Background: Retinol influences the process of keratinization of the epidermis, which improves stratum corneum structure and reduces transepidermal water loss. It also significantly enhances mature skin by brightening hyperpigmentation and reducing the signs of photoageing. Cosmeceuticals are intended to both provide aesthetic effects for the skin and allow dermatological treatment. The aim of the study was to assess the rejuvenating effect of retinol serum on facial skin at concentrations of 0.3 and 0.5%, as well as any improvements in skin brightening and elasticity. Materials and Methods: Thirty-seven volunteers were included in the study, after confirming tolerance. The novel formula was applied once daily to the face for a period of 12 weeks: one retinol concentration on the left side and the other on the right. The initial study with liquid crystal formula (study vehicle) was carried out for 8 weeks on 28 volunteers. Treatment efficiency was evaluated at baseline, and 56 and 84 days following treatment using the multi probe adapter and Fotomedicus imaging system. PRIMOS was used to measure skin surface roughness. The visual analogue scale method enabled the results to be determined by 3 independent specialists. Results: Skin hyperpigmentation, unevenness, and wrinkles gradually decreased over the course of treatment, both on the left and right parts of the face. Adverse events were predominantly mild or moderate skin irritation. More frequent and more intense symptoms were observed on the left side (0.5%). Conclusion: Retinol in liquid crystal formulation is safe and provides significant clinical benefits associated with unification of skin colour, overall skin tone, skin elasticity, and moisture. Regular use of retinol typically results in brightening of the skin and reduced signs of ageing. The objective findings confirmed the effectiveness of the procedures.
Cosmeceuticals are intended to provide both aesthetic effects and act as dermatological treatments, and are described as cosmetic products with drug-like activities. They can be regarded as topical cosmetic-pharmaceutical hybrids used to maintain and improve appearance; in addition, they are externally applied, produce useful and expected results, and have both aesthetic properties and meet medical, physical, and chemical standards. Retinol products are over-the-counter alternatives to topical anti-ageing treatments; however, retinoic acid is not used for topical application because it is registered as a drug, as is the case with synthetic retinoids such as adapalene, tazarotene, and bexarotene. In contrast, retinyl esters, retinol, retinaldehyde, and the oxoretinoids can be used in topical cosmeceutical preparations [1‒3].
In order to increase the penetration of the active ingredient into deeper layers of the skin, promoters of sorption are used to accelerate its transport through the stratum corneum where it can accumulate. Retinol has the ability to effectively penetrate the stratum corneum due to its lipophilic nature. After reaching a keratinocyte, retinol moves towards the interior and binds with an appropriate receptor: cytosolic retinol-binding protein (CRBP) is one such group of receptors with high affinity for retinol. The liquid crystal structure is an organized arrangement of surfactant and oil suspended in oil and water; however, the precise composition of the formula must be adjusted depending on the active ingredients that will be suspended and the preparation process itself. Retinol in a serum based on a liquid crystal formulation is a new cosmetic product believed to offer improved penetration. The liquid crystal formula resembles the structure of the lipid layer of the skin, the stratum corneum. The serum contains skin-compatible membrane lipids and lamellar systems, thus allowing the active ingredient to penetrate more actively through the epidermal layers and enabling better absorption. It is possible to use the alcoholic form of retinol in the active emulsion and still observe its active functioning [4, 5].
The aim of the study was to compare the effects of 2 concentrations of retinol (0.3 and 0.5%) in the novel liquid crystal formula with regard to their anti-ageing effectiveness and their tolerance by the user. An initial study was first performed to confirm the tolerability of the liquid crystal formula, and then significant tests were performed with the active ingredient.
Following an initial study on a group of 28 healthy women volunteers (mean age: 41 years), 37 healthy women volunteers (mean age: 46 years) were included for the in vivo split-face study with 0.3 and 0.5% retinol (Fig. 1). Dermatological patch test was conducted for a group of 30 volunteers. Each participant was selected in accordance with the guidelines of the Helsinki Declaration of 1964 and its later amendments or comparable ethical standards as well as with the approval of the Bioethics Commission (Nos. RNN/210/17/KE and KE/229/19). The inclusion and exclusion criteria for participation in the study are given in Table 1.
