Gene knockdown in HaCaT cells by small interfering RNAs entrapped in grapefruit-derived extracellular vesicles using a microfluidic device

Materials

HaCaT cells were purchased from Cell Line Service GmbH (Eppelheim, Germany). Dulbecco’s modified Eagle’s medium, trypsin–EDTA solution, and RIPA buffer were purchased from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan). Fetal bovine serum (FBS) was obtained from Sigma Aldrich (MO, USA). Negative control siRNAs (21-mer; 5′-CUUACGCUGUCAUGAUCGAtt-3′; 5′-UCGAUCAUGACAGCGUAAGtt-3′), anti-luciferase siRNAs (21-mer; 5′-CUUACGCUGAGUACUUCGAtt-3′; 5′-UCGAAGUACUCAGCGUAAGtt-3′), and TAMRA-labeled siRNAs (21-mer; 5′-TAMRA-UAUUGCGUCUGUACACUCAtt-3′; 5′-TAMRA-UGAGUGUACAGACGCAAUAtt-3′) were synthesized by Fasmac Co., Ltd. (Kanagawa, Japan). Luciferase-pcDNA3 was obtained from the William Kaelin Lab (Addgene; plasmid #18,964). Furthermore, 3,3′-dioctadecyloxacarbocyanine perchlorate (DiO) and 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI) were obtained from Promo Cell GmbH (Heidelberg, Germany). SYBR GOLD, LysoTracker™ Green DND-26, Hoechst 33342, and Pierce™ Silver Stain Kit were purchased from Thermo Fisher Scientific (MA, USA). Proteinase K (ProK) was obtained from Kanto Chemical Co., Inc. (Tokyo, Japan).

Isolation and characterization of GEVs

Commercially available grapefruits (grown in South Africa) were used in this study. The grapefruits were assigned a voucher no. Experimental of plants, including the collection of plant material, comply with relevant institutional, national, and international guidelines and laws. The grapefruit juice used for GEVs collection was prepared using the one-step sucrose cushion ultracentrifugation method45. Specifically, grapefruit juice was centrifuged at 3,000 × g for 10 min, followed by centrifugation at 10,000 × g for 30 min. The supernatant was filtered through a 0.22-µm filter. For sucrose cushion-based isolation, 40 mL of the filtrate was loaded over 4 mL of 30% sucrose in phosphate-buffered saline (PBS) and centrifuged at 141,000 × g for 90 min at 4 °C using a Himac CP80WX machine with swing rotor 28S (Eppendorf Himac Technologies Co., Ltd, Ibaraki, Japan). The supernatant was discarded, and the sucrose layer was resuspended in PBS, followed by ultracentrifugation at 141,000 × g for 90 min at 4 °C to wash and pellet the GEVs. The pellet was resuspended in PBS and ultracentrifuged at 16,500 × g for 10 min at 4 °C. The size of GEVs was measured using qNano, which is a TRPS technique (Meiwaforsis, Tokyo, Japan). The PdI and zeta potentials of the GEVs were measured using a Zetasizer Nano ZS (Malvern Panalytical Ltd, Worcestershire, UK). The protein concentration of the GEV solution was determined using a BCA protein assay kit (FUJIFILM Wako Pure Chemical Corporation).

Preparation of siRNA-GEVs using an MD

A 5 Input 3D Chip (150 nm) and a P-pump (Dolomite, Blacktrace Holdings Ltd., Royston, UK) were used as the MD (Fig. S2). Because the pump used in this study was of the non-pulsating type, stable liquid feeding can be maintained. Approximately 20 µg/mL (protein concentration) GEVs in ethanol and 400 nM siRNAs in PBS were injected into channels A and B, respectively, as shown in Fig. S2. The pressure conditions of pump A and pump B were selected on the basis of a DoE built using a central composite design, as shown in Table S1. GEVs and siRNAs were injected into the microchannels using 10 conditions. The particle size and PdI were evaluated using a Zetasizer Nano, dynamic light scattering (DLS) technique. To evaluate siRNA loading efficiency (LE%), the quantity of siRNAs was calculated by analysis of fluorescence intensity. The siRNAs were stained using SYBR Gold after ultrafiltration using a Nanosep device (10 kDa MWCO; Pall Corporation, NY, USA).

