Visible Light Emitted Quantum Dots Targeting EGFR For In Vitro And In Vivo Imaging Of Triple-negative Breast Cancer

Objective: Triple-negative breast cancer has become one of the research hotspots of contemporary oncology due to the lack of therapeutic target.At present,there are various studies of different imaging modalities for breast cancer imaging diagnosis.Among them,optical imaging is characterized by strong sensitivity.However,the researches focused on the application of near-infrared light imaging,and the exploration of visible light was rare.In this study,we prepared a novel targeting probe consisting of visible light emitted quantum dot QD625 and the EGFR monoclonal antibody Cetuximab,which could be used for cell fluorescent imaging and in vivo imaging of triple-negative breast cancer tumor-bearing nude mice.The aim of this study was to explore the feasibility and imaging conditions of using visible light emitted quantum dot as a carrier to target EGFR in vitro/in vivo,which could provide the basis of visible light emitted quantum dot for in vivo imaging,and the prospective application in fluorescent imaging diagnosis of triple negative breast cancer.Methods: Carboxyl groups on the surface of water-soluble QD625-COOH were activated by EDC and NHS,then QD625 could covalently combined with the amino groups of Cetuximab,and the probe QD-Cetuximab was synthesized.The morphology,particle size,stability and fluorescence properties of probe were detected by TEM(transmission electron microscope)、DLS(dynamic light scattering)and multi-function microplate reader.Human breast cancer cells 468 were incubated for 24 h with a concentration gradient of QD-Cetuximab and QD,and cytotoxicity experiment was performed by using CCK8 kit and microplate reader.Confocal fluorescence microscopy was used to observe the red fluorescence of 468 after incubating with QDCetuximab and QD for 2 h,and fluorescence quantitative analysis was performed.After 468 and 453 were incubated for 2 h with different concentrations of QD-Cetuximab and QD,the relationship between cell uptake and different incubation concentrations of two kinds of probes was analyzed by inverted fluorescence microscopy and flow cytometry.468 tumor-bearing mice were cultured for about one month.100 μL of QD-Cetuximab and QD(both contain 150 pmol equivalent QD625)were injected into the 468 tumorbearing mice via the tail vein,and the imaging and probe distribution at different time points were observed by In-Vivo Imaging System FX PRO.Results: QD-Cetuximab had a particle size of about 40.34 ± 2.44 nm detected by TEM,a hydrated particle size of 57.85 ± 4.69 nm detected by DLS,and it had a stable structure.The cytotoxicity of QD-Cetuximab was not obvious within a certain concentration range.When the concentration of QD-Cetuximab was ≤50 nmol/L,the relative survival rate of cells was more than 90%,and when the concentration exceeded 100 nmol/L,the relative survival rate of cells was reduced to 72.52% ± 4.91%,P <0.05.The red fluorescence of 468 incubated with QD-Cetuximab was stronger than that of 468 incubated with QD and 453 incubated with QD-Cetuximab or QD observed by the inverted fluorescence microscope.It was the same under confocal fluorescence microscope.Image-Pro Plus 6.0 software analysis showed that the IOD(integrated optical density)of the experimental group and the control group were 863.356 ± 37.206 and 169.971 ± 20.374,respectively(n=3,P <0.05),and the IOD of the former was 5.1 times than that of the latter.Flow cytometry showed that the uptake of QD-Cetuximab and QD by 468 both increased with incremental incubating concentration,and the former was more significantly.Flow Jo software analysis showed that when the concentration of QD-Cetuximab and QD were 25,50,100,and 200 nmol/L,the quantified average fluorescence intensities were 128.0 ± 11.7 vs 23.8 ± 8.9,221.3 ± 15.1 vs 32.1 ± 6.4,420.3 ± 14.5 vs 57.1 ± 7.9,606.8 ± 22.7 vs 98.1 ± 7.5,respectively(n=3,P <0.05),and the ratios were 5.4,6.9,7.4,6.2,respectively.The QD-Cetuximab and QD probes mainly accumulated in the liver in vivo.The fluorescence emitted by tumor was not obvious under the background of the fluorescence of liver.However the fluorescence was visible in the isolated tumor tissue,and the fluorescence of the experimental group was stronger than that of the control group.Bruker MI SE software analysis showed that the average fluorescence intensities of the isolated tumor tissues of the QD-Cetuximab group and the QD group were(2.46 ± 0.60)×104 and(1.29 ± 0.05)×104,respectively(n=4,P=0.015).Conclusion: In this study,the feasibility of visible light emitted QD-Cetuximab targeting EGFR for in vitro and in vivo fluorescent imaging of triple negative breast cancer was initially confirmed by cytology and in vivo imaging.Cetuximab could increase the targeting ability of the probes and promote cell uptake.Excess QD have a certain degree of cytotoxicity.The in vivo contrast imaging of QD-Cetuximab being used in triple negative breast cancer tumor-bearing mice was not ideal,but the isolated tumor tissue imaging results were acceptable.Therefore,further modification of QD should be considered to reduce the liver uptake and improve in vivo fluorescence imaging efficiency.