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Development of Inhalable Dry Gene Powders
for Pulmonary Drug Delivery
by Spray-Freeze-Drying
Edina Vranić, Merima Sirbubalo, Amina Tucak, Jasmina Hadžiabdić,
Ognjenka Rahić, and Alisa Elezović
Abstract
There is considerable potential for pulmonary gene
therapy as a treatment for a number of conditions for
which current treatment is inadequate. Delivering genes
directly to the lungs by dry powder inhalers (DPIs) have
attracted much attention due to better stability of genes.
Formulating genes as powders for aerosol delivery is a
challenge as it requires not only flowability and dis-
persibility of the powders but also maintaining gene
stability and biological activity during manufacturing and
delivery. In this review, we aim to provide an overview
about the potentials of spray-freeze-drying (SFD) for the
development of inhalable dry gene powders for pul-
monary drug delivery. We will discuss the main steps
involved within the production process (i.e., spraying,
freezing and drying) and introduce different SFD methods
which can successfully be used for the production of
porous particles whose physical and aerosol characteris-
tics are considered to be ideal for use in pulmonary drug
delivery.
Keywords
Pulmonary drug delivery Gene therapy Dry gene
powders Spray-freeze-drying
1 Introduction
In recent years, the lungs have been studied as a very
attractive target for drug delivery [1]. Pulmonary drug
delivery offers several advantages over injectable, transder-
mal or oral methods of delivery. Inhalables provide a
non-invasive method of delivering drugs into the blood-
stream, they enable effective drug targeting to the lungs for
relatively common respiratory tract diseases and provide
very rapid absorption similar to the intravenous route
because of an enormous surface area and a relatively low
enzymatic, controlled environment for the systemic absorp-
tion of medications. Delivering drug to the lungs can also
help avoid gastrointestinal tract problems such as poor
solubility, low bioavailability, gut irritability, unwanted
metabolites, food effects and dosing variability [2,3].
Gene therapy has as its central principle the addition of
gene function through gene transfer. It has been the subject
of a great deal of information, and misinformation, over the
past decades [4]. However, there is considerable potential for
pulmonary gene therapy as a treatment for a number of
conditions for which current treatment is inadequate [5].
Gene delivery in humans requires carriers that will transfer
genes into the nuclei of target cells. These carriers (viral or
non-viral systems) first must be safe for human use, they
must be efficient in transfection, and protect the genes from
degradation before arriving at the target cell. Both viral and
non-viral gene delivery systems have been used in the lungs,
however, both have limitations associated with their bio-
logical properties [5]. Viral vectors are highly effective
delivery agents, but their high immunogenicity has led
researchers to seek safer alternatives [6]. Very popular
non-viral delivery systems are biodegradable and biocom-
patible poly (D,L-lactide-co-glycolide) (PLGA) polymers,
cationic liposomes, polyethyleneimine (PEI) etc. They are
much safer than viral delivery systems, but their transfection
efficiency in vivo is limited [7].
Pulmonary administration is a powerful tool for achieving
effective pulmonary gene therapy against several lung dis-
eases, such as cystic fibrosis, a1-antitrypsin deficiency, and
lung cancer [8]. These diseases could be treated by
high-level and long-term expression of the corresponding
gene of interest [6]. The lungs possess inherent advantages
for gene therapy since they are easily accessible via the
airways, offer a large surface area for transfection and reduce
E. Vranić(&)M. Sirbubalo A. Tucak J. Hadžiabdić
O. RahićA. Elezović
Department of Pharmaceutical Technology, Faculty of Pharmacy,
University of Sarajevo, Zmaja od Bosne 8, 71 000 Sarajevo,
Bosnia and Herzegovina
e-mail: evranic@yahoo.com
©Springer Nature Switzerland AG 2020
A. Badnjevic et al. (eds.), CMBEBIH 2019, IFMBE Proceedings 73,
https://doi.org/10.1007/978-3-030-17971-7_79
533
the risk of systemic side effects. The most important thing in
pulmonary gene therapy is to deliver a therapeutic gene to
the lungs. To achieve this goal, several intravenous formu-
lations have been developed. However, delivery to the lungs
via intravenous injection is limited since genes are rapidly
degraded by endonucleases in the systemic circulation.
