Fluorescent pH-Sensing Probe Based on Biorefinery Wood

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Article

Fluorescence pH-Sensing Probe Based on Biorefinery Wood Lignosulfonate and Its Application in Human Cancer Cell Bioimaging Yuyuan Xue, Wanshan Liang, Yuan Li, Ying Wu, Xinwen Peng, Xueqing Qiu, Jinbin Liu, and Run-Cang Sun J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b04583 • Publication Date (Web): 04 Dec 2016 Downloaded from http://pubs.acs.org on December 5, 2016

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Journal of Agricultural and Food Chemistry

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Fluorescence pH-Sensing Probe Based on Biorefinery Wood Lignosulfonate and Its

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Application in Human Cancer Cell Bioimaging

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Title running header: Bioimaging agent based on Lignosulfonate

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Yuyuan Xue,†,

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Xueqing Qiu,†, ‡, * Jinbin Liu† and Runcang Sun,§

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† School of Chemistry and Chemical Engineering, South China University of Technology, Guangzhou,

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China.

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‡ State Key Laboratory of Pulp and Paper Engineering, South China University of Technology,

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Guangzhou, China.

‡

Wanshan Liang,†,

‡

Yuan Li,†,

‡,

* Ying Wu,†,

‡

Xinwen Peng,‡,*

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§Institute of Biomass Chemistry and Utilization, Beijing Forestry University, Beijing, China.

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* Corresponding authors:

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Dr. Yuan Li

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School of Chemistry and Chemical Engineering

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South China University of Technology

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Wu Shan Road, Guangzhou 510640, China

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E-mail: [email protected], Tel.: +86-020-87114033.

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Dr. Xinwen Peng

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State Key Laboratory of Pulp and Paper Engineering

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E-mail: [email protected]

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Dr. Xueqing Qiu

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School of Chemistry and Chemical Engineering

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25


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E-mail: [email protected] 1 ACS Paragon Plus Environment


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ABSTRACT

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A water-soluble, ratiometric fluorescent pH probe, L-SRhB, was synthesized via grafting

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spirolactam rhodamine B (SRhB) to lignosulfonate (LS). As the ring-opening product of

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L-SRhB, FL-SRhB was also prepared. The pH-response experiment indicated that L-

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SRhB showed a rapid response to pH changes from 4.60 to 6.20 with a pKa of 5.35,

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which indicated that L-SRhB has the potential for pH detection of acidic organelle. In

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addition, the two probes were internalized successfully by living cells through the

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endocytosis pathway and could distinguish normal cells from cancer cells by different

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cell staining rate. In addition, L-SRhB showed obvious cytotoxicity to cancer cells, while

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it was nontoxicity to normal cells in the same condition. L-SRhB might have prospective

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potential in cancer therapy. L-SRhB might be a promising ratiometric fluorescent pH

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sensor and bio-imaging dye for the recognition of cancer cells. Our results also provided

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a new perspective to the high-value utilization of lignin.

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KEYWORDS: biomass, rhodamine B, ratiometric sensor, FRET, cancer sensing

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2 ACS Paragon Plus Environment

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INTRODUCTION

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Lignosulfonate (LS), a water soluble lignin derivative, obtained from the biorefinery in

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a sulfite pulp mill, shows widespread application potential in many fields.1-8 High-value

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utilization of lignin based on its inherent features has attracted wide attention.9-10

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Fluorescence under ultraviolet (UV) or visible light excitation is an intrinsic property of

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lignin and this feature has been applied as a sensitive probe for lignin constituent

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analysis.11-12

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chromophore, namely, lignin has been found many years ago, the application based on

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the fluorescent property is rarely reported.11,

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measurement, fluorescence detection has been widely applied in various fields such as H+,

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metal ion detection and cell imaging, for its sensitivity, selectivity, lossless detection and

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real-time monitoring.14-28 Especially, ratiometric fluorescence probes are widely

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investigated because this kind of probes can avoid environmental factors by the self-

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calibration of two emission bands. Ratiometric probes have been widely reported in

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various systems including small molecules15-19 and nanoparticle-based sensors such as

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supramolecular aggregates,20, polymer particles,21-22 quantum dots29 and silica.30-31 It is

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noteworthy that nanoparticle probes have unique advantages in cell imaging, such as

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endocytic marker and predominant permeability.25 Meanwhile, water solubility and low

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toxicity are important aspects to be considered, while the probes are used in biomedical

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and environmental detection. It is well known that LS has good water-solubility,

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biocompatibility, polymeric nanoparticle structure31 and intrinsic fluorescent emission.11-

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13, 33

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sensor and bioimaging agent.34

Despite

an

inexpensive

and

13

environmental-friendly

fluorescent

As a powerful tool for quantitative

All of these features above motivate us to study the potential application of LS as



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In this work, we demonstrated a novel strategy to achieve the high-value utilization of

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lignin by constructing a ratiometric probe based on LS-dye system, for H+ detection

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(Scheme 1). LS shows a wide fluorescence emission between 300-700 nm, which

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relatively well matches the absorption spectrum of rhodamine B (Figure 1). LS serves as

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the donor whereas the SRhB works as a recognition element for H+ and acts as the energy

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acceptor when the long-wavelength fluorophore, rhodamine B is generated. Meanwhile,

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based on the good biocompatibility of LS, the pH probe L-SRhB was successfully

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applied for bioimaging. Interestingly, the LS-based pH probe could distinguish normal

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cells (HL-7702) from cancer cells (SMMC-7721, SPC-A-1 and HepG2). The cytotoxicity

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of the probes were also investigated and discussed.

