Boosting Fluorescence-Photoacoustic-Raman Properties in One Fluorophore for Precise Cancer Surgery

Article

Boosting Fluorescence-Photoacoustic-Raman Properties in One Fluorophore for Precise Cancer Surgery Ji Qi, Jun Li, Ruihua Liu, ..., Dingbin Liu, Dan Ding, Ben Zhong Tang [email protected] (D.D.) [email protected] (B.Z.T.)

HIGHLIGHTS Fluorescence, photoacoustic, and Raman properties can be boosted in one molecule Preoperative fluorescence/PA imaging deciphers comprehensive tumor information Intraoperative fluorescence/ Raman imaging helps to delineate tiny residual tumors The one-for-all organic agent provides a unique platform for precise cancer surgery

A one-for-all organic agent for integrated triple-modality imaging-guided cancer surgery has been developed, in which the fluorescence, photoacoustic (PA), and Raman properties could be precisely tuned and boosted by tuning the molecular structure and intramolecular motions. By taking advantage of the merits of each mode, the organic nanoagent helps to decipher tumor information at different surgical stages and improve cancer surgery outcomes significantly. The preoperative fluorescence and PA imaging provide comprehensive information about tumors, while intraoperative fluorescence and Raman imaging accurately delineate tiny residual tumors.

Qi et al., Chem 5, 1–21 October 10, 2019 ª 2019 Elsevier Inc. https://doi.org/10.1016/j.chempr.2019.07.015

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Article

Boosting Fluorescence-Photoacoustic-Raman Properties in One Fluorophore for Precise Cancer Surgery Ji Qi,1,5 Jun Li,2,5 Ruihua Liu,2 Qiang Li,3 Haoke Zhang,1 Jacky W.Y. Lam,1 Ryan T.K. Kwok,1 Dingbin Liu,3 Dan Ding,2,* and Ben Zhong Tang1,4,6,*

SUMMARY

The Bigger Picture

Multi-modality organic imaging agents hold great potential for comprehensive disease diagnosis and treatment by taking advantage of each mode, yet the development is less than satisfied. Here, we develop the one-for-all agent with boosted fluorescence, photoacoustic (PA), and Raman properties by tuning the molecular structure and intramolecular motion for triple-modality imagingguided cancer surgery. Compared with the other analogs, compound OTPATQ3 can generate the highest fluorescence, PA, and Raman (2,215 cm 1) signals, which presents the first organic molecule integrating with these optical imaging modalities. In vivo experiments with the OTPA-TQ3-based nanoagent help to decipher tumor information at different surgical stages and improve cancer surgery outcomes. The pre-operative fluorescence and PA imaging are capable of providing comprehensive tumor information, while the intraoperative fluorescence and Raman imaging delineate tumor margins in a sensitive high-contrast manner. This one-for-all organic molecular agent allows for accurate cancer imaging and resection, rendering great promise for integrated multi-modality imaging applications.

High-performance molecular agent enables maximal diagnosis and treatment outcomes (e.g., cancer surgery). The ideal imageguided cancer surgery calls for diverse imaging methods at different stages of the complex cancer operation, yet there is no individual imaging technique meeting all the requirements throughout the entire surgery process. Here, we report a kind of one-for-all organic agent, in which the fluorescence, photoacoustic, and Raman properties can be simultaneously boosted by tuning the molecular structure and intramolecular motion, for triplemodality imaging-guided precise cancer surgery. The preoperative fluorescence and photoacoustic imaging help to decipher comprehensive tumor information, while intraoperative fluorescence and Raman imaging delineate the tumor margins accurately. This work highlights a new strategy to develop multifunctional organic agents with one-for-all signature for comprehensive cancer surgery, rendering great promise for clinical translation.

INTRODUCTION Complete removal of tumor tissues is decisively important for prolonging the patients’ lifetime and even thoroughly curing cancers.1,2 Image-guided cancer surgery that aims to employ molecular imaging techniques to help the surgeon catch and remove all the tumor nodules thus arise at the historic moment and has been used clinically in recent years.3–5 Ideal image-guided cancer surgery calls for diverse imaging methods at the different stages of cancer operation.6,7 Before surgery, the basic information such as the size, number, and location of the tumors inside the body has to be confirmed, which requires the imaging technique possessing excellent spatial resolution and high sensitivity.8,9 Nevertheless, during surgery, the surgeon faces several conundrums such as identification of tiny tumors (e.g., < 1 mm) and the margin between normal and tumorous tissues, as well as judgment of whether residue tumors exist after main tumor resection.10,11 Hence, the imaging technique with superb sensitivity and prominent signal-to-background ratio is highly desirable intraoperatively. Unfortunately, there is no individual imaging technique meeting all the requirements throughout the entire surgery process. Taking versatile optical imaging techniques for examples, fluorescence imaging possesses excellent sensitivity, but the penetration capability and spatial resolution are limited.12,13 Photoacoustic (PA) imaging can provide large penetration depth beyond the optical diffusion limit meanwhile maintaining high spatial resolution yet the sensitivity is

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not that satisfied.14–17 Besides, Raman imaging is a complementary optical imaging technique, featuring a cell-silent region (1,800–2,800 cm 1), which permits highcontrast imaging with zero interference of biological background and thus holds great potential for precise intraoperative inspection of residual tumors.18–20 As a consequence, the combined advantages of fluorescence, PA, and Raman imaging modalities decidedly promote the cancer surgical outcomes, which calls for highly efficient fluorescence-PA-Raman triple-modality imaging agents. At present, the most commonly used strategy for preparing multi-modality imaging agents is to combine various components into one platform (all-in-one strategy) to make use of their respective functions.21–25 Although effective, this method is hindered by the complicated composition, reduced reproducibility and uncertain pharmacokinetics, hence less accessible for clinical translation.26,27 Alternatively, one-for-all organic agents with multiple imaging capacities in one molecule have received more attention due to the lower complicity, simpler preparation, defined structure, and far better reproducibility than the all-in-one agents.28,29 To our knowledge, however, one-for-all organic agents with simultaneous fluorescence, PA, and Raman imaging capabilities have been scarcely reported since it is considerably hard to develop a molecular guideline to enable and boost every optical imaging efficacy at the same time. All the three imaging modalities stem from external light excitation, e.g., fluorescence and PA are associated with the radiative and nonradiative decay pathways from the excited state to the ground state, respectively, while Raman signal originates from the relaxation of virtual energy state (Scheme S1). The radiative and nonradiative pathways are expected to be greatly impacted by the molecular motions (e.g., rotation, vibration, and twisting) that could consume the excited-state energy, and Raman signal is a kind of molecular vibration and rotation.18,30 These processes are competitive to each other, so it is really difficult to simultaneously boost them in one organic molecule. Since the aforementioned three optical imaging capacities and the corresponding photophysical processes are closely related to intramolecular motions, we wonder whether organic molecules with rotation and vibration units can serve as a high-performing fluorescence-PA-Raman triple-modality imaging agent, which has never been explored before. Molecular motions that play a pivotal role in determining many fundamental physical and chemical processes hold stupendous potential for advancing the biomedical field, as controllability and utilization of dynamic molecular motions can lead to functional or smart materials with accurately tunable properties, benefiting to precision medicine and personalized theranostics.31–33 For example, our recent studies about the aggregation-induced emission (AIE) luminogens clearly demonstrate that intramolecular motions contribute greatly to the photophysical energy dissipation pathways and that restriction of intramolecular motions significantly promotes fluorescence in the aggregates.34–36 However, so far, there have been few reports on the design of multi-functional bioagents for precision medicine by fully taking advantage of active intramolecular motion after light absorption, as it is indeed challenging to command and unify microcosmic molecular dynamic behaviors to determine macroscopic biomedical function and optimize the efficacy. This motivates us to develop advanced optical bioprobes with biomedical effectiveness not achievable by currently available ones. In this contribution, we report for the first time that boosted fluorescence, PA, and Raman properties can be integrated into one organic fluorophore, in which

