Rational Design of a Multifunctional Molecular Dye with Single Dose

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Rational Design of a Multifunctional Molecular Dye with Single Dose and Laser for Efficiency NIR-II Fluorescence/ Photoacoustic Imaging Guided Photothermal Therapy Ruiping Zhang, Zhenjun Wang, Liying Xu, Yuling Xu, Yi Lin, Ying Zhang, Yao Sun, and Guang-Fu Yang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.9b03152 • Publication Date (Web): 02 Sep 2019 Downloaded from pubs.acs.org on September 2, 2019

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Analytical Chemistry

Rational Design of a Multifunctional Molecular Dye with Single Dose and Laser for Efficiency NIR-II Fluorescence/Photoacoustic Imaging Guided Photothermal Therapy Ruiping Zhang†+, Zhenjun Wang†‡+, Liying Xu#+, Yuling Xu‡+, Yi Lin&, Ying Zhang‡, Yao Sun*‡ and Guangfu Yang‡ †Shanxi

Da Yi Hospital, Shanxi Medical University, Taiyuan 030001, China. laboratory of Pesticides and Chemical Biology, Ministry of Education, International Joint Research Center for Intelligent Biosensor Technology and Health, Center of Chemical Biology, College of Chemistry, Central China Normal University, Wuhan 430079, China. # Department of Radiology, Zhongnan hospital of Wuhan University, Wuhan 430071, China ‡Key

Key Laboratory of Analytical Chemistry for Biology and Medicine, Ministry of Education, Wuhan University, Wuhan 430074, China &

+

These authors contributed equally to this work.

KEYWORDS: NIR-II imaging, Molecular Dye, Photoacoustic, Multimodal Imaging, Photothermal Therapy ABSTRACT: Multifunctional probes integrating accurate multi-diagnosis and efficient therapy hold great prospect in biomedical research. However, the sophisticated construction and difficulties in matching the ratios of doses and laser triggers of probes for each modal imaging and therapy are still hindered the extensive practice of multifunctional probes in biomedicine. We herein rationally designed an organic dye SY1080 with intrinsic multifunction by both introducing 3,4-ethylenedioxy thiophene (EDOT) and the selenium containing acceptor unit into the backbone to balance the fluorescence brightness and emission wavelength. Under single dose and 808 nm laser irradiation conditions, SY1080 not only carried out NIR-II fluorescence/photoacoustic imaging of real-time and noninvasive tumor delineation with excellent contrast, but also effectively ablated tumors with laser irradiation to perform photothermal therapy under the guidance of dual-modal imaging. These exciting results highlighted SY1080 as a multifunctional and universal phototheranostic platform for potential applications.

Within the last decade, optical imaging in the second nearinfrared channel (NIR-II, 1.0-1.7 µm) has arisen much attention of multidisciplinary researchers owing to its remarkable merits such as diminished auto-fluorescence and reduced tissue scattering relative to traditional visible and NIR-I channels (400-950 nm).1-15 Considering the translational ability of NIRII modality into clinical research, the development of smallmolecule NIR-II emitters with high biocompatibility, optimal pharmacokinetics, and well-defined architectures were highly demanded.16-26 More recently, a series of small-molecule emitters with donor-acceptor-donor (D-A-D) backbone have been rationally designed, which facilitates the electronic delocalization and minimizes the energy gap between the hybridized highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO), leading to red shift with emission in NIR-II channel.27-36 Exploiting these dyes’ advantage of brilliant photo-stability and fluorescence features, NIR-II fluorescence imaging has already achieved noninvasive diverse biomedical applications including visualizing tumor angiogenesis and lymphatic metastasis during

tumor growth as well as providing surgical guidance with excellent contrast and spatiotemporal resolution.37-42 Despite these significant progress in NIR-II channel, the biological utilization potential with these promising D-A-D NIR-II dyes for multimodal imaging as well as phototheranostics are rarely explored. Multifunctional fluorescence probes have evolved as a fastgrowing research tools with goals of multidimensional visualization of early events in cancer diagnostics and therapy.43-46 However, the extensive utilization of multifunctional probes on biomedicine are still heavily hampered by several barriers. For example, integration of multiple functional units into one molecular platform usually involves abundant and complex chemistry.47-50 Moreover, matching well the dose and laser trigger of probes for fluorescence and other imaging modality or imaging guided therapy remains a challenge for biomedicine.31,36 Hence, continuous efforts should be employed for exploiting multifunctional fluorescence platform with simplified preparation procedures, with controlled ratios of multiple dose

