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In Vivo Photoacoustic Detection and Imaging of Peroxynitrite Xialing Qin, FAN LI, Yifan Zhang, Tao Feng, Gongcheng Ma, Yongxiang Luo, Peng Huang, and Jing Lin Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b01992 • Publication Date (Web): 02 Jul 2018 Downloaded from http://pubs.acs.org on July 3, 2018

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Analytical Chemistry

In Vivo Photoacoustic Detection and Imaging of Peroxynitrite Xialing Qin, Fan Li, Yifan Zhang, Gongcheng Ma, Tao Feng, Yongxiang Luo, Peng Huang,* Jing Lin* Guangdong Key Laboratory for Biomedical Measurements and Ultrasound Imaging, Laboratory of Evolutionary Theranostics, School of Biomedical Engineering, Health Science Center, Shenzhen University, Shenzhen 518060, China. ABSTRACT: Photoacoustic detection is an emerging non-invasive and non-ionizing detection technique with the merits of rich contrast, high resolution and deep tissue penetration, especially for in vivo detection and imaging. Herein, we developed a photoacoustic molecular imaging probe (denoted as nanonaps) composed of a naphthalocyanine dye and a heptamethine dye as the internal standard with unchanged signals at 860 nm and the sensing component with peroxynitrite (ONOO−) target-decreased signals at 775 nm, respectively. The as-prepared nanonaps displayed high sensitivity and specificity of ONOO− both in vitro and in vivo. The PA860/PA775 ratio was increased as a function of the concentration of ONOO− (0~150 nM). More interestingly, our ratiometric nanonaps could be used for in vivo detection and imaging of ONOO−.

Peroxynitrite (ONOO−) is one of the reactive oxide species (ROS) that modulate essential functions in living organisms.1 Specifically, it could easily penetrate cell membrane and oxidize a series of critical biomolecules, such as proteins, lipids, nucleic acids, glycogen, iron-sulfur clusters, and thiols.2 Thus, ONOO− plays crucial roles in many diseases, such as anti-viral, vascular diseases, diabetes, cangiocardiopathy, neurodegenerative diseases, cerebral ischemia-reperfusion injury, and inflammatory lung diseases.3-7 Recent studies also show that ONOO− is implicated closely in the immunosuppression of tumor.8-10 Therefore, the development of techniques for detecting and monitoring ONOO− is important for understanding the pathophysiology and early diagnosis of ONOO− related diseases. Current detection techniques of ONOO− are mainly included fluorescence detection, proteins staining, amperometric detection and magnetic resonance imaging (MRI) detection.11-14 Among these techniques, fluorescence detection is widely used due to its advantages like high sensitivity, short acquisition time and high temporal resolution. However, the excitation light of fluorescence imaging ranging from 400 to 700 nm, which limits tissue penetration of less than 10 mm. 15,16 Furthermore, fluorescence imaging generally relies on fluorescence as the signal readout and autofluorescence hampers its further application in vivo.17-19 Besides, protein staining contained nitrated tyrosine residues is an indirect technique and

lacked compatibility with living biological specimens.12 Amperometric detection only used in simulated environment that was not suitable in vivo. 13 MRI detection bears low spatial resolution.12 Therefore, it remains a big challenge to design robust assays for detecting ONOO− level in vivo. Photoacoustic detection (PAD), a new detection technique, is based on the interaction between photoacoustic (PA) probes and analytes that causes the changes of PA signals as indicators, which can be detected by PA imaging. In comparison with other imaging modalities, PA imaging is an emerging non-invasive and non-ionizing imaging with the merits of rich contrast, high resolution and deep tissue penetration.20-25 So far, there are few PA probes to measure the concentration of ONOO−. For instance, Pu and coworkers developed Near infrared (NIR) light absorbing semiconducting polymer nanoparticles as ratiometric PA probe for ROS imaging in living mice with a limitation of 50 nM.26 Recently, they used an organic semiconducting nanoprobes doped with bulky borane for real-time ratiometric PA imaging of ONOO− in the tumor of living mice with a limitation of 100 nM.27 However, their PA probes are which involved sophisticated organic preparation. Therefore, there is still a high demand for simple and facile PA probes for detection and imaging of ONOO−. In this study, we reported a new ratiometric nanoprobe based on one-step nanoprecipitation of commercial mol-

