Evaluation of Factors Impacting Nicotine Permeation in Oral Nicotine Pouch Products

Abstract

In recent years, oral nicotine pouches or “tobacco-free nicotine pouches” have emerged as potential reduced-risk alternatives to traditional tobacco products. The aim of this research was to evaluate and further develop laboratory tools to measure the nicotine release rate from nicotine pouches and its permeation through the oral mucosa. For this, we evaluated the nicotine dissolution rate and buccal permeability of three nicotine pouch brands: Zyn, Velo, and Dryft nicotine pouches. Dissolution testing was conducted using the United States Pharmacopeia 4 (USP-4) apparatus and an in-house developed sinker method in three different artificial salivas, one laboratory-made saliva (Buffer saliva) and two commercially available salivas (Salivea and Orthana). Zyn nicotine pouches yielded the fastest nicotine release rate when compared to Velo and Dryft nicotine pouches. Nicotine permeability was assessed using the Side-Bi-Side diffusion cell with the porcine buccal mucosa model. Using neat nicotine solutions, we examined the effects of nicotine concentration (0.05–0.8 mg/mL), pH (5–10) (which covers the nicotine concentration and pH range in the three product extracts), and the three artificial saliva compositions on buccal permeability over 2 h (15, 30, 60, 90, and 120 min). While nicotine concentration did not affect the apparent permeability coefficient (P _app) within the tested range, higher pH resulted in a higher P _app value. Moreover, Buffer saliva and Salivea showed higher P _app values than those of Orthana. We further studied nicotine permeability by testing extracts from each nicotine pouch prepared by the sinker dissolution method in biorelevant, mucin containing Orthana saliva. Zyn nicotine pouches provided the highest permeability (38.24 × 10^–6 cm/s), followed by Dryft and Velo nicotine pouches (31.06 × 10^–6 cm/s and 14.84 × 10^–6 cm/s, respectively). Overall, our data indicate that the pH, artificial saliva composition, and product formulation can affect nicotine buccal absorption under laboratory conditions. This research provides a valuable dissolution/permeation tool to measure the nicotine release rate from nicotine pouches and its permeation through the oral mucosa, which can inform product development and adult tobacco consumers’ sensory experience.

1. Introduction

Nicotine pouches, also known as “tobacco-free nicotine pouches”, represent a burgeoning category of oral nicotine products that have gained popularity worldwide and are potential reduced-risk alternatives to traditional tobacco products. , Unlike traditional tobacco products, nicotine pouches are preportioned consumer products that do not contain cut, ground, powdered, or leaf tobacco but do contain nicotine derived from tobacco or synthesized, a base filler material, flavors, sweeteners, pH adjusting agents, modifiers, and other ingredients. Nicotine pouches typically contain substantially fewer toxicants compared to traditional tobacco products. Brands such as Zyn, Velo, Dryft, and many others offer a variety of nicotine pouch products, each with different flavors and nicotine levels.

When a nicotine pouch is placed in the mouth between the gum and lip, nicotine is extracted by saliva and diffuses through the oral mucosa into the bloodstream. Studies suggest that the nicotine absorption rate from nicotine pouches is similar to that of traditional smokeless tobacco products and nicotine gums.

The oral mucosa consists of the buccal, sublingual, gingival, palatal, and labial mucosa. The buccal and sublingual mucosa are nonkeratinized, whereas the gingival mucosa is keratinized. Nicotine released from nicotine pouches is mainly permeated through the nonkeratinized buccal mucosa into the bloodstream. The buccal nicotine delivery bypasses first-pass metabolism and avoids presystemic elimination in the gastrointestinal (GI) tract. The rate and efficiency of nicotine buccal permeation depend on factors such as pH, nicotine concentration, formulation, saliva production, pouch placement, and contact time with the mucosa.

Nicotine is a weak base, and its absorption is impacted by pH. At higher pH levels, more nicotine exists in its nonionized (free base) form, which is more readily absorbed through the mucosa. Saliva production rate can also influence nicotine dissolution and permeation. More saliva can increase nicotine extraction; however, excessive saliva may lead to swallowing, reducing local absorption, and increasing negative sensory impact such as throat irritation. Positioning the pouch in an area with a high mucosal surface area (such as the upper lip) can increase the absorption. These factors can impact adult tobacco consumers’ sensory experience when using nicotine pouches.

In recent years, several well-established methodologies have been developed to study nicotine dissolution release from various oral tobacco products, including nicotine pouches. These methodologies have been rigorously developed and validated using a range of apparatus, such as the United States Pharmacopeia (USP) apparatus USP-1, USP-2, USP-4, and the μDiss profiler. −

Given the intricate nature of buccal permeation, various in vitro and ex vivo models have been utilized to investigate the mechanisms of nicotine absorption and to forecast its bioavailability in humans. These models encompass the porcine buccal mucosa model, the TR146 cell-based model, and the PermeaPad artificial membrane model. , Among these models, porcine buccal mucosa is widely used to predict human buccal absorption due to its similar buccal characteristics, enzyme activity, and permeability to human buccal mucosa. Additionally, it is easier to acquire and more cost-effective compared to human buccal mucosa. , Several experimental tools are available to assess nicotine buccal permeation, with the Franz diffusion cell being one of the most widely used for evaluating transmembrane transport. It was originally designed for transdermal studies and has been adapted for buccal permeability studies by using excised buccal mucosa to simulate the human mucosal barrier. In the Franz cell, the donor compartment contains the nicotine formulation and the acceptor compartment is filled with a suitable medium, typically a buffer solution designed to mimic human body fluid. The setup allows for continuous sampling of the acceptor solution to quantify the amount of nicotine that permeates over time, providing valuable insights into the permeation rate of nicotine. In addition to the Franz cell, the Side-Bi-Side diffusion cell is another commonly used method for evaluating nicotine transport through mucosal membranes. These cells use a similar setup to the Franz diffusion cell but feature a configuration that allows stirring and heated water jackets in both the donor and the acceptor compartments.

In this study, we provided a comprehensive analysis of the dissolution and permeation characteristics of nicotine from three commercially available nicotine pouch products, namely, Zyn, Velo, and Dryft, to evaluate and further develop tools for nicotine pouches assessment. Initially, we characterized the nicotine content, pH, and nicotine dissolution release profiles using a well-established USP-4 methodology in these nicotine pouches. , Inspired by the work of Knopp et al., we further developed a biorelevant sinker (low-volume) dissolution method to study nicotine release from the nicotine pouches.

