Materials
Negatively-charged peptide-4-formylbenzoic acid (NP-FA) and CPP peptides (Ac-CGehGehGehGehG and GrrrrrrrrGC) were ordered at > 95% purity from GL Biochem (Shanghai, China). 1,2-Distearoyl-sn-glycero-3- phosphoethanolamine-N-maleimide (polyethylene glycol) (DSPE-PEG2000-MAL) was purchased from NOF (Tokyo, Japan). Dioleoyl phosphoethanolamine (DOPE) was purchased from NOF (Tokyo, Japan), and cholesterol (Chol) was purchased from Wako (Tokyo, Japan). Cholesteryl hemisuccinate (CHEMS) and SA-R8/SA-H8 were synthesized in our laboratory. DTX was purchased from Nuorui (Beijing, China). Methyl thiazolyl terazolium (MTT) was purchased from TCI (Shanghai, China). The negative control siRNA (siN.C.), 6-carboxyfluoresceinaminohexyl (FAM)-labeled negative control siRNA (FAM-siRNA) (antisense strand, 5′-ACGUGACACGUUCGGAGAATT-3′), and siRNA targeting the polo-like kinase 1 (PLK-1) messenger RNA (mRNA) (siPLK-1, antisense strand, 5′-GUGAUCUUCUUCAUCAAGGdTdT-3′) were custom-synthesized by GenePharma (Shanghai, China). All primers were synthesized by AuGCT Biotechnology (Beijing, China). Fluorescent probes such as rhodamine-phalloidin, CellLight Early Endosomes-RFP BacMam 2.0, and LysoTracker Red were obtained from Invitrogen/Thermo Fisher Scientific (Waltham, MA, USA). Roswell Park Memorial Institute (RPMI) 1640 medium and fetal bovine serum (FBS) were purchased from Gibco/Thermo Fisher Scientific. Hoechst 33,258 and 4% formaldehyde were supplied by Macgene (Beijing, China). Triton X-100 was purchased from Amresco (Solon, OH, USA).
Synthesis of DPRP conjugates
The synthesis of the DPRP conjugates is displayed in Fig. 1a. The compound DPRP was synthesized as described by Yang et al. [53], with minor modifications. Briefly, CPP (MW 1571.87, 95.9%) in anhydrous methanol was mixed with NP-FA (MW 1672.63, 93.6%) in the presence of a fivefold molar amount of acetic acid. The reaction mixture was stirred continuously under nitrogen at 30 °C for 12 h. After the product was precipitated in cold anhydrous diethyl ether (DEE)/acetone (8:2, v/v), the resulting solid was collected by centrifugation, rinsed with the same solvent systems, and treated under reduced pressure until dry.
The molecular weight distribution of DPRP (MW 3226.48) was determined using an Applied Biosystems 5600 QTRAP mass spectrometer (ABSciex, Framingham, MA, US) and analyzed in positive ion mode.
Kinetics of the DPRP hydrolysis
The peptide, final concentration of 2.0 mg mL−1, was incubated in phosphate-buffered saline (PBS; 10 mM phosphate, 150 mM NaCl) at pH 6.5 and 7.4 to evaluate the acidic pH-mediated degradation of the imine-containing DPRP. During incubation in buffer solutions at 37 °C, aliquots of the mixture were removed and analyzed using high-performance liquid chromatography (HPLC) at discrete time points (0, 3, 6, 9, 12, 24, and 48 h).
The HPLC analysis was performed using a Kromasil 100–5 C18 column (250 × 4.6 mm, pore size 5 μm) and a Shimadzu LC-20AT HPLC system (Shimadzu, Kyoto, Japan), and the chromatograms were recorded at 220 nm using methanol: water (8:92, v/v) containing 0.01% trifluoroacetic acid (v/v) as a solvent, with a flow rate of 1.0 mL min−1 at 30 °C.