Another problem is nontargeted distribution, subsequently
leading to poor therapeutic effects and adverse effects [8,9].
Delivering genes directly to the lungs by nebulizers, pres-
surized metered-dose inhalers (pMDIs), or dry powder
inhalers (DPIs) can solve these problems. Among these
aerosol inhalation systems, DPIs have attracted much
attention due to their low cost, portability, no propellant, and
ease of handling [10]. Dry powder formulation also offers
the better stability of genes by minimizing the exposure to
high shear stress (during nebulization) and avoids the com-
patibility issues with propellants (in metered dose inhaler
(MDI) [11]. Particle engineering methods that are suitable
for preparing inhaled powder formulation of genes are spray
drying, spray-freeze-drying and supercritical fluid technol-
ogy [12]. Spray-freeze-drying (SFD) is powderization tech-
nique used to produce highly porous low-density powders,
with high dispersibility and reachability to the lungs.
2 Spray-Freeze-Drying (SFD)
Spray-freeze-drying is biopharmaceutical powder production
method, which has been attracting increasing interest in
various areas of research [13]. The process has been widely
used in pharmaceutical research, as well as food science and
technology [14]. This technology can enhance the apparent
solubility of poorly water-soluble drugs [15–18]. It is used as
an approach that facilitates the development of dosage forms
for alternative delivery pathways (pulmonary, nasal routes
and delivery to the epidermis by needle-free injection) [19].
Some research groups have used SFD for preprocessing the
protein/peptide ingredient prior to encapsulation in poly
(lactic-co-glycolic acid) (PLGA) microspheres [20,21]. It is
also used very successfully to improve the storage stability
of protein/peptide active ingredients [13,22]. Spray-
freeze-drying is preferred over classical spray-drying or
freeze-drying for many reasons. First, it is possible to
produce very porous powders with controlled particle-size
distributions, and the technology offers the possibility to
process thermosensitive active ingredients.
The term “spray-freeze-drying”(SFD) refers to processes
with three steps in common: dispersion of bulk liquid
solutions into droplets, droplet freezing, and sublimation
drying of the frozen material. Schematic diagram of the
spray-freeze-drying process is shown in Fig. 1. The first step
in SFD is the dispersion of bulk liquid and the formation of
droplets by using various types of nozzles and droplet stream
generation systems. In the next step, droplets are being
frozen either by transfer of thermal energy from the liquid to
a cold gas, another immiscible liquid or a solid in contact
with the droplet surface or by the diffusion of energy rich
volatiles into the surroundings at low vapor pressure. The final
step of the process is sublimation drying in which mobile
solvent molecules separate from the surface of the frozen solid
when they have acquired sufficient energy. This final step is
significantly different from sublimation drying of frozen
solutions in vials during freeze-drying because the specific
surface area of frozen droplets exceeds the ratio of the surface
area available for the escape of solvent molecules [19].
Spray-freeze-drying covers different production methods:
atmospheric freezing, spray-freezing into vapor over a liquid
cryogen (SFV), spray-freezing into liquid cryogen (SFL),
spray-freezing onto solid surfaces (thin film freezing, TFF)
[13,19]. Spray freezing into vapor over liquid cryogen
(SFV) was first reported in 1948 and was performed by
Benson and Ellis to investigate the surface area of protein
particles [23]. The liquid feed was atomized into the vapor
over a cryogenic liquid, such as liquid nitrogen or propane
using either two-fluid or ultrasound nozzles. The droplets
begin to freeze during the time of flight through the cold
vapor phase and completely freeze upon contact with the
cryogenic liquid phase itself [13]. Spray-freezing into liquid
cryogen (SFL) is one of the most commonly used spray-
freeze-drying techniques and it involves the atomization of a
drug solution mostly via a two-fluid or an ultrasonic nozzle
into a spray chamber filled with a cryogenic liquid [24]. The
spraying process can be performed beneath or above the
surface of the cryogenic liquid, depending on the position of
the nozzle. Upon contact with the cryogenic liquid, the
droplets solidify rapidly (in milliseconds) because of the
high heat-transfer rate. After the spray freezing process is
completed, the whole content can be lyophilized, with con-
ventional freeze-drying. The frozen solvent is removed, as in
the case of freezing with cryogenic liquids, by vacuum or
atmospheric freeze-drying. The large surface area of the
frozen powder and loose porous structure of the powder
allow relatively fast and homogeneous drying compared
with a standard lyophilization process [25].
