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Aspirin-Based Carbon Dots, a Good Biocompatibility of Material Applied for Bioimaging and Anti-Inflammation Xiaowei Xu, Kai Zhang, Liang Zhao, Chen Li, Wenhuan Bu, Yuqin Shen, Zhongyi Gu, Bei Chang, Changyu Zheng, Chongtao Lin, Hongchen Sun, and Bai Yang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b12252 • Publication Date (Web): 09 Nov 2016 Downloaded from http://pubs.acs.org on November 12, 2016

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ACS Applied Materials & Interfaces

Aspirin-Based Carbon Dots, a Good Biocompatibility of Material Applied for Bioimaging and Anti-Inflammation Xiaowei Xu,¶,§,‡,† Kai Zhang,‡ Liang Zhao,¶ Chen Li,# Wenhuan Bu,¶ Yuqin Shen,§ Zhongyi Gu,§ Bei Chang,¶ Changyu Zheng,ǁ Chongtao Lin,§,* Hongchen Sun,¶,†,* and Bai Yang‡,* ¶

Department of Oral Pathology, School and Hospital of Stomatology, Jilin University,

Changchun 130021, P. R. China §

Department of Periodontology, School and Hospital of Stomatology, Jilin University,

Changchun 130021, P. R. China †

Jilin Provincial Key Laboratory of Tooth Development and Bone Remodeling, Jilin University,

Changchun 130021, P. R. China ‡

State Key Laboratory of Supramolecular Structure and Materials, College of Chemistry, Jilin

University, Changchun 130012, P. R. China #

Department of Oral Medicine, School and Hospital of Stomatology, Jilin University, Changchun

130021, P. R. China ǁ

Molecular Physiology and Therapeutics Branch, National Institute of Dental and Craniofacial

Research, National Institutes of Health, Bethesda, MD, USA *Address correspondence to [email protected], [email protected], [email protected]

1 ACS Paragon Plus Environment


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Abstract The emerging photoluminescent carbon-based nanomaterials are promising in various fields besides cell imaging and carrier transport. Carbon nanomaterials with specific biological functions, however, are rarely investigated. Aspirin is a very common anti-inflammatory medication to relieve aches and pains. In this study, we have tried to create a carbon nanoparticle with aspirin and we expect that this new carbon nanoparticle will have both anti-inflammatory and fluorescent biomarker functions. Fluorescent aspirin-based carbon dots (FACD) were synthesized by condensing aspirin and hydrazine through a one-step microwave-assisted method. Imaging data demonstrated that FACD efficiently entered into human cervical carcinoma and mouse monocyte macrophage cells in vitro with low cell toxicity. Results from quantitative polymerase chain reaction and histological analysis indicated that FACD possessed effective anti-inflammatory effects in vitro and in vivo compared to aspirin only. Hematology, serum biochemistry and histology results suggested that FACD also had no significant toxicity in vivo. Our results clearly demonstrate that FACD has dual functions; cellular imaging/bioimaging and anti-inflammation, and suggest that FACD have great potential in future clinical applications.

KEYWORDS: aspirin, carbon dots, bioimaging, anti-inflammation, biocompatibility


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INTRODUCTION Carbon dots (CDs) were discovered through purifying carbon nanotubes from arc-discharge soot in 2004.1 CDs are less than 10 nm of carbon particles with fluorescence property.2 They have good solubility, high photoluminescence quantum yield (PLQY), excellent resistance to photobleaching and low cytotoxicity.3-7 It has been reported that in vivo administration of CDs had no significant effects on blood biochemistry or hematology analysis, and caused no histopathological abnormality on the main organs.8 The CDs could be excreted from the body efficiently and rapidly after injection via intravenous injection, intramuscular injection or subcutaneous injection.9 Little or no toxicity, high PLQY and good biocompatibility of CDs lead to widely possible clinical applications, such as bioimaging, photothermal therapy and drug or gene delivery.10-13 CDs can be synthesized by two major methods: top-down cutting from different carbon sources and bottom-up carbonization methods from polymer and small molecule.14-18 These two methods can only prepare CDs with solo photoluminescence (PL) property, which needs further modification to endow biofunction. For the bottom-up carbonization method, three groups of materials, chemical reagents, raw resources and biologically active substances, are used. Chemical reagents include citric acid, glycerol, glycol, glucose, sucrose and other similar vehicles.19-22 Raw resources, such as orange juice, banana juice and watermelon peel, are used for CDs synthesis with the merits of facileness and environmental friendliness.21,23,24 Lastly, biologically active substances, such as protein-based biopolymer Bombyx mori silk,25 DNA26 and similar materials, have been used as carbon resources for CDs synthesis. The biological activities, however, can disappear during the process of hydrothermal treatment at high



