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Methotrexate coated AZA-BODIPY nanoparticles for chemotherapy, photothermal and photodynamic synergistic therapy
Dyes and Pigments 179 (2020) 108351
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Dyes and Pigments journal homepage: http://www.elsevier.com/locate/dyepig
Methotrexate coated AZA-BODIPY nanoparticles for chemotherapy, photothermal and photodynamic synergistic therapy Ruonan Li, Yingying Du, Weihan Guo, Yu Su, Yang Meng, Zhongqiang Shan, Yaqing Feng, Shuxian Meng * School of Chemical Engineering and Technology, Tianjin University, Yaguan Road 135, Tianjin, 300350, PR China
A R T I C L E I N F O
A B S T R A C T
Keywords: Chitosan AZA-BODIPY Methotrexate Chemotherapy Photothermal therapy Photodynamic therapy
Multifunctional chitosan-based nanoparticles were designed and synthesized by photosensitizer (PS) AZABODIPY grafting water-soluble chitosan as nanoshells and then loaded drug methotrexate (MTX) into the nanoshells, which generated chemotherapy, photothermal therapy (PTT), photodynamic therapy (PDT) and imaging into one system for synergistic therapy in vitro. The nanoshells in this designed nanostructure can serve as drug carriers to increase solubility, reduce toxicity and enhance the efficacy of synergistic PTT and PDT. Cellviability indicated the low toxicity and safety of chitosan-based AZA-BODIPY nanoshells as carrier materials and the low-drug dosage of chitosan-based nanoparticles (half-maximal inhibitory concentration, IC50 ¼ 34.5 μg/mL) is lower than that of pure drug MTX (IC50 ¼ 56.9 μg/mL). Under light irradiation, photothermal temperature and MTT studies show that nanoparticles produce reactive oxygen species and exhibit photothermal conversion ef ficiency (38.3%). Thus, this study provides a multifunctional nanoparticle with chemotherapy, photothermal, photodynamic and imaging, synergistic therapy and diagnose for cancer treatment.
1. Introduction Cancer, a malignant tumor, is the second most deadly factor in the world [1]. Compared with traditional methods of cancer treatment, in recent years, some new therapeutic methods such as photothermal therapy (PTT), photodynamic therapy (PDT), gene-targeted therapy, and immunotherapy have gradually became good complements to traditional treatments for cancer [2–5]. Among these therapies, PDT and PTT have received extensive attention due to theirselves non-invasive, rapid therapeutic process, good cell killing effect, and reproducible operation. As for PTT, it refers to achieving therapeutic effects by illu minating the nanomaterials to generate heat, increasing the temperature of the tumor region, destroying the function of the cancer cells to induce their apoptosis or thermally ablating the tumor [6–9]. PDT involves the conversion that the PS molecules absorb light energy and transter to an excited state after the PS molecules are excited by light. Due to the unstable excited state, the PS molecules transfer energy to the sur rounding oxygen molecules or matrix during the return to the ground state, producing reactive oxygen species (ROS), such as singlet oxygen, hydroxyl radicals, and superoxide anion. Excessive ROS can interact with biomolecules (proteins, nucleic acids, etc.) in cancer cells,
destroying their structures, thereby affecting their functions, ultimately inducing cancer cell death and achieving cancer treatment [10]. Some of the PS molecules, which are mostly composed of near-infrared fluores cent dyes. BODIPY as one of them, has significant photophysical prop erties, such as long excitation/emission wavelength, high molar absorption coefficient and fluorescence quantum yield. In recent years, researchers are interested in transferring the maximum absorption value of related fluorescent dyes to near infrared (NIR) region [11,12]. One of the most effective methods to obtain the maximum absorption red shift is to replace methoxy bridge carbon atoms with nitrogen, which obtaining AZA-boron dipyrrolide (AZA-BODIPY). Compared with similar derivatives of BODIPY, the red shift of wavelength is usually about 90 nm [13,14]. Herein, a photosensitizer (ABDP-SI) based on AZA-BODIPY with high absorption efficient and good photo-stability was designed and synthesized. Most PS molecules are hydrophobic and readily aggregate in aqueous solution, which can affect biological activity, water solubility and singlet oxygen generation (SOG) [15]. Moreover, the accumulation of non-selective PS may have an adverse effect on normal cells. By stabi lizing PS into target delivery carries comprising polymer nanoparticles, liposomes, and polymer micelles, the water stability and selective
* Corresponding author. E-mail address: [email protected] (S. Meng). https://doi.org/10.1016/j.dyepig.2020.108351 Received 2 January 2020; Received in revised form 10 February 2020; Accepted 10 March 2020 Available online 2 April 2020 0143-7208/© 2020 Elsevier Ltd. All rights reserved.
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Scheme 1. The preparation of MTX@CABS, cellular uptake and phototherapy.
accumulation of PS can be improved in the targeted tissue [16–20]. Therefore, a hydrophilic chitosan chain was introduced to AZA-BODIPY to construct a hydrophilic unit of nanoparticle structure, and in which a water-insoluble drug methotrexate was entrapped. In vitro experiments have shown that Methotrexate coated AZA-BODIPY nanoparticles (MTX@CABS) with absorption in the NIR region has great photothermal and photodynamic effects (Scheme 1). In the light irradiation below, MTX@CABS exhibits excellent photodynamic performance, and the particle conversion effectively increases while increasing the irradiation power. Cellular uptake of MTX@CABS was studied by confocal laser scanning microscopy (CLSM). In addition, studies have been conducted on phototoxic and cytotoxic (HeLa) cell lines of human cervical cancer, indicating a obvious inhibitory effect on cancer cells.
MHz NMR spectrometer at 298 K. UV–vis spectra and Fluorescence spectra were recorded on Hitachi spectrophotometer. Particle size were acquired via DLS on Zetasizer Nanoseries (Nano ZS). Using EnSpire Multilabel Reader (PerkinElmer, USA) to measure cell viability. The invitro cellular uptake images were obtained from CLSM (UltraView Vox, PerkinElmer, USA). Photothermal temperature was recorded via a thermal infrared imager (FLIR E6). 2.2. Synthesis of BF2 chelate of [5-(4-hydroxyphenyl)-3-phenyl-1Hpyrrol-2-yl]-[5-(ethyl-6-(4-phenoxy)hexanoate)-3-phenylpyrrol-2ylidene]amine (a) ABDP (0.513 g, 1 mmol), Ethyl-6-bromohexanoate (1 eq, 0.223 g, 1 mmol) and potassium carbonate (3 eq, 0.414 g, 3 mmol) was dissolved in acetone (40 mL) and heated under reflux for 14 h. After cooling to room temperature, the solvent was evaporated and the crude product was partitioned between H2O (100 mL) and EtOAc (100 mL). The organic layer after dried with sodium sulfate was concentrated and purified by silica gel column chromatography (CH2Cl2/MeOH 100:1 v/ v) to get product a. Product was recrystallized from hexane/CH2Cl2 as green solid (0.295 g, 45%). 1H NMR (400 MHz, CDCl3) δ 8.97-8. 85 (m, 8 H), 7.37-7.20 (m, 9 H), 6.92 (s, 1 H), 6.83-6.74 (m, 3 H), 4.02 (m, 2 H), 3.87 (t, J ¼ 6.4 Hz, 2 H), 2.17 (t, J ¼ 6.0 Hz, 2 H), 1.66-1.51 (m, 2 H), 1.42 (m, 4 H), 1.18 (t, J ¼ 6.8 Hz, 3 H). Electro-Spray ionization triple quadrupole tandem mass spectrometry (ESI-TQ MS) m/z [MHþ] found 656.2970, calculated 656.3085.
