Solamargine (SM; CAS No., 20311-51-7; purity, >98%) was purchased from MedChemExpress (Monmouth Junction, NJ, USA).

The chemical precipitation method (23) was adopted to prepare Fe₃O₄. The molar ratio of Fe²⁺:Fe³⁺=1:2 (a certain amount of FeSO₄ and FeCl₃) was dissolved in distilled water. Next, 4 mol/ml NaOH (pre-heated to 60°C) was incubated with FeSO₄ and FeCl₃ mixture with mechanical agitation. The Fe₃O₄ precipitate was then formed. The lecithin/Fe₃O₄ nanoparticle (quality ratio, 10:1) mixture was dissolved in water (the volume of water was equal to 1/5 of the mixture). In brief, the nanoparticles were added into the SM solution and underwent ultrasonic treatment for 6 h. The mixture was then further mixed with ether solution, which contained lecithin and cholesterol. Following rotation for 1 min, the mixture underwent rotary evaporation in a water bath at 37°C. Following emulsion being performed three times, the magnetic nanoparticles were aggregated. Fe₃O₄-SM was separated and purified. A transmission electron microscope (magnification, ×120,000) (2000 FX; JEOL, Ltd., Tokyo, Japan) was used to record TEM images. X-ray diffraction (XRD) was performed using Rigaku D/max 2550V (Rigaku Corporation, Tokyo, Japan). Particle size (PCS) analysis was performed using LS13320 (Beckman Coulter, Inc., Brea, CA, USA). The SM content in Fe₃O₄-SM nanocomplex was determined using inductively coupled plasma-mass spectrometry (ICPMS; Optima 5300DV, PerkinElmer, Inc., Waltham, MA, USA).

The pancreatic cancer BxPC-3 cell line (American Type Culture Collection, Manassas, VA, USA) was cultured in RPMI-1640 medium (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.) in a humidified incubator with 5% CO₂ at 37°C. For the subsequent experiments, the cell grouping was as follows: Mock group, tumor cells without any treatment; SM group, tumor cells were treated with SM for 16 h; and Fe₃O₄-SM group, cells were treated with Fe₃O₄-SM for 16 h. The final concentration of SM in the latter two groups was set at 4.8 µM.

The cells were seeded into 96-well plates at a density of 1x10⁴ cells/well, prior to being treated with SM or Fe₃O₄-SM for 16 h. The final concentrations of SM were 2.4, 4.8 and 9.6 µM. Cell viability was determined using a CCK-8 assay (Beyotime Institute of Biotechnology, Haimen, China). Absorbance was read on an automated plate reader (Bio-Rad Laboratories, Inc., Hercules, CA, USA) at 450 nm. Cell growth inhibition is presented as the percentage of untreated controls. Growth inhibition was also determined when the cells were treated with SM or Fe₃O₄-SM (final concentration of SM, 4.8 µM) for 12, 18, 24 and 48 h. All determinations were performed in triplicate.

Apoptosis was tested using an Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) kit, according to the manufacturer's protocol. In brief, the tumor cells treated with SM or Fe₃O₄-SM were collected and re-suspended in PBS. Following incubation with Annexin V-FITC for 15 min and with PI for 10 min, cell apoptosis was analyzed using a FACScan flow cytom-eter (BD Biosciences, Franklin Lakes, NJ, USA). In order to determine cell cycle distribution, the cells were first fixed with 4% paraformaldehyde for 30 min at 4°C, prior to being collected and stained with PI for 30 min at 4°C. FACScan (BD Biosciences) with CELLQuest™ software version 3.3 (BD Biosciences) was used for data analysis.

At a density of 2×10⁶ cells/well, the cells were incubated with SM or Fe₃O₄-SM at 37°C for 16 h in a 96-well plate. Colorimetric substrate (Ac-DEVD-pNA) was used to detect the activities of caspase-3. The caspase-3 detection kit (cat. no. G007) was purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). The samples were maintained at 37°C and the optical density at 405 nm was measured using an ELISA reader (Multisken Ascent; Thermo Labsystems, Santa Rosa, CA, USA).

As previously described (24), a scratch assay was performed to detect cell migration. The cells (1.0×10⁶ cells) were seeded onto the dishes and maintained in an incubator for 8 h. A P200 pipette tip was used to scratch the monolayer. Next, the cells were incubated for another 12 h. The gap distance between the scratch edges was measured by cellSens software (Olympus Corporation, Tokyo, Japan) to determine the cell migration ability.

