Highly Crystalline Multicolor Carbon Nanodots for Dual-Modal

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Highly Crystalline Multicolor Carbon Nanodots for DualModal Imaging-Guided Photothermal Therapy of Glioma Min Qian, Yilin Du, Shanshan Wang, Chengyi Li, Huiling Jiang, Wei Shi, Jian Chen, Yi Wang, Ernst Wagner, and Rongqin Huang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b19716 • Publication Date (Web): 12 Jan 2018 Downloaded from http://pubs.acs.org on January 12, 2018

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ACS Applied Materials & Interfaces

Highly Crystalline Multicolor Carbon Nanodots for Dual-Modal Imaging-Guided Photothermal Therapy of Glioma Min Qian a, Yilin Du a, Shanshan Wang a, Chengyi Li a, Huiling Jiang a, Wei Shi d, Jian Chen d, Yi Wang b,*, Ernst Wagner c and Rongqin Huang a,* a

Department of Pharmaceutics, School of Pharmacy, Key Laboratory of Smart Drug Delivery,

Ministry of Education, Fudan University, Shanghai 201203, China. b

Center for Advanced Low-dimension Materials, Donghua University, Shanghai 201620, China.

c

Pharmaceutical Biotechnology, Center for System-based Drug Research, Center for

Nanoscience (CeNS), Ludwig-Maximilians-Universität München, Munich 81377, Germany. d

Department of Neurosurgery, Affiliated Hospital of Nantong University, Nantong 226001,

China. KEYWORDS: glioma, solid-state synthesis, graphitic carbon nanodots, dual-modal imaging, photothermal therapy

ACS Paragon Plus Environment

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ABSTRACT: Imaging-guided site-specific photothermal therapy (PTT) of glioma and other tumors in central nervous system (CNS) presents a great challenge for the current nanomaterial design. Herein, an in-situ solid-state transformation method was developed for the preparation of multicolor highly crystalline carbon nanodots (HCCDs). The synthesis yields 6-8 nm sized HCCDs containing a highly crystalline carbon nanocore and a hydrophilic surface, which therefore simultaneously provide strong photoacoustic and photothermal performance as well as tunable fluorescence emission. In vitro and in vivo results demonstrate that the novel HCCDs have high water dispersity, good biocompatibility, but potent tumor cell killing upon NIR irradiation. As demonstrated in U87 glioma-bearing mice, HCCDs specifically accumulate in brain tumors and facilitate dual-modal imaging-guided PTT, with therapeutic antitumoral effects without any apparent damage to normal tissues.

1. INTRODUCTION Glioma is considered to be the most malignant tumor in central nervous system (CNS) with high morbidity and mortality. Although surgical resection, alone or combined with chemotherapy/radiotherapy, has been considered to be the state-of-the-art clinical technology for current glioma treatments, the prognosis is unsatisfactory, with the median survival still less than 2 years.1,2 This might be mainly attributed to the facts that the deep location of glioma in CNS with infiltrative growth accompanied by the low specificity of current treatments lead to unclear therapeutic borders, consequently causing severe adverse effects to normal tissues.3 Additionally, the blood-brain barrier (BBB) largely impedes drug accumulation in glioma, contributing to the failure of therapies.4,5 To improve the therapeutic outcome, reengineering of theranostic


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modalities, which can simultaneously image the glioma and deliver therapeutic agents to overcome the BBB, is an urgent demand. Recently, nanoparticle-based drug carriers have been tested for treatment of various malignant tumors. Especially, nanoparticles with sizes below 12 nm (nanodots) have the capability of penetrating the BBB gaps and accumulating in brain tumors.6,7 Among them, fluorescent carbon nanodots are of great potential because of their uniformed diameter, high water dispersity and good biocompatibility, as well as the stable fluorescence emission for highly sensitive tumor imaging.8,9 Nevertheless, most of the current carbon nanodots can only serve as either fluorescence imaging agents or chemotherapeutic vehicles.10 They were rarely applied for noninvasive Near Infrared (NIR) - activated photothermal therapy (PTT), where a deep-tissue penetration of NIR and the controllability of light dose would be extremely desirable for glioma treatment.11,12 The lack of application might be attributed to the low photothermal ability of traditional carbon nanodots, resulting from the large amounts of lattice defects via hydrothermal synthesis.13 Meanwhile, the insufficient spatial depth via mere fluorescence imaging of common carbon nanodots might also misguide the NIR irradiation,14 and consequently cause harm to the fragile healthy CNS. Therefore, in this work, an in-situ solid-state synthesis was developed to prepare highly crystalline carbon nanodots (HCCDs). These HCCDs not only exhibit tunable full-color emissions but also show high photothermal ability and strong photoacoustic effect. They

can

cross

the

BBB

and

specifically

accumulate

in

glioma,

and

achieve

photoacoustic/fluorescent dual-modal imaging-guided PTT. 2. EXPIRIMENTAL SEECTION 2.1. Chemicals and materials. Pluronic F127 was bought from Sigma-Aldrich (St. Louis, MO, U.S.A.). Phenol, formaldehyde, sodium hydroxide and other reagents, if not specified, were


