Switchable Photoacoustic Imaging of Glutathione Using MnO2

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Biological and Medical Applications of Materials and Interfaces

Switchable Photoacoustic Imaging of Glutathione Using MnO2 Nanotubes for Cancer Diagnosis Chang Liu, Depeng Wang, Ye Zhan, Lingyue Yan, Qian Lu, Michael YZ Chang, Jingwen Luo, Lidai Wang, Dan Du, Yuehe Lin, Jun Xia, and Yun Wu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b14944 • Publication Date (Web): 30 Nov 2018 Downloaded from http://pubs.acs.org on November 30, 2018

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ACS Applied Materials & Interfaces

Switchable Photoacoustic Imaging of Glutathione Using MnO2 Nanotubes for Cancer Diagnosis

Chang Liu1,‡, Depeng Wang1,‡, Ye Zhan1‡, Lingyue Yan1‡, Qian Lu2, Michael Yu Zarng Chang1, Jingwen Luo3, Lidai Wang3, Dan Du2, Yuehe Lin2,*, Jun Xia1,*, Yun Wu1,*

1

Department of Biomedical Engineering, University at Buffalo, The State University

of New York, Buffalo, New York 14260, United States 2

School of Mechanical and Material Engineering, Washington State University,

Pullman, Washington 99164, United States 3

Department of Mechanical and Biomedical Engineering, City University of Hong

Kong, Hong Kong, P. R. China

‡ These

authors contribute equally

*Corresponding

authors

E-mail: [email protected] (Y. H. Lin). E-mail: [email protected] (J. Xia). E-mail: [email protected] (Y. Wu).

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Table of Content Graphic


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ABSTRACT Glutathione is overexpressed in tumor cells and regulates cancer growth, metastasis and drug resistance. Therefore, detecting glutathione levels may greatly facilitate cancer diagnosis and treatment response monitoring. Photoacoustic (PA) imaging is a noninvasive modality for high-sensitivity, high-resolution, deep-tissue optical imaging. Switchable PA probes that offer signal on/off responses to tumor targets would further improve the detection sensitivity and signal-to-noise ratio of PA imaging. Here we explore the use of MnO2 nanotubes as a switchable and biodegradable PA probe for dynamic imaging of glutathione in cancer. Glutathione reduces black MnO2 nanotubes into colorless Mn2+ ions, leading to decreased and “signal off” PA amplitude. In phantoms, we observed a linear response of reduced PA signals of MnO2 nanotubes to increased glutathione concentrations. Using melanoma as the disease model, we demonstrated that MnO2 nanotubes-based PA imaging of glutathione successfully distinguished B16F10 melanoma cells from BEAS-2b normal cells and discriminated B16F10 tumors from healthy skin tissues. Our results showed that MnO2 nanotubes are a potent switchable and biodegradable PA probe for glutathione imaging in cancer diagnosis.

Keywords: photoacoustic imaging, switchable contrast reagent, glutathione, MnO2, nanotubes, cancer, melanoma



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INTRODUCTION As the most abundant non-protein molecule in the cells, glutathione (GSH) plays a number of physiological roles, such as immune modulation, anti-oxidation, and detoxification. An elevated level of GSH is typically found in many types of human cancers, including melanoma, breast cancer, lung cancer, and leukemia

1-2.

The high

level of GSH not only promotes cancer growth, but also increases the resistance of cancer cells to chemotherapeutic drugs 1, 3. Thus, in vivo imaging of glutathione level will provide important insights for cancer diagnosis and therapeutic responses. However, current techniques for in vivo imaging of GSH level all have their inherent limitations. For instance, fluorescence imaging, while possessing high sensitivity, has limited tissue penetration depth and low spatial resolution 4. Positron emission tomography (PET) of glutathione transferase activity is associated with health risks due to ionizing radiation 5-6. Magnetic resonance imaging (MRI) is quite expensive and not suitable for continuous imaging over a long period of time 7. To overcome these challenges, we propose to develop MnO2 nanotubes-based, switchable photoacoustic imaging (PAI) to detect GSH for cancer diagnosis. Taking advantage of the low acoustic scattering in tissue, PAI offers advantages of high sensitivity, deep tissue penetration capability (up to ~11 cm in ex vivo tissue 8), low system cost, and no radiation risk. Thus, it has been increasingly used in biomedical imaging

9-10.

Among many endogenous or exogenous contrast agents, switchable PA

probes detect tumor targets using the “signal off/on” mechanism instead of relying on the concentration difference between tumor and normal tissues. Therefore, they provide a higher detection sensitivity in discriminating between benign and malignant lesions 11.

The development of switchable PA probes is still in the very early stage, and most

studies focused on activatable PA probes that offer “signal on” responses to tumor


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targets. For example, peptide-based activatable PA probes were synthesized to detect the matrix metalloproteases (MMP-2 and MMP-9)

12-13

and furin 14. Semiconducting

nanoparticles have been developed as activatable PA probes for in vivo imaging of reactive oxygen species

15-17.