Each participant received 2 kinds of products (serum 3 and serum 4) consisting of 0.3 or 0.5% retinol in the same liquid crystal formula. Each product was applied once daily to the face at night, one formulation to one side and the other formulation to the other side: 4 pumps of 2 mL were spread on each side using a special applicator. The protocol was continued for 12 weeks: skin assessments were measured on day 0 (baseline) (D 0), after 56 days (D 56), and after 84 days (D 84). All participants agreed to use sunscreen products with SPF = 50+ and avoid sunbathing and prolonged exposure to the sun during the study. The participants were advised not to use new skincare products or other medications during the entire course of the study. The study characteristic card was presented and handed out to each of the volunteers. They were informed about the risk of potential adverse effects. On the first visit, each of the volunteers received a card with clearly specified recommendations regarding morning and evening care, related to the application of retinol. The volunteers were asked not to change the daily care routine. The same treatment regimen was used for the initial study but with no retinol component in the formulation.
All measurements were conducted in a draught-free measurement room with controlled humidity (H = 30–50%) and temperature (T = 19–21°C). During the study, participants were cared for by a dermatologist. A precise analysis of the effects of the tested product on the tone, moisture, sebum level, elasticity, firmness, and smoothness of the skin was performed as follows .
Skin Tone Evaluation
Skin tone was measured with an MX18 Mexameter® (Courage & Khazaka). It is used to measure erythema, vascular changes, and skin discoloration. The probe emits radiation of a certain length, which is absorbed by dyes. A receiver in the device measures the amount of reflected light not absorbed by chromophores, with more radiation being absorbed by darker skin. The probe was gently pressed to the skin, respectively, on the left and right parts of the face (cheek and forehead) and the measurement was taken. Triplicate measurements were taken of each place [7, 8].
Skin Hydration Evaluation
Skin hydration was measured with a CM825 Corneometer® (Courage & Khazaka). The measurement time was about 1 s and the frequency of measurement 0.9–1.2 MHz. The accuracy of the measurement was ±3%. Three measurements were performed on the left and right sides of the face, that is, within the central part of the cheek and forehead, and the mean value was taken as the final result. The probe for the measurement was set vertically and the measurements were not repeated in 1 place [9, 10].
Skin Sebum Evaluation
The level of skin sebum was measured using a Sebumeter® (Courage & Khazaka). A special tape was applied to the skin which reacted only to skin sebum, independent of the water content. The time of application was 30 s. Accuracy of the measurement was ±5%. Data were collected from the forehead and cheek, on the left and right sides of the face .
Skin Elasticity Evaluation
Skin elasticity and firmness were measured by an SEM 575 Cutometer® (Courage & Khazaka). Resistance of the skin to being sucked up by negative pressure (firmness) and its ability to return into its original position (elasticity) are displayed as curves. Suction was applied for 3 s as a constant negative pressure of 450 mbar, and this was followed by a 3-s relaxation period; 3 repetitions were performed for each measurement. Of the 2 measured parameters, R2 reflects the ratio of total skin elasticity to total stretch, while R8 indicates the ability of the skin to return to its original position. The measurements were taken from the left and right sides of the forehead and cheek .
The depth and volume of wrinkles were measured by PRIMOS GFMesstechnik GmbH. PRIMOS is an optical system that creates three-dimensional measurements of selected areas of the skin surface. A sensitive high-definition camera records the image using an optical sensor. The lighting conditions were standardized and repeated for each picture. The system measures variations of up to 10 mm in the surface of the skin .
