Folic acid-chitosan coated stylosin nanostructured lipid carriers: fabrication, in vitro–in vivo assessment in breast malignant cells

Abstract Synthesis of targeted nanostructure lipid carriers for stylosin (STY-CFN-NPs) delivery to MCF-7 cells. STY-CFN-NPs were formulated via the homogenization and ultra-sonication technique. After evaluating the amount of drug encapsulation and FA binding, the toxicity effect of the STY and STY-CFN-NPs on MCF-7 cells was measured by the MTT method. Cell cycle analysis, AO/PI staining and qPCR to assess the inducing of apoptosis as well as Tubo cancer cell inoculated mouse model for antitumor properties of STY-CFN-NPs were used. Significant increases in nanoparticle size and changes in zeta potential were observed after FA-CS coating on nanoparticles. Slow release of the STY within 144 h as well as the acceptable rate for STY encapsulation efficiency (92.4% and FA binding (52.5%) to the STY-CFN-NPs (PS: 66.26 ± 3.02 nm, ZP: 29.54 ± 1.01 mV and PDI: 0.32 ± 0.01) was reported. STY-CFN-NPs exhibited higher toxicity compared to STY suspension and treatment with STY-CFN-NPs was lead to increased apoptotic cells, stopped cells in the SubG1 phase, and also increased caspase and BAX expression and decreased BCL-2 and BCL-XL expression in in vitro and decreased the size of murine tumors (54.57% in 16 days) in in vivo. The results showed STY-CFN-NPs have good potential for breast cancer management.


Introduction
Breast cancer (BC) is caused by the proliferation of malignant cells in the lining of the mammary ducts and is known as the most common solid tumor in women and accounts for about 30% of all cancers [1]. In this type of malignancy, various factors such as family history, age, sex, estrogen level, and breast condition play an important role [2]. Studies have shown that the incidence is less than 4% in women under 35, about 7% in women under 40, and the highest risk in postmenopausal women [3]. Tumor removal, radiotherapy, chemotherapy, etc. are among the common treatments to control this type of malignancy. However, drug instability, poor pharmacokinetics, restrictions on the use of high doses, drug toxicity on cells and tissues normal and, most importantly, drug resistance impairs the function of treatment [4][5][6]. Therefore, acquiring new treatments as well as new chemotherapy drugs is an important priority for the treatment of this malignancy [7].
Terpenes are known as a group of natural organic compounds with a wide range of medicinal activities such as anti-cancer properties [8]. Stylosin is a terpene compound capable of inducing apoptosis and cytotoxic effects on cancer cells and has been extracted from various Ferula species including Ferula Fraxinus, stylosa, and ovina. Its anti-cancer effects against 5637, CH1, SKMEL-28, and A549 cancer cells [9,10] have been reported in various studies. The insolubility of Stylosin in water [9], while reducing the bioavailability and chemical stability of this compound, limits its clinical applications. Today, new drug delivery systems (DDS) have provided an effective way to increase the solubility and stability of compounds. The use of DDS can lead to controlled release, and increase bioavailability, solubility, and drug uptake [11]. Modifiable structures and advanced functions of nanosystems such as micelles, nanoemulsions, liposomes, and nanoparticles have led to their widespread use in various fields such as imaging, diagnosis, treatment, drug delivery, etc. [12]. Lipid nanoparticles and nanostructures are two highly efficient carriers in increasing bioavailability and transport of water-insoluble compounds to target cells. However, nanostructured lipid carriers (NLCs) have more advantages over SLNs including improved drug release control, greater stability, and higher encapsulation efficiency [13,14]. NLC is a new generation of lipid DDS that protects the loaded drug and increases its half-life, Increases bioavailability, and reduces the need for high doses of the drug [15,16]. These nanocarriers (NCs) are lacking some of the disadvantages of lipid colloidal systems such as liposomes, SLNs, etc. The presence of a solid lipid matrix in conditions such as room temperature or body temperature causes more order in the NC structure [16]. In addition, NLC has the property of mucosal adhesion, which makes it efficient to use for oral administration. The high efficiency of encapsulation and loading of lipophilic and hydrophilic drugs on NLC-NCs is another advantage of using these NCs [16,17].
The possibility of modifying the surface of lipid nanostructures has provided a good opportunity to prepare formulations with specific functions. In various studies, NLCs with complex surface has been designed to increase oral bioavailability and intestinal absorption [18]. Chitosan (CS) as a natural cationic biopolymer has been widely used due to its properties such as the ability to open tight attachments of intestinal epithelial cells [19] and its adhesion to mucosal surfaces to modify the surface of NLC nanoparticles [20]. Due to the specific properties of chitosan, its use to modify the surface provides a great opportunity for the oral adsorption of lipid nanoparticles including SLN [21], NLC [22] and liposomes [23]. Targeted drug delivery and reduction of treatment side effects are among the most important goals in cancer treatment. The use of chitosan due to its positive charge can play a key role in the transfer of NCs to negatively charged cancer cells and increase their internalization [24]. Other methods of targeting include the use of special ligands such as transferrin, FA, etc. at the nanoparticle level. Since receptors for some ligands, such as FA, are overexpressed in malignant cells, modifying the surface of nanoparticles with such ligands can increase the binding of NCs to cancer cells and their internalization by receptors [25][26][27]. In this study, STY was first loaded on NLC nanoparticles and then the surface of nanoparticles was coated with FA conjugated CS. Finally, its anti-cancer effects against MCF-7 cells were evaluated

