Quantitative Monitoring of Hypoxia-Induced Intracellular Acidification

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Quantitative Monitoring of Hypoxia-Induced Intracellular Acidi#cation in Lung Tumor Cells and Tissues Using Activatable Surface-Enhanced Raman Scattering Nanoprobes Dandan Ma, Jing Zheng, Pinting Tang, Weijian Xu, Zhihe Qing, Sheng Yang, Jishan Li, and Ronghua Yang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b03590 • Publication Date (Web): 01 Nov 2016 Downloaded from http://pubs.acs.org on November 3, 2016

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Analytical Chemistry

Quantitative Monitoring of Hypoxia-Induced Intracellular Acidification in Lung Tumor Cells and Tissues Using Activatable Surface-Enhanced Raman Scattering Nanoprobes Dandan Ma,a Jing Zheng,a,* Pinting Tang,a Weijian Xu,a Zhihe Qing,b Sheng Yang,b Jishan Li,a Ronghua Yang,a,b,* a

State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha, 410082, China; bSchool of Chemistry and Biological Engineering, Changsha University of Science and Technology, Changsha, Hunan, 410004 China.

*To whom correspondence should be addressed:

E-mail: [email protected], [email protected]. Fax: +86-731-8882 2523

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ACS Paragon Plus Environment


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ABSTRACT: Hypoxia is considered to contribute to pathophysiology in various cells and tissues, a clear understanding about the relationship between hypoxia and intracellular acidification will help to elucidate the complex mechanism of glycolysis under hypoxia. However, current researches are mainly focused on overexpressing of intracellular reductases accelerated by hypoxia, and the investigations focusing on the relationship between hypoxic degree and intracellular acidification remained to be explored. For this vacuity, we report herein a new activatable nanoprobe for sensing pH change under different degrees of hypoxia by surface enhanced Raman spectroscopy (SERS). The monitoring was based on the SERS spectra changes of 4-nitrothiophenol (4-NTP)-functionalized gold nanorods (AuNR@4-NTP) resulting from the nitroreductase (NTR)-triggered reduction under hypoxic conditions while the as-generated 4-aminothiophenol (4-ATP) is a pH-sensitive molecule. This unique property can ensure the SERS monitoring of intracellular acidification in living cells and tissues under hypoxic conditions. Dynamic pH analysis indicated the pH decreased from 7.1 to 6.5 as functions of different degree of hypoxia (from 15% to 1%) due to the excessive glycolytic activity triggered by hypoxia. Given the known advantages of SERS sensing, these findings hold promise in studies of pathophysiological pathways involving hypoxia.

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INTRODUCTION Hypoxia, a condition of decreased oxygen availability, is considered to contribute to pathophysiology in various cells and tissues.1 Specifically, hypoxia can cause pulmonary oxidative damage and imbalance of redox state in lung whose important function is to maintain an adequate oxygenation in the oragnism.2 Most importantly, the excessive glycolytic activity in lung triggered by hypoxia can result in vast production of lactate and thus accompany by the intracellular acidification, which would in turn result in inhibition of glycolysis.3 Therefore, demonstrating the hypoxia-induced intracellular acidification in living systems is of great biomedical importance. However, current researches have been mainly focused on overexpressing of intracellular reductases such as DT-diaphorase, azoreductase, and nitroreductase (NTR) accelerated by hypoxia,4-9 few effort has been explored for quantitative intracellular acidification monitoring in living cells under the different hypoxic degree. Since a clear understanding about the relationship between the hypoxia and intracellular acidification will contribute to elucidate the complex mechanism of glycolysis under hypoxic conditions, efficient pH indicators which can function well in living cells and tissues under hypoxia is thus a challenging task. To date, several excellent techniques including fluorescence and nuclear magnetic resonance (NMR), have been developed for the detection of pH under hypoxia.10-13 For instance, Jongsma et al. proposed the

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P magnetic resonance spectroscopy to monitor

cerebral energy metabolism and acid-base homeostasis during impaired oxygen supply in fetal sheep while Hori et al. prepared a fluorescent probe by linking p-nitro benzyl moiety to SNARF for monitoring intracellular pH of tumor cells under hypoxia.14,15 In spite of the promising application capability, insufficient water solubility make these organic small molecules-based strategies still difficult to implement for the intracellular sensing of pH in a