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1Department

of Chemistry, The Hong Kong Branch of Chinese National Engineering Research Center for Tissue Restoration and Reconstruction, Institute for Advanced Study, and Department of Chemical and Biological Engineering, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, China

2State

Key Laboratory of Medicinal Chemical Biology, Key Laboratory of Bioactive Materials, Ministry of Education, and College of Life Sciences, Nankai University, Tianjin 300071, China

3College

of Chemistry, Research Center for Analytical Sciences, State Key Laboratory of Medicinal Chemical Biology, and Tianjin Key Laboratory of Molecular Recognition and Biosensing, Nankai University, Tianjin 300071, China

4Center

for Aggregation-Induced Emission, SCUT-HKUST Joint Research Institute, State Key Laboratory of Luminescent Materials and Devices, South China University of Technology, Guangzhou 510640, China

5These 6Lead

authors contributed equally

Contact

*Correspondence: [email protected] (D.D.), [email protected] (B.Z.T.) https://doi.org/10.1016/j.chempr.2019.07.015

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Scheme 1. Schematic Illustration of the One-for-All Organic Agent for Image-Guided Surgery The one-for-all agent can be used for comprehensive preoperative fluorescence and PA imaging of tumors, and intraoperative fluorescence and Raman imaging of tiny residual tumors to boost cancer surgery outcomes. ex, excitation light.

these optical imaging properties are significantly impacted by the molecular structure and intramolecular motion. We start with the design and synthesis of a series of near-infrared (NIR)-absorbing organic fluorophores (named OTPATQ1-3) with different substituted units (phenyl, phenyl-alkyne, and phenylalkyne-phenyl, respectively, Scheme S2), followed by significant enhancement of the water solubility of these hydrophobic molecules via formulation into welldispersed nanoparticles (NPs). Compared with the other two analogs, compound OTPA-TQ3 with the large phenyl-alkyne-phenyl substitutes can generate the highest fluorescence, PA, and Raman (in cell-silent region) signals, which presents the first organic molecule integrating with these optical imaging modalities. Lastly, the feasibility of such one-for-all multi-functional OTPA-TQ3 NPs in intricate biomedical application such as image-guided cancer surgery was investigated. In vivo study reveals that precise cancer surgical treatment can be achieved under the guidance of OTPA-TQ3 NPs permitting preoperative NIR fluorescence and PA imaging as well as intraoperative fluorescence and Raman imaging (Scheme 1). This study suggests that the one-for-all organic agent with optimized imaging performance holds great promise for practical applications.

RESULTS Synthesis, Characterization, and Photophysical Properties The push-pull or donor-acceptor (D-A) approach, in which the electron-donating and electron-withdrawing moieties are alternatively arranged along the conjugated structure, is effective to reduce the bandgap.37,38 In this work, we employ alkoxysubstituted triphenylamine (OTPA) as the donor and thiadiazoloquinoxaline (TQ) as the acceptor to construct the NIR chromophores. The strong D-A interaction ensures efficient intramolecular charge transfer (ICT), which is beneficial to realizing small electronic band gap and thus NIR absorption and emission. The octyloxy substitutes in triphenylamine unit could increase the electron-donating nature, as well as endow the resultant compounds with good solubility and processability. Moreover, the long side chains are designed to retain some room between the conjugated backbones, which is favorable for intramolecular motions in aggregated state.39,40 A series of analogs with different substituted groups (i.e., phenyl, phenyl-alkyne, and phenyl-alkyne-phenyl) in TQ core have been synthesized (Figure 1A) to investigate their influence on the optical imaging properties in terms of fluorescence, PA,

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Figure 1. Structure, Calculation, and Photophysical Properties of OTPA-TQs (A and B) Shown in (A) are chemical structures and in (B) are optimized molecular geometries of OTPA-TQ1-3 in the ground state. (C) Absorption spectra of OTPA-TQ1-3 in THF (20 mM). (D) PL spectra of OTPA-TQ3 (20 mM) and (E) a AIE value versus water fraction (f w ) in THF-water mixtures. aAIE is defined as the ratio of the PL intensities of the compounds in THF-water mixtures and pure THF (fw = 0).

and Raman. The synthetic route to OTPA-TQ molecules is presented in Scheme S2. Key synthesis steps include Stille cross-coupling reaction between the tributyltinsubstituted OTPA (5) and the dibromo-molecule (6) to produce the dinitro-compound (7) as a dark purple solid, followed by the iron-catalyst nitro reduction and subsequent cyclization with benzils to obtain the final compounds. The benzil derivatives (9–11) with different substitutes were prepared in advance. The intermediates and final compounds are characterized by 1H NMR, 13C NMR, and high-resolution mass spectrum (HRMS). Detailed synthesis processes and characterizations are shown in the Supplemental Information (Figures S1–S22). To gain in-depth understanding about the molecular geometry, we conducted density functional theory (DFT) calculations with Gauss 09 program at B3LYP/6-31G(d)

