Aggregation-Induced Emission Nanoparticles Encapsulated with

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Aggregation-Induced Emission Nanoparticles Encapsulated with PEGylated Nano Graphene Oxide and Their Applications in Two-photon Fluorescence Bioimaging and Photodynamic Therapy in vitro and in vivo Xianhe Sun, Abudureheman Zebibula, Xiaobiao Dong, Guanxin Zhang, Deqing Zhang, Jun Qian, and Sailing He ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b05546 • Publication Date (Web): 06 Jul 2018 Downloaded from http://pubs.acs.org on July 7, 2018

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ACS Applied Materials & Interfaces

Aggregation-Induced Emission Nanoparticles Encapsulated with PEGylated Nano Graphene Oxide and Their Applications in Two-photon Fluorescence Bioimaging and Photodynamic Therapy in vitro and in vivo Xianhe Sun1, Abudureheman Zebibula2, Xiaobiao Dong3, Guanxin Zhang3, Deqing Zhang3, Jun Qian1, Sailing He1,4, * 1

State Key Laboratory of Modern Optical Instrumentations, Centre for Optical and

Electromagnetic Research, Zhejiang University, Hangzhou, Zhejiang, 310058, China 2

Department of Urology, Sir Run Run Shaw Hospital, College of Medicine, Zhejiang University,

Hangzhou, Zhejiang, 310016, China 3

Beijing National Laboratory for Molecular Sciences, CAS Key Laboratories of Organic Solids

and Analytical Chemistry for Living Biosystems, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, China 4

School of Electrical Engineering, Royal Institute of Technology, OSQULDAS VÄG 6, SE-100

44 Stockholm, Sweden KEYWORDS: aggregation-induced emission, nanographene oxide, nanoparticles, bioimaging, photodynamic therapy, tumor

ACS Paragon Plus Environment

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ABSTRACT. Aggregation-induced emission (AIE) nanoparticles have been shown promise for fluorescence bioimaging and photodynamic therapy due to the good combination of nanoparticles and organic dyes or photosensitizers. Among several kinds of AIE nanoparticles, those that are capsulated with nanographene oxides (NGO) are easy to make, size-tunable, and have proven to be very stable in deionized water. However, the stability in saline solution still needs improvement for further applications in chemical or bio-medical fields, and the efficacy of photodynamic therapy using NGO-capsulate AIE photosensitizers has not been evaluated yet. Herein, we modified NGO with polyethylene glycol (PEG) to improve the stability of NGOcapsulated AIE nanoparticles in phosphate buffer saline. Furthermore, by combining with a typical AIE and photosensitizing dual functional molecule, we performed both two-photon fluorescence bioimaging and photodynamic therapy in vitro and in vivo. Our work shows that AIE nanoparticles capsulated with PEGylated nanographene oxide can be a powerful tool for future bio-imaging and photodynamic therapy applications.

Introduction With the rapid developments of nanotechnology, different types of nanoparticles have been developed for a wide range of applications, especially in bioimaging and photodynamic therapy (PDT)1-6. Since nanoscale size is associated with a high surface-to-volume ratio, nanoparticles are suitable for specific surface modification and feature a large drug-loading capacity. Specific modification can greatly improve the behaviors of nanoparticles, such as targeting ability7-9 and hydrophilicity10, while their large drug-loading capacity can decrease the side effects of using nanoparticles. These advantages make nanoparticles one of the hotspots in recent research.


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Many materials have been used to form nanoparticles, such as organic molecules11, quantum dots12, noble metals13, rare earth ion doped materials14, etc. Among them, organic materials, such as organic dyes and organic photosensitizers, have played an essential role in bio-imaging and PDT due to their good biocompatibility and simple synthesis processes15-18. However, most commonly-used organic dyes and organic photosensitizers are planar-shaped and suffer from aggregation-caused quenching19. Due to the π-π stacking interactions among the large conjugated aromatic structures, most conventional organic dyes and photosensitizers lose efficacy in their aggregated state when dissolved in a poor solvent or made into nanoparticles20, 21. Fortunately, since they were first discovered by Tang’s group, a new type of organic dyes, with the property of aggregation induced emission (AIE), have been developed22, 23. Thanks to the propellershaped structures, nonradiative transitions of AIE molecules can be suppressed, which result in the sharply grows of quantum yield with the increase of aggregation degree24. Beyond fluorescent dyes, photosensitizers grafted with AIE properties have also recently been developed and applied on PDT25-29. Making AIE molecules into nanoparticles simply conforms to their nature and has become an advantage rather than obstacle. So far, a series of methods have been introduced to make AIE nanoparticles for biological applications, including the Reprecipitation method, silica coating methods, amphiphilic molecule encapsulating methods etc.23, 30-32. Previously, our group reported AIE nanoparticles capsulated with Nano graphene oxide (NGO) as an alternative method31. These nanoparticles are sizetunable, easy to fabricate, and have been shown to be stable in deionized water. In addition, as a derivative of graphene, NGO has an sp2-hybridized planar structure grafted with rich functional groups (e.g., -OH, C=O)33. The planar structure means that NGOs are easy to combine through π bond conjugation, and the functional groups allow the opportunity for modification through


