NIR-Ⅱ Fluorescence Imaging Of Sentinel Lymph Nodes Based On Dual-NIR-Ⅱ Probes In Breast Cancer

Breast cancer has a high incidence and mortality rate in the female population,which seriously threat the life and health of women.Despite being the primary method for diagnosing metastasis of breast cancer,the currently used sentinel lymph node biopsy（SLNB）has limitations,such as the lack of an ideal contrast agent and prolonged period for pathological analysis.Thus,we propose to develop NIR-Ⅱ contrast agents with high stability,high resolution,and good biocompatibility that can facilitate excellent lymph node imaging.Moreover,our study demonstrates that the use of dual probes in NIR-Ⅱ fluorescence imaging enables real-time,non-invasive,and accurate identification of the metastasis state of SLNs.This paper carries out research from the following three aspects:1.The IR-FFCHP probe was constructed by S-D-A-D-S structural optimization.We revealed that the prepared water-soluble IR-FFCHP probe processed high absorption coefficient(ε=17.0×10³M^-1·cm^-1)and 0.73%quantum yield in aqueous when normalized with the quantum yield of IR-FE in toluene（which is 3.1%）.Compared with clinical ICG,IR-FFCHP exhibited excellent imaging ability with improved imaging time and signal-to-noise ratio.The IR-FFCHP also demonstrated excellent passive tumor targeting ability,making it as a promising tumor imaging agent.We further employed the IR-FFCHP and NIR-Ⅱb（Pb S/Cd S quantum dots or Er@Y-PEG）probes,which do not overlap in emission spectra,as dual probes for imaging of tumor and SLNs,as well as for fluorescence-guided surgery（FGS）under the same excitation.Compared with the blue dye or nuclide labeled nanoparticles used in current SLNB,our imaging approach greatly improved the imaging quality and has better biological safety.2.The NIR-Ⅱ ratiometric fluorescence dual probes with targeted and non-targeted moieties were constructed using Nd or Er-doped rare earth nanoparticles with emission wavelengths at 1060 nm and 1525 nm,respectively.We modified Nd-NPs with folic acid as the targeted probe and Er-NPs with PEG as the non-targeted probe.Due to the different levels of targeted probes enrichment in healthy/metastatic lymph nodes,real-time,non-invasive diagnosis of tumor-infiltrating sentinel lymph nodes in the breast cancer tumor model was achieved based on the ratio of targeted/untargeted fluorescence intensity in the lymph nodes.The diagnostic results of ratiometric fluorescence imaging for 28 tumor-bearing mice were verified by the clinical gold standard,with an accuracy of 92.8%for the ratiometric fluorescence detection strategy.Compared with clinical diagnostic procedures,our detection method was more convenient,non-invasive and time-saving.Furthermore,the mice without metastasis detected by the NIR-Ⅱ ratiometric fluorescence dual probes were treated with four different treatment regimens.The prognostic data of each group proved that the resection of non-metastatic SLNs in the early stage of cancer is unnecessary.Our study also revealed that the NIR-Ⅱ ratiometric dual probes have the ability of real-time feedback during the operation,which can assist in developing of treatment plans.3.Building upon the previous chapters,this chapter expands the imaging ability and applications of NIR-Ⅱ ratiometric dual probes.The NIR-Ⅱ ratiometric fluorescence dual probes showed superior imaging performance compared to ICG,IR-800CW,and the Pb S/Cd S quantum dots.Experiments on 4T1 model of toe lymph node metastasis demonstrated the unique advantage of the NIR-Ⅱ ratiometric dual probes in intraoperative diagnosis.In addition,this section provides ex vivo and high-magnification fluorescence imaging of SLNs to illustrate the effectiveness and reliability of the ratiometric strategy in multiple states.Ratiometric fluorescence imaging was performed on SLNs with differently metastatic degrees,proving that this strategy is capable of distinguishing inflammatory SLNs from metastatic SLNs and aiding in the diagnosis of SLNs metastatic degree.The ratiometric dual probes have versatile applications.By studying the peritumoral lymphatic system in the process of tumor growth,we were able to realize fluorescence imaging of numerous new lymphatic vessels and in vivo detection of the metastasis status of inguinal lymph node.In summary,this paper has developed a series of NIR-Ⅱ dual probes,and successfully carried out sentinel lymph node imaging,surgical navigation,and non-invasive detection in mice with breast cancer.Our approach not only realized high-contrast in vivo imaging and improved imaging quality,but also overcame the limitations of single-channel fluorescence detection,providing a new concenpt for non-invasive,accurate,and rapid visualization of lymph node detection.