FotoMedicus software was used to take and collect photos documenting the course of therapy and changes in the appearance of the patient. The system allows greater repeatability of shots between different sessions and visits. Pictures were taken in a sitting position. Standard lighting conditions were used for all pictures. The software also allows for a deeper analysis of the condition of the skin, that is, to accurately photograph various types of discolorations, or wrinkles.
Visual Analogue Scale
Due to the high quality of the FotoMedicus analysis, visual analogue scale (VAS) evaluation was carried out by 3 independent researchers on the images. The VAS ranged from 0 to 100%. It consists of vertical lines marked with no change and total improvement at the bottom and top of the line, respectively. Clinical changes were compared before the series of treatment procedures and again after 8 and 12 weeks.
A subjective evaluation was performed by the participants after 8 and 12 weeks, as well as after the initial study. The survey was designed to assess the perception of the products (0.3 and 0.5% retinol) (n = 37) and the liquid crystal base (N = 28). The participants rated the products on a 5-point scale defined as 5 – definitely satisfied, 4 – satisfied, 3 – not able to assess, 2 – not satisfied, and 1 – definitely not satisfied. The participants rated hydration, tone, elasticity, softness, smoothness, evenness of complexion, and overall skin condition improvement.
Side effects were evaluated on a 4-point scale (0 – none, 1 – mild, 2 – moderate, and 3 – severe). The following side reactions were assessed: burning, eye conjunctival irritation, watery eyes, skin irritation around the eyes, feeling of dry skin, feeling of tightness of the skin, peeling of the skin, burning sensation and stinging of the skin, rash, redness of the skin, itching, pimples, papules, urticaria bubbles, general discomfort, and others.
The assumption of normality and equal variances was assessed by the use of the Shapiro-Wilk test and Levene’s test, respectively. For normally distributed variables, the mean and standard deviation (mean ± SD) was used, while for non-normally distributed variables, median and interquartile range (median [LQ–UQ]) were applied. The differences in percentage changes of analysed skin parameters between the 2 retinol concentrations were determined by means of the non-parametric Mann-Whitney U test. Differences in measured skin parameters between the 3 measured time points were evaluated using ANOVA with repeated measures (with the Greenhouse-Geisser or Huynh-Feldt correction when appropriate) followed by Bonferroni post hoc tests. For the Cutometer®, data were presented as mean and standard deviation (mean ± SD). For normally distributed data with equal variances, the differences between groups were determined by means of the paired t test and Wilcoxon signed rank test. VAS data were presented as median and interquartile range and evaluated by means of the non-parametric Mann-Whitney U test. Differences were considered statistically significant when p values were <0.05.
By 12 weeks, the skin parameters had significantly improved following treatment with 0.3 or 0.5% retinol. Overall skin condition, moisture level, and alignment of skin colour were improved compared to baseline. The hyperpigmentation spots decreased with both 0.3 and 0.5% retinol serum treatments and a significant overall improvement in skin condition resulted at 56 and 84 days. However, no clinical or significant differences in efficacy were observed between the 2 concentrations of retinol.
Medical History Outcomes
Twenty-eight participants completed the initial study. Two subjects were lost to follow-up. The skin phototypes of the participants were scored as I, II, and III, using the Fitzpatrick skin phototype criteria. The results of the group of volunteers (N = 65) are as follows: I = 9, II = 46, and III = 10.
Patch Test Results
The results of patch tests were negative, among the group of 30 patients, which means that the products did not provoke irritative or allergenic reactions in the subjects. After patch testing, it was determined that liquid crystal serum does not have irritating or allergic effects also with retinol.
Results from Initial Study
No participants reported any subjective complaints, that is, feelings of pruritus, burning, or other symptoms that may indicate a negative impact of the evaluated product on their skin. None (100%) indicated that the serum changed colour, consistency, or smell during application, and 88.9% of participants reported fast absorption of the serum. The skin was not found to be irritated, rough, or itchy after 2 months of application. On the basis of the good tolerance results with the liquid crystal base, further studies with the active ingredient were started.
