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Biological and Medical Applications of Materials and Interfaces

Transfection Potency of Lipid Nanoparticles Containing mRNA Depends on Relative Loading Levels
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  • Suiyang Liao
    Suiyang Liao
    Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada
    Michael Smith Laboratories, University of British Columbia, Vancouver, British Columbia V6T 1Z4, Canada
    School of Biomedical Engineering, University of British Columbia, Vancouver, British Columbia V6T 2B9, Canada
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  • Shuangyu Wang
    Shuangyu Wang
    Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada
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  • Abishek Wadhwa
    Abishek Wadhwa
    Department of Microbiology and Immunology, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada
    Life Sciences Institute, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada
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  • Alexandra Birkenshaw
    Alexandra Birkenshaw
    Faculty of Pharmaceutical Sciences, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada
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  • Kevin Fox
    Kevin Fox
    Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada
    Faculty of Pharmaceutical Sciences, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada
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  • Miffy Hok Yan Cheng
    Miffy Hok Yan Cheng
    Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada
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    Orcidhttps://orcid.org/0000-0002-0261-4642
  • Irafasha C. Casmil
    Irafasha C. Casmil
    Michael Smith Laboratories, University of British Columbia, Vancouver, British Columbia V6T 1Z4, Canada
    School of Biomedical Engineering, University of British Columbia, Vancouver, British Columbia V6T 2B9, Canada
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    Orcidhttps://orcid.org/0000-0003-1890-7930
  • Armando Alcazar Magana
    Armando Alcazar Magana
    Michael Smith Laboratories, University of British Columbia, Vancouver, British Columbia V6T 1Z4, Canada
    Life Sciences Institute, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada
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  • Nuthan Vikas Bathula
    Nuthan Vikas Bathula
    Michael Smith Laboratories, University of British Columbia, Vancouver, British Columbia V6T 1Z4, Canada
    School of Biomedical Engineering, University of British Columbia, Vancouver, British Columbia V6T 2B9, Canada
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  • Chia Hao Ho
    Chia Hao Ho
    Michael Smith Laboratories, University of British Columbia, Vancouver, British Columbia V6T 1Z4, Canada
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    Orcidhttps://orcid.org/0000-0003-3670-2680
  • Jin-Yu Cheng
    Jin-Yu Cheng
    Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada
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  • Leonard J. Foster
    Leonard J. Foster
    Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada
    Michael Smith Laboratories, University of British Columbia, Vancouver, British Columbia V6T 1Z4, Canada
    Life Sciences Institute, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada
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    Orcidhttps://orcid.org/0000-0001-8551-4817
  • Kenneth W. Harder
    Kenneth W. Harder
    Department of Microbiology and Immunology, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada
    Life Sciences Institute, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada
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  • Colin J. D. Ross
    Colin J. D. Ross
    Faculty of Pharmaceutical Sciences, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada
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  • Pieter R. Cullis*
    Pieter R. Cullis
    Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada
    *Email: [email protected]
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    Orcidhttps://orcid.org/0000-0001-9586-2508
  • Anna K. Blakney*
    Anna K. Blakney
    Michael Smith Laboratories, University of British Columbia, Vancouver, British Columbia V6T 1Z4, Canada
    School of Biomedical Engineering, University of British Columbia, Vancouver, British Columbia V6T 2B9, Canada
    *Email: [email protected]
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    Orcidhttps://orcid.org/0000-0002-5812-9689
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ACS Applied Materials & Interfaces

Cite this: ACS Appl. Mater. Interfaces 2025, 17, 2, 3097–3105
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https://doi.org/10.1021/acsami.4c20077
Published December 31, 2024

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Abstract

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When formulating mRNA into lipid nanoparticles (LNP), various copy numbers of mRNA are encapsulated, leading to a distribution of mRNA loading levels within the LNPs. It is unclear whether the mRNA loading level affects the functional delivery of the message. Here we show that depending on the mRNA loading level, LNPs exhibit distinct mass densities and can be fractionated via ultracentrifugation. Upon fractionation, we investigated if mRNA loading levels influence LNP sizing, lipid composition, and morphology. We further conducted in vitro and in vivo functional delivery of mRNA and found that the LNP fraction with the highest mRNA loading levels was the least transfection competent.

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Introduction

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Ribonucleic acid (RNA) nanomedicine has been clinically proven in recent years with the approval of small interfering RNA (siRNA) therapies such as Onpattro, (1) which treats transthyretin-induced amyloidosis (hATTR) and the Pfizer/BioNTech (2) and Moderna (3) mRNA vaccines against COVID-19, and most recently, the Moderna (4) mRNA vaccine against Respiratory Syncytial Virus. All four of these medicines use lipid nanoparticle (LNP) formulations to deliver the RNA. However, there are missing links between LNP structure and performance in RNA functional delivery, including how the mRNA copy number in LNPs impacts translation into the intended proteins.
The formulation of lipid nanoparticles is driven by the fusion of nanosized vesicles (5) in a random fashion. As a result, in the same batch of lipid nanoparticles, one LNP could contain n copies of mRNA with n = 0, 1, 2, 3, 4, etc. (6,7) As the final size of LNPs is largely dictated by the surface concentration of hydrophilic poly(ethylene glycol) (PEG) lipids to terminate the fusion induced by the conversion of ionizable lipids to the neutral state, the size of the LNP does not necessarily reflect the number of mRNA copies it contains. Thus, separation based on size is unlikely to reflect the mRNA loading level.
Alternatively, nucleotides and lipids possess distinct mass/buoyant densities. Single-stranded RNA has a density of ∼1.6 g/cm3 (measured in potassium iodine gradient (8) and free mRNA was found to sediment during centrifugation (9)). The lipid species generally have a mass density in the range of 0.8–1.1 g/cm3, depending on their chemical composition and molecular configuration. The empty LNPs (eLNPs) formed using the lipid compositions applied in Onpattro/Comirnaty/Spikevax were found to be lighter than the aqueous buffer (see below the literature update on using analytical ultracentrifugation (AUC) to measure LNPs). Thus, the mRNA–LNP formulation’s mass density depends on the mRNA loading level. The LNPs with low mRNA loading will likely be less dense than the aqueous buffer (e.g., phosphate-buffered saline (PBS) buffer has a density of ∼1.013 g/cm3 at 25 °C (10)), while the highly loaded LNPs could be heavier. The density contrast between LNPs with various mRNA loading levels and their aqueous environment thus provides the basis for a centrifugation-based fractionation method.
A previous study used a centrifugation-based technique, called analytical ultracentrifugation (AUC) (11) to analyze LNPs. Henrickson et al. (11) measured the nucleotide drug (siRNA) loading in LNPs with density-matching AUC. In the experiment, they tuned the density of the aqueous buffer by varying the fraction of heavy water and then measured the sedimentation coefficient of the LNP samples. They found that under certain buffer conditions, empty LNPs (eLNPs) float, LNPs with an N/P ratio of 6 (N/P ratio is the ratio between the positively charged nitrogen groups from the ionizable lipids and the negatively charged phosphate group from RNA) also float, while LNPs formulated at an N/P ratio of 1, thus higher RNA loading, sediment. Similar approaches were applied by Bepperling et al. (12) to estimate an average loading of five copies of mRNA in one LNP particle. Previously, our group (Leung et al. (13)) applied sucrose density gradient centrifugation to separate LNPs containing siRNA. Vaidya et al. (14) demonstrated the utilization of the same technique (sucrose gradient) to separate LNPs based on their mRNA loading level. Recently, Zhao et al. (15) have performed sedimentation velocity AUC on Pfizer/BioNTech mRNA LNPs and found the majority float in a centrifugal field, in agreement with the work from Calzolai (9) and co-workers.
Here we show the feasibility of fractionating LNPs with distinct mRNA loading levels by centrifugation in the original storage buffer. We use DLin-KC2-DMA (KC2) (16) as the ionizable lipid to formulate the model LNPs at a benchmark composition of KC2/distearoylphosphatidylcholine (DSPC)/cholesterol/PEG-DMG (50/10/38.5/1.5 mol %). Upon separation, we characterized the morphology of LNPs subpopulations with cryoEM. We further investigated the in vitro and in vivo functional delivery by fractionated LNP subpopulations.

Results and Discussion

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Centrifugal Fractionation of LNPs and the Integrity of LNP/mRNA

Under centrifugation, nanoparticles experience three major forces: centrifugal force, frictional force, and buoyant force. Balancing the three forces yields the so-called Svedberg equation, (17) describing nanoparticles’ terminal velocity under a unit centrifugal field. For lipid nanoparticles from the same batch, we assume the constant size and shape (thus, constant volume vLNP). We transform the Svedberg equation to show that the particle moving direction (or the sign of the sedimentation coefficient) depends on the density contrast between the particle and the buffer (for complete mathematical analysis, see the Supporting Information).
sLNPasLNPb≈vLnPρLNPa−ρsolρLNPb−ρsol
(1)
LNPs’ terminal velocity (magnitude and direction) in a centrifugal field largely depends on their mass density. (s: Svedberg coefficient; vLNP: volume of LNPa/LNPb; ρLNPa, ρLNPb, ρsol: densities of LNPa, LNPb, and solvent, respectively.)
As shown in eq 1, for LNPa and LNPb, when one is heavier, while the other is lighter than the solvent, their moving direction diverges on centrifugation.
In brief, we rapidly mixed firefly luciferase mRNA (mFluc) and the lipid cocktail through a T junction at an N/P ratio of 6. The mixture was dialyzed overnight in PBS buffer to produce the final form of LNPs. We then centrifuged the LNPs at ∼300 000 RCF for 2 h. Upon terminating the ultracentrifugation, we collected upper one-third and lower two-thirds by volume from the centrifuge tube and further refer the subpopulations as low-density LNPs (LDLNPs) and high-density LNPs (HDLNPs), respectively. The original batch is referred to as OLNP hereafter. Figure S1 depicts LNPs (labeled with DiD, thus appearing blue to provide visual inspection) before and after centrifugation. The workflow of the fractionation process is demonstrated schematically in Figure 1A. We validated the structural integrity of LNPs upon ultracentrifugation via DLS (Figure 1B, size distribution by intensity) and the RiboGreen assay to show that the subpopulations are within the size range of the original batch and no release of mRNA with encapsulation efficiency over 98% (Figure S2). As the RiboGreen assay does not characterize the integrity of mRNA, we evaluated the intactness of payload mRNA via capillary electrophoresis. No indication of mRNA degradation was found (Figure 1C). The percentage integrities of encapsulated mRNAs were found to be 91.7, 94.4, and 91.0% for OLNP, LDLNP, and HDLNP, respectively.

