摘要Background:Targeted delivery of biological macromolecules to the small intestine remains challenging due to their susceptibility to degradation in the hostile gastric environment.Methods:This study introduces a minimally invasive,in situ injection technique for the murine small intestine that facilitates localized luminal delivery while circumventing gastric barriers.The procedure involves a small abdominal incision for direct injection into the duodenum near the pylorus.Postsurgical monitoring of physiological parameters,systemic inflammatory markers,liver function,and intestinal integrity was conducted over 72 h.Histopathological analysis was performed.The delivery of the functional protein TAT-EGFP(Tat protein fused to enhanced green fluorescent protein)to intestinal epithelial cells was evaluated and compared with oral gavage.As a proof of concept,single-cell RNA sequencing of the intestinal epithelium was performed after high-mobility group box 1 administration.Results:Postsurgical monitoring indicated only transient,anesthesia-related hypo-thermia and minor behavioral alterations.No significant changes were observed over 72 h in body weight,core temperature,clinical severity scores,systemic inflammatory markers(C-reactive protein and leukocytes),liver function(alanine aminotransferase),or intestinal integrity.Histopathological analysis confirmed preserved tissue architec-ture and normal digestive,absorptive,and barrier functions.The model successfully delivered TAT-EGFP to intestinal epithelial cells,an outcome not achievable via oral gavage due to gastric degradation.Single-cell RNA sequencing of the intestinal epi-thelium after high-mobility group box 1 administration revealed inflammatory gene expression patterns in specific epithelial subpopulations.Conclusions:Compared to traditional methods such as oral gavage or organoid cul-ture,this technique offers precise,degradation-resistant delivery of macromolecules in a physiological context.The model's versatility makes it a powerful platform for intestinal research,with applications in drug delivery assessment,gene therapy evalu-ation,and host-microbiota interaction studies.