Document Type : Original Research Article
Subjects
Introduction
Breast cancer remains the most commonly diagnosed cancer among women worldwide and represents the second leading cause of cancer-related death in this population [1]. Common therapeutic drugs include anthracyclines (e.g., doxorubicin/DOX), taxanes (paclitaxel, docetaxel), fluorouracil, and cyclophosphamide. While DOX is a potent chemotherapeutic option, its clinical efficacy is limited by poor blood-brain barrier permeability, low biological stability, and a pronounced first-pass metabolism. Because of these limitations, large administered dose concentrations is required which result in severe side effects for healthy tissues [2]. Chemoresistance is also a problem that limits the effectiveness of chemotherapy and leads to metastasis and relapse of cancer [3]. The possible mechanism of multi-drug resistance in breast cancer cells includes increased resistance to apoptosis, increased DNA repair capacity, overexpression of drug efflux transporters, enzyme systems inactivating antitumor drugs by affecting their metabolism, etc. [4]. All the chemoresistance mechanisms mentioned above were identified in a small subpopulation of cells within tumors with self-renewal and differentiation capabilities named cancer stem cells (CSCs) [4, 5]. In conclusion, undesired side effects, drug resistance and recurrence following longer exposure to the drugs, are still the main drawbacks of current chemotherapy drugs.
In order to solve the weaknesses of cancer therapy by chemotherapy drugs, various studies have been done to design drug delivery systems including nano micelles, microemulsions liposomes, polymer-based vesicles, hydrogel, nanoparticulate and niosomes systems for anti-cancer agents such ad DOX [6-10]. These nanocarriers improve absorption, permeability, water solubility, stability, bioavailability distribution, and metabolism of drug and enhance the efficiency of tumor targeting, simultaneously decrease the DOX adverse effects and solve the problem of multidrug resistance [11-13]. In recent years, advancements in nanotechnology within the pharmaceutical field have led to increased research on niosomes as drug delivery systems for cancer therapy. Niosomes are vesicular nanocarriers capable of encapsulating hydrophilic and hydrophobic drugs. These nanocarriers are composed of non-ionic surfactant and lipid such as cholesterol and have biodegradation and biocompatibility properties, adequate encapsulation ability, cost effective preparation and suitable stability [14, 15].
In this study, we loaded DOX into noisome by thin-film hydration (THF) method. To the best of our knowledge, this study is the first one to compare the inhibitory effect of free DOX and Nio-DOX on down regulation of some cancer stem cell markers in MCF-7 cell line.
Niosomes were prepared by film hydration method. A mixture of 300 μM Tween 40 and Span 40/cholesterol (at a 50:50 weight ratio) was dissolved in chloroform for 30 min. The organic solvent was then removed using a rotary evaporator (Heidolf, Germany) at 60 °C, resulting in the formation of a thin film on the bottom of the round-bottom flask. This thin film was subsequently hydrated with phosphate-buffered saline (PBS, pH 7.4) containing DOX (304 mg/ml) and stirred (120 rpm, 40 min). Then, to obtain a uniform niosomes, the hydrated niosomes was sonicated using a sonicator (sonopuls DK 255 P, Bandelin, Germany) with 10 kHz frequency for 3 min (30s on and 10s off). The niosomal suspensions were kept at room temperature for 24 hours to ensure thorough annealing and drug distribution within the lipid bilayer, after which they were refrigerated for further analysis [16, 17]
Size, morphology, and zeta potential assessment
The morphology of niosomes was executed by SEM (KYKY SEM China; EM-3200). The mean size of the niosomes was conducted using a dynamic light scattering (DLS) technique. The evaluation of the zeta potential of the niosomes was undertaken utilizing the Zetasizer Nano ZS (Malvern Instruments). The stability of drug-loaded niosomes was evaluated under different conditions. Samples were stored for three months at 4–8 °C and 25 °C. Particle size and drug entrapment efficiency were measured to assess stability. Each procedure was repeated three times.
The entrapment efficiency of the niosomes was assessed through centrifugation. Specifically, 1 mL of the niosomal suspension was centrifuged at 13,000 rpm for 120 minutes using a Hettich centrifuge (Germany). After centrifugation, the supernatant was separated, and the pellet containing the niosomal lipid bilayers was disrupted with isopropyl alcohol. The concentration of DOX in both the supernatant and the pellet was measured using a UV-spectrophotometer at 491 nm (UV 1800, Shimadzu Co., Japan). The drug entrapment percentage was calculated using the formula:
EE% = (Cp × 100) / (Cp + Cs),
where Cp is the concentration of DOX in the pellet and Cs is the concentration in the supernatant.
