A Promising Ultra-Small Unilamellar Carrier System For Enhanced Skin Delivery Of α-Mangostin As An Anti-Age-Spot Serum Part 1

Abstract: If it can be effectively delivered to its site of action, α-mangosteen has the potential to develop novel cosmeceuticals due to its melanogenesis-blocking activity. This study aimed to develop an ultra-small unilamellar carrier system for α-mangosteen and to evaluate its effectiveness as an anti-age-spot serum on humans in vivo. The ultra-small unilamellar carrier bases were optimized using a 25-factorial design, with five factors (virgin coconut oil, soy lecithin, Tween 80, and stirring duration and speed) and two levels (low and high); the response of droplet size was analyzed using Design Expert 12®. The anti-spot examination was conducted by capturing digital images of the human skin after topical application of an α-mangosteen-loaded ultra-small unilamellar carrier at night for two consecutive weeks. The results thereof were analyzed using Motic Live Imaging 3.0  and a standard red, green, and blue score. The optimized serum formula was confirmed with a  composition of 2.3% virgin coconut oil, 1% lecithin, and 28.3% Tween 80 (polysorbate 80) at a stirring speed of 1500 revolutions per minute for 15 min. Incorporation of 3% α-mangostin to the optimized base formula produced an ultra-small unilamellar carrier globule size of 16.5 nm, with a zeta potential of −25.8 mV and a polydispersion index of 0.445. Physical characterization of an α-mangosteen-loaded ultra-small unilamellar carrier comprised 90.94% transmittance, a pH value of 6.5, a viscosity of 38 cP,  specific gravity of 1.042 g/mL and 72.46% entrapment efficiency. A transmission electron microscope confirmed spherical nanosized droplets in the system. Topical application of an α-mangosteen-loaded ultra-small unilamellar carrier at night for 2 consecutive weeks demonstrated anti-age-spot activity shown through a significant reduction in intensity and area of spots in human volunteers (p < 0.05).

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【For more info:george.deng@wecistanche.com / WhatApp:86 13632399501】

Keywords: α-mangosteen; nanocarrier; ultra-small unilamellar carrier; cosmeceutical serum; nanoemulsion

1. Introduction

Age spots (liver spots) are the most common form of skin hyperpigmentation [1]. They are most common on skin that has had sun exposure over many years, such as the backs of the hands, tops of the feet, face, shoulders, and upper back and are often an unwelcome sign of aging. Consequently, there is a wide range of skin-whitening products available, with varied efficacy and side effects. An ongoing focus is to develop optimized cosmeceutical products that provide a highly concentrated skin-brightening effect in less time than that taken by conventional products [2]. Another focus is on developing natural-based cosmeceuticals, as they are often perceived more positively by the public and therefore provide marketing advantages. The challenge in developing a  phytocompound-based formula is to effectively deliver the active ingredient to its target sites through the stratum corneum barrier layer [3]. Nanocosmeceuticals offer the potential for enhanced skin permeation with relatively simple application procedures and the potential for targeted delivery. Examples of phytocompound-based nano cosmeceuticals [4–7] include those for vitamin E and D-panthenol Nanotopes™ [8,9].

α-mangostin (1,3,6,trihydroxy-7-methoxy-2,8-bis(3-methylbut-2-en-1-yl)-9H-xanthan- 9-one has the potential to inhibit the process of melanin formation (dark spots) [10]. The compound showed strong anti-melanogenic activity against B16F1 melanoma cells through suppression of the activity of the tyrosinase enzyme, an important enzyme in melanin synthesis, and exertion of a de-pigmentation effect on normal human epidermal melanocytes (NHEMs) [11]. Therefore, α-mangostin can be used as a component of cosmetics or drugs for the treatment of spots, chloasma, or melanosis. 

As a polyphenolic compound, α-mangosteen is susceptible to oxidation that could limit effectiveness when applied to the skin. An effective formulation approach, therefore, needs to enhance both skin permeation and stability of the active compound. Several approaches have shown enhanced stability and skin delivery of α-mangosteen [12], including liposomes [13], niosomes [14], and proteasomes [15].

Our study is focused on the ultra-small unilamellar carrier (USUC) matrix: a nanocarrier system also termed NanotopeTM. This system is characterized by a much smaller droplet size (≤40 nm) in comparison to other unilamellar or multilamellar liposomes (100–300 nm) [8]. Smaller droplets are produced by a fixed ratio of an oily phase, a surfactant, a cosurfactant, and an aqueous phase; ratios are obtained from an optimization procedure. In the USUC, a dispersed phase is surrounded by a single layer of phosphatidylcholine (surfactant) stabilized by a cosurfactant in the dispersing medium [16]. Smaller droplet size is beneficial for deep penetration into the stratum corneum [8].

