In Vitro And In Vivo Targeting Imaging Of Pancreatic Cancer Using A Fe₃O₄@SiO₂ Nanoprobe Modified With Anti-mesothelin Antibody

Pancreatic cancer（PC）is one of the most malignant and highly invasive tumors in the world.PC patients have poor prognosis and 5 year survival rate is below 5%.The symptom of PC in early stage is not specific;over 80% patients diagnosed with PC are in the advanced stage,and lost the opportunity of operation;patients did not respond well to the main treatment,which are the most important reasons of poor prognosis.Molecular imaging,a hot area of research in recent years,its technology and method can be used to diagnose and treat PC,expecting to increase correct early diagnosis rate and improve prognosis of PC.It should be noted that molecular imaging researches are restricted to the laboratory or animal experiment.New biomarkers mentioned in recent studies are still at exploring stage and are not used in clinical yet.CA19-9 is the only biomarker used in PC patients in clinic.But it lacks sensitivity and specificity when used to diagnose PC.It is often used to observe the response to treatment of PC patients.Mesothelin（MSLN）,one of the cell surface glycopteins,is a new PC biomarker.MSLN is overexpressed in malignant pancreas tissue,but is not expressed in normal pancreas tissue,acute or chronic pancreatitis or benign tumors of pancreas.Superparamagnetic iron oxide（SPIO）nanoparticle is the most common used of MRI negative contrast agent due to its excellent superparamagnetism,nontoxicity and high biocompatibility.In this research,we synthesized a novel MRI negative contrast agent,which was modified with MSLN antibody in the surface of Fe₃O₄@SiO₂ nanoprobe.Then analyze its characterization,side effects and target ability to PC in vitro and in vivo.ObjectiveThis research aims to synthesize a new type nanoprobe,Fe₃O₄@SiO₂ modified with MSLN antibody（A-MFS）,as MRI negative contrast agent.Then to observe its characterization,side effects and to verify the feasibility and possibility as MRI negative contrast agent to diagnose PC,according to its in vitro and in vivo target studies to PC cells and nude mice PC xenografts.Materials and Methods1.Synthesis of Fe₃O₄@SiO₂ probe modified with anti-MSLN antibodySolvothermal reaction was used to prepare magnetic microspheres（Fe₃O₄）.Coat SiO₂-NH₂ in its surface to prepare Fe₃O₄@SiO₂-NH₂ nanoprobe（FS）.Finally modify MSLN antibody on FS surface to synthesize targeting nanoprobe（A-MFS）.Morphological characteristics of A-MFS nanoprobe were observed by transmission electron microscope（TEM）.Measure its hydrodynamic diameter,relaxation rate,and test its stability.2.Cell toxicity of A-MFSThe cytotoxicity of the various concentrations nanoprobe A-MFS against human pancreatic cancer SW1990 cells at different time was determined by measuring the inhibition of the cell growth using a Cell Counting Kit-8（CCK-8）assay.When the in vivo targeting test was done,major organs of mice were collected to hematoxylin and eosin（H&E）staining to observe their pathological changes.3.In vitro targeting test of A-MFSWith the purpose of testing the targeting ability of the A-MFS to SW1990 cells in vitro,red fluorescence quantum dots（QDs）,which are water-soluble,were added to the probes FS and A-MFS to synthesize fluorescence nanomaterials.The SW1990 cells in the logarithmic phase were incubated with fluorescent nanoprobes FS and A-MFS,respectively,for 30 min.LM-3,human liver cancer cell line,was incubated with fluorescent nanoprobe A-MFS（1 mg/m L）for 30 min as control.Then cells were visualized through fluorescence microscope to determine the different effects of FS and A-MFS on SW1990 and LM-3 cells.After SW1990 and LM-3 cells were digested with pancreatin,they were incubated with nanoprobes FS and A-MFS for 1 h at 37 ℃.Finally the sample was analyzed by flow cytometry,to observe the targeting ability of nanoprobes