Synthesis, characterization and biological evaluation of a novel biscarboxymethyl-modified tetraphenylchlorin compound for photodynamic therapy

Abstract

A novel photosensitizer trans-2,3-dihydro-2,3-bis(carboxymethyl)-5,10,15,20-tetraphenylchlorin (BCTC) was synthesized. Its photophysical and photochemical properties, intracellular localization, photocytotoxicity in vitro and vivo were also investigated. BCTC displays a characteristic long wavelength absorption peak at 652 nm and shows a singlet oxygen quantum yield of 0.68 in DMF. Without light activation, BCTC was nontoxic to human esophageal cancer cells. However, upon light activation, BCTC exhibited significant photocytotoxicity. After PDT treatment, the growth of Eca-109 tumor in nude mice was significantly inhibited. This study suggests that BCTC is an effective photosensitizer for PDT to tumors.

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Introduction

Photodynamic therapy (PDT) is a novel cancer therapy that relies on the selective uptake of a photosensitizer into cancer cells followed by irradiation with red light, which produces singlet oxygen and other reactive oxygen species,^1–3 then highly cytotoxic singlet oxygen readily react with tumor cells and damage to them. PDT is a well-recognized modality for selected tumor destruction without damaging surrounding healthy tissues.^4–8 Because of the known limited diffusion of singlet oxygen through tissues, the PDT effects are largely localized to the photosensitizer-containing cells, thus reducing potential damage to normal cells in the vicinity of the tumor. An ideal photosensitizer should have absorption spectra at long wavelengths (the PDT “therapeutic window” is 600 to 800 nm), which allows deeper tissue penetration and decreased nonspecific lesions.⁹ In addition, an ideal photosensitizer should also have minimal toxicity in the dark, a high quantum yield of triplet state formation in the presence of light, high selectivity for tumor cells over normal cells, rapid clearance from normal tissues.^10–12

Over the past decade, a substantial effort has been put into the development of various classes of photosensitizers that present better absorption, greater tumor specificity. Thousands of photosensitizers have been tested in vitro and in vivo, but only a few of them managed to reach clinical trials.¹³ As mentioned by Wagner, chlorin-based photosensitizers have been found to have applications as phototoxic drugs for PDT.^14,15 Compared to porphyrins, chlorins are particularly promising photosensitizers,¹⁶ they offer increased absorption in the farthest-red side band (above 650 nm), thus enabling the use of a light with deeper penetration in organic tissues. Moreover, chlorins present good singlet oxygen quantum yields, making them overall better photosensitizers and attractive for use in PDT. Among chlorins, temoporfin (Foscan) is a compound that has been proven to be effective in treatment of head and neck cancers.^17,18 The red-most absorption is found at 650 nm in methanol and 652 nm in fetal-bovine serum. The drug carries four highly aromatic phenyl groups, which do not lie planar to the chlorin macrocycle, but are positioned in an angle of 60°.¹⁹ However, Foscan have significant drawbacks such as easily to be oxidized and difficult to be synthesized with high purity. So developing new chlorins are still appealing.

Novel photosensitizers are usually developed starting from already available photosensitizing compounds, and modulated structure to improve their photodynamic properties, solubility. However, tetraphenyl-chonrin with two carboxyl groups for PDT application has been seldom reported before. The present work aims to investigate the PDT effect of BCTC, which was designed based on the idea that a chlorin contained two carboxyl groups on the β position. The synthesis and characterization, photophysical properties, subcellular localization, photodynamic activities in vivo and vitro of the compound are reported herein.

Results and discussion

Synthesis and characterization

Two methods to prepare BCTC were explored. One synthesis route of BCTC involved a two-step process, as shown in Scheme 1.

Scheme 1 Synthesis of the BCTC. Reagents and conditions: (a) NCCH₂COOCH₃, NaH, DMSO, 60 °C, 1 h; then 80 °C, 3 h; H₂O and NaCl, 140 °C, 14 h, 40.6%. (b) HCl_aq (37%), reflux 20 h, 69%.

