Brighter, More Stable, and Less Toxic: A HostâGuest Interaction-Aided

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Biological and Medical Applications of Materials and Interfaces

Brighter, More Stable, and Less Toxic: A Host-Guest Interactions Aided Strategy for Fabricating Fluorescent Silica Nanoparticles and Applying Them in Bioimaging and Biosensing at the Cellular Level Chunxiu Liu, Hao Yu, Qiang Li, Changqing Zhu, and Yunsheng Xia ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b03034 • Publication Date (Web): 23 Apr 2018 Downloaded from http://pubs.acs.org on April 24, 2018

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ACS Applied Materials & Interfaces

Brighter, More Stable, and Less Toxic: A Host-Guest Interactions Aided Strategy for Fabricating Fluorescent Silica Nanoparticles and Applying Them in Bioimaging and Biosensing at the Cellular Level Chunxiu Liu†, Hao Yu‡, Qiang Li§, Changqing Zhu*,†, and Yunsheng Xia*,† †Key Laboratory of Functional Molecular Solids, Ministry of Education; College of Chemistry and Materials Science, Anhui Normal University, Wuhu, 241000, China. ‡Anhui Province Key Laboratory of Active Biological Macromolecules Research; Central Laboratory, Wannan Medical College, Wuhu, 241000, China. §Department of Biochemistry, Wannan Medical College, Wuhu, 241000, China.

ABSTRACT: The exploration of fluorescent tools with distinguished optical properties and favorable biocompatibility is significant for bio-sensing and bio-imaging. We herein present a host-guest interactions aided strategy for fabricating fluorescent silica nanoparticles (FSNPs), which is enabled by cyclodextrin (CD) supermolecules. Compared with conventional FSNPs, the modified products (are named as fluorophore@CD@SNPs) possess several advantages. First, the incorporated fluorophores can thoroughly get rid of their intrinsic aggregation due to CD’s inclusion effect, and the fluorescence intensity of the obtained fluorophore@CD@SNPs can enhance 48-67%. Then, the fluorophores can be well fixed by the host CD molecules. As a result, the leak rates of the incorporated fluorophores are only 15-17%, which is about 3 times lower than that of conventional ones (42-48%). Notably, the as-prepared fluorophore@CD@SNPs show observable lower cytotoxicity as compared with their conventional counterparts, probably due to the substantially decreased leakage of the incorporated fluorophores. Because of prominent properties and versatile fabrication, the proposed fluorophore@CD@SNPs not only possess better performances for cell-imaging but are competent for ratiometric sensing of pH value at living cell using (indicator-reference) integrative silica NPs.

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KEYWORDS: fluorescent silica nanoparticles, cyclodextrin-fluorophore complexes, host-guest interactions, cell imaging, ratiometric sensing

1. INTRODUCTION Fluorescent silica nanoparticles (FSNPs) have attracted considerable attention in the fields of nanoscience and nanotechnology.1-8 In addition to well combining the advantages of silica NPs (transparency

for

visible

light,

porosity,

good

biocompatibility,

versatile

surface

modification/functionalization) and small organic fluorophores (tunable emission wavelength, uniform fluorescence intensity as compared with quantum dots and/or carbon dots) into one entity, FSNPs possess a few superior properties/functions including enhanced fluorophore photostability, increased brightness due to multiple dye encapsulation, multicolor fluorescent coding, and so on.8-11 Therefore, FSNPs show great potential in the applications for bio-imaging, bio-sensing, drug delivery, gene carriers, photodynamic therapy, biocatalysis, etc.3,8,12-21 Despite these substantial achievements, there are a few concerns and even problems should be concerned. First of all, FSNPs often suffer from aggregation-induced fluorescence quenching as multiple fluorophore molecules are incorporated into one silica NP.10,22-23,25-26 Whether reverse microemulsion or stöber fabrication approaches, the aggregation of fluorophores within silica NPs is almost inevitable due to their intrinsic hydrophobic effects. Second, the incorporated fluorophores tend to leak out from the host silica NPs,11,27-31 which not only causes the decreased reliability of signal output but results in additional biotoxicity. To overcome this problem, fluorophores can be covalently conjuncted onto host silica NPs.11,29-33 In spite of effectivity, this approach should face two problems. First, the conjunction processes are tedious. Second, for covalent conjunction, a few active groups (amino, hydroxyl, carboxyl, etc.) should be essential, 2


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which is obviously impractical for all kinds of fluorophores. As a result, it is urgent to explore a facile fabrication strategy, in which the above two problems (aggregation-induced quenching, dye leakage) can be well concerned simultaneously. Such strategy would greatly enrich the kit of nano-fabrication, and well promote the applications of FSNPs in the fields of nano-biology. In this study, we presented a modified strategy for fabricating brighter, more stable, and lower toxic FSNPs assisted by supramolecular host-guest interactions. Before silica incorporation, fluorophores (such as rhodamine-B (RB), rhodamine-6G (R-6G), fluorescein (FL)) are first included by cyclodextrin (CD) molecules. By rational choice of supramolecular cavity size, the reaction ratio of fluorophores and CD can be well controlled 1:1. Compared with conventional FSNPs, the modified products (are named as fluorophore@CD@SNPs in this paper) possess several advantages. First, the incorporated fluorophores can thoroughly get rid of their intrinsic aggregation due to CD’s inclusion effect, and the fluorescence intensity of the obtained fluorophore@CD@SNPs can enhance 48-67%. Then, the fluorophores can be well fixed by the inclusion effect of host CD molecules. As a result, the leak rates of the incorporated fluorophores are only 15-17% within 24 h, which is about 3 times lower than that of conventional ones (42-48%). Notably, the obtained fluorophore@CD@SNPs show observable lower cytotoxicity as compared with their conventional counterparts, probably due to the substantially decreased leakage of the incorporated fluorophores. Because of prominent properties and versatile fabrication, the proposed fluorophore@CD@SNPs not only possess better performances for cell-imaging but are competent for ratiometric sensing of pH value at living cell using (indicator-reference) integrative silica NPs.

