Ceramide

The aim of using ceramides or pseudoceramides in a cosmetic formulation is to improve the barrier function, i.e., the maintenance of the lipid layer in stratum corneum. The reason why the pseudoceramides improves barrier function in the skin is mainly due to its physical characteristics to form the multi-lamellar structure with other components in the emulsion. In the phase study of pseudoceramides / fatty acid / cholesterol complex system, their ability to form lamellar structure was determined. Finally, it was demonstrated that pseudoceramides had the high ability to form the lamellar structure with emulsifier and with or without oils. When it was observed under the cross-polarized microscope, the optical anisotropy of Maltese cross, a typical configuration of multi-lamellar mesophase texture was shown. And it was demonstrated that this aggregation structure in the emulsion was similar to the intercellular lipid bilayer in the skin.

pseudoceramides with Oil

pseudoceramides without Oil

The cross-polarized light microscopic photography of a pseudo-Stratum Corneum emulsion (x 400)

Pseudoceramides, An Oil Core with Multi-Lamellar Liquid Crystal Layers

When the pseudoceramides with oils was squeezed by force, the emulsion droplet was destroyed and the oil components at the core of the droplet leaked out into the water phase. From this result, we can deduct that the 3D structure of the pseudoceramides are "an oil core with multi-lamellar liquid crystal layers".

The squeezing demonstration of pseudoceramides

Pseudoceramides Formula Design
The next table represents the comparison of natural skin system & pseudoceramides compositions. Because the structure and composition of pseudoceramides are very similar to those of natural skin system, the pseudoceramides can contribute to the barrier recovery of the damaged skin, which suggests the potential of clinical use of pseudoceramides to the chronic skin diseases originated from barrier abnormality, such as atopic dermatitis, dry skin, psoriasis, etc.

STABILITY TEST The stability test of the pseudoceramides was performed for four weeks within a cyclic temperature incubator (C/C, -20), in room temperature, and at 45. , There was no case of destruction or phase separation of the pseudoceramides, which demonstrate that the pseudoceramides was very stable.

After a day	After 4 weeks at RT
After 4 weeks at C/C	After 4 weeks at 45

Application of Pseudoceramides into Drug Delivery System (DDS) The most common formula for DDS was liposome. However, due to its weak stability, it is well known that the liposome has the limitation on the wide application on DDS system.

Pseudoceramides can be used to stabilize the active lypophilic materials within its oil core. The next figure shows that the pseudoceramides is very good for the stabilization of retinol palmitate, which is known as an effective material for the anti-aging & wrinkle of the skin

1) Target : How to Stabilize Retinols ? 2) Solution : Retinols in MLE Stabilize it in the oil pool of MLE

Effectiveness of Pseudoceramides in an In vivo System: Barrier Function Recovery We first assessed whether or not the pseudoceramides has effects on the damaged skin by measuring the TEWL changes in the acute disruption model in comparison with placebo and air exposed non-treatment skin of the hairless mice. The pseudoceramides and placebo have nearly the same compositions except pseudo-ceramide (pseudoceramides) and were prepared with the same method. The figure below shows main components of the pseudoceramides and the placebo and the optical texture of the emulsions, which were prepared by the method reported by our R&D group previously. 17, 18

(A) Pseudoceramides	(B) Pseudoceramides -2
Cross-polarized light microscopic photography of MLE cream (A) and the placebo system (B) (X 400)

Next, we used the acute barrier disruption model described earlier with tape-stripped hairless mice to test the effect of pseudoceramides on barrier recovery. We measured the TEWL changes in pseudoceramides -treated mouse skin and compared them with both placebo-treated skin and air-exposed, non-treated skin. In a comparison test of pseudoceramides -treated and non-treated (NT) skin, pseudoceramides -treated skin appeared to show a faster barrier-function repair rate compared with the air-exposed NT skin. The same is true of the comparison of the pseudoceramides -treated skin and the placebo-treated skin. We concluded only that the lamellar structure increase the intrinsic barrier function recovery.

( A )	( B )
The effect of pseudoceramides on barrier recovery, comparing with air exposed non-treated (N.T) skin (A : p>0.05) and the placebo system (B : p>0.05). The p values were calculated by Student’s t-test.

Effectiveness of pseudoceramides in an in vivo system: Histopathological studies Comparison of Epidermal Cell Thickness It has previously been reported that an occlusive moisturizer, such as vaseline, could develop a non-permeable barrier on damaged skin19, 20 and that it is effective in improving the water-holding capacity.21 However, it has also been reported that the occlusion with water-impermeable membranes, which prevents water-loss, could not prevent the epidermal hyperplasia.22 The degree of epidermal hyperplasia is correlated with the level and duration of barrier disruption and likewise, the epidermal mitotic index increases with repeated disruption, indicating that the hyperplasia could be ascribed to increased cell proliferation. Thus, the damaged skin19 could be better treated when the TEWL value decreases more rapidly and the epidermal cell thickness increases less rapidly. In the acute disruption model, the epidermal cell thickness changes after 24 hours and 48 hours from barrier function disruption, and the pseudoceramides treated skin showed the lowest increase in epidermal cell thickness and the air exposed non-treated skin showed the highest.

( A )	( B )
(A) The effect of pseudoceramides on the epidermal cell thickness 48 hours after barrier disruption; The relative thicknesses are calculated on the basis of pseudoceramides treated skin thickness. The p values are calculated by Student’s t-test. (B) Skin samples were stained with hematoxylin and eosin In the biopsy samples in the chronic disruption model, the pseudoceramides treated skin showed the lowest increase in epidermal cell thickness and the air exposed non-treated skin showed the highest (data not shown). To verify these changes, morphological changes of the SC layers in the chronic disruption model were examined using the OsO4 staining method with an electron microscope, and its results are shown in the figure below, where the pseudoceramides treated skin had the fewest numbers of SC layers, and the air exposed non-treated skin had the most.

The electron microscopic view of (A) pseudoceramides, (B) placebo treated and (C) air exposed non-treated skin (X 5,000). The samples were stained by the OsO4 staining.Finally, we examined the structure of the lamellar multi-layers stained by the RuO4 with an electron microscope. The result informed us that the structure played an important role to the barrier function. In the below figure, the pseudoceramides treated skin showed an almost normal intercorneocyte lipid multi-layer structure, whereas placebo treated skin and non-treated skin showed disordered intercorneocyte lipid structures. From these, it can be concluded that the pseudoceramides must have a positive effect on the skin barrier in the