Ag2S Quantum Dots Conjugated Chitosan Nanospheres toward

School of Pharmacy, Shanghai Jiao Tong University, Shanghai 200240, China .... Caiping Ding , Xuanyu Cao , Cuiling Zhang , Tangrong He , Nan Hua ...
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Ag2S Quantum Dots Conjugated Chitosan Nanospheres toward Light-Triggered Nitric Oxide Release and Near-Infrared Fluorescence Imaging Lianjiang Tan,† Ajun Wan,*,† and Huili Li‡ †

School of Chemistry and Chemical Engineering, Shanghai Jiao Tong University, Shanghai 200240, China School of Pharmacy, Shanghai Jiao Tong University, Shanghai 200240, China

‡

S Supporting Information *

ABSTRACT: Nanoscaled light-triggered nitric oxide (NO) delivery vehicles with the ability of near-infrared (NIR) fluorescence imaging was presented, which consisted of chitosan (CS)-based S-nitrosothiols (SNO) and encapsulated silver sulfide quantum dots (Ag2S QDs). CS-SNO compounds that bore NO-storing functional groups were prepared via amino modification of chitosan. Water-soluble Ag2S QDs were synthesized and conjugated with the CS-SNO compounds with the aid of ethylenediaminetetraacetic acid (EDTA). The biocompatible Ag2S-CS-SNO nanospheres, with dimension of ∼117 nm, exhibited bright NIR fluorescence and satisfactory photostability under NIR irradiation. The Ag2S-CS-SNO nanospheres could release NO under irradiation of UV or visible light at physiological pH and temperature yet would hardly release NO if NIR irradiation was applied. Cell imaging was successfully performed, demonstrating that the Ag2S-CS-SNO nanospheres could emit readily observable NIR fluorescence and release NO in living cells. The NIR fluorescence imaging of the Ag2S-CS-SNO nanospheres did not interfere with the light-triggered NO release from them, which would provide new perspectives for the application of multifunctional nanostructured materials in diagnostics and imaging.

1. INTRODUCTION Nitric oxide (NO) is a multifaceted signaling and bioregulatory agent effective in mediating multiple biological events. It is always involved in the regulation of the cardiovascular system, the central and peripheral nervous systems, and the immune system.1−3 The development of NO delivery systems has attracted considerable attention owing to the physiological features of NO in both normal and disease states.4−9 We have developed chitosan-based NO donors that can spontaneously release NO molecules in physiological environment and simultaneously detect the NO release in situ with the aid of conjugated fluorescent agents.10,11 It is critical, however, for a therapeutic NO delivery system to be capable of controlled NO release.12 In addition, incorporation of fluorophores that are highly effective for in vivo fluorescence imaging plays an important role in therapeutic applications. Quantum dots (QDs), also termed semiconductor nanocrystals, have attracted significant interest and attention during the past decades due to their potential applications for biological labeling and imaging.13−16 QDs have distinct advantages over conventional organic dyes for both in vitro and in vivo imaging.17,18 For in vivo fluorescence imaging, nearinfrared (NIR) fluorescent quantum dots possess superior properties over those emitting in visible region due to reduced autofluorescence and negligible tissue scattering in this region as well as great penetration depth for deep tissue imaging with high temporal resolution and feature fidelity.19−21 In the past © 2013 American Chemical Society

few years, efforts have been devoted to development of NIR fluorescent QDs for in vivo imaging applications. A variety of NIR fluorescent QDs have been successfully designed, such as PbS,22 PbSe,23 heterostructured QDs (CdSe/ZnTe, CdTe/ CdSe, etc.),24,25 and Si quantum dots.26 The Cd-, Pb-, or Hgcontaining QDs are intrinsically toxic, which limits their use for in vivo applications. The Si quantum dots have low toxicity, but they were found to gather and stay in the livers and spleens of mice, which resulted in inflammation and spotty death of liver cells.26 Silver chalcogenides are appealing narrow-bandgap semiconductor materials for preparing low-toxicity NIR fluorescent QDs.27−32 In these studies, NIR fluorescent Ag2S QDs synthesized in organic phase exhibited excellent optical properties and good biocompatibility. More recently, functionalized Ag2S QDs used for targeted small animal imaging have been reported.33,34 The water-soluble Ag2S QDs synthesized with facile methods served as excellent NIR imaging probes, which provides new perspectives for nanodiagnostics and imaging in vivo. So far, controlled delivery of NO from fluorescent complexes with reduced toxicity and ability of efficient cell uptake remains challenging. There is an urgent need for biocompatible NO donors that provide therapeutically effective NO release and Received: August 6, 2013 Revised: October 18, 2013 Published: November 13, 2013 15032

