Open AccessArticle

Evaluation of Permeability, Safety, and Stability of Nanosized Ketoprofen Co-Spray-Dried with Mannitol for Carrier-Free Pulmonary Systems

Heba Banat

Ilona Gróf

²,

Mária A. Deli

Rita Ambrus

^1,*

and

Ildikó Csóka

Institute of Pharmaceutical Technology and Regulatory Affairs, Faculty of Pharmacy, University of Szeged, Eötvös u. 6, H-6720 Szeged, Hungary

Institute of Biophysics, HUN-REN Biological Research Centre, Temesvári Blvd. 62, H-6726 Szeged, Hungary

Author to whom correspondence should be addressed.

Appl. Sci. 2025, 15(3), 1547; https://doi.org/10.3390/app15031547

Submission received: 30 December 2024 / Revised: 30 January 2025 / Accepted: 1 February 2025 / Published: 3 February 2025

(This article belongs to the Topic Applied System on Biomedical Engineering, Healthcare and Sustainability 2024)

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Figure 1
Cell viability of (A) A549 alveolar and (B) CFBE bronchial epithelial cells after 1-h treatments with KTP raw and the developed F0, F1 samples measured by impedance. Values are presented as means ± SD, n = 5–6. Statistical analysis: ANOVA followed by Dunett’s test. *** p < 0.001 compared to the control group. C, control; KTP, ketoprofen raw; TX-100, Triton X-100 detergent. "> Figure 2
Permeability of KTP raw, F0, and F1 DPIs (50 μg/mL KTP concentration in the donor compartment) across the co-culture model of (A) alveolar epithelial cells and (B) bronchial epithelial cells after 30- and 60-min assay time. Values are presented as means ± SD, n = 4. Statistical analysis: ANOVA followed by Dunett’s test. ** p < 0.01, *** p < 0.001. "> Figure 3
Evaluation of the tightness of the A549 alveolar and the CFBE bronchial epithelial co-culture models after the KTP raw, F0, and F1 DPIs permeability experiment by (A,C) transepithelial electrical resistance (TEER) measurement and the (B,D) permeability of fluorescein and albumin marker molecules. Values are presented as means ± SD, n = 4. Statistical analysis: ANOVA followed by Dunett’s test. "> Figure 4
Validation of barrier integrity in A549 alveolar epithelial and CFBE bronchial epithelial cell layers after permeability assays with KTP raw, F0, and F1. Immunostaining for (A) β-catenin and (B) zonula occludens protein-1. Cyan: cell nuclei; red: junctional proteins; scale bar: 20 μm. C, control; KTP, ketoprofen raw. "> Figure 5
Laser diffraction results of particle size (D[0.5] (µm)) and particle size distribution (Span) for sample F1 (freshly prepared; F1—0m, and after one month; F1—1m and 3 months; F1—3m of storage). Results are presented as means ± SD, n = 3., **** p < 0.0001 significantly different. "> Figure 6
SEM images of F1 sample at (a) 0 months, (b) 1 month, and (c) 3 months. "> Figure 7
DSC thermal analysis of (A) raw ketoprofen (KTP) and the physical mixture (PM), and (B) sample F1, analyzed as a freshly prepared sample (F1—0m) and after 1 (F1—1m) and 3 (F1—3m) months of storage. "> Figure 7 Cont.
DSC thermal analysis of (A) raw ketoprofen (KTP) and the physical mixture (PM), and (B) sample F1, analyzed as a freshly prepared sample (F1—0m) and after 1 (F1—1m) and 3 (F1—3m) months of storage. "> Figure 8
XRPD structural analysis of (A) raw ketoprofen (KTP) and the physical mixture (PM), and (B) sample F1, analyzed as a freshly prepared sample (F1—0m) and after 1 (F1—1m) and 3 (F1—3m) months of storage. The circles highlight the characteristic peaks of KTP in the raw drug, physical mixture, as well as fresh and stored formulations. "> Figure 9
In vitro aerodynamic characteristics (MMAD, FPF, and EF) of sample F1 were analyzed as a freshly prepared sample (F1—0m), and after 1 (F1—1m) and 3 (F1—3m) months of storage, at a flow rate of 60 L/min. Data are presented as means ± SD (n = 3 independent measurements). Statistical significance is indicated, **** p < 0.0001 significantly different. "> Figure 10
In vitro aerodynamic distribution of the F1 sample was analyzed as a freshly prepared sample (F1—0m), and after 1 (F1—1m) and 3 (F1—3m) months of storage, at a flow rate of 60 L/min. Data are presented as means ± SD (n = 3 independent measurements). Statistical significance is indicated as follows: * p < 0.05, ** p < 0.01. "> Figure 11
In vitro release study of raw KTP, and spray-dried sample (F1) was analyzed as a freshly prepared sample (F1—0m) and after 1 (F1—1m) and 3 (F1—3m) months of storage, in simulated lung media (SLM). Results are expressed as mean ± SD (n = 3 independent measurements). ">

Versions Notes

Abstract

Pulmonary drug delivery presents a promising approach for managing respiratory diseases, enabling localized drug deposition and minimizing systemic side effects. Building upon previous research, this study investigates the cytotoxicity, permeability, and stability of a novel carrier-free dry powder inhaler (DPI) formulation comprising nanosized ketoprofen (KTP) and mannitol (MNT). The formulation was prepared using wet media milling to produce KTP-nanosuspensions, followed by spray drying to achieve combined powders suitable for inhalation. Cell viability and permeability were conducted in both alveolar (A549) and bronchial (CFBE) models. Stability was assessed after storage in hydroxypropyl methylcellulose (HPMC) capsules under stress conditions (40 °C, 75% RH), as per ICH guidelines. KTP showed good penetration through both models, with lower permeability through the CFBE barrier. The MNT-containing sample (F1) increased permeability by 1.4-fold in A549. All formulations had no effect on cell barrier integrity or viability after the impedance test, confirming their safety. During stability assessment, the particle size remained consistent, and the partially amorphous state of KTP was retained over time. However, moisture absorption induced surface roughening and partial agglomeration, leading to reduced fine particle fraction (FPF) and emitted fraction (EF). Despite these changes, the mass median aerodynamic diameter (MMAD) remained stable, confirming the formulation’s continued applicability for pulmonary delivery. Future research should focus on optimizing excipient content, alternative capsule materials, and storage conditions to mitigate moisture-related issues. Hence, the findings demonstrate that the developed ketoprofen–mannitol DPI retains its quality and performance characteristics over an extended period, making it a viable option for pulmonary drug delivery.