Spray-freeze-drying is highly promising technology for
the production of porous particles whose physical and
aerosol characteristics are considered to be ideal for use in
pulmonary drug delivery. The particles produced by SFD are
typically amorphous and homogeneous. Spray freeze-dried
particles can be engineered to the desired respiratory size
range (below 5 µm) or even down to nano-scale [25]. The
large porous particles have relatively small aerodynamic
diameters usually smaller than 5 lm and large geometrical
diameters usually larger than 10 lm.
Spray-freeze-drying has become very popular for pro-
cessing of biologicals such as therapeutic proteins,
534 E. Vranićet al.
monoclonal antibodies, and vaccines because of its ability to
produce highly porous particles at sub-ambient temperatures
with or without excipients. Spray-freeze-drying has been
used to formulate a significant number of thermolabile and
highly potent therapeutic proteins/peptides into dry powder
inhalation products, including recombinant-derived human-
ized anti-IgE monoclonal antibody [26], recombinant human
deoxyribonucleases [26], insulin [27], small interfering RNA
(siRNA) [11] and plasmid DNA pSG5lacZ [28]. It is
important to mention that the application of SFD is not
limited to the production of porous materials; the technique
is equally capable of producing high-density particles. It is
also worth noting that the application of SFD is not only
limited to aqueous solutions because most volatile organic
solvents can be processed [25]. Despite all, SFD methods
have many disadvantages. Almost all SFD methods are still
highly experimental and only scaled for laboratory purposes
[13]. Methods are not as well established due to their high
complexity and high cost.
3 Development of Dry Gene Powders by SFD
Formulating genes as powders for aerosol delivery is a
challenge as it requires not only flowability and dispersibility
of the powders but also maintaining gene stability and bio-
logical activity during manufacturing and delivery [29].
Development of inhalable aerosol systems for pulmonary
gene delivery is critical for clinical use. The mean problem
in the formulation is destabilization of the gene or delivery
system caused by several stresses: heating, freezing, spray-
ing and shearing stress in the nozzle [8,9]. These forms of
physical stress during the preparation might cause a critical
loss of the gene. Therefore, a stable preparation of dry gene
powder is necessary to achieve the clinical application of
pulmonary gene therapy. Critical factors that can affect
inhalability of the particles are also morphology and particle
size of prepared powders [11]. To allow efficient lung
deposition, prepared powders must have optimal particle
size. Only particles that exhibited aerodynamic diameter
between 1 and 5 µm can reach the deep lung. Aerodynamic
diameter is affected by the geometric diameter as well as the
density of the particles. Particles with a geometric diameter
of 1–5µm usually have poor flowability and dispersibility
due to the strong cohesion force. On the other hand, porous
particles with large geometric size tend to have small aero-
dynamic size because of the low density. These types of
particles are desirable for inhalation due to the good
flowability and dispersibility. In general, aerosol particles
with aerodynamic diameters between 1 and 5 lm can
achieve good lung deposition and the choice of inhaler
device, therefore plays an important role in determining the
success of aerosol delivery [1,25].
Very porous particles produced by spray-freeze-drying
has made this technology an attractive method for preparing
inhaled powders. Porous particles with large physical size
and low density exhibit small aerodynamic size, which can
promote high flowability. In addition, porous particles have
high specific surface area, thereby enhancing dissolution rate
in the lungs. The porosity of the particles could be controlled
by altering the solute concentration of the feed solution for
spray-freeze-drying [11].
