1. Introduction
Terbinafine hydrochloride (TEB) (
Figure 1A) is an allylamine antifungal agent used for the treatment of various infectious diseases, such as onychomycosis, tinea corporis, and tinea cruris, through inhibiting squalene epoxidase and interfering with ergosterol synthesis [
1,
2]. However, with continuous research in clinical pharmacy, the oral administration of terbinafine has induced many adverse reactions, including drug interactions, hepatotoxicity, gastrointestinal, and systemic side effects [
3,
4,
5]. Fortunately, local administration can improve patient compliance, such as targeted therapy, sustained release, and prolonged drug action time. Moreover, it can overcome various limitations and side effects associated with oral administration. Therefore, the local administration of terbinafine has become a primary focus in pharmaceutical formulation development [
6,
7,
8]. However, its inadequate transdermal permeability poses challenges in achieving effective therapeutic concentrations at the site of local treatment. Addressing this issue requires further research and intervention to enhance the efficacy of TEB in local antifungal therapy [
9,
10,
11]. Moreover, more research needs to be explored to understand how to improve the local delivery mechanism of TEB, enhance its permeability, and ensure that the drug is continuously released at the treatment site [
12,
13,
14]. These efforts may help to overcome the limitations of TEB treatment and thereby improve the effectiveness of antifungal therapy.
Terbinafine is currently available in free base and hydrochloride formulations [
15], as well as topical preparations, including creams, gels, and sprays. TEB is commonly administered as terbinafine hydrochloride in clinics, with the molecular formula C
21H
26ClN and the molecular weight (MW) of 327.9 g/mol. Its melting point is 204–208 °C, and it has an oil–water partition coefficient (LogP) of 3.3. TEB•HCl is a white powder, easily soluble in solvents like methanol, dichloromethane, and ethanol, and has a slightly lower solubility in water [
16]. However, the limited local concentration of terbinafine poses a significant obstacle in effectively treating deep-skin fungal infections [
15]. In order to address this challenge, researchers have proposed various strategies. One approach involves the use of liposomes as carriers to enhance skin permeability, encapsulation efficiency, and drug stability [
17]. Another promising avenue of exploration is the utilization of nanovesicles as potential carriers [
18]. However, these approaches often entail complex preparation processes, have limited stability, and rely on passive drug transport, leading to variable therapeutic effects among individuals.
Due to the barrier function of the stratum corneum, skin administration is often limited, which impedes the passage of most drug molecules, allowing only a few with specific physicochemical properties to penetrate the skin [
19]. Iontophoresis is a technique that enhances the transdermal delivery of compounds by applying a safe and small electric current, primarily used for the delivery of large and charged molecules [
20,
21]. It is an active non-invasive drug delivery technology that facilitates the transport of charged and neutral molecules into and across biological membranes [
22,
23]. The preparation of drugs into suitable formulations and their compatibility with iontophoresis can enhance the transdermal permeation and therapeutic efficacy of the drugs [
24,
25]. By adjusting the pH, terbinafine can be transformed into an ionic drug. Currently, there are no relevant products or reports on TEB hydrogel patches that are assisted by iontophoresis systems.
This study aims to develop terbinafine into hydrogel patches (
Figure 1B) and adapt terbinafine hydrogel patches to an iontophoresis device to increase the skin drug concentration of terbinafine. Hydrogel patches have the advantages of high drug loading, precise dosing, and broad applicability, and they exhibit good compatibility with human skin. The back of the hydrogel patch is covered with a conductive backing layer, which prevents reductions in the effective drug dosage due to friction from clothing or other external factors. It is expected that this approach will significantly improve the local therapeutic concentration of the drug.
4. Materials and Methods
4.1. Rats and Reagents
Female SD rats, 200 ± 50 g, were sourced from the Laboratory Animal Center of Zhejiang Province (Hangzhou, China). They were accompanied by a production license with the number SCXK (Zhejiang 2019-0002), as well as a use license with the number SYXK (Zhejiang 2019-0011).
Terbinafine hydrochloride (purity: 99.0%, Macklin (Shanghai, China), #C12641041), Methanol (Chengdu Kelong (Chengdu, China), #2021121301), glacial acetic acid (Chengdu Kelong, #2021031601), Triethylamine (Aladdin, China, #11309090), sodium polyacrylate (Showa Denko, #161870A, Tokyo, Japan), gelatin (Rousselot (Ghent, Belgium), #1456799), Povidone K90 (IPS, America, #03600162502), glycerin (Macklin, China, #C10087952), aluminum glyoxyl (Macklin, China, #C12871941), and EDTA-2Na (Chengdu Kelong, China, #2021032201) were also obtained.