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Materials and methods

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Chemicals

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LS was supplied by the Tranlin paper mill (Shandong province, China). Chloride salts of

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metal ion (K+, Ca2+, Mg2+, Cr3+, Hg2+ and Co2+), sulfate salts of metal ion (Mn2+, Ni2+,

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Zn2+ and Cu2+), nitrate salts of metal ion (Fe3+ and Pb2+), tris(hydroxymethyl)-

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aminomethane (Tris), sodium dihydrogen phosphate, sodium hydrogen phosphate, citric

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acid, trisodium citrate, sodium hydroxide (NaOH), Rhodamine B (C28H31ClN2O3),

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epichlorohydrin (C3H5OCl), diethylenetriamine (C4H13N3), dichloromethane (CH2Cl2)

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and anhydrous methanol were analytical grade. Dulbecco's Modified Eagle Medium

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(DMEM), Fetal bovine serum (FBS), penicillin, and 3-(4,5-dimethylthiazol-2-yl)-5-(3-

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carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) were purchased from

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Sigma-Aldrich. Mito-Tracker Green was purchased from Beyotime Biotechnology.

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Human hepatocyte cell line (HL-7702), human hepatocellular liver carcinoma cell line

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(SMMC-7721, HepG2) and human lung adenocarcinoma cancer cells (SPC-A-1) were

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obtained from Sun Yat-Sen University (Guangzhou, China).

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Synthesis of SRhB

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Rhodamine B (1g, 1.717 mmol) was dissolved in 20 mL of anhydrous methanol under

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nitrogen atmosphere, and then diethylenetriamine (2 mL) was added at 40

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Subsequently, the temperature was slowly raised to 65 oC. After 8 h, methanol was

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evaporated under reduced pressure. Then, CH2Cl2 (50 mL) and water (100 mL) were

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added and the organic layer was separated, washed 8 times with water. The solvent was

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removed and dried in vacuum to give SRhB (1g). Yield: 1.05 g, 89%. The spectra of


o

C.


98

1

H/13C-NMR and High Resolution Mass Spectrometer (HRMS with ESI source) were

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presented in Supporting Information (Figure S1, S2, S3).

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Epoxide of LS

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LS (15.0 g) was dissolved in a solution of NaOH (3.64 g) in distilled water (30 mL). The

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solution was stirred at 50 °C for half an hour to activate phenolic hydroxyl groups. Then

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C3H5OCl (0.750 g) was added dropwise, and the mixture was stirred at 50 °C for 8 h.

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When it was cooled to room temperature, the mixture was extracted three times with

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dichloromethane to remove the remaining C3H5OCl. The E-LS was obtained with a 30

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wt% solid content.

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SRhB Modified E-LS

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E-LS (5 mL aqueous solution, 30 wt%) was dissolved in distilled water (50 mL) and the

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pH of solution was adjusted to 12 using NaOH. 0.3 g of SRhB in THF (2 mL) was added

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dropwise, and then the mixture was stirred at 60 °C for another 8 h. The reaction was

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gradually cooled to room temperature. THF was removed under reduced pressure. The

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mixture was filtered and extracted by dichloromethane for three times to remove the

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residual SRhB. The aqueous phase was collected and dialyzed in distilled water for 3

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days in order to remove salt and unreacted regents. Finally, a brown powder of L-SRhB

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(0.7 g) was obtained by lyophilization.

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Synthesis of FL-SRhB

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L-SRhB (1.0 g) was dissolved in distilled water (30 mL). Subsequently, dilute HCl

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solution was added to adjust the pH value of solution to 4 and then stirred for 30 min. L-

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SRhB was fully changed into FL-SRhB until the UV-vis absorption intensity at 567 nm


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was stable. Purple FL-SRhB (1.0 g) was obtained after lyophilization (Scheme 1, Figure

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S4).

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Preparation of L-SRhB probe aqueous solutions.

