Characterization of Liquid Formulations for Enhanced Buccal Permeation: Exploring Key Attributes

TL;DR

This study shows that buccal drug formulations with higher adhesion and viscosity improve drug retention and delivery through the mouth mucosa.

Contribution

The study introduces a combined qualitative and quantitative method to assess buccal formulation attributes and their impact on drug delivery.

Findings

Increasing HPC concentration improved adhesion and viscosity, leading to longer residence time and higher drug retention.

Formulation F2 showed a 1.6-fold increase in drug flux compared to the polymer-free formulation when residence time was considered.

Adding benzalkonium chloride further enhanced permeation, increasing apparent permeability by 1.5-fold compared to F2.

Abstract

Background: Buccal administration offers direct access to systemic circulation, improving drug bioavailability when compared with the conventional oral route. This advantage depends on the formulation’s ability to remain in contact with the buccal mucosa. Attributes such as adhesion and viscosity are suggested to be correlated and contribute to enhanced residence time at the administration site. Methods: Buccal formulations with varying hydroxypropyl cellulose concentrations were prepared. Adhesion, viscosity, and residence time were assessed using a novel combined qualitative and quantitative approach. Drug permeation was evaluated in vitro using a biomimetic membrane and ex vivo using porcine buccal tissue, and it was further enhanced by adding the permeation enhancer benzalkonium chloride. Permeability measurements were integrated with residence time to estimate effective drug delivery. Results: Increasing HPC concentration improved both adhesion and viscosity, with 2% HPCs (F2) showing the strongest effect (45.5 ± 13.7 g), correlating with longer residence time (43.4% drug retained at 2 min vs. ~20% for 0–1% HPC). Although the polymer slightly reduced apparent permeability, when residence time was considered, drug flux increased 1.6-fold compared to the polymer-free formulation (F0), rising from 12.9 × 10−5 cm/min (F0) to 19.4 × 10−5 cm/min (F2) after 2 min. The addition of BKC further enhanced permeation, with apparent permeability increasing 1.5-fold vs. F2 and 2.5-fold vs. F0. Conclusions: Buccal liquid preparations’ efficacy is influenced by residence time and subsequent drug permeation. Residence time benefits from the synergistic effects of adhesion and viscosity, highlighting the importance of experimentally assessing these parameters during the development of oromucosal products.

Figures

Tables

Table 2. Summary of the characterization of formulations F0, F1, and F2.

Formulation	HPCs (%)	Appearance	pH	Adhesiveness(Fmax, g)	Viscosity (mPa·s)	DSretained (%) After 2 min
F0	0%	Comply *	4.36	3.20 ± 1.7	3.32	20.3
F1	1%	Comply *	4.66	30.5 ± 10.9	319	19.4
F2	2%	Comply *	4.80	45.5 ± 13.7	3565	43.4

Full text

1. Introduction

The pharmaceutical industry is strongly focused on overcoming the poor bioavailability of many drug products, which remains one of the main limitations to achieving effective orally administered therapies [1,2,3]. In this context, the oral mucosa has gained increasing recognition as an attractive alternative route of administration [4,5,6]. By enabling direct entry of the drug into the systemic circulation and bypassing hepatic first-pass metabolism, this pathway can enhance bioavailability and provide a faster onset of action [7,8]. In addition, buccal delivery offers advantages in terms of convenience, non-invasiveness, and improved patient compliance, particularly for populations with swallowing difficulties such as pediatric and geriatric patients [1,9,10,11]. Within the mucosal regions of the oral cavity, the buccal mucosa—lining the area between the gums and inner cheeks—exhibits robust vascularization, high permeability, and elevated local tolerance, with relatively low enzymatic activity [2,5]. The primary challenges associated with buccal delivery likely arise from the several factors that can impact buccal absorption, namely, the following: (i) the relatively small surface area of the buccal mucosa restricts the amount of drug that can be applied and absorbed [7,12]; (ii) the salivary washout contributes to dilution of the formulation, mechanical washout, and premature removal of the drug from the absorption site [13,14]; and (iii) the presence of mucus and membrane barriers can hinder drug permeation, particularly for molecules with unfavorable physicochemical properties. Moreover, the dynamic oral environment, characterized by mastication, speech, and swallowing, introduces mechanical stresses that further reduce formulation retention [11,14]. These factors often lower drug concentrations at the mucosal surface below therapeutic levels, thereby limiting clinical efficacy [11,15]. For these reasons, the successful development of buccal drug products must prioritize strategies that maintain prolonged contact between the dosage form and the mucosa, thereby increasing residence time and enhancing drug permeation across the buccal membrane [3,4,9,16].

While dosage forms such as tablets and films are often preferred for longer residence time, liquid formulations can provide a faster onset of action, flexible dosing, ease of administration, and improved patient comfort—features that are particularly relevant for pediatric and geriatric populations [1,17]. However, liquid systems face additional challenges, including rapid clearance and limited residence time compared to solid or semi-solid dosage forms, which have constrained their development and systematic evaluation [18,19]. Several strategies have been employed to optimize residence time and consequently enhance drug permeation, which are critical considerations in the development of effective liquid buccal drug products. Importantly, the incorporation of adhesive polymers has proven to be one of the more effective strategies [4,9].

