Isolation And Analysis Of Tumor Cell Subpopulations Based On Biomimetic Immuno-fluorescent Magnetic Multifunctional Nanoprobes

Tumor heterogeneity is an important characteristic of malignant tumor,which is manifested as the difference from genotype to phenotype between different patients with the same malignant tumor or between tumor cells in different parts of the same patient.Heterogeneity among tumor cells leads to differences in tumor proliferation capacity,invasion capacity and drug sensitivity,and ultimately affects the diagnosis,treatment and progression of tumor patients.Therefore,tumor cell subpopulations analysis is very necessary for individualized tumor treatment.Currently,studies on tumor heterogeneity are mainly conducted by single-cell sequencing.However,this method is complicated and expensive.Other researchers used microfluidic chips and magnetic nanoparticles to isolate and analyze subgroups of tumor cells,but the preparation of microfluidic chips is complicated.In order to achieve easily and quickly capture and separate multicomponent tumor cells,a new type of multifunctional nanoprobe with fluorescence,magnetic,biomimetic and targeting functions were constructed in this thesis.And following the nanoprobes were applied to the separation and analysis of tumor cell subpopulations,based on their magnetic and fluorescence properties of the probe.The main contents of this thesis are as follows:Part 1:It mainly constructs a multifunctional nano-probe integrating fluorescent,magnetic,biomimetic and targeting functions.Firstly,Fluorescent-magnetic nanoprobes（FMNPs）were prepared by combining embedding and self-assembly methods,ie.The γ-Fe₂O₃ nanoparticles were surfactant-based cetyltrimethylammonium bromide（CTAB）based on the embedding method forms micelles.Then（3-Mercaptopropyl）trimethoxysilane（MPS）is coated with a layer of thiolated silicon dioxide on the surface,and then through the metal positional action assembles quantum dots（QDs）onto the surface of the micelle to obtain FMNPs.In order to improve the purity of capture of tumor cells by reducing nonspecific adsorption of probe to white blood cells,FMNPs were camouflaged with leukocyte membrane（LFMNPs）through physical extrusion.The cell membrane of LFMNPs was then coupled to protein G（PG）using the lipophilicity of the coupling agent DSPE-PEG-NHS.Since the non-immune mechanism of PG can bind to the Fc region of immunoglobulin（IgG）,the Her2 antibody is coupled to LFMNPs-PG,and finally a multifunctional nanoprobe（LFMNPs-Ab）integrating fluorescent,magnetic,biomimetic and targeting functions is obtained.Moreover,the hydrated particle size,surface charge,morphology,magnetic and fluorescent properties of the probe were characterized by dynamic light scattering（DLS）,transmission electron microscopy（TEM）,vibrating sample magnetometer（VSM）,fluorescence inverted microscope,etc.The results showed that the prepared probes had good magnetic and fluorescent properties,and the tumor cell subpopulations with different surface Her2 protein expression could be specifically identified.Part 2:The main content is isolation and analysis of tumor cell subpopulations based on LFMNPs-Ab multifunctional probe.Firstly,a carboxypolystyrene microballoons model with different amounts of protein L（PL）（MBs-PL）were constructed to simulate the tumor cells subpopulations with different expression of Her2.After incubation with LFMNPs-Ab,the MBs-PL exhibit different magnetic gradients as MBs-PL combines different amounts of probes.Therefore,the MBs-PL could be captured and separated by different magnetic adsorption times,which demonstrates the feasibility of the magnetic responsed-based separation.Then,the reaction conditions such as incubation time,the concentration of LFMNPs-Ab and tumor cells were optimized.Secondly,LFMNPs-Ab was used to magnetically separate the breast tumor cell lines BT474（Her2+++）,MDA-MB-453（Her2++）and MDA-MB-231（Her2-）with different expression levels of Her2.And further the capture performance of probes for single component cells,two component cells,and three component cells were investgaed.The results showed that LFMNPs-Ab could effectively isolate tumor cell subpopulations with different Her2 expression levels.Finally,the subsets of tumor cells in the blood sample were further capture and isolate by employing LFMNPs-Ab.