The in vitro release study used Franz cells with a diameter of 1. 5 cm and a 20 mL receptor phase volume (Ashke-shisheh CO., Iran). A dialysis membrane (Sigma, MWCO 12400) was soaked in 50% ethanol for 24 h then placed on top of the Franz cells. Nio-DOX and a blank noisome (1 cc) were added to a dialysis bag, and the donor compartment contained 50% ethanol, stirred at 37 ± 0. 2 ˚C. Samples were taken at intervals for 5 hours and analyzed with a UV spectrophotometer at 491 nm [16].
Stability studies involved storing samples at 4° C and 25° C for three months. Measurements of particle size, Zeta potential, and encapsulation efficiency occurred right after preparation and after 30 days.
The human breast MCF-7 cell line was obtained from Pasteur Institute Cell Bank, Tehran, Iran. All cells were markedly cultured in high glucose DMEM medium supplemented with 10% FBS, and 1% penicillin/streptomycin. MCF-7 cells were cultured in incubator with 5% CO2 and at 37 °C. Upon reaching 90% confluency, the cells underwent a washing process using sterile phosphate-buffered saline, followed by detachment from the culture surfaces through trypsinization employing 0.25% trypsin–EDTA. Finally, cells were re-suspended in a complete DMEM medium and used for MTT.
The effect of different concentrations of free DOX and Nio-DOX on MCF7 cell viability was assessed by using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide). Briefly, around 12,000 MCF-7 cells were seeded into each well of 96-well plate with 100 mL cell media (DMEM, 10% FBS). For cell adherence, 96-well plate incubated overnight at 37°C and 5% CO2. Different concentrations of free DOX and the Nio-DOX (0.1, 0.2, 0.4, 0.8, 1.6, 3.2, 6.4 mM) were added in different wells to see their cytotoxic effects. Control wells for free DOX were treated with medium and for Nio-DOX were treated with blank noisome. After 48 h of exposure to free DOX or Nio-DOX, 10 mL of MTT solution (5 mg/mL PBS) was added to each well. After incubation for 4h, cell media was removed and 100 mL of DMSO was added to dissolve the formazan crystals and then read the absorbance at 570 nm. All experiments were performed in triplicates. The half maximal inhibitory concentration (IC50) values of agents were calculated using Prism 6.0 (GraphPad Software, Inc., San Diego, California, USA).
Cultured MCF-7 cells were seeded into three groups: a control group (untreated group), a group treated with IC50 concentration of DOX, a group treated with IC50 concentration of Nio-DOX.
Gene expression analysis by real-time PCR
RNA extraction and cDNA synthesis
Total RNA isolation kit (DENAzist Asia, Mashhad, Iran) was used to extract total RNA from untreated and treated cells and then quantity and quality of RNA were determined using nanodrop and agarose gel electrophoresis. For reverse transcription–polymerase chain reaction, mix 1 μg of the template RNA, 0.5 μg oligo (dt)18 (Cat. No. YT4551; Yekta Tajhiz Azma), 1 mM dNTPs (Cat. No. YT5553; Yekta Tajhiz Azma), and 200 U of M-MuLV reverse transcriptase (Cat. No. EP0441; Thermo Scientific) and DEPS-treated water up to final reaction volume of 20 μl. Mix the above mixture by quick vortex then incubate at 42°C for 60 min and then terminate the reaction by incubation at 70°C for 15 min.
The specific primers for OCT4, NANOG, ALDH1A1, ABCB1, CD133, CD44, SOX2 and B2m (as internal control) were designed using Oligo 7 software (Table 1). Experimental reaction was performed by adding the components in this order, 2x SYBR Green master mix (Cat. No. C101021; Parstous, Iran) (5 ml), primer F and R mix 10 mM (1 ml), cDNA (1 ml), PCR-grade H2O (3 ml). The reaction microtubes were placed in Rotor-Gene real-time thermal cyclers, and the appropriate program according to the manufacturer’s instructions was run, (1 Cycles, 15 min Duration of a cycle, 95 °C Temperature), and (40 Cycles, 20 s duration of cycle in 95 °C Temperature, and 20 s duration of cycle in 62 °C Temperature and 10 s duration of cycle in 72 °C). After real-time RT-PCR, the temperature increased from 60 °C to 95°C at a rate of 1°C/5s to construct a melting curve. The specificity of amplified target genes were verified by agarose gel electrophoresis and melting curve analysis. The 2−ΔΔCT method was used to analyze the relative changes in gene expression between control and treat group from real-time quantitative PCR experiments. β2M was used as housekeeping genes in real-time PCR data analysis.
