br Fig Cumulative release of
Fig. 5. Cumulative release of NR-loaded NCs from gel-NCs formulation at 37 °C in PBS medium, GSK1838705A = 7.4. Concentration of gelator in all systems is 2% w/v and NCs used in this experiment are: HA 80 nm NCs of 4 and 8% w/v and HA 40 nm NCs in concentration of 4% w/v. Gels were prepared with 0.5 mL volume of NCs and gel was incubated with 12.5 mL of PBS. Mean values are shown; error bars represent standard deviations of 3 independent trials.
negligible diﬀerence (10–20%) in release from their gel (Moysan et al., 2014). % released decreases at 72 h as there is some partitioning of the NR into the release medium.
Following the release of NCs from the gel, the size of NCs released in the medium was examined. Released NCs of 172 ± 24 nm were ob-served, which is larger than the initial size of NCs (∼80 nm). Hence the integrity of NCs was evaluated under conditions of gel preparation. In Fig. 6, the size of NCs in the presence or absence of gelator after heating at 60 °C is presented. NCs heated to 60 °C exhibited only a slight in-crease in size to 55 ± 3 and 98 ± 17 nm for HA 40 and HA 80 nm formulations respectively. However, in the presence of gelator, the size of NC for both systems, increased up to ∼200 nm. It seems that it is not the temperature that causes increase in size of NCs, but the presence of gelator around NCs. PDI values for all samples were ≤0.2 suggesting that no aggregation occurred. How much this increase in size would limit the diﬀusion in the tumour mass is diﬃcult to predict. There is limited data in the literature describing the diﬀusion of nanoparticles across the tumour. These data indicate that nanoparticles with a size of 250 nm or a positive surface charge may have a limited diﬀusion (Laprise-Pelletier et al., 2018; Nomura et al., 1998). According to this, the NCs described in the manuscript are expected to have adequate diﬀusion across the tumour.
Irrespective of the size observed by light scattering, using TEM imaging it was possible to confirm the presence of NCs < 100 nm diameter, dispersed in the gel (Fig. 6B). The presence of fibrous network is clearly seen with homogenous distribution of spheroid particles in between fibers. Therefore, Coincidental evolution could be inferred that the increase in size observed by light scattering is not due to the aggregation of the NCs but rather associated to the presence of fibers around the NCs.
When considering a formulation for direct intra-tumoural delivery, injectability of the system is a crucial characteristic to evaluate. Our group has previously reported 70% gel recovery, after a time-dependent rheology test, for the N4-octanoyl-2′-deoxycytidine. In Fig. 7, an image of a syringe loaded with gel-NCs composite formulation, and the ap-pearance after its contents were dispensed through the needle is shown. The gel formulation with NCs reformed instantly and remained in its gel form. The vial has been inverted to show that the gel has retained its viscoelastic properties after passing through the needle. Furthermore,
Fig. 6. A) Change of initial size of HA 40 or HA 80 nm NCs after being exposed to heat at 60–70 °C with and without gelator. Black bars present size of NCs alone before the heating, black bars with grey lines NCs alone after the heating at 60 °C and grey lines NCs in presence of solubilised gelator after the heating to 60 °C. Mean values are shown; error bars represent standard deviations of 3 independent trials. B) cryo-TEM imaging of 2% w/v gelator – 8% w/v HA 80 nm NCs.
their ability to be placed in tissue was tested in an in vitro simulation of intra-tumoural delivery i.e. chicken thigh. To mimic a resection site and intra-tumoural delivery, we bored out a hole in chicken thigh and used a syringe (without needle) to place the gel inside and observed the shape of the gel. Formulations with the same gelator concentration (2% w/v) and diﬀerent amounts of NCs (8 and 4% w/v) were compared in terms of injectability. While the gel containing 4% w/v NCs retained its structure, the gel containing 8% w/v NCs did not reform after dispen-sing through a syringe (images in ES1-S5).