Keywords:

nanotechnology; particle engineering; spray drying; carrier-free; DPI; stability; permeability; combined powders

1. Introduction

Pulmonary drug delivery has emerged as a vital approach for treating respiratory and systemic diseases due to the lung’s unique physiological features. Direct drug administration to the lungs offers significant advantages, including avoidance of first-pass metabolism, rapid therapeutic onset, and localized drug action, which minimizes systemic side effects [1]. Furthermore, non-invasive administration enhances patient compliance, making this route particularly appealing for chronic conditions such as asthma, chronic obstructive pulmonary disease (COPD), tuberculosis, and cystic fibrosis (CF) [2,3,4,5,6].

Among the various inhalation devices, dry powder inhalers (DPIs) are preferred due to their portability, ease of use, and the solid-state stability of the formulations [7]. Traditionally, carrier-based DPIs, which rely on excipients like lactose for drug dispersion, have faced limitations in achieving efficient drug deposition in the respiratory tract [8]. To address these challenges, carrier-free DPI systems have been developed, incorporating active pharmaceutical ingredients (APIs) and excipients into a single, cohesive formulation [9]. Particle size and aerodynamic properties play critical roles in the success of pulmonary formulations, with particles in the range of 1–5 µm being essential for deep lung deposition.

Particle engineering plays a pivotal role in DPI development by tailoring the surface properties of drugs, carriers, or excipients. This approach balances interparticle forces, enhances stability during processing and storage, and improves aerodynamic performance [10]. Wet media milling is a well-established pharmaceutical technique for producing stable nanosuspensions of poorly water-soluble APIs. The scalability of wet milling can be enhanced using modeling techniques to optimize industrial applications, reducing costs and improving efficiency [11]. Spray drying, another scalable and efficient particle engineering technique, has been employed to produce inhalable powders with favorable properties, including ease of use and extended shelf-life stability [12]. Recently, combining these two approaches has garnered attention for developing DPI formulations.

Stability is a fundamental consideration in pharmaceutical product development, especially for inhalable formulations like DPIs, where storage conditions can significantly influence particle morphology and aerodynamic performance [13]. While solid dosage forms generally exhibit better stability than liquid formulations, nanosized APIs in solid forms are susceptible to aggregation, which can affect their functionality. Crystalline APIs are often preferred for their superior physical stability compared to amorphous forms [14], though amorphous APIs may provide enhanced dissolution and bioavailability. Striking a balance between these properties is essential to address stability challenges such as crystal growth, sedimentation, and agglomeration, ensuring both the safety and efficacy of the final product over its shelf life [15].

Our research group previously developed a carrier-free DPI formulation combining nanosized ketoprofen (KTP) with mannitol (MNT), targeting pulmonary inflammation and improving mucus clearance [16]. By employing wet media milling and spray drying, we successfully produced an optimized formulation with favorable particle size, morphology, and aerodynamic performance. The dual functionality of this formulation allows KTP nanoparticles to address inflammation while MNT enhances mucus clearance. The promising results of this approach warrant further investigations into its pulmonary applicability, particularly its permeability through lung cell lines and long-term stability.

Building upon this foundation, the current study aims to extend the investigation of this optimized formulation. Specifically, we sought to evaluate its cytotoxicity, permeability, and stability under stress conditions (40 °C and 75% RH) as per ICH guidelines. To explore the influence of MNT on lung permeation, an MNT-free reference sample was included. Two lung cell lines, A549 (alveolar epithelial cells) and CFBE (bronchial epithelial cells) were utilized to assess cellular permeability and safety. Furthermore, the physical and aerodynamic properties of the stored formulation were examined using advanced analytical techniques, including laser diffraction, scanning electron microscopy (SEM), differential scanning calorimetry (DSC), X-ray powder diffraction (XRPD), drug release, and Andersen cascade impaction. By addressing these critical aspects, this study seeks to validate the formulation’s suitability for pulmonary drug delivery and to guide future optimization for clinical applications.

2. Materials and Methods

2.1. Materials

KTP (TCI Chemicals, Shanghai, China) served as the model drug. Polyvinyl alcohol (PVA) (ISP Customer Service GmbH, Cologne, Germany) and sodium dodecyl sulfate (SDS) (VWR Chemicals, Leuven, Belgium) functioned as the stabilizers. MNT (Mo-lar Chemicals Ltd., Budapest, Hungary) and leucine (AppliChem GmbH, Darmstadt, Germany) were incorporated into the spray drying process. The distilled water used throughout the study was sourced from a Milli-Q system (Millipore, Merck KGaA, Darmstadt, Germany). All reagents used in cell line measurements were purchased from Merck Life Sciences Ltd., Budapest, Hungary, unless otherwise indicated.

2.2. Preparation Techniques

KTP, due to its low water solubility, requires a pre-dispersion step. A KTP-containing nanosuspension was prepared following our previously established method [16]. Briefly, the drug particles were dispersed in a water-based stabilizer solution composed of polyvinyl alcohol (1% w/v) and sodium dodecyl sulfate (0.1% w/v). Wet media milling was then conducted using a planetary ball mill (Retsch Planetary Ball Mill PM 100 MA, Retsch GmbH, Haan, Germany) with 20.00 g of 0.3 mm zirconium dioxide (ZrO₂) beads. Milling took place in a 50 mL chamber at 400 rpm for 60 min.

