Characterization of nanoparticles
Dynamic light scattering
MCM-41 NPs were successfully synthesized using CTAB, as a precursor, and TEOS, as the silicate source. The synthesized NPs were first characterized in terms of size. The size of NPs is a determinant factor affecting the efficiency of loaded therapeutics in that smaller NPs are more internalized into cells, thereby increasing the intracellular concentration of the loaded therapeutics (21). NPs with a size below 300 nm are efficiently internalized into target cells and exhibit their pharmaceutical effects (37). The resulting NPs, in the present study, were found with the size below 300 nm (220 ± 11.5 and 293 ± 8.7 nm, for MCM-41 NPs and ABZ-loaded MCM-41 NPs, respectively, Fig. 1) (46, 59). Also, size distribution is a critical factor that affects various properties of NPs, such as biological effects, (27) reproducibility, (9) and stability (15). Particles with varying sizes have various properties in terms of i) blood circulation, ii) cellular uptake, and iii) biodistribution (27). Also, increasing the particle size and size distribution can result in the physical instability of NPs (15). The size distribution of MCM-41 and ABZ-loaded MCM-41 NPs were equal to 0.214, and 0.282, respectively, indicating that these NPs were homogenous and monodisperse (57, 26). Also, the zeta potential of NPs is an important factor to determine the stability of NPs suspension as NPs with the same charge (positive or negative) in aqueous solutions with low ionic strength repulse each other, and this restrains their aggregation (21). The zeta potential of MCM-41 and ABZ-loaded MCM-41 NPs were found to be -36.3 ± 4.57 and -33.0 ± 4.93 mV, respectively.
Transmission electron microscopy
TEM was used to evaluate the size and structural features of ABZ-loaded MCM-41 NPs. The results of TEM confirmed the results of DLS and demonstrated that the NPs were synthesized at the nanoscale size. As Fig. 2a shows, the NPs were formed in uniform nanospheres with the hexagonal array of channels. Also, the porous structure of these NPs can be clearly observed.
Scanning electron microscopy
SEM was used to evaluate the surface morphology and size distribution of ABZ-loaded MCM-41 NPs. The SEM image of the NPs demonstrated that these particles were synthesized as homogenous and spherical NPs, which had a smooth surface (Fig. 2b). Also, it demonstrated that ABZ-loaded MCM-41 NPs were well dispersed without aggregation.
Fourier Transformed Infrared Spectroscopy
Fig. 3 demonstrates the FTIR spectroscopy of pure ABZ. The peak assigned to 3323 cm−1 was related to the stretching vibration mode of amide N-H. The absorption band observed at around 2960 cm−1 was related to the aliphatic hydrocarbon group (C-H). The ester C=O bond of the carbamate portion was observed at about 1713 cm−1. The peak related to the aromatic C=C bond was observed at around 1623 cm−1, which along with the amide N-H bond, constitute the benzimidazole portion of the drug. The peak observed at around 1523 cm−1 indicated the stretching vibration mode of the C=N group (3). Overall, the FTIR spectrum indicated normal bands of pure ABZ (3). Also, the bands related to asymmetric and symmetric Si-O-Si stretches of MCM-41 were observed at 1110 and 825 cm-1, respectively. A peak at around 980 cm-1 was also related to the tension of the Si-OH bonds of the MCM-41 compound (23). The existence of ABZ related peaks in ABZ-loaded NPs (e.g., 1623 cm−1, demonstrate by arrows in Fig. 3) confirmed that ABZ was loaded into the NPs. According to the results, ABZ preserved its chemical bonds in MCM-41 NPs, indicating the physical loading of ABZ into the NPs.
Thermogravimetric analysis and differential scanning calorimetry
To determine the drug loading efficiency and thermal stability of ABZ, the TGA curves (mass vs. temperature) of ABZ, MCM-41, and ABZ-loaded MCM-41 NPs were measured. According to the results (Fig. 4a), an initial weight loss occurred in MCM-41 NPs at around 250 ℃ due to the evaporation of absorbed water molecules, indicating the hydrophilic nature of this carrier, which is an advantage for loading of poorly water-soluble drugs (32). Also, ABZ-loaded MCM-41 NPs started to be degraded at 150 ℃ and continued up to 900 ℃. This resulted in a mass loss of 30%, which was related to the degradation of ABZ. Based on the results, the amount of ABZ adsorbed onto the NPs was 30%. ABZ was almost completely degraded at 900 ℃ (Fig. 4a).
DSC analysis can be used to investigate the existence or absence of a crystalline drug (e.g., ABZ) in the pores of mesoporous NPs (4, 29). Also, this method can be used to study the glass transition temperature (Tg) of the samples, where at the temperature above Tg, various physical properties of a material, such as free molecular volume, heat capacity, thermal expansion coefficient, dielectric coefficient, and viscoelastic features, suddenly change (32, 8). Fig. 4b illustrates the DSC profiles of the standard ABZ, MCM-41, and ABZ-loaded MCM-41 NPs. As MCM-41 NPs did not have any transitions in the temperature range of 50-350 ℃, only the thermal transition of ABZ was observed (32). Thus, a melting endothermic peak at around 200 ℃ was observed in the thermogram of ABZ, which is indicative of a crystalline anhydrous state of the drug. Also, the DSC profile of ABZ-MCM-41 demonstrated a melting endothermic peak around 170 ℃, which was related to the crystalline anhydrous state of ABZ, confirming drug loading into NPs.
