Ce6-Modified Carbon Dots for Multimodal-Imaging-Guided and Single

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Biological and Medical Applications of Materials and Interfaces

Ce6-Modified Carbon Dots for Multimodal-Imaging-Guided and Single-NIR-Laser-Triggered Photothermal/Photodynamic Synergistic Cancer Therapy by Reduced Irradiation Power Shan Sun, Jingqin Chen, Kai Jiang, Zhongdi Tang, Yuhui Wang, Zhongjun Li, Chengbo Liu, Aiguo Wu, and Hengwei Lin ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b19042 • Publication Date (Web): 16 Jan 2019 Downloaded from http://pubs.acs.org on January 17, 2019

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ACS Applied Materials & Interfaces

Ce6-Modified Carbon Dots for Multimodal-Imaging-Guided and Single-NIR-Laser-Triggered Photothermal/Photodynamic Synergistic Cancer Therapy by Reduced Irradiation Power

Shan Sun,†,§,‖ Jingqin Chen,‡,‖ Kai Jiang,† Zhongdi Tang,† Yuhui Wang,† Zhongjun Li,Δ Chengbo Liu,*,‡ Aiguo Wu,† and Hengwei Lin*,†

†Key

Laboratory of Graphene Technologies and Applications of Zhejiang Province,

Ningbo Institute of Materials Technology & Engineering, Chinese Academy of Sciences, Ningbo 315201, P. R. China

‡Institute

of Biomedical and Health Engineering, Shenzhen Institutes of Advanced

Technology, Chinese Academy of Sciences, Shenzhen 518055, P. R. China

ΔCollege

of Chemistry and Molecular Engineering, Zhengzhou University, Zhengzhou,

45001, P. R. China

§University

‖These

of Chinese Academy of Sciences, Beijing, 100049, P. R. China

two authors contributed equally to this work.

*[email protected] (C.L.); [email protected] (H.L.)

Keywords: carbon dots, photothermal, photodynamic, synergistic therapy, multimodal imaging, single-NIR-laser irradiation, reducing irradiation power

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ABSTRACT Photo-mediated cancer therapy, mainly including photothermal therapy (PTT) and photodynamic therapy (PDT), have received tremendous attention in recently years thanks to their noninvasive and stimuli-responsive features. The single mode of PTT or PDT, however, shows obvious drawbacks, either requiring high power laser irradiation to generate enough heat or only providing limited efficacy due to the hypoxia nature inside tumors. In addition, the reported synergistic PTT/PDT usually utilized two excitation sources to separately activate PTT and PDT, and the problem of high power laser irradiation for PTT was still not well solved. Herein, a new concept, loading a small amount of photosensitizers (PSs) onto a PTT agent (both of them can be triggered by a single near-infrared (NIR) laser) was proposed to evade the shortcomings of PTT and PDT. To validate this idea, minute quantities of photosensitizer chlorin e6 (Ce6) (0.56% of mass) were anchored onto amino-rich red emissive carbon dots (RCDs) that possessing superior photothermal (PT) character under 671 nm NIR laser (PT conversion efficiency to be 46%), and meanwhile the PDT of Ce6 can be activated by this laser irradiation as well. The findings demonstrate that Ce6-modified RCDs (named Ce6-RCDs) offer much higher cancer therapy efficacy under a reduced laser power density (i.e., 0.50 W·cm-2 at 671 nm) in vitro and in vivo than that of the equivalent RCDs or Ce6 under the same irradiation conditions. Besides, the Ce6-RCDs also exhibit multimodal imaging capabilities (i.e., fluorescence (FL), photoacoustic (PA) and PT), which can be employed for guidance of the phototherapy process. This study suggests not only a strategy to enhance cancer phototherapy efficacy, but also a promising candidate (i.e., Ce6-RCDs) for multimodal FL/PA/PT imaging-guided and single-NIR-laser-triggered synergistic PTT/PDT for cancers by a reduced irradiation power.

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INTRODUCTION The high mortality of cancer makes it one of the most refractory and fatal diseases worldwide.1-2 According to the World Cancer Report, the cancer cases may increase to 15 million in the year of 2020.3 Therefore, novel diagnostic methods and effective therapies are urgently needed to cope with this intricate disease. Compared to the traditional cancer therapies such as surgery, radiotherapy and chemotherapy,4-6 phototherapy (e.g., photothermal therapy (PTT) and photodynamic therapy (PDT)) has received considerable attention due to its noninvasive and stimuli-responsive features.7-14 As one of the important modalities of light-triggered treatment, PTT, utilizing photo-absorbing molecules or nanoparticles to convert absorbed energy to heat and consequently causing local hyperthermia, is considered to be one of ideal methods for cancer therapy due to its high accuracy and low side effects.15-18 However, most of the previously reported PTT agents utilize visible light as excitation source, and this not only limited tissue penetration depth, but also led to severe phototoxicity.18-19 In addition, to deliver adequate heat into the internal site of a tumor, relatively high power densities of laser (e.g., 2 W·cm-2) were frequently applied.3,20-23 It’s obvious that the high power laser irradiation injures normal tissues adjacent to the tumor and reduces therapeutic accuracy, and as well as brings pains to patients during the treatment process.24 Hence, reduction of laser irradiation power for PTT is highly desirable for its clinical applications. Another important phototherapy method, PDT, which utilizes photosensitizers (PSs) to convert absorbed photons’ energy to produce reactive oxygen species (ROS), has also attracted lots of attention in recent years.25 The therapeutic outcome of this approach, however, is generally limited due to photo-decomposition of PSs under laser irradiation,11 the short lifetime and diffusion distance of ROS,26-28 and impaired singlet oxygen (1O2) production from local hypoxia in tumor.12,29-30 Thus, to compensate the consumption of PSs, large amounts of PSs were usually used, but this would induce additional side effects.31 In order to overcome the drawbacks of single-mode phototherapy and enhance its efficacy, combination of multiple therapy methods had been introduced.32-38 Among such attempts, great efforts were devoted to the fabrication of synergistic PDT/PTT platforms.11,39-42 Most of these examples, however, employed two excitation sources (for 3



