Chemicals
Chrysin (>99%), Tris HCl, 5-fluorouracil (>99%), and CS (Mw = 100–300 kDa, 70–75% deacylated). Trisodium pentapolyphosphate (TPP, 99%), Tween 80, and mitochondria isolation buffer (mannitol (>95%), 4-(2-hydroxyethyl) piperazine-1-ethane sulfonic acid, N-(2-hydroxyethyl), and piperazine-N-(2-ethane sulfonic acid) [HEPES]), and MTT [3–(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]. All these chemicals were of analytical grade and purchased from Sigma-Aldrich Company in Germany. Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were purchased from (GIBCO, New York, USA). L-Glutamine, trypsin-EDTA, and trypan blue were purchased from Invitrogen (VIC, Australia). Penicillin/streptomycin was purchased from Thermo Fisher Scientific (Waltham, MA, USA).
In silico studies
The protein structure of SDH was retrieved and processed for in silico molecular docking investigations using the RCSB Protein Data Bank (PDB) (www.rcsb.org). The PDB contains data on three-dimensional structures of biomacromolecules. For SDH, a grid box was created around the co-crystallized ligand binding site [13]. The experimentation on molecular docking was performed using Docking Server 2009 and Discovery Studio Software Tools 2019 [14].
Synthesis of chrysin chitosan nanoparticles (CCNPs)
After pure chrysin powder was dissolved in 96% methanol, the solution was combined with 0.1% w/v chitosan solution in 0.1% acetic acid under magnetic stirring. Then, 40 mL TPP solution (0.1% w/v) and 1.075 mL Tween-80 solution (0.01% v/v) were added dropwise to the solution under magnetic stirring at 1000 rpm to produce chrysin-loaded chitosan nanoparticles [15].
Physical characterization of CCNPs
The ultraviolet (UV) absorption of the supernatant of CCNPs subjected to ultracentrifugation was used to the measure encapsulation efficiency (EE) of chrysin. By analysis of the supernatants of standard chrysin solutions (10–100 g/mL) in a UV/Vis spectrophotometer (Shimadzu, Japan), the relevant calibration curves were created. Measurements of chrysin were performed at 348 nm (λmax) [16].
The encapsulation efficiency (EE) of chrysin was calculated from the following formula:
$$\mathrm{EE}\ \left(\%\right)=\left(\frac{\ \mathrm{Amount}\ \mathrm{of}\ \mathrm{chrysin}\ \mathrm{encapsulated}\ \mathrm{in}\ \mathrm{NPs}}{\mathrm{Initial}\ \mathrm{amount}\ \mathrm{of}\ \mathrm{chrysin}\ \mathrm{used}}\right)\times 100$$
(1)
All measurements were performed in triplicate and the results were reported as the mean ± standard deviation [17].
In vitro release study
For the in vitro release study, CCNPs (0.1 g) were placed in 1× PBS, pH 7.4 (50 mL). The complete release system was maintained at 37°C under 100 rpm continuous stirring. Every 2 h for 24 h, 1 mL of solution was removed and replaced with 1 mL of buffer [15]. The amount of loaded chrysin was analyzed by UV spectrophotometry. The amount of chrysin released from microspheres over a given time was determined via the following equation: Chrysin release (%) = amount of chrysin released from microspheres/total loading amount of chrysin in microspheres × 100 [15].
UV analysis
A Shimadzu (UV-3101 PC) spectrometer attached to a shaking water bath (Julabo SW20C) was used to measure electronic absorption spectra of chrysin [18].
Fourier transform infrared spectroscopy (FTIR) analysis
The molecular bonding formation between chrysin and chitosan nanoparticles was studied using the infrared (IR) spectra recorded on KBr disks using a Perkin Elmer 1720 spectrophotometer in the range of 4000–400 cm−1. Pure chitosan nanoparticles and chitosan nanoparticles loaded with chrysin were measured [19].
X-ray powder diffraction (XRD) spectroscopy
A Philips X PERT-PRO X-ray diffractometer (GNR, APD2000PRO, Italy) equipped with a monochromatic Cu K (1.5406 A) x-ray source was used to produce the XRD patterns of chitosan and CCNPs. The voltage employed was 45 kV and the angle range was scanned from 5 to 35° with a step size of 0.0167° [20].