To study nicotine permeation, we employed the Side-Bi-Side diffusion cell with a porcine buccal mucosa model. We first explored the influence of the concentration, pH, and artificial saliva composition on nicotine permeability in neat solutions. Subsequently, we utilized the in-house developed sinker dissolution method to prepare sufficiently concentrated nicotine extracts from Zyn pouches in a wide range of artificial saliva compositions. These extracts then served as donor solutions to study the impact of artificial saliva composition on the permeation of nicotine. Ultimately, we studied the effect of product content on nicotine permeability by comparing extracts from Zyn, Velo, and Dryft nicotine pouches, prepared by the sinker dissolution method in Orthana saliva. These data provided valuable insights into the factors (e.g., nicotine concentration, pH, artificial saliva composition, and product content) that affect the rate of nicotine absorption, allowing us to assess the performance of these products under simulated oral conditions and gain a better understanding of their behavior. Overall, this work presents a valuable dissolution/permeation tool for product development that can provide insightful information about the performance of nicotine pouch products.

2. Materials and Methods

2.1. Materials

Optima grade acetonitrile, methanol, and water, ACS grade potassium phosphate dibasic anhydrous, potassium chloride anhydrous, calcium chloride dihydrate, sodium chloride, magnesium chloride hexahydrate, potassium carbonate anhydrous, sodium carbonate anhydrous, sodium bicarbonate, concentrated hydrochloric acid, 5N sodium hydroxide solution, and caffeine (98.5% purity) internal standard were purchased from Thermo Fisher Scientific (Wyman, MA). 6N ammonium hydroxide was purchased from RICCA (Pocomoke, MS) and 1 M acetic acid was purchased from Fluka (Pittsburgh, PA). ISO 17034 certified solution of nicotine (10 mg/mL) was provided by SPEX Certiprep (Metuchen, NJ). DL-nicotine (methyl-d ₃, 98%), methylene blue, and Gibco PBS solution (pH 7.4) were purchased from Fisher Scientific (Wyman, MA). DL-nicotine was acquired from Sigma-Aldrich (St. Louis, MO). H3 and H9 stirrers for diffusion cells were acquired from PermeGear (Hellertown, PA). Orthana saliva was purchased from Biofac A/S (Kastrup, Denmark). Salivea saliva was provided by Laclede Inc. (Rancho Dominguez, CA).

2.2. Methods

2.2.1. Artificial Saliva Preparation

Three artificial saliva solutions (Orthana saliva, Salivea saliva, and Buffer saliva) were used in this study. Buffer saliva was prepared according to the method described in the German Institute for Standardization (DIN) Recipe listed in the German standard DIN V Test Method 53160-1 2002-10. Briefly, the pH of 1 L of Type 1 water was lowered to 2.5 by adding 2 mL of 12N hydrochloric acid. The Buffer saliva solution was then prepared by dissolving 0.68 g of potassium hydrogen phosphate anhydrous, 0.33 g of sodium chloride anhydrous, 0.15 g of calcium chloride dihydrate, 0.75 g of potassium chloride anhydrous, 0.53 g of potassium carbonate anhydrous, and 0.17 g of magnesium chloride hexahydrate in acidified Type 1 water. If needed, the pH of the solution was further adjusted to 6.8 ± 0.1 through incremental additions of either 5N sodium hydroxide or 12N hydrochloric acid, using a disposable glass Pasteur pipet.

Orthana saliva is a commercially available lubricating mouth spray with a neutral pH. It (per 100 mL of aqueous solution) contains porcine gastric mucin 3500 mg, methyl-4-hydroxybenzoate 0.1 g, benzalkonium chloride 2 mg, EDTA disodium salt, H₂O (E386) 0.05 g, H₂O₂ 250 ppm, xylitol 2.0 g, peppermint oil 5 mg, spearmint oil 5 mg, NaCl 0.045 g, KCl 0.063 g, CaCl₂ 0.030 g, K₂HPO₄ 0.010 g, and KOH 0.076 g.

Salivea saliva is also a commercially available dry mouth mouthwash containing purified water, xylitol, propylene glycol, hydrogenated starch hydrolysate, poloxamer 407, sodium benzoate, hydroxyethyl cellulose, benzoic acid, mutanase, disodium phosphate, zinc gluconate, lactoferrin, lysozyme, lactoperoxidase, potassium thiocyanate, aloe vera, calcium lactate, glucose oxidase, dextranase, flavors, and six natural enzymes. The buffer capacity was increased by adding 0.149 g of potassium chloride, 0.117 g of sodium chloride, and 2.1 g of sodium bicarbonate to 1 L of Salivea saliva. The pH of Salivea saliva was adjusted to 6.8 ± 0.1 through incremental additions of either 5N sodium hydroxide or concentrated hydrochloric acid.

2.2.2. Nicotine Determination

The nicotine content in pouch products was quantified in triplicate using a previously validated and published liquid chromatography system coupled with a mass spectrometry (LC–MS/MS) method. Briefly, one nicotine pouch product was cut in half, and both the pouch material and filler were added to an extraction vessel. The sample was then fortified with 100 μL of internal standard nicotine methyl-d ₃ (10 mg/mL). After fortification, 15 mL of 2 M NaOH was added to the extraction vessel and shaken using a Geno grinder at 1500 rpm for 10 min. This was followed by the addition of 10 mL of acetonitrile and approximately 1 g of NaCl to the extraction vessel, which was shaken again at 1500 rpm for 10 min and immediately followed by centrifugation at 4000 rpm for 5 min to separate the organic layer from the aqueous layer. Postextraction, an aliquot of the organic phase was diluted 40 times with 90% acetonitrile, filtered using 0.2 μm PTFE Whatman Mini UniPrep Syringeless filters, and analyzed using an ultraperformance liquid chromatography system coupled with a mass spectrometry (UPLC-MS/MS) instrument. The instrument included a Waters Acquity I-Class UPLC system coupled to a Xevo-TQD triple-quadrupole mass spectrometer. Chromatographic separation was achieved using a Waters Acquity C18 column (50 × 2.1 mm, 2.5 μm). The flow rate was set to be 0.4 mL/min for a 5 min run-time. The mobile phases used were 10 mM ammonium acetate buffer (mobile phase A, pH = 10) and acetonitrile (mobile phase B). The initial elution composition was 95% ammonium acetate buffer and 5% acetonitrile and changed to a gradient of 55% acetonitrile over the first 2.5 min. The acetonitrile was then increased to 90% at a 2.75 min period and was held constant for 0.75 min. The eluents were returned to the original condition of 5% acetonitrile at 4 min and held constant for 1 min to allow re-equilibration of the system. The column and autosampler temperatures were maintained at 45 (±1) and 10 (±1) °C, respectively, and the standards and sample extracts injection volume were set to 1 μL. Analysis was performed by electrospray ionization using multiple reaction monitoring (MRM) in the positive-ion mode. The mass spectrometry (MS) parameters were set to a capillary voltage of 0.5 kV, 150 °C ion source temperature, 350 °C desolvation temperature, 20 L/h cone gas, and 650 L/h desolvation gas.

