AIEgen Nanoparticles of Arylamino Fumaronitrile Derivative with High

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AIEgen Nanoparticles of Arylamino Fumaronitrile Derivative with High Near-infrared emission for Two-photon Imaging and in vivo Cell Tracking Zhonghua Liu, Fang Liu, Yijian Gao, Weixia Qing, Yongwei Huang, Shengliang Li, and Dongzhu Jin ACS Appl. Bio Mater., Just Accepted Manuscript • DOI: 10.1021/acsabm.8b00643 • Publication Date (Web): 06 Dec 2018 Downloaded from http://pubs.acs.org on December 10, 2018

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ACS Applied Bio Materials

AIEgen Nanoparticles of Arylamino Fumaronitrile Derivative with High Near-Infrared Emission for Two-Photon Imaging and in vivo Cell Tracking

Zhonghua Liu,† Fang Liu,‡ Yijian Gao,† Weixia Qing,† Yongwei Huang,†,* Shengliang Li,§,* Dongzhu Jin†

†Laboratory

for NanoMedical Photonics, School of Basic Medical Sciences, Henan

University, Kaifeng 475004, China. E-mail: [email protected] ‡The

First Affiliated Hospital, Henan University, Kaifeng 475004, China.

§Department

of Nanomedicine, Houston Methodist Research Institute, Houston, TX

77030, United States. E-mail: [email protected]

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ABSTRACT Developing of two-photon materials for live-cell imaging and in vivo analysis in-deep have been received great attention, and it is still urgent so that such microscopy technique could be promoted and advanced into new progress using the powerful probs. Herein, a new arylamino fumaronitrile derivative NPAPF was synthesized and transferred as aggregation-induced emission luminogen (AIEgen) fluorescent nanoparticles (AF-NPs) via assembly technique. This AF-NPs exhibited two-photon absorption cross-section at 2.6×106 GM with 19.5% of fluorescence quantum yield. Moreover, utilizing the great potential of AF-NPs, two-photon imaging of live cells with good cytocompatibility is realized upon two-photon microscopy. By in vivo long-term tracing studies of mesenchymal stem cells, we demonstrated the tremendous advantage of AF-NPs tracer in monitoring the stem cells transplant. Therefore, our unique AF-NPs provided an efficient two-photon-absorbing probe for investigating biological mechanism and behavior, and also opened a new avenue for spatiotemporal visualization of transplanted stem cells.

KEYWORDS: organic nanoparticles; mesenchymal stem cell; aggregation-induced emission; two-photon; in vivo imaging

INTRODUCTION 2


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Fluorescence imaging techniques, especially two-photon excitation microscopy, have received extensive attention because of their potential in biosensor and diagnosis.1-9 Fluorescent materials that can be excited with the near-infrared (NIR) light by two-photon are particularly useful because biological tissues is optically transparent in NIR region, which possesses numerous of superiority over one-photon microscopy (OPM), mainly including low autofluorescence, high penetration depth (4500 μm), reduced photo-damage and photo-bleaching effects.10-15 Therefore, two-photon microscopy (TPM) has shown promise as visual tools for imaging and diagnosis in the biomedical field, and enormous efforts were devoted to developing nanoprobe that has two-photon emission capacity for bio-imaging and sensor design. 16-21

However, the small cross-section of two-photon absorption (TPA) and serious

cytotoxicity place a big limit on the advance in TPM. The exploitation of novel fluorescent nanoprobe with large TPA cross-section and high quantum yield, therefore, is urgently needed. Recently, the advancement in organic fluorescent nanoparticles have greatly prompted the development of TPM probes for bio-imaging in the past decade.22-26 These organic fluorescent nanoparticles possess ultra-bright fluorescence emission and low cytotoxicity. Nevertheless, the aggregation-caused quenching (ACQ) effect badly hindered the further biomedical exploitations in TPM. Fortunately, Tang’ group exploited a novel aggregation-induced emission (AIE) molecule, which launched strong fluorescence when aggregated or restricted.27 AIE molecules greatly settle the challenge of low quantum yield and fluorescence quenching by ACQ effect.