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purchased from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China). Fetal bovine serum (FBS) was bought from Biological Industries (Kibbutz Beit Haemek, Israel). Phosphate buffer solution (PBS), penicillin, and streptomycin were obtained from Hyclone (Logan, Utah, U.S.A.). Dulbecco's modified Eagle's medium (DMEM), trypsin, and L-glutamine were purchased from Gibco. Cell Counting Kit-8 (CCK-8) was bought from Dojindo Laboratories (Kumamoto, Japan). TdT-mediated dUTP nick end labeling (TUNEL) apoptosis detection kit (FITC-labeled) was purchased from KeyGEN Biotech (Nanjing, China). Live/Dead cell imaging kits were bought from Life Technologies Corporation (Carlsbad, CA, U.S.A.). 2.2. Preparation of HCCDs and their derivatives. Pluronic (F127)-contained mesoporous phenolic resin (F127-mPR) was prepared as previously described.15 Typically, the mixture of phenol (0.6 g), formalin aqueous solution (2.1 mL, 37 wt%) and NaOH solution (15 mL, 0.1 M) was stirred with pluronic F127 solution (0.064 g mL-1, 15 mL) at 66 °C for 2 h. Then, distilled water (50 mL) was added and agitated for another 17 h. After dilution with 260 mL water, the solution was heated at 130 °C for 24 h. The yellow powder (F127-mPR) was collected via freeze-drying after washing three times by deionized water. To prepare HCCDs, catalysts (NaCl, LiCl and KNO3) were homogeneously dispersed into F127-mPR via water-assisted stirring and freeze-drying. Then, a brownish black powder (HCCDs@MCN) was obtained via calcination of the freeze-dried powder under air atmosphere at 350 °C for 3 h. To remove MCN and some agminated carbon nanoparticles, the brownish black powder was dispersed in water via strong ultrasonication. In the following, the dispersion was centrifuged at 8,000 rpm for 5 min (three times) and subsequently at 16,000 rpm for 5 min (only once) to recover the supernatant containing the HCCDs. Finally, the supernatant was dialyzed (MW: 14,000) against water for 48 h to remove any low-molecular weight impurities. HCCDs were obtained via freeze-drying of


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the fluid in dialysis bag. For comparison, derivatives of HCCDs were synthesized via the method mentioned above but with different calcination temperatures (150, 250, 350 and 450 °C) and time periods (1, 2, 3 and 4 h), respectively. 2.3. Characterization. Transmission electron microscopy (TEM) measurements were performed on JEOL-2100F electron microscope (Japan). X-ray diffraction patterns (XRD) were analyzed using a BrukerD8 Advance & Davinci Design diffractometer (Germany). X-ray photoelectron spectroscopy (XPS) was conducted by RBD-upgraded PHI-5000C ESCA system with Al Ka radiation (hv = 1486.6 eV) as the X-ray source for excitation. Raman spectra were performed with a SPEX-403 spectrometer (France). Infrared spectra (IR) were carried out with Thermo Nicolet AVATAR 360FT-IR (U.S.A.) using KBr pellet method. Zeta potentials and DLS particle size distribution were measured by Zeta Potential/Particle Sizer Malvern 3600 (U.K.). Fluorescent emission spectra were operated on an Angilent-G9800A fluorescence spectrophotometer (U.S.A.). UV-vis spectra were obtained from a UV-vis absorption spectrometer (UV-2401PC, SHIMADZU, Japan).

13

C and 1H NMR were done on a DMX 500

(Bruker, Germany) with deuteroxide as solvent. NIR-mediated photothermal imaging of different concentrations and laser powers was recorded with a thermal imaging equipment (VarioCAM, JENOPTIK, Germany). The data of TG-MS were acquired by SDT Q600-GSD 301 T2 (U.S.A., Germany) from 25 °C to 800 °C in air. The photothermal effect of HCCDs was measured under irradiation (808 nm) by a semiconductor laser unit (Changchun Laser Optoelectronics Technology Co., Ltd. China). 2.4. Photothermal evaluation and thermal imaging of HCCDs. The temperature data of HCCDs dispersion with various concentrations or different laser power densities were obtained