In one of the first studies, Maa et al. successfully used
spray-freeze-drying to produce protein powders containing
Fig. 1 Schematic diagram of the spray-freeze-drying process [11]
Development of Inhalable Dry Gene Powders …535
recombinant human desoxyribonuclease (rhDNase) and
anti-IgG monoclonal antibody (anti-IgG Mab) for dry
powder inhalation. Maa et al. revealed that a dry powder
produced by SFD had superior inhalation characteristics
compared to dry powders prepared by spray drying. Spray-
freeze-drying can also guarantee the high-level recovery of
produced dry powders, even if the initial amount of the
formulation is small, which is very important for the study of
dry gene powders on a laboratory scale since the employed
genes are relatively expensive [26].
Kuo and Hwang first reported about the dry gene powders
prepared by SFD. However, its gene transfection character-
istics in vivo have remained unknown [30]. Mohri et al.
successfully prepared inhalable dry plasmid DNA (pDNA)
powders by SFD. They examined the stability of pDNA
obtained through SFD and the morphology of prepared dry
pDNA powders. All powders had spherical and highly
porous particles about 20–40 lm in diameter. The ternary
structure of pDNA was evaluated by electrophoresis to
investigate the integrity of pDNA in the powders prepared
by SFD. In the preparation without chitosan, the pDNA was
almost completely degraded through SFD. However, the
addition of chitosan improved the destabilization of pDNA
caused by the physical stress during SFD. Chitosan has been
reported to show high tolerability in the body. Unfortu-
nately, on the other hand, it has been reported that the
transfection efficiency of chitosan is low compared to that of
polyethyleneimine (PEI), a non-biodegradable polycation
[9]. Kuo and Hwang also reported the destabilization of
pDNA through SFD and a protective effect of poly-
ethyleneimine, a polycation, but not mannitol, supporting
their results in part [30].
Okuda et al. also used SFD to prepare inhalable dry gene
powders. In order to achieve higher gene transfection effi-
ciency, authors have synthesized poly(aspartamide) deriva-
tives with an ethylenediamine unit as a side chain (poly{N
[N-(2-aminoethyl)-2-aminoethyl]aspartamide} (PAsp(DET))
and their block copolymers with poly(ethylene)glycol
(PEG-PAsp(DET)) as vectors. These novel biodegradable
polycations have superior efficiencies with minimal cyto-
toxicity compared to PEI. The final product had spherical
and porous structures with a 5–10 lm diameter, and they
showed that the integrity of plasmid DNA could be main-
tained during powder production. Both PAsp(DET)- and
PEG-PAsp(DET)-based dry gene powders could achieve
higher gene transfection efficiencies in the lungs compared
with chitosan-based dry gene powders [8].
Liang et al. demonstrated in their study that dry gene
powder could be stably prepared by SFD without the loss of
plasmid DNA integrity and that the powder exhibited a gene
expressing effect in the lungs of mice following pulmonary
administration [31]. For clinical application, however, a
higher gene transfection efficiency in the lungs is necessary.
In their last study, Liang et al. employed spray-freeze-drying
to prepare dry powder of small interfering RNA (siRNA) to
treat lung diseases. Mannitol and herring sperm DNA were
used as bulking agent and model of small nucleic acid
therapeutics, respectively. The gel retardation and liquid
chromatography assays showed that the siRNA remained
intact after spray-freeze-drying even in the absence of
delivery vector. The powder formulation exhibited a high
emitted fraction (EF) of 92.4% and a modest fine particle
fraction (FPF), of around 20%. Authors successfully
demonstrated that spray-freeze-drying can be used to pro-
duce naked siRNA formulation with intact integrity [11].
4 Conclusion
Spray-freeze-drying is powderization technique used to
produce a highly porous low-density powders. The powders
produced by SFD are considered more suitable for inhalation
compared with those produced by conventional techniques.
