Preparation Of D-A-D-Based Organic NIR-Ⅱ Fluorescent Molecules For NIR-Ⅱ Fluorescence Imaging And Photothermal Therapy Of Tumors

Fluorescence imaging（FI）in the second near-infrared optical window（NIR-Ⅱ,1000-1700 nm）as a new field of molecular imaging has received increasing attention from researchers.NIR-Ⅱ FI exhibited deep penetration depth（2-7 mm）and high spatio-temporal resolution owing to reduced photon scattering,reduced tissue absorption and reduced tissue auto-fluorescence compared to NIR-I FI.However,NIR-Ⅱ fluorescent organic small molecules,including D-A-D structure dyes and some organic dyes based on cyanine,squarine,BODIPYs,and rhodamine,have been reported so far.To satisfy the requirements of NIR-Ⅱ FI,the design and development of new fluorescent molecules with simple structure and facile synthesis still remain challenging.In addition,Single-component nanoplatforms have been increasingly applied to imaging and photothermal therapy（PTT）of tumors.However,most of the reported fluorescent organic small molecules are difficult to achieve the effect of PTT.Besides,the highly efficient NIR-Ⅱ PTT,such as 1064 nm laser whose MPE is 1 W cm^-2,is rarely reported.Considering the two issues mentioned above,firstly,three D-A-D type NIR-Ⅱ molecules based on TTDT were designed and developed.Meanwhile,TTDT-TF NPs can achieve NIR-Ⅱ FI of vascular system and tumor with high imaging quality.What’s more,three double-acceptors conjugated polymers with bright NIR-Ⅱ fluorescence signal and superior photothermal effect were designed and developed by adopting the strategy of the two acceptors（DPP and TTQ）.Meanwhile,P1 NPs successfully achieve NIR-Ⅱ FI and NIR-Ⅱ PTT of tumor models in vivo.The specific contents of this paper are as follows:（1）The design and development of thienothiadiazole-based NIR-Ⅱ fluorescent organic small molecules for NIR-Ⅱ FI:Firstly,a novel acceptor TTDT with small molecular weight(423.46 g mol^-1,the maximum absorption peak at 536 nm)and simple synthesis was designed and developed.Subsequently,three electron donor units（TF,SF,and TSF）were introduced and combined with the acceptor TTDT to develop three TTDT-based NIR-Ⅱ fluorophores with D-A-D structure by stille coupling reaction.All of them exhibit NIR-I absorption（600-900 nm）and NIR-Ⅱ fluorescence emission（900-1200 nm）.Then,three small molecules were self-assembled with amphiphillic polymers(m PEG-DSPE₅₀₀₀)into their corresponding nanoparticles（TTDT-TF NPs,TTDT-SF NPs,and TTDT-TSF NPs）through nano-precipitation.According to the photophysical behaviors experiments,TTDT-TF NPs(absorption at 600-1000,emission at 900-1300 nm and molar extinction coefficient of 15.8 L g^-1 cm^-1)with strong NIR-Ⅱ fluorescence signals and superior photostability were selected for in vivo NIR-Ⅱ FI of vascular system and tumor.Dynamic light scattering（DLS）and transmission electron microscope（TEM）data suggested that the hydrodynamic diameter（D_h）is 120±2 nm and have uniform spherical morphology.In addition,at the same absorbance,NIR-Ⅱ fluorescence intensity of TTDT-TF NPs and TTQ-TF NPs is similar by the photophysical behaviors experiments.Finally,the results of NIR-Ⅱ FI of blood vessels and tumor in vivo proved that TTDT-TF NPs can achieve high-quality and high-signal-to-noise NIR-Ⅱ FI of blood vessels and tumor,and can achieve superior tumor enrichment at 36 h post injection,which was beneficial to find the best window in tumor resection or PTT.This work also further proves that the acceptor TTDT has superior potential application prospect in the field of NIR-Ⅱ FI.（2）Double-acceptor conjugated polymers for NIR-Ⅱ FI and NIR-Ⅱ PTT:This chapter adopted the strategy of introducing double acceptors to prolong absorption wavelength and TC as an electron donor to enhance solubility.Three double-acceptor conjugated polymers were synthesized by stille coupling reaction.All of them exhibited strong NIR absorption at the 1064 nm and fluorescence emission in the NIR-Ⅱ region（1200-1400 nm）.Subsequently,these polymers were used to fabricate nanoparticles（P1 NPs,P2 NPs,and P3 NPs）with m PEG-DSPE₅₀₀₀ as a carrier through nano-precipitation.Through studying their photophysical and photothermal behaviors,P1 NPs(absorption at 600-900,emission at 900-1300 nm,molar extinction coefficient of 26.7 L g^-1 cm^-1 and QY of0.1%)have strong NIR-Ⅱ fluorescence signals,excellent photostability,and superior photothermal properties(photothermal conversion efficiency（η）of 55.1%,1064 nm and 1 W cm^-2)and can act as a superior thermal agent.Thus,P1 NPs were selected for in vivo and in vitro NIR-Ⅱ FI and NIR-Ⅱ PTT.MTT cytotoxicity and confocal experiments exhibited that P1 NPs has good biocompatibility and low toxicity.After continuous irradiation of 1064 nm laser for 5 min,it is found that P1 NPs can kill 78.44%of 4T1 tumor cells and have obvious inhibitory effect for 4T1 Tumor cells.Subsequently,P1 NPs can achieve high-quality and high-spatio-temporal resolution NIR-Ⅱ FI of blood vessels and tumor under the 808 nm laser and exhibit obvious enrichment in the tumor site at 24 h post injection.Finally,the in vivo PTT experiments revealed that P1 NPs can significantly inhibit tumors under 1064nm laser and display superior NIR-Ⅱ PTT efficiency.Overall,it has provided a promising approach to design and develop photothermal agents applied for NIR-Ⅱ FI and NIR-Ⅱ PTT.