Figure 1

Figure 1. Centrifugal fractionation of LNPs based on their mRNA loading levels. (A) Schematic illustration of LNP fractionation via ultracentrifugation. Depending on the LNPs’ density contrast with the aqueous system, two fractions (LDLNP and HDLNP) are generated from the fractionation process. Together with the original batch, three samples are compared throughout this paper; (B) particle size distribution by intensity measured with DLS; payload mRNA integrity by Bioanalyzer shown as electropherograms in (C1) and bands in (C2).

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Characterization of Lipid Composition and N/P Ratio

To further characterize the properties of the fractionated LNPs, we quantified the cholesterol and mRNA concentration, respectively. The bar chart in Figure 2A uses the ratio of mRNA/lipid to characterize the mass of mRNA formulated in the subpopulation of LNPs containing unit mass of total lipids. The higher the ratio of mRNA/lipid, the higher the mRNA loading in that fraction.

Figure 2

Figure 2. Validation of centrifugal fractionation, characterization of lipid composition, and calculation of N/P ratio. (A) Bar chart of RNA/total lipid ratio to represent the mRNA loading levels and validate the fractionation method; (B) pie charts showing lipid composition of OLNP, LDLNP, and HDLNP as characterized by ultrahigh-performance liquid chromatography–mass spectrometry (UHPLC-MS).

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The encapsulation of mRNA into LNP relies on self-assembly (18) events triggered primarily by the electrostatic attraction between nucleic acids and the ionizable lipids. We question whether the variation of mRNA loading could lead to significant variation in lipid compositions between fractions with different densities. To determine this, we performed UHPLC-MS; however, no significant difference between the original batch and the subpopulations (Figure 2B) was observed. This agrees with previous studies characterizing LNP lipid composition using SANS, (19) where it is shown that using different N/P ratios during formulation does not alter the final lipid composition. Combining the lipid composition and mRNA/lipid ratio, the actual N/P ratios are calculated to be 2.9, 7.5, and 0.5, for OLNP, LDLNP, and HDLNP, respectively.

LNP Morphology Depends on mRNA Loading Level

Currently, we lack an understanding of how the loading levels of mRNA influence the morphology of LNPs. (20) Previous work has suggested that the mRNA-loaded LNPs at neutral pH are coated with phospholipid mono- (21,22) or bilayers (23) with mRNA encapsulated within polar environments surrounded primarily by helper lipids that can form monolayers and bilayers such as DSPC and cholesterol. (24) This is consistent with the fact that the presence of helper lipids is essential for encapsulation. (1) The neutral form of the ionizable lipid has limited solubility in monolayer or bilayer lipid structures, as evidenced by phase separation into the hydrophobic “oil droplet” electron-dense core. The polar environments can frequently be visualized as “bleb” structures, (25−27) local aqueous compartments within the LNPs. It would be expected that the presence of larger numbers of mRNA within an LNP would lead to larger or more bleb structures. We therefore examined the LNP subpopulations using cryoEM (Figure 3A) and analyzed the images to investigate how LNPs’ morphologies (size and anisotropy) depend on mRNA loading level.

Figure 3

Figure 3. mRNA loading levels can influence the distribution of LNP morphology. (A1–A3) Representative cryoEM images of OLNP, LDLNP, and HDLNP (scale bar: 100 nm); (B) pie charts showing the composition of LNPs with various number of blebs; (C) schematic illustration of measuring LNPs and blebs with FIJI; (D1–D3) scatter plots mapping the dimension of LNPs and blebs (purple dots: LNPs without blebs; green dots: LNPs with blebs) (E) raincloud plot showing the size distribution of blebs.

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In Figure 3A1, we show that the as-prepared LNPs are composed of spherical LNPs without blebs and nonspherical LNPs due to the formation of blebs. (26,27) LDLNPs, with relatively lower mRNA loading, the majority (∼70%) of LNPs is found without blebs, while the rest exhibit single small blebs; the particles from HDLNPs, when possessing blebs, however, exhibit larger blebs and occasionally multiple blebs docking on the same particle (Figure 3A2,A3). The composition of LNPs with various numbers of blebs is visualized as pie charts in Figure 3B.
To characterize the size and anisotropy of LNPs and the size of blebs, we measured the LNPs from the cryoEM images with FIJI. To reflect the anisotropy of LNPs, we measured both the polar diameter (the longest distance within the LNPs; denoted as X) and the equatorial diameter (denoted as Y) of LNPs. To reflect the size of the blebs, we measured the width of the lipid–water interface (denoted as X) and the height of the blebs (denoted as Y). The measurement scheme is illustrated in Figure 3C.
In Figure 3D, we mapped the polar diameter X and the equatorial diameter Y of LNPs as scatter plots to display the absolute size of LNPs (the edge color of the marker is consistent with the color scheme used for OLNP/LDLNP/HDLNP while the face color choice reflects the number of blebs, purple for particles without blebs, green in various shades for particles with blebs, in consistency with Figure 3B). The slope of the linear trend line by fitting X and Y of LNPs serves as an indicator of the anisotropy of LNPs. Dimensions in X and Y were also projected as histograms to indicate the size distribution of LNPs. First, we point out the correlation between the percentage of particles with blebs and the mRNA loading level (50, 27, and 51% for OLNP, LDLNP, and HDLNP, respectively). Second, we note that particles without blebs are anisotropic (isotropy being 0.98, 1.00, and 1.02) and share very similar sizes (52.1, 55.1, and 49.1 nm) in all three types of LNPs; for particles with blebs, the size (69.6, 68.7, and 74.6 nm) and isotropy (1.19, 1.08, and 1.23) depend on mRNA loading levels. (The overall size distribution as measured by cryoEM is visualized in Figure S3.) Third, we found that the mRNA loading level also influences the size distribution of bleb size. The average sizes of blebs from OLNP/LDLNP/HDLNP are 37.1, 33.8, and 42.0 nm (bleb size distribution shown in Figure 3E). Finally, the observation of multibleb LNPs motivated us to calculate the average number of blebs on the same LNP and found the value to be 1.23, 1.11, and 1.48 for OLNP, LDLNP, and HDLNP, respectively.
Taken together, we conclude that the mRNA loading level can influence the morphology of the particles with blebs but not the particles without blebs. Higher loading level leads to larger LNPs with higher anisotropy, larger blebs, and/or more multibleb LNPs. The statistics from cryoEM, together with the effective N/P ratio, are summarized in Table 1 to present the reader with a quantitative viewpoint. More representative cryoEM images can be found in Figure S4.
Table 1. Quantification of LNP Morphologies Based on CryoEM Images
  OLNPLDLNPHDLNP
composition (LNP with vs without blebs)50:5073:2749:51
particles without blebssize/nm52.155.149.1
isotropy0.981.001.02
particles with blebssize/nm69.668.774.6
isotropy1.191.081.23
bleb size/nm37.133.842.0
average number of blebs per particle (with blebs)1.231.111.48
 N/P ratio2.97.50.5

In Vitro mRNA Delivery with OLNP, LDLNP, and HDLNP

We studied the cellular interactions of OLNP/LDLNP/HDLNPs using mRNA encoding enhanced green fluorescence protein with covalently conjugated cyanine 5 (Cy5-mEGFP) as a reporter gene. We incubated the Cy5-mEGFP-loaded OLNP/LDLNP/HDLNP with HEK293 cells and inspected both Cy5 and EGFP signals with confocal laser scanning microscopy (CLSM). As the dose of Cy5-mEGFP is controlled, the Cy5 signal reports the cellular uptake of mRNA/LNPs while the EGFP signal reports the actual expression, namely, the functional delivery of the mRNA payload.
In Figure 4A, we show that while the level of cellular uptake of mRNA (indicated by the Cy5 channel) is comparable across three types of LNPs, the level of mEGFP expression varies. OLNP and LDLNPs express significantly more green fluorescence protein (GFP) than HDLNP. Semiquantification was further performed by line analysis of intensity with FIJI and plotted on top of the GFP and Cy5 channels. This result was reproduced with Huh7 cells (Figure S5). We also segmented each cell and quantified the mean fluorescent intensities, as shown in Figure S6. We hypothesize that this disparity between cell uptake and functional delivery can be attributed to particles’ ability to escape from the endolysosomal systems. Thus, we studied the colocalization between LNP and late endosome. We labeled the late endosome with Rab7a fused with RFP before incubating with DiD-labeled LNPs. The visual inspection from Figure 4B indicates a significantly higher degree of colocalization between RFP/Rab7a and DiD/LNP in the case of HDLNPs, in comparison to OLNP/LDLNP. The colocalization analysis was performed, and we found that the Pearson’s coefficient for HDLNP is ∼0.75, while the values are around 0.35 for either OLNP or LDLNP (Figure S7).

Figure 4

Figure 4. Cellular uptake and functional delivery of mRNA with OLNP, LDLNP, and HDLNP. (A) CLSM images of OLNP, LDLNP, and HDLNP ((A1), (A2), and (A3), respectively) delivering Cy5-labeled mEGFP into HEK293 cells (scale bar: 50 μm); (B1–B3) CLSM study on the colocalization between LNP colocalization and Rab7a, representing late endosomes (scale bar: 50 μm).

