Design of a mimetic stratum corneum

This natural liquid crystalline structure has a bi-continuous structure of water with amphiphilic lipids and it simultaneously possesses important moisturising and water retention effects.3 In the past years, there has been a great deal of interest in emulsion preparations using the self-organisation of structures formed in mixed systems of surfactants and amphiphilic lipids. These include fatty alcohol, fatty acid and lecithin, ranging from very fluid hardsphere vesicles or microemulsions to very viscous gel-like liquid crystals.4,5 Figure 1 shows an example of a schematic skin model of oil droplets surrounded by LC forming LCE. It was scientifically reported that by using this kind of system a greater moisturising effect and an improvement in the barrier function of the stratum corneum could be achieved. There are, however, only few successful results due to the poor stability of the LC structure in actual application on human skin. In other words, LCs surrounding the oil droplet vanish quickly at skin temperature.

The aim of this study is to optimise preparations of LCEs, stable even at above skin temperature, and to confirm the efficacy and functions of emulsions which mimic the structure of intercellular lipids. In this study, hydrogenated lecithin (HL) is used to form LCs. PSE and PS can be expected to improve the flexibility of a lamellar layer structure and stabilise the LC form due to their flat molecular structures.

Materials and methods

Materials
PS, PSE and HL for a preparation of the liquid crystalline base (LCB) were supplied by Nikko Chemicals, Japan. Oils (squalane, caprylic/capric triglyceride, etc) and waxes (fatty alcohol, etc) were also obtained from Nikko Chemicals, Japan. All materials in the experiments were used without further purification.

Preparation of the liquid crystalline base (LCB)
LCB was prepared in a wax form by mixing HL, PSE, PS and fatty alcohols (C16-C22) above their melting points and then cooling down to room temperature.

Preparation of the LCEs and O/W emulsions (O/W-Es)
LCEs were prepared with LCB, oils, polyols and water by a high speed homogenising emulsification at 70±5°C using a T K Homomixer (Tokushu Kika Kogyo, Japan). The LCB was firstly added to an oil phase and agitated to dissolve. Then this mixture of LCB with oils was added into a water phase to obtain LCEs. Meanwhile, O/W-Es were prepared as the controls, using polysorbate-60 as an emulsifier following the same procedure.

Confirmation of the liquid crystalline structure
The structure of LCEs was confirmed by two kinds of microscopic observations: polarised light microscopic observation (BX50 – Olympus, Japan) attached to a CCD digital camera FX380; and a TEM (JEM1200EX – JEOL, Japan) analysis.6 The structural stability was also confirmed at 25, 37 and 40°C and on a human skin (human volunteer tests).

Measurement of bound water content
The bound water content in LCEs or O/W-Es was measured by DSC analysis (DSC meter 220C – Seiko instruments, Japan).7

Human volunteer evaluations

Moisturising effect

2.5mg/cm2 of LC/W-Es or O/W-Es was applied respectively on the inside forearms of six male and female volunteers (25-45 years old) and the volunteers were kept for 15 minutes in an incubation room (22°C/45% RH). The conductance (µS) of the applied and non-applied areas was measured at 22°C/45%RH by SKICON 200 (IBS). The moisturising effect was measured 60 minutes after application. The moisturising effect was evaluated by comparing the changes of the conductance between initial level and each interval.8

Trans epidermal water loss reductive effect

LCEs and O/W-Es were applied twice a day on the inner thighs of 12 male volunteers (28-55 years old) respectively for 21 days and the TEWL of the area measured. The measurement interval was three weeks after application. The TEWL was evaluated using the AS-TW2 (Asahi Biomed, IBS) in the incubation room at 21°C and 50% relative humidity (RH).9

The condition of the skin on the inner thighs before and after application was evaluated by tape stripping method. A few layers of stratum corneum were stripped off using cellophane tapes, and the skin condition observed by the BG dye method.10

Results and discussion

Confirmation of the LC structure
Polarised light microscopic observation Test samples of LCEs or O/W-Es were observed to confirm the existence of the LC structure by a polarised light microscopy.

As illustrated in Figure 2, the LCEs clearly show the Maltese cross image of emulsion droplets (Fig. 2C and 2D). However, the O/W-Es show no Maltese cross images (Fig. 2A and 2B). Therefore, LCEs based on the LCB were confirmed to form LC structures, which might be identified as a lamellar form. On the other hand, the O/W-Es prepared with polysorbate 60, instead of the LCB, did not form a LC structure.

