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Biocompatible Chitosan-Carbon Dots Hybrid Nanogels for NIRImaging-Guided Synergistic Photothermal/Chemo-Therapy Hui Wang, Sumit Mukherjee, Jinhui Yi, Probal Banerjee, Qianwang Chen, and Shuiqin Zhou ACS Appl. Mater. Interfaces, Just Accepted Manuscript • Publication Date (Web): 09 May 2017 Downloaded from http://pubs.acs.org on May 9, 2017

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ACS Applied Materials & Interfaces

Biocompatible Chitosan-Carbon Dots Hybrid Nanogels for NIR-Imaging-Guided Synergistic Photothermal/Chemo-Therapy Hui Wang,a,b Sumit Mukherjee,a Jinhui Yi,a Probal Banerjee,a Qianwang Chen,b and Shuiqin Zhoua* a

Department of Chemistry of The College of Staten Island, The City University of New York,

Staten Island, NY 10314, USA; Ph.D. Program in Biochemistry and Chemistry, The Graduate Center of the City University of New York, New York, NY 10016,USA b

High Magnetic Field Laboratory, Hefei Institutes of Physical Science, Chinese Academy of

Sciences, Hefei 230031, China ABSTRACT: Multifunctional nanocarriers with good biocompatibility, imaging function, and smart drug delivery ability are crucial for realizing imaging-guided highly efficient chemotherapy in vivo. This paper reports a type of chitosan-carbon dots (CDs) hybrid nanogels (CCHNs,～65 nm) by integrating pH-sensitive chitosan and fluorescent CDs into a single nanostructure for simultaneous NIR imaging and NIR/pH dual-responsive drug release to improve therapeutic efficacy. Such CCHNs were synthesized via a nonsolvent-induced colloidal nanoparticle formation of chitosan-CDs complexes assisted by ethylenediaminetetraacetic acid (EDTA) molecules in aqueous phase. The selective crosslinking of chitosan chains in the

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nanoparticles can immobilize small CDs complexed in the chitosan networks. The resultant CCHNs display high colloidal stability, high loading capacity for doxorubicin (DOX), bright and stable fluorescence from UV to NIR wavelength range, efficient NIR photothermal conversion, and intelligent drug release in response to both NIR light and pH change. The results from in vitro tests on cell model and in vivo tests on different tissues of animal model indicate that the CCHNs are nontoxic. The DOX-loaded CCHNs can permeate into the implanted tumor on mice and release drug molecules efficiently on site to inhibit tumor growth. The additional photothermal treatments from NIR irradiation can further inhibit the tumor growth benefited from the effective NIR photothermal conversion of CCHNs. The demonstrated CCHNs manifest a great promise toward multifunctional intelligent nanoplatform for imaging guided highly efficient cancer therapy with low side effect. KEYWORDS: carbon dots; chitosan; CCHNs; responsive release; NIR imaging 1. INTRODUCTION Although great progress has been achieved about the diagnosis and therapy in last decades, cancer remains one of the leading death causes.1 Patients treated by conventional chemotherapy commonly suffer from severe side effects including suppression of hematopoiesis, gastrointestinal, and cardiac toxicity when it is administered in vivo directly.2-3 One of the key attributes of chemotherapy is to develop a kind of drug delivery system that can intelligently trigger the drug release to reduce its side effects and improve overall therapeutic efficacy.4-5 So far, many kinds of drug carriers have been studied to improve chemotherapeutic outcomes, including inorganic nanoparticles, liposomes, metal-organic framework, smart nanogels and so on.6-19 Among these nanocarriers, smart polymer nanogels are not only tunable in porous structure for responsive drug loading and release, but also highly biocompatible promoted by


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their high content of water and physicochemical similarity to extracellular matrices.20-21 On the other side, multifunctional nanocarrier that combines imaging function and therapeutic capability have great advantages over traditional chemotherapy because the imaging function can help to precisely identify location of tumor region and monitor the treatment progress.22-24 In particular, fluorescent NIR imaging is an attractive technique for disease detection and analysis because NIR light reduces background effects of autofluorescence and scattering in vivo.25 Many naturally abundant polymers including chitosan, lignin and starch have demonstrated extensive applications in the biomedical and biotechnological fields owing to its low-cost, nontoxic, biodegradable and non-immunogenic properties.26-28 In particular, pH-sensitive chitosanbased composite nanocarriers have widely been explored for controlled release formulations to deliver genes, proteins, and drugs.29-38 On the other hand, carbon dots (CDs) or graphene oxide quantum dots with unique optical properties have been incorporated into different nanomaterials or polymer matrix for applications as high quality membrane, catalysts, drug carriers, MRI contrast agents and nanodevice.39-44 When these optically active nanoparticles are embedded into intelligent materials such as environmentally sensitive polymers, the resultant composite materials can be applied in sensing field.45-46 Compared with other optically active nanoparticles, CDs are less toxic and cheaper. Furthermore, CDs can be used for photothermal therapy and NIR fluorescence imaging due to their NIR absorption and emission nature.6, 47-48 Considering these unique material properties of chitosan and CDs, it is meaningful to design chitosan-CDs hybrid nanogels (CCHNs) to combine the NIR imaging function with NIR-responsive drug delivery to improve treatment outcomes. In this manuscript, we have successfully designed and synthesized a type of multifunctional nanocarriers by combining biocompatible chitosan and fluorescent CDs into a single system.


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With a rational design, such CCHNs can be easily synthesized using a non-solvent induced chitosan colloidal formation followed by an in situ crosslinking in aqueous solution. Specifically, the chitosan bearing –NH2/–OH groups, CDs bearing –OH/–COOH groups, and EDTA molecules carrying –COOH groups can form associated chitosan-CDs-EDTA complexes via hydrogen bonding interactions. At appropriate pH values, the introduction of an unfavorable solvent to chitosan like ethanol can induce the resulted complexes to collapse, forming compact nanoparticles stabilized by the surface charges from EDTA molecules and partially protonated amine groups of chitosan. The in situ chemical crosslinking of the chitosan chains will randomly encapsulate the CDs and EDTA molecules in the resultant chitosan networks. When these crosslinked particles are dispersed into large amount of neutral water, the small EDTA molecules will diffuse out. Thus, the dialysis of the synthesized product against frequently changed water can remove the small EDTA molecules, free CDs, and ethanol molecules, resulting in stable CCHNs. Such prepared CCHNs are expected to display cooperative properties from both chitosan and CDs and integrate several important functions as depicted in Figure 1. While the CDs can provide fluorescent NIR imaging and photothermal effects, the small nanogels can permeate into tumor site to serve as a transmembrane carrier for delivering doxorubicin (DOX) drug molecules into cancer cells for chemotherapy on tumor site. The favorable interactions between the embedded CDs in CCHNs and DOX molecules through π-π stacking and electrostatic attractions allow CCHNs to load high amount of drug molecules. Furthermore, the pH-/NIR dual responsive properties of CCHNs enable both endogenous pH-triggered drug release on tumor site and exogenous NIR light enhanced drug release to realize a synergistic therapy for high therapeutic efficacy.