Spray-freeze-drying involves multiple steps: liquids are first
sprayed into a cryogen such as liquid nitrogen and the
droplets, which are frozen immediately, are then transferred
into a freeze dryer to allow the sublimation of ice, resulting
in the formation of highly porous powders. Spray-freeze
drying has shown to be a feasible method if good particle
size control, spherical particle shape and a high product yield
are essential. The porous particles produced by spray-
freeze-drying has made this technology an attractive method
for preparing inhaled gene powders. Many authors reported
the use of spray-freeze-drying technology to produce dry
powder of gene complexes that are suitable for inhalation.
These dry powders produced by SFD could very success-
fully reach to intrapulmonary region and exhibit gene
expression in the lungs. Since the aerosol performance of a
powder formulation is also affected by the design of inhaler,
a careful selected inhaler device could improve the delivery
efficiency.
Conflict of Interest The authors have no conflicts of interest to
disclose.
References
1. Labiris, N., Dolovich, M.: Pulmonary drug delivery. Part I:
Physiological factors affecting therapeutic effectiveness of aero-
solized medications. Br. J. Clin. Pharmacol. 56(6), 588–599
(2003a)
2. Patton, J., Byron, P.: Inhaling medicines: delivering drugs to the
body through the lungs. Nat. Rev. Drug Discov. 6(1), 67–74
(2007)
3. Swarbirck, J., Boylan, J.: Encyclopedia of Pharmaceutical Tech-
nology, 3rd edn, pp. 1279–1287. M. Dekker, New York (2000)
536 E. Vranićet al.
4. West, J., Rodman, D.M.: Gene therapy for pulmonary diseases.
Chest 119(2), 613–617 (2001)
5. Jenkins, R.G., McAnulty, R.J., Hart, S.L., Laurent, G.J.: Pul-
monary gene therapy. Realistic hope for the future, or false dawn
in the promised land? Monaldi Arch. Chest. Dis. 59,17–24 (2003)
6. Bivas-Benita, M., Romeijn, S., Junginger, H.E., Borchard, G.:
PLGA–PEI nanoparticles for gene delivery to pulmonary epithe-
lium. Eur. J. Pharm. Biopharm. 58(1), 1–6 (2004)
7. Roth, J.A., Cristiano, R.J.: Gene therapy for cancer: what have we
done and where are we going? J. Natl. Cancer Inst. 89(1), 21–39
(1997)
8. Okuda, T., Suzuki, Y., Kobayashi, Y., Ishii, T., Uchida, S., Itaka,
K., Kataoka, K., Okamoto, H.: Development of biodegradable
polycation-based inhalable dry gene powders by spray freeze
drying. Pharmaceutics 7(3), 233–254 (2015)
9. Mohri, K., Okuda, T., Mori, A., Danjo, K., Okamoto, H.:
Optimized pulmonary gene transfection in mice by spray-freeze
dried powder inhalation. J. Control. Release 144(2), 221–226
(2010)
10. Labiris, N., Dolovich, M.: Pulmonary drug delivery. Part II: The
role of inhalant delivery devices and drug formulations in
therapeutic effectiveness of aerosolized medications. Br. J. Clin.
Pharmacol. 56(6), 600–612 (2003b)
11. Liang, W., Chan, A., Chow, M., Lo, F., Qiu, Y., Kwok, P., Lam,
J.: Spray freeze drying of small nucleic acids as inhaled powder for
pulmonary delivery. Asian J. Pharm. 13(2), 163–172 (2017)
12. Pfeifer, C., Hasenpusch, G., Uezguen, S., Aneja, M.K., Reinhardt,
D., Kirch, J., Schneider, M., Claus, S., Friess, W., Rudolph, C.:
Dry powder aerosols of polyethylenimine (PEI)-based gene
vectors mediate efficient gene delivery to the lung. J. Control.
Release 154,69–76 (2011)
13. Schiffter, H.: Spray-freeze-drying in the manufacture of pharma-
ceuticals. Eur. Pharm. Rev. 3,1–7 (2007)
14. Ishwarya, S.: Spray-freeze-drying: a novel process for the drying
of food and bioproducts. Trends Food Sci. Technol. 41, 161–181
(2015)
15. Rogers, T.L., Overhoff, K.A., Shah, P., Yacaman, M.J., Johnston,
K.P.: Micronized powders of a poorly water soluble drug produced
by a spray-freezing into liquid-emulsion process. Eur. J. Pharm.