FA-CS preparation
For this purpose, the FA-NHS solution was first prepared by the following method. The FA was added to the DMSO solvent and then EDC and NHS were added to the above solution. The resulting solution was then incubated in the dark for one hour and finally filtered. To bind folic acid to chitosan, the FA-NHS solution was added dropwise to the CS dissolved in acetic acid, and the resulting solution was incubated for 24 h under continuous stirring. To terminate the reaction, the pH of the solution was adjusted to 9, and the precipitate was centrifuged, dialyzed, and finally lyophilized.

Preparation of NLC loading STY
Oleic acid and glyceryl monostearate as liquid and solid lipids respectively as well as lecithin were selected to prepare the lipid matrix. The drug was then added to the lipid matrix and the obtained mixture was incubated at 80 C until melting. To prepare the aqueous phase, Tween 80 was dissolved in 20 ml of distilled water (DW) and incubated at 80 C. Finally, the blue phase was quickly added to the lipid phase and the resulting mixture was homogenized and then sonicated.

STY-NLCs characterization
The mean size, dispersion, and f-p of the formulation were analyzed by the DLS method (ZEN3600, Malvern Instruments Ltd., UK). Samples were analyzed in a ratio of 1-10 in dispersed DW and at 25 C under a 175 scattering angle in three replications.

Fabrication of STY-CFN-NPs
FA-NHS was prepared by mixing 6 mg of FA, 12 mg of EDC and 7.6 mg of NHS in DMSO. The resulting mixture was filtered after 1 h of incubation, and then a solution containing 1% acetic acid and chitosan was added to it, and after 24 h of incubation, the pH was adjusted to 9 and the precipitate was centrifuged, dialyzed and lyophilized. In order to bind FA-CS to the surface of NLC-NPs, FA-CS powder (5 mg/ml) was dissolved in acetic acid (1%) solvent under continuous stirring conditions. Then, CF solution was gently added to the solution containing NLC-NPs. The sample was stirred continuously for 2 h and then centrifuged at 10,000 rpm for 5 min. The pellet was rinsed three times with acetic acid (1%) solution to remove not-reacted FA-CS. Finally, the pellet was lyophilized by a freeze dryer machine and the STY-CFN-NP powder was fabricated as its final formulation.
2.6. DLS, f-potential, FTIR, and SEM of STY-CFN-NPs The size, dispersion index, and surface charge of the samples were measured similarly to the method in Section 2.4. To prepare the samples for FTIR, a mixture containing nanoparticles and KBr powder was prepared in the form of compressed tablets and analyzed in the range of 500-4000 cm À1 . SEM was used to examine the morphology of the sample along with the particle size. First, a suspension containing nanoparticles dissolved in distilled water was prepared and after spraying the samples on aluminum foil and drying them, the surface of the samples was covered with gold and examined microscopically.