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level. In order to simplify the calculations, we abbreviated the substituted aliphatic groups as methoxy units. The optimized molecular geometries of the compounds in ground state are presented in Figure 1B. They possess similar geometry with largely twisted intramolecular rotors, which would dissipate the excited-state energy as the free high-frequency rotation in solution and be beneficial for realizing the AIE effect and bright emission in aggregation and solid state. As shown in Figures S23–S25, the wave function of highest occupied molecular orbital (HOMO) distributes along both OTPA and TQ units, whereas the lowest unoccupied molecular orbital (LUMO) is mostly localized on electron-deficient TQ core, indicating D-A characteristic and efficient ICT.41 Further study on other molecular orbitals (HOMO-1, HOMO-2, HOMO3, and HOMO-4 and LUMO+1, LUMO +2, LUMO +3, and LUMO +4, Figures S26– S28) suggests that the two phenyl-alkyne-phenyl groups in OTPA-TQ3 enable better conjugation and electronic transition. The absorption spectra of compounds OTPA-TQ1-3 in tetrahydrofuran (THF) show strong absorption in the range of 600–750 nm (Figure 1C), matching well with the excitation light sources of commercially available fluorescence and PA imaging systems. The maximal absorption coefficients increase in the order of OTPATQ2 < OTPA-TQ1 < OTPA-TQ3, which is in the same trend as the calculated oscillator strengths (Table S1). The higher absorption coefficient of OTPA-TQ3 would be favorable for efficient light excitation. The photoluminescence (PL) maxima of compounds OTPA-TQ1-3 in THF solution are in sequence at 894, 911, and 910 nm, respectively (Figure S29; Table S1). The large Stokes shifts of about 200 nm efficaciously avoid the overlap between excitation and emission spectra, allowing for efficient utilization of the fluorescent light. We then studied their fluorescence property by adding water (poor solvent) into THF (good solvent) solution. For all three compounds, the PL intensities slightly decrease when gradually increasing water fraction from 0% to 30% (Figures 1D, 1E, and S30) due to the solvent polarity effect and therefore the transformation to twisted intramolecular charge transfer (TICT) state.42 The emission intensities largely intensify when further increasing water fraction to 95%, representing typical AIE signature. Interestingly, the aAIE value (defined as the ratio of maximal PL intensity in aggregate state and solution state) of OTPATQ3 in THF-water mixtures with 95% water fraction is higher than the other two derivatives (Table S1), attributing to that the large twisted rotors (phenyl-alkynephenyl) on OTPA-TQ3 result in reduced intermolecular interactions such as p-p stacking in the aggregated state.43 This result reveals that the size of intramolecular rotors would significantly influence the fluorescence property. Preparation, Characterization, and Fluorescence Properties of the NPs To render the hydrophobic compounds with good in vivo biocompatibility, OTPATQs were formulated into stable and small-nanosized NPs through nanoprecipitation method using an amphiphilic lipid-PEG2000 co-polymer as the encapsulation matrix (Figure 2A). During this process, the hydrophobic organic molecule randomly assembles in the core, and the amphiphilic surfactant forms the shell to result in water-soluble NPs.15 The size and morphology of OTPA-TQ1-3 NPs, respectively, were characterized by transmission electron microscopy (TEM) and dynamic light scattering (DLS) measurements. As depicted in Figures 2B and S31, the DLS data suggest that the hydrodynamic diameters of OTPA-TQ1-3 NPs are 141, 142, and 144 nm, respectively, and the TEM images indicate that all the three NPs possess approximately spherical morphology with similar average diameters of 110–120 nm (Table S2). The smaller sizes obtained from TEM measurement is probably due to the shrinkage of the hydration layer in the dried TEM samples.37 The obtained NPs are a kind of green solution with good transparency, which is similar as

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Figure 2. Characterization and Photophysical Properties of the NPs (A) Schematic illustration of the nanoprecipitation process. (B) Representative DLS result and TEM image of OTPA-TQ3 NPs. (C and D) Shown in (C) is the absorption and in (D) PL spectra of the NPs (20 mM). (E) PLE mapping of OTPA-TQ3 NPs in aqueous dispersion (20 mM).

the molecules in THF (Figure S32). The absorption maxima of OTPA-TQ1-3 NPs are centered at 684, 701, and 705 nm, respectively (Figure 2C; Table S1), which are redshifted slightly as compared with those in solution states. On the contrary, the PL spectra of OTPA-TQ1-3 NPs are centered at 880, 897, and 895 nm, respectively (Figure 2D; Table S1). The hypsochromic shifts of NPs emission spectra for about 15 nm when compared with those in THF solutions may be attributed to that the fluorophoric molecules in NPs are surrounded by the same molecules with less polarity than THF. The PL spectra of the compounds in toluene (Figure S29B) exhibit hypsochromic shifts of about 20 nm as compared to that of THF solution, which is similar as the NPs form and confirms the influence of polarity effect. By using indocyanine green (ICG) as the reference (with a nominal photoluminescence quantum yield [PLQY] of 13% in dimethyl sulfoxide [DMSO]),44 the PLQYs of OTPA-TQ1-3 NPs are measured to be 2.5%, 1.8%, and 2.7%, respectively. These values are comparable to the recent reported organic emitters with similar emission range, and much higher than the widely used carbon nanotubes (0.4%).45,46 The changes of PLQYs are in the same trend as the aAIE values (Table S1), suggesting that AIE characteristic is vital for realizing highly luminescent organic NPs. To better understand the AIE characteristics and photophysical processes of the molecules, we measured the PL lifetime in solution and NPs forms (Figure S33; Table S3), and the corresponding radiative and nonradiative decay rates are presented in Table S4. Of note, the radiative decay rates increase slightly from solution to NPs

