Materials
50/50 poly(d,l-lactic-co-glycolic acid), ester terminated (PLGA 50:50), PURAC PDLG 5004 (inherent viscosity 0.41 dl/g) was purchased from Purac Biomaterials (Netherlands); docetaxel trihydrate, pharm. EP 7.5 (Doc) was purchased from QUILU Pharmaceutical Co. Ltd. (China); polyvinyl alcohol (PVA), 87–90% hydrolyzed, average mol wt 30,000–70,000, was purchased from Sigma-Aldrich (USA); dimethyl sulfoxide (DMSO), methylene chloride, and puriss. (MeCl2) were purchased from Chimmed (Russia); folic acid–dodecylamine (FoAD) was kindly provided by IREA Institute (Moscow, Russia, http://www.irea.org.ru). Acetonitrile used as the mobile phase in high-performance liquid chromatography (HPLC) was purchased from Macron (Poland). All other chemicals used were of reagent grade. DMEM culture medium, trypsin–EDTA, gentamicin, and rabbit polyclonal antibodies against the folate receptor α were purchased from Thermo Fisher Scientific (USA). Fetal bovine serum (FBS) was obtained from HyClone (USA). Alexa Fluor® 488-conjugated goat anti-rabbit secondary antibodies were purchased from Abcam (USA). FITC Annexin V Apoptosis Detection Kit with PI was obtained from BioLegend (USA). Other reagents were purchased from Sigma-Aldrich (USA).
Preparation of polymeric nanoparticles (NP–Doc, F–NP–Doc, F–NP–FDA)
PLGA nanoparticles were prepared using a single-emulsion solvent-evaporation method. All phases were filtered through 0.45-μm nylon membrane filters before use. The polymeric nanoparticles (NPs) were formulated by mixing of Doc (NP–Doc), FoAD and Doc (F–NP–Doc), FoAD and fluorescein diacetate (F–NP–FDA) with PLGA 50:50 solution in MeCl2. This organic phase was mixed with aqueous PVA solution and sonicated on ice. We used the Sonics Vibra-Cell VCX 750 sonicator (Sonics & Materials Inc., USA) equipped with #630-0220 probe and #630-0420 microtip with 6 mm tip diameter. Sonication was performed in 100-mL glass beaker using the following settings: energy—45 J, probe amplitude—45% (108 μm), pulsation (pulse/clear)—2 s/2 s, sonication time: 1-min sonication (2-min overall pulse/clear)—1-min rest—1-min sonication (2-min overall pulse/clear). MeCl2 was evaporated from obtained emulsion at room temperature for 12–16 h with constant stirring. Suspension of polymeric particles was centrifuged (30,000g, 4 °C) and resuspended in water with addition of d-mannitol. Suspension with additive was freeze-dried and stored in refrigerator (4 °C) before use.
Preparation of NP–Doc
5.0 mg of Doc and 100.0 mg of PLGA 50:50 were dissolved in 2.5 mL of MeCl2 and mixed with 17.5 mL of 1.0% PVA solution, then processed as described above. 50.0 mg of d-mannitol was added to the suspension before lyophilization.
Preparation of F–NP–Doc
0.2 mg of FoAD, 10.0 mg of Doc, and 200.0 mg of PLGA 50:50 were dissolved in 5.0 mL of MeCl2 and mixed with 35.0 mL of 1.0% PVA solution, then processed as described above. 70.0 mg of d-mannitol was added to the suspension before lyophilization.
Preparation of F–NP–FDA
2.0 mg of FDA and 200.0 mg of PLGA 50:50 were dissolved in 5.0 mL of MeCl2 and mixed with 20.0 μL of FoAD solution in MeCl2 (5 mg/mL). This organic phase was added to 35.0 mL of 0.25% PVA solution and then processed as described above. d-Mannitol was added to the suspension before lyophilization. FDA content was determined by UV spectroscopy at 209 nm.
Characterization of NPs
Particle size analysis and zeta potential measurements
The mean hydrodynamic diameter (Z-average), particle size distribution and polydispersity index (PdI) were measured by dynamic light scattering. Zeta potentials were determined by electrophoretic light scattering using Zetasizer Nano ZS ZEN 3600 (Malvern Instruments Ltd., UK) at 25 °C. The NP samples were prepared in deionized water at a concentration of about 0.2 mg NP/mL and measured in triplicates.
