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Mn porphyrins as efficient photosensitizers for magnetic resonance imaging and photodynamic therapy
Dyes and Pigments 166 (2019) 189–195
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Mitochondria-targeting Pt/Mn porphyrins as efficient photosensitizers for magnetic resonance imaging and photodynamic therapy
T
Mengqian Yanga, Jingran Denga, Ding Guoa, Qi Sunc,∗∗, Zejiang Wanga, Kai Wangb, Fengshou Wua,∗ a Key Laboratory for Green Chemical Process of the Ministry of Education, School of Chemical Engineering and Pharmacy, Wuhan Institute of Technology, Wuhan, PR China b Hubei Collaborative Innovation Center for Advanced Organic Chemical Materials, College of Chemistry and Chemical Engineering, Hubei University, Wuhan, PR China c School of Chemistry and Environmental Engineering, Wuhan Institute of Technology, Wuhan, PR China
A R T I C LE I N FO
A B S T R A C T
Keywords: Photodynamic therapy Magnetic resonance imaging Mitochondria-targeted Porphyrin
Photodynamic therapy (PDT), as a minimally invasive and highly efficient anticancer approach, has received extensive attention. However, the search for more effective and less toxic photodynamic photosensitizers with good biocompatibility and high specificity are still highly expected. Herein, we synthesized and characterized a new porphyrin-based photosensitizer Pt-MnPor-PPh3, which integrated the magnetic resonance imaging (MRI) and fluorescence diagnostics and mitochondrial-targeted photodynamic therapy. The cell viabilities reduced with the increase of photosensitizer dose. The IC50 of Pt-MnPor-PPh3 was calculated to be 30.74 μg/mL under light irradiation for 10 min. In contrast, the cell viabilities of the control group, HeLa cells treated without laser irradiation, were still over 80% even at 50 μg/mL of Pt-MnPor-PPh3. The cellular uptake and subcellular localization of the conjugate were further evaluated through a confocal laser scanning microscope. The results showed that the conjugate had good mitochondrial-targeted properties in the cancer cells. Moreover, the presence of paramagnetic metal Mn ions in Pt-MnPor-PPh3 endowed them with good MRI performances, which provided a feasible synthetic strategy to develop new medicines for MRI-guided and organelle-targeted PDT applications in cancer theranostics.
1. Introduction It is well known that early accurate diagnosis of cancer not only improves patient survival, but also helps clinicians determine the most appropriate treatment strategy for the patient, thereby avoiding underor over-treatment. Therefore, it is urgent to establish an early diagnosis method for cancer with high sensitivity and specificity [1]. Among the imaging technologies, magnetic resonance imaging (MRI) has been widely used for early diagnosis of cancer due to its non-invasive, high spatial resolution and three-dimensional imaging advantages [2–7]. MR often relies on ‘‘contrast-enhancing agents’’ to improve inherent contrast between normal and diseased tissue. MR signal intensity or signal intensity differences (contrast) are dependent upon longitudinal (1/T1) and transverse relaxation rates (1/T2) of tissue protons. Agents containing paramagnetic ions had been shown to effectively alter longitudinal (1/T1) and/or transverse relaxation rates (1/T2) by changing the local magnetic field when they come in close proximity to water
∗
protons [8]. The coordination of porphyrin with manganese exhibited excellent MRI performance due to the planar structure of porphyrin, which allowed the water molecules to fully contact the paramagnetic metal manganese [9,10]. More importantly, the manganese complexes showed lower toxicity than that of commercially available contrast agent Gd-DTPA [11]. On the other hand, photodynamic therapy (PDT) has become one of the most promising non-invasive and safe treatment options for cancer treatment [12–15]. As a highly localized treatment, PDT involves focusing extracorporeal light on photosensitizers (PSs) in disease tissue in the body to produce reactive oxygen species (ROS) that effectively and selectively destroy diseased tissue without damage to surrounding healthy tissue [16–18]. Although PDT is widely employed in most countries with good effect for two decades, the selectivity of commercial PDT agents for tumor tissues still offers considerable opportunities for further improvement [19–21]. In the organelles of mammalian cells, mitochondria are
Corresponding author., Corresponding author. E-mail addresses: [email protected] (Q. Sun), [email protected], [email protected] (F. Wu).
∗∗
https://doi.org/10.1016/j.dyepig.2019.03.048 Received 25 December 2018; Received in revised form 16 March 2019; Accepted 18 March 2019 Available online 20 March 2019 0143-7208/ © 2019 Elsevier Ltd. All rights reserved.
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2.3. Synthesis of triphenylphosphonium-conjugated porphyrin (Por-PPh3)
indispensable organelles responsible for producing the energy supply of most cells. They also play a key role in programmed cell death, such as apoptosis, and are easily damaged by ROS formation [22,23]. When the mitochondria became carcinogenic, the potential of mitochondrial membrane increased, which will induce preferential accumulation and retention of cationic mitochondrial targeting agents in cancer cells comparing with normal cells [24,25]. Thus, in order to deliver the PSs to the mitochondrial of cancer cells, the lipophilic cations, such as triphenylphosphine could be introduced to the photosensitizer as they can cause them to undergo passive diffusion and benefit from the electrochemical potential present between the inner and outer layers of the cell membrane. The electrochemical potential energy facilitates the diffusion of cations, thereby enhancing their cellular uptake. Therefore, conjugating a PDT agent with a cationic mitochondrial targeting agent can result in rapid damage to cancer cells, improve therapeutic effects, and reduce unnecessary side effects [26]. Herein, we designed and synthesized a multifunctional porphyrinbased photosensitizer Pt-MnPor-PPh3 that integrated MRI and fluorescence diagnostics and mitochondrial-targeted photodynamic therapy. Specifically, a lipophilic triphenylphosphonium cation (TPP) as a good mitochondria-targeted agent was conjugated with manganese porphyrin (MnPor) through an alkyl chain. The MnPor-TPP, bearing pyridine ring, was further coordinated with transplatin to yield Pt-MnPorPor. Pt-MnPor-Por displayed high ROS yield due to the heavy atomic effect of platinum [27]. Due to the presence of paramagnetic metal manganese, the as-synthesized Pt-MnPor-PPh3 showed good magnetic resonance imaging performance. Finally, the cytotoxicity and subcellular localization of the Pt-MnPor-PPh3 were evaluated through MTT method and confocal laser scanning microscope, respectively.
The Por 2 (100 mg, 0.157 mmol) was dissolved in a mixture of DMF and anhydrous potassium carbonate (44 mg, 0.314 mmol). After stirring for 30 min at room temperature, (4-Bromobutyl)triphenylphosphonium bromide (113 mg, 0.236 mmol) was added dropwise to the resulting solution within 30 min. The mixture was then stirred and heated to reflux for 4 h. After filtration, the filtrate was evaporated in vacuo and the residue was purified by silica gel column chromatography (MeOH in CH2Cl2, 1–2% v/v) to get the desired product, yield 70%. 1H NMR (400 MHz, CDCl3) δ 9.03 (d, 3J = 5.7 Hz, 6H, CH pyridyl), 8.93 (d, 3J = 4.7 Hz, 2H, β-pyrrole), 8.82 (s, 4H, β-pyrrole), 8.79 (d, 3J = 4.8 Hz, 2H, β-pyrrole), 8.08 (d, 2H, CH phenoxy), 8.12 (d, 3J = 8.3 Hz, 6H, CH pyridyl), 7.78 (m, 15H, CH Phenyl), 7.53 (d, 2H, CH phenoxy), 4.41 (d, 2H, OCH2), 4.09 (t, 2H, CH2), 3.77 (t, 2H, CH2), 1.85 (d, 2H, CH2), −2.90 (s, 2H, NH); MALDI-TOF MS: calcd for: 950.371, found: 950.380; UV–Vis (DMF): λ = 418, 514, 549, 589, 646 nm. 2.4. Synthesis of bromobutane porphyrin (Por-Butyl) The Por 2 (100 mg, 0.157 mmol) was dissolved in a mixture of DMF and anhydrous potassium carbonate (43.4 mg, 0.314 mmol). After stirring for 30 min at room temperature, bromobutane (25.4 μL, 0.236 mmol) was added dropwise to the solution. The mixture was stirred and heated to reflux for another 4 h. The solid was removed by filtration, and the filtrate was evaporated in vacuo. The residue was then purified through silica gel column chromatography (MeOH in CH2Cl2, 1–2% v/v) to get the desired product, yield 85%. 1H NMR (400 MHz, CDCl3) δ 9.03 (d, 6H, CH pyridyl), 8.95 (d, 2H, β-pyrrole), 8.85 (s, 6H, β-pyrrole), 8.15 (d, 6H, CH phenoxy), 8.11 (d, 2H, CH pyridyl), 7.29 (d, 2H, CH phenoxy), 4.26 (d, 2H, OCH2) 1.98 (t, 2H, CH2), 1.68 (t, 2H, CH2),1.12 (d, 3H, CH3), −2.87 (s, 2H, NH); UV–Vis (DMF): λ = 418, 514, 549, 589, 646 nm.
