Human foreskin fibroblast (HFF) and MCF-7 breast cancer cell lines were purchased from the cell bank at the Research Institute of Biotechnology, Ferdowsi University of Mashhad, Mashhad, Iran. To ensure the authenticity of the cells, DNA profiling of short tandem repeats (STRs) was utilized and regular screening for mycoplasma contamination was conducted. Stearic acid, CS (LMW), non-ionic surfactant (Tween 80), lecithin, N-hydroxysuccinimide (NHS), ethanol (EtOH), 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), potassium persulfate (PPS), dimethylsulfoxide (DMSO), 2,2-Diphenyl-1-picrylhydrazyl (DPPH), and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) were obtained from Merck (Darmstadt, Germany). AC, propidium iodide (PI), acridine orange (AO), phosphate-buffered saline (PBS), and 2,5-diphenyl-2H-tetrazolium bromide (MTT) were purchased from Sigma-Aldrich (St. Louis, MO, USA). FBS was bought from Gibco (Franklin Lakes, NJ, USA). Cell culture medium, antibiotics, and trypsin EDTA were obtained from Invitrogen (Carlsbad, CA, USA).

SLN, in which the AC was passively loaded, was prepared using homogenization and ultrasonication. Stearic acid was used as a lipid component, and Tween 80 and lecithin were used as surfactants and co-surfactants. To prepare the lipid phase, 10 mg of AC, dispersed in distilled water, was added to the mixture containing stearic acid and lecithin, and the temperature of the mixture was raised to 85 °C. The aqueous phase was prepared by mixing water and Tween 80. After the isothermalization of lipid and aqueous phases (at 85 °C), the aqueous phase was added to the lipid phase, and the mixture was homogenized using a high-pressure homogenizer (HPH) at 85 °C for 5 minutes. After 24 h of storage at room temperature, the final mixture was centrifuged using a high-speed centrifuge and the collected supernatant was used for HPLC analysis and to evaluate the amount of free AC. The HPLC system (Alliance 2695, Waters, Milford, MA, USA) was equipped with a UV detector and consisted of C18 as a stationary phase. The column was operated in isocratic mode (50:50 MeOH: H${}_2$O) at a flow rate of 0.5 mL/min. The UV detector was set at 254 nm for the measurement of AC.

To modify the surface of AC-SLN, a CS coating attached to FA was used. For this purpose, CS attached to FA was first prepared, and then, added to the mixture containing the NPs. To prepare CS-FA, FA powder was dissolved in DMSO, and EDC and NHS were added to it. After incubating for 1 h, the resulting mixture was filtered. To connect CS to FA, CS powder was dissolved in 1% acetic acid, and after ensuring its dissolution, the resulting mixture was added to the FA-NHS mixture and incubated for 24 h on a magnetic stirrer. Next, by adjusting the pH to 9, a CS precipitate of FA was formed, which was dialyzed, and then, lyophilized to remove free FA. To decorate the NP surfaces, CS-FA powder was dissolved in 1% acetic acid and added to SLNs dissolved in distilled water (DW). The final mixture was mixed for 2 h under continuous stirring and after centrifugation, was lyophilized. The supernatant was used for HPLC analysis and to determine the percentage of FA binding to the surface of the NPs.

Average size and surface charge along with the dispersion distribution of AC-SLN-CS-FA were evaluated by DLS. Firstly, a suspension containing AC-SLN-CS-FA dissolved in DW (1:100) was prepared. Then, after ensuring that the NPs were dispersed, the resulting suspension was analyzed under a scattering angle of 170 degrees at a temperature of 25 °C. FESEM was used to examine the morphology of the samples. For this purpose, NPs were dispersed in DW and sonicated. Next, a drop of the resulting solution was sprinkled on aluminum foil and left to dry. After the solvent had dried, the surface of the sample was coated with gold and subjected to microscopic examination. The samples for FTIR were prepared by compressing NPs with potassium bromide (KBr) into pellets, which were analyzed in the FTIR device.