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Scheme 1. Schematic Illustration for the Fabrication Processes of the Proposed Fluorophore@CD@SNPs. (A) The Fabrication Processes of RB@β-CD@SNPs or R-6G@β-CD@SNPs. (B) The Fabrication Processes of FL/RB@β-CD@SNPs.

2. EXPERIMENTAL SECTION 2.1. Instruments and Characterizations. Fluorescence and absorption spectra were recorded with a Hitachi F-4600 fluorescence spectrophotometer and a Hitachi-U-3100 spectrophotometer, respectively. Transmission electron microscopy (TEM) photographs were taken with a HT-7700 Hitachi microscope at an accelerating voltage of 100 kV. Characterizations of scanning electron microscopy (SEM) were carried out on a Hitachi S-4800 instrument under an accelerating voltage of 5 kV. Fourier transform infrared (FT-IR) spectra were measured from a KBr window on a 4


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PerkinElmer PE-983 FT-IR spectrophotometer. All pH values were measured with a model pHs-3c meter. Human alveolar basal epithelial (A549) adenocarcinoma cells were observed by a confocal spectral microscopy imaging system (Leica TCS SP8) and a inverted fluorescence microscopy imaging system (Olympus IX71), respectively. A549 cells viability experiments analyzed with a Synergy2 Multi-Mode Microplate Reader (BIO-TEK, INC). 2.2. Materials. RB, R-6G, tetraethyl orthosilicate (TEOS), cyclohexane, hexanol, aqueous ammonia solution (NH4OH, 75 wt% water, 25 wt% ammonia), TritonX-100, paraformaldehyde, 4′,6-diamidino-2-phenylindole (DAPI), nigericin and cell counting kit-8 (CCK-8) were purchased from Sigma-Aldrich. FL and HCl were obtained from Shanghai Reagent Company. KCl and β-CD were acquired from Sinopharm. Cell culture media and supplements, trypan blue and live-dead cell staining kit were purchased from HyClone. All solutions were prepared with double deionized water. 2.3. Synthesis of Fluorophore@CD@SNPs. Fluorophore@CD@SNPs were synthesized using a water-in-oil microemulsion method.18,27 We herein employed RB@CD@SNPs as an example for demonstrating the fabrication processes. The microemulsion consisted of a mixture of 1.77 mL of Triton X-100, 1.8 mL of hexanol, 7.5 mL of cyclohexane, 480 µL of a 0.002 M aqueous RB solution in 0.02 M β-CD aqueous solution, and 100 µL of NH4OH that was stirred for 20 min at room temperature, and then 100 µL of TEOS was added. The reaction was stirred for 24 h followed by addition of ethanol to break the microemulsion and recover the SNPs. The SNPs were separated from the reaction mixture by centrifugation at 4500 rpm for 8 min and washed three times with ethanol and one time with water. R-6G@β-CD@SNPs, FL/RB@β-CD@SNPs (the ratio of FL and RB is 1:0.9) and conventional FSNPs (without β-CD molecules) were fabricated 5



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using the same microemulsion system. 2.4. Study the Leakage of the Fluorophore@CD@SNPs and Conventional FSNPs. First, the fluorophore@CD@SNPs and conventional FSNPs were dispersed in PBS solutions (pH 7.4) and incubated for different time, respectively. The concentrations of both two kinds of SNPs were 100 µg mL-1, and the incorporated fluorophores’ concentrations were 4.0 × 10-7 M. Then the liquid supernatant was collected by centrifugation (5000 r/min, 10 min). Third, the supernatant was again centrifuged (1000 r/min, 30 min) with an ultrafiltration tube. Finally, the ultrafiltrates’ fluorescence emission was measured. For conventional FSNPs, the incubation time was 24 h. To better study the stability of the as-prepared fluorophore@CD@SNPs, different incubation time (24 h, 2d, 4d, and 6d) was adopted. 2.5. Cell Incubation. A549 cells were grown in a 100 mm Petri dish in culture medium, which was prepared by mixing Dulbecco’s modified Eagle’s medium (DMEM, 89%, v/v), fetal bovine serum (FBS, 10%, v/v) and penicillin−streptomycin (1%, v/v). The cells were incubated under an atmosphere of 5% CO2 and 95% air at 37 °C for 24 h before the cell experiments. 2.6.

Cell

Viability

Experiments.

