Study On Construction Of Magnetic Transfection Vector For Silencing CXCR4

Nowadays,the morbidity and mortality of malignant tumors are gradually increasing,which has become a major problem that needs to be solved urgently in the field of life sciences,medicine and even sociology.The development of more efficient and less expensive new technologies for cancer diagnosis and treatment has outstanding scientific significance and social benefits.This research majored in the biomedical application of nanomaterials,and the purpose was to develop efficient nucleic acid carriers for tumor-related gene expression regulation.Specifically,heptafluorobutyric anhydride was used to prepare fluorinated polyethylene glycol-polyethyleneimine（F₇-PEG-PEI,FPP）,which coated ferric tetroxide（Fe₃O₄）nanoparticles as to be the novel magnetic nucleic acid carriers.The novel carriers were used to transfect small interfering RNA（siRNA）into tumor cells for knocking down CXC chemokine receptor 4（CXCR4）.In this study,oleic acid-modified Fe₃O₄ magnetic nanoparticles（MNPs）were prepared by high-temperature pyrolysis of iron acetylacetonate.Then,dimercaptosuccinic acid（DMSA）was used to prepare aqueous-dispersed DMSA@MNPs by ligand exchange.Transmission electron microscopy（TEM）images show that the nanoparticles were nearly spherical particles with the size of about 12 nm.High-resolution TEM（HRTEM）and selected area electron diffraction（SAED）show that the nanoparticles were Fe₃O₄ nanoparticles with inverse spinel structures.The magnetization curves and alternating magnetic field heating curves confirmed that the prepared MNPs have strong superparamagnetic and magnetothermal effects.F₇-PEG-PEI was prepared by the reaction of heptafluorobutyric anhydride with the amine groups of bifunctional polyethylene glycol（H₂N-PEG-COOH）,followed by amidation with polyethyleneimine（PEI）.Characterization by elemental analysis,Fourier transform infrared spectroscopy（FT-IR）and ¹⁹F-nuclear magnetic resonance(¹⁹F-NMR)proved that F₇-PEG-PEI was synthesized.Subsequently,F₇-PEG-PEI was modified on the surface of DMSA@MNPs by electrostatic adsorption to construct fluorinated magnetic transfection carriers（FPP@MNPs）.The results of dynamic light scattering（DLS）and zeta potential show that F₇-PEG-PEI was coated on the surface of DMSA@MNPs,and the energy dispersive spectroscopy shows that FPP@MNPs nanoparticles contained fluorine,and the spatial distribution of fluorine and iron was highly correlated.The measurement of cell viability and apoptosis verified that FPP@MNPs had good biosafety within the range of dosage used.The results of confocal microscopy observation of cellular uptake indicate that fluorination was an effective strategy to overcome the"PEG dilemma"and thereby improved the cellular uptake efficiency of magnetic nanoparticles.Then the delivery efficiency of FPP@MNPs as siRNA carriers was verified in a variety of cell lines,indicating that FPP@MNPs had good versatility.Moreover,the transfection efficiency could be further improved by external magnetic fields.In cell transfection experiments in vitro,FPP@MNPs were used to transfect siRNA for silencing CXCR4.The results of RT-PCR and flow cytometry confirmed that the expression of CXCR4 in 4T1 breast cancer cells was significantly inhibited.In order to construct more efficient and targeting transfection vectors,the CXCR4-targeted E5 peptide was modified on the surface of FPP@MNPs to construct E5@FPP@MNPs.The results of DLS and zeta potential measurements show that the constructed E5@FPP@MNPs had good aqueous dispersibility and colloidal stability.The results of flow cytometry show that compared with FPP@MNPs,E5@FPP@MNPs could significantly inhibit the binding of 12G5antibody to CXCR4 on HL60 cells,indicating that E5@FPP@MNPs could specifically bind to CXCR4 and played a blocking role.In vitro delivery efficiency studies show that E5@FPP@MNPs could deliver siRNA to HL60 cells more efficiently.Finally,flow cytometry was used to detect the efficiency of E5@FPP@MNPs transfecting siRNA and the effect of knocking down the target protein.The results confirmed that the combined application of inhibitors and RNAi achieved synergistic inhibition of CXCR4 expressed on HL60 cells.The innovation of this study is that the"PEG dilemma"was successfully overcome by fluorination modification,which effectively improved the cellular uptake efficiency of the nanoparticles.Additionally,the efficiency of siRNA transfection mediated by fluorinated magnetic carriers could be further enhanced by external magnetic fields.Furthermore,E5peptide modification of carriers were performed to achieve targeted gene silencing in suspension cells effectively,and provided a beneficial method for the development of targeted regulation of gene expression in tumor cells.