GraphPad prism 5 software was used for data analysis. Data are expressed as the mean ± standard deviation of triplicate measurements. One-way ANOVA analysis was also used to determine significant differences between treatment groups and untreated control. Data were presented as the mean ± standard deviation and the significance level was considered at * p < 0.05 and ** p < 0.01.
Characterizations of the niosomes
The size and morphology of niosomes are shown in figure 1a, b, c. The niosomes showed a spherical vesicular structure with an average size of 67.81 ± 3.25 nm. The zeta potential of the optimized niosomes was -3.48 ± 0.16 mV, which suggests that the charged niosomes are stable against aggregation. Zeta potential was an key factor in drugs penetration since typically negatively charged particles exhibit strong interactions with cell membranes and drug [18].
In this investigation, the optimal DOX niosomes entrapment efficiencies were around 92.03 % ± 2.95. A perfect drug delivery system would have a compact structure, high encapsulation efficiency, and be able to carry sufficient drugs to enter the cell.
Figure 1d shows the in vitro drug release profile of DOX from the loaded noisome. The formulation showed a sustained release for it. In vitro release after 5 h from niosomal dispersion was 51.0 % while about 88.81 % of DOX were released from free solutions. The current experiments obviously showed that the drug amount released from niosomal formulation is effectively delayed compared to the free drug solution selected as control.
The stability of Nio-DOX in terms of vesicle particle size, zeta potential, and entrapment efficacy after 3 months storage at both 4-8 and 25 °C have been reported in Table 2.
Cytotoxic effects of free DOX and Nio-DOX on MCF-7 cells were assessed by MTT assay. MCF-7 were exposed to various concentrations of free DOX and Nio-DOX (0.1, 0.2, 0.4, 0.8, 1.6, 3.2, 6.4 mM) and cell proliferation was assessed after 48 h. As shown in figure 2, free DOX and Nio-DOX significantly inhibited the proliferation of MCF-7 cells in a dose-dependent manner. When comparing the same concentration in both exposures, it clearly appears that the Nio-DOX has enhanced growth inhibitory effects in MCF7 cells in comparison to free DOX. IC50 doses of free DOX and Nio-DOX were determined and Nio-DOX (IC50 value 1.6 mM) exhibited a 3.5-fold reduction in IC50 compared to free DOX (IC50: 5.6 mM). Although formulation of Nisome was optimized to have the least toxicity, it showed little toxicity due to its constituent detergent compounds (IC50 value of empty noisome was more than 12.8 mM) (Figure 3).
Expression analysis of CSC markers
QRT-PCR was performed to investigate expression values of some important CSC markers at mRNA level after MCF-7 treatment with free DOX and Nio-DOX. As shown in Figure 4, expressions of OCT4, NANOG and SOX2 increased about 2-fold after treatment with free DOX. However, treatment with Nio-DOX didn’t change expression of OCT4 and NANOG significantly and reduced expression of SOX2 about 2-fold. Furthermore, as shown in Figure 5, expressions of CD44, CD133, ALDH1 and ABCB1 genes increased 1.5-2.9-fold after treatment with free DOX. However, our results demonstrated that the expression of CD44: 52%, CD133: 95%, ALDH1: 92% and ABCB1: 97% significantly decreased in MCF-7 cells after Nio-DOX treatment compared with control group. Therefore, our results showed that noisome nanoparticles increased the inhibitory effects of DOX.
In this study we prepared noisome with spherical structure as a promising delivery system with size, zeta potential, and entrapment efficiency values of 67.81 ± 3.25 nm, -3.48 ± 0.16 mV, and 92.03±2.95 %, respectively which indicates an efficient encapsulation of DOX. The prepared noisomes remained stable for three months at -4 ˚C. The release profiles of DOX loaded in niosomes revealed the extended release over of 5 h. Considering the physical and chemical properties of Nio-DOX, they demonstrated a stable, permeable, and cost-effective nanocarrier for delivering DOX to cancer cells. Based on cytotoxicity studies, blank noisome showed no significant toxicity effect on cells confirming cytocompatibility of the nanocarrier. Moreover, Nio-DOX showed a 3.5-fold reduction in IC50 values compared with free DOX which show better cytotoxic effects of Nio-DOX against breast cancer cells compared with free administered DOX, probably due to better internalizations of niosomes and a sustained release of DOX. Present data also indicates a greater effect of Nio-DOX on down regulation of CSCs markers including CD44, CD133, ALDH1 and ABCB1 in comparison with free DOX. Among pluripotent transcription factors, although, SOX-2 down regulate two-fold after Nio-DOX treatment, expression of OCT4 and NANOG didn’t change.