Polymer-related properties, such as molecular weight, flexibility, swelling, and charge, among others [4], are expected to influence the adhesion capacity and mechanisms involved. The most widely investigated groups of adhesive polymers are predominantly hydrophilic [7] and are believed to swell and allow chain interactions—by Van der Walls, electrostatic, or hydrophobic reactions—with the mucin molecules on the buccal mucosal surface [7,8,20]. Nonetheless, the mechanisms of adhesion remain unclear due to their inherent complexity. Improving residence time in liquid formulations may ultimately be achieved through the synergistic effects of combining adhesive polymers with the increased solution viscosity [15,20]. Both strategies are expected to delay the removal of the formulation from the application site. Typical and well-studied adhesive polymers for this purpose include carbomers, chitosan, sodium alginate, and cellulose derivatives [16]. In particular, hydroxypropyl cellulose (HPC), a cellulose derivative, has been widely explored in oral and topical adhesive formulations [21,22]. Following an adequate residence time of the formulation, the drug substance (DS) must be able to cross the buccal mucosal membrane and reach the systemic circulation. Most compounds permeate by passive diffusion [6], adopting the route that offers the least resistance, which is influenced by drug lipophilicity. Drug lipophilicity is affected by the formulation’s pH, as it impacts the ionization degree of the DS and, consequently, its affinity for the buccal membrane. However, it is important to note that both low (pH < 4) and high (pH > 8) pH values can provoke mucosal irritation [10,23]. Additionally, other properties of DS, such as molecular weight, the number of rotational bonds, and the number of hydrogen bond donors, have been directly correlated with its permeability [24]. Formulation scientists have studied the incorporation of permeation enhancers to improve unfavorable properties of molecules or to act on the membrane itself, consequently enhancing the permeability of formulations [25].

Overall, residence time is a critical property for buccal formulation efficacy, particularly for liquid preparations, as it ensures that the formulation remains in place upon application despite the additional challenges these systems face. Adhesion and viscosity are key contributors to residence time, and their optimization may enhance drug retention and permeation. Most methodologies for assessing residence time for solid or semi-solid systems may not be easily applicable to low-viscosity liquids. Approaches to correlate formulation properties such as adhesion and viscosity remain, consequently, limited. In the present paper, we aim to evaluate the combined use of hydroxypropyl cellulose grades M and G (HPC-M and HPC-G) as adhesive polymers to improve adhesiveness, viscosity, and, consequently, residence time in liquid buccal formulations. Additionally, the potential of benzalkonium chloride (BKC) to enhance drug permeation is evaluated through in vitro and ex vivo models, correlating residence time with absorption. A combination of qualitative and quantitative approaches is employed to monitor residence time and its effect on drug delivery, providing a framework for the rational design of effective buccal formulations.

2. Materials and Methods

2.1. Materials

Drug substance model—Naloxone Hydrochloride—was purchased from Noramco Inc., Wilmington, DE, USA. Klucel^TM^ Hydroxypropyl cellulose (HPC) MXF (HPC-M) Pharm (Ashland, OR, USA). Klucel^TM^ Hydroxypropyl cellulose GXF (HPC-G) Pharm (Ashland, OR, USA). Purified water (Bluepharma, Coimbra, Portugal); ethanol absolute anhydrous Carlo Erba (Val de Reuil, France); benzalkonium chloride (BKC) Spectrum Chemical MFG Corp. (New Brunswick, NJ, USA); Permeapad^®^ phospholipid-based Barrier membranes (innoME GmbH, Espelkamp, Germany); collagen artificial gut SaborPlus (Leiria, Portugal); sodium chloride (NaCl) Merck (Darmstadt, Germany); potassium chloride (KCl), Merck (Darmstadt, Germany); disodium hydrogen phosphate (Na_2_HPO_4_), Panreac AppliChem ITW Reagents (Milano, Italy); potassium dihydrogen phosphate (KH_2_PO_4_), Panreac AppliChem ITW Reagents (Milano, Italy); hydrochloric acid 37% Fisher (Fontenay-sous-Bois, France) and ortho-phosphoric acid 85% Panreac (Darmstadt, Germany). Ammonium acetate (NH_4_CH_3_CO_2_), Merck (Darmstadt, Germany); ammonia solution 25% (NH_4_OH), Merck (Algés, Portugal); hydrochloric acid 37% (HCl), Fisher Chemical (Leicestershire, UK).

2.2. Solution Preparations

Phosphate-buffered saline (PBS) at pH = 7.4 was used as the receptor medium in the permeability studies. It was prepared by weighing 8.0 g of NaCl, 0.20 g of KCl, 1.44 g of Na_2_HPO_4_, and 0.24 g of KH_2_PO_4_ into a 1000 mL clean and dry volumetric flask. Upon dissolution in ultrapure water, the flask was made up with the same solvent. The pH was adjusted to 7.4 with HCl 37%, using a surface electrode (VWR International, Radnor, PA, USA); the osmolality was verified to be within 275–290 mOsm/kg, measured by Semi-Micro Osmometer K7400, Herbert Knauer GmbH, Berlin, Germany.