FA coupling and STY entrapment efficiency
STY encapsulation efficiency (EE) and FA binding capacity to NLCs were determined by the indirect method with a UV spectrophotometer (k ¼ 220 and 282 nm respectively). %EE and FA coupling were finally assessed as presented below: Total drug-unbound drug=Total drug Ã 100

STY release assay
To evaluate the release rate of the drug during specific times, a colloidal suspension of nanoparticles with a dose of 100 mg/10 ml was prepared and poured into a ready dialysis bag. The dialysis bag (12-14 kD, Sigma Aldrich) was suspended in phosphate buffer (pH 7.2) in beaker after sealing. Beaker was subjected to continuous agitation (100 rpm at 37 C) for144 h. At specified times; 1 ml of the solution around the dialysis bag was removed for analysis and replaced with 1 ml of fresh PBS. By evaluating the absorption of the supernatant at a wavelength of 220 nm and substituting the numbers in the formula obtained from the standard curve and using the following formula, the rate of drug release was evaluated: % cumulative of release: STY in each sampling time point (M t ) / initial weight of the STY-loaded in the sample (M 0 ) Â 100 [28].

MTT procedure for cytotoxic assay
Initially, the cells were seeded in 96 well plates and after 24 h of incubation, their culture medium was emptied and replaced with a treatment medium containing serial doses of NPs. 48 h after treatment, the treatment medium was drained, MTT solution (0.5 mg/ml) was added and the cells were incubated in the incubator for 4 h. Finally, the MTT medium was replaced with DMSO and the wavelength of each well was recorded at 570 nm [29], and by substituting the sample adsorption in the following formula, the percentage of cell viability at each concentration was evaluated. %CV ¼ (sample absorbance/control absorbance) Â 100.

AO/PI staining assay
For this purpose, the MCF-7 cells were transferred to 6 wells plate and after 24 h, they were treated with the IC 50 concentrations. After 48 h, the treatment medium was drained and a mixture of dye containing AO and PI dye dissolved in PBS was added to each well. The samples were immediately examined by an inverted fluorescent microscope.

Cell cycle analysis
Initially, the logarithmic phase cells were seeded in 6 wells plate and after 24 h, their culture medium was drained and replaced with a treatment medium containing concentrations obtained from MTT assay. On the second day after treatment, the treatment medium was drained, and after washing, 1 ml of trypsin was added to each well. After ensuring that the cells separated from the bottom of the plate, 1 ml of culture medium was added to each well and the suspension containing cells was transferred to separate micro tubes. After centrifugation, the supernatant was drained and 300 ll of PI dye was added to the cell sediment for 10 min and then analyzed by flow cytometry.

Quantitative PCR assay
Changes in gene expression were assessed by qPCR methods. For that, the MCF7 cells were cultured in 25 cm 2 flasks and after ensuring the connection of the cells to the bottom, were treated with various concentrations of NPs. After 48 h of holding the flasks in the incubator, all total RNA of the cells were isolated and after determining their amount by nanodrop method, they were used as a template for cDNA synthesis. Finally, using CDNA, specific primer (Table 1), water, and Syber green, the reaction mixture was prepared and analyzed under a specific temperature schedule.

Murine model for breast cancer
For this purpose, 18 female BALB/c mice (5-6 weeks, 20-22 g) were purchased and 100 ll suspension of Tubo mouse tumor cells (5 Â 10 5 /ml) was injected subcutaneously into each sample. After the emergence of tumors, the samples were divided into three groups including a control group (phosphate buffer recipient) and two experimental groups receiving STY and STY-CFN-NPs. The samples were treated by intraperitoneal injection every two days and on the day of treatment, the tumor dimensions were measured using a caliper, and finally the tumor volume was calculated using the following equation: Tumor volume (mm 3 After the treatment period, all specimens were sacrificed and the tumors were removed and placed in 10% formalin. After placement in paraffin, tissue sections were prepared from the tumor and stained with hematoxylin and eosin methods and examined by a pathologist.

