Aggregation-Induced Emission Luminogen-Embedded Silica

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Aggregation-Induced Emission Luminogen-Embedded Silica Nanoparticles Containing DNA Aptamers for Targeted Cell Imaging Xiaoyan Wang, Panshu Song, Lu Peng, Aijun Tong, and Yu Xiang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.5b09644 • Publication Date (Web): 10 Dec 2015 Downloaded from http://pubs.acs.org on December 22, 2015

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ACS Applied Materials & Interfaces

Aggregation-Induced Emission Luminogen-Embedded Silica Nanoparticles Containing DNA Aptamers for Targeted Cell Imaging Xiaoyan Wang,1 Panshu Song,2 Lu Peng,1 Aijun Tong1 and Yu Xiang1* 1

Department of Chemistry, Beijing Key Laboratory for Microanalytical Methods and Instrumentation, Key Laboratory of Bioorganic Phosphorus Chemistry and Chemical Biology (Ministry of Education), Tsinghua University, Beijing 100084, China. Email: [email protected] 2

National Institute of Metrology, Beijing 100029, China.

ABSTRACT: Conventional fluorophores usually undergo aggregation-caused quenching (ACQ), which limits the loading amount of these fluorophores in nanoparticles for bright fluorescence imaging. On the contrary, fluorophores with aggregation-induced emission (AIE) characteristics are strongly fluorescent in their aggregate states, and have been an ideal platform for developing highly fluorescent nanomaterials, such as fluorescent silica nanoparticles (FSNPs). In this work, AIE luminogens based on salicylaldehyde hydrazones were embedded in silica nanoparticles through a facile noncovalent approach, which afforded AIE-FSNPs emitting much brighter fluorescence than some commercial fluoresceindoped silica and polystyrene nanoparticles. These AIE-FSNPs displaying multiple fluorescence colors were fabricated by a general method, and they underwent much less fluorescence variations to environmental pH changes compared with fluorescein-hybridized FSNPs. In addition, a DNA aptamer specific to nucleolin was functionalized on the surface of AIEFSNPs for targeted cell imaging. Fluorescent microscopy and flow-cytometry studies both revealed highly selective fluorescence staining of MCF-7 (a cancer cell line with nucleolin overexpression) over MCF-10A (normal) cells by the aptamer-functionalized AIE-FSNPs. The fluorescence imaging in different color channels was achieved using AIE-FSNPs containing each of the AIE luminogens, as well as photoactivatable fluorescent imaging of target cells by the caged AIE fluorophore.

Keywords: Aggregation-induced emission, Silica nanoparticle, DNA aptamer, Photoactivatable fluorescence, Cell imaging

Introduction Fluorescent silica nanoparticles (FSNPs) have been widely utilized as biosensors for cellular imaging because of their superior properties for cellular studies including biocompatibility, hydrophilicity, transparency, chemical stability and ease of surface functionalization.1-2 However, conventional fluorescent dyes, such as fluorescein, undergo aggregation-caused quenching (ACQ) due to their selfquenching effect,3 thereby making it very difficult to fabricate FSNPs with brighter fluorescence by loading more fluorophores beyond the critical amount at which ACQ occurs. In contrast, a family of fluorophores (luminogens) with aggregation-induced emission (AIE) characteristics, discovered in 2001, are strongly fluorescent even in their aggregate or solid states.4 These AIE-active molecules are weakly fluorescent or non-fluorescent when dispersed in

solution due to the intramolecular motions that cause non-radiative decay of the excited states, while they are highly fluorescent in aggregates and solids since the intramolecular motions are restricted.5 So far, many fluorophores in the AIE family composed of either hydrocarbons, heteroatoms or metal complex have been reported and applied as bioprobes and stimuli-responsive materials.6-8 Among them, several hydrocarbon- or silole-based AIE luminogens have been covalently hybridized with FSNPs for biosensing and imaging applications.9-11 These AIE-FSNPs composites not only provide strong fluorescence in aqueous solution, but also facilitate morphology control and surface modification of the nanoparticles. For example, AIE-FSNPs functionalized by folic acid or biotin have been prepared for cell imaging.10-11 Nevertheless, the use of biomolecules, which are usually more specific to targets of interest than small molecules, on AIE-FSNPs for targeted cell imaging are still challenging and have not

