Highly efficient gene silencing activity of sirna embedded in a nanostructured gyroid cubic lipid matrix
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1 Highly efficient gene silencing activity of sirna embedded in a nanostructured gyroid cubic lipid matrix Cecília Leal *, Nathan F. Bouxsein, Kai K. Ewert and Cyrus R. Safinya * Physics, Materials, and Molecular, Cellular & Developmental Biology Departments, University of California, Santa Barbara, CA 93106, USA Correspondence and requests for materials should be addressed to C.L. (cecilal@mrl.ucsb.edu) or C.R.S. (safinya@mrl.ucsb.edu). Supplementary information S1
2 Figure S1: Chemical structure of the lipids used in this work. All lipids are commercially available. They bear similar, oleic acid tails to prevent lateral demixing of the membranes. Similar to conventional lipids, GM (also termed monoolein) has a very low solubility in water (10-6 M) but is able to swell in water giving rise to a variety of liquid crystalline mesophases 1. GM H H DTAP N + Cl - DPC P - N + Figure S2: In order to investigate the effect of the nucleic acid length on the stability of the cubic phase structure, we used the same lipid system (DTAP/GM) to prepare complexes with long DNA (highly polymerized calf thymus (HPCT) DNA, USB). For DTAP/GM HPCT DNA complexes, no cubic phase was observed at any Φ GM. (a) Synchrotron SAXS scans of DTAP/GM DNA complexes at varied Φ GM, displaying the inverted hexagonal (H C II ) and the lamellar (L C α ) phase. In the lamellar phase (L C α ), the characteristic correlation between DNA molecules gives rise to a broad peak at q DNA that shifts to lower q (larger interhelical differences) as the content of neutral lipid increases. The lamellar phase is obtained up to Φ GM =0.70. At this point, coexistence with the inverse hexagonal phase (H C II ) is observed (Φ GM = 0.70 and 0.75). At higher Φ GM, the H C II phase is found exclusively. At no Φ GM did we find evidence for a cubic phase of CL DNA complexes. Generally, gyroid cubic phases have a lattice spacing of the order of 100 Å 2. The sirna used in this study (19 bp with 2nt 3 overhangs) is a short, rod-like molecule with a length ( 52 Å) much shorter than its persistence length (between 500 Å and 1000 Å), and an S2
3 aspect ratio of about two. Thus, its inclusion in a bicontinuous lipid matrix can be performed without disruption of the cubic structure. In contrast, the average length of HPCT DNA molecules is many times their persistence length of 500 Å to 1000 Å and the energetic cost associated with bending the semi-flexible DNA to incorporate it within the curved water channels of the cubic phase appears to inhibit formation of this phase. Instead, the 2D inverse hexagonal phase, which requires minimal DNA bending, is retained even at very high Φ GM. (b) Unit cell spacing (d and a H ) obtained for the DTAP/GM DNA complexes at ρ = 1 in ptimem buffer. The lattice spacings obtained for the DTAP/GM DNA complex phases are a few Angstroms smaller than those obtained for the corresponding sirna complexes. At Φ GM = 0.50, the lamellar phase spacing d = 2π/q 001 equals Å for DNA complexes and Å for sirna complexes, with the difference closely matching the difference in diameter (sirna = 26 Å and DNA = 20 Å) of the nucleic acids. S3
4 Figure S3: Unit cell dimensions (a, a H, and d, for the cubic, inverse hexagonal, and lamellar phases, respectively) obtained for DTAP/GM-siRNA complexes at charge ratios ρ = 1 (blue triangle), ρ = 0.5 (red square), ρ = 2 (black circle) in two different aqueous environments (low conductivity water (left) and ptimem cell culture medium (right)). The amount of sirna was kept constant in all samples, while the amount of lipid was varied to achieve the desired charge ratio. There is no significant difference between the spacings obtained for the different aqueous environments. In addition, the various phases display essentially the same lattice spacings for all charge ratios. G, However, the phase transition from the cubic phase (Q sirna II ) to the inverse hexagonal phase (H sirna II ) occurs at higher molar fractions of GM for negatively charged complexes (ρ = 0.5) than for electroneutral (ρ = 1) and positively charged complexes (ρ = 2). In other words, the cubic phase is stabilized for a narrower range of Φ GM (Φ GM 0.80 at ρ = 0.5 vs. Φ GM 0.75 at ρ = 1 and ρ = 2 in ptimem). This fact is even more evident in water, where the cubic phase is only stabilized for Φ GM 0.85 at ρ = 0.5. The cell culture medium ptimem contains salts, and the resulting electrostatic screening seems to allow the cubic phase to form for a wider range of Φ GM. It appears that the cubic phase is less able than the inverted hexagonal phase to accommodate a lipid/sirna charge mismatch in the regime of excess sirna (ρ = 0.5). S4
5 Table S1 Small-angle X-ray results for experiments presented in Figure 2 q (1/Å) b a (Å) e hkl a h + k + l LIPIDS c Lipid-siRNA d LIPIDS c Lipid-siRNA d not observed not observed / Lipid-siRNA d. Dilute regime (Fig. 2e) a hkl are the lattice indices, b q is the X-ray wave vector, c LIPIDS refers to DTAP/GM (Φ GM =0.85) with 30 wt% water, d Lipid-siRNA is the DTAP/GM sirna (Φ GM =0.85) complex, and e a is the lattice spacing of the Ia3d cubic phase given by a = π h + k + l q References 1. Larsson, K. Lipids-molecular organization, physical functions and technical applications. (The oily press Ltd, Dundee; 1994). 2. Larsson, K. Two cubic phases in monoolein-water system. Nature 304, (1983). 3. Complete author list for reference 9 in the manuscript: Hacein-Bey-Abina, S.; Garrigue, A.; Wang, G. P.; Soulier, J.; Lim, A.; Morillon, E.; Clappier, E.; Caccavelli, L.; Delabesse, E.; Beldjord, K.; Asnafi, V.; Macintyre, E.; Dal Cortivo, L.; Radford, I.; Brousse, N.; Sigaux, F.; Moshous, D.; Hauer, J.; Borkhardt, A.; Belohradsky, B. H.; Wintergerst, U.; Velez, M. C.; Leiva, L.; Sorensen, R.; Wulffraat, N.; Blanche, S.; Bushman, F. D.; Fischer, A.; Cavazzana-Calvo, M. S5
6 S6
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