2. Experimental sections 2.1. Synthesis of propionic ester of tripyridylporphyrin (Por 1) The Por 1 was synthesized according to the method reported in the literature with minor modification [28]. To a 250 mL three-neck flask, 4-hydroxybenzaldehyde (2.45 g, 20 mmol), 4-pyridinecarboxaldehyde (7.72 g, 72 mmol), acetic acid anhydride (10 mL, 105 mmol) and 180 mL propionic acid were added. The resulting solution was then heated to reflux, followed by the addition of freshly distilled pyrrole (6.17 g, 92 mmol) within 15 min. After refluxing for another 1.5 h, the solvent was evaporated in vacuo. The residue was dissolved in CH2Cl2 and extracted with saturated aqueous NaHCO3 solution until the organic layer was neutral. The organic layer was then concentrated and the residue was purified by silica gel column chromatography (EtOH in CH2Cl2, 0–8% v/v) to get the desired product, yield 6%. 1H NMR (400 MHz, CDCl3) δ 9.05 (d, 6H, CH pyridyl), 8.96 (d, 2H, β-pyrrole), 8.86 (s, 6H, β-pyrrole), 8.21 (d, 2H, CH phenoxy), 8.17 (d, 6H, CH pyridyl), 7.53 (d, 2H, CH phenoxy), 2.81 (q, 2H, CH2), 1.43 (t, 3H, CH3), −2.90 (br s, 2H, NH); MALDI-TOF MS:calcd for: 690.762, found: 690.262; UV–Vis (DMF): λ = 418, 514, 548, 589, 646 nm.
2.5. Synthesis of manganese triphenylphosphonium-conjugated porphyrin complex (MnPor-PPh3) The Por-PPh3 (158 mg, 0.153 mmol) was dissolved in 15 mL DMF and heated to 100 °C, followed by the addition of collidine (0.2 mL, 1.53 mmol) and Mn(OAc)2.4H2O (376 mg, 1.53 mmol). The reaction mixture was then heated to 130 °C for 12 h in the dark under a nitrogen atmosphere. After cooling, the solution was diluted with CH2Cl2 and washed with water. The organic layer was concentrated and the residue was purified by silica gel column chromatography (MeOH in CH2Cl2, 2–3% v/v) to get the desired product, yield 95%. MALDI-TOF MS: calcd for: 1004.004, found: 1003.309; UV–Vis (DMF): λ = 467, 571, 609 nm. 2.6. Synthesis of manganese bromobutane porphyrin complex (MnPorButyl) The Por-Butyl (105.57 mg, 0.153 mmol) was dissolved in 15 mL DMF and heated to 100 °C, followed by the addition of collidine (0.2 mL, 1.53 mmol) and Mn(OAc)2.4H2O (376 mg, 1.53 mmol). The mixture was then heated to 130 °C for 12 h in the dark under a nitrogen atmosphere. After cooling to the room temperature, the residue was purified through silica gel column chromatography (MeOH in CH2Cl2, 2–3% v/v) to get the desired product, yield 95%. MALDI-TOF MS: calcd for: 742.73, found: 743.5599; UV–Vis: (DMF) λ = 466, 566, 601 nm. Synthesis of trans-Pt manganese triphenylphosphonium-conjugated porphyrin complex (Pt-MnPor-PPh3). To a 10 mL flask, transplatin (39 mg, 0.129 mmol), silver nitrate (22 mg, 0.129 mmol) and 2 mL DMF were added. After stirring at room temperature for 24 h, the resulted turbid solution was centrifuged to remove white silver chloride. The obtained light yellow solution was added to the suspension of MnPor-PPh3 (31 mg, 0.0286 mmol) in 2 mL
2.2. Synthesis of hydroxy tripyridylphenoxyporphyrine (Por 2) The Por 2 was synthesized according to the method reported in the literature [28]. The propionic ester of Por 1 (105 mg, 0.152 mmol) was saponified by dissolving the compound in a mixture of 15 mL dioxane, 4 mL MeOH and 2 mL 2 N NaOH. After stirring for 30 min in the dark, the reaction mixture was quenched by addition of 5 mL 1 N HCl solution. A saturated solution of NaHCO3 was then added until the porphyrin precipitated from the solution. The precipitate was isolated by centrifugation (3700 rpm, 5 min) and washed two times with water. The acquired solid was then dried under high vacuum (0.4 mbar) for a prolonged period to yield 92 mg (yield 95%) of a purple crystalline compound which was used immediately in the next step. 190
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2.10. Confocal laser scanning microscope studies
DMF. The mixture was stirred at 50 °C for another 48 h. After cooling down to room temperature, 5 mL diethyl ether was added. The solid was collected by filtration, followed by washed with methanol, dichloromethane and diethyl ether, respectively, and then dried in vacuum to acquire 51 mg pure product, yield 90%. MS (ESI): m/ z = 1607.11 [M-{PtCl(NH3)2}-2(NO3)-4NH3]+, 1334.87 [M-{PtCl (NH3)2}-PPh3-2(NO3)-Br]+, 1116 [M-{Py-PtCl(NH3)2}-NO3-PPh3-Br{O-Bu}-2Cl], 1003 [M-3{PtCl(NH3)2}-3(NO3)-Br]+, 409[M-2{PtCl (NH3)2}-2(NO3)-Br-2Cl-2NH3]3+; UV–Vis (DMF): λ = 467, 574, 621 nm.
HeLa cells were cultured in RPMI 1640 medium (GibcoTm) containing 10% fetal calf serum and 1% antibiotic penicillin and streptomycin (P/S), and cultured at 37 °C in a humidified 5% CO2 atmosphere. Cells (6 × 103 cells per well) were seeded in 6-well plates (with sterile coverslips in each well) and incubated for two days at 37 °C in a humidified 5% CO2 atmosphere. After renewal with the new medium, the cells were incubated with a photosensitizer at a concentration of 1.0 μM for 12 h at 37 °C. After that, the supernatant was carefully removed and the cells were washed three times with PBS. Subsequently, the slides were mounted and observed by a Zeiss Laser Scanning Confocal Microscope (LSM7 DUO), and then analyzed using ZEN 2009 software (Carl Zeiss). Co-staining of porphyrin, MitoTracker Green and LysoTracker Green: HeLa cells were first incubated with porphyrin for 12 h at 37 °C and then further incubated with MitoTracker Green (50 nM) or LysoTracker Green (50 nM) for 10 min at 37 °C. The cells were washed three times with PBS solution before imaging on a confocal microscope.
2.7. Synthesis of trans-Pt bromobutane porphyrin manganese complex (PtMnPor-Butyl) To a 10 mL flask, transplatin (39 mg, 0.129 mmol), silver nitrate (22 mg, 0.129 mmol) and 2 mL DMF were added. After stirring at room temperature for 24 h, the resulted turbid solution was centrifuged to remove white silver chloride. The obtained light yellow solution was added to the suspension of MnPor-Butyl (21 mg, 0.0286 mmol) in 2 mL DMF. The mixture was stirred at 50 °C for another 48 h. Then the solution was cooled down to room temperature and precipitated with diethyl ether. The solid was filtered, washed with methanol, dichloromethane and diethyl ether, respectively, and then dried in vacuum to acquire 42 mg pure product, yield 90%. MS (ESI): m/z = 1116 [M-{Py-PtCl(NH3)2}-NO3-{O-Bu}-2Cl]+; UV–Vis (DMF): λ = 466, 573, 613 nm.
2.11. In vitro T1-weighted MR imaging HeLa cells were incubated with Pt-MnPor-PPh3 aqueous solutions at varied concentrations of 0, 0.1, 0.25, 0.5, 0.75, 1.0 mM, respectively, for 4 h at 37 °C in 5% CO2. Then, the cells were washed three times with PBS [32]. Before imaging on a 3.0 T MR imaging system (Discovery MR750, GE Healthcare, USA), the cells were dispersed and fixed in 0.5% agarose with 100 mL eppendorf tubes. After that the cell suspension in each tube was placed in a wrist receiver coil. T1W images were acquired using an SE/2D sequence with the following parameters: TR = 400 ms, TE = 12.2 ms, NEX = 4.00, matrix = 256 × 256, slice thickness = 2 mm, slice space = 0.8 mm, and FOV = 12 cm.
