Preparation and Application of Decanoic Acid/Arginine Hydrogels to a Transdermal Formulation

Abstract

We prepared a supramolecular hydrogel composed of decanoic acid and arginine (C10/Arg gel) and evaluated its application to a transdermal formulation. C10/Arg gel adjusted to pH 7 with 1 M NaOH aq or 1 M HCl aq provided a translucent hydrogel with a lamellar liquid crystal structure in the concentration region of decanoic acid ≥12% and arginine ≤9%. Rheological measurements showed that C10/Arg gel is a viscoelastic material with both solid and liquid properties, with elasticity being dominant over viscosity in the low shear stress region. The skin permeability of hydrocortisone (HC) and indomethacin (IM) from C10/Arg gels was investigated in vitro using hairless mouse skin and compared to control formulation drug suspensions (IM or HC) in water. The cumulative permeation amount of HC and IM from the C10/Arg gel at 10 h after application was approximately 16 and 11 times higher than that of the control, respectively. On the other hand, the flux of IM decreased with increasing arginine concentration, likely due to the acid–base interaction between Arg and IM in C10/Arg gel. Adequate drug skin permeation enhancement by C10/Arg gel requires optimizing the gel composition for each specific drug.

INTRODUCTION

Methods of administering drugs include injection, oral, transmucosal, intranasal and transdermal routes. Transdermal administration has several advantages, such as avoiding hepatic first-pass effects and the simplicity of administration to elderly and infant patients who have difficulty with oral administration. The skin protects the body from external stimuli, with the stratum corneum functioning as the main physical barrier.^1,2) The stratum corneum restricts the penetration of drugs into the skin³⁾ and thus transdermal absorption enhancers are often used to improve the transdermal absorption of drugs.

Examples of transdermal absorption enhancers include alcohols, fatty acids, amines, esters, amides, hydrocarbons, surfactants, terpenes, sulfoxides, and phospholipids.⁴⁾ Fatty acids, another type of absorption enhancer, enhance the permeation of biomembranes. Skin corrosivity tests of fatty acids with carbon chain lengths of 3–12 using rat or human skin showed that decanoic acid (C10) and dodecanoic acid are less corrosive than fatty acids with shorter carbon chains.⁵⁾ Sodium decanoate, a salt of decanoic acid, is used medically as an absorption enhancer in suppositories in Japan.⁶⁾ The fatty acid sodium salt is used because C10 is insoluble in water, but the pH of the sodium salt is over 9, which is not suitable for application to the skin. The application of a mixture of fatty acids and propylene glycol to the skin promotes the absorption of low-molecular-weight drugs.^7–11)

Low-molecular weight gelling agents amenable to spontaneous supramolecular hydrogel formation were reported.^12–15) Verma et al. reported that the morphology of self-assembled structures formed by an aqueous mixture of cetyltrimethylammonium bromide and the hydrophobic amino acid mimic anthranilic acid change with pH and form highly viscous supramolecular hydrogels at appropriate pH values.¹²⁾ Kaneko et al. reported that aqueous solutions of arginine oleate, obtained by mixing arginine (Arg) and oleic acid, formed molecular aggregates such as micelles, vesicles, and lamellar liquid crystals as the concentration of arginine oleate was increased.¹³⁾ Li et al. investigated the aggregation behavior of mixtures of lysine and fatty acids with different chain lengths in aqueous solutions; these mixtures formed micelles, vesicles, sponge structures, and fibers upon varying the composition and chain length of the fatty acids.¹⁴⁾ They also reported that when two fatty acids, octanoic acid and C10 were separately mixed with Arg in aqueous solution, self-assembled structures such as micelles, vesicles, and sponge structures were formed, depending on the composition of the fatty acids and their chain length.¹⁵⁾

Arg is widely used in cosmetics and personal care products and is considered safe by the U.S. Food and Drug Administration at the concentrations used for cosmetic applications, based on information provided by the Voluntary Cosmetic Registration Program.^16,17)

The aim of this study was to utilize C10. C10 is difficult to handle but promotes drug absorption when combined with the basic amino acid Arg and applied as a transdermal formulation. Evaluation of the structure and rheological properties of the resulting hydrogels and the usefulness of the gels as a transdermal formulation were investigated by skin permeation tests using hairless mouse skin. Hydrocortisone (HC), a neutral drug, and indomethacin (IM), a weakly acidic drug, were used as model compounds for the transdermal formulation. The effect of drug properties on the skin permeation of supramolecular hydrogels formed using C10 and Arg was investigated.

