Application Of Rare Earth Nanomolecular Probe Targeting PD-L1 In Near Infrared II Fluorescence Imaging Of Triple Negative Breast Cancer

Objective:To explore the ability of rare earth molecular probe targeting PD-L1 in vitro and in vivo and to reflect the expression level of PD-L1 under Near infrared II fluorescence imaging window,so as to provide a molecular imaging method for early prediction of the immunotherapy effect of triple-negative breast cancer and the efficacy of non-invasive monitoring.Methods:①The rare earth nanometer material NaYF4@NaNdF4:20%Yb was synthesized by high temperature co-precipitation method,and PD-L1 antibody was conjured after the surface modification of PAA(poly acrylic acid)to obtain the rare earth nanometer fluorescent molecular probe RENPS-PD-L1.②High resolution transmission electron microscope was used to observe the morphology of the probe and roughly estimate its particle size.The particle size and Zeta potential of the probe were measured by multi-angle particle size and high sensitivity Zeta potential analyzer.③The emission spectrum of the molecular probe under excitation light of 808nm was detected by fluorescence spectrometer.The absorption spectrum of the probe was measured using an ULTRAVIOLET spectrophotometer to verify the successful connection of the antibody.④To test the cytotoxicity of probes in vitro by CCK-8:In vitro toxicity of RENPS-PD-L1 probe was detected using normal breast epithelial cells and triple negative breast cancer cells.⑤In vivo safety test of rare earth molecular probe RENPS-PD-L1:Toxicity of RENPS-PD-L1 probe was detected in healthy female Balb/c mouse model.⑥Immunofluorescence was used to verify the targeting of RENPS-PD-L1 to PD-L1 at the cellular level:RENPS-PD-L1 was connected with the red fluorescent dye Rhodamine,and then the probe was co-incubated with cells with different PD-L1 expression levels to observe the strength of red fluorescent signal in each group and analyze its relationship with PD-L1 expression levels.⑦In vivo targeting of RENPS-PD-L1 was verified by MMTV-PyVT spontaneous tumor-forming breast cancer model:The mice models were randomly divided into the targeted groups and non-targeted groups,respectively by tail intravenous RENPs-PD-L1 and RENPsIgG.NIR fluorescence imaging was performed at 1,6,12,24,48,72,96,and 120 hours after probe injection,exploring fluorescence signal intensity between the targeted group and the non-targeted group,and the expression of PD-L1 was detected by immunohistochemical staining of the tumor tissues of the two groups.⑧To explore the biological distribution of rare earth molecular probe RENPS-PD-L1:healthy female Balb/c mice were injected with RENPS-PD-L1 probe.At 2,6,12,24,48,and 96 hours after the injection of RENPS-PD-L1 probe,the main organs were taken for in vitro NIRⅡ fluorescence imaging,then the fluorescence intensity of each organ at different time points was analyzed.Results:①The preparation of RENPs-PD-L1 was successful.②The particle size of RENPs-PD-L1 was 32 nm,the hydrodynamic size was 50 nm,The Zeta potential of RENPS was 43.09 mV,and the Zeta potential of RENPS-PD-L1 and RENPS-IgG after antibody connection was-38.46 mV and-38.35 mV.③The emission peak of RENPsPD-L1 was measured by fluorescence spectrometer at 1060 nm under the excitation of 808nm laser.④At the cell toxicity experiment,even under the concentration of 100μg/mL of RENPs-PD-L1,the cells remain more than 80%survival rate,showing low cytotoxicity and good security in vitro experiment.Compared with the control group,the weight of experimental group mice did not significantly lower,function indicators,such as ALT and as AST,did not see obvious difference neither.⑤Immunofluorescence assay showed that the probe could target cells with high expression of PD-L1.⑥Triple negative breast cancer MMTV-PyVT transgenic mice fluorescence imaging experiments show RENPs-PD-L1 probe in animals with good targeting,the signal-to-noise ratio of fluorescence imaging in 24 hours after injection probe reached the highest,up to 4.26.⑦Immunohistochemical staining verified again RENPs-PD-L1 targeting PD-L1 in vivo biological distribution experiment showed that the main distribution sites of probes is the liver and spleen.Conclusion:The rare earth nanomolecular probe RENPS-PD-L1 has a small particle size,high fluorescence stability and good biosecurity.It has a good targeting ability to cells and tumor tissues with high PD-L1 expression,and can detect the expression level of PD-L1 in vivo,which can provide a powerful means for in vivo prediction and evaluation of the efficacy of immunotherapy for triple negative breast cancer.