| Background and purpose Glioma is the most common intracranial tumor, most with infiltrative growth characteristics.Due to blood brain barrier(Brain blood barrier, BBB),the contrast agent can not enter tumor, and its boundary is difficult to determine during surgery.Therefore,the glioma has the tend to recur after surgey,and the prognosis is poor. Early diagnosis and the clear definition of tumor boundary are the key factors affecting the prognosis. With the rapid development of nanotechnology, the contrast agent combined with nanopolymer can be made into targeted nanoparticle contrast agent, which can penetrate the blood brain barrier and tumor specific binding, prolong the half-life of contrast agent, may provide the early diagnosis of brain gliomas.Therefore, It may greatly promote the development of early dignosis of brain tumor. In this study, we synthesized a kind of multi-functional targeted contrast agent, including ligand Angiopep2, magnetic resonance imaging part(Gd3+) and fluorescence imaging in vitro part(FITC). The purpose of this experiment was to synthetize a targeted contrast agent which can penetrate the blood-brain barrier and accumulate in the tumor.At the same time,non-invasive MRI technology was used to track the progress of cell uptaking in vitro, dynamically observe the tumor boundary. This double targeting function nanotechnology helps the early diagnosis of tumor and evaluates tumor prognosis using MRI(including preoperative and postoperative residual effect for diagnosis, follow-up). This research provides some new ideas of diagnosis of central nervous system diseasesbefore operation,which helps to improve the prognosis of glioma.Materials and methods1. the synthesis and characters of Angiopep-2 mediated dual-targeting nanoparticle:the synthesis of CD-PDEA-b-P(OEGMA-co-Gd-co-ANG-co-FITC);TE was fixed,measure the signal intensity at different concentration of Gd3+ of Ang-NP-Gd and alkymyl-DOTA-Gd by changing TR, and compare the longitudinal relaxation rate between them;C6 cells was cultured with different concentration of NP and Ang-NP,48 h later,the absorbance was recorded at 570 nm by a microplate reader,then we compare the relationship between cell viability and concentration.2. Evaluation of dual-targeting nanoparticle in vito:C6 cells and endothelial cells was incubated with Ang-NP-FITC and NP-FITC at 30 min, 4h and 24 h by flow cytometry; C6 glioma spheroid was incubated with FITC labeled Ang-NP or NP at 30 min, 4h and 24 h.For the competition assay, Angiopep-2 was added in advance,after 60 min incubation, FITC labeled Ang-NP was set into the wells at 30 min, 4h and 24 h.3. C6 glioma model of rats:18 male SD rats, about 250-280 g; C6 glioma cells were injected into the right caudate nucleus of rat to establish C6 glioma model. We measured the the volume of tumor at different time, and made a curve; 4-20 days after the establishment of glioma model, contrasted imaging was collected by injecting Ang-NP-Gd and NP-Gd respectively by the 1.5T MRI scanning. Image was transmitted to the adw4.4 workstation, we measured the degree of enhancement of tumor, the tumor boundary and CNR; then the rat brain tumor was completely stripped down, embedded in paraffin,and HE staining was done.Result 1. The longitudinal relaxation rate of Ang-NP-Gd is higher than alkymyl-DOTA-Gd;the cytoxicity of targeted and non-targeted contrast agent was negligible.2. C6 cells and endothelial cells uptook plenty of Ang-NP-FITC;The order of fluorescence intensity of C6 glioma spheroids was Ang-NP-FITC>NP-FITC>Ang-NP-FITC+Angiopep-2。3. The function between the volume of tumor and time was exponential relationship. Ang-NP-Gd made tumor early enhanced,and the boundary was more clear;CNR was increased by time,and reaches the top at 12 h.Conclusion The nanoparticles contrast agent modified with Angiopep2 specific binding of low density lipoprotein receptor related protein-1(LRP-1) receptor, which is expressed in tumor cells and the blood-brain barrier endothelial cells, has the very strong ability to penetrate the BBB and enter the brain tumor location.The nanoparticles contrast agent helps the early diagnosis and clear boundary of glioma, and provides a new research direction to further improve the prognosis of glioma. |
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