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Figure 1. Schematic depiction of DOX-loaded CCHNs for therapeutic application. 2. EXPERIMENTAL SECTION D(t)-Glucose was product of ACROS. All other chemicals were product of Aldrich. Doxorubicin hydrochloride (DOX), glutaraldehyde (GA, 25%), HCl (37%), and ethylene diamine tetraacetic acid (EDTA) were used as received. Chitosan (Mn = 5000) was purified by dissolution in acetic acid, filtration, and dialysis against water. The final concentration of chitosan solution is adjusted to 5 mg/mL. 2.1. Synthesis of CCHNs. Fluorescent CDs with surface –OH and –COOH groups were firstly prepared using our previously reported method.49 20 mg of such prepared CDs were dissolved into 10 mL of chitosan solution. After being stirred for 30 min at 70.0 oC, 6 mg of EDTA was added and the solution was continuously stirred for 60 min. Then, nonsolvent ethanol (1 mL) was slowly introduced drop by drop under stirring to form opalescent suspension of complex nanoparticles. At last, 90 µL of GA solution was added to crosslink the chitosan chains at ambient temperature for 6 h, leading to the formation of CCHNs. The obtained CCHNs were


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purified by repeated centrifugation (20,000 rpm for 30 min), decantation, and redispersion in water for three times, followed by a dialysis (Mw cutoff 12000-14000) for 72 h against frequently changed water to remove EDTA, ethanol and free CDs at ambient temperature. 2.2. Characterization of CCHNs. The shape of CCHNs was observed using field emission scanning electron microscopy (SEM, AMRAY 1910) and transmission electron microscopy (TEM, FEI TECNAI system). The nanostructure of CCHNs was observed using high-resolution TEM (HRTEM, JEOL JEM 2100 system). Elemental analysis was obtained with energydispersive X-ray (EDX) detector installed on the same HRTEM. The absorption spectra in UVVisible wavelength range were collected on a Thermo Electron Co. Helios β UV-vis Spectrometer. The photoluminescence (PL) spectra were collected on a JOBIN YVON Co. FluoroMax®-3 Spectrofluorometer. The photothermal experiments were conducted using a NIR laser (808 nm, 1.5 W/cm2). The hydrodynamic size and size distribution of the dust-filtered CCHNs were analyzed on a light scattering spectrometer (BI-200SM) with a He-Ne red laser (35 mW) as the light source. 2.3. In vitro DOX Loading and Release of CCHNs. To load DOX molecules into the CCHNs, a DOX solution at a concentration of 5.0 mg/mL and pH = 7.4 was firstly prepared. 0.4 mL of such made DOX solution was then added drop by drop into 10 mL of CCHNs dispersion (0.1 mg/mL) under stirring. The mixture was continuously stirred for 24 h to allow DOX loading process to reach equilibrium. Afterwards, DOX-loaded CCHNs were separated via centrifugation at 20,000 rpm for 20 min and washed multiple times with distilled water. The free DOX from the supernatant and all washing solutions were combined and quantified using the characteristic absorption peak of DOX at 480 nm. The content of DOX loaded into the CCHNs was calculated by (Mo – Mf)/MHN ×100%, where Mo is the mass of DOX contained in the original solution


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before loading experiment, Mf is the mass of free DOX remained in the supernatant and washing solutions, and MHN represents the mass of the CCHNs used for loading experiment. To test the releasing kinetics of DOX molecules from CCHNs in response to the pH change and NIR irradiation, two sets of experiments were respectively carried out. DOX-loaded CCHNs were redispersed in 25 ml of 0.005 M PBS solution (pH=7.4). In the first set, two dialysis bags holding 5 mL of DOX-loaded CCHNs dispersion in PBS were placed into 50 mL of 0.005 M PBS (pH = 7.4) at 37 o C. At certain releasing stages, a NIR irradiation (808 nm at 1.5 W/cm2) was applied to one of the releasing bag. In the second set, three dialysis bags containing 5 mL of DOX-loaded CCHNs dispersion were placed into 50 mL of 0.005 M PBS solutions at pH = 7.4, 6.2 and 5.0, respectively. The exterior PBS solutions containing the released DOX molecules were sampled at specific releasing stages. The amount of released DOX was analyzed based on the characteristic absorption peak of DOX at 480 nm. The releasing kinetics is expressed by the total percentage of DOX released from dialysis bags at different releasing stages. 2.4. Cellular Uptake by Flow Cytometry Analysis. Mouse 4T1 breast tumor cells were firstly incubated with CCHNs (50 µg/mL) for specified time lengths and washed with cold PBS repeatedly. These cells were then digested by Trypsin-EDTA, washed with PBS, and fixed in 2% paraformaldehyde at 4 oC overnight. After separated from the fixative medium by centrifugation (500 g, 3 min) and washed with PBS, cells were finally resuspended in PBS for flow cytometry analysis on a FACSCanto II flow cytometer (BD Biosciences). The signals were collected at the emission wavelength of 530 nm, excited with a laser of 488 nm. 2.5. In Vitro Cellular Imaging Ability. After seeded onto glass cover slips and incubated overnight in culture medium in a 6-well plate, 4T1 cells were further incubated with CCHNs (0.05 mg/mL) for 2 h. These cells treated with CCHNs were then washed with cold PBS triple


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times and fixed in 4% paraformaldehyde at 37°C for 15 min. Finally, these cells treated with CCHNs were mounted onto glass slides with ProLong® Gold Antifade Mountant (Life Technologies Inc., Gaithersburg, MD) for acquisition of images with a Leica SP8x Microscope (Leica Microsystems GmbH, Germany). Three excitation wavelengths were used (405, 488 and 546 nm). The emission regions were 425-480 nm, 508-540 nm and 566-700 nm, respectively. 2.6. In Vitro TPF Imaging. After seeded into collagen-coated glass bottom dishes (4000 cells/cm2) and grown in culture medium for 24 h, 4T1 cells were incubated with CCHNs (0.05 mg/mL) for 2 h. These cells treated with CCHNs were then washed, fixed, and mounted onto glass slides as described above for acquisition of two-photon fluorescence (TPF) images with a Leica SP8 2-Photon/FLIM microscope. 2.7. Ex Vivo NIR Imaging. 200 µL of the PBS (0.005 M, pH=7.4), CDs (0.01 mg/mL) and CCHNs (0.05 mg/mL) were intravenously injected into different tumor naive female C57BL/6 mice, respectively. The mice were allowed to recover for two hours followed by an anesthesia with intraperitoneal (IP) infusion of ketamine: xylazine at 100 mg/Kg: 10 mg/Kg. Subsequently, mice were sacrificed and their brain was removed and washed with PBS. Ex vivo NIR images of the brains were acquired on the Odyssey® Imaging System (LI-COR Biosciences) with excitation wavelength of 800 nm. The NIR fluorescence originated from CDs or CCHNs was pseudo colored green and the autofluorescence from the brain tissue was pseudo colored red. 2.8. Biodistribution and Histopathological Evaluation. Biodistribution of the CCHNs were analyzed by monitoring the fluorescence intensity. Female BALB/c mice receiving no injection were used as controls. Mice receiving intravenous injection of 200 µL CCHNs at 1 mg/mL were euthanized after 24, 48, 72 and 96 h, respectively. Their whole organs, including liver, spleen, kidneys, lungs, and heart were collected. Fluorescence intensity was acquired for each type of