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state, while the nonradiative decay rates decrease significantly. In solution, the molecules undergo active intramolecular motions, which would consume the excitedstate energy and result in dominated nonradiative relaxation. In contrast, the molecular environment in the aggregate state imposes higher structural rigidity, which blocks the nonradiative decay channel of the excited state and the radiative one opens, giving rise to increased PL efficiency.34,47 The highest fluorescent brightness and aAIE value of OTPA-TQ3 NPs should be attributed to the most twisted molecular structure, which could reduce the intermolecular interactions within NPs. PL excitation (PLE) mapping was also performed to gain deep understanding about the excitation-emission relationships, and PLE maps of the organic NPs (Figures 2E, S34, and S35) manifest eligible excitation and emission in NIR region. Photoacoustic Properties of the Compounds and NPs We next studied the structure-PA property relationships of OTPA-TQ molecules and the influence of rotor size in PA signal output. Firstly, glycerol was employed to elevate solution viscosity on the intramolecular motions of the compounds, which has been confirmed by the increased PLQYs in viscous environments (Table S5). Noteworthy, OTPA-TQ3 exhibits the largest brightness enhancement, probably attribute to the prominent AIE signature. As shown in Figure 3A, the PA intensities of all three molecules decrease upon increasing the glycerol fraction in N,N-dimethylformamide (DMF)-glycerol mixtures, revealing the key role of intramolecular motions for PA signal output.48 It is also noted from Figure 3A that the PA amplitude of OTPA-TQ3 profoundly declines of 65% from pure DMF to 90% glycerol fraction, which is more pronounced than that of the other two derivatives (40% for OTPATQ1 and 50% for OTPA-TQ2). This result suggests that the excited-state intramolecular motions of larger molecular rotors contributes greater on PA generation. The PA spectra of OTPA-TQ1-3 NPs were recorded by measuring the PA intensity at different wavelengths from 680 to 950 nm. As shown in Figure 3B, the PA spectra of OTPA-TQ1-3 NPs match well with the absorption profiles (Figure 2C), indicating that the PA signals are produced from the NIR absorption of the molecules. Under the irradiation of 680 nm NIR pulsed laser, for each OTPA-TQ molecule (50 mM), its NPs state (140 nm by DLS) shows around 2.5-fold higher PA intensity than its bare aggregate (without lipid-PEG2000) state in THF/water mixture with 95% water fraction (the aggregates possess broad size distribution of >400 nm measured by DLS). As the amphiphilic co-polymer lipid-PEG2000 acting as the surfactant can essentially improve the water solubility of the hydrophobic molecules and provides much larger specific surface area, which permits more effective excited-state intramolecular motions in aqueous media, and this result further validates that there is a positive correlation between excited-state intramolecular motion and PA signal output,29,48 in good accordance with that of Figure 3A. When comparing the PA amplitudes among the three OTPA-TQ NPs, as depicted in Figure 3C, OTPA-TQ3 NPs exhibit the highest PA intensity, which is about 1.4-fold higher than the other two counterparts NPs. This could be ascribed to that the large phenyl-alkyne-phenyl rotors lead to stronger intramolecular motions43 and thus generate stronger PA signal, agreeing well with Figure 3A. The higher absorption coefficient of OTPA-TQ3 might also account for the pronounced fluorescence and PA performance. The PA amplitude of OTPA-TQ NPs was also compared with the well-known high-performing PA imaging agents including semiconducting polymer NPs (SPNs) (Figure S36) and methylene blue (MB).15,49 Since MB, SPNs, and OTPATQ1-3 NPs have similar absorption at about 680 nm (Figure S37), rational comparison in the same concentration (50 mM) was allowed using a 680 nm pulsed laser. As

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Figure 3. PA Properties of the Compounds and NPs (A) Relative PA intensity of OTPA-TQ molecules (50 mM) in DMF-glycerol mixtures with different glycerol fractions at 680 nm. (B) PA spectra of OTPA-TQ1-3 NPs (50 mM). (C) PA intensity of MB, semiconducting polymer NPs (SPNs), and OTPA-TQ1-3 NPs with the same concentration (50 mM) at 680 nm. The molar concentration of SPNs is based on the repeating unit. Data are presented as the means G SD (n = 3). (D) PA amplitudes of OTPA-TQ3 NPs as a function of concentration. Data are presented as the means G SD (n = 3).

shown in Figure 3C, under the same experimental condition, the PA amplitude of OTPA-TQ3 NPs is 1.7-fold and 2.4-fold higher than that of SPNs and MB, respectively. Furthermore, the PA intensity of OTPA-TQ3 NPs shows a good linear relationship with the molar concentration of OTPA-TQ3 (Figure 3D), indicating the potential for quantitative analysis. Raman Properties of the Compounds and NPs The alkyne group is introduced into OTPA-TQ2 and OTPA-TQ3 molecules as it has been well accepted to produce Raman signature in the cell-silent region.50,51 Figure 4A displays the Raman spectra of the OTPA-TQ1-3 NPs. Noteworthy, OTPATQ3 NPs other than the other two NPs possess rather strong Raman signal at 2,215 cm 1, which is in the cell-silent region and refers to the typical carbon-carbon triple bond stretching and vibration signature of alkyne groups. The strong Raman signal of OTPA-TQ3 is probably ascribed to the large conjugation of the phenylalkyne-phenyl units as confirmed by DFT calculation (Figure S28),52,53 warranting it an efficient biomarker for highly specific Raman imaging with negligible

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Figure 4. Raman Properties of the Compounds and NPs (A) Raman spectra of OTPA-TQ1-3 NPs (100 mM, excitation: 532 nm). (B) Raman intensity of OTPA-TQ3 (50 mM, excitation: 532 nm) in pure THF, THF-water mixture with 10% THF fraction, and encapsulated NPs.

background. Furthermore, the influence of intramolecular motions on Raman property was studied by investigating the Raman spectra of OTPA-TQ3 in the forms of free molecule (in THF solution), large aggregate (in THF-water mixture with 90% water fraction) and NPs. As depicted in Figure 4B, the OTPA-TQ3 NPs show much stronger Raman intensity at 2,215 cm 1 than the large aggregates, whereas the free molecule has the highest Raman signal, which is in accord with previous reports that molecular motion brings about Raman scattering.54,55 As the polymer surfactant lipid-PEG2000 favors improved solubility of OTPA-TQ3 in water, benefiting to promoted intramolecular motions, this result implies that the Raman intensity from phenyl-alkyne-phenyl in the unconfined free-motion form would be stronger than the restricted aggregation. From the basic theory of Raman scattering, after light absorption, the molecules are excited to a virtual energy state.56 In our case, the wavelength of the incident light (532 nm) could also lead to the electronic transition of OTPA-TQ3 molecule. Therefore, the result in Figure 4B suggests that intramolecular motions in the aforementioned high energy state would significantly enhance the Raman signal from phenyl-alkyne-phenyl. The interplay of fluorescence, PA and Raman properties, and the corresponding photophysical processes in different states are depicted in Scheme S3. By gradually activating the intramolecular motions (e.g., from large aggregate to NPs state and further to THF solution), the PA and Raman properties are enhanced, while the fluorescence is influenced adversely. The twisted 3D molecular structure and pronounced AIE effect enable high NIR fluorescent brightness in aqueous media. The strong excited-state intramolecular motions and high absorption coefficient of OTPA-TQ3 are responsible for the strong PA generation capability. Moreover, the conjugated phenyl-alkyne-phenyl units and intramolecular motions warrant OTPATQ3 strong Raman signal in the cell-silent region. Accordingly, the best imaging performance of OTPA-TQ3 can be attributed to both the large conjugated substitutes and intramolecular motions. Stability of the NPs Photostability is of critical importance for optical imaging agents; hence, we investigated the photobleaching resistance capacities of OTPA-TQ1-3 NPs under light irradiation and the clinically used MB was used as a control. After exposure to

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Figure 5. Stability of the NPs (A) Photostability of the NPs and MB under continuous light (650 nm, 200 mW cm 2 ) irradiation. A and A0 are the maximal PL intensity of OTPA-TQs NPs and MB without and with light irradiation. (B) Plot of I/I 0 versus RONS (ClO and ,OH, 1 mM) treatment. I and I 0 are the maximal PL intensity of OTPA-TQs NPs and ICG (black, OTPA-TQ1 NPs; blue, OTPA-TQ2 NPs; red, OTPA-TQ3 NPs; green, ICG) in PBS solutions in the presence and absence of RONS, respectively. Data are presented as the means G SD (n = 3). (C) Plots of photoacoustic intensity of OTPA-TQ3 NPs in a phantom (50 mM) against number of laser pulses at 700 nm (2.4 3 10 4 pulses; 17.5 mJ cm 2 laser and 10 Hz pulse repetition rate).

