Antitumor Effect of n-Butylidenephthalide Encapsulated on B16/F10 Melanoma Cells In Vitro with a Polycationic Liposome Containing PEI and Polyethylene Glycol Complex

Advanced melanoma can metastasize to distal organs from the skin and yield an aggressive disease and poor prognosis even after treatment with chemotherapeutic agents. The compound n-Butylidenephthalide (BP) is isolated from Angelica sinensis, which is used to treat anemia and gynecological dysfunction in traditional Chinese medicine. Studies have indicated that BP can inhibit cancers, including brain, lung, prostate, liver, and colon cancers. However, because BP is a natural hydrophobic compound, it is quickly metabolized by the liver within 24 h, and thus has limited potential for development in cancer therapy. This study investigated the anticancer mechanisms of BP through encapsulation with a novel polycationic liposome containing polyethylenimine (PEI) and polyethylene glycol complex (LPPC) in melanoma cells. The results demonstrated that BP/LPPC had higher cytotoxicity than BP alone and induced cell cycle arrest at the G0/G1 phase in B16/F10 melanoma cells. The BP/LPPC-treated cell indicated an increase in subG1 percentage and TUNEL positive apoptotic morphology through induction of extrinsic and intrinsic apoptosis pathways. The combination of BP and LPPC and clinical drug 5-Fluorouracil had a greater synergistic inhibition effect than did a single drug. Moreover, LPPC encapsulation improved the uptake of BP values through enhancement of cell endocytosis and maintained BP cytotoxicity activity within 24 h. In conclusion, BP/LPPC can inhibit growth of melanoma cells and induce cell arrest and apoptosis, indicating that BP/LPPC has great potential for development of melanoma therapy agents.


Introduction
The compound n-Butylidenephthalide (BP) can be isolated from Angelica sinensis-also called dong quai-which is primarily grown in Asia and has major applications in the treatment of anemia and gynecological dysfunction in traditional Chinese medicine [1,2]. Moreover, BP is a natural liposoluble compound with a low molecular weight (188.226 g/mol) that possesses multiple pharmacological activities, such as reducing injury and fibrosis in the liver and neuroprotective activity [3][4][5]. Studies have revealed that BP exerts cytotoxic or inhibitory effects on various cancers, including brain, lung, colon, prostate, and liver cancers [6][7][8][9][10][11][12]. However, no anti-melanoma effect was recorded until a study of BP on melanoma in vitro.
Although BP has anticancer effects, the clinical application for BP is limited by its properties, namely poor dissolution, forming of dimers, easy hydration, and oxidization influencing bioavailability [13]. To overcome these limitations and create an effective therapeutic agent, drug carriers have been developed. For example, a polycationic liposome complex containing PEI and PEG (Lipo-PEG-PEI complex; LPPC) is a drug carrier comprising DOPC (1,2-Dioleoyl-sn-glycero-3-phosphocholine), DLPC (1,2-dilauroyl-sn-glycero-3-phosphocholine), PEG (Polyethylene glycol), and PEI (polyethylenimine), and with properties including a positive charge with 40 mV, protein capture, drug entrapment into lipophilic or hydrophilic structures, and components that can be metabolized by the liver. Moreover, curcumin-loaded LPPC can improve the cytotoxic activity of curcumin, resulting in cancer cell death [14][15][16][17]. Here, we wondered whether the modification of a drug delivery system with LPPC may overcome the drawbacks of BP and reduce the required dosage to enhance drug efficacy.
Studies have indicated that nanoparticles with positive charges provide a method for entrapping drugs into cells through means such as the phagocytosis, clathrin-mediated, caveolae-mediated, clathrin-independent, caveolae-independent, and macropinocytosis pathways [18,19]. However, the antimelanoma mechanism of an LPPC-encapsulated BP system has not been elucidated. The present study demonstrated that the anticancer effect of BP in melanoma cells is mediated through cell cycle arrest and apoptosis mechanisms. In addition, this study established a novel BP nanoparticle system to overcome poor water solubility and ameliorate the anticancer effect for comparison with free BP.

