Astragaloside II-loaded neutrophil nanocarrier drug delivery system for liver cancer treatment

Cell culture

The human liver cancer cell line Huh-7 (CL-0120) was purchased from Procell Life Science & Technology Co., Ltd. (Wuhan, China). Huh-7 cells were cultured in DMEM complete medium supplemented with 10% fetal bovine serum (FBS), 100 μg/mL streptomycin, and 100 U/mL penicillin. Cells were maintained at 37°C in a humidified atmosphere with 5% CO₂. Experiments were conducted using cells in the logarithmic growth phase.

Isolation and culture of neutrophils

Human bone marrow-derived neutrophils (IMP-H188) were purchased from SHRBIO (Nanjing, China). The purchased neutrophils were suspended in RPMI 1640 medium supplemented with 1% HEPES buffer (Gibco, USA). Freshly isolated neutrophils were seeded in 6-well plates (3 × 10⁶ cells per well) and stimulated with 100 nM phorbol 12-myristate 13-acetate (PMA) (Sigma-Aldrich, USA) to induce the formation of NETs. ¹⁶

CCK-8 assay for cell viability

Huh-7 cells were seeded in 96-well plates (1 × 10⁵ cells/mL), with 100 μL per well. After 24 h of incubation at 37°C and 5% CO₂, cells were treated with media containing different concentrations of ASII (20, 40, 80, and 120 μM). After 24 h, 5% CCK-8 reagent (Elabscience, China) was added to each well, and cells were incubated for an additional 2–4 h. Absorbance was measured at 450 nm using a microplate reader (Thermo Fisher, USA), and cell viability and the half-maximal inhibitory concentration (IC₅₀) of the drug were calculated.

Apoptosis detection

Cell apoptosis was assessed using an Annexin V-FITC/PI Apoptosis Detection Kit (Beyotime, China). Huh-7 cells treated with ASII or co-cultured with PG@AS-Neu (including both suspended and adherent cells) were collected by centrifugation, gently resuspended in PBS, and counted. According to the manufacturer’s protocol, 1 × 10⁵ resuspended cells were centrifuged at 1000 rpm for 5 min, and the supernatant was discarded. Cells were resuspended in 195 μL Annexin V-FITC binding buffer, followed by the addition of 5 μL Annexin V-FITC and 10 μL propidium iodide (PI) staining solution. After gentle mixing, cells were incubated in the dark at room temperature for 10–20 min. Apoptosis levels were analyzed using a BD LSR Fortessa II flow cytometer (Agilent, USA).

Scratch wound assay

Huh-7 cells were seeded in 6-well plates (1 × 10⁶ cells per well) and cultured for 12 h. A scratch was made using a 10 μL pipette tip, and detached cell clusters were removed by washing with PBS. After replacing the medium with serum-free medium containing ASII, cells were cultured for an additional 24–48 h (with serum-free medium without ASII as the control). Cell migration distance was photographed using an IX73 microscope (Olympus, Japan), and migration rates were calculated for statistical analysis.

Transwell assay

Transwell chambers with 8 μm pores (Corning, USA) were used for cell migration and invasion assays. For the migration assay, 1 × 10⁵ Huh-7 cells were resuspended in 100 μL serum-free medium and added to the upper chamber. The lower chamber was filled with 600 μL medium containing 20% FBS as a chemoattractant. After 24 h of incubation, cells that migrated through the membrane were fixed with 4% paraformaldehyde for 30 min and stained with 0.1% crystal violet for 10 min. For the invasion assay, the upper chamber was pre-coated with diluted Matrigel before cell seeding. Non-migrated cells and excess dye were removed using a cotton swab, and migrated cells were photographed and counted using an IX73 microscope (Olympus, Japan).

Preparation of PG@AS-Neu

First, 40 mg of PEG-PLGA was dissolved in 2 mL of chloroform and sonicated until the solution became clear. Then, 200 μL of deionized water was added, and the mixture was sonicated for 5–10 min to form a primary emulsion. Next, 4 mL of 4% polyvinyl alcohol (PVA) solution was added, and the mixture was sonicated again for 5–10 min to form a double emulsion. The organic solvent was evaporated by stirring at room temperature for 5 h. The resulting product was collected by centrifugation at 10,000 rpm for 15 min and washed 2–3 times with deionized water to obtain blank nanomicelles.

