Magnetofluorescent Carbon Quantum Dot Decorated Multiwalled

Article Cite This: ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

Magnetofluorescent Carbon Quantum Dot Decorated Multiwalled Carbon Nanotubes for Dual-Modal Targeted Imaging in ChemoPhotothermal Synergistic Therapy Ming Zhang,†,∥ Wentao Wang,‡ Yingjun Cui,∥ Ninglin Zhou,*,†,§ and Jian Shen*,† †

Jiangsu Collaborative Innovation Center for Biological Functional Materials, College of Chemistry and Materials Science, Jiangsu Key Laboratory of Biofunctional Materials, Jiangsu Engineering Researche, Center for Biomedical Function Materials, Nanjing 210023, China ‡ Jiangsu Key Laboratory for Molecular and Medical Biotechnology, College of Life Sciences, Nanjing Normal University, Nanjing 210023, China § Nanjing Zhou Ninglin Advanced Materials Technology Company Limited, Nanjing 211505, China ∥ Department of Biological Sciences, Florida International University, Miami, Florida 33199, United states S Supporting Information *

ABSTRACT: Magnetofluorescent nanoparticles with diagnostic and therapeutic functions show great promise in nanomedicine. Here, we report the magnetofluorescent carbon nanotubes (CNTs)/doxorubicin (DOX) nanocomposites and their functions act in synergetic chemo-photothermal synergistic therapy (Chemo/PTT) in cancer excision. Magnetofluorescent CNTs conjugated with a folic acid (FA-GdN@CQDs-MWCNTs) were targets for dual-modal fluorescence (FL)/magnetic resonance (MR) imaging. Experiments in vitro and in vivo identified FAGdN@CQDs-MWCNTs with low toxicity, and good biocompatibility. Moreover, FA-GdN@CQDs-MWCNTs whose release can be fostered by pH and NIR light dual-stimuli had been proved to be available for loading DOX. Following nuclear translocations, FA-GdN@CQDs-MWCNTs were engineered to deliver DOX that targeted the nuclei. In vivo experiment indicates that the Chemo/PTT, as compared with the respective single treatment, can significantly control tumor growth. In addition, Chemo/PTT was not shown to render any appreciable toxicity. These findings suggest that the FA-GdN@CQDs-MWCNTs/DOX could function as a multifunctional platform for simultaneous FL/MR imaging, PTT therapy, and drug delivery. KEYWORDS: carbon nanotubes, magnetofluorescent, folic acid, photothermal therapy cancers.7 As compared to traditional chemotherapy, PTT can locally and selectively target in tumor fields while being friendly to normal tissues.8 Multiwalled CNTs (MWCNTs) have attracted tremendous interests in numerous domains (such as hydrogen storage, reinforce, electric conduction, and biomedicine) because of their excellent chemical and physical properties.9 Most recently, CNTs have been performed as nanovectors for targeted drug delivery systems.10,11 Meanwhile, CNTs have been reported as a hopeful NIR hyperthermia agent for PTT of cancer extensively.12 For example, pluronic F-127 conjugated MWNTs have displayed highly resultful peculiarity as a photothermal agent in PTT.13 The MCWNTs provide a serviceable material for targeting delivery of drugs and heat to

1. INTRODUCTION The worldwide increasing of incidence of cancer should be treated carefully.1 The effect of using medicine commonly is limited by the lack of targeting specificity and its potential side effects to the healthy tissues.2 Thus, to improve the targeting specificity of conventional medicine and its therapeutic effects are one of the key requirements. Moreover, it will be more helpful to understand the treatment effect timely and accurately as long as biological labeling of tumor tissue could be achieved.3 Chemotherapy, which based on chemical substances as antitumor drugs to therapy cancers, is a general method in current clinical applications.4 Fallaciously, chemotherapy has some limitations. For example, it does not work sometimes or failed to completely destroy cancer cells.5 Drug resistance is the major issue of chemotherapy.6 As an infancy cancer treatment, PTT has been verified as an effective strategy for cancer ablation in the mechanism of using hyperthermia generated by electromagnetic radiation to inhibit © XXXX American Chemical Society

Received: July 27, 2017 Accepted: December 5, 2017

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DOI: 10.1021/acsbiomaterials.7b00531 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

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ACS Biomaterials Science & Engineering

Figure 1. Schematic diagram of the preparation process for (A) GdN@CQDs and (B FA-GdN@CQDs-MWCNTs/DOX). (C) TEM images of GdN@CQDs. (D) XPS survey scan. (E) (a) TEM and (b) HRTEM images of MWCNTs-COOH. (F) (a) TEM and (b, c) HRTEM images of GdN@CQDs-MWCNTs.

neous therapy and imaging is, therefore, clearly a definite requirement to improving nanomaterials. To investigate the effect of magnetofluorescent carbon quantum dots (CQDs), research teams have lately performed a series of work to develop its function as nanotheranostic for FL/MR and cancer ablation.21,22 The synergetic therapy based on magnetofluorescent CQDs, MWCNTs, and antitumor drugs for drug delivery and dual imaging, however, has not been studied yet.23 The concept aims to develop an easy one-pot method what can be applied into fabrication of a magnetofluorescent GdN@ CQDs-MWCNTs, which act on FL and MR dual-modal imaging, and cancer therapy on the base of excellent magnetic and fluorescence properties. Therefore, in the current study, we developed a multifunctional nano drug carrier that covering the magnetic MWCNTs surface with GdN@CQDs and FA. We produced FA-GdN@ CQDs-MWCNTs have prominent stability and good water dispersibility, while these properties are with long-term potential for FL/MR imaging, excellent photothermal effect, and high DOX loading capacity. FA-GdN@CQDs-MWCNTs/ DOX possess both pH and NIR light dual-stimuli responsive

