| Green fluorescent protein(GFP)is a class of natural proteins derived from the bioluminescent jellyfish Aequorea victoria and has many advantages,such as large fluorescence quantum yield,large Stokes shift,good biocompatibility and good light stability.Therefore,it is often used as a biological genetic marker.However,the emission wavelengths of most fluorescent protein chromophore analogs are in the visible region and rarely reach to the nearinfrared region.Photodynamic therapy(PDT)is a photochemical-based treatment modality commonly used to treatneoplastic diseases.Traditional photosensitizers such as porphyrins,Bodipy derivatives,naphthalimide derivatives,and cyanines have some inherent defects,such as small Stokes shift,poor photostability,low signal-to-noise ratio and so on.Besides that,fluorescent proteins chromophore analogs photosensitizers are rarely reported.Thus,the construction of near-infrared(NIR)fluorescence imaging fluorescent protein chromophore analogs is important.In this paper,based on imidazolidinone,three fluorescent protein chromophore analogs were constructed and applied to PDT.A phenothiazine fluorescent protein was synthesized by [2+3] cycloaddition reaction using N-butylphenothiazine-3-carbaldehyde,tert-butyl glycine hydrochloride and imide ester as raw materials.Ptz-FP was a heavy-atom free photosensitizer that promoted by S atom with excellent singlet oxygen generation which can be used for PDT.Due to the introduction of Nbutylphenothiazine-3-carbaldehyde containing a sulfur atom,the emission wavelength of the photosensitizer Ptz-FP was located at 626 nm,the Stokes shift was as high as 190 nm,and the singlet oxygen yield was 33.1 %.The photosensitizer has low cell dark toxicity(≥89.9 %)and high phototoxicity measured by MTT method,and can kill more than 50 % of A-549 cells within 30 min.In addition,the production of reactive oxygen species in A-549 cells was successfully detected within 15 min by reactive oxygen species detection kit DCFH-DA,and the apoptosis process of A-549 cells was monitored by AO/EB live dead cell stain.Finally,the photosensitizer Ptz-FP was successfully applied to two-photon fluorescence(TPF)imaging.Therefore,the photosensitizer Ptz-FP is expected to lay the foundation for the future development of fluorescent protein chromophore-based photosensitizers guided by fluorescence imaging.Two NIR fluorescent protein chromophore photosensitive dyes RFP-OH and RFP-Ac based on red fluorescent protein chromophore analogue was synthesized for PDT in A-549 cells and zebrafish.Compared with Ptz-FP,the photosensitive dyes RFP-OH and RFP-Ac have improved in terms of maximum absorption and emission wavelengths,Stokes shifts,and singlet oxygen yields.Firstly,the maximum emission wavelengths of RFP-OH and RFP-Ac were reached to the NIR band,which were 721 nm and 758 nm,respectively.Secondly,the stokes shift of RFP-OH and RFP-Ac was as large as 228 nm and 260 nm,respectively.Thirdly,the singlet oxygen yield of RFP-OH and RFP-Ac was 35.6 % and 28.8 %,respectively.Tumor cells are rich in biological thiols,such as Cys.After RFP-Ac encountered with Cys,the singlet oxygen yield of the products increased to 34.1 %.In addition,it can be seen from DFT calculations that RFP-OH can transition to the triplet state through intersystem crossing after being excited to the singlet state,thereby enhancing the singlet oxygen yield.Photosensitizer RFP-OH exhibited superior two-photon fluorescence imaging properties in zebrafish.Besides that,both photosensitizers RFP-OH and RFP-Ac showed low dark toxicity(≥91.02 %)and high photo toxicity.Photosensitizers RFP-OH and RFP-Ac exhibited good PDT effects in A-549 cells and zebrafish,and both produced reactive oxygen species under light irradiation.Futhermore,photosensitizer RFP-Ac could inhibit the migration of A-549 cells under light irradiation.In a word,considering to the positive results of the experiments,RFP-OH and RFPAc had a great potential for PDT in anti-cancer therapy. |
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