Biomimicking of intercellular lamellar phase using new types of ceramides, 1-o-stearoyl ceramide NP and ceramide EOP with ultra-long chain (C24-C32)
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Presented by: Boroung Lee
Introduction
Stratum corneum (SC), the outer layer of skin barrier, plays an important role in preventing epidermal water loss as well as protecting the skin against pathogens. It has been well known that the ceramide plays the most critical role in determining the skin barrier function in the intercellular lipid phase of SC. In order to strengthen the skin barrier function to a level similar to that of the skin, new types of ceramides are being developed. A good example can be found in 1-o-stearoyl ceramide NP (1OS Cer), containing acyl chains in both N- and 1-O-position of the sphingoid base component, and ceramide EOP with ultra-long chain (C24-C32) (ULC Cer). A systematic study on what functions 1OS Cer and ULC Cer perform in the intercellular lamellar layer and how it affects the skin barrier function is needed. If these studies are carried out successfully, we could achieve great technological advances in the field of skin care.
Methods
A 2-D ceramide-based lipid membrane system was constructed by using an equivalent molar ratio of ceramide, stearic acid and cholesterol. The ceramide composition contained 100 wt% of ceramide NP (Cer NP) or a ceramide mixture (Cer NP and 1OS Cer or ULC Cer). The ceramide mixture was formulated in the weight fraction of 0.8-0.95 of Cer NP and 0.05-0.2 of 1OS Cer or ULC Cer with stearic acid and cholesterol. Then, the ceramide-based lipid mixture was dissolved with 1 mg/ml concentration in chloroform/methanol (2:1, v/v). The π-A isotherm of 2-D lipid membranes was obtained by using the Langmuir-Blodgett trough (KSV-NIMA, KN-2002). For this, we spread the lipid solution at the air-water interface on a Teflon trough subphase. The solvents were evaporated for 10 min. The lipid monolayer was compressed at 10 mm/min at 26 ℃ by closing and opening the barriers. The surface pressure was measured by using a Wilhelmy plate at least in triplicate. The lipid membrane at the air-water interface was compressed to 40 mN/m of surface pressure and then transferred onto a mica substrate (20 x 20 mm2, agar scientific, AGG250-2) by raising the mica support vertically through the air-water interface at 1 mm/min.
Results
The π-A isotherm of ceramide-based lipid membranes revealed that the mean value of a molecular area was expanded in the presence of 1OS Cer. By contrast, the mean value of a molecular area was rather compressed as the content of ULC Cer increased. This implies that 1OS Cer disturbed the molecular association while forming the lipid membrane, whereas ULC Cer led to close packing of lipid molecules in the membrane. We observed that ULC Cer causes the phase transition from liquid to solid to occur at a surface pressure as low as 15 mN/m. It is very interesting to compare it with 1OS Cer, which induces a phase transition at a relatively high surface pressure of 25 mN/m. From topology and surface roughness observations of lipid monolayer films through AFM and contact angle analyses, we could figure out that formulation of Cer NP and 1OS Cer made the membrane rough and hydrophobic, which was quite comparable to the case with ULC Cer or 100 wt% of Cer NP.
Discussion and conclusion
In this study, we propose a new ceramide-based lipid membrane system in which novel ceramides, 1OS Cer and ULC Cer, were associated with Cer NP, cholesterol and stearic acid. The asymmetric alkyl chains of 1OS Cer enabled occupation more area per one molecule during membrane formation, thereby impeding tight molecular association of the lipid membrane. While ULC Cer led to formation of a closely packed lipid membrane due to their strong hydrophobic interaction. Furthermore, the incorporation of 1OS Cer slowed down the phase transition compared with the case of ULC Cer in same surface pressure. These results highlight that the new ceramides, 1OS Cer and ULC Cer directly affect the formation of the lipid lamellar phase, which would be deeply involved in the regulation of skin barrier function.
Stratum corneum (SC), the outer layer of skin barrier, plays an important role in preventing epidermal water loss as well as protecting the skin against pathogens. It has been well known that the ceramide plays the most critical role in determining the skin barrier function in the intercellular lipid phase of SC. In order to strengthen the skin barrier function to a level similar to that of the skin, new types of ceramides are being developed. A good example can be found in 1-o-stearoyl ceramide NP (1OS Cer), containing acyl chains in both N- and 1-O-position of the sphingoid base component, and ceramide EOP with ultra-long chain (C24-C32) (ULC Cer). A systematic study on what functions 1OS Cer and ULC Cer perform in the intercellular lamellar layer and how it affects the skin barrier function is needed. If these studies are carried out successfully, we could achieve great technological advances in the field of skin care.
Methods
A 2-D ceramide-based lipid membrane system was constructed by using an equivalent molar ratio of ceramide, stearic acid and cholesterol. The ceramide composition contained 100 wt% of ceramide NP (Cer NP) or a ceramide mixture (Cer NP and 1OS Cer or ULC Cer). The ceramide mixture was formulated in the weight fraction of 0.8-0.95 of Cer NP and 0.05-0.2 of 1OS Cer or ULC Cer with stearic acid and cholesterol. Then, the ceramide-based lipid mixture was dissolved with 1 mg/ml concentration in chloroform/methanol (2:1, v/v). The π-A isotherm of 2-D lipid membranes was obtained by using the Langmuir-Blodgett trough (KSV-NIMA, KN-2002). For this, we spread the lipid solution at the air-water interface on a Teflon trough subphase. The solvents were evaporated for 10 min. The lipid monolayer was compressed at 10 mm/min at 26 ℃ by closing and opening the barriers. The surface pressure was measured by using a Wilhelmy plate at least in triplicate. The lipid membrane at the air-water interface was compressed to 40 mN/m of surface pressure and then transferred onto a mica substrate (20 x 20 mm2, agar scientific, AGG250-2) by raising the mica support vertically through the air-water interface at 1 mm/min.
Results
The π-A isotherm of ceramide-based lipid membranes revealed that the mean value of a molecular area was expanded in the presence of 1OS Cer. By contrast, the mean value of a molecular area was rather compressed as the content of ULC Cer increased. This implies that 1OS Cer disturbed the molecular association while forming the lipid membrane, whereas ULC Cer led to close packing of lipid molecules in the membrane. We observed that ULC Cer causes the phase transition from liquid to solid to occur at a surface pressure as low as 15 mN/m. It is very interesting to compare it with 1OS Cer, which induces a phase transition at a relatively high surface pressure of 25 mN/m. From topology and surface roughness observations of lipid monolayer films through AFM and contact angle analyses, we could figure out that formulation of Cer NP and 1OS Cer made the membrane rough and hydrophobic, which was quite comparable to the case with ULC Cer or 100 wt% of Cer NP.
Discussion and conclusion
In this study, we propose a new ceramide-based lipid membrane system in which novel ceramides, 1OS Cer and ULC Cer, were associated with Cer NP, cholesterol and stearic acid. The asymmetric alkyl chains of 1OS Cer enabled occupation more area per one molecule during membrane formation, thereby impeding tight molecular association of the lipid membrane. While ULC Cer led to formation of a closely packed lipid membrane due to their strong hydrophobic interaction. Furthermore, the incorporation of 1OS Cer slowed down the phase transition compared with the case of ULC Cer in same surface pressure. These results highlight that the new ceramides, 1OS Cer and ULC Cer directly affect the formation of the lipid lamellar phase, which would be deeply involved in the regulation of skin barrier function.