SOFW Journal 2014 paper on Tagra's encapsulated retinol (Tagravit™ R1): 7% retinol, 98% entrapment, far better stability and ~9% wrinkle reduction.
S. Dochi and D. Goldstein, Tagra Biotechnologies Ltd. SOFW Journal, 140, 8-2014.
Abstract
Retinol has an important role in the regulation of epithelial cell growth and differentiation; it stimulates collagen production, accelerates cell reproduction and normalizes skin keratinisation, resulting in younger, clearer and healthier looking skin. However, while retinol is among skincare's most popular ingredients, its well known instability poses certain problems to product developers and brings many challenges to skincare formulations.
In this paper we describe the preparation of retinol microcapsules using the solvent extraction method in order to increase retinol stability while ensuring its efficacy. Retinol microcapsules were investigated for their physicochemical properties, stability and efficacy.
The well-established microcapsule manufacturing method yielded spherical microcapsules loaded with 7% retinol having an average particle size of 35 µm. The stability results obtained indicate that the mean relative retinol content was significantly higher when formulating with encapsulated retinol compared to free retinol. In addition, the in-vivo clinical study clearly shows that the encapsulation process does preserve the dermatological activity of retinol as an anti-aging/wrinkle agent.
Introduction
In recent years, as part of the trend towards a healthier way of living, consumers have become more aware of the benefits of active ingredients on skin health (1, 2). Therefore cosmetics formulators constantly seek opportunities to innovate and design new skin care products by using various unique actives such as vitamins, peptides, essential oils and antioxidants, which can all improve skin health, maintain its youthful glow and prevent skin disorders (3).
Such innovative products present new challenges to the cosmetic industry since most of those active ingredients tend to degrade once incorporated into the cosmetic formulation, resulting in activity loss. Exposure to heat, extreme pH conditions, oxidants and incompatibilities with other ingredients composing the formulation can all lead to insufficient efficacy and reduced stability, which will eventually damage consumer loyalty.
To overcome those formulation challenges and to give formulators and product developers the technical edge they seek, different delivery systems are being used. By using a delivery system the formulator can enhance the function of the personal care or cosmetic formulation through stabilizing the active ingredients, enabling incompatible ingredients in the same formula, and providing targeted delivery for improved effects.
Microencapsulation is a common technology for the protection and stabilization of active ingredients from the surrounding environment, and it can control and determine the release of the active in the final application (4, 5). The aims of this study were to increase retinol's stability, to ensure its efficient delivery and to preserve its therapeutic effect for cosmetics use.
Retinol is one of the forms of vitamin A. It has an important role in the regulation of epithelial cell growth and differentiation; it stimulates collagen production, accelerates cell reproduction and normalizes skin keratinisation. It has been found to be an effective treatment for different dermatological conditions and skin disorders such as photo-aging, acne vulgaris, psoriasis and wrinkles (6, 7, 8, 9). Retinol significantly improves skin elasticity, smoothness and softness, resulting in younger, clearer and healthier looking skin.
While retinol is among skincare's most popular ingredients, its well known instability (10, 11) poses certain problems to product developers and brings many challenges to skincare formulations. Various delivery systems such as liposomes, micelles, emulsions and lipid nanoparticles were designed to improve retinol stability (12, 13, 14), but with limited success.
In this paper we describe the preparation of retinol microcapsules, named Tagravit™ R1, by the patented microencapsulation technology of Tagra Biotechnologies. Those retinol microcapsules were investigated for their physicochemical properties, stability and efficacy.
Materials and Methods
Retinol 5°C was purchased from BASF. Polymethyl methacrylate (PMMA) and polyvinyl alcohol (PVA) were purchased from Sigma. 1,2,3-Trioctanoylglycerol (tricaprylin) was purchased from Abitec. Butylated hydroxytoluene was procured from Lanxess GmbH. All other reagents used were of high purity or HPLC grade.
Tagravit™ R1 Microcapsule Preparation
Tagravit™ R1 microcapsules containing retinol were prepared by the solvent extraction method (15, 16). The microcapsules were prepared by dissolving PMMA in the organic solvent to form a homogeneous polymer solution. The retinol was added during mixing to the polymer solution, followed by the addition of tricaprylin and BHT to the organic phase.