2.8. Reactive oxygen species detection The reactive oxygen species generation of the porphyrin conjugates was determined using DCFH method [29]. Specifically, DCFH-DA was treated by 0.1 M NaOH for 30 min to convert to dichlorofluorescin. Irradiation of activated DCFH solutions in the presence of photosensitizers solutions (1 × 10−6 M in PBS buffer with 0.5% DMSO) results in the transformation of non-fluorescent activated into highly fluorescent 2′,7′-dichlorofluorescein with emission peak at 532 nm. The mixed solutions were exposed to light irradiation (LED lamp, 50 W) for different time intervals. The fluorescence spectra of DCFH solutions were recorded in 500–580 nm emission range under an excitation of 488 nm. The PBS buffer was used as control.
3. Results and discussion 3.1. Synthesis The synthetic routes of Pt-MnPor-PPh3 and Pt-MnPor-Butyl were demonstrated in Scheme 1. Firstly, stoichiometric pyrrole was condensed with 4-hydroxybenzaldehyde and 4-pyridinecarboxaldehyde to yield 5, 10, 15-tri-(4-pyridyl)-20-(4-hydroxy-phenyl) porphyrin, which was then conjugated with (4-bromobutyl)-triphenylphosphonium bromide under alkaline conditions to afford Por-PPh3 in 70% yield. The Por-PPh3 was then coordinated with Mn(OAc)2.4H2O to obtain MnPorPPh3. Finally, the Pt-MnPor-PPh3 was synthesized following the procedure described in the literature [27,33]. In parallel, an analogous compound without conjugation of triphenylphosphonium (Pt-MnPorButyl) was also prepared for comparison using a similar method.
2.9. Cell viability assays Human cervical carcinoma (HeLa) cells were cultured in DMEM (Dulbecco Modified Eagle Medium) supplemented with 5% FCS (fetal calf serum), 100 U/mL penicillin, 100 μg/mL streptomycin at 37 °C and 6% CO2. HeLa cells were first seeded in 96-well plates at 3 × 103 cells per well for 24 h. Cells were treated with samples overnight in the dark. Cytotoxicity was determined by MTT reduction assay [30,31]. The cell monolayer was washed twice with phosphate buffered saline (PBS) and then incubated with 50 μL of MTT solution (0.5 mg/mL) for 3 h at 37 °C. After removing the medium, 100 μL of DMSO was added. The solution was shaken for 30 min to dissolve the formed crystal in living cells. Absorbance was measured at a dual wavelength of 540 nm and 690 nm on a Labsystem Multiskan plate reader (Merck Eurolab, Switzerland). Each dosing concentration was performed in triplicate wells and repeated twice for MTT assay. The photocytotoxicity of samples was assessed by a similar protocol. HeLa cells were first seeded in 96-well plates at 3 × 103 cells per well for 24 h. The cells were treated with samples in the dark overnight. Afterwards, the cell was exposure to yellow light (4 J/cm2, 10 min) produced from a 400 W tungsten lamp fitted with a heat-isolation filter and a 500 nm long-pass filter. The fluence rate was 6 mW/cm2. Cell viability was determined by the MTT reduction assay.
3.2. Photophysical properties The photophysical properties of Por-Butyl, Por-PPh3, MnPor-Butyl, MnPor-PPh3, Pt-MnPor-Butyl and Pt-MnPor-PPh3 were studied in DMF solutions. The UV–vis absorption spectrum of Por-Butyl and Por-PPh3 (Fig. 1) exhibited a sharp Soret band centered at 418 nm and weak Q bands at 514, 548, 589 and 646 nm, respectively. After the metal complexation, the two peaks in the Q bands of MnPor-Butyl, MnPorPPh3, Pt-MnPor-Butyl and Pt-Mn-Por-PPh3 disappeared, probably due to the increase of molecular symmetry. Meanwhile, the Soret band of porphyrins showed a certain redshift after coordination with metal. As shown in Fig. 1a, the Soret band of Por-Butyl or Por-PPh3 was located at 418 nm, while the MnPor-Butyl, MnPor-PPh3, Pt-MnPor-Butyl and PtMnPor-PPh3 shifted to 467 nm. This is because after the manganese complexed with porphyrin ring, the d orbitals of metal overlapped with the π* orbitals of porphyrin ring, forming a π anti-bond orbital, which leads to a decrease of energy gap of metal complexes [34]. The 191
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Scheme 1. Synthetic routes of Por 1, Por 2, Por-PPh3, Mn-Por-PPh3 and Pt-Mn-Por-PPh3 (i) acetic anhydride, propionic acid, 130 °C, 1.5 h, 6%; (ii) p-dioxane, MeOH, NaOH (aq), 30 min, 95%; (iii) K2CO3, (4-Bromobutyl)triphenylphosphonium bromide, DMF, 65 °C, 4 h, 70%; (iv) Mn(OAc)2·4H2O, collidine, DMF, 130 °C, 12 h, 95%; (v) transplatin, silver nitrate, DMF, 50 °C, 48 h, 90%; (vi) K2CO3, bromobutane, DMF, 65 °C, 4 h, 95%; (vii) Mn(OAc)2·4H2O, collidine, DMF, 130 °C, 12 h, 95%; (viii) a) transplatin, silver nitrate, 24 h, b) DMF, 50 °C, 48 h, 90%.
fluorescence spectrum of the synthesized porphyrins was tested in DMF solutions. As shown in Fig. 1b, Por-Butyl and Por-PPh3 exhibited intense red emission, with maximum peak around at 655 and 723 nm, respectively. However, after coordination with metals, the corresponding manganese and platinum complexes (MnPor-Butyl, MnPorPPh3, Pt-MnPor-Butyl and Pt-MnPor-PPh3) showed the inhibition of fluorescence to some extent. The fluorescence quantum yield of PorButyl and Por-PPh3 were calculated to be 0.207 and 0.261, respectively, using TPP as reference, while their manganese and platinum complexes were all lower than 0.002 (Table 1).
3.3. Reactive oxygen species detection The reactive oxygen species (ROS) generation of the photosensitizers was investigated by a commercial probe 2′, 7′-dichlorofluorescin diacetate (DCFH-DA). Firstly, DCFH-DA was treated with NaOH solution to transfer to DCFH before being used in vitro. The nonfluorescent DCFH will be rapidly oxidized to a green fluorescent DCF in the presence of reactive oxygen species. The green fluorescence (λem = 532 nm) of DCFH will increase quantitatively upon reacting with ROS generated from photosensitizers. As shown in Fig. 2, the fluorescence intensity of DCFH displayed a time-dependent enhancement upon the irradiation with a LED lamp. Under the same conditions,
Fig. 1. UV–vis absorption and fluorescence spectrum of the intermediates and targeting compounds. 192
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Table 1 The photophysical properties of Por-Butyl, Por-PPh3, MnPor-Butyl, MnPor-PPh3, Pt-MnPor-Butyl and Pt-MnPor-PPh3 in DMF solution. Sample
Por-Butyl Por-PPh3 MnPor-Butyl MnPor-PPh3 Pt-MnPor-Butyl Pt-MnPor-PPh3 a
Absorption peaks (nm) Soret band
Q-band
418 418 466 467 465 467
514,548,590,645 514,549,589,645 568, 605 570, 609 574, 615 574, 616
Emission peaks(nm)
ε (Lmol−1cm−1)
ϕf a
649, 648, 666, 652, 650, 649,
2.76 × 105 2.75 × 105 3.49 × 104 8.14 × 104 5.09 × 104 9.64 × 104
0.207 0.261 0.011 0.014 0.004 0.007
712 714 715 713 711 712
The fluorescence quantum yield (φf) of the synthesized compounds were calculated using as TPP as reference according to the method in supporting information.