IR806-pyridine dithioethylamine was synthesized as an

activatable probe for GSH imaging in cervical cancer 18. In this study, we have developed the MnO2 nanotubes-based, switchable PA probe that, for the first time, utilizes “signal off” mechanism for in vivo imaging of GSH for cancer diagnosis. MnO2 nanotubes absorb a wide spectrum of light, therefore they can serve as an effective contrast reagent for PAI. Once MnO2 nanotubes are uptaken by cells, intracellular GSH will convert black MnO2 nanotubes into colorless Mn2+ ions, leading to decreased PA amplitude. The higher the GSH concentration, the faster the PA amplitude decreases, enabling dynamic visualization of GSH level. Over the past few years, various MnO2-based nanomaterials have been developed for cancer imaging. For instance, MnO2 nanoparticles 19, MnO2 nanotubes 20 and MnO2 nanosheets 7, 21 were used as GSH-activated MRI contrast reagents for cancer imaging. MnO2 nanotubes and MnO2 nanosheets are excellent fluorescence quenchers. They were used together with fluorescence dye labeled DNA sensing probes and two-photon mesoporous silica nanoparticles for GSH-activated fluorescence imaging of tumor cells

7, 20, 22.

The

applications of MnO2 nanotubes in switchable PAI have never been explored. Here, we tested our switchable PAI approach in the diagnosis of melanoma, which is the most serious type of skin cancer and it accounts for most of skin cancer deaths. Dermatoscopy, skin biopsy, and lymph node biopsy are current diagnostic methods for melanoma, however, they are invasive and are limited by low sensitivity and specificity 23.

PAI has great potential to become a non-invasive and sensitive diagnostic modality

for melanoma. It has successfully demonstrated imaging of melanoma vasculature


24-


26,

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thickness 26, angiogenesis 27, and metastasis 28. However, because melanin exists in

both benign nevi and melanoma, it is quite challenging to distinguish benign and malignant lesions solely through PAI signal amplitude. Because GSH is closely related with tumor growth, metastasis and drug resistance, the dynamic imaging of GSH via MnO2 nanotube-based, switchable PAI may provide insights at molecular level about tumor malignancy, tumor burden, metastasis potential and treatment response. On the contrary to activatable, “signal on” probes (such as IR806-pyridine dithioethylamine18) with which signals may be interfered by increased melanin levels due to tumor growth, MnO2 nanotubes-based PA probe uses “signal off” mechanism, therefore it may better distinguish GSH-induced signals from melanin-induced signals and offers higher diagnostic accuracy. Besides, MnO2 nanotubes are biodegradable, the resulted Mn2+ ions can be quickly excreted by kidneys and thus should induce little toxicity

29-30,

especially when they are applied locally for melanoma diagnosis. Here we have demonstrated the effective detection of GSH via MnO2 nanotubes in vitro and in vivo using custom-built photoacoustic computed tomography (PACT) systems.

RESULTS AND DISCUSSION Set up of photoacoustic computed tomography (PACT) systems Custom-built, linear-transducer-array-based photoacoustic computed tomography (PACT) systems were used in this study (Figure 1). There were two configurations of systems: one for phantom imaging and the other for in vivo animal imaging. As shown in Figure 1a, the phantom experiment involved a tube filled with contrast agents. The tube was immersed in a water tank and its photoacoustic signals were detected by a linear ultrasound array located on the left side of the tube. For in vivo imaging, the animal was placed underneath a water tank (the bottom was sealed with a plastic


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membrane) and its photoacoustic signals were detected from the top by the linear transducer array. Light illumination was provided from the side by two linear fiber bundles. We used different setups for phantom and in vivo studies, because our previous study showed that side illumination did not work well for objects immerged in a clear medium 31. For both phantom and animal imaging, signal excitation was achieved by a 10 Hz pulsed laser emitting 750 nm wavelength light. This wavelength was chosen because MnO2 nanotubes had good absorption at this wavelength 20 and the OPO laser output is relatively strong and uniform. In addition, 750 nm laser diodes are commercially available, allowing for future system miniaturization. To account for energy fluctuation, we measured the light energy in each experiment and post calibrated the PA amplitude. Compared to previous activatable PAI studies which used commercialized PAI systems with a fixed imaging platform, our setup can be easily reconfigured for different imaging needs. The actual imaging performance is also superior because we can adjust various receiving parameters, such as gain, filter, and input impedance, for the best photoacoustic image quality. The translational impact of our PACT system is high because we used a clinical ultrasound transducer array (ATL/Philips L7-4), which can be better adapted by the current practice compared to custom-made curved or high frequency transducers. Besides, our setup involved only a single imaging wavelength with temporal data analysis, which not only simplified system setup and data collection but also reduced the overall cost. Details of other system parameters can be found in the methods section.



Figure 1. Schematic diagrams and photos of linear-array-based photoacoustic computed tomography (PACT) systems. (a) Phantom and in vitro PACT system. (b) In vivo animal PACT system.

Synthesis and characterization of MnO2 nanotubes The MnO2 nanotubes were synthesized by reducing potassium permanganate (KMnO4) by poly (diallyldimethylammonium chloride) (PDDA) with one-pot reaction, following our previously reported method

20.

The MnO2 nanotubes showed tubular

structures in water (Figure 2a) with hydrodynamic diameter of 66.4 nm and polydispersity of 0.23. The MnO2 nanotubes carried positive surface charge with zeta potential of 12.59±1.84mV.


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Switchable PAI of GSH in phantoms We first characterized the PA signal strength of MnO2 nanotubes at various concentrations (0.05, 0.1, 0.25, 0.5, 1.0, 1.5, and 2.0 mg/mL). A serial dilution of MnO2 nanotube suspension was prepared with deionized (DI) water. These suspensions were then loaded into phantom tubes and sequentially imaged using the PAI system shown in Figure 1a. For each sample, 100 frames were acquired for averaging to eliminate the effect of laser intensity fluctuation. Figure 2b showed averaged PA images for each sample. As expected, PA amplitude increased as the concentrations of MnO2 nanotubes increased. For quantitative analysis, we extracted the average PA amplitude within the white box (Figure 2b), and then normalized the values in each tube with that of the 2.0 mg/mL suspension. The results, shown in Figure 2c, clearly indicated a linear relationship between the PA amplitude and the concentration of MnO2 nanotubes. These results demonstrated that the relative concentration of MnO2 nanotubes can be straightforwardly predicted from the PA amplitude.

