Synthesis and characterization of smart stimuli-responsive herbal drug-encapsulated nanoniosome particles for efficient treatment of breast cancer

2.1 Extraction

The extract of Glycyrrhiza glabra was obtained from its root, while the extract of Hedera helix was obtained from its leaves. Before extracting, the type and species of plants were confirmed by the botanists of Yazd University. At first, 500 g of the mentioned parts of the plants were dried in ambient temperature, and were made into powder. Extraction was performed by Soxhlet method. First, 50 g of the powder obtained from each plant was compressed in a cartridge chamber and then placed in a Soxhlet column and by attaching a balloon containing 500 mL of ethanol (Merck, Germany) to the Soxhlet and installing a condenser and water inlet and outlet pipes, extraction was started and continued for 6 h to obtain plant extracts. Finally, in order to evaporate the solvent, the extract was dried away from sunlight at room temperature.

2.2 The maximum absorbance wavelength (λ _max) and standard curves

In order to figure out the maximum absorbance wavelength of each extract, stock solution of each extract with a concentration of 1 mg/mL in PBS (Sigma, USA) and Isopropanol buffer (Merck, Germany) was prepared, and by using diluent method, various concentration of each extract from its stock solution were obtained. Then, the absorbance of each sample was measured by spectrophotometer (Epoch, USA) in the range of 200–800 nm and the wavelength which had the highest absorption in all sample was reported as λ _max. The standard curves in PBS and Isopropanol buffer for each extract was plotted by measuring absorption of various concentration of each extract at λ _max using spectrophotometer and the equation line of each curve was also calculated.

2.3 Synthesize of nanoniosomes containing extracts

To synthesize Nio-GGE and Nio-HHE and select the optimal formulations, the following steps were performed briefly:

• synthesizing of nano-niosomes by thin film method,

• evaluation and comparison of the effect of the type of non-ionic surfactants used in the synthesis of niosomes as well as different molar ratios of cholesterol:surfactant in the encapsulation efficiency (EE%) of niosomes,

• selection of optimal formula from various formulations based on (EE%), extract release rate, and niosome size,

• adding 5% distearoyl phosphoethanolamine-polyethylene glycol (DSPE-PEG) (Ludwigshafen, Germany) to enhance the stability of nanosystems and evaluate its effect,

• adding 20% 1,2-dipalmitoyl-sn-glycero-3-phosphocholine phospholipid (DPPC) (Ludwigshafen, Germany) to induce temperature sensitivity as a stimulus for drug delivery to cancer tissue.

The thin film method was used to synthesize the niosomes to deliver the extracts to the cancer cells. In this study, different formulations of niosomes were synthesized to encapsulate each of the extracts and then the optimal formulation was selected from them. In summary, in the first stage, different ratios of cholesterol, non-ionic surfactants (Tween-60 and Span-60) (DaeJung Chemicals & Metals, South Korea), and DPPC were dissolved in solvent Chloroforms (Sigma, USA), according to Tables 1 and 2 for synthesizing Nio-GGE and Nio-HHE, respectively. The synthesizing of niosome by the thin film method has two phases. In the first or organic phase, cholesterol and surfactants using chloroform solvent were dissolved in a round bottom balloon. Then, in order to remove the solvent, the rotary instrument (Heidolph, Hei-VAP) at 50°C, 150 rpm, and vacuum conditions were used. After evaporation of chloroform as solvent, a thin lipid layer is formed at the bottom of the balloon. Finally, in order to ensure the removal of the solvent, the balloon was aerated with nitrogen gas for a few seconds and held at 4°C for 24 h. In the aqueous phase, Glycyrrhiza glabra extract (GGE) and Hedera helix extract (HHE) were inactively loaded into the niosomes. For this purpose, synthesized niosomes dissolved with GGE and HHE at a concentration of 2 mg/mL in PBS buffer at pH = 7 and were hydrated by a rotary apparatus at 60°C and 150 rpm. Reducing the size of the synthesized nanoniosomes were also done by using probe sonicator microtype (ChromeTech, UH1200B) with 60% power that was alternatively on for 15 s and off for 10 s every 45 min. Also, during the sonication, by placing the samples on the container containing ice, their temperature was prevented from increasing. Finally, the un-entrapped extracts were separated from the niosomes containing the extracts using a 12 kDa cut off dialysis bag.