Analysis of morphology and particle size of nanoparticles
The morphological features of the CCNPs were investigated using a JEOL JEM-2100 high-resolution TEM operated at an accelerating voltage of 200 keV. Gatan Digital Micrograph software was used to view, collect, and interpret the TEM images. A dilute suspension of CCNPs in ethanol was dropped onto copper grids to prepare the samples for TEM examination [21, 22]. SEM was performed to determine the morphology, size, and shape of the surface of the particles (TESCAN VEGA 3 SBH model). The sample was placed in an ion sputter on an electron microscope of a metal stub and coated in gold. A random scan of the stub was taken for the CCNPs [23].
Determining zeta potential
The electrophoretic mobility (UE) of CCNPs was measured using a folded capillary cell and the zeta potential (ζ) was measured using a Zetasizer Nano Series (Brookhaven, USA). For the experiment, 1 mL of diluted CCNPs was used. All measurements were performed in triplicate [24].
Isolation of mitochondria from normal adult mice
Five adult male Swiss albino mice, weighing 28–30 g, were purchased from the Faculty of Pharmacy, Alexandria University, Egypt. The mice were maintained under suitable temperature conditions and a 12-h light/dark cycle. The experimental animals were kept in accordance with the National Institute of Health’s Guidelines for the Care and Use of Laboratory Animals. The experiments on animals were monitored by the Egyptian Ethical Committee of Tanta University’s Faculty of Science (IACUC-SCI-TU-0165) [25]. At the end of the experimental period, the mice were euthanized by cervical dislocation after intraperitoneal (i.p.) administration of sodium pentobarbital anesthesia (300 mg/kg) [26]. The liver was removed from the peritoneal cavity. The gallbladder was then located and removed using a scalpel as previously described [27, 28].
Determining IC50 of chrysin and CCNPs on succinate-ubiquinone oxidoreductase activity (complex II) in normal adult mice
The MTT technique was used to determine the activity of succinate dehydrogenase and to determine IC50 by examining the inhibitory effect of different concentrations of chrysin (5–100 μM), CCNPs (5–160 μM), CNPs (200–2000 μg/mL), and 5-fluorouracil (5–40 μM) on the substrate of succinate (400 mM), with rotenone (40 mg/mL), antimycin A (2 mg/mL), and KCN (40 mM) added into the mitochondrial suspension (50 μL) [29].
Determination of protein content
The mitochondrial protein content was detected using the Bradford assay [30].
Determining the effect of the IC50 values of chrysin, CCNPs, CNPs, and 5-fluorouracil on succinate dehydrogenase and coenzyme Q reductase activities (complex II)
Effect of IC50 values of chrysin, CCNPs, 5-fluorouracil on SDH activity by the MTT test in normal adult mice
The MTT test was used to determine succinate dehydrogenase activity. Briefly, the mitochondrial suspension (50 μL) was incubated with the IC50 concentrations of chrysin (34.66 μg/mL), CNPs (184.1 μg/mL), CCNPs (12.2 μg/mL), and 5-fluorouracil (0.05 μg/mL) in normal cell lines; then, succinate (400 mM) was added as a substrate and rotenone (40 mg/mL), antimycin A (2 mg/mL), and KCN (40 mM) were added as inhibitors of complex Ι, ΙΙΙ, and ΙV of the ETC, respectively. The reaction volume was made up to 1 mL with 50 mM phosphate buffer and then incubated at 37°C for 30 min. Then, 50 μL of MTT (0.025%) was added to the mitochondrial suspension. In the next step, 50 μL of dimethyl sulfoxide (DMSO) was used to dissolve the formed formazan crystals. Finally, the absorbance at 570 nm was assayed using an ELISA reader (Tecan, Rainbow Thermo, Austria) [29].
Determination of succinate dehydrogenase–coenzyme Q oxidoreductase activity (CII)
The mitochondrial suspension (50 μL) obtained from normal adult mice was incubated with the IC50 values of chrysin, CNPs, CCNPs, and 5-fluorouracil, and the specific activity was detected as described in recent methods [31, 32]. The activity of complex ΙΙ was expressed as nmol/min/mg protein using the standard DCPI curve [33].