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temperature. It is a fascinating challenge to keep the biological activities of any drug’s molecule during CDs synthesis. Aspirin, a representative of non-steroidal anti-inflammatory drugs, has a more than one hundred year history with a side-effect of gastric damage and poor solubility in the physiological environment.27-29 Recently, some studies also suggest that aspirin has the possibility to decrease the risk of cancer and heart disease.30-34 Therefore, it will still be useful to alleviate gastric damage and improve solubility for efficient clinical applications of aspirin. CDs are usually water-soluble, and easier to circulate in vivo.35 It was reported that aspirin could covalently bond with the cyclooxygenase (COX) family of enzymes and consequently decrease the production of prostaglandin E2 (PGE2) in order to achieve its anti-inflammatory effect.36-38 The acetyl of aspirin irreversibly acetylates a serine residue of COX-1 or COX-2, thereby blocking the approach of fatty acid substrate to the active site for its oxygenation,39-41 which makes it possible to retain aspirin’s anti-inflammatory effects without the involvement of the ester of aspirin in the reaction. Moreover, synthesized CDs generally possess more functional groups than the original reagents, and these groups may give rise to synergistic effects in anti-inflammation. In this study, our goal is to create fluorescent aspirin-based carbon dots (FACD) and retain all aspirin bioactivities with lower or no side-effect of gastric damage and good solubility. We used a “one-step” microwave-assisted strategy which condensed aspirin and hydrazine to synthesize FACD, and further characterized their physicochemical properties by X-ray photoelectron spectroscopy (XPS), Fourier transform infrared spectroscopy (FTIR), quantitative polymerase chain reaction (QPCR), and histology. Interestingly, our data demonstrated that the FACD not only retained anti-inflammatory effects, but also had stronger anti-inflammatory effects than that of aspirin alone. Therefore, our results suggest that FACD is an efficient formula to deliver


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aspirin with good solubility, bio-imaging and anti-inflammation, and possesses good potential for use in clinical practice.



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RESULTS AND DISCUSSION Synthesis and Characterization of FACD. In this study, FACD was synthesized by a one-step microwave-assisted strategy through condensation of aspirin and hydrazine carbonization (Figure 1a). Hydrazine assists the aspirin to resolve and disperse in water due to ionization of -COOH into -COO-. After microwave treatment, FACD was obtained and re-dispersed in water. To obtain more uniform-sized FACD, the as-prepared FACD solutions were first centrifuged to remove large aggregates, and further dialyzed in deionized water using a membrane for 2 days to get rid of unreacted small molecules, such as highly toxic hydrazine. After purification, morphology and structure of the FACD were characterized by transmission electron microscopy (TEM) and X-ray powder diffraction (XRD). Figure 1b is TEM image which shows a uniform dispersion without apparent aggregation. Sizes of FACD particles were 2-5.5 nm in diameter (Figure 1d). High-resolution a TEM (HRTEM) image demonstrated that FACD formed well-resolved lattice planes with ~ 0.34 nm spacing between two adjacent lattices, consisting with the (002) crystallographic facet of graphite (Figure 1c). The lattice parameters of FACD fitted well to the XRD pattern (Figure 1e), which was consistent with previous reports about CDs. 22 FTIR spectra was adopted to further analyze the chemical structure of FACD (Figure 2). Our data showed that the FTIR spectrum of aspirin exhibited C=O stretching vibration of acetyl and carboxyl at 1755 cm-1 and 1690 cm-1, and N2H4 exhibited N-H bending vibration at 1618 cm-1. After forming FACD, the C=O of acetyl was partially preserved through its FTIR spectrum. Meanwhile, the C=O stretching vibration of carboxyl shifted to 1623 cm-1, becoming consistent with the position of the amide I band. The N-H bending vibration shifted to 1550 cm-1, which is consistent with the amide II band of secondary acyclic hydrazide. A new band appeared at 1295 cm-1, which was assigned to the C-N stretching vibration of hydrazide.


