Facile Preparation Of Uniform FeSe₂ Nanoparticles For PA/MR Dual-modal Imaging And Photothermal Therapy In Tumors

Part Ⅰ Preparation of FeSe₂ nanoparticles and toxicity studyObjective Develope a new kind of magnetic nanomaterials, iron diselenide（FeSe₂）nanoparticles and explore toxicity study and in vivo biodistribution study of FeSe₂-PEG nanoparticles.Methods（1）FeSe₂ nanoparticles were synthesized by a simple solution-phase method and functionalized by polyethylene glycol（PEG）. And then, the stability, characterization and magnetic property of the obtained FeSe₂-PEG nanoparticles were evaluated.（2）4T1murine breast cancer cells, He La human cervical cancer cells, and 293 T human embryo kidney cells were incubated with different concentrations of FeSe₂-PEG for 24 h. The standard methyl thiazolyl tetrazolium（MTT） assay was then used to determine their relative viabilities.（3）Healthy Balb/c mice were intravenously injected with FeSe₂-PEG（dose = 20 mg/kg）. Blood samples as well as main organs and tissues and were collected at different time points for serum biochemistry complete, blood panel analysis and hematoxylin and eosin（HE） staining.（4）These organs collected in（3） were weighted. Then we used inductively coupled plasma atomic emission spectrometry（ICP-AES） to determine the total amount of Fe in each measured organ.Results（1）The transmission electron microscopy（TEM） and high resolution TEM（HR-TEM） images of the sample in solution indicated the good uniformity of FeSe₂-PEG nanoparticles, and the size was about 8-10 nm. Compared with dopamine modified Fe3O4 nanoparticles, FeSe₂-PEG exhibited much stronger broad band absorbance in the NIR region. After modified with PEG, the FeSe₂-PEG nanoparticles exhibited excellent stability in various physiological solutions. Heating curves of FeSe₂-PEG solutions indicated the good photothermal property. The hysteresis loop measurement suggested thesuperparamagnetic nature of our FeSe₂-PEG nanoparticles. The r2 relaxivity of FeSe₂-PEG was measured to be 133.38 m M-1 s-1 based on the Fe concentration, which was much higher than that of Fe3O4-DA（88.99 m M-1 s-1）.（2） A series of in vitro cytotoxicity assay showed no significant cytotoxic response of cells even at high nanoparticle concentrations.（3）Through longterm toxicity study of FeSe₂-PEG nanoparticles, we concluded that all the serum and blood detection indexes remained within the normal ranges. We did not notice obvious morphology damage on organs in Hematoxylin and eosin（H&E） stained tissue slices.（4）In vivo biodistribution suggested iron contents mainly cumulated in liver and spleen organs. Notably, a persistent decrease of iron levels in all measured major organs with time extending was observed, indicating the gradual clearance of FeSe₂-PEG from mice.Conclusion Ultra-small superparamagnetic FeSe₂-PEG nanoparticles were stable in physiological environments and exhibited excellent magnetic property and optical property.In addition, FeSe₂-PEG could be effectively eliminated from the treated animals and showed no obvious toxicity.Part Ⅱ FeSe₂ nanoparticles for PA/MR dual-modal imaging and photothermal therapy in tumorsObjective To study the feasibility of FeSe₂ nanoparticles for magnetic response imaging（MRI）,photoacoustic imaging（PA） and photothermal therapy.Methods（1）MTT assay and Calcine AM & propidium iodide（PI） co-staining assay were used to detect in vitro PTT effect of FeSe₂-PEG nanoparticles to 4T1 cancer cells.（2）Generate the 4T1 tumors mouse model. T2-weighted MR images and PA images of 4T1tumor-bearing mice were taken before and after FeSe₂-PEG intratumorally（i.t.） injection.In addition, the signal intensity of tumors from MR and PA images were measured.（3）To verify the in vivo PTT effect of FeSe₂-PEG nanoparticles, an Infrared（IR） thermal imaging camera was used to monitor the tumor temperature when the mice were exposed to the 808 nm laser. Tumor-bearing mice i.t injected with the same volume of saline were used as the control.（4） Mice bearing 4T1 tumors were randomly divided into four groups（n =4 per group）: saline only without laser; laser only without FeSe₂-PEG injection; injection ofFeSe₂-PEG without laser irradiation; injection of FeSe₂-PEG with laser irradiation. Tumor sizes were recorded after therapy.Results（1）In vitro PTT results showed decreasing cell viabilities as the laser power was increased.（2）After intratumorally injected with FeSe₂-PEG,tumor region presents darker effects in MRI and remarkably enhanced signals in PA imaging.（3）With the increase of laser power density and irradiation time, the temperature of the tumor surface obviously increased. In contrast, the temperature of tumors without FeSe₂-PEG injection showed no significant increase under laser irradiation at the same irradiation condition.（4） It was found that tumors after injection of FeSe₂-PEG followed by laser irradiation were eradicated without recurrence within 40 days. The tumors of all the other groups, by comparison, did not show any trend of growth inhibition. Therefore, our results demonstrated the excellent in vivo cancer therapeutic effect of the PTT mediated by FeSe₂-PEG.Conclusion FeSe₂-PEG nanoparticles have effective PTT effects, excellent MR and PA imaging ability, which demonstrate the great potential of FeSe₂-PEG as a tumors PTT agents and multimodal imaging contrast agent.