Development of biocompatible polymer film patches for the delivery of kratom (Mitragyna speciosa) extract as a natural analgesic

2.1. Materials

Mitragynine standard was purchased from Chromadex (California, USA). Chitosan, pectin, agar, SA, PVA, acetic acid, and d-limonene (Li) were purchased from Chemipan Corporation Co., Ltd. (Bangkok, Thailand). Phenoxyethanol, Eucalyptol (Eu), laurocapram (Lau), and glycerin were obtained from Chanjao Longevity Co., Ltd. (Bangkok, Thailand). HPLC-grade acetonitrile, methanol, ethanol, and chloroform were bought from RCI Labscan Limited (Bangkok, Thailand). Ammonium acetate, dibutyl phthalate (DBP), polyethylene glycol 400, PG, polysorbate 80, glycerin, polyvinyl pyrrolidone K30 (PVPK30), and hydroxy propyl methyl cellulose (HPMC) were bought from P.C. Drug Center Co., Ltd. (Bangkok, Thailand).

2.2. Workflow of the experiment

To provide a clear overview of the experimental procedures, the workflow of this study is summarized into a flow diagram (Fig. 1). The experimental design was structured to ensure systematic development, characterization, and evaluation of biocompatible polymer films containing kratom extract.

Figure 1. Workflow of the experiment. [Click here to view]

2.3. Preparation of kratom extract

Kratom leaves were collected from Namphu subdistrict, Ban Na San, Surat Thani, in the southern region of Thailand, from October to November 2021. The voucher specimens (J. Wungsintaweekul N5/001) were deposited at the Department of Biology, Faculty of Science, Prince of Songkla University.

Fresh kratom leaves were washed, dried at 40°C, and ground into fine powder using a Retsch grinder. The extraction process was performed as follows: 1 kg of powdered kratom leaves was boiled in 10 l of water for 1 hour. The decoction was filtered, and the moist residue was then macerated in 20 l of ethanol for 3 days. The mixture was filtered, and the maceration process was repeated two more times. All filtrates were pooled and evaporated under reduced pressure to obtain a concentrated extract. The ethanolic extract was stored at 4°C for 24 hours, allowing for phase separation. The precipitate was discarded, and the supernatant was collected and evaporated to dryness. Final drying was achieved using freeze-drying. The yield of the extract was approximately 140 g, corresponding to a 14% yield based on the initial dry weight of the plant material (w/w).

2.4. Determination of mitragynine content in kratom extract

For establishing the linearity of the standard calibration curve, five concentrations of mitragynine (5–80 µg/ml) were prepared, and high-performance liquid chromatography or HPLC analysis was conducted using an Agilent 1260 Infinity II photodiode-array detector, California, USA. A 250 × 4.6 mm reversed-phase C18 column (VertiSep UPS, Vertical Chromatography Co., Ltd., Bangkok, Thailand) with a five-micron particle size was used for the HPLC. The mobile phase used was a buffer made using ammonium acetate with a pH of 6, and acetonitrile in a ratio of 35:65, with detection performed at an absorbance wavelength of 225 nm. For the determination of mitragynine content, a 100 ml volumetric flask was used to dissolve 10 mg of Kratom extract in methanol. The HPLC method was validated according to the International Council for Harmonization (ICH) Q2(R1) guidelines for linearity, range, limit of detection (LOD), limit of quantitation (LOQ), precision, and accuracy (Table 1).

Table 1. HPLC parameters of validation method according to the ICH Q2(R1) guidelines for mitragynine determination.

2.5. Preparation of biocompatible polymer films containing kratom extract

First, blank films were prepared using the solvent casting method [24]. Various types and concentrations of polymers were selected for film preparation and categorized into four formulation groups, where each group was composed of different types and concentrations of plasticizers (Table 2). Water or chloroform was used as the solvent to dissolve the polymer and all other ingredients, depending on the properties of the polymer. For obtaining a clear solution, a magnetic stirrer was used to blend all ingredients. To form the film, a petri dish was taken and the solution containing polymers was poured into it; thereafter, the petri dish was kept in the hot air oven for 15 hours at 45°C. Each group of blank films was subsequently used to incorporate kratom extract (130 mg) into the solution. In the case of the polymer solution containing kratom extract, the film was formed by keeping the petri dish holding the solution at 30°C for 2–5 days until it dried. For transdermal delivery, various types and concentrations of penetration enhancers were added to two selected types of kratom extract-containing films (Table 3). All of the film formulations contained phenoxyethanol as a preservative.

Table 2. Four types of film formulations containing various ingredients and amounts.

Table 3. Type 3 and Type 4 of film formulations containing various penetration enhancers and concentrations.