We describe here the development of a USUC nanocarrier system for α-mangosteen, physical characterization, and in vivo testing in human volunteers. Formula optimization was undertaken using a 25-factorial design [6,17] and was essential to providing a good-quality USUC with optimal droplet size and stability.

2. Materials and Methods 

2.1. Materials

α-mangostin (purity ≥ 90%) was purchased from Institut Teknologi Bandung (Mark Herb, Bandung, Indonesia). Other components included soy lecithin (food-grade; Shankar Soya Products, Indore, India), virgin coconut oil (VCO; Wahana, Padang, Indonesia), and Tween 80 (Bratachem, Jakarta, Indonesia). All other chemicals were of pro-analysis grade.

2.2. Experimental Design 

Optimization was carried out using a 25-factorial design consisting of three composition variables (Tween 80 (X1), soy lecithin (X2), and VCO (X3) concentrations) and two process variables: stirring duration (X4) and speed (X5) (with two levels—high and low) [18]. Thirty-two USUC base formulae were prepared, employing the factors and levels described in Table 1. The dependent variable was the droplet size of the USUC. The size was required in the range of 0–40 nm [19]. The effects of each variable and its interactions were  determined using a factorial equation in the following form:

2.3. USUC Base Formula Optimization

The USUC base formulae were prepared with spontaneous aqueous-phase titration. Soy lecithin and Tween 80 were stirred with a magnetic stirrer (IKA® C-MAG HS 7, Staufen, Germany) at a temperature of 75 ◦C, with VCO added during stirring. Water was titrated slowly into the mixture until a transparent solution was produced [8]. The 32 bases were characterized in terms of their particle (droplet) size using a particle size analyzer (Shimadzu SALD 2300, Tokyo, Japan).

Data were analyzed using Design Expert® version 12 computer software (StatEase®, Minneapolis, MN, USA) to fit the factorial equation with added interactions and correlate the response with the examined variables [17]. The effect and the interactions between the independent variables were described with 3D surface and contour plots [19].

Preparation of α-Mangostin USUC

α-mangostin (3% w/v) was dissolved in VCO before USUC preparation, as described above. The composition of VCO, soy lecithin, and Tween 80 and the conditions of the mixing process were the same as those for the optimized USUC base [8,20,23].

2.4. Characterization of an α-Mangostin-Loaded USUC 

2.4.1. Physicochemical Properties

Characterization of a USUC involves organoleptic examination, pH value, % transmittance, physical stability via freeze-and-thaw cycles, viscosity, specific gravity, droplet size, polydispersity index (PDI), and zeta potential [21,24,25]. The pH of USUC formulae was measured using a pH meter (Hanna Instrument, Woonsocket, RI, USA) that was previously calibrated. This measurement was carried out once a week during 8-week storage at room temperature [26]. Transmittance was measured with a UV-visible spectrophotometer (SHIMADZU UV-1601, Tokyo, Japan) at a wavelength of 650 nm. The transmittance of close to 100% indicates the transparency of liquid samples [27,28].

The physical stability of the USUC bases was evaluated using a freeze-and-thaw cycling test: the USUCs were kept in storage at a temperature of −5 ◦C for 24 h and then at 25 ◦C  for another 24 h. This test was repeated for three cycles. The physicochemical properties of the USUC, such as the pH, viscosity, specific gravity, and transmittance, were evaluated after three cycles [29].

The viscosity of USUC bases was measured with a cup-and-bob viscometer (Brookfield DV2T, Middleboro, MA, USA) using spindle number 3 at a speed of 100 rpm in triplicate. The specific gravity of USUC bases was determined using a pycnometer at 25 ◦C [20,30].

Droplet size, the polydispersity index (PDI), and zeta potential of the α-mangostin USUCs were determined using a particle size analyzer (HORIBA Scientific SZ-100, Kyoto, Japan) at 25 ◦C [31].

2.4.2. Determination of Encapsulation Efficiency (EE)

The amount of α-mangostin entrapped in the USUC formula was released via extraction of an α-mangosteen-loaded USUC with ethyl acetate (1:2), followed by sonication of it for 10 min (Elmasonic S 80 (H), Singen, Germany). The concentration of α-mangostin in the ethyl acetate solution was measured using a validated spectrophotometric analytical method at a λ max of 314 nm [24].

Entrapment efficiency (EE) was calculated using the following equation:

2.4.3. Micros   copic Analysis via Transmission Electron Microscope (TEM)

Morphology of the α-mangosteen-loaded USUC was observed using a TEM (JEOL JEM 1010, Tokyo, Japan) at 80.0 KV and 30,000× magnification. A 10 µL sample was dropped on a grid, dyed with uranyl acetate, and dried. The observation was carried out at room temperature [8,24,32].

