The 50:50 PLGA(poly lactic-co-glycolic acid), MW 30–70 kDa with an inherent viscosity of 0.59 dL/g, polyvinyl alcohol (PVA), MW 12–23 kDa, 87–89% hydrolyzed, N-hydroxysuccinimide (NHS), EDC (1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide), and penicillin/streptomycin solution were obtained from Sigma-Aldrich (St. Louis, MO). Ethyl acetate and all other reagents used were supplied by Fisher Scientific (Fairlawn, NJ). Paclitaxel was obtained from Wako Chemicals. All reagents were of analytical grade. Cell cultures chemicals-DAPI, trypan blue, trypsin (0.25%), and MTT assay kit were purchased from Sigma-Aldrich. Alamar blue stain was supplied by Invitrogen.
Aptamer AS1411 (NH2-5′-(GGTGGTGGTGGTTGTGGTGGTGGTGG)-3′) and non-specific aptamer (NH2 -5′-(CCTCCTCCTCCTTCTCCTCCTCCTCC) - 3′) were purchased from Operon.
2.1 Formulation of paclitaxel-loaded nanoparticles
Effective encapsulation of chemotherapeutic drug within the nanoparticle depends on various factors like preparation method, polymer and drug composition, solvent and drug solubility, and stabilizer composition (Fonseca et al. 2002). We have adopted the nanoprecipitation-solvent evaporation method to synthesis paclitaxel-loaded PLGA nanoparticles with minor modifications. PVA, a widely known stabilizer for PLGA nanoparticles (Feng 2001), was employed in the synthesis along with a hydrophobic eight-carbon spacer chain of homo-bifunctional chemical cross-linker, bis(sulfosuccinimidyl) suberate (BS3). This carbon spacer chain will align itself on the nanoparticle surface with its COOH groups on the terminal hydrophilic side extending towards the outside of the nanoparticle surface (Thamake et al. 2011). Paclitaxel, a highly potent chemotherapeutic drug widely used against various tumors, showed limited clinical success owing to its low therapeutic efficiency and low solubility in many pharmaceutical solvents (Jin et al. 2009). Upon incorporation within PLGA nanoparticles, paclitaxel demonstrated enhanced therapeutic index of the drug and lack of the toxic effects caused by its commercial adjuvant Cremophor®EL (Gradishar et al. 2005).
Nanoparticles were prepared using a single-emulsion technique/solvent evaporation method that has been reported elsewhere with slight modification (Thamake et al. 2011; Cartiera et al. 2010). Briefly, 65 mg of PLGA dissolved in 1 ml of ethyl acetate was added to 2.2% aqueous solution of PVA containing 0.5 mg ml−1 of BS3. This mixture was sonicated at room temperature using an ultrasonic processor UP200H system (Hielscher Ultrasonics GmbH, Germany) at 40% amplitude for 2 min in continuous mode. The excess solvent was evaporated by continuous stirring for 45 min to 1 h followed by centrifugation at 10,500 rpm for 15 min to remove excess of aqueous solution. The separated nanoparticles were washed by resuspending in water three times. One milligram of paclitaxel in 1 ml of ethyl acetate was added along with the polymer/solvent mixture to prepare drug-loaded nanoparticles. Surface morphology and size were also determined by high-resolution scanning electron microscopy and atomic force microscopy.
2.2 Preparation of aptamer-conjugated drug-loaded nanoparticles
In order to achieve an aptamer–nanoparticle bio-conjugate capable of targeting desired cells, the binding protocol must preserve the biological activity of the aptamer (Balamurugan et al. 2008). Hence, special care was taken in choosing a suitable covalent binding procedure for functionalizing the aptamer on nanoparticle surface with effective binding by maintaining its biological activity (Janas and Janas 2011). Amine-modified AS1411 aptamer was conjugated on the carboxyl group carrying nanoparticles using the common conjugation strategy of carbodiimide chemistry (cross-linking of the carboxylic acid group on the nanoparticle surface and the amine group of the aptamer to form an amide linkage). The carboxyl groups on the nanoparticle surface were converted to its succinimide by using EDC and NHS, which was then allowed to react with NH2-AS1411 aptamer. This method is well studied by many researchers which effectively carried out the conjugation of aptamer on to the polymer nanoparticles (Davies et al. 2010; Ling et al. 2011; Farokhzad et al. 2004; Dhar et al. 2008).