2.2.3. pH of Nicotine Pouch Extracts

The pH of nicotine pouch extracts was determined using a SevenMulti pH meter according to the Center for Disease Control (CDC) pH method for smokeless tobacco products. The pH meter was fitted with a pH Sensor InLab Expert Pro-ISM probe (Mettler Toledo, Columbus, OH). Briefly, two pouches were used for the product pH measurement. The nicotine pouch product was cut in half and was emptied into a container along with 20 mL of deionized water. The mixture was stirred with a magnetic stir bar for 5 min premeasurement, with the sample extracts being continuously stirred throughout the measurement period. Three replicates were conducted for each nicotine pouch product.

2.2.4. Dissolution Testing Using USP-4 Apparatus

Dissolution testing was carried out using a USP-4 flow-through cell apparatus (SOTAX, Westborough, MA, USA) following our previously published procedures and in accordance with the FDA guidance for industry. ,, Nicotine pouch products were tested in 12 replicates. Buffer saliva for this testing was prepared according to the German Institute for Standardization (DIN) standard DIN V Test Method 53160-1 2002-10.

Briefly, the dissolution testing and fraction collection with Buffer saliva were conducted using the USP-4 apparatus in an open loop and offline configuration. The setup included seven flow-through cells, a cell holder with a water bath, a reservoir and pump for Buffer saliva, and a fraction collection rack. The pump was set to deliver a steady flow of Buffer saliva (4 mL/min) through the flow-through cells (22.6 mm diameter). The cells were immersed in a water bath maintained at 37 ± 0.5 °C. Each sample cell had a 5 mm ruby bead check valve at the bottom, and approximately 6.6 g of 1 mm glass beads were added to ensure laminar flow of Buffer saliva. Nicotine pouches were directly placed in each flow-through cell. Additionally, around 6.6 g of 3 mm glass beads were used to keep the pouch in place and prevent it from floating within the cell.

The dissolution testing followed FDA guidelines, with 12 replicates of one product and dissolution profiles recorded at intervals up to 15 min. Each replicate was dissolved into 9 fractions. Fractions 1–5 were collected every 4 min each, resulting in a final volume of 16 mL per fraction. Fractions 6–9 were collected every 10 min each, resulting in a final volume of 40 mL per fraction. The total dissolution time was 60 min. After collecting all 9 fractions from each sample replicate, 0.9 mL of each dissolution fractions 1–5 were transferred to an autosampler vial. Then 0.1 mL of caffeine (1 mg/mL) was added as the internal standard. For fractions 6–9, 0.5 mL were transferred to an autosampler vial and then 0.1 mL of caffeine (1 mg/mL) was added and followed by 0.4 mL of buffer saliva.

The concentrations of nicotine (μg/mL) in all collected fractions from the 12 replicates were quantified using our previously described ultraperformance liquid chromatography system with a photodiode array (UPLC-PDA) method. The milligram/pouch nicotine concentration was determined using the instrument (μg/mL) calculated nicotine concentration, the weight of the sample analyzed, and the volume of the dissolution fraction. The cumulative concentrations of nicotine (mg/pouch) for each tested product were calculated by summing the average nicotine released at each fraction time point from all 12 replicates. This sum represents the total amount of nicotine released up to each time point. The percentage relative to the total nicotine released at each time point was then calculated and plotted to provide the total release profile. The relative percentage to the total nicotine released was calculated by dividing the amount of nicotine released up to each time point for each fraction by the cumulative amount released in 60 min. This calculation provided a comprehensive release profile for each product, illustrating the percentage of nicotine released over time.

2.2.5. Dissolution Testing Using the Sinker Method

Dissolution testing using the sinker method was conducted using a novel lab-made, low-volume sinker dissolution setup and was built as a nonstandardized, secondary approach to meet the sampling needs for permeation testing. This setup was inspired by the in vitro nicotine release testing conducted by Knopp et al. The sinker’s outer cell was a standard 80 mL glass collection tube, and the sinker was 3D-printed using polylactic acid with dimensions 12 × 21 × 2 mm in length (tube height), breadth (tube diameter), and width (tube thickness), respectively. A mesh with a sieve size of ∼0.84 mm was glued to the top of the sinker to hold the nicotine pouch product in place. The dissolution method was set up such that each nicotine pouch was individually weighed and centered within the confines of the sinker. The sinker was then placed at the bottom of an 80 mL glass collection tube, such that the mesh faced the top. A 20 mm magnetic cross-stir bar was placed on top of the mesh, and rotation was set up at 100 rpm. The dissolution test was timed, and the measurement was initiated with the addition of 10 mL of saliva. For permeation analysis, the dissolution testing was stopped at 30 min, where 0.1 mL of sample was collected for nicotine quantitation, and the remainder of the sample was stored in tightly capped centrifuge tubes at 4 ± 0.5 °C. The cumulative dissolution nicotine release profile was studied by following a similar setup, where, following the addition of 10 mL of saliva to the sinker setup, 0.25 mL of sample was aliquoted out at time points t = 4, 8, 12, 16, 20, 30, 40, 50, and 60 min and replaced with 0.25 mL of fresh saliva. From each collected dissolution fraction, 0.1 mL of the sample was pipetted into autosampler vials. This was followed by the addition of 0.1 mL of the caffeine internal standard (1 mg/mL) and 0.8 mL of DI water. The samples were then vortexed for 3–5 s and analyzed for nicotine content using an UPLC-PDA.

