Morphology-driven zinc oxide biointeractive carriers with biological barrier modulating effects for pulmonary delivery of liraglutide

Jin-Hyuk Jeong; Chang-Soo Han; Ji-Hyun Kang; Dong-Wook Kim; Chun-Woong Park

doi:10.1186/s12951-026-04085-y

. 2026 Feb 1;24:446. doi: 10.1186/s12951-026-04085-y

Morphology-driven zinc oxide biointeractive carriers with biological barrier modulating effects for pulmonary delivery of liraglutide

Jin-Hyuk Jeong ¹, Chang-Soo Han ², Ji-Hyun Kang ³, Dong-Wook Kim ⁴, Chun-Woong Park ^1,^✉

PMCID: PMC13200438 PMID: 41622258

Abstract

Pulmonary delivery provides a noninvasive route for systemic administration of biologics, yet efficient lung deposition and permeation across pulmonary barriers remain major challenges. In this study, morphology-engineered zinc oxide (ZO) biointeractive carriers were fabricated and evaluated as inhalable carriers for liraglutide (LG). Three distinct morphologies were obtained: smooth spherical ZO-1 (5–7 μm), spiky sea-urchin-like ZO-2 (5–8 μm with elongated ~ 3.5 μm tips), and compact spiky ZO-3 (1–3 μm with short ~ 1.3 μm tips). Particle image velocimetry (PIV) revealed morphology-dependent aerodynamic behaviors, where ZO-3 exhibited turbulence-driven dispersion favoring distal lung deposition. At the cellular level, ZO-3 demonstrated enhanced mucus penetration and reduced macrophage uptake, maintaining prolonged contact with the epithelial surface. Following intratracheal administration, LG@ZO-3 achieved bioavailability of ~ 60% relative to subcutaneous injection in healthy rats and ~ 51% in diabetic rats, far exceeding the < 2% oral bioavailability of semaglutide. These results suggest that morphology-controlled modulation of aerodynamic and biological interactions can overcome multiple pulmonary barriers, offering a promising strategy for effective inhalable delivery of peptide therapeutics.

Graphical Abstract

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Supplementary Information

The online version contains supplementary material available at 10.1186/s12951-026-04085-y.

Keywords: Zinc oxide, Morphology engineering, Pulmonary delivery, Barrier modulation, Mucus penetration, Macrophage evasion, Peptide absorption

Introduction

Pulmonary drug delivery with systemic effects has gained increasing attention over the past decade [1–3]. The unique physiological and structural features of the lungs, including a thin alveolar epithelium and extensive surface area, facilitate high systemic bioavailability, bypass first-pass metabolism, enable rapid therapeutic onset, and offer ample surface area for drug absorption [4, 5]. Consequently, pulmonary administration often leads to higher systemic drug concentrations than other noninvasive routes [3]. Although these advantages are predominantly applied to small molecules, pulmonary drug delivery is also a viable route for administering biologics in both local and systemic therapies [6]. Notably, many proteins and peptides with molecular weights below 30 kDa can rapidly cross the alveolar membrane and enter the systemic circulation, achieving high absorption efficiency without the need for penetration enhancers commonly required by other non-invasive delivery routes [7].

Various formulation approaches and inhalation devices are available for pulmonary drug delivery [8–11]. Biologics require precise dose consistency, physicochemical stability, and efficient, reproducible deposition in the respiratory tract [12–14]. Dry powder formulations present an attractive and highly valued option for biological administration as they circumvent many solubility and stability issues, enable a broad range of single-dose administrations per inhalation, exhibit lower susceptibility to microbial growth, and accommodate both hydrophilic and hydrophobic drugs [15].

However, several challenges must be addressed to achieve high absorption of biologics via pulmonary delivery and dry powder inhalers (DPIs). First, for aerosolized drugs to be effectively delivered to the lungs, the mass median aerodynamic diameter (MMAD) should typically fall within the range of 0.5–5 μm. However, powders of this size often exhibit high cohesiveness, which leads to poor flowability [16–19]. Thus, extensive particle engineering is required at the particulate level to balance interparticle forces, ensure stability during manufacturing and storage, and facilitate optimal dispersion and aerosolization upon inhalation [20, 21]. Second, once deposited in the lungs, particles may dissolve slowly or incompletely [22, 23]. Additionally, the mucus ciliary escalator, pulmonary mucus layer, and biofilm serve as significant diffusion barriers that limit drug penetration [24–27]. Pulmonary mucus, a complex viscoelastic gel composed of glycoproteins, is an environment where drugs typically exhibit low diffusion rates and are prone to inactivation. The physicochemical properties of inhaled particles significantly influence their ability to traverse mucus barriers and biofilms, ultimately affecting drug delivery efficiency [28–31]. Third, the pulmonary mononuclear phagocytic system, particularly alveolar macrophages, plays a critical role in immune surveillance, inflammation initiation, and the subsequent removal of inhaled particles through phagocytosis. Alveolar macrophages, monocytes, and dendritic cells engulf and eliminate foreign particles, thereby reducing the pharmacological efficacy of the inhaled formulations [32]. Thus, evading alveolar macrophage uptake is a key strategy for enhancing drug bioavailability [33–35].

Among the various strategies used to overcome the pulmonary delivery barriers, carrier-based blends remain the most widely employed and commercially viable approach for DPI formulations [20, 36]. Recent studies have focused on optimizing carrier-based blends by modulating drug carrier adhesion. Well-balanced adhesion ensures formulation stability while allowing effective drug detachment upon inhalation, improving aerodynamic performance. Drug-carrier interactions, which are influenced by electrostatic forces, van der Waals interactions, mechanical interlocking, and moisture-related properties, are critical and continuous. These characteristics are determined by the physicochemical properties of the carrier, including size, density, surface roughness, morphology, and surface energy [37–40]. Moreover, carrier size and morphology not only influence aerosolization efficiency but also play a critical role in governing drug transport across the pulmonary mucus barrier. While both muco-adhesion and muco-penetration may offer advantages depending on therapeutic objectives, they serve distinct functions within inhalation delivery [41, 42]. Muco-adhesive systems can prolong residence time at the epithelial surface, which is beneficial for locally acting therapeutics; however, for systemic peptide delivery, rapid and deep traversal of the mucus layer is essential to ensure efficient absorption before enzymatic degradation or macrophage clearance occurs [43–45]. Therefore, when the goal is to achieve meaningful systemic exposure, muco-penetrating behavior is generally more favorable than strong muco-adhesion, as supported by recent conceptual and experimental studies [46, 47]. Particle shape, density, and surface roughness affect macrophage uptake, cellular adhesion, biodegradability, residence time, and systemic absorption. Irregularly shaped or high-aspect-ratio particles tend to resist macrophage phagocytosis more effectively than spherical counterparts [48, 49]. Moreover, porous or wrinkled particles with rough surfaces adhere more efficiently to the cell membranes and exhibit enhanced diffusion. Notably, some studies suggest that surface roughness within the range of 0.2–1 μm is optimal for cell adhesion, although material composition and surface energy also play influential roles [50–52].

Despite increasing emphasis on particle engineering for inhalation, the number of commercially available excipients remains limited to approximately 40. Among these, zinc oxide (ZO) has been approved as a pulmonary excipient in solution form and has demonstrated potential to enhance aerosol performance as a force control agent [53, 54]. However, to the best of our knowledge, in-depth studies of the role of ZO microparticles in inhaled drug delivery and absorption are scarce. ZO exists in multiple stable crystalline forms, including hexagonal wurtzite and cubic zinc blende structures [55, 56]. Differences in atomic arrangement across crystal facets result in variations in surface energy, which influence the relative growth rates of different facets, ultimately dictating the particle’s final morphology [57]. Moreover, because each facet exhibits distinct chemical properties, growth modifiers can selectively alter the facet-specific growth rates, allowing the controlled fabrication of ZO particles with tailored sizes, shapes, and densities [58, 59].

Additionally, ZO has the potential to interact electrostatically with biologics, such as peptides and RNA, thereby influencing their stability [60, 61]. Biologics are generally more susceptible to degradation due to temperature and humidity variations, which can significantly affect their clinical utility [62, 63]. By stabilizing peptide interactions, ZO may protect biologics from external degradation and structural changes [61, 64–66]. Furthermore, ZO-peptide complexes have been reported to modulate PepT1 and ZnT1 mRNA expression, potentially affecting their mutual absorption [67, 68]. The role of Zn in lung protection and injury has also been documented, highlighting its relevance to pulmonary drug delivery [69–72].

Therefore, this study aimed to (i) fabricate inhalable ZO biointeractive carriers with aerodynamic properties favorable for pulmonary delivery, specifically particle sizes within the respirable range and morphologies optimized for deep lung deposition, loaded with the peptide drug liraglutide; (ii) evaluate their ability to stabilize the drug, enhance aerosol performance, penetrate pulmonary mucus, evade macrophage uptake, and promote epithelial adhesion; and (iii) investigate their potential to improve the systemic absorption of liraglutide following pulmonary administration. To this end, ZO biointeractive carriers were engineered with a central core and radially extended tip-like structures to generate aerodynamic turbulence, thereby improving inhalation efficiency. Furthermore, tip-containing structures with aspect ratios close to 1 have been designed to reduce mucus adhesion and facilitate cell attachment via optimal surface roughness. These investigations aim to provide a particle-engineering-based framework for developing stable and effective pulmonary delivery systems for biologics, focusing on peptide-based inhalation therapies [73].