MATERIALS AND METHODS

Materials

C10 and Arg were Wako special grade (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan). HC and IM were biochemistry grade and obtained from FUJIFILM Wako Pure Chemical Corporation. Water was Japanese Pharmacopoeia water for injection. Hydroxypropyl-β-cyclodextrin (HP-β-CD) was purchased from Nihon Shokuhin Kako Co., Ltd. (Tokyo, Japan). Other reagents were analytical grade.

Preparation of Supramolecular Hydrogels

The required amount of Arg (0–10%) was dissolved in water and the pH of the final supramolecular hydrogel was adjusted to 7 with 1 M NaOH aq or 1 M HCl aq. C10 was heated and melted, and the required amount (10–25%) was added to the stirring Arg solution maintained at 60 °C. Air bubbles generated during stirring were removed by centrifugation (3000 rpm). The obtained hydrogel (C10/Arg gel) was kept in an air thermostatic bath at 25 °C for several days prior to use.

Construction of Phase Diagrams

Hydrogels were prepared in the range of C10: 10–25%, Arg: 0–10%, water: 75–90%, at intervals of approximately 1–5% and were visually observed at room temperature (15–25 °C). We defined a gel as a mixture that did not flow 2 s after inverting the vial holding the mixture.

Polarizing Microscopy

Polarizing microscopy was performed using a Nikon ECLIPSE E600W POL (Nikon Corporation, Tokyo, Japan). The sample was applied to a glass slide, covered with a cover glass, sealed with silicone grease (G-501, Shin-Etsu Chemical Co., Ltd., Tokyo, Japan), and left to stand for 1 week prior to observation at 25 °C.

Small-Angle/Wide-Angle X-Ray Scattering (SAXS/WAXS) Measurements

SAXS/WAXS measurements were carried out using the BL40B2 beamline (Structural Biology II Beamline) at SPring-8 (Hyogo, Japan). Samples were placed in a 2 mm diameter quartz glass capillary. The camera distance was 2000 mm for SAXS and 580 mm for WAXS. A PILATUSS 3S 2M (large area pixel detector) was used as a detector. The exposure time was approximately 100 s. All measurements were performed at 25 °C and using an X-ray wavelength of 0.1 nm. The morphology of each molecular assembly and the interlayer distance in each gel were determined from the SAXS/WAXS scattering intensity I(q) versus the wave vector q (q = (4π/λ) sin θ, λ: X-ray wavelength, 2θ: scattering angle) profile.

Rheological Measurements

Rheological measurements were performed using a stress-controlled rheometer (HAKKE MARS III Thermo Fisher Scientific Inc. MA, U.S.A.). A sandblasted parallel plate (35 mm diameter, 0.5 mm gap) was used. The temperature was maintained at 32 °C using a Peltier-based temperature control device. Dynamic rheological measurements were conducted by subjecting the sample to strain (γ) at a frequency of 1 Hz, and the storage modulus (G′) and loss modulus (G″) were determined from the stress (σ). A solvent trap prevented the evaporation of water from the sample.

Skin Permeation Experiments

The C10/Arg gel formulations used for the skin permeation tests are shown in Table 1. The sum of the C10 and Arg concentrations was used as the gelling agent concentration, and to each formulation 0.5% HC or 1% IM was added. These drugs were dissolved in C10/Arg gels. The control formulation drug suspension comprised 0.5% HC or 1% IM in water. The pH of the IM suspension was adjusted to pH 7 with 1 M NaOH aq. Frozen skin from hairless mice (Laboskin^®, Hos: HR-1 Male, 7 weeks old, Hoshino Laboratory Animals Inc., Ibaraki, Japan) was thawed and mounted on a Franz diffusion-type cell (diffusion area of 1.74 cm² and 7 mL of receptor phase). The receptor phase was phosphate buffer saline (PBS, pH 7.4) kept at 32 °C. For the skin permeation test for HC, 0.5% HP-β-CD was added to the receptor solution to maintain sink conditions. The receptor phase was stirred at 550 rpm using a magnetic stirrer. A 1 g sample was applied to the skin in the donor compartment and the top of the donor compartment was closed. Half milliliter of the receptor phase was collected at predetermined times and replaced with fresh PBS. The drug concentrations in the receptor phase were determined using HPLC.