Determination of docetaxel loading
Lyophilized NP–Doc and F–NP–Doc nanoparticles were dissolved in DMSO, and Doc content was measured using the Agilent 1200 HPLC system with a UV detector (Agilent Technologies, USA). Chromatographic separation was performed on a reverse-phase column Agilent ZORBAX Eclipse XDB-C18, 250 × 4.6 mm, 5 µm, thermostated at 30 °C. Flow rate: 1.2 mL/min. Chromatography was carried out in the gradient conditions: phase A—water, phase B—acetonitrile. Gradient was programmed as follows: isocratic 72% A, 28% B (0–9 min), then linear (9–30 min) from 72% A, 28% B to 28% A, 72% B, then linear back to 72% A, 28% B (30–50 min). The Doc peak was measured at a wavelength of 290 nm and quantitatively determined by comparing with a standard curve. Approximate retention time for Doc was about 27 min.
Particle morphology examination
The morphological examination of NPs was performed by transmission electron cryomicroscopy (TEM). The samples were diluted in 1.0 mL of purified water, vortexed for 1 min and placed on previously hydrophilized supporting mesh. After vitrification in liquid ethane, the samples were transferred in liquid nitrogen to the pressing station and then placed in a cassette holder under cryogenic conditions. Imaging was performed using Titan Krios TEM FEI (Thermo Fisher Scientific, USA) at 5000×–18,000× magnification in a low-dose mode using a Falcon II electron detector.
In vitro docetaxel release studies
In vitro drug release study was performed using dialysis method. 1.0 mL of NP suspension containing 77.85 mg of NPs was placed into the dialysis membrane tubings OrDial D14 (Orange Scientific, Belgium) with pore diameter of 12–14 kDa and dialyzed against 2000 mL of phosphate buffered saline (PBS), pH 7.4, containing 0.5% Tween 80 at 37 °C on an orbital shaker stirring at a rate of 50 rpm. At the indicated times (the total incubation time was 48 h), the samples were quantitatively transferred from dialysis bags to glass vials, frozen, and lyophilized. Docetaxel loading in the lyophilized samples was determined by HPLC. Time-dependent curve of docetaxel release from polymeric nanoparticles was built according to the obtained results.
Cell lines
HeLa human cervical carcinoma cells, MDA-MB-231 human breast adenocarcinoma cells and LECH human embryonic lung fibroblasts were obtained from the Russian collection of cell cultures (St. Petersburg, Russia). MCF7R human breast adenocarcinoma cells and SKOV3 human ovarian carcinoma cells were kindly provided by Dr. V. Yu. Alakhov (Supratek Pharma Inc. Quebec, Canada). MCF7 human breast adenocarcinoma cells were purchased from the American Type Culture Collection (ATCC). Cells were maintained in plastic flasks (Corning, USA) in DMEM medium supplemented with 10% FBS and 50 µg/mL gentamicin in a CO2 incubator at 37 °C in a humidified atmosphere containing 5% CO2. Cells were passaged twice a week using trypsin–EDTA solution.
Evaluation of folate receptor α expression
The folate receptor expression levels in cells were assessed by flow cytometry. Cells were harvested using trypsin–EDTA solution, washed once with PBS and fixed with 2% paraformaldehyde. Fixed cells were permeabilized with 0.3% Triton X-100, blocked with 2% fatty acid-free bovine serum albumin (BSA) in PBS and incubated with rabbit polyclonal antibodies against folate receptor α (1:200 dilution) in blocking buffer for 1 h. After washing, the cells were incubated in blocking buffer with Alexa Fluor 488-labeled secondary antibodies (1:200 dilution). Afterwards, cells were washed, resuspended in PBS and analyzed on a BD FACSCalibur flow cytometer (BD Biosciences, USA). 104 cells were analyzed in each sample. The staining index (SI) was calculated according to the formula:
$${\text{SI}} = \frac{{{\text{MFIst}} - {\text{MFIa}}}}{{2 \times {\text{SDa}}}},$$
where MFIst and MFIa are medium fluorescence intensity of stained cells and an autofluorescence, respectively; SDa is standard deviation of the MFIa.
In vitro cellular uptake study
HeLa cells were seeded onto coverslips in 24-well plates 1 day prior to the experiment for attachment and adaptation. Cells were incubated with F–NP–FDA (12 μM in fluorescein diacetate equivalent) for 1 h in folic acid-free DMEM supplemented with 0.1% BSA in a CO2 incubator under standard conditions. At the end of incubation, cells were washed three times with PBS and analyzed on a BD FACSCalibur flow cytometer equipped with a 488-nm argon laser in the fluorescence channel FL1 or Axiovert 40 CFL fluorescence microscope (Carl Zeiss, Germany). To disclose the potential mechanisms of nanoparticle uptake, the endocytosis inhibitors including genistein (1 μg/mL, caveolae-mediated endocytosis inhibitor), monodansylcadaverine (300 μM, clathrin-mediated endocytosis inhibitor) and amiloride (1.25 mM, macropinocytosis inhibitor) were added to HeLa cells 30 min prior to incubation with F–NP–FDA.
