Synthesis And Characterization Of New-type Dual-modalities Probe For MRI And Fluorescence Imaging

Part I synthesis and Characterization of dual-modalities probe SPIO@PEG/HIDObject:synthesis of Fe3O4and modalities of PEG and linked the HID, the magnetic and fluorescence dual-modalities probe, and characterized the probe.Material and method:FeC13-6H2O (0.54g), Na aciykte and Na sodium acetate (NaOAc) were dissolved in EG (5mL) and DEG (15mL) under magnetic stirring. The obtained solution was transferred to a Teflon-lined stainless-steel autoclave and sealed to heat at200℃for10h. After reaction, the autoclave was cooled to room temperature. The obtained Fe3O4nanoparticles were washed6times with defonized water, and distributed in ethanol (6mg/mL).Coating formation was done by adding PEG solutions to iron oxide suspension in weight ratios of1/5PEG/iron oxide and stirring for48h via mechanical stirrer at300rpm. PEG-coated nanoparticles were separated over a strong magnet and the supernatant was decanted. The product was washed6times by alcohoL5mL PEG-iron oxide and lmL IR808(1mg/mL) was dissolved in alcohoL The obtained sohition was stirring for24h at300rpm, subsequently the product washed by H2O until no further fluorescence was observed in the supernatant, and dispersed in50mL H2O by using a bath ultrasonic for30min.CharacterizationThe characteristics of nanoparticfes were examined by different methods. Fourier transform infrared spectroscopy (FTIR, Thermo Nicofet) was carried out to assess the surface bands of nanoparticles. Consequently, dried nanoparticfes were mixed with KBr powder and then were pressed to produce pellets. Nanopartictes size and morphology were investigated by transmission electron microscope (TEM, JEM-2100). The adsorbed PEG layer on the surface of nanoparticles was measured by thermogravimetirc analysis,(TGA, Pyris I DSC/0.2UW) scanning from200℃to700℃at10℃/min heating rate. The photo luminescence (PL) spectra were measured using an Edinburgh FLSP920fluorescence spectrophotometer with Xe900xenon arc lamp). Fluorescence images were acquired with an FVTS spectrum system (Caliper Life Sciences, IVIS1000) with an excitation maximum of770nm and emission maximum of800nm.ResultThe SPIO core was synthesized follow by the previously reported method[1], the TEM image (Fig. la, Fig.1b) shows spherical shape and clusters structure. The diameter of the SPIO was100±20nm. TEM images (Fig.1c, Fig.1d) indicated that the diameter of the SPIO@PEG is about130nm. Fig.1e shows the FTIR spectra of SPIO, SPIO@PEG, SPIO@PEG/HE). For these nanoparticles the characteristic adsorption band of SPIO magnetite at590cm-1is found. The PEG modified SPIO spectrum comprises the main absorbance of ether stretch band at the1105cm-1and-CH2vibrational band at1261cm-1. Moreover, the C-O-C ether stretching and vibration bands at1065cm-1and1346cm-1, OH stretching/vibration band at around3400cm-1, C-H asymmetric/symmetric stretching and vibration band at2950cm-1and 2860cm-1were observed. Blue shifts of Fe-O vibration of coated nanoparticles suggest the new band formation between iron oxide surfaces and PEG/HID coating. And as Fig S3shows, SPIO shows a12.5%weight tost at the temperature range between200to700℃, due to the organic composition of SPIO (mainly polyactylte). While, SPIO@PEG/HID had a22.5aweight lost. The difference between two sum at400℃is attributed to the formation of organic modification of PEG/HID. The optical character of SPIO@PEG/HID’s the emission wave peak was around800nm in H2O, The insert photograph shows the SPIO@PEG/HDD captured by a0.2T magnet. The partial fluorescence sign taken by IVIS optical system clearly showed the partial fluorescence sign, which confirmed the conjugation of SPIO@PEG and HID. Loop test (Fig. SI) shows both SPICK SPIO@PEGand SPIO@PEG/HID has no hysteresis on the room-temperature magnetization curves. Further, SPIO’s relaxation efficiencies are evaluated by measuring the transverse relaxation rates, using a clinical3.0T MRI. The dark signal enhancement in the T2-weighted MR images, which revealed a progressive decrease as the concentration of SPIO@PEG/HDDD decreased, and the T2relaxivites of SPIO@PEG/HID nanoparticles linearly increased depend to the concentration of the nanoparticles (Fig.2b). And the value of R was62.9ms’’i which is much higher than normal human tissues. This study demonstrated the potential value of SPIO@PEG/HID as a contrast agent for optical and MR imaging. Conclusion:we synthesis the dual-modalities probe SPIO@PEG/HID, which has a T2relaxivites of62.9Ms"1and emission around800nm. The production is stable and has a great character in magnet and fluorescence field, more bio-tests will be done in the next part. Part II:The Diffidence of MCF-7and293T Cells Cultured with the Dual-modalities Probe SPIO@PEG/HIDObject:to verify the character of SPIO@PEG/HID in vitro.Material and method:Human Breast cancer cell line (MCF-7), and human embryo kidney cell line (293T) cell were obtained from Shanghai Institutes for