Self-Immolative Fluorescent and Raman Probe for Real-Time Imaging

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Biological and Medical Applications of Materials and Interfaces

A Self-Immolative Fluorescent and Raman Probe for Real-Time Imaging and Quantification of #-Glutamyl Transpeptidase in Living Cells Yang Zhang, Gengwu Zhang, Peng Yang, Basem Moosa, and Niveen M. Khashab ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.9b07186 • Publication Date (Web): 10 Jul 2019 Downloaded from pubs.acs.org on July 17, 2019

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ACS Applied Materials & Interfaces

A Self-Immolative Fluorescent and Raman Probe for Real-Time Imaging and Quantification of γ-Glutamyl Transpeptidase in Living Cells Yang Zhang,† Gengwu Zhang,† Peng Yang,† Basem Moosa,† Niveen M. Khashab*,† †Smart

Hybrid Materials Laboratory (SHMs), Advanced Membranes and Porous Materials Center,

King Abdullah University of Science and Technology (KAUST), Thuwal 23955-6900, Saudi Arabia

KEYWORDS: plasmonic nanomaterials, sensor, fluorescence enhancement, SRES, cancer cell

ABSTRACT: Characterizing over-expressed enzymes or biomarkers in living cells is critical for the molecular understanding of disease pathology and consequently for designing precision medicines. Herein, a “switch-on” probe is designed to selectively detect γ-glutamyl transpeptidase (GGT) in living cells via a unique ensemble of enhanced fluorescence and surface-enhanced Raman scattering (SERS). In the presence of GGT, the γ-glutamyl bond in the probe molecule is cleaved, thereby activating a fluorescent probe molecule as well as a Raman reporter molecule. Consequently, the detection of GGT is achieved based on both plasmonic fluorescent enhancement (PFE) and SERS with a limit of detection as low as 1.2 x10-3 U/L (normal range for GGT levels in the blood is 9-48 U/L). The main advantage of this platform is that on the occasion of fluorescence signal interference, especially in the presence of free metal ions in cells, the SERS signals still hold high stability as a backup. This work highlights the benefits of marrying two complimentary sensing techniques into one platform that can overcome the major obstacles of 1

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real-time biomarkers detection and imaging in living cells. INTRODUCTION Enzymes constitute the major class of natural biomarkers in our bodies for catalyzing more than five thousand biochemical reactions with specificity and fleetness.1-3 γ-Glutamyl transpeptidase (GGT),4 a cell-surface-bound enzyme, has been recognized as a potential biomarker of malignant tumors as it selectively catalyzes the cleavage of γ-Glutamate (γ-Glu) in glutathione (GSH) and other γ-glutamyl compounds.5-9 The activity of GGT is intimately related to many essential physiological processes as well as a vast range of diseases (e.g., asthma, diabetes and cancers).10-11 Recent advances have disclosed an aberrant expression of GGT in a certain type of cancers such as liver, ovarian, and cervical and its possible role in the process of drug resistance of cancer cells.12 As a result, real-time and accurate detection of GGT in living cells can be highly valuable to improve the diagnosis and predict possible drug resistance activity. To date, a multitude of fluorescence imaging probes for the detection of GGT activity have been designed by the linkage of a GGT activated site.13-16 Other than the common hurdles for live-cell imaging such as photo-stability, cell penetration, and specific binding to the biomolecule of interest, probes with high sensitivity that are suitable to detect the low abundant GGT in complex biological media are still rare. Anchoring probes by a near-infrared fluorescence (NIR) moiety to overcome the shortcoming of a proper excitation wavelength have resulted in low photo-thermal damage and high cell penetration.13,

17

However, the low brightness of NIR fluorescence,18-19

encouraged more research on plasmon enhanced fluorescence (PEF) as an effective technique to greatly intensify the fluorescence signals thereby improving the sensitivity of detection considerably.20-24 This signal boosting effect can be attributed to the enhanced fluorescence via the 2