The organic phase was mixed with a water phase containing 0.2% of PVA and agitated to form an emulsion. The emulsion was transferred into the extraction water phase. At this stage the droplets subsequently solidified into rigid microcapsules due to the extraction of the organic solvent. After the sedimentation of the microcapsules they were collected by filtration and washed with water to remove any impurities. The Tagravit™ R1 microcapsules were dried using lyophilization to obtain a free flowing powder.
Determination of Retinol Content, Entrapment Efficiency and Yield
Retinol was quantified by reverse-phase HPLC using a JASCO HPLC UV-975 system equipped with a variable ultraviolet detector (JASCO Analytical Instruments, Japan). Separations were carried out using a Eurospher-100 column (4 nm, 25 cm × 4 mm I.D.), with an isocratic mobile phase containing 95% v/v methanol and 5% v/v THF, at a flow-rate of 1.0 ml/min. Retinol was detected at 325 nm.
Forty milligrams of Tagravit™ R1 microcapsules were weighed and dissolved in 5 ml dichloromethane (DCM), followed by appropriate dilution with methanol. The filtered samples were analyzed by HPLC. The retinol content (w/w %) in the microcapsules was defined as the measured retinol content in the microcapsules divided by the microcapsule weight. The entrapment efficiency level was calculated using the following equation:
Encapsulation efficiency = estimated content / theoretical content × 100
Production yield was determined and calculated by %Yield = W/Wo × 100, where W is the weight of microcapsules and Wo is the total expected weight of retinol, BHT, tricaprylin and polymer.
Size and Morphological Characterization
Microcapsules were analyzed for their size and size distribution using a HORIBA LA-300 laser diffraction particle size distribution analyzer (HORIBA Ltd., Japan). The microcapsules were suspended in soft distilled water before each measurement. All measurements were made at constant scattering angles (six wide-angle detectors) at 25°C. All data analysis was performed in automatic mode.
The morphology of Tagravit™ R1 microcapsules was characterized by scanning electron microscopy (SEM 505, Philips, Eindhoven, Netherlands). Specimens for SEM observation were made by mounting the microcapsules directly on an SEM grid using double-sided sticking tape, coated with platinum under reduced pressure and then placed in the scanning electron microscope (Magellan™ 400L).
Stability Study
The stability of Tagravit™ R1 was evaluated and compared to free retinol. Stability was evaluated by measuring retinol content (% of initial) over time. In addition, physical observations of formulations incorporated with either Tagravit™ R1 or free retinol were used for qualitatively measuring product stability.
The encapsulated and free retinol were incorporated in an o/w base cream and in a water gel base formulation (pH 7.3) and incubated at 4°C, 25°C and 40°C in closed glass vessels protected from light.
In order to measure the retinol content within the formulation, the following extraction method was used. Cream and gel samples (2 g) were weighed and dissolved by mixing with 5 ml DCM for 2 min, followed by separation by centrifugation for 5 min, 6K rpm at 25°C. The supernatant was removed and transferred into a separate vessel. The extraction procedure was repeated twice. The collected supernatants were diluted with an appropriate amount of methanol. All the samples were filtered and analyzed by HPLC.
In-Vivo Efficacy Test
A randomized blind clinical study was performed to evaluate the anti-aging activity of the encapsulated retinol. The clinical study was carried out according to the Declaration of Helsinki. The study was performed using the Fast Optical in vivo Topometry of Human Skin (FOITS) technique (17, 18).
The subjects applied either the base night cream (n=28) or base night cream containing retinol microcapsules (n=25), according to randomization, twice daily for four weeks. Twenty-eight subjects were used as a negative control without any treatment. During this study no additional cosmetic products were allowed to be used by participants.
The periorbital regions (two regions per subject) were tested. Each tested region was divided into five areas, from the stronger pronounced wrinkles (crow's feet, area 1) to the less marked morphologies (farther from the eye, area 5). Two parameters were determined in order to analyze the morphological structure of the periorbital area: Rz (average depth of roughness) and Ra (arithmetic mean roughness) (19).