Fig. 2. The changes of fluorescence intensity at the characteristic peak of DCFH (525 nm) as a function of irradiation time. Fig. 3. Concentration dependence of the cytotoxicity of Pt-MnPor-PPh3 against HeLa cells, as determined by a MTT assay. All experiments were carried out in triplicate, and the error bars are standard deviations.
the fluorescence intensity of DCFH that mixed with Pt-MnPor-PPh3 was enhanced over ten times after irradiation for 270 s, which is apparently higher than that of Pt-MnPor-Butyl, suggesting Pt-MnPor-PPh3 had the higher efficiency of ROS generation due to the tethered triphenylphosphonium cation. Meanwhile, Pt-MnPor-PPh3 showed the higher efficiency of ROS generation than that of MnPor-PPh3 under the same condition. Since the capacity of photosensitizer to generate ROS is dependent on the efficiency of the intersystem crossing (ISC) from 1PS* to 3 PS* [35], the higher efficiency of ROS generation for Pt-MnPor-PPh3 is probably due to the heavy-atom effect of platinated porphyrin.
3.5. Confocal fluorescence imaging To investigate the organelle-targeted properties of Pt-MnPor-PPh3 in cancer cells, 2 μM Pt-MnPor-PPh3 and Pt-MnPor-Butyl were incubated with human cervical carcinoma (HeLa) cells, respectively. As shown in Fig. 4b, the HeLa cells displayed red fluorescent signals after 8 h of incubation with two photosensitizers. To further figure out the subcellular localization of Pt-MnPor-PPh3 and Pt-MnPor-Butyl in HeLa cells, LysoTracker Green and MitoTracker Green staining (Fig. 4c) was applied to visualize cell lysosomes and mitochondria, respectively. As shown in Fig. 4d, the red emission from Pt-MnPor-PPh3 is almost overlapped with that of green fluorescence of MitoTracker Green. The corresponding Pearson Correlation (PC) coefficient was 0.95 of PtMnPor-PPh3, indicating that it could target the mitochondria of cancer cells, probably due to the presence of lipophilic triphenylphosphonium cation (TPP). Meanwhile, the red fluorescence of Pt-MnPor-Butyl was also observed in the cytoplasm, indicating the accumulation of sample in cancer cells. However, the Pt-MnPor-Butyl was mainly localized in the lysosomes of cells with the PC value for lysosomes localization was 0.70, as shown in Fig. 4d.
3.4. In vitro dark cytotoxicity and photocytotoxicity Since the Pt-MnPor-PPh3 exhibited the high efficiency of ROS generation, its cytotoxicity against HeLa cells was further investigated using a standard MTT assay. As shown in Fig. 3, Pt-MnPor-PPh3 displayed the low dark toxicity after 24 h incubation with HeLa cells in a dose-dependent manner. When the concentration of Pt-MnPor-PPh3 was less than 50 μM, it did not lead to any significant decrease in survival fraction, with cell viability higher than 80%. Meanwhile, the photocytotoxicity of synthesized porphyrin was also evaluated by a similar protocol. As shown in Fig. 3, after light irradiation, the cell survival fraction decreased significantly as the concentration of PtMnPor-PPh3 increased from 0 to 50 μM, indicating the significant PDT effects due to the high efficiency of ROS generation of Pt-MnPor-PPh3. The IC50 of Pt-MnPor-PPh3 was calculated to be 30.74 μg/mL under light irradiation for 10 min. These results clearly confirmed that the synthesized Pt-MnPor-PPh3 could be used as an efficient photosensitizer for cancer therapy.
3.6. MR contrast properties Since the synthesized Pt-MnPor-PPh3 contained the paramagnetic metal ions, its magnetic resonance imaging (MRI) signal was further evaluated. The MR contrast performance of Pt-MnPor-PPh3 was measured at different concentrations of 0, 0.1, 0.25, 0.5, 0.75, 1.0 mM. As shown in Fig. 5, T1-weighted images of Pt-MnPor-PPh3 became brighter 193
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Fig. 4. Laser scanning confocal microscopy images of HeLa cells treated with Pt-MnPor-PPh3 and Pt-MnPor-Butyl (2 μM) for 8 h. The lysosome was stained with LysoTracker Green. The mitochondria was stained with MitoTracker Green (The scale bar is 20 μm). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
PPh3, which was conjugated with lipophilic triphenylphosphonium cation (TPP), could selectively enter the mitochondria of cancer cells, as analyzed using the confocal laser scanning microscope. Besides, the presence of paramagnetic metal Mn ions in Pt-MnPor-PPh3 endowed them with good MRI performances. Thus, the as-synthesized Pt-MnPorPPh3 provided a feasible synthetic strategy to develop new medicines for MRI-guided and mitochondria-targeted PDT applications in cancer theranostics. Acknowledgments We are thankful for the support from National Natural Science Foundation of China (21601142, 21804102) and Natural Science Foundation of Hubei Province (2017CFB689, 2017CFB222). Appendix A. Supplementary data Supplementary data related to this article can be found at https:// doi.org/10.1016/j.dyepig.2019.03.048. References [1] Wu F, Su H, Cai Y, Wong WK, Jiang W, Zhu X. Porphyrin-implanted carbon nanodots for photoacoustic imaging and in vivo breast cancer ablation. ACS. Appl. Bio. Mater. 2018;1:110. [2] Yang K, Hu LL, Ma XX, Ye SQ, Cheng L, Shi XZ. Multimodal imaging guided photothermal therapy using functionalized graphene nanosheets anchored with magnetic nanoparticles. Adv Mater 2012;24:1868. [3] Feshitan JA, Vlachos F, Sirsi SR, Konofagou EE, Borden MA. Theranostic Gd(III)lipid microbubbles for MRI-guided focused ultrasound surgery. Biomaterials 2012;33:247. [4] Lee SM, Song Y, Hong BJ, MacRenaris KW, Mastarone DJ, O'Halloran TV. Modular polymer-caged nanobins as a theranostic platform with enhanced magnetic resonance relaxivity and pH-responsive drug release. Angew Chem Int Ed 2010;122:10156. [5] Sanson C, Diou O, Thevenot J, Ibarboure E, Soum A, Brulet A. Doxorubicin loaded magnetic polymersomes: theranostic nanocarriers for MR imaging and magnetochemotherapy. ACS Nano 2011;5:1122. [6] Yang XQ, Grailer JJ, Rowland IJ, Javadi A, Hurley SA, Matson VZ. Multifunctional stable and pH-responsive polymer vesicles formed by heterofunctional triblock copolymer for targeted anti-cancer drug delivery and ultrasensitive MR imaging. ACS Nano 2010;4:6805. [7] Yang J, Lee CH, Park J, Seo S, Lim EK, Song YJ. Antibody conjugated magnetic PLGA nanoparticles for diagnosis and treatment of breast cancer. J Mater Chem 2007;17:2695. [8] Kadish KM, Patel N, Chen Y, Joshi P, Pera P, Baumann H. The effect of metallation
Fig. 5. a) In vitro T1-weighted MRI of Pt-MnPor-PPh3 at varied concentrations; b) corresponding T1-weighted MR signal intensity versus concentration of PtMnPor-PPh3.
with the increase of Mn concentration, indicating that Pt-MnPor-PPh3 could be served as a good positive contrast agent. 4. Conclusions In summary, we have successfully synthesized a new porphyrinbased photosensitizer Pt-MnPor-PPh3, which could be used as a multifunctional theranostic agent. The Pt-MnPor-PPh3 exhibited the high efficiency of ROS generation, probably due to the heavy-atom effect of the platinated complexes. Comparing with Pt-MnPor-Butyl, Pt-MnPor194
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[23] Murphy MP. Understanding and preventing mitochondrial oxidative damage. Biochem Soc Trans 2016;44:1219. [24] Wang X, Peralta S, Moraes CT. Mitochondrial alterations during carcinogenesis: a review of metabolic transformation and targets for anticancer treatments. Adv Cancer Res 2013;119:127. [25] Chandel NS. Mitochondria and cancer. Cancer Metabol 2014;2:8. [26] Noh I, Lee D, Kim H, Jeong CU, Lee Y, Ahn JO. Enhanced photodynamic cancer treatment by mitochondria-targeting and brominated near-infrared fluorophores. Adv Sci 2018;5:233. [27] Naik A, Rubbiani Riccardo, Gasser Gilles, Spingler Bernhard. Visible‐light‐induced annihilation of tumor cells with platinum–porphyrin conjugates. Angew Chem Int Ed 2014;53:6938. [28] van Dongen SF, Clerx J, Nørgaard K, Bloemberg TG, Cornelissen JJ, Trakselis MA. A clamp-like biohybrid catalyst for dna oxidation. Nat Chem 2013;5:945. [29] Yang K, Zhang X, Yang F, Wu F, Zhang X, Wang K. DNA photocleavage and binding modes of methylene violet 3RAX and its derivatives: effect of functional groups. Aust J Chem 2017;70:830. [30] Wu F, Yue L, Su H, Wang K, Yang L, Zhu X. Carbon dots @ platinum porphyrin composite as theranostic nanoagent for efficient photodynamic cancer therapy. Nanoscale. Res. Lett. 2018;13:357. [31] Yao S, Zheng Y, Jiang L, Xie C, Wu F, Huang C, Wang K. Methylene violet 3RAXconjugated porphyrin for photodynamic therapy: synthesis, DNA photocleavage, and cell study. RSC Adv 2018;8:4472. [32] Wu F, Chen J, Li Z, Su H, Leung KC, Wang H, Zhu X. Red/Near-Infrared emissive metalloporphyrin-based nanodots for magnetic resonance imaging-guided photodynamic therapy in vivo. Part Part Syst Char 2018;35:1800208. [33] Zhu S, Yao S, Wu F, Jiang L, Wong K, Zhou J, Wang K. Platinated porphyrin as a new organelle and nucleus dual-targeted photosensitizer for photodynamic therapy. Org Biomol Chem 2017;15:5764. [34] Wu F, Yang M, Zhang J, Zhu S, Shi M, Wang K. Metalloporphyrin-indomethacin conjugates as new photosensitizers for photodynamic therapy. J Biol Inorg Chem 2019;24:53. [35] Jiang J, Liu D, Zhao Y, Wu F, Yang K, Wang K. Synthesis, DNA binding mode, singlet oxygen photogeneration and DNA photocleavage activity of ruthenium compounds with porphyrin-imidazo[4,5-f] phenanthroline conjugated ligand. Appl Organomet Chem 2018;32:e4468.
