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Stable and Size-Tunable Aggregation-Induced Emission Nanoparticles Encapsulated with Nano Graphene Oxide and Applications in Three-Photon Fluorescence Bioimaging Zhenfeng Zhu, Jun Qian, Xinyuan Zhao, Wei Qin, Rongrong Hu, Hequn Zhang, Dongyu Li, Zhengping Xu, Ben Zhong Tang, and Sailing He ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.5b05606 • Publication Date (Web): 07 Dec 2015 Downloaded from http://pubs.acs.org on December 8, 2015

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Stable and Size-Tunable Aggregation-Induced Emission Nanoparticles Encapsulated with Nano Graphene Oxide and

Applications

in

Three-Photon

Fluorescence

Bioimaging Zhenfeng Zhu, †

[+],†

Jun Qian, §

[+],†

§

Li, Zhengping Xu, Ben Zhong Tang, †

‡

φ

†

Xinyuan Zhao, Wei Qin, Rongrong Hu, Hequn Zhang, Dongyu *,‡

and Sailing He

*†

State Key Laboratory of Modern Optical Instrumentation, Centre for Optical and Electromagnetic

Research, Zhejiang Provincial Key Laboratory for Sensing Technologies, JORCEP (Sino-Swedish Joint Research Center of Photonics) Zhejiang University, Hangzhou, 310058, China.

§

Institute of

Environmental Health, School of Public Health, Zhejiang University, Hangzhou, 310058, China.

‡

Department of Chemistry, the Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, China. φSCUT-HKUST Joint Research Laboratory, Guangdong Innovative Research Team, State Key Laboratory of Luminescent Materials and Devices, South China University of Technology (SCUT), Guangzhou 510640, China.

[+]

These authors contributed equally to this work.

* Address correspondence to: [email protected], [email protected]. KEYWORDS: Nano graphene oxide • aggregation-induced emission • nanoparticles • three-photon luminescence • bioimaging ABSTRACT: Organic fluorescent dyes with high quantum yield are widely applied in bioimaging and biosensing. However, most of them suffer from a severe effect called aggregation-caused quenching (ACQ), which means that their fluorescence is quenched at high molecular concentrations or in the

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aggregation state. Aggregation-induced emission (AIE) is a diametrically opposite phenomenon to ACQ, and luminogens with this feature can effectively solve this problem. Graphene oxide has been utilized as a quencher for many fluorescent dyes, based on which biosensing can be achieved. However, using graphene oxide as a surface modification agent of fluorescent nanoparticles is seldom reported. In this manuscript, we used nano graphene oxide (NGO) to encapsulate fluorescent nanoparticles, which consisted of a type of AIE dye named TPE-TPA-FN (TTF). NGO significantly improved the stability of nanoparticles in aqueous dispersion. In addition, this method could control the size of nanoparticles flexibly, as well as increase their emission efficiency. We then used the NGO-modified TTFnanoparticles to achieve three-photon fluorescence bioimaging. The architecture of ear blood vessels in mice and the distribution of nanoparticles in zebrafish could be observed clearly. Furthermore, we extended this method to other AIE luminogens and showed it was widely feasible.

Organic fluorescent dyes have been widely used in biosensing and bioimaging due to their advantages of high quantum yield, sensitivity, selectivity, and biocompatibility.1-4 Among them, deep red/nearinfrared dyes are highly desired in in vivo bioimaging.5,6 However, due to a notorious phenomenon called aggregation-caused quenching (ACQ),7 most organic dyes are fluorescent only in dilute solutions, and their fluorescence is quenched when they are in the aggregation state or at relatively high molecular concentrations.8 The main reason for the ACQ effect is that conventional dyes are typically comprised of planar aromatic rings which have strong π–π stacking interactions, and the excited states of the dyes in aggregation form often decay via non-radiative pathways.9,10 In 2001, Tang’s group discovered a phenomenon opposite to ACQ. A series of silole molecules were found non-luminescent in benign solvents but emissive in the aggregation state (e.g. as nanoparticle suspensions in poor solvents or as solid-state thin films), and this novel phenonemon is called the aggregation-induced emission (AIE) effect.11,12 They identified restrictions in intramolecular rotation (RIR) in the aggregates as a main reason for the AIE effect.12 Due to their novel properities, AIE luminogens have been widely utilized ACS Paragon Plus Environment

