Subchronic Toxicity Study of Nitric Oxide Nanobubbles Injection in Sprague-Dawley Rats

Abstract Following the success of hydrogen nanobubble technology, development has expanded to include similar products with other gases, such as nitric oxide (NO). In previous studies, NO donors have been frequently used for both therapeutic and diagnostic purposes. However, the use of NO in nanoformulations has received little attention thus far. Thus, 90 days of subchronic toxicity testing in Sprague-Dawley rats were used in this study to assess potential adverse effects of intravenous administration of NO nanobubbles (NONB). Hematological factors, serum biochemistry, and histology of the liver, kidney, heart, lung, and spleen were investigated. The findings of the intravenous NONB injection trial in rats, using graded doses of 0.01 mL, 0.04 mL, and 0.06 mL, demonstrated no deaths during the 90-day treatment period. Biochemical parameters for liver function, lipid profile, kidney function, and serum electrolyte levels remain within acceptable limits, with slight variations in sodium and potassium levels. This condition was also supported by histological findings in the liver, kidney, and spleen, indicating mild to moderate damage, while the heart and lungs were normal. Thus, the intravenous administration of NONB at a dose of up to 0.06 mL in this subchronic toxicity test remains safe. Further studies with adjustments to the test formulation are highly encouraged until the clinical trial stage in humans. Competing Interest Statement The authors have declared no competing interest. Footnotes Injection material The injection material was obtained from the Indonesian Molecular Innovation (IMI) Foundation as an infusion fluid containing NONB at a concentration of 26 million bubbles/mL. The NONB solution was prepared in reverse osmosis. A buffer containing 1% ascorbic acid and 1% sodium bicarbonate was used to maintain pH stability. All materials were prepared under laboratory protocols, and all preparations were conducted aseptically to minimize contamination(11).