Materials and methods

Materials

Liraglutide was purchased from Sinopep-Allsino Biopharmaceutical Co., Ltd. (China). Zinc acetate dihydrate, zinc oxide, and potassium citrate monohydrate were purchased from DUKSAN Co., Ltd. (Republic of Korea). The ammonia solution was obtained from Daejung Chemical Co. (Republic of Korea). D-mannitol was purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). The water used in the experiments was purified using an Ultrapure Water Purification System (Milli-Q^® Eq. 7000, Merck KGaA, Germany). High-performance liquid chromatography (HPLC)-grade solvents were used for the analysis, with HPLC-grade acetonitrile (ACN) purchased from Honeywell Burdick & Jackson (USA). Unless otherwise specified, all other chemicals and solvents were of analytical grade and were purchased from Merck KGaA (Germany) and used without further purification.

Cell lines and maintenance

Calu-3 human lung adenocarcinoma cells (Korean Cell Line Bank, Republic of Korea) were cultured in Minimum Essential Medium (MEM, Gibco™, Thermo Fisher Scientific Inc., USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS, Gibco™, Thermo Fisher Scientific Inc., USA) and 1% Penicillin-Streptomycin (PS, Gibco™, Thermo Fisher Scientific Inc., USA). Calu-3 cells at passage numbers 23–29 were used in the experiments.

Raw264.7 mouse macrophages were purchased from the American Type Culture Collection (ATCC, USA) and cultured in high-glucose Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% FBS and 1% PS. Raw264.7 cells at passage numbers 11–20 were used in the experiments.

A549 human alveolar epithelial cells were purchased from ATCC and cultured in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% FBS and 1% PS. A549 cells at passage numbers 21–29 were used in the experiments.

All cells were maintained in a humidified atmosphere of 5% CO₂ at 37 °C.

Preparation method

Fabrication of ZO microparticles for inhalation carrier

ZO for inhalation was synthesized using zinc acetate dihydrate and ammonia solution (25.0–30.0 wt%) as precursors for zinc cations and hydroxide anions, respectively. First, a transparent aqueous solution (100 mL) of zinc acetate dihydrate (0.01 M) and ammonia (~ 0.3 M) was prepared at room temperature.

For ZO-1, potassium citrate monohydrate was added as a crystal growth modifier to achieve a final concentration of approximately 1 mM, and the mixture was subjected to hydrothermal treatment in a dry oven set at 100 °C for 1 h. ZO-2 was prepared without a crystal growth modifier and underwent hydrothermal treatment in a dry oven set at 100 °C for 4 h. Similarly, ZO-3 was prepared without a crystal growth modifier and was hydrothermally treated in a dry oven at 100 °C for 1 h.

After the reaction, the solution was passed through a nylon membrane filter with a pore size of 450 nm. The residual powder was washed multiple times with deionized water and dried in an oven at 60 °C for 12 h. The dried powders were then calcinated at 800 °C for 30 min using a muffle furnace (Dae Heung Science, Republic of Korea).

Preparation of liraglutide microparticles and loading onto inhalable ZO

Liraglutide microparticles for inhalation (LG) were prepared using a Nano Spray Dryer B-90 (BÜCHI Labortechnik AG, Switzerland). A solution of liraglutide at a concentration of 53.4 µM was mixed with an aqueous solution of 11.0 mM D-mannitol, followed by spray drying under controlled conditions (inlet temperature: 120 °C, gas flow: 110–140 L/min, pressure: 55–65 mbar, pump rate: 10%, spray rate: 40%). The resulting LG consisted of 9.1% liraglutide and 90.9% mannitol by weight.

For carrier-based formulations, LG were physically blended with each ZO type—ZO-1, ZO-2, or ZO-3—using a Turbula^® Model T2F Shaker Mixer (WAB Group, Switzerland) for 10 min. A blending ratio of 66.7% LG to 33.3% ZO by weight was selected based on optimization experiments demonstrating improved liraglutide stability (Figure S1). The individual formulations were designated as LG@ZO-1 (LG blended with ZO-1), LG@ZO-2 (LG blended with ZO-2), and LG@ZO-3 (LG blended with ZO-3).

Preparation of FITC-labeled ZO and FITC-labeled liraglutide

For the fluorescein isothiocyanate (FITC) labeling of ZO, 40 mg of ZO particles were dispersed in 30 mL of dimethylformamide (DMF). A diluted APTS solution consisting of 5 µL of amino-propyl-triethoxy-silane (APTS) in 250 µL of DMF was added to the suspension, followed by sonication and stirring at room temperature for 20 h [74–76]. The particles were collected by centrifugation (2,000 g, 10 min), washed with DMF, and resuspended in 5 mL of DMF. Subsequently, a FITC solution prepared by dissolving 10 mg of FITC in 5 mL of DMF was added, and the mixture was stirred for 4 h. The FITC-labeled ZO particles were recovered by centrifugation, washed thoroughly with DMF, dried under vacuum, and stored as dry powders.

For FITC labeling of LG, 50 mg of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDAC, TCI) was added to 5 mL of a 1% aqueous LG solution and incubated for 2 h at room temperature. The FITC solution (5 mg in 5 mL methanol) was then added under continuous stirring, and the reaction mixture was incubated in the dark for 3 h. The labeled LG was purified by dialysis (MWCO 3.5 kDa) against water for 7 d in the dark [77, 78]. The resulting FITC-labeled LG solution was lyophilized and stored at 4 °C in the dark until further use for microparticle preparation, as described in Sect. "Preparation of liraglutide microparticles and loading onto inhalable ZO".

Physicochemical properties of ZO microparticles

Scanning electron microscope (SEM) and energy-dispersive X-ray spectroscopy (EDS)

The surface morphologies of the inhalable ZO- and LG-loaded formulations were visually examined using a scanning electron microscopy (SEM; ZEISS-GEMINI LEO 1530, Zeiss, Germany). In addition, elemental mapping of ZO was performed using energy-dispersive X-ray spectroscopy (EDS) equipped with Ultim Max and Ultim Extreme detectors (Oxford Instruments, UK) to visualize the spatial distribution of Zn and oxygen within each inhalable ZO particle type (ZO-1, ZO-2, and ZO-3) and reagent-grade raw ZO. This analysis confirmed the elemental uniformity and absence of detectable impurities across the particle morphologies. Furthermore, the morphology of the deposited powder was observed at Stages 2 and 4 in an inhalation simulation study using a Next Generation Impactor (NGI, Copley Scientific Limited, UK). The samples were fixed onto carbon tape and coated with platinum using a Hummer VI sputtering device (Anatech Ltd, USA), achieving a coating thickness of 200 Å. The analysis was conducted at an accelerating voltage of 3 kV with magnifications of 2,000× or 10,000×. For image processing and analysis, particle counting and coadherence rate measurements were performed using ImageJ software (v1.54i, National Institutes of Health, USA) [79].

Powder X-ray diffraction (PXRD)

Powder X-ray diffraction (PXRD) patterns were obtained using an X’Pert PRO MRD^® (PANAlytical Ltd., Netherlands) with Cu Kα radiation at 50 mA and 40 kV. The synthesized ZO particles were then placed on plates at room temperature for further analysis. 2θ scans were collected over a range of 5° to 60°.

Elemental and surface compositional analysis

The elemental composition was evaluated using nondestructive X-ray fluorescence spectroscopy (XRF) to determine the overall elemental distribution in the synthesized inhalable ZO microparticles. Samples of each type of ZO (ZO-1, ZO-2, and ZO-3) and a reagent-grade raw ZO reference standard were prepared for XRF by flattening their surfaces with a coverslip, and the spectra were acquired using a ZSX Primus II spectrometer (Rigaku Co., Ltd., Japan).

To investigate surface-specific composition, X-ray photoelectron spectroscopy (XPS) was performed on the ZO type with the largest surface area (ZO-3) using a PHI Quantera-II system (ULVAC-PHI, Inc., Japan) equipped with an Al Kα Mono source (Ep: 1486.6 eV). The analysis was conducted under a chamber pressure of 4.6 × 10⁻⁸ Pa (base) and 5.0 × 10⁻⁷ Pa (working), providing compositional information for approximately the top 40 nm of the particle surface. XPS was used to quantify the relative proportions of Zn and carbon to assess the extent and distribution of carbonate species, a common byproduct of ZO synthesis, and to compare the effects of calcination on surface carbonate removal. These results were also compared with those of reagent-grade raw ZO to benchmark the surface purity of inhalable ZO.

Brunauer-emmett-teller (BET)

The Brunauer–Emmett–Teller (BET) method with nitrogen adsorption was used to measure the specific surface area. The analysis was performed using an ASAP^® 2425 analyzer (Micromeritics Instruments Corp., USA). Each sample cell, containing approximately 2 g of powder, was degassed at 120 °C for 2 h to remove gas. The micropore area and volume were calculated using the t-plot method [80].

Surface energy of ZO particles

The surface energy of the fabricated ZO was evaluated by measuring its static contact angle. All the samples were compressed into flat circular discs using a hydraulic press (Handtap, ENERPAC, USA) at a force of 0.5 kN. The static contact angle was measured using a contact angle goniometer (Phoenix 300, Surface Electro Optics, Republic of Korea) and the sessile drop method. The test was conducted at room temperature by dispensing 5 µL of deionized water onto the surface. The surface energy (solid surface tension) was calculated using software provided by the instrument manufacturer. The surface free energy was determined using the Girifalco-Good-Fowkes-Young approach [81, 82].

ZO release analysis

Zinc ion (Zn²⁺) release from the synthesized inhalable ZO particles was comprehensively evaluated at the microscale level to elucidate the influence of particle morphology on dissolution behavior.

