Rheological Properties and Composition Affecting the Skin Permeation of a Model of a Hydrophilic Drug in Lecithin Reverse Wormlike Micelles

Abstract

We investigated the effect of the rheological properties and composition of lecithin reverse wormlike micelles (LRWs) on the skin permeation of a model of a hydrophilic drug to determine whether LRWs support uniform hydrophilic drug/oil-based formulations and good drug penetrate into skin. Here, we prepared LRWs with D (−)-ribose (RI) or glycerol (GL) as polar compounds, liquid paraffin (LP) or isopropyl myristate (IPM) as oils, and 6-carboxyfluorescein (CF) as a model for a hydrophilic drug, and evaluated the rheological properties and skin penetration characteristics of the preparations. The LRWs showed moderate viscosity at 25 °C, a typical storage temperature, but decreasing viscosity at 32 °C, the surface temperature of human skin, suggesting that the LRWs would penetrate the microstructure of skin (e.g., wrinkles and hair follicles). The highest skin permeability of CF was observed when IPM was used as the oil, suggesting that both the stratum corneum and hair follicle routes are involved in drug permeation. The penetration of CF into hair follicles is influenced not only by the rheology of the formulation but also by the interaction between IPM and sebum in the hair follicles.

INTRODUCTION

Hydrophilic drugs are typically incorporated into water-containing topical preparations such as lotions and creams for application to the skin. However, some drugs are unstable in water, and thus oil-based formulations without water are used to ensure the stability of these drugs. Vaseline, a commonly used oil-based vehicle, allows only low drug skin permeation, and thus oil-based formulations amenable to the formulation of homogeneous hydrophilic drugs and exhibiting good drug permeation into the skin are required.

Lecithin reverse wormlike micelles (LRWs) are highly viscoelastic bodies formed by a mixture of three components: lecithin, polar compound, and oil. Lecithin and the polar compound associate in oil to form long flexible cylindrical reverse micelles, which entangle to form a three-dimensional network that holds a large amount of oil in the inter-micelle gaps.¹⁾ Various drugs, such as scopolamine,²⁾ broxaterol,²⁾ indomethacin,³⁾ diclofenac,³⁾ and testosterone,⁴⁾ have been solubilized in LRWs and exhibit enhanced skin permeability, suggesting that LRWs are potentially useful for transdermal applications.^5,6)

We therefore used LRWs as vehicles for a model of a hydrophilic drug in the absence of water and observed good drug permeation. Water is the most common polar compound for LRWs, but it can affect the stability of drugs. Various polar compounds form LRWs, typically by using n-decane as an oil.^7–11) In this study, D (−)-ribose (RI) and glycerol (GL) were selected as polar compounds to substitute for water to form non-aqueous formulations, as both are considered safe ingredients for application to the skin. RI is the backbone sugar of RNA, and GL is a polyol. Both compounds are used in cosmetics and medicals as humectant, skin protectant agents.¹²⁾

n-Decane is commonly used as an oil to prepare LRWs. We previously revealed that when mixed with water, liquid paraffin (LP), often used as an oil in skin-application formulations, forms LRWs. The molecular weight of LP affects the ease of formation and the viscoelasticity of the LRWs.¹³⁾ Isopropyl myristate (IPM) reportedly forms LRWs when mixed with water,¹⁾ RI, or tetraglycerol.⁴⁾ IPM is a well-tolerated skin permeation enhancer when used topically and is incorporated into many pharmaceutical and cosmetic preparations.¹⁴⁾ Thus, we investigated the use of LP and IPM as the oil and the phase state of LRWs, with RI or GL as the polar compound. 6-Carboxyfluorecein (CF), a model for a hydrophilic drug, was incorporated into LRWs and the rheological properties of the LRWs were investigated, together with the permeation of CF through pig ear skin.

