The discovery and enhanced properties of trichain lipids in lipopolyplex gene delivery systems

Novel trichain lipids have been identified with enhanced transfection properties in lipopolyplexes.


General materials and methods S3
Synthetic procedures S4

Scrambled peptide A synthesis (sc) S14
HPLC Methods S15 1 H and 13 C NMR spectra of compounds S17

Formulation, transfection and biophysical studies S33
Lipid transesterification studies S33

Formulation of lipopolyplexes S33
Dynamic light scattering S34

In vitro transfection experiments S34
Gel retardation, release and protection assay S35

Transmission electron microscopy S36
Small angle neutron scattering S36

General materials and methods
Synthesis Unless otherwise noted, solvents and reagents for synthesis were reagent grade from commercial suppliers and used without further purification. Dry CH2Cl2 and MeCN were obtained using anhydrous alumina columns. 1 All moisture-sensitive reactions were performed under a nitrogen or argon atmosphere using oven-dried glassware. Reactions were monitored by TLC on Kieselgel 60 F254 plates with detection by UV, potassium permanganate, and phosphomolybdic acid stains. Flash column chromatography was carried out using silica gel (particle size 40-63 μm). 1

Formation of disulfide bonds via iodine oxidation:
To the resin-bound, fully protected linear peptide was added I2 (10 eq.) in DMF (3 mL). The reaction syringe was agitated for 2 h at room temperature.
The iodine solution was drained from the syringe under vacuum and the resin washed with DMF (5 x 1.5 mL), 2% ascorbic acid in DMF (2 x 1.5 mL) and DMF (5 x 1.5 mL). The N-terminal Fmoc group was removed (as described) and the peptide cleaved from the resin.

S15
Peptide Cleavage: TFA/TIPS/EDT/H2O (94:2.5:2.5:1; 3 mL) was added to the reaction syringe. The syringe was then agitated for 3 h at room temperature. The cleavage cocktail was drained from the vessel under vacuum and diethyl ether (~10-15 mL) added to the filtrate. The resulting precipitate in solution was spun at 4000 rpm for 10 mins at 4 °C to produce a crude peptide pellet. The supernatant (diethyl ether) was decanted off and the peptide washed a further three times with diethyl ether. The crude peptide pellet was then re-dissolved in minimum water and freeze-dried for storage prior to purification.

Formation of disulfide bonds via aerial oxidation:
Crude peptides were re-dissolved in water (1 mg per 10 mL) and stirred at room temperature for 5 days. The solution was then concentrated and freeze-dried for storage prior to HPLC purification. The purified peptide was analyzed by analytical HPLC using an Onyx monolithic C18 column (Phenomenex ® ; 100 x 3.0 mm, 2 μm macropore size, 13 nm mesopore size, flow rate 0.85 mL/min).
The analysis of the chromatograms was conducted using Star Chromatography Workstation software Version 1.9.3.2.
ESI-MS analysis was performed on a Waters Acquity Ultra Performance LC/MS machine.

HPLC Methods:
Preparative high performance liquid chromatography:

Transfection procedures and biophysical studies Preparation of vesicles
Cationic di-chain and tri-chain lipids were formulated into vesicles at a 1:1 molar ratio with DOPE (and in some instances lysoPE) and a final cationic lipid concentration of 1 mg/mL (or 2 mg/mL for neutron scattering experiments). The required amount of lipids was weighed into a vial and dissolved in chloroform, and the solvent evaporated in vacuo overnight to produce a thin lipid film.
The film was then hydrated with the required amount of water (D2O was used for neutron scattering experiments). The resulting vesicle suspension was then probe sonicated for 5 min using a Lucas-Dawes probe sonicator (model: 7535A) operating at 50% maximum output. The vesicles were then centrifuged at 13,000 rpm (~16,000 g) for 5 min to remove any titanium particles shed from the probe.

Formulation of lipopolyplexes
Unless otherwise stated, lipid:peptide:DNA (LPD) complexes were prepared at 0.25:6.5:1 charge ratios. This was equivalent to approximately 0.75-1:4:1 LPD weight ratios. Previous studies showed that the order of mixing of the three components was crucial for the activity of the complex. The highest transfection efficiency was obtained when peptide was added to the lipid first, at equal volumes to each other, followed by the addition of and an equal volume of DNA to the lipid:peptide S34 mixture at the intended concentrations. 8 For example, 50 µL of 0.08 mg/mL peptide was added to 50 µL containing 0.75-1 µg of lipid, then 100 µL of 0.1 mg/mL DNA was added to the lipid:peptide mixture.

Dynamic light scattering
1 mL samples of vesicles or PD and LPD complexes, using gWIZ luciferase plasmid (pDNA), were prepared and transferred to a plastic clear-sided cuvette. Size and zeta potential of the complexes were measured by dynamic light scattering using a Zetasizer Nano-ZS series, Malvern Instruments Ltd, UK.

Cell culture
Rat neuroblastoma B104 cells were obtained as a gift from the Institute of Child Health, University All measurements were carried out in triplicate, and the error bars represent the standard deviation calculated from three different measurements carried out in one experiment.