Figure 2. Morphology and PA properties of MnO2 nanotubes. (a) TEM image of MnO2 nanotubes. (b) PA images of phantom tubes filled with MnO2 nanotubes at different concentrations from 0.05 to 2.0 mg/mL. (c) Linear relationship between MnO2 nanotube concentrations and normalized PA amplitudes for phantom tubes in (b). (Mean ± standard deviations for n=3.)



Next, we investigated the effect of GSH on PA signals from MnO2 nanotubes. GSH was added to 2 mg/mL MnO2 nanotubes. The GSH concentrations in the final mixtures were 0, 3.90, 7.62, 11.16, 14.55 and 17.78 mmol/L, respectively. To ensure that the reaction between GSH and MnO2 nanotubes was complete, each mixture was stirred and then kept at room temperature for 10 min before imaging. The MnO2 nanotubes/GSH mixtures were loaded into the phantom tubes, and 100 frames of images were acquired for each sample. Averaged PA images were shown in Figure 3a. MnO2 nanotubes without GSH treatment exhibited the highest PA amplitude. The amplitude gradually decreased with increased GSH concentrations. When the concentration of GSH was higher than 14.54 mmol/L, PA signals of MnO2 nanotubes could not be differentiated from the water background. Similar to Figure 2, within the white box, we quantified the average PA amplitude of each mixture (Figure 3a), and then normalized these PA amplitudes to the control (i.e. 0 mmol/L GSH treatment). A nearly linear relationship was observed between the normalized PA amplitude (PAafter GSH exposure/PAbefore GSH exposure)

and the GSH concentration (0 ~ 15 mmol/L), suggesting

that MnO2 nanotube-based, switchable PAI may be used to quantify GSH levels based on the signal reduction before and after interaction with GSH (Figure 3b).

Figure 3. Evaluation of MnO2 nanotubes as a switchable, “signal off” PA probe for GSH imaging in phantoms. (a) PA images of phantom tubes filled with a mixture of MnO2 nanotubes and GSH at GSH concentrations of 0, 3.9, 7.62, 11.16,


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14.54 and 17.78 mmol/L, respectively. (b) Relationship between GSH concentrations and normalized PA amplitudes for phantoms in (a). (Mean ± standard deviations for n=3.)

Switchable PAI of GSH in B16F10 melanoma cells and BEAS-2B normal cells We investigated the use of MnO2 nanotubes as a switchable, “signal off” PAI contrast reagent for intracellular GSH detection. B16F10 melanoma cells and BEAS2B normal epithelial cells were incubated with 0.5 mg/mL MnO2 nanotubes for 6 h. Then, one portion of the cells was collected (i.e. cells harvested at 0 h post incubation), and the other portion of cells was transferred to fresh cell culture medium containing no MnO2 nanotubes and harvested at 18 h post incubation. Untreated B16F10 cells and BEAS-2B cells were used as the controls. All cells were imaged with our PACT system designed for phantom tube imaging (Figure 1a). As shown in Figure 4 and Figure S1, untreated cells showed the lowest PA amplitudes due to the absence of MnO2 nanotubes inside the cells. The cells collected right after the incubation with MnO2 nanotubes showed the highest PA amplitudes. At 18 h post incubation, the PA signals of B16F10 cells dropped significantly, only ~12% higher than the control, while the PA signals of BEAS-2B cells remained unchanged, ~2.2-fold higher than the control. The significant decrease of PA amplitudes in melanoma cells but not in normal cells indicated that MnO2 nanotubes were effectively reduced by GSH in tumor cells, and MnO2 nanotubes could be used to distinguish melanoma cells from normal cells. It should be noted that for these experiments, we pushed cells to the center of the tubes, thus the photoacoustic amplitudes were not uniform along the tubes. However, since our data analysis focused on the central region, the uniformity did not affect the final results.



Figure 4. Evaluation of MnO2 nanotubes as a switchable, “signal off” PA probe for GSH imaging in cells. B16F10 melanoma cells and BEASs-2B normal epithelial cells were incubated with 0.5 mg/mL MnO2 nanotubes for 6 h. (a) PA images of B16F10 cells were acquired at 0 h and 18 h post incubation. Normalized PA amplitudes for B16F10 cells (b) and BEAS-2B cells (c). The PA amplitude of each sample was quantified by averaging PA signals within the white box and was then normalized to the control. PA signals increased at 0 h post incubation for both B16F10 cells and BEAS-2B cells, indicating the uptake of MnO2 nanotubes by both cells. At 18 h post incubation, decreased PA signals were only observed in B16F10 cancer cells but not in BEAS-2B normal cells, demonstrating that MnO2 nanotubes can be used as a switchable PA probe for intracellular GSH imaging to detect melanoma. (Mean ± standard deviations for n=3. *: p90% viability when the concentrations of MnO2 nanotubes were below or equal to 50 g/mL. When the concentrations of MnO2 nanotubes were further increased to much higher levels, such as 750 g/mL, we observed that ~65% cells were still viable. There was no significant difference in cell viability between B16F10 melanoma cells and BEAS-2B normal cells.

