Reagents and antibodies
PTX, 5,5’,6,6’-tetrachloro-1,1’,3,3’-tetraethyl-benzimidazolyl carbocyanine iodide (JC-1), 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT), Annexin V-FITC/PI Kit and DAPI were purchased from Sigma-Aldrich (St. Louis, MO, USA). BEZ235(Dactolisib) was obtained from MedChem Express (Monmouth Junction, NJ, USA). RPMI-1640 was obtained from Hyclone (Salt Lake City, UT, USA). FBS was purchased from Hangzhou Sijiqing Company. PI3K Antibody Kit (9655#), Akt Antibody Kit (9916#), mTOR Antibody Kit (9964#), Bcl-2 Family Antibody Kit (9942#), Apoptosis Antibody Kit (9915#), secondary goat anti-rabbit and anti-mouse antibodies were purchased from Cell Signal Technology (Danvers, MA, USA). Cyclin B1 and Cyclin-dependent-kinase (CDK1) were purchased from Abcam Biological Technology (USA). β-actin was obtained from Cell Signal Technology (Danvers, MA, USA).
Cell lines and cell culture
Human HepG2 cell line was bought from the American Type Culture Collection (Manassas, VA, USA). HepG2R, a cell line resistant to paclitaxel by HepG2 cells. The process of inducd HepG2R was as follows: When the cells were in the logarithmic growth phase, added PTX with a lower concentration for 24h, then performed cell inheritance, and repeatedly stimulated with this concentration until it was stable. Increasing the concentration of PTX continued to stimulate to a stable state. When the resistant strain treated with PTX reached 4–5 times the IC50 of the sensitive strain, the resistant strain was obtained.
Synthesis of HPG-PCL
The amphiphilic HPG derivative PCL-HPG was synthesized by anionic ring-opening polymerization as follows: First 1.5g (0.10 mmol) of dry PCL was weighed into a 50mL Schlenk flask and repeatedly evacuated several times with nitrogen gas using an anhydrous oxygen-free. Then 0.8mL of 25% potassium methanol solution was added and the temperature was gradually increased to 95°C while stirring. 10min later, the excess methanol was removed by re-evacuation. Subsequently, 4.5g (60.7mmol) of epichlorohydrin was added dropwise to the reaction system using a micro syringe pump under the protection of nitrogen. After the dropwise addition, the reaction was continued for 5h. At the end of the reaction, the product was dissolved with an appropriate amount of methanol and the acidified cation exchange resin was added to remove the potassium ions. The crude product was removed by rotary evaporation and lyophilised using a dialysis bag with a cut-off molecular weight of 3000 to give a clear yellow viscous solution.
Preparation of HPG-PCL-PTX nanoparticles
Weigh 500mg of HPG-PCL in 4mL of water, 10mg of PTX in 2mL of acetone. The PTX solution was added to the HPG-PCL solution drop by drop (as the PTX solution was added, the system gradually became cloudy, and the solution was clarified by adding acetone) and stirred at room temperature overnight. The drug-loaded nanoparticles were lyophilised by dialysis.
characterization of HPG-PCL-PTX
The average particle-diameter distribution and zeta potential of the nanospheres were tested by a Malvern Zetasizer Nano ZS (Malvern, Worcester, UK). The morphology of the NPs was observed with a transmission electron microscope (TEM, Tecnai F30, FEI Company, Hillsboro, OR, USA). The structure of the synthesized PLGA-PEG in CDCl3 was confirmed by the 1H NMR spectra (Varian Unity Inova 400 Mhz, Agilent Technologies, Inc., Santa Clara, CA, USA). To analysis drug loading content (LC%) of NPs, high-performance liquid chromatography (HPLC, LC 1200, Agilent Technologies, Santa Clara, CA, USA) was applied. Calculate LC% by the following equation : LC%=(BEZ235weight inNPs)/(totalNPsweight)×100%
Molecular targeted
The HPG-PCL-PTX was labeled with FITC, then co-cultured with HepG2 and HepG2R cells. The nanomedicine with unmodified targeting molecules used as a control, observe the endocytosis effect of the two cell lines on nano-drugs at 0.5h, 1h, 2h and 4h, respectively.
Cell viability
HepG2 and HepG2R cells (5000–10000 per well) were plated into 96-well plates and incubated overnight in a 5% CO2 incubator at 37°C. After indicated drugs exposured for 24 h, cell viability was measured using MTT assay according to the manufacturer’s instructions. Absorbance was measured at 450 nm using a spectrophotometer. IC50 was calculated by Graphpad Prism Version 5.0 software. The resistant index (RI) was calculated utilizing the following formula: RI = IC50 of resistant cells/IC50 of parental cells.
