Studies Of Nucleolin Aptamer-Modified Triptolide’s Stability And Cells Selectivity In Vitro

Objective: Nucleolin which is a glycoprotein present on the surface of tumor cells have important tumor target potential application. The subject used oligonucleotides aptamer which can specifically bind with nucleolin as targeting molecules. The aptamer coupled with triptolide by acid sensitive key to construct a new type of triptolide-aptamer conjugate cell delivery system targeted to pancreatic cancer cells. The aim was to discuss the stability of triptolide-aptamer conjugate by simulating physiological environment of human body in vitro, then speculated whether the conjugate can be delivered to the target site or not. Moreover, pancreatic cancer cell line BxPC-3as the experimental group, normal pancreatic cell line HPC-Y5and normal liver cell line L-02as control group, to investigate the affinity of the delivery system.Methods:(1) propiolic acid allyl ester coupled with triptolide through the formation of vinyl ether bond by the synthetic method of Michael addition. Intermediate carboxylic acid groups with high activity were obtained under catalytic conditions of tetrakispalladium. Finally, the targeted triptolide-aptamer conjugate was formed via amide bond linking with triptolide intermediates and aptamer by condensation agent DMT-TT.(2) Through simulating human physiological environment in vitro, triptolide-aptamer conjugate was detected at different time points in the buffer with different pH and in plasma by reversed phase high performance liquid chromatography, which was proved by the free triptolide, calculate the release rate of free triptolide, thus speculated that whether the conjugate can be delivered to the target site or not.(3) Pancreatic cancer cell line BxPC-3as the experimental group, normal liver cell line L-02and normal pancreas cell line HPC-Y5as control group, the combining ability of triptolide-aptamer conjugate with pancreatic cancer cells was detected by flow cytometry. The uptake efficiency of conjugate into tumor cells was observed using fluorescence microscopy.Results:(1)52%yield of white solid3-(Tripterygium Wilfordii Hook based ether)-propiolic acid allyl ester was obtained under catalytic conditions in N-methyl morpholine, then obtained white solid3-(Tripterygium Wilfordii Hook based ether)-acrylic acid, finally in the catalytic condensation agent,50%yield of white powder triptolide-aptamer conjugate was obtained. In the synthesis stage, through the LC-MS, NMR, HPLC and other analytical methods to monitor and identification of the target product, the graph was consistent with target product features.(2) Triptolide-aptamer conjugate are stable under physiological conditions and in the plasma, along with a decrease in pH, free triptolide content increased continuously. When the pH value to6, more than60%of free triptolide released from conjugate.(3) The fluorescence intensity enhanced obviously after pancreatic cancer cell BxPC-3incubation with nucleolin aptamer, higher than the random control. While the fluorescence intensity is consistent with randomized consensus after the control group cell L-02and HPC-Y5incubation with nucleolin aptamer. The fluorescence intensity is consistent with nucleolin aptamer after BxPC-3incubation with triptolide-aptamer conjugate.finallyConclusion:In this study, small molecular compound propiolic acid allyl ester reacted with triptolide, so as to construct the intermediate propenyl ether bond with acid sensitivity, finally formation of the targeted triptolide-aptamer conjugates. This is the first time to synthesis triptolide-aptamer conjugate with acid sensitive vinyl ether bond. The conjugate was stable in physiological conditions and in the plasma, along with a decrease in pH, free triptolide content increased continuously. Vinyl ether bond can effectively ensure triptolide-aptamer conjugate stable in plasma and the physiological environment, so as to ensure the success of the delivery system to reach and enter the target site.Nucleolin aptamer on pancreatic cancer cells has good targeting, which can effectively distinguish between target cells and non target cells. Aptamers coupled with triptolide did not change its target, still can specifically bind to pancreatic cancer cell, as a carrier conduct a specific targeting pancreatic cancer cells and entering the cells, so as to reduce the toxic side effect on normal cells and hepatocytes.