Figure 6. Cell viability of B16F10 cells and BEAS-2B cells after incubation with MnO2 nanotubes at various concentrations for 24 h. No significant toxicity was observed when the concentration of MnO2 nanotubes was below or equal to 50 g/mL. (Mean ± standard deviations for n=3.)


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Switchable PAI of GSH in melanoma tumors Finally, we demonstrated the feasibility of using MnO2 nanotubes as a switchable, “signal off” PA probe for in vivo melanoma detection. CD-1 mice bearing B16F10 melanoma tumors (~100 mm3) were imaged using our PACT system (Figure 1b) before (negative control) and 0, 24 and 48 h after the intra-tumor injection of 100 L 2 mg/mL MnO2 nanotubes (Figure 7). Ultrasound imaging was also used to assist in identifying tumor regions (Figure S3). PA signals within tumor regions were averaged and then normalized with that of negative control. The PA amplitudes of control tumors with no injection were the lowest. Immediately after the injection of MnO2 nanotubes, the PA amplitudes of tumors increased by 2.2-fold comparing with that of controls. Then, the PA amplitudes decreased gradually over time due to the continuous reduction of MnO2 nanotubes by GSH. At 24 h post injection, the PA amplitudes were reduced by ~20% and were 1.8-fold higher than that of control. At 48 h post injection, the PA amplitudes were only slightly higher than negative controls, indicating that most of the injected MnO2 nanotubes were reduced to Mn2+ ions. To verify that the PA signal decrease was indeed caused by the reduction of MnO2 nanotubes from reaction with GSH, instead of the clearance or degradation of MnO2 nanotubes, the same amount of MnO2 nanotubes (100 L 2 mg/mL) were injected subcutaneously to CD-1 mice with no tumors. We measured the PA signals in healthy tissues before (negative control) and at 0, 24 and 48 h post injection. As shown in Figure 7b, the PA amplitude reduced by ~8% at 24 h post injection and was ~2.12-fold higher than that of negative control. At 48 h post injection, the PA amplitude reduced by ~25%, which was still ~1.73-fold higher than that of negative control. The much higher PA amplitudes observed in healthy tissues than tumors demonstrated that MnO2



nanotubes were a sensitive PA probe to GSH levels and could be used to distinguish melanoma tumors from healthy tissues. In order to demonstrate that the PA signal reduction observed in tumors was not caused by the dilution effect due to tumor growth, we also imaged the tumors injected with 100 L 2 mg/mL multiwall carbon nanotubes (MWCNT). These MWCNT had outer diameter of 8-15nm and length of 0.5-2um, which showed some similarity in morphology as MnO2 nanotubes but would not degrade inside the tumors. As expected, the PA signals increased immediately after the MWCNT injection, however, the PA signals only decreased slightly (~11%) at 48 h post injection, which may be due to the slight tumor growth. Together, these results demonstrated that MnO2 nanotubes are an effective, switchable PA probe for in vivo GSH imaging in melanoma diagnosis.


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Figure 7. Evaluation of MnO2 nanotubes as a switchable PA probe for GSH imaging in vivo. (a) CD-1 mice bearing B16F10 tumors were administrated with MnO2 nanotubes and MWCNT via intratumor injection (100 L, 2 mg/mL). CD-1 mice with no tumors were injected subcutaneously with 100 L 2 mg/mL MnO2 nanotubes. PA images were acquired before (control) and at 0, 24, and 48 h after injection. The dashed ovals indicate the approximate tumor region or injection region. (b) Normalized PA amplitudes for tumors and healthy tissues. PA amplitudes were quantified by averaging PA signals in the region of interest, and then the averaged values were normalized to the control. A significant decrease in PA signals was observed in tumors injected with MnO2 nanotubes but not in healthy tissues injected with MnO2 nanotubes or in tumors injected with MWCNT, demonstrating that MnO2 nanotubes were a sensitive switchable PA probe for in vivo GSH imaging in melanoma diagnosis. (Mean ± standard deviations for n=3.*: p90% viability when they were incubated with up to 50 g/mL MnO2 nanotubes for 24 h (Figure 6). These results agreed with our previous observation in MCF7 human breast cancer cells

20.

Zhu et al. also showed that MnO2 nanoparticles did not induce toxic

effects in 4T1 murine breast cancer cells 30. In vivo, manganese is an essential element for human health and abundantly exists in human body 32. Besides, the generated Mn2+ ions can be rapidly cleared by kidneys. Therefore, MnO2 nanotubes should have low toxicity 29-30, especially when they are applied locally for melanoma diagnosis. (2) In our PACT setup, we employed a single-wavelength approach with temporal data analysis, which avoids the hurdle of wavelength switching or multi-laser imaging used in other studies 12-13, 15-16, 18. In addition, 750 nm laser diodes are commercially available and may enable portable PAI. (3) For skin cancer, similar as we did in our animal study, the MnO2 nanotubes can be administrated topically via direct injection into unusual moles or suspicious spots. By measuring the PA signal reduction we may detect the levels of GSH and assist in cancer screening and diagnosis. Compared with systemic administration, the local administration may realize much lower effective dose, and thus reduce the potential risk of toxicity and may facilitate clinical translation 33. (4) Since melanin absorbs light and generates strong PA signals, with a “signal on” probe, it may be difficult to tell whether the signal increase is caused by the activated probes or due to melanoma progression. On the contrary, MnO2 nanotube-based “signal off” probe has reduced PA signals in tumor cells, thus it better distinguishes GSH-induced signals from melanin-induced signals, minimizes the “noise” due to tumor growth, and does not cause unnecessary anxieties to the patient with suspicious melanoma. Moving forward, our method may address the unmet clinical need for quantitative characterization of GSH dynamics in melanoma

34.