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interaction of fluorophores with the localized plasmons.25-27 Generally, such an enhancement originates from two paths, namely, the excitation and emission enhancement by means of increasing light absorption or altering the non-radiative and radiative decay rates of a molecule.28-29 These are contingent upon i) position and orientation of the molecules; ii) the local field from the plasmonic structures; and iii) the extent of overlap between the spectra of the plasmonic structure and the fluorescent molecule.30-31 An alternative method to overcome photobleaching and narrow emission peaks is to employ surface-enhanced Raman scattering (SERS), which has been recognized as a powerful tool for sensing and detection.32-34 Nonetheless, SERS bio-application remained largely limited due to the overlap of the bands of most of the Raman reporters with the biological system interface. Most recently, alkyne, azide, cyano and carbon deuterium (C-D) bonds have shown unique Raman emission in the cell Raman-silent region (around 1800 cm-1 to 2800 cm-1),35 where the Raman signals of the interface substrate is negligible without improving much on the detection speed that remains quite slow as compared to fluorescence-based techniques.36 Thus, leveraging the advantages of SERS (higher molecular recognition ability and photostability) and fluorescence (higher detection speed) techniques would evolve a new generation of smart or switchable nano-probes that hold better chances for translation and actual clinical applications.34, 37-38 In this work, a switchable γ-glutamyl transpeptidase probe based on both fluorescence and SERS platforms is prepared and mounted on silica-protected gold nanorods (AuNRs) (Scheme 1). The fluorescence is originally quenched until the γ-glutamyl bond in the probe molecules is cleaved and the self-immolative fluorescent probe (FP) is released (Scheme 1 in purple). As a result, the NIR fluorescence is switched on with significant enhancement via plasmon coupling. 3



Simultaneously, the spontaneous intramolecular cyclization yields a cyano functionalized Raman reporter (RR) (Scheme 1 in green). The RR is readily absorbed by the silica layer to interact with the gold core, by virtue of its sulfur moiety, giving rise to quantitative SERS signals in the cell Raman-silent region. To the best of our knowledge, this is the first “turn-on” sensing and imaging platform in living cells based on plasmon prompted fluorescence enhancement as well as Raman signals in the cell Raman-silent region.

Scheme 1. Schematic illustration of the “turn-on” probe for detecting γ-glutamyl transpeptidase (GGT) based on PEF and SERS techniques.

RESULTS AND DISCUSSION The AuNRs were synthesized following a seed-mediated method with homogenous structure (Figure S1a), displaying an average length of ~49.1 nm and a diameter of ~18.5 nm (Figure S2), with an aspect ratio of 2.6. AuNRs possess a great advantage that their localized surface plasmon resonance can be easily tuned from the visible to the NIR range by tailoring their aspect ratio.39 UV-vis spectra showed that the gold nanorods have a plasmon band maximum at ~680 nm (Figure S3). The AuNRs were then coated with mesoporous silica according to a reported procedure with minor modifications (Figure S1b). 40 The thickness of the silica shell was modulated by adjusting 4


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the concentration of CTAB mentioned in the experimental section. Here, the thickness was tuned to ca. 16 nm in order to get the best enhancement (Figure S4). After the silica coating, a slight red shift of the absorbance peak is observed resulting from the change of refractive index around the AuNRs (Figure S3).40 The mesoporous silica-coated AuNRs were then modified with azide groups to click the probe molecules (Scheme 2a). The zeta-potential measurements support the azide functionalization of the mesoporous silica-coated AuNRs (Figure S5). Another proof of the azide modification was displayed by Fourier-transform infrared spectroscopy (FTIR) (Figure S6). The nanorods were washed and centrifuged many times to make sure that the free azide was removed. A new peak was observed at ~2100 cm-1 in the spectrum of azide-modified AuNRs further confirming the azide modification. The probe molecule is designed with a GGT cleavable bond

41-42

and a self-immolative

backbone that affords dually a fluorescent probe (FP) and a Raman reporter (RR). The probe molecule is clicked on AuNRs via a two-steps functionalization method (P1 and P2) as outlined in Scheme 2b. The detailed synthetic and characterization procedures for all compounds are included in the ESI. The successful surface modification of mesoporous silica-coated gold nanorods was confirmed by the FTIR spectrum (Figure S6). The peaks at 1500 cm-1 were associated with the characteristic peaks of the aromatic ring, indicating the successful functionalization of mesoporous silica-coated gold nanorods with P1. The disappearance of the signal at ~2100 cm-1 also indicated the consumption of azide groups by cyclization. After the P2 connection (Scheme 2b), new peaks appeared at around 1600 cm-1 corresponding to the carbonyl bonds from P2, and the characteristic peak of the cyano group was observed at ~2200 cm-1. After the successful functionalization of the probe molecules, the obtained AuNRs showed slight fluorescence due to the hydroxyl acylation of 5



FP (Figure S7).

