Boosting Radioimmunotherapy by Functionalized Self-Assembled EGCG Nanoparticles Enhances Antitumor Effect for FLASH-RT

Background: With the ability to achieve ideal efficacy while significantly reducing radiation damage to normal tissues, ultra-high dose rate radiotherapy (FLASH-RT) is considered one of the most innovative technologies for cancer treatment in the era of precision medicine. However, compared with conventional radiotherapy (CONV-RT), FLASH-RT has not demonstrated superior efficacy in treating tumors. Methods: We found that the tea polyphenol EGCG could observably promote FLASH-RT X-ray-induced ROS production and DNA damage compared to CONV-RT. A radiosensitizer was further designed by functionalized self-assembled EGCG nanoparticles (named BENPs), aiming to strengthen the anti-tumor effect of FLASH-RT. In vitro experiments such as CCK-8 assay and DNA damage experiment were carried to verify the sensitising effect of BENPs to 4T1 cells. It was further validated in vivo and the molecular mechanism was analyzed using immunofluorescence staining. Biosafety was evaluated by hematoxylin and eosin (H&E) staining and blood routine experiments. Flow cytometry was used to investigate the in vivo immune status of mice triggered by BENPs synergized with FLASH-RT. RNA sequencing assay was employed to estimate the immune response in the spleen of mice. Results: This combined strategy markedly induced apoptosis and necrosis in tumor cells, which availably inhibited the malignant progression of tumors with good biosafety. More than that, BENPs-assisted FLASH-RT facilitated dendritic cell maturation and increased CD8 + Cytotoxic T cells, B lymphocytes, natural killer and memory T cells differentiation, implying the induction of “positive regulation” of the immune microenvironment, with a better immune prognosis. Meanwhile, the activation of immune regulation was confirmed by effectively upregulation of proinflammatory cytokines in the serum. Conclusion: Our study suggests that the potential application of BENPs as a sensitizer for FLASH-RT that brings new inspiration for the future clinical application of FLASH-RT therapy. Keywords: ultra-high dose rate radiotherapy, radiosensitizer, nanoparticles, immune response, tea polyphenol