DOX is a potent chemotherapy drug approved by the US Food and Drug Administration for the treatment of breast cancer [13, 19-23]. Despite the positive effects of it, severe side toxic effects including fatigue, alopecia, nausea, hair loss, vomiting, and oral sores and long treatment durations are vexing drawbacks in the use of this drug [24]. DOX has also adverse effects on the immune system, making the patient susceptible to infections [13]. Physical barrier to diffusion of drugs and insufficient drug delivery within the tumor is another limiting factor. Moreover, resistance to DOX has frequently appeared in reports on breast cancer [25, 26]. Breast CSCs are a subpopulation of breast cancers that express stem cell- associated markers and have high capacity for tumor formation [27-29]. Drug resistance of Breast CSCs to traditional chemotherapy (e.g. DOX) lead to anti-cancer treatment failure and cancer recurrence. [30, 31].
According to our results, expression of most CSCs markers that were investigated increased following treatment with free DOX which show drug resistance of cancer stem-like cells to DOX. Similarly, another study showed an increase in expression of stem-cell marker in spheroids formed from doxorubicin-treated cells when compared to non-treated cells [32]. Moreover, in another research, a DOX-resistant MCF-7 cell line was established through a series of long-term DOX in vitro treatment and data revealed that DOX-resistant MCF-7 cells showed increased cancer stemness, multi-drug resistance compared and invasiveness compared with parental cells [29].
Role of nanocarriers in enhancing chemotherapy efficacy of drugs and overcoming the limitations of conventional chemotherapy for cancer treatment is demonstrated in a lot of research [33]. Noisome is considered as a new drug delivery systems that are preferable to other nanocarriers, due to simple preparation, low cost and stability, safety, biodegradability, biocompatibility and prominent quality for solubilizing hydrophobic agents [34]. Noisome can preserve drugs from degradation, metabolism and clearance, enzyme inhibition by encapsulating them and thus improving the bioavailability of drugs [35-39]. Therefore, controlled-release Nuisomes lead to increased drug persistence and stability in the target tissue while simultaneously reducing side effects, including toxicity and hemolysis [39, 40].
In this study, we encapsulated DOX into noisome as a safe nanocarrier with little cytotoxicity and indicated that noisome can intensify the cytotoxicity of DOX against MCF-7 cells. Encapsulation of DOX inside noisome were also carried out in previous studies. Nio-DOX demonstrated a slight decrease in the IC50 value when assessed against the doxorubicin-resistant ovarian cell line, while simultaneously exhibiting an enhanced anticancer efficacy against the MCF-7, MDA MB 468 cell lines, as well as KG-1 acute myeloblastic leukemia cells; notably, the findings of this investigation align with those previously reported in the literature [2, 41, 42]. Niosomal DOX showed lower IC50 value compared with free administered DOX against MCF-7 cells which was similar to our result [43]. In another study, niosomal formulations of DOX overcame the limitation of DOX to cross the blood-brain barrier and ultimately improved the delivery of DOX into the brain for the treatment of brain tumors [44]. In vitro cytotoxicity studies showed improved cytotoxicity of Tamoxifen- and DOX-loaded niosomes on MCF-7 cells as well as better synergistic effect compared to the free drug combination [45]. Furthermore cationic PEGylated niosomes co-loaded with DOX, quercetins, and short interfering RNAs have also yielded promising results for treatment of gastric cancer [46]. Melittin-loaded niosomes has been shown to down-regulate the expression of Bcl2, MMP2, and MMP9 genes while up-regulate the expression of Bax, Caspase3 and Caspase9 genes in breast cell lines. They have also enhanced the apoptosis rate and inhibited cell migration, invasion compared to the free melittin. Therefore, melittin-loaded niosomes showed more anti-cancer effects than free melittin and niosomes are proper vesicle carriers for melittin [17]. Furthuremore, a number of researches have reported the use of niosomes as drug carriers for the delivery of many anti-cancer drugs such as artemisone, estradiol, gemcitabine, cisplatin, 5-FU, paclitaxel, gamma oryzanol, balanocarpol and tamoxifen for the treatment of cancers. In all studies, niosomal formulation of drugs can resolve their inadequate aqueous solvability and restricted permeability and enhance the solubility of drugs and help to drug retention which is important for long-term delivery [34, 47-58].