The

viability

of

various

staining

agents

(the

fluorophore@CD@SNPs, the fluorophore@SNPs, the fluorophores) treated A549 cells was evaluated by using a CCK-8 assay. First, A549 cells were seeded in 96-well microplates at a density of 5 × 104 cells per mL in 100 µL of culture medium. After 24 h incubation, the culture supernatants were removed. Then, the solution (10 µL) containing different concentrations (0, 125, 250, 500, 1000, 2000, 4000 µg mL-1) of the various staining agents was added to culture medium, respectively (For RB and R-6G fluorophores, the used concentrations just corresponded to the incorporated fluorophores within the fluorophore@CD@SNPs). Third, each staining agents 6


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containing culture medium was averagely divided into 6 parts, which were then severally added into the microplates for parallel tests. After additional 24 h incubation, the culture supernatants were removed and the microplates were washed three times by PBS solution (pH 7.4). Fourth, additional 100 µL of culture medium and 10 µL of CCK-8 were added into each well of 96-well microplates and incubated for 2 h at 37 °C. Finally, the resulting microplates were measured by a Synergy2 Multi-Mode Microplate Reader (BIO-TEK, INC) for cell viability. In addition, for comprehensively and visually detecting cell viability, live-dead cell staining kit assay and trypan blue staining assay were also employed. 2.7. Cell Imaging. A549 cells were grown overnight in a 35 mm Petri dish. The living cells were treated with the fluorophore@CD@SNPs or conventional fluorophore@SNPs and with a final concentration of 200 µg mL-1, respectively. Then these cells incubated for 10 h in a 35 mm Petri dish under an atmosphere of 5% CO2 and 95% air at 37 °C in serum-free medium.3,23,24 The treated cells were then washed three times with PBS and fixed with 4% paraformaldehyde at room temperature for 20 min. After that cell nuclei were stained with 1 µg mL-1 DAPI in H2O for 5 min. The samples were observed by a confocal spectral microscopy imaging system. The images of the cells were captured using the dedicated photomultiplier channel. 2.8. Ratiometric pH Sensing at Living Cell Level. The living cells were treated with FL/RB@β-CD@SNPs (200 µg mL-1) in a 35 mm Petri dish in serum-free medium under an atmosphere of 5% CO2 and 95% air at 37 °C for 10 h. The treated cells were then washed three times with PBS. Finally, 20 µL of 1 mM nigericin, 100 µL of 2.4 M KCl, 880 µL of DMEM culture medium, and 1 mL of PBS solution with different pH value (pH 4.4, 5.4, or 7.4) were added sequentially, which were incubated with A549 cells for 30 min. Fluorescence imaging 7



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experiments in living A549 cells were conducted by a confocal spectral microscopy imaging system. The green and red emission signals were collected selectively using the dedicated photomultiplier channel.

3. RESULTS AND DISCUSSION 3.1. Fabrication and Properties of the Fluorophore@CD@SNPs. Water-in-oil microemulsion system was adopted for the fabrication of the proposed fluorophore@CD@SNPs. We herein employed RB fluorophore as an example for demonstrating the fabrication processes and structure characterizations of the target products. As shown in Scheme 1A, RB and β-CD are premixed each other and form RB@CD complexes firstly. β-CD molecules were chosen here because they can react with RB and form 1:1 inclusion complex.34-38 To include RB as fully as possible, excess β-CD molecules (CD:RB = 10:1) were employed. The observable enhancement of absorbance and fluorescence intensity (3.23% and 24.8%) preliminarily indicates that RB fluorophores are successfully included into CD molecules by host-guest interactions (Figures S1 and S2 in Supporting Information). Our experiments indicated that large excess β-CD molecules did not impact the formation of silica NPs (see below). The water solution containing RB@β-CD complexes was then introduced in the ready water-in-oil microemulsion system, and NH4OH and TEOS were added orderly. The formation of RB@β-CD@SNPs can be performed by 24 h at room temperature. As shown in Figures 1A and 1B, the formed silica NPs are highly monodisperse and spherical, and their size is 70 ± 3 nm with only about ± 4% size distribution. The size and morphology of the products are similar with the ones (Figure S4 in Supporting Information.) obtained by 8


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conventional water-in-oil microemulsion system (without β-CD molecules). Furthermore, the particle size can be easily tuned by the amounts of the added NH4OH (Figure S5 in Supporting Information), which is similar with that of conventional silica NPs fabrication system.27 The FT-IR data (Figure 1C) can give some further information on the host-guest interactions of RB and β-CD molecules. For RB fluorophores, we can concern three sets of absorption bands that relate to CD induced inclusive effects. The first one (1590, 1489, 1415 cm-1) is the stretching vibration absorption bands of C=C bonds within RB’s phenyl groups, and the second (1702, 810 cm−1) and the third one (2971 cm−1) result from C-H bending vibration of the phenyl groups and the stretching vibration of -CH2 group in the alkyl chain, respectively. In terms of the physical mixture of RB and β-CD (green curve in Figure 1C), these absorption bands keep almost invariable. In contrast, for RB@β-CD products, these stretching/bending vibration signals show an obvious intensity decrease and even completely disappeared.39-42 According to the previous reports,34-38,49 RB can enter the macrocyclic cavity of β-CD molecules due to matchable size and hydrophobic effects. Such host-guest interactions well limit the vibration and/or rotation of the inclusive RB molecules. At the same time, R-6G based system (Figure 1F) exhibits a high consistency as compared with RB one, and similar FSNPs can be obtained (Figures 1D and 1E). These data demonstrate that the proposed fabrication system can be employed for different kinds of fluorophores, preliminarily demonstrating its versatility.

