Glypican-1-targeted and gemcitabine-loaded liposomes enhance tumor-suppressing effect on pancreatic cancer

Characterization and in vitro drug release of GPC1-LP (GEM)

As shown in Figure 1A, DLS revealed that the hydrodynamic diameter of GPC1-LP (GEM) was observed to be around 100 nm, which suggested the successful establishment of GPC1-LP (GEM). Next, the amount of GEM released from LP (GEM) and GPC1-LP (GEM) was examined at pH 7.4 and pH 5.0. Cumulative drug release profiles revealed that both LP (GEM) and GPC1-LP (GEM) exhibited a burst release of GEM within 5 h and a slow release from 5 h to 24 h at pH 7.4 (blood plasma) and pH 5.0 (tumor endocytic compartment) (Figure 1B). Notably, almost 80 % of GEM was released from both LP (GEM) and GPC1-LP (GEM) within 24 h at pH 5.0, which was significantly higher than that at pH 7.4 (30%) (Figure 1B), indicating more GEM releasing into the tumor environment.

Figure 1. Characterization of GPC1-LP (GEM). (A) Size distributions of GPC1-LP (GEM) determined by dynamic light scattering. (B) Cumulative drug release profiling of LP (GEM) and GPC1-LP (GEM) in phosphate-buffer saline (PBS, pH 7.4 and pH 5.0).

Expression of GPC1 in PANC-1 cells

GPC1 was highly expressed in pancreatic cancer, as predicted by GEPIA, a tumor-related database(Figure 2A). The expression levels of GPC1 The expression levels of GPC1 were detected in PANC-1, SW1990 and 293T cells. Both qRT-PCR and western blotting showed higher expression level of GPC1 in PANC-1 and SW1990 cells than in 293T cells (Figure 2B, 2C). Consistently, cell immunofluorescence showed the positive expression of GPC1 in PANC-1 but not 293T cells (Figure 2D). These results suggested that PANC-1 and SW1990 cells were GPC1-overexpressed cells.

Figure 2. Expression of GPC1 in 293T cells, sw1990 cells and PANC-1 cells. (A) GPC1 expression in pancreatic cancer was predicted by GEPIA. The mRNA and protein levels of GPC1 in 293T cells, SW1990 and PANC-1 cells by (B) qRT-PCR and (C) western blotting. (D) The expression of GPC1 in 293T cells and PANC-1 cells by cell immunofluorescence. bar = 20 μm. *p<0.05,***p<0.001.

In vitro anti-proliferation effect of GPC1-LP (GEM)

Cell proliferation and apoptosis were used to evaluate the anti-tumor effect of GPC1-LP (GEM). MTT assay showed that 293T cells treated with LP (GEM) exhibited similar cell viability with 293T cells treated with GPC1-LP (GEM) at 24 h and 48 h, while both were reduced compared with cells treated with free GEM (Figure 3A). Compared with PANC-1 cells with free GEM, cell viability was decreased in PANC-1 cells treated with LP (GEM), and cells treated with GPC1-LP (GEM) showed lower cell viability than cells treated with LP (GEM) at 24 h and 48 h (Figure 3A). Consistently, colony formation results also revealed that compared with control cells, the clone number was decreased in 293T cells treated with free GEM (Figure 3B). The clone number was similar between 293T cells treated with LP (GEM) and GPC1-LP (GEM), while which were decreased compared to 293T cells treated with free GEM (P < 0.05, Figure 3B). In PANC-1 and SW1990 cells, the clone number in cells treated with GPC1-LP (GEM) was the lowest, followed by cells treated with LP (GEM), cells treated with LP (GEM), cells treated with free GEM, and control cells (P < 0.05, Figure 3B). These data indicated that GPC1-LP (GEM) had superior anti-proliferation effect than LP (GEM) and free GEM in PANC-1 and SW1990 cells. (Figure 3).