4.2. Establishment and Verification of TEB HPLC Analysis Method
Following the Chinese pharmacopeia (2020) and the characteristics of transdermal drug delivery preparations, this study improved the high-performance liquid chromatography (HPLC) method for TEB detection. The chromatographic conditions were as follows. Column: Diamonsil-C18 (4.6 mm × 250 mm, 5 μm), injection volume: 20 μL, column temperature: 30 °C, mobile phase: methanol–water (0.2% triethylamine and 1% acetic acid) = 7:3, detection wavelength: 282 nm, and detection time: 10 min. To ensure the scientific validity and accuracy of the experimental research, the specificity, linearity, precision, accuracy, detection limit, and quantitation limit of this method were validated.
4.3. Solubility Measurement of TEB in Different Media
The solubility of TEB in different solution media was determined using the saturated solution method. Excess TEB was added to various media and shaken overnight at 25 °C, and then the saturated solution was filtered, diluted, and subsequently injected into HPLC for detection.
4.4. Preparation of Hydrogel Patch
The TEB gel patches were prepared using a two-phase mixing method. In total, 10 g of glycerol was weighed into beakers, and the corresponding amounts of sodium polyacrylate, aluminum glycerolate, and 0.0125 g EDTA-2Na were slowly added and stirred to obtain the glycerol phase. In another beaker, 50 mL phosphate-buffered solution (20 mM) was combined with the prescribed amounts of gelatin and PVP K90, which was heated and stirred to achieve complete swelling. After cooling, 0.125 g tartaric acid was added, followed by 0.1 g TEB, which was stirred evenly into the matrix. The glycerol phase was then slowly added to the water phase and stirred at a speed of 300 r·min
−1. The mixture was centrifuged at 5000 r·min
−1 for 15 min to remove any bubbles, coated onto a non-woven backing, and dried at 50 °C. The final product, a hydrogel patch, was obtained by applying an anti-cohesion layer to the coated surface (
Figure 6).
4.5. Optimization of TEB Hydrogel Patch
In this study, we conducted a preliminary screening and identified sodium polyacrylate/aluminum hydroxide as the skeleton material for the hydrogel patch, glycerin as the humectant, gelatin as the excipient, PVP K90 as the adhesive, and the phosphate buffer as the solvent for pH adjustment and for increasing the conductivity of the gel patch. Based on previous experiments [
39], it was determined that the contents of sodium polyacrylate/aluminum hydroxide, gelatin, and PVP K90 had the greatest impact on the hydrogel patch. To screen and optimize the prescription, a factorial design method was employed, utilizing a 3-factor 2-level test (
Table 6).
Furthermore, we designed an evaluation index for the TEB hydrogel patches by referring to the relevant literature on hydrogel patches and considering the characteristics of the prepared TEB hydrogel patches. The evaluation index is presented in
Table 7 and served as the basis for assessing the prescription of the TEB hydrogel patches. The method used to evaluate initial adhesion was carried out according to the Chinese pharmacopeia (2020). This method determined the maximum number of standard steel balls that the patch could adhere to. The moisturizing properties of the hydrogel patch were assessed by measuring its ability to retain moisture at a certain temperature, known as moisture retention. The specific experimental method involved placing the hydrogel patch in an oven at 40 °C for 24 h, weighing it before and after, and calculating the weight change. The calculation formula for moisture retention was as follows:
The initial adhesion and moisture retention scores were combined with a quantitative weight standard score for somatosensory evaluation to calculate a comprehensive score for the patch.
