Abstract: Background and Objective: Thymoquinone (TQ) has been reported for its efficacy to inhibit various cancer stages. Nanoparticles (NPs), showed great promise as drug carriers for cytotoxic agents. This study aims to investigate the ability of zein based NPs to enhance TQ cytotoxicity in MCF-7 cells. Materials and Methods: TQ was loaded on ZN NPs using the antisolvent phase separation technique. The prepared TQ ZN NPs were investigated for size, shape, in vitro release and cytotoxicity activity in MCF-7 cells. The presentation of data was performed using mean±SD. The IBM Statistical Package for Social Science (SPSS) statistics software (version 25) (SPSS Inc., Chicago, IL, USA) was utilized for carrying out statistical analysis. Results: TQ-Zein NPs revealed spherical shaped NPs with in vitro TQ sustained release for over 36 hrs and enhanced cytotoxicity activity in MCF-7 cells when compared with either pure TQ or ZN. Cell cycle analysis results showed accumulation of MCF-7 cells in G2/M and pre-G1 phases were observed in MCF-7 cells challenged with TQ ZN NPs. A significant increase in the % of cells for early and late apoptosis in addition to the total cell death as shown by cells stained with annexin V. Conclusion: Formulation of TQ in the form of ZN based NPs improved cellular permeation and apoptotic activity of TQ that leads to potentiation of its cytotoxic activities against MCF-7cells.
INTRODUCTION
Thymoquinone (TQ) is a monoterpene and the main active volatile oil ingredient of black cumin (black seed) Nigella sativa L. (NS) family Ranunculaceae. Black seed is used as herbal (natural) medicine and condiment1,2. NS oil is used for the treatment of various health problems as bronchial asthma, gastrointestinal problems, hypertension and obesity. Clinical investigations on black seed extract taken orally have shown promising antioxidant and anti-inflammatory effects3,4.
Besides, the efficacy of NS extract has shown results of cancer prevention and treatment5. TQ as a volatile oil ingredient of NS has been reported in various studies to have pharmacological properties6-9. Among these studies, the anticancer effects of TQ with selective cytotoxicity for cancer cells7,10-16. Besides, TQ augments chemotherapy and radiotherapy treatments through the sensitization of cancer cells affecting the resistance mechanisms17,18. The promising data for TQ as an antitumor agent is hindered by the low bioavailability of TQ.
Zein (ZN) a protein from natural plant origin is approved by the FDA and is generally recognized as a safe excipient19-21. The hydrophobic properties of ZN, because of its high content of hydrophobic amino acids, allow its use as a moisture barrier in the food industry22-28. Besides, ZN has been reported in formulation studies to form colloidal aggregates in physiological conditions that allow its use as a matrix for nano/ micro-particles formation26,29-31. Reports have indicated liver targeting characteristics of ZN as a drug carrier32,33. ZN nanospheres have been studied for sustained drug delivery applications for small drug molecules and macromolecules for oral and parenteral routes30,34-40. ZN has shown antioxidant and cytotoxic activities41,42. This work aimed to formulate the TQ loaded ZN nanoparticles (NPs) to enhance TQ cytotoxic activity against breast cancer cells and evaluate the role of ZN as a drug carrier for TQ.
MATERIALS AND METHODS
Study area: This study was carried out at the Nanotechnology Lab and cell culture lab, Department of Pharmaceutics, King Abdulaziz University, Jeddah, Saudi Arabia and from June, 2019-August, 2020.
Drug and chemicals: The TQ, ZN and ethanol were all purchased from Sigma-Aldrich (St. Louis, MI, USA). Dulbecco’s Modified Eagle Medium (DMEM), streptomycin, penicillin, Fetal Calf Serum (FCS), trypsin-EDTA (0.05%) and phosphate buffer (PBS pH 7.4) were purchased from Thermo Fisher Scientific Inc. (Waltham, MA, USA).
TQ-ZN NPs formulation: TQ-ZN NPs as previously reported was prepared with slight modification32. In short, TQ was dissolved in absolute ethanol and ZN was dissolved in 90% ethanol. Both alcoholic solutions were mixed and then added to an aqueous solution of PVA (1 % w/v) and stirred for 4 h and then subjected to evaporation of ethanol. The TQ-ZN NPs dispersion was separated by centrifugation at 20,000 rpm for 30 min at 8°C then washed with deionized water (two cycles), then lyophilized and stored until fully characterized.
TQ-ZN NPs size and zeta potential evaluation: A sample from the prepared TQ-ZN NPs was dispersed in deionized water and then analyzed for size and zeta potential using Zetasizer Nano ZSP (Malvern Panalytical, Malvern, UK). The average of five runs was used for the determination of TQ-ZN NPs size and zeta potential.