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reliable and convenient way under hypoxia.16 Recently, luminescent nanoparticles have been tremendously developed for intracellular pH detection. For instance, Ma et al. reported a ratio pH sensor based on carbon nanodots for the detection of the intracellular pH, whose noticeable feature is the facile adjustment of pH response range, and He et al. currently reported a kind of fluorescent silicon nanoparticles (SiNPs)-based pH sensor which has a wide-pH response, and strong fluorescence coupled with good photostability.17,18 Nevertheless, when directly applied in biological environments, it may suffer from interference of endogenous biological molecules-triggered background signal. Based on the (i) ultrahigh detection sensitivity up to single molecular level, (ii) narrow and sharp emission peaks for multiplex detection, (iii) interference-free in diverse environment, such as oxygen, humidity, and foreign species,19,20 surface-enhanced Raman spectroscopy (SERS) become more attractive to apply in the detection of biological species in living cells and tissues.21-22 Among these applications, SERS is especially extensively used in sensing inorganic species in living cells such as carbon monoxide, hydrogen sulfide, nitric oxide integrated with metal nanoparticles.23-26 However, to the best of our knowledge, quantitative monitoring pH under different degree of hypoxia in living cell with SERS has not been previously reported. In this paper, we describe a new strategy based on the SERS spectra changes of nitroaromatic substrate resulting from the irreversible reaction of nitroreductase (NTR) to monitor pH change under different degree of hypoxia. According to the previous report,27 the expression level of NTR directly corresponds with the hypoxic status, while the nitroaromatic compounds have been proved to be superior substrates for NTR with reduced nicotinamide adenine dinucleotide (NADH) serving as an electron donor under hypoxic conditions.28 On the basis of such a NTR-catalyzed reduction reaction, herein, we chose 4-nitrothiophenol (4-NTP) as the nitroaromatic substrate. Meanwhile, considering the high SERS enhancement capacity of gold nanorods (AuNRs), we functionalized 4-NTP onto the surface of AuNRs to fabricate an

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activatable SERS nanoprobe (AuNR@4-NTP). Under hypoxic conditions, the nitro groups in 4-NTP could ultimately be reduced to an amino group and termed as 4-aminothiophenol (4-ATP). It is noteworthy that the as-generated 4-ATP exhibit NH3+ group in acidic solutions and can response well as functions of different pH.29 Thus, the pH dependence of 4-ATP reduced by NTR under hypoxic conditions allows tracking the pH changes in living cells and tissues. On the basis of this, the nanoprobe in our design can monitor the dynamic of pH change at high spatial resolution and offer a potentially rich opportunity to understand the glycolytic mechanism under hypoxic conditions, thus further facilitate the antitumor drugs screening and optimization.

EXPERIMENTAL SECTION Preparation of AuNRs@4-NTP Nanoprobes. AuNRs with good rod shape and a very high yield were produced by a seed-mediated growth method.30 The solution was cleaned by centrifugation (8000 rpm, 10 min) to remove CTAB monolayer which has a highly cell toxicity and hinder the formation of Au-S bound between 4-NTP and AuNRs and then redispersed in deionized water. The solution was functionalized with 4-NTP to form the AuNR@4-NTP nanoprobes by adding 100 µL 4-NTP (5 mM) aqueous solution dropwise to 5 mL of as-prepared AuNRs solutions at room temperature for 3 h, following by centrifuged at 8000 rpm for 10 min to remove free 4-NTP and then redispersed in deionized water. The details of AuNRs@4-NTP@CPPs synthesis were shown in supporting information. SERS Detection in Solution. The nitroreductase power was dissolved into ultrapure water and stored in -20 ℃ to keep the enzyme activity. In 2 mL tubes, the prepared AuNRs@4-NTP@CPPs solution (0.5 nM) incubated with NADH (500 µM) and different concentrations of NTR in 10 mM citric acid-phosphate buffer. After incubation for 60 min at 37 °C in a water bath, a portion of the reaction solution was transferred to capillary to conduct SERS detection. 5



