br exclusively Rios de la Rosa et al b
exclusively (Rios de la Rosa et al., 2017b). Cells were pelleted (1000 rpm, 5 min, 25 °C) and re-suspended in 400 µL PBS. The inter-nalization of DY547-NP was determined on 10,000 individual and live 4hydroxy Nonenal with the BD LSRFortessa cytometer (BD Bioscience, San Jose CA, USA) equipped with the FACSDiva software (v8.0.1). Data were ana-lyzed with FlowJo (vX.0.7, Tree Star, Ashland, OR, USA) after gating single and live events in the FSC-A/FSC-H and FSC/SSC windows, re-spectively. Untreated cells were used as autofluorescence control in order to calculate the median fluorescence intensity (MFI) fold change over time, as well as the percentage of positive events for each cell line.
with laser scanning confocal microscope.
2.5. Co-culture experiments: HCT-116 and fibroblasts
Co-culture experiments were performed using TCP 6-well plates with flat bottom (Prod. No. 3513, Corning, UK). HCT-116 cells were seeded at a density of 10,000 cells/cm2 on the bottom of the wells, whereas fibroblasts (HDFa) were seeded on transwell inserts (MW6 Transwell Inserts, 0.4 µm PET Membrane, Corning, UK) at a density of 10,000 cells/cm2. Cells were left adhere for 12 h in separate multi-well plates (37 °C, 5% CO2), then inserts culturing HDFa cells were trans-ferred to the 6-well plates culturing cancer cells and 3 mL of complete DMEM was added in each well to allow media exchange between the two compartments (overnight, 37 °C, 5% CO2). Cells were co-cultured up to 48 h (37 °C, 5% CO2).
2.5.1. Nanoparticle kinetics of internalization in co-culture: flow cytometry Cells were incubated with DY547-NP (125 µg/mL in complete cell culture media) corresponding to a final 40 nM siRNA concentration, up to 48 h (37 °C, 5% CO2). After each time point (4, 12, 24 and 48 h), NP containing medium was removed, cells were washed with PBS (n = 3) and detached using Trypsin-EDTA solution (#59417C, Sigma-Aldrich, UK) for 10 min at room temperature. Note that trypsin was used to remove any residual membrane-bound nanoparticle, enabling the de-tection of internalized DY547-NP only. Cells were pelleted (1000 rpm, 5 min, 25 °C) and re-suspended in 400 µL PBS. The internalization of DY547-NP was determined on 10,000 individual and live cells with the BD LSRFortessa cytometer (BD Bioscience, San Jose CA, USA) equipped with the FACSDiva software (v8.0.1). Data were analyzed with FlowJo (vX.0.7, Tree Star, Ashland, OR, USA) after gating single and live events in the FSC-A/FSC-H and FSC/SSC windows, respectively. Untreated International Journal of Pharmaceutics 561 (2019) 114–123
cells were used as autofluorescence control in order to calculate the median fluorescence intensity (MFI) fold change over time, as well as the percentage of positive events for each cell line. Untreated cells were also used as a control.
2.6. Imaging: CD44, nanoparticle internalization and siRNA localization
2.6.1. CD44 expression: inverted microscope
Images of IF samples were acquired using an inverted microscope (Leica DMI6000B, Leica Microsystems, UK) coupled with a 5.5 Neo sCMOS camera (Andor, UK) and the EL6000 fluorescent lamp (Leica Microsystem, UK), all controlled by μManager software (v.1.46, Vale Lab, UCSF, USA). For acquisitions, immersion oil 63X/1.40-0.60 HC PL Apo objective was used, using I3 filter cube (Leica Microsystem, UK). Images were post-processed using ImageJ adjusting brightness/contrast for a better visualization of CD44 (v1.51h, http://imagej.nih.gov/ij).
2.6.2. Nanoparticle internalization: confocal microscope
An inverted SP5 laser scanning confocal microscope (Leica TSC SP5 AOBS, Leica Microsystem, UK) was used to acquire volumetric datasets of IF stained cells. Acquisitions were performed using the immersion oil 63X/1.40 HCX PL Apo objective. Images were acquired with sequential scan using 405, 488, 546 and 594 nm laser lines. Images were acquired with different settings accordingly to each experiment, in particular pinhole was kept to 1 airy unit aperture, pixel size adjusted to 150–165 nm, laser lines settings were adjusted to the dyes, frequency scan and averaged line were modified accordingly to the sample, whether live or fixed. Images were post-processed using ImageJ ad-justing brightness/contrast for a better visualization of components (v1.51h, http://imagej.nih.gov/ij). Large field images were also used to quantify the amount of siRNA internalized: briefly, the maximum pro-jection of the siRNA channel was obtained, Otsu threshold was applied, the area of the signal was measured and expressed as % with respect to the scanned area.
2.7. siRNA delivery and KRAS silencing
NP (0.125 mg/mL, final concentration in complete cell culture media) were used for KRAS silencing experiments. NP were prepared loading 40 nM (corresponding to 0.5 µg/mL) of L3-siRNA sequence (sense 5′-3′: GGACUCUGAAGAUGUACCU[dT][dT] 21nt, standard purification, Sigma-Aldrich, UK). Refer to the Supplementary Information for detailed description of siRNA sequences tested for KRAS silencing (Section SI.2). Briefly, HCT-116 cells were seeded at a density of 10,000 cells/cm2 in TCP 12-well plates with flat bottom (Thermo Scientific, NUNC MULTIDISH 12, #150628) and left adhere overnight. Cells were then incubated in complete media containing NP (HA-coated) for 48 h in a humidified 5% (v/v) CO2 air atmosphere at 37 °C. In the case of Nanocin NP, the media was changed after 24 h, which has been found to improve the silencing efficiencies. Nanocin is compatible with repeat transfection due to relatively low cell toxicity. Lipofectamine™ 2000 was used as control. NP containing media were removed and cells thorough rinsed with PBS (n = 3). The total RNA was extracted using phenol-chloroform method with: TRI Reagent (Cat. No.: T9424, Sigma-Aldrich). Reverse Transcription Reaction was performed using kit: High Capacity RNA-to-cDNA kit, Applied Biosystems and fi-nally amplification (qPCR) was performed using kit: 2xqPCRBIO Sy-Green Mix Lo-ROX (Cat.No. PB20.11-05, Applied Biosystems, UK) fol-lowing manufacturer’s instructions.