Novel porphyrin–Schiff base conjugates: synthesis, characterization and in vitro photodynamic activities

Abstract

Three novel porphyrin–Schiff base conjugates derived from tetra(4-aminophenyl)porphyrin (TAPP), namely, tetra[4-(4-hydroxy benzylideneamino)]phenyl porphyrin (3a), tetra[4-(2-thienyldeneamino)]phenyl porphyrin (3b) and tetra[4-(2-pyridyldeneamino)]phenyl porphyrin (3c), were synthesized and characterized by IR, UV-vis, ¹H NMR, HRMS and elementary analysis. Their biological activities against human epidermoid carcinoma (A431) cells were evaluated with an MTT assay. As we expected, the porphyrin conjugates showed negligible cytotoxicity to A431 cells in the absence of light, while their phototoxic activities were improved after irradiated with LED lamp (425 nm) and increased significantly with increased doses. The fluorescence microscope pictures revealed that the three porphyrin–Schiff conjugates could diffuse into skin cancer cells, demonstrating that these compounds are potential candidates for photodynamic therapy agents.

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1. Introduction

Cancer is one of the deadliest diseases nowadays. The traditional cancer treatments, including surgery, chemotherapy and radiation therapy, may cause serious side effects resulting from the loss of normal organ function.^1,2 In contrast, photodynamic therapy (PDT) can selectively destroy malignant cells by the administration of a non-toxic photosensitizer and holds the potential for minimal side effects.^3,4 Upon irradiation with light of a specific wavelength, photosensitizers transfer energy to triplet oxygen (³O₂), and then generate singlet oxygen (¹O₂) and other reactive oxygen species (ROS) near the tumor. These generated molecules can cause intracellular oxidative damages and cell death through apoptosis or necrosis.^5,6

Porphyrins and their derivatives, as promising photosensitizers, have attracted extensive attention in the field of PDT for cancer treatment.^7–10 This is owing to the obvious advantages of porphyrins, such as favourable wavelength, intense absorption in the visible region of the electromagnetic spectrum and low intrinsic toxicity.¹¹ Moreover, it has been demonstrated that porphyrins prefer to accumulate in tumor cells in vitro and in vivo rather than in normal tissue. Notably, the first-generation photosensitizer Photofrin is a complex mixture of hematoporphyrin derivatives and has been approved by the United States Food and Drug Administration (FDA) for PDT treatment of various cancers, such as oesophageal cancer, early- and late-stage lung cancer, bladder cancer and malignant, nonmalignant and early-stage cervical cancer.^12–14 There has been growing interest in the synthesis and functionalization of porphyrins amongst chemists.^15,16

Schiff bases are an important class of organic compounds, and have been reported to have a wide range of biological activities, including antiviral, anticancer, cytotoxic, and antimicrobial.^17,18 Thus, the development and synthesis of novel Schiff base derivatives as potential chemotherapeutics has attracted the attention of organic and medicinal chemists.¹⁹ Furthermore, the hydroxyl group endows compounds with good solubility in aqueous environments, and is a commonly used hydrophilic moiety in dissolution of some photosensitizers. Thereby, porphyrin conjugates with hydroxyl groups are expected to show enhanced therapeutic efficacy in vitro.^20–22 Besides, heterocyclic rings, such as thiophene, furan and pyrrole, in the compounds have played a major role in expanding their pharmacological properties. For example, pregnenolone derivatives containing heterocyclic moieties were found to exhibit anticancer activity.^23,24 In addition, suitable photosensitizer candidates were obtained by attaching various substituents to the peripheral positions of the porphyrin core.

Herein, we report the design and synthesis of porphyrin–Schiff base conjugates bearing hydroxyl groups, thienyl and pyridyl groups on the porphyrin peripheral substituents positions 3a–3c (Scheme 1). In addition, we evaluated their cytotoxic activity against human skin cancer cell lines and normal skin cells. Furthermore, the photodynamic therapy and cellular uptake of the porphyrins in tumor cells were also investigated.