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Scheme 1. Synthetic Strategy of Ag2S-CS-SNO Nanospheres (a); Light-Triggered NO Release Mechanism of Ag2S-CS-SNO Nanospheres (b)

fluorescence imaging. In the present work, we have designed a light responsive NO releasing vehicle with the function of NIR fluorescence emission for therapeutic applications. Chitosan was used as the NO carrier, since it has excellent biocompatibility and chelating characteristics due to the high hydrophilicity and activity of amino groups.35 S-Nitrosothiols (RSNO), which are generally viewed as the reservoir for NO,36−39 were conjugated to chitosan for light-triggered NO release. Furthermore, integration of Ag2S QDs endowed the chitosan-based vehicles with superior NIR fluorescence emission that penetrates tissue deeply. The use of Ag2S QDs was intended to provide high-quality imaging for in vivo applications.

deionized water. The Ag2S QDs terminated with carboxylic acid groups were obtained. 2.3. Preparation of CS-S-Nitrosothiols Compounds. The amino groups of chitosan were the reactive sites for the formation of chitosan-based S-nitrosothiols. N-Acetyl-DL-penicillamine was dissolved in pyridine at 0 °C under stirring, followed by the addition of acetic anhydride for reaction at 25 °C for 24 h. The product was heated to 60 °C under reduced pressure to remove the pyridine and then washed and dried to obtain a thiolactone. The thiolactone was added to methylene chloride, followed by the addition of chitosan at a mass ratio to the thiolactone of 1:1. The mixture was stirred at 25 °C for 6 h under the protection of nitrogen. The resultant product was centrifuged at 8000 rpm for 5 min, washed by methylene chloride, and vacuum-dried at 25 °C for 3 h to obtain CS-SH compounds. 100 mg of as-prepared CS-SH was added to 2 mL of 1 M hydrochloric acid under stirring, followed by dropwise addition of a sodium nitrite aqueous solution (0.5 M) at 0 °C. The mixture was allowed to react for 30 min. The resultant CS-SNO compounds were centrifuged for 10 min, washed by methanol, and vacuum-dried at 25 °C for 3 h. 2.4. Preparation of Ag2S-CS-SNO Nanospheres. The Ag2S-CSSNO nanospheres were synthesized with a nonsolvent-aided counterion complexation method. 7.5 mg of EDTA was added to 10 mL of assynthesized Ag2S QDs aqueous solution and stirred until full dissolution. The use of EDTA was aimed at facilitating the conjugation of Ag2S QDs with CS-SNO, as EDTA can effectively bind Ag2S QDs via chelation and can mix with CS. The as-prepared CS-SNO compounds were then added into the above solution at a molar ratio to the EDTA of 1:2.8 and stirred for 3 min. After that, ethanol, a nonsolvent for both CS-SNO and EDTA, was added dropwise to the solution under stirring until the clear solution turned cloudy, which signified the formation of Ag2S-CS-SNO colloidal particles, with the Ag2S QDs spontaneously encapsulated in the nanospheres. The resultant colloidal solution was filtered and centrifuged at 8000 rpm for 5 min. The sediment was redispersed into deionized water and dialyzed against deionized water for 12 h to remove EDTA. The Ag2SCS-SNO nanospheres were then obtained, which were capable of releasing NO under irradiation of UV or visible light and emitting NIR fluorescence excited by NIR light (Scheme 1b). 2.5. Characterization. Fourier transform infrared spectroscopy (FTIR) tests were conducted on a Spectrum 100 FTIR spectrometer (PerkinElmer). Samples were dried, powdered, and made into films by mixing them with KBr. Samples were dissolved in D2O and analyzed by 15N NMR spectrometry. The RSNO structure was identified on an AVANCE III NMR spectrometer (Bruker, Switzerland) at 400 MHz. Chemical shifts are given in parts per million relative to neat