2.4 Investigating the EE%

After separating un-entrapped extracts from niosomes, in order to destroy the membrane of niosomes for releasing encapsulated extract, niosome were lysed by using isopropanol buffer. Then, using spectrophotometric method at the maximum wavelength of each extract, the concentration of the encapsulated extract was calculated by the standard curve of the extract in isopropanol given by equation (1):

2.5 Investigation of the in vivo release rate of extracts from nanoniosomes in response to temperature and pH stimuli

The temperature and pH of cancer cells (42°C and pH = 4.5 and 5.2) are different from the temperature and pH of normal cells (37°C and pH = 7.4), so temperature and pH can be used as stimulus to target drug delivery in cancer therapy. In this study, in order to investigate the release pattern of extracts from synthesized nanoparticles, the drug release pattern in different conditions in response to stimuli was investigated. To evaluate the release profile of the herbal extracts, 1 mL of the niosomes containing extracts was poured into a cellulose dialysis bag and placed in 10 mL of PBS buffer and stirred at different temperatures and pH (physiological conditions of the body and conditions of cancer cells). Buffer sampling was performed at 1, 2, 4, 6, 8, 24, and 48 h and the concentration of the extract in sample was evaluated by spectrophotometer at the maximum wavelength of each extract and standard curve of each extract at PBS buffer. It should be noted that after each sampling, the same amount of buffer was replaced with the same temperature and pH.

2.6 Investigation of size, dispersion index (PDI), and zeta potential

Using a nano-sizer (Brookhaven Instruments Corporation, Germany), the particle size distribution range as well as the peak particle size range were determined. The range of particle size distribution as well as the diameter of nanoparticles is determined using DLS, for which the Brookhaven Instruments Corporation (Germany) nanosayer was used. For this purpose, laser light was irradiated to the niosomes at a wavelength of 657 nm at angles 90° and 25°C. The zeta potential of nanosystems were measured using an instrument of Brookhaven Instruments Corporation (Germany) at 25°C. 2,000 µL of sample with a concentration of 0.1 mg/mL was used to determine the surface charge.

2.7 Microscopic analysis

Microscopic studies are always a reliable way to study the morphology and structure of nanoparticles. In this study, to analyze the morphology, 3D structure, and shape of nanoparticles, atomic force microscope (AFM) (Nanowizard II; JPK instruments; Germany), scanning electron microscope (SEM) (EM3200-KYKY, China), and Transmission electron microscopy (TEM) (FEI Tecnai 20, type Sphera, Lake Oswego, OR) were used. To prepare the sample for imaging by SEM, 5 µL of the sample was poured on a glass plate and after drying in air, it was covered with a thin layer of gold to provide electrical conductivity. To study the nanoparticles with AFM, the niosomes were diluted with water (1:1,000) and sonicated for 20 min in order to homogenize and reduce the size of the nanoparticles and after that the samples were placed on a mica sheet and imaging was performed. For TEM imaging, samples were placed on 200 Cu-coated grid and imaging was carried out by a cryogenic TEM.

2.8 FTIR evaluation

The functional groups of synthesized nanoniosomes were analyzed by infrared spectroscopy method and were compared with the functional groups of blank niosomes. In the infrared spectrum, two areas are mainly considered. The area from 1,550 to 4,000 cm⁻¹ is the area where most of the bonding tensions occur. This area usually has a relatively small number of peaks, but many of its peaks identify functional groups. To ensure the absence of free drug and additives in the sample, the dialyzed sample was used and in order to reduce the humidity, the sample was placed in an oven at a temperature of approximately 60°C for about half an hour. Also, to investigate the non-interaction of the free form of extracts with niosomes, the spectrum of the extracts and blank niosome were assayed and compared with each other. To investigate the FTIR spectrum, 1 mg of the sample was first added to potassium bromide (KBr) in a ratio of 1:100, and then compressed under a pressure of 5–8 tons/cm, and after the cubes were compressed, the FTIR assay was carried out by FTIR spectrophotometer (Brucker, Germany) at a range of 400–4,000 cm⁻¹.