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covalent interactions34-36. However, there are still shortcomings. Since PBS has the same osmotic pressure as the biological internal environment, good stability in PBS is essential for many biological applications. Thus far, however, NGO-capsulated AIE nanoparticles are not adequately stable in PBS. In addition, the efficacy of PDT using NGO-capsulate AIEphotosensitizer nanoparticles has not been evaluated yet. Herein, we modified NGO with polyethylene glycol (PEG), denoted as NGP, to improve the nanoparticles’ stability in PBS. The improvement was significant and demonstrated to be feasible for other kinds of AIE molecules. Furthermore, by combining with a typical AIE and photosensitizing dual functional molecule, we first achieved fluorescent bio-imaging of mouse blood vessels as well as UMUC3 cells. Afterwards, we evaluated the PDT effects of the nanoparticles both in vitro and in vivo. The therapeutic effects were significantly different from those of the control groups. To our knowledge, this is the first time NGP (NGO) capsulated AIE nanoparticles have been used in the application of in vitro and in vivo PDT.

Experimental Section Materials. CH3-PEG-SH (methoxy PEG Thiol, MW5000) and 1,2-distearoyl-sn-glycero-3phosphoethanolamine-N- [methoxy (polyethylene glycol)-5000] (mPEG-DSPE-5000) were obtained from JenKem Technology (Beijing, China). The 2- ((4-(2,2-bis(4-methoxyphenyl)-1phenylvinyl) phenyl) (phenyl)methylene) malononitrile (TPE-red) used in this article was prepared according to our previous work37. The 9,10-anthracenediylbis (methylene) dimalonic acid (ABDA), chloroacetic acid, sodium bisulfide, and 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) were all obtained from Sigma-Aldrich (Shanghai, China). Minimum essential media (MEM), trypsin-EDTA solution, and fetal bovine serum (FBS) were


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purchased from Gibco. Hydrochloric acid, sodium hydroxide, tetrahydrofuran, PBS (1×), and dimethyl sulfoxide were obtained from the Chemical Reagent Department of Zhejiang University. Deionized water was used in all experiments. Characterization. Transmission electron microscopy (TEM) images were obtained using a JEOL JEM-1200 microscope, which was operated at 80 kV in bright-field mode. The atomic force microscopy (AFM) images were taken by a CSPM5500A atomic force microscope. The hydrodynamic size distribution was measured with a Malvern Zetasizer Nano ZS-90. The absorption spectra of molecules and nanoparticles were measured on a Shimadzu 2550 UV-vis scanning spectrophotometer. The FTIR spectra were measured with a Nicolet is10 infrared spectrometer by Thermal Fisher Co., Ltd. The one-photon excited fluorescent spectra were obtained on an F-2500 HITCH fluorescence spectrophotometer. Synthesis of Nano graphene oxide modified with PEG (NGP). NGO was synthesized using a modified Hummer’s method, which was illustrated in our previous report38, 39. Afterwards, the as-prepared NGO was modified with PEG using method introduced by Ji40. The PEGylation procedure consists of two major components: carboxylation and PEGylating. First, 10 mL of NGO solution (2 mg/mL) was mixed with 1.2 g sodium hydroxide and 1.0 g chloroacetic acid. After 3 hours of sonication, the solution was neutralized with acid and then purified by dialyzing for two days. The NGO-COOH solution was collected by further filtration through a 0.22 μm microporous membrane. Afterwards, the PEG chains were modified onto NGO by forming disulfide bonds via air oxidation41, 42. Specifically, 50 mg of mPEG-SH was first added into NGO-COOH solution (0.5 mg/mL, 20 mL). After 10 min of sonication, 220 mg of sodium bisulfide was added gradually


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under vigorous stirring, and another 1 h of sonication was performed. Then, the resulting solution was stirred at 55 ℃ overnight before another 50 mg of mPEG-SH was added. The solution was stirred for 1.5 days, and the reaction product was dialyzed, filtrated, and collected for further use. Fabrication of NGP (NGO)-TPEred (AIE) nanoparticles. The NGP (NGO)-TPEred (AIE) nanoparticles were fabricated by the method described in our previous paper31. Briefly, 0.4 mL NGP (or NGO) solution, 0.4 mL TPE-red (or other AIE molecules) solution (in THF, 0.5 mM), and 1.2 mL deionized water were quickly mixed, followed by dialyzing for two days to remove excess THF. The resulting NGP (NGO)-TPEred (AIE) nanoparticles were collected by centrifugation at 1.32k rpm for 20 mins. Measurement of fluorescence quantum yield. The fluorescence quantum yield was measured by comparing with that of Rhodamin B (RhB, a common dye). The integral of the spectral envelopes was used as the fluorescence intensity. The quantum yield of NGP-TPEred nanoparticles was calculated according to Eq. (1)44

