Chemicals
Iron (III) chloride hexahydrate (FeCl3·6H2O), ammonium hydroxide (NH4OH), ferrous sulfate heptahydrate (FeSO4.7H2O), nisin, rutin, sodium citrate dihydrate, and ethanol were all acquired from Sigma-Aldrich, USA. Gibco (USA) was the supplier of dimethyl sulfoxide (DMSO).
Methods
Preparation of rutin-capped iron oxide nanoparticles (R-IONP)
Fe (III) and Fe (II) salts with a molar ratio of 2:1 were dissolved in 40 ml of water with vigorous mechanical stirring. At 40 °C, 1 ml of Rutin solution (30 mg Rutin dissolved in 1 ml DMSO) was added dropwise. At 80 °C, 5 ml of 28% ammonium hydroxide was added, and the mixture was magnetically stirred for 30 min until a distinct brown shade appeared (R-IONP). A permanent magnet was used to isolate Rutin-IONP and was washed many times with deionized water and ethanol to eliminate unreacted contaminants. After 4 h of drying in the oven at 60 °C, INPs were finally obtained. After separation of R-IONP with a permanent magnet, the concentration of the collected free Rutin in the supernatant was calculated at an excitation wavelength of 380 nm and emission wavelength of 490 nm using a spectrofluorometer (Shimadzu, RF5301pc, Japan). Then, the coating efficiency was calculated using the following formula:
$$Coating{\text{ }}efficiency{\text{ }}\left( \% \right)=\frac{{Initial{\kern 1pt} \, amount\,\,of\,\,Ru\, – \,Supernatent\,free\,\,amount\,\,of\,\,Ru}}{{Initial\,\,amount\,\,of\,\,Ru}}$$
Preparation of nisin-capped iron oxide nanoparticles (N-IONP)
N-IONP might be made via post-functionalization synthesis of citrate IONP capped with nisin. Fe (III) and Fe (II) salts with a molar ratio of 2:1 were dissolved in 40 ml of water with vigorous mechanical stirring. At 80 °C, 5 ml of 28% ammonium hydroxide was added dropwise, then 4.9 g of sodium citrate was added. This was accomplished by combining 30 µl of nisin with the IONP. A permanent magnet was used to isolate N-IONP, and it was washed many times with deionized water and ethanol to eliminate unreacted contaminants. After 4 h of drying in the oven at 60 °C, INPs were finally obtained. The coating efficiency of nisin was calculated with the same previous method at an excitation wavelength of 280 nm, and observing emission at approximately 350 nm.
Physical characterization
Transmission electron microscope (TEM)
A transmission electron microscope (TEM) (JEM 1230 electron microscope, Jeol, Tokyo, Japan) was used to determine the size and morphology of the produced nanocomposites. TEM is a technique that involves passing an electron beam through an extremely thin object. It operates on the same principles as a light microscope, except that rather than light, it uses electrons. A drop of solution was applied to a copper-coated carbon grid. Before the examination, the grid had to be dried at room temperature for 5 min.
Atomic force microscope (AFM)
Topographic information was acquired with the atomic force microscope (AFM) (Wet—SPM9600, Japan). A tip scans a sample for AFM, and while scanning, the deflection of the cantilever on which the tip is positioned determines the force between the tip and the sample. Drawing the deflection of the cantilever against its position on the sample produces a topographic image.
Dynamic light scattering (DLS)
DLS measures the hydrodynamic size of particles and molecules in suspension. The measured hydrodynamic diameter reflects the dimension of the NP along with a layer of surface-bound solvent. The size measured by DLS was determined to be the “hydrodynamic diameter,” which is the size of a hypothetical hard sphere that diffuses in the same manner as the particle being measured. The Zeta Sizer (NICOMP TM 380 ZLS, USA) was used to measure the size distribution of the generated nanoformulations.
Zeta potential
Zeta potential was assessed utilizing the zeta sizer nano series (Nano ZS, Malvern Instruments, Malvern, UK) within a potential range of -200 to 200 mV. The potential evaluation was conducted at 25 °C, and the zeta potential value was derived by analyzing the direction and velocity of the prepared nanoformulations in the applied electric field.
Fourier transform infrared (FTIR) spectroscopy
FTIR spectroscopy was used to determine the functional groups of the active ingredients in the produced nanoformulations by examining the peak value in the infrared radiation area. An FTIR spectroscope (FTIR Edwards High Vacuum, Craeley Sussex, England) was used to get the transmitted peaks in the range of 449.33 to 4000.6 cm− 1.
