English

• 1.   Introduction

  Tumor is a great life threat to human. The past decade we have witnessed the burgeoning of optical imaging and its wide applications for biomedicines such as genomics, proteomics, cell biology, and drug discovery [1].

  As we all know, early-stage tumor has been detected by the invention of kinds of probes, especially the invention of radiationless fluorescence probes [2, 3]. Fluorescence probe which can be used to improve tumor early detection is still a popular topic among related experts and researchers. Recently, the near-infrared (NIR) fluorescence probes are increasingly popular in biological imaging and sensing for the reason that longwavelength (650–900 nm) excitation and emission have the advantages of minimum photodamage to biological samples, deep tissue penetration, and minimum interference from background to fluorescence by biomolecules in the living systems [4–6]. However, the development of NIR fluorescence probes has some troubles, such as photostabilities and fluorescence quantum yields of probes, targeting of tumors. Up to now, the common NIR fluorophores only have cyanine dyes, such as Cy5 and Cy7 [7, 8].

  Porphyrins [9], due to the unique properties of photophysical and photochemical, have a special relevance to photodiagnosis (PD) and in photodynamic therapy (PDT) of oncological and non oncological diseases [10]. Moreover, we used porphyrins as fluorescence probes because they are relatively stable and have good photostability [11].

  Tumor-penetrating internalizing RGD peptide (iRGD) is known to have a relatively high and specific affinity for αvβ3 - integrin receptor [12–14], which is increased vascular and tissue permeability in a tumor-specific and neuropilin-1-depenendent manner, allowing co-administered drugs to penetrate into extravascular tumor tissue. The CendR motif, a class of cell and tissue-penetrating peptides with a specific R-x-x-R carboxylterminal motif, is not active unless it occupies a C-terminal position in the peptide after the cleavage, recognizing neuropillin-1(the receptor for the CendR peptides) and facilitating the penetration into the tumor of iRGD and co-administered drugs [15].

  In this study, the iRGD [16], which having tumor-penetrating ability and good cell membrane permeability, was used to improve the permeability of cell membrane firstly, and then the fluorescent probe TPP-glycine was apt to enter into cells.

  2.   Results and discussion

  2.1   The synthesis of TPP-glycine

  The synthetic process of TPP-glycine was shown in Scheme 1. The TPP and TPP-glycine were identified by analyzing the absorption of hydrogen in Fig. 1.

  Scheme 1

  

  图 Scheme 1  Synthesis of the probe.

  Scheme 1.  Synthesis of the probe.

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  图 1

  

  图 1  ¹H NMR spectra of TPP (left) and TPP-glycine (right).

  Figure 1.  ¹H NMR spectra of TPP (left) and TPP-glycine (right).

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  ¹H NMR (400MHz, CDCl₃): δ 8.91 (6H), 8.83 (d, 2H, J=4.7Hz), 8.48 (d, 2H, J=8.0Hz), 8.35 (d, 2H, J=8.0Hz), 8.26 (3H), 8.25 (3H), 7.85–7.80 (m, 6H), 7.80–7.76 (m, 4H), 4.15 (s, 3H), -2.73 (s, 2H). MALDI-TOF MS C₄₆H₃₂N4O₂ calcd. for 672.25, found 672.21.

  ¹H NMR (400MHz, CDCl₃): δ 8.91–8.74 (m, 8H), 8.34–8.17 (m, 10H, ArH), 7.83–7.70 (m, 9H, ArH), 4.44 (d, 2H, CH₂), 3.89 (s, 3H, CH₃), -2.78 (s, 2H). MALDI-TOF MS C₄₈H₃₅N₅O₃ calcd. for 729.27, found 730.26.

  2.2   The optical properties of TPP, TPP-COOH, TPP-glycine

  The absorption, emission intensity and fluorescence quantum yield results were displayed in Fig. 2 and Table 1, respectively.

  图 2

  

  图 2  Absorption (a) and fluorescence (b) spectra of TPP, TPP-COOH, TPP-glycine in CH₂Cl₂ solution (3 × 10^-6 mol/L).

  Figure 2.  Absorption (a) and fluorescence (b) spectra of TPP, TPP-COOH, TPP-glycine in CH₂Cl₂ solution (3 × 10^-6 mol/L).

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  表 1

  

  表 1  Absorption wavelength, B band-excited fluorescence maxima of the porphyrins, fluorescence quantum yield and fluorescence lifetime in CH₂Cl₂ solution.

  Table 1.  Absorption wavelength, B band-excited fluorescence maxima of the porphyrins, fluorescence quantum yield and fluorescence lifetime in CH₂Cl₂ solution.

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  These results indicate that introduced carboxyl group and further glycine methyl ester has no significant influence on the optical properties of the porphyrin core.

