2.3. Liposome Characterization Studies

The mean size, associated polydispersity index (PDI) and zeta potential of all liposome preparations were measured following dialysis in PBS by dynamic light scattering and relative electrophoretic mobility in water using the Zetasizer Nano ZS instrument (Malvern Instruments Ltd., Worchestershire, UK).

The siRNA encapsulation and retention of all liposome preparations was determined using the Quant-iT RiboGreen RNA Assay Kit (Thermofisher Scientific). Following dialysis purification from un-encapsulated and/or un-retained siRNA, liposomes were denatured and solubilized in 1% Triton X-100 at 37 °C for 15 min to release siRNA cargo. Released siRNA was mixed with RiboGreen reagent for fluorescent labeling and emission was read at 525 nm. Fluorescence units of solubilized liposomes was fit to a known standard curve of siRNA in 1% Triton X-100. EE% of each liposome formulation was calculated as (pmols siRNA encapsulate/total original pmols siRNA used) × 100.

The stability of siRNA-loaded liposomes was assessed using multiple gel electrophoresis techniques. Stability assays were run using 1% agarose gels with 1 ug/mL Ethidium Bromide (EtBr) and 1× Tris-Boric-EDTA (TBE) buffer for gel preparation and running. All siRNA bands were visualized by EtBr binding via transillumination through a GFP filter.

For a qualitative analysis of EE%, siRNA-loaded liposome sample aliquots were taken prior to purification by dialysis and equal fractions were loaded onto agarose gels. Un-encapsulated, un-complexed siRNA, free in solution, was ran into gel at 100 V for 30 min while liposomes with encapsulated/complexed siRNA were excluded from gel penetration and isolated to the wells.

To determine minimum heparin concentration necessary for complete siRNA displacement from liposome surface, siRNA-loaded liposomes from 100:1 lipid:siRNA wt-to-wt samples were exposed to varying concentrations of heparin (0–400 ug/mL) for 30 min at room temperature. Equal fractions were loaded onto agarose gels and displaced siRNA was ran into gel at 100 V for 30 min. In follow-on experiments, to examine the proportion of lipoplexed siRNA that was outer associated and vulnerable to heparin displacement, liposome samples were taken after purification by dialysis, exposed to 100 ug/mL heparin and equal fractions were loaded and ran as described. To examine total encapsulated siRNA and total lipoplexed siRNA, liposome samples were taken after purification by dialysis, exposed to 1% Triton X-100 and 100 ug/mL heparin to denature liposome nanoparticles and release all complexed siRNA and equal fractions were loaded and ran as described. To examine the proportion of encapsulated siRNA that was completely contained within the core of the liposome, liposome samples were taken after purification by dialysis, treated with 100 ug/mL heparin and re-dialyzed overnight to remove previously complexed siRNA from solution, then exposed to 1% Triton X-100 and 100 ug/mL heparin to denature liposome nanoparticles and release all siRNA within the core.

To determine minimum RNase necessary for complete siRNA degradation, free siRNA controls were exposed to varying concentrations of RNase A enzyme (0.001–100 ug/mL) for 30 min at 37 °C. Equal fractions were loaded onto agarose gels and the remaining, undigested siRNA was ran into gel at 100 V for 30 min. In follow-on experiments, siRNA-loaded liposomes were prepared with and without the exposure to 0.5 ug/mL RNase A, RNase digestion was stopped by flash freezing in liquid N₂ and liposome samples were exposed to 1% Triton X-100 and 100 ug/mL heparin to denature liposome nanoparticles and release all complexed siRNA. The protection of outer associated siRNA demonstrated via heparin displacement was validated by the protection from RNase enzyme degradation compared to the degradation of free siRNA controls.

Liposome morphology and lamellarity were investigated by STEM using a negative-stain method. Liposomes were applied dropwise to a carbon film coated copper grid and allowed to air dry. Liposome films were then stained with 2% phosphotungstic acid and air-dried for 1 min at room temp. Samples were visualized with Zeiss Auriga 40 STEM scope and images were acquired by SmartSEM image acquisition software (Carl Zeiss, Inc., Oberkochen, Germany).