Construction Of Polymer-cell Conjugates With Double-click Fluorescent Probe

Among the currently used tumor treatment methods,molecular targeted therapy and immunotherapy are considered to be precise and effective treatment methods.However,molecular targeted therapy has high requirements on the site specificity of tumor cells,and long-term use will lead to drug resistance;immunotherapy such as CAR-T therapy is difficult to operate,expensive,and not easy to be widely promoted.Therefore,it is of great significance to develop tumor cell-specific treatments using simple and effective methods.Cell surface engineering is a method of designing on the cell surface to modulate cell function and cell interactions with other materials.Orthogonal reactive groups can be introduced on the surface of tumor cells using cell surface engineering.The double "click" pre-fluorescent carrier developed by orthogonal reaction can construct bioconjugates modularly,which can not only couple proteins,polymers and antibodies,but also monitor the conjugation process in real time and realize in situ optical detection.Therefore,we designed a fluorescent probe DBCO-Cy7-Nor with double click properties,using it as a pre-fluorescent carrier to construct polymer-cell conjugates through two different click chemistry reactions,and realize the control of the coupling process.For real-time optical monitoring,it is hoped that with the help of double-click fluorescent probes as a bridge to connect with cells,simple and efficient binding of polymers,antigens,peptides,etc.to tumor cells can be developed,bringing more possibilities for further tumor treatment.Our main work is as follows:1.The fluorescent probe DBCO-Cy7-Nor and polyethylene glycol with tetrazine as orthogonal reactive groups were chemically synthesized.2.The fluorescence intensity changes of the fluorescent probe DBCO-Cy7-Nor when single-click and double-click were tested were tested,and whether the fluorescence changes of small molecule coupling and polymer coupling were the same,and whether the polymer chains of different molecular weights were the same,still have the same fluorescence changes.The test results show that the fluorescence is significantly enhanced only when the two orthogonal groups on DBCO-Cy7-Nor react.During the click reaction,all have the characteristics of significantly enhanced fluorescence.3.In vitro experiments of fluorescent probe DBCO-Cy7-Nor: The cell viability was tested by MTT and Live/Dead experiments of He La cells,which proved that DBCO-Cy7-Nor has good biocompatibility;in vitro experiments were carried out with He La cells.In the click chemistry experiment,we found that when DBCO-Cy7-Nor double clicks,there is a layer of red fluorescent substance on the surface of He La cells,which proves that DBCO-Cy7-Nor can visually construct polymer-cells on the surface of tumor cells conjugate.