Apolipoprotein E Binding Drives Structural and Compositional Rearrangement of mRNA-Containing Lipid Nanoparticles - PubMed (original) (raw)

. 2021 Apr 27;15(4):6709-6722.

doi: 10.1021/acsnano.0c10064. Epub 2021 Mar 23.

Marianna Yanez Arteta 2, Michael Lerche 2, Lionel Porcar 3, Christian Lang 4, Ryan A Bragg 5, Charles S Elmore 6, Venkata R Krishnamurthy 7, Robert A Russell 8, Tamim Darwish 8, Harald Pichler 9 10, Sarah Waldie 1 11 12, Martine Moulin 11 12, Michael Haertlein 11 12, V Trevor Forsyth 11 12 13, Lennart Lindfors 2, Marité Cárdenas 1

Affiliations

Apolipoprotein E Binding Drives Structural and Compositional Rearrangement of mRNA-Containing Lipid Nanoparticles

Federica Sebastiani et al. ACS Nano. 2021.

Abstract

Emerging therapeutic treatments based on the production of proteins by delivering mRNA have become increasingly important in recent times. While lipid nanoparticles (LNPs) are approved vehicles for small interfering RNA delivery, there are still challenges to use this formulation for mRNA delivery. LNPs are typically a mixture of a cationic lipid, distearoylphosphatidylcholine (DSPC), cholesterol, and a PEG-lipid. The structural characterization of mRNA-containing LNPs (mRNA-LNPs) is crucial for a full understanding of the way in which they function, but this information alone is not enough to predict their fate upon entering the bloodstream. The biodistribution and cellular uptake of LNPs are affected by their surface composition as well as by the extracellular proteins present at the site of LNP administration, e.g., apolipoproteinE (ApoE). ApoE, being responsible for fat transport in the body, plays a key role in the LNP's plasma circulation time. In this work, we use small-angle neutron scattering, together with selective lipid, cholesterol, and solvent deuteration, to elucidate the structure of the LNP and the distribution of the lipid components in the absence and the presence of ApoE. While DSPC and cholesterol are found to be enriched at the surface of the LNPs in buffer, binding of ApoE induces a redistribution of the lipids at the shell and the core, which also impacts the LNP internal structure, causing release of mRNA. The rearrangement of LNP components upon ApoE incubation is discussed in terms of potential relevance to LNP endosomal escape.

Keywords: ApoE; lipid nanoparticles; mRNA delivery; protein corona; small-angle scattering.

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Conflict of interest statement

The authors declare the following competing financial interest(s): M.Y.A., M.L., C.S.E., and L.L. are employed by AstraZeneca R&D Gothenburg, R.A.B. is employed by AstraZeneca R&D Macclesfield, and V.R.K. was employed by AstraZeneca R&D Boston during the development of this work.

Figures

Figure 1

Figure 1

SANS data collected with sample MCH at four different contrasts (A), MMC at five different contrasts (B), and MCHPC at four different contrasts (C). The legends in panels A, B, and C describe the percentages of d-PBS present in the solvent. The black solid lines are the result of model fitting. The curves were shifted for clarity. The exact composition of the LNP formulations is given in Table 1. The peak due to internal structure is clearly visible for MCH and MCHPC (d-PBS > 60%). For MMC, however, a small deviation from the model around q = 0.1 Å–1 for all solvent contrasts is possibly due to internal structure, which was not included for this particular data set modeling. In panel D, the scattering intensity averaged over the q values 0.004–0.007 Å–1 is plotted against the percentage of D2O content in the buffer for the MMO sample, showing that this sample is invisible in solvents where proteins are also invisible to neutrons. Schematic drawing of the core–shell structure including the distribution of the components in the LNP; water is not represented in the schematics; the core has an average water volume fraction of 18 ± 5% (E). In the insets of panels A, B, and C the LNP schematics have the components colored according to their SLD values (i.e., deuterated components are black).

Figure 2

Figure 2

ApoE binding to LNP as measured by LNP immobilized particles on a QCM-D sensor. The raw frequency shift for overtone 5, 7, and 9 is reported as a function of ApoE concentration (A). The frequency shift has been offset by the equilibrium value obtained after LNP injection/rinsing. The overlap for all overtones suggests that the ApoE is a rigid film adsorbed on the LNP (no change in dissipation occurs). Note that a negative change in frequency is related to an increase in adsorbed wet mass. The number of ApoE molecules per LNP as a function of ApoE concentration is calculated assuming hexagonal (gray squares) and random (black circles) packing by using the Sauerbrey equation (B). Transition of free ApoE (PDB ID: 2l7b) into proposed hairpin-like configuration adapted to fit on an 80 nm diameter LNP (green). Domains bound to the LNP surface are shown in red with hydrophobic leucines and isoleucines shown as sticks (C). The illustration was prepared using the PyMOL Molecular Graphics System, version 2.0, Schrödinger, LLC.

Figure 3

Figure 3

SANS data collected for the LNPs prepared with a mixture of deuterated and hydrogenated components (MMO) that allows the LNPs to be matched out in a buffer with 46% D2O content and to enhance the structural effect of ApoE incubation for 3 h: solvent containing 46% d-PBS (black symbols), ApoE (red symbols), MMO with (light blue symbols) and without (blue symbols) ApoE (A). LNPs prepared with dMC3 (MMC) and measured at 46% d-PBS with (red symbols) and without (black symbols) ApoE (B). SANS data for LNP prepared with 100% d-cholesterol (MCH) measured in 39% d-PBS with (red symbols) and without (black symbols) ApoE (C). LNP prepared with 100% d-cholesterol and 32% dDSPC (MCHPC) measured in 39% d-PBS with (red symbols) and without (black symbols) ApoE (D). Schematics of how the particle composition changes upon apolipoprotein binding: cholesterol moves toward the surface while MC3 partitions to the core (E). Solid lines are best fits to the experimental data. The nominal LNP composition is provided in Table 1. In the insets of panels B, C, and D the LNP schematics have the components colored according to their SLD values (i.e., deuterated components are black).

Figure 4

Figure 4

LNP volume and composition in the presence (gray) and absence of ApoE (black). The histograms in the top row show the volumes of shell and core calculated from the radius and thickness obtained fitting the SANS data respectively for MCH (A), MCHPC (B), and MMC (C). In the middle row, the histograms show the volume fractions for the LNP components present in the core when solvent is excluded: MCH (A), MCHPC (B), and MMC (C). In the bottom row, the histograms show the volume fractions for the LNP components present in the shell: MCH (A), MCHPC (B), and MMC (C). LNP samples were prepared according to Table 1. Changes in DSPC, cholesterol, and MC3 composition are significant.

Figure 5

Figure 5

Stability of fully hydrogenated LNPs upon incubation with ApoE in terms of core structure measured by SAXS (A): SAXS patterns were measured at 1:1 wt % ApoE/mRNA at no ApoE (black), 0 h (blue), 3 h (red), and 15 h (gray) of incubation time. Size measured by DLS (B, top) and encapsulation efficiency (B, bottom). Increasing ApoE/mRNA weight ratios were used in B, and size and encapsulation efficiency were measured at day 0 (blue circles), 1 (red squares), and 3 (gray triangles) of incubation time. Error bars are almost always within the size of the symbols for SAXS, DLS, and encapsulation data.

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