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nowdays in various research fields, such as organic light emitting diode (OLED),13 chemical/biological sensing,14 lasing,12 bioimaging,15 etc. As a derivative of graphene, graphene oxide (GO) is a single layer material consisted of sp2hybridized carbon atoms arranged in a honeycomb two-dimensional (2-D) crystal lattice.16 Singlelayered GO has every atom (two sides), as well as rich functional groups (e.g. -OH, C=O), exposed on its surface, giving it an extremely large surface area and many opportunities to link drugs and other functional molecules through covalent/noncovalent interaction.17,18 Nano graphene oxide (NGO) is a nanoscale form of graphene oxide, which keeps most properities of GO. NGO also possesses its own unique characters due to the size effect, such as good aqueous-dispersity and fluorescence emission around 500 nm under one-/two-photon excitation.19 In addition, since GO is mainly comprised of carbon, it is relatively biocompatible, making it an ideal candidate for biological applications. A commonly used way to generate AIE-nanoparticles is the reprecipitation method, in which AIE molecules in benign solvents (e.g. THF, DMSO, DMF) were mixed with their poor solvent (e.g. water).20 However, in this approach, the benign solvent is usually poisonous, which limits the applications of AIE-nanoparticles in biological environments. In addition, AIE-nanoparticles are quite unstable in these solution systems, and they are prone to aggregate and deposit spontaneously after a certain short time. Thus, many methods have been proposed to synthesize biocompatible and stable AIE-nanoparticles via certain surface modification.21,22 TPE-TPA-FN (TTF) is a typical deep-red emitted AIE luminogen. Herein, we used NGO as a surface modification agent to encapsulate TTF-nanoparticles. The acquired NGO modified TTF-nanoparticles (TTF-NGO NPs) showed very good stability in aqueous dispersion, and their size could be tuned by controlling the amount of added NGO. Surprisingly, the surface modification of NGO increased the emission efficiency of TTF-nanoparticles instead of quenching their fluorescence, since previously GO has been widely utilized as a quencher for most fluorescent probes.23,24 The stable and emission enhanced TTF-NGO NPs could also emit bright three-photon luminescence (3PL) due to the high ACS Paragon Plus Environment

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nonlinear optical (NLO) efficiency of TTF, and so they were used as fluorescent probes for 3PL bioimaging. With the help of strong 3PL signals from TTF-NGO NPs, the architecture of blood vessels in mice ears and the distrubution of nanoparticles in zebrafish could be observed clearly. Furthermore, we popularized this method to other AIE luminogens and proved it was widely feasible. RESULTS AND DISCUSSION Preparation and Characterization of TTF-NGO NPs. NGO was synthesized with a modified Hummer’s method based on previous reports.19,25,26 According to TEM and dynamic light scattering (DLS) analysis (Figure 1A and B), the as-synthesized NGO had an average size of ~30 nm. The absorption and emission spectra of NGO in aqueous dispersion were shown in Figure S1, and it emitted green fluorescence under the excitation of a hand-held ultraviolet lamp (inset), with the emission peak at 500 nm. TTF was synthesized according to the previous report,27 and its chemical structure is shown in Figure 1C. It is a typical donor-π-acceptor-π-donor structure with extended π-conjugation, giving it distinct NLO effects, as well as causing it to emit deep-red fluorescence upon excitation.27,28 The AIE feature of TTF is attributed to the two TPE groups on the two ends of the molecule.27,28 The absorption and (onephoton) fluorescence spectra of TTF in a THF/water mixture [with a water volume fraction (fwater) of 80 vol%] are shown in Figure 1D. Its absorption peak was at 520 nm while its emission spectrum centered at 625 nm. Before using hydrophobic TTF in a hydrophilic biological environment, certain surface modifications are required, among which sophiscated PEG and silica coating have been widely used.28,29 Here, we proposed a new and simple method to modify TTF-nanoparticles with NGO (as shown in Scheme 1). The TEM (Figure 1E) and DLS (Figure 1F) analyses showed that the TTF-NGO NPs had an average size of ~250 nm.


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Figure 1. A) A TEM image of NGO. B) DLS number-weighted diameter of NGO. Inset: a photograph of aqueous dispersion of NGO under daylight. C) Molecular structure of TTF. D) Absorption and fluorescence spectra of TTF in a THF/water mixture with fwater=80%. E) A TEM image of TTF-NGO NPs (concentration of TTF [TTF]=0.1 mM, and concentration of NGO [NGO]=20 µg/ml). F) DLS number-weighted diameter of TTF-NGO NPs ([TTF]=0.1 mM, and [NGO]=20 µg/ml). Inset: a photograph of aqueous dispersion of TTF-NGO NPs under daylight. Scare bar in TEM images: 200 nm.