Compared to existing medical


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imaging modalities, PAI is also advantageous because of its non-ionizing nature (compared to positron emission tomography 5), deeper imaging depth (compared to fluorescence imaging 4), and cheaper and portable imaging platform (compared to MRI 7).

CONCLUSIONS In summary, we have demonstrated that MnO2 nanotubes are a promising switchable and biodegradable PA probe for dynamic imaging of GSH for cancer diagnosis. Using melanoma as the disease model, we have shown that MnO2 nanotubes based PAI successfully distinguished B16F10 cells from BEAS-2B cells and differentiated B16F10 tumors from healthy skin tissues in mice. For future clinical studies, we could administrate the MnO2 nanotubes topically, which may significantly facilitate the regulatory approval procedure. In the future, we will further enhance the sensing capability of MnO2 nanotubes by optimizing their physiochemical properties, such as width, length, and surface charges. We will investigate how the physiochemical properties of MnO2 nanotubes affect the cellular uptake efficiency, the response time to GSH, and subsequently, the detection sensitivity and specificity. As for the PACT setup, we need further development to mitigate the system size. Currently, the main bottleneck is the illumination source, which is flash-lamp based and bulky. Recent studies have pointed a promising direction using compact laser diode arrays

35

and LED arrays

36

as the illumination source.

Combined with portable ultrasound data acquisition systems, we can potentially achieve a laptop-sized setup to be used in dermatology clinics. Multi-wavelength imaging may also be implemented in the future to quantify blood oxygenation or to be combined with other probes for ratiometric or molecular imaging. Since our previous



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study has also demonstrated the feasibility of MnO2 nanotubes-based MRI and fluorescence imaging of microRNAs

20,

we can further combine various imaging

modalities and explore the possibility and benefits of MnO2 nanotube-based multimodal, multiparametric imaging of melanoma and other types of cancers. As a noninvasive and quantitative GSH characterization tool, our study will open new avenues in the field of cancer imaging and diagnosis.

EXPERIMENTAL SECTION MnO2 nanotubes synthesis MnO2 nanotubes were synthesized based on our previously reported protocol Briefly,

potassium

permanganate

(KMnO4)

was

reduced

by

20.

poly

(diallyldimethylammonium chloride) (PDDA) with one-pot reaction. In 25 mL of aqueous solution, PDDA and KMnO4 were mixed at the mass ratio of 1: 1. The mixture was heated in a 120 °C oil bath and continuously stirred for 2 h. When the color of the solution became dark brown, Mn7+ of KMnO4 was reduced to Mn4+. Then, the asprepared MnO2 nanotubes suspension was centrifuged, washed with ultrapure water and lyophilized. After that, the MnO2 nanotubes were formulated as an aqueous dispersion at a concentration of 2 mg/mL and stored at 4 °C.

MnO2 nanotubes characterization MnO2 nanotubes were dispersed in deionized water. The size, size distribution and zeta potential measurements were acquired using a NanoBrook Omni Particle Size and Zeta Potential Analyzer (Brookhaven Instruments). The morphology of MnO2 nanotubes was observed by transmission electron microscopy (TEM).


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PACT system for tube phantom imaging The silicon tube has 1 mm inner diameter and 1.6 mm outer diameter. During experiments, the tube was immersed in a water tank with a window on the left side. The window was sealed with an acoustically transparent plastic membrane, allowing good PA signal penetration. Photoacoustic signals were detected through this window, using a 128-element linear array transducer (ATL/Philips L7-4, 5 MHz central frequency). Photoacoustic excitation (750 nm) was provided by an Nd:YAG pumped optical parametric oscillator (OPO) laser (SureliteTM OPO Plus, Continuum) with 10 Hz pulse repetition rate and 10 ns pulse duration. The light was delivered to the tube through an optical fiber bundle with 1.4 cm diameter and 60% coupling efficiency. The maximum light intensity at the tube surface is around 15 mg/cm2, which is lower than the American National Standards Institute (ANSI) safety limit (25 mJ/cm2) at 750 nm. The received PA signals were amplified (by 54 dB) and digitized by a 128-channel ultrasound data acquisition (DAQ) system (Vantage, Verasonics) with 20 MHz sampling rate. After each laser pulse, the raw channel data was reconstructed using the universal back-projection algorithm

37,

and was displayed in real-time during

experiments.

PACT system for animal imaging All animal experiments were performed in compliance with the animal protocol approved by Institutional Animal Care and Use Committee at University at Buffalo. In this experiment, a water tank with a window at the bottom was used. Again, the window was sealed with the thin plastic membrane. During experiments, mice were placed underneath the window and coupled to the membrane with ultrasound gel. To image the whole tumor, we scanned the L7-4 array over 4 cm with a step size of 0.1 mm. The



excitation light (750 nm) was routed to the imaging region through a bifurcated optical fiber bundle with one circular input and two linear outputs. The light intensity on the skin surface of mice is around 12 mJ/cm2, which is also below the ANSI safety limit. After each laser pulse, one frame was acquired with the 128-channel Verasonics system.

