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. 2021 Aug 30;18(6):953–962. doi: 10.1007/s13770-021-00379-4

Stabilization of Lipid Lamellar Bilayer Structure of Stratum Corneum Modulated by Poly (2-methacryloyloxyethyl phosphorylcholine) in Relation to Skin Hydration and Skin Protection

Ae-Ri Cho Lee 1,✉, Heekyung Moon 1, Kazuhiko Ishihara 2
PMCID: PMC8599549  PMID: 34460064

Abstract

Background:

One crucial factor in skin tissue engineering is to understand the hydration and barrier property of skin. We investigated the skin hydration and stabilization strategy of inter-lamellar structure of stratum corneum (SC) using poly (2-methacryloyloxyethyl phosphorylcholine) (PMPC).

METHODS:

The unique hydration and stabilization potency of PMPC on the barrier function of the SC examined using freshly excised hairless mouse skin as a model membrane and the relationship between the stabilization of the lipid lamellar bilayer (LLB) and its enhanced water holding capacity was established.

RESULTS:

Differential scanning calorimeter based on the phase-transition temperature of lipid domain of SC demonstrated that PMPC stabilized the LLB. The ratio of the heat of lipid phase transition (△H) of SC exposed to water and PMPC for 24 h was 1.51. X-ray crystallography showed the presence of well- organized lipids in intercellular membranes exhibiting short and long periodicity of lamellar phases. The peak at 4.4 nm attributed to the long periodicity phase (LPP) was missing in water-treated SC, where, the presence of 4.2- 4.4 nm peak in PMPC treated SC indicated that PMPC stabilized LPP. Transmission electron microscopy study demonstrated that the LLB structure became more rigid and orderly in PMPC treated SC.

CONCLUSION:

The unique ion paired structure of PMPC enhances the barrier function of the SC by stabilizing LLB structure and hydration by inducing weakly bound water. The unique hydration state and stabilization effect from extended water exposure could provide a valuable information to prepare reliable artificial skin matrix and skin tissue.

Supplementary Information

The online version contains supplementary material available at 10.1007/s13770-021-00379-4.

Keyword: MPC, Phospholipid polymer, Skin hydration, Skin protection, Stratum corneum, Lipid lamellar bilayer

Introduction

Water is an essential component of the stratum corneum (SC) influencing its elasticity and barrier function. The SC lipids in the bi-laminate structure of the epidermis barrier appears to be responsible for the SC hydration [1–3]. Healthy SC is composed of 15–20 (wt %) water; which is presents in three forms: free, weakly bound and very strongly bound form [4–6].

A synthetic poly (2-methacryloyloxyethyl phosphorylcholine) (PMPC, MW 140 K) was reported to enhance the free water content on the surface of polymer and lower the nonspecific protein adsorption [7, 8]. The high proportion of free water in PMPC treated surface maintains the structural integrity of proteins without any 3-D conformational changes. PMPC could enhance the retention amount of moisture and prevent water evaporation upon application with PMPC on dry skin [9].

A clinical study with 20 volunteers on water holding capacity of PMPC via measuring the hydration state of the SC showed 20–40 folds increase in water holding capacity [10, 11]. As the hydration rate of the skin significantly affects the permeation rates of a drug [12], it is integral to investigate the effects of PMPC on the skin permeation rate.

In previous studies, a marked reduction of the skin permeation rate of nicotinic acid (NA) was observed with the presence of PMPC (0.5–5%) in the donor solution [13]. The permeation profile of a model drug, NA, without PMPC in donor solution followed the biphasic manner: first steady phase and second hydration phase. The abrupt, more than tenfold inflation in skin permeation rate from the first steady state (43.5 ± g/cm2/h) to the second hydration phase (457.3 ± g/cm2/h) supports the interruption of barrier function due to intensive skin hydration. The skin permeation profile of NA in the presence of PMPC (3 wt.-%) in the donor solution followed the monophasic mode: the steady state flux (10.9 ± g/cm2/h) without rapid raise of the flux. As the concentration of PMPC increased, the skin permeation rate decreased accordingly [13].