Table 1. Composition of C10/Arg Gel

C10/Arg gel	Gelling agent (%)			C10/Arg molar ratio
	C10	Arg	Total
DA-1	14	2.4	16.4	6
DA-2	17	2.9	19.9	6
DA-3	20	3.4	23.4	6
DA-4	17	0	17	—
DA-5	17	1.9	18.9	9
DA-6	17	3.9	20.9	4
DA-7	15	2.9	17.9	5
DA-8	20	2.9	22.9	7

The flux was determined from the slope of the linear part of the skin permeation profile.

HPLC Analysis of Drugs

The HPLC system used for HC quantification consisted of a UV detector (UV-2075 Plus, JASCO Corporation, Tokyo, Japan), HPLC pump (PU-2080 Plus, JASCO Corporation), autosampler (AS-2051 Plus, JASCO Corporation), and a chromatographic data processor (ChromNAV, JASCO Corporation). The column was an L-Column 2ODS (4.6 × 150 mm, Kanto Chemical Co., Inc., Tokyo, Japan), and the column temperature was 40 °C. The mobile phase was degassed acetonitrile : water = 35 : 65 (v/v), flowing at a rate of 1.0 mL/min. The sample injection volume was 20 µL and the detection wavelength was 254 nm.

The HPLC system used for IM quantification consisted of a UV detector (UV-2075 Plus, JASCO Corporatioon), HPLC pump (LC-10AD, Shimadzu Corporation, Kyoto, Japan), autosampler (SIL-6B, Shimadzu Corporation), and a chromatography data processing system (Chromato-PRO, Runtime Instruments Co., Tokyo, Japan). The column was a Mightysil RP-18 GP (4.6 × 250 mm, 5 µm, Kanto Chemical Co., Inc.). The mobile phase was degassed acetonitrile: 0.1 mol/L acetate buffer (pH 3.3) = 3 : 2 (v/v), flowing at a rate of 1.0 mL/min. The sample injection volume was 20 µL and the detection wavelength was 278 nm.

The concentration of each drug was determined from the peak area using an external calibration curve.

Data Analysis

Data obtained from the skin permeation experiments are presented as the mean ± standard deviation (S.D.) of three to six experiments. A t-test was used for comparisons between two groups and ANOVA followed by Tukey–Kramer’s HSD test for multiple comparisons. A p-value less than 0.05 indicated a significant difference.

RESULTS AND DISCUSSION

Preparation and Structure of Hydrogels

Figure 1 shows a partial triangular phase diagram for a C10/Arg gel adjusted to pH 7. The figure was created using 65 points (C10: 10–25%, Arg: 0–10%, water: 75–90% at 1–5% intervals). Details are given as supplementary material, and show the actual measured compositions and their conditions (Supplementary Fig. S1). Translucent gels could be prepared in the range of C10 ≥ 12% and Arg ≤ 9%. At C10 ≥ 12%, a translucent gel was also formed with C10 alone; when Arg was added, no gelation or cloudiness was observed at low C10 concentrations, and at higher Arg concentrations, the gel was not translucent but clouded white.

Fig. 1. Partial Phase Diagram of C10/Arg/Water System

represents the translucent gelation area, represents the cloudy gelation area, represents the cloudy solate area, and □ indicates not tested. Numbers in the figure are the formulation number of C10/Arg gels, described in Table 1.