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and laser reducing the interferences between each modality and therapy,

Figure 1. a) Chemical structures, NIR-II fluorescence signals and electronic properties of SY1080 and previous reported NIR-II fluorophore H1. The organic fluorophores were calculated using Gaussian09 software. b) A schematic illustration to show that prepared SY1080 based probes displays its multifunction for PA/NIR-II/PTT theranostic on tumor-bearing mice with laser irradiation.

which could speed up the development of biomedicine. Herein, we rationally designed a novel D-A-D molecular dye SY1080 with intrinsic multifunction (Figure 1), and it would exhibit the following merits. First, SY1080 can achieve better fluorescent performance than several existing NIR-II fluorophores with the introduction of 3,4-ethylenedioxy thiophene (EDOT) as bridging unit into the D-A-D backbone, which could distort the conjugation of backbone as well as prevent intermolecular and intramolecular interactions.34 Introducing EDOT yet caused hypochromatic shift in the emission,51 therefore, a heavier selenium was used instead of sulfur atom in the acceptor unit to realize red-shift in the emission wavelength. More importantly, the D-A-D architecture endowed SY1080 with strong fluorescence absorption around 800 nm. This result suggested that under 808 nm laser, the optical energy absorbed by SY1080 could be partly converted into heat to trigger the cancer cells death by highly effective and noninvasive photothermal therapy (PTT).52,53 In addition to ablate tumor cells, the photothermal effect of SY1080 could also generate acoustic waves and converted into photoacoustic (PA) signals.54 As hybrid imaging modes, the NIR-II/PA imaging meet the requirement of both superior contrast and high penetration depth for delineating tumor margin, as well as determine the best photothermal treatment time after administration to realize optimal photothermal effect in cancer therapy. Based on the designed

SY1080 with single dose and NIR laser trigger, PA/NIR-II tumor imaging and precisely PTT have been well performed with excellent results, which could speed up the utilization of D-A-D based NIR-II molecular dyes for multifunctional biomedical applications in the near future.

EXPERIMENTAL SECTION General procedure for the synthesis of SY1080 NPs. SY1080 was facilely prepared in 16% yield (Supported information). It was well confirmed by NMR and MS (Figure S1-S3). Further, the SY1080 NPs was prepared via matrix-encapsulation method. To a solution of DSPE-PEG5000 (4.5 mg in dd water) added SY1080 (0.5 mg in THF) under ultrasonic for 1 min. Then, THF was dried over by N2 and centrifuged to obtain SY1080 NPs with 50 kDa centrifugal filter. Absorption/emission spectrum and in vitro photo-stability measurement. NIR absorbance of the SY1080 was recorded on NIR spectrophotometer. Emission spectrum was taken using Applied NanoFluorescence spectrometer. The fluorescence quantum yield (QY) measurements were using IR-26 in 1,2dichloroethane (DCE) (QY=0.5%) as reference. In a standard manner, a series of solutions of IR-26 in DCE and SY1080 in THF was tested and the integrated fluorescence was plotted against absorbance at 785 nm for IR-26 and SY1080.