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Meanwhile, a series of the fluorescent probes with AIE characteristics have been developed for bio-imaging and sensor in recent years.28-37 Moreover, the AIE probe with large cross-section and high quantum yield also attracts much attention used as TPM probe in the past years.38-42 Herein, we explored an AIEgen-based nanoparticle (AF-NPs) with near-red emission and high TPA cross-section for two-photon fluorescence imaging and in vivo tracking. An arylamino fumaronitrile derivative, bis(4-(N-(2-naphthyl)pheny -lamino)phenyl)fumaronitrile (NPAPF) was selected as the luminogen and transferred into AF-NPs via assembly technique. AF-NPs demonstrated here possessed excellent good biocompatibility and good stability in various mediums covering wide pH. Moreover, the AIEgen-based AF-NPs exhibited ultra-high TPA cross-section of 2.6×106 GM and quantum yield at 19.5%. AF-NPs have been successfully applied in vivo cell tracing and demonstrated the potentiality served as efficient TPM probe.

EXPERIMENTAL SECTION Materials. Dulbecco’s Modified Eagle’s Medium (DMEM) and fetal bovine serum (FBS) were purchased from Hyclone (Logan, UT, USA). 1,2-distearoyl-sn-glycero-3phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG2000) was obtained from Avanti Polar Lipids (Alabaster, AL). 3-(4,5-dimethylthiazol-2 -yl)- 2,5diphenyltetrazolium bromide (MTT) was obtained from Sigma-Aldrich (Tokyo, Japan). Human breast cancer cell lines MDA-MB-231 were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA). Mesenchymal stem cells were isolated from Sprague Dawley rat. Unless specified, all of the commercial 4


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products were used without further purification. NPAPF

Synthesis.

Bis(4-(N-(2-naphthyl)phenylamino)phenyl)-fumaronitrile

(NPAPF) was prepared by using our previously procedures with little modification.43 The 4-bromophenylacetonitrile and iodine was firstly purged with nitrogen and then cooled to -78 °C. Then, sodium methoxide (52.6 mmol) and methanol (40 mL) were slowly put into above mixture and sharply mixed for other 40 min. Reaction mixture was then remained an ice-water bath with 4 h stirring. hydrochloric acid solution (5% in v/v) was added dropwise and then filtered. The filtrate was obtained through further enrichment. Then, a mixture of bis(4-bromophenyl)fumaronitrile (2.03 mmol), Cs2CO3 (5.98 mmol), N-phenyl-substituted amine (4.47 mmol), and toluene (15 mL) was purged with nitrogen, the catalyst Pd/P(t-Bu)3 were added into above mixture and further was purged and heated at 110 °C for 24 h. After cooled to room temperature, the mixture of water and dichloromethane were then added for organic extraction. The product was purified by HPLC (Figure S1) and determined by mass spectrometry (Figure S2). Preparation of nanoparticles. The near-infrared AF-NPs were obtained from DSPE-PEG2000 and NPAPF by the thin-film hydration way. NPAPF and DSPE-PEG2000 in 10 mL of chloroform at mass ratio of 1:5. The dry NPAPF-containing lipid film formed by removed the organic, and then was hydrated with PBS at 60 °C for 30 min. After purified through a 0.2 µm filter, the resulted sample was stored for the further usage. Characterization. The concentration of NPAPF in nanoparticles was measured by

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UV-vis spectrophotometry (Perkin Elmer Lambda 850). The size distribution and zeta potential of the AF-NPs in buffer with different pH were measured with a ZetaSizer Nano series Nano-ZS (Malvern Instruments Ltd, Malvern, UK). The morphology was recorded with transmission electron microscopy (TEM, FEI Tecnai F20). The absorption and fluorescence spectra were respectively recorded by using a Perkin Elmer Lambda 850 UV-vis-NIR spectrophotometer and an LS-55 Fluorescence Spectrometer. Two-photon Absorption Measurements. TPA fluorescence spectra were measured using two-photon induced fluorescence (TPIF) spectroscopy. In detail, the samples were excited with 100 fs laser pulses from the commercial cw mode-locked Ti:sapphire laser, and a femtosecond optical parametric amplifier (OPA) was employed within the spectral range of 800-960 nm. The emission of AF-NPs aqueous was collected by a high numerical aperture lens for further sending to a spectrometer’s entrance slit. Meanwhile, for calculating the quantum yield, Rhodamine B was selected as the reference. The concentration of AF-NPs suspension is calculated based on NP. The TPA cross-section of NP was calculated from the following equation: (1) The r and s represent the reference and sample, respectively; S is the integral area of the TPIF; Φ was denote as the fluorescence quantum yield; φ represent the overall fluorescence collection efficiency of the instrument, and c represents the concentration of sample. 6


