MR Molecular Imaging Diagnosis Of Hepatic Tumor-targeting SPIO/Ferritin Plasmid Complex Nanoparticles

Hepatocellular carcinoma(HCC)is one of the diseases that threats human health and life.Magnetic resonance imaging(MRI)contrast agents for HCC are almost non-targeting,which are difficult for diagnosing the early tumors.This project based on the previous study of target research of HCC and aims to develop a contrast agent for the specific dual-source targeting the hepatocellular carcinoma cells.Results from this study increased the diagnosis rate of early HCC and achieve the specific diagnosis of early HCC in molecular level.First,NH2-PEG-SA was firstly synthesized through the reaction between amine group of NH2-PEG-NH2 and carboxyl group of stearic acid in the presence of EDC.A54-PEG-SA was then synthesized through the coupling reaction between amine group of NH2-PEG-SA and carboxyl group of A54 in the catalytic condition of EDC.SPIO-loaded nanostructured lipid carrier(SNLC)with hepatocyte passive targeting function and A54 modefied SNLC(A54-SNLC)with specific hepatoma cell active targeting function were prepared by emulsion-diffusion method.The physicochemical characteristics of the SNLC and A54-SNLC were evaluated.The results of particle size,potential and multi-dispersion index demonstrated that the particle sizes were around 46.4±3.44 nm and 45.8±3.96 nn,while zeta potential were around-20.9±1.05 mV and-21.5±1.13 mV.Thus,almost no changes of size and zeta potential in charge with the addition of peptide A54,TEM showed that the particle size distribution of the two magnetic nanoparticles was unifonn and spherical,the magnetic Fe3O4 nanoparticles were wrapped in lipid nanoparticles.The results are consistent with the results of particle size potentiometer.An AFP-Fth plasmid(DNA)with an endogenous specific ferritin expression on hepatocellular carcinoma cells was constructed.Magnetic nanostructures lipid carriers and DNA was combined through electrostatic interaction using protamine as the medium.An active dual-source T2-weighted MRI contrast agent(A54-SNLC/Protamine/DNA ternary complexes,A54-SNPD)with specific targeting of hepatocellular carcinoma cells was prepared.At room temperature for 2 weeks the magnetic nanoparticles solutions showed no aggregation,this demonstrated that A54-SNPD ternary complexes prepared by this study are highly dispersive and stable.TEM was conducted to investigate the morphology and structure and particle size distribution of ternary complexes,further to verify the successful formation of the A54-SNPD ternary nanoparticles.The cytotoxicity of the SNLC,A54-SNLC,and A54-SNPD with di?erent weight ratios of A54-SNLC to protamine/DNA(DNA,2?g/well)on LO2 and Bel-7402 cells were measured by MTT assay.The results manifested the SNLC,A54-SNLC and A54-SNPD ternary nanoparticles had a relatively high biocompatibility and low toxicity for both tumor cells and normal cells,which can be used safely for clinical magnetic resonance imaging.Vitro gene transfection test of ternary nanoparticles indicates that A54-SNPD with weight ratio of 20/4/1 on Bel-7402 cells was optimal and compatible for transfection in Bel-7402 cells.The AFP protein expression in Bel-7402 cells and LO2 cells were analyzed by performing Western Blot analysis.The results showed that AFP protein expression can be detected in Bel-7402 cells,whereas little AFP protein was detected in LO2 cells.Furthermore,the Fth expression level in Bel-7402 cells and L02 cells before and after gene transfection of A54-SNPD(20/4/1)ternary nanoparticles were also analyzed by Western Blot analysis.The results showed that Fth protein expression level in A54-SNPD(20/4/1)transfected AFP-positive Bel-7402 cells was prominently higher than that in untransfected Bel-7402 cells.In contrast,there was no remarkable di? erence between the A54-SNPD transfected L02 cells and the untransfected L02 cells.The results show that the reporter gene plasmid AFP-Fth under the control of AFP promoter can be expressed in the AFP-positive Bel-7402 cells definitely and specifically.In contrast,the reporter gene with AFP promoter had little e?ect in AFP-negative L02 cells.This attributed to the endogenous expressive ability of A54-SNPD toward Bel-7402 cells.Qualitative or quantitative cell uptake experiments were conducted to further investigate the targeting ability of A54-SNPD on Bel-7402 cells and L02 cells,further to confirm the exogenous targeting ability of the A54-SNPD to Bel-7402 cells.Subcutaneous transplanted model and orthotropic hepatoma model were established successfully,which were confirmed by anatomical and histopathological results.The histopathological results also found that the Bel-7402 tumor tissues were observed with positive staining of AFP.In contrast,the normal liver tissues were detected with negative staining of AFP.The result firrther to vertify that the Bel-7402 tumor was considered as AFP-positive tumor,which was consistent with the above cell results.Left and right sides of the nude mices were separately transplanted with HepG2 and Bel-7402 tumor and observed by the small animal fluorescence living imager.The results showed that A54-SNLC could reach and accumulate in the Bel-7402 tumor specifically and effectively.The results of the study on the targeting uptake of A54-SNPD and S:NPD in nude mice with orthotropic hepatoma were also indicated that the targeting ability of A54-SNPD nanoparticles to Bel-7402 cells were better than that of SNPD by active targeting mechanism.A54-SNPD showed a strong targeting ability in Bel-7402 cells.MRI results of magnetic nanostructure lipid carriers(A54-SNLC,SNLC)show that the A54-SNLC prepared by our study has negative imaging effect,and there is a certain concentration effect relationship in certain range of concentration.As the concentration of A54-SNLC increased,the negative enhancement effect of T2WI was more obvious,so A54-SNLC can be used as MR T2 contrast agent.MR imaging results of A54-SNLC to Bel-7402 cells in vitro showed that the signal intensity decreased most obviously when the A54-SNLC was uptaked by Bel-7402 cells,confirmed the exogenous targeting effect of A54-SNLC on Bel-7402 cells.After SNLC and A54-SNPC were injected into nude mices in vivo separately,the signal:intensity decreased in tumor of A54-SNLC group was obviously higher than that of SNLC group,confirmed the exogenous targeting effect of A54-SNLC on Bel-7402 tumors.The study of MR molecular imaging in ternary complex nanoparticles(A54-SNPD,A54-SNP,SNPD,SNP)showed the ternary complex nanoparticles have obvious negative enhancement effect.Because A54-SNPD contains exogenous targeting function of the iron accumulation and endogenous expressive function of the Fth protein,T2 imaging intensity values of hepatic tumors were successfully decreased via the T2 contrast enhancement of ternary nanoparticles,and the targeting effect can lasts from 6 h to 48 h.The A54-SNPD group had the most significant reduction in SNR value of the tumor compared with the other three groups.It is emphasized that the novel A54-SNPD termary nanoparticle as active dual-source T2-weighted MRI contrast agent were able to increase the diagnostic sensitivity and specificity of hepatic cancer.In suumary,This study successfully prepared dual-source ternary complex nanoparticles(A54-SNPD),which has specific targeting function on Bel-7402 cells and tumors.Vitro and vivo experiments confirmed that A54-SNPD had a good application prospect in targeted MRI imaging of liver cancer.