1 A0 F1 I 0 n12 = • • • 0 A1 F0 I1 n02

（1）

Where η stands for the fluorescence quantum yield, A stands for absorption at 450 nm, F stands for the fluorescence intensity, I stands for the intensity of excitation light, n stands for the refractive index of the solvent, and subscripts 1 and 0 stands for the sample (NGP-TPEred nanoparticles) and the reference (RhB in methanol), respectively. Reactive oxygen species (ROS) detection ex vivo. The ROS producing ability of NGPTPEred was verified by a common indicator, ABDA. Briefly, 2 mL of NGP-TPEred (0.1 mM for


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TPE-red molecule) and 0.2 mL ABDA solution (1 mg/mL in DMSO) were mixed evenly and exposed to 450 nm continuous wave laser light (40 mW, 200 mW/cm2). The absorption spectra were measured every 2 min, and the decrease of the OD value at 377 nm was used as the indicator of ROS production. The control group was processed identically, except without laser irradiation. Stability of NGP (NGO)-TPEred (AIE) nanoparticles in PBS (1×). The stability of NGP (NGO)-TPEred (AIE) nanoparticles was evaluated in PBS (1×). NGP-TPEred (AIE) nanoparticles were dissolved in PBS (1×) with a concentration of 0.1 mM for TPE-red (or other AIE) molecules and divided into three equal parts. The absorption spectrum of each part was recorded at the beginning, and 24 hours later, and the amount of stable nanoparticles was indicated by the peak OD values. Meanwhile, NGO-TPEred (AIE) nanoparticles in PBS (1×) were processed identically and used as a control group. Two-photon excited fluorescence property evaluation. Two-photon excited fluorescence spectra of NGP-TPEred were recorded using a lab-built fluorescence detection system. As illustrated in Figure S6, a 1040-nm fs laser45 was used as the excitation light and focused on the sample by a lens with a focal length of 50 mm. On the side of the sample, a 20× objective lens (NA = 0.75, Olympus) was used to collect the fluorescence signals into a spectrometer (PG2000, Ideaoptics). A series of spectra of NGP-TPEred were recorded with different excitation powers, and the fluorescence intensity was calculated by the integral of each spectrum envelope. Finally, we also constructed a scatter plot and the linear fitting of fluorescence intensity and the square of the incident power, which verifies the linear relationship between them.


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The two-photon absorption cross-section (TPACS) was also measured by the comparison with RhB. The two-photon excited fluorescence intensities of NGP-TPEred nanoparticles and RhB were recorded using the method described above. The TPACS of NGP-TPEred nanoparticles could be calculated using Eq. (2)46

1 F10 c0 n0 = （2）  0 F01c1n1 where δ stands for the TPACS, η stands for the fluorescence quantum yield, F stands for the two-photon excited fluorescence intensity, c stands for the molar concentration, n stands for the refractive index of the solvent, and subscripts 1 and 0 stand for the sample (NGP-TPEred nanoparticles) and the reference (RhB in methanol), respectively. Cell culture. UMUC3 cells (human bladder cancer cell line) were purchased from the Cell Culture Center of the Institute of Basic Medical Science, Chinese Academy of Medical Sciences (Shanghai, China). MEM medium with 10% FBS was used as the culture media (1% penicillin solution and 1% amphotericin B solution were added into the medium to protect from bacterial infection), and the incubation environment was kept at 37 ℃ and with 5% CO2. Animal preparation. In this work, all animal experiments were conducted in compliance with the requirements of Zhejiang University Animal Study Committee for the care and use of laboratory animals in research. The mice were housed at the Animal Experimentation Center of Zhejiang University, with the condition of 24 °C and 12 h light/dark cycle. During the experiments, the mice were provided with standard laboratory chow and water. Two-photon excited fluorescence bioimaging. Two-photon excited fluorescence bio-imaging was applied to UMUC3 cells and mice ear blood vessels using an upright scanning microscope


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(Olympus, BX61+FV1200) and a 1040 nm fs laser. As illustrated in Figure S7, a 20×/0.75 or a 60×/1.00 water-immersed objective lens (Olympus) was used to achieve beam focusing and signal collection. After passing a series of specific dichroic mirrors and optical filters, the fluorescence signal was finally detected by a photomultiplier tube (PMT 2 in Figure S7) work in nondescanned detection mode. For in vitro experiment, UMUC3 cells in logarithmic growth phase were seeded in 35-mm cultivation dishes at confluence of 50% to 60%. Next day, the cells were treated with NGPTPEred nanoparticles in a concentration of 15 μg/mL for about 2 hours. Afterwards, the cells were washed with PBS (1×) three times and imaged directly. For in vivo experiment, male BALB/c mice (8 weeks old, about 25 g) were used for ear blood vascular imaging. The mouse ear was fixed on a plate by glue after intravenous injection of NGP-TPEred nanoparticles solutions (1 mg/mL in PBS, 200 μL) through the tail vein. Then, sterile water was smeared between the objective and the mouse ear before imaging. MTT assay. To evaluate cytotoxicity of NGP-TPEred nanoparticles, MTT assay was applied on UMUC3 cells. The cells in logarithmic growth phase were seeded in a 96-well culture plate (5 × 103 per well) and cultured at 37 ℃ with 5% CO2 for 24 hours. Then, the medium was replaced by 200 μL of fresh MEM media containing different amount of NGP-TPEred nanoparticles (0, 5, 10, 20, and 50 μg/mL for TPE-red molecules). For experimental groups, the cells were irradiated with 450 nm light (200 mW/cm2, 5 min) after 2-hour incubation. For control groups, the cells were not irradiated. The cells in experimental and control groups were then cultured for additional 48 h at 37 °C and with 5% CO2. Afterwards, MTT solution (20 μL, 5 mg/mL) was added into each well, and the cells were incubated for another 4 hours. The medium