Hasegawa and coworkers also reported the decrease in skin permeation rate of ρ-hydroxybenzoic acid and its esters (parabens) by MPC-co-butylmetacrylate (PMB). Addition of PMB decreased the skin permeation rate of paraben and the decreasing ratio was dependent on the PMB concentration [14]. By preventing paraben skin flux from the topical formulation to our body, addition of PMB prevents adverse effects by paraben while maintaining antibacterial effect of paraben in pharmaceutical formulation.

Stratum corneum, which consists of 10–20 overlays of the dead keratinized cells embedded in the lipid lamellar bilayer (LLB) structure, has an integral role in maintaining water holding and barrier function in addition to prevention of the influx of exogenous compounds to the skin. Disruption of the LLB structure in the SC causes the deterioration of the water holding property of SC.

Skin diseases, such as atopic dermatitis and xerosis, cause dryness, itching and cracking of the skin [15, 16]. These symptoms are associated with impairment of the SC function whose lipid composition affects the hydration state and permeability of water through the skin [17].

In this study, we investigated the stabilization potency of PMPC on the barrier function of the SC and established the relationship between the stabilization of LLB and its enhanced water holding capacity using a small angle X-ray crystallography (SAXS), a wide angle X-ray crystallography (WAXS), a differential scanning calorimeter (DSC) and a transmission electron microscopy (TEM). Freshly excised hairless mouse skin used as a model membrane. This study will bring new insight into the importance of lipid composition in SC for the elucidation of skin barrier function.

Materials and methods

PMPC solution

PMPC (100% MPC; Mw = 940 kDa, Mn = 252 kDa) was obtained from NOF Co. Ltd., Tokyo, Japan as 5 wt % aqueous solutions without any additives. PMPC solution employed as 0.5–3% aqueous solution after dilution. All other chemical ingredients were used as purchased.

Skin model

Female hairless mouse (Jackson Lab., 5–8 week age, 20 ± 2 g weight) were obtained from Hanlimwon (Seongnam, Korea). Freshly excised abdominal parts of the skin employed as a model membrane.

Skin sample preparation

Freshly excised skin was mounted between the donor and receptor half-cells of side-by-side diffusion cells; the diffusion area was 0.64 cm2. The donor and receptor solutions were placed in each cell compartment; cell volume 3.5 mL [4].

After completing 24 h in vitro skin permeation study, the exposed diffusion area washed with water and excised. Then the excised skin area photographed and processed for TEM.

TEM

Samples were prepared as described in previous [13]. In brief, fixation of the skin specimen was processed with 2.5% glutaraldehyde in 0.1 M cacodylate buffer solution for 3 h. As a post-fixation procedure, settled skin sample treated with 0.2% osmium tetraoxide (OsO4) in cacodylate buffer solution for 1 h. The sliced sections stained and investigated with electron microscopy (JEM-1200EX-II. JEOL Co., Tokyo, Japan).

DSC study

Separation of stratum corneum from viable skin was carried out according to our previous reports [13]. In brief, freshly excised hairless mouse skin was mounted on the filter paper, wetted with 0.1% (pH 7.4 phosphate buffer) trypsin solution in covered Petri dish for 14 h at room temperature. Stratum corneum layer was carefully separated from viable skin with a forceps. Separated SC, which is clear and thin layer, was placed either into water or PMPC solutions for equilibration. Equilibrated SC cooled to − 40 ºC within the sealed capsule at the rate of 0.3–0.4 °C per min, and held for 10 min prior to measurements. The sample temperatures were elevated to 150 °C at a rate of 5 °C per min and the phase transition property measured.

X-ray crystallography study

SC sheets separated by placing whole skin (1 × 2 cm2) on 0.5% trypsin (in PBS) soaked filter paper for 7 h. The separated SC dried on silica gel for 1 h and dried SCs put into the tested solutions: 1% PMPC, 3% PMPC, and water 5 mL. Three pieces of SC placed in each tested solution, shaken for 12 h at 100 rpm at 34 °C before loaded into SAXS (small angle X-ray scattering) chamber. All measurements on isolated SC samples performed at Korean Institute of Science and Technology (KIST), Seoul, Korea. The scattering intensities measured as a function of the scattering angle (θ). From the scattering angle, the scattering vector (Q) was calculated as Q = 4π(sinθ)/λ, where the wavelength is 0.154 nm at the sample position.

