DCPO based nanoparticles as a near-infrared fluorescent probe for Cathepsin B

Abstract

A novel NIR fluorescent probe for CTB was designed and synthesized based on DCPO. The newly synthesized probe was quite water-soluble and formed nanoparticles which has potential in tumor-targeted imaging. When treated with CTB, the probe released the fluorescent molecule DCPO. Imaging of different tumor cells also indicated that the probe was efficient in detecting CTB.

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Introduction

Cathepsin B (CTB), a cysteine protease, is involved in numerous pathological processes including cell death, inflammation, and toxic peptide production.^1,2 CTB is known to be overexpressed in various cancers,³ particularly in aggressive cancers,^4–7 making it an attractive target for tumor-specific prodrug design.^8–10 A successful example was brentuximab vedotin,¹¹ an antibody-drug conjugate approved by the FDA for relapsed Hodgkin lymphoma and relapsed systemic anaplastic large cell lymphoma. Given the importance of CTB in the diagnosis and treatment of cancer, particularly in the design of antibody-drug conjugates, a probe is required for its detection. Enormous progress in the field of bioimaging has been made in recent years through the use of fluorescence imaging techniques,¹² and enzyme-activated fluorescent probes are of particular value for their efficiencies and specificities.¹³

Enzyme-sensitive probes can be divided into two classes: Förster resonance energy transfer (FRET)-based probes and intramolecular charge transfer (ICT)-based probes. A FRET-based probe typically consists of two fluorescent molecules with overlapping absorption spectra and a cleavable linker.¹⁴ An ICT-based probe typically has an electron donor group that switches off fluorescence emission when blocked.¹⁵ Several fluorescent probes for CTB have been developed based on these two mechanisms.^16–21 However, they show some limitations, such as difficult synthetic chemistry, poor water solubility, or low tumor specificity. A novel probe to overcome these disadvantages is required.

Nanoparticle technology has been widely used in tumor-targeted drug delivery since the enhanced permeability and retention (EPR) effect was discovered. PEGylation is a common strategy used for designing nano-micelles. Our group recently developed a CTB-sensitive nano prodrug (Fig. 1) which exhibited good anti-tumor activity both in vitro and in vivo.²² The prodrug released 7-ethyl-10-hydroxycamptothecin (SN-38) in the presence of CTB. We hypothesized that an appropriate fluorescent molecule could replace SN-38, generating a nano fluorescent probe for CTB.

Fig. 1 Design of probe 1 and mechanism of DCPO release.

Near-infrared (NIR) fluorescent probes with emission wavelengths of 650–900 nm are favored for their minimum photo-damage to biological samples, deep tissue penetration, minimum interference from the background, and auto-fluorescence by biomolecules in living systems.^23–26 Dicyanomethylene-4H-pyran derivatives such as DCPO are widely used in fluorescence imaging because they are more photostable than other NIR fluorophores such as squaraine and cyanine.^27–31 In addition, DPCO is hydrophobic and has similar physicochemical properties to SN-38, making it a good replacement in the design of a nano fluorescent probe. In this study, we designed a nano fluorescent probe for CTB utilizing the prodrug strategy. The probe was soluble in water and self-assembled into micelles with average sizes of 100–200 nm. In the presence of CTB, the probe released DCPO following 1,6-elimination and cyclization.

Experimental

General procedures

¹H NMR and ¹³C NMR spectras were recorded in CDCl₃, CD₃OD, and DMSO-d₆ on a Bruker DRX-400 (400 MHz) using TMS as internal standard. Chemical shifts were reported as δ (ppm) and spin−spin coupling constants as J (Hz) values. The mass spectra (MS) were recorded on a Finnigan MAT-95 mass spectrometer or 4800 MALDI TOF/TOF analyzer. Cathepsin B from human liver and mPEG2000-N₃ were purchased from Sigma-Aldrich. Other chemicals were purchased from Sinopharm Chemical Reagent Co., Ltd. Human colon cancer cell line HCT-116 and ovarian carcinoma cell line SKOV-3; and human hepatoma cell line HepG2, and human cervical cancer cell line HeLa were purchased from the Shanghai Institute of Biochemistry and Cell Biology (Shanghai, China). The cell culture fluid and FBS were purchased from Thermo Fisher Scientific. UV-vis absorption spectra were recorded on a Varian Cary 100 spectrophotometer. Fluorescence spectra was measured with a Hitachi F-4500 Fluorescence spectrophotometer.