Optimization of microfluidics conditions

The conditions of sending pressure of pump A and pump B were optimized using the DoE and RSM53,54. The optimal preparation conditions and their credible ranges were estimated using the RSM incorporating thin-plate spline interpolation (RSM-S) and bootstrap (BS) resampling53,54, which was performed using dataNESIA (version 3.0, Azbil Corp., Tokyo, Japan).

The optimal solution was to obtain preparation conditions that minimized particle size and PdI and maximized LE%. The accuracy of the optimal solution was confirmed by valuation of the BS optimal solution and standard division. The frequency numbers of resampling with replacements were set at 1000, 2000, 3000, and 4000.

Cryo-transmission electron microscopy

The morphology of GEVs and siRNA-GEVs was observed using a cryogenic transmission electron microscope (cryo-TEM). Nanoparticles (1 μL of 5 mM lipid concentration) were applied to hydrophilized copper grids (200 mesh; JEOL Ltd., Akishima, Tokyo, Japan) and blotted. The samples were rapidly frozen using a rapid freezing system (EM-CPC, Leica Microsystems, Tokyo, Japan) and then observed using an EM-3100FEF cryo-TEM (JEOL Ltd.) at an accelerating voltage of 300 kV. Observations were performed using a cooling holder (model 626; Gatan, CA, USA).

Evaluation of cellular uptake of nanoparticles

The GEVs were labeled with DiO (Ex: 484 nm, Em: 501 nm). In brief, 2 µL of the 500 µM DiO-ethanol solution was added to the GEV suspension containing 50 µg of protein. The suspension was incubated for 10 min at room temperature, followed by washing by ultracentrifugation to remove unbound dye. Approximately 4 × 104 HaCaT cells/well were seeded in 24-well plates. After overnight incubation at 37 °C, the cells were treated with 20, 40, or 80 µg/mL of DiO-labeled GEVs for 3 or 6 h at 37 °C. To investigate the effect of endocytosis on cellular uptake, cells were treated with FITC-labeled transferrin (20 µg/mL) and DiO-labeled GEVs (40 µg/mL) in the presence of 0.4 M sucrose, which is a clathrin-mediated endocytosis inhibitor. Transferrin is a protein taken up by clathrin-mediated endocytosis. The cellular uptake of GEVs was evaluated using a flow cytometer (CytoFLEX, Beckman Coulter, Inc., CA, USA). The mean fluorescence intensity ratio (MFIR) was calculated by dividing the fluorescence intensity obtained from the sample by that obtained from the untreated cells.

Evaluation of intracellular trafficking of GEVs or siRNA-GEVs

The GEVs were labeled with DiI (Ex: 550 nm, Em: 565 nm), as described above. Approximately 2.0 × 105 HaCaT cells were seeded in a Poly-L-Lysine-coated glass-bottomed dish (Matsunami, Osaka, Japan). After overnight incubation at 37 °C, the cells were treated with 40 µg/mL of DiI-labeled GEVs for 6 h at 37 °C. Endosomes/lysosomes and nuclei were stained using LysoTracker™ Green DND-26 (Ex: 504 nm, Em: 511 nm) and Hoechst 33,342 (Ex: 352 nm, Em: 461 nm), respectively. Cellular images were observed using a confocal laser scanning microscope (CLSM) FV-3000 (Olympus, Tokyo, Japan) equipped with an oil-immersion objective lens (Plan-Apochromat 63x/NA 1.4). Moreover, to investigate the intracellular localization of the siRNA-GEVs, TAMRA-labeled siRNAs and lipids were labeled with DiO, as described above. Hoechst 33,342, LysoTracker™ Green DND-26, and DiO-TAMRA were excited by 405, 488, and 561 nm laser wavelengths, respectively.

Sucrose density gradient fractionation

siRNA-GEVs were prepared using TAMRA-labeled siRNAs, as described above. The labeled siRNA-GEVs were applied to a discontinuous sucrose density gradient (5–60%) and then ultracentrifuged at 220,000 × g for 2 h at 4 °C using a Himac CS100GXL machine with a swing rotor S55S (Eppendorf Himac Technologies Co., Ltd, Ibaraki, Japan). As a result, 11 fractions were obtained. Sucrose was removed by dialysis using 10,000 MWCO Slide-A-Lyzer dialysis cassettes (Thermo Fisher Scientific) at 4 °C overnight. The fluorescence emitted by TAMRA-siRNAs was measured using a fluorescence spectrophotometer (FP8300, JASCO Corporation, Tokyo, Japan) set at an excitation wavelength of 565 nm and fluorescence wavelength of 580 nm. The particle numbers for all the fractions were measured using qNano (Meiwaforsis, Tokyo, Japan).

