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  • 103-82-2 further investigated the potential of selective

    2020-08-30

    further investigated the potential of selective delivery of HA-decorated nanoparticles to cancer cells (HCT-116: CD44+, CD44v6high) when the latter were co-cultured with CD44low fibroblasts (HDFa).
    2. Materials and methods
    2.1. Nanoparticles and siRNA loading
    2.1.1. Material preparation
    All materials used in this study were handled under RNase free conditions: solutions were prepared with nuclease-free solvents, and  International Journal of Pharmaceutics 561 (2019) 114–123
    materials were either purchased RNase free or made RNase free by sequential washing with RNaseZap® RNase Decontamination Solution (Ambion, LifeTechnologies, UK), 70% v/v EtOH in water, and finally sterile nuclease-free water (Ambion, Life Technologies, UK) prior to use. Both polymeric nanotechnologies were manufactured under con-trolled mixing conditions using round-bottom vials (2 mL Safe-Lock Tubes, 2 mL, round bottom, PCR clean, Eppendorf, UK) and magnetic stirring bars (micro 7 mm × 2 mm, Fisher Scientific, UK). Polycations: Chitosan from crab shells with viscosity average molecular weight of 656 kDa (Almalik et al., 2013a), was purchased from Sigma Aldrich (Basingstoke, UK; Product code: 51009219, Lot#WE44069811), and purified prior to use as described elsewhere (Mao et al., 2004). Poly (hexamethylene biguanide), also known as polyhexanide and as Na-nocin, with average molecular weight approximately of 3.2 kDa (www. tecrea.com) was prepared as a sterile solution with a concentration of 1 mg/mL in RNase free water. Hyaluronic 103-82-2 (HA) with weight average molecular weight of 183 kDa (GPC with SLS, viscometer and RI detectors) was purchased from Contipro (Czech Republic). Selected anti-KRAS sequence (siRNA): 1 mg/mL stock solutions of siRNA were prepared in RNase-free water for both L3-siRNA (sense 5′-3′: GGACU CUGAAGAUGUACCU[dT][dT] 21nt, standard purification, Sigma-Al-drich, UK) and DY547-labeled L3-siRNA (5′-DY547 GGACUCUGAAGA UGUACCU-3′, Dharmacon, UK). Polymeric solutions used for the manufacturing of nanoparticles were prepared as follows. Chitosan was dissolved overnight at a concentration of 0.69 mg/mL in 4.6 mM HCl (aq) in nuclease-free water and then pH adjusted to 5 by adding nu-clease-free 0.1 M NaOH (aq). Chitosan solution was sterile filtered using 0.45 μm PDVF syringe filters. Nanocin sterile solution was diluted to
    0.69 mg/mL with RNase free sterile water. HA was dissolved overnight in RNase free water at a concentration of 1.5 mg/mL, and then the pH was adjusted to 5 by adding nuclease-free 0.1 M HCl (aq). HA solution was sterile filtered using 0.22 μm PES syringe filters. HA solutions with different concentrations were obtained by diluting the 1.5 mg/mL so-lution in nuclease-free water. Sterile siRNA solutions were prepared at the desired concentration by diluting the 100 μM stock solution (aq) with RNase free and sterile water and stored at −20 °C until use.
    2.1.2. Preparation of nanoparticles
    HA-coated nanoparticles were prepared with a concentration of 1 mg/mL in water, using a procedure similar to the one already de-scribed by Lallana et al. (Lallana et al., 2017) for the preparation of RNA-loaded HA/chitosan ternary complexes. In a typical procedure, a given volume of the 0.69 mg/mL polycation (chitosan or Nanocin) so-lution was gently pipetted over the same volume of the siRNA solution (with concentration adjusted the targeted % wt. loading) under mag-netic stirring (1,000 rpm, 25 °C). After 20 min, the polycation/siRNA complex dispersion was gently pipetted to the same volume of a 1.5 mg/mL HA solution under magnetic stirring (1,000 rpm, 25 °C). The final mixture was stirred for further 30 min to obtain siRNA-loaded nanoparticle formulations of ca. 1 mg/mL (calculated from the nano-particle polyelectrolyte feed ratio). A typical loading of 2.3% wt. siRNA (compared to the polycation) was used for nanoparticle characteriza-tion and cell culture experiments. Please note that from now the term nanoparticles (NP) refers to both HA-coated Nanocin or chitosan na-noparticles.
    2.1.3. Preparation of nanoparticles varying polycation:HA ratio
    NP were prepared varying polycation:HA ratio to identify the op-timal formulation in terms of size and ζ potential. NP were prepared using HA solutions with the following concentration (polycation:HA ratio): 0.375 mg/mL (1:1), 0.75 mg/mL (1:2), 1.125 mg/mL (1:3) and 1.5 mg/mL (1:4). For uncoated NP (1:0 ratio), RNase free water (pH adjusted to 5) was used. NP were prepared using the same procedure described above (Section 2.1.2). Briefly, the polycation/siRNA complex dispersion was pipetted in an equivalent volume of HA at different concentrations to vary the polycation:HA weight ratio. Note that in this