Development, optimization, and characterization of bioactive monoterpenoid benzoquinone-loaded cubosomes as a novel nanocomposite material for hemostatic applications.

Rapid blood clotting is essential for controlling traumatic hemorrhage to save the lives of sufferers. For this purpose, hemostatic cubosomal nanoparticles loaded with thymoquinone (TQ) were formulated. The concentrations of the constituents of thymoquinone-loaded cubosomes (TQCs), i.e., the concentrations of glyceryl monooleate (GMO), Poloxamer 188 (P188), and polyvinyl alcohol (PVA), were optimized using a response-surface methodology, considering the optimum values of the polydispersity index (PDI), zeta potential (ζ), entrapment efficiency (EE), and in vitro blood clotting efficiency. The optimized TQC exhibited a PDI of 0.214 ± 0.044, a ζ potential of 68.75 ± 2.01 mV, an EE of 73.61% ± 16.75%, and an in vitro clotting time of 150.22 ± 16.21 s. The particles were found to be highly stable, exhibiting a burst release pattern consistent with the Gallagher-Corrigan pattern. Optimized drug-loaded cubosomes, due to their highly positive ζ potential, led to platelet aggregation, and the released TQ from the TQC was shown to induce platelet activation. This phenomenon ultimately led to a stable, firm blood clot, as observed in surface morphological studies. This effect was further validated using the rat tail injury model, showing a blood loss of 48.07 ± 6.22 mg in the TQC-treated group, which was significantly (p < 0.0001) lower than that in the standard treatment group (273.4 ± 19.70 mg). Additionally, the optimized TQC demonstrated an optimum antimicrobial activity, especially against Staphylococcus aureus, as well as potent in ovo angiogenic activity, supporting their application in open wounds as a potent hemostatic agent with additional advantages for wound healing.