Abstract: This research aimed at the key issue that chemical drugs and Chinese medicine hydrophilic small molecule anti-tumor drugs were difficult to break through the dense interstitial permeability barrier of pancreatic cancer to achieve the key problem of drug efficacy in the deep part of tumor tissue. To solve this problem, the lipophilic molecule squalene(SQ) and the hydrophilic anti-tumor drug chidamide(CHI) were linked by a trypsin responsive bond to form a prodrug(SQ-CHI) and a folic acid modified prodrug self-assembled nanoparticles(FA-SQ-CHI NPs) were further developed. The feature of prodrug molecules and nanoparticles were characterized.The in vitro release characteristics and cytotoxicity of blank vector were investigated. The efficacy and permeability of the prodrug nanoparticles in the PSN-1 monolayer cell and PSN-1/HSPC co-cultured tumor spheroids model was evaluated. The results showed that SQ-CHI prodrug molecules and FA-SQ-CHI NPs were successfully developed.The nanoparticles were regular spherical, well-dispersed, with a particle size of(173.3±1.5) nm, a drug load of(59.02±0.8)% and showed trypsin responsive release ability. The prodrug nanoparticles can significantly enhance the penetration and anti-proliferation effects of CHI in the PSN-1/HSPC tumor spheroids. In conclusion, the construction of folic acid-modified SQ-CHI prodrug self-assembled nanoparticles can significantly enhance the penetration of CHI in the pancreatic cancer microenvironment in vitro. This research would provide a new idea for the construction of targeted drug delivery system for chemical drugs and Chinese medicine hydrophilic small molecule drugs in the application of anti-pancreatic cancer.