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Figure 1. (a) Chemical structures of 1-3 and the UV-induced conversion of 3 to 4. (b) Fluorescence spectra of 1 and 2 at 100 μM in DMF and Reaction Buffer (0.2 M NaCl, 0.1 M sodium phosphate buffer, pH 7.3), as well as those of 3 in Reaction Buffer before and after UV irradiation at 365 nm for 1 h. Inset is the part of the figure at low fluorescence intensity range. Excitation was at 375 nm.

been reported yet. In addition, the fabrication of AIEFSNPs in previous studies requires covalent attachment of hydrocarbons or siloles as AIE-active molecules to silicate esters,12 making the synthesis complicated. DNA aptamers, first identified through a combinatorial technique called systematic evolution of ligands by exponential enrichment (SELEX) in 1990s,13-15 are an attractive class of biomolecules for the recognition of specific targets, ranging from metal ions and small molecular metabolites to biomolecules and even whole cells.16 The affinity of aptamers to target molecules is comparable or sometimes even better than that of some antibodies to the same targets.17 In recent two decades, many DNA aptamers have been developed into sensors for highly selective and sensitive biosensing and imaging,18-28 as well as potential targeted cancer therapeutics.29-33 The use of traditional fluorophores and aptamers for targeted cancer cell imaging was also successful achieved.30, 34-38 However, traditional fluorophores encounter ACQ, while AIE molecules conjugated with aptamers for target recognition were scarcely studied. In this work, taking advantages of the bright fluorescence of AIE-FSNPs and the specific target recognition of DNA aptamers, we prepared DNA aptamer-functionalized FSNPs containing AIE luminogens (Apt-AIE-FSNPs) for targeted cell imaging. These Apt-AIE-FSNPs, with tunable and uniform sizes, were facilely fabricated by a general method simply through non-covalent embedment of AIE luminogens inside the nanoparticles and subsequent functionalization of a nucleolin-specific DNA aptamer on the surface. Depending on the AIE fluorophores used, they could display multiple fluorescence colors ranging from green to orange, as well as photoactivatable fluorescence upon UV irradiation. The as-prepared Apt-AIEFSNPs were also successfully applied for targeted cell imaging, through which cancer cells (MCF-7 with membrane

nucleolin overexpression) were visualized over normal cells (MCF-10A) with a high selectivity.

Results and Discussion Fluorescent properties of the AIE luminogens Aggregation-induced emission (AIE) luminogens 1-3 (Figure 1a) used in this study were synthesized by modifying substituted salicylaldehydes with alkyl/benzyl bromides and then followed by reaction with hydrazine. They are derivatives of salicylaldehyde hydrazones39-42 known to be fluorescent due to excited state intramolecular proton transfer (ESIPT) and usually exhibit AIE fluorescence characteristics, e.g. fluorescent in aggregate states but non-fluorescent in solution, because the nonradioactive decay of the excited state by intramolecular motions in solution was prohibited when their molecules are closely packed in aggregates.5 As illustrated in Figure 1b, 1 and 2 displayed green and yellow fluorescence, respectively, as aggregates in the poor solvent of Reaction Buffer (0.2 M NaCl, 0.1 M sodium phosphate buffer, pH 7.3), while they are almost non-fluorescent in the good solvent of DMF (Figure 1b). The difference in maximum emission wavelength for 1 and 2 is due to the effect of substitution groups on salicylaldehyde hydrazones.39, 43-44 Compound 345 was nearly non-fluorescent even in the poor solvent of Reaction Buffer because of the 2-nitrobenzyl caging group.46 Upon UV irradiation at 365 nm that converts 3 to 4 (Figure 1a), photoactivatable47 AIE fluorescence in orange was observed (Figure 1b). Synthesis and characterization of functionalized AIE-FSNPs containing 1-3

DNA-

To prepare the AIE luminogen-embedded fluorescent silica nanoparticles (AIE-FSNPs), 1-3 were non-covalently embedded into silica nanoparticles during the polymerization of silicate ester monomers in the presence of ammonium, tetrahydrofuran and ethanol (Figure 2, step (a)).

