The Experiment Study Of A New Pattern Bimodal Probe For Targeting Imaging Of CD133 Positive Glioma Stem Cells

Part Ⅰ The preparation and characterization of paramagnetic bimodalprobe(fluorescence/magnetic resonance)Objective To synthesize a kind of non-targeting bimodal probe based on the properties of fluorescence and MRI(Magnetic Resonance Imaging).Materials and methods1.Synthesis of NIR QDs 1) The mixture of 50.8 mg of Te(0.4 mmol) and 75.6 mg of Na BH4(2 mmol) were loaded into a round-bottom flask(10 m L) with well sealed. Then, 10 ml of DW was injected into the flask by a syringe. The reaction was stirred in bath water of 80°C, generating the Na HTe solution for future studies. 2) 228 mg of Cd Cl2(0.5 mmol) was dissolved in 40 ml DW, and then 128μL of MPA was added into the solution with stirring. Shortly, the p H degree was adjusted to 11–12 with Na OH. The reaction was connected with condensator, with the continuous protection of N2. When the solution was heated to 90 °C, prepared Na HTe(1.3 m L) was injected. The mixture was heated to 100 °C. After 4 hours, the samples taken from reacted solution were monitored every 15 min. When the expected Cd Te(750 ± 20 nm) was formed, the mixture was cooled to room temperature for future studies.2.Synthesis of BSA-Gd3+-DTPA 1)The BSA solution was prepared by dissolving 200 mg of BSA in 3 m L of Na HCO3(0.1 M, p H 8.5). 200 mg of DTPAA was dissolved in DMSO(1 m L) in the condition of vacuum glove boxes. The solution of DTPAA was gradually added to BSA 100 μL once a time, meanwhile the p H was quickly adjusted to 8.5 with Na OH(5 M) after each addition, the reaction was stirred at room temperature for 2 hours. To remove extra DTPAA and DMSO, the product(BSA- DTPA) was dialyzed with citrate buffer(0.1 M, p H 6.5) three times. 2)1 m L of Gd Cl3, which was prepared by dissolving 100 mg of Gd Cl3 in 1 m L of Na-acetate buffer(0.1 M, p H 6.5), was added to BSA-DTPA slowly, the mixture was stirred for 24 hours. Finally, the complex BSA-Gd-DTPA was completely dialyzed with citrate buffer(0.1 M, p H 6.5) 3 times, and lyophilized for stored at 4 °C temperature.3. Synthesis and purification of non-targeting Cd Te·BSA-Gd3+ The solution of EDC·HCl and NHS were prepared with 0.2 m L DW. The EDC was added to Cd Te, and then the NHS added, reacting for 15 min at room temperature. The activated Cd Te was added to lyophilize BSA–Gd3+–DTPA(5 mg). The reaction was adjusted at a Cd Te/BSA/EDC·HCL/NHS molar rate of 1:15:4000:8000, when the p H degree was controlled at 8- 9 by Na OH(1 M). 2 hours later, the resulting solution was purified by super–centrifuge at the speed of 85,000 g for 30 min, 3 times, and washed by borate saline buffer(p H 8.2) to remove excess BSA. Cd Te·BSA–Gd3+ was dispersed in PBS(10 m M, p H 7.4).4. Characterization of materials Particle size was detected by Nano Particle Analyzer. Particle distribution and morphology was carried out by Transmission Electron Microscopy(TEM).The wavelength of Cd Te and Cd Te·BSA–Gd3+ was compared by Fourier transform infrared spectroscopy(FTIR) spectrometer. The longitudinal and transverse relaxation times were measured by using a 3.0 T MRI.Result The size of NIR Cd Te QDs was 2.5 – 3.0 nm in average with a perfect monodisperse. The wavelength of Cd Te·BSA–Gd3+ was 729 nm, which was almost the same compared with that of Cd Te(740 nm).Observing by UV lights, the Cd Te·BSA-Gd3+ emerged a bright red fluorescence. The Cd Te·BSA-Gd3+ showed high longitudinal and transverse proton relaxation rates(r1, r2) with 17.874 and 26.975 s-1 per m M of Gd3+ respectively, and the r2/r1 ratios was 1.515. In vitro MRI results showed that the Cd Te·BSA–Gd3+ has a great signal enhancement in T1 – weighted imaging.Conclusion The experimental results present the prepared Cd Te·BSA–Gd3+ probe capable of fluorescent and MR bimodal imaging, which laid a foundation of targeting imaging of CD133+ GSCs. Part Ⅱ The Experiment of Targeting Imaging of Cd Te·BSA-Gd3+·Abprobe Against CD133+ Glioma Stem Cells in VitroObjective To confirm the capabilities of bio-imaging and targeting ability of the Cd Te·BSA-Gd3+·Ab probe in vitro.Materials and methods1. The cultivation of cells The cell lines of SU2 s were confirmed as a kind of CD133 positive glioma stem cells which were plenty of CD133 receptors. The SU2 s were cultured with in DMEM/Ham’s F-12 supplemented, containing 20 ng/ml of basic fibroblast growth factor(b EGF), and 20 ng/ml of epidermal growth factor(EGF) and 2 % of N2. SU2 s cells were cultured at 37 °C in 5% CO2 incubator. Observing by inverted microscope the cells were floating in the bottom.2. Cytotoxicity study The cytotoxicity was evaluated by an adjusted MTT assays with SU2 s cells. Comparing the biomaterial toxic of Cd Te with Cd Te·BSA-Gd3+·Ab.3. Synthesis of Cd Te·BSA–Gd3+·Ab The Cd Te·BSA-Gd3+ was reacted with CD133 monoclonal antibodies and EDC·HCl at a rate of 1: 15: 4000 for 30 min. The functionalized products were purified by super-centrifuge at the speed of 85,000 g for 30 min, 2 times, and washed with borate saline buffer(p H 8.2). Finally, the resulting Cd Te·BSA–Gd3+·Ab was dispersed in PBS(10 m M, p H 7.4) at 4 °C temperature for storage.4. Targeting imaging of Florescence in vitro The SU2 s cells were cultured with Cd Te·BSA-Gd3+·Ab for 5, 10, 20, 30 min, when the Cd Te·BSA-Gd3+ were set as a contrast group. The results of samples were oberserved by fluorescence microscopy.5. Targeting imaging of MRI in vitro The SU2 s cells were respectively cultured with Cd Te·BSA-Gd3+, Cd Te·BSA–Gd3+·Ab, Magnevist(Gd–DTPA) and NS for 30 min.T1-weighted MRI was performed by 3.0 T MRI.Result The cell viability of cells with Cd Te·BSA-Gd3+ was higher than that of Cd Te. According to the performance of fluorescence microscopy, the SU2 s cells with Cd Te·BSA–Gd3+·Ab had more fluorescence distribution than the group of Cd Te·BSA–Gd3+. The results of MRI demonstrate the SU2 s cells with Cd Te·BSA–Gd3+·Ab had a higher signal enhancement, compared with that of non–targeted cell group by Cd Te·BSA-Gd3+, Magnevist(Gd–DTPA) and NS.Conclusion The experimental results present the prepared Cd Te·BSA–Gd3+·Ab probe新型MRI荧光双模态探针实现CD133+capable of fluorescent and MR bimodal imaging,and the probe can selectively located in the CD133+GSCs with lower toxicity.This study provides a promising strategy for early diagnosis and targeted therapies of glioma.