International Journal of Pharmaceutics 643 (2023) 123225

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International Journal of Pharmaceutics

journal homepage: www.elsevier.com/locate/ijpharm

Engineered microparticles of hyaluronic acid hydrogel for controlled

pulmonary release of salbutamol sulphate
Dariush Nikjoo a, b, 1, *, Irès van der Zwaan a, 1, Jonas Rudén c, Göran Frenning a, *
a
Department of Pharmaceutical Biosciences, Uppsala University, P.O. Box 591, 751 24 Uppsala, Sweden
b
Division of Material Science, Department of Engineering Science and Mathematics, Luleå University of Technology, 971 87 Luleå, Sweden
c
Pharmaceutical Development, Orexo AB, 751 05, Uppsala, Sweden

A R T I C L E I N F O A B S T R A C T

Keywords: Most pulmonary drugs are immediate-release formulations with short duration of action. Controlled release
Hyaluronic acid systems provide the ability to deliver drugs at a controlled rate, which helps maintain drug concentrations within
Salbutamol sulphate the therapeutic window for a longer period of time. This study aimed to produce microparticles (MPs) of hy­
Hydrogel
aluronic acid hydrogel (HAGA) loaded with salbutamol sulphate (SS) for controlled release in the lung. The drug-
Controlled release
Pulmonary
loaded MPs were prepared via spray drying and underwent extensive characterization, which revealed that SS
was successfully encapsulated in the HAGA matrix. The prepared MPs (denoted as HASS) ranged in size from 1.6
± 0.4 μm to 1.7 ± 0.5 μm with a fine particle fraction (FPF) of 48–56% and showed improvement in aerodynamic
properties compared to unloaded HAGA hydrogel MPs. In vitro drug release studies performed in a Transwell
system confirmed the potential of the particles to release the drug in a sustained manner. The drug release was
delayed for all formulations, with a t63 between 5 and 30 min, compared to < 1min for pure SS. This study
advances our understanding of the formulation of a highly soluble drug to achieve controlled release in the lung.

1. Introduction metabolism, preventing immediate uptake (Levy 1973).

However, it is unable to sustain a constant drug concentration at the
Drugs play a fundamental role in global healthcare, with nearly half intended site of action and therefore, has limitations for drugs with
of the world’s population consuming more than one dose of medication narrow therapeutic windows (Wen, et al. 2015). In contrast, zero-order
per day in 2020, up from 33% in 2005 (Aitken and Kleinrock 2020). delivery systems are better suited for drugs with narrow therapeutic
Although medications offer undeniable advantages, they also have windows or hepatic toxicity. They achieve this by adjusting the release
drawbacks such as side effects and inconvenient dosing schedules, rate to match the elimination rate, ensuring a steady drug concentration
which frequently lead to poor patient compliance (Sabate 2003). and releasing a lower overall amount of the drug in the body (Wischke
Immediate-release formulations are the most prevalent drug products and Schwendeman 2012; Zhao et al., 2017).
available, which release their active component promptly upon Contemporary drug delivery systems rely on either drug diffusion or
administration. This delivery method is suitable for drugs with wide release from ruptured structures. Biodegradable carriers use polymers
therapeutic windows and long biological half-lives. However, its phar­ that break down through hydrolysis or enzymatic action, forming sol­
macokinetic profile usually has limitations, leading to rapid fluctuations uble products that can be eliminated from the body. Diffusion-based
in drug concentration (Maiti and Sen 2017). systems are driven by the concentration gradient between the formu­
To overcome these limitations, controlled drug-delivery techniques lation and the external environment (Peppas and Brannon-Peppas 2001;
can be implemented (Griffith 2000). FDA-approved controlled release Von Burkersroda, et al. 2002). Microchips, osmotic pumps, and hydrogel
systems exhibit early, rapid release followed by first-order release drug delivery systems function via diffusion. However, microchips and
(Huang and Brazel 2001; Zhong, et al. 2018), where the release rate is osmotic pumps have zero-order release kinetics and drawbacks
proportional to the remaining amount of drug in the formulation. The including limited applicability, higher manufacturing costs, and com­
first-order release kinetics introduce a delay in drug clearance and plex dosing (Laracuente, et al. 2020). On the contrary, the release of

* Corresponding authors.
E-mail addresses: [email protected] (D. Nikjoo), [email protected] (G. Frenning).
1
These authors equally contributed to the present work.

https://doi.org/10.1016/j.ijpharm.2023.123225
Received 11 May 2023; Received in revised form 30 June 2023; Accepted 11 July 2023
Available online 12 July 2023
0378-5173/© 2023 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-
nc-nd/4.0/).
D. Nikjoo et al. International Journal of Pharmaceutics 643 (2023) 123225