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matching the different adsorption ranges of PSs and photothermal agents) to separately activate PTT and PDT,10,20-22,43 resulting in complicated setups and prolonged treatment time, and meanwhile not yet well solving the problem of high power laser irradiation for PTT. Therefore, the development of synergistic PDT/PTT strategies using a single near-infrared (NIR) laser by low power irradiation is highly desirable.11,25,44 Carbon dots (CDs), a newly emerged type of fluorescence (FL) nanomaterials, have received much attention in recent years thanks to their numerous merits (e.g., facile preparation, high photostability, excellent biocompatibility, easy surface modification and potential photoacoustic (PA), photothermal (PT) and photodynamic (PD) functions).45-54 Based on these superior properties, CDs are deemed to be promising candidates for construction of multifunctional cancer phototheranostic platforms.50 In order to take full advantages of CDs in biomedical applications, modulation of their absorption and emission in red and even NIR regions are significant for offering both FL imaging in deep tissues and potential PA and PT functions.55 Although several papers had been published recently utilized CDs to fabricate multifunctional theranostic platforms through combining FL/PA/PT imaging and PTT/PDT, the use of high power laser to trigger PTT cannot generally be avoided.21, 56 To further demonstrate the potential applications of CDs in cancer phototheranostics, herein, amino-rich red emissive CDs (RCDs) was prepared via a solvothermal method referring to our previous report.57 Furthermore, photosensitizer chlorin e6 (Ce6), that can effectively generate singlet oxygen (1O2) under light irradiation, was anchored onto the RCDs by a facile amide condensation reaction (Scheme 1a). Note that to decrease additional side effects and preserve the inherent features of the RCDs, only small amounts of Ce6 were loaded (i.e., 0.56% of mass). Except for the high photostability and superior biocompatibility, the Ce6 modified RCDs (named Ce6-RCDs) also hold the merits of PA and PT functions (PT conversion efficiency η=46% at 671 nm laser). Consequently, Ce6-RCDs are demonstrated to behave as a potential multifunctional phototheranostic agent featured FL/PA/PT imaging and synergistic PTT/PDT for cancers both in vitro and in vivo. As shown in Scheme 1b, after intravenous (i.v.) injection of the Ce6-RCDs into tumor-bearing mice via tail vein, the materials are prone to accumulate in tumor by 4


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enhanced permeability and retention (EPR) effects. Then, the Ce6-RCDs can enter into tumor cells through an internalization process and particularly localize in the cellular nucleoli. More importantly, due to the possibility of activation of the PTT (RCDs) and PDT (Ce6) by a single NIR laser (671 nm), the Ce6-RCDs were found to offer much higher cancer therapy efficacy with a reduced laser power (i.e., 0.5 W·cm-2) than that of the equivalent RCDs or Ce6 under the same irradiation conditions. In addition, it is worthy to note that the Ce6-RCDs are found to be able to penetrate through nuclear pores and selectively accumulate in RNA-rich cellular nucleoli, which might be partially responsible for their high efficacy for cancer. This study suggests a strategy to enhance cancer phototherapy efficacy by a relatively low laser irradiation power (i.e., loading a small amounts of PSs onto a PTT agent and triggering both of them by a single laser), and meanwhile proposed a promising multifunctional theranostic agent (i.e., Ce6-RCDs) for multimodal imaging-guided and single-NIR-laser-triggered cancer therapy.

Scheme 1. a) Schematic representation for the preparation of the amino-rich red emissive carbon dots (RCDs) and Ce6 modified RCDs (Ce6-RCDs); b) Schematic illustration of the Ce6-RCDs for simultaneous FL, PA and PT imaging and synergistic PTT/PDT process in vivo.

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RESULTS AND DISCUSSION Synthesis and characterization of the Ce6-RCDs Based on our previous report,57 the amino-rich RCDs were facilely synthesized through a solvothermal reaction of citric acid and polyethyleneimine in formamide. Ce6, selected as the PDT agent, can be conjugated onto the RCDs by a mild amide condensation reaction and then Ce6 modified RCDs (i.e., Ce6-RCDs) were obtained (detailed synthetic procedures being available in the Experimental Section). The morphologies of RCDs and Ce6-RCDs were firstly characterized by transmission electron microscopy (TEM). As shown in Figure 1a, the RCDs and Ce6-RCDs are monodispersed nanoparticles with average diameters about 3.4 nm and 3.7 nm, respectively. Their high resolution TEM (HR-TEM) images (insets in Figure 1a) present the same crystalline structure of the two materials with a lattice spacing of 0.21 nm, which is consistent with the (100) facet of graphene. Thus, we infer that the morphology of RCDs did not change obviously after the conjugation by Ce6. To further illustrate the particle sizes, diameters of the RCDs and Ce6-RCDs were investigated by dynamic light scattering (DLS). As shown in Figures S1 (Supporting Information, SI), the mean hydrodynamic diameters of RCDs and Ce6-RCDs are found to be around 9.8 nm and 9.9 nm, respectively. The relatively larger particle sizes of nanoparticles observed from DLS than that of the TEM are generally attributed to the formation of hydration layers on the surface of CDs in aqueous media and particles shrinkage during the preparation process for TEM testing.58-60 To analyze the composition and surface states of the RCDs and Ce6-RCDs, their Fourier transform infrared (FT-IR) spectra, X-ray photoelectron spectroscopy (XPS), Raman spectrum and Zeta potential were measured. In Figure 1b, the FT-IR spectrum of RCDs shows characteristic peaks at 1100, 1390, 1590 and 1650 cm-1, assigning to CO, CN, C=C and C=N/C=O stretching vibrations, respectively. The broad absorption band between 3200 and 3400 cm-1 can be attributed to amino (NH2) and hydroxyl (OH) groups on the surface. In comparison with the RCDs, the FT-IR absorption peak of –NH2 at ~3200 cm-1 was found to decrease from the Ce6-RCDs, confirming successfully coupling Ce6 onto the RCDs. XPS results of Ce6-RCDs further verified the FT-IR analysis. For example, the wide scan XPS spectrum (Figure S2a, SI) of Ce6-RCDs 6


