Cellular Uptake
The cellular uptake properties of the proteins labeled with FITC were analyzed by flow cytometry. The uptake of HFtn by tumor cells was mediated by TfR1 has been proved previously. As shown in Figure 3, cellular uptake of HFtn was much higher than that of free FITC, which is probably attributed to the TfR1 receptor-mediated endocytosis. Significantly, the mean fluorescence intensity of FITC-tLyP-1-HFtn was approximately three-fold higher than FITC-HFtn, suggesting that the enhanced intracellular uptake of tLyP-1-HFtn was probably attributed to TfR1 and NRP-1 dual receptor-mediated endocytosis. In contrast, FITC-M-tLyP-1-HFtn showed similar mean fluorescence intensity as the FITC-HFtn, indicating that the mutation at the tLyP-1 peptide sequence resulted in the inactivation of the NRP-1 receptor-mediated endocytosis.