Polypeptide-Conjugated Second Near-Infrared Organic Fluorophore

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Polypeptide Conjugated Second Near-Infrared Organic Fluorophore for Image-Guided Photothermal Therapy Tuanwei Li, Chunyan Li, Zheng Ruan, Pengping Xu, Xiaohu Yang, Pan Yuan, Qiangbin Wang, and Lifeng Yan ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.9b00452 • Publication Date (Web): 21 Feb 2019 Downloaded from http://pubs.acs.org on February 21, 2019

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Polypeptide Conjugated Second Near-Infrared Organic Fluorophore for Image-Guided Photothermal Therapy

Tuanwei Li1§, Chunyan Li2§, Zheng Ruan1, Pengping Xu3, Xiaohu Yang2, Pan Yuan1, Qiangbin Wang2,4*, Lifeng Yan1*

1CAS

Key Laboratory of Soft Matter Chemistry, iChEM, and Department of

Chemical Physics, University of Science and Technology of China. Hefei, 230026, P.R.China. 2CAS

Key Laboratory of Nano-Bio Interface, Division of Nanobiomedicine and i-

Lab, CAS Center for Excellence in Brain Science, Suzhou Institute of Nano-Tech and Nano-Bionics, Chinese Academy of Sciences, Suzhou 215123 China 3Department

of Materials Science and Engineering, University of Science and Technology of China. Hefei, 230026, P.R.China.

4College

of Materials Sciences and Opto-Electronic Technology, University of Chinese Academy of Sciences, Beijing 100049, China

Corresponding

author:

Lifeng

Yan:

[email protected];

[email protected]

ACS Paragon Plus Environment

Qiangbin

Wang:

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Abstract Imaging-guided photothermal therapy (PTT) is an attractive strategy to improve the diagnosis accuracy and treatment outcomes by monitoring the accumulation of photothermal agents in tumors in real-time and determining the best treatment window. Taking advantage of the superior imaging quality of NIR II fluorescence imaging and remote-controllable phototherapy modality of PTT, we developed a facile macromolecular fluorophore (PF) by conjugating small molecule NIR-II fluorophore (Flav7) with amphiphilic polypeptide. The PF can form uniform micelles in aqueous solution, which exhibit slight negative charge. In vitro experimental results showed that the PF nanoparticles showed satisfactory photophysical properties, prominent photothermal conversion effciency (42.3%), excellent photothermal stability, negligible cytotoxicity and photothermal toxicity. Meanwhile, the PF can visualize and feature the tumors by NIR-II fluorescence imaging owing to prolonged blood circulation time and enhanced accumulation in tumors. Moreover, in vivo studies revealed that the PF nanoparticles achieved excellent photothermal ablation effect to tumors with low dose of NIR-II dye and light irradiation, and the process can be traced by NIR fluorescence imaging.

Keywords: second near-infrared (NIR-II) fluorescence, photothermal therapy (PTT), imaging-guided, polypeptide, nanomedicine.


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Fluorescence imaging can visualize the biological events and has received considerable attention in disease detection and fluorescence guided therapy for its noninvasion, high efficiency and low cost.1,2 Very recently, fluorescence imaging in the second near-infrared (NIR-II, 1000-1700 nm) window has attracted heightened attention for its superior imaging quality owing to reduced tissue absorption and scattering, and minimized intrinsic tissue autofluorescence.3,4 To date, the most active NIR-II fluorophores are limited to carbon nanotubes,5,6 rare earth materials7,8 and quantum dots (QDs).9-12 However, the deficiencies of unknown long-term toxicity, potential metal ion leakage and limited clearance from the body impede their translation towards clinical applications.13 Organic fluorophores with emission peaks covering the NIR-II window are the most potential candidates for their designability of the chemical and physical properties, minimal biotoxicity, in vivo biocompatibility and biodegradation.14 The intrinsic hydrophobicity of most organic fluorophores is a bottleneck that must be breached when it comes to biological applications.15-17 In spite of few water-soluble organic NIR-II molecules,15,18 the hydrophobicity organic fluorophores are commonly conjugated with or encapsulated by polymer to expand their application in biology.19,20 Organic fluorophores loaded by polymer carriers obtain decent biocompatibility, extended blood circulation time and responsiveness or targeting depending on the polymers, which endows organic NIR-II agents with increasing application in fluorescent imaging as well as fluorescence-guided surgery and photothermal therapy.21,22 Photothermal therapy (PTT) is an emerging noninvasive and remote-controllable


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therapeutic modality, which can make use of the local heating generated from absorbed and converted external near-infrared (NIR) light energy of photothermal agents (PTAs) to ablate tumors.23 To improve the therapeutic outcomes of PTT, it is necessary to apply appropriate laser dosage, determine the best treatment time, develop excellent photothermal agents with enhanced absorption and photothermal conversion efficiency (PCE) and increase the accumulation of PTAs in tumors.24 A series of inorganic and organic photothermal agents, such as noble metal materials,25 upconversion nanoparticles (UCNPs),26 transition-metal chalcogenides,27 oxides nanoparticles,28 carbon nanotubes,29 semiconducting polymer nanoparticles