Scheme 1 Synthetic routes for TAPP and 3a–3c.

2. Experimental

2.1 Materials and instruments

¹H NMR spectra were collected in a Varian Inova 400 MHz NMR spectrometer. Deuterated dimethylsulfoxide (d₆-DMSO) was used as the solvent and TMS as the internal reference. Elemental analyses (C, H and N) were performed on a Vario EL-III CHNOS instrument. Mass spectra (MS) were recorded on a matrix-assisted laser desorption/ionization time of flight mass spectrometer (MALDI-TOF MS, Krato Analytical Company of Shimadzu Biotech, Manchester, Britain). FT-IR (4000–400 cm⁻¹) spectra were obtained with samples in KBr matrix for the title complexes on A BEQUZNDX-550 series FT-IR spectrophotometer. The UV-vis spectra were recorded on a Shimadzu UV1800 UV-vis-NIR spectrophotometer using CHCl₃ as the solvent. Fluorescence spectra were recorded in CHCl₃ with a HITACHI F-4500 spectrophotometer. Chromatographic purification was performed with silica gel (100–120 mesh). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) was purchased from Sigma-Aldrich Japan (Chiba, Japan). Pyrrole and DMF were distilled before use. All the other solvents and reagents were used without further purification.

2.2 Synthesis of porphyrins

2.2.1 Tetra(4-nitrophenyl)porphyrin (TNPP). The synthetic method for TNPP was presented in a previous report,^25,26 as shown in Scheme 1. In a 250 mL three-necked flask, p-nitrobenzaldehyde (5.5 g, 36 mmol) and acetic anhydride (6 mL, 64 mmol) were added to a refluxing mixture of propionic acid (150 mL), then freshly distilled pyrrole (2.5 mL, 36 mmol) in 10 mL of propionic acid was dropwise added (ca. 30 min) to the mixture with stirring. The reaction mixture was brought to reflux for 2 h. The mixture was allowed to cool overnight for efficient precipitation, and a dark residue was collected by filtration. The dark solid was washed with water and dried under vacuum. The solid was purified in 40 mL of refluxing pyridine for 1 h. After that, the solution was cooled to room temperature and stored at −4 °C overnight. The mixture was then filtered under vacuum and washed with acetone until the eluent became colorless. The yield of the purified red brown product was 12%.

2.2.2 Tetra(4-aminophenyl)porphyrin (TAPP). The above-obtained TNPP (1.5 g, 1.9 mmol) was dissolved in 75 mL of concentrated HCl solution, and the aqueous solution was heated to 75–80 °C in a water bath. Then another concentrated HCl solution (25 mL) with SnCl₂·2H₂O (10.0 g, 44 mmol) was added slowly into the aforementioned solution under stirring, followed by heating to 75–80 °C for 2 h. After finishing the heating treatment, the reaction mixture was cooled with a cold-water bath and then an ice bath, and neutralized to weak base by adding concentrated NH₃·H₂O slowly. Then solid was collected by filtration, and mixed vigorously with 5% NaOH (150 mL). The mixture was filtered and washed with distilled water repeatedly. The residue was extracted by Soxhlet extraction with 200 mL of chloroform and then the solvent was removed under vacuum. The crude product was purified by SiO₂ column chromatography (CH₂Cl₂/EtOH = 2 : 1) to give purple porphyrin TAPP. Yield: 30.0%. Mp > 250 °C; anal. calcd for C₄₄H₃₄N₈ (%): C, 78.32; H, 5.08; N, 16.60. Found: C, 77.89; H, 5.14; N, 16.27. MS, m/z: 676.00 [M + H]⁺ amu; UV-vis (CHCl₃) λ_max (nm): 428 (Soret band), 523 (Q_I), 563 (Q_II), 604 (Q_III), 655 (Q_IV); IR (KBr) (cm⁻¹): 3443 (ν_{vs N–H}, amino), 3375 (ν_N–H, pyrrole), 1512 (δ_N–H, amino), 1465 (ν_C=N, pyrrole), 1288 (ν_C–N, amino). ¹H NMR (d₆-DMSO, 300 MHz): H, ppm 8.89 (s, 8H, β position of the pyrrole moiety), 7.86 (d, 8H, ArH), 7.01 (d, 8H, ArH), 5.58 (s, 8H, amine), −2.74 (s, 2H, inner NH).