Synthesis of functional conjugates
DSPE-PEG2000-DPRP and DSPE-PEG2000-CPP were synthesized using our published methods [15]. Briefly, the designated peptide (20 mg of thiol-containing DPRP or 10 mg of thiol-containing CPP) was dissolved in 4 mL of methanol. DPRP or CPP solutions were added to a methanol solution of DSPE-PEG2000-MAL and gently stirred at room temperature. After 48 h of stirring under nitrogen protection, the resulting solution was incubated with L-cysteine (10 times the molar ratio of maleimide residues) for 4 h to cap the unreacted maleimide groups. The reaction mixture was dialyzed in a membrane with a molecular weight cutoff of 25,000 (Spectra/Por, Spectrum Labs, Rancho Dominguez, CA) against distilled water (adjusted to pH 10.5–11.0 using 1 M NaOH) for 48 h to remove the excess peptides and quencher. The solution was lyophilized and stored at 20 °C.
The molecular weight distributions of DSPE-PEG2000-DPRP and DSPE-PEG2000-CPP were determined by performing matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (Bruker Daltonics, Bremen, Germany), using alpha-cyano-hydroxycinnamic acid as the matrix for mass spectrometry analysis.
Preparation of liposomes
A lipid composition (molar ratio) of DOPE (56%), CHEMS (22%) and Chol (22%) was used to prepare the basic liposomes (SUV). The lipid film was formed in a round bottom flask after evaporation in a rotary evaporator for 60 min at 37 °C. After drying with nitrogen for 1 min to remove the residual solvent, the thin film was hydrated using 10 mL of 10 mM HEPES buffer (HBS) at pH 4.0 (pretreated with DEPC). The lipid dispersion was extruded 5 times through polycarbonate membranes (Whatman, Kent, UK) with a 0.2 μm pore size using an NLI TBX 001 liposome extruder device (Northern Lipids, Burnaby, BC, Canada) to control the size.
The SA-R8/siRNA and SA-H8/siRNA complexes were attained by mixing the siRNA with an SA-R8 (0.1 mg mL−1, in HBS) or SA-H8 solution (0.12 mg mL−1, in HBS) with vortexing, respectively. Following the incubation of the complexes with SUV for 10 min at room temperature, the system was sonicated for 2 min and cooled in an ice-bath. A NaOH solution (0.1 M) was later added dropwise to the above-mentioned system to adjust the pH value to 7.4.
N-L, C-L and D-L were formed using the postinsertion method. Briefly, a lipid film of DSPE-PEG2000, DSPE-PEG2000/DSPE-PEG2000-CPP or DSPE-PEG2000/DSPE-PEG2000-DPRP was formed in the same manner as described above, and hydrated with HBS (pH 7.8, pretreated with DEPC) to induce the formation of micelles. For the N-LR/si, C-LR/si or D-LR/si preparations, 0.5 mL of micelle solution was added to 3 mL of SA-R8/siRNA loaded SUV at the required molar ratio (3% DSPE-PEG and 5% DSPE-PEG-peptides of total lipids) and incubated for 4 h at 37 °C. D-LH/si was prepared using the procedure described above, except that SA-R8 was replaced by SA-H8. In addition, D-LH/si-DTX was formulated by incorporating of DTX into the lipid mixture of the SUV film. All of the resulting liposomes were permitted to cool to room temperature before use. Encapsulation efficiency (EE) and drug loading content (DLC) of DTX was determined by HPLC. The HPLC analysis was performed using a Kromasil 100–5 C18 column (250 × 4.6 mm, pore size 5 μm) and a Shimadzu LC-20AT HPLC system (Shimadzu, Kyoto, Japan), and the chromatograms were recorded at 230 nm using methanol: acetonitrile: water (30:50:20, v/v) as a solvent, with a flow rate of 1.0 mL min−1 at 30 °C. The EE and DLC values were calculated according to the following equations:
$${\text{EE}}\,\left( \% \right)\, = {\text{A}}_{1} /{\text{A}}_{2} \times 100\%$$
$${\text{DLC}}\,\left( \% \right)\, = {\text{A}}_{1} /{\text{B}} \times 100\%$$
A1 is the weight of drug in the liposomes, A2 is the weight of drug added, and B is the weight of whole liposomes.