2.6. Characterization of physicochemical properties of film containing kratom extract

2.7. In vitro release of films containing kratom extract

A modified Franz diffusion cell (Model Hanson 57-6M, California, USA) was used to conduct this experiment. The receptor chamber capacity was 11 ml, and the available area was 1.77 cm² [25]. The tested films were used on both the air-exposed and plate-attached sides. First, the kratom extract films were cut to 3.14 cm² pieces and placed directly on the receptor cells without the use of a synthetic membrane. Methanol solution (20% v/v) weighing 11 ml was loaded into the receptor compartment. The modified Franz diffusion cell’s temperature was controlled during the experiment by using a circulating water bath set to 37°C. A magnetic stirrer was used to stir the receptor compartment continuously at 500 rpm throughout the experimental duration. Then, 1 ml of samples were withdrawn at 15 and 30 minutes, and also at 1, 2, 3, 4, 6, 8, and 12 hours. After the collection of each sample, 1 ml of fresh 20% v/v methanol solution was added immediately. Samples collected for HPLC analysis were filtered through a 0.45 µm membrane prior to injection, and the membrane was replaced at each sampling point, after which they were analyzed by HPLC using the following equation:

${\mathit{Q}}_{\mathit{t}}=({\mathit{C}}_{\mathit{n}}\mathit{V}+{\sum }_{\mathrm{i}-1}^{\mathrm{n}-1}{\mathrm{C}}_{\mathrm{i}}\mathrm{S}\mathrm{)/}\mathit{A}(4)$

where Q_t is the cumulative released amount of mitragynine, C_n is a receiver solution concentration of mitragynine at each sampling time, C_i is the concentration of mitragynine in the receiving medium at the ith sampling time, V receptor compartment volume (11 ml), S is the withdrawn sample’s volume, and A is diffusion cell’s effective release area (1.77 cm²).

2.8. In vitro skin permeation of films containing kratom extract

Freshly excised ears from domestic pigs (Sus scrofa domesticus) were obtained from a local abattoir in Hat Yai, Thailand. These ears were transported to the laboratory on ice within 2 hours of slaughter. The skin from the dorsal side of the ear was carefully separated from the underlying cartilage. Hair was removed using an electric clipper, and any remaining subcutaneous fat and connective tissue were meticulously scraped off the dermal side. The full-thickness skin was then washed with phosphate-buffered saline (pH 7.4), cut into sections of approximately 4 × 4 cm, wrapped in aluminum foil, and stored at −20°C before use. On the day of the experiment, skin sections were thawed at room temperature and visually inspected for integrity. The use of post-mortem tissue from a commercial food source did not require specific institutional animal ethics committee approval; however, all handling procedures adhered to institutional biosafety guidelines and were declared as exempt determination research (MHESI 68014/602, April 04, 2024). Pig ear skin is a well-established model for in vitro human skin permeation studies due to its anatomical and physiological similarities to human skin [21,27,28]. The Franz diffusion cell’s upper compartment covered the skin onto which the films were placed. The samples of 1 ml from the receptor chamber were withdrawn at 0.5, 1, 2, 4, 8, 16, and 24 hours, and an equal volume of fresh receptor medium (20% v/v of methanol) was filled to replace it. The mitragynine content in the receptor fluid samples was analyzed by HPLC. The cumulative amount of mitragynine permeated per unit area and function of time was used to plot the permeation profile. The following equation, eq. (5) was used to calculate the cumulative amount (Q_t) of mitragynine permeation per unit area.

${\mathit{Q}}_{\mathit{t}}=({\mathit{C}}_{\mathit{n}}\mathit{V}+{\sum }_{\mathrm{i}-1}^{\mathrm{n}-1}{\mathrm{C}}_{\mathrm{i}}\mathrm{S}\mathrm{)/}\mathit{A}(5)$

where Q_t is the cumulative amount of mitragynine permeated per unit area of skin (µg/cm²), C_n is the receiver solution concentration of mitragynine at each sampling time, C_i is the mitragynine concentration (µg/ml) measured at sampling interval No.i, V is the individual Franz diffusion cell’s volume (ml), S is the sampling aliquot’s volume, and A is the effective diffusion surface area, which is 1.77 cm².

J_ss = P × C_d (6)

where J_ss is the steady state flux, P is the permeability coefficient, and C_d is the donor phase mitragynine concentration.

T_lag = L²/6D (7)

where T_lag is obtained from the time difference between the start of permeation and the point at which the permeation reaches a steady state, L is the skin thickness, and D is apparent diffusivity through the skin.

ER = J_ss of film with enhancer/J_ss of film without enhancer (8)

where ER is the enhancement ratio and J_ss is the steady state flux of film containing enhancer or without enhancer.

2.9. Deposition of mitragynine in the skin

After completing the in vitro permeation study (24 hours), the amount of mitragynine accumulated in the skin was analyzed [26]. A centrifuge tube containing 5 ml of methanol was used to deposit the skin that was taken out from the diffusion cell and cut into small pieces. Thereafter, this solution was vortexed for 2 minutes, after which sonication was done for 15 minutes, and centrifugation was done at 10,000 rpm (10 minutes at 4°C). This supernatant was finally evaluated with HPLC to determine the mitragynine content.