Inhalable dry powders were produced using a Mini Spray Dryer (Büchi B-191, Flawil, Switzerland) with parameters optimized based on our previous study [16]: inlet temperature 90 °C, outlet temperature 60 °C, aspirator at 80%, pump rate at 2%, and a flow rate of 600 L/min. MNT, widely used as a mucolytic agent in cystic fibrosis therapy, was combined with KTP in a 1:1 mass ratio to evaluate its therapeutic potential. To improve dispersibility, leucine was also incorporated alongside MNT, resulting in a formulation designated as F1. For comparison, a reference formulation without MNT or leucine was prepared and labeled as F0.

2.3. Cell Line Measurement

2.3.1. Cell Culture

The A549 cells (ATCC, Manassas, VA, USA), a human immortalized alveolar type II-like lung epithelial cell line, were grown in Dulbecco’s modified Eagle medium (DMEM; Gibco, Life Technologies, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS, Pan Biotech, Aidenbach, Germany) and 50 μg/mL gentamicin. The CFBE human bronchial epithelial cells were grown in Minimum Eagle Medium (MEM; Gibco, Life Technologies, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (Pan-Biotech GmbH, Aidenbach, Germany), stable glutamine (Glutamax, 2 mM), puromycin (2 μg/mL) and 50 μg/mL gentamycin. Both cell lines were grown in a humidified incubator with 5% carbon dioxide (CO₂) at 37 °C. All plastic surfaces were coated with 0.05% rat tail collagen in sterile, distilled water before cell seeding in culture dishes and the medium was changed every 2 days. For permeability measurements, the alveolar and the bronchial epithelial cells were cultured in the presence of human vascular endothelial cells to create co-culture models. The endothelial culture media (ECM-NG, Sciencell, Carlsbad, CA, USA) was supplemented with 5% FBS, 1% endothelial growth supplement (ECGS, Sciencell, Carlsbad, CA, USA), and 0.5% gentamicin. For permeability assays the epithelial cells were cultured on inserts (CellQuart, PET membrane, 3 μm pore size, 1.12 cm²) placed in 12-well plates and kept in co-cultures for 10 days. To prepare the co-culture model, endothelial cells (<P9) were passaged (8 × 10⁴ cells/cm²) to the collagen-type-IV (100 μg/mL) coated bottom side of tissue culture inserts and epithelial cells were seeded (1 × 10⁵ cells/cm²) to the upper side of the membranes which were coated with rat tail collagen.

2.3.2. Dilution of Dry Powders for the Cellular Assays

KTP and carrier-free dry powders were re-dispersed in 5 mL cell culture medium for the cell viability measurements or in Ringer-HEPES buffer (118 mM sodium chloride (NaCl), 4.8 mM potassium chloride (KCl), 2.5 mM calcium chloride (CaCl₂), 1.2 mM magnesium sulfate (MgSO₄), 5.5 mM D-glucose, 20 mM Hepes, pH 7.4) containing 1% FBS for the permeability experiments. The final KTP concentration was 0.5 mg/mL in these samples. After 25 min of sonication, the samples were further diluted for the cell viability and permeability experiments.

2.3.3. Cell Impedance Kinetics

Kinetics of the alveolar and bronchial epithelial cell reaction to the active agent and DPIs was monitored by impedance measurement at 10 kHz (RTCA-SP instrument; ACEA Biosciences, San Diego, CA, USA). Impedance measurement is a label-free, real-time, non-invasive method, and correlates linearly with adherence, growth, number, and viability of cells [17]. For background measurements 50 µL cell culture medium was added to the wells, then cells were seeded at a density of 5 × 10³ cells/well to rat tail collagen-coated 96-well plates with integrated gold electrodes (E-plate 96, ACEA Biosciences). Cells were cultured for 4 days and monitored every 10 min until the end of experiments. At the beginning of plateau phase of growth, cells were treated with suspensions of KTP raw and DPI samples (50-300-500 μg/mL) for 48 h. Triton X-100 detergent (10 mg/mL) was used as a reference compound inducing cell toxicity. Cell index was defined as Rn–Rb at each time point of measurement, where Rn is the cell–electrode impedance of the well when it contains cells and Rb is the background impedance of the well with the medium alone.

2.3.4. Permeability Measurements

For the permeability experiments, the inserts were transferred to 12-well plates containing 1.5 mL Ringer-HEPES buffer in the lower (acceptor) compartments. In the apical (donor) compartments 0.5 mL buffer was pipetted with suspensions of KTP raw or DPI samples. To avoid the unstirred water layer effect, the plates were kept on a horizontal shaker (120 rpm) during the assay. The assays lasted for 60 min. Samples from both compartments were collected and the KTP concentration was measured by UV spectrophotometry. The apparent permeability coefficient (P_app) was calculated as previously described [18]. The cleared volume was determined based on the concentration of the tracer in the acceptor compartment (Δ[C]_A) after 60 min (Δt), the concentration in the donor compartment at time zero ([C]_D), the volume of the acceptor compartment (V_A; 1.5 mL), and the surface area available for permeability (A; 1.12 cm²), using Equation (1):

\begin{matrix} P_{a p p} (cm / s) = \frac{∆ {[C]}_{A} \times V_{A}}{A \times [C] D \times ∆ t} \end{matrix}

(1)

2.3.5. Barrier Integrity Measurements After Permeability Experiments

Transepithelial electrical resistance (TEER) reflects the tightness of the intercellular junctions closing the paracellular cleft, Therefore, the overall tightness of cell layers of biological barriers. TEER was measured to check the barrier integrity by an EVOM volt-ohmmeter combined with STX-2 electrodes (World Precision Instruments, Sarasota, FL, USA), and was expressed relative to the surface area of the cell layers as Ω × cm². Resistance of cell-free inserts was subtracted from the measured values. TEER was measured before and after the permeability experiments.

