Study Of Nanoprobes Used For The Capture Of Drug-resistant Cells In Acute Myeloid Leukemia

The drug resistance and recurrence is the bottleneck in the treatment of acute myeloid leukemia(AML).One of the key technologies to solve the problem is the capture and detection of drug-resistant cells.The current mainstream cell sorting methods are flow cytometry cell sorting and immunomagnetic bead cell sorting.However,flow cytometry instruments are expensive and difficult to operate,and usually require more than 105 cells as the initial sample load.Immunemagnetic bead cell sorting usually requires high gradient magnetic field,which can reach strong magnetic field and may result in non-specific capture of cells with magnetic properties inherently.Here,we report a highly efficient magnetic nanoprobe based microfluidic cell sorting system(MMCS).MMCS system combines magnetic nanoprobe and microfluidic for the capture and detection of drug-resistant cells under simple permanent magnets,breaking the limitations of existing cell sorting methods.Firstly,multiple sizes of Fe3O4@OA nanoparticles(10,15,20 nm)were successfully prepared by thermal decomposition method.By improving the preparation process,the synthesis yield of Fe3O4 nanoparticles was increased to 1 g,with a yield greater than 89%.Water-soluble Fe3O4@PEG nanoparticles were obtained by the modification of amphiphilic DSPE-PEG,which has high saturation magnetization,good stability and superior batch-tobatch consistency.By comparision,20 nm Fe3O4 nanoparticle which was stable and had the highest saturation magnetization(93 emu/g [Fe])was selected for subsequent cell sorting.Secondly,two kinds of different nanoprobes were constructed and their cell capture ability were studied to select a high-performance nanoprobe.One was peptide-based nanoprobe.Streptavidin-biotin amplification system was used to assemble streptavidin and peptide on the surface of Fe3O4 nanoparticles in the construction of peptide-based nanoprobe,which maximized the loading of fluorescent molecules and peptides.Prussian blue staining results showed that the unmodified fluorescent molecule Fe3O4@PEG@SA@E5 nanoprobe had excellent specific targeting ability to CXCR4,while the cell targeting ability of the modified fluorescent molecule Fe3O4@PEG@SA-Cy5@E5nanoprobe was significantly reduced.It was found that Cy5 can covalently bind to E5,which can quench the fluorescence of Cy5 and change the spatial structure of the peptide,resulting in a significant reduction of the targeting ability of peptide E5.Therefore,the silica-coated iron oxide nanoparticles Fe3O4@SiO2/Cy5 were prepared,in which Cy5 was coupled in the middle of the silica layer.After mixing with E5,the fluorescence intensity of the mixture remained unchanged,which verified the feasibility of constructing nanoprobe to achieve cell capture and detection.The other was an antibody-based nanoprobe.Monoclonal antibody 12G5 and fluorescent molecule F647 was modified on the surface of iron oxide nanoparticles successively to construct Fe3O4@PEG@12G5-F647 nanoprobe,which was proved to have excellent specific cell targeting ability.The optimal cell sorting conditions were determined as follows: 40 μg of Fe3O4@PEG@12G5-F647 nanoprobe(12 antibodies conjugated)was added to 5*105 HL-60 cells and incubated at 37°C for 1 h.The Fe3O4@PEG@12G5-F647 nanoprobe had a high cell capture efficiency(96% ~ 100%),and the cell sorting purity was as high as 98%.Besides,the capture and detection of target cells has nothing to do with the number of negative cells,indicating that this method has strong anti-interference ability.Among the two kinds of nanoprobes,antibody-based nanoprobe was demonstrated to have distinguished cell-targeting ability with a cell capture efficiency of more than 96%.Therefore,antibody-based nanoprobe was selected to combine with the microfluidic chip,achieving the capture and detection of AML drug-resistant cells.Finally,the combination of magnetic nanoprobe and microfluidic(MMCS)was used to capture drug-resistant cells under a permanent magnet,and the number of drug-resistant cells was detected by a fluorescence quantitative analyzer.A total of two versions of microfluidic chip were designed.The optimized version of microfluidic chip can capture target cells more effectively,with a higher cell capture effiency compared with the original version of microfluidic chip.The optimal cell flow rate was determined to be 10 μL/min,and the cell capture rate reached to 90.4%.The linear range of microfluidic cell detection was 104~5*105cells(R2 was 0.990).A standard curve for microfluidic cell capture and detection was also established.The detection limit was determined to be 1.53*103.In the cell capture and detection of simulated samples,the detected cell number was consistent with the actual cell number of HL-60(R2 was greater than 0.990),and the relative deviation is less than 10%,proving that the detection of drug-resistant cells by microfluidic had a high accuracy.In conclusion,MMCS system for the capture and detection of drug-resistant cell in acute myeloid leukemia has been successfully developed,which overcomes the limitations of current cell sorting methods.MMCS system has the advantages of low cost,simple operation,no strong gradient magnetic field,high cell sorting efficiency,high cell sorting purity,and automated sorting process,which is portable and has potential for widespread clinical application.