The effect of mesoporous silica nanoparticles loaded with epirubicin on drug-resistant cancer cells

Introduction

Cancer is one of the main reasons of death, and chemotherapy is one of the most common cancer treatment approach that uses chemotherapeutic agents to kill cancer cells [1, 2]. The major impediment in chemotherapy cancer treatment is multi drug resistance (MDR). This resistance mechanistically is related to the adenosine triphosphate (ATP)-binding cassette (ABC) transporters such as P-glycoprotein (P-gp or ABCB1). These transporters are located in the cell membrane and their function is pumping drugs out of cancer cells, forming drug efflux. Therefore, the intracellular concentration of drugs is reduced that in turn, leading to drug resistance [3, 4]. Several strategies have been developed to overcome this hurdle. Recently, nanoparticles have attracted increasing attention in drug delivery fields.  Nanoparticulate drug delivery systems (NDDSs) have been proposed to execute “efflux circumventing strategy” to overcome MDR by intracellular drug delivery [5-8]. Upon arriving at tumor regions through enhanced permeability and retention (EPR) effects, NDDSs are uptake by tumor cells and transported into endosomal compartments, that are in perinuclear regions physically away from the membrane ABC transporters [9]. This location advantage allows drugs released within these endosomal compartments to evade the efflux pumping, acting to kill tumor cells [3, 10-15].

     Over the past decades, several nanoparticles have been developed and applied for targeted drug delivery to cancer cells, such as liposomes [16-21], polymeric micelles [22-25], dendrimers [26-28], carbon nanotubes [29-32], inorganic nanoparticles [33-36], nanographene [37, 38] and silica-based materials [39-43].

     Mesoporous silica nanoparticles (MSNs) have attracted much attention due to their unique physiochemical properties, including large specific surface area and high pore volume, controllable particle size, easy surface modification, considerable stability and biocompatibility, and high drug-loading capacity. In 1992, scientists succeeded to synthesize ordered mesoporous silica nano­materials for the first time. This discovery in material science lead to a diversity of applications ranging from food manufacturing to pharma­ceutical technology [44]. In 2001, Vallet-Regí et al reported the application of MSNs as a delivery system for ibuprofen for the first time. They showed that up to 30 wt% of this drug could be loaded into the nanoparticles and sustained drug release from MSNs [45]. In 2015, Malaekeh-Nikouei and coworkers used functionalized MSNs for delivery of epirubicin (EPI). They proved that phosphonated MSNs have suitable pH-dependent release pattern as at pH=5.5 about 70% of EPI was released from MSNs in 24 h, while at pH=7.2, the amount of drug release dropped down to about 8% at the same time [41].

     In the present study, we synthesized and functionalized MSNs by phosphonate group in order to achieve in negatively charged nanoparticles. The negative surface charge of MSNs and the mesoporous nature of these nanoparticles would allow good encapsulation of positively charged EPI. After that, we evaluated the cytotoxicity of the prepared nanoparticles on drug-sensitive human breast cancercell line (MCF-7) and drug-resistant cancer cells (MCF-7/ADR).

 Materials and methods

Tetraethylorthosilicate (TEOS), 3-(trihydroxysilyl) propyl methylphosphonate (42%), cetyltrimethylammonium bromide (CTAB) and Pluronic®F127 were purchased from Sigma-Aldrich (USA). Cell culture medium RPMI 1640, fetal bovine serum (FBS) and penicillin/streptomycin were obtained from Gibco (USA).

 Synthesis and functionalization of MSNs

The synthesis was performed as described in our previous study [41]. Briefly, deionized water and NaOH (2 M) were mixed. Then, Pluronic® F127 and CTAB were dissolved in this solution. This mixture was heated to 80 °C under stirring. After 30 min, TEOS was added into the solution. 20 min later, under argon atmosphere, the 3-(trihydroxysilyl) propyl methylphosphonate was added to the solution and stirred for another 2 h at 80 °C. Finally, the solution was cooled down to room temperature to obtain phosphonated MSNs (Ph-MSNs).

 Surfactant removal

For removing of CTAB, dialysis method was used as follows. After nanoparticles synthesis, 30 ml of Ph-MSN nanoparticles suspension was transferred to a dialysis tube and dialyzed for 24 h against a mixture containing water:ethanol (50:50) and HCl. Next, Ph-MSNs were dialyzed against double distilled water for another 24 h and this process repeated twice [46, 47].

 Characterization of MSNs

In order to determine the shape and structure, the nanoparticles were subjected to transmission electron microscopy (TEM) at 120 kV (LEO 912AB Zeiss electron microscope-Germany). One droplet of ethanolic dispersion of nanoparticles was placed on the carbon-coated copper grid and dried at room temperature. The particle size and zeta potential of the samples were determined by the Zetasizer (Nano-ZS, Malvern, UK) after dilution with deionized water.

For determination of carbon, hydrogen and nitrogen content, elemental analysis was carried out. Measurements were done using Costech ECS 4010 (Italy). FTIR spectroscopy was carried out using a Perkin–Elmer (USA) in the absorption mode in the range of 4000–450 cm^-1.