drugs from hydrogels is typically governed by Fickian diffusion, which is a need to conduct practical and theoretical investigations using
leads to first-order release kinetics, unless sophisticated formulation advanced dissolution and release tools to replicate the inhaled drug
techniques are employed to modify the release kinetics to zero-order. (Li delivery environment, considering key parameters for effective delivery.
and Mooney, 2016; Prabhu et al., 2015; van de Manakker et al., 2009). Recently we have reported HA-based hydrogels to engineer inhalation
Hydrogels have unique benefits: they are inexpensive to produce with powders for controlled pulmonary drug delivery. The MPs presented a
biodegradable polymers, easily administered, and shield therapeutic spherical shape, swelling, and stability in water with desirable size and
agents from degradation, enabling minimally invasive delivery (Hoare aerodynamic performance (Nikjoo, et al. 2021).
and Kohane 2008; Larrañeta, et al. 2018; Mariani, et al. 2013). Some In this work, we show how HA hydrogel can be used to design for­
hydrogel-based formulations have priority for FDA approval (Patel and mulations for the sustained release of SS as a short-acting bronchodi­
Dalwadi 2013) but further improvements are required to achieve zero- lator, using spray drying to encapsulate the drug in a hydrogel matrix.
order release. The effect of drug composition on the release profile is discussed and an
Drug delivery to the lungs by inhalation offers a targeted therapy optimal range for the preparation of formulation is identified. The
option for respiratory diseases, and it comes with the convenience of produced MPs are characterized in terms of particle size and size dis­
portable inhalers (Du, et al. 2013; Patil and Sarasija 2012; Liang, et al. tribution, morphology, chemical composition, solid dispersion, thermal
2015; Loira-Pastoriza, et al. 2014). Inhalation enables local delivery, stability, aerosolization and powder storage performance and in-vitro
reducing doses and systemic side effects, while improving the drug’s release. Mathematical models are utilized to analyze and describe the
therapeutic index. Moreover, the alveoli’s extensive absorption area release kinetics as well.
enhances efficient absorption of drug molecules via this noninvasive
route (Moore, et al. 2017). Nonetheless, drug molecules not only un­ 2. Materials and methods
dergo rapid clearance from the lungs (Srichana, et al. 2005), but they
also experience substantial local metabolism (Lipworth and Clark 1997). 2.1. Materials
However, due to the short residence time of drug in the lungs, patients
may need to take frequent doses, which can potentially reduce patient Sodium hyaluronate, a biopolymer with a molecular weight ranging
compliance. from 1.01 to 1.8 million Da and a Lot number of 027362, was procured
Sustained drug-release hydrogels have the potential to enhance the from Lifecore (USA). Merck (Germany) provided the charge modifying
effectiveness of inhaled medications and overcome drawbacks of pul­ agent L-Lysine, the crosslinking agent Glutaraldehyde (GA) in a 50%
monary drug administration. However, there is currently no commer­ aqueous solution, and solvents such as ethanol (EtOH), 2-propanol, and
cially available formulation for sustained drug release in the lungs. hydrochloride acid (HCl). Other organic solvents like methanol and
Nonetheless, there are ongoing clinical trials for liposome-based sus­ acetonitrile, which were at least of HPLC grade, were obtained from
tained-release formulations (Loira-Pastoriza, et al. 2014). VWR (France). The buffer used was potassium phosphate with a pH of
Salbutamol sulphate (SS) is a short-acting β2-adrenergic agonist, a 7.5. Salbutamol sulphate, a European Pharmacopoeia (EP) Reference
bronchodilator used to treat asthma and chronic obstructive pulmonary Standard with a D50 of 1.87 ± 0.02 µm and Span of 2.03, as well as
disease (COPD). Physicochemically, SS is an amphoteric compound that Trifluoroacetic acid (TFA) that was at least 99% pure, were both pro­
can occur in aquatic solutions in four different species depending on pH cured from Sigma-Aldrich (Germany). Ultrapure water from PURELAB
(protonated, zwitterionic, neutral, and deprotonated) with pK± a1 = 9.22, flex with a resistivity of 18.2 MΩ and a total organic carbon (TOC) level
pK±a2 = 10.22, pKa1 = 9.60 and pKa2 = 9.84 (Ijzerman, et al. 1984). Prior
◦ ◦
of ≤ 4 ppb was used throughout all experiments.
research indicates that SS has an exceptionally fast onset of action, with
a Tmax between 15 and 30 min (Lipworth and Clark 1997; Moore, et al. 2.2. Preparation of drug-loaded hyaluronic acid hydrogel microparticles
2017; Srichana, et al. 2005). The drug’s rapid onset of action is bene­
ficial as it helps to alleviate symptoms quickly. Nevertheless, owing to The hydrogels based on HA were produced in accordance with our
SS’s high water solubility, the drug’s effects are brief, necessitating more prior research (Nikjoo, et al., 2021). In summary, a mixture of aqueous
frequent administration to sustain symptom relief. Presently, clinical solutions containing 1%w/v HA and 8%v/v GA crosslinker was blended
formulations necessitate repetition every 4 to 6 h (GlaxoSmithKline Inc. and agitated until HA-based hydrogels were formed. The reaction was
2017). The effectiveness of SS can be enhanced by creating a controlled- catalyzed by acid with a pH of 2.6 at room temperature for 24 h. The
release formulation that gradually dispenses the drug over an extended resulting hydrogels were mixed with an appropriate amount of ultrapure
period, leading to more efficient symptom relief. This, in turn, can water and then subjected to centrifugation. The acidic supernatant was
reduce the frequency of dosing, increase patient compliance, and replaced with purified water, and this process was repeated 3–5 times
improve overall treatment outcomes. until the pH was stabilized close to neutral (above 6). The solution was
A nanoliposomal controlled-release formulation of SS demonstrated further assessed using UV spectrophotometry to achieve a zero-
sustained release for up to 14 h (Honmane, et al. 2019). However, the absorbance reading, indicating the absence of residual materials in the
reported 4 h dissolution time for pure SS by the authors suggests that the solution. The purified hydrogel solution produced through chemical
membrane used in the diffusion cell may have had a notable impact on crosslinking was subsequently spray-dried to generate hydrogel micro­
the results. Another study demonstrated the mucoadhesive properties of particles (HAGA).
HA-based formulations of SS. In vitro release of SS lasted for 20 h and In order to incorporate SS into the HA-based hydrogel and create
showed prolonged retention and reduced systemic exposure in vivo (Li inhalable microparticles, the purified hydrogel solutions (prior to spray
et al. 2017). Microparticles of poly (D, L-lactic-co-glycolic acid) and drying) were diluted with ultra-pure water (0.1% w/v) to produce a
respirable grade lactose have also been used for the sustained release of homogeneous solution. This was then mixed with the appropriate
SS (Kumaresan and Sathishkumar 2016). Although diffusion bag studies amount of charge modifying agent (L-Lysine; 5% w/w of hydrogel) and
indicated sustained release for up to 12 h, no control (pure SS) was dissolved SS (to achieve weight ratios of 4:1, 4:2, and 4:4 for HAGA:SS)
included, making it difficult to assess the influence of the dialysis bag on before being fed into a mini spray dryer (B-191, Büchi Labortechnik AG,
the release profile. Switzerland) with a nozzle orifice diameter of 0.7 mm to generate solid
Further investigation is required to explore modified structures of microparticles. The resulting dry powder microparticles loaded with the
hyaluronic acid as a potential biopolymer for controlled inhalation of SS, drug were named HASS (4:1), HASS (4:2), and HASS (4:4), respectively.
a highly soluble drug, such as hydrogel structures obtained through The spray drying process had been optimized in our previous research
spray drying, which are not covered in the literature. Additionally, there (Nikjoo, et al., 2021) and was conducted with a feed rate of 0.1 mL/min,
aspiration gas flow of around 30 L/h, inlet temperature of 110 ◦ C, and