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In Vivo mRNA Delivery with OLNP, LDLNP, and HDLNP

As in vitro results have been repeatedly shown to be poorly predictive of in vivo performance, (28,29) we decided to test OLNP, LDLNP, and HDLNP in mice. We are trying to address two key research questions. Does the mRNA loading level in LNPs influence how well (potency) and where (biodistribution) the mRNA payloads are being translated in vivo? We applied albino B6 (B6(Cg)-Tyrc-2J/J) mice as the mouse model for our animal work as the original C57BL/6J strain has been widely used for nanomedicine study, while the albino substrain offers higher signal-noise ratio when performing bioluminescence experiments. In brief, for each LNP formulation, we injected albino B6 mice (n = 4) through their tail vein at the dose of 0.3 mg mRNA/kg of mouse weight. Three hours post-injection, the mice were dosed with d-luciferin through intraperitoneal route and then anesthetized for in vivo imaging. Two major organs that are relevant to “Onpattro-like” LNP formulations, liver and spleen, were harvested after euthanizing the mice for ex vivo imaging.
We found, in accordance with the in vitro assay, that HDLNPs are the least potent formulation in the functional delivery of mRNA in vivo. The expression of the mFluc payload is 20-fold less by HDLNPs than those encapsulated in OLNP/LDLNP (Figure 5B). The same trend is maintained for liver and spleen luminescence (Figure 5C). For a visual presentation of liver–spleen tropism, we project the liver radiance against spleen radiance by making a scatter plot in Figure 5D. No significant alteration of tissue distribution can be observed. While the HDLNPs express 20-fold less than OLNP/LDLNP, the bioluminescence signal is still 1000-fold above the saline control. The LDLNPs produce ∼50% more luminescence than OLNPs. The trend is likely due to the absence of the less potent HDLNP fraction in LDLNP from OLNP but could also be attributed to the enrichment of empty LNPs, whose role in mRNA therapeutic delivery is largely unknown.

Figure 5

Figure 5. In vivo functional delivery of mFluc. (A) Schematic illustration of the animal study; (B) in vivo bioluminescence images of mice at 3 h after IV injection at mFluc dosage of 0.3 mg/kg (n = 4) and bar chart showing whole-body bioluminescence quantification from IVIS imaging. Data are presented as the mean ± standard deviation (SD) (n = 4). (C) Representative ex vivo bioluminescence images of extracted livers and spleens from mice dosed with OLNP/LDLNP/HDLNP (top to bottom) and bar chart showing bioluminescence of extracted livers and spleens. Data are presented as the mean ± SD (n = 4). (D) Scatter plot by projecting ex vivo liver radiance readout against spleen readout to indicate tissue tropism.

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Conclusions

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We have shown that ultracentrifugation is an effective technique to fractionate LNPs based on their mRNA loading level without disturbing the integrity of neither LNP nor mRNA. The mRNA loading level has an influence on LNPs’ overall morphology. As shown above by the cryoEM images, the LNP fraction with a relatively lower mRNA loading level displays smaller and fewer blebs, while the LNP fraction with high mRNA loading exhibits larger and multibleb constructs. Thus, taken together, the distribution of the mRNA loading level within the same batch of LNPs translates to a distribution of N/P ratios. Although intuitive thought leads one to prefer the LNP fraction with high mRNA loading, our in vitro and in vivo experiments revealed that they are the least potent population in the formulation, likely due to their deviation from the optimal N/P ratio. Moreover, we have shown that the mRNA loading level mainly alters LNPs’ potency but not the biodistribution of the proteins expressed by the mRNA payload.
However, we must point out several weaknesses and unanswered questions of this work. First, this centrifugal fractionation has low resolution in separating, as it is solely based on the density contrast of LNPs against the aqueous buffer. The low resolution is mainly due to the requirement of density contrast with the aqueous buffer and also partly due to nanoparticles’ fast diffusion. One could add more resolution to such separation by building a sucrose gradient as recently shown. (14) However, the added viscosity requires a higher centrifugal field and/or extended centrifugation time, not to mention the possible unwanted sucrose-lipid interactions. These factors might alter the identities of LNPs. Furthermore, what is the role of empty LNPs? The fractionation process presented herein could not remove eLNPs from either of the subpopulations. Thus, the role of empty LNPs in the functional delivery of mRNA therapeutics is still unclear. We hope to share our insights into the role of eLNPs in another work in the future. Finally, although we have shown (Figure 1) that the fractionation does not damage neither LNP nor mRNA, this does not necessarily indicate no alteration on the molecular level. Does the mechanical stress, especially the centrifugal force and the frictional force, alter the LNPs by rearranging lipids or a phase redistribution? This would require a meticulous study using small-angle X-ray scattering and small-angle neutron scattering (with selectively deuterated lipids).
To conclude this work, we point out several promising directions to optimize LNP formulations. Since the mRNA loading level could impact LNPs’ potency, there is substantial interest in developing better fractionation with higher granularity and understanding if there is and what is the optimal copy number of mRNA (or N/P ratio) inside one LNP. With the understanding of the ideal N/P ratio, we should design LNP formulating strategies to encapsulate mRNA in the most optimal subpopulation of the intrinsically heterogeneous LNPs. Most ideally, one should aim to develop formulation strategies to generate homogeneous LNPs with the optimal mRNA loading level.

Methods

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Materials

The lipids DSPC and PEG-DMG-2000 were purchased from Avanti Polar Lipids. The ionizable lipids DLin-KC2-DMA were purchased from Dr. Marco Ciufolini. Cholesterol was purchased from Sigma-Aldrich. Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were purchased from Thermo Fisher Scientific. All other chemicals were purchased from Sigma-Aldrich unless otherwise stated. Firefly luciferase mRNA (mFluc) was purchased from NanoVation Therapeutics, Inc. by S.L. and A.K.B. Cy5-mEGFP was provided by Dr. Andrew J. Varley (RNA and Formulation Core of UBC).

LNP Formulation and Characterization

All LNPs were formulated by first dissolving lipids (ionizable lipid, DSPC, cholesterol, PEG-DMG-2000) into ethanol to a final concentration of 10 mM and at a molar ratio of 50/10/38.5/1.5%, respectively. Then, the lipid–ethanol mixture was rapidly mixed with mRNA in aqueous buffer (25 mM sodium acetate at pH 4) at an amine-to-phosphate ratio of 6. The rapid mixing was achieved using a T junction at a 1:3 ethanol/water volume ratio and a total flow rate of 20 mL/min. The resulting mixture was dialyzed overnight against >500-fold volume of PBS at pH 7.4. Upon dialysis, the mixture was then sterile filtered through Supor membrane syringe filter (Pall Corporation, Mississauga, ON, Canada) at the size cutoff of 0.2 μm and then concentrated via ultrafiltration using Amicon ultracentrifugation units with 100k molecular weight cutoff (EMD Millipore Corporation, Billerica, MA). Total lipid content was determined by extrapolation using the Cholesterol E Kit (Wako Diagnostics, Mountain View, CA). Particle size was measured via dynamic light scattering using the Malvern Zetasizer Nano ZS.

Bioanalyzer

mRNA extracted from LNPs was prepared according to the Agilent RNA 6000 Nano kit guide and run on the Agilent Bioanalyzer 21000 using the mRNA Nano assay protocol. Percentage of intact mRNA was determined by obtaining the area under the curve of the mFluc peak on the electrogram.

Cryo-TEM

The freshly prepared LNPs were concentrated to ∼20 mg/mL of total lipid concentration, added to glow-discharged copper grids (3–5 μL), and plunge-frozen using an FEI Mark IV Vitrobot (FEI, Hillsboro) to generate vitreous ice. Grids were moved into a Gatan 70° cryotilt transfer system preequilibrated to at least −180 °C and then inserted into the microscope. An FEI LaB6 G2 TEM (FEI, Hillsboro, OR) operating at 200 kV under low-dose conditions was used to image all samples with an FEI Eagle 4k CCD camera. All samples were imaged at 55 000× magnification with a nominal underfocus of 1–2 μm to enhance contrast. All sample preparation and imaging were performed at the UBC Bioimaging Facility (Vancouver, BC).

Liquid Chromatography–Mass Spectrometry (LC-MS) Analysis

The LNP formulation was further diluted to a 10 μM concentration in a solution of 70% acetonitrile and 30% isopropyl alcohol before LC-MS analysis. The analysis was conducted using an Impact II high-resolution spectrometer (Bruker Daltonics, Bremen, Germany) coupled with an Elute UHPLC system (Bruker Daltonics). Compounds were separated using an Acquity CSH C18 column (130 Å, 1.7 μm, 100 × 2.1 mm2) with a CSH C18 VanGuard FIT Cartridge (1.7 μm, 2.1 × 5 mm2) (Waters, Milford, MA) and a multigradient method. The analysis utilized mobile phase A (acetonitrile/water 60:40 v/v) and mobile phase B (isopropanol/acetonitrile 90:10 v/v), both containing 0.1% formic acid and 10 mM ammonium formate. The gradient profile was as follows: 0 min, 15% B; 0–2 min, 30% B; 2–2.5 min, 50% B; 2.5–12 min, 80% B; 12–12.5 min, 99% B; 12.5–13.5 min, 99% B; 13.5–13.7 min, 15% B; 13.7–17 min, 15% B. The column temperature was set at 65 °C, flow rate at 0.5 mL/min, injection volume at 1 μL, and autosampler at 4 °C. Data-dependent acquisitions were acquired in positive (ESI+) ionization mode. The settings were as follows: capillary voltage, 4500 V; nebulizer gas pressure, 2.0 bar; dry gas flow, 9 L/min; dry gas temperature, 220 °C; mass scan range, 100–1700 m/z; spectra acquisition rate, 3 Hz; and cycle time, 0.7 s.

Cell Culture and Treatments

A human hepatocarcinoma cell line (Huh7) was cultured and maintained in DMEM media supplemented with 10% FBS, at 37 °C under 5% CO2. For transfection with LNPs, ∼10 000 cells per well were seeded into 96-well microplates for 24 h before treating with the LNP containing mFluc at mRNA dosage from 0 to 0.3 μg/mL. Upon 24 h of incubation, the medium was removed, and cells were lysed with 100 μL of Glo Lysis buffer (Promega, Madison, WI). The lysates were mixed at a 1:1 ratio with luciferase substrate (Promega, Madison, WI) and luminescence was quantified using a Spark Multimode Microplate reader (Tecan, Zürich, Switzerland). Luminescence readings were then normalized to the total protein content per well and determined using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, Rockford, IL), and transfection was calculated by normalizing to the luminescence values for untreated cells.