PAI of MnO2 nanotubes MnO2 nanotubes was suspended in DI water at concentrations of 0.05, 0.1, 0.25, 0.5, 1.0, 1.5 and 2.0 mg/mL. 100 uL of MnO2 nanotubes were loaded in phantom tubes and imaged with PACT system for tube phantom imaging. One hundred frames were acquired for each sample. To investigate the stability of MnO2 nanotubes in serial PA imaging, PA signals from 2.0 mg/mL MnO2 nanotubes were measured after the nanotubes were exposed to 100, 2000, 4000, 8000 and 16000 laser pulses. To investigate the serum stability, MnO2 nanotubes was suspended in 50% FBS at concentrations of 2.0 mg/mL and incubated at room temperature for 0 min, 15 min, 1 h, 4 h and 24h. Then 100 uL of MnO2 nanotubes/FBS mixture was loaded in phantom tubes and imaged with PACT system for tube phantom imaging. One hundred frames were acquired for each sample.

Effect of GSH on PA signals of MnO2 nanotubes GSH was added to 100 uL 2mg/mL MnO2 nanotube suspensions to reach final concentrations of 0, 3.90, 7.62, 11.16, 14.54 and 17.78 mmol/L. The mixture was stirred and incubated at room temperature for 10 min to allow complete reaction between GSH and MnO2 nanotubes. Then, the samples were loaded in phantom tubes and imaged with our PACT setup. One hundred frames were acquired for each sample.


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Cell culture B16F10 mouse melanoma cells and BEAS-2B normal human epithelial cells were obtained from the American Type Culture Collection (Manassas, VA). The cells were cultured in RPMI 1640 media (Life Technologies; Grand Island, NY; 11875-093) supplemented with 10% fetal bovine serum (FBS; Life Technologies; 26140-079) and 1× penicillin streptomycin (PS; Life Technologies; 15140-122). The cells were seeded into the petri dishes (Greiner Bio-one Monroe NC) at the concentration of 2×105 cells/mL and incubated in a CO2 incubator at 37 ºC. The cells were subcultured every 2 days.

Cell viability B16F10 cells and BEAS-2B cells were seeded at 105 cells/well in 12-well plates and incubated in a CO2 incubator at 37 ºC overnight. The cells were then incubated with MnO2 nanotubes at concentrations of 0 (negative control), 5, 10, 20, 50, 100, 200, 500 and 750 g/mL. At 24 h post incubation, alamarBlue assay (Thermo Fisher Scientific, DAL1025) was used to measure cell viability following manufacturer’s protocol. Briefly, one part of alamarBlue reagent was added to 10 parts of cell culture medium. The mixture was added to cells and incubated at dark for 3 h. Then the fluorescence intensity was measured by a microplate reader (Tecan, San Jose, CA) with excitation wavelength of 560 nm and emission wavelength of 590 nm. The cell viability was normalized to negative controls.

Evaluate MnO2 nanotubes as a switchable PA probe for GSH imaging in cells. B16F10 cells and BEAS-2B cells were seeded into 6-well plates at the concentration of 2×105 cells/well and incubated in a CO2 incubator at 37 ºC overnight.



Then the cells were treated with 0.5 mg/mL MnO2 nanotubes suspended in RPMI 1640 basal medium without FBS and PS for total 6 h. Then the MnO2 nanotubes were removed by transfering the cells into fresh, regular culture medium. The cells were harvested at 0 h and 18 h post treatment by trypsinization and fixed in 4% paraformaldehyde. The cell number was counted and the same number of cells were loaded in the phantom tubes for PAI. One hundred frames were acquired for each sample.

Evaluate MnO2 nanotubes as a switchable PA probe for GSH imaging in vivo. Total 4 million B16F10 cells were subcutaneously injected into the lower flank of the CD-1 mice. When the tumor volume reached ~150 mm3, 100 L 2 mg/mL MnO2 nanotubes were injected intratumorally. Same amount (100 L, 2 mg/mL) of multiwall carbon nanotubes (Cheap Tubes Inc., COOH functionalized, outer diameter was 8-15 nm; length was 0.5-2.0 m) were injected to the tumors and used as the control. Meanwhile, 100 L 2 mg/mL MnO2 nanotubes were injected subcutaneously in CD-1 mice with no tumor as the second control. The PA signals from the region of interest (i.e. tumors or the injection regions of healthy mice) were collected before (negative control) and at 0, 24 and 48 h post injection. At each time point, the region of interest was imaged with the PACT system for animal imaging. Unlike the 2D tube imaging study, animal imaging involved 3D scanning. We acquired 400 frames over a 4 by 4 cm2 region and then the 3D data was projected over depth to form a maximum intensity projection (MIP) image. All images were then normalized according to the maximum amplitude in the negative control. For better illustration of changes, signal amplitudes below one were plotted in gray scale, while the others were plotted in color scale.


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ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. PA images of BEAS-2B cells after incubation with MnO2 nanotubes. Stability of MnO2 nanotubes in serum. Tumor imaging using both PAI and ultrasound imaging.

AUTHOR INFORMATION *Corresponding

authors

E-mail: [email protected] (Y. H. Lin). E-mail: [email protected] (J. Xia). E-mail: [email protected] (Y. Wu).

Author contributions Y.W. and J. X. conceived the study; Q. L., D. D. and Y. L. prepared the MnO2 nanotubes; C. L., D. W., Y. Z., L. Y., Q. L., J. L., and M. Y. Z. conducted the experiments and collected experimental data; C. L., D. W., Y. Z., L. Y., J. X. and Y. W. analyzed and interpreted the data; and Y.W., J. X., D. W. and C. L. wrote the paper with contributions from all other authors who commented on drafts and approved the final version of the manuscript.