2.10. Stability of films containing kratom extract

The films were stored for 6 months at a room temperature of 30°C ±1°C, and a RH of 75%. The amount of mitragynine remaining in the films was measured using HPLC at 0 and 6 months. In addition, the physical characteristics of the films, such as color and thickness, were observed.

2.11. Microbiological testing of films containing kratom extract

Microbiological testing was performed to assess the safety and stability of the film formulations, following acceptance criteria outlined in relevant pharmacopeial guidelines [29,30]. The total viable count of microorganisms (including molds, yeasts, and bacteria) in each gram of sample was required to be less than 1,000 colony-forming units (CFUs). In addition, the product had to be free from specified pathogenic microorganisms, namely Clostridium spp, Pseudomonas aeruginosa, Candida albicans, and Staphylococcus aureus.

For determining total aerobic microbial count (TAMC) and total combined yeast and mold count (TYMC), each sample of 10 mg was diluted using 90 ml of soybean-casein digest broth and agitated vigorously for 30 minutes. Subsequently, for TAMC, a 1 ml aliquot of the dilution was plated on soybean-casein digest agar, and for TYMC, it was plated on Sabouraud dextrose agar (SDA). Incubation of TAMC plates was done for 3–5 days at 30°C–35°C, whereas incubation of TYMC plates was done for 5–7 days at 20°C–25°C. Colonies were then counted and reported as CFU per gram of sample.

Detection of P. aeruginosa and S. aureus involved diluting a sample of 10 g using soybean-casein digest broth (90 ml), followed by filtration through a sterile membrane. The same broth (100 ml) was used to immerse the filter, which was then incubated for 24 hours at 30°C–35°C. Post-incubation, aliquots were streaked onto Cetrimide agar for P. aeruginosa and Baird–Parker agar for S. aureus, and incubation was done for 18–72 hours at 30°C–35°C to observe colony formation.

For the detection of Clostridium spp., the sample (10 mg) was diluted using thioglycollate medium (90 ml), and then it was heated for 1 minute at 80°C, and incubated under CO₂ for 48 hours at 30°C–35°C. After incubation, sub-culturing was performed under anaerobic conditions on reinforced clostridial agar, and incubated for 48–72 hours at 30°C–35°C for detecting colony growth.

For C. albicans, 10 g of sample was diluted with 90 ml of Sabouraud dextrose broth and incubated at 30°C–35°C for 3–5 days. Following incubation, a portion of the broth was streaked onto SDA plates and incubation was done for 24–48 hours at 30°C–35°C. Plates were examined to detect characteristic colony growth.

2.12. Skin irritation test of prototype biocompatible polymer film patch

The skin irritation test was conducted by the Thailand Institute of Scientific and Technological Research according to the test method ISO 10993-23:2021, 1^st Edition, Biological evaluation of medical devices Part 23: Tests for irritation. This test was certified with certificate number TS-66009 for animal raising and use in accordance with the “Guide for the Care and Use of Laboratory Animals” [31] and OECD GLP guidelines. Evaluation of the skin irritation of the patch on rabbits was performed with three male New Zealand White rabbits. The test sample patch (with kratom extract and permeability enhancers) and the control group patch (without kratom extract and permeability enhancers), each of which measured 2.5 × 2.5 cm, were applied on the rabbit skin at specified locations for each rabbit for 4 hours. Next, any abnormalities observed on the rabbit skin in each location were scored at 1, 24, 48, and 72 hours. Rabbit skin abnormality scores at 24, 48, and 72 hours, and the normal skin symptoms of the animals involved in the experiment were reviewed to calculate the primary irritation index (PII). The PII value of the patch on the rabbit skin indicates the nature of the rabbit skin reaction, with a high value indicating severe irritation of the rabbit skin, as shown in Table 4.

Table 4. PII values and skin reaction characteristics of rabbits.

2.13. Preparation of prototype biocompatible polymer film patch product

The product consisted of two parts and was prepared followingly: The first part was a pre-made patch without an active ingredient for medical use, consisting of different layers such as the barrier layer, adhesive layer, and peel-off sheet, as shown in Figure 2, numbers (1, 2, and 4). The backing sheet is the top layer when the patch is placed on the skin. The adhesive layer is responsible for holding the patch to the skin. The peel-off sheet (Protective liner) is a sheet that prevents the active ingredient from being released before use. The pre-made patch is usually made of polymer materials or natural fibers, or a mixture of polymers and natural fibers. The pre-made patch without an active ingredient can be selected from nonwoven polyester, polyurethane, polyurethane foam, nonwoven polyurethane, or polyethylene, polyethylene foam. In this study, nonwoven silicone was selected for packing the film containing kratom extract. The second part was the active ingredient layer, as shown in Figure 2. Number 3 is the film with the main active ingredient components, which are the kratom extract, the film-forming agent, plasticizer, the skin permeability enhancer, the preservative, and the solvent, resulting in a clear brown solution before drying to form the film.

