| Part ?THE SYNTHESIS AND CHARACTERIZATION OF SUPERPARAMAGNETIC IRON OXIDE BIMODAL NANOPROBEObjectiveTo synthesize magnetic nanoparticles modified by phosphatide polyethylene glycol(DSPE-PEG)and construct MRI/fluorescent dual-modality molecular probe,the characterization of the probe was detected.Materials and methods1.Synthesis of magnetic nanoparticle(Fe3O4): 2mmol Fe(acac)3,20 ml dioctyl ether,oleic acid and oleyl amine(3.4:0.6,total amount was 12mmol)were mixed and purged with nitrogen in a 50 ml three-necked bottle.The temperature of the system was increased to 110?.Opened the cork and eliminated water and impurities from lower boiling point in solution,then kept the temperature under 110°C for an hour.Then the mixture was heated to 220°C for 2h,the solution turned to brightly black,then heated to 290°C for an hour.After the system cooling to room temperature,the synthesized magnetic nanoparticles were purified under the attracting of the magnetic field with ethanol for several times,and dissolved in chloroform at room temperature.Mixed magnetic nanoparticles and phosphatide polyethylene glycol,evaporated the chloroform in the solution using rotary evaporation apparatus and dissolved the magnetic nanoparticles into deionized water.The product was stored under the room temperature at last.2.Synthesis of Fe3O4-PEG: Dispersing a small amount of Fe3O4-OA solution in chloroformic solution,and then added DSPE-PEG-2000 chloroformic solution,and then added 5ml ironed water.Finally,put the mixed solution into the hot pot with a temperature of 70°C.The mixture was shaken for 5min at a rate of 3000rpm/min.Filtered the above mixed solution.Finally,the mixture was stored in preservation solution under 4°C.3.Synthesis of Fe3O4-PEG-Cy7-EMO: Gly-Phe-Leu-Gly was mixed with EDC/N HS solution.After 20 minutes of activation,the EMO was added to the mixture and GFLG-EMO was obtained.Then GFLG-EMO was mixed with Cy7,Fe3O4-PEG activated liquid(EDC/NHS activated)for 12 h.Fe3O4-PEG-Cy7-EMO can be obtained by the purification of the product.4.The characterization of Fe3O4-PEG-Cy7/Fe3O4-PEG-Cy7-EMO: The morphology and core size of nanoparticles were characterized by TEM.The magnetic property of nanoparticles was performed with a VSM and a 1.5T MRI scanner.DLS was adopted to measure the hydrodynamic size of magnetic nanoparticles and their colloidal stability in different p H of saline solution was observed.Fluorescence imaging of magnetic nanoparticles was adopted to detect the capability of fluorescence imaging.ResultsThe TEM results demonstrated that Fe3O4 had a suitable size(9.1±1.7nm)and a good dispersion.The VSM results demonstrated that the saturation magnetization of Fe3O4 was 55.18em?g-1,when the applied external magnetic field was zero,indicating that Fe3O4 was suitable for T2 WI imaging.The DLS results showed that the hydrodynamic size of Fe3O4-PEG,Fe3O4-PEG-Cy7,Fe3O4-PEG-Cy7-EMO were 27.59±8.26 nm,29.81±9.79 nm,and 28.58±8.47 nm,and their hydrodynamic size kept stable in different p H of saline solution.Zeta analyzer results showed that the surface charge of Fe3O4-PEG,Fe3O4-PEG-Cy7,Fe3O4-PEG-Cy7-EMO were-40.2±6.83 m V,-39.8±7.12 m V,-38.7±6.32 m V.The relaxation curve showed that the r2 value of Fe3O4-PEG,Fe3O4-PEG-Cy7,Fe3O4-PEG-Cy7-EMO were 95.0m/Ms,86.9m/Ms,and 103.5m/Ms.Magnetic nanoparticles didn't show a significant precipitation in different concentrations and different p H values of saline for 7 days,indicated that the magnetic nanoparticle had a stability of colloid.MRI imaging in vitro showed that T2 WI signal decreased