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was then removed carefully, and the precipitates were dissolved by DMSO (200 μL, per well). Finally, OD570 of solution in each well was recorded on an enzyme-linked immunosorbent assay (ELISA) microplate reader after shaking the plate for 10 min. To study the relationship between the cell viability and the time period of continuous irradiation, the UMUC3 cells were first incubated with 20 μg/mL NGP-TPEred nanoparticles for 2 hours and then irradiated for different time periods (2, 4, 6, and 8 min). The cells were cultured for another 48 hours. Afterwards, the cell viability was measured using the method introduced above. In vivo toxicity. 12 male mice (8 weeks old, about 25 g) were randomly divided into 4 groups with different treatments: (1) PBS (1×), sacrificed 24 hours post injection; (2) NPG-TPEred (10 mg/kg), sacrificed 24 hours post injection; (3) PBS (1×), sacrificed 15 days post injection; (4) NPG-TPEred (10 mg/kg), sacrificed 15 days post injection. Afterwards, major organs of mice were collected for further hematoxylin and eosin (H&E) staining. In vivo PDT of tumors. Cells suspension (5×106 cells/mL) was first prepared using UMUC3 cells in logarithmic growth phase. Then the cells suspension was subcutaneously injected at the armpit site of the male nude mice (5 weeks old, purchased from Slaccas Co., Ltd., Shanghai, Chinese Academy of Science). Afterwards, the mice were housed under the condition of 24 °C and 12 h light/dark cycle. The tumor volume was determined by Volume = Length × Width2 × 0.5, in which Length and width were measured using a Vernier caliper every day. When the tumor volume reached about 75 mm3, 12 tumor-bearing mice were randomly divided into 4 groups with different treatments: (1) PBS (1×), without irradiation; (2) NGP-TPEred nanoparticles (in PBS (1×), 1 mg/mL, 100 μL), without irradiation; (3) PBS (1×), with irradiation;


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(4) NGP-TPEred nanoparticles (in PBS (1×), 1 mg/mL, 100 μL), with irradiation. PBS or NGPTPEred nanoparticles were intratumorally injected, and the irradiation (450 nm, 200mW/cm2, 20 min) was applied at 4 hours and 4 days post-injection. The tumor volumes and body weights of the mice in each group were recorded every day, and the observation period lasted for 15 days. The tumors of the mice were dissected out for H&E staining after sacrifice. Histological examination. The major organs and tumors were immediately fixed in 10% formalin after sacrifice and kept in 4 ℃ for overnight. Then the samples were embedded in paraffin and sectioned. Afterwards, the sections were stained with hematoxylin and eosin. The histological slices were imaged and analyzed.

Results and discussion Synthesis of Nano graphene oxide and the modification of PEG. The NGO was synthesized using a modified Hümmers method, which was illustrated in our previous work31, 38. The morphology of NGO was first checked by atomic force microscopy (AFM). Figure 1a shows the AFM image of NGO particles, and the cross section of the coarse dashed line in the insert figure shows more details. Since the substrate is inclined, a fine dashed line for the substrate plane was drawn, base on which we could find that the NGO has a height of about 2 nm. The planar size of NGO was characterized by TEM images. As shown in Figure 1b, NGO particles have a diameter of about 30 nm, which coincidences well with the results of dynamic light scattering (DLS) measurements (insert in Figure 1b). The modification of PEG was achieved using the method introduced by Ji40, and the entire procedure is illustrated in Figure S1. Ultra-violet-visible (UV) absorption spectra, Zeta potential, and FTIR spectra were used to confirm the successful modification of PEG. From Figure 1c


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(detailed by insert), we could find that the near infrared absorption of NGP is larger than that of NGO, which coincidences well with previous reports40, 47. Figure 1d shows zeta potentials of NGO and NGP. As we could see, NGO was negatively charged (-35.5 mV) while NGP was less negatively charged (-17.9 mV) due to the neutral PEG chains. FTIR spectra indicate the intramolecular vibration modes and could be used as “fingerprints” of molecules. From Figure 1e, we could recognize that NGO has strong signals for –OH and -C=O, while NGP has weak signals for –OH or –C=O but has strong signals for –C-O-C-, which indicates the successful covalent modification of PEG47, 48. Furthermore, as shown in Figure 1f, the fluorescence properties of NGO and NGP were also checked using a fluorescence spectrophotometer, and the fluorescence spectrum of NGP was found to have more “long wavelength components” than that of NGO. As illustrated in previous literature49, the fluorescence of GO or GO derivatives roughly consist of two major components, which results from the recombination of electron-hole pairs localized within sp2 clusters and the existence of oxygen-containing functional groups. During the carboxylation process in PEGylation, the opening of epoxide groups and hydrolysis of esters on the NGO led to new oxygen-containing functional groups and changed the microenvironment of NGO34. This process would increase the weight of long wavelength component in fluorescence, and result in the spectrum shown in Figure 1f.