continuous red light (650 nm, 200 mW cm 2) irradiation for 60 min, the absorption and emission properties of all three NPs remain constant, whereas MB dye with similar absorption maximum is easily photobleaching, as evidenced by the PL intensity decreasing to 55% of the original value (Figure 5A). Additionally, reactive oxygen and nitrogen species (RONS) are a kind of important signaling molecules closely related to body health, which are also known to overexpress in many diseased regions, e.g., cancer, inflammation, and cardiovascular diseases.57,58 As a consequence, stable optical probes that are resistant to RONS are momentous for in vivo disease detection. As presented in Figure 5B, all the OTPA-TQ1-3 NPs show excellent resistance to various RONS. In contrast, the FDA-approved ICG is severely destroyed in the presence of ClO and ,OH, which is likely due to the degradation of alternatively arranged single-double bonds. We further studied the probe stability under PA experiment condition. After exposure to 2.4 3 104 pulses (17.5 mJ cm 2 laser and 10 Hz pulse repetition rate), nearly no PA signal loss is observed (Figure 5C), indicating good photostability and suitability for PA imaging. Noteworthy, the NPs also show good colloidal stability, as no precipitation is observed after storage at room temperature for 2 weeks, and the average diameters nearly do not change as well (Figure S38). Taken together, these results reveal that the organic nanoagents possess superb stability with various treatments, which are suitable for the long-term in vivo applications. Preoperative Fluorescence and PA Imaging of Tumors The aforementioned results have reasonably demonstrated that OTPA-TQ3 NPs possess the optimized fluorescence, PA, and Raman signals in aqueous environment. Thus, the OTPA-TQ3 NPs were used for the following in vivo utilization to investigate whether such an optical agent with excellent fluorescence-PA-Raman properties could be beneficial to precise image-guided cancer surgery. As preoperative imaging requires both good spatial resolution and high sensitivity to reveal the size, number, and location of tumors in vivo, both NIR fluorescence imaging and PA imaging were performed before surgery with 4T1 subcutaneous tumor-bearing mice by intravenous administration of OTPA-TQ3 NPs. After NPs injection, the

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Figure 6. Preoperative Fluorescence and PA Imaging of Tumor-Bearing Mice In Vivo (A) Representative fluorescence images of tumor-bearing mice after intravenous injection of OTPA-TQ3 NPs (200 mL, 650 mM based on OTPA-TQ3) at different time points as indicated. (B) Fluorescence intensity of the tumor site as a function of post-injection time. Data are presented as the means G SD (n = 3 mice). (C) Fluorescence intensities of slices of tumor and main organs (heart, liver, spleen, lung, and kidneys) resected from the tumor-bearing mice at 24 h post-injection. All the tissues were sliced with the same thickness of 1 mm for fluorescence imaging and quantitative analysis in order to better eliminate the influence of tissue penetration depth. Data are presented as the means G SD (n = 3 mice). (D and E) Shown in (D) are representative PA images and in (E) the corresponding PA intensity of tumor site after intravenous administration of OTPATQ3 NPs at different time points as indicated. Data are presented as the means G SD (n = 3 mice).

tumor-bearing mice were concurrently scanned by IVIS imaging system and smallanimal opt-acoustic tomography system (MOST) at designated time intervals. The time-dependent in vivo non-invasive NIR fluorescence images are shown in Figure 6A, and the corresponding fluorescence intensity-time relationship in tumor is depicted in Figure 6B, which reveal that the NIR fluorescence signal at tumor site becomes intense gradually as the time elapses stemming from the passive enhanced permeability and retention (EPR) effect.59 The outstanding EPR effect of OTPATQ3 NPs should be benefited from their appropriate size and surface chemistry. Besides tumor, the reticuloendothelial system (RES) organs including liver and spleen

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where the NPs are prone to accumulate are also lighted up (Figures 6C and S39).60 At 24 h post-injection, the fluorescence signal at tumor site reaches the maximum and the tumor signal-to-background (surrounding normal skin autofluorescence) ratio is as high as 9.2, which is considered to be very high according to the literatures.61 On the other hand, the mice were also imaged by PA instrument within 24 h study duration. As shown in Figure 6D, the PA signal in tumor gradually amplifies over time and arrives at the peak at 24 h post-injection, coinciding well with the trend of time-dependent fluorescence imaging results (Figures 6A and 6B). Noteworthy, the average PA signal in tumor at 24 h is 7.0 times higher than the background (0 h) before NPs injection (Figure 6E), giving better performance than many highperforming PA imaging agents.37,62 Both preoperative NIR fluorescence imaging and PA imaging indicate that the OTPA-TQ3 NPs can detect the tumors in vivo in an extremely high-contrast manner at 24 h post-injection, providing the surgeon with important information on surgical plan. Intraoperative Fluorescence and Raman Imaging of Tiny Residual Tumors Next, tumor resection surgery was conducted by a surgeon from Tianjin First Central Hospital (Tianjin, China) based on the information by preoperative imaging. In the clinic, the most challenging issues for the surgeon during surgery are to evaluate whether there are residual tumors left behind post-major tumor excision as well as to differentiate the boundaries between normal and tumor tissues.2,63 Thereby, the combination of fluorescence and Raman imaging holding the integrated advantages of fast and real-time mode, excellent sensitivity and high signal-to-background ratio are desirable during cancer operation. In our case, the first surgery (S1) was performed by the surgeon with his experience after the OTPA-TQ3 NPs were intravenously injected into 4T1 tumor-bearing mice for 24 h, which was followed by NIR fluorescence and Raman imaging at the same time. In case that the tumors are totally removed by the surgeon, as confirmed by hematoxylin and eosin (H&E) histological analyses, there are no fluorescence and Raman signals at the surgical incision sites. In other cases that there are tiny tumors left behind, a certain degree of fluorescence signal can be observed (Figure 7A). Although OTPA-TQ3 NPs possess rather high NIR fluorescence, the volume of residue tumors is tiny, leading to only small amount of NPs in them. Besides, the normal tissue autofluorescence compromises the signal-to-background ratio as well. Thus, the surgeon cannot accurately assess whether the weak fluorescent areas are indeed tumors. Even so, intraoperative fluorescence imaging is quite necessary, as it is fast, sensitive, and real time and can rapidly point out the suspicious areas of residual tumors. To pursue precise surgical treatment, Raman imaging with microscopic resolution was conducted in the suspicious areas with faint fluorescence. As illustrated in Figure 7B, the OTPA-TQ3 NPs with strong Raman signal (2,215 cm 1) in the cell-silent region can sensitively visualize the residual tumors and their boundaries to normal tissues by Raman imaging with ‘‘yes-or-no’’ signature. Such excellent effectiveness of residual tumor detection during surgery should be attributed to both the high Raman signal of OTPA-TQ3 NPs and the innate zero-background nature of Raman imaging in cell-silent region. It is noted that 94% of the tested tiny areas with Raman signal are confirmed as tumors when consulting the H&E histological analysis (Figure 7C). As Raman imaging is much faster than histological analysis, it holds great promise for intraoperative residual tumor inspection.6,64 Noteworthy, the intraoperative fluorescence-Raman imaging with OTPA-TQ3 NPs can clearly delineate tiny residual tumors after S1 with diameters of about 450 mm (Figure S40). After demonstrating the existence of residual tumors, the surgeon could perform the second surgery (S2) to remove the residual tumors until there are no fluorescence and Raman signals (Figures 7D–7F).