2.9 Investigating the stability of synthesized nanoniosome

The poor stability of nanoniosomes over time is among main challenges in the application of nanoniosome as a drug delivery carrier. In this study, the EE% (for evaluating the extracts leakage), size maintaining, zeta potential, PDI, and stability of the synthesized nanoniosomes were evaluated in 180 days. For evaluating the stability, synthesized nanoniosomes were kept at 4°C for 180 days and the stability of the mentioned items was investigated at various time intervals by using spectrophotometry and DLS methods as previously described.

2.10 Evaluation of antimicrobial activity of synthesized nanoniosomes

The antibacterial activity of Nio-GGE and Nio-HHE were investigated and compared with free form of extracts, on Escherchia coli and Staphylococcus aureus bacteria obtained from Pasture institute of Iran, Tehran. Then, to evaluate the antibacterial activity of nanosystems, niosomes containing each extract and the free form of the extract were assayed by spotting from 2.5 to 100 µL of nanoparticle suspensions (20 mg/mL) on a Tryptone Soya Agar (TAS) (Merck, Germany) plate seeded with 10⁷ cells/mL, S. aureus, and E. coli, and was incubated at 37°C for 24 h. As positive control, Amplicine and Tetracycline disks (Sigma, USA) were used to evaluate the antibacterial activity of the samples and finally to assay the antibacterial activity of the samples, the non-growth zone around each sample was studied.

2.11 Determination of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC)

The minimum concentration of an antibacterial agent that is sufficient to prevent bacterial growth in the laboratory is called MIC). In this study, the broth dilution method was used to evaluate the MIC according to the Clinical and Laboratory Standard Institute guideline. In this method, successive dilutions of the niosome containing each extract and the free form of that extract were prepared separately in TSB (Tryptic soy broth; Merck, Germany). Bacterial suspensions were then added to each tube to obtain a final inoculation of 8 logs CFU/mL. Then, the tubes were incubated at 37°C for 24 h. The growth medium alone was a positive control and the growth medium containing bacteria without any antibacterial agent was used as a negative control. Finally, the MIC of each sample was examined using spectrophotometry method at wavelength of 620 nm and the lowest concentration of each sample that inhibited the growth of bacteria was reported as MIC. The lowest concentration of an antimicrobial agent that can kill 99% of the initial inoculated microorganism is called the MBC. In order to determine the MBC, after determining the MIC, 100 µL of suspension was separated from any tube in which bacterial growth was not observed and cultured on a TSA plate and incubated for 24 h at 37°C and after that the number of grown colonies was counted. The concentration at which the counts were reduced to one-thousandth of the initial suspension colonies was then reported as the MBC concentration.

2.12 Cell culture

In this study, MCF-7 and BT-474 cell lines were used as breast cancer cells and MCF-10A cell line was used as normal breast cells, all of which were prepared by Pasteur Institute of Iran and were cultured based on American Type Culture Collection (ATCC) cell culture protocol. Human foreskin fibroblastic (HFF) cell line as normal cells and control group were obtained from Stem cell biology research center, Yazd, Iran. All cells were cultured in Dulbecco’s modified Eagle Medium (DMEM) (Sigma, USA) containing 10% fetal bovine serum (FBS) (Sigma, USA) and penicillin-streptomycin (Gibco, Grand Island, NY). All cultures were performed under standard conditions (37°C, 95% humidity, and 5% CO₂ to create and maintain physiological pH).

2.13 In vivo cellular uptake assay

Fluorescence microscopy technique was used to evaluate the cellular uptake of the synthesized nanoniosomes and also to evaluate the intracellular localization of penetrated nanoniosomes and the cellular uptake of nanoniosome in cancer cells compared to normal cells. MCF-7 cells as cancer cells and HFF cells as normal cells were seeded in a 6-wall plate (10 × 10⁵ cells/well) and treated with niosomes containing the extracts and the free form of each extract for 6 h, then rinsed for 3 times using PBS buffer cells and 4% paraformaldehyde (Sigma, USA) solution was used to fix them. Fluorescence property was obtained by adding 0.1% mol of Dil (1,10-dioctadecyl-3,3,30,30-tetramethylindocarbocyanine Perchlorate) (Thermo Fisher Scientific Waltham, MA) to GGE and HHE. 4',6'-diamidino-2-phenylindole (DAPI) (Thermo Fisher Scientific Waltham, MA) (1 mg/mL) was also used to stain cell nuclei and cells’ imaging was done by fluorescence microscope (BX61, Olympus, Japan).