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Figure 1. Characterization of NGO and NGP. a) An AFM image of as-prepared NGO (insert: cross section of the dashed line); b) A TEM image of NGO (insert: hydrodynamic size distribution of NGO); c) Absorption spectra of NGO and NGP (insert: details between 400 – 800 nm); d) zeta potentials of NGO and NGP; e) FTIR spectra of NGO and NGP; f) 325 nm light excited fluorescence of NGO and NGP. Scale bar in b): 100 nm. Error bars indicate SD. Synthesis of NGP-TPEred (AIE) nanoparticles. A commonly-used AIE and photosensitizing dual functional molecule, TPE-red37, 50 (molecular structure is shown in Figure S2), was used to fabricate NGP-AIE nanoparticles. The nanoparticle fabrication procedure is illustrated in Figure 2a, and the reaction products were recorded as NGP-TPEred. We first studied the morphology of NGP-TPEred by TEM images, and from Figure 2b we could find that the sizes of NGP-TPEred nanoparticles are mainly distributed in the range of 50 - 100 nm. From the absorption spectrum (blue curve in Figure 2c), we could find that the peak absorption is located at about 450 nm, and NGP-TPEred could be excited with wavelengths up to 550 nm


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(absorption value at 550 nm is 0.221, and the fluorescence exited by 550 nm light was very weak, as shown in Figure S3). From the fluorescence spectrum (red curve in Figure 2c), we could find that the peak fluorescence intensity appears at about 630 nm, and the fluorescence signal covers wavelengths between 520 - 850 nm. Furthermore, by using a comparison method, we calculated the quantum yield as 3.55%. The photodynamic effects of NGP-TPEred were then evaluated using a common ROS probe, ABDA51. The absorption of the NGP-TPEred and ABDA mixture with/without 450 nm laser irradiation were measured, and the decrease of absorption at 377 nm was used as the indicator of ROS production. From Figure 2d, we could recognize that the absorption value at 377 nm drops to 70% of the original value in the presence of 450 nm laser irradiation, whereas a slight increase of absorption could be observed in the control group. This phenomenon confirmed the capability of NGP-TPEred to produce ROS under 450 nm laser irradiation. As we know, there has been works confirming the ROS generation ability of NGO52 or using NGO in PDT system53 before, thus herein we also studied the effect of NGP on ROS production in our cases. From the discussion in Supporting Information (section “Study on the effect of NGP on ROS production”), we concluded that: 1) the NGP we fabricated has good ability of producing ROS; 2) NGP-TPEred remains stable with a few NGP, and only little fraction of ROS production of NGP-TPEred could be attributed to the NGP alone; 3) the NGP can further enhance the ROS production of TPE-red nanoparticles. Finally, we checked the stability of NGP-TPEred in PBS (1×). TPE-red nanoparticles capsulated with NGO were also synthesized and recorded as NGO-TPEred. Both NGO-TPEred nanoparticles and NGP-TPEred nanoparticles were dissolved in PBS (1×) and left for 24 hours. The peak absorption value at 450 nm was used as an indicator of stability. From Figure 2e, after 24 hours, the absorption of NGP-


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TPEred dropped slightly, while that of NGO-TPEred dropped almost to zero, which indicates better stability of NGP-TPEred than NGO-TPEred.

Figure 2. Synthesis and characterization of NGP-TPEred. a) Illustration scheme of the synthesis procedure of NGP-TPEred; b) A TEM image of NGP-TPEred; c) Absorption spectrum and 450 nm-light excited fluorescence spectrum of NGP-TPEred; d) Normalized absorption value of


