Oxyhydrogen nanobubbles attenuate angiogenesis, inflammation, and immunosuppression in Wistar rats hepatocellular carcinoma model

Abstract

Hepatocellular carcinoma (HCC) remains a major cause of cancer-related mortality worldwide, while currently available first-line therapies are often constrained by systemic toxicity, resistance, and limited single-target mechanisms. Therefore, safer and broader-spectrum therapeutic strategies are urgently needed. This study evaluated the antitumor efficacy of oxyhydrogen nanobubbles (HHOnbs), an innovative multigas nanosystem that simultaneously delivers O₂, H₂, and low-dose H₂O₂, and hypothesized that HHOnbs would suppress HCC progression through multitarget modulation of the tumor microenvironment. A true experimental study was conducted using diethylnitrosamine/carbon tetrachloride (DEN/CCl₄)-induced HCC in male Wistar rats. Six groups were assigned: negative control, positive control, HHOnbs (200 million particles/mL; 0.1 mL per intravenous injection) administered every 2 days for a total of 12 or 24 treatments, and Lenvatinib (1.2 mg/kg body weight, orally) administered in 3- or 5-dose regimens. Antitumor activity was evaluated by immunohistochemical analysis of CD31, CD68, and FoxP3 expression, representing angiogenesis, macrophage-mediated inflammation, and regulatory T-cell-associated immunosuppression, respectively. In addition, TERT gene amplification was quantified using PCR. Compared with the positive control, HHOnbs significantly reduced CD31 by 42.07–43.55%, CD68 by 42.61–48.02%, and FoxP3 by 44.96–48.71% (all p < 0.001), with greater effects observed at higher treatment frequencies. TERT gene amplification was suppressed by up to 47.89% in the 24-administration group. These effects were comparable to those of Lenvatinib, while HHOnbs showed more consistent frequency-dependent responses across all biomarkers. In conclusion, HHOnbs demonstrated significant multitarget antitumor activity in experimental HCC and represent a promising nanotherapeutic candidate for liver cancer management.