Outcomes from the Courage-Khazaka Multi Probe Adapter
The 12-week retinol treatment resulted in greater skin smoothness, elasticity, and colour (less hyperpigmentation), with an overall improvement in the appearance of the skin and a reduction in apparent skin ageing. Overall, visible signs of photoageing, such as hyperpigmentation, and elasticity improved on both sides of the face. A statistically significant reduction in mean severity scores was observed compared to baseline.
Results after 8 and 12 Weeks of Treatment
Data on percentage change in moisture (a), sebum (b), hyperpigmentation (c), and erythema (d) after 8 weeks of therapy with regard to baseline values obtained before retinol administration are presented in Figure 2. The figure demonstrates medians (horizontal bar with a diamond sign) and lower-upper quartile ranges (box) for 0.3% (light grey boxes) and 0.5% (dark grey boxes) retinol, applied to either the cheek or forehead.
Data are also presented of percentage change in moisture (a2), sebum (b2), hyperpigmentation (c2), and erythema (d2) after 12 weeks of therapy with regard to initial values obtained before retinol was administered. The figure demonstrates medians (horizontal bar with a diamond sign) and lower-upper quartile ranges (box) for 0.3% (light grey boxes) and 0.5% (dark grey boxes) retinol, applied to either the cheek or forehead. No statistically significant differences were observed between the 2 retinol concentrations for any parameters, regardless of the location on the face. However, significant differences were observed between 8 and 12 weeks of treatment, on both the left and right sides of the face.
Results of Skin Tone
Retinol 0.3 and 0.5% improved an overall colour of the skin and decreased the pigment intensity at weeks 8 and 12. Figure 3 presents the results of pigmentation and erythema measurements on the cheek (a and c, respectively) and forehead (b and d, respectively) before (white bars) the application of retinol (0.3 or 0.5%) and after 8 (light grey bars) or 12 (dark grey bars) weeks. The values represent mean ± SD (*p < 0.05, **p < 0.01, ***p < 0.0001 vs. baseline; #p < 0.05, ##p < 0.01, ###p < 0.0001 vs. 8 weeks). No significant differences were observed between the mean effects of treatment with 0.3 versus 0.5% retinol.
Results of Skin Hydration and Sebum Levels
Retinol significantly improved skin hydration on the right and left sides (p < 0.0001). No significant change in sebum level was observed after 12 weeks of treatment on the left side of the face.
Figure 4 presents the results of moisture and sebum measurements of the cheek (a and c, respectively) and forehead (b and d, respectively) before (white bars) the application of retinol (0.3 or 0.5%) and after either 8 (light grey bars) or 12 (dark grey bars) weeks. The values represent mean ± SD (*p < 0.05, **p < 0.01, ***p < 0.0001 vs. baseline; ##p < 0.01 vs. 8 weeks).
Results of Elasticity
Gross elasticity was found to improve: values closer to 1 indicate a better result. On both parts of the cheek, skin elasticity (R2) improved continuously: 0.735 ± 0.091 to 0.767 ± 0.077 (p = 0.017) for the right and 0.753 ± 0.070 to 0.785 ± 0.096 (p = 0.081) for the left. A significant improvement in elasticity was only observed on the right side of the face (0.3%). The parameter R8 decreased on the forehead and on the cheek, indicating that the skin is better able to return to its original position (Table 2).
Results of Skin Profilometry
All PRIMOS parameters for skin roughness improved significantly over the course of the study (Fig. 5). Instrumental tests indicated favourable effects of the liquid crystal formula with retinol on photoageing skin. Improvement was recorded among each of 5 included participants.