MATERIALS AND METHODS

Materials

Soybean lecithin (Phospholipon 90G; phosphatidylcholine content ≥94.0%) was purchased from H. Holstein Co., Ltd. (Hamburg, Germany). LP was kindly donated by Kaneda Co., Ltd. (K-140N, Tokyo, Japan). IPM was purchased from NOF Co., Ltd. (Tokyo, Japan). White vaseline was purchased from KENEI Pharmaceutical Co., Ltd. (Osaka, Japan). Distilled water was purchased from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan). RI, GL and CF were purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). 1,1′-Dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI) was purchased from Sigma-Aldrich (St. Louis, MO, U.S.A.). Other reagents were reagent grade and obtained from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan).

Sample Preparation

The concentrations of lecithin, CF and DiI were fixed at 20, 0.1, and 0.1%, respectively. Table 1 shows the actual weighed quantities of the main formulas. The name of each formulation is a combination of the polar compound, its % concentration and oil, e.g., GL5.6LP. The preparation method followed the procedure used by Imai et al.⁴⁾ The specific method used in the present study is described below.

Table 1. Composition of Samples

	GL2.4LP	GL5.6IPM	RI3.0IPM
Lecithin	2.00 g	2.00 g	2.00 g
GL	0.24	0.56	—
RI	—	—	0.30
LP	7.74	—	—
IPM	—	7.42	7.68
CF	0.01	0.01	0.01
DiI	0.01	0.01	0.01

Composition of GL2.4LP, GL5.6IPM and RI3.0IPM correspond to a★, b★ and c★ in Fig. 1, respectively.

Lecithin/GL/LP systems: The required amounts of lecithin and CF were weighed in a vial to which was added 2 mL methanol to obtain a solution. The methanol was evaporated in a draft chamber for more than 12 h, followed by drying in a decompression desiccator for more than 48 h. The required amounts of GL and LP (containing DiI) were added to the residue in the vial, which was then capped tightly and mixed using a magnetic stirrer until the mixture was homogeneous (more than 12 h).

Lecithin/GL/IPM systems: The required amounts of lecithin, GL, CF and IPM (containing DiI) were weighed in a vial, which was then capped tightly and mixed using a magnetic stirrer for more than 12 h until the mixture was homogeneous.

Lecithin/RI/LP and lecithin/RI/IPM systems: The required amounts of lecithin, RI and CF were weighed in a vial and 2 mL of methanol was added to obtain a solution. The methanol was evaporated in a draft chamber for more than 12 h, then dried in a decompression desiccator for more than 48 h. The required amount of LP or IPM containing DiI was added to the residue in the vial, which was then capped tightly and mixed using a magnetic stirrer for more than 12 h until the mixture was homogeneous.

The samples were maintained at 25 °C for several days to allow equilibration.

As a control formulation (VA), 10 g of vaseline, 0.01 g of CF and 0.01 g of DiI were weighed in a glass beaker and stirred well using a glass rod for more than 5 min until the mixture was homogeneous.

Visual Observation and Phase Diagrams

CF and DiI were not added in the samples for the phase diagram study. Samples were observed visually in vials for their turbidity, fluidity, and birefringence at 25 °C. The results were used to construct phase diagrams. The solubility of CF in LRWs was observed using a polarizing microscope (ECLIPSE E600 POL; Nikon Co. Ltd., Tokyo, Japan).

Small-Angle X-Ray Scattering (SAXS) Analysis

SAXS measurements were conducted using the BL40B2 SAXS beamline (Structural Biology II Beamline) at SPring-8 (Hyogo, Japan) with a 2 m camera distance and a large area pixel detector (PILATUS-3S 2M; Dectris, Baden, Switzerland), as previously reported.¹³⁾ The wavelength of the beam was 0.1 nm and the exposure time was about 100 s. The samples were injected into a quartz capillary (ϕ2 mm). All measurements were performed at 25 °C. The cross-section radius of a rod-like particle can be calculated from cross-sectional plots of the SAXS profile.⁷⁾

Rheological Measurements

Dynamic rheological measurements were performed on a MCR302 modular compact rheometer (Anton Paar GmbH, Glaz, Austria) with a cone-plate geometry (diameter, 35 mm; cone angle, 1°). The temperature was maintained at 25 or 32 °C and a solvent trap was used to prevent solvent evaporation from the samples.

