Magnetic and fluorescent carbon nanotubes for dual modal imaging and photothermal and chemo-therapy of cancer cells in living mice

Carbon 123 (2017) 70e83

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Magnetic and fluorescent carbon nanotubes for dual modal imaging and photothermal and chemo-therapy of cancer cells in living mice Ming Zhang a, b, Wentao Wang d, Fan Wu a, b, Ping Yuan a, b, Cheng Chi a, b, Ninglin Zhou a, b, c, * a Jiangsu Collaborative Innovation Center for Biological Functional Materials, College of Chemistry and Materials Science, Nanjing Normal University, Nanjing, 210023, China b Jiangsu Key Laboratory of Biofunctional Materials, Jiangsu Engineering Research, Center for Biomedical Function Materials, Nanjing, 210023, China c Nanjing Zhou Ninglin Advanced Materials Technology Company Limited, Nanjing, 211505, China d Jiangsu Key Laboratory for Molecular and Medical Biotechnology, College of Life Sciences, Nanjing Normal University, Nanjing, 210023, China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 12 May 2017 Received in revised form 5 July 2017 Accepted 11 July 2017 Available online 14 July 2017

Multi-walled carbon nanotubes (MWCNTs) have drawn increasing attention in biomedical fields because of their unique structures and properties, including good photothermal performance, large surface areas, strong near-infrared (NIR) absorbance, and size stability on the nanoscale. However, big challenge for this platform is to achieve fluorescence/magnetic resonance (MR) imaging and photothermal therapy (PTT) therapy in single nanotube. In this work, Multi-walled carbon nanotubes-magnetofluorescent carbon quantum dots/doxorubicin nanocomposites was prepared. The nanocomposite was then used as carriers for targeted drug transport in cancer therapy. These nanocomposites possess high heat-generating ability, pH and NIR responsive drug delivery, and heat-induced high drug release as well. Experiments in vitro and in vivo show that this platform can deliver anti-cancer drugs to targeted cells, releasing them intracellular upon NIR irradiation, and eliminate tumors effectively through chemo/photothermal synergistic therapeutic effect. Based on the findings of this work, further development of using other CNTs as highly efficient NIR agents can be achieved for vivo tumor imaging and chemo/photothermal synergistic therapeutic. © 2017 Published by Elsevier Ltd.

Keywords: Multi-walled carbon nanotubes NIR absorbance Magnetofluorescent Chemo/photothermal

1. Introduction Multifunctional nano-theranostics materials that work in the form of combining therapeutic and diagnostic functions into a single hybrid nanomaterial have attracted substantial attentions [1,2]. Carbon nanotubes (CNTs), formed by carbon atoms, have structures of one-dimensional hollow tubular bodies [3]. Since CNTs displayed unique structures and remarkable physical properties, various applications of CNTs have emerged in materials, catalysis and life sciences etc, especially in the matter of tumor therapy [4e6]. As a consequence, they have a dominant position in carrying multiple diagnostics and target deliveries into a specific area of complex biological systems [7]. Furthermore, they are

* Corresponding author. Jiangsu Collaborative Innovation Center for Biological Functional Materials, College of Chemistry and Materials Science, Nanjing Normal University, Nanjing, 210023, China. E-mail address: [email protected] (N. Zhou). http://dx.doi.org/10.1016/j.carbon.2017.07.032 0008-6223/© 2017 Published by Elsevier Ltd.

monitored noninvasively with the applying of bio-imaging tools both drugs and their accumulation at the target sites as long as therapeutic and imaging agents are incorporated into CNTs in vitro and in vivo conditions. Present imaging modalities, including fluorescence, magnetic resonance (MR), photoacoustic, positron emission tomography, and ultrasound images, have significant benefits and limitations [8e10]. Magnetic resonance imaging (MRI) is regarded as one of the most powerful techniques among modern diagnostic medicines as it can penetrate deeply into tissues, providing anatomical details and high quality three-dimensional images of soft tissues in a non-invasive monitoring manner [11]. Fluorescence imaging (FI), in contrast, has the capacity for singlecell sensitivity and subcellular resolution [12]. Apparently, combining the advantages of MRI and FI can bridge gaps in sensitivity and depth between these two modalities, and consequently, leading to an improved reliability in diagnoses [13]. Gadolinium (Gd) has been confirmed to exhibit excellent contrast efficiency because of its unique magnetic property [14]. Many Gd-containing nanoparticles and chelates have been devel-

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oped as effective probes for MR diagnosis. With abilities of inhibiting calcium channels, causing cardiovascular and neurologic toxicity, the free Gd3þ is known as highly toxic [15,16]. Therefore, some groups have developed magnetofluorescent carbon quantum dots (CQDs) by doping or conjugating a paramagnetic gadolinium ion for fluorescence and magnetic resonance imaging in vitro and in vivo [17,18]. However, some of this magnetofluorescent CQDs have limited practical applications, because of low quantum yield and multiple time consuming synthesis steps. There has been a no report of magnetofluorescent CQDs as a nano-theranostic for MR/ fluorescence and chemotherapy. It has been shown that doping of hetero elements (including N) during synthesis would significantly improve the fluorescence properties of CQDs [19]. Thus, the purpose of this work is to develop a simple one-pot method to fabricate a magnetofluorescent GdN@CQDs, with excellent magnetic and fluorescence properties, for MR and fluorescence dual-modal imaging [20]. Several researchers have developed magnetofluorescent CNTs by doping or conjugating a paramagnetic carbon quantum dots for fluorescence and magnetic resonance imaging in vitro and in vivo. However, some of these magnetofluorescent CNTs have the nature of limited practical applications, because of low loading yield and multiple time consuming synthesis steps [21,22]. Therefore, an original strategy to derivatize CNTs with magnetic/fluorescence CQDs is described. With the chemical inertness, the modification of CNTs was typically carried out with noncovalent functionalization. Thus, the purpose of this work aims to develop a magnetofluorescent CNTs, with excellent magnet and fluorescence properties, for MR and fluorescence dual-modal imaging. There is an increasing interest in using photothermal therapy (PTT) induced by near-infrared (NIR) laser as a highly effective alternate to conventional cancer treatment approaches [23,24]. Imaging-guided photothermal therapy (PTT), a novel adjuvant therapeutic method of precision therapy, could be used to eliminate remaining cancer cells completely with precise guidance in the future [25,26]. Owing to the unique physical and chemical properties of carbon nanotubes (CNTs), CNTs have generated substantial interest in nanomedicine for applications in thermal therapy. CNTs can absorb NIR laser light to produce a local high temperature, which may kill cancer cells [27e29]. CNTs have emerged as promising candidates for highly efficient delivery of drugs and biomolecules due to their unique structure and properties [30]. CNTs can be conjugated with anti-cancer drugs by covalent and noncovalent interaction methods. Inspired by the properties of CNTs, we pay increasing attention to the combination of PTT and chemotherapy in cancer treatments. Although previous studies have demonstrated that PTT is efficient in cancer therapy with the help of the above CNTs, single PTT cannot always eradicate cancers completely, leading to cancer recurrence [31,32]. However, study shows that heat derived from CNTs have efficiency to enhance the effect of chemotherapy. The combination of photothermal therapy (PTT) and chemotherapy, termed chemo-thermotherapy, can achieve enhanced anti-cancer efficacy via synergistic effects. EGFR antibody is an emerging class of targeting ligands which also serve as biological drugs that can be used to treat various diseases [33]. Comparing to other targeting agents, EGFR antibody possess distinctive advantages: low synthesis cost, low-immunogenicity, small size that makes it easy to penetrate into solid tumors, and high affinity comparable to monoclonal antibodies for binding almost any molecules [34]. As escort molecules, EGFR antibody is able to deliver drugs or nanoparticles encapsulating drugs, target cells via high-affinity and specific binding [35]. Here we report the magnetofluorescent MWCNTs as a nanotheranostic for MR/fluorescence imaging and chemo/PTT therapy. The GdN@CQDs-MWCNTs/DOX-EGFR possess, we revealed, strong heat-generating ability. Drug delivery experiment reveals that the