The images in Figure 6 present the left and right sides of the face before treatment and after 8 and 12 weeks of serum application. The VAS results indicate that the mean/median change in the colour of the facial skin was 23.3% on the left and 24.3% on the right after 8 weeks, and 26.2 and 27.9%, respectively, after 12 weeks. No significant differences were observed between the left and right sides (Table 3). The highest percentage change observed in the VAS assessment was found for smoothing the surface of the skin: by 26.7 and 29.6% for 8 and 12 weeks on the left, and by 28.5 and 31.2% on the right. A slightly greater reduction in discoloration was observed on the right side (24.6% after 8 weeks and 28.9% after 12 weeks) than on the left (22.4% after 8 weeks and 26.4% after 12 weeks). In addition, overall skin condition improved by 26.2 and 28.5% after 8 and 12 weeks on the left side, and by 26.7 and 29.3% on the right.
Side effects were perceived as greater on the left side than on the right side; however, no significant difference in effectiveness was observed between the sides. However, the volunteers noticed a greater improvement after 12 weeks of therapy with 0.3% retinol. The absolute values and percent changes in self-assessment are presented in Figures 7 and 8.
Significantly more side effects were noted in the 0.5% retinol group. The self-assessments demonstrated a significantly higher level of tolerability with 0.3% retinol than 0.5% retinol at both 8 and 12 weeks. Peeling, irritation, and itchiness were reported at 8 and 12 weeks on the left side (0.5%) and the right (0.3%). Significantly more reports of side effects were associated with the 0.5% retinol (Table 4).
Our study provides an initial analysis of a new cosmetic product consisting of retinol in a novel liquid crystal formula intended for topical treatment with the aim of improving the appearance of ageing skin; the studied formula includes the maximum concentration of retinol allowed for use in cosmetic products, that is, 0.3% , and compares it with 0.5%. Previous publications have evaluated the medical use of various derivatives of vitamin A (tretinoin, adapalene, and tazarotene) in the therapy of various forms of acne, including youth acne and acne vulgaris; as an anti-ageing medication; and in the treatment of diseases associated with keratosis disorders, ichthyosis, and psoriasis [14‒16].
Griffiths et al. evaluated whether 0.1 and 0.025% tretinoin cream causes irritation or promotes any improvement in skin parameters, and which concentration maximizes clinical response with minimal side effects. It was found that both 0.1 and 0.025% tretinoin produce similar clinical and histological changes in patients with photoageing; however, significantly greater side effects were observed at the higher concentration.
The occurrence of side effects during the course of treatment may have a negative effect on the assessment of skin conditions by the participants, and can contribute to the abandonment of the treatment or force changes which may lead to failure. Our present findings indicate that the tested liquid crystal serums with retinol demonstrate few reported side effects at the 2 tested time points; however, more intensive side effects were observed with the concentration of 0.5% retinol than 0.3%. In addition, the 2 concentrations gave comparable results after 12 weeks. The lower concentration yielded better results than the higher concentration.
Kang et al.  assessed the efficacy and safety of 4 concentrations of tazarotene cream in the therapy of facial photodamage. Tazarotene cream (0.01, 0.025, 0.05, and 0.1%) was applied daily and compared with 0.05% tretinoin emollient cream and its vehicle. The products decreased fine lines, hyperpigmentation, and pore size, and improved elasticity and overall appearance. The greatest efficacy was demonstrated by 0.1% tazarotene cream. However, the higher concentrations of tazarotene were associated with a higher incidence of adverse events .
Our study used only products and recipes that were created on the basis of a new formula and are owned by the researchers. Using the commercial products, it would not be possible to fully assess whether the formula has an impact on the improvement of skin parameters or influences the penetration of the active ingredient.
In particular, the skin colour significantly improved, and hyperpigmentation reduced, by 23% on the left and by 19% on the right cheek (p < 0.0001); it also decreased by 15% on the left forehead and 18% on the right forehead (p < 0.0001). Skin hydration was significantly enhanced by 18% on the left and 26% on the right (p < 0.0001). Skin elasticity improved after 12 weeks of treatment, but not significantly; this might be due to the time that the skin needs to regenerate its deeper layers. However, the skin was reported to have generally better elasticity and smoothness in self-assessment.
