Co-delivery of Aurora-A inhibitor XY-4 and Bcl-xl siRNA enhances antitumor efficacy for melanoma therapy

Materials

N-[1-(2,3-dioleoyloxy) propyl]-N,N,N-trimethylammonium methyl sulfate (DOTAP) and cholesterol were purchased from Avanti Polar Lipids (Alabaster, AL, USA). DMEM and MTT were obtained from Sigma-Aldrich Co., (St Louis, MO, USA) and used without further purification. Chloroform and other organic solvents were purchased from ChengDu KeLong Chemical Co., Ltd (ChengDu, People’s Republic of China) Hoechst 33258 was purchased from Beyotime, (Shanghai, People’s Republic of China). XY-4 was previously synthesized in our laboratory. Mouse B16 melanoma cell line was purchased from the American Type Culture Collection (Manassas, VA, USA). Cells were cultured in DMEM medium supplemented with 10% fetal calf serum, incubated at 37°C with 5% CO₂ humidified air atmosphere. Antibodies against mouse cytochrome c, β-actin, Bcl-xl, caspase 3 and caspase 9 were purchased from Abcam (Cambridge, MA, USA). C57 mice were obtained from Beijing HFK Biotechnology Co., Ltd (Beijing, People’s Republic of China) and maintained under specific pathogen-free conditions. All animal procedures were approved and controlled by the Institutional Animal Care and Treatment Committee of Sichuan Provincial People’s Hospital and carried out according to the Animal Care and Use Guidelines of Sichuan Provincial People’s Hospital.

Preparation of liposomes

XY-4 loaded cationic liposomes were prepared as previously described.³⁴ Briefly, DOTAP, cholesterol and XY-4 were co-dissolved at 15:5:1 mass ratio in chloroform, and the lipid film was formed by evaporation using a rotary evaporation for 2 hours. To remove the residual organic solvent, the flask was placed on a vacuum pump for 20 minutes. The dried film was subsequently rehydrated in distilled water to a final drug concentration of 0.5 mg/mL, which was finally filtered through a 0.45 µm nylon filter to remove unloaded XY-4.

XY-4/Bcl-xl siRNA co-loaded cationic liposomes (XY-4/siBcl-xl-CLP) were prepared by further incubating Bcl-xl _targeting siRNAs with prepared XY-4 loaded cationic liposomes in fresh serum-free medium before administration.

Due to its specific fluorescence property and similar hydrophobic nature to XY-4, coumarin-6 (Sigma-Aldrich Co.) was used as a model drug to evaluate the cell uptake efficiency. The preparation method for coumarin-6 loaded cationic liposomes was the same as that of XY-4 loaded liposomes. The coumarin-6 loaded neutral liposomes were also prepared in a similar way by substituting DOTAP with soybean phospholipid.

Characterization of XY-4 loaded cationic liposomes

Size distribution and zeta potential of the XY-4 loaded cationic liposomes were measured using a Zetasizer Nano ZS (Malvern Instruments, Malvern, UK). Measurements were performed at 25°C after equilibration for 2 minutes.

All results were the mean of three test runs

The concentration of XY-4 was determined by high performance liquid chromatography (HPLC; Waters Alliance 2695; Waters, Milford, MA, USA). The solvent delivery system was equipped with a column heater and a plus autosampler. Detection was carried out on a Waters 2996 detector. Chromatographic separations were performed on a reversed phase C18 column (4.6×150 mm-5 mm, Sunfire Analysis column; Waters) with the column temperature kept at 28°C. Methanol/water (75/25, v/v) was used as eluent at a flow rate of 1 mL/min.

Drug loading (DL) and encapsulation efficiency (EE) of prepared liposomes were determined as follows. Briefly, 10 mg of lyophilized XY-4 loaded cationic liposomes were dissolved in 0.1 mL dichloromethane (DCM) and diluted with methanol. The amount of XY-4 in the solution was determined by HPLC. Finally, the DL and EE of XY-4 loaded cationic liposomes were calculated according to Equations 1 and 2:

In vitro release study

In vitro release behavior of XY-4 loaded liposomes was studied using a dialysis method. XY-4 cationic liposomes and equivalent free XY-4 were loaded into dialysis tubes (molecular weight cutoff 3 kDa) which had been soaked in PBS, and the ends were tightened. The dialysis tubes were incubated in 30 mL PBS (pH =7.4) containing 0.5% of Tween-80 at 37°C with gentle shaking. Release media were collected at a fixed time point and replenished with the same amount of fresh dialysis solution. The samples were determined by HPLC to analyze the concentration of XY-4. All results were the mean of the three samples.

