One-step synthesis of peptide conjugated gold nanoclusters for the high expression of FGFR2 tumor targeting and imaging

Abstract

An effective method to synthesize gold nanoclusters that can specifically recognize fibroblast growth factor receptor2 (FGFR2) was reported. We designed a new peptide sequence, cysteine–cysteine–tyrosine–leucine–glutamine–leucine–glutamine–alanine–glutamic–glutamic–arginine–NH₂, and then used it as the precursor to synthesize red luminescent gold nanoclusters (GNCs) in one step. In order to better understand the properties of the GNCs, ultraviolet-visible spectra, transmission electron microscopy, Fourier transform infrared spectroscopy, X-ray photoelectron spectroscopy and matrix-assisted laser desorption/ionization time of flight mass spectrometry were used to characterize the GNCs. We also successfully applied the GNCs in fluorescence imaging of esophageal cancer KYSE510 cells that express in a high level of the FGFR2. The results demonstrated that the GNCs possess good luminescence, high stability, nontoxicity and good biocompatibility. We expect that the GNCs could find more applications in overexpression of the FGFR2 tumor cells imaging.

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Introduction

The family of fibroblast growth factor receptors (FGFRs) that contains four closely related members FGFR1–4 is considered to contain important biomarkers. It had been found that the FGFRs expressed abnormally in breast cancer, prostate cancer, gastric cancer, and bladder cancer.¹ For example, the FGFR1 and the FGFR2 were amplified in triple negative breast cancer;² the FGFR3 was high expression in bladder cancer;³ and the FGFR4 was high expression in prostate cancer too.⁴ Therefore, targeting the FGFRs for tumor imaging and therapy could be an important research subject. Some traditional therapeutic strategies aiming at interfering with FGFR or FGF activity had been developed, which included small-molecule tyrosine kinase inhibitors, monoclonal antibodies and FGF ligand traps.⁵ In recent years, scientists try to introduce nanotechnology for the FGFR2 high expression tumor imaging and therapy. Anna et al.⁶ employed a FGF1 mutant that can target the FGFRs and used it to synthesize gold nanoparticles (FGF1_v–AuNPs) for the first time. Their results showed that the FGF1_v–AuNPs were uptake specifically by FGFRs high expression tumor cells and also had a good therapeutic effect under infrared irradiation on tumor cells. Their work demonstrated that the nanomaterial technology could be effective in this field but there is room for improvement. For example, it involved a multi-step process to make the gold nanoparticles at first and then modified them with the FGF1 mutants, which makes the process of preparation complicated and expensive. Furthermore, gold nanoparticles are not fluorescent themselves and they need complicated modification for fluorescent imaging to tumor cells. Therefore, it is valuable to develop a one-step method to synthesize fluorescent gold nanoclusters with targeting ability. Compared with gold nanoparticles, gold nanoclusters possess smaller particle size, lower toxicity, better biocompatibility and more excellent luminescent properties. And previous research results^7,8 also showed that it is feasible to synthesize gold nanoclusters by using one-step method.

In this paper, we report a one-step method for the synthesis of gold nanoclusters that can specifically target the FGFR2 high expression tumor cells. We used a new peptide drug,⁹ which can specifically target the FGFR2 high expression tumor cells, as template to design a new peptide sequence cysteine–cysteine–tyrosine–leucine–glutamine–leucine–glutamine–alanine–glutamic–glutamic–arginine–NH₂ (CCYLQLQAEER–NH₂). The peptide contains two functional domains, the CCY and the LQLQAEER–NH₂ (we named it JNP5 for short). The CCY can play the role of both reducing and protecting agents in the synthesis process,^7,10 and the JNP5 can specifically target the FGFR2 high expression tumor cells.⁹ We synthesized red luminescent gold nanoclusters which conjugated with the CCYLQLQAEER–NH₂ (abbreviated as CCYJNP5). For the convenience of narrating, we named the gold nanoclusters CCYJNP5–GNCs. The CCYJNP5–GNCs were applied in the fluorescence imaging of the esophageal cancer KYSE510 cells. To the best of our knowledge, our approaches were reported for the first time.

