Polyethylene glycol-block-poly(D, L-lactic acid) (PEG5k–b-PLA8k) was purchased from Advanced Polymer Materials, Inc. (Montreal, Canada). Gef (184475-35-2) and CsA (59865-13-3) were purchased from J&K Scientific (Beijing, China). All other compounds and solvents were purchased from J&K Chemical (Shanghai, China) and utilized without further purification or dilution.
Preparation and characterization of NPs
Polymeric NPs were prepared via the nanoprecipitation method52,53,54. To fabricate CsA/Gef-NPs, 1 mg of CsA and 40 mg of PEG-PLA were dissolved in 2 ml of acetone, while 1 mg of Gef was dissolved in 0.1 ml of dimethyl sulfoxide (DMSO). Next, the solution was mixed and added dropwise into 10 ml of DI water while stirring. After stirring for 30 min, the solution was evaporated in a rotary evaporator at reduced pressure at 30 °C for ~20 min to remove acetone, yielding a final polymer concentration of 0.1 mg/mL. The transparent solution containing NPs was concentrated with an Amicon Ultra-4 centrifugal filter (MWCO 10 kDa; UFC800324, Millipore, Germany) and washed with DI water. The concentration of copolymer was determined by high-performance liquid chromatography. CsA-NPs and Gef-NPs were prepared by the same method. The DH
, polydispersity index (PDI), and ζ potential of the drug-loaded micelles were measured using a Malvern Nano-ZS 90 laser particle size analyzer at 25 °C. The morphological characteristics of the NPs were observed by transmission electron microscopy (TEM; H-6009IV, Hitachi, Japan). Samples for TEM were stained with 1% uranyl acetate.
Cell lines and cell culture
The NSCLC cell lines PC-9, which possesses an EGFR-activating mutation exon 19 deletion and H1975, which harbors the EGFR-activating mutation L858R and the resistant mutation T790M, were purchased from the cell bank of the Chinese Academy of Science (Shanghai, China). PC-9-GR cells were obtained according to our previous report.35 Cells were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM; Gibco, Carlsbad, CA, USA) at 37 °C in a 90% humidified atmosphere with 5% CO2. All media were supplemented with 10% FBS, penicillin (100 units/mL), and streptomycin (100 mg/mL). Antibiotics, FBS, 0.25% (w/v) trypsin, and the 0.03% (w/v) EDTA solution were purchased from HyClone.
Cell viability and proliferation assays
Cells were seeded in 96-well plates (5000 cells and 100 μL of media per well) and incubated at 37 °C overnight. Subsequently, the adherent cells were treated with different concentrations of free Gef, free CsA, CsA-NPs, Gef-NPs, or CsA/Gef-NPs. Cells treated with medium that contained the same amount of DMSO as the drug treatment group were included as controls. After 48 h of incubation, 30 μL of 3-[4,5-dimethylthiazol-2-yl]-3,5-diphenyl tetrazolium bromide (MTT) solution (5 mg/mL in PBS) was added to each well. After 4 h of incubation at 37 °C, the MTT solution was removed from the wells, followed by the addition of 100 μL of DMSO per well. The absorbance of individual wells was then measured at 492 nm using a microplate reader (Multiskan FC, Thermo Scientific) after 2 h of incubation. All experiments were conducted in triplicate. The IC50 was calculated using GraphPad Prism 6.0. Untreated cells served as controls, and their viability was defined as 100%. Cell viability was calculated using the following formula: cell viability = (A sample/A control) × 100%, where A represents the absorbance at 492 nm. The results were from three repeated independent experiments.
LIVE/DEAD staining assay
For the LIVE/DEAD staining assay, cells were seeded in 6-well plates at a cell density of 2 × 105/well. After treatment with different concentrations of NPs for 48 h, the cells in each well were collected and re-suspended in 200 μL of PBS. Thereafter, the cells were stained with 1 μL of staining solution containing calcein-AM (2 μM) and 1 μL of PI (4 μM), which stains apoptotic/dead cells. After 30 min of incubation in the dark at 37 °C, the cells were imaged using a fluorescence microscope. The percentage of red (dead) cells among all cells (green + red) represents the cell death ratio.