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These results demonstrate FACD is passivated by N2H4 through the amidation reaction, and the acetyl group preserves its roles in reducing fever and inflammation. Next, XPS analysis of C 1s, N 1s and O 1s spectra (Figure 3a-c) confirmed formation of hydrazide and the reservation of acetyl. Figure 3b presents the C 1s spectrum of FACD. The C 1s spectrum could be fitted into four peaks: O=C−O peak at 288.5 eV, O=C−N peak at 287.8 eV, C−O peak at 286.3 eV and C−C peak at 284.7 eV, whereas the C 1s spectrum of aspirin was only fitted into three peaks without the O=C−N peak at 287.8 eV (Figure 3d). These results strongly support the amidation of the −COOH of aspirin. The O=C−O peak in FACD could belong to the remaining carboxyl and/or acetyl. However, it could not be concluded that acetyl exists in FACD even if there was a peak of C-O (286.3 eV), because C-O could have originated from the hydrolysis of aspirin into salicylic acid. Note that the phenolic hydroxy group is easily oxidized into quinine by oxygen during the microwave process resulting in loss of the C-O, although aspirin could be hydrolyzed into a salicylic acid containing a phenolic hydroxy group (i.e. C−O). To further prove the absence of the phenolic hydroxy group in our FACD, the color reaction between ferric chloride and phenols was applied (Figure S1). Furthermore, the combination of the peaks at 288.5 and 286.3 eV demonstrated the existence of acetyl in FACD. In addition, control experiments were performed by replacing aspirin with salicylic acid to further prove this conclusion. Figure S2a-e show the comparisons between aspirin and salicylic acid in synthesizing FACD. Equimolar aspirin and salicylic acid are added into water, respectively, followed by the addition of hydrazine. Then, these two mixtures are heated by microwave oven for 8 min separately and produced Figure S2a and b products. After adding water, the apparent colors of these solutions are obviously different (Figure S2c). Through further purification, it is found that the aspirin-based solution is highly fluorescent, while the salicylic acid-based solution



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is not (Figure S2d and e). N 1s XPS spectrum of FACD was fitted into three peaks, −NO3- peak at 406.9 eV, −NH3+ peak at 401.1 eV and −NH− peak at 399.4 eV (Figure 3a). Likewise, the O 1s XPS spectrum of FACD was fitted into three peaks. The O=C−N peak at 531.7 eV confirmed the existence of the amidation process (Figure 3c). Therefore, all these data indicate that FACD is passivated by N2H4 through the amidation reaction and possesses intense photoluminescence. PL Properties and Stability of FACD. Figure 4 is the relevant optical analysis of FACD. UV-vis absorption spectrum of aspirin in alkaline solution showed that there were two peaks, π-π* peak at 230 nm and n-π* peak at 295 nm (Figure 4a). After microwave processing, in the UV-vis absorption spectrum of FACD, there was one more absorption shoulder at 360 nm assigned to surface state of FACD, implying that the PL originated from the hybrid structure between the surface groups and the carbon core.42 The peak at ~289 nm indicated a functional group, like C=O. In the PL spectra of FACD, the excitation spectrum was focused on 360 nm, which was consistent with the absorption peak of the surface state (Figure 4a and b). The optimal emission wavelength of FACD was located at 432 nm (360 nm excitation). The inserts of Figure 4b are photographs of aqueous solutions of FACD taken under sunlight and UV light. The bright blue emission color was clearly observable under UV light. Full width at half maximum (FWHM) of FACD was ~ 60 nm. PLQY of FACD was measured to be 23% using quinine sulfate as a reference,

which

is

comparable

to

reports

in

previous

literature.2,6,7

Furthermore,

excitation-dependent PL behavior could be observed just like other fluorescent carbon materials, but not obviously (Figure S3). Generally, the excitation-dependent PL behavior is understandable in terms of two or more PL centers induced by the complicated structures of FACD.43 Thus, our data suggest that FACD possesses two PL centers, a carbon core and the surface state that is the predominate center.43