Figure 2. Composition of prototype biocompatible polymer film patch product. [Click here to view]

2.14. Statistical analysis

Physicochemical characterization tests were performed in triplicate (n = 3), in vitro release studies were conducted with five replicates (n = 5), and skin permeation studies were performed with four replicates (n = 4) per formulation. The data were expressed as mean ± SD. The significant differences were calculated by ANOVA and multiple comparison test with p value < 0.05.

3.1. Mitragynine content in kratom extract

The kratom extract was prepared at the laboratory of Associate Professor Juraithip Wungsintaweekul. Quantitative analysis of the active compound, mitragynine, revealed a concentration of 10.96% ± 0.51% w/w. Extraction was carried out using maceration with methanol as the solvent [21,32]. In this study, only mitragynine content was considered for quality control, as it is the principal alkaloid associated with the extract’s analgesic activity. Compared to the ethanolic extract of kratom, which was black in color [21], the methanolic extract appeared brown. This difference in color may be attributed to variations in the chemical composition of the extracts, resulting from differences in extraction techniques, solvents, and extraction temperatures [32]. The extraction protocol involved evaporation under reduced pressure followed by freeze-drying, which is highly effective in removing solvents. However, for Good Manufacturing Practice-compliant manufacturing, a formal test for residual solvents would be a necessary quality control step.

3.2. Biocompatible polymer films containing kratom extract and physicochemical properties

The four types of blank films with different concentrations of polymers and plasticizers had different characteristics. The film made using PVA was clear in appearance. However, when PVPK 30 was mixed in the film, it caused the film to become cloudy. In addition, when PVPK30 was used as the only polymer in film preparation, film formation was not successful. The films prepared from ethyl cellulose and PVPK30, agar, pectin, and SA groups were cloudy, especially the natural polymer group, and they had a rough texture. The amount of polymer in the film affected the thickness of the film; for instance, higher polymer content resulted in the film being thicker. On the other hand, the amount of plasticizer in the film affected the flexibility and stiffness of the film; for instance, films with high plasticizer content were softer and easily absorbed moisture into the film. The appropriate amount of plasticizer depended on the type of polymer used to prepare the film. Kratom extract was added to blank films from the four types. The appearance of blank film and film containing kratom extract is shown in Figure 3. The initial formulation of the film was prepared using 130 mg of kratom extract per 78 cm² (per film), containing approximately 10–15 mg of mitragynine, to evaluate the film-forming capability and the characteristics of the resulting film. The method used for the preparation of the film was the solvent casting method, but heat was avoided during the drying process due to the thermal instability of the kratom extract. Methanol (3 ml) was used to dissolve the extract, after which it was introduced to each film formulation’s polymer solution. Then the mixtures were dried at a controlled temperature of 30°C ± 2°C. Films of Types 1, 2, and 4 required 2 days to dry completely, whereas Type 3 required 4 days to form a film. It was observed that films of Types 3 and 4 were compatible with the kratom extract, forming uniform yellow films. In contrast, Type 1 film exhibited unevenly distributed brown patterns, while Type 2 film showed aggregation of the extract at the center. The properties of the blank films and kratom extract films are summarized in Table 5. The thickness and moisture uptake of kratom film Type 1 and Type 2 could not be measured due to the uneven dispersion of the kratom extract. Natural extracts were highly compatible with natural polymers in film formulations due to their chemical affinity, structural compatibility, and synergistic bioactivity. This compatibility supported uniform dispersion, enhanced film functionality, and maintained the stability of bioactive compounds. The shared biodegradability and safety profiles further supported their combined application in pharmaceutical products. However, natural polymers also present certain limitations, including susceptibility to microbial contamination and relatively low TS, which may compromise the mechanical integrity and shelf life of the final product [25,33,34]. As shown in Figure 3, the gross nonuniformity and phase separation of the extract in Type 1 and 2 films rendered them unsuitable for development as a dosage form. In contrast, Type 3 and 4 films demonstrated excellent compatibility and homogeneity and were therefore selected for further optimization with permeation enhancers.