In vitro gene transfection

B16 cells were seeded into 6-well plates at a density of 5×10⁴ cells per well in 2 mL DMEM medium containing 10% FBS, before transfection. The 5′-carboxyfluorescein labeled siRNA (FAM siRNA) labeled negative control siRNA was used as a reporter gene. After replacing the medium with 1 mL of fresh serum-free medium, gene transfer complexes including 100 pmol of siRNA in serum-free medium were pipetted into each well, and the mass ratio of liposome/siRNA and polyetherimide 25K (PEI25K)/siRNA were 5:1 and 2:1, respectively. After incubation for 4 h, the media were replaced by complete medium. Twenty-four hours later, pictures of each well were taken under a microscope and luciferase activity was measured by flow cytometry (Epics Elite ESP; Coulter Corp., Miami, FL, USA).

In vitro gene silencing study

siRNA _targeting mouse Bcl-xl (_target sequence: 5′-AAG GAU ACA GCU GGA GUC AGU-3′), scrambled siRNA (_target sequence: 5′-AAU UCU CCG AAC GUG UCA CGU-3′) and FAM-labeled negative control siRNA were purchased from GenePharma Co., Ltd (Shanghai, People’s Republic of China) in unprotected, desalted, annealed form.

To determine the level of Bcl-xl mRNA, total RNA was extracted from B16 cells using TRIzol™ Reagent (Thermo Fisher Scientific, Waltham, MA, USA), and individual cDNAs were synthesized with a SuperScript II reverse transcriptase assay (Thermo Fisher Scientific). Real-time quantitative polymerase chain reaction (PCR) was performed with an SYBR GreenER quantitative PCR SuperMix Universal kit (Thermo Fisher Scientific). Reactions were run with a standard cycling program: 50°C for 2 minutes, 95°C for 10 minutes, 40 cycles of 95°C for 15 seconds, and 60°C for 1 minute, on an AB7500 real-time PCR system (Applied Biosystems, Foster City, CA, USA). The PCR primers to detect mouse Bcl-xl (forward; 5′-TCG GGA TGG AGT AAA CTG GG-3′, reverse; 5′-CCA CGC ACA GTG CCC C-3′) and GAPDH (forward; 5′-ATG GGG AAG GTG AAG GTC G-3′, reverse; 5′-TAA AAG CAG CCC TGG TGA CC-3′) were synthesized and purified by TSINGKE Biological Technology (Chengdu, People’s Republic of China).

Cellular uptake in vitro

B16 mice melanoma cancer cells were cultured on 6-well plates for 24 hours to achieve approximately 70% confluence. Cationic liposomes or neutral liposomes encapsulating coumarin-6 were added to the designated wells. After incubation for 6 hours, the cells were collected for measurement of coumarin-6-derived green fluorescence. The fluorescence was observed using a Zeiss M2Bio fluorescence microscope (Carl Zeiss Meditec AG, Jena, Germany). To identify each cell, we simultaneously stained cell nucleus with blue fluorescent dye Hoechst 33258.

Cell proliferation study

The anti-proliferation abilities of free XY-4, cationic liposome delivered Bcl-xl siRNA, and drug loaded liposomes to B16 cell lines were evaluated by the MTT method. Briefly, B16 cells were plated at a density of 5×10³ cells per well in 100 µL of DMEM containing 10% FBS in 96-well plates and grown for 24 hours. Cells were then exposed to a series of free XY-4 or cationic liposome delivered Bcl-xl siRNA or drug loaded liposomes at different concentrations for 72 hours. The viability of cells was measured using the MTT method. Results were the mean of six test runs.

Western blotting analysis

Cellular proteins were extracted by using RIPA cell lysate containing 1% (v/v) phenylmethane sulfonyl fluoride (PMSF) and collected by centrifugation at 13,000 rpm for 15 minutes at 4°C. The total protein concentration was determined using the BCA Protein Assay Kit (Vazyme Biotech Co., Ltd, Nanjing, People’s Republic of China). Proteins were separated using 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and transferred to a polyvinylidene fluoride membrane. _target proteins were then detected with primary antibodies and horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse secondary antibodies. All these bands are detected on the same gel, and after each exposure we stripped the membrane and incubated it with another antibody. Protein blots were visualized with SuperLumia ECL Plus (Thermo Fisher Scientific).