2-Nitro-5,10,15,20-tetraphenylporphyrin (compound 1) was synthesised as the literature.²⁰ The compound 2 was synthesised through a Michael addition reaction of compound 1 with methylcyanoacetate using NaH as a base in anhydrous DMSO. Firstly, compound 1 and methylcyanoacetate at 80 °C afforded a mixture of trans-chlorin diastereomers,²¹ then decarboxymethylation of diastereomers with NaCl in wet DMSO at 140 °C yielded compound 2 which was purified by column chromatography and characterized by ¹H NMR, MALDI-TOF mass spectral anslysis (Fig. S1–S2†).

Compound 2 underwent hydrolysis in the presence of concentrate hydrochloric acid at reflux temperature to produce BCTC.²² BCTC was purified using column chromatography and characterized by ¹H NMR, ¹³C NMR and HRMS (Fig. S5–S8†).

Since decarboxymethylation of trans-chlorin diastereomers needed long reaction time, high reaction temperature and generated 2 in relative low yield (40.6%), another synthesis pathway was explored (Scheme 2).

Scheme 2 Synthesis of the BCTC. Reagents and conditions: (a) NCCH₂CN, K₂CO₃, THF, reflux 7 h, 62.5%. (b) HCl_aq (37%), reflux 20 h, 65.2%.

Reaction between compound 1 and methylene compound malononitrile in the presence of K₂CO₃ in anhydrous THF at reflux afforded compound 3. Monitored by TLC, a cyclopropyl intermediate can be clearly observed, the intermediate compound underwent a ring-opening reaction with a second equivalent of malononitrile anion nucleophile to produce compound 3.²¹

In order to simplify the synthesis procedure, the reaction mixture of compound 1 to compound 3 was cooled, filtered and evaporated to affored the crude 3 without further purification. The crude 3 underwent hydrolysis in the presence of concentrate hydrochloric acid at reflux temperature to produce BCTC. However, high purity BCTC cannot be obtained by column chromatography or recrystallization from dichloromethane/petroleum ether. Therefore, crude 3 was purified by column chromatography to afford the tittle pure product (yield: 62.5%), and characterized by ¹H NMR, MALDI-TOF mass spectral analysis (Fig. S3–S4†). The pure 3 underwent hydrolysis in the presence of concentrate hydrochloric acid at reflux temperature to produce BCTC in 65.2% yield.

UV-visible absorption and fluorescence spectra

As shown in Fig. 1a, BCTC had the characteristic Soret and Q band absorptions at 421 nm (Soret), 521 nm, 548 nm, 598 nm and 652 nm (Q band) in DMSO solution, respectively. When excited at 421 nm, BCTC showed strong emission peaks at 653 nm and 718 nm (Fig. 1b).

Fig. 1 Spectra and the singlet oxygen quantum yield of BCTC. (a) UV-vis absorption spectrum of BCTC (10 μM in DMSO). (b) Fluorescence spectrum of BCTC, excition wavelength is 421 nm (10 μM in DMSO). (c) Photodecomposition of DPBF by ¹O₂ after irradiation of BCTC in DMF. (d) First-order plot for the photodecomposition of DPBF photosensitized by BCTC (monitoring the maximum absorption of DPBF at 410 nm).

Singlet oxygen quantum yield

The singlet oxygen quantum yield (Φ_Δ) value was determined by quenching the fluorescence intensity of DPBF. The disappearance of DPBF spectra were monitored at 410 nm using UV-vis spectrophotometer, the absorption intensity of DPBF (λ = 410 nm) continuously decreased with the irradiation time increasing (Fig. 1c). After the data were plotted as ln[DPBF₀]/[DPBF_t] versus irradiation time t, straight lines were obtained for the sensitizers, and the slope for each compound was obtained after fitting with a linear function (Fig. 1d). The ¹O₂ quantum yield Φ_Δ of BCTC in DMF was 0.68 which is higher than temoporfin (0.43).²³