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by a thermocouple probe at certain time points. At the same time, thermal images of HCCDs dispersion were recorded by a thermal imaging equipment (VarioCAM, JENOPTIK, Germany). 2.5. Fluorescence stability of HCCDs. The HCCDs were dispersed in water or PBS with various pH and different UV exposure periods for photostability measurements at room temperature. The fluorescence data were collected by an Agilent-G9800A fluorescence spectrophotometer (U.S.A.). 2.6. Measurement of photothermal performance of HCCDs. In order to evaluate the photothermal efficiency, 0.2 mL of deionized water and HCCDs aqueous dispersion (150 µg mL1

) were irradiated by an 808 nm NIR laser (2 W cm-2) to reach a temperature platform. A

thermocouple probe was inserted into the aqueous dispersions with the temperature evolution recorded every 10 s. Then the laser was turned off, and the decrease of temperature was also recorded by the same method. The photothermal conversion efficiency (η) was calculated by eq 1: η =

hS(T max − Tsurr ) − Q S I(1 − 10 − A ) 808

(1)

where h and S are the heat transfer coefficient and the surface area of the container, respectively. Tmax and Tsurr denote the maximum system temperature and the surrounding temperature, Qs is related to specific heat capacity of the solvent, I is the laser power (2 W cm-2), and A808 is the absorbance of HCCDs at 808 nm. The value of hS is derived by eq 2: τS =

m DC D hS

(2)

where ߬s is the sample system time constant and mD and CD are the mass (0.2 g) and heat capacity (4.2 J g-1 °C-1) of water, respectively. Qs is measured independently to be 12.6 mW within pure water. In order to obtain the value of hS, θ is introduced, which is defined as follows:


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θ =

T − Tsurr Tmax − Tsurr

(3)

where T is the real-time temperature of HCCDs aqueous dispersion. Then, hS can be calculated by linearizing the time and -lnθ during the cooling period. 2.7. Cell culture and animal model. U87 glioblastoma cells were obtained from American Type Culture Collection (ATCC, Rockville, MD, U.S.A.) and maintained in DMEM media supplemented with 10% FBS, 1% L-glutamine, 1% penicillin and 1% streptomycin stock solution, at 37 °C and under a humidified atmosphere containing 5% CO2. The media were changed every two days, and the cells were passaged by trypsinization before confluence. Male nude mice with body weight about 20 g were maintained under specific pathogen free conditions at the Department of Experimental Animals, Fudan University. Glioma-bearing mice were established by implanting about 5 × 105 U87 cells into the right caudatoputamen with a stereotactic fixation device. All animal experiments were performed in accordance with guidelines evaluated and approved by the Ethics Committee of Fudan University. 2.8. Cellular uptake. U87 cells were planked in 96-well microplates at a density of 1 × 104 cells per well with 200 µL of media for 24 h. After removing the medium, HCCDs with various concentrations from 50 to 150 µg mL-1 were added into the wells and incubated for 2 h. In order to explore the influence of incubation time, HCCDs with a concentration of 150 µg mL-1 were incubated with U87 for different time periods. Then the cells were rinsed twice and fixed with 4% paraformaldehyde for 15 min before taking multi-color confocal images (Ex: 405, 458, 488 and 514 nm) with a Carl Zeiss LSM710 instrument (Germany). 2.9. In vivo imaging. For in vivo fluorescence imaging, the glioma-bearing mice were fasted one day to reduce food autofluorescence at the 19th day after implantation. Then the mice were intravenously injected with HCCDs dispersion (at the dose of 50 mg kg-1) and normal saline,


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respectively. The images were taken by an IVIS Spectrum in vivo imaging system (Caliper, U.S.A.) at different time points after administration (excited at different wavelength via spectral unmixing for the final results). After that, the mice were sacrificed by decapitation with the whole brains and main organs removed for further imaging. For in vivo photoacoustic imaging, glioma-bearing mice at the 15th day after tumor implantation were intravenously injected with HCCDs at the dose of 50 mg kg-1. Then, the 3D images of the whole brains were gathered by Vevo LAZER (VisualSonics FujiFilm, Canada) at different time points. Before the in vivo imaging, photoacoustic absorptions and photoacoustic imaging of HCCDs with different concentrations in photoacoustic-free tubes were carried for calibration. 2.10. Cytotoxicity in vitro. CCK-8 assays were carried out to quantify the cytotoxicity of glioma cells under HCCDs exposure. U87 cells (1 × 104, 100 µL) were cultured in a 96-well plate for 24 h to allow attachment, then treated with 100 µL medium containing various concentrations of HCCDs separately for 24 h. Then the culture media were replaced with fresh media and 10 µL CCK-8 solution per well. After incubated for further 2 h at 37 °C, the absorbance at 450 nm (A450) was recorded with a microplate reader (BioTek Synergy 2, U.S.A.). Survival rate = (Asample/Acontrol) × 100%, where Asample and Acontrol represent the absorbencies of the sample and control wells, respectively. 2.11. PTT in vitro. After 24 h attachment in 96-well plate, U87 cells (1 × 104, 100 µL) were incubated with various concentrations of HCCDs for 2 h. Then media in different groups with and without irradiation by an 808 nm laser (2 W cm-2, 5 min) were replaced with fresh media for another 12 h incubation. After that, 10 µL CCK-8 solution was added per well for further 2 h incubation at 37 °C before measuring the absorbance at 450 nm (A450) by the microplate reader.