The Zn²⁺-selective fluorescent probe FluoZin™−3, AM (Thermo Fisher Scientific, USA) was employed for microscale visualization. This probe exhibits strong fluorescence upon binding Zn²⁺, with excitation/emission maxima of approximately 494/516 nm. A 3 mM stock solution of FluoZin™−3, AM was diluted 1:1000 in Hank’s Balanced Salt Solution (HBSS), followed by adding esterase to initiate intracellular ester hydrolysis. The mixture was incubated for 30 min at 37 °C under 5% CO₂ to ensure complete probe activation. Separately, a 0.15% agarose gel was prepared in phosphate-buffered saline (PBS, pH 7.4) into which the activated probe solution was incorporated before gelation. The solution was melted using microwave heating and dispensed into 12-well plates. Immediately before gel solidification, the ZO particles (ZO-1, ZO-2, and ZO-3) were gently scattered onto the surface. The number of particles was adjusted based on morphology-dependent size differences to ensure comparable initial particle volumes across formulations. Time-lapse confocal laser scanning microscopy (CLSM, LSM 980, ZEISS, Germany) was performed at 0, 10, 20, 30, 40, 50, and 60 min to monitor the fluorescence activation around individual particles (1–3 particles per image). The fluorescence intensity profiles were quantified to determine localized Zn²⁺ release kinetics at the particle level.

Particle image velocimetry (PIV) analysis

The particle dispersion dynamics of the inhalable ZO formulations (ZO-1, ZO-2, and ZO-3) were evaluated using a particle image velocimetry (PIV) system (HAS-D71M, DITECT Corporation, Japan) equipped with a diode-pumped green laser (532 nm, Laserlab, Korea), following previously described methods with modifications. Each formulation (5 mg) was loaded into a hydroxypropyl methylcellulose (HPMC) hard capsule (size 3) and aerosolized using an RS01^® dry powder inhaler (Plastiape, Italy).

The RS01^® device used in this study corresponds to the same medium-resistance variant supplied with the commercial inhalation product Fluterol^® (Hanmi Pharmaceutical Co., Ltd., Republic of Korea), exhibiting a measured pressure drop of approximately 0.016 kPa^0.5/(L·min⁻¹) at 60 L/min with a size #3 capsule.

The device was mounted at the inlet of a clear acrylic cube (200 mm × 200 mm × 200 mm) with an outlet orifice diameter of 50 mm, and aerosolization was performed using a vacuum pump (Edwards, UK) at a controlled flow rate of 60 L/min [38].

A laser sheet was aligned parallel to the aerosol flow, and the particle motion was recorded at 8,000 frames/s with a resolution of 640 × 480 pixels. Representative frames were selected 30, 60, and 90 ms after the onset of aerosol emissions. These time points were chosen to capture unimpeded primary aerosol plumes before the particles rebound from the far end of the acrylic cube, which typically occurs after approximately 100–150 ms.

Velocity vector analysis was performed using the Flownizer 2D software (ver. 1.2.1.2, DITECT Corporation), using a recursive cross-correlation algorithm. To assess spatial flow characteristics, particle velocity components in the horizontal (U-axis) and vertical (V-axis) directions were extracted every 0.3 ms for 100 ms at three measurement points: the orifice center and two points located 9 cm from each lateral edge of the orifice. The 9 cm distance corresponds to the induction port length of the NGI, representing the anatomical length of the human oropharyngeal region. In addition, cross-sectional velocity vector distributions across the entire 9 cm transect were mapped at 30, 60, and 90 ms to visualize the dispersion profile of each formulation [83].

In-vitro aerodynamic performance

In accordance with the aerosol testing standards specified in USP Chapter < 601>, the aerodynamic performance of raw liraglutide, LG, LG@ZO-1, LG@ZO-2, and LG@ZO-3 formulations was evaluated using a NGI and an RS01^® DPI device. To ensure consistent dosing, each sample was loaded into HPMC hard capsules (size 3) at a liraglutide-equivalent content of 1 mg.

The RS01^® device used in this study was the same medium-resistance variant supplied with the commercial product Fluterol^®, exhibiting a measured pressure drop of 0.016 kPa^0.5/(L·min⁻¹) at 60 L/min for a size #3 capsule.

To prevent particle bounce and re-entrainment, the NGI stage collection plates were pre-coated with 3% silicone oil dissolved in hexane. The capsule was inserted into an RS01 DPI device connected to the mouthpiece of the induction port. The experiment was conducted by air inhalation at a controlled flow rate of 60 L/min for 4 s. For an NGI flow rate of 60 L/min, the aerodynamic cutoff diameters of each stage were determined as 8.06 μm, 4.46 μm, 2.82 μm, 1.66 μm, 0.94 μm, 0.55 μm, 0.34 μm, and 0.14 μm for stages 1–7 and Micro Orifice Collector (MOC). The quantity of the sample remaining in the capsule and deposited on each collection plate at each stage was quantified using HPLC. The parameters of the aerosolization performance were calculated by Inhalytix^® software (Copley Scientific Ltd., Nottingham, UK), i.e., fine particle fraction (FPF), fine particle dose (FPD), extra fine particle fraction (eFPF), extra fine particle dose (eFPD), mass median aerodynamic diameter (MMAD), and geometric standard deviation (GSD). The equations for ED, FPF, and eFPF are as follows:

MMAD and GSD were calculated using the guidelines provided in USP Chap. 601. The MMAD was determined from a plot of the mass fraction smaller than the aerodynamic diameter, specified as D50%, on a logarithmic probability scale. GSD was calculated using the following equation:

In vitro study on the behavior of the formulation in mucus

Diffusion behavior of LG in artificial mucus

Artificial mucus was prepared by dissolving Mucin from porcine stomach (FUJIFILM Wako Pure Chemical Corp., Japan) in DPBS at a concentration of 5% w/v. FITC-labeled LG, prepared according to the method described in Sect. 2.3.3, was added onto the surface of the artificial mucus at a loading amount equivalent to approximately 0.1 mg of LG per 5 mL of mucus. The diffusion behavior of LG across the mucus layer was subsequently evaluated under the same imaging and quantification conditions described below.

The movement of FITC-labeled LG in the mucus was analyzed using multiple particle tracking (MPT) as previously described. The particles were observed and tracked using confocal laser scanning microscopy (CLSM; LSM 980, ZEISS, Germany). Particle movement was recorded for 30 min at 1-minute intervals. The tracking videos were analyzed using Tracker video analysis software [30]. The coordinates of the particle centroids were transformed into the time-averaged mean-squared displacement (MSD), which was calculated using the following equation:

where x(t) and y(t) represent the particle coordinates at a given time, and t is the time scale or time lag.

Mucus penetration study in Calu-3 cells

To evaluate the mucus penetration behavior of LG in the presence of inhalable ZO formulations, Calu-3 cells were cultured on Corning^® Transwell^® inserts (12-mm diameter, 1.12 cm² growth area) with polycarbonate membranes (0.4 μm pore size; pore density 4 × 10⁶ pores/cm²). Cells were seeded at a density of 5 × 10⁵ cells/cm², and the air–liquid interface (ALI) was initiated on day 3. Cultures were maintained for 10–14 days, and ALI maturation was confirmed when the transepithelial electrical resistance (TEER) reached 300–500 Ω·cm² and remained stable for at least three consecutive days. Before the experiment, the cells were equilibrated with pre-warmed HBSS at 37 °C for 30 min and then exposed to the FITC-labeled LG-loaded zinc oxide formulation for 1, 2, and 4 h. After exposure, the cells were washed three times with cold HBSS, fixed with 4% paraformaldehyde (PFA), and stained with Hoechst 33,342 for nuclear staining, followed by two additional washes with HBSS. Subsequently, the cells were stained with Alexa Fluor 488-labeled wheat germ agglutinin (WGA) and washed twice with cold HBSS. Finally, the samples were observed using CLSM [30].

Macrophage adhesion and uptake studies

The adhesion of inhalable ZO particles to macrophages was evaluated using RAW264.7 cells. The cells were seeded in 96-well plates at 1 × 10⁴ cells/well and cultured for 24 h under standard conditions. Before exposure, cells were equilibrated with pre-warmed Hank’s balanced salt solution (HBSS) at 37 °C for 30 min. The FITC-labeled ZO formulations were then applied and incubated for 2 h. After exposure, the unbound particles were removed by washing three times with cold HBSS. Cells were fixed with 4% paraformaldehyde (PFA), counterstained with Hoechst 33,342, washed twice with HBSS, and imaged using confocal laser scanning microscopy (CLSM). The fluorescence intensities were quantified to assess particle adhesion.

To assess the resistance of liraglutide (LG)-loaded ZO formulations to macrophage phagocytosis, RAW264.7 cells were seeded in 96-well plates at the same density and cultured for 24 h. Nuclei were stained with Hoechst 33,342 and washed twice with HBSS, followed by equilibration in pre-warmed high-glucose DMEM at 37 °C for 30 min. Cells were then exposed to FITC-labeled LG-loaded ZO formulations for 2 h. Live-cell CLSM images were acquired every 10 min under controlled conditions (37 °C, 5% CO₂) using a Cell Discoverer 7 system (ZEISS, Germany). Fluorescence intensities were quantified to evaluate the cellular uptake. Additional visualization was performed at the 2 h endpoint using a phase-contrast microscope (CKX53, Olympus, Japan).