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GdN@CQDs-MWCNTs/DOX-EGFR exhibit both pH and heat responsive drug release behaviors. Furthermore, experiments in vitro and in vivo demonstrate the excellent cancer ablation ability of GdN@CQDs-MWCNTs/DOX-EGFR. The strategy of material synthesis combining PTT and chemotherapy is illustrated in Fig. 7A. Our research demonstrates the as-synthesized GP-GdN@CQDsMWCNTs/DOX-EGFR are promising cancer ablation nanoplatforms for combining PTT and chemotherapy. 2. Materials and methods 2.1. Materials and chemicals MWCNTs (i.d./o.d. ¼ 2e4 nm/10e20 nm) were purchased from Shenzhen Nanotech Port Co. Ltd. Citric acid (C6H8O7, CA), gadolinium chloride (GdCl3) and diethylenetriamine (C4H13N3, DETA) were obtained from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China). Dialysis membranes of 500e1000 Da (USA, Spectrumlabs) were purchased from Toscience Biotechnology Co, Ltd. (Shanghai, China). All chemicals were analytical grade and used as received without further purification. Distilled water was used throughout the whole experiment. Hydroxy-2,5-dioxopyrrolidine-3-sul fonicacid sodium salt (Sulfo-NHS, 97%) and 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC, 99%) were acquired from Aladdin. Chemical Co. (Shanghai, China). Fetal bovine serum (FBS) and DMEM was obtained from SunShine Biotechnology Co., Ltd. (Nanjing, China). The A549, H522, MDA-MB-231, and MCF-10A were purchased from the Cell Bank of Culture Collection of Chinese Academy of Sciences (Shanghai, China). All other reagents and solvents were of analytical grade and used as received. 2.2. Characterization The photoluminescent (PL) spectra were recorded using molecular fluorescence spectrometer (Cary Eclipse, varian, USA). The infrared spectrum was performed on a (FT-IR) Nexus 670 FTIR type (Nicolet). The X-ray diffraction (XRD) analysis was performed using a D/Max 2500V/PC diffractometer (Rigaku Corporation, Japan). The thermogravimetric (TG) measurements were performed on a Perkin-Elmer TG 7 instrument. UVeVis spectroscopy measurements were performed on a Cary 5000 UVeViseNIR spectrometer (Varian). The surface composition and element analysis of the samples were recorded using X-ray photoelectron spectroscopy (XPS, EscaLab-250, Thermo, USA). The hydrodynamic size and zetapotential were measured on a Malvern ZEN 3600 Zetasizer (Malvern Instruments, UK). The transmission electron microscopy (TEM) images were acquired on a JEM-2100F transmission electron microscope. MR images were acquired by a 7 T BioSpec 70/30 experimental scanner (70/30 Bruker BioSpin; Ettlingen, Germany). The fluorescent images of cells were acquired by confocal laser scanning microscope (TI-E-A1R, Nikon, Japan). Details of methods used for material characterization are described in the “Experimental Section” (Supporting Information). 2.3. Synthesis of GdN@CQDs GdN@CQDs were synthesized according to previous a CQDs method with fine modification. Briefly, 0.5 g of CA, 0.3 g of polyglutamic acid, and 0.1 g of GdCl3 were dispersed into 10 mL of double distilled water (DDW) in sequence under vigorous stirring. Subsequently, the above mixture solution was added into a 25 mL Teflonlined stainless steel autoclave and heated to 200  C for 6 h. The dark brown products were obtained after cooling to room temperature. The large and agglomerated nanoparticles were removed by centrifuging at 12,000 rpm for 10 min. The supernatant

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containing GdN@CQDs was filtered through a 0.22 mm microporous membrane to further remove residual large particles. Final purification of the GdN@CQDs solution was conducted through a dialysis membrane (MWCO: 500e1000 Da) for 24 h (changed deionized water every 4 h). After this process, a solid product was collected after freeze-drying and could be dissolved again for further applications. 2.4. Synthesis of MWCNTs-PEI Multiwalled carbon nanotubes (MWCNTs) with 10e20 nm outer diameter and 10e25 mm average length, which were kindly provided by Shenzhen Nanotech Port Co. Ltd, were heated and refluxed in a mixture of concentrated sulfuric and nitric acid (v/v ¼ 3:1) for 24 h. The obtained carboxyl-terminated MWCNTs (MWCNT-COOH) were then dispersed into methanol for further use. For synthesis of MWCNTs-PEI, N-(3-dimethylaminopropyl)-N0 ethylcarbodiimidehydrochloride (EDC, 0.2 g) and N-hydroxysuccinimide (NHS, 0.2 g) were introduced to a methanol suspension (150 mL) of MWCNT-COOH (30 mg) to activate the carboxylic acid groups of MWCNT-COOH, and the mixture was stirred gently at room temperature for 3 h. Then 0.2 g PEI (branched, Mw 60000) was added to the activated MWCNTs-COOH solution, followed by another 24 h of stirring. The resulting MWCNTs-PEI was separated by centrifuging, rinsed with methanol repeatedly to remove excess polymer, and then dried under vacuum. 2.5. Synthesis of GdN@CQDs-MWCNTs For synthesis of GdN@CQDs-MWCNTs, EDC (0.2 g) and NHS (0.2 g) were introduced to a methanol suspension (10 mL) of MWCNT-COOH (10 mg) to activate the carboxylic acid groups of MWCNT-COOH, and the mixture was stirred gently at room temperature for 3 h. Then MWCNT-PEI (20 mg) was added to the activated MWCNT-COOH solution, and the mixture was stirred at room temperature in dark for 24 h. The resulting GdN@CQDsMWCNTs was recovered by centrifuging, washed in sequence with ethanol and water, and then dispersed in water. 3. Results and discussion 3.1. Preparation and properties of GP-GdN@CQDs-MWCNTs/DOXEGFR As shown in Fig. S1, Gd-doped CQDs were synthesized via a onestep hydrothermal method using citric acid (CA), polyglutamic acid, and gadolinium chloride (GdCl3) as starting materials [36,37]. CA was selected as carbon source because it can provide more carboxyl groups which may chelate gadolinium ions during and after formation of CQDs than other nontoxic acids [38]. Moreover, CA is more easily polymerized in the presence of polyglutamic acid [39]. The morphology and size distribution of the synthesized GdN@CQDs were characterized using TEM. Fig. 1B shows that GdN@CQDs are approximately spherical in shape. A histogram in Fig. S2 shows the size distribution taken from over 100 nanoparticles and has an average size of 3.2 ± 1.2 nm. The highresolution TEM (HRTEM) image of GdNS@CQDs indicated that the lattice spacing were 0.21 nm (Fig. 1A), which matches well with the (100) facet of graphite [39]. As-prepared Gd-doped CQDs (GdN@CQDs) exhibits excellent water solubility and cellmembrane permeability, which provides potentials for dualmodal MR/fluorescence imaging in vitro and in vivo. Then, we prepared GdN@CQDs-MWCNTs with the controlled conjugation onto the acid-treated surface of MWCNTs. Fig. 1A illustrates the procedure used a traditional EDC/Sulfo-NHS chemical

