Polymers and reagents
TPGS-b-(PCL-ran-PGA) copolymer (Mw approximately 24,000) was synthesized in our laboratory (Tsinghua University, China). Poly (vinyl alcohol) (PVA; (80% hydrolyzed), dichloromethane, branched polyethylenimine (MW approximately 25,000, BPEI25k) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) kit were purchased from Sigma-Aldrich Corporation (MO, USA). 4',6-diamidino-2-phenylindole (DAPI) was purchased from VECTOR (Burlingame, USA). Dulbecco's Modified Eagles' Medium (DMEM), penicillin-streptomycin, fetal bovine serum (FBS), and Dulbecco's phosphate buffered saline (DPBS) were purchased from Invitrogen-Gibco (Carlsbad, CA). Plasmid vectors pShuttle2, pIRES-EGFP, and pDsRED-E1 were acquired from Invitrogen Corporation (Clontech, USA).
Preparation of expression vectors
Human endostatin gene was amplified by PCR using the following primers: hEndostatin-F: 5′-GCTCTAGA(XbaІ)gccaccatgggaattcatgcacagccaccgcgacttcc-3′ and hEndostatin-R: 5′-GGGGTACC (KpnІ)ttacttggaggcagtcatg-3′. PCR products were digested with Xba I and KpnІ and inserted into pShuttle2 vector (Clontech). The recombinant plasmid pShuttle2-endostatin was verified by DNA sequencing. The expression of endostatin in HeLa cells transfected with PEI or TPGS-b-(PCL-ran-PGA)-based NPs was analyzed by Western blot analysis.
Preparation of docetaxel-loaded TPGS-b-(PCL-ran-PGA) NPs and determination of drug contents
Docetaxel-loaded and blank TPGS-b-(PCL-ran-PGA) NPs were prepared using a modified water-in-oil-in-water solvent evaporation as described previously . Briefly, TPGS-b-(PCL-ran-PGA) copolymer (50 mg) and a certain amount of docetaxel (0 to 25mg) were dissolved in 2 ml methylene chloride, followed by 30-s sonication to emulsify the mixture (UW 70/HD 70; tip, MS 72/D; Bandelin Electronic GmbH & Co., Berlin, Germany). After the addition of 3 ml of 7% (w/v) aqueous solution of PVA, the emulsion was sonicated again for 20 s. The resulting double emulsion was then poured into 50 ml 1% (w/v) aqueous PVA solution containing 2% isopropanol and then maintained under mechanical stirring for 1 h at 600 rpm. The residual methylene chloride was then evaporated by vacuum. Next, the 2 ml aliquots of the nanosphere suspension were washed twice with 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)/NaOH (pH 7.0), and the NPs were harvested by centrifugation at 7,000 rpm for 10 min. In addition, the fluorescent coumarin-6 loaded TPGS-b-(PCL-ran-PGA) NPs were prepared in the same way except that 0.1% (w/v) coumarin-6 was entrapped instead of docetaxel.
The docetaxel-loading content, defined as the mass percentage of docetaxel entrapped in nanoparticles, was quantified using UV–vis analysis (UV-2000 UV–vis spectrophotometer, Unico Inc., WI, USA). First, docetaxel-loaded nanoparticle solutions were lyophilized to yield the solid nanoparticle samples. Then, the dried nanoparticle samples were weighed and redissolved in a mixture of chloroform and DMSO (1:1, v/v). The absorbance of docetaxel at 482.5 nm was measured, and the drug content in the solution was calculated based on a previously established calibration curve.
Preparation of PEI-modified TPGS-b-(PCL-ran-PGA) NPs/DNA complexes
The NPs were prepared as described previously . Briefly, the particles were formulated with a ratio of the polymer nitrogen to the DNA/phosphate (N/P) equal to TPGS-b-(PCL-ran-PGA) NP solution (0.2 ml) and were mixed with 2 mg of PEI in sterile HEPES-buffered saline. The PEI-modified TPGS-b-(PCL-ran-PGA) NP solution was then added to the plasmid DNA solution at different N/P ratios and vortexed gently. The PEI-modified TPGS-b-(PCL-ran-PGA) NP/DNA complexes were incubated in sterile PBS for 20 min at room temperature.
Characterization of nanoparticles
The mean particle size and size distribution were measured using dynamic light scattering (DLS) (Zetasizer Nano ZS90, Malvern Instruments Ltd., Malvern, UK). In brief, the NPs were suspended in deionized water at a concentration of 0.1 mg/ml. The mean hydrodynamic diameter was determined via cumulative analysis. The DLS determinations were predicated based on the electrophoretic mobility of the NPs in an aqueous medium. The electrophoretic mobility was evaluated using folded capillary cells in an automatic mode. Zeta potential of the NPs was detected using laser Doppler anemometry (LDA; Zetasizer Nano ZS90, Malvern Instruments Ltd.). Samples were prepared in PBS and diluted 1:3 with deionized water to ensure that the measurements were performed under conditions of low ionic strength where the surface charge of the particles can be measured accurately. The final concentration of the polymer was 0.01 mg/ml. All data represent five measurements from one sample.