Experimental procedures

Chemicals

All the chemicals were of reagent grade without further purification. HAuCl₄·3H₂O was purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Peptides JNP5 and CCYJNP5 were purchased from China Peptides Co., Ltd. (Shanghai, China). Sodium hydroxide was purchased from Damao Chemical (Tianjin, China). The esophageal tumor KYSE510 cell line and the human embryonic kidney 293A cell line were provided by Institute of Biomedicine & Cell Biology Department of Jinan University, P. R. China. Dulbecco Modified Eagle's Medium (DMEM) and RPMI-1640 were purchased from Gibco-BRL Life Technologies. 3-(4,5-Dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) and Cytotoxicity Assay kit were supplied by Omega Bio-Tek. Bovine Serum Albumin (BSA) was purchased from Sigma. Ultra pure water of 18.0 MΩ was used for all experiments.

Preparation of the CCYJNP5–GNCs and the BSA–GNCs

For the synthesis of the CCYJNP5–GNCs, 1 mL of 4.428 mM HAuCl₄ solution was added into the same amount of the aqueous solution of the CCYJNP5 peptides under magnetic stirring. At the same time, the colorless solution turned deep yellow. When adjusted the pH to 11–12 by adding the sodium hydroxide solution (130 μL, 1 M), the color of the solution changed from deep yellow to pale yellow. Finally, the brown CCYJNP5–GNCs were formed after 18 h under magnetic stirring at room temperature. The solution containing the CCYJNP5–GNCs was dialyzed with a dialysis membrane (8000 molecular weight cutoff) for 12 h to remove excess reactants. In the end, the product powder was obtained by the freeze drier. The BSA stabilized Au clusters (BSA–GNCs) were fabricated using the simple, one-step synthetic route which was reported before.¹¹ In a typical experiment, HAuCl₄ solution (5 mL, 10 mM) was added to BSA solution (5 mL, 50 mg mL⁻¹) under vigorous stirring at 37 °C for 12 h. During this period, the color of the solution changed from light yellow to deep brown. The solution containing the BSA–GNCs was dialyzed with a dialysis membrane (8000 molecular weight cutoff) for 12 h to remove excess reactants. In the end, the product powder was obtained by the freeze drier.

UV, FTIR, fluorescence, TEM, and XPS spectroscopic studies

The ultraviolet-visible (UV) spectra were measured on a UV 3150 Spectrophotometer (Shimadzu, Japan) using 1 cm path length quartz cuvettes with a resolution of 0.1 nm. Nicolet Avatar FTIR model 330 spectrometer (Thermo, America) was used to measure Fourier Transform Infrared (FTIR) spectra of the CCYJNP5 and the CCYJNP5–GNCs in the range from 500 cm⁻¹ to 3800 cm⁻¹. Fluorescence spectra were taken on a RF-5301 Fluorospectro-photometer (Shimadzu, Japan). The measurements of the quantum yield and the fluorescent lifetime were both performed on FLS980 photoluminescence spectrometer (Edinburgh Instruments Ltd). The quantum yield was obtained by measuring the intensity of emitting fluorescence and absorption light through the integrating sphere. Transmission electron microscope (TEM) samples were prepared by dropping the CCYJNP5–GNCs solution onto a carbon-coated copper grid, drying at room temperature, and then characterized by a TEM (JEM-2100F, JEOL, Japan) operated at an accelerating voltage of 200 kV. X-Ray photoelectron spectroscopy (XPS) measurements were carried out on an ESCALab250 X-ray photoelectron spectroscopy (Thermo, America).

MALDI-TOF MS measurement

The matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass measurement of the CCYJNP5–GNCs was obtained on a Bruker ultrafleXtreme MALDI-TOF/TOF system (Bruker Daltonics, America) in positive mode with 2,5-dihydroxybenzoic acid as the matrix.