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To further understand the origin of FACD PL, the time-correlated single-photon counting (TCSPC) assay was performed at 375 nm excitation (Figure 4c). Resultant average lifetime was 3.82 ns and contained two lifetime components with different ratios, 0.68 ns (~14.54 %) and 4.36 ns (~85.46 %). These further demonstrated that there were two PL centers in FACD.43 The PL center with 0.68 ns of lifetime was responsible for the excitation-dependent PL behavior, which only took small proportions in the whole lifetime, leading to the unobvious excitation-dependent behavior. However, the second PL center, a surface state center, was an obvious steady emission. In the experiment, we found that aspirin in pure water possessed unobvious PL emission owing to its low solubility, whilst in strong alkaline solution (pH>14) it could improve solubility and PL emission of aspirin.44 Thus, we compared PL properties of aspirin with those of FACD in order to further understand the PL mechanism of FACD. Results showed that aspirin and FACD were different. PL emission wavelength of aspirin in hydrazine was at 410 nm, and its PLQYs were 6% (Figure S4a). In addition, nanosecond fluorescence lifetime of aspirin was 2.74 ns at 375 nm excitation by TCSPC (Figure S4b). Noticeably, aspirin was not able to be applied for cell imaging, since its obvious PL emission was only present in strong alkaline solution. To understand PL stability of FACD, effects of ionic strength and pH and UV exposure on FACD were evaluated (Figure S5a-c). The PL peak position and intensity remained nearly unchanged when the concentration of KCl increased from 0 to 2.0 M (Figure S5a), which is a characteristic of great significance because it is necessary for FACD to be functional in the physiological salt concentrations in order to use FACD in clinical application. Next, we found that PL intensities of FACD decreased in high (˃7) or low ( 0.05) (Figure 8b). Next, the implant sites were stained with H&E and the inflammatory situation evaluated. Indeed, H&E staining indicated that the carrageenan treated control group had clear inflammatory cell layers, with numerous neutrophils infiltration underneath the epidermis (Figure 8c). The inflammatory cell layer, however, was much smaller in FACD treated rats, only scattered neutrophils infiltration was underneath the epidermis (Figure 8c). These data clearly demonstrate that FACD also possesses effective anti-inflammatory function in vivo. In Vivo Biocompatibility of FACD. Whether or not FACD can eventually be used in clinical applications, depends on whether the FACD cause worse side effects in vivo. To evaluate biosafety of FACD, hematological analyses, serum biochemistries and histological analyses were performed for control (saline group), aspirin and FACD groups. First, all rats used in this



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experiment were alive until experiment termination. Second, hematological analyses, red blood cell count (RBC), platelet count (PLT), white blood cell count (WBC), hemoglobin (HGB), hematocrit (HCT), and mean corpuscular hemoglobin (MCH) from samples on days 1, 3 and 7, did not show any statistically significant differences (Figure S8). These data indicate that FACD has no side effect on hematology. Third, serum biochemistry assays, alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), blood urea nitrogen (BUN), creatinine (CREA), and total bilirubin (TBIL) from samples on days 1, 3 and 7, demonstrated that there was no significant difference for all tests in all three groups (Figure S8). Tests of ALT, AST and ALP are for liver function, TBIL for gallbladder, BUN and CREA for kidney. Therefore, our results suggest that FACD has no toxicity for liver, gallbladder or kidney. Last, histological analyses demonstrated that no histological abnormity was found in heart, liver, spleen and kidney from all samples of the three groups (Figure 9). All of the above data strongly suggest that FACD is safe for in vivo applications.


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CONCLUSIONS In summary, we have synthesized aspirin-based FACD using a one-step microwave-assisted strategy by condensing aspirin and hydrazine. FACD can efficiently internalize into RAW264.7 cells and HeLa cells with low cytotoxicity in vitro. FACD retains or has stronger anti-inflammation effects in vivo and in vitro. Importantly, the FACD did not show any toxicity in vivo, which indicates that the dual functions of FACD have very good biocompatibility. Our results suggest that FACD is a useful clinical and biomedical nanoparticle.



