Studying Membrane Transport Of EGFR Targeted Nano-drug By Atomic Force Microscopy

In cancer treatment,delivery of anticancer drugs to specific sites through drug delivery systems（DDSs）will provide more precise drug administration and higher the treatment effects.In order to improve the efficiency of DDSs,many researchers use ligand modified nano-drugs to specifically target corresponding receptors.Epidermal Growth Factor Receptor（EGFR）,is considered to be a reasonable target for drug delivery.Through ligand-receptor interactions,targeted anticancer nano-drugs can be specifically delivered to cancer cells to achieve therapeutic effects.The drug entry the cell is the first and most important step in the delivery process,and understanding its transmembrane dynamics is crucial for screening EGFR-targeted nano-drugs.In this study,the anticancer drug camptothecin（CPT）was coated with 7-generation polyamide-amine dendritic macromolecules（G7-PAMAM）as drug carriers.Epidermal Growth Factor（EGF）ligand and tumor cell Epidermal Growth Factor peptide GE11（YHWYGYTPQNVI）were modified,and PAMAM-CPT-EGF and PAMAM-CPT-GE11were synthesized.The dynamic mechanism of endocytosis of different targeted nano-drugs was studied by using force tracing technology based on atomic force microscopy（AFM）and nano-indentation technology combined with fluorescence imaging technology under near-physiological conditions,and the targeting effect and therapeutic effect were discussed.The main research contents are as follows:（1）.Dynamic endocytosis of single PAMAM-CPT-EGF targeted nano-drug was tracked by force tracing technique.The force required for endocytosis of PAMAM-CPT-EGF cells was 52±18 p N and the required time was 94.4±35.9 ms.The endocytosis displacement of PAMAM-CPT-EGF cells was 26.6±3.2 nm and the average velocity was 0.2μm·s^-1.Competitive inhibition experiments with free EGF and Genistein treatment showed that the probability of force signal in the force curve decreased from13.6%in the control group to 2.1%and 2.4%,respectively.Dynamic parameters of PAMAM-CPT（EGF without targeted ligand）endocytosis by A549 cell and PAMAM-CPT-EGF endocytosis by Vero cell（EGFR receptor low expression on normal somatic membrane）were recorded.The above indicated that the force signal detected by force tracing came from the endocytosis process of targeted nano-drug mediated by EGF-EGFR interaction.In addition,the endocytosis required by PAMAM-CPT-EGF targeted nano-drug were both larger and slower than those of non-targeted nano-drug.The endocytosis of PAMAM-CPT-EGF targeted nano-drug is mainly mediated by clathrin,caveolae and macropinocytosis by different endocytosis inhibitors.The results were verified by fluorescence imaging experiments.（2）.Dynamic parameters of the endocytosis process of single PAMAM-CPT-GE11targeted nano-drug were studied by force tracing technique.The probability of force signal generation in the force curve was 6.9%,which was lower than the 13.6%probability of force signal generation in the endocytosis process of PAMAM-CPT-EGF targeted nano-drug.These results indicate that PAMAM-CPT-EGF has better membrane transport efficiency due to the better affinity between EGF and EGFR.In addition,the endocytosis of PAMAM-CPT-GE11 nano-drug was measured to be 42±10 p N,the required time was87.8±20.7 ms,the endocytosis displacement was 25.7±1.4 nm,and the average speed was0.3μm·s^-1.It was found that the endocytosis process of single PAMAM-CPT-GE11nano-drug was shorter and faster.（3）.The membrane transport effect of PAMAM-CPT-EGF and PAMAM-CPT-GE11targeted nano-drugs were evaluated by nano-indentation technology based on AFM.It was found that after treatment with PAMAM-CPT-EGF and PAMAM-CPT-GE11 targeted nano-drugs,the young’s modulus of the cells decreased from 2.0±1.1 k Pa to 0.4±0.2 k Pa and 0.6±0.3 k Pa,respectively.After PAMAM-CPT-EGF targeted nano-drug treatment,the young’s modulus of cells decreased more than PAMAM-CPT-GE11 treatment,and the cell hardness decreased significantly.PAMAM-CPT-EGF targeted nano-drug has better membrane transport efficiency than PAMAM-CPT-GE11.