2.2.3 General procedure for the synthesis of the porphyrin–Schiff base conjugates 3a–3c. TAPP (0.2 g, 0.2959 mmol), dissolved in 20 mL of anhydrous ethanol, was heated to reflux, and another ethanol solution (30 mL) with aldehyde (9 mmol) was added dropwise into the solution, followed by refluxing the mixture for 3 h. The progress of the reaction was monitored by TLC. After finishing the reaction, the mixture was fully cooled down and the precipitate was filtered off and washed with ethanol repeatedly. The solid was dried under vacuum. The obtained violet product was dissolved in dichloromethane and purified by column chromatography (the silica gel column was pretreated with alkaline triethylamine to prevent the dissociation of the product) using a binary eluent of dichloromethane and ethanol.
Tetra[p-(p-hydroxy benzylideneamino)]phenyl porphyrin (3a). Compound 3a was prepared from TAPP (0.2 g, 0.2959 mmol), and p-hydroxybenzaldehyde (1.1 g, 9 mmol) according to the above given procedure. Yield 72%. Mp > 250 °C; anal. calcd for C₇₂H₅₀N₈O₄ (%): C, 79.25; H, 4.62; N, 10.27. Found: C, 78.91; H, 4.27; N, 9.91. MALDI-TOF-MS: m/z 1091.4018 for [M + 1]⁺ (calcd 1091.3955); UV-vis (CHCl₃) λ_max (nm): 426 (Soret band), 521 (Q_I), 559 (Q_II), 603 (Q_III), 654 (Q_IV); IR (KBr) (cm⁻¹): 3314 (ν_N–H, pyrrole), 3423 (ν_O–H, phenyl), 1580 (ν_C=N, Schiff base), 1512 (ν_C=C, phenyl), 1159 (δ_C–N, pyrrole). ¹H NMR (d₆-DMSO, 300 MHz): H, ppm 10.22 (s, 4H, –OH), 8.92 (s, 8H, β position of the pyrrole moiety), 8.83 (s, 4H, Schiff base), 8.20–8.22 (d, J = 7.1, 8H, phenyl), 7.94–7.96 (d, J = 7.1, 8H, phenyl), 7.62–7.64 (d, J = 7.1, 8H, phenyl), 6.97–6.99 (d, J = 7.8, 8H, phenyl), −2.83 (s, 2H, inner NH).
Tetra[p-(2-thienyldeneamino)]phenyl porphyrin (3b). Compound 3b was prepared from TAPP (0.2 g, 0.2959 mmol), and 2-thenaldehyde (1.0 g, 9 mmol) according to a procedure similar to that for compound 3a. Yield 48%. Mp > 250 °C; anal. calcd for C₆₄H₄₂N₈S₄ (%): C, 73.11; H, 4.03; N, 10.66. Found: C, 73.58; H, 3.95; N, 10.28. MALDI-TOF-MS: m/z 1051.4948 for [M + 1]⁺ (calcd 1050.2415); UV-vis (CHCl₃) λ_max (nm): 425 (Soret band), 519 (Q_I), 558 (Q_II), 596 (Q_III), 651 (Q_IV); IR (KBr) (cm⁻¹): 3445 (ν_N–H, pyrrole), 1618 (ν_C=N, Schiff base), 1585 (ν_C=C, phenyl), 1169 (δ_C–N, pyrrole), 716 (ν_C–S, thienyl). ¹H NMR (d₆-DMSO, 300 MHz): H, ppm 9.18 (s, 4H, Schiff base), 8.94 (s, 8H, β position of the pyrrole moiety), 8.22–8.26 (d, J = 7.3, 8H, phenyl), 7.92–7.94 (d, J = 5.0, 4H, thienyl), 7.85 (s, 4H, thienyl), 7.72–7.74 (d, J = 7.3, 8H, phenyl), 7.33 (s, 4H, thienyl), −2.84 (s, 2H, inner NH).
Tetra[p-(2-pyridyldeneamino)]phenyl porphyrin (3c). The compound 3c was also prepared from compound TAPP (0.2 g, 0.2959 mmol), and 2-pyridinecarboxaldehyde (1.0 g, 9 mmol) according to aforementioned procedures. Yield 72%. Mp > 250 °C; anal. calcd for C₆₈H₄₆N₁₂ (%): C, 79.20; H, 4.50; N, 16.30. Found: C, 78.97; H, 4.27; N, 16.46. MALDI-TOF-MS: m/z 1032.3773 for [M + 1]⁺ (calcd 1031.1732); UV-vis (CHCl₃) λ_max (nm): 420 (Soret band), 520 (Q_I), 558 (Q_II), 596 (Q_III), 649 (Q_IV); IR (KBr) (cm⁻¹): 3437 (ν_N–H, pyrrole), 3053 (ν_C–H pyridyl), 1628 (ν_C=N, Schiff base), 1569 (ν_C=C, phenyl), 1173 (δ_C–N, pyrrole), 800 (δ_C–H, pyridyl). ¹H NMR (d₆-DMSO, 300 MHz): H, ppm 8.93–8.98 (dd, J = 6.5 J = 6.2, 8H, β position of the pyrrole moiety), 8.83 (s, 4H, Schiff base), 8.31–8.37 (m, 8H, phenyl), 8.03–8.09 (d, J = 7.5, 4H, pyridyl), 7.88–7.94 (m, 4H, pyridyl), 7.83 (s, 8H, phenyl), 7.72 (t, 4H, pyridyl), 7.63 (t, 4H, pyridyl), −2.83 (s, 2H, inner NH).