Flow cytometry assay

The cellular apoptosis and cell cycle status were tested via propidium iodide (PI) staining. In brief, B16 cells were seeded into 6-well plates and exposed to a series of free XY-4, cationic liposome delivered Bcl-xl siRNA, and drug loaded liposomes, separately. The treated cells were collected, washed, and stained with PI at room temperature for 15 minutes. The percentage of apoptotic cells and the property of cell cycle arrest were analyzed by flow cytometry (Becton Dickinson, Franklin Lakes, NJ, USA).

In vivo tumor xenograft study

For the abdominal cavity metastatic model, 1×10⁵ B16 cells were injected subcutaneously into the right flanks of female C57 mice (6–8 weeks old) on day 0. On day 3, the mice were randomized into four groups (five mice per group) and numbered. The four groups were intratumorally injected with five groups of normal saline (control), XY-4 loaded cationic liposome (XY-4: 10 mg/kg), cationic liposome delivered Bcl-xl siRNA (siRNA: 12 µg), or XY-4/Bcl-xl-CLP (siRNA: 12 µg; XY-4: 10 mg/kg) every other day. The weight of mice was recorded every day. Tumor burden was measured every 2 days with a caliper (calculated volume [mm³] =π/6× length × width × width). On day 20, when the mice in the control group were very weak, all mice were euthanized by cervical vertebra dislocation, and their tumors were immediately harvested, weighed, and analyzed.

Histological analysis

The excised tissues were fixed in 4% neutral-buffered formalin solution for more than 24 hours and embedded in paraffin. Sections of the tissues (3–5 µm) were stained with hematoxylin and eosin. This analysis was performed following the manufacturer’s protocol, and the samples were examined with a fluorescence microscope (400×).

A commercially available TdT-mediated dUTP nick-end labeling (TUNEL) kit (Promega, Madison, WI, USA) was used to analyze the apoptotic effects in B16 mouse melanoma xenograft tumor tissues. This analysis was performed following the manufacturer’s protocol.

Statistical analysis

Data are expressed as the mean value ± SD. Statistical analysis was performed with one-way analysis of variance (ANOVA) using SPSS software (IBM Corporation, Armonk, NY, USA). P-values less than 0.05 were considered to be statistically significant.

Preparation and characterization of XY-4/Bcl-xl siRNA co-loaded cationic liposomes

To combine hydrophobic Aurora-A kinase inhibitor XY-4 with Bcl-xl _targeting siRNA, we developed a drug and gene co-delivery system based on cationic liposomes. A general view of XY-4/siBcl-xl-CLP is presented in Figure 2. In this system, the hydrophobic XY-4 was encapsulated in the core area of liposomes while positive charges provided by DOTAP were utilized to bind and transfer Bcl-xl _targeting siRNA. The prepared liposomes were characterized in detail. As presented in Figure 3A, the prepared liposomes were monodispersed (PDI =0.183) with a mean particle size of 91.3±4.5 nm, indicating that they had a very narrow particle size distribution. The spectrum of co-delivery liposomes is presented in Figure 3B with a zeta potential of 38.5±0.5 mV. The DNA-binding ability of prepared cationic liposomes was evaluated by gel retardation assay, and is shown in Figure 3C. When the N/P ratio was 8, complete retardation of DNA was achieved. It was also found that XY-4 could be efficiently encapsulated into cationic liposomes, creating XY-4 loaded cationic liposomes with encapsulation efficiency of 84.6% and drug loading rate of 4.76%.

To confirm whether cationic liposome-encapsulated XY-4 could be released, the release profile of XY-4 loaded liposomes was studied using a dialysis method in vitro. Our results showed a slow-releasing behavior of XY-4 loaded liposomes in vitro with about 64.9% of total XY-4 released in 4 days. Compared to the free XY-4 group, the release of liposomal XY-4 was much slower (Figure 4).

Further to biophysical characterization, we compared the transfection efficiency of prepared cationic liposomes with PEI25K (the “gold standard” transfection agent) in vitro. The FAM labelled negative control siRNA was used as a reporter nucleotide. As can be seen from Figure 5A, B16 cells could be efficiently transfected by cationic liposome/FAM negative control (FAM-NC) siRNA complexes. The transfection efficiency on B16 cells was 32%±3.8% compared to that of 40%±2.6% for PEI25K. In addition, Figure 5B and C demonstrate the transfection ability of cationic liposomes with an FAM siRNA-based reporter gene. The high siRNA transfection performance suggested that cationic liposomes may be efficient in siRNA delivery.

