Paramagnetic Albumin Decorated CuInS₂/ZnS QDs For CD133⁺ Glioma Bimodal Targeted Imaging

Part Ⅰ The preparation and characterization of non-targeted MR/fluorescence bimodal nanoprobesObjective To synthesize a new kind of non-targeted MR/fluoscence bimodal nanoprobes and to characterize the properties.Materials and methods1. Synthesis of CuInS₂/Zn S QDs: 1) 26.2 mg of Cu（acac）2, 41.2 mg of In（acac）3 and absolute excess of DDT were mixed in a 25 m L three-neck flask. The flask was connected with ventilation equipment and the whole device was sealed. Then the sealed system was vacumized and refilled with inert argon to remove oxygen. The mixture was heated to 230 °C with magnetic stirring to form the CuInS₂ core QDs. With temperature increasing, the color of solution changed progressively from colorless to pale yellow, red, and finally dark reddish-brown and CuInS₂ generates. 2) Dissolve Zn（OAc）2 into 4 m L of ODE, 1 m L of OA and 1m L of OLA to prepare the Zn S precursor. Then the Zn S precursor was injected dropwise slowly into the mixture at intervals of 20 min and still keep it under argon atmosphere. The final liquid was cooled to room temperature and precipitated by adding excess ethanol followed by centrifugation at 10,000 rpm for 15 min. The resulting precipitation was dispersed into chloroform. The whole purification process was repeated at least three times to obtain a pure product for further use.2. Synthesis of BSA-DTPAGd: 1) 1 g of BSA was dissolved in 15 m L of borate saline buffer（50 m M, p H 8.2）. Then 1 g of DTPA completely dissolved in 5 m L of DMSO was added into the prepared BSA solution, make the p H= 8-8.5 using borate saline buffer（50 m M） and Na OH（0.5 M）. The mixture was stirred for at least 4 h and then purified by dialyzing against 5×4 liter of citrate buffer（0.1 M, p H= 6.5）. 2) A little amount of Gd Cl3·6H₂O（approximately 1.5 mmol, 0.5 g） dissolved in 5 m L of sodium acetate buffer（0.1 M, p H= 6.5） was added slowly into the mixture prepared above. Keep this blend reacting for 24 h with mechanical agitation and then dialyze against 5×4 liter of sodium citrate buffer（0.1 M, p H= 6.5） for 3 days and then against distilled water. The resulting product was lyophilized to give BSA doped with gadolinium as white powder. Make it stored at 4 °C for further use.3. Preparation of paramagnetic albumin decorated CuInS₂/Zn S QDs: About 0.5 g of the prepared BSA-DTPAGd compound was dissolved in 30 m L of deionized water in a 50 m L beaker. Put the beaker under the probe of ultrasonic cell crushing equipment and make sure that the probe insert below the liquid level of BSA mixture at least 5 mm. Then the 0.5 m L of QDs/chloroform mixture was injected into the above BSA water solution using a syringe with ultrasonic crushing done every 10 seconds for intervals of the same time at 200 W. The obtained white emulsion was evaporated via a rotary evaporation machine to remove redundant chloroform. Ultimately, the resulting optical clear solution was purified twice by ultra-centrifugation at 70,000 g for 15 min to remove the residual free BSA-DTPAGd compound. The precipitation was dispersed in p H=8.2borate saline buffer（50 m M） for further experiments. This process also implemented phase transfer of QDs through ligand exchange from oil phase to water phase during the process of ultrasonic crushing, which was also suitable for in vivo use.4. Characterization: The ultraviolet–visible absorption spectra of nanomaterials were recorded using a fluorescence spectrophotometer. The transmission electron microscopy（TEM） pictures were taken to observe its morphology and size after dilution and drying on copper grids. The hydrodynamic diameters（HD） of nanocrystals were measured with a dynamic laser scattering（DLS） to characterize its size. The relaxation rate of CuInS₂/Zn S@BSA-DTPAGd QDs（p QDs）, Magnevist and BSA-DTPAGd were calculated on a 1.41 T MR scanner and the in vitro MR imaging of p QDs and Magnevist were scanned on a 0.5 T MR scanner.Results The size of CuInS₂/Zn S@BSA-DTPAGd QDs was 45 nm in average with a uniform monodisperse. The fluorescence curve of nanoprobe was a single narrow symmetrical peak and its maximum emission wavelength was at about 630 nm excited at 470 nm with bright red fluorescence under UV light. The p QDs showed high longitudinal and transverse proton relaxation rates（r1, r2） with 15.233 and 26.058 s-1 per m M of Gd3+ respectively, and the r2/r1 ratio was 1.71. In vitro MRI results showed that the p QDs has a great signal enhancement in T1 weighted imaging.Conclusion All of these studies fully illustrate the prepared CuInS₂/Zn S@BSA-DTPAGd QDs is very suitable for fluorescent imaging and MRI applications, which laid a solid foundation for targeting dual modal imaging of CD133⁺ GSCs. PartⅡThe Preparation of Targeting MR/fluorescence Nanoprobes and in vivo and in vitro Bimodal ImagingObjective To prepare paramagnetic albumin decorated CuInS₂/Zn S targeted bimodal nanoprobes and to perform MR/fluorescence imaging in vivo and in vitro.Materials and methods1. Cultivation of SU2 s and establishment of animal model: SU2 s were cultured using serum free DMEM/F12 medium, containing growth factors for this neural stem cells（20 ng/ml EGF, 20 ng/ml b EGF, 2% N2） and moderate amount of antibiotics, 37 °C in a 5% CO2 atmosphere. Cell growth was observed under an inverted microscope. Glioma was subcutaneous inoculated to nude mice, the tumor