Back to Journals » International Journal of Nanomedicine » Volume 13

Development of liposomal pemetrexed for enhanced therapy against multidrug resistance mediated by ABCC5 in breast cancer

Authors Bai F, Yin Y, Chen T, Chen J, Ge M, Lu Y, Xie F, Zhang J, Wu K, Liu Y

Received 29 August 2017

Accepted for publication 7 December 2017

Published 6 March 2018 Volume 2018:13 Pages 1327—1339

DOI https://doi.org/10.2147/IJN.S150237

Checked for plagiarism Yes

Review by Single anonymous peer review

Peer reviewer comments 3

Editor who approved publication: Dr Linlin Sun

Download Article [PDF] 

Fang Bai,1–3,* You Yin,4,* Ting Chen,1,* Jihui Chen,1 Meixin Ge,2 Yunshu Lu,2 Fangyuan Xie,5 Jian Zhang,1 Kejin Wu,3 Yan Liu1,6

1Department of Pharmacy, Xinhua Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, 2Department of General Surgery, Xinhua Hospital, Affiliated to Shanghai Jiao Tong University, School of Medicine, Shanghai, 3Department of Breast Surgery, Obstetrics and Gynaecology Hospital, Fudan University, Shanghai, 4Department of Neurology, Changzheng Hospital Affiliated to Second Military Medical University, Shanghai, 5Department of Pharmacy, Shanghai Eastern Hepatobiliary Surgery Hospital, Shanghai, 6Department of Pharmacy, Changzheng Hospital Affiliated to Second Military Medical University, Shanghai, People’s Republic of China

*These authors contributed equally to this work

Purpose: Breast cancer is the most common cancer among women. Pemetrexed, a new generation antifolate drug, is one of the primary treatments for breast cancer. However, multidrug resistance (MDR) in breast cancer greatly hampers the therapeutic efficacy of chemotherapies such as pemetrexed. Nanomedicine is emerging as a promising alternative technique to overcome cancer MDR. Thus, pemetrexed-loaded d-alpha tocopheryl polyethylene glycol 1000 succinate (vitamin E TPGS) liposomes (liposomal pemetrexed) were developed as a strategy to overcome MDR to pemetrexed in breast cancer.
Materials and methods: Liposomal pemetrexed was developed using the calcium acetate gradient method. The cytotoxic effects, apoptosis-inducing activity, in vivo distribution, and antitumor activity of liposomal pemetrexed were investigated.
Results: Liposomal pemetrexed was small in size (160.77 nm), with a small polydispersity of <0.1. The encapsulation efficacy of liposomal pemetrexed was 63.5%, which is rather high for water-soluble drugs in liposomes. The IC50 of liposomal pemetrexed following treatment with MDR breast cancer cells (MCF-7 cells overexpressing ABCC5) was 2.6-fold more effective than pemetrexed. The in vivo biodistribution study showed that the liposomes significantly accumulated in tumors 24 h after injection. The antitumor assay in mice bearing MDR breast cancer xenograft tumors confirmed the superior antitumor activity of liposomal pemetrexed over pemetrexed. It was also found that the improved therapeutic effect of liposomal pemetrexed may be attributed to apoptosis through both extrinsic and intrinsic pathways.
Conclusion: Liposomal pemetrexed represents a potential therapeutic approach for overcoming breast cancer MDR.

Keywords:
cancer nanotechnology, breast cancer, multidrug resistance, pemetrexed, liposomes

Introduction

Data from GLOBOCAN indicate that there were 1,677,000 new cases and 522,000 deaths from breast cancer worldwide in 2012, making it the most frequent cancer among women.1 Chemotherapy remains one of the primary treatments for breast cancer. Pemetrexed is a new generation antifolate drug that has multitargeted activity toward different enzymes, specifically thymidylate synthase, dihydrofolate reductase, and glycinamide ribonucleotide formyl transferase.2 Pemetrexed has shown superior efficacy in five Phase II trials in locally advanced or metastatic breast cancer.3 This drug has demonstrated a response rate of ~30% in advanced breast cancer patients with minimal or no prior chemotherapy.3

Despite evidence of efficacy, development of multidrug resistance (MDR) to chemotherapy represents a major obstacle.4 MDR in cancer, referring to the ability of cancer cells to survive following treatment with a wide range of chemotherapeutics, is one of the most important problems in chemotherapy.4 In drug-resistant cancer cells, there exists overexpression of drug efflux pump proteins, comprising members of the ATP-binding cassette (ABC) superfamily, including P-glycoproteins (P-gp/ABCB1), breast cancer resistance proteins (BCRP, also named ABCG2) and ABCC subfamily (such as ABCC1-12).57 These proteins significantly decrease the intracellular concentration of chemotherapeutics, which is widely thought to be the most common underlying basis of MDR.8

Drug efflux pump proteins, including BCRP and ABCC11/MRP8, have been reported to be associated with drug resistance to pemetrexed in a variety of cancers including breast cancer.9,10 ABCC5, an ATP-dependent transporter, has been found to be overexpressed in breast cancer skeletal metastasis relative to primary breast tumors.11 Studies also showed that patients with high expression of ABCC5 tended to have a worse prognosis.12,13 In our previous studies, we found that ABCC5 was significantly associated with breast cancer chemotherapy resistance to pemetrexed, and breast cancer cells transfected with ABCC5 demonstrated MDR properties (unpublished data, 2015).