2.14 Cytotoxicity assay

The MTT assay was carried out to evaluate the cytotoxicity effect of the synthesized nanosystems in this study on cancer cells and normal cells. In this study, the effect of Nio-GGE toxicity on breast cancer cells was investigated and compared with its free form and also the cytotoxicity effect of Nio-HHE was evaluated and compared with its free form. Then, the toxicity of Nio-HHE and Nio-GGE on breast cancer cells was compared to select a drug with stronger anti-cancer effect. Finally, the toxicity of the synthesized nanosystems on normal breast cells (MCF 10A) and normal HHF cells was investigated in order to evaluate the side effect of the synthesized nanoniosomes on normal cells. After culturing the cells on 96-well plates and after reaching an approximate number of 10 × 10⁴ cells/per well, the cells were treated with different concentrations (1,000, 750, 500, 250, 125, and 62.5) of each sample separately for 48 h and at the end of this stage, 20 µL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT salt) (Sigma, USA) (concentration of 0.5 µL) was added to them and were incubated for 4 h. After the supernatant was separated, 150 µL of dimethyl sulfoxide (DMSO) (Sigma, USA) was added to each well to eliminate the formazan crystals and incubation was done for 30 min. The absorbance of each well at wavelength of 570 nm was then read by ELISA reader (Synergy HTX, BioTek, USA) and finally the cell viability rate was calculated.

2.15 Gene expression analysis

In order to investigate the effects of the synthesized niosomes on the expression of genes involved in breast cancer and compare with the effect of free form of extracts on the expression of these genes, the quantitative real-time PCR technique was applied. The genes studied in this study were p53 and MCL-1 genes. Also, β-actin gene was used as housekeeping gene (data normalize). After MCF-7 cells as cancerous cells, and MCF 10A as normal cells, were cultured and treated with Nio-GGE, Nio-HHE, GGE, HHE and blank niosome for 48 h. Total mRNA of each sample was isolated by RiboEx LS reagent (GeneAll, South Korea). cDNA was synthesized from 1 μg of isolated RNA by cDNA synthesized kit (Thermo-Fisher Scientific) according to kit protocol. Real-time PCR amplification was carried out by SYBR Premix Ex Taq II (Takara, Dalian) in 35 cycles based on the following:

• 1–95°C for 5 min, 2–95°C for 30 s, 3–60°C for 30 s, 4–72°C for 30 s, and 5–72°C for 5 min.

The sequences of primers used in RT and real-time PCR are listed in Table 3.

2.16 Western blot analysis

Bradford assay was used to assay the concentration of proteins. MCF-7 cells were first cultured in a cell culture flask and after reaching 80% confluence were treated with Nio-HHE, Nio-GGE, GGE, HHE, and empty niosomes for 48 h and were lysed by HES buffer (Sigma, USD). Then, 50 µg samples were applied to a 10% SDS-PAGE and then sent into a nitrocellulose membrane. The membranes were incubated for 2 h at 25°C in BSA 5% (Merck, German) in order to barricade non-specific binding. In the next step, the membranes were incubated for 12 h at 4°C with 1:1,000 diluted primary β-actin antibody. Then, by using 1:2,000 diluted horseradish peroxidase-conjugated secondary antibody at 25°C, blots were probed for 2 h and at the end, the visualization were done by applying a visualization system (Syngene GBOX Gel Documentation 680×) along with ECL kit (GE Healthcare).

2.17 Statistical analysis

In this study, all results were gathered with three repetitions and the values were displayed as mean value ± standard deviation for all three repetitions. Also, a student t-test was used to compare two independent groups and one-way ANOVA test was used to compare multiple samples. Data analysis was also performed using GraphPad PRISM version 8 software (GraphPad, San Diego, CA) and EXCEL.

