Efficacy of a Novel Lyophilized Arginine Lysine Polypeptide in the Treatment of Acne: In Vitro Experiments and a Split-Face Controlled Clinical Trial

Huanga S, Chen Y, Qiao Z, Hu Y, Zhang H, Zhang X, Huang Y and Xiang Q

Published on: 2021-01-01

Abstract

Acne lesions are divided into three types: erythema, pigmentation, and scar, which can damage skin barrier function. And proliferations of keratinocytes are essential for maintenance of epidermal layers. The objective of this study was to assess the potential efficacy of a novel lyophilized cosmetic composed of arginine lysine polypeptide (LCAL) on improving skin condition. We evaluated the efficacy of LCAL in vitro by cell proliferation and adhesion assay, and in vivo by VISIA, CK and dermoscopy. The cell assays result showed that LCAL enhanced the proliferation of normal human primary fibroblast cells and adhesion of HaCaT cells. We also assessed the improvements of skin condition by sebum secretion, ITA and erythema index using CK at 0 (baseline), 2, and 4 week. Compared with the baseline, sebum secretion decreased by 40% and ITA increased at 4 wk?P<0.01?. Post-acne erythema was ameliorated, with erythema index decreasing by 18% after 4 wk?P<0.01?. The imaging captured using the VISIA and dermatoscope confirmed the reduction of post-acne erythema area and erythema index. These results indicated that LCAL was effective for improving skin barrier function which is a potential therapeutic strategy in improving skin barrier function after acne.

Keywords

Acne; Arginine lysine polypeptide; Lyophilized cosmetic; Sebum secretion; Skin barrier

Introduction

Acne is the most common skin disease that affects the appearance of adolescents?affecting more than 80% of people aged 11-30 year. Besides, acne often persists into adulthood, with more than 20% of people reporting acne in their 40s [1,2]. Post-acne erythema, pigmentation, and scarring, often occur during the healing of active acne [3]. These problems can be emotionally distressing to people and can affect all aspects of their lives. Indeed, acne is a difficult financial burden to carry due to its recurrent episodes . Addressing this issue has become a hot topic in clinical practice in recent years [5]. Erythema is caused by excessive expansion of capillaries in the skin, and increased levels of melanin in the epidermis or dermis lead to pigmentation [6]. Moreover, many studies have shown that acne patients have abnormal skin barrier function: changes in sebum membrane composition, higher sebum secretion, etc. [7]. Therefore, repairing skin lesions after acne not only needs to remove the extra deposited melanin, but also accelerate healing of the skin barrier and recover the normal metabolism of the skin.

Currently, for mild to moderate scarring, microneedling and/or sublative or microneedling and chemical peeling are usually sufficient [2]. However, once the scars appear, treating them can be difficult, expensive, and time consuming. This is a disease in which“an ounce of prevention is worth a pound of cure [8].” Therfore, using

Cosmetics with protective and therapeutic functions to repair the skin barrier have caused the industries to search for alternative active ingredients [2,9]. Bioactive polypeptides are ideal candidates for development of these cosmeceutical products due to its wide spectrum of bioactivities [10]. Moreover, bioactive polypeptides not only promote the proliferation of skin cells, but also promote the repair of skin wounds [11]. In addition, arginine/lysine polypeptide have low molecular weights, which can penetrate into the basal layer of epidermis, promote proliferation of keratinocytes and skin repairing [12]. However, most bioactive polypeptides have poor stability and are prone to denaturation at room temperature, which affects their clinical effects [13]. In order to solve this problem, a lyophilized preparation was prepared using vacuum freeze-drying technology. The lyophilized preparation had low water content and inhibited the growth of pathogenic microorganisms under vacuum conditions, in the absence of any preservatives. This compound was produced at ultralow temperatures, and could perfectly preserve the activity of bioactive polypeptides. Due to its portability and ability to get transported at ambient temperatures, the product was favored by general consumers [14].