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Scheme 1. Schematic illustration for the synthesis of TTF-NGO NPs. Effects of NGO on Chemical Stability and Size Tunability of TTF Nanoparticles. Most AIE-nanoparticles synthesized via the reprecipitation method20 is quite unstable and prone to precipitate quickly. To demonstrate the advantages of our method, the THF solution of TTF was mixed with water (fwater=80 vol%), and NGO with various quantities was then added to the above system to form TTF-NGO NPs. The final concentration of TTF was 0.1 mM. TTF-nanoparticles without the addition of NGO was used as the control. We then monitored the absorbance of various nanoparticles at 520 nm for 14 days, from which we could determine their stability. As shown in Figure 2A, for TTF-nanoparticles without the addition of NGO, the absorbance decreased by 85% after only two days. However, the absorbance of nanoparticles with the addition of NGO decreased gently, indicating that the stability of nanoparticles had been improved. TTF-NGO NPs were very stable when the concentration of added NGO was higher than 20 µg/ml, since in those cases, their absorbance decreased by less than 25% even after 14


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days. In addition, we also monitored the peak fluorescence intensities of TTF-nanoparticles with the concentration of added NGO ranging from 0 to 40 µg/ml, and the same tendency could be observed (Figure 2B). These results illustrated that the surface modification of NGO could effectively stabilize the TTF-nanoparticles.

Figure 2. A) Temporal evolution of the absorbance at 520 nm of 0.1 mM TTF in THF/water mixture (fwater=80 vol%), with varying quantities of added NGO. B) Temporal evolution of the peak fluorescence intensity of 0.1 mM TTF in THF/water mixture (fwater=80 vol%), with varying quantities of added NGO. C-E) TEM images of TTF-NGO NPs ([TTF]=0.1 mM), with varying quantities of added NGO: C) 5 µg/ml NGO, D) 10 µg/ml NGO and E) 20 µg/ml NGO. F) A TEM image of TTF-NGO NPs ([TTF]=0.01 mM), with the addition of 10 µg/ml NGO. Scale bar in TEM images: 500 nm. Furthermore, by controlling the quantity of NGO, the size of the TTF-nanoparticles could also be


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tuned. Figure 2 C-E show the TEM images of TTF-NGO NPs ([TTF]=0.1 mM) with different quantities of NGO, and the average diameter of the nanoparticles were ~400 nm ([NGO]=5 µg/ml), ~300 nm ([NGO]=10 µg/ml) and ~200 nm ([NGO]=20 µg/ml), respectively. In addition, the size of TTF-NGO NPs could also be tuned by changing the concentration of TTF (before dialysis and centrifugation) while keeping the quantity of NGO the same. The average diameter of TTF-NGO NPs ([TTF]=0.01 mM, and [NGO]=10 µg/ml) was ~100 nm (Figure 2F), which was much smaller than that of TTF-NGO NPs ([TTF]=0.1 mM, and [NGO]=10 µg/ml), as shown in Figure 2D. The DLS number-weighted diameter characterization also showed the same tendency as the TEM images (Figure S4). Enhanced Emission Efficiency of TTF Nanoparticles after Surface-modification of NGO. Previously, GO has been widely utilized as an efficient quencher for many fluorescent probes, via fluorescence resonance energy transfer or charge transfer.23 To determine whether the quenching effect of outer layers of NGO would influence the fluorescence efficiency of TTFnanoparticles, we prepared several types of nanoparticles, which were TTF-nanoparticles (fwater=80, 90, and 95 vol% in THF/water system) without the addition of NGO, TTF-NGO NPs ([TTF]=0.1 mM and [NGO]=20 µg/ml), and DSPE-mPEG coated TTF nanoparticles (abbreviated as TTF-PEG NPs). It is worth mentioning that using DSPE-mPEG to encapsulate hydrophobic dyes is a simple and popular way to synthesize fluorescent nanoparticles,30 and TTF-PEG NPs have been widely applied in bioimaging.28,31 For each sample, the absorbance at peak wavelength was kept the same (Figure 3A). In this case, the concentration of TTF in each sample could be considered to be the same according to Beer's Law. 520 nm-light was then used to excite the fluorescence of these samples. Surprisingly, TTF-NGO NPs had the brightest fluorescence among all of them (Figure 3B). To determine the reason for this phenomenon, DLS