BP/LPPC-Induced Cell Cycle Arrest (G0/G1) in B16/F10 Melanoma Cells
The cell cycle distribution of the B16/F10 cells was treated with BP (0-120 µg/mL; 0-48 h) or BP/LPPC (0-45 µg/mL; 0-12 h), resulting in cell cycle arrest at the G0/G1 phase in a time course and dosage-dependent manner (p < 0.05), as shown in Table 2. Comparison of conditions of BP and BP/LPPC induced 66% cell cycle arresting at the G0/G1 phase, BP needed 80 µg/mL treated for 24 h and BP/LPPC needed only 30 µg/mL treated for 6 h. From this result, BP/LPPC induced cell cycle arresting more efficiently than BP in low dose or short time conditions. BP and BP/LPPC treatment reduced the proportion of cell cycles in the S and G2/M phase. Moreover, BP-and BP/LPPC-treated cells showed decreased protein expression of RB, p-RB, CDK4, and cyclin D1 and increased protein expression of P53, p-P53, and P21, which led cell cycle arrest at the G0/G1 phase, as shown in Figure  2A(i) to (iii). After BP and BP/LPPC treatment for time course and dosage, the cells were collected and analyzed for the subG1 phase using flow cytometry. The results showed that the percentage of The cell cycle distribution of the B16/F10 cells was treated with BP (0-120 µg/mL; 0-48 h) or BP/LPPC (0-45 µg/mL; 0-12 h), resulting in cell cycle arrest at the G 0 /G 1 phase in a time course and dosage-dependent manner (p < 0.05), as shown in Table 2. Comparison of conditions of BP and BP/LPPC induced 66% cell cycle arresting at the G 0 /G 1 phase, BP needed 80 µg/mL treated for 24 h and BP/LPPC needed only 30 µg/mL treated for 6 h. From this result, BP/LPPC induced cell cycle arresting more efficiently than BP in low dose or short time conditions. BP and BP/LPPC treatment reduced the proportion of cell cycles in the S and G 2 /M phase. Moreover, BP-and BP/LPPC-treated cells showed decreased protein expression of RB, p-RB, CDK4, and cyclin D1 and increased protein expression of P53, p-P53, and P21, which led cell cycle arrest at the G 0 /G 1 phase, as shown in Figure 2A(i) to (iii). After BP and BP/LPPC treatment for time course and dosage, the cells were collected and analyzed for the subG1 phase using flow cytometry. The results showed that the

Morphological Evaluation and Mechanism of BP/LPPC-Induced Apoptosis
To investigate drug-induced cell death through the apoptosis pathway, the cells were stained using a TUNEL assay after BP or BP/LPPC treatment. The BP-and BP/LPPC-treated cells indicated a positive TUNEL result and apoptotic morphology, including chromatin condensation, DNA fragmentation, and presence of apoptotic bodies, as shown in Figure 3A. The immunocytochemistry staining results indicated that BP and BP/LPPC activated extrinsic (Fas, FasL and Claved-Cas-8) and intrinsic (Bax, AIF, and Cleaved-Cas-9) apoptosis pathways and triggered downstream Cleaved-Cas-3 activity, as shown in Figure 3B. Moreover, Caspase-3, -8, and -9 were activated after BP and BP/LPPC treatment in time course and dosage-dependent manners using western blotting analysis, as shown in Figure 3C,D.
To determine whether caspase cascade was activated by BP or BP/LPPC, the cells were pretreated with Caspase-3 inhibitor before BP and BP/LPPC treatment. The results revealed that activation of Caspase-3 was blocked when the cells were pretreated with an inhibitor, as shown in Figure 3E. These results demonstrated that BP-and BP/LPPC-induced cell death through activation of extrinsic and intrinsic apoptosis pathways.

Morphological Evaluation and Mechanism of BP/LPPC-Induced Apoptosis
To investigate drug-induced cell death through the apoptosis pathway, the cells were stained using a TUNEL assay after BP or BP/LPPC treatment. The BP-and BP/LPPC-treated cells indicated a positive TUNEL result and apoptotic morphology, including chromatin condensation, DNA fragmentation, and presence of apoptotic bodies, as shown in Figure 3A. The immunocytochemistry staining results indicated that BP and BP/LPPC activated extrinsic (Fas, FasL and Claved-Cas-8) and intrinsic (Bax, AIF, and Cleaved-Cas-9) apoptosis pathways and triggered downstream Cleaved-Cas-3 activity, as shown in Figure 3B. Moreover, Caspase-3, -8, and -9 were activated after BP and BP/LPPC treatment in time course and dosage-dependent manners using western blotting analysis, as shown in Figure 3C,D. To determine whether caspase cascade was activated by BP or BP/LPPC, the cells were pretreated with Caspase-3 inhibitor before BP and BP/LPPC treatment. The results revealed that activation of Caspase-3 was blocked when the cells were pretreated with an inhibitor, as shown in Figure 3E. These results demonstrated that BP-and BP/LPPC-induced cell death through activation of extrinsic and intrinsic apoptosis pathways.  before BP/LPPC treatment. Cleaved-Caspase-3 was detected using western blotting. The columns showed mean ± SD. # p < 0.05 versus control with significant decrease. * p < 0.05 combination group versus BP or BP/LPPC only with significant increase. (E) The activation of Caspase-3 was blocked when B16/F10 cells were pretreated with an inhibitor. # p < 0.05 combination group versus BP or BP/LPPC only with significant decrease. * p < 0.05 combination group versus BP or BP/LPPC only with significant increase.

