D,L-lactide (3,6-dimethyl-1,4-dioxane-2,5-dione, C6H8O4) with a purity greater than 99% was purchased from Aldrich, Milwaukee, USA. They were recrystallized at least three times from ethyl acetate before reaction to remove the residual impurities in the monomer. d-α-tocopheryl polyethylene glycol 1000 succinate (TPGS, C33O5H54 (CH2CH2O)23) was from Sigma (St. Louis, MO, USA). Stannous octoate (Sn(OOCC7H15)2), glycolide (1,4-Dioxane-2,5-dione, C4H4O4) (purity >99%) and PLGA (50:50, MW 50,000) were also purchased from Sigma (St. Louis, MO, USA). Docetaxel powder of purity 99.8% was purchased from Shanghai Jinhe Bio-Technology Co. Ltd (Shanghai, China). Acetonitrile and methanol were purchased from EM Science (ChromAR, HPLC grade, Mallinckrodt Baker, USA). All other chemicals used were of the highest quality commercially available. Ultrahigh pure water produced by Boon Environmental Tech. Industry Co., Ltd. (Tianjin, China) was utilized throughout all experiments. Human cervix carcinoma HeLa cells were provided by American Type Culture Collection (ATCC, Rockville, MD).
Synthesis of PLGA-TPGS Random Copolymer
PLGA-TPGS random copolymers were synthesized by ring-opening bulk polymerization of lactide and glycolide monomer with TPGS in the presence of stannous octoate as catalyst. Briefly, weighted amounts of glycolide, lactide, TPGS and 0.5 wt% stannous octoate (in distilled toluene) were added in a reaction flask. The mixture was heated to 145°C and allowed to react for 16 h. Synthesis was carried out under an oxygen- and moisture-free environment. The resulting product was dissolved in DCM and then precipitated in excess cold methanol to remove unreacted monomers and TPGS. The final product was collected by filtration and dried under high vacuum at 45°C for 48 h.
Characterization of PLGA-TPGS Random Copolymer
FTIR spectrophotometer (Thermo Nicolet, Madison, WI, USA) was used to investigate the molecular structure of PLGA-TPGS copolymer. In brief, the samples for FTIR analysis were prepared by grinding 99% KBr with 1% random copolymer and then pressing the mixture into a transparent tablet. The TPGS content and number-averaged molecular weight of the copolymer were determined by 1H NMR in CDCl3 at 300 Hz (Bruker ACF300). The weight-averaged molecular weight and molecular weight distribution were determined by gel permeation chromatography (Waters GPC analysis system with RI-G1362A refractive index detector, Waters, Milford, USA) column was used. The mobile phase was THF delivered at a flow rate of 1 ml/min. The injection volume was 50 μl sample solution (0.1% w/v polymer in mobile phase). The calibration curve was established by using polystyrene standard sample (molecular weights: 5.51 × 104, 1.39 × 104, 2.97 × 103 and 162, respectively). Thermogravimetric analysis (TGA, TGA 2050 thermogravimetric analyzer, USA) was carried out to investigate the thermal properties of the copolymers. During the TGA analysis, about 5–15 mg of the random copolymer sample was heated from 30 to 600°C at a rate of 10°C/min.
Preparation of Docetaxel-Loaded PLGA-TPGS Nanoparticles
The docetaxel-loaded PLGA-TPGS nanoparticles were prepared by a modified solvent extraction/evaporation method . In brief, a given amount of docetaxel and 100 mg PLGA-TPGS copolymer were dissolved in 8 ml dichloromethane (DCM). The formed solution was poured into 120 ml 0.03% TPGS pure water solution under gentle stirring. The mixture was sonicated for 120 s at 25 W to form O/W single emulsion. The emulsion was then evaporated overnight to remove DCM. The particle suspension was centrifuged at 20,000 rpm for 15 min and then washed three times to remove the unloaded drug and surfactant. The resulted particles were resuspended in 10 ml water and freeze-dried for 2 days. In addition, the fluorescent coumarin-6–loaded nanoparticles were prepared in the same way, except 0.1% (w/v) coumarin-6 was encapsulated instead of docetaxel.
Characterization of Drug-Loaded PLGA-TPGS Nanoparticles
Size and Size Distribution
The particle size and size distribution of the nanoparticles were measured by Dynamic Light Scattering (Zetasizer Nano ZS90, Malvern Instruments LTD., Malvern, UK). One milligram of particles was suspended in 3 ml of pure deionized water before measurement. The measurement was performed in triplicate.