In this study, a type of lyophilized preparation, LCAL, containing arginine lysine polypeptide, oligopeptide-1, tripeptide-1 copper, soluble collagen and other active polypeptide components exerting repairing activities were prepared by vacuum freeze-drying for the conditioning of facial skin after acne. In order to evaluate the potential efficacy of the LCAL on the repair of acne lesions, the effect on cell proliferation and adhesion were investigated. In addition, the effectiveness of the LCAL for the repair of skin lesions was confirmed in healthy adults by using VISIA and dermoscopy to detect biophysical skin parameters. Moreover, we considered that this study could provide scientific data and an objective evaluation towards the development and application of active powder compounds of protein polypeptides.

Materials and Methods

The experimental protocols were approved by the Ethics Committee of Jinan University (Guangdong, China). The efficacy of the LCAL on acne lesions was evaluated on the basis of the EU EEMCO standard and Safety and Technical Standards for Cosmetics (2015 edition). Each volunteer gave written informed consent to participate in the study and for the use of their clinical photographs.

Cell Proliferation Assay and Cell Adhesion Assay

Human fibroblast primary cells were extracted from circumcised prepuces of infants (provided by the Reproductive Surgery department of the First Affiliated Hospital of Jinan University). Proliferation of Human fibroblast primary cells was assessed using an MTT assay and the concentrations of LCAL are 0.25 μg/mL, 0.0625 μg/mL, 0.0156 μg/mL, 0.0039 μg/mL, 0.0009 μg/mL. Absorbance at 570 nm was measured in a microplate reader (MK3; Thermo, Waltham, MA, UnitedStates). Each assay was performed in triplicate.

Cell adhesion activity on fibronectin (FN) (25 μg/mL) and various concentrations of LCAL (40 μg/mL, 10 μg/mL, 2.5 μg/mL, 0.625 μg/mL) was measured in 96-well plates. Images were then captured with an MF53 microscope (Mshot, Guangzhou, China) at 20X magnification and analysed with ImageJ to calculate the cell adhesion area. The experiment was performed in triplicate, and the number of adherent cells was calculated from the average of images captured at nine positions per well [15].

Clinical Evaluation in Humans

Subject Selection

A total of 30 subjects (20 women and 10 men) with post-acne erythema and Fitzpatrick skin phototypes III-IV were recruited. This study was conducted in accordance with the Declaration of Helsinki. The age limit was balanced between 18 and 45 y (mean 23.67 ± 4.03 y). Exclusion criterion were mental illness; serious diseases of the heart, liver, and kidney; autoimmune diseases; history of skin cancer and severe herpes simplex infection; chemical peeling; skin treatment within the past 3 mo; any type of laser or intense pulsed light treatments within the previous 6 mo; and refusal to give informed consent.

Study Design

The volunteers were instructed to apply an amount of 12 mL of the LCAL (40 μg/mL) on their right facial skin twice daily in the morning and evening after cleaning their face for 4 wk, while left facial skin were treated with 6% glycerinum as control group. Saline (6 mL) should be first added to the LCAL, and then shaken gently to fully dissolve the powder. Clinical (subjective as well as objective) evaluations [16] were made at 0 (baseline), 2, and 4 wk. Upon arrival, subjects were instructed to complete a product efficacy questionnaire on erythema, pigmentation and pain using a 10-point scale (1, none; 2 - 4, mild; 5 - 7, moderate; 8 - 10, severe). Besides, participants were also asked to fill out a questionnaire of the subject Global Aesthetic Improvement Scale (GAIS) [17] which evaluating the parameters (spreadability, smell, absorption, freshness, silkiness, tightness) of product after applying. And using a point system from 1 to 5 (1, highly unsatisfied; 2, unsatisfied; 3, ok; 4, satisfied; 5, highly satisfied) at week 2. Scores of 4 and 5 were regarded as positive answers. After finishing the questionnaire, each subject will be tested for objective evaluation of the product efficacy by using VISIA and dermoscopy.

