Research On AFP Aptamer-modified Magnetic Nanoparticles Targeted Liver Cancer Via Magnetic Resonance Imaging

Hepatocellular Carcinoma（HCC）is one of the most common malignancies in clinic.It has a high incidence rate,high mortality and occult condition.There are no symptoms in the early stage,and the degree of malignancy is high and the prognosis is poor.Therefore,research on new technologies and methods for early warning and targeted diagnosis of liver cancer is of great significance to improve the early diagnosis and quality of life of liver cancer.It has been reported that AFP has a high sensitivity for diagnosis of liver cancer.Usually,the AFP concentration in the serum has a close correlation with the size of liver cancer,the smaller the cancer tissue,the smaller the positive detection rate of AFP and the relationship with liver cancer.As a tumor marker of liver cancer,AFP is of great value for clinical diagnosis,it plays an important role in the early diagnosis,prediction of recurrence,curative effect,differential diagnosis and prognosis of liver cancer.With the rapid development of molecular imaging in the diagnosis and treatment of diseases,based on its ability to display specific molecules at the tissue level and the cellular level,nanomaterials are surface-modified and combined with molecular probes that are recognized for non-invasive and real-time accurate positioning and tracing of tumors or lesions.At the same time,due to the nanomaterials have high permeability and retention effect（EPR）on tumor tissues,they can be phagocytosed by tumor cells in vitro and in living tissues,thereby providing in vivo imaging of small lesions and even individual tumor cells,it contributes to research on the occurrence,progression and metastasis of tumors,and the realization of accurate treatment of tumors and dynamic monitoring of therapeutic efficacy.Magnetic Resonance Imaging（MRI）has the advantages of high resolution and accurate anatomical location,MRI technology based on molecular targeting is expected to become an ideal non-invasive detection method with tumor targeting.How to find more effective target of liver cancer and construct liver cancer targeting MRI imaging probe has become a hot spot in the field of MRI molecular imaging of liver cancer.In this study,a magnetic nanoprobe（Fe₃O₄@KCTS-AFP-apt）modified with AFP aptamer was constructed,and its magnetic resonance imaging characterization and targeting were analyzed to determine the MRI of liver cancer.Molecular Imaging provides theoretical basis for further reference imaging and target therapy.Objective:In this work,the magnetic Fe₃O₄nanoparticle was prepared by the hydrothermal method as a core.After the activation of carbodiimide,cross-linking withα-ketoglutarate chitosan（KCTS）,the core-shell type Fe₃O₄@KCTS was successfully prepared.AFP aptamer was cross-linked with magnetic nanomaterials to construct a magnetic nanoparticle targeting probe specifically targeted to the tumor.Method:In order to prevent the reaction system from containing too much components,we prepared magnetic Fe₃O₄ nanoparticles by oxidative hydrothermal method using ferrous sulfate（Fe SO₄·7H₂O）and oxidized by hydrogen peroxide（H₂O₂）.Magnetic Fe₃O₄@KCTS with a certain carboxyl group on the surface was prepared by the carboxylation of Fe₃O₄as the nucleus and the carbodiimide reaction.Then,the AFP aptamer was coupled to Fe₃O₄@KCTS under the aid of EDC/NHS,and a functionalized magnetic Fe₃O₄@KCTS-AFP-apt nanoprobe was successfully constructed.The morphology,particle size,dispersion index and potential of AFP-MNPs-apt were analyzed by transmission electron microscopy（TEM）,dynamic light scattering（DLS）respectively.X-ray diffraction（XRD）was used to analyse the microstructure of Fe₃O₄@KCTS-AFP-apt,the Fourier transform infrared spectrometer（FTIR）was introduced into to discover the groups on the nanoparticles and material composition of Fe₃O₄@KCTS-AFP-apt.The Cell Counting Kit-8（CCK-8）was used to detect the cytotoxicity of Fe₃O₄@KCTS-AFP-apt.The laser scanning confocal microscope was introduced to observe the fluorescence imaging of Fe₃O₄@KCTS-AFP-apt was incubated with AFP-positive cells.The Prussian blue staining was used to detecte the iron content engulfed by cells.The MR imaging capability of Fe₃O₄@KCTS-AFP-apt targeted to Hep G2 cells was analyzed by GE 3.0T MRI.Results:A functional nanoprobe Fe₃O₄@KCTS-AFP-apt was successfully prepared which could effectively target to the tumor.TEM results showed that the probe exhibited round or oval,dispered uniformly and the surface charged negative,the diamater was about 100 nm.Fe₃O₄@KCTS-AFP-apt could specifically bind to Hep G2 cell in vitro and tumor tissue.The MR imaging was performed on a GE 3.0T MRI instrument.The T₂-weighted imaging was effective and the signal was strong,which could effectively shorten the T₂-weighted relaxation time,which was used as a T₂ contrast agent to enhance the imaging contrast of the Hep G2 cell.Conclusion:We successfully constructed the Fe₃O₄@KCTS-AFP-apt probe that could effectively target tumor of liver cancer.This was an early molecular image of aptamer-targeted tumor of diagnosis and treatment with providing early molecular imaging diagnosis of liver cancer targeting probe system with broad application prospects.