Cell proliferation and DNA synthesis were determined using a Click-iT® EdU Alexa Fluor® 488 Assay Kit (Invitrogen) according to the manufacturer’s protocol. Briefly, cells (2 × 104 cells/well) were seeded in 48-well plates and cultured overnight before being exposed to different concentrations of CsA-NPs, Gef-NPs or CsA/Gef-NPs for 48 h. Cells treated with DMEM alone were used as the control. Next, 100 μL of EdU (10 μM) was added to each well, followed by 4 h of incubation at 37 °C. After washing with PBS three times, the cells were fixed with 4% paraformaldehyde in PBS for 30 min and permeabilized with 0.5% Triton X-100 in PBS for 30 min at room temperature. Thereafter, 100 μL of Alexa Fluor 488 staining solution was added to each well, followed by incubation for 30 min in the dark at room temperature. After the solution was removed, 100 μL of Hoechst 33342 (5 μg/mL) nuclear staining solution was added to each well, followed by incubation for 10 min. Finally, the cells were visualized by fluorescence microscopy. The ratio of EdU-positive cells (green) to all Hoechst-positive cells (blue) represents the proliferation ratio.
Apoptosis analysis using flow cytometry with Annexin V-FITC
To analyze apoptosis, cells were seeded in 6-well plates at a density of 2.0 × 105 cells/well and allowed to grow overnight. Next, the cells were treated with different drugs at different concentrations for 48 h at 37 ℃. Untreated cells were used as the control. After incubation, the cells were harvested and washed twice with cold PBS. Next, 1 × 105 cells were dispersed in 100 μL of 1 × Annexin V binding buffer. Subsequently, 5 μL of Annexin V-FITC and 5 μL of PI were added, and the cells were incubated at room temperature in the dark for 15 min. Finally, 400 μL of 1 × Annexin V binding buffer was added under gentle mixing, and the samples were analyzed by flow cytometry (BD Biosciences, San Jose, CA).
Western blot analysis
Cells were cultured at a density of 2.0 × 105 cells/well in a 6-well plate and allowed to grow overnight. Next, following treatment with free Gef, free CsA, CsA-NPs, Gef-NPs, or CsA/Gef-NPs, the cells were harvested and lysed in RIPA lysis buffer supplemented with complete protease inhibitor cocktail tablets on ice. Protein was also extracted from the tumor tissues of mice in the in vivo study. Protein concentrations were determined by the bicinchoninic acid protein assay. An equivalent amount of protein was taken from each sample, separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, transferred onto polyvinylidene difluoride membranes, and then incubated with antibodies (Cell Signaling Technology) against cleaved poly(ADP-ribose) polymerase (c-PARP), phosphorylated STAT3 (p-STAT3), total STAT3 (t-STAT3), B cell lymphoma-2 (Bcl-2), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The blots were developed with horseradish peroxidase-conjugated secondary anti-rabbit IgG or anti-mouse IgG antibody (Cell Signaling Technology) and visualized with a chemiluminescent substrate on X-ray films (Kodak). Western blotting of each protein was performed at least three times.
In vivo antitumor activity
Four- to five-week-old male BALB/c nude mice were purchased from the Shanghai Experimental Animal Center of the Chinese Academy of Science and used for NSCLC implantation. In total, 100 μL of the PC-9-GR and H1975 cell suspensions (5 × 106 cells) was subcutaneously injected into the right flank of each mouse. When the tumor volume reached approximately 120 mm3, the mice were randomized into treatment groups. Mice bearing H1975 xenografts were assigned into five groups for histological study, whereas mice bearing PC-9-GR xenografts were randomized into 11 groups (6 groups for tumor growth evaluation and 5 groups for histological study). Each group contained five to seven mice. NP solutions containing the combination of CsA and Gef (at a dose of 10 mg/kg for each drug) were IV injected every other day three times, while the combination of the two free drugs was administered by gavage (gav) (at a dose of 20 mg/kg for each drug) successively for 6 days. CsA-NPs (IV, three times, 10 mg/kg CsA equivalence), Gef-NPs (IV, three times, 10 mg/kg Gef equivalence), Gef (gav, six times, 20 mg/kg), and saline were included as references. The tumor volume (V) was calculated using the following formula: V = (L × W2) × 0.5, where L represents the length, and W represents the width. The weight of each mouse was measured for the evaluation of systemic toxicity. At the end of the study, the mice were sacrificed by CO2 inhalation, and the tumor tissues were fixed in formalin and embedded in paraffin. All the animal protocols were conducted in compliance with the National Institute’s Guide for the Care and Use of Laboratory Animals.
Histopathological analysis of tumor tissues
Immunohistochemical (IHC) staining using primary antisera and avidin-biotin-peroxidase complex methods were performed using formalin-fixed tumor sections. p-STAT3 expression was monitored using antibodies and visualized by light microscopy. Six fields of view for each sample (magnification, 400×) were randomly selected and analyzed by three pathologists.
All the data are presented as means ± SD and were analyzed using SPSS 17.0 software. The significance of differences was assessed using one-way ANOVA combined with Student’s t-test (*p < 0.05; **p < 0.01; ***p < 0.001).