Targeting And Multimodal Molecular Imaging Of Prostate Cancer Mediated By Recombinant Dual Reporter Gene System P_DD3-TfR-WPRE-P_CMV-Luc

Objective:to construct DD3 promoter-driven dual reporter gene system using genetic recombination technique and investigate the expressing specificity of dual reporter gene.Meanwhile,we aimed to fabricate magnetic nanoparticles（Tf-USPION）and explore cellular toxicity and the capability of in vitro MR imaging.Furthermore,utilizing the above molecular probes,we study the in vivo targeting and multimodality imaging of prostate cancer.Our study integrated molecular imaging techniques mediated by specific receptors and reporter gene,and provided co-registration of structural and molecular-genetic information for the early detection and dynamic monitor therapy of prostate cancer.Methods:The recombinant plasmidwith dual promoter and dual reporter gene were constructed using genetic recombination technique.After inserted by WPRE enhancer,the constructed plasmid was packaged and amplified in HEK293 cells to acquire large amounts of adenovirus vectors.Normal cells,prostate cancer cells and other tumor cells were cultured and treated with the recombinant adenovirus.TfR and Luc protein expression levels were estimated through Western blotting.Ultrasmall superparamagnetic iron oxide nanoparticles were synthesized in reacting kettles via a one-step approach.Tf-USPION nanoprobe was fabricated by the conjugation of as-prepared USPION and transferrin.After characterition,in vitro MR imaging and MTT assays are carried out after cells are incubated with the same concentrations of USPION or Tf-USPION.22RV1 and LNCaP cells were subcutaneously transplanting into the left shoulder of BALB/c nude mice.Fluorescence imaging of animals was carried out during an observation period of 2 weeks.Then dynamical MR imaging was performed after tail vein administration of Tf-USPION.Tumor-bearing mice with injection of Tf-USPION without the recombinant adenovirus were set as control group.2 weeks later,animals were sacrificed and hematoxylin and eosin staining of the dissected organs was examined to visualize the potential toxicity in vivo.Result:（1）The recombinant plasmid expressing transferrin and luciferase under the control of DD3 promoter,was successfully constructed.The expression of TfR and Luc elevated significantly in prostatic cancar cells,but not in normal cells and various non-prostatic tumor cells.（2）The magnetic nanoparticle Tf-USPION was successfully fabricated via one step method and surface modification techniques.The prepared Tf-USPION had a narrow size distribution with the mean diameter of 10nm,85.33mmol/L^-1S^-11 in T₂ relaxivity value and no obvious cytotoxicity.（3）In vivo imaging of 22RV1and LNCaP bearing mice showed that bright fluorescence signals could be visualized in tumor region.The fluorescent intensity increased progressively over the time,reached the maximum on the 5-7 day post-injection,and then fluorescence in the tumor region gradually diminished with time in the next 7 days.LNCaP bearing mice showed better performance but no obvious fluorescence could be observed in normal mice treated with subcutaneous injection of recombinant adenovirus.（4）At 0.5 h post-injection,enhanced hypotensive T₂-weighted images could be observed only in tumor region,with clearly visualized location,size and boundary.T₂ enhancing effect reached the maximum at 4h.T2WI of the control group showed that,tumor region had dark signal from 0.5 h post-injection.The enhancing effect increased slowly over the time but was too low to define the tumor’s border.（5）HE examinationsof the control group and normal mice showed no obvious injury in cellular structures of major organs.Conclusion:（1）The adenovirus vector Ad-PDD3-TfR-WPRE-PCMV-Luc that could express transferrin and luciferase under the control of DD3 promoter,was successfully constructed.（2）The magnetic molecular probe Tf-USPION is successfully fabricated and is promising for in vivo applications because of the advantages of suitable size,high relaxation,excellent MR performance and low cytotoxicity.（3）The recombinant adenoviral vector Ad-P_DD3-TfR-WPRE-P_CMV-Luc was a very ideal candidate for targeted multimodality imaging-based detection of PCa and its metastasis in vivo with the combined utilization of Tf-USPION.The excellent bio-compatibility and imaging capability of adenovirus vector Ad-P_DD3-TfR-WPRE-P_CMV-Luc and Tf-USPION indicated that further clinical translation of this imaging strategy could be expected.