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Photostability. To compare the photostability of AF-NPs, 200 µL aqueous solutions containing AF-NPs with various pH (the range of 1-13) were added into 96-well plates, respectively. Then, the Maestro in vivo fluorescence imaging system (Cri Inc.) was employed to collect the fluorescence images. Meanwhile, the fluorescence intensity of the above samples at various pH and incubated with PBS at different times were recorded at 640 nm using a microplate reader (Infinite M200, Tecan, USA). All experiments were repeated for three times. Cell culture. MDA-MB-231 cell line was cultured in DMEM supplemented with 10% FBS and 100 U·mL-1 penicillin/streptomycin at 37 °C in a 5% CO2 incubator with the humidity of 95%. The mesenchymal stem cell (MSC) was obtained from female SD mice of 8-10 weeks old, and were cultured in L-DMEM medium. The purified MSCs were expanded every 3 days for the next experiments. Cytotoxicity assays. Cells were seeded into 96-well plates at a density of 5000 cells per well and incubated overnight for reaching 80% confluency. After that, the old medium was discarded and refilled with 100 μL of fresh medium containing various concentrations of AF-NPs. After 24 h treatment, 0.5 mg·mL-1 MTT was added and incubated for other 3 h. Then, 100 mL DMSO was added to dissolve the resultant of MTT. The optical density (OD) of 570 nm was recorded by microplate reader (Infinite M200, Tecan, USA). Meanwhile, untreated cells in the normal medium were chosen as control group. All results are showed as mean ± SD compared to the control cells. One-photon and two-photon excited in vitro cellular imaging.

MSC and

MDA-MB-231 cell lines were treated with AF-NPs for 18 h at 37 °C incubator. After

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three times wash by PBS, the cells sample were observed on a confocal microscope (LSM 710, Zeiss). An excitation wavelength of laser set at 525 nm for capturing the emission of 600-700 nm (single channel). For two-photon images, an excitation of the pulse laser located at the wavelength of 830 nm. In vivo imaging. Female mice were obtained from the Animal Laboratory of Henan University (Kaifeng, China). All animal experiments were executed under the regulations of animal ethics that rooting from Henan University Animal Studies Committee. In order to the in vivo imaging, nude mice were injected with AF-NPs and the Maestro in vivo optical imaging system was employed to capture the imaging figure under green excitation. And also, AF-NPs at the dose of 10 mg.kg-1 were injected into the tail vein of nude mice for study the systemic distribution in different times. Statistical analysis. All data are showed as the mean ± standard deviation (SD). One-way analysis of variance (ANOVA) and t-test was used to analyze the differences between groups using the statistical software of SPSS. In statistical data, p < 0.05 was identified to be significant difference. RESULTS AND DISCUSSION The molecule structures and quantum yield of NPAPF. AIE luminogen NPAPF with AIE characteristic and near infrared emission was successfully synthesized according to previously reported work.43 The molecule and electronic structures were firstly investigated, as shown in Figure 1a and 1b, its HOMO-LUMO distribution shows that the NPAPF molecules possess an intrinsic character of intramolecular 8


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charge transfer (ICT). The AIE effect of NPAPF was also investigated by recording its fluorescence change in the mixture of tetrahydrofuran (THF)/water with different water fraction (fw). With the gradual addition of water into THF (fw≤50%), the emission intensity at 640 nm for NPAPF becomes increasingly weak (Figure 1c). The quenching effect of fluorescence that caused by the augment of solvent polarity indicates the property of twisted intramolecular charge transfer (TICT). The fluorescence emission radically enhanced with a further increase of water fw ≥ 50%, indicating the AIE characterization of NPAPF. We also study the quantum yield (ΦF) of NPAPF at various water fw. As shown in Figure 1d, when the water fw were less than 60%, NPAPF had negligibly small ΦF values (less than 1%) evaluated using Rhodamine B (RhB) methanol solution as a reference (ΦF = 89%). The ΦF value of NPAPF then dramatically increased when more water (fw≥80%) was added into the THF solution. More importantly, when the water fw reached to 99%, the ΦF values of NPAPF is 17.6%. These results demonstrated that NPAPF luminogen possesses TICT and AIE character simultaneously. The unique AIE feature makes NPAPF as a good AIEgen-based luminogen for construction of NIR nanoparticles.