Phospholipid analysis by liquid chromatography-tandem mass spectrometry (LC–MS/MS)

To analyze the lipid components in GEVs and siRNA-GEVs, the LC–MS/MS Method Package for Phospholipid Profiling (Shimadzu Corporation, Kyoto, Japan) was used, according to the manufacturer’s instructions. The library of phospholipid targets in the method package includes phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylinositol (PI), phosphatidylserine (PS), and sphingomyelin (SM). Briefly, 1.0 × 1011 particles of GEVs and siRNA-GEVs (prepared using GEV fractions with the same particle number) were added to methanol containing 0.1% formic acid for mass spectral analysis. Next, 5 µL of the sample solution was injected into a Kinetex C8 column (internal diameter 2.1 mm, length 150 mm, and size 2.6 µm; Phenomenex) at a flow rate of 0.3 mL/min. The samples were eluted using a gradient of mobile phases A (20 mM ammonium formate in water) and B (isopropanol: acetonitrile = 1:1 v/v). The concentration of mobile phase B was programmed at 20% (0 min)–20% (1 min)–40% (2 min)–92.5% (25 min)–92.5% (26 min)–100% (35 min)–20% (38 min). The oven temperature was 45 °C. Data processing and lipid identification/quantification were performed using LabSolutions software (version 5.99 SP2; Shimadzu Corporation, Kyoto, Japan). The analytical results were obtained by multiple reaction monitoring of transitions. The peak area ratio was calculated by dividing the area of the peak obtained for the sample by that obtained for the internal standard (IS); 17:0–20:4 PI (Avanti Polar Lipids) was added as an IS to each sample to obtain a final concentration of 0.38 µmol/L.

ProK treatment

ProK treatment was carried out according to the previously reported method 46. GEVs (250 µg/mL) were treated with ProK (50 µg/mL) for 30 min at 37 °C, and then the GEVs were incubated with 5 mM phenylmethylsulfonyl fluoride (PMSF) for 10 min at 37 °C.

Evaluation of protein by SDS-PAGE

The protein contents of GEVs and siRNA-GEVs were evaluated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). First, 100 µg of GEVs and siRNA-GEVs was condensed by ultracentrifugation. The pellets were dissolved in RIPA buffer and the solutions were centrifuged at 21,500 × g for 10 min at 4 °C. The supernatants were mixed with 6 × sample buffer containing 2-mercaptoethanol and incubated at 95 °C for 4 min. Next, the samples were loaded on 12% polyacrylamide gels (TGX™ FastCast™ Acrylamide kit, Bio-Rad Laboratories, CA, USA). Electrophoresis was performed at 150 V, and then the gels were silver-stained (Pierce™ Silver Stain Kit). Protein Ladders covering 10–180 kDa ranges (NIPPON Genetics Co., Ltd.) were loaded.

Gene knockdown assay

HaCaT cells were seeded at 2.0 × 104 cells/well in 24-well plate and incubated at 37 °C overnight. The cells were transfected with luciferase-pcDNA3 (0.2 µg) in the presence of lipofectamine 2000 (LFN2000) and incubated at 37 °C for 12 h. Next, the cells were washed with PBS. siRNA-GEVs (containing 10 pmol siRNAs) were added and then the cells were incubated for 24 h at 37 °C. After incubation, luciferase activity was evaluated using the luciferase assay system (Promega, WI, USA) in accordance with the manufacturer’s protocol. The luminescence intensity was detected (by compensating for the protein concentration) using a microplate reader (Synergy H1, Agilent Technologies, CA, USA). The gene knockdown effect was evaluated after treatment with 10 µg/mL RNase A (NIPPON GENE CO., LTD. Tokyo, Japan) at 37 °C for 30 min using the same methods as described above.

Statistical analysis

Statical analysis was performed using ANOVA and Dunnett test with JMP Pro version 16.0.0 (SAS Institute, Cary, NC, USA). The reported p-values were considered statistically significant at p < 0.05.

Read more here: Source link