Spectrophotometer absorption was used to evaluate the AC encapsulation and FA binding percentages. For this purpose, the amount of free substance was measured and subtracted from the total substance. The remainder was entered into the reaction and the amount of trapped substance was observed. This method is called the indirect method. A serial concentration of AC was prepared, and then, the absorption of each sample was measured and the related standard graph was drawn. Next, the absorbance of the supernatant (Section 2.2) was measured at the same wavelength. By inserting it into the formula obtained from the standard curve, the amount of free AC in the supernatant was obtained, and finally, the percentage of encapsulation efficiency (EE%) of the substance was calculated using the formula below:

EE (%) = (total amount of drug – the amount of free drug)/total amount of drug $\times$ (100).

The same method was used to evaluate the level of FA binding. Standard curves related to different FA concentrations were drawn, and the amount of FA binding was obtained by placing the amount of absorption obtained from the supernatant in Section 2.3 and replacing the numbers in the above formula.

Dialysis method and absorption spectrophotometry were used to evaluate drug release within 5 days. Firstly, the free AC and NPs were dispersed in the phosphate buffer and poured into the dialysis bag, which had already been activated. The lid of the bag was closed, and the bag was suspended in the phosphate buffer at 37 °C and 100 rpm. At certain time intervals, 1 mL of the phosphate buffer was removed from around the bag to check the amount of released substance, and 1 mL of fresh buffer was re-added to the beaker. Then, the collected samples were analyzed by spectrophotometry. The drug release graphs were drawn in Microsoft Excel.

Firstly, ABTS and DPPH free radicals were prepared. To prepare the ABTS free radicals, a solution containing distilled water, PPS, and ABTS was prepared and incubated in the dark for 24 hours. Then, the solution was diluted with DW until it reached an absorbance of 0.7 at a wavelength of 734 nm. DPPH free radicals were prepared using 96% EtOH, in which DPPH powder was dissolved. Next, different concentrations of NPs were prepared and equal volumes of the desired free radical were added. Then, the samples containing ABTS free radicals were incubated for one hour in the dark at room temperature, after which, their absorbance was recorded at a wavelength of 734 nm. The samples containing DPPH free radicals were incubated for 30 minutes in the incubator and analyzed at a wavelength of 517 nm.

To conduct the cytotoxic assay, cells were seeded in 96-well plates for 24 h, after which, they were treated with different doses of AC-SLN-CS-FA for 48 h. Next, the medium containing AC-SLN-CS-FA was drained from each well, and 20 µL of MTT solution was added. After 4 h, the required time for the reaction to be completed, the plates containing the cells were removed from the incubator, and the MTT medium was drained. The formazan crystals, formed by the MTT reaction with living cells, were dissolved by adding DMSO (100 µL) to each well. Then, the absorbance of the samples was measured at a wavelength of 570 nm. By replacing the numbers in the formula below, the cell viability following the treatment with the NPs was calculated and compared to the untreated cells.

Viability of cells (%) = OD ${}_{\text{sample}}$/OD ${}_{\text{control}}$ $\times$ (100).

For staining, cells were first cultured in 6-well plates, and then, treated with different concentrations of AC-CFS-NPs. After the treatment period, the treatment medium was drained and 1 mL of PBS containing AO (1 µL; 1 mg/mL) and PI (1 µL; 1 mg/mL) dye was added to each well. Finally, images of the cells were taken using a fluorescent inverted microscope.

Firstly, the cells were transferred to a 6-well plate. After incubating for 24 h, the cells were subjected to different concentrations of AC-SLN-CS-FA for 48 h. Afterward, the medium containing the treatment was drained, the cells were washed with PBS, and trypsin was added to each well. After ensuring that the cells were separated, the cell suspension was transferred to separate microtubes and centrifuged. After the supernatant was removed, 300 microliters of PI dye were added to the cell sediment and analyzed by flow cytometry (Becton Dickinson, Maryland, MD, USA) after 15 min of incubation.

The effects of AC-SLN-CS-FA treatment on expression levels of apoptotic genes BAX, Bcl-2, and caspase-8 were evaluated. Table 1 shows the primer list. Cells were treated (1 $\times$ 10${}^6$) with various doses of AC-SLN-CS-FA for 48 h. Next, the cells were separated from the bottom of the flask, and the total RNA was isolated using the Biotech kit. The amount of RNA was assessed by the nanodrop method, and then, cDNA was synthesized using the RNA strand. The reaction mixture was prepared to contain cDNA with water, SYBR green, and special primers, in a volume of 20 microliters, and analyzed using the following temperature–time conditions in the Bio-Rad device: the holding step included 95 °C, 15 min for 1 cycle; the annealing step included 95 °C, 15 s, 58.5 °C, 20 s, and 39 cycles; the extension step included 72 °C, 20 s, and 39 cycles; and the melting step included 65–95 °C for 1 cycle. The delta–delta Ct ($\mathrm{\Delta }$$\mathrm{\Delta }$Ct) method was used to evaluate the relative changes in gene expression. GAPDH was used as a reference gene.

