Abstract: To construct nanostructured lipid carriers(NLCs)with different particle sizes but the same other physicochemical properties, central composite design was adopted. Coumarin-6(C-6)was selected as the model drug due to its high lipophilicity and high fluorescence intensity. Physicochemical properties of NLCs with 100 nm, 200 nm and 300 nm in particle size could remain stable during certain time in K-R solution and PBS. Release experiments in vitro showed that cumulative release of C-6 in NLCs was less than 7% after 24 h. The MTT assay indicated that both blank NLCs and C-6 loaded NLCs showed low toxicity. To confirm the integrity of NLCs in gastrointestinal tract, DiR-loaded NLCs were prepared and the distribution in vivo was monitored by fluorescence imaging. After 6 h oral administration, intact DiR-loaded NLCs could stiu be found, suggesting that NLCs could be used to characterize the uptake in gastrointestinal tract.