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tissue using a Xenogen IVIS imaging system. For histopathological analysis, organs of heart, kidneys, liver, lungs and spleen from female BALB/c mice were harvested at 120 h after receiving injection. These organs were fixed in 10% buffered formalin and embedded in paraffin. Sections were cut into 5 µm slices and stained with hematoxylin and eosin (H&E). Images of these tissues were collected under an optical microscope. 2.9. In Vitro Cytotoxicity. The MTT assay was used to analyze the cytotoxicity of the CCHNs and DOX-loaded CCHNs with or without an exposure to NIR light. 4T1 cells were cultured in 96-well microplate at a seeded density of 2000 cells per well with 100 µL medium. After an overnight incubation, the medium in each well was replaced with 100 µL fresh medium containing drug-free CCHNs or DOX-loaded CCHNs at a variety of concentrations. The wells containing no samples but normal medium were used as control. To perform the NIR photothermal treatments, 5 min of NIR irradiations at 1.5 W/cm2 were applied on the cells. After incubated with the samples for 24 h, the cells were continuously incubated for 2 h with the addition of 10 µL MTT solution (5 mg/mL in PBS) at 37 °C in a humidified environment of 5% CO2. The medium was then discarded and 100 µL of DMSO solution was added to lyse the cells. The absorbances at 570 nm and 690 nm were measured at a microplate reader. 2.10. In Vivo Tumor Therapy. The tumors were implanted by subcutaneous injection of 4T1 cells (2 × 106 cells/mL in PBS) into right flanks of 5-week old female BALB/c mice. Five days after tumor inoculation, mice were administered intravenously four times for every three days with PBS, empty CCHNs or DOX-carried CCHNs (200 µL, 2 mg/mL per mouse per injection), respectively. After 24 h of every injection, tumors were exposed or not exposed to 5 min of NIR irradiation (808 nm, 1.5 W/cm2). Tumor sizes and mice weight were monitored every 3 days for 18 days.


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3. RESULTS AND DISCUSSION

Figure 2. Structure and property of CCHNs. (a) SEM image of CCHNs, the inset shows the solution appearance of CCHNs in water without (left) and with (right) irradiation of UV light (365 nm). (b) TEM image of CCHNs, the inset is SAED pattern of single CCHN. (c) HRTEM image of part of single CCHN; (d) Energy dispersive spectrum and 2D lattice fringe of single CD in CCHNs. (e) Diameter variation of CCHNs in culture medium over a time period of 12 days. (f) Excitation spectra of the CDs and CCHNs with emission wavelength = 450 nm. The size and shape of as-prepared CCHNs was characterized by electron microscopy. Both SEM and TEM images (Figure 2a and b) show that CCHNs have a quasisphere morphology in an average diameter about 65 nm. The CCHNs dispersion in water appears in yellow color. When a UV light (wavelength = 365 nm) was applied on the solution, bright blue light was observed (inset in Figure 2a). The inset in Figure 2b shows the SAED pattern of single CCHN. The sharp diffraction spots should be attributed to the highly crystallized CDs embedded in the CCHNs because chitosan nanogel alone is amorphous. The high-resolution TEM image of single


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CCHN (Figure 2c) and single nanocrystal CD with clear 2D lattice fringe (inset in Figure 2d) confirms the presence of crystallized CDs randomly immobilized in the chitosan matrix. The 2D interplanar distance of 0.333 nm in the nanocrystal can be indexed to (002) lattice plane of graphitic carbon. The EDS of single nanocrystals buried in CCHNs (Figure 2d) only shows the copper peak originated from TEM grid and carbon peak, further confirming that the crystallized domains are composed of CDs. The size, stability, and optical properties of CCHNs in aqueous phase were further characterized. The light scattering results reveals that the hydrated CCHNs are nearly monodispersed in size with an average hydrodynamic diameter (Dh) of 78 nm in water (Figure S1 in Supporting Information). Furthermore, the CCHNs are very stable and well-dispersed in cell culture medium containing 10 % serum, which is important for their applications in biological systems. The measured average Dh values of CCHNs remain unchanged (within experimental error) over the monitored 12 days (Figure 2e). The comparison of the UV–vis absorption spectra from free CDs, chitosan matrix, and CCHNs (Figure S2) reveals that the CCHNs exhibits the characteristic absorption peaks from both chitosan nanogels (peak centered at 260 nm) and CDs (a shoulder peak at 235 nm resulting from absorption of aromatic Pi system)50, proving the successful incorporation of CDs in chitosan nanogels. Figure 2f demonstrates the excitation spectra from both CDs and CCHNs. For an emission wavelength of 450 nm, both spectra show a maximum excitation peak position around 363 nm. No obvious peak shift was observed, indicating that the CCHNs used the same wavelength as that of CDs to produce the fluorescence at 450 nm.


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Figure 3. PL properties and cellular uptake. (a and b) PL and upconverted PL spectra of CCHNs collected using a broad range of excitation wavelengths; (c) Flow cytometric analysis of cellular uptake of CCHNs by 4T1 cells over different incubation time; (d) Mean fluorescence intensities obtained from flow cytometric analysis of CCHNs. Figure 3a shows the PL spectra of CCHNs over an excitation wavelength (λex) range of 320540 nm. The increase in λex causes a red-shift of the emission peak of CCHNs, indicating that the CDs embedded in CCHNs possess different surface energy traps.51 The most intensive fluorescence occurs at 472 nm excited at λex = 400 nm. The fluorescence quantum yields (QYs) of the CD and CCHNs were determined to be 9.1% and 3.9%, respectively, using RB as a standard. The decrease in QY of CCHNs compared to the free CDs could be due to the surface binding of the CDs with the crosslinked chitosan chains, which would affect the surface property


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of the CDs and quench the emitted photons. The photostability of CCHNs was further quantified by monitoring the fluorescence intensity change of CCHNs over a 2 h continuous excitation with λex = 360 nm. The negligible change (～5.1 %) (Figure S3) in maximum PL intensity proves an excellent photostability of the CCHNs, an important feature necessary in bioimaging. Figure 3b shows the upconversion PL spectra of CCHNs excited with red/NIR light (λex = 620-980 nm). Strong upconverted emissions were correspondingly detected in the wavelength range of 517464 nm, resulting from the multiphoton activation process of the embedded CDs as previously reported.52 The bright fluorescence observed from the CCHNs enables us to evaluate the cellular uptake ability for the CCHN particles using flow cytometry. Figure 3c shows that the fluorescence intensity of 4T1 cells is dramatically enhanced after being treated with the CCHNs for 2 h at 50 µg/mL. The continuous incubation with the CCHNs further enhances the fluorescence intensity from cells, demonstrating a time dependent nonspecific cellular uptake process of CCHNs. The quantitative analysis in Figure 3d confirms that the cellular uptake of CCHNs by 4T1 cells mainly occurs at the initial 2 h. After 2 h, the continuous incubation from 2 h to 8 h only increases the fluorescence intensity of the 4T1 cells by approximately 12%. The fast and successful cellular uptake of these CCHNs endow them great potential as efficient nanocarriers for delivery of payloads through cell membrane. Figure 4a-c displays the confocal fluorescent images of 4T1 cells loaded with CCHNs, obtained respectively under the excitation of three lasers (405, 488, and 546 nm). The CCHNs containing CDs emitted strong fluorescence and highlighted the 4T1 cells in multicolor forms. No fluorescence intensity change is observable on the 4T1 cellular images after a continuous exposure to 488 nm laser for 30 min (Figure S4), revealing the excellent photostability of