Biopharm. 55, 161–172 (2003)
16. Kondo, M., Niwa, T., Okamoto, H., Danjo, K.: Particle charac-
terization of poorly water-soluble drugs using a spray-freeze
drying technique. Chem. Pharm. Bull. 57(7), 657–662 (2009)
17. Leuenberger, H.: Spray-freeze-drying—the process of choice for
low water soluble drugs. J. Nanoparticle Res. 4, 111–119 (2002)
18. Hu, J., Johnston, K., Williams, R.: Rapid dissolving high potency
danazol powders produced by spray freezing into liquid process.
Int. J. Pharm. 271(1–2), 145–154 (2004)
19. Wanning, S., Süverkrüp, R., Lamprecht, A.: Pharmaceutical spray
freeze drying. Int. J. Pharm. 488(1–2), 136–153 (2015)
20. Costantino, H., Johnson, O., Zale, S.: Relationship between
encapsulated drug particle size and initial release of recombinant
human growth hormone from biodegradable microspheres.
J. Pharm. Sci. 93(10), 2624–2634 (2004)
21. Leach, W., Simpson, D., Val, T., Anuta, E., Yu, Z., Williams, R.,
Johnston, K.: Uniform encapsulation of stable protein nanoparti-
cles produced by spray-freezing for the reduction of burst release.
J. Pharm. Sci. 94(1), 56–69 (2005)
22. Wang, S.H., Kirwan, S.M., Abraham, S.N., Staats, H.F., Hickey,
A.J.: Stable dry powder formulation for nasal delivery of anthrax
vaccine. J. Pharm. Sci. 101(1), 31–47 (2012)
23. Benson, S., Ellis, D.: Surface areas of proteins. I. Surface areas and
heats of absorption 1. J. Am. Chem. Soc. 70(11), 3563–3569
(1948)
24. Rogers, T.L., Johnston, K.P., Williams III, R.O.: Solution based
particle formation of pharmaceutical powders by supercritical or
compressed fluid CO
2
and cryogenic spray-freezing technologies.
Drug Dev. Ind. Pharm. 27, 1003–1015 (2001)
25. Chow, A., Tong, H., Chattopadhyay, P., Shekunov, B.: Particle
engineering for pulmonary drug delivery. Pharm. Res. 24(3), 411–
437 (2007)
26. Maa, Y.F., Nguyen, P.A., Sweeney, T., Hsu, C.C.: Protein
inhalation powders: spray drying versus spray freeze drying.
Pharm. Res. 16, 249–254 (1999)
27. Bi, R., Shao, W., Wang, Q., Zhang, N.: Spray-freeze-dried dry
powder inhalation of insulin-loaded liposomes for enhanced
pulmonary delivery. J. Drug Target. 16(9), 639–648 (2008)
28. Yu, Z., Garcia, A.S., Johnston, K.P., Williams III, R.O.: Spray
freezing into liquid nitrogen for highly stable protein nanostruc-
tured microparticles. Eur. J. Pharm. Biopharm. 58, 529–537 (2004)
29. Lam, J., Liang, W., Chan, H.: Pulmonary delivery of therapeutic
siRNA. Adv. Drug Deliv. Rev. 64(1), 1–15 (2012)
30. Kuo, J.H., Hwang, R.: Preparation of DNA dry powder for
non-viral gene delivery by spray—freeze-drying: effect of protec-
tive agents (polyethyleneimine and sugars) on the stability of
DNA. J. Pharm. Pharmacol. 56,27–33 (2004)
31. Liang, W., Kwok, P.C.L., Chow, M.Y.T., Tang, P., Ma-son, A.J.,
Chan, H.K., Lam, J.K.W.: Formulation of pH responsive peptides
as inhalable dry powders for pulmonary delivery of nucleic acids.
Eur. J. Pharm. Biopharm. 86,64–73 (2014)
Development of Inhalable Dry Gene Powders …537