tumor sites, sequentially improving the entire efficacy of cancer ablation for optimum clinical effect. Currently, many groups synthesized that MWCNTs/anticancer drugs nanocomposites could be used as a nanoplatforms for synergetic cancer Chemo/ PTT therapy.14 For example, DOX can be easily bound to MWCNTs noncovalently by strong van der Waals and electrostatic interaction.15 Such tumor-antibiotic formulations can not only improve their anticancer efficiency in cancer cells but also minimize the potential toxic side effects. Magnetic fluorescent nanoparticles, with the advantages of magnetic materials and fluorescent materials, can realize a variety of functions such as the magnetic separation, targeting identification, fluorescence imaging, and magnetic resonance imaging at the same time.16 Although it has a good application prospect and draw much attention of scientific researchers in numerous fields, its biological toxicity and biological compatibility needs to be addressed.17,18 Additionally, magnetic fluorescent nanoparticles synthesis have localizations related to the necessities of toxic, costly chemicals, the multistep, tanglesome procedure and time-consuming.19,20 The current trend in terms of theranostics nanoplatforms for coinstantaB


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Gd, 11.84% N, and 16.47% O. No Cl element was discovered by XPS analysis, suggesting that unreacted GdCl3 was removed completely. GdN@CQDs displayed extra characteristic peaks consistent with Gd 3d (1189.1 eV), and Gd 4d (142.6 eV), affirming the oxidation state of Gd3+ in GdN@CQDs (Figure 1D).26,27 The N 1s peak at 399.5 eV was attributed to N in an NH2 state and aromatic N, but, the N 1s peak of GdN@CQDs at 398.8 eV was attributed to N at the surface and NH2 (Figure S2).28 The XRD patterns of GdN@CQDs did not show welldemarcated diffraction peaks, possibly due to an amorphous carbon structure (Figure S3). Different from the XRD pattern of GdCl3, no diffraction peaks were detected in responding to free Gd3+, reveling that Gd3+ was tightly chelated with the NCQDs and the coordination between Gd3+ and N@CQDs caused the preparation of [email protected] These results verified that the GdN@CQDs were N@CQDs-passivated Gd3+ via the coordination between Gd3+ and the surface groups on N@CQDs. The MWCNTs were initially oxidized for introducing carboxylic acid groups onto the side walls of MWCNTs. As indicated by TEM images, the original MWCNTs (Figure S4) have been cut shorter after the oxidation (Figure 1E). First, the GdN@CQDs were covalently grafted on the shortened MWCNTs via the formation of NH2 between the COOH of MWCNT-COOH and the amine of GdN@CQDs. TEM images from the magnetically modified CNTs were obtained for the sake of affirm the joint of the GdN@CQDs to the surface of MWCNTs. A carefully chosen micrograph in Figure 1F indicated a sporadic ornamentation of MWCNTs-COOH with dark dots. The GdN@CQDs scattered along the surface of CNTs, and crystalline structure of the GdN@CQDs can be discovered, which is dramatically other than the case of MWCNT-COOH. Further, surface conjugation of GdN@ CQDs nanoparticles to the MWCNT-COOH gave rise to an augment in the negative zeta potential which is associated with the incremental COO- group on the surface of the GdN@ CQDs.19 Next, the GdN@CQDs-MWCNTs with PEI were synthesized and proved. PEI-covered GdN@CQDs-MWCNTs (GdN@CQDs-MWCNTs-NH2) were terminated with NH2. The initial GdN@CQDs-MWCNTs have a zeta potential of −22.4 mV, which turns to 17.91 mV when modified with PEI (Figure S6). PEI content in GdN@CQDs-MWCNTs-NH2 was determined using thermal gravimetric analysis (TGA) (Figure S5). The difference between GdN@CQDs-MWCNTs-NH2 and GdN@CQDs-MWCNT-COOH in the weight loss at 400 °C showed that the PEI grafting amount is about 13.6%.24 The NH2 portion in GdN@CQDs-MWCNTs-NH2 is favorable for further conjugating the needed receptors and biomolecules. GdN@CQDs-MWCNTs-NH2 coated with FA by a traditional EDC/Sulfo-NHS chemical reaction so that to target for dual FL/MR. Prevailing wisdom suggests majorities of human cancer cells express surface-FA-receptors which increase with the growing progression of diseases. Used the zeta potential and UV−vis absorption analysis to affirm the FA molecules conjugated on the surface of the GdN@CQDsMWCNTs-NH2 or not.19 GdN@CQDs-MWCNTs-NH2 at pH 7.4 possessed the zeta potential value of 17.91 ± 2.8 mV (Figure S6). Negative groups presented as OH- and COO- on the GdN@CQDs-MWCNTs-NH2 surface became the mainly reasons about leading to a negative zeta potential values. Ulteriorly, increasing in the negative zeta potential caused by augmented COO- groups on the surface of the GdN@CQDsMWCNTs-NH2. The UV−vis absorption spectrum of GdN@

DOX release behavior, and building on this function, it can be used to foster drug accumulation intracellular so as to enhancement of the efficiency in killing cancer cells. The FL/ MR imaging effects of FA-GdN@CQDs-MWCNTs/DOX was investigated, and the nanocomposites were then used for in vivo targeted FL/MR imaging. In addition, the therapeutic effect of the FA-GdN@CQDs-MWCNTs/DOX were further investigated in vivo with tumor-bearing mice model, and the tumors were significantly inhibited in certain cases.