Combination of BP/LPPC and 5-FU had a Synergistic Effect
The B16/F10 cells were treated with a serial concentration of BP/LPPC combined with 5-FU (0.2 µg/mL) or a serial concentration of 5-FU combined with BP/LPPC (10 µg/mL) for 48 h. The cell viability of the combination group (56.01 ± 0.78% to 6.63 ± 0.16% in BP/LPPC combined with 0.2 µg/mL 5-FU; 70.81 ± 3.18% to 27.34 ± 0.31% in 5-FU combined with 10 µg/mL BP/LPPC) was lower than that of the single drug group (100 ± 1.39% to 6.88 ± 0.19% in BP/LPPC only; 100 ± 3.14% to 32.73 ± 1.58% in 5-FU only) in a dosage-dependent manner, with a combination index (CI) of 0.42, as shown in Figure 4A,B. Moreover, the cell viability of the combination group significantly decreased compared with the BP/LPPC or 5-FU drug only-treated group (p < 0.05). These results suggested that BP/LPPC combined with 5-FU had a synergistic effect on the cytotoxicity of melanoma cells.

Combination of BP/LPPC and 5-FU had a Synergistic Effect
The B16/F10 cells were treated with a serial concentration of BP/LPPC combined with 5-FU (0.2 µg/mL) or a serial concentration of 5-FU combined with BP/LPPC (10 µg/mL) for 48 h. The cell viability of the combination group (56.01 ± 0.78% to 6.63 ± 0.16% in BP/LPPC combined with 0.2 µg/mL 5-FU; 70.81 ± 3.18% to 27.34 ± 0.31% in 5-FU combined with 10 µg/mL BP/LPPC) was lower than that of the single drug group (100 ± 1.39% to 6.88 ± 0.19% in BP/LPPC only; 100 ± 3.14% to 32.73 ± 1.58% in 5-FU only) in a dosage-dependent manner, with a combination index (CI) of 0.42, as shown in Figure 4A,B. Moreover, the cell viability of the combination group significantly decreased compared with the BP/LPPC or 5-FU drug only-treated group (p < 0.05). These results suggested that BP/LPPC combined with 5-FU had a synergistic effect on the cytotoxicity of melanoma cells.

Protection Effect of BP Encapsulated with LPPC
To investigate whether LPPC encapsulation protected and maintained BP′s cytotoxic activity, BP was encapsulated with and without LPPC and stored in ultra pure and sterile water (H2O) at 4 °C or in a protein-rich environment at 37 °C and incubated for 0, 4, 8, and 24 h. The activity of BP was evaluated based on its cytotoxicity in B16/F10 cells. BP/LPPC exhibited a higher cytotoxicity (IC50 =

Protection Effect of BP Encapsulated with LPPC
To investigate whether LPPC encapsulation protected and maintained BP s cytotoxic activity, BP was encapsulated with and without LPPC and stored in ultra pure and sterile water (H 2 O) at 4 • C or in a protein-rich environment at 37 • C and incubated for 0, 4, 8, and 24 h. The activity of BP was evaluated based on its cytotoxicity in B16/F10 cells. BP/LPPC exhibited a higher cytotoxicity (IC 50 = 15.21 ± 0.04 µg/mL in H 2 O; 29.55 ± 0.78 µg/mL in 10% fetal bovine serum (FBS) at 24 h) than did BP without LPPC encapsulation (IC 50 = 194.83 ± 4.11 µg/mL in H 2 O; 191.57 ± 2.30 µg/mL in 10% FBS at 24 h) in all environments, as shown in Figure 5A,B, thereby indicating that LPPC encapsulation protected and maintained BP activity to enhance the cytotoxicity of BP in melanoma cells.

