Research On NQO1 Biosensing And Photodynamic Therapy Based On Conjugated Oligomer Fluorescent Probes

In organism,enzymes participate in almost every process of life activities.The development of new technologies to monitor enzymes plays a vital role in the early diagnosis,treatment and prevention of diseases.Human NAD(P)H:Quinone oxidoreductase 1(NQO1)is an important reductase,which is related to tumor.It is over-expressed in many cancer cells(lung,colon,liver,breast,prostate,pancreatic and etc.).Thus,it is significant to establish a highly selective and sensitive method with high signal-to-background ratio for NQO1 detection and cancer diagnosis.Recently,conjugated oligomers have been widely applied in biosensing,cell imaging,drug release,and integration of cancer diagnosis and treatment due to their low toxicity,stable photo stability,good biocompatibility,and high sensitivity.In addition,the side chain of the conjugated oligomers is modifiable.Water-soluble groups can be modified to expand its application in biological systems.Also,it can be modified with functional groups to satisfy requirements of imaging,tumor treatment,and integration of diagnosis and treatment.Herein,an activatable biosensor based on water-soluble conjugated oligomer(OPFV-TLQ)was developed for highly sensitive detection of NQO1 and visualization imaging of cancer cells.Oligo(ethylene glycol)(OEG)and the trimethyl lock quinone(TLQ)group were modified on the side chain of OPFV-TLQ,which makes the probe possess good water-solubility and low fluorescence background signal,respectively.Interestingly,in the presence of NQO 1,the benzoquinone was reduced to hydroquinone,followed by the removal of the quinone propionic acid(QPA)with an intramolecular cyclization reaction,which dramatically activates the fluorescence of OPFV-TLQ.Importantly,about 141-fold enhancement in fluorescence intensity of the probe was obtained,which provides one of the highest signal-to-background ratios for the detection of NQO 1 compared to the reported methods.The detection limit was as low as 0.068 ng/mL.Additionally,the catalytic constant was 67.71 ?M-1 s-1,which is significantly higher than those of other probes,indicating the excellent catalytic efficiency of NQO1.Molecular docking certifies that the probe is a good substrate of NQO1.Additionally,we further studied the endogenous NQO1 imaging of the cells,and we found that the fluorophores after enzymatic hydrolysis were mainly distributed on the lysosomes through colocalization imaging,which indicates that the probe product has the lysosome-targeted ability.The probe can identify different cancer cells,which indicates that the probe has broad applications in the cancer cell/tissue imaging and cancer differentiation.Furthermore,after the probe being hydrolyzed by NQO1,the fluorophore OPFV-NH2 also has has high production capacity of reactive oxygen species,which enables it to apply in stimulus-responsive photodynamic therapy.When the concentration of OPFV-TLQ was 500 nM,the cell death rates of A549,MCF-7 and MDA-MB-231 cells were 93%,95%,and 84%,respectively.Therefore,our probe can simultaneously achieve multiple functions such as fluorescence imaging,PDT,and stimulus response,providing new methods for cancer diagnosis and treatment.We further studied the structure-activity relationship of fluorescent probes.The neutral conjugated polymer PFV-TLQ and cationic conjugated oligomer OPFV-TLQ-NM3+ were then synthesized.We found that the fluorescence of PLV-TLQ and OPFV-TLQ-NMe3+ only recovered by 1.9-fold and 5.6-fold,respectively,after being activated by NQO1.The detection limit of OPFV-TLQ-NMe3+was 33 ng/mL.However,the fluorescence intensity of OPFV-TLQ enhanced by 141-fold with high sensitivity.It is known that the conjugated polymer has a large molecular weight and may loosely aggregate in the buffer,which could hinder the probe to interact with the active site of NQO1.Because the isoelectric point of NQO1 is 8.91,it is positively charged in the experimental conditions.Thus,the electrostatic repulsion between the cationic oligomer and NQO1 will affect the activation of the oligomer by the enzyme,resulting in the low detection sensitivity.Therefore,the OPFV-TLQ demonstrates the best response for NQO1 due to its appropriate structure.These results provide an important guideline for the design of other enzyme probes.