Characterization of liposomes
The size distribution and zeta-potential of each formulation were determined using DLS (Malvern Zetasizer Nano ZS 90, Malvern, UK). A 1 mL suspension was placed in a DLS cuvette and measured with detection optics arranged at 90°. Three serial measurements were performed for each sample.
The formation of the SA-R8-siRNA complex was evaluated using AGE. Briefly, 2 g of agarose were dissolved in 100 mL of 0.5 × Tris–Borate-EDTA buffer (TBE) with heating and then 10 μL of Exred solution (10,000 ×) were added. The solution was poured into a plate when the temperature was 60 °C. An appropriate amount of 0.5 × TBE was added after 30 min. Subsequently, the comb was gently pulled out vertically and the plate was transferred to an electrophoresis tank. Next, SA-R8 and siRNA (molar ratios: 0, 1, 3, and 5) were mixed with loading buffer and added to the wells. Electrophoresis was performed at 100 V for 30 min. Then the results were visualized and photographed with a gel imager (Bio-Rad).
The morphology of the D-L liposomes was examined using TEM and AFM. For the TEM analysis, 10 μL of the liposomal formulation were uniformly loaded onto a carboncoated copper grid for 1 min, and then the films were negatively stained with 10 μL of a 1% phosphotungstic acid solution for 1 min. Excess sample and stain were absorbed with filter paper and the copper grid was dried for imaging using a HITACHI H-7500 transmission electron microscope (Hitachi, Tokyo, Japan) at 50,000 × magnification. The D-L sample solution was dropped onto the surface of mica and dried under nitrogen for 2 h to acquire AFM topographic images. Particles were observed using a Bioscope Resolve atomic force microscope (Bruker Nano Surfaces, Santa Barbara, CA, USA) in PeakForce quantitative nanomechanical imaging mode. The D-L suspension was imaged at a scan rate of 1 Hz. A Bruker silicon–nitride ScanAsyst Air probe with a spring constant of 0.4 N m−1 was used. All images were processed using NanoScope Analysis software (Nanoscope Analysis, Bruker-AXS, Santa Barbara, CA, USA) and Young’s modulus of D-L was calculated by fitting the retract curve using the Derjaguin–Muller–Toropov model.
Cell culture
The human breast adenocarcinoma cell line (MCF-7 cells) was obtained from the Cell Bank of the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China) and was grown in RPMI 1640 medium supplemented with 10% FBS, 100 IU mL−1 penicillin, and 100 mg mL−1 streptomycin. The cells were cultured in a 37 °C humidified incubator with a 5% CO2 atmosphere.
Through serial passages, some cells were adapted to grow in the low pH medium (pH 6.5), adjusted with 1 M HCl.
In vitro cellular uptake
MCF-7 cells grown in pH 6.5 or 7.4 medium were seeded into 6-well plates at a density of 2 × 105 cells per well in 2 mL of complete RPMI 1640 medium and cultured at 37 °C in a 5% CO2 humidified atmosphere for 24 h. After the attachment period, the cells were rinsed with PBS and then incubated with pH 6.5 or 7.4 medium containing free siRNA or FAM-siRNA-loaded N-L, C-L and D-L, which were preincubated in serum-free medium at the corresponding pH for 5 h. The ultimate concentration of FAM-siRNA was 100 nM. After treatment for 2 h at 37 °C, the cells were trypsinized and washed with cold PBS containing heparin (500 U mL−1). Following two washes with cold PBS, cells were filtered and a flow cytometry analysis was performed with BD FACSCalibur flow cytometer (BD Biosciences, San Jose, CA, USA).