evidently with the increasing concentration of probes.The fluorescence experiment showed that the sample had a strong fluorescence,and can be preserved for 4~6 months.ConclusionThe superparamagnetic iron oxide molecular probe synthesized in this article has a good dispersion,colloidal stability,good optical property and magnetic property.Most importantly,the probe is suitable for the MR T2 WI imaging and fluorescenc e imaging.Part ?THE PANCREATIC CANCER CELLS CULTURED WITH THE MOLECULAR PROBE IN VITROObjectiveThe pancreatic cancer cell line Bx PC-3,XPA-1,PANC-1,human lung fibroblast cell line MRC-5 and human pancreatic duct epithelial cell line HTERT-HPNE were cultured with Fe3O4-PEG,Fe3O4-PEG-Cy7,Fe3O4-PEG-Cy7-EMO.The targeting capabilities and cytotoxicity of the molecular probe in vitro were researched.Materials and methods(1)The pancreatic cancer cell line Bx PC-3,XPA-1,PANC-1 were cultured with Fe3O4-PEG-Cy7-EMO,prussian blue staining was adopted to observe the phagocytosis of the probe in cells,the control group were human lung fibroblast cell line MRC-5 and human pancreatic duct epithelial cell line HTERT-HPNE.(2)The pancreatic cancer cell line Bx PC-3 was cultured with Fe3O4-PEG-Cy7-E MO,TEM was adopted to observe the distribution of the probe in cells,the control group was human pancreatic duct epithelial cell line HTERT-HPNE.(3)The pancreatic cancer cell line Bx PC-3 was cultured with Fe3O4-PEG-Cy7-E MO,the labeled cells were prepared with 1% agarose in 1.5 ml eppendorf tubes and then scanned under a 1.5T MR scanner,to verify the phagocytosis of cells,the control group was human pancreatic duct epithelial cell line HTERT-HPNE.(4)The pancreatic cancer cell line Bx PC-3 was cultured with Fe3O4-PEG-Cy7-E MO,the labeled cells were prepared with 1% agarose in 1.5 ml eppendorf tubes and then scanned under a fluorescent device,to verify the phagocytosis of cells,the control group was human pancreatic duct epithelial cell line HTERT-HPNE.(5)The pancreatic cancer cell line Bx PC-3,XPA-1,PANC-1 were cultured with Fe3O4-PEG,Fe3O4-PEG-Cy7,Fe3O4-PEG-Cy7-EMO,MTT assay was utilized to detect the cytotoxicity of probes.(6)The pancreatic cancer cell line Bx PC-3,XPA-1,PANC-1 were cultured with Fe3O4-PEG,Fe3O4-PEG-Cy7,Fe3O4-PEG-Cy7-EMO,flowcytometry was utilized to detect the apoptosis of cells,the control group were human lung fibroblast cell line MRC-5 and human pancreatic duct epithelial cell line HTERT-HPNE.(7)The pancreatic cancer cell line Bx PC-3,XPA-1 were cultured with Fe3O4-P EG-Cy7-EMO,Hoechst staining was adopted to observe the inhibition effect of nanoparticles on cells.(8)The pancreatic cancer cell line Bx PC-3,XPA-1,PANC-1 were cultured with Fe3O4-PEG-Cy7-EMO,reactive oxygen staining was adopted to observe the inhibition effect of nanoparticles on cells.Results(1)Prussian blue staining results showed significant blue particles in pancreatic cancer cells compared with normal cell lines,indicating that pancreatic cancer cells have a stronger phagocytosis for probes.(2)TEM results demonstrated that more nanoparticles can be seen in BxPC3 cell line compared with normal cell lines,indicating that the probe has a better targeting ability for pancreatic cancer cells.(3)The mean signal of Bx PC-3 cell line decreased more compared to HTERTHPNE cell line on T2 mapping,indicating that the probe has a better targeting ability for pancreatic cancer cells.(4)The fluorescence imaging demonstrated that Bx PC-3 cell line had a stronger fluorescence intensity compared to HTERT-HPNE cell line,indicating that the probe has a better targeting ability for pancreatic cancer cells.