The invasive ability of the cells was determined using a Transwell assay with Matrigel. In brief, the cells were starved overnight. The cells at a density of 2×10⁵ cells/ml were seeded with Matrigel (BD Biosciences) into the upper chamber of the Transwell. The upper chamber was filled with RPMI-1640 medium without FBS. RPMI-1640 medium containing 15% FBS was plated into the lower chamber of the Transwell. The Transwell was maintained at 37°C for 24 h, allowing the cells to invade into the lower chamber. The invaded cells were harvested and then fixed with 4% paraformaldehyde at 4°C for 30 min. The cells were then stained with 0.1% crystal violet dye for 20 min at room temperature. The cells were observed under an inverted microscope (magnification, ×40).

RNAiso Plus (Takara Bio, Inc., Otsu, Japan) was used to isolate total RNA. The RNA was reverse transcribed using M-MLV reverse transcriptase (Promega Corporation, Madison, WI, USA). The synthesized cDNA was subject to subsequent PCR quantification. PCR was performed using SYBR qPCR mix (Toyobo Life Science, Osaka, Japan) on an iCycler (Bio-Rad Laboratories, Inc.). The thermocycling conditions were as follows: 95°C for 3 min; 33 cycles of 95°C for 15 sec, 60°C for 30 sec; a final extension at 72°C for 10 min. The 2^−∆∆Cq method was used for data analysis (25). β-actin mRNA expression was used as a reference. The Primer-BLAST-based sequences are listed in Table I.

Cells were lysed in NP40 lysis buffer (Beyotime Institute of Biotechnology) containing protease inhibitors. Following centrifugation at 12,000 × g for 5 min at 4°C, the protein concentration was detected using a bicinchoninic acid protein quantitative analysis kit (Thermo Fisher Scientific, Inc.). Proteins (20 µg) was separated by 8% SDS-PAGE and transferred onto polyvinylidene difluoride membranes. In order to block non-specific binding, the membranes were incubated with 5% skimmed milk at room temperature for 2 h. The membranes were incubated with primary antibodies against the following: XIAP (cat. no. ab28151; dilution, 1:100), survivin (cat. no. ab208938; dilution, 1:1,000), Ki-67 (cat. no. ab16667, 1:100), PCNA (cat. no. ab29; dilution, 1:200), cyclin D1 (cat. no. ab134175; dilution, 1:10,000), MMP-2 (cat. no. ab92536; dilution, 1:2,000), MMP-9 (cat. no. ab38898; dilution, 1:1,000), TIMP-2 (cat. no. ab1828; dilution, 1:1,000), p-Akt (cat. no. ab131443; dilution, 1:800), Akt (cat. no. ab188099; dilution, 1:2,000), p-mTOR (cat. no. ab109268; dilution, 1:1,000), mTOR (cat. no. ab2732; dilution, 1:2,000) and GAPDH (cat. no. ab8245; dilution, 1:1,000; all Abcam, Cambridge, UK) at 4°C overnight. The next day, the membranes were incubated with a goat anti-rabbit horseradish peroxidase-conjugated IgG H&L secondary antibody (cat. no. ab6721; dilution, 1:2,000; Abcam). Bands were developed on X-ray film by enhanced chemiluminescence (Beyotime Institute of Biotechnology). The density of the blots was read by using the Quantity One software version 2.4 (Bio-Rad Laboratories, Inc.).

The Balb/c nude mice (n=25, 4-6 weeks old, 12-15 g, male) were obtained from Shanghai Animal Center. Animals were housed at 22°C with 40-50% humidity. After being acclimatized, the animals were approved for the experiments. BxPC-3 cells (1.0×10⁷/0.2 ml) were implanted into the hypoderm of the armpit of the mice to produce pancreatic cancer xenografts. When the diameter reached 3-4 mm, the mice were distributed into the following 4 groups (6 animals/group): Mock group, mice were considered as control; saline group, mice were injected with 0.9% saline by caudal vein injection; SM group, mice were injected with SM by caudal vein injection; Fe₃O₄-SM group, mice were injected with 0.2 ml Fe₃O₄-SM and a round magnet (magnet size was 0.3T; diameter, 25.40 mm; thickness 6.35 mm) was placed externally on the mouse (the magnet was tied using a steel wire under the armpit). The final concentration of SM was 4.8 µM (according to the data from the growth inhibition assay). The largest subcutaneous tumor detected in the present study had a diameter of 1.8 cm and no mice exhibited multiple subcutaneous tumors. According to previous studies (26,27), the humane endpoints were judged by the mouse weight loss (>20% of total body weight) or mouse activity assessment (hunching, stationary, ruffling and poor grooming) and mice were euthanized by O₂/CO₂-asphyxiation and dissected. All the protocols in the animal studies were approved by the Ethics Committee of Jiangsu Cancer Hospital (Nanjing, Jiangsu, China).