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Analytical Chemistry Equivalent SY1080 NPs and indocyanine green (ICG) aqueous solution were continuously irradiated for 1h (808 nm, 0.33 W/cm2). NIR-II ﬂuorescent images (1000 LP, 10 ms) were collected at every 15 min interval on a NIR-II imaging system. Then the ﬂuorescence intensity at various time points was analyzed by ImageJ. In vitro photothermal imaging and photothermal stability test. SY1080 NPs aqueous solution was illuminated under laser (808 nm, 5 min and 1.0 W/cm2). The solution temperatures were calendared at 10 s interval using camera (Fluke, Ti400, USA). In order to further explore photothermal stability of SY1080 NPs, SY1080 NPs and ICG solution were illuminated under the aboved conditions and naturally cooled down. The camera calendared the temperature of 6 heating-cooling cycles. Then the photothermal conversion efficiency (η) of SY1080 NPs and ICG was calculated. In vitro Photoacoustic effect. All PA signal was calendared by multispectral optacoustic tomographic (MSOT) imaging system. To measure PA signal of SY1080 NPs, phantoms which filled with the different concentration (12.5-200 μg/mL) of SY1080 NPs were suspended into a water tank and imaged at pulsed laser ranging from 680 to 980 nm. PA intensities were measured in regions of interests (ROIs) for each sample using MSOT imaging system software and the correlation curve between PA intensity and concentration was plotted. Cell and animal model. 4T1 cells were chosen and 1 × 106 of 4T1 cells (in 100 μL PBS) were subcutaneously implanted into the nude mice. Until tumor volume reached ~80 mm3, the tumor models are using for imaging/therapy studies. All animal experiments were performed in agreement to the guidelines of the Institutional Animal Care and Use Committee.

In vivo PTT and Histological analysis. We randomly divided 4T1 models (n = 5 each group) into four groups: (a) SY1080 NPs (100 µg) injection with laser irradiation group, (b) SY1080 NPs (100 µg) injection group, (c) PBS (100 µL) injection with laser irradiation group, and (d) PBS (100 µL) injection group. For (a) and (c) groups, at 12 h post-injection, 808 nm/1.0 W/cm2 NIR laser irradiated the tumor for 5 min to carry out PTT. In order to investigate the therapeutic ability of SY1080 NPs, the tumor volume and body weight after various treatment were documented every other day until 14 days for all groups. To further evaluate the effect of in vivo PTT, one nude mouse for each group was sacrificed randomly and then tumor tissues and major organs were excised for histological study by hematoxylin-eosin (H&E) staining at 12 h after various treatment. In vivo PTT on orthotopic breast tumor models. All orthotopic breast tumor models were bought from Servicebio. As dealing with subcutaneous tumor models, the orthotopic breast tumor models were first injected with SY1080 NPs (100 μL, 1 mg/mL) to determine the best PTT time through NIR-II fluorescence images, then randomly divided into the same four groups (n = 3 each group) : (a) SY1080 NPs (100 µg) injection with laser irradiation group, (b) SY1080 NPs (100 µg) injection group, (c) PBS (100 µL) injection with laser irradiation group, and (d) PBS (100 µL) injection group. The tumor volume and body weight were also documented every other day until 14 days for all groups to evaluate the effect of PTT on orthotopic breast tumor models.

RESULTS AND DISCUSSION

In Vitro Cytotoxicity and Photothermal Study. SY1080 NPs were tested through cell counting kit-8 (CCK-8) assay. 1 × 104 of 4T1 cells/per well was incubated for 12 h and added with SY1080 NPs with 0-40 μg/mL for 2 h. Then cells were handled in the presence and absence laser for 5 min. After continued incubation for another 12 h, the supernatant was replaced by PBS (90 μL) and CCK-8 (10 μL) followed by incubation for 4 h, which were analyzed by a microplate reader under 450 nm. To further evaluate photothermal effect for 4T1 cell lines of SY1080 NPs, live/dead cells (green/red) were also distinguished by Calcein-AM/PI double staining assay. NIR-II Bioimaging of lymphatic/tumor vascular. For lymphatic system bioimaging, SY1080 NPs PBS solution (50 μL, 1 mg/mL) was injected into the hindfoot pad of Balb/c mice (n = 3) intradermally. The lymphatic system was recorded after injection. Further, SY1080 NPs (100 µL, 1 mg/mL) was intravenously injected into 4T1 tumor models. Fluorescence images were obtained under 808 nm laser (0.1 W/cm2, 1000 LP, 50 ms). Imaging analysis was performed using Image J. NIR-II fluorescence/PA dual-modal in vivo imaging. For visualizing the tumor site and determining the best photothermal treatment time, 4T1 models (n=5) were intravenously injected with one dose of SY1080 NPs (100 μL, 1 mg/mL). NIR-II fluorescence/PA images were acquired at 0, 2, 6, 12, 24 h. The analysis of imaging data was carried out by Image J and MSOT imaging system software, respectively.