(5)MTT showed that the probe can inhabit proliferation of cells effectively,and the inhibition was relevant to the concentration of the probe.(6)Flowcytometryall results demonstrated that Fe3O4-PEG-Cy7-EMO had a stronger toxicity for pancreatic cells compared to control groups.(7)Hoechst sating trials indicated that the Fe3O4-PEG-Cy7-EMO can inhibit the life vitality of pancreatic cell lines,the morphology of cells changedcdue to the toxicity of the probe.(8)ROS results disclosed the mechanism of apoptosis of the cell lines may be related to the reactive oxygen produced in cells associating with Fe3O4-PEG-Cy7-E MO.ConclusionsThe results proved that the probe Fe3O4-PEG-Cy7,Fe3O4-PEG-Cy7-EMO can label pancreatic cells effectively,they can inhibit the proliferation of pancreatic cells.What's more,the probe was suitable for MRI/fluorescence imaging.It was proved to be a dual modality targeting molecular probe.Part ?VIVO IMAGING OF ORTHOTOPIC NUDE MOUSE MODEL OF PANCREATIC CANCER WITH THE MOLECULAR PROBEObjectiveThe blood compatibility of probes was detected.The molecular probes were injected into the established orthotopic nude mouse model of pancreatic cancer,MRI imaging,fluorescence imaging,HE staining and Prussian blue staining method were adopted to investigate the distribution of the probe in the mouse.The ability of the probe for MRI/fluorescence imaging was detected and the best time point for imaging was determined.Materials and methods(1)The human pancreatic cancer cell line Bx PC-3 transfected with GFP(Bx PC-3-GFP)was cultured,then the cell line was digested with pancreatic enzymes,and the digested cells were injected into the mouse.When the tumor formed,the tumor tissues were removed and cut into 1.0×1.0 mm pieces,then sutured to the pancre as of nude mouse.(2)Twelve orthotopic implanted onto nude mice were divided into 3 groups,T2 WI scanning was adopted,then the probes of Fe3O4-PEG,Fe3O4-PEG-Cy7,Fe3O4-PEG-Cy7-EMO were injected into mice,T2 WI scanning was adopted at the point of 2,4,6,24 and 48 h.After MRI imaging at 6h,24 h,48h,the main viscus and tumors were collected and examined by HE staining and Prussian blue staining.(3)Fluorescence imaging was adopted for the rest 3 tumor-bearing mice,and after the probe of Fe3O4-PEG-Cy7-EMO was injected into mice,fluorescence imaging was adopted again at the point of 2,4,6,24 and 48 h.The viscus and tumor of the mice were used for fluorescence imaging.ResultsBoth MRI and fluorescence imaging can show the distribution of probes in the tumor,and the MRI results further suggested that at the 6h time point,the tumor tissue T2 signal dropped most obviously.The fluorescence imaging indicated that the liver and spleen also had the accumulation of probes at 2h after intravenous injection,indicating that the liver and spleen had a phagocytic effect on the probe.The pathological HE staining indicated that there was no obvious abnormality in the main organ cells,indicating that the probe had no obvious toxicity on the organs.And Prussian blue staining results showed that the probe could escape from the phagocytosis of liver and spleen,and accumulate in the tumor at last.ConclusionsBoth MRI and fluorescence imaging showed that the probe could escape from the phagocytosis of liver and spleen,and accumulate in the tumor at last.T2 WI signal of the tumor decreased most at 2h,and the intensity of the tumor was strong at 780 nm,indicating that the probe was suitable for MRI/fluorescence imaging. |
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