Cell viability

The cytotoxicity of the inhalable zinc oxide–based LG formulation toward bronchial and alveolar epithelial cells was assessed using a 3-(4,5-dimethyl-2-thiazolyl)−2,5-diphenyl-2 H-tetrazolium bromide (MTT) assay. A549 cells were seeded in 96-well plates at a density of 1 × 10⁴ cells/well and cultured for 24 h to allow cell adhesion and spreading. The cells were then treated with the formulation at liraglutide-equivalent concentrations of 0, 0.08, 0.31, 1.25, and 5.00 µg/mL for 48 h.

After treatment, the culture medium was replaced with 100 µL of MTT solution (0.5 mg/mL), and the cells were incubated at 37 °C for 4 h. The supernatant was then removed, and 100 µL of DMSO was added to dissolve the formazan crystals, followed by orbital shaking for 10 min. Absorbance was measured at 540 nm using a microplate reader (SpectraMax iD3, Molecular Devices, USA). Although MTT formazan exhibits its primary absorbance maximum near 570 nm, preliminary system-suitability testing in our laboratory showed that measurements at 540 nm provided more stable baselines and lower signal variability under our assay conditions; therefore, 540 nm was selected as the analytical wavelength for this study [84].

In vivo Pharmacokinetic study in Sprague-Dawley rats

All animal experiments were conducted in accordance with the ARRIVE guidelines and the Guide for the Care and Use of Laboratory Animals, and were approved by the Institutional Animal Care and Use Committee (IACUC) of Chungbuk National University (approval number: CBNUA-25-0015−01). Male Sprague-Dawley (SD) rats (8 weeks old, 220–280 g; Samtako, Republic of Korea) were housed under controlled environmental conditions (temperature: 23 ± 1 °C; relative humidity: 50 ± 10%; 12 h light/dark cycle) with ad libitum access to a standard pellet diet and purified water. Animals were acclimatized for seven days before experimentation.

For the pharmacokinetic (PK) study in healthy rats, animals were randomly assigned to three groups (n = 4 per group): SC_LG (subcutaneous injection of liraglutide solution), ITI_LG (intratracheal insufflation of liraglutide powder), and ITI_LG@ZO-3 (intratracheal insufflation of liraglutide-loaded ZO-3 powder). All formulations were administered at a liraglutide-equivalent dose of 0.5 mg/kg. For SC injection, liraglutide was dissolved in saline immediately prior to dosing. For intratracheal dosing, dry powder formulations were loaded into a Dry Powder Insufflator™ (Penn-Century Inc., USA) and delivered under isoflurane anesthesia.

Blood samples (500 µL) were collected from the retro-orbital sinus into heparinized tubes at predetermined time points (0.5, 1, 2, 4, 6, 8, 12, 24, 48, and 72 h). Blood sampling was performed under light anesthesia to minimize stress. Plasma was obtained by centrifugation at 4,000 rpm for 10 min at 4 °C and stored at − 80 °C until analysis. Plasma liraglutide concentrations were quantified using liquid chromatography–tandem mass spectrometry (LC-MS/MS; 1290 Infinity II, Agilent Technologies, USA; QTRAP 6500, Sciex, Germany).

In the alloxan-induced carbohydrate metabolism disorder model, liraglutide was administered at a higher dose (1 mg/kg) to ensure adequate plasma exposure for pharmacokinetic profiling. Disease-associated physiological variability in peptide absorption and systemic disposition can lead to lower and more variable circulating peptide levels; therefore, a higher dose was selected to maintain concentrations within the validated quantification range of the LC–MS/MS assay. The same dosing and sampling procedures as in the healthy rat study were used.

Randomization was performed using a simple random number generator. Blinding was not applied; however, sample processing and analysis followed a predefined protocol to minimize bias. Humane endpoints were established, and animal discomfort was minimized through appropriate anesthesia and CO₂ euthanasia at study termination.

In vivo biodistribution study in Sprague-Dawley rats

Male Sprague–Dawley (SD) rats (8 weeks old, 220–280 g; Samtako, Korea) were housed under controlled temperature (23 ± 1 °C), relative humidity (50 ± 10%), and a 12 h light/dark cycle. Rats were fed an AIN-76 A purified diet (Research Diets Inc., NJ, USA) and purified water ad libitum, and were maintained on this diet for 10 days prior to experimentation. All procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of Chungbuk National University (approval number: CBNUA-25-0015−01).

Animals were randomly assigned to four groups (n = 3 per group), except for a naive control group (n = 1). Randomization was performed using a simple random number generator. FITC-labeled ZO-based powder formulations were administered via intratracheal insufflation (ITI) at a liraglutide-equivalent dose of 0.5 mg/kg using a Dry Powder Insufflator™ (Penn-Century Inc., USA) under isoflurane anesthesia.

At predetermined time points (0, 24, and 48 h post-dose), animals were euthanized by CO₂ asphyxiation and perfused transcardially with phosphate-buffered saline (PBS, pH 7.4). PBS perfusion was selected instead of in situ fixation to avoid potential quenching of FITC fluorescence during the early fixation phase. After excision, the lungs, heart, kidneys, and liver were fixed in 10% neutral-buffered formalin, embedded in paraffin, and sectioned at a thickness of 4 μm.

Fluorescence imaging of tissue sections was performed using an in vivo optical imaging system (IVIS; VISQUE InVivo Smart-LF, Vieworks, Korea). Tissue slices were used instead of whole-organ imaging because, given the sensitivity limitations of the imaging equipment, thin sections allowed improved signal-to-noise ratio and more reliable visualization of intraparenchymal fluorescence distribution. Fluorescence intensity was quantified using CleVue™ software (Vieworks, Korea).

All procedures were conducted to minimize animal discomfort, and tissue handling followed standard histopathological preparation protocols. Sample size was determined based on feasibility considerations for preliminary biodistribution assessment.

High-performance liquid chromatography (HPLC) -UV analytical method

The HPLC method for the quantitative analysis of liraglutide was performed using a Vanquish Core HPLC system (Thermo Scientific, USA). The analytical column used was an Aegispak C8 250 × 4.6 mm, 5 μm HPLC analytical column (Young Jin Biochrom Co., Ltd., Republic of Korea). The mobile phase was prepared by dissolving 7.1 g of Na₂HPO₄ in 1,000 mL of water, adjusting the pH to 3.5 with phosphoric acid, and mixing with 78% acetonitrile at a 40:60 (v/v) ratio. The mobile phase was filtered through a 0.45 μm membrane filter (Whatman, UK) and degassed before use. The mobile phase was pumped through the column at a 1.0 mL/min flow rate, and the column temperature was maintained at 45 °C. The detection wavelength was set to 215 nm, and the injection volume for each sample was 50 µL. The HPLC method was validated for quantitative analyses.

Statistics

All statistical analyses were performed using GraphPad Prism 8 (version 8.4.2; San Diego, CA, USA). P-values are reported in the corresponding figure legends, with statistical significance defined as p < 0.05.

Results and discussion

Physicochemical characterization of inhalable ZO particles and formulations

Morphology of inhalable ZO particles

Representative SEM images of inhalable ZO (ZO) particles designed for deep-lung drug delivery are shown in Fig. 1A–F, and the corresponding quantitative morphological parameters are summarized in Fig. 1G. ZO-1 exhibited a spherical morphology with a relatively uniform particle size distribution in the 5–7 μm range. In contrast, ZO-2 displayed a spiky, sea-urchin-like morphology, consisting of a central core with radially protruding elongated tips, with an overall particle size of 5–8 μm, similar to ZO-1. ZO-3, prepared using the same method as ZO-2 but with a shorter reaction time, exhibited a smaller particle size distribution of 1–3 μm while maintaining the spiky morphology. The average aspect ratios of ZO-1, ZO-2, and ZO-3 were within the narrow range of 1.04–1.10, indicating that the particles were primarily based on a spherical framework.

Although ZO-2 and ZO-3 were synthesized using the same strategy except for the reaction time, their morphological characteristics differed not only in overall size but also in tip geometry. While the average tip diameters were comparable, the tip length of ZO-2 (3.5 μm) was approximately three times longer than that of ZO-3 (1.3 μm), suggesting differences in structural robustness. Consistent with this, the SEM images frequently revealed fractured tips in ZO-2, whereas ZO-3 retained intact tips without apparent breakage. The inter-tip distance also differed significantly, with ZO-2 exhibiting an average spacing of ~ 1.8 μm compared to ~ 600 nm for ZO-3. Such variations in tip density and spacing are likely to influence surface roughness, which may, in turn, affect cell adhesion. Previous reports have demonstrated that surface roughness within the 0.2–1 μm range promotes enhanced cell adhesion [52, 85].

Elongated or porous particles tend to evade macrophage uptake, but how particles with low aspect ratios and sharp, complex surfaces, such as ZO-2 and ZO-3, are taken up remains unclear. These morphological differences also impact mucus penetration. While particle size governs both Brownian motion and steric hindrance, surface area and protrusion geometry modulate interactions with the mucus network. Sharp protruding tips may disrupt the nonlinear cohesive forces of the mucus and generate asymmetric forces that facilitate particle transport.

In summary, ZO-1 and ZO-2 shared comparable size distributions but exhibited markedly different morphologies. On the contrary, ZO-2 and ZO-3, despite being synthesized under similar conditions, showed a two-fold difference in particle size and statistically significant differences in tip-to-tip distance and tip-to-center length. These morphological disparities are expected to critically affect the aerodynamic performance, mucus mobility, and interactions with macrophages, influencing the overall in vivo behavior of inhalable formulations.