Notes The authors declare no competing financial interest.

ACKNOELEDGEMENTS The authors thank the support from the National Center for Advancing Translational



Sciences of the National Institutes of Health under award number UL1TR001412 to University at Buffalo. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

REFERENCES 1. Balendiran, G. K.; Dabur, R.; Fraser, D., The role of glutathione in cancer. Cell Biochemistry and Function 2004, 22 (6), 343-352. 2. Estrela, J. M.; Ortega, A.; Obrador, E., Glutathione in cancer biology and therapy. Critical Reviews in Clinical Laboratory Sciences 2006, 43 (2), 143-181. 3. Traverso, N.; Ricciarelli, R.; Nitti, M.; Marengo, B.; Furfaro, A. L.; Pronzato, M. A.; Marinari, U. M.; Domenicotti, C., Role of glutathione in cancer progression and chemoresistance. Oxidative Medicine and Cellular Longevity 2013, 2013: 972913 4. Jiang, X.; Chen, J.; Bajić, A.; Zhang, C.; Song, X.; Carroll, S. L.; Cai, Z.-L.; Tang, M.; Xue, M.; Cheng, N., Quantitative real-time imaging of glutathione. Nature Communications 2017, 8, 16087. 5. Leide-Svegborn, S., Radiation exposure of patients and personnel from a PET/CT procedure with 18F-FDG. Radiation Protection Dosimetry 2010, 139 (1-3), 208-213. 6. Huang, Y.-C.; Huang, H.-L.; Yeh, C.-N.; Lin, K.-J.; Yu, C.-S., Investigation of brain tumors using 18F-fluorobutyl ethacrynic amide and its metabolite with positron emission tomography. OncoTargets and Therapy 2015, 8, 1877. 7. Zhao, Z.; Fan, H.; Zhou, G.; Bai, H.; Liang, H.; Wang, R.; Zhang, X.; Tan, W., Activatable Fluorescence/MRI Bimodal Platform for Tumor Cell Imaging via MnO2 Nanosheet–Aptamer Nanoprobe. Journal of the American Chemical Society 2014, 136 (32), 11220-11223. 8. Zhou, Y.; Wang, D.; Zhang, Y.; Chitgupi, U.; Geng, J.; Wang, Y.; Zhang, Y.; Cook, T. R.; Xia, J.; Lovell, J. F., A Phosphorus Phthalocyanine Formulation with Intense Absorbance at 1000 nm for Deep Optical Imaging. Theranostics 2016, 6 (5), 688. 9. Wang, L. V.; Yao, J., A practical guide to photoacoustic tomography in the life sciences. Nature Methods 2016, 13 (8), 627-638. 10. Wang, D.; Wang, Y.; Wang, W.; Luo, D.; Chitgupi, U.; Geng, J.; Zhou, Y.; Wang, L.; Lovell, J. F.; Xia, J., Deep tissue photoacoustic computed tomography with a fast and compact laser system. Biomedical Optical Express 2017, 8 (1), 112-123. 11. Valluru, K. S.; Wilson, K. E.; Willmann, J. K., Photoacoustic Imaging in oncology: translational preclinical and early clinical experience. Radiology 2016, 280 (2), 332-349. 12. Levi, J.; Kothapalli, S.-R.; Bohndiek, S.; Yoon, J.-K.; Dragulescu-Andrasi, A.; Nielsen, C.; Tisma, A.; Bodapati, S.; Gowrishankar, G.; Yan, X., Molecular photoacoustic imaging of follicular thyroid carcinoma. Clinical Cancer Research 2013, 19 (6), 1494-1502.


