Materials
All of the compounds that were used were reagent grade or better, and the solvents were used as they were received unless otherwise specified. The following reagents were used as received: C60 (99.5%, SES RESEARCH, USA), gemcitabine (Sigma Aldrich), 2-amino-1,3-propanediol (AK Scientific), DBU (1,8-diaza-bicyclo[5.4.0]undec-7-ene, Sigma Aldrich), glycine (POCH, Poland), p-toluenesulfonic acid (POCH, Poland), acetic anhydride (Acros Organics), malonic acid (Acros Organics), CBr4 (Sigma Aldrich), EDCI hydrochloride [N-ethyl-N′-(3-dimethylaminopropyl)carbodiimide hydrochloride, Acros Organics], N-hydroxysuccinimide (Sigma Aldrich) and sodium hydride (Acros Organics). All of the solvents that were used to prepare the carbon nanomaterials were prepared according to literature procedures by distilling them with calcium hydride and were used immediately. The nuclear magnetic resonance spectra were measured on a Bruker Avance III 500 MHz NMR Spectrometer with TMS as the internal standard. The MS spectra for the water-insoluble fullerenes were collected using an Autoflex II MALDI-TOF mass spectrometer, and for the water-soluble [60]fullerene derivatives using an MS electrospray ionization time-of-flight (ESI-microTOF) mass spectrometer, both instruments from Bruker Daltonics Inc (Fremont, CA, USA). The final dialysis purification of the water-soluble nanomaterial nanoC60GEM was performed on Pall Microsep™ centrifugal membranes with molecular cut-offs 1 and 3 kDa. The purity of all of the compounds was assessed using an Agilent1260 equipped with a DAAD detector at 260 nm, RP-column: Eclipse plus C18 (3,5 μm); flow 0.5 mL/min. The Fourier transform infrared (FTIR) measurements were carried out using an Agilent Cary 640 FTIR spectrometer equipped with a standard source and a DTGS Peltier-cooled detector. The nanoconjugate powder was mixed with KBr and measured in the transmittance mode in the 700–4000 cm−1 range. The spectrum was recorded at 32 accumulations with a spectral resolution of 4 cm−1. The obtained data were analyzed by the baseline, water, and carbon dioxide correction. The chemical analysis of the sample surface was performed using X-ray photoelectron spectroscopy (XPS). The measurements were performed on a PHI5700 Physical Electronics spectrometer. The XPS studies were carried out with using an Al Kα monochromatized X-ray source (hν = 1486.6 eV). The survey spectrum was measured at pass energy 187.75e eV, and the high-resolution core levels of C1s, F1s, N1s, O1s and Si2 were measured at pass energy 23.5 eV. The analysis of the chemical states of the detected elements, as well as the calculations of the atomic concentration was performed using MULTIPAK (version 9.6.7.1, 2015, Ulvac-phi Inc.) software. The near-infrared luminescence (1270 nm) was measured perpendicular to the excitation beam in the photon-counting mode using a thermoelectric-cooled NIR PMT module (H10330-45; Hamamatsu, Japan) equipped with a 1100 nm cut-off filter and an additional dichroic narrow-band filter NBP, and was selectable from the spectral range 1150–1355 nm (NDC Infrared Engineering Ltd., Essex, UK). The data were collected using a computer-mounted PCI-board multichannel scaler (NanoHarp 250; PicoQuant GmbH, Berlin, Germany). Data analysis, including the first-order luminescence decay fitted using the Levenberg–Marquardt algorithm, was performed by custom-written software. The acquisition time for obtaining the singlet oxygen phosphorescence signals was 20 s. The EPR measurements were taken using a Bruker EMX-AA EPR spectrometer (Bruker BioSpin, Rheinstetten, Germany). EPR samples were run using microwave power of 10.6 mW, a modulation amplitude of 0.05 mT, center field 339.0 mT, scan width 8 mT and scan time 21 s. The light for photodynamic therapy was delivered from royal blue 100 W COB source (440 nm, FWHM 17.4 nm) (Chanzon, Shenzhen, China). The cell plate was placed in a holder above light source. Distance from the lens was set to achieve high uniformity of light power distribution on whole plate area. The cells were irradiated with 20 mW/cm2 for 1000 s for a total dose of 20 J. Temperature of cells during irradiation did not exceed 32 °C.
