Methotrexate hydrate, gemcitabine, cytosine β-d-arabinofuranoside, doxorubicin, and 2′-deoxycytidine hydrochloride were obtained from Aladdin (Shanghai, China). DSPE-PEG2k-COOH was supplied by Ruixi Biological Technology Co., Ltd (Xi’an, China). Soy lecithin and cholesterol were purchased from A.V.T Pharmaceutical Co., Ltd. (Shanghai, China). Dialysis tubes (MW: 3500 D) were from Spectrum Laboratories, Inc. (CA, USA). Dimethyl sulfoxide (DMSO), IR-780 iodide, and MTT were obtained from Sigma Aldrich (MO, USA). DMEM, RPMI-1640, fetal bovine serum (FBS), penicillin G sodium, and streptomycin sulfate were obtained from Gibco BRL (MD, USA). The FITC Annexin V Apoptosis Detection Kit I and the PI/RNase Staining kit were from BD Biosciences (CA, USA). MitoProbeTM JC-1 Assay kit and DAPI were purchased from Thermo Fisher Scientific (MA, USA). Protein extraction kit is obtained from Beyotime Biotechnology Co., Ltd. (Shanghai, China). All other compounds were analytical grade, and a Millipore system was used to purify water.
Cell Culture and Animals
JEG-3 (human chorionic carcinoma cells) and HepG2 (human liver cancer cells) were cultured in Eagle's Minimum Essential Medium or DMEM containing 10% FBS and penicillin/streptomycin (100 U/mL), respectively. Pricell-0051 (normal human placental trophoblast cells) and MCF-7 (Human breast cancer cells) cell lines were cultured in RPMI-1640 containing FBS and penicillin/streptomycin (100 U/mL) in a 37 °C 5% CO2 incubator. Female Sprague-Dawley (SD) rats (220±20 g) and female nude BALB/c mice (20±2 g) were from the Laboratory Animal Center of Zhejiang Chinese Medical University. The Scientific Investigation Board of Zhejiang Chinese Medical University approved all animal studies, which were consistent with NIH ethical guidelines.
High expression of ENT1 in JEG-3 cells
Firstly, the mRNA expression of various kinds of cell membrane transporters were evaluated in JEG-3 cells, including human equilibrative nucleoside transporter 1 (ENT1), concentrative nucleoside transporters (CNT), organic anion transporters (OATs), organic cation transporters (OCTs), carnitine/organic cation transporters (OCTNs), multidrug resistance-associated protein (MRPs), p-glycoprotein (p-gp), and breast cancer resistance protein (BCRPs). JEG-3 cells were grown to about 80% confluence in 10 cm dish and collected for RT-PCR analysis. Total RNA was extracted from cultured JEG-3 cells using TRIzol reagent (Ambion, Thermo Fisher Scientific, USA). High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Carlsbad, USA) was employed to prepare cDNA from RNA (2 µg), and cDNA was analyzed via RT-PCR with SYBR Premix Ex Taq (Takara, Dalian, China). The PCR primers for ENT1 were 5′-ATCTGCGCTATTGCCAGTG-3′ (forward) and 5′-TCCAACTTGGTCTCCTGCTC-3′ (reverse). For mRNA analysis of other transporters in JEG-3 cells refer to the above steps.
For western blot analysis, JEG-3 cells, MCF-7 cells, and HepG2 cells were plated overnight in 6-well plates. The cell lysates were collected and assessed via 13% denaturing polyacrylamide gel electrophoresis. An ultrasonic cell disruptor was used to extract proteins from cells, after which a Bio-Rad Electrophoresis instrument was used for quantification. Proteins were then separated via SDS-PAGE, transferred to PVDF membranes, and incubated with primary antibodies overnight. Secondary antibodies were then used to probe blots. A gel imaging system was then used for protein detection.
Specific uptake of cytarabine into the cells
JEG-3 cells, MCF-7 cells, and HepG2 cells were plated overnight in 6-well plates. Then, the adhered cells are treated with Cy (5 μM) for 30 mins or 60 mins. Then, cells were washed with PBS for five times for moving the free drug in medium. After that, the cells are lysed and treated with acetonitrile for precipitating protein. Finally, the content of Cy was detected by LC/MS (8050, SHIMADZU, Japan).