Page 28 of 30

Page 29 of 30 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


13. Levi, J.; Kothapalli, S. R.; Ma, T.-J.; Hartman, K.; Khuri-Yakub, B. T.; Gambhir, S. S., Design, synthesis, and imaging of an activatable photoacoustic probe. Journal of the American Chemical Society 2010, 132 (32), 11264-11269. 14. Dragulescu-Andrasi, A.; Kothapalli, S.-R.; Tikhomirov, G. A.; Rao, J.; Gambhir, S. S., Activatable oligomerizable imaging agents for photoacoustic imaging of furinlike activity in living subjects. Journal of the American Chemical Society 2013, 135 (30), 11015-11022. 15. Pu, K.; Shuhendler, A. J.; Jokerst, J. V.; Mei, J.; Gambhir, S. S.; Bao, Z.; Rao, J., Semiconducting polymer nanoparticles as photoacoustic molecular imaging probes in living mice. Nature Nanotechnology 2014, 9 (3), 233-239. 16. Zhang, J.; Zhen, X.; Upputuri, P. K.; Pramanik, M.; Chen, P.; Pu, K., Activatable Photoacoustic Nanoprobes for In Vivo Ratiometric Imaging of Peroxynitrite. Advanced Materials 2017, 29 (6), 1604764. 17. Yin, C.; Zhen, X.; Fan, Q.; Huang, W.; Pu, K., Degradable semiconducting oligomer amphiphile for ratiometric photoacoustic imaging of hypochlorite. ACS Nano 2017, 11 (4), 4174-4182. 18. Yin, C.; Tang, Y.; Li, X.; Yang, Z.; Li, J.; Li, X.; Huang, W.; Fan, Q., A Single Composition Architecture‐Based Nanoprobe for Ratiometric Photoacoustic Imaging of Glutathione (GSH) in Living Mice. Small 2018, 14 (11), 1703400. 19. Song, M.; Liu, T.; Shi, C.; Zhang, X.; Chen, X., Bioconjugated manganese dioxide nanoparticles enhance chemotherapy response by priming tumor-associated macrophages toward M1-like phenotype and attenuating tumor hypoxia. ACS Nano 2015, 10 (1), 633-647. 20. Lu, Q.; Ericson, D.; Song, Y.; Zhu, C.; Ye, R.; Liu, S.; Spernyak, J. A.; Du, D.; Li, H.; Wu, Y., MnO2 Nanotubes-based NanoSearchlight for Imaging of Multiple MicroRNAs in Live Cells. ACS Applied Materials & Interfaces 2017, 9 (28), 2332523332. 21. Fan, H.; Zhao, Z.; Yan, G.; Zhang, X.; Yang, C.; Meng, H.; Chen, Z.; Liu, H.; Tan, W., A smart DNAzyme–MnO2 nanosystem for efficient gene silencing. Angewandte Chemie 2015, 127 (16), 4883-4887. 22. Meng, H.-M.; Jin, Z.; Lv, Y.; Yang, C.; Zhang, X.-B.; Tan, W.; Yu, R.-Q., Activatable two-photon fluorescence nanoprobe for bioimaging of glutathione in living cells and tissues. Analytical Chemistry 2014, 86 (24), 12321-12326. 23. Merlino, G.; Herlyn, M.; Fisher, D. E.; Bastian, B. C.; Flaherty, K. T.; Davies, M. A.; Wargo, J. A.; Curiel‐Lewandrowski, C.; Weber, M. J.; Leachman, S. A., The state of melanoma: challenges and opportunities. Pigment Cell & Melanoma Research 2016, 29 (4), 404-416. 24. Zhang, H. F.; Maslov, K.; Stoica, G.; Wang, L. V., Functional photoacoustic microscopy for high-resolution and noninvasive in vivo imaging. Nature Biotechnology 2006, 24 (7), 848-851. 25. Oh, J.-T.; Li, M.-L.; Zhang, H. F.; Maslov, K.; Stoica, G.; Wang, L. V., Threedimensional imaging of skin melanoma in vivo by dual-wavelength photoacoustic microscopy. Journal of Biomedical Optics 2006, 11 (3), 034032-034032-4.



26. Zhou, Y.; Xing, W.; Maslov, K. I.; Cornelius, L. A.; Wang, L. V., Handheld photoacoustic microscopy to detect melanoma depth in vivo. Optical Letters 2014, 39 (16), 4731-4734. 27. Wang, Y.; Xu, D.; Yang, S.; Xing, D., Toward in vivo biopsy of melanoma based on photoacoustic and ultrasound dual imaging with an integrated detector. Biomedical Optical Express 2016, 7 (2), 279-286. 28. Langhout, G. C.; Grootendorst, D. J.; Nieweg, O. E.; Wouters, M. W. J. M.; Hage, J. A. v. d.; Jose, J.; Boven, H. v.; Steenbergen, W.; Manohar, S.; Ruers, T. J. M., Detection of melanoma metastases in resected human lymph nodes by noninvasive multispectral photoacoustic imaging. Journal of Biomedical Imaging 2014, 2014, 5. 29. Chen, Q.; Feng, L.; Liu, J.; Zhu, W.; Dong, Z.; Wu, Y.; Liu, Z., Intelligent Albumin–MnO2 Nanoparticles as pH‐/H2O2‐Responsive Dissociable Nanocarriers to Modulate Tumor Hypoxia for Effective Combination Therapy. Advanced Materials 2016, 28 (33), 7129-7136. 30. Zhu, W.; Dong, Z.; Fu, T.; Liu, J.; Chen, Q.; Li, Y.; Zhu, R.; Xu, L.; Liu, Z., Modulation of hypoxia in solid tumor microenvironment with MnO2 nanoparticles to enhance photodynamic therapy. Advanced Functional Materials 2016, 26 (30), 54905498. 31. Wang, Y.; Lim, R. S. A.; Zhang, H.; Nyayapathi, N.; Oh, K. W.; Xia, J., Optimizing the light delivery of linear-array-based photoacoustic systems by double acoustic reflectors. Scientific Reports 2018, 8 (1), 13004. 32. O’Neal, S. L.; Zheng, W., Manganese toxicity upon overexposure: a decade in review. Current Environmental Health Reports 2015, 2 (3), 315-328. 33. Anselmo, A. C.; Mitragotri, S., A review of clinical translation of inorganic nanoparticles. The AAPS Journal 2015, 17 (5), 1041-1054. 34. Meyskens Jr., F. L.; Farmer, P.; Fruehauf, J. P., Redox Regulation in Human Melanocytes and Melanoma. Pigment Cell Research 2001, 14 (3), 148-154. 35. Daoudi, K.; van den Berg, P. J.; Rabot, O.; Kohl, A.; Tisserand, S.; Brands, P.; Steenbergen, W., Handheld probe integrating laser diode and ultrasound transducer array for ultrasound/photoacoustic dual modality imaging. Optical Express 2014, 22 (21), 26365-26374. 36. Zhu, Y.; Xu, G.; Yuan, J.; Jo, J.; Gandikota, G.; Demirci, H.; Agano, T.; Sato, N.; Shigeta, Y.; Wang, X., Light emitting diodes based photoacoustic imaging and potential clinical applications. Scientific Reports 2018, 8 (1), 9885. 37. Xu, M.; Wang, L. V., Universal back-projection algorithm for photoacoustic computed tomography. Physical Review E 2005, 71 (1), 016706.


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