30

and organic dyes,31,32

have been developed for photothermal therapy. Organic photothermal agents, such as ICG,33 IR1048-MZ 34 and polypyrrole (PPy),35 can be applied to PTT for their inherent biodegradability and biocompatibility. However, the poor photothermal stability and unsatisfactory photothermal conversion efficiency, compared to their inorganic counterparts, of organic PTAs impel people to improve their photothermal properties.24 And great efforts have been made to develop organic PTAs with higher photothermal conversion efficiency and better photothermal stability. Accurate therapeutic is expected to minimize the damage to the surrounding healthy tissues during the PTT.24 Although the photoacoustic imaging (PAI), a symbiotic function of photothermal effect as well as PTT, can serve as an alternative imaging method to assist the PTT, different imaging modalities are necessary to improve the diagnosis accuracy and enhance therapeutic efficiency.24, 36 Here, a NIR-II fluorescence probe (PF) was developed by conjugating small


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molecule NIR-II fluorophore with amphiphilic polypeptide to sever as fluorescent and photothermal agent simultaneously. The as-synthesized polymeric fluorophore can form uniform micelles (90 nm) in aqueous solution, which exhibit slight negative charge (-12.5 mV). Meanwhile, PF with inherent biodegradability and biocompatibility showed prominent photothermal conversion effciency (42.3%) and excellent photothermal stability. The experiment in vivo showed that the PF can be accumulated at the tumor site effectively via the enhanced permeability and retention (EPR) effect as well as nanomaterials-induced endothelial leakiness (NanoEL),37-41 which can be visualized by the NIR-II fluorescence imaging. Moreover, the PF can also be used as superior organic PTAs to implement NIR-II fluorescence image-guided PTT in vivo (Scheme 1).

Results and Discussion Synthesis and characterization As illustrated in Scheme 2, the Flav7 was synthesized by a widely applied synthetic route for cyanine dye from commercially available raw material. The intermediates (1, 2, and 3) and Flav7 were obtained with high purity and yield, and also can be confirmed by nuclear magnetic resonance (1H NMR and 13C NMR) and ESI-MS (see ESI). As an extremely hydrophobic organic fluorophore, Flav7 can well dissolve in common organic solvents (DMF, DMSO, CH2Cl2, methanol et al.) and exhibited similar UV-vis-NIR absorption to previous report42 (Figure S2). The spectral regions of the Flav7 (λmax, abs=1026 nm, λmax, em=1081 nm excited by a 785 nm laser diode in


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CH2Cl2, Figure 1) were included in the second near-infrared window (NIR-II; 1000– 1700 nm). Meanwhile, it demonstrated high molar extinction coefficient (ε = 5.49 × 105 M−1 cm−1 in DMSO), acceptable fluorescence quantum yield (Φ = 0.303%, λex=808 nm, in DMSO), and quantum efficiency (QE = εΦ =1663 M−1 cm−1). All of those outstanding photophysical properties make Flav7 a promising candidate for second near-infrared fluorescence imaging in vivo. To design a covalent linkable fluorophore, here we introduced a carboxyl group into the Flav7 by superseding the reactive chloro-group at the central position with a thio-substituent. The functionalized Flav7 (FS) can be confirm by NMR and mass spectrometry (see ESI). Meanwhile, the disappearance of the peak at 663 cm-1 (C-Cl) for Flav7 and the emergence of the peak at 1797 cm-1 (C=O) for FS in the Fourier transform infrared (FT-IR) spectra further corroborated the success of the reaction (Figure S3). The optical properties of FS (λmax,

abs=1026

nm, λmax,

em=1065

nm in

CH2Cl2. ε = 5.09 × 105 M−1 cm−1, Φ = 0.305% in DMSO) have no obvious change in comparison with Flav7 (Figure 1a), indicating a negligible PET process due to the weak electron-donating capability of the thio-substituent 43. The slightly blue shift of FS in fluorescence emission spectra (λmax, em=1065 nm in CH2Cl2) may be induced by the increase in molecular rigidity after introducing the functional substituent.44 Hydrophobicity of the FS is an inevitable obstacle when applied in vivo. Here an amphiphilic copolymer of P(OEGMA)21-P(Asp)16 synthesized and was used to load the FS through an amido linkage to enhance the solubility and biocompatibility of FS. Besides, the proportion of the FS to polymer is another issue to consider for the self-


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quenching effect of polymeric dyes and a small feeding ratio (the molar ratio of FS to polymer is 1:10) was applied.45 The ultimate portion of FS to polymer was calculated based on the UV-vis-NIR absorbance measurements (7.89%, mole fraction). However, the 1H NMR signal is hard to accurately identify the proportion of FS for the low content (Figure S4a). GPC traces show a slightly attenuated elution time for PF, indicating a higher molecular weight (Figure S4b). The spectra of the dye is not affected by the polymer support (λmax,

abs=1026nm,

λmax,

em=1064nm

in CH2Cl2, Figure 1a.). More

importantly, the PF can form polymeric micelles when dispersed in PBS. Dynamic light scattering (DLS) was used to evaluate the size of micelles. As shown in Figure S5, the size of the micelles is about 90 nm (PDI=0.148), which means it can accumulate in tumors preferentially through the EPR effect. The slight negative charge property (12.5 mV) endows the micelles with prolonging retention time in body 46 and enhanced NanoEL.38 As presented in Figure 1b (PF in PBS), the absorbance around the 800900nm is attributed to the molecular aggregates,13 and an obvious aggregation-induced quenching (AIQ) effect based on the mechanics of single-molecule fluorescence resonance energy transfer (smFRET) can be found for the weak fluorescence. After replacing the solvent with FBS, the size of the micelles increased to 124 nm and the fluorescence recovered significantly (Figure 2b and S5b). FRET is sensitive to intraand intermolecular separation in the 1–10 nm range,47,48 and the increase in micelle size can separate the fluorophores from aggregation state and alleviate fluorescence quenching (Figure S5).