Cell culture

The KYSE510 and 293A cells were cultured in 1640 and DMEM medium with 10% Fetal Bovine Serum (FBS), respectively. All cell lines were maintained at 37 °C in an atmosphere of 5% CO₂ for 24 h. The cells were collected and implanted onto the confocal dishes, and the culturing was continued for one day.

Cytotoxicity assay

Cytotoxicity assay were carried out to evaluate the potential cytotoxicity of the CCYJNP5–GNCs in normal cells and cancer cells. Equal amounts of KYSE510 and 293A cells (5 × 10³ cells per well) were seeded onto each well of 96-well plates and incubated for 24 h under culture conditions. Then the CCYJNP5–GNCs concentrations 2.5, 1.2, 0.62, 0.31, 0.16 mg mL⁻¹ were added to each well of 96-well plates and incubated for at 37 °C for 1 h, respectively. The cells without adding of the CCYJNP5–GNCs were used as the control group and their viabilities were set as 100%. A volume of 10 μL of the MTS was added to each well and incubated for at 37 °C for 30 min. The optical density (OD) values at 490 nm were measured with a microplate reader (Diatek). Each concentration was assayed for three times and the final values were expressed as a percentage of the control.

Targeting ability evaluation of the CCYJNP5–GNCs

The KYSR510 and 293A cells were seeded in confocal dishes at a density of 2.5 × 10⁴ cells per dish and subsequently incubated in culture medium for 48 h (the medium was changed every 24 h). Both the KYSE510 and 293A cells were incubated with 1 mL solutions of the CCYJNP5–GNCs (2.5 mg mL⁻¹) for 1 h, respectively. In addition, the KYSE510 cells were also incubated with 1 mL solutions of the BSA–GNCs (2.5 mg mL⁻¹) for 1 h. Subsequently, these cells were washed with phosphate buffer solution (PBS) for three times to remove free and physically absorbed GNCs. Then these cells were imaged by a confocal fluorescence microscopy (LEICA TCS-SP5, laser: 405 nm, detector: HyD) at 20 × 10 × 4 magnification. All the experiments were performed under the same conditions that the percentage of the voltage value was 208.9% and the size of the pinhole was 60.7 μm.

Competitive inhibition assay

The competitive inhibition control experiments of the KYSE510 were also performed by culturing cells with free CCYJNP5 peptides (1 mL, 100 μg mL⁻¹) for 1 h primarily and then with the CCYJNP5–GNCs (2.5 mg mL⁻¹) for 1 h. Finally, these cells were washed with PBS for three times and tested by a confocal fluorescence microscopy (LEICA TCS-SP5, laser: 405 nm, detector: HyD) at 20 × 10 × 4 magnification. All the experiments were performed under the same conditions that the percentage of the voltage value was 208.9% and the size of the pinhole was 60.7 μm.

Results and discussion

Synthesis and characterization of the CCYJNP5–GNCs

Fig. 1A and B show the results by observing the as-prepared solution in room light and under a UV light source emitting 365 nm light, respectively, which indicates that the CCYJNP5–GNCs were fabricated successfully. In the synthesis process, we had optimized the conditions including the reaction temperature, the pH, the ratio of CCYJNP5/HAuCl₄ and the reaction time (ESI†). We found that the intensity of the fluorescence become weak with the increase of the reaction temperature (Fig. S1†). Although the luminescence of the CCYJNP5–GNCs was much stronger under lower reaction temperature, it needed much longer time to complete the reaction. In order to guarantee the fluorescence intensity of the GNCs with shorter reaction time, we chose room temperature for the reaction (Fig. S1†). In addition to the reaction temperature, the ratio of CCYJNP5/HAuCl₄ and the pH also had a great influence on the fluorescence intensity of the GNCs. The luminescence of the CCYJNP5–GNCs became weaker when the ratio of CCYJNP5 : HAuCl₄ was not 1 : 1 (Fig. S2†). The optimum pH was 11–12 (Fig. S3†). The reason might be that the phenolic group can be converted into a negative phenolic ion, which plays a role of reducing gold ions under basic conditions. The other results of the optimization processes were shown in ESI (Fig. S4†).