2. Materials and methods 2.1. Materials and instrumentation O-Carboxymethyl chitosan (OCMC, Mw ¼ 40 K, deacetylation de gree ¼ 80%) was purchased from Sybridge Co., Ltd. (Shanghai, China). Benzaldehyde, acetophenone, ammonium acetate, nitromethane boron fluoride ethyl ether (48%), N-hydroxy-succinimide (NHS), Ethyl-6bromohexanoate were obtained from Taitan (Shanghai, China). Gen eral chemicals were all of analytical grade and used without further purification. Methotrexate (MTX) was provided by Chemart Tianjin Co., Ltd. 1,3-diphenylisobenzofuran (DPBF), 20 ,70 -dichlorfluorescein-diac etate (DCFH-DA), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), 40 ,6-Diamidino-2-phenylindole (DAPI) were bought from Beyotime Biotechnology. Fetal bovine serum (FBS) was purchased from HyClone by Thermo Fisher (Suzhou, China). Phosphate buffered saline (PBS), penicillin/streptomycin, Trypsin-EDTA, Dulbecco’s modi fied Eagle medium (DMEM) were obtained from Gibco by Thermo Fisher (Suzhou, China). BF2 chelate of [5-(4-hydroxyphenyl)-3-phenyl-1Hpyrrol-2-yl]-[5-phenyl-3-phenylpyrrol-2-ylidene]amine was synthe sized as reported [21]. The NMR spectra were measured on a Bruker AVANCE III HD 400
2.3. Synthesis of BF2 chelate of [5-(4-hydroxyphenyl)-3-phenyl-1Hpyrrol-2-yl]-[5-(ethyl-6-(4-phenoxy)hexanoic acid)-3-phenylpyrrol-2ylidene]amine (b) Compound a (0.131 g, 0.2 mmol) and KOH (20 eq, 0.224 g, 4 mmol) was dissolved in a mixture solution of THF (6 mL), ethanol (6 mL) and H2O (3 mL). The mixture was stirred and heated at 86 � C for 2 h. The solvent was removed after reaction, left H2O only and then added THF (5 mL). The reaction mixture was neutralized with 4 M HCl adjusted pH 2
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Scheme 2. Synthesis of CABS.
to 5 and was extracted with CH2Cl2. It was then further purified by column chromatography (CH2Cl2/MeOH 50:1 v/v) to yield blue com pound b (80%, 0.100 g). 1H NMR (400 MHz, DMSO‑d6) δ 12.29 (s, 1 H), 8.18-8.13 (m, 6 H), 7.98 (d, J ¼ 7.4 Hz, 2 H), 7.80 (s, 1 H), 7.65 (t, J ¼ 7.7 Hz, 2 H), 7.58-7.39 (m, 9 H), 7.22 (d, J ¼ 8.4 Hz, 2 H), 4.14 (t, J ¼ 5.9 Hz, 2 H), 2.32 (t, J ¼ 7.0 Hz, 2 H), 1.83 (m, 2 H), 1.66 (m, 2 H), 1.53 (q, 2 H). Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) m/z [MHþ] found 627.25, calculated 628.32.
was obtained after centrifugation and purified with dialysis membrane in water for 24 h, and then dried in vacuum to get blue precipitation. 2.6. Preparation of MTX@CABS CABS (20 mg) was dissolved in deionized water (20 mL) and was vortexed at 40 � C for 4 h. A MTX solution with 5 mL DMSO was added to CABS solution drop by drop under vigorous stirring at 37 � C for 3 h. Then the mixture solution was incubated at 37 � C for over 15 h. To unloaded drug (MTX), the solution was dialyzed using dialysis mem branes at r.t. for 48 h (MWCO: 4000 Da). The product was dried in a lyophilizer at 20 � C for 10 h and was obtained as a powdery state characterization of polymers (Scheme 2). The encapsulation efficiency of MTX was 90%(w/w).
2.4. Synthesis of BF2 chelate of [5-(4-hydroxyphenyl)-3-phenyl-1Hpyrrol-2-yl]-[5-(2,5-dioxopyrrolidin-1-yl-6-phenoxyhexanoate)-3phenylpyrrol-2-ylidene]amine (ABDP-SI) To a solution of compound b (0.125 g, 0.2 mmol), NHS (4 eq, 0.921 g, 0.8 mmol) and DMAP (2 eq, 0.489 g, 0.4 mmol) in CH2Cl2 (50 mL) were added EDC�HCl (2 eq, 0.763 g, 0.4 mmol) and stirred for 4 h at r.t. in the dark. The reaction mixture was washed with water and brine for 3 times and CH2Cl2 layer was dried with sodium sulfate. Then the crude product was concentrated and purified by silica gel column chromatography (CH2Cl2/MeOH 100:3 v/v) to get blue compound ABDP-SI. 1H NMR (400 MHz, DMSO‑d6) δ 8.17 (d, J ¼ 8.3 Hz, 2 H), 8.09 (t, J ¼ 11.8 Hz, 4 H), 7.96 (d, J ¼ 8.3 Hz, 2 H), 7.80 (s, 1 H), 7.64-7.60 (t, J ¼ 15.8 Hz, 2 H), 7.51-7.35 (m, 8 H), 7.2 (d, J ¼ 7.7 Hz, 2 H), 4.14 (t, J ¼ 12.1 Hz, 2 H), 2.82 (s, 1 H), 2.74 (t, J ¼ 14.7 Hz, 2 H), 1.83 (q, 2 H), 1.73 (q, 2 H), 1.56 (p, 2 H). Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) m/z [MHþ] found 724.27.
2.7. Characterization of CABS and MTX@CABS 1
H NMR spectra were recorded from a Bruker AVANCE III HD 400 MHz NMR spectrometer (400 MHz) at 298 K in DMSO‑d6. The infrared spectrum was obtained using KBr using a FTIR spectrometer (Bruker, TENSOR 27), and a spectrum of 500–4000 cm 1 (scanning speed of 4 cm 1) was recorded. UV–visible absorption spectra were performed on a Hitachi spectrophotometer. Fluorescence spectra were measured on a Hitachi F-2500 fluorescence spectrophotometer. The amount of ABDP-SI conjugated to CABS was determined by a calibration curve of ABDP-SI standard solution (Fig. S1) in DMSO using UV–vis absorption measure ments at 606 nm. The morphology of MTX@CABS was observed using a transmission electron microscope (TEM, JEM-2100 F, Japan). The average particle size of CABS and MTX@CABS and polydispersity index (PDI) were measured by a dynamic light scattering (DLS) method at 25 � C using Zetasizer Nanoseries (Nano ZS).
2.5. Synthesis of CABS CABS was synthesized as following procedures. 31 mg of OCMC was dissolved in deionized water (30 mL) and was stirred at 37 � C for 30 min. Then ABDP-SI (20 mg, 0.0276 mmol) in DMSO (1 mL) was added in dropwise into OCMC solution under vigorous stirring for 4.5 h. CABS 3
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2.8. Stability studies
2.9. The release of ABDP-SI and MTX from MTX@CABS
The photostability of MTX@CABS was investigated under normal light or continuous dark conditions at 37 � C for 30 days. The UV–vis absorption measured samples at 606 nm at different time points (5, 10, 15, 20, 25, and 30 days). The stability of MTX@CABS was investigated in various media to investigate its ability to resist PBS and seruminduced dissociation. MTX@CABS was prepared to a concentration of 100 μg/mL solution and diluted ten times with PBS, DMEM or DMEM with 10% FBS for 48 h, and the mixed solution was determined by the DLS method.
ABDP-SI, which was weak or not grafted onto OCMC, and MTX were released from MTX@CABS using a dialysis method. MTX@CABS in the dissolution medium (15% DMSO-PBS, 20 mg/mL) was added into a dialysis bag which was adequately immersed in a bottle within 100 mL of different dissolved solution (pH ¼ 5.0 or 7.4), and the bottle was shaken at 100 rpm in the dark. 1.5 mL of the release solution was taken at intervals of time to measure the drug release concentration. The concentration of released ABDP-SI and MTX were detected by UV–vis absorption measurement and were analyzed by the free ABDP-SI and MTX calibration curves in the dissolution medium.
Fig. 1. (A) Normalized UV–vis absorption spectra of OCMC, ABDP-SI and CABS. (B)Fluorescence absorption spectra of ABDP-SI, CABS(C) MTX@CABS stability in PBS over time and (D) in different solutions for 48 h(E) TEM and size distribution of MTX@CABS.(F). Release profiles (stable storage) of MTX and ABDP-SI from the MTX@CABS in PBS pH ¼ 5.0 or 7.4 at 37 � C. 4
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2.10. Cell culture
mg/mL of MTX@CABS for 12 h. DCFH-DA (1:1000) diluted in serumfree cell culture medium was added to the cells for 20 min in a 37 � C cell culture incubator. After washing thoroughly with serum-free cell culture medium [25]. The level of ROS was observed by measuring the oxidation of the probe to 20 ,70 -dichlorofluorescein (DCF) by fluorescence microscopy.