Artificial saliva (phosphate buffer, pH = 6.8) was used for the hydration process of the collagen gut. It was prepared by weighing 1.19 g of Na_2_HPO_4_, 95.0 mg of KH_2_PO_4_, and 4.0 g of NaCl into a 500 mL clean and dry volumetric flask. Upon dissolution in ultrapure water, the flask was made up with the same solvent. The pH was adjusted to 6.8 using ortho-phosphoric acid 85%.

Ammonium acetate buffer (50 mM ammonium acetate, pH = 9.0) was used for the mobile phase of the quantification method of residence time and the permeation evaluation. It was prepared by putting 7.7 g of ammonium acetate (CH_3_COONH_4_) into a 2000 mL clean and dry volumetric flask and dissolving it in ultrapure water. Upon dissolution, the flask was filled up with ultrapure water. The pH was adjusted to 9.0 using ammonia solution 25%.

2.3. Methods

2.3.1. Formulations

The formulations used for the studies comprised a BCS class III model drug—Naloxone Hydrochloride—in an HPC-based liquid composition containing organic co-solvents. Distinct percentages of HPC’s—0% (F0), 1% (F1), and 2% (F2 and F2a)—were formulated, maintaining a ratio of 60:40 (w/w) of HPC-G and HPC-M grades. The formulation compositions are presented in Table 1. The model drug concentration was 40 mg/mL.

The formulations were prepared using a mechanical mixing stirrer (VWR International, Radnor, PA, USA). The process started with the dissolution of the DS in purified water for 30 min at room temperature (RT). Following this initial step, HPC-G and HPC-M were sequentially added, under continuous stirring, to the initial solution at a percentage of 1% (F1) or 2% (F2 and F2a) (approx. 4 h). For F2a, BKC was added in a percentage of 0.01% and stirred for 10 min. Following these steps, ethanol was added to all formulations (F0, F1, F2, and F2a) and stirred for 1 h.

Pre-stability studies of F2 were conducted over a 1-month period to provide preliminary evidence of physical and chemical stability. Assay, pH, and visual appearance were evaluated under long-term (25 °C/60% RH) and accelerated (40 °C/75% RH) conditions for climatic zone II, according to ICH Q1A(R2). The assay method was the same as mentioned for the residence time quantitative measurement (see Section 2.3.6. Residence Time).

Pre-formulation studies are described in Appendix A to provide the scientific rationale and experimental evidence for the drug formulation’s final composition used in the manuscript.

2.3.2. Appearance

The appearance evaluation was performed against a white background, under daylight, using a 1.5 mL clear glass vial filled with the formulation. The visual evaluation was performed with the vial at vertical and horizontal positions over a white surface. This visual inspection assesses the clarity of the formulation (presence of any turbidity), the coloration, the presence or absence of particles of foreign matter, and the occurrence of phase separation. The visual assessment methodology applied in this study was based on established principles outlined in the European Pharmacopoeia, USP, and ICH guidelines [26,27,28,29,30].

2.3.3. pH Value Determination

The pH of each formulation was determined using a calibrated pH meter with a surface electrode (VWR International, Radnor, PA, USA), immersed directly into the sample. Calibration was performed prior to use with standard buffer solutions covering the expected pH range, and measurements were taken once stabilized. The average of three readings was reported as the pH value of each formulation. All measurements were performed at room temperature (20–25 °C).

2.3.4. Adhesiveness

The adhesiveness of each formulation was qualitatively measured using a Texture Analyser (TA) TA. XTplus (Stable Micro Systems, Surrey, UK), equipped with a 5 kg load cell. TA was assembled with a P/10 Delrin cylinder probe (10 mm diameter), and the Mucoadhesion Rig (A/MUC) accessory was used to perform the measurement. A schematic representation of this methodology is available as Supplementary Information (Figure S1). The software used for data analysis was Exponent Software (version 7.0.1.0, Stable Micro Systems, Surrey, UK). The equipment settings were: pre-test speed—0.80 mm/s; test speed—0.10 mm/s; post-test speed—0.80 mm/s; applied force—2.5 g; maximum return distance: 10,000 nm; contact time: 60 s; trigger type: auto; and trigger force—5.0 g. For the measurement, an artificial mucosa pre-hydrated in artificial saliva (phosphate buffer pH = 6.8) at 37 °C was used and placed in the mucoadhesion rig accessory. The formulation was placed above the hydrated mucosa and, after 2 min, the maximum detachment force (F_max_—expressed in g, is the force performed by the equipment arm to have a total detachment of the probe from the sample placed over the mucosa), the work of adhesion (expressed in g·s, is the total amount of forces involved in the separation between the probe and sample placed over the mucosa), and the return distance (expressed in mm, is the distance performed by the probe until the total separation from the sample) were measured and registered. During method development, it was determined that a force value below 20 g is categorized as non-adhesive, while a value between 20 g and 40 g indicates detectable adhesion (though unquantifiable). Force values exceeding 40 g enabled adhesion quantification, and therefore, they were classified as being adhesive. The average of three readings was reported as the adhesiveness value.