Figure 2 shows a typical sample and polarized light micrographs of the translucent gel DA-2. Figure 2(a) shows an external view of DA-2 with the vial placed upside down; the gel is located at the top of the picture. DA-2 is a gel-like substance that does not flow when its container is turned upside down. The polarized light micrograph of DA-2 in Fig. 2(b) exhibits a mosaic pattern, suggesting that C10/Arg gel has a liquid crystal structure consisting of molecular aggregates with polarizing properties.

Fig. 2. Photograph of DA-2 in the Vial Placed Upside Down (a) and Polarized Light Microscopy Image of DA-2 (b)

Observation of a cloudy gelation region (Fig. 1) under a polarizing microscope confirmed polarization, but needle-like crystals were observed. When the hydrogel containing the crystals was heated at 32 °C, the melting point of C10, the crystals disappeared. Therefore, the needle-like crystals in the cloudy gelatinous region are considered to be derived from C10.

SAXS/WAXS analysis was used to clarify the morphology of the DA-2 molecular assembly. The SAXS profile with q in Fig. 3(a) shows repeated peaks in the low-q region, with q values of 0.51, 1.02 and 1.56 nm⁻¹, giving a q-value ratio of 1 : 2 : 3. This indicates that DA-2 has a lamellar liquid crystal structure. In the high-q region (WAXS), the observed broad scattering peak indicates that C10 in the hydrogel is not crystallized (Fig. 3(b)).

Fig. 3. SAXS (a) and WAXS (b) Patterns for DA-2 and the Lamella Interlayer Distance (c)

The d-spacing represents the distance between the lamellar layers and is calculated from the q-value for the first peak in the low-q region using equation (1), derived from Bragg’s equation.

  

(1)

Figure 3(c) shows the relationship between the d-spacing and the gelling agent concentration, with d tending to shorten with increasing gelling agent concentration. As the concentration of gelling agent increases, the water content decreases and the water layers between the lamellar layers narrow, indicating a denser packing structure.

C10 is a weakly acidic compound containing carboxyl groups, and Arg is a basic amino acid containing amino and guanidino groups. Since both C10 and Arg are weak electrolytes, their ionization state depends on the pH of the aqueous environment and the ratio of the molecular and ionic forms.

When the pH is not adjusted, C10 is in a molecular form because the pK_a of C10 is 4.9, and C10 and water are separated. When C10 sodium salt is added to water, the pH of the solution is approximately 10, and C10 exists as monomers and micelles in ionic form and does not form gel. From the Henderson-Hasselbalch equation, about 1% of C10 is in the molecular form and the remainder is in the ionic form at pH 7, forming a lamellar layer and a translucent gel (Figs. 1, 3).

Arg is a basic amino acid with an isoelectric point around 11, so adding Arg to water raises the pH. When the pH was not adjusted to pH 7, gelation occurred in the range of C10 ≥ 13% and 6% ≤ Arg ≤ 8% (data not shown). The pH was 5–8 depending on the composition, and a translucent gel was formed when the C10/Arg molar ratio was in the range of 2 to 3. This suggests the involvement of C10-Arg salts in gelation.

The pK_a of Arg is pK_a1 = 1.82, pK_a2 = 8.99, and pK_a3 = 12.48. At pH 7, the carboxy group (negative), amino group (positive), and guanidino group (positive) of Arg are all ionized and Arg has an overall monovalent positive charge. When the C10/Arg molar ratio is 6, 1 out of 6 mol of C10 may interact with 1 mol of Arg to form a C10-Arg salt, and the remaining 5 mol of C10 may exist as ions. When the C10-Arg salt and C10 ions, formed by electrostatic interaction of C10 in the hydrophobic part and Arg in the hydrophilic part, are present in water in an optimal balance, a lamellar liquid crystal structure is considered to be formed and a translucent gel is created. If the ratio of Arg increases, the ratio of C10-Arg salt also increases and a translucent gel cannot be obtained.

