Functionalization Of Immunomagnetic Beads And Its Application In Circulating Tumor Cells Enrichment

Circulating tumor cells(CTCs),which are malignant tumor cells shed by solid tumor lesions(primary and metastatic)into the peripheral blood.Entering the peripheral blood,a small number of CTCs may escape and anchor at other organs,developing metastases and increasing the risk of death in patients with malignant tumors.Biopsies and subsequent pathological analysis are important standards for cancer diagnosis.However,tumor biopsy is highly invasive and there is a risk of promoting tumor metastasis,and it is difficult to take multiple samples for real-time tumor monitoring.In addition,tumor biopsy also has shortcomings like sampling bias and difficulty in deep tumors sampling.By contrast,CTCs can be isolated from readily available peripheral blood by the noninvasive "liquid biopsies",and the amount and biological information of CTCs can be effectively applied to the early diagnosis of tumors,rapid assessment of chemotherapy drugs,individualized treatment(including clinical screening drugs,drug resistance detection,tumor recurrence monitoring as well as the development of new drugs),etc.Therefore,CTCs detection becomes an important part of "liquid biopsy".For the rare occurrence of CTCs in blood(a few to hundreds of CTCs per milliliter),the development of efficient CTCs enrichment platform is critical to CTC-based liquid biopsies.In various enrichment methods,immunomagnetic separation has attracted extensive attention because of advantages such as easy operation,simple functionalization,relatively good enrichment performance and convenient coupling with other identification methods.Although existing immunomagnetic platforms have achieved some good performance,the magnetic beads on the captured CTCs cannot be released,which may damage the structural integrity of CTCs,interfere with the cell microenvironment,and make it difficult to proliferate and culture CTCs in vitro.In addition,the purity of CTCs isolated by most immunomagnetic beads is low(0.01%-0.1%),making the CTCs identification process difficult.And the protein and genetic material carried by white blood cells seriously interferes the molecular biological study of CTCs.Therefore,it is of great significance to realize the nondestructive recovery and high purity enrichment of CTCs while ensuring high capture efficiency.Glutathione-mediated disulfide bond breakage has been widely used in drug/gene stimulative release.The introduction of disulfide bond between the magnetic core and CTCs targeting antibody can achieve the detachment of magnetic beads under relatively mild condition.In addition,fluorescent labeling of the magnetic beads can visualize the separation process and help determine whether the beads are completely detached.In order to improve the anti-nonspecific adsorption property,lipids and proteins are widely applied in the functionalization of immunomagnetic beads.For example,liposomes are naturally passivating,have strong anti-nonspecific adsorption capacity against proteins and cells.The fluidic property of the lipid bilayer enables the redistribution of modified antibodies,which can effectively enhance the enrichment performance of the magnetic beads.The cell membrane not only has lipid bilayer,but also has abundant membrane proteins.Specific membrane proteins endow the cell membrane-camouflaged nanoparticles unique tumor cell targeting ability,anti-leukocyte nonspecific adsorption ability and immune escape ability.It has been reported that graphene nanosheets can extract a large number of phospholipid molecules from the cell membrane due to the strong dispersion force between graphene nanosheets and lipid molecules.In addition,graphene nanosheets not only have hydrophobic interaction with proteins,but also have strong π-π stacking between aromatic protein residues,resulting in strong protein adsorption capacity.Therefore,graphene nanosheets can be used as a functional component to fix lipids and proteins,providing a new method for the introduction of lipids and proteins on the surface of magnetic beads.Around the above content,this paper mainly carries out the following work.According to reports,graphene nanosheets can destructively extract much phospholipids from the cell membrane smoothly due to the strong dispersion force between graphene nanosheets and lipid molecules.Accordingly,the main research content is as follows.1.Quantum dot modified immunomagnetic beads for capture and release of circulating tumor cellsMagnetic nanoparticles(MNPs)with uniform size and regular morphology were prepared through hydrothermal method.The MNPs are hydrophilic,superparamagnetic and has a particle size of about 300 nm.With the layer-by-layer assembly strategy,quantum dots(QDs)can be introduced,and QDs-MNPs composite can be constructed.After