664 (R2 = 0.9678) dot-
ted line; mesh nebulizer y = 2.9912x + 2.372 (R2 = 0.9634) dashed line

Drug Delivery and Translational Research
1 3
Evaluation ofAAT activity uponnebulization
When dealing with inhaled products, three doses are com-
monly defined: the dose on the label (metered), the dose
released by the device, and the dose that reaches the deep
lung. If the active ingredient is a protein, a fourth dose
value must also be considered, i.e., the dose that reaches the
deep lung in a still biologically active form. Therefore, we
measured the specific elastase inhibition activity of solu-
ble AAT (i.e., the activity divided by the amount of protein
measured in each fraction obtained from DD/DDR experi-
ments) to evaluate possible inactivation due to oxidation or
denaturation upon nebulization. The relative AAT activity
was expressed as a percentage of that measured immediately
after resuspension. As reported in Fig.3, the activity of the
protein tended to decrease with increasing nebulization time,
with a comparable trend for both nebulizers. No significant
differences could be detected between the two nebulizers
at the same time points. After 1min of nebulization, the
activity was not significantly different (t-test) from that of
the starting solution with both nebulizers, being 84% (± 4%)
for the jet nebulizer and 87% (± 11%) for the mesh nebulizer.
The slight reduction of activity could be at least partially
ascribed to the procedure of recovery from the filter, that
cannot be discriminated from the effect of nebulization.
Anyway, after the same recovery procedure, at the end
of nebulization with the jet nebulizer, the activity decreased
to 72% (± 15%), not significantly different from that of the
starting solution, while with the mesh nebulizer, the final
activity was 60% (± 11%) statistically lower than the starting
solution, pointing at the nebulization procedure as the main
cause of loss of activity. For both nebulizers, the AAT activ-
ity in the residual solution in the ampoule was significantly
lower with respect to both the starting solution and the final
nebulized product: this is probably ascribable to the fact
that the solution in the ampoule is repeatedly stressed. The
decrease in activity over time could either be associated with
denaturation or to methionine oxidation.
To our knowledge, this is the first paper reporting data
on AAT activity upon both jet and mesh nebulization. The
effect of both nebulizers on protein integrity is in agree-
ment with a paper on the nebulization of insulin-like growth
factor I, which showed comparable performances by using
jet or mesh nebulizers [38]. In both cases, a reduction in
the activity of the protein was observed with respect to the
starting solution. In this sense, the mesh nebulizer does
not seem to provide a better preservation with respect to
the jet nebulizer. The data relevant to the latter differ from
what previously reported about the residual activity of AAT
(95 ± 11.3%) after nebulization with eFlow® [39, 40]. It is
worth underlining that the conditions at which protein solu-
tion is exposed during DD/DDR test should be regardedasa
worst-case scenario, considering the continuous air exposure
and the collection on a solid dry support.
Evaluation ofAAT aggregation uponnebulization
To ascertain the cause of the partial loss of specific activ-
ity over time, we evaluated potential protein aggregation
of AAT before and after nebulization by means of DLS.