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MATERIALS AND METHODS Synthesis of FACD. Briefly, 2 g of aspirin (SIGMA-Aldrich, St. Louis, MO, USA) and 22.2 mmol of N2H4·H2O (Beijing Chemical Reagents Company, Beijing, China) were dissolved in 8.92 mL of deionized water, then microwaved in a domestic 500 W microwave oven for 8 min. During this process, the color of the solution changed from a colorless liquid to a light brown and, finally, to a dark brown clustered solid resulting in the formation of FACD. The FACD was cooled at room temperature, dissolved in 80 mL of deionized water, centrifuged to remove agglomerated particles at 8000 rpm for 20 min, then filtered through a 0.22 µm membrane to remove large particles. To further purify the FACD, the solution was dialyzed in deionized water using a membrane (MWCO=3.5-5 KD, Float-A-Lyzer G2, Spectrum Laboratories, Rancho Dominguez, CA, USA) for 2 days. Finally, the clear, yellow-brown FACD was obtained. Characterization of FACD. UV-vis absorption spectra were obtained using a Lambda 800 UV-vis spectrophotometer. PL spectroscopy was performed with a Shimadzu RF-5301 PC spectrophotometer. The excitation wavelength was between 340 and 400 nm. The PLQY of FACD and aspirin were estimated at room temperature using quinine in 0.5 mol/L H2SO4 aqueous solution as the PL reference. In order to minimize reabsorption effects, absorption value in the 10 mm fluorescence cuvette was kept below 0.10 at the excitation wavelength. Transmission electron microscopy (TEM) was conducted using a Hitachi H-800 electron microscope at an acceleration voltage of 200 kV with a CCD camera. High-resolution TEM (HRTEM) imaging was implemented by a JEM-2100F electron microscope at 200 kV. An Olympus BX-51 fluorescence microscope with a CCD camera was used to examine the photoluminescent self-assembly materials. Fourier transform infrared (FTIR) spectra were performed with a Nicolet AVATAR 360 FTIR instrument. X-ray photoelectron spectroscopy


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(XPS) was investigated using a VG ESCALAB MKII spectrometer with a Mg KR excitation (1253.6 eV). Binding energy calibration was based on C 1s at 284.6 eV. X-ray powder diffraction (XRD) investigation was carried out using a Siemens D5005 diffractometer. In Vitro Cell Imaging and In Vivo Fluorescence Bioimaging and Cytotoxicity Assays. HeLa and RAW264.7 cell lines were obtained from the Chinese Academy of Sciences Cell Bank (Shanghai, China). Both cell lines were grown in Dulbecco’s Modified Eagle Medium (DMEM) with high glucose, containing 10% fetal bovine serum (FBS), 100 U/mL penicillin and 100 mg/mL streptomycin. Cells were incubated at 37 °C in humidified 5% CO2. Medium was changed every 2-3 days. To evaluate the cell internalization ability of the FACD, HeLa, BMSC, RAW264.7 and KB cells were seeded at 1.5×105 cells/well in 6-well plates, cultured at 37 °C in a humidified 5% CO2 atmosphere for 24 h, then all medium was removed and FACD was added at 100 µg/mL into the well. After 4 h incubation, the cells were washed three times with PBS, then fixed in 4% formaldehyde at 37 °C for 10 min, washed twice with PBS, and observed by confocal laser scanning microscopy (CLSM, Olympus, Japan) using a 405 nm excitation filter to obtain images. All experimental animals used in this study were approved by Jilin University Animal Care and Use Committee. Six nude mice (6 weeks old) were used for in vivo fluorescence bioimaging assays, three for the FACD treated group and three for PBS control group. For each mouse, 300 µL of FACD at 1 mg/mL or 300 µL of PBS was injected on the middle of the back subcutaneously. Then, mice were anesthetized and imaged using the Kodak in vivo imaging system with an excitation/emission wavelength at 390/535 nm. Similarly, ex vivo bioimaging of heart, liver, spleen and kidney from those mice were imaged using the same system after 300 µL of FACD at 1 mg/mL or 300 µL of PBS was injected intraperitoneally.