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Figure 2. Synthesis of AIE-FSNPs-1 (a) and the subsequent conjugation of DNA aptamers on the silica nanoparticles (b-d). The AIE-FSNPs containing 2 or 3 are synthesized and conjugated with DNA aptamers following the same procedure.

Figure 3. (a) Fluorescence spectra of 0.05 mg/mL (particle mass per mL solution) AIE-FSNPs containing 1-3 in Reaction Buffer, where AIE-FSNPs-3 is irradiated by 365 nm UV light for 1 h to activate the fluorescence. (b) Fluorescence spectra of 0.05 mg/mL AIE-FSNPs-1 in Reaction Buffer before and after multiple times of washing using 30% DMF/water. Excitation was at 375 nm. (c) TEM images of AIE-FSNPs-1 of different diameters (from left to right: 60, 100, 140 nm).

Figure 4. (a) Zeta potential of AIE-FSNPs-1 at each stage of DNA conjugation. (b) Fluorescence spectra of 0.05 mg/mL Apt-AIE-FSNPs containing 1-3 in Reaction Buffer, where AptAIE-FSNPs-3 is irradiated by 365 nm UV light for 1 h to activate the fluorescence. Excitation was at 375 nm.

These AIE-FSNPs showed nearly the same fluorescence or photoactivatable characteristics as the AIE luminogens alone (Figure 3a), and the fluorescence of AIE-FSNPs containing 1 (AIE-FSNPs-1) was found almost identical after multiple times of washing using even 30% dimethylformamide (DMF)/water (Figure 3b), suggesting the full preservation of the luminogen’s AIE properties in AIEFSNPs and the stable embedment of the dyes inside the nanoparticles. This non-covalent approach was more facile than the covalent approach used in previous reports for the fabrication of AIE-FSNPs.12, 48 In fact, we initially carried out the reaction of 1 with 3aminopropyltriethoxysilane (APS) to serve as the precursor for the preparation of AIE-FSNPs by the conventional covalent approach. However, it only afforded AIE-FSNPs-1 with a much lower fluorescence intensity (less than 10% when normalized by silica mass) compared with those prepared by the non-covalent approach above, probably because the formation of fluorescent aggregates was much less efficient for APS-modified 1 in the covalent approach than unmodified 1 in the non-covalent approach. The diameter of spherical AIE-FSNPs-1 was tunable from 60 to 140 nm by varying the amount of ammonia used for the synthesis, as illustrated in the transmission electron microscope (TEM) images (Figure 3c) and dynamic light scattering (DLS) data (Figure S1, Supporting Information) of AIE-FSNPs-1 synthesized under different conditions. The average diameters estimated by DLS were larger than those measured by TEM, because the nanoparticles had a certain extent of swelling and hydration in solution and DLS measured the hydration diameters. The AIE fluorophore 1 inside the 140 nm nanoparticles was found very stable upon UV irradiation (Figure S2, Supporting Information), and the colloid solution was found to be stable for days as indicated by fluorescence spectra upon aging (Figure S3, Supporting Information). Compound 2-3 were also successfully embedded in the uni-