Data were analyzed by one-way ANOVA using the GraphPad Prism software (version 8) (GraphPad Software, Inc., San Diego, CA, USA). Variance was analyzed and means were compared using the LSD method. A significance level of p 0.05 was used to determine the statistical significance of the results.

Fig. 1A and B show acceptable physicochemical properties for AC-SLN-CS-FA. As indicated, NPs had a Z-average of 245.7 $\pm$ 10.7 nm with an average diameter of 86.7 $\pm$ 9.4 nm, reposted by number. The NPs had a uniform size distribution with a PDI of 0.31. The small size of the particles along with the dispersion index of less than 0.5 indicated their stability and lower tendency to accumulate [26, 27]. The zeta potential of the NPs was +21.3 $\pm$ 7.9 mV. The surface charge of the nanoparticles can also provide information on the stability of particles, considering that a higher surface charge of $\pm$ 20 mV in lipid NPs was sufficient to prevent the agglomeration of the particles [28, 29]. Thus, it can be said that the synthesized NPs have ideal stability. In addition to the size, distribution, and surface charge of the NPs, their morphology also affected some of their characteristics, such as EE%, ligand-receptor binding ability, and cellular absorption [30, 31]. Microscopic images (Fig. 1C) show spherical NPs with smooth surfaces. The prepared images also show NPs with dimensions below 100 nm, which are comparable to the DLS results. In Fig. 1D, the characteristic peaks of SLN-NPs are shown as strong peaks at 2862.7, 2919.6, and 1466 cm${}^{-1}$, corresponding to O-H, C-H, and C=C bonds, respectively [32]. The peak present at 1723 cm${}^{-1}$ is related to C=O bonds, which indicates the formation of CS-FA and confirms the successful surface modification of NPs with CS.

Fig. 1.

Characterization of AC-SLN-CS-FA. (A) Results for size. (B) Results for zeta-potential of AC-SLN-CS-FA. (C) FESEM image of AC-SLN-CS-FA; and (D) FTIR spectrum of AC-SLN-CS-FA. PDI, polydispersity index.

According to the formula obtained from the standard curve for AC (y = 0.3631x + 0.0763, R${}^2$: 0.99) and FA (y = 0.8361x + 0.2414, R${}^2$: 0.97), and by replacing the absorption of the supernatant in these formulations, the amounts of AC and free FA were calculated. Next, the EE of AC was reported to be 86.3%, and the amount of bound FA was 63%.

Fig. 2 shows the drug release profile. The release of free AC from the dialysis tubing reached 100% during the first 24 h of the study. The result exhibited 70.5% cumulative drug release after 5 days. This graph shows more drug release during the first 24 h because of the release of drug molecules located on the surface of the NPs [22]. The slow release of the drug after the initial burst release indicates the entrapment of the drug throughout the nanocarrier and the effectiveness of the formulation in the continuous and controlled release of the drug.

Fig. 2.

Release Study. Release of free AC from dialysis tubing and AC from AC-SLN-CS-FA. Data were presented as mean $\pm$ SD.

Investigation of ABTS and DPPH free radicals inhibition rates in the presence of AC-SLN-CS-FA by spectrophotometer absorption method at 734 and 517 nm wavelengths revealed the high inhibitory effects of AC-SLN-CS-FA on DPPH free radicals. As shown in the graph (Fig. 3), NPs are capable of inhibiting both free radicals in a concentration-dependent manner. Increasing the concentration of the NPs to about 250 µg/mL caused more than an 80% inhibition of both free radicals; however, in low concentrations, a greater inhibition of DPPH free radicals was observed. These results show the antioxidant power of NPs in inhibiting free radicals and preventing damage by oxidative stress.

Fig. 3.

Percentage inhibition of ABTS and DPPH free radicals in the presence of AC-SLN-CS-FA. Data were presented as mean $\pm$ SD. (n = 3, *** p 0.001). DPPH, 2,2-diphenyl-1-picrylhydrazyl; ABTS2, 2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid).