After the 60-min permeability assay, the tightness of the respiratory co-culture models was determined by two passive permeability marker molecules with different sizes. In the donor compartments, 0.5 mL Ringer-HEPES was added containing sodium fluorescein (SF; 10 μg/mL; Mw: 376 Da) and Evans blue labeled albumin complex (EBA; 167.5 μg/mL Evans blue dye and 10 mg/mL bovine serum albumin; MW: 67.5 kDa). The inserts were kept in 12-well plates on a horizontal shaker (120 rpm) for 30 min. The concentrations of the marker molecules in the samples from the acceptor compartments were determined by a spectrofluorometer (Fluorolog 3, Horiba Jobin Yvon, Palaiseau, France; fluorescein: λ_ex: 485 nm, λ_em: 520 nm; EBA: λ_ex: 584 nm, λ_em: 680 nm). P_app values were calculated as described above for KTP except for the 30-min assay length.

2.3.6. Immunocytochemistry

To evaluate morphological changes in A549 and CFBE cells caused by KTP raw or the developed carrier-free DPIs, immunostaining for junctional proteins β-catenin and zonula occludens protein-1 (ZO-1) was performed. After the permeability experiments, cells on culture inserts were washed with phosphate buffered saline (PBS) and fixed with 3% paraformaldehyde solution for 15 min at room temperature then washed with PBS. The cells were permeabilized by 0.2% TX-100 solution for 10 min and the nonspecific binding sites were blocked with 3% bovine serum albumin in PBS. Primary antibodies rabbit anti-β-catenin (AB_476831, 1:200) and rabbit anti-ZO-1 (AB_138452, 1:400; Life Technologies, USA) were applied as overnight treatment at 4 °C. Incubation with secondary antibodies anti-rabbit IgG conjugated with Cy3 (AB_258792, 1:400) lasted for 1 h and Hoechst dye 33342 was used to stain cell nuclei. After mounting the samples (Fluoromount-G; Southern Biotech, Birmingham, AL, USA) staining was visualized by Leica TCS SP5 confocal laser scanning microscope (Leica Microsystems GmbH, Wetzlar, Germany).

2.4. Stability Study

The stability study was conducted in a Binder KBF 240 constant-climate chamber (Binder GmbH, Tuttlingen, Germany). This chamber, equipped with an electronically controlled APT.line™ preheating and cooling system, ensured precise temperature and reproducible results within a range of 10 to 70 °C and a relative humidity (RH) range of 10 to 80%. Following ICH guidelines, the stability test was performed at 40 °C with 75% RH. The formulations were stored in size 3 Ezeeflo™ hydroxypropyl methylcellulose capsules (ACG-Associated Capsules Pvt. Ltd., Mumbai, India) for 3 months. An MNT-containing sample (F1) was selected for the stability study. Sampling was conducted immediately after preparation (F1—0m), as well as after 1 month (F1—1m) and 3 months (F1—3m) of storage.

2.4.1. Particle Size

Laser diffraction analysis was conducted to determine the particle size and distribution of the samples (Fresh, and after 1 month and 3 months) using a Mastersizer Scirocco 2000 (Malvern Instruments Ltd., Worcestershire, UK). The refractive index for KTP was set at 1.592. A dry dispersion unit was employed, with 0.5–1.0 g of the sample placed in the feeder tray. The air pressure was set to 3.0 bar, with the vibration feed at 75%. Each sample was measured in triplicate. Particle size distribution was represented by D[0.1] (indicating the size which below 10% of the volume distribution falls), D[0.5] (median particle size), and D[0.9] (indicating 90% of the volume distribution is below this size). Furthermore, the span of the size distribution was calculated using Equation (2).

\begin{matrix} Span = \frac{D [0.9] - D [0.1]}{D [0.5]} \end{matrix}

(2)

2.4.2. Scanning Electron Microscopy

The morphology of freshly prepared and stored spray-dried samples was analyzed using a scanning electron microscope (SEM) (Hitachi Scientific Ltd., Tokyo, Japan). The SEM was operated at 10 kV, and the samples were coated with a gold–palladium layer using a Bio-Rad sputter coater, Bio-Rad, Hercules, CA, USA (2.0 kV, 10 mA, 10 min). The coating process was performed under an air pressure range of 1.3–13.0 mPa. Particle size was calculated using ImageJ 1.53e software, with measurements taken from 50 to 100 particles per sample.

2.4.3. Differential Scanning Calorimetry

Differential scanning calorimetry (DSC) was performed using the Mettler Toledo DSC 821e system, managed by STARe thermal analysis software version 9.3 (Mettler Inc., Schwerzenbach, Switzerland). Approximately 2–3 mg of each sample was placed in hermetically sealed aluminum pans with pierced lids, with an empty pan used as the reference. Analysis was conducted over a temperature range of 25 °C to 300 °C, at a heating rate of 10 °C/min, with argon as the carrier gas flowing at 10 L/h.

2.4.4. X-Ray Powder Diffraction

The samples and raw materials were examined using a BRUKER D8 Advance X-ray powder diffractometer (Bruker AXS GmbH, Karlsruhe, Germany). The analysis employed a Cu Kα1 radiation source (λ = 1.5406 Å) and a VÅNTEC-1 detector. Scans were conducted with a copper target and nickel filter, operating at 40 kV and 40 mA, across a range of 3° to 40°, with a step time of 0.1 s and a step size of 0.01°. The crystallinity index (Xc) was calculated with raw KTP serving as the 100% crystalline reference standard (Equation (3)), where A represents the area under the diffraction curve.

\begin{matrix} X_{c} (%) = \frac{A c r y s t a l l i n e}{A c r y s t a l l i n e + A a m o r p h o u s} \times 100 % \end{matrix}

(3)