2. MATERIALS AND METHODS 2.1. Materials. Chitosan (CS, weight-average molecular weight Mw = 108 kDa, degree of acetylation ≥90%) was purchased from Sinopharm Chemical Reagent Co., Ltd., China, and used without further purification. Silver acetate (AgAc), sodium nitrite, hydrochloric acid (1 M), methylene chloride, methanol, ethonal, and acetic anhydride were all provided by Sinopharm Chemical Reagent Co., Ltd., China. N-Acetyl-DL-penicillamine, glutathiose (GSH), pyridine, indocyanine green (ICG), and RPMI 1640 cell culture medium were all purchased from Sigma-Aldrich. Ethylenediaminetetraacetic acid (EDTA) was purchased from Aladdin. Fetal bovine serum (FBS) was purchased from the Institute of Biochemistry and Cell Biology, CAS. CuFL, a NO-sensitive fluorescent molecule, was synthesized in our own lab via the route reported in our previous work.11 PBS (phosphate buffered saline) buffers (0.01 and 0.2 M, pH = 7.4) were prepared in our own lab. All raw chemicals were analytical grade unless otherwise stated. 2.2. One-Pot Synthesis of Ag2S Quantum Dots. Scheme 1a depicts the typical synthetic procedure of Ag2S QDs in our work. AgAc and GSH, with a molar ratio of 4:3, were mixed with EG in a threeneck flask at 120 °C under argon flow and kept at this temperature for 1 min to form a white cloudy mixture. The mixture was then heated to 150 °C and held for 10 min, changing from white cloudy to light yellow clear and finally to yellow. Once the reaction was finished, the mixture was cooled to room temperature and was dialyzed against deionized water for 24 h to remove unreacted molecules and ions. The resultant colloidal solution was precipitated by ethanol and centrifuged at 8000 rpm for 10 min, followed by redispersion of the sediment in 15033

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Figure 1. FTIR spectra of CS, CS-SNO, and Ag2S-CS-SNO nanospheres (a). 15N NMR spectrum of Ag2S-CS-SNO nanospheres (b). nitromethane (δ = 0) as the external standard. Surface analysis was conducted on an ESCALAB 250 X-ray photoelectron spectrometer (XPS, Thermo Scientific, US) with nonmonochromatic Al Kα X-ray (1486.6 eV). The analyzer was operated at 20 eV pass energy with an energy step size of 1 eV (full spectra) and 0.1 eV (high-resolution spectra). Binding energy calibration was based on C 1s at 284.6 eV. Powder X-ray diffraction (XRD) patterns were collected using a D/ max-2200/PC X-ray diffractometer (Rigaku, Japan) fitted with nickelfiltered Cu Kα radiation. The data were collected at 0.02° intervals with counting for 0.2 s at each step. The freeze-dried Ag2S-CS-SNO nanospheres were weighted and analyzed by thermogravimetry (TG), which was performed on a Q5000IR thermogravimetric analyzer (TA Instruments) under N2 flow from room temperature to 800 °C at a heating rate of 20 °C/min. Transmission electron micrographs (TEM) were recorded on a JEM-2100 transmission electron microscope (JEOL, Japan) at 200 kV. Samples were suspended in ethanol, fully dispersed by ultrasonic wave, and deposited on a 300 mesh copper grid prior to observation. The size distribution of the Ag2S-CS-SNO nanospheres was determined by a Nano ZS90 particle size and zeta potential analyzer (Malvern, UK) based on dynamic light scattering (DLS) at a scattering angle of 90°. Vis−NIR absorption spectra were recorded by a Lambda 750S UV−vis−NIR spectrophotometer (PerkinElmer), the measured range being 750−1050 nm. NIR fluorescence spectra were collected on a Fluorolog-3 fluorescence spectrophotometer (Horiba Jovin Yvon, France) equipped with liquid nitrogen cooled InGaAs detector (800−1600 nm), applying the excitation laser of 808 nm. NIR images of the Ag2S-CS-SNO nanospheres in PBS buffer and mouse whole blood were acquired under 808 nm excitation using a liquid nitrogen cooled twodimensional InGaAs camera (Princeton Instruments) with a sensitivity ranging from 800 to 1700 nm. 2.6. Measurement of NO Release. Quantitative detection of the NO molecules released from the Ag2S-CS-SNO nanospheres was carried out by a TBR 4100/1025 free radical analyzer equipped with an ISO-NOP sensor (WPI Ltd.). The sensor was calibrated by the addition of 50 μM KNO2 in a mixture of 0.33 g of KI and 20 mL of 0.1 M H2SO4. The NO release was measured at 37 °C, where the detection sensitivity was determined to be 2.55 pA/nM. The details are as follows: 0.1 mg of nanospheres was dispersed in 1 mL of a 0.2 M PBS buffer (pH = 7.4), forming a stable suspension. Then the suspension was rapidly injected into 19 mL of the PBS buffer when the ISO-NOP sensor had reached a low, stable current level. The NO probe was immersed about 2 cm into the suspension under magnetic stirring. 2.7. Cell Assay. L929 cells (mouse fibroblast cells) purchased from Institute of Biochemistry and Cell Biology, CAS, were cultured in RPMI 1640 medium supplemented with 10 wt % FBS, 100 IU/mL penicillin and 100 μg mL−1 streptomycin in a humidified incubator with 5 vol % carbon dioxide at 37 °C. The medium was refreshed every 2 or 3 days according to cell density. Cytotoxicity of the Ag2S-CS-SNO nanospheres was evaluated by 3(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium chloride (MTT)