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demonstrates the presence of three main elements (i.e., C, N and O with the molar ratio to be 1.0:0.29:0.11). Figure S2b-d (SI) presents the corresponding high resolution XPS spectra of C1s, N1s and O1s, respectively. The C1s XPS spectrum can be fitted into five binding energies at 284.6, 285.3, 286.4, 287.6 and 288.8 eV, arising from CC/C=C, CN, CO, C=N/C=O and O=COH bonds, respectively.55,57 The N1s XPS spectrum can be deconvoluted into pyridine N (399.6 eV) and graphitic N/amino N (400.6 eV), respectively.55,57 The O1s XPS spectrum contains two fitting peaks at 531.6 and 532.9 eV, which can be ascribed to the C=O and COH/COC bonds.55,57 Moreover, Raman spectrum was measured to provide more structure information for the Ce6-RCDs. Figure S3 (SI) illustrates two typical Raman bands at around 1350 and 1630 cm-1, indicating the presence of disordered (D band) and graphitized (G band) structure in the Ce6-RCDs. Several minor peaks were also observed from this Raman spectrum at about 795, 965, 1100 and 1480 cm-1, which can be ascribed to the isopropyl stretching, pyridine-ring breathing, pyrrole-ring breathing and OCH2/OCH3 transforming, respectively.57 Subsequently, surface charges of the samples were evaluated. As shown in Figure 1c, Zeta potential of the RCDs was measured to be 25.3 mV, attributing to the existence of –COOH/OH groups on their surface. The Zeta potential of Ce6-RCDs, however, was observed to negatively shift to 32.5 mV, probably due to containing multiple –COOH groups on Ce6. Since aggregation is one of the most common problems for application of nanomaterials in biomedical fields, the stability of Ce6-RCDs dispersion in water and cell culture medium were evaluated and found no aggregation occur even standing for 3 days (Figure 1d). Finally, the loading capacity of Ce6 on the RCDs was determined to be 0.56% (mass ratio) (see more details for the calculation method in the Experimental Section). Such a low loading efficiency of Ce6 was chosen not only aiming to decrease influence to the intrinsic properties of RCDs, but also to lessen potential phototoxicity of excessive amounts of PSs.

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-NH 2

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Figure 1. a) TEM images of the RCDs and Ce6-RCDs (inset: HR-TEM images and particle sizes distribution of the RCDs and Ce6-RCDs); b) FT-IR spectra of the RCDs and Ce6-RCDs; c) Zeta potentials of the RCDs and Ce6-RCDs; d) Photographs of the RCDs and Ce6-RCDs dispersions in water and cell culture medium.

Optical properties of the Ce6-RCDs Figure 2a presents the UV-Vis absorbance spectra of Ce6, RCDs and Ce6-RCDs. As the previous report, Ce6 shows two apparent absorption peaks at 405 and 640 nm.61 Both of the RCDs and Ce6-RCDs exhibit broad absorption band (centered at ~540 nm) starting from 440 nm and tailed up to ca. 750 nm. Such an extension of UV-Vis absorption to NIR region is considered to be beneficial for their potential functions of PA and PT. Besides, the Ce6-RCDs also show an absorption peak at ∼405 nm in comparison with the RCDs, confirming again the successful conjugation of Ce6. Then, the FL emission spectra of Ce6-RCDs under different excitation wavelengths were measured and a slightly excitation-dependent feature was observed, i.e., with the excitation wavelengths altering from 470 to 590 nm, their corresponding emission maxima shifting from 632 to 653 nm (Figure 2b). Note that similar FL behavior of the RCDs was observed as that of the Ce6-RCDs (Figure S4, SI), demonstrating that the intrinsic emission properties of RCDs were well preserved even with conjugation of Ce6. As shown in Figure 2c, RCDs and Ce6-RCDs hold the same optimal excitation and emission wavelengths at ∼550 nm and 640 nm, respectively, but no obvious FL emission was 8


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observed from equivalent Ce6 at 550 nm excitation. This confirms that the emission of Ce6-RCDs should be primarily resulted from the RCDs. Furthermore, the photostability assay of Ce6-RCDs was carried out under continuous irradiation by a xenon lamp. As compared with free Ce6, Ce6-RCDs present superior photostability and only slight FL intensity decrease was observed (Figure 2d). Free Ce6 RCDs Ce6-RCDs

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Light-trigged generation of 1O2, heat and PA signals from Ce6-RCDs To demonstrate potential applications in bio-imaging and cancer therapy, light-trigged production of 1O2, heat and PA signals from the Ce6-RCDs were further investigated. The light trigged 1O2 production from Ce6-RCDs and equivalent concentration of Ce6 was evaluated using singlet oxygen sensor green (SOSG) as indicator. Under the 671 nm laser irradiation for 15 min, similar SOSG emission intensity was observed, implying that no significant influence for 1O2 production was caused even after modification of Ce6 onto the RCDs, and therefore the Ce6-RCDs can also be regarded as a PDT agent (Figure 3a). Note that the FL intensity of SOSG induced by equivalent concentration of Ce6 is 9



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obviously higher than that of Ce6-RCDs before the laser irradiation, confirming that the Ce6-RCDs hold lower phototoxicity than the free Ce6 under daylight circumstance. Given the extraordinarily broad absorption (up to NIR region), the feasibility of utilizing Ce6-RCDs for PA imaging was investigated. As shown in Figure 3b, the PA signals were found to increase with the increasement of Ce6-RCDs concentrations from 0 to 1.0 mg·mL-1. In addition, a good linear relationship between PA intensity and Ce6-RCDs concentration was also revealed. Subsequently, photothermal properties of the Ce6-RCDs were thoroughly evaluated. First, the laser power-dependent temperature variations of Ce6-RCDs (200 μg·mL-1) under the irradiation of 671 nm laser were measured. As displayed in Figure 3c, the temperature of the Ce6-RCDs dispersion increases quickly with the enhancement of laser power density from 0.13 to 2.53 W·cm-2. Based on the previous studies that carcinoma cells could be destroyed efficiently at a temperature higher than 50 °C within a few minutes,21 500 mW·cm-2 could be chosen as the optimal laser power for cancer phototherapy. Second, the temperature changes of Ce6-RCDs aqueous dispersion with various concentrations (0200 μg·mL-1) were measured. As revealed in Figure 3d, under the continuous laser irradiation by 671 nm laser (500 mW·cm-2), the temperatures of Ce6-RCDs dispersion were found to elevate from 7.3°C (25 μg·mL-1) to 27.3°C (200 μg·mL-1). In contrast, temperatures of pure water and free Ce6 solution (1.9 μM, an equivalent Ce6 concentration of 200 μg·mL-1 of the Ce6-RCDs) only increased by 0.2 and 0.3 °C, respectively. These results manifested that Ce6-RCDs can quickly and efficiently convert the absorbed laser energy into heat. Third, in order to calculate photothermal conversion efficiency (η) of the Ce6-RCDs, the temperature changes of Ce6-RCDs dispersion (50 μg·mL-1) as a function of time were recorded (insert of Figure 3e). By using the fitting parameters of the cooling stage in Figure 3e, the η of Ce6-RCDs was calculated to be 46% (see more details in the Experimental Section). Finally, the photothermal stability of Ce6-RCDs dispersion (200 μg·mL-1) was examined. As presented in Figure 3f, the limit temperature of Ce6-RCDs dispersion keeps constant even experienced nine cycles, indicating their excellent heat reproducibility and photothermal stability.