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L-SRhB was dissolved in redistilled water to make 30 mg/mL stock solution. During the

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preparation of the solution for spectral measurement, 20.0 μL L-SRhB stock solution was

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added into the flask containing 2.98×103 μL buffer solutions, and then the system was

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stirred for 30 min. The buffer solutions were prepared as follows: citric acid buffer saline

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for pH 3.8, 4.2, 4.6, 5.0, 5.2, 5.4, 5.6, 5.8, phosphate buffer saline for pH 6.0, 6.2, 6.4, 6.6,

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6.8, 7.0, 7.2, 7.4, respectively. The test of the metal irons was prepared from

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ZnSO4·7H2O, CuSO4·5H2O, AgNO3, CoCl2·6H2O, KCl, HgCl2, MnSO4·H2O, CaCl2,

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NiSO4·6H2O, MgCl2·6H2O, CrCl3·6H2O, Fe(NO3)3·9H2O, Pb(NO3)2 by the Tris

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(tris(hydroxymethyl) aminomethane) buffer solution at pH=7.00 with final concentrations

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of 5×10-4 M for all of them. 20.0 μL L-SRhB stock solution was added into the flask

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containing 2.98×103 μL metal ions solutions, and then the system was stirred for 30 min.

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Finally, fluorescence and absorption measurements were performed.

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Reversible response to CO2 and N2

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20mL L-SRhB (1 mg/mL) redistilled water solution were prepared and charged to a vial,

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which was bubbled with CO2 (10 mL/min) for 10 min until the fuchsia solution was

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obtained. The aqueous solution was then bubbled with N2 (80 mL/min) for 48 min to

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remove CO2 until the pH was stable. The brown solution was obtained and the cycles

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were repeated for two times.

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Cell culture and cytotoxicity experiments



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In order to compare the difference of cell staining between normal cancer cells with our

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probes, HL-7702, SMMC-7721, SPC-A-1 and HepG2, which were the universal and

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common cell lines in the cytotoxicity study, were chose in this work. The cell lines HL-

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7702, SMMC-7721, SPC-A-1 and HepG2 (Sun Yat-Sen University, China) were cultured

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in DMEM in a humidity incubator at 37 oC with 5% CO2. The medium contained 10%

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FBS and 100 units/mL penicillin. The MTS test (3-(4,5-dimethylthiazol-2-yl)-5-(3-

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carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) was used to analyse the

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cytotoxic effect of L-SRhB and FL-SRhB on the cells. Before the experiments, the cells

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were washed with phosphate buffered saline (PBS) for two times, Briefly, HepG2 cells

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were seeded in 96-well microplates at a density of 1×104 cells/mL in 100 μL of DMEM

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with different concentrations of L-SRhB and FL-SRhB (0.010 mg/mL, 0.050 mg/mL,

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0.10 mg/mL, 0.25 mg/mL, 0.50 mg/mL, 1.0 mg/mL and 0 mg/mL as control). Plates were

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incubated for 48 h at 37 °C in a 5% CO2 humidified incubator. HL-7702, SMMC-7721,

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SPC-A-1 cells were treated with L-SRhB and FL-SRhB at concentration of 0.25 mg/mL,

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and the three cell lines were incubated for 5 min, 30 min, 1 h, 4 h, 8 h, 12h at 37 °C in a

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5% CO2 humidified incubator, respectively. After the incubation period, the cell medium

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was changed with fresh medium (200 μL/ well) after washing three times with PBS. Then,

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20 μL MTS agent was added to each well. After being incubated for further 4h at 37 °C,

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the plate was shaken for 10 min, and the absorbance was measured at 490 nm by a

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microplate reader (Thermo Fisher Scientific). The cell viability was calculated by the

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following equation: cell viability=(OD490treated

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OD490blank well).

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Imaging experiments

well-OD490blank well)/(OD490control well-


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Cells were seeded in a 35 mm petri dish with a glass cover slide. After 24 h incubation,

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the cells were stained with 0.25 mg/mL solution of L-SRhB and FL-SRhB, respectively.

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Then, they were incubated additionally for 5 min, 30 min, 1 h, 4 h, 8 h, 12 h, respectively.

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Before imaging, the cells were washed with PBS (pH 7.4) solution for three times.

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Channel: excitation: 485 nm, emission collected: 565-580 nm.

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Uptake mechanism of L-SRhB in HepG2 cells.

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The uptake mechanism of L-SRhB and FL-SRhB were determined by culturing cells in

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different temperature with L-SRhB. HepG2 cells was treated with L-SRhB at

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concentration of 0.25 mg/mL for 8 h at 4 °C and 37 °C in a 5% CO2 humidified incubator,

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respectively. Before imaging, the cells were washed with PBS (pH 7.4) solution for three

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times.

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Colocalization-imaging experiments of mitochondrion in HepG2 cells.

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After being incubated with L-SRhB (0.25 mg/mL) for 8h at 37 °C, HepG2 cells were

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washed with PBS (pH 7.4) for three times. Then the cells were incubated with Mito-

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Tracker Green (25 nM) for 15 min. The medium was removed and cells were washed

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with PBS (pH 7.4) for three times. Channel: excitation: 485 nm, emission collected: 495-

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520 nm.

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Measurements and statistic test.