Cellular uptake of cationic liposome delivered cargo by B16 cells

The capacity of cationic liposomes to deliver XY-4 into B16 melanoma cancer cells was determined. The cellular uptake ability of cationic liposomes was compared with that of neutral liposomes in vitro. As a fluorescence probe, coumarin-6 was encapsulated into cationic liposomes or neutral liposomes. After B16 melanoma cancer cells were exposed to coumarin-6 containing cationic liposomes or neutral liposomes for 2 hours, cells were collected for analysis of coumarin-6-derived fluorescence. As shown in Figure 6, B16 cells took up cationic liposomes in preference to neutral liposomes. After incubation for 2 hours, more cells with stronger green fluorescence were observed in the former group. These results suggested that cationic liposomes were more efficient than neutral liposomes for delivering their cargo to melanoma cells. This result is consistent with our previous reports in which bladder cancer cells exhibited higher drug cell uptake when treated with cationic polymeric nanoparticles rather than neutral nanoparticles. In this work, our results also indicated that the cationic surface of cationic liposomes could improve the cellular uptake of cargos, suggesting that the delivery of hydrophobic XY-4 into cancer cells was facilitated by the cationic carrier.

Anticancer activity in vitro

To further enhance the therapeutic ability of XY-4, the combination of XY-4 with Bcl-xl _targeting siRNA was analyzed. As an Aurora-A kinase inhibitor, XY-4 is capable of inducing cell cycle arrest in cancer cells. Thus, we first evaluated this property on B16 melanoma cells by flow cytometry. As shown in Figure 7A, after 24 hours’ exposure to XY-4, a significant inhibition of cell cycle progression could be observed.Com pared with the control group, the percentages of G2/M fraction in the XY-4 treated group were increased from 11.8% to 79% (p<0.05). Meanwhile, the cell cycle distribution of XY-4 treated cell in G1 and S phase were obviously decreased (p<0.05), demonstrating significant G2/M phase arrest (Figure 7B). We studied the potential anti-cancer ability of XY-4 on B16 melanoma cells in vitro. As presented in Figure 8A, according to our MTT results, XY-4 could inhibit the proliferation of B16 melanoma cells with a IC₅₀ of 5.5 µM. On the other hand, in this study, a Bcl-xl _targeting siRNA was designed to silence the expression of Bcl-xl gene. As a member of the Bcl-2 family of proteins, Bcl-xl acts as an anti-apoptotic protein by preventing the release of mitochondrial contents such as cytochrome c, which leads to caspase activation.³⁵ According to our real-time quantitative PCR (qPCR) results, the designed siRNA (50 nM and 100 nM) delivered by cationic liposomes efficiently reduced Bcl-xl mRNA level (p<0.05, Figure 8B). As expected, after 72 hours exposure, the cationic liposome delivered Bcl-xl siRNA (50 nM) showed obvious anti-proliferation ability (Figure 9). We then evaluated the possible enhanced anticancer effect of free XY-4 and Bcl-xl siRNA combination on B16 cell lines. In our study, cationic liposome delivered Bcl-xl siRNA (50 nM) was combined with XY-4 at different molar ratios (1:25, 1:50, 1:100, 1:200, 1:400 and 1:800). By evaluating cell proliferation status, the combination treatment showed obvious anti-proliferation effects on B16 cells at the ratio of 1:400 and 1:800 (Figure 9). In these two groups, cell survival rates were much lower than either free XY-4 or the Bcl-xl siRNA group. These results suggested that possible enhanced therapeutic effect might exist by the combination of XY-4 and Bcl-xl siRNA.

Despite their anti-cancer ability, the poor water solubility of XY-4 still limited the further application of this combination. In order to address this issue, we utilized cationic liposomes to co-deliver XY-4 and Bcl-xl siRNA, forming XY-4/siBcl-xl-CLP. In this formulation, the hydrophobic XY-4 was encapsulated in the core area while Bcl-xl siRNA was bound to the surface by electronic interaction. The anti-cancer abilities of XY-4/siBcl-xl-CLP (Bcl-xl siRNA/XY-4 mass ratio: 1/400; XY-4 concentration: 2.5–80 µM) on the B16 cell line were then studied. According to our results, XY-4/siBcl-xl-CLP showed strong anti-cancer effects in an obvious concentration dependent manner (Figure 10). It showed better anti-cancer effects than free XY-4 or cationic liposome delivered Bcl-xl siRNA (siBcl-xl-CLP) alone on B16 cells. In addition, the XY-4/siBcl-xl-CLP had similar anti-cancer ability to free XY-4 and siBcl-xl-CLP in combination. Our results indicated that co-delivery of the two agents by cationic liposomes not only retained their cancer controlling ability, but also demonstrated enhanced efficacy when administrated together.