reaction. Subsequently, the MWCNTs were initially oxidized and shortened to introduce carboxylic acid groups onto the side walls of CNTs. For the preparation of the composites consisting of carbon nanotubes and CQDs, the MWCNTs surfaces need to be functionalized in order to meet the required amount of oxygen containing groups (OCGs) on the tubes as well as shorten the length of the asreceived tubes. As indicated by the typical transmission electron microscope (TEM) images (Fig. 1D), the original MWCNTs (Fig. 1C) have been cut short after the oxidation (as marked in Fig. 1D). The length of the resulting MWCNT-COOH (statistical results of 100 nanotubes in TEM images) is in the range of 50e500 nm with a mean of 180 nm (Fig. S2B and Fig. S3). The branched polyetherimide (PEI) was covalently grafted on the shortened CNTs via the formation of amides between the carboxylic acid of MWCNTs-COOH and the amine of PEI. Representative TEM images of MWCNTs-PEI are shown in Fig. 1E. In the high-resolution image, the coreeshell structure could be observed clearly. The outer wall of the MWCNTs was coated with a polymer shell along the stretched direction of the tube and there were obvious differences between graphite sheets and polymer shell [40]. The thickness of polymer shell was ca. 2e3 nm (as marked in Fig. 1E). Such core-shell nanostructures were same as those of polymer-grafted MWCNTs reported previously. Fig. 2A shows the Fourier transform infrared (FT-IR) spectra of the as-prepared MWCNT-PEI. The MWCNT-PEI presents an absorption band at which are corresponding to the amide carbonyl vibration and the N-H bending vibration, respectively. The new peaks at 2922 and 2836 cm1 ascribed to C-H stretch modes also confirmed the existence of PEI. Moreover, the weak absorption band at 1718 cm1 attributed to C¼O of -COOH was presented, indicating the consumption of most -COOH groups in the carboxyterminated MWCNTs due to the formation of amide structure after the graft of PEI. The initial MWCNTs have a zeta potential of 18.9 mV, which changes to 12.3 mV when modified with PEI (Fig. S4). PEI content in MWCNT-PEI was determined using thermal gravimetric analysis (TGA) (Fig. S5). The difference in the weight loss at 400  C between MWCNT-PEI and MWCNT-COOH showed that PEI grafting amount was about 18%. The amine moiety in MWCNT-PEI is favorable for further conjugating the desired functional groups. Herein, a portion of the primary amine groups were used to react with GdN@CQDs to introduce the magnetofluorescent nanoparticles. The as-prepared multiamino-functionalized MWNTs can be used to assemble negatively charged GdN@CQDs used a traditional EDC/Sulfo-NHS chemical reaction. When GdN@CQDs was attached to MWCNTsPEI, the polymer grafts on the surfaces of MWCNTs may form a self-standing film because of the high density of grafting polymer and high solubility of quaternized PEI in water, and GdN@CQDs may be dispersed uniformly in the film, forming a GdN@CQDs-MWCNTs. This phenomenon can be observed by TEM. As shown in Fig. 1F,a, a self-standing film composed of functionalized MWCNTs was observed on the TEM grid. Nevertheless, the GdN@CQDs was not seen clearly, probably due to the coverage of polymer chains (Fig. 1F,b). It can be found that the as-prepared GdN@CQDsMWCNTs were well loaded spots with a lattice distance of ca. 0.21 nm, which was consistent with the facet of GdN@CQDs (1F,c). The initial MWCNTs had a zeta potential of 12.3 mV, which changed to 4.7 mV when modified with GdN@CQDs (Fig. S4). GdN@CQDsMWCNTs with quantum dots were well-dispersed individual units that facilitated the monitoring of real-time fluidic behavior and fluorescent imaging in biosystems. These results proved that GdN@CQDs were successfully attached to MWCNTs-PEI and a GdN@CQDs-MWCNTs hybrid film was fabricated. DOX was selected as a model drug to investigate the feasibility of GdN@CQDs-MWCNTs as a drug delivery carrier. DOX was mixed with GdN@CQDs-MWCNTs in PBS buffer at room temperature for

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Fig. 1. (A) Assay of Working Protocol of the GP-GdN@CQDs-MWCNTs/DOX-EGFR Drug Delivery System (B) TEM images of GdN@CQDs. (B) TEM images of original MWCNTs. Inset: HTEM images of single MWCNTs. (D) D TEM images of MWCNTs-COOH. (E) TEM images of MWCNTs-PEI. (F) TEM images of GdN@CQDs-MWCNTs (a). HTEM images of GdN@CQDsMWCNTs (b, c). (G) TEM images of GP-GdN@CQDs-MWCNTs/DOX. (A colour version of this figure can be viewed online.)

24 h in the dark. We speculated that DOX was probably conjugated with GdN@CQDs-MWCNTs through physicochemical interactions, including (p-p) stacking, electrostatic, and hydrophobic interactions. Meanwhile, it had reported that DOX could be directly adsorbed onto the surface of Gd doped CQDs via electrostatic, hydrophobic interaction and p-p stacking [19]. It was known that, the standard “bottom-up” approach consisted of using self-assembled structures (liposomes, micelles, dendrimers) to obtain a synthetic nanovector capable of encapsulating, protecting, transporting, and delivering a therapeutic agent [41]. Here we prepared a new micelle conjugated to MWCNTs, with ability to allow fluorescence/magnetic resonance (MR) imaging and chemophotothermal synergistic therapeutic effect. Genipin (GP), a natural and non-toxic crosslinking reagent was used to cross-link GdN@CQDs/DOX. The GPGdN@CQDs-MWCNTs/DOX were characterized by transmission electron microscopy (TEM). Fig. 1G demonstrated strong attachment of the GdN@CQDs/DOX micelles to the MWCNTs surfaces, and the MWCNTs was evenly borne with micelles. Interestingly, there were many dark dots wrapped in the micelle (as marked in dotted red circles) as shown in TEM image of single GdN@CQDs/DOX micelles, suggesting that GdN@CQDs were wrapped after chemical

crosslinking reaction. The results of TEM characterization revealed single GdN@CQDs/DOX particles were about 32 nm. It was observed that the color of the GP cross-linked GdN@CQDs nanospheres turned dark-bluish (Fig. S6A). The color was deepened with the increase of cross-linking time. Bluish color attributed to double bonds in the GP cross-linking GdN@CQDs-MWCNTs/DOX. Lately, novel targeting agents, including EGFR antibody, short peptides and other small molecules, have become the new generation targeting molecules. EGFR antibody is small strands of DNA or RNA that could form unique 3-dimensional structures that specifically combine to molecular targets with high affinity [35]. To examine the presence of the EGFR antibody on particle surface, we used EGFR antibody modification to yield fluorescent CNTs-EGFR. The acid group on the particle surface was first converted to NHS ester in the presence of EDC and subsequently was covalently coupled to the EGFR antibody (Fig. 1A). Further studies of the optical properties were performed to see the interaction between the MWCNTs-COOH, GdN@CQDs, and GdN@CQDs-MWCNTs. Fig. 2B showed photoluminescence (PL) results. The PL spectrum clearly showed that there were not any significant emission wavelength shifts between GdN@CQDs and

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Fig. 2. (A) FT-IR spectra of MWCNTs-PEI. (B) Photoluminescence spectra for GdN@CQDs, MWCNTs GdN@CQDs-MWCNTs (in DI water) recorded at lex ¼ 350 nm. (C) Photoluminescence emission spectra of GdN@CQDs-MWCNTs (progressively longer excitation wavelengths from 295 nm to 435 nm with a 20 nm increment). (D) N2 adsorption/ desorption isotherms and corresponding pore-size distribution curve (inset) of the GdN@CQDs-MWCNTs sample. (A colour version of this figure can be viewed online.)

GdN@CQDs-MWCNTs. Meanwhile, GdN@CQDs attached to the MWCNTs result in substantially decreased PL intensity. As previously demonstrated, the quenching suggested an energy transfer from GdN@CQDs into MWCNTs and the transfer can occur in three possible ways: hole transfer, electron transfer, or both [42]. At present, we believe the dominant mechanism is most likely due to electron transfer from the conduction band of the GdN@CQDs to the empty electronic states of the MWCNTs, since MWCNTs are known to have a high electron affinity, as also seen by many previous studies. GdN@CQDs are brought together with carbon nanotubes, intensity of the excitonic emission of GdN@CQDs decreases, a process called fluorescence quenching. Our observation of rapid charge transfer between the GdN@CQDs-MWCNTs hybrid structure was consistent with prior literature explaining the charge transfer from the excited semiconductors to the CNTs [42]. The control experiment with the non MWCNTs and GdN@CQDs showed almost no GdN@CQDs in the structure as shown earlier with TEM, the GdN@CQDs presented in the luminescent emission can arise from adsorbed carbon quantum dots on the surface as the luminescent peak was at 425 nm not shifted. As shown in Fig. 2C, exploring the optical properties of the as-synthesized GdN@CQDsMWCNTs, the PL spectra were investigated. The red and black lines in Fig. S6B were the PL spectra of GdN@CQDs-MWCNTs, which can be seen that the excitation (lex) and emission (lem) peaks were