Gel retardation assay
The binding of pDNA with free TPGS-b-(PCL-ran-PGA)/PEI NPs was determined by 0.8% agarose gel electrophoresis. A series of different weight ratios (w/w) of pDNA to NPs was loaded on the agarose gel (10 μl of the sample containing different amounts of pDNA). The pDNA bands were then visualized under a UV transilluminator at a wavelength of 365 nm.
Evaluation of loading efficiency of pDNA to the NPs
The loading efficiency of pDNA to the NPs was evaluated by measuring unbound pDNA in the NPs solution. The supernatant in the loaded docetaxel or free TPGS-b-(PCL-ran-PGA)/PEI NPs solution was recovered by removing the NPs by centrifugation. Free pDNA concentration in the supernatant was determined by measuring absorbance at 260 nm using a UV-2550 spectrophotometer (Shimadzu Corp., Kyoto, Japan). Loading efficiency of pDNA in NPs was determined by subtracting the amount of pDNA recovered in the washing solutions from initial amount of pDNA added. Encapsulation efficiency was defined as the percentage of pDNA encapsulated in NPs with respect to the initially added amount of pDNA.
In vitro drug release
NPs with 15 mg docetaxel-loaded TPGS-b-(PCL-ran-PGA) were dispersed in 5 ml release medium (phosphate buffer solution of pH 7.4 containing 0.1% w/v Tween 80) to form a suspension. Tween 80 was used to increase the solubility of docetaxel in the buffer solution and avoid the adherence of docetaxel to the tube wall. The suspension was transferred into a dialysis membrane bag (Spectra/Por 6, MWCO = 1,000, Spectrum Laboratories, Inc., TX, USA). Then, the closed bag was put into a centrifuge tube and immersed in 15-ml release medium. The tube was put in an orbital water bath at 37°C under a 120 rpm horizontal shaking. Ten milliliters of solution was periodically removed for analysis and was replaced with fresh release medium. The collected samples were mixed in a mixture of chloroform and DMSO (1:1, v/v). The analysis procedure was the same as described above.
To investigate the in vitro release of pDNA, 5 mg of TPGS-b-(PCL-ran-PGA)/PEI-25K (n = 6) and coumarin-6-loaded TPGS-b-(PCL-ran-PGA)/PEI (n = 6) were incubated in 1 ml of DPBS buffer (pH 7.4) in a micro-centrifuge tube in shaking incubator at 37°C. After incubating for 24 h, half of the samples (n = 3) were transferred into a 25 mM sodium acetate buffer (pH 5.0) to simulate acidication of the endolysosome of the cell. Samples were taken periodically in microcentrifuge tubes and were centrifuged at 14,000 rpm for 10 min to obtain pellet NPs. The supernatants were removed and replaced with fresh buffer, and NPs were resuspended by vigorous pipetting. The supernatants were stored at −70°C until analysis by UV measurement. Unmodified TPGS-b-(PCL-ran-PGA) NPs were also analyzed as a background control.
HeLa and 293T cells (ATCC, VA, USA) were cultured in DMEM, pH 7.4, supplemented with 25 mM NaHCO3, 10 μg/ml streptomycin sulfate, 100 μg/ml penicillin G, and 10%(v/v) FBS. Cells were maintained at 37°C in a 5% CO2, 95% air incubator.
The cytotoxicity of the NPs was determined using MTT assay. The culture medium was removed and replaced with 20 μl/well MTT (5 mg/ml) solution in each well, followed by 4-h incubation at 37°C in a fully humidified atmosphere with 5% CO2. MTT was taken up by active cells and transformed into insoluble purple formazan granules in the mitochondria. Subsequently, the medium was discarded, the precipitated formazan was dissolved in DMSO (150 μl/well), and optical density of the solution was evaluated using a microplate spectrophotometer at a wavelength of 570 nm. The analytic assays were performed every day, and at least 4 wells were randomly taken into examination each time. The determination of cell viability depends on these physical and biochemical properties of cells.
Cellular uptake of PEI-modified TPGS-b-(PCL-ran-PGA) NPs
The cells were plated in a 6-well (3 × 105 per well) plate, cultured at 37°C in 5% CO2, rinsed twice, and pre-incubated for 1 h with 2 ml of serum-free medium at 37°C. Coumarin-6-loaded TPGS-b-(PCL-ran-PGA)/PEI NPs with or without pDsRED or pIRES-EGFP were added in a particle concentration of 0.01 to 0.2 mg/ml and incubated for 1 to 4 h at 37°C. The cells were then washed three times with 1 ml of PBS (pH 7.4) to remove free PEI/plasmids-modified TPGS-b-(PCL-ran-PGA) NPs, resuspended in PBS, and then analyzed by fluorescent microscopy.