Fig. 1 Photographs of the prepared aqueous solution of the CCYJNP5–GNCs (A) in room light and (B) under a UV light source emitting 365 nm light. Fluorescence excitation and emission spectra (C) of JNP5 (P₁) and CCYJNP5 (P₂) peptides prepared GNCs (excitation at 380 nm and emission at 640 nm).

We also investigated the feasibility of the JNP5 and the CCYJNP5 peptide in the synthesis of gold nanoclusters, respectively. As shown in Fig. 1C, the fluorescence intensity of the GNCs produced by the peptide modified with a CCY sequence was much larger than that of the GNCs produced by the peptide without a CCY sequence. Thus, the CCY plays an important role in the synthesis of the CCYJNP5–GNCs. The reason might be that after Au ions were introduced into the peptide solution, some Au ions interact with the thiol groups of the CC to form bidentate Au thiolate intermediates, e.g., –SR–[Au–SR–]₂.^7,12 When the pH was adjusted to 11–12, the phenolic group of Y was converted into a negative phenolic ion which played a role of reducing gold ions, and the phenolic group then changed to a phenoxide structure.¹³ As shown in Fig. 2A, the absorption peak at 274 nm of the CCYJNP5, which comes from the tyrosine (Y), disappeared in the spectrum of the CCYJNP5–GNCs, which demonstrated that the phenolic group participated in the reaction. Finally, Au atoms are captured by the SH groups of the peptide and aggregated to form the CCYJNP5–GNCs.

Fig. 2 UV-visible absorption spectra of as-prepared the CCYJNP5–GNCs and CCYJNP5 (P₂) peptides (A); luminescence lifetime of the CCYJNP5–GNCs (B).

The fabricated CCYJNP5–GNCs had a wide excitation wavelength with the maximum excitation wavelength at 380 nm and the maximum emission wavelength at 640 nm as shown in Fig. 1C. The quantum yield (QY) of the clusters was measured to be 2.86%.

The lifetime measurement of the CCYJNP5–GNCs in Fig. 2B presents a biexponential fluorescence decay: τ₁ = 378.3 ns (20.52%) and τ₂ = 1530 ns (79.48%). The results show that the CCYJNP5–GNCs have a long lifetime. It could be that the complex contributes to the luminescence of the CCYJNP5–GNCs due to ligand–metal charge transfer and Au(I)–Au(I) interactions.^14,15

Fig. 3A shows the TEM image of the CCYJNP5–GNCs. One can see that the CCYJNP5–GNCs were well dispersed and the average particle diameter was about 2.0 nm. The small size of the CCYJNP5–GNCs makes them more attractive for cell imaging.¹⁶

Fig. 3 (A) TEM image of the CCYJNP5–GNCs. Scale bar 10 nm; (B) FTIR spectra of the CCYJNP5–GNCs (a) and the CCYJNP5 peptides (b). The spectra between them are enlarged 20 units for the contrast distinctly.

In order to obtain further information about the peptide conjugated on the surface of the CCYJNP5–GNCs, we measured the FTIR of the CCYJNP5 and the CCYJNP5–GNCs. As shown in Fig. 3B, the IR absorption peaks at 1627 cm⁻¹ (amide I, carbonyl stretch vibration) and 1395 cm⁻¹ (amide III, C–N stretch vibration)¹⁷ were found in both the CCYJNP5 and the CCYJNP5–GNCs, indicating the successful binding of the CCYJNP5 to the GNCs. Additionally, the skeleton vibration of benzene in tyrosine (Y)¹⁸ at 1540 cm⁻¹ was found in the spectrum of the peptide, but disappeared in the spectrum of the CCYJNP5–GNCs, which also demonstrated that the phenolic group of Y had taken part in the reaction.