Animal rights

All animal experiments were conducted in accordance with policies of the NIH Guide for the.

Care and Use of Laboratory Animals (NIH Publications No. 8023, revised 1978) and the Institutional Animal Care and Use Committee (IACUC) of Duksung Women’s University. Specific protocols used in this study were approved by the Duksung Women’s University IACUC (approved protocol number: 2020-012-007).”

Results

Water holding capacity of PMPC

Figure 1 shows the photographs of exposed diffusion area just after completing in vitro skin permeation study. Skin hydration state perfused with water and 2% PMPC for 24 h observed. Part of free water drops presents outside on the skin surface (A), while PMPC treated skin exhibited a greater ability to retain water (B). As PMPC increase the weakly bound secondary water as shown in DSC thermal graph, PMPC increased the water holding capacity of the SC.

Fig. 1.

Fig. 1

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Photograph of skin hydration state perfused with water and 2% PMPC for 24 h. A Part of free water stays outside on the skin surface; B PMPC treated skin exhibited a greater ability to retain water. As PMPC increase the weakly bound secondary water as shown in DSC thermo-grams, PMPC increased the water holding capacity of the SC. Exposed skin area is 0.64 cm2. Scale bar represents 0.1 cm

DSC study

DSC analysis was carried out between 0 and 150 °C with temperature elevation rate of 5 °C per minutes. Figure 2A shows DSC thermo-grams focusing on the phase-transition temperature of water domain of stratum corneum perfused for 12 h with tested solutions. In pure water treated SC, the endothermic peak showed around 100 °C, which corresponding to the boiling point of free water. In PMPC treated SC, the transition temperature of water shifts to 129 °C, corresponding to the weakly bound secondary water. Water in SC presents in three different forms-free water, weakly bound secondary water and strongly bound primary water. 2% PMPC solution treated SC showed phase transition at 129 °C which indicates a secondary water. Pure water treated SC and untreated control SC showed phase transitions at 107 °C (free water) and 144 °C (strongly bound primary water), respectively. As water in SC presents in three forms-free water, weakly bound bulk water and strongly bound primary water, PMPC treatment increases the weakly bound water content in the SC.

Fig. 2.

Fig. 2

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A DSC thermos-grams focusing on the phase-transition temperature of water domain of stratum corneum perfused for 12 h with tested solutions: Water in SC presents in three forms-free water, weakly bound bulk water and strongly bound primary water. 2% PMPC solution treated SC showed phase transition at 129 °C which indicates a secondary water. Pure water treated SC and untreated control SC showed phase transitions at 107 °C (free water) and 144 °C (strongly bound primary water), respectively. As water in SC presents in three forms-free water, weakly bound bulk water and strongly bound primary water, PMPC treatment; B DSC thermogram focusing on the phase-transition temperature of lipid domain of SC. The presence of PMPC stabilizes the LLB: the temperature of transition temperature and heat of transition (DH) increase; C, D Transmission electron micrograph of the stratum corneum and dermis layer from a 360 m hairless mouse skin sections perfused for 12 h with water (C) and 2% PMPC (D). A: Wider separation depicted as an arrow B: Corneocytes are still cohesive (as arrows depict). Scale bar represents 1 m

In the SC, water presents as three types: very strongly bound primary water that never freezes, even below –40 °C, less tightly bound secondary water mostly affected by the changes in atmosphere and free water. Primary water content hardly affected by the environment and remains constant as ~ 5% per dry SC weight.

Figure 2B shows DSC thermo-gram of SC, directing on the phase-transition temperature of lipid portion. The presence of PMPC stabilizes the LLB: the transition temperature and heat of transition (△H) increase. Endothermic peaks near 60 °C correspond to lipid phase transition of SC. Lipid phase transition of water treated SC shows at 62 °C while SC from 3% MPC treated shows at 65 °C. The increase in phase transition temperature in MPC treated SC as compared to water treated SC implies the stabilization effect of MPC.