Figure 3. GPC1-LP (GEM) inhibited cell growth in293T cells, PANC-1 cells and SW1990 cells. (A) Cell viability of 293T cells, PANC-1 cells and SW1990 cells treated with different doses of GEM, LP (GEM) and GPC1-LP (GEM) at 24h and 48 h by MTT assay. (B) Clone number of 293T cells, PANC-1 cells and SW1990 cells treated with PBS (control), GEM, LP (GEM) and GPC1-LP (GEM) by colony formation assay. *p<0.05.

In vitro pro-apoptosis effect of GPC1-LP (GEM)

Flow cytometry analysis found similar cell apoptosis in control cells, cells treated with LP, and cells treated with GPC1-LP, while the rate of apoptotic cells was increased in cells with free GEM compared with control cells, cells with LP, and cells with GPC1-LP; meanwhile, compared with cells with free GEM, cells with LP (GEM) showed the increased rate of apoptotic cells, and then GPC1-LP (GEM) treatment further increased the rate of apoptotic cells (Figure 4A). Consistently, western blotting showed that compared with control cells, free GEM treatment distinctly inhibited Bcl-2 level as well as promoted the expression of Bax, cleaved-caspase-3 and cleaved-caspase-9 (P < 0.01), and both LP (GEM) and GPC1-LP (GEM) further inhibited Bcl-2 level as well as promoted the expression of Bax cleaved-caspase-3 and cleaved-caspase-9, especially GPC1-LP (GEM) (P < 0.05, Figure 4B). These findings suggested that superior pro-apoptosis effect of GPC1-LP (GEM) than LP (GEM) and free GEM in PANC-1 and SW1990 cells.

Figure 4. GPC1-LP (GEM) induced cell apoptosis in PANC-1 cells and SW1990 cells. (A) Cell apoptosis rate of PANC-1 cells treated with PBS (control), LP, GPC1-LP, GEM, LP (GEM) and GPC1-LP (GEM) by flow cytometry analysis. (B) The expression of apoptosis-related proteins (Bcl-2, Bax, cleaved-caspase-3, and cleaved-caspase-9) in PANC-1 cells and SW1990 cells treated with PBS (control), GEM, LP (GEM) and GPC1-LP (GEM) by western blotting. *p<0.05.

In vivo therapeutic effect of GPC1-LP (GEM)

In vivo experiments showed that the bodyweight of mice with different treatments, including LP, GPC1-LP, GEM, LP (GEM) and GPC1-LP (GEM), was in comparison to that of the orthotopic PDAC mice (Figure 5A). Notably, the tumor weight was similar in control cells, cells with LP, and cells with GPC1-LP, while the tumor weight was lowest in cells with GPC1-LP (GEM), followed by cells with LP (GEM) and free GEM (Figure 5B), which suggested a better anti-tumor effect of GPC1-LP (GEM) on orthotopic PDAC mice. The correlation between GPC1 mRNA and hypoxia in pancreatic cancer was analyzed by TCGA database, the result showed that GPC1 level was positive correlation with hypoxia (Figure 5C). Moreover, we detected the mRNA expression of the hypoxic-related genes in tumor tissues through qRT-PCR, and found that GPC1-LP (GEM) could significantly inhibit the expression of the hypoxic-related genes (Figure 5D).

Figure 5. GPC1-LP (GEM) inhibited tumor growth of the orthotopic pancreatic cancer mice. (A) The weight changes of the orthotopic pancreatic cancer mice treated with saline (control), LP, GPC1-LP, GEM, LP (GEM) and GPC1-LP (GEM). (B) The tumor weight of the orthotopic pancreatic cancer mice treated with saline (control), LP, GPC1-LP, GEM, LP (GEM) and GPC1-LP (GEM). (C) The correlation between GPC1 mRNA and hypoxia in pancreatic cancer was detected by TCGA database. (D) qRT-PCR was used to detect mRNA expression levels of tumor tissues and genes associated with hypoxia. *p<0.05.