Figure 3. Appearance of blank film and kratom extract film of Type 1 to Type 4. [Click here to view]

Kratom film Types 3 and 4 were selected for the addition of skin penetration enhancers for transdermal delivery. Three enhancers were used: limonene, eucalyptol, and laurocapram. Limonene and eucalyptol are in the terpenes group that enhance skin penetration through similar mechanisms, while laurocapram functions through a different mechanism. Table 3 presents the amounts of kratom extract and penetration enhancers-both individually and in combination, in each film type. The appearance of the resulting films is shown in Figure 4. The physical properties, thickness, and percentage of moisture uptake of the two types of films are shown in Table 6. The addition of the permeability enhancer did not affect the thickness or moisture absorption of either film formulation, but it influenced the appearance of each film type. The kratom Type 3 film exhibited significantly higher moisture uptake compared to the Type 4 film, primarily due to the presence of pectin in its composition. Pectin is known to possess poor physical properties, high hydrophilicity, and brittleness [35].

Figure 4. Appearance of Type 3 and Type 4 kratom extract film containing enhancers. [Click here to view]

Table 5. Properties of blank films and kratom extract films of various types of polymers.

Table 6. Properties of kratom extract films Type 3 and Type 4 containing various skin enhancers.

The surface morphology of the two types of films, when observed using SEM, revealed no noticeable differences between the blank films and those containing kratom extract. This indicates that the kratom extract was well integrated into both types of film matrices (Type 3 and Type 4). The results are shown in Figure 5 at magnifications of 1,500× and 10,000×, respectively. The surface morphology of kratom film Type 3 and Type 4, both containing various skin penetration enhancers, is shown in Figures 6 and 7 at magnifications of 1,500× and 10,000×, respectively. Film Type 3, formulated with kratom extract and composed mainly of SA, pectin, and agar, exhibited a rough surface texture. The addition of the three different penetration enhancers revealed that limonene and eucalyptol had minimal impact on the film’s surface, which remained similar to the film without any enhancers. In contrast, laurocapram and its combinations with limonene or eucalyptol caused noticeable changes in surface morphology, consistent with visible physical changes. Films containing laurocapram, either alone or in combination, displayed a glossier appearance, as if coated with an oily layer. For the kratom film Type 4, which used PVA as the main polymer, the original surface was smooth. The inclusion of penetration enhancers, particularly laurocapram, increased surface roughness. However, when laurocapram was combined with either limonene or eucalyptol, the increase in roughness was less pronounced. This observation aligned with the physical characteristics, where films containing laurocapram or its combinations showed uneven distribution of yellow color across the film. These findings will serve as a basis for further investigation into the skin permeability properties of mitragynine, the active compound, in subsequent studies.

Figure 5. Surface texture of the blank films and kratom films Type 3 and Type 4 using SEM. [Click here to view]

Figure 6. Surface texture of kratom films Type 3 with various enhancers using SEM. [Click here to view]

Figure 7. Surface texture of kratom films Type 4 with various enhancers using SEM. [Click here to view]

The structural analysis of the different film types using Differential scanning calorimetry (DSC) showed that the polymer peaks in the films containing kratom extract differed from those in the blank films for both film types (Type 3 and Type 4). The results are illustrated in Figure 8. Furthermore, in case of the structure of film Type 3 and film Type 4 containing kratom extract with individual skin penetration enhancers (three types) and combinations of two enhancers (limonene with laurocapram and eucalyptol with laurocapram), no noticeable changes were observed in the polymer peaks of film Type 3, which was primarily composed of SA, pectin, and agar, compared to the kratom film without enhancers. In contrast, in film Type 4, which uses PVA as the main polymer, changes in the DSC polymer peaks were observed in the films containing laurocapram and the combinations of laurocapram with the other enhancers.

Figure 8. DSC profiles of blank film and kratom extract film of Type 3 and Type 4. [Click here to view]

In the study of changes in mitragynine crystals in the patch using XRD, it was found that both the blank film and the film containing kratom extract showed no crystalline structures. The substances in both types of films exhibited an amorphous form, as shown in Figure 9. In addition, when kratom extract films containing each of the three individual permeation enhancers, as well as the films containing combinations of two enhancers, were observed, crystalline structures of the extract or other components were not found. These films also exhibited an amorphous form, similar to the previously mentioned films. The physicochemical characterization provided critical insights into the performance of the formulations. The amorphous state of mitragynine within the polymer matrix, confirmed by the absence of crystalline peaks in the XRD patterns (Fig. 9), is highly advantageous. This ensures that the active ingredient is molecularly dispersed, maximizing its thermodynamic activity and eliminating the need for dissolution from a stable crystal lattice prior to release.

