Fluorescence Imaging Of Liver Diseases Related Enzyme Markers In Cells And In Vivo

Liver diseases are lesions that occurred in the liver,including liver injury,hepatitis,liver fibrosis,cirrhosis and liver cancer.Especially,liver fibrosis is almost the inevitable pathological process for the development of various hepatitis.And liver cancer is the final form.That leads the high incidence of liver fibrosis and liver cancer.According to statistics,these two diseases have a high mortality,thus seriously threaten human health.In fact,patients will obtain the desirable therapeutic effect and prognosis in early stage of these two diseases.Since no obvious clinical symptoms during early stage and the low resolution of traditional diagnostic ways,diagnosing liver diseases in early stage are difficult.As a result,when diagnosis is often in the middle and late stages.Thereby,development of effective methods to achieve the early detection of liver fibrosis and liver cancer are urgent requisite.Fortunately,enzyme markers which are overexpressed in abnormal tissues and intracellular cells can act as screening tools for early diagnosis of diseases.Enormous literatures have found that there is a high level of Monoamine oxidase B（MAO-B）in patients with early liver fibrosis.Therefore,it is considered to be a useful marker of early liver fibrosis.Because of the abnormally elevated activity ofα-L-fucoidase（AFU）in the early stage of liver cancer even before forming a lesion,so it has been suggested as an ideal marker for early liver cancer.Meanwhile,alkaline phosphatase（ALP）is also widely recognized as a marker of liver cancer.Therefore,precise imaging dynamics of these related enzyme markers will be expected to open up new avenues for the early diagnosis of liver fibrosis and liver cancer.Fluorescence microscopy imaging techniques have the advantages of non-invasivenes,high sensitivity,spatio-temporal resolution and contrast ratio.Due to long-wavelength excitation,two-photon and near-infrared fluorescence imaging methods also can effectively avoid autofluorescence,reduce light scattering,improve signal-to-noise ratio and penetration depth.Benefitting from these advantages,they have become powerful tools for in situ dynamic imaging of molecular events in cell and in vivo.Especially enzyme activated fluorescence imaging analysis that constructed based on enzyme specific reactions,has become one of the most promising ways for the early diagnosis of asymptomatic diseases.However,two-photon or near-infrared fluorescence probes for early diagnosis of liver fibrosis and liver cancer via in vivo detection of their early markers,MAO-B,AFU,ALP have rarely been reported.So,there is an urgent need to develop new methods for accurate in situ monitoring of these enzyme markers,which is of great significance for studying their biological role in the development of diseases and promoting early diagnosis of diseases.Based on the aforementioned concerns,aimming at the existing obstacles in early diagnosis of liver fibrosis and liver cancer,this dissertation constructed a series of simple fluorescence,near-infrared fluorescence（NIR）/photoacoustic（PA）dual mode and chemiluminescence（CL）imaging probes which possess excellent properties of good photostability,high sensitivity and selectivity.They realized in situ and rapid analysis of enzyme markers（MAO-B,AFU,ALP）related with early-stage liver diseases.These probes not only provide a highly sensitive,fast,and accurate imaging materials for effective diagnosis of liver diseases in early stage,but also can act as ideal tools for the deep demystification of the specific biological functions of these enzymes in organisms.The main results of this dissertation were shown as follows:1.Based on the specific reaction of benzylamine with MAO-B,we constructed a two-phone fluorescence probe termed BiPhAA for in vivo detection of MAO-B.BiPhAA could be easily got in one reaction step.In the presence of MAO-B,the benzylamine part of BiPhAA was catalyzed and oxidized within 10 min,then generating an aldehyde group,subsequently reacting with the amino group on the adjacent benzene ring.As a result,a larger conjugated system was produced and emitted a bright fluorescence.According to our work,BiPhAA could rapidly sense MAO-B activity with high selectivity and sensitivity.Expectedly,the imaging results of HL-7702 and LX-2 cells proved that BiPhAA possessed the ability for real-time monitoring of endogenous MAO-B fluctuates.Through two-photon fluorescence imaging,BiPhAA was successfully accomplished in situ imaging of MAO-B activity in mice,indicating liver fibrosis.2.We presented a first two-photon turn-on fluorescence reporter,named HcyCl-F,for fast sensing lysosomal AFU in living system.In our design strategy,AFU could fast hydrolyze the2-chloro-4-phenyl-α-L-fucoside bond of HcyCl-F,then generating a hydroxyl group.As a result,a donor-π-acceptor（D-π-A）type fluorophore HcyCl-OH was produced and fluoresced brightly.According to our work,HcyCl-F could rapidly detect AFU with high selectivity and sensitivity.Using Two-photon fluorescence microscope,HcyCl-F successfully accomplished to real-time monitor the fluctuation of AFU in lysosomes.Furthermore,in situ imaging of AFU in mice showed the high level of AFU in liver tumor tissue.Notably,HcyCl-F can clearly detect stage I liver tumors.3.Based onα-L-fucoidosidase（AFU）,we developed a small molecule DCBOD-F for the early diagnosis of liver cancer with NIR and PA imaging dual modes.The product DCBOD-OH was rapidly generated after DCBOD-F reacting with AFU.Thereby,the fluorescence at 670 nm was significantly enhanced under the excitation of 650 nm.In vitro experiments showed that DCBOD-F can highly selective and specific respond to AFU with NIR signal.Profit from the NIR fluorescence,DCBOD-F visualized the floating of AFU in lysosomes.Relative to other normal cells,we found a higher level of AFU in cancer cells.More importantly,DCBOD-F achieved accurate in situ imaging AFU to diagnose early liver tumor through NIR mode.4.Owing to high level of alkaline phosphatase（ALP）in cancer tissue and taking advantage ofitsavailablechemiluminescence（CL）substrate3-[2-spiroadamatane]-4-methoxy-4-[3-phosphoryloxy]-phenyl-1,2-dioxetane)dioxetane（AMPPD）,we assembled an integrated platform named MSN@H₆L@β-CD@AMPPD NPs for liver tumor-specific diagnose and therapy.AMPPD as energy donor was bond to cyclodextrin（β-CDs）by interaction of host-guest,and then covalently attached to the mesoporous silica（MSNs）surface loaded with energy receptor（4-carboxyphenyl）porphyrin（H₆L）.Overexpressed ALP can specifically hydrolyse the phosphoester bond of AMPPD accompanying energy release,then H₆L accepts the energy and is excited which not only emits a bright light at 670 nm but also creates ¹O₂.MSN@H₆L@β-CD@AMPPD NPs were easily constructed and possessed excellent stability and reproducibility.According to our work,these NPs could emit NIR fluorescence and produce ¹O₂ after activated by ALP in vitro.Excitedly,the CL imaging and therapy data fully prove that MSN@H₆L@β-CD@AMPPD NPs could target liver tumor for its high-sensitivity imaging diagnosis and growth inhibition.