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form 140 nm AIE-FSNPs using the same procedure (Figure S4, Supporting Information). To serve as a control, fluorescein isothiocyanatehybridized FSNPs (FITC-FSNPs) of 140 nm diameter were also synthesized in this study by the standard method16,35 through covalent attachment of FITC to APS. Such FITCFSNPs were then compared with AIE-FSNPs-1. As shown in Figure S2 (Supporting Information), upon increasing the amount of FITC-APS used in the synthesis of FITCFSNPs, the fluorescence of the resulting FITC-FSNPs containing more dyes increased to a maximum and then decreased, in accordance with the aggregation-caused quenching (ACQ) effect of fluorescein when exceeding the critical loading amount for ACQ to occur. However, such drawback was not observed for AIE-FSNPs-1 (Figure s5a, Supporting Information), because the fluorescence of AIE-FSNPs-1 increased with the amount of 1 used for the synthesis in a wide range without reaching a plateau. Although the absolute quantum yield of fluorescein (~0.9 at low concentrations) is higher than AIE luminogen 1 (~0.25 in aggregates or solids),39, 45 the fluorescence intensity of AIE-FSNPs-1 could be enhanced by embedding more compound 1 to overcome the maximum fluorescence of FITC-FSNPs (normalized by the same mass of silica), achieving at least 4-fold brighter fluorescence in the concentration range of this study (Figure S5a, Supporting Information). The concentration-dependent fluorescence of 1 and fluorescein (Figure S6, Supporting Information) also demonstrated that the highest possible fluorescence of 1 could exceed that of fluorescein. In addition to fluorescence intensity, another characteristic of AIE luminogens is its fluorescent aggregates, so that their fluorescence should be much more insensitive to environmental variations such as pH than conventional fluorophores,

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because most of the AIE molecules inside the aggregates are inaccessible to the solvent molecules. The insensitivity to pH changes can minimize the effect of cellular pH variations on the imaging of targets other than pH.49 To testify this hypothesis, the fluorescence of AIE-FSNPs-1 and FITC-SFNPs was monitored in a serial of buffer solutions under different pH conditions. The fluorescence intensity of AIE-FSNPs-1 in aggregates was found insensitive (within 5% variation) to pH in the range of 5-9, while that of FITC-FSNPs showed a large variation over 300% in the same pH range (Figure S2b, Supporting Information), because fluorescein is a pH sensitive molecule in solution switching between its lactone (at low pH, low fluorescence) and ring-open (at high pH, strong fluorescence) tautomers.50 In addition to the FITC-FSNPs above, commercial fluorescein-doped silica nanoparticles and polystyrene beads were also studied and their fluorescence properties were compared with that of AIE-FSNPs-1. The fluorescence of AIE-FSNPs-1 was found the highest among them (Figure S7 and Table S2, Supporting Information). To conjugate the nanoparticles with DNA aptamers, AIE-FSNPs-1 were first reacted with APS to introduce amine groups to the surface of the nanoparticles (amineAIE-FSNPs-1, Figure 2, step (b)), which were subsequently treated by sulfo-succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (Sulfo-SMCC) to convert the amines to thiol-reactive maleimides on the nanoparticles (maleimide-AIE-FSNPs-1, Figure 2, step (c)).51 Finally, thiol-modified DNA aptamers specific to nucleolin were attached to the nanoparticles through the thiol-maleimide reaction51 to provide the aptamer-functionalized nanoparticles (Apt-AIE-FSNPs-1, Figure 2, step (d)).

Figure 5.Fluorescence images of MCF-7 and MCF-10A cells incubated with Apt-AIE-FSNPs-1or rDNA-AIE-FSNPs-1. (a) Fluorescence channel (excitation by 405 nm laser and emission within 500-550 nm); (b) Merge of fluorescence channel and bright channel.

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Figure 6. (a) Cytotoxicity of Apt-AIE-FSNPs-1 at different concentrations to MCF-7 cells after 8 h incubation. (b) Flowcytometry analysis of MCF-7 and MCF-10A cells incubated with Apt-AIE-FSNPs-1 and AIE-FSNPs-rDNA-1.