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ecules for detection and imaging of ONOO− (Scheme 1). The nanoprobes consist of 5,9,14,18,23,27,32,36octabutoxy-2,3-naphthalocyanine (ONc), IR780 iodide, and poly(oxyethylene)-poly(oxypropylene)poly(oxyethylene) (PEO-PPO-PEO, Pluronic F127). In the addition of Pluronic F127, the ONc and IR780 could assemble into stable ONc/IR780@F127 nanoprobes (named as nanonaps). The ONc acted as an internal standard with unchanged PA signals at 860 nm, and IR780 was the sensing component with decreased PA signals at 775 nm. The ratio of the ratiometric PA value (PA860/PA775) could be used as an indicator for ONOO−detection.

accomplished by a Vevo 2100 LAZR system (FUJIFILM Visual Sonics, USA) with the following parameters: Frequency: 40 MHz; 2D gain: 0 dB; PA gain: 40 dB; the excitation wavelengths: 775 and 860 nm. Preparation of nanonaps. The nanonaps were prepared via a modified nanoprecipitation method according to previous references. 28,29 The hydrophobic ONc and IR780 in CH2Cl2 were added dropwise into F127 (10% W/V) under ice-water bath. Different ratios of ONc and IR780 (10:10, 10:5, 10:2, 10:1, and 10:0.5) were tested to achieve the highest homogeneity and maximum loading efficiency. This mixture solution was sonicated for 6 min, and stirred for 4 h at room temperature. The nanonaps were purified with centrifugal filtration to remove all free F127 at 4 °C, and stored at fridge for further use. Preparation of analytes. Solutions of ONOO−, •OH, O2, NO•, O2•−, ROO• ions were prepared according to previously reported methods.1,30,31 ClO−, H2O2, Cys and Arg solutions were prepared as follows: 1

1) ClO−: The concentration of the ClO− stock solution was determined by measuring the absorbance at 292 nm with a molar extinction coefficient of 350 M-1cm-1.1 Diluted solutions were used as standard analytes. 2) H2O2: The stock H2O2 solution (9.8 M) was purchased from commercial reagent company. Diluted solutions were used as standard analytes. Scheme 1. (A) Molecular structures of IR780, Pluronic F127 and ONc used for the preparation of nanonaps. (B) Illustration of a ratiometric nanoprobe composed of ONc, IR780, and Pluronic F127.

EXPERIMENTAL SECTION Chemicals and Materials. All regents and chemicals were commercial available. The ONc was purchased from Sigma-Aldrich (St. Louis, MO, USA). NaNO2, NaClO, H2O2, and KO2 were purchased from Aladdin Reagent (Shanghai, China). IR780 iodide, 2,2'-Azobis(2methylpropionamidine) dihydrochloride (98%), Iron(II) sulfate heptahydrate (99.5%, AR), L-cysteine (Cys) and Larginine (Arg) were purchased from J&K Chemical Ltd. (Shanghai, China). Manganese dioxide, Sodium molybdenum oxide and Sodium nitroferricyanide (III) dehydrate were purchased from Macklin Biochemical Co., Ltd (Shanghai, China). Deionized water (18.2 MΩ·cm) was used in all experiments, which was produced by a Milli-Q Academic water purification system (Millipore Corp., Billerica, MA, USA). Reagents for cell culture were supplied by Gibco (Tulsa, USA). Equipment. UV-Vis-NIR spectra were recorded on a Varian UV-Vis-NIR spectrophotometer (Cary 60 Bio, China) and a SYNERGY H1 microplate reader (BioTEK, USA). Dynamic light scattering (DLS) was performed on the Malvern Nano-ZS Particle Size (Zetasizer Nano, UK). Transmission electron microscopy (TEM) images were obtained on a JEM-1230 instrument (Nippon Tekno, Japan). The mass spectra were recorded by using a LC-MS AGILENT instrument (Agilent, USA). PA imaging were