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Figure 3. a) Florescence intensities of the Ce6-RCDs (50 μg·mL-1) and equivalent concentration of Ce6 (0.47 μM) in SOSG solution under different irradiation time by 671 nm laser; b) PA imaging and PA signals of the Ce6-RCDs at different concentrations; c) Temperature elevation of the Ce6-RCDs dispersion (200 μg·mL-1) as the function of laser power; d) Temperature rise of water, Ce6 solution (1.9 μM), and various concentration of Ce6-RCDs dispersions under the 671 nm laser irradiation at 500 mW·cm-2 for 10 min; e) Plot of cooling time data (collected from the cooling stage shown in the inset) versus –lnθ and the corresponding linear fitting curve; f) Temperature variation of the Ce6-RCDs dispersion (200 μg·mL-1) with 671 nm laser (500 mW·cm-2) on-and-off for nine cycles.

In vitro imaging and PDT/PTT Since the primary issue for exploring potential applications of nanomaterials in biomedical field is their cytotoxicity, the viabilities of three kinds of typical cancer cells (i.e., HeLa, MCF-7 and 4T1) treated with free Ce6, RCDs, and Ce6-RCDs were investigated and compared. As shown in Figure S5 (SI), very low cell mortality rates were observed for all the three types of cells after incubated with the three materials at various concentrations (i.e., Ce6 at 08 μM and both of RCDs and Ce6-RCDs at 0200 μg·mL-1). These results indicate that not only the RCDs and Ce6, but also the Ce6-RCDs possess low cytotoxicity even at relatively high concentrations. The outstanding optical properties and negligible cytotoxicity of Ce6-RCDs encouraged us to examine their capability for imaging in vitro. Figure 4a shows confocal images of HeLa, MCF 7 and 4T1 cells cultured with Ce6-RCDs and then co-stained with Hoechst 33258 (a typical staining agent for nuclear DNA). As can be seen from the 11



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Ce6-RCDs channel (under excitation at 552 nm), bright red FL was observed from the entire nuclear region, and particularly in certain sites that had been proven to be the nucleoli (mainly containing ribosomal RNA).57 The good complementation of the Ce6-RCDs-labeled sites (red) and Hoechst-labeled sites (blue) from the merged images further verified such conclusion. In addition, the general nucleolus recognition capability of Ce6-RCDs was confirmed from the findings that all kinds of the selected carcinoma cells exhibit similar imaging feature (Figure 4a). It is worth mentioning that the imaging properties of Ce6-RCDs are identical to the RCDs (Figure S6, SI), demonstrating that the low loading of Ce6 has negligible influence on their imaging performance. Furthermore, 1O

2

generation capability of the Ce6-RCDs in vitro was examined using the FL ROS

Assay Kit (DCFH-DA). As shown in Figure 4b, no FL emission was observed from HeLa cells in both DCFH-DA and RCDs plus DCFH-DA groups before and after irradiation by 671 nm laser. An intense FL, however, can be found in the Ce6-RCDs plus DCFH-DA group after laser irradiation, verifying efficient 1O2 production by Ce6-RCDs under the laser irradiation and consequently playing PDT function. To further evaluate the photostability of Ce6-RCDs in vitro, HeLa cells were separately incubated with Ce6-RCDs and free Ce6. As displayed in Figure 4c, the red FL from Ce6-RCDs (arising from the RCDs moiety of the Ce6-RCDs) still keeps intense even after continuous irradiation for 12 min under the excitation of 552 nm, but the FL intensity of Ce6 rapidly faded to an invisible level after 3 min irradiation at 405 nm laser. These results demonstrate that the Ce6-RCDs could be employed as both a potential PDT agent and superior bio-imaging agent with nucleolus staining and high photostability features.

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Figure 4. a) Confocal fluorescence images of living HeLa, MCF-7 and 4T1 cells after co-staining with Ce6-RCDs and Hoechst; b) Fluorescence images of 1O2 detection probe (DCFH-DA) in HeLa cells after incubation with DCFH-DA alone, and RCDs and Ce6-RCDs in the presence of DCFH-DA by exposing to the 671 nm laser for 10 min; c) Confocal fluorescence images of HeLa cells separately incubated with Ce6-RCD and free Ce6 under irradiation at 552 nm and 405 nm, respectively, for different time. Conditions for confocal fluorescence images: Hoechst channel, λex=405 nm, λem=420−480 nm; DCFH-DA channel, λex=488 nm, λem=508-600 nm; Ce6-RCDs channel, λex=552 nm, λem=600-725 nm; free Ce6 channel, λex=405 nm, λem=625-750 nm; All scale bar: 10 μm.

