Fig. 7
Lyophilisation of RALA/ALN NPs were prepared with a range of trehalose concentrations. The a mean hydrodynamic size and zeta potential of L-RALA/ALN NPs at mass ratio of 10:1 containing 1 μg ALN. Particles were made up to a 50 μL volume and left to incubate for 30 min at RT. Subsequently, particles were lyophilised to achieve a final trehalose concentration of 1%, 2.5% and 5% w/v before reconstitution in 50 μL of UltraPure DNase/RNase-Free Distilled Water. Particles were measured using the Malvern Zetasizer Nano ZS instrument at 20 °C through DLS and laser doppler electrophoresis, respectively. b The sample quality of L-RALA/ALN NPs was measured through corresponding PDI and the mean count rate (kcps). Results are displayed as mean ± SEM, n = 3. TEM images of c RALA/ALN NPs taken at 30 K magnification, and (D) L-RALA/ALN NPs were taken at 80 K magnification. NPs were loaded onto carbon reinforced 400 mesh copper grids (TAAB Laboratories, UK) which were subsequently stained with 5% uranyl acetate before drying. Grids were imaged using a JEM-1400Plus Transmission Electron Microscope (JEOL Inc., USA) at an accelerating voltage of 120 kV to provide high resolution images. (Scale bars = C: 100 nm, D: 50 nm). Cell viability of e LN229 and f T98G cells treated with L-RALA/ALN NPs as measured by MTS. Cells were treated with uncomplexed ALN to achieve a final concentration ranging from 5 μM to 1 mM and L-RALA/ALN NPs (1 μg ALN) were prepared at a 10:1 mass ratio such that the final concentration of ALN per well ranged between 5 μM to 75 μM. Lyophilised NPs were reconstituted in 50 μL of UltraPure DNase/RNase-Free Distilled Water prior to use. Cells were treated for 6 h before medium was replaced with complete media and left to incubate for 72 h. Post-incubation, cell viability was assessed using MTS reagent and cell viability was expressed as a percentage of the untreated control (considered to be 100% viable). The IC50 values were calculated using commercial software (GraphPad Prism®, 8.1.2, GraphPad Software, USA). Results are displayed as mean ± SEM, n = 3