McDaniel et al.  reported that AHA-Ret was more tolerable than 1.0% retinol and 0.025% tretinoin, suggesting that a higher concentration of retinol could cause some adverse effects; unfortunately, the formula of the product was not given in the study. The present study examined whether the liquid crystal formula with retinol (0.3 and 0.5%) could provide both better penetration and skin moisturization, and be better tolerated over a long-term study. A previous evaluation of the efficacy of retinol 0.5% cream in combination with niacinamide 4.4%, resveratrol 1%, and hexylresorcinol 1.1% by Farris et al.  identified a visible improvement of overall skin condition after 2 weeks of first application. After 4 weeks, significant improvements in hyperpigmentation, evenness of skin tone, and wrinkles were observed compared to baseline. No dryness, itching, or tingling was observed after 10 weeks of treatment .
Another randomized, double-blinded study by Dhaliwal et al.  evaluated the efficacy of bakuchiol 0.5% cream applied twice daily (N = 21) or retinol 0.5% cream applied daily (N = 23) over 12 weeks. A facial photograph was taken to analyse the effects at 0, 4, 8, and 12 weeks. The retinol product was found to cause more side effects, such as itching, burning, and scalding, than bakuchiol. Both bakuchiol and retinol significantly decreased hyperpigmentation and wrinkles, but with no significant difference between the compounds .
Our present findings indicate that the liquid crystal formula is well tolerated and absorbed quickly through the skin. While there were few subjective reports of itching or burning with 0.3% retinol, more side effects were observed with 0.5% retinol and these were more intense. Hence, it is recommended that products with 0.5% retinol should first be applied every second, or even third, day to assess tolerance and efficacy.
Our unpublished pilot study (early view publication)  comparing 0.15 and 0.3% retinol confirms that the 0.3% liquid crystal formula improves the condition of the skin while being well tolerated during 2 months of treatment: a patch test found 0.15 and 0.3% liquid crystal serum to not cause any irritating or allergic effects. We may assume that the better effect of 0.3% retinol, according to the volunteers, is due to the fact that the use of higher concentration (0.5%) was unpleasant and less convenient. Hence, both subjective and objective methods of measurement were employed, and a range of equipment was used to objectively assess the effects of treatment. In addition, 3 independent specialists assessed the effects of treatment on a VAS.
Although cosmeceuticals are not drugs, they claim to have drug-like effects and, hence, require testing for efficacy and safety. The application of 0.3 or 0.5% retinol in liquid crystal formula was found to provide significant improvements for all efficacy end points. Treatment with 0.3% retinol in liquid crystal formula represents a new advancement in skin rejuvenation, delivering optimal retinoid efficacy with minimal irritation in subjects with skin affected by signs of ageing.
Financial support from the Medical University of Lodz (Grant No. 502-03/3-066-02/502-34-094 and Grant No. 503/3-066-02/503-31-001-18-EB) was gratefully acknowledged. Thanks to the Dr Koziej company for enabling the formulation of cosmetics tested.
Statement of Ethics
The test was conducted under the supervision of a dermatologist matched in accordance with the Declaration of Helsinki and the recommendations and guidelines of Cosmetics Europe. Moreover, it was approved by the Bioethics Committee Nos. RNN/210/17/KE and KE/229/19. Informed consent was obtained from all the participants of the study.
The authors have no conflicts of interest to declare.
Substantial contributions to the conception or design of the work: Malwina Zasada. Acquisition, analysis, or interpretation of data for the work: Malwina Zasada, Anna Erkiert-Polguj, and Elżbieta Budzisz. Drafting the manuscript or revising it critically for important intellectual content: Malwina Zasada, Anna Erkiert-Polguj, and Elżbieta Budzisz. Final approval of the version to be published: Malwina Zasada, Anna Erkiert-Polguj, and Elżbieta Budzisz. Agreement to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved: Malwina Zasada, Anna Erkiert-Polguj, and Elżbieta Budzisz.