Animal Studies

All animal protocols were approved by the Canadian Animal Care Committee and conducted in accordance with the relevant UBC guidelines and regulations. Mice were maintained on a regular 12 h light/12 h dark cycle in a modified barrier animal facility at UBC. C57BL/6J-TyrC2J mice aged between 8 and 12 weeks were used throughout. The mice were individually weighed and imaged prior to tail vein intravenous (i.v.) injection of LNP containing mFluc at an mRNA dose of 0.3 mg/kg of mouse weight. At 3 h post-injection, mice were administered d-luciferin substrate solution (150 mg/kg of mouse weight) intraperitoneally for an incubation period of 10 min before in vivo bioluminescence imaging. Mice belonging to the same dosing groups (n = 3/4) were anesthetized with 3% isoflurane and placed on the imaging platform in a supine position with the inhalation of 3% isoflurane via nose cones. The mice were imaged 10 min post administration of d-luciferin and imaged on the LagoX IVIS Spectrum In Vivo Imaging System, with an exposure time of 10 s. Quantification of bioluminescence was performed by measuring the radiance (photons/s/cm2/steradian) from the regions of interest (abdominal region) using the Aura Imaging Software (Spectral Instruments Imaging).
Immediately following the IVIS imaging (3 h post-injection), the mice were euthanized, and livers and spleens were collected and imaged ex vivo. Bioluminescence values were quantified by the radiance (photons/s/cm2/steradian) for each tissue using the LagoX IVIS Spectrum In Vivo Imaging System.

Statistical Analysis

All data were reported as the mean ± the standard error of the mean (SEM). Comparisons were made between distinct groups by analyzing with one-way analysis of variance (ANOVA). Statistical significance, or P values, were reported directly on the figure. P values below 0.5 were considered nonsignificant (ns). Data analysis and visualization were performed by python scripting.

Supporting Information

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The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsami.4c20077.

  • Mathematical deduction of eq 1; photos of DiD-labeled LNPs before and after ultracentrifugation (Figure S1); RiboGreen assay results show encapsulation efficiency (Figure S2); LNP size distribution by cryoEM (Figure S3); other representative cryoEM images (Figure S4); cellular uptake and functional delivery of mRNA in Huh7 cells (Figure S5); quantification of CLSM images (Figure S6); and colocalization analysis shown as Pearson’s coefficient (Figure S7) (PDF)

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Author Information

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  • Corresponding Authors
  • Authors
    • Suiyang Liao - Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada;  Michael Smith Laboratories, University of British Columbia, Vancouver, British Columbia V6T 1Z4, Canada;  School of Biomedical Engineering, University of British Columbia, Vancouver, British Columbia V6T 2B9, Canada
    • Shuangyu Wang - Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada
    • Abishek Wadhwa - Department of Microbiology and Immunology, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada;  Life Sciences Institute, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada
    • Alexandra Birkenshaw - Faculty of Pharmaceutical Sciences, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada
    • Kevin Fox - Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada;  Faculty of Pharmaceutical Sciences, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada
    • Miffy Hok Yan Cheng - Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada;  Orcidhttps://orcid.org/0000-0002-0261-4642
    • Irafasha C. Casmil - Michael Smith Laboratories, University of British Columbia, Vancouver, British Columbia V6T 1Z4, Canada;  School of Biomedical Engineering, University of British Columbia, Vancouver, British Columbia V6T 2B9, Canada;  Orcidhttps://orcid.org/0000-0003-1890-7930
    • Armando Alcazar Magana - Michael Smith Laboratories, University of British Columbia, Vancouver, British Columbia V6T 1Z4, Canada;  Life Sciences Institute, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada
    • Nuthan Vikas Bathula - Michael Smith Laboratories, University of British Columbia, Vancouver, British Columbia V6T 1Z4, Canada;  School of Biomedical Engineering, University of British Columbia, Vancouver, British Columbia V6T 2B9, Canada
    • Chia Hao Ho - Michael Smith Laboratories, University of British Columbia, Vancouver, British Columbia V6T 1Z4, Canada;  Orcidhttps://orcid.org/0000-0003-3670-2680
    • Jin-Yu Cheng - Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada
    • Leonard J. Foster - Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada;  Michael Smith Laboratories, University of British Columbia, Vancouver, British Columbia V6T 1Z4, Canada;  Life Sciences Institute, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada;  Orcidhttps://orcid.org/0000-0001-8551-4817
    • Kenneth W. Harder - Department of Microbiology and Immunology, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada;  Life Sciences Institute, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada
    • Colin J. D. Ross - Faculty of Pharmaceutical Sciences, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada
  • Author Contributions

    The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

  • Funding

    This work was supported by a NanoMedicine Innovative Network (NMIN) Grand Challenge grant to A.K.B., P.R.C., K.W.H., C.J.D.R., and L.J.F.; NMIN Graduate Award─Doctoral to ICC; MSFHR Scholar Award and Tier II Canada Research Chair to A.K.B.; NMIN Graduate Award─Doctoral and the UBC Four Year Doctoral Fellowship (4YF) Award to A.W.

  • Notes
    The authors declare no competing financial interest.

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This article references 29 other publications.