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CCHNs for potential long-term cellular imaging. The top-down Z-scanning confocal images of B16F10 cells treated by CCHNs (Figure S5) confirm their ability to cross the cell membrane and enter into intracellular regions.53 The CCHNs do not appear in the karyons, but distribute randomly throughout the cytoplasm. Figure 4d-f shows transmission (d), TPF (e), and overlaid (f) images of the 4T1 cells incubated with CCHNs excited with a NIR laser (820 nm). Obviously, the endocytosed CCHNs could also successfully highlight the 4T1 cells under NIR laser because of the upconverted fluorescence produced from CCHNs. Compared to images acquired in visible light wavelength range, those taken in the NIR wavelength

range

would

have

much

better

tissue

penetration

and

lower

autofluorescence. Therefore, NIR imaging performance of CCHNs was evaluated ex vivo. 200µL of PBS, CDs in PBS (0.01 mg/mL), and CCHNs in PBS (0.05 mg/mL) were intravenously injected into three different mice, respectively. After 2 h, the brain sections were removed from the mice and scanned using the Odyssey scanner. As shown in Figure 4g-i, no NIR fluorescence signals were observed on the brain from the mouse receiving injection of PBS only. When the free CDs or CCHNs were injected, the bright fluorescent signals were observed on the brain tissues, which should result from the excellent NIR imaging ability of CDs.54 Several factors can influence nanoparticles to cross blood brain barrier (BBB), including size, shape, surface charge type and density, surface ligand property, and composition of nanocarriers.55 It was proposed that adsorptive transcytosis is the mechanism for nanocarriers coated with albumin or chitosan to cross the BBB. For our CCHNs with hydrodynamic diameter of ~ 78 nm, the positive surface charges (+ 20.2 mV) from chitosan and the local hydrophobic carbon domains from the CDs should be the major factor, thus the adsorptive transcytosis could be the main transport mechanism for our CCHNs to cross the BBB.


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Figure 4. In vitro and ex-vivo bioimaging ability. (a-c) confocal images of 4T1 cells treated with CCHNs excited by different lasers of (a) 405, (b) 488, and (c) 546 nm, respectively. (d) Transmission, (e) TPF, and (f) overlaid images of 4T1 cells treated with CCHNs excited by a NIR laser of 820 nm. (g-i) Ex-vivo NIR images of brain sections removed from tumor naive female C57Bl-6 mice 2 h after receiving injection with PBS (d, 0.005 M, pH=7.4), CDs dispersed in PBS (e, 0.01 mg/mL) and CCHNs in PBS (f, 0.05 mg/mL), obtained from the Odyssey scanner with excitation laser of 800 nm. After investigating the optical properties and bioimaging function, the functionality of the CCHNs as drug carriers was assessed with the anticancer drug of doxorubicin (DOX) as a model. As shown in Figure S6, the characteristic absorption peak of DOX molecules at 480 nm appears


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in the CCHNs after incubated with the DOX solution in PBS and isolation, confirming the successful drug loading of CCHNs. The DOX loading contents of the CCHNs and chitosan nanogels were determined to be 17.8% and 14.5%, respectively. The higher drug loading capability of CCHNs than the chitosan nanogels containing no CDs might be explained by the favorable π-π interactions between DOX molecules and the CDs embedded in CCHNs.56-57 Figure 5a displays the release kinetics of DOX from CCHNs in PBS medium (pH = 7.4, 37 °C) subjected or not subjected to NIR irradiations. Without exposure to NIR light, the CCHNs release DOX molecules slowly and steadily after initial fast release. A momentary (5 min) exposure to NIR irradiation could significantly enhance the drug release rate from CCHNs. When NIR irradiation was taken away, the DOX release returned back to the slow and steady rate. Such NIR-induced enhancement of the drug releasing rate should be originated from the local heat produced by CDs in CCHNs due to their efficient conversion of NIR light to heat. For example, the temperature of water containing 0.05 mg/mL CDs can increase by ~ 20 °C within 5 min when exposed to NIR irradiation (808 nm, 1.5 W/cm2) (Figure S7), demonstrating a highly efficient photothermal conversion ability. The measured photothermal conversion efficiency of CDs is about 25.2%, similar to the one exhibited by gold nanorods (24.4%).58 Relatively, the temperature of water containing no CDs (control) only increases by ~ 3.2 °C under identical conditions. The local heat produced by the CDs in CCHNs could weaken the drug-carrier interactions (e.g., hydrogen bonding and π-π stacking between DOX molecules and CCHNs) and increases the mobility of DOX molecules.59 Such a NIR light-sensitive drug release will provide an exogeneous way to improve the therapeutic efficacy of the CCHNs carrier.


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Figure 5. In vitro drug release and therapy. (a) DOX releasing kinetics from CCHNs in PBS (pH = 7.4) at 37 °C under 5 min exposure to NIR irradiation (808 nm, 1.5 W/cm2) at the indicated time points; (b) DOX releasing kinetics from CCHNs in buffers with different pH at 37 °C; (c) In vitro cytotoxicity of 4T1 cells after receiving treatments with drug-free and DOXloaded CCHNs with/without 5 min exposure to NIR irradiation; (d) Therapeutic efficacies of


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CCHNs nanocarrier for NIR photothermal, DOX chemo, and their combined treatments. (e) A scheme for chemotherapy alone or combined photothermal/chemo therapy on cancer cells using CCHNs as drug carriers. Considering the pH-sensitivity of chitosan, we expect that the CCHNs nanocarriers will also display pH-responsive drug release. Figure 5b manifests the releasing profiles of DOX molecules from CCHNs dispersed in PBS solution with different pH values at 37 oC. Results show that only about 50.8% and 56.4% of preloaded DOX molecules were released from the CCHNs over 78 h at pH = 7.4 and 6.2, respectively. When the pH was adjusted to 5.0, a much faster DOX releasing rate was observed, with about 72.2% of the preloaded drug was released from CCHNs over the same time period. The significantly enhanced drug releasing rate of CCHNs at pH = 5.0 should be mainly attributed to the pH-sensitivity of chitosan with a pKa ~ 6.2. When the pH decreases from 6.2 to 5.0, the –NH2 groups of the chitosan chains reach to a nearly completed protonation, inducing a full swelling of the positively charged chitosan nanogels. Meanwhile, the acidic pH in releasing medium also enhances the solubility of DOX molecules (pKa ~ 8.2) in water. The electrostatic repulsions between both positively charged DOX molecules and chitosan networks reduce the drug-carrier interactions. Therefore, the highly soluble DOX could diffuse out the swollen (or open) chitosan networks at a much faster rate at pH = 5.0. Such a pH-triggerable drug release will provide an endogenous stimulus to intelligently regulate the drug release from the carriers under acidic tumor microenvironments.60-61 Figure 5c manifests the 4T1 cell viability after receiving treatments with drug-free and DOX-loaded CCHNs in the absence or presence of 5 min NIR irradiation. Clearly, the 24 h treatments with CCHNs at 100 µg/mL or below have no effects on the cell viability, indicating no cytotoxicity of CCHNs. The addition of 5 min exposure to NIR light onto this treatment with