BCTC cytotoxicity on human esophageal cancer cells

The effect of BCTC on the viability of cultured Eca-109 cells was evaluated by MTT assay. As shown in Fig. 2a, there was almost no dark cytotoxicity observed when exposed up to 10 μM BCTC, and the Eca-109 cells' viability was more than 80% when incubation with the highest concentration (10 μM) BCTC in dark. After irradiation with laser light (Fig. 2b), a significant cytotoxicity was detected. Low concentration (0.001 μM) caused moderate damage at the largest light dose (8 J cm⁻²). Raising the concentration from 0.001 μM to 10 μM, the cell viability was decreased sharply at the same light dose. Incubation with the same concentration BCTC, the cell viability was decreased with the increase of light dose. The data in Fig. 2 indicate that the efficacy of PDT depends on the irradiation energy and the concentration of BCTC. The cell viability of Eca-109 cells that were exposed to light without BCTC preincubation was similar to that of cells without any treatment (data were not shown).

Fig. 2 In vitro photodynamic activity of BCTC. (a) Dark toxicity of each concentration assayed. Data correspond to mean values ± SD from three different experiments. (b) Light dose dependence effect on Eca-109 cell viability after incubation with different concentration of BCTC for 24 hours, followed by irradiation. Data correspond to mean values ± SD from at least three different experiments.

Intracellular localization

The intracellular localization of BCTC in Eca-109 cells was investigated by confocal microscopy after incubating Eca-109 cells for 12 hours and staining them with fluorescent dye (Hoechst 33342) for cell nucleus. As shown in Fig. 3, BCTC was mainly found in cytoplasm and nuclear membranes corresponding to the red fluorescent and nucleus colocalized with the blue fluorescence of the nucleus probe.

Fig. 3 Intracellular location of BCTC in Eca-109 cells. Eca-109 cells were incubation with 5 μM BCTC for 4 hours in dark, and then stained with Hoechst 33342. Red fluorescence corresponds to BCTC, blue fluorescence represents the signal for Hoechst 33342.

In vivo PDT efficacy

The in vivo therapeutic efficacy of PDT using BCTC was evaluated in Eca-109 tumor bearing BABL/c nude mice. When tumor sizes had reached 5–7 mm in diameter, mice were given intravenous injection via tail vein of BCTC at a dose of 2.5 mg kg⁻¹. Then, the tumor site was irradiated with 100 J cm⁻², 650 nm, 180 mW cm⁻². As shown in Fig. S9,† in BCTC-PDT group, the tumors began swelling at 1 day post PDT. The tumors became dark, necrotic and a scar formed at 3 days post PDT. Then the scar of tumors fell off and normal healthy skin reconstructed 14 days post PDT. The volume of tumors in control group was larger than that in BCTC-PDT group at the same time. The volume growth curves of tumors were provided in Fig. 4, the tumor volume increased about 10-fold for 14 days in control group. BCTC-PDT decreased the tumor volume at the 5th day post treatment, and the tumor growth was slower than that in control group.

Fig. 4 Tumor volume at different time points post-PDT. The tumors in control group continued to grow and were significantly larger than in PDT-treated group after 6 days post-treatment. The date shown are the means ± SD of three independent experiments.

Furthermore, to confirm the PDT efficacy of BCTC for tumor therapy, histology analysis with hematoxylin–eosin (H&E) and Tunel reagents of tumor tissues were performed after irradiation with laser. As shown in Fig. 5, H&E and TUNEL staining clearing indicated severe apoptosis in large areas of tumor in BCTC-PDT group. The tumor cells from the control group were round in shape, and exhibited well-delineated margins with very limited signs of local infiltrations.

Fig. 5 Light microscopic observation of histology analysis with hematoxylin–eosin (H&E) and Tunel reagents of T24 tumor tissues. Severe apoptosis in large areas of tumor was observed in BCTC-PDT group.