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    Leung, A. K. K.; Hafez, I. M.; Baoukina, S.; Belliveau, N. M.; Zhigaltsev, I. V.; Afshinmanesh, E.; Tieleman, D. P.; Hansen, C. L.; Hope, M. J.; Cullis, P. R. Lipid Nanoparticles Containing siRNA Synthesized by Microfluidic Mixing Exhibit an Electron-Dense Nanostructured Core. J. Phys. Chem. C 2012, 116 (34), 18440– 18450,  DOI: 10.1021/jp303267y
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    Lipid Nanoparticles Containing siRNA Synthesized by Microfluidic Mixing Exhibit an Electron-Dense Nanostructured Core
    Leung, Alex K. K.; Hafez, Ismail M.; Baoukina, Svetlana; Belliveau, Nathan M.; Zhigaltsev, Igor V.; Afshinmanesh, Elham; Tieleman, D. Peter; Hansen, Carl L.; Hope, Michael J.; Cullis, Pieter R.
    Journal of Physical Chemistry C (2012), 116 (34), 18440-18450CODEN: JPCCCK; ISSN:1932-7447. (American Chemical Society)
    Lipid nanoparticles (LNP) contg. ionizable cationic lipids are the leading systems for enabling therapeutic applications of siRNA; however, the structure of these systems has not been defined. Here we examine the structure of LNP siRNA systems contg. DLinKC2-DMA(an ionizable cationic lipid), phospholipid, cholesterol and a polyethylene glycol (PEG) lipid formed using a rapid microfluidic mixing process. Techniques employed include cryo-transmission electron microscopy, 31P NMR, membrane fusion assays, d. measurements, and mol. modeling. The exptl. results indicate that these LNP siRNA systems have an interior lipid core contg. siRNA duplexes complexed to cationic lipid and that the interior core also contains phospholipid and cholesterol. Consistent with exptl. observations, mol. modeling calcns. indicate that the interior of LNP siRNA systems exhibits a periodic structure of aq. compartments, where some compartments contain siRNA. It is concluded that LNP siRNA systems formulated by rapid mixing of an ethanol soln. of lipid with an aq. medium contg. siRNA exhibit a nanostructured core. The results give insight into the mechanism whereby LNP siRNA systems are formed, providing an understanding of the high encapsulation efficiencies that can be achieved and information on methods of constructing more sophisticated LNP systems.
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    Rational design of cationic lipids for siRNA delivery
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    Nature Biotechnology (2010), 28 (2), 172-176CODEN: NABIF9; ISSN:1087-0156. (Nature Publishing Group)
    We adopted a rational approach to design cationic lipids for use in formulations to deliver small interfering RNA (siRNA). Starting with the ionizable cationic lipid 1,2-dilinoleyloxy-3-dimethylaminopropane (DLinDMA), a key lipid component of stable nucleic acid lipid particles (SNALP) as a benchmark, we used the proposed in vivo mechanism of action of ionizable cationic lipids to guide the design of DLinDMA-based lipids with superior delivery capacity. The best-performing lipid recovered after screening (DLin-KC2-DMA) was formulated and characterized in SNALP and demonstrated to have in vivo activity at siRNA doses as low as 0.01 mg/kg in rodents and 0.1 mg/kg in nonhuman primates. To our knowledge, this represents a substantial improvement over previous reports of in vivo endogenous hepatic gene silencing.
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    Self-assembled mRNA vaccines
    Kim, Jeonghwan; Eygeris, Yulia; Gupta, Mohit; Sahay, Gaurav
    Advanced Drug Delivery Reviews (2021), 170 (), 83-112CODEN: ADDREP; ISSN:0169-409X. (Elsevier B.V.)
    A review. MRNA vaccines have evolved from being a mere curiosity to emerging as COVID-19 vaccine front-runners. Recent advancements in the field of RNA technol., vaccinol., and nanotechnol. have generated interest in delivering safe and effective mRNA therapeutics. In this review, we discuss design and self-assembly of mRNA vaccines. Self-assembly, a spontaneous organization of individual mols., allows for design of nanoparticles with customizable properties. We highlight the materials commonly utilized to deliver mRNA, their physicochem. characteristics, and other relevant considerations, such as mRNA optimization, routes of administration, cellular fate, and immune activation, that are important for successful mRNA vaccination. We also examine the COVID-19 mRNA vaccines currently in clin. trials. mRNA vaccines are ready for the clinic, showing tremendous promise in the COVID-19 vaccine race, and have pushed the boundaries of gene therapy.
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    Gilbert, J.; Sebastiani, F.; Arteta, M. Y.; Terry, A.; Fornell, A.; Russell, R.; Mahmoudi, N.; Nylander, T. Evolution of the Structure of Lipid Nanoparticles for Nucleic Acid Delivery: From in Situ Studies of Formulation to Colloidal Stability. J. Colloid Interface Sci. 2024, 660, 66– 76,  DOI: 10.1016/j.jcis.2023.12.165
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    Forchette, L.; Sebastian, W.; Liu, T. A Comprehensive Review of COVID-19 Virology, Vaccines, Variants, and Therapeutics. Curr. Med. Sci. 2021, 41 (6), 1037– 1051,  DOI: 10.1007/s11596-021-2395-1
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    A Comprehensive Review of COVID-19 Virology, Vaccines, Variants, and Therapeutics
    Forchette, Lauren; Sebastian, William; Liu, Tuoen
    Current Medical Science (2021), 41 (6), 1037-1051CODEN: CMSUCM; ISSN:2523-899X. (Huazhong University of Science and Technology)
    A review. Summary: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative pathogen of the coronavirus disease 2019 (COVID-19), has caused more than 179 million infections and 3.8 million deaths worldwide. Throughout the past year, multiple vaccines have already been developed and used, while some others are in the process of being developed. However, the emergence of new mutant strains of SARS-CoV-2 that have demonstrated immune-evading characteristics and an increase in infective capabilities leads to potential ineffectiveness of the vaccines against these variants. The purpose of this review article is to highlight the current understanding of the immunol. mechanisms of the virus and vaccines, as well as to investigate some key variants and mutations of the virus driving the current pandemic and their impacts on current management guidelines. We also discussed new technologies being developed for the prevention, treatment, and detection of SARS-CoV-2. In this paper, we thoroughly reviewed and provided crucial information on SARS-CoV-2 virol., vaccines and drugs being used and developed for its prevention and treatment, as well as important variant strains. Our review paper will be beneficial to health care professionals and researchers so they can have a better understanding of the basic sciences, prevention, and clin. treatment of COVID-19 during the pandemic. This paper consists of the most updated information that has been available as of June 21, 2021.
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    Heinz, F. X.; Stiasny, K. Distinguishing Features of Current COVID-19 Vaccines: Knowns and Unknowns of Antigen Presentation and Modes of Action. npj Vaccines 2021, 6 (1), 104  DOI: 10.1038/s41541-021-00369-6
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    Distinguishing features of current COVID-19 vaccines: knowns and unknowns of antigen presentation and modes of action
    Heinz, Franz X.; Stiasny, Karin
    npj Vaccines (2021), 6 (1), 104CODEN: VACCBC; ISSN:2059-0105. (Nature Research)
    A review. COVID-19 vaccines were developed with an unprecedented pace since the beginning of the pandemic. Several of them have reached market authorization and mass prodn., leading to their global application on a large scale. This enormous progress was achieved with fundamentally different vaccine technologies used in parallel. mRNA, adenoviral vector as well as inactivated whole-virus vaccines are now in widespread use, and a subunit vaccine is in a final stage of authorization. They all rely on the native viral spike protein (S) of SARS-CoV-2 for inducing potently neutralizing antibodies, but the presentation of this key antigen to the immune system differs substantially between the different categories of vaccines. In this article, we review the relevance of structural modifications of S in different vaccines and the different modes of antigen expression after vaccination with genetic adenovirus-vector and mRNA vaccines. Distinguishing characteristics and unknown features are highlighted in the context of protective antibody responses and reactogenicity of vaccines.
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    Buschmann, M. D.; Carrasco, M. J.; Alishetty, S.; Paige, M.; Alameh, M. G.; Weissman, D. Nanomaterial Delivery Systems for mRNA Vaccines. Vaccines 2021, 9 (1), 65  DOI: 10.3390/vaccines9010065
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    Nanomaterial delivery systems for mRNA vaccines
    Buschmann, Michael D.; Carrasco, Manuel J.; Alishetty, Suman; Paige, Mikell; Alameh, Mohamad Gabriel; Weissman, Drew
    Vaccines (Basel, Switzerland) (2021), 9 (1), 65CODEN: VBSABP; ISSN:2076-393X. (MDPI AG)
    The recent success of mRNA vaccines in SARS-CoV-2 clin. trials is in part due to the development of lipid nanoparticle delivery systems that not only efficiently express the mRNA-encoded immunogen after i.m. injection, but also play roles as adjuvants and in vaccine reactogenicity. We present an overview of mRNA delivery systems and then focus on the lipid nanoparticles used in the current SARS-CoV-2 vaccine clin. trials. The review concludes with an anal. of the determinants of the performance of lipid nanoparticles in mRNA vaccines.
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    Tenchov, R.; Bird, R.; Curtze, A. E.; Zhou, Q. Lipid Nanoparticles─From Liposomes to mRNA Vaccine Delivery, a Landscape of Research Diversity and Advancement. ACS Nano 2021, 15 (11), 16982– 17015,  DOI: 10.1021/acsnano.1c04996
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    Lipid Nanoparticles-From Liposomes to mRNA Vaccine Delivery, a Landscape of Research Diversity and Advancement
    Tenchov, Rumiana; Bird, Robert; Curtze, Allison E.; Zhou, Qiongqiong
    ACS Nano (2021), 15 (11), 16982-17015CODEN: ANCAC3; ISSN:1936-0851. (American Chemical Society)
    A review. Lipid nanoparticles (LNPs) have emerged across the pharmaceutical industry as promising vehicles to deliver a variety of therapeutics. Currently in the spotlight as vital components of the COVID-19 mRNA vaccines, LNPs play a key role in effectively protecting and transporting mRNA to cells. Liposomes, an early version of LNPs, are a versatile nanomedicine delivery platform. A no. of liposomal drugs have been approved and applied to medical practice. Subsequent generations of lipid nanocarriers, such as solid lipid nanoparticles, nanostructured lipid carriers, and cationic lipid-nucleic acid complexes, exhibit more complex architectures and enhanced phys. stabilities. With their ability to encapsulate and deliver therapeutics to specific locations within the body and to release their contents at a desired time, LNPs provide a valuable platform for treatment of a variety of diseases. We present a landscape of LNP-related scientific publications, including patents and journal articles, based on anal. of the CAS Content Collection, the largest human-curated collection of published scientific knowledge. Rising trends are identified, such as nanostructured lipid carriers and solid lipid nanoparticles becoming the preferred platforms for numerous formulations. Recent advancements in LNP formulations as drug delivery platforms, such as antitumor and nucleic acid therapeutics and vaccine delivery systems, are discussed. Challenges and growth opportunities are also evaluated in other areas, such as medical imaging, cosmetics, nutrition, and agrochems. This report is intended to serve as a useful resource for those interested in LNP nanotechnologies, their applications, and the global research effort for their development.
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    Kloczewiak, M.; Banks, J. M.; Jin, L.; Brader, M. L. A Biopharmaceutical Perspective on Higher-Order Structure and Thermal Stability of mRNA Vaccines. Mol. Pharmaceutics 2022, 19 (7), 2022– 2031,  DOI: 10.1021/acs.molpharmaceut.2c00092
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    A Biopharmaceutical Perspective on Higher-Order Structure and Thermal Stability of mRNA Vaccines
    Kloczewiak, Marek; Banks, Jessica M.; Jin, Lin; Brader, Mark L.
    Molecular Pharmaceutics (2022), 19 (7), 2022-2031CODEN: MPOHBP; ISSN:1543-8384. (American Chemical Society)
    A review. Preservation of the integrity of macromol. higher-order structure is a tenet central to achieving biol. drug and vaccine product stability toward manufg., distribution, storage, handling, and administration. Given that mRNA lipid nanoparticles (mRNA-LNPs) are held together by an intricate ensemble of weak forces, there are some intriguing parallels to biol. drugs, at least at 1st glance. However, mRNA vaccines are not without unique formulation and stabilization challenges derived from the instability of unmodified mRNA and its limited history as a drug or vaccine. Since certain learning gained from biol. drug development may be applicable for the improvement of mRNA vaccines, we present a perspective on parallels and contrasts between the emerging role of higher-order structure pertaining to mRNA-LNPs compared to pharmaceutical proteins. In a recent publication, the location of mRNA encapsulated within lipid nanoparticles was identified, revealing new insights into the LNP structure, nanoheterogeneity, and microenvironment of the encapsulated mRNA mols. We extend those findings by considering the effect of encapsulation on mRNA thermal unfolding with the observation that encapsulation in LNPs increases mRNA unfolding temps.
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    Brader, M. L.; Williams, S. J.; Banks, J. M.; Hui, W. H.; Zhou, Z. H.; Jin, L. Encapsulation State of Messenger RNA inside Lipid Nanoparticles. Biophys. J. 2021, 120 (14), 2766– 2770,  DOI: 10.1016/j.bpj.2021.03.012
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    Encapsulation state of messenger RNA inside lipid nanoparticles
    Brader, Mark L.; Williams, Sean J.; Banks, Jessica M.; Hui, Wong H.; Zhou, Z. Hong; Jin, Lin
    Biophysical Journal (2021), 120 (14), 2766-2770CODEN: BIOJAU; ISSN:0006-3495. (Cell Press)
    Understanding the structure of mRNA (mRNA) lipid nanoparticles, and specifically the microenvironment of the mRNA mols. within these entities, is fundamental to advancing their biomedical potential. Here, we show that a permeating cationic dye, thionine, can serve as a cryogenic electron microscopy contrasting agent by binding selectively to encapsulated mRNA without disturbing lipid nanoparticle morphol. Cryo-electron microscopy images identify the mRNA location, revealing that mRNA may exist within solvent-filled cavities or may be substantially lipid assocd.
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    Cheng, M. H. Y.; Leung, J.; Zhang, Y.; Strong, C.; Basha, G.; Momeni, A.; Chen, Y.; Jan, E.; Abdolahzadeh, A.; Wang, X.; Kulkarni, J. A.; Witzigmann, D.; Cullis, P. R. Induction of Bleb Structures in Lipid Nanoparticle Formulations of mRNA Leads to Improved Transfection Potency. Adv. Mater. 2023, 35 (31), 2303370  DOI: 10.1002/adma.202303370
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    Induction of Bleb Structures in Lipid Nanoparticle Formulations of mRNA Leads to Improved Transfection Potency
    Cheng, Miffy Hok Yan; Leung, Jerry; Zhang, Yao; Strong, Colton; Basha, Genc; Momeni, Arash; Chen, Yihang; Jan, Eric; Abdolahzadeh, Amir; Wang, Xinying; Kulkarni, Jayesh A.; Witzigmann, Dominik; Cullis, Pieter R.
    Advanced Materials (Weinheim, Germany) (2023), 35 (31), 2303370CODEN: ADVMEW; ISSN:0935-9648. (Wiley-VCH Verlag GmbH & Co. KGaA)
    The transfection potency of lipid nanoparticle (LNP) mRNA systems is critically dependent on the ionizable cationic lipid component. LNP mRNA systems composed of optimized ionizable lipids often display distinctive mRNA-rich "bleb" structures. Here, it is shown that such structures can also be induced for LNPs contg. nominally less active ionizable lipids by formulating them in the presence of high concns. of pH 4 buffers such as sodium citrate, leading to improved transfection potencies both in vitro and in vivo. Induction of bleb structure and improved potency is dependent on the type of pH 4 buffer employed, with LNP mRNA systems prepd. using 300 mM sodium citrate buffer displaying max. transfection. The improved transfection potencies of LNP mRNA systems displaying bleb structure can be attributed, at least in part, to enhanced integrity of the encapsulated mRNA. It is concluded that enhanced transfection can be achieved by optimizing formulation parameters to improve mRNA stability and that optimization of ionizable lipids to achieve enhanced potency may well lead to improvements in mRNA integrity through formation of the bleb structure rather than enhanced intracellular delivery.
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    Whitehead, K. A.; Matthews, J.; Chang, P. H.; Niroui, F.; Dorkin, J. R.; Severgnini, M.; Anderson, D. G. In Vitro–In Vivo Translation of Lipid Nanoparticles for Hepatocellular siRNA Delivery. ACS Nano 2012, 6 (8), 6922– 6929,  DOI: 10.1021/nn301922x
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    In Vitro-In Vivo Translation of Lipid Nanoparticles for Hepatocellular siRNA Delivery
    Whitehead, Kathryn A.; Matthews, Jonathan; Chang, Philip H.; Niroui, Farnaz; Dorkin, J. Robert; Severgnini, Mariano; Anderson, Daniel G.
    ACS Nano (2012), 6 (8), 6922-6929CODEN: ANCAC3; ISSN:1936-0851. (American Chemical Society)
    A significant challenge in the development of clin. viable siRNA delivery systems is a lack of in vitro-in vivo translatability: many delivery vehicles that are initially promising in cell culture do not retain efficacy in animals. Despite its importance, little information exists on the predictive nature of in vitro methodologies, most likely due to the cost and time assocd. with generating in vitro-in vivo data sets. Recently, high-throughput techniques have been developed that have allowed the examn. of hundreds of lipid nanoparticle formulations for transfection efficiency in multiple exptl. systems. The large resulting data set has allowed the development of correlations between in vitro and characterization data and in vivo efficacy for hepatocellular delivery vehicles. Consistency of formulation technique and the type of cell used for in vitro expts. was found to significantly affect correlations, with primary hepatocytes and HeLa cells yielding the most predictive data. Interestingly, in vitro data acquired using HeLa cells were more predictive of in vivo performance than mouse hepatoma Hepa1-6 cells. Of the characterization parameters, only siRNA entrapment efficiency was partially predictive of in vivo silencing potential, while zeta-potential and, surprisingly, nanoparticle size (when <300 nm) as measured by dynamic light scattering were not. These data provide guiding principles in the development of clin. viable siRNA delivery materials and have the potential to reduce exptl. costs while improving the translation of materials into animals.
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    Paunovska, K.; Sago, C. D.; Monaco, C. M.; Hudson, W. H.; Castro, M. G.; Rudoltz, T. G.; Kalathoor, S.; Vanover, D. A.; Santangelo, P. J.; Ahmed, R.; Bryksin, A. V.; Dahlman, J. E. A Direct Comparison of in Vitro and in Vivo Nucleic Acid Delivery Mediated by Hundreds of Nanoparticles Reveals a Weak Correlation. Nano Lett. 2018, 18 (3), 2148– 2157,  DOI: 10.1021/acs.nanolett.8b00432
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    A direct comparison of in vitro and in vivo nucleic acid delivery mediated by hundreds of nanoparticles reveals a weak correlation
    Paunovska, Kalina; Sago, Cory D.; Monaco, Christopher M.; Hudson, William H.; Castro, Marielena Gamboa; Rudoltz, Tobi G.; Kalathoor, Sujay; Vanover, Daryll A.; Santangelo, Philip J.; Ahmed, Rafi; Bryksin, Anton V.; Dahlman, James E.
    Nano Letters (2018), 18 (3), 2148-2157CODEN: NALEFD; ISSN:1530-6984. (American Chemical Society)
    Endothelial cells and macrophages play active roles in disease and as a result are important targets for nucleic acid therapies. While thousands of chem. distinct lipid nanoparticles (LNPs) can be synthesized to deliver nucleic acids, studying more than a few LNPs in vivo is challenging. As a result, it is difficult to understand how nanoparticles target these cells in vivo. Using high throughput LNP barcoding, we quantified how well LNPs delivered DNA barcodes to endothelial cells and macrophages in vitro, as well as endothelial cells and macrophages isolated from the lung, heart, and bone marrow in vivo. We focused on two fundamental questions in drug delivery. First, does in vitro LNP delivery predict in vivo LNP delivery. By comparing how 281 LNPs delivered barcodes to endothelial cells and macrophages in vitro and in vivo, we found in vitro delivery did not predict in vivo delivery. Second, does LNP delivery change within the microenvironment of a tissue. We quantified how 85 LNPs delivered barcodes to eight splenic cell populations, and found that cell types derived from myeloid progenitors tended to be targeted by similar LNPs, relative to cell types derived from lymphoid progenitors. These data demonstrate that barcoded LNPs can elucidate fundamental questions about in vivo nanoparticle delivery.
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  • Abstract