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Figure 1. Characterizations of fluorophore@β-CD@SNPs. SEM (A) and TEM (B) images of RB@β-CD@SNPs, respectively. (C) FT-IR spectra of the β-CD, physical mixture of RB and β-CD, RB@β-CD and RB. SEM (D) and TEM (E) images of R-6G@β-CD@SNPs, respectively. (F) FT-IR spectra of the β-CD, physical mixture of R-6G and β-CD, R-6G@β-CD and R-6G.

We then studied the properties of the obtained fluorophore@β-CD@SNPs. As shown in Figure 2A, the fluorescence intensity of the RB@β-CD@SNPs is enhanced 48% as compared with their conventional counterparts. Furthermore, it is noted that the fluorescence peak of the RB@β-CD@SNPs locates at 597 nm, which is 3.4 nm shorter than that of RB@SNPs (Figure 2B). Generally, in water solution or in conventional SNPs, RB molecules tend to aggregate each other and result in a fluorescence quenching and bathochromi-shift due to their intrinsic hydrophobicity. Because of inclusive effects of β-CD molecules, the aggregation of RB molecules within the host silica NPs is well avoided. As a result, a shorter wavelength emission and a fluorescent enhancement (Based on Figure S2 in Supporting Information, the fluorescence enhancement partially result from the inclusive effects of β-CD molecules.) are observed for the obtained RB@β-CD@SNPs. The fluorescence emission of the RB@β-CD@SNPs can keep almost 10


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invariable even after 6 days incubation in PBS solution (Figure S7 in Supporting Information).

Figure 2. The optical properties and stability of the proposed fluorophore@CD@SNPs and conventional FSNPs. (A) Fluorescence spectra of the RB@β-CD@SNPs and the RB@SNPs in PBS solutions. The inset shows a photograph of the the RB@β-CD@SNPs (left cuvette) and the RB@SNPs (right cuvette) in H2O. (B) Normalized intensity fluorescence spectra of the RB@β-CD@SNPs and the RB@SNPs in PBS, respectively. (C) The fluorescence spectra of the ultrafiltrates from the RB@β-CD@SNPs and the RB@SNPs, respectively. (D) Photostability of the RB@β-CD@SNPs and the RB@SNPs. Figures 2E-2H are the corresponding data for R-6G fluorophores. The concentrations of the incorporated fluorophores are 4.0 × 10-7 M.

Then, the fluorophore leakage from the RB@β-CD@SNPs and conventional RB@SNPs was studied. For easier comparison, the two kinds of silica NPs with same concentration (The concentrations of the incorporated fluorophores are 4.0 × 10-7 M, which were quantified by their absorbance based on the calibration curves showing in Figure S13 in Supporting Information.) were dispersed in PBS solutions (pH 7.4) and incubated for different time. Then, the two systems were ultra-filtrated, and the ultrafiltrate’ fluorescence was then measured. As shown in Figure 2C, the fluorescence intensity (black curve) of the ultrafiltrate from the RB@β-CD@SNPs is only about one half of the one from the RB@SNPs (red curve) after 24 h incubation. Further calculations (According to Figures 2A and 2C) indicate that the leakage rate of the 11



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RB@β-CD@SNPs is only 15%, which is about 3 times lower than that of the RB@SNPs (42%). Furthermore, based on Figure S8 in Supporting Information, the fluorophore leakage for the RB@β-CD@SNPs mainly occurred in the first 24 h incubation. After then, the leakage rate well decreased. Obviously, for the RB@β-CD@SNPs, fluorophore leakage was well decreased by the inclusive effects of β-CD molecules. In addition, the introduction of the host-guest interactions can also enhance the photostability of the incorporated fluorophores. As shown in Figure 2D, under UV-illumination, the photostability of RB@β-CD@SNPs is obviously higher than that of conventional RB@SNPs. Towards R-6G fluorophores, CD molecules’ inclusion can lead to similarly optical property promotion, as shown in Figures 2E-2H. 3.2. Cell Cytotoxicity Study. The cytotoxicity experiments were then conducted for assessing the biological application potentials of the as prepared fluorophore@CD@SNPs. As shown in Figure 3, in terms of the RB@ or R-6G@β-CD@SNPs treated A549 cells, the viability is nearly 100% after exposure to 125-1000 µg mL-1 NPs containing solutions for 24 h, and the death rate is only about 10% even the SNPs’ concentration is as high as 4000 µg mL-1 (red bars). In comparison, for conventional fluorophore@SNPs, the cell livability exhibits a distinct decrease at same conditions (green bars). Further experiments indicate that nude fluorophores, whether RB or R-6G molecules, have more serious cytotoxicity (blue bars). It should be noted that the cytotoxicity could keep almost unchanged as the as-prepared fluorophore@CD@SNPs were stored for 3 days (Figure S20 in Supporting Information). As a result, the excellent biocompatibility of the proposed fluorophore@CD@SNPs probably results from the decreased leakage of the incorporated fluorophores due to the inclusive effects of CD molecules.

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Figure 3. (A) Cell cytotoxicity of the RB@β-CD@SNPs, RB@SNPs and RB. (B) Cell cytotoxicity of the R-6G@β-CD@SNPs, R-6G@SNPs and R-6G. The concentrations of RB and R-6G just corresponded to the incorporated fluorophores within the fluorophore@CD@SNPs.