A stock solution of ASII was prepared by dissolving 2 mg of ASII in 1 mL of DMSO. Under continuous stirring, 1 mL of the ASII stock solution was slowly added to 5 mL of blank nanomicelle solution (2 mg/mL) to obtain the PG@AS solution. The PG@AS solution was added to a neutrophil culture dish, allowing the neutrophils to uptake the PG@AS nanoparticles. After 12 h, the final PG@AS-Neu material was obtained.

Drug release

The release of ASII was quantified using high-performance liquid chromatography (HPLC). ASII was separated using a C18 column with a mobile phase of acetonitrile:water (32%:68%) and detected at a UV absorption wavelength of 203 nm. A standard curve of ASII was established using HPLC to calculate the drug loading capacity (Figure 3(a)). PG@AS-Neu was encapsulated in a dialysis bag (1000 Da) and immersed in solutions with pH 7.4, pH 5.5, and pH 5.5 + IL-6. At different time points, the solutions were collected, and the absorbance of ASII was measured using HPLC. The cumulative release percentage was calculated as follows:

Hemolysis assay

Fresh blood collected from adult Kunming mice placed in EP tube was mixed with ten volumes of 0.9% NaCl solution, shaken for several minutes, and centrifuged at 1500 rpm/min for 10 min at 4°C. The supernatant was discarded, and the red blood cells (RBCs) were resuspended in 0.9% NaCl solution. This process was repeated until the supernatant became clear, and a 2% RBC suspension was prepared. For the experimental groups, 0.5 mL of PEG-PLGA nanoparticle solutions at different concentrations was mixed with 0.5 mL of the 2% RBC suspension. Negative and positive controls were prepared by mixing 0.5 mL of the 2% RBC suspension with 0.5 mL of deionized water and 0.9% NaCl solution, respectively. The mixtures were incubated at 37°C for 0.5 h, followed by centrifugation at 1500 rpm/min for 15 min at 4°C. Hemolysis was observed and recorded.

Data analysis

All data are presented as mean ± standard deviation. Each experiment was performed in triplicate, and at least three independent experiments were conducted. Statistical analysis was performed using GraphPad Prism 7.0 (La Jolla, CA, USA) and SPSS 18.0 (SPSS, Chicago, IL, USA). One-way analysis of variance was used to determine statistical significance, with p < 0.05 (*) or p < 0.05 (#) considered statistically significant.

Cytotoxic effects of ASII on liver cancer cells

As the primary active component of the nanomaterial, the inhibitory effects of ASII on liver cancer were initially investigated in vitro. As shown in Figure 1(a), treatment of Huh-7 liver cancer cells with increasing concentrations of ASII resulted in a significant dose-dependent reduction in cell viability. IC₅₀ of ASII for Huh-7 cells was determined to be 62.69 μM, providing a critical reference for subsequent nanomaterial preparation and dosage optimization. To investigate the effects of ASII on Huh-7 cell apoptosis, Annexin V/PI staining was performed. ASII treatment significantly increased the apoptosis rate of cancer cells from 5% in the control group to 16% (Figure 1(b)). These findings indicated that ASII exhibited potent cytotoxic effects on liver cancer cells. Additionally, the impact of ASII on cancer cell migration and invasion was evaluated using scratch wound and Transwell assays. The scratch wound assay (Figure 1(c)) revealed that ASII treatment significantly inhibited cancer cell migration. At 48 h, the tumor cell migration rate in the ASII group was only 33%, significantly lower than that in the PBS group. The Transwell assay (Figure 1(d)) demonstrated that ASII treatment reduced cancer cell migration and invasion by approximately 50%. Collectively, these results indicated that ASII exerted significant cytotoxic effects on liver cancer cells and effectively inhibited their migration and invasion.

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Figure 1.

ASII exhibits potent anticancer effects in vitro. (a) CCK-8 assay determining the IC₅₀ of ASII on Huh-7 liver cancer cells. (b) Annexin V/PI staining showing apoptosis in cancer cells after ASII treatment. (c) Scratch wound assay evaluating the migratory ability and quantification of migration rates of cancer cells at 24 h and 48 h after ASII treatment. (d) Transwell assay assessing changes in cancer cell migration and invasion after ASII treatment. *p < 0.05 vs. PBS; #p < 0.05 vs. Neutrophil.