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was used to determine the size of these nanoparticles, since the emission efficiency of AIE luminogens is proportional to their aggregation degree. As shown in Figure 3C, TTFnanoparticles without NGO (fwater= 95 vol%) had the largest size. TTF-NGO NPs were smaller than the "naked" TTF-nanoparticles, but their average size was larger than that of TTF-PEG NPs (~100 nm, since larger-sized TTF-PEG NPs were difficult to prepare31). TTF is a molecule that exhibits the twisted intramolecular charge transfer (TICT) effect,28 which is always observed in organic fluorophores with donor-π-acceptor structure, and featured with increased emission intensity with decreasing solvent polarity.15 For "naked" TTF-nanoparticles, they were exposed to a large polar environment, which arose from the THF/water mixture. For TTF-NGO NPs, the surface modification of NGO provided a relatively less polar environment for TTF molecules. Thus, although the aggregation degree of TTF-NGO NPs was much smaller than that of "naked" TTF-nanoparticles, the TICT effect helped them achieve higher emission efficiency due to the modification of NGO. We further investigated the quenching effect of NGO on TTFnanoparticles via fluorescence lifetime measurement. As we know, the fluorescence lifetime of fluorophores would shorten when fluorescence quenching takes place. However, the fluorescence lifetime of TTF-NGO NPs was almost the same as that of TTF-PEG NPs (Figure 3D). Since DSPE-mPEG is not a fluorescence quencher, it would not affect the lifetime of TTFPEG NPs obviously. The lifetime result illustrates that no obvious fluorescence quench occurred on TTF-nanoparticles when NGO was attached onto their surface. The high quenching efficiency of GO for traditional organic fluorophores in previous studies was mainly due to the strong π-π stacking effect and electrostatic interaction between the fluorophore molecules and GO, and this would induce the formation of tight complexes to quench the fluorescence of organic fluorophores via an efficient energy/charge transfer process.32-34 In our case, TTF is a type of


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AIE luminogen with non-planar and highly twisted propeller-shaped molecular conformation, as well as neutral electric charge, and these would weaken hamper the π-π stacking effect and electrostatic interaction with NGO. It provided a relatively large distance between the NGO and TTF molecules when they formed TTF-NGO NPs, which could restrict to some extent the quenching effect towards the outer layers of TTF NPs. Another possible reason was that the particle size of NGO in our study was much smaller than those of GO in previous reports, and its fluorescence quenching capability should be much weaker. Furthermore, the size of TTF NPs was very large (~250 nm) while NGO (~30 nm) was just a single layer on their surface. Even NGO may quench the fluorescence of TTF molecules at some outer layers of TTF-NGO NPs, the emission of most TTF molecules inside the NPs, which dominates the fluorescence behavior of the whole TTF-NGO NP, should not be affected. TTF-NGO NPs with larger size had brighter fluorescence than TTF-PEG NPs, and it was due to the reason that higher concentration of TTF molecules (with AIE feature) aggregated inside the TTF-NGO NPs. Compared with those existing approaches to fabricate AIE-nanoparticles (e.g. silica or PEG coating),29,31,35 our proposed method is faster, simpler and more effective.


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Figure 3. A) Absorbance and B) fluorescence spectra of TTF-NGO NPs, TTF-PEG NPs and TTF in THF/water mixtures with different water volume fractions (fwater=80, 90 and 95 vol%). C) DLS number-weighted diameter of TTF-NGO NPs, TTF-PEG NPs and TTF in THF/water mixture (fwater=95 vol%). D) Time-resolved decay profiles of TTF-NGO NPs and TTF-PEG NPs. 3PL of TTF-NGO NPs and Applications in Bioimaging. Compared to ACQ dye, AIEactive TTF can be designed as nanoparticles with very high doping concentration, possessing high linear optical (LO) and NLO efficiency. Herein, we studied the NLO effect of TTF-NGO NPs ([TTF]=0.1 mM and [NGO]=20 µg/ml) with a scanning microscope equipped with a 1560 nm femtosecond (fs) laser (Figure S5), and two types of NLO signals were observed (Figure 4A). One was a sharp spectrum with its peak at 520 nm. As 520 nm equals to one-third of 1560