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In vitro cytotoxicity of FACD was assessed by MTT assay. RAW264.7 cells were seeded at 8 × 104 cells/well in 96-well plates and cultured for 24 h. Culture medium was replaced with 200 µL growth medium containing FACD at 0, 10, 20, 50 or 100 µg/mL. After 24 h incubation, the media were removed and washed twice with PBS, then 200 µL medium was added containing 20 µL 3- (4, 5-dimethyl-2-thiazolyl) -2, 5-diphenyl-2-H-tetrazolium bromide (MTT, 5 mg/mL) into each well and incubated for another 4 h until a purple precipitate was visible. Finally, the supernatant was removed, and 150 µL/well dimethyl sulfoxide (DMSO) was added to dissolve the formazan crystals. The absorbance was measured at 570 nm using a microplate reader. The cell viability was calculated according to the following equation:

Cell viability (%) =

absorbance of the CDs treated group ×100 % absorbance of the control group

In Vitro Anti-Inflammatory Analysis of FACD by QPCR Assays. In vitro anti-inflammatory effects of FACD were assessed by examining expressions of TNF-α and IL-1β using QPCR assay. RAW264.7 cells were seeded at 2×105 cells/well in 6-well plates and cultured for 24 h. The media were removed, aspirin or FACD were added at 0, 50 or 100 mg/mL into the well and incubated for 1 h, then we added LPS at 1 µg/mL into each well. After 24 h and 48 h, cells were collected and used to extract total RNA by Qiagen RNeasy mini purification kit (Qiagen, Valencia, CA, USA). One µg of total RNA was used for each reverse transcription reaction using iScript™ cDNA synthesis kit (Takara Bio, Tokyo, Japan). QPCR was carried out to determine the expression level of TNF-α, IL-1β and β-actin (used as internal control) using SYBR-Green Premix Ex Taq (Takara Bio) by MxPro Mx3005P real-time PCR detection system (Agilent

Technologies,

Santa

Clara,

CA,

USA).

Primers

TNF-α

(5’-TATGGCCCAGACCCTCACA-3’)/TNF-α (5’-GGAGTAGACAAGGTACAACCCATC-3’),

F R

primers

IL-1β

F

(5’18

ACS Paragon Plus Environment

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TCCAGGATGAGGACATGAGCAC-3’)/IL-1β GAACGTCACACACCAGCAGGTTA-3’) CATCCGTAAAGACCTCTATGCCAAC-3’)/

R and

primers β-actin

(5’β-actin R

F

(5’(5’-

GAACGTCACACACCAGCAGGTTA-3’) were used for QPCR assays. In Vivo Anti-Inflammatory Analysis of FACD by Histology and ELISA Assays. Twelve male Wistar rats (160-200 g) were randomly divided into three groups for FACD, aspirin or control group (n = 4). Rats were anesthetized with ether when 1 mL of FACD at 25 mg/kg, aspirin at 25 mg/kg or PBS (control) was given intraperitoneally. Thirty minutes later, rats were subcutaneously implanted with polyester sponges soaked with 1 % carrageenan in sterile PBS. After 5 h, blood samples were collected for PGE2 detection using a PGE2 ELISA kit (R&D Systems, Minneapolis, USA). Then, rats were anesthetized and euthanized by heart perfusion with 4% of paraformaldehyde, and tissues specimens were collected and fixed in 4% of paraformaldehyde for an additional 2 days. The samples were dehydrated in a graded series of ethanol and embedded in paraffin, then 5 µm sections were prepared for hematoxylin and eosin staining (H&E staining). In Vivo Toxicity Assay of FACD. Thirty-six male Wistar rats (160-200 g) were randomly divided into three groups for FACD, aspirin or control (n = 12). Rats were anesthetized with ether, then 1 mL of FACD at 25 mg/kg, aspirin at 25 mg/kg or PBS (control) was given intraperitoneally. Blood samples were collected for hematology and serum biochemistry analysis at 1, 3, and 7 days. ALT, AST, ALP, BUN, CREA, and TBIL commercial kits (Roche Diagnostics, Mannheim, Germany) were used for serum biochemistry analysis. Hematological parameters were measured by a hematology analyzer (MEK-7222K; Nihon Kohden, Tokyo, Japan). Then, rats were euthanized by heart perfusion with 4% of paraformaldehyde. Heart, liver,



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spleen and kidney were harvested and continued to fix in 4% of paraformaldehyde for an additional 24 h. Tissues were dehydrated in a graded series of ethanol and embedded in paraffin, and 5 µm sections were prepared for H&E staining to evaluate if the FACD caused any toxicity. Statistical Analysis. Each experiment was repeated three times. Data was presented as mean ± standard deviation (SD). Statistical analyses were performed by one-way ANOVA. Differences with a P < 0.05 were considered significant.