Biological Sciences, SIBS. Cells were maintained in PRLM-1640,supplemented with10%FBS,100μg/mL penicillin, and incubated at37℃with5%CO2.The cytotoxicity of the nanoparticles was assessed by the MTT assay.293T cells were grown in a96-well plate and treated with different concentrations for12h and48h. MTT (Sigma-Aldrich) was added to each well to a final concentration of0.5mg/mL for the measurement of formazan formation. The absorbance was measured at570nm in a Versa Max microtiter plate reader (Molecular Devices, Sunnyvale, CA). Dat a are presented as mean±standard deviation of triplicate samples.The uptake of iron oxide nanoparticles by the cells can be probed by Prussian blue staining method and TEM. Briefly,293Tand MCF-7cells were seeded into6well plates (105/well) and grown until50-60%confluence, SPIO@PEG/HID was added at final contraction50μg/mL, after24h incubation, the cell was fixed by4%formaldehyde and stained by Prussian blue, observed on IR-microscope. Meanwhile,293T and MCF-7cells were seeded into6well plates (105/welI) and grown until50-60%confluence. Coated SPIO were added to the wells so that the final Fe concentration were20、50、100μg/mL. The cells were incubated for24h, then, washed with PBS3times. The samples were collected and Fe contraction was measured by ICP (PE, OPTIMA5300DV). Meanwhile, the cells were concluded and bio luminescence image was taken by IVIS system, then fixed in1%agarose in4mL eppendorff tubes and then scanned by3.0T MR.Results:The SPIO@PEG/HID incubated with293T at six concentrations up to100μg/mL(p>0.05) for12h and48h. more than90%of cells were alive after12h incubation. These results indicate that the SPIO@PEG/HID did not possess a highly toxic nature, which suggest that the SPIO@PEG/HID was safe for human at cell leveL The nanoparticles were mainly found in the cytoplasm of the cells, as the fluorescent spots were evident in focus. After60min the nanoparticles uptake were identified in several endosomes and cell member (figure.4k). Furthermore, Prussian blue stain, confirmed the localization in cytoplasmic and cell member. Heterogeneity in cellular accumulation of particles was also apparent following Prussian blue staining, confirming findings obtain by direct observation of IR sign in MCF-7by fluorescence microscopy. It is worth mentioning that under the incubation conditions used no sign of cell suffering or morphological changes was observed by TEM. This was done to evaluate the potential of SPIOs as a targeted MR contrast agent to cancer cells during to the special character of IR808and so-called "targeted". The T2-weighted MR phantom images are shown in Fig.5. The mean date of the cellular iron contents in293Tcells are3.1pg/mL,2.54pg./mLcell,1.05pg/mL for the incubation contention was100μg/mL,50μg/mL,20μg/mL. Meanwhile, in MCF-7cells the mean dates were6.42pg/mL,3.5pg/mL,1.48pg/mL.Conclusions:As previously report, IR808shows a strong ability of tumor-targeted, we assumed FE would inherit the ability. The Fe contention was different, and the tumor was highly. Part Ⅲ:the efficacy of dual-modalities probe in vivo evaluated by MRI and fluorescence imagingObject:verify the efficacy of dual-modalities probe SPIO@PEG/HED by MRI and fluorescence imaging in vivoMaterial and method:All animal experiments were in agreement with the guidelines of the Institutional Animal Care and Use Committee. The euthymic mice (female,6-7weeks old) were maintained in an aseptic environment Tumor implantation was carried out by subcutaneous injection of0.1mL of MCF-7cell suspension (5million cells); tumor growth was monitored daily till tumor size between2-3mm. The colloidal dispersions of SPIONs were subcutaneous ly injected by tail vein of mouse with a dose of0.5mg Fe/kg body weight. MR imaging was performed1h postnanoparticle administration. For MR imaging, the mouse were fixed in a supine position. MR studies were performed using a3.0T with animal coil. In vitro fluorescence imaging was conducted on a cryogenically cooled IVIS system During image acquisition, isofluorane anesthesia was maintained using a nose cone delivery system and animal body temperature was regulated using a digitally thermostated bed integrated within the IVIS system. Signal intensity was quantified as the sum of all detected photon counts with a region of interest prescribed over the mouse using the Living view4.2software package.Results:Results showed the NIR and MR image of breast model after injection SPIO@PEG/HID via tail vein. The NIR sign was mainly accumulated in tumor region, made the tumor easily distinguished from other tissue. We also examined the NIR sign of tumor and several ex vivo organ to confirm the accumulation of tumor. And result also shows the magnetic contrast between after-injection and post-injection1h, T2MAP was performed, and the color map shows the SPIO@PEG/HID was accumulated from the outsides. Those confirmed the SPIO@PEG/HID as a potential tumor probe.Conclusions:the dual-modalities probe SPIO@PEG/HID turn to be efficacy in vivo.