The linear viscoelastic region was obtained by strain sweep measurements under a constant frequency (1 Hz) prior to frequency sweep measurements. The frequency sweep measurement, angular frequency (ω) dependence of the storage modulus (G′) and loss modulus (G″) were then investigated using the strain in the linear viscoelastic region in the strain sweep measurements. Complex viscosity (η*) is the viscosity measured in an oscillatory experiment and is calculated using Eq. 1.

  

(1)

Skin Permeation Study

Frozen edible pig ear skin (KAC Co., Ltd., Kyoto, Japan) was used in the permeation experiments. Skin samples were thawed in lukewarm water and carefully washed with purified water. The outer surface of each pig ear was carefully shaved with an electric clipper and shaver, then the shaved skin was excised from the cartilage. Excess fat was carefully removed and the skin was placed on a Franz-type diffusion cell (effective diffusion area: 0.64 cm², Perme Gear, Inc., Hellertown, PA, U.S.A.) with a receptor volume of about 5 mL. Phosphate buffer solution (1/15 mol/L, pH 7.4, PBS) was added to the receptor cell and maintained at 32 °C and stirred using a magnetic stirrer during the study. Saline was added to the donor chamber, and after 20 min the skin impedance was measured using an impedance meter (AS-TZ1; Asahi Techno Lab Co. Ltd., Kanagawa, Japan). About 60 min after the application of saline, the saline was removed from the donor chamber and the skin surface was carefully wiped with a laboratory wipe (KimWipes; Nippon Paper Crecia Co., Ltd., Tokyo, Japan). Next, 0.2 mL samples containing CF and DiI were placed in the donor compartment on the skin, then 0.15 mL aliquots of the receptor fluid were collected at predetermined times and immediately replaced with fresh PBS. The collected PBS samples were filtered through a polytetrafluoroethylene (PTFE) filter (Millex-LCR, 0.45 mm; Merck Millipore Ltd., Burlington, MA, U.S.A.) and the concentration of CF in each collected PBS sample was determined using HPLC.

After withdrawing the 24 h sample of receptor fluid, the skin surface was wiped with a laboratory wipe and the skin was stripped twice using adhesive tape (Cellulose Tape; NICHIBAN Co., Ltd., Tokyo, Japan) to remove samples remaining on the skin surface. The treated area of the skin was punched out and the skin samples with high impedance were divided into two parts under a stereomicroscope. One half of each sample was used to quantify CF in the skin and the other half was used for cross-sectional observation. The skin sample used for quantification was cut into small pieces and CF in the skin was extracted with 0.5 mL of methanol by sonication for 30 min. The extract was filtered through a PTFE filter, the solvent from 150 µL of the filtrate was evaporated, then the residue was dissolved in 150 µL of PBS by sonication for 10 min. The solution was filtered through a PTFE filter and the CF concentration in the filtrate was determined by HPLC. The amount of CF in the skin per unit area (ng/cm²) was calculated using the effective diffusion area of the divided skin.

HPLC Conditions

CF concentration was determined using an HPLC system (Acquity UPLC H-Class; Waters Co., Ltd., Milford, MA, U.S.A.) equipped with a fluorescence detector (Acquity FLR; Waters Co., Ltd.). A 1 µL aliquot of sample was injected into the HPLC system equipped with an Aqcuity BEH C18 column (1.7 µm, 100 × 2.1 mm i.d.; Waters Co., Ltd.) maintained at 40 °C using 20 mM potassium dihydrogen phosphate aqueous solution–acetonitrile (60: 40, v/v) as the mobile phase at a flow rate of 0.2 mL/min. Eluting compounds were detected using an excitation wavelength of 492 nm and an emission wavelength of 512 nm.

