Aptamer-conjugated Pegylated Quantum Dots Targeting EGFRvⅢ For Fluorescence Imaging Of Glioma

Objective: To investigate the application of aptamer-conjugated PEGylated Quantum Dots(QD-Apt)targeting epidermal growth factor receptor variant III(EGFRv III)for fluorescence imaging of Glioma.Methods: 1.Synthesis,characterization and safety evaluation of QD-Apt.1)Synthesis and characterization of QD-Apt: Biotinylated aptamer 32(A32)conjugated streptavidin-PEG-Cd Se/Zn S QDs(SA-QD)by the biotin-streptavidin cross-linking technique and then the aptamer-conjugated PEG-Cd Se/Zn S QDs(QD-Apt)was required.The sizes of nanoparticles were analyzed by Zetasizer Nano ZS particle size;Photoluminescent measurement was performed with a fluorescence analyzer.To validate the connection between aptamer and QDs,QDs and QD-Apt were analyzed by agarose gel electrophoresis.2)Safety evaluation of QDs and QD-Apt: CCK-8 was applied to evaluate the cell viability of HUVEC,U87,U87-EGFRv III cells after treatment with various concentrations of QD-Apt in vitro.After injected QD-Apt, the weights of mice were recorded at different time.All organs were harvested and stained with hematoxylin and eosin(H&E)for histopathological study by light microscopy.2.QD-Apt targets to glioma cell lines in vitro 1)EGFRv III expression in human glioma cell lines: To detect the expression of EGFRv III in HUVEC,U87,and U87-EGFRv III cell lines,Western Blot,RT-PCR and immunofluorescence were applied.2)QD-Apt targets to glioma cell lines: QD-Apt was used to label the cells and then observed them with laser scanning confocal microscope.Flow cytometry was used to validate the labeled effect of the QD-Apt.3.The biodistribution and targeted imaging for mice glioma in situ.Orthotopic glioma models were established and MRI was used to monitor the growth of tumors.After 4 weeks,tail-vein injection of QDs or QD-Apt for six hours,all mice were observed by the IVIS Imaging System.Tumor and normal brain tissues’ frozen sections were observed by laser confocal microscopy.All organs were dissected from mice and measured the fluorescence intensity of them by fluorescence plate reader.Results: 1.QD-Apt nanoprobe was constructed successfully.The sizes of QD-Apt were slightly increased compared with QD,but all of their sizes were about 20 nm;Photoluminescent measurement reveals the maximum luminescent wavelength of QD and QD-Apt both were 605±5 nm,and their absorption peak were 330 nm.The result of electrophoresis suggested that biotinylated aptamer and streptavidin-QD had been connected.The safety studies suggested that QD-Apt was nontoxic and enjoyed optimal biocompatibility.2.There was no expression of EGFRv III in HUVEC and U87 while U87-EGFRv III had a strong positive expression of EGFRv III.QD-Apt can specially bind to the U87-EGFRv III cells and the binding rate of QD-Apt conjugated U87-EGFRv III reached up to 81.53% in vitro.3.Fluorescence imaging in orthotopic glioma model mice bearing U87-EGFRv III showed that QD-Apt could cross the blood-brain barrier and then selectively accumulated in the tumors depending on binding to EGFRv III and generated strong fluorescence signal compared with normal brain tissues,which contributed to the margins of gliomas were visualized clearly by QD-Apt.The QD-Apt predominantly accumulated in the tumors(EGFRv III +),liver and kidneys,low levels were observed in the para-tumor,normal brain,spleen,heart and lungs.Conclusion: The QD-Apt can specially bind to EGFRv III for Fluorescence Imaging of Glioma.