viability assay. The L929 cells were seeded in 96-well culture plates at a density of 4000 cells per well and incubated at 37 °C for 24 h for cell attachment. The culture medium in each well was then replaced by a fresh medium containing the Ag2S-CS-SNO nanospheres at different concentrations (0.01−1 mg/mL). One row of the 96-well plates was used as control. After further incubation for 24 or 48 h, the culture plates were rinsed with a 0.01 M PBS buffer (pH = 7.4) to remove unattached cells and the remaining cells were treated with 5 mg mL−1 MTT stock solution in PBS for 4 h. The medium containing unreacted MTT was then carefully removed. The obtained formazan was dissolved in DMSO, and the absorbance of individual wells was recorded at 570 nm using a Multiskan MK3 enzyme-labeled Instrument (Thermo Scientific). The cell survival rate was determined by the following equation:

cell survival rate (%) =

absorbance of test cells × 100% absorbance of control cells (1)

2.8. In Vitro Cell Imaging. A suspension of L929 cells incubated with Ag2S-CS-SNO nanospheres (0.1 mg/mL) for 5 h was transferred to an eight-well Lab-Tek II chamber slide (Nalge Nunc, Naperville, IL). The medium was then aspirated from the wells, and the cells were rinsed with fresh culture medium three times before observation. NIR cell imaging was performed using a liquid nitrogen cooled twodimensional InGaAs camera (Princeton Instruments) with a sensitivity ranging from 800 to 1700 nm. The excitation light was provided by an 808 nm laser diode, and the emitted photons were collected in the 1000−1700 nm NIR range by applying a 1000 nm long-pass filter. The NIR light captured by the camera was shown in pseudocolor. 2.9. Imaging of Intracellular NO Release. L929 cells were incubated with Ag2S-CS-SNO nanospheres (0.1 mg/mL) for 5 h. After thorough washing with fresh culture medium, 10 μg of CuFL was added and incubated for 2 h at 37 °C. Then the cells were rinsed three times with fresh culture medium. The cell fluorescence was observed by a confocal laser scanning microscope (Zeiss LSM 710, Germany) equipped with a 488 nm excitation source by the green channels. The 488 nm laser acted as both NO triggering and exciting laser source.

3. RESULTS AND DISCUSSION 3.1. Chemical Structure. Fourier transform infrared (FTIR) spectra of CS, CS-SNO, and Ag2S-CS-SNO nanospheres are illustrated in Figure 1a. The CS showed a broad absorption peak at ∼3381 cm−1, typical of the stretch vibration of O−H and N−H. The peaks situated at 2920 and 2870 cm−1 were ascribed to the C−H stretching. For the CS-SNO compounds, two peaks appeared at 1730 and 1665 cm−1, ascribed to the presence of −COCH3 and −NHCO− groups. The absorption at 1922 cm−1 was derived from the S−N stretching, indicating the formation of −SNO groups in the compounds. For the Ag2S-CS-SNO nanospheres, the peak at 15034

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Figure 2. XPS spectrum of Ag2S-CS-SNO nanospheres (a). S 2p signals (b) and Ag 3d signals (c) recorded for Ag2S-CS-SNO nanospheres. UV−vis spectrum of Ag2S-CS-SNO nanospheres in water (d).

Figure 3. XRD patterns of CS and Ag2S-CS-SNO nanospheres (a). TG curve of Ag2S-CS-SNO nanospheres (b).

1735 cm−1 was attributable to the CO stretching of both the carboxylic acid groups at the surface of Ag2S QDs and the ester groups in CS-SNO. No obvious absorption peak corresponding to free thiol group (2490 cm−1)40 appeared in the spectrum, suggesting that GSH molecules were bound to the surface of Ag2S QDs via the Ag−thiol bond. The nitrogen NMR technique, an effective tool in both organic chemistry and biochemistry, was employed to further confirm the presence of −SNO groups in the Ag2S-CS-SNO nanospheres. As indicated in Figure 1b, a sharp peak was situated at 790.1 ppm in the 15N NMR spectrum, characteristic of tertiary RSNOs.41 It could be concluded that the NO-storing functional groups did exist in the Ag2S-CS-SNO nanospheres. The X-ray photoelectron spectrometry (XPS) technique is effective in determining the surface chemical composition and element valence state of a sample. The full XPS spectrum shown in Figure 2a indicates the presence of the elements Ag and S as well as N, C, and O from the CS-SNO compounds. The high-resolution XPS spectrum in Figure 4b shows a