Figure 2. In vitro size, optical/PA features of SY1080 NPs. a) TEM images. b) The average diameters of SY1080 NPs in PBS (0.5 mg/mL) and FBS after various incubation times were determined by DLS. c) NIR absorption/fluorescence. d) Photostability of SY1080 NPs and ICG (808 nm, 1 W/cm2, 1h). e) PA spectra of SY1080 NPs (100 µg/mL). f) PA intensity of SY1080 NPs with 10 to 200 µg/mL concentrations.

A novel NIR-II small-molecule dye, SY1080 was designed by using an optimized D-A-D scaffold including 3,4-



ethylenedioxy thiophene (EDOT) as bridging unit and acceptor unit with selenium, which contributed to enhanced fluorescence intensity along with the red-shift in emission. First, through introducing EDOT as bridging unit into the D-A-D backbone, the fluorescence brightness was distinctly enhanced owing to EDOT distorted the backbone as well as prevents intermolecular and intramolecular interactions. Nonetheless, hypochromatic shift in the absorption and emission spectra was observed after introducing EDOT. Considering the NH2 group in the donor group of D-A-D scaffold could both increase the energy of HOMO and LUMO, and slightly affected the absorption/emission wavelength of dyes 55, we take no account of the NH2 group and focus on the change of the acceptor core to prolong the absorption or emission wavelength. 56 Therefore, heavier selenium (Se) was substituted in place of sulfur (S) atom in the acceptor core was utilized to extend the emission wavelength. SY1080 was synthesized with 16% total yield (Supported information) and well confirmed by NMR and MS (Figure S1-S3). Density functional therapy (DFT) calculations analyzed the electronic description of the fluorophores SY1080 and H1 on the Gaussian09 software using B3LYP method. As shown in Figure 1a, through the obtained (LUMO/HOMO), SY1080 (1.34 eV) exhibited a larger energy band gap compared with H1 (1.18 eV) 30, which causing brightness of fluorescence signals. Moreover, brightness NIR-II fluorescence signal of SY1080 higher than H1 in THF should be attributed to the introduction of EDOT unit protecting the conjugated backbone from intermolecular and intramolecular interactions (Figure 1a). The quantum yield (QY) of SY1080 in THF was determined to be 1.5%, using IR-26 in DCE (QY = 0.5%) as a reference (Figure S4). Furthermore, hydrophobic SY1080 was enveloped into DSPE-PEG5000, a PEGylated surfactant, for facile preparation of water soluble and biocompatible SY1080 NPs for in vivo applications. The prepared SY1080 NPs exhibited an average size of ~120 nm characterized by transmission electron microscopy (TEM) (Figure 2a), which was in agreement with the results of dynamic light scattering (DLS) (Figure S5). After 48 h incubation, no obvious change in diameter was detected for SY1080 NPs in PBS or FBS (Figure 2b), indicating good media stability of SY1080 NPs. SY1080 displayed the major absorption peak at 820 nm and emission peak at 1080 nm, resulting in a large Stoke shift (~260 nm, Figure 2c). Subsequently, the photostability of SY1080 NPs was also investigated and it demonstrated the desirable photostability when exposed at continuous 808 nm irradiation for 60 min relative to Indocyanine Green (ICG) (Figure 2d). Here, we also utilized DSPE-PEG5000 to envelop ICG to serve as the control group (in the below article, ICG means ICG NPs). Besides, SY1080 NPs in different media (FBS and PBS) exhibited negligible photobleaching (808 nm/60 min, Figure S6). For long-term storage, the fluorescence intensity of SY1080 NPs in PBS and FBS displayed no decay over 7 days, confirming long-term stability (Figure S7). Further, PA spectrum of SY1080 NPs showed strong PA signals extending from 800 to 950 nm in the NIR region (Figure 2e). Additionally, the PA intensity of SY1080 NPs had a good linear correlation with its concentration within a range from 10 to 200 µg/mL through in vitro phantom tests (Figure 2f), which suggesting excellent feasibility of PA signal quantification. Finally, the amount of SY1080 encapsulated in the SY1080 NPs was also measured using an NIR spectrophotometer analysis. The SY1080 encapsulation efficiency of SY1080 NPs was about 81.6 ± 2.4 % (Figure S8).