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SERS Imaging and pH Calibration for Living cells. First, A549 cells were incubated with fresh medium containing 0.5 nM AuNRs@4-NTP@CPPs under normoxic conditions (20% O2) and different hypoxic (15%, 10%, 5%, and 1% O2) conditions for 3 h, respectively. Then, cells were washed three times with phosphate buffered saline (PBS) to remove the nanoprobes loosely

attached

to

the

cell

surface

and

then

treated

with

high

K+

4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)-buffered solution (30 mM NaCl, 120 mM KCl, 0.5 mM CaCl2, 0.5 mM MgCl2, 5 mM glucose, and 20 mM HEPES) at various pH values (pH 4.5, 5.5, 6.0, 6.5, and 7.5) in the presence of 10.0 µM nigericin.17 After 15 min, the SERS monitoring of A549 cells was carried out on a Raman microscope. For the mechanism of glycolysis experiment, A549 cells were incubated with fresh media containing 0.5 nM AuNRs@4-NTP@CPPs nanoprobes under a typical hypoxic condition (1% O2) for 3 h and then treated with 10 mM glucose and 5 mM 2-DG for 30 min and then washed three times by PBS. For each SERS spectrum, SERS signal collected with a 633 nm laser, a 50×(NA 0.45) microscope objective with a long working distance, the laser output power (2 mW) and the acquisition time (3 s). SERS Imaging of Hypoxia in Lung Tissue Slices. Frozen tissue slices were prepared from the lungs of the nude mice. The tissue slices incubated with AuNRs@4-NTP@CPPs (0.5 nM) under normal (20%) and hypoxia (1%) for 3 h and then nigericin-treated with tissue slices exposed to external media at pH 7.5 for 15 min, or was first cultured with AuNRs@4-NTP@CPPs (0.5 nM) under hypoxia (1%) for 3 h and treated with 10 mM glucose and 5 mM 2-DG for 30 min at room temperature, respectively, and finally washed with PBS for three times. For each SERS spectrum, SERS signal collected with a 633 nm laser, a 50×(NA 0.45) microscope objective with a long working distance, the laser output power (2 mW) and the acquisition time (3 s).

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RESULTS AND DISCUSSION The underlying chemistry involved in our design is based on the transformation between 4-NTP and 4-ATP catalyzed by NTR with the aid of NADH, as shown in Scheme 1A. The chemical reaction may go through a process in which the nitro group of 4-NTP is converted stepwise by NTR into amino group. Before sensing exploration of our constructed SERS nanoprobe, this transformation was first investigated by using high performance liquid chromatography (HPLC) and 1H nuclear magnetic resonance (NMR). One can see from Figure 1 that the standard samples of 4-ATP, NADH, and 4-NTP all showed distinctly different retention times, which located at 4.15, 6.53 and 10.28 min, respectively. When 4-NTP was mixed with NTR in the presence of NADH, a new peak located at about 4.15 min which was identical to the retention time of 4-ATP appeared, while the peak of 4-NTP at 10.28 min gradually decreased. On the contrary, when 4-NTP was mixed with inactived NTR, the two peaks attributed to the original substrate of 4-NTP and NADH demonstrated no obvious change. In addition, the products attained from the transformation between 4-NTP and 4-ATP catalysed by NTR, and the pure 4-NTP and 4-ATP compounds were characterized by 1H NMR respectively (Figure S1), giving consistent results with the HPLC results. These collective results indicated that the 4-NTP could be effectively and specifically reduced by NTR, thus guaranteeing the desirable transformation between 4-NTP and 4-ATP under hypoxic conditions. Inspired by the transformation between 4-NTP and 4-ATP reduced by NTR, we then fabricated a new hypoxia-triggered pH-sensitive SERS nanoprobes by functionalizing AuNRs with 4-NTP (AuNRs@4-NTP). AuNRs employed in our design were synthesized through the seed-mediated growth method, and the average aspect ratio was 3.0 (Figure S2A). To eliminate the obvious cytotoxity caused by the surfactant molecules CTAB on the surface of AuNR, the solution of AuNRs was cleaned by centrifugation to remove CTAB monolayer.