symmetric peak at 161.3 eV, assignable to the 2p electrons of S. No peak of element S corresponding to higher energy appeared, confirming that no unbound thiol group existed on the surface of the nanospheres. In Figure 2c, the peaks of Ag 3d5/2 (367.3 eV) and Ag 3d3/2 (373.3 eV) indicate that the oxidation state of Ag ion is univalent in the Ag2S nanocrystals.42 Figure 2d shows the UV−vis spectrum of Ag2S-CS-SNO nanospheres. A strong absorption situated at 339.6 nm is attributed to the n0 → π* electronic transition, which is common to S-nitrosothiols and is usually used as a reliable quantitative assessment of their decomposition.43 Another absorption peak found at 587.2 nm is ascribed to the nN → π* electronic transition of S-nitrosothiols in the Ag2S-CS-SNO nanospheres. These results further confirm the formation of Snitrosothiols in Ag2S-CS-SNO nanospheres. The X-ray diffraction (XRD) patterns of CS and Ag2S-CSSNO nanospheres are shown Figure 3a. As a typical noncrystalline polymer, CS showed only an amorphous diffraction peak at 2θ ≈ 20°. When Ag2S QDs were 15035

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Figure 4. TEM micrograph (a), high-resolution TEM micrograph (b), and size distribution of as-synthesized Ag2S QDs stabilized by GSH (c). TEM micrographs (d, e) and size distribution (f) of Ag2S-CS-SNO nanospheres.

Figure 5. Vis−NIR absorption spectra (a) and fluorescence spectra (b) of Ag2S QDs and Ag2S-CS-SNO nanospheres in water.

Ag2S crystal. The size distribution histogram obtained by measuring the diameter of selected nanoparticles in the TEM image is shown in Figure 4c. The average size of the Ag2S QDs turned out to be 7.6 ± 0.4 nm, in good accordance with the value determined by XRD. The TEM images of Ag2S-CS-SNO nanospheres are shown in Figure 4d,e. The nanospheres with spherical outline had narrow size distribution (Figure 4d). Hardly any free-standing QDs outside the nanospheres were observed, indicating the Ag2S QDs were entrapped in the polymeric networks (Figure 4e). The size distribution of the nanospheres acquired based on DLS test is shown in Figure 4f. The diameter of the nanospheres was in the range of 20−300 nm, and the Z-average diameter was 117 nm. 3.3. Optical and NO Releasing Properties. Figure 5a shows the Vis−NIR absorption spectrum of GSH-stabilized Ag2S QDs and Ag2S-CS-SNO nanospheres dispersed in water. The colloidal solution of Ag2S QDs exhibited a discernible absorption peak in the NIR region at 883 nm, which is the lowest-energy excitonic absorption of the Ag2S QDs. For the Ag2S-CS-SNO nanospheres, the absorption peak was situated at 895 nm, a red-shift from that of the Ag2S QDs. The absorption edge was at ∼970 nm, corresponding to an energy gap of approximately 1.28 eV. The NIR fluorescence (FL) spectra of Ag2S QDs and Ag2S-CS-SNO nanospheres excited with an 808 nm laser diode are illustrated in Figure 5b. A

incorporated, distinct peaks were observed, the positions and relative intensities of which matched well those of monoclinic Ag2S (acanthite-type crystal). The broadening of the diffraction peaks may suggest the nanocrystalline nature of the sample. It should be noted that the resolution of the diffraction signals was a little lower than that of Ag2S QDs,28 likely due to the influence of CS. The crystal size calculated based on Scherrer equation44 was 8 nm. The XRD results confirm that there were indeed Ag2S QDs encapsulated in the Ag2S-CS-SNO nanospheres. Thermogravimetry (TG) analysis is useful for determining the Ag2S QDs content in the Ag2S-CS-SNO nanospheres, for the chitosan matrix would decompose and volatilize at high temperature, and the residues are Ag2S QDs. As can be seen in Figure 3b, the Ag2S-CS-SNO nanospheres gradually decomposed with the increase of temperature until ca. 550 °C. The TG curve then leveled off, and the remaining mass was 9.9% of the total mass. Therefore, loading of Ag2S QDs in the nanospheres was 9.9 wt %. 3.2. Morphology and Size Distribution. The morphology of as-prepared Ag2S QDs and Ag2S-CS-SNO nanospheres was observed by TEM. As shown in Figure 4a, the Ag2S QDs were monodisperse nanoparticles. The high-resolution TEM (HRTEM) micrograph in Figure 4b clearly shows lattice fringes of the nanocrystals with an interplanar spacing of ∼0.25 nm, which could be assigned to the (−1, 1, 2) facets of monoclinic 15036

