Early Diagnosis Of Pancreatic Cancer In Vivo By Plectin1 Molecular Targeted Probe With Fluorescent And MR Imaging

Part ? Design and characterization of Dual-modal Molecular Probe Targeted Plectin1Objective To design SPIONs(Superparamagnetic Iron Oxide nanoparticles)modified by amination of phosphatide polyethylene glycol(NH2-PEG),combine it with the Plectin1 antibody and Cy7-NHS to synthesize a dual-modal Molecular probe anti-Plectin1-NH2-PEG·SPIONs-Cy7,and detect it's characterization.Materials and methods 1.Design of NH2-PEG·SPIONs 1.1 The synthesis of oil soluble SPIONs: Moderate Fe(acac)3,oleyl amine and oleic acid were blended under nitrogen and the mixture was heated to 220°C for 30 min,and to 290°C for 1h,respectively,then,it was cooled to room temperature.The SPIONs were purified by ethanol under the attracting of magnetic field,and then stored 4°C after dissolving in chloroform.1.2 Synthesis of NH2-PEG·SPIONs: NH2-PEG was dispersed in chloroform and mixed it with the soluble SPIONs above,rotated and evaporated at 65°C with deionized water for 13 min.The solution was spined by centrifuge and dispersed in deionized water after ultrasonic oscillation.The mixture was filtrated for several times before storing.2.Design of anti-Plectin1-NH2-PEG·SPIONs-Cy7 The NH2-PEG·SPIONs were dispersed in deionized water and mixed with MES,then 2.68 ml EDC and 4.8ml sulfo-NHS were added into the solution.The mixture was shaken in a swing bed following centrifugation.Disposing the mixture with borate buffer(BB)and 70 ?l 0.5mg/ml anti-Plectin1 antibody,700 ?g Cy7 wereadded afterwards.The final solution was shaken in a swing bed for 12 h,and then stored away from light at 4 ?.3.The characterization of NH2-PEG·SPIONs and anti-Plectin1-NH2-PEG· SPIONsCy7 The morphology of NH2-PEG·SPIONs was examined by Transmission electron microscopy(TEM)and the core size of NH2-PEG·SPIONs was displayed on HRTEM.The colloid stability of NH2-PEG·SPIONs was surveyed for 1 week.The hydrodynamic size and zeta potential of NH2-PEG·SPIONs and anti-Plectin1-NH2-PEG· SPIONs-Cy7 were detected.The magnetic property was determined with a Vibrating Sample Magnetometer(VSM)and 3.0T MR.The toxicological test was studied with NH2-PEG·SPIONs and anti-Plectin1-NH2-PEG· SPIONs-Cy7 in healthy mouse.Results NH2-PEG·SPIONs had a suitable size(12nm)and great monodisperse on TEM and HRTEM.The colloid stability of NH2-PEG·SPIONs was proved good.The residual magnetism of NH2-PEG·SPIONs was zero and the saturation magnetization was 80em?g-1 which was suitable as MR imaging contrast agents.The T2 signal of the mixture decreased evidently according to the increasing concentration of NH2-PEG· SPIONs on MR imaging.The measured r2 value of the relaxation curve of NH2-PEG ·SPIONs was 115.95 m M/s.The hydrodynamic size of NH2-PEG·SPIONs?NH2-PEG·SPIONs-Cy7 and anti-Plectin1-NH2-PEG· SPIONs-Cy7 were about 28.85 nm,55.80 nm and 84.34 nm,respectively.The particle sizes were suitable for MRI contrast agent.The surface charge of NH2-PEG·SPIONs?NH2-PEG·SPIONs-Cy7 and antiPlectin1-NH2-PEG· SPIONs-Cy7 were about 15.9m V??24.8m V and?11.3m V,respectively.These testify that the fluorescent material Cy7-NHS and(or)antibody anti-Plectin1 successfully connected to the surface of magnetic nanoparticles.No evident for toxicity of NH2-PEG·SPIONs and anti-Plectin1-NH2-PEG· SPIONs-Cy7 were observed in vivo experiment.Conclusion The molecular probe anti-Plectin1-NH2-PEG·SPIONs-Cy7 synthesized in this study showed well dispersion,proper magnetic property as well as low toxicity which is proved to be a promising probe for MR imaging.Part ? Fluorescence?MR Imaging of Pancreatic Cancer in Animal Models Using Double Modal Targeted ProbeObjective The efficacy of targeted probe as negative contrast and its dispersion in mice were evaluated by MRI?Fluorescence imaging and histological staining.Materials and methods 1.The human pancreatic cancer cell line transfected with RFP(red fluorescent protein,RFP)was cultured and then digested with pancreatin,and the suspension of cells was injected subcutaneously to the mice.When the tumor formed,the tumor tissues were removed and cut into 1.0×1.0 mm and sutured to the pancreas of nude mouse.2.Before injecting with targeted probe through tail vein,the tumor-bearing mice were scanned pre-contrast administration.The enhancement scanning was performed at the following time-point 8h,24 h and 48 h.Four mice were sacrificed at each time-point.The liver,spleen,kidney,pancreas and tumor were collected and examined by HE and Prussian blue staining.Results After injecting the probe at 8h?24h,Prussian blue staining showed that a few blue particles disbursed in pancreatic tumors,especially in the periphery of pancreatic tumor,at 48 h,lots of blue particles disbursed in the center of pancreatic tumor indicating that the particles targeted probe can targetly distributed in the tumor.After injecting the probe at 48 h,T2WI showed the T2 signal of pancreatic tumor was lower than that of pre-contrast administration.The near-infrared fluorescence imaging,at 8 h and24 h after probe injection,showed the fluorescence of probe were mainly distributed in nude mice liver and spleen The probe fluorescence mostly distributed in the tumor area and equivalent to that of tumor at 48 h of near-infrared fluorescence imaging.T2 signal in liver and spleen decrease obviously after the probe injected,and the lower signals were still maintained at 48 h.Prussian blue staining showed thatdifferent amounts of blue particles disbursed in the liver and spleen.T2 signal in kidney has no obvious change compared to that of pre-contrast administration.Prussian blue staining showed that only little or no obvious blue particles disbursed in the kidney.Conclusion The MR/Fluorescence molecular probe reported here can target to the pancreatic cancer and make a reduction in the T2 signal intensity,Prussian blue staining,further demonstrates that the probe anti-Plectin1-NH2-PEG·SPIONs-Cy7 is a potential specific probe for pancreatic tumors.However,the change of T2 signal intensity is weak and vulnerable to many factors such as the tumor necrosis,hemorrhage and cystic degeneration.Further researches are needed for real clinical application.