The nude mice bearing xenografts were injected with saline (0.9%), SM or Fe₃O₄-SM by caudal vein injection. On the seventh, fourteenth and twenty first days after the injection, MRI scans were performed on the mice. MRI was conducted using a 1.5 Tesla scanner (INTERA ACHIEVA 1.5T; Philips Medical Systems) with SENSE-body coil. The tumor exhibited a high-signal intensity on T2-weighted images. The longitudinal diameter (d1) on the sagittal images, the anteroposterior diameter (d2) on the sagittal images and the largest lateral diameter (d3) on the axial images were measured. The diameter-based calculations for tumor volume were calculated as d1 × d2 × d3 × π/6.

The animals were sacrificed and the tumor mass were excised. Following fixing with 4% paraformaldehyde overnight at 4°C, the samples were dehydrated in a graded ethanol series, followed by routine paraffin embedding and sectioning (3-4 µm). The paraffin-embedded tissue sections were subjected to H&E staining and IHC. The sections were subjected to deparaffinization by washing with xylene and rehydration in a graded ethanol series. Slides were boiled by immersing them in a sodium citrate buffer (pH 6.0, 10 mM) and heated to 95°C for antigen retrieval. The cooled sections were then incubated in 3% hydrogen peroxide for 10 min at room temperature. Following incubation with 10% normal goat serum (Beyotime Institute of Biotechnology) for 30 min at 37°C, the sections were maintained with primary anti-Ki-67 antibody (cat. no. ab15580; dilution, 1:100, Abcam) at 4°C overnight. Biotin-labeled secondary antibodies were the incubated with the sections at room temperature for 1 h, prior to incubation with horseradish peroxidase-conjugated streptavidin for 30 min at room temperature. Slides were stained with diaminobenzidine (DAB) for 5 min at room temperature. Next, Mayer's hematoxylin (Sangon Biotech Co., Ltd., Shanghai, China) was incubated with the slides for 2 min at room temperature. The sections were observed using a light microscope (magnification, ×100). Finally, the sections were mounted with neutral balsam (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China).

TUNEL was conducted using TUNEL assay kit (Roche Diagnostics, Basel, Switzerland), according to the manufacturer's protocol. In brief, the tissues were fixed with 4% paraformaldehyde overnight at 4°C. Xylene was used for deparaffinization of the paraffin-embedded sections. Terminal deoxynucleotidyl transferase (TdT) enzyme was incubated with sections for 1 h at 37°C. The sections were incubated with 0.3% H₂O₂ for 3 min at room temperature. The nuclei were stained with 50 µl DAB working solution for 10 min at room temperature. The slides were counterstained with hematoxylin and mounted with neutral balsam. A light microscope was used to observe the cell staining (magnification, ×100). The percentage of apoptotic cells was determined by counting TUNEL-positive cells. The brown staining demonstrated apoptotic cells and the blue staining demonstrated non-apoptotic cells. Five randomly selected fields was observed.

P<0.05 was considered to indicate a statistically significant difference. Prism Graphpad version 6.0 software (GraphPad Software, Inc., La Jola, CA, USA) was used to analyze the data. Data was shown as mean ± standard deviation. One-way analysis of variance followed by Tukey's multiple comparisons test.

As demonstrated in Fig. 1A, the average particle size of Fe₃O₄-MLP was 11.9 nm. The results from XRD demonstrated that the diffraction peaks of Fe₃O₄-MLP were 210, 320, 410, 435, 520, 450 and 533. The results were in line with the characteristic peak of Fe₃O₄ nanoparticles (Fig. 1B). The average particle size of Fe₃O₄-SM was 5-6 nm (Fig. 2A and B).The diffraction peaks of Fe₃O₄-SM were similar to that of Fe₃O₄-MLP (Fig. 2C).

