(M1130-02-14) The Effect of the Edge Activator Combinations in Transethosomal Formulations for Skin Delivery of Thymoquinone

Purpose: Thymoquinone is a bioactive compound found in the seeds of Nigella sativa. It has been extensively studied for its various pharmacological properties, including anti-inflammatory, antioxidant, and antimicrobial effects. Although liposomes and ethosomes have been used as carrier systems for thymoquinone, their efficacy in delivering thymoquinone to the skin is limited. Transethosomes have been shown to improve the skin penetration and bioavailability of various drugs. Transethosomes are vesicular carrier system that are composed of phospholipids, ethanol, and water, and are characterized by their high deformability. The objective of this study is to investigate the use of transethosomes as a potential carrier system for the transdermal delivery of thymoquinone. Specifically, we aim to examine the effect of combining ionic and non-ionic surfactants on the performance of transethosomal formulation. To achieve this, we conducted Langmuir monolayer studies to investigate various lipid-surfactant pairs, to optimize the interfacial and rheological properties of the formulation. We evaluated the use of both anionic and non-ionic surfactants, as well as mixed surfactant combinations to determine the most effective formulation for the transdermal delivery of thymoquinone.

Methods: In this study, Langmuir monolayer studies were conducted to determine the optimal combination of surfactants for the transethosomal formulation. Specifically, Tween 20 was tested in combination with two ionic surfactants, sodium lauryl sulfate and sodium lauroyl glutamate, at a ratio of 4:1 nonionic surfactant to ionic surfactant, respectively. This approach allowed for the selection of the most effective surfactant combination for the transethosomal formulation. Next, the transethosomal formulation was prepared using the optimized surfactant combination. The aqueous phase, containing the drug and edge activator, was added dropwise to the lipid-ethanol mixture while stirring and followed by ultrasonication. The resulting formulation was composed of 3% Phospholipon 90G, 200 mg thymoquinone, 50% ethanol, and 0.05% surfactant for the transethosome formulation and without surfactant for the ethosome formulation. The optimized formulation was then evaluated for its particle size, zeta potential, TEM imaging, entrapment efficiency, transepidermal water loss (TEWL), in vitro human cadaver skin permeation, and antimicrobial activity. The permeation study was performed using vertical glass Franz diffusion cells with dermatomed human cadaver skin samples. The antimicrobial activity was assessed using the disc diffusion method against Staphylococcus aureus ATCC 25923.

Results: The results of the permeation study revealed that the (F2) formulation, which contained a combination of SLS and Tween 20, exhibited the highest permeation of 297.17 (ug/cm²) after 24 hours. This was followed by the (F1) formulation, which contains only Tween 20 as a surfactant, with a permeation value of 267.13 (ug/cm²). The (F3) formulation, containing GLS and Tween 20, showed a permeation value of 177.0 (ug/cm²) after 24 hours. All transethosomal formulations showed higher permeation than the (E) ethosomal formulation, which had a permeation value of 149.0 (ug/cm²) after 24 hours. The control group, which was a hydroethanolic solution, had the lowest permeation value of 77.0 (ug/cm²) after 24 hours. The permeation profiles of thymoquinone over 24 hours are shown in Figure 1. Figure 2 shows the thymoquinone deposition in the epidermal and dermal layers. In addition, Figure 3 demonstrates the antimicrobial effects of the transethosomal formulations compared to thymoquinone alone, azithromycin as a positive control, and blank solution as a negative control.

Conclusion: In the case of thymoquinone, the combination of SLS and Tween 20 in the transethosomal formulation showed the highest permeation rate, indicating the synergistic effect of these surfactants in enhancing transdermal delivery. The F2 formulation, which contained SLS and Tween 20 in a ratio of 1:4, exhibited the highest permeation rate compared to other formulations containing only one type of surfactant or a different combination of surfactants. The use of a combination of sodium lauroyl glutamate and Tween 20 in transethosomal formulations can lead to a more sustained release effect and higher skin deposition of the encapsulated drug. Sodium lauroyl glutamate, an ionic surfactant, can promote the formation of smaller vesicles with higher stability, which can lead to a slower release rate of the encapsulated drug. Additionally, this surfactant can also improve the interaction of the vesicles with the skin, leading to higher deposition of the drug in the skin layers. On the other hand, Tween 20, a nonionic surfactant, can improve the solubility and permeability of lipophilic drugs, leading to a higher concentration of the drug in the vesicles and a higher diffusion rate through the skin which explains F1 formulation behavior. Transethosomal formulations increased the antimicrobial effect of thymoquinone compared to free thymoquinone. The enhanced antimicrobial effect of thymoquinone when delivered in transethosomes can be attributed to several factors. Firstly, the smaller size of transethosomes allows for better penetration of the drug through the bacterial cell membrane, increasing its ability to disrupt the membrane and induce cell death.Secondly, transethosomes can protect the drug from degradation by enzymes, allowing for a higher concentration of active drug.

Figure 1. Skin permeation profile of thymoquinone over 24 hours in Franz diffusion cells containing human cadaver skin samples (N=5)

Figure 2. Amounts of thymoquinone in ug/mg of skin deposited in the epidermis and dermis of human skin samples after a 24-hour permeation study (N=5).

Figure 3. Bacterial inhibition study. Inhibition of bacterial growth on agar plates by control negative; Azithromycin 15ug positive control; F1, F2, F3, E and TQ 40ug.