Experimental

Materials and instrumentations

All solvents and reagents were obtained from commercial suppliers and used without further purification unless otherwise stated. Melting points were obtained on a “stuart” Bibby apparatus and are uncorrected. ¹H NMR and ¹³C NMR spectra were recorded on a Bruker 400 MHz spectrometer. Chemical shifts were reported as δ values relative to the internal standard tetramethylsilane. ESI-MS spectra were recorded on a Micromass triple quadrupole mass spectrometer. MADLI-TOF mass spectra were recorded on a AB SCIEX 4800 Plus MALDI TOF/TOF™. HRMS spectra were recorded on a Brucker Daltonics APEXIII 7.0 tesla FT mass spectrometer (Bruker Daltonik GmbH, Bremen, Germany). Column chromatography was performed using silica gel H (300–400 mesh). UV-vis absorption spectrum was recorded on an ultraviolet visible spectrophotometer (Model V-530, Japan). Fluorescence spectra were measured on a fluorescence spectrophotometer (FluoroMax-4, France).

Synthesis of trans-2,3-dihydro-2,3-bis(cyannomethyl)-5,10,15,20-tetraphenyl-chlorin (compound 2)

The mixture of sodium hydride (702 mg, 29.25 mmol) and methylcyanoacetate (3.25 mL, 36.56 mmol) in anhydrous DMSO (100 mL) was stirred at 60 °C. After 1 h, compound 1 (2 g, 3.04 mmol) was added in. The mixture was stirred at 80 °C for 3 h. Then H₂O (1.3 mL) and NaCl (1.78 g, 30.40 mmol) were added, and the temperature was increased to 140 °C. After 14 h, the mixture was cooled, diluted with dichloromethane (800 mL), washed with H₂O (6 × 200 mL), dried over anhydrous Na₂SO₄, filtered, and evaporated to dryness. The crude chlorin was purified by a silica gel column (ethyl acetate : petroleum ether = 1 : 10). The eluent was collected, condensed to give a purple red solid (856.9 mg, 40.6%). Mp > 300 °C. ¹H NMR (400 MHz, CDCl₃): δ ppm 8.67 (d, J = 4.7 Hz, 2H), 8.50 (s, 2H), 8.28 (dd, J = 17.0, 6.0 Hz, 6H), 7.78 (m, 16H), 4.91 (dd, J = 8.1, 4.2 Hz, 2H), 2.86 (dd, J = 17.2, 4.1 Hz, 2H), 2.57 (dd, J = 17.2, 8.3 Hz, 2H), −1.67 (s, 2H). MS (MALDI-TOF): m/z 695.4 [M + H]⁺.

Synthesis of trans-2,3-dihydro-2,3-bis(dicyannomethyl)-5,10,15,20-tetraphenyl-chlorin (compound 3)

A mixture of K₂CO₃ (672 mg, 4.83 mmol) and malononitrile (44 mg, 0.668 mmol) in anhydrous THF (10 mL) was stirred for 1 h at reflux under N₂. The reaction mixture was cooled to room temperature, and compound 1 (400 mg, 0.607 mmol) was added to the mixture. The mixture was allowed to stir for 6 h at reflux under N₂ until all starting material and intermediate cyclopropychlorin had disappeared. The reaction mixture was cooled, filtered and evaporated to dryness. The crude chlorin was purified by column chromatography over silica gel using 20% ethylacetate/petroleum ether as eluent to give purified compound 3 (282.4 mg, 62.5%) as a purple red solid. Mp > 300 °C. ¹H NMR (400 MHz, CDCl₃): δ ppm 8.74 (d, J = 4.9 Hz, 2H), 8.55 (s, 2H), 8.35 (t, J = 5.8 Hz, 4H), 8.27 (s, 2H), 8.03 (d, J = 6.4 Hz, 2H), 7.94 (d, J = 7.7 Hz, 4H), 7.90–7.83 (m, 4H), 7.77 (t, J = 10.8 Hz, 6H), 5.42 (d, J = 3.9 Hz, 2H), 4.39 (d, J = 3.9 Hz, 2H), −1.80 (s, 2H). MS (MALDI-TOF): m/z 745.4 [M + H]⁺.

