Construction And Biological Imaging Application Of Activatable Second Near-infrared Window Fluorescent Probes With BODIPY Skeleton

Benefiting from the inherent merits of non-invasive,high resolution,real-time visualization,and fast feedback,fluorescence imaging has become a promising approach for the investigation of biological and physiological processes.Comparing with optical imaging in the visible（400-700 nm）and near-infrared I window（NIR-I,700-900 nm）,fluorescence imaging in the second near-infrared window（NIR-II,1000-1700 nm）is highly attractive for in vivo application because of its increased temporal-spatial resolution,deeper tissue penetration,lower light scatter and auto-fluorescence.As the essential part in NIR-II fluorescence imaging,the inherent properties of NIR-II materials determine its application prospects.Especially,organic molecule dyes are promising to transition rapidly into a clinical setting,as they featured high biocompatibility,flexible structure,and facile modification.However,most reported NIR-II molecule dyes belong to“always-on”mode.The sensing and imaging of the region of interest relies on molecular accumulation and retention instead of specific recognition of target,which inevitably causes high background signal,poor sensitivity,and influences the accuracy of diagnostics.Furthermore,to obtain enough imaging contrast,long-time circulation and metabolism to remove excess or unbound dyes is inevitable for these always-on probes,which also presents a big challenge to the dynamic monitoring of biological processes.As an alternative approach,NIR-II probes that can be specifically activated by biomarkers are able to provide higher signal-to-noises（SBRs）and real-time information,exhibiting great significance for imaging and exploring biological processes at the living level.Nevertheless,as an emerging field（only 3 years in development so far）,the development of activatable NIR-II molecule probe is hindered by some issues.Firstly,due to the difficulty in constructing novel NIR-II fluorophore and the commonly used responsive mechanism face challenge in the long-wavelength region,activatable NIR-II fluorescent probes are rarely reported,and their excitation/emission maxima are mainly located between 900 and 1000 nm,failing to show the advantages of NIR-II probes in imaging contrast and penetration depth.Secondly,reported NIR-II fluorescent probes were single-target responsive,which may face the issue of non-specific activation and even cause“false positive”signal to influence the accuracy of diagnostic and therapeutic outcomes.In summary,this dissertation initially aims to construct the novel NIR-II fluorophore.Afterward,to face the issues of limited emission wavelength and non-specific activation,several novel pathological parameter activatable NIR-II fluorescent probes are obtained through reasonable design strategy and precise architecture regulation.Finally,based on the Vivo imaging systems,these probes are applied for real-time monitoring,precise diagnosis,and treatment of diseases at the living level.The main contents are as follows:1:Based on BODIPY skeleton,the novel NIR-II fluorophore has constructed.In our experiments,the difluoroboron dipyrromethene（BODIPY）dye has been selected as fluorophore considering its high stability and facile modification.As the electron withdrawing unit,naphthylamine derivative was connected to the BODIPY group through the vinyl to enlargeπ-conjugated system.Afterward,we incorporated different groups（including,thioether,alkyl,amino,piperazinyl,etc）into the third position of the BODIPY,five kinds of BODIPY fluorophores（namely WD-S-C₄H₆O₂、WD-Cl、WD-C₂H₅、WD-NHCH₃、and WD-CH₃）with D-π-A structure were constructed.Experiments have shown that WD-CH₃with the strongest electron-donor（piperazinyl）group exhibited maximal emission over 1000 nm（1024 nm）,which can be ascribed to enhanced intramolecular electron transfer process.Furthermore,WD-CH₃ not only has large Stokes shift（over 200 nm）and high molar absorption coefficient,but also has good chemical and photostability,indicating that WD-CH₃ fluorophore has the potential as the effective platform for activatable NIR-II fluorescent probe design.2:On the basis of regulating conjugate structure strategy:construction of activatible NIR-II molecular probe for in vivo elucidation of disease-related viscosity variations.Principally,in the media with varying viscosities,the flexible linker between the donor and acceptor will alter the conjugatied skeleton of the molecule and its energy dissipation approach,giving viscosity-dependent fluorescence response.Inspired by this,WD-CH₃,as the viscosity-activated precursor probe was initially obtained.Then,aiming to obtain optimal viscosity response,we investigated the structure-activity relationship.In our experiments,based on the WD-CH₃ scaffold,we