Materials
Peptide H and R were purchased from Kelowna Biotech (Taipei, Taiwan) at > 95% purity. miR-139 and FAM-miR139 were purchased from Genepharma (Shanghai, China). DSPE-PEG2000-NHS was obtained from Nanocs Inc. (Boston, MA, USA). Afatinib and PLGA were purchased from Sigma-Aldrich (St. Louis, MO, USA). Monoclonal antibodies (Mab) of Bax, Bcl-2, β-catenin, caspase 9, caspase 3, PARP, RIP1, RIP3 and E-cadherin were purchased from Cell Signaling Technology (Beverly, MA, USA). Slug Mab was from Abcam (Cambridge, UK). β-actin goat anti-mouse and rabbit IgG antibody was purchased from Millipore (Billerica, MA, USA). All cell culture medium and reagents were bought from Promega (Madison, WI, USA), Invitrogen (Carlsbad, CA, USA), Gibco BRL (Grand Island, NY, USA), or Hyclone (Logan, UT, USA). All other chemical reagents were obtained from either Merck (Darmstadt, Germany) or Sigma-Aldrich (St. Louis, MO, USA).
Preparation of afatinib-loaded LPN-HR or miR-139-loaded LPN-HR
Peptide-conjugated lipids were firstly sythesized by mixing peptide H or R and DSPE-PEG-NHS at a molar ratio 1:1 for 24 h at room temperature. The resulting compounds were dialyzed against water by dialysis bag (3.5–5 kDa MWCO membrane, Spectrum Inc., CA, USA). The purified peptide-conjugated lipids were obtained by freeze-drying and characterized by mass.
Afatinib/LPN-HR were prepared by an o/w emulsion method. 3 mg/ml of mPEG-DSPE or peptide-conjugated lipid and lecithin were prepared separately in ethanol/water solution. PLGA (50:50 LA:GA; MW 35,000–45,000) was prepared at a concentration of 2 mg/ml in acetone. For example, to prepare 10 nM Afa/LPN-HR, 2 µl of 20 μM afatinib in DMSO was added into PLGA stock solution. mPEG-DSPE or peptide-conjugated lipid and lecithin were added into PBS buffer to prepare aqueous dispersion. Then, afatinib/PLGA solution was carefully added dropwise into the aqueous lipid medium. The resulting dispersion was sonicated for 10 min using a bath sonicator. The organic solvent was removed by evaporation. The nanoparticles were filtered by 0.45 μm pore size filter and stored at 4 °C.
Preparation of miR-139/LPN-HR was similar to that of afatinib-loaded nanoparticles. For example, to prepare 100 nM miR-139/LPN-HR, 20 µl of 20 μM miR-139 was added into PLGA solution. The miR-139/PLGA solution was then added into the aqueous lipid dispersion. The following steps were the same as the preparation of afatinib-loaded nanoparticles.
Characterization of afatinib or miR-139 in various LPN formulations: size distribution, zeta potential, EE%, DL% and TEM images
Size distribution and zeta potential of nanoparticles were measured by Zetasizer Nano ZS90 (Malvern Instruments Ltd., Malvern, Worcestershire, UK) at 25 °C with a scattering angle of 90.0°. PDI was calculated by Zetasizer family software v7.11. Records were analyzed from three individual measurements.
The morphology of nanoparticles was observed by TEM (JEM-1400Plus, Japan). The samples were dropped on the grid for 1 min, and the excess of solution was drawn off from the edge with a filter paper. After that, the specimens were stained by 1% phosphotungstic acid for 45 s. Excess solution was blotted with a filter paper, and the samples were dried in the air at room temperature.
A dispersion of Afa- or miR-139 containing nanoparticles was centrifuged at 15,000 rpm and 4 °C through an ultracentrifuge filter (Amicon®, MW: 10 kDa). The filtrate was collected and analyzed by HPLC–UV detector and microplate reader (TECAN Sunrise, Männedorf, Schweiz), respectively. Each sample was detected in triplicate. EE% or DL% of Afa or miR-139 in LPN or LPN-HR were calculated by the following formula.
$$ {\text{EE}}\% = \left[ {\left( {{\text{W}}_{\text{e}} {-}{\text{W}}_{\text{f}} } \right)/{\text{W}}_{\text{e}} } \right] \times 100\% $$
(1)
$$ {\text{DL}}\% = \left[ {\left( {{\text{W}}_{\text{e}} {-}{\text{W}}_{\text{f}} } \right)/{\text{W}}_{\text{t}} } \right] \times 100\% $$
(2)
where We is the weight of added Afa or miR, Wf is the weight of Afa or miR in the filtrate, and Wt is the total nanoparticle weight.