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metal ions, S-nitrosothiols are stable under physiological conditions. The dissociation of S−N bond in S-nitrosothiols results in NO generation. Usually, the energy of UV and visible light is sufficient for the S−N bond cleavage. To investigate the NO release from the Ag2S-CS-SNO nanospheres induced by light, quantitative detection of released NO was conducted, and the results are depicted in Figure 6. The released NO was proven by electron spin resonance (ESR) analysis (Figure S1 in Supporting Information). The ESR signal denoting NO release appeared under irradiation of 488 laser. When the irradiation was removed, the signal intensity decreased. Figure 6a shows instant NO release of the Ag2S-CS-SNO nanospheres in PBS buffer under excitation of different wavelengths. The samples excited by 365 nm UV light and 488 nm visible light exhibited steady NO release at increasing rate during the initial 80 min, reaching a maximum instant release of 990 and 892 nM, respectively. Then a gradual decrease in the NO release was observed for both the two cases, and the instant NO release still remained at relatively high levels (843 and 670 nM, respectively) even after 3 h irradiation. The sample excited by 808 nm NIR light showed a much smaller amount of NO release, with the maximum instant NO release of only 154 nM. The sample without irradiation did not show observable NO release. It is evident that the NO release from the Ag2S-CSSNO nanospheres needed light triggering in UV and visible regions, and the NIR light could not induce substantial NO release. The cumulative NO release profiles in Figure 6b indicate ever-increasing amount of released NO in the data range. Although more NO molecules were generated by the Ag2S-CS-SNO nanospheres under 365 nm excitation, the 488 nm light source was preferred in therapy since it causes less injury to living tissues. We further examined the light-triggered NO release by switching on and off light irradiation at 488 nm. As illustrated in Figure 6c, burst NO release occurred once the

symmetric emission peak centered at 1106 nm with an impressive full width at half-maximum (fwhm) of 65 nm was detected for the Ag2S QDs. For the Ag2S-CS-SNO nanospheres, the NIR emission peak was found at 1102 nm, and the fwhm of the peak was similar to that of the Ag2S QDs. The Stokes shift of Ag2S QDs and Ag2S-CS-SNO nanospheres were 220 and 205 nm, respectively. We can conclude that the conjugation of chitosan would hardly influence the optical properties of the Ag2S QDs, except enhancing the fluorescence intensity by 7.6%. It is most likely that conjugation with chitosan increases electron delocalization of the QDs. The Ag2S-CS-SNO aqueous solution was light yellow without irradiation. When excited by 808 nm NIR light, bright photoluminescence occurred. The fluorescence quantum yield of the Ag2S-CS-SNO nanospheres was also evaluated. As listed in Table 1, four samples of Ag2S-CS-SNO nanospheres Table 1. Size and Fluorescence Quantum Yield of Ag2S-CSSNO Nanospheres sample

Z-average diameter (nm)

A B C D

119.9 117.0 123.5 118.6

QYa (%) 2.7 3.3 2.7 3.0

± ± ± ±

1.1 1.6 1.3 1.2

QY = fluorescence quantum yield, measured against indocyanine green (ICG) in DMSO (QY = 13%). a

exhibiting a quantum yield of ∼3% (calculated following the equation in Supporting Information) qualified for NIR imaging in vivo. The size of the Ag2S-CS-SNO nanospheres seemed not to have a direct relationship with their quantum yield. Photochemical release of NO from RSNO derivatives is an intriguing phenomenon. In the absence of light and transition

Figure 6. NO generation with time from Ag2S-CS-SNO nanospheres in PBS buffer (pH = 7.4, 37 °C) triggered by light of different wavelengths (a). Cumulative NO release profiles of Ag2S-CS-SNO nanospheres triggered at 365 and 488 nm (b). Controlled on-and-off No release behavior of Ag2SCS-SNO nanospheres by 488 nm light (c). The laser power applied was 30 W. 15037

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Figure 7. Changes of fluorescence emission of Ag2S-CS-SNO nanospheres in PBS buffer with the NO release triggered by 488 nm irradiation for 0, 2, 4, and 6 h from top to bottom (a). Fluorescence intensity changes of Ag2S-CS-SNO nanospheres with time subject to continuous irradiation of 808 and 488 nm light at the power of 100 W (b).