Sebum Level, ITA and Erythema Index Measurement

Appropriate instruments were used to check skin-associated parameters every 2 wk during the research: The sebum level [18] of skin were measured using the Sebumeter SM810 probes (Courage & Khazaka `Electronic GmbH, Germany). The color of post-acne erythema expressed as a* (red-green chromatic) and individual topology angle (ITA) values was measured using the Skin-Colorimeter CL 400 probe (Courage & Khazaka Electronic GmbH, Germany). The color of the skin was calculated using the L*a*b* color space related values, in which the a* value is proportional to erythema. Accordingly, ITA ( ITA° = [arctan ([L* – 50)/b*) * 180/π)]) [19] classifies skin color from light to dark. The levels of erythema of post-acne erythema were measured using the Mexameter® MX 18 probe (Courage & Khazaka Electronic GmbH, Germany). All these probes were connected to the Cutometer® dual MPA 580 multi probe adapter system (Courage & Khazaka Electronic GmbH, Germany).

Baseline (pre-treatment) data from all subjects were used to evaluate the relation between image analysis-based measurements. In the auto-classification technique, large fluorescence spots and acne-specific inflammation were the main features used in classifying the post-acne erythema. Using the Baseline measurements, the association of the area of the fluorescence spots with respect to the subjects’ post-acne erythema was evaluated.

Changes in the red area of acne lesions and post-acne erythema throughout the study were measured using the VISIA Photograph System (Canfield®, USA), which allows comparison of the photographs taken before and after the experimental procedure. To observe the changes in post-acne erythema in more detail, images magnified by 50 times were taken with the CBS-807 Dermatoscope (CBS, Taiwan).

Evaluation Criterion

The change in cutaneous physiological properties induced by the product was evaluated by assessing skin sebum level parameters at 0 (baseline), 2, and 4 wk after applying the product [20]. The efficacy of the product was also evaluated on the levels of erythem and the ITA value at 0, 2, and 4 wk. Finally, evaluating the skin sensation of the applied product by the GAIS using a 5-point scale.

Data Analysis

All data were expressed as the mean ± standard deviation (SD) of at least three independent experiments and were assessed using the SPSS13.0 software. Student’s t-tests were used to compare improvements between groups, where P < 0.05 was considered statistically significant. GraphPad Prism 6 software (GraphPad Software Inc., La Jolla, CA, United States) was used for statistical analysis.

Results

Cell Proliferation Assay and Cell Adhesion Assay

The proliferation of varying concentrations of LCAL on the proliferation of normal human primary fibroblast cells was assessed. The maximum level of proliferation is 77% over control, and it is dose dependent with an EC50 = 0.0161 μg/mL (Figure 1A). These experiments demonstrated that LCAL promote the proliferation of human primary fibroblast cells.

The cell adhesion activity of HaCaT cells on various concentrations of LCAL were evaluated. Various concentrations of LCAL and FN (25 μg/mL) significantly promoted cell adhesion compared with the control group. Moreover, compared with control group, the LCAL (40 μg/mL ) significantly enhanced cell adhesion (P < 0.001, Figure 1B). We also determined the average number of cells attached to monolayers representing the control and varying concentrations of LCAL groups. The result of Quantitative analysis showed that LCAL (40 μg/mL ) had the highest number of attached cells that were well-spread (Figure 1C). The largest number of cells was attached to LCAL, with approximately 140 cells attached per field of view. When compared with the control group, cell attachment was significantly lower at 70 - 80 cells per field (Figure 1D).

Figure 1: Effects of stereo repair freeze-dried powder (LCAL) on cell proliferation and cell adhesion. A. Effects of varying concentrations of LCAL on the proliferation of normal human primary fibroblast cells at 72 h. The maximum level of proliferation is 77% over control, and it is dose dependent with an EC50 = 0.0161 μg/ml. B. Quantitative detection of the percentage of adherent HaCaT cells. C. Optical micrographs of HaCaT cells with varying concentrations of the LCAL and FN (25 μg/ml) adhering to monolayers. n = 3, means ± SD. *P<0.05, **P<0.01, ***P < 0.001 vs control group.