To confirm each step of surface modification was efficient in the above conjugation procedure, we carried out zeta potential analysis of the nanoparticles at each step including AIE-FSNPs-1, amine-AIE-FSNPs-1, maleimideAIE-FSNPs-1 and Apt-AIE-FSNPs-1. As depicted in Figure 4a, AIE-FSNPs-1 were negatively charged (−53.1 mV), which was ascribed to the surface Si-OH groups undergoing partial deprotonization. However, after introducing amines, amine-AIE-FSNPs-1 were positively charged (+2.3 mV) because of the protonization of amines in neutral conditions. The formation of maleimide-AIE-FSNPs-1 dismissed the positive charges and made the surface slightly less negatively charged (−49.8 mV) compared to AIE-FSNPs-1. The further enhanced negative charges (−78.9 mV) on the surface of Apt-AIE-FSNPs-1 suggested the successful conjugation of DNA aptamers on the nanoparticles. To further conform the successful conjugation of DNA, modified fluorescein-labeled DNA aptamer was also linked onto AIE-FSNPs-1 by using the same conjugation procedure (Figure S8). The TEM image of AptAIE-FSNPs-1 was very similar to those of AIE-FSNPs-1 (Figure S9, Supporting Information), indicating the nanoparticle underwent almost no shape change of the inorganic silica component during the DNA conjugation. However, the diameter of Apt-AIE-FSNPs-1 from DLS analysis increased about 30 nm compared with AIEFSNPs-1, suggesting the attachment of DNA and linkers increased the hydration diameter of the particles. The amount of 1 and DNA in or on the 140 nm Apt-AIEFSNPs-1 (prepared using 1 mmol 1 and 1.25 mL ammonium for the synthesis) was measured as around 881 nmol and 3.5 nmol per mmol SiO2, respectively, by the element analysis of AIE-FSNPs-1 before amine-functionalization and the UV260 measurement of DNA concentrations during the conjugation between thiol-DNA and maleimideAIE-FSNPs-1. More efficient embedment of AIE luminogen 1 was observed in larger AIE-FSNPs-1 (517, 804 and 881 nmol per mmol SiO2 for 60, 100 and 140 nm nanoparticles, respectively). Through the above method, Apt-AIEFSNPs containing 1-3 were all successfully prepared, and their fluorescent properties were still fully preserved as

those of the corresponding AIE-FSNPs (Figure 4b), suggesting the optical properties of these AIE-FSNPs were compatible with the conjugation chemistry. Targeted cell imaging using Apt-AIE-FSNPs Encouraged by the advantages of AIE-FSNPs and the successful DNA conjugation, we further applied the 140 nm Apt-AIE-FSNPs for targeted cell imaging based on the specific recognition of the DNA aptamer to nucleolin,35-36, 52-55 a membrane protein overexpressed in cancer cell lines. It was demonstrated that the ideal size of nanoparticles for cellular uptake should be no more than 200 nm in diameter,56 so all the three different sizes of nanoparticles (60, 100 and 140 nm) in our work could be used for cellular study in principle. According to a study showing that non-modified silica nanoparticles around 50 nm was of highest efficiency for cell uptake while that of 110 nm is of lower efficiency,57 we therefore used the largest silica nanoparticles (140 nm) to minimize the non-specific cellular uptake of the nanoparticles for a more efficient aptamerenhanced uptake. Toward MCF-7 (a breast cancer cell line with nucleolin overexpression) and MCF-10A (a normal cell line) cells, Apt-AIE-FSNPs containing each of 1-3 were added to the culture media and the cells were incubated to allow the binding as well as uptake of the nanoparticles into the cells. The culture media was then discarded and the cells were washed to remove unbound Apt-AIE-FSNPs, then the cells were imaged under a confocal fluorescence microscope equipped with a laser for excitation at 405 nm and a green, yellow or orange channel for emission detection. As displayed in Figure 5, green fluorescence was clearly observed inside MCF-7 cells incubated with AptAIE-FSNPs-1 (Figure 5, column 1 and 2), while almost no fluorescence signal was found for MCF-10 cells after incubation with Apt-AIE-FSNPs-1(Figure 5, column 3), suggesting the high selectivity of Apt-AIE-FSNPs-1 to fluorescently stain the target cells. The fluorescence inside MCF7 cells should be because of the efficient uptake of AptAIE-FSNPs-1 by the cells, through the selective binding of the DNA aptamer to the nucleolin on MCF-7 cells as well