 Drug loading

Drug loading was carried out as follows. 1 mg of Ph-MSNs was dispersed in 0.5 ml of EPI solution (2 mg/ml) and stirred for 24 h in dark environment at room temperature. After that, Ph-MSNs were centrifuged and washed with deionized water to remove free drug molecules. To determine EPI loading efficiency, the fluorescence of the EPI supernatant and washed solutions was measured (ex: 488 nm/ em: 555 nm) in a microplate reader (Synergy H4- Hybrid, USA). The EPI content was calculated using a serial dilutions of EPI and eventually the drug loading content and drug loading efficiency were calculated as follows:

Drug loading content=(weight of loaded drug in Ph-MSNs/weight of drug and Ph-MSNs) ×100%

Drug loading efficiency= (weight of loaded drug in Ph-MSNs/weight of feeding drug)×100%

 Cell Culture

Drug-sensitive human breast cancercell line (MCF-7) and drug-resistant cancer cells (MCF-7/ADR) were cultured in complete medium (RPMI-1640 medium supplementedwith 10% FBS, 100 U/ml penicillin, and 100 μg/mlstreptomycin) and maintained at 37 °C in a humidifiedmediaand 5% CO₂ incubator.

 Evaluation of Ph-MSN and cell viability

In vitro biocompatibility of Ph-MSNs and cytotoxicity of EPI-loaded Ph-MSNs (Ph-MSNs-EPI) was measured using the MTT viability assay. Cells were seeded in 96-well plates at a density of 5×10³ cells per well in 100 µl of complete medium and incubated for 24 h. The cells were incubated with Ph-MSNs, Ph-MSNs-EPI and free EPI at different concentrations for 72 h. After the incubation, the MTT assay was performed by removing the medium and adding of 180 µl of fresh medium and 20 µl of MTT solution (5 mg/ml in PBS). The plates were incubated for 4 h at 37 °C. Subsequently, the medium was carefully removed, and then 200 µl of dimethyl sulfoxide (DMSO) was added to each well in order to dissolve the cells and MTT formazan. The plates were gently shaken for 5 min, and the absorbance of the obtained DMSO solution was determined at 570 nm by the microplate reader (Synergy H4-Hybrid, USA). The untreated cells were considered as control with 100% viability [48]. The viability of cells was calculated as follows.

 Viability= (Absorbance of each well/Average of absorbance of control wells) ×100

 Data analysis

Every experiment was repeated at least three times. IC₅₀ values were achieved using Prism (GraphPad software, San Diego, CA).

 Result and discussion

Synthesis and characterization of MSNs

MSNs are promising inorganic nanoparticles to deliver hydrophilic and hydrophobic anticancer drugs, gene, and siRNA into different cancer cell lines [49]. Because of their intrinsic high stability, excellent biocompatibility, and good degradability, these nanoparticles  exhibited great potential to overcome MDR [50, 51]. Herein we used this inorganic carrier to improve antitumor efficacy of EPI. MSNs were synthesized and modified to possess phosphonate groups on its surface, forming the phosphonate functionalized MSNs (Ph-MSNs), followed by the template extraction using a dialysis method. The mean nanoparticles size and zeta potential were measured in deionized water. In order to achieve smaller and more stable Ph-MSNs in aqueous media for biological applications, we used CTAB and Pluronic® F127 as templating agents to reach better nanoparticles. This synthesis approach is very effective in obtaining smaller and monodispersed nanoparticles [41]. The hydrodynamic size of Ph-MSNs was 98.1±5.2 nm and the conjugation of phosphonate groups onto the surface of MSNs donates a high negative charge to the nanoparticles (-19.5± 0.6 mV). Regarding the positive charge of CTAB, the negative zeta potential of nanoparticles revealed that the CTAB removed successfully from the structure. The porous structure and size of Ph-MSNs were characterized using TEM. As shown in Fig. 1, the morphology was nearly spherical. FTIR spectrum showed typical peaks of silica nanoparticles (Fig. 2). The peaks at 1078 cm^-1, 802 cm^-1 and 467 cm^-1 are pertaining to Si–O–Si and Si–O. The peak at 952 cm^-1 attributed to the vibration band of Si–OH. Also a broad peak around 3300 cm^-1 is related to adsorbed water and Si–OH groups on the surface of MSNs.

     The results achieved by elemental analysis are another evidences for the removal of CTAB from the pore of nanoparticles. Elemental analysis showed that most of CTAB was removed from the structure as the nitrogen content was very low (Table 1).

EPI loading

EPI, an antitumor drug, was selected as a model drug and penetrated into the pores of Ph-MSNs by soaking nanoparticles in a solution of EPI. The electrostatic interaction between positively charged EPI and negatively charged Ph-MSNs is the driving force of the loading process. The loading content and loading efficiency of Ph-MSNs were 9.45% and 18.90%, respectively.