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nm, it was determined to be a third harmonic generation (THG) signal (from TTF-NGO NPs), which is a typical third-order NLO effect. The other was a 3PL signal, with its emission peak at 660 nm. Its spectrum envelope was very similar to that of one-photon luminescence (Figure 1D). We measured the three-photon absorption (3PA) cross-section of single TTF molecule at the wavelength of 1560 nm, and the measured value was ~3.82×10-78 cm6 s2. Based on this value, we further calculated the 3PA cross-section of single TTF-NGO NP (σNP), and the result was ~1.45×10-68 cm6 s2. The three-photon action cross-section of single TTF-NGO NP (equal to η σNP, where the measured fluorescence quantum yield η of TTF-NGO NPs had a value of 34.2%) was calculated to be 4.96×10-69 cm6 s2. Figure 4B shows the 3PL image of a capillary glass tube filled with aqueous dispersion of TTF-NGO NPs, taken with the aforementioned scanning microscope. The 3PL signals from TTF-NGO NPs were very distinct, which allowed the morphology of the capillary glass tube to be discriminated easily from the background. We further explored the application of TTF-NGO NPs in 3PL bioimaging, under the excitation of a 1560 nm fs laser. Considering the fact that 3PA cross-section of endogenous fluorophores inside an animal body was very small while exogenous TTF-NGO NPs possess high 3PL efficiency, TTF-NGO NPs-assisted 3PL in vivo microscopy could give high SBR (signal to background ratio), and 3PL images with high contrast could be achieved. In addition, 1560 nm is very close to an optical tissue window ranging from 1600 to 1800 nm,6 and light at this wavelength has much lower tissue scattering than the wavelength of commonly used fs laser sources (e.g. 700-1000 nm for Ti: sapphire fs laser). Thus, 3PL microscopy based on 1560 nm fs excitation could achieve much larger imaging depth for in vivo application. TTF-NGO NPs were intravenously injected into a mouse via its tail vein, and the ear of the mouse was imaged with


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the scanning microscope. By virtue of the bright 3PL from TTF-NGO NPs, the structure of blood vessels in the mouse ear at different vertical depths (Figure 4D) was clearly revealed, and Figure 4C shows a reconstructed 3D image of the vascular architecture. TTF-NGO NPs were also microinjected into the embryos of zebrafish. After being cultured for 48 h, the zebrafish were used for 3PL imaging. Compared with the transmission image of zebrafish in Figure S7, we found TTF-NGO NPs distributed uniformly in the zebrafish according to their 3PL signals (Figure 4E). Even after allowing the zebrafish to grow, no obvious aggregation of TTF-NGO NPs was observed since no abnormally bright fluorescent spots could be observed, indicating they were chemically and optically stable in the zebrafish. TTF-NGO NPs with high biocompatibility, chemical stability, and bright NLO signals can be used as promising bioprobes for in vivo functional imaging in the future.


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Figure 4. A) 3PL and THG spectra of aqueous dispersion of TTF-NGO NPs. B) A 3PL image of aqueous dispersion of TTF-NGO NPs in a capillary glass tube. C) A reconstructed 3D image of the blood vessels of a mouse ear, which was intravenously injected with TTF-NGO NPs. D) 3PL


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images of ear blood vessels of the mouse, which were taken at depths of 5 µm, 25 µm, 45 µm, and 65 µm. E) A 3PL image of zebrafish, 48 h after the addition of TTF-NGO NPs into its embryos. All the spectra and images were taken under a scanning microscope equipped with a 1560 nm-fs laser. Scale bar: 500 nm. Study on Other AIE-NGO NPs. In addition to TTF, we also tried to verify whether the NGO modification could improve the performance of other AIE-nanoparticles. Herein, we selected four types of AIE molecules, HPS,11 TPE,12 BODIPY-TPE-3 (BT3),36 and TPEPT137 (Figure S8), to fabricate corresponding AIE-nanoparticles (fwater=80 vol% in THF/water system, [AIE molecule]=0.1 mM). By monitoring the absorption spectra of AIE-nanoparticles with/without the addition of NGO, we could determine their stability in aqueous dispersion. According to Figure 5A-D, AIE-nanoparticles without the addition of NGO were not stable, and they precipitated completely in water two days after preparation. The absorption spectra of AIE-nanoparticles with the addition of NGO did not change much after two days, indicating that their stability improved after the surface-modification of NGO. The samples were further dialyzed and centrifuged, and their TEM images are shown in the insets of Figure 5A-D. The nanoparticles were spherical and had uniformly distributed sizes. Figure 5E-F show the photographs of the as-synthesized aqueous dispersion of AIE-NGO NPs under daylight and UV irradiation, respectively. They were clear and transparent, and their emitted fluorescence was uniform and bright, indicating the AIE-NGO NPs dispersed well in water and no obvious fluorescence quenching occurred after the modification of NGO. The good colloidal stability, as well as the uniform distribution of the aqueous dispersion of all the AIE-NGO NPs, was further confirmed by the Zeta-potential analysis in Table 1. For all the nanoparticles, their Zeta-potentials were between -20 and -33 mV, which are very close to that of pure NGO (~-24.5 mV), and we could verify the presence of