Figure 9. X-ray diffraction patterns of the different film types are shown, with the left image representing Film Type 3, composed of agar, pectin, and SA, and the right image representing Film Type 4 (PVA). The blue line indicates the film containing kratom extract, while the red line represents the blank film without the extract. [Click here to view]

High-performance liquid chromatography, or HPLC, was used to determine the amount of the active compound mitragynine. Based on the chromatographic analysis of the film samples, it was found that the mitragynine peaks (which appeared at approximately 13.9–14.2 minutes) of the kratom extract films containing each of the three individual penetration enhancers, as well as the combined enhancer formulations, showed no interference from peaks of other film components. The content of mitragynine in various film formulations and the distribution of the active compound within the films were determined by extracting them with methanol and analyzing the amount of mitragynine. Both types of films containing different penetration enhancers were evaluated, as shown in Table 7. The recovery content of mitragynine was generally high, with most values exceeding 80%. However, the distribution of the active compound within the films varied, particularly in film Type 3, which contained laurocapram or a combination of laurocapram and limonene as penetration enhancers. These types of films showed relatively high variability, with SDs as wide as 12%–14%. In contrast, film Type 4 exhibited a more uniform distribution of the active compound throughout the film. The high variability is not just a random error, but evidence of a fundamental formulation incompatibility. The lipophilic enhancer (laurocapram) is likely immiscible with the hydrophilic natural polymer matrix, leading to phase separation. The extract, being more soluble in the lipophilic phase, partitions into these enhancer-rich domains, causing a nonuniform distribution.

Table 7. Mitragynine content and distribution of kratom extract films Type 3 and Type 4 containing various skin enhancers.

From the elongation at break (EB) and TS tests, it was found that films prepared from natural polymer blends comprising agar, pectin, and SA (Type 3 film) were fragile and exhibited low tensile resistance, with poor adhesive properties. However, this product development utilized a commercially available medical-grade adhesive layer with a drug-resistant backing layer, specifically designed for patch formulation. Therefore, the issues related to adhesive performance and tensile resistance of the active substance-loaded films were not considered problematic in this context. In contrast, the films prepared using PVA (Type 4 film) demonstrated excellent TS and EB, indicating good mechanical durability. In both types of films, the incorporation of kratom extract resulted in decreased TS and elongation compared to films without the extract, as shown in Table 8. Furthermore, the molecular structure of pectin in Type 3 film played a critical role in influencing mechanical properties. Pectin is known to possess low TS; therefore, to enhance the mechanical performance and reduce excessive flexibility in pectin-based film matrices, additional biopolymers (agar, SA) were incorporated [35].

Table 8. TS and EB values of films Type 3 and Type 4.

3.3. In vitro release of films containing kratom extract

The release kinetic models, which included zero-order, first-order, and the Higuchi model, were used to analyze the cumulative release of the active compound, mitragynine, over a 12-hour period in two selected film formulations. The release profiles (Fig. 10) were then evaluated to determine which model best described the release behavior. This analysis was conducted to guide the design of appropriately sized transdermal patches and to calculate the mitragynine release rate in accordance with the identified release mechanism. The receptor medium contained 20% methanol, which was selected to maintain sink conditions for mitragynine. After 12 hours of experimentation, the film structure remained largely intact with only minor deterioration, indicating that the receptor solvent did not completely dissolve the film matrix. Film Type 3 and Type 4 exhibited comparable mitragynine release profiles, with a cumulative release percentage of 11.92% and 12.13% of the total mitragynine content in the films, respectively. Furthermore, the cumulative release amount of film Type 3 and Type 4 was 46.65 ± 7.62 and 48.31 ± 12.31 µg, respectively. The release followed a first-order kinetic model, as implied by the highest R² values among the models tested. This suggests that the remaining concentration of mitragynine within the film matrix influences the release rate. During the initial phase, a faster release rate was observed due to the higher concentration of the active compound present in the film at the beginning of the release period, but the release gradually decreased over time.

Figure 10. Release profiles of Type 3 and Type 4 film containing kratom extract at various points of time in 12 hours. Error bar show SD for n = 5. [Click here to view]