to cells quantitatively.4.In vivo MRI targeting test of A-MFSA 0.1 m L of the cell suspension was injected subcutaneously,respectively,into the right axillas of nude mice,then held in captivity.These mice continued to be raised under SPF conditions in the Laboratory Animal Center.The growth of tumors was observed.Suitable mice with tumors were divided into 3 groups and to be scanned in a Siemens 3.0 T magnetic resonance imaging machine under anesthesia.3 groups of mice were received intravenous injection of saline,FS and A-MFS via caudal veins,respectively.Images were obtained before and after injection of solution.Then contrast the T2 WI signal intensity of tumors.Measure the biodistribution of Fe in organs and tumors.Tumor tissue was then observed under a TEM to find nanomaterials.All these tests were used to verify targeting ability in vivo.Results1.Characterization of A-MFSThe nanoprobe Fe₃O₄@SiO₂ modified with anti-MSLN antibodies（A-MFS）was successfully prepared.It is spherical and regular in shape.The size of the probe is uniform,and most of the hydrodynamic diameters range between 110 nm and 130 nm.The black central part of the probe is the magnetic nucleus（Fe₃O₄）,with a larger electron density.The surrounding gray portion is the amorphous Si O₂ wrap.XPS detected that Si and antibodies were coated on the nanoprobe.A-MFS has high stability of diffusion.The T2 relaxation rate of A-MFS nanoprobe is 59.435 mM/s and its T1 relaxation rate is 0.549mM/s.High ratio of r2/r1 stands for good negative contrast.2.Cytotoxicity of A-MFSWith the increase of the concentration of A-MFS nanoprobe,the viability of cells was above 86 %,irrespective of the incubation period.On the basis of our experiment,circulation time of A-MFS nanoprobe in vivo was below 24 h,so the nanoprobe was little toxicity.H&E result showed no significant differences in histology between the experimental group of A-MFS and the control group of saline.Therefore,the targeting nanoprobe was nontoxic to mice,and manifested potential for clinical application.3.In vitro targeting testThe red fluorescent dots were more frequently observed on the surface of SW1990 cells,after incubation with A-MFS.The other two groups,SW1990 cells with FS nanoprobe and LM-3 cells with A-MFS nanoprobe showed only a little red fluorescent dots.Flow cytometric analysis showed that the combination rate of A-MFS nanoprobe with SW1990 cells was much higher than other groups.4.In vivo targeting by A-MFSFrom the T2 WI images of nude mice tumors,we could clearly see that significant signal drop in A-MFS groups,while not in FS and saline groups.The T2 WI signal values of experimental group xenografts were decreased by 342.533±42.6 after injection of AMFS soultion,and control group of the saline solution was decreased by-61.233±33.9,while the FS group decreased by-58.7±19.4 at 2.5h.The decrease of tumor signal by AMFS was much more significant than that by saline and FS solution（p<0.05）.The Fe levels in tumor and lung tissue in A-MFS group were obviously higher than in the FS group.The content of Fe in other organs（heart,liver,spleen and kidney）was approximately similar in both A-MFS and FS groups.TEM of transplanted tumors treated with A-MFS revealed A-MFS accumulation in the tumor cells.ConclusionThe Fe₃O₄@SiO₂ probe was successfully prepared and co-loaded with anti-MSLN antibody.Results showed stable and nontoxic A-MFS with regular morphology.Our team demonstrated that A-MFS was capable of specifically recognizing the MSLN-expressing cells in vitro.In addition,A-MFS also exhibits excellent MRI properties and targeting potential to human pancreatic cancer xenografts of nude mice in vivo.Therefore,A-MFS nanoprobe could effectively target pancreatic cancer in vitro and in vivo.In conclusion,AMFS nanomaterial represents a potential T2 negative contrast agent for diagnosis of pancreatic cancer and its metastasis.