Physicochemical properties of STY-NLCs and STY-CFN-NPs
The particle size, PDI, and zeta potential of the STY-NLCs were observed to be 45.4 ± 0.52 nm, 0.3 ± 0.02, and À14.0 ± 1.49 mV, respectively. After surface modification of STY-NLCs with CS-FA, The particle size showed a significant increase (66.26 ± 3.02 nm). Also, the surface charge changed from negative to positive (þ29.54 ± 1.01), and in addition, the dispersion index of nanoparticles increased slightly (0.32 ± 0.01) (Figure 1(A)). Particle size and dispersion index are among the effective features in improving drug delivery. Nanoparticles with low PDI show significant homogeneity and this improves the biological properties of nanoparticles [7]. As the results show, the size of nanoparticles with chitosan coating has increased, however, the STY-CF-NLCs have an acceptable size for clinical applications [30,31].  On the other hand, the cationic nature of CS caused a change in surface charge from negative to positive, which leads to the active delivery of nanoparticles and increases their tendency to bind to cancer cells with negative surface charge and can play an effective role in drug delivery to the target cell. Previous studies have also reported improved drug delivery by chitosan-coated nanoparticles [32]. Micrographs obtained from SEM also showed a spherical morphology with a smooth surface and size below 100 nm of STY-CFN-NPs, which clearly confirms the DLS results (Figure 1(B)). Figure 2 shows the recorded FTIR spectra for STY-CF-NLCs. These were recorded at 3417 cm À1 for the combined effect of the NH2 and OH group. The CH3/CH2 CeH stretching peaks appeared at 2920.59, 2865.46 cm À1 [33]. The presence of a peak at 1732 cm À1 (C ¼ O stretching) demonstrations that the FA-CS is formed and CS has successfully covered NLC-NPs [26,34].

FA coupling, encapsulation efficiency, and release of STY
The amount of STY loading and FA binding was measured indirectly by UV-spectrophotometry (Figure 3(A,B)). The formula obtained from the standard diagram for STY and FA was reported as Y ¼ 0.836X þ 0.214 and Y ¼ 0.55X þ 0.207 respectively. Using this formulation and the formula mentioned in section 2.7, % STY loading and FA binding were obtained to be 92.2% and 52.5%, respectively. Examination of the drug release chart shows a slow and stable release of STY over 144 h (Figure 3(C)). The reason for the slow release of STY can be attributed to the drug being trapped in the inner core of the lipid matrix, which is slowly released. On the other hand, the presence of CS-FA around the nanoparticles can further protect the drug from rapid excretion [35]. In order to investigate the STY's release dependence in the pathophysiological pH conditions, both physiologic (pH 7.2) and pathologic (pH 6.5) conditions were selected as the normal blood and tumor tissue environment, respectively. The result shows a significantly greater releasing process in shorter periods at acidic conditions (Figure 3(C)).

Cytotoxic effects of STY and STY-CFN-NPs
The cytotoxic potential of STY and STY-CFN-NPs on MCF-7 cells is represented in Figure 4. These results clearly exhibited that STY and STY-CFN-NPs have a dosedependent cytotoxic effect. The results also showed the effect of higher toxicity of STY-CFN-NPs compared to STY and also the effect of selective toxicity of STY-CFN-NPs against cancer cells. Figure 4 shows the effect of STY toxicity on MCF-7 cancer cells. As can be seen, STY is able to inhibit MCF-7 cells only at a concentration of 400 lg/ml, and at lower concentrations, the cell viability is reported to be 100%. STY-containing nanoparticles from 50 lg/ml showed a significant inhibitory effect on cells and more than 90% of cells were inhibited by increasing the concentration to 100 lg/ml. These results show higher toxicity effects of STY-CFN-NPs compared to free STY. Due to the inhibitory effect of nanoparticles on cancer cells, in order to investigate possible side effects and the effect of nanoparticle toxicity on normal cells, an HFF cell was used as a normal cell and the toxicity of nanoparticles against it was investigated. The results showed that nanoparticles were able to inhibit about 50% of normal cells only at a concentration of 400 lg/ml and no inhibitory effect was observed at lower concentrations. According to the results, concentrations of 100 and 200 lg/ml STY-CFN-NPs can be used as safe concentrations to inhibit breast cancer cells without affecting normal cells.