2. EXPERIMENTAL SECTION 2.1. Materials. Except specified, the other chemicals were acquired from Aladdin. Chemical Co., Ltd. (Shanghai, China). MWCNTs were obtained from XF Nanomaterials Chemicals Co., Ltd. (Nanjing, China). Gadolinium(III) chloride (GdCl3, 99.99%), 1-Ethyl-3-(3(dimethylamino)propyl) carbodiimide (EDC, 99%) and Hydroxy-2,5dioxopyrrolidine-3-sul fonicacid sodium salt (Sulfo-NHS, 97%) were purchased from Thermo Fisher HyClone Co., Ltd. (USA). Folic acid (FA, > 98%) was obtained from Aladdin. Chemical Co., Ltd. (Shanghai, China). Penicillin, streptomycin and Dulbecco’s modified Eagle medium (DMEM) were purchased from Thermo Fisher HyClone Co., Ltd. (USA). Phosphate Buffer solution (PBS) and fetal bovine serum (FBS) was purchased from Hyclone Co., Ltd. (USA). All of solvents and reagents were of analytical grade and used as received. 2.2. Instrumentation. Photoluminescence (PL) spectra was collected using a molecular fluorescence spectrometer (Cary Eclipse, USA). The X-ray diffraction (XRD) analysis was performed using a D/ Max 2500 V/PC diffractometer (Rigaku Corporation, Japan). The thermogravimetric analyze (TGA) measurements was collected on a PerkinElmer TG 7 instrument. UV−vis−NIR spectroscopy measurements were performed on a Cary 5000 UV−vis−NIR spectrometer. The ζ potential measurement and dynamic light scattering (DLS) analysis were recorded on a Zetasizer Nano ZS90 Analyzer. The transmission electron microscope (TEM) images were acquired on a JEM-2100F transmission electron microscope. MR images were acquired by a 3 T MRI scanner (70/30 Bruker BioSpin, Germany). The fluorescent images of cancer cells were recorded by upright fluorescent microscope (XSP-63X, Nikon, Japan) and confocal laser scanning microscope (TI-E-A1R, Nikon, Japan). The methods used for material characterization were displayed in the Experimental Section in the Supporting Information).

3. RESULTS AND DISCUSSION 3.1. Characterization. Multifarious carbon-stabilized metal nanocomposites have been synthesized in the manner of molecular organic precursors.24 Similarly, our research tried to prepare carbon-passivated gadolinium nanocomposites as an MRI contrast agent by one-pot hydrothermal treatment of the mixture of poly lysine, citric acid, and GdCl3 as citric acid is readily polymerized with the existent of amine group (Figure 1A).25 A clear yellow solution was gained. After centrifugation, dialysis, and lyophilization, the solid powder was dispersed again in deionized water. The morphology and particle size distribution of GdN@CQDs fabricated are presented in Figure. 1C and Figure S1A. It can thus be seen that the GdN@CQDs are isolated from each other and exhibit an almost roundshapes with the narrow size distribution (3.8−5.5 nm). DLS analysis (inset of Figure S1) showed a single narrow peak at ca. 4.8 nm, which is corresponded with the TEM result and revels that the prepared GdN@CQDs are highly dispersible in deionized water. The crystalline structure of the GdN@CQDs are identified by the well-resolved crystal lattice, with labeled lattice spaces of 0.223 nm (inset in Figure 1C), compliance with the (100) lattice spacing of graphite. XPS result recorded the entity of Gd in the GdN@CQDs with 62.51% C, 21.70% C


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Figure 2. (A) Fluorescence spectra of GdN@CQDs, FA-GdN@CQDs-MWCNTs, FA-GdN@CQDs-MWCNTs/DOX, and DOX. The inset shows photographs of GdN@CQDs, FA-GdN@CQDs-MWCNTs, FA-GdN@CQDs-MWCNTs/DOX, and DOX aqueous solution under UV light (365 nm). (B) Fluorescence spectra of FA-GdN@CQDs-MWCNTs with different excitation wavelengths. (C) Upconversion PL spectra of FA-GdN@ CQDs-MWCNTs obtained with different excitation wavelength. (D) Comparison on the photobleaching characteristics of FA-GdN@CQDsMWCNTs and FITC under a 500 W xenon lamp.

the upconversion PL performance of the FA-GdN@CQDsMWCNTs could also devote to the multiphoton active process. 34 Importantly, the FA-GdN@CQDs-MWCNTs could emit light in the NIR range under the excitation of NIR light, which is highly satisfactory for biological applications.35,36 Similarly, the PL behavior in λex-dependent was beneficial to multicolor in vitro biological imaging applications (Figure S8).37 Furthermore, the FA-GdN@ CQDs-MWCNTs exhibited excellent photostability. As shown in Figure 2D, after UV lamb irradiation for 1 h, the FL intensity of the FA-GdN@CQDs-MWCNTs could still retain 88.2%, while the FL intensity of FITC lessened to 27.3% of the starting value. Our work manifested that the FA-GdN@ CQDs-MWCNTs not only demonstrated tunable excitation and upconversion PL properties, showed excellent photostability likewise. 3.3. In Vitro Biocompatibility of FA-GdN@CQDsMWCNTs. To explore the biological applications of FAGdN@CQDs-MWCNTs, their potential cytotoxicity needed to be surveyed beforehand. HeLa and MCF-7 cells were first incubated with FA-GdN@CQDs-MWCNTs at various concentrations for 24 h, then their survival rate were ascertained by MTT assay. Figure 3A, B shows that FA-GdN@CQDsMWCNTs did not exhibit higher toxicity than single MWCNTs, which were produced during the preparation of contrast agents even at a highly concentration of 600 μg/mL. The excellent biocompatibility to cancer cells reminded us to deduced that the FA-GdN@CQDs-MWCNTs possess a high

