Investigation On The Dynamic Mechanism Of Transferrin Promoting Membrane Transport Of T7 Peptide-Decorated Nano-Drugs

Chemotherapy drugs for the treatment of cancer can eliminate rapidly dividing tumor cells,but their poor water solubility,pharmacokinetics,and strong systemic cytotoxicity limit their therapeutic efficacy.In order to reduce the toxicity of anticancer drugs to healthy cells and increase the therapeutic effect on cancer cells,many studies are focusing on exploring drugs that specifically treat cancer.Transferrin receptor（Tf R）is a cell membrane associated glycoprotein involved in iron homeostasis and cell growth.Due to its high expression on malignant cells,Tf R has been used as a target for therapeutic drug delivery to cancer cells.The T7（HAIYPRH）peptide targeting TfR,which can enter cells through endocytosis with the help of endogenous transferrin（Tf）,has become a promising targeting ligand.The entry of nano-drugs into cells is an important part of the therapeutic effect of drugs.Understanding the dynamic mechanism of Tf promoting the cell membrane transport of T7 coupled nano-drug will help us to more accurately apply targeted nano-drug.In this study,the five-generation polyamide-amine dendrimer（G5-PAMAM）was used as a drug carrier to physically encapsulate the anticancer drug Camptothecin（CPT）.The targeting ligand T7 was modified on the carrier by-NH₂ on the surface of G5-PAMAM to synthesize PAMAM-CPT-T7 targeted nano-drug.The dynamic mechanism of Tf promoting PAMAM-CPT-T7 into cells was investigated by force tracing technology based on atomic force microscopy（AFM）and nano-indentation technology combined with fluorescence imaging technology.The main research contents are as follows:（1）Force tracing technique was used to capture the transmembrane dynamic process of single PAMAM-CPT-T7 into cells and detect its transmembrane dynamic parameters.When A549 cells were incubated in serum-free medium（SFM）for 1 h,the force of entry into cells was 57±18 p N,the distribution of endocytosis was 188.4±92.9 ms,and the probability of endocytosis force signal was 9.0%.When A549 cells were incubated with 25μM Tf for 1 h,the force and distribution of entering the cells were 43±12 p N and166.3±70.0 ms,respectively.The probability of endocytosis force signal was 14.5%.The concentration and time of Tf incubation in A549 cells were changed to measure the force,distribution and force signal probability of PAMAM-CPT-T7 entry into cells.It was found that Tf at the final concentration of 25μM for 1 h could significantly increase the probability of PAMAM-CPT-T7 entry into A549 cells,and the force and distribution of PAMAM-CPT-T7 entry into A549 cells were significantly decreased after Tf incubation.This indicated that incubation of cells with 25μM Tf for 1 h was the optimal condition to promote the entry of PAMAM-CPT-T7 into cells.To understand the pathway of T7nano-drug entry into cells,clathrin-mediated endocytosis was inhibited by CPZ,caveolin-mediated endocytosis was inhibited by Filipin,and endocytosis pathway was inhibited by EIPA.The probability of force signal was significantly reduced after inhibition,indicating that T7 nano-drug entered into cells through the above pathways.The above conclusions were supported by fluorescence imaging experiments.（2）Nano-indentation technique was further used to detect the membrane transport efficiency of PAMAM-CPT-T7 under different conditions.A549 cells were incubated in SFM for 1 h and then treated with PAMAM-CPT-T7.The Young’s modulus of A549 cells was 1.1±0.5 k Pa.When A549 cells were incubated with Tf at the final concentrations of 15,25 and 35μM for 1 h,the Young’s modulus was 1.0±0.5 k Pa,0.6±0.3 k Pa and 0.5±0.2 k Pa,respectively.When A549 cells were incubated with Tf at the final concentration of 25μM for 10 min,1 h,and 2 h,the Young’s modulus was 1.1±0.5 k Pa,0.6±0.3 k Pa,and 0.6±0.3k Pa,respectively.It was found that the Young’s modulus of A549 cells decreased significantly after incubation with 25μM Tf for 1 h,but there was no significant change in the Young’s modulus measured by incubation with a higher concentration of Tf or a longer incubation time,indicating that 25μM Tf for 1 h is the optimal condition for promoting the membrane transport of PAMAM-CPT-T7.（3）To investigate the promoting effect of Tf on PAMAM-CPT-T7 entry in Vero cells.It was found that the probability of endocytosis force signal detection in Vero cells treated with SFM and 25μM Tf for 1 h was 3.3%and 3.1%,respectively,and there was no significant change.The results of nano-indentation showed that the Young’s modulus of the cells obtained in SFM was 1.6±0.8 k Pa,and that of the cells incubated with 25μM Tf for 1h was 1.5±0.7 k Pa,which showed the same trend as the results of force tracing experiment.These results indicated that Tf did not significantly promote PAMAM-CPT-T7 entry in healthy cells with low Tf R expression,which was also confirmed by fluorescence imaging experiments.