3.1 The maximum absorbance wavelength and standard curves

Based on Figure 1, it could be concluded that the λ _max for GGE and HHE is at 310 nm and 350 nm, respectively (Figure 1a and d). Figure 1 also demonstrates the standard curves of GGE and HHE in PBS and Isopropyl buffer (Figure 1b, c, e, and f). These data were obtained by analyzing absorbance spectrum of each extract at the range of 200–800 nm. The results of this analysis show that the equation line in isopropyl buffer for GGE and HHE is Y = 0.001725X − 0.005714 and 0.005436X + 0.003732, respectively, and square value (R ²) is 0.9966 and 0.9993, respectively, while the equation line in PBS buffer for GGE and HHE is Y = 0.001219X – 0.003801 and Y = 0.004267X – 0.002001 and R ² is 0.997 and 0.9968, respectively.

Figure 1

The data obtained from spectrophotometry analysis. (a) Maximum wavelength of GGE is at 310 nm. (b) Standard curve of GGE in Isopropyl buffer, the equation line is Y = 0.001725X − 0.005714 and R ² = 0.9966. (c) Standard curve of GGE in PBS buffer, the equation line is Y = 0.001219X − 0.003801 and R ² = 0.997. (d) Maximum wavelength of HHE is at 350 nm. (e) Standard curve of HHE in Isopropyl buffer, the equation line is Y = 0.005436X + 0.003732 and R ² = 0.9993. (f) Standard curve of HHE in PBS buffer, the equation line is Y = 0.004267X − 0.002001 and R ² = 0.9968.

3.2 Choosing the optimal formulation

Tables 4 and 5 show the different effects of molar ratio of surfactant; cholesterol, DPPC, and DSPE:PEG in EE%, maximum extract release in 72 h, and the size of nanoparticle in Nio-GGE and Nio-HHE, respectively. EE% and drug release from nanoparticles play critical roles in selecting the nanoparticle as a drug delivery carrier. Based on Table 1, F6 formula with EE% of 69 ± 1.2%, maximum release of 60.8% at 72 h, and size 111 nm is the optimal formula among other formulation for delivery of GGE to targeted tissue. According to Table 5, the optimal formula for delivery of HHE to cancerous tissue is F5 formula with the size of 97.7 nm, maximum release rate of 63.5 at 72 h, and EE% of 58 ± 2.4%. So, after selecting these formulations as optimal formulations, other analyses were performed on these nanoparticles.

3.3 Size, PDI, and electrical charge of nanoparticles

Figure 2 illustrates the size, PDI, and zeta potential of optimal formula after extracting loading. DSPE-PEG reduces the size of nanoparticles and averts their accumulation by increasing the negative charge in nanoparticles and creating a stronger repulsive force. According to Figure 2, the Nio-GGE is anionic type with a zeta potential of −23.5 ± 4.5 mV (Figure 2b), a size of 111 ± 8.5 nm, and PDI of 0.113 (Figure 2a). Based on Figure 2, Nio-HHE is also anionic type with zeta potential of −19.9 ± 6.7 mV (Figure 2d), a PDI of 0.35, and a size of 97.7 nm (Figure 2c).

Figure 2

Investigating the optimal formulation of synthesized niosomes after loading extract in terms of size and zeta potential. (a) Size of Nio-GGE. (b) Zeta potential of Nio-GGE. (c) Size of Nio-HHE. (d) Zeta potential of Nio-HHE. Data obtained from DLS analysis.

3.4 Microscopy analysis

As mentioned previously, microscopic assay is the best way to examine the morphology of the nanosystems. In this study, AFM, SEM, and TEM were used to study the morphology of the nanoparticles. SEM (Figure 3a and c) shows a spherical shape with a homogeneous size of nanoparticles. The results of this analysis are consistent with the DLS results and confirm its data and, as was anticipated, confirmed the mean nanoparticle diameter measured by the DLS method. AFM data (Figure 3b and d) also show the appropriate 3D structure of the nanoparticles. In general, by examining the microscopic data, it can be concluded that the synthesized nanoparticles have a spherical shape, smooth and uniform surface, and do not have any aggregation. Images taken by TEM microscopy intelligibly show that the synthesized nanoparticles are spherical-elliptical in shape with a size of about 100 nm (Figure 4), which once again confirms the results of DLS.

Figure 3

The microscopy imaging obtained by SEM and AFM; (a) SEM imaging of Nio-GGE. (b) AFM imaging of Nio-GGE. (c) SEM imaging of Nio-HHE. (d) AFM imaging of Nio-HHE.

