Chemical and reagents
Custom ordered FITC labeled poly L-lysine of molecular weight 40k Da was purchased from Fisher Scientific Inc. 3-(4,5-dimethylthiazolyl-2)- 2,5-diphenyl tetrazolium bromide (MTT) was purchased from Invitrogen (catalog number V13154). Anti-mir-10b RNA major (sequence: 5′-CACAAAUUCGGUU-CUACAGGGUA-3′) and anti-mir-10b RNA minor (sequence: 5′-ACAGAUUCGAUU-CUAGGGGAAU-3′) were ordered from IDT (Coralville, IA). Negative RNA control (catalog AM4611) was ordered from Applied Biosystems (Austin, TX). The RNA cy5 labeling kit (catalog number MIR 3700) was ordered from Mirus Bio LLC (Madison, WI). Breast cancer cell MDA-MB-231 was purchased from ATCC (catalog number HTB-26). DMEM medium and FBS were purchased from ATCC (catalog 30-2008). Antibody for RHOC was purchased from Santa Cruz Biotechnology (catalog sc-26480). Antibody for beta-Actin was purchased from Abcam (catalog ab8229).
Cy5 labeling of RNA
Following the kit instructions, 1 mM RNA ( including major anti-micro-10b, minor anti-micro-10b, and negative RNA) was mixed with 2 mM Cy5 labeling reagent and incubated at room temperature (21 °C ± 1 °C) for 2 hours. The labeled RNA molecules were purified by standard procedures following instructions from the kit. The Cy5-labeled RNA molecules were aliquoted as 10 μM and stored at −80 °C before experiments.
Preparation and characterization of PLL-RNA nanoparticles
Anti-mir-10b RNA molecules and PLL were both dissolved into pure RNAse, DNAse-free water (Fisher Scientific, catalog number BP2484-100) and auto-claved at 121 °C for 30 min before experiments. Multiple tubes containing 0.5 mL of various concentration of FITC labeled PLL (2 μM to 2 mM) were added with a 0.5 mL of constant anti-mir-10b RNA (0.5 mM) solution, then vortexed for 5 min and incubated for 1 hour at room temperature. For the fluorescence measurement, excitation wavelength was at 485 nm, emission wavelength at 516 nm, slit widths at 5 nm, accumulation time of 0.4 sec. Multiple tubes containing 0.5 mL various concentrations (2 μM to 2 mM) of Cy5-labeled anti-mir-10b RNA was added with 0.5 mL fixed concentration of PLL (0.5 mM), then vortexed for 5 minutes and incubated for 1 hour at room temperature. The fluorescence was measured with a Fluoro Max-2 fluoresce photometer with the excitation at 630 nm, emission wavelength at 660 nm, slit widths at 5 nm, accumulation time of 0.4 sec.
Based on the titration of PLL to anti-mir-10b above, the 1:1 molar ratio was used to produce PLL-anti-mir-10b nanoparticles. 2 mM of anti-mir-10b was mixed with an equal volume of 2 mM PLL, and vortexed at room temperature for 5 min. The RNA loading were estimated by measuring the solution absorbance at 260 nm. During the reaction, RNA molecules bound to PLL and formed micelle, no detectable free RNA in the solution can be found by the solution absorbance at 260 nm. The final RNA concentration of stock of RNA-PLL was estimated as 1 mM. The particle size of anti-mir-10b-PLL was analyzed by dynamic light scattering (DLS).
DLS measurements of the nanoparticle size
The dynamic light scattering (DLS) instrument was home-built. A 532 nm laser was focused into a sample cell and the scattering light was collected by an optical fiber. A D532/10X filter (Chroma Technology, Brattleboro, VT) was used to exclude possible autofluorescence. To minimize the effect of after pulse, the scattering light output from the optical fiber was split by a cube beam splitter ( Thorlabs, Newton, NJ), and detected by a pair of SPCM-AQR-14 avalanche photodiodes (Perkin-Elmer Optoelectronics, Vaudreuil, Canada). The output was fed into a Flex01-05D multi-tau correlator (Correlator.com, Bridgewater, NJ), and the correlation function was calculated in real time. An NIST traceable particle size standard (Catalog 64010, Polysciences Inc. Warrington PA) was used to calibrate the setup.
MDA-MB-231 cells were counted and plated 1 × 105/well in 24-well tissue culture plates to achieve 90% confluence. A vertical or horizontal wound was created using a 1000-μL pipette tip. The wounded cells were washed three times with PBS and DMEM medium with 10% FBS, and covered with either anti-mir-10b contained medium or control medium. The cells were then placed onto a Nikon Eclipse TE300 inverted microscope equipped with an environmental chamber (In Vivo Scientific, LLC) maintained at 37 °C for imaging. Random fields in each well were selected for imaging with a Nikon Plan Apo 4X/0.2 objective using a Retiga 1300 cooled CCD camera (QImaging) controlled by Volocity Acquisition (Perkin Elmer) software. The location of the imaged cells were recorded and saved with the Volocity software (Velocity Software, Inc.) allowing the same location to be imaged at 0 and 24 hours. Images were captured at designated times to assess wound closure. The inhibition effectiveness was estimated by the relative distance of wound closure.
To obtain 3-dimensional images of the cell uptake of RNA-nanoparticles, we used a Zeiss 710 laser scanning confocal microscope. A 20X NA 1.0 water-dipping objective was used. The FITC and Cy5 fluorophores were excited by the 488-nm and 633-nm lasers simultaneously. The emission band of 493–628 nm for the FITC-labeled PLL channel and 638–759 nm band for the Cy5-labeled RNA channel were recorded simultaneously.
In vitro toxicity assay
MDA-MB-231 cells were plated in a 96-well plate at a seeding density of 1×106 cells per well in 0.1 mL of growth medium consisting of DMEM plus 10% FBS. Cells were grown at 37 °C for 24 hours, and the medium was removed. Medium containing designed PLL-anti-mir-10b nanoparticles was incubated for an additional 24 hours, 100 μL of fresh medium containing 50 μg of MTT was added to each well, and cells were incubated for 4 hours. Lysis buffer (10 (W/v) SDS, 45% DMF, pH 4.7) was then added and incubated for 24 hours. Absorbance was measured at 570 nm in a microplate reader (Molecular Devices Corp.). Survival percentage was calculated as compared to no-treatment cells (100% survival).
Cells were harvested in RIPA lysis buffer (150 mM NaCl, 10 mM Tris-HCl, pH 7.5, 1% NP40, 1% deoxycholate, 0.1% SDS, protease inhibitor cocktail (Roche)). Proteins from total cell lysate were analyzed by NuPAGE 4%–12% Bis-Tris gradient gel (Invitrogen), transferred to the PVDF membrane, blocked with 2% non-fat milk in PBS/Tween-20, blotted with the antibodies for RhoC 1:200 and beta-actin (1:10,000), and developed with anti-mouse IgGHRP 1:10,000 using the ECL kit from Invitrogen.
All wound healing experiments were repeated at least three times. The representative experiments were reported in the figures. For the Western blot, three repeated experiments were carried out. The ratio of intensity of the RhoC bands to the actin bands were quantified using the software of LumiAnalyst 3.1 (Roche). The significant difference of anti-mir-10b compared to controls was based on the t-test P value < 0.05 (n = 3).