Highly Stable NIR Fluorescent Sensors And Their Application In Bioimaging

Imaging, especially fluorescent imaging has attracted considerable attention because of the high sensitivity, resolution as well as the simple operation which has been widely utilized in the field of biomedical and life science. Considering the advantages of NIR light such as deep tissue penetration and low auto-fluorescence background and the high density excitation of the contrast agent during the bioimaging, fluorophores with NIR emission and high photostability are especially preferable in bioimaging. However, up to date, the various available fluorophores suffer from either short wavelength emission or poor photostability. Therefore, this dissertation focuses on the development of constructing highly stable fluorescent contrast agents via encapsulation or utilization of novel fluorophores, especially in exploring its parcticle application in biological labelling, tumor imaging and monitoring of controlled drug release.Firstly, the concept and principle of imaging especially fluorescence imaging, the challenge for the fluorophores used in the imaging and the major response mechanism of fluorescent sensors were introduced.Considering the advantage of the enhanced permeability and retention (EPR) effect in bioimaging, fluorescent nanoparticles have attracted considerable attention. Cyanine is one of the most commonly used near-infrared (NIR) dyes, whose practical application is critically hindered by the poor stability. In chapter2, a system of cyanine-encapsulated nanoparticles CyN-12@NHs was designed and synthesized, in which cyanine dye molecules were encapsulated by the nanomicelles via the self-assembly of the amphiphilic block copolymer PS-b-PAA, then subsequently cross-linked by MPTMS to present core-shell type nanoparticles with diameter of35nm. The isolation of the dye molecules from environmental factors endows the encapsulated cyanine dye several advantages, such as high hydrophilicity, extremely strong fluorescence in water with large Stokes shift (110nm),50-fold and95-fold higher photostability than the free dye and ICG， respectively. Moreover, the NIR cyanine-based nanoparticles exhibit nearly no cytotoxicity to cells and outstanding performance in living cell imaging. Finally, in the in vivo imaging with tumor-bearing mouse model, the nanoparticles has a long retention in tumor via intratumor injection because of the EPR effect. Therefore, the NIR silica-cyanine hybrid nanocomposites have been successfully developed as highly qualified contrast agent for bioimaging, along with a breakthrough in photostability and bright fluorescence with large Stokes shift.Glutathione (GSH) plays a critical role in maintaining oxidation-reduction homeostasis in biological systems. Considering the detection of GSH by fluorescence sensors is limited to either the short wavelength emission or the poor photostability, a highly stable colorimetric and ratiometric NIR fluorescent sensor (DCM-S) for GSH detection was constructed on basis of dicyanomethylene-4H-pyran (DCM) chromophore. The specific disulfide bond is incorporated via a carbamate linker as the GSH responsive group, which simultaneously blue-shifts and quenches the fluorescence. Upon addition of GSH, DCM-S exhibits the outstanding colorimetric (from yellow to red) and ratiometric fluorescent response with the6-fold enhancement of NIR fluorescence at665nm in quantum yield. More importantly, the GSH-treated DCM-S (DCM-NH2actually) possesses20-fold longer fluorescence half-life period as well as much better photostability than the FDA-approved ICG. Finally, the ratiometric detection of GSH was also successfully operated in the living cell imaging, exhibiting NIR fluorescence and large Stokes shift (215nm) with nearly no background fluorescence interference. As a consequence, DCM-S can be utilized as colorimetric and ratiometric NIR fluorescent sensor for GSH, with a great potential in the development of GSH-induced drug delivery system.Theranostic prodrugs equipped with fluorophores has become attractive to monitor the biodistribution and activation of prodrugs. To overcome the disadvantages of exsiting prodrugs such as the short wavelength fluorescence and poor photostability of fluorophores, in Chapter4, an anticancer drug CPT is further conjugated with DCM-S to construct novel NIR DCM-based theranostic prodrug platform DCM-S-CPT (including the PEG-PLA nanoparticles) for cancer treatment in vivo with high photostability. Upon the interaction of GSH, NIR fluorescence centered at665nm is distinctly enhanced along with the GSH-induced release of active CPT. Importantly, in vitro evaluation illustrates that the GSH-treated DCM-S-CPT have significantly better photostability (19-fold enhancement in the fluorescence half-life period) than the commercial ICQ which is highly desirable for in situ fluorescence-tracking of cancer chemotherapy. For the first time, DCM-S-CPT has been successfully utilized for in vivo and in situ tracking of drug release and cancer therapeutic efficacy in living animals by NIR fluorescence. We systematically studied the cytotoxicity, flow cytometry, inhibition rates of tumor growth (IRT) and pharmacokinetic features of the prodrug DCM-S-CPT. In bioimaging in vivo, DCM-S-CPT exhibits fantastic tumor-activatable performance when intravenously injected into tumor-bearing nude mice, as well as specific cancer therapy with little side effects. DCM-S-CPT loaded in PEG-PLA nanoparticles shows even higher antitumor activity than free CPT, and is also retained longer in the plasma. The tumor-targeting ability and the specific drug release in tumors make DCM-S-CPT a promising prodrug to achieve high efficacy and low side-effects, providing significant advances towards deeper understanding and exploration of theranostic drug-delivery systems.Copper is a crucial nutrient for life and its homeostasis connected to severe diseases such as Menkes and Alzheimer’s disease. In Chapter5, the donor-acceptor system of indoline-benzothiadiazole is established as the novel and reactive platform for generating amine radical caitons with the interaction of Cu2+, which has been successfully exploited as the building block to be highly sensitive and selective NIR colorimetric and fluorescent Cu2+sensors. Upon addition of copper ion, an instantaneous red shift of absorption spectra as well as the quenched NIR fluorescence of the substrates is observed, which establishs the two-channel response of IBT and IBTM for Cu2+. The feasibility and validity of the radical cation generation are confirmed by cyclic voltammetry and electron paramagnetic resonance spectra. Moreover, the structure modification (methyl in IBTM to aldehyde unit in IBT) endows IBT+’around40-fold longer half-life period than IBTM+’via the extention of the electron spin density and change of the charge distribution. Therefore, IBT has been proved to be a highly qualified NIR colorimetric and fluorescent sensor for Cu2+with high sensitivity and selectivity, which benefits for the further design of novel radical cation based fluorescent sensors for copper ion.Considering the relatively poor stability of radical cations generated during the detection of Cu2+, in Chapter6, four diindoline-acceptor based molecules have been synthesized for copper ion detection via the same detection mechanism with much higher stability of radical cations. The four newly developed sensors exhibit similar two-channel response in absorption spectra (red shift) and fluorescence spectra (quench of fluorescence). The selectivity is also guaranteed by the specific detection mechanism. Outstandingly, because of the introduction of two indoline units, the stability of the radical cations is significantly improved even compared with IBT+.Therefore, the diindoline-acceptor type structure has the potential for constructing NIR colorimetric and fluorescent sensor for copper ion with high sensitivity, selectivity and stability.In Chapter7, two fluorescence sensors for Cu2+were designed and synthesized based on benzothiadiazole and Schiff base unit. The fluorescence of BTS and BTDS in the visible region is significantly enhanced upon the addition of Cu2+with outstanding sensitivity and selectivity, demonstrating the potential application as OFF-ON fluorescent sensors for Cu2+. Moreover, the ability of commercially available4-ClNBD as colorimetric sensor for H2S and OFF-ON fluorescent sensor for N3-simultaneously was investigated.