Synthesis of trans-2,3-dihydro-2,3-bis(carboxymethyl)-5,10,15,20-tetraphenyl-chlorin (compound BCTC)

The mixture of compound 2 or compound 3 (0.2 mmol) and concentrate hydrochloric acid (10 mL) was refluxed for 20 h under N₂. The resulting mixture was cooled to room temperature, diluted with water (20 mL), neutralized with 1 mol L⁻¹ NaOH. The suspension was extracted with dichloromethane (3 × 20 mL). The combined organic layers were washed with water (3 × 40 mL) and dried over anhydrous Na₂SO₄. The organic solvent was removed under reduced pressure and the residue was performed by column chromatography over silica gel using 5% MeOH/CH₂Cl₂ as eluent to give purified chlorin BCTC as a purple red solid. Mp > 300 °C. UV-vis (DMSO): λ_max nm 421, 521, 548, 598, 652. ¹H NMR (400 MHz, DMSO-d₆): δ ppm 12.25 (s, 2H), 8.59 (d, J = 4.8 Hz, 2H), 8.34 (s, 2H), 8.25 (d, J = 7.2 Hz, 4H), 8.15 (d, J = 4.9 Hz, 2H), 7.95 (d, J = 7.2 Hz, 2H), 7.90–7.56 (m, 14H), 4.85 (dd, J = 9.7, 3.4 Hz, 2H), 2.60 (dd, J = 15.7, 3.3 Hz, 2H), 2.38 (dd, J = 14.3, 11.4 Hz, 2H), −1.71 (s, 2H). ¹³C NMR (100 MHz, DMSO-d₆): δ ppm 173.30, 166.73, 152.29, 141.66, 141.59, 140.76, 134.91, 134.13, 134.00, 132.88, 132.60, 128.54, 128.49, 128.38, 127.86, 127.48, 124.57, 122.69, 113.40, 49.38, 26.81. HRMS (ESI): m/z calcd for C₄₈H₃₇N₄O₄ [M + H]⁺, 733.2809; found, 733.2820.

Photophysical and photochemical measurements

Absorption and emission spectra. UV-visible absorption spectrum was recorded on an ultraviolet visible spectrophotometer (Model V-530, Japan). Fluorescence spectra were measured on a Fluorescence Spectrophotometer (FluoroMax-4, France). Slits were kept narrow to 1 nm in excitation and 1 or 2 nm in emission. Right angle detection was used. All the measurements were carried out at room temperature in quartz cuvette with path length of 1 cm. BCTC was dissolved in dimethyl sulfoxide (DMSO) to get 10 μM solution.

Singlet oxygen generation detection. The singlet oxygen ability of BCTC was monitored by chemical oxidation of 1,3-diphenylisobenzofuran (DPBF) in the DMF solution. BCTC (5 × 10⁻⁶ M) and DPBF (2 × 10⁻⁵ M) were mixed and irradiated. The reaction was monitored spectrophotometrically by measuring the decrease in optical density every 10 s at an absorbance maximum of 410 nm of DPBF. The natural logarithm values of absorption of DPBF at 410 nm were plotted against the irradiation time and fit by a first-order linear leastsquares model to get the singlet oxygen generation rate of the photosensitized process.²⁴ The singlet oxygen quantum yield (Φ_Δ) of BCTC in DMF was calculated using methylene blue as a standard.

In vitro experiments

Cell line and culture conditions. Human esophageal cancer cell line Eca-109 was obtained from the Type Culture Collection of the Chinese Academy of Sciences. All cell culture related reagents were purchased from Shanghai Ming Rong Bio-Science Technology Co., Ltd. Cells were cultured in normal RPMI-1640 culture medium with 10% fetal bovine serum (FBS), 50 units per mL penicillin and 50 μg mL⁻¹ streptomycin in 5% CO₂ at 37 °C.

MTT cell viability assay. Eca-109 cells were cultured in RPMI-1640 medium with 10% (v/v) FBS, collected with 0.25% (w/v) trypsin, and seeded in 96-well plates at 6 × 10³ cells per well. The cells were allowed to attach to the bottom of the wells for 24 hours prior to start the experiment. RPMI-1640 medium containing BCTC in different concentrations (range from 0.001 to 10 μM) was administered to cells and allowed to uptake for 24 hours. RPMI-1640 medium containing BCTC was removed and cells were washed with fresh PBS before irradiation with different light doses (range from 1 to 8 J cm⁻²) using an Nd:YAG laser at 650 nm. The cell viability was evaluated by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-terazolium bromide (MTT) colorimetric assay 24 hours after treatment. In parallel, non-irradiated cells were used to investigate the dark cytotoxicity.