Preparation and characterization of ASII-Loaded neutrophil nanocarrier (PG@AS-Neu)

In previous experiments, we validated the cytotoxic effects of ASII on cancer cells. However, due to its poor solubility and short circulation time in vivo, ASII is rapidly metabolized and struggles to accumulate at tumor sites. To address this, we constructed PEG-PLGA nanomicelles to encapsulate ASII and utilized neutrophils for targeted drug delivery. The morphology of the PEG-PLGA nanomicelles was observed using transmission electron microscopy (TEM). As shown in Figure 2(a), the nanomicelles exhibited a large hollow hydrophobic structure, suitable for encapsulating the hydrophobic drug ASII. Figures 2(b) and 2(c) show that the nanomicelles had an appropriate hydrodynamic diameter of approximately 220.6 nm and a zeta potential of −25.1 mV. Finally, the drug-loaded nanomicelles were internalized by neutrophils to obtain the final carrier, PG@AS-Neu.

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Figure 2.

Characterization of PG@AS-Neu. (a) TEM image of PEG-PLGA nanomicelles. (b) Hydrodynamic diameter of PEG-PLGA nanomicelles. (c) Zeta potential of PEG-PLGA nanomicelles.

Drug release and biosafety evaluation of the nanocarrier

Next, we evaluated the drug release performance of the nanocarrier under different conditions. First, based on the standard curve (Figure 3(a)), we calculated the encapsulation efficiency and drug loading rate of the PEG-PLGA carrier for ASII to be 82.3% and 12.6%, respectively. Additionally, the effective drug concentration in PG@AS-Neu was determined based on the uptake of PG@AS by neutrophils. As shown in Figure 3(b), under pH 7.4 conditions, the drug release rate was only 8.41% at 24 h. In contrast, under pH 5.5 conditions, the drug release rate significantly increased to 29.64% at 24 h. Notably, in the presence of inflammatory factors at pH 5.5, the release rate of ASII reached 74.32% at 24 h, indicating that the nanocarrier can selectively release drugs in inflammatory environments. To assess the biosafety of the nanomicelles, hemolysis assays were performed at different concentrations. As shown in Figures 3(c) and 3(d), no significant hemolysis was observed in any group. Even at a nanomicelle concentration of 2 mg/mL, the hemolysis rate remained below 5%, demonstrating the excellent biosafety of the prepared nanomicelles.

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Figure 3.

Drug release and biosafety evaluation of PG@AS-Neu. (a) ASII standard curve. (b) Drug release profile of PG@AS-Neu. (c) Hemolysis assay of PEG-PLGA. (d) Hemolysis rate of PEG-PLGA.

Significant anticancer effects of the nanocarrier in vitro

After successfully preparing the nanocarrier PG@AS-Neu, we investigated its antitumor efficacy in vitro. Huh-7 liver cancer cells were co-cultured with neutrophils, PEG-PLGA, ASII, or PG@AS-Neu, and cell viability was assessed using the CCK-8 assay. As shown in Figure 4(a), neutrophil and PEG-PLGA treatment did not significantly affect cancer cell viability, whereas both ASII and PG@AS-Neu treatments significantly reduced cell viability. Notably, although both ASII and PG@AS-Neu exhibited potent anticancer effects, no significant difference was observed between the two groups. This suggested that the cytotoxicity of PG@AS-Neu primarily originated from ASII and that the preparation process did not compromise the anticancer activity of ASII. Similar results were observed in apoptosis assays (Figures 4(b) and 4(c)). After treatment with neutrophils, PEG-PLGA, ASII, and PG@AS-Neu, the apoptosis rates were approximately 4.6%, 4.7%, 14.1%, and 14.5%, respectively, with no significant difference between the ASII and PG@AS-Neu groups. Finally, the Transwell assay (Figure 4(d)) revealed that PG@AS-Neu treatment reduced cancer cell migration and invasion by approximately 50%. These results demonstrated that the ASII-loaded neutrophil nanocarrier exhibited potent anticancer effects in vitro, and the nanomaterial preparation process did not affect the anticancer activity of ASII.

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Figure 4.

Neutrophil nanocarrier exhibits significant anticancer effects in vitro. (a) CCK-8 assay evaluating the viability of Huh-7 liver cancer cells after co-culture with PBS, Neutrophil, PEG-PLGA, ASII, or PG@AS-Neu. (b) Annexin V/PI staining showing apoptosis levels in Huh-7 cells after different treatments. (c) Quantitative flow cytometry results of apoptosis assays. (d) Transwell assay evaluating the effects of PG@AS-Neu treatment on cancer cell migration and invasion. *p < 0.05 vs. PBS; #p < 0.05 vs. Neutrophil.