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drug-free CCHNs slightly decreases the cell viability, implying that the local heat produced from the CDs embedded in CCHNs under NIR irradiation has no significant impact on cytotoxicity. Similarly, 5 min NIR irradiation alone also has negligible toxicity to 4T1 cells (Figure S8). In a different scenario, when the 4T1 cells received 24 h treatments with DOX-loaded CCHNs at 100 µg/mL, about 40% cells were killed, indicating that active DOX drug can be successfully released from CCHNs to kill cancer cells. Moreover, the addition of 5 min NIR light irradiation onto this treatment can further reduce the cell viability. For example, 57.4% of the 4T1 cells could be killed after receiving 24 h treatment with DOX-loaded CCHNs (100 µg/mL) in a combination with 5 min NIR light treatment. Figure 5d compares the in vitro therapeutic efficacies of 4T1 cells after receiving treatments described above. The additive therapeutic efficacies (Tadditive) was estimated based on the equation of Tadditive = 100 - (fchemo × fphotothermal) × 100 with f being the fraction of survived cells after each treatment.62-63 Clearly, the therapeutic efficacy from combined treatments (Tcombined) of DOX-loaded CCHNs plus NIR irradiation was significantly higher than Tadditive obtained from additive chemo- and photothermal treatment alone. All p-values of the t-test analysis on the comparison of Tcombined and Tadditive values, obtained at each treatment concentration of CCHNs, are lower than 0.01. This indicates that the Tcombined and Tadditive are significantly different. Figure 5e illustrates the synergistic effect of NIR photothermal treatment in addition to the chemotherapy with DOX-loaded CCHNs. When pH-responsive DOX-loaded CCHNs enter into tumor cells, DOX molecules will be released at an enhanced rate because of the acidic intracellular lysosomes and endosomes in cancer cells. The small DOX molecules released in cells will then enter into the nuclei and kill the cancer cells. On the other hand, the additional NIR irradiation on the DOX-loaded CCHNs can further speed up the drug release because the


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CDs in CCHNs can efficiently convert NIR light to local heat, which weakens the drug-carrier interactions and increases the mobility of DOX molecules. Therefore, the CCHNs can facilitate both endogeneous and exogeneous regulation of drug delivery to release more DOX drug to kill cancer cells efficiently for high therapeutic efficacy.64

Figure 6. Biocompatibility test of CCHNs. (a) Cytotoxicity of CCHNs on noncancerous HEK293T cells. (b) Biodistributions of CCHNs in different organs from nude mice after treated with different time. (c) Histological tissue sections of heart, kidneys, liver, lungs, and spleen 120 h post-treatment with CCHNs at 1 mg/mL. The scale bar = 100 µm. While the CCHNs-based nanocarriers have demonstrated high therapeutic efficacy against cancer cells in vitro, it is necessary to examine their toxicity and biodistribution on normal cells and healthy animal. Figure 6a shows that more than 97% of HEK293T cells are still alive after receiving 72 h treatment with CCHNs of different concentrations, indicating that CCHNs are


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safe to the normal cells at concentrations up to 100 µg/mL. To look into biodistribution of CCHNs in animal body, the fluorescence intensity of major organs from tumor-free mice receiving the injection of CCHNs was monitored. Results in Figure 6b indicates that liver was the dominant organ in accumulation of CCHNs at 24 h post-injection because of reticuloendothelial system uptake, followed by the kidneys, spleen, lungs, and heart. At 96 h post-injection, less than 20% of initially-accumulated CCHNs remained in liver and minimal amounts of CCHNs were found in heart, kidney, lung, and spleen. In order to test in vivo toxicity of CCHNs on clean organs of healthy animal, histological analysis on tissues of heart, kidneys, liver, lungs and spleen of mice 120 h post-treatment with CCHNs at 1 mg/mL via tail vein injection was performed. Results in Figure 6c show that the tissues from mice receiving treatment of CCHNs are similar to those from non-injected mice, which reveals the nontoxicity to mice and excellent biocompatibility of CCHNs. To examine in vivo drug carrier function of CCHNs for cancer therapy, tumor bearing mice (implanted with 4T1 cells) were intravenously injected with empty CCHNs and DOX-loaded CCHNs, respectively. Tumors were then subjected or not subjected to 5 min exposure to NIR irradiation at 24 h after each dose of injection. Four random groups of tumor-bearing mice with four in each group were treated four times for every three days with PBS (control), CCHNs plus NIR irradiation (photothermal treatment only), DOX-loaded CCHNs without NIR irradiation (chemotherapy only), and DOX-loaded CCHNs plus NIR irradiation (photothermo-chemo combined therapy), respectively. Tumor sizes in mice were measured every three days for 18 days post-treatment. The picture in Figure 7a demonstrates that the in vivo bright NIR fluorescence originated from the injected CCHNs can light up the tumor site of treated mouse when a NIR laser of 710 nm was applied on day 18. The PL spectrum of CCHNs confirmed their


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NIR emissions with a maximum emission located at 806 nm when λex = 710 nm (Figure S9). The PL quantum yield of CCHNs excited at 710 nm was determined to be 1.6%, using Cy5.5 as a standard. The bright NIR fluorescence emitted on the tumor site of the mouse treated with CCHNs provides a promise for CCHNs to be used for NIR imaging-guided chemotherapy.

Figure 7. In vivo tumor therapy of DOX-loaded CCHNs. (a) NIR image of tumor site from a mouse intravenously treated with DOX-loaded CCHNs at day 18 post-treatment. (b) Tumor growth curves of mice after receiving different treatments including control, photothermal therapy, chemotherapy, and combined photothermo-chemo therapy. (c) Mass and picture of tumors obtained from mice on day 18 after receiving different treatments. (d) Monitoring curves


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on the body weight of mice over the whole treatment period. (e) H&E-stained sections of tumor obtained from mice after different treatments including control, photothermal therapy, chemotherapy, and combined therapy. The tumor development of the mice experiencing different treatment strategies was investigated. As demonstrated in Figure 7b and Figure S10, tumors in mice receiving treatment with PBS solution (control) grew rapidly. The NIR irradiation alone did not show observable effect on tumor growth. In addition, tumors in mice injected by CCHNs continue to grow rapidly no matter whether NIR irradiation was applied or not, indicating that CCHNs alone have no function to inhibit tumor growth. Tumors in mice received chemotherapy with DOX-loaded CCHNs show a dramatically reduced tumor growth rate. Notably, the chemo-photothermal combined treatment with addition of 5 min NIR exposure to DOX-loaded CCHNs at 24 h after each new dose further inhibits tumor growth significantly. Figure 7c shows the picture and mass of tumors harvested from mice receiving different treatments ended on day 18. Obviously, the therapeutic outcome achieved by chemo-photothermal combinatory therapy is much better than additive result from separated photothermal therapy and chemotherapy based on the tumor growth reduction ratio, suggesting a synergistic effect by the combinatory chemo-photothermal therapy. It should be mentioned that no weight loss was observed in this group of mice as compared to other groups (Figure 7d). The histological analysis on the tumor tissues (Figure 7e) also show that tumors cells from control group and photothermal therapy group are largely not damaged with normal membranes and nuclear structures. In contrary, most of the tumor cells from the chemotherapy treated mice, especially those receiving the chemo-photothermal combined treatment, were severely damaged with loss of nuclei. Such high therapeutic efficacy from combined treatments should be attributed to excellent tumor permeability, fast cell