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NGO on the surface of the AIE-nanoparticles. We proved that NGO modification is a widely feasible way to fabricate stable and bright AIE-nanoparticles, which may have potential applications in many areas.

Figure 5. A-D) Temporal evolution of absorption spectra of different kinds of samples: A) TPE B) HPS C) BT3 and D) TPETP1 in THF/water mixture (fwater=80 vol%, [AIE molecule]=0.1 mM) with/without the addition of 20 µg/ml NGO. (insets: TEM images of HPS-NGO NPs, TPE-NGO


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NPs, BT3-NGO NPs, and TPEPT1-NPs, respectively.) E-F) Photographs of aqueous dispersion of the five types of AIE-NGO NPs, under E) daylight and F) UV irradiation. Table 1. Zeta-potential characterization of AIE-NGO NPs and NGO NPs: Nanoparticles

Zeta potential water (mV)

NGO NPs

-24.5±2.3

TTF-NGO NPs

-32.9±2.6

TPEPT1-NGO NPs

-28.7±2.7

BT3-NGO NPs

-29.0±2.5

TPE-NGO NPs

-21.1±2.3

HPS-NGO NPs

-23.4±2.4

in

DI

CONCLUSION In summary, surface-modification of NGO could greatly improve the stability of TTFnanoparticles in a THF/water mixture. In addition, the size of the TTF-nanoparticles could be tuned easily by adjusting the amount of NGO. TTF-NGO NPs with low toxicity, high chemical stability and rich NLO effects were further used for 3PL bioimaging. The architecture of blood vessels in mice ears and the distribution of nanoparticles in zebrafish could be observed clearly. We also expanded this method to other AIE luminogens and proved it was widely feasible and applicable. In the future, AIE-NGO NPs can be conjugated with various bio-molecules and drugs though the rich groups and large surface area of NGO, and achieve various biomedical applications.

MATERIALS AND METHODS


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Materials. NGO was synthesized following our previous work.19 TTF27 and other AIE luminogens used in this manuscript were prepared in Prof. Ben Zhong Tang’s11,12,36 and Prof. Jianli Hua’s group.37 DSPE-mPEG2000 was purchased from Creative PEGWorks, Inc. Tetrahydrofuran (THF) was obtained from Sinopharm Chemical Reagent Co., Ltd. Reagents for mice and zebrafish culture were purchased from Invitrogen Co. Deionized (DI) water was used in all the experimental procedures. Characterization. TEM images were taken by a JEOL JEM-1230 transmission electron microscope operating at 160 kV in bright-field mode. Absorption spectra were recorded with a Shimadzu

2550

UV-vis

scanning

spectrophotometer

at

room

temperature.

The

photoluminescence spectra were obtained by a fluorescence spectrophotometer (F-2500, HITACHI, Japan) with a xenon lamp excitation ranging from 300 to 800 nm. The size distribution and zeta potentials of nanoparticles were determined by laser light scattering with a particle size analyzer (90 Plus, Brookhaven Instruments Co. USA) at a fixed angle of 90°. The time-resolved decay profiles were recorded on Edinbergh Instrument FLS920. Synthesis of TTF (AIE)-NGO Nanoparticles. TTF (or other AIE molecules) was dissolved in THF, and the solution was then mixed with aqueous dispersion of NGO, to keep a final fwater=80 vol%. The mixture was dialyzed against DI water with an 8-14 kDa cutoff cellulose membrane for 50 h to remove THF. The dialyzed solution was then centrifuged at a certain speed to remove the excess NGO, and the obtained TTF (AIE)-NGO NPs were redispersed into DI water. HeLa Cell Culture. HeLa cells were cultured in DMEM at 37 ℃ in an atmosphere of 95% air and 5% CO2. They were incubated for 24 h for further cell viability analysis.