3) Cys and Arg: A 10 mM stock solution of Cys or Arg was prepared in deionized water and then diluted into standard analytes. In Vitro Detection of ONOO−. 50 μL of nanonaps stock solution was added quickly into different concentration of ONOO− solutions (0, 10, 25, 50, 75, 125 and 150 nM) with total volume of 0.5 mL. The final concentration of nanonaps was fixed at 3 μg/mL of ONc. The 100 μL of above reaction solutions were added into 96-well plates and then their absorbance at 780 and 863 nm were recorded by using a microplate reader, respectively. For PAD, reaction solutions (0.2 mL) were also prepared by the above same process. The final concentration of nanonaps was fixed at 25 μg/mL of ONc. PA imaging was conducted under two different excitation wavelengths at 775 and 860 nm. Selectivity Experiments. Analytes solutions (ONOO−, ClO−, H2O2, •OH, 1O2, NO•, O2•−, ROO•, Cys and Arg) were prepared by deionized water. Then a 50 μL of nanonaps stock solution was added quickly in previously prepared analytes solutions (450 μL) to reach a final concentration of 3 μg/mL of ONc, and the final concentrations of ONOO− and ClO− were 1 mΜ, other analytes concentrations were 50 μM. The absorbance and PA imaging of above reaction solutions were measured by the above mentioned method. Cell Cytotoxicity. HeLa, 4T1 and MCF 10a cells were obtained from the Chinese Academy of Science. HeLa cells were cultured in Dulbecco’s Modified Eagle’ s Medium (DMEM) containing 10% (v/v) fetal bovine serum (FBS), penicillin (100 μg/mL) and streptomycin (100


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Analytical Chemistry μg/mL) at 37 °C with 5% CO2. 4T1 and MCF10a cells were cultured in RPMI 1640 Medium containing 10% (v/v) FBS, penicillin (100 μg/mL) and streptomycin (100 μg/mL) at 37 °C with 5% CO2. The cytotoxicity of nanonaps was evaluated on HeLa, 4T1 and MCF10a cells. Cells were seeded at a density of 1 × 104 cells per well in 96-well plates and incubated for 12 h. Then the culture medium was replaced and the cells were exposed to different concentrations of nanonaps (0-10 μg/mL of ONc) for another 24 h under the same conditions. Then cell viabilities were detected by a standard methyl thiazolyl tetrazolium (MTT) assay. In vivo PA imaging of ONOO−. The animal experiments were conducted in accordance with the Guidelines of the Shenzhen University Animal Care and Use Committee. Female BALB mice aged 6-8 weeks (weighted 2530 g) were obtained from Guangdong Medical Laboratory Animal Center (Guangdong, China). To establish tumorbearing mice, 4T1 cells (2 × 106) suspended in 100 μL of PBS were subcutaneously injected in right rear flank of mice. When the tumor size reached ∼60 mm3, nanonaps (25 μg of ONc, 250 μL) were locally injected in tumor tissues, and then in vivo PA imaging of ONOO− was carried out. As control, nanonaps were injected in left thigh of normal mice. PA images were acquired at 0, 10, 20, 30, 40, 50, 60 and 120 min after injection. RESULTS AND DISCUSSION To obtain uniform nanonaps, several different ratios of ONc and IR780 (10:10, 10:5, 10:2, 10:1, and 10:0.5) were investigated. As shown in Table S1, the polydispersity Index (PDI) of 10:2 was less than 0.2, so this ratio of 10:2 was chosen for further use. The nanonaps display well-defined sphere (Figure 1A) with average hydrodynamic diameter of 184.7 ± 17.3 nm (Figure 1B, Table S1, Supporting information ), which remains no change after storage in PBS (pH7.4) for 7 days (Figure S1, Supporting Information). The UV-Vis-NIR spectra indicate that there are two absorption peaks at 780 and 863 nm, corresponding to IR780 and ONc, respectively (Figure 1C). As shown in Figure 1D, the PA peaks of nanonaps site at 775 and 860 nm, corresponding to IR780 and ONc, respectively.