2.2.6. Determination of Nicotine in Dissolution Fractions

For the USP-4 dissolution and sinker methods, nicotine was determined using an Acquity I-Class UPLC-PDA instrument from Waters (Milford, MA, USA). The UPLC system was equipped with a BEH C18 analytical column (2.1 × 100 mm, 1.7 μm) and a BEH C18 VanGuard precolumn (2.1 × 5 mm, 1.7 μm) (Waters, Milford, MA, USA). The optimized PDA detector parameters included a sampling rate of 20 points/second, a 250–410 nm scan range, and 4.8 nm resolution. The mobile phases were 10 mM ammonium acetate buffer (mobile phase A) and 100% acetonitrile (mobile phase B), with a flow rate of 0.4 mL/min throughout an 8 min run-time. The elution initial composition was maintained at 95% ammonium acetate buffer and 5% acetonitrile for the first 2 min, changing by linear gradient to 70% ammonium acetate buffer and 30% acetonitrile over the course of the next 3.5 min. The acetonitrile was then increased to 95% over a 1.5 min period and held constant at 95% ammonium acetate buffer and 5% acetonitrile for the last minute, to allow for re-equilibration of the system. The injection volume of standards and sample extracts was 10 μL, and the column and autosampler temperatures were maintained at 45 (±1) and 10 (±1) °C, respectively. The nicotine calibration standards were prepared using ISO 17034 certified nicotine stock solution (10 mg/mL) with a calibration range of 0.5, 2, 5, 10, 20, and 40 μg/mL for standards level 1 to 6, respectively.

2.2.7. Preparation of Solutions for Permeability Studies

For donor solutions, to prepare 100 g (equivalent to 100 mL) of neat nicotine solutions with various nicotine concentrations and pH values, we first weighed 90 g of PBS and added the target amount of nicotine liquid. Next, a pH buffer (1 M NaHCO₃–Na₂CO₃ or HCl, depending on the target pH) was added to adjust the solution to the desired pH. Once the target pH was achieved, additional PBS was added to bring the total weight to 100 g. A similar procedure was used to prepare neat nicotine solutions in three different artificial saliva samples.

For the acceptor solutions, PBS (pH 7.4) was used consistently across all permeability studies. To ensure isotonicity with the corresponding donor solutions, NaCl was added to the PBS, and the osmolality was measured using a Semi-Micro Osmometer K-7400 S (Icon Scientific, North Potomac, MD).

2.2.8. Ex Vivo Permeability Studies

The porcine buccal mucosa, collected immediately after the pigs (6–9 month-old) had been processed, was obtained from Animal Technologies (Tyler, TX). The animals were sacrificed, and the buccal tissue from the inner cheek area was collected, frozen immediately, and shipped on dry ice. Upon arrival, the tissue was kept in a freezer (−20 °C) and used within one month.

For permeation studies, the frozen porcine buccal mucosa was thawed and cut into 500 ± 50 μm-thick sheets using an electric dermatome (model S, Integra Lifesciences, Princeton, NJ) and trimmed into adequate pieces with surgical scissors. This thickness corresponds to that of buccal epithelial, which contributes to the diffusion barrier. The permeability studies were carried out in the Side-Bi-Side diffusion cells (PermeGear, Hellertown, PA). Both the donor and the acceptor chambers held a volume of 2 mL and were exposed to a surface area of the porcine buccal mucosa barrier of 0.63 cm² (orifice diameter of 9 mm). Using the membrane holder, each porcine buccal membrane was mounted between the donor and the acceptor compartments, with the epithelium facing the donor chamber and the connective tissue region facing the acceptor chamber. Prior to conducting the experiments, both chambers of diffusion cells were filled with PBS solution (pH 7.4) and incubated for 1 h in a water bath to equilibrate the porcine buccal mucosa tissue in all of the cells at 37 °C. After equilibration, both donor and acceptor were emptied. At time point zero (t = 0, start of the experiment), the donor chamber was filled with various nicotine solutions or dissolution extracts, while the acceptor chamber contained isotonic PBS (pH 7.4) in all of the experiments. The acceptor compartment was continuously stirred at 600 rpm with a Teflon-coated bar magnet placed inside it. Sink conditions (nicotine concentrations in the acceptor compartment never exceed 10% of those in the donor throughout the permeation experiment) were maintained in all experiments. The permeability studies were carried out over 2 h at 37 °C. Samples of 200 μL were withdrawn from the acceptor chamber at predetermined time intervals (15, 30, 60, 90, and 120 min) and immediately replenished with an equal amount of fresh PBS buffer at each respective time point. At the end of the experiment (t = 120 min), 200 μL of sample from the donor compartment was withdrawn and analyzed. The pH values of the acceptor and donor solutions were measured prior to and after the permeability experiments. Each experiment was conducted in 12 replicates (n = 12).

To determine whether the integrity of the barrier was maintained throughout the permeation study, 2 mL of 0.05% methylene blue solution was applied to the donor chamber postexperiment, following the method described by Jacobson et al. Twenty minutes later, the acceptor solution was visually inspected for blue staining (see Supporting Information, Figure S1). The absence of staining was taken as evidence that the barrier remained intact. The sample with blue staining in the acceptor solution was excluded from the final calculation. Nicotine from collected samples was determined using the same UPLC-MS/MS method as that described in Section .

2.2.9. Permeability Calculations

For each permeation experiment, the cumulative amount of permeated nicotine (dQ, μg) in the acceptor compartment was plotted as a function of time (dt, s). The steady-state flux (J, slope of the linear portion of the curve) of nicotine through the barrier normalized by the surface area (A, 0.63 cm²) was calculated according to eq :

$$J=\frac{\mathrm{d}Q}{A\times \mathrm{d}t}$$ 1

The lag time was calculated by extrapolating the linear portion of the curve to the intersection with the x-axis. The apparent permeability coefficient (P _app, cm/s) was calculated by normalizing the flux (J) measured over the concentration of nicotine in the donor compartment at t = 0 s (C ₀) as described by eq :

$$P_{\mathrm{a}\mathrm{p}\mathrm{p}}=\frac{J}{C_0}$$ 2

Calculations proved that the sink conditions were upheld. Each permeability experiment was repeated 12 times, and the final value of P _app was determined from the average of the individual P _app calculated from each replicate.

2.2.10. Statistical Analysis

Data are presented as the mean ± standard deviation (SD). Significant changes in permeability were evaluated by a two-sided Student’s t-test. A significance level of p ≤ 0.05 was adopted in all cases. GraphPad Prism version 10 from GraphPad Software Inc. (Boston, MA) was used for statistical calculations and plotting.

