THE INFLUENCE OF RETINOL CONCENTRATION IN LIQUID CRYSTAL FORMULA ON EPIDERMAL GROWTH FACTOR, INTERLEUKIN-6 AND TRANSGLUTAMINASE-1 mRNA EXPRESSION IN THE EPIDERMIS

A key stage in the successful development of new formulations intended for human use is the assessment of percutaneous penetration of lipophilic substances as retinol with a high LogP value 5.68 (1). The cosmetic industry has undergone great progress over the past three decades, and the use of in vitro human epidermis skin tissue equivalents is gaining popularity as an in vitro alternative to human and pig skin for percutaneous assessment (2, 3).

By using modern tissue culture technology it is now possible to generate reconstructed human epidermis in vitro. These reconstructed skin models are used to analyze the efficacy and safety of cosmetics and medicines, as well as their constituent compounds (4, 5). In the reconstructed skin model, keratinocytes are arranged in four-layer cell structures. Epidermal thickness can be determined in histological section as the degree of skin atrophy. Full differentiation of the epidermis from the stratum basale to stratum corneum can be observed.

The epidermis is a keratinized stratified squamous epithelium formed by keratinocytes; these are nucleated cells which divide mitotically and are responsible for the process of epidermis keratinization, during which they are transformed into anucleated corneocytes. One layer of the epidermis, the stratum granulosum, contains granules known as keratohyalins and determines the degree of epidermal permeability. It is permeable to fat-soluble vitamins, including vitamin A (retinol). Outside this layer lies the stratum corneum, the outermost layer of the skin formed from a combination of horny cells and lamellar lipid layers and acts as the decisive barrier between the body and the environment; it prevents water loss from the body and protects it against penetration by exogenous substances. The hydrolipid coating is responsible for the maintenance of the basic protective function of the skin. It helps maintain the firmness, elasticity, hydration and softness of the skin by protecting against the penetration of water and hydrophilic substances into the epidermis and limiting transepidermal water loss (TEWL) (6, 7).

The aim of the present study was to determine the effects of liquid crystal formula containing 0.15%, 0.3% and 0.5% retinol on cell proliferation in the human epidermis in vitro. The study also evaluates the effects of the different formulas on the morphological and histologic characteristics of the skin, and compares the effects of 0.3% retinol in liquid crystal formula with 0.3% in lipid. Retinol and its derivatives cause severe local irritation manifested as mild erythema and stratum corneum peeling of the skin, despite many beneficial effects on dermatological applications. To determine potential anti- inflammation effect in vehicles, the expression of interleukin 6 (IL-6) was evaluated. IL-6 is a pleiotropic cytokine, which, has a role in activating and maintaining the inflammatory response (8, 9). Epidermal growth factor (EGF) is polypeptide produced mainly by keratinocytes and is known to influence skin epidermal cell regeneration by modulating the migration and proliferation of keratinocytes (10). Moreover to examined the possibility to reinforces the structure of the epidermis by forming strong cross-links between the structural proteins that make up the cornified cell envelope the transglutaminase-1 (TGM1) was used (11).

Liquid crystals have numerous applications, such as liquid crystal displays, solar cells and sensors. They also have many applications in medicine and biology. DNA, proteins and other molecules of the human body can also form liquid crystal phases. Liquid crystal-based formulas offer great promise in cosmetology as an option for recovering cutaneous tissues, particularly in antiaging treatments. The study used liotropic crystals whose basic units are lamellas. Lamellar crystal system is easiest to form in oil-in-water emulsions using a combination of a high hydrophile-lipophile balance (HLB) emulsifier and a low or medium HLB coemulsifier. Each of them play a specific function in the emulsion. The primary hydrophilic emulsifier reduces the interfacial tension and allows the formation of oil dispersions with small drops, while the hydrophobic coemulsifier creates a lamellar network, which after lowering the temperature below the Krafft temperature transforms into a gel phase. Liquid crystal is composed of amphiphilic lipids that self-assembly in solvents, has attracted extensive attention due to its good performance of drug loading and drug release (12).