The HPLC system is composed of a PM1110 pump (Hitachi, Tokyo, Japan), an autosampler (Primaide 1210), a C18 column (Phenomenex), and a L2420 UV–VIS detector (Hitachi). The mobile phase was composed of water, acetonitrile, and methanol (55:25:20 v/v). The solution was degassed by a sonicator before detection. The flow rate was 1.0 ml/min, and UV detection was performed at wavelength 254 nm.
In vitro drug release of afatinib from LPN
The release of afatinib from nanoparticles was performed by dialysis method. 1 ml of afatinib formulation was added into a dialysis bag (3.5–5 kDa MWCO membrane, Spectrum, Inc., Rancho Dominguez, CA, USA). The dialysis bag was placed into a 37.5 ml pH 7.4 or 6.5 PBS with stirring at 400 rpm, and the temperature was maintained at 37 °C. At the indicated time, an aliquot of 0.1 ml sample was obtained from dialyzer, and the same volume of PBS was replaced into dialyzer to maintain the original volume of solution. The nanoparticles were broken by acetonitrile with sonication. The afatinib concentration in each sample was detected by HPLC. The cumulative release of afatinib was then calculated.
Hemolysis test
Blood (1.5 ml) was collected from SD rat by cardiac puncturing with heparin. RBCs were separated by centrifugation of whole blood at 1500 rpm for 10 min at 4 °C. This step was continued until the supernatant was clear. The supernatant containing plasma and platelets was discarded. The RBC pellet was suspended in 12 ml PBS and divided into 12 tubes (1 ml per tube). Different formulations were added to the tubes, which were then incubated at 37 °C for 24 h. Triton X was used as a positive control. Each group of samples was mixed with Drabkin’s reagent (Sigma, St. Louis, MO, USA). Hemoglobin was oxidized to cyanmethemoglobin after the reaction with Drabkin’s reagent (Sigma). The concentration of cyanmethemoglobin was measured at 540 nm by using Tecan Infinite microplate reader (Männedorf, Switzerland).
Cell culture
Rat small intestinal epithelial IEC-6 cells and human colorectal adenocarcinoma Caco-2 cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), penicillin/streptomycin and sodium bicarbonate. All these cells were maintained in an incubator with 5% CO2 at 37 °C.
Cytotoxicity of various Afa/LPN or miR-139/LPN-HR on Caco-2 or IEC-6 cells
Caco-2 and IEC-6 cells were seeded at the density of 1 × 104 cells/well and 4 × 103 cells/well in 96-well plates. Different concentrations of afatinib and formulations were added to the medium and incubated for 72 h for Caco-2 cells and 24, 48, or 72 h for IEC-6 cells. After treatments, the cultured medium was removed, and the cytotoxicity was determined by sulforhodamine B assay. The absorbance was measured at 540 nm by Tecan microplate reader.
Transfection efficiency of miR-139/LPN-HR
Caco-2 cells were seeded at the density of 1 × 105 cells/well in a 24-well plate and cultured for 48 h. FAM-miR-139/LPN-HR and other formulations were added into the plate and incubated for 24 h. After treatments, the cells were collected and centrifuged at 300g at 4 °C and then suspended in cold PBS. The samples were analyzed by FACSCalibur flow cytometer. Fluorescence was detected through a FL1 filter for FAM-miR139. Data acquirement and computation were calculated by BD Biosciences software. Each treatment was performed in triplicate.