3. Results and Discussion

3.1. Nicotine Determination

The nicotine content in the three nicotine pouches (Zyn 6 mg cool mint, Velo 4 mg mint, and Dryft 7 mg spearmint) was determined, as shown in Table , according to a previously validated and published salting-out assisted liquid–liquid extraction method. The test was performed in triplicate, and the average total amount of nicotine in each pouch product was found to be 5.58, 5.94, and 4.16 mg/pouch for Zyn, Dryft, and Velo, respectively. The calculated mean percent (%) recovery from the target nicotine content was found to be 93%, 85%, and 104% for Zyn, Dryft, and Velo, respectively (Table ).

1. Mean Nicotine Content and Weights of Nicotine Pouches (n = 3) .

product	pouch weight (g) (n = 3)	labeled nicotine (mg/pouch)	calculated nicotine content (mg/pouch) (SD)	percent (%) nicotine yield
Zyn 6 mg cool mint	0.41	6.0	5.58 (0.31)	93
Dryft 7 mg spearmint	0.38	7.0	5.94 (0.50)	85
Velo 4 mg mint	0.44	4.0	4.16 (0.24)	104

^aResults are reported as mean ± standard deviation (n = 3).

3.2. pH of Sample Extracts

The pH of the nicotine pouch extracts was measured using the CDC method for smokeless tobacco products and was found to be 8.22, 7.40, and 8.32 for Zyn, Velo, and Dryft nicotine pouches, respectively. The pH of these pouch products was also measured postdissolution in various salivas, namely, Orthana saliva, Salivea saliva, and Buffer saliva, to understand its potential effect on product solubility and oral absorption (Table ).

2. pH of 3 Nicotine Pouch Products and Their Extracts in Three Different Artificial Salivas in the Donor Chamber before (T = 0 min) and after Permeation (T = 120 min), and pH of PBS Buffer Solutions in the Acceptor Chamber after Permeation Study (T = 120 min) .

product name	product pH (SD)	donor sample	donor pH (T = 0 min) (SD)	donor pH (T = 120 min) (SD)	acceptor pH (T = 120 min) (SD)
Zyn 6 mg cool mint	8.22 (0.02)	Zyn in Buffer saliva	8.06 (0.03)	8.46 (0.02)	7.02 (0.41)
Zyn 6 mg cool mint	8.22 (0.02)	Zyn in Salivea saliva	8.04 (0.05)	8.36 (0.04)	7.14 (0.13)
Zyn 6 mg cool mint	8.22 (0.02)	Zyn in Orthana saliva	7.79 (0.09)	8.09 (0.02)	7.06 (0.03)
Dryft 7 mg spearmint	8.32 (0.05)	Dryft in Orthana saliva	7.74 (0.02)	7.83 (0.01)	7.03 (0.10)
Velo 4 mg mint	7.40 (0.02)	Velo in Orthana saliva	6.94 (0.05)	6.83 (0.00)	7.03 (0.02)

^aResults are reported as mean (standard deviation) (n = 12).

3.3. Dissolution Testing

3.3.1. Dissolution Testing Using USP-4 Apparatus

The nicotine dissolution release profiles of the three commercially available products in a lab-made, near-neutral (pH 6.8) Buffer saliva were characterized as shown in Figure , following the FDA guidance for industry using an established methodology that has been validated for smokeless tobacco and nicotine pouch products. Figure A shows the cumulative nicotine release profiles of Zyn, Dryft, and Velo nicotine pouches on a per-pouch basis. The data show that 93%, 79%, and 102% of labeled nicotine were released in Buffer saliva, under the same experimental conditions, within 60 min, for Zyn, Dryft, and Velo, respectively. The amount of nicotine released based on quantified nicotine values in pouches was found to be 100%, 93%, and 98% for Zyn, Dryft, and Velo, respectively. The cumulative nicotine dissolution release profiles were normalized to better understand and compare the nicotine release rates of each pouch product in the given Buffer saliva, as shown in Figure B. To achieve this, nicotine release rates were calculated as a function of its percent total release, where the percentage of total release was calculated by dividing the amount of nicotine released at each time point (per fraction) by the cumulative amount released at 60 min. The fastest release profile was obtained by Zyn, whereas Velo and Dryft nicotine pouches exhibited release rates similar to one another but slower than those of Zyn nicotine pouches. This observation was further confirmed by calculating the difference factor (f ₁) and similarity factor (f ₂) by adopting the methodology referenced in the guidance for industry from FDA’s Center for Drug Evaluation and Research (CDER). , Generally, f ₁ values up to 15 (0–15) and f ₂ values of 50 or greater (50–100) demonstrate equivalency of the two curves. Velo and Dryft nicotine pouches were found to be equivalent to one another with an f ₁ value of 11.2 and a f ₂ value of 54.2. Zyn was not equivalent to either Dryft or Velo with an f ₁ value of 22.3 and 20.8 and an f ₂ value of 37.5 and 39.9, respectively.

1.

(A) Cumulative and (B) percent of total dissolution release profiles of nicotine collected from Zyn 6 mg cool mint, Dryft 7 mg spearmint and, Velo 4 mg mint pouches (n = 12) (error bars ± 1 SD) using the USP-4 methodology in Buffer saliva.

3.3.2. Dissolution Testing Using the Sinker Method

The dissolution of Zyn, Dryft, and Velo nicotine pouch products was conducted using the sinker method in various artificial salivas where Zyn nicotine release was studied across the three artificial salivas, namely, Buffer saliva, Orthana saliva, and Salivea saliva, while Dryft and Velo nicotine release was only studied in Orthana saliva. The dissolution testing using this method was conducted in two phases, where the first set of experiments were designed to cater to the sampling needs for permeation testing. For this, the three nicotine pouch products were tested using the sinker method in 10 mL of saliva at a speed of 100 rpm for 30 min. Table shows the average nicotine concentration obtained from triplicate runs for each product. The final 30 min dissolution fractions were further used for nicotine permeation testing.

3. Measured Nicotine Concentrations of Nicotine Pouch Products in Three Different Artificial Salivas after 30 min of Dissolution Using the Sinker Method.

sample	nicotine conc. (mg/mL) (SD)
Zyn 6 mg cool mint in Buffer saliva	0.60 (0.02)
Zyn 6 mg cool mint in Salivea saliva	0.48 (0.05)
Zyn 6 mg cool mint in Orthana saliva	0.60 (0.01)
Dryft 7 mg spearmint in Orthana saliva	0.45 (0.01)
Velo 4 mg mint in Orthana saliva	0.38 (0.04)

The second set of experiments involved the characterization of the nicotine released from the nicotine pouch products in 10 mL of fresh Orthana saliva, with aliquots of nicotine analyzed within 60 min, at time points similar to that of the USP-4 method (but with different artificial saliva). USP-4 used Buffer saliva, whereas the sinker method used Orthana saliva. Figure A shows the cumulative nicotine release from the three nicotine pouch products in Orthana saliva on a per-pouch basis. Data show that when a nicotine pouch is extracted in Orthana saliva, which is a commercially purchased product that is known to be biorelevant, the amount of nicotine released within 60 min corresponds to 95%, 67%, and 92% of the nicotine target for Zyn, Dryft, and Velo nicotine pouches, respectively. The amount of nicotine released based on quantified nicotine values was found to be 102%, 79%, and 89% for Zyn, Dryft, and Velo, respectively.