CQDs-MWCNTs-NH2, FA and FA-GdN@CQDs-MWCNTs was shown in Figure S7. The free GdN@CQDs-MWCNTsNH2 showed characteristic peaks at 263 and 375 nm attributing to (π → π*) and (n → π*) transition, respectively. The same two GdN@CQDs-MWCNTs-NH2 peaks were discovered for FA conjugated GdN@CQDs-MWCNTs-NH2, with a blue shift from 263 to 260 nm, suggesting GdN@CQDs-MWCNTs-NH2 successfully coated with FA. Compared with free GdN@CQDs, the fluorescent intensity of FA-GdN@CQDs-MWCNTs decreased owing to the quenching effect of CNTs (Figure 2A). Using quinine sulfate to standardize,33 the fluorescence quantum yield (ΦS) of the as-prepared GdN@CQDs and FAGdN@CQDs-MWCNTs to be 8.9% and 6.4%, respectively. The FA-GdN@CQDs-MWCNTs still showed notable fluorescence, facilitating the in vitro studies of CNT-based formulations using fluorescence imaging. 3.2. Fluorescence Spectra of FA-GdN@CQDsMWCNTs. In pursuit of further studying the optical properties of the as-obtained FA-GdN@CQDs-MWCNTs, the λexdependent PL behavior in fluorescent carbon materials, was investigated and displayed in Figure 2B. As shown in Figure 2B, With increasing excitation wavelength from 290 to 420 nm, the emission peak shifted from 420 to 480 nm, which indicated the related distribution of surface energy traps of the CQDs.30−32 The maximum PL emission situated at 350 nm gained with λex = 450 nm. Figure 2C showed the PL spectra of the FA-GdN@ CQDs-MWCNTs stimulated by long-wavelength light from 900 to 650 nm demonstrated the upconverted emissions from 520 to 420 nm. Consistent with the formerly reported CQDs, D


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Figure 3. Relative viability of (A) HeLa and (B) MCF-7 cells incubated with different concentrations of MWCNTs-COOH and FA-GdN@CQDsMWCNTs (0−600 μg/mL). (C) T1-weighted and T2-weighted MR images of FA‑GdN@CQDs-MWCNTs at different concentrations of Gd3+ ions. (D) Linear relationship between r1 and corresponding Gd3+ concentration of FA-GdN@CQDs-MWCNTs. (E) Linear relationship between transverse relaxivities (r2) and equivalent Gd3+ concentration of FA-GdN@CQDs-MWCNTs.

3.5. In Vitro NIR Effect of FA-GdN@CQDs-MWCNTs. To identify the proper concentration in vivo, the NIR effect of the FA-GdN@CQDs-MWCNTs was tested. Deionized water acted as the negative control. Previous data proved that cancer cells can be ablated as soon as the temperature exceeds 42.5 °C.38 Given their efficient NIR absorption characteristics between 800 and 1000 nm (Figure 4A), the potential of FA-GdN@ CQDs-MWCNTs photothermal ablation therapy of cancer were investigated by employing a 808 nm laser. Following increasing laser power, the maximum temperature could approach approximately 47 °C, even up to 58 °C under 2 W/cm 2 . The finding indicated that FA-GdN@CQDsMWCNTs dispersed in deionized water at various concentrations are available in eliminating cancer cells even with a pretty low concentration. The solution temperature increased with the growing FA-GdN@CQDs-MWCNTs concentrations as shown in Figure 4B and Figure S9. The temperature of the deionized water group had no distinct changes under different laser power while experimental group did not. These data provide evidence that FA-GdN@CQDs-MWCNTs can rapidly and efficiently transfer laser energy into heat in the way of efficient photoabsorption at 808 nm. In light of the good NIR absorption and photothermal conversion of FA-GdN@CQDsMWCNTs, we then recorded the temperature of solution (50 μg/mL, 0.5 mL) as a representation of time under continuous

promise for drug loading and target delivery of a drug that has an extreme possibility to cause cancer cells death.2 3.4. In Vitro Targeting MRI Imaging. The longitudinal (T1) and transverse (T2) relaxation times were measured with a 3T clinical MR imaging system to explore the possibility of using FA-GdN@CQDs-MWCNTs as a diagnostic MRI contrast agent. In vitro MR images showed distinguished MR property of FA-GdN@CQDs-MWCNTs. Figure 3C showed the T1 weighted MR images of FA-GdN@CQDs-MWCNTs solutions. Positive and increased contrast enhancement of MR signals clearly came out with the increasing of Gd 3+ concentration, whereas T2-weighted MRI intensity gradually decreased (Figure 3D). These results provided compelling evidence that FA-GdN@CQDs-MWCNTs magnified the longitudinal proton relaxation process. Figure 3C, D showed the 1/T1 and 1/T2 relaxation rates of water protons in the FA-GdN@CQDs-MWCNTs solutions at different Gd3+ concentrations. The relaxation values r1 and r2 were obtained by the slopes of the linear regression fits from the relaxation plots. The r1 and r2 values of FA-GdN@CQDsMWCNTs were 8.77 mM−1s−1 and 12.32 mM−1s‑1, respectively. In addition, the r2/r1 ratio of FA-GdN@CQDs-MWCNTs was 1.40, indicating that FA-GdN@CQDs-MWCNTs is a adaptive paramagnetic material as a T1 contrast agent. E


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Figure 4. Photochemical and photophysical properties of FA-GdN@CQDs-MWCNTs. (A) UV−vis−-NIR absorbance spectra of FA-GdN@CQDsMWCNTs solutions at varied concentration. (B) Effects of varying the FA-GdN@CQDs-MWCNTs concentration and laser-irradiation power from 2 W/cm2 are shown. (C) Temperature IR images of FA-GdN@CQDs-MWCNTs and FA-GdN@CQDs-MWCNTs/DOX aqueous solution (50 μg/ mL) under 808 nm laser (2 W/cm2) irradiation recorded with an IR camera. (D) Photothermal effect of FA-GdN@CQDs-MWCNTs aqueous solution (50 μg/mL) when illuminated with a 808 nm laser (2 W/cm2). (E) Plot of DOX loading amount for FA-GdN@CQDs-MWCNTs versus the drug concentration. The cumulative release rate of FA-GdN@CQDs-MWCNTs/DOX at various conditions (pH 7.5, pH 5.5 and pH 5.5 + NIR) after 24 h.