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Figure 1. Aggregation-induced emission (AIE) characterization of NPAPF. (a) Chemical structure and (b) HOMO and LUMO energy level of NPAPF. The change of (c) the emission intensity and (d) quantum yield of NPAPF with the increment of water fraction of the mixture of THF/water.

Morphology and characterization of AF-NPs. To facilitate further applied in biomedical imaging, the aggregation- induced emission luminogen (AIEgen) fluorescent nanoparticles (AF-NPs) were fabricated via the modified film hydration method (Figure 2a).44 DSPE-PEG2000 (molecular structure as shown in Figure S3) was used to encapsulate NPAFA to self-assembly fluorescent nanoparticles (AF-NPs). As shown in Figure 2b, TEM images show that AF-NPs are uniform spheroidal particle with the mean diameter around 70 nm. Dynamic light scattering (DLS) was employed to confirm their hydrodynamic diameter and size distribution at 95±2.1 nm (Figure 2c). We also studied their photophysical properties, as described in Figure 2d,

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AF-NPs have a maximum emission at 640 nm when excited at 495 nm, and confirmed a large Stokes shift (>140 nm). Importantly, the quantum yield of AF-NPs in water reaches 19.5%, highlighting the compact aggregation of NPAPF in the interior of nanoparticles. In addition, the fluorescence decay curves of NPAPF in various aggregation statues were tested and showed that the fluorescent lifetime of free NPAPF, NPAPF aggregation, and AF-NPs possesses a similar fluorescence lifetime (Table S1). These results indicated that the aggregation degree of AIE molecular only affects the fluorescence intensity, not fluorescence delay. The photo-stability and physical stability of AF-NPs. In order to apply in bio-imaging, the stability of AF-NPs was further investigated. As shown in Figure 2e, the present AF-NPs show a good photostability in water, phosphate buffer and solutions with various pH over 1 to 13. Meanwhile, as previous report,45,

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the

fluorescent intensity of AF-NPs remains unchanged over 24 days when stored at 4 °C (Figure 2f). These results suggested that the AF-NPs possessed well photostability in various buffer solutions, particularly in various pH. Moreover, the hydrodynamic diameters of AF-NPs keep unchanged after being stored for 30 days at 4 °C (Figure 2g), suggesting the super-stability of AF-NPs. This super-stable AF-NPs with high emission have a great potential in bio-imaging as a fluorescence-enhanced probe.

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Figure 2. Preparation and representation of AIEgen-based AF-NPs. (a) Schematic representation of the preparation of AF-NPs. (b) TEM image and (c) hydrodynamic diameter of AF-NPs. (d) Absorption and fluorescence spectra of AF-NPs. Inset: Tyndall effect (left) and fluorescence (right) images of AF-NPs. (e) Fluorescence variation of AF-NPs incubated with different medium with various pH buffer and serum. Inset: the corresponding fluorescence graphics of AF-NPs at various buffer solutions. (f) Fluorescence change of AF-NPs after 24 days storage. (g) Size stability of AF-NPs after 30 days storage.

In vitro imaging: Before bio-imaging, the biocompatibility of the resulting AIEgen-based NPs was firstly studied by the standard MTT assay. Figure 3 showed

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that AF-NPs were nontoxic and safe in MSC. And the similar biocompatibility was also received in MDA-MB-231 cells (Figure S4). Benefited from the enhanced emission, the concentrations of AF-NPs used in a follow-up experiment were obviously lower than the tested concentration of MTT analysis. For bio-imaging, in vitro one-photon imaging was studied by confocal laser scanning microscopy (CLSM). As shown in Figure 3b, bi-color images revealed that the red fluorescent AF-NPs expressly located in the cell cytoplasm when the nucleus of MSC was labeled by with nucleus dye DAPI (blue fluorescence). To further analyze the intracellular localization of AF-NPs, colocalization of AP-NPs and Lysotracker green was checked and showed that AP-NPs could merged with lysosome after the entrance of cells, which indicated the uptake pathway of AP-NPs (Figure S5). And CLSM imagines of MDA-MB-231 cell verified the above conclusion (Figure S6). These results indicated that AF-NPs was an excellent probe for cellular imaging.