2.3 Photodynamic activity experiments in vitro

2.3.1 Cell culture and incubation conditions. The A431 cells and human keratinocyte (HaCaT) cells were cultured in a humidified incubator at 37 °C with 5% CO₂ in DMEM/F-12 medium supplemented with 10% fetal bovine serum, 100 U mL⁻¹ penicillin and 100 μg mL⁻¹ streptomycin. The medium was replaced with fresh cell culture medium every 48 h. When ∼80% the confluence was reached, the medium was removed and washed with 5 mL of phosphate buffer saline (PBS) three times, and then cells were replaced with fresh medium for subculture. The porphyrin–Schiff base conjugates were dissolved in DMSO at a concentration of 5 mM as the stock solution and then diluted with complete medium to the required concentration immediately prior to use. The final DMSO concentration was always below 0.5% v/v, which showed no effects on cell viability.

2.3.2 Cellular treatment with porphyrins 3a–3c. To evaluate the cytotoxicity of compounds 3a–3c in dark (cytotoxicity without light exposure), cells in the exponential growth phase were seeded at a density of 2 × 10⁴ cells per mL in 96-well culture plates containing 200 μL of culture medium per well. The cells were then incubated for 24 h at 37 °C to allow for cell adherence. The medium was then replaced with the 3a–3c solutions in cell culture medium (10 μM) and incubated for 24 h in the dark. Every plate had three wells without complexes as the control and cells treated with each compound were assessed in triplicate. HaCaT cells were treated in the same conditions just for comparison. Phototoxicity experiments were performed with A431 cells in 96-well plates similar to the procedures described above. Subsequently, the compound solutions were removed, and the cells were washed with 5 mL of PBS three times and exposed to blue light.

2.3.3 Irradiation. Irradiation was carried out using a blue light source, which was an LED equipped with a 150 W lamp. A wavelength range between 400 and 450 nm was selected using optical filters. The light intensity at the treatment site was 100 mW cm⁻². Exposure times were 0 s and 480 s, in which cells were delivered 0 J cm⁻² and 48 J cm⁻².