Confocal Microscope Observation

Skin samples for cross-sectional observation were embedded in an embedding agent (Tissue-Tek O.C.T. compound; Sakura Finetek Japan Co., Ltd., Tokyo, Japan) and sliced into 20 µm thick slices using a Frozen tissue section preparation device (Tissue-Tek POLAR-DM; Sakura Finetek Japan Co., Ltd., Tokyo, Japan). Each sliced skin sample was observed using a confocal microscope system (A1 with LU-N4 lase unit; Nikon Co. Ltd., Tokyo, Japan) to evaluate the penetration of CF (excitation wavelength: 488 nm, detection wavelength range: 500–550 nm) and DiI (excitation wavelength: 561 nm, detection wavelength range: 570–620 nm).

Polypropylene (PP) Filter Permeation Study

A PP filter plate with a 70 µm pore size and a thickness of 2 mm (AS ONE Co., Ltd., Osaka, Japan) and filter paper (No.2; ADVANTEC Co., Ltd., Tokyo, Japan) were used in the permeation experiments. DiI-saturated IPM solution was used as a control.

A filter paper was placed on a glass plate, then a 2 × 2 cm piece of PP filter was placed on the filter paper. A Franz-type diffusion donor cell with a diffusional area of 0.28 cm² was placed on the PP filter and fastened with clips. A 0.2 mL aliquot of sample was placed in the donor cell on the PP filter. The filter paper was collected at predetermined times and placed in a glass vial, then an appropriate amount of methanol (5 to 12 mL) was added and the vial was sonicated for 30 min. The concentration of DiI in the methanol was determined using a fluorescence spectrophotometer (FP-8350; JASCO Co., Ltd., Tokyo, Japan) using an excitation wavelength of 549 nm and an emission wavelength of 562 nm.

Statistical Analysis

All data for the skin permeation studies are expressed as the mean ± standard deviation (S.D.). After ANOVA, differences between groups were tested using the Tukey–Kramer honestly significant difference test. A p-value less than 0.05 was considered statistically significant.

RESULTS AND DISCUSSION

Visual Observation and Phase Diagrams

Here, we used vaseline as a control vehicle and LRWs containing the polar compounds RI and GL as non-aqueous, oil-based, semi-solid vehicles. LRWs in n-decane and containing RI reportedly have higher viscosity and viscoelasticity than LRWs with GL.⁴⁾ LP and IPM were selected as oils for the LRWs because they are commonly used in external skin formulations. The concentration of lecithin was fixed at 20%.

The samples prepared were visually observed and phase diagrams were prepared. Figure 1 (a) shows a photograph of LRWs at 25 °C. Immediately after the LRWs are placed upside down, they are semi-solid and do not flow. However, when left to stand, they become viscous. These are typical viscoelastic characteristics of LRWs. Figure 1 (b) shows phase diagrams of lecithin/polar compounds/oil systems at 25 °C. Samples with high viscoelasticity and no birefringence are defined as LRWs in the phase diagrams. When LP was used as the oil, samples with a GL concentration between 1.0 to 2.4% and a RI concentration between 0.1 to 2.4% were in the LRW state. For both polar compounds (GL and RI), the LRWs had low fluidity and no remarkable difference was observed visually in the LRW formation range, although a small increase in fluidity was observed at a GL concentration of 2.4%. When IPM was used as the oil, samples with a GL concentration between 3.0 to 5.6% and a RI concentration between 2.0 to 5.2% were in the LRW state. For both polar compounds, the fluidity of the LRWs decreased as the amount of GL and RI increased in the LRW formation range, similar to that observed using n-decane as an oil.^8,10) The LRW formation using IPM as an oil exhibited a wider GL and RI concentration range and higher fluidity than samples using LP as an oil.

