Glioblastoma multiforme targeting nano magnetic resonance contrast agent as well as preparation and application thereof

[0039] Example 1 Preparation and Characterization of Targeted Nano Magnetic Resonance Contrast Agent

[0040] Preparation of PEG‐USPIO nanoparticles: ultrasmall superparamagnetic iron oxide particles (USPIO), phosphatidylethanolamine‐polyethylene glycol (DSPE‐PEG), phosphatidylethanolamine‐polyethylene glycol‐active ester (DSPE‐PEG) ‐NHS) and immunofluorescence-labeled coumarin 6 or Cy7.5 were mixed in a weight ratio of 4:20:2:1, dissolved in dichloromethane, ultrasonicated for 10 minutes, and 37°C rotary steamed for 60 minutes to remove dichloromethane and formed a film covering the The inner wall of the bottle, water-dispersed film, to obtain water-dispersed nanoparticles;

[0041] Preparation of PEG‐USPIO‐PEPHC1 nanoparticles: Add 50 μL of sulfhydryl-activated short peptide PEPHC1 20 mg / mL to 4 ml of prepared deionized water-dispersed nanoparticles (0.25 mg / mL USPIO), sonicate for 10 min, centrifuge at 12000 rpm at 4 ° C, discard clear, and disperse the precipitate with ...

[0043] Example 2 Characterization diagram of magnetic sensitivity and fluorescence characteristics of targeted nano magnetic resonance contrast agent

[0044] Take an appropriate amount of nano-magnetic resonance contrast agent solution samples, prepare different concentrations of PEG-USPIO-PEPHC1 with deionized water, the iron concentration is 0, 5, 10, 20, 40ug / mL, and place them in 2ml centrifuge tubes respectively. Then PEG-USPIO with corresponding iron concentration was configured as a control. The tubes are placed sequentially in plastic test tube racks and placed in boxes filled with water. Deionized water was used as blank control. The clinical 3.0T MR system (Discovery MR750, GE Medical System, USA) and supporting small animal coils were used for T2Map sequence scanning to evaluate the effect of negative reinforcement. The T2Map images were post-processed using GE Function tool 4.6 special software.

[0045] PEG-USPIO-PEPHC1-Cy7.5 Fluorescence Intensity Detection...

[0048] Example 3 In vitro cell antibody and short peptide to detect the expression of EGFRvIII mutants respectively

[0049] Detection of cell surface antigen expression

[0050] 1) Inverted fluorescence microscope analysis:

[0051] U87 or U87‐EGFRvIII cells were treated with 1x10 5 The cells were seeded in a 12-well plate at a density, adhered to the wall for 24 hours, discarded the culture medium, washed 3 times with PBS, 3 minutes each time; fixed with 4% paraformaldehyde for 20 minutes, dried naturally for 10 minutes, washed 3 times with PBS, 5 minutes each time; Add immunostaining blocking solution and place at room temperature for 1 hour. After absorbing the blocking solution, add primary antibody and incubate overnight at 4°C; the next day, wash thoroughly with PBS, add secondary antibody, and incubate for 1 hour at room temperature in the dark. After fully washing with PBS, add 1% DAPI staining solution, cell nuclei were stained in the dark for 5 minutes at room t...