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D. Nikjoo et al. International Journal of Pharmaceutics 643 (2023) 123225

atomized air flow rate of 357 L/h. The dried microparticles were were heated from 25 ◦ C to 500 ◦ C at a uniform heating rate of 10 ◦ C/min
collected and stored in a desiccator (using phosphorus pentoxide under a flow of N2 (50 mL/min).
desiccant) at room temperature (with about 1% relative humidity)
before undergoing further analysis. 2.5.3. Thermogravimetric analysis (TGA)
To conduct thermogravimetric analysis (TGA) on the specimens, a
2.3. Particle size and shape portion weighing 5–10 mg was heated from 20 to 500 ◦ C at a rate of
10 ◦ C/min under an argon atmosphere using the STA 449C Jupiter in­
2.3.1. Particle size analysis strument from NETZSCH, Germany. The obtained data was processed
A laser diffraction apparatus (Coulter LS230, Coulter Corp, Miami, using NETZSCH Ta software.
USA) was used to monitor the size distribution of the samples. To obtain
a homogeneous suspension, 4 mg of microparticles were mixed with 4 2.6. Zeta potential
mL of isopropanol and agitated for 10 min. The emulsion was then
sonicated in a water bath (Branson ultrasonic cleaner, B5210E-MT, Studying the surface charge of MPs by analyzing electrophoretic
Danbury, USA) for 10 min at a frequency of 47 kHz ± 6%. The result­ mobility and calculating the resulting zeta potential is a highly effective
ing well-dispersed admixture was injected into the measurement cell method. The charging characteristics of the samples were studied using
filled with isopropanol, and a particle refractive index of 1.66, a a Malvern Zetasizer Nano-ZS, a widely used instrument from Malvern
dispersant refractive index of 1.37, and an imaginary index of 0.1 were Instruments in Worcester-shire, UK. The zeta potential of the MPs was
used for particle size analysis. Particle diameter was determined using determined in NaCl electrolyte (10 mM) with a pH of 6, at a temperature
the Fraunhofer theory, and the median diameter and SPAN were of 25 ◦ C, following a previously established protocol (Delgado, et al.
calculated and presented as the average of five measurements. The SPAN 2007; Nikjoo, et al. 2021).
provides an indication of the width of the particle size distribution and
was calculated using the following equation: 2.7. Water-sorption measurement
d90 − d10
SPAN = , (1) To measure water vapor adsorption/desorption, a Micromeritics
d50
ASAP 2020 volumetric gas adsorption analyzer (Norcross, GA, USA) was
where the values d90 , d50 , and d10 represent the 90th, 50th, and 10th utilized. Prior to measurement, all samples underwent degassing using a
percentile, respectively, of the particle size distribution by volume. Micromeritics Smart VacPrep060 under dynamic vacuum (1 × 10-4 Pa)
for 5 h at 75 ◦ C. In this study isothermal condition at 298.15 ± 0.1 K
2.3.2. Scanning electron microscopy (SEM) using a water bath was used to perform the adsorption and desorption
SEM analysis was used to examine the morphology of the specimens. tests, under vapor pressures ranging from 0.3 to 2.7 kPa and relative
To prepare the samples, an appropriate amount of material was placed humidity levels ranging from 10 to 85%.
on metal rods covered with Leit-C double-adhesive conductive carbon
tape (Plano GmbH, Wetzlar, Germany). Next, a thin Au/Pd layer was 2.8. Assessment of aerosolization performance
deposited on the samples using a sputter coater from Polaron, Quorum
Technologies Ltd. (Newhaven, UK). The microparticles were then The Fast-Screening Impactor (FSI; Copley Scientific, UK) was utilized
examined using a Leo/Zeiss 1550 microscope (Jena, Germany), which to determine the fine particle fraction (FPF) of MPs, as described pre­
was equipped with SmartSEM software. An acceleration voltage of 2–3 viously (Rudén, et al. 2021). A Screenhaler device (Thalberg, et al.
kV was used to capture images of MPs at varying magnification levels. 2016) was loaded with approximately 1 mg of the formulations, and the
To achieve high-resolution imaging of the surface, the InLens SE de­ FSI experiment was conducted with a flow rate of 60 L/min and a suc­
tector was used. tion time of 4 sec. The quantity of drug in the impactor was measured at
three stages: the throat, the pre-separator, and the filter stage. Following
2.4. Chemical characterization FSI experimentation, 20 mL of H2O with 0.03% TFA was added to the
throat, the pre-separator, and the filter (from the filter stage). After a 20-
The chemical structure of hydrogel MPs was analyzed using Atten­ minute incubation, 500 µL samples were obtained from each of the three
uated Total Reflectance Fourier Transform-Infra-Red (ATR-FTIR) spec­ stages, and the samples were subsequently filtered utilizing an Eppen­
troscopy, performed with a Bruker VERTEX 80v FTIR spectrometer dorf filter by centrifuging at 14500 rpm for 15 min. The samples were
(Germany). The procedure was conducted over a range of 500–4000 quantified using UPLC-UV after being centrifuged.
cm− 1, with a resolution of 4 cm− 1 and 128 scans, and the outcomes were By employing the subsequent formula, the FPF of all formulations
utilized for analysis purposes. was determined:
Filter stage
2.5. Solid state characterization and thermal stability FPF = × 100. (2)
ED
2.5.1. X-ray diffraction (XRD) The fine particle fraction (FPF) is expressed as a percentage. The
The XRD data, utilized for the phase identification of a crystalline SS filter stage corresponds to the quantity of drug that settled on the filter
within a hydrogel structure, were recorded using a PANalytical Empy­ (comprising particles having an aerodynamic diameter of<5 µm), while
rean instrument that had a PIXcel3D detector. The instrument was the emitted dose (ED) denotes the overall drug amount present across all
configured with Cu-Kα radiation at 40 kV and 45 mA settings. High three stages. All measurements were conducted in triplicate.
Score Plus software (v. 3.0.1) was utilized to analyze the data.
2.9. Assessment of drug release
2.5.2. Differential scanning calorimetry (DSC)
For conducting DSC and TGA analyses, samples were retrieved from 2.9.1. In vitro drug release measurements
a desiccator that was maintained at a room temperature with a humidity To assess the release profiles of SS embedded formulations, a Corning
level of roughly 1%. The thermal transition-related enthalpy alterations Transwell system (24 mm inserts, polycarbonate membranes with 0.4
of the samples were investigated using a DSC Q1000 (TA Instruments, µm pore size, obtained from Sigma-Aldrich, Germany) was utilized. The
USA). An aluminum pan, left empty, was employed as a reference. wells were pre-filled with 2.3 mL of medium, and subsequently, the
Samples weighing around 2 mg, which were not sealed hermetically, Transwell were placed containing the formulation (~150 µg), with a