Methods
Synthesis of nanoC60GEM
2,2′-[(1,3-Dioxopropane-1,3-diyl)diimino]diacetic acid or malonyl-diglycine (1).
Glycine (9.75 g, 0.130 mol) was suspended in of 10 mL water and dissolved by adding NaOH to the solution (5.21 g, 0.130 mol). A solution of diethyl malonate in 30 mL of ethanol (10.4 g, 0.065 mol) was added and the whole mixture was placed in a 100-mL flask and heated under reflux for 3 h. The solvent was then removed on a rotary evaporator. Then, 12 M hydrochloric acid was being added to the residue until the pH of the mixture reached 1. The flask with the mixture was placed in a refrigerator for 12 h; during which the product precipitated. The resulting white solid was filtered off, washed with cold water and ethanol and dried. The final product was obtained as a white solid with a 19% yield (2.83 g) with m.p. 233 °C.
1H-NMR (d6-DMSO, 400 MHz, ppm): 8.35(t, J = 5.8 Hz, 2H, NH); 3.78(d, J = 5.8 Hz, 4H, CH2–NH); 3.17 (s, 2H, –CO–CH2–CO).
13C-NMR (d6-DMSO, 100 MHz, ppm): 171.5; 167.5; 42.9; 42.7; 41.2.
Diethyl 2,2′-[(1,3-dioxopropane-1,3-diyl)diimino]diacetate or diethyl malonyl-diglycinate (2).
Compound 1 (8.00 g, 36.67 mmol) was dissolved in 150 mL of ethanol and 348.8 mg (1.83 mmol, 5% molar ratio) of p-toluenesulfonic acid monohydrate was added. The reaction mixture was heated under reflux for 90 h). The solvents were then removed on a rotary evaporator to form solidifying oil. This product was purified using the extraction technique (DCM:NaHCO3 solution). The organic phase was dried using anhydrous CaCl2 and evaporated in vacuo to form a slightly yellowish oil. The final product was obtained with an 80% yield (8.09 g).
1H-NMR (d6-DMSO, 500 MHz, ppm): 8.44 (t, J = 5.7 Hz, 2H, NH);4.09 (m, 4H, CH2–CH3); 3.85(d, J = 5.7 Hz, 4H, CH2–NH); 3.18(bs, 2H, –CO–CH2–CO); 1.18(t, J = 7 Hz, 6H, CH3).
13C-NMR (d6-DMSO, 125 MHz, ppm): 170.1; 167.6; 61.0; 42.9; 42.5; 41.3; 14.4, 14.3
Synthesis of diserinol malonate acetate (3)
Diserinol malonate and its peracetylated version were synthesized on a large scale using a modified protocol (Serda et al. 2018). Briefly, 2-amino-1,3-propanediol (100 g, 1076 mmol) and dimethyl malonate (57.4 mL, 500 mmol) were dissolved in dry isopropanol, heated with vigorous stirring at 50 °C for 30 min and then stirred at room temperature, under a nitrogen atmosphere for 14 days. After that time, the solid precipitate was filtered off and washed with cold isopropanol. After recrystallization from isopropyl alcohol, the white solid was obtained and dried under lyophilization (m.p. 132 °C, yield 92%). Serinol malonate (25.0 g, 99.9 mmol) was suspended in pyridine (100 mL, 1240 mmol) at 0 °C, to which acetic anhydride (150 mL, 1590 mmol) was added dropwise over the course of an hour. After reaching 25 °C, dissolution occurred and the solution was stirred continuously for 48 h. At 0 °C, methanol (50 mL, 1225 mmol) was added to quench the remaining acetic anhydride and the solution was stirred for 1 h, after which the solvents were removed in vacuo and the residue (yellow oil) was dissolved in dichloromethane and recrystallized for 12 h in hexane. The precipitate was collected as white crystals via filtration, washed with hexane and dried in vacuo (m.p. 91 °C, lit. 91–92 °C (Clark et al. 1972)).