Synthesis of compound 2

To a solution of DCPO (485 mg, 1.55 mmol) and TEA (315 mg, 3.10 mmol) in 5 ml DMSO was added bis(4-nitrophenyl) carbonate in batches. The mixture was stirred at room temperature for 1 h before the addition of N,N′-dimethylethylenediamine (770 mg, 2.33 mmol). The resulting mixture was stirred at room temperature for another 16 h and poured into water and extracted with DCM. The organic layer was washed with water and brine, dried over Na₂SO₄ and concentrated under vacuum. The crude product was purified by column chromatography using PE/EA (10/1–1/1, v/v) as eluent to afford 650 mg compound 2 as a yellow solid, which was used directly for the next step.

Synthesis of compound 3

Compound 2 (450 mg, 0.67 mmol) was added in batches to a solution of TFA in DCM (1/10, v/v). The mixture was stirred at room temperature for 20 min and poured into cold Et₂O. The precipitate was filtered and washed with Et₂O to give pure compound 3 (340 mg, 61% yield from DCPO) as a brown solid. Mp 183–186 °C, ¹H NMR (400 MHz, DMSO-d₆) δ 8.74 (dd, J = 8.4 Hz, 1.0 Hz, 1H), 8.66 (s, 1H), 8.56 (s, 1H), 7.98–7.90 (m, 1H), 7.87–7.77 (m, 3H), 7.75 (s, 1H), 7.68–7.58 (m, 1H), 7.50 (d, J = 16.1 Hz, 1H), 7.35–7.26 (m, 2H), 7.04 (s, 1H), 3.71 (t, J = 5.6 Hz, 1H), 3.59 (t, J = 5.6 Hz, 1H), 3.27–3.12 (m, 2H), 3.07 (s, 2H), 2.95 (s, 1H), 2.68–2.59 (m, 3H). ¹³C NMR (100 MHz, DMSO-d₆) δ 158.0, 154.2, 152.9, 152.0, 137.7, 135.4, 132.0, 130.5, 129.1, 126.2, 124.6, 122.6, 122.4, 119.4, 119.0, 117.0, 115.7, 111.6, 106.7, 89.5, 60.4, 51.2, 46.3, 45.9, 45.2, 34.4, 32.7.

Synthesis of compound 5

Compound 2 (340 mg, 0.65 mmol) and compound 4 (403 mg, 0.54 mmol) were dissolved in anhydrous DMF and DIPEA (209 mg, 1.62 mmol) was added into the solution. The mixture was stirred at room temperature for 16 h. Then the mixture was poured into water and extracted with DCM, the organic layer was washed with water and brine and dried over Na₂SO₄. The solvent was removed under decreased pressure to give the crude product which was purified by column chromatography using DCM/MeOH (15/1, v/v) as eluent. The product collected was further purified by Pre-HPLC with ACN/water (70/30, v/v) as eluent to give the title compound 150 mg as a brown solid (157 mg, 27%). Mp 142–145 °C, ¹H NMR (400 MHz, DMSO-d₆) δ 10.10 (s, 1H), 8.73 (d, J = 8.2 Hz, 1H), 8.11 (d, J = 7.0 Hz, 1H), 7.93 (t, J = 7.7 Hz, 1H), 7.72–7.86 (m, 4H), 7.67–7.44 (m, 4H), 7.33(dd, J = 13.7, 6.4 Hz, 7H), 7.13 (dd, J = 30.3, 7.8 Hz, 2H), 7.04 (s, 1H), 5.98 (s, 1H), 5.41 (s, 2H), 5.15–4.94 (m, 4H), 4.67–4.50 (m, 2H), 4.44–4.34 (m, 1H), 4.24–4.11 (m, 2H), 3.95–3.85 (m, 1H), 3.12–2.72 (m, 9H), 2.02–1.88 (m, 1H), 1.76–1.52 (m, 2H), 1.49–1.28 (m, 2H), 1.05 (t, J = 7.0 Hz, 1H), 0.84 (dd, J = 16.8, 6.6 Hz, 6H). ¹³C NMR (100 MHz, DMSO-d₆) δ 171.2, 170.6, 158.8, 158.0, 156.1, 152.8, 151.9, 144.1, 138.8, 137.7, 137.0, 136.6, 135.4, 131.9, 129.5, 129.2, 128.3, 127.7, 127.6, 126.1, 124.6, 122.3, 119.4, 119.0, 118.2, 117.1, 117.0, 115.7, 106.7, 82.7, 80.0, 78.8, 77.5, 72.7, 68.2, 66.7, 65.4, 64.2, 63.7, 62.3, 60.3, 60.1, 57.2, 57.1, 55.1, 53.1, 52.4, 34.5, 31.5, 30.3, 29.8, 29.4, 28.8, 26.7, 24.8, 19.2, 18.8, 18.1. HRMS (ESI) m/z calcd for C₅₆H₆₀N₉O₁₁ [M + H]⁺ 1034.4412, found 1034.4397.