2.3.5. Viscosity

The viscosity of each formulation was determined at 21 ± 1 °C using a rotational viscometer ROTAVISC lo-vi (IKA^®^-Werke GmbH & Co. KG, Staufen, Germany). The spindles VOL-SP-6.7 (93 rpm), VOL-SP-9.4 (19 rpm), and VOL-SP-4 (17.5 rpm) were used to evaluate formulations F0, F1, and F2, respectively. Measurements were performed as single determinations due to the high reproducibility of the instrument. This approach was considered sufficient, as the analysis aimed to capture relative differences between formulations rather than absolute rheological values across different polymer concentrations.

2.3.6. Residence Time

The residence time of each formulation was determined using an acrylic-based apparatus with a movable angle, inspired by the Lubrizol Life Science Health apparatus [31]. A schematic representation of this equipment is available as Supplementary Information (Figure S2). To determine the residence time, the formulation was placed on an initial application site over a portion of hydrated artificial mucosa (12.5 × 5.2 cm), with the platform at a 30° angle. Afterward, a continuous flux of artificial saliva, at 37 °C ± 0.5 °C to mimic the in vivo environment, was maintained using a peristaltic pump (PP 3300D, 4-400 RPM 0.1HP, VWR, Radnor, PA, USA) at a flow rate of 0.8 mL/min to mimic a healthy human salivary washout [14].

The residence time was assessed at 2, 6, and 10 min after test initiation. The test is initiated when the formulation is applied, T_initial_ (5 s after application). The DS was quantified at the mentioned timepoints and qualitatively evaluated through visual inspection and photographic documentation, capturing the trajectory of the formulation along the 12.5 cm length of artificial mucosa.

The percentage of DS retention (DS_retained_) is calculated relative to the initial amount of DS at the beginning of the test (DS_initial_), which is quantified at a specified timepoint (DS_t=x_) (Equation (1)):

$$[eqn]$$

The quantification of DS was performed using a reversed-phase HPLC. It consists of a Shimadzu, Kyoto, Japan, Ultra-Fast Liquid Chromatography (UFLC) equipped with a quaternary pump, an autosampler unit, an oven, and a PDA detector (SPD-M2OA). The column used was a YMC-Triart C18 column (150 mm × 4.6 mm; 5 µm) with the autosampler at 20 °C. The isocratic elution was carried out with a mobile phase containing 50 mM ammonium acetate buffer, pH 9.0: ACN (44:56 v/v), with a flow rate of 1.3 mL/min, and the column temperature was maintained at 30 °C, the injection volume of 20 µL, and a run time of 7 min, and the detection was monitored at 229 nm using a PDA detector. Data was integrated using Waters Empower^®^ 3 software. HPLC method validation tests were performed considering the ICH regulations on validation of analytical procedures, including selectivity, precision, linearity, limits of detection and quantification, accuracy, and stability assessment, according to ICH Q2 (R1) [32]. Representative chromatograms, including blank, DS standard solution, excipients, and excipients spiked with the standard, are provided in Supplementary Figures S3–S6 to demonstrate selectivity, absence of interference, and method specificity.

2.3.7. Permeation Evaluation

Drug permeation studies were performed on vertical Franz diffusion cells (1.77 cm^2^ diffusion area, 12 mL volume) placed on a V6A-02 stirring system, which maintains the receptor medium (PBS pH 7.4, 37 ± 0.5 °C) at a 500 rpm stirring speed. Both system and diffusion cells were purchased from PermeGear, Inc. (Hellertown, PA, USA). A schematic representation of the apparatus is available as Supplementary Information (Figure S7). Studies were performed under infinite dose conditions, collecting samples of 500 µL from the receptor solution. The sampling occurred at the following timepoints: 5, 10, 15, 20, 25, 30, 40, 50, 60, 90, 120, 180, 240, 300, 360, 480, and 600 min; each collection was followed by reposition with fresh buffer. The concentration in the receptor medium at each timepoint of the steady state phase was kept under sink conditions. The presence of air bubbles under the membrane was consistently monitored and removed according to the membrane supplier’s procedure, as well as any signs of evaporation. Samples were analyzed by HPLC. The separation technique and the parameters of the HPLC methodology were the same as mentioned for the residence time quantitative measurement (see Section 2.3.6. Residence Time). No additional treatment was done to the aliquots taken during the timepoints.

For each permeation experiment, the amount of DS permeated over the surface area (dQ/A) was calculated for the respective time interval (dt). The flux (J) is represented by the linear regression slope of the steady-state phase in the plot containing the cumulative amount of permeated DS as a function of time (Equation (2)). To minimize subjectivity, a steady-state slope was determined based on criteria adapted from an algorithm described in the literature [23]:

$$[eqn]$$

The apparent permeability coefficient (P_app_) was calculated by dividing the flux with the initial concentration of the permeated drug (C_0_), according to Equation (3):

$$[eqn]$$

The enhancement ratio (ER) of P_app_ was measured by the ratio between the P_app_ values obtained in the presence and absence of the permeation enhancer.

In Vitro Permeation Studies

For the in vitro permeation studies, the Permeapad^®^ Barrier membrane, exhibiting no observable signs of physical damage, was meticulously positioned between the receptor and donor chamber of the Franz cell. To induce a liposomal gel-like structure, thereby enhancing its biomimetic characteristics, the membrane was stabilized for 30 min, in contact with the receptor medium, before initiating the test. No additional membrane treatment steps were necessary as this biomimetic membrane is considered ready-to-use.

