Construction And Performance Study Of High Selective Capture Surface Of Tumor Cells

Detection of circulating tumor cells(CTC)is critical for early diagnosis,treatment,and metastasis of cancer.However,due to the extremely low levels of CTC in the blood of early cancer patients,the isolation and enrichment is challenging.Herein,a cell membrane mimetic surface(CMMS)is constructed on glass and polystyrene substrates to repel blood cell adhesion.Furthermore,tumor cell targeting ligands,folate(FA)and a cyclic arginine-glycine-aspartic acid(RGD)peptide are tethered on CMMS to enhance tumor cell binding and capture.This surface modification strategy of tumor cell capture and blood cell repulsion,will play an important role in biomedicine such as cancer diagnosis and targeted therapy,since this method is a general-purpose,low-cost and effective technical solution.This thesis mainly includes the following three parts:(1)Construction and performance study of folic acid(FA)targeting ligand coating.A polydopamine(PDA)layer with mussel-like universal adhesion was deposited on material-independent substrates such as glass and polystyrene as a mediated layer.An anti-adhesion cell membrane structure coating(CMMS)composed of PMEN91(containing active ester group and phosphorylcholine zwitterionic random copolymer)and polyethylene glycol(PEG,5 k Da)was constructed on the surface of PDA.The ligand FA was then coupled to the terminal carboxyl group of the PEG chain on the CMMS to enhance tumor cell binding with the coating(CMMS/FA).The coatings were constructed either off-line or on-line using a surface plasmon resonance(SPR)instrument and analyzed quantitatively.The static water contact angle(WCA)and atomic force microscopy(AFM)were used to characterize the coating.The results showed that each coating was successfully constructed.Quantitative analysis of cell adhesion showed that the He La cell capture efficiency of CMMS/FA coating from mixed cells(L929:He La=100:1)was 23.5%and the purity was 94.4%.(2)Construction and performance study of peptide(RGD)ligand coating.The folate ligand was replaced with arginine-glycine-aspartic acid(RGD)and coupled to the surface of the CMMS to construct an RGD ligand coating(CMMS/RGD).Firstly,the effects of concentration,temperature,solvent and reaction time on the construction of peptide coating were explored.The coatings formed under various conditions were characterized by static water contact angle(WCA),atomic force microscopy(AFM)and photoelectron spectroscopy(XPS).The results of all the characterization results indicated that the construction of the polypeptide coating was successful.The results of cell adhesion analysis confirmed that the CMMS/RGD coating not only resisted the adhesion of normal cells(L929),but also increased the He La cells capture efficiency from mixed cells(L929:He La=100:1)upto 60.1%with a purity of 93.8%.(3)Construction of dual ligands coating and its application in tumor cell capture.Under the optimal reaction conditions,FA and RGD were anchored to the outwardly extending PEG carboxyl group on the surface of CMMS to construct dual ligand content controllable coating(CMMS/FA-RGD).By screening optimization of cancer cell binding by regulating the surface density of the two ligands,optimal CMMS/FA-RGD coating was prepared.Systematic characterization of CMMS/FA-RGD coatings revealed that the static contact angle of the dual ligand coating was 8-12°higher than that of any single ligand coating,and the surface topography roughness increased by approximately 3.2 nm.Surface plasmon resonance(SPR)instrument on-line measurements demonstrated that the ligand densities of FA and RGD are 22 ng/cm~2 and 9 ng/cm~2 respectively on the optimized CMMS/FA-RGD coating.The He La cell capture efficiency of CMMS/FA-RGD coating was 91.4%from mixed cells of L929:He La=100:1 suspension and 90.6%from whole blood spiked with cancer cells(WBC:He La=2120:1).Both of the capture purity is higher than 80%.Compared to normal cultured He La and MCF-7 cells,the CMMS/FA-RGD coating captured cells showed 89%viability.The high viability of the captured cells could be promising for single tumor cell analysis.