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The 1H/13C-NMR spectra of samples were recorded with a suitable amount of each

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sample dissolved in 0.5 mL of DMSO-d6, CDCl3 or D2O at room temperature by DRX-

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400 spectrometer (400 MHz 1H-NMR frequency, 125 MHz 13C-NMR frequency, Bruker

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Co., Ettlingen, Germany).The fouriertrans form infrared spectroscopy (FT-IR) was tested

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by Auto system XL/I-series/Spectrum 2000 spectrometry (Thermo Nicolet Co, Madison,



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WI, USA) between 4000 and 400 cm-1. Elemental analyses of LS, L-SRhB-5% and L-

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SRhB-15% were measured by Elementar Vario EL cube. The retention time of LS, L-

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SRhB-5% and L-SRhB-15% were determined by a Waters 1515 Isocratic HPLP pump

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(Waters, USA) with an Agilent 1100 series gel permeation chromatography system

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(Agilent Technologies Corp., Santa Clara, USA) with PLgel 5 μm 1000 Å and PLgel 5

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μm 500 Å columns. The eluent was 0.10 mol/L NaNO3 solution (pH=10.4) with a flow

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rate of 0.50 mL/min. All samples were filtered by a 0.22 μm filter. The fluorescence

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emission spectra were recorded at excitation wavelengths of 330. The UV-via absorption

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was recorded at wavelength from 450 to 650 nm. The pH and metal ions response

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experiments were repeated at least three times and the average values were obtained. For

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the cytotoxicity assay experiment, three sets of parallel experiments were carried out and

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the average values were obtained.

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RESULTS AND DISCUSSION

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Design and application of the L-SRhB probe

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On the one hand, LS shows a wide emission spectrum from 350 to 650 nm under UV

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excitation. It indicates that LS has the potential as a donor in the FRET-based probe.

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Meanwhile, in order to achieve the water-solubility of probes, LS is a good candidate. On

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the other hand, the structure of SRhB derivative as fluorescent turn-on probes has

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attracted many researchers due to its high sensitivity, fluorescence quantum efficiency

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and relatively long-wavelength emission. For the relatively well match between the

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fluorescence of LS and the UV-vis absorption of rhodamine B, a FRET-based ratiometric

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probe, L-SRhB, was built up by grafting SRhB to LS. The pKa of L-SRhB was

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determined as 5.35 by Henderson-Hasselbalch equation. This value is suitable for the pH

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detection of acidic organelles (about 4.50-6.00). Meanwhile, lignin, as one of the main

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constituent parts of plant, has a fine biocompatibility. L-SRhB was utilized in cell

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imaging. Moreover, for the nanoparticle structure of LS due to its intramolecular

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aggregation property,32 endocytosis is the main pathway when L-SRhB gets into cells and

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this is good for the acidic organelles detection by the fusion of cytomembrane.

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Structure analyses of L-SRhB

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FT-IR was used to study the change of functional groups in LS, SRhB and L-SRhBs to

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confirm the effective covalent bonding between LS and SRhB (Figure S5). The

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adsorption intensities of the aromatic skeleton in L-SRhB-5% and 15% both increased

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comparing with that of LS. The areas of absorption at 2940 cm-1 and 2820 cm-1 enhanced

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because of the introduction of diethylenetriamine building block.35 The enhanced broad

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absorption at 3420 cm-1 is ascribed to the N-H and -OH groups from SRhB and



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epichlorohydrin. Meanwhile, in the spectrum of L-SRhB, the enhanced sharp absorption

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at 1265 cm-1 is ascribed to the stretching vibrational absorptions of C-N groups.

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To further illustrate the chemical structure change in LS, the NMR spectra of LS, SRhB

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and L-SRhB-5%, 15% with quantitative samples were shown in Figure S6, S1 and S2. In

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the spectrum of LS, the signals between 7.4-6.4 ppm and 4.0-2.7 ppm are from LS’s

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aromatic and methoxyl protons, respectively.36 Comparing the spectra of L-SRhBs with

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those of LS and SRhB, the clear aromatic proton signals at 7.82, 7.52, 7.00, 6.62 and 6.36

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ppm are contributed to SRhB’s aromatic protons. The aliphatic proton signals at 2.60,

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1.89, 1.06 and 0.98 ppm are contributed to SRhB’s aliphatic protons. From

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spectra of LS and L-SRhBs, significant difference was observed between 0-80 ppm

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(Figure S7). There was none of obvious difference in low field between LS and L-SRhBs

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because of the low grafting content of SRhB. Comparing the spectrum of 5%-L-SRhB

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with that of 15%-L-SRhB, there was an obvious enhancement of SRhB’s signals which

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was in good agreement with the elemental analysis results that the increasing nitrogen

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content was appeared in Table S1. It confirmed the successful synthesis of L-SRhB. In

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the spectrum of FL-SRhB, the signals at 8.80, 8.37, 8.24, 7.88, 7.36, 7.24 and 7.11 ppm

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are ascribed to the aromatic protons of rhodamine B.

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The sulfonation degrees of LS, 5% L-SRhB and 15% L-SRhB were 1.48, 1.56 and 1.37

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mmol/g, respectively, which were obtained from the element analysis (Table S1). The

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retention times of 15% L-SRhB, 5% L-SRhB and LS were successively larger with the

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decreasing grafting rate of SRhB. Meanwhile, the cross-link effect of epichlorohydrin in

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lignosulfonate also resulted in the decrease of the retention time of the three materials.


