br Preparation of nanoparticles varying siRNA loading
2.1.4. Preparation of nanoparticles varying siRNA loading
2.2. Nanoparticle characterization
NP (1 mg/mL in water) hydrodynamic diameter (Z-average size), size polydispersity (PDI), and ζ potential were measured at 25 °C (pre-equilibration for 2 min; 1 mg/mL) using a Zetasizer Nano ZS (model ZEN3600, Malvern Instruments Ltd., UK) equipped with a solid state HeNe laser (λ = 633 nm) at a scattering angle of 173°. Size distribu-tions were calculated by applying the general-purpose algorithm and are presented as the average of the Z-average values of three in-dependent samples.
2.2.2. Payload protection against RNAse
2.2.3. Nanoparticle stability
NP were stored at 4 °C for one week and then tested to check var-iations in size, surface charge and efficacy (for silencing experiments details refer to Section 2.7).
2.3. Cell culture and CD44 characterization
All cell culture experiments and following procedures were per-formed at the University of Manchester (UK), unless otherwise speci-fied. The human colorectal carcinoma cell line HCT-116 (CCL-247) was purchased from ATCC (Manassas, VA, USA) and the adult human dermal fibroblast (HDFa, #C0135C) cell line was purchased from Thermo-Fisher Scientific (UK). Cells were cultured in a humidified 5% (v/v) CO2 air MethoxyX04 at 37 °C in complete medium, cell culture growth media were supplemented with 10% (v/v) fetal bovine serum (FBS, F7524), 2 mM L-glutamine (G7513) and 1% (v/v) penicillin–-streptomycin (P4333). McCoy’s 5A medium (M8403) and DMEM (D5671) were used for HCT-116 and HDFa, respectively. Please note that cells were regularly tested for mycoplasma and used at passage numbers below 20, and that all cell culture products were purchased from Sigma- Aldrich (Gillingham, UK). International Journal of Pharmaceutics 561 (2019) 114–123
Cells were grown in T-75 flasks until reaching ∼70% confluency and harvested using pre-warmed Enzyme-Free, Phosphate Buffer solu-tion (PBS)-based Cell Dissociation Buffer (#13151-014, Gibco®/ Invitrogen, UK). Individual cell samples were prepared in 1.5 mL Eppendorf tubes by suspending approximately 100,000 viable cells in 100 μL Fluorescence-Activated Cell Sorting (FACS) buffer (PBS, 5% (v/
v) FBS, 0.1% (m/v) NaN3) and stained for 30 min at room temperature with the primary antibody mouse anti-human CD44 (1:100) (156-3C11, Cell Signalling Technology, UK) or IgG1/IgG2 control (1:10) (AbD Serotec, UK). Excess primary antibody was removed by centrifugation and cells were incubated for further 30 min at room temperature with the secondary antibody: goat anti-mouse IgG H&L, AlexaFluor®647-conjugated (1:2000) (ab150115, Abcam, UK). The expression of total CD44 (CD44pan) was recorded for 10,000 live, individual cells using a 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 live cells in the FSC/ SSC window and cell singlets in the FSC-H/FSC-A window, respectively. The median fluorescence intensity (MFI) of the isotype control for each cell line was used to calculate the MFI fold change for each marker.
2.3.3. CD44 expression: immunofluorescence staining
v) BSA/PBS on ice for 30 min, gently washed with PBS, and incubated with 1:250 goat anti-mouse IgG H&L, AlexaFluor®488-conjugated (ab150117, Abcam, UK) on ice for additional 30 min. Cells were finally washed with PBS (twice), fixed with 4% PFA solution (5 min, RT), washed with PBS and stored in 1 mg/mL ascorbic acid solution in PBS at 4 °C in the dark until further use.
2.4. Nanoparticle internalization
NP (0.125 mg/mL, final concentration) for cell experiments were prepared in complete cell growth medium as follows, a final siRNA concentration of 40 nM (0.5 µg/mL) was obtained. HA-coated chitosan NP (1 mg/mL in water) were diluted after preparation to a concentra-tion of 250 µg/mL with sterile and nuclease-free water. The final con-centration of 125 µg/mL used for cell culture experiment was obtained by addition of an equal volume of two-fold cell culture medium (refer to Supporting Information SI.1 for the preparation of concentrated cell culture medium). Nanocin/HA NP (1 mg/mL) were diluted after pre-paration by adding 11.6 µL of nanoparticles to complete cell culture media to a final volume of 1 mL. Note that kinetic of internalization studies were performed loading DY547-siRNA (L3 sequence: 5′- DY547 GGACUCUGAAGAUGUACCU-3′; Dharmacon, UK) in NP (i.e. DY547-NP).
2.4.1. Quantification of nanoparticle internalization: flow cytometry Cells were plated in Costar tissue culture polystyrene (TCP) 12-well plates with flat bottom (#3513, Corning, UK) and incubated (37 °C, 5% CO2) with 125 µg/mL DY547-NP for specific time points: 4, 12, and 24 h. Untreated cells were used as a negative control. After each in-cubation time, nanoparticle-containing medium was removed, cells were washed with PBS (n = 3) and detached using Trypsin-EDTA so-lution (#59417C, Sigma-Aldrich, UK) for 10 min at room temperature. Trypsin was used to remove any residual membrane-bound nano-particle, enabling the detection of internalized nanoparticles