Studying protein stability directly in the aerosol would be
ideal but unfeasible. Among the apparatuses proposed by
European Pharmacopoeia to assess aerodynamic perfor-
mances of nebulized products, we selected the Twin Stage
Impinger. The choice was driven by two main reasons: the
first is that the collection of the nebulized product directly
in liquid, in our opinion, better represents the wet environ-
ment found in the lungs by nebulized droplets; the volume
of epithelial lung fluid is estimated between 10 and 30mL
[27], a volume that is close to the volumes prescribed by
Eur. Ph. to fill the impingement chambers. The second rea-
son is that the use of an impinger rather than an impactor
could reduce the interferences in the protein state due to
prolonged exposition to air and, specifically for NGI, to the
contact with the metal components or, in DD/DDR tests, to
the recovery from filters. DLS analysis of samples collected
in impingement chambers indicated that, right after disso-
lution, the AAT solution presented a single population of
macromolecules having a hydrodynamic diameter of 7nm,
broadly consistent with previous reports on monomeric,
monodispersed AAT [23, 41]. The graph in Fig.4 shows
the size up to 100nm to highlight any difference in the
samples. Nebulization with both nebulizers did not modify
Table 2 Mass median aerodynamic diameter and geometrical stand-
ard deviation obtained from the two nebulizers after 2, 4, 6min or at
the end of nebulization by NGI (mean ± standard deviation; n = 3)
Time (min) Jet nebulizer Mesh nebulizer
MMAD GSD MMAD GSD
23.3 ± 1.2 2.70 ± 0.01 3.2 ± 0.3 1.6 ± 0.2
43.9 ± 0.1 2.5 ± 0.5 3.3 ± 0.3 1.7 ± 0.1
63.1 ± 0.5 3.4 ± 0.1 3.1 ± 0.1 1.6 ± 0.1
93.9 ± 0.1 2.8 ± 0.1 3.2 ± 0.1 1.6 ± 0.1
End 3.3 ± 0.5 2.4 ± 0.5 3.1 ± 0.2 1.7 ± 0.1
Table 3 Delivery values of AAT solutions using the two nebulizers
(mean ± standard deviation; n = 3)
Jet nebulizer Mesh nebulizer
DDR (mg/min) 2.8 ± 1.2 2.8 ± 0.4
DD (mg) 35.0 ± 3.4 51.0 ± 8.5
Estimated FPD (mg) 25.1 ± 3.3 39.9 ± 7.4
Nebulization time (min) 17 ± 3 15 ± 3

Drug Delivery and Translational Research
1 3
the aggregation state of the protein, as no other populations
appeared in ampoules as well as in fractions collected in the
stages of the Twin Stage Impinger.
SEC analysis—which, in our configuration was sensitive
in the 17–670kDaMW range (up to 15-mers of AAT)—
indicated a major band with an elution time around 8.5min
consistent with monomeric, glycosylated AAT (around
54kDa) (Fig.5). Multiple low-intensity bands with elu-
tion times of 5–7min were consistent with aggregates with
MWs ranging from 150 to 670kDa. The chromatograms of
nebulized and non-nebulized AAT were essentially super-
imposable for both mesh and jet nebulizers, confirming
that nebulization did not affect aggregation. Moreover, in
our tests, we could not find any evidence of an increase in
the concentration of the solution ascribable to nebulization
by both jet or mesh nebulizers, differently from what was
reported by Steckel for jet nebulizers [37]. Overall, aggrega-
tion did not seem to be the cause of loss of activity for AAT,
leaving methionine oxidation over time as the most likely
cause of inactivation.
Evaluation ofAAT denaturation uponnebulization
As temperature is known to affect protein stability, it was
monitored in the ampoule before and after nebulization.
A significant increase (+ 10.0°C ± 3.1°C) was observed
with the mesh nebulizer, whereas a non-significant increase
(+ 0.4°C ± 2.3°C) was observed in the ampoule of the jet
nebulizer. To assess whether these temperature changes
might be responsible for protein denaturation, and thus a
Fig. 3 Activity of AAT
obtained after nebulization of
the solution with the jet nebu-
lizer and the mesh nebulizer in
comparison with the starting
protein activity (dotted). # with
respect to starting solution; §
with respect to F1; **p < 0.01,
***p < 0.001
AB
Fig. 4 Size distribution analysis by volume of AAT dissolved in water before and after nebulization with jet (A) or mesh (B) nebulizers deriving
from Twin Stage Impinger experiments

Drug Delivery and Translational Research
1 3
partial loss in its specific activity, we measured CD spectra
of the protein before and after nebulization. As reported in
Fig.6A, the spectra were completely superimposable.
Secondary structure analysis using the Dichroweb server
confirmed almost identical structures (Table4).
To confirm that the temperature to which AAT is
exposed during nebulization does not affect its folding, we
performed thermal denaturation experiments monitored
by CD at 220nm. A transition temperature (Tm) of 69°C
(Fig.6B, inset) was calculated. However, the transition
associated with this Tm was not associated with full loss
of secondary structure, as indicated by CD spectra con-
sistent with partial secondary structure preservation up to
90°C (Fig.6B). Moreover, a CD spectrum almost identi-
cal to that of the starting solution of AAT was obtained
upon heating to 90°C followed by slow cooldown, indi-
cating that the secondary structure changes are reversible
(Fig.6B).