  2.3   In vitro imaging

  As shown in Fig. 3, the aggregation degree of TPP and TPPglycine had no obvious change. However, aggregation degree of TPP-glycine D4 is much strongerthanTPP D2 when interactedwith iRGD[17]. Therefore, iRGD has the function which promotes probes to penetrate GLC-82 cytomembrane easily. The results were identical with those reported in literature [18].

  图 3

  

  图 3  Fluorescence microscope images of living GLC-82 cells. (B1) GLC-82 cells; (B2) GLC-82 cells were incubated with TPP for 1 h; (B3) GLC-82 cells were incubated with TPP-COOH for 1 h; (B4) GLC-82 cells were incubated with TPP-glycine for 1 h. (D1–D4): GLC-82 cells were pre-incubated with iRGD for 1 h, and then treated with the probe (D1-none, D2-TPP, D3-TPP-COOH, D4-TPP-glycine) for 1 h. For DAPI and BODIPY, the excitation wavelength is set as 488 nm and 633 nm, respectively.

  Figure 3.  Fluorescence microscope images of living GLC-82 cells. (B1) GLC-82 cells; (B2) GLC-82 cells were incubated with TPP for 1 h; (B3) GLC-82 cells were incubated with TPP-COOH for 1 h; (B4) GLC-82 cells were incubated with TPP-glycine for 1 h. (D1–D4): GLC-82 cells were pre-incubated with iRGD for 1 h, and then treated with the probe (D1-none, D2-TPP, D3-TPP-COOH, D4-TPP-glycine) for 1 h. For DAPI and BODIPY, the excitation wavelength is set as 488 nm and 633 nm, respectively.

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  2.4   MTT assay

  MTT assay [19] can be used to detect the toxicity of cells. The results in Fig. 4a–c showed that the cell viability decreased with the increase of concentration of probe. Compared with TPP-glycine and TPP, the toxicity of TPP-COOH is obviously bigger than the others when the concentration of probes is higher than 5 μmol/L. The date has showed that added iRGD has no significant influence on cells. In a practical application, the safe concentration of probes for cells are lesser than 1 μmol/L. From Fig. 4, the values of cell viability are greater than 90% when the concentration of probes is lesser than 1 μmol/L. Therefore, theprobescan be applied todetect tumor cells.

  图 4

  

  图 4  The MTT assay results of TPP (a), TPP-COOH (b) and TPP-glycine (c).

  Figure 4.  The MTT assay results of TPP (a), TPP-COOH (b) and TPP-glycine (c).

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  3.   Conclusion

  In this paper, TPP-glycine was synthesized and characterized by UV–spectrophotometer, fluorescence spectrophotometer, fluorescence microscope, and MTT assay. Experimental results showed that the porphyrins modified with glycine methyl ester had a good affinity with GLC-82 cells than other porphyrins when interacted with iRGD. And it also indicated that iRGD can penetrate GLC-82 cell membranes contribute to enter into cells of fluorescent probe TPP-glycine.

  4.   Experimental

  4.1   Materials

  Silica gel (200–300 mesh, 300–400mesh), Liquid NMR (Bruker AVANCE Ⅲ 400M), MALDI-TOF-MS (Bruker Autoflex tof/tof Ⅲ), UV–vis spectrophotometer (Shimadzu UV-1800), Fluorescence spectrophotometer (Varian Cary Eclipse), Transient Fluorescence (Horiba JY Fluorolog-3), Flow Cytometry (FACScalibur).

  Pyrrole(AR, Nanjin Tianzun Chemical Ltd.), methyl p-formylbenzoate (AR, Nanjin Tianzun Chemical Ltd.), trifluoroacetate (AR, Northern Chemical Reagent Chemical Ltd), benzaldehyde (AR, Tianjin Guangfu Chemical Limited Corporation), DMF (AR, Tianjin Guangfu Chemical Ltd.), iRGD (96%, GL Biochem Ltd.).

  4.2   Synthesis of TTP-glycine

  A solution of phenyl pyrrolidine (146.2 mg, 1 mmol), methyl 4-formylbenzoate (82.1 mg, 0.5 mmol), and aromatic aldehyde (0.5 mmol) in CH₂Cl₂ (100 mL) were added F₃CCOOH (0.6 mmol, 44 μL). The mixture was stirred in the dark for 3 h and then DDQ (0.9 mmol, 200 mg) was added. After the solution was further stirred at room temperature for 1 h. NaHCO₃ (106 mg, 1 mmol) was added and the solvent was removed from reduced pressure. The product was isolated by column chromatography (silicagel) using CH₂Cl₂/hexane = 1/2 as eluent. Next, A mixture of TPP (0.1 mmol) and KOH (2 mmol) in THF (5 mL), EtOH (1 mL) and water (1 mL) was refluxed for 2 h. After the solution was cooled to room temperature, the residue was dissolved in water (50 mL) and acidified to pH 3 with 1 mol/L HCl solution. CH₂Cl₂ was added and the organic layer was collected. The solvent was removed in vacuo. Recrystallization from CH₂Cl₂ gave TPP-COOH (0.95 mmol, 95%) C₄₅H₃₀N₄O₂ calcd. for 658.24, found 659.20.