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as the endocytosis activated by the binding, while AptAIE-FSNPs-1 could hardly bind MCF-10A owing to the lack of nucleolin overexpression on the cells. Instead of the DNA aptamer, another random DNA sequence (rDNA) that should not bind to nucleolin was used as a control to modify AIE-FSNPs-1 following the same procedure to form rDNA-AIE-FSNPs-1. MCF-7 cells incubated with such rDNA-AIE-FSNPs-1 showed little fluorescence (Figure 5, column 4), indicating the essential role of the DNA aptamers for efficient MCF-7 cell binding. Maleimide-AIE-FSNPs-1 nanoparticles without DNA conjugation were found to exhibit strong cellular uptake, very likely because that the maleimide groups on the particle surface efficiently reacted with the thiols on membrane proteins to form stable covalent bonds (Figure S10, Supporting Information), suggesting the essential role of DNA-modification to reduce non-specific cellular binding or uptake of the silica nanoparticles. In fact, it has been reported that the internalizing and uptake rates of nanoparticles was dependent on many factors such as particle type, size, shape, surface charge, modification, cell type, as well as culture time56, 58. A study compared the uptake of silica nanoparticles for HUVEC and HeLa cells, and the intracellular accumulation of silica nanoparticles was up to 10 times higher for HUVEC than HeLa cells.58 The aptamer AS1411 was highly specific to bind with nucleolin overexpressed on many cancer cells, including MCF-7, HeLa, HepG2 and so on.59 There are other reports that successfully distinguished MCF-7 cells from normal cells.35, 60-61 Aptamer-modified gold nanoparticles can assist their cellular uptake for ATP detection inside live cells.62 These results suggested that the uptake of nanoparticles by cells are complicated and determined by a serial of factors, while aptamer functionalization could

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indeed induce much stronger cellular uptake compared to random DNA sequences. The cytotoxicity of Apt-AIE-FSNPs-1 was studied using the MTT assay and the cells were found mostly alive upon incubation with Apt-AIE-FSNPs-1 in a concentration range of 0-0.05 mg/mL for at least 8 h (Figure 6a), demonstrating the good biocompatibility of Apt-AIEFSNPs-1 for cellular studies. To further confirm the selectivity of the nanoparticles to MCF-7 cells over MCF-10A cells, in addition to fluorescence imaging, flow-cytometry analysis was also carried out using MCF-7 andMCF-10A cells incubated with AptAIE-FSNPs-1 or rDNA-AIE-FSNPs-1. As demonstrated in Figure 6b, a large amount of strongly fluorescent cells (>200 fold over blank) were only observed for MCF-7 cells incubated with Apt-AIE-FSNPs-1, while the signal of MCF-7 cells with rDNA-AIE-FSNPs-1 was much weaker (10 fold over blank). MCF-10A cells with either Apt-AIEFSNPs-1 or rDNA-AIE-FSNPs-1 were found similar to the blank. These results supported that the efficient cellular binding and uptake of the nanoparticles only occurred when the recognition of MCF-7 cells by the DNA aptamers was present. To testify whether the binding between Apt-AIEFSNPs-1and MCF-7 cells was originated from the specific interaction between nucleolin and the aptamer, a nucleolin antibody co-localization study was carried out. First, fluorescein-labeled aptamer (fluorescein-aptamer) and cyanine5 -labeled aptamer (Cy5-aptamer) were found to overlap almost completely with each other in MCF-7 cells (Figure S11, Supporting Information), which served as a positive control to confirm the instrument setup was suitable for the co-localization study. Further, fluoresceinaptamer and Cy5-labeled antibody (Cy5-antibody)

Figure 7. Antibody co-localization study of Cy5-labeled nucleolin antibody with (a) fluorescein-labeled aptamer and (b) AptAIE-FSNPs-1.Excitation laser and emission range are 405/488/638 nm and 500-550/500-550/663-738 nm for 1, fluorescein and Cy5, respectively.