In addition to the FTIR spectroscopic results, we used the XPS to examine the valence of Au. Fig. 4A shows that the binding energies of Au 4f_7/2 and Au 4f_5/2 were centered at 83.6 eV and 87.4 eV, respectively. The spectrum of Au 4f_7/2 was deconvoluted into two distinct components (purple and green curves) at binding energies of 83.5 eV and 84.1 eV, which suggested that both Au(0) and Au(I) exist in the clusters. And there was a certain amount (∼33%) of Au(I) on the surface of gold core, which helped the stabilization of clusters.^19,20

Fig. 4 (A) XPS spectra of the Au 4f of the CCYJNP5–GNCs (the original spectrum – black, the fitted result – red). The Au 4f_7/2 binding energy was deconvoluted into two components, which showed peaks at 83.5 eV (Au(0) – purple) and 84.1 eV (Au(I) – green); (B) MALDI-TOF MS of the CCYJNP5–GNCs recorded in the positive mode. Matrix: DHB.

As shown in Fig. 4B, the MALDI-TOF mass measurement provided the number of Au atoms. The prepared CCYJNP5–GNCs contained 21 gold atoms, and the pace between adjacent main peaks was consistent with the molecular weight of a peptide missing. It was consistent with the published literature that the gold nanoclusters containing 21, 25, and 28 gold atoms are relatively stable.²¹

Stability of the CCYJNP5–GNCs in PBS buffer solution

To examine the fluorescence stability of the CCYJNP5–GNCs, we measured the fluorescence spectra of the CCYJNP5–GNCs that were dispersed in PBS buffer solution and water at same concentrations, respectively. As shown in Fig. 5, no obvious change of the emission spectra and fluorescence intensities were observed, which indicated that the prepared CCYJNP5–GNCs possessed good fluorescence stability in physiological status.

Fig. 5 Fluorescence spectra of the CCYJNP5–GNCs dispersed in PBS buffer solution (blue line) and water (red line).

Cytotoxicity and targeting ability of the CCYJNP5–GNCs

The CCYJNP5–GNCs exhibit red emission, which can eliminate the interference of cell autofluorescence and reduce the damage to cells.²² Thus, it is a potential biological imaging fluorescence probe. In order to verify the conjecture above, we chose the KYSE510 cell line that expresses the FGFR2 in high level as the test group and the 293A cell line that expresses the FGFR2 at a very low level as control group. The cytotoxicity of the CCYJNP5–GNCs was measured first (Fig. S5†) and we found that the viability of the KYSE510 and 293A cells remained over 95% after incubation with 2.5 mg mL⁻¹ of CCYJNP5–GNCs for 1 h. The results indicated that the prepared CCYJNP5–GNCs were almost nontoxicity and suitable for tumor imaging.

We assessed the uptake of the CCYJNP5–GNCs by both cell lines after 1 h of incubation by the LEICA TCS-SP5 confocal fluorescence microscopy with a 405 nm laser. As shown in Fig. 6, both (A) and (B) exhibit red fluorescence, indicating that the CCYJNP5–GNCs were successfully uptake by both kind of cells. However, the fluorescence intensity of (A) (the KYSE510 cell line) is much stronger than that of (B) (the 293A cell line), which means that there is larger amount of the CCYJNP5–GNCs in the KYSE510 than that in the 293A. The results demonstrated that there is no influence on targeting ability of the JNP5 to the FGFR2 although the CCYJNP5–GNCs were fabricated by the peptide CCYJNP5, which is consistent with our original expectation. Therefore, the entry process relies on the recognition between the FGFR2 and the JNP5 conjugated on the GNCs, and then the CCYJNP5–GNCs are taken up by receptor-mediated endocytosis pathway for tumor imaging.^23,24

Fig. 6 Confocal fluorescence images of targeting ability evaluated assay (A–C) and competitive inhibition assay (D). (A and B) represent images of the KYSE510 cells and the 293A cells after incubated with the CCYJNP5–GNCs, respectively. (C) represents images of the KYSE510 cells after incubated with the BSA–GNCs. (D) represents that the KYSE510 cells were preincubated with the free CCYJNP5 and then incubated with the CCYJNP5–GNCs. (A₃–D₃) are the overlapping images of bright (A₁–D₁) and fluorescent images (A₂–D₂), respectively. All of the scale bars are 15 μm.