Figure 4

Image taken by TEM from Nio-GGE. This image shows clearly two-layer structure of niosome and its spherical shape.

3.5 Evaluation of the interaction between extracts and nanoparticles

FTIR spectrum were obtained in order to investigate the interaction between the extract and synthesized nanoniosome. The FTIR pattern (Figure 5) shows characteristic peaks in the range of 400–4,000 cm⁻¹. The peak at 3,445 cm⁻¹ in fact represents the hydroxyl bands vibrating in stretching and bending motion which repeated with slight difference in Nio-GGE and Nio-HHE, respectively, at 3,437 and 3,228 cm⁻¹. The peak at 2,925 cm⁻¹ is related to the symmetric and asymmetric stretching of carbon of CH3 group, which can be seen in the niosomes’ FTIR patterns after loading the extracts. Also, the peaks at 1,638 and 1,223 cm⁻¹ show the stretching around the C═O axis and the bending at the CH2 axis in lipids and surfactants, which can be traced after loading the extract with a slight difference in the FTIR spectrum of the niosomes. The peak at 1,100 cm⁻¹ is also due to the presence of Tween-60 and DSPE-PEG in the structure of niosomes, which indicates the stretching in the ester and ether groups of these compounds. Most peaks were repeated with a slight difference after the extracts were loaded into the niosomes, a slight difference due to intermolecular forces such as the hydrogen bond in Tween-60. Also, by examining the FTIR spectra, it can be concluded that the extracts and the nanoparticles have retained their chemical nature and integrity after loading, and no effective chemical interaction that changes their natural nature has occurred between them.

Figure 5

FTIR spectra of (a) Nio-GGE and (b) Nio-HHE.

3.6 The stability of nanoparticles

Figure 6 shows the stability of the synthesized niosomes after 180 days storage at −4°C. Based on Figure 6a, alteration in nanoparticle size during 180 days have been negligible, although nanoniosomes have increased slightly in size after 180 days so these data indicate that these particles are stable in terms of size. Examination of electrical charge changes in the nanoparticles (Figure 6b) also showed that the charge of the synthesized nanosystems tends to become more negative over time with a constant slope. Nanoparticle PDI has also increased over time, although this increase in Nio-HHE has occurred irregularly compared to Nio-GGE (Figure 6c). By examining the amount of extract loaded into the nanoparticles over 180 days, we concluded that GGE leakage from nanosystems occurred more rapidly than HHE leakage from nanosystems, which could be due to the chemical nature of GGE, which is more volatile than HHE (Figure 6d). Although this amount of nanoparticle leakage in this time period can be ignored. Overall, with this evaluation, it can be concluded that the synthesized niosomes have appropriate stability.

Figure 6

The stability analysis of Nio-GGE and Nio-HHE: (a) in terms of size; (b) zeta potential; (c) PDI; and (d) drug leakage over 180 days storage at −4°C.

3.7 Antibacterial activity

Antibacterial studies in this study showed that encapsulation of GGE and HHE increased their antibacterial properties. Also, by studying the antibacterial properties of Nio-GGE and Nio-HHE on E. coli and S. aureus bacteria, it can be concluded that Nio-HHE has stronger antibacterial properties compared to Nio-GGE. The results of this comparison are shown in Table 6.

3.8 Extract release profile

The results of extract release from nanoniosome after 72 h at various pH (4.5, 5.2, and 7.4) and temperatures are presented in Figure 7. Based on Figure 7, it can be concluded that increasing the temperature and decreasing the pH can increase the release of the drug from the nanoparticles, so that the highest release rate in Nio-GGE and Nio-HHE was related to the temperature of 42°C and pH = 4.5. However, by comparing the graphs, it can be concluded that the increase in temperature had a greater effect on drug release than the increase in pH. Accordingly, the highest rate of GGE release from niosomes in 72 h at 42°C and pH 4.5 and at 37°C and pH 7.2 was 71.3 and 50.1%, respectively. This rate was 76.2 and 48.2% for HHE, respectively. Therefore, considering these data, it can be concluded that the synthesized niosomes in this study had temperature-pH sensitivity. Responses of the synthesized nano-niosomes to different pH and temperatures also could be understood from Figure 7. According to this figure, the release of extracts from nanoniosomes occurred in a triphasic pattern, such that in the initial phase, the extract was burst released, in the second phase, it was released slowly, and in the third phase, release occurred close to zero.