PTX-PLGA NPs (10 μg/μL) was washed three times with 250-μL aliquots of a 10 mM phosphate buffered saline (PBS) (pH 7.4) and incubated with 200 μL of 400 mmol/L EDC and 200 μL of100mmol/L NHS for 15 min at room temperature with gentle shaking. The resulting NHS-activated particles are covalently linked to amine-modified AS1411 aptamer (1 μg/μL). The sample was allowed to react for 2 h with constant mixing at room temperature, and three final washes were performed using the 20 mM Tris–HCl, 5 mM MgCl2 at pH 8.0. The resulting aptamer–nanoparticle bio-conjugates were resuspended and preserved in suspension form in DNase–RNase-free water at 4°C before use.
2.3 Surface morphology characterization
The shape and surface morphology of paclitaxel-loaded PLGA NPs were analyzed using a scanning electron microscope (SEM) (FESEM, JSM-6700F, JEOL, Japan) at an accelerating voltage of 3–5 kV. Nanoparticles were fixed to sample stubs with double-sided carbon tape and sputter-coated with platinum which was carried out by an Auto Fine Coater (JEOL, Tokyo, Japan) for 50 s for viewing by SEM. For atomic force microscopy (AFM), drug-loaded PLGA solution (200 μL) was deposited on a glass surface and vacuum-dried. The sample was characterized by AFM (Digital Instruments 3000AFM) in tapping mode. Three-dimensional imaging of the drug-loaded nanoparticles was done using transmission electron microscopy (TEM, JEM 2200 FS, JEOL, Japan). One drop of the sample solution was deposited onto a carbon-coated copper grid that had been previously hydrophilized under UV light and air-dried at room temperature prior to examination under TEM.
2.4 Surface chemistry characterization
The aptamer labeling on the surface of paclitaxel-loaded PLGA NPs was confirmed from the surface chemistry measured by X-ray photoelectron spectroscopy (XPS, AXIS His-165 Ultra, Kratos Analytical, Shimadzu Corporation, Japan). Five microliters of the sample was applied on a clean silicon substrate and dried in vacuum. The binding energy spectrum was recorded from 0 to 1,000 eV with pass energy of 80 eV under the fixed transmission mode.
2.5 Cell culture studies
GI-1 cells obtained from Riken Bio Resource Center, Japan, were cultured in monolayers to 80% confluence by maintaining in Dulbecco’s minimal essential medium (DMEM, Gibco) supplemented with 10% fetal bovine serum and 1% penicillin–streptomycin solution in a 5% CO2-humidified atmosphere at 37°C. Normal human mammary epithelial cells obtained from Gibco were maintained in HuMEC-ready medium (Gibco) supplemented with growth supplements and antibiotics in 5% CO2-humidified atmosphere at 37°C. For use in experiments, 1 × 104cells/ml per well were seeded in glass-based dish for confocal studies; approximately 5,000–8,000 cells were seeded in 96-well plates for cytotoxic studies; 3 × 104 cells were plated in a 25-mL flask for phase contrast studies, and 2.5 × 106 cells per well was seeded in glass plate for flow cytometry studies.
2.6 Confocal microscopy
GI-1 cells were seeded in glass-based bottom well dish at a density of 1 × 104 cells/ml. The plates were incubated at 37°C and grown to 70% confluency. Cells were treated with a fixed concentration, i.e., 100 μg/ml, of aptamer-conjugated Nile red dye-tagged paclitaxel-loaded PLGA NPs (Apt-NR-PTX-PLGA NPs) and aptamer-conjugated paclitaxel-loaded PLGA nanoparticles (Apt-PTX-PLGA NPs) for different time periods. The cells were incubated with the dye-loaded particles and subjected to confocal microscopy after 2 and 120 h. The anticancer drug-loaded PLGA nanoparticles were incubated with the cells and subjected to confocal microscopy at 24, 72, and 120 h.
