New Molecular Imaging Probes For Accurate Diagnosis Of Idiopathic Pulmonary Fibrosis And Lung Cancer

Objective:Idiopathic pulmonary fibrosis(IPF)is a pulmonary interstitial disease.Because of its complicated etiology and unclear pathogenesis,its diagnosis and treatment face enormous difficulties.Matrix metalloproteinases(MMPs),especially MMP-2,MMP-9 and MMP-14,are currently thought to play roles in the progression of the disease in IPF.In this paper,we designed a new type of near-infrared probe can be used for real-time dynamic monitoring of MMPs in IPF process changes,and inhibit the activity of MMPs to alleviate the purpose of IPF disease progression.Methods:The target probe was chemically synthesized,the cyanine was fluorophore,and the hydroxamic acid as an inhibitor of MMPs,was the targeting group.The two were chemically linked by click chemistry.The ability of the probe to recognize MMPs was assessed by fluorescence of the probe and co-localization with the fluorescence of fluorescent primary antibody of MMP-2,MMP-9,MMP-14.Observe the change of fluorescence intensity of probe in cell fibrosis model induced by TGF-β1 for 24,48 and 72 h.The concentration of MMP-2,MMP-9 and MMP-14 were determined by enzyme-linked immunosorbent assay(ELISA).The mice in normal group and bleomycin group were established at 3,7,9,14,21 and 28 days.The changes of the fluorescence intensity of the probe were observed at different stages of pulmonary fibrosis.Results:Compound IRGP-710 was identified by nuclear magnetic resonance and mass spectrometry as the designed target product.In the colocalization experiments,the probe fluorescence were fitted very well with the fluorescent primary antibody which were labeled by FITC.Fluorescence intensities gradually increased at cell fibrosis model which stimulated at different times.And the ELISA assay also showed that MMPs levels were gradually increased.In in vivo experiments,the leves of MMPs were different in mice when stimulated different days,and IRGP-710 was able to sensitively detect this change.Conclusion:Probe IRGP-710 localization group is hydroxamic acid and therefore with high specificity for MMP-2,MMP-9 and MMP-14.It not only can be applied to qualitatively detect the concentration of MMPs in cell experiments,but also can be applied to the in vivo imaging of MMPs during the progression of fibrotic diseases.Because IRGP-710 can bind to the active site of MMPs and inhibit MMPs activity,it can alleviate the promotion of MMPs on pulmonary fibrosis.Objective: To design and synthesize a fluorescent probe that can target to MMP-2,MMP-9 and MMP-14 and to differentiate cancer tissues from adjacent normal tissues in lxmg cancer surgery.By inhibiting the activity of MMP-2,MMP-9 and MMP-14,blocking tumor angiogenesis,invasion and metastasis.To achieve the purposes of accurate imaging and therapeutic.Methods: The type of inhibitor was identified by literature search.The probe was modified by cyanine to 8-aminooctanoic acid as a positioning group and hydroxamic acid as a targeting group to obtain the target structural probe by chemical synthesis.The Probe localization was detected by fitting the fluorescence of the probe to the fluorescence of the fluorescent primary antibody of MMP-2,MMP-9,MMP-14.The probes were applied to different tumor cells and collected the fluorescence intensity of probe.Finally,the enzyme-linked immunosorbent assay(ELISA)was used to determine the concentration of MMPs in each cell line.A nude mouse model was constructed to assess whether the probe could be localized to the tumor site and to perform in vivo MMPs imaging.Simultaneously,evaluated the therapeutic effect of the probe.By establishing orthotopic lung cancer mice model,probe localization was verified by in vivo imaging and 3D imaging.Determine whether the probe can distinguish the cancer site from the normal site.Results: The final compound was identified by nuclear magnetic resonance and mass spectrometry as the target probe and responded only to MMP-2,MMP-9,and MMP-14.Confocal experiments show that the probe can qualitatively detect the difference in the leves of MMPs in different cells.In the nude mice experiment,the probe was injected into the body through the tail vein and could locate the tumor site within 2 hours and emit fluorescence.Two-dimensional and three-dimensional imaging results of in orthotopic lung cancer mice showed that the probe could be injected into the lung cancer site via the tail vein and the fluorescence intensity was significantly higher than that of adjacent normal tissues.Conclusion: The probe utilizes hydroxamic acid as a targeting group to accurately target MMP-2,MMP-9 and MMP-14.It not only can be applied to the qualitative detection of intracellular differences in the concentration of MMPs in different cells,but also can be used for in vivo imaging of MMPs.Probe not only locate MMPs but also inhibit their activity,enabling imaging and therapy to be combined.Objective: Cysteine(Cys)plays significant roles in many physiological process,although its normal concentration is maintained at the micromolar level.Abnormally high levels of intracellular Cys can lead to many diseases including cancer.Herein,we designed and synthesized a ratiometric near-infrared(NIR)fluorescent probe for the detection of mitochondrial Cys in cells and in orthotopic lung cancer mice.Methods: Chemically synthesized fluorescent probe Cy-OAcr.Cy-OAcr has a lipophilic iminium cation unit as the mitochondrial guider and an acrylate group as the Cys recognition unit as well as a fluorescence modulator for rearranging the conjugated7t-electron system of cyanine fluorophore.We employed two commonly used commercally mitochondria-specific