Firstly, as the structure of the liquid crystal vehicle is similar to that of the lipids of the stratum corneum, characterized by layered arrangements similar to those present in cell membrane lipid bilayers. It increases the degree of order of the alkyl chains of the skin lipid film; by contrast, this order is reduced by a conventional W/O cream. Liquid crystal systems are also more stable than other emulsions, such as O/W and W/O, because they stimulate greater hydration in the external layers of the epidermis. They retain water in the stratum corneum and could improve the sustained release of active ingredients to target areas. Liquid crystal emulsions characterized by high chemical-physical durability and high affinity with the skin (13, 14). These liquid crystal structures have been reported to offer various benefits when used as vehicles for medication (15). The ideal liquid crystal vehicle for substance incorporation is non-comedogenic, specifically prepared and does not include minerals in its formula. It forms a rigid structure, thus providing the formulation with a higher stability and promoting real skin hydration (16). In the present study, a hydrophobic lipid formula (oil solution) was created to act as a point of reference to the studied liquid crystal formula. Lipid-based vehicle has ability to improve the bioavailability and solubility of water insoluble active ingredients. Improving drug absorption, mostly in oral delivery, in the recent years gained much importance. They may include emulsions, self-micro or self-nano emulsifying drug delivery systems (SMEDDS/SNEDDS) as well as oil suspensions and solution (17).

MATERIALS AND METHODS

Materials

Four kinds of original vehicle formulas with retinol were used: three consisting of liquid crystal bases with 0.15%, 0.3% and 0.5% retinol, and one of a conventional natural lipid base with 0.3% retinol. In the study was used liotropic crystals, whose basic units are lamellase. The lipid-based formulation was oil solution consists of emollients.

1. Preparation of tested retinol formulas

Retinol 15D (retinol in Caprylic/Capric Trigliceryde) was delivered from BASF Group; it’s molecular weight is 286.5 g/mol, density 0.954 g/cm3. All solutions were prepared in ultra-high purity deionised water, and all the reagents used were of analytical grade. To form the liquid crystal base, three sets of ingredients were used: A (Caprylic/Capric Triglyceride, Stearyl Alcohol, Tocopheryl Acetate), B (Water, Xanthan Gum) and C (Cetearyl glucoside, Sorbitan Olivate, Cetearyl alcohol), in appropriate concentration presented in Table 1. Lamellar crystal system was made of a high HLB emulsifier as Cetearyl glucoside, Sorbitan Olivate, Cetearyl alcohol (HLB = 9.5) and a low/medium coemulsifier as Stearyl Alcohol, Caprylic/Capric Triglyceride, Tocopheryl Acetate (HLB = 5.25). Oil phase A components mixed and heated to 70 – 80°C. The phase B components were mixed to form a gel and heated like phase A. The C ingredients were then added to B in a propeller stirrer. To the B + C mixture A components were added, stirring continuously. The homogenisation process was carried out twice (10.000 rpm/min): in a high shear mixer at 65 – 75°C then after 20 minutes water bath at 80°C, and then allowed to cool to room temperature while stirring. A combination of 1.5% preservatives (Phase D) was added at room temperature to each cooled formula (Phenoxyethanol, Methylparaben, Butylparaben, Ethylparaben, Propylparaben). To prepare the lipid formula, we use only oil phase and substances that are soluble in nonpolar solvents as Isopropyl Palmitate (HLB = 11.5) and Caprylic/Capric Triglyceride (HLB = 5), Tocopheryl Acetate (HLB = 6) were mixed cold (Table 2), in 19–21°C. Retinol 15D was added, one day after the base formulas were prepared, in red light to each type of vehicle, respectively to liquid crystal (Phase E) and lipid (18). The samples were stored in airtight, dark glass cosmetics jars until used. All products were tested for microbiological purity.

Table 1. Composition of the liquid crystal-based vehicle with retinol. Retinol in 0.15%*; 0.3%**; 0.5%*** concentration.

Table 2. Composition of the lipid-based vehicle with retinol.

