br where Xi is the amount
where Xi is the amount initially loaded into NCs and Xt the amount that is in NCs at time point. As drug released was below the detection limit, by measuring the drug retained within NCs, the amount of drug re-leased could be indirectly calculated. The small amount discarded at each time point when removing sample and replacing it with PBS was added to the amount released. Quantification of GEM C14 by HPLC was carried out using a method
2.2.3. In vitro activity of drug-loaded NCs in cancer cells
GEM C14-loaded NCs were manufactured as described above and were assessed for in vitro cell growth inhibition on cancer cell lines. Eight formulations (HA 80, HA 40, PGA 80 and PGA 40 nm NCs blank or loaded with GEM C14) were tested and compared with GEM C14 and GEM hydrochloride (GEM-HCl). The initial concentration of GEM C14 in NCs was ∼25 µM. Blank NCs did not contain drug. Human carci-noma cell lines used in experiments were the HCT 116 colorectal and MIA Paca-2, Panc-1 and GEM-resistant Panc-1 pancreatic cancer cell lines. Human carcinoma cell lines (HCT 116, MIA Paca-2 and Panc-1) were all sourced from the American Type Culture Collection (ATCC).
General cell culture involved incubation of serially sub-cultured cells at 37 °C in a humidified L-NAME hydrochloride of 95% air and 5% CO2. Cells were grown in RPMI 1640 medium supplemented with 10% v/v FBS. Once cells had reached approximately 70% confluence they were used for the experiments. A pancreatic GEM-resistant cell line was generated by continuous culture of parental Panc-1 cells in medium supplemented with incremental gemcitabine concentrations, and then maintained in medium spiked with 1 μM Gemcitabine. This cell line was developed and kindly donated by Dr Lodewijk Dekker (University of Nottingham).
220.127.116.11. Growth inhibition assay. The MTT assay was used for mitochondrial activity evaluation, a surrogate marker for viable cell number. Briefly, cells were seeded into 96-well plates at a density of 3 x103 cells/well in 180 µL of culture medium. An extra plate was prepared containing cells from each cell line in order to carry out an MTT assay at the time the cells were treated. This allows the initial optical density (OD) to be determined. After 24 h, cells were treated with drug alone (GEM C14 and GEM-HCl), blank NCs and GEM C14-loaded NCs starting with GEM concentration from 25 µM followed by 10-fold serial dilutions. Four wells were used for each condition. Control cells were treated with medium alone. Following 72 h incubation, 50 µL of 2 mg/mL MTT solution were added to each well. Cells were incubated for a further 2.5 h to allow formazan production. The aqueous medium was then aspirated and the formazan product solubilised in DMSO (150 µL/well). Absorbance at 570 nm (optical density - OD570) was read using an EnVision 2104 Multilabel Reader. The concentrations at which cell growth is inhibited by 50% (GI50) were calculated from the dose-response curves estimated by linear interpolation between two closest values after taking into account the initial optical density acquired at the time of treatment. MTT assays were carried out on ≥3 separate occasions and mean GI50 values were calculated.
2.2.4. Gel-NC composites for localised cancer therapy
N4-octanoyl-2′-deoxycytidine was used as LMWG as it was reported
to gel in pure water using a heating-cooling cycle as the method for gel formation (Skilling et al., 2016). A defined amount of gelator was added to water and heated in a water bath up to 60 °C until the compound completely dissolved. The sample was then allowed to cool to room temperature for ∼30 min. For the preparation of gel-NCs composite, the procedure was the same except that the gelator was dissolved in NCs formulation instead of in pure water. The following concentrations of LMWG were investigated: 0.5, 1, 1.5, 2, 2.5, 3, 3.5 and 4% (w/v). All 4 NCs formulations were tested (HA 80, HA 40, PGA 80 and PGA 40 nm NCs). HA 80 nm NCs were tested at concentrations of 8; 4 and 2% w/v.