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Figure 7. Intraoperative Fluorescence and Raman Imaging of Tiny Residual Tumors (A) Representative fluorescence images of the OTPA-TQ3 NPs-treated tumor-bearing mice before and after S1 treatment. (B and C) Shown in (B) is Raman imaging and in (C) H&E-stained tissues of the operative incision site after S1. (D) Representative fluorescence images of the OTPA-TQ3 NPs-treated tumor-bearing mice before and after S2 treatment. (E and F) Shown in (E) is Raman imaging and in (F) are H&E-stained tissues of the operative incision site after S2. (G) Survival curves for the tumor-bearing mice after various treatments as indicated (n = 10 mice per group). (H and I) Representative H&E-stained images of the lung slices from the dead mice are shown in (H) Control, and in (I) are S1 groups. The black dashed lines circle the tumor areas. (J) Representative H&E-stained images of the lung slices from the mice in S2 group that survived 60 days.

The survival rates of mice after S1 (with fluorescence and Raman signals at the surgical incision sites) and S2 (without any signals), respectively, were monitored with the mice received no surgical treatment as the control. As shown in Figure 7G, the mice without surgical treatment and only undergoing S1 all died within 40 days post-surgery. In contrast, the 10 mice in S2 group survived 40 days, however, 2 mice were then dead on day 43 and 47 after surgery, respectively. The other 8 mice survived during 60-day study duration. Given that it has been well accepted that 4T1 tumor model has aggressive metastatic potential,65 the influence of lung metastasis on survival rates was assessed. In this experiment, the lung organ of each dead mouse in the 3 groups was excised, sliced, and stained with H&E for histological analysis. As displayed in Figures 7H and 7I, it is found that almost all the dead mice in the Control and S1 groups suffer from lung metastases, as confirmed by the H&E staining. Moreover, the primary tumors from mice in these two groups also grew bigger as the time elapsed. Therefore, the mice in Control and S1

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cohorts died probably attributed to both the lung metastases and primary tumor growing. On the other hand, for S2 group, since there was no primary tumor recurrence observed for the 2 dead mice, the lung metastases were examined. As shown in Figure S41, obvious lung metastases are observed, which should be the main cause of death for these 2 mice. Furthermore, the other 8 surviving mice in S2 group were sacrificed at the end of the study (on day 60 after surgery), and the histological analyses reveal neither primary tumor recurrence nor obvious lung metastases (Figure 7J). We then investigated the possible reason for the less lung metastases in S2 group. Since the surgeries (both S1 and S2) were carried out on day 9 after the 4T1 cancer cells were injected subcutaneously into the mouse axillary space, the lung tissues were examined with H&E on day 9 post-cancer cell inoculation. As shown in Figure S42A, the H&E-stained lung slices show negligible tumor metastases on this day, implying that the lung metastases may not significantly occur at this time point. It is also found that distinct lung metastases can be observed on day 21 after 4T1 cancer cell inoculation (Figure S42B). Therefore, the complete tumor resection was performed at the relatively early stage of cancer, which would greatly reduce the risk of subsequent lung metastases, leading to 8 of 10 mice in S2 group surviving 60 days. Biocompatibility is critically important for molecular probes, so we studied the biosafety and excretion of OTPA-TQ3 NPs. The cellular viability was evaluated by co-culturing the NPs with different cell lines (4T1 cancer cells, NIH 3T3 cells, and Detroit 551 human fibroblast cells, respectively). As depicted in Figure S43, all the cells treated with a high concentration of NPs (30 mM) display good viability of higher than 90%, suggesting low cytotoxicity. The hepatic and renal function analyses (Figure S44) and blood routine examination (Figure S45) of the OTPA-TQ3 NPs-treated mice also show no noticeable abnormalities, indicating the low in vivo toxicity. To evaluate how the NPs are cleared from the body, after intravenous administration of OTPA-TQ3 NPs into the healthy mice, the feces and urine of the mice were then collected and imaged at designated time intervals. As shown in Figure S46, significant fluorescence signal from the OTPA-TQ3 NPs can be observed in the collected feces within 7 days after injection, demonstrating that the NPs are mainly excreted from the body through biliary pathway, i.e., from liver to bile duct, intestine, and finally to feces.37,66 On the other hand, there is negligible fluorescence signal in the urine, indicating that the NPs would not be cleared via renal pathway. Taken together, these results indicate that the NPs have good biocompatibility and no obvious side effect is observed. Systematic investigation may be required to study the long-term fate and biocompatibility of the organic nanoprobes in the future. To study the influence of agent dose on the imaging performance, different concentrations (0, 81, 162, 325, and 650 mM) of OTPA-TQ3 NPs with the same volume of 200 mL were intravenously injected into the tumor-bearing mice, and the PA, fluorescence and Raman imaging were investigated. To make a rational comparison, the imaging was carried out in the same condition as indicated in the experimental procedures. Generally, the signal intensity intensifies with increased concentration (Figures S47 and S48). For preoperative PA imaging, the high concentration of 650 mM can provide detailed information about tumor size in a high-contrast manner, while other lower concentrations are not able to illuminate the tumor site clearly. The intraoperative fluorescence imaging could only illuminate residual tumors with the high-concentration nanoagent, possibly because there is tissue autofluorescence and a low NPs concentration in the tiny residual tumor nodules, while Raman imaging can provide a better signal in a relatively low nanoagent concentration, probably because of the zero-background signature in the cell-silent region. As a

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consequence, a concentration of 650 mM could provide the most comprehensive information about tumor at different surgical stages, and no obvious side effect was observed, representing the optimal experimental condition for image-guided tumor surgery. We hope that these comparisons would provide useful guidance for other organic molecular probes.