2. Human skin in vitro

The study employed a multilayered, highly-differentiated model of human epidermis consisting of a culture of normal, human-derived epidermal keratinocytes (NHEK) (19), commercially known as EpiDerm (MatTek, Ashland USA). The epidermis, consisting of a basal, spinous, granular layer and a stratum corneum, was well defined in the study material. The thickness of the tissue ranged from 70 – 130 µm (20).

The reconstructed skin was cultured on specially-prepared cell culture inserts using vehicle-free medium containing KGF (Keratinocyte Growth Factor) to preserve the histology (MatTek in vitro Life Science Laboratories, s.r.o., Bratislava II Slovak Republic). As the in vitro model closely resembles natural human skin on the ultrastructural level it is eminently suited for assessing the efficacy and irritancy of cosmetic products. Its morphological and growth characteristics are uniform and highly reproducible. The tissues and culture media are also tested for bacterial, viral, fungal and mycoplasma contamination (21). Each tissue sample was divided in two: one half was used for histological analysis and the other for mRNA expression analysis.

Maintenance of skin samples and treatment with retinol formulas

After delivery, the tissues were incubated overnight (‘pre-equilibration’) to allow them to fully recover from the stress of shipping. Under sterile conditions, the package was opened and the inserts containing the tissues were removed from the agarose. The tissues, at refrigerator temperature (4°C), were then transferred to 6-well plates containing pre-warmed assay medium. The plates were placed into the incubator at 37°C and 5% CO2 for one hour prior to medium exchange. The assay media contained within the 6-well plates was then aspirated off and replaced with 0.9 ml (per well) of pre-warmed, fresh assay media. Following overnight incubation, 35 µl of test material was added into the cell culture insert on top of the EpiDerm sample. All of the following samples were applied as two inserts each: 0.15%, 0.3%, 0.5% liquid crystal vehicles, 0.3% lipid vehicle, and DPBS (Dubelco’s Phosphate Buffered Saline, Gibco) as negative control. All samples (35 µl/cm2) were applied topically to the skin once with a positive displacement pipette. After the test substance was applied, a nylon mesh (diameter 1.2 cm; 8 mm) was placed over the surface of the skin to improve the spread of vehicles during the exposure period. Nylon mesh did not affect the morphology of skin samples tested by light microscopy. To allow liquid interface feeding of the EpiDerm sample, the experiment was performed in 12-well plates with a hanging top (HNG-TOP-12), which was placed in the 12-well plate with a tissue insert and covered by standard top. The medium was replaced every other day and preheated. The skin samples were exposed for 10 days to the 0.15%, 0.3% and 0.5% liquid crystal formula and the 0.3% lipid formula. After exposure of the EpiDerm samples to the test materials, any liquid remaining on top of the tissues was removed. Each insert was individually rinsed gently three times with DPBS.

Description of analytical methods

The IL-6, TGM1 and EGF mRNA expression of the skin-equivalent tissue were measured after exposure for 10 days. Three independent experiments were performed.

1. Total RNA extraction and cDNA generation

The skin-equivalent tissues were placed into 200 µl of RNAlater solution (Ambion) and stored at –20°C until RNA isolation. Total RNA extraction was carried out using RNeasy Mini kits (Qiagen) and a TissueRuptor homogenizer (Qiagen) according to the manufacturer’s instructions. RNA content and purity was measured using a PicoDrop spectrophotometer (Picodrop Limited). The quality of RNA samples was determined according to absorption ratio at 260/280 nm. The purified total RNA was immediately used for cDNA synthesis or stored at –80°C.

cDNA was generated with the Maxima First Strand cDNA Synthesis Kit (Thermo Fisher Scientific Inc) according to the manufacturer’s protocol, with 200 ng of total RNA being used as starting material. Reverse transcription was performed according to the guidelines specified by the kit (25°C for 10 min, 50°C for 30 min, 85°C for 5 min). The cDNA samples were kept frozen at –20°C (22-24).