Structural and compositional characteristics of inhalable ZO particles

The physicochemical properties of the inhalable ZO particles before and after calcination, including their purity and elemental distribution, are shown in Fig. 2. PXRD patterns were recorded for raw ZO and the synthesized ZO-1, ZO-2, and ZO-3 particles before and after calcination (Fig. 2A). Prior to calcination, ZO-1 exhibits diffraction patterns identical to those of raw ZO. In contrast, ZO-2 and ZO-3 showed additional diffraction peaks that did not match those of raw ZO, although these additional peaks were identical for ZO-2 and ZO-3. Representative mismatched peaks appeared at 2θ values of 20°, 21°, 27°, 28°, 33°, 39°, 41°, and 58°, which are characteristic of carbonate impurities that arise when the calcination process is incomplete or absent [86, 87]. After calcination, all ZO samples (raw ZO, ZO-1, ZO-2, and ZO-3) displayed identical diffraction patterns corresponding to wurtzite ZO, with reflections indexed at (100), (002) (101), (102), (110), (103), (200), (112), (201), (004), and (202) planes. These results are consistent with Joint Committee on Powder Diffraction Standards (JCPDS) card No. 36–1451, confirming that calcination successfully eliminated carbonate impurities and facilitated the formation of pure ZO crystals [88]. This indicates that calcination is essential to achieve structural homogeneity and crystallinity comparable to those of standard ZO.

The presence of surface carbonate species was further examined using XPS (Fig. 2B). Across all the samples, the near-surface region (~ 1 nm) contained a lower proportion of Zn and a correspondingly higher level of carbon. Notably, pre-calcined ZO-3 exhibited a 10–20% lower Zn signal and a 5–30% higher C signal up to a depth of 40 nm compared to both calcined ZO-3 and raw ZO. Following calcination, ZO-3 showed Zn and C levels nearly identical to those of raw ZO, with elemental deviations of less than 1% beyond a depth of 2.5 nm. These findings confirm that calcination effectively removed surface-bound carbonate impurities and restored the surface composition to that of raw ZO.

Although XPS depth profiling was performed for all ZO formulations, ZO-3 is presented as the representative dataset in Fig. 2B. This is because ZO-1 and ZO-2 exhibited depth profiles nearly identical to that of ZO-3, resulting in curves that were visually indistinguishable when plotted together. Therefore, ZO-3 was selected as the most interpretable representative sample, while the text describes the common trends observed across all formulations.

Elemental mapping using SEM-EDS was performed to visualize the distribution of Zn within the particles (Fig. 2C). Raw ZO, ZO-1, ZO-2, and ZO-3 exhibited elemental maps that matched their particle morphology, confirming the homogeneous distribution of Zn. In the case of ZO-1, the spherical morphology led to shading artifacts at the particle edge because of the angled detection of the EDS relative to the SEM beam. Nevertheless, no compositional inhomogeneity was detected across the particles, indicating consistent bulk purity.

XRF analysis was conducted to quantify the elemental purity (Fig. 2D). Raw ZO contained 75.5% Zn, 23.7% O, and ~ 0.7% C by mass. ZO-1, ZO-2, and ZO-3 had nearly identical compositions, with differences within 0.5% for each element. ZO content exceeded 95% in all cases when expressed in oxide form, while CO₂ content differed by less than 1.5%. These results confirm that all inhalable ZO particles were synthesized with high chemical purity and consistent elemental composition.

In summary, the PXRD, XPS, SEM-EDS, and XRF analyses consistently confirmed that the carbonate impurities present in the precalcined samples were eliminated after calcination, resulting in phase-pure ZO with uniform composition and high crystallinity. Such purity and structural consistency are critical for ensuring the reproducible physicochemical behavior, biocompatibility, and performance of ZO-based inhalable formulations.

Surface physicochemical properties and ion release behavior of inhalable ZO particles

The surface properties and ion release behavior of the inhalable ZO particles were evaluated using BET analysis, surface energy measurements, and zinc ion release studies (Fig. 3). BET analysis revealed pronounced differences in the specific surface areas and pore characteristics among ZO-1, ZO-2, and ZO-3 (Fig. 3A). The spherical ZO-1 exhibited an extremely low BET surface area (0.17 m²/g) and micropore area (0.27 m²/g). In contrast, ZO-2, despite having a comparable particle size distribution to ZO-1, demonstrated a markedly larger BET surface area (2.62 m²/g, ~ 15-fold higher than ZO-1) and micropore area (1.48 m²/g, ~ 5-fold higher). ZO-3, with its smaller particle size range (1–3 μm) and sea-urchin-like morphology, displayed the largest BET surface area (9.48 m²/g, ~ 55-fold higher than ZO-1) and micropore area (5.07 m²/g, ~ 18-fold higher). These findings suggest that particle size reduction combined with a spiky surface morphology synergistically increases the available surface area and porosity.

Surface energy measurements further confirmed the morphological effects on physicochemical properties (Fig. 3B). ZO-1 exhibited the lowest surface energy (66.84 mN/m), whereas ZO-2 and ZO-3 showed significantly higher values (87.72 and 88.03 mN/m, respectively). The increased surface energy of spiky particles likely reflects stronger wettability and enhanced interaction with aqueous environments, which may, in turn, influence the dispersion behavior and biological interactions.

Zinc ion release behavior was assessed using Fluozin™−3, which fluoresces upon binding zinc ions, allowing visualization and quantification at the single-particle microenvironment level (Fig. 3C and D). ZO-1 and ZO-2 exhibited negligible increases in fluorescence over 60 min, and slight decreases were observed, likely due to ion diffusion beyond the localized detection region. In contrast, ZO-3 displayed a sharp increase in the fluorescence intensity after 30 min, indicating a burst release of zinc ions. Quantitative analysis confirmed that ZO-3 released significantly higher levels of zinc ions than ZO-1 and ZO-2. This behavior can be attributed to the higher specific surface area and pore volume of ZO-3, which facilitates rapid hydration and ion diffusion once the particle surface is wetted.

In summary, although ZO-1 and ZO-2 share similar size distributions, their distinct morphologies result in substantially different surface properties, with ZO-2 exhibiting a larger surface area and higher surface energy than ZO-1, yet showing similar ion-release patterns. In contrast, ZO-3, despite having a morphology identical to that of ZO-2, exhibited a markedly greater surface area and porosity, which was attributed to its smaller size, ultimately leading to enhanced zinc ion release. These findings indicate that particle size and morphology synergistically dictate the surface physicochemical properties that govern drug–particle interactions, cellular adhesion, and potential biological responses [67, 89, 90].

Aerosolization and dispersion behavior of inhalable ZO particles

The aerosolization patterns and dispersion behavior of inhalable ZO particles (ZO-1, ZO-2, and ZO-3) were investigated using particle image velocimetry (PIV) to assess the influence of particle morphology on aerodynamic performance and potential pulmonary deposition (Fig. 4).

Fig. 4 — Aerodynamic flow analysis by particle image velocimetry (PIV). (A–C) PIV particle (top) and vector (bottom) images of ZO-1, ZO-2, ZO-3 at 30, 60, 90 ms. (D–F) U–V velocity phase maps at 9 cm downstream from the orifice for upper, middle, and central planes, respectively (V vs. U velocity over 0–100 ms). (G–I) PIV-derived velocity distributions at the same cross-section and time points, displayed across the full range or within ± 0.1 mm ms⁻¹. Black boxes highlight broadened transverse dispersion fields indicating enhanced turbulent mixing

Figure 4A–C present particle and vector images at 30, 60, and 90 ms. Particle images clearly showed that ZO-3 produced a more explosive release of fine particles than ZO-1 or ZO-2 across all time points. This dense release limited vector resolution in overlapping regions, but meaningful differences among morphologies were still evident, with red boxes highlighting representative regions of interest. At 30 ms, just before reaching the distal observation window, ZO-1 and ZO-2 displayed relatively linear U-axis trajectories. ZO-1 released particles with higher velocities, whereas ZO-2 exhibited more streamlined flow patterns indicative of velocity decay. In contrast, ZO-3 generated extensive particle–particle collisions, leading to multidirectional dispersion and vortical flows. These behaviors persisted at 60 and 90 ms.

At later time points, ZO-1 propagated almost exclusively along the U-axis with high velocities, including ~ 9 cm from the orifice, a distance corresponding to the NGI induction port length, mimicking the oropharyngeal airway. This suggests that ZO-1’s morphology promotes a jet-like plume prone to inertial impaction at the oropharynx. ZO-2 also predominantly maintained U-axis trajectories but at lower velocities, consistent with its spine-like projections generating variable drag and smoother, more streamlined aerosolization [91]. ZO-3 combined both features, but owing to extensive collisions, produced broad angular dispersion and turbulence that persisted across all time points.

Figure 4D–F quantify U- and V-velocity distributions at three positions (orifice edges and central axis at 9 cm) up to 100 ms. ZO-1 exhibited clustered U-velocities at 4–6 mm/ms and V-velocities < 1 mm/ms, consistent with a focused jet. ZO-2 showed more evenly distributed U-velocities (0–6 mm/ms) and symmetric V-velocities (–1.3 to 1.3 mm/ms), reflecting its uniform, laminar-like dispersion. ZO-3 exhibited the broadest distribution range, including negative U-velocities (–5.3 mm/ms) and V-velocities (–2.4 to 4.5 mm/ms), confirming the formation of vortices and bidirectional flow patterns.

