| Objective:Breast cancer,as one of the most common malignant tumor and the primary cause of cancer mortality in women,is seen as a serious threat to human health.At the initial diagnosis,10-40%of breast cancer patients have metastatic lymph node(MLN),which is considered as the first step toward distant metastases and is related to poor outcomes for patients.Therefore,the early detection of MLN plays a crucial role in guiding breast cancer treatment and improving prognosis of patients.This study developed and synthesized a novel breast cancer-targeting and adenosine triphosphate(ATP)-responsive molecular probe,which was applied to detect MLN in a preclinical animal model through dual-modality fluorescence molecular imaging(FMI)and magnetic particle imaging(MPI)in vivo.Materials and methods:The targeting peptide CREKA and ATP-responsive aptamer(dsDNA-Cy5.5)with specific fluorescence response properties were modified to the surface of superparamagnetic iron oxide nanoparticles(SPIOs)through cross linker sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane 1-carboxylate(sulfo-SMCC),and the prepared molecular probe was abbreviated as SPIOs@A-T.In the characterization investigation of SPIOs@A-T,the morphologies,ultraviolet(UV)absorption spectrum,particle size and Zeta potential of SPIOs@A-T were analyzed by transmission electron microscope(TEM),ultraviolet-visible spectrum(UVVis)and dynamic light scattering(DLS)equipment,for evaluating whether SPIOs@A-T was successfully synthesized.In the study of the imaging properties of SPIOs@A-T,a series concentration of ATP solutions was set up to evaluate the fluorescence response properties of SPIOs@A-T,and the same concentrations of CTP,GTP,and UTP were seen as controls.Then the fluorescent signal was acquired by an IVIS Lumina System(Perkin Elmer).The MPI imaging property of SPIOs@A-T was also examined using MOMENTUMTM Imager(Magnetic Insight Inc).The cytotoxicity and the cellular uptake of SPIOs@A-T were assessed.Since CREKA can specifically bind to fibrinfibronectin complex in breast cancer,we performed Western Blot experiments on MLN of breast cancer and normal lymph node(NLN),and further compared the expression levels of fibronectin between them.In the in vivo imaging experiment by FMI and MPI,4T1 cells were injected into the left rear footpad of each mouse to establish breast cancer with MLN animal model,and healthy mice with NLN were used as a control group.Both were injected with SPIOs@A-T,then followed by performing dualmodality FMI and MPI in vivo.Mice were sacrificed 24 hours later,and lymph node tissue was dissected to obtain FMI and MPI images ex vivo,after which the tissue was fixed for further HE staining and Prussian blue staining analysis.To validate that MPI is not limited by tissue depth,we performed the dual-modality imaging scan on the same mice with MLN both before and after skin incisions and compared the change of signal.The biosafety assessment in vivo was achieved by HE staining and serological analysis of the major tissues and organs of mice.Results:1)Preparation and characterization of SPIOs@A-T nanoprobe:The TEM image showed that SPIOs@A-T were mono-dispersed and homogeneous.The hydrodynamic sizes of SPIOs and SPIOs@A-T were(51.63±16.30)nm and(58.58±19.34)nm,respectively.After modification with CREKA and dsDNA-Cy5.5,the Zeta potential changed from a positive potential of(4.24±0.83)mV to a negative of(-9.36±2.04)mV.The UV-vis absorption spectrum showed that SPIOs@A-T had an absorption peak at 260280 nm.The above results proved the successful synthesis of SPIOs@A-T.2)Evaluation of imaging performance in vitro and cellular uptake ability of SPIOs@A-T:The results of FMI in vitro showed that,within a certain range,the fluorescence signal intensity of SPIOs@A-T increased significantly with the increase of ATP concentration,whereas the weaker fluorescence intensity and no obvious signal change was visualized in the response with the control groups of CTP,GTP or UTP.Additionally,MPI in vitro results suggested that SPIOs@A-T possessed good MPI imaging performance.In the concentration range of 12.5-400.0 nM,the cell viability of 4T1 cells treated with SPIOs@A-T remained at 90-100%,without obvious cytotoxicity.The confocal images indicated SPIOs@A-T was uptaken great by 4T1 cells due to its ATP-responsive and targeting property.Western Blot results suggested that the expression level of fibronectin in MLN was 1.8 times higher than that in NLN.3)Dual-modality imaging of MLN in breast cancer by SPIOs@A-T nanoprobe:Due to the existence of ATP-responsive fluorescence design,after injection with SPIOs@A-T,FMI in vivo showed that the fluorescence intensity in the left popliteal lymph node region of MLN model mice increased first and then decreased with time.The signal background ratio(SBR)peaked at 12 h after injection,which was 4.99 times than that of NLN.The signal change trend of MPI in vivo was similar to that of FMI,and at 12 h after injection,the MPI signal value of SPIOs@A-T group was 1.87 times than that of NLN.At the same time,the HE and Prussian blue staining of lymph nodes further verified the above imaging results.Compared to before skin incision,the fluorescence signal was significantly higher after skin incision,while there was no remarkable difference for MPI signal,suggesting that MPI is not limited by tissue depth.In vivo biosafety experiments showed that no obvious pathological changes were found in major organs and serological indexes.Conclusion:In conclusion,a novel FMI/MPI dual-modality molecular imaging probe SPIOs@A-T was synthesized and manifested the feasibility for distinguishing MLN from NLN in a preclinical animal model.On the one hand,the unique ATP-responsive fluorescence design allows FMI to specifically light up the lymphatic metastasis.On the other hand,MPI can compensate for the limited imaging depth defect of FMI,thus it could ensure sensitive imaging of deeper metastatic tumor cells.Due to superior tumor-targeting and specificity,SPIOs@A-T can be a powerful tool for early detection and accurate diagnosis of MLN in vivo. |
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