Transmittance electron microscopic (TEM) analysis

The structure of the LCs surrounding the oil droplet of LCEs (Fig. 2C) was observed by a TEM. As shown in Figure 3, the existence of multi-lamellar structures around the oil droplets was confirmed (Fig. 3A) and this multi-lamellar structure is similar to intercellular lipid layers found in the stratum corneum (Fig. 3B).11 This result can be explained by the equation (1) known as critical packing parameter (CPP).12

Critical packing parameter (CPP)
= v/(ao lc) ——————————— (1)
ao: the optimal surface area
v: hydrocarbon chain volume
lc: critical length of lipids

The CPP determines whether lipids will form spherical micelles (CPP<1/3), nonspherical micelles (1/3<CPP<1/2), bilayers (vesicles) (1/2<CPP<1) or lamellar (CPP~1). Here, HL itself is in the range of 1/2<CPP<1 which is not inclined to form lamellar structures. PS itself has CPP>1. By mixing HL with PS, the CPP of the mixture will be close to 1, which is favourable for lamellar formation.

Phase behaviour study

The schematic phase diagram of LCB/squalane/water system in the whole concentration range at 25°C is illustrated in Figure 4. As shown in the line of water/LCB (Fig. 4), a lamellar LC phase was formed at 3-30wt% of LCB and this LC phase was confirmed to change to a hexagonal phase with an increasing concentration of the LCB up to 70wt% of LCB. Above 71wt% of LCB, a reverse micellar phase having gel formation appeared. Therefore it was suggested that LCEs based on LCB (HL mixed with PS) could form sufficiently stable lamellar structures even at a low LCB concentration.

Stability of the liquid crystalline structure

Stability against temperature

LCE stability (Fig. 2C) at different temperatures is illustrated in Figure 5. Each photograph shows the change of the LC form at 25, 37 and 40°C. The LCEs were confirmed to maintain the LC form at between 37-40°C (higher than actual skin temperature). Therefore, the LCEs can be expected to keep their LC form during practical application on the skin.

Stability against skin temperature

As shown in Figure 6, LCEs recovered (Fig. 2C) from the inside forearm of a volunteer could still demonstrate Maltese cross images after six hours. This suggests that the LC form is stable at skin temperature. It is expected that the LCEs may act as an artificial stratum corneum lipid.

Bound water content

Generally, water in intercellular lipids, (bound water) exists in a different form from free water, which easily evaporates from the skin’s surface. Bound water in intercellular lipids is retained tightly in the lipids’ structure and protects the skin against drying. The bound water content in LCEs was quantified and compared with those in O/W-Es by measuring the melting enthalpy of the test samples. Here, the total content of water (bound water + free water) in the test samples was quantified by the Karl Fisher method. The result is shown in Figure 7. In the LCEs the bound water was 12.7 %, and in the O/W-Es, the bound water was 8.3%. LCEs therefore are estimated to have almost a 53% higher bound water content. Consequently, LCEs can be said to have a much more effective moisturising effect than conventional emulsion of O/W-Es.

Human volunteer evaluation

Moisturising effect

The moisturising activity of LCEs (Fig. 2C) was evaluated and compared with O/W-Es (Fig. 2A) by measuring skin conductance of volunteers.

Figure 8 shows the changes of skin conductance as a function of time. After one hour, LCEs conductance was 100.0 µS±9.6. In the case of O/W-Es, it was 61.2µS±8.2. The student t-test confirms that LCE conductance is significantly higher than that of O/W-Es (*p<0.05). Hence, LCEs are considered to act much more effectively as a moisturiser than conventional O/W-Es.

Reducing ability of transepidermal water loss (TEWL)

TEWL was measured at the point when LCEs (Fig. 2D) and O/W-Es (Fig. 2B) were separately applied on the skin (Fig. 9). After three weeks of application (twice a day), the TEWL of LCEs was significantly less than the TEWL of O/W-Es.

Improvement of skin condition

Skin conditions after application were observed by the BG dye method, and the results depicted in Figure 10. As shown in Fig. 10 (before application, left), the size of corneocytes was not uniform prior to the application of O/W-Es. After the four weeks of applying LCEs, corneocytes tended to be a uniform size. However, there was no change on the size of corneocytes in the four weeks following O/WE application (Fig. 10). This indicates that it is possible to improve skin turnover by using LCEs.