Subject’s Satisfaction

During the experiment, all 30 subjects completed the required measurements and image analysis. Results of self-questionnaires regarding skin sensation and efficacy of the product after application are shown in Figure 2. The smell, spreadability, absorption, freshness, silkiness, and tightness of the product were assessed among participants (n = 30) who responded positively to the conditions "spreadability" (mean 4.29) and "absorption" (mean 4.06) (Figure 2A). As shown in Figure 2B, the self-assessment regarding the decrease in post-acne erythema, pigmentation, and pain was determined on a 10-point scale (1, none; 2 - 4, mild; 5 - 7, moderate; 8 - 10, severe). After 4 wk of product use, there was a significant decrease in post-acne erythema and pigmentation compared to that in baseline (P < 0.01).

Figure 2: Subjective evaluation of participants based on the skin sensation and the effects after using the LCAL. A. Skin sensation was evaluated using a 1-5 point score based on 6 aspects. The size of the blue area represents the pros and cons of the overall skin sensation. B. Amelioration of erythema, pigmentation and pain was assessed based on semi-quantitative statistics on a 10-point visual analog scale (VAS) (1, none; 2-4, mild; 5-7, moderate; 8-10, severe), at baseline (W0), after 2 (W2) and after 4 (W4) wk. Data are expressed as mean±SD (n = 30). *P < 0.05, **P < 0.01, vs W0 group.

Measurement of sebum levels, skin color and erythema index

As shown in Figure 3A, there were no significant differences between the experiment groups and control group at baseline in sebum levels. Sebum levels were lower in the experiment groups (mean ± SD, 27.6 ± 1.07 vs 35.4 ± 2.71 μg/cm2 W0, P=0.1680) than in the control group (30.23 ± 1.65 vs 35.9 ± 0.29 μg/cm2 W0, P=0.2873) for 2 wk. After 4 wk, sebum levels significantly decreased by 40% (P<0.01, vs W0) in the experiment groups, than in the control group during the study period. This finding indicated that LCAL exhibited a certain effect leading to reduced sebum levels on the surface of the skin (Figure 3A).

The presence of skin color and erythema index between W0, W2, and W4 were shown in Figure 3B, C, and D. As shown in Figure 3B, the a* value significantly decreased both in experiment groups and control group compared with W0, Respectively. After treatment with LCAL for 2 wk, the a* value of the skin decreased significantly to 15.58 ± 0.25, which was 9.14% lower than baseline (P < 0.05). In addition, the percentage of a* value between W0 and W4 was significantly decreased in the experiment groups than in the control group (6.38% vs 12.36%). After 4 wk, the ITA value increased by 7.38 % compared with baseline (43.40 ± 0.98) in experiment groups and increased by in control group. This finding suggested that treatment with the product led to a significant increase of the whitening degree and overall improvement of post-acne erythema.

The erythema indices of post-acne erythema exhibited a reduction after 4 wk of treatment (Figure 3D). After 4 wk, the erythema indices decreased significantly in the experiment groups (316.89 ± 12.84, P<0.01) than in the control group (328.25 ± 20.68, P<0.05) between W0 and W4 . This result indicated that post-acne erythema exhibited a significant improvement after use of the product.

Figure 3: A series of tests on skin and selected post-acne lesions before (W0), after 2 (W2) and after 4 (W4) wk. A. The mean level of sebum at different time points after treatment with the 6% glycerinum (control group) and LCAL. B The a* value of post-acne lesions at different time points after treatment with the 6% glycerinum and LCAL. C. The ITA value of post-acne lesions at different time points after treatment with the 6% glycerinum and LCAL. D. The mean erythema index at different time points after treatment with the 6% glycerinum and LCAL. Data are expressed as mean ± SEM (n = 30). *P < 0.05, **P < 0.01 vs W0 group.