The exclusion of AAT precipitation, aggregation, and
thermal denaturation in the ampoule suggested that the
decrease in specific AAT activity of up to 50% was likely
associated with the oxidation of AAT, particularly at resi-
dues Met358 and Met351 [42]. Oxidation of the protein is
expected to occur to a greater extent when nebulized by jet
nebulizer, due to recirculation of protein solution, which
leads to repeated exposure of the protein to the air–liquid
interface. For this reason, the residual in the ampoule has
been considered a worst case for jet nebulization [43], and a
consistent reduction in activity was expected. On the other
hand, the stability of the residual solution in the ampoule of
the mesh nebulizer in principle should not represent a major
issue since it does not recirculate [7]. Differently from what
expected, residual AAT activity in the ampoule of the mesh
nebulizer was low and comparable to that in the ampoule
of the jet nebulizer, hinting again at oxidation as the main
degradation mechanism during nebulization.
Fig. 5 A SEC analysis of sam-
ples deriving from nebulization
with the jet nebulizer compared
with the starting solution. B
SEC analysis of samples deriv-
ing from nebulization with the
mesh nebulizer compared with
the starting solution. The insert
of panel A shows the calibra-
tion curve used to estimate
the molecular weights. The
band with elution time around
8.5min correspond to a protein
of 54kDa consistent with gly-
cosylated AAT
Fig. 6 A CD analysis of samples
deriving from nebulization
with jet and mesh nebulizers
compared to starting solution.
B Circular dichroism spectra
of AAT (solid line), AAT upon
incubation at 90°C (dashed line)
and after incubation at 90°C
and slow return to 20°C (dotted
line). Inset: temperature ramp,
with a calculated Tm of 69°C

Drug Delivery and Translational Research
1 3
Overall, no advantages were observed by using the jet
nebulizer instead of the mesh nebulizer. The time required
to complete the nebulization was not significantly differ-
ent between the two nebulizers, being 17 ± 3min for the jet
nebulizer, as determined by hearing the typical sputtering
sound, and 15 ± 3min for the mesh nebulizer, defined by
the automatic stop of the device. However, considering the
higher dose delivered by the mesh nebulizer in the same
nebulization time, the latter is overall more advisable in
terms of efficiency.
Conclusions
The results above provide a characterization of AAT activity
after nebulization. Both nebulizers guarantee comparable
and acceptable preservation of the activity of the nebulized
protein and did not induce aggregation or changes in its con-
formation. This is of particular importance given the pos-
sible immunogenicity determined by aggregated proteins.
The two nebulizers demonstrated equivalent aerosolization
performance except for the fact that, with respect to the jet
nebulizer, the mesh nebulizer provides higher efficiency in
delivering the dose. This allows us to conclude that nebu-
lization of a solution, even though not optimized for this
administration way, may represent a suitable administration
strategy in delivering the protein directly to the lungs in
AATD patients, either as a support therapy to parenteral
administration or, for subjects with a precocious diagnosis,
to prevent the onset of pulmonary symptoms.
Acknowledgements The authors would like to thank Mr. Giovanni
Mingardi for the technical support in aerosol characterization tests.
Author contribution AB study conception and design, data collection
and analysis, draft writing, VV data collection, AM data collection,
RG data collection, GB data collection, FB draft revision, MA, AAC,
SB study conception, and design, supervising, draft revision and RB
funding acquisition, supervision, and draft revision.
Funding Open access funding provided by Università degli Studi
di Parma within the CRUI-CARE Agreement. This research was
partially funded by a Regione Emilia Romagna POR-FESR grant
(PG/2015/731196) to R.B.
Data availability The datasets generated during and/or analyzed dur-
ing the current study are available from the corresponding author on
reasonable request.
Declarations
Ethics approval Not applicable.
Consent for publication All authors read and approved the final manu-
script and agree with its publication.
Competing interests The authors declare no competing interests.
Open Access This article is licensed under a Creative Commons Attri-
bution 4.0 International License, which permits use, sharing, adapta-
tion, distribution and reproduction in any medium or format, as long
as you give appropriate credit to the original author(s) and the source,
provide a link to the Creative Commons licence, and indicate if changes
were made. The images or other third party material in this article are
included in the article's Creative Commons licence, unless indicated
otherwise in a credit line to the material. If material is not included in
the article's Creative Commons licence and your intended use is not
permitted by statutory regulation or exceeds the permitted use, you will
need to obtain permission directly from the copyright holder. To view a
copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/.
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