Ex Vivo Permeation Studies

For the ex vivo permeation studies, the buccal mucosa tissue, sourced from pigs, was obtained from a local meat supplier. The buccal mucosa was transported in containers filled with ice-cold PBS, pH = 7.4. Within a timeframe of 6 h after collection, visible fat and a substantial portion of the submucosa were surgically excised using appropriate instruments. The buccal epithelium was isolated utilizing a heat treatment technique (0.9% sodium chloride solution for 60–90 s at 60 °C), which is considered the most ideal method [33]. Subsequently, microscopic examination confirmed the thickness of the isolated epithelium to range from 250 µm to 500 µm, following the recommended thickness for permeation studies [12,33]. Ensuring utmost care to prevent any damage, the epithelium was carefully positioned with the outer surface facing upwards between the receptor and donor chamber of the Franz cell.

Indirect Estimation of Permeation Flux

An indirect estimation of the permeation flux (J) was performed using the in vitro apparent permeability coefficient (P_app_) and the concentration of drug substance at 2 min (DS_retained t=2min_) (µg/g), both experimentally determined.

The estimated flux (J_est_) was calculated according to Equation (4):

$$[eqn]$$

This calculation is based on the following assumptions:

1. The retained drug at 2 min reflects the fraction available for permeation over the exposure period.
2. The apparent permeability coefficient (P_app_) measured in vitro was calculated under steady-state conditions and subsequently applied to describe the early flux, assuming similar permeation behavior over this short time frame.
3. A formulation density of approximately 1 g/cm^3^ is assumed, allowing the retained drug amount (µg/g) to be considered equivalent to a concentration (µg/cm^3^) for the purposes of flux calculation.
4. Dynamic changes in membrane properties or drug depletion over time, including the effects of salivary flow and enzymatic activity, are not considered, being assumed to remain the same for all formulations under study.

This evaluation is here presented as an innovative approach to compare drug formulations by estimating drug flux, but it is not a direct measurement of in vivo flux.

2.3.8. Statistical Analysis

Statistical comparisons were performed using Student’s paired or unpaired t-test to assess differences in adhesion, viscosity, or residence time between the same formulation at different timepoints (paired t-test) or between different formulations (unpaired t-test). A p-value < 0.05 was considered statistically significant.

3. Results

The study described herein was conducted in two stages. Initially, three oromucosal formulations—F0, F1, and F2—were prepared with increasing percentages of HPCs (0%, 1%, and 2%, respectively). These formulations were characterized in terms of adhesion, viscosity, and residence time. In the second stage, a similar F2 formulation (F2a) was prepared, incorporating a permeation enhancer (BKC) to improve the permeation profile. According to the literature, the addition of 0.01% of BKC is not expected to affect the adhesion or viscosity characteristics; therefore, F2a was considered similar to F2 [34,35].

3.1. Appearance and pH Assessment

Formulations’ appearance is an important stability indicator. Visual inspection of all formulations revealed clear solutions, with no turbidity, visible particles, or phase separation (Figure 1).

The assessment of formulation pH is crucial for evaluating the tolerability of the buccal mucosa upon contact with the solution. The pH values of the evaluated formulations ranged from 4.36 to 4.80 (n = 3) (Table 2). These values fall within the recommended range [10,36], indicating that pH is not a limiting factor in the development of these formulations.

3.2. Adhesiveness and Viscosity

Adhesiveness and viscosity are important characteristics of buccal liquid formulations, as they can impact their performance. The influence of the presence and increasing quantities of HPCs on formulation adhesion and viscosity was evaluated and is shown in Figure 2.

The results indicate that F0, containing 0% polymer, exhibited an adhesion value of 3.20 g. For F1, with 1% polymer, the adhesion was 30.5 g, whereas F2, containing 2% polymer, exhibited an adhesion of 45.5 g (mean ± SD, n = 3). These results indicate a direct correlation between increasing HPC concentration and the adhesive capacity of the formulation. Furthermore, formulation viscosity increased proportionally with polymer content (3.32 mPa·s for F0, 319 mPa·s for F1, and 3565 mPa·s for F2). Although viscosity was measured as a single determination, the pronounced differences observed among formulations were sufficient to establish clear trends in the relationship between polymer concentration and viscosity.

3.3. Residence Time

It has been proposed that the residence time of a formulation is enhanced by its adhesiveness and viscosity, which allow the formulation to remain at the site of action for longer periods [15,20].

The analysis of residence time was conducted both quantitatively and qualitatively, as illustrated in Figure 3. A schematic representation of the apparatus is shown in Figure 3A. During the 10 min testing period, photographic documentation was captured, while the formulation was simultaneously collected at the end of the apparatus and subsequently quantified at different timepoints.

