Preparation of SMV and miR–21i-loaded PLGA/PEI nanoparticles
The poly (D,L-lactide-co-glycolide) (PLGA, 50:50) and Polyethylenimine (PEI) were purchased from Sigma-Aldrich, China. The PLGA (30 mg) and PEI (0.1 mg) was dissolved in 1ml of chloroform. The simvastatin (SMV, 10% w/w) was dissolved in the organic phase and stirred for 15 min. Followed by a mixture of bovine serum albumin (BSA, 5 mg) and miRNA–21i (300 µg) in 200 µl of EDTA buffer was added to the organic phase slowly and immediately vortexed for 2 min. The mixture was probe-sonicated for 6 min (60% Amplitude) in an ice-bath resulting in the formation of water-in-oil (W/O) emulsions. The so-formed W/O emulsion was added to 5 ml of 2.5% polyvinyl alcohol solution and again sonicated for 4 min in the ice-bath. The emulsion was stirred for 15h at room temperature to allow evaporating all of the organic solvents. The SMV/miRNA-loaded nanoparticles were collected by ultracentrifugation at 12000 rpm for 10 min using a sophisticated centrifuge. The nanoparticles were washed twice and stored at 4°C until further use.
Gel retardation analysis
Gel retardation assay was performed in 2% Agarose gel pre-stained with 0.5 mg/ml of ethidium bromide. Briefly, 2% Agarose gel was prepared in TRIS-acetate EDTA buffer and condensation ability of different formulations in N/P ratio was evaluated. The experiment was carried out at 80 V for 30 min and the retardation at different N/P ratio was analyzed by gel image analysis system.
Nanoparticle characterization
The hydrodynamic particle size and zeta potential of nanoparticles were evaluated by laser particle analyzer (Fritsch ANALYSETTE 22, Germany). The experiments were performed at 25°C in triplicate numbers. The morphology of particles was evaluated by transmission electron microscope (TEM) using PHILIPS TECHNAL–10 (Holland). The TEM was carried out at 100 kV. The diluted samples were placed in a copper grid containing 300-mesh and excess water was removed and in turn stained with 2% uranyl acetate solution.
In vitro SMV/miRNA release study
The release study was performed by dialysis method. To evaluate the release of miRNA, miR–21i-FAM was used. The nanoparticle dispersion was mixed with 1 ml of release medium (pH 7.4, phosphate buffered saline) and packed in a dialysis membrane (MWCO 3000 Da) which is in turn placed in a rotary shaker (100 rpm) at 37°C. At fixed time interval, 1 ml of release medium was collected and replaced with equal amount of fresh buffer. The release of SMV was determined by HPLC method and miR–21i-FAM was evaluated by measuring the fluorescence intensity of FAM (λex 488 nm and λem 520 nm) using a microplate reader (Tecan, Durham, USA). WATERS (E2695) HPLC was used with a C18 column. The mobile phase consisted of acetonitrile/water (20:80) at a constant flow rate of 1.2 ml/min at 242 nm.
Cellular uptake analysis of PPN-S21i
The cellular uptake analysis was performed by confocal laser scanning microscopy (CLSM) and flow cytometer. BGC–823 cells (ATCC, USA) were cultured in RPMI–1640 media supplemented with 10% FBS. 2×105 cells were seeded in each well of 12-well plate and kept aside for 24h. The cells were treated with fresh medium containing the PPN-S21i nanoparticle containing rhodamine-B as a fluorescent tracker. The nanoparticles were incubated for 3h and then washed twice with PBS and then stained with Lysotracker Green for 10 min. The cells were again washed and fixed with 4% paraformaldehyde. The cells were observed under Leica Microsystems, Mannheim, Germany. The cellular uptake was further studied by flow cytometer (BD Biosciences, San Diego, CA). The cells were treated in the same manner as mentioned above and then collected by scrapping and 10,000 events were recorded.
Cell viability assay
The cell viability assay was performed by cell counter kit–8 (CCK8) assay. To begin this assay, 8×103 BGC–823 cells (ATCC, USA) were seeded in each well of 96 well plates and incubated for 24h in 100 µl volume. The cells were then treated with a SMV, SMV+miR–21i and PPN-S20i nanoparticles in a concentration range from 1–100 µg/ml. For all concentration of SMV, miR–21i was fixed at 50 µg/ml. The cells were incubated for 24h. The cells were washed carefully and 10 µl of CCK–8 solution was added to each well of 96 well-plate and incubated for 1h at 37°C. The respective absorbance was measured using a microplate reader at 460 nm using BioTek, USA.
Flow cytometer-based apoptosis assay
The apoptosis assay was performed by Annexin-V/PI staining using flow cytometer. To begin this assay, 2×105 BGC–823 cells were seeded in each well of 12 well plates and incubated for 24h. The cells were then treated with a free SMV, free miR–21i, SMV+miR–21i and PPN-S20i nanoparticles in a fixed concentration. The cells were incubated for 24h and then cells were collected by trypsinization process and pellet was collected by centrifugation process. The cells were resuspended in 200 µl of binding buffer and stained with 3 µl of Annexin-V and PI solution and incubated for 15 min in dark conditions. The cell apoptosis was evaluated by recording 10,000 events in flow cytometer.
Live/Dead assay
The anticancer effect was further studied by Live/Dead assay. To begin this assay, 2×105 cells were seeded in each well of 12 well plates and incubated for 24h. The cells were then treated with a free SMV, free miR–21i, SMV+miR–21i and PPN-S20i nanoparticles in a fixed concentration. The cells were incubated for 24h. The cells were washed and stained with Acridine Orange (AO, 5 µg/ml) and PI (2.5 µg/ml) and incubated for 15 min and then images were captured using a fluorescence microscope (Olympus, USA).
Pharmacokinetic analysis in animal
The Sprague-Dawley (SD) rats were obtained from Animal Facility Center of China-Japan Union Hospital of Jilin University, China. The animal study was approved by Institutional Animal Ethics Committee of China-Japan Union Hospital of Jilin University, Changchun. The experimental protocol for animals was approved by China-Japan Union Hospital of Jilin University Ethical guidelines for Small Animals. The SD rats were given free access to food and water and maintained under ambient conditions with 12h dark/light cycle. The rats were administered with a fixed SMV dose of 7.5 mg/kg of SD rats. The SD rats were divided into two groups with six rats in each group and formulations were administered by tail vein injection. Blood samples were collected in a periodical manner from 0.25–24h. The plasma was separated from whole blood and stored in –80°C until further analysis. At the end of study period, mice were sacrificed with the exposure to CO2.
Statistical analysis
Results are expressed as mean ± standard deviation (SD). Comparison between two groups was performed using Student’s t-test. P<0.05 was considered statistically significant.