located at 355 and 460 nm, respectively. The photograph of GdN@CQDs-MWCNTs dispersion under 365 nm UV light exhibits strong blue emission as shown in the inset of Fig. S6A. In quest of further exploring the optical properties of the as-synthesized GdN@CQDs-MWCNTs, the lex-dependent PL behavior, which was mutual in fluorescent carbon materials, was investigated and displayed in Fig. 2D. This behavior was attributed to the surface states which affect the band gap of GdN@CQDs. The surface state is similar to a molecular state, whereas the size impact was a result of quantum dimensions, both of which were attributed to the complexity of the excited states of GdN@CQDs-MWCNTs. The lexdependent PL behavior was useful for multicolor in vitro and in vivo biological imaging applications (Fig. S7) [43]. As shown in the high-resolution TEM image, the pores were perpendicular to the surface of MWCNTs. This kind of feature would facilitate the mass transportation between inner and outer surfaces of the GdN@CQDs coating to allow drug loading and release. The single point surface area, pore volume (P/P0), and average pore diameter of GdN@CQDs-MWCNTs were determined to be 50.65 m2/ g, 0.30 cm3/g, and 1.69 nm, respectively, by Brunauer-EmmettTeller (BET) measurement (Fig. 2D). The in vitro drug loading and release performance plays a key role in a drug delivery system. Herein, DOX, a widely used anti-cancer drug for chemotherapy treatment, was used as a model drug to study the drug loading and

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release efficiency of GP-GdN@CQDs-MWCNTs. The drug loading was carried out by incubating GP-GdN@CQDs-MWCNTs with different concentrations of DOX in PBS (pH 7.4). The UVevis spectra, photoluminescence spectra, and zeta potential were used to confirm the conjugation. The surface zeta potential of GdN@CQDs-MWCNTs changed from 4.7e5.8 mV after DOX conjugation (Fig. S4). It suggested that the negative charge (COO-) of the GP-GdN@CQDs-MWCNTs conjugated with a positive charge (-NH2) on DOX, which may be due to electrostatic interaction. The UVevis absorption spectra of free DOX, GdN@CQDs-MWCNTs, GPGdN@CQDs-MWCNTs/DOX-EGFR are shown in Fig. 3A. There was a notable difference in the spectra of GP-GdN@CQDs-MWCNTs/DOXEGFR; its UVevis absorption peak was near 488 nm, which corresponded to the absorption wavelength of DOX. The data suggested that the formation of GdN@CQDs-MWCNTs/DOX occurred. Since GdN@CQDs-MWCNTs and DOX had different fluorescent properties when excited at different wavelengths, fluorescence spectroscopy was a great method to characterize the Conjugates [44]. As seen in Fig. S8, the fluorescence of the conjugate (GdN@CQDs-MWCNTs/ DOX) showed the same excitation peaks all two reactants displayed when they were tested independently (Fig. 2 for DOX alone), and this confirmed that the conjugate product contained the GdN@CQDs-MWCNTs and DOX. As shown in Fig. 3A, the red shift of the peak at 260 nm of GP-GdN@CQDs-MWCNTs/DOX-EGFR compared to DOX could be due to the interaction between the

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EGFR antibody and the carboxylic groups of GdN@CQDs-MWCNTs/ DOX, indicating the successful conjugation of EGFR antibody to GPGdN@CQDs-MWCNTs/DOX. For biomedical applications, good dispersion of nanomaterials in a physiological medium was a prime merit. In the current study, we investigated the physical stabilities of GP-GdN@CQDs-MWCNTs/DOX-EGFR in deionized water, and GPGdN@CQDs-MWCNTs/DOX-EGFR solution under 365 nm UV light exhibits strong blue emission as shown in Fig. S6A. Fig. 3A showed the UVevis spectrum of GP-GdN@CQDsMWCNTs/DOX-EGFR, with broad absorption from 650 to 1000 nm. Their absorption in the NIR region from 780 to 900 nm made the as-prepared GP-GdN@CQDs-MWCNTs/DOX-EGFR highly promising for PTT. Given their efficient NIR absorption features between 750 and 850 absorption wavelengths, the GP-GdN@CQDsMWCNTs/DOX-EGFR were investigated for their potential in photothermal ablation therapy of cancer using a 808 nm laser [37]. Under continuous irradiation of 808 nm laser (2 W/cm2), the temperature elevation of the aqueous dispersions containing GdN@CQDs-MWCNTs and GP-GdN@CQDs-MWCNTs/DOX-EGFR (100 mg/mL) was measured (Fig. S9A). The control experiment demonstrated that the temperature of pure water only increased by less than 2.6  C from room temperature. However, with the addition of GP-GdN@CQDs-MWCNTs/DOX-EGFR (100 mg/mL), the temperature of the aqueous dispersions increased by 23.6  C after a 5 min irradiation. These results indicated that GdN@CQDs-

Fig. 3. (A) UVeViseNIR spectra of DOX, GdN@CQDs-MWCNTs, and GP-GdN@CQDs-MWCNTs/DOX-EGFR solutions. (B) Photothermal response of GdN@CQDs-MWCNTs aqueous solution (100 mg/mL) under irradiation for 5 min with an NIR laser (808 nm, 2 W/cm2) and then the laser was shut off. (C) XPS survey spectra of GdN@CQDs-MWCNTs. (D) Linear correlation between longitudinal relaxivities (r1) and equivalent Gd concentration of nanoprobe. Inset: T1-weighted MR images of GdN@CQDs-MWCNTs with various concentrations (equivalent Gd concentration: 0, 0.2, 0.4, 06, 0.8, 1.0, 1.2 mM). (A colour version of this figure can be viewed online.)

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MWCNTs can rapidly and efficiently convert laser energy into heat via efficient photoabsorption at 808 nm. To further study the photothermal effect of the GdN@CQDs-MWCNTs, we recorded the change in temperature of the solution (100 mg/mL, 1 mL) as a function of time in continuous irradiation of 808 nm laser (2 W/ cm2) until the solution reached a steady-state temperature (Fig. 3B). According to the obtained data and the previous literature, the photothermal conversion efficiency can reach approximately to 45.3%. The successful formation of GdN@CQDs-MWCNTs was further verified by XPS spectra (Fig. 3C). GdN@CQDs-MWCNTs showed additional characteristic peaks corresponding to Gd 4d (142.7 eV) and Gd 3d (1182.0 eV), confirming the successful Gd3þ chelation onto the surface of MWCNTs [39]. The introduction of Gd3þ renders GdN@CQDs-MWCNTs the MRI modality because it can accelerate longitudinal (T1) relaxation of water protons and exert bright contrast in regions where the nanoprobes localize. Thus, we investigated the T1 contrast capability of GdN@CQDs-MWCNTs as MRI contrast agents on a 1.2 T MRI instrument and the results were present in Fig. 3D. Obviously, longitudinal relaxivity of GdN@CQDsMWCNTs was enhanced linearly along with the increase of equivalent Gd concentration (Fig. 3D). The specific relaxivity values (r1) 3þ evaluated from a plot of the relaxation time (T1 1 ) versus the Gd 1 1 ion concentration is determined to be 7.178 mM s , which was much larger than commercially available MRI agent Magnevist (r1 ¼ 4.2 mM1 s1, 1.2 T). In the case of a nanoparticle based MRI contrast agent, small size is important to reach a high longitudinal relaxivity because longitudinal relaxivity is largely dependent upon direct interactions between Gd3þ and hydrogen protons. The ultrasmall GdN@CQDs-MWCNTs with high specific surface ratios could maximize dipole-dipole interactions between Gd3þ and hydrogen protons and, thus, were responsible for the high r1. Consistently, the gray-scaled T1-weighted images of GdN@CQDs-MWCNTs with different Gd3þ concentrations clearly showed a positive contrast enhancement, and the brightness became more prominent as increasing Gd3þ concentration from 0 to 1.2 mM (inset of Fig. 3D). But, no such signal enhancement was observed for bare MWCNTs. Therefore, we can conclude that GdN@CQDs-MWCNTs synthesized in this study hold greater potential as fluorescence/MR bimodal nanoprobe in medical imaging for a better diagnosis. 3.2. T2-weighted MR imaging in vivo and vitro The GdN@CQDs-MWCNTs distribution in tissue is vital to evaluate their imaging efficiency and side effects. In vivo MR imaging and the tissue distribution of GdN@CQDs-MWCNTs were tested with mice as model. GdN@CQDs-MWCNTs entered the blood circulation of mice immediately after injection of GdN@CQDsMWCNTs (Fig. S9B). The liver was highly visualized by the excellent contrast MR signal and was substantially distinguished from other organs after 4 h post injection. It is clear that the brightness of the liver region increases from 1 to 4 h, and then decreases gradually at 12 h post injection in the T1-weighted MR images. These results indicate that the developed GdN@CQDs-MWCNTs can be used for T1-weighted MR imaging of mouse liver after intravenous injection. 3.3. Cell internalization and optical imaging ability of GdN@CQDsMWCNTs The intracellular behavior of our GdN@CQDs-MWCNTs after endocytosis was also investigated. A549 cells were incubated with or without GdN@CQDs-MWCNTs for 0.5 h and imaged with confocal microscope. According to the lex-dependent PL behavior of GdN@CQDs-MWCNTs, GdN@CQDs-MWCNTs emitted red emis-