As shown in Figure 3A, at the beginning of the test (T_initial_, 5 s after application), F0 rapidly reached the endpoint as soon as the formulation drop was applied, indicating an almost negligible residence time. This phenomenon was not observed in the presence of HPC (F1 and F2), particularly at higher HPC concentrations. After 2 min, a portion of F2 was still visually present at the application site, whereas F0 and F1 had spread across the mucosa, leaving only a faint colored trace. By 6 min, a similar faint trace was observed for all three tested formulations, with no visual differences between 6 and 10 min. These qualitative results were consistent with the quantitative analysis of the amount of DS retained at each timepoint (Figure 3B; mean ± SD, n = 3). After 2 min, F2 retained approximately double the amount of DS_retained_ (43.4%) compared to F0 (20.3%) and F1 (19.4%). At subsequent timepoints, the DS_retained_ by F2 became comparable to the other formulations, which remained consistent until the end of the study.

To summarize, a review of the results for appearance, pH, adhesiveness, viscosity, and DS retained at the 2 min timepoint is compiled in Table 2.

3.4. Pre-Stability Studies

F2 was the formulation with the most favorable properties in terms of residence time, adhesiveness, and viscosity. Pre-stability studies were conducted on a formulation with the same composition as F2 over a 1-month period under long-term (25 °C/60% RH) and accelerated (40 °C/75% RH) conditions, corresponding to climatic zone II. The results are presented in Table 3.

After 1 month of storage (T1M), F2 formulation (freshly prepared) maintained the same appearance—a clear solution, with no evidence of turbidity, visible particles, or phase separation at any evaluated timepoint. During this period, the pH remained close to that recorded at T0, and importantly, both conditions showed assay values within the accepted pharmacopeial limits (95–105%). Assay results are expressed as mean ± SD, n = 2.

3.5. Permeation

The subsequent test focused on the evaluation and optimization of F2 buccal permeation.

To optimize the permeation properties of the formulation, benzalkonium chloride (BKC), a widely used permeation enhancer, was added to F2, referred to in this article as F2a (similar to F2 but containing BKC). This cationic surfactant has been reported to enhance mucosal permeation through mechanisms such as emulsification and epithelium disruption [37,38,39], which are expected to be transient at the relatively low concentration used here.

The results for apparent permeability (P_app_) comparing F2 and F2a are presented in Figure 4. The in vitro permeability of the F0 formulation is provided as Supplementary Material (Figure S8). F2a exhibited a significantly improved P_app_, with an enhancement ratio of 1.5 (P_app_ F2a = 14.8 cm/min (×10^−5^); mean ± SD, n = 3) compared to F2 (P_app_ F2 = 9.68 cm/min (×10^−5^); mean ± SD, n = 3). The results are consistent with previous reports in the literature using different types of surfactants for the same purpose [35,40].

To validate the impact of BKC, the permeation of both F2 and F2a was assessed using an ex vivo membrane (Figure 5). Porcine buccal epithelium was selected for these studies, as it closely resembles the morphological and structural characteristics of the human buccal mucosa [41,42].

Consistent with the in vitro permeation assessment, the presence of BKC (F2a) increased the formulation P_app_ by an enhancement ratio of 1.5 (Figure 5B) across the ex vivo epithelium. Although relatively higher variability and lower P_app_ values were observed compared to the in vitro results, the permeation profile (Figure 5A) consistently showed increased cumulative drug permeation for F2a (mean ± SD, n = 3).

In an in vivo context, the permeability flux of a formulation is influenced by the amount of drug remaining on the mucosa after administration. To enable a more meaningful comparison between formulations, an indirect estimation of the permeation flux (J_est_) was performed by combining the experimentally determined P_app_ with the DS_retained_ at 2 min.

Table 4 presents the experimental P_app_ and DS_retained_ at 2 min, as well as the resulting estimated permeation flux values.

According to the permeability results, the inclusion of 2% HPCs (F2) appears to decrease the velocity (P_app_) at which the drug crosses the membrane (P_app_ F0 = 12.9 cm/min (×10^−5^); P_app_ F2 = 9.68 cm/min (×10^−5^)). However, as shown in Table 4, when considering the amount of DS retained, the J_est_ of F2 is 1.5× higher than that of F0 (J_est_ F0 = 1.05 µg/(cm^2^·min); J_est_ F2 = 1.68 µg/(cm^2^·min)).

For F2a, the inclusion of BKC resulted in an estimated 1.5-fold increase in flux relative to F2 (higher P_app_) and a 2.5-fold increase relative to the formulation without polymer or BKC (F0).

4. Discussion

This study aimed to evaluate how varying quantities of a cellulose-based adhesive polymer can affect the formulation’s ability to maintain contact with the site of application and thus promote permeation across transmucosal membranes. Appearance and pH were also assessed, as these factors can impact the suitability of the buccal delivery system and influence patient compliance. Although the buccal mucosa is generally recognized for its relatively high local tolerance, minimizing adverse effects [5], maintaining an optimal pH is essential. The pH values of the formulations investigated in this study fall within the range considered acceptable for buccal administration [10,36], although they are slightly below the physiological buccal pH (6.2–7.6). While prolonged exposure to formulations with pH ~4.5 could potentially affect mucosal comfort or integrity, the short residence time associated with buccal naloxone administration in an emergency context substantially minimizes this risk, making acute mucosal damage unlikely [43,44]. Both properties were therefore considered to be compliant with the intended behavior.