Synthesis and characterization of DSPE-PEG2k-Cy
Firstly, 140.00 mg of DSPE-PEG2k-COOH was dissolved in dimethylformamide. Then, cytarabine, 2-(7-Azabenzotriazol-1-yl)-N, N, N', N'-tetramethyluronium hexafluorophosphate (HATU), triethylamine, and 1-hydroxybenzotriazole (HOBt) were added to the dimethylformamide system, and stirred for 3-5 h in an ice bath. The molar ratio of DSPE-PEG2k-COOH, cytarabine, HATU, HOBt, and triethylamine is 1:1.2:1.2:1.2:2. After that, the reaction solution was moved to a dialysis bag (MWCO = 3500 Da), and dialyzed in 1000 mL of deionized water for 72 h. Finally, DSPE-PEG2k-Cy was freeze-dried and stored in 4℃ for further usage. Molecular weight and structure of DSPE-PEG2k-Cy was analyzed by the MALDI-TOF-MS (GCT-Premier, Waters, USA) and the Nicolet 6700 FT-IR spectrophotometer (Thermo Electron Corporation, USA).
Preparation and characterization of [email protected] and [email protected]
[email protected] was prepared by thin film hydration method combined with high pressure homogenization method. In brief, 22 mg physical mixture of DSPE-PEG2k-COOH and DSPE-PEG2k-Cy, 94 mg of lecithin, 21 mg cholesterol powder were added to a round-bottom flask and dissolved with 10 mL chloroform. Then, a yellow thin film was formed by rotating on a rotary evaporator for 2 h. Then, the round-bottom flask was placed in a vacuum drying oven overnight to remove of residual chloroform. After that, 10 mL of MTX solution (2 mg/mL) was added to the pear-shaped flask round-bottom flask and hydrate for about 30 mins at 40°C. During this process, the clear yellow solution gradually turned into a yellow colloidal solution. Then, the hydrated liquid was homogenized 3 times by a high-pressure homogenizer (AH110D, ATS Engineering Inc., Canada). Finally, the dispersion was freeze-dried (FreeZone 2.5, LABCONCO, USA) with 5% (w/v) mannitol as lyoprotectant. The preparation of [email protected] is to replace DSPE-PEG2k-Cy with an equivalent molar amount of DSPE-PEG2k-COOH, and the other preparation methods are described above. The preparation of fluorescein (doxorubicin or IR780)-labeled liposomes is to replace MTX with fluorescein solution, and the other preparation methods are described above.
Drug loading and in vitro drug release
To measure the amount of MTX encapsulated within [email protected], the lyophilized [email protected] and [email protected] were ultrasonically dissolved in anhydrous DMSO. Samples were filtered through a 0.22 µm filter membrane, after which HPLC (1100, Agilent, USA) was used to determine drug concentrations . The drug loading rate (DL%) and entrapment efficiency (EE%) were assessed as follows:
DL%= (amount of MTX in the sample/total weight of formulations)×100%
EE%= (amount of MTX in the sample/total amount of MTX added in preparation)×100%
In vitro release profiles of MTX containing formulations were determined at 37°C in PBS or acetate buffered saline with pH of 7.4 and 5.5. Typically, 5 mL of [email protected] or [email protected] were added to a dialysis bag (MWCO 3500 Da) that was placed in 250 mL of buffer saline and constantly stirred (100 rpm) at 37°C. At specific time points, 2 mL of the external buffer was extracted and an equal amount of fresh medium was added. HPLC was used to assess the content of MTX.
Cell uptake studies
JEG-3 cells were used for uptake analyses. Briefly, cells were plated overnight in 6-well plates, and then treated with media without or with nanocarriers (Dox-labeled Lipo or Dox-labeled Cy-Lipo). The fluorescence intensity was determined via flow cytometry (FACSCalibur, BD Biosciences, USA). Dox-labeled nanocarrier localization within cells was assessed via confocal laser scanning microscopy (CLSM, FV1200, Olympus, Japan). JEG-3 cells were grown overnight in chambered coverslips, followed by treatment for certain time with Dox-labeled Lipo or Dox-labeled Cy-Lipo containing culture medium. Finally, cells were fixed with 4% (v/v) paraformaldehyde and nuclei were DAPI stained before testing.
The mechanism of ENT1-mediated endocytosis
To explore the role of ENT1 in the uptake of Cy grafted nanocarriers, two kinds of high-affinity substrates of ENT1 were selected for competitive inhibition experiments. Briefly, different concentration (0.2-5.0 μM) of 2′-deoxycytidine or gemcitabine was cultured with the adherent JEG-3 cells for 2 h. Then, Dox-labeled Lipo or Dox-labeled Cy-Lipo was added and co-cultured for another 4 h. Subsequent steps were same as those in uptake assay.