TQ-ZN NPs encapsulation efficiency: The prepared TQ-ZN NPs formula sample was dissolved in ethanol and then injected into high-performance liquid chromatography (HPLC) analysis was obtained by an Agilent 1100 liquid chromatography (Wilmington, DE, USA) that contained a micro vacuum degasser (model G1379A), quaternary pump (model G1311A), multiple wavelength detector (model G13658) and analyzed for TQ content as previously reported43. Equation 144 was used to determine the Encapsulation Efficiency (EE) percentage of TQ:
TQ-ZN NPs in vitro diffusion study: An automated vertical Franz diffusion cell (MicroettePlus, Hanson Research, Chatsworth, CA, USA) was used for the diffusion study as previously described45. A diffusion buffer (pH 7.0) containing Tween 20 (0.5%w/v) stirred at 400 rpm and a diffusion membrane (0.1 μm) were utilized. Samples were collected at specified time intervals. TQ content in the withdrawn aliquots was analyzed by HPLC as previously reported46.
Scanning electron microscopy: The sample preparation for investigation was carried out after securing these metallic stubs by utilizing the sticky tape (double-sided). The sticky tape was then fused to aluminum stubs and the vacuum was utilized for performing gold coating. The scanning electron microscope, model JEM 100-CX, JEOL (Tokyo, Japan) was utilized for assuring the availability of the selected TQ-ZN formula’s surface morphology for carrying out analysis.
Cells and culture of cells: The MCF-7 cells were procured from the Vacsera located in Giza, Egypt. The maintenance of cells was assured out by using Dulbecco’s Modified Eagle Medium (DMEM). 100 units mL–1 of Penicillin, 100 μg mL–1 of streptomycin and 10% of heat-inactivated fetal bovine serum (10%) were utilized for supplementing the culture medium. Moreover, 37°C temperature and CO2humidified (v/v) atmosphere (5%) was considered for keeping the cells within the state of sub-confluence.
Anti-proliferative activity assessment: The assessment of the prepared plain formula of TQ and ZN and TQ-ZN NPs against the MCF-7 liver cancer cells was carried out by using Sulforhodamine B (SRB). Moreover, the assessment of considered preparations’ anti-proliferative activities against the human esophageal epithelial cells (HEEpiC) was also carried out. The seeding of cells was carried out into 96 well plates having 1000-2000 cells per well and trypsinization was carried out by using 0.25% Trypsin-EDTA. The treatment of cells was carried out by using the serial concentrations of the isolated compounds for 72 hrs. Afterward, 10% of trichloroacetic acid (TCA) was utilized for fixing the cells for 1 h, at a temperature of 4°C. All cells were splashed multiple times with water, which was followed by the utilization of 0.4% SRB solution was f or staining of cells. This process was followed by keeping them in the dark at room temperature for 10 min. The washing of cells was carried out by using 1% glacial acetic acid. After which the plates were left for drying overnight and Tris-HCl was utilized for dissolving the SRB-stained cells. The determination of color intensity (OD) was carried out by using the monochromator SpectraMax® M3 plate reader (Molecular Devices, Sunnyvale, CA, USA) at 540 nm. OD measurements were then utilized for calculating the growth inhibition (IC50) percentage. The measurement of concentration was carried out three times and the complete experiment was carried out three times.
Progression analysis of cell cycle: Approximately 3×105cells per well into N = 6 well-culture plates were seeded. The controlled incubation was performed and the cells were treated within the drug-free media, which was followed by challenging the cells with TQ-ZN NPs (5.3 μg mL–1) and equal concentrations of TQ and ZN for 24 hrs. The investigation of the cell cycle was performed by using CycleTESTTM PLUS DNA Reagent Kit (Becton Dickinson Immunocytometry Systems, San Jose, CA, USA). Those cells which have a known DNA content (PBMCs) were considered as a control for test samples’ DI (DNA index). According to instructions provided by the manufacturers, the staining process was performed by using Propidium iodide. Moreover, for carrying out cell cycle distribution analysis, the DNA cytometer and CELLQUEST software (Becton Dickinson Immunocytometry Systems, San Jose, California, USA) were considered by the researcher.
Caspase 3 enzyme assay: As explained within the experiment of analysis of the cell cycle, the treatment of cells was carried out by using the same preparations for the duration of the same period (24 hrs). Later on, the cells were lysed, which was followed by the extraction and subjection of cells to caspase 3 content determination assay, by consuming a commercial kit. The entire procedure was performed according to the instructions provided by the manufacturers (USCN Life Science Inc., Wuhan, Hubei, China).
