Thymoquinone-loaded Nanosized Ethosomal-based Hydrogels: Their Preparation, Characterization, in Vitro, Ex Vivo, and Antimicrobial Evaluation against Staphylococcus aureus
Authors: Pathak Pratibha , Almalki Waleed H. , Alruwaili Nabil K. , Alzahrani Abdulaziz , Almujri Salem Salman , Alhamyani Abdulrahman , Altamimi Abdulmalik Saleh Alfawaz , Sahoo Ankit , Singh Tanuja , Barkat Md. Abul , Kumar Vikas , Verma Amita , Rahman Mahfoozur 
Published: January 2025

Background:
Thymoquinone, nigella sativa, bioavailability, solubility, stability, ethosome, hydrogel, S. aureus.

Aim:
The present study focused on a nanosized ethosome formulation of thymoquinone with potent antimicrobial
activity against Staphylococcus aureus.

Methods:
This study aimed to develop nanosized TQ-loaded ethosome-based hydrogels and evaluate their antimicrobial
effects. The methods included UV‒VIS spectrophotometer analysis, solubility studies, preparation
of TQ-loaded ethosomes, thermodynamic stability, in vitro drug release, characterization, preparation of ethosome-
based hydrogels, ex vivo skin permeation, skin drug retention, and antimicrobial studies against S. aureus.

Results:
UV‒VIS analysis revealed that thymoquinone (TQ) demonstrated a maximum absorbance peak at
λmax 254 nm. TQ has the highest solubility in ethanol (60 mg/mL) and soy lecithin (65 mg/mL). TQ solubility
in PBS 7.4 (75 mg/mL) with ethanol (50:50% w/v) was found to be crucial for determining in vitro and ex
vivo drug release. Ethosomes were developed using soy lecithin (1.5-4.5% w/w), cholesterol (0.20-0.42%
w/w), and ethanol (30-47% w/w) across ten formulations (E1-E10). These ethosomes were further evaluated
for physical stability. Formulations E6-E10, with optimal concentrations of soy lecithin, cholesterol, and high
ethanol, showed thermodynamic stability for up to 6 weeks. These materials were selected for further study because
of their stability and in vitro drug release. E6 resulted in the greatest drug release and permeation due to
the optimal lipid, cholesterol, and higher ethanol concentrations. E6, with a particle size of 114.8 nm, a PDI of
0.247, and a zeta potential of -0.497 mV, showed optimal stability and drug encapsulation, and the TEM results
confirmed the presence of spherical vesicles. The addition of carbopol-940 (1% w/w) resulted in the formation
of a gel, enhancing the topical application and sustained release of the drug. Compared with the TQCG
hydrogel, the E6 hydrogel showed superior TQ permeation and flux over 24 hours. The first-order model
fits the release kinetics, confirming the suitability of the E6 hydrogel for further study. The E6 hydrogel retained
3.6 times more drugs than TQ-CG, enhancing skin penetration and drug delivery. The TQ-loaded ethosome
(E6 in D3) demonstrated the second-highest antimicrobial action, followed by the E6 hydrogel [D2],
with the Clinsol gel as a control [C] showing the maximum inhibition against S. aureus. The efficacy of E6 is
likely due to better diffusion. The slower diffusion of the hydrogel provides sustained action, making it effective
for prolonged topical drug delivery.

Conclusion:
The E6 hydrogel shows promise for local therapeutic benefits and sustained drug release and
could be a superior herbal option for managing skin infections.