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Photothermal properties and photothermal stability PF and most of other near infrared II fluorophores can absorb energy (light) effectively for their high molar extinction coefficient, while only a tiny fraction of those energy was released in the form of luminescence emission. Quite naturally, a reasonable speculation is that considerable fraction of the light energy was converted into heat (nonradiative decay). So, the photothermal conversion efficiency (PCE) of the PF in aqueous solutions was evaluated taking the method reported by Roper’s et al.49 As shown in Figure 2a, the PF exhibited prominent photothermal conversion effciency (42.3%), which is significantly above the PCE of organic photothermal transduction agents (PTAs) have been reported earlier such as ICG( ∼3.1%),33 IR1048-MZ (20.2%),34 and dopamine-Melanin CNSs (40%).30 What's more, the PCE of the PF is also comparable and even higher than many inorganic PTAs (Pt nanodots, 46.9%;50 Polypyrrole@Fe3O4, 39.15%;51 csUCNP@C, 38.1%;26 Au nanorods, 21%;52 black phosphorus quantum dots, 28.4%53 et al.). The high PCE indicated that the PF is a potential PTA to apply to PTT with low light and/or PTA dosage. Photothermal stability is another important parameter to evaluate the PTA. The solution of PF in cuvette was exposed to an 808nm NIR laser at a power density of 0.5 Wcm-2 to measure the cycles of heating/cooling processes. The solution was irradiated for 5 min (laser on), and then was cooled to room temperature (laser off) for five cycles. As shown in Figure 2b, heating behavior of the solution showed negligible deterioration during the recycling. Meanwhile, the UV-vis-NIR spectra have no obvious change in optical absorption even after eight cycles (Figure 2c and Figure S6a). The PF exhibits


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durable stability in water, PBS and FBS at ambient temperature for 15 days (Figure S6c), however, the absorbance for PF in DMSO diminished significantly in just five days (Figure S6f). Furthermore, PF in DMSO was also bleached rapidly when radiated with an 808nm laser (Figure S6d and S6e), which is ascribed to the longish conjugated structure of the fluorophore.54 The significantly enhanced stability of PF in aqueous solution may benefit from the fortification of molecular rigidity. In an aqueous solution, the PF can form polymeric micelles, and the local concentration of the dye in the micelles is high enough to aggregate via strong π-stacking. The highly ordered molecular stacking not only can increase the molecular rigidity and then improve photostability, but also result in the distinct hypsochromic absorption shift (Haggregates) and therefore promote the PCE by promoting nonradiative decay.24 In short, the PF showed excellent photothermal conversion efficiency and photothermal stability comparable to inorganic PTAs in aqueous solution. Both of the advantages together with the inherent biodegradability and biocompatibility of organics highlighting the potential of PF as an excellent PTA for PTT.

Cytotoxicity and photothermal toxicity of PF in vitro The standard MTT assay against 4T1 and HepG2 cancer cells was used to evaluate the cytotoxicity and PTT effect of PF at the cellular level. Meanwhile, the temperature changes of the culture medium were monitored during the irradiation. As shown in Figure 2d, the culture medium without PF demonstrated negligible temperature rise (~1.5 oC), while the temperature in experiment groups increased markedly (9.3 oC for


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0.5 μg mL-1, 14.5 oC for 1.0 μg mL-1, 19.2 oC for 1.5 μg mL-1, 22.7 oC for 2.0 μg mL-1 and 29.8 oC for 2.5 μg mL-1) in 5 min. It has been reported that the hyperthermia (> 48 oC)

can induce cell necrosis, while relatively mild temperature (41-45 oC) can inhibit

cell activity and stimulate the overexpression of heat shock proteins.55 In view of the lower environmental temperature (~30 oC) compared with body temperature, the temperatures rising by 11 oC can affect the cell activity and a 18 oC increase can kill the cancer cells effectively. Cell viabilities of 4T1 and HepG2 cells versus PF of different concentrations were presented in Figure 2e and 2f. It can be found that the cell viability decreased dramatically under irradiation as the concentration of the PF (on FS basis) reached to 1.5 μg mL-1, indicating a prominent photothermal cytotoxicity of PF. Moreover, the PF possessed of no significant cytotoxicity even at a high dosage of PF in the dark owing to its excellent biocompatibility. To visualize the photothermal effect, live/dead staining assay was performed to distinguish the live/dead cells (green/red) with a fluorescence microscope. As shown in Figure 3a and Figure S7, almost all of the cells showed strong green fluorescent emission when the cells were treated without irradiation, indicating negligible cytotoxicity of PF in dark. When exposed to an 808nm laser at 0.5 Wcm-2 for 5 min, the cells incubated with 1.5 μg mL-1 of PF were killed completely for the significant increase in temperature (+19.2 oC), while the control cells appeared largely unaffected for the tiny temperature rise (+1.5 oC). But it should be noted that the cells incubated with 0.5μg mL-1 of PF did not possess higher mortality compared with control cells although they were conducted hyperthermia (~44.5 oC, Figure 3d). This is because a