2.4.4. Visual Evaluation of an α-Mangostin-Loaded USUC in Human Volunteers 

Patch testing was conducted on the inner forearm skin of 10 volunteers and left for 24 h to check potential irritation reactions, such as red, itchy rashes on the skin. An assay of the anti-spot effect of the α-mangostin USUC was carried out on 2 female volunteers,  aged 57 years and 40 years, respectively. The experiment was performed by the ethical clearance issued by the Faculty of Medicine, Andalas University (document No. 181/UN.16.2/KEP-FK/2020 on 23 December 2020). The α-mangosteen-loaded USUC  was applied thinly on spots and all over the face at night for 2 consecutive weeks. Both volunteers filled out informed consent to participate in this study and agreed not to use any facial lightening cream during the test. Before being photographed, volunteers cleaned their faces with commercial oil-free makeup remover. The volunteers’ faces were photographed using a single-lens reflex digital camera (Nikon D 810, Melville, NY, USA) before and after treatment. Each facial image was taken from a distance of 30–40 cm, using a camera equipped with 36 megapixels and dimensions of 7360 × 4912. The intensity of dark spots was validated by measuring the values of red, green, and blue (RGB color model) with Adobe® Photoshop for Windows and OS X (Microsoft Corp, Redmond, WA, USA). The size of the dark spots was determined using a microscope (Olympus, Ningbo, China) equipped with Motic Live Imaging 3.0. [33–35].

2.5. Statistical Analysis

Data are shown as mean ± standard deviation. A simple t-test was used to confirm the optimized formula against the predicted response; a paired t-test was conducted to identify significant improvements in the parameters of skin conditions. A two-way ANOVA (α = 0.05) was employed to establish a significant difference between means, followed by a Duncan multiple range test at the 5% significance level [18].

3. Results 

3.1. Base Formula Optimization

Thirty-two USUC base formulae were prepared with various factors, as described in Table 2. Measurement of the response variable showed a wide droplet-size range of 11.3 nm–184.5 µm. Only six formulae (F1, F3, F9, F12, F25, and F28) met the criteria of a USUC system, with droplet size ≤ 40 nm [8]. Data in Table 2 were analyzed further with Design Expert® software to obtain a regression model followed by the determination of the optimized formula.

3.2. Response (Y): Effect of Independent Variables on Particle Size

Evaluation of droplet size as the response (Y) was performed. Five factors, or independent variables, were modeled in the experimental design stage, followed by the generation of a  response surface for droplet size. Figure 1 depicts 3D response-surface plots of independent variables’ effects on particle size. The factorial equation obtained is given in Equation (3):

The droplet-size response increased when the composition of Tween 80 and soy lecithin was at higher levels (Figure 1a). Decreasing the level of VCO and increasing the level of Tween 80 resulted in a droplet-size response of 20–70 nm (Figure 1b). The effect of the relationship between Tween 80 and stirring time on droplet-size response showed that a  longer stirring time resulted in a droplet-size response of 20–100 nm (Figure 1c). A droplet size response of 20,000 nm was achieved with a stirring speed of 1200–1350 rpm and a Tween 80 content of 20%. An increase in the Tween 80 level with a lower level of stirring speed increased the droplet-size response by up to 80,000 nm, indicated by the blue-to-green area (Figure 1d). Increasing content of soy lecithin and VCO resulted in larger values of droplet size, as marked with the blue-to-green area (50,000–100,000 nm) (Figure 1e). Increasing lecithin content and using longer stirring time resulted in larger droplet size,  as indicated by the blue-to-green area (50,000–100,000 nm) (Figure 1f). Increasing soy lecithin content with high stirring speed increased droplet size, as marked by the green-to-yellow region (50,000–150,000 nm) (Figure 1g). Evaluation of VCO content and stirring time on droplet-size response showed that a stirring time of 15 min and a VCO level of <1.5% resulted in a droplet size of 40 nm (Figure 1h). The droplet-size profiles were varied,  with different levels of combination for both VCO versus stirring speed and stirring speed versus stirring time (Figure 1i,j).

3.3. Determination of Optimal Formula by Software Design Expert®

Analysis was carried out using Design Expert® to generate superimposed contour plots. In Figure 2, the yellow area represents the prediction area of the optimum base formula, with the droplet-size response. An estimated optimal base formula was found at a concentration of 28.2 Tween 80% and 1% soy lecithin, with a desirability value of 1.00. A desirability value closer to 1 indicates the model’s ability to produce the optimized formula. The optimized conditions for manufacturing the USUC base were a stirring speed of 1500 rpm for 15 min. This composition and these conditions obtained the predicted response of 34.04 nm. Preparation of the optimized formula for confirmation (Table 3)  resulted in a droplet size of 36 nm, which is not significantly different from the predicted response (p ≥ 0.05).

【For more info:george.deng@wecistanche.com / WhatApp:86 13632399501】