sion under red illumination. After 0.5 h incubation, only cell membrane and cytoplasm exhibited strong red emission, while nuclei were not infiltrated significantly, as shown in Fig. 4A and B. This observation confirms that GdN@CQDs-MWCNTs could be internalized by A549 cells and pass through cell membranes to enter the cytoplasm but not into the cell nuclei under 0.5 h incubation time. GdN@CQDs-MWCNTs exhibited excellent photostability, suggesting that it can be served as a cellular imaging agent. 3.4. pH and NIR responsive drug release of GP-GdN@CQDsMWCNTs/DOX-EGFR The DOX loading and release properties of DOX loaded GdN@CQDs-MWCNTs in various conditions were evaluated. The amount of DOX loaded into the GdN@CQDs-MWCNTs was estimated by measuring the absorbance at 480 nm (Fig. S10). It is found that the loading amount of DOX increased with the increasing in drug concentration and finally reached a saturation value (270 mg/ g) when the initial drug concentration is higher than 600 mg/mL (Fig. 4C), suggesting the controlled loading of DOX onto the GdN@CQDs-MWCNTs surfaces. It has been reported that many aromatic drug molecules including DOX could be directly adsorbed onto the outer-wall of MWCNTs via hydrophobic interaction and pep stacking [45]. Next, release of the loaded DOX was observed in different pH conditions (pH 5.0 and pH 7.4) by measuring UVeVis absorption of DOX from a supernatant after precipitating GPGdN@CQDs-MWCNTs/DOX-EGFR. Several pulses of NIR laser irradiation (808 nm, 2 W/cm2, 5 min for each pulse) were applied on those samples. The cumulative release of DOX was then measured based on UVeVis absorption at various time points. As shown in Fig. 4D, GP-GdN@CQDs-MWCNTs/DOX-EGFR was relatively stable at pH 7.4, so that DOX was scarcely released during 4 h. In contrast, at pH 5.0, around 24.2% of the DOX was released in 4 h, which was due to the increased water solubility of DOX under acidic condition. These results suggest that DOX-loaded GdN@CQDs-MWCNTs nanocarriers were stable at neutral pH environment, and could release DOX under a cancer cell environment (at pH 5.0). DOX release was boosted to 15% after 4 h NIR irradiation, but the nonirradiated group showed small amount of DOX release in a nontriggered manner. Therefore, DOX release from GP-GdN@CQDsMWCNTs/DOX-EGFR can be controlled by NIR laser triggering and pH change. Next, we wondered whether NIR light trigger drug release behavior of GP-GdN@CQDs-MWCNTs/DOX-EGFR would also exist inside cells. Firstly, A549 cells were incubated with GP-GdN@CQDsMWCNTs/DOX-EGFR for 2 h and then washed with fresh cell culture to remove nano-carriers and drugs outside cells. After being irradiated by the 808 nm laser (1 W/cm2) for 5 min, cells were imaged via confocal fluorescence microscope. An interesting phenomenon is that fluorescence of DOX was quenched after loading on the GdN@CQDs-MWCNTs (Fig. 4E), the reoccurrence of DOX fluorescence could be an indicator of drug release. From confocal fluorescence images, no dramatic discrepancy was found in free DOX treated cells after laser irradiation, and drug molecules were uniformly distributed in the cytoplasm and nuclei. In contrast, remarkably enhanced DOX fluorescence was observed in the cells incubated with GP-GdN@CQDs-MWCNTs/DOX-EGFR post laser exposure, suggesting release of DOX from the nano-carriers. 3.5. Targeting efficiencies of GP-GdN@CQDs-MWCNTs/DOX-EGFR The conjugation of the anti-EGFR antibody to GP-GdN@CQDsMWCNTs/DOX was confirmed by the dot blotting method with secondary antibody-HRP detection on the nitrocellulose mem-

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Fig. 4. Confocal fluorescence microscopic images of A549 cells treated with PBS (A) and GdN@CQDs-MWCNTs (B) for 0.5 h all the scale bars are 30 mm. (C) Plot of DOX loading amount for GdN@CQDs-MWCNTs versus the drug concentration. (D) The cumulative release rate of GP-GdN@CQDs-MWCNTs/DOX-EGFR at different conditions (pH 7.5, pH 5.5, pH 7.4 þ NIR, and pH 5.5 þ NIR) after 4 h. The insets: UVevis absorbance spectra drug release after incubating GP-GdN@CQDs-MWCNTs/DOX-EGFR at pH 5.5 þ NIR. (E) Confocal fluorescence images of A549 cells incubated with GP-GdN@CQDs-MWCNTs/DOX-EGFR (or free DOX) ([DOX] ¼ 25 mM) for 2 h, washed with PBS to remove extracellular nanoparticles, and then treated with laser irradiation (808 nm, 1 W/cm2, 10 min) (Lþ). Un-irradiated cells were used as the controls (L). Red colors represent DOX fluorescence. The scale bar: 25 mm. (A colour version of this figure can be viewed online.)

brane. Therefore, the optimal cell line for verifying the targeting ability of GP-GdN@CQDs-MWCNTs/DOX-EGFR, we evaluated the expression of EGFR in A549, H522, MDA-MB-231, and MCF-10A cells. As shown in Fig. 5A and B, A549 and MDA-MB-231 cells overexpressed EGFR, however, H522, and MCF-10A was null for EGFR. This result is consistent with previous reports that investigated the quantity of EGFR in lung or breast cancer cells [37]. To examine whether anti-EGFR antibody conjugation to MWCNTs regulated the EGFR antibody-induced endocytosis of EGFR in

colorectal cancer cell lines of variable EGFR expression, we used GPGdN@CQDs-MWCNTs/DOX and GP-GdN@CQDs-MWCNTs/DOXEGFR to track EGFR antibody induced EGFR binding by confocal microscopy. When cells were incubated with GP-GdN@CQDsMWCNTs/DOX-EGFR at 37  C for 1 h (Fig. 5C), clear localization of membrane-bound GP-GdN@CQDs-MWCNTs/DOX-EGFR was observed in cells incubated GP-GdN@CQDs-MWCNTs/DOX. Moreover, the PL intensity of a single cell incubated in GP-GdN@CQDsMWCNTs/DOX and GP-GdN@CQDs-MWCNTs/DOX-EGFR was

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Fig. 5. (A) Comparisons of EGFR levels in A549, H522, MDA-MB-231, and MCF-10A cells. Bands are representative of three individuals from each group. (B) Densitometric analysis of western blots (lower location). Each bar represents the mean ± SEM (n ¼ 3). (C) Confocal microscopy fluorescence images of A549 cell treated with GP-GdN@CQDs-MWCNTs/DOX and GP-GdN@CQDs-MWCNTs/DOX-EGFR. The inset: The fluorescence strength of single cells was measured using image J software. (D) Cytotoxicity of A549 cells after treatment with GP-GdN@CQDs-MWCNTs/DOX and GP-GdN@CQDs-MWCNTs/DOX-EGFR. P values were calculated by Tukey's post-test (**p < 0.01, or * p < 0.05). (A colour version of this figure can be viewed online.)

measured using image J software, indicated that GP-GdN@CQDsMWCNTs/DOX-EGFR presented a uniformly distributed and intensive red fluorescence. To validate this result, we used MTT to further quantify the cellular binding (Fig. 5D), and the result indicated that the cellular binding amount of GP-GdN@CQDs-MWCNTs/DOXEGFR was dependent on the expression level of EGFR of the cells. The binding of GP-GdN@CQDs-MWCNTs/DOX-EGFR in A549 cells was higher than that in cells incubated GP-GdN@CQDs-MWCNTs/ DOX.