Figure 8. Optical image (a) and an NIR fluorescence image (b) of Ag2S-CS-SNO nanospheres in PBS buffer and mouse whole blood at a concentration of 0.112 mg/mL. Mean fluorescence intensities of the two samples determined based on the NIR fluorescence image (c). Fluorescence stability of the Ag2S-CS-SNO nanospheres in mouse whole blood within 48 h (d).

Ag2S-CS-SNO nanospheres were irradiated. When the light source was removed, the instant NO release decreased rapidly. Once again the light was on after 3 min, and the release of NO recovered. The NO release in response to alternating irradiation and darkness exhibited a “zig-zag” profile without any sign of fatigue effect. These results supported our notion that light-triggered release of NO can be very useful in photodynamic therapy. Irradiated by 488 nm light, the Ag2S-CS-SNO nanospheres emitted fluorescence at nearly the same wavelength as that under 808 nm irradiation, as can be seen in Figure 7a. With prolonged irradiation time, the fluorescence intensity decreased, yet the decreasing trend became less obvious with the time. To elucidate the reason for these phenomena, we traced the time-dependent fluorescence of the Ag2S-CS-SNO nanospheres irradiated by 808 and 488 nm light, respectively. As illustrated in Figure 7b, the fluorescence intensity of the sample irradiated by 808 nm light showed a slow decrease in

the data range, maintaining 73.6% of the original value after 8 h irradiation. This result indicates that continuous irradiation would not exert great influences on the fluorescence properties of the Ag2S-CS-SNO nanospheres. For the sample irradiated by 488 nm light, the fluorescence decrease was more significant in the first 4 h and gradually leveled off thereafter. More importantly, the fluorescence intensity of this sample was 59.5% of the original value, smaller than that of the sample irradiated by 808 nm light. It has been mentioned above that the Ag2S-CS-SNO nanospheres would release NO under 488 nm irradiation in a PBS buffer, while a much smaller amount of NO would be released under 808 nm irradiation. It is likely that some NO molecules generated under 488 nm irradiation diffused to the surface of the encapsulated Ag2S QDs through the chitosan network and coordinated with Ag sites of the QDs, taking advantage of their lone pair electrons. Consequently, the fluorescence was reduced to some extent, yet the quenching extent was tiny compared with that of the chitosan 15038

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Figure 9. TEM images at varied magnifications of the uptake of Ag2S-CS-SNO nanospheres by L929 cells.

Figure 10. Bright-field microscopic images of L929 cells incubated for 24 h with Ag2S-CS-SNO nanospheres at varied concentrations (mg/mL): 0.01 (a), 0.05 (b), 0.1 (c), 0.5 (d), and 1.0 (e). Viability of L929 cells incubated with different concentrations of Ag2S-CS-SNO nanospheres (f). The cells without treatment of Ag2S-CS-SNO nanospheres were used as controls.

encapsulated CdTe QDs by NO.10 This explanation was supported by the fluorescence response experiment (Figure S2 in Supporting Information). On the basis of all of these results, we can conclude that irradiation with NIR light will excite the Ag2S-CS-SNO nanospheres to emit NIR fluorescence but will not trigger appreciable NO release. Only when visible or UV light is applied will the NO release be triggered in the PBS buffer resembling physiological environment. This provides the feasibility of monitoring the distribution of the nanospheres while avoiding unscheduled NO release. As UV or visible light for NO triggering does not penetrate tissue effectively, a

controllable laser source can be provided along with the Ag2SCS-SNO nanospheres when they are used for in vivo imaging. Furthermore, we observed the physical stability and nearinfrared photoluminescence of the Ag2S-CS-SNO nanospheres in both PBS buffer and mouse whole blood. As shown in Figure 8a, the Ag2S-CS-SNO nanospheres were physically stable in the two mediums without any sign of precipitation or cloudness for 48 h. In fact, the two samples were stable even after 8 weeks. The NIR image in Figure 8b indicates that the Ag2S-CS-SNO nanospheres in the mouse blood maintained high fluorescence after 48 h. Figure 8c shows the fluorescence intensity of the two 15039

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Figure 11. Bright-field image (a), NIR fluorescence image acquired immediately upon 808 nm irradiation (b), NIR fluorescence image acquired 3 h after 808 nm irradiation (c), and dark-field image without irradiation (d) of L929 cells incubated with 0.1 mg/mL Ag2S-CS-SNO nanospheres for 5 h. CLSM images (488 nm excitation) of the NO detection of CuFL stained cells without the Ag2S-CS-SNO nanospheres (e) and containing the Ag2S-CS-SNO nanospheres (f). NIR image (g) and merged image (CLSM and NIR) (h) of the CuFL stained cells containing Ag2S-CS-SNO nanospheres.