DISCUSSION We have developed a kind of one-for-all molecular agent for comprehensive imageguided surgery application. The fluorescence, PA, and Raman properties in one organic molecule are greatly impacted by the molecular structure and intramolecular motion, as they have an effect on the photophysical properties, and the corresponding optical imaging performance. As compared with the other two compounds, OTPA-TQ3 with the largest intramolecular rotation units has the most twisted 3D molecular structure and the strongest AIE effect, benefiting to the highest NIR fluorescent brightness in aqueous media. Moreover, the strong excited-state intramolecular motions and high absorption coefficient endow OTPA-TQ3 with the strongest PA signal generation capability. Furthermore, the phenyl-alkyne-phenyl units warrant OTPA-TQ3 NPs strong Raman signal at 2215 cm 1 in the cell-silent region, and the intramolecular motions in high energy state after light excitation are demonstrated to significantly enhance the Raman signal. Taking advantages of the high sensitivity of fluorescence imaging and good spatial resolution and penetration depth of PA technique, the intravenously administrated OTPA-TQ3 NPs enable tumor detection preoperatively and then guidance of surgical plan. Further intraoperative imaging during cancer surgery manifests that the OTPA-TQ3 NPs can help the surgeon accurately remove all of the tiny residual tumors by virtue of the fast, real-time, and sensitive fluorescence imaging and high-contrast Raman imaging with zero background, which greatly prolong the lifetimes of mice post-surgery. Moreover, the organic nanoprobe shows good biocompatibility, and no side effect was observed from both the in vitro and in vivo tests. This work represents the first example of boosting fluorescence-PA-Raman properties in one organic fluorophore, which allows for accurate cancer imaging and resection, rendering great promise for integrated multi-modality imaging applications. There are still several aspects that can be considered for meeting the clinical translation of multi-modality optical imaging agent. First, the probes with longer excitation and emission wavelengths would be better, for example, to increase the absorption and emission wavelength of the probes to the recently developed second NIR window would enable larger penetration depth, and higher spatial resolution. Second, other post-surgery treatment (e.g., immunotherapy) may be needed to combine with the multi-modality image-guided precision surgery, especially for patients with metastases, to inhibit both the primary tumors and metastatic tumors effectively. Third, new instrumentation that integrating the complex measurements and data analyses in one platform could definitely facilitate in situ monitoring and improve the performance of multi-modality imaging, benefiting for clinical use.

EXPERIMENTAL PROCEDURES Materials and Characterizations All the chemicals and reagents were purchased from chemical sources and were used as received. The nuclear magnetic resonance (NMR) spectra were recorded on a Bruker AV 400 spectrometer. HRMS were measured with a GCT premier CAB048 mass spectrometer in matrix assisted laser desorption ionization-time of flight (MALDI-TOF) mode. The theoretical calculation was carried out at the level of B3LYP/6-31G* using DFT method with the Gaussian 09 program package (the

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Cartesian coordinates of OTPA-TQ1-3; see Tables S6–S8). The ultraviolet-visible (UV-vis) absorption spectra were performed using a Shimadzu 2550 UV-vis scanning spectrophotometer. The steady-state PL spectra were conducted on a Horiba Fluorolog-3 spectrofluorometer. Transient PL at room temperature was measured using an Edinburgh FLS1000 fluorescence spectrophotometer with a photodetector of PMT-980. Raman spectra were acquired by a confocal Raman microspectroscopy with a 532 nm excitation (Renishaw). DLS was measured on a 90 plus particle size analyzer. TEM images were obtained from a JEM-2010F transmission electron microscope with an accelerating voltage of 200 kV. Preparation of the NPs 1 mg of the organic compound and 2 mg of amphiphilic lipid-PEG (DSPE-PEG2000) were dissolved in 1 mL of THF. The obtained THF solution was poured into 9 mL of deionized water under sonication with a microtip probe sonicator (XL2000, Misonix Incorporated, NY). The mixture was then sonicated for another 1 min and violently stirred in fume hood overnight at room temperature to evaporate residue THF, and the obtained NPs solution was used directly. Photoacoustic Properties The PA properties were studied by using a multi-spectral optoacoustic tomography (iTheraMedical, Germany), which was equipped with a wavelength-tunable (680–980 nm) optical parametric oscillator pumped by a Nd:YAG laser with excitation pulses of 7 ns duration at a repetition rate of 10 Hz. The light from the fiber covered an area of 4 cm2 with a maximum incident pulse energy of approximately 70 mJ (100 mJ, 70% fiber coupling efficiency). This generated an optical fluence of 17.5 mJ cm 2, which was well within the safe exposures according to the American National Standard for Safe Use of Lasers. The PA intensity was measured by finely analyzing the regions of interest of acquired images. PA spectra of the NPs solutions were obtained by recording the PA signals at different wavelengths from 680 nm to 950 nm (10 nm wavelength for each slice). The relationship between PA intensity and NPs concentration was established by using various concentrations of NPs solutions (10, 25, 50, 100, and 200 mM). The comparison of PA intensity of different agents (50 mM) was conducted by the excitation of 680 nm pulsed laser. The probe stability during PA experiment was evaluated by scanning OTPA-TQ3 NPs in a phantom (50 mM) with 2.4 3 104 of laser pulses at 700 nm (17.5 mJ cm 2 laser and 10 Hz pulse repetition rate). Cytotoxicity Study 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-tetrazolium bromide (MTT) assay was used to evaluate the cytotoxicity of the NPs in different cell lines. 4T1 breast cancer cells, NIH 3T3 cells and Detroit 551 human fibroblast cells were respectively harvested in a logarithmic growth phase and seeded in 96-well plates (5000 cells per well with 100 mL suspension) for 24 h and grew to 80% confluence. Then the culture medium was replaced with 100 mL of fresh culture medium containing OTPA-TQ3 NPs with various concentrations (the concentrations based on OTPA-TQ3 are: 0 mM, 1.5 mM, 3 mM, 6 mM, 15 mM, and 30 mM), separately. After incubating for 24 h, the culture medium was removed and the wells were washed three times with PBS, and 100 mL of MTT dissolved in serum-free culture medium (0.5 mg/mL) was added into each well. After 4 h, the MTT solution was removed cautiously and 100 mL of DMSO was added into the wells, followed by gently shaking for 10 min. Then, the absorbance of MTT was measured by a Bio-Rad 680 microplate reader at 490 nm to evaluate the viability of cells inside.