2.

(A) Cumulative and (B) percent of total dissolution release profiles of nicotine collected from Zyn 6 mg cool mint, Dryft 7 mg spearmint, and Velo 4 mg mint pouches (n = 12) (error bars ± 1 SD) using the sinker method in Orthana saliva.

The cumulative dissolution release profiles of these nicotine pouches in Orthana saliva were normalized to better understand their nicotine release rates, as shown in Figure B. The fastest nicotine release profile was obtained for Zyn pouches, with an average rate of release of 99% within 30 min. This was followed by Velo, with an average release rate of 92%, and last by Dryft, with the slowest rate of release at 83% within 30 min. These observations indicate that the dissolution of nicotine from the nicotine pouch products across both the USP-4 and sinker methods shows the same trend, with Zyn showing the fastest rate of release and Velo and Dryft nicotine pouches showing equivalent release rates, despite the different artificial saliva used in each methodology.

We further studied the impact of various artificial salivas on the dissolution of nicotine from the nicotine pouch products and its impact on permeability. The main initiative behind this experiment was to account for the differences in the artificial saliva compositions we encounter across individual demographics, in vitro. We conducted dissolution testing using the sinker method where the Zyn nicotine pouch product was studied in Buffer saliva, Salivea saliva, and Orthana saliva. The data obtained, as shown in Figure , indicate that changes in saliva composition do indeed impact nicotine release, with Zyn in Salivea saliva showing the slowest rate of nicotine release, followed by Zyn in Orthana saliva and Zyn in Buffer saliva showing the fastest rate of nicotine release.

3.

(A) Cumulative and (B) percent of total nicotine dissolution release profiles of Zyn 6 mg cool mint nicotine pouches in three different salivas using the sinker method (error bars ± 1 SD).

3.4. Ex Vivo Permeability Testing

3.4.1. Impact of Donor Nicotine Concentration on Buccal Permeability

The permeability studies were carried out in the Side-Bi-Side diffusion cells, as shown in Figure .

4.

Diagram of ex vivo permeability experiment using Porcine Buccal Mucosa tissue.

In this study, nicotine concentrations in the product extracts obtained from sinker dissolution testing ranged from approximately 0.3 to 0.6 mg/mL (Table ). These differences reflect both different nicotine contents across products and the distinct nicotine release profiles associated with each commercial formulation. To evaluate whether donor concentration influences nicotine permeability, we conducted ex vivo buccal permeability studies using neat nicotine solutions in PBS across a range of donor concentrations (0.05–0.8 mg/mL). This range encompasses the nicotine concentrations observed in the product extracts. All neat nicotine solutions used in this evaluation were maintained at a constant pH of 8.5 to eliminate potential confounding effects of pH, thereby allowing the study to focus solely on the impact of the nicotine concentration on permeation.

We observed a dose-dependent increase in flux following the increase in nicotine concentration in the donor compartment (Figure B). To calculate the final P _app, the flux was normalized by individual donor concentration at t = 0 (C ₀) (eq ). After normalization, the P _app revealed minimal change in the tested range of 0.05–0.8 mg/mL of nicotine concentration (Figure A).

5.

(A) Apparent permeability coefficients (P _app) and (B) cumulative amount of nicotine permeating through porcine buccal mucosa measured by employing different donor concentrations at pH 8.5 in PBS buffer solution. Results are reported as mean ± standard deviation (n = 12).

This suggests that, within this range (0.05–0.8 mg/mL), the P _app of nicotine is independent of donor concentration. As mentioned in Nair et al.’s study, nicotine is typically anticipated to permeate the buccal mucosa barrier through passive diffusion, meaning that the permeability coefficient remains independent of the concentration gradient (ΔC) across the epithelium, assuming that factors such as drug solubility, molecular activity, and the epithelial barrier remain constant. Nicotine is soluble in water, and given the relatively low concentration range of tested nicotine (0.05–0.8 mg/mL), nicotine solubility and molecular activity are likely constant. Furthermore, the diffusion area (A) is not expected to change due to the Side-Bi-Side cell setup.

Additionally, the permeability of nicotine at donor concentrations higher than 0.8 mg/mL was assessed (1, 2, and 5 mg/mL of nicotine) (see Supporting Information, Figure S2), and P _app was found to be decreased following the increase of donor concentration. This finding was consistent with previous reports. , Zorin et al. attributed this phenomenon to a change in the partitioning coefficient, as higher concentrations reduce the affinity of nicotine for the buccal epithelium, thereby decreasing the partitioning coefficient and further reducing the permeability coefficient.

3.4.2. Impact of Donor pH on Buccal Permeability of Nicotine

To study the impact of pH on nicotine buccal permeability, we made neat nicotine solutions across a range of pH, from 5.0 to 10.0, maintaining a constant donor concentration at 0.5 mg/mL nicotine in PBS solution. The results in Figure indicated that nicotine permeability was strongly influenced by pH, with higher pH resulting in a higher P _app, aligning with previous reports. , One of these reports showed that increasing the pH of nicotine solutions significantly increased P _app in the TR146 cell culture model, and the other one showed the same trend in the porcine oral mucosa model. Nicotine, an alkaline alkaloid with well-separated pK _a of 3.26 and 7.90, exists in mono- and diprotonated forms at acidic pH and in unprotonated form at basic pH. At pH values above 8, the proportion of unprotonated nicotine molecules increases, leading to a higher fraction of uncharged molecules. Free-base nicotine exhibits a greater affinity for phospholipid bilayers on cell surfaces, facilitating faster transcellular passage and consequently more rapid absorption through the oral mucosa (pH-partition theory). This enhanced permeability results in quicker nicotine delivery to the bloodstream and higher blood concentrations. Conversely, at lower pH, the proportion of unprotonated nicotine decreases, reducing its affinity for the cell membrane and resulting in slower permeation.

6.