In vitro confocal imaging
MCF-7 cells (1 × 105 cells) were seeded into a sterile glass-bottomed dish (35 × 10 mm) and incubated in complete RPMI 1640 medium at pH 6.5 and 7.4 for 48 h to assess the cellular uptake by CLSM. After the cells were washed five times with PBS, serum-free medium containing free or liposomal FAM-siRNA was introduced as described in the earlier paragraph (N-L, C-L, and D-L (D-LR/FAM-si and D-LH/FAM-si)). The final concentration of FAM-siRNA in the culture medium was 200 nM. The cells were then incubated at 37 °C for 3.5 h and washed four times with cold PBS containing heparin (500 U mL−1). Cells were fixed with 4% formaldehyde for 10 min, followed by three 5-min rinses with cold PBS. Cells were stained with rhodamine-phalloidin for 20 min after permeabilization with 0.1% Triton X-100 in PBS to image F-actin. Finally, the nuclei were labeled with Hoechst 33,258 at 37 °C for another 20 min and imaged using a Leica TCS SP8 confocal platform (Leica Microsystems Inc., Mannheim, Germany). FAM-siRNA, rhodamine-phalloidin, and Hoechst 33,258 were excited with 494, 540, and 352 nm lasers, respectively.
The internalization and endosomal release of the liposomal FAM-siRNA were characterized by performing CLSM. MCF-7 cells were seeded in a sterile glass-bottomed dish (35 × 10 mm) at a density of 6 × 104 cells per well and cultured in complete RPMI 1640 medium at pH 6.5. MCF-7 cells were preincubated with CellLight Early Endosomes-RFP BacMam 2.0 (20 particles per cell, as recommended by the supplier) for 24 h at 37 °C to stain the early endosomes. Late endosomes/lysosomes were labeled with 500 nM LysoTracker Red (Invitrogen/Molecular Probes, CA, USA) for 0.5 h. The following day, the cells were incubated with acid-pretreated D-L (SA-R8/FAM-siRNA or SA-H8/FAM-siRNA) for 3 or 6 h at pH 6.5. Subsequently, the cells were rinsed three times with cold PBS containing heparin (500 U mL−1) and then fixed and subjected to nuclear staining.
MCF-7 cells were incubated with double-labeled liposomes (containing 400 nM FAM-siRNA and 680 ng mL−1 DiD) that had been pretreated as described above at 37 °C for 3 h and 6 h to observe the time-dependent changes in the intracellular uptake and distribution of FAM-siRNA or liposomes, respectively. Images of fluorescent cells were also recorded.
Confocal microscopy images were acquired using a Leica confocal platform with a 63 × oil immersion objective at an excitation wavelength of 494 nm for FAM-siRNA, 555 nm for CellLight Early Endosomes-RFP BacMam 2.0, 561 nm for LysoTracker Red, 620 nm for DiD dye, and 352 nm for Hoechst 33,258.
In vitro transfection and analysis of gene silencing
MCF-7 cells were seeded into 25 cm2 tissue culture flasks at a density of 1.5 × 106 cells/flask in 4 mL of complete RPMI 1640 medium. After a 24 h incubation at 37 °C in a humidified atmosphere with 5% CO2, the medium was replaced with fresh serum-free medium containing free siRNA or siRNA-loaded samples (N-L, C-L, or D-L (D-LR/si and D-LH/si)). The final concentration of the siRNA (siPLK-1 or siN.C.) utilized in the experiment was 100 nM. Following a 5 h incubation, the medium was replaced with complete medium and cells were cultured for an additional 48 h (for mRNA assay) or 72 h (for protein quantification) at 37 °C. Subsequently, the PLK-1 mRNA and protein levels were determined using quantitative reverse transcription polymerase chain reaction (qRT–PCR) and western blot analysis, respectively.