For confocal laser microscopy analysis, cells were pre-incubated for 1 h at 37°C in serum-free medium and then for 30 min to 4 h in the presence of PEI/plasmids pIRES-EGFP or/and pDsRED gene-modified TPGS-b-(PCL-ran-PGA) NPs with a final particle concentration of 0.05 mg/ml. The samples were mounted in fluorescent mounting medium (Dako, Glostrup, Denmark), and fluorescence was monitored. Images were acquired using a confocal laser microscope (Eclipse TE 2000, Nikon Corporation, Tokyo, Japan).
The cells were seeded into 6-well plates overnight, allowed to attach the bottom. Cells were cultured at 37°C in 5% CO2, rinsed twice, and pre-incubated for 1 h with 2 ml of serum-free medium at 37°C. Endostatin gene-modified TPGS-b-(PCL-ran-PGA) NPs were added in a particle concentration of 0.01 to 0.2 mg/ml and incubated for 1 to 4 h at 37°C. The cells were then washed three times with 1 ml PBS (pH 7.4) to remove any free PEI/plasmid-modified TPGS-b-(PCL-ran-PGA) NPs. After being cultured in fresh complete medium for 48 h, the cells were lysed with lysis buffer (Beyotime Institute of Biotechnology, Haimen, Jiangshu, China) containing PMSF (Sigma Chemical Co., St. Louis, MI, USA) for 30 min at 4°C. The lysate was then centrifuged for 20 min at 13,000 rpm and 4°C. The proteins were then separated through SDS-PAGE and transferred onto the PVDF membrane (Immobion-P Transfer Membrane, Millipore Corp., Billerica, MA, USA). Membranes were blocked in a tris-buffered saline with 0.1% Tween 20 solution containing 5% non-fat dry milk and incubated overnight with anti-human endostatin antibody as primary antibody at 4°C. Anti-β-actin antibody was used as loading control.
Mice maintenance and subcutaneous tumor model
Female BALB/c-nu/nu mice were supplied by the Medical Experimental Animal Center of Guangdong Province (Guangdong, China). A subcutaneous tumor model of nude mice was constructed. Six-week-old female nude mice (18 ± 2 g) were subcutaneously inoculated with 1.5 to 2 × 106 HeLa cells. When the tumor size reached about 100 mm3, the mice were randomly divided into six groups with six mice each: PBS, TPGS-b-(PCL-ran-PGA) (group ANP), TPGS-b-(PCL-ran-PGA)/PEI(group BNP), TPGS-b-(PCL-ran-PGA)/PEI-pEndostatin (group CNP), docetaxel-loaded TPGS-b-(PCL-ran-PGA) (group DNP), and docetaxel-loaded TPGS-b-(PCL-ran-PGA)/PEI-pEndostatin (group FNP), of which each dose of NPs contained 0.2 mg PCL-PGA-TPGS, 10 μg PEI, and 50 μg pDNA for treatment. The groups were treated once every 3 days with intratumoral injections of PBS or NPs. Tumor size was measured with a caliper, and the weight of each mouse was measured with a scale. Tumor volume was calculated as the equation ‘volume = length × width2 / 2’. The mean tumor volume and mouse weight were used to construct the curves of tumor growth versus mouse growth to evaluate therapeutic efficiency and toxicity.
At the end of the treatment, the mice in each group were killed and dissected. The heart, liver, lungs, spleen, kidneys, and other organs were checked for signs of toxicity. Tumor specimens were then prepared as paraffin-embedded sections for histopathological analysis. The study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of Institutional Animal Care and Use Committee of Graduate School at Shenzhen, Tsinghua University. The protocol was approved by the Animal Welfare and Ethics Committee of Graduate School at Shenzhen, Tsinghua University, China (no. 2011-YZ-BG).
Measurement of microvessel density in tumor tissues
Microvessel density (MVD) in tumor tissue was evaluated immunohistochemically using a monoclonal anti-mouse CD31 antibody (rat anti-mouse CD31 monoclonal antibody, clone MEC 13.3; BD Biosciences, NJ, USA). Tumor samples were collected after the therapy regimen was finished. Immunohistochemical staining was performed as described previously . MVD (%) was calculated from the ratio of the CD31-positive staining area to the total observation area in the viable region. Three to six fields per section were randomly analyzed, excluding necrotic areas. Positive staining areas were calculated using imaging analysis software (Win Roof; Mitani Corporation, Fukui, Japan).
Statistics and data analysis
All cell experiments were repeated at least three times unless otherwise indicated. Images of Western blot results from representative experiments were presented. The figures were created using Adobe Photoshop CS graphics program. All data were analyzed by paired t test using SPSS 11.0 software. Differences were considered statistically significant at P < 0.05.