A Novel Phytosphingosine Based 1-O-Acylceramide: Synthesis, Physicochemical Chracterization and Role in The Lipid Lamellar Organization
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Presented by: Chang Seo Park
Introduction: Sphingosine based 1-O-Acylceramides were first identified by Rabionet M. et.al. in 2013(J Lipid Res 54) and was speculated to contribute to the stability of lipd lamella phase organization in the human stratum corneum. No reports on the finding of phytosphingosine based 1-O-Acylceramide and of functional characterization of this new class of ceramide have been reported yet.
Purpose: In this study, we report a synthesis of phytosphingosine based 1-O-Acylceramide (1-O-Acyl-ceramide NP). Series of physicochemical analyses and molecular dynamics (MD) simulation were peformed to characterize its molecular behaviours in a lipid lamellar.
Methods: Optimization of 1-O-acylation process was done to obtain phytosphingosine based 1-O-Acylceramide. LC/MS and 1H NMR analyses were conducted to identify the structure and conformation of the 1-O-Acylceramides. Multilayed lamellae structures were fabricated through equimolar molecular assembly of Cers (Cer NP and 1-O-Acylceramide), cholesterol, and stearic acid. The lamellar structure and phase properties of the fabricated multilayed lamellae were investigated by using DSC, XRD, and cryo-TEM analyses. MD simulation was performed to investigate the effect of 1-O-Stearoyl-ceramic NP on the LPP structure and skin barrier characteristics.
Results: A selective 1-O-acylation condition was optimized to yield 1-O-Stearoyl-ceramide NP or 1-O-Palmitoyl-ceramide NP. LC/MS and 1H NMR analyses confirmed that it has correct 1-O-Acyl conformation. A scale up production process for 1-O-Stearoyl-ceramide NP was successfully eastablished. DSC analysis revealed that the incorporation of 1-O-Stearoyl-ceramide NP increased the main transition temperature of the 2D multilayerd lamellar membranes while lowering the total heat capacity. 3-D multi-lamellar vesicles as a SC mimetic-nanovesicle (SCNV) system were prepared using ceramide NP/cholesterol/fatty acid(model SC lipids) in combination with 1-O-Stearoyl-ceramide NP. The average particle size of the nanovesicles was 200∼300 nm. TEM images and polarized microscopy images showed multi-lamellar structure regardless of the presence of 1-O-Stearoyl-ceramide NP. The SCNVs showed significantly improved long-term stability with the increase in the incorporated 1-O-Stearoyl-ceramide NP. These findings indicate that 1-O-Stearoyl-ceramide NP is important for stabilizing the structure of SCNVs containing SC lipids. MD simulation was conducted using a sandwich-structured LPP model. 5 wt% 1-O-Stearoyl-ceramide NP was placed between the bilayer and the slab layer. The chain structure of 1-O-Stearoyl-ceramide NP was extended to both the bilayer and slab layer, leading to a more disordered lipid layer structure compared to the model without 1-O-Stearoyl-ceramide NP. The disordered structure allowed the higher density arrangement of the lipid layer structure. The result was also confirmed by the radial distribution function (RDF). We also analyzed the skin barrier properties by calculating the potential of mean force (PMF) required for ethanol molecules to pass through the lipid layers using the Umbrella Sampling method. The model with 1-O-Stearoyl-ceramide NP exhibited a higher energy barrier, which suggests that 1-O-Stearoyl-ceramide NP suppresses the passage of hydrophilic molecules and contributes to a moisturizing effect on the skin.
Discussion and Conclusion: This the first report on the development of a novel 1-O-Acylceramides, specifically 1-O-Stearoyl-ceramide NP. Based on the molecular structure of 1-O-Stearoyl-ceramide NP, we postulated that it might play a critical role in stabilizing the SC multilamellar organization through bidirectional anchoring neighboring lipid lamellars. The results obtained in this study highlight that 1-O-Stearoyl-ceramide NP is important and play a role in stabilizing the biophysical behavior of SC lamellar organization. The results from MD simulation further support that 1-O-Stearoyl-ceramide NP can enhance permeability barrier function of SC lamellar organization via bidirectional anchoring mode of action.
