Application Of Activatable Near-Infrared Small Molecular Probe In Fluorescence/Photoacoustic Imaging

Accurate and highly sensitive detection and imaging of biomolecules can specifically distinguish abnormal pathological processes,which is the key to the early diagnosis of disease.Hydrogen peroxide（H₂O₂）is a prominent reactive oxygen species（ROS）with relative stability,which makes it a potential diagnostic marker for pathological states.Cathepsin B（CTB）,which is overexpressed in tumor cells,can directly or indirectly degrade the components of the outer matrix and basal membrane,and promote tumor invasion and metastasis.CTB is a cysteine-degrading protease that mainly exists in cytolytic enzymes.It is involved in the co-degradation of various proteins in human intracellular lysosomes and can be used as an important tumor marker.Near infrared fluorescence（NIR FL）imaging can reduce the spontaneous fluorescence of organisms and the damage of living tissues.Although it has the characteristics of high sensitivity and high controllability,the tissue penetration is limited.As a new technology,photoacoustic（PA）imaging combines high-contrast optical imaging（OI）with ultrasound imaging with high resolution and deep tissue penetration.It can overcome tissue scattering and has better spatial resolution and penetration depth than OI.FL/PA dual modes can combine the advantages of each other to achieve high sensitivity and high spatial resolution at the same time.Therefore,the design of FL/PA bimodal imaging probe has great application potential in biomedical and molecular diagnostics.In this paper,an excellent NIR FL dye-hemicyanine（HCy-OH/NH₂）was synthesized.Based on this small molecule dye,two kinds of activated probes with FL/PA dual-modal imaging capability were designed:（1）Mitochondria-targeted NIR probe TPP-HCy-BOH,were designed and synthesized for FL/PA dual-modal detection and imaging of H₂O₂in an inflamed mouse model.Compared with probe HCy-BOH which cannot target mitochondria,TPP-HCy-BOH has a lower detection line（LOD=0.348μM）and higher pseudo-first-order kinetic constants(k_obs=4.72×10^-3s^-1)in vitro.TPP-HCy-BOH has excellent imaging effect on exogenous H₂O₂in cervical cancer（HeLa）cells,endogenous H₂O₂in leukemia cellsin mouse macrophage（RAW264.7）cells,and lipopolysaccharide（LPS）-induced acute inflammation model.（2）The NIR probes HCy-Cit-Val and HCy-Gly-Leu-Phe-Gly toword CTB were designed and synthesized for FL/PA dual-mode imaging and detection for the overexpressed CTB activity in living tumors with high sensitivity and spatial resolution.CTB-specific NIR molecular probes HCy-Cit-Val and HCy-Gly-Leu-Phe-Gly were designed and synthesized to accurately and highly sensitive FL/PA dual-modal imaging and detection for the overexpressed CTB activity in living tumors.The sensitivity and specificity of CTB for the recognition of molecular probes HCy-Cit-Val and HCy-Gly-Leu-Phe-Gly were evaluated effectively,and the effects of different molecular probes HCy-Cit-Val and HCy-Gly-Leu-Phe-Gly for the early diagnosis of CTB-associated tumors were observed and distinguished.Among them,the kinetic constant(k_cat/K_M=30.17μM^-1min^-1)of the enzymatic reaction of HCy-Cit-Val was higher than that of HCy-Gly-Leu-Phe-Gly(8.71μM^-1min^-1),and the LOD（0.292 U/L for FL,0.407 U/L for PA）obtained by HCy-Cit-Val was lower than that of HCy-Gly-Leu-Phe-Gly（0.532 U/L for FL,0.723 for PA）.In addition,HCy-Cit-Val was significantly better than HCy-Gly-Leu-Phe-Gly in FL/PA dual-modal diagnosis of HeLa and the mouse tumor region constructed by HeLa.In addition,HCy-Cit-Val was significantly better than HCy-Gly-Leu-Phe-Gly in FL/PA dual-modal diagnosis of HeLa and mouse tumor region constructed by HeLa.