Preparation Of Magnetic-Flourescent Nanoparticles And Isolation Performance On Circulating Tumor Cells

Circulating tumor cells（CTCs）,which are closely related to tumor development,have been considered as an important biomarker in liquid biopsy,showing great promise for clinical applications in the early cancer diagnosis,patient prognosis,drug efficacy monitoring and personalized therapy.Immunomagnetic separation has been widely used for the detection and separation of CTCs due to its simplicity,rapidity and specificity.The current immunomagnetic separation techniques mainly utilize the chemical affinity between specific ligands（e.g.,antibodies,aptamers and peptides）coated on magnetic beads and the biomarkers overexpressed on the cell surface.The ligands,which are very expensive and easy to deactivate,can’t achieve broad-spectrum capture of heterogeneous CTCs.In addition,the cell viability is susceptible to conventional three-color immunofluorescence identification and destructive cell-release process,seriously affecting their downstream analyses.It is found that there are strong hydrogen bonding and hydrophobic interactions between abundant galloyl groups of tannic acid（TA）and the glycocalyx structure of tumor cells.And TA exhibits excellent resistance to the non-specific adhesion of blood cells,making it an ideal candidate for broad-spectrum capture of heterogeneous CTCs.As a novel metal-organic framework,zeolitic imidazolate framework-8（ZIF-8）possesses unique merits including convenient synthesis,high porosity,controllable size,and rapid degradation in response to multiple physiological stimuli（ATP/p H）,providing the possibility for in situ labeling and non-destructive releasing of CTCs.Inspired by these,we prepared a magnetic-flourescent nanoparticle（FR@Z-pTANPs）by successively constructing ZIF-8 shell encapsulating rhodamine B（Rhm B）and poly（tannic acid）（p TA）coating on the surface of Fe₃O₄nanoparticles.This platform can not only achieve broad-spectrum capture and in situ fluorescence labeling of various heterogeneous CTCs in blood,but also gently release the captured CTCs by cell-friendly stimuli（ATP and p H）.Firstly,we synthesized Fe₃O₄ NPs using the solvothermal method,then successively prepared ZIF-8 shell encapsulating Rhm B and pTAcoating on their surface through the one-pot method and enzyme-catalyzed oxidative polymerization to obtain FR@Z-pTANPs.The particle size and morphology of the nanoparticles were characterized by dynamic light scattering（DLS）and transmission electron microscopy（TEM）.The results demonstrated that FR@Z-pTANPs with distinct core-shell structure showed a hydrodynamic diameter of 313.6 nm.The chemical composition of the nanoparticles was characterized by Fourier transform infrared spectroscopy（FT-IR）and thermogravimetric analysis（TGA）,which verified the successful modification of ZIF-8 shell and pTAcoating.The magnetic responsiveness and fluorescence properties of the nanoparticles were further characterized by vibrating sample magnetometer（VSM）and fluorescence microscope（FM）.And the results showed that the saturation magnetization intensity of FR@Z-pTANPs was42.7 emu/g,indicating their excellent superparamagnetism.In addition,bright red fluorescence of FR@Z-pTANPs could be observed in the Rhoda channel.Then,the CTC-capture performance of FR@Z-pTANPs was investigated.The good cytocompatibility of FR@Z-pTANPs was demonstrated by the Alamar Blue method,and the results of cell incubation experiments showed that FR@Z-pTANPs exhibited excellent properties of recognizing and fluorescent labeling towards CTCs.FR@Z-pTANPs exhibited quite high capture efficiency（>88%）for both Ep CAM-positive（Hep G2,MCF-7）and Ep CAM-negative（He La,MDA-MB-231）cancer cells at a concentration of 100μg/m L after incubating for 25 min.Additionally,the non-specific adhesion of 86.9%white blood cells was effectively inhibited.For the artificial blood samples containing 10-200 CTCs/m L,the capture efficiencies of He La and Hep G2 by FR@Z-pTANPs were 72.3%and 80.9%,indicating their high detection sensitivity.Moreover,CTCs in blood samples of different cancer patients（colon,ovarian,esophageal,lung,stomach and lymphoma）could be effectively detected by FR@Z-pTANPs.Finally,the ATP/p H-responsive cell-release performance of FR@Z-pTANPs,the viability and proliferative capacity of the released cells were systemically studied.It was shown that the cumulative release of Zn²⁺from FR@Z-pTANPs was 65.9%and65.1%after incubation in PBS solution（ATP=4 m M）for 2 h and acidic PBS solution（p H=6）for 5 h,respectively.And the release efficiency of captured CTCs was higher than 80%under the above conditions.Besides,the released CTCs showed intact structures,high viabilities（>90%）and good proliferation ability.