Cell culture
MDA-MB-231: Breast cancer was obtained from Nawah Scientific Inc. (Mokatam, Cairo, Egypt). Cells were maintained in DMEM media supplemented with 100 mg/mL of streptomycin, 100 units/mL of penicillin, and 10% of heat-inactivated fetal bovine serum in a humidifier, 5% (v/v) CO2 atmosphere at 37 °C.
Cytotoxicity assay
Cell viability was assessed by the sulforhodamine B (SRB) assay. Aliquots of 100 µL cell suspension (5 × 103 cells) were placed in 96-well plates and incubated in complete media for 24 h. Cells were treated with another aliquot of 100 µL media containing drugs at various concentrations. After 48 h of drug exposure, cells were fixed by replacing the media with 150 µL of 10% TCA and incubated at 4 °C for 1 h. The TCA solution was removed, and the cells were washed 5 times with distilled water. Aliquots of 70 µL SRB solution (0.4% w/v) were added and incubated in a dark place at room temperature for 10 min. Plates were washed 3 times with 1% acetic acid and allowed to air-dry overnight. Then, 150 µL of TRIS (10 mM) was added to dissolve protein-bound SRB stain; the absorbance was measured at 540 nm using a BMG LABTECH®- FLUOstar Omega microplate reader (Ortenberg, Germany).
Statistical analysis
By triplet repetition of each experiment, results were collected as average ± SD. Using SPSS, one-way analysis of variance (ANOVA) was performed for the comet test and oxidative stress parameters. Scheffe’s test was used to compare the independent and dependent parameters at statistically significant values (P < 0.05) as a post-hoc comparison.
In silico analysis
GRP78 structural preparation
The structure of GRP78 was retrieved from the protein data bank using PDB ID 5E8413. Water molecules and ions were removed except for the two Zn ions and the ATP molecule in the N-terminal. Only one chain was used from the downloaded structure. To add the glycans to the protein at T85, T151, T166, T184, and T20314, the CHARMM-GUI website was utilized14,15,16,17,18.
MD simulation
After adding the glycans to the protein, the CHARMM-GUI webserver was used to prepare the systems and generate the input files for the GROMACS MD engine19,20. A standard unbiased MD simulation lasting 500 ns was performed in GROMACS 2021 to sample the conformation of the protein. We utilized the solution builder module of the CHARMM-GUI server to generate the input files21,22,23,24. The GRP78 protein was solvated in a 13.2 nm long cubic box. After solvating the system using the transferable intermolecular potential 3 points (TIP3P) water model with a padding of 1 nm from the furthest distant atom, the systems were neutralized by adding NaCl ions at a concentration of 0.154 M. The CHARMM36m force field was used to obtain the amino acid parameters of the protein, as well as those of the TIP3P water model and the ions. The ATP molecule was parameterized using the CHARMM general force field (CGenFF).
Periodic boundary conditions (PBC) were used in all three dimensions throughout the simulation. To prevent atomic collisions, the potential energy was minimized using the steepest descent algorithm. Following that, the temperature and pressure in the systems were equilibrated in two steps. When the maximum force applied to any atom was less than 100 KJ/(mol.nm), or when the number of reduction steps reached 100,000, the minimizing process was regarded to be converged. The NVT ensemble and the velocity rescale technique were utilized to reach an average temperature of 310 K during the first phase of the equilibration process. The NPT ensemble, the Berendsen barostat, and the velocity rescale algorithms were utilized in the second phase to maintain an atmospheric pressure of 1 atm and an average temperature of 310 K25. For the 500 ns production run, an NPT ensemble was utilized, and the temperature and pressure were regulated by a Nose-Hoover thermostat and a Parrinello-Rahman barostat, respectively. The temperature was kept at 310 degrees Celsius, while the pressure was maintained at one atmospheric pressure26. The LINear Constraint Solver (LINCS) was used to impose length constraints on the hydrogen-bonded atoms27. We utilized Particle Mesh Ewald (PME) summation to calculate the electrostatics with a threshold of 1.2 nm28. By using a time step of 1 femtosecond during equilibration and 2 femtoseconds throughout the production run, the Newtonian equations of motion were integrated using the leap-frog approach. We took 5,000 frames at 0.1 ns intervals during the simulation.