irradiation of 808 nm laser (2 W/cm2) until the temperature came to a steady-state. The temperature change in centrifuge tube under laser irradiation was tested using an infrared thermal imager. As shown in Figure 4C, the FA-GdN@CQDsMWCNTs/DOX aqueous solution temperature up to 52 °C within 5 min, which is sufficient to kill the cancer cells. As a control, the deionized water temperature is slightly altered under the same laser condition. Given the further interest in the photothermal effect of the FA-GdN@CQDs-MWCNTs, we undertook the investigation of relative relationship between time and temperature of the solution (50 μg/mL, 1.0 mL) under continuous irradiation of 808 nm laser (2 W/cm2) before the temperature of solution achieved a steady-value (Figure 4D). According to the earlier work,38 the photothermal conversion efficiency can achieve approximately 44.5%.

3.6. Drug Loading and Releasing. A series of work has been completed in vitro to identify the drug performance about loading and release. We conjectured that DOX could be potentially conjugated with FA-GdN@CQDs-MWCNTs through electrostatic and van der Waals interaction force.39 The zeta potential and UV−vis spectra were applied to verify the conjugation. In the UV−vis spectrum of FA-GdN@CQDsMWCNTs (Figure S10), a characteristic absorption peak at about 485 nm attributable to DOX was discovered, implying the successful loading of DOX. The surface zeta potential of FA-GdN@CQDs (Figure S11) changed from −26.18 ± 2.32 to −16.20 ± 4.51 mV after DOX conjugation. It signified that the negative charge of the FA-GdN@CQDs-MWCNTs interplayed with a positive charge on DOX under the role of electrostatic interaction. As seen in Figure 2A, the fluorescence of FAF


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Figure 5. (A) Western blot analysis of FA receptor expression and (B) the ratio of FA receptor to β-actin quantified on (a) MCF-7 and (b) HeLa cell membrane (n = 3). (C) Hemolysis percentages of RBCs by FA-GdN@CQDs-MWCNTs at different concentrations. Inset: optical images of RBCs from (a) PBS (10 days) and (b, c) FA-GdN@CQDs-MWCNTs (b, 10 h; c, 10 days). (D) ALT, AST, TB, creatinine, and Hb at different doses and different time intervals. Relative viabilities of (E) MCF-7 and (F) HeLa cells after incubation with free DOX, FA-GdN@CQDsMWCNTs/DOX, FA-GdN@CQDs-MWCNTs (808 nm, 2 W/cm2, and 5 min), and FA-GdN@CQDs-MWCNTs/DOX (808 nm, 2 W/cm2 and 5 min) at various concentrations for 24 h.

GdN@CQDs-MWCNTs/DOX showed the same excitation peaks as FA-GdN@CQDs-MWCNTs and DOX, which confirmed that the conjugate product contains these two things.40 The capability of drug loading was tested by incubating the FA-GdN@CQDs-MWCNTs and various concentrations of DOX in PBS. The relationships between the loading amount of DOX and drug concentration and final saturation value (250 mg/g) (Figure 4E), revealing the controlled loading of DOX onto the MWCNTs and GdN@ CQDs and surfaces. Making adequate understanding about the underlying behavior of FA-GdN@CQDs-MWCNTs function as a drug donor was meaningful for evaluating the efficacy of chemotherapy in cancer elimination. PBS solutions (pH 7.5 and 5.5) were employed to simulate microenvironments of normal tissue and tumor, respectively. As shown in Figure 4F, for FA-GdN@ CQDs-MWCNTs in PBS solution (pH 7.5), the accumulative drug released rate reaches 13.4% in which DOX dissociates from FA-GdN@CQDs-MWCNTs voluntarily via Brownian movement. In the present situation, a higher release rate of DOX from FA-GdN@CQDs-MWCNTs was achieved when in

the acidic environment, in which it dissolved quickly via a process of protonation. A fast DOX release and high release rate were achieved 57.8% by applying 808 nm laser. Extremely increases appear at time points of 0.5, 2, and 8 h during this process, which in responding to the release rate of 17.4, 10.8, and 5.8%, respectively. The DOX molecule can be released with exogenous NIR irradiation, and the endolysosome with lower pH would boost anticancer efficacy and minimize the side effect of drug.41 To investigate the pH-triggered and NIR drug release behavior, the FA-GdN@CQDs-MWCNTs/DOX was dialyzed at pH 5.0+NIR condition. UV−vis spectrum was recorded with different intervals, from 1 to 24 h. As shown in Figure S12, the absorption intensity of DOX rose dramatically at pH 5.0+NIR, compliance with the dissolution of the FA-GdN@CQDsMWCNTs and dissociation of drug-MWCNTs nanocomposites in the thermal and acidic circumstance. 3.7. Expression of FA Receptor among Cancer Cell Lines. FA receptors are known as overexpressed and actively internalized through FA receptor-mediated endocytosis in different cancer cell lines, so, FA has been allowed for being G


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Figure 6. (A) CLSM images of HeLa cells treated with FA-GdN@CQDs-MWCNTs for 2 h. (B) CLSM of HeLa cells treated with FA-GdN@ CQDs-MWCNTs/DOX for 2 h. (C) IR thermal images of HeLa tumor-bearing mice after injection with FA-GdN@CQDs-MWCNTs, FA-GdN@ CQDs-MWCNTs/DOX and PBS. (D) Temperature variations of tumors recorded by the IR thermal camera in various groups during 808 nm laser irradiation. The arrows refer to the fluorescence intensity of the individual cells. (E) In vivo T1-weighted MR images and color-mapped images of tumor sites after injection with FA-GdN@CQDs-MWCNTs/DOX for 0−4 h.