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internalization, and enhanced DOX release rate of CCHNs in acidic microenvironments of tumor sites and under NIR irradiation.65 4. CONCLUSIONS A multifunctional nanocarrier for simultaneous NIR imaging and combined photothermalchemo therapy has been developed based on biocompatible CCHNs. The CCHNs can be prepared in aqueous phase through the nonsolvent-induced colloidal nanoparticle formation of the hydrogen bonded EDTA-chitosan-CDs complexes at suitable pH values followed by in situ crosslinking of chitosan chains. While the hydrophilic chitosan network endows excellent stability and porous space for high loading capacity of CCHNs, the CDs embedded in CCHNs provide bright and stable fluorescence and NIR photothermal effects. The small CCHNs particles can permeate into tumor site, enter into cells quickly, and emit bright fluorescence to highlight the cells or tumor tissues when visible or NIR laser was applied. The CCHNs can release the preloaded drug efficiently triggered by both endogenous (acidic medium) and exogenous (NIR light) activations to kill 4T1 cancer cells. In vivo studies confirmed that CCHNs are safe to normal tissues and manifest high therapeutic efficacy as DOX nanocarrier against implanted 4T1 tumors on mice. The combined chemo-photothermal treatments with DOX-loaded CCHNs subjected to NIR irradiation exhibit the best therapeutic efficacy benefited from the NIR photothermal effects of CDs in CCHNs. ASSOCIATED CONTENT Supporting Information The supporting information is available free of charge via the Internet at http://pubs.acs.org. Hydrodynamic size distribution, UV-Vis absorption spectrum, photostability, NIR-excited PL spectrum of CCHNs, Z-Scanning confocal fluorescence images of CCHNs-labeled cells, UV–


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Visible absorption spectra of CCHNs, DOX, and DOX-loaded CCHNs, NIR photothermal conversion curves of CDs in water, cell viability in culture medium with/without an exposure to NIR light, in vivo tumor growth curves treated by drug-free CCHNs with or with no NIR irradiation. AUTHOR INFORMATION *E-mail address: [email protected] (S. Zhou) Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. ACKNOWLEDGEMENTS The authors gratefully acknowledge the financial support from the PSC-CUNY Research Award and American Diabetes Association (Basic Science Award 1-12-BS-243). REFERENCES (1) Siegel, R. L.; Miller, K. D.; Jemal, A. Cancer Statistics, 2015. CA-Cancer J. Clin. 2015, 65, 5-29. (2) MacDonald, V. Chemotherapy: Managing Side Effects and Safe Handling. Can. Vet. J. 2009, 50, 665-668. (3) Burgess, D. J. Therapy: Enhancing Efficacy by Reducing Side Effects. Nat. Rev. Cancer 2012, 12, 377-377. (4) Mura, S.; Nicolas, J.; Couvreur, P. Stimuli-Responsive Nanocarriers for Drug Delivery. Nat. Mater. 2013, 12, 991-1003. (5) Caldorera-Moore, M. E.; Liechty, W. B.; Peppas, N. A. Responsive Theranostic Systems: Integration of Diagnostic Imaging Agents and Responsive Controlled Release Drug Delivery Carriers. Acc. Chem. Res. 2011, 44, 1061-1070. (6) Wang, H.; Sun, Y.; Yi, J.; Fu, J.; Di, J.; del Carmen Alonso, A.; Zhou, S. Fluorescent Porous Carbon Nanocapsules for Two-Photon Imaging, NIR/pH Dual-Responsive Drug Carrier, and Photothermal Therapy. Biomaterials 2015, 53, 117-126. (7) Zhao, Y.; Luo, Z.; Li, M.; Qu, Q.; Ma, X.; Yu, S.-H.; Zhao, Y. A Preloaded Amorphous Calcium Carbonate/Doxorubicin@Silica Nanoreactor for pH-Responsive Delivery of an Anticancer Drug. Angew. Chem. Int. Ed. 2015, 54 (3), 919-922.


25


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 26 of 30

(8) Sur, S.; Fries, A. C.; Kinzler, K. W.; Zhou, S.; Vogelstein, B. Remote Loading of Preencapsulated Drugs into Stealth Liposomes. Proc. Natl. Acad. Sci. U. S. A. 2014, 111, 22832288. (9) Meng, H.; Wang, M.; Liu, H.; Liu, X.; Situ, A.; Wu, B.; Ji, Z.; Chang, C. H.; Nel, A. E. Use of a Lipid-Coated Mesoporous Silica Nanoparticle Platform for Synergistic Gemcitabine and Paclitaxel Delivery to Human Pancreatic Cancer in Mice. ACS Nano 2015, 9, 3540-3557. (10) Wang, Y.; Bansal, V.; Zelikin, A. N.; Caruso, F. Templated Synthesis of SingleComponent Polymer Capsules and Their Application in Drug Delivery. Nano Lett. 2008, 8, 17411745. (11) Wang, H.; Yi, J.; Mukherjee, S.; Banerjee, P.; Zhou, S. Magnetic/NIR-Thermally Responsive Hybrid Nanogels for Optical Temperature Sensing, Tumor Cell Imaging and Triggered Drug Release. Nanoscale 2014, 6, 13001-13011. (12) Liu, Z.; Fan, A. C.; Rakhra, K.; Sherlock, S.; Goodwin, A.; Chen, X.; Yang, Q.; Felsher, D. W.; Dai, H. Supramolecular Stacking of Doxorubicin on Carbon Nanotubes for in vivo Cancer Therapy. Angew. Chem. Int. Et. 2009, 48, 7668-7672. (13) Mu, Q.; Jeon, M.; Hsiao, M. H.; Patton, V. K.; Wang, K.; Press, O. W.; Zhang, M. Stable and Efficient Paclitaxel Nanoparticles for Targeted Glioblastoma Therapy. Adv. Healthc. Mater. 2015, 4, 1236-1245. (14) Liu, Z.; Chen, K.; Davis, C.; Sherlock, S.; Cao, Q.; Chen, X.; Dai, H. Drug Delivery with Carbon Nanotubes for in vivo Cancer Treatment. Cancer Res. 2008, 68, 6652-6660. (15) Mo, R.; Jiang, T.; DiSanto, R.; Tai, W.; Gu, Z. ATP-Triggered Anticancer Drug Delivery. Nat. Commun. 2014, 5, 3364. (16) Li, N.; Yu, Z.; Pan, W.; Han, Y.; Zhang, T.; Tang, B. A Near-Infrared Light-Triggered Nanocarrier with Reversible DNA Valves for Intracellular Controlled Release. Adv. Funct. Mater. 2013, 23, 2255-2262. (17) Wang, D.; Guo, Z.; Zhou, J.; Chen, J.; Zhao, G.; Chen, R.; He, M.; Liu, Z.; Wang, H.; Chen, Q. Novel Mn3[Co(CN)6]2@SiO2@Ag Core–Shell Nanocube: Enhanced Two-Photon Fluorescence and Magnetic Resonance Dual-Modal Imaging-Guided Photothermal and Chemotherapy. Small 2015, 11, 5956-5967. (18) Della Rocca, J.; Liu, D.; Lin, W. Nanoscale Metal–Organic Frameworks for Biomedical Imaging and Drug Delivery. Acc. Chem. Res. 2011, 44, 957-968. (19) Wang, D.; Zhou, J.; Chen, R.; Shi, R.; Zhao, G.; Xia, G.; Li, R.; Liu, Z.; Tian, J.; Wang, H.; Guo, Z.; Wang, H.; Chen, Q. Controllable Synthesis of Dual-MOFs Nanostructures for pHResponsive Artemisinin Delivery, Magnetic Resonance and Optical Dual-Model ImagingGuided Chemo/photothermal Combinational Cancer Therapy. Biomaterials 2016, 100, 27-40. (20) Das, M.; Zhang, H.; Kumacheva, E. Microgels: Old Materials with New Applications. Annu. Rev. Mater. Res. 2006, 36, 117-142. (21) Doring, A.; Birnbaum, W.; Kuckling, D. Responsive Hydrogels--Structurally and Dimensionally Optimized Smart Frameworks for Applications in Catalysis, Micro-system Technology and Material Science. Chem. Soc. Rev. 2013, 42, 7391-7420. (22) Molina, M.; Asadian-Birjand, M.; Balach, J.; Bergueiro, J.; Miceli, E.; Calderon, M. Stimuli-Responsive Nanogel Composites and Their Application in Nanomedicine. Chem. Soc. Rev. 2015, 44, 6161-6186. (23) Feng, L.; Gao, M.; Tao, D.; Chen, Q.; Wang, H.; Dong, Z.; Chen, M.; Liu, Z. CisplatinProdrug-Constructed Liposomes as a Versatile Theranostic Nanoplatform for Bimodal Imaging Guided Combination Cancer Therapy. Adv. Funct. Mater. 2016, 26, 2207-2217.


