Biomimetic Fluorescent Magnetic Multifunctional Probes With Different Magnetic Susceptibility For Near-simultaneous Isolation And Detection Of Multiple Circulating Tumor Cells

At the early stage of cancer,a small number of circulating tumor cells(CTCs,>5 CTCs/7.5 mL blood)exist in the peripheral blood,and they will undergo epithelial-mesenchymal transformation(EMT)during the process of metastasis,producing heterogeneous subtypes,mainly manifested by changes in surface marker types and their expression levels on the cell surface.Previous research showed that there was a close connection between the numbers and subtypes of CTCs in peripheral blood and progression of cancer.Therefore,isolating and screening for heterogeneous CTCs in peripheral blood is of great importance for early cancer diagnosis,personal treatment and therapeutic effect evaluation.At present,the methods to detect heterogeneous CTCs are mainly flow cytometry and microfluidic chip.Flow cytometry equipment is expensive and operated by professional and technical personnel,it also requires numerous cell number.The complexity of microfluidic chip also limits its application.In view of the problems raised above,based on the previous work in our laboratory,a series of probes with magnetism of different gradients,fluorescence,biomimetic and targeting functions were constructed to effectively and accurately isolate and enrich multiple circulating tumor cells with different surface markers at the same time.The main research contents are as follows:Part 1:Construction of biomimetic fluorescence magnetic probes with different magnetic responses.Firstly,the magnetic precursor particles with different magnetic reponse and fluorescent quantum dots were assembled respectively into a series of fluorescent magnetic nanocomposites S/M/W-FMNPs(strong/medium/weak magnetism fluorescent magnetic nanoparticles)via embedding and layer-by-layer self-assembly approach,followed by preparing the leukocyte membrane fragment-coated S/M/W-FMNPs(S/M/W-LFMNPs)through physical extrusion method.Furthermore,DSPE(distearyl phosphatidylthanolamine)was applied as a bridge to coupled with different specific antibodies to obtain the S/MW-LFMNPs-Ab(strong/medium/weak magnetism fluorescent magnetic probes)which exhibited good dispersion,good stability and strong magnetic manipulability.Part 2:Studying the enrichment of heterogenous CTCs by biomimetic fluorescence magnetic probes with different magnetic susceptibility.Three types of tumor cells with different surface markers and gradually decreased expression levels(BT474(Her2+++),LNCaP(PSMA++)and MDA-MB-231(Vimentin+))were selected as study models.Firstly,the feasibility of constructed probes sequentially separating of heterogeneous CTCs was performed with modified MPs to mimick tumor cell subpopulations.Carboxylic microparticles(MPs)with different proteins and expression levels were constructed(protein-modified MPs)followed by coupling with prepared probes respectively to obtain microparticles with different magnetic responses.Under the external magnetic field,the three microparticles combined with the probes could be separated at different magnetic separation times,which proved the feasibility of the method.Later,the above selected three kinds of tumor cell subpopulations were used as positive cells and macrophages RAW 264.7 as negative control cells to investigate the isolation and detection performance of S/M/W-LFMNPs-Ab for tumor cell subpopulations.The results showed that the probes could detect about 90%of CTCs in the simple PBS buffer system with almost no white blood cells(WBC)in the background within 3 min.After mixing two or three types of cells together,the probes could capture the tumor cells,and each type of the tumor cells could be sequentially magnetically collected at different magnetic separation time points under an external magnetic field.The results of isolation of tumor cells in mimic clinical samples showed that multiple tumor cells could be separated and collected with S/M/W-LFMNPs-Ab in complex systems without complicated pre-processing.Further studies showed that the extremely low detection limit of the nanoprobes for rare tumor cell was 1 cell/mL.Therefore,this method has potential important value in early cancer diagnosis,personal treatment and prognosis evaluation.