Materials
Docetaxel (DTX) was purchased from Guilin Huiang Biopharmaceutical Co., Ltd. (Guilin, China). HSA (20%) was purchased from Octapharma (Vienna, Austria). Dimethylsulfoxide (DMSO), 3-[4,5-dimethylthiazol-2-yl]-2,5,-diphenyltetrazolium bromide (MTT), and 4′,6-diamidino-2-phenylindole (DAPI) were supplied by Shanghai Yuanye Bio-Technology Co., Ltd. (Shanghai, China). Fluorescein isothiocyanate (FITC) was obtained from Beijing Dingguo Bio-Technology Co., Ltd. (Beijing, China). Human lung cancer cell line A549 was acquired from American Type Culture Collection (ATCC, Logan, UT, USA). Organic solvents of chromatography grade were purchased from Sinopharm Chemical Reagent Beijing Co., Ltd. (Beijing, China).
Methods
Preparation of the NPs
DTX-loaded HSA NPs (DTX-NPs) were prepared via a self-assembly method. Specifically, DTX (10–40 mg) was initially dissolved in 1 mL of ethyl alcohol. This solution was then mixed thoroughly with 0.65 mL of 12% disodium hydrogen phosphate and 100 mg of HSA, which had been pre-heated to 65 °C. After incubation for 3 min at 65 °C, the solution was slowly injected into 50 mL of 0.05 M sodium tartrate or sodium gluconate buffer with stirring (900 rpm) at 65 °C. This solution was immediately chilled in an ice bath. The remaining unencapsulated DTX was subsequently removed via ultrafiltration. The samples were freeze-dried without the use of additional cryoprotectants.
Single-factor optimization of DTX-NP preparation
Three parameters, including the drug-to-HSA (w/w) ratio, the duration of HSA incubation, and the choice of stabilizer, were selected as independent variables. The selected drug-to-HSA (w/w) ratios were 10, 20, 30, and 40 mg/mL. Meanwhile, the incubation times of HSA were 10, 20, and 30 min. In addition, the choice of stabilizer included either sodium tartrate or sodium gluconate buffer. The preparation of DTX-NPs is described above in “Preparation of the NPs” section. The effects of the three factors on the particle diameter and loading capacity (LC) of DTX-NPs were independently evaluated. Finally, DTX-NPs that were synthesized via the optimal conditions were used in the subsequent investigations.
Particle diameter and morphological characterization
The mean particle diameters of the NPs were determined via dynamic light scattering (DLS) measurements using a Zetasizer Nano ZS 90 (Malvern Instruments, Ltd., Malvern, UK) [49]. The morphologies of the NPs were investigated via scanning electron microscopy (SEM) using a JSM-6700F microscope manufactured by JEOL (Tokyo, Japan). One droplet of diluted suspension was deposited onto a patch of silicon wafer and dried at room temperature. The sample was subsequently sputter-coated with platinum and then examined by SEM.
Determination of the drug LC and EE
The drug concentration was initially determined by high-performance liquid chromatography (HPLC) using a Shimadzu-20AD system (Kyoto, Japan). The HPLC system was equipped with a Diamonsil C18 reverse-phase column (particle size 5 µm, 4.6 × 150 mm) manufactured by Dikma Technologies (Beijing, China). The detection wavelength was set at 232 nm and the column temperature was maintained at 40 °C. A mixture of 0.043 M ammonium acetate and acetonitrile (45:55, v/v) was used as the mobile phase at a flow rate of 1 mL/min. The drug content in the DTX-NPs was then determined via a literature, which is briefly summarized as follows [50]. The NPs were diluted in acetonitrile and sonicated for 15 min. The supernatant obtained after centrifugation at 12,000 rpm for 10 min was then injected into the HPLC system in a volume of 20 μL to quantify the amount of DTX. The equations used for calculating LC (%) and EE (%) are as following.
