Biominerlized Co₉S₈ Nanodots For Simultaneous Cancer Photoacoustic Imaging And Photothermal Therapy

In recent years, nano-materials have attracted widespread concern in the field of cancer research because of their unique physical, chemical and biological features. A variety of nano-materials with good adsorption of near-infrared radiation（NIR） has achieved some success in cancer photoacoustic imaging and photothermal therapy. However, nano-materials with low toxicity, good histocompatibility, high photothermal conversion efficiency and excellent photodynamic features are still under investigation.Objective : To synthesize Co₉S₈ nanomaterial（Co₉S₈-NDs） by biomineralization method via protein induction and to evaluate their potential in photoacoustic imaging by measurement of its characteristics and to explore their photothermal therapeutic effects on cancer through in vivo and in vitro experimentsMethods:（1） Co₉S₈-NDs were synthesized by protein induced biomineralization method. The optimum conditions for synthesis were explored. The size, morphology and composition of the as-prepared Co₉S₈-NDs were characterized by high-resolution transmission electron microscopy（HRTEM）, X-ray diffraction（XRD） sperctroscopy, X-ray photoelectron sperctroscopy（XPS）. Thephotoacoustic effect of Co₉S₈-NDs was assessed by photothermal and photothermal cycle experiment in vitro.（2） The standard methyl tetrazolium（MTT） assay was carried out to determine the cytotoxicity of Co₉S₈-NDs on 4T1（murine breast cancer cells）, Hela（human cervical carcinoma cells） and 3T3（murine fibroblast cells）. The generation of ROS in 4T1 cells induced by Co₉S₈-NDs before and after NIR light irradiation（808nm, 0.75W/cm2 for 10min） was evaluated. The uptake of Co₉S₈-NDs by 4T1 cells was examined. Laser scanning confocal microscopy（LSCM） was employed to observe the intracellular distribution of Rhodamin B-labeled Co₉S₈-NDs in 4T1 cells stained by LysoTracker Green DND-26. 4T1 cells were incubated with Co₉S₈-NDs for 6h and then irradiatedwith 808-nm laser with different power densities to demonstrate the in vitro cytotoxicity of Co₉S₈-NDs on 4T1 cells with NIR irradiationby differential staining with calcein AM（live cells, green fluorescence） and propidium iodide（dead cells, red fluorescence）（3） Co₉S₈-NDs were injected into the mice via tail vein and photoacoustic tomography was used for tumor imaging and photothermal treatment was employed..THE growth and survival of tumor-bearing mice were noted and photothermal therapeutic effect was observed at in vivo level. Tumors tissues collected at one day after the corresponding treatments were examined by hematoxylin and eosin（H&E） staining to observe the histological changes of tumors tissues in each group.Results:（1） The as-prepared Co₉S₈-NDs displayed crystalline structures of monodisperse spheres with a mean diameter of 2.1nm..DLS measurement show that Co₉S₈-NDs exhibited the hydrodynamic size of 26.21 nm with good size stability. Moreover, the obtained Co₉S₈-NDs exhibited significant optical absorption from visible to NIR region.（2）No significant cytotoxicity of Co₉S₈-NDs was observed for all three types of cells even at high concentrations up to 100ug/mL. However, NIR laser light treatment（808nm, 0.75W/cm2 for 5min.） of 4T1 cells incubated with Co₉S₈-NDs at various concentrations for 24 h led to significantly enhanced cytotoxicity. Compared to the Co₉S₈-NDs group, ROS levels of the Co₉S₈-NDs-NIR group increased by 43.2% after treatment with NIR laser light（808nm, 0.75W/cm2 for 10 min.） suggesting that Co₉S₈-NDs were involved in the photodynamic process and induced the production of ROS in the presence of NIR light. Co₉S₈-NDs with red fluorescence exhibited a co-localization percentage of 75% in the lysosomes with green fluorescence, suggesting that Co₉S₈-NDs are preferably internalized into the lysosomes via endocytosis.The cytocidal effect positively correlated with laser density..The majority of cells were destroyed after being incubated with Co₉S₈-NDs and exposed to the NIR laser at 0.75W/cm2 for 10 min.（3） The surface temperature of tumors in Co₉S₈-NDs group reach up to 50℃ after only 5min laser irradiation. Whereas, the increase of surface temperature was less than 5℃after irradiation in the saline group. The tumor volumes were monitored in the subsequent 21 days. In the control group（PBS） irradiation（+） and irradiation（-）resulted in 16-fold and 18-fold increases of tumor volumes. Co₉S₈-NDs group without irradiation also exhibited similar tumor growth with those of control group.Most importantly, in the Co₉S₈-NDs group, irradiation with NIR resulted in significant damage to the tumors. Two days after irradiation, the tumors displayed necrosis and complete tumor ablation sustained without recurrence till 21 days after irradiation. H&E statining showed that Co₉S₈-NDs treatment and subsequent NIR irradiation led to servere cell necrosis and hemorrhagic inflammation In constrast, whether irradiated or not, no obvious tumor necrosis was observed in control which received PBS injection.Conclusions: BSA stabilized Co₉S₈-NDs exhibit intense NIR absorbance, ultrafine size, good biocompatiblity, desirable photothermal conversion efficiency and unique photodynamic under NIR irradiation. Moreover, Co₉S₈-NDs assumed multiple imaging features including PA and MR,which facilitates multimodal imaging with precise tumor localization. We conclude that the photodynamic properties of Co₉S₈-NDs make them a promising nanophotosensitizer allowing both PTT and PDT for cancer.