The human cervical cancer cell lines HeLa cells were grown in DMEM (GIBCO) containing 10% FBS (HyClone), 100 μg/mL streptomycin, and 100 U/mL Penicillin at 37 � C in 5% CO2 and 95% air in a CO2 humidified incubator. After culturing 24 h of HeLa cell, a series of concentrations of CABS or MTX@CABS were added, and the cells were cultured for an additional 24 h [22].
3. Results and discussion
2.11. Imaging for HeLa cells and drug uptake efficiencies
3.1. Synthesis and characterization
CLSM (UltraView Vox, PerkinElmer, USA) analysis was performed to observe the uptake and imaging ability of the MTX@CABS in injected cells. HeLa cells (5 � 105) were cultured on 8 mm2 glass coverslips in 48well plates after 24 h and were treated with 20 μg/mL of MTX@CABS for 4 h at 37 � C in the dark. The cells were washed with PBS after removing the supernatant, and then immediately fixed with 4% para formaldehyde. Furthermore, the nucleus was stained with DAPI. MTX@CABS was stimulated at 561 nm and monitored at 610–640 nm. DAPI was excited at 405 nm and monitored at 450–550 nm. The drug uptake efficiency was measured by treating HeLa cells with MTX@CABS. HeLa cells implanted in a six-well plate were treated with CABS or MTX@CABS (20 μg/mL) and cultured at 37 � C for 2, 4, and 6 h in the dark. After being rinsed with PBS and gathered in tubes, HeLa cells were subjected to flow cytometry (BD, FACSAria III, USA) with a 561 nm laser. The fluorescence intensity of the cells was examined by flow cytometry.
The synthetic procedure of MTX@CABS is shown in Scheme 2. Firstly, ABDP was synthesized as reported [21]. As for ABDP-SI, the purpose of the introduction of the long alkyl chains and the NHS group into ABDP was to keep the large conjugate structure at a certain distance from the AZA-BODIPY chromophore which can ensure the successful grafting of ABDP-SI onto the OCMC backbone and maintain the optical properties of the chromophore. Compounds were characterized by 1H NMR, Matrix-assisted laser desorption ionization time-of-flight mass spectrometry, and Electro-Spray ionization triple quadrupole tandem mass spectrometry (Figs. S2–4). The integration ratios of peaks and m/z signals in the MS spectrum agreed well with that of theoretical calculation. OCMC, ABDP-SI, and CABS were characterized by FTIR (Fig. S5). In the case of ABDP-SI, the broad band at 3419 cm 1 was F⋯H-N (pyrrole) hydrogen bonds, and the pyrrole N-H stretching vibration band shifted to 3382 cm 1. Bands at 1727 cm 1 and 1253 cm 1 indicated the pres ence of the C¼O stretching vibration of the ester group and C-N stretching vibration. The wild bands at 3473 cm 1 were H-O overlapped with H-N stretching vibration, while at 1620 cm 1 was C¼O stretching vibration overlapped with N–H bend and the band at 1172-1028 cm 1 was C–O overlapped with C-O-C stretching vibration in OCMC, respec tively. Compared with that of OCMC and ABDP-SI, the CABS FTIR spectra show the appearance of the H-N (–NH band) at 1615 cm 1 and the disappearance of the H-N (–NH2 band) at 1620 cm 1 which indi cated that the –NH2 of OCMC was partly converted into –NH groups. There was a weak absorption band at 1741 cm 1 attributed to the C¼O stretching vibration of ABDP-SI, and a slight sharp band at 1255 cm 1 was the C-N bending peak. Besides, OCMC and ABDP-SI, the CABS was assessed by UV spectroscopy and fluorescence spectroscopy for more accurate evaluation (Fig. 1A and B). OCMC UV–vis absorption and fluorescence emission from 250 nm to 700 nm were very low and ignored. Compared with ABDP-SI, the CABS UV–vis absorption at 308 nm shifting to 300 nm, and maximum absorption at 606 nm shifting to 583 nm indicates that the OCMC-grafted interaction had some effect on the electron density of both conjugated ABDP-SI and OCMC sections. Further characterized by spectroscopic measurement of CABS, the fluorescence emission changed significantly from 612 nm (ABDP-SI) to 604 nm. 1H NMR, FTIR (Fig. S5), UV–vis (Fig. 1A), and fluorescence spectra (Fig. 1B) confirmed the successful conjugation of ABDP-SI onto OCMC. The particle size characterized by DLS (Fig. S6) showed that MTX@CABS (540.4 nm) was dramatically larger than CABS (176.1 nm) due to the encapsulation of MTX enlarging the particle diameter. TEM and DLS characterization of MTX@CABS showed that the average hy drodynamic diameter determined by DLS was approximately 540.4 nm, which is larger than the data obtained by TEM (Fig. 1E). The margin of error may be due to solvation effects and concentration dependence. As shown in Fig. 1C, the size and PDI changed a little at room temperature in 30 days, indicated high stability in PBS. MTX@CABS was stable in different solutions for 48 h shown in Fig. 1D. In the meantime, the so lutions’ colors (Fig. S7) of CABS and MTX@CABS were confirmed the successful inclusion of MTX. The release of MTX and ABDP-SI from nanoparticles was investi gated. As can be observed from Figure 1F, MTX was released faster, and ABDP-SI released slowly, and both were nearly saturated after 70 h. It was balanced, so the amount of release was basically no longer increased
2.12. In vitro cytotoxicity studies The cytotoxicity of three different reagents on HeLa cells was tested using standard MTT methods. After incubating HeLa cells in 96-well plates for 24 h, a series of concentrations of CABS, MTX, MTX@CABS were added, and cultured for another 24 and 48 h, respectively. At the same time, in the case of the same MTX concentration (free MTX which was equivalent to the grafting amount of MTX on MTX@CABS), we used pure drug MTX and MTX@CABS as the control group. The medium of the control group and the test group were set to have the same volume. Cell viability was assessed by MTT assay. The inhibition rate was calculated according to a previous method [23]. To investigate the photothermal conversion efficiency, a series of concentrations of CABS aqueous solution were laser irradiated (808 nm, 1.2 W/cm2) and the temperature within 10 min was recorded. Besides, at the same concentration of CABS, HeLa cells were exposed to the 808 nm laser lamp with different excitation powers (0.2, 0.4, 0.6, 0.8, 1.0, and 1.2 W/cm2) to obtain the temperature change with time. The appropriate irradiation conditions can be selected by then. The CABS solution was irradiated with a red irradiation lamp (808 nm, 1.2 W/ cm2). When the temperature of the solution reached a steady-state, the lamp was extinguished, allowing the solution to naturally cool. During this process, the temperature is recorded every 10 s. The photothermal conversion efficiency can be calculated by reference to the literature [24]. In vitro photodynamic and photothermal treatment of HeLa cells. To investigate the photothermal effect, different concentrations of CABS and MTX@CABS were added to HeLa cells seeded onto 96-well plates. After 6 h of incubation, NIR irradiation or no irradiation for 10 min was performed by an 808 nm infrared lamp having a power density of 1.2 W/ cm2. To assess photodynamic efficacy, cells were treated with different concentrations (0–80 μg/mL) of CABS and MTX@CABS and with or without irradiated with light at 660 nm (250 mW/cm2) with an output of 250 mW for 8 min. After 24 h, the cells were subjected to MTT assay. 2.13. Intracellular reactive oxygen species (ROS) assay HeLa cells were cultured in six-well plates and pretreated with 20 5
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Fig. 2. UV–vis spectra of (A) the DPBF (DMF 30 μL,1 mg/mL) in 2 mL water solution after 0–10 min with 660 nm irradiation, (B) the DMF 30 μL and 2 mL water solution containing 100 μg/mL CABS (without DPBF) after 0–20 min with 660 nm irradiation, (C) the DPBF (DMF 30 μL,1 mg/mL) in 2 mL water solution containing 100 μg/mL CABS with 660 nm irradiation, and (D) Consumption rito of DPBF in CABS solution and DPBF itself over time.