3.3. Cell Imaging and Ratiometric pH Sensing. Cell imaging performances were then explored and measured by confocal fluorescence image system. The cells were first incubated with the proposed fluorophore@CD@SNPs and conventional fluorophore@SNPs, respectively. Then, for easier observation, cell nucleus was further stained by DAPI (Figures 4A, 4E, 4I, 4M). The bright field images (Figures 4D, 4H, 4L, 4P) indicate that all cells can well keep their normal morphology. Then, as shown in Figures 4B, 4F, 4J, 4N, all the cellular cytoplasm can be stained by fluorophores incorporated silica NPs.

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Figure 4. Confocal fluorescence images of A549 cells stained by the fluorophore@CD@SNPs and conventional fluorophore@SNPs. Cell nuclei were stained with DAPI (A, E, I and M). Cell cytoplasm were stained by the fluorophore@β-CD@SNPs (B and J) and conventional fluorophore@SNPs (F and N), respectively. C, G, K and O are the overlays of A and B, E and F, I and J, M and N, respectively. D, H, L, and P are the corresponding bright field images. Cells were incubated with 200 µg mL-1 of the fluorophore@CD@SNPs and conventional fluorophore@SNPs for 10 h, respectively.

However, there are a few differences in staining performances for two kinds of FSNPs. For the RB@ and R-6G@β-CD@SNPs treated cells (Figures 4B, 4J), all cell cytoplasm parts are brighter and more uniform, and the cells’ outlines are more well-defined, as compared with the ones treated by their conventional counterparts (the RB@ and R-6G@SNPs). Obviously, the better staining performances of the as-prepared fluorophore@CD@SNPs result from the inclusive 14


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effects of CD molecules. Because of CD molecules assisted host-guest interactions, the resulting fluorophore incorporated silica NPs are brighter, more stable, and lower toxic. To assess the application versatility of the proposed fluorophore@CD@SNPs, analyte targets responsive SNPs were designed and their sensing performances were studied at living cell level. We herein employed pH ratiometric assay as an example for demonstrating the sensing potential of the proposed SNPs’ based system. FL and RB fluorophores were adopted for pH reporter and reference, respectively. Because FL can also be included by β-CD molecules,34,43 the proposed CD aided strategy is then extended for fabricating two kinds of fluorophores containing pH ratiometric probes (The fabrication processes are shown in Scheme 1B, and the procedures are provided in Experimental.). Herein, the pH responsive products are named as FL/RB@β-CD@SNPs. As shown in TEM images (Figure 5A), the as-prepared FL/RB@β-CD@SNPs are well monodisperse and their size is also about 70 nm. FL molecules are one of weak acids (pKa = 6.4), and their monoanion and dianion possess a low (Φ = 0.36) and higher (Φ = 0.93) fluorescence quantum yields, respectively.44-47 So, FL fluorophores are often used for pH sensing in biological systems.44-45 As shown in Figure 5B, for the as-prepared FL/RB@β-CD@SNPs, the fluorescence emission intensity from FL fluorophores (emission peak at about 536.6 nm) gradually increases with the enhancement of pH values. These results indicate that the inclusive effects of CD molecules do not impact their pH responses, probably due to that the host-guest interactions do not disturb the FLs’ binding sites for H+ ions. In comparison, RB molecules’ fluorescence intensities (emission peak at 597 nm) keep almost constant in all time (Figure 5C) because they are pH inert. As described in Figure 5D, the ratio of IFL/IRB gradually increases from 0.467 to 3.25 as pH is continuously increased from 3.0 to 8.0, indicating the application potential for ratiometric pH 15



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sensing. The pH sensing performances of the proposed probes at living cell level were then conducted. A549 cells were first stained by the FL/RB@β-CD@SNPs, and then incubated in different pH (pH 4.4, 5.4, 7.4) mediums. The bright field images showing in Figures 5H, 5L, 5P indicate that all living cells can well keep their normal morphology. The viability of the staining cells is at least 98% according to trypan blue staining assay. (Figure S23 in Supporting Information). These data demonstrate that the as-prepared FL/RB@β-CD@SNPs are highly biocompatible. Then, as shown in Figures 5E-5N, the ﬂuorescence images were collected by using green and red detector channels of the confocal microscope, for selectively monitoring the evolution of FL and RB fluorescence, respectively. These sets of images show that the green fluorescence intensities from FL fluorophores become stronger and stronger with the enhancement of pH value (Figures 5E, 5I and 5M); in contrast, the red ones from RB ones are almost invariable in all time (Figures 5F, 5J and 5N). Their merge images (Figures 5G, 5K and 5O) provide visualized color changes for different pH values. These data demonstrate that the proposed FL/RB@β-CD@SNPs can act as ratiometric sensing probes for pH assay at living cell level.

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Figure 5. Ratiometric pH sensing at living cell level by the FL/RB@β-CD@SNPs. (A) TEM image of the as-prepared FL/RB@β-CD@SNPs. The emission spectra of the probes excited by 450 (B) and 520 (C) nm at different pH values, which correspond to FL (sensor) and RB (reference) emission, respectively. (D) IFL/IRB versus pH value. Confocal fluorescence microscopy images for pH sensing in A549 cells showing sensor dye channel (E, I, M), reference dye channel (F, J, N), overlay images (G, K, O), and bright field images (H, L, P).

