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  • br investigated Fig br In the stability study neither

    2019-10-22


    investigated Fig. 2.
    In the stability study, neither aggregation nor precipitation of na-noparticles were observed during storage ≤1 month. This good col-loidal stability profile, which could be attributed to the electrostatic repulsion between the negatively charged NCs agrees with the stability data reported for other polymeric based NCs (Lozano et al., 2013; González-Aramundiz et al., 2015; Zanotto-Filho et al., 2013; Abellan-Pose et al., 2016). Although, changes in particle size of the nano-particles throughout the entire course of the study were minimal, PGA 
    NCs exhibited significant (1-way ANOVA Dunnett’s multiple compar-ison test, p < 0.05) increase in size after 4 weeks storage for both PGA 40 and 80 nm NCs. This could be the result of film thinning and NCs coalescence due to the lower polymer coverage (assessed by ZP) which led to weak repulsive forces between PGA NCs compared to HA NCs.
    The size of HA 80 nm NCs did not change significantly after in-cubation in PBS nor in RPMI. However, a slight increase in the PDI value (p < 0.05, Dunnett’s multiple comparison test) was observed upon incubation of the NCs in RPMI medium. This could be a
    consequence of higher salt content (2× potassium ions) in the RPMI medium and is more likely due to the potential interaction of the pro-teins from the FBS serum in the RPMI, with the NCs. The presence of polymers and other molecules on the surface of particles is a well-known approach for conferring electrostatic and steric stabilisation to NCs (Tan et al., 2010; Storm et al., 1995; Moore et al., 2015).
    3.1.3. In vitro activity of GEM C14 loaded polymeric NCs in cancer Y-27632 Human colorectal and pancreatic carcinoma cells were exposed to serial dilutions of NCs (blank or loaded with the drug) and to drug alone for 72 h. Subsequently, MTT assays were carried out to assess cell via-bility and GI50 values are presented in Fig. 3. GEM C14-loaded NCs as well as free drugs (GEM-HCl and GEM C14) gave nanomolar GI50 values in all wild type cancer cell lines (HCT 116, MIA PaCa-2 and Panc-1), demonstrating high potency. On the other hand, blank NCs (no drug) exhibited a 10 to 100-fold higher GI50 values than drug containing NCs implying that growth inhibition of these cancer cells is mainly due to the drugs activity. Representative dose response curves are shown in ESI-Fig. S6 for HCT 116 and MIA PaCa-2 cell lines and Fig. S7 for Panc-
    1 and GEM resistant Panc-1 cell lines.
    A pancreatic GEM-resistant cell line (GEM R Panc-1) was obtained by continuous culture of parental Panc-1 cells in gemcitabine and 
    maintained with gemcitabine at a dose of 1 μM. In this cell line, free drug in the form of either GEM C14 or GEM-HCl exhibited negligible growth inhibitory effects with GI50 values in the µM range confirming its resistance towards not only commercial GEM-HCl but also towards the modified lipophilic GEM C14. Activity of GEM C14 loaded NCs in GEM resistant Panc-1 cell line showed 20–50 times lower GI50 values compared to those of GEM C14 and GEM-HCl respectively. Interestingly, in contrast to data on wild type cancer cell lines, blank NCs in GEM resistant Panc-1 cell line showed similar activity to drug loaded NCs. For this drug resistant cell line, growth inhibition appears to be a consequence of the NC formulation which includes both non-ionic surfactants and a small amount of a cationic surfactant i.e CTAB. CTAB has been described as a cytotoxic agent (He et al., 2011; Ito et al., 2009). The presence of CTAB in blank NCs may possibly explain the similar toxicity of empty and loaded NCs on the GEM R pancreatic cell line.
    3.2. Association of polymeric NCs and LMWGs
    N4-octanoyl-2′-deoxycytidine was chosen as a gelator on account of the absence of solvents used in its formulation, thus its compatibility with NCs. Initially, hydrogels consisting of only gelator and water were
    rapidly formed after a heating-cooling cycle. The storage modulus (G′) derived from the LVE region of amplitude sweep tests was used as a measure of gel strength (Fig. S3). Results indicated that the con-centration of gelator can influence gel strength in the hydrogel only and in nanocomposite hydrogels with NCs (Fig. 4). For pure hydrogels, G′ values increased from 148 Pa to 1760 Pa in gelator concentration range between 0.5 and 2% w/v. However, for the highest concentration (2.5% w/v), the value of the elastic modulus dropped to 517 Pa. The influence of the gelator concentration on the value of the elastic modulus has been reported elsewhere (Friggeri et al., 2004; Frith et al., 2015). With increases in gelator concentration the elastic modulus will increase until it reaches a plateau (Mohmeyer and Schmidt, 2005) or a decrease in G′ values can be observed above certain concentrations. At higher concentrations the gelation process can almost be instantaneous re-sulting in shorter fibers and consequently reduced elasticity of the system or can lead to precipitation and the disruption of the gelator network (Menger and Caran, 2000). In the case of gel-NCs composite, it is important to note that the gelator, N4-octanoyl-2′-deoxycytidine, at a concentration of 0.5% w/v failed to gel in the presence of NCs. Whereas with 1% w/v gelator concentration a gel was macroscopically formed but with a short LVE region. However, further increases in gelator concentration improved the linearity of the LVE region and resulted in increased gel strength (Fig. S3B). When pure hydrogel and gel-NCs composite are compared at the same gelator concentration (2.5% w/v) a 10-fold increase in G′ values in the presence of NCs is noted. Ad-ditionally, gel strength was independent of NCs’ size (40 or 80 nm) or polymeric coating (hyaluronic acid or poly-glutamic acid) as their G′ were not significantly different (ESI-S4).