a fitable ligand for fostering drugs and macromolecules being absorbed into cells. As a consequence, the level of FA receptor in HeLa and MCF-7 cell lines was tested by the Western blot in Figure 5A. Obviously, the expression of FA receptor showed a cell line-dependent manner as expression of FA receptor was a low level in MCF-7 cells, whereas showed 1.3-fold higher in HeLa cells (Figure 5B). 3.8. In Vitro Hemolysis Effect of FA-GdN@CQDsMWCNTs. Hemolysis rate evaluating identifies the solubility of the blood cells in touch with foreign matters and acts as a vigoroso in vitro test to access the hemolysis property of biological materials. As blood cells are destroyed, hemoglobin is subsequently handed in from the cells. Herein, the hemolysis experiments were conducted to evaluate the blood compatibility of FA-GdN@CQDs-MWCNTs. It can be seen from Figure 5C that FA-GdN@CQDs-MWCNTs hemolysis percentages were 0.211, 0.463, 0.492, 0.618, and 0.842% for dosage of 50, 100, 200, 400, and 600 mg/L, respectively. It is found that the hemolysis rates increase slightly with the increase of FAGdN@CQDs-MWCNTs concentration. Therefore, FA-GdN@ CQDs-MWCNTs has negligible hemolytic activity, which is an important character for intravenous administration.24,42 The interaction of the FA-GdN@CQDs-MWCNTs with the RBC membrane enacted a momentous role in vivo application.

In this work, female C57BL/6 mice were used as the experiment subject to study in vivo toxicity of FA-GdN@ CQDs-MWCNTs. During the entire experimental period, the observed behaviors of all the treated mice were normal, indicating a negligible acute toxicity of FA-GdN@CQDsMWCNTs. At the given time points after administration (10 h and 10 days), the blood was collected for red blood cell morphology analysis and biochemistry assays. The mice were sacrificed immediately after blood collection. Hematological analyses were implemented to quantify the underlying in vivo toxicity of FA-GdN@CQDs-MWCNTs after administration. The optical images of the RBS (inset of Figure 5C) indicate that the treatment of FA-GdN@CQDs-MWCNTs does not alter the shape of red blood cells in the test mice. Meanwhile, serum biochemistry tests (SBT) were also performed for quantitative evaluation about total bilirubin (TB), aspartate aminotransferase (AST), alanine aminotransferase (ALT), creatinine, and hemoglobin (Hb). Figure 5D showed that these indicators are at similar levels for the mice injected to FAGdN@CQDs-MWCNTs and for the control mice. This means that the FA-GdN@CQDs-MWCNTs do not induce an inflammatory response in mice. Furthermore, the histopathological observations are also conducted to determine the migration of FA-GdN@CQDs-MWCNTs by the lung. For 10 h H


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(Figure S16) and previous literature.44 Easily to find from Figure 6B that colocalization of between FA-GdN@CQDsMWCNTs (merged as green color) and DOX fluorescence (merged as red color) in HeLa nuclei. Furthermore, the plot of intensity values across the individual cell was tested by ImageJ, and we could see that a dense mass of DOX enters into the cell nuclei, resulting in a dramatic increase in fluorescence. Overall, the cellular uptake and intracellular trafficking mechanisms of FA-GdN@CQDs-MWCNTs/DOX can be delivered as follows: First, FA-GdN@CQDs-MWCNTs/DOX have internalized the cells endocytosis. Second, the FA-GdN@CQDs-MWCNTs/ DOX are introduced into the lysosomes. Finally, DOX is released and then accesses and accumulates in the nuclei under the acidic environment of lysosomes and endosomes.45 Overall, the results in Figure 6A, B clearly demonstrate that FA-GdN@ CQDs-MWCNTs can promote ultrahigh cellular uptake and DOX transfection efficiencies, which can get ready for the development of highly biocompatible and resultful nanocarriers for DOX delivery applications. 3.11. In Vivo NIR Imaging. NIR imaging of FA-GdN@ CQDs-MWCNTs/DOX was applied in the living mice so as to clarify tumor sites and a suitable healing time for PTT. Tumor temperature was recorded by IR thermal camera. The temperature of the tumor in the FA-GdN@CQDs-MWCNTs and FA-GdN@CQDs-MWCNTs/DOX-treated group up to 43 °C after laser irradiation (2 W/cm2, 2 min). The temperature further up to 53 °C after 4 min (Figure 6C). No obvious variation in control group (PBS) when it was steady at approximately 27−28 °C. Figure 6D shows the increased noticeably temperature of the tumor, which is adequate to arouse nonreversible destroy to cancer cells.46 3.12. In Vivo Targeting MRI Imaging. The application of MR imaging in vivo for small animals was also demonstrated by the C57BL/6 mice after intravenous injection with FA-GdN@ CQDs-MWCNTs/DOX. The positive-contrast enhancement of nanoparticles was studied anatomically by transversal MR imaging in vivo. The precontrast and postcontrast MR imaging of T1-weighted for the tumor of the experimental mouse was recorded at the beginning, and after 0.5−4 h min following injection with FA-GdN@CQDs-MWCNTs/DOX. T1 MR imaging of each mouse was acquired on 3 T scanner. For the tumor, the MR signal was positively enhanced after intravenous injection with FA-GdN@CQDs-MWCNTs/DOX. The intension of MRI (Figure 6E) signal in living mice was gradually improved with an increasing injection time. Red box of Figure 6E showed images of the T1 distribution of the tumor, in which the region of the tumor becomes red with time. These results suggested that FA-GdN@CQDs-MWCNTs/DOX have nice targeting ability in tumor sites. The results indicated that FAGdN@CQDs-MWCNTs/DOX have specificity for T1 enhanced MR imaging in tumor sites. 3.13. Combined Chemo-Phototherapy Synergistic Effect of FA-GdN@CQDs-MWCNTs/DOX in Vivo System. To testify the potential employ of FA-GdN@CQDsMWCNTs/DOX for in vivo therapy further, we studied their practicable for FL imaging-guided PTT using HeLa tumorbearing nude mice. The mice were cured with the subcutaneous injection of FA-GdN@CQDs-MWCNTs/DOX, when the tumor size reached to 60 mm3. The facility of FA-GdN@ CQDs-MWCNTs/DOX dispersed in PBS for in vivo FL imaging was tested by injecting into the tumor, and then monitored by a Maestro 2 Multispectral Small-Animal Imaging. In Figure 7A, the subcutaneous injection site (tumor) displays a