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Mitochondria-targeting Pt/Mn porphyrins as efficient photosensitizers for magnetic resonance imaging and photodynamic therapy
T
Mengqian Yanga, Jingran Denga, Ding Guoa, Qi Sunc,∗∗, Zejiang Wanga, Kai Wangb, Fengshou Wua,∗ a Key Laboratory for Green Chemical Process of the Ministry of Education, School of Chemical Engineering and Pharmacy, Wuhan Institute of Technology, Wuhan, PR China b Hubei Collaborative Innovation Center for Advanced Organic Chemical Materials, College of Chemistry and Chemical Engineering, Hubei University, Wuhan, PR China c School of Chemistry and Environmental Engineering, Wuhan Institute of Technology, Wuhan, PR China
A R T I C LE I N FO
A B S T R A C T
Keywords: Photodynamic therapy Magnetic resonance imaging Mitochondria-targeted Porphyrin
Photodynamic therapy (PDT), as a minimally invasive and highly efficient anticancer approach, has received extensive attention. However, the search for more effective and less toxic photodynamic photosensitizers with good biocompatibility and high specificity are still highly expected. Herein, we synthesized and characterized a new porphyrin-based photosensitizer Pt-MnPor-PPh3, which integrated the magnetic resonance imaging (MRI) and fluorescence diagnostics and mitochondrial-targeted photodynamic therapy. The cell viabilities reduced with the increase of photosensitizer dose. The IC50 of Pt-MnPor-PPh3 was calculated to be 30.74 μg/mL under light irradiation for 10 min. In contrast, the cell viabilities of the control group, HeLa cells treated without laser irradiation, were still over 80% even at 50 μg/mL of Pt-MnPor-PPh3. The cellular uptake and subcellular localization of the conjugate were further evaluated through a confocal laser scanning microscope. The results showed that the conjugate had good mitochondrial-targeted properties in the cancer cells. Moreover, the presence of paramagnetic metal Mn ions in Pt-MnPor-PPh3 endowed them with good MRI performances, which provided a feasible synthetic strategy to develop new medicines for MRI-guided and organelle-targeted PDT applications in cancer theranostics.
1. Introduction It is well known that early accurate diagnosis of cancer not only improves patient survival, but also helps clinicians determine the most appropriate treatment strategy for the patient, thereby avoiding underor over-treatment. Therefore, it is urgent to establish an early diagnosis method for cancer with high sensitivity and specificity [1]. Among the imaging technologies, magnetic resonance imaging (MRI) has been widely used for early diagnosis of cancer due to its non-invasive, high spatial resolution and three-dimensional imaging advantages [2–7]. MR often relies on ‘‘contrast-enhancing agents’’ to improve inherent contrast between normal and diseased tissue. MR signal intensity or signal intensity differences (contrast) are dependent upon longitudinal (1/T1) and transverse relaxation rates (1/T2) of tissue protons. Agents containing paramagnetic ions had been shown to effectively alter longitudinal (1/T1) and/or transverse relaxation rates (1/T2) by changing the local magnetic field when they come in close proximity to water
∗
protons [8]. The coordination of porphyrin with manganese exhibited excellent MRI performance due to the planar structure of porphyrin, which allowed the water molecules to fully contact the paramagnetic metal manganese [9,10]. More importantly, the manganese complexes showed lower toxicity than that of commercially available contrast agent Gd-DTPA [11]. On the other hand, photodynamic therapy (PDT) has become one of the most promising non-invasive and safe treatment options for cancer treatment [12–15]. As a highly localized treatment, PDT involves focusing extracorporeal light on photosensitizers (PSs) in disease tissue in the body to produce reactive oxygen species (ROS) that effectively and selectively destroy diseased tissue without damage to surrounding healthy tissue [16–18]. Although PDT is widely employed in most countries with good effect for two decades, the selectivity of commercial PDT agents for tumor tissues still offers considerable opportunities for further improvement [19–21]. In the organelles of mammalian cells, mitochondria are
Corresponding author., Corresponding author. E-mail addresses: [email protected] (Q. Sun), [email protected], [email protected] (F. Wu).
∗∗
https://doi.org/10.1016/j.dyepig.2019.03.048 Received 25 December 2018; Received in revised form 16 March 2019; Accepted 18 March 2019 Available online 20 March 2019 0143-7208/ © 2019 Elsevier Ltd. All rights reserved.
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2.3. Synthesis of triphenylphosphonium-conjugated porphyrin (Por-PPh3)
indispensable organelles responsible for producing the energy supply of most cells. They also play a key role in programmed cell death, such as apoptosis, and are easily damaged by ROS formation [22,23]. When the mitochondria became carcinogenic, the potential of mitochondrial membrane increased, which will induce preferential accumulation and retention of cationic mitochondrial targeting agents in cancer cells comparing with normal cells [24,25]. Thus, in order to deliver the PSs to the mitochondrial of cancer cells, the lipophilic cations, such as triphenylphosphine could be introduced to the photosensitizer as they can cause them to undergo passive diffusion and benefit from the electrochemical potential present between the inner and outer layers of the cell membrane. The electrochemical potential energy facilitates the diffusion of cations, thereby enhancing their cellular uptake. Therefore, conjugating a PDT agent with a cationic mitochondrial targeting agent can result in rapid damage to cancer cells, improve therapeutic effects, and reduce unnecessary side effects [26]. Herein, we designed and synthesized a multifunctional porphyrinbased photosensitizer Pt-MnPor-PPh3 that integrated MRI and fluorescence diagnostics and mitochondrial-targeted photodynamic therapy. Specifically, a lipophilic triphenylphosphonium cation (TPP) as a good mitochondria-targeted agent was conjugated with manganese porphyrin (MnPor) through an alkyl chain. The MnPor-TPP, bearing pyridine ring, was further coordinated with transplatin to yield Pt-MnPorPor. Pt-MnPor-Por displayed high ROS yield due to the heavy atomic effect of platinum [27]. Due to the presence of paramagnetic metal manganese, the as-synthesized Pt-MnPor-PPh3 showed good magnetic resonance imaging performance. Finally, the cytotoxicity and subcellular localization of the Pt-MnPor-PPh3 were evaluated through MTT method and confocal laser scanning microscope, respectively.