Purpose: In this study, we report a synthesis of phytosphingosine based 1-O-Acylceramide (1-O-Acyl-ceramide NP). Series of physicochemical analyses and molecular dynamics (MD) simulation were peformed to characterize its molecular behaviours in a lipid lamellar.
Methods: Optimization of 1-O-acylation process was done to obtain phytosphingosine based 1-O-Acylceramide. LC/MS and 1H NMR analyses were conducted to identify the structure and conformation of the 1-O-Acylceramides. Multilayed lamellae structures were fabricated through equimolar molecular assembly of Cers (Cer NP and 1-O-Acylceramide), cholesterol, and stearic acid. The lamellar structure and phase properties of the fabricated multilayed lamellae were investigated by using DSC, XRD, and cryo-TEM analyses. MD simulation was performed to investigate the effect of 1-O-Stearoyl-ceramic NP on the LPP structure and skin barrier characteristics.
Results: A selective 1-O-acylation condition was optimized to yield 1-O-Stearoyl-ceramide NP or 1-O-Palmitoyl-ceramide NP. LC/MS and 1H NMR analyses confirmed that it has correct 1-O-Acyl conformation. A scale up production process for 1-O-Stearoyl-ceramide NP was successfully eastablished. DSC analysis revealed that the incorporation of 1-O-Stearoyl-ceramide NP increased the main transition temperature of the 2D multilayerd lamellar membranes while lowering the total heat capacity. 3-D multi-lamellar vesicles as a SC mimetic-nanovesicle (SCNV) system were prepared using ceramide NP/cholesterol/fatty acid(model SC lipids) in combination with 1-O-Stearoyl-ceramide NP. The average particle size of the nanovesicles was 200∼300 nm. TEM images and polarized microscopy images showed multi-lamellar structure regardless of the presence of 1-O-Stearoyl-ceramide NP. The SCNVs showed significantly improved long-term stability with the increase in the incorporated 1-O-Stearoyl-ceramide NP. These findings indicate that 1-O-Stearoyl-ceramide NP is important for stabilizing the structure of SCNVs containing SC lipids. MD simulation was conducted using a sandwich-structured LPP model. 5 wt% 1-O-Stearoyl-ceramide NP was placed between the bilayer and the slab layer. The chain structure of 1-O-Stearoyl-ceramide NP was extended to both the bilayer and slab layer, leading to a more disordered lipid layer structure compared to the model without 1-O-Stearoyl-ceramide NP. The disordered structure allowed the higher density arrangement of the lipid layer structure. The result was also confirmed by the radial distribution function (RDF). We also analyzed the skin barrier properties by calculating the potential of mean force (PMF) required for ethanol molecules to pass through the lipid layers using the Umbrella Sampling method. The model with 1-O-Stearoyl-ceramide NP exhibited a higher energy barrier, which suggests that 1-O-Stearoyl-ceramide NP suppresses the passage of hydrophilic molecules and contributes to a moisturizing effect on the skin.
Discussion and Conclusion: This the first report on the development of a novel 1-O-Acylceramides, specifically 1-O-Stearoyl-ceramide NP. Based on the molecular structure of 1-O-Stearoyl-ceramide NP, we postulated that it might play a critical role in stabilizing the SC multilamellar organization through bidirectional anchoring neighboring lipid lamellars. The results obtained in this study highlight that 1-O-Stearoyl-ceramide NP is important and play a role in stabilizing the biophysical behavior of SC lamellar organization. The results from MD simulation further support that 1-O-Stearoyl-ceramide NP can enhance permeability barrier function of SC lamellar organization via bidirectional anchoring mode of action.