6
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[26]. The ABDP-SI released by MTX@CABS was the part that was not bonded to the OCMC. The release rate of the substance was different under various solution environments, and the release rate under the condition of pH ¼ 7.4 was faster than pH ¼ 5.0.
degradation was caused by singlet oxygen produced by CABS rather than by CABS itself, the UV–Vis spectra record CABS absorbance changes in aqueous solutions without DPBF stimulation over 20 min (Fig. 2D). The change in the absorption spectrum was small, indicating CABS itself was not degraded. In order to study the uptake of HeLa cells to MTX@CABS, the flow cytometry was used to detect the fluorescence intensity of cells at different incubation times (2, 4, 6 h) in order to find a suitable incu bation time. As shown in Fig. 3, as the cell culture time increasing, the fluorescence intensity increased, indicating that more MTX@CABS was taken up by the cells. After 4 h of incubation of HeLa cells, the rate of cellular uptake was reduced. So HeLa cells were supplemented with MTX@CABS for 4 h. Intracellular fluorescence imaging was examined using CLSM. Fig. 4a showed the green fluorescence in the cytoplasm, again demonstrating that MTX@CABS can be taken up by cells. To verify that MTX@CABS can produce singlet oxygen in HeLa cells, DCFH-DA was used as a detector. In an aerobic environment, non-fluorescent DCFH-DA was oxidized to fluorescent dichlorofluorescein (DCF). In the dark environment, almost none green fluorescence was emitted in all control groups because sufficient singlet oxygen could not be produced [27]. In contrast, bright green fluorescence appeared in the illumination group (Fig. 4b). The results confirmed that MTX@CABS can effectively produce singlet oxygen in HeLa cells and can be used as a photodynamic agent. In order to investigate the photothermal conversion efficiency, a series of concentrations of CABS solution was irradiated with an 808 nm laser of various laser power. The photothermal curve of CABS shows an intense solution concentration and laser power density dependence. Photothermal heating was carried out at room temperature to 54.2 � C and 57.3 � C(Fig. 5), indicating that CABS can effectively convert the 808 nm laser into heat. The photothermal effect of CABS was quantitatively evaluated by photothermal conversion experiments. The multiple laser irradiation caused a continuous temperatures’ rise/fall of CABS in order to study the photothermal stability of CABS. As shown in Fig. 6, the maximum temperature variation fluctuation of the five cycles of was small, indi cating that CABS had good photostability [28]. A quantitative analysis of a heating/cooling process (Fig. S8) yields a photothermal conversion efficiency of 38.3%.
3.2. Spectroscopic properties In order to study the PDT and PTT of MTX@CABS, the photochemical properties of CABS and MTX@CABS were further studied. CABS or MTX@CABS 1O2 generation capabilities were tested in aqueous and HeLa cells to verify their potential for PDT, respectively. As a singlet oxygen detector, DPBF detected changes in the amount of singlet oxygen produced by CABS over time. Under 10 min of laser irradiation (250 mW/cm 2, 660 nm), the change in absorbance of DPBF itself in the mixed aqueous solution at the highest absorption at 425 nm was negligible (Fig. 2A). As shown in Fig. 2B and C, the DPBF solution to which CABS was added to presented a significant decrease in absorbance at 425 nm within 10 min. The absorbance of DPBF was reduced, indi cating that DPBF was degraded. To further demonstrate that DPBF
Fig. 3. The flow cytometry of HeLa cells costained with MTX@CABS with different times (2, 4, and 6 h).
Fig. 4. The CLSM images (a) of HeLa cells costained with MTX@CABS (20 μg/mL); (b) The generation of intracellular ROS mediated by MTX@CABS samples. 7
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Fig. 5. Temperature rise curves of (a) CABS (60 μg/mL) under various irradiation intensities and (b) under different concentrations(808 nm 1.2 W/cm2) in water.
3.3. Phototherapy effect evaluation Toxicity assessments were performed on CABS, MTX, and MTX@CABS. Fig. S9 showed the cell viability of HeLa cells incubated with a series of concentrations (0–320 μg/mL) of MTX, CABS, and MTX@CABS for 24 h and 48 h. Under different incubation conditions, we found that even with a CABS concentration of up to 320 μg/mL, the cell viability of HeLa cells remained above 70%. These results demon strate the low cytotoxicity and safety of CABS as the carrier materials. The IC50 values of HeLa cells were calculated by cytotoxic MTT re sults of MTX and MTX@CABS. As shown in Table S1, the IC50 value of MTX@CABS was significantly lower than MTX. The inhibition of HeLa cells by MTX@CABS containing equal amounts of MTX was enhanced compared to free MTX (Fig. S9 and Table S1). The results show that CABS as a carrier material can more effectively encapsulate drugs on cancer cells. Cytotoxicity experiments investigated the phototherapy effects of CABS and MTX@CABS on HeLa cells [29]. Different concentrations of CABS and MTX@CABS were added to HeLa cells, and the cells were irradiated with or without irradiation for 10 min with different combi nations of 808 nm and 660 nm lasers. After 24 h the MTT value was measured. An 808 nm, 1.2 W/cm2 laser was utilized for PTT. A laser of 660 nm and 250 mW/cm2 was used for PDT. As a control group, the toxicity of CABS to cells at different concentrations was negligible in the
Fig. 6. Heating reproducibility stability of CABS (808 nm, 0.8 W/cm2, 60 μg/ mL) in H2O.
Fig. 7. In vitro photodynamic and photothermal treatment of HeLa cells: stained with CABS and MTX@CABS of different concentrations without and with irradiation by a 660 nm laser (a) PDT or an 808 nm laser (b) PTT and (c) combined PDT&PTT. 8
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absence of irradiation (Fig. 7). This is consistent with the previous experimental results. When irradiated with lasers at 808 nm, 660 nm, or 808 nm along with 660 nm for 10 min, both HeLa cells died more than both non-irradiated CABS and MTX@CABS. The IC50 values of CABS and MTX @CABS for HeLa cells were lower than those of unirradiated cells, respectively. And with the increase in CABS and MTX@CABS concen trations from 20 to 80 μg/mL, the cells were significantly reduced, which showed significant photothermal and photodynamic efficacy. Under the same conditions, MTX@CABS had a better phototherapy effect than CABS because MTX@CABS also had a chemotherapy effect. The PDT and PTT of the same dosing conditions were compared separately, and the PTT was more evident by cell survival rate. These results for PTT and PDT treatment alone are auspicious, and a combination of the two ap proaches is further employed for enhanced cancer treatment. As shown in Fig. 7C, after combining the two therapies, even at lower doses of MTX@CABS (40–80 μg/mL), the cells were significantly reduced, which was more effective than treatment alone. These results indicated that MTX@CABS can be used as an effective PDT and PTT nanomedicine to kill cancer cells. This significantly improved therapeutic effect was not only due to the photothermal effect of CABS but also to the efficacy of photodynamics to produce singlet oxygen [30].
[4] [5]
[6] [7]
[8]
[9] [10]
4. Conclusion
[11]
In this study, we designed and synthesized a new amphiphilic pho totheratic agent MTX@CABS, which could also do as drug delivery system. The cytotoxicity assays have proved the excellent biocompati bility and low toxicity of CABS nanostructure. The particles with high stability can be internalized by cancer cells via endocytosis and have good photodynamic and photothermal effect to cancer cells. This work highlights the potential of amphiphilic drug delivery system used to chemotherapy, photodynamic and photothermal synergistic therapy.
[12] [13]
[14]
Declaration of competing interest
[15]
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
[16]
[17]
Acknowledgments This research was supported by the National Natural Science Foun dation of China (NSFC, No. 21676187, 21476162, 21773168), National Key R&D Program of China (21761132007), China International Science and Technology Project (No. 2016YFE0114900).
[18]
[19]
Appendix A. Supplementary data
[20]
1
H NMR and ESI-TQ mass spectra of a, b,and ABDP-SI, FTIR absor bance spectra, photothermal curve of CABS, Cell viability of HeLa cells incubated with MTX, CABS and MTX@CABS, IC50 values of MTX and MTX@CABS (PDF) Supplementary data to this article can be found online at https://doi. org/10.1016/j.dyepig.2020.108351.