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NGP-TPEred and ABDA mixture with/without irradiation; e) Relative absorption of NGP(NGO)-TPEred in PBS(1×) at 0 h and 24 h. Error bars indicate SD. In addition to TPE-red, three other kinds of typical AIE molecules: TPE24, TTF54, and BT355 (molecular structures are shown in Figure S2), were chosen to check whether the NGP capsulation method could be popularized to other AIE molecules. Among them, TPE is a “star” AIE molecule, and many AIE molecules are derived from TPE structure24; TTF has been widely used for applications in biomedical field because of its large multiphoton absorption cross section and far red/NIR fluorescence54, 56; BT3 is a typical AIE compound with high fluorescence quantum yield as the combination of TPE and BODIPY (a common dye)55. These three kinds of AIE molecules all have outstanding properties and are highly representative. After the same capsulation procedure described above, the NGP-AIE nanoparticles were all dissolved in PBS (1×). As shown in Figure 3a and 3b, the as-prepared NGO-AIE/NGP-AIE nanoparticles were all well-dispersed and could emit strong fluorescence under ultra-violet light irradiation. All the solutions were then left undisturbed for 24 hours, and absorption spectra were measured at the beginning and the end of this period. From Figure 3c-3e, we could find that NGP-TPE, NGP-TTF, and NGP-BT3 remain stable after 24 h, while NGO-TPE, NGO-TTF, and NGO-BT3 suffered from irreversible aggregation, which caused the sharp decline of absorption values. This confirmed that the modification of PEG could be a universal way to improve the stability of NGO capsulated AIE nanoparticles in PBS (1×).


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Figure 3. NGO- or NGP-capsulated TPE, TTF, and BT3 nanoparticles a) under daylight; b) under irradiation of ultra-violet light; c) - e) Absorption spectra in PBS (1×) at 0 h and 24 h. Two-photon excited fluorescence property evaluation. Two-photon excited fluorescence has become an essential tool in bioimaging due to its unique advantages, such as great penetration ability, weak background of autofluorescence, and little photodamage to tissues57. The two-photon fluorescence properties of NGP-TPEred was studied using a lab-built fluorescence detection system. The two-photon fluorescence spectrum of NGP-TPEred was obtained under the excitation of a 1040 nm fs laser. From Figure 4a, we could find that the twophoton fluorescence spectrum is the same as that under 450 nm excitation. By changing the incident power, we obtained a series of spectra. We then constructed a scatter plot and the linear fitting of fluorescence intensity and the square of incident power, which confirms the secondorder nonlinear process (Figure 4b). By using a comparison method, the two-photon action cross section of NGP-TPEred was calculated as 2.27×103 GM for each molecule. According to a


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previous literature58, as product of two-photon absorption cross-section and fluorescence quantum yield, the two-photon action cross section could be enhanced when forming AIE nanoparticles. Herein, as a specific kind of AIE nanoparticles, NGP-TPEred can also enhance the two-photon action cross section of TPEred. Intuitively, combined with the two-photon absorption cross section and quantum yield we measured above, the two-photon action cross section of NGP-TPEred could be calculated as 80.585 GM; on the other hand, TPE-red molecules even have no ability to fluoresce37, which indicate negligible two-photon action cross section. This comparison confirms the enhanced two-photon action cross section of NGP-TPEred. Next, we utilized a scanning microscope to perform two-photon fluorescence bioimaging. We first explored the two-photon fluorescence imaging on mice ear blood vessels. The NGP-TPEred nanoparticles were injected intravenously with a dose of 10 mg/kg, and the imaging integral period of each pixel was set to 10 μs. The same view was imaged 40 times, and we put these pictures together to enhance the contrast. According to the imaging result shown in Figure S8b, in addition to large blood vessels, we could even recognize fine blood vessels, which show the good blood vascular imaging capability of NGP-TPEred. As shown in Figure S8d, when the same procedures were applied on NGO-TPEred, only large vessels could be recognized and the signal points were distributed sparsely. Since the fluorescence intensities of NGP-TPEred/NGOTPEred checked by two-photon fluorescence imaging of capillary tubes show insignificant difference (shown in Figure S8a, S8c), the disparity in imaging results should be attributed to better stability of NGP-TPEred in blood environment. To verify the ability of NGP-TPEred nanoparticles to mark cells, we applied in vitro twophoton fluorescence imaging on UMUC3 cells. In this work, we used a 20× water-immersive objective lens to focus and collect fluorescence signals. As shown in Figure 4c-4e, the two-


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photon fluorescence signals coincidence well with cells in bright field images, which indicate that NGP-TPEred nanoparticles have good ability to mark cells.

Figure 4. a) Two-photon excited fluorescence spectrum of NGP-TPEred; b) the power dependence relationship under 1040 nm fs-excitation; c) – e) two-photon excited fluorescence image, bright field image, and merged image of UMUC3 cells using the 20×objective lens. Scale bar: 50 μm. In vivo toxicity. Since we have injected NGP-TPEred into mice bodies intravenously, the in vivo toxicity must be studied. 12 mice were divided randomly into four groups with different treatments: (1) PBS (1×), sacrificed 24 hours post injection; (2) NPG-TPEred (10 mg/kg), sacrificed 24 hours post injection; (3) PBS (1×), sacrificed 15 days post injection; (4) NPGTPEred (10 mg/kg), sacrificed 15 days post injection. Afterwards, major organs of the mice were dissected out for H&E staining. As shown in Figure 9S, no obvious inflammation, injury or


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abnormality could be observed, which indicates negligible in vivo toxicity of NGP-TPEred nanoparticles. MTT assay. To evaluate the cytotoxicity and PDT efficacy of NGP-TPEred, MTT assay was applied on UMUC3 cells59. As shown in Figure 5a, the relative cell viability remains higher than 90% even with NGP-TPEred concentration of 50 μg/mL, which indicates low cytotoxicity of NGP-TPEred. At the same time, when the cells incubated with NGP-TPEred nanoparticles were put under 450 nm laser irradiation (40 mW, 5 min), the relative cell viabilities dropped with the increase of concentration. We then changed the irradiation time while keeping the concentration at 20 μg/mL. From the result shown in Figure 5b we could find the relative cells viability dropped substantially with the increase of irradiation time. These phenomena indicate that NGPTPEred has low toxicity in the dark, but higher toxicity under the irradiation of 450 nm laser light.