2.3.4 Determination of cytotoxicity and phototoxicity of the porphyrin–Schiff base conjugates. The determination of cell survival was documented by MTT assay.^27,28 Briefly, this method is based on the reduction of MTT to a coloured formazan compound (λ_max = 570 nm) by the active mitochondrial hydrogenase in living cells. After 24 h incubation with each compound, 20 μL of MTT (5 mg mL⁻¹) in PBS was added to each well followed by incubation for another 4 h in a humidified incubator with 5% CO₂ at 37 °C. The MTT solution was then removed and 150 μL of DMSO was added to each well to lyse the released formazan dye. The data were recorded by optical density (OD) at 570 nm. The relative cell viability was expressed as the OD absorbance of the tested compound versus that of the control. The same procedures were carried out without irradiation to determine the toxicity of normal skin cells in the dark.

2.4 Cellular uptake of porphyrin conjugates

The HaCaT and A431 cells were seeded on 24-well culture plates in complete culture medium and allowed to adhere overnight. The porphyrin–Schiff base conjugates (10 μM) were added to each well and incubated at 37 °C in the dark for 24 h followed by washing twice with PBS. The cellular uptake of porphyrin–Schiff base conjugates was examined under fluorescence microscopy with a filter set at 535 nm excitation light (BP 515–560, FT 580, LP 590).

3. Results and discussion

3.1 Synthesis of porphyrin–Schiff base conjugates

Three novel porphyrin–Schiff conjugates 3a–3c were successfully synthesized according to the synthetic route in Scheme 1 and characterized by elemental analysis, mass spectroscopy, UV-vis spectroscopy and ¹H NMR spectroscopy. In order to get the target porphyrin–Schiff base conjugates, TNPP was firstly obtained through the reaction of pyrrole and p-nitrobenzaldehyde with a molar ratio of 1 : 1 under refluxing propionic acid by Adler's strategy. Then TNPP was reduced by SnCl₂·2H₂O in concentrated HCl solution to get the key intermediate TAPP. Finally, the three conjugates were obtained from condensation of amine groups of TAPP with different aldehydes in a mixture of dichloromethane and ethanol under reflux. The formation of the C=N bond can be greatly accelerated by addition of anhydrous magnesium sulfate to the solution, which improved the yields of conjugates in the meanwhile.

The synthesis of three porphyrin–Schiff base conjugates was accomplished using an easy-operation and fast reaction, while their separation and purification was difficult by column chromatography because of the breakdown of the C=N bond in acid medium. Therefore, the silica gel column was treated with alkaline triethylamine in advance to prevent the dissociation of the product and exhibit pretty good results.

3.2 Spectral properties of porphyrin–Schiff base conjugates

The UV-vis absorption spectra of 3a–3c in dichloromethane solution showed one intense Soret band around 426 nm, followed by four weak Q-bands in the visible spectral region of 519–652 nm (Fig. 1). Their spectra data were collected and summarized in Table 1. It can be seen that the absorption peaks of porphyrins 3a–3c exhibited no obvious wavelength difference and showed slightly blue shifts compared with TAPP. This might be attributed to the chemical groups changing from primary amines to Schiff bases. The electronic effect from the –N=C– and phenyl groups in the porphyrin–Schiff base conjugates was similar to that of amino groups and phenyl groups in TAPP,²⁹ and in the meanwhile the p–π conjugation between N atoms and the porphyrin ring was weakened, which caused the blue shifts of the Soret and Q bands. Furthermore, the changes were almost the same, which indicated that the position of the peripheral substituents was far from the porphyrin ring and had little effects on the distribution of electrons. Therefore, it did not produce noticeable effects upon the UV-vis absorption properties of TAPP.²⁶

Fig. 1 UV-vis absorption spectra of TAPP and 3a–3c.

