Vesicles of poly-?-glutamate (?-PGA) were capsulated in order to improve skin efficacy and increase an adsorption into the epidermis. The capsulating method of ?-PGA was investigated to demonstrate the formation of self-assemblies by passing through a high pressure microfluidiser, rheological properties and physiological activity of stratum corneum (SC) by using ?-PGA vesicles.
In these compounds were ?-PGA, soy hydrogenated lecithin (HL), soy sterols (SS), cationic agent, which could be stably formed to the suitable vesicles. Barrier function and physiological behaviour of SC were evaluated by in vitro and in vivo tests. Skin moisture activity of volunteers was significantly improved and TEWL was simultaneously reduced. Following skin improvement test of application of PGA liposome, the corneocyte shapes tended to be uniform. These results suggested that ?-PGA vesicles were effective for external ointments and skin care cosmetics.
Poly-?-glutamate (?-PGA) is one of the good natural moisturisers of amino acid types (Fig. 1) achieved from biofermentation of soy beans (Fig. 2).1 However, ?-PGA has still not featured widely in the cosmetic industry, because of its odour and colour instability at high temperatures.1 Also ?-PGA is not able to give an inner moisturising effect, although it has an amino acid backbone as a natural moisture factor (NMF).2 Several cosmetic research papers have already been devoted to the effectiveness on skin epidermis using a ?-PGA formulation compared to sodium hyaluronate (Na- HA).3 But the papers could not isolate the actual inner moisturising effect as well as strengthening of the barrier function.2,4 The generally high molecular polymer like hyaluronic acid (HA) did not penetrate into the skin because of the large gel size. Nowadays, capsulated vesicles of high molecular polymers, like a ?-PGA, are not easy to find in terms of studies related to the application of capsulation technology.
Materials and methods
Materials
Key materials of poly-?-glutamate (Gelprotein A-8002, Idemitsu, Japan), hydrogenated lecithin (Lipoid S-75-3N, Lipoid, Germany) soy sterols (Phytosterol FK-P, Tama biology, Japan) were required for a preparation of the cosmetic and food grade liposome. Polyquaternium-11 (PQ-11, Gafquat 440) a cationic agent was supplied by ISP, USA. All materials in the experiments were applied as a technical grade without further purification.
Preparation of g-PGA liposome
?-PGA liposome was prepared with a selforganisation form by mixing ?-PGA, HL, SS, PQ-11 above their melting points and then cooling down until room temperature (about 25°C). This sample was passed through HPMF (passage times: 1~10 times). The optimum weight ratio of ?-PGA liposome component was made of 1:1:0.2:0.1±0.05. The structure of ?-PGA liposome was confirmed by microscopic observations.
Measurement of physiological activity (in vivo and in vitro test)
Measurements were taken of physiological activity including the moisturising effect, TEWL reduction activity, and skin improvement after application using the ?-PGA liposome applied to the inside forearms of 14 volunteers (aged between 20-40). Water holding capacity (WHC) of ?-PGA liposome against each HA, ?-PGA, HL (same concentration) was measured by Karl-Fisher (in vitro) method.
• Water holding capacity Two grams of each sample containing an equal amount of water was put in a dish and placed in the condition of 37°C±2°C/10%RH. The water holding capacity of ?-PGA liposome compared with non-capsulated ?-PGA was measured by the loss of weight of the samples stored in incubator over a period of time. The water loss content of samples at hour intervals was measured by weighing over a period of 1 to 8 hours.5
• Moisturising effect 2 mg/cm2 of cosmetic essence was applied respectively on the inside forearms of five male and female volunteers (aged 21-40). The volunteers were initialised for 15 minutes in an incubation room (25°C/45% RH). The conductance (?S) of applied and non-applied areas was measured by Corneometer CM825 (CK Koln-Germany).6
• TEWL reductive effect Essence of 1% ?-PGA included liposome was treated twice a day on the forearms of seven male volunteers (aged 21-40) respectively for 28 days and TEWL reduction was measured. The measurement interval was initial then 1, 2, 3, 4 and 6 weeks after application. TEWL was evaluated by using the AS-TW2 (Asahi Biomed, I.B.S. Co., Ltd. Japan) in the incubation room maintained at 21°C and 50% RH.7
• Skin improvement measurement Skin condition of the forearm before and after application was evaluated by tape stripping method.8 A few layers of stratum corneum were stripped off by using cellophane tapes. Then the skin condition was observed by BG dye method.9,10
Statistical analysis
Means and standard deviations were presented and compared by Student’s t-test with significant probability levels of p<0.05.