Nanomedicine offers an unprecedented opportunity for rational drug delivery in cancer treatment and has emerged as an innovative and promising alternative technique to overcome MDR.12 Nanomedicine possesses the intrinsic ability to overcome MDR through direct inhibition of P-gp using surfactants, evasion of MDR, that depends on drug efflux pump proteins via endocytosis or phagocytosis, the effect on cell apoptosis, inhibition of cellular defensive mechanisms, drug detoxification system, or DNA synthesis, replication, and repair. The nonionic surfactants, including d-alpha tocopheryl polyethylene glycol 1000 succinate (vitamin E TPGS) that can form hydrogen bonds with P-gp may be used to avoid recognition by P-gp, thereby increasing cellular uptake of the formulated drug.12 Several nanocarriers such as liposomes and polymeric or metallic nanoparticles have been engineered to evade or overcome drug efflux through drug transporters, resulting in enhanced drug retention in cancer cells.8 Thus, we aimed to develop pemetrexed-loaded TPGS liposomes to overcome drug resistance in breast cancer.

Pemetrexed disodium is a water-soluble drug, and evidence suggests that the encapsulation efficacy (EE) of water-soluble drugs in liposomes is very low when formulated using conventional lipid-hydration methods, due to the small aqueous internal volume of liposomes.14 Active loading approaches, including pH gradient loading, is a promising method to load drugs with high EE.15 One pH gradient loading technique, the calcium acetate gradient method, has been found to be suitable for loading amphipathic weak acids into liposomes.15 Considering that pemetrexed disodium is an amphipathic weak acid, we hypothesized that this compound can be effectively loaded into liposomes with high EE in response to transmembrane calcium acetate gradients.

In this study, pemetrexed-loaded TPGS liposomes were developed using the calcium acetate gradient method. The pemetrexed-loaded TPGS liposomes were characterized according to size, zeta potential, and EE. The cytotoxic effects, apoptosis-inducing activity, in vivo distribution, and antitumor activity of these liposomes were also investigated.

Materials and methods

Materials

Pemetrexed disodium was purchased from Jiangsu Simcere Pharmaceutical R&D Co., Ltd (Nanjing, People’s Republic of China). Hydrogenated soybean phosphatidylcholine (HSPC) was bought from Shanghai Toshisun Biology & Technology Co., Ltd. (Shanghai, People’s Republic of China). Cholesterol was purchased from Sigma-Aldrich Co. (St Louis, MO, USA). Tocopheryl polyethylene glycol 1000 succinate (TPGS) was bought from Sigma-Aldrich Co. Cell Counting Kit-8 (CCK-8) was purchased from Dojindo Laboratories (Kumamoto, Japan).

Cell lines and culture

Human breast cancer MCF-7 cells (Shanghai Institutes for Biological Sciences, Chinese Academy of Medical Sciences, Shanghai, People’s Republic of China) and HEK293 cell lines (ATCC® CRL1573™, purchased from American Type Culture Collection [Manassas, VA, USA]) were grown in Dulbecco’s Modified Eagle’s Medium (Hyclone Co., Ltd., Shanghai, People’s Republic of China) supplemented with 10% fetal bovine serum (Sigma-Aldrich Co.) and 1% antibiotics (100 U/mL penicillin and 100 mg/mL streptomycin) at 37°C under 5% CO2.

Preparation of liposomes

The liposomes were prepared using the film dispersion method. Briefly, liposomes were prepared using a molar ratio of 47.4:42.6:10 for HSPC, cholesterol, and TPGS, respectively. These materials were dissolved in chloroform and mixed together. The mixed solution was evaporated at 65°C for 2 h to form a thin lipid film. Subsequently, 0.6 mM calcium acetate solution (dissolved in distilled water) was slowly injected and distilled for 1 h. Then, the liposomes were subsequently extruded through membrane filters with pore sizes of 200 nm (twice) and 100 nm (twice) nm using an extruder device (Avestin, Ottawa, ON, Canada) at 55°C. Following this, excess calcium acetate was removed by dialyzing against saline for 12 h using a dialysis membrane (molecular weight cut-off [MWCO] 3500, Spectra/Por®) (Spectrum Laboratories Inc., Rancho Dominguez, CA, USA). Thereafter, the pemetrexed solution (10 mg/mL; dissolved in distilled water) was injected into the dialysis membrane and heated at 60°C for 30 min. After loading, free pemetrexed was removed by dialyzing against phosphate-buffered saline (PBS; 0.01 M, pH 7.4) for 2 h using the dialysis membrane (MWCO 3500) at 25°C. The final liposomes were collected and stored at 4°C.

The size and zeta potential of the liposomes, dispersed in deionized water, were analyzed using the dynamic light scattering (DLS) method, with the Zeta sizer Nano S 90 (Malvern Instruments, Malvern, UK). The pemetrexed EE and drug loading was measured using high-performance liquid chromatography (HPLC; L2000, Hitachi Co., Ltd., Tokyo, Japan) with a C18 column (Nova-Pak C18, 4.6×150 mm, Waters, Guyancourt, France), operated at 25°C at a wavelength of 226 nm. The liposomes were destroyed by a 10.0-fold volume of methanol, and the mobile phase was composed of water with 0.02 mol/L disodium hydrogen phosphate/acetonitrile (85:15) at a flow rate of 1.0 mL/min. The amount of pemetrexed in the liposomes was determined from the peak area correlated with the standard curve. The standard curve of pemetrexed was prepared under conditions identical to those described above. All analyses were performed in triplicate, with an injection volume of 10 μL in the HPLC. The morphology of the liposomes was observed using a transmission electron microscope (TEM; H-600, HITACHI Company, Tokyo, Japan). A drop of dispersed liposome was stratified into a carbon-coated grid and adhered onto the carbon substrate for 30 min. The excess material was removed, and phosphotungstic acid hydrate (2%) was added. After drying, the grid was observed using TEM.

