The Fe3O4@LDH multicore-shell nanostructure was synthesized in two steps: firstly, magnetite nanoparticles were synthesized by solvothermal and Ostwald ripening method, and then layered double hydroxide nanoflakes were prepared on the magnetic nanoparticles by in-situ coprecipitation method and used as the new nanocarrier. Ethylene glycol was used for three reasons: solvent, reduction agent of Fe3+ to Fe2+, and synthesis of nanoparticles with monodisperse nanoparticles [21].
Characterization of synthesized nanomaterials
Fig. 1 depicts the schematic of the synthesis of Fe3O4@LDH multicore@shell nanostructure. Fig. 2 confirms and demonstrates surface morphologies and also determines particle sizes of (a) Fe3O4 nanospheres, (b) Fe3O4@LDH multicore-shell nanostructure, and (c) Fe3O4@LDH-ibuprofen. Fig. 2a and Fig. 2b obviously display the monodisperse structure of Fe3O4 nanospheres and Fe3O4@LDH multicore-shell nanostructure. The diameter of the Fe3O4 nanostructure is determined 80-130 nm, and the thickness of the LDH nanoflakes shell is about 70-110 nm (Fig. 2d). Furthermore, the Fe3O4@LDH-ibuprofen, has a morphology, even after loading of drug into the LDH layer structure (Fig. 2c).
The TEM was used to prove the core-shell structures and to show the porous and multicore nanoparticles created by the Ostwald ripening method. This method leads to formation of magnetic sphere particles in self-assembly form from smaller particles. According to the obtained TEM image, the size of core-forming nanoparticles is 10-13 nm and the size of magnetic spheres is approximately 80-130 nm. With regard to (Fig. 3a) Fe3O4@LDH multicore-shell nanostructure and (Fig. 3b) Fe3O4@LDH-ibuprofen, as the drug-loading nanostructure. Fig. 3b confirms that the multicore and core-shell structure of nanoparticles are completely stable after loading of the drug.
The Fourier transform infrared spectra of (a) Fe3O4 nanospheres, (b) Fe3O4@LDH multicore-shell nanostructure, (c) Fe3O4@LDH-ibuprofen, and (d) Fe3O4@LDH-diclofenac are shown in Fig. 4. The FT-IR spectra of Fe3O4 nanospheres (Fig. 4a), two highest peaks linked to metal-oxygen bonds, were observed. The first band detected in the range of 385–540 cm-1 is normally apportioned to octahedral–metal stretching, whereas the highest one detected in the 500–600 cm-1 range is consistent with basic stretching vibrations of the metal at the tetrahedral site. The higher frequency band at 574 cm-1 and lower frequency band at 448 cm-1are assigned to the tetrahedral and octahedral, respectively. Additionally, the peak at ~3360 cm-1 is attributed to the stretching vibrations of hydroxyl allocated to hydroxyl absorbed by magnetic nanospheres, and the existence of water is evidenced by the appearance of the bending mode at 1645 cm-1 and the stretching mode at 3476 cm-1. Compared with the spectrum of Fe3O4@LDH multicore-shell nanostructure with Fe3O4@LDH-ibuprofen and Fe3O4@LDH-diclofenac, there are particular similar peaks in their spectra (Fig. 4c and 4d). The principal peaks were between 2800 and 3000 cm−1 due to the alkyl stretching of drugs, especially in Ibuprofen due to the existence of many methyl groups in its structure compared with Diclofenac. Two peaks also appeared at approximately 1421 and 1576 cm-1, recognized to the symmetric and asymmetric stretch of the carboxyl group, respectively. The interaction between the metal atom and the carboxylate groups was classified into three types: monodentate, bridging, and chelating [22, 23]; the major difference (200–320 cm-1) was related to the monodentate interaction, and the lowest difference (<110 cm-1) was for the chelating bidentate. The medium-range difference (140–190 cm-1) was for the bridging bidentate. The Δ (1576– 1421 = 155 cm-1) was ascribed as bridging bidentate. Moreover, the peaks at 1440 and 1519 cm−1 are related to C–C stretching vibration in benzene rings. These outcomes provided subsequent assistance that Ibuprofen and Diclofenac have been loaded into the layered double hydroxide nanoflakes in the anionic form.