In order to assess the potential effect of retinol on the skin profile, the frequency distributions of depth (FDD) were calculated for baseline and end values in the range from -600 µm to +600 µm using 5 µm intervals. The sections of depth were represented as micro-structure (0-50 µm), fine-structure (55–170 µm) and rough-structure (>170 µm). A positive effect was concluded if a decrease occurs at higher depth (>170 µm: rough-structure), while an increase in FDD is measured for lower depth (<170 µm: fine-structure).
Results
Microcapsule Characterization
The well-established microcapsule manufacturing method yielded microcapsules containing 7% retinol with a production yield ranging between 87-93%. The concentration of retinol was monitored using HPLC, and 95% recovery was observed (i.e., final observed concentration of 6.7% w/w) as indicated in Table 1.
Yield (%) | Content (% w/w) | Encapsulation efficiency (%) |
|---|---|---|
91.3 ± 2.9 | 7.4 ± 0.1 | 98.0 ± 1.7 |
Table 1: Mean yield (%), content (% w/w) and entrapment efficacy (%) of retinol in Tagravit™ R1 microcapsules (mean ± SD, n=4).
Morphology
The developed retinol microcapsules appear as a free flowing powder which is light yellow in color. Microcapsules were visualized using SEM, confirming that the microcapsules are spherical in shape as shown in Fig. 1.
Fig. 1: SEM micrograph of retinol microcapsules (Tagravit™ R1). Magnification: (A) 5K/bar 3 µm and (B) 0.5K/bar 10 µm.
Particle Size
The mean size and size distribution of the developed retinol microcapsules were determined by the HORIBA LA-300 laser diffraction particle size distribution analyzer (HORIBA Ltd., Japan). Particle size analysis results are presented in Table 2 and Fig. 2. The mean size value of the microcapsules containing retinol was 35 ± 5 (mean ± SD diameter, µm). The presence of one narrow peak indicates that the microcapsule population is homogenous in size.
Mean size | 90% particle population | 50% particle population | 10% particle population |
|---|---|---|---|
35.25 ± 5.21 | 56.73 ± 8.33 | 32.40 ± 4.68 | 17.27 ± 2.40 |
Table 2: Mean microcapsule size and size distribution by volume (diameter, µm, mean ± SD, n=4). Fig. 2: Representative graph for the mean size and size distribution of Tagravit™ R1 microcapsules.
Stability Results
The stability of encapsulated retinol compared to free retinol, both incorporated into a topical base gel formulation (pH 7.3), was evaluated. The relative content of retinol (% of initial) in each sample was evaluated at 25°C and 40°C and is shown in Fig. 3–4.
The stability results show that the mean relative content was significantly higher when formulating with encapsulated retinol compared to free retinol. The results obtained from samples stored for 550 days at 25°C show that the mean content of retinol was higher in encapsulated form as compared to free retinol, 49% and 0% of initial, respectively (Fig. 4).
Also, the results show that retinol encapsulated in Tagravit™ R1 was more stable after a storage period of 300 days at 25°C. The mean content of retinol was higher in Tagravit™ R1 microcapsules compared to naked retinol, 82% and 10% of initial, respectively.
Storage at 25°C (base gel, pH 7.3) | Tagravit™ R1 (% of initial) | Free retinol (% of initial) |
|---|---|---|
300 days | 82% | 10% |
550 days | 49% | 0% |
Tagravit™ R1 stability was physically observed and compared to free (non-encapsulated) retinol in a gel formulation. The gel was incorporated with 0.1% of retinol (1.45% of Tagravit™ R1) at 40°C for 360 days. It was clearly noticed that by formulating with Tagravit™ R1 the discoloration of the gel formulation was prevented. Tagravit™ R1 microcapsules successfully protect retinol from oxidation and degradation as an active raw material in cosmetic formulations.
Fig. 3: Stability of Tagravit™ R1 microcapsules compared to free retinol incorporated in a base gel formulation (pH 7.3) after 100 storage days at 40°C. Data are represented as mean retinol content % of initial (n=3).