Statistical analysis: The presentation of data was performed using mean±SD. The IBM Statistical Package for Social Science (SPSS) statistics software (version 25) (SPSS Inc., Chicago, IL, USA) was utilized for carrying out statistical analysis. The means were compared by using the analysis of variance (ANOVA), which was then followed by Tukey’s post hoc test. The statistical significance was indicated by p<0.05.
RESULTS
Preparation and characterization of TQ-TPGS NPs: Particle size results of the prepared TQ-ZN formulation showed an average size of (175±8.2 nm) as measured by Zetasizer Nano ZSP. TEM micrograph of the prepared TQ-ZN (Fig. 1) showed spherical nanospheres with comparative results with that obtained by the zetasizer.
In vitro release of TQ-ZN NPs formula: The in vitro release profile of optimized TQ-ZN is shown in Fig. 2. The TQ-ZN NPs showed a burst release of TQ was observed. After 6 hrs, around 25% of TQ was released from NPs. At the end of 24 hrs, 80% of TQ released.
Determination of TQ-ZN NPs IC50 values: The data in Fig. 3 demonstrated the IC50 values acquired for the samples. TQ-ZN NPs had the least IC50 value, i.e., 2.5±1.2 μM, whereas TQ-raw had an IC50 value of 17.6±3.2 μM. Thus, when TQ was loaded in the NPs, there was a decline to about half of the value of IC50.
| Fig. 1: | Scanning electron microscope image of TQ-ZN NPs (×10,000) |
| Fig. 2: | In vitro release profile of TQ-ZN NPs in phosphate-buffered saline (PBS) buffer pH 7.4 at 37±0.5°C |
| Results are presented as mean±SD, n = 3 | |
Analysis of the cell cycle: The results of cell cycle analysis were presented in Fig. 4, demonstrating a substantial difference within the cell cycle. Observations revealed that the performance of TQ-ZN in all phases was according to expectations and there were no significant effects of plain-M on the G0-G1 phase. The cells percentage within the G2-M phase demonstrated noticeable improvement in incubation with TQ-ZN. Thus, as compared to other samples, these effects were more noteworthy. The pre-G1 apoptosis also demonstrated a similar effect.
| Fig. 3: | IC50 of the ZN, TQ-raw, or TQ-ZN NPs in the MCF-7 cells |
| *Significantly different from control at p<0.05, #Significantly different from plain-M at p<0.05, $ significantly different from TQ-raw at p<0.05 | |
Annexin V staining: The analysis of apoptosis determination by flow cytometry was performed by using the Annexin V-FITC apoptosis detection kit. The data of Fig. 5 presented that in comparison with the other treatments, TQ-ZN exhibited higher and distinct early, late, as well as total cell apoptosis and cell death by necrosis.
Caspase-3 assay: TQ-ZN contributed to caspase-3 content’s substantial enhancement (Fig. 6), resulting in approximately a two-fold increment, as compared to cells treated by TQ-raw. Moreover, caspase-3 content was induced by Plain-NPs, in comparison to the control value.
| Fig. 4: | Assessment of MCF-7 cell death using flow cytometric analysis after annexin V staining |
*Significantly different from control at p<0.05, #Significantly different from plain-M at p<0.05, $significantly different from TQ-raw at p<0.05 |
|
| Fig. 5: | Impact of ZN, TW-raw, TQ-ZN NPs treatments on the annexin-V FITC positive-staining of MCF-7 cells. N.B.: G1, S, G2 and M. The S represents cell cycle phases that gica an indicator for cell death via apoptosis |
*Significantly different from control at p<0.05, #Significantly different from plain-M at p<0.05, $Significantly different from TQ-raw at p<0.05 |
|
| Fig. 6: | Effect of TQ-ZN on caspase-3 content in MCF-7 cells |
*Significantly different from control at p<0.05, #Significantly different from plain-M at p<0.05, $Significantly different from TQ-raw at p<0.05 |
|
DISCUSSION
In this study, the formulation of TQ loaded ZN NPs to evaluate the role of ZN as a drug carrier and sustain the release of TQ and enhance TQ cytotoxic activity against breast cancer cells. TQ-Zein NPs revealed spherical shaped NPs with in vitro TQ sustained release for over 36 hrs and enhanced cytotoxicity activity in MCF-7 cells when compared with either pure TQ or ZN.
