Fabrication Of Molecular Probe Targeting NRP-1 And Magnetic Resonance Molecular Imaging Of Gliomas

Objective: To detect the relative expression of neuropilin-1 in different glioma cell lines and screen a proper object for our experiment.To synthesis a novel magnetic resonance molecular probe USPIO-PEG-tLyP-1 by labeling the neuprolin-1 targeting peptide tLyP-1(CGNKRTR)with ultrasmall superparamagnetic iron oxide and identify its physicochemical property and biotoxicity for further application in clinical.To verify USPIO-PEG-tLyP-1's ability to target glioma cells and yield T2 WI signal decrease.Methods:1 Glioma cell lines(U87?U251?H4 and C6)were cultured in 10% fetal bovine serum and passaged when necessary.Neuropilin-1 expression levels were detected by RT-PCR and Western blot compared with HUVEC and normal rat brain.The subcellular localization of neuropilin-1 was detected by Immunofluorescence staining.2 Peptide tLyP-1(CGNKRTR)was purified by high performance liquid chromatogram and verified by mass chromatogram.The novel magnetic resonance molecular probe USPIO-PEG-tLyP-1 was coupled by USPIO-PEG and tLyP-1 using EDC as the coupling reagent.The diameter of USPIO-PEG-tLyP-1 was detected by TEM and dynamic light scattering.Then the T2 WI signal strength variation of USPIO-PEG and USPIO-PEG-tLyP-1 was detected by 3.0T MRI.At last,cell-toxicity of USPIO-PEG-tLyP-1 was assessed by MTS assay in vitro.3 The uptake efficiency of USPIO-PEG-tLyP-1 was measured by Prussian blue staining and MR imaging in vitro.Value of T2 transverse relaxation time of glioma cells incubated with USPIO-PEG and USPIO-PEG-tLyP-1 was calculated by T2 map sequence.4 Distribution of USPIO-PEG and USPIO-PEG-tLyP-1 in U87 glioma cells was detected by TEM imaging.Results:1 Neuropilin-1 was significantly overexpressed in these glioma cell lines(U87 ? U251 ? H4 and C6).Among these cell lines,the expression of neuropilin-1 in U87 glioma cells was the highest.The difference was statistically significant.In addition,Neuropilin-1 were mainly located in cytoplasma and membrane.2 The solution of USPIO-PEG-tLyP-1 was stable in PBS and the hydrodynamic size was 43.84 nm.In addition,there was no significant difference in R2 values of USPIO-PEG and USPIO-PEG-tLyP-1 with different Fe concentrations ranging from 0 to 5 ?g/mL.Compared with control group,there was no statistically significant difference in the survival rate of cells that were treated with USPIO-PEG-tLyP-1 at a concentration ranging from 0 to 50 ?g Fe/mL.3 Prussian blue stain showed that when incubated with USPIO-PEG-tLyP-1,U87 glioma cells clearly exhibited deeper blue staining than those incubated with USPIO-PEG.Vitro MRI showed that USPIO-PEG-tLyP-1 significantly enhanced the negative contrast effect than USPIO-PEG.A further quantitative analysis showed that R2 of U87 glioma cells incubated with USPIO-PEG-tLyP-1 was higher than those incubated with USPIO-PEG.The difference was statistically significant.4 The TEM studies suggested that USPIO-PEG-tLyP-1 was taken up by U87 glioma cells and mainly distributed in cytoplasm,endoplasmic reticulum,mitochondria and lysosome.Conclusions: We successfully synthesized a novel type of magnetic resonance molecular probe named USPIO-PEG-tLyP-1 and verified it's good biocompatibility at the given concentration range.Furthermore,our work demonstrated USPIO-PEG-tLyP-1 as an ideal magnetic resonance molecular probe for targeting glioma cells by prussian blue staining and vitro MRI,thus providing a sufficient basic for further study.