Table 1 Photophysical data of TAPP and 3a–3c (CHCl₃, 25 °C)

Compounds	Soret band (nm)	Q bands (nm)	λem (nm)
TAPP	428.0	523.0, 563.5, 598.0, 656.0	665
3a	426.5	520.5, 559.0, 594.5, 652.0	657, 711
3b	426.5	520.0, 557.0, 594.0, 650.5	655, 712
3c	425.5	519.5, 557.0, 593.5, 651.0	655, 712

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The fluorescence emission spectra of TAPP and 3a–3c in chloroform were obtained upon excitation at λ_ex = 415 nm, as shown in Fig. 2. All compounds gave their emission spectra with different λ_em (Table 1) and the λ_em of the porphyrin conjugates were shorter than that of TAPP, which was owing to the effect of the peripheral substituents around the porphyrin chromophores. After changing from primary amine to Schiff base, the p–π conjugation between N atoms and the porphyrin ring was changed to π–π conjugation and the decrease of the π-electron cloud density of the porphyrin ring resulted in a blue shift of λ_em. Comparing with 3b and 3c, the outermost substituted hydroxyl groups (–OH) in compound 3a contributed to the electron cloud density of the conjugated porphyrin ring by electron donating effects, making the longer emission wavelength. New weak emission peaks of the porphyrin conjugates around 712 nm appeared, while those peaks are absent on the spectra of TAPP, which might be attributed to electron transition from the central H atom to the conjugated system of porphyrins.^29,30

Fig. 2 The fluorescence spectra of TAPP and 3a–3c. Concentrations of the porphyrins were 1 × 10⁻⁶ mol L⁻¹ in CHCl₃.

The vibrational spectroscopy can provide ample information about the structure of porphyrin. The tentative IR assignments of the TAPP and porphyrin–Schiff base conjugates are reported in Table 2. The characteristic amino bands were found in TAPP at 1288 and 3443 cm⁻¹, while the characteristic amino bands were absent in compounds 3a–3c. However, three new bands ascribed to the stretching vibration of –C=N– at 1580, 1618 and 1628 cm⁻¹ were observed in the IR spectra of compounds 3a–3c, respectively, which indicated that the expected porphyrin–Schiff base conjugates were successfully synthesized by the condensation of TAPP with the selected p-hydroxybenzaldehyde, 2-thenaldehyde and 2-pyridinecarboxaldehyde, respectively. A broad band that appeared at 3423 cm⁻¹ in the spectra of 3a was assigned to ν_O–H of the phenolic. The band at 716 cm⁻¹ was ascribed to the ν_C–S of the thiophene group of 3b. The typical C–H peak of the pyridine ring of 3c was observed at 3053 and 800 cm⁻¹. In addition, all of the obtained compounds, including TAPP and porphyrin–Schiff base conjugates, showed a broad absorption band in the range 3445–3314 cm⁻¹, which belonged to the ν(NH) of the porphyrin ring.

Table 2 ¹H NMR and IR spectral bands of TAPP and 3a–3c

Comp.	^1H NMR spectra (ppm)				IR spectra (cm^−1)
	δinnerNH	δβ–H	δAr–H	δNH or δCH=N	νNH	νC=N (imine)	νC=N (ring)
TAPP	−2.74	8.89	7.01–7.86	5.58	3375	—	1465
3a	−2.83	8.92	6.97–8.22	8.83	3314	1580	1468
3b	−2.84	8.94	8.22–8.26	9.18	3445	1618	1425
3c	−2.83	8.94	7.82–8.37	8.83	3437	1628	1470