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Cell Viability Analysis. The effects of TTF-NGO NPs on cell viability were determined by cell counting kit 8 (CCK8, Dojindo Laboratories in Japan). HeLa cells were seeded in 96-well plates with 4000 cells per well. After incubated for 24 h, cells were treated with TTF-NGO NPs at various concentrations (1, 5, 50 and 100 µg/ml) for 12 h and 24 h. Cells without the treatment of TTF-NGO NPs were used as control. The absorbance ratio of sample to control could be used to evaluate the cell viability. Fish Line and Embryos Microinjection. Zebrafish fish line (wild type AB line) was raised and maintained in Zebrafish Resource Center (Core facilities, School of medicine, Zhejiang University) according to the standard Zebrafish Unit as described previously.38 Briefly, fish were seeded under a constant 14-hour-on/10-hour-off light cycle at 28 ℃. The embryos of zebrafish were collected at single-cell stage, and transferred into an injection tray containing the system water. TTF-NGO NPs and phosphate buffered saline (PBS) were injected into the yolks of embryos separately. Animals. 8-week-aged female BALB/c mice were obtained from the Laboratory Animal Center of Zhejiang University (Hangzhou, China). Mice were housed in cages and fed with standard mouse chow and water. The cages were maintained in a room with controlled temperature (25±1 °C) and a 12 h light/dark cycle. The protocol of animal experiments was approved by the Institutional Ethical Committee of Animal Experimentation of Zhejiang University in China, and the experiments were performed strictly according to governmental and international guidelines on animal experimentation. According to requirements for Biosafety and Animal Ethics, all efforts were made to minimize the number of animals used and their suffering.


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Experimental Set-up of a Scanning Microscope Equipped with a 1560 nm-fs Laser. As shown in Figure S5, a 1560 nm-fs laser (FLCPA-01C, Calmar Laser, 1 MHz, 400 fs) was coupled into an upright confocal microscope (BX61+FV1000, Olympus). The beam was reflected by DC1 (dichroic mirror: 1000-1600 nm reflection, 400-950 nm transmission, customized by Chroma Technology Corp), and then scanned by two computer-driven galvanometers. After passing through a scan lens and a tube lens, the laser beam was focused into the sample by a water-immersed microscope objective (XLPLN25XWMP2, Olympus, 25× 1.05 NA). Signals were epi-collected with the same objective. For 3PL imaging, they passed through DC2 (dichroic mirror: 1000-1600 nm reflection, 400-950 nm transmission, customized by Chroma Technology Corp), an optical filter (590 nm long-pass), and were collected by a photomultiplier tube (PMT, HPM-100-50, Becker & Hickl GmbH) for computer processing. For NLO spectra measurement, signals passed through DC2, and were finally recorded with an optical fiber spectrometer (PG 2000, Ideaoptics Instruments) Quantum yield measurement. The absolute quantum yield of TTF-NGO NPs in aqueous dispersion was measured with a commercial system consisted of an integrated sphere (Labsphere Inc.) and a spectrometer (ocean optics E6500). Measurement of 3PA cross-section of single TTF molecule. We firstly used the nonlinear transmissivity method to measurement the 3PA cross-section of single TTF molecule at the wavelength of 1560 nm. The nonlinear transmissivity of a 3PA medium can be described as39

T (I0 , λ ) =

1 1 + 2γ ( λ ) I 02 L

where I0 is the peak intensity of the incident light, L is the path length of the sample, γ(λ) is the


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3PA coefficient (in units of cm3/GW2), which is a macroscopic parameter depending on the concentration of the 3PA molecules. It can be further expressed as