In vitro drug release

The in vitro release of the liposomes was performed as described below. Briefly, 1 mL of the liposome solution was mixed with 9 mL PBS (0.01 M, pH 7.4) and placed in a dialysis membrane (MWCO 3500). Then, the dialysis membrane was introduced into a vial containing 100 mL PBS (0.01 M, pH 7.4). The vial of samples was fixed in a water bath, which was kept at 37°C with constant stirring. At predetermined time intervals, 100 μL of the dialysate was taken and replaced with 100 μL PBS (0.01 M, pH 7.4). The amount of pemetrexed released into the dialysate was determined using the reversed-phase HPLC method. Experiments were carried out for up to 48 h.

Cellular uptake analysis by confocal microscopy

Confocal studies were conducted to compare the in vitro cellular uptake rates between native pemetrexed and liposomal pemetrexed through an endocytic pathway. Briefly, pemetrexed was labeled with europium, which is a radioactive element with excitation and emission at 365 and 610 nm, respectively. MCF-7 cells (2×104) were seeded onto Livefocus culture plates (Jiangsu Biomagsystem Medical Co., Ltd., Jiangsu, People’s Republic of China). After 24 h, the medium was changed with 1 mL of either pemetrexed or liposomal pemetrexed labeled with a concentration of 1 μg/mL and incubated for 1, 2, and 4 h at 37°C. After washing with PBS (0.01 M, pH 7.4) twice, the cells were fixed with 4% paraformaldehyde. Following this, cells were washed twice with PBS (0.01 M, pH 7.4) and stained with 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI; Sigma-Aldrich Co.) for 20 min to visualize the nuclei. Finally, the cells were carefully washed three times with PBS (0.01 M, pH 7.4) and mounted under glass coverslips. The fluorescence was visualized with a confocal microscope (Leica TCS SP5; Leica Microsystems, Wetzlar, Germany) using the 63× oil immersion lens for the detection of the three fluorescent dyes at 370 nm excitation/470 nm emission (blue, DAPI) and 365/610 nm (red, europium).

Generation of adenovirus

For recombinant adenovirus construction, the ABCC5 gene cDNA (Ad-ABCC5) and the green fluorescence protein gene cDNA (Ad-GFP; control) were cloned by polymerase chain reaction and inserted into the pDC315-EGFP vector (Hanbio Co., Ltd., Shanghai, People’s Republic of China), under the control of the mouse cytomegalovirus promoter. The pDC315-ABCC5 and pBHGloxE1,3Cre (33, 34) were cotransfected into HEK293 cells using the LipoFiter™ transfection reagent (Hanbio Co., Ltd.) to generate the recombinant adenoviruses. The adenoviruses were propagated in the HEK293 cells. The recombinant Ad-ABCC5 and Ad-GFP adenoviruses produced in the HEK293 cells were purified, and the virus titer was measured using plaque assays. The stock solutions of Ad-ABCC5 and Ad-GFP used were both 1×1010 plaque formation unit (PFU)/mL.

Cytotoxicity assay

The cytotoxic effects of the liposomal pemetrexed and pemetrexed against the cells were measured using the CCK-8 (Dojindo Laboratories). Briefly, MCF-7 cells were seeded in 96-well plates with a density of 5×103 cells per well. ABCC5 was added to the appropriate wells to obtain a multiplicity of infection (MOI) of 1,000. After 4 h, a series of concentrations of liposomal pemetrexed or pemetrexed were added to these cells. Then, the cells were incubated for 96 h at 37°C. Following this, cytotoxicity was evaluated by adding 10 μL of CCK-8 solution to each well of the plate. After incubation for 4 h, the absorbance was measured at 450 nm using the BioTek ELx800 Universal Microplate Reader (BioTek, Winooski, VT, USA). The cell viability was calculated by the formula: [(AE − AB)/(AC − AB)] ×100%, where AE, AC, and AB were defined as the absorbance of experimental samples, untreated samples, and blank controls, respectively. The IC50 values for liposomal pemetrexed and pemetrexed were calculated using GraphPad Prism version 5 (GraphPad Software, Inc., La Jolla, CA, USA).

Western blot

The effects of liposomal pemetrexed and pemetrexed on MCF-7 cells after 72 h were investigated using Western blot analysis through the detection of bax and caspase-3. Briefly, extracted proteins (80 μg) were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes. Rabbit antihuman bax mAb (final dilution 1:1,000; Abcam, Cambridge, UK), rabbit antihuman caspase-3 mAb (final dilution 1:1,000; Cell Signaling Technology, Danvers, MA, USA), cleaved caspase-3 mAb (final dilution 1:1,000; Cell Signaling Technology), caspase-8 mAb (final dilution 1:1,000; Abcam, Cambridge, UK), cleaved caspase-8 mAb (final dilution 1:1,000; Abcam), caspase-9 mAb (final dilution 1:1,000; Cell Signaling Technology), or cleaved caspase-9 mAb (final dilution 1:1,000; Cell Signaling Technology) were used as the primary antibodies, and the goat anti-rabbit IgG (final dilution 1:2,000; LI-COR Biosciences, Lincoln, NE, USA) was used as the secondary antibody. The bands were visualized using the Image Studio system (LI-COR Biosciences, Lincoln, NE, USA).