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D. Nikjoo et al. International Journal of Pharmaceutics 643 (2023) 123225

Whatman glass microfiber filter (21 mm, binder-free, grade GF/C) function is here substituted for the fraction of dissolved drug so that Eq.
positioned at the bottom. The experiment was initiated by adding 700 µL (4) takes the form
of medium onto the drug, which was considered as time point 0. The [ ]
du
Transwell was then placed on a shaking table (Heidolph Unimax 1010) tperm + u = A 1 − e− (t/t63 )n
. (5)
dt
and shaken at a speed of 150 rpm. Samples of 200 µL were taken at
various time points, and compensated with 200 µL of fresh medium. At This equation was solved numerically for u and the result was fitted
the end of the experiment, 3 mL of methanol was added to determine the to the experimental release data to obtain estimates of the parameters A,
total dissolved fraction. Prior to quantification of the samples using t63 and n, which henceforth will be referred to as the corrected param­
UPLC-UV (as detailed in Section 2.9.2), excess HA hydrogel was eters. The characteristic permeation time was kept fixed at an inde­
removed from the samples by centrifugation using a centrifugal filter pendently determined value. It is worth noting that the Weibull
unit, at 14500 rpm for 15 min. distribution function reduces to a power law at short times;
To investigate the diffusion profile of pure SS, the same procedure as
1 − e− (t/t63 )n
≃ (t/t63 )n (6)
outlined above was employed. However, in this case, instead of adding
powdered SS onto the Whatman glass microfiber filter, a SS solution was for t≪t63 . Hence, the exponent n may provide some indication of the
pipetted onto it. For each diffusion experiment, approximately 10 µg of release mechanism, similar to the widely used Korsmeyer-Peppas model
SS in solution was added. (Korsmeyer, et al. 1983; Ritger and Peppas 1987).

