(T1330-03-21) Ultrasound Responsive Violacein Loaded Liposomes for Sonodynamic Therapy against Broad-Spectrum Bacteria and Melanoma: In Vitro Studies

Purpose: Violacein, a bis-indole compound with known anticancer and antimicrobial properties, faces significant clinical limitations due to its poor water solubility. This study aimed to overcome this challenge by evaluating the efficacy of both free and liposome-encapsulated violacein as a novel sonosensitizer for sonodynamic therapy (SDT). The core objective was to determine if this approach could enhance violacein's therapeutic effects against melanoma cells and various microorganisms by encapsulating it in liposomal nanocarriers. A key part of the study was to elucidate the underlying mechanisms, particularly the role of oxidative stress in the observed therapeutic outcomes. Ultimately, this research provides a comprehensive assessment of a novel strategy that could lead to new, more effective treatments in oncology and infectious diseases, bypassing the inherent physicochemical limitations of violacein.

Methods: To achieve the study's objectives, a multi-faceted experimental approach was employed. A liposomal formulation of violacein was developed using a microfluidics-based method, a technique known for producing uniform nanoparticles with high encapsulation efficiency. The liposomes were composed of EggPC, cholesterol, and DOTAP, carefully selected to create a stable and effective delivery system. The physicochemical properties of these liposomes, including their size, zeta potential, and encapsulation efficiency, were thoroughly characterized using dynamic light scattering and high-performance liquid chromatography. The study then proceeded to evaluate the cytotoxic effects of both free and liposome-encapsulated violacein on two distinct human melanoma cell lines: MICH-2 (primary) and HTB-140 (metastatic). The cell viability was assessed using the MTT assay, a standard colorimetric method that measures cellular metabolic activity. To investigate the sonodynamic aspect, the melanoma cells were treated with violacein and subsequently exposed to ultrasound. The role of oxidative stress in the therapeutic mechanism was quantified using a Muse Oxidative Stress Kit, which allowed for the precise measurement of superoxide radical positive cells via flow cytometry. In addition to the anticancer studies, the antimicrobial efficacy of violacein was tested against a broad spectrum of microorganisms, including both bacteria and fungi. The antimicrobial activity was evaluated in both planktonic and biofilm forms, providing a comprehensive assessment of its potential to combat microbial infections. The study also included a detailed analysis of the interactions between violacein and ultrasound, examining the compound's stability and its ability to generate reactive oxygen species.

Results: The results of this study provide compelling evidence for the enhanced therapeutic efficacy of violacein when combined with sonodynamic therapy. The liposomal formulation of violacein was successfully developed, yielding nanoparticles with a small size (approximately 38 nm) and a high encapsulation efficiency of around 70%. A key finding was that the combination of violacein and SDT led to a dramatic reduction in the viability of melanoma cells, with a decrease of over 90%. This synergistic effect was particularly pronounced in the MICH-2 cell line, where the combined treatment was significantly more effective than either violacein or ultrasound alone. The study also revealed a substantial increase in oxidative stress in the treated cells, indicating that the generation of reactive oxygen species plays a crucial role in the observed anticancer effects. The antimicrobial investigations yielded more varied results, with the efficacy of violacein being highly dependent on the specific microbial strain. Notably, potent bactericidal and fungicidal effects were observed against Vibrio vulnificus and Candida auris, both in their planktonic and biofilm forms. The liposomal formulation of violacein, when activated by ultrasound, achieved up to a 5-log reduction in V. vulnificus, demonstrating its potential as a powerful antimicrobial agent. The study also highlighted the complex interplay between the liposome's surface charge and its interaction with different microbial membranes, offering insights into the nuanced nature of its antimicrobial activity.

Conclusion: In conclusion, this study successfully demonstrates that violacein, particularly when encapsulated in a liposomal formulation and combined with ultrasound, exhibits potent anticancer and antimicrobial properties. The liposomal delivery system effectively addresses the challenge of violacein's poor water solubility, enabling higher and more stable concentrations of the compound. The synergistic action of liposomal violacein and sonodynamic therapy represents a highly promising approach for the treatment of melanoma, as evidenced by the significant reduction in cell viability and the induction of oxidative stress. The findings also underscore the potential of this combination therapy to combat challenging microbial infections, with particularly strong effects observed against Vibrio vulnificus and Candida auris. While the antimicrobial efficacy was found to be strain-dependent, the results highlight the versatility of this therapeutic strategy. The study provides a solid foundation for further research, including in vivo studies, to explore the full clinical potential of this novel approach. The development of an animal model for the preclinical evaluation of SDT with violacein against cancers and microbes is a logical next step. Overall, this research paves the way for the development of new, more effective therapies that harness the power of sonodynamic activation to enhance the therapeutic effects of natural compounds like violacein.

Acknowledgements: Authors acknowledge the funding provided by the National Science Centre, Poland under Grant No. 2023/07/X/NZ6/00520