Preparation of CD59sp-conjugated CDDP/miRNA-loaded liposomes
At first, CDDP-loaded liposome was prepared by hydration-sonication method. Briefly, distearoylphosphatidylcholine (DSPC) succinylphosphatidylethanolamine (DSPE-mPEG), distearoyl-N-(3-carboxy-propionoylpoly (ethyleneglycol) succinl) phosphatidylethanolamine (DSPE-PEG-COOH), and 1,2-dioleoyl–3-trimethyl-ammoniumpropane (DOTAP) were added in a molar ratio of 40:20:10:2 in the chloroform (along with 10% w/w of CDDP). The organic solvent was evaporated in a rotary evaporator at 55°C for 2h. The dried lipid-film was hydrated by incubating in a 1X PBS for 1h. The crude dispersion was sonicated for 5 min using a probe sonicator. To this liposome, 2 mg EDC was added per 1 mg/ml of liposome and stirred for 1h; followed by CD59sp (1 mg/ml) was added to the solution and stirred in dark condition for 4h. The unconjugated initial ingredients and unconjugated CD59 were removed by centrifugation at 5000 xg for 15 min at 4°C. The cationic liposome was then incubated with required amount of miRNA overnight and it is loaded on the surface of liposome.
Characterization of CD59sp-conjugated CDDP/miRNA-loaded liposomes
The particle size and polydispersity index (PDI) and zeta potential was determined by dynamic light scattering (DLS) technique using ZetaSizer (Nano series- Nano-ZS ZEN3600, Malvern Instruments, UK). Before the measurement, 0.1 ml of samples was diluted to 1 ml and measured triplicate at 25°C. The particle morphology was evaluated using transmission electron microscopy (TEM, JEM–2100, JEOL, Japan). The drop of samples were placed in the copper grid and stained with 2% phosphotungistic acid and dried using infrared light. The particles were then observed under electron microscope.
Drug loading study
The drug loading and quantification was performed by OPDA-derivatization method. For this purpose, known quantity of dried liposome was added to 100 µl of DMF and vortexed. To this, 100 µl of OPDA and 200 µl of PBS were added and the mixture was heated at 100°C for 15 min. The samples were cooled in ice-bath and 1.6 ml of DMF was added to make it to 2 ml as a final volume. Earlier, a calibration curve was performed at different concentration of CDDP and analyzed through UV-Vis spectrophotometer (JASCO-V–730) at a wavelength of 706 nm.
Cellular uptake of CD59sp-conjugated CDDP/miRNA-loaded liposomes
The cellular uptake study was first performed by confocal laser scanning microscopy (CLSM) method. Briefly, HeLa cells (ATCC, USA) were seeded in 6-well plate with a cover-slip on it and the cells were incubated for 24h at 37°C. Next day, cells were treated with LP-miCDDP and CD/LP-miCDDP formulations, respectively for 2h. The cells were washed 2 times and immediately incubated with Lysotracker GreenTM for 10 min. The cells were washed and fixed with 4% paraformaldehyde and stained with DAPI as a nuclear staining. The cover-slip containing cells were mounted on a glass slide and observed under microscope (Leica SP8).
The cellular uptake was further evaluated by flow cytometer. Briefly, HeLa cells were seeded in 6-well plate and the cells were incubated for 24h at 37°C. Next day, cells were treated with LP-miCDDP and CD/LP-miCDDP formulations, respectively for 2h. The cells were washed and detached with trypsin/ethylenediaminetetraacetic acid mixture and centrifuged a 1200 rpm for 3 mins. The pellet cells were reconstituted with the PBS and studied in BD FACSCalibur flow cytometer (BD FACS, NJ, USA).
In vitro anticancer effect analysis
The in vitro anticancer effect of free miRNA and the formulations were evaluated by MTT assay. Briefly, briefly, 1×104 HeLa cells were seeded in 96-well plate and the cells were incubated for 24h at 37°C. The cells were either treated with increasing concentration of lipofectamine-miRNA complex or free CDDP, miCDDP and CD/LP-miCDDP formulations or incubated for 24h. The cells were washed two times with PBS and added with 20 µl of MTT solution that has a concentration of 5 mg/ml and incubated for 4h at 37°C. 100 µl of DMSO was added and incubated for 15 min and then absorbance was read at 570 nm using a microplate reader. The untreated cells were taken as a control and IC50 value were calculated from GraphPad Prism software.
Apoptosis analysis—Nuclear staining
The nuclear morphology of cancer cells after treatment was evaluated by Hoechst 33342 staining. Briefly, 2×105 HeLa cells were seeded in 96-well plate and the cells were incubated for 24h at 37°C. The cells were then treated with free CDDP, miCDDP and CD/LP-miCDDP formulations and incubated for 24h. The cells were washed with PBS twice and fixed with 4% paraformaldehyde for 10 min. The cells were washed and then stained with 10 µg/ml of Hoechst 33342 solution for 15 min. The cells were washed three times and morphology was observed through IN Cell Analyzer 2000 (GE Healthcare Life Science, USA).
Apoptosis analysis—Flow cytometer
The quantitative apoptosis was performed by Annexin V-FITC and PI double staining followed by flow cytometer analysis. Briefly, 2×105 HeLa cells were seeded in 96-well plate and the cells were incubated for 24h at 37°C. The cells were then treated with free CDDP, miCDDP and CD/LP-miCDDP formulations and incubated for 24h. After 24h, cells were scrapped gently and centrifuged at 1200 rpm for 3 min. The pellet cells were reconstituted with 100 µl of binding buffer and stained with 5 µl of Annexin V-FITC and PI and subjected to incubation for 15 min in the dark atmosphere. The volume was made upto 1 ml and studied using the in BD FACSCalibur flow cytometer (BD FACS, NJ, USA).
Pharmacokinetic analysis
The Sprague Dawley rats were obtained from In-House Animal Facility, Weifang No.2 People’s Hospital, Weifang, China. The experimental animal study protocol was approved by Institutional Animal Ethics Committee of Weifang No.2 People’s Hospital, Weifang, China. The pharmacokinetic analysis of free drug and drug-loaded formulations were studied in Sprague Dawley rats (200+20 g). The rats were given free access to food and water until the study and caged in a standard atmosphere as per the guidelines set by the Institutional committee for animal care and handling. The rats were divided into 3 groups with 5 rats in each group. All the rats received the drug and formulation treatment via the tail vein injection. The CDDP is administered at a dose of 5 mg/kg while formulations were given 5 mg/kg equivalent dose. The blood samples were collected from the rats from retro-orbital plexus after 0.25h, 0.5h, 1h, 2h. 4h, 6h, 8h, 12h, 24h, 36h and 48h, respectively. At the end of study period, mice were sacrificed with the exposure to CO2. The heparin mixed samples were centrifuged at 10000 rpm for 10 min and plasma was stored for further analysis in –80°C. 150 µl of plasma and 150 µl of ethanol was mixed and vortexed for 1h. The mixture was centrifuged at 12000 rpm for 15 min and the supernatant was used to calculate the CDDP concentration. The CDDP was measured by ion coupled plasma mass spectrometry (ICP-MS, Perkin-Elmer Corporation, USA).
Statistical analysis
P value < 0.05 was considered statistically significant. Quantitative data were expressed as mean ± SD. Statistical comparisons were made by one-way ANOVA analysis and Student’s t-test.