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    Figure 1

    Figure 1. Centrifugal fractionation of LNPs based on their mRNA loading levels. (A) Schematic illustration of LNP fractionation via ultracentrifugation. Depending on the LNPs’ density contrast with the aqueous system, two fractions (LDLNP and HDLNP) are generated from the fractionation process. Together with the original batch, three samples are compared throughout this paper; (B) particle size distribution by intensity measured with DLS; payload mRNA integrity by Bioanalyzer shown as electropherograms in (C1) and bands in (C2).

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    Figure 2

    Figure 2. Validation of centrifugal fractionation, characterization of lipid composition, and calculation of N/P ratio. (A) Bar chart of RNA/total lipid ratio to represent the mRNA loading levels and validate the fractionation method; (B) pie charts showing lipid composition of OLNP, LDLNP, and HDLNP as characterized by ultrahigh-performance liquid chromatography–mass spectrometry (UHPLC-MS).

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    Figure 3

    Figure 3. mRNA loading levels can influence the distribution of LNP morphology. (A1–A3) Representative cryoEM images of OLNP, LDLNP, and HDLNP (scale bar: 100 nm); (B) pie charts showing the composition of LNPs with various number of blebs; (C) schematic illustration of measuring LNPs and blebs with FIJI; (D1–D3) scatter plots mapping the dimension of LNPs and blebs (purple dots: LNPs without blebs; green dots: LNPs with blebs) (E) raincloud plot showing the size distribution of blebs.

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    Figure 4

    Figure 4. Cellular uptake and functional delivery of mRNA with OLNP, LDLNP, and HDLNP. (A) CLSM images of OLNP, LDLNP, and HDLNP ((A1), (A2), and (A3), respectively) delivering Cy5-labeled mEGFP into HEK293 cells (scale bar: 50 μm); (B1–B3) CLSM study on the colocalization between LNP colocalization and Rab7a, representing late endosomes (scale bar: 50 μm).

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    Figure 5

    Figure 5. In vivo functional delivery of mFluc. (A) Schematic illustration of the animal study; (B) in vivo bioluminescence images of mice at 3 h after IV injection at mFluc dosage of 0.3 mg/kg (n = 4) and bar chart showing whole-body bioluminescence quantification from IVIS imaging. Data are presented as the mean ± standard deviation (SD) (n = 4). (C) Representative ex vivo bioluminescence images of extracted livers and spleens from mice dosed with OLNP/LDLNP/HDLNP (top to bottom) and bar chart showing bioluminescence of extracted livers and spleens. Data are presented as the mean ± SD (n = 4). (D) Scatter plot by projecting ex vivo liver radiance readout against spleen readout to indicate tissue tropism.

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  • References

    This article references 29 other publications.