Apoptosis assays

Apoptosis measurements were conducted using the Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) apoptosis detection technique for MCF-7 cell lines and standard methods provided by Dojindo Laboratories. Briefly, 1×106 cells were plated in 6-well plates. After drug treatment with native pemetrexed or liposomal pemetrexed (3 μg/mL) for 72 h, cells were harvested by trypsinization and centrifugation at 1,000 rpm for 3 min. Additionally, the cells were washed twice with PBS (0.01 M, pH 7.4). Following this, 100 μL of Annexin V-binding buffer was added to the cells followed by the addition of 5 μL of Annexin V-FITC (1 μg/μL) and 5 μL of PI (10 μg/μL). Cells were then incubated at 25°C room temperature in the dark for 15 min. Apoptosis was determined using flow cytometry (FACSCalibur; BD Biosciences). All experiments were performed in triplicate.

Hoechst 33342 staining

Cell apoptosis caused by liposomal pemetrexed or pemetrexed was analyzed by nuclear staining using Hoechst 33342. MCF-7 cells were seeded in 6-well plates with a density of 1×106 cells per well. ABCC5 was added to the appropriate wells at an MOI of 1,000. After 24 h, liposomal pemetrexed, with pemetrexed at a concentration of 3 μg/mL, was added to the wells. Following 72 h, the cells were washed with PBS for 5 min at room temperature. Then, these cells were fixed with a mixture of methyl alcohol and acetic acid (4:1, 4°C) for 15 min and incubated with Hoechst 33342 (100 ng/mL in PBS) in the dark for 15 min at room temperature. Finally, the cells were washed with PBS for 5 min and observed using a fluorescence microscope.

Animal studies

All Balc/b nude mice (female, 5 weeks, 18 g) were purchased from the Shanghai Super B&K Laboratory Animal Corp. Ltd. (Shanghai, People’s Republic of China) and were placed in a specific pathogen-free environment with free access to food and water. All the mice received care in compliance with the criteria of the National Regulation on the Management of Laboratory Animals. The animal study was approved by the Research Ethics Committee of Xinhua Hospital affiliated with Shanghai Jiao Tong University (Shanghai, People’s Republic of China).

In vivo antitumor assays in mice

On day 0, 1×107 MCF-7 cells were injected subcutaneously into the right armpit of each athymic nude mouse. When tumors were ~100 mm3 in size on day 30, adenoviruses containing ABCC5 (5×109 PFU) were injected into the tumors of the mice to facilitate overexpression of ABCC5 in the tumor cells. As expected, examination of the frozen sections showed the overexpression of ABCC5 in tumors, as indicated by diffused green fluorescence (Figure S1). When tumors were ~150 mm3 in size in each group on day 35, the mice were treated with intravenous injections (via the tail vein) of liposomal pemetrexed (20 mg/kg pemetrexed), pemetrexed (20 mg/kg pemetrexed), or the same dose of PBS from day 35 to 46. The tumor volumes (V) were measured using a caliper once every day (V = width2 × length/2).

In vivo imaging

MCF-7 cells (1×107) were injected subcutaneously into the right armpit of nude mice. When tumors were ~500–600 mm3 in size, the in vivo fluorescence imaging system was used to observe the real-time distribution of DiR-loaded liposomes. Mice were administered the DiR-loaded liposomes, at DiR doses of 0.2 mg/kg, via tail vein injection, and scanned at 1, 4, and 24 h using the Bruker MI SE (Bruker Biospin Corporation, Woodbridge, CT, USA). Meanwhile, free DiR at a dose of 0.2 mg/kg was injected via the tail vein of mice and scanned at 24 h. Then, the mice were killed via cervical dislocation at 24 h, and the tumor, heart, liver, lungs, and kidneys were harvested for ex vivo imaging.

Statistical analysis

Data in this study were analyzed using the statistic package SPSS 18.0 (SPSS Inc., Chicago, IL, USA). Student’s t-test (two-tailed) was used to conduct statistical analyses. Data are expressed as mean ± SD and P-values <0.05 were considered significant.

Results

Characterization of liposomes

The data for size, zeta potential, drug loading, and EE of liposomes are summarized in Table 1. Liposomal pemetrexed were small in size (160.77 nm) and had a small polydispersity of <0.1, suggesting that the size distribution of liposomal pemetrexed was rather homogenous (Table 1). TEM revealed that liposomal pemetrexed was spherical in shape, with a size of ~100 nm and good dispersion (Figure 1A). Notably, the size of liposomal pemetrexed observed by TEM was smaller than that obtained from the DLS experiment (approximately one-third decrease). The reason is that the size of liposomal pemetrexed obtained from the DLS experiment reflected the hydrodynamic size, whereas TEM reflected the size of dried liposomal pemetrexed. Thus, the observed decrease in size was expected, since the hydration shell portion of the liposomal pemetrexed was eliminated in TEM. The nearly neutral zeta potential (−1.40 mV) indicated that liposomal pemetrexed are less likely to interact with environmental factors through electrical interaction, resulting in good stability in circulation. The liposomal pemetrexed EE was found to be 63.5%, which is rather high for water-soluble drugs in liposomes.

Table 1 Characteristics of liposomes
Note: Data are expressed as mean ± SD (n=3).
Abbreviations: EE, encapsulation efficacy; PDI, polydispersity.

Figure 1 Characterization of liposomal pemetrexed. (A) Transmission electronic microscopy image of liposomal pemetrexed. Scale bar represents 200 nm. (B) In vitro release kinetics of pemetrexed from liposomal pemetrexed dissolved in PBS (0.1 M, pH 7.4). The release of pemetrexed was monitored using reversed-phase HPLC at different time periods. Data are presented as mean ± SD (n=3).
Abbreviations: HPLC, high-performance liquid chromatography; PBS, phosphate-buffered saline.