Then, we systematically investigated the in vitro photothermal features of SY1080 NPs. The photothermal effect of SY1080 NPs aqueous solution documented by camera showed concentration/power density-dependence. After illumination for 5 min, the temperature of SY1080 NPs rose rapidly along with the increased concentration (0 to 50 µg/mL) (Figure 3a, Figure S9). As seen in Figure 3a, the maximum photothermal temperature of SY1080 NPs was up to 52.7 ℃ (25 µg/mL). Meanwhile, increasing of laser power density (0.5-2.0 W/cm2), the maximum photothermal temperature of SY1080 NPs was also enhanced (Figure S10). We also discovered that after six cycles of heating-cooling treatment lasting for the period of 60 min, the maximum photothermal temperature of ICG of each cycle significantly declined, while that of SY1080 NPs didn’t show any obvious change. This result indicated SY1080 NPs exhibited distinctly better photothermal stability than easily photobleached ICG (Figure 3b). Then, the photothermal conversion efficiency (η) of SY1080 NPs was calculated to be 22.3 % (Figure S11), being higher than ICG according to our experiments (Figure S12) and previous reports.57 Considering the excellent photothermal performance of SY1080 NPs under in vitro conditions, the cytotoxicity and photothermal ablation ability of SY1080 NPs were evaluated by CCK-8 assay and Calcein-AM/PI double staining assay on 4T1 breast cancer cells (Figure 3c and 3d). Cell viabilities were negligible affected under laser free condition, with the addition of SY1080 NPs at different concentrations, even at higher concentration of 40 µg/mL cell viabilities were not considerable affected (Figure 3c). This result suggests the high biocompatibility of SY1080 NPs. Only upon incubation in SY1080 NPs and then irradiation for 5 min, it was clearly observed that 4T1 cells were significantly ablated. Especially SY1080 NPs was over 40 µg/mL, the viabilities of 4T1 cells was only ~3 % under laser irradiation, indicating SY1080 NPs possessed promising photothermal ablation ability. These exciting results from cell experiments further confirmed SY1080 NPs was a desirable PTT agent thanks to its high photothermal conversion efficacy (η).

Figure 3. Photothermal effect of SY1080 NPs in the solution. a) Photothermal features of SY1080 NPs at various concentrations (808 nm, 1 W/cm2). b) The cycles of photothermal heating/cooling for SY1080 NPs and ICG. c) Cell viabilities under the incubation of SY1080 NPs in the presence or absence 808 nm laser. d) Fluorescence images of live/dead 4T1 cells (green/red) with Calcein-AM and PI staining after various treatments.