The capacity of the formulation to persist at the administration site for a specified duration is assessed by its residence time, a metric influenced by the viscosity and adhesive properties of the formulation. The use of mucoadhesive polymers is commonly described as a significant strategy for enhancing adherence to mucosal surfaces. HPCs are among the most widely used polymers in pharmaceutical formulations due to their ease of handling, availability, water solubility, and non-toxicity [45].

Beyond their favorable mucoadhesive characteristics, the selection of HPCs as the primary polymeric matrix is further justified by their suitability for applications requiring a rapid onset of action, minimal diffusional barriers, and high formulation flexibility. The non-ionic nature of these polymers minimizes the risk of charge-mediated drug–polymer interactions, allowing for the systematic evaluation of alternative drug candidates without the need for extensive reformulation. This feature renders HPCs particularly well-suited for buccal delivery systems designed to achieve fast therapeutic action and broad applicability across chemically diverse active pharmaceutical ingredients [46,47,48].

Although an increase in polymer concentration is generally associated with enhanced mucoadhesion [49], several studies have shown that this effect is not linear and may reach a plateau or even decrease at higher polymer levels [8]. Pre-formulation studies, detailed in Appendix A [35,50,51,52,53,54], were conducted to rationally select the optimal formulation composition. Compatibility and stability assessments guided the formulation toward an HPC-based system. Through a Design of Experiments approach, it was demonstrated that the selected HPC combination provided adequate mucoadhesive performance and suitable viscosity characteristics. The selected polymer levels fall within the safety limits established in the FDA Inactive Ingredient Database (IID), ensuring regulatory acceptability for oromucosal administration. Overall, the chosen HPC composition was considered optimal to evaluate its impact on formulation residence time and naloxone permeation.

In the present paper, higher HPC percentages were observed to not only improve adhesion but also enhance viscosity, which was particularly evident at a concentration of 2%. This correlation was previously demonstrated by Stamatialis et al. using the same polymer in gels, where an increase in HPC concentration led to higher formulation viscosity [55]. HPCs exhibit viscoenhancing properties as well, corroborating the apparent positive association between adhesion and viscosity. While the ability to modulate viscosity and adhesion is beneficial, particularly for preventing drug removal by the salivary washout, it is also important to consider that excessively high viscosity can lead to handling difficulties and local irritation [56].

Some authors have established a correlation indicating that both adhesion and viscosity can significantly influence residence time [8,15,57], which aligns with our findings. The selected residence time assessment points (2, 6, and 10 min) were chosen to reflect the behavior of the liquid formulations in the buccal cavity while maintaining the clinical requirement for rapid onset of action in naloxone administration. The early time point (2 min) represents the clinically most relevant window for absorption, whereas the later time points (6 and 10 min) provide additional discriminatory power between formulations. The formulation containing 2% HPCs, which presented the highest adhesion and viscosity values, exhibited prolonged residence time at the application site (43.4% of retained DS) compared to formulations with lower HPC concentrations (~20% of retained DS), particularly after 2 min of application. Moreover, the impact on residence time was negligible when 1% HPC was used (19.4% of retained DS after 2 min), which was similar to the formulation without adhesive polymer (20.3% of retained DS after 2 min). This reinforces the idea that residence time benefits from the synergistic effects of adhesion and viscosity, underlining the importance of experimentally assessing residence time to determine the optimal polymer percentage. A formal statistical correlation analysis was not included due to the limited number of formulations, and therefore, the relationships between adhesiveness, viscosity, and residence time were interpreted based on consistent experimental trends and mechanistic considerations rather than inferential statistics.

Extending the residence time at the administration site is expected to impact drug permeation by prolonging the interaction between the polymeric matrix and the buccal epithelium. This, in turn, contributes to the improved efficacy of oromucosal products [3,58]. Prior to proceeding with the permeability studies, pre-stability studies were conducted with the 2% HPC formulation to assess the physical and chemical stability of the drug product. The results demonstrated no discernible alterations in drug content, appearance, or pH over the 1-month storage period. This suggests that incorporating higher amounts of HPC does not adversely affect drug stability, nor promote significant drug–excipient interactions, even under accelerated conditions (40 °C/75% RH). These findings reinforce the suitability of HPC as an inert and compatible polymer for oromucosal formulations, ensuring that enhanced mucoadhesive properties can be achieved without compromising the integrity or efficacy of the active pharmaceutical ingredient [21,22,59]. The robustness of the formulation is maintained, supporting its potential for further development and clinical application.

To enhance drug permeability, BKC was incorporated into the formulation containing 2% HPC. BKC is a permeation enhancer known to facilitate drug permeation by inducing transient epithelium disruption [37,39]. Evidence from the literature indicates that, at low concentrations such as 0.01%, the effect of BKC on epithelium integrity is reversible, with mucosal structure and barrier function typically recovering after cessation of exposure, depending on both the concentration and the duration of exposure [60,61,62,63]. While repeated or prolonged exposure at higher concentrations may lead to cumulative or irreversible epithelial changes [63,64,65], these scenarios are not representative of the short-term, acute administration of naloxone evaluated in this study. The 0.01% concentration was selected based on previous studies and regulatory guidance, demonstrating efficacy as a permeation enhancer while remaining within safe and well-tolerated limits for mucosal application [39,63,66]. This concentration range is commonly used in oromucosal and nasal products and is further supported by regulatory assessments. EMA has issued a comprehensive report addressing the utilization of BKC as an excipient in medicinal products, where a warning statement such as “May cause local irritation” is considered sufficient for oromucosal products containing BKC [61]. Hsu et al. also reported that 0.01% BKC is compatible with naloxone [35].