2.4.5. Andersen Cascade Impactor Measurement

The aerosolization properties of both freshly prepared and stored spray-dried samples were evaluated using an Andersen Cascade Impactor (ACI) from Copley Scientific Ltd., Nottingham, UK. An inhalation flow rate of 60 L/min was generated by a vacuum pump (High-capacity Pump Model HCP5, coupled with a Critical Flow Controller Model TPK, Copley Scientific Ltd.) and verified with a mass flow meter (Flow Meter Model DFM 2000, Copley Scientific Ltd.). Samples were loaded into size 3 Ezeeflo™ hydroxypropyl methylcellulose capsules (ACG-Associated Capsules Pvt. Ltd., Mumbai, India) and placed in a Breezhaler^® single-dose device. The device was activated, and inhalation was performed over 4 s. To replicate lung adhesive properties, the stage plates were pre-coated with a Span 85 and cyclohexane mixture (1:99 v/v%), which was allowed to dry. After inhalation, all ACI parts were rinsed with 50% methanol (v/v%) to retrieve deposited KTP. The KTP concentration at each stage was measured via UV/VIS spectrophotometry (ATI-UNICAM UV/VIS Spectrophotometer, Cambridge, UK) at 258 nm. Inhalytix™ software (Copley Scientific Ltd., Nottingham, UK), (https://www.copleyscientific.com/inhaler-testing/apsd-data-analysis-software/inhalytix/, accessed on 10 October 2024), was used to analyze the in vitro aerodynamic properties of the samples. Aerodynamic profiles were characterized by several metrics: MMAD (Mass Median Aerodynamic Diameter) representing true particle size during inhalation; FPF (Fine Particle Fraction), the percentage of drug particles under 5 µm; and EF (Emitted Fraction), denoting the percentage of drug mass reaching the impactor compared to the initially loaded KTP amount.

2.4.6. In Vitro Release Study

A modified paddle method (Hanson SR8 Plus, Teledyne Hanson Research, Chatsworth, CA, USA) aligned with the European Pharmacopeia was used to assess KTP release. Instead of the standard 1000 mL vessels, 100 mL vessels and smaller paddles were employed. Simulated lung fluid (SLF) was prepared with NaCl, sodium bicarbonate (NaHCO₃), CaCl₂, sodium dihydrogen phosphate (NaH₂PO₄), sulphuric acid (H₂SO₄), and glycine at a pH of 7.4. Each vessel was filled with 50 mL of SLF and maintained at 37 °C to simulate human airway conditions. The samples containing 5 mg of KTP were dispersed in the SLF, with the paddle rotating at 50 rpm for 60 min. At intervals of 5, 10, 15, 30, and 60 min, 5 mL samples were taken and replaced with fresh medium. The samples were filtered through a Millex-HV syringe-driven filter unit with a 0.45 µm pore size (Millipore Corporation, Bedford, MA, USA) and analyzed using UV/VIS spectrophotometry at 258 nm (ATI-UNICAM UV/VIS Spectrophotometer, Cambridge, UK). Measurements were conducted in triplicate.

2.4.7. Statistical Analysis

Statistical analysis was performed using GraphPad Prism version 8.0.1 (GraphPad Software, La Jolla, CA, USA). A two-way ANOVA test was used to assess statistical significance, with results considered significant at p ≤ 0.05 (* p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001).

3. Results and Discussion

3.1. Cell Line Measurements

3.1.1. Cell Impedance Assays

Evaluating the safety of dry powder formulations and their compatibility with lung epithelial cells is crucial to ensure they do not adversely affect pulmonary tissues. To address potential toxicity concerns related to the developed formulations, a cell viability assay was conducted. Real-time impedance measurement, a sensitive method for detecting changes in cell barrier function and viability, was utilized. In A549 cells, only the MNT-containing sample (F1) caused a slight decrease in cell impedance at high concentrations (Figure 1A). Conversely, neither the F0 nor F1 carrier-free DPIs affected the cell impedance of CFBE epithelial cells (Figure 1B). These results indicate well-preserved cell viability and barrier integrity across all groups and nearly all KTP concentrations, consistent with previously published findings [19]. Moreover, these findings suggest that the developed formulations can be safely administered via the pulmonary route. In contrast, the detergent Triton X-100 induced cell death, sharply reducing impedance to nearly zero. Based on the impedance results, suspensions of KTP raw and DPI formulations containing 50 μg/mL KTP were selected for the permeability experiments.

3.1.2. Ketoprofen Permeability Across the Respiratory Co-Culture Models

The permeability of KTP raw, F0, and F1 DPIs was tested on the co-culture models of the alveolar and bronchial epithelial barriers at 30- and 60-min time points (Figure 2). KTP demonstrated good penetration across both models; however, its permeability was lower across the CFBE bronchial epithelial barrier compared to the alveolar epithelial barrier (Figure 2B), likely due to structural differences between the two cell lines [20]. F0 and F1 DPIs enhanced the permeability of KTP in the A549 alveolar co-culture model at the 30- and 60-min time points.

Furthermore, the MNT-containing sample (F1) increased the permeability by 1.4-fold compared to raw KTP at the 30-min time point (Figure 2A). This improvement may be attributed to the presence of MNT, which could enhance drug diffusion by modifying the surface properties of the particles or interacting with epithelial cells to facilitate better permeation [21]. It is important to note that different dosage forms can influence permeability. For instance, Sipos et al. demonstrated a 5-fold increase in meloxicam permeability through a nasal cell line when formulated as a liquid with polymeric micelles, which facilitated faster transport [22]. In contrast, a 2.5-fold enhancement in permeability was recorded when a freeze-dried formulation for nasal delivery was developed [23].

3.1.3. Barrier Integrity Measurements After Permeability Experiments

The barrier integrity of the respiratory epithelial models after permeability assays was assessed using transepithelial electrical resistance (TEER) measurements and permeability assays with two marker molecules: sodium fluorescein and Evans Blue-labeled albumin (Figure 3). No changes in barrier integrity were observed for either the tested active agent or the DPI formulations in the culture models, as reflected by the unaltered TEER values of the cell layers and permeability values for two marker molecules compared to the control group (Figure 3A–D). These results indicate that neither raw KTP nor the developed DPI formulations exerted harmful effects on the barrier, suggesting their potential for safe application. Our findings are in line with previously published results on nasal epithelial cells [22], where TEER values remained at the level of the control group after 1-h treatment with meloxicam nanoformulation indicating that it does not damage the barrier function. This further supports the safety of our DPI formulations.

