Background

Gasotransmitters such as nitric oxide (NO), carbon monoxide (CO), and hydrogen sulfide (H₂S) are small gas molecules produced endogenously in the body and play a crucial role in various physiological processes, including vascular regulation and protection against oxidative stress. Although initially known as toxic substances, at low doses, gasotransmitters have therapeutic benefits through the hormesis mechanism, where small doses provide protective and stimulatory effects, while high doses become toxic. Imbalances in gasotransmitter production due to aging, chronic diseases, or unhealthy lifestyles increase the need for exogenous gasotransmitter supplementation. However, challenges in their application include low gas stability and the risk of toxicity at high doses.

The innovation of nanobubble technology offers a solution to enhance the efficiency and stability of gasotransmitter delivery within the body. With a size of less than 1 µm, nanobubbles can penetrate target tissues such as tumors, evade the immune system, and increase accumulation at therapy sites. Additionally, nanobubbles exhibit high stability in biological fluids and possess a large capacity for carrying gases, making them effective in the treatment of hypoxic tissue oxygenation, cancer, and cardiovascular disorders. This delivery system not only improves the effectiveness of gasotransmitter-based therapies but also minimizes the risk of toxicity.

In addition to gasotransmitters, oxyhydrogen gas (HHO) has also shown potential as a therapeutic agent due to its antioxidant, anti-inflammatory, and antitumor properties. HHO has been proven to inhibit cancer cell proliferation, induce apoptosis, and reduce oxidative stress in various studies. This research is designed to evaluate the impact of HHO nanobubbles on liver cancer progression using Wistar rats (Rattus norvegicus) as the experimental animal model.

However, before clinical applications can be implemented, a comprehensive evaluation of the toxicity of this gasotransmitter combination and the impact of HHO nanobubbles on liver cancer progression is necessary. The optimal gasotransmitter ratio must be maintained to avoid toxic effects, and this study aims to assess the toxicity of gasotransmitter nanobubbles through IC50 measurements (in vitro) and LD50 measurements (in vivo) as a foundation for developing safe and effective gasotransmitter-based therapies. Additionally, through molecular and histopathological analyses, this research on HHO is expected to provide new insights into the working mechanisms of HHO nanobubbles, while also opening opportunities for the development of more effective and low-risk nanotechnology-based liver cancer therapies.

Goals:

1. Evaluation of Gasotransmitter Nanobubble Toxicity and Therapeutic Potential:

• Assess the toxicity of the gasotransmitter nanobubble combination (NO, CO, H₂S)   through IC50 (cell viability) and LD50 (acute toxicity in animal models) measurements.
• Analyze pathological impacts, toxicity symptoms, and potential side effects to ensure the safety of gasotransmitter-based nanotechnology therapies.
• Compare the effectiveness of the gasotransmitter nanobubble combination in influencing cell viability and explore its potential as a safe and efficient therapeutic agent.

2. Assessment of HHO Nanobubble Efficacy in Liver Cancer Therapy:

• Investigate the effects of HHO nanobubbles on liver cancer progression, focusing on cell proliferation, apoptosis, and telomere length in Wistar rat models.
• Compare the therapeutic efficacy of HHO nanobubbles with conventional liver cancer treatments used as positive controls, aiming to develop a more effective and safer nanotechnology-based cancer therapy.

Expected result 

This study is expected to generate comprehensive data on the toxicity and therapeutic effectiveness of the nanobubble gasotransmitter combination (NO, CO, H₂S), including safe dosage thresholds and potential side effects, serving as a foundation for developing safe and effective gasotransmitter-based therapies. Additionally, the research aims to advance the application of nanotechnology in medical treatments, particularly for targeted therapies in cancer, hypoxia, and cardiovascular diseases. By exploring the mechanisms of HHO nanobubbles, the study also seeks to open new opportunities for the development of nanotechnology-based liver cancer therapies that are more effective and have minimal side effects, ultimately contributing to safer and more efficient cancer treatments