13

C-NMR

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The result of retention time indicated that the molecular dimensions of our probes were

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relatively larger than that of LS.

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In addition, the dialysis experiment presented in Figure S4 was to prove the successful

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synthesis of L-SRhB and FL-SRhB but no physical mixing because the molecular weight

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of RhB or SRhB was much less than the molecular weight cut-off of the dialysis tube.

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Optical response of L-SRhB to pH

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UV-vis spectra of L-SRhB at various pH values between 3.80 and 7.40 were shown in

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Figure 2. When the pH value is higher than 7, SRhB are non-fluorescent and colorless.

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On the contrary, ring-opening of the corresponding spirolactam gives rise to fluorescence

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emission and a pink color, when the pH value is smaller than 7. With the decrease of pH

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value, the absorption intensity at about 560 nm increased, which was contributed to the

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absorption of rhodamine B. The linear relationship of absorption intensity at 566 nm and

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pH values from 4.20 to 6.20, and the R2 was 0.9754 (Figure 2b). The maximal absorption

259

intensity at 566 nm was reached at pH=4.2 and the absorption intensity had almost no

260

change with the decrease of pH (Figure 2b). Moreover, the pH-response of L-SRhB was

261

sensitive and its absorption intensity saturation was reached within several minutes at pH

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5.40 and 6.40 (Figure S9). In addition, L-SRhB also displayed a good reversibility by the

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CO2/N2 bubbling experiment (Figure S10).

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The overlap between the absorption spectrum of rhodamine B and the fluorescence

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spectrum of LS was significant and beneficial for the FRET from the excitation state of

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LS to the ground state of rhodamine B (Figure 1).

267

Then, the fluorescence response of L-SRhB to pH was explored at various pH values in

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PBS buffers (Figure 3). LS exhibited blue fluorescence emission peaking at about 410 nm



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on excitation of 330 nm. When the pH changed from 7.40-4.20, the emission at 410 nm

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from LS backbone decreased and the new emission band at 587 nm gradually emerged.

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The ratio of fluorescence intensities R (I594/I400) showed an observable enhancement (up

272

to 13.9-fold) from 0.045 at pH 7.4 to 0.626 at pH 4.2. The ratio of fluorescence intensities

273

from the two fluorophores (I594/I400) was therefore ideal for the estimation of pH. The

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dual-emission fluorescence intensity ratio R increased linearly (Y=-0.3051×X+1.978,

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R2=0.9949) with the pH value in the range of 4.60-6.20 as displayed in Figure 3.

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Meanwhile, the pKa of L-SRhB was determined as 5.35 by Henderson-Hasselbalch

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equation: lg[(Rmax-R)/(R-Rmin))]=pH-pKa.37-38 In addition, the pH-dependent fluorescence

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spectra of SRhB and L-SRhB were investigated to evaluate the sensitivity effect from LS.

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As presented in Figure S11, LS almost showed no effect on the sensitivity of SRhB in

280

aqueous solution.

281

Besides sensitivity, selectivity of L-SRhB was also investigated. It is well known that the

282

ring-opening reaction is induced by Lewis acid. Therefore, different metal ions solutions

283

were prepared to study the L-SRhB response to various metal ions in aqueous solution. It

284

was obvious that all the common metal ions such as Na+, K+, Mg2+, Ca2+, Mn2+, Co2+,

285

Ni2+, Zn2+, Cd2+, Cr3+, Hg2+, Pb2+ exhibited no response at 5×10-4 M comparing with the

286

control test (Figure S12). In the case of Fe3+ and Cu2+ ion, a little L-SRhB could

287

flocculate with them at high concentration. It indicated that L-SRhB had a good

288

selectivity for pH detection.

289

Performance in photobleaching and photostability

290

Photobleaching and photostability makes against the sensitivity and stability of sensors

291

and are also two important factors to be considered. Meanwhile, considering the effect of


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pH, the photobleaching and photostability of our probe were measured in pH=5 and 6.2

293

buffer solution, respectively, at the concentration of 0.25 mg/mL under relatively strong

294

UV-irradiation (365 nm) of 12 W. It was noteworthy that the light power in the cell

295

imaging was 0.3 W.