Cellular uptake and endocytic mechanism of coumarin-6 (C-6) loaded LPNs on Caco-2 cells
Caco-2 cells (1 × 105 cells/well) were seeded in the 24 well plate overnight and incubated with coumarin-6 loaded LPN formulations at pH 7.4 and 6.5 for 6 h. After treatments, the cells were washed, detached, and then collected. The cells were centrifuged and suspended in cold PBS. The samples were analyzed by FACSCalibur flow cytometer (BD Biosciences, San Jose, CA, USA). Fluorescence was detected through a FL-1 filter for coumarin-6 and fluorescence signals were represented on a logarithmic scale. Data acquisition and analysis were calculated by BD FACStation™ software (BD Biosciences). Each group was analyzed in triplicate.
Endocytic mechanism of C-6/LPNs on Caco-2 cells was determined by pre-treating cells with different endocytosis inhibitors including poly-lysine (P-L; 10 µM, positive charge inhibitor), chlorpromazine (CPZ; 10 µM, clathrin-mediated endocytosis inhibitor), 5-(N,N-dimethyl) amiloride (DMA; 10 µg/ml, micropinocytosis inhibitor), nystatin (NYS; 20 µg/ml, and caveolae-mediated endocytosis inhibitor) for 60 min. The cells were then treated with coumarin-6 loaded nanoparticles for 6 h. After treatments, the harvested cells were centrifuged at 300g at 4 °C and then suspended in cold PBS. The samples were analyzed by FACSCalibur flow cytometer (BD Biosciences).
Coumarin-6/LPN and miR-FAM/LPN intracellular localization
Caco-2 cells (2 × 105 cells/well) were seeded in the 6 well plate. Coumarin-6/LPN and FAM-miR139/LPN-HR were added to the plate, treated for 1 or 6 h, respectively. The medium was removed and the cells were stained with LysoRed at 37 °C. After staining, cells were washed with PBS, and fixed with 4% paraformaldehyde at room temperature. Cells were permeabilized by Triton-X at room temperature. DAPI was used to stain cell nuclei at 37 °C. After mounting the samples, the cells were visualized by CLSM (Olympus FV10i, Olympus America Inc., Center Valley, PA, USA).
Annexin V/PI staining
Caco-2 cells were seeded in a 6-well plate. Different treatments were added into the plate and incubated for 72 h. After treatments, the cells were collected, centrifuged at 300g at 4 °C for 8 min twice, and then suspended in PBS. Cells were stained by Annexin V-FITC Apoptosis Detection Kit (Strong Biotech Corporation, Taipei, Taiwan) in the dark at room temperature. The samples were analyzed by FACSCalibur flow cytometer.
Cell cycle assay
Caco-2 cells (2 × 105 cells/well) were seeded in a 6-well plate. Different treatments were added into the plate and incubated for 72 h. After treatments, the cells were harvested, centrifuged, and then suspended in PBS. Cells were fixed by 70% ethanol and stored in − 20 °C overnight. After that, cells were washed by PBS and centrifuged at 4 °C and re-suspended in cold PBS. Cells were stained by PI solution in the dark at room temperature. The samples were analyzed by FACSCalibur flow cytometer.
Cell migration assay
Cell migration ability was measured using the Transwell inserts (Greiner, Frickenhausen, Germany). 2 × 105 cells/insert were seeded in the upper chamber and incubated with different treatments for 48 h. After treatment, the medium on the upside chambers was removed carefully and fixed with 70% ethanol for 10 min at room temperature. Cells that remained on the upside of insert membrane were removed by cotton swabs. Inserts were immersed into 0.2% crystal violet and stained for 30 min at room temperature. Cells in five randomly selected visual fields (magnification, ×40) were counted in each Transwell chamber. The relative migration % was calculated by counting the number of cells that had migrated to the lower side of the membrane compared to the control group.
Western blot assay
Caco-2 cells (2 × 105 cells/dish) were seeded in the 6 cm dish, and various treatments were added and cultured for 72 h. Cells were lysed by RIPA buffer (Cell Signaling, Beverly, MA, USA) and protein was extracted. Protein quantification was determined by BCA TM Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA). Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene difluoride membrane. After blocking, the membranes were immersed in primary antibodies at 4 °C overnight, followed by incubation with secondary antibodies at room temperature for 1 h. The relative protein expression was detected by chemiluminescent imaging system (ImageQuant LAS 4000).
Statistical analysis
Experimental data were expressed as the mean ± standard deviation (SD) and analyzed by Student’s t-test. P < 0.05 was considered statistically significant.