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Qualitative evaluation was conducted using live/dead cell kits. U87 cells were cultured in 96well plates at a density of 1 × 104 per well for 24 h, allowing the cells to attach. Media in relevant groups were replaced by HCCDs (150 µg mL-1) and incubated for another 2 h. At the end of incubation, cells in corresponding groups were exposed to an 808 nm laser (2 W cm-2, 5 min). Then, cells were washed with PBS, and placed in fresh media for further 12 h incubation. Finally, the cells were stained by live/dead cell kits according to the manufacturer’s instruction. The pictures of live (green) and dead (red) cells were collected by confocal microscopy. 2.12. Therapeutic effect and toxicity in vivo. U87 glioma-bearing mice were randomly divided into 4 groups (n = 6 per group): (a) saline group; (b) laser group; (c) HCCDs group; and (d) HCCDs+PTT group. Then 150 µL saline and HCCDs (5 mg mL-1) were intravenously injected on the 7th and 10th day after implantation. For PTT groups, the glioma sites were exposed to an 808 nm laser at 1.0 W cm-2 for 5 min after the mice were anesthetized. After treatments, body weight was recorded every other day. One mouse was sacrificed randomly per group at day 10 after designated treatments. Major organs and whole brains were taken out for in vivo toxicity assays. Optical photographs were taken using a common Canon camera. TUNEL and H&E staining were conducted according to the manufacturer’s instruction. Frozen sections were obtained with a Leica CM3050 S Cryostat (Germany) and observed under a Leica DMI4000 D fluorescent microscope (Germany). 3. RESULTS AND DISCUSSION To prepare the HCCDs, freeze-dried mesoporous phenolic resin generated from Pluronic F127

(poly-ethylene

oxide-poly-2-propylene

oxide-polyethylene

oxide),

phenol

and

foemaldehyde was used as the carbon resource, which can be crystallized via solid-state transformation under 350 °C with NaCl/LiCl/KNO3 as catalysts (Scheme 1, Figure S1 and


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experimental section). Calcination results in formation of mesoporous carbon nanospheres (MCN) with HCCDs attached (HCCDs@MCN). After ultrasonication, HCCDs can be collected by removing MCN and fragments by centrifugation. This synthesis has several virtues as compared to previous methods. First, the solid-state synthesis under high temperature prompted the

high

crystallization

of

carbon

cores,

which

is

responsible

for

the

strong

photothermal/photoacoustic effects. Second, the in-situ transformation of polymer happened from core to shell by Ostwald ripening,16,17 which guaranteed the residual surface groups via optimized synthesis conditions. This is beneficial for the water dispersity, biocompatibility and full-colored surface state fluorescence of HCCDs. Third, a mesoporous phenolic resin which would be transformed into mesoporous carbon nanospheres (MCN) at 350℃ was employed as template supports, assuring the uniformity and small size of HCCDs.18 It was favorable for the nanodots to target glioma through the small BBB alveolus or paracellular transport via the narrow lesioned glioma BBB.19,20 In addition, the solid-state synthesis is simple and the carbon resource is a FDA-approved pharmaceutical excipient,21,22 which would be in favor for pharmaceutical applications of HCCDs. As shown by TEM images (Figure 1A, B and S2A), HCCDs were uniformed sphere-like particles with narrow size distribution from 6 to 8 nm. As compared with the other carbon nanodots, the much more obvious aterrimus and brilliant whiteness of HCCDs in bright-field and dark-field TEM images, respectively, implies their high degree of crystallinity. HRTEM images (Figure 1C, D) showed clear crystalline lattice fringes with d-spacing values of 0.24 and 0.21 nm, which could be indexed as the (1120) and (100) planes of graphitic carbon, respectively.23,24 Fast Fourier transform (FFT) patterns validated the single crystallinity of an individual nanodot, while the inverse fast Fourier transform (IFFT) confirmed the high crystallinity without obvious lattice defects (Figure 1E). XRD pattern (Figure