At the end of the incubation period, the cell monolayers were rinsed three times with 1 ml of PBS buffer (0.01 M, 7.4) to remove excess nanoparticles or free dye. Apt-NR-PLGA NPs-treated cells were stained with lysotracker (Sigma) to mark the location of endosomes within the cells. Nanoparticles gain entry into the cells by means of endosome-mediated transport. The Apt-PTX-PLGA NPs treated cells were stained with tubulin marker to selectively mark the micro-spindles. This is to evaluate the action of paclitaxel released from the PLGA nanoparticles. Fresh PBS (0.01 M, pH 7.4) buffer was added to the plates, and the cells were viewed and imaged under a confocal laser scanning microscope (Leica TCS SP5, Leica Microsystems GmbH, Germany) equipped with an argon laser using FITC filter (Ex 488 nm, Em525 nm) and Red filter (Ex 561 nm). The images were processed using Leica Application Suite software.
2.7 In vitro cell viability assay
Bioassay of cell viability was investigated by means of the cellular mitochondrial activity (using methylthiazolyldiphenyl-tetrazolium bromide or MTT assay). GI-1 cell line and normal HMEC cells were exposed to plain paclitaxel(Taxol), plain PLGA nanoparticles(Plain-PLGA NPs), paclitaxel-loaded PLGA nanoparticles(PTX-PLGA), specific aptamer-labeled paclitaxel-loaded PLGA nanoparticles (AS1411-PTX-PLGA NPs), and non-specific aptamer-labeled paclitaxel-loaded PLGA nanoparticles (NS-PTX-PLGA NPs) at a concentration of 100 μg/ml for a 24-h duration. The NPs were sterilized with UV irradiation for 30 min before use. At given time interval, the cultured cells were assayed for cell viability with MTT (Sigma). The wells were washed twice with PBS, and 10 μl of MTT (5 mg/mL, Sigma) supplemented with culture medium was added. After 4 h incubation in the incubator, the culture medium was removed, and the precipitate (formazan crystals) was dissolved in 100 μL of dimethylsulfoxide. Relative percentage of metabolically active cells relative to untreated controls was then determined on the basis of the mitochondrial conversion of 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide to formazan by cellular mitochondrial dehydrogenase present in viable cells. The amount of 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide that is converted to formazan indicates the number of viable cells. The results were assessed in a 96-well format micro plate reader by measuring the absorbance at a wavelength of 490 nm.
Another 96-well plate of GI-1 cells (5,000 cells/100 μl per well) was plated to make a comparison of the anti-proliferative effect of PTX-PLGA NPs and Apt-PTX-PLGA NPs on the cancer cells. Assays based on the cellular metabolic activity (using Alamar blue or AB), was performed after treating the cells with varying concentrations (0.001 μg–1 μg/ml) of the PTX-PLGA NPs and Apt-PTX-PLGA NPs for 24, 48, and 72 h. Alamar blue assay evaluates the proliferation and metabolic activity of cells. In living cells, the mitochondrial reductase enzymes are active and reduce Alamar blue to form a different-colored product from the blue dye. This reducing ability of the cells explains the active metabolism taking place within the cells. When the samples added to the cells are toxic in nature, the reducing ability of the cells to reduce the dye decreases. By measuring the fluorescence intensity of Alamar blue dye at 590–620 nm, the cell viability was determined. This colorimetric cell proliferation assays allow for easy and reliable colorimetric determination of viable cell numbers with excellent sensitivity. All the experiments were repeated in triplicate. Just before adding MTT reagent, representative phase contrast microscope images of cells were taken using an Olympus BX 41 microscope (Olympus, Center Valley, PA, USA).
2.8 Flow cytometry
GI-1 cells and HMEC cells were plated at a density of 2.5 × 106 cells per well in DMEM medium and HuMEC-Ready medium, respectively, and incubated at 37°C until it attained 70% confluence. The culture medium was replaced with Apt-NR-PLGA NPs suspension medium for 1–3 h at 37°C. The incubated cells were washed three times with cold PBS and trypsinized. The pellet was washed with PBS three times and fixed with 1% (w/v) para-formaldehyde solution. DAPI staining was done to stain the nucleus. The cellular uptake of nanoparticles by the cells was investigated by flow cytometry (FACScan, Becton Dickinson).