Endocytosis was studied by adding JEG-3 cells to 12 well-plates and pre-treating them for 30 min with inhibitors of various endocytic pathways including the clathrin-dependent endocytosis inhibitor chlorpromazine (50 μM), the caveolin-dependent endocytosis inhibitor indomethacin (50 μM), the micropinocytosis inhibitor colchicine (10 μM), sodium azide (10 μM), and quercetin (10 μM) as tools for inhibiting caveolae- and clathrin-independent endocytosis. Next, the medium was removed, and fresh medium containing Dox-labeled Cy-Lipo were added into the plate and incubated for 4 h. Subsequent steps were same as those in uptake assay.
Cy-Lipo on ENT1 regulatory effects were evaluated on protein (western blot) and mRNA (RT-PCR) level. The JEG-3 cells were added to 6-well plates at 105 cells/well. At 24 h post-plating, 5 μg/mL of Cy-Lipo was added for 0, 0.5, 1, 2, 4, 8, 12, and 24 h. Cells were then isolated and a protein extraction kit (Beyotime, China) was used to isolate cytosolic or membrane proteins. Segregated proteins were assessed via western blotting as above, with β-actin cadherin as respective cytosol and membrane controls. Furthermore, JEG-3 cells treated as described above were used for RNA isolation for subsequent RT-PCR as previous described.
In vitro cytotoxicity
MTX formulation cytotoxicity was assessed via MTT assay. The groups were as follows: free MTX, MTX plus DSPE-PEG2k-Cy (abbreviated as MTX+Cy-lipid and the molar ratio of Cy-lipid to MTX is about 1:5.0), [email protected], and [email protected] JEG-3 cells or Pricell-0051 cells were added to 96-well plates (1×104 cells/well). After 24 h, cells were rinsed using PBS and treated with MTX (0.001-30 μg/mL) for 48 h at 37 °C. Viability was assessed by adding 20 μL of MTT (5 mg/mL) per well for 4 h. Media was then removed and formazan crystals were dissolved via the addition of 150 μL of DMSO. A microplate reader (Varioskan Flash 3001, Thermo Fisher Scientific, USA) was then used to assess absorbance at 490 nm.
To investigate the effect of ENT1 on MTX formulations induced cytotoxicity, 2′-deoxycytidine and gemcitabine, as competitive inhibitors of ENT1, were cultured with adherent JEG-3 cells for 2 h. Following an additional 48 h culture with MTX formulations (containing 20 μg/mL of MTX), the medium was removed, and PBS was used to wash cells thrice. MTT reagent was then added for 4 h as above, and absorbance was assessed via microplate reader.
Cell cycle and apoptosis studies
For cell cycle analysis, JEG-3 cells (105/well) were added to 6-well plates for 24 h, and were then treated with PBS, free MTX, MTX+Cy-lipid, [email protected], and [email protected] at an equivalent MTX concentration (5 μg/mL) for another 24 h. Then, JEG-3 cells were collected (1000 rpm, 5 min) and fixed using 70% ethanol for 8 h at 4℃. After resuspended in PI/RNase Staining buffer for 30 min, the cell cycle was determined with flow cytometry.
Apoptosis of JEG-3 cells were detected using the FITC Annexin V Apoptosis Detection Kit I. The cells (105/well) were seeded in 6-well plates. Following culture for 24 h, cells were respectively treated with PBS, free MTX, MTX+Cy-lipid, [email protected], and [email protected] at an equivalent MTX concentration (5 μg/mL) for 24 h. All other protocols were conducted based on provided directions. The cells were analyzed by flow cytometry. The cell cycle and apoptosis studies were performed in triplicate in different days.
Mitochondrial membrane potential changes and cell structure damage studies
Mitochondrial membrane potential changes induced by various MTX formulations were evaluated by JC-1 probe. Briefly, JEG-3 cells were added to 6-well plates. Following culture for 24 h, the cells were treated with PBS, free MTX, MTX+Cy-lipid, [email protected], and [email protected] at an equivalent MTX concentration (5 μg/mL) for 12 h. JC-1 solution was exchanged for cold PBS following two JC-1 staining buffer washes, after which samples were assessed by CLSM at λex (488 nm)/λem (530 nm) for green fluorescence and λex (488 nm)/λem (590 nm) for red fluorescence.