LPPC with Positive Charge Triggered Cell Uptake of BP through the Endocytosis Pathway
After the cells had been incubated with BP or BP/LPPC, BP (blue fluorescence) in the cells was observed at 15 min in the BP/LPPC group and 30 min in the BP group, as shown in Figure 6A. In the quantitative analysis, the BP value of cell uptake in the BP/LPPC group (6.52 ± 3.89 to 20.32 ± 0.36 µg per 2.5 × 10 5 cells) was higher than that in the BP only group (1.51 ± 1.22 to 16.19 ± 0.01 µg per 2.5 × 10 5 cells) for melanoma cells, as shown in Figure 6B. These data indicated that BP/LPPC penetrated the cells more quickly than did BP. Therefore, the pathway of cell uptake induced by LPPC encapsulation was investigated next. Through use of different endocytosis inhibitors, the BP value of cell uptake was decreased after pretreatment with all endocytosis inhibitors, namely AHH (2.53 ± 0.46 to 8.38 ± 0.25 per 2.5  10 5 cells; micropinocytosis pathway), FIII (5.69 ± 1.28 to 12.77 ± 0.06 per 2.5  10 5 cells; caveolae-mediated endocytosis pathway), and CPZ (2.42 ± 0.25 to 12.42 ± 0.52 per 2.5 × 10 5 cells; clathrin-mediated endocytosis pathway), as shown in Table 3. This suggested that BP/LPPC with a positive charge induced varying degrees of endocytosis to enhance cell uptake of BP in melanoma cells.

LPPC with Positive Charge Triggered Cell Uptake of BP through the Endocytosis Pathway
After the cells had been incubated with BP or BP/LPPC, BP (blue fluorescence) in the cells was observed at 15 min in the BP/LPPC group and 30 min in the BP group, as shown in Figure 6A. In the quantitative analysis, the BP value of cell uptake in the BP/LPPC group (6.52 ± 3.89 to 20.32 ± 0.36 µg per 2.5 × 10 5 cells) was higher than that in the BP only group (1.51 ± 1.22 to 16.19 ± 0.01 µg per 2.5 × 10 5 cells) for melanoma cells, as shown in Figure 6B. These data indicated that BP/LPPC penetrated the cells more quickly than did BP. Therefore, the pathway of cell uptake induced by LPPC encapsulation was investigated next. Through use of different endocytosis inhibitors, the BP value of cell uptake was decreased after pretreatment with all endocytosis inhibitors, namely AHH (2.53 ± 0.46 to 8.38 ± 0.25 per 2.5 × 10 5 cells; micropinocytosis pathway), FIII (5.69 ± 1.28 to 12.77 ± 0.06 per 2.5 × 10 5 cells; caveolae-mediated endocytosis pathway), and CPZ (2.42 ± 0.25 to 12.42 ± 0.52 per 2.5 × 10 5 cells; clathrin-mediated endocytosis pathway), as shown in Table 3. This suggested that BP/LPPC with a positive charge induced varying degrees of endocytosis to enhance cell uptake of BP in melanoma cells.

Preparation of BP/LPPC
n-Butylidenephthalide (BP), (E) + (Z), 95% was purchased from Alfa Aesar, Thermo Fisher Scientific (Waltham, MA, USA), and its chemical structure was described in a previous study [1], as shown in Figure 1A. The LPPC used in this study was provided by National Chiao Tung University, Hsinchu, Taiwan. A total of 100 µL of LPPC was added to 900 µL of ultra pure and sterile water (H2O) and the mixture was centrifuged (Microcentrifuges, Force 1624, Select BioProducts, Edison, NJ, USA) at 9000 rpm for 5 min to remove the supernatant. The pellet was resuspended in 100 µL of H2O and strongly vortexed after addition of 20 µL of 1 M BP mixed with 10 µL of dimethylsulfoxide (DMSO). After incubation for 30 min at room temperature, the unencapsulated BP was removed and the pellet was resuspended in a solution containing 750 µL of H2O and 750 µL of PEG-1500 solution (100 mg/mL, Acros Organics, Morris Plains, NJ, USA) for 30 min at room temperature. The pellet (BP/LPPC) was stored at 4 °C and treated for cells for 1 day. The BP value of the LPPC encapsulation equaled the total BP value minus the BP value of the LPPC unencapsulated in the supernatant. The BP value in the supernatant was measured using a fluorescence spectrophotometer at 350 nm (Hitachi F4500, Hitachi Instruments Inc., Tokyo, Japan). The BP/LPPC were prepared according to a