2.9.2. Analytics: Ultrahigh pressure liquid chromatography (UPLC) 3. Results and discussion
To quantify the specimens, a Waters Acquity UPLC-UV I-Class system
equipped with a BEH C18 column (2.1 × 50 mm) with 1.7 µm particle 3.1. Particle size and shape
size was employed. The mobile phases comprised water with 0.03% TFA
(Mobile phase A) and acetonitrile with 0.03% TFA (Mobile phase B). An Particle size is crucial for inhalation powders as only 1–5 μm parti­
isocratic method was utilized with a mobile phase composition of 97:3 cles can deposit effectively in the lungs. In our previous study, it was
(A:B), a flow rate of 0.8 mL/min, and a total run time of 1.5 min. For all demonstrated that HA hydrogels could be utilized as feed in spray drying
samples, an injection volume of 2 µL was used, while the sample tem­ to generate microparticles (Nikjoo, et al. 2021). Herin, the previously
perature was appointed to 18 ◦ C and the column temperature was optimized spray drying process was employed to encapsulate SS in HA
maintained at 40 ◦ C. The wavelength selected for detection was 224 nm. hydrogels. Table 1 displays that all obtained MP varieties exhibited
The Quantification of SS was accomplished by using a calibration fairly narrow particle size distributions, with a span ranging from 1.1 to
curve using an external standard. To validate the UPLC-UV method, the 1.3. The drug-loaded MPs [HASS (4:1) – HASS (4:4)] had a similar size
inter- and intraday variation of calibration curve samples were deter­ (1.6 ± 0.4 μm to 1.7 ± 0.5 μm) and were slightly smaller than the
mined within a range of 0.1 to 10 µg/mL. unloaded MPs (HAGA; the size of 2.3 ± 1.1 μm). These results demon­
strate that the previously optimized process for chemical synthesis and
2.9.3. Data analysis spray drying can be successfully used to engineer drug-containing par­
The in vitro drug release measurements were used to determine the ticles of suitable size for inhalation.
fraction of released drug u as a function of time t. Microsoft Excel, Particle shape was assessed by SEM. The drug-containing formula­
Matlab (R2022b), and GraphPad Prism 9 were used to analyze and plot tions (HASS), depicted in Fig. 1a–c, exhibited folded and fractured MPs
the dissolution data. Two different methods were utilized to analyze the without uniform spherical geometry. Furthermore, the absence of an
measured release profiles. The first involved fitting the release profiles internal cavity indicates that the MPs underwent further shrinkage post
to the Weibull distribution function, which can be expressed as follows: water evaporation and solidification at the surface of the microparticles.
[ n ]
u = A 1 − e− (t/t63 ) . (3) This morphology is in agreement with prior findings on particles pro­
duced by spray drying from polymers and active ingredients (Carrigy,
In the Weibull distribution function, A represents a constant that takes et al. 2019; Fallacara, et al. 2019; Li et al. 2017; Liu, et al. 2018; Mar­
into account the possibility that some drugs may not be released. t63 is a tinelli, et al. 2017). A surface with corrugations reduces the contact area
characteristic time such that u/A = 1 − e− 1 ≈ 0.63 for t = t63 , and n is a and minimizes agglomeration, thus improving the aerosolization per­
nondimensional exponent. The parameters obtained from this function formance of particles (Shahin, et al. 2019). On the other hand, the empty
describe the kinetics resulting from the dissolution of the drug in the microparticles (HAGA; Fig. 1d) had a spherical shape with smooth
donor compartment and the diffusional release of the dissolved drug surfaces and no visible macroporosity. This is due to the increased sta­
across the membrane into the acceptor compartment. Therefore, these bility of the hydrogel resulting from its crosslinked structure, which
parameters will henceforth be referred to as effective parameters in the minimizes MPs collapse during the drying process (Nikjoo, et al. 2021).
subsequent analysis. Moreover, the presence of polydispersity is evident in all SEM images,
A second analysis was performed based on a mechanistic model which is expected in spray drying using a twin-fluid atomizer. To sum­
previously developed for membrane-type dissolution tests of inhaled marize, the presence of SS in the MPs leads to a smaller particle size and
drugs (Frenning, et al. 2020; van der Zwaan, et al. 2022). This model a corrugated particle surface, which does not seem to depend on the
considers that transport across the membrane (for simplicity henceforth proportion of SS in the studied range (20–50% w/w).
referred to as permeation) is proportional to the drug concentration in
the donor compartment. In this case, the fraction of dissolved drug 1 − s
is related to the fraction of released drug u by an ordinary differential
equation. Table 1
Different formulations and particle size analysis of microparticles.
du
tperm + u = 1 − s. (4) Sample Mean (µm) SDa (µm) Median (µm) Span
dt
HAGA 2.3 1.1 2.2 1.3
Here, tperm is a characteristic time for permeation (referred to as the HASS (4:1) 1.7 0.5 1.8 1.3
characteristic diffusion time in our previous work) and defined so that HASS (4:2) 1.6 0.4 1.7 1.1
the drug concentration in the donor compartment decays as e− t/tperm HASS (4:4) 1.7 0. 5 1.7 1.2

when no solid drug remains. Specifically, the Weibull distribution a

Pooled standard deviation (Killeen 2005).

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D. Nikjoo et al. International Journal of Pharmaceutics 643 (2023) 123225

Fig. 1. SEM images of MPs produced by spray drying of drug-loaded hydrogels: (a) HASS (4:1), (b) HASS (4:2) and (c) HASS (4:4) as well as (d) unloaded
(HAGA) hydrogel.

3.2. Chemical composition carboxylate salts (st) at 1402 cm− 1, a C-O-C (st) band at 1021 cm− 1, and
a band at 1730 cm− 1 indicating the presence of o-ester groups, which
The process of preparing drug-loaded HA hydrogel MPs involved the arise from either crosslinking through the use of epoxides or ion ex­
chemical crosslinking of HA, which was followed by spray drying. To change of the carboxyl groups of HA from –COO-Na + to –COOH
analyze the chemical composition of the crosslinked MPs and potential (Antunes, et al. 2010; Nikjoo, et al. 2021; Zhao, 2006).
interactions with the embedded SS, Fig. 2 depicts the results of FTIR Several characteristic principal peaks were identified in the FTIR
spectroscopy. The FTIR spectrum of the empty (HAGA) MPs displayed spectrum of SS, including sharp peaks resulting from C-O stretching at
the same characteristic peaks as previously reported for HA crosslinked around 1100 cm− 1 and O–H bending at around 1500 cm− 1 (Nath, et al.
with GA, indicating that successful crosslinking did occur (Nikjoo, et al. 2010).
2021). Some of the identified peaks in the FTIR spectrum include a The FTIR spectra of the drug-loaded (HASS) MPs showed the char­
characteristic peak at 2993 – 3716 cm− 1 corresponding to the stretching acteristic peaks of SS without any significant shifts, indicating the lack of
vibration of –OH and –NH functional groups, an alkane –CH band (st) at chemical interactions between the drug and polymer in the solid state
2920 cm− 1, a C = O (st) band at 1600 cm− 1, symmetric bands of (Kumaresan and Sathishkumar 2016). As expected, the intensity of
peaks originating from SS increased with increasing SS content [from
20% for HASS (4:1) to 50% for HASS (4:4)], while the intensity of peaks
originating from HAGA decreased.