The Por 2 (100 mg, 0.157 mmol) was dissolved in a mixture of DMF and anhydrous potassium carbonate (44 mg, 0.314 mmol). After stirring for 30 min at room temperature, (4-Bromobutyl)triphenylphosphonium bromide (113 mg, 0.236 mmol) was added dropwise to the resulting solution within 30 min. The mixture was then stirred and heated to reflux for 4 h. After filtration, the filtrate was evaporated in vacuo and the residue was purified by silica gel column chromatography (MeOH in CH2Cl2, 1–2% v/v) to get the desired product, yield 70%. 1H NMR (400 MHz, CDCl3) δ 9.03 (d, 3J = 5.7 Hz, 6H, CH pyridyl), 8.93 (d, 3J = 4.7 Hz, 2H, β-pyrrole), 8.82 (s, 4H, β-pyrrole), 8.79 (d, 3J = 4.8 Hz, 2H, β-pyrrole), 8.08 (d, 2H, CH phenoxy), 8.12 (d, 3J = 8.3 Hz, 6H, CH pyridyl), 7.78 (m, 15H, CH Phenyl), 7.53 (d, 2H, CH phenoxy), 4.41 (d, 2H, OCH2), 4.09 (t, 2H, CH2), 3.77 (t, 2H, CH2), 1.85 (d, 2H, CH2), −2.90 (s, 2H, NH); MALDI-TOF MS: calcd for: 950.371, found: 950.380; UV–Vis (DMF): λ = 418, 514, 549, 589, 646 nm. 2.4. Synthesis of bromobutane porphyrin (Por-Butyl) The Por 2 (100 mg, 0.157 mmol) was dissolved in a mixture of DMF and anhydrous potassium carbonate (43.4 mg, 0.314 mmol). After stirring for 30 min at room temperature, bromobutane (25.4 μL, 0.236 mmol) was added dropwise to the solution. The mixture was stirred and heated to reflux for another 4 h. The solid was removed by filtration, and the filtrate was evaporated in vacuo. The residue was then purified through silica gel column chromatography (MeOH in CH2Cl2, 1–2% v/v) to get the desired product, yield 85%. 1H NMR (400 MHz, CDCl3) δ 9.03 (d, 6H, CH pyridyl), 8.95 (d, 2H, β-pyrrole), 8.85 (s, 6H, β-pyrrole), 8.15 (d, 6H, CH phenoxy), 8.11 (d, 2H, CH pyridyl), 7.29 (d, 2H, CH phenoxy), 4.26 (d, 2H, OCH2) 1.98 (t, 2H, CH2), 1.68 (t, 2H, CH2),1.12 (d, 3H, CH3), −2.87 (s, 2H, NH); UV–Vis (DMF): λ = 418, 514, 549, 589, 646 nm.
2. Experimental sections 2.1. Synthesis of propionic ester of tripyridylporphyrin (Por 1) The Por 1 was synthesized according to the method reported in the literature with minor modification [28]. To a 250 mL three-neck flask, 4-hydroxybenzaldehyde (2.45 g, 20 mmol), 4-pyridinecarboxaldehyde (7.72 g, 72 mmol), acetic acid anhydride (10 mL, 105 mmol) and 180 mL propionic acid were added. The resulting solution was then heated to reflux, followed by the addition of freshly distilled pyrrole (6.17 g, 92 mmol) within 15 min. After refluxing for another 1.5 h, the solvent was evaporated in vacuo. The residue was dissolved in CH2Cl2 and extracted with saturated aqueous NaHCO3 solution until the organic layer was neutral. The organic layer was then concentrated and the residue was purified by silica gel column chromatography (EtOH in CH2Cl2, 0–8% v/v) to get the desired product, yield 6%. 1H NMR (400 MHz, CDCl3) δ 9.05 (d, 6H, CH pyridyl), 8.96 (d, 2H, β-pyrrole), 8.86 (s, 6H, β-pyrrole), 8.21 (d, 2H, CH phenoxy), 8.17 (d, 6H, CH pyridyl), 7.53 (d, 2H, CH phenoxy), 2.81 (q, 2H, CH2), 1.43 (t, 3H, CH3), −2.90 (br s, 2H, NH); MALDI-TOF MS:calcd for: 690.762, found: 690.262; UV–Vis (DMF): λ = 418, 514, 548, 589, 646 nm.
2.5. Synthesis of manganese triphenylphosphonium-conjugated porphyrin complex (MnPor-PPh3) The Por-PPh3 (158 mg, 0.153 mmol) was dissolved in 15 mL DMF and heated to 100 °C, followed by the addition of collidine (0.2 mL, 1.53 mmol) and Mn(OAc)2.4H2O (376 mg, 1.53 mmol). The reaction mixture was then heated to 130 °C for 12 h in the dark under a nitrogen atmosphere. After cooling, the solution was diluted with CH2Cl2 and washed with water. The organic layer was concentrated and the residue was purified by silica gel column chromatography (MeOH in CH2Cl2, 2–3% v/v) to get the desired product, yield 95%. MALDI-TOF MS: calcd for: 1004.004, found: 1003.309; UV–Vis (DMF): λ = 467, 571, 609 nm. 2.6. Synthesis of manganese bromobutane porphyrin complex (MnPorButyl) The Por-Butyl (105.57 mg, 0.153 mmol) was dissolved in 15 mL DMF and heated to 100 °C, followed by the addition of collidine (0.2 mL, 1.53 mmol) and Mn(OAc)2.4H2O (376 mg, 1.53 mmol). The mixture was then heated to 130 °C for 12 h in the dark under a nitrogen atmosphere. After cooling to the room temperature, the residue was purified through silica gel column chromatography (MeOH in CH2Cl2, 2–3% v/v) to get the desired product, yield 95%. MALDI-TOF MS: calcd for: 742.73, found: 743.5599; UV–Vis: (DMF) λ = 466, 566, 601 nm. Synthesis of trans-Pt manganese triphenylphosphonium-conjugated porphyrin complex (Pt-MnPor-PPh3). To a 10 mL flask, transplatin (39 mg, 0.129 mmol), silver nitrate (22 mg, 0.129 mmol) and 2 mL DMF were added. After stirring at room temperature for 24 h, the resulted turbid solution was centrifuged to remove white silver chloride. The obtained light yellow solution was added to the suspension of MnPor-PPh3 (31 mg, 0.0286 mmol) in 2 mL
2.2. Synthesis of hydroxy tripyridylphenoxyporphyrine (Por 2) The Por 2 was synthesized according to the method reported in the literature [28]. The propionic ester of Por 1 (105 mg, 0.152 mmol) was saponified by dissolving the compound in a mixture of 15 mL dioxane, 4 mL MeOH and 2 mL 2 N NaOH. After stirring for 30 min in the dark, the reaction mixture was quenched by addition of 5 mL 1 N HCl solution. A saturated solution of NaHCO3 was then added until the porphyrin precipitated from the solution. The precipitate was isolated by centrifugation (3700 rpm, 5 min) and washed two times with water. The acquired solid was then dried under high vacuum (0.4 mbar) for a prolonged period to yield 92 mg (yield 95%) of a purple crystalline compound which was used immediately in the next step. 190
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2.10. Confocal laser scanning microscope studies
DMF. The mixture was stirred at 50 °C for another 48 h. After cooling down to room temperature, 5 mL diethyl ether was added. The solid was collected by filtration, followed by washed with methanol, dichloromethane and diethyl ether, respectively, and then dried in vacuum to acquire 51 mg pure product, yield 90%. MS (ESI): m/ z = 1607.11 [M-{PtCl(NH3)2}-2(NO3)-4NH3]+, 1334.87 [M-{PtCl (NH3)2}-PPh3-2(NO3)-Br]+, 1116 [M-{Py-PtCl(NH3)2}-NO3-PPh3-Br{O-Bu}-2Cl], 1003 [M-3{PtCl(NH3)2}-3(NO3)-Br]+, 409[M-2{PtCl (NH3)2}-2(NO3)-Br-2Cl-2NH3]3+; UV–Vis (DMF): λ = 467, 574, 621 nm.
HeLa cells were cultured in RPMI 1640 medium (GibcoTm) containing 10% fetal calf serum and 1% antibiotic penicillin and streptomycin (P/S), and cultured at 37 °C in a humidified 5% CO2 atmosphere. Cells (6 × 103 cells per well) were seeded in 6-well plates (with sterile coverslips in each well) and incubated for two days at 37 °C in a humidified 5% CO2 atmosphere. After renewal with the new medium, the cells were incubated with a photosensitizer at a concentration of 1.0 μM for 12 h at 37 °C. After that, the supernatant was carefully removed and the cells were washed three times with PBS. Subsequently, the slides were mounted and observed by a Zeiss Laser Scanning Confocal Microscope (LSM7 DUO), and then analyzed using ZEN 2009 software (Carl Zeiss). Co-staining of porphyrin, MitoTracker Green and LysoTracker Green: HeLa cells were first incubated with porphyrin for 12 h at 37 °C and then further incubated with MitoTracker Green (50 nM) or LysoTracker Green (50 nM) for 10 min at 37 °C. The cells were washed three times with PBS solution before imaging on a confocal microscope.