296

The fluorescence intensities at about 400 nm and 580 nm, which were ascribed to LS and

297

rhodamine, respectively, were both monitored before and after difference time UV-

298

irradiation. A marked decrease of fluorescence intensity at 394 nm was observed,

299

especially when the pH value was 6.2 (Figure 4b). After UV-irradiation for 50 min, the

300

fluorescence intensity at 394 nm dropped about 40 % and 15 % in pH=6.2 and 5.0 buffer

301

solution, respectively (Figure 4a-4b). It indicated that LS showed an obvious

302

photobleaching at high pH solution under relatively strong UV-irradiation. The

303

comparative rate of photobleaching of L-SRhB at 394 nm was not linear and appeared

304

first quick back slow trend. Meanwhile, the emission at 577 nm of L-SRhB also slightly

305

decreased whether the pH value was 5 or 6.2. However, RhB showed a good stability

306

against photobleaching after 50 min UV-irradiation in the control experiment (Figure

307

S19). For the FRET system in L-SRhB, photobleaching of LS, which was the energy

308

donor in this system, resulted in a decreasing intensity at 577 nm and the changes of PL

309

intensity at 394 and 577 nm had the similar trend (Figure 4c-4d).39

310

In the experiment of photostability, L-SRhB showed a good photostability during

311

prolonged illumination (14 h) in the buffer solution of pH=6.2 under different

312

temperature from 25 to 60 oC (Figure 5b). However, in the buffer solution of pH=5, the

313

photostability at 566 nm decreased with the temperature increasing. After prolonged



314

illumination, the absorption intensity at 566 nm dropped 2 %, 18%, 33 % and 46 %, when

315

the ambient temperature was 25, 40, 50 and 60 oC, respectively.

316

Stability in the solution of cell culture media

317

To demonstrate that L-SRhB has a fine stability against agglomeration in cell culture

318

media, the UV-vis absorptions of L-SRhB in PBS (pH=7.4) solution at the concentration

319

of 0.25 mg/mL were measured after different storage times from 0 h to 72 h (Figure 5a).

320

The aggregation of lignin has a great influence on the absorption spectrum, which had

321

been reported by our previous studies.32-33 It was noteworthy that the absorption intensity

322

of L-SRhB at 280 nm was too large to get an accurate result at 0.25 mg/mL. The

323

absorption intensity at 300 nm was chose to determine the stability of L-SRhB solution.

324

Expectedly, after 72 h storage time, the absorption spectra almost had no changes at 300

325

nm comparing with the original state (see Figure 5a). It indicated that L-SRhB has a fine

326

stability against agglomeration in cell culture media because of the strong electrostatic

327

interaction and good solubility in PBS (pH=7.4) solution.

328

Cytotoxicity of L-SRhB and FL-SRhB

329

Encouraged by the pH-responsive property of L-SRhB, the biological application of the

330

probe was studied. Initially, we should investigate the biocompatibility and cytotoxicity

331

of the probe by MTS method as they are very important factors to be considered. For the

332

pH reversibility of L-SRhB, the ring-opening product (FL-SRhB) was also investigated in

333

the biological region.

334

Firstly, the cytotoxicity of LS was studied on HepG2 cells after 48 h incubation. It could

335

be seen in Figure 6a that the cell viability of LS was changed in the manner of

336

concentration of LS. Interestingly, the cell viability increased to 1.3 folds, when the


Page 16 of 40

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337

concentration of LS increased from 0.01 mg/mL to 0.5 mg/mL. Nevertheless, when LS

338

concentration was up to 1.0 mg/mL, the cell viability slightly decreased comparing with

339

the control experiment. It was proposed that the three-dimensional aggregation of LS

340

would induce the dynamic viscosity of cells at relatively high concentrations, which

341

would reduce the cell viability.40 This result indicated that LS exhibited a very low

342

cytotoxicity when the dose of LS was below 1 mg/mL.

343

Secondly, the cytotoxicity of L-SRhB and FL-SRhB was evaluated on HepG2 cells after

344

48 h incubation with different concentration from 0 to 1.0 mg/mL (Figure 6b). The

345

cytotoxicity of the probes on HepG2 cells showed that the toxicity was enhanced with the

346

increasing concentration from 0 to 1.0 mg/mL and the toxicity of L-SRhB was higher

347

than that of FL-SRhB during our test, especially in the 8 h cytotoxicity study (Figure 7).

348

FL-SRhB was synthesized by adding dilute HCl into L-SRhB solution to fully open the

349

corresponding spirolactam of SRhB, which would have no effect on the backbone of L-

350

SRhB, namely, LS. It indicated that the difference of cytotoxicity to cancer cells between

351

L-SRhB and FL-SRhB was not caused by LS. It was proposed that the remarkable

352

difference of cytotoxicity between L-SRhB and FL-SRhB was connected with the

353

structural difference introduced by the ring-opening of spirolactam of SRhB. The

354

cytotoxicity of the probes was relatively high after 48 h cell culture and the cell viability

355

was almost lower than 0.3 after 48 h cell culture at 0.25 mg/mL. Meanwhile, the two

356

probes could stain HepG2 cells quickly and it was no need for a 48 h cell culture with L-

357

SRhB and FL-SRhB.

358

Finally, in order to further evaluate the performance of L-SRhB and FL-SRhB in

359

biological application, the cytotoxicity of L-SRhB and FL-SRhB was studied on HL-



360

7702, SMMC-7721, SPC-A-1 cells and HepG2 after 8 h incubation with the

361

concentration of 0.25 mg/mL (Figure 7).