3.1.4. Immunocytochemistry

Morphological changes in the cell–cell junctions of A549 alveolar and CFBE bronchial epithelial cell layers after permeability experiments were assessed by immunostaining for β-catenin on A549 cells (Figure 4A) and zonula occludens protein-1 (ZO-1) on CFBE cells (Figure 4B). The results confirmed that KTP raw, F0, and F1 formulations did not impair barrier integrity, as evidenced by the continuous, belt-like immunostaining of β-catenin and ZO-1 at the epithelial cell borders, which was similar across all groups (Figure 4).

3.2. Stability Study

3.2.1. Particle Size

As reported in our previous paper [16], the particle size of KTP in the nanosuspension was reduced to the nano range (238.30 ± 1.42 nm), which enhanced the surface area and promoted a faster dissolution rate. Following spray-drying, and after incorporation of MNT particles, the D [0.5] value of the F1 sample increased to 2.325 ± 0.14 µm, indicating the formation of a nano-in-micro particle system. Further particle size analysis showed that the particle size of the formulation remained almost consistent throughout the testing period (Table 1), confirming the absence of particle agglomeration. Notably, the size range remained within the optimal 1–5 µm range required for effective pulmonary delivery [24].

While statistical analysis showed no significant variation in particle size during storage (Figure 5), the span values increased significantly. This suggests that particle size distribution was affected, likely due to humidity-induced uneven moisture absorption [24,25], which may have caused varying degrees of particle swelling or surface roughening, thereby broadening the distribution. Span, a parameter defining the breadth of size distribution, is critical in dry powder formulations. A wide particle size distribution (high span) can result in poor powder flow and uneven distribution during inhalation, potentially compromising inhaler performance and leading to drug deposition in non-target areas.

3.2.2. SEM Images

The morphology of the stored samples was analyzed using SEM to evaluate potential changes in particle structure. SEM images of the spray-dried formulation (F1) demonstrated a near-spherical morphology with a wrinkled surface (Figure 6). Spray-dried powders intended for pulmonary delivery must adhere to specific shape and size criteria to ensure effective deposition in the bronchioles or alveoli. The spherical morphology facilitates a uniform size distribution, while the wrinkled surface improves contact angles with lung tissues, enhancing aerodynamic performance [26]. After storage, ImageJ 1.53e software analysis indicated a slight increase in particle size (Table 2), which likely resulted from moisture absorption under humid conditions. Similar findings were corroborated by laser diffraction measurements. Despite this increase, the particle size remained below 5 µm, which is ideal for pulmonary delivery [27]. Additionally, SEM images of F1_3m revealed a slightly rougher surface, potentially caused by solid-state change during storage under high humidity [13]. This change may have led to uneven swelling or surface roughening.

3.2.3. Thermal Analysis by DSC

DSC was used in this study to monitor changes in the thermal stability and crystallinity of the samples over time after storage under stress conditions. The initial melting point of KTP was 97.83 °C, while in the physical mixture (PM)—comprising raw materials of KTP, stabilizers, MNT, and leucine—it was 96.50 °C (Figure 7A). In sample F1, the melting point was reduced to 91.50 °C, mostly due to the milling process. Moreover, the less intensive peaks in sample F1 compared to KTP raw indicated that some particles partially converted to amorphous after spray drying. After storage (Figure 7B), the endothermic peak’s position and intensity remained consistent, with melting points recorded at 90.84 °C after 1 month and 90.39 °C after 3 months. Overall, the DSC curves indicate that the thermal stability of the samples remained generally unchanged throughout the testing period, which is in line with previously published results [28].

3.2.4. Structural Analysis by XRPD

XRPD enables monitoring of structural changes in spray-dried samples during storage, provided the XRPD patterns of KTP and the excipients are known. The solid-state form of the active ingredient is crucial, as differences between crystalline and amorphous states can affect particle morphology, interparticle interactions, and aerodynamic performance. The XRPD diffractograms (Figure 8A) reveal characteristic peaks of raw KTP and the physical mixture (PM), with raw KTP displaying intense 2-theta (2θ) peaks at 13.5°, 14.53°, 17.23°, 18.48°, and 22.84°, indicative of its crystalline structure. After formulation, the spray-dried sample (F1) retained these characteristic peaks at reduced intensity (Figure 8B), suggesting partial amorphization with a crystallinity index (Xc) of 44.54%. During storage, KTP’s characteristic peaks persisted, supporting prior studies indicating that formulations stored in HPMC capsules maintain structural integrity under stress [29]. However, rougher diffractograms observed in stored samples suggest a partial loss of crystalline order, reflected in reduced Xc values (40.17% for F1_1m and 33.81% for F1_3m). This partial transition to an amorphous state may impact both aerodynamic performance and drug release profiles.

3.2.5. In Vitro Lung Deposition by Aerodynamic Measurements

The deposition profile of the spray-dried samples was evaluated using Andersen Cascade Impactor (ACI), and the in vitro aerodynamic results were analyzed with Inhalytix™ V 2.0 software (Figure 9). The freshly prepared F1 formulation demonstrated a high fine particle fraction (FPF) (~64%), surpassing those of commercially available formulations used with the Breezhaler^® device [30]. Additionally, the emitted fraction (EF) was notably high (~95%), reflecting efficient powder release from the inhaler. This enhanced aerosolization performance can be attributed to the inclusion of MNT and leucine, which improve powder cohesion and adhesion to hydroxypropyl methylcellulose (HPMC) capsules, ensuring consistent dose delivery [28].