4.6. Iontophoresis-Assisted Transdermal Permeation In Vitro
The SD rats were anesthetized and euthanized, and their backs were prepared by removing the hair. The skin was then carefully excised and placed on a glass plate. Then, the subcutaneous tissue and adhesions were meticulously removed. The skin was cut into 1.5 cm × 1.5 cm squares. Next, the skin squares were positioned at the opening of the diffusion cell, with the epidermis facing outward. TEB patches were applied to the rat skin’s epidermis. The hydrogel from the ion electroosmotic group was transferred to a carbon cloth electrode, which was connected to the positive electrode of the electroosmometer. The diffusion cell cover was secured using a clamp. In the receiving cell, 4 mL of a 20% PEG400-NaH2PO4 solution was added and connected to the negative electrode of the electroosmometer. The diffusion cell was maintained at a constant temperature of 32 °C, with the stirring speed set at 600 r·min−1. At designated time points (0.5, 1, 2, 4, 6, and 8 h), 1 mL samples was collected, and an equal volume of blank acceptor was added after each collection. The samples were subsequently filtered using a 0.22 μm microporous filter membrane and then injected into the HPLC for detection.
The calculation formula is as follows:
where “
n“ represented the specific sampling time point in a series of sequential sampling intervals, and 0.64 cm
2 was the area of the diffusion cell mouth.
Subsequently, the transdermal penetration of TEB was evaluated by conducting skin penetration experiments on isolated rats to investigate the effects of current density, hydrogel patch substrate pH, and drug concentration. The transdermal penetration performance of TEB was compared with that of a commercial TEB cream as a reference.
4.7. Microdialysis and Subcutaneous Tissue Pharmacokinetics In Vivo
In vitro recovery rate: Different concentrations (1.0, 10.0, and 20.0 μg·mL−1) of TEB saline solution were placed in a double-channel beaker. A linear probe with specific dimensions (φ = 15 μm, L = 200 μm, and 20 KDa) was immersed in the TEB saline solution through one channel of the beaker, while magnetic stirrers were placed in the beaker to ensure thorough mixing. The solution in the beaker was completely exchanged with the linear probe. Subsequently, blank normal saline was injected at different flow rates (1.0, 2.0, and 3.0 μL·min−1) using a microsyringe. After allowing for a 0.5 h equilibrium period, dialysate samples were collected. Four samples were collected for each flow rate. The recovery rate of TEB microdialysis was determined by calculating the ratio between the concentration of the receiving solution and the concentration of the drug in the beaker.
In vivo recovery rate: In vivo microdialysis experiments employed the reverse method, as it was not possible to directly measure the drug concentration outside the probe in the unique in vivo environment. The SD rats were anesthetized using an intraperitoneal injection of urethane (1.25 g∙kg−1). After removing the hair at the administration site on the abdomen, the rats were placed on a thermostatic pad at 37 °C. The linear probe was implanted into the deep dermis using a guide needle, with the probe membrane remaining in the subcutaneous tissue. Terazosin hydrochloride normal saline solutions with different concentrations (1.0, 5.0, and 10.0 μg·mL−1) were perfused at different flow rates (1.0, 2.0, and 3.0 μL·min−1). Four samples were collected at each flow rate, and a 0.5 h balance period was maintained before each collection. The recovery rate was determined by calculating the ratio between the concentration of the dialysate and the concentration of the original drug.
Transcutaneous pharmacokinetic study: The microdialysis probe membrane remained in the subcutaneous tissue of the rat, while a self-made hydrogel patch (containing 1 mg·cm−2, with an administration area of 2 cm × 2 cm) was applied to the hairless abdomen of the rat, directly above the microdialysis probe. The blank normal saline solution was perfused at a flow rate of 1.0 μL·min−1. After allowing for a 0.5 h equilibrium period, one acceptor solution was collected every hour, resulting in eight samples collected at each flow rate over a total of eight hours. The subcutaneous TEB concentration correction was determined by calculating the ratio between the dialysate concentration and the recovery rate at the corresponding time points in vivo.
The corrected concentration data were analyzed using DAS 2.0 to derive relevant parameters and generate the subcutaneous drug concentration–time curve.
4.8. Effects on Skin Microstructure
Transmission electron microscopy (TEM) was employed to examine the ultrastructure influences and pathological changes in the longitudinal subcutaneous tissue. The skin from the rats was removed, fixed overnight in a 2.5% glutaraldehyde solution at a temperature of 4 °C, and then processed. After cleaning, the samples were treated with osmic acid for 1 h, followed by ethanol and isoamyl acetate treatment, drying, coating, and observation under the scanning electron microscope.
4.9. Statistical Analysis
The statistical analysis was performed by using GraphPad Software 9.0. The data were expressed as means ± standard deviations (SDs). The statistically significant differences were determined by one-way analysis of variance (ANOVA) testing. Significance was defined as p < 0.05.