XY-4/Bcl-xl siRNA co-loaded cationic liposomes induced apoptosis in B16 melanoma cells

To determine whether the XY-4/siBcl-xl-CLP-induced loss of proliferation capacity and cell viability of melanoma cancer cells was associated with the induction of apoptosis, B16 cells were treated with free XY-4 (10 µM), siBcl-xl-CLP (Bcl-xl siRNA concentration: 50 nM) and XY-4/siBcl-xl-CLP (10 µM for XY-4 and 50 nM for Bcl-xl siRNA). The numbers of apoptotic cells were assessed using flow cytometry. As shown in Figure 11A and B, after 48 hours of treatment, the apoptosis rates of free XY-4 and siBcl-xl-CLP treated cells were 7.7% and 14.6%, respectively, while that of the XY-4/siBcl-xl-CLP group was 30.4% (vesus 1.5% of control group, p<0.05). The apoptosis assay results were consistent with the MTT results above, demonstrating enhanced effects between XY-4 and Bcl-xl siRNA, and suggesting inducing of apoptosis as a possible mechanism.

We further studied the possible anti-cancer mechanism by Western blotting. The protein levels of several related proteins suggested that the observed apoptosis might be induced through triggering a mitochondria-dependent apoptotic signaling pathway. As showed in Figure 12, compared to free XY-4 or the siBcl-xl-CLP treated group, XY-4/siBcl-xl-CLP obviously increased the cytochrome c levels in B16 cells, which is a critical step in the mitochondrial-dependent signaling pathways of apoptosis. Treatment of cells with the XY-4/siBcl-xl-CLP group also resulted in an obvious reduction in the levels of the anti-apoptotic proteins Bcl-xl. After the treatment with the combination group, increased protein levels of cleaved caspase-9 and cleaved caspase-3 were observed, while the expression of caspase-9 was decreased. This indicated that the XY-4/siBcl-xl-CLP had stronger effect on activating the mitochondrial-based apoptosis pathway than the single drug formulation. Our results suggested that apoptosis activation might be a possible explanation for the enhanced anti-cancer effects of the co-loaded liposome.

Anticancer effect of XY-4/Bcl-xl siRNA co-loaded cationic liposomes in vivo

We then examined the in vivo antitumor effect of the co-delivering liposomes in C57 mice. The anticancer activity through intratumoral injection on the xenograft model of B16 mouse melanoma is illustrated in Figure 13. According to our results, the mean tumor volume for the control group increased from 100.43±15.44 to 610.6±54.6 mm³, XY-4 loaded cationic liposome group increased from 100.5±14.68 to 358.1±54.92 mm³ and Bcl-xl siRNA group increased from 90.45±15.24 to 451.4±52.92 mm³, whereas the XY-4/siBcl-xl-CLP group only increased from 100±13.52 to 201.83±54.2 mm³, demonstrating higher anticancer efficacy (Figure 13A). At the end of the experiment, the mean tumor weight of the control group, the XY-4 loaded cationic liposome group and the Bcl-xl siRNA group were 0.82±0.09 g, 0.59±0.061 g and 0.54±0.06 g, respectively, while that of XY-4/siBcl-xl-CLP group was 0.25±0.12 g (p<0.01 versus control group, Figure 13B). Compared with other treatment groups, the co-delivery formulation group achieved a statistically significant reduction in tumor weight (P<0.001). In addition, gross changes such as weight loss, ruffling of fur, and changes in behavior were not seen in XY-4/siBcl-xl-CLP treated mice (data not shown). No obvious pathological changes of heart, liver, kidney, lung and spleen were found by histological analysis of tissue sections (Figure 13D). The in vivo gene silencing efficiency of Bcl-xl in each group was also evaluated (p<0.1 versus control group, Figure 13C).

To study the mechanism associated with the anticancer activity of XY-4/siBcl-xl-CLP in vivo, a TUNEL assay was carried out. As shown in Figure 13E, numerous positive nucleus identified as apoptosis could be observed in the XY-4/siBcl-xl-CLP treated tumor tissue, whereas such nuclei were rare in other groups. This again implies that apoptosis induction may be an important mechanism of inhibiting melanoma tumor growth. This co-delivery liposomal formulation demonstrated much stronger apoptosis inducing capacity in vivo, which is consistent with our in vitro results above.

These results suggested that intratumoral injection of XY-4/siBcl-xl-CLP could efficiently inhibit the growth of B16 melanoma tumor in vivo through inducing apoptosis. Meanwhile, this co-delivery formulation demonstrated obviously higher therapeutic efficacy with high safety.