Cell Membrane Transport Of Folic Acid Targeted Nano-Drugs Among Different Cell Lines

Targeted drug delivery systems（TDDS）deliver anticancer drugs to specific sites through ligand-receptor interactions,it can improve the selectivity of drugs to tumor tissue and cells and reduce its side effects.The specific interaction between folic acid（FA）and folic acid receptor（FR）has attracted the attention of many researchers.FA is inexpensive,readily available,non-toxic,and non-immunogenic.It can be modified on the surface of nano-drugs to accurately target FR expressed on cell membranes and improve the efficiency of nano-drugs entering cells.The different levels of FR expression in the cell membrane of tumor cells and normal cells will lead to the difference in the cell membrane transport kinetics of nano-drugs.Cell membrane transport is the first and most important step in the process of targeted nano-drug receptors entering cells.The analysis of cell membrane transport is of great significance for screening ideal targeted nano-drugs for cancer treatment in different organs.In this study,FA as a targeting molecule,it was modified on the surface of 5th generation polyamid-amine（G5-PAMAM）carrier coated with anticancer drug Doxorubicin（DOX）.The dynamic cell membrane transport of targeted nano-drug into different tumor cells（He La and A549）and normal cells（Vero）was studied in real time using force tracing technique by atomic force microscopy（AFM）.The membrane transport efficiency and mechanism of three cell lines were further investigated by AFM-based nano-indentation and fluorescence imaging.The main research contents are as follows:（1）The endocytosis of single FA-PAMAM-DOX targeted nano-drugs in different cell lines was measured by force tracing technique.The force required for endocytosis of FA-PAMAM-DOX in He La cells is 53±13 p N,the time required is 159.2±62.8 ms,and the average speed is 0.174μm·s^-1.The force required for endocytosis of FA-PAMAM-DOX in A549 cells is 62±17 p N.The required time is 153.2±67.9 ms,and the average speed is 0.189μm·s^-1.The force required in the dynamic process of endocytosis in Vero cells is 55±9 p N,the required time is 141.6±48.7 ms,and the average speed is 0.194μm·s^-1.（2）He La,A549 and Vero cells pretreated with free FA were subjected to competitive inhibition experiments,and the probability of force signals in all force curves decreased from14.2%to 3.3%,9.3%to 2.9%,and 4.9%to 2.9%,respectively.The results indicate that the inhibitory effect of free FA on tumor cells with high expression level of FR is more effective than that of normal cells.The dynamic process of endocytosis of PAMAM-DOX（non-targeted ligand FA）in three cell lines was recorded,and the probability of endocytosis signal is reduced by 76.0%,74.2%and 43.8%,respectively,which indicate the greater endocytosis ability for FA conjugated nano-drugs in tumor cells.Fluorescence imaging experiments were used to support the above conclusions.（3）The effects of FA-PAMAM-DOX targeted nano-drugs on the cell stiffness of different cell lines were further compared using AFM-based nano-indentation technique.It was found that Young’s modulus of FA-PAMAM-DOX targeted nano-drugs in He La,A549 and Vero cells decreased by 49.5%,36.7%and 23.0%,respectively.The results indicated that the changes in cell stiffness is most significant in tumor cells,especially in He La cells.This suggests that FA-PAMAM-DOX targeted nano-drugs have the highest membrane transport efficiency in He La cells,further confirming the results of force tracing experiments.（4）Different endocytosis pathway inhibitors were used to study the transmembrane transport mechanism of FA-PAMAM-DOX targeted nano-drug into He La cells.It was found that the endocytosis process of FA-PAMAM-DOX targeted nano-drug is mainly through clathrin-mediated endocytosis and caveolin-mediated endocytosis and macropinocytosis.Among them,clathrin-mediated endocytosis is dominant.