Figure 4 shows the changes in the subjects’ post-acne erythema area and porphyrin counts. When comparing the average percent change in the subjects’ post-acne erythema area with respect to Baseline (W0), at W2, experiment groups showed about 31.63% more reduction compared to control group (26.14%). After 4 wk, experiment groups showed a stronger reduction compared to control group. When comparing the porphyrin counts, both treatments at W2 and W4 showed a reduction. However, at W4, while control group continued to maintain almost the same amount of reduction, the reduction due to treament of LCAL lowered to about 6%.

Figure 4: Treatment effect of LCAL : average percent change of lesion area and porphyrin at W2 and W4 with respect to Baseline (W0) for 6% glycerinum (control group) and LCAL groups. After treatment with LCAL for 4 wk, the lesion area decreased by 49.36%, while decreased by 34.77% in control group. And the porphyrin counts treated with LCAL also decreased faster than in control group.

Image Analysis

Photographs of 3 representative cases are shown in Figure 5. The VISIA photograph system was used to observe the size and color of post-acne erythema areas before and after using the product. After treatment with LCAL for 4 wk, the result showed that there is a decrease of post-acne erythema areas both in experiment groups and control group (Figure 5A). Moreover, the post-acne erythema areas decrease faster than control group between W0 and W4. This result indicated that the acne and post-acne erythema of the subjects were improved after after application of the product.

Compared and analyzed the skin imaging pictures before and after use to judge whether the product has the effect of repairing skin barrier. The images enlarged 50 times were captured using a dermatoscope (Figure 6A). Comparing the circled post-acne erythema before and after treatment, it was found that post-acne erythema became lighter and scars diminished at 4 wk. Statistical results showed that there is a significant difference in experiment groups between W0 and W4 (Figure 6B; P < 0.01). It can be seen that erythema area decreased by 42.83% in experiment groups (P < 0.01) and decreased by 32.25% in control group after treatment for 4 weeks compared with W0. The product had a significant effect on repairing skin lesions after acne.

Figure 5: Images captured using the VISIA photograph system before (W0), after 2 (W2) and after 4 (W4) wk using 6% glycerinum (control group) and the LCAL group. A. QJL, female, 23 y. B. LJY, female, 20 y. C. LDD, female, 25 y. Compared with W0, post-acne lesions and erythema area decreased after using the LCAL at W2, W4 and the rate of decline was faster than in the control group. The dotted yellow lines indicate the left and right face areas. The areas in black represent the areas for analysis.

Figure 6: Representative images of 3 cases taken by dermatoscope (×50) . A. QJL, female, 23 y. B. LJY, female, 20 y. C. LDD, female, 25 y. Post-acne lesions decreased after using the LCAL at W0, W2, W4. After using the product for 4 wk, post-acne erythema became lighte, and the acne scar disappeared. Compared areas are shown in the blue circle. The arrow in image points out an acne scar. Scale bar represents 1 mm. D. Quantitative analysis of erythema area at W0, W2, W4 measured using ImageJ software. n = 30, means ± SD, *P < 0.05 vs W0 group.

Conclusion

In acne patients, due to the high facial sebum secretion, the keratinocyte lipid bilevel structure is destroyed and the skin barrier function is impaired, which will aggravate the severity of acne. [21,22]. It is also known that some acne treatments such as physical treatments like peelings compromise epidermal permeability, and change its physiological activity [7,23]. The use of cosmetic products to improve skin protection and skin barrier function maintenance is essential for keeping the skin in good condition [24,25]. Ly Thi Huong Nguyena proved that Astragali Radix alleviated particulate matter (PM) induced skin barrier defects through regulation of apoptosis and proliferation in keratinocytes [26]. Much of skin barrier function is provided by the stratum corneum, which acts in the homeostatic control of water [27]. The balance between proliferation and apoptosis of keratinocytes is essential for maintenance of epidermal layers and stratum corneum formation [26]. In vitro assays, the LCAL exhibited significant activities in promoting normal human primary fibroblast cells proliferation and HaCaT cells adhesion.