3.3. Solid state characteristics and thermal stability

The solid state and thermal stability were investigated by a combi­

nation of XRD, DSC, and TGA. As seen in Fig. 3, the diffractograms of the
drug-containing (HASS) and empty (HAGA) hydrogel MPs exhibited a
broad hump around 20◦ but no distinct peaks, demonstrating their
amorphous structure. In contrast, the diffractogram of pure SS displayed
sharp peaks (at 10.54 , 15.23 , 18.38 , 22.76 , 23.05 , 28.53 , and
◦ ◦ ◦ ◦ ◦ ◦

29.34 ), indicating its crystalline nature (Honmane, et al. 2019). As

◦

expected for spray-dried formulations, the particle formation process led

to the amorphization of SS (Li et al. 2017).
Fig. 4 shows the further investigation of the solid state using DSC.
The DSC thermogram of pure SS displayed a sharp endothermic peak at
200 ◦ C due to melting, and a second endothermic transition at 270 ◦ C,
which is associated with the decomposition of SS (Honmane, et al. 2019;
Li et al. 2017). The thermogram of empty (HAGA) microparticles pre­
sented a broad endothermic peak below 100 ◦ C, due to the loss of
Fig. 2. FTIR spectra of SS along with the drug-loaded (HASS) and unloaded moisture remaining after the initial drying process, and two exothermic
(HAGA) hydrogel MPs. peaks at higher temperatures (200–250 ◦ C), related to degradation

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D. Nikjoo et al. International Journal of Pharmaceutics 643 (2023) 123225

Fig. 3. XRD spectra of the pure drug (SS), drug-loaded (HASS), and unloaded
Fig. 5. TGA thermograms of SS along with the drug-loaded (HASS), and
(HAGA) hydrogel MPs. unloaded (HAGA) hydrogel MPs.

evaporation in the range of 20–150 ◦ C. Additionally, degradation of the

backbone was observed at 200 ◦ C (Nikjoo, et al. 2021). Melting and
subsequent degradation of pure SS also occurred around 200 ◦ C (cf.
Fig. 5) (Felix, et al. 2009). The major weight loss of the drug-containing
(HASS) MPs around 200 ◦ C can thus be interpreted as a combination of
degradation of both SS and HA. The results suggest that the MPs can
withstand heating during spray drying, up to at least 100–120 ◦ C, if such
high temperatures occur. Therefore, the MPs are also expected to be
stable during storage at room temperature.

3.4. Zeta potential

The calculated zeta potential reflects the electrical potential near the
particle surface at the shear (or slip) plane and was found to be: − 12.8
± 10.3, − 7.0 ± 1.3, and − 2.5 ± 1.0 mV for HASS (4:1), HASS (4:2), and
HASS (4:4) formulations, respectively. The results indicate a lower
negative charge of drug-containing MPs compared to the HAGA
formulation without L-lysine (-17.4 ± 4.2). The zeta potential is a
measure of the surface charge in the liquid state. A correlation between
Fig. 4. DSC thermograms of the pure drug (SS), drug-loaded (HASS), and the surface charge in liquid (zeta potential) and in the gas phase has
unloaded (HAGA) hydrogel MPs. been established for polymer nanoparticles (Ouadah, et al. 2013) and
the zeta potential could therefore be used as an indication of the surface
(Collins and Birkinshaw 2008). In contrast, the thermograms of the charge of particles in the dry state. The surface charge of MPs plays a
drug-containing (HASS) MPs exhibited relatively featureless curves. The crucial role in their aerodynamic properties as well as their mucoad­
magnitude of the low-temperature endothermic event, attributed to hesive properties, mucopenetration, and cellular uptake during mucosal
water evaporation, decreased with increasing drug content. No clear drug delivery. Studies have indicated that positively charged MPs
glass transition could be observed, and any melting peak, if present, was exhibit greater mucoadhesion, while negatively charged MPs are known
not prominent, likely due to overlapping with the exothermic peak to enhance mucopenetration (Poinard, et al. 2019; Russo, et al. 2016;
arising from hydrogel degradation. Nevertheless, these results are in line Sandri, et al. 2010). Although HA-based MPs have been proven to in­
with the high dispersion and amorphization of SS in the HASS formu­ crease mucoadhesivity and drug penetration, further studies are
lations (Li et al. 2017). required to calculate the effect of surface charge on each parameter in
The moisture content and degradation stages of the MPs were further the system.
analyzed by TGA; see Fig. 5. The drug-containing formulations exhibited
a moisture content of 1.2 – 2.7% (w/w), which is significantly lower 3.5. Water-sorption
than the 6.4% observed for the empty (HAGA) MPs. Typically, low
moisture content (<10% w/w) leads to enhanced stability and aero­ The hygroscopic nature of the MPs was confirmed by water vapor
solization properties (Shahin, et al. 2019). The higher moisture content sorption measurements (Fig. 6), which showed a reversible sorption
observed in the empty (HAGA) MPs can be attributed to their hygro­ mechanism with no chemical interaction between water molecules and
scopic nature, which is due to the presence of various hydrophilic the formulations. In this regard, HAGA showed higher water absorption
functional groups, including –OH, –COO, and –NH. Furthermore, the (35 mmol/g) compared to the drug-loaded formulations (HASS; be­
absence of SS in HAGA microparticles may prevent the internal desic­ tween 8 and 14 mmol/g), indicating higher hygroscopicity. The
cant effect, which could contribute to the higher moisture content. The decrease in hygroscopicity of the HASS formulations from HASS (4:1) to
TGA analysis showed that the HAGA microparticles underwent water HASS (4:4) can be explained by the decrease of HAGA hydrogel (with
hydrophilic functional groups) in favor of SS in the formulations.