Due to the superior PD and PT functions, bimodal PDT/PTT efficacy of the Ce6-RCDs for cancer is subsequently evaluated in vitro by MTT assay. First of all, phototoxicity of the laser irradiation conditions (i.e., 500 mW·cm-2 of 671 nm laser that being applied for the following phototherapy) was examined. As shown in Figure S7 (SI), negligible cytotoxicity was found to MCF-7 and 4T1 cells, but about 14% cell mortality was caused by such laser irradiation to HeLa cells. These results reflect different survivability of cell lines to laser irradiation. To further compare synergetic PDT/PTT with single PDT or PTT, experiments were set and carried out using Ce6-RCDs and equivalent concentrations of Ce6 and RCDs. As illustrated in Figure 5a-c, only small portions of cells were killed in the PDT group (i.e., free Ce6 under 500 mW·cm-2 of 671 nm laser), this might be because only little 1O2 being produced by the low equivalent concentration of Ce6. The PTT group (i.e., RCDs under 500 mW·cm-2 of 671 nm laser), 13



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however, revealed enhanced effects than that of the PDT group, probably due to the high PT property of the RCDs. Nevertheless, the cancer cells viabilities were still maintained more than 55% even with the highest applied concentration of RCDs (i.e., 200 μg·mL-1). These results demonstrate that the cancer cells killing capability of either PDT or PTT group is inadequate. Astonishingly, the cell viabilities decreased sharply with increasement of Ce6-RCDs concentrations under the same irradiation conditions (i.e., 500 mW·cm-2 of 671 nm laser), confirming outstanding synergistic effects for killing cancer cells. These results also verified that activation of PTT and PDT under a single laser irradiation could boost their effects from each other and represent advantageous than that of separately triggering PTT and PDT using two laser sources. Moreover, the synergistic effects of the Ce6-RCDs on HeLa cells were further confirmed by green Calcein-AM and red propidium iodide (PI) co-staining experiments. As shown in Figure 5d, nearly no cells death appeared in blank group (PBS) under laser irradiation, and only small portions of cells were killed in the PDT (free Ce6) or PTT (RCDs) group. While in the PDT+PTT group (Ce6-RCDs), nearly all cells were destroyed.

Figure 5. a) HeLa, b) MCF-7, and c) 4T1 cells viabilities at various concentrations of free Ce6 (PDT only), RCDs (PTT only) and Ce6-RCDs (PDT+PTT) with 671 nm laser irradiation (500 mW·cm-2) for 20 min; d) Calcein-AM/PI co-staining fluorescence images of HeLa cells incubated with PBS (blank), free Ce6 (PDT), RCDs (PTT) and Ce6-RCDs (PDT+PTT) under the irradiation by 671 nm laser (500 mW·cm-2) for 20 min, scale bar = 50 μm. 14


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To assess possible roles of the nucleolus accumulation property of Ce6-RCDs for facilitating their therapeutic efficiency to carcinoma cells, the morphology changes of HeLa cells with different laser irradiation times were investigated. As shown in Figure S8 (SI), sharp cellular outline and bright red FL in the whole nuclear region, especially nucleolus, can be observed in the control group (without laser irradiation). However, a certain degree of damage to nucleolus, nucleus and cells membrane gradually appeared with prolonged irradiation time, such as nucleolus ablation, chromatin fragmentation, membrane disruption and surface blebbing. In addition, Hoechst 33258 co-staining (for nucleus) experiments displayed the dye leakage from the nuclear region and nuclei shrinkage with the irradiation time increasing (Figure S9, SI). These phenomena imply that the destruction of cancer cells by the Ce6-RCDs under laser irradiation may be initiated from the “control center” of cells (i.e., RNA and DNA in the nucleolus and nucleus, respectively), and this is believed to be responsible, at least partially, for the high PTT/PDT efficacy. In vivo FL/PA imaging Based on the interesting experimental results in vitro, feasibility of the Ce6-RCDs for FL and PA imaging in vivo (4T1 tumor-bearing Balb/c nude mice being selected as the animal model in all the in vivo experiments) was followed investigated. When the tumor reached a volume of approximately 5060 mm3, the mice were intravenously injected with Ce6-RCDs (100 μL, 1.0 mg·mL-1) and PBS as experimental and control groups, respectively. As shown in Figure 6a and 6b, FL intensity of the tumor area (marked by a white circle in Figure 6a) was observed to increase quickly within 28 h post-injection, indicating gradual accumulation of the Ce6-CDs in the tumor via the EPR effects. Given the NIR absorption feature of Ce6-CDs, PA and ultrasound (US) dual-modality imaging of tumor cross section were simultaneously measured.62 The US images are used to help clarifying the tumor boundary and location. From the PA images, an obviously enhanced PA contrast was observed at the 4 h post-injection (Figure 6c), indicating abundant accumulation of Ce6-RCDs in tumor. The average PA signal intensity of the region of interest (ROI) increases over time till 8 h (Figure 6d), which is in good accordance with the FL imaging results. Based on these findings, on one hand the Ce6-RCDs are verified 15



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to be a potential FL and PA imaging agent in vivo, and on the other hand the appropriate time point to implement phototherapy is determined (i.e., 8 h post-injection). Note that only very weak FL and PA signals were observed after 24 h injection of the Ce6-RCDs (Figure 6a-d), implying that the materials can be excreted from the mice quickly. To further confirm this conclusion, mice were sacrificed at 24 h post-injection of the material and their major organs (heart, liver, spleen, lung and kidney) and tumors were excised. The observed very weak FL emissions from these organs and tumor demonstrate that most of the Ce6-RCDs were removed from the mice in 24 h (Figure 6e). Furthermore, the hematoxylin and eosin (H&E) staining of these excised organs were also examined. As shown in Figure 6f, no obvious inflammation and damage were found in the experimental group (i.e., Ce6-RCDs) as compared with the control group (i.e., PBS), confirming again the excellent biocompatibility of the Ce6-CDs.

Figure 6. a) In vivo FL images at different time points after i.v. injection of Ce6-RCDs; b) Average FL intensities of tumor region (white circle) at different time points (n=3); c) Real-time in vivo PA and US images of Ce6-RCDs in tumor area after i.v. injection; d) Average PA signal intensities of tumor region at different time points (n=3); e) Ex vivo images of mice organs and tumor after 24 h post-injection of the Ce6-CDs; f) H&E staining of major organ sections gathered from control and Ce6-RCDs treatment groups at 24 h post-injection. 16