Figure 3. (a) Viability of MSC after incubated with various concentrations of AF-NPs for 24 h. (b) Confocal images of MSC after treatment with 5 µg·mL-1 AF-NPs for 18 h. The excitation was seted at 405 and 525 nm for DAPI and AF-NPs, respectively. The scale bar was 20 µm. 13



Two-photon imaging potential of AF-NPs was investigated in this work. We first study the TPA spectrum of AF-NPs in the region from 790 to 880 nm. As described in Figure 4a, the maximum TPA cross-section (δ) appears at 860 nm, and after calculated using Rhodamine B as the reference, AF-NPs exhibited a high value of 2.6×106 GM (based on nanoparticle concentration). Meanwhile, the emission spectrum of AF-NPs was investigated under the two-photon excitation and the emission peak was positioned at 630 nm, indicated that AF-NPs possessed near-red excitation and emission capacity simultaneously (Figure 4b). The excitation power dependent emission intensities at 630 nm further confirmed that the emission is due to a two-photon excitation process (Figure S7). Moreover, two-photon imaging of MSC and MDA-MB-231 cells after incubation with AF-NPs were exhibited in Figure 4c. The red fluorescence from the cytoplasm is distinctly captured, indicating that AF-NPs exhibit the great potential in two-photon fluorescence imaging. These results further confirmed that AF-NPs effectively internalized into cells, which was consistent with the results shown in one-photon imaging.

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Figure 4. Two-photon imaging capacity of AF-NPs. (a) TPA spectra and (b) the corresponding TPA-induced emission spectrum of AF-NPs aqueous. (c) Two-photon imaging of MSC and MDA-MB-231 cells after incubated with 5 µg·mL-1 AF-NPs for 18 h. The excitation wavelength of two-photon imaging was 830 nm. Scale bar was defined as 20 µm. In vivo stem cell imaging. Prompted by the aforementioned results, AF-NPs were employed as long-term probes for in vivo stem cell tracking. MSC that pre-labeled by AF-NPs (5 µg·mL-1) for 18 h was seeded into the right thigh root of the mouse by subcutaneous injection. In vivo imaging was performed from at 0.5 h to 22 days post injection using a Maestro in vivo optical imaging system. As exhibited in Figure 5, the injected site showed strong fluorescence at 0.5 h post injection. And impressively, after treatment of 22 days, the injected site still exhibited an obvious fluorescence signal while background is blue background, which definitely confirm the enormous prospect of AF-NPs in long-term cell tracking in vivo. Additionally, using the model of tumor-bearing mice, AF-NPs was demonstrated their advantage in tumor-targeting ability after intravenous injection. (Figure S8).

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Figure 5. in vivo fluorescence images of 1×106 of MSC pre-labeled by AF-NPs (5 µg·mL-1) in the mouse.

CONCLUSIONS In summary, we successfully synthesized the ultra-bright NIR AIE nanoparticles based on NPAPF, which possessed excellent colloidal stability and photostability. Cytotoxicity analyses showed that AF-NPs have a favorable biocompatibility. More importantly, these resultant AIE nanoparticles possess large TPA cross-section at 2.6×106 GM and high quantum yield at 19.5%. Two-photon imaging and long-term tracing analysis in vivo demonstrated that AF-NPs has enormous potential in TPM imaging with deeper tissue-penetration and high signal-to-noise ratio. This study provides a novel TPM probe for further potential biomedicine application.

Supporting Information Detailed experimental procedures including HPLC analysis, mass spectrometry, fluorescence decay, the cell viability of MDA-MB-231 cells by MTT assay, confocal microscopy images of intracellular localization, excitation-dependence emission, and tumor-targeting in vivo imaging

Corresponding author *E-mail: [email protected] (Yongwei Huang) *E-mail: [email protected] (Shengliang Li)

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Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This work was supported by the National Natural Science Foundation of China (No: 21572045), the Natural Science Foundation of Henan Province (No: 162300410017 and 162102310074), Foundation for Science & Technology Innovation Talents in the Universities of Henan Province (No: 16HASTIT008), the Fundamental Research Funds of Henan University (No. 2014XXJC0019 and 2014YQPY0084).

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from

Tetraphenylethene

Building

Blocks:

Synthesis,

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AIEgen Nanoparticles of Arylamino Fumaronitrile Derivative with High

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