Scheme 2. (a) Preparation of AuNRs with mesoporous silica coating and azide functionalization. (b) Further modification of AuNRs with probe molecules, consisting of two steps (P1 and P2 connection).

Many research groups have studied the interaction between the plasmon resonance and fluorophores.43-45 It was reported that the fluorescence enhancement optimally relies on the stronger overlap between the absorption/emission spectrum of the fluorophores and the surface plasmon peak of the plasmonic materials employed.46-47 AuNRs with a specific aspect ratio of 2.6 was chosen for this system. As shown in Figure 1a, the normalized emission spectrum of FP overlapped well with the surface plasmon absorption spectrum of AuNRs. With the purpose of evaluating the PEF of the probe functionalized AuNRs, the fluorescence intensity of FP alone in a water suspension was carried out as a control sample. The number of FP molecules linked to the mesoporous silica shell was calculated through the absorption of the combined supernatant. The total concentration of FP on AuNRs was estimated to be almost the same as that of the free molecules in the water suspension. The fluorescence enhancement was observed by a factor of 5.6 6


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times at a silica thickness of ~16 nm (Figure 1b).

Figure 1. (a) Absorption (green) and emission (red) spectra of FP in water. The Uv-vis spectra of AuNRs was labeled by a shaded curve. (b) Fluorescence spectra of probe AuNRs (green line) and that of the control sample with the same amount of FP in water (black line). (c) Fluorescence response of probe AuNRs (5 μg / mL) to GGT at different concentrations following incubation at 37 oC at for 30 min. (d) The linear relationship of fluorescence intensity (λem = 710 nm) toward the concentration of GGT from 0-1 U / L.

The system was then tested in the presence of GGT in PBS buffer and the response was recorded by measuring the fluorescence spectrum. Upon interaction with GGT, the γ-glutamyl bond in the 7



probe molecules is cleaved, which triggered a spontaneous intramolecular cyclization, thus liberating the FP that has NIR fluorescence at ~710 nm. The fluorescence emission at ~710 nm increased with the incubation time, and an approximate plateau was reached after 30 mins (Figure S8). The probe AuNRs were incubated with different concentrations of GGT in PBS buffer for 30 mins at 37 oC, and the resulting fluorescence spectra were obtained (Figure 1c). As shown in Figure 1c, without GGT, probe AuNRs were very stable in PBS buffer after 30 mins with negligible fluorescence. After the incubation with GGT, the intensity of the signal at ~710 nm is enhanced with increasing the concentration of GGT. The plot of fluorescence intensity at ~710 nm against the concentration of GGT showed a linear relationship in the range of 0-1 U/L (Figure 1d). The limit of detection was calculated to be ~1.2 x10-3 U/L (based on 3 δ/s, where s is the slope of the linear equation and δ is the standard deviation of 15 times of blank measurements), which is significantly sensitive (Table S1) to detect the amount of GGT in serum of cancer patients (5-85 U/L).48 The selectivity of the probe AuNRs was evaluated by testing a variety of substrates including inorganic salts (NaCl, KCl, MgCl2, ZnSO4), glucose, glutathione, 10% fetal bovine serum (FBS), trypsin, and reactive oxygen species (H2O2). As shown in Figure 2, the probe AuNRs exhibited exclusive selectivity to GGT.