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In vivo PT imaging and PDT/PTT To evaluate PDT/PTT efficacy of Ce6-RCDs in vivo, 4T1-tumor-bearing nude mice were randomly divided into four groups (five mice per group) and administered different treatments as follows: i) blank group (PBS + laser), ii) PDT group (free Ce6 + laser), iii) PTT group (RCDs + laser), and iv) PDT+PTT group (Ce6-RCDs + laser). When the tumor size reached around 150 mm3, corresponding materials were respectively injected into the mice belonging to different groups via the tail vein, and irradiated by the 671 nm laser (500 mW·cm-2 for 10 min) at 8 h post-injection. The intratumoral temperatures of these treated groups were initially recorded using an IR thermal camera. As shown in Figure 7a,b, the limit temperatures of tumor in blank (PBS) and PDT (free Ce6) groups increased to only about 4041 °C after laser irradiation (671 nm laser at 500 mW·cm-2), demonstrating low photothermal response of such laser irradiation and negligible thermal therapy of Ce6.63-64 However, tumor temperatures of the mice from PDT + PTT group (Ce6-RCDs) elevated rapidly and reached a plateau within 2 min with the maximum temperature of 48.8°C. A similar temperature change curve was obtained in the PTT group (RCDs), indicating the PTT effects of Ce6-RCDs mainly arising from the RCDs component. The tumor size and mice body weight were then measured regularly after the above four group treatments. As illustrated in Figure 7c, the mice in PDT group exhibited negligible tumor growth inhibition from comparing with the blank group. The tumor volumes in both control and PDT groups reached about 6.5 times as the initial size at the end of the monitor period (13 days), indicating that the PDT alone nearly has no effects to tumor. Mice in PTT group, however, showed partial inhibition in tumor growth, but the efficacy was far from sufficient (tumor volume still increased to ~3.5 times as the initial size at day 13). In contrast, the PDT+PTT group exhibited remarkable restriction in tumor growth and the tumor volume shrank to only ~30% of the initial size at day 13. These tumor volume alterations on the day 13 from the four group treatments can be clearly seen in Figure 7d. Note that no obvious variations of the mice body weight were found among all these group treatments during the whole observed period (Figure S10, SI), indicating insignificant adverse effects of Ce6-RCDs. Furthermore, the mice in PDT+PTT group 17



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showed tumor necrosis and black scar on tumor site after 3 days of the treatment, and no substantial tumor relapse was observed till day 13 (Figure 7e). Although local necrosis and black scar on tumor site were also found from PTT group on the day 3 post-treatment, the tumor recurrence obviously appeared at day 13. In contrast, no apparent damages to the tumor were observed in both blank and PDT groups on day 3, and tumors grew rapidly during the monitor period (Figure 7e). Overall, these in vivo experiments clearly verified that the efficacy of single NIR-light-triggered synergistic PDT/PTT being much better than either single mode of PDT or PTT under a relatively low power laser irradiation. To evaluate instant therapy efficacy, H&E staining of tumors collected from different groups was carried out after 1 day treatment. As shown in Figure 7f, tumor tissues in PDT group displayed thriving tumor cells with minor damage in comparison with the blank group. Partial tissue destruction and reduced highly active tumor cells are observed from the PTT groups, indicating a certain degree of necrosis of the tumors. The PDT + PTT group, however, showed significant tissue loss, nuclei shrinking and pyknosis from the tumor. The discrepancy of tumor tissue morphology among various group treatments further confirmed the superior anticancer efficacy of synergistic PDT/PTT than either PDT or PTT alone. To show the long term detrimental effects of Ce6-RCDs, the major organs (heart, liver, spleen, lung, and kidney) of mice randomly selected from blank and PDT+PTT groups were collected and subjected to H&E staining after 13 days treatment. Compared with the blank group, neither apparent inflammation nor damage from the PDT+PTT group was observed, demonstrating low toxicity of Ce6-RCD in vivo (Figure S11, SI).

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Figure 7. a) Thermal images of tumor-bearing mice before and after laser irradiation (671 nm at 500 mW·cm-2) for 5 min from the four group treatments at 8 h post-injection; b) Temperature elevation curves of tumor regions with laser irradiation time (671 nm at 500 mW·cm-2) from the four group treatments at 8 h post-injection; c) Tumor growth curve recorded after the four group treatments from 0 to 13 days; d) Digital photographs of tumors gathered from the four group treatments at day 13; e) Digital photographs of tumor-bearing mice at day 0, 3 and 13 after the four group treatments; f) H&E staining of tumors collected from the four group treatments after 1 day, scale bar=100 μm.

CONCLUSIONS In summary, a new concept to reduce the frequently used high laser irradiation power for cancer PTT was proposed and verified in this study (i.e., loading a small amount of PSs onto a PTT agent and triggering both of them by a single laser). The selection of low loading of PSs was aiming to decrease influence to the intrinsic properties of the PTT 19



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agent, and meanwhile to lessen potential phototoxicity of excessive amounts of PSs. Specifically, minute quantities of Ce6 (0.56% of mass) were anchored onto amino-rich RCDs that possessing superior PT character under 671 nm NIR laser (Ce6 can also be activated by this laser irradiation). The in vitro and in vivo experiments demonstrate that the Ce6-RCDs can offer excellent cancer therapy efficacy under a relatively low laser power density (i.e., 0.50 W·cm-2 at 671 nm), but efficacies by the equivalent RCDs or Ce6 were far from sufficient under the same irradiation conditions. Note that the specific nucleolus accumulation feature of the Ce6-RCDs may also play a role, at least partially, to the observed high treatment effects. Besides, the Ce6-RCDs also exhibit multimodal imaging capabilities (i.e., FL, PA and PT), which could be employed for guidance of the phototherapy process. Overall, this study on one hand suggested a strategy to enhance cancer phototherapy efficacy by a reduced irradiation power, and on the other hand proposed an ideal candidate (i.e., RCDs, of which holding features of high photostability, excellent biocompatibility, abundant surface amino groups for functionalization, red emission, and extended absorption to the NIR region) for further exploring potential applications of CDs in cancer theranostics.

EXPERIMENTAL Materials. All chemicals were from commercial sources and used without further purification. Formamide was acquired from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China).