[60]Fullerene hexakis adduct (6)
The C60 (360 mg, 0.500 mol) was added to a freshly distilled toluene (400 mL), mixed for 20 min using a magnetic stirrer and suspended using an ultrasonic bath. Then, CBr4 (248.7 mg, 0.75 mmol) and malonate 2 (137.1 mg, 0.500 mmol), both of which were suspended in dichloromethane (7 mL), were added. Next, 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU, 95.1 mg, 0.625 mmol) in 5 mL of DCM was added and the reaction was allowed to proceed for 3 h at room temperature. The final product was isolated using the flash chromatography technique, (silica: Mallinckrodt, 75–250 μm particles, 150 Å pore size). The unreacted residue and impurities were removed with toluene and dichloromethane and then the product was eluted with CH2Cl2:MeOH 5:1 (240 mL) and CH2Cl2:MeOH 2:1 (225 mL) mixtures. The dark brown filtrate was evaporated in vacuo to form a brown solid (powder). This final product was obtained with a 64% yield (315 mg) and the mass was confirmed using MALDI-TOF (Additional file 1: Fig. S1). The obtained monoadduct 4 (183 mg, 0.184 mmol), diserinol malonate acetate (771.1 mg, 1.843 mmol) and CBr4 (1222.3 mg, 3.686 mmol) were dissolved in 20 mL of DCM; to which 170 mL of freshly distilled toluene was added and stirred for 5 min. Next, 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU, 337 mg, 2.212 mmol) in dichloromethane (3 mL) was added slowly over the course of 4 h and the reaction was allowed to proceed with mixing at room temperature for 34 h. Flash chromatography was used (silica: Mallinckrodt, 75–250 μm particles, 150 Å pore size) to isolate the hexakis product. The unreacted residue and some of the impurities were removed with toluene and dichloromethane and then the product was eluted with a CH2Cl2:MeOH 5:1 (360 mL) mixture. The brown filtrate was evaporated (100 mL flask) in vacuo to form a dark brown grease (2.003 g), which was hydrolysed in order to obtain unprotected derivative 6. For this purpose, the protected [60]fullerene derivative 5 (1.966 g) was dissolved in 20 mL of dry DCM and then sodium hydride (200.0 mg, 8.33 mmol) was carefully added in portions and stirred for 15 min. Next, methanol was added (8 mL, in portions of 0.2–0.5 mL) and during this period, intense bubbling was observed (at the bottom of the flask, however, unreacted so it remained). The flask was tightly closed and the reaction with mixing was allowed to proceed at room temperature for 60 h. Then, 1.5 mL of methanol was added to the brown mixture in the flask; no bubbling was observed. The solvents were evaporated in vacuo to form a black solid (1.735 g). Next, a partially cleaved fullerene nanomaterial was placed in a 100-mL flask and then 1,4-dioxane (18 mL) and concentrated hydrochloric acid (3 mL, 35%) were added (the black solid product immediately dissolved). The flask was tightly closed and the reaction mixture was allowed to proceed for further hydrolysis at room temperature for 7 days. The solvents were evaporated in vacuo to form a brown solid of compound 6 (2.092 g). The final product was purified by dialysis of an aqueous solution of fullerene 6 using cellulose membranes (molecular weight exclusion limit 1.0 kDa; Spectrum Labs, USA). The dialysis lasted 7 days, and the final fullerene nanomaterial 6 was concentrated, frozen and lyophilized. The final product was obtained as a brown solid with a 35% yield (137.8 mg).