In vitro and ex vivo permeability studies were performed. The in vitro model relied on a ready-to-use biomimetic membrane (Permeapad^®^ Barrier) that, when hydrated, forms a liposomal gel-like structure, allowing the assessment of both paracellular and transcellular routes of passive diffusion [67,68]. This system mimics cellular membranes and biological tissues, providing a stable and robust platform for in vitro drug permeability [69]. Complementing these data, ex vivo studies used a porcine buccal tissue, which is widely recognized as a suitable model for ex vivo studies. It closely resembles human buccal mucosa in morphology, structure, composition, and enzymatic activity [12,70,71], allowing reliable prediction of drug transport and formulation performance in early development studies, while minimizing the need for animal experiments. Importantly, several studies have reported a strong correlation between porcine and human buccal permeability [6,72,73]. Although in vivo studies to assess potential mucosal damage were not conducted at this stage, the excipients were selected based on established safety data and prior use in approved oromucosal products. Further evaluation of in vivo safety and pharmacokinetics of the formulations will be considered in future clinical development.

The presence of BKC influenced P_app_, resulting in a 1.5-fold increase using either the biomimetic in vitro membrane (P_app_ increased from 9.688 cm/min (×10^−5^) to 14.8 cm/min (×10^−5^) with BKC) or the ex vivo membrane (P_app_ increased from 2.256 cm/min (×10^−5^) to 3.497 cm/min (×10^−5^) with BKC). As expected, ex vivo results showed higher variability and lower P_app_ values when compared to the in vitro results. Animal-based tissues (ex vivo) are structurally and morphologically more complex than biomimetic in vitro membranes, which, despite providing greater bio-relevance, the processes of tissue preparation and irregular tissue surfaces are challenging, leading to higher variability [25,56]. Histological assessment of the isolated epithelium is available in the Supplementary Material (Figure S9).

Interestingly, the presence of HPCs appears to reduce the velocity (P_app_) at which the drug crosses the membrane (P_app_ F0 = 12.9 cm/min (×10^−5^); P_app_ F2 = 9.68 cm/min (×10^−5^)) (Figure S8), possibly due to the potential entrapment of the drug in the polymeric matrix and inherent limitations of the permeation setup. To assess this impact, a comprehensive analysis was conducted to determine the drug flux (J) by correlating the residence time values with the permeation results, with a specific focus on the amount of DS retained after 2 min. Naloxone, classified as a BCS III drug, is intended for emergency treatment of opioid overdose, where rapid onset of action and prompt systemic availability are critical [44]. Therefore, prolonged residence time in the oral cavity is neither desirable—given the need for fast onset—nor expected, due to salivary washout, enzymatic activity, and mechanical forces, which limit drug exposure in the buccal environment [74,75].

Our findings indicate that higher polymer concentrations increase residence time at 2 min, likely influencing drug permeation. Although overall results suggested that the inclusion of 2% HPC (F2) could reduce the drug’s permeability rate (F2 vs. F0), focusing on the 2 min residence time, DS permeation flux was 1.6-fold higher for F2 than F0. Furthermore, BKC increased flux by 1.5-fold compared to F2 (polymer only) and 2.5-fold compared to F0 (without enhancer or polymer). Though this evaluation is an indirect estimation, not a direct measurement of in vivo flux, it reinforces the need to evaluate residence time and its positive impact on permeation flux. To our knowledge, this was the first time a qualitative and quantitative method was developed to assess the residence time of oromucosal formulations and use it to estimate flux.

5. Conclusions

In the present investigation, the combined use of HPC-M and HPC-G as adhesive polymers significantly enhanced both adhesiveness and viscosity in a concentration-dependent manner. At 2% polymer concentration, these properties translated into a marked increase in formulation residence time (43.4% of retained DS after 2 min), underscoring the close relationship between adhesion, viscosity, and mucosal retention. Prolonged residence at the administration site enhanced interaction between the formulation and the buccal epithelium, thereby facilitating drug permeation. The incorporation of BKC further improved permeability, as confirmed by both in vitro and ex vivo models, which is consistent with its known mechanism of action on epithelial membranes and the previous literature and regulatory reports demonstrating safety at this concentration. Notably, despite a reduction in apparent permeation rate in the presence of the polymer, the higher retention compensated for this effect, ultimately resulting in increased drug flux.

Overall, these findings demonstrate that the efficacy of buccal liquid formulations is strongly influenced by residence time, which, in turn, depends on the synergistic effects of polymer-induced adhesion and viscosity. Importantly, this work introduces, for the first time, a combined qualitative and quantitative approach to assess residence time, providing a valuable tool to guide the rational design and optimization of oromucosal drug delivery systems.