In vitro release

In vitro release of the drug from liposomal pemetrexed was measured in physiological buffer conditions. As shown in Figure 1B, the release kinetics occurred in a biphasic manner with an initial quick release followed by a slow and continuous release. The total cumulative amount of pemetrexed released from the liposome pemetrexed was ~76% during the 48-h experimental period.

Cellular uptake analysis by confocal microscopy

In an effort to compare the cellular uptake efficiency between liposomal pemetrexed and pemetrexed, a time-dependent cellular uptake study was performed on MCF-7 cells using confocal microscopy. As shown in Figure 2A, the red fluorescence signal for free pemetrexed was barely detectable, suggesting that uptake of pemetrexed was minimal when in the form of free pemetrexed. Notably, when encapsulated in liposomes, the red fluorescence of pemetrexed significantly increased (Figure 2B). There were no significant discrepancies in red fluorescence of liposomal pemetrexed among the different time points (1, 2, and 4 h), suggesting that liposomal pemetrexed entered MCF-7 cells and reached a plateau within a short period of time. Similar results were obtained in MCF-7+ABCC5 cells, which were transfected by adenoviruses containing ABCC5 (Figure 3).

Figure 2 In vitro cellular uptake of (A) pemetrexed and (B) liposomal pemetrexed in MCF-7 breast cancer cells examined using a confocal microscope. Nuclei were stained with DAPI (blue). Pemetrexed was labeled with europium (red). Scale bars represent 50 μm.
Abbreviation: DAPI, 4′,6-diamidino-2-phenylindole dihydrochloride.

Figure 3 In vitro cellular uptake of (A) pemetrexed and (B) liposomal pemetrexed in MCF-7+ABCC5 breast cancer cells examined using a confocal microscope. Nuclei were stained with DAPI (blue). Pemetrexed was labeled with europium (red). Scale bars represent 50 μm.
Abbreviation: DAPI, 4′,6-diamidino-2-phenylindole dihydrochloride.

Transfection of adenovirus

As shown in Figure S2, Western blot demonstrated that ABCC5 expression was significantly upregulated in MCF-7 cells, after adenoviruses containing ABCC5 were transfected into these cells. The MCF-7 cells transfected with ABCC5 were defined as MCF-7+ABCC5. The upregulation of ABCC5 confers MDR in MCF-7 cells.57

Cytotoxicity assays

The cytotoxic effects of drugs were evaluated in MCF-7 and MCF-7+ABCC5 cells using the CCK-8 assay. Both liposomal pemetrexed and pemetrexed showed dose-dependent inhibition of cell proliferation in MCF-7 and MCF-7+ABCC5 cells (Figure 4). Compared with pemetrexed, liposomal pemetrexed showed a left shift of the dose-response curve, suggesting that the cytotoxic effect of liposomal pemetrexed was higher than pemetrexed.

Figure 4 The dose-dependent cytotoxicity induced by pemetrexed and liposomal pemetrexed in (A) MCF-7 and (B) MCF-7+ABCC5 cells. The inhibition rate was calculated relative to the respective controls. Data are presented as mean ± SD (n=3). *P<0.05 for pemetrexed vs liposomal pemetrexed.

To calculate the cytotoxic effects more precisely, we calculated the IC50 of drugs in Table 2. The IC50 of pemetrexed in MCF-7+ABCC5 cells was increased by 2.2-fold compared to that in MCF-7 cells, suggesting that MCF-7 cells showed significant drug resistance following ABCC5 transfection. Notably, the IC50 of liposomal pemetrexed (0.026 μg/mL) was reduced by 6.4-fold compared to that of pemetrexed (0.167 μg/mL) in MCF-7 cells, whereas the IC50 of liposomal pemetrexed (0.145 μg/mL) was reduced by 2.6-fold compared to that of pemetrexed (0.37 μg/mL) in MCF-7+ABCC5 cells. Taken together, the cytotoxic effect of liposomal pemetrexed was significantly higher than pemetrexed in both MCF-7 and MCF-7+ABCC5 cells.

Table 2 IC50 values (μg/mL) of the CCK-8 assay performed during pemetrexed and liposomal pemetrexed treatment for 5 days in MCF-7 and MCF-7+ABCC5 cells
Notes: The drug concentration that caused a 50% inhibition of the control growth rate (IC50) was calculated from a sigmoid plot by nonlinear regression analysis using the generated equation. Data are expressed as mean ± SD.

Apoptosis assays

The effect of pemetrexed on induction of apoptosis in MCF-7 cells was measured using the Annexin V-FITC/PI staining technique. As shown in Figure 5A, treatment with liposomal pemetrexed induced a significantly higher percentage of cell apoptosis (20.7%) than pemetrexed (11.2%). To further investigate the detailed mechanism underlying apoptosis induced by liposomal pemetrexed, the expression levels of the caspase-3, cleaved caspase-3, and bax were measured using Western blot. As shown in Figure 5B, the expression levels of the caspase-3, cleaved caspase-3/8/9, and bax increased after treatment with liposomal pemetrexed, suggesting that liposomal pemetrexed induced apoptosis via extrinsic and intrinsic pathways.

