2.1. Materials
Iron (III) chloride (99%, molecular weight 162.20 Mwt), iron (II) chloride (99%, Molecular weight 126.20 Mwt), and ammonia 25% were purchased from Merck (Kenilworth, NJ, USA) with laboratory purity. Polyvinyl alcohol was obtained from Acros Organics (PVA, Fair Lawn, NJ, USA, average M.W. 6000). For the synthesis of Zn/Al-LDH, aqueous zinc nitrate six-hydrated, aqueous aluminum nitrate nine-hydrated, and sodium hydroxide (99%, ChemAR, Kielce, Poland) were used. 5-fluorouracil (5-FU) (an anti-cancer drug, C4H3FN2O2, 98%)) from AKSci (Union City, CA, USA) and dimethylsulfoxide ((CH3)2SO, DMSO) were purchased from Sigma Aldrich (St. Louis, MO, USA). All chemicals were used with laboratory purity and without previous purification. Deionized water was used in all stages of the experiment.
2.2. The Provision of Samples
The co-precipitation method was used to synthesize Fe
3O
4 nanoparticles [
15]. The reaction process (1) by which Fe
3O
4 nanoparticles are formed from two soluble salts is as follows:
FeCl3·6H2O (0.99 g) and FeCl2·4H2O (2.4 g) were separately prepared in distilled water with a ratio of 2:1 and mixed in a glass multi-wall reactor. After a few minutes, 6 mL ammonia was immediately added to the reactor and stirred with a magnetic stirrer for 15 min. In this stage, a black precipitate formed, indicating the formation of Fe3O4. Then, the sample was washed three times. After that, 2% of polyvinyl alcohol was dissolved in the black precipitate by placing it in an autoclave at 150 °C for one day. The uncoated nanoparticles were eliminated by washing them using deionized water (DI) water and dried at 70 °C for 8 h. Typically, a base solution (5-FU dissolved in dimethyl sulfoxide) was added to the prepared black powder under the magnetic mixer for the whole day. To prepare the Zn/Al-LDH using the co-precipitation method, at first, a solution of Al(NO3)3·9H2O and Zn(NO3)2·6H2O salts with a molar ratio of Zn/Al = 4 in a four-mouth flask under a nitrogen gas environment was prepared. The NaOH solution was added into the solution by dropwise addition at a rate of 1 drop per minute. The adding process of NaOH was continued until a solution with a pH of 7 was reached. Then, 3 g of prepared nanoparticles was mixed with Zn/Al-LDH. Upon completion, sample sediment was collected by centrifugation, washed three times, and dried in an oven.
2.3. Characterization
The XRD analysis was performed to determine the type of phase present in the sample. A Shimadzu XRD PW-6000 Model (Kyoto, Japan) using monochromatic CuK𝛼 radiation (𝜆 = 1.5406 Å) at 40 kV-30 mA in the range of 2–80° was used. To determine the surface coating entity of the nanoparticles, Fourier transformed infrared spectroscopy (FTIR) was used. The analysis was performed using a Thermo Nicolet 6700 (the Auxiliary Experiment Module (AEM), Madison, WI, USA) device at a resolution of 0.09 cm−1 in the range of 500 to 4000 cm−1. Samples were assessed using the potassium bromide (KBr) disk method. To investigate the thermal behavior and change in the amounts of the drug, polymer, Fe3O4 nanoparticles, and layered double hydroxides (LDH) weight loss, thermogravimetric analysis and differential thermogravimetric analysis were conducted to identify the mass reduction using a Mettler–Toledo model (TGA/DTG, Greifensee, Switzerland) in the range of 20 to 1000 °C.
All the samples were tested in a powder form. Pure iron oxide nanoparticle, polymer, drug, and LDH samples were also evaluated as reference samples. The average particle size and the particle size distribution plot were examined via dynamic light scattering, using a MALVERN (NanoS, Malvern, UK) device.