Figure 1. Characterizations of nanonaps. A) Representative TEM image of nanonaps (Scale bar:200 nm); B) Size distribution of nanonaps in the solution (Inset: a photograph of nanonaps solution; C) UV-Vis-NIR spectra of nanonaps, free ONc and IR780; D) PA spectra of nanonaps, free ONc and IR780.

Next, the selectivity of nanonaps was investigated. As shown in Figure 2A, only ONOO− could induce remarkable absorption decrease of nanonaps at 780 nm, which is attributed to ONOO− mediated the breakage of the IR780 structure (Figure S2, Supporting information). The similar reaction mechanism was established in previous works.21,32 As shown in Figure 2B, the absorption (A863/A780) ratios of nanonaps were calculated. For ONOO− (1 μM), the A863/A780 ratio is ~2.64. For other analytes, the A863/A780 ratio is less than 1.80. Afterwards, the PA response of nanonaps was evaluated in Figure 2C. The PA amplitude at 775 nm decreased significantly in the presence of ONOO−, and kept unchanged at 860 nm. In contrast, in the presence of other analytes, such as ClO−, and H2O2, the PA spectra were as same as blank control. As shown in Figure 2D, the PA860/PA775 ratio of ONOO− group was ~2.88. For other analytes, the PA860/PA775 ratio is less than 1.5. PA images showed high selectivity of nanonaps against ONOO− (Figure 2E). More interestingly, photographic images also demonstrated that nanonaps are in response to ONOO−, changing its colors from green into orange (Figure 2F). These results suggested that the as-prepared nanonaps showed good selectivity to ONOO−.



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cated that the as-prepared nanonaps had great potential for in vitro detection and imaging of ONOO−. Encouraged by the in vitro results, the in vivo detection and imaging of exogenous ONOO− were performed on subcutaneous 4T1 xenograft model. The normal mice were used as the control group. After intratumoral injection of nanonaps (25 μg ONc in 250 μL), PA signals of tumor tissues at 775 and 860 nm were monitored. The PA signal at 775 nm significantly decreased over time (Figure 4A), indicating the existence of ONOO− in tumor. For the control group, after local injection of nanonaps (25 μg ONc in 250 μL) into normal thigh of mice, the PA860/PA775 ratio remained negligible change over time (Figure 4B). As shown in Figure 4C, quantitative analysis revealed that PA860/PA775 ratio gradually increased to 3.10 ± 0.084 within 120 min post-injection for nanonaps-treated tumorbearing mice. In the control group, the PA signals at 775 and 860 nm kept almost unchanged (1.36 ± 0.001) within 120 min post-injection. Therefore, nanonaps could be effectively used to detect in vivo ONOO− through ratiometric PA imaging. −

Figure 2. The selectivity of nanonaps toward ONOO . A) Representative absorption spectra of nanonaps (3 μg/mL of − − ONc) in the absence and presence of ONOO (1 μM), ClO (1 μM) or H2O2 (50 μM); B) The A863/A780 ratios of nanonaps (3 μg/mL of ONc) in presence of analytes; C) Representative PA spectra (25 μg/mL of ONc) of nanonaps in the presence of − − ONOO (1 μM), ClO (1 μM) or H2O2 (50 μM) ;D) The PA860/PA775 ratios of nanonaps (25 μg/mL of ONc) in the presence of analytes; E) PA images of nanonaps (25 μg/mL of ONc) in the presence of analytes; F) Photograph of nanonaps (25 μg/mL of ONc) before and after reaction with analytes. (n=3 per group).