Bio-TEM was applied for observing the cell structure and mitochondrial damage effect induced by MTX formulations. Briefly, JEG-3 cells were seeded in a 6 cm dish. After culture for 24 h, the cells were treated with PBS, free MTX, MTX+Cy-lipid, [email protected], and [email protected] at an equivalent MTX concentration (5 μg/mL) for another 24 h. Then, cells were digested, centrifuged, and fixed with 2.5% glutaraldehyde solution at 4°C for more than 4 h. Finally, the bio-TEM observation was performed after sample preparation.
In vivo fluorescence studies
BALB/c nude mice were subcutaneously injected in the flank with 1×107 JEG-3 cells. When JEG-3 tumors in BALB/c mice were 200-300 mm3 in size, IVIS imaging systems (PerkinElmer) were used to assess mice at specified time points following intravenous IR780- labeled Lipo and IR780-labeled Cy-Lipo administration, respectively. The fluorescence image was collected at pre-treatment or 0.5 h, 2 h, 6 h, 12 h, and 24 h after treatment.
Pharmacokinetic and biodistribution studies
The advanced Automatic Blood Collection System (Instech, USA) was applied to study the pharmacokinetic feature of various MTX formulations. Female Wistar rats (200-220 g) were acclimatized at 25±2°C for 1 week before the experiments. Animals were randomized into 4 groups. Group I was intravenously administered saline as control group, and groups II, III and IV were intravenously given free MTX solution, [email protected], and [email protected] injections respectively at 5 mg/kg dosage of equivalent MTX. Blood (200 μL) was obtained at 0.25, 0.5, 1, 2, 4, 8, 12, 24, 48 and 72 h through carotid artery after MTX administration. 150 μL of plasma was combined with 450 μL acetonitrile to precipitate proteins, and samples were analyzed for drug content estimation by LC/MS. The kinetic software (Thermo-Scientific, USA) was used to assess key PK parameters including mean residence time (MRT), area under the curve (AUC), peak plasma concentration (Cmax), half-life (t1/2), and time to maximal plasma concentration (Tmax) with a compartmental model.
JEG-3 tumor-bearing mice were also used for organ distribution studies. Animal groups and dosage are consistent with pharmacokinetic studies. For bio-distribution of MTX formulations, three animals per group were sacrificed at 2, 8, and 24 h following treatment, and tumors, lungs, kidneys, livers, and spleens were isolated, weighed, and frozen. These tissues were then homogenized, spun down for 1 min, rested for 45 min, and combined with 100 µl of 10% trichloroacetic acid solution followed by vortexing for 1 min, adding 5 mL of acetonitrile, and incubating for 10 min. Samples were then spun down for 10 min at 6,000 rpm, and supernatants were isolated, combined with mobile phase, and passed through 0.22 µm membrane filters. Finally, the drug content was estimated by LC/MS.
In vivo antitumor studies
BALB/c nude mice were subcutaneously injected in the flank with 1×107 JEG-3 cells. When tumors were 50-100 mm3 in size, nude mice were intravenously administrated with saline or MTX formulations (free MTX, MTX+Cy-lipid, [email protected], and [email protected], 5 mg/kg MTX equivalent) on days 9, 12, 14, 18, 21. At the 24th day, the mice were sacrificed, and the tumors as well as the main organs were excised, weighed, washed three times using saline, and subjected to fixation with 10% neutral buffered formalin. The primary organs were harvested for H&E staining and the tumor tissues were collected for H&E, Tunel, and Ki67 staining. All immunohistochemical sections were observed with digital scanning microscope imaging system (OCUS-100117, Grundium, Finland). The survival of the remaining mice (n = 5) was analyzed via Kaplan-Meier analysis.
In vivo biocompatibility analysis
In this section, healthy mice were administered the [email protected] or [email protected] dispersion via intravenous tail vein (10 mg/kg MTX every three days) for five times. Saline was injected as a control. After the administration, routine blood assessments were conducted and measurements were made of blood biochemical indices. During the in vivo antitumor studies, the body weight and activity status of mice in each group were monitored for evaluating the side effects of the MTX formulations. After in vivo antitumor studies, the primary organs of each group were harvested for H&E staining.
Data were means ± SD, and were assessed with SPSS v17.0 (IBM Inc., IL, USA). P < 0.05 was the significance threshold.