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Animal Experiments All animal studies were conducted under the guidelines set by Tianjin Committee of Use and Care of Laboratory Animals, and the overall project protocols were approved by the Animal Ethics Committee of Nankai University. Tumor-Bearing Mice 6-Week-old female BALB/c mice were obtained from the Laboratory Animal Center of the Academy of Military Medical Sciences (Beijing, China). To establish the xenograft 4T1 tumor-bearing mouse model, 4T1 breast cancer cells (1 3 106) suspended in 30 mL of RPMI-1640 medium were injected subcutaneously into the right axillary space of the BALB/c mouse. After about 9 days, mice with tumor volumes of about 80–120 mm3 were used subsequently. Preoperative Fluorescence and Photoacoustic Imaging The xenograft 4T1 tumor-bearing female mice were used for the following experiments (n = 3 mice for quantitative analyses of all imaging modalities). The tumorbearing mice were anesthetized using 2% isoflurane in oxygen, and OTPA-TQ3 NPs (200 mL, 650 mM based on OTPA-TQ3) were intravenously injected into the tumor-bearing mice using a microsyringe. Then in vivo NIR fluorescence and PA imaging were concurrently carried out to provide comprehensive information about the tumor. For fluorescence imaging, it was performed with the Maestro EX fluorescence imaging system (CRi, Inc.) with excitation at 704 nm and signal collection in the spectral region of 740–950 nm. For PA imaging, it was performed with the same mice as fluorescence imaging on a commercial small-animal opt-acoustic tomography system (MOST, iTheraMedical, Germany). The PA data were acquired at 700 nm excitation (17.5 mJ cm 2 laser and 10 Hz pulse repetition rate), after which the images were reconstructed using the model-based algorithm supplied within the ViewMSOT software suite (V3.6, iThera Medical). The fluorescence and PA images were recorded at designated time intervals after injection. Tumor Resection and Intraoperative Fluorescence-Raman Imaging Based on the information provided by preoperative fluorescence and PA imaging at 24 h post-injection, the tumors were resected (n = 20 mice). Briefly, the tumorbearing mice were anesthetized using 2% isoflurane in oxygen. With the experience of a surgeon, the tumor tissues were aseptically prepped and sterile instruments were employed to excise the tumors (S1). The operative incision sites were followed by NIR fluorescence imaging to detect if there were residual tumors left behind. The fluorescence imaging was performed similar to the aforementioned in vivo case (Maestro EX fluorescence imaging system with excitation at 704 nm and signal collection in the spectral region of 740–950 nm). The tissues at the operative incision sites were subsequently dissected and sliced, and the frozen sections were used for Raman microscopy and H&E staining. For Raman microscopy, frozen sections were placed on quartz slides (Ted Pella, Inc.) and air-dried. A 503 or 123 objective lens was used, and Raman spectral maps and correlating white light images were acquired using the Renishaw Streamline function (excitation at 532 nm, the irradiated power was 2 mW and the exposure time for each line was 1 s). The Raman spectra were analyzed by least squares analysis using Wire 2.0 software (Renishaw). At the same time, H&E staining was performed to confirm whether there were residual tumors in the operative sites left behind after surgery, and the obtained slices were examined by a digital microscope (Leica QWin). After demonstrating the existence of residual tumors after the first surgery (S1), the second surgery (S2) was performed to remove the tumors until there were no fluorescence and Raman signals, as well as confirmed by H&E staining.

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Histological Analysis After S1 and the intraoperative imaging with fluorescence and Raman techniques, tissues at the operative sites were collected and fixed in 4% paraformaldehyde at 4 C overnight. Then the samples were embedded in paraffin, sliced at a thickness of 5 mm, and the slices were stained with H&E, and imaged by an optical microscopy (Leica QWin). After S2, the surgical sites were also examined by H&E staining in a similar manner. Survival Rate Study The xenograft 4T1 tumor-bearing mice were randomly selected and then divided into three groups (n = 10 mice per group), which were named ‘‘Control,’’ ‘‘S1 with FL-RA signals,’’ and ‘‘S2 without any signals,’’ respectively. For the ‘‘Control’’ group, the mice were intravenously injected with OTPA-TQ3 NPs (200 mL, 650 mM based on OTPA-TQ3). For the ‘‘S1 with FL-RA signals’’ group, after the intravenous injection of OTPA-TQ3 NPs (200 mL, 650 mM based on OTPA-TQ3) for 24 h, the tumor-bearing mice were imaged with fluorescence and PA imaging, and tumor resection surgery was conducted by a surgeon with his experience (S1), but there were still fluorescence and Raman signals at the operative sites. For the ‘‘S2 without any signals’’ group, after the intravenous injection of OTPA-TQ3 NPs (200 mL, 650 mM based on OTPA-TQ3) for 24 h, the tumor-bearing mice were imaged with fluorescence and PA imaging, and tumor resection surgery was conducted by a surgeon with his experience (S1). With the information provided by the intraoperative fluorescence and Raman imaging, the second surgery (S2) was performed, after which no fluorescence and Raman signals could be detected. The survival rates were monitored within 60-day study duration. Serum Biochemistry Assay and Complete Blood Count Healthy BALB/c mice were randomly assigned to two groups (n = 4 per group). On day 0, mice in one group were intravenously injected with 200 mL of OTPA-TQ3 NPs (650 mM based on OTPA-TQ3), and mice in another group was not treated for control. On day 7, all mice were sacrificed, and their blood was collected from the arteriae ophthalmica. The blood samples were utilized for blood routine examination and hepatic and renal function analyses, respectively. Statistical Analysis Statistical analysis was performed by GraphPad Prism. Quantitative data were expressed as means G standard deviation (SD). One-way ANOVA and unpaired Student’s t test were utilized for statistical analyses. p Value < 0.05 was considered statistically significant.

SUPPLEMENTAL INFORMATION Supplemental Information can be found online at https://doi.org/10.1016/j.chempr. 2019.07.015.

ACKNOWLEDGMENTS This work was supported by the National Science Foundation of China (51622305, 21788102, and 51873092), the National Basic Research Program of China (2015CB856503), the Research Grants Council of Hong Kong (C6009-17G, C2014-15G, A-HKUST605/16, 16308016, and 2018YFE0190200), the Innovation and Technology Commission (ITC-CNERC14SC01 and ITS/254/17), the Fundamental Research Funds for the Central Universities, Nankai University, and the

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Science and Technology Plan of Shenzhen (JCYJ20160229205601482 and JCY20170818113602462).

AUTHOR CONTRIBUTIONS D.D. and B.Z.T. conceived and designed the study. J.Q. synthesized and characterized the compounds. J.Q. and J.L. performed the NPs preparation and in vitro experiments. J.L., R.L., and Q.L. performed the in vivo experiments. H.Z. provided technical assistance with the theoretical calculation. J.Q., J.L., J.W.Y.L., R.T.K.K., D.L., D.D., and B.Z.T. analyzed the data and participated in the discussion. J.Q., J.L., D.D., and B.Z.T. contributed to the writing of this paper.

DECLARATION OF INTERESTS The authors declare no competing interests. Received: December 4, 2018 Revised: January 28, 2019 Accepted: July 17, 2019 Published: August 8, 2019

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