3.4. In vitro skin permeation of films containing kratom extract

The skin permeation study results of film Type 3 and film Type 4 using pig ear skin as a model, both with and without individual and combined permeation enhancers, are shown in Table 9. It was found that the incorporation of limonene and eucalyptol into both film types significantly enhanced the transdermal permeation of mitragynine compared to formulations without permeation enhancers. Among the enhancers tested, eucalyptol demonstrated the highest efficiency in facilitating mitragynine permeation into the receptor fluid, which serves as an indicator of potential systemic absorption. In contrast, the use of laurocapram alone resulted in reduced mitragynine permeation in film Type 4. Moreover, combinations of laurocapram with limonene or eucalyptol still led to decreased permeation compared to formulations without any enhancers. However, in film Type 3, both individual and combined permeation enhancers were more effective in promoting mitragynine permeation than formulations without enhancers. These results indicate laurocapram or azone exhibited a retardation effect when mitragynine passes through the skin in Type 4 film, which corresponds to the work of Tuntiyasawasdikul et al. [21]. The superior performance of the terpene enhancers, eucalyptol and limonene, compared to laurocapram in the Type 4 PVA matrix can be attributed to their distinct mechanisms of action. Terpenes are small, lipophilic molecules known to partition into the stratum corneum, where they disrupt the highly ordered intercellular lipid bilayers, thereby increasing their fluidity and creating pathways for mitragynine diffusion. In contrast, the retardation effect observed with laurocapram in the hydrophilic PVA film suggests a negative formulation interaction. It was hypothesized that laurocapram formed a reservoir within the film matrix, reducing the thermodynamic activity of mitragynine and thus lowering its partitioning from the hydrophilic vehicle into the lipophilic stratum corneum [36]. In the development of transdermal patches intended for systemic pain relief, it is essential to incorporate skin permeation enhancers to facilitate the absorption of the active compound through the skin and into systemic circulation at levels sufficient to exert a therapeutic effect. In this study, the selected permeation enhancers included compounds from the terpene group (limonene and eucalyptol) and the azone group (laurocapram). These agents have been previously reported to have effectively enhanced the transdermal delivery of various drugs while demonstrating good safety profiles and low skin irritation potential [36]. As these two classes of permeation enhancers act via different mechanisms to enhance skin permeability, the concept of combining them was proposed to improve drug/active ingredient absorption efficiency while minimizing toxicity and skin irritation. Two binary combinations were evaluated: laurocapram with limonene and laurocapram with eucalyptol. The total concentration of permeation enhancers used was 5% w/w in both single-agent and combined formulations. In the combined formulations, each enhancer was present at 2.5% w/w. The selected concentration range was based on literature reports, which indicate that effective concentrations for transdermal patch formulations typically range from 0.1% to 5% [15]. Understanding the permeation characteristics of mitragynine through the skin and into systemic circulation provides valuable insights for optimizing and designing formulations to ensure effective transdermal delivery. Furthermore, the surface morphology observed via SEM (Figs. 6 and 7) directly correlated with permeation outcomes. In Type 4 (PVA) films, the addition of laurocapram resulted in a rougher surface, suggesting some degree of immiscibility. This likely contributed to the observed retardation effect on mitragynine permeation, possibly by isolating the mitragynine within enhancer-rich domains and reducing its partitioning into the skin.

Table 9. The transdermal permeation parameters of mitragynine at 24 hours for the various film formulations.

*Indicates a statistically significant difference compared to the control group (p < 0.05).

3.5. Deposition of mitragynine in the pig ear skin

Based on the study of mitragynine accumulation in pig ear skin after 24 hours, the effects of Type 3 and Type 4 films containing kratom extract with and without each of the three individual permeation enhancers, as well as combinations of two enhancers, were investigated. The results are presented as bar graphs comparing the amount of mitragynine accumulated in the skin and in the systemic circulation, as shown in Figure 11. The findings indicated that in the case of Type 3 film, the permeation enhancer eucalyptol was able to provide the most effective increase in mitragynine accumulation in both the skin and the systemic circulation. In contrast, in Type 4 film, both limonene and eucalyptol were able to effectively enhance the mitragynine accumulation in the skin and circulation. However, Type 3 film consistently demonstrated significantly higher accumulation of mitragynine in both the skin and systemic circulation compared to Type 4 film (p < 0.05). Therefore, Type 3 film containing eucalyptol as a permeation enhancer showed the greatest potential for development into an effective transdermal analgesic patch potential for systemic delivery.

Figure 11. Accumulated amount of mitragynine in the skin and systemic circulation from Type 3 film and Type 4 film containing different permeation enhancers after 24 hours. Error bar show SD for n = 4. [Click here to view]

3.6. Stability of films containing kratom extract

Considering the desirable physical properties and high skin permeation rates, a stability study was conducted on nine selected film formulations from both Type 3 and Type 4 films. From Type 3, six film formulations with and without different types of permeation enhancers were selected. From Type 4, three film formulations were chosen: one without a permeation enhancer, and two containing single enhancers, limonene and eucalyptol. The films were stored at room temperature (30°C ± 1°C) and 75% RH for 6 months. The remaining amount of mitragynine in the films was analyzed using HPLC at 0 and 6 months, along with observations of physical changes such as film color and thickness, as shown in Table 10. The results showed that films from both Type 3 and Type 4 retained more than 81% of mitragynine content after when they were freshly prepared (0 month). However, Type 4 film, which used PVA, exhibited a higher remaining mitragynine content compared to films made from agar, pectin, and SA. This is likely due to the natural polymers’ tendency to absorb moisture, which negatively affects the stability and solubility of the kratom extract in the films, especially since the extract is poorly water-soluble. After a certain period, the films became greasy or sticky to the touch and gradually darkened in color with extended storage. Furthermore, the mitragynine content significantly decreased over time. After six months, the amount of mitragynine had dropped by 10%–25% in all nine film formulations, indicating poor stability of the extract within the product. Therefore, protective measures are needed to shield the films from environmental factors. These may include using packaging that prevents light and moisture penetration, increasing the initial amount of extract in the film to compensate for degradation during storage, or limiting the shelf life to ensure the product is freshly prepared before use. These approaches would help improve both the effectiveness and practical usability of the final product.