Besides the selectivity of nanonaps, their sensitivity was also investigated. The adding of ONOO− resulted in significantly decreased of the absorption at 780 nm in a dose-dependent manner (Figure 3A). There is a good linear correlation (R2 = 0.97943) between A863/A780 ratios of nanonaps and the concentration of ONOO− (0-250 nM), with a limit of detection of 10 nM (see the inset in Figure 3B). For in vitro PA detection, the PA amplitude at 775 nm decreased with the concentration increase of ONOO− (Figure 3C). As shown in Figure 3D, there is a good linear relationship between PA860/PA775 ratios and the concentration of ONOO− (0-250 nM) (R2 = 0.98996). The limit of PA detection is 10 nM. Meanwhile, PA images demonstrated that PA signals at 775 nm decreased with ONOO− (Figure 3E). According to previous studies,33-35 the rates of in vivo ONOO− production have been estimated to be about 50-100 μM per min in tumor microenvironment. In our case, the limit of ONOO− detection by nanonaps was lower than the physiological steady-state concentration of ONOO− (at the nanomolar level)36. Moreover, nanonaps could work in pH 5.0-7.4 which similar with tumor acidic microenvironment (Figure S3, Supporting information). Additionally, no obvious cytotoxicity was observed for nanonaps incubated with MCF 10a, HeLa and 4T1 cells (Figure S4, Supporting information). These results indi-

−

Figure 3. The sensitivity of nanonaps toward ONOO . A) The UV-Vis-NIR spectra of nanonaps (3 μg/mL of ONc), with − different concentrations of ONOO (0-1 μM); B) A863/A780 ratios of nanonaps (3 μg/mL of ONc) as a function of the − concentration of ONOO . Inset: The plot of ratiometric ab− sorption A863/A780 ratios of nanonaps against the ONOO concentration between 0 to 250 nM; C) PA spectra of nanonaps (3 μg/mL of ONc) with different concentrations of − ONOO (0-1 μM); D) PA860/PA775 ratios of nanonaps (25 μg/mL of ONc) as a function of the concentration of − ONOO . Inset: The plot of ratiometric PA amplitude intensi− ty PA860/PA775 ratios of nanonaps against the ONOO concentration between 0 to 250 nM; E) PA images of nanonaps − (25 μg/mL of ONc) with different concentrations of ONOO ; (n=3 per group).


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Analytical Chemistry Figure S2, mass spectra of IR780 before and after adding ONOO−; Figure S3, UV-Vis-NIR spectra of nanonaps at various pH values, and in the presence of ONOO−; Figure S4, cell viability of nanonaps.

AUTHOR INFORMATION Corresponding Author E-mail: [email protected]; [email protected] Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS This work is financially supported by the National Natural Science Foundation of China (31771036, 51573096, 51703132), the Basic Research Program of Shenzhen (JCYJ20170412111100742, JCYJ20160422091238319), and Fok Ying-Tong Education Foundation for Young Teachers in the Higher Education Institutions of China (161032).

REFERENCES

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Figure 4. In vivo ratiometric PA imaging of ONOO . PA/ultrasound merged images of A) 4T1 tumor tissues and B) left thighs of mice after injection of nanonaps (25 μg ONc in 250 μL). C) PA860/PA775 ratios as a function of time postinjection. (n = 3 per group)

CONCLUSIONS In summary, we developed a ratiometric PA nanonaps for ONOO− detection both in vitro and in vivo. The nanonaps were prepared by using one-step nanoprecipitation of commercial molecules including ONc, IR780 and F127. The ONc was used as the internal standard with unchanged signals at 860 nm. The IR780 was used as the sensing component with ONOO− target-decreased signals at 775 nm. More importantly, the as-prepared nanonaps were able to detect in vivo ONOO−. The nanonaps held great potential for real-time in vivo monitoring of ONOO− with deep tissue penetration and high spatial resolution.

Supporting Information The Supporting Information is available free of charge on the ACS Publications website at xxxx. Table S1, Sizes of nanonaps with different ratios of ONc and IR780; Figure S1, size change of nanonaps in PBS;

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