Although no significant changes in the MMAD were observed after storage, a decline in FPF values was recorded (Figure 9). This reduction can likely be attributed to surface morphology changes during storage, such as increased roughness or partial agglomeration due to moisture absorption under stressed conditions. The decrease in FPF suggests heightened cohesiveness among aerosol particles post-storage, potentially caused by moisture uptake, which increases their tendency to form agglomerates midair. Such changes may disrupt flow dynamics and reduce aerosolization efficiency. These findings align with those of a previous study by Tran et al. (2020), which reported a significant decrease in FPF after storage while observing no substantial changes in MMAD [31].

Figure 10 illustrates the particle distribution across the stages of the ACI. The ACI stages numbered −1 to 6, are used to assess the particle size distribution of aerosolized formulations. Each stage corresponds to a specific cut-off diameter based on the aerodynamic diameter of the particles. The cut-off diameters (in µm) for each stage are as follows: −1: 8.6, −0: 6.5, 1: 4.4, 2: 3.2, 3: 1.9, 4: 1.2, 5: 0.55, and 6: 0.26 [28]. Effective KTP deposition was predominantly observed in the second, third, and fourth stages, which correspond to the bronchial and upper alveolar regions [32]. A significant portion also reached the filter, representing deposition in the deeper lung regions, emphasizing the powder’s capability to target smaller airways.

An important aspect to consider is the impact of MNT concentration on aerodynamic characteristics. As demonstrated in our previous study [16], high MNT content can lead to crystallization, phase separation, and an increase in particle size, potentially compromising the aerosol performance of MNT-containing formulations. Consistent with these findings, Arte et al. (2024) reported that MNT concentration significantly influenced the physical and aerosolization stability of spray-dried protein formulations [15]. However, assessing the specific effects of MNT concentration on the stability of the current formulation lies beyond the scope of this study.

To address the reduced aerodynamic performance after storage, future studies should explore different capsule materials and storage conditions, particularly varying relative humidity levels, to enhance stability and maintain aerosolization properties.

3.2.6. In Vitro Dissolution Study

To simulate lung conditions, an in vitro dissolution test was conducted using a simulated lung medium (pH 7.4). As shown in Figure 11, sample F1 demonstrated fast KTP release, attributed to the nanosized form of KTP after the milling process, which increases the surface area [33]. The freshly prepared sample (F1) achieved complete drug release (100%) within the first 10 min, facilitated by the presence of MNT, which promotes disintegration and accelerates drug release [34]. Fast dissolution is particularly advantageous in pulmonary delivery as it minimizes the likelihood of particle elimination by mucociliary clearance [35]. Additionally, the rapid release profile persisted after storage, with samples F1_1m and F1_3m releasing approximately 75% of the drug within 5 min. However, after 10 min, the release from F1_3m declined, potentially due to solid-state change or increased interparticle interactions, reducing the effective surface area and compromising the consistent drug release [36]. This observation aligns with previously published results [37].

4. Conclusions

This study evaluated the cytotoxicity and permeability of a ketoprofen–mannitol carrier-free DPI formulation, alongside its stability when stored in HPMC capsules under stress conditions following ICH guidelines (40 °C and 75% relative humidity). The developed DPI represents a promising strategy for drug repositioning, offering potential therapeutic benefits for managing lung inflammation while saving time and resources in drug development. KTP showed good penetration across both alveolar and bronchial models, with lower permeability through the CFBE bronchial barrier than the A549 alveolar barrier, probably due to structural differences. The MNT-containing F1 formulation enhanced permeability by 1.4-fold through the A549 alveolar cells compared to raw KTP at the 30-min time point, likely because MNT improves drug diffusion or interacts with epithelial cells to facilitate permeation. While F1 caused a slight decrease in cell impedance at high concentrations in A549, neither the F0 nor F1 carrier-free DPIs affected the cell impedance of CFBE epithelial cells. Moreover, no changes in barrier integrity were observed for either the tested active agent or the DPI formulations in both culture models, which further supports the safety of our DPI formulations. The formulation retained favorable properties after storage, owing to the carefully selected excipients and optimized technological processes. Stability testing demonstrated consistent particle size and the persistence of KTP’s partially amorphous nature over time. Dissolution studies confirmed rapid initial drug release, indicating effective delivery potential. Aerosol performance analysis revealed no significant change in MMAD values after storage, although a reduction in FPF was observed, likely due to moisture absorption affecting particle surface morphology. Future studies should focus on exploring alternative capsule types and storage conditions, particularly at lower relative humidity levels, to further improve stability and maintain optimal aerosolization properties.

Author Contributions

Conceptualization, H.B., R.A. and I.C.; methodology, H.B., I.G., M.A.D., I.C. and R.A.; software, H.B. and I.G.; validation, H.B., R.A. and I.C.; formal analysis, H.B., I.G., M.A.D., R.A. and I.C.; investigation, H.B. and I.G.; resources, R.A. and I.C.; data curation, H.B., R.A. and I.C.; writing—original draft preparation H.B.; writing—review and editing, H.B., R.A., I.G., M.A.D. and I.C.; visualization, H.B., R.A. and I.C.; supervision, R.A. and I.C.; project administration, R.A. and I.C.; funding acquisition, R.A. and I.C. All authors have read and agreed to the published version of the manuscript.

Funding

This research was supported by National Research, Development and Innovation Office, NKFIH_OTKA K_146148.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data is contained within the article.

Acknowledgments

Project no TKP2021-EGA-32 has been implemented with the support provided by the Ministry of Culture and Innovation of Hungary from the National Research, Development and Innovation Fund, financed under the TKP2021-EGA funding scheme.

Conflicts of Interest

The authors declare no conflicts of interest. The authors alone are responsible for the content and writing of this article.

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Figure 1. Cell viability of (A) A549 alveolar and (B) CFBE bronchial epithelial cells after 1-h treatments with KTP raw and the developed F0, F1 samples measured by impedance. Values are presented as means ± SD, n = 5–6. Statistical analysis: ANOVA followed by Dunett’s test. *** p < 0.001 compared to the control group. C, control; KTP, ketoprofen raw; TX-100, Triton X-100 detergent.