AO/PI staining
In this study, to investigate the occurrence of apoptosis, nucleic acid-binding dyes including AO and PI were used. AO dye penetrates healthy cells to produce a green fluorescence dye, whereas PI stains only cells with destroyed membranes, dead and necrotic cells with a red fluorescence dye. According to the results ( Figure 5), in the control group, all cells are green and have normal morphology, while in the experimental groups treated with different concentrations of STY-CFN-NPs, alteration in the morphology of the cells was observed. In addition, with increasing treatment concentration, the number of red cells increased and the number of green cells decreased, which indicates an increase in apoptotic cells with increasing treatment concentration.

Cell cycle analysis
During apoptosis, chromosomal DNA is broken down into nucleosomal fragments by the enzyme endonuclease. These components leak from the cell and the DNA content of apoptotic cells is lower than the DNA content of cells in phase G1 of the cell cycle. Thus apoptosis is usually associated with the SubG1 or pre-G1 phase of the cell cycle.

Molecular assay
The Bcl-2 family includes important regulators of Peru and anti-apoptosis, of which BCL2-and BCL-XL are considered anti-apoptotic factors and BAX as pro-apoptotic factors. Pro-apoptotic factors, by disrupting mitochondrial integrity, cause the release of apoptogenic agents such as cytochrome c (Cytc) from the mitochondria and activates downstream signals to promote apoptosis. While anti-apoptotic factors, by maintaining mitochondrial integrity, prevent the release of apoptogenic agents and  subsequent apoptosis. Release of cytochrome C from mitochondria activates apoptotic pathways, the most important of which is the caspase-related pathway, which ultimately leads to cell death [36]. The results showed that STY-CFN-NPs induced apoptosis signaling in treated cells by inhibiting anti-apoptotic genes (BCL-2 and BCL-XL), activating pro-apoptotic factors (BAX), and activating caspases (Figure 7).

STY-CFN-NPs anti-tumor effects
The effectiveness of treatment was evaluated by measuring tumor volume and histopathological studies of tumors. Comparison of the percentage of tumor inhibition in samples treated with STY (23.22%) and samples treated with STY-CFN-NPs (54.57%) shows the effectiveness of treatment with nanoparticles compared to free drugs. Histopathological examination of tumor tissue in the control sample showed the presence of carcinoma islets that were separated by stroma (S) (connective tissue). In experimental samples, in some islets of cells, apoptosis occurred and is observed in a dense and dark color. A comparison of sections obtained from samples treated with STY and STY-CFN-NPs shows that in the nanoparticles-treated group, more cells occurred apoptosis, which shows the efficiency of drug delivery systems to increase the effectiveness of treatment (Figure 8).