introduced strong electron-drawing（Nitro）or electron-donating groups（methoxy or diethylamino）into the ortho-position of the BODIPY fluorophore.Therefore,altogether four derivations,namely WD-CH₃,WD-NO₂,WD-OCH₃ and WD-NME₂ were synthesized.After screening,WD-NO₂ with the electron-withdrawing（nitro）group at the ortho-position of BODIPY displayed the best combination of properties,including highest sensitivity to viscosity（31-fold fluorescence enhancement at 982 nm）,high stability against environment factors（p H,polarity）,and relatively high quantum yield（1.6%in glycerol）,which could be ascribed to its biggest intramolecular dihedral angle.Finally,as the first viscosity-activated NIR-II fluorescent probe,WD-NO₂ was able to monitor the viscosity changes in some interesting and less explored pathological processes in situ,including DOX-mediated solid tumor apoptosis and diabetes-induced liver injury.3:Flexible designing strategy to construct activatable NIR-II fluorescent probes with emission maxima beyond 1200 nm.Emission wavelength is one of the most important cutting-edge topics in the field of fluorescent probes.Notably,longer fluorescence emission affords higher imaging contrast and tissue penetration depth since it decreased photon scattering and tissue auto-fluorescence,allowing more accurate biological species mapping in vivo.Unfortunately,the emission maximal of the currently reported activatable NIR-II probes was mainly located between 900 and 1000 nm,thus,developing new activatable fluorescent platform with emission maxima beyond 1200 nm is highly significant.After exhaustively evaluated the strategies for bathochromic shift of wavelength,we thus consider that enlargeπ-conjugated region and enhance the electron density of donor group may lead to optical bathochromic shifts.In our experiments,we still elected BODIPY as fluorophore.Then,integration of above strategies（enlargeπ-conjugated region and enhance the electron density of donor group）into one system,and combining with the tuning of the push-pull electronic effect before and after stimulation by analytes,four proof-of-concept probes WH-X（WH-1、WH-2、WH-3,and WH-4）were synthesized.All probes could be specifically activated by H₂S,and the maximal emission of reaction products was red-shifted from 925 nm to 1205nm,and such optical bathochromic shifts may attribute to the cooperation of enlargingπ-conjugated region and enhancing the electron-donating ability of the donor unit.Screening results showed that WH-3 exhibited the ideal combination of properties,including high sensitivity,fast response,acceptable quantum yield,and long absorption/emission wavelength（peak lies at 925/1140 nm）after H₂S activation.Finally,benefiting from the inherent merits of the deep penetration and high spatial resolution of NIR-II imaging,WH-3 was able to real-time track endogenous H₂S generation and fluctuation in tumor-bearing mice with high-imaging contrast.Moreover,we can reasonably expect that our approach can be generalized to construct probes for various biomarkers by flexible regulation of the recognition sites.4:“Dual-lock-dual-key”controlled NIR-II molecular probe for specific discrimination of orthotopic colon cancer and imaging-guided tumor excision.Benefiting from the advantages of depth tissue penetration and high resolution,NIR-II imaging has become a promising approach in tumor diagnosis and therapy.While a considerable number of NIR-II fluorescent probes have been reported for tumor application,most of them are major in subcutaneous tumor recognition and treatment.The depth of these regions is limited and visible,failing to display the merits of NIR-II fluorescence imaging.The main reason is as follows:currently available NIR-II tumor diagnostic agents are based on“always on”modality or single biomarker activation,which are subject to limited imaging contrast,non-specific activation,and even false positive diagnosis.Focusing on above issue,based on BODIPY skeleton,we developed a H₂S/H⁺dual-stimuli responsive NIR-II fluorescent probe WH-N₃ for precise delimitation of tumor and intraoperative fluorescence-guided surgical resection.WH-N₃ itself was non-fluorescent,and it can only be lighted up through the synergistic activation by H₂S and tumor acidic environment（TEM）.Such a“dual-lock-dual-key”strategy based activatable probe exhibited significantly higher tumor-to-normal tissue（T/N）ratios than“always on”agent（ICG）and single parameter responsive counterpart probes in the imaging of colon tumor,which over-expresses H₂S.Meanwhile,WH-N₃was able to track tumor-drived endogenous H₂S fluctuation and accurately differentiate colon tumor based on H₂S content discrepancy.More excitingly,under the guidance of highly specific NIR-II fluorescence of the probe,tiny orthotopic colon tumor with diameter down to 0.8 mm was facilely resected by WH-N₃.