The XRD patterns of the Fe3O4 nanospheres (5a) in the 2θ range of 2–70° are shown in Fig. 5. Besides, with loading the drugs, the regenerated matrix indicates representative diffraction peaks of the LDH-drugs, representing two sharp basal reflections indexed as (003) and (006) reflections in line with the well-crystallized lamellar construction in synthesized nanocarrier with 3R rhombic proportion. The important diffraction peaks of Fe3O4@LDH-ibuprofen (5b) and Fe3O4@LDH-diclofenac (5c) are achieved at 2θ value of 7.6° and 7.8°. The d003 spacing of Fe3O4@LDH-ibuprofen and Fe3O4@LDH-diclofenac were found to be 2.62 nm and 2.22 nm, respectively.
The magnetic properties of magnetic nanospheres (Fig. 6a) and Fe3O4@LDH multicore-shell nanostructure (Fig. 6b) were specified using a vibrating sample magnetometer (VSM). The magnetic saturation values of the magnetic nanospheres and Fe3O4@LDH multicore-shell nanostructure were 59 and 32 emu g-1, respectively. After the LDH shell packing of Fe3O4 (curve (b)), the saturated magnetization of the Fe3O4@LDH multicore-shell nanostructure decreases because of the shield of the LDH nanoflakes.
Inhibition zones for the two pathogen bacteria including Bacillus cereus strain ATCC11778T (Fig. 7a) and Klebsiella pneumonia strain PTCC10031T (Fig. 7b) were observed in the presence of the nanoparticles. Most nanoparticles and nanostructures exert their antibacterial properties with different mechanisms such as destruction of bacterial membranes, inhibition of biofilm formation, or other multiple mechanisms [24], indicating that the nanoparticle has antimicrobial feature which can be considered as an extra benefit for drug delivery.
MTT assay analysis showed that Fe3O4@LDH multicore-shell nanostructure in 0.001 g concentration had a less negative effect on C2C12 cells as upon 90% of the cells treated viable in comparison to the control group (Fig. 8). The non-toxicity of nanoparticle can be considered a positive point for using them in drug delivery to eukaryotic organisms, especially humans. In addition, no valuable difference has been observed between 0.001 and 0.005 g concentrations of Fe3O4@LDH on cell viability. However, it was observed that the viability of about 20%-30% of cells decreases in the presence of the high concentration of the nanostructure (0.05 and 0.01 g).
Based on the literature, the nanoparticle size plays an important role in cellular uptake and intracellular trafficking of drug encapsulated in LDH nanoparticles. Several studies have demonstrated that FITC-LDHs are internalized into cells through the clathrin-mediated endocytosis [25]. However, it has been noticed that the mechanism of selectively permeating into cell is effective only at LDH nanoparticle size of 300 nm or less [26]. As the size of Fe3O4@LDH nanoparticles is 80-130 nm, probably these nanoparticles penetrate into C2C12 cell via selectively clathrin-mediated endocytosis.
UV-Vis spectrum of Ibuprofen and Diclofenac release assay
Drug release is specified as the speed of mass transport from a solid phase into the broth media under normal conditions. The major phase in drug delivery is an interaction between the drug carrier and PBS (pH=7.4) that happens at the interface of carrier and buffer solution, and absorption was measured via UV-Vis Spectrophotometer in specified intervals. According to the previous studies, the suitable pH for releasing drug in oral nanoparticles is in physiological buffers condition (pH 7.4) [27]. Each segment of the gastrointestinal (GI) tract maintains its own characteristic pH level from the acidic stomach lumen (pH 1–3) for digestion to the alkaline duodenum and ileum (pH 6.6–7.5) for the neutralization of chyme. Oral nanoparticles retain a hydrophobic and collapsed state in the stomach due to the protonation of hydroxyl groups and increase the zeta potential. After gastric passage, an increase in pH leads to activation of nanoparticles due to decrease of zeta potential and hydrogen bond breakage of interlayers of LDH [27-30]. It should be noted that Ibuprofen and Diclofenac are weak acids that are not be soluble in acidic media, but should be soluble at pH higher than 6.8 [31-33]. Moreover, at a pH above 9, the increase in the concentration of competing OH- anions is responsible for the observed decrease in the recovery [33]; over ~90% of the drugs are released at pH 7.4 in the first several hours. Designed nanoparticles for oral drug delivery such as our nanoparticle undergo a surface charge reversal and decrease zeta potential after gastric passage, hoping that drug release will possibly occur in the alkaline intestinal tract instead. Using our inorganic materials with different densities of positively-charged facilitated loading and trapping of anionic drugs such as Ibuprofen (an anti-inflammatory prodrug for bowel disease) in acidic environments (pH<3). When the drug-loaded nanoparticles were placed in physiological buffers (pH 7.4), a partial negative surface charge on the nanoparticle was generated; this electrostatic repulsion triggered the sustained release of loaded drugs. According to the previous studies, the release assays were, hence, carried out in physiological buffers condition (pH 7.4) [10, 18] at 37 ºC which is similar to the normal body temperature.