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1F) showed an intense peak at 2θ of approximately 25.5° accompanied by two weak peaks 2θ of about 41° and 53°, which could be assigned to the typical (002), (100) and (004) diffractions of graphitic carbon, respectively.25,26 Raman spectrum of HCCDs gave obvious signals similar to the symmetry A1g mode and E2g mode of sp2-hybridized graphitic carbon atoms at 1385 cm-1 (D-band) and 1600 cm-1(G-band), respectively.27 The relatively lower peak intensity ratio (ID/IG) of 0.75 than those of the other high-defected carbon nanomaterials also indicated the high crystallization of HCCDs.25,27,28 These results show that small-sized HCCDs can be synthesized via the in-situ solid-state transformation under 350 °C. Usually, calcination under high temperature will reduce the water dispersity of carbon nanomaterials and produce agglomeration. However, this did not occur in HCCDs synthesized by the catalyst- and pluronic F127 template-assisted solid-phase transformation, as revealed by the dynamic light scattering (DLS) plot and dispersity measurements. As shown in Figure S2A, S2B and S3, the almost identical DLS particle size compared with that measured by TEM if hydrophilization is considered, indicated the mono-dispersity of HCCDs in water. Meanwhile, HCCDs can freely disperse in different physiological media without apparent sediment even after a super centrifugation of 10,000 rpm for 10 min. It’s known that Ostwald ripening is a classic crystallization process for nanoparticles.17,29 We assumed that the in-situ solid-phase formation of HCCDs followed this manner and crystallization started from the core of the polymer. Therefore, as the endgame of the crystallization under the optimized synthesis conditions, hydrophilic surface groups (such as pluronics-derived ethylene glycol fragments and oxidation products) would form as the shell of HCCDs, which ensured the good water dispersity. To ascertain this, FTIR and NMR spectroscopy was employed to investigate surface functional groups, both of which clearly indicated the presence of carboxyl, hydroxyl, and some (nitrate-


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derived) amide groups on HCCDs’ surfaces (Figure S4). HCCDs possess a negative zeta potential of -28 mV (Figure S2C) consistent with carboxylate surface groups. Moreover, XPS survey spectrum (Figure S5A) showed the existence of C, O, N elements on HCCDs’ surfaces, corresponding to atomic percentages of 74.15%, 24.65%, and 1.20%, respectively. And the deconvoluted spectra (Figure 1G, S5B and S5C) all revealed carbon-bonded oxygen/nitrogen configurations on HCCDs in addition to the sp2-hybridized graphitic carbon. Notably, the low but significant incorporation of the element nitrogen may originate from the catalyst (KNO3), which was doped into the carbon surface and carbon framework of HCCDs under high temperature to form pyridinic (398.4 eV), pyrrolic (400.0 eV) and graphitic (401.1 eV) structures, respectively.13,30 In addition, DTG curve of HCCDs (Figure S6A) showed two distinct weight loss at 395 °C and 454 °C, suggesting the decomposition of surface groups and crystallized carbon cores, respectively.31,32 Accordingly, MS spectra (Figure S6B) also gave the C and N signals associated with heterothermic decomposition from the shell and the core. All these results suggested that HCCDs with hydrophilic groups as shell and highly crystalline carbon as core can be fabricated via the simple solid-state synthesis. Owing to the special structure, HCCDs exhibited unique optical and photothermal properties. As shown in UV-Vis spectra (Figure 2A), the gradually decayed wide-absorption of HCCDs from UV to visible region suggested the different surface states that originated from the plentiful surface groups.33,34 The two slightly raised shoulder peaks at 255 and 340 nm implied the π - π* and n - π* transitions of dominated sp2 hybridized carbons (C=C) and carbonyl groups (C=O) respectively.35,36 Meanwhile, HCCDs also had a much stronger absorption in the NIR region (808 nm) as compared to GO nanodots at the same concentration. This can be explained by the presence of the highly crystalline cores, which produce a larger conjugated π electron system to