Synthesis of probe 1

Compound 5 (120 mg, 0.116 mmol), mPEG-2000-N₃ (278 mg, 0.139 mmol), CuSO₄·5H₂O (6 mg, 0.023 mmol), and sodium ascorbate (5.00 mg, 0.023 mmol) were dissolved in a mixture of t-BuOH and water (2/1, v/v). Then it was heated to 60 °C under N₂ for 2 h. Subsequently, the mixture was concentrated under reduced pressure and purified by column chromatography to afford the title compound (272 mg, 77%) as a brown solid. ¹H NMR (400 MHz, CDCl₃) δ 9.57 (s, 1H), 8.92 (d, J = 7.6 Hz, 1H), 7.89–7.71 (m, 3H), 7.67–7.50 (m, 5H), 7.45 (t, J = 7.8 Hz, 1H), 7.37–7.28 (m, 5H), 7.18–6.79 (m, 5H), 5.55 (s, 2H), 5.22–4.82 (m, 7H), 4.74–4.48 (m, 6H), 4.09 (s, 1H), 3.94–3.41 (m, 175H), 3.37 (s, 3H), 3.16–2.85 (m, 8H), 2.18–2.06 (m, 1H), 1.71–1.39 (m, 4H), 1.34–1.15 (m, 3H), 0.92 (dt, J = 17.2, 8.5 Hz, 6H). The purity of probe 1 was above 96% according to HPLC. MALDI-TOF-ESI-MS showed the center of peak at m/z 3051 (M + Na).

Water solubility assay

Excess amount of DCPO, compound 5 and probe 1 was added into 0.1 mL water or saline and dissolved thoroughly. The undissolved substance was filtered by a 0.22 μm filter and the concentration of the saturated solution was measured by HPLC. The concentrations of all compounds were transferred into the relative concentration of DCPO.

Particle size distribution

Probe 1 was dissolved in acetone and added into water, the resulting mixture was dialyzed for 12 h and the size distribution was measured on a dynamic light scattering (DLS) (ZetasizerNano ZS, Malvern Instruments, UK). The morphology of the nanoparticles was observed by transmission electron microscopy (TEM) (JM-2100, Japanese). A drop of aqueous nanoparticles suspension was deposited onto a 300 mesh copper grid coated with a thin carbon film. The grids were dried at room temperature and observed by TEM.

CTB assay

CTB (25 μg) from human liver was dissolved in 3.75 mL buffer containing 50 mM sodium acetate (pH = 5.0) and 1 mM EDTA at −80 °C before use. The above stock solution (240 μL) was activated with 760 μL PBS buffer (0.4 M, pH = 6.0) containing 8 mM L-cysteine and 4 mM EDTA for 15 min at 37 °C. The final concentration of CTB in this solution was 1.6 μg mL⁻¹. Then the tested probe 1 in 5 μL DMSO (10 mM) was added to CTB solution and incubated at 37 °C for 24 h. For the control group, the 240 μL of CTB solution was replaced with equivoluminal PBS buffer. The sample (50 μL) was collected during 24 h and the concentration of probe 1 as well as DCPO was determined by HPLC using external standard method. At last, the solution was adjusted to pH 7–8 with a slight amount of NaHCO₃ (aq.) and equivoluminal DMSO was added then the fluorescence emission was recorded.

Cell culture assay

HCT-116, SKOV-3, HepG2 and Hela cells were employed for in vitro cell imaging. The cells were cultured at 37 °C, 5% CO₂ and 95% humidity in McCoy's 5A (for HCT-116 and SKOV-3), or MEM (for HepG2), or DMEM (for Hela) medium with 10% FBS and 1% penicillin/streptomycin (Thermo Fisher Scientific). The cells were seeded in a 96-well imaging plate with an amount of 2000 (for HCT-116) or 5000 (for SKOV-3) or 15 000 (for HepG2) or 5000 (for Hela) cells per well overnight. Probe 1 was co-cultivated with cells at variant concentrations for different time. Before observation, the cells were washed three times with PBS buffer (pH = 7.4). Finally, the cells were taken photos on a inverted fluorescence microscope with blue exciting light. The average fluorescence intensity was given by the flow cytometry assay on a Guava easyCyte HT system.