8


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Figure 2. Fluorescence response of the probe AuNRs (5 μg / mL)to different substrates. KCl (150 mM), NaCl (150 mM), MgCl2 (2.5 mM), ZnSO4 (0.1 mM), glucose (10 M), glutathione (2 mM), FBS (0.1%, v/v), trypsin (0.05 U / L), H2O2 (10 mM), GGT (0.4 U / L). The applicability of this probe as a SERS sensor for detecting GGT was first investigated in aqueous solution. In the presence of GGT, the RR with the cyano group is released which showed signals in the cell Raman-silent region. The presence of sulfur together with the cyano group accelerates the absorption of RR on AuNR resulting in the SERS signals of a cyano group at ~2250 cm-1, as shown in the SERS spectra (Figure 3a). With increasing of the concentration of GGT, the peak intensity at ~2250 cm-1 increased. There was a linear correlation between the peak intensity and the concentration of GGT over the range 0-60 U/mL, with a limit of detection of 0.095 U/mL (based on 3 δ/s) (Figure 3b). This limit of detection is orders of magnitude higher than the fluorescence method due to the relatively weak Raman scattering response of the cyano group. However, the SERS method showed better stability than the fluorescence method in the presence of metal cations especially Cu2+ which drastically interferes with the fluorescent signal as demonstrated in figure S9.

9



Figure 3．(a) SERS spectra of probe AuNRs (10 μg / mL) upon the addition of GGT at different concentrations following incubation at 37 oC for 60 min. (b) The linear fitting curve of SERS intensity to the concentration of GGT.

HepG2 is a cancer cell line that overexpresses GGT. Thus, HepG2 cells were chosen as a model cell system to investigate the feasibility of the probe AuNRs to image GGT in cells. First, a cell viability test was performed with HepG2 cancer cells to evaluate the toxicity of the probe AuNRs. After increasing the concentration of probe AuNRs, cell viability decreased negligibly, indicating the good biocompatibility of the probe AuNRs (Figure 4a). The HepG2 cells were then incubated with the probe AuNRs for 1h at 37 oC, after that the NIR fluorescence inside cells was obtained. As shown in figure 4b, the HepG2 cells showed intracellular fluorescence after incubation with probe AuNRs. In contrast, when NIH-3T3 cells with lower expression of GGT were pretreated with probe AuNRs at the same conditions, as a control sample, the fluorescence was negligible (Figure S10). Raman imaging was then performed in the live HepG2 cells. As it can be seen from figure 4c, the SERS signals at ~2250 cm-1 marked with the green color indicated the overexpression of GGT in HepG2 cells, which showed excellent localized quantification for screening of GGT in live cells at real-time. 10


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Figure 4. (a) Cell viability of HepG2 cancer cells after treatment with different concentrations of probe AuNRs with incubation time of 12 h at 37 oC. (b) Confocal fluorescence images of HepG2 cells after incubation with probe AuNRs. (c) SERS images of HepG2 cancer cells after incubation with probe AuNRs, the cell area was outlined by red lines.

CONCLUSIONS A smart “turn-on” nano-probe has been assembled for the sensitive detection of GGT in live cells. This unique platform combines the advantages of fluorescence together with Raman by designing a self-immolative molecule that provides signals that can be accurately decoded by these two techniques. In the presence of GGT, the probe molecule is able to convert into a NIR fluorescent probe (FP) with a remarkable fluorescence enhancement (λem, 710 nm), accompanied by the release of a Raman reporter (RR) to produce a SERS signal at ~2250 cm-1. This probe displayed NIR fluorescence with high sensitivity in solution as well as in HepG2 cancer cells. Simultaneously, the probe AuNRs can yield Raman signals in the cell Raman-silent region without any interference. This stimulus responsive platform combines the unique advantages of 11



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both fluorescence (fast detection speed and very low detection limit) and SERS (high stability and anti-interference) techniques, promising a feasible outlet for direct medical applications and clinical diagnosis.