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satisfy the most probable long-wavelength absorption (Figure 2E).24,25 As a result of these special absorptions, HCCDs gave an interesting emission. As demonstrated in Figure S7, HCCDs exhibited excitation-dependent full-color fluorescence, where the emission could be tuned from blue to green through increas-ing the excitation wavelength, and the optimal emission was centered about 506 nm at 440 nm-excitation (Figure 2B). This special emission might be attributed to the plentiful surface states, which will produce a series of emissive traps.18,37 Furthermore, under a certain excitation wavelength illumination, a surface state emissive trap will dominate the emission (Figure 2E).38,39 The tunable fluorescence behavior is beneficial for bioimaging and many other important applications such as photodetectors or solar cells. In addition, HCCDs had remarkable photothermal effect. As demonstrated by photothermal curves and images (Figure 2C, 2D and S8), HCCDs aqueous solution could be heated under the 808-nm laser irradiation, which exhibited a HCCDs concentration- and laser power-dependent manner. The photothermal conversion efficiency of HCCDs (Figure S9) measured according to a previously reported method could reach 42.3%, which was much higher than those of graphene nanodots (28.6%), carbon nanospheres (35.1%) and gold nanorods (21%).40,41,42 Particularly, the temperature of HCCDs solution (150 µg mL-1) could reach 54.6 °C after 2.0 W cm-2 NIRirradiation for 5 min, exceeding the lethal limit of cells (about 50 °C).43 Moreover, HCCDs in aqueous solution had a good photothermal stability, the temperature change by heating via repetitive on-off NIR irradiation for 3 times did not exhibit any obvious reduction of heating capacity (Figure 3B and 3D), and the optical absorption kept almost the same before and after irradiations (Figure S9). This advanced photothermal ability of HCCDs might come from their highly crystallized cores that contributed to the intense NIR absorption and strong π-conjugated lattice vibrations.44 These characteristics will be advantageous for photothermal-related


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applications such as therapy, imaging, and heating. Furthermore, HCCDs can produce strong photoacoustic effect due to their highly crystalline carbon cores. As shown in Figure 4C and S10, HCCDs in aqueous solution exhibited linearly elevated photoacoustic signals (R2 = 0.9978) with the increased concentration from 0 to 1.0 mg mL-1 at 705 nm (in agreement with the observed NIR absorption in Figure 2A). Correspondingly, gradually enhanced contrasts were acquired. Since the remarkably increased depth and spatial resolution of photoacoustic imaging compared to traditional in vivo optical imaging,45 HCCDs-contrasted photoacoustic imaging offers a significant potential for glioma visualization. To consolidate the origin of this desirable photoacoustic/photothermal and fluorescence performance of the solid-state derived highly crystalline cores with hydrophilic surface of HCCDs under the optimal conditions, a series of parallel control experiments were carried (Experimental Section). First, the fluorescence emission of HCCDs was stable at wide range of pH and different UV irradiation time periods (Figure S11). Second, all the fluorescence intensities via different wavelength excitations initially increased and then decreased when varying the crystallization temperature from 150 °C to 450 °C or the crystallization time from 1 h to 4 h (Figure 3A, 3C, S12 and S13). Third, the photothermal heating ability gradually enhanced with the increase of the crystallization temperature or the prolongation of the crystallization time (Figure 3B, 3D and S14). These results could be explained as the outcomes of the special in-situ solid-phase crystallization of carbon nanodots from core to shell under high temperatures. At the low temperature (150 °C), carbon cores did not crystallized well, and correspondingly, the multiple surface chromophores did not form. Thus, negligible fluorescence/photothermal effects were observed. With the elevation of the calcination temperature, the crystallized carbon cores accompanied by the multiple surface states would be


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formed for the enhanced fluorescence/photothermal effects. However, overheating extended the crystallization from the cores to the whole nanoparticle without remaining hydrophilic surface groups, resulting in absence of fluorescence, high photothermal activity but also low water dispersity. Conversely, at the initial stage of crystallization (1 h), although the numerous surface states had already formed sufficient for the fluorescence emission, the insufficiently low degree of crystallization led to low photothermal ability. As prolonging the crystallization time, the surface groups could be further regulated for a slightly increased fluorescence. Meanwhile, the photothermal effect was enhanced via the growth of crystallized cores. However, solid-state transformation at 350 °C for over 3 h further promoted crystallization of carbon cores at expense of the surface groups, which consequently caused a high photothermal heating performance but an obviously decreased fluorescence. In addition, the protonation or deprotonation of surface groups at harsh pH could also depress the fluorescence via interfering the surface state emissive traps.46,47 These findings suggested that highly water-dispersible and uniformly small-sized HCCDs with simultaneous FL and photoacoustic/photothermal functions can be synthesized by our optimal in situ solid-state transformation procedure. The advantageous properties of HCCDs synthesized by this protocol might be extremely useful for glioma and other cancer treatment, as further demonstrated by the following experiments. HCCDs were found to display negligible cytotoxicity even at the concentration of 1000 µg mL-1 (Figure S15), suggesting their good biocompatibility. Nevertheless, they were rapidly taken up by glioma cells in concentration- and time-dependent manner, and mediate the apparent blue, green, yellow, and red emissions for multicolor imaging of glioma cells under excitation at 405, 458, 488, and 514 nm, respectively (Figure 4A and S16). The good biocompatibility and broad fluorescence tunability offer HCCDs an opportunity for glioma-targeted fluorescence imaging in