grew up about 3 weeks later.2. Cytotoxicity research: Cytotoxicity of p QDs was evaluated via MTT cell proliferation assay. Briefly, human glioma stem cell line SU2 s cells in logarithmic phase were inoculated into a 96-well cell-culture plate at a density of ca. 5×103 cells per well and incubated at 37 °C in a 5% CO2 atmosphere for 24 h. The cells were washed with PBS and cultured with p QDs at different Cu2+ concentrations（100 μL per well） for 24 h and 48 h under 37 °C. The cells were washed again with PBS and fresh medium was added. Subsequently, 20 μL per well of MTT at a concentration of 5 mg/m L was added to each well and incubated for another 4 h at 37 °C under a 5% CO2 atmosphere. After low-speed centrifugation and carefully removal of the supernatant, the obtained precipitation was dissolved into 100μL of DMSO. Blank group without QDs was set as the control. The absorbance was monitored by a microplate reader at a wavelength of 570 nm. The cytotoxicity was finally expressed as the percentage of cell viability of the treatment group relative to control group.3. Synthesis of CuInS₂/Zn S@BSA-DTPAGd-CD133 m Ab（p QDs-CD133 m Ab）: EDC·HCl was used as the cross-linker to prepare QD-antibody bioconjugates. The BSA-coated p QDs were reacted with m Ab at a QDs/m Ab/EDC·HCl molar ratio of 1:10:4000 in borate saline buffer（50 m M, p H= 8.2） for 2 h at room temperature. The final bioconjugate products（p QDs-CD133 m Ab） were purified by centrifugation at 100, 000 g for 30 min and washed with 0.01 MPBS（p H= 7.4） two times and then dispersed in phosphate saline buffer（0.01 M, p H 7.4, 0.5% BSA, 0.02% sodium azide） for further use.4. In vitro targeted bimodal imaging: Select SU2 s in logarithmic phase and disperse in serum free DMEM/F12 medium after centrifugal and dispersion. 20 μL of SU2 s cell suspension was smeared on several glass slices and fixed with 4% paraformaldehyde. And they were rinsed with fresh PBS several times. Then they were divided into experimental groups and control groups, and incubated with p QDs-CD133 m Ab and p QDs without antibody at the same Cu2+ concentration for 5 min, 10 min, 20 min, and 30 min, respectively. The targeted binding in vitro was observed under a fluorescence microscope after all the glass slices were washed with PBS more than twice and dried naturally.The SU2s（1 × 106/ml）in logarithmic phase were seeded into 6-well plate with 1 m L per well, and incubated with p QDs-CD133 m Ab, p QDs, and Magnevist at the same Gd3+ concentration, respectively, at 37 °C for 1 h. Then the cells were washed several times and redispersed in 200 μL of PBS. Meanwhile, normal saline（NS） was used as the blank control group. 200 μL of these four groups of liquid were transferred into a removable 96-well plate and taken MR T1- weighted imaging in vitro on a GE MR system. The specific scanning parameters were as follows: T1 spin echo sequence, TR= 2000 ms, TE= 108 ms, Matrix= 256×192, FOV= 80 mm×80 mm, FOV phase of 40%, slice width= 2.0 mm, at room temperature.5. In vivo toxicity analysis and biodistribution: Experimental group treated with p QDs via tail vein and control group without any injection were set up for in vivo toxicity study of the prepared p QDs. The mice were sacrificed on the seventh day and their main organs（including heart, liver, spleen, lung, kidney and intestine） were taken out to slice, fix with 4% paraformaldehyde and stain with H&E. The results were observed and analyzed with an optical microscope. The biodistribution of p QDs in main organs and tumors can be obtained through inductively coupled plasma mass spectrometry（ICP-MS） analysis of Cu2+ at 6 h, 24 h, 48 h, 4 d and 7 d, respectively. Experimental mice injected with p QDs intravenously, and the blank ones without injection were sacrificed at different time intervals and their main organs（including heart, liver, spleen, lung, kidney and intestine） were removed to be frozen, crushed and digested by concentrated acid, then sent for ICP-MS analyzing. The subcutaneous glioma also have done the same treatment.6. In vivo targeting bimodal imaging: All the disposals of mice were in accordance with the guidelines of the Institutional Animal Care and Use Committee of Soochow University. Imaging was conducted when the tumor size reached ca. 0.5-1.0 mm after inoculation. To assess the target specificity of the as-prepared bimodal nanoprobes, the mice were divided into two groups and injected them with the same dosage of p QDs-CD133 m Ab and p QDs, respectively. The in vivo MR imaging was performed on a magnetic resonance imaging system before and after the mice were injected of p QDs-CD133 m Ab, p QDs and Magnevist at the dosage of 0.05 mmol Gd3+/Kg body weight via tail vein. T1-weighted imaging scanning parameters were as follows: TR= 200 ms, TE= 18.2 ms, Matrix= 256×192, FOV= 80 mm×80 mm, slice thickness= 2.0 mm, at room temperature. The in vivo fluorescence imaging were acquired on an in vivo imaging system before and after the intravenously injection of the same dosage of p QDs-CD133 m Ab and p QDs.Conclusion The experimental results present the prepared p QDs-CD133 m Ab probe is capable of fluorescence and MR bimodal imaging, and this probe can selectively located in the CD133⁺ GSCs with lower toxicity. This study provides a promising strategy in the accurate diagnosis of glioma. The biodistribution and toxicity analysis in vivo indicate that the fabricated p QDs are low toxic.