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      Akinc, A.; Maier, M. A.; Manoharan, M.; Fitzgerald, K.; Jayaraman, M.; Barros, S.; Ansell, S.; Du, X.; Hope, M. J.; Madden, T. D.; Mui, B. L.; Semple, S. C.; Tam, Y. K.; Ciufolini, M.; Witzigmann, D.; Kulkarni, J. A.; van der Meel, R.; Cullis, P. R. The Onpattro Story and the Clinical Translation of Nanomedicines Containing Nucleic Acid-Based Drugs. Nat. Nanotechnol. 2019, 14 (12), 1084– 1087,  DOI: 10.1038/s41565-019-0591-y
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      The Onpattro story and the clinical translation of nanomedicines containing nucleic acid-based drugs
      Akinc, Akin; Maier, Martin A.; Manoharan, Muthiah; Fitzgerald, Kevin; Jayaraman, Muthusamy; Barros, Scott; Ansell, Steven; Du, Xinyao; Hope, Michael J.; Madden, Thomas D.; Mui, Barbara L.; Semple, Sean C.; Tam, Ying K.; Ciufolini, Marco; Witzigmann, Dominik; Kulkarni, Jayesh A.; van der Meel, Roy; Cullis, Pieter R.
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      The regulatory approval of Onpattro, a lipid nanoparticle-based short interfering RNA drug for the treatment of polyneuropathies induced by hereditary transthyretin amyloidosis, paves the way for clin. development of many nucleic acid-based therapies enabled by nanoparticle delivery.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXitlWit7vO&md5=3184ac1c3c74e6924fb1bb14b5229e95
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      Polack, F. P.; Thomas, S. J.; Kitchin, N.; Absalon, J.; Gurtman, A.; Lockhart, S.; Perez, J. L.; Pérez Marc, G.; Moreira, E. D.; Zerbini, C.; Bailey, R.; Swanson, K. A.; Roychoudhury, S.; Koury, K.; Li, P.; Kalina, W. V.; Cooper, D.; Frenck, R. W.; Hammitt, L. L.; Türeci, Ö.; Nell, H.; Schaefer, A.; Ünal, S.; Tresnan, D. B.; Mather, S.; Dormitzer, P. R.; Şahin, U.; Jansen, K. U.; Gruber, W. C. Safety and Efficacy of the BNT162b2 mRNA Covid-19 Vaccine. N. Engl. J. Med. 2020, 383 (27), 2603– 2615,  DOI: 10.1056/NEJMoa2034577
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      Safety and efficacy of the BNT162b2 mRNA Covid-19 vaccine
      Polack, Fernando P.; Thomas, Stephen J.; Kitchin, Nicholas; Absalon, Judith; Gurtman, Alejandra; Lockhart, Stephen; Perez, John L.; Marc, Gonzalo Perez; Moreira, Edson D.; Zerbini, Cristiano; Bailey, Ruth; Swanson, Kena A.; Roychoudhury, Satrajit; Koury, Kenneth; Li, Ping; Kalina, Warren V.; Cooper, David; Frenck, Robert W., Jr.; Hammitt, Laura L.; Tureci, Ozlem; Nell, Haylene; Schaefer, Axel; Unal, Serhat; Tresnan, Dina B.; Mather, Susan; Dormitzer, Philip R.; Sahin, Ugur; Jansen, Kathrin U.; Gruber, William C.
      New England Journal of Medicine (2020), 383 (27), 2603-2615CODEN: NEJMAG; ISSN:1533-4406. (Massachusetts Medical Society)
      A review. Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and the resulting coronavirus disease 2019 (Covid-19) have afflicted tens of millions of people in a worldwide pandemic. Safe and effective vaccines are needed urgently. methods In an ongoing multinational, placebo-controlled, observer-blinded, pivotal efficacy trial, we randomly assigned persons 16 years of age or older in a 1:1 ratio to receive two doses, 21 days apart, of either placebo or the BNT162b2 vaccine candidate (30μg per dose). BNT162b2 is a lipid nanoparticle-formulated, nucleoside-modified RNA vaccine that encodes a prefusion stabilized, membrane-anchored SARS-CoV-2 full-length spike protein. The primary end points were efficacy of the vaccine against lab.-confirmed Covid-19 and safety. results A total of 43,548 participants underwent randomization, of whom 43,448 received injections: 21,720 with BNT162b2 and 21,728 with placebo. There were 8 cases of Covid-19 with onset at least 7 days after the second dose among participants assigned to receive BNT162b2 and 162 cases among those assigned to placebo; BNT162b2 was 95% effective in preventing Covid-19 (95% credible interval, 90.3 to 97.6). Similar vaccine efficacy (generally 90 to 100%) was obsd. across subgroups defined by age, sex, race, ethnicity, baseline body-mass index, and the presence of coexisting conditions. Among 10 cases of severe Covid-19 with onset after the first dose, 9 occurred in placebo recipients and 1 in a BNT162b2 recipient. The safety profile of BNT162b2 was characterized by short-term, mild-to-moderate pain at the injection site, fatigue, and headache. The incidence of serious adverse events was low and was similar in the vaccine and placebo groups. conclusions A two-dose regimen of BNT162b2 conferred 95% protection against Covid-19 in persons 16 years of age or older. Safety over a median of 2 mo was similar to that of other viral vaccines.
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    3. 3
      Baden, L. R.; El Sahly, H. M.; Essink, B.; Kotloff, K.; Frey, S.; Novak, R.; Diemert, D.; Spector, S. A.; Rouphael, N.; Creech, C. B.; McGettigan, J.; Khetan, S.; Segall, N.; Solis, J.; Brosz, A.; Fierro, C.; Schwartz, H.; Neuzil, K.; Corey, L.; Gilbert, P.; Janes, H.; Follmann, D.; Marovich, M.; Mascola, J.; Polakowski, L.; Ledgerwood, J.; Graham, B. S.; Bennett, H.; Pajon, R.; Knightly, C.; Leav, B.; Deng, W.; Zhou, H.; Han, S.; Ivarsson, M.; Miller, J.; Zaks, T. Efficacy and Safety of the mRNA-1273 SARS-CoV-2 Vaccine. N. Engl. J. Med. 2021, 384 (5), 403– 416,  DOI: 10.1056/NEJMoa2035389
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      Efficacy and safety of the mRNA-1273 SARS-CoV-2 vaccine
      Baden, L. R.; El Sahly, H. M.; Essink, B.; Kotloff, K.; Frey, S.; Novak, R.; Diemert, D.; Spector, S. A.; Rouphael, N.; Creech, C. B.; McGettigan, J.; Khetan, S.; Segall, N.; Solis, J.; Brosz, A.; Fierro, C.; Schwartz, H.; Neuzil, K.; Corey, L.; Gilbert, P.; Janes, H.; Follmann, D.; Marovich, M.; Mascola, J.; Polakowski, L.; Ledgerwood, J.; Graham, B. S.; Bennett, H.; Pajon, R.; Knightly, C.; Leav, B.; Deng, W.; Zhou, H.; Han, S.; Ivarsson, M.; Miller, J.; Zaks, T.
      New England Journal of Medicine (2021), 384 (5), 403-416CODEN: NEJMAG; ISSN:1533-4406. (Massachusetts Medical Society)
      Vaccines are needed to prevent coronavirus disease 2019 (Covid-19) and to protect persons who are at high risk for complications. The mRNA-1273 vaccine is a lipid nanoparticle-encapsulated mRNA-based vaccine that encodes the prefusion stabilized full-length spike protein of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus that causes Covid-19. This phase 3 randomized, observer-blinded, placebo-controlled trial was conducted at 99 centers across the United States. Persons at high risk for SARS-CoV-2 infection or its complications were randomly assigned in a 1:1 ratio to receive two i.m. injections of mRNA-1273 (100μg) or placebo 28 days apart. The primary end point was prevention of Covid-19 illness with onset at least 14 days after the second injection in participants who had not previously been infected with SARS-CoV-2. The trial enrolled 30,420 volunteers who were randomly assigned in a 1:1 ratio to receive either vaccine or placebo (15,210 participants in each group). More than 96% of participants received both injections, and 2.2% had evidence (serol., virol., or both) of SARS-CoV-2 infection at baseline. Symptomatic Covid-19 illness was confirmed in 185 participants in the placebo group (56.5 per 1000 person-years; 95% confidence interval [CI], 48.7 to 65.3) and in 11 participants in the mRNA-1273 group (3.3 per 1000 person-years; 95% CI, 1.7 to 6.0); vaccine efficacy was 94.1% (95% CI, 89.3 to 96.8%; P<0.001). Efficacy was similar across key secondary analyses, including assessment 14 days after the first dose, analyses that included participants who had evidence of SARS-CoV-2 infection at baseline, and analyses in participants 65 years of age or older. Severe Covid-19 occurred in 30 participants, with one fatality; all 30 were in the placebo group. Moderate, transient reactogenicity after vaccination occurred more frequently in the mRNA-1273 group. Serious adverse events were rare, and the incidence was similar in the two groups. The mRNA-1273 vaccine showed 94.1% efficacy at preventing Covid-19 illness, including severe disease. Aside from transient local and systemic reactions, no safety concerns were identified.
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      Wilson, E.; Goswami, J.; Baqui, A. H.; Doreski, P. A.; Perez-Marc, G.; Zaman, K.; Monroy, J.; Duncan, C. J. A.; Ujiie, M.; Rämet, M.; Pérez-Breva, L.; Falsey, A. R.; Walsh, E. E.; Dhar, R.; Wilson, L.; Du, J.; Ghaswalla, P.; Kapoor, A.; Lan, L.; Mehta, S.; Mithani, R.; Panozzo, C. A.; Simorellis, A. K.; Kuter, B. J.; Schödel, F.; Huang, W.; Reuter, C.; Slobod, K.; Stoszek, S. K.; Shaw, C. A.; Miller, J. M.; Das, R.; Chen, G. L. Efficacy and Safety of an mRNA-Based RSV PreF Vaccine in Older Adults. N. Engl. J. Med. 2023, 389 (24), 2233– 2244,  DOI: 10.1056/NEJMoa2307079
      There is no corresponding record for this reference.
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      Kulkarni, J. A.; Witzigmann, D.; Leung, J.; van der Meel, R.; Zaifman, J.; Darjuan, M. M.; Grisch-Chan, H. M.; Thöny, B.; Tam, Y. Y. C.; Cullis, P. R. Fusion-Dependent Formation of Lipid Nanoparticles Containing Macromolecular Payloads. Nanoscale 2019, 11 (18), 9023– 9031,  DOI: 10.1039/C9NR02004G
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      Fusion-dependent formation of lipid nanoparticles containing macromolecular payloads
      Kulkarni, Jayesh A.; Witzigmann, Dominik; Leung, Jerry; van der Meel, Roy; Zaifman, Josh; Darjuan, Maria M.; Grisch-Chan, Hiu Man; Thony, Beat; Tam, Yuen Yi C.; Cullis, Pieter R.
      Nanoscale (2019), 11 (18), 9023-9031CODEN: NANOHL; ISSN:2040-3372. (Royal Society of Chemistry)
      The success of Onpattro (patisiran) clearly demonstrates the utility of lipid nanoparticle (LNP) systems for enabling gene therapies. These systems are composed of ionizable cationic lipids, phospholipid, cholesterol, and polyethylene glycol (PEG)-lipids, and are produced through rapid-mixing of an ethanolic-lipid soln. with an acidic aq. soln. followed by dialysis into neutralizing buffer. A detailed understanding of the mechanism of LNP formation is crucial to improving LNP design. Here we use cryogenic transmission electron microscopy and fluorescence techniques to further demonstrate that LNP are formed through the fusion of precursor, pH-sensitive liposomes into large electron-dense core structures as the pH is neutralized. Next, we show that the fusion process is limited by the accumulation of PEG-lipid on the emerging particle. Finally, we show that the fusion-dependent mechanism of formation also applies to LNP contg. macromol. payloads including mRNA, DNA vectors, and gold nanoparticles.