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Then, the purified AuNRs were incubated with 4-NTP to form AuNRs@4-NTP and the corresponding TEM image was shown in Figure S2B. From these results, one can see the morphology of AuNRs have no significant difference after incubation with 4-NTP. In Figure S3A, UV-vis absorption spectrum of AuNRs showed the longitudinal surface plasmon resonance (LSPR) and the transverse surface plasmon resonance (TSPR) peak at 662 nm and 520 nm, respectively. In addition, UV-vis absorption spectrum of 4-NTP in ethanol solution is showed in Figure S3B. When 4-NTP molecules were absorbed on the surfaces of AuNRs via Au-S bond, the LSPR blue shifted to 658 nm which indicating the replacing of CTAB monolayer. Additionally, Zeta potential measurements of AuNRs@4-NTP significantly shifted from 29.8 mV to 8.2 mV compared with that of the AuNRs because some positive-charged CTAB molecules on the AuNRs surface were occupied by 4-NTP, which confirmed the successful fabrication of AuNRs@4-NTP (Figure S4). With the AuNRs@4-NTP in hand, we then explored its SERS spectra and NTR responsiveness in 10 mM citric acid-phosphate buffer. Considering that the gold nanoparticles-based SERS substrates may suffer from relatively poor reproducibility due to aggregation,31 we functionalized AuNRs@4-NTP with cell penetrating peptides (CPPs, CAAAAAAAK(ME)3) to form a biocompatible protective layer. Since steric effect and repulsion provided by CPPs can provide an effective protection layer and prevent the aggregation of AuNRs, we thus can obtain a stable SERS pH nanosensor.32,33 The characterization of AuNRs@4-NTP@CPPs was shown in Figure S5. As shown in Figure 2A, the SERS spectrum of AuNRs@4-NTP@CPPs exhibited characteristic bands at 1069 and 1331 cm-1 that were assigned to C-S stretching and O-N-O stretching mode, respectively (Table S1),34 while no SERS signal can be observed for AuNRs without 4-NTP (Figure S6). Upon NTR was introduced to the mixed solutions of AuNRs@4-NTP@CPPs, the SERS spectrum changed significantly. Specifically, the peak at 1331 cm-1 assigned to O-N-O stretching

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decreased, and concomitantly two characteristic bands of 4-ATP at 1380 and 1438 cm-1 attributed to the b2 modes of 4-ATP emerged, which was in excellent agreement with the SERS spectrum of AuNRs@4-ATP@CPPs. These changes further revealed the successful transformation of nitro group in 4-NTP into amino group with the aid of NTR-mediated catalysis. Furthermore, since the NTR level directly corresponds with the hypoxic status in the solid tumors, we next investigated the dose-dependent response of AuNRs@4-NTP@CPPs to NTR. The SERS spectra of AuNRs@4-NTP@CPPs with varied concentrations of NTR were depicted in Figure S7. As the concentration of NTR increased, the SERS peak at 1438 cm-1 increased while 1331 cm-1 clearly decreased, which was accordant to the expected increase in the number of N=N groups and decrease in the number of NO2 groups. It is worth noting that the peak at 1069 cm-1 corresponded to C-S stretching in both 4-NTP and 4-ATP is nearly independent on the reaction and remained comparable in the presence and absence of NTR, we thus used the signal ratios of the 1438 cm-1 (or 1331 cm-1) to 1069 cm-1 to infer information on the concentration of NTR. As shown in Figure 2B, there is an approximately linear relationship between I1438/I1069 (and I1331/I1069) and the concentration of NTR over the range of 0.5-9.6 µg mL-1 and the detection limit for NTR is calculated to be 0.3 µg mL-1 (upon I1438/I1069) and 0.4 µg mL-1 (upon I1331/I1069). To investigate the response selectivity of our constructed AuNRs@4-NTP@CPPs nanoprobe, potential interferents were examined, such as salts (Na+, Mg2+, Ca2+, K+), amino acids (glycine, arginine), vitamin C, H2O2, glucose. As shown in Figure S8, when various potential interferents were incubated with AuNRs@4-NTP@CPPs (0.5 nM) nanoprobes, only NTR could induces a significant SERS peak increasing at 1438 cm-1, indicating potential interfering species no reactivity toward AuNRs@4-NTP@CPPs nanoprobes under the same conditions. Furthermore, as the reduzate, it is noteworthy that two modes of 4-ATP under different pH values can be distinguished by their spectra: the NH3+ form in acidic solutions and the

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N=N one in neutral and alkaline solutions. As the pH value became lower, the quinonoidic surface imine form of 4-ATP was changed to the aromatic amine state and then further protonated to the ammonium one.35 Based on this unique properties, we next investigated the SERS spectra of AuNRs@4-ATP@CPPs in citric acid-phosphate buffer solutions of various pH values from 3.0 to 8.0 in steps of 1.0 pH (Figure S9A). With the varying of pH, the SERS intensity ratios of peaks at 1438 cm−1 and 1069 cm−1 shown that 4-ATP is strongly dependent on the pH values (Figure S9B). It could be observed that the value of I1438/I1069 has an excellent response in a wide pH range from 3.0 to 8.0 with small deviations. In contrast, no obvious SERS spectra change could be observed for AuNRs@4-NTP@CPPs under different pH, which attributed to both C-S bound and nitro of 4-NTP are not sensitive to the pH (Figure S10A). Therefore, the SERS intensity ratios of I1438/I1069 of AuNRs@4-NTP@CPPs have not obvious changed with pH increase from pH 3.0 to pH 8.0 (Figure S10B). Then, to study the feasibility of our constructed nanoprobes for pH sensing under hypoxia, Figure 2C shows the SERS spectra changes of AuNRs@4-NTP@CPPs response to different pH in presence of 7 µg mL−1 NTR. Upon NTR-catalyzed cleavage reaction, the SERS spectrum changes significantly, standard SERS pH titrations of the as-generated 4-NTP were then performed in citric acid-phosphate buffer solutions with different pH values. All of them displayed sensitive SERS spectra response to the changes of pH values. Figure 2D shows the calibration curve of SERE ratio versus pH performed in citric acid-phosphate buffer solutions with a wide pH range from 3.0 to 8.0. It could be observe that the ratio value of I1438/I1069 has excellent response behavior in pH range from 3.0 to 8.0 in ensemble solution measurement with small deviations. Based on this ratio calibration curve, pH values can be inferred. The ratio peak intensity increased with the increasing pH in the citric acid-phosphate buffer solution, which was caused by the increased number of the deprotonated N=N. Inspired by this

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result, it can be inferred that our constructed SERS nanoprobe has great potential for serving good NTR-activated pH probe which can function well under hypoxia. After investigating the pH response characteristics of the AuNRs@4-NTP@CPPs SERS nanoprobe in citric acid-phosphate buffer solution, we then explored its potential application in lung tumor cells and tissues, as shown in Scheme 1B. According to the previous report, CPPs could transport nanoparticles into the plasma membrane in an energy-independent way and avoid confining them by the endocytosis pathway to the endosome or lysosomes.36,37 This is mainly attributed that the cell membrane could generate a nanoscale hole to assist the spontaneous translocation of cationic AuNRs@CPPs

(CPPs

are

positively

charged)

to

cytoplasm

side

under

a

transmembrane (TM) potential. Thus, the AuNRs@CPPs can move freely in the “cytoplasm” region and the membrane reseals itself with in a microsecond after translocation, while the TM potential is strongly diminished.38 In order to evaluate the cytotoxicity of AuNRs@4-NTP@CPPs-based SERS nanoprobe in biological system, we used classical MTT assay in A549 cells. Figure 3A showed that the cell viability decreased obviously with the increasing dose of AuNRs@4-NTP from 0.1 to 1.0 nM, while no obvious cytotoxicity was observed for AuNRs@4-NTP@CPPs. The result indicated that the biocompatibility of the proposed AuNRs@4-NTP@CPPs has been improved by peptides functionalization. Following further investigation of cellular uptake of AuNRs@4-NTP@CPPs nanoprobes in A549 cells was performed using dark-field resonant light scattering imaging.39 Dark-field microscopy (DFM) images show a crowd of spots, evidencing rapid internalization of the AuNRs@4-NTP@CPPs nanoprobes into the cells and mostly distributed in the cytoplasm (Figure 3B1). In contrast, when A549 cells incubated with AuNRs@4-NTP, a few spots can be observed in Figure 3B2. These data confirmed that AuNRs@4-NTP@CPPs efficiently

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transported across the plasma membrane without being endocytosed. With the guidance of the DFM, spectra can be performed readily from a single tested cell in which the positions of the AuNRs@4-NTP@CPPs can be localized. In order to investigate the transformation of nanoprobes in living cells, we recorded the SERS spectra in A549 cells incubated with AuNRs@4-NTP@CPPs under normoxic condition and a typical hypoxic condition. First, A549 cells were equally divided into two dishes and incubated with 0.5 nM AuNRs@4-NTP@CPPs under 20% O2 and 1% O2 for 3 h, respectively. Then A549 cells were treated with nigericin and then exposed to the external medium at pH 7.5, which was well used for homogenizing the intracellular pH and culture medium. As shown in the Figure 4A, bright-field images show that A549 cells have good morphology after the incubation of AuNRs@4-NTP@CPPs under 20% O2 and 1% O2. Under normoxic condition, no obvious the peak intensity mapping images of 1438 cm-1 can be observed, which indicated the transformation between 4-NTP and 4-ATP has not occurred because NTR cannot be expressed under normoxic condition. However, under hypoxic conditions, the peak intensity mapping images of 1438 cm-1 clearly appears, thus, the ratio peak intensities mapping of I1438/I1069 increased in the cells. Considering the absolute difference between intracellular environment and buffer solution, if applied in cellular sensing, a standard calibration curve about oxygen levels versus pH should be established in advance. First, when O2 concentration was set as 1%, the cells in each dish were collected respectively and equally divided into 5 new dishes and all the cells were treated with nigericin and then exposed to fresh external media at pH from 4.5 to 7.5. The peak intensity mapping images of 1438 cm-1 in cell increased with pH increasing, while the peak intensity mapping images of 1069 cm-1 demonstrated no obvious change. According to the color bar, one can see that the ratio peak intensity mapping increased with the pH from 4.5 to 7.5. Meanwhile, when oxygen concentration was set as 5%, 10%, 15% respectively, the

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ratiometric mapping images have a similar trend under pH from 4.5 to 7.5. While when O2 concentration was 20%, the trend of ratio mapping images has nothing to do with pH (Figure S11). The typical Raman intensity from the strongest to the weakest were chosen according to the color bar of ratio mapping images and corresponding sites 1, 2 and 3 marked in ratio mapping images and SERS spectra collected from the sites of 1, 2 and 3 were shown in Figure 4B (a1-a5) and Figure S12, respectively. The ratio value of I1438 to I1069, which demonstrated a characteristic pH-dependent signal in Figure 4C, indicated that the AuNRs@4-NTP@CPPs could be effectively used to measure pH with a good linear calibration curve in the pH range from 4.5 to 7.5 under 1%, 5%, 10%, and 15% O2 concentrations. Based on this ratio peak intensity and pH curve, the pH values of the A549 cells could be inferred under different O2 concentrations. Collectively, the developed AuNRs@4-NTP@CPPs nanoprobes exhibit a powerful ability to study the relationship between pH changes and degree of hypoxia. To further investigate the dynamic changes of local pH in A549 cells under hypoxia, we used the AuNRs@4-NTP@CPPs nanoprobes went through slight hypoxic condition (15% O2) to extreme hypoxic condition (1%), then gradually increased O2 concentration to 15% O2. With the decreasing of O2 concentrations, the Raman image with the band of 1438 cm-1 started to change while the band of 1069 cm-1 no obvious change, and the corresponding the pH started to change in A549 cells. Then, the typical Raman intensity from the strongest to the weakest were chosen according to the color bar of ratio mapping images (I1438/I1069) and corresponding sites 1, 2 and 3 marked in mapping images (Figure S13 b1-b6) and SERS spectra collected from the sites of 1, 2 and 3 were shown in Figure S14, respectively. Moreover, the intensity ratios of Raman peak 1438 cm-1 to 1069 cm-1 at the three sites were analyzed and summarized in Figure 5A. From the constructed I1438/I1069 versus pH calibration curves, pH alterations under different oxygen levels can be quantitatively determined (pH = 6.46+0.04 [O2], r2 = 0.98). Figure 5A showed that pH decreased from 7.1 to 6.5 through slight

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hypoxia conditions to extreme hypoxia condition and then gradually recover to pH 7.1 with the increase O2 concentration to 15%. In addition, since the excessive glycolytic activity triggered by hypoxia can result in vast production of lactate and thus accompany by the intracellular acidification, we further investigated the mechanism of intracellular glycolytic activity under hypoxia in living cells. Specifically, the average pH values of A549 cells were measured when the cells were exposed to different stimuli, such as 2-deoxy-D-glucose (2-DG) and glucose. 2-DG is a glucose analogue, which can inhibit glycolysis since glucose-6-phosphate inhibits phosphoglucose isomerase, while glucose can be used to increase glycolysis.40 Figure 5B confirmed that the addition of 2-DG could cause pH increase while glucose could induce a decrease in pH, which was reflected by the change in the I1438/I1069. The results demonstrated that the nanoprobe could be successfully applied to estimation of pH fluctuations associated with different stimuli under hypoxia, and also confirmed that the pH fluctuations is caused by intracellular glycolytic activity which has a potential application for studying the pathway of cell metabolism under hypoxia.41 Finally, we try to further examine the feasibility of our proposed nanoprobe to sense pH of lung cancer tissue under different hypoxic levels. Tissue slices were equally divided into two dishes and incubated with AuNRs@4-NTP@CPPs under normoxic condition (20% O2) and a typical hypoxic condition (1% O2) for 3 h, respectively. We performed the measurement of SERS peak on tissue slices treated with nigericin and then exposed to the external media at pH 7.5. As depicted in Figure 6A, slices treated in the normal O2 concentration served as a control group. When the O2 concentrations decreased from 20% to 1%, the SERS peak intensity of 1438 cm-1 increased, whereas that of 1069 cm-1 demonstrated no obvious change. Additionally, as for the lung cancer tissue slice kept under 1% O2, we used 2-DG and glucose to treat them respectively. The Raman mapping image of 1438 cm-1 showed the SERS signal

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at 1438 cm-1 increased slightly for the 2-GD-treated group, while the SERS signal at 1438 cm-1 decreased significantly for the glucose-treated one (Figure 6B). Moreover, the Z-scanning confocal overlay mapping imaging shows that SERS signal can still be clearly observed up to 500 µm of penetration depth (Figure 6C). Overall, our experimental results suggested that the proposed SERS nanoprobe can be used for high-contrast SERS imaging of pH under hypoxia in deep tumor tissues. Conclusion. In summary, we have presented a new SERS nanoprobe for quantitative monitoring the dynamic change of intracellular acidification in live cells and tissues under different oxygen concentrations. By functionalizing 4-NTP on AuNRs, both NTR responsiveness and SERS activity are integrated into the nanoprobes. The specific transformation of the nitro group of 4-NTP into an amino group in the presence of NTR could result in SERS spectra changes, thereby making the nanoprobes comes into play under hypoxic condition. Due to the pH-sensitive properties of the as-generated 4-ATP, the dynamic change of pH in live cells and tissues could be followed. The dynamic pH analysis indicated that pH was unevenly decreased as functions as O2 concentrations decrease in living cells. In particular, we have also demonstrated that our SERS nanoprobes can perform well for the quantitative monitoring of local pH values in lung cancer cells and tissues under 2-DG and glucose stimulation, verifying the inhibiting effect of 2-DG and activing effect of glucose for glycolysis under hypoxia. Taken together, we expect it will offer a potentially rich opportunity to understand the glycolytic mechanism under hypoxic conditions and further facilitate the antitumor drugs screening and optimization. Acknowledgment. The work was supported by the financial support through the National Natural Science Foundation of China (21405038, 21575018, 21475036) and the Fundamental Research Funds for the Central Universities. Supporting Information Available: Materials and apparatus, synthsis of AuNRs, preparation

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of AuNRs@4-NTP@CPPs, etc. This material is available free of charge via the Internet at http://pubs.acs.org

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Figure Captions Scheme 1. (A) The transformation between 4-NTP and 4-ATP catalyzed by NTR with the aid of NADH. (B) Schematic illustration of monitoring hypoxia-induced intracellular acidification using the AuNRs@4-NTP@CPPs-based SERS nanoprobe. Figure 1. HPLC profiles of (a) 4-ATP (100 µM), (b) NADH (100 µM), (c) 4-NTP (100 µM), (d) the products of the transformation between 4-NTP and 4-ATP catalyzed by NTR with the aid of NADH, and (e) the products of the transformation between 4-NTP and 4-ATP catalyzed by inactive NTR with the aid of NADH. Figure 2. (A) SERS spectra of AuNRs@4-NTP@CPPs (red curve), AuNRs@4-ATP@CPPs (pink curve), and AuNRs@4-NTP@CPPs treated with 10 µg mL-1 NTR (green curve) in the presence of 500 µM NADH at 37 ℃ for 60 min. (B) Plots of I1438/I1069 (red curve) and I1331/I1069 (green curve) as a function of the concentration of NTR in 10 mM citric acid-phosphate buffer solution (pH 7.4). (C) SERS spectra of AuNRs@4-NTP@CPPs (0.5 nM) reacted with NTR (7 µg mL−1) in the presence of 500 µM NADH at 37 ℃ for 60 min under different pH buffer solutions. (D) Histogram of I1438/I1069 of AuNRs@4-NTP@CPPs (0.5 nM) reacted with NTR (7 µg mL−1) in the presence of 500 µM NADH at 37 ℃ for 60 min under different pH buffer solutions. Error bars represent variations between three measurements. Figure 3. (A) Cell relative viability of A549 cells after treated with AuNRs@4-NTP (blue bars) and AuNRs@4-NTP@CPPs (grass green bars). 1-10 represent the concentration of AuNRs@4-NTP or AuNRs@4-NTP@CPPs: 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 and 1.0 nM, respectively. (B1-B2) DFM images of A549 cells incubated with AuNRs@4-NTP@CPPs and AuNRs@4-NTP for 3 h after washing with PBS for three times, respectively (The concentrations of AuNRs@4-NTR@CPPs and AuNRs@4-NTP were 0.5 nM). Orange scale bar: 2.5 µm, white scale bar: 10 µm. 20


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Figure 4. (A) SERS images of A549 cells which incubated with AuNRs@4-NTP@CPPs for 3 h and then kept under 20% and 1% O2 for 3 h (All the A549 cells were nigericin-treated and exposed to the external media at pH 7.5). (B) SERS imaging of A549 cells which kept under 20% and 1% O2 for 3 h and then incubated with AuNRs@4-NTP@CPPs, finally treated with nigericin and exposed to external media at pH 4.5, 5.5, 6.0, 6.5 and 7.5, respectively. (C) Plots of I1438/I1069 as a function of pH under (a) 1% O2, (b) 5% O2, (c) 10% O2, and (d) 15% O2 conditions. Scale bar: 5 µm. Figure 5. (A) Histogram of the local pH of A549 under different O2 concentrations. (B) Histogram of pH of A549 cells where were kept under 1% O2 condition and then treated with stimuli glucose (a), the untreated control group (b) and 2-DG (c). Error bars represent the standard errors of the mean. Figure 6. (A) SERS imaging of nigericin-treated tissues (pH 7.5) under 20 % and 1% O2 concentrations. (B) SERS imaging of 2-DG (5 mM) and glucose (10 mM)-treated tissues under 1% O2. (C) Z-scanning confocal SERS imaging of tissues under 1% O2 concentration at different penetration depths ( 0-500µm). Scale bar: 100 µm.

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A

AuNRs Laser 633 nm

B

NTR

Glucose

O2

960 H+

glycolysis

1080 1200 1320 1440

4-NTP

H+

4-ATP

pyruvate TCA cycle

960

1080 1200 1320 1440

Lactate

4-ATP+

H+

1%

H+ H+

H+

960 1080 1200 1320 1440

R a m a n S h ift / c m - 1

Scheme 1

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Glucosetransporter CPPs

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a Absorance unit

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4-ATP

b

NADH

c

4-NTP 4.15

d

6.53

10.28

e 0

2

4

6

8

10

12

Time / min

Figure 1

23


14


40000 32000

-1 1331 cm-1 1438 cm

AuNRs@4-NTP +NTR

24000 16000

AuNRs@4-ATP

3.6

B

1.2

2.7 0.9 1.8 0.6 0.9 0.3

8000

AuNRs@4-NTP

0.0

0

0.0 1040

1170

1300

0

1430

2

4

Raman Shift / cm 6000

8

10

12

7

8

1.5

C

D

1438 cm-1

1069 cm-1

1.2

3600

I1438/I1069

4800

6

NTR / µg

-1

pH 8.0 pH 7.0

2400

pH 6.0 pH 5.0

1200

0.9

0.6

pH 4.0 pH 3.0

0

0.3

1040

1170

1300

3

1430 -1

4

5

6

pH

Raman Shift / cm

Figure 2

24


I1331/I1069

1069 cm-1

I1438/I1069

SERS intensity / a.u.

A

SERS intensity / a.u.

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

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A 100

Cell viability (%)

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75 50 25 0

1

2

3

4

5

6

7

8

9

B2

B1

Figure 3

25


10


A

Bright field

1069 cm-1

1438 cm-1

pH 4.5

pH 5.5

pH 6.0

Overlay

Ratio mapping

20% O2

1% O2

B

pH 6.5

pH 7.5

1069 cm-1

1438 cm-1

Overlay

Ratio mapping

a12

3

a2 3

1

2

Weak

a3 3 1

a4 1 2

2

1

3

a5

3

2

Ramam intensity

C

1

Strong

1 % O2 5 % O2 10 % O2 15 % O2

1 .6 I /I 1438 1069

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

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1 .2 0 .8 0 .4

4 .8

5 .6

6 .4

7 .2

pH

Figure 4

26


8 .0

2

3

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8.4

7.8

B

A 7.7

pH

7.2

pH

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


6.6

7.0 6.3

6.0 5.6

5.4 15%

1%

5%

10%

a

15%

[O2]

Figure 5

27


b

c


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A

1438 cm-1

1069 cm-1

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overlay

C

20% O2

0 µm

100 µm 1% O2

200 µm

300 µm

B 400 µm (-) 2-DG 500 µm

(+)Glucose

Figure 6

28


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For TOC Only

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Quantitative Monitoring of Hypoxia-Induced Intracellular Acidification

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