2.7. Synthesis of trans-Pt bromobutane porphyrin manganese complex (PtMnPor-Butyl) To a 10 mL flask, transplatin (39 mg, 0.129 mmol), silver nitrate (22 mg, 0.129 mmol) and 2 mL DMF were added. After stirring at room temperature for 24 h, the resulted turbid solution was centrifuged to remove white silver chloride. The obtained light yellow solution was added to the suspension of MnPor-Butyl (21 mg, 0.0286 mmol) in 2 mL DMF. The mixture was stirred at 50 °C for another 48 h. Then the solution was cooled down to room temperature and precipitated with diethyl ether. The solid was filtered, washed with methanol, dichloromethane and diethyl ether, respectively, and then dried in vacuum to acquire 42 mg pure product, yield 90%. MS (ESI): m/z = 1116 [M-{Py-PtCl(NH3)2}-NO3-{O-Bu}-2Cl]+; UV–Vis (DMF): λ = 466, 573, 613 nm.
2.11. In vitro T1-weighted MR imaging HeLa cells were incubated with Pt-MnPor-PPh3 aqueous solutions at varied concentrations of 0, 0.1, 0.25, 0.5, 0.75, 1.0 mM, respectively, for 4 h at 37 °C in 5% CO2. Then, the cells were washed three times with PBS [32]. Before imaging on a 3.0 T MR imaging system (Discovery MR750, GE Healthcare, USA), the cells were dispersed and fixed in 0.5% agarose with 100 mL eppendorf tubes. After that the cell suspension in each tube was placed in a wrist receiver coil. T1W images were acquired using an SE/2D sequence with the following parameters: TR = 400 ms, TE = 12.2 ms, NEX = 4.00, matrix = 256 × 256, slice thickness = 2 mm, slice space = 0.8 mm, and FOV = 12 cm.
2.8. Reactive oxygen species detection The reactive oxygen species generation of the porphyrin conjugates was determined using DCFH method [29]. Specifically, DCFH-DA was treated by 0.1 M NaOH for 30 min to convert to dichlorofluorescin. Irradiation of activated DCFH solutions in the presence of photosensitizers solutions (1 × 10−6 M in PBS buffer with 0.5% DMSO) results in the transformation of non-fluorescent activated into highly fluorescent 2′,7′-dichlorofluorescein with emission peak at 532 nm. The mixed solutions were exposed to light irradiation (LED lamp, 50 W) for different time intervals. The fluorescence spectra of DCFH solutions were recorded in 500–580 nm emission range under an excitation of 488 nm. The PBS buffer was used as control.
3. Results and discussion 3.1. Synthesis The synthetic routes of Pt-MnPor-PPh3 and Pt-MnPor-Butyl were demonstrated in Scheme 1. Firstly, stoichiometric pyrrole was condensed with 4-hydroxybenzaldehyde and 4-pyridinecarboxaldehyde to yield 5, 10, 15-tri-(4-pyridyl)-20-(4-hydroxy-phenyl) porphyrin, which was then conjugated with (4-bromobutyl)-triphenylphosphonium bromide under alkaline conditions to afford Por-PPh3 in 70% yield. The Por-PPh3 was then coordinated with Mn(OAc)2.4H2O to obtain MnPorPPh3. Finally, the Pt-MnPor-PPh3 was synthesized following the procedure described in the literature [27,33]. In parallel, an analogous compound without conjugation of triphenylphosphonium (Pt-MnPorButyl) was also prepared for comparison using a similar method.
2.9. Cell viability assays Human cervical carcinoma (HeLa) cells were cultured in DMEM (Dulbecco Modified Eagle Medium) supplemented with 5% FCS (fetal calf serum), 100 U/mL penicillin, 100 μg/mL streptomycin at 37 °C and 6% CO2. HeLa cells were first seeded in 96-well plates at 3 × 103 cells per well for 24 h. Cells were treated with samples overnight in the dark. Cytotoxicity was determined by MTT reduction assay [30,31]. The cell monolayer was washed twice with phosphate buffered saline (PBS) and then incubated with 50 μL of MTT solution (0.5 mg/mL) for 3 h at 37 °C. After removing the medium, 100 μL of DMSO was added. The solution was shaken for 30 min to dissolve the formed crystal in living cells. Absorbance was measured at a dual wavelength of 540 nm and 690 nm on a Labsystem Multiskan plate reader (Merck Eurolab, Switzerland). Each dosing concentration was performed in triplicate wells and repeated twice for MTT assay. The photocytotoxicity of samples was assessed by a similar protocol. HeLa cells were first seeded in 96-well plates at 3 × 103 cells per well for 24 h. The cells were treated with samples in the dark overnight. Afterwards, the cell was exposure to yellow light (4 J/cm2, 10 min) produced from a 400 W tungsten lamp fitted with a heat-isolation filter and a 500 nm long-pass filter. The fluence rate was 6 mW/cm2. Cell viability was determined by the MTT reduction assay.
3.2. Photophysical properties The photophysical properties of Por-Butyl, Por-PPh3, MnPor-Butyl, MnPor-PPh3, Pt-MnPor-Butyl and Pt-MnPor-PPh3 were studied in DMF solutions. The UV–vis absorption spectrum of Por-Butyl and Por-PPh3 (Fig. 1) exhibited a sharp Soret band centered at 418 nm and weak Q bands at 514, 548, 589 and 646 nm, respectively. After the metal complexation, the two peaks in the Q bands of MnPor-Butyl, MnPorPPh3, Pt-MnPor-Butyl and Pt-Mn-Por-PPh3 disappeared, probably due to the increase of molecular symmetry. Meanwhile, the Soret band of porphyrins showed a certain redshift after coordination with metal. As shown in Fig. 1a, the Soret band of Por-Butyl or Por-PPh3 was located at 418 nm, while the MnPor-Butyl, MnPor-PPh3, Pt-MnPor-Butyl and PtMnPor-PPh3 shifted to 467 nm. This is because after the manganese complexed with porphyrin ring, the d orbitals of metal overlapped with the π* orbitals of porphyrin ring, forming a π anti-bond orbital, which leads to a decrease of energy gap of metal complexes [34]. The 191
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Scheme 1. Synthetic routes of Por 1, Por 2, Por-PPh3, Mn-Por-PPh3 and Pt-Mn-Por-PPh3 (i) acetic anhydride, propionic acid, 130 °C, 1.5 h, 6%; (ii) p-dioxane, MeOH, NaOH (aq), 30 min, 95%; (iii) K2CO3, (4-Bromobutyl)triphenylphosphonium bromide, DMF, 65 °C, 4 h, 70%; (iv) Mn(OAc)2·4H2O, collidine, DMF, 130 °C, 12 h, 95%; (v) transplatin, silver nitrate, DMF, 50 °C, 48 h, 90%; (vi) K2CO3, bromobutane, DMF, 65 °C, 4 h, 95%; (vii) Mn(OAc)2·4H2O, collidine, DMF, 130 °C, 12 h, 95%; (viii) a) transplatin, silver nitrate, 24 h, b) DMF, 50 °C, 48 h, 90%.
fluorescence spectrum of the synthesized porphyrins was tested in DMF solutions. As shown in Fig. 1b, Por-Butyl and Por-PPh3 exhibited intense red emission, with maximum peak around at 655 and 723 nm, respectively. However, after coordination with metals, the corresponding manganese and platinum complexes (MnPor-Butyl, MnPorPPh3, Pt-MnPor-Butyl and Pt-MnPor-PPh3) showed the inhibition of fluorescence to some extent. The fluorescence quantum yield of PorButyl and Por-PPh3 were calculated to be 0.207 and 0.261, respectively, using TPP as reference, while their manganese and platinum complexes were all lower than 0.002 (Table 1).
3.3. Reactive oxygen species detection The reactive oxygen species (ROS) generation of the photosensitizers was investigated by a commercial probe 2′, 7′-dichlorofluorescin diacetate (DCFH-DA). Firstly, DCFH-DA was treated with NaOH solution to transfer to DCFH before being used in vitro. The nonfluorescent DCFH will be rapidly oxidized to a green fluorescent DCF in the presence of reactive oxygen species. The green fluorescence (λem = 532 nm) of DCFH will increase quantitatively upon reacting with ROS generated from photosensitizers. As shown in Fig. 2, the fluorescence intensity of DCFH displayed a time-dependent enhancement upon the irradiation with a LED lamp. Under the same conditions,
Fig. 1. UV–vis absorption and fluorescence spectrum of the intermediates and targeting compounds. 192
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Table 1 The photophysical properties of Por-Butyl, Por-PPh3, MnPor-Butyl, MnPor-PPh3, Pt-MnPor-Butyl and Pt-MnPor-PPh3 in DMF solution. Sample
Por-Butyl Por-PPh3 MnPor-Butyl MnPor-PPh3 Pt-MnPor-Butyl Pt-MnPor-PPh3 a
Absorption peaks (nm) Soret band
Q-band
418 418 466 467 465 467
514,548,590,645 514,549,589,645 568, 605 570, 609 574, 615 574, 616
Emission peaks(nm)
ε (Lmol−1cm−1)
ϕf a
649, 648, 666, 652, 650, 649,
2.76 × 105 2.75 × 105 3.49 × 104 8.14 × 104 5.09 × 104 9.64 × 104
0.207 0.261 0.011 0.014 0.004 0.007
712 714 715 713 711 712
The fluorescence quantum yield (φf) of the synthesized compounds were calculated using as TPP as reference according to the method in supporting information.