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short period of the mild hyperthermia (less than 2 min) can’t induces immediate cell death through necrosis although the viabilities of the cells may be inhibited (Figure 2e and 2f). After photothermal ablation, the morphologies of the tumor cells were changed significantly and cells collapse can be found in Figure 3b. Bright field and fluorescence staining images showed that there is a legible boundary to differentiate the dead cells (left) and alive cells (right). Meanwhile, the boundary also served as a temperature threshold to dominate cell death modes (through either necrosis or apoptosis) for the temperature-driven treatment mode of PTT. On the left side of the boundary, the temperature is high enough to induce necrocytosis immediately. On the other side, however, slightly lower temperature is incapable to destroy plasma membrane integrity and kill the cells immediately56 (consistent with Figure 3a). These results confirmed the high-efficiency photothermal toxicity and acceptable cytotoxicity induced by PF.

NIR-II imaging for biodistribution and tumor accumulation in vivo Encouraged by the excellent photothermal effect and anticipated NIR-II fluorescence performance in vitro, tumor accumulation and photothermal ablation ability of the PF was evaluated by administering the drugs intravenously. NIR-II fluorescence images were acquired by using an in vivo NIR-II imaging system (Suzhou NIR-Optics Technology Co., Ltd., China) at predetermined time points under the conditions of irradiation with an 808 nm laser diode at ~45 mWcm-2. As shown in Figure 4a, representative fluorescence images were used to visualize the distribution and accumulation of PF in vivo. In the beginning of the imaging stage (0 h), the mice


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emitted faint fluorescence signal for the unavoidable aggregation-induced quenching. However, the bodies of the mice were lighted gradually for the fluorescence recovery in 6 h after injection and the fluorescence intensity of the whole body peaked in 12 h. Polymeric micelles can be accumulated in tumor via the EPR effect and the fluorescent images (Figure 4a) and surface plot (Figure 4b) revealed outstanding fluorescence signal in tumors and distinct difference comparing to the surrounding tissue. As shown in Figure 4c, fluorescence signals in the tumors were increased rapidly within 6 h and peaked within 24 h. Furthermore, the PF maintained high accumulation in the tumor over the next 36 h, which means an extended therapeutic window. The tumors and major organs (kidney, liver, spleen, lung and heart) were collected to evaluate the biodistribution of PF by ex vivo fluorescence imaging. As shown in Figure 4d, the liver and lung possessed the strongest fluorescence signal while the tumor exhibited moderate intensity at the 24th hour. Although both the EPR and NanoEL effects can accelerate the accumulation of PF in the tumor, the liver is still the foremost organ of drug accumulation57. Therefore, biocompatibility, biodegradability are anticipated for PTAs. NIR-II fluorescence imaging performs higher spatial and temporal resolution than the imaging in the NIR-I window for the reduced absorption and scattering, and avertible tissue autofluorescence in vivo. From the grayscale images as shown in Figure 4e, the tumor could be distinguished from the surrounding tissue visually. Fluorescence intensity of the line profile in tumor also revealed an increased uptake of PF and tumorto-background ratio (Figure 4f) with time prolonging within 24h after intravenous


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injection. The tail vein and neck vascular were immediately imaged after intravenous injection of PF and the vasculatures is legible (Figure S8 and S9).

In Vivo Infrared Thermography and Photothermal therapy The PF nanoparticles demonstrated excellent photothermal performance in vitro and can accumulate in the local tumor efficiently via the EPR effect. Combined with tumor localization ability by NIR-II imaging, the PF can serve as NIR-II fluorophore and PTA simultaneously to perform a NIR-II fluorescence image-guided photothermal therapy. To evaluate the PTT effect in vivo, PBS, FS and PF were injected intravenously into 4T1 tumor-bearing BALB/c mice. Then the tumors were irradiated with a laser (808nm, 0.65 Wcm-2) for 10 min at 16h post-injection to ensure a high concentration of PF in tumor (Figure 4d). In situ thermal images and real-time temperature were recorded by an infrared camera (ICI7320, USA) to visualize the temperature rise period (Figure 5a). As shown in Figure 5b, the temperature of PF treated group raised quickly by 16.2 oC during the first 200 s and then raised slowly to 51.8 oC within 10 min. Whereas, the group of PBS and FS showed negligible increase in temperature and the final temperature is 34.8 oC and 36.4 oC, respectively. After 24 h post photothermal treatment, the tumors of the mice were excised and observed with H&E staining for histology analysis. Severe cytoclasis and obvious karyolysis can be observed in group of PF with irradiation (Figure 5f) while the cells remained intact in the other groups (Figure 5c, 5d and 5e), indicating a high hyperthermia therapeutic efficacy of the PF in vivo.