NanoC60GEM
In a small vial, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI, 6.60 mg, 0.034 mmol), N-hydroxysuccinimide (NHS, 3.96 mg, 0.034 mmol) and fullerene 6 (30.0 mg, 0.0014 mmol) were dissolved in 2 mL of a MES buffer (10% 2-(N-morpholino)ethanesulfonic acid in water, pH = 4.5). After 5 min of stirring, the whole mixture was added (rinsing the vial twice with 0.5 mL of the MES buffer) into a reaction vessel (10 mL) containing gemcitabine (GEM, 10.32 mg, 0.034 mmol). The [60]fullerene derivative 6 was dissolved instantly and the reaction vessel was placed on a magnetic stirrer at room temperature for 92 h. Afterwards, the final product was purified by dialysis against an aqueous solution, using a cellulose membrane (10 mL, molecular weight exclusion limit 1.0 kDa; Spectrum Labs, USA). The dialysis lasted 10 days and the water was changed twice a day. Finally, the dialysate was concentrated, frozen and lyophilized. The final product was obtained as a brown solid with a 79% yield (29.42 mg) and its purity was confirmed by HPLC (Additional file 1: Fig. S7).
Time-resolved singlet oxygen detection
Phosphate-buffered (pD 7.4, 10 mmol) D2O solutions of nanoC60-GEM and TMPyP in a 1-cm-optical path quartz fluorescence cuvette (QA-1000; Hellma, Mullheim, Germany) was excited by light pulses that were generated by an integrated nanosecond DSS Nd:YAG laser system, equipped with a narrow bandwidth optical parametric oscillator (NT242-1k-SH/SFG; Ekspla, Vilnius, Lithuania). The laser system delivered pulses at a 1-kHz repetition rate, with a pulse energy up to several hundred microjoules in the visible region, and up to several tens of microjoules in the UVA–UVB region. The [60]fullerene photosensitizers were photoexcited using a 427 nm wavelength. The absorbance of the samples was set to 0.27 in that wavelength. In order to adjust the photoexcitation energy in the experiments, a laser beam was attenuated with three pieces of wire mesh (light transmission 40% each). The quantum yield of singlet oxygen formation was calculated using 5,10,15,20-tetrakis(1-methyl-4-pyridinio)porphyrin tetra(p-toluenesulfonate) (TMPyP) as the reference compound (ϕ = 0.75) (Snyder et al. 2006).
Electron paramagnetic resonance spin trapping studies
EPR spin trapping was used with DMPO as the spin trap at a concentration of 100 mM. Samples containing 0.1 mg/mL nanoC60GEM in 80% DMSO were irradiated in EPR quartz flat cells in the resonant cavity with 402- to 508-nm (24 mW/cm2) light, which was produced by a 300-W high-pressure compact arc xenon lamp (Cermax, PE300CE13FM/Module300W; PerkinElmer Optoelectronics, GmbH, Wiesbaden, Germany). The irradiation setup was equipped with a water filter, heat reflecting hot mirror, cut-off filter that blocked light below 390 nm and a blue additive dichroic filter 505FD64-25 (Andover Corporation, Salem, NH, USA).
Cell lines and culture methods
The human breast carcinoma; MCF-7 and human lung carcinoma epithelial-like A549 were obtained from the American Type Culture Collection (ATCC). The human pancreas adenocarcinoma AsPc-1 and pancreatic carcinoma of a ductal origin PANC-1 were obtained from Sigma-Aldrich. The murine pancreatic ductal adenocarcinoma; PAN02 was obtained from NCI-Frederick Cancer Research Facility and the normal human fibroblasts NHDF was obtained from PromoCell. The cell lines; MCF-7, A549, PANC-1 and NHDF were grown in Dulbecco’s modified Eagle’s medium (DMEM). The DMEM for MCF-7, A549 and PANC-1 were supplemented with 12% heat-inactivated fetal bovine serum (Sigma) and for the NHDF with 15% non-inactivated fetal bovine serum (Sigma). The AsPc-1 and PAN02 cell lines were cultured in a RPMI 1640 medium (Gibco, UK), which had been supplemented with 10% heat-inactivated fetal bovine serum (Sigma). All of the culture media were supplemented with the antibiotics penicillin/streptomycin (Gibco) (1% v/v). The cell lines were grown as monolayer cultures in 75 cm2 flasks (Nunc) under standard conditions at 37 °C in a humidified atmosphere at 5% CO2. All of the cell lines were tested for Mycoplasma contamination.