To our knowledge, no studies have been conducted to investigate the effect of Nio-DOX on expression of CSC markers. According to our results, while Nio-DOX treatment showed significant effect on down regulation of some CSC markers including CD44, CD133, ALDH1, ABCB1 and SOX-2, treatment with free DOX increased them. CSCs in solid tumors like breast tumors are distinguished by expression of certain cell surface biomarkers, such as CD133, CD44 [59]. CD133 and CD44 are membrane glycoproteins involved in migration, cell adhesion, and drug resistance and originally classified as a CSC marker in many solid tumors [60-64]. High ALDH1 activity is defined as a marker for CSC populations in many cancer types [65, 66]. Increased expression of ATP binding cassette (ABC) transporter superfamily which actively pump various chemotherapeutic drugs out of cancer stem cells, leads to multidrug resistance (MDR) in these cells by reducing intracellular drug accumulation and thus impairing chemotherapy effectiveness [67]. The expression of NANOG, OCT4 and SOX2 is crucial for stemness maintenance in normal and cancer stem cells and considered as integral regulators of pluripotency [68, 69].
Encapsulating DOX in niosomes increases its effectiveness in reducing CSC markers in MCF-7 cells primarily due to enhanced drug delivery and sustained release. The niosome’s surface characteristics, including its size and composition, can influence its uptake by cells, including CSCs. Therefore, concentration of drugs within the cell can be increased, potentially overcoming the effect of ABC transporters that are trying to remove the drug. This contributed to better drug efficacy and potentially overcome drug resistance. Furthermore, Niosomes act as a nano-carrier, improving stability and protecting DOX from degradation and allowing it to be selectively transported to the target cells. This results in higher DOX concentrations at the site of action, thus improving its ability to inhibit CSC markers [70, 71]. Finally, CSCs are notorious for pumping drugs out via efflux pumps like ABC transporters. Encapsulation can sometime bypass these mechanisms because the noisome get inside by endocytosis, delivering the drugs inside the cell where it is harder to pump out. Furthuremore, niosomes can indirectly affect the activity of ABC transporters, which are also involved in drug efflux and drug resistance in CSCs. Inhibition of P-glycoprotein (P-gp) by noisome lead to enhanced bioavailability of anticancer drugs and higher efficiency in drug delivery to the CSCs in comparison with free drugs [12, 13, 34, 54].
Various nanodrug delivery systems such as solid lipid nanoparticles, magnetic nanoparticles, polymeric nanoparticles, dendrimers, micelles, liposomes, quantum dots, etc are utilized for chemotherapeutic drug delivery. These nanocarriers help to overcome drug resistance by bypassing the drug efflux mechanisms mediated by ABC transporters, which typically pump drugs out of cells and reduce their effectiveness [72]. For example, some research revealed the potential of DOX solid lipid nanoparticles (SLNs) in overcoming P-gp-mediated multi drug resistance in resistant leukemia cells and Adriamycin-resistant breast cancer cell line. Greater accumulation of DOX SLNs in resistant cells with enhanced apoptotic cell death and decreased cell viability compared to plain DOX revealed the potential of DOX SLNs to overcome chemoresistance [73-75]. According to another study, DOX liposomes increased cytotoxicity on vincristine resistant HL60 cells due to directly interaction with P-gp and inhibition of P-gp through endocytosis [76]
In conclusion, noisome are promising nanocarriers for encapsulating anti-cancer drugs to improve their release and, thus therapeutic affect and biological activity. Noisome encapsulated drugs significantly enhanced antitumor activity and cellular uptake of drugs as compared to free solution of the drug [34, 47-55]. According to our results, Nio-DOX have greater cytotoxicity against MCF-7 cells and inhibitory effect on CSC markers thus rendering noisome a promising nanocarrier for the potential treatment of metastatic breast cancer. However, niosomal applications as a delivery systems for drugs have some difficulties due to some disadvantages such as aggregation, leakage of the entrapped drug, time-consuming preparation procedure, high cost of preparation, difficulties for large-scale production, large amount of drugs that remain unencapsulated during the synthesis and other challenges that still have to be solved to make niosomes clinically applicable [43, 48, 71].
The authors thank Vice Chancellor for Research and Technology, Shahid Bahonar University of Kerman, Kerman, Iran for financial support
The authors declare that they have no conflict of interest.