METHODS Materials. Gold(III) chloride hydrate (HAuCl4·xH2O), sodium borohydride (NaBH4), sodium azide,

sodium

hydroxide,

hexadecyltrimethylammonium

silver

nitrate,

bromide

L-ascorbic

(CTAB),

acid,

sulfuric

tetraethoxysilane

acid, (TEOS),

(3-iodopropyl)trimethoxysilane, acryloyl chloride, Boc-Cys(Trt)-OH, γ-Glutamyltranspeptidase, 3-amino-propionitrile,

Boc-Glu-OtBu,

1-bromo-3,5-dimethoxybenzene,

IR780-iodide,

trimethylamine and all other chemicals are purchased from Sigma Aldrich. All solvents are obtained from VWR chemicals. All chemicals were used without further purification. Deionized water (Millipore Milli-Q grade) prepared in the lab, was used in all experiments, with a resistivity of 18.2 MΩ. The TEM samples were prepared by drop AuNRs on carbon-coated Cu grids. Transmission electron microscopy (TEM) was acquired on

FEI machine operating at 200 kV. The size of

AuNRs was calculated by using ImageJ software (150 AuNRs). UV-vis spectra were obtained from SHIADZ UV-vis spectrophotometer (UV-2600). Fluorescence spectra were detected by using a VARIAN fluorescence spectrophotometer. Zeta potential was measured by a Zeta Sizer produced by Malvern Instruments. Raman spectra and imaging were carried out on a Raman spectrometer (Horiba Jobin Yvon, Labram Aramis) equipped with a 785 nm excitation wavelength (32 mW laser power). Confocal fluorescence imaging was obtained using a Zeiss LSM 710 12


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upright confocal microscope. Synthesis of gold nanorods. The gold nanorods were synthesized based on a seed mediated method as reported earlier with a slight modification.49-50 Firstly, the seed solution was prepared by adding 100 μL of HAuCl4 (24 mM) into 7.5 mL of CTAB (0.1 M), then 0.6 mL of ice-cold NaBH4 (0.01 M) was injected under vigorous stirring for 2 minutes. The seed solution was kept for 2-3 h before use. Lastly, 240 μL of the seed solution was added into a growth solution prepared by mixing 2 mL of HAuCl4 (24 mM), 2 mL of H2SO4 (0.5 M), 450 μL of AgNO3 (10 mM), and 800 μL of L-ascorbic acid (0.1 M) in 100 mL of CTAB (0.1 M). The mixture was kept at 30 oC for 12 h and purified by centrifugation. Synthesis of silica-coated gold nanorods. The stöber method was followed with minor modifications.51-53 The gold nanorods were centrifuged with a final CTAB concentration of 1 mM and a 3.5 times increase in the concentration of gold nanorods, while compared with the stock solution before centrifugation. NaOH (0.1 M, 40 μL) was added to 10 mL of gold nanorods to adjust the pH to ~10.5, and the solution was stirred for 30 min. TEOS (90 μL, 20% in methanol) was added in 3 batches at an interval of 30 min. Then the solution was stirred gently for 24 h at room temperature. The nanorods were purified with centrifugation and dispersed into 5 ml of ethanol. Synthesis of azide-functionalized silica-coated gold nanorods. 3-azidopropyltrimethoxysilane was

synthesized

from

(3-Iodopropyl)trimethoxysilane.50

Then,

150

μL

of

3-azidopropyltrimethoxysilane (25% in DMF) was added to 5 mL of the silica-coated gold nanorods in ethanol. The solution was heated to reflux for 12 h. The AuNRs were purified by centrifugation to remove the excess azide. 13



Probe molecule attachment. P1 (100 μL, 1 mg/ mL) was added to 2 mL of the silica-coated gold nanorods with azide groups in H2O. Then 0.1 mL of CuSO4 (0.01 M) and 0.2 mL of sodium ascorbate were added and stirred for 24 h. The solution was centrifuged and washed with sodium citrated solution, then suspended in 2 mL of methanol. The supernatant was collected to calculate the amount of P1 attached to the nanorods by UV-vis spectra. The P1 attached nanorods (2 mL) were added to the mixture of NaHCO3 with P2 (10 mg) in water (4 mL) and the mixture was stirred for 36 h at room temperature. The final probe AuNRs were purified by centrifugation. Fluorescence detection of γ-glutamyl transpeptidase. Enzyme buffer (U/L, pH=7.4, PBS, 1X) was added to the solution of probe AuNRs, then incubated at 37 oC. Different concentrations of the enzyme buffer (U/L, pH=7.4, PBS, 1X) were added to the solution of probe AuNRs (5 μg/mL), then incubated at 37 oC for 30 min. The fluorescence spectra were obtained with different concentration of GGT. SERS detection of γ-glutamyl transpeptidase. Different concentrations of γ-glutamyl transpeptidase were prepared freshly with PBS buffer. In a 0.5 mL tube, 50 μL of γ-glutamyl transpeptidase solution was mixed with 250 μL of probe AuNRs (10 μg/mL). The mixture was incubated at 37 oC for 1h. Finally, the mixture solution was concentrated and transferred into capillary tubes for SERS detection. Fluorescence imaging of γ-glutamyl transpeptidase in living cells. Two cell lines (HepG2, NIH-3T3) were selected, which overexpress and lower-express γ-glutamyl transpeptidase, respectively. Cells in Dulbecco’s modified Eagle’s medium (DMEM) (with 10% fetal bovine serum (FBS), 1% penicillin-streptomycin) were seeded on a cover glass and allowed to adhere 14