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Analytical Chemistry NIR-II imaging of blood vessels of the tumor site and lymphatic system were carried out in subcutaneous 4T1 models (Figure S13). The excellent images were observed clear blood vessels as well as lymph vessel from the surrounding background tissues with high SBR. The PA/NIR-II imaging ability of SY1080 NPs was further studied in xenograft 4T1 models. After intravenously injection with single-dose of SY1080 NPs in PBS (100 µg), NIR-II fluorescence/PA images of living mice were continually documented under 808 nm laser. Both signals on the tumor region from the NIR-II/PA images in SY1080 NPs treated group were gradually highlighted from the surrounding background tissue with the increasing time, reaching maximum at 12 h post-injection (Figure 4a). The imaging data indicated that SY1080 NPs could passively target and accumulate in the tumor region efficiently. Quantified analysis of NIR-II fluorescence and PA images further verified that the accumulation of probe in tumor regions increased within 24 h, obtaining max peak at 12 h (Figure 4b). As shown in Figure 4b, we also evaluated SBR of dual-modal tumor images. The SBR of NIR-II fluorescence/PA images reached a maximum value of ～7.5/7.1 respectively (12 h postinjection). Ex vivo biodistribution studies were conducted to validate the results from in vivo imaging (24 h post-injection). The NIR-II Ex-vivo imaging of tumors/major organs indicated that the tumor and liver exhibited the strongest fluorescence signals, corresponding with in vivo data (Figure S14). SY1080 NPs was mainly accumulated through liver, suggesting that SY1080 NPs chiefly excreted through the hepatobiliary system. All imaging data demonstrated that SY1080 NPs could passively target and accumulate in the tumor sites. More important, both single dose and laser trigger strategy for dualmodal imaging based on the SY1080 could efficiency reduce the interferences between NIR-II and PA imaging modalities and side effects from the traditional multiple doses and different laser triggers. Therefore, both NIR-II and PAI modalities can be synergistically combined together using single dose and laser of SY1080 to provide complementary information on 4T1 tumors and have ability to precisely guided the further photothermal therapy.

Figure 4. In vivo dual-modal imaging with SY1080 NPs. a) NIRII fluorescence/PA images of 4T1 models at 0, 2, 6, 12, 24 h. b) The signal to background ratio and semi-quantified fluorescence/PA intensities over time. Inspired by the impressive results of dual-modal imaging, we further explored the image-guided photothermal therapeutic function of SY1080 NPs. According to the time-point of maximum signals of the in vivo dual-modal imaging in tumor site, PTT in living mice bearing xenograft 4T1 breast tumors was carried out (12 h post-injection). The tumor region in living mice was irradiated with or without 808 nm laser (1 W/cm2/5 min) after intravenously administration of SY1080 NPs (100 µL, 1 mg/mL, the same dose as dual-modal imaging) or PBS (100 µL) for 12 h. The camera recorded temperature change of laser-treated groups (Figure 5a). The temperature of the tumor surface (SY1080 NPs and laser irradiation group) rose rapidly from 31 ℃ to 50 ℃ (Figure 5b). As for the control group (injection PBS and laser irradiation), however, the tumor temperature only had a slight increase (Figure 5b) which was insufficient to induced tumor cells death. To further investigate the antitumor effect of SY1080 NPs under laser irradiation in vivo, the body weight and tumor growth for all groups of nude mice were checked every other day till 14 d. No obvious weight loss was observed in SY1080 NPs and laser irradiation treatment group (Figure 5c and 5d), suggesting high biocompatibility of PTT with SY1080 NPs injection treatment. Meanwhile, the tumor growth was significantly inhibited in SY1080 NPs and laser irradiation treatment group compared with other control groups, which suggested efficient in vivo photothermal therapeutic function of SY1080 NPs (Figure 5e). Besides, survival rate was also documented till mice death (Figure 5f). As for SY1080 NPs based PTT group, the survival rate remained to be 100% after 60 d, which was higher than other control groups. Furthermore, H&E staining was carried out on tumor/normal tissues at 12 h after various treatment to verify the PTT effect of SY1080 NPs. Negligible histopathological abnormalities were observed in major organs (liver, lung, heart, spleen and kidneys) of all groups (Figure S15). Tumor cells damage with cell shrinkage and nuclear condensation were only found in



SY1080 NPs and laser irradiation combination treatment, while were not obvious in other control groups (Figure S16).

Figure 5. In vivo PTT effects on 4T1 models using SY1080 NPs. a) IR thermal images. b) Temperature changes on the subcutaneous tumor regions in Figure 5a treated of SY1080 NPs/PBS. c) Figures of SY1080 NPs/laser irradiation treated tumor model for 0, 2, 6, 14 days. Black dotted circles indicate tumor sites. d) Average body weights were analyzed. e) Tumor growth inhibition curves. f) Survival curves after different treatments.