Conclusion

This study was conducted to find an optimum way to enhance the skin barrier functions of cosmetic formulations. This study showed that skin barrier functions of cosmetic formulations can be improved by focusing on the relationship between the structure and the functions of emulsion films without any special active ingredients. Utilising hydrogenated lecithin, phytosterol emulsifier (PSE), phytosterol (PS) and fatty alcohols, it is possible to produce an advanced liquid crystalline base (LCB). The LCB is an amphiphilic substance complex, which can be easily made into a liquid crystal emulsion (LCE). This structure was confirmed as a multi-layer liquid crystalline form surrounding oil droplets. In addition, LCEs were confirmed to be stable when existing as a multi-layer liquid crystalline structure on skin surface in actual applications. LCEs possess both high moisturising and water retaining effects. In clinical tests, the skin moisture condition of the volunteers was improved and TEWL was simultaneously reduced. Therefore, this study can contribute to the development of new tools for formulating advanced skin care cosmetics.

References

1 S. Nishiyama, H. Komatsu, and M. Tanaka: A study on skin hydration with cream. J. of Soc. Cosmet. Chem. Japan 16(2): 136-143, 1983.

2 L. Norlen, Skin barrier Formation: The membrane folding model. The J. of Investigative Dermatology 117(4): 823-829, 2001.

3 T. Okamoto, Y. Matsushita, E. Matsuura, M. Masuda: The preparation of visible emulsion and its applications to cosmetics. J. of Soc. Cosmet. Chem. Japan 39(4): 290-297, 1983.

4 T. Suzuki, J.Fukasawa, H. Iwai: Multi-lamellar emulsion of stratum corneum lipid. J. of Soc. Cosmet. Chem. Japan 27(3): 193-205, 1993.

5 Y. Aoki and Y. Sumida: Enhancement of moisturising abilities of skincare products by a novel water retaining system. 22nd IFSCC congress, Edinburgh, Podium 38: 1- 8, 2002.

6 Inoue T, Tsujii K, Okamoto K, Toda K: Differential scanning calorimetric studies on the melting behavior of water in stratum corneum. J. Invest Dermatol 86: 689-693, 1986.

7 R. S. Summers, B. Summers, P. Chandar, C. Feinberg, R. Gursky, and A. V. Rawlings: The effect of lipids with and without humectant on skin xerosis. J. Soc. Cosmet. Chem 47: 27-39, 1996.

8 I. Y. Kim, C.K. Zhoh, and H.C. Ryoo: Liquid crystalline technology of cosmetic industry and moisturising effect of skin. J. of the society of cosmetic scientists of Korea 30(2): 279-294, 2004.

9 J. Levin and H.I Maibach: Correlation transepidermal water loss and percutaneous absorption. Cosmetics and Toiletries 120(7): 28-31, 2005.

10 R. Shukla, V. Bansal, M. Chaudhary, A. Basu, R. R. Bhonde, M. Sastry: Biocompatibility of gold nanoparticles and their endocytotic fate inside the cellular compartment. Langmuir 21: 10644-10654, 2005.

11 T. Suzuki, J. I. Fukasawa, H. Iwai, I. Sugai: Multilamellar emulsion of stratum corneum lipid. 16th IFSCC Yokohama, 3-28, 1994.

12 K.Kratzat and H. Finkelmann, Liq. Cryst.,

13: 691-699, 1993.

ABSTRACT
The assembly behaviour of hydrogenated lecithin (HL) and phytosterol (PS) with phytosterol emulsifier (PSE) in water was investigated and it was discovered that at optimum condition, their mixture forms a lamellar crystalline (LC) structure. This LC structure has been confirmed by transmittance electron microscopy (TEM) to be similar to the structure of intercellular lipids in the stratum corneum. Therefore, stable oil-in-water emulsions, which have the structure of LC, were prepared successfully by surrounding the oil droplets with this kind of LC structure. The efficacy and functions of LC emulsion(s) (LCE) were studied in vitro and in vivo. In comparison with conventional emulsions without an LC structure, the LCE showed a bound water content of 53% higher by DSC measurement. Furthermore, some trans epidermal water loss (TEWL) reduction increased by 2.5 times and the skin condition was predominantly improved after a four-week application on human volunteers. It is suggested that an LC structure formed by an optimised mixture of HL, phytosterol and PSE not only produces stable LCEs but also moisturises the skin and improves skin barrier functions. Keywords: Barrier function, liquid crystalline, multi-layer, bound water, moisturising effect.