Laser-driven VHEE pulsed fast fractionation (PFF): second-scale inter-pulse timing differentially modulates normal tissue and tumour toxicity

Radiotherapy (RT) is constrained by the narrow therapeutic window between tumour control and normal-tissue toxicity. While FLASH RT has been reported to reduce normal-tissue toxicity at ultra-high dose rates (UHDR) with preserved tumour control in several preclinical models, the radiobiological potential of temporal regimes intermediate between conventional fractionation and FLASH remains largely unexplored. Here, we use a laser-plasma accelerator (LPA) to access this regime by delivering sub-picosecond very high-energy electron (VHEE, 50-300 MeV) pulses at ultra-high instantaneous dose rates (UHIDR; > 109 Gy s-1) with externally programmable inter-pulse intervals. We introduce Pulsed Fast Fractionation (PFF): ultrashort (fs-ps) pulses delivered under UHIDR conditions with second-scale inter-pulse intervals and implementation-dependent dose per pulse. In this work, we vary the inter-pulse interval from 1 to 10 s while keeping dose per pulse and total dose constant, and identify an interval of 1 s that enhances normal-tumour separation: human fibroblasts exhibit reduced toxicity, whereas colorectal carcinoma cells show increased cytotoxicity under matched dose conditions. In vivo, zebrafish embryos reproduce the interval-dependent normal-tissue protection, supporting relevance in a whole-organism context. These results establish second-scale pulse timing as a biologically active degree of freedom for VHEE delivery, positioning PFF as a temporally optimised approach that complements spatial dose modulation and extends the current fractionation-FLASH framework.