Figure 4G-I illustrates the temporal evolution of V-axis velocity distributions at 9 cm. ZO-1 displayed a peak-like profile, with central velocities reaching ~ 7 mm/ms at all time points. Rapid velocity drops above 10 mm and below 20 mm suggest particle convergence and confinement, while overall spread remained limited (–20 to 30 mm). ZO-2 exhibited a stepwise profile, with a plateau region (~ 5–6 mm/ms) near 3 mm that extended across 10–20 mm, followed by slower layers (~ 1 mm/ms) above and below, producing stable stratified dispersion. ZO-3 showed a turbulence-modulated stepwise profile, similar to ZO-2 but with oscillating peaks and valleys within each layer. A broad high-velocity zone (~ 5 mm/ms) extended 16–20 mm vertically, accompanied by secondary (1 mm/ms, 20–25 mm) and tertiary (0.1–0.3 mm/ms, 20–40 mm) layers. The wide spread (–40 to 45 mm) indicates extensive turbulence-induced dispersion.

Together, these findings highlight the distinct aerodynamic signatures shaped by particle morphology. ZO-1 behaves as a narrow, high-velocity jet, which may render it more prone to oropharyngeal impaction and, therefore, less favorable for efficient transport into the distal lung. ZO-2 exhibited a streamlined, layered dispersion with stable flow fields, suggesting its potential to enhance pulmonary deposition by reducing upper airway losses and supporting a more uniform inhalation profile. ZO-3 generates extensive turbulence and multidirectional flows, which, despite the reduced linear velocity, may facilitate redistribution within branching airways, thereby offering the greatest potential for reaching peripheral lung regions. Notably, such turbulence combined with moderate velocities is generally regarded advantageous for pulmonary delivery because vortical motion reduces particle impaction in the upper airways and enhances penetration into the distal lung regions [83, 92]. Collectively, these results suggest that while ZO-1 morphology may be less favorable for distal deposition, ZO-2 could offer the potential for a more balanced and consistent delivery profile, and ZO-3, through turbulence-driven dispersion, may hold particular promise for enhancing deep lung deposition when employed in dry powder inhaler formulations [37, 83].

Morphology of the formulation loaded with liraglutide

Figure 5 shows the morphology of LG and the formulations blended with the zinc oxide carriers ZO-1, ZO-2, and ZO-3, denoted as LG@ZO-1, LG@ZO-2, and LG@ZO-3, respectively. At a magnification of 10,000×, the particle surface features are clearly resolved. The co-adherence ratios of LG to each ZO carrier were quantified (n = 15) and are summarized in Fig. 5E.

Fig. 5 — Morphology and drug–carrier adhesion of liraglutide formulations. (A–D) SEM images of (A) LG, (B) LG@ZO-1, (C) LG@ZO-2, (D) LG@ZO-3 (10 000×). (E) Co-adherence rate of LG on ZO particles (n = 15). One-way ANOVA with Tukey’s test (***p < 0.001, ****p < 0.0001)

Figure 5A shows the LG particles, which appear predominantly spherical with relatively smooth surfaces. The particle size distribution ranged from 0.3 to 3 μm, indicating a comparatively uniform morphology. Some particles exhibited fissures or surface irregularities, likely resulting from beam-induced damage or the intrinsic fragility of the spray-dried particles.

Figure 5B shows LG@ZO-1, where the LG particles were electrostatically co-attached to ZO-1. The co-attachment ratio was 5.3% ± 2.9, the lowest among the three carriers. While this weak interaction facilitates detachment, the low degree of co-attachment also suggests that the LG particles may not sufficiently accompany ZO during aerosolization. Therefore, it may be difficult to expect additional functional contributions from ZO in this formulation.

Figure 5C corresponds to LG@ZO-2, which exhibited the strongest co-attachment, with a 58.7% ± 14.0 ratio. This pronounced interaction likely reflects the favorable particle size and surface area of ZO-2, which enables extensive electrostatic association. However, excessive adhesion could hinder efficient drug detachment from the respiratory tract, indicating that high co-attachment is not necessarily synonymous with optimal aerosolization performance.

Figure 5D depicts the LG@ZO-3. The smaller particle size and larger specific surface area of ZO-3 promoted effective adhesion; however, the limited carrier size constrained the overall number of attached LG particles. The co-attachment ratio was 17.7% ± 2.8. This intermediate level of co-attachment may provide a balance, ensuring sufficient association for ZO to accompany LG particles, while still allowing detachment under inhalation conditions.

The morphological analysis indicates that the ZO carriers differ substantially in their capacity for electrostatic co-attachment with liraglutide particles. Although ZO-2 exhibited the highest association, an intermediate level of co-attachment, as seen with ZO-3, may be more desirable, allowing ZO to accompany LG particles without excessively compromising drug detachment. These observations highlight the importance of tuning carrier–drug adhesion strength to optimize inhalation formulations, although further aerodynamic and cellular studies are required to validate these implications.

In-vitro aerodynamic performance and morphology analysis using NGI

The in vitro aerodynamic performances of the formulations were evaluated using NGI at 60 L/min (Fig. 6; Table 1). Figure 6A shows a schematic diagram of the NGI stages corresponding to the anatomical regions of the respiratory tract. The deposition profiles (Fig. 6B) revealed that raw liraglutide exhibited predominant deposition in the induction port and stage 1, whereas spray-dried LG was mainly distributed in stages 2–4. The incorporation of ZO carriers markedly altered the deposition behavior. LG@ZO-1 facilitated efficient release from the capsule and device, achieving a high emission dose (~ 96%). However, owing to the lubricating effect of its low-surface-energy morphology, a substantial portion of the particles was retained in the pre-separator, thereby limiting the FPD. In contrast, LG@ZO-2 and LG@ZO-3, despite lacking a lubricating effect because of their larger surface areas, reduced deposition in the induction port, pre-separator, and stage 1, and enhanced delivery to deeper lung regions. LG@ZO-3 demonstrated the most favorable inhalation efficiency across all parameters (FPF, eFPF, FPD, and eFPD), suggesting its superior potential for pulmonary delivery.

Table 1.

Aerodynamic performance of inhalable liraglutide microparticles

Formulation	ED (%)	FPF (%) < 4.46 μm	eFPF (%) < 1.66 μm	FPD (µg)	eFPD (µg)	MMAD (µm)	GSD
Raw liraglutide	88.67 ± 4.01	3.48 ± 0.54	0.86 ± 0.24	18.10 ± 5.85	4.25 ± 0.73	-	-
LG	51.74 ± 3.89	77.69 ± 8.91	27.43 ± 0.33	276.38 ± 59.17	96.87 ± 9.36	3.38 ± 0.17	1.58 ± 0.01
LG@ZO-1	96.3 ± 0.35 **	60.63 ± 5.88	38.88 ± 5.29	271.82 ± 38.18	156.41 ± 48.00	2.46 ± 0.11 **	1.74 ± 0.09
LG@ZO-2	76.01 ± 4.14 **	71.49 ± 2.28	28.14 ± 1.39	408.86 ± 6.84	161.12 ± 11.80 **	3.58 ± 0.22	1.95 ± 0.04 **
LG@ZO-3	75.73 ± 3.67 **	88.85 ± 1.90	45.44 ± 4.57 *	423.91 ± 21.27 *	216.00 ± 8.34 ***	2.84 ± 0.21	1.79 ± 0.12

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Aerodynamic parameters including emitted dose (ED), fine particle fraction (FPF), effective FPF (eFPF), fine particle dose (FPD), effective FPD (eFPD), mass median aerodynamic diameter (MMAD), and geometric standard deviation (GSD) are presented as mean ± standard deviation (n = 3). Statistical significance was analyzed using two-way ANOVA followed by Tukey’s multiple comparison test against the liraglutide control (LG). *p < 0.05, **p < 0.01, ***p < 0.001

The inhalation parameters summarized in Table 1 corroborate these findings, highlighting the distinct performance of each formulation. LG@ZO-1 showed an enhanced emitted dose but limited deep-lung delivery, LG@ZO-2 increased the FPD and eFPD by reducing early stage deposition, and LG@ZO-3 achieved the highest overall inhalation efficiency.

To further investigate the particle behavior at different pulmonary deposition sites, the morphology of particles collected from NGI Stages 2 and 4 was examined by SEM (Fig. 6C). SEM analysis revealed surface cracks and deformation of LG particles across all formulations, attributable to electron beam exposure during imaging. At stages 2 and 4, no particles corresponding to ZO-1 were observed, suggesting limited penetration beyond the upper stages, whereas ZO-2 and ZO-3 particles were clearly detected. In Stage 2, both carriers retained their characteristic sea-urchin-like morphology, indicating their ability to bypass the upper airways. In Stage 4, LG@ZO-2 showed partial fragmentation of its protruding spikes, suggesting potential structural fragility during aerodynamic transport. In contrast, LG@ZO-3 maintained a morphology closely resembling its pre-storage state, underscoring its robustness and stability during inhalation.

The combined aerodynamic and morphological analyses suggested distinct pulmonary behaviors for each ZO carrier type. ZO-1 was not detected at NGI Stages 2 or 4, indicating that it may have a limited ability to reach the bronchi or bronchioles. This implies that the liraglutide carried by the ZO-1 particles may not effectively access the primary absorption regions of the lungs. In contrast, both ZO-2 and ZO-3 were observed at stages 2 and 4, suggesting their ability to reach the bronchial and bronchiolar regions. For ZO-2, the partial fragmentation of its elongated spikes in stage 4 raises the possibility that its structural stability may be compromised under aerodynamic stress, which could reduce the consistency of its functional contributions in vivo. In comparison, ZO-3 not only reached deeper lung regions but also preserved its morphology across stages, supporting the expectation that it could provide more consistent effects at the sites of drug absorption. These findings indicate that while both ZO-2 and ZO-3 show potential as inhalable carriers, ZO-3 may offer a more reliable performance because of its combination of efficient pulmonary deposition and morphological robustness.