[21]
[22]
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[23]
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Dyes and Pigments journal homepage: http://www.elsevier.com/locate/dyepig
Methotrexate coated AZA-BODIPY nanoparticles for chemotherapy, photothermal and photodynamic synergistic therapy Ruonan Li, Yingying Du, Weihan Guo, Yu Su, Yang Meng, Zhongqiang Shan, Yaqing Feng, Shuxian Meng * School of Chemical Engineering and Technology, Tianjin University, Yaguan Road 135, Tianjin, 300350, PR China
A R T I C L E I N F O
A B S T R A C T
Keywords: Chitosan AZA-BODIPY Methotrexate Chemotherapy Photothermal therapy Photodynamic therapy
Multifunctional chitosan-based nanoparticles were designed and synthesized by photosensitizer (PS) AZABODIPY grafting water-soluble chitosan as nanoshells and then loaded drug methotrexate (MTX) into the nanoshells, which generated chemotherapy, photothermal therapy (PTT), photodynamic therapy (PDT) and imaging into one system for synergistic therapy in vitro. The nanoshells in this designed nanostructure can serve as drug carriers to increase solubility, reduce toxicity and enhance the efficacy of synergistic PTT and PDT. Cellviability indicated the low toxicity and safety of chitosan-based AZA-BODIPY nanoshells as carrier materials and the low-drug dosage of chitosan-based nanoparticles (half-maximal inhibitory concentration, IC50 ¼ 34.5 μg/mL) is lower than that of pure drug MTX (IC50 ¼ 56.9 μg/mL). Under light irradiation, photothermal temperature and MTT studies show that nanoparticles produce reactive oxygen species and exhibit photothermal conversion ef ficiency (38.3%). Thus, this study provides a multifunctional nanoparticle with chemotherapy, photothermal, photodynamic and imaging, synergistic therapy and diagnose for cancer treatment.
1. Introduction Cancer, a malignant tumor, is the second most deadly factor in the world [1]. Compared with traditional methods of cancer treatment, in recent years, some new therapeutic methods such as photothermal therapy (PTT), photodynamic therapy (PDT), gene-targeted therapy, and immunotherapy have gradually became good complements to traditional treatments for cancer [2–5]. Among these therapies, PDT and PTT have received extensive attention due to theirselves non-invasive, rapid therapeutic process, good cell killing effect, and reproducible operation. As for PTT, it refers to achieving therapeutic effects by illu minating the nanomaterials to generate heat, increasing the temperature of the tumor region, destroying the function of the cancer cells to induce their apoptosis or thermally ablating the tumor [6–9]. PDT involves the conversion that the PS molecules absorb light energy and transter to an excited state after the PS molecules are excited by light. Due to the unstable excited state, the PS molecules transfer energy to the sur rounding oxygen molecules or matrix during the return to the ground state, producing reactive oxygen species (ROS), such as singlet oxygen, hydroxyl radicals, and superoxide anion. Excessive ROS can interact with biomolecules (proteins, nucleic acids, etc.) in cancer cells,
destroying their structures, thereby affecting their functions, ultimately inducing cancer cell death and achieving cancer treatment [10]. Some of the PS molecules, which are mostly composed of near-infrared fluores cent dyes. BODIPY as one of them, has significant photophysical prop erties, such as long excitation/emission wavelength, high molar absorption coefficient and fluorescence quantum yield. In recent years, researchers are interested in transferring the maximum absorption value of related fluorescent dyes to near infrared (NIR) region [11,12]. One of the most effective methods to obtain the maximum absorption red shift is to replace methoxy bridge carbon atoms with nitrogen, which obtaining AZA-boron dipyrrolide (AZA-BODIPY). Compared with similar derivatives of BODIPY, the red shift of wavelength is usually about 90 nm [13,14]. Herein, a photosensitizer (ABDP-SI) based on AZA-BODIPY with high absorption efficient and good photo-stability was designed and synthesized. Most PS molecules are hydrophobic and readily aggregate in aqueous solution, which can affect biological activity, water solubility and singlet oxygen generation (SOG) [15]. Moreover, the accumulation of non-selective PS may have an adverse effect on normal cells. By stabi lizing PS into target delivery carries comprising polymer nanoparticles, liposomes, and polymer micelles, the water stability and selective
* Corresponding author. E-mail address: [email protected] (S. Meng). https://doi.org/10.1016/j.dyepig.2020.108351 Received 2 January 2020; Received in revised form 10 February 2020; Accepted 10 March 2020 Available online 2 April 2020 0143-7208/© 2020 Elsevier Ltd. All rights reserved.
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Scheme 1. The preparation of MTX@CABS, cellular uptake and phototherapy.
accumulation of PS can be improved in the targeted tissue [16–20]. Therefore, a hydrophilic chitosan chain was introduced to AZA-BODIPY to construct a hydrophilic unit of nanoparticle structure, and in which a water-insoluble drug methotrexate was entrapped. In vitro experiments have shown that Methotrexate coated AZA-BODIPY nanoparticles (MTX@CABS) with absorption in the NIR region has great photothermal and photodynamic effects (Scheme 1). In the light irradiation below, MTX@CABS exhibits excellent photodynamic performance, and the particle conversion effectively increases while increasing the irradiation power. Cellular uptake of MTX@CABS was studied by confocal laser scanning microscopy (CLSM). In addition, studies have been conducted on phototoxic and cytotoxic (HeLa) cell lines of human cervical cancer, indicating a obvious inhibitory effect on cancer cells.
MHz NMR spectrometer at 298 K. UV–vis spectra and Fluorescence spectra were recorded on Hitachi spectrophotometer. Particle size were acquired via DLS on Zetasizer Nanoseries (Nano ZS). Using EnSpire Multilabel Reader (PerkinElmer, USA) to measure cell viability. The invitro cellular uptake images were obtained from CLSM (UltraView Vox, PerkinElmer, USA). Photothermal temperature was recorded via a thermal infrared imager (FLIR E6). 2.2. Synthesis of BF2 chelate of [5-(4-hydroxyphenyl)-3-phenyl-1Hpyrrol-2-yl]-[5-(ethyl-6-(4-phenoxy)hexanoate)-3-phenylpyrrol-2ylidene]amine (a) ABDP (0.513 g, 1 mmol), Ethyl-6-bromohexanoate (1 eq, 0.223 g, 1 mmol) and potassium carbonate (3 eq, 0.414 g, 3 mmol) was dissolved in acetone (40 mL) and heated under reflux for 14 h. After cooling to room temperature, the solvent was evaporated and the crude product was partitioned between H2O (100 mL) and EtOAc (100 mL). The organic layer after dried with sodium sulfate was concentrated and purified by silica gel column chromatography (CH2Cl2/MeOH 100:1 v/ v) to get product a. Product was recrystallized from hexane/CH2Cl2 as green solid (0.295 g, 45%). 1H NMR (400 MHz, CDCl3) δ 8.97-8. 85 (m, 8 H), 7.37-7.20 (m, 9 H), 6.92 (s, 1 H), 6.83-6.74 (m, 3 H), 4.02 (m, 2 H), 3.87 (t, J ¼ 6.4 Hz, 2 H), 2.17 (t, J ¼ 6.0 Hz, 2 H), 1.66-1.51 (m, 2 H), 1.42 (m, 4 H), 1.18 (t, J ¼ 6.8 Hz, 3 H). Electro-Spray ionization triple quadrupole tandem mass spectrometry (ESI-TQ MS) m/z [MHþ] found 656.2970, calculated 656.3085.