Supramolecular hydrogels are formed when amphiphilic materials such as low-molecular-weight gelling agents self-assemble through non-covalent bonds such as hydrogen bonds and van der Waals forces to construct a three-dimensional network structure.^12,18–21) Examples of low-molecular weight gelling agents include surfactants, fatty acid derivatives, and amino acid derivatives¹⁹⁾ and these agents are generally produced through chemical synthesis following molecular design to self-assemble and gel through intermolecular interactions.^18–22)

Here we showed that a supramolecular hydrogel with a lamellar liquid crystal structure is formed by the C10 ions and C10-Arg salt generated by stirring and mixing C10 and Arg under pH 7 conditions, without the need for chemical synthesis.

Rheology Measurements

We investigated the effect of gelling agent (C10 + Arg) concentration on the dynamic viscoelastic behavior of C10/Arg gel, and the results for a fixed C10/Arg molar ratio of 6.0 are shown in Fig. 4. Figure 4(a) shows the dependence of G′ and G″ on the stress σ at a frequency of 1 Hz. G′ and G″ are independent of σ in the low-σ region, regardless of the gelling agent concentration. This stress range is called the linear viscoelasticity (LVE) region. The internal structure of the gel does not break down within the LVE region. The gradual decrease in G″ with increasing σ indicates that the internal structure of the gel gradually breaks down as σ increases. G′ in the LVE region is larger for a higher gelling agent concentration. The value of σ at the intersection of G′ and G″ is shown in Fig. 4(b). Below this value of σ, G′ is larger than G″ (G′ > G″), and the material retains its gel shape, whereas above this value of σ, G″ is larger than G′ (G″ > G′), and the material changes to a sol and begins to flow.²³⁾ These results indicate that the hydrogel behaves as a viscoelastic material. As the concentration of the gelling agent increases, the σ value at the intersection tends to increase (Fig. 4(b)). As the concentration of the gelling agent increases, the water content decreases, suggesting that the supramolecular hydrogel has a more intact gel structure because the water layer between the lamellar layers decreases and the molecular aggregates pack more densely. Hydrogels for topical application require appropriate viscoelastic properties so that the gel can be easily removed from the container and applied to the skin. C10/Arg gel exhibits both solid and liquid properties and has rheological properties suitable for transdermal formulations.

Fig. 4. Dynamic Viscoelastic Behavior (a) and Shear Stress in Crossover (b) of C10/Arg Gel at Different Gelling Agent Concentrations

G′: Storage modulus, G′′: loss modulus, σ : shear stress.

Skin Permeability of Drugs

We investigated the usefulness of C10/Arg gels as a base for transdermal formulations by conducting in vitro skin permeation studies of HC and IM using hairless mouse skin. Addition of 0.5% HC or 1% IM did not change the physical properties of the C10/Arg gels.

Figure 5 shows the drug skin permeation profiles for HC or IM incorporated into DA-2 formulations and from a water suspension (control). The skin permeation curves showed a linear relationship between time and drug permeation after 7 h, indicating a steady state. The cumulative permeated amounts of HC and IM from DA-2 at 10 h after application were approximately 16 and 11 times higher than that of the control, respectively. The flux of IM from DA-2 was 9 times higher than that of the control. Note that the flux of HC from DA-2 could not be compared with that of the control because the HC concentration of the control was below the detection limit until after 6 h of transmission. These findings indicate that C10/Arg gel promotes the skin permeation of drugs.

Fig. 5. Skin Permeation Profiles for HC (a) and IM (b) from Suspension (□) and DA-2 (●)

Each point represents the mean  ±  S.D. of three to six experiments.

We investigated the effects of formulations of C10/Arg gels on drug permeation through the skin. Figure 6(a) shows the effect of the concentration of the gelling agent on the flux of HC and IM. The molar ratio of C10 and Arg was fixed at 6 and the concentration of the gelling agent was varied from 16.4 to 23.4%. As the gelling agent concentration increased, the fluxes of HC and IM tended to decrease but the lag time remained approximately 5 h (Fig. 6(b)). This suggested that changes in gelling agent concentration between 16.4 to 23.4% did not affect the diffusion of HC and IM in skin. The increased solubility of a drug decreases thermodynamic activity and flux,^24,25) from which we can infer that as the gelling agent concentration increases, the maximum amount of drug that can be solubilized also increases. Thus, the solubility of the drug likely increased without altering the concentration of the drug in the formulation, resulting in a decrease in both the thermodynamic activity and flux. This suggests that there might be a range of concentrations below the gelling agent concentration of DA-1 (16.4%) that would maximize the permeation effect.