6
D. Nikjoo et al. International Journal of Pharmaceutics 643 (2023) 123225

loaded MPs in comparison to empty ones can be attributed to the

corrugated surface of the former. The fact that the amount of SS did not
affect the aerosolization is consistent with this view; the particle shape
was insensitive to the amount of SS in the investigated range (20 – 50%
w/w; cf. Sec. 3.1). The FPF of the formulations showed promising
aerosol performance compared to clinical formulations of SS with an
FPF of around 20% (Palander, et al. 2000).
Additionally, the lower negative charge observed for drug-loaded
MPs, as determined by their zeta potential, may contribute to the
reduced aggregation and higher FPF values. The deposition of inhaled
powder in the lungs is controlled by a number of mechanisms, one of
which is electrostatic charge. When it comes to HA-based MPs, a strong
negative charge can lead to high cohesion during deposition. It is worth
noting that the electrostatic charge of the plastic material used in inhaler
manufacturing and operation can also have a significant impact on
material loss (Karner and Urbanetz 2011; Martinelli, et al. 2017). To
address this problem, charge-modifying agents have been found effec­
tive in neutralizing the negative charge of MPs, improving their flow
characteristics, and reducing particle cohesiveness. These agents can
consist of either neutral molecules or positively charged excipients
Fig. 6. Water sorption/desorption isotherms of MPs. (Martinelli, et al. 2017; Singh, et al. 2012). In this study, a fixed con­
centration of L-lysine (5% w/w) was added to the HAGA hydrogel so­
Overall, the formulations remained physically stable despite exposure to lution before spray drying to neutralize the negative surface charge.
high humidity levels for a brief duration.
As was the case for pure HAGA microparticles (Nikjoo, et al. 2021), 3.7. In vitro drug release
the shape of the isotherms (consistent with Type IV) points towards
multilayer water sorption with a weak interaction between adsorbent In vitro drug, dissolution/release from the drug-containing (HASS)
and adsorbate. Moreover, the presence of hysteresis loops (of H3 type) formulations was determined using the Transwell setup and compared
suggests a mesoporous structure with slit-shaped pores (Rouquerol, et al. to that of pure SS. The selection of SS as the model drug for the HA
1999; Sing, et al. 1985; Thommes, et al. 2002). formulations was based on its high solubility and rapid onset of action.
However, its fast dissolution rate poses a challenge in determining its
3.6. Aerosolization performance dissolution profiles in standard dissolution media for in vitro pulmonary
dissolution tests (Tay, et al. 2018). Therefore, water was used as a
To evaluate the aerosolization efficiency of the formulated MPs, the dissolution medium in this work. By not using a more biorelevant
fine particle fraction (FPF) was measured using the FSI. The FPF refers to dissolution medium, such as PBS or simulated lung fluid (SLF), the
the proportion of the administered dose that reaches the lungs and has dissolution profiles could possibly correlate less to the in vivo situation.
an aerodynamic size of 5 µm or less. With an emitted dose (ED) of 143, However, as the focus of this research was on the development of a
223 and 365 µg for HASS (4:1), HASS (4:2) and HASS (4:4), respectively, controlled-release formulation, it was considered acceptable. It was
all three drug-loaded formulations (HASS) exhibited a similar FPF be­ more important to be able to obtain a full dissolution profile of all for­
tween 48 and 56% (Fig. 7) that was significantly larger than that of the mulations, including the pure SS, to verify if there was a sustained
empty formulation (HAGA). The higher FPF values observed for drug- release created by the HA formulations.
To validate that the dissolution profiles of the formulations were not
restricted by transport through the membrane of the Transwell system, a
80 diffusion profile of SS in solution was also recorded in the Transwell
system (data not shown). The solution exhibited an expected permeation
profile, with the fraction absorbed being proportional to 1 − e− t/tperm , with
a tperm of 8.2 min.
60
3.7.1. Effective dissolution profiles
The effective dissolution profiles of the drug-containing formulations
FPF (%)

(HASS) and the pure drug (SS) can be seen in Fig. 8. The experimental
40 data were fitted with the Weibull distribution equation (Eq. (3), repre­
sented by solid lines, and the effective dissolution parameters were
extracted from the fits. It can be observed that the dissolution profile of
pure SS is the fastest overall with an effective t63 of 8.4 min. This value
20 was not significantly different from the characteristic permeation time
(8.2 min), indicating that transport across the Transwell membrane
(permeation) was the rate-limiting process for pure SS. All formulations
showed a significantly slower effective dissolution compared to pure SS.
0 Moreover, all three formulations showed a different dissolution profile
and dissolution rate compared to each other. The slowest effective
A