Table 10. The chemical and physical properties of kratom extract films after storage at room temperature (30°C ± 1°C) and 75% relative humidity (RH) for 6 months.

3.7. Microbiological testing of films containing kratom extract

In the prepared kratom extract film, a preservative (phenoxyethanol) was added to extend the product’s shelf life and maintain microbiological stability throughout its usage or until the product is fully consumed. This was done in accordance with medical product standards for herbal-based formulations, which require testing to ensure the absence of harmful microorganisms, as specified by product safety regulations. Microbial limit testing was performed following the methods outlined in the Thai Herbal Pharmacopoeia 2019. The results showed that 1 g of the kratom extract film contained no detectable aerobic bacteria, yeast, or mold. Furthermore, no pathogenic microorganisms were found, including Clostridium spp, P. aeruginosa, C. albicans, and S. aureus.

3.8. Skin irritation potential of the patch in rabbits

The test results showed that none of the rabbits exhibited any signs of redness or swelling on the skin, and no deaths or abnormal symptoms were observed. The PII was assessed, and its value was 0. Therefore, it can be concluded that the test sample, kratom extract patch containing eucalyptol as a permeation enhancer, did not create any irritation of skin in rabbits under the conditions of this study.

3.9. Prototype biocompatible polymer film patch product

Based on the study data on the film’s properties including mitragynine release and permeation through the skin and into systemic circulation, chemical and physical stability, microbiological stability, and skin irritation in rabbits it was found that the film formulated with agar, pectin, and SA as the polymer base, combined with eucalyptol as a permeation enhancer and containing kratom extract at a concentration of 1.625 mg/cm² (0.1625 mg/cm² of mitragynine), was the most suitable for development as a transdermal kratom extract patch intended potential for systemic delivery. The prototype film containing kratom extract was designed to be cut into a 4 × 4 cm² piece and adhered to a white nonwoven silicone backing sheet with an adhesive layer, which was cut into a 6 × 6 cm² piece. The entire assembly was then covered with a release liner, as shown in Figure 12. The preparation method for the prototype of the kratom extract patch has been registered as a petty patent in Thailand, with two application numbers: 2103000840 and 2303001978. Pharmacokinetic data of oral mitragynine consumption show that a single dose (6.65–53.20 mg) produces a Cmax ranging from 17.1 to 125 ng/ml [37]. This prototype kratom extract patch, containing 2.6 mg of mitragynine per patch and supplemented with eucalyptol as a skin penetration enhancer, may be capable of delivering sufficient amounts of mitragynine to achieve therapeutic levels for analgesic effects. Furthermore, mitragynine acts on opioid receptors without activating the beta-arrestin-2 pathway, which is responsible for adverse effects like respiratory depression seen with traditional opioids [37]. This provides a strong justification for why developing alternative delivery systems for this compound is a promising area of research. The development of an alternative product incorporating kratom extract, supported by scientific evidence, represents a promising formulation for further preclinical evaluation in pain management. Such innovation not only enhances the value of the kratom plant but also generates income opportunities across sectors involved in its beneficial applications. Nevertheless, clinical studies remain essential to confirm its analgesic efficacy and ensure the advancement of an effective therapeutic product.

Figure 12. The transdermal patch containing kratom extract. On the left side, the topmost layer is the white nonwoven silicone backing sheet, followed by the adhesive layer, the kratom extract film, and the release liner at the bottom. The image on the right shows the side of the patch that comes into contact with the skin, covered by the release liner. Beneath it is the kratom extract film, adhesive layer, and the backing sheet. [Click here to view]

3.10. Study limitations and future directions

While this study successfully identifies a promising lead formulation, several limitations had to be acknowledged. First, this study was conducted using an ex vivo porcine skin model; although it is a well-accepted surrogate, it may not perfectly predict in vivo pharmacokinetics in humans. Second, the use of 20% methanol in the receptor fluid, while necessary to maintain sink conditions for the poorly soluble mitragynine, is nonphysiological and may overestimate the achievable permeation rate. Third, the stability study was preliminary, conducted under a single storage condition. Finally, the safety assessment was limited to a primary irritation screening test in rabbits, which does not assess the potential for sensitization or long-term irritation.

Therefore, future research should proceed along a clear translational path, and the next step would be conducting in vivo pharmacokinetic studies in a suitable animal model (e.g., rats or minipigs) to determine the plasma concentration profile of mitragynine delivered from the lead patch formulation, and establishing an in vitro-in vivo correlation. Concurrently, a comprehensive stability program according to ICH guidelines, including accelerated storage conditions and photostability testing, is required. Furthermore, extensive preclinical safety studies, including skin sensitization assays, must be performed before this formulation can be considered for human clinical trials to evaluate its efficacy in pain management.