Fig. 6. Effect of Gelling Agent Concentration on Flux (a) and Lag Time (b)

The ratio of C10/Arg was fixed at 6. Gelling agent concentration: DA-1 (□: 16.4%), DA-2 (■: 19.9%), DA-3 (: 23.4%). Each point represents the mean  ±  S.D. of three to six experiments. * Significant difference (p < 0.05).

The effects of C10 and Arg concentration in gels were investigated and are shown in Fig. 7. Figure 7(a) shows the flux of HC and IM for C10/Arg gels with the C10 concentration fixed at 17% and with Arg concentrations of 0% (DA-4), 1.9% (DA-5), 2.9% (DA-2), and 3.9% (DA-6). The drug flux increased even at an Arg concentration of 0% (DA-4), indicating that C10 is responsible for the enhanced drug skin permeation effect of C10/Arg gel. The Arg concentration had no effect on the HC flux but the IM flux decreased significantly as the Arg concentration was increased. HC is a neutral drug, whereas IM is a weakly acidic drug. Release studies of IM from supramolecular hydrogels composed of bis-imidazole-based surfactants showed that strong affinity with the gel resulted in less release.¹⁸⁾ An increase in Arg concentration in C10/Arg gels increases the acid–base interaction of Arg with the C10 fatty acids and with the carboxy groups of IM. This may increase the solubility of IM in the vehicle and decrease the IM flux. It was suggested that Arg had no enhancement effect on drug skin permeation, and so we did not conduct experiments with formulations containing Arg concentrations higher than 3.9%.

Fig. 7. Effect of Arg (a) and C10 Concentration (b) on Flux

(a) C10 concentration was fixed 17%. Arg concentration: DA-4 (: 0%), DA-5 (: 1.9%), DA-2 (■: 2.9%), DA-6 (: 3.9%), (b) Arg concentration was fixed 2.9%. C10 concentration: DA-7 (: 15%), DA-2 (■: 17%), DA-8 (: 20%). Each point represents the mean  ±  S.D. of three to six experiments. * Significant difference (p < 0.05).

Figure 7(b) shows the flux of each drug for C10/Arg gels with the Arg concentration fixed at 2.9% and the C10 concentration at 15% (DA-7), 17% (DA-2), and 20% (DA-8). The flux slightly decreased as the C10 concentration increased, but not significantly. Fatty acids have been proposed as chemical permeation enhancers, for example to disrupt the stratum corneum barrier function,^11,26) increase the skin/vehicle partitioning of drugs,²⁷⁾ and increase solvent transport across the skin.²⁸⁾ These actions of C10 may promote skin permeation of the drug from the C10/Arg gel. On the other hand, simply increasing the concentration of C10 does not help increase the drug’s promoting effect. The above results indicate that the contribution of C10 to the skin permeation enhancement effect of C10/Arg gel is significant, and that a C10 concentration of about 15% is adequate to achieve the desired effect.

CONCLUSION

By mixing C10 and Arg in water and adjusting the pH to 7, translucent gels could be easily prepared in the range of C10 ≥ 12% and Arg ≤ 9%. The C10 ions and C10-Arg salt in the resulting supramolecular hydrogel act as amphiphiles to form molecular aggregates and construct a lamellar liquid crystal structure that is likely a stable gel. Furthermore, C10/Arg gels can promote the skin permeation of HC and IM. This drug skin permeation promoting effect may be due to C10, a fatty acid. In addition, acid–base interactions between Arg and acidic drugs in the C10/Arg gels may affect the solubility of the drugs in the vehicle. C10/Arg gel can promote the skin permeation of drugs upon selecting the appropriate gel composition for each drug.

Acknowledgments

The SAXS measurements were performed using the BL40B2 beamline of the large synchrotron radiation facility SPring-8 (2018A1200, 2020A1076, 2022A1143).

Thanks are due to Ms. Haruna Ohmori for her technical assistance with the experiments.

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

This article contains supplementary materials.

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