362

After incubation for 8 h, no apparent toxicity effect was found on normal and cancer cells

363

by FL-SRhB when the concentration was 0.25 mg/mL. On the contrary, L-SRhB showed

364

an obvious decrease of cell viability of cancer cells, while almost had no killing effect on

365

normal cells in the same condition. This phenomenon might be related with the difference

366

of cellular uptake ability between normal and cancer cells in 8 h cell culture. The strong

367

cellular uptake ability of cancer cells to L-SRhB resulted in the high cytotoxicity. The

368

mechanism was investigated and discussed in details in the following section.

369

Fluorescence imaging in Living cells

370

The cell imaging of L-SRhB and FL-SRhB was studied with the similar cell incubation

371

condition as cytotoxicity test by Confocal Laser Scanning Microscope (Figure 8, 9 and

372

S13-S18).

373

Generally, cells take in small particles (about 200 nm) through the endocytosis pathway.

374

In order to further determine that the internalization of L-SRhB and FL-SRhB were

375

mediated by endocytosis, the uptake experiment was performed at low temperature,

376

which was a classical endocytosis blocker (Figure 8). When HepG2 cells were incubated

377

at 4 oC, the uptakes of L-SRhB and FL-SRhB were significantly reduced and almost no

378

red fluorescence were observed in the cells, compared to the cells at 37 oC. It indicated

379

that the uptakes of the probes were mediated by endocytosis.

380

Rhodamine B is known as the mitochondria localization agent. However, the pH of

381

mitochondrion is alkalescent. The alkaline environment would have the negative effect

382

on the cell staining experiment because the pKa of the reversible ring-opening reaction of


Page 18 of 40

Page 19 of 40


383

L-SRhB is 5.35. In order to confirm whether our probes localized in mitochondria, the

384

colocalization imaging experiments of mitochondrion in HepG2 cells were performed

385

(Figure S18). It was obvious that the fluorescence brightness of Mito-Tracker Green

386

within the cells was relatively concentrated on the mitochondria. Obviously, partial

387

fluorescence overlap was detected on the mitochondrion between these of Mito-Tracker

388

Green and our two probes. It indicated that the ring-opening product of L-SRhB

389

accumulated in mitochondria. The underlying reason is the five-member ring of the

390

corresponding spirolactam was not closed in alkalescent environment. We proposed the

391

ring-opening product of L-SRhB with red fluorescence might be stabilized by the

392

interaction between -SO3- and quaternary ammonium salt, which came from LS and the

393

ring-opening product of L-SRhB, respectively, in alkalescent condition. Meanwhile, the

394

chemical bond between SRhB and lignosulfonate is stable and the rhodamine B will not

395

leak from our probes. As a result, despite our probe will also localize in the mitochondria,

396

there was little negative effect on the cell staining experiment.

397

In order to compare the difference of cell staining between normal cancer cells with our

398

probes, HL-7702, SMMC-7721, SPC-A-1 and HepG2 cell lines were chose in this work.

399

As expected, L-SRhB and FL-SRhB were internalized successfully by these four living

400

cell lines and could stain them after 12 h cell culture (Figure S15-S17). L-SRhB and FL-

401

SRhB would stain cancer cells (SMMC-7721, SPC-A-1) quickly in about 5 min owing to

402

the endocytic marker (Figure S14). The red fluorescence increased obviously with culture

403

time (Figure S15-S17). Despite L-SRhB and FL-SRhB had the ability to stain normal cell

404

(HL-7702), the red fluorescence from the probes was not visible until 12 h cell culture

405

and its staining time was much longer than that in cancer cells (Figure S15). However,



406

the pKa of L-SRhB is 5.35, which is valuable for studying the acidic organelles pH (about

407

4.50-6.00). Since both cancer and normal cells have acidic organelles, the probes could

408

stain all eukaryocyte in theory. It was proposed that the different staining time for cancer

409

and normal cells might mainly result from endocytic rate. The endocytosis of cancer cells

410

(SMMC-7721, SPC-A-1) was much stronger than that of normal cells (HL-7702) to the

411

probes. Meanwhile, the red fluorescence of probes increased with the decreasing pH

412

value. It is well known that cancer cells are more acidic than normal cells. This property

413

would also affect the difference in cell staining between cancer and normal cells.

414

Interestingly, L-SRhB and FL-SRhB could distinguish normal cells from cancer cells

415

owing to the different staining time. There was almost no red fluorescence in the normal

416

cells after incubation for 8 h with L-SRhB and FL-SRhB, respectively, while the obvious

417

red fluorescence from the probes was observed in the cancer cells in the same condition

418

(Figure 9). Our result provides an interesting design strategy for bio-imaging dyes based

419

on lignin, a renewable biomass.