Preparation of BP/LPPC
n-Butylidenephthalide (BP), (E) + (Z), 95% was purchased from Alfa Aesar, Thermo Fisher Scientific (Waltham, MA, USA), and its chemical structure was described in a previous study [1], as shown in Figure 1A. The LPPC used in this study was provided by National Chiao Tung University, Hsinchu, Taiwan. A total of 100 µL of LPPC was added to 900 µL of ultra pure and sterile water (H 2 O) and the mixture was centrifuged (Microcentrifuges, Force 1624, Select BioProducts, Edison, NJ, USA) at 9000 rpm for 5 min to remove the supernatant. The pellet was resuspended in 100 µL of H 2 O and strongly vortexed after addition of 20 µL of 1 M BP mixed with 10 µL of dimethylsulfoxide (DMSO). After incubation for 30 min at room temperature, the unencapsulated BP was removed and the pellet was resuspended in a solution containing 750 µL of H 2 O and 750 µL of PEG-1500 solution (100 mg/mL, Acros Organics, Morris Plains, NJ, USA) for 30 min at room temperature. The pellet (BP/LPPC) was stored at 4 • C and treated for cells for 1 day. The BP value of the LPPC encapsulation equaled the total BP value minus the BP value of the LPPC unencapsulated in the supernatant. The BP value in the supernatant was measured using a fluorescence spectrophotometer at 350 nm (Hitachi F4500, Hitachi Instruments Inc., Tokyo, Japan). The BP/LPPC were prepared according to a previously described manufacturing process and analyzed the characteristics, including particle sizes (200 nm to 280 nm), average zeta-potential (~38 mV), drug release, and encapsulation capacity [13].

Cell Culture and Reagent
The cancer cells used were B16/F10 (mouse melanoma cells) and K-Blab (mouse fibroblast sarcoma cells). The control cells were Blab/3T3 (mouse fibroblast), MDCK (canis kidney epithelial cells), and SVEC (mouse endothelial cells), all of which were purchased from the Food Industry Research and Development Institute (Hsinchu, Taiwan). The medium used for all cells was Dulbecco's Modified Eagle's Medium, containing 10% heat inactivated fetal bovine serum (FBS; Gibco BRL, Gaithersburg, MD, USA), HEPES (10 mM; Gibco), pyruvate (1 mM; Gibco), and P/S (100 U/mL penicillin and 100 µg/mL streptomycin; Gibco). Cells were cultured and incubated in a growth medium in a 37 • C humidified atmosphere with 5% CO 2 . BP (Alfa Aesar, Haverhill, MA, USA) and 5-Fluorouracil (5-FU; Sigma, Setagaya, Tokyo, Japan) were dissolved in DMSO and stored at 4 • C or −20 • C in each in vitro experiment.

BP/LPPC-Induced Cytotoxicity
The cytotoxicity of the drugs was estimated based on cell viability and detected by modified 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay (MTT). The cells were plated on 96-well culture plates (5 × 10 3 per well) and incubated overnight, and then treated with a serial concentration of BP (0-400 µg/mL) or BP/LPPC (0-100 µg/mL) dissolved in a medium for 24 or 48 h, respectively. After removal of the medium, the cells of each concentration were reacted with 100 µL of MTT solution (400 µg/mL, Sigma) for 6-8 h. The MTT solution was replaced with 50 µL of DMSO to dissolve formazan crystals and detect optical density (O.D.) values using a microplate reader (Molecular Device, Spec384, San Jose, CA, USA) at 550 nm. The viability of the cells in the medium only was used as a control and regarded as 100% viable.