Fig. 9 illustrates that the drug release occurred at intervals within 15 min to 72 h in the wavelength of 264 nm for Ibuprofen and wavelength of 276 nm for Diclofenac. Both drug releases gently increased within 15 min to 6 h interval, and the concentration of drug was fixed within 6 h to 72 h interval. The release rate of Ibuprofen was 90% within 24 h, 94% in 48 h, and 96% in 72 h. The values for Diclofenac in 24 h 78%, within 48 h of 81% in 72 h and 82%, respectively, indicating less Diclofenac release in comparison to Ibuprofen per unit of time. This can be due to lower solubility in water [34], highly lipophilicity [35] of Diclofenac, small size, and more sterile effect of Diclofenac compared to Ibuprofen [36] that cannot be easily released between layers. The release gradually arrived the maximum amount of 90 % for Ibuprofen and 78% for Diclofenac in the first 6 h. On the other hand, the most absorbed drug in the outer layer of the Fe3O4@LDH and bonded drug to the substrate by its hydrogen bonds release in the 24 h especially in first 6 h which is useful for quickly developing as a therapeutic dose. Other remaining drugs in the structure less than 10% for Ibuprofen and 20% for Diclofenac which were in the interlayers of the LDH release slowly. The slower delivery rate can be utilized as a therapeutic dose in longer time for decreasing the number of doses required. The cumulative release kinetic of Ibuprofen and Diclofenac from Fe3O4@LDH nanostructure in phosphate buffer saline (PBS) at pH=7.4 showed a sustained release of up to 72 h that closely resembled first order release kinetics through a combination of drug diffusion and dissolution of LDH under physiological conditions (Fig. S3).
Drug loading between LDH layers leads to different release rates of drug and enhances the solubility of the drug and also, reduces its side effects, compared with old and industrial methods.
As other anionic drugs, it seems that the drug release mechanism of Diclofenac and Ibuprofen from the LDH nanoparticles is probably surface diffusion and bulk diffusion via anionic exchange of the drugs anions on, or in, the LDHs with anions in the PBS solution [37]. Fe3O4/LDHs nanocomposites have been also noticed for drug delivery in different studies due to their layered structure and unique properties [38]. Komarala et al. developed LDH–Fe3O4 magnetic nanohybrids by a mixed method (coprecipitation synthesis and hydrothermal method) within the range of 10–15 nm for magnetic hyperthermia and delivery of Doxorubicin to cancer cells (HeLa cells) [39]. They showed that Doxorubicin was successfully loaded into the nanohybrids (drug-loading efficiency; ∼99%) and released by pH dependent manner. The concentration of 0.94 mg ml-1 (R2 = 0.957) Dox-loaded nanocomposites decreased the viable cell population by 50% and prevented their proliferation [40]. In another study, Fe3O4@MTX-LDH/Au nanoparticles were developed by Zhao et al. through coprecipitation electrostatic interaction strategy to deliver the anticancer drug of methotrexate (MTX). Likewise, the cumulative percent of the prepared sample and some previously reported materials for Diclofenac and Ibuprofen are compared in Table 1.