Mannitol as a Critical Excipient in Spray-Dried Chitosan Microspheres for Nasal Donepezil Delivery: Insights from Integrated Biomimetic Models

Abstract

Background/Objectives: The aim of this study was to develop and apply a novel integrated approach for predicting local mucosal tolerability of spray-dried chitosan/mannitol microspheres previously developed for nose-to-brain donepezil delivery.

Methods: Microspheres were prepared by ultrasonic spray-drying, and process reproducibility was evaluated based on particle size distribution, entrapment efficiency, and process yield across independent batches. A lactose-based formulation served as a comparative control. A novel biomimetic model was developed to investigate water evaporation under simulated nasal conditions, enabling prediction of formulation dehydration and crust-like layer formation on the nasal mucosa during nasal residence time. Donepezil-loaded chitosan microspheres and their physical mixture with mannitol were used as controls. Analyses were complemented by solid-state and rheological characterization to elucidate the effects of formulation composition and processing on the observed behavior. Irritation potential was further assessed using the established slug mucosal irritation (SMI) assay.

Results: Reproducible microsphere size distribution (Dv10 11.5 ± 1.1 µm, RSD 9.6%; Dv50 28.4 ± 3.9 µm, RSD 13.7; Dv90 61.3 ± 8.4 µm, RSD 8.4%), entrapment efficiency (99.6 ± 1.8%, RSD 1.8%) and process yield (40.9 ± 5.5%, RSD 13.3%) confirmed the robustness of the ultrasonic spray-drying. Replacing mannitol with lactose failed to achieve the desired particle size distribution, highlighting the key role of mannitol under the investigated processing conditions. The biomimetic model coupled with rheological studies demonstrated that chitosan-based gels formed by microsphere swelling in simulated nasal fluid, maintain viscosity, resist dehydration, and undergo rehydration. Additionally, mannitol enhanced water retention and reduced evaporation without increasing occlusivity or the risk of mucosal dehydration. Furthermore, powders containing mannitol exhibited a lower irritation potential in the SMI assay compared to chitosan microspheres alone.

Conclusions: Mannitol is a critical determinant of the performance of donepezil-loaded chitosan-based microspheres, contributing to the desired particle size distribution, process reproducibility, favorable hydration and improved mucosal tolerability, thereby supporting the suitability of this platform for nasal donepezil delivery.

Introduction

Development of nose-to-brain delivery systems constitutes a rapidly evolving research field addressing key limitations in drug solubility, permeability, stability, and nasal residence time, to better exploit the potential of the nasal route [1]. Among advanced delivery platforms, systems that are simple to manufacture and readily scalable, such as swellable powder formulations, have attracted considerable attention. These systems can incorporate mucoadhesive polymers [2], permeation enhancers [3], and/or nanocarriers [4], thereby optimizing nasal residence time as well as drug release and absorption profiles.

Compared with liquid formulations, nasal powders offer several important advantages, including improved stability without the need for preservatives, prolonged retention at the nasal mucosa, higher local drug concentrations, and reduced dependence on patient coordination during administration [5,6,7]. In addition, powder formulations minimize formulation runoff into the throat, thereby reducing discomfort and unpleasant aftertaste. The potential of nasal powders for nose-to-brain drug delivery has been demonstrated in animal studies [3,8,9,10,11]. Moreover, recent reports indicate more efficient olfactory deposition of nasal powders [2,12,13] compared to results reported for nebulized liquid formulations [14,15].

Spray-drying, as a single-step, rapid, and scalable technique, has become a key method for the preparation of nasal powders [7]. It enables precise control over microparticle size and morphology, which is critical for nasal administration. By fine-tuning formulation and process parameters, the radial distribution of components during drying can be controlled, thereby influencing particle size, density, and morphology through changes in evaporation rate and heat-mass transfer [16]. Consequently, geometric and aerodynamic diameter of spray-dried particles can be tailored by selecting appropriate excipients and optimizing process parameters, including feed solid content, inlet temperature, atomizing capacity, and feed rate [7,17,18].

According to current regulatory recommendations for localized nasal delivery, the majority of aerosolized particles should exceed 10 μm in size to ensure deposition within the nasal cavity [19]. While geometric particle size describes the physical dimensions of particles, aerodynamic diameter is a more relevant predictor of the site and efficiency of particle deposition in the respiratory tract because it incorporates the effects of particle density and shape [20,21]. Ultimately, assessment of nasal deposition pattern using anatomically relevant nasal cast models provides direct and physiologically relevant aerodynamic assessment of nasal powder formulations, as it integrates the combined effects of particle size, density, morphology, delivery device, and administration conditions on regional deposition [22]. In our previous work, we developed a free-flowing, spray-dried platform of donepezil-loaded chitosan/mannitol microspheres with particle size optimized for nasal delivery. This formulation demonstrated promising in vitro performance, indicating its potential for efficient brain-targeted delivery of donepezil, as evidenced by high olfactory deposition (65.5%) in a 3D-printed nasal cast, along with favorable swelling, mucoadhesion, drug release, and permeation-enhancing properties [13]. The schematic representation of the microspheres is presented in Figure 1.

Despite these encouraging results, another critical aspect of nasal powder delivery warrants consideration at this stage of formulation development, namely, the prediction of local mucosal tolerability. Reliable biomimetic models are essential for evaluating such effects. It is well recognized that deposited powder particles may induce irritation, sneezing, itching, or pain [23]. However, to date, very few studies have addressed this issue.