TUNEL Assay
Drug-induced apoptosis was determined using an In Situ Cell Death Detection Kit, POD (Roche, Mannheim, Germany). The B16/F10 cells were seeded on 10 cm 2 dishes overnight and incubated with 80 µg/mL of BP for 24 h and 30 µg/mL of BP/LPPC for 6 h. The cells were harvested, washed, and fixed with 10% formaldehyde, and dried on silane-coated glass slides (Matsunami, Tokyo, Japan). After being washed, the cells on the slides were treated with 3% H 2 O 2 to decrease the activity of endogenous peroxidase, and then incubated with cold 0.1% Triton X-100 in 0.1% sodium citrate to increase the permeability of the cells on ice. The cells were washed with PBS, incubated with the TUNEL reaction mixture for 2 h at 37 • C, and then counterstained with PI (10 µg/mL, Sigma). The morphology of cell apoptosis (apoptotic body, DNA fragmentation, and DNA condensation) was observed using a fluorescence microscope (ZEISS AXioskop2, Carl Zeiss, Munich, Germany) at a magnification of ×400.

Inhibition Caspase-3 Activity Assay
The B16/F10 cells were incubated on 6-well culture plates (5 × 10 5 cells per well) overnight, and then the medium of each well was replaced with a Caspase-3 inhibitor (1 µM Z-DEVD-FMK, G-Biosciences, Louis, MO, USA) and incubated for 2 h. After removal of the medium, the cells were treated with BP (80 µg/mL) for 24 h or BP/LPPC (30 µg/mL) for 6 h. The protein expression level of Caspase-3 was detected using western blotting.

Protection of BP Activity through LPPC Encapsulation
To determine the protection of BP activity through LPPC encapsulation, BP and BP/LPPC were resolved in 200 µL of ultra pure and sterile water (H 2 O) or a protein-rich environment solution (10% FBS in PBS), with a final BP concentration of 3 mg/mL in each group; the cells were then stored at 4 • C or 37 • C for 0, 4, 8, or 24 h. After incubation, the cells were treated with incubated BP or BP/LPPC and IC 50 was calculated using MTT. The protective effect on BP activity was evaluated through the cytotoxicity of BP in the tumor cells.

Cell Uptake of BP/LPPC in Qualitative and Quantitative Analysis
To analyze the cell uptake of BP, the cells were seeded on 15 mm microscope cover glasses (Assistent, Glaswarenfabrik Karl Hecht GmbH & Co KG, Sondheim, Germany) in a 3.5 cm 2 dish (5 × 10 5 cells per dish) and incubated overnight. The medium in each dish was replaced with a medium containing 50 µg/mL of BP or BP/LPPC and incubated for 0, 15, 30, 45, or 60 min. After incubation, the cover glasses were removed, washed, and fixed with 10% neutral formalin. The cell uptake of BP was observed through blue fluorescence on an upright fluorescence microscope (ZEISS AXioskop2) at a magnification of ×400. The bright-field was observed under differential interference contrast at a magnification of ×400.
For quantitative analysis of the cell uptake of BP, the cells (2.5 × 10 5 per well) were incubated in each well of the 24-well culture plate overnight, and the mediums were replaced with 50 µg/mL of BP or BP/LPPC and incubated for 0, 15, 30, 45, and 60 min. Finally, the BP values of the cells were extracted with phenol-chloroform and calculated using a fluorescence spectrophotometer (HITACHI F-4500) at 350 nm [13].
To investigate the endocytosis pathway induced by LPPC encapsulation, the B16/F10 cells were plated on 24-well culture plates (2.5 × 10 5 per well) and incubated overnight. To remove the medium, 300 µL of medium was added to each well, which contained endocytosis inhibitors of amiloride hydrochloride hydrate (AHH; 13.31 µg/mL, Sigma), Filipin III (FIII; 1 µg/mL, Sigma), or chlorpromazine hydrochloride (CPZ, 10 µg/mL, Sigma) for 1 h incubation. The medium in the well was replaced with 50 µg/mL BP or BP/LPPC and treated for 0, 15, 30, 45, and 60 min. The treated cells were harvested, the BP was extracted with phenol-chloroform, and the BP value in the cells was calculated using fluorescence spectroscopy at 350 nm.

Statistics
In this paper, all results are presented as mean ± standard deviation. The statistical analysis utilized Student's t test to define statistical significance, which was defined as p < 0.05.