injected group, some black dots can be seen in the lung, but after 10 days, black aggregates in the lung significantly lessen both in dimensions and number, hinting that the FA-GdN@ CQDs-MWCNTs can be removed from the lung. There are no obvious FA-GdN@CQDs-MWCNTs observed in other organs (Figure S13). The distribution of FA-GdN@CQDs-MWCNTs in spleen, heart, lung, kidney, and liver was quantified by ICP-AES after the mice were injected with FA-GdN@CQDs-MWCNTs (Figure S14). The high amount of injected dose (ID) per gram tissue of the FA-GdN@CQDs-MWCNTs accumulated in the spleen and liver at 4 h post injection. However, the amount of FA-GdN@CQDs-MWCNTs was decreased in the spleen and liver after 4 h. Therefore, the in vivo distribution studies of FA-GdN@CQDs-MWCNTs suggest that this nanocomposites have potential as low-toxicity materials for in vivo biological application. 3.9. In Vitro Cell Viability Test by FA-GdN@CQDsMWCNTs/DOX in NIR-Triggered PTT. As discussed above, FA-GdN@CQDs-MWCNTs is a multifunctional nanoplatforms for drug carrier and PTT. MCF-7 (Figure 5E), HeLa (Figure 5F), and HepG2 cells (Figure S15) were treated with various concentrations of free FA-GdN@CQDs-MWCNTs, DOX, and FA-GdN@CQDs-MWCNTs/DOX for 24 h. The survival rate of all cell types was shown in the increasingincubation-times manner with increasing-concentrations of free FA-GdN@CQDs-MWCNTs/DOX and DOX. The FA-GdN@ CQDs-MWCNTs/DOX could release DOX as soon as it was absorbed into cancer cells, which may lead to a statistically higher cytotoxic efficacy than DOX. Interestingly, HeLa cells exhibited higher cytotoxicity than MCF-7 or HepG2 cells, which could be indicated by the higher level of FA receptors on surface, which is consistent with the Western blot results. In contrast, the FA-GdN@CQDs-MWCNTs/DOX with NIR irradiation showed the lowest survival compared to nontargeted DOX (chemotherapy), a single treatment of FA-GdN@CQDsMWCNTs/DOX without NIR irradiation (chemotherapy), and FA-GdN@CQDs-MWCNTs with NIR irradiation (PTT). It was valuable to be recorded that the nanoparticles be targets for cancer cells, meanwhile made efforts to enhance the therapy efficiency together with individual chemotherapy or PTT. The therapy and Chemo/PTT act synergistically causing a remarkable reduction to the survival rate instead of any use of method alone. 43 The hyperthermia effect on the physiological function and chemotherapeutic efficacy of DOX improved with photothermal act and had a noteworthy synergistic effect at raised temperatures. 3.10. Cellular Uptake of the FA-GdN@CQDs-MWCNTs/ DOX. The crucial aspect of a drug delivery system is rapid internalization of FA-GdN@CQDs-MWCNTs/DOX by in vitro cells, which was studied by CLSM. On the basis of the λex-dependent PL behavior of FA-GdN@CQDs-MWCNTs, FA-GdN@CQDs-MWCNT exhibit green and red fluorescence under 488 and 543 nm illumination. After 2 h of incubation, different from the not-infiltrated-cell nuclei, cell membrane and cytoplasm exhibited strong green and red fluorescence, as shown in Figure 6A. This observation verifies that FA-GdN@ CQDs-MWCNTs could pass through cell membranes and enter into HeLa cells by the work of endocytosis so as to access the cytoplasm and even the nuclei. Figure 6B reveals that the distribution of DOX in HeLa nucleus, meaning that DOX are entered into cells via FA-GdN@CQDs-MWCNTs/DOX. This result is absolutely accord with free DOX intracellular imaging I


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Figure 7. (A) Real-time in vivo red FL images obtained after subcutaneous injections of FA-GdN@CQDs-MWCNTs/DOX in nude mice at various time points. 0 h refers to the time of injection. (B) Typical results of nude mice after 14th day of inoculation. (C) The size of tumors in various groups of mice after various treatments (a, PBS, b, DOX; c, FA-GdN@CQDs-MWCNTs/DOX; d, FA-GdN@CQDs-MWCNTs+Laser; and e, FAGdN@CQDs-MWCNTs/DOX+Laser). (D) Relative tumor volumes were normalized to their initial sizes. (E) Relative body weight of mice after different treatments.