2. Real time PCR

The mRNA quantification was performed using the following standard TaqMan® Gene Expression Assays (Applied Biosystems): epidermal growth factor (EGF) (assay ID: Hs01099990_m1), transglutaminase-1 (TGM1) (assay ID: Hs00165929_m1) (25), interleukin 6 (IL-6) (assay ID: Hs00174131_m1) (26); ACTB, a member of the actin protein family, (assay ID: Hs01060665_g1) was used as a control. Skin treated with DPBS alone was used as a reference group.

TaqMan PCR assays were performed in 10 µl reactions comprising 10 ng cDNA, 5 µl TaqMan Universal PCR Master Mix and 0.5 µl appropriate TaqMan Gene Expression Assay (×20). All reactions were run in triplicate on a 7900HT Fast Real-time PCR System (Applied Biosystems) in FastGene Fast 96-well PCR plates (Nippon Genetics Europe GmbH). PCR was performed according to the following sequence: 20 s at 95°C, 40 cycles of 3 s at 95°C, and 30 s at 60°C.

Expression values were calculated using Sequence Detection System 2.3 and Data Assist Software (Applied Biosystems). ΔΔCt values were calculated for all genes using the untreated sample as a reference.

Histopathological analysis

The epidermal models underwent histological analysis at the end of the study. Sections were taken with a scalpel and fixed in 10% buffered formalin. They were then placed in a cassette, dyed with a drop of eosin and then subjected to standard histological preparation. Paraffin sections about 2 µm thick were stained with hematoxylin and eosin (H & E) to contrast the cytoplasm and cell nuclei, which allowed the entire structure of the tissue to be examined. Epidermal thickness was measured. The samples were fixed in neutral formalin. The obtained preparations were examined using an Olympus BX41 light microscope (4, 27).

Statistical analysis

Results were expressed as mean with standard deviation (mean ± SD). The Shapiro-Wilk test was used to determine normality, and Levene’s test was used to determine variance. The standard Student’s t-test was used to compare data showing no departures from normality and equal variances. For multiple comparisons (more than two groups) one-way ANOVA with post hoc Scheffe tests was applied. Any identified relationship was assumed to be significant at P < 0.05.

RESULTS

Gene expression in reconstructed epidermis

The cultures treated with 0.5% liquid crystal formula or 0.3% lipid formula demonstrated reduced expression of TGM1 mRNA compared to control i.e. tissues treated with DPBS. Those treated with 0.15% and 0.3% liquid crystal formula demonstrated greater TGM1 expression.

Samples treated with 0.5% liquid crystal formula demonstrated 1.5 times lower TGM1 mRNA expression than DPBS controls, while a decrease of more than 18-fold was observed in those treated with 0.3% lipid formula. Expression increased by 5.0 times in the 0.3% liquid crystal formula and 4.3 times in the 0.15% liquid crystal formula (P < 0.0001) (Fig. 1).

Fig. 1. Expression of transglutaminase-1 (TGM1); P < 0.05 versus DPBS control.

EGF mRNA expression was the greatest for cultures treated with 0.5% liquid crystal formula, being 23.7 fold higher than the DPBS standard. In addition, 0.3% liquid crystal formula demonstrated a 6.5-fold increase and 0.15% formula a 13.5-fold increase (P < 0.0001) (Fig. 2). Effect of 0.15%, 0.3% and 0.5% liquid crystal vehicle and 0.3% lipid vehicle on EGF mRNA expression was evaluated in reconstructed skin epidermis in vitro, after 10 days of experiment. Results are given as means and standard deviations (all reactions were run in triplicate): 9.068 ± 1.215, 14.197 ± 1.618, 54.719 ± 5.737 and 2.707 ± 0.234, respectively.

Fig. 2. Expression of epidermal growth factor (EGF); P < 0.05 versus DPBS control.

None of the cultures treated with any of the liquid crystal or lipid formulas demonstrated any significant difference from the DPBS reference standard (differences are 0.077 – 1.052) with regard to IL-6 expression. A P-value of 0.136 was observed for the 0.3% lipid formula (Fig. 3).

Fig. 3. Expression of interleukin 6 (IL-6); P < 0.05 versus DPBS control.