  The EDC·HCl (1.53 mg, 8 mmol) and NHS (1.15 mg, 10 mmol) were added to the solution of TPP-COOH (2.63 mg, 5 mmol) in anhydrous DMF (2 mL). The mixture was stirred at room temperature for 12 h. And then glycine (2.4 mg, 4 equiv.) was dissolved in anhydrous DMF (2 mL) and a large excess of K₂CO₃ was added. The reaction was kept for 12 h at room temperature and then poured into CH₂Cl₂ (30 mL), washed with water. The product was collected by filtration and purified by flash chromatography (CH₂Cl₂) to obtain the final TPP-glycine as a solid.

  4.3   Measurement of fluorescence quantum yield

  The concentration 3 × 10^-6 mol/L of TPP, TPP-COOH, or TPPglycine were prepared which the porphyrins dissolved in CH₂Cl₂ solution. And then the values of UV absorption and emission intensity were tested by UV–spectrophotometer and fluorescence spectrophotometer.

  4.4   Cell culture and in vitro imaging

  The GLC-82 cells were cultured with RPMI 1640 medium in the incubator (37 ℃, 5% CO₂). The cultured cells were washed by PBS, and added trypsin. Then put them into the 15 mL centrifuge tube. Finally, centrifuging (500 rpm, 5 min), making suspension of cells and counting cells.

  Cover glasses were plated into 48-multiwell plate, and then a density of 5 × 10⁴ cells each well were plated and grown on cover glasses, cultured 24 h in the incubator (37 ℃, 5% CO₂). After 24 h, the iRGD (2 μmol/L) was plated in single well and cultured 1 h firstly. Next 2 μmol/L TPP, TPP-COOH, TPP-glycine was plated in wells which including iRGD and no iRGD, respectively and cultured 1 h. Then the cells were washed by PBS three times and added paraformaldehyde to fix cells for room temperature 5 min and washed by PBS three times again. And then cellular nuclei were stained by DAPI for room temperature 5 min. The DAPI was washed by PBS three times. Finally, the cover glasses were taken out from wells, added anti-fade fluorescence mounting medium and fixed on the glass slides with transparent nail polish. The made-samples were measured with Confocal Microscope (Ultra View Vox).

  4.5   MTT assay

  Cell viability measurements were performed by MTT analysis. Cells were plated at a density of 5 × 10⁴ cells each well into 96-multiwell plate for 24 h in the incubator (37 ℃, 5% CO₂). Making up 2 μmol/L iRGD and different concentration TPP, TPP-COOH, TPP–glycine. Concentrations were as follows: 10 μmol/L, 5 μmol/L, 1 μmol/L, 10 nm, 100 nm. First, adding 100 μL iRGD into cells and cultured for 1 h. Next, different concentration TPP, TPP-COOH, TPP-glycine were added to the wells which including iRGD and wells without iRGD, respectively. After 48 h, 10 μL MTT (5 μg/mL) was added into every single well and incubation was continued for 4 h at 37 ℃. Then, 90 μL DMSO was added to single wells to dissolve the blue formazan in the live cells. Finally, the value of OD were measured at 490 nm using a microplate reader (Thermo Scientific).

  Acknowledgments

  This work is supported by National Natural Science Foundation of China (No. 21076147). We are grateful to Prof. H. Huang and Prof. L. Yang for providing GLC-82 cells and some support.

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• 

  Scheme 1  Synthesis of the probe.

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  Figure 1  ¹H NMR spectra of TPP (left) and TPP-glycine (right).

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  Figure 2  Absorption (a) and fluorescence (b) spectra of TPP, TPP-COOH, TPP-glycine in CH₂Cl₂ solution (3 × 10^-6 mol/L).

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  Figure 3  Fluorescence microscope images of living GLC-82 cells. (B1) GLC-82 cells; (B2) GLC-82 cells were incubated with TPP for 1 h; (B3) GLC-82 cells were incubated with TPP-COOH for 1 h; (B4) GLC-82 cells were incubated with TPP-glycine for 1 h. (D1–D4): GLC-82 cells were pre-incubated with iRGD for 1 h, and then treated with the probe (D1-none, D2-TPP, D3-TPP-COOH, D4-TPP-glycine) for 1 h. For DAPI and BODIPY, the excitation wavelength is set as 488 nm and 633 nm, respectively.

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  Figure 4  The MTT assay results of TPP (a), TPP-COOH (b) and TPP-glycine (c).

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  Table 1.  Absorption wavelength, B band-excited fluorescence maxima of the porphyrins, fluorescence quantum yield and fluorescence lifetime in CH₂Cl₂ solution.

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•