3.6. Cell viability of in vitro chemo, photothermal, and chemophotothermal treatments We next studied the therapy efficacy of the prepared GPGdN@CQDs-MWCNTs/DOX-EGFR in vitro. Live and dead cells can be differentiated by employing calcein AM (green) and PI (red) costaining methods. In laser-only (2 W/cm2) and PBS groups, strongly green fluorescence of all cells showed a absolutely survival (Fig. 6A). This phenomenon illustrated the inability of the heat generated from pure water in irradiation to cause cancer cell death. However, GP-GdN@CQDs-MWCNTs/DOX-EGFR incubated cancer cells were largely killed after laser irradiation, showing decreasing cell viabilities as the sample concentration was increased (Fig. 6A), as indicated by the intense homogeneous red fluorescence obtained. This finding is attributed to the fact that at high power doses, GP-GdN@CQDs-MWCNTs/DOX-EGFR can simultaneously release DOX and produce heat to efficiently kill cells by simultaneous Chem/PTT using a single red laser. We further quantitatively evaluated the bimodal Chem/PTT efficacy of GP-GdN@CQDsMWCNTs/DOX-EGFR using MTT assay (Fig. 6B,C and Figs. S10B and C). Fig. 6B and C showed that high concentration (200 mg/mL)

of GP-GdN@CQDs-MWCNTs alone did not affect the survival of A549 cells obviously as well as MDA-MB-231 cells without laser irradiation. However, a number of the cells were destroyed once incubated with GP-GdN@CQDs-MWCNTs/DOX-EGFR (200 mg/mL) and then exposured to 808 nm laser (2 W/cm2, 5 min). By contrast, upon 2.0 W/cm2 of laser irradiation, the viabilities of both A549 cells and MDA-MB-231 cells decreased significantly as the concentration of GP-GdN@CQDs-MWCNTs/DOX-EGFR increased, and approximately 90% cancer cell mortality rate was achieved at a concentration of 200 mg/mL, in accordance with the results from the calcein AM and PI co-staining. The results showed that, for GPGdN@CQDs-MWCNTs/DOX-EGFR, the high cytotoxicity comes from both the heat via NIR irradiation and the released DOX [38]. As can be predicting from drug release experiment, DOX release can be largely promoted by laser irradiation, leading to higher chemotherapy toxicity than that without NIR irradiation. Clearly, the combination of photothermal and chemotherapy achieves high cancer ablation efficacy with low administration dose, otherwise a higher dose or stronger laser power should be applied in either single cancer treatment.

3.7. In vivo photothermal imaging and Chemo/PTT Using an appropriate time, contrast agent concentration, and irradiation power, the mouse groups were treated with PBS, GdN@CQDs-MWCNTs, and GP-GdN@CQDs-MWCNTs/DOX-EGFR. Variations in temperature during irradiation were monitored using IR thermal-imaging system. The temperature of the tumor in the GP-GdN@CQDs-MWCNTs/DOX-EGFR-treated group increased to 38  C after laser irradiation at 2 W/cm2 for 1 min. The temperature further increased to 51.8  C after irradiation for 5 min

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Fig. 6. (A) Fluorescent images of propidium iodide (red, dead cells) and calcein AM (green, live cells) costained A549 cells after laser irradiation. Relative viability of A549 (B) and MDA-MB-231 cells (C) treated with PBS, GdN@CQDs-MWCNTs, and GP-GdN@CQDs-MWCNTs/DOX-EGFR of different concentrations (0, 5, 10, 20, 25, 50, 100, and 200 mg/mL), respectively, for 24 h and treated with GdN@CQDs-MWCNTs, GP-GdN@CQDs-MWCNTs/DOX-EGFR of different concentrations (0, 5, 10, 20, 25, 50, 100, and 200 mg/mL), respectively, under NIR irradiation (808 nm, 2 W/cm2) for 5 min (E) IR thermal images of PBS, GdN@CQDs-MWCNTs, and GP-GdN@CQDs-MWCNTs/DOX-EGFR treated mice under 5-min NIR irradiation. (F) Temperature changes of tumors monitored by the IR thermal camera in different groups during laser irradiation as indicated in (E). (A colour version of this figure can be viewed online.)

(Fig. 6E). Whereas, temperature of the control group showed no significant change while it was stable approximately at 31e33  C. Fig. 6F showed that the temperature of the tumor increased noticeably. Cancer cells were killed due to high temperature. In order to further assess the in vivo therapeutic effect of GPGdN@CQDs-MWCNTs/DOX-EGFR, A549 tumor-bearing nude mice were randomly divided into four groups (five mice per group): saline, DOX, GP-GdN@CQDs-MWCNTs/DOX-EGFR, and GdN@CQDsMWCNTsþirradiation. Mice in the treatment group were intratumorally injected with GP-GdN@CQDs-MWCNTs/DOX-EGFR at the

dosage of (100 mL, 100 mg/mL). After 1 h post injection, tumors were exposed to an 808 nm laser at 2 W/cm2 for 5 min in order to construct a steadily ambient temperature area at around 51.8  C. By the way, we recorded the slightly changes of temperature by infrared thermal camera. In marked contrast, the photothermal effect of the mice treated with GdN@CQDs-MWCNTs showed relatively smaller temperature range than GP-GdN@CQDsMWCNTs/DOX-EGFR. Mice were sacrificed, and tumors were excised and weighed at 14 days. A photograph and a line graph of mice after various treatments on the 14 day are shown in Fig. 7B,C

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Fig. 7. (A) Schematic diagram showing the strategy for the synthesis and combined photothermal and chemo-therapy of GP-GdN@CQDs-MWCNTs/DOX-EGFR. (B) Representative photographs of tumor-bearing mice and tumor after different treatments (a: PBS, b: DOX, c: GP-GdN@CQDs-MWCNTs/DOX, d: GP-GdN@CQDs-MWCNTsþlaser, e: GP-GdN@CQDsMWCNTs/DOX-EGFRþlaser). (C) Time-dependent tumor growth curves observed after different treatments. (D) Weight of different groups of mice after various treatments. (E) H&E staining of tumor sections gathered from various treatment groups (a: PBS, b: DOX, c: GP-GdN@CQDs-MWCNTs/DOX-EGFR, d: GP-GdN@CQDs-MWCNTsþlaser, e: GP-GdN@CQDsMWCNTs/DOX-EGFRþlaser) of mice on day 14. (A colour version of this figure can be viewed online.)

and showed clearly sharp differences in the effects on inhibition of tumor volume among the four groups. Meanwhile, most of tumors were ablated after treatment and effectively suppressed upon PTT treatment (Fig. 7A). Compared to treatment with free DOX, GPGdN@CQDs-MWCNTs/DOX-EGFR without laser irradiation showed improved therapeutic effect, probably owing to the enhanced

tumor uptake by the active targeting and the enhanced permeability retention (EPR) effect. Although enhanced inhibition of tumor growth was observed with injection of only GP-GdN@CQDsMWCNTs/DOX-EGFR or GdN@CQDs-MWCNTs with NIR laser irradiation, tumor growth was only partially delayed (Fig. 7B,C and Fig. S11), indicating the poor efficacy in inhibiting tumor growth. In

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Fig. 8. In vivo distribution of GP-GdN@CQDs-MWCNTs/DOX-EGFR in tumor slices. Green: GP-GdN@CQDs-MWCNTs. Red: DOX. Red arrows indicated orange merged by green and red suggesting GP-GdN@CQDs-MWCNTs co-localized with DOX sited on the inside of vessels. Green arrows indicated green fluorescence suggesting GP-GdN@CQDs-MWCNTs sited on the wall of vessels. (A colour version of this figure can be viewed online.)