The initial TQ release is related to the surface-bound release of TQ. The NPs showed sustained TQ release as a result of diffusion of TQ from NPs core through the hydrophobic ZN matrix33. The results of raw TQ against MCF-7 cells are per the results reported previously47. On the other hand, IC50 data agree with a previous report17. The reduction in the IC50 value of TQ loaded ZN NPs indicating that the nanocarrier system improved the activity of TQ. This finding is similar to the reported data of TQ in the human lung cancer line (MCF-7 cells)35. Our findings showed that TQ efficacy as a cytotoxic agent was augmented by loading in ZN NPs. This could be related to improved TQ’s cellular permeability by the nanocarrier, modification of the TQ cellular uptake mechanism. Raw TQ is transported into cells by passive transport, while the TQ-ZN NPs transport mechanism is endocytosis. Besides, the encapsulation of drugs by ZN as hydrophobic nanospheres could improve cellular uptake across the cell membrane that leads to increased intracellular TQ concentration48.
Apoptosis is a mechanism that regulates cell death at a genetic level, leads to the removal of harmed cells. The TQ-TQ-ZN NPs effect on G2-M phases of the MCF-7 cell line indicated that TQ loaded ZN NPs formula augmented TQ efficacy. The improved MCF-7 cell percentage in the pre-G1 cell cycle phase indicates apoptotic potential39. The apoptosis induction with the increased TQ efficiency for arresting at different cell cycle stages can be correlated to the capability of modulating the cellular components expressions, during the pathways of cell signaling, cell-cycle arrest and apoptosis. The evidence reported similar findings related to cancer cells49.
Based on the findings and the previous outcomes of the analysis of the cell cycle, it can be stated that the increment in TQ’s apoptotic potential is due to the arresting of different phases of the cell cycle. The present data is supported by the outcomes of previous reports and demonstrated that TQ can contribute to the arrest of colorectal cancer cells (HCT 116) within the G2-M phase. Moreover, in vitro, TQ contributed to a G2/M phase cell cycle arrest in TFK1 as well as HuCCT-1 cells and decreased the expression of G2/M checkpoint protein cyclin B1. Moreover, TQ also caused a significant increment in the percentage of the apoptotic cells in the pre-G phase23,27. The available evidence revealed that TQ blocks the cells in the G0-G1 phase14,16. Thus, in support of the previously published studies, it can be stated that apoptosis induction can be referred to as a mechanism of TQ’s anti-proliferative properties40. Regarding annexin V, the current data acquire support from the previously published studies demonstrating that enhanced staining of melanoma cells with annexin V, after being challenged with TQ. Moreover, TQ-ZN NPs demonstrated a significant increment in the proportion of the MCF-7 cells with positive annexin staining. In the present research, the caspase-3 content was signi cantly enhanced by TQ-ZN NPs. Thus, along with the previous reports, the present outcomes indicated that TQ can enhance cellular caspase-334-39. Previous researches have demonstrated that TQ-ZN NPs’ enhanced effects on the cleaved content of caspase-3 in A543 cells39. The cleaved content caspase-3’s elevation is considered as the latter cytosolic occurrence before apoptosis. Thus, the formulation of TQ in a nano-structured system improves the content of caspase-3. The advancement of activity of cleaved caspase-3 by the anti-tumor agents’ nano-structured systems has also been previously reported27. Thus, the outcome also indicates the observed enhancement of caspase-3 content. Finally, this study confirms the efficiency of TQ in the suppression of breast cancer cells via induction of cancerous cell apoptosis especially by using ZN as a carrier. Clinical studies should be applied to confirm these findings.
CONCLUSION
The formulated TQ-ZN NPs were almost spherical. In vitro release of TQ from NPs was markedly delayed-release, predicting a better presentation to the tumor cells. TQ-ZN NPs significantly enhances their cytotoxic activities against MCF-7 cells. This is mediated, at least partly, by enhanced apoptosis as evidenced by cell cycle analysis, annexin V staining and determination of caspase 3 which improved cytotoxicity of TQ-ZN Nps.
SIGNIFICANCE STATEMENT
This study revealed the importance of ZN NPs as a novel system for improved delivery and augment the effect of TQ in cancer therapy. The results indicated the improved cytotoxic effects of TQ loaded ZN NPs when compared with raw TQ. This can be beneficial for the treatment of breast cancer with minimum side effects as TQ is approved as a safe drug. This study will shed light on researchers and oncologists to identify the importance of nanocarriers in cancer therapy.
ACKNOWLEDGMENT
This Project was funded by the Deanship of Scientific Research (DSR) at King Abdulaziz University, Jeddah, under grant no. (G-170-166-1441). The authors, therefore, acknowledge with thanks DSR for technical and financial support.