$$ {\text{LC}}\,\left( \% \right) = \frac{{{\text{The}}\;{\text{amount}}\;{\text{of}}\;{\text{DTX}}}}{{{\text{The}}\;{\text{weight}}\;{\text{of}}\;{\text{lyophilized}}\;{\text{nanoparticles}}}} \times 100\% $$
$$ {\text{EE}}\,\left( \% \right) = \frac{{{\text{DTX}}\;{\text{total}} - {\text{DTX}}\;{\text{free}}}}{{{\text{DTX}}\;{\text{total}}}} \times 100\% $$
In vitro release studies [51]
To investigate the kinetics of DTX release from the DTX-NPs, a suspension of DTX-NPs containing 2 mg of DTX was transferred into a dialysis bag with a MWCM of 8000 Da. This dialysis bag was subsequently placed into 80 mL of release medium (PBS) containing 0.5% v/v Tween 80. The temperature was maintained at 37.0 ± 0.5 °C and the medium was stirred at a speed of 100 rpm. DTX (1 mg/mL) which had been dissolved in polysorbate 80 and 13% (w/w) ethanol (free DTX) was used as a control and was treated in the same manner). During a period of 48 h, the surrounding environment was maintained by replacing 0.5 mL of the release medium with an equal volume of fresh medium at regular intervals. The extraction solution was solubilized in 500 μL of acetonitrile and assayed by HPLC.
Differential scanning calorimetry (DSC)
Thermograms were obtained via DSC measurements. The samples of DTX, lyophilized HSA, physical mixtures of DTX and HSA, and lyophilized DTX-NPs were placed in sealed aluminum pans. During these measurements an empty pan was also used as a reference. These samples were heated from 20 to 250 °C at a rate of 10 °C/min under a flow of nitrogen gas (20 mL/min). Each sample was prepared and analyzed in triplicate.
In vitro cytotoxicity assays
The cytotoxicity of the DTX-NPs was determined in A549 cells using the MTT assays. Briefly, the cells were plated at a density of 12,000 cells per well and incubated with 100 µL of serial dilutions of DTX-NPs (DTX concentrations of 1, 10 and 50 μg/mL), free DTX (dissolved in polysorbate 80 and 13% (w/w) ethanol) and drug-free NPs. After another 24 h, 20 µL of MTT (5 mg/ml in PBS, pH = 7.4) was added and incubated at 37 °C for 4 h. The medium was then removed and 100 µL of DMSO was added into each well to dissolve the crystals. OD was then measured at 490 nm using a microplate reader (Synergy4, multi-mode microplate reader, BioTek, Winooski, VT, USA).
Cellular uptake determination of DTX-NPs in vitro
To detect the uptake of the NPs by A549 cells, FITC-labeled HSA was used to prepare the NPs (FITC-DTX-NPs). Cells were seeded at a density of 7 × 104 cells per well onto 24-well plates and incubated with FITC-DTX-NPs for 1, 2, or 4 h. Untreated cells were used as a control. After the cells had been gently washed twice with PBS, they were re-suspended and fixed with 4% paraformaldehyde and analyzed using a EPICS XL flow cytometer (Beckman Coulter Corp, Tokyo, Japan) to measure the fluorescence intensities.
To prepare the samples for fluorescence microscopy imaging, 7 × 104 cells per well were incubated with FITC-DTX-NPs for 4 h and fixed with 4% paraformaldehyde. The nuclei were stained with DAPI for 3 min. Fluorescence microscopy images were obtained using a Zeiss 710 LSMNLO Confocal Microscope (Carl Zeiss; Jena, Germany).
Assessment of the maximum tolerated dosage (MTD)
Male BALB/c Mice were purchased from Beijing Vital River Laboratory Animal Technology Co. Ltd. (Beijing, China). Prior to the experiments, the entire animal protocol was reviewed and approved by the Institution Animal Ethics Committee (Jilin University, license No.SCXK-(JI) 2011–0003) and we adhered to the Guidelines on Humane Treatment to Lab Animals (published in 2009) [39]. The mice were randomly separated into six groups (n = 6). DTX-NPs were dispersed in 0.9% saline solution with the DTX concentrations to be 60, 75 and 90 mg/kg (corresponding to groups i, ii and iii, respectively). Meanwhile, free DTX was dispersed into Tween 80 and 13% (w/w) ethanol in water at a ratio of 1:3 (w/w), at DTX concentrations of 20, 30 and 40 mg/kg to yield groups iv, v and vi, respectively. The mice were injected intravenously via their tail vein on days 1, 3 and 5. They were weighed prior to each injection and they continued to be weighed on days 10, 15 and 20. The dosage that resulted in a 20% loss in weight was defined as the MTD [52].
), free DTX (
), DTX-NPs (
) and drug-free NPs (
) against A549 cell line at various DTX concentrations. Data are expressed as mean ± SE (n = 6). **p < 0.01 and ***p < 0.001, in comparison with the control