marked contrast, tumor in the combination therapy group (GPGdN@CQDs-MWCNTs/DOX-EGFRþlaser irradiation) was completely eradicated without regrowth in 14 days. The potential adverse effect of the five groups is evaluated by fluctuations of mice body weight. The weights of all groups of mice were relatively stable (Fig. 7D). In addition, hematoxylin and eosin (H&E) staining of tumor slices confirmed that whole cells in PBS groups of tumors largely retained their normal morphology with distinctive nuclear structures and membrane. Severe necrosis and apoptosis of tumor cells could be observed in the tumors of the DOX-treated groups as compared to the control groups. Although the tumors were significantly inhibited after GP-GdN@CQDs-MWCNTs-EGFRþlaser irradiation (Fig. S11), some amount of remaining tumor tissue and surrounding fibrous tissue were observed by optical microscope in tissue section, but there almost no tumor cells in the group receiving GP-GdN@CQDs-MWCNTs/DOX-EGFR injection and NIR laser irradiation (Fig. 7E). Finally, the in vivo cytotoxicity GP-GdN@CQDs-MWCNTs/DOXEGFRþlaser irradiation were evaluated in major organs by H&E staining. No pathological changes were observed in spleen, kidney, lung, and heart for the mice in GP-GdN@CQDs-MWCNTs/DOXEGFRþlaser irradiation group as compared to those in the control groups (Fig. S12). Taken together, Chem/PTT in vivo results indicated that the GP-GdN@CQDs-MWCNTs/DOX-EGFR induced

apparent tumor inhibition and no obvious damage to major organs in tumor-bearing mice, revealing a potential platform for treatment of malignant cancers [46]. A qualitative analysis of DOX in Fig. 8 aimed to provide direct evidence of DOX accumulation in the tumor tissue induced by photothermal effect of GP-GdN@CQDs-MWCNTs. It was shown that 14 days chemo/PTT therapy administration, a large number of DOX was observed in the GP-GdN@CQDs-MWCNTs/DOX-EGFR and GP-GdN@CQDs-MWCNTs/DOX-EGFRþNIR group. Furthermore, confocal microscopy images of tumor sections shows that, after injection, the GP-GdN@CQDs-MWCNTs/DOX-EGFR accumulate in the tumor area through the EPR effect, and significant green fluorescence was observed in the tumor area in comparision with control groups (Fig. 8). Which further suggested that the effect of MWCNTs could destroy tumor cells effectively in deep-seated tissues. As the retention of MWCNTs persisted for a long period in the tumor site, we speculated that the extensive distribution of MWCNTs in both intravascular and extravascular spaces would not only induce thrombus formation in vessels to block blood flow to lack in nutrient and oxygen of tumor cells, but kill tumor cells directly by the powerful photothermal effect. Thus achieve favorable tumor growth inhibition efficacy when tumor oxygen grafts receive NIR illumination again. Before moving forward to study in vivo tumor combination

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therapy by using GP-GdN@CQDs-MWCNTs/DOX-EGFR, we firstly studied the in vivo behaviors of such agent in animals utilizing the intrinsic physical properties of MWCNTs. Blood was extracted from nude mice intravenous injected GP-GdN@CQDs-MWCNTs/DOXEGFR (100 mL, 100 mg/mL) at various time points post injection [38]. The concentrations of nanocomposite in blood were determined by measuring the Raman signals of MWCNTs. As shown in Fig. S13B, the level of GP-GdN@CQDs-MWCNTs/DOX-EGFR in blood decreased gradually over time. The MWCNTs signals in blood remained at a reasonably high level even at 24 h post injection. Then, the leakage of free Gd3þ was tested after GP-GdN@CQDsMWCNTs/DOX-EGFR were incubated in serum at 37  C for 0e2 weeks. The samples were centrifuged to collect the supernatant, and the Gd3þ released into the supernatant was detected by ICPAES. The results in Fig. S13C indicate a small amount of Gd3þ in the supernatant probably due to incomplete centrifugation or by the release of weakly bound Gd3þ in GP-GdN@CQDs-MWCNTs/ DOX-EGFR, because Gd leakage did not increase even after GPGdN@CQDs-MWCNTs/DOX-EGFR were incubated in serum for 2 weeks. CQDs chelation is effective in minimizing the leakage of Gd3þ, so GP-GdN@CQDs-MWCNTs/DOX-EGFR are stable sufficiently to be used as probes because of their low toxicity. 4. Conclusion In conclusion, the GP-GdN@CQDs-MWCNTs/DOX-EGFR was developed as NIR adsorbed theranostic agents to realize the combination of photoacoustic imaging and chemo-photothermal therapy of tumor in vitro and in vivo. UVeViseNIR spectra, PL spectra, FT-IR, Zeta potential, and TEM analyses confirmed the successful synthesis of GP-GdN@CQDs-MWCNTs/DOX-EGFR conjugate. As the high mass extinction coefficient at 808 nm and lower excitation energy loss, GP-GdN@CQDs-MWCNTs/DOX-EGFR shows photothermal conversion efficiency up to 45.3%. The loaded DOX could be triggered by external NIR light and lower intracellular pH value. In vivo chemo-photothermal treatment of GP-GdN@CQDsMWCNTs/DOX-EGFR was also studied and 100% tumor elimination was realized in irradiation at the laser power density of 2 W/ cm2, and no recurrent was observed. Acknowledgements This project was supported by the Jiangsu six category outstanding talent (2012-NY-031), Jiangsu province science and technology support plan (BE2014327, BE2015367), the Jiangsu Agricultural Science and Technology Innovation Fund CX (15) 1016, JHB05-21 and Nanjing-321, the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.carbon.2017.07.032. References [1] Q. Chen, X. Wang, C. Wang, et al., Drug-induced self-assembly of modified albumins as nano-theranostics for tumor-targeted combination therapy, ACS Nano 9 (5) (2015) 5223e5233. [2] Z. Liu, Nano-theranostics for imaging-guided phototherapy: fighting cancer metastasis, Nanomed Nanotechnol. Biol. Med. 12 (2) (2016) 462. [3] Y. Chen, X. Li, K. Park, et al., Hollow carbon-nanotube/carbon-nanofiber hybrid anodes for Li-ion batteries, J. Am. Chem. Soc. 135 (44) (2013) 16280e16283. [4] H. Cheng, C. Hu, Y. Zhao, et al., Graphene fiber: a new material platform for unique applications, NPG Asia Mater. 6 (7) (2014) e113. [5] N. Dilbaghi, H. Kaur, R. Kumar, et al., Nanoscale device for veterinay technology: trends and future prospective, Adv. Mater. Lett. 4 (2013) 175e184.

