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) while preserving tumour control in several preclinical models, the radiobiological potential of intermediate temporal regimes between conventional fractionation and FLASH remains largely unexplored. Here, we show that temporal separation of ultrashort (fs-ps) dose-delivery pulses delivered at ultra-high instantaneous dose rate, with second-scale inter-pulse intervals, can differentially modulate biological response in vitro and in vivo. Samples were irradiated with very high-energy electrons (VHEEs, 50 MeV to 300 MeV) generated by a laser-plasma accelerator (LPA), using a temporal irradiation modality we term pulsed fast fractionation (PFF), with instantaneous dose rates exceeding 10^12 Gy/s. Varying the inter-pulse interval from 1 s to 10 s while maintaining dose per pulse and total dose constant modulated biological response in both in vitro and in vivo models. At the shortest investigated inter-pulse interval ({theta} = 1s), MRC5-hTERT human fibroblasts showed higher viability, whereas HCT116 colorectal carcinoma cells showed lower viability under matched dose conditions. In vivo, the same interval was associated with reduced radiation-induced growth impairment in zebrafish embryos, consistent with the response pattern observed in the non-tumour cell model and extending this timing-dependent response to a whole-organism context. These findings identify second-scale pulse timing as a biologically active degree of freedom for VHEE delivery, positioning PFF as a distinct temporal irradiation regime with the potential to complement spatial dose modulation through temporal optimisation and extend the current fractionation-FLASH framework.