Intracellular localization. Eca-109 cells grown on coverslips were incubated with the BCTC solution (5 μM) for 4 hours at 37 °C in the dark. Then removing the solution with ice-cold PBS three times, cells were stained with fluorescent dye (Hoechst 33342) for cell nucleus diluted in the culture medium without serum. After washing with PBS, coverslips were fixed for 10 min at −4 °C with 4% paraformaldehyde and cells were then examined by fluoresceence with a confocal microscope (LSM 410, Zeiss, Germany). BCTC was excited at 421 nm and its emitted light was monitored through a 600–700 nm band-pass filter, and Hoechst 33342 was excited at 350 nm and blue fluorescence was detected through a 450–500 nm band-pass filter.

In vivo experiments

Animal models. Five-week-old male BALB/c nude mice (5 weeks old) were anesthetized and 5 × 10⁶ Eca-109 cells were injected subcutaneously in 200 μL PBS into right forelimb. When implanted tumor sizes were more than 10 mm in diameter, tumors were excised and small pieces of the tumor (approximately 2 mm square pieces) were implanted subcutaneously into the right dorsal area of male BALB/c nude mice (5 weeks old). When tumor sizes had reached 5–7 mm in diameter after implantation (14–21 days), the male BALB/c nude mice were used for studies of PDT efficacy of BCTC.

In vivo PDT efficacy. When the tumor reached 5–7 mm in diameter, the mice were injected with BCTC at a dose of 2.5 mg kg⁻¹. Then the mice were restrained in plastic plexiglass holders without anesthesia and treated with laser light (650 nm, 100 J cm⁻², 180 mW cm⁻²). The power was monitored during the entire treatment. Post-PDT, the mice were observed daily for tumor regrowth or tumor cure. Visible tumors were measured using two orthogonal measurements L and W (perpendicular to L), and the volumes were calculated using the formula V = LW²/2 and recorded.

Histology examination. To confirm the PDT efficacy of BCTC for tumor therapy, histology analysis of tumor tissues was performed after irradiation with laser light (650 nm, 100 J cm⁻², 180 mW cm⁻²). Tumor tissues in the control group and PDT treatment groups were separated and fixed with 10% neutral buffered formalin and embedded in paraffin (n = 5). The sliced tissues were stained with hematoxylin–eosin (H&E) and Tunel reagents, and then examined under a microscope.

Human & animal welfare. All experiments were performed in triplicate and the data were expressed as mean plus and minus the standard error of the mean. Analysis of variance (ANOVA) and Student's t-test were used to determine the statistically significant difference among different groups when appropriate.

The experiments were carried out in accordance with the guidelines issued by the Ethical Committee of Donghua University.

Conclusions

In summary, a novel chlorin BCTC was synthesized via two different pathways and its photodynamic activities were evaluated in vitro and in vivo. It had an absorption peak at 652 nm and excellent photocytotoxicity but low dark cytotoxicity towards human esophageal cancer cells. As shown by confocal microscopy, the staining of the cytoplasmic and nuclear membrane through BCTC was detected. In vivo therapeutic efficacy of PDT using BCTC, after exposed to 650 nm laser light irradiation, the growth of Eca-109 tumor in nude mice was significantly inhibited. The overall results show that compound BCTC is a highly promising antitumor agent for photodynamic therapy.

Acknowledgements

The authors thank the Chinese National Natural Science Foundation (21372042, 81301878, 21402236), Foundation of Shanghai Science and Technology Commission (No. 15XD1523400, 14140903500, 15431904100, 201370, 13ZR1441000, 13ZR144090, 14ZR1439900, 14ZR1439800, 15ZR1439900, 13431900700, 14431906200) and Foundation of Songjiang Science and Technology Commission (No. 14SJGGYY08, 15SJGG45).

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c6ra01813k

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