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ASSOCIATED CONTENT Supporting Information. Ferric chloride assay for demonstrating phenol; comparisons during synthesizing FACD between aspirin and salicylic acid; excitation-dependent PL spectra of FACD; the physiological stability of FACD in PBS, cell culture media and serum; evaluation of FACD biocompatibility in vivo on days 1, 3 and 7 after rats treated with PBS, aspirin at 25 mg/kg or FACD at 25 mg/kg. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author * E-mail: [email protected], [email protected]; Fax: +86-431-88975348. Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. ACKNOWLEDGMENT We would like to thank Cindy Clark and Brigit S. Sullivan, NIH Library Editing Service, for reviewing and editing the manuscript. This work was supported by grants from the National Key Research and Development Program of China 2016YFC1102800, and the Natural Science Foundation of China (81320108011, 81600879, 81371153, 81500820 and 81400488).



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Figure 1. (a) Schematic illustration of microwaved FACD via “one-step” method. Characteristics of FACD: (b) TEM image, (c) HRTEM image, (d) size distributions, (e) XRD pattern of the FACD. The average size and interplanar distance is 4.1 and 0.34 nm, respectively.


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Figure 2. FTIR spectra of aspirin (black), N2H4 (red), and the as-prepared FACD (blue).



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Figure 3. XPS spectra of (a-c) the as-prepared FACD and (d) aspirin.


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Figure 4. The optical properties of the FACD. (a) UV-vis absorption spectra of aspirin (black) and FACD (red). (b) The excitation spectrum (black) and emission spectrum (red) of FACD. (c) Fluorescence decay at 432 nm of the FACD dilute aqueous solution at 25 °C (excitation at 375 nm; IRF = instrument response function). The average lifetimes were calculated using the n

equation τ ave = ∑ aiτ i . i =1



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Figure 5. Cell images of FACD treated cells and cytotoxicity assay. (a1, b1, c1 and d1) are CLSM images. (a2, b2, c2 and d2) are optical images. (a3, b3, c3 and d3) are merged images of optical and CLSM images. (a) RAW264.7 cells, (b) HeLa cells, (c) KB cells, (d) BMSC, (e) cytotoxicity data obtained from treated RAW264.7 cells at various doses. Data are represented as means ± SD from three experiments.


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Figure 6. In vivo fluorescence bioimaging of FACD in nude mouse. Left mouse is PBS treated as a control and right mouse is FACD treated in the panels a and b. The photographs were taken under (a) X-ray and (b) visible light at 390/535 nm wavelength of excitation/emission. The circles in (b) represent the injection sites.



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Figure 7. In vitro anti-inflammation effect of aspirin and FACD evaluated by real-time PCR. (a) Expression of TNF-α at 24 h after treated with aspirin or FACD. (b) Expression of IL-1β at 24 h after treated with aspirin or FACD. (c) Expression of TNF-α at 48 h after treated with aspirin or FACD. (d) Expression of IL-1β at 48 h after treated with aspirin or FACD. Data are represented as means ± SD from three experiments. *: P < 0.05, **: P < 0.01.


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Figure 8. Anti-inflammation effect of aspirin and FACD in vivo. (a) Scheme of drug administration and anti-inflammation effect evaluation. (b) PGE2 level of rats after treated with PBS, aspirin or FACD for 5 h. (c) Histological evaluation of anti-inflammation effect induced by carrageenan after administration of aspirin or FACD (each at 25 mg/kg, intraperitoneally), and the control group was treated with sterile PBS at the same volume at 5 h. P: polyester sponge implant interface. *: P < 0.05, **: P < 0.01.



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Figure 9. Histological evaluation of FACD toxicity in vivo. Heart, liver, spleen and kidney were examined 7 days after intraperitoneal injection of FACD at 25 mg/kg.


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