The slug mucosal irritation (SMI) assay, developed by Lenoir et al., represents a promising approach for predicting sensory effects on human nasal mucosa, based on the correlation between mucus production in slugs and the incidence of stinging, itching, and burning sensations in humans [24]. This method has been applied to assess the irritancy of nasal liquid formulations [25,26] and nasal powder excipients [23], enabling highly reproducible differentiation between the samples. SMI assay aligns with the principles of the 3Rs (Replacement, Reduction, and Refinement) by supporting the development of alternative testing methods and the use of lower organisms, such as invertebrates, instead of vertebrate animals [24,26]. To the best of our knowledge, the SMI assay is currently the only approach capable of screening sensory effects on the nasal mucosa without the use of vertebrate animal or human studies.

Furthermore, in the case of swellable particles that form a gel layer at the site of deposition, potential formulation dehydration and crust-like layer formation during nasal residence time may lead to discomfort and reduced patient adherence [27]. Nevertheless, as far as we are aware, no studies have investigated the potential dehydration of gel-forming nasal formulations, under biorelevant conditions, that could reduce mucosal tolerability of developed formulations.

The aim of this study was to develop a novel integrated approach enabling overall prediction of mucosal tolerability of swellable donepezil-loaded chitosan/mannitol microspheres previously developed by our group, throughout their interaction with the nasal mucosa, from initial contact to the expected residence period. These microspheres were originally prepared by ultrasonic spray-drying, a challenging and rarely used approach successfully employed to achieve the target particle size for nasal delivery [28]. In the present study, process reproducibility was assessed across independent batches alongside evaluation of mucosal tolerability. Attention was given to the role of mannitol as a key excipient in the microsphere formulation. Mannitol is widely used as a pharmaceutical excipient due to its favorable physicochemical properties such as low hygroscopicity, chemical inertness towards the active pharmaceutical ingredient, and excellent biocompatibility [29]. Owing to its osmotic activity, mannitol is also used as an active inhalation agent in patients with cystic fibrosis, where it increases mucus hydration, thereby enhancing mucociliary and cough clearance [30].

Current in vitro characterization of nasal powder formulations primarily focuses on aerosol performance, particle deposition, drug release, permeability, and mucoadhesion [31]. Beyond these conventional performance attributes, the hydration state of swellable nasal powder formulations is also an important consideration. The hygroscopic properties of individual excipients in powders intended for nasal administration have been investigated using dynamic vapor sorption [23], while swelling behavior of nasal powder formulations in contact with simulated nasal fluid has been evaluated using Franz diffusion cells [13,32]. In other pharmaceutical applications, gravimetric assays of evaporative water loss and water vapor transmission rate (WVTR) measurements have been employed to characterize water transport and dehydration behavior of hydrated formulations, particularly in the context of (trans)dermal delivery [33,34].

However, these approaches do not capture the complex water-transfer processes that may occur when a swellable nasal powder formulation is deposited onto the nasal mucosal surface. To address this gap, we developed a biomimetic experimental approach that enables integrated investigation of water evaporation, water transport through the swollen gel, resistance to dehydration, and rehydration through an available aqueous source under simulated nasal conditions. A climate chamber coupled with an immersion cell, serving as a formulation carrier, was used to establish a biomimetic model incorporating physiologically relevant temperature and humidity together with a mucin-coated interface. This setup enabled controlled simulation of water transfer between the mucosa and the formulation, allowing formulation-dependent differences in water exchange behavior to be characterized. Such differences may affect the hydration state and rheological properties of the swollen formulation, its water retention and behavior during nasal residence, as well as local mucosal hydration and, consequently, formulation performance and tolerability. Characterization of these processes is therefore relevant to the rational development and optimization of swellable nasal powder systems.

In this study, the proposed methodology was applied to donepezil-loaded chitosan microspheres, donepezil-loaded chitosan/mannitol microspheres and a corresponding donepezil-loaded chitosan microspheres/mannitol physical mixture. The biomimetic assessment of formulation behavior was complemented by solid-state and rheological characterization to elucidate the impact of formulation composition and the production process on the observed behavior. In addition, local tolerability was evaluated using the established slug mucosal irritation (SMI) assay. Together, these complementary biomimetic models provide mechanistic insight into formulation performance and enable prediction of local mucosal tolerability.

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Materials

Donepezil hydrochloride (hereafter referred to as donepezil) was obtained from Carbosynth Ltd. (Compton, UK). Low-molecular-weight chitosan (50–190 kDa, 75–85% deacetylated; hereafter referred to as chitosan) was sourced from Sigma-Aldrich (Darmstadt, Germany). Mannitol was obtained from VWR International Ltd. (Lutterworth, Leicestershire, UK). Lactose monohydrate (GranuLac®; further denoted as lactose) was purchased from Meggle (Wasserburg am Inn, Germany). Simulated nasal fluid (SNF) was prepared by dissolving NaCl (Kemig, Zagreb, Croatia), KCl (Kemig, Zagreb, Croatia), and CaCl2·2H2O (Sigma-Aldrich, Darmstadt, Germany) in distilled water to a concentration of 150.0 mM, 40.0 mM and 5.3 mM, respectively. Mucin type III (bound sialic acid 0.5–1.5%, partially purified powder) was obtained from Sigma-Aldrich (Darmstadt, Germany).

All other chemicals and solvents used were of analytical grade and were purchased from Kemika (Zagreb, Croatia).

Perkušić, M.; Nižić Nodilo, L.; Jug, M.; Jakobušić Brala, C.; Scherließ, R.; Hafner, A. Mannitol as a Critical Excipient in Spray-Dried Chitosan Microspheres for Nasal Donepezil Delivery: Insights from Integrated Biomimetic Models. Pharmaceutics 2026, 18, 1023. https://doi.org/10.3390/pharmaceutics18081023

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Read also our introduction article on Mannitol here: