A hemicyanine-based optical probe for biomembranes and intracellular pH sensing

Author’s Accepted Manuscript A hemicyanine-based optical probe biomembranes and intracellular pH sensing

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Qingyun Gao, Jinya Du, Han Liu, Shuang Lu, Xinwen Zhou, Changying Yang www.elsevier.com/locate/jlumin

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S0022-2313(18)30552-0 https://doi.org/10.1016/j.jlumin.2018.05.046 LUMIN15628

To appear in: Journal of Luminescence Received date: 26 March 2018 Revised date: 3 May 2018 Accepted date: 17 May 2018 Cite this article as: Qingyun Gao, Jinya Du, Han Liu, Shuang Lu, Xinwen Zhou and Changying Yang, A hemicyanine-based optical probe for biomembranes and intracellular pH sensing, Journal of Luminescence, https://doi.org/10.1016/j.jlumin.2018.05.046 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

A hemicyanine-based optical probe for biomembranes and intracellular pH sensing Qingyun Gao, Jinya Du, Han Liu, Shuang Lu, Xinwen Zhou, Changying Yang* College of Biological and Pharmaceutical Science, China Three Gorges University, Yichang443002, P R China

*

Corresponding author. Tel: 86-717-6395643; Fax: 86-717-6395580. E-mail address: [email protected].

Abstract The hemicyanine-based fluorescent probe L3 was demonstrated as a suitable membrane physiological pH probe. L3 easily interacted with the lipid components and anchored in inner membrane and gave a red fluorescence signal (~ 650 nm) in neutral or acidic pH, while the emission switching off when intramembrane pH varied to basic. Moreover, the absorption color of L3 changed from purple red to colorless with increasing pH. Thus L3 served as the pH-dependent modulator, a dual optical biomembranes pH sensing probe, showing good linear relationship in physiological pH range 7.0 ~ 8.5 or 7.5 ~ 9.0. The liposomes prepared under random pH and suspensions of gastric cancer cells (HGC-27) samples were used for verification of proposed intracellular pH sensing method, and satisfied results were obtained. Key words: Intracellular pH, Fluorescence, Ratiometric probe, Liposomes, Gastric cancer cell

1. Introduction The pH of biomembrane or intracellular pH (pHi) has a significant effect on cell cycle-related life activities, such as endocytosis [1], enzymatic action [2], tissue activities [3], cell apoptosis [4], and material transfer [5]. Abnormal pHi always leads to aberrant cellular function and cellular growth, which easily result in some diseases such as cancer [6] and Alzheimer’s [7]. Changes on membrane pH can be observed in these diseases such as cancer whose cellular pH is always lower than normal cell [8]. Hence, accurate measurement of cellular pH can be an available way of providing vital information for physiological and pathological studies. It is essential for health professional to have simple and effective methodologies for the rapid, selective and sensitive determination of pHi. With the development of scientific and technological progress, various methods in detection membrane pH have been developed. Spectroscopy [9 -11], nuclear magnetic resonance [12, 13] and microelectrodes [14, 15] are three more common ways used for measuring pHi. Fluorescence spectroscopy with its high spatial and temporal resolution attracted more attention over other methods [16], making it popular in researchers to seeking probe with both excellent optical properties and good biocompatibility [17, 18]. Moreover, fluorescence techniques have high sensitivities, which tend to be operationally simple, and are in most cases nondestructive to cells. In these years, pH-sensitive fluorescent probes based on small molecule, nano material, fluorescent protein and nucleic acid have been widely developed, such as AP-Cy [19], lipobeads [20], green fluorescent protein [21] and I-switch [22]. Small molecular fluorescent probes which are always fluorescence “on-off” (“off-on”) or ratiometric, win out due to their easy to get and can be modified into a new probe with prospective properties [23, 24]. For instance, a piperazine-linked naphthalimide derivative operates well in a mitochondrial environment within whole cells and displays a desirable off-on fluorescence response to mitochondrial acidification [25]. However, many of them suffer from various drawbacks, such as photobleaching, rather short wavelength, leakage from cells, and low sensitivity upon pH change in

complex biosystem. Future efforts need to be concentrated on developing more sensitive pH fluorescence probes, which owing longer emission wavelength (> 600 nm) for avoiding the interference from biological autofluorescence. The development of red and NIR fluorescent probes for pH has attracted increasing attention due to the central role of pH in many cellular events and the implication on abnormal cell growth and division noted in diseases such as inflammation and cancer [26-28]. Fluorescence behaviors of the free pH indicators are often different inside cells and probably non-sensitive to cellular pH change. The pH-sensitive NIR aza- BODIPY dye, synthesized by Chen et al., was highly fluorescent only under acidic conditions in bulk solution, but not strictly pH sensitive when was incorporated with micelles, liposomes and hydrophobic pockets of proteins [29]. The biological pH probes reported recently worked for either cytosol (6.80 - 7.40) or the acidic organelles (4.50 - 6.00). A NIR fluorescent probe Tpy-Cy responded rapidly to minor pH fluctuations within the range of 6.70 - 7.90 and was achieved successfully for the real-time imaging of cellular pH in living HepG2 and HL-7702 cells [30, 31]. CQ-Lyso as a lysosome-targeting ratiometric fluorescent probe had been developed for the sensitive detection of pHi (4.0 - 6.0) in living cells [32]. The desirable probes should respond remarkably to a minor pH change, give dependable results, and meanwhile avoid interference from native cellular species particularly. Herein, a hemicyanine-based small molecular fluorescent probe was presented as a biomembrane pH chemosensor within physiological neutral to basic variation. The fluorescence of the probe was strengthened and more sensitively respond to the pH change in biomembrane media. pKa did not really change in biomembrane. The quantitatively determination of pH in the range of 7.5 to 9.0 was constructed to measure the real samples, including liposomes and gastric cancer cells. The no-wash stable living-cell pH sensing made it an excellent choice for in vivo-cell tracking instead of pH indicators.

Scheme 1. Schematic drawing of L3

2. Experimental 2.1 Materials (E)-2-(4-(diphenylamino)styryl)-1,1,3-trimethyl-1H-benzo[e]indol-3-ium iodide (L3) (Scheme 1), which was synthesized and purified using the method from our previous work [33]. 2-Oleoyl-1-palmitoyl-sn-glycero-3-phosphocholine (POPC, and 2-Oleoyl-1-palmitoyl-sn-glycero-3-phospho-L-serine sodium salt (POPS,

99.0%) 75%)

were purchased from Sigma-Aldrich. Bovine serum albumin (BSA, fraction V, ≥ 96%), hemoglobin (Hb, from swine, ≥ 98%), human serum albumin (HSA, recombination in rice, ≥ 96%), lysozyme (Lys, from chicken egg white, ≥ 98%), myogolobin (Mb, from equine skeletal muscle, ≥ 95%), pepsin (from porcine gastric mucosa, ≥ 2,500 units/mg), trypsin (from bovine pancreas, ≥10,000 BAEE units/mg) and albumin (Alb, from chicken egg white ≥ 98%) were all purchased from Sigma and used as received. Undifferentiated gastric cancer cells (HGC-27) were presented by Natural Product Laboratory of China Three Gorges University. Unless otherwise noted, all solvents and other reagents used were of analytical grade without further purification. Redistilled water was used throughout the experiments. Britton-Robinson (B-R) buffer was prepared with the mixture of 0.04 M H3PO4, H3BO3, and HAc in advance. The pH of which was adjusted by addition of 0.2 M aqueous NaOH. 2.2 Spectrum measurements The fluorescences of probe L3 (10 μM) in various media were measured on F-4600 spectrofluorimeter (Hitachi, Japan) with the excitation and emission slits were both 5.0 nm. The excitation wavelength was 530 nm. Before measuring, incubation of L3 in liposomes was conducted for 1 h at room temperature to equilibrium the interaction between probe and liposomes. The absorbance spectra were recorded on Shimadzu

UV-2600 spectrophotometer in the wavelength range of 700-200 nm. For fluorescent pictures in paper were photographed under 365 nm UV lamp. 2.3 Liposome preparation Unilamellar liposomes were prepared as described reference [34, 35]. Briefly, the components containing various molar amounts of POPS and POPC (1:9, 5:5, 9:1) were dissolved in 5 mL chloroform followed by the solvent removal by rotary evaporation at 45 °C, then kept overnight under N2. The thin lipid film was hydrated at 55°C in B-R buffer for 30 min. The obtained turbid suspension was vortexed for 2 min and then sonicated 15 min with probe sonic disintegrator (600 w). One millilitre of the obtained suspension was then extruded 15 times through a 100 nm polycarbonate membrane using a mini extruder (Avanti Polar Lipids Inc., USA). And pH responses of the probe in liposomes were obtained by measuring the fluorescence intensity of 10 μM L3 in liposomes with different pH. 2.4 Gastric cancer cells samples Suspension of gastric cancer cells were obtained in the process of subculture for standby application. Following, part of cell suspension was treated by mixing pH buffer at a volume ratio of 1:1 to create cell environments with various pH. To find out the optical properties of L3 in cancer cells, stock solution of L3 in ethanol was added to 1 mL cell suspension to give a final probe concentration of 10 μM. All the measurements were performed within one hour so that the cells were still alive.

3. Results and discussion 3.1 Optical properties of L3 in liposomes Phosphatidylcholine is the main phospholipid in cell membranes [36], and mixed anionic/zwitterionic lipid bilayers are always used in membrane related studies [37]. We used liposomes containing different fractions of POPC and POPS to construct a simple artificial model biomembrane. As shown in Fig. 1a, the probe L3 featured an absorption band between 420-650 nm with a maximum at 530 nm in neutral aqueous buffer (pH 7.0). In liposomes, the absorption band changed more intensely and narrowly and the absorbance was related with the constituents of lipids. L3 emitted

extremely weak fluorescence in aqueous buffer because of the twisted intra-molecular charge transfer (TICT) excited state [38] induced by water. While >70-folds enhancements of L3 emission at 650 nm were found in liposomes (Fig. 1b). This Off-On emission mechanism might be attributed to the interaction of the probe with lipid bilayers. As shown in Fig. 2 inset, the color of L3 in liposomes has no obvious difference with in buffer in day light, but L3 exhibited bright red fluorescence emission under 365 nm UV lamp in liposomes while showed dark in aqueous. The impedance of the rotation of triphenylamine moieties in L3 molecule by lipid bilayers led to the inhibition of TICT effect and the recovery of fluorescence intensity.

Fig. 1. Absorbance (a) and Fluorescence (b) spectra of 10 μM L3 in buffer and liposomes composed of different mole ratios of POPS: POPC at pH 7.0.

It is known that lipid composition of the cell membrane closely related to their characteristics, which can be a good feature to classify the types of cell lines [39]. The emissions of the probe L3 in three different liposomes were similar (Fig. 1b), indicated that L3 anchored into hydrophobic tails and the luminescence was not affected by lipid constituents and the charges of the polar head. The time course of fluorescence intensity at 650 nm (Fig. 2) showed that L3 instantly interacted with liposomes and the fluorescence intensity kept stable at least 30 min. The fast response and good photostability enabled L3 acting as a sensitive and stable biomembrane probe.

To find out whether the probe was anchored to the interior or external membrane, liposomes were then sonicated after interacted with L3, and their fluorescence were shown in fig. S1. The emission intensity did not change with ultrasound time, which was significantly different with our previous work on the probe H3, whose fluorescence intensity greatly decreased after a sonication time [40]. It was implied that the probe L3 was located in the inner membrane so that its fluorescence could not be affected by mechanical ultrasound.

Fig. 2. Time course of 10 μM L3 in liposomes (POPS: POPC, 1:9) (pH 7.0). Inserts: Images of L3 in buffer and liposomes under the irradiation of a UV lamp at 365 nm.

On account of the complexity of cellular environments, photo-stability and anti-interference of the probe were particularly important. The anti-jamming capability of the probe was then tested under kinds of interferents might exist in biological environments. Eight kinds of proteins (10 μM) (BSA, HSA, Hb, Mb, Alb, Trp, Lys and pepsin) and ten kinds of metal ions (100 μM) (Na+, K+, Ca2+, Co2+, Ni2+, Cu2+, Fe3+, Mg2+, Mn2+, Pb2+) were selected to measure the effect of normal coexistence substance on the probe characteristic in biomedia. As shown in Fig. 3, most of interferents exhibited no remarkable effects on the fluorescence of L3 in lipid bilayer, which enabled L3 as a promising NIR fluorescent membrane probe.

Fig. 3. Fluorescence responds of 10 μM L3 in 1: 9 POPS: POPC liposomes (gray) upon addition of 10 μM proteins (blue) or 100 μM metal ions (green) at pH 7.0.

3.2 pH-Response of L3 in liposomes It is interesting that L3 undergoes distinct luminescence switching from On to Off (Scheme S1 and Fig. 4) when pH increase in liposomes (POPS: POPC, 1:9). L3 emitted bright red in acid to neutral lipid and became weak or even disappeared in alkali liposomes (Fig. 4a), demonstrating that L3 acted as a sensitive OH- responsive sensor, the fluorescence quenching was due to the reaction of OH- with the positively charged indolinium group and broken of molecular conjugation [41]. Fig. 4 show the pH dependence on the emission spectrum of L3 that now displays fluorescent pH sensing activity in the physiological pH range in biomembrane media. Furthermore the change of fluorescence intensity based on the pH variation is over 20-fold. Liposomes containing different fractions of anionic POPS and zwitterionic POPC were prepared for further study of the effect in lipid constituents on the probe pH response. Although the charges of the polar head in lipid has no influence on L3 optical properties in neutral liposomes, it is reasonable that charged lipid species effect the reaction of L3 with OH-, which could shift the pKa of L3 in a certain sense [42]. As shown in Fig. 6a and Figs. S2 S4 S6, the shape of pH titration curves of the probe in three liposomes were very similar, fluorescence appeared as a platform at pH 2.0 - 7.0 and 9.7 - 13.0 and there was a gap occurred at pH range 7.0 - 9.7 in

liposomes. The values of pKa were obtained according to the titration cures where I/Imax was 0.5 [43]. Similar gaps existed in L3 fluorescence in various liposomes led to no apparent shift on pKa (8.5 ± 0.1). This might due to the probe were in the same interior hydrophobic microenvironment after full interaction with liposomes [29]. It is worth noting that pKa of L3 shifted more than one unite compared with the response of L3 to pH in aqueous buffer (pKa = 7.23). It is also an evidence for L3 locating into the intramembranes. Surrounded by hydrophobic fatty alkyl chain, L3 is more stable and not sensitive to OH-, thus shifting the equilibrium of the reaction towards the reactant side and suppressing the addition reaction to take place under the neutral condition (Scheme S1).

Fig. 4. (a) Emission spectra and (insert) fluorescence images under 365 nm UV illumination of 10 μM L3 in liposomes composed of 1: 9 POPS: POPC as a function of pH. (b) Plot of emission intensities at 650 nm versus pH.

Fig. 5. (a) Absorbance spectra and (insert) photographs under natural light of 10 μM L3 with different pH values in the presence of liposomes (POPS: POPC, 1:9).

Fig. 6. (a) Fluorescence (b) absorbance based pH titration curve of L3 (10 μM) in buffer (black) and liposomes composed of mole ratio POPS: POPC at 1: 9 (red), 5: 5 (blue) and 9: 1 (olive). I/Imax denote the observed and maximal emission intensities at 650 nm when excited at 530 nm, respectively.

It is noted that absorption of the probe in liposomes showed similar trends with pH changes. As shown in Fig. 5, it featured two obvious bands with a maximum at around 538 nm and 358 nm, respectively. With pH value increasing, the characteristic peak at 538 nm progressively diminished, accompanied by enhancement of absorbance at 358 nm, which could be a good feature for a ratiometric absorptive probe. The phenomenon can be ascribed to the increased formation of alkaline form of the probe [43]. The ratios of the absorbance at 538 nm vs 358 nm in various pH were provided (Fig. 5b), which were constant regardless of the change of fluorophore concentration by photobleaching or change of the external environment [44]. The nonlinear absorbance ratio regression analysis of L3 in liposomes (POPS: POPC, 1:9) (Fig. 5) gave a pKa of 7.19 [45]. Meanwhile, the color of the solution changed from purple red to colorless, which indicated that L3 could also serve as a visual indicator for pH [46]. Compared to the pKa value of L3 in aqueous buffer based on the same ratiometric absorbance method (6.53), the similar shift tendencies were found (Fig. 6b and Figs. S3 S5 S7), about 1 unit increase (from 6.53 to 7.19 in 1: 9, 7.56 in 9: 1 liposomes (POPS: POPC), respectively) [47]. All of the results showed that the pKa of the L3 changed about 1 unit by its interaction with biomembranes, but L3 was

really a sensitive dual-way-respond intracellular pH probe, based both on fluorescence intensity and ratiometric absorbance. 3.3 Quantitative detection of pH in membranes

Fig. 7. The linear relationship between pH and relative fluorescence (blue)/absorbance ratio (red) of L3 in liposomes (POPS: POPC, 1: 9).

More detailed experiments about standard fluorescence pH titrations were performed within the pH range 7.5-9.0. It was showed that there was a good linearity and the regression equation was

with correlation

coefficient 0.99 (Fig. 7), which indicated L3 as a potential probe for quantitative detection of inner membrane pH. The pH-dependent ratiometric absorbance curve was also conducted and featured similar changes, and the linear relationship was also existed within the scope of 6.8 - 8.5 of pH. The relationship can be described as (R2 = 0.99). Both the fluorescence intensities and absorbance ratio of L3 in liposomes showed good reactivity to pH changes, which reflecting good applicability of the probe in detection of pH in complicated membrane environments. In order to evaluate the ability of the proposed optical sensor in the application for biomembrane pH measurement, two types of samples including liposomes and suspensions of gastric cancer cells were investigated.