Figure 5 Cell apoptosis induced by pemetrexed or liposomal pemetrexed. (A) Cell apoptosis rate of MCF-7 cells measured by flow cytometry. MCF-7 cells were treated with pemetrexed or liposomal pemetrexed (3 μg/mL) for 72 h and stained with Annexin V-FITC and PI. Data are presented as mean ± SD (n=3). *P<0.05. (B) The expression of bax, caspase-3, -8, and -9 proteins in MCF-7 cells were detected after incubation with pemetrexed or liposomal pemetrexed with a 3 μg/mL concentration for 72 h. An equal amount of cell lysate (20 μg of protein/lane) was analyzed using Western blot with glyceraldehyde-3-phosphate dehydrogenase as the loading control. (C) Images of MCF-7 cells stained with Hoechst 33342. MCF-7 cells were treated with pemetrexed or liposomal pemetrexed (3 μg/mL) for 48 h and then stained with Hoechst 33342. Scale bars represent 50 μm.
Abbreviations: Blank, no drugs; Pem, pemetrexed; Lipo-pem, liposomal pemetrexed; FITC, fluorescein isothiocyanate; PI, propidium iodide.

Further, we also employed Hoechst 33342, a fluorescent dye that specifically stains nucleic acids, to measure apoptosis induced by pemetrexed and liposomal pemetrexed. As shown in Figure 5C, untreated MCF-7 and MCF-7+ABCC5 cells showed regular and round nuclei. However, after treatment with pemetrexed or liposomal pemetrexed, the nuclei of the cells became crimped and condensed, displaying a bright blue color. Compared with pemetrexed, liposomal pemetrexed clearly induced more apoptotic cells displaying condensed, bright-colored nuclei, suggesting that liposomal pemetrexed possessed greater apoptotic-inducing activity than pemetrexed.

In vivo imaging in mice

To trace the distribution of liposomal pemetrexed in nude mice bearing tumor xenografts, we loaded the fluorescent DiR dye into the liposomes. DiR dye is a superior fluorescent dye that we used to trace the distribution of nanoparticles in our previous studies.15 Figure 6A shows the real-time distribution of DiR-loaded liposomes or free DiR in nude mice bearing tumor xenografts. Mice that did not receive any DiR dye were used as controls. The free DiR injected into mice did not significantly accumulate in the tumors. Notably, after intravenous injection of DiR-loaded liposomes, accumulation in tumors occurred within 3 h. This gradually increased and reached significant accumulation at 24 h, indicated by the strong fluorescence signal observed around the tumor mass.

Figure 6 Distribution of DiR-loaded liposomes in nude mice bearing MCF-7 tumors. MCF-7 cells (1×107) were injected subcutaneously into the right armpit of nude mice. When tumors were ~500–600 mm3 in size, the in vivo fluorescence imaging system was used to observe the real-time distribution of DiR-loaded liposomes. Mice were administered DiR-loaded liposomes or free DiR, with a DiR dose of 0.2 mg/kg, via tail vein injection, and scanned at 1, 4, and 24 h using the Bruker MI SE (Bruker Biospin Corporation, Woodbridge, CT, USA). Then, the mice were sacrificed via cervical dislocation at 24 h, and the tumor, heart, liver, lungs, and kidneys were harvested for ex vivo imaging. (A) The real-time distribution of DiR or DiR-loaded liposomes in nude mice. (B) Ex vivo fluorescence images of different organs harvested from the mice at 24 h postinjection.
Abbreviation: Blank, no drugs.

To clearly observe fluorescence signals, the tumors and major organs were excised and collected at 24 h following injection. The ex vivo fluorescent images further demonstrated the significant accumulation of DiR-loaded liposomes in excised tumors (Figure 6B). No obvious fluorescence uptake was found in the hearts, lungs, spleens, and kidneys, with primarily background-to-moderate signals observed.

The inhibition of tumor growth by liposomes in vivo

The therapeutic effects of liposomal pemetrexed and pemetrexed were evaluated in mice bearing MCF-7+ABCC5 tumor xenografts (Figure 7). As shown in Figure 7A, liposomal pemetrexed treatment resulted in a 115.5% decrease in tumor volume, whereas pemetrexed treatment resulted in a 30.9% decrease in tumor volume. By the end of the experiment, compared with the initial tumor volume (150 mm3), the tumor volume in untreated mice had increased by 5.3-fold, and the tumor volume in the pemetrexed-treated mice had increased by 4.0-fold. The tumor volume in the liposomal pemetrexed-treated mice increased by 2.5-fold. The tumor volume of the liposomal pemetrexed-treated group was significantly smaller than that of the other groups (untreated =793 mm3, pemetrexed =606 mm3, liposomal pemetrexed =368 mm3; liposomal pemetrexed vs untreated: P<0.01, liposomal pemetrexed vs pemetrexed: P<0.01, pemetrexed vs untreated: P<0.01). Additionally, the tumor weight of the liposomal pemetrexed-treated group was significantly lower than the other two groups (P<0.01) (Figure 7B).

Figure 7 In vivo antitumor study in mice bearing subcutaneous breast cancer xenografts. For therapeutic experiments, tumor growth was induced by subcutaneous injection of 1×107 MCF-7 cells on the right armpit. After 30 days, when tumor growth was over 100 mm3, adenovirus containing ABCC5 was injected into the tumors of the mice. After 35 days, when tumor growth was over 150 mm3, mice were injected with the drugs (20 mg/kg pemetrexed or liposomal pemetrexed) via the tail vein once a day for 10 days. Tumors were measured with a caliper. (A) The tumor growth curve. (B) The excised tumors were weighed at the end point. The tumor weights of each group were compared using two-tailed unpaired t-tests. Data are presented as mean ± SD (n=6). (C) Images of excised tumors of each group at the end point. *P<0.01.
Abbreviations: Pem, pemetrexed; Lipo-pem, liposomal pemetrexed.