Evaluation of cytotoxic activity of NPs
Cells were seeded in folic acid-free DMEM supplemented with 10% FBS and 50 μg/mL gentamicin in 96-well plates 1 day prior to the experiment for attachment and adaptation. Docetaxel and nanoparticles were added in a wide range of concentrations in triplicates for each concentration and incubated under standard conditions for 24–72 h. Cell survival was measured by the MTT assay (Alley et al. 1988) and the sulforhodamine B (SRB) assay (Skehan et al. 1990). MTT assay. 2 h before the end of incubation, 50 μL of MTT at a concentration of 1 mg/mL in culture medium was added to each well. After color development, the medium was removed, the precipitated formazan crystals were dissolved in 150 μL of DMSO, and the color intensity was measured by absorbance at 570 nm using an iMark microplate absorbance reader (Bio-Rad, USA). SRB assay. After the end of incubation, the medium was removed, and the cells were fixed with 10% trichloroacetic acid (TCA) for 1 h. Then TCA was removed, and the samples were dried in air and stained for 30 min with SRB solution (0.4% SRB in 1% acetic acid). Next, the plates were washed five times with 1% acetic acid and dried in air, and protein-bound SRB was dissolved in unbuffered 10 mM Tris (pH 10.5). The color intensity was measured by absorbance at 570 nm on a microplate reader. Cell survival was assessed as a percentage of the untreated control.
Quantitation of apoptosis by flow cytometry
HeLa cells were seeded in six-well plates (Corning, USA) 1 day prior to the experiment and cultured in FA-free DMEM supplemented with 10% FBS and 50 μg/mL gentamicin. On the day of the experiment, the culture medium was replaced with fresh medium containing docetaxel and polymeric nanoparticles in concentration of 2.5 and 10 nM in docetaxel equivalent. After the incubation was completed, cells were harvested using trypsin–EDTA, washed twice in PBS and stained with FITC Annexin V Apoptosis Detection Kit with PI according to the manufacturer’s instructions. Cells were incubated for 15 min in a staining buffer containing Annexin V-FITC and propidium iodide. Samples were analyzed without washing on a BD FACSCalibur flow cytometer in the fluorescence channels FL1 (for FITC) and FL2 (for PI).
In vivo anticancer activity of F–NP–Doc
All animal studies were conducted according to the European Convention for the Protection of Vertebrate Animals (Strasbourg 1986). The protocols of this study and experimental procedures were approved by the Ethical Committee of the National Research Center “Kurchatov Institute”. Specific pathogen-free (SPF) female C57BL/6 mice were obtained from Pushchino Nursery of Laboratory Animals (Pushchino, Russia). Ca755 mammary adenocarcinoma cells (1 × 106) were injected subcutaneously into the right flank of each animal (the day of tumor implantation was designated as day 0). The mice were randomized into vehicle control (saline) and treatment groups (n = 9 per group), and the treatments were started 48 h after tumor implantation. Docetaxel was dissolved in Tween 80/ethanol/saline (PRODUCT MONOGRAPH, Taxotere®), and F–NP–Doc was dissolved in saline. Drugs at a dosage of 15 mg/kg/dose in 0.4 mL of corresponding solvent were intravenously administered via the lateral tail vein on 3, 6 and 9 days after tumor inoculation. Tumor sizes were measured every 2–3 days, and tumor volume (V, mm3) was calculated using the formula:
$$V\, = \,a^{ 2} \, \times \,b/ 2,$$
where a and b refer to the short and long diameters of tumor, respectively.
The tumor growth inhibition (TGI) was calculated using the formula:
$${\text{TGI}}\left( \% \right)\, = \,(V_{\text{C}} {-}V_{\text{T}} )/V_{\text{C}} \, \times \,100,$$
where Vc and Vt refer to the mean tumor volume in control and experimental groups, respectively.
Statistical analysis
Plotting and statistical data processing were performed using OriginPro 8.5 (OriginLab Corporation) and Microsoft Excel (Microsoft Corporation). The accuracy of the results was evaluated using the Student’s t-test. The data in the tables and graphs are presented as an average of at least three experiments ± SE.