difference from Figure 11b. In a control experiment, L929 cells without incubation with the Ag2S-CS-SNO nanospheres showed no fluorescence is (Figure 11d). These results indicate that the Ag2S-CS-SNO nanospheres can be well used for NIR cell imaging with an NIR laser for excitation. To examine the NO release of the nanospheres inside living cells, we introduced NO-sensitive CuFL into the nanosphere-containing cells and placed them under 488 nm irradiation. As shown in Figure 11f, green fluorescence typical of the CuFL encountering NO appeared upon irradiation, indicating rapid NO release from the nanospheres in the cells. In comparison, the cells without containing nanospheres showed little visible fluorescence signal (Figure 11e). The cells in Figure 11f emitted NIR fluorescence under NIR excitation (Figure 11g). The merged image of Figures 11f and 11g shown in Figure 11h further demonstrates that NO release from the Ag2S-CS-SNO nanospheres can be achieved in living cells irradiated by visible light.

samples determined by analyzing the image in Figure 8b. The Ag2S-CS-SNO nanospheres in the mouse blood emitted stronger fluorescence. The histogram in Figure 8d depicts the fluorescence stability of the Ag2S-CS-SNO nanospheres in the mouse blood. The NIR fluorescence decreased marginally with time and retained 82% of the original fluorescence intensity after 48 h storage. 3.4. Cytotoxicity and in Vitro Cell Fluorescence Imaging. For an ideal NO delivery vehicle, the ability to permeate through the cell membrane for efficient cell uptake is extremely important. We thus investigated the uptake of Ag2SCS-SNO nanospheres by L929 cells by the TEM technique. The TEM images in Figure 9 reveal that some Ag2S-CS-SNO nanospheres entered the cells and were mostly localized in the cytoplasm after 1 h incubation. The uptake of the Ag2S-CSSNO nanospheres by cells was fairly rapid, which favors the NO delivery and fluorescence imaging in cells. It is necessary to examine the accurate cytotoxicity of Ag2SCS-SNO nanospheres before applying them to living cells for NO delivery and fluorescence imaging.45 Figure 10a−e shows the bright-field microscopic images of L929 cells incubated with different concentrations of Ag2S-CS-SNO nanospheres for 24 h. In each case, the cells adhered to the plates and maintained normal morphology. The increasing concentration of the Ag2SCS-SNO nanospheres seemed not to exert a remarkable influence on the proliferation of the cells. The results of MTT assay, a standard colorimetric assay for evaluating cytotoxicity, are shown in Figure 10f. The cell viability decreased slightly with the increase of nanospheres concentration. At the concentration of 1.0 mg/mL, 88% and 83% of the cells survived relative to the control group after incubation for 24 and 48 h, respectively, suggesting the low cytotoxicity of the Ag2S-CS-SNO nanospheres. In comparison with the Ag2S QDs (Figure S3 in Supporting Information), the cells incubated with the Ag2S-CS-SNO nanospheres had a little higher viability at high concentrations (0.5 and 1 mg/mL). In vitro cell imaging of the Ag2S-CS-SNO nanospheres was investigated. NIR fluorescence was observed from the L929 cells incubated with the Ag2S-CS-SNO nanospheres for 5 h upon irradiation of 808 nm light (Figure 11b), indicating excellent imaging ability of the Ag2S-CS-SNO nanospheres. After continuous irradiation for 3 h, the fluorescence still remained at a high level (Figure 11c), without displaying great

4. CONCLUSIONS To sum up, Ag2S QDs-chitosan nanospheres containing Snitrosothiol functional groups were prepared using facile methods. Under physiological conditions, the prepared nanospheres could release NO under irradiation of UV or visible lights and would emit NIR fluorescence if excited by NIR light source. The two functions can be utilized without interference with each other. This unique feature makes the use of the Ag2SCS-SNO nanospheres more convenient. With excellent biocompatibility and photostability, the Ag2S-CS-SNO nanospheres hold great potential as a NO delivery and fluorescence imaging platform for biomedical applications.

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ASSOCIATED CONTENT

S Supporting Information *

Relevant experimental results and data not included in the article. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel/Fax +86-21-34201245; e-mail [email protected] (A.W.). 15040

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The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by Specialized Research Fund for the Doctoral Program of Higher Education (20130073120087), the National Natural Science Foundation (21076124 and 51173104), and the Nanometer Technology Program of Science and Technology Committee of Shanghai (11 nm0503500).

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