Fig. 1. Representative Photograph of LRW (a) and Phase Diagrams of Lecithin/Polar Compound/Oil Systems (b) at 25 °C

(a) Lecithin/RI3.0%/IPM system, (b) □: Not tested, : Turbid, ■: LRW, : Two-phase, : Solution. The lecithin concentration was fixed at 20%. a★, b★ and c★ correspond to GL2.4LP, GL5.6IPM and RI3.0IPM, respectively.

Having confirmed that LRWs can be prepared using any of the tested polar compounds and oils, LRWs containing 0.1% of CF (molecular weight (MW): 376, logarithm of partition coefficient (log P): −3.5) and DiI (MW: 843, log P: 23.2) were prepared. CF was used as a model for a hydrophilic drug and DiI was used as a tracer for oil. Hereinafter, the various LRWs used are denoted by the combination of the type and concentration of polar compound and the type of oil, i.e., GL1.2LP, GL2.4LP, GL3.4IPM, GL4.0IPM, GL5.6IPM, RI3.0IPM, RI4.0IPM and RI5.2IPM.

LRWs containing CF and DiI could be prepared using any of the tested polar compounds or oils, and there was little visible difference in fluidity between LRWs containing CF and DiI and those lacking CF and DiI. We predicted low skin permeation of CF from LRWs with very low fluidity and thus in subsequent studies we used LRWs comprising GL2.4LP, GL5.6IPM and RI3.0IPM, which have moderate and similar fluidities.

We confirmed the solubility of CF in the LRWs by observing GL2.4LP, GL5.6IPM, RI3.0IPM and VA under a polarizing microscope. CF crystals observed in VA were not observed in the LRWs, confirming that LRWs can solubilize CF in non-aqueous, oil-based, semi-solid vehicles. CF crystals did not appear in LRWs stored at 25 °C for more than a year (Fig. 2).

Fig. 2. Polarization Micrographs of Samples Stored at 25 °C for More than a Year

(a) VA, (b) GL2.4LP, (c) GL5.6IPM, (d) RI3.0IPM.

SAXS is useful for the structural analysis of micelle particles several nanometers to several tens of nanometers in diameter. The profile of the intermediate q region provides information on particle shape.¹⁵⁾ Figure 3 shows the SAXS profile for RI3.0IPM as an example. The slope of the intermediate q region (q = 0.07 to 0.8 nm⁻¹) of −1 is indicative of cylindrical particles (i.e., the presence of reverse wormlike micelles), supporting the formation of LRWs. The cross-section radii of the LRWs obtained in this study were all less than 2.0 nm, which is comparable to those of other LRW systems.^7,9)

Fig. 3. SAXS Profile (Scattering Intensity versus the Wave Vector q) for RI3.0IPM at 25 °C

The area between the dotted lines indicates the intermediate q region.

Rheological Measurements

Rheological properties were measured at two temperatures: 25 °C to characterize the formulation during storage and removal from a container, and 32 °C to clarify properties after application to the skin.

Figure 4 (a) shows the relationship between the complex viscosity (η*) and the angular frequency of VA and LRWs at 25 °C. The η* of VA decreased with increasing angular frequency, which is characteristic of a non-Newtonian fluid. In contrast, the η* of LRWs remained constant up to a certain angular frequency, characteristic of a Newtonian fluid, then decreased at higher angular frequency, characteristic of a non-Newtonian fluid. In general, LRWs show a decrease in η* in the high angular frequency region, called “shear thinning,”^1,10,16) confirming that GL2.4LP, GL5.6IPM and RI3.0IPM were LRWs. The constant values of η* for GL2.4LP, GL5.6IPM and RI3.0IPM were approximately 4, 3 and 36 Pa·s, respectively.