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¹H NMR spectra of the compounds (TAPP and 3a–3c) were recorded in d⁶-DMSO and the peak assignment is summarized in Table 2. As for TAPP, a sharp singlet was observed at 5.58 ppm and was assigned to the H-atoms of the NH₂ groups. This amino proton signal was absent in the ¹H NMR spectra of compounds 3a–3c. On the contrary, the azomethine protons (–CH=N–) appeared as a singlet at 8.83–9.18 ppm, which revealed that the porphyrin–Schiff base derivatives were successfully synthesized by the condensation reaction. In addition, ¹H NMR spectra of compound 3a showed a peak at 10.22 ppm, which belonged to the hydroxyl protons of the phenol.³¹ Similarly, the multiplets for thiophene protons of 3b and pyridyl protons of 3c were detected in the range of 7.33–7.94 ppm and 7.63–8.09 ppm, respectively.³² Furthermore, the broad multiplet observed in the range 6.97–8.37 ppm for all of the obtained compounds was assigned to the aromatic protons. The ¹H NMR spectra of TAPP and the porphyrin–Schiff base derivatives all exhibited a weak singlet at approximately −2.83 ppm, which was the characteristic signal of the inner protons of the porphyrin ring. The formation of all compounds was also confirmed by their mass spectral data given in the Experimental section.

3.3 Photostability of compounds

The rate of photodegradation exhibited by a compound under light conditions is a very important parameter to assess when we consider their potential application as photosensitizers.^33,34 In order to establish if porphyrin–Schiff base conjugates 3a–3c could undergo photobleaching, we performed photostability studies in the same conditions of irradiation used for photocytotoxicity in the A431 cells (fluence rate 100 mW cm⁻²). The UV-vis and fluorescence emission spectra of each porphyrin (1 μM) were measured after different times of irradiation (0, 5, 10, 15, 20, 25, and 30 min). However, we observed minimal decay of absorption and emission intensity. As an example, the UV-vis and fluorescence emission spectra of porphyrin–Schiff base conjugate 3a at different irradiation times are shown in Fig. 3. The results point out that all compounds (3a–3c) showed high photostability under the conditions of the experiment, which is the desired property of a PDT photosensitizer.

Fig. 3 UV-vis absorbance spectra (A) and emission spectra (B) of porphyrin 3a after different irradiation times.

3.4 Cytotoxicity assay

The cytotoxicity of the porphyrins 3a–3c was evaluated by two kind of human skin cell lines, human keratinocyte HaCaT and epidermoid carcinoma A431. The cells seeded in well plates were treated with 10 μM of 3a–3c solutions in the dark. As shown in Fig. 4, the relative cell viability of A431 cells for 3a–3c was in the range of 74.8–75.5%, while that of HaCaT cells for 3a–3c maintained from 86.5% to 109.8%. From the histogram of the cytotoxicity comparison in Fig. 4, it was not hard to see that the cell viability of HaCaT and A431 cells was almost the same and maintained around 92% and 82% when cultured with TAPP, revealing that TAPP was almost harmless to both normal cells and cancer cells. However, porphyrins 3a–3c showed higher cytotoxicity in dark to A431 cancer cells when compared with TAPP, especially for porphyrin 3c, which showed the highest cytotoxicity with the A431 cell viability of 74.8%. Conversely, negligible cytotoxicity was detected on HaCaT cells for compounds 3a–3c with cell viability of over 80% and even exceeding 100%. It was worth noting that 3b and 3c were observed without cytotoxicity in dark for normal HaCaT cells and 3a showed very slight cell damage. The cell survival histogram demonstrated that the synthesized porphyrin conjugates were more effective to kill the cancerous A431 cells than the normal HaCaT cells at a concentration of 10 μM after 24 h of incubation and the antitumor activities of all of the porphyrin–Schiff base conjugates were evidently higher than that of TAPP with the same culture conditions. The results were attributed to the presence of the Schiff base, which showed conspicuous inhibitory activity to the growth of cancer cells.

Fig. 4 Cytotoxicity of TAPP and 3a–3c on A431 cells and normal skin cells.

3.5 Photodynamic effect

In vitro phototoxicity studies were performed using A431 cells. The A431 cells adhered on the culture plate were incubated with porphyrins conjugates 3a–3c solutions at a concentration of 10 μM for 24 h. After finishing the incubation, the samples were irradiated with blue light with a wavelength of 400–450 nm, a fluence rate of 100 mW cm⁻² and light doses from 0 to 48 J cm⁻². Cell viability was determined by MTT assay. As shown in Fig. 5, all of the tested compounds exhibited strong photocytotoxicity after exposure to the blue light for 0–480 s, and the cell viability decreased dramatically with the increase of the light dose. The cell survival reduced sharply at the beginning of irradiation, and the decreasing tendency of cell viability levelled off when the irradiation dose exceeded 12 J cm⁻².

