1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000] (DSPE-PEG2K) was purchased from Shanghai Advanced Vehicle Technology Pharmaceutical Co., Ltd. Roswell Park Memorial Institute (RPMI-1640), trypsin, 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) and fetal bovine serum (FBS) were purchased from Dalian Meilun Biotechnology Co., Ltd, China. DTX-LA and DTX-S-LA were synthesized and characterized by former work in our lab [12, 29]
2.2 Preparation of DL NPS and DSL NPS
One-step precipitation method was used to prepare the nanoassemblies. In short, 4 mg of DTX-LA or DTX-S-LA and 20% (w/w) DSPE-PEG2K were accurately weighed and dissolved in 100 µL ethanol. Then this miscible solvent containing formulation components was cautiously added dropwise into 2 mL deionized water and continuously stirred for 2 minutes (800 rpm, K-MSH-Pro-6A, JKI, Shanghai, China). Apply vacuum-rotary evaporation procedure for almost 10 min to remove ethanol. Finally, volume with deionized water to 2 mL. Particle size and polydispersity index (PDI) of conjugate NPS were measured by a Zetasizer (Nano ZS, Malvern, UK) in triplicate.
2.3 Cell lines and cell culture
The murine breast cancer cell line (4T1) was bought from the cell bank of Chinese Academy of Medical Sciences (Beijing, China). 4T1 cells were cultured in RPMI-1640 medium supplemented with 10% FBS, penicillin (30 mg/L) and streptomycin (100 mg/L) in a humid atmosphere containing 5% CO2 at 37°C.
2.4 In vitro cell viability assay
To explore whether conjugate NPS could effectively inhibit tumor cells’ growth in vitro, and more importantly, to provide guidance for in vivo experiments, MTT assay was performed in 4T1 cells. A certain density of 4T1 cells (1000 cells/100 µL/well) were incubated in 96 well plates for 12 h to allow cell attachment. Then fresh medium containing a series of concentrations of DTX solution, DL NPS and DSL NPS was added to each well to replace old RPMI-1640 medium. Cultivated for another 48 h or 72 h, the drug-contained medium was replaced by 100 µL fresh medium and 20 µL MTT solutions (5 mg/mL) which would be discarded after incubating for 4 h. 200 µL DMSO was added to each well to dissolve the formazan. The absorbance value of each hole in 96 well plates at 570 nm was selected for measurement on a microplate reader (Model500, USA). The Eq. 1 was utilized to calculate the inhibition rate. And the half maximal inhibitory concentrations (IC50) was evaluated by nonlinear regression analysis.
Equation 1: inhibition rate (%) = (1-Asample/Acontrol) × 100
2.5 Animal study
The BALB/c mice and SD rats in this study were offered by the Laboratory Animal Center of Shenyang Pharmaceutical University. All the animal experiments were performed in compliance with the Guide for Care and Use of Laboratory Animals which were approved by the Institutional Animal Ethical Care Committee (IAEC) of Shenyang Pharmaceutical University.
2.6 In vivo antitumor efficacy study
To further explore whether these conjugate nanoassemblies could improve the therapeutic index of DTX in vivo, anticancer efficacy study should be performed. This study was performed in the female BALB/c mice weighed 18–22 g. In short, the 4T1 cells, suspended in PBS (5×106 cells per mouse), were subcutaneously injected into the right auxiliary flank of mice to build tumor-bearing mice models. The mice were divided evenly into 4 groups (n = 5) when the tumor volumes reached almost 120–150 mm3. Then each group of mice were subjected to treat every two days with saline, Home-made Taxotere (10 mg/kg), DL NPS (10 mg/kg, DTX equivalence), and DSL NPS (10 mg/kg, DTX equivalence) via tail vein injection, respectively. The tumor volumes and body weights were monitored and recorded every two days. Tumor volumes were calculated by Eq. 2. After 4 times administration followed by 2 days observation, the mice were sacrificed to collect its’ tumors and calculate the tumor burden after the last treatment by Eq. 3.
Equation 2: V (mm3) = (a2 × b)/2 (a represents the shortest width and b represents the longest length)
Equation 3: B (%) = w / W × 100% (B represents the tumor burden, w represents the tumor weight and W represents the body weight)
2.7 Physical stability and drug release behavior studies
The physical stability of nanoassemblies was carried out at 4°C for 3 months. The particle size and PDI were determined on a specific time point to monitor the variations. The drug release behavior of DL NPS and DSL NPS was performed in plasma. Briefly, the certain concentration of NPS solution was incubated with mice plasma in an air bath (CHA-S, Guohua Electric Applance Co., Ltd. Jiangsu, China) with the shaking of 100 rpm at 37°C. Plasma samples, at pre-determined time interval, were withdrawn and extracted by acetonitrile for the sedimentation of protein. The supernatant was assessed periodically by HPLC assay on a reverse ODS Cosmosil-C18 column (150 mm × 4.6 mm, 5 µm) with acetonitrile/water (55:45, v/v) for DTX detection and acetonitrile/water (90:10, v/v) for DTX-LA and DTX-S-LA detection. The flow rate was 1.0 mL/min and the detection wavelength was 230 nm.
2.8 Pharmacokinetic properties study
The pharmacokinetic (PK) experiment was carried out on Sprague–Dawley (SD) rats weighed 200–220 g which were divided into 3 groups (n = 5). Administrated with a single intravenous injection of Home-made Taxotere, DL NPS and DSL NPS to deliver a DTX equivalent dose of 5 mg/kg, blood samples were withdrawn at pre-determined time intervals via orbital venous plexus. The plasma was collected by centrifugation at 13,000 rpm for 10 min. Precipitation of protein method was applied to extract the drugs. The quantitative analysis was assessed by HPLC-MS/MS with C18 column (100 mm × 2.1 mm, 5 µm). Acetonitrile/water (95:5, v/v) as mobile phase at 0.2 mL/min was used to analyze DTX-LA and DTX-S-LA. The concentration of DTX was determined by elution: 0-0.5 min, 70% water; 0.51–2.5, 5% water; 2.6-3.0, 70% water.
2.9 Bio-distribution study
BALB/c mice bearing 4T1 malignant tumor were used to fulfill biodistribution study. The mice model was built according to antitumor efficacy study’s method. The mice were randomly divided into 3 groups (n = 6) and treated with Home-made Taxotere (10 mg/kg), DL NPS (10 mg/kg, DTX equivalence) and DSL NPS (10 mg/kg, DTX equivalence) via tail vein injection, respectively. After four and twenty-four hours postinjection, three mice of each group were sacrificed to harvest major organs (heart, liver, spleen, lung and kidney) and tumors. Then the free DTX in tissue homogenate were quantified by HPLC-MS/MS on an ACQUITY UPLC system (Waters Corp). The methods of sample extraction and quantification were in accordance with that of PK study as mentioned above.