On the Formation and Morphology of Lipid Nanoparticles Containing Ionizable Cationic Lipids and siRNA

Kulkarni, Jayesh (University of British Columbia)


In recent years the mantra surrounding gene therapy has been “delivery, delivery, delivery”,1-3 meaning that intracellular delivery of macromolecular RNA and DNA constructs into target cells was the primary impediment to practicing gene therapy in vivo. Viral vectors suffer from immunogenicity, manufacturing and other concerns, whereas non-viral vectors have toxicity and potency issues. However, the recent successful Phase III trial of a lipid nanoparticle (LNP) formulation of siRNA to treat transthyretin (TTR)-induced amyloidosis suggests that non-viral vectors are starting to overcome the delivery barrier.4 A key advance has been identification and  incorporation of an optimized ionizable cationic lipid in the LNP-siRNA systems. Examples of such lipids are heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate (DLin-MC3-DMA or MC3) and 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA or KC2).5-6 These lipids exhibit acid-dissociation constants (pKa) below 7 ensuring a near neutral surface charge in the circulation upon intravenous administration, yet a strong positive charge at acidic pH to allow entrapment of nucleic acid polymers. Lipids such as MC3 and KC2 have been optimized for in vivo gene silencing in hepatocytes following intravenous (i.v.) administration and exhibit pKa values in the range 6.2-6.7.5

Here, we re-examine the mechanism of LNP formation and the nature of the electron-dense structures formed using cryo-TEM and small angle X-ray approaches. We show that LNP-siRNA formed using ethanol dilution/rapid-mixing techniques display small multilamellar structure at high siRNA contents where the nucleic acid is trapped between closely apposed lipid bilayers. At lower (clinically relevant) siRNA contents, LNP siRNA systems exhibit a combination of siRNA-bilayer structure and an amorphous electron dense core, likely arising from an oil droplet consisting primarily of the neutral form of the ionizable cationic lipid.

Materials and Methods

Cryogenic transmission electron microscopy

LNPs were concentrated to a final concentration of 15-25 mg/mL of total lipid. 2-4 µL of LNP suspension was added to glow-discharged copper grids, and plunge-frozen using a FEI Mark IV Vitrobot (FEI, Hillsboro, OR) to generate vitreous ice. Grids were stored in liquid nitrogen until imaged.

For 200 kV imaging: Grids were moved into a Gatan 70° cryo-tilt transfer system pre-equilibrated to at least -180°C, and subsequently inserted into the microscope. An FEI LaB6 G2 TEM (FEI, Hillsboro, OR) operating at 200 keV under low-dose conditions was used to image all samples. A bottom-mount FEI Eagle 4K CCD camera was used to capture all images. All samples (unless otherwise stated) were imaged at a 55,000x magnification with a nominal under-focus of 1-2 µm to enhance contrast. Sample preparation and imaging was performed at the UBC Bioimaging Facility (Vancouver, BC).

For 300 kV imaging: Grids were transferred to an FEI Titan Krios (FEI, Hillsboro, OR) operating at 300 keV fitted with a Falcon III direct electron detector. All samples (unless otherwise stated) were imaged at a 47,000x magnification with a nominal under-focus of 1-2 µm to enhance contrast. Sample imaging was performed at the UBC Life Sciences Centre (Vancouver, BC).

Small Angle X-ray Scattering

Small angle X-Ray scattering (SAXS) experiments were conducted on the SAXSLAB Ganesha 300XL SAXS system at 4D Labs (SFU, Burnaby, BC). The sample to detector distance was adjustable across 1.4 m to allow measurements from q = 0.0025 Å-1 to q = 2.8 Å-1. The x-ray beam has a wavelength of 0.154 nm generated by a Cu-Ka X-ray source. Concentrated LNP suspensions were loaded into quartz capillary tubes purchased from Charles Supper Company (Natick, MA) which are approximately 80 mm long, 1.5 mm in diameter, and 0.01 mm thick. After transfer, the tubes were sealed using capillary wax. Samples were loaded into a temperature-controlled Linkam heater stage which maintained a constant temperature of 22.7°C throughout all experiments.


The results of this investigation demonstrate that at high siRNA contents (N/P = 1), the LNP siRNA systems formed at both pH 4 and 7.4 adopt a small multilamellar vesicle structure consisting of siRNA sandwiched between closely apposed concentric lipid bilayers. Conversely, LNP formed in the absence of siRNA at pH 7.4 exhibit an amorphous hydrophobic lipid core consistent with an oil-in-water dispersion. At intermediate siRNA contents corresponding to N/P values of 3 and 6 (which corresponds to formulations used clinically) where there is an excess of ionizable cationic lipid, siRNA remains sandwiched within bilayer lipid assemblies (as indicated by small angle X-ray studies) whereas the LNP core displays amorphous structure consistent with the presence of oil-phase lipid. For systems where there is only a slight excess of ionizable cationic lipid (N/P values of 1.1 and 1.5) outer regions of the LNP display concentric ring structure whereas the LNP centre displays amorphous structure. On the basis of these observations we propose a revised model of LNP-siRNA structure for therapeutically active formulations, where the bulk of the ionizable cationic lipid segregates into a central oil phase and stacked bilayers of lipid-siRNA aggregates are located towards the periphery of the LNP.

Discussion and Conclusion

The major finding of this investigation is that LNP-siRNA do not show evidence of inverted micellar structures but rather closely-apposed lipid layers sandwiching siRNA molecules. Excess ionizable cationic lipid appears to form an oil-droplet phase generating a lipid-containing core. These findings have major implications for the design of ionizable cationic lipids, design of nanoparticles for siRNA delivery to hepatic and extrahepatic targets, and finally, improving particle stability by understanding to role of each of the components.


1. Kaczmarek, J. C. et al. Advances in the delivery of RNA therapeutics: from concept to clinical reality. Genome Med 2017.

2. Cullis, P. R. et al. Lipid Nanoparticle Systems for Enabling Gene Therapies. Mol Ther 2017.

3. Elsabahy, M. et al. Non-viral nucleic acid delivery: key challenges and future directions. Curr Drug Deliv 2011.

4. Alnylam Pharmaceuticals, I., Alnylam and Sanofi Report Positive Topline Results from APOLLO Phase 3 Study of Patisiran in Hereditary ATTR (hATTR) Amyloidosis Patients with Polyneuropathy.

5. Jayaraman, M. et al.,  Maximizing the potency of siRNA lipid nanoparticles for hepatic gene silencing in vivo. Angewandte Chemie 2012.

6.Semple, S. C. et al., Rational design of cationic lipids for siRNA delivery. Nat Biotechnol 2010.

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