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composition (Abellan-Pose et al., 2016). Although the majority of the work in the literature describes the intravenous administration of na-noparticles, intratumoural injection (the intended application of our nanocomposite hydrogel) of small NCs locally also has the potential to give increased penetration through the tumour as described for Pegy-lated gold nanoparticles (50 nm), an emulsion (85 nm) and neutral li-posomes (120 nm). Whilst an 250 nm emulsion and 100 nm liposomes with positive surface charge had limited diﬀusion (Laprise-Pelletier et al., 2018; Nomura et al., 1998).
We progressed to the release studies of GEM C14 without under-taking drug stability studies as very similar alkanoyl gemcitabine de-rivatives were previously established as being stable in vitro (Immordino et al., 2004). In addition, for other N-alkanoyl gemcitabine derivatives the amide MIK665 (S-64315) was also demonstrated to be stable be-tween 6 and 8 in vitro (Wickremsinhe et al., 2013; Bender et al., 2009). The release studies were carried out under sink conditions, by in-cubating the NCs in PBS at 37 °C for 1 month. The amount released from HA 40 and HA 80 nm was evaluated by analysis of the amount re-maining in the NCs and then subtracting this from the initial amount encapsulated. Both formulations showed a similar biphasic drug release profile (Fig. 1), characterised by an initial burst release of ∼20% fol-lowed by a second phase in which further ∼40% of drug was released over 1 month. The initial burst release maybe attributed to the non-ionic surfactants solubilising drug at the oil water interface (Youm et al., 2014). The total amount of GEM C14 released from the NCs after one month was 63 ± 8 and 66 ± 10% from HA 40 and HA 80 nm NCs respectively. The amount of drug that remained encapsulated in NCs during the release studies is notable (∼35% after 1 month) thus showing its stability inside the nanocarrier and the ability of the na-nocarrier to maintain sustained release. Similarly, Bastiancich et al. reported 56% of lauroyl-gemcitabine (GEM C12) released in the first 48 h and 77% released after 1 month (Bastiancich et al., 2016). Delayed release of GEM C14 (with a longer carbon chain compared to C12) can be attributed to its higher aﬃnity towards the oil core and the diﬀusion between the hydrophobic core and surrounding environment can be hindered by the packing density of the surfactant molecules and the presence of the polymer coating (Lamprecht et al., 2002; Poletto et al., 2008). These data suggest the potential for sustained release in future in vivo studies.
3.1.2. Stability of NCs upon storage and after incubation in bio-relevant media
From an industrial and translational perspective, it is important to obtain formulations that are stable upon storage. Additionally, a critical aspect of physiological relevance is their stability in bio-relevant media as it is known that stability in biological media can be a barrier in the development of many nanoformulations (Moore et al., 2015). There-fore, storage stability at RT and in the fridge as well as the stability of the NCs in PBS, pH = 7.4 and in RPMI cell culture medium was
Fig. 2. A) Influence of the storage conditions (room or fridge temperature) on the size of NCs after 4 weeks. Diameters of HA 80 and PGA 80 NCs are presented after storage in a cupboard at RT or in the fridge at 8 °C. B) Physico-chemical characteristics (diameter and PDI) of HA 80 nm NCs after incubation with PBS and RPMI medium. Measurements were performed on the day of the incubation at RT. Mean values are shown; error bars represent standard deviations of 3 independent trials. 1 way ANOVA Dunnett’s multiple comparison test, diameter of NCs was compared with initial size, *(p < 0.05).