Trichain cationic lipids: the potential of their lipoplexes for gene delivery

Lipoplexes (LDs) have been prepared from DNA, DOPE and either a dichain oxyethylated cationic lipid or their novel trichain (TC) counterpart.


Formulation of Lipid:DNA Complexes S3
Dynamic Light Scattering and Zeta Potential S3

Small Angle Neutron Scattering S4
Cell Culture S5

Transfection Experiments S5
Figures S7  (Beckman Microfuge, UK) at 13,000 rpm (~16,000 g) for 10 min to remove any titanium particles that had been shed from the probe.

Gel Retardation Experiments
The pDNA binding efficiency of LD complexes was determined using agarose gel electrophoresis (0.8% w/v agarose gel containing Gel Red in Tris-acetate-edetate (TAE) buffer at pH 7.4). LDs to be tested were formulated at L:D charge ratios of 1:1 to 4:1 and to a final pDNA concentration of 1 μg per 10 μL per well. 2 μL of gel loading buffer (prepared using 0.25% w/v bromphenol blue in a 40% w/v sucrose solution) was added to each sample before loading onto each well. The gel was run at 80 mV for one hour (Fisher Brand Model HU12 electrophoreses chamber, Loughborough, UK) after which the gel was visualized under UV light illumination using an Alphalmage EP Multi-Image Light Cabinet (Randpark Ridge, South Africa). Uncomplexed, free pDNA was used as a control. Each gel was repeated on more than one occasion to ensure reproducibility.

Picogreen Experiments
In an attempt to determine the extent of pDNA complexation, picogreen-binding to the LDs was measured. 50 µL of picogreen reagent (namely 1:150 v:v picogreen in 3 x Tris-EDTA (TE) buffer) was added to 100 µL of LD suspension (containing 0.2 µg of DNA) and incubated in 96-well black plates at room temperature for 5 min before fluorescence was determined. The fluorescence associated with the LDs was measured at an excitation wavelength of 485 nm and an emission wavelength of 520 nm with a gain of 1000 using a FLUOstar Omega fluorimeter (BMG LABTECH GmbH, Ortenberg, Germany) equipped with a plate reader. Any pDNA not incorporated in the LDs was quantified and expressed as relative fluorescence units (RFU) compared to the free (naked) pDNA control, which was denoted as 100% RFU in order to normalise the fluorescence signal. All experiments were performed in triplicate and the mean and SD calculated.

Transmission Electron Microscopy
Vesicle suspensions and LDs were negatively stained with 4 % w/v uranyl acetate by placing a drop of sample on a formvar 200 mesh copper grid for one minute followed by drying with filter paper.
The grid was then placed on a drop of uranyl acetate for approximately five minutes and washed in Due to the size and polydispersity of the vesicles, the SANS data for the vesicles were analysed using the mixed 'sheet and stack model' in which the vesicles were assumed to be a mixture of infinite S5 planar sheets (taken to be the bilayer of any unilamellar vesicles present in the preparation) and one-dimensional stacks (taken to be the bilayer and repeat distance for any multilamellar vesicles present). 1 In all cases the model was constrained to yield the same mean thickness of the bilayer (L) for both the unilamellar and multilamellar vesicles. The SANS data was modelled by the least square refinement of up to 6 parameters, namely bilayer (L), Lorentz correction factor (Rσ), the number of bilayers in a stack (M), their mean separation (D) and the absolute scale factors for the unilamellar and multilamellar vesicles. If no multilamellar vesicles were present only 3 parameters were refined.
In the present study fitted value of Rσ (related to the curvature and extent of rigidity for the sheets) reporter lysis buffer (provided in the kit) for 1 hour at 37 °C before freezing at -70 °C for at least 30 minutes followed by thawing at room temperature to ensure more efficient cell lysis. 50 μL of the S6 lysate was then transferred to a white 96-well plate and the luciferase activity was measured for 10 seconds using an MLX Microtitre ® Plate Luminometer (Dynex Technologies, USA) with an automatic feeding system delivering 100 μL of the reconstituted luciferase assay reagent into each well. The amount of protein in each transfection lysate was measured using a BCA protein assay kit according to manufacturer's instructions. Luciferase activity was expressed as Relative Light Units (RLU) per milligram of protein (RLU mg -1 protein).