Results and discussion
Rheological optimisation of the ?-PGA liposome
The design of the vesicle is shown in Figure 3a. It shows the vesicle capsulated ?-PGA inside hole. Figure 3b shows the capsulated ?-PGA product. Appearance was semi-transparent, having a good texture and good adsorption. It is important that the stability of ?-PGA liposome was dependent on the passage time of HPMF. Passing pressure was controlled at 20,000 psi and the passing temperature was set up at less than 30±5°C. As shown in Figure 4, both normal ?-PGA and ?-PGA liposome saw significantly decreased viscosity after passing from 3 times to 10 times due to the passing HPMF power. Therefore, the best condition of passage time considering economical efficiency was homogeneously stabilised above 3 times passing. Hence, the ?-PGA liposome could be expected to absorb into the skin epidermis in a practical application on the skin surface. The vesicle size after 3 passage times was about 500 nm-1 mm. Figure 5c shows the viscometric results for the 1% ?-PGA solution. Although the plot was widely viscose due to the relatively high torque, the viscosity curves with ascending and descending shear rate showed a clockwise hysteresis. ?-PGA liposome (Fig. 5b) showed similar torque but shear rate was decreased against ?-PGA solution. Whereas although the HA solution showed similar torque, the viscosity at low shear rate increased quickly (Fig. 5a).
Confirmation of liposome vesicles
Test samples of ?-PGA liposome were observed to confirm the existence of a bi-layer structure by measuring microscope. As shown in Figure 6, ?-PGA liposome clearly shows the vesicles having bi-layer structures of droplets. The vesicle size was about 100 nm to 1500 nm (average size 700 nm). These capsules could offer excellent long-term stability, even in a high temperature (stored for 4 weeks at 45°C incubation). Therefore, ?-PGA liposome based on HL and PS was confirmed to have vesicle structures, which might be identified to form a lamellar structure as seen in Figure 6.
Water holding capacity of the ?-PGA liposome
The water holding capacity (WHC) was evaluated and compared with various conditions by using the Karl Fisher method (Fig. 7). The measuring condition of WHC was at 50°C/70RH%. As shown in Figure 7, water holding capacity of ?-PGA liposome 4 hours after application was higher than normal ?-PGA and HL at same concentration. Whereas, 1% of HA showed similar capacity as the ?-PGA. Therefore, the use of barrier restoration LC is significantly more effective than that of ?-PGA and HA (p<0.05). The main reason for the increase of WHC could be explained by keeping water content due to formed ?-PGA liposome vesicles with blending HL, SS.?
Clinical evaluation
Moisturising effect
The moisturising activity with ?-PGA liposome compared 1% ?-PGA with 1% HA was observed by measuring skin conductance using seven volunteers. Figure 8 shows the changes of skin conductance over time. After 8 hours, the conductance of ?-PGA liposome was 55.54 mS±1.8. On the other hand, in the case of 1% ?-PGA the conductance was 52.79 mS±1.7. 1% HA was 49.36 mS±1.7. The moisture effect of ?-PGA liposome by 12.5% higher than HA (p<0.05). In addition, according to the Student t-test, the conductance of ?-PGA liposome was confirmed to be significantly higher than that of HA (p<0.05) and ?-PGA (p<0.05). Hence, capsulated ?-PGA liposome was shown to perform much more effectively as a moisturiser than conventional HA.
Reducing ability of trans-epidermal water loss (TEWL)
As shown in Figure 9, the TEWL reducing effect was measured when ?-PGA liposome, non-capsulated ?-PGA, and HA (respectively 1% concentration) were separately applied on the skin. Following 2 weeks to 4 weeks of application (twice a day), both HA and ?-PGA showed slightly decreased TEWL after 3 weeks. In the case of ?-PGA liposome after 4 weeks was significantly decreased in comparison to TEWL reducing activity of non-capsulated ?-PGA. Total average TEWL level after 6 weeks was remarkably decreased by 20.16%. Therefore, the reason for ?-PGA liposome having high TEWL reducing effect, is caused by promoting a synergic effect of water holding capacity forming a bi-layer between HL and SS.