Fig. 4. Frequency Sweep Measurements at 25 and 32 °C

(a), (c) Complex-viscosity (η*) vs. angular frequency curves. ●: VA, ◇: GL2.4LP, □: GL5.6IPM, △: RI3.0IPM. (b), (d) Variation in G′ and G″ as a function of angular frequency (ω). ●G′, ○G″: VA, ◆G′, ◇G″: GL2.4LP, □G′, □G″: GL5.6IPM, ▲G′, △G″: RI3.0IPM.

Figure 4 (b) shows the results of frequency sweep measurements, G′ and G″, for samples at 25 °C. The elastic component G′ of VA was dominant throughout the measurement range. In contrast, for GL2.4IPM and GL5.6IPM, the viscous component G″ was dominant throughout the measurement range. For RI3.0IPM, G′ and G″ intersected near 10 rad/s, and G″ was dominant on the low frequency side of the intersection. This behavior is observed in the Maxwell model, a basic model of viscoelastic bodies, and is characteristic of LRWs.

At 32 °C, a decrease in η* was observed with increasing temperature especially in VA and RI3.0IPM, whereas the relationship between η* and the angular frequency showed the same trend to that at 25 °C (Fig. 4 (c)). It has been reported that the viscosity of LRWs decreases as the temperature increases due to a decrease in the hydrogen bonding of the polar sites of lecithin.¹⁷⁾ The results of frequency sweep measurements show that G’ was dominant in VA, whereas in all LRWs including RI3.0IPM, G″ was dominant at 32 °C (Fig. 4 (d)).

LRWs show moderate viscosity at 25 °C, and their viscosity decreases at 32 °C, the temperature to which the product increases when applied on the skin. Consequently, LRWs are semi-solid at 25 °C, facilitating removal from a container, and liquid at 32 °C, likely enabling penetration of fine skin structures (e.g., wrinkles and hair follicles), and may be useful for transdermal applications.

Skin Permeation Study

Figure 5 shows the time course of the cumulative amount of CF that permeated through the pig ear skin samples. The permeation of hydrophilic drugs through skin with low impedance is high and thus the data with skin impedance greater than 10 kΩ·cm² were used.¹⁸⁾ The cumulative amount of CF at 24 h after the application of VA was 17.2 ± 11.3 ng/cm² whereas for GL2.4LP and RI3.0IPM, they were 3.97 ± 0.80 and 32.0 ± 23.4 ng/cm², respectively, which is not significantly different from VA. In contrast, the application of GL5.6IPM resulted in significantly higher permeation of CF than the other formulations (131 ± 130 ng/cm² at 24 h after application).

Fig. 5. Skin Permeation Profiles of CF from Samples at 32 °C

●: VA, ◇: GL2.4LP, □: GL5.6IPM, △: RI3.0IPM. Each point represents the mean ± S.D. (n = 6–8). a) Significant difference relative to VA, GL2.4LP and RI3.0IPM (p < 0.05). b) Significant difference relative to VA and GL2.4LP (p < 0.05).

Figure 6 (a) shows the amount of CF in the skin at 24 h after application. The amount of CF after the application of VA was 22.9 ± 15.7 ng/cm² and was similarly low for GL2.4LP. In contrast, when GL5.6IPM was applied, the amount of CF in the skin was significantly higher than those of VA and GL2.4LP. The use of RI3.0IPM resulted in a significantly higher amount of CF in the skin compared to the use of GL5.6IPM, in contrast to the trend from the skin permeation profiles, where GL5.6IPM was the highest.

Fig. 6. Amount of CF Penetration at 24 h after Application of the Dosage

(a) Amount in the skin. (b) Total penetrated amount of CF in the skin and receptor fluid. Each point represents the mean ± S.D. (n = 6–8). a) Significant difference relative to VA, GL2.4LP and GL5.6IPM (p < 0.05). b) Significant difference relative to VA and GL2.4LP (p < 0.05).