Preparation of GPC1-LP (GEM)

LP (GEM) was firstly prepared. In brief, 120 mg of hydrogenated soybean phospholipid (HSPC) and 34 mg of cholesterol were dissolved in 6 mL of chloroform, and 2 mL of GEM hydrochloride solution (3 mg/mL) was added into the above lipid solution. Subsequently, a uniform emulsion was formed by the ultrasonic shaking for 10 min, and then chloroform was removed by reduced pressure distillation at 45°C for 1h. After the temperature was raised to 60°C, 2 mL of isothermal PBS solution was added to hydrate for 1 h, followed by ultrasonication for 3 min, and repeated freeze-thaw for 4 cycles. Lastly, LP (GEM) was were obtained by incubating with 12 mg of carboxylated distearoyl phospho-ethanolamine-polyethylene glycol (DSPE-PEG2000) for 15 min in a 60°C water bath. Similarly, unloaded LPs were prepared as described above but without the addition of GEM.

Afterwards, 900 μL of LP (GEM) solution was incubated with 100 μL of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (10 mg/mL) and 50 μL of N-Hydroxysuccinimide (10 mg/mL) by shaking at 400 rpm in a constant temperature shaker at 25° C for 3 h, then, 10 μL of anti-GPC1 antibody (10 nmol, Abcam, Cambridge, MA, USA) was added dropwise into the above mixture. After shaking for 2 h, the mixture was blocked with bovine serum albumin (BSA), and GPC1-LP (GEM) was obtained by shaking for 10 h.

Material characteristics

The size distribution of GPC1-LP (GEM) were observed by dynamic light scattering (DLS). DLS was performed by Zetasizer Nano Z (Worcestershire, UK).

In vitro drug release analysis

The drug release profiles of LP (GEM) and GPC1-LP (GEM) at different pH values were analyzed. In brief, 200 μL LP (GEM) and GPC1-LP (GEM) were separately loaded into a dialysis bag (molecular retention of 8,000-12,000 Da), and the dialysis bag was immersed in 35 mL of PBS buffer (pH 7.4 and 5.0, respectively). PBS buffer with pH 7.4 simulated blood plasma environment and pH 5.0 simulated tumor endocytic compartment. The entire dialysis system was shaken at 200 rpm in a constant temperature shaker at 37°C in the dark. Subsequently, 1 mL of dialysate was taken at 0.5 h, 1 h, 2 h, 4 h, 6 h, 9 h, 12 h, and 24 h, respectively. Finally, the concentration of GEM in the dialysate was determined by UV spectrophotometer, and the in vitro release profile was calculated.

Cell culture

Human pancreatic cancer cell line PANC-1 and human embryonic kidney cell line 293T were obtained from Shanghai Obio Technology Co., Ltd (China), and maintained in RPMI-1640 medium (Gibco, Carlsbad, CA, USA) containing 10% fetal bovine serum (Gibco) with standard incubation conditions (5% CO₂ and 37°C).

Cell immunofluorescence

Expression of GPC1 was detected in PANC-1 and 293T cells. PANC-1 and 293T cells were fixed in 4% paraformaldehyde overnight, respectively. Then cells were incubated with GPC1 antibody, followed by the incubation of FITC-labelled second antibody. Cell nucleus was stained with DAPI for 5 min. Meanwhile, cells without FITC-labelled second antibody were served as control group. Last, inversion fluorescence microscope was used to observe the expression of GPC1.

MTT assay

PANC-1 and 293T cells were grown in 96-well plates for 24 h, and then incubated with GEM, LP (GEM) and GPC1-LP (GEM) BTZ (with GEM concentrations of 2, 5, 10, 20, 50 and 100 μg/mL), respectively, for 24 h and 48 h. Next, MTT (10 μL, Sigma, St Louis, MI, USA) was added to incubate with cells for 4 h, and dimethyl sulfoxide (150 μL, Sigma) was then used to dissolve formazan precipitates. The zero hole (medium, MTT, DMSO) and blank hole were set up. The absorbance at 450 nm were read by microplate reader (Molecular Devices, USA).

Colony formation assay

PANC-1 and 293T cells were grown in 96-well plates for 24 h, and then incubated with PBS (control), GEM, LP (GEM) and GPC1-LP (GEM) BTZ (with GEM concentrations of 10 μg/mL), respectively, for 24 h. Next, 0.5% (w/v) crystal violet in ethanol was added into cells for 5 min. The mean number of colonies was calculated under 10 different fields of vision.