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Accordingly, the photothermal inhibition effect of PF to tumors was further estimated upon the tumors having reached around 150 mm3. The 4T1-tumor-bearing mice on both sides were randomly divided into three groups (n=5): PBS (only or laser treated), FS (only or laser treated) and PS (only or laser treated). Then the tumors at the left each group were irradiated with a laser (808nm, 0.65 Wcm-2) for 10 min after 16 h post-injection. Tumor volumes and body weights were measured every other day (Figure 7a and 7b), and digital photos of the mice were taken passingly during subsequent 21 days after the treatments (see ESI). Tumor volumes in the groups of PBS with and without irradiation, FS with and without irradiation, and PF without irradiation resulted in 6.6-fold, 7.0-fold, 8.0-fold, 5.7-fold and 7.1-fold increases respectively compared to original volumes, indicating the result is not significant (P>0.05). In comparison, the tumors in the group of PF with irradiation were severely inhibited as compared with control groups (P < 0.001) (Figure 6a). Mice in the group of PF demonstrate mild loss of weight (~5%) in the first two days, caused by tumor ablation, and then restore weight to normal. The other mice demonstrate negligible weight fluctuations as shown in Figure 6b. During treatment, the tumors treated by irradiation in the group of PF began to scab in the second day and the crusts fall off gradually in the end of the treatment (Figure 6c and Figure S11). After 21 days, the mice were sacrificed and tumors were photographed and weighed accurately (Figure 6d and 6e), further verifying that the PF exhibited excellent photothermal ablation effect in vivo. H&E stains of the main organs were examined and demonstrated the PF has no discernible damage on normal tissues (Figure 7). Excellent photothermal theranostic


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capability, noticeable biosecurity and impressive tumor fluorescence imaging capability in vivo make PF a promising candidate for image-guided photothermal therapy.

Conclusions In summary, we developed a macromolecular NIR-II fluorescence probe by conjugating small molecule fluorophore with amphiphilic polypeptide, while it can serve as efficient organic photothermal agent simultaneously. The PF can form uniform micelles (90 nm) in aqueous solution and exhibit prominent photothermal conversion effciency (42.3%) and excellent photothermal stability. The PF nanoparticles emits faint fluorescence signal for the unavoidable aggregation-induced quenching in PBS. However, after injected into the mice through caudal vein, the probe emits intense NIRII fluorescence for the fluorescence recovery, which can be used to distinguish the tumors accurately and monitor the accumulation of photothermal agents in tumors in real-time. Moreover, the PF provided a prolonged treatment time window and achieved successful ablation of tumors without obvious biotoxicity. Therefore, the PF with inherent biodegradability and biocompatibility is a promising candidate for accurate diagnosis by NIR-II fluorescence imaging and/or remote-controllable photothermal therapy of tumor and other diseases.

Experimental Section Materials


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Acetophenone,

N,N-dimethyl-3-aminophenol,

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3-mercaptopropionic

acid,

aluminium chloride, dicyclohexylcarbodiimide (DCC), fluorescein diacetate (FDA), Nhydroxysuccinimide, propidium iodide (PI), thiazolyl blue tetrazolium bromide (MTT) and 4-(dimethylamino)-pyridin (DMAP) were purchased from Aladdin Corporation (China). Fetal bovine serum (FBS), dulbecco’s modified Eagle’s medium (DMEM), and dialysis bag (cutoff Mw = 7000) were purchased from Hyclone. Perchloric acid, 1, 2-dichloroethane and other chemicals were obtained from Sinopharm Chemical Reagent Co., Ltd. Poly[oligo(ethylene glycol) methyl ether methacrylate]-blockpoly(L-aspartic acid) (P(OEGMA)21-P(Asp)16) was synthesized in our laboratory.58 The solvents of N, N-dimethylformamide (DMF) and dichloromethane were dried with CaH2. Ultrapure water was prepared by Milli-Q Synthesis System (18.2 M, Millipore). All of the other organic solvents and chemicals (AR purity) were used without any further purification.

Characterization 1H

NMR and

13C

NMR spectra were carried out on a Bruker 400 M NMR

spectrometer and the CDCl3 or DMSO-d6 were used as the solvent. Biorad iMark microplate reader was used to record the absorbance of the samples for the MTT assay. Dynamic light scattering (DLS) spectra were recorded on a Malvern Zetasizer NanoZS90 to measure the hydrodynamic size and zeta-potential of the micelles.

Synthesis of 7-dimethylamino flavyliym heptamethine dye (Flav7)


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Flav7 was synthesize according to the references 42 and 59 with some changes.

Acetic acid 3-dimethylamino-phenyl ester (1) N, N-Dimethyl-3-aminophenol (5.0g, 36.4 mmol), triethylamine (7 mL, 50.4 mmol) and DMAP (0.2g, 1.6 mmol) were dissolved in dichloromethane (DCM) and stirred for 5 min with nitrogen in ice bath. Then acetyl chloride (3.2 mL, 45.0 mmol) in DCM was added via a constant pressure drop funnel in more than 20 min. Later on, the reaction mixture was moved to room temperature and stirred for another 4 hours. After quenched by water, the aqueous phase was extracted with DCM (40 mLx3). The dichloromethane solution was collected, washed with saturated salt water and dried over anhydrous sodium sulfate. The solvent was removed by vacuum-rotary evaporation to obtain a dark red liquid (6.34g, 97% yield). ESI-MS m/z: [M+Na] calcd for C10H13O2NaN, 202.08; found, 202.08. 1H

NMR (300 MHz, CDCl3) δ 7.21 (d, J = 7.9 Hz, 1H), 6.62 (s, 1H), 6.45 (s, 2H),

2.95 (s, 6H), 2.29 (d, J = 1.0 Hz, 3H). 13C

NMR (75 MHz, CDCl3) δ 169.69, 151.78, 151.66, 129.65, 109.94, 109.20,

105.41, 40.43, 21.22.