The lamellar de-lamination and corneocytes separation

Figure 2C and D show TEM of the epidermis and dermis layers from a hairless mouse skin Sects. (360 µm) perfused for 12 h with water (C), which shows wide separation between corneocytes and stratum basal layer. Wide separation between collagen fibers and other dermal components observed in TEM study explains the increased skin permeation rate with the presence of the hydrating vehicle in donor and receptor solution while performing in vitro skin permeation study [18]. As shown in Fig. 2D, PMPC treated samples appears that corneocytes are closely adhesive and epidermis is tightly adherent with dermis. As low as (< 1–3%) of PMPC in water can stabilize the protection capacity of the skin by minimizing the disruption of LLB structure by water.

X-ray crystallography study

Figure 3A shows small angle X-ray crystallography of stratum corneum equilibrated with water, control and 3% PMPC solution. Both peaks at 23.21 and 4.37 nm were disappeared in water treated SC. With the addition of PMPC to the separated SC, the diffraction peaks in the X-ray measurements became sharp and pronounced as compared with that of water only treated SC. This change of the peaks indicates that the lamellar structure become more rigid and orderly as it stabilizes following addition of PMPC [13]. As water content increased, the interlayer spacing (d) of the lamellar increased. As the water in the mixture replaced with PMPC solution, the d value increase was smaller than that of water only treated one. The lipids arranged in intercellular membranes appearing short- and long-periodicity lamellar distinct stage. The peak at 4.4 nm ascribed to the long periodicity phase was missing in SC in water sample. A lack of o4.19–4.37 nm peak and 23.21–25.09 nm peak f in water treated SC attributed to the decrease of the fraction of the long periodicity phase (LPP) and an alteration of LPP. A peak at 3.4 nm ascribed to crystalline cholesterol. PMPC penetrates into the lamellar bi-layer due to a specific interaction with phospholipids and stabilizes the lamellar structure. Figure 3B shows small angle X-ray diffraction patterns of stratum corneum equilibrated with water (left), control (center) and 3% PMPC solution (right). A typical diffraction pattern of hairless mouse SC observed. At low angle (towards the center) three diffraction circle patterns appeared, which are the third, fourth and fifth order diffraction peaks of the LLB arrangement. In Hydrated SC sample, the diffraction pattern shows the decrease in intensity especially at low angle diffraction rings, which might be due to amorphous keratin or to lipids, in which arranged in the liquid state in the bilayers [19–23]. PMPC treated SC shows that the diffraction pattern maintains its intensity of low angle diffraction rings as untreated control SC. Diffraction ring depicted as an arrow. This result suggests PMPC stabilize the keratin and inter lamellar lipid structure of SC.

Fig. 3.

Fig. 3

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A Small angle X-ray crystallography of stratum corneum equilibrated with water, control and 3% PMPC solution. The lipids organized in intercellular membranes exhibiting short- and long-periodicity lamellar phases. 4.44 nm peak attributed to the long periodicity phase was missing in SC in water sample. A lack of 4.2–4.44 nm peak and 23.2–25.09 nm peak in water treated SC attributed to the decrease of the fraction of LPP and an alteration of LPP. A 3.4 nm peak attributed to crystalline cholesterol; B Wide angle X-ray diffraction patterns of stratum corneum equilibrated with water (left), control (center) and 3% PMPC solution (right). Diffraction ring depicted as an arrow in control and PMPC treated stratum corneum

Figure 4A shows the proposed ion pairing between two PMPC molecules. PMPC is ‘Inner salt’ with negative and positive charges together in one molecule. Ion pairing between positive charged nitrogen in tri methyl ammonium and negative charge of oxygen in phosphate group in PMPC molecule formed. Ion pairing occurs only in circumstances, where water molecule does not approach and form hydrogen bonds with nearby molecule. In PMPC, tri-methyl functional groups in quaternary ammonium poses hydrophobic property, thus shield the water molecule approach. By ion pairing interaction, PMPC molecule could form dimer, which has lost ionic property of inner salt. In benzene molecule, conjugation causes depolarization. However, the presence of oxygen near methyl functional group in PMPC increases the polarity. Due to an oxygen presence, electron resonance occurs and the conjugation increases polarity; it leads to positively charged methyl group and negatively charged oxygen; hydrogen bonds could be formed between water molecule and PMPC polymer [25, 26]. PMPC can bind water molecules due to hydrogen bond.