420

In summary, we successfully synthesized a novel ratiometric fluorescent pH sensitive

421

probe by grafting SRhB to LS and gave positive results when tested the spectrum

422

response both in aqueous solution and in cell imaging. The ratio of fluorescence

423

intensities R (I594/I400) showed a 13.9-fold enhancement from pH 7.40 to pH 4.20 and

424

increased linearly with the pH value in the range of 6.20-4.60. The pKa of L-SRhB is 5.35,

425

which is valuable for studying the acidic organelles. In the biological application, L-

426

SRhB and FL-SRhB showed a good biocompatibility. In addition, the probes could

427

distinguish normal cells from cancer cells in the cellular stain test by different cell

428

staining time. Despite L-SRhB showed potential in the sensor and cellular uptake, there


Page 20 of 40

Page 21 of 40


429

were also many challenges on the commercialization process of LS-based probes owing

430

to the structural diversity, including the consistency and repeatability of performance. To

431

sum up, the probe proposed here demonstrated that a novel strategy was presented to

432

achieve the high-value utilization of lignin, and might be a promising ratiometric

433

fluorescent pH sensor and bioimaging dye for cancer cell.

434

Supporting Information Available

435

Supporting information included all the 1H/13C-NMR, FT-IR, HRMS spectra, elemental

436

analyses, bioimage, the response to ion metals and additional spectroscopic data. This

437

material is available at free of charge via the Internet at http://pubs.acs.org

438

ACKNOWLEDGMENT

439

The authors would like to acknowledge the financial support of the National Natural

440

Science Foundation of China (21402054, 21436004). The Fundamental Research Funds

441

for the Central Universities

442



443

REFERENCES

444

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lignin polymers. Ind. Crop. Prod. 2011, 33, 259-276.

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dispersing cellulase aggregates. Bioresource Techn. 2015, 185, 165-170.

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2012, 41, 1538-1558.

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efficient inverted perovskite solar cells with sulfonated lignin doped PEDOT as hole

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probe for Fe (III) and its application in living cells. Tetrahedron Lett. 2011, 52, 2840-

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fluorescent probes: rational design and bioimaging applications. Accounts Chem. Res.

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(20) Xu, M. Y.; Wu, S. Z.; Zeng, F.; Yu, C. M. Cyclodextrin supramolecular complex as

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a water-soluble ratiometric sensor for ferric ion sensing. Langmuir 2009, 26, 4529-4534.

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temperature imaging of living cells with polymeric ratiometric fluorescent thermometers.

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application in living cells. Chem. Commun. 2012, 48, 4486-4488.

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Birimzhanova, D.; Yu, Y.; Tang, B. Z. Fluorescent chemosensor for detection and

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Mao, L. Q. Ratiometric fluorescent probe based on gold nanoclusters and alizarin red-

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553



554

FIGURE CAPTIONS

555

Scheme 1 Synthesis of SRhB, E-LS, L-SRhB and FL-SRhB.

556

Figure 1 (a) The mechanism of ratiometric probe L-SRhB to H+ in aqueous media. (b)

557

Normalized absorption spectra (LS and Rhodamine B) and fluorescence spectrum (LS) in

558

PBS solutions (pH=7). The gray area was the overlap between the fluorescence spectrum

559

of LS and the absorption spectrum of Rhodamine B.

560

Figure 2 (a) Absorption spectra of L-SRhB in PBS buffers at various pH values. (b)

561

Absorption intensity at 566 nm in PBS buffers at various pH. Inset is the linear plots of

562

absorption intensity at 566 nm to pH (4.20-6.00).

563

Figure 3 Fluorescence spectra of L-SRhB in PBS buffers (0.2 mg/mL) at various pH

564

values. Inset is the linear plots of fluorescence intensity to pH (4.60-6.20). λex=330 nm.

565

Figure 4 The photobleaching of L-SRhB (0.25 mg/mL) in the buffer solution of pH=5 (a)

566

and 6.2 (b) at 25 oC, respectively, on irradiation at 365 nm with an ultraviolet lamp (P=12

567

W); Changes in the PL intensity at 394 nm (c) and 577 nm (d), respectively, with

568

irradiation at 365 nm in different time; ΔI=(I-I0 min)/I0 min. λex=330 nm.

569

Figure 5 (a) The stability of L-SRhB (0.25 mg/mL) in the solution of cell culture media

570

(PBS, pH=7.4) and the absorption intensity at 300 nm after different storage times from 0

571

h to 72 h, respectively. (b) The photostability of L-SRhB (0.25 mg/mL) at 566 nm, on

572

irradiation at 365 nm with an ultraviolet lamp (P=12 W).

573

Figure 6 (a) Cytotoxic effect of lignosulfonate at different concentrations in HepG2 cells.

574

(b) Cytotoxic effects of L-SRhB and FL-SRhB in HepG2 cells. HepG2 cells (1×105

575

cells/mL) were cultured with different concentration of L-SRhB and FL-SRhB for 48 h,

576

respectively. 28 ACS Paragon Plus Environment

Page 28 of 40

Page 29 of 40


577

Figure 7 Viability of HL-7702, SMMC-7721, SPC-A-1 and HepG2 cells in L-SRhB and

578

FL-SRhB solution at 0.25 mg/mL after 8 h. All the data were obtained from three

579

independent experiments (p

Fluorescent pH-Sensing Probe Based on Biorefinery Wood

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