 In vitro cytotoxicity of Ph-MSNs

In order to study the efficiency of EPI loaded Ph-MSNs, the standard MTT assay was carried out to assess the cellular viability of MCF-7 cells. Free EPI was set as positive control. As shown in Table 2, in sensitive cell lines, the IC₅₀ of free EPI was lower than the IC₅₀ of Ph-MSN-EPI, indicating that free-EPI is marginally more effective than Ph-MSN-EPI, likely due to EPI’s higher water solubility and consequently higher cellular uptake [52].

     The major barrier to the successful chemotherapy is MDR which is mainly related to drug efflux from cancer cells mediated by P-gp. P-gp is overexpressed in many human cancers, which exports drugs from the cells and resulted in reduction of the effective concentration of intracellular drug, that in turn lead to the failure of therapy. However, it is important to note that the theoretic and crystallography studies showed that the molecular size of P-gp is ∼160 A° long and 45 × 65A° wide, with the core consisting of two nucleotide binding domains (NBDs) and two transmembrane domains (TMDs). The residues in transmembrane segments form a funnel-shaped drug-binding domain, which is narrow at the cytoplasmic side, wide at the extracellular side and 9∼25 A° in the middle. Because of this unique feature, macromolecules such as some kinds of proteins remain effective against MDR, due to the large size of protein molecules impedes pumping them out by the P-gp [53-55]. Accordingly, we synthesized Ph-MSNs with the size of 98 nm to deliver EPI to MDR cells, with the aim of overcoming MDR. On the other hand, we anticipated that after cellular internalization, EPI loaded Ph-MSNs rapidly enter lysosomes in the perinuclear regions, which are far away from P-gp transporters in the resistant cell lines thus possibly bypassing drug efflux [56].

     MCF-7 is a drug-sensitive human breast cancer cell line, and MCF-7/ADR is its drug-resistant counterpart, that is widely used as the MDR experimental model cell line because of its high expression level of P-gp [57, 58]. The MTT assay showed that the cell viability was still over 60% after treatment with 5 μg/ml EPI, which proved the resistance of MCF-7/ADR cells to EPI (Fig. 3). Therefore, we selected the resistant cell line MCF-7/ADR to determine whether MSNs containing EPI could reverse MDR. Through MTT assay, IC₅₀ values of EPI and MSNs containing EPI against the sensitive MCF-7 cells and resistant MCF-7/ADR cells were evaluated and thereby the drug resistance index (DRI) was calculated and listed in Table 2. The DRI value suggests a level of drug-resistance of the MCF-7/ADR cell compared to its counterpart sensitive cell MCF-7 (59). According to the DRI value, cancer cells can be classified into three categories: drug-sensitive one with DRI ranging from 0 to 2, moderate drug-resistant one with DRI from 2 to 10, and highly drug-resistant one with DRI higher than 10 [59, 60]. From DRI point of view, the DRI value in the EPI treated breast cancer cells is 3.963 for 72 h incubation, manifesting moderate drug-resistant cancer cells. For MSNs containing EPI (Ph-MSN-EPI), the IC₅₀ value of EPI against sensitive MCF-7 cells is 8.705 µg/ml for 72 h incubation, while that against resistant MCF-7/ADR cells is 4.854 µg/ml (72 h), which is around 2-fold decrease over that of EPI.The DRI value for MSNs containing EPI (Ph-MSN-EPI) after 72 h incubation was 1.8. The values of IC₅₀ and DRI indicate that MSNs containing EPI treated the moderate resistant MCF-7/ADR as sensitive drug-resistant cancer cells (Fig. 4 and Table 2).

 The viability of sensitive MCF-7 and resistant MCF-7/ADR cells after incubation with pH-MSN-EPI at concentration of 5µg/ml was about 75% and 44%, respectively. On the other hand, the viability of sensitive MCF-7 and resistant MCF-7/ADR cells after incubation with EPI at concentration of 5 µg/ml was about 48% and 60%, respectively (Fig. 3 and Fig. 4). These results proved that the MSNs containing EPI are more effective on resistance cells than sensitive cells. In addition, the MSNs containing EPI but not EPI were capable of effectively killing drug-resistant cells.

 Therefore, all of these results indicate that MSNs containing EPI are a good candidate for effectively cancer drug resistance reversal. Probably, one can attribute it to the size-exclusion effect of P-gp, as small drug molecules can easily pump out of cell, whereas, nanoparticles remain inside.

 Conclusion

In the present study, in order to achieve effective drug-resistance reversal, we have developed the MSN based drug delivery system to deliver EPI. Surface functionalization of MSNs with phosphonate group reduces nanoparticles aggregation due to electrostatic repulsion of negatively charged phosphonate groups on the surface of MSNs. Considering the size of these nanoparticles, the drug loaded MSNs showed acceptable toxicity against moderate resistant cancer cell line MCF-7/ADR. Although further efforts are necessary to reveal the exact efficacy of these nanoparticles for MDR cancer treatment, but this study suggests that MSNs containing EPI could serve as a practical example to overcome MDR.

 Acknowledgments

This study was supported by a Grant from the Vice Chancellor for Research of Mashhad University of Medical Science, Mashhad, Iran.

 Conflict of interest

There is no conflict of interest in this study.