)
:1

:2

:4

dissolution (t63 of 35.8 min) was observed for HASS (4:1) with 20% drug
G

(4

(4

(4
A

loading. A significantly faster effective dissolution (t63 of 22.5 min) was

H

SS

SS

SS

seen for HASS (4:2), with 33% loading. The fastest effective dissolution
A

A
H

(t63 of 16.6 min), which still was significantly slower than that of pure
Fig. 7. Fine particle fraction (FPF) of MPs. SS, was found for HASS (4:4), with 50% drug loading. Therefore, it can

7
D. Nikjoo et al. International Journal of Pharmaceutics 643 (2023) 123225

1.0 1.0
(a)

Fraction permeated (-)

Fraction released (-)

0.5 SS 0.5 SS
HASS (4:1) HASS(4:1)
HASS (4:2) HASS(4:2)
HASS (4:4) HASS(4:4)
0.0 0.0
0 100 200 1.0
Time (min) (b)

Fraction dissolved (-)

Fig. 8. Release profiles obtained for SS and drug-containing microparticles
(HASS). The solid lines represent fits of the Weibull distribution function.

0.5 SS
be concluded that all HA hydrogel based formulations exhibited
extended drug release, and this effect became more pronounced as the HASS(4:1)
drug content decreased. It is plausible that a less dense network will be HASS(4:2)
formed in the hydrogel structure for the formulations containing less
HA. HASS(4:4)
To normalize the dissolution profiles, the total amount of dissolved 0.0
0 100 200
drug was determined by adding methanol after the final time point.
However, this method resulted in the extraction of some drugs from the Time (min)
drug-containing HASS formulations, as well as an increase in the amount
Fig. 9. Determination of the corrected dissolution profiles: (a) Fit of the
of pure SS in solution. The extracted rate parameters (the t63 values) are transformed Weibull distribution function to the experimental release data and
independent of the normalization used. (b) fraction dissolved SS.
Due to a limited amount of each formulation, the formulations as
well as the pure SS were manually deposited on the filter in the Trans­
well rather than dispersed by using an impactor (such as the Andersen 40
Cascade Impactor). The amount of powder that was deposited on the
filters was similar for all formulations (~150 µg for each of the three Effective t63
replicas) and was spread out over the filter as much as possible to avoid 30 Corrected t63
the formation of large agglomerates on the filter. Given that the amount
t63 (min)

of powder used was minimal and was evenly distributed across the filter,
20
it did not form a layer that could obstruct the pores in the Transwell
system. Additionally, since all the formulations and pure SS were sub­
jected to the same treatment, this procedure is not expected to have a 10
significant impact on the observed differences between the dissolution
profiles.
0
0 50 100
3.7.2. Corrected dissolution profiles
A second analysis was used to minimize the influence of the Trans­ % API
well membrane on the dissolution profiles. To this end, the fraction
Fig. 10. Comparison between effective (accounting for dissolution and
absorbed drug u, as obtained from Eq. (5), was fitted to the experimental permeation) and corrected (dissolution only) t63 .
data, to yield the results presented in Fig. 9a. From the fits, corrected
dissolution parameters were extracted for each of the drug-containing
release rate reduces upon encapsulation of SS with HAGA hydrogel, yet
formulations and pure SS. The corrected dissolution profiles, obtained
there is no explanation provided for the underlying mechanisms causing
by using the corrected dissolution parameters in the Weibull distribution
this effect. Some insights may be gained from the release exponent n,
[Eq. (3)], are displayed in Fig. 9b. As can clearly be seen, the dissolution
extracted from the fits displayed in Fig. 9a. The release exponent ranged
of pure SS can for all practical purposes be considered to be instanta­
from 0.35 ± 0.04, obtained for HASS (4:2) via 0.42 ± 0.09 for HASS
neous. On the contrary, the release of SS from the formulations is
(4:4) to 0.53 ± 0.06 for HASS (4:1). The findings align with the n values
retarded, and the extent of retardation decreases with the increasing
documented in the literature for microparticles (Agnihotri, et al. 2004;
amount of SS in the formulation, as would be anticipated from the un­
Dodov, et al. 2009; Kumbar, et al. 2002). In the case of monodisperse
corrected profiles discussed above. As clearly seen in Fig. 10, drug
swellable spherical systems, a value of n = 0.43 corresponds to Fickian
release is retarded for all formulations, with corrected t63 ranging from
diffusion, a value of n = 0.89 indicates Case-II transport, and 0.43 < n
about 5 min for HASS (4:4), with 50% drug, to about 30 min for HASS
< 0.89 is characteristic of anomalous diffusion (both diffusion and
(1:4), with 20% drug compared to < 1min for pure SS. Moreover, a
swelling controlled release) (Dodov, et al. 2009; Ritger and Peppas
comparison between the corrected and effective t63 indicates that the
1987). Therefore, it seems that the obtained values signify Fickian
effect of the membrane decreases with decreasing dissolution rate, as
diffusion, except for the HASS (4:1) formulation, where the value
anticipated.
strongly suggests both swelling-controlled and diffusion-controlled
release. The reason for this is the larger proportion of HA hydrogel in
3.7.3. Mechanisms of drug release and prolongation
this particular formulation compared to SS. It is worth noting, though,
The outcomes presented above demonstrate that the dissolution/
that if there is polydispersity, the release exponent values would

8
D. Nikjoo et al. International Journal of Pharmaceutics 643 (2023) 123225

significantly decrease, making it difficult to exclude Case-II transport as Dodov, M.G., Calis, S., Crcarevska, M., Geskovski, N., Petrovska, V., Goracinova, K.,
2009. Wheat germ agglutinin-conjugated chitosan–Ca–alginate microparticles for
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