Colony-formation assay
Cells were cultured into 6-well plates at a density of 1000 cells per well to adhere overnight, then the cells were treated with the drugs for 24h. After about two weeks, the cell colonies were clearly visible. The medium was aspirated, colonies were fixed in 4% paraformaldehyde for 15min and stained with 0.1% crystal violet for 30min. Cell colonies were counted, and the number cultured with drugs was compared with the control.
Cell cycle analysis
Propidium iodide (PI) was used to stain the DNA content. After drug interventions for 24 h, cells were collected, washed with PBS, and fixed with 70% precooled ethanol. Before testing, cells were washed with PBS three times, added 50µg/ml PI and 100µg/ml RNase A in the dark for 30min. Dye was removed, the cells were resuspended with PBS, followed by flow cytometry analysis (BD FACSCalibur, USA). The distribution of cells at specifific cell cycle stages was assessed with ModFit Version 3.0 software (Verity Software House, Topsham, ME).
Apoptosis detection
Cells were seeded in 24-well plates and incubated for overnight. After drug treatment for 24h, cells were collected after digestion and centrifugation, double-stained with Annexin V-FITC(10µL) and PI(5µL) at room temperature for 20min in the dark. Cell apoptosis was detected by flow cytometry within 1 h (BD Biosciences) and analyzed apoptotic rates using the Flow Jo software.
Mitochondrial membrane potential (ΔΨm) measurement
JC-1 is a sensitive probe for measuring mitochondrial membrane potential. The high/low mitochondrial membrane potential of cells treated with dugs determined red/green fuorescence intensity excited by polymer/monomer of JC-1, from which the mitochondrial apoptosis level of the cells was analyzed. Cells were cultured in 24-well plates overnight, treated with drugs for 24h, counterstained with DAPI and JC-1, examined with fluorescence microscope. Besides, cells treated with drug were stained with 10µg/ml JC-1 for 30min at room temperature and analyzed with flow cytometry for changes in ΔΨm.
Western blot
Total protein was extracted from cells lysed with radioim munoprecipitation bufer (RIPA, Beyotime Biotechnology, Shanghai, China) containing a protease inhibitor cocktail (Beyotime Biotechnology, Shanghai, China) and centrifuged. The BCA protein assay kit (Biosharp, Hefei, China) was used for measuring protein concentration. Soluble lysates containing about 20 µg proteins per sample were resolved with sodium dodecyl sulfate–polyacrylamide gel electrophoresis(SDS-PAGE) and transferred to a polyvinylidene fluoride membrane (Millipore, Sigma, USA). After blocking using 5% skim milk, membranes were probed with primary antibodies (dilutions were 1:1000) at 4°C overnight and secondary antibodies (1:2000) at room temperature for 1 h. Protein bands were visualized with an ECL luminescent detection kit (Thermo Fisher Scientific Waltham, MA, USA) and images were captured with a gel imager Bio-Rad (Hercules, CA, USA). Quantified with Image J Version 1.48 software (NIH, Bethesda, MD). The protein content of β-actin was used as a loading control.
Xenograft studies
All animal experiments were approved by the Animal Experimental Ethics Committee of Anhui University of Science and Technology and were carried out in accordance with appropriate procedures. Animal experiments were carried out in the SPF-level animal room of the Central Laboratory of Medical School Anhui University of Science and Technology (NO: AUST201810088). 100µl of HepG2 cell suspension (2×107/ml) were injected subcutaneously into the dorsal right side of 5-week-old female BALB/c nude mice (Vital River Laboratories, Beijing, China). When tumor volume reached 100 mm3, mice were randomized into five groups: control group: 100µl saline daily, ip; HPG-PCL-PTX group: 5mg/kg weekly, ip; BEZ235 group: 45mg/kg daily, oral treatment. (The dose of BEZ235 used in vivo was based on the specifications of Selleck Chemicals); HPG-PCL-PTX + BEZ235 group: PTX (5mg/kg weekly, ip) plus BEZ235 (45mg/kg daily, oral treatment). Each group of rats received treatment for 28 days, and tumor size and body weight were measured every 3 days. Tumor volume was calculated according to the formula: V = L×W2×1/2 (V, volume; L, length of tumor; W, width of tumor). Mice were sacrificed after the treatment; tumor tissues were isolated; protein was extracted for WB detection.
Statistical analysis
All analyses were performed with SPSS version 18.0 (SPSS Inc., Chicago, IL, USA). All experiments were repeated three times independently and analyzed by Student t-test or two-way ANOVA using GraphPad Prism 5 (GraphPad Software, Inc., San Diego, CA, USA). Data are expressed as mean ± SD. p < 0.05 was taken as statistically significant.