For qRT–PCR assessment, cells were collected and total RNA was extracted from transfected cells using TRIzol reagent (Tiangen, China) according to the manufacturer’s protocol. First strand cDNAs were synthesized from 2 μg of total RNA with a Quantscript RT Kit (first strand cDNA synthesis kit) (Tiangen, China). After cDNA synthesis, 4 μL of cDNA templated were subjected to qRT–PCR analysis of PLK-1 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression using the SuperReal Premix SYBR Green kit (Tiangen, China). Following analysis using the CFX 96 Touch Real-Time PCR Detection System (Bio-Rad), the relative gene expression was quantified using the 2−∆∆Ct method. Data are presented as the fold change in PLK-1 expression normalized to the housekeeping gene GAPDH as the endogenous reference and relative to the untreated control cells. The primers used for PCR amplification were as follows: GAPDH forward: 5ʹ-GGGTGTGAACCATGAGAAGT-3ʹ; GAPDH reverse: 5ʹ-GACTGTGGTCATGAGTCCT-3ʹ; PLK-1 forward: 5ʹ-CGAGGTGCTGAGCAAGAAAGGGC-3ʹ; and PLK-1 reverse: 5ʹ-CCACGGGGTTGATGTGCTTGGGA-3ʹ. The cycling procedure was as follows: 95 °C for 15 min, followed by 40 cycles of 95 °C for 10 s and 61 °C for 30 s. The specificity was verified by performing a melting curve analysis and agarose gel electrophoresis.
For the western blot assay, the transfected cells were first washed with ice-cold PBS three times and then lysed in radioimmunoprecipitation assay buffer (Bestbio. Co. Ltd., Shanghai, China) containing phenylmethanesulfonylfluoride (PMSF). The resulting cell suspension was incubated on ice for 30 min with vortexing every 5 min. The lysates were collected by centrifugation at 14,000 rpm for 10 min at 4 °C. Subsequently, the protein concentration was determined using the bicinchoninic acid protein assay (MultiSciences Biotech, Beijing, China). After separation by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis, the total protein (50 mg) was transferred (at 250 mA for 2.5 h) to Immobilon-P membranes (Millipore, Bedford, MA, USA). Membranes were blocked with 5% bovine serum albumin (BSA) in Tris-buffered saline with Tween-20 (TBST) for 1 h at room temperature and incubated overnight at 4 °C with an anti-PLK-1 monoclonal antibody (Cell Signaling Technology Inc., Danvers, MA, USA; 1:1,000) or rabbit anti-beta-actin antibody (Antibody Revolution Inc., San Diego, CA, USA; 1:2,000) as the internal control in TBST containing 5% BSA. Next, the membrane was incubated with a horseradish peroxidase-linked anti-rabbit IgG antibody (Cell Signaling Technology Inc.; 1:3,000) in 5% BSA for 1 h at ambient temperature, followed by imaging using the Molecular Imager ChemiDoc XRS + system (Bio-Rad).
Cell apoptosis assay
MCF-7 cells growing in pH 6.5 or 7.4 medium were cultured in 25 cm2 tissue culture flasks at a density of 6 × 105 cells per flask in 4 mL of complete RPMI 1640 medium, as mentioned above. After 24 h of culture at 37 °C in a 5% CO2 humidified atmosphere, the cells were washed with PBS and exposed to fresh serum-free medium containing free siPLK-1 or siPLK-1 (or siN.C., only for D-L)-loaded N-L, C-L and D-L. The final concentration of siRNA used in the experiment was 100 nM. Following a 6 h incubation, the medium was replaced with complete medium for a routine culture of 72 h at 37 °C. Subsequently, the cells were collected and stained with the Annexin V-FITC apoptosis detection kit (KeyGEN, Nanjing, China) according to the manufacturer’s instructions and were immediately analyzed using a BD FACSCalibur flow cytometer by collecting 10,000 events (excitation 488 nm; emission 530 nm).
Cells were incubated with D-LH/si-DTX for 48 h and then processed using the same procedures described above to assess the effect of the codelivery of DTX and the siRNA on apoptosis. The final concentrations of siPLK-1 and DTX were 100 nM and 0.1 µg mL−1, respectively.