2. Materials and methods 2.1. Materials and instrumentation O-Carboxymethyl chitosan (OCMC, Mw ¼ 40 K, deacetylation de gree ¼ 80%) was purchased from Sybridge Co., Ltd. (Shanghai, China). Benzaldehyde, acetophenone, ammonium acetate, nitromethane boron fluoride ethyl ether (48%), N-hydroxy-succinimide (NHS), Ethyl-6bromohexanoate were obtained from Taitan (Shanghai, China). Gen eral chemicals were all of analytical grade and used without further purification. Methotrexate (MTX) was provided by Chemart Tianjin Co., Ltd. 1,3-diphenylisobenzofuran (DPBF), 20 ,70 -dichlorfluorescein-diac etate (DCFH-DA), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), 40 ,6-Diamidino-2-phenylindole (DAPI) were bought from Beyotime Biotechnology. Fetal bovine serum (FBS) was purchased from HyClone by Thermo Fisher (Suzhou, China). Phosphate buffered saline (PBS), penicillin/streptomycin, Trypsin-EDTA, Dulbecco’s modi fied Eagle medium (DMEM) were obtained from Gibco by Thermo Fisher (Suzhou, China). BF2 chelate of [5-(4-hydroxyphenyl)-3-phenyl-1Hpyrrol-2-yl]-[5-phenyl-3-phenylpyrrol-2-ylidene]amine was synthe sized as reported [21]. The NMR spectra were measured on a Bruker AVANCE III HD 400
2.3. Synthesis of BF2 chelate of [5-(4-hydroxyphenyl)-3-phenyl-1Hpyrrol-2-yl]-[5-(ethyl-6-(4-phenoxy)hexanoic acid)-3-phenylpyrrol-2ylidene]amine (b) Compound a (0.131 g, 0.2 mmol) and KOH (20 eq, 0.224 g, 4 mmol) was dissolved in a mixture solution of THF (6 mL), ethanol (6 mL) and H2O (3 mL). The mixture was stirred and heated at 86 � C for 2 h. The solvent was removed after reaction, left H2O only and then added THF (5 mL). The reaction mixture was neutralized with 4 M HCl adjusted pH 2
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Scheme 2. Synthesis of CABS.
to 5 and was extracted with CH2Cl2. It was then further purified by column chromatography (CH2Cl2/MeOH 50:1 v/v) to yield blue com pound b (80%, 0.100 g). 1H NMR (400 MHz, DMSO‑d6) δ 12.29 (s, 1 H), 8.18-8.13 (m, 6 H), 7.98 (d, J ¼ 7.4 Hz, 2 H), 7.80 (s, 1 H), 7.65 (t, J ¼ 7.7 Hz, 2 H), 7.58-7.39 (m, 9 H), 7.22 (d, J ¼ 8.4 Hz, 2 H), 4.14 (t, J ¼ 5.9 Hz, 2 H), 2.32 (t, J ¼ 7.0 Hz, 2 H), 1.83 (m, 2 H), 1.66 (m, 2 H), 1.53 (q, 2 H). Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) m/z [MHþ] found 627.25, calculated 628.32.
was obtained after centrifugation and purified with dialysis membrane in water for 24 h, and then dried in vacuum to get blue precipitation. 2.6. Preparation of MTX@CABS CABS (20 mg) was dissolved in deionized water (20 mL) and was vortexed at 40 � C for 4 h. A MTX solution with 5 mL DMSO was added to CABS solution drop by drop under vigorous stirring at 37 � C for 3 h. Then the mixture solution was incubated at 37 � C for over 15 h. To unloaded drug (MTX), the solution was dialyzed using dialysis mem branes at r.t. for 48 h (MWCO: 4000 Da). The product was dried in a lyophilizer at 20 � C for 10 h and was obtained as a powdery state characterization of polymers (Scheme 2). The encapsulation efficiency of MTX was 90%(w/w).
2.4. Synthesis of BF2 chelate of [5-(4-hydroxyphenyl)-3-phenyl-1Hpyrrol-2-yl]-[5-(2,5-dioxopyrrolidin-1-yl-6-phenoxyhexanoate)-3phenylpyrrol-2-ylidene]amine (ABDP-SI) To a solution of compound b (0.125 g, 0.2 mmol), NHS (4 eq, 0.921 g, 0.8 mmol) and DMAP (2 eq, 0.489 g, 0.4 mmol) in CH2Cl2 (50 mL) were added EDC�HCl (2 eq, 0.763 g, 0.4 mmol) and stirred for 4 h at r.t. in the dark. The reaction mixture was washed with water and brine for 3 times and CH2Cl2 layer was dried with sodium sulfate. Then the crude product was concentrated and purified by silica gel column chromatography (CH2Cl2/MeOH 100:3 v/v) to get blue compound ABDP-SI. 1H NMR (400 MHz, DMSO‑d6) δ 8.17 (d, J ¼ 8.3 Hz, 2 H), 8.09 (t, J ¼ 11.8 Hz, 4 H), 7.96 (d, J ¼ 8.3 Hz, 2 H), 7.80 (s, 1 H), 7.64-7.60 (t, J ¼ 15.8 Hz, 2 H), 7.51-7.35 (m, 8 H), 7.2 (d, J ¼ 7.7 Hz, 2 H), 4.14 (t, J ¼ 12.1 Hz, 2 H), 2.82 (s, 1 H), 2.74 (t, J ¼ 14.7 Hz, 2 H), 1.83 (q, 2 H), 1.73 (q, 2 H), 1.56 (p, 2 H). Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) m/z [MHþ] found 724.27.
2.7. Characterization of CABS and MTX@CABS 1
H NMR spectra were recorded from a Bruker AVANCE III HD 400 MHz NMR spectrometer (400 MHz) at 298 K in DMSO‑d6. The infrared spectrum was obtained using KBr using a FTIR spectrometer (Bruker, TENSOR 27), and a spectrum of 500–4000 cm 1 (scanning speed of 4 cm 1) was recorded. UV–visible absorption spectra were performed on a Hitachi spectrophotometer. Fluorescence spectra were measured on a Hitachi F-2500 fluorescence spectrophotometer. The amount of ABDP-SI conjugated to CABS was determined by a calibration curve of ABDP-SI standard solution (Fig. S1) in DMSO using UV–vis absorption measure ments at 606 nm. The morphology of MTX@CABS was observed using a transmission electron microscope (TEM, JEM-2100 F, Japan). The average particle size of CABS and MTX@CABS and polydispersity index (PDI) were measured by a dynamic light scattering (DLS) method at 25 � C using Zetasizer Nanoseries (Nano ZS).
2.5. Synthesis of CABS CABS was synthesized as following procedures. 31 mg of OCMC was dissolved in deionized water (30 mL) and was stirred at 37 � C for 30 min. Then ABDP-SI (20 mg, 0.0276 mmol) in DMSO (1 mL) was added in dropwise into OCMC solution under vigorous stirring for 4.5 h. CABS 3
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2.8. Stability studies
2.9. The release of ABDP-SI and MTX from MTX@CABS
The photostability of MTX@CABS was investigated under normal light or continuous dark conditions at 37 � C for 30 days. The UV–vis absorption measured samples at 606 nm at different time points (5, 10, 15, 20, 25, and 30 days). The stability of MTX@CABS was investigated in various media to investigate its ability to resist PBS and seruminduced dissociation. MTX@CABS was prepared to a concentration of 100 μg/mL solution and diluted ten times with PBS, DMEM or DMEM with 10% FBS for 48 h, and the mixed solution was determined by the DLS method.
ABDP-SI, which was weak or not grafted onto OCMC, and MTX were released from MTX@CABS using a dialysis method. MTX@CABS in the dissolution medium (15% DMSO-PBS, 20 mg/mL) was added into a dialysis bag which was adequately immersed in a bottle within 100 mL of different dissolved solution (pH ¼ 5.0 or 7.4), and the bottle was shaken at 100 rpm in the dark. 1.5 mL of the release solution was taken at intervals of time to measure the drug release concentration. The concentration of released ABDP-SI and MTX were detected by UV–vis absorption measurement and were analyzed by the free ABDP-SI and MTX calibration curves in the dissolution medium.
Fig. 1. (A) Normalized UV–vis absorption spectra of OCMC, ABDP-SI and CABS. (B)Fluorescence absorption spectra of ABDP-SI, CABS(C) MTX@CABS stability in PBS over time and (D) in different solutions for 48 h(E) TEM and size distribution of MTX@CABS.(F). Release profiles (stable storage) of MTX and ABDP-SI from the MTX@CABS in PBS pH ¼ 5.0 or 7.4 at 37 � C. 4
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2.10. Cell culture
mg/mL of MTX@CABS for 12 h. DCFH-DA (1:1000) diluted in serumfree cell culture medium was added to the cells for 20 min in a 37 � C cell culture incubator. After washing thoroughly with serum-free cell culture medium [25]. The level of ROS was observed by measuring the oxidation of the probe to 20 ,70 -dichlorofluorescein (DCF) by fluorescence microscopy.