strong fluorescence signal. The area of the FL signal progressively amplifies from the tumor, which means that FAGdN@CQDs-MWCNTs/DOX have good distribution ability in the tumor. This result verifies that FA-GdN@CQDsMWCNTs/DOX act on the tumor location through the active targeting and enhanced permeability and retention (EPR) effect, meanwhile valid FL could be detected around the field. Simulated by the above results, we then followed on evaluating the synergistic anticancer efficiency of FA-GdN@ CQDs-MWCNTs/DOX on nude mice bearing tumors. When the tumor achieved about 100−120 mm3 in size, mice were fallen into five groups (6 mice per group), as follows: PBS (Group 1), free DOX (Group 2), free FA-GdN@CQDsMWCNTs/DOX (Group 3), FA-GdN@CQDs-MWCNTs +NIR laser irradiation (Group 4), andFA-GdN@CQDsMWCNTs/DOX+NIR laser irradiation (Group 5). Expectedly, the temperature of tumor area treated with FA-GdN@CQDsMWCNTs and FA-GdN@CQDs-MWCNTs/DOX promptly increased and kept at 50 °C during NIR laser irradiation. This temperature is enough to arouse nonreversible destroy cancer cells.47 Representative photographs of the nude mice with tumors were shown in Figure 7B, C. The tumor volumes (Figure 7D) and body weights were then tested (Figure 7E). On the 14th day, three mice were sacrificed and excised while the remains were observed for 28 days. Compared to the free DOX, FA-GdN@CQDs-MWCNTs/DOX without NIR laser irradiation displayed enhanced therapeutical effect, perhaps due to the improved cancer cells uptake by the EPR effect and active targeting. Although improved induction of tumor damage was shown with the injection of FA-GdN@CQDs-MWCNTs/ DOX or FA-GdN@CQDs-MWCNTs with 808 nm laser irradiation (Figure 7C), the tumor growth was shown, suggesting the low efficacy in inducting tumor damage. In

contrast, tumors in the synergistic therapy group (FA-GdN@ CQDs-MWCNTs/DOX+NIR laser irradiation) were totally eradicated. Additionally, eosin (H&E) staining and hematoxylin of tumor slices affirmed that intact cells in the FA-GdN@ CQDs-MWCNTs/DOX+ NIR laser irradiation group sharply differed with PBS groups and partial groups in the portion of morphology about nuclear structures and membrane (Figure 7F). The in vivo anticancer results forcefully suggested FAGdN@CQDs-MWCNTs/DOX under 808 nm laser irradiation could achieve effective Chemo/PTT combinative purpose for cancer ablation. Therefore, the combination of Chemo/PTT has been certified to be more functional in cancer therapy. In addition to heat that can directly damage the cancer cells, the photothermal effect could be exploited to excite drug release improvement and enhance chemotherapy efficacy, which provided a conjunctively improved therapeutic efficacy superior to that gained by single therapy. We also monitored the mice body weight in the course of the treatment and did surprised found that the FA-GdN@CQDsMWCNTs/DOX+NIR laser irradiation did not affect this index (Figure 7E), suggesting that multiple injections of FA-GdN@ CQDs-MWCNTs/DOX never caused obvious side effects, which is ascribed to the enrichment effect of FA-GdN@CQDsMWCNTs in tumor tissue and NIR and pH-controlled release DOX drug from FA-GdN@CQDs-MWCNTs/DOX. side effects. In addition, organ indices were tested that offer information on the basic toxicity. The organ index is defined as the ratio of the organ wet weight to the body weight (g/g).45 There is no obvious difference in the lung, heart, and kidney indices of the experimental and other groups (Figure S17), showing that the FA-GdN@CQDs-MWCNTs/DOX+NIR laser irradiation groups never cause obvious toxicity and side effects on the heart, lung, spleen, liver, and kidney. Also, given J



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indication by the H&E staining results (Figure S18), no pathological changes among the main organs for the mice in FA-GdN@CQDs-MWCNTs/DOX+NIR irradiation laser group were detected, either other groups. Building on the results of photothermal in vivo and chemotherapy therapy, what we found was that FA-GdN@CQDs-MWCNTs/DOX made great contribution to tumor inhibition while was played out damage major organs in tumor-bearing mice. Above all, we proposed FA-GdN@CQDs-MWCNTs/DOX as a potential platform for cure of malignant cancers.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsbiomaterials.7b00531.

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REFERENCES

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4. CONCLUSION In summary, a multifunctional CNTs drug delivery (FA-GdN@ CQDs-MWCNTs) with narrow size distribution, great photostability, high DOX loaded, and solubility in water was prepared in the current study. The as-synthesized FA-GdN@CQDsMWCNTs/DOX own high thermal efficiency and enhancement of DOX release under NIR (808 nm) light irradiation. FA-GdN@CQDs-MWCNTs/DOX had prominent stability, bimodal contrast imaging functionality, NIR and pH-sensitive drug release, and drug targeting characteristics. In vivo tumor, MR imaging results suggested that FA-GdN@CQDsMWCNTs/DOX has specificity for T1 enhanced MR imaging in the tumor area. Investigations in vitro and in vivo display much higher tumor destroy ability of the FA-GdN@CQDsMWCNTs than single chemotherapy or PTT. It makes great sense that FA-GdN@CQDs-MWCNTs work as a perfect nanoplatforms for dual model imaging and Chemo/PTT with properties in fluorescence and paramagnetic. The FA-GdN@ CQDs-MWCNTs/DOX might be applied to MRI therapeutic efficiency of tumors in routine preclinical studies in near future. This study manifests the possibility of FA-GdN@CQDsMWCNTs/DOX as a hopeful nanoplatforms for efficient combined Chemo/PTT in cancer ablation. What’s more, this work implies that admirable tuning of the surface functional group of nanomaterials is crucial to ameliorating their behaviors in vivo as well as applications in vivo.

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Detailed description of experimental methods and Figures S1−S18 (PDF)

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Ninglin Zhou: 0000-0002-5530-8294 Funding

The authors gratefully acknowledge the support of this work by Jiangsu province science and technology support plan (BE2015367), and the Jiangsu Collaborative Innovation Center of Biomedical Functional Materials. Notes

The authors declare no competing financial interest. K


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