A comparison of the effects of different concentrations of retinol on mRNA levels in keratinocytes of human reconstructed skin is presented in Table 3. Total mRNA was extracted from epidermal keratinocytes (NHK) and quantified by qRT-PCR. Control samples are those exposed to DPBS.

Table 3. A comparison of different concentrations of retinol with regard to mRNA expression of IL-6, EGF and TGM1, in keratinocytes of human reconstructed skin. Significance between vehicles a, b, c and d for each gene expression is P < 0.0001 but between a and b for both IL-6 and EGF is P = 0.012.; TGM1 is P = 0.106.

All retinol formulas induced an insignificant decrease in IL-6 level. Interestingly, the 0.5% and 0.3% liquid crystal formulas demonstrated lower induction of IL-6, a pro-inflammatory cytokine, than the 0.3% lipid vehicle.

Histopathology observations

The epidermis was well differentiated, consisting of basal, spinous, granular layer and stratum corneum. At least four viable cell layers were present. Tissue morphology and thickness were within acceptable ranges and indicated the appropriate formation of an epidermal barrier, the presence of a functional stratum corneum, a viable basal cell layer, and intermediate spinous and granular layers (Fig. 4).

Fig. 4. Cross-section through the Epiderm reconstructed epidermis model used in the study, H & E, zoom ×100 (A) and ×200 (B). →← s.c. - stratum corneum; the most superficial layer of the epidermis usually consisting of 15 – 20 layers of dead cells, reduced to flattened scales filled with keratin (in general, the stratum corneum has a thickness between 10 and 40 µm).

However, the correct positioning of the collected section inside the paraffin block during sectioning presented a serious technical problem. The resulting section was sometimes several cell layers thick, and despite the use of staining solution, the spatial orientation of the examined tissue was inadequate for accurate measurement; hence, the sections do not represent the entire thickness of the epidermis. Nevertheless, all collected images of skin treated with 0.15% and 0.3% retinol (liquid crystal and lipid) indicate the presence of a thicker stratum corneum; this may be attributed to an intensive process of keratinization in the epidermis resulting from accelerated cell renewal stimulated by retinol (Fig. 5).

Fig. 5. Epidermis tested with various retinol concentrations (A1) (A2) 0.3% lipid, (B1) (B2) 0.3%, (C1) (C2) 0.15%, (D1) (D2) 0.5% liquid crystal formulas, formalin fixed H & E Stained, zoom ×40 and ×100, respectively. Clearly thicker stratum corneum (s.c.) comparing to the Fig. 4.

DISCUSSION

Despite their wide availability, few studies have used commercially-available models of human skin to examine the effects of vehicle on percutaneous penetration. However, some have compared the use of reconstructed full-thickness skin models with normal human skin in testing anti-aging compounds (3, 28).

Epidermal tissues treated with 0.15%, 0.3% and 0.5% liquid crystal formulation did not demonstrate significant differences in IL-6 gene expression; however, this expression mostly increased in those treated with 0.3% lipid formulation. It has been proposed that IL-6, a pro-inflammatory cytokine produced by irradiated keratinocytes, may have a role in metalloproteinase expression activation in dermal fibroblasts, leading to the reconstruction of connective tissues (29, 30).

Kirchmayer report that all-trans retinoic acid suppresses interleukin-6 expression in interleukin-1-stimulated synovial fibroblasts (31). Our present findings indicate that liquid crystal formula was associated with lower IL-6 expression than the 0.3% lipid formula, suggesting lower activation of the inflammatory response. The results presented by Milian-Ciesielska et al. indicate the interesting anti-inflammatory therapeutic strategies for lichen sclerosis (LS). The study pointed to the damage to the small nerve fibers in the disease process. In LS treatment, are used ointments with vitamin A, as well as others. It would be interesting to determine the effect of various vehicles using retinol on aging skin to assess intraepidermal nerve fiber density in skin biopsies (32).

In the present study, the most significant increase of IL-6 was observed following the application of 0.3% lipid vehicle. The decreases observed after application of 0.5%, 0.3% and 0.15% liquid crystal vehicle may suggest that the liquid crystal formula protects against inflammation; this is supported by clinical observations of lower levels of irritation, redness and peeling (own unpublished results).