Drug release from nanoparticles
Several daily doses of a drug are needed to attain and preserve the therapeutic concentration of the drug. This might lead to significant fluctuations in the plasma concentration of the drug, (18) resulting in a decrease in the concentration beyond the minimum effective concentration, or an increase in the concentration above the minimum toxic concentration, and consequently, resulting in the lack of therapeutic effects or unfavorable toxic effects (21). These fluctuations in the plasma drug concentration can be reduced using sustained-release and controlled-release drug delivery systems, leading to improvements in the therapeutic outcome of the drug (49).
In the present study, in order to simulate the pH changes in the gastrointestinal tract, the drug release was evaluated at pH 1.9 and 7.4, corresponding to the pH values of the human stomach (31) and intestinal fluids (12), respectively. The results demonstrated that the release of ABZ from MCM-41 NPs was initiated with a burst release, in which in the first 15 min of the study, 62 and 70% of the loaded ABZ was released at pH 1.9 and 7.4, respectively (Fig. 5). The burst drug release could stem from the release of the adsorbed drug onto the NPs or weakly bound between the drug and the NPs surface (20). In addition, by increasing the surface area of NPs, the amount of initial burst release increased (7). The pattern of the drug release continued with a gradually increasing trend at both pH values, in which 75 and 80% of the loaded drug were released after 12 h, indicating a sustained and controlled drug release pattern.
Also, according to the results, the drug release was pH-dependent; however, the difference in the amount of the drug release between two pHs was not statistically significant. These results were approximately similar to the results of Nguyen et al. study (44), where the difference in the amount of drug (prednisolone) release from MSNs at pH 1.9 and pH 7.4 was ~ 2%. In the current study, the difference in the amount of drug release in two pHs could be related to various factors, such as MSNPs agglomeration at acidic pH and variation in the surface charge of the nanoformulation in different pH (62, 60). At acidic pH (pH 1.9), the drug release from the pores of the agglomerated particles is inhibited, resulting in lower drug release (62). Also, to determine the kinetics of the drug release, the profiles of drug release were analyzed using different kinetic models, including zero and first order, Higuchi, and Korsmeyer Peppas models, and the correlation coefficient values were determined for the linear curves. Based on the results, the higher R2 values (0.3766 and 0.4156 at pH 1.9 and 7.4, respectively) were obtained in the Higuchi model compared to other drug release models, thus the drug release from the nanoformulation at both pH followed the Higuchi kinetic model.
Brunauer-Emmett-Teller surface area analysis
The N2 adsorption/desorption isotherms of the calcined MCM-41 and ABZ-loaded MCM-41 NPs are demonstrated in Fig. 6. MCM-41 and ABZ-loaded MCM-41 NPs demonstrated the behavior of the mesoporous material and type IV isotherm, based on the IUPAC isotherm classification system. These isotherms can be divided into three steps, including i) a linear increase in N2 adsorption, occurring at relatively low pressure owing to the monolayer adsorption of N2 on the wall of MCM-41 and ABZ-loaded MCM-41 NPs; ii) capillary condensation of N2 inside the mesopores, which is indicative of narrow pore size distribution; and iii) saturation step, presented as a long plateau at higher pressures owing to the low N2 adsorption onto the external surface of the calcined MCM-41 and ABZ-loaded MCM-41 NPs (32). Based on these results, the surface volume for MCM-41 and ABZ-loaded MCM-41 NPs was found to be 540 and 380 m2/g, respectively, while the pore size for these formulations was found to be 2.5 and 2.1 nm, respectively. In addition, the pore volume of MCM-41 and ABZ-loaded MCM-41 NPs was determined to be approximately 0.75 and 0.46 cm3/g, respectively.
Biological effects of the nanoparticles
The cytotoxicity effects of ABZ, MCM-41, and ABZ-loaded MCM-41 NPs were investigated on HepG2 cells using MTT assay to determine if ABZ loading into NPs caused enhanced cytotoxicity effects or not. For this purpose, ABZ at the concentrations of 0, 3.1, 6.3, 12.5, 25, 50, and 100 µM were used as these concentrations encompass the reported serum concentration of ABZ (4.3 µM), when administered at the standard doses (10 mg/kg/day). Also, a significantly higher drug concentration (100 µM) was used to make the toxicity more pronounced. The results demonstrated that MCM-41 NPs, at the concentration of 25 µg/mL, had no toxicity effects on the cells. It was found that ABZ loading into NPs caused a significant increase in the cytotoxicity effects of the drug in a concentration-dependent manner (Fig. 7), in which the IC50 values for ABZ and ABZ-MCM-41 NPs were estimated 23 and 7.9 µM, respectively.
Migration is a distinctive feature of cellular behavior that contributes to embryogenesis, tissue remodeling, wound healing, and pathologies, such as cancer metastasis and invasion (24, 25). The cell migration was determined using HepG2 cells at different time intervals to recognize the distance at which cancer cell invasion happened. A scratch with a width of 500 µm was generated and treated with ABZ and ABZ-loaded MCM-41 NPs at the drug concentration of 25 µM. The cancer cell migration was monitored by taking pictures at times 0 and 24 h (Fig. 8). As the results demonstrated, both ABZ and ABZ-loaded MCM-41 NPs inhibited cell migration. However, ABZ-loaded MCM-41 NPs, compared to ABZ, seemed to be more potent to inhibit the migration as according to the observation, the scratch width corresponded to ABZ was less than that corresponded to ABZ-loaded MCM-41 NPs. This indicated the potency of MCM-41 NPs to increase the antiproliferative effects of ABZ. These results were in agreement with the results of cytotoxicity evaluation, where the NPs caused an increase in the cytotoxicity effects of ABZ against HepG2 cells.