4-N, N-dimethylamino-2-hydroxyacetophenone (2) Compound 1 (6.0g, 33.5 mmol) and aluminium chloride (15g, 111.9 mmol) were dissolved in 1,2-dichloroethane (50 mL) and refluxed at 80 oC for 1 h. Then the temperature of the reaction was raised to 140 oC for another 6 h. The reaction was


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quenched by adding 1 M HCl (100 mL) slowly, and the mixture was extracted with DCM (100 mL2). After removing the solvent, the raw product was purified using column chromatography (gradient elution from n-hexane to n-hexane/ethyl acetate = 10:1) to get faintly yellow crystals (2.83g, 47% yield). ESI-MS m/z: [M+Na] calcd for C10H13O2NaN, 202.08; found, 202.08. 1H

NMR (400 MHz, CDCl3) δ 12.89 (s, 1H), 7.54 (d, J = 9.1 Hz, 1H), 6.22 (dd, J

= 9.0, 2.6 Hz, 1H), 6.09 (d, J = 2.5 Hz, 1H), 3.05 (s, 6H), 2.49 (s, 3H). 13C

NMR (75 MHz, CDCl3) δ 200.79, 164.76, 155.81, 132.34, 110.26, 103.85,

97.79, 39.97, 25.59.

7-N, N-dimethylamino-4-methyl-flavylium perchlorate (3) 4-N,

N-dimethylamino-2-hydroxyacetophenone

(2.5g,

14.0

mmol)

and

acetophenone (7.5 mL, 64.4 mmol) were dissolved in acetic acid (20 mL), and 70% perchloric acid (25 mL) was added dropwise at room temperature. Then the reaction was raised to 60 oC for 6 h and 110 oC for another 12 h. After cooled to room temperature, 100 mL water was poured in to facilitate a red precipitate. The precipitate was filtered, washed with ethyl acetate and dried under vacuum to obtain the compound 3 without further purification (4.50 g, 90% yield). ESI-MS m/z: [M-ClO4] calcd for C18H18NO+, 264.14; found, 264.14. 1H

NMR (300 MHz, DMSO-d6) δ 8.44 – 8.29 (m, 2H), 8.19 (d, J = 9.0 Hz, 2H),

7.74 (dq, J = 14.3, 6.8 Hz, 3H), 7.54 – 7.44 (m, 1H), 7.32 (d, J = 2.5 Hz, 1H), 3.33 (s, 6H), 2.84 (s, 3H).


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13C NMR (75 MHz, DMSO-d

6) δ 164.13, 163.73, 158.16, 157.44, 134.04, 129.61,

129.37, 128.75, 127.64, 118.14, 117.56, 111.95, 96.03, 40.60, 19.55.

7-dimethylamino flavyliym heptamethine dye (Flav7) A solution of 3 (4.0g, 11.5 mmol) and 2-Chloro-1-formyl-3-(hydroxymethylene)cyclohex-1-ene (0.86g, 5 mmol) in a mixture solvent of n-butanol (50 mL) and benzene (25 mL) was stirred at 110 oC for 10 h under a nitrogen atmosphere.60 After cooled to room temperature, the solvent was evaporated under reduced pressure, and the crude product was washed with little of DCM. The Flav7 was purified by column chromatography (gradient elution from DCM to DCM/ methanol = 10:1) to get Flav7 as a furvous solid (2.35g, 56% yield). ESI-MS m/z: [M-ClO4] calcd for C44H40ClO2 N2+, 663.27; found, 663.27. 1H

NMR (400 MHz, DMSO-d6) δ 8.11 (d, J = 13.7 Hz, 2H), 8.02 (dd, J = 10.3, 8.3

Hz, 6H), 7.68 – 7.46 (m, 8H), 6.98 – 6.83 (m, 4H), 6.68 (d, J = 2.5 Hz, 2H), 3.09 (s, 12H), 2.77 (s, 4H), 1.89 (s, 2H). 13C

NMR (101 MHz, DMSO-d6) δ 156.76, 155.75, 154.50, 145.30, 144.26, 132.02,

131.49, 131.20, 129.54, 126.56, 124.56, 113.75, 113.70, 112.33, 102.23, 97.45, 43.36, 29.53, 29.05, 26.68.

Synthesis of functionalized Flav7 (FS) Flav7 (193mg, 0.25 mmol) was dissolved in dry DCM and stirred at 0 oC bubbling with nitrogen. Next the 3-mercaptopropionic acid (300 μL, 2.8 mmol) and triethylamine


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(400 μL) in DCM were added dropwise, successively. Then the reaction was moved to room temperature and stirred continuously for 48 h. After removing the solvent, the FS was purified by column chromatography (136mg, 65% yield). ESI-MS m/z: [M-ClO4] calcd for C47H45N2O4S+, 733.31; found, 733.31. 1H

NMR (400 MHz, DMSO-d6) δ 8.17 – 7.76 (m, 8H), 7.69 – 7.30 (m, 8H), 6.96

– 6.69 (m, 4H), 6.61 – 6.47 (m, 2H), 3.05 (t, J = 14.6 Hz, 12H), 2.91 – 2.82 (m, 2H), 2.72 (s, 2H), 2.00 (q, J = 7.5 Hz, 2H), 1.87 (s, 2H). 13C

NMR (101 MHz, CDCl3) δ 178.08, 161.25, 160.38, 159.16, 149.03, 145.25,

142.55, 136.54, 136.30, 135.78, 134.40, 134.35, 131.25, 118.35, 118.18, 116.95, 48.12, 38.95, 37.36, 31.86, 31.72.