Cytotoxicity studies
The cells were seeded in 96-well plates (Nunc) at a density of 5 × 103 cells/well for the cancer cell lines and 4 × 103 cells/well for the normal cell line (NHDF) and incubated at 37 °C for 24 h. An MTS assay (CellTiter 96® AQueous One Solution Cell Proliferation Assay from Promega) was performed after a 72 h incubation of the cells using nanoC60GEM (2–0.1 mg/mL) and gemcitabine (0.01–25 μM). The medium was then removed and 20 mL of a CellTiter96®AQueousOne Solution-MTS (Promega) was added to each well (with 100 mL DMEM without phenol red) and incubated at least for 1 h at 37 °C. The absorbance of the samples was measured at 490 nm using a multi-plate reader (Synergy 4, BioTek). The results which were expressed as a percentage of the control were calculated as the inhibitory concentration (IC50) values using GraphPad Prism 7. Each experiment was repeated at least three times, each in triplicate.
Cell cycle assay
The PAN02 cells were seeded in 3 cm Petri dishes (Nunc) at a density of 1.5 × 105 cells/dish and incubated at 37 °C for 24 h. Then, solutions of the nanoC60GEM (35 and 17 μM) and gemcitabine (1.5 and 0.75 μM) were added. After a 48-h treatment, an assay was performed using a Muse Cell-Cycle Kit (Millipore) according to the manufacturer’s instructions. Briefly, the cells were collected, washed with cold PBS and centrifuged at 300g. Next, the cells were fixed in ice-cold 70% ethanol and stored at − 20 °C overnight. Afterwards, the cells were centrifuged and resuspended in 200 μL of a Muse™ Cell Cycle Reagent and incubated for 30 min at room temperature in the dark. After staining, the cells were processed for the cell cycle analysis using a Muse Cell Analyzer (Millipore). The experiments were performed at least three times, each in triplicate.
Annexin V binding assay
The PAN02 cells were seeded in 3-cm Petri dishes (Nunc) at a density of 1,5 × 105 cells/dish and incubated at 37 °C for 24 h. Then, solutions of the nanoC60GEM (35 and 17 μM) and gemcitabine (1.5 and 0.75 μM) were added. After 48 h, an assay was performed using an Annexin V & Dead Cell Kit (Millipore) according to the manufacturer’s instructions. Briefly, both detached and adherent cells were collected and centrifuged at 500 g for 5 min. Afterwards, the resuspended cells were incubated with 100 μL of a Muse™ Annexin V & Dead Cell Reagent for 20 min at room temperature in the dark. After staining, the events for live, early and late apoptotic cells were counted using a Muse Cell Analyzer (Millipore). The experiments were performed at least three times, each in triplicate.
Photodynamic therapy on the PAN02 cell line
The PAN02 line cells were plated in 24-well plates at density of 35,000/well. Twenty-four hours after plating, the cells were incubated with high-glucose DMEM that contained nanoC60GEM at different concentrations. Feeding was repeated two more times, at 24-h intervals. The day after the final feeding the cells were washed twice with PBS that contained calcium and magnesium ions, then irradiated for 15 min using a blue led light (440 nm) at a fluence rate 20 mW/cm2. Dark control cells were kept in the same conditions except light exposure. After irradiation, the cells were provided with DMEM with 10% FBS. The cytotoxic effect of the photodynamic treatment was quantified 24 h after irradiation, using a MTT assay for the mitochondrial redox function. The MTT solution in DMEM with 10% FBS was added to the treated and control culture wells (final concentration of 0.5 mg/mL). After incubation for 30 min at 37 °C, the culture medium was removed and the remaining blue precipitate was solubilized in DMSO, followed by reading the absorbance at 560 nm in a plate reader (GENios Plus, Tecan Austria GmbH). The results are reported as the percentage of the paired untreated controls. The experiments were repeated a minimum of three times.
Statistical analysis
The results are expressed as the mean ± standard deviation (SD) from at least three independent experiments. A statistical analysis was performed using the one-way ANOVA with a Bonferroni post hoc test. A p value of 0.05 or less was considered to be statistically significant. GraphPad Prism v.7.0 software (GraphPad Software, USA) was used for the analysis (Fig. 1).