Figure 7

Thermo-pH sensivity release of extracts from nanoniosomes assay. (a) Release of GGE from nanosystem at constant temperature and variable pH. (b) Statistical analysis data of GGE release from nanosystems at constant temperature and variable pH. (c) Release of GGE from nanosystem at constant pH and variable temperature. (d) Statistical analysis data of GGE release from nanosystems at constant pH and variable temperature. (e) Release of HHE from nanosystem at constant temperature and variable pH. (f) Statistical analysis data of HHE release from nanosystem at constant temperature and variable pH. (g) Release of HHE from nanosystem at constant pH and variable temperature. (h) Statistical analysis data of HHE release from nanosystems at constant pH and variable temperature. *, **, ***:P-value <0.05.

3.9 Cellular uptake

Figure 8 illustrates the successful delivery of Nio-GGE and Nio-HHE to MCF-7 and HFF cells. The in vivo transfection data was obtained by fluorescence microscopy. Based on Figure 8, the optimal nanoparticle had proper ability to deliver the drugs to the target tissue, which can confirm its efficiency in drug delivery to cancer cells.

Figure 8

Intracellular delivery of niosomes containing extracts to MCF-7 cancerous cells and HFF normal cells. The nuclei is counterstained with DAPI (blue fluorescence). Dil dye is used in order to stain the phospholipid (green). Merge is the result of the integration of DAPI and Dil images which indicates the successful entry of nanoparticles into the cells.

3.10 Cell viability study

Figure 9 demonstrates the cell viability rate of MCF-7, MCF 10A, BT-474, and HFF cell lines after treatment with different doses of GGE, Nio-GGE, HHE, Nio-HHE, and blank niosomes in 48 h. According to Figure 9, encapsulation of extracts in nanoniosome improves their therapeutic properties. The data of this analysis show that HHE IC₅₀ on MCF-7 and BT-474 cancer cells was 264.3 µg/mL and 128 µg, respectively, while after encapsulation in nanoniosomes, Nio-HHE IC₅₀ on these cells decreased to 180.1 and 62.89 µg/mL, respectively, which indicate decrease in the usage dose of the extract and thus increase in its anti-cancer properties after encapsulation. Similarly, the IC₅₀ of GGE on MCF-7 and BT-474 cancer cells was 309.3 and 274.1 µg/mL, while the IC₅₀ of Nio-GGE on these cells was 206.5  and 148 µg/mL, respectively. As mentioned, this decrease in IC₅₀ of the extract indicates improvement in its anti-cancer properties after encapsulation. Also, in order to measure the cytotoxicity of GGE and HHE and compare it with the cytotoxicity of Nio-GGE and Nio-HHE on normal cells, normal HHF and MCF 10A cells were treated with different concentrations of extracts and niosomes containing extracts for 48 h. The results of this study showed the non-toxicity of extracts and nanosystems containing extracts on normal cells. Based on these results, it can be concluded that the use of plant extracts instead of conventional methods in the treatment of cancer can reduce the adverse side effects. To evaluate the compatibility of niosomes with normal body cells and to evaluate their non-toxicity on normal cells, different dilutions of blank niosomes were affected on HFF and MCF 10A normal cell lines, and the results demonstrate the non-toxicity of the synthesized nanosystems on normal cells. It can also be concluded from Figure 9 that Nio-HHE has much stronger anti-tumor properties than Nio-GGE.