Diffusion and mucus-penetrating behavior of the formulations

The ability of biointeractive carriers to move actively within the viscous mucus enhances the probability of drug particles traversing this biological barrier. The mobility of ZO particles is governed by their size, morphology, and surface properties. Representative CLSM tracking trajectories (Fig. 7A) and MSD analysis (Fig. 7B) demonstrated that ZO-3 exhibited a markedly more dynamic motion than ZO-1 and ZO-2. All the samples showed time-dependent displacement; however, the mobility of ZO-3 was significantly higher, indicating an improved potential for overcoming the mucus barrier.

Fig. 7 — Mucus penetration and epithelial transport of liraglutide formulations. (A) Representative particle trajectories of ZO formulations in mucus. (B) Ensemble-averaged geometric mean square displacement (MSD) over time (mean ± SD, n = 3). (C) CLSM X–Z images showing time-dependent FITC-labeled drug transport at the mucus–cell interface in Calu-3 cells for (i) LG, (ii) LG@ZO-1, (iii) LG@ZO-2, (iv) LG@ZO-3. (D) Mean fluorescence intensity in the mucus layer and (E) at the cell membrane (mean ± SD, n = 3). Two-way ANOVA with Dunnett’s post hoc test vs. LG at each time: *p < 0.05, **p < 0.01, ***p < 0.001

FITC-labeled LG and LG@ZO formulations were applied to Calu-3 membranes cultured under ALI conditions to evaluate penetration under physiologically relevant conditions (Fig. 7C). The X–Z CLSM images at 1, 2, and 4 h revealed distinct behaviors (Fig. 7C). At 1 h, most formulations were confined to the mucus or lost during washing with negligible epithelial deposition. At 2 h, LG was nearly absent, whereas LG@ZO-1, LG@ZO-2, and LG@ZO-3 were still detected in the mucus. After 4 h, LG@ZO-2 and LG@ZO-3 had accumulated in the Calu-3 epithelium, whereas LG@ZO-1 remained largely restricted to the mucus. Quantitative analyses (Fig. 7D and E) confirmed that LG@ZO-1 was localized primarily in the mucus, whereas LG@ZO-2 and LG@ZO-3 penetrated the epithelial layer. Overall, the permeability followed the order of ZO-1 < ZO-2 < ZO-3, consistent with the diffusion behavior observed in artificial mucus. These findings suggest that ZO-2 and ZO-3 can improve the epithelial delivery of drug particles; however, the contribution of zinc oxide to in vivo absorption may still be constrained by mucociliary clearance and immune surveillance.

Morphological differences explain these trends. ZO-1, a 6.5 μm smooth sphere with the lowest specific surface area, likely exhibited strong adhesive interactions with mucin fibers, consistent with prior reports that smooth-surfaced particles adhere extensively to mucin networks and show restricted mobility in viscoelastic gels [93, 94]. By contrast, ZO-2 (7.3 μm) and ZO-3 (3.3 μm) featured spiky, sea-urchin-like morphologies. Such protruding tips can locally disrupt the mucin mesh, reduce adhesive trapping, and facilitate passage, in line with literature demonstrating superior transport of anisotropic or spiky particles through mucus-like gels [94–96]. Among them, ZO-3 showed the highest mobility, which is attributable to its small size, which reduces steric hindrance within the mucus mesh, coupled with a spiky surface that promotes local network disruption.

Taken together, the enhanced transport of ZO-3 arises from a synergistic interplay between the reduced particle size and spiky morphology, two features widely recognized to facilitate particle diffusion across viscoelastic barriers such as mucus gels [31, 94].

Macrophage adhesion and uptake of formulation

FITC-labeled inhalable ZO particles and FITC-labeled LG-containing formulations were administered to Raw264.7 cells, and their cellular interactions were examined either at the 2 h time point or dynamically in live-cell conditions using optical microscopy and CLSM (Fig. 8).

Representative CLSM images of Raw264.7 cells after 2 h of incubation with FITC-labeled ZO formulations are shown in Fig. 8A. In the ZO-1 and ZO-2 groups, fewer than five particles per cell were observed, typically in the range of 1–3. In sharp contrast, ZO-3, characterized by its sea-urchin-like morphology and smaller size (~ 3 μm), exhibited extensive interactions with cells, with more than 50 small particles surrounding and adhering to the cell surface. Quantitative analysis of cellular fluorescence (Fig. 8B) supported these observations: ZO-3 showed a 3.1-fold and 2.4-fold increase in mean fluorescence intensity compared with ZO-1 and ZO-2, respectively. These results indicate that the sea-urchin-like architecture, combined with the smaller particle size of ZO-3, markedly enhanced the cell-surface association relative to its larger counterparts.

The optical and CLSM images of the LG-based formulations are shown in Fig. 8C. Raw LG exhibited negligible interactions with macrophages. LG@ZO-1 produced spherical particles (~ 6 μm) with a density of approximately 4–5 particles per cell, whereas LG@ZO-2 displayed a similar spherical but spiky morphology (~ 6 μm), attaching at 2–3 particles per cell and occasionally shedding detached needle-like fragments. In contrast, LG@ZO-3, consisting of smaller (~ 3 μm) sea-urchin-like particles, adhered at densities exceeding 10 particles per cell, though their small size limited clear morphological distinction in CLSM images.

Live-cell CLSM imaging revealed distinct uptake behaviors of the formulations. Raw LG underwent gradual internalization, which became evident at 120 min. A similar pattern was observed for LG@ZO-1, with the particle entry beginning as early as 30 min. Conversely, LG@ZO-2 and LG@ZO-3 exhibited minimal uptake, with LG@ZO-3 showing almost no internalization, even at 120 min. Quantitative fluorescence intensity measurements at 2 h (Fig. 8D) were consistent with these observations, showing that LG@ZO-3 exhibited more than 50% reduction in mean fluorescence compared to pure LG, whereas LG@ZO-2 showed a 1.5-fold decrease. These results clearly indicate that the presence of sea-urchin-like ZO, particularly in the smaller ZO-3 configuration, suppresses macrophage uptake despite enhanced cell surface adhesion.

Taken together, these results suggest that the sea urchin-like morphology of ZO, when combined with a reduced particle size (~ 3 μm), is highly effective in suppressing macrophage phagocytosis. The pronounced adhesion of ZO-3 particles, despite their resistance to internalization, points to the decoupling of the binding and uptake processes. This phenomenon is consistent with previous reports showing that particles with high aspect ratios or roughened porous surfaces hinder phagocytosis by disrupting membrane curvature and phagocytic cup formation. In particular, particles with surface roughness features of 0.2–1 μm are known to promote adhesion, and the ~ 600 nm tip-to-tip spacing of ZO-3 spikes likely explains their enhanced attachment [52, 85].

Previous studies have consistently demonstrated that non-spherical and high-aspect-ratio particles resist macrophage-mediated clearance. Champion and Mitragotri reported that wormlike polymeric particles with very high aspect ratios (> 20) exhibited negligible phagocytosis by macrophages compared to spherical particles of equal volume, with internalization events 6–20 times lower [97]. Likewise, they demonstrated that microparticles with irregular or protruding surfaces disrupted the symmetry required for efficient engulfment, substantially lowering the uptake efficiency [35]. In line with these findings, the spiky and compact architecture of ZO-3 particles (~ 3 μm, ~ 600 nm spike spacing) likely interferes with phagocytic engulfment, leading to strong adhesion but suppressed internalization.

Overall, these observations demonstrate that nanoscale surface topography, rather than morphology alone, governs macrophage–particle interactions. Specifically, ZO-3 combines strong surface adhesion with markedly reduced phagocytosis, suggesting a promising strategy for modulating the macrophage-mediated clearance of inhalable formulations.

Cell viability study

The cytotoxicity of the formulations was assessed using an MTT assay on A549 lung epithelial cells (Figure S2). Across the tested concentration range (0.08–5.00 µg/mL), all formulations maintained high levels of cell viability (> 80%), comparable to LG. Importantly, the tested range encompassed concentrations higher than those expected to be encountered by cells under physiological inhalation exposure; however, the ZO-containing formulations did not reduce cell viability. No pronounced morphology-dependent differences were observed among the ZO carriers, indicating an overall favorable cytocompatibility.

These results suggest that the zinc oxide-based formulations exhibited good in vitro safety, maintaining cell viability at levels comparable to those of LG across all tested concentrations. However, cell viability assays only provide a partial view of the safety of inhalation. Further in vivo studies are necessary to evaluate potential pulmonary toxicity under actual inhalation conditions, complementing the cellular data presented here [67, 69, 98–101].

Pharmacokinetics and pulmonary biodistribution in Sprague-Dawley rats

The pharmacokinetics of liraglutide were first evaluated in healthy SD rats following subcutaneous (SC) injection of LG solution, intratracheal insufflation (ITI) of LG powder, and ITI of LG@ZO-3 powder at a dose equivalent to 0.5 mg/kg. As shown in Fig. 9A, B, the plasma concentration–time profiles and corresponding PK parameters revealed distinct absorption characteristics among the formulations. SC_LG produced the highest systemic exposure and served as the reference. ITI_LG resulted in markedly reduced exposure (AUC ≈ 23% of SC_LG), consistent with limited pulmonary absorption of liraglutide alone. In contrast, ITI_LG@ZO-3 achieved substantially improved exposure, with an AUC nearly 2.5-fold greater than ITI_LG and reaching approximately 60% of the systemic exposure obtained after SC administration. ITI_LG@ZO-3 also exhibited a shorter Tmax, indicating more rapid absorption. These findings demonstrate that the ZO carrier system effectively enhances pulmonary delivery and systemic availability of liraglutide.