Gel retardation, release and protection assay
The binding, release and protection of pDNA from DNAse I degradation that the LPD complexes and run at 80 mV for one hour. The gel was visualized using a Herolab EASY UV transilluminator. The intensity of the pDNA bands were analysed using GelAnalyzer program.

Transmission electron microscopy
LPD complexes were prepared at 0.25:6.5:1 charge ratios at a final volume of 50 µL and a final pDNA concentration of 0.02 mg/mL. A drop of sample was placed on a Formavar 200 mesh copper grid for a few minutes, after which the sample was dried using a filter paper. The sample was stained by placing the grid sample face down onto a drop of 4% uranyl acetate solution for several minutes.
The grid was then washed in ethanol and water and left to dry before visualisation on an FEI Tecnai T12 transmission electron microscope, USA. where θ/2 is the scattering angle, was determined by normalising the scattering to the appropriate sample transmission after subtraction of the scattering from the relevant solvent also normalised to its corresponding transmission. The fitting of the SANS data always included flat background corrections to allow for any mismatch in the incoherent and inelastic scattering between the sample and the solvent, with the levels of the fitted background being checked to ensure that they were physically reasonable. The SANS data for the DOTMA:DOPE vesicles and LPR complexes dispersed in D2O were routinely modeled either assuming a mixture of (isolated/single) infinite planar (lamellar)

Small angle neutron scattering
sheets with or without one-dimensional paracrystals (stacks) to account for the presence in the sample of any multilamellar vesicles. When modeling the vesicles and LPR complexes dispersed in D2O as (single) lamellar sheets, the fits to the SANS data were obtained by the least-squares S37 refinement of three parameters, namely L, Rσ, and the absolute scale factor (together with the background, as described above), where Rσ is the Lorentz correction factor which provides information about the extent of rigidity/curvature of the lamellar sheets. In this study the polydispersity on the thickness of the bilayer (σ(L)/L) was fixed at 10-6. When stacks were added to the (single) lamellar sheet model, the fit to the SANS data was obtained by least-squares refinement of seven parameters, namely, the mean bilayer thickness (L), the Lorentz factor (Rσ), the number of bilayers in the stack (M), their mean separation or d-spacing (D), the width of the Gaussian distribution in the plane, (σ(D)/D), and the absolute scale factors for the unilamellar and multilamellar vesicles. Where a Bragg peak was clearly observed in the data, it was fitted using bilayers of 5 and 10. In the present study, σ(L)/L was again fixed as 0.1, σ(D)/D at 0.05, and Rσ at 300. In addition, when modeling the SANS data using a mixed population of sheets and stacks, L, σ(L)/L, and Rσ were constrained to be the same for the isolated/single and stacked lamellae, a not unreasonable assumption. If no Bragg peak was seen in the SANS data, it was fitted using a stack with a maximum of 2 bilayers. In such cases the data was fitted using a higher number of bilayers comprising the stacks to ensure that it did not improve the quality of the fit obtained. For all models, the least-squares refinements were performed using the model-fitting routines provided in the FISH 9 software.

Circular dichroism
The circular dichroism (CD) spectra of LPD complexes prepared in D2O (used because of its better optical transparency than H2O) was measured using a Chirascan Plus spectrophotometer (Applied Photophysics, UK) at 20 °C, a scan speed of 30 nm/min, bandwidth of 1 nm and time per point of 2 sec. Complexes were prepared using the di-chain lipid DOSEG3 and the equivalent tri-chain lipid TC-DOSEG3 formulated with either the targeting peptide A or non-targeted K16 peptide. All LPDs were prepared at 0.25:6.5:1 charge ratio and a final ctDNA concentration of 0.05 mg/mL. Spectra were obtained at 200-320 nm and 20 o C using a scan speed of 30 nm/min using a 0.5 cm path length cell.
Appropriate D2O background subtraction was performed for all spectra. The spectra of free ctDNA, lipid vesicles alone, and peptides alone were also obtained as a reference.  Figure S2. % transfection in B104 cells of LPDs prepared using the lipids DODEG4, TC-DODEG4, DOSEG3 and TC-DOSEG3 with peptide A and pDNA at increasing lipid ratios in the LPD complex from 0.5:4:1 to 3:4:1 weight ratios. The 0.25:6.5:1 charge ratio chosen for subsequent experiments is equivalent to 0.7-1:4:1 weight ratio, and therefore corresponds to the range in which highest transfection efficiency was observed. Data is the mean of three measurements ± standard deviation. Figure S3. % Protein content compared to blank (untreated cells) of LPD complexes at 0.25:6.5:1 charge ratio prepared either fully in OptiMEM (grey bars) or in 25% v/v water then diluted with OptiMEM (black bars) in B104 cells using the various di-and trichain group 1 and 2 cationic lipids, peptide A and gWiz plasmid. DOTMA and DOTAP LPDs were also tested with a scrambled version of peptide A (sc). Peptide A:pDNA (PD) complexes at 6.5:1 charge ratio in the absence of lipid were also tested together with lipofectamine 5:1 and L2K 4:1 weight ratios as controls. Data is the mean of three measurements ± standard deviation.