[6] L. Wen, W. Ding, S. Yang, et al., Microwave pumped high-efficient thermoacoustic tumor therapy with single wall carbon nanotubes, Biomaterials 75 (2016) 163e173. [7] B.R. Smith, E.E.B. Ghosn, H. Rallapalli, et al., Selective uptake of single-walled carbon nanotubes by circulating monocytes for enhanced tumour delivery, Nat. Nanotechnol. 9 (6) (2014) 481e487. [8] R. Weissleder, M. Nahrendorf, M.J. Pittet, Imaging macrophages with nanoparticles, Nat. Mater. 13 (2) (2014) 125e138. [9] M. Zhang, W. Wang, N. Zhou, et al., Near-infrared light triggered phototherapy, in combination with chemotherapy using magnetofluorescent carbon quantum dots for effective cancer treating, Carbon 118 (2017) 752e764. [10] G.J. Vrooijink, M. Abayazid, S. Patil, et al., Needle path planning and steering in a three-dimensional non-static environment using two-dimensional ultrasound images, Int. J. Robot. Res. 33 (10) (2014) 1361e1374. [11] C.K. Kuhl, S. Schrading, K. Strobel, et al., Abbreviated breast magnetic resonance imaging (MRI): first postcontrast subtracted images and maximumintensity projection-a novel approach to breast cancer screening with MRI, J. Clin. Oncol. 32 (22) (2014) 2304e2310. [12] L. Wang, J. Lee, M. Zhang, et al., Fluorescence imaging technology (FI) for highthroughput screening of selenide-modified nano-TiO2 catalysts, Chem. Commun. 52 (14) (2016) 2944e2947. [13] G. Gollavelli, Y.C. Ling, Magnetic and fluorescent graphene for dual modal imaging and single light induced photothermal and photodynamic therapy of cancer cells, Biomaterials 35 (15) (2014) 4499e4507. [14] J. Garcia, T. Tang, A.Y. Louie, Nanoparticle-based multimodal PET/MRI probes, Nano Med. 10 (8) (2015) 1343e1359. [15] P.E. Buffet, L. Poirier, A. Zalouk-Vergnoux, et al., Biochemical and behavioural responses of the marine polychaete Hediste diversicolor to cadmium sulfide quantum dots (CdS QDs): waterborne and dietary exposure, Chemosphere 100 (2014) 63e70. [16] Y. Zhao, X. Wang, Q. Wu, et al., Translocation and neurotoxicity of CdTe quantum dots in RMEs motor neurons in nematode Caenorhabditis elegans, J. Hazard. Mater. 283 (2015) 480e489. [17] V. Georgakilas, J.A. Perman, J. Tucek, et al., Broad family of carbon nanoallotropes: classification, chemistry, and applications of fullerenes, carbon dots, nanotubes, graphene, nanodiamonds, and combined superstructures, Chem. Rev. 115 (11) (2015) 4744e4822. [18] K. Hola, Y. Zhang, Y. Wang, et al., Carbon dots-Emerging light emitters for bioimaging, cancer therapy and optoelectronics, Nano Today 9 (5) (2014) 590e603. [19] S.H. Chiu, G. Gedda, W.M. Girma, et al., Rapid fabrication of carbon quantum dots as multifunctional nanovehicles for dual-modal targeted imaging and chemotherapy, Acta Biomater. 46 (2016) 151e164. [20] Y. Shi, Y. Pan, J. Zhong, et al., Facile synthesis of gadolinium (III) chelates functionalized carbon quantum dots for fluorescence and magnetic resonance dual-modal bioimaging, Carbon 93 (2015) 742e750. [21] K.A. Zimmermann, D.L. Inglefield, J. Zhang, et al., Single-walled carbon nanohorns decorated with semiconductor quantum dots to evaluate intracellular transport, J. Nanopart. Res. 16 (1) (2014) 2078. [22] Z.S. Qian, X.Y. Shan, L.J. Chai, et al., DNA nanosensor based on biocompatible graphene quantum dots and carbon nanotubes, Biosens. Bioelectron. 60 (2014) 64e70. [23] X. Huang, I.H. El-Sayed, W. Qian, et al., Cancer cell imaging and photothermal therapy in the near-infrared region by using gold nanorods, J. Am. Chem. Soc. 128 (6) (2006) 2115e2120. [24] Y. Liu, K. Ai, J. Liu, et al., Dopamine-melanin colloidal nanospheres: an efficient near-infrared photothermal therapeutic agent for in vivo cancer therapy, Adv. Mater. 25 (9) (2013) 1353e1359. [25] T. Liu, S. Li, Y. Liu, et al., Mn-complex modified NaDyF 4 :Yb@ NaLuF 4: Yb, Er@ polydopamine coreeshell nanocomposites for multifunctional imagingguided photothermal therapy, J. Mater. Chem. 4 (15) (2016) 2697e2705. [26] S. Gao, L. Zhang, G. Wang, et al., Hybrid graphene/Au activatable theranostic agent for multimodalities imaging guided enhanced photothermal therapy, Biomaterials 79 (2016) 36e45. [27] M. Zhang, N. Zhou, P. Yuan, et al., Graphene oxide and adenosine triphosphate as a source for functionalized carbon dots with applications in pH-triggered drug delivery and cell imaging, RSC. Adv. 7 (15) (2017) 9284e9293. [28] X. Liang, W. Shang, C. Chi, et al., Dye-conjugated single-walled carbon nanotubes induce photothermal therapy under the guidance of near-infrared imaging, Cancer Lett. 383 (2) (2016) 243e249. [29] K. Cherukula, K. Manickavasagam Lekshmi, S. Uthaman, et al., Multifunctional inorganic nanoparticles: recent progress in thermal therapy and imaging, Nano Mater. 6 (4) (2016) 76. [30] M.L. Chen, Y.J. He, X.W. Chen, et al., Quantum dots conjugated with Fe3O4filled carbon nanotubes for cancer-targeted imaging and magnetically guided drug delivery, Langmuir 28 (47) (2012) 16469e16476. [31] X. Tu, L. Wang, Y. Cao, et al., Efficient cancer ablation by combined photothermal and enhanced chemo-therapy based on carbon nanoparticles/doxorubicin@SiO2 nanocomposites, Carbon 97 (2016) 35e44. [32] Z. Li, P. Huang, X. Zhang, et al., RGD-conjugated dendrimer-modified gold nanorods for in vivo tumor targeting and photothermal therapy, Mol. Pharm. 7 (1) (2009) 94e104. [33] S.D. Jayasena, Aptamers: an emerging class of molecules that rival antibodies in diagnostics, Clin. Chem. 45 (9) (1999) 1628e1650. [34] M. Khedri, H. Rafatpanah, K. Abnous, et al., Cancer immunotherapy via nucleic

M. Zhang et al. / Carbon 123 (2017) 70e83 acid aptamers, Int. Immunopharmacol. 29 (2) (2015) 926e936. [35] M.K. Yu, D. Kim, I.H. Lee, et al., Image-guided prostate cancer therapy using aptamer-functionalized thermally cross-linked superparamagnetic iron oxide nanoparticles, Small 7 (15) (2011) 2241e2249. [36] J. Ge, Q. Jia, W. Liu, et al., Red-emissive carbon dots for fluorescent, photoacoustic, and thermal theranostics in living mice, Adv. Mater. 27 (28) (2015) 4169e4177. [37] M.S. Noh, S. Lee, H. Kang, et al., Target-specific near-IR induced drug release and photothermal therapy with accumulated Au/Ag hollow nanoshells on pulmonary cancer cell membranes, Biomaterials 45 (2015) 81e92. [38] J. Liu, C. Wang, X. Wang, et al., Mesoporous silica coated single-walled carbon nanotubes as a multifunctional light-responsive platform for cancer combination therapy, Adv. Funct. Mater. 25 (3) (2015) 384e392. [39] Y. Xu, X.H. Jia, X.B. Yin, et al., Carbon quantum dot stabilized gadolinium nanoprobe prepared via a one-pot hydrothermal approach for magnetic resonance and fluorescence dual-modality bioimaging, Anal. Chem. 86 (24) (2014) 12122e12129. [40] H. Gong, Y. Chao, J. Xiang, et al., Hyaluronidase to enhance nanoparticle-based photodynamic tumor therapy, Nano Lett. 16 (4) (2016) 2512e2521.

83

[41] M. Zhang, C. Chi, P. Yuan, et al., A hydrothermal route to multicolor luminescent carbon dots from adenosine disodium triphosphate for bioimaging, Mater. Sci. Eng. C 76 (2017) 1146e1153. [42] S. Azoz, J. Jiang, G. Keskar, et al., Mechanism for strong binding of CdSe quantum dots to multiwall carbon nanotubes for solar energy harvesting, Nanoscale 5 (15) (2013) 6893e6900. [43] X. Gong, Q. Zhang, Y. Gao, et al., Phosphorus and nitrogen dual-doped hollow carbon dot as a nanocarrier for doxorubicin delivery and biological imaging, ACS Appl. Mater. Inter. 8 (18) (2016) 11288e11297. [44] S. Li, D. Amat, Z. Peng, et al., Transferrin conjugated nontoxic carbon dots for doxorubicin delivery to target pediatric brain tumor cells, Nanoscale 8 (37) (2016) 16662e16669. [45] H. Wu, H. Shi, H. Zhang, et al., Prostate stem cell antigen antibody-conjugated multiwalled carbon nanotubes for targeted ultrasound imaging and drug delivery, Biomaterials 35 (20) (2014) 5369e5380. [46] X. Song, L. Feng, C. Liang, et al., Ultrasound triggered tumor oxygenation with oxygen-shuttle nanoperfluorocarbon to overcome hypoxia-associated resistance in cancer therapies, Nano Lett. 16 (10) (2016) 6145e6153.