Figure 6 (b) shows the sum of CF in the skin and in the receptor fluid at 24 h after application. The sum of CF in the skin and in the receptor fluid after the application of VA or GL2.4LP was similar, about 40 ng/cm², whereas those after the application of GL5.6IPM and RI3.0IPM were similar and significantly higher by comparison.

Skin permeation through the stratum corneum is described as passive diffusion. Flux through the skin is correlated with the partition coefficient and the diffusion coefficient. A higher skin concentration indicates higher partition, so flux should be higher if the diffusion rate remains constant. In this case, the skin concentration of CF after application of RI3.0IPM was higher than that of GL5.6IPM. These results suggest the involvement of mechanisms other than partition and passive diffusion through the stratum corneum. Skin permeation occurs not only through the stratum corneum, but also through appendages such as hair follicles.^19,20) We confirmed the route of permeation by observing skin cross-sections at 24 h after application.

Figure 7 shows representative images of vertically cross-sectioned skin at 24 h after application. DiI has a relatively high molecular weight and is a highly lipophilic fluorescent dye (MW: 843, log P: 23.2), and it only penetrates skin together with oil. Thus, DiI was used as a tracer of oils.²¹⁾ After the application of VA, weak fluorescence from CF (green) and DiI (red) was observed in some parts of the stratum corneum, indicating low penetration of CF into the skin. For GL2.4LP, CF and DiI fluorescence was observed in the stratum corneum, indicating that CF penetrated the stratum corneum together with LP. The application of GL5.6IPM and RI3.0IPM resulted in strong CF and DiI fluorescence in the stratum corneum, and CF was observed in the epidermis and dermis. We conclude that CF penetrated the stratum corneum with IPM (yellow in the merged images), then penetrated the epidermis and dermis by partition and diffusion.

Fig. 7. Cross-Section of the Skin Samples, Observed by Confocal Microscopy at 24 h after Application

Green indicates the distribution of fluorescence from CF. Red indicates the distribution of fluorescence from DiI. HF: hair follicle, SC: stratum corneum.

Focusing on the hair follicles, little fluorescence was observed in VA, whereas fluorescence was observed in LRWs, although its intensity varied. In the case of GL2.4LP, only weak fluorescence of DiI was observed. In the cases of GL5.6IPM and RI3.0IPM, CF and DiI were observed in a hair follicle, indicating that LRWs themselves may penetrate relatively deeply. CF penetrated the skin both through stratum corneum and hair follicle routes after application of GL5.6IPM and RI3.0IPM.

Figure 8 shows the relationship between skin impedance before the permeation study and the amount of CF permeated in 24 h. This figure shows data for all the samples, including samples not used in the analysis because of their low skin impedance (less than 10 kΩ·cm²). Using VA and GL2.4LP, only a small amount of CF permeated regardless of skin impedance, while for LRWs using IPM as an oil (GL5.6IPM and RI3.0IPM), high CF permeation was observed in skin whose impedance was less than 10 kΩ·cm². A similar relationship was observed for the amount of CF in the skin samples.

Fig. 8. Relationship between Skin Impedance before the Permeation Study and the Amount of CF Permeated in 24 h

●: VA, ◇: GL2.4LP, □: GL5.6IPM, △: RI3.0IPM.

The above relationship and cross-sectional observations of the skin samples indicated that CF penetration through the hair follicle was promoted in the cases of GL5.6IPM and RI3.0IPM. DiI was observed in the hair follicle, so the vehicle itself penetrated the hair follicle. The penetration of substances into capillaries is affected by the viscosity of the substance, the diameter of the capillary, and the wettability between the substance and the capillary. A PP filter with a pore size of 70 µm was selected as a model membrane because the diameter of hair follicles at the skin surface of pig ear is reportedly 113 ± 43 µm, similar to that of human skin.²²⁾ In addition, the surface free energy and its dispersion components of PP which affect the wettability or adhesion of a formulation to a skin surface,²³⁾ are similar to that of human and pig skin.²⁴⁾ The permeation of DiI was used as a tracer of vehicle permeation through PP filter.