Figure 5. a) Relative viabilities of UMUC3 cells incubated with different concentrations of NGP-TPEred nanoparticles solutions (0, 5, 10, 20 and 50 μg/mL), blue for dark treatment, red for light irradiation; b) Relative viabilities of UMUC3 cells incubated with NGP-TPEred


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nanoparticles solutions (20 μg/mL) under different irradiation times (0, 2, 4, 6 and 8 min). Error bars indicate SD. In vivo PDT of tumors. The UMUC3 tumor xenograft models were employed to evaluate the in vivo PDT efficacy of tumors. Due to the high malignancy and representativeness60, 61, UMUC3 was chosen as the type of tumor cells. When the tumor volume grew to about 75 mm3, 12 mice were divided randomly into 4 groups with different treatments: (1) PBS (1×), without irradiation; (2) NGP-TPEred nanoparticles, without irradiation; (3) PBS (1×), with irradiation; (4) NGPTPEred nanoparticles, with irradiation. During the 15 days of observation, the tumor volumes and body weights of the mice were recorded every day. As Figure 6a shows, with the presence of both NGP-TPEred nanoparticles and 450 nm laser irradiation, the growth of tumor was totally inhibited at once, and shows a noticeable difference from the other 3 groups. During the observation, the body weights were recorded as an indicator of health. As shown in Figure 6b, not much difference could be recognized among the 4 groups. After the observation, all mice were sacrificed, and four representative mice were picked out for photos (Figure 6d-6g). The tumors were then collected for photos (Figure 6c) and sent to H&E analysis. The photos show the comparison of therapeutic effects in different groups intuitively, and from Figure 6h-6k we could find most of the tumor tissue of group (4) necrosed, while tumor tissues in other 3 groups shows no sign of cell death, which further confirmed the PDT efficacy of NGP-TPEred nanoparticles.


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Figure 6. In vivo PDT of UMUC3 xenograft tumor. a) Relative tumor volume under different treatments during 15-day-observation; b) Body weights of the mice under different treatments during 15-day-observation; c) A photo of typical tumor masses after different treatments; d) - g) Photos of mice after different treatments; h) - k) H&E stained sections of tumor tissue after different treatments. The treatments: group (1) PBS (1×), without irradiation; group (2) NGPTPEred nanoparticles, without irradiation; group (3) PBS (1×), with irradiation; group (4) NGPTPEred nanoparticles, with irradiation. Scale bar: 50 μm. Error bars indicate SD.

Conclusion


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In this work, we modified NGO with PEG chains to improve the behaviors of NGO-AIE nanoparticles in PBS. Afterwards, by making a typical dual-functional AIE molecule, TPE-red, into NGP-TPEred nanoparticles, we first achieved two-photon fluorescence imaging of both mouse ear blood vessels and UMUC3 cells. We then evaluated the in vitro and in vivo PDT effects by simultaneously applying NGP-TPEred nanoparticles and 450 nm laser irradiation. The therapeutic effects shew noticeable differences from control groups. This work shows two major innovations and advantages: 1) modification of PEG on NGO obviously improves stability of NGO-AIE nanoparticles in PBS, and the method could be popularized to different kinds of AIE molecules; 2) it is the first attempt to use AIE nanoparticles capsulated with NGP (NGO) in in vitro and in vivo PDT, and the growth of tumors has been inhibited for several days. Compared with commonly used PEG capsulation method, NGP capsulation might be less direct and feasible. However, NGP is naturally a chemically versatile template with high surface-to-volume ratio. Besides a wide variety of functional groups, NGP also has large π-bond conjugation areas, which means the modification could be achieved not only through covalent interactions, but also through π-π stacking interaction62. Therefore, we think NGP capsulation method shows more opportunities and potentials in multifunctional applications. In addition, as we illustrate in supporting information (“Study on effect of NGP on ROS production”), NGP-TPEred also shows enhanced ROS production ability when compared to the PEG capsulated TPE-red nanoparticles, which indicates its greater potential in the application of photodynamic therapy. In summary, our work has demonstrated the capability of NGP-AIE nanoparticles as a potential powerful tool in bioimaging and PDT.