Fig. 4.

Fig. 4

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A Proposed ion paired structure of PMPC for water-holding & inter-lamellar lipid stabilization. PMPC is methacrylate with a phosphorylcholine group in the side chain. The phosphorylcholine has its zwitterionic structure, consisting of a phosphate anion and a trimethylammonium cation. PMPC has about 3186 functional residues (Mw = 940) per molecule. Hydration of PMPC may occur by hydrophobic hydration of the three methyl groups in the trimethylammonium group. B Proposed schematic diagram of the unique hydration (depicted as two blue dots and a green dot as water molecule, H2O) and protection of stratum corneum barrier property of PMPC. PMPC depicted as an arrow

Figure 4B illustrates a proposed mechanism of the unique hydration (depicted as two blue dots and a green dot as water molecule, H2O) and protection of stratum corneum barrier property of PMPC. The interaction between PMPC and water molecules and networks of PMPC on the surface of the skin illustrated and depicted as an arrow. The Zwitterion charges of the residues could interact each other to form a cross-linked structure so that the surface covered with PMPC network [25, 26]. PMPC molecules may bind on the surface of stratum corneum through ionic interaction between positive charge of quaternary ammonium ion and negative charge of the skin surface. The network is supposed to be a 3-dimentional architecture and multiple layer of PMPC. The surface of stratum corneum laminated with PMPC film protected from outer environment such as water activity [26].

In this mode, PMPC could reduce the permeation rate of drugs penetrating skin. By forming hydrogen bonds between oxygen atoms of PMPC and water molecules, PMPC may serve as a humectant. Previous study supports our results. Fluorescent –labelled MPC applied on the mouse skin and the skin biopsy revealed that PMPC remained on the surface of the skin [11, 26]

Discussion

PMPC is a zwitterionic polymer; that is, both the anionic group and cationic group bind covalently in one molecule. The trimethylammonium cation and phosphate anion are situated close to one another, with tree methyl groups bound to the nitrogen atom of the MPC unit located outside of the polymer chains. This provides a site that can form favorable interactions with water through hydrophobic hydration, inducing a more ordered water structure that is similar to that of free water in the bulk phase [24, 25]. The water structure is more stable and take on an ice-like clathrate structure. In our DSC study, phase transition temperature of water in MPC treated SC showed transition temperature at 129 °C, which suggest water presents as a more ordered form.

In 2017, Ishihara et.al. reported that the phosphorylcholine groupsin PMPC possess a hydrophobic hydration layer that did not disturb the hydrogen bonding between water molecules. The number of hydrogen bonds is lower in the hydrophobic region than in pure water, and water-water hydrogen bonds are stabilized, especially around the alkyl chains, by hydrophobic hydration [26]. It induces an increase in clathrate cage structure of surrounding water molecules, I, e, the ammonium ice-like water state is formed. With this unique hydration, PMPC could not make strong interactions with proteins and cells. Some biomedical applications have used PMPC as a protein and solid surface modification agents. The hydrophobic main chain of PMPC does not make contact with water molecules directly when the PMPC is dissolved in aqueous media. The phosphorylcholine group has a unique hydration state and proteins hardly adsorb on the PMPC brush surface [27].

Kanekura et al., also reported that PMPC can increase the retention of moisture and prevention of water after application of PMPC on dry skin [11]. The effect of poly (MPC-co-BMA) on the water barrier function of SC, changes in trans epidermal water loss (TEWL) in response to poly (MPC-co-BMA) were monitored by measuring TEWL. TEWL was 4.25 ± 0.1 g/m2/h on the back skin of the mice prior poly MPC treatment. The Mean difference between treated and control group was 1.20 (0.44–1.96) at day 1. Poly (MPC-co-BMA) reduced TEWL significantly compared with the control. Water holding capacity was measured on volar forearms of 21 healthy volunteers after poly MPC-co-BMA application or distilled water. Water retention capacity in the skin treated with poly (MPC-co-BMA) was much higher [11].