In vitro cell viability studies using the MTT assay
Cell culture
Frozen human normal fibroblast cells (ATCC-CRL-2524) were cultured in accordance with standard cell culture protocols. The cells were cultured in DMEM containing 4.5 g/L glucose and 2% L-glutamine and supplemented with 10% FBS and 1% penicillin-streptomycin solution. Initially, the cells were seeded at low density and maintained at 37°C in a 5% CO2 incubator with 95% humidity.
Cell viability using MTT assay
The MTT tests were used to assess cell proliferation (viability) in accordance with the method of Denizot and Lang [34]. The cells were counted and then re-seeded in 96-well plates to a final concentration 1 × 105 cells/mL (3 × 104 cells/well). The cells were treated with different concentrations of chrysin (12.5–100 μM), CNPs (500–2000 μg/mL), CCNPs (12.5–100 μM), and 5-fluorouracil as a parallel control (3.13–100 μM). After 48 h, the culture medium was removed and the cells were washed twice gently with ice-cold PBS, and 20 μL of MTT (5 mg/mL) was removed from the 96-well microplates. The plates were placed in a cell culture incubator at 37°C for 4 h. The microplates were then shaken at the highest speed for 20 min at room temperature; after the incubation, the medium/MTT was removed and added 100 μL of DMSO was added to each well to dissolve the formazan produced. A negative control well, containing 10 μL of MTT stock solution control added to 100 μL of medium, was used. An ELISA reader was used to determine the absorbance of the plate at 570 nm (StatFax-2100, Awareness Technology, Inc., USA) [35].
The cell viability was determined as a percentage using the following equation:
$$\% { Viability}=\mathrm{Sample}\ \mathrm{absorbance}/\mathrm{Control}\ \mathrm{absorbance}\times 100$$
IC50 = the concentration of chrysin, CCNPs, or 5-fluororacil that causes 50% inhibition of cell proliferation.
Isolation of crude mitochondria from normal fibroblast cell lines
To isolate mitochondria from cells, 4 × 106 normal fibroblasts were subjected to the protocol of Wieckowski et al. [36].
Determination of the two activities of mitochondrial CΙΙ in normal fibroblast cell lines
Determination of SDH activity by MTT test with IC50 of chrysin, CCNPs, and 5-fluorouracil in normal cell lines
The MTT test was used to evaluate succinate dehydrogenase activity. Briefly, mitochondrial suspension (50 μL) was incubated with IC50 of chrysin (129 μg/mL), CCNPs (156 μg/mL), and 5-fluorouracil (8.07 μg/mL) in normal cell lines; then, succinate (400 mM) was added as a substrate and rotenone (40 mg/mL), antimycin A (2 mg/mL), and KCN (40 mM) were added as inhibitors of complex Ι, ΙΙΙ, and ΙV of the ETC, respectively. The reaction volume was made up to 1 mL with 50 mM phosphate buffer, then incubated at 37°C for 30 min. Then, 50 μL of MTT (0.025%) was added to the mitochondrial suspension. In the next step, 50 μL of DMSO was used to dissolve the formed formazan crystals. Finally, the absorbance at 570 nm was assayed using an ELISA reader (Tecan, Rainbow Thermo, Austria) [29].
Determination of succinate dehydrogenase–coenzyme Q oxidoreductase activity (CII) in normal human cell lines
The mitochondrial suspension (50 μL) obtained from normal cell lines was incubated with the IC50 of chrysin (129.1 μg/mL), CCNPs (1556 μg/mL), and 5-fluorouracil (8.07 μg/mL). Specific activity was detected according to the method of Taylor et al. [31]. The calculated CII activity was expressed as nmol/min/mg protein using the standard DCPI curve [33].
Statistical analysis
The results are shown as the mean ± SEM (n = 3). GraphPad Prism, v.6, was used for all the statistical analyses. The tests were repeated three times and two-way ANOVA tests followed by post hoc Tukey tests were used to establish statistical significance. To assess SDH activity, we used ANOVA tests as a specialized statistical analysis test. The threshold for statistical significance was fixed at P < 0.05.