As demonstrated in Fig. 3A, the Fe₃O₄-MLP treatment did not decrease cell growth. Compared with the Mock group, the cell viability was first depressed by SM (P<0.05) and was then further inhibited by Fe₃O₄-SM (P<0.05). The effect of SM occurred in a dose-dependent manner. Furthermore, CCK-8 results demonstrated that during 12-18 h, the cell growth inhibition effect produced by SM was stronger than that generated by Fe₃O₄-SM. However, the inhibitory effect of Fe₃O₄-SM was greater than that of SM after 24 h (Fig. 3B; P<0.05). The results demonstrated that the Fe₃O₄-SM exerted its antitumor effect slowly.

Subsequently, apoptosis and cell cycle progression were determined. Flow cytometric results demonstrated that apoptosis was first induced by SM (P<0.05) and then further enhanced by Fe₃O₄-SM (P<0.05; Fig. 3C and D). Furthermore, compared with the mock group, the cell numbers in the G1 phase during the cell cycle progression were higher in the SM (P<0.05) and Fe₃O₄-SM (P<0.01) groups. However, the cell percentage in G2 phase was reduced in the SM and Fe₃O₄-SM groups (P<0.05). Fe₃O₄-SM was revealed to significantly enhance the effect of SM (Fig. 3E and F; P<0.05). To further confirm the pro-apoptotic effect of Fe₃O₄-SM, the expression of proliferation-related and apoptosis-related molecules was determined. The results demonstrated that the expression of XIAP, survivin, Ki-67, PCNA and cyclin D1 were decreased at the transcriptional and translational levels in the SM and Fe₃O₄-SM groups. Treatment with Fe₃O₄-SM further increased the effect of SM (P<0.05; Fig. 4A–C). The activity of caspase-3 was also tested, and data from ELISA revealed that the active caspase-3 activity was also increased in the Fe₃O₄-SM group, compared with the SM group (P<0.01; Fig. 4D).

Metastasis is also a common and typical phenotype of cancer. Therefore, the effect of Fe₃O₄-SM on cell migration and invasion ability was examined. Fig. 5A demonstrated that the wound thickness was larger in the SM and Fe₃O₄-SM (P<0.01) groups, suggesting that the cell migration ability was depressed. Furthermore, it was demonstrated that the cell migration ability was dampened more by Fe₃O₄-SM than by SM. Additionally, cell invasion was further inhibited by Fe₃O₄-SM, compared with the SM group (P<0.01; Fig. 5B). The expression of molecules associated with tumor metastasis was also detected. The results of the present study demonstrated that the expression of MMP-2 was decreased more in the Fe₃O₄-SM group than in SM group (P<0.05). However, the expression of MMP-9 exhibited no significant changes among these groups. By contrast, the expression of TIMP-2 was higher in the Fe₃O₄-SM group than that in the SM group (P<0.05; Fig. 5C–E).

To illustrate the underlying mechanisms, the activity of the Akt/mTOR signaling pathway was determined. The results revealed that the expression of p-Akt and p-mTOR was decreased by SM (P<0.05), compared with the Mock group. Furthermore, treatment with Fe₃O₄-SM further enhanced the effect of SM (P<0.01; Fig. 6A and B).

The effect produced by Fe₃O₄-SM was estimated in vivo. MRI was employed to assess tumor size and volume. MRI images revealed that the tumor volume increased as time progressed. However, the tumor growth rates in the SM and Fe₃O₄-SM groups were slower than those in the mock and saline groups. After 21 days, the tumor volume, size and weight in the Fe₃O₄-SM group was the smallest and lightest among these four groups (P<0.01; Figs. 7A and B and 8A). Furthermore, H&E staining demonstrated that, compared with the SM group (Fig. 8B), the tumor malignance was first decreased by SM (P<0.05), and then further inhibited by Fe₃O₄-SM (P<0.05). To further confirm the antitumor effect of Fe₃O₄-SM, the expression of Ki-67 was determined by IHC. The assay demonstrated that the expression of Ki-67 was suppressed by SM and Fe₃O₄-SM (P<0.01; Fig. 9A). In addition, the TUNEL assay demonstrated that SM significantly induced apoptosis, and Fe₃O₄-SM further enhanced the apop-tosis mediated by SM (P<0.01; Fig. 9B).