Fig. 2. The changes of fluorescence intensity at the characteristic peak of DCFH (525 nm) as a function of irradiation time. Fig. 3. Concentration dependence of the cytotoxicity of Pt-MnPor-PPh3 against HeLa cells, as determined by a MTT assay. All experiments were carried out in triplicate, and the error bars are standard deviations.
the fluorescence intensity of DCFH that mixed with Pt-MnPor-PPh3 was enhanced over ten times after irradiation for 270 s, which is apparently higher than that of Pt-MnPor-Butyl, suggesting Pt-MnPor-PPh3 had the higher efficiency of ROS generation due to the tethered triphenylphosphonium cation. Meanwhile, Pt-MnPor-PPh3 showed the higher efficiency of ROS generation than that of MnPor-PPh3 under the same condition. Since the capacity of photosensitizer to generate ROS is dependent on the efficiency of the intersystem crossing (ISC) from 1PS* to 3 PS* [35], the higher efficiency of ROS generation for Pt-MnPor-PPh3 is probably due to the heavy-atom effect of platinated porphyrin.
3.5. Confocal fluorescence imaging To investigate the organelle-targeted properties of Pt-MnPor-PPh3 in cancer cells, 2 μM Pt-MnPor-PPh3 and Pt-MnPor-Butyl were incubated with human cervical carcinoma (HeLa) cells, respectively. As shown in Fig. 4b, the HeLa cells displayed red fluorescent signals after 8 h of incubation with two photosensitizers. To further figure out the subcellular localization of Pt-MnPor-PPh3 and Pt-MnPor-Butyl in HeLa cells, LysoTracker Green and MitoTracker Green staining (Fig. 4c) was applied to visualize cell lysosomes and mitochondria, respectively. As shown in Fig. 4d, the red emission from Pt-MnPor-PPh3 is almost overlapped with that of green fluorescence of MitoTracker Green. The corresponding Pearson Correlation (PC) coefficient was 0.95 of PtMnPor-PPh3, indicating that it could target the mitochondria of cancer cells, probably due to the presence of lipophilic triphenylphosphonium cation (TPP). Meanwhile, the red fluorescence of Pt-MnPor-Butyl was also observed in the cytoplasm, indicating the accumulation of sample in cancer cells. However, the Pt-MnPor-Butyl was mainly localized in the lysosomes of cells with the PC value for lysosomes localization was 0.70, as shown in Fig. 4d.
3.4. In vitro dark cytotoxicity and photocytotoxicity Since the Pt-MnPor-PPh3 exhibited the high efficiency of ROS generation, its cytotoxicity against HeLa cells was further investigated using a standard MTT assay. As shown in Fig. 3, Pt-MnPor-PPh3 displayed the low dark toxicity after 24 h incubation with HeLa cells in a dose-dependent manner. When the concentration of Pt-MnPor-PPh3 was less than 50 μM, it did not lead to any significant decrease in survival fraction, with cell viability higher than 80%. Meanwhile, the photocytotoxicity of synthesized porphyrin was also evaluated by a similar protocol. As shown in Fig. 3, after light irradiation, the cell survival fraction decreased significantly as the concentration of PtMnPor-PPh3 increased from 0 to 50 μM, indicating the significant PDT effects due to the high efficiency of ROS generation of Pt-MnPor-PPh3. The IC50 of Pt-MnPor-PPh3 was calculated to be 30.74 μg/mL under light irradiation for 10 min. These results clearly confirmed that the synthesized Pt-MnPor-PPh3 could be used as an efficient photosensitizer for cancer therapy.
3.6. MR contrast properties Since the synthesized Pt-MnPor-PPh3 contained the paramagnetic metal ions, its magnetic resonance imaging (MRI) signal was further evaluated. The MR contrast performance of Pt-MnPor-PPh3 was measured at different concentrations of 0, 0.1, 0.25, 0.5, 0.75, 1.0 mM. As shown in Fig. 5, T1-weighted images of Pt-MnPor-PPh3 became brighter 193
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Fig. 4. Laser scanning confocal microscopy images of HeLa cells treated with Pt-MnPor-PPh3 and Pt-MnPor-Butyl (2 μM) for 8 h. The lysosome was stained with LysoTracker Green. The mitochondria was stained with MitoTracker Green (The scale bar is 20 μm). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
PPh3, which was conjugated with lipophilic triphenylphosphonium cation (TPP), could selectively enter the mitochondria of cancer cells, as analyzed using the confocal laser scanning microscope. Besides, the presence of paramagnetic metal Mn ions in Pt-MnPor-PPh3 endowed them with good MRI performances. Thus, the as-synthesized Pt-MnPorPPh3 provided a feasible synthetic strategy to develop new medicines for MRI-guided and mitochondria-targeted PDT applications in cancer theranostics. Acknowledgments We are thankful for the support from National Natural Science Foundation of China (21601142, 21804102) and Natural Science Foundation of Hubei Province (2017CFB689, 2017CFB222). Appendix A. Supplementary data Supplementary data related to this article can be found at https:// doi.org/10.1016/j.dyepig.2019.03.048. References [1] Wu F, Su H, Cai Y, Wong WK, Jiang W, Zhu X. Porphyrin-implanted carbon nanodots for photoacoustic imaging and in vivo breast cancer ablation. ACS. Appl. Bio. Mater. 2018;1:110. [2] Yang K, Hu LL, Ma XX, Ye SQ, Cheng L, Shi XZ. Multimodal imaging guided photothermal therapy using functionalized graphene nanosheets anchored with magnetic nanoparticles. Adv Mater 2012;24:1868. [3] Feshitan JA, Vlachos F, Sirsi SR, Konofagou EE, Borden MA. Theranostic Gd(III)lipid microbubbles for MRI-guided focused ultrasound surgery. Biomaterials 2012;33:247. [4] Lee SM, Song Y, Hong BJ, MacRenaris KW, Mastarone DJ, O'Halloran TV. Modular polymer-caged nanobins as a theranostic platform with enhanced magnetic resonance relaxivity and pH-responsive drug release. Angew Chem Int Ed 2010;122:10156. [5] Sanson C, Diou O, Thevenot J, Ibarboure E, Soum A, Brulet A. Doxorubicin loaded magnetic polymersomes: theranostic nanocarriers for MR imaging and magnetochemotherapy. ACS Nano 2011;5:1122. [6] Yang XQ, Grailer JJ, Rowland IJ, Javadi A, Hurley SA, Matson VZ. Multifunctional stable and pH-responsive polymer vesicles formed by heterofunctional triblock copolymer for targeted anti-cancer drug delivery and ultrasensitive MR imaging. ACS Nano 2010;4:6805. [7] Yang J, Lee CH, Park J, Seo S, Lim EK, Song YJ. Antibody conjugated magnetic PLGA nanoparticles for diagnosis and treatment of breast cancer. J Mater Chem 2007;17:2695. [8] Kadish KM, Patel N, Chen Y, Joshi P, Pera P, Baumann H. The effect of metallation
Fig. 5. a) In vitro T1-weighted MRI of Pt-MnPor-PPh3 at varied concentrations; b) corresponding T1-weighted MR signal intensity versus concentration of PtMnPor-PPh3.
with the increase of Mn concentration, indicating that Pt-MnPor-PPh3 could be served as a good positive contrast agent. 4. Conclusions In summary, we have successfully synthesized a new porphyrinbased photosensitizer Pt-MnPor-PPh3, which could be used as a multifunctional theranostic agent. The Pt-MnPor-PPh3 exhibited the high efficiency of ROS generation, probably due to the heavy-atom effect of the platinated complexes. Comparing with Pt-MnPor-Butyl, Pt-MnPor194
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