4. CONCLUSIONS In summary, we presented a host-guest interactions aided strategy for fabricating fluorescent silica NPs. The inclusive effects of CD molecules not only prevent the aggregation of incorporated fluorophores but efficiently decrease fluorophore leakage from the host silica NPs. As a result, the 17



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as-prepared fluorophore@CD@SNPs are brighter, more stable, and lower toxic as compared with their conventional counterparts, and they exhibit enhanced performances in bio-imaging and bio-sensing at cell level. On the one hand most small molecule fluorophores have hydrophobic parts, and on the other hand the dimension of supramolecular cavity can be well modulated by their varieties (CD, pillararene, calixarene, cucurbituril, etc.) and/or the numbers of the building units (α-, β-, γ-CD, etc.).48-53 So, the proposed host-guest interactions aided strategy is potentially versatile for fabricating fluorophore@supermolecule@SNPs for biological applications.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: Absorption spectra, fluorescence spectra and FT-IR spectra analysis for the fluorophore@β-CD. SEM images, TEM images and photograph of fluorophore@supermolecule@SNPs.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] (Y.X.). *E-mail: [email protected] (C.Z.).

Notes The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work is financially supported by the National Natural Science Foundation of China (Nos. 21775004 and 21422501), Wanjiang Scholar program, and Foundation for Innovation Team of Bioanalytical Chemistry.

REFERENCES (1) Ghosh Chaudhuri, R.; Paria, S. Core/Shell Nanoparticles: Classes, Properties, Synthesis Mechanisms, Characterization, and Applications. Chem. Rev. 2012, 112, 2373–2433. (2) Burns, A.; Owb, H.; Wiesner, U. Fluorescent Core–Shell Silica Nanoparticles: Towards ‘‘Lab on a Particle’’ Architectures for Nanobiotechnology. Chem. Soc. Rev. 2006, 35, 1028–1042. (3) Lee, J. E.; Lee, N.; Kim, H.; Kim, J.; Choi, S. H.; Kim, J. H.; Kim, T.; Song, I. C.; Park, S. P.; Moon, W. K.; Hyeon, T. Uniform Mesoporous Dye-Doped Silica Nanoparticles Decorated with Multiple Magnetite Nanocrystals for Simultaneous Enhanced Magnetic Resonance Imaging, Fluorescence Imaging, and Drug Delivery. J. Am. Chem. Soc. 2010, 132, 552–557. (4) Van Blaaderen, A.; Vrij, A. Synthesis and Characterization of Monodisperse Colloidal Organo-silica Spheres. J. Colloid Interface Sci. 1993, 156, 1–18. (5) Tang, L.; Cheng, J. Nonporous Silica Nanoparticles for Nanomedicine Application. Nano Today 2013, 8, 290–312. (6) Bae, S. W.; Tan, W.; Hong, J. I. Fluorescent Dye-Doped Silica Nanoparticles: New Tools for Bioapplications. Chem. Commun. 2012, 48, 2270–2282. (7) Montalti, M.; Prodi, L.; Rampazzo, E.; Zaccheroni, N. Dye-Doped Silica Nanoparticles as Luminescent Organized Systems for Nanomedicine. Chem. Soc. Rev. 2014, 43, 4243–4268. 19



Page 20 of 26

(8) Korzeniowska, B.; Nooney, R.; Wencel, D.; McDonagh, C. Silica Nanoparticles for Cell Imaging and Intracellular Sensing. Nanotechnology 2013, 24, 442002. (9) Ow, H.; Larson, D. R.; Srivastava, M.; Baird, B. A.; Webb, W. W.; Wiesner, U. Bright and Stable Core−Shell Fluorescent Silica Nanoparticles. Nano Lett. 2005, 5, 113–117. (10) Larson, D. R.; Ow, H.; Vishwasrao, H. D.; Heikal, A. A.; Wiesner, U.; Webb, W. W. Silica Nanoparticle Architecture Determines Radiative Properties of Encapsulated Fluorophores. Chem. Mater. 2008, 20, 2677–2684. (11) Ma, D.; Kell, A. J.; Tan, S.; Jakubek, Z. J.; Simard, B. Photophysical Properties of Dye-Doped Silica Nanoparticles Bearing Different Types of Dye-Silica Interactions. J. Phys. Chem. C 2009, 113, 15974–15981. (12) Slowing, I. I.; Trewyn, B. G.; Giri, S.; Lin, V. S. Y. Mesoporous Silica Nanoparticles for Drug Delivery and Biosensing Applications. Adv. Funct. Mater. 2007, 17, 1225–1236. (13) Yang, P.; Gai, S.; Lin, J. Functionalized Mesoporous Silica Materials for Controlled Drug Delivery. Chem. Soc. Rev. 2012, 41, 3679–3698. (14) Radu, D. R.; Lai, C. Y.; Jeftinija, K.; Rowe, E. W.; Jeftinija, S.; Lin, V. S. Y. A Polyamidoamine Dendrimer-Capped Mesoporous Silica Nanosphere-Based Gene Transfection Reagent. J. Am. Chem. Soc. 2004, 126, 13216–13217. (15) Bharali, D. J.; Klejbor, I.; Stachowiak, E. K.; Dutta, P.; Roy, I.; Kaur, N.; Bergey, E. J.; Prasad, P. N.; Stachowiak, M. K. Organically Modified Silica Nanoparticles: A Nonviral Vector for in Vivo Gene Delivery and Expression in the Brain. Proc. Natl. Acad. Sci. U. S. A. 2005, 102, 11539–11544. (16) Kim, S.; Ohulchanskyy, T.; Pudavar, H.; Pandey, R.; Prasad, P. Organically Modified Silica 20


