Activatable Near-Infrared Fluorescent Probes For Highly Sensitive Imaging Of Tiny Breast Tumors And Metastatic Lesions

Breast cancer is one of the world’s leading causes of death,with low five-year survival rate,which is mainly due to missing the best timing of treatment and cancer metastasis in the clinical management.The precise diagnosis of tiny cancers and metastatic cancers from breast cancer greatly increases the chances of effective treatment and can help reduce further suffering and mortality.Advances in the near-infrared fluorescence imaging provide great opportunities for identifying cancer at its early stages and ultimately optimizing patient-specific therapies,owing to its considerable advantages over traditional molecular imaging,such as high signal-to-background ratio(SBR),high sensitivity,low cost.The improvement of imaging sensitivity is crucial for the detection of tiny cancers and metastatic cancers.The improving of SBR is the key to gaining the higher sensitivity and realizing accurate tumor detection.It is an effective strategy to improve SBR by reducing the signal from non-tumor parts.Therefore,the design of activated near-infrared(NIR)fluorescent probes is more promising.Activatable probes are designed to stay in the"OFF" state in non-target tissues or blood,and signals can be turned "ON" only in response to specific targets or events.Consequently,elimination of the background signal dramatically improves the SBR,thereby making activatable probes a preferential choice for cancer imaging with high specificity and sensitivity.To design activatable probes for cancer detection,choosing a suitable stimulus plays a significant role in design.There is growing recognition that cancer is a metabolic disease.Although tumors are highly heterogeneous and complex,dysregulated energy metabolism is commonly observed in nearly all types of cancer cells.A potential target for imaging in breast cancers is acidic extracellular pH(pH 6.5-7.0),which mainly results from the abnormal aerobic glycolysis of tumor cells,has been recognized as a hallmark of different subtypes of breast cancers and metastatic tumors.Therefore,we aim to develop pH-activatable probes to targeting tiny breast tumors and metastatic tumors.The detailed works of dissertation are listed below.(1)A metabolic acidity-activatable calcium phosphate probe with fluorescence signal amplification capabilities for non-invasive imaging of tiny breast tumors.In this work,we report a robust strategy to create a metabolic acidity-activatable calcium phosphate(CaP)fluorescent probe(hereafter referred to as MACaP)based on the self-assembly of CaP and the NIR dye IR780 iodide mediated by a poly(acrylic acid)(PAA).By delicately modulating the synthesis conditions of the MACaP probe,its pH-responsive range could be adjusted to perfectly match the extracellular pH of the tumor microenvironment,with rapid and sharp responsiveness(pH range:6.8-7.0,ΔpH=0.2).The fluorescent probe remained silent in blood and normal tissue,owing to an aggregation-caused quenching(ACQ)effect(OFF state).However,upon reaching the tumor sites,the probe underwent fast disassembly in the acidic tumor pH range,which caused the release of IR780 and significantly enhanced the fluorescence signal(ON state),enabling tumor-specific imaging against a dark background.The MACaP probe benefited from its OFF/ON design and its ultra-pH sensitivity exhibited superior ability for in vivo tiny breast tumors imaging.(2)An acidic-microenvironment activatable probe for ultrahigh specific fluorescence imaging of lung metastases.We further optimized the synthetic conditions of the probe and obtained acidic-microenvironment activatable CaP fluorescent probe,SCaPI,with improved SBR value.Meanwhile,the superior sensitivity and specificity of SCaPI enabled high-contrast visualization of breast cancer with single/multiple lung metastases,as well as lung adenocarcinoma in situ.(3)Sensitive and specific identification of sentinel lymph node metastasis using a pH-activatable probe.In this work,we designed a tumor acidity-sensitive,NIR probe(termed ICPPs)for specific and sensitive identification of sentinel lymph nodes metastasis in vivo,based on calcium phosphate matrix and a NIR fluorophore,indocyanine green(ICG).We investigated the capability and sensitivity of this probe for tumor extracellular pH imaging in vitro and in vivo using metastatic sentinel lymph nodes model.Our results showed that the new probe provides non-invasive pH-activatable precise detect metastatic lymph nodes in vivo.In summary,through a self-assembly strategy,as well as rational regulation of the preparation conditions,we successfully developed two pH-ultrasensitive CaP-based fluorescent probes.Unlike traditional CaP nanoprobes,which usually display broad pH responses inside endosomes or lysosomes at a low pH(4.0-6.0),probes could sharply respond to the subtle pH difference between the tumor microenvironment and normal tissues(pH 6.8-7.0,ΔpH=0.2),leading to the activation of fluorescence for in vivo tumor imaging.Owing to its extreme sensitivity to the tumor extracellular pH,probes would not produce any noticeable fluorescence signal until reaching the tumor site,thereby significantly increasing the SBR.Our results demonstrated that probes could not only be used for imaging various tumors with high specificity but also enabled robust visualization of smaller tumor lesions and metastatic lesions.This study provides a robust strategy to modulate the pH response range of activatable NIR probes,which is particularly important for their applications in precise tumor diagnosis and treatment.