Results and discussion

The synthetic route of probe 1 is shown in Scheme 1 and 2. DCPO and intermediate 4 were synthesized as previously described. The key DCPO derivative 3 was synthesized from DCPO, which was treated with bis(4-nitrophenyl) carbonate followed by N-trityl-N,N′-dimethylethylenediamine to afford compound 2. The trityl protecting group was removed in the presence of TFA. As shown in Scheme 2, compound 3 reacted with intermediate 4 using DIPEA as a base to obtain compound 5, which was PEGylated through classical Click reaction to afford probe 1.

Scheme 1 Reagents and conditions: (a) (i) bis(4-nitrophenyl) carbonate, TEA, DMF; (ii) N-trityl-N,N′-dimethylethylenediamine; (b) TFA, DCM, 61% yield from DCPO.

Scheme 2 Reagents and conditions: (a) compound 3, DIPEA, DMF; (b) mPEG2000-N₃, CuSO₄·5H₂O, sodium ascorbate, t-BuOH, water, 60 °C.

We first evaluated the water-solubility of probe 1, compound 5, and DCPO. As shown in Table 1, compound 5 and DCPO were nearly water-insoluble, whereas probe 1 showed good water solubility. Probe 1 formed nanoparticles in water, sizes of which were measured by dynamic light scattering and the TEM image of the nanoparticles was taken. According to Fig. 3, probe 1 showed a narrow size distribution; the main hydrodynamic diameters of micelles were 100–200 nm (Fig. 3). The size data suggest the passive targeting potential to tumor tissue because of the EPR effect.

Table 1 Relative solubility in water and saline

Solubility^a	DCPO	Compound 5	Probe 1
a Not detectable by HPLC.
Water	1	0.004	520
Saline	0.03	NA^a	597

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Fig. 2 (a and b) Relative concentrations of probe 1 remained and DCPO released in PBS buffer with/without CTB. (c) Fluorescence emission spectrum (λ_e = 560 nm) of the two solutions above by adjusting the pH to 7–8.

Fig. 3 Size distribution of nanoparticles formed by probe 1 in water and TEM image.

Subsequently, the absorption and emission spectrum of probe 1 was measured (Fig. S1†). Probe 1 did not show NIR fluorescence emission because the ICT process was suppressed. In the presence of CTB in PBS with a pH of 6.0, probe 1 released DCPO as expected; this process was monitored by HPLC, because the concentration of DCPO was too low and fluorescence emission was dramatically decreased under acidic conditions, as we reported previously. After incubation for 24 h, the conversion rate of probe 1 was 50%; however, the control group without CTB showed that only a small amount of DCPO was released. Upon adjusting the pH to 7–8 with NaHCO₃ (aq.), the experimental solution turned dark red and NIR emission was observed by a fluorescence detector, whereas no distinct change was observed in the control group (Fig. 2).

Based on these results, probe 1 was employed to detect CTB in tumor cells. HeLa, HCT-116, HepG2, SKOV-3 cells were used to evaluate CTB levels. In order to determine the proper concentration and incubation time, HepG2 cells were incubated with different concentrations of probe 1 for 12 or 24 h. Next, the probe remaining in the cell culture was washed with PBS three times and photos were taken using a fluorescence inversion microscope system. As shown in Fig. 4, only when the cells were incubated with 100 μM of probe 1 for 24 h, clear red fluorescence was observed, which was in keeping with the average fluorescence intensity given by the flow cytometry assay.

Fig. 4 HepG2 cells were incubated with different concentrations of probe 1 for different time.

Probe 1 was used to image the other cell lines using the same concentration and incubation time. According to Fig. 5, the three cell lines showed different fluorescence brightness levels, which may related to the different CTB levels. However, all three cell lines showed medium fluorescent brightness because of the slow intracellular release of DCPO from probe 1.

Fig. 5 Hela, SKOV-3 and HCT-116 cells were incubated with 100 μM probe 1 for 24 h.

Conclusions

In summary, a novel NIR fluorescent probe for CTB was designed and synthesized. The probe exhibited good water solubility. Moreover, the probe self-assembled into nanoparticles, indicating its potential for passive tumor-targeting. When incubated with different cell lines, the probe showed different imaging effects. Thus, probe 1 is very useful for tumor imaging and can be a guide for studying CTB-activated prodrugs.

Acknowledgements

This work was supported by the State Key Laboratory of Fine Chemicals (KF1517).

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c6ra14628g

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