26

Page 27 of 30

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(24) Song, X.-R.; Wang, X.; Yu, S.-X.; Cao, J.; Li, S.-H.; Li, J.; Liu, G.; Yang, H.-H.; Chen, X. Co9Se8 Nanoplates as a New Theranostic Platform for Photoacoustic/Magnetic Resonance DualModal-Imaging-Guided Chemo-Photothermal Combination Therapy. Adv. Mater. 2015, 27, 3285-3291. (25) Chang, Y. T.; Liao, P. Y.; Sheu, H. S.; Tseng, Y. J.; Cheng, F. Y.; Yeh, C. S. Near-Infrared Light-Responsive Intracellular Drug and siRNA Release using Au Nanoensembles with Oligonucleotide-Capped Silica Shell. Adv. Mater. 2012, 24, 3309-3314. (26) Bhattarai, N.; Gunn, J.; Zhang, M. Chitosan-Based Hydrogels for Controlled, Localized Drug Delivery. Adv. Drug Deliv. Rev. 2010, 62, 83-99. (27) Ma, Y.; Dai, J.; Wu, L.; Fang, G.; Guo, Z. Enhanced Anti-Ultraviolet, Anti-Fouling and Anti-Bacterial Polyelectrolyte Membrane of Polystyrene Grafted with Trimethyl Quaternary Ammonium Salt Modified Lignin. Polymer 2017, 114, 113-121. (28) Builders, P. F.; Arhewoh, M. I. Pharmaceutical Applications of Native Starch in Conventional Drug Delivery. Starch 2016, 68, 864-873. (29) Chen, C. K.; Wang, Q.; Jones, C. H.; Yu, Y.; Zhang, H.; Law, W. C.; Lai, C. K.; Zeng, Q.; Prasad, P. N.; Pfeifer, B. A.; Cheng, C. Synthesis of pH-Responsive Chitosan Nanocapsules for The Controlled Delivery of Doxorubicin. Langmuir 2014, 30, 4111-4119. (30) Wei, W.; Yuan, L.; Hu, G.; Wang, L.-Y.; Wu, J.; Hu, X.; Su, Z.-G.; Ma, G.-H. Monodisperse Chitosan Microspheres with Interesting Structures for Protein Drug Delivery. Adv. Mater. 2008, 20, 2292-2296. (31) Wang, C.; Ravi, S.; Garapati, U. S.; Das, M.; Howell, M.; MallelaMallela, J.; Alwarapan, S.; Mohapatra, S. S.; Mohapatra, S. Multifunctional Chitosan Magnetic-Graphene (CMG) Nanoparticles: a Theranostic Platform for Tumor-targeted Co-delivery of Drugs, Genes and MRI Contrast Agents. J. Mater. Chem. B 2013, 1, 4396-4405. (32) Guliyeva, U.; Oner, F.; Ozsoy, S.; Haziroglu, R. Chitosan Microparticles Containing Plasmid DNA as Potential Oral Gene Delivery System. Eur. J. Pharm. Biopharm. 2006, 62, 1725. (33) Guo, L.; Yan, D. D.; Yang, D.; Li, Y.; Wang, X.; Zalewski, O.; Yan, B.; Lu, W. Combinatorial Photothermal and Immuno Cancer Therapy Using Chitosan-Coated Hollow Copper Sulfide Nanoparticles. ACS Nano 2014, 8, 5670-5681. (34) Chen, R.; Zheng, X.; Qian, H.; Wang, X.; Wang, J.; Jiang, X. Combined Near-IR Photothermal Therapy and Chemotherapy using Gold-Nanorod/Chitosan Hybrid Nanospheres to Enhance The Antitumor Effect. Biomater. Sci. 2013, 1, 285-293. (35) Agnihotri, S. A.; Mallikarjuna, N. N.; Aminabhavi, T. M. Recent Advances on Chitosanbased Micro- and Nanoparticles in Drug Delivery. J. Control. Release 2004, 100, 5-28. (36) Zhang, W.; Gilstrap, K.; Wu, L.; K. C, R. B.; Moss, M. A.; Wang, Q.; Lu, X.; He, X. Synthesis and Characterization of Thermally Responsive Pluronic F127−Chitosan Nanocapsules for Controlled Release and Intracellular Delivery of Small Molecules. ACS Nano 2010, 4, 67476759. (37) Wang, H.; Di, J.; Sun, Y.; Fu, J.; Wei, Z.; Matsui, H.; del C. Alonso, A.; Zhou, S. Biocompatible PEG-Chitosan@Carbon Dots Hybrid Nanogels for Two-Photon Fluorescence Imaging, Near-Infrared Light/pH Dual-Responsive Drug Carrier, and Synergistic Therapy. Adv. Funct. Mater. 2015, 25, 5537-5547. (38) Shao, L.; Chang, X.; Zhang, Y.; Huang, Y.; Yao, Y.; Guo, Z. Graphene Oxide CrossLinked Chitosan Nanocomposite Membrane. Appl. Surf. Sci. 2013, 280, 989-992.


