br Table br Excipients and Physico chemical characteristics
Excipients and Physico-chemical characteristics of diﬀerent NCs loaded with GEM C14 (0.1% w/w). Mean values (n = 3) are shown, error bars represent standard deviation of the mean, where n is number of independent trials. All NC formulations contained 0.25 mg CTAB The aqueous phase:oil phase ratio was 1:8 (w:w). All amounts are expressed in mg per formulation, where final formulation volume is 1.5 mL. HA 80 nm NCs represent hyaluronic AMG-176 coated NCs with a diameter of 80 nm; whilst PGA 80 nm NCs are poly-L-glutamic acid coated NCs with a diameter of 80 nm; HA 40 nm NCs are hyaluronic acid coated NCs with a diameter 40 nm; and PGA 40 nm NCs are poly-L-glutamic acid coated NCs with a diameter of 40 nm.
Formulation name NC Excipients
3.1. Production of HA and PGA NCs
HA or PGA NCs were produced by self-emulsification, without the use of any solvents. Considering the first objective of this work, to re-duce their size below 100 nm, the influence of the amount of oil (Miglyol® 812 N), and the amount of surfactants (Tween® 80 and Kolliphor® HS 15) was investigated as described in the Supplementary Information (ESI-S1). The excipients and physico-chemical character-istics of HA and PGA NCs of two sizes (i.e. 40 and 80 nm) and loaded with GEM-C14 are given in Table 1. All formulations exhibited low polydispersity (PDI ≤ 0.2). The charge of the NCs depended on the polymer coating with zeta potential values closer to neutral when PGA was used (−10 to −4 mV) while more negative charge was observed for HA NCs (−19 to −9 mV). Our lab has previously reported the preparation of polymeric NCs (Protamine) with a size close to 200 nm, using a self-emulsifying technique (Jakubiak et al., 2017), however, here we describe for the first time the production of HA and PGA NCs below 100 nm using a self-emulsification process. The formation of nanoemulsions with a globule size < 100 nm, first at elevated tem-peratures (85 °C) (Heurtault et al., 2003) and later at 30 °C has also been reported (Lefebvre et al., 2017; Saberi et al., 2013). However, these emulsions did not have a polymer shell as those reported in this work.
A high encapsulation eﬃciency (> 90%) of GEM C14 is obtained in all formulations indicating compatibility between the drug and the core of NCs. High encapsulation eﬃciency (> 70%) of lipophilic compounds is a characteristic for NCs (Mora-Huertas et al., 2010) and our report is in accordance with other self-emulsifying NCs (Jakubiak et al., 2017). The size and spherical shape of the NCs were additionally confirmed by TEM imaging (see ESI – Fig. S2).
The drug loading (0.1 w/w) is suﬃcient for intra-tumoural delivery as will be illustrated by comparison to the closest literature example of Lee et al. These authors demonstrated that a polymeric film designed for treatment of gastrointestinal cancer contained 150 μg of GEM per im-plant and showed appropriate loading as it induced complete regression of CT-26 colon carcinoma s.c. model in mice (Lee et al., 2012). With a drug loading of 0.1% (w/w) or 80 μg/mL of GEM C14 (assuming 8% w/ w oil/surfactant in the nanoemulsion) we can achieve 16 μg per tumour (max volume that can be injected is typically 200 μL). This drug loading could potentially be suﬃcient for GEM C14 as compared to GEM as the lipophilic derivative will have increased retention at the administration site and also has nanomolar potency as demonstrated below (see Fig. 3).
The importance of the NCs formulations developed herein is re-flected by the known influence of the physicochemical properties of nanocarriers on their diﬀusion through the tumour stroma (Gratton et al., 2008; Ernsting et al., 2013). (Lee et al., 2010; Jiang et al., 2008). Generally, smaller particles i.e. ≤100 nm perform better in terms of accumulation and distribution in tumours (Chauhan et al., 2012; Saw et al., 2018; Cabral et al., 2011; Meerovich et al., 2008). Moreover, in a recent publication by our group, PGA-PEG NCs of 100 nm were found to be drained into lymph nodes faster than the 200 nm NCs of the same