that,disulfide bond was introduced between the magnetic core and the anti-epithelial cell adhesion molecule(Ep CAM)antibody.In blood samples(containing 5-200 CTCs model cells per milliliter),as prepared immunomagnetic beads achieved capture efficiency above 90%,with a 2-min incubation.Incubating the enriched CTCs model cells with low concentration glutathione for 15 min,the fluorescent signals on cells disappeared,indicating that all magnetic beads were detached.In addition,the viability of the recovered CTCs model cells was 98%,and they could be directly used for in vitro culture,their invasion and migration characteristics remained nearly unchanged.In addition,the immunomagnetic beads successfully enriched CTCs from peripheral blood samples of 11 tumor patients,with an average of 8.6 ± 5.8 CTCs detected per milliliter of blood.In blood samples from 9 healthy volunteers,no CTC was detected.2.Leukocyte membrane analogue camouflaged biomimetic immunomagnetic beads for high purity enrichment of circulating tumor cellsGraphene nanosheets can be combined with the magnetic core with the layer-by-layer assembly strategy(MNPs/GNs).Utilizing the interactions between the graphene nanosheets and lipids,proteins,simple incubation of the MNPs/GNs with white blood cells,biomimetic magnetic nanoparticles coated with white membrane analogue can be constructed.Through the high affinity between biotin-streptavidin,biotin-modified CTCs-targeting antibodies can be grafted by inserting a biotin-modified lipid linker.The detection limit of the obtained biomimetic nanoplatform is as low as 3 CTCs per milliliter.In whole blood sample with few CTCs model cells(20-200 cells/m L),the platform achieved capture efficiency above85.0% and purity above 94.4%.The enrichment process had little effect on the survival rate of CTCs model cells(cell viability: 98.0%),enriched cells could be directly cultured in vitro.In addition,the biomimetic nanoplatform captured 2-48 CTCs from peripheral blood sample of 8 tumor patients(1.5 m L)with good reproducibility(mean relative standard deviation 8.7 ± 5.6%).In blood samples from5 healthy volunteers,no CTC was detected.3.Phospholipid-protein functionalized immunomagnetic beads for efficient separation of circulating tumor cellsCell membrane substitutes(CMS)can be constructed with commercialized lipids and proteins,which has clear composition and structure.CMS can avoid the shortcomings of some cell-derived materials,and CMS coating may reduce the interference from blood on the surface structure and function of magnetic beads to some extent.It is reported that modification of human serum albumin,a non-reactive protein,can reduce the adsorption of opsin and prolong the cycle time of nanoparticles.Its pre-adsorption may shield some non-specific binding sites of magnetic beads and increase the CTCs targeting specificity.Through incubation of MNPs/GNs with biotinylated phospholipids and human serum albumin(HSA),magnetic beads with phospholipid-protein modification can be obtained,the components of which are simple and well-defined.Grafting antibody via the interaction of streptavidin and biotin,immunomagnetic beads can be constructed.With the modification of CMS,the anti-nonspecific adsorption ability of the enrichment platform was improved,and it could realize efficient and high-purity enrichment of various CTC model cells(capture efficiency above 90.5%,capture purity above 86.6%)from the mixed cell samples(CTCs model cells: interference cells =1:100)in serum.In whole blood sample with 15 CTCs model cells per 0.5 m L,the capture efficiency was about 70.0%,and 411-529 white blood cells were nonspecifically detected.4.Protein camouflaged immunomagnetic beads and its application in circulating tumor cells isolationPre-absorption of protein on immunomagnetic beads,can occupy the surface physically,may help reduce protein corona adsorption.In order to make the magnetic beads more stable in blood,blood protein pre-camouflage of the platform is an ideal implementation strategy.Taking the advantage of the interaction between graphene nanosheets and proteins,magnetic beads with different protein camouflage could be prepared by incubating MNPs/GNs with serum and the four most abundant protein in plasma(HSA,fibrinogen,immunoglobulin,and transferrin).With further antibody grafting,protein camouflaged magnetic beads(CIMNPs)can be obtained.Among which,HSA modification layer can significantly improve the anti-nonspecific adsorption ability of the material.As for the mixed cell samples in serum(CTCs model cells: interfering cells =1:100),the HSA-CIMNPs showed good capture efficiency(>85.5%)and excellent capture purity(94.5%)for CTCs model cells.In whole blood sample with 25-55 CTCs model cells per 0.5 m L,the capture efficiency was about 84.0%-90.0%,and 164-264 white blood cells were nonspecifically detected.