ASSOCIATED CONTENT


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Supporting Information. Supporting Figures: Schematic illustration of the synthetic procedure of PEGylated nanographene oxide; Schematic illustration of the structure of TPE, TPE-red, BT3 and TTF; Normalized spectrum of NGP-TPEred and water (control) exited by 550 nm light ; Absorption spectra of NGP-TPEred, ABDA mixture solutions; Study on effects of NGP on ROS production; Normalized absorption value of ABDA at 377 nm when mixed with different solutions under 450 nm light irradiation; Schematic illustration of lab-built fluorescence detection system; Schematic illustration of the two-photon excited fluorescence imaging system; Two-photon excited fluorescence images of capillary glass tube and mouse ear blood vessels; H&E stained sections of major organs of mice with different treatment. AUTHOR INFORMATION Corresponding Author *Sailing He 1. State Key Laboratory of Modern Optical Instrumentations, Centre for Optical and Electromagnetic Research, Zhejiang University, Hangzhou, Zhejiang, 310058, China 2. School of Electrical Engineering, Royal Institute of Technology, OSQULDAS VÄG 6, SE100 44 Stockholm, Sweden E-mail: [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. Notes


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The authors declare no competing financial interest. Funding Sources The National Key Research and Development Program of China (No. 2017YFA0205700) The National Natural Science Foundation of China (11621101). ACKNOWLEDGMENT This work was supported by the National Key Research and Development Program of China (No. 2017YFA0205700) and the National Natural Science Foundation of China (11621101). ABBREVIATIONS AIE, aggregation-induced emission; PDT, photodynamic therapy; NGO, nanographene oxide; PEG, polyethylene glycol; PBS, phosphate buffer saline; TPE-red, 2- ((4-(2,2-bis(4methoxyphenyl)-1-phenylvinyl) phenyl) (phenyl)methylene) malononitrile; ABDA, 9,10anthracenediylbis (methylene) dimalonic acid; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide; MEM, minimum essential media; FBS, fetal bovine serum; TEM, transmission electron microscopy; AFM, atom force microscopy; NGP, nanographene oxide modified with PEG; Rh B, rhodamine B; ROS, reactive oxygen species; TPACS, two-photon absorption cross-section; H&E, hematoxylin and eosin. REFERENCES

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Figure 1. Characterization of NGO and NGP. a) An AFM image of as-prepared NGO (insert: cross section of the dashed line); b) A TEM image of NGO (insert: hydrodynamic size distribution of NGO); c) Absorption spectra of NGO and NGP (insert: details between 400 – 800 nm); d) zeta potentials of NGO and NGP; e) FTIR spectra of NGO and NGP; f) 325 nm light excited fluorescence of NGO and NGP. Scale bar in b): 100 nm. Error bars indicate SD. 99x57mm (300 x 300 DPI)



Figure 2. Synthesis and characterization of NGP-TPEred. a) Illustration scheme of the synthesis of NGPTPEred; b) A TEM image of NGP-TPEred; c) Absorption spectrum and 450 nm-light excited fluorescence spectrum of NGP-TPEred; d) Normalized absorption value of NGP-TPEred and ABDA mixture with/without irradiation; e) Relative absorption of NGP(NGO)-TPEred in PBS(1×) at 0 h and 24 h. Error bars indicate SD. 180x184mm (300 x 300 DPI)


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Figure 3. NGO- or NGP-capsulated TPE, TTF, and BT3 nanoparticles a) under daylight; b) under irradiation of ultra-violet light; c) - e) Absorption spectra in PBS (1×) at 0 h and 24 h. 99x56mm (300 x 300 DPI)



Figure 4. a) Two-photon excited fluorescence spectrum of NGP-TPEred; b) the power dependence relationship under 1040 nm fs- excitation; c) – e) two-photon fluorescence image, bright field image, and merged image of UMUC3 cells using the 20×objective lens. Scale bar: 50 µm. 99x66mm (300 x 300 DPI)


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Figure 5. a) Relative viabilities of UMUC3 cells incubated with different concentrations of NGP-TPEred nanoparticles solutions (0, 5, 10, 20 and 50 µg/mL), blue for dark treatment, red for light irradiation; b) Relative viabilities of UMUC3 cells incubated with NGP-TPEred nanoparticles solutions (20 µg/mL) under different irradiation times (0, 2, 4, 6 and 8 min). Error bars indicate SD. 70x27mm (300 x 300 DPI)



Figure 6. In vivo PDT of UMUC3 xenograft tumor. a) Relative tumor volume under different treatments during 15-day-observation; b) Body weight of mice under different treatments during 15-day-observation; c) A photo of typical tumor masses after different treatments; d) - g) Photos of mice after different treatments; h) - k) H&E stained sections of tumor tissue after different treatments. The treatments: group (1) PBS (1×), without irradiation; group (2) NGP-TPEred nanoparticles, without irradiation; group (3) PBS (1×), with irradiation; group (4) NGP-TPEred nanoparticles, with irradiation. Scale bar: 50 µm. Error bars indicate SD. 129x96mm (300 x 300 DPI)


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TOC 50x44mm (300 x 300 DPI)


Aggregation-Induced Emission Nanoparticles Encapsulated with

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