Since the MTT reaction utilizes the succinate dehydrogenase enzyme of living cells and the production of purple formazan crystals, it was used to evaluate the percentage of cell viability in the presence of AC-SLN-CS-FA. The cytotoxicity effects of free AC were evaluated against normal HFF and MCF-7 cancer cells, and no toxicity was observed in the free AC data compared to the HFF cells. However, significant cytotoxicity was observed in the effects of free AC against MCF-7 cells (p 0.01 and p 0.001, respectively). The results shown in Fig. 4A–D reveal the higher inhibitory effect that AC-SLN-CS-FA had against MCF-7 cells compared to normal cells. Additionally, AC-SLN-CS-FA exhibited no detectable toxicity effect on HFF cells at any of the investigated concentrations, whereas, AC-SLN-CS-FA had an inhibitory IC${}_{50}$ of ~20 µg/mL in the MCF-7 cells. These results show the selective toxicity of AC-SLN-CS-FA on malignant cells and its safe application on normal cells.

Fig. 4.

Cytotoxicity study. MTT results: (A) Cytotoxicity effects of AC against HFF cells; (B) cytotoxicity effects of AC against MCF-7 cells; (C) cytotoxicity effects of AC-SLN-CS-FA against HFF cells; (D) cytotoxicity effects of AC-SLB-CS-FA against MCF-7 cells. Data were presented as mean $\pm$ SD. (n = 3, ** p 0.01 and *** p 0.001).

Fluorescent dyes were used to examine the cell morphologies and their changes during treatment. AO dye can penetrate living cells, and after penetration, it causes a green color to be emitted, while PI penetrates damaged cells and causes a red color to be emitted. Fig. 4 shows the morphological images of the stained cells. As can be seen, all the cells in the untreated group are green and have a normal morphology; in the treated groups, however, the number of cells emitting an orange color gradually increases as the treatment concentration increases, thereby indicating an increase in the presence of apoptotic cells. Furthermore, the morphological change of the cells to a spherical state is a characteristic of apoptotic cells, and can be observed in Fig. 5. The staining results exhibit that apoptosis increases in accordance with the increasing treatment concentrations.

Fig. 5.

AO/PI double-staining. The increase in red-color-emitting cells during the increase in treatment concentrations indicates the proapoptotic effects of AC-SLN-CS-FA on MCF-7 cells.

Fig. 6 shows that 4.15%, 65%, 9.17%, and 15.8% of the cells in the control group are in the sub-G1, G1, S, and G2–M phases, respectively. However, in the AC-SLN-CS-FA-treated cells, a decrease is observed in the percentage of cells in all phases of the cell cycle, except the sub-G1 phase, which shows the effect of AC-SLN-CS-FA on increasing the percentage of cells in this phase. Since sub-G1 phase cells are known to be apoptotic cells, these findings show the effect of AC-SLN-CS-FA on the induction of apoptosis in treated cells. No arrest was observed in any cell cycle stages other than sub-G1 in the treated cells.

Fig. 6.

The flow cytometry diagram shows the cells arresting in the sub-G1 phase after treatment with AC-CFS-NPs.

The results of the RNA analysis showed a change in BAX and Bcl-2 gene expressions, leading to the activation of the internal apoptotic pathway. Upregulation of the BAX gene and downregulation of Bcl-2 represents the activation and suppression of pro- and antiapoptotic genes, respectively, thereby indicating the induction of apoptosis in the treated cells. The extrinsic apoptotic pathway was investigated by analyzing caspase 8 gene expression levels. The caspase 8 expression levels showed no significant change compared to the control, which indicates the inactivity of the external pathway and the occurrence of apoptosis from the internal pathway (Fig. 7).

Fig. 7.

Real-time qPCR analysis. Molecular analysis showed an increase in (A) BAX gene expression and a decrease in (B) Bcl-2 expression along with no change in (C) caspase 8 expression using the delta–delta Ct ($\mathrm{\Delta }$$\mathrm{\Delta }$Ct) method (mean $\pm$ SD, n = 3, * p 0.05, ** p 0.01, and *** p 0.001; 0, 15, 20, and 25 are the concentrations of AC-SLN-CS-FA in µg/mL).