Fig. 5 Relative cell viability of A431 cells irradiated with light doses from 0 to 48 J cm⁻².

The cells lines treated with 3a–3c at different concentrations were irradiated with blue light for 360 s. The compounds showed remarkable photodynamic effects at all concentrations. Fig. 6 showed the histogram of the cell viability at different compound concentrations, which indicated that the viability of tumoral A431 cells reduced significantly with the increasing porphyrin concentration. The IC₅₀ values, defined as the porphyrin concentration at which 50% of tumor cells were killed after the photoirradiation as compared to the control, were calculated based on the results of Fig. 6 and are summarized in Table 3. From the IC₅₀ values listed in Table 3, it was obvious that the conjugate 3a was the most phototoxic under the above irradiation conditions with an IC₅₀ of 0.57 μM, which was followed by 3b with an IC₅₀ of 1.19 μM and then 3c with an IC₅₀ of 1.31 μM. Notably, 3a showed a 2.4 times enhanced photocytotoxicity compared to that for TAPP, which might be attributed to the attached phenolic group. The phenolic group is easily oxidized to a benzoquinonyl group in cellular metabolic pathways,^35–37 which finally causes obvious cell damage.

Fig. 6 Relative cell viability of A431 cells at different concentrations of porphyrins TAPP and 3a–3c.

Table 3 IC₅₀ values of TAPP and 3a–3c for A431 cells

Compounds	IC50 (μM)
TAPP	1.36
3a	0.57
3b	1.19
3c	1.31

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3.6 Cellular uptake of porphyrin conjugates

It is a quite crucial question as to whether photosensitive compounds could penetrate into tumor cells for the therapeutic effect of PDT. Cellular uptake of porphyrins could be directly observed by fluorescence microscope without fluorescence labeling^38–40 because porphyrins uptaken by tumor cells would emit red fluorescence when excited. Based on this, we could judge whether the porphyrins entered the cells. In the fluorescence microscopic images (summarized in Fig. 7), it was obvious that all the compounds could enter the A431 cells causing intracellular fluorescence. The 3b and 3c samples displayed slightly brighter red fluorescence spots, while cells incubated with 3a did not reveal enough fluorescence plots. These findings indicated that the conjugates 3a–3c were different in cellular uptake and not in the nucleus. Furthermore, these intracellular levels of conjugates resulted in different photodynamic activities. Interestingly, the conjugate 3a with the greatest photodynamic activity corresponded with the lowest intracellular uptake of all the conjugates tested.

Fig. 7 Fluorescence microscopy of A431 cells incubated with TAPP and 3a–3c (10 μM, 24 h) (Left) white light and (Right) fluorescence.

4. Conclusion

In conclusion, three novel porphyrin conjugates 3a–3c were synthesized with excellent yields and their biological activities were evaluated by A431 cells with MTT assay. The conjugates 3b and 3c exhibited high selective inhibition against A431 cells in the absence of light. The results revealed that these conjugates could easily and selectively accumulate in tumor cells and had more cytotoxic effects on them. Incubation of A431 cells gave rise to an efficient cellular uptake of porphyrin conjugates, which was determined by fluorescence microscopy. The fluorescent image revealed that cells displayed an uptake of conjugates and appeared to be localized in the cytoplasm. The result was consistent with the strong photocytotoxic activity of porphyrin compounds after irradiation. The facts indicated that these porphyrin conjugates were potential candidates for anticancer drugs in PDT, but their promising behavior needed further investigation in both in vitro and in vivo experiments.

Acknowledgements

The authors acknowledge the research grant provided by the National Nature Science Foundation (21271148).

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