The nanoparticles were imaged by a field emission scanning electron microscopy (FESEM) system at an accelerating voltage of 5 kV. The particle surface was coated with platinum layer before being observed.
The samples were prepared by diluting the nanoparticles suspension with pure deionized water. Zeta potential of the nanoparticles was measured by Laser Doppler Anemometry (LDA; Zetasizer Nano ZS90, Malvern Instruments LTD., Malvern, UK). The data were obtained with the average of three measurements.
Drug Loading and Encapsulation Efficiency (EE)
The docetaxel entrapped in the drug-loaded PLGA-TPGS nanoparticles was measured by HPLC (LC 1200, Agilent Technologies, Santa Clara, CA). A reverse-phase Inertsils C-18 column (150 × 4.6 mm, pore size 5 mm, GL science Inc, Tokyo, Japan) was used. Briefly, 5 mg nanoparticles were dissolved in 1 ml DCM. After DCM was evaporated in nitrogen atmosphere, the samples were extracted by 5 ml mobile phase (50/50 (v/v) acetonitrile/water solution). Twenty microliters of the clear solution was used for HPLC analysis. The flow rate of mobile phase was 1 ml/min. Retention time was controlled at 15 min. The column effluent was detected at 227 nm with a UV/VIS detector. The drug encapsulation efficiency was defined as the ratio between the amount of docetaxel entrapped in nanoparticles and that added in the process.
Differential Scanning Calorimetry (DSC)
The physical status of docetaxel inside the drug-loaded PLGA-TPGS nanoparticles was investigated by differential scanning calorimetry (DSC 822e, Mettler Toledo, Greifensee, Switzerland). The samples were purged with dry nitrogen at a flow rate of 20 ml/min. The temperature was ramped at a rate of 10°C/min.
In Vitro Drug Release
In brief, 15 mg drug-loaded PLGA-TPGS nanoparticles 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. The suspension was transferred into a Regenerated Cellulose Dialysis Membrane (Spectra/Por 6, MWCO = 1000, Spectrum, Houston, 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 shaking at 120 rpm at 37.0°C. Ten milliliters of solutions was periodically removed for HPLC analysis and replaced with fresh medium. The collected samples were extracted with 2 ml DCM and reconstituted in 5 ml mobile phase. The DCM was evaporated by nitrogen stream. The analysis procedure was the same as for the measurement of encapsulation efficiency.
Cellular Uptake of Coumarin-6–Loaded PLGA-TPGS Nanoparticles
Human cervical adenocarcinoma cancer cell lines HeLa cells were cultured in Dubelco’s modified essential medium (DMEM) supplemented with 10% fetal bovine serum and antibiotics. The culture was maintained in incubator containing 5% CO2 at 37°C. The cells were incubated with 250 μg/ml coumarin-6–loaded nanoparticles at 37°C for 4 h, rinsed with cold PBS for three times and then fixed by methanol for 10 min. Cells were stained with DAPI for 15 min and washed three with PBS. Finally, cells were observed by confocal laser scanning microscope (CLSM, Olympus Fluoview FV-1000, Olympus Optical. Co., Ltd., Tokyo, Japan). The images of the cells were determined with differential interference contrast (DIC) channel, and the images of coumarin-6–loaded nanoparticles and the nuclei of the cells stained by DAPI were recorded with following channels: blue channel (DAPI) with excitation at 340 nm and green channel (coumarin-6) with excitation at 488 nm [18, 19].
In Vitro Cytotoxicity of PLGA-TPGS Nanoparticles
HeLa cells were seeded in 96-well plates at the density of 5,000 viable cells per well and incubated 24 h to allow cell attachment. The cells were incubated with docetaxel-loaded PLGA-TPGS nanoparticle suspension at 0.025, 0.25, 2.5, 10 and 25 μg/ml docetaxel concentrations and drug-free PLGA-TPGS nanoparticle suspension with equivalent nanoparticle concentrations of 0.25, 2.5, 25, 100 and 250 μg/ml for 24, 48 and 72 h, respectively. At the determined time, the formulations were replaced with DMEM containing MTT (5 mg/ml), and cells were then incubated for additional 4 h. MTT was aspirated off, and DMSO was added to dissolve the formazan crystals. Absorbance was measured at 570 nm using a microplate reader (Bio-Rad Model 680, UK). Untreated cells were taken as control with 100% viability, and cells without the addition of MTT were used as blank to calibrate the spectrophotometer to zero absorbance.
The data are expressed as mean ± SD. The significance of differences was assessed by Student’s t test and was considered significant when p = 0.05.