Figure 9 shows the time course of the amount of permeated DiI at 25 °C. IPM solution was used as a control, DiI permeated quickly, with approximately 90% permeated within 2 min (data not shown). No DiI permeated in the case of VA. VA has elastic properties and did not penetrate capillaries of the PP filter. LRWs prepared using GL as a polar compound (GL2.4LP and GL5.6IPM) resulted in about 16% of the DiI permeating at 20 min after application of the formulation, which is higher than that of RI3.0IPM (about 7% of the dose). The order of PP filter permeability of DiI at 25 °C was RI3.0IPM < GL2.4LP ≈ GL5.6IPM, which was the same order as that of LRWs’ η*. LRWs have viscoelastic or viscous properties and penetrated capillaries of the PP filter at a rate dependent on the rheological properties of LRWs. The η* at 32 °C of LRWs are similar, so the PP filter permeation at 32 °C should be similar for all LRWs. Based on this, all LRWs should exhibit similar hair follicle penetration, but this is not consistent with the permeation profile or the amount in the skin observed in the present study.

Fig. 9. PP Filter Permeation Profiles of DiI from Samples at 25 °C

●: VA, ◇: GL2.4LP, □: GL5.6IPM, △: RI3.0IPM. Each point represents the mean ± S.D. (n = 3–6).

Low penetration of CF from GL2.4LP cannot explain the result of PP filter permeation. LP and IPM may have different actions on skin. Sebum is secreted in hair follicles and the interaction between sebum and the transdermal dosage affects the hair follicle permeability of drugs.²⁵⁾ Various vehicles affect the phase transition of sebum. IPM has a large effect on the phase transition of sebum,²⁶⁾ and thus LRWs incorporating IPM may interact highly with sebum. Differences in the interaction between LP and IPM in LRWs and sebum may affect the hair follicle penetration of samples. LRWs using IPM as an oil may penetrate hair follicles due to their rheological properties and the interaction between IPM and sebum.

In comparisons of GL5.6IPM and RI3.0IPM, the total amount in skin and permeated was similar (Fig. 6 (b)), and was consistent with the PP filter permeation expected at 32 °C. However, these results cannot explain why GL5.6IPM enhances CF permeation through the skin, and why RI3.0IPM shows high CF concentration in the skin (Figs. 5, 6 (a)). Factors not identified in the present study, such as those affecting contribution rates via the stratum corneum route and the follicular route, may be involved, and further study is necessary.

CONCLUSION

We attempted to develop a semisolid oily vehicle for drugs that are unstable in water to support the high skin permeation of such drugs. LRWs were prepared using GL and RI as polar compounds instead of water, LP and IPM as oils, CF as a model of a hydrophilic drug, and DiI as a tracer for oils. The LRWs solubilized CF and were stable for over a year. Skin permeation experiments demonstrated that LRWs using IPM as an oil showed higher CF permeability than VA and LRWs formed using LP, suggesting that permeation occurs through both the stratum corneum and hair follicle routes. The penetration of CF into hair follicles may be affected not only by the rheology of the dosage form but also by the interaction between IPM and the sebum in hair follicles.

LRWs incorporating IPM may be a good oil-based vehicle for hydrophilic drugs and support good drug permeation into the skin.

Acknowledgments

Thanks are due to Mr. Kankei Nakaharu for his technical assistance with the experimental work. This work was supported by JSPS KAKENHI Grant No. JP22K06566. The synchrotron radiation experiments were performed at the BL40B2 of SPring-8 with the approval of the Japan Synchrotron Radiation Research Institute (JASRI) Proposal No. 2022A1143. We thank Kaneda Co., Ltd. for providing the LP sample.

Conflict of Interest

The authors declare no conflict of interest.

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