(A) Apparent permeability coefficients (P _app) and (B) cumulative amount of nicotine permeating through porcine buccal mucosa measured by employing different pH with the donor nicotine concentration at 0.5 mg/mL in PBS solution. Results are reported as mean ± standard deviation (n = 12).

3.4.3. Impact of Different Artificial Salivas on Buccal Permeability of Nicotine

To investigate the influence of artificial saliva composition on nicotine buccal permeability, neat nicotine solutions were made in three artificial saliva solutions, namely, Buffer saliva, Salivea saliva, and Orthana saliva, while maintaining a constant donor concentration at 0.5 mg/mL nicotine and constant pH at 8.5. Nicotine permeability was found to be the highest in Buffer saliva (59.19 × 10^–6 cm/s), followed by Salivea saliva (49.65 × 10^–6 cm/s), while Orthana saliva showed the lowest permeability (31.32 × 10^–6 cm/s) (Figure ). The observed difference may be attributed to the varying formulations, viscosity, or other specific features of these artificial saliva solutions.

7.

(A) Apparent permeability coefficients (P _app) and (B) cumulative amount of nicotine permeating through porcine buccal mucosa measured by employing different artificial saliva solutions at a concentration of 0.5 mg/mL and pH 8.5. Results are reported as mean ± standard deviation (n = 12).

3.4.4. Permeability of Nicotine Released from Commercially Available Nicotine Pouches

To understand the potential effect of artificial saliva compositions on buccal absorption of nicotine from nicotine pouch products, we evaluated the permeability of nicotine released from Zyn 6 mg Cool mint nicotine pouches in the three different artificial saliva compositions mentioned above. The product’s extraction solutions were obtained using the sinker method after 30 min of dissolution. Nicotine concentrations were measured to be 0.60, 0.48, and 0.60 mg/mL, for extraction solutions in Buffer saliva, Salivea saliva, and Orthana saliva, respectively (Table ). As shown in Table , the three Zyn extraction solutions also showed different pH levels. The pH values of Zyn extract in Buffer saliva and Salivea saliva were similar (8.06 and 8.04, respectively), whereas the Zyn extract in Orthana saliva showed a lower pH of 7.79, which could be attributed to the different buffer capacities of these artificial saliva solutions.

The nicotine permeability values for Zyn extracts in Buffer saliva and Salivea saliva were slightly different, measured at 46.29 × 10^–6 cm/s and 44.63 × 10^–6 cm/s, respectively. In contrast, the extract prepared in Orthana saliva exhibited a noticeably lower P _app (38.24 × 10^–6 cm/s) (Figure A). It is possible that nicotine permeability is influenced by the type of artificial saliva, the pH of the product extracts, the nicotine concentration, or a combination of these factors. As described in Section , nicotine permeability is highly dependent on pH, with higher pH values resulting in an increased P _app, as shown in Figure . Additionally, Figure demonstrates that P _app is independent of donor nicotine concentration within the range of 0.05 to 0.8 mg/mL. Therefore, the differences observed in nicotine permeability among the artificial saliva extracts are less likely to be attributed to variations in nicotine concentration.

8.

(A) Apparent permeability coefficients (P _app) measured by employing dissolution extraction solutions of Zyn 6 mg cool mint pouches in different artificial salivas and (B) P _app of nicotine in different artificial salivas and different pH at a nicotine concentration of 0.5 mg/mL (neat solution). Results are reported as mean ± standard deviation (n = 12).

Diverse types of artificial saliva may exhibit varying extraction efficiencies for ingredients other than nicotine, such as flavors, present in the product. These variations can result in differing concentrations of these ingredients in the extract, which may, in turn, influence nicotine permeation. Furthermore, the ingredients released from the product may interact with components in different artificial salivas, potentially altering nicotine permeability. To eliminate these potential confounding effects, we prepared three solutions containing neat nicotine and the respective three types of artificial saliva, each with a nicotine concentration of 0.5 mg/mL. Additionally, we adjusted the pH of each solution to match the pH of its corresponding extraction solution from Zyn nicotine pouches: Buffer saliva at pH 8.06, Salivea saliva at pH 8.04, and Orthana saliva at pH 7.79.

Nicotine permeability in these three solutions is presented in Figure B. To analyze these results, we first compared them with the permeability data shown in Figure A, where the pH was consistently 8.5 across all donor solutions. Overall, a decrease in pH led to a reduction in the nicotine permeability of all three solutions. Specifically, for the Buffer saliva, nicotine permeability decreased from 59.19 × 10^–6 cm/s to 52.69 × 10^–6 cm/s as the pH dropped from 8.5 to 8.06. Similarly, for the Salivea saliva, nicotine permeability declined from 49.65 × 10^–6 cm/s to 44.44 × 10^–6 cm/s when the pH decreased from 8.5 to 8.04. The Orthana saliva exhibited the most significant reduction, with nicotine permeability decreasing from 31.32 × 10^–6 cm/s to 22.69 × 10^–6 cm/s, as the pH fell from 8.5 to 7.79. This pronounced drop in nicotine permeability in the Orthana saliva is likely attributed to the larger pH decrease (from 8.5 to 7.79). These findings align with the observed relationship between pH and nicotine permeability, as illustrated in Figure . Overall, the results in Figure B reflect the combinational effects of both the types of artificial saliva and the pH levels.

Next, we compared the permeability data presented in Figure A,B. Both figures featured the same artificial saliva solutions at the same pH levels. The key difference lay in the preparation of the donor solutions: Figure A shows data for saliva solutions that underwent the dissolution process, whereas Figure B presents data for saliva solutions with neat nicotine directly added (with a small amount of pH buffer added to achieve the desired pH level). We observed varying degrees of differences in nicotine permeability between the two sets of solutions. For Buffer saliva, the P _app of nicotine from the Zyn pouch extract was 46.29 × 10^–6 cm/s, compared to 52.69 × 10^–6 cm/s for the neat nicotine solution at the same pH of 8.06. In the case of Salivea saliva, the P _app of nicotine from the Zyn pouch extraction was 44.63 × 10^–6 cm/s, closely matching the value of 44.44 × 10^–6 cm/s observed for the neat nicotine solution at the same pH of 8.04. The most significant difference was observed in the Orthana saliva, where P _app was 38.24 × 10^–6 cm/s for the Zyn pouch extraction solution as compared to 22.69 × 10^–6 cm/s for the neat solution. This substantial difference may be explained by the fact that the ingredients released from the Zyn nicotine pouch may interact with components in Orthana saliva, potentially increasing nicotine permeability.