Conjugation of Flav7 and P(OEGMA)21-P(Asp)16 (PF) P(OEGMA)21-P(Asp)16 (577mg, 0.04 mmol), FS (3.2mg, 0.004 mmol), and Nhydroxysuccinimide (2.0mg, 0.017 mmol) were dissolved in dry DCM in ice bath and then DCC (4mg, 0.019 mmol) in dry DCM was added dropwise. After a 48 h reaction at room temperature, the solvent was removed by vacuum-rotary evaporation. Next the sample was redissolved in acetone and the insoluble precipitate was removed by centrifugal separation. The PF was further purified by dialysis and freeze-drying (490mg, 85% yield). The efficiency of FS loaded on the amphiphilic polymer was calculated by a UV-vis method with DCM as the solvent (7.89%, mole fraction).

Characterization


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Absorbance and Emission Spectra The absorption spectra of the samples were taken on an UV1700PC ultraviolet spectrophotometer. The NIR-II fluorescence emission spectra were recorded on an Applied Nano-Fluorescence spectrometer (USA) equipped with a 785nm laser diode. DCM, PBS and FBS were used as the media to correct the background.

Calculation of the NIR-II Quantum Yield The absolute fluorescence quantum yield was measured on a Quantaurus-QY PLUS C13534 (Hamamatsu Photonics (China) Co., Ltd) and an 808nm laser was chosen as the external excitation light source. First, all of the photons were recorded with an integrating sphere after the laser through a blank reference, then the reference was replaced by the samples dissolved in DMSO and the spectra (750-1400nm) were measured again. The quantum yield was calculated by the equation below:

where QYsample is the quantum yield of Flav7 and FS, F represents the fluorescence intensity, and A is the light intensity of the laser after through the reference or the samples (Figure S12).

Measurements of Photothermal Effect and Stability To evaluate the photothermal process of PF in aqueous solutions, the solutions of PF (1 mL, 2 μg mL-1 on FS basis) in a quartz cuvette was irradiated with an 808nm


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diode-laser (Hi-Tech Optoelectronices Co. Ltd) for 300 s at a laser power density of 0.5 W cm-2. Then turning off the laser and a natural cooling process was continued for another 900 s. Temperature of the solution was recorded on a digital thermometer at selected intervals. Repetitive irradiation was implemented for 5 times to evaluate the photothermal stability. Simultaneously, the absorption spectra of the solution was measured.

Calculation of the Photothermal Conversion Efficiency The photothermal conversion efficiency of PF in water was calculated by the equation described by Roper’s et al.49

Where η is the photothermal conversion efficiency, h is the heat transfer coefficient, S is the surface area of the facula area, ΔT is the temperature difference to the surroundings, I is the power density of laser, Aλ is the absorbance intensity of the solution at 808 nm and the Qdis represents the heat induced by solvent absorption. Among which, hS can be evaluated by the equation below:

Where the θ is defined as (T-Tamb)/ΔTmax, t is the time of cooling down, m is the mass of the solution, Cp is the heat capacity of solvent. In our measurements, ΔTmax is 41.3 oC (ca. 60.1-19.8), Qdis is 0.0242 J s-1 (measured independently), I is 1.0 W cm-2, A808 is 0.905, m is 1 g and Cp is 4.2 J (g•


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oC)-1.

The photothermal conversion efficiency of PF in water was calculated to be

42.3%.

Cell Culture and Cyotoxicity Assays Murine breast cancer cell lone (4T1) cells and human hepatoma cell line (HepG2) cells were cultured in complete DMEM medium supplemented with 10% FBS at 37 oC and in a 5% CO2 atmosphere. The cytotoxicity was measured by methyl tetrazolium (MTT) and FDA/PI co-staining assay. To evaluate the photothermal ablation to cancer cells in vitro, 4T1 or HepG2 were plated in 96-well plates at a density of 3500 cells per well for 24 h. Subsequently, the culture medium was removed and fresh DMEM with PF at various concentrations (0, 0.5, 1.0, 1.5, 2.0, 2.5 μg mL-1 on FS basis) was supplemented and incubated for 4 h. Then the cells were irradiated with a laser (808nm, 0.5 W cm-2) for 5 min and the temperature of the culture medium was recorded by a non-contact infrared thermometer. After incubation for additional 20 h, a standard MTT assay was used to evaluate the cells viability. Live/dead cell staining assay against 4T1 and HepG2 cells was performed on an Olympus IX71 fluorescence microscope to observant the phototoxicity directly. Simply put, the cells of 4T1 or HepG2 were seeded in 6-well plates and incubated for 24 h under dark conditions before treatment. After that the medium was replaced by fresh DMEM containing PF (0, 0.5, 1.5 μg mL-1 on FS basis) and incubated for another 24 h.


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Then the cells were treated with irradiation (808nm, 0.5 W cm-2, 5 min), and stained with fluorescein diacetate (FDA) and propidium iodide (PI) half an hour later.