dyes,Mito-Tracker Green and Rhodaminel23,to carry out the colocalization experiments.We further used Lysosome-specific dye LysoTracker Green DNA-26 to verify the localization capability of our probe Cy-OAcr in mitochondria.We investigated whether the probe Cy-OAcr could quickly respond to intracellular Cys by confocal laser scanning microscope(CLSM)and flow cytometry analysis.The A549,HI650 and PC9 cell lines were treated with N-ethylmaleimide(NEM),Cys,glutathione monoethyl ester(GSH-MEE),and homocysteine(Hey)to construct intracellular without thiol,high Cys concentration,high glutathione(GSH)concentration,and high Hey concentration cell models to observe the probe’s ability to selective response to Cys.A549,H1650 and PC9 cells were injected subcutaneously in the armpit of nude mice to form tumor-bearing mouse model and urethane stimulated FVB mice to establish the orthotopic lung cancer mice model to evaluate whether the probe could be applied to Cys detection in vivo.Results: The synthesized probe was characterized by lK and 13 C NMR spectroscopy and mass spectrometry.The results showed that the probe was in accordance with the molecular structure of the design.The probe only offered a very high level of ratiometric fluorescence emission towards Cys.The co-localization experiment results verified that probe possessed excellent mitochondrial-targeting ability.Laser confocal and flowcytometry results showed that the probe was able to respond to Cys in cells within 30 minutes without interference from intracellular GSH and Hey.Animal experiments show that the probe could emit different fluorescence intensity ratio values consistent with the diffremt concentration of tumor-bearing mice and in orthotopic lung cancer mice.Conclusion: The probe Cy-OAcr was able to quickly react with Cys and produce a shift in fluorescence emission profile,meanwhile avoiding the interference from other small endogenous reactive small molecules.Probe Cy-OAcr was capable to detect and image Cys in three living cancer cell lines and their corresponding tumor-bearing mice models,and the iminium cation of this fluorophore platform could facilitate the probe accumulating in mitochondria.In vivo imaging analyses reveal that the probe can discriminate tumor lesions from normal tissues,indicating its significant potential applications for clinical auxiliary diagnosis of cancer.Objective: Cys-SSH plays an important role in the dynamic regulation of redox in mammals,and its dynamic changes and effects in some imbalanced redox diseases have not yet been fixlly elucidated.Early clinical diagnosis and treatment of lung cancer and pulmonary fibrosis disease has been faced with tremendous difficulties.Near-infrared ratio fluorescent probes are increasingly being considered and developed for in vivo biomolecule detection because of their unique advantages.This article aims to use fluorescent probe technology to detect Cys-SSH in vivo to reveal its biological role in lung cancer and pulmonary fibrosis.Methods: Design and synthesis of a near-infrared ratio fluorescent probe that is sensitive to Cys-SSH.Determination of probe selectivity to determine if the probe can be used to detect Cys-SSH.TGF-pl was used to construct different degrees of cell fibrosis model.Laser confocal and flow cytometry were used to verify whether the probe could detect Cys-SSH changes.The probes were incubated with different tumor cell lines and to assess whether the probes could distinguish Cys-SSH levels in different cell lines.The cells were treated with different reagents,which were N-ethylmaleimide(NEM),a thiol scavenger;CysSSCys,a raw material for Cys-SSH synthesis;and the key enzyme inhibitor,AOAA,using probes to verify Cys-SSH intracellular sources.Constructing tumor-bearing mouse model to verify whether the probe can be used for Cys-SSH imaging in vivo.Constructing pulmonary fibrosis and orthotopic lung cancer models to reveal the potential role of Cys-SSH in these two diseases.Results: The nuclear magnetic resonance and mass spectrometry confirm that the synthesized compound is consistent with the target molecule.Probes have good selectivity for Cys-SSH.By laser confocal and flow detection in the cell fibrosis model,the Cys-SSH concentration gradually increased with the prolonged stimulation time.And the probe can reveal the different concentration in different tumor cells through different fluorescence ratio values.When the cells were treated with NEM,the probe failed to react with Cys-SSH to release channel 2 fluorescence,so channel 1 fluorescence was enhanced.However,with the addition of foreign aid CysSSCys,intracellular Cys-SSH production increased and the fluorescence of channel 2 was significantly increased.Cystathionine y lyase(CSE)and cystathionine P-synthase(CBS)are key enzymes for the in vivo synthesis of the Cys-SSH pathway.When these two enzyme inhibitors AOAA are added,Cys-SSH synthesis was reduced and probe channel 2 fluorescence was reduced.Tumor-bearingmouse model with A549,PC9,and H1650 was constructed,and the probe could sensitively detect changes in Cys-SSH content at the tumor site.The difference in Cys-SSH content can be observed through lung fibrosis and orthotopic lung cancer models,which helps to reveal the potential application value of Cys-SSH in clinical treatment.Conclusion: The designed and synthesized probes have superior near-infrared ratio imaging performance.When reacted with Cys-SSH,the spectrum before and after the reaction changes significantly.The probe can be used not only for the detection of simple Cys-SSH in vitro,but also for the detection of Cys-SSH in complex biological samples.It plays an important role in revealing the biological value of Cys-SSH.