Cell proliferation assay
MCF-7 cells were cultured with the samples over a wide range of concentrations to evaluate the cytotoxicity of different D-L samples. Cell viability was measured using the MTT assay. Cells (5 × 103 cells/well) were seeded into 96-well plates and incubated at 37 °C and 5% CO2 before the experiment, and a total volume of 200 μL was used. The old medium was replaced with medium containing D-L (100 nM siRNA and 0.01 µg mL−1 DTX) or the controls after 12 h of incubation. Untreated cells cultured in growth medium were used as the blank control. After 72 h, 20 μL of MTT solution (5 mg mL−1) were added to each well and incubated with cells for another 4 h. Next, the solution was removed, and 100 μL of DMSO were added to dissolve the MTT formazan crystals. The absorbance was measured at a wavelength of 490 nm using a microplate reader (Synergy 4, Bio Tec, USA). Cell viability (%) was defined as the percentage of the absorbance of the wells containing the cells incubated with the sample suspension to the blank control. All data are presented as the means of four measurements (± SD). The experiment was repeated three times.
Penetration and inhibition of three-dimensional tumor spheroids
Three-dimensional tumor spheroids of MCF-7 cells were prepared using hanging drop method, as previous described [54]. Briefly, 200 μL of agarose gel solution (2%, w/v) were heated at 80 °C and added to a 48-well plate. After cooling and solidifying, 500 μL of complete RPMI 1640 medium (pH 6.5 or 7.4) were added to each well. Twenty microliters of a cell suspension (1 × 103 cells) were suspended on the lid of a 48-well culture plate to induce sufficient cell aggregation. The cells were incubated in a 37 °C humidified incubator with a 5% CO2 atmosphere followed by 72 h of culture. The resulting cellular aggregates were transferred to the corresponding wells and grown for another 48 h.
MCF-7 tumor spheroids (300 μm in diameter) were incubated for 24 h with D-L (400 nM FAM-siRNA and 1.33 μg mL−1 DiD) at pH 6.5 and 7.4 to evaluate the penetration ability. After two washes with cold PBS, the tumor spheroids were transferred to a chambered coverslip and examined using CLSM. The tumor spheroids were scanned from the top to the equatorial plane to acquire Z-stack images. Each scanning layer was 8 µm thick, and the total scan depth was 64 µm.
MCF-7 tumor spheroids were incubated with PBS, free siPLK-1 and siPLK-1- or siN.C.-loaded N-L, C-L and D-L (200 nM siRNA and 2 μg mL−1 DTX) for 5 days to evaluate the inhibitory effect on proliferation. The spheroid volume was monitored with a 10 × objective lens using an inverted phase microscope (Motic, Xiamen, China). The major (dmax) and minor (dmin) diameters of each tumor spheroid were recorded, and the volume was calculated with the formula: V = 0.5 × dmax × dmin2. The percent change in the tumor spheroid volume ratio was calculated using the following equation: R = (Vi/V0) × 100%, where Vi is the tumor spheroid volume after treatment and V0 is the tumor spheroid volume before treatment.
Animals
Female BALB/c nude mice (6 weeks old) were purchased from the Vital River Laboratory Animal Center (Beijing, China) and fed a rodent diet. All procedures involving animal housing and treatment were approved by the Institutional Authority for Laboratory Animal Care of Hebei Medical University.
In vivo imaging
MCF-7 cells were subcutaneously injected into BALB/c mice to establish a subcutaneous xenograft breast tumor-bearing mouse model. When the tumor volume reached approximately 200 mm3, the mice were intravenously injected with 200 μL of free Cy5-siRNA or different liposomes (N-L, C-L, or D-L) containing Cy5-siRNA at 0.28 mg kg−1 and formulated DiD (C-L and D-L) at 0.1 mg kg−1. Subsequently, the acquisition of images of fluorescence signals from the whole body was performed using a Kodak in vivo imaging system (Kodak In Vivo Imaging System FX Pro, Carestream Health, USA) at the indicated time points (3, 6, 12, 24 and 36 h after injection). At the end point, the mice were sacrificed by cervical dislocation, and the major organs and tissues, including the heart, lung, liver, spleen, kidney, stomach, intestine and tumor, were collected and examined as described above.
