Study On The Trans-membrane Transport Process Of Aptamer Targeting Nano-drug

Targeted nano-drug delivery system is a new strategy for diagnosis and therapy of cancer,which shows great potential in the delivery of anti-tumor drugs.Targeted nano-drug can reduce the dosage of drug,improve the tumor selectivity of anticancer drugs,reduce the side effects of anticancer drugs,and improve the therapeutic effect of drugs.However,the important dynamic process of targeted nano-drug into cells remains to be studied.Targeted nano-drug are identified the receptors on the surface of cell membrane and transport into the cell,then they can play a further therapeutic role.Understanding its transmembrane dynamic process is very important for the design and application of targeted nano-drug.In this paper,the five generation polyamide dendrimer(G5-PAMAM)was used as drug carrier to load anticancer drug camptothecin(CPT).Then,the aptamer AS1411 was used as a target ligand to modify the surface of G5-PAMAM to synthesize targeted nano-drug PAMAM-CPT-AS1411.The dynamic process of single PAMAM-CPT-AS1411 nanoparticles entry into cells was studied under physiological conditions using force tracing technique based on atomic force microscope(AFM).Meanwhile,the transmembrane transport pathway was studied by force tracing and fluorescence microscopy.The main research contents are as follows:1.The force tracing technique was used to record the transmembrane dynamic process of a single targeted drug PAMAM-CPT-AS1411 into cancer cells(A549 cells).Meanwhile,the parameters such as force,time,probability and average speed of transmembrane transport were measured.The force distribution ranged from 41.15 to 158.49 p N,with an average of 85.86 p N.The corresponding duration ranged from 52.15 ms to 351.75 ms,with an average of 171.11 ms.The probability of generating force signal was 17.78±3.07%,and the average speed was 0.11 μm/s.2.The dynamic parameters of PAMAM-CPT(without the targeting ligand AS1411)entering A549 cells and PAMAM-CPT-AS1411 into Vero cells(normal somatic cells expressing few nucleolin proteins on the cell membrane)were studied.The force was85.04±19.09 p N and 88.60±17.92 p N,respectively.The duration was 85.16±28.39 ms and81.20±18.98 ms,respectively.The probability of the force signal was 6.88±1.72% and4.09±1.52%,the average speed was 0.22 μm/s and 0.23 μm/s,respectively.The results showed that the specific interaction of AS1411 and nucleolin which was overexpressed on cancer cells enhanced the probability of the PAMAM-CPT-AS1411 cell entry.Moreover,the specific interaction induced receptor-mediated endocytosis prolonged the duration and decreased the average speed of single PAMAM-CPT-AS1411 nano-drugcell entry.However,the force for PAMAM-CPT-AS1411 cell entry was not changed.3.The transmembrane transport mechanism of PAMAM-CPT-AS1411 nano-drug into A549 cells was studied by using different inhibitors of endocytosis.The results showed that PAMAM-CPT-AS1411 entry A549 cells through a multiple of pathways,but the main endocytosis pathway was clathrin-mediated endocytosis and macropinocytosis.