Discussion

MDR in cancer is one of the most significant obstacles in chemotherapy.4 Thus, overcoming MDR has great potential in increasing the therapeutic efficacy of cancer therapy. Recently, nanomedicine approaches have emerged as a novel strategy to overcome cancer drug resistance. In this study, we developed pemetrexed-loaded TPGS liposomes using the calcium acetate gradient method as a potential strategy to overcome MDR to pemetrexed for breast cancer treatment. Liposomal pemetrexed was observed to be small in size and possessed high pemetrexed EE, homogenous distributions, and sustained release. Most notably, liposomal pemetrexed was found to efficiently deliver pemetrexed to both breast cancer cells and MDR breast cancer cells, resulting in enhanced therapeutic effects compared to free pemetrexed.

Among the nanocarriers, liposomes are the dominant class of clinically approved nanomedicine products.16 Liposomes, spherical lipid vesicles with single or multiple lipid bilayers, have wide-ranging applications, owing to their high biocompatibility, favorable pharmacokinetic profiles, ease of surface modifications, and long circulation time following surface modification with poly(ethylene glycol).14 Recently, several liposomes have been approved for clinical use, including Doxil®, which was first approved in 1995.17 However, liposomes do have limited clinical utility as a consequence of properties such as uncontrollable drug release, instability in storage, and insufficient drug loading.13 Specifically, due to their small aqueous internal volumes, liposomes have a rather low EE for water-soluble drugs.14 In this study, for the first time, we encapsulated pemetrexed disodium in liposomes with a high EE (63.5%) using the calcium acetate gradient method, which is known to be suitable for loading amphipathic weak acids into liposomes.15 Considering that this method is suitable for industrialized large-scale production, liposomal pemetrexed has great pharmaceutical potential in this respect.

The data presented here confirmed that liposomal pemetrexed was efficiently bound and delivered to both MCF-7 and MCF-7+ABCC5 cells. Due to more effective internalization, liposomal pemetrexed also demonstrated higher cytotoxic effects on both MCF-7 and MCF-7+ABCC5 cells. Compared with pemetrexed, the IC50 of liposomal pemetrexed was reduced by 6.4-fold in MCF-7 cells and 2.2-fold in MCF-7+ABCC5 cells, suggesting that liposomal pemetrexed showed enhanced cytotoxic activity against both breast cancer cells and MDR breast cancer cells. The detailed molecular mechanisms underlying the enhanced cytotoxic effects of liposomal pemetrexed could at least be partially attributed to apoptosis via both extrinsic and intrinsic pathways.

The in vivo biodistribution study showed that DiR-loaded liposomes significantly accumulated in tumors 24 h after injection. In animal studies, liposomal pemetrexed treatment in MCF-7+ABCC5 xenograft models was associated with higher anti-tumor effects than free pemetrexed treatment. Our findings further elucidated the mechanisms by which liposomal pemetrexed function to exert its antitumor activity. First, long circulating liposomal pemetrexed accumulated in tumors due to the enhanced permeability and retention effect. Subsequently, liposomal pemetrexed was bound to and internalized in tumor cells. After internalization, liposomal pemetrexed readily released pemetrexed into the cytoplasm, resulting in significant therapeutic effects.

One of the strengths of our study was that the MCF-7 cells overexpressing ABCC5 were used as a breast cancer resistance cellular model. In our previous studies, we found that ABCC5 was significantly associated with breast cancer chemotherapy resistance to pemetrexed, and breast cancer cells transfected with ABCC5 exhibited MDR properties (unpublished data, 2015). Although MCF-7ADR cells, characterized by long durations of adriamycin (Dalian Meilun Biotech Co., Ltd., Dalian, People’s Republic of China) stimulation, are typically used as breast cancer MDR cellular models, we have found that MCF7/ADR cells are hard to culture and undergo gradual cell death when cultured for long periods of time. In contrast, our established MCF-7+ABCC5 cells were easy to culture with good cellular conditions. Importantly, these cells demonstrated MDR properties, suggesting that they are feasible candidates for MDR cell models.

Employing liposomes to overcome MDR in breast cancer may be a novel and effective approach to reversing MDR. Common strategies to overcome MDR include modification of chemotherapy regimens and using inhibitors of the ABC family to reverse related drug resistance. However, modification of chemotherapy regimens may not ameliorate adverse events and has even been found to enhance the side effects associated with chemotherapy.4 As well, many ABC transporter inhibitors have shown negative or modestly positive efficacy. This may be due to the fact that inhibitors are generally weak and nonspecific, and undesirable pharmacokinetic interactions exist between chemotherapeutics and the ABC transporter inhibitors.4,12,18 Nanomedicine offers an innovative and promising alternative approach due to its intrinsic ability to overcome MDR and avoid recognition by the ABC protein family via nonionic surfactants such as TPGS. Notably, nanomedicine has the great potential to reduce the side effects associated with chemotherapy drugs, whereas conventional strategies to overcome cancer MDR, including combinations of chemotherapy drugs and ABC transporter inhibitors, have a greater risk of producing side effects. Thus, liposomal pemetrexed represents a potential therapeutic approach to overcome cancer MDR.

Conclusion

Liposomal pemetrexed offers the possibility of delivering pemetrexed to MDR cells and demonstrated an improved therapeutic effect against MDR cells. Our results suggest that the improved therapeutic effect of liposomal pemetrexed in MDR cells may be attributed to apoptosis through both extrinsic and intrinsic pathways. Thus, it is reasonable to suggest that MDR treatment with liposomal pemetrexed may achieve favorable clinical efficacy, since liposomal pemetrexed can effectively destroy MDR cells. We believe that liposomal pemetrexed can shed new light on MDR cancer in the near future, and patients with MDR cancer may significantly benefit from this treatment strategy.