Cell apoptosis assay

Annexin V-FITC Apoptosis Detection kit was used to evaluate the cell apoptosis. PANC-1 cells were treated with PBS (control), LP, GPC1-LP, GEM, LP (GEM) and GPC1-LP (GEM) BTZ (with GEM concentrations of 10 μg/mL), respectively, for 24 h. Next, cells were digested with Trypsin and washed with PBS, followed by resuspending in 1 × Binding Buffer, and stained with PI and FITC-Annexin V for 15 min at 25°C in the dark. Cells were finally detected using flow cytometer (Beckman Coulter, Fullerton, CA, USA).

Quantitative reverse transcription (qRT)-PCR

Total RNA from PANC-1 and 293T cells was obtained by Trizol (Invitrogen), respectively, and then reverse transcription of RNA was performed using PrimeScript™ RT reagent Kit (Takara, Dalian, China). The qRT-PCR was carried out by the SYBR Premix Ex Taq TM II (Takara) on Rotor-Gene RG-3000A (Corbett Life Science, Sidney, Australia). The PCR primers for GPC1 sense primer was 5′-TACAGAGGAGGCCTCAAAGC-3′ and antisense primer was 5′-GGCATCATGCATCATCTCAG-3′; and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) sense primer was 5′-GTGGATCAGCAAGCAGGAGT-3′ and antisense primer was 5′-AAAGCCATGCCAATCTCATC-3′. The PCR parameters were set as follows: 95°C for 10 min, 40 cycles of 94°C for 30 s, 58°C for 30 s, and 72°C for 15 s. GAPDH were served as the internal control, and data were analyzed with 2-ΔΔCt method.

Western blotting assay

PANC-1 cells were treated with PBS (control), GEM, LP (GEM) and GPC1-LP (GEM) (with GEM concentrations of 10 μg/mL), respectively, for 24 h. Total proteins were obtained using lysis buffer, and then quantitated by bicinchoninic acid kit (Beyotime, Shanghai, China). Following sample separating and transferring into PVDF membranes, membranes were immerged in 5% nonfat milk for 1 h. Next, primary antibodies of GPC1, apoptosis-related proteins (including Bcl-2, Bax, cleaved-caspase-3 and cleaved-caspase-9) (1: 800, Abcam), as well as β-actin (1: 1000, Beyotime), respectively, were used for immunoblotting of the membranes overnight at 4°C. Then, membranes were reacted with secondary antibody (1: 1000, Beyotime) for 2 h keeping in dark at room temperature. The signals were revealed using enhanced chemiluminescence Plus reagent (Beyotime) to image blots. The band quantification was carried out using Image J software.

Animal model

Approval from the local animal Ethics Committee of the animal laboratory center of Zhengzhou University was obtained prior to experiments. Healthy male BALB/c nude mice (4 weeks old and weighing 18-22g, purchased from Charles River, Beijing, China) were used for the following experiments after one week of acclimation. Orthotopic PDAC model was established as previously described [38]. Briefly, PANC-1 cells were suspended in RPMI-1640 medium, and then 5 × 10⁵ PANC-1 cells were subcutaneously injected into mice. After 4 weeks, the tumor could reach to about 8 mm in diameter. Next, tumors were resected into fragments (about 1 mm³), and then transplanted into the pancreas of nude mice. Orthotopic PDAC model was successfully obtained when the diameter of tumor reached to 5-10 mm.

In vivo therapeutic performance

A total of 48 BALB/c nude mice with orthotopic PDAC were randomly and equally divided into 6 groups, and treated, respectively, with saline (control), LP, GPC1-LP, GEM, LP (GEM) and GPC1-LP (GEM) by intravenous injection twice a week for 2 weeks. On day 15, the bodyweight of mice was monitored every 3 days to plot body weight curve and the mice were euthanized. In addition, the tumor was resected and tumor weight was measured.

Statistical analysis

Statistical analysis was carried out using statistical analysis software (SPSS 19.0, SPSS Inc., Chicago, IL, USA). Data were expressed as the mean ± S.D and analyzed by one-way analysis of variance. A P-value of < 0.05 was considered to indicate a statistically significant result.