Citric acid, polyethyleneimine (PEI, MW=600), 4-(4,6-dimethoxy-

1,3,5-triazin-2-yl)-4-methylmorpholinium

chloride

(DMT-MM),

dimethylsulfoxide

(DMSO), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and Hoechst 33258 were obtained from Aladdin Industrial Inc. (Shanghai, China). Reactive oxygen species assay kit (DCFH-DA) was bought from Suolaibao Bio-Technology Co., Ltd. (Shanghai, China). Dulbecco’s modified Eagle’s medium (DMEM) and Roswell Park Memorial Institute (RPMI-1640) medium were purchased from Thermo Fisher Scientific Inc. (USA). Fetal bovine serum (FBS) was from PAN-Seratech (Germany). Penicillin-streptomycin and trypsin-EDTA were acquired from KeyGEN BioTECH Corp., Ltd. (Jiangsu, China). Singlet Oxygen Sensor Green (SOSG) was from Invitrogen, 20


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(Carlsbad, USA). Calcein-AM/PI Double Stain Kit was bought from Yeasen Biological Technology Co., Ltd. (Shanghai, China). Chlorin e6 (Ce 6) was purchased from J&K Scientific Ltd. (Beijing, China). Characterizations. Transmission electron microscopy (TEM) and high-resolution TEM (HR-TEM) images were taken on a Tecnai F20 microscope. Fourier transform infrared (FT-IR) spectra were recorded on a Nicolet 6700 FT-IR spectrometer. X-ray Photoelectron Spectroscopy (XPS) were carried out with ESCALAB 250Xi (Thermo Scientific). Raman spectrum was obtained on the Renishaw inVia Reflex spectroscopy (Renishaw, UK) at the excitation wavelength of 532 nm. UV−Vis absorption spectra were performed on a PERSEE T10CS UV−Vis spectrophotometer. Fluorescence emission and excitation spectra were recorded on a Hitachi F-4600 spectrophotometer. Zeta potentials and dynamic light scattering (DLS) were measured on Zetasizer Nano ZS (Malvern, UK). The photodynamic/photothermal performance of the Ce6-RCDs was measured with the irradiation of 671 nm laser (Changchun New Industries Optoelectronics Technology Co. Ltd., China). The temperature change and photothermal images were performed by a photothermal imaging system (Ti400, Fluke, USA). Cellular images were taken with a Leica confocal laser fluorescence microscope (TSCSPS II, Germany). MTT results were collected by a microplate reader (Imark 168−1130, Biorad, USA). In vivo florescence images were recorded by CALIPER IVIS SPECTRUM system (Hengjia Co., Ltd., Beijing). PA experiments were carried out using the home-made AR-PAM system.62 Synthesis of RCDs. The RCDs was prepared referring to our previous report with slight modification.57, 65 Briefly, 10 g of citric acid and 6.3 g of PEI were added and dissolved in 200 mL of formamide. This solution was then transferred into a Teflon autoclave and kept in air oven at 160 °C for 4 h. When the autoclave was naturally cooling down to the room temperature, the viscous reaction solution was poured out and filtered with 0.22 μm membrane filter to remove lager particles. After one week of dialysis (MWCO 2000 Da), rotary evaporation was used to remove water, and the RCDs were finally isolated as a dark red powder.

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Synthesis of Ce6-RCDs. In a typical procedure, 1.0 mg of Ce6 and 2.1 mg of DMT-MM were firstly dissolved in 67.5 mL of methanol to form a transparent solution. Then, 50 mg of the RCDs that pre-dissolved in 7.5 mL DMSO was added into the above methanol solution and stirred for 24 h at room temperature. The obtained solution was purified by dialysis (MWCO 2000 Da) for 3 days (the first day with 0.1 M NaHCO3 to remove residual Ce6 and DMT-MM, and the following 2 days with deionized water to replace NaHCO3). Finally, the Ce6-RCDs were collected through lyophilization. Calculation of Ce6 loading capacity. Due to the broad absorption of RCDs in the range of 320420 nm, it is not appropriate to apply the absorbance of Ce6 (at 405 nm) to determine its loading capacity. Consequently, the FL emission of Ce6 at 656 nm (λex=405 nm) was taken for such calculation. Firstly, a typical standard curve between Ce6 concentration and its emission intensity at 656 nm (λex=405 nm) was created (Figure S12a, SI). Then, the loading capacity of Ce6 on the RCDs can be facilely calculated to be 0.56% (mass ratio) according to the emission intensity of Ce6-RCDs at 656 nm under excitation at 405 nm (Figure S12b, SI). Evaluation of photodynamic performance. SOSG was utilized to evaluate the singlet oxygen (1O2) generation in aqueous solution. In general, 1.0 mL of Ce6-RCDs aqueous dispersion (50 μg·mL-1) and the equivalent concentration of free Ce6 solution (0.47 µM) were loaded in a cuvette, respectively. After the addition of SOSG (2.5 μM), the samples were exposed to a laser of 671 nm with a power density of 500 mW·cm-2 for 15 min. Then, the FL intensity of SOSG was recorded at different irradiation time under the excitation wavelength of 488 nm. Evaluation of photothermal performance. The digital photothermal imaging system was utilized to monitor the following temperature changes. First, 1.0 mL of Ce6-RCDs aqueous dispersion at a concentration of 200 μg·mL-1 was loaded in a cuvette and irradiated by the laser of 671 nm at different power densities. Second, the Ce6-RCDs aqueous dispersion with various concentrations (0200 μg·mL-1) were added in cuvettes and separately irradiated by 671 nm laser (500 mW·cm-2) for 10 min. The determination

22


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of photothermal conversion efficiency (η) was referring to the previous reports.57,

66

In

briefly, the η of Ce6-RCDs can be calculated and obtained by the following equation:

where h: heat transfer coefficient; A: surface area of the cuvette; ΔTmax,mix and ΔTmax,water: temperature change of Ce6-RCDs dispersion and water (solvent) at the limit temperature, respectively; I: laser power; Aλ: absorbance of Ce6-RCDs dispersion at 671 nm. PA experiments. Different concentrations of the Ce6-RCDs (01 mg·mL-1) were mixed with matrigel matrix at the mass ratio of 1:1 (04 °C). AR-PAM system was used to obtain the PA signal and image (with the excitation wavelength of 700 nm).62 Cell culture. MCF 7, HeLa and 4T1 cells were cultured in various medium (MCF 7 and HeLa in DMEM, 4T1 in RPMI-1640) containing 10% FBS and 1% penicillin/ streptomycin in a 37 °C incubator with 5% CO2. Cell staining and the detection of 1O2 in vitro. For all cell imaging experiments, different types of cancer cells were seeded in glass culture dishes (1105 cell per dish) and cultured with various kinds of materials under different conditions (i.e., 50 μg·mL-1 RCDs and 50 μg·mL-1 Ce6-RCDs were dispersed in culture medium and incubated with cells for 2 h; Ce6 was dispersed in culture medium at the concentration of 16.6 μM and co-cultured with cells for 6 h). For co-staining experiments, living cells were first incubated with 50 μg·mL-1 RCDs or Ce6-RCDs in culture medium for 2 h, followed by staining of 5.0 μM Hoechst 33258 for 30 min. All samples were rinsed with PBS twice before imaging with confocal microscope. The imaging parameters are set as follows: for RCDs or Ce6-RCDs channel, λex=552 nm and λem=600750 nm; for Ce6 channel, λex=405 nm and λem=600750 nm; for Hoechst channel, λex=405 nm and λem=420480 nm. For the detection of 1O2 in vitro, DCFH-DA was employed as an indicator. Three groups of HeLa cells were co-cultured with PBS, 200 μg·mL-1 RCDs and 200 μg·mL-1 Ce6-RCDs for 2 h, respectively. Materials were removed and cells were washed with PBS before the incubation with 10 μM DCFH-DA for another 30 min. Samples were then