γ (λ) = σ3' (λ)N0 = σ3' (λ)NAd0 ×10−3 where N0 is the molecular density (in units of 1/cm3), NA is Avogadro’s number, and d0 is the molar concentration of the absorbing molecules (in units of M). Here σ'3(λ) is the molecular 3PA cross-section (in units of cm6/GW2). The 3PA cross-section can also be defined as σ'3(λ)= σ3(λ)•(hν)2 (σ3(λ) is in units of cm6 s2). To measure the 3PA coefficient γ(λ) of TTF molecule at 1560 nm, we performed nonlinear transmission measurements based on a fs OPA system (Libra– USP-HE, 1 kHz, 160 fs). The laser beam was focused onto a 1-cm long cuvette containing 2mM TTF molecule in toluene, and the transmissivity of 1560 nm fs laser passing through the TTF molecule sample was measured accordingly. Calculation of 3PA cross-section of single TTF-NGO NP. Since TTF-NGO NPs of large size have distinct light scattering, it was not suitable to use the nonlinear transmissivity method to directly measure the 3PA cross-section of single TTF-NGO NP. Herein, we used an indirect way to estimate it, and the quantity of TTF molecules in each TTF-NGO NP needs to be calculated first. The concentrations of TTF and NGO in final aqueous dispersion of TTF-NGO NPs were 0.1 mg/mL and 20 µg/ml, respectively. As the TTF-NGO NPs were stable in water, the density of the suspension could be estimated as ~1 g/cm3. As the average size of TTF-NGO NPs determined from TEM was ~250 nm, total number of TTF-NGO NPs in 1 mL of suspension can be calculated from the following formula:


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(0.1 + 0.02) × 10 −3 g Total volume of TTF -NGO NPs 1 g / mL = = 1.47 × 1010 4 Average volume of each NP π × (125 × 10−7 )3 mL 3 Finally, the quantity of TTF molecules (MW=1074) in each TTF-NGO NP is estimated as: Total number of TTF molecules 0.093 mmol × 6.022 ×10 23 / mol = = 3.81× 109 Total number of TTF -NGO NPs 1.47 × 1010 Thus, the 3PA cross-section of single TTF-NGO NP could be obtained by the product of the quantity of TTF molecules in each TTF-NGO NP and the 3PA cross-section of single TTF molecule.

In vivo 3PL Imaging of Ear Blood Vessel of Mice. Mice were intravenously injected with aqueous dispersion of TTF-NGO NPs (1 mg/mL in 1×PBS, 250 µL solutions per mouse). The scanning microscope system equipped with the 1560 nm-fs laser was adopted for the imaging of blood vessels in the mouse ear. The mouse ear was fixed on a cell culture dish by glue. Deuteroxide was smeared between the water-immersed objective (XLPLN25XWMP2, Olympus, 25×1.05 NA) and the mouse ear. The scan speed was set to 10 µs/pixel (512×512 pixels per frame).

3PL Imaging of Zebrafish Treated with TTF-NGO NPs. Embryos of zebrafish was microinjected with 0.5 nL TTF-NGO NPs, and then cultured for 48 h. The grown zebrafish was put on a dish. The scanning microscope system equipped with the 1560 nm-fs laser was adopted for

imaging.

Deuteroxide

was

smeared

between

the

water-immersed

objective

(XLPLN25XWMP2, Olympus, 25×1.05 NA) and zebrafish. The scan speed was set to 10 µs/pixel (512×512 pixels per frame).


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Conflict of Interest: The authors declare no competing financial interest. Acknowledgements. This work was supported by National Basic Research Program of China (973 Program; 2013CB834704 and 2011CB503700), the National Natural Science Foundation of China (61275190 and 91233208), the Program of Zhejiang Leading Team of Science and Technology Innovation (2010R50007), the Open Fund of the State Key Laboratory of Luminescent Materials and Devices (South China University of Technology), the Swedish Research

Council,

SOARD,

and

The

Research

Grants

Council

of

Hong

Kong

(HKUST2/CRF/10). We thank Prof. Jianli Hua for providing AIE materials and Dr. Haiyan Qin for the assistance on fluorescence lifetime and quantum yield measurement. Supporting Information Available: Additional investigation of the function of NGO on the stability of TTF-nanoparticles and its size tunable effects though TEM observation. The results of toxicity analysis of TTF-NGO NPs in cells and zebrafish. The Supporting Information is available free of charge on the ACS Publications website at DOI:

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Table of Contents Stable and Size-Tunable Aggregation-Induced Emission Nanoparticles Encapsulated with Nano Graphene Oxide and Applications in Three-Photon Fluorescence Bioimaging Zhenfeng Zhu[+], Jun Qian[+], Xinyuan Zhao, Wei Qin, Rongrong Hu, Hequn Zhang, Dongyu Li, Zhengping Xu, Ben Zhong Tang*, and Sailing He* We proposed a new method to fabricate AIE-nanoparticles (NPs), which were surface modified with nano graphene oxide (NGO). The size-tunable AIE-NGO NPs possess good chemical stability and rich nonlinear optical effects. We then successfully achieved their three-photon fluorescence imaging in the blood vessel of mice ears and zebrafish.


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