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vivo. As shown in Figure 4B, intravenously administrated HCCDs gradually accumulated in the tumor region and mediated a brightest imaging at 30 min post injection under 465 nm excitation in glioma-bearing nude mice. Interestingly, the contrast imaging persisted at 2 h post injection, remaining in the glioma site (Figure 4B), consistent with a prolonged accumulation of fluorescent HCCDs within glioma. This might be attributed to their hydrophilic surfaces and small particle sizes, which guaranteed the relatively long circulation time and passive targeting via the elevated enhanced permeability and retention (EPR) effect, utilizing paracllular transport across the small leaky vasculatures on BBB and tumor interstitium.6,20 Notably, some accumulation of HCCDs within the liver and kidney observed in ex vivo imaging indicated that HCCDs might be mostly eliminated by these two organs. These demonstrated the high potential of HCCDs for glioma visualization via fluorescence imaging. Moreover, this targeted glioma visualization was consolidated via photoacoustic imaging due to the strong photoacoustic effect of HCCDs. As revealed by photoacoustic images in Figure 4D, intravenously administrated HCCDs had a time-dependent targeted accumulation in glioma-bearing mice similar to the observation from fluorescence images. At 30 min after intravenous injection, the glioma could be distinctly identified and accurately positioned from different photoacoustic imaging sections. Therefore, accompanying with the photothermal ability and targeted accumulation of HCCDs, the sensitive fluorescence imaging and deep space-resolved photoacoustic imaging can guide a visualized photothermal therapy of tumor, especially for the deep-seated glioma in CNS, where the time period and position of NIR irradiation after injection could be well regulated to minimize the side effect. Next, the in vitro and in vivo photothermal therapy of glioma were examined on U87 cells and glioma-bearing mice, respectively. As shown by live-dead stain and CCK results in Figure 5A


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and 5B, neither HCCDs nor the 808-nm laser irradiation alone could mediate any cytotoxicity, due to the biocompatibility of HCCDs and the inability of only NIR irradiation for heat generation on cells, respectively. However, HCCD-incubated cells upon irradiation with an 808nm laser for 5 min exhibited an obvious death and cytotoxicity which remarkably increased when increasing the HCCD concentration. Upon the 808-nm laser irradiation, HCCDs resulted in an approximately 80% cancer cell death rate, suggesting their great potential for PTT. Further on, in vivo glioma PTT was performed guided by fluorescence & photoacoustic imaging, thus, treatment could be focused onto the tumor region at 30 min after intravenous injection (Figure 5C). As demonstrated in Figure 5D, neither HCCDs nor the 808-nm laser irradiation alone resulted in apparent glioma cell death, comparable to the saline control treatment. In contrast, combining HCCD treatment with laser irradiation (HCCDs+PTT) mediated noticeable glioma cell death in vivo, in agreement with the in vitro cytotoxicity results. Meanwhile, ex vivo images also clearly indicated that only the HCCDs+PTT treatment could lead glioma to burnt escharosis and vanishing (Figure 5E). Correspondingly, survival and body weight curves showed that HCCDs+PTT-treated glioma-bearing mice had minimal body weight loss and the longest survival time (Figure 5F and 5G). As compared to the untreated group (saline), the median survival time of HCCDs+PTT-treated mice (26 days) was far longer than that of the other groups (saline, 19 days; laser alone, 19 days; and HCCDs alone, 19.5 days). These desirable performances validated the impactful PTT of glioma using HCCDs as the carrier. In addition, the ex vivo images and H&E stained images (Figure S17 and S18) indicated that HCCDs+PTT treatment did not cause apparent inflammation, cell necrosis or apoptosis in normal tissues including heart, liver, spleen, lung, kidney and brain, evidencing the low side effects. The excellent photothermal therapeutic outcomes might be attributed to the facts: (1) accurate


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guidance by fluorescence & photoacoustic imaging for optimal therapeutic site and time window; (2) highly water-dispersed and small-sized HCCDs for the enhanced targeted accumulation across the BBB; (3) high crystallinity of HCCDs for strong photothermal effect. Therefore, the solid-state derived HCCDs have great potential for biomedical application. 4. CONCLUTIONS In summary, HCCDs simultaneously possessing a high crystallinity and good water dispersity were synthesized via an in situ solid-state transformation procedure using F127 and phenol resin as the carbon source for formation of a mesoporous carbon template. Origination from the larger conjugated π system in crystallized carbon cores and the multiple surface states in hydrophilic surface

groups,

HCCDs

have

tunable

fluorescence

emissions

and

strong

photoacoustic/photothermal efficiency. Meanwhile, attributing to the uniform small size of 6-8 nm and their high hydrophilicity, HCCDs apparently can permeate the glioma BBB and specifically accumulate in glioma cells. Therefore, dual-modal to imaging-guided PTT targeted to glioma in mice was achieved. For this, the exceptional characteristics of the novel theranostic nanodots provide a multicolor and deep-spatial resolution necessary for the best-possible therapeutic outcome. In addition, HCCDs are highly biocompatible and avoid, together with optimal focusing on the treatment site guided by imaging, side effects to the normal tissues.