Table 1 Recovery of pH obtained by fluorescence method in liposome samples. Sample

Real pH

Detected pH

SD %

RSD %

Recovery %

1

7.52

7.79

19

2.4

103

2

7.62

7.68

4

0.5

101

4

7.89

7.92

2

0.3

100

5

8.18

8.08

7

0.9

99

6

8.48

8.24

17

2.1

97

7

8.67

8.45

16

1.8

97

Table 2 Recovery of pH obtained by absorbance ratiometric method in liposome samples. Sample

Real pH

Detected pH

SD %

RSD %

Recovery %

1

6.97

7.63

47

6.1

109

2

7.15

7.48

23

3.1

105

3

7.26

7.48

15

2.1

103

4

7.35

7.46

8

1.0

101

5

7.52

7.13

28

3.9

95

6

8.18

7.88

21

2.7

96

7

8.48

8.19

20

2.5

97

Various liposomes with random pH were prepared to examine the accuracy of pH detection based on two quantitative methods. Considering the same inside and outside pH of simple composed membrane, the intramembrane pH of the liposome samples were measured by a portable pH meter for comparation, and the results were shown in Tables 1 2. As for fluorescence method, the recoveries were in the range of 97% - 103% with a maximum RSD of 2.4%, which were satisfied values for complex media pH determinations [48]. While for absorptive ratiometric, recoveries range from 94% to 109%, and the maximum RSD was 6.1%, which was rather lower rationality. Thus, the fluorescence probe method was more accurate and suitable for lipid bilayer membrane pH quantitative measurements.

Fig. 8. (a) Time dependence of fluorescence intensity of 10 μM L3 in gastric cell suspension (HGC-27). (b) Emission spectra of 10 μM L3 in HGC-27 with different pH value.

pH sensitive response of visible and near-infrared fluorescence of L3 for monitoring the intracellular pH was furtherly performed in live cells. The L3 was incubated with the live cells, and the fluorescence was measured directly. Considering the high incidence and mortality rate of gastric cancer in worldwide [49], undifferentiated HGC-27 cells were used in the experiment. HGC-27 cells were normally cultured in pH 7.4 physiological media, and the fluorescence of L3 in HGC-27 cells suspensions was measured. As shown in Fig. 8a, probe interacted with cell membrane at around 2 minutes, which was consistent with the statement that transmembrane time of small molecules continues for seconds or even minutes [50]. This implies that the probe was in the inner membrane and measuring the intracellular pH, which reinforced the previous hypothesis. The intracellular pH of HGC-27 cell was determined to be 9.09 based on the built linear equations, which has not been tested until now within the scope of the author’s knowledge. Wu et. al reported that the intracellular pH of poorly differentiated gastric cancer cells (AGS) was obviously alkalified to about 8.60 [51]. When pH of HGC-27 cells suspensions were manmade modulated, the fluorescence of probe changed sensitively (Fig. 8b), which can also be used for simple identification of cells with different intracellular pH, such as cancer cells whose intracellular pH was more alkaline than normal cells [52]. These results demonstrate that L3 is promising as a vis-NIR fluorescent sensor for monitoring the intracellular pH.

4. Conclusion In summary, we have developed an organic optical probe with TICT characteristics, L3, for biomembranes and intracellular pH sensing. The probe located into hydrophobic interior fatty acid chains and emitted intense red fluorescence, based on the inhibition of TICT excited state. L3 is pH sensitive in liposomes or living cells by dual way. The absorption and emission color changes both with increasing pH. So that two quantitative methods based on fluorescence intensities and absorbance ratio were developed for the pH sensing. The linear physiological pH dependence of the vis-NIR fluorescence or ratio absorbance of L3 had been successfully explored for monitoring intracellular pH in liposomes samples and cancer gastric cells. It was verified that the range of L3 for pHi sensing thus covered the physiological pHi window from 7.0 to 9.0. Upon interacting with the lipid components, L3 shifts its pKa values (from 6.53 to 7.19 based on ratio absorbances, from 7.23 to 8.38 based on fluorescence intensities, respectively). However, the pKa of L3 in membranes was not affected by lipid components or the charges of the polar head, which demonstrated that the probe could be stably used to visualize intracellular pH changes with negligible interference. Therefore, the probe proposed here could be broadly applicable to the detection and quantification of pH changes in biological systems. The reported results herein are important for further applications of the vis-NIR fluorescence of L3 in bioimaging.

Acknowledgments We are grateful for the financial support from the National Natural Science Foundation of China (21473101).

Appendix A. Supplementary data Supplementary data associated with this article can be found in the online version.

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