Fig. 4: Stability of Tagravit™ R1 microcapsules compared to free retinol incorporated in a base gel formulation (pH 7.3) after 550 days storage at 25°C. Data are represented as mean retinol content % of initial (n=3).
Fig. 5: Physical observation of base gel formulation incorporated with Tagravit™ R1 microcapsules (left) compared to naked retinol (right). Samples were incubated at 40°C for (A) 1 day, (B) 100 days and (C) 360 days.
In-Vivo Efficacy Results
For the efficacy evaluation of retinol microcapsules on skin, the Fast Optical in vivo Topometry of Human Skin (FOITS) technique was used. Parameters Rz (average depth of roughness) and Ra (arithmetic mean roughness) were determined.
Fig. 6 illustrates the mean changes of Rz and Ra for each tested area after 4 weeks of treatment, while Fig. 7 presents the mean changes in the periorbital zone (average of the 5 subareas) following 4 weeks of application of the different treatments. The base night cream induced an increase in the roughness parameters of about 3%.
The results show that base night cream incorporated with Tagravit™ R1 microcapsules induced a significant reduction of both Rz and Ra of about 9%, compared to the group treated with base night cream and the untreated group.
The mean absolute changes in depths as difference to initial value obtained from subjects in the three experimental groups are shown in Fig. 8. The results obtained from subjects treated with base night cream with Tagravit™ R1 microcapsules show positive results, as the treatment induced an increase in FDD values in the micro-structure (about 6%) and a decrease of about 12% in the rough-structure.
Fig. 6: Mean changes of parameters Rz and Ra at areas 1 to 5 after 4 weeks (t4w) of treatment with base night cream and base night cream incorporated with Tagravit™ R1 microcapsules, as compared to untreated volunteers. Area 1 in proximity, area 5 in distance to the eye.
Fig. 7: Mean changes of parameters Rz and Ra in the periorbital zone (average of the 5 subareas) after 4 weeks of treatment with base night cream and base night cream incorporated with Tagravit™ R1 microcapsules, as compared to untreated volunteers.
Fig. 8: Mean FDD difference to initial value (%) after 4 weeks of treatment with base night cream and base night cream incorporated with Tagravit™ R1 microcapsules, as compared to untreated volunteers.
Conclusions
While retinol is among skincare's most popular ingredients, it nevertheless poses certain problems to product developers and brings many challenges to skincare formulations.
In an attempt to increase retinol stability and to ensure its efficient delivery while preserving its potency and efficacy, retinol microcapsules, having the commercial name Tagravit™ R1, were successfully prepared and produced by the solvent removal method. Tagravit™ R1 was designed to release the retinol following mechanical pressure. Upon application, while rubbing the formulation into skin, the polymeric shell collapses and the microcapsules begin to fracture, releasing the encapsulated retinol. Microcapsules break down to invisible particles, but still retain a portion of the payload to continue and deliver over time.
Retinol microcapsules, which come as a free flowing powder, are spherical in shape and exhibit an average particle size of about 35 µm. Tagravit™ R1 microcapsules are loaded with 7% of retinol with an encapsulation efficiency of 98%. Since retinol is known to be sensitive to light, pH, oxidants, heavy metals and air, its stability is one of the most important factors when formulating a cosmetic product. The results of this study indicate that the Tagravit™ R1 microcapsules made by Tagra Biotechnologies are significantly more stable than the free form of retinol in various formulations and conditions. In addition, the results obtained from the in-vivo clinical study clearly show that the encapsulation process does preserve the dermatological activity of retinol as an anti-aging/wrinkle agent.
Thus, Tagravit™ R1 used for retinol delivery would play a crucial role in eliciting its efficacy, as retinol is extremely unstable and easily gets degraded to biologically inactive forms on exposure to light and air.
Authors' address: Dr. Shaher Dochi and Dr. Danny Goldstein, Tagra Biotechnologies Ltd., Northern Industrial Park Tel-Hai, N. Galilee, 12100, Israel.
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