To further confirm the minimal impact of nicotine concentration on permeability, we conducted an additional experiment measuring nicotine permeability at 0.6 mg/mL using a neat nicotine solution in Buffer saliva with a pH of 8.06. These concentration and pH values match those found in Zyn extraction solutions prepared in Buffer saliva. The resulting permeability was compared to that of a neat solution in Buffer saliva at the same pH (8.06) but with a nicotine concentration of 0.5 mg/mL, as shown in Figure B. Only a minimal difference in permeability was observed (see Supporting Information Figure S3), reinforcing the conclusion that small variations in nicotine concentration within the range of 0.05–0.8 mg/mL are unlikely to affect permeation outcomes, as discussed in Section .

More importantly, we observed only a small variation in the nicotine permeability results among the three Zyn pouch extraction solutions (Figure A). These differences are significantly smaller compared to those observed with neat solutions in different artificial saliva samples (Figures A and B). This suggests that, in addition to nicotine, other compounds extracted from the products during dissolution testing have a significant impact on nicotine permeability of the extraction solutions. These effects appear to be substantial enough to outweigh the influence of saliva type and pH, as demonstrated in Figures A and B.

3.4.5. Impact of Pouch Product Formulation on Buccal Permeability of Nicotine

We further evaluated the permeability of nicotine released from different nicotine pouch products, namely, Zyn 6 mg cool mint, Dryft 7 mg spearmint, and Velo 4 mg mint in Orthana saliva, to understand the potential effect of product formulation on buccal absorption of nicotine. Similarly, the three nicotine pouch extracts were collected from the sinker method after 30 min of dissolution. The nicotine concentrations of the three nicotine pouches extracts in Orthana saliva were found to be 0.60 mg/mL, 0.38 mg/mL, and 0.45 mg/mL for Zyn, Velo, and Dryft, respectively (Table ). As shown in Table , the three nicotine pouch extracts exhibited different pH levels. The pH values of Zyn 6 mg Cool mint and Dryft 7 mg spearmint extracts in Orthana saliva were similar (7.79 and 7.74, respectively), whereas the Velo 4 mg mint extract in Orthana saliva showed a lower pH of 6.94. The highest nicotine permeability was observed with Zyn pouches extract, with an average P _app of 38.24 × 10^–6 cm/s (Figure ). This was followed by Dryft, with an average P _app of 31.06 × 10^–6 cm/s, and last by Velo, which had the lowest P _app at 14.84 × 10^–6 cm/s (Figure ). The lowest permeability of the Velo nicotine pouch extract might be attributed to its low pH (6.94) and product formulation. As shown in Figure , the P _app of nicotine was independent of donor concentration in the range of 0.05 to 0.8 mg/mL of nicotine. For this reason, the differences observed in nicotine permeability in this study should not be attributed to the different concentrations of nicotine. Furthermore, the pH in Zyn and Dryft extracts were similar. Therefore, the observed difference in the Zyn and Dryft extracts may be attributed to their specific product formulations.

9.

(A) Apparent permeability coefficients (P _app) and (B) cumulative amount of nicotine permeating through porcine buccal mucosa measured by employing extraction solutions of different commercial nicotine pouch products in Orthana saliva. Results are reported as mean ± standard deviation (n = 12).

4. Conclusion

This study provides a comprehensive evaluation of the factors influencing nicotine release from nicotine pouch products and its permeation through buccal mucosa, offering valuable insights into their performance under simulated oral conditions. By employing advanced permeation methodologies such as the Side-Bi-Side diffusion cell with the porcine buccal mucosa model and dissolution methodologies such as USP-4 and sinker methods, we were able to systematically investigate the impact of nicotine concentration, pH, artificial saliva composition, and product formulation on the nicotine dissolution rate and buccal absorption. Our findings highlight the critical role of formulation and environmental factors in determining the rate and extent of nicotine release and buccal permeability. For instance, Zyn pouches demonstrated the fastest nicotine release and highest permeability compared to Velo and Dryft nicotine pouches, which can be attributed to differences in their filler formulations and pH. Additionally, the type of artificial saliva significantly influenced both the nicotine release and permeability, with Buffer saliva facilitating the fastest release and highest permeability, while Salivea and Orthana saliva exhibited slower rates. These results underscore the importance of considering variations in saliva composition in evaluating nicotine pouch performance. The study also revealed that while nicotine concentration did not affect permeability within the tested range (0.05–0.8 mg/mL), pH played a pivotal role, with higher pH levels leading to increased permeability. Pharmacokinetic (PK) studies have demonstrated significant variability in nicotine absorption rates among individuals, which can be attributed to differences in saliva pH, oral mucosa barrier, and genetic factors affecting nicotine metabolism. For example, PK studies show that saliva pH directly impacts the proportion of nicotine in its freebase form, which is more readily absorbed through the oral mucosa. Additionally, individual differences in saliva composition, such as different buffering capacities, may further contribute to variability in nicotine permeability. These findings highlight the importance of considering both physiological factors and product design in understanding variability in bloodstream nicotine levels. Overall, this research not only advances our understanding of the dissolution and permeation behavior of nicotine pouch products but also establishes a robust experimental framework for their evaluation. By bridging the gap between product formulation, nicotine release, and nicotine absorption, this study lays the groundwork for future research to assess pouch performance and enhance their role as viable alternatives to traditional tobacco products. While this experimental model is informative in product characterization and comparison, we believe clinical PK studies are still needed for a robust assessment of nicotine intake and abuse liability.

5. Limitations

The current model is based on in vitro–ex vivo testing and, as such, does not fully emulate the complex physiological conditions of the human oral cavity. While the findings from this study provide a valuable foundation for laboratory models of dissolution and permeation, future in vitro–in vivo correlation (IVIVC) investigations will help bridge the gap between these laboratory models and real-world conditions.

All data generated or analyzed during this study are included in this published article.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.5c06007.

• Image of the blue dye staining experiment and apparent permeability coefficients and cumulative amounts of nicotine permeating through porcine buccal mucosa under different pH and buffer conditions (PDF)

X.L. and A.S. contributed equally to this research. They developed the methods, executed all experiments, prepared the figures, and wrote the first draft of the manuscript. F.A. served as the principal investigator of dissolution in this research, designed the dissolution experiments, and wrote and reviewed the manuscript. M.Z. served as the principal investigator of permeation in this research and reviewed the manuscript.

This research was solely funded by Altria Client Services, LLC.

The authors declare no competing financial interest.