The surface morphology and elemental compositions of the particles were recorded using a NOVA NANOSEM 230 model field emission-scanning electron microscope (Los angeles, CA, USA) and energy-dispersive X-ray spectroscopy, respectively. Energy-dispersive X-ray spectroscopy (EDX) was used to probe and determine the chemical composition and identify the elements that were present in the samples.
For understanding the magnetic properties of the nanoparticles, a vibrating sample magnetometer device—model Lakeshore 4704 of the Lakeshore cryotronics company (Westerville, Ohio, United States of America)—measured in the range of −10,000 to 10,000 Oe was used. The morphology and size of the nanoparticles of the synthesized samples were evaluated using high-resolution scanning transmission electron microscopy (HRTEM, Hitachi H-7100, Tokyo, Japan). The Image J software was used to obtain the particle size distribution of the sample by choosing more than 100 particles. The drug loading and drug release behavior were then measured by a UV-visible spectrophotometer using a Perkin Elmer, Lambda 35, UV-Visible spectrometer at λmax = 214 nm. Inductively coupled plasma-optical emission spectrometry (Optima 8300, Perkin Elmer, Wellesley, MA, USA) was employed to identify the elements Mg, Al, and Fe and their concentrations. A CHN analyzer (LECO, TruSpec, Stockport, UK) was used to measure the percentage of N, H, and C elements. The magnetic characteristic was investigated using a vibrating sample magnetometer analysis in a measuring range of 20,000 to 20,000 G.
For evaluation of cellular toxicity and cell viability assay, normal human fibroblast (3T3) and human hepatocellular carcinoma cells (HepG2) that were obtained from American Type Culture Collection (ATCC) (Manassas, VA, USA) were used. The cells were cultured in Roswell Park Memorial Institute, 1640 medium (Nacalai Tesque, Kyoto, Japan) containing 10% fetal bovine albumin (Sigma-Aldrich, St. Louis, MO, USA) along with 1% antibiotics (included 10,000 units/mL penicillin/10,000 μg/mL streptomycin0 (Nacalai Tesque, Kyoto, Japan) in A T75 flask in a 37 °C 5% CO2 incubator. When the cell layers were harvested via 0.25% trypsin/1mM-EDTA (Nacalai Tesque, Kyoto, Japan), they were seeded in a plate of a 96-well tissue culture with 1.0 × 104 cells per well for one day to attach and about 80% confluence was attained for treatment. After one day of attachment to the respective wells, cells were treated with specific values of the treatment sample (1.25 to 100 μg/mL) consisting of Fe3O4 nanoparticles-polyvinyl alcohol (FPVA), Fe3O4 nanoparticles-polyvinyl alcohol-zinc/aluminum layered double hydroxide (FPVA-ZLDH), pristine 5-FU, and FPVA-5FU-ZLDH. The treatment solutions were prepared by dissolving the compound in 1% DMSO and RPMI at a ratio of 1:1 and then diluting it in the same media to produce various concentrations from 1.25 to 100 μg/mL. After incubation at 24 and 72 h, the viability of the cells and cytotoxicity were assessed by the methylthiazol tetrazolium (MTT)-based assay. For this purpose, 10 μL of MTT solution (5 mg/mL in PBS) was added to each well and placed in an incubator for 3 h before being aspirated. Then, purple formazan salt formed was dissolved in 100 μL DMSO solution in the dark and at room temperature. The absorption rate of the microplate reader device (Biotek LE800, Winooski, VT, USA), which reflects cell growth, was characterized at 570 nm. The intensity of the color is proportional to the number of cells that are metabolically active and therefore to the viability of the cells. For the computation of the half-maximal inhibitory concentration (IC50), the x-axis against the y-axis was the plot. Plotting the xy graph using the logarithmic value of their concentration and the following curve fitting (nonlinear regression) under the xy analysis to find a straight line equation fit (y = ax + b) was used to calculate the IC50 values. Several pieces of software were used for statistical analysis, including SPSS, ANOVA, and Duncan’s Multiple Range Test. The pristine 5-FU and FPVAFU-ZLDH (nanoparticles) were significantly different (* p value < 0.5).