To evaluate performance under disease-associated physiological conditions, a parallel PK assessment was conducted in an alloxan-induced carbohydrate-metabolism-disorder model (Figs. 9C, D). Similar trends were observed, with ITI_LG@ZO-3 achieving an AUC corresponding to approximately 51% of SC_LG—substantially exceeding the exposure from ITI_LG and ITI_LG@ZO-2. Although somewhat reduced compared with healthy rats, likely due to altered peptide disposition in the disease state, the systemic bioavailability remained robust and therapeutically meaningful. This level of pulmonary absorption is notable, considering that oral semaglutide (Rybelsus^®) exhibits an absolute bioavailability below 2% [102].

Pulmonary biodistribution was further examined using FITC-labeled ZO formulations (Fig. 9E, F). Strong lung fluorescence was observed immediately after ITI administration for all groups, confirming efficient deposition. Fluorescence intensity decreased progressively, and most ZO particles were cleared from the lungs by 48 h, consistent with the near-complete disappearance of signals in extrapulmonary organs (Supplementary Figure S3). Although the specific clearance routes could not be fully determined, these data suggest that ZO carriers undergo rapid elimination, supporting their favorable short-term safety profile.

Clear morphology-dependent differences were observed in intrapulmonary distribution. ZO-1 remained largely confined to proximal airway regions, whereas ZO-2 and ZO-3 reached deeper lung structures. ZO-3 demonstrated the most homogeneous and widespread pulmonary distribution, consistent with its superior aerodynamic performance.

Collectively, the integrated PK and biodistribution findings highlight the potential of ZO-3 as an effective pulmonary carrier for liraglutide. ITI_LG@ZO-3 not only enhanced systemic exposure to near-SC levels but also exhibited favorable biodistribution and rapid clearance without prolonged retention. While these results support the translational promise of the ZO-3 morphology, further studies evaluating long-term biocompatibility, exact clearance mechanisms, and therapeutic outcomes will be essential for clinical development.

Conclusion

This study demonstrates that pulmonary administration can serve as a practical route for systemic delivery of biologics, extending beyond GLP-1 analogs to encompass a wide range of peptide and nucleic acid therapeutics. By modulating the morphology of ZO carriers, we established that morphology-driven control of pulmonary deposition and biological barrier interactions enables therapeutically relevant systemic exposure while addressing major challenges such as inefficient aerosolization, limited mucus penetration, and rapid macrophage clearance.

The three ZO morphologies exhibited fundamentally different aerodynamic and biological behaviors. Smooth, spherical ZO-1 (5–7 μm) generated narrow, high-velocity plumes in PIV studies, leading to predominant deposition in the proximal airways with minimal reach into distal lung regions. Spiky ZO-2 (5–8 μm with elongated ~ 3.5 μm tips) improved deposition efficiency and produced stratified laminar flows. However, its elongated tips were structurally fragile and prone to breakage during aerodynamic transport, potentially compromising reproducibility. In contrast, compact spiky ZO-3 (1–3 μm with short ~ 1.3 μm tips) generated turbulence-driven dispersion and multidirectional redistribution in PIV analysis, promoting homogeneous deposition throughout the bronchial and bronchiolar regions while preserving structural integrity.

At the cellular level, these aerodynamic distinctions translate into morphology-specific interactions with the lung barrier. ZO-1 adhered strongly to the mucus but remained trapped in the proximal layers, limiting epithelial access. ZO-2 exhibited moderate penetration; however, partial tip breakage reduced its consistency. ZO-3 displayed superior mucus penetration and wide intrapulmonary distribution, and despite strong adhesion to macrophages, it resisted phagocytic uptake, thereby prolonging drug residence at the epithelial interface. This unique decoupling of adhesion and uptake underscores how the nanoscale surface topography can be exploited to evade macrophage clearance while maintaining intimate drug–cell contact.

Pharmacokinetic studies further validated the translational relevance of this approach. Intratracheal administration of liraglutide-loaded ZO-3 (LG@ZO-3) achieved pulmonary bioavailability of approximately 60% relative to subcutaneous injection in healthy rats and 51% in diabetic rats, far exceeding the < 2% oral bioavailability of marketed semaglutide (Rybelsus^®). These data confirm that morphology-controlled ZO carriers can achieve therapeutically meaningful systemic delivery via the lungs. Biodistribution analysis indicated that ZO particles were rapidly cleared from pulmonary and extrapulmonary tissues within 48 h, supporting a favorable short-term safety profile. Although long-term clearance mechanisms warrant further study, the transient pulmonary residence of ZO carriers suggests low risk of accumulation.

Nevertheless, ZO inherently possesses toxicological limitations stemming from its poor solubility, limited degradability, and potential for dose-dependent inflammatory responses. Although micro-scale ZO used in this study demonstrated rapid short-term clearance, long-term retention, chronic exposure toxicity, and cumulative inflammatory effects were not assessed and remain essential considerations for any future translational work. For this reason, the present study should be regarded strictly as a proof-of-concept demonstration of morphology-driven pulmonary delivery, and future research must investigate alternative biocompatible materials capable of reproducing the advantageous aerodynamic and biointeractive properties observed here while minimizing toxicological risk [103–106].

Overall, this work establishes a biologically oriented strategy in which morphological control of ZO biointeractive carriers governs aerodynamic dispersion, mucus penetration, macrophage evasion, and systemic absorption. Among the tested morphologies, ZO-3 consistently achieved the most balanced performance, integrating efficient lung deposition with favorable biointerface interactions. These findings highlight the potential of morphology-engineered excipients as barrier-modulating carriers for the inhalable delivery of biologics, advancing the development of noninvasive pulmonary therapeutics.

Supplementary Information

Supplementary Material 1^{(314.8KB, docx)}

Acknowledgements

We would like to thank Editage (http://www.editage.co.kr) for the English language editing.

Author contributions

J.-H.J.: Formal analysis, investigation, visualization, writing—original draft preparation, and writing—review and editing. C.S.H.: Formal analysis and data curation. J.-H.K.: Conceptualization and writing- original draft. D.-W.K.: Funding acquisition and supervision. C.-W.P.: Conceptualization, methodology, funding acquisition, and supervision. All the authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the National Research Foundation of Korea Grant funded by the Korean Government(NRF-2021R1A2C4002746), and this research was supported by a grant of the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (grant number : RS-2024-00335798). This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (No. RS-2025-02273102). This research was supported by the Regional Innovation System & Education (RISE) program through the (Chungbuk Regional Innovation System & Education Center), funded by the Ministry of Education (MOE) and the (Chungcheongbuk-do), Republic of Korea (2025-RISE-11-014-03).

Data availability

The datasets used in this study are available from the authors upon request.

Declarations

Declarations of generative AI and AI-assisted technologies in the writing process

During the preparation of this manuscript, the authors used ChatGPT-5 (OpenAI, San Francisco, CA, USA) to check grammatical accuracy and correct typographical errors. After using this tool, the authors carefully reviewed and edited the content as necessary and take full responsibility for the final content of the article.

Competing interests

The authors declare no competing interests.

Footnotes

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Material 1^{(314.8KB, docx)}

Data Availability Statement

The datasets used in this study are available from the authors upon request.

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PERMALINK

Morphology-driven zinc oxide biointeractive carriers with biological barrier modulating effects for pulmonary delivery of liraglutide

Jin-Hyuk Jeong

Chang-Soo Han

Ji-Hyun Kang

Dong-Wook Kim

Chun-Woong Park

Abstract

Graphical Abstract

Supplementary Information

Introduction

Materials and methods

Materials

Cell lines and maintenance

Preparation method

Fabrication of ZO microparticles for inhalation carrier

Preparation of liraglutide microparticles and loading onto inhalable ZO

Preparation of FITC-labeled ZO and FITC-labeled liraglutide

Physicochemical properties of ZO microparticles

Scanning electron microscope (SEM) and energy-dispersive X-ray spectroscopy (EDS)

Powder X-ray diffraction (PXRD)

Elemental and surface compositional analysis

Brunauer-emmett-teller (BET)

Surface energy of ZO particles

ZO release analysis

Particle image velocimetry (PIV) analysis

In-vitro aerodynamic performance

In vitro study on the behavior of the formulation in mucus

Diffusion behavior of LG in artificial mucus

Mucus penetration study in Calu-3 cells

Macrophage adhesion and uptake studies

Cell viability

In vivo Pharmacokinetic study in Sprague-Dawley rats

In vivo biodistribution study in Sprague-Dawley rats

High-performance liquid chromatography (HPLC) -UV analytical method

Statistics

Results and discussion

Physicochemical characterization of inhalable ZO particles and formulations

Morphology of inhalable ZO particles

Fig. 1.

Structural and compositional characteristics of inhalable ZO particles

Fig. 2.

Surface physicochemical properties and ion release behavior of inhalable ZO particles

Fig. 3.

Aerosolization and dispersion behavior of inhalable ZO particles

Fig. 4.

Morphology of the formulation loaded with liraglutide

Fig. 5.

In-vitro aerodynamic performance and morphology analysis using NGI

Fig. 6.

Table 1.

Diffusion and mucus-penetrating behavior of the formulations

Fig. 7.

Macrophage adhesion and uptake of formulation

Fig. 8.

Cell viability study

Pharmacokinetics and pulmonary biodistribution in Sprague-Dawley rats

Fig. 9.

Conclusion

Supplementary Information

Acknowledgements

Author contributions

Funding

Data availability

Declarations

Declarations of generative AI and AI-assisted technologies in the writing process

Competing interests

Footnotes

References

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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