The human cervical cancer cell lines HeLa cells were grown in DMEM (GIBCO) containing 10% FBS (HyClone), 100 μg/mL streptomycin, and 100 U/mL Penicillin at 37 � C in 5% CO2 and 95% air in a CO2 humidified incubator. After culturing 24 h of HeLa cell, a series of concentrations of CABS or MTX@CABS were added, and the cells were cultured for an additional 24 h [22].
3. Results and discussion
2.11. Imaging for HeLa cells and drug uptake efficiencies
3.1. Synthesis and characterization
CLSM (UltraView Vox, PerkinElmer, USA) analysis was performed to observe the uptake and imaging ability of the MTX@CABS in injected cells. HeLa cells (5 � 105) were cultured on 8 mm2 glass coverslips in 48well plates after 24 h and were treated with 20 μg/mL of MTX@CABS for 4 h at 37 � C in the dark. The cells were washed with PBS after removing the supernatant, and then immediately fixed with 4% para formaldehyde. Furthermore, the nucleus was stained with DAPI. MTX@CABS was stimulated at 561 nm and monitored at 610–640 nm. DAPI was excited at 405 nm and monitored at 450–550 nm. The drug uptake efficiency was measured by treating HeLa cells with MTX@CABS. HeLa cells implanted in a six-well plate were treated with CABS or MTX@CABS (20 μg/mL) and cultured at 37 � C for 2, 4, and 6 h in the dark. After being rinsed with PBS and gathered in tubes, HeLa cells were subjected to flow cytometry (BD, FACSAria III, USA) with a 561 nm laser. The fluorescence intensity of the cells was examined by flow cytometry.
The synthetic procedure of MTX@CABS is shown in Scheme 2. Firstly, ABDP was synthesized as reported [21]. As for ABDP-SI, the purpose of the introduction of the long alkyl chains and the NHS group into ABDP was to keep the large conjugate structure at a certain distance from the AZA-BODIPY chromophore which can ensure the successful grafting of ABDP-SI onto the OCMC backbone and maintain the optical properties of the chromophore. Compounds were characterized by 1H NMR, Matrix-assisted laser desorption ionization time-of-flight mass spectrometry, and Electro-Spray ionization triple quadrupole tandem mass spectrometry (Figs. S2–4). The integration ratios of peaks and m/z signals in the MS spectrum agreed well with that of theoretical calculation. OCMC, ABDP-SI, and CABS were characterized by FTIR (Fig. S5). In the case of ABDP-SI, the broad band at 3419 cm 1 was F⋯H-N (pyrrole) hydrogen bonds, and the pyrrole N-H stretching vibration band shifted to 3382 cm 1. Bands at 1727 cm 1 and 1253 cm 1 indicated the pres ence of the C¼O stretching vibration of the ester group and C-N stretching vibration. The wild bands at 3473 cm 1 were H-O overlapped with H-N stretching vibration, while at 1620 cm 1 was C¼O stretching vibration overlapped with N–H bend and the band at 1172-1028 cm 1 was C–O overlapped with C-O-C stretching vibration in OCMC, respec tively. Compared with that of OCMC and ABDP-SI, the CABS FTIR spectra show the appearance of the H-N (–NH band) at 1615 cm 1 and the disappearance of the H-N (–NH2 band) at 1620 cm 1 which indi cated that the –NH2 of OCMC was partly converted into –NH groups. There was a weak absorption band at 1741 cm 1 attributed to the C¼O stretching vibration of ABDP-SI, and a slight sharp band at 1255 cm 1 was the C-N bending peak. Besides, OCMC and ABDP-SI, the CABS was assessed by UV spectroscopy and fluorescence spectroscopy for more accurate evaluation (Fig. 1A and B). OCMC UV–vis absorption and fluorescence emission from 250 nm to 700 nm were very low and ignored. Compared with ABDP-SI, the CABS UV–vis absorption at 308 nm shifting to 300 nm, and maximum absorption at 606 nm shifting to 583 nm indicates that the OCMC-grafted interaction had some effect on the electron density of both conjugated ABDP-SI and OCMC sections. Further characterized by spectroscopic measurement of CABS, the fluorescence emission changed significantly from 612 nm (ABDP-SI) to 604 nm. 1H NMR, FTIR (Fig. S5), UV–vis (Fig. 1A), and fluorescence spectra (Fig. 1B) confirmed the successful conjugation of ABDP-SI onto OCMC. The particle size characterized by DLS (Fig. S6) showed that MTX@CABS (540.4 nm) was dramatically larger than CABS (176.1 nm) due to the encapsulation of MTX enlarging the particle diameter. TEM and DLS characterization of MTX@CABS showed that the average hy drodynamic diameter determined by DLS was approximately 540.4 nm, which is larger than the data obtained by TEM (Fig. 1E). The margin of error may be due to solvation effects and concentration dependence. As shown in Fig. 1C, the size and PDI changed a little at room temperature in 30 days, indicated high stability in PBS. MTX@CABS was stable in different solutions for 48 h shown in Fig. 1D. In the meantime, the so lutions’ colors (Fig. S7) of CABS and MTX@CABS were confirmed the successful inclusion of MTX. The release of MTX and ABDP-SI from nanoparticles was investi gated. As can be observed from Figure 1F, MTX was released faster, and ABDP-SI released slowly, and both were nearly saturated after 70 h. It was balanced, so the amount of release was basically no longer increased
2.12. In vitro cytotoxicity studies The cytotoxicity of three different reagents on HeLa cells was tested using standard MTT methods. After incubating HeLa cells in 96-well plates for 24 h, a series of concentrations of CABS, MTX, MTX@CABS were added, and cultured for another 24 and 48 h, respectively. At the same time, in the case of the same MTX concentration (free MTX which was equivalent to the grafting amount of MTX on MTX@CABS), we used pure drug MTX and MTX@CABS as the control group. The medium of the control group and the test group were set to have the same volume. Cell viability was assessed by MTT assay. The inhibition rate was calculated according to a previous method [23]. To investigate the photothermal conversion efficiency, a series of concentrations of CABS aqueous solution were laser irradiated (808 nm, 1.2 W/cm2) and the temperature within 10 min was recorded. Besides, at the same concentration of CABS, HeLa cells were exposed to the 808 nm laser lamp with different excitation powers (0.2, 0.4, 0.6, 0.8, 1.0, and 1.2 W/cm2) to obtain the temperature change with time. The appropriate irradiation conditions can be selected by then. The CABS solution was irradiated with a red irradiation lamp (808 nm, 1.2 W/ cm2). When the temperature of the solution reached a steady-state, the lamp was extinguished, allowing the solution to naturally cool. During this process, the temperature is recorded every 10 s. The photothermal conversion efficiency can be calculated by reference to the literature [24]. In vitro photodynamic and photothermal treatment of HeLa cells. To investigate the photothermal effect, different concentrations of CABS and MTX@CABS were added to HeLa cells seeded onto 96-well plates. After 6 h of incubation, NIR irradiation or no irradiation for 10 min was performed by an 808 nm infrared lamp having a power density of 1.2 W/ cm2. To assess photodynamic efficacy, cells were treated with different concentrations (0–80 μg/mL) of CABS and MTX@CABS and with or without irradiated with light at 660 nm (250 mW/cm2) with an output of 250 mW for 8 min. After 24 h, the cells were subjected to MTT assay. 2.13. Intracellular reactive oxygen species (ROS) assay HeLa cells were cultured in six-well plates and pretreated with 20 5
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Fig. 2. UV–vis spectra of (A) the DPBF (DMF 30 μL,1 mg/mL) in 2 mL water solution after 0–10 min with 660 nm irradiation, (B) the DMF 30 μL and 2 mL water solution containing 100 μg/mL CABS (without DPBF) after 0–20 min with 660 nm irradiation, (C) the DPBF (DMF 30 μL,1 mg/mL) in 2 mL water solution containing 100 μg/mL CABS with 660 nm irradiation, and (D) Consumption rito of DPBF in CABS solution and DPBF itself over time.