Discussion
Cancer treatment using nanotechnology has shown great progress in recent years [37]. The use of this technology causes the delivery of drug molecules without affecting healthy cells to the target sites [38][39][40] and while increasing the stability, solubility, half-life, and bioavailability of many chemotherapy drugs causes the increased drug accumulation in the tumor microenvironment and decrease drug dose [41].
Various types of drug delivery systems (DDS) including protein, liposomal, polymeric, metallic, and carbon nanocarriers have been purposefully used for research on breast cancer cells [42]. Among these, nanostructured lipid carriers (NLCs) are one of the most useful drug delivery systems. These systems are based on a solid matrix made of biodegradable and biocompatible materials that, while increasing drug loading, cause its stability in the gastrointestinal tract and controlled drug release [43]. The physicochemical properties of the drug play a key role in selecting the appropriate technique for the synthesis of NLC nanoparticles. Loading of hydrophobic compounds has been reported in NLC in a variety of ways [16]. High-and low-energy techniques have been used to synthesize NLC nanoparticles in various studies. Methods such as ultrasound or high pressure homogenization are considered as high energy techniques and have been widely used in more recent studies [44]. In contrast, micro emulsification methods, double emulsion, phase inversion temperature, etc. are known as low energy methods and are based on the spontaneous formation of droplets by modifying the surfactant/oil/water ratio [45]. In pharmaceutical applications, the size and dispersion of nanoparticles affect the stability, safety and drug delivery by nanocarriers. Ultrasound and high pressure homogenization in high energy methods [46] and temperature in low energy methods [47] are the determining factors in reducing the size and dispersion of nanoparticles. In this study, NLC-NPs containing stylosin were synthesized using homogenization and probe-sonication methods, and their physicochemical properties were evaluated. The results exhibited the formation of nanoparticles with a hydrodynamic diameter of 45.04 nm, single dispersion (PDI $ 0.3), and stability (ZP $ À14.01 mV). Similarly, in a 2017 study using highpressure homogenization method, NLC-NPs containing red ginger extract with an average diameter of 131-154 nm, dispersion index of 0.1-0.17 and surface charge of À33 to 46 mV were synthesized [48] which compared to STY-NLC-NPs, has a larger size with a smaller and more stable dispersion index. In a 2014 study, phase inversion temperatures were used to load ferulic acid on NLC nanoparticles. In this study, particles with a hydrodynamic diameter <50 nm and a dispersion index of 0.3 were synthesized [49]. In another study, the synthesis of NLC with size <100 nm and PdI > 0.3 was reported by phase inversion temperature method [50], which is comparable to the physicochemical properties of STY-NLC-NPs synthesized by homogenization and ultra-sonication method.
NLC is a hydrophobic nanocarrier that due to its hydrophobic nature [51] is detected by the reticuloendothelial system (RES) and deleted by opsonin proteins [52]. Therefore, modifying their surface with a hydrophilic compound can play an effective role in increasing the concealing properties of nanocarriers. Natural cationic polymers such as chitosan play an important role in the stability of nanocarriers in vivo due to their mimicry of extracellular matrix [53][54][55][56]. The use of chitosan on the surface of nanoparticles increases mucosal adhesion, increases carrier penetration into cells and tissues, and controls drug release [57]. On the other hand, due to the positive surface charge, it increases the carrier uptake into cancer cells (with a negative charge) and increases the internalization of the drug and the effectiveness of treatment [58]. Previous studies have shown that the use of chitosan in nanocarrier structures can provide a combination of the effect of EPR (inactive target) and target ligand (active target) [59,60]. Also, the conjugation of specific ligands of cancer cells such as transferrin and folic acid with chitosan can increase the uptake of nanocarriers into cancer cells, causing them to be internalized by the receptor and provide active targeting [61].
In this study, due to the hydrophobic nature of STY and the possibility of its removal by immune system opsonization, nanocarriers were used to transfer this compound to breast cancer cells. Since high-efficiency hydrophobic compounds are loaded on liposomal nanoparticles, NLC nanocarriers were used to deliver this compound. The surface of the nanoparticles was then modified with chitosan bound to folate. Examination of the physicochemical properties of nanoparticles showed the presence of spherical and stable (ZP: 29.54 mV) nanostructures, with diameter of 66.26 nm and a dispersion index of 0.32 (Figure 1(A)). Increasing the size and changing the surface charge from negative to positive in the coated nanoparticles compared to STY-NLC-NPs is related to the presence of chitosan and folic acid coating on the surface of the nanoparticles. Similar to the present study, many studies have reported surface charge change from negative to positive and increase in size after nanoparticle surface modification with chitosan [62][63][64]. In a study conducted in 2021 [7] similar to the present study, a slight increase in particle size, as well as a change in surface charge from negative to positive, were reported after coating of NLC-NPs by chitosan. The encapsulation efficiency of STY in nanoparticles by adsorption method at 220 nm was reported to be 92.2%, which shows a higher rate compared to the encapsulation efficiency (83.74%) of luteolin in chitosan-coated NLC nanoparticles (LTN-CS-NLCs) [7]. A comparison of drug release efficiencies in STY-CF-NLCs and LTN-CS-NLCs nanoparticles indicates a lower drug release rate in STY-CF-NLCs. As Figure 3(C) shows, the drug release rate after 144 h has reached about 87%, while the lutein release efficiency after 25 h has been reported to be about 80% [7]. The reduction in drug release in STY-CF-NLCs can be related to the type of drug as well as the presence of FA in its structure.