The uptake of the BSA–GNCs and the CCYJNP5–GNCs by the KYSE510 cell line also demonstrated the same results. One can see from Fig. 6C that only weak red fluorescence was observed indicating that few BSA–GNCs had entered the KYSE510 cells. By comparison, the CCYJNP5–GNCs in the KYSE510 cells showed strong red fluorescence (Fig. 6A). The reason could be that there is no recognition between the FGFR2 and the BSA, thus the BSA–GNCs could not be taken up by specific endocytosis pathway. However, the BSA–GNCs could enter the tumor cells by the pathway of enhanced permeability and retention effect (EPR).^25,26 Therefore, the weak red fluorescence in the KYSE510 cells was observed.

Competitive inhibition assay

In competitive inhibition assay, the KYSE510 cells were incubated with the free CCYJNP5 peptides (100 μg L⁻¹) for 1 h first, then washed with PBS buffer solution for three times and incubated with the CCYJNP5–GNCs (2.5 mg L⁻¹) for another 1 h (Fig. 6D), while the control group was only incubated with the CCYJNP5–GNCs (2.5 mg L⁻¹) for 1 h (Fig. 6A). In contrast, the fluorescence intensity in the KYSE510 cells which incubated with the CCYJNP5–GNCs directly was obviously stronger than that of another group, which demonstrated that the free peptides could compete for binding to the FGFR2 on the cell membrane and blocked the internalization of the CCYJNP5–GNCs with the FGFR2 which is expressed in high level in the KYSE510 cells. As a result, they may have an influence on the availability of the FGFR2 for the CCYJNP5–GNCs and reduce the uptake of the CCYJNP5–GNCs. The result further confirmed that the CCYJNP5 peptides still possess the targeting ability for the FGFR2 after fabricated the gold nanoclusters, and the CCYJNP5 peptides conjugated on the surface of the clusters facilitated the uptake by the KYSE510 cells through receptor-mediated endocytosis.

Table 1 shows the quantitative determinations of the four groups in Fig. 6 to compare the amounts of the CCYJNP5–GNCs taken up by cells. About 2000 μm² was selected in each group and the corresponding average intensity was presented. One can find that the intensity of group A was much larger than that of other groups. The data was consistent with the results in Fig. 6, indicating that there were much more CCYJNP5–GNCs entered in the KYSE510 cells in group A.

Table 1 Spectra of the fluorescence intensity of the selected area (about 2000 μm²). Group A–D in Table consistent with (A–D) in Fig. 6. The ratio was calculated by A/(A–D) of the average fluorescence intensity, respectively

Groups	A	B	C	D
Area spectra
Area/(μm)^2	2012.54	2005.77	2007.95	2013.92
Average intensity	75.01	10.04	15.61	8.55
Ratio	1.00	7.47	4.80	8.77

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Conclusion

In summary, we have successfully developed a simple, one-step method to synthesize red luminescence gold nanoclusters, the CCYJNP5–GNCs. The prepared GNCs possess good luminescence, high stability, nontoxicity, and good biocompatibility. The successful application of the CCYJNP5–GNCs to the targeting of the FGFR2 which is expressed in high level in the KYSE510 tumor cells confirm our previous expectation that the CCYJNP5 peptides still possess the targeting ability for the FGFR2 after being used to synthesize the gold nanoclusters. The results show that the CCYJNP5–GNCs could be a potential material for the FGFR2 high expression tumor imaging and diagnosis. Meanwhile, the design and synthesis of the CCYJNP5–GNCs also provide a new idea for tumor diagnosis materials.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (20875106), Guangdong Natural Science Found Committee (9151027501000003), and State Key Laboratory of Chemo/Biosensing and Chemometrics, Hunan University (4299001).

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c5ra20113f

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