Figure 2. Permeability of KTP raw, F0, and F1 DPIs (50 μg/mL KTP concentration in the donor compartment) across the co-culture model of (A) alveolar epithelial cells and (B) bronchial epithelial cells after 30- and 60-min assay time. Values are presented as means ± SD, n = 4. Statistical analysis: ANOVA followed by Dunett’s test. ** p < 0.01, *** p < 0.001.

Figure 3. Evaluation of the tightness of the A549 alveolar and the CFBE bronchial epithelial co-culture models after the KTP raw, F0, and F1 DPIs permeability experiment by (A,C) transepithelial electrical resistance (TEER) measurement and the (B,D) permeability of fluorescein and albumin marker molecules. Values are presented as means ± SD, n = 4. Statistical analysis: ANOVA followed by Dunett’s test.

Figure 4. Validation of barrier integrity in A549 alveolar epithelial and CFBE bronchial epithelial cell layers after permeability assays with KTP raw, F0, and F1. Immunostaining for (A) β-catenin and (B) zonula occludens protein-1. Cyan: cell nuclei; red: junctional proteins; scale bar: 20 μm. C, control; KTP, ketoprofen raw.

Figure 5. Laser diffraction results of particle size (D[0.5] (µm)) and particle size distribution (Span) for sample F1 (freshly prepared; F1—0m, and after one month; F1—1m and 3 months; F1—3m of storage). Results are presented as means ± SD, n = 3., **** p < 0.0001 significantly different.

Figure 6. SEM images of F1 sample at (a) 0 months, (b) 1 month, and (c) 3 months.

Figure 7. DSC thermal analysis of (A) raw ketoprofen (KTP) and the physical mixture (PM), and (B) sample F1, analyzed as a freshly prepared sample (F1—0m) and after 1 (F1—1m) and 3 (F1—3m) months of storage.

Figure 8. XRPD structural analysis of (A) raw ketoprofen (KTP) and the physical mixture (PM), and (B) sample F1, analyzed as a freshly prepared sample (F1—0m) and after 1 (F1—1m) and 3 (F1—3m) months of storage. The circles highlight the characteristic peaks of KTP in the raw drug, physical mixture, as well as fresh and stored formulations.

Figure 9. In vitro aerodynamic characteristics (MMAD, FPF, and EF) of sample F1 were analyzed as a freshly prepared sample (F1—0m), and after 1 (F1—1m) and 3 (F1—3m) months of storage, at a flow rate of 60 L/min. Data are presented as means ± SD (n = 3 independent measurements). Statistical significance is indicated, **** p < 0.0001 significantly different.

Figure 10. In vitro aerodynamic distribution of the F1 sample was analyzed as a freshly prepared sample (F1—0m), and after 1 (F1—1m) and 3 (F1—3m) months of storage, at a flow rate of 60 L/min. Data are presented as means ± SD (n = 3 independent measurements). Statistical significance is indicated as follows: * p < 0.05, ** p < 0.01.

Figure 11. In vitro release study of raw KTP, and spray-dried sample (F1) was analyzed as a freshly prepared sample (F1—0m) and after 1 (F1—1m) and 3 (F1—3m) months of storage, in simulated lung media (SLM). Results are expressed as mean ± SD (n = 3 independent measurements).

Table 1. The particle sizes of the F1 sample as freshly prepared and after storage according to the laser diffraction analysis.

Sample *	D[0.1] (µm)	D[0.5] (µm)	D[0.9] (µm)	Span
F1_0m	1.073 ± 0.07	2.325 ± 0.14	13.867 ± 2.34	5.503 ± 0.68
F1_1m	1.065 ± 0.12	2.337 ± 0.38	143.822 ± 5.91	61.073 ± 3.20
F1_3m	1.125 ± 0.45	2.444 ± 0.93	297.506 ± 10.36	121.275 ± 11.51

* Sampling was conducted immediately after preparation (F1—0m), as well as after 1 month (F1—1m) and 3 months (F1—3m) of storage.

Table 2. The particle sizes of the F1 sample as freshly prepared and after storage according to the SEM images. Sampling was conducted immediately after preparation (F1—0m), after 1 month (F1—1m) and 3 months (F1—3m) of storage.

Sample	Particle Size (µm)
F1_0m	2.32 ± 0.12
F1_1m	2.35 ± 0.39
F1_3m	2.47 ± 0.89

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MDPI and ACS Style

Banat, H.; Gróf, I.; Deli, M.A.; Ambrus, R.; Csóka, I. Evaluation of Permeability, Safety, and Stability of Nanosized Ketoprofen Co-Spray-Dried with Mannitol for Carrier-Free Pulmonary Systems. Appl. Sci. 2025, 15, 1547. https://doi.org/10.3390/app15031547

AMA Style

Banat H, Gróf I, Deli MA, Ambrus R, Csóka I. Evaluation of Permeability, Safety, and Stability of Nanosized Ketoprofen Co-Spray-Dried with Mannitol for Carrier-Free Pulmonary Systems. Applied Sciences. 2025; 15(3):1547. https://doi.org/10.3390/app15031547

Chicago/Turabian Style

Banat, Heba, Ilona Gróf, Mária A. Deli, Rita Ambrus, and Ildikó Csóka. 2025. "Evaluation of Permeability, Safety, and Stability of Nanosized Ketoprofen Co-Spray-Dried with Mannitol for Carrier-Free Pulmonary Systems" Applied Sciences 15, no. 3: 1547. https://doi.org/10.3390/app15031547

APA Style

Banat, H., Gróf, I., Deli, M. A., Ambrus, R., & Csóka, I. (2025). Evaluation of Permeability, Safety, and Stability of Nanosized Ketoprofen Co-Spray-Dried with Mannitol for Carrier-Free Pulmonary Systems. Applied Sciences, 15(3), 1547. https://doi.org/10.3390/app15031547

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