Supplying active substances to deeper layers of the skin is very challenging. Most cosmetic preparations do not penetrate through the stratum corneum to deeper layers of the skin. It is assumed that carriers of active substances should be composed of biodegradable components that undergo enzymatic degradation to the compounds naturally occurring in the human body. According to the published results, carriers exceeding 40 nm are not able to penetrate the living cells of the epidermis or pass into the bloodstream, and so cause no adverse effects [31
]. The use of an active substance, and especially its uncontrolled release, can lead to a hypersensitivity reaction. An extremely important factor determining safety is the proper choice of surfactants, which means that only compounds that have been approved by the American Food and Drug Administration (FDA) and are on the Generally Recognized as Safe (GRAS) list can be used in the preparation of carriers. Besides particle size and biodegradability, attention should be paid to the route of administration. The design of active substance carriers was intended to enable the delivery of hydrophobic substances to the deeper layers of the skin. The high lipophilicity of active compounds is a major obstacle to their use. However, this problem has been solved by introducing active substances into the core of a hydrophobic nanoemulsion system with a modified hydrophilic surface. In this way, it is possible to dermally transport a lipophilic substance and then release it.
3.1. Optimization, Characterization and Stability of Smart SEDDS Systems Obtained through Nanoemulsion Structural Design
The combination of biomaterials—especially biosurfactants and active substances—enables the development of intelligent formulations and opens up enormous possibilities for biomedical applications. The production of optimal SEDDS requires relatively high concentrations (generally over 30% w/w
) of surfactants. Co-solvents, such as ethanol, propylene glycol and polyethylene glycol, can act as co-surfactants in emulsion systems. Solvents can help to dissolve large amounts of hydrophilic surfactants or a hydrophobic drug in the lipid base. Alcohols and other volatile solvents evaporate, which can lead to the precipitation of the active substances encapsulated in SEDDS. The nanoemulsions obtained in this study were stabilized with a natural biosurfactant, i.e., a sodium salt of SF, and prepared using the SEDDS technique. SF is a natural biosurfactant characterized by high surface activity, low toxicity and high biodegradability, whereby it is highly suitable for use in epidermal formulations. Oil is one of the most important substances in creating SEDDS. It enables the dissolution of lipophilic active compounds, facilitates self-emulsification and increases the transport of active substances to the deeper layers of the skin. Capmul—a mixture of mono and diglycerides of medium chain (mainly caprylic) fatty acids—was used as the model oil phase for the construction of phase diagrams. It is an excellent solvent for many organic compounds and a useful emulsifier for water–oil (w/o) systems. Pure surfactants often organize themselves well at the liquid–liquid boundary, which results in relatively stiff interfaces and even liquid–crystal phases. Co-surfactant Transcutol HP was used to achieve ultralow interfacial tension. It was added to the process to enhance the effectiveness and the oil-solubilizing capacity of the nanoemulsion systems. Phase diagrams were constructed with various concentrations of the selected oil, SF and the co-surfactant (the SF, co-surfactant and oil phase weight ratios of 0.01–96.99:0.01–96.99:3–70% w/w
were tested). As nanoemulsions form upon dilution in aqueous media, they are often described as microemulsion pre-concentrates. A ternary phase diagram was constructed and used to identify the region of efficient self-emulsification. By constructing phase diagrams, one can study the phase behavior of surfactant systems. In this way, one can obtain information on the different phase boundaries as a function of the composition variables, and more importantly one can choose the compositions, which after spontaneous dilution (50 mg of a given composition is added to 10 mL of distilled water) form o/w nanoemulsions. This method represents an effective approach to nanoemulsion preparation. It is a low-energy emulsification technique, making use of the chemical energy stored in the components. As a consequence, low-energy emulsifications offer advantages in terms of their low cost, higher energy efficiency and simplicity of implementation [32
]. The choice of an appropriate surfactant is extremely important for controlling the functional properties of nanoemulsions, including their long-term colloidal stability and further interactions with biological systems. The surfactant is adsorbed at the oil–water interface, reducing the interfacial energy, as well as providing a mechanical barrier to coalescence or other nanoemulsion destabilization processes. As a model for further research, a phase triangle with CA as the oil phase was developed. Antioxidant vitamins, including vitamins C and E, play important roles in the cosmetics industry. The antioxidant properties of vitamin C help to delay the aging of skin cells and the sealing of blood vessels. Vitamin C also has anti-inflammatory and discoloration brightening properties. Vitamin E is known as the “vitamin of youth” because it inhibits the aging process of the body. Vitamin E also has valuable nutritional, moisturizing, oiling and regeneration properties. Since these compounds are some of the strongest antioxidants, it was decided to use them as the oil phase in the prepared formulations. CA had already been used in the development of SEDDS systems for oral drug delivery and it might enhance the solubility of poorly water-soluble substances [34
]. In the present study, optimized SF, TR, CA, vitamin C and vitamin E contents were selected, considering the self-emulsification properties of the formulations upon their addition to water under mild agitation conditions. Visual observations were carried out for transparent and easily flowable oil-in-water (o/w) nanoemulsions after dilution. Favorable weight ratios were selected for the individual components on the basis of the developed phase triangle (S3). The amounts were 10–60:10–90:10–65 w/w