A similar study found resveratrol in multipurpose liotropic liquid crystal emulsion to show limited effects as an antiaging product, and only influenced pore size on the forehead to a minor extent. Even so, 95.8% of the study participants were satisfied with the product (33). Nonetheless the other study showed increased expression of the genes involved in keratinocyte differentiation, indicating that the liquid crystal formula induces faster renewal of epidermal cells than the lipid base.

A key role in cell differentiation and growth is played by a small polypeptide called epidermal growth factor produced mainly by keratinocytes in the skin. Noteworthy, the stimulation of human keratinocytes with angiotensin II, which the epidermal expression has been reported in the skin, is comparable to EGF (34). The basement membrane of the dermal-epidermal junction is implicated in the proliferation, differentiation, and adhesion of keratinocytes as well as storage of growth factors as EGF (35). EGF is known to influence skin epidermal cell regeneration by modulating both the migration and proliferation of keratinocytes (10, 36, 37). Greater increases in EGF expression were observed in tissues treated with 0.5% and 0.3% retinol in liquid crystal than 0.3% retinol in lipid. To provide general statement and prove the effectiveness that liquid crystals improve retinol effect further research could be carried out using more conventional vehicle as O/W, W/O. Both EGFR and EGF play important roles in wound healing by stimulating epidermal and dermal regeneration (38). The involvement of EGF expression found GLUT1 to influence UVB-induced epidermal hyperplasia by enhancing proliferation in epidermal keratinocytes. It can be hence implied that the retinol used in the present study has a stronger influence on the keratinization process when delivered in liquid crystal medium (39).

Compensated by keratinocyte proliferation in epidermal basal layer, the superficial desquamation puts epidermis in constant self-renewal. Cohesion in the epidermis depends on the presence of specialized adhering junctions, desmosomes, and their modified version encountered in the horny layer (35). Transglutaminase-1, 3 and 5 are expressed in the spinous and granular layers of the epidermis. TGM1 is located in both the cytosol and the membrane. TGM1 expression is differentiation dependent and initiates in the spinous layer together with the envelope precursors involucrin and cornifin, among others, and the keratins K1 and K10. Transglutaminase-1 reinforces the structure of the epidermis by forming strong cross-links between the structural proteins that make up the cornified cell envelope (11, 40). Our findings indicate that the liquid crystal formula forms a denser cell envelope than the lipid formula, liquid crystal is composed of amphiphilic lipids that self-assembly in solvents, has attracted extensive attention due to its good performance of drug loading and drug release (12). Their structures and chemical properties are similar to those of cell membranes. Acting like the phospholipids and ceramides found in the skin, these emulsifiers do not disrupt the barrier properties and function because of their compatibility with skin lipids (41) and that this may act as a more effective protective barrier between the body and its environment. More specifically, 0.3% and 0.15% liquid crystal formula induced higher TGM1 expression than the 0.5% liquid crystal and the 0.3% lipid formulas, the latter of which demonstrated the lowest expression; this may result in the formation of a thinner outermost epidermal layer, which is comprised of terminally-differentiated keratinocytes (corneocytes). Therefore, it is possible that this base may increase the sensitivity of the skin when used for formulations with retinol. Shao et al. note that topical application of 0.4% retinol increases epidermal thickness and dermal angiogenesis by stimulating the proliferation of epidermal keratinocytes and dermal endothelial cells, as well as the TGF-β/CTGF pathway (42).

Conclusion the liquid crystal formula was associated with significantly greater increases in EGF expression than the lipid formula. Our findings indicate that the choice of base might have an influence on the release rate of hydrophilic active substances such as retinol. Thus, it may improve the efficacy of therapy. Furthermore, higher concentrations of retinol in biocompatible skin bases might have better antiaging results and probably it could decrease inflammation reaction and further examination has to be carried out. Indeed, it is important to balance between the effectiveness of a given formula and the risk of causing skin irritation.

Acknowledgements: We would like to offer our thanks to the Dr. Koziej company for assisting with the formulation of the tested cosmetics.

Conflict of interests: None declared.

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