Zhang et al., investigated the effect of PMPC on fibrous tissue formation and cell adhesion (CAP) forming reactions. Silastic elastomer plates coated and uncoated PMPC were implanted subcutaneously in the rat dorsal region. The thickness of the fibrous tissue capsule and the amount of collagen at 4,8,12 weeks were lower in the PMPC treated group. MPC polymer- coated materials exhibited inhibition of fibrous tissue formation and collagen synthesis and showed higher synthesis rates of non-collageneous proteins, such as elastin and proteoglycans, without an active inflammatory response. MPC polymer could regulate the fibrous tissue formation [28]. As the PMPC prevents non-specific interactions with biological molecules and cells, the immobilized bioactive molecules play an important role for the specific capture.

In transdermal drug delivery, the principal resistance to drug transport and water evaporation reside in the diffusion process through the stratum corneum [29–32]. Two important functions of stratum corneum are prevention of water loss and retention of moisture. The construction of a potent water barrier including corneocyte enhancing lipid production, and natural moisturizing factor (NMF) generation is strongly depend on the level of SC hydration [8]. Preserving an adequate SC lipid bi-laminate structure is very crucial to balance the skin moisture state. The role of SC lipids has investigated and the bi-laminate structure of the epidermis barrier lipids appear to be responsible for the hydration.

A NMF, a different parts of mixture of low molecular weight, hydrophilic compounds first generated within the corneocytes by degradation of filagrin, the histidine-rich protein, has an important role for balancing skin hydration. In an untreated control skin sample, water presents as three different forms; free water, physically bound water (weakly bound water) and chemically strong bound water. Thus in control skin sample, the mixtures of three-types of water give broad endothermic phase transition profiles. Water treated skin: sharp endothermic peak: 100 °C phase transition temp. In 2% PMPC treated SC, water presents mostly as a weakly bound form.

Shaku and coworkers reported that bound water content of a lamellar structure was increased by addition of PMPC even after the appearance of free water. PMPC mechanism to increase bound water is different from that of proline and sphingolipid. The ability of PMPC to increase bound water of the freeze-dried hairless mouse SC was greater than that of typical moisturizers such as sodium hyaluronate and sodium pyrrolidonecarboxylate. Clearly, PMPC acts on both intercellular lipids and hygroscopic components to enhance the retention of water in SC by increasing bound water [27].

In this study, we proposed ion paired structure of PMPC for water-holding and inter-lamellar lipid stabilization and illustrated in Fig. 4A. PMPC (Mw = 940 kDa) has about 3.0 × 103 phosphorylcholine groups as a polar group in the polymer and each monomer unit is a zwitterion which has both positive charge and negative charge. Solubility of PMPC is quite high more than 40 wt % of polymers can be dissolved in water. The water molecules prefer to bind to the PMPC, however, they detach rapidly from the polymer chains. Due to this unique hydrophilic nature, the PMPC may serve as a humectant.

Each progression stage leads to the production of an efficient moisture barrier and NMF production influenced by the level of SC hydration. Thus, the hydration property of PMPC could enhance the SC hydration and LLB stabilization.

The role of PMPC for maintaining a proper SC lipid bi-laminate structure is very important to balance the skin hydration state and skin barrier property. The presence of PMPC in water can help to maintain the barrier property of the skin by preventing the disruption of LLB structure caused by extended water exposure. Zwitterion nature of the residues can make PMPC molecules interact each other to form a cross-linked conformation, so that the surface covered by PMPC network. Understanding the unique hydration state and water holding capacity of PMPC and membrane stabilization effect from extended water exposure could provide a valuable information to prepare a reliable artificial skin matrix and smart scaffold in skin tissue engineering.

In this study, we hypothesized the unique ion paired structure of PMPC influences water-holding capacity and inter-lamellar lipid stabilization in SC. TEM and X-ray crystallography studies demonstrate that the lamellar structures became more rigid and orderly as PMPC stabilizes LLB. PMPC in hydrophilic solution enhances the barrier property of the SC by stabilizing LLB structure and skin hydration by increasing the secondary bound water.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary file1 (PDF 686 KB) (686.1KB, pdf)

Acknowledgements

This work was supported in part by the Duksung Women’s University Research Grants 2020.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical statement

The animal experiments were performed under animal protocol number (no. 2020-012-007) approved by the Duksung Women’s University Institutional Animal Care and Use Committee (IACUC).

Footnotes

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