27


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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(39) Jung, N.; Kim, S. M.; Kang, D. H.; Chung, D. Y.; Kang, Y. S.; Chung, Y.-H.; Choi, Y. W.; Pang, C.; Suh, K.-Y.; Sung, Y.-E. High-Performance Hybrid Catalyst with Selectively Functionalized Carbon by Temperature-Directed Switchable Polymer. Chem. Mater. 2013, 25, 1526-1532. (40) Xie, Z.; Wang, F.; Liu, C. Y. Organic-Inorganic Hybrid Functional Carbon Dot Gel Glasses. Adv. Mater. 2012, 24, 1716-1721. (41) Ge, J.; Jia, Q.; Liu, W.; Guo, L.; Liu, Q.; Lan, M.; Zhang, H.; Meng, X.; Wang, P. RedEmissive Carbon Dots for Fluorescent, Photoacoustic, and Thermal Theranostics in Living Mice. Adv. Mater. 2015, 27, 4169-4177. (42) Lim, S. Y.; Shen, W.; Gao, Z. Carbon Quantum Dots and Their Applications. Chem. Soc. Rev. 2015, 44, 362-381. (43) Li, X.; Rui, M.; Song, J.; Shen, Z.; Zeng, H. Carbon and Graphene Quantum Dots for Optoelectronic and Energy Devices: A Review. Adv. Funct. Mater. 2015, 25, 4929-4947. (44) Wang, H.; Revia, R.; Wang, K.; Kant, R. J.; Mu, Q.; Gai, Z.; Hong, K.; Zhang, M. Paramagnetic Properties of Metal-Free Boron-Doped Graphene Quantum Dots and Their Application for Safe Magnetic Resonance Imaging. Adv. Mater. 2017, 29, 1605416. (45) Jin, H.; Chen, Q.; Chen, Z.; Hu, Y.; Zhang, J. Multi-LeapMotion Sensor Based Demonstration for Robotic Refine Tabletop Object Manipulation Task. CAAI Trans. Intell. Technol. 2016, 1, 104-113. (46) Alippi, C. A Unique Timely Moment for Embedding Intelligence in Applications. CAAI Trans. Intell. Technol. 2016, 1, 1-3. (47) Yang, S.-T.; Wang, X.; Wang, H.; Lu, F.; Luo, P. G.; Cao, L.; Meziani, M. J.; Liu, J.-H.; Liu, Y.; Chen, M.; Huang, Y.; Sun, Y.-P. Carbon Dots as Nontoxic and High-Performance Fluorescence Imaging Agents. J. Phys. Chem. C 2009, 113, 18110-18114. (48) Sk, M. A.; Ananthanarayanan, A.; Huang, L.; Lim, K. H.; Chen, P., Revealing The Tunable Photoluminescence Properties of Graphene Quantum Dots. J. Mater. Chem. C 2014, 2, 6954-6960. (49) Wang, H.; Zhuang, J.; Velado, D.; Wei, Z.; Matsui, H.; Zhou, S. Near-Infrared- and Visible-Light-Enhanced Metal-Free Catalytic Degradation of Organic Pollutants over CarbonDot-Based Carbocatalysts Synthesized from Biomass. ACS Appl. Mater. Interfaces 2015, 7, 27703-27712. (50) Wang, H.; Wei, Z.; Matsui, H.; Zhou, S. Fe3O4/Carbon Quantum Dots Hybrid Nanoflowers for Highly Active and Recyclable Visible-Light Driven Photocatalyst. J. Mater. Chem. A 2014, 2, 15740-15745. (51) Wang, F.; Pang, S.; Wang, L.; Li, Q.; Kreiter, M.; Liu, C.-y. One-Step Synthesis of Highly Luminescent Carbon Dots in Noncoordinating Solvents. Chem. Mater. 2010, 22, 4528-4530. (52) Cao, L.; Wang, X.; Meziani, M. J.; Lu, F.; Wang, H.; Luo, P. G.; Lin, Y.; Harruff, B. A.; Veca, L. M.; Murray, D.; Xie, S.-Y.; Sun, Y.-P. Carbon Dots for Multiphoton Bioimaging. J. Am. Chem. Soc. 2007, 129, 11318-11319. (53) Parmesan, C.; Yohe, G., A Globally Coherent Fingerprint of Climate Change Impacts across Natural Systems. Nature 2003, 421, 37-42. (54) Tao, H.; Yang, K.; Ma, Z.; Wan, J.; Zhang, Y.; Kang, Z.; Liu, Z. In vivo NIR Fluorescence Imaging, Biodistribution, and Toxicology of Photoluminescent Carbon Dots Produced from Carbon Nanotubes and Graphite. Small 2012, 8, 281-290.


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(55) Saraiva, C.; Praça, C.; Ferreira, R.; Santos, T.; Ferreira, L.; Bernardino, L. NanoparticleMediated Brain Drug Delivery: Overcoming Blood–Brain Barrier to Treat Neurodegenerative Diseases. J. Control. Release 2016, 235, 34-47. (56) Ma, M.; Chen, H.; Chen, Y.; Wang, X.; Chen, F.; Cui, X.; Shi, J. Au Capped Magnetic Core/Mesoporous Silica Shell Nanoparticles for Combined Photothermo-/chemo-therapy and Multimodal Imaging. Biomaterials 2012, 33, 989-998. (57) Sun, X.; Liu, Z.; Welsher, K.; Robinson, J.; Goodwin, A.; Zaric, S.; Dai, H. NanoGraphene Oxide for Cellular Imaging and Drug Delivery. Nano Res. 2008, 1, 203-212. (58) Tian, Q.; Jiang, F.; Zou, R.; Liu, Q.; Chen, Z.; Zhu, M.; Yang, S.; Wang, J.; Wang, J.; Hu, J. Hydrophilic Cu9S5 Nanocrystals: A Photothermal Agent with a 25.7% Heat Conversion Efficiency for Photothermal Ablation of Cancer Cells in Vivo. ACS Nano 2011, 5, 9761-9771. (59) Chen, J.; Guo, Z.; Wang, H. B.; Gong, M.; Kong, X. K.; Xia, P.; Chen, Q. W. Multifunctional Fe3O4@C@Ag Hybrid Nanoparticles as Dual Modal Imaging Probes and NearInfrared Light-Responsive Drug Delivery Platform. Biomaterials 2013, 34, 571-581. (60) Maciel, D.; Figueira, P.; Xiao, S.; Hu, D.; Shi, X.; Rodrigues, J.; Tomás, H.; Li, Y. RedoxResponsive Alginate Nanogels with Enhanced Anticancer Cytotoxicity. Biomacromolecules 2013, 14, 3140-3146. (61) Kato, Y.; Ozawa, S.; Miyamoto, C.; Maehata, Y.; Suzuki, A.; Maeda, T.; Baba, Y. Acidic Extracellular Microenvironment and Cancer. Cancer Cell Int. 2013, 13, 89. (62) Wu, W.; Shen, J.; Banerjee, P.; Zhou, S. Water-Dispersible Multifunctional Hybrid Nanogels for Combined Curcumin and Photothermal Therapy. Biomaterials 2011, 32, 598-609. (63) Hahn, G. M.; Braun, J.; Har-Kedar, I. Thermochemotherapy: Synergism between Hyperthermia (42-43 degrees) and Adriamycin (of bleomycin) in Mammalian Cell Inactivation. Proc. Natl. Acad. Sci. U. S. A. 1975, 72, 937-940. (64) Wang, C.; Xu, H.; Liang, C.; Liu, Y.; Li, Z.; Yang, G.; Cheng, L.; Li, Y.; Liu, Z. Iron Oxide @ Polypyrrole Nanoparticles as a Multifunctional Drug Carrier for Remotely Controlled Cancer Therapy with Synergistic Antitumor Effect. ACS Nano 2013, 7, 6782-6795. (65) Wang, H.; Wang, K.; Tian, B.; Revia, R.; Mu, Q.; Jeon, M.; Chang, F.-C.; Zhang, M. Preloading of Hydrophobic Anticancer Drug into Multifunctional Nanocarrier for Multimodal Imaging, NIR-Responsive Drug Release, and Synergistic Therapy. Small 2016, 12, 6388-6397.


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