Finally, to further demonstrate the universal ability of SY1080 NPs for PTT, orthotopic breast tumor models were established. As shown in Figure S17, the highest fluorescent intensity of tumor site in orthotopic 4T1 tumor mice treated with SY1080 NPs was monitored at 12 h, thus subsequent PTT was performed at 12 h after injection of SY1080 NPs. Same as the therapeutic experiments on xenograft models, orthotopic breast tumor mice treated with PBS, PBS under laser irradiation and SY1080 NPs were set as control groups. The temperature of models treated with SY1080 NPs under 808 laser irradiation increased apparently compared with mice treated with PBS under 808 laser irradiation during the period of PTT (Figure 6ab). To exclude the systematic toxicity of PTT with SY1080 NPs, body weights were documented every 2 d after various treatments. All groups showed an increasing body weight, indicating a decent biocompatibility and non-obvious systematic toxicity of PTT with SY1080 NPs (Figure 6c-d). The efficiency of PTT was reflected by the tumor size which was recorded every 2 d after various treatments. As demonstrated in Figure 6e, the inhibition efficiency for PTT treated-mice is more obvious than that of other groups. Based on these results, the utilization of SY1080-based multifunctional probe with single dose and laser trigger for image-guided photothermal therapy on cancer will overcome the side effects from the multiple dose of traditional theranostic agents, which will greatly implicate the applications of multifunctional probes in preclinical and clinical research.

Figure 6. In vivo PTT on orthotopic 4T1 tumor models by using SY1080 NPs. a) IR thermal images of mice injected of SY1080 NPs and PBS under continuous laser irradiation. b) Temperature changes for the orthotopic tumor sites treated with SY1080 NPs and PBS under continuous laser irradiation. c) Figures of orthotopic 4T1 models after different treatments at 0, 4, 8, 14 days. Black dotted circles indicate tumor sites. d) Body weights of orthotopic 4T1 models for various treatments. e) Tumor growth inhibition curves.

CONCLUSIONS In short, we have demonstrated a rational designed dye SY1080 by introducing Se substituted acceptor core into the backbone and EDOT as bridging unit. SY1080 has been identified as a multifunction theranostic platform and SY1080 NPs has been facilely prepared with high biocompatibility, stability and tumor targeting capability and achieved PA/NIRII imaging guided PTT with a single-dose. Based on advantage of both NIR-II fluorescence/PA imaging, we realized real-time, non-invasive tumor delineation with high resolution at deep tissue as well as determined the best PTT time after administration of SY1080 NPs. Moreover, with the help of multimodal imaging, the superior PTT function of SY1080 NPs was performed in efficient inhibition on tumor growth with laser irradiation owing to its good photothermal conversion efficacy. These excellent results highlighted SY1080 as a multifunctional platform for cancer theranostic in the future. According to previous reports and our experiments, tumor recurrence was an urgent problem after PTT in vivo. Even precise imaging guide can noticeably boost PTT efficiency, PTT is still limited by incomplete ablation of certain tumor area, that attribute to the restricted penetration depth of laser illumination and the overexpression of heat shock proteins. Therefore, the construction of photothermal transduction agents of long emission wavelength is essential to achieve killing tumors deep inside skin by the use of longer wavelength laser irradiation. In addition, taking advantage of chemotherapy/photodynamic therapy/gene therapy to remedy the deficiency of PTT, the result combination therapy could optimize anti-tumor effects. 58-60


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Analytical Chemistry

ASSOCIATED CONTENT Supporting Information 1HNMR, 13CNMR, MALDI-TOF-MS, Excretion routes, tumor imaging and therapy, Ex-vivo biodistribution, H&E analyses of tumor tissues and chemical synthesis.

AUTHOR INFORMATION Corresponding Author * [email protected]

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This work was supported by grants from the National Key Research and Development Program of China (2017YFA0505203), the National Natural Science Foundation of China (21708012), Wuhan Morning Light Plan of Youth Science and Technology (201705304010321), the financial support by self-determined research funds of CCNU from the colleges, basic research and operation of MOE (1100-30106190234). Finally, thanks to the Open Research Fund of Key laboratory of Analytic Chemistry for Biology and Medicine (ACBM2019002) from Wuhan University.

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Rational Design of a Multifunctional Molecular Dye with Single Dose

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