In this study, we carried out a 4-wk efficacy evaluation of a LCAL on 30 subjects with acne lesions. Yonezawa Kaori found that moisturizing skincare was effective for improving skin barrier function and assessed the skin barrier function (transepidermal water loss, stratum corneum hydration, skin pH and sebum secretion) as a primary outcome [20]. Yamamoto Ayako analyzed several parameters in patients with and without acne, and observed an increased sebum secretion rate and reduction in horny layer conductance [28]. According to evaluation of skin parameters during the experimental period, a* value and erythema of the specified acne lesions were significant improved after treatment of product for 4 wk. G. Micali evaluated acne lesions and erythema using standard erythema-directed digital photography (VISIA- CR™ system), which enables a better assessment of minimal lesions and of the background erythema compared with standard photography [29]. In our study, the imaging captured using the VISIA and dermatoscope confirmed the reduction of post-acne erythema area and erythema index. These results demonstrated that LCAL not only accelerated the repair process of acne lesions, but also reduced post-acne erythema and pigmentation.

At present, the main clinical treatment methods for acne lesions are alpha hydroxy acids (AHAs) and laser treatment [30,31]. Although these methods have immediate effects in the short term, they carry the risk of adverse reactions. Long-term use of fruit acid has been reported to cause keratin thinning, barrier weakening and the skin sensitivity [32]. Photoelectric therapy, such as fractional laser and photorejuvenation may lead to secondary skin pigmentation [33]. All above methods have caused damage to the skin, accompanied by varying degrees of pain, skin dryness, tightness and other symptoms. Meanwhile, consumers are required to spend a lot of money for these risky treatments. Therefore, it is particularly important for patients with acne to choose a safe, moderate and sustainable skin care strategy to prevent sequelae after acne.

The core functional ingredients of LCAL are various protein polypeptides, including arginine-lysine polypeptide, soluble collagen, tripeptide-1 copper and oligopeptide-1. Arginine-lysine polypeptide not only promote cell proliferation and improve cell metabolism, but also repair the skin barrier [34]. Copper tripeptide-1, known as copper peptide (GHK-Cu), can accelerate cell renewal and promote the regeneration of the epithelial tissue of the skin [35]. Soluble collagen, containing a large number of hydrophilic groups, has the ability of moisturizing and replenishing the water of the skin [36]. Thus, the synergistic effect of various proteins accelerates the repair of skin barrier function.

Limitations of this study, among other things included the restricted number of test cases, the lack of gender distinction and the lack of distinguishing treatment effects on different grades of acne lesion. Acne lesion are usually divided into 3 types: erythema, pigmentation and scarring. The aim of this study was to mainly investigate the efficacy of the product in reducing erythema after acne. Perhaps a longer-term follow-up would be more fitting for evaluating the effects of treatment on pigmentation and scarring, which would be included in future research. In the present study, we observed a significant improvement in skin condition after the treatment of LCAL. However, it takes longer for the skin barrier to fully recover after acne. LCAL can improve skin barrier function, but it should be noted that it is a daily-use consumer item with restorative function, not a medicine. We still need acne medications to prevent exacerbation of inflammatory acne before utilizing the product to repair acne lesions.

Conclusions

Our research validated that the LCAL was effective in improving the skin condition after acne. This finding was also confirmed by questionnaires, focusing on the self-evaluation of participants and instrumental results. After 4 wk of everyday application of the LCAL solution, erythema index decreased by 18% after 4 wk, and the ITA value of the specified lesion area was increased. We also observed the visible improvement of skin condition after acne examined with VISIA and CBS dermatoscopes.

In summary, the above mentioned research confirmed that lyophilized powder of bioactive polypeptides was effective in the treatment of skin with imperfect functional barrier after acne. It was able to accelerate the healing of acne lesions and reduce post-acne erythema, as well as prevent post-inflammation pigmentation.

Conflict Of Interest: None declared.

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