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irradiated by 671 nm laser at 500 mW·cm-2 for 10 min, and immediately imaged through a confocal microscope (λex=488 nm and λem=505545 nm). Measurement of cell cytotoxicity. Standard MTT assay was applied to evaluate the cell cytotoxicity of the relevant materials. Briefly, different types of cancer cells were seeded in 96-well plate (1104 cell per well) and allowed to attach overnight. Then, the cells were incubated with different concentration of RCDs (0200 μg·mL-1), Ce6-RCDs (0200 μg·mL-1) and free Ce6 (01.9 μM) for 24 h. Afterward, 10 μL MTT was added into each well and incubated with cells for an additional 4 h. Finally, the culture medium was discarded and replaced with 100 μL DMSO per well, the absorbance of each well was measured by a microplate reader at 550 nm. Calcein-AM/PI assay. Calcein-AM/PI co-staining was performed to evaluate the synergistic efficacy of Ce6-RCDs. Four groups of HeLa cells were incubated with PBS, 100 μg·mL-1 RCDs, 100 μg·mL-1 Ce6-RCDs and 0.93 μM free Ce6 for 2 h, respectively. Then, the cells were washed twice with PBS before exposing to the irradiation of 671 nm laser at the power density of 500 mW·cm-2 for 20 min. Afterward, all samples were co-stained with Calcein AM (2 μM) and PI (4.5 μM) for 5 min and immediately observed by a confocal microscope. Morphology changes of cells during irradiation process. To monitor the morphology changes of cells during the laser irradiation process, HeLa cells were seeded in glass culture dishes and counterstained with Ce6-RCDs (200 μg·mL-1, 2 h) and Hoechst (5 μg·mL-1, 30 min). Then, all dishes were rinsed with PBS twice and exposed to the irradiation of 671 nm laser at the power density of 500 mW·cm-2 for different times (020 min). Samples were fixed with 4% paraformaldehyde before imaging. In vitro PDT/PTT. Various types of cancer cells were seeded in 96-well plate and incubated with different concentration of materials (free Ce6, RCDs and Ce6-RCDs) for 2h. The group without the addition of material was set as control. Then, all samples were irradiated by 671 nm laser at 500 mW·cm-2 for 20 min, standard MTT assay was employed to measure the cell viabilities. 24


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Animal experiments. All animal experiments were carried out by complied with the regulations of Animal Study Committee of Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences. Female Balb/c nude mice (4 weeks) were purchased from the Medical Experimental Animal Center of Guangdong Province (Guangzhou, China). 4T1 cells were selected to build tumor model. Briefly, 100 μL 4T1 cells (1106) in PBS were subcutaneously injected into upper thigh of every mouse. After injection for 7 days, the tumor volume reached the size of around 50-60 mm3. In vivo FL/PA imaging. FL images were taken at different time intervals (0, 2, 4, 6, 8 and 24 h) after intravenous injection of Ce6-RCDs (100 μL, 1.0 mg·mL-1) with the CALIPER IVIS SPECTRUM system (excitation wavelength of 550 nm). The mice were sacrificed at 24 h after injection, their major organs and tumors were excised to examine the FL intensities. Besides, the PA and US dual-modality images of tumor cross section were simultaneously obtained using AR-PAM system (excitation wavelength of 700 nm).52 In vivo PT imaging and PDT/PTT. The PT imaging and PDT/PTT experiments were implemented until the tumor volume reached around 150 mm3. Tumor-bearing nude mice were randomly classified into four groups (five mice per group) and administered different treatments as follows: i) blank group (PBS ,150 μL+laser), ii) PDT group (19 μM free Ce6, 150 μL+laser), iii) PTT group (2 mg·mL-1 RCDs, 150 μL+laser), and iv) PDT+PTT group (2 mg·mL-1 Ce6-RCDs, 150 μL+laser). All materials were intravenously injected into mice via the tail vein and the irradiation condition of 671 nm laser was set as 500 mW·cm-2 for 10 min. When the tumors of different treated groups were exposed to laser irradiation, their intratumoral temperature were recorded by an IR thermal camera every 30 s. The tumor size and body weight were measured regularly after the treatment. And the tumor volume (V) was calculated with the following equation: V=ab2/2, where a and b represent the length and width of the tumor, respectively. Hematoxylin and eosin (H&E) staining. After 1 day of treatment, the tumors collected from different groups were preserved in 4% formalin solution. The major organs of mice in blank group and Ce6-RCDs treated group were gathered at day 13 post-treatment. All

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tissues were embedded in paraffin blocks, sliced and stained with H&E before observation under bright-field microscopy.

ASSOCIATED CONTENT Supporting Information Additional data including DLS, XPS, Raman, FL emission, MTT, cell imaging, mice body weight, H&E staining of major organ section and plots for calculating Ce6 loading capacity (Figure S1-S12). The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/xxxxxx.xxxxxxx.

AUTHOR INFORMATION Corresponding Author [email protected] (C.L.); [email protected] (H.L.) Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS The authors acknowledge the financial support from the National Natural Science Foundation of China (51872300, U1832110, and 91739117), Ningbo Science and Technology Bureau (2016C50009), and the W. C. Wong Education Foundation (rczx0800). The authors would also like to sincerely thank Prof. Qing Huang at NIMTE for allowing us accessing the fluorescence spectrophotometer in his lab.

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Ce6-Modified Carbon Dots for Multimodal-Imaging-Guided and Single

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