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Figure 8. Fluorescence images of MCF-7 cells incubated with Apt-AIE-FSNPs-2 and Apt-AIE-FSNPs-3 (before and after UV irradiation). (a) Fluorescence channel (excitation by 405 nm laser, and emission within 525-575 nm and 550-600 nm for Apt-AIEFSNPs-2 and Apt-AIE-FSNPs-3, respectively); (b) Merge of fluorescence channel and bright channel.

were incubated with MCF-7 cells in the same solution, and the antibody stain was found to co-localize within that of the aptamer (Figure 7a), with a higher staining efficiency for the aptamer mainly due to the smaller size and higher concentration of the aptamer conjugate used. Finally, Apt-AIE-FSNPs-1 and Cy5-antibody were mixed for incubation with MCF-7 cells, and the Apt-AIE-FSNPs-1 stain was within that of the antibody (Figure 7b), with less staining area for the nanoparticles likely because of the much larger nanoparticles than the antibody and their different endocytosis efficiency. Collectively, these data strongly indicated that the aptamer and aptamerfunctionalized AIE-FSNPs bound to MCF-7 cells through the nucleolin overexpressed on the cell membrane. Using Apt-AIE-FSNPs-2 instead of Apt-AIE-FSNPs-1, MCF-7 cells were also successfully imaged in the yellow channel instead of the green channel (Figure 8, column 1), enabling the fluorescent imaging of target cells in multiple channels (colors) using different AIE luminogens in this study. More interestingly, when photoactivatable Apt-AIE-FSNPs-3 were applied for MCF-7 cell imaging, the cells after incubation with Apt-AIE-FSNPs-3 were initially non-fluorescent (Figure 8, column 2) and the fluorescence in the orange channel only showed up after the sample was irradiated by UV light at 365 nm (Figure 8, column 3),45-46, 63 though the non-fluorescent Apt-AIEFSNPs-3 before the irradiation should have already bound to the cells efficiently via the DNA aptamer and underwent cellular uptake. The efficiency of the photo-induced conversion of 3 to 4 in these AIE-FSNPs-3 was found to be as high as 91%, according to the quantification of 3 in the nanoparticles by HPLC analysis (Figure S12, Supporting

Information). Such photoactivatable characteristics of Apt-AIE-FSNPs-3 can be applied for selective imaging of target cells at a specific location of interest by controlling the site of UV irradiation.49

Conclusion In summary, we have prepared a serial of aptamerfunctionalized and AIE luminogen-embedded fluorescent silica nanoparticles (Apt-AIE-FSNPs) by a general method, and applied them for targeted cell imaging. Compared with conventional FSNPs such as commercial fluoresceindoped silica and polystyrene nanoparticles, these AIEFSNPs are synthesized through a facile non-covalent approach, and are much brighter in fluorescence. The fluorescence of AIE-FSNPs can be further enhanced by loading more AIE luminogens into the nanoparticles without encountering any ACQ effect. It is also less sensitive to environmental changes such as pH, which may reduce the bias of imaging for the fluorescent nanoparticles at different cellular environment. Moreover, the Apt-AIE-FSNPs functionalized by DNA aptamers specific to nucleolin are successfully applied for targeted cell imaging of MCF-7 cells over MCF-10A cells with a high selectivity. Multiplecolor and photoactivatable cell imaging have also been achieved using the Apt-AIE-FSNPs containing each of the AIE luminogens 1-3 as well. We believe the conjugation of other biomolecules such as proteins and polysaccharides to these AIE-FSNPs will provide more useful imaging reagents with advantageous fluorescent properties for cellular studies.

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AUTHOR INFORMATION Corresponding Author Yu Xiang* Department of Chemistry, Beijing Key Laboratory for Microanalytical Methods and Instrumentation, Key Laboratory of Bioorganic Phosphorus Chemistry and Chemical Biology (Ministry of Education), Tsinghua University, Beijing 100084, China. Email: [email protected]

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 thank for the financial support from the National Natural Science Foundation of China (Nos. 2142200199, 21390410 and 21375074), the National Key Scientific Instrument and Equipment Development Project of China (No. 2012YQ030111), the Tsinghua University Initiative Scientific Research Program (No. 20131089220), and the Recruitment Program of Global Youth Experts of China.

SUPPORTING INFORMATION AVAILABLE Experimental procedures and additional figures. This information is available free of charge via the Internet at http://pubs.acs.org/

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Aggregation-Induced Emission Luminogen-Embedded Silica

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