ICG-loaded Black Phosphorus Nanosheets For Fluorescence Imaging-guided Breast Cancer Therapy

Objective:To improve the photothermal stability and tumor targeting ability of ICG,PEG-modified ICG-loaded black phosphorus nanocomplex(ICG@BPNS-PEG)was prepared in the study.Targeting tumor imaging to trace tumor site was used to guided breast cancer therapy.And the synergistic photothermal effect of ICG@BPNS-PEG was prepared inhibit tumor growth.In this study,a multifunctional nanometer imaging diagnosis and treatment system is constructed to realize the integration of tumor diagnosis and treatment.Methods:1.Synthesis and characterization of ICG@BPNS-PEG:First,a small amount of black phosphorus(BP)was prepared by heating red phosphorus(RP)under closed condition at high temperature.Then black phosphorus nanosheets(BPNSs)were separated from the bulk BP in the liquid phase.BPNS and ICG are mixed and dissolved in different proportions,and then mixed and centrifuged with polyethylene glycol(PEG)to prepare ICG@BPNS-PEG.2.In vitro experiment:under 808nm NIR irradiation(1.65W/cm~2,5min),the temperature changes of ICG@BPNS-PEG solution with the concentration of 2.5,5,10 and20ug/ml were collected by Thermal Infrared Camera,and the optimal concentration of ICG@BPNS-PEG was selected.Then the temperature changes of PBS,ICG,BPNS-PEG and ICG@BPNS-PEG solution at the same concentration were compared to evaluate the excellent photothermal conversion efficiency of ICG@BPNS-PEG.The temperature changes of ICG@BPNS-PEG solution under four light cycles were recorded to evaluate the photothermal stability of ICG@BPNS-PEG.3.Cell assay:the cytotoxicity of ICG@BPNS-PEG was quantified by MTT assay.FDA/PI dead and live cell staining as well as MTT were used to qualitatively and quantitatively detect the photothermal efficacy of ICG@BPNS-PEG.Flow cytometry was used to observe the uptake of ICG and ICG@BPNS-PEG in breast cancer cells and evaluate the tumor cell targeting of ICG@BPNS-PEG.4.Animal experiment:First,4T1 breast cancer mice model was established.Then ICG solution and ICG@BPNS-PEG solution were injected from the tail vein.Its distribution and metabolism in the tumor sites of mice were observed by small animal live imaging system.Then 20tumor-bearing BALB/c nude mice with tumor were divided into 5 groups.The photothermal efficacy of ICG and ICG@BPNS-PEG,including PBS,ICG@BPNS-PEG,NIR,BPNS-PEG+NIR and ICG@BPNS-PEG+NIR,with 4 mice in each group were observed.And the changes of tumor volume and body weight over 14 days were recorded to evaluate the effect of photothermal therapy on tumor inhibition and the biocompatibility of ICG@BPNS-PEG.Finally,the heart,liver,spleen,lung and kidney of mice were taken out for HE section staining to evaluate the toxicity of ICG@BPNS-PEG in vivo.Results:ICG@BPNS-PEG nanocomposite with a diameter of about 240±28nm was successfully prepared in this study.The results of photothermal assay in vitro showed that ICG@BPNS-PEG has higher photothermal conversion efficiency and photothermal stability than free ICG and BPNS-PEG.The results of cytotoxicity assay showed that ICG@BPNS-PEG had no obvious cytotoxicity on normal RPE cells,MCF-7 as well as 4T1 breast cancer cells(P>0.05).The results of cell uptake assay showed that ICG@BPNS-PEG was more easily ingested by tumor cells than ICG,indicating that it has a strong ability to target tumor cells.The results of FDA/PI dead and live cell staining and MTT assay showed that the synergistic photothermal effect of ICG@BPNS-PEG is significantly better than that of free ICG(P<0.05).The results of Fluorescence imaging in mice showed that compared with free ICG,ICG@BPNS-PEG stayed for a long time in vivo and the tumor site was the best enriched at 12h after the injection,indicating that ICG@BPNS-PEG improved the tumor targeting ability of ICG and reduced its metabolic rate.Under the NIR light irradiation of808 nm,the tumor size of mice injected with the ICG@BPNS-PEG solution significantly reduced compared with that injected with PBS and BPNS-PEG.It showed that ICG@the synergistic effect of field of BPNS-PEG effectively inhibit the growth of the tumor(P<0.05).The weight of mice may had no obvious difference(P>0.05),and there was no significant difference in HE staining results of heart,liver,spleen and kidney sections among all groups,indicating good biocompatibility of ICG@BPNS-PEG in vivo.Conclusion:The ICG@BPNS-PEG prepared by us has tumor targeting,synergistic photothermal effect and good biocompatibility.It improves the photothermal stability of ICG and the ability to target and trace tumor sites,and enhances ICG.And the photothermal effect of BPNS-PEG,to achieve ICG@BPNS-PEG targeting and tracing the tumor site to guide photothermal therapy.Therefore,using ICG@BPNS-PEG to target and trace the tumor site fluorescence imaging to guide treatment is expected to realize the integration of breast cancer diagnosis and treatment.