Design And Synthesis Of CD22 Targeted Nano-materials And Its Application In Tumor Targete Tracing

Objective: Based on receptor-ligand mutual recognition principle,by the chemical and biological coupling method,we designed,synthesized a fluorescent nano-materials with CD22 targeting function,characterized the fluorescent nano-materials property,and completed its tracing on CD22 over-expression cell line through a series of vitro cell targeting experiments,providing a biological and powerfultool for CD22 diagnosis.Method: 1.Based on the receptor-ligand mutual recognition principle and Nacetylneuraminic acid(NANA)had CD22 recognition structure,we derived NANA with two amino carbon imine(EDC),then the unstable derivation of O-acyl radical ester(NANA-EDC)was obtained,the NANA carboxyl was actived in NANA-EDC and used for biological coupling with amino quantum dots(NH2-QDs),then NANA-QDs nanocomposites were prepared completely.2.The NANA-QDs nanocomposite material properties were characterized by measuring NANA ligand coupling degree,particle size distribution,Zeta potential,quantum yield,spectroscopic properties,biological toxicity and other properties,the results proved that NANA-QDs nanocomposites had uniform structure,good fluorescence properties and safety.3.In vitro experiments,NANA-QDs nanocomposites targeting performance was evaluated by fluorescence distribution on CD22 over-expression Daudi cell line,competitive binding experiments,nanoparticle endocytosis assay and immunofluorescence co-localization experiments,provingits specificity and affinity to CD22.4.NANA-QDs nanocomposites were used for detecting CD22 expression level for a variety of tumor cells,and the results were verified by the traditional methods of Western blotting,flow cytometry and immunofluorescence.Result: The TEM and DLS assay results showed that NANA-QDs nanocomposites had about 10 nm average particle size and uniform distribution.The average NANA coupling degree was per NH2-QDs nanoparticles molecular surface could be coupled with 294 NANA molecules,the high ligand concentration made it conducive to the next targeting experiment.The quantum yield kept in about 70% stably after coupling,and the fluorescence spectrum was symmetry,proving NANA-QDs with productive fluorescence performance.In the cell targeting assay,NANA-QDs nanoparticles generated intense red fluorescence signal on Daudi cells surface,and the initial fluorescence signal distribution is similar to the structure of cell membrane;while the negative control with weak fluorescence and nonspecific distribution.Competitive binding with free NANA assay and immunofluorescence co-localization experiments showed that the combination principle between NANA-QDs nanocomposite and Daudi cell was of NANA’s ring structure had specific CD22 recognition ability.In the NANA-QDs nanocomposite kinetics experiment,the fluorescence distribution was from the Daudi cells surface to Daudi cells cytoplasm gradually,which indicated that NANA-QDs could enter the cell through the CD22 endocytosis,above a series of experiments showed that NANA-QDs had a specific identification and targeting function to CD22 receptor.The NANA-QDs nanoparticles were used for the CD22 expression determination of six kinds of tumor cells of lymphoma,liver cancer,lung cancer and breast cancer,showed that the CD22 expression in lymphoma cell was high,while hepatocellular carcinoma,lung cancer and breast cancer cells showed low CD22 expression,and in lung cancer cell A549,the CD22 expression increased with passaging.The reliability of CD22 targeting experimental results was verified by conventional methods such as immunofluorescence,flow cytometry and WesternblottingConclusion: In this study,using the principle of NANA ring structure specific recognition of CD22 receptor,NANA-QDs fluorescent nanomaterial was prepared,NANA-QDs had the uniform particle size and distribution,fluorescence properties,low toxicity,and strong targeting ability to transmembrane glycoprotein CD22,providing a biological and powerful tool for CD22 diagnosis.