Animals and Tumor Model Female Balb/c mice (Beijing HFK Bioscience Co., Ltd, 5 weeks) received care in compliance with the guideline approved by Institutional Animal Care and Use Committee of the University of Science and Technology of China. Murine mammary carcinoma tumor models were established by subcutaneous injection of 4T1 cells in the flank region of the back. Tumor models established only in the right side of Balb/c nude mice were used for live-imaging, while tumor models established in both sides of the Balb/c mice was used to evaluate the photothermal ablation efficiency. The tumorbearing mice were used for subsequent NIR-II fluorescence imaging in vivo and photothermal therapy until the tumor volume reached 150 mm3: tumor volume = (tumor length) × (tumor width)2/2.

In vivo NIR-II imaging for biodistribution and accumulation of PF The whole-body NIR-II fluorescence imaging was performed by using an in vivo NIR-II imaging system (Suzhou NIR-Optics Technology Co., Ltd., China). Briefly, an 808nm diode laser was used to excite the PF at a power density of ~120 mW cm-2 and an InGaAs camera with a 640 × 512 pixel focal plane array was used to image the small animal models. Following intravenous injection of PF (2.5 mg kg-1 on FS basis) into mice, NIR-II images were obtained immediately. Then, the whole-body imaging was


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carried out at predetermined time points to visualize the biodistribution and accumulation of PF in vivo. After 24 or 60 h, the mice for imaging was sacrificed and their major organs and tumors was collected for visualization the drug metabolism. The images were analyzed by image J software.

In vivo infrared thermography and photothermal therapy 4T1 tumor-bearing mice on both sides were randomly divided into three groups (n = 5): PBS (only or laser treated, 150 μL), FS (only or laser treated, 2.5 mg/kg) and PS (only or laser treated, 2.5 mg kg-1 on FS basis). The mice in the group of FS and PF were given an equivalent FS dose of 2.5 mg kg-1 via tail vein injection. After 16 h postinjection, the left tumors of the mice in all groups were exposed to light (0.65 W cm-2, 808nm) for 10 minutes, while the tumors on right side without further treatment as controls. Infrared images of the mice were photographed and the real time temperature changes at the tumor region were recorded simultaneously by an infrared camera (ICI7320, Infrared Camera Inc., Beaumont, Texas, USA). The drug administration and irradiation-treatment was only given once. The volume of the tumors and the weight of the mice were measured using a vernier caliper and an electronic balance every other day. After in vivo photothermal therapy, the mice were sacrificed and their tumor tissues were harvested and weighed up. The tumors for all of the group were photographed for comparison. For histology examination, the main organs and tumors for each group were observed with hematoxylin & eosin (H&E) stained pathological sections.


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Statistical analysis The statistical significance of the results were estimated by a one-tailed Student’s t-test. *P < 0.05 was used as the statistically significant.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. NMR spectra and mass spectra of 1, 2, 3, Flav7 and FS. Absorption and fluorescence emission spectra of the probes. Live/dead cell staining images of cells. Optical images of the mice during the photothermal treatment stage.

AUTHOR INFORMATION Corresponding Authors *E-mail: [email protected] *E-mail: [email protected]

ORCID Tuanwei Li: 0000-0003-3168-8584 Chunyan Li: 0000-0002-1155-6050 Zheng Ruan: 0000-0002-2759-0587 Pengping Xu: 0000-0002-7527-3332 Xiaohu Yang: 0000-0002-8491-0882


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Pan Yuan: 0000-0002-0719-4843 Qiangbin Wang: 0000-0001-6589-6328 Lifeng Yan: 0000-0002-6063-270X

Author Contributions §The

authors contributed equally to this work.

Acknowledgements The research is supported by the National Natural Science Foundation of China (51673180, 51873201, 81401464, 21425103, 21778070), the National Key Research and Development Program (2016YFA0101503, 2017YFA0205503). The authors thank Suzhou NIR-Optics Technology Co., Ltd. for its instrumental and technique support on the in vivo NIR-II imaging.

Conflict of Interest The authors declare no conflict of interest.

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Scheme 1. Schematic illustration of the mechanism of the NIR Image-Guided Photothermal Therapy by PF.


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Scheme 2. Synthesis Route of Flav7, FS and PF.


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Figure 1. Absorption and fluorescence emission spectra of Flav7, FS and PF in DCM (a). Absorption and fluorescence emission spectra of PF in PBS and FBS (b).


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Figure 2. (a) Calculation of the Photothermal Conversion Efficiency (1.0 Wcm-2, 808 nm). Black line: The temperature curve of PF in water with laser on for 900 s, followed by natural cooling. Red line: Linear time data versus –Ln θ during the cooling period. (b) Temperature change curves of PF in water under irradiation of 808 nm laser (0.5 Wcm-2). (c) Absorbance at 808 nm of PF in water upon irradiation under an 808 nm laser (0.5 Wcm-2). (d) The temperature changes of the cell culture medium containing PF (0, 0.5, 1.0, 1.5 2.0 and 2.5 μg/mL on FS basis) in the 96-well plates, obtained from non-contact infrared thermometer (5 min, 0.5 Wcm-2, 808 nm, n=4). MTT assays of PF on 4T1 (e) and HepG2 (f) cancer cells without (black column) or with (red column) light irradiation (808 nm, 0.5 Wcm-2, 5 min). One-tailed Student’s t-test, *compared with control group P

Polypeptide-Conjugated Second Near-Infrared Organic Fluorophore

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