Page 21 of 26 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


Nanoparticles Co-encapsulating Photosensitizing Drug and Aggregation-Enhanced Two-Photon Absorbing Fluorescent Dye Aggregates for Two-Photon Photodynamic Therapy. J. Am. Chem. Soc. 2007, 129, 2669–2675. (17) Roy, I.; Ohulchanskyy, T. Y.; Bharali, D. J.; Pudavar, H. E.; Mistretta, R. A.; Kaur, N.; Prasad, P. N. Optical Tracking of Organically Modified Silica Nanoparticles as DNA Carriers: A Nonviral, Nanomedicine Approach for Gene Delivery. Proc. Natl. Acad. Sci. U. S. A. 2005, 102, 279–284. (18) Tan, W.; Wang, K.; He, X.; Zhao, X.; Drake, T.; Wang, L.; Bagwe, R. P. Bionanotechnology Based on Silica Nanoparticles. Med. Res. Rev. 2004, 24, 621–638. (19) He, X. X.; Wang, K.; Tan, W.; Liu, B.; Lin, X.; He, C.; Li, D.; Huang, S.; Li, J. Bioconjugated Nanoparticles for DNA Protection from Cleavage. J. Am. Chem. Soc. 2003, 125, 7168–7169. (20) Argyo, C.; Weiss, V.; Bräuchle, C.; Bein, T. Multifunctional Mesoporous Silica Nanoparticles as a Universal Platform for Drug Delivery. Chem. Mater. 2014, 26, 435–451. (21) Popat, A.; Hartono, S. B.; Stahr, F.; Liu, J.; Qiao, S. Z.; Lu, G. Q. Mesoporous Silica Nanoparticles for Bioadsorption, Enzyme Immobilisation, and Delivery Carriers. Nanoscale 2011, 3, 2801–2818. (22) Van Blaaderen, A.; Vrij, A. Synthesis and Characterization of Colloidal Dispersions of Fluorescent, Monodisperse Silica Spheres. Langmuir 1992, 8, 2921–2931. (23) Wang, Z.; Xu, B.; Zhang, L.; Zhang, J.; Ma, T.; Zhang, J.; Fu, X.; Tian, W. Folic Acid-Functionalized Mesoporous Silica Nanospheres Hybridized with AIE Luminogens for Targeted Cancer Cell Imaging. Nanoscale 2013, 5, 2065–2072. (24) Lu, F.; Wu, S. H.; Hun,. Y.; Mou, C. Y. Size Effect on Cell Uptake in Well-Suspended, Uniform Mesoporous Silica Nanoparticles. Small 2009, 5, 1408-1413. 21



Page 22 of 26

(25) Zhang, X.; Zhang, X.; Yang, B.; Liu, L.; Hui, J.; Liu, M.; Chen, Y.; Wei, Y. Aggregation-Induced Emission Dye Based Luminescent Silica Nanoparticles: Facile Preparation, Biocompatibility Evaluation and Cell Imaging Applications. RSC Adv. 2014, 4, 10060–10066. (26) Wang, X.; Song, P.; Peng, L.; Tong, A.; Xiang, Y. Aggregation-Induced Emission Luminogen-Embedded Silica Nanoparticles Containing DNA Aptamers for Targeted Cell Imaging. ACS Appl. Mater. Interfaces 2016, 8, 609–616. (27) Bagwe, R. P.; Yang, C.; Hilliard, L. R.; Tan, W. Optimization of Dye-Doped Silica Nanoparticles Prepared Using a Reverse Microemulsion Method. Langmuir 2004, 20, 8336–8342. (28) Lin, Y. S.; Tsai, C. P.; Huang, H. Y.; Kuo, C. T.; Hung, Y.; Huang, D. M.; Chen, Y. C.; Mou, C. Y. Well-Ordered Mesoporous Silica Nanoparticles as Cell Markers. Chem. Mater. 2005, 17, 4570–4573. (29) Leventis, N.; Elder, I. A.; Rolison, D. R.; Anderson, M. L.; Merzbacher, C. I. Durable Modification of Silica Aerogel Monoliths with Fluorescent 2,7-Diazapyrenium Moieties. Sensing Oxygen near the Speed of Open-Air Diffusion. Chem. Mater. 1999, 11, 2837–2845. (30) Suratwala, T.; Gardlund, Z.; Davidson, K.; Uhlmann, D. R.; Watson, J.; Peyghambarian, N. Silylated Coumarin Dyes in Sol-Gel Hosts. 1. Structure and Environmental Factors on Fluorescent Properties. Chem. Mater. 1998, 10, 190–198. (31) Sokolov, I.; Kievsky, Y. Y.; Kaszpurenko, J. M. Self-Assembly of Ultrabright Fluorescent Silica Particles. Small 2007, 3, 419–423. (32) Baker, G. A.; Pandey, S.; Maziarz, E. P. III; Bright, F. V. Toward Tailored Xerogel Composites: Local Dipolarity and Nanosecond Dynamics within Binary Composites Derived from Tetraethylorthosilane and ORMOSILs, Oligomers or Surfactants. J. Sol.-Gel Sci. Technol. 1999, 22