Acknowledgment

The authors thank Dr Jie Gao and Dr Chuan Yin for editing the entire manuscript. This work was financially supported by the Shanghai Municipal Commission of Health and Family Planning Foundation (No 20154Y0077), the Science and Technology Commission of Shanghai Municipality (No 12JC1406800, No 14411950206 and No 17411950104), the Shanghai Key Specialty Project of Clinical Pharmacy (No 2016-40044-002), the National Natural Science Foundation of China (No 81771964) and the Chinese Naval Medical University military medical innovation (No 2017JS07).

Disclosure

The authors report no conflicts of interest in this work.


References

1.

Ferlay J, Soerjomataram I, Dikshit R, et al. Cancer incidence and mortality worldwide: sources, methods and major patterns in GLOBOCAN 2012. Int J Cancer. 2015;136(5):E359–E386.

2.

Chattopadhyay S, Moran RG, Goldman ID. Pemetrexed: biochemical and cellular pharmacology, mechanisms, and clinical applications. Mol Cancer Ther. 2007;6(2):404–417.

3.

Martin M. Clinical experience with pemetrexed in breast cancer. Semin Oncol. 2006;33(1 Suppl 2):S15–S18.

4.

Dong X, Mumper RJ. Nanomedicinal strategies to treat multidrug-resistant tumors: current progress. Nanomedicine (Lond). 2010;5(4):597–615.

5.

Gottesman MM, Fojo T, Bates SE. Multidrug resistance in cancer: role of ATP-dependent transporters. Nat Rev Cancer. 2002;2(1):48–58.

6.

Schinkel AH, Jonker JW. Mammalian drug efflux transporters of the ATP binding cassette (ABC) family: an overview. Adv Drug Deliv Rev. 2003;55(1):3–29.

7.

Ravna AW, Sylte I, Sager G. A molecular model of a putative substrate releasing conformation of multidrug resistance protein 5 (MRP5). Eur J Med Chem. 2008;43(11):2557–2567.

8.

Dean M, Fojo T, Bates S. Tumour stem cells and drug resistance. Nat Rev Cancer. 2005;5(4):275–284.

9.

Li L, Sham YY, Bikadi Z, Elmquist WF. pH-Dependent transport of pemetrexed by breast cancer resistance protein. Drug Metab Dispos. 2011;39(9):1478–1485.

10.

Uemura T, Oguri T, Ozasa H, et al. ABCC11/MRP8 confers pemetrexed resistance in lung cancer. Cancer Sci. 2010;101(11):2404–2410.

11.

Mourskaia AA, Amir E, Dong Z, et al. ABCC5 supports osteoclast formation and promotes breast cancer metastasis to bone. Breast Cancer Res. 2012;14(6):R149.

12.

Gao J, Feng SS, Guo Y. Nanomedicine against multidrug resistance in cancer treatment. Nanomedicine (Lond). 2012;7(4):465–468.

13.

Wicki A, Witzigmann D, Balasubramanian V, Huwyler J. Nanomedicine in cancer therapy: challenges, opportunities, and clinical applications. J Control Release. 2015;200:138–157.

14.

Akbarzadeh A, Rezaei-Sadabady R, Davaran S, et al. Liposome: classification, preparation, and applications. Nanoscale Res Lett. 2013;8(1):102.

15.

Clerc S, Barenholz Y. Loading of amphipathic weak acids into liposomes in response to transmembrane calcium acetate gradients. Biochim Biophys Acta. 1995;1240(2):257–265.

16.

Peer D, Karp JM, Hong S, Farokhzad OC, Margalit R, Langer R. Nanocarriers as an emerging platform for cancer therapy. Nat Nanotechnol. 2007;2(12):751–760.

17.

Zhu Y, Yu F, Jiao Y, et al. Reduced miR-128 in breast tumor-initiating cells induces chemotherapeutic resistance via Bmi-1 and ABCC5. Clin Cancer Res. 2011;17(22):7105–7115.

18.

Greenberg PL, Lee SJ, Advani R, et al. Mitoxantrone, etoposide, and cytarabine with or without valspodar in patients with relapsed or refractory acute myeloid leukemia and high-risk myelodysplastic syndrome: a phase III trial (E2995). J Clin Oncol. 2004;22(6):1078–1086.

Supplementary materials

Figure S1 Viruses transfection into the tumors of nude mice (×200). Thirty days after injection when the tumor growth was over 100 mm3, adenovirus contained ABCC5 was injected into the tumors of the mice (twice for each tumor, each time with 5×10^9 PFU). The frozen sections of the tumors demonstrated the overexpression of ABCC5 in the tumors by the diffused green GFP fluorescence.
Abbreviations: DAPI, 4′,6-diamidino-2-phenylindole dihydrochloride; PFU, plaque formation unit; GFP, green fluorescent protein; Blank, no drugs.

Figure S2 Expression of ABCC5 protein of MCF-7 cells transfected with adenoviruses containing ABCC5 and the MCF-7 cells without transfection. GAPDH is used an internal control. The expression of ABCC5 was measured by Western blot. The bands were visualized with the Image Studio system (LI-COR Biosciences; Lincoln, NE, USA).
Abbreviation: GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Creative Commons License This work is published and licensed by Dove Medical Press Limited. The full terms of this license are available at https://www.dovepress.com/terms.php and incorporate the Creative Commons Attribution - Non Commercial (unported, v3.0) License. By accessing the work you hereby accept the Terms. Non-commercial uses of the work are permitted without any further permission from Dove Medical Press Limited, provided the work is properly attributed. For permission for commercial use of this work, please see paragraphs 4.2 and 5 of our Terms.

Download Article [PDF]