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[26]. The ABDP-SI released by MTX@CABS was the part that was not bonded to the OCMC. The release rate of the substance was different under various solution environments, and the release rate under the condition of pH ¼ 7.4 was faster than pH ¼ 5.0.
degradation was caused by singlet oxygen produced by CABS rather than by CABS itself, the UV–Vis spectra record CABS absorbance changes in aqueous solutions without DPBF stimulation over 20 min (Fig. 2D). The change in the absorption spectrum was small, indicating CABS itself was not degraded. In order to study the uptake of HeLa cells to MTX@CABS, the flow cytometry was used to detect the fluorescence intensity of cells at different incubation times (2, 4, 6 h) in order to find a suitable incu bation time. As shown in Fig. 3, as the cell culture time increasing, the fluorescence intensity increased, indicating that more MTX@CABS was taken up by the cells. After 4 h of incubation of HeLa cells, the rate of cellular uptake was reduced. So HeLa cells were supplemented with MTX@CABS for 4 h. Intracellular fluorescence imaging was examined using CLSM. Fig. 4a showed the green fluorescence in the cytoplasm, again demonstrating that MTX@CABS can be taken up by cells. To verify that MTX@CABS can produce singlet oxygen in HeLa cells, DCFH-DA was used as a detector. In an aerobic environment, non-fluorescent DCFH-DA was oxidized to fluorescent dichlorofluorescein (DCF). In the dark environment, almost none green fluorescence was emitted in all control groups because sufficient singlet oxygen could not be produced [27]. In contrast, bright green fluorescence appeared in the illumination group (Fig. 4b). The results confirmed that MTX@CABS can effectively produce singlet oxygen in HeLa cells and can be used as a photodynamic agent. In order to investigate the photothermal conversion efficiency, a series of concentrations of CABS solution was irradiated with an 808 nm laser of various laser power. The photothermal curve of CABS shows an intense solution concentration and laser power density dependence. Photothermal heating was carried out at room temperature to 54.2 � C and 57.3 � C(Fig. 5), indicating that CABS can effectively convert the 808 nm laser into heat. The photothermal effect of CABS was quantitatively evaluated by photothermal conversion experiments. The multiple laser irradiation caused a continuous temperatures’ rise/fall of CABS in order to study the photothermal stability of CABS. As shown in Fig. 6, the maximum temperature variation fluctuation of the five cycles of was small, indi cating that CABS had good photostability [28]. A quantitative analysis of a heating/cooling process (Fig. S8) yields a photothermal conversion efficiency of 38.3%.
3.2. Spectroscopic properties In order to study the PDT and PTT of MTX@CABS, the photochemical properties of CABS and MTX@CABS were further studied. CABS or MTX@CABS 1O2 generation capabilities were tested in aqueous and HeLa cells to verify their potential for PDT, respectively. As a singlet oxygen detector, DPBF detected changes in the amount of singlet oxygen produced by CABS over time. Under 10 min of laser irradiation (250 mW/cm 2, 660 nm), the change in absorbance of DPBF itself in the mixed aqueous solution at the highest absorption at 425 nm was negligible (Fig. 2A). As shown in Fig. 2B and C, the DPBF solution to which CABS was added to presented a significant decrease in absorbance at 425 nm within 10 min. The absorbance of DPBF was reduced, indi cating that DPBF was degraded. To further demonstrate that DPBF
Fig. 3. The flow cytometry of HeLa cells costained with MTX@CABS with different times (2, 4, and 6 h).
Fig. 4. The CLSM images (a) of HeLa cells costained with MTX@CABS (20 μg/mL); (b) The generation of intracellular ROS mediated by MTX@CABS samples. 7
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Fig. 5. Temperature rise curves of (a) CABS (60 μg/mL) under various irradiation intensities and (b) under different concentrations(808 nm 1.2 W/cm2) in water.
3.3. Phototherapy effect evaluation Toxicity assessments were performed on CABS, MTX, and MTX@CABS. Fig. S9 showed the cell viability of HeLa cells incubated with a series of concentrations (0–320 μg/mL) of MTX, CABS, and MTX@CABS for 24 h and 48 h. Under different incubation conditions, we found that even with a CABS concentration of up to 320 μg/mL, the cell viability of HeLa cells remained above 70%. These results demon strate the low cytotoxicity and safety of CABS as the carrier materials. The IC50 values of HeLa cells were calculated by cytotoxic MTT re sults of MTX and MTX@CABS. As shown in Table S1, the IC50 value of MTX@CABS was significantly lower than MTX. The inhibition of HeLa cells by MTX@CABS containing equal amounts of MTX was enhanced compared to free MTX (Fig. S9 and Table S1). The results show that CABS as a carrier material can more effectively encapsulate drugs on cancer cells. Cytotoxicity experiments investigated the phototherapy effects of CABS and MTX@CABS on HeLa cells [29]. Different concentrations of CABS and MTX@CABS were added to HeLa cells, and the cells were irradiated with or without irradiation for 10 min with different combi nations of 808 nm and 660 nm lasers. After 24 h the MTT value was measured. An 808 nm, 1.2 W/cm2 laser was utilized for PTT. A laser of 660 nm and 250 mW/cm2 was used for PDT. As a control group, the toxicity of CABS to cells at different concentrations was negligible in the
Fig. 6. Heating reproducibility stability of CABS (808 nm, 0.8 W/cm2, 60 μg/ mL) in H2O.
Fig. 7. In vitro photodynamic and photothermal treatment of HeLa cells: stained with CABS and MTX@CABS of different concentrations without and with irradiation by a 660 nm laser (a) PDT or an 808 nm laser (b) PTT and (c) combined PDT&PTT. 8
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absence of irradiation (Fig. 7). This is consistent with the previous experimental results. When irradiated with lasers at 808 nm, 660 nm, or 808 nm along with 660 nm for 10 min, both HeLa cells died more than both non-irradiated CABS and MTX@CABS. The IC50 values of CABS and MTX @CABS for HeLa cells were lower than those of unirradiated cells, respectively. And with the increase in CABS and MTX@CABS concen trations from 20 to 80 μg/mL, the cells were significantly reduced, which showed significant photothermal and photodynamic efficacy. Under the same conditions, MTX@CABS had a better phototherapy effect than CABS because MTX@CABS also had a chemotherapy effect. The PDT and PTT of the same dosing conditions were compared separately, and the PTT was more evident by cell survival rate. These results for PTT and PDT treatment alone are auspicious, and a combination of the two ap proaches is further employed for enhanced cancer treatment. As shown in Fig. 7C, after combining the two therapies, even at lower doses of MTX@CABS (40–80 μg/mL), the cells were significantly reduced, which was more effective than treatment alone. These results indicated that MTX@CABS can be used as an effective PDT and PTT nanomedicine to kill cancer cells. This significantly improved therapeutic effect was not only due to the photothermal effect of CABS but also to the efficacy of photodynamics to produce singlet oxygen [30].
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4. Conclusion
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In this study, we designed and synthesized a new amphiphilic pho totheratic agent MTX@CABS, which could also do as drug delivery system. The cytotoxicity assays have proved the excellent biocompati bility and low toxicity of CABS nanostructure. The particles with high stability can be internalized by cancer cells via endocytosis and have good photodynamic and photothermal effect to cancer cells. This work highlights the potential of amphiphilic drug delivery system used to chemotherapy, photodynamic and photothermal synergistic therapy.
[12] [13]
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Declaration of competing interest
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The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
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Acknowledgments This research was supported by the National Natural Science Foun dation of China (NSFC, No. 21676187, 21476162, 21773168), National Key R&D Program of China (21761132007), China International Science and Technology Project (No. 2016YFE0114900).
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Appendix A. Supplementary data
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1
H NMR and ESI-TQ mass spectra of a, b,and ABDP-SI, FTIR absor bance spectra, photothermal curve of CABS, Cell viability of HeLa cells incubated with MTX, CABS and MTX@CABS, IC50 values of MTX and MTX@CABS (PDF) Supplementary data to this article can be found online at https://doi. org/10.1016/j.dyepig.2020.108351.
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