for the biosurfactant, the co-surfactant and the oil phase, respectively. The strategy used for the assembly of the self-emulsifying drug delivery systems formulated by mixing surface, matrix and cargo components is presented in Scheme 1
Considering that this type of surfactant-stabilized carrier was new and had not been described in the literature before, we decided to carry out studies to determine the impact of its composition and subsequent dilutions on the physicochemical properties of the formulations. For this purpose, we selected three model compositions stabilized with increasing amounts of SF, namely 10, 30 and 50%, tested both at 100-fold and 1000-fold dilutions. The selected composition samples were characterized by their droplets size (DH
), polydispersity (PdI) and ζ-potential. As can be seen in Figure 1
A, the presence of both 30 and 50% SF in the composition leads to a drastic reduction in the size of the obtained nanoemulsions at the 100-fold dilution. At the 1000-fold dilution, this effect is also visible, although the difference is smaller. It is worth noting that the composition with 10 SF and the one with 30% SF contained the same amount of oil phase (50%). This phenomenon suggests that the biosurfactant plays a key role in stabilizing this type of structure, and is indicative of the excellent compatibility of all of the components in the amounts used. As expected, a higher dilution resulted in a proportional decrease in the next considered parameter, namely PdI (Figure 1
B). The best parameters, expressed by the lowest PdI value, are for compositions containing 50% SF, which indicates their excellent homogeneity. On the other hand, the increasing SF content lowers the zeta potential, which is further reduced as a result of further dilution (Figure 1
C). Nevertheless, the obtained values are very high (e.g., in the range of −75.9–−92.6 mV for the 100-fold dilution), which may suggest their high stability. The size distributions of the nanoemulsions with vitamin C and vitamin E after preparation and after 195 days are shown in Figure S4
. The nanoemulsion containing curcumin dissolved in CA was characterized by a small particle size. However, as can be seen in Table 1
, the PdI of this formulation is higher than the Pdl values of the other formulations. Additionally, aggregation of molecules was observed in the nanoemulsions. Smaller particle sizes ranging from 20 to 50 nm were obtained by Yousef et al. for a nanoemulsion with curcumin [36
]. In their study they used other surfactants, which suggests that the choice of surfactant may influence the size of the particles.
On the basis of the above results, for further research we chose the 50:30:20 (SF/TR/oil w/w
%) composition, proving its universality by using a different oil phase (Tables S3 and S5
) and a different dilution. Table 1
presents data describing o/w nanoemulsions, containing vitamin C or E and CA as the oil phase at a 200-fold dilution. It can be seen that the use of these oils resulted in an increase in the size of the obtained nanostructures and an increase in PdI. Additionally, the zeta potential increased, which is a very positive phenomenon. In summary, it can be stated that despite the larger particle sizes of the nanoemulsions, they are still within the acceptable size range for skin applications and are characterized by high zeta potential values, indicating high stability, which is very important during the production process.
Transmission electron microscopy and scanning electron microscopy were used to determine the morphology of the obtained nanocarriers. Representative images obtained for the systems containing CA, vitamin C or vitamin E showed the presence of well-separated (practically with no aggregation) spherical droplets, with a relatively narrow size distribution (Figure 2
). The obtained results indicate that the droplets are homogenous and sufficiently stabilized. The morphology of the unloaded nanoemulsion system (Figure 2
A) indicates a more spherical carrier shape than in the case of the system loaded with the additional active substance (Figure 2
B). As for the carrier with encapsulated curcumin, its shape differs from that of the other tested formulations. After loading curcumin into the CA and preparing the nanoemulsion, it was observed that the droplets lost their spherical shape. The morphology of the CAC nanoemulsions (Figure 2
B) was characterized by a more spindle-like shape, and the spherical structures became flattened and elongated. This feature and the specific zeta potential may suggest instability of the prepared nanoemulsion. Representative transmission microscopy images are shown in Figure 2
The stability of a nanoemulsion is one of the most important factors for any potential biological application. Such nanostructures when not stabilized electrostatically are usually metastable due to the short-range van der Waals attraction. In order to avoid aggregation due to their low colloidal stability, steric or electrostatic repulsion can be applied for stabilization. The nanoemulsions with the 50:30:20 composition (SF/TR/oil w/w
%, where oil is either CA, Vit C, Vit E) were first visually assessed and no changes in the physical appearance of the formulations were observed. After 7 days, they remained clear with no signs of creaming, sedimentation, flocculation or coalescence. The samples were stored at 25 °C for 195 days and then their stability was evaluated based on the particle size (DH
), particle size distribution and zeta potential. The literature data indicate that sufficiently high and sufficiently low potential zeta values (up to 30mV and below −30mV, respectively) enhance the stability and functionality [37
]. Unfortunately, the storage period had a destructive effect on the systems with solubilized curcumin in CA—flocculation was observed, while systems with Vit C and Vit E remained clear, with slight opacity. The hydrodynamic diameter of the nanoemulsion with Vit C slightly increased, reaching 190.57 ± 0.3 after 195 days. In the case of the system with Vit E, a decrease in diameter down to 147.6 ± 1.9 was observed. Nevertheless, both the decrease of 19.74% for Vit E and the increase of 7.99% for Vit C resulted in system stabilization. Both formulations were characterized by good PdI values of 0.183 ± 0.06 and 0.126 ± 0.016 for Vit E and Vit C, respectively. The zeta potentials (−77.57 ± 0.8 and −89.7 ± 1.14) suggested that the systems had good stability. The formulations containing the vitamins were found to be physically and chemically stable for about 195 days at the room temperature of 25 °C.