Discussion
As several studies have indicated the anticancer activity of BP, our study was the first to demonstrate the anti-melanoma effects of BP with dose-dependent reduction of cell viability at indicated time intervals. Furthermore, BP also exhibited a better inhibitory drug concentration on K-balb, which is mouse fibroblast sarcoma. After that, the effects of BP on different types of cells was examined and the results revealed that the cytotoxic activity of BP toward control cells was lower than tumor cells such as fibroblasts, endothelial cells, and kidney epithelial cells; consequently, the property of BP was selective between tumor and control cells. It is noted that the tumor microenvironment is full of stromal cells, and among them, fibroblasts are the main cell types. For melanoma treatment, the tumor microenvironment affected the therapeutic efficacy due to immunosuppression that was contributed by the tumor associated fibroblasts [20][21][22]. Our results revealed that BP inhibited not only fibroblast sarcoma (K-balb) but also normal fibroblasts (Balb/3T3) in vitro, suggesting BP might have a possibility in targeting fibroblast cells to modulate the tumor microenvironments contributing to a clinical response.
Moreover, another strategy for melanoma treatment has described that lipid nanoparticles provide advantages as a drug delivery system, such as physical stability, controlled release, and good skin permeation. A previous study has found that the LPPC delivery system offered better skin penetration and drug accumulation to improve the therapeutic efficacy in breast cancer [16]. After LPPC carrier encapsulation, LPPC was able to maintain the antimelanoma activity of BP and enhance the tumor cytotoxicity of melanoma cells. Moreover, LPPC encapsulation reinforced the inhibitory effects on both fibroblast sarcoma (K-balb) but also normal fibroblasts (Balb/3T3), indicating LPPC encapsulated BP could improve the anti-fibroblast activity, which ranged from 3.35 to 3.7-fold.
To further investigate the anti-melanoma mechanisms of BP and BP/LLPC, the results of our study indicated that BP and BP/LPPC induced cell cycle arrest by mediated cell cycle regulators' protein expressions. The data revealed that BP suppressed the protein expression of total RB and phosphorylated RB and downstream CDK4 as well as cyclin D1, contributing to cell cycle arrest at G 0 /G 1 phase. Additionally, BP also activated phosphorylated p-P53 and P21 protein expressions that both led to cell cycle progression blocking. Meanwhile, after BP/LPPC or BP treatment, BP as well as BP/LPPC was observed to induce cell death via cell apoptosis and BP was able to regulate the extrinsic and intrinsic apoptotic protein expressions that contribute to caspase cascade activation. Moreover, comparing the drug concentration of BP and BP/LPPC, BP/LPPC was lower in tumor cells that indicated LPPC encapsulation, and thus antitumor efficiency was enhanced. In contrast, BP/LPPC was quicker than BP to induce cell cycle arrest and activate cell apoptosis. The clinical drug 5-FU usually induces strong side effects in patients such as bone marrow suppression; however, BP alone and BP encapsulated by LPPC were less toxic to normal cells. When BP/LPPC was combined with 5-FU, the same concentration of 5-FU led to a decrease in the cell viability of melanoma cells, demonstrating that BP/LPPC had synergistic effects when combined with 5-FU.
Although drawbacks of BP, such as poor water solubility and interaction with serum protein, reduce its scope of medicinal application, nanoparticle drug delivery systems are an alternative method to solve these problems. Several nanoparticles have been developed to improve water solubility and reduce drug interactions with serum albumin; such interactions enhance the stability of the drug [13][14][15][16][17]. The role of LPPC in BP encapsulation was to prevent BP from hydrated or protein-rich environments to stabilize its anticancer activity. The data revealed that during BP/LPPC incubation, LPPC with a positive charge was more easily internalized by tumor cells than control cells. The endocytosis pathway was considered to have been activated by LPPC with a positive charge; therefore, the data indicated that LPPC may have caused the clathrin-mediated, caveolae-mediated, clathrin-independent, caveolae-independent, and micropinocytosis pathways to absorb more BP into tumor cells, thereby causing death. The present study demonstrated that BP and BP/LPPC induced cell cycle arrest and cell apoptosis. The drawbacks of BP were alleviated through LPPC encapsulation to enhance the bioavailability and cytotoxicity of BP to tumor cells.
In conclusion, our study demonstrated that BP possessed cytotoxic activity to melanoma cells and fibroblast sarcoma cells. LPPC carrier encapsulation indeed improves the bioavailability of BP and enhances the toxic activity of BP on tumor cells. Taken together, BP and BP/LPPC inhibited melanoma cells through blocking cell cycle progression and activating cell apoptosis. As a result, BP and BP/LPPC provided a new way to treat melanoma.