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overnight at 37oC in an incubator containing 5% CO2 and 95% humidity. Then the cells were incubated with the culture medium containing probe AuNRs (5 μg/mL) for 1 h. After that, the medium was removed and the cells were washed with phosphate-buffered saline solution (PBS, 1X) three times and then fixed with 4% paraformaldehyde (PFA) for 20 mins followed by washing with PBS three times. Finally, the cover glass was fixed on a slide for cell imaging. SERS imaging of γ-glutamyl transpeptidase in living cells. HepG2 cells were cultured in DMEM medium (with 10% FBS, 1% penicillin-streptomycin) and maintained in an incubator at 37 oC containing 95% humidity and 5% CO2. Then the cells were seeded on a cover glass and allowed to adhere for 12h followed by incubation with medium containing probe AuNRs (10 μg/mL) for 2 h. After that, the medium was removed and the cells were washed with PBS (1X) three times and then fixed with 4% PFA for 20 mins followed by washing with PBS three times. Finally, the cover glass was fixed on a slide for Raman mapping. Cell viability assay. The biocompatibility of nano-probe was obtained by the cell counting kit-8 (cck-8) assay. HepG-2 cells were seeded in 96-well plates and incubated for 24 h at 37 oC. Then 100 μL of various concentrations of the probe were added and incubated for another 12 h. Finally, 10 μL of CCK-8 solution was added to each well of the plate and incubated for an extra 2 h. The absorbance at 450 nm was measured by using a microplate reader.

ASSOCIATED CONTENT Supporting Information. The supporting information is available free of charge on the ACS publications website at DOI: Structure characterization of AuNRs (TEM), UV-vis and fluorescence spectrum. The synthetic 15



procedures of P1 and P2, and their characterization. AUTHOR INFORMATION Corresponding Author *Email: [email protected]. Tel: +966 2 808-2410. Fax: +966 2 802-1172. ORCID Basem Moosa: 0000-0002-2350-4100 Niveen M. Khashab: 0000-0003-2728-0666 Notes The authors declare no competing financial interest. ACKNOWLEDGMENT We thank King Abdulaziz City for Science and Technology (KACST) for their generous funding through the MERS-CoV research grant program (number 20-0004), which is a part of the Targeted Research Program (TRP). REFERENCES (1) Johnson, C. H.; Ivanisevic, J.; Siuzdak, G. Metabolomics: Beyond Biomarkers and Towards Mechanisms. Nat. Rev. Mol. Cell Biol. 2016, 17, 451. (2) Cohen, L.; Walt, D. R. Highly Sensitive and Multiplexed Protein Measurements. Chem. Rev. 2019, 119, 293-321. (3) Gillette, M. A.; Mani, D. R.; Carr, S. A. Place of Pattern in Proteomic Biomarker Discovery. J. Proteome Res. 2005, 4, 1143-1154. (4) Hu, X.; Legler, P. M.; Khavrutskii, I.; Scorpio, A.; Compton, J. R.; Robertson, K. L.; Friedlander, A. M.; Wallqvist, A. Probing the Donor and Acceptor Substrate Specificity of the Gamma-Glutamyl 16


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Self-Immolative Fluorescent and Raman Probe for Real-Time Imaging

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