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Figure 1. (A) Bright-field and (B) dark-field TEM images of HCCDs. (C, D) HRTEM images and (E) corresponding FFT pattern of HCCDs. The inset in (C) is inverse Fourier transform of the HRTEM image. (F) XRD pattern and Raman spectrum (inset) of HCCDs. (G) Highresolution C1s XPS spectra of HCCDs.


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Figure 2. (A) UV-Vis spectrum of HCCDs and the corresponding comparison of NIR absorption with GO nanodots (inset). (B) Excitation-emission map of HCCDs. (C) Temperature changes of HCCDs solution with different concentrations under the irradiation of an 808-nm laser (2.0 W cm-2). (D) Thermal images of HCCDs solution (150 µg mL-1) irradiated by an 808-nm laser with various power intensities. (E) Schematic illustration of the simultaneous photothermal heating and fluorescence mechanisms of HCCDs.


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Figure 3. (A, C) Fluorescence intensities and (B, D) temperature changes of HCCDs derivatives (150 µg mL-1 ) synthesized at (A, B) different temperatures and (C, D) different calcination time periods, tested via various excitations (A,C) and repeatedly turning on/off the 808 nm NIR laser for 3 times (B,D), respectively.


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Figure 4. (A) Confocal fluorescence images of U87 glioma cells incubated with 150 µg mL-1 HCCDs for 2 h under different excitations. Bar = 100 µm. (B) In vivo fluorescence images of glioma-bearing mice at different time points after intravenous administration of saline and HCCDs, respectively. The ex vivo images of major organs were taken at 120 min after administration. (C) Photoacoustic images of HCCDs solution with different concentrations. (D) Real-time photoacoustic images of glioma in mice at different time points after intravenous administration of HCCDs.


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Figure 5. (A) Confocal images of U87 cells after different treatments. Green: live cells; red: dead cells. Bar = 100 µm. (B) Cytotoxicity of U87 cells incubated with different concentrations of HCCDs with or without an 808-nm laser irradiation (2.0 W cm-2, 5 min). (C) The photo of the dual imaging guided PTT on glioma-bearing mice. (D) Glioma apoptosis results (on 20th day post injection) of mice with different treatments. Blue: DAPI-stained nucleus; green: FITClabeled apoptosis cells. Bar = 100 µm. (E) Photos of brains in glioma-bearing mice with different treatments. The solid lines circled the glioma, while the dotted lines circled the escharosis (disappeared glioma) via PTT.(F) Kaplan-Meier survival curves of glioma-bearing mice via different treatments (n = 6). (G) Body weight curves of glioma-bearing mice in different groups (n = 6).


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Scheme 1. Illustration of solid-phase synthesis and biological effects of highly crystalline carbon nanodots (HCCDs). Calcination of mesoporous phenol resin (mPR) results in formation of mesoporous carbon nanospheres (MCN) with HCCDs attached (HCCDs@MCN). After ultrasonication, HCCDs can be collected by removing MCN and fragments by centrifugation.


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ASSOCIATED CONTENT Supporting Information. The following files are available free of charge. Characterization data (DLS, FTIR, NMR, XPS, XRD); fluorescence, photothermal and photoacoustic effect of HCCDs and its derivatives; cytotoxicity results both ex and in vivo. (PDF) AUTHOR INFORMATION Corresponding Author * Email for R.H.: [email protected] * Email for Y.W.: [email protected] Author Contributions Y.W. and M.Q. designed and synthesized the multicolor carbon nanodots. R.H. and M.Q. designed and performed biological studies. M.Q. and H.J. performed the ex vivo studies. M.Q., Y.D., S.W. and C.L. performed the in vivo studies. All authors contributed with discussions of the work. The manuscript was written with contributions of all authors. All authors have given approval to the final version of the manuscript. Notes The authors declare no competing financial interest. ACKNOWLEDGMENT This work was supported by National Natural Science Foundation of China (21675032, 81573002 and 81773280) and Shanghai Pujiang Program (17PJD002). Rongqin Huang and Ernst


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Table of Contents (TOC)


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Highly Crystalline Multicolor Carbon Nanodots for Dual-Modal

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