Construction Of Optical Probe Based On Energy Transfer And Its Application In Tumor And Liver Injury Imaging

Bioimaging technology is an important research method to study the structure of tissue and physiological functions of the organisms.At present,the main imaging technologies include fluorescence imaging(FI),magnetic resonance imaging(MRI),computed tomography(CT),emission computed tomography(ECT),photothermal imaging(PTI),raman imaging(RI),ultrasound imaging(USI)and photoacoustic imaging(PAI).Among them,fluorescence imaging has attracted much attention due to its low cost,simple operation,high sensitivity,high spatial-temporal resolution and non-invasive imaging.However,the application of most fluorescent imaging probes in organisms is limited by short excitation wavelength,high tissue background signal and complex environmental factors.Therefore,the near-infrared ratiometric fluorescent probe has become a research hots pot because of its deep tissue penetration,low tissue background signal and self-tuning ability to reduce the influence of environmental factors.In this paper,a series of near-infrared ratiometric fluorescent probes based on energy transfer have been co nstructed for tumor and early liver injury imaging,and the specific work carried out as follows:(1)Compared with"aways-on"fluorescent probes,the activated molecular probes that constructed based on specific chemical reactions with the markers of tumor tissue can significantly improve the signal-to-background ratio of tumor imaging and have better application prospects.However,although most tumor tissue markers are mainly distributed in the tumor area,they may also be present ed in the blood and normal tissues.As a result,these probes may be activated in the blood and normal tissues owing to continuous interaction with the target in the non-tumor area,which makes it difficult to achieve high specificity and high signal-to-background imaging of the tumor.In contrast,the reversible response probe can effectively avoid the cumulative response of the probe in the blood circulation process,thereby effectively achieving high specificity and high signal-to-background imaging of tumors.In this chapter,we developed an ATP and H⁺dual-stimulus response near infrared reversible probe ATP-p H based on energy transfer for tumor specific fluorescence/photoacoustic ratiometric imaging.The probe was constructed by silicon rhodamine as donor and Changsha(CS)dye connected with N-aminoethyl piperazine as receptor and tumor target response group.It could only be activated in tumor region with high concentration of H⁺and ATP,so as to realize the near-infrared ratiometric fluorescence imaging of 700/780 nm under 660 nm excitation.In addition,the response of the probe ATP-p H to both H⁺and ATP were reversible,thus avoiding the false signal caused by the non in situ response of the probe before reaching the tumor area.We had successfully achieved tumor specific fluorescence/photoacoustic ratiometric imaging in mice and surgical resection of tumor under the guidance of dual-wavelength fluorescence.(2)Except for tumor,liver disease also threatens human health.Early diagnosis of liver disease is critical to its treatment.Peroxynitrite(ONOO^-)is considered to be a key pathogenic factor in acute liver injury and neurodegeneration.In our previous work,we had developed a cyanine dye grafted upconversion nanoprobe(UCNP)for ONOO^-detection in drug-induced liver injury mice.However,we found that cyanine dyes could also be responsive to light and some other targets in liver injury,leading to false positives signals.In response to these problems,we designed and synthesized two kinds of fluorescent probes,E-CC and H-CC,which have high selectivity to ONOO^-with absorption peaks at 540 nm and 660 nm,respectively.In order to improve the water solubility and prolong the excitation wavelength of the probes,we covalently grafted them onto the upconversion nanoparticles(3-UCNPs),obtaining two ratiometric fluorescence probes 3-UCNPs@PEI@E-CC and3-UCNPs@PEI@H-CC with high selectivity to ONOO^-.The 3-UCNPs@PEI@E-CC was successfully realized the imaging of endogenous ONOO^-in hepatoma cell(Hep G2 cell)and normal liver cell(BRL cell)stimulated by lipopolysaccharide(LPS).The 3-UCNPs@PEI@H-CC was successfully used in high fidelity imaging of CCl ₄induced acute liver injury in mice.(3)Although we have achieved early acute liver injury imaging in mice,we couldn't realise the reversible imaging of early liver injury and repair in mice.If we wanted to judge the liver repair status of mice,we need to compare the normal group,liver injury group and liver repair group at the same time.This method was not only complicated,but also might bring misleading results due to the individual differences in mice.At the same time,we couldn't obtain real-time imaging results of early liver injury and repair process.To this end,we developed a small molecule probe NB3which could reversibly respond to the related analytes in early liver injury(peroxynitrite,ONOO^-)and liver repair(glutathione,GSH)and then integrated with a core-shell upconversion nanoparticle to build the sophisticated nanoprobe4-UCNPs@PEI@NB3.The nanoprobe exhibited high sensitivity and selectivity to ONOO^-,and required millimolar GSH to reduce the product when the nanoprobe oxidized by micromolar ONOO^-,which were well matched with the ONOO^-and GSH levels in liver.Moreover,this nanoprobe had a relatively long retention time in liver,which laid the foundation for its application to real-time imaging of liver injury and repair.Finally,the novel nanoprobe successfully realised real-time reversible imaging among the different degrees of liver injury and repair in vivo.(4)It has been reported in literature that the hepatocytes cannot synthesize HOCl during acute liver injury because the lacking of myeloperoxidase(MPO)in hepatocytes,which can catalyze the production of HOCl.Even if HOCl does not exist in liver cells,is there no HOCl in liver injury?To explore this problem,we first developed a small molecule fluorescent probe(CSQ)with high selectivity and sensitivity to HOCl through a combination of"design and screening strategies".Then we connected CSQ to the surface of upconversion nanomaterials,and constructed an upconversion nanoprobe 5-UCNPs@PEI@CSQ that could detect HOCl with high selectivity.Finally,in order to improve the water solubility and cell recognition ability of the probe,we further modified polyethylene glycol(PEG)or PEG-galactose(Gal,liver parenchymal cell targeting group)on the surface of the nanoprobe,and constructedtheprobe5-UCNPs@PEI@CSQ@PEGand5-UCNPs@PEI@CSQ@PEG-Gal.Cell imaging results showed that5-UCNPs@PEI@CSQ-@PEG could enter both hepatic macrophages and hepatic parenchymal cells at the same time,while 5-UCNPs@PEI@CSQ@PEG-Gal could only enter hepatic parenchymal cells,and 5-UCNPs@PEI@CSQ@PEG only detected HOCl in hepatic macrophages,but not in hepati c parenchymal cells.However,when the probes 5-UCNPs@PEI@CSQ@PEG-Gal and 5-UCNPs@PEI@CSQ@PEG were used for acute liver injury imaging in vivo,their fluorescence signals were all significantly enhanced.Combining the results of cells,in vivo experiments,and related literature studies,we speculated that the drug first induces oxidative damage to liver macrophages,causing MPO to leak into the interstitial tissue fluid of liver cells,which in turn leads to the production of HOCl in the interstitium of li ver cells.Follow-up work is underway.