Page 23 of 26 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


15, 37–48. (33) Frantz, R.; Carbonneau, C.; Granier, M.; Durand, J. O.; Lanneau, G. F.; Corriu, R. J. P. Studies

of

Organic–Inorganic

Solids

Possessing

Sensitive

Oligoarylene–Vinylene

Chromophore-Terminated Phosphonates. Tetrahedron Lett. 2002, 43, 6569–6572. (34) Politzer, I. R.; Crago, K. T.; Hampton, T.; Joseph, J. Effect of β-Cyclodextrin on the Fluorescence, Absorption and Lasing of Rhodamine 6G, Rhodamine B and Fluorescein Disodium Salt in Aqueous Solutions. Chem. Phys. Lett. 1989, 159, 258–262. (35) Liu, Y.; Chen, Y.; Liu, S. X.; Guan, X. D.; Wada, T.; Inoue, Y. Unique Fluorescence Behavior of Rhodamine B

upon Inclusion Complexation with Novel Bis(β-cyclodextrin-6-yl)

2,2′-Bipyridine-4,4′-dicarboxylate. Org. Lett. 2001, 3, 1657–1660. (36) Willner, I.; Eichen, Y.; Frank, A. J. Tailored Semiconductor-Receptor Colloids: Improved Photosensitized H2 Evolution from Water with TiO2-β-Cyclodextrin Colloids. J. Am. Chem. Soc. 1989, 111, 1511–1884. (37) Degani, Y.; Willner, I.; Haas, Y. Lasing of Rhodamine B in Aqueous Solutions Containing β-Cyclodextrin. Chem. Phys. Lett. 1984, 104, 496–499. (38) Lincoln, S. F.; Coates, K. T.; Schiller, R. L. Inclusion of Rhodamine B by β-Cyclodextrin. An Equilibrium and Kinetic Spectrophotometric Study. J. Inclusion Phenom. 1987, 5, 709–716. (39) Lin-Vien, D.; Colthup, N. B.; Fateley, W. G.; Grasselli, J. G. The Handbook of Infrared and Raman Characteristic Frequencies of Organic Molecules; Academic Press: New York, 1991. (40) Farag, A. A. M.; Yahia, I. S. Structural, Absorption and Optical Dispersion Characteristics of Rhodamine B Thin Films Prepared by Drop Casting Technique. Opt. Commun. 2010, 283, 4310–4317. 23



Page 24 of 26

(41) Liu, J.; Li, C.; Li, F. Fluorescence Turn-On Chemodosimeter-Functionalized Mesoporous Silica Nanoparticles and Their Application in Cell Imaging. J. Mater. Chem. 2011, 21, 7175–7181. (42) Seçkin, T.; Gültek, A.; Kartaca, S. The Grafting of Rhodamine B onto Sol-Gel Derived Mesoporous Silicas. Dyes Pigm. 2003, 56, 51–57. (43) Flamigni, L. Inclusion of Fluorescein and Halogenated Derivatives in α-, β-, and γ-Cyclodextrins. A Steady-State and Picosecond Time-Resolved Study. J. Phys. Chem. 1993, 97, 9566–9572. (44) Han, J.; Burgess, K. Fluorescent Indicators for Intracellular pH. Chem. Rev. 2010, 110, 2709–2728. (45) Burns, A.; Sengupta, P.; Zedayko, T.; Baird, B.; Wiesner, U. Core/Shell Fluorescent Silica Nanoparticles for Chemical Sensing: Towards Single Particle Laboratories. Small 2006, 2, 723–726. (46) Zanker, V.; Peter, W. Die Prototropen Formen Des Fluoresceins. Chern. Ber. 1958, 91, 572–580. (47) Bogdanova, L. N.; Mchedlov-Petrossyan, N. O.; Vodolazkaya, N. A.; Lebed, A. V. The Influence of β-cyclodextrin on Acid–Base and Tautomeric Equilibrium of Fluorescein Dyes in Aqueous Solution. Carbohydr. Res. 2010, 345, 1882–1890. (48) Schneider, H. J. Binding Mechanisms in Supramolecular Complexes. Angew. Chem. Int. Ed. 2009, 48, 3924–3977. (49) Dsouza, R. N.; Pischel, U.; Nau, W. M. Fluorescent Dyes and Their Supramolecular Host/Guest Complexes with Macrocycles in Aqueous Solution. Chem. Rev. 2011, 111, 7941–7980. (50) Douhal, A. Ultrafast Guest Dynamics in Cyclodextrin Nanocavities. Chem. Rev. 2004, 104, 24


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1955–1976. (51) Nau, W. M.; Florea, M.; Assaf, K. I. Deep Inside Cucurbiturils: Physical Properties and Volumes of their Inner Cavity Determine the Hydrophobic Driving Force for Host–Guest Complexation. Isr. J. Chem. 2011, 51, 559–577. (52) Arunkumar, E.; Forbes, C. C.; Smith, B. D. Improving the Properties of Organic Dyes by Molecular Encapsulation. Eur. J. Org. Chem. 2005, 19, 4051–4059. (53) Liow, S. S.; Zhou, H.; Sugiarto, S.; Guo, S.; Chalasani, M. L. S.; Verma, N. K.; Xu, J.; Loh, X. J. Highly Efficient Supramolecular Aggregation-Induced Emission-Active Pseudorotaxane Luminogen for Functional Bioimaging. Biomacromolecules 2017, 18, 886–897.

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