Clustering and docking of nisin, rutin, and citrate to GRP78
The simulation of GRP78 was clustered based on the RMSD of Cα atoms using the TTClust Python library to obtain a representative frame for each cluster22. The number of clusters was determined based on the elbow method, which generated two clusters. The representative frame for each cluster was selected as the middle frame. Two representative frames were obtained for GRP78. For each frame, it was loaded in Autodock Tools V 1.5.7 and was prepared by removing nonpolar H-atoms and adding Gasteiger charges. For GRP78, the searching box was defined to encompass the SBDβ domain with dimensions and center of x = 70, y = 120, z = 120, and x = 20.464, y = 53.937, z = 53.097, respectively, with a spacing of 0.686. We include enough space for small molecules and nisin conformational sampling30,31. The exhaustiveness in AutoDock Vina was set at 1000 for an elaborate search of the best pose30,31. Rutin, citrate, and nisin (PDB ID: 1WCO) were prepared using the same program and were prepared using the same steps. However, for nisin, only ten dihedrals (Ile1 Φ and ψ, Dhb2 Φ and ψ, D-Ala3 Φ, Cys7 ψ, D-Abu8 Φ, Cys11 ψ, Lys12 Φ and ψ) were allowed to rotate while the rest were kept non-rotatable32. The results from docking were examined by eye, and the best pose in each of the six systems was used as a starting point to perform the MD simulation for 200 ns using GROMACS by utilizing the same options as mentioned above. The CHARMM36 force field parameters of nisin were obtained from a previous work33.
MM-GBSA
The binding free energy (ΔG) between each ligand and the GRP78 protein was computed using the Molecular Mechanics/Generalized Born Surface Area (MM-GBSA) method, as implemented in the gromacs molecular mechanics Poisson Boltzmann surface area (gmx_MMPBSA) software package version 1.6.4 (https://valdes-tresanco-ms.github.io/gmx_MMPBSA/dev/)34,35. This approach integrates molecular mechanics energies with continuum solvation models to estimate the free energy of binding. To elucidate the contributions of individual amino acid residues to the overall binding affinity, per-residue energy decomposition analysis was carried out for all residues located within a 1 nm cutoff from the ligand.
All calculations were performed under physiological ionic strength conditions (0.154 M NaCl), employing a Generalized Born (GB) solvation model with the GB parameter (igb) set to 5. The internal and external dielectric constants were assigned values of 1.0 and 78.5, respectively, to reflect the polarizability differences between the solute and the surrounding aqueous environment. The fundamental formulation used for the binding free energy calculation is described by Eq. 1:
$$\Delta{{G\; = \; < \;Gcomplex\; – \;(Greceptor\; + \;Gligand) > \;\;\;\;}}$$
(1)
Here, < > denotes the ensemble average of the free energies for the complex, receptor, and ligand calculated across the analyzed simulation frames. In this study, the entire 200 ns trajectory was utilized, with snapshots sampled every 10 frames, resulting in a total of 200 frames for the energy calculations. The various energy components can be determined according to Eq. 2 through 6:
$$\Delta{{Gbinding\; = \;}}{{{\Delta}H\; – \;T}}\Delta{{S\;\;}}$$
(2)
$$\Delta{{H\; = \;}}{{E_{gas}}}{{\; + \;}}{{E_{sol}\;\;}}$$
(3)
$$\Delta{{E_{gas}\; = \;}}\Delta{{E_{ele}}}{{\; + \;}}\Delta{{E_{vdw}\;\;}}$$
(4)
$$\Delta{{E_{sol}\; = \;}}\Delta{{E_{GB}}}{{\; + \;}}\Delta{{E_{SA}\;\;}}$$
(5)
$${{ESA\; = \;\gamma }}\cdot{{SASA\;\;\;\;}}$$
(6)
where: ΔH represents the enthalpy, which is derived from the gas-phase energy (Egas) and the solvation-free energy (Esol). The term −TΔS accounts for the entropic contribution to the binding free energy, and it was not calculated in this study. The gas-phase energy (Egas) comprises electrostatic (Eele) and van der Waals (Evdw) energy terms. The solvation-free energy (Esol) is calculated as the sum of the polar solvation energy (EGB) and the nonpolar solvation energy (ESA), with the latter estimated based on the solvent-accessible surface area36,37.