Tumor suppression study
A xenograft tumor model was built by subcutaneously injecting MCF-7 cells, as described above, to assess the antitumor efficacy. After the tumors had grown to approximately 100 mm3, the mice were randomly divided into 10 groups (n = 6–7) and treated with 5% glucose (Control), free siPLK-1, various liposomal formulations (N-L, C-L, and D-L) carrying siRNA or/and DTX by intravenous injection once every other day for 10 d. The doses of siRNA and DTX in each injection were 0.28 mg kg−1 and 0.86 mg kg−1, respectively. The body weight and tumor size were measured at least once every 2 days throughout the postexposure period. The major (Dmax) and minor (Dmin) diameters of each tumor were recorded, and the volume was calculated with the formula: VT = 0.5 × Dmax × Dmin2. Relative tumor volume (RTV) was calculated using the formula RTV = (Va/V0) × 100%, where Va is daily tumor volume, V0 is initial tumor volume.
Detection of PLK-1 expression in tumor tissues
Tumor tissues were removed 24 h after the last administration to analyze PLK-1 expression in vivo. Tumor fragments (100 mg) were processed for total mRNA or protein extraction followed by qRT–PCR and western blot assays, respectively. The extracted mRNA samples were standardized to the same absorbance value of 260 nm and the expression of the PLK-1 mRNA was detected using qRT–PCR as described above. The selected tumor tissues were homogenized in 1 mL of RIPA lysis buffer (20 mM Tris–HCl (pH 7.4), 150 mM NaCl, 1% Triton X-100, 10 mM KCl, 1.5 mM MgCl2, 1 mM DTT, 100 mM AEBSF, 100 μM aprotinin, 5 mM bestatin, 1.5 mM E64, 2 mM leupeptin, 1.5 mM pepstatin A, cyclosporin A, sodium fluoride, beta-glycerophosphoric acid disodium salt, sodium orthovanadate, disodium molybdate dihydrate, and sodium pyrophosphate) (BestBio, China) supplemented with PMSF (1 mM) to evaluate PLK-1 protein levels. The lysates were incubated on ice for a total of 30 min and vortexed every 5 min. After purification and quantification, the protein levels were determined using western blot analysis as described above.
Pathological evaluation
For the histological analysis of tumor tissues and organs, the mice were sacrificed 24 h after the last administration, and both the tumor tissues and the major organs, including heart, lung, liver, spleen and kidney, were excised from one mouse randomly selected from each group. After 4% formaldehyde fixation, the sectioned specimens underwent H&E staining and histological evaluation using an optical microscope.
TUNEL staining for apoptosis was conducted on the specimens using the TRITC staining in situ Apoptosis Detection Kit (KeyGEN, Nanjing, China) according to the manufacturer’s protocol. Briefly, frozen sections were fixed with 4% formaldehyde for 30 min at room temperature and washed three times with PBS. Following an incubation in permeabilization solution (freshly prepared 1% Triton X-100 in PBS, pH 7.4) for 5 min, the sections were rinsed three times with PBS followed by treatment with 3% hydrogen peroxide (diluted in methanol) for 10 min at ambient temperature. Each sample was labeled with 50 μL of reaction mixture (45 μL of equilibration buffer, 1.0 μL of TRITC-5-dUTP and 4.0 μL of the TdT enzyme) at 37 °C in a dark and humidified atmosphere. For a positive control, 100 μL of DNase I reaction solution were added to the sample and treated for 30 min at 37 °C before the introduction of the TdT enzyme. For the negative control, the TdT enzyme was excluded from the labeling reaction mixture. Nuclei were stained with Hoechst 33,258 for 25 min at 37 °C, and the sections were examined under a confocal laser scanning microscope (Leica, Heidelberg, Germany).
Statistical analysis
All data are presented as the means ± standard deviations (SD) from at least three repeated experiments. Differences between any two groups were determined using ANOVA. P < 0.05 was considered statistically significant.