Figure 9

Cytotoxicity studies: (a) the cell viability rate (%) of MCF-7 cells after treatment with various concentrations of GGE and Nio-GGE in 48 h. (b) Comparative study of IC₅₀ GGE and Nio-GGE on MCF-7 cancer cells. (c) The cell viability rate (%) of MCF-7 cells after treatment with various concentrations of HHE and Nio-HHE in 48 h. (d) Comparative study of IC₅₀ HHE and Nio-HHE on MCF-7 cancer cells. (e) The cell viability rate (%) of BT-474 cells after treatment with various concentrations of GGE and Nio-GGE in 48 h. (f) Comparative study of IC₅₀ GGE and Nio-GGE on BT-474 cancer cells. (g) The cell viability rate (%) of BT-474 cells after treatment with various concentration of HHE and Nio-HHE in 48 h. (h) Comparative study of IC₅₀ HHE and Nio-HHE on BT-474 cancer cells. (i) The cell viability rate (%) of MCF 10A cells after treatment with various concentrations of GGE and Nio-GGE in 48 h. (j) The cell viability rate (%) of MCF 10A cells after treatment with various concentrations of HHE and Nio-HHE in 48 h. (k) The cell viability rate (%) of HFF cells after treatment with various diluent of blank niosome in 48 h. (l) The cell viability rate (%) of MCF 10A cells after treatment with various diluent of blank niosome in 48 h. ns: no significant difference. *, **, **: P-value <0.05.

3.11 Gene expression

In this study, real-time PCR technique was used to evaluate the effect of GGE and HHE on the expression of p53 and MCL-1 genes as genes involved in the development of breast cancer and compare them with their encapsulated form. Figure 10 illustrates the effect on GGE, HHE, Nio-GGE, Nio-HHE, and blank niosome on the expression of p53 and MCL-1 genes in MCF-7 and MCF 10A cells. According to this figure, after treatment of MCF-7 cells with HHE, the expression of p53 and MCL-1 genes increased by 60% and decreased by 33% compared to the house keeping gene, respectively. While the expression of these genes after treatment of cancer cell with Nio-GGE compared to the control group increased by about 100% and decreased by 49.8%, respectively. Expression of p53 and MCL-1 genes in cancer cells after GGE treatment increased by 38% and decreased by 15% compared to the control group, respectively. While this rate after treatment of cells with Nio-HHE has reached to 86% increase in gene expression and 35% decrease in gene expression, respectively. Blank niosomes had no effect on the expression of these genes in breast cancer cells. On the other hand, by examining the effect of extracts and niosomes containing the extract on the expression of p53 and MCL-1 genes in normal MCF 10A cells, it was found that the extracts and niosomes of the extract had no effect on the expression of genes in normal cells. In other words, the results of this study consistent with the results of MTT assay show that niosomes containing extracts have no side effects on normal cells as non-target tissue.

Figure 10

p53 and MCL-1 gene expression in MCF-7 cells as cancerous cells and in MCF 10A cells as normal cells. (a) Changes in expression of p53 gene after treatment with specific concentration of HHE and Nio-HHE and blank niosome. (b) Changes in expression of p53 gene after treatment with specific concentration of GGE and Nio-GGE and blank niosome. (c) Changes in expression of MCL-gene after treatment with specific concentration of HHE and Nio-HHE and blank niosome. (d) Changes in expression of MCL-1 gene after treatment with specific concentration of GGE and Nio-GGE and blank niosome; ns: no significant difference. *, **: P-value <0.05.

3.12 Western blot analysis

In order to investigate the specific effect of GGE, Nio-GGE, HHE, and Nio-HHE on the expression of p53 and MCL-1 proteins, MCF-7 cells were treated with extracts and niosomes containing extracts for 48 h. As displayed in Figure 11, the p53 proteins remarkably increase after treatment with extracts and niosomes containing extract, while the MCL-1 proteins are notably less after treatment. Also, by analyzing western blot data, it could be concluded that the expression of these proteins is more affected by niosomes containing extracts than their free form. Also, the effect of Nio-HHE compared to Nio-GGE on the expression of these proteins in treated cells is significantly higher, which indicates that Nio-HHE is outstandingly effective than Nio-GGE in the treatment of breast cancer.

Figure 11

(a) Changes in expression of p53 protein in MCF-7 cells after treatment with GGE and Nio-GGE. (b) Changes in expression of p53 protein in MCF-7 cells after treatment with HHE and Nio-HHE. (c) Changes in expression of MCL-1 protein in MCF-7 cells after treatment with GGE and Nio-GGE. (d) Changes in expression of MCL-1 protein in MCF-7 cells after tratment with HHE and Nio-HHE. (e) Gel retardation assay to evaluate the genes expression in MCF-7 cells after treatment with GGE and Nio-GGE. (f) Gel retardation assay to evaluate the gene expression in MCF-7 cells after treatment with HHE and Nio-HHE. ns: no significant difference. *, **: P-value <0.05.