Some physical parameters such as spherical morphology, uniform size distribution, and surface charge above ±30 mV provide acceptable properties for the stability and toxicity of nanoparticles [65]. In the current investigation, due to the presence of FA in the structure of STY-CF-NLCs, MCF-7 cells have been chosen as FA-positive receptor cells [66,67] to examine the toxicity of STY-CF-NLCs. MTT results clearly exhibited that STY-CF-NLCs have higher dose-dependent toxicity compared to free STY at 48 h. The IC 50 concentration of nanoparticles in normal cells is notably larger than that in MCF-7 cells. The reason can be due to the mechanism of cellular uptake in cancerous cells. In other words, considering the overexpression of folate receptors at the MCF-7 cells' membrane [68], receptor-mediated endocytosis has the potential to significantly reduce cell survival in the cancerous cell lines. The above discus was added in the section. Moreover, the free STY is not soluble and accessible enough to easily be uptake by cells. The lower toxicity of free STY than that with its encapsulated form verifies the role of bio-accessibility in improving its bioactivity.
In different studies similar to the present study, improved drug toxicity has been reported after loading on various nanocarriers [7,61,62] which can be attributed to more efficient drug delivery by DDS. In this study, the occurrence of apoptosis was evaluated as an effective mechanism in suppressing cancer cells. Examination of cell cycle arrest in treated cells showed that STY-CF-NLCs at a concentration of 85 lg/ml induces apoptosis in the cancer cell population by up to 80.7% in the Sub G1 phase, which confirms the occurrence of apoptosis in treated cells ( Figure 6). In other words, the increased SubG1 peaks show the occurrence of apoptosis, which cause death before arriving at the G1, S, or G2 phase. In other words, STY's toxicity is not compensable enough to induce cell cycle arrest. In a 2016 study, the effect of cell cycle arrest and induction of apoptosis was reported in cells treated with melatoninloaded NLC nanoparticles in combination with low-dose tamoxifen. The results of this study showed a twofold increase in the percentage of apoptosis and a decrease in cell proliferation by 10% [69]. Similar results are shown by Aghazadeh et al. They exhibited the pro-apoptotic effects of kaempferol-loaded NLC-NPs by flow cytometry as well as molecular analysis [70]. They reported a 22% increase in apoptotic cells as well as decreased expression of BCL-2 and mcl-1 genes (anti-apoptotic genes) and increased expression of the BAD gene (pro-apoptotic gene) in treatment with kaempferol-NLCs [70].
In this study, the anti-tumor effects of nanoparticles in vivo were evaluated due to the slow release of the drug from the nanocarriers. Quantitative results showed a 54.57% reduction in breast tumor growth in the nanoparticle-treated mouse model, and the apoptotic regions in tissue samples isolated from treated mice compared with controls clearly showed the nanoparticle anti-tumor effects. In a 2017 study, the antitumor effects of RIPL-modified liposomes containing DTX in nude BALB/c mice with SKOV3 cell tumors were reported [71]. In another study, the anti-tumor effects of DTX-loaded NLCs (EE $ 95-98%) on a mouse model inoculated with SKOV3 ovarian cancer cells were evaluated and the results exhibited 91% inhibition of tumor growth in treated specimens [72]. Similarly in the present study, stronger antitumor effects of nanoparticles were shown compared to the drug, which can be attributed to long-term retention and stable drug release. In addition, the targeting of nanoparticles with folic acid ligand and electrostatic interactions due to the presence of the cationic polymer chitosan on the surface of nanoparticles and their binding to the surface of negatively charged cancer cells cannot be ignored.

Conclusion
Side effects and drug resistance during common cancer treatments highlight the need for alternative therapies from natural sources. The anti-cancer effects of many plant bioactive compounds have been reported in various studies. However, conventional drug delivery methods do not have the necessary efficiency to transport the drug to the target site and causing the drug to accumulate in non-target tissues and cause side effects. In this study, stylosin was selected as a natural active compound and then nanostructure lipid carriers were used to increase its solubility, stability, and targeted delivery to breast cancer cells. The results showed an increase in the effectiveness of treatment during DDS use. Our findings on the cytotoxic, pro-apoptotic, and antitumor effects of STY-CF-NLCs suggest that this formulation is a promising therapeutic agent for the prevention and treatment of breast cancer.

Acknowledgment
This work was supported by Islamic Azad University, Mashhad, Iran and thus is appreciated by the author.

Disclosure statement
No potential conflict of interest was reported by the author(s).

Funding
This research was performed at personal expense in the laboratory of Islamic Azad University of Mashhad.