      >> More from SciFinder ®
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      Li, S.; Hu, Y.; Lin, J.; Schneiderman, Z.; Shao, F.; Wei, L.; Li, A.; Hsieh, K.; Kokkoli, E.; Curk, T.; Mao, H.-Q.; Wang, T.-H. Single-Particle Spectroscopic Chromatography Reveals Heterogeneous RNA Loading and Size Correlations in Lipid Nanoparticles. ACS Nano 2024, 18, 15729,  DOI: 10.1021/acsnano.4c02341
      There is no corresponding record for this reference.
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      Li, S.; Hu, Y.; Li, A.; Lin, J.; Hsieh, K.; Schneiderman, Z.; Zhang, P.; Zhu, Y.; Qiu, C.; Kokkoli, E.; Wang, T.-H.; Mao, H.-Q. Payload Distribution and Capacity of mRNA Lipid Nanoparticles. Nat. Commun. 2022, 13 (1), 5561  DOI: 10.1038/s41467-022-33157-4
      7
      Payload distribution and capacity of mRNA lipid nanoparticles
      Li, Sixuan; Hu, Yizong; Li, Andrew; Lin, Jinghan; Hsieh, Kuangwen; Schneiderman, Zachary; Zhang, Pengfei; Zhu, Yining; Qiu, Chenhu; Kokkoli, Efrosini; Wang, Tza-Huei; Mao, Hai-Quan
      Nature Communications (2022), 13 (1), 5561CODEN: NCAOBW; ISSN:2041-1723. (Nature Portfolio)
      Lipid nanoparticles (LNPs) are effective vehicles to deliver mRNA vaccines and therapeutics. It has been challenging to assess mRNA packaging characteristics in LNPs, including payload distribution and capacity, which are crit. to understanding structure-property-function relationships for further carrier development. Here, we report a method based on the multi-laser cylindrical illumination confocal spectroscopy (CICS) technique to examine mRNA and lipid contents in LNP formulations at the single-nanoparticle level. By differentiating unencapsulated mRNAs, empty LNPs and mRNA-loaded LNPs via coincidence anal. of fluorescent tags on different LNP components, and quant. resolving single-mRNA fluorescence, we reveal that a commonly referenced benchmark formulation using DLin-MC3 as the ionizable lipid contains mostly 2 mRNAs per loaded LNP with a presence of 40%-80% empty LNPs depending on the assembly conditions. Systematic anal. of different formulations with control variables reveals a kinetically controlled assembly mechanism that governs the payload distribution and capacity in LNPs. These results form the foundation for a holistic understanding of the mol. assembly of mRNA LNPs.
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      De Kloet, S. R.; Andrean, B. A. G. Buoyant Density Gradient Centrifugation of RNA and DNA in Alkali Iodide Solutions. Biochim. Biophys. Acta, Nucleic Acids Protein Synth. 1971, 247 (4), 519– 527,  DOI: 10.1016/0005-2787(71)90688-5
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      Guerrini, G.; Mehn, D.; Scaccabarozzi, D.; Gioria, S.; Calzolai, L. Analytical Ultracentrifugation to Assess the Quality of LNP-mRNA Therapeutics. Int. J. Mol. Sci. 2024, 25 (11), 5718  DOI: 10.3390/ijms25115718
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      Schiel, J. E.; Hage, D. S. Density Measurements of Potassium Phosphate Buffer from 4 to 45 °C. Talanta 2005, 65 (2), 495– 500,  DOI: 10.1016/j.talanta.2004.06.029
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      Henrickson, A.; Kulkarni, J. A.; Zaifman, J.; Gorbet, G. E.; Cullis, P. R.; Demeler, B. Density Matching Multi-Wavelength Analytical Ultracentrifugation to Measure Drug Loading of Lipid Nanoparticle Formulations. ACS Nano 2021, 15, 5068,  DOI: 10.1021/acsnano.0c10069
      11
      Density Matching Multi-wavelength Analytical Ultracentrifugation to Measure Drug Loading of Lipid Nanoparticle Formulations
      Henrickson, Amy; Kulkarni, Jayesh A.; Zaifman, Josh; Gorbet, Gary E.; Cullis, Pieter R.; Demeler, Borries
      ACS Nano (2021), 15 (3), 5068-5076CODEN: ANCAC3; ISSN:1936-0851. (American Chemical Society)
      Previous work suggested that lipid nanoparticle (LNP) formulations, encapsulating nucleic acids, display electron-dense morphol. when examd. by cryogenic-transmission electron microscopy (cryo-TEM). Critically, the employed cryo-TEM method cannot differentiate between loaded and empty LNP formulations. Clin. relevant formulations contain high lipid-to-nucleic acid ratios (10-25 (wt./wt.)), and for systems that contain mRNA or DNA, it is anticipated that a substantial fraction of the LNP population does not contain a payload. Here, we present a method based on the global anal. of multi-wavelength sedimentation velocity anal. ultracentrifugation, using d. matching with heavy water, that not only measures the std. sedimentation and diffusion coeff. distributions of LNP mixts., but also reports the corresponding partial sp. vol. distributions and optically separates signal contributions from nucleic acid cargo and lipid shell. This makes it possible to reliably predict molar mass and anisotropy distributions, in particular, for systems that are heterogeneous in partial sp. vol. and have low to intermediate densities. Our method makes it possible to unambiguously measure the d. of nanoparticles and is motivated by the need to characterize the extent to which lipid nanoparticles are loaded with nucleic acid cargoes. Since the densities of nucleic acids and lipids substantially differ, the measured d. is directly proportional to the loading of nanoparticles. Hence, different loading levels will produce particles with variable d. and partial sp. vol. An UltraScan software module was developed to implement this approach for routine anal.
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      Bepperling, A.; Richter, G. Determination of mRNA Copy Number in Degradable Lipid Nanoparticles via Density Contrast Analytical Ultracentrifugation. Eur. Biophys J. 2023, 52, 393,  DOI: 10.1007/s00249-023-01663-y
      12
      Determination of mRNA copy number in degradable lipid nanoparticles via density contrast analytical ultracentrifugation
      Bepperling, Alexander; Richter, Gesa
      European Biophysics Journal (2023), 52 (4-5), 393-400CODEN: EBJOE8; ISSN:0175-7571. (Springer International Publishing AG)
      Lipid nanoparticles as delivery system for mRNA have recently attracted attention to a broader audience as COVID-19 mRNA vaccines. Their low immunogenicity and capability to deliver a variety of nucleic acids renders them an interesting and complementary alternative to gene therapy vectors like AAVs. An important quality attribute of LNPs is the copy no. of the encapsulated cargo mol. This work describes how d. and mol. wt. distributions obtained by d. contrast sedimentation velocity can be used to calc. the mRNA copy no. of a degradable lipid nanoparticle formulation. The detd. av. copy no. of 5 mRNA mols. per LNP is consistent with the previous studies using other biophys. techniques, such as single particle imaging microscopy and multi-laser cylindrical illumination confocal spectroscopy (CICS).
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      Leung, A. K. K.; Hafez, I. M.; Baoukina, S.; Belliveau, N. M.; Zhigaltsev, I. V.; Afshinmanesh, E.; Tieleman, D. P.; Hansen, C. L.; Hope, M. J.; Cullis, P. R. Lipid Nanoparticles Containing siRNA Synthesized by Microfluidic Mixing Exhibit an Electron-Dense Nanostructured Core. J. Phys. Chem. C 2012, 116 (34), 18440– 18450,  DOI: 10.1021/jp303267y
      13
      Lipid Nanoparticles Containing siRNA Synthesized by Microfluidic Mixing Exhibit an Electron-Dense Nanostructured Core
      Leung, Alex K. K.; Hafez, Ismail M.; Baoukina, Svetlana; Belliveau, Nathan M.; Zhigaltsev, Igor V.; Afshinmanesh, Elham; Tieleman, D. Peter; Hansen, Carl L.; Hope, Michael J.; Cullis, Pieter R.
      Journal of Physical Chemistry C (2012), 116 (34), 18440-18450CODEN: JPCCCK; ISSN:1932-7447. (American Chemical Society)
      Lipid nanoparticles (LNP) contg. ionizable cationic lipids are the leading systems for enabling therapeutic applications of siRNA; however, the structure of these systems has not been defined. Here we examine the structure of LNP siRNA systems contg. DLinKC2-DMA(an ionizable cationic lipid), phospholipid, cholesterol and a polyethylene glycol (PEG) lipid formed using a rapid microfluidic mixing process. Techniques employed include cryo-transmission electron microscopy, 31P NMR, membrane fusion assays, d. measurements, and mol. modeling. The exptl. results indicate that these LNP siRNA systems have an interior lipid core contg. siRNA duplexes complexed to cationic lipid and that the interior core also contains phospholipid and cholesterol. Consistent with exptl. observations, mol. modeling calcns. indicate that the interior of LNP siRNA systems exhibits a periodic structure of aq. compartments, where some compartments contain siRNA. It is concluded that LNP siRNA systems formulated by rapid mixing of an ethanol soln. of lipid with an aq. medium contg. siRNA exhibit a nanostructured core. The results give insight into the mechanism whereby LNP siRNA systems are formed, providing an understanding of the high encapsulation efficiencies that can be achieved and information on methods of constructing more sophisticated LNP systems.
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    14. 14
      Vaidya, A.; Parande, D.; Khadse, N.; Vargas-Montoya, N.; Agarwal, V.; Ortiz, C.; Ellis, G.; Kaushal, N.; Sarode, A.; Karve, S.; DeRosa, F. Analytical Characterization of Heterogeneities in mRNA-Lipid Nanoparticles Using Sucrose Density Gradient Ultracentrifugation. Anal. Chem. 2024, 96, 5570,  DOI: 10.1021/acs.analchem.4c00031
      There is no corresponding record for this reference.
    15. 15
      Zhao, H.; Sousa, A. A.; Schuck, P. Flotation Coefficient Distributions of Lipid Nanoparticles by Sedimentation Velocity Analytical Ultracentrifugation. ACS Nano 2024, 18, 18663,  DOI: 10.1021/acsnano.4c05322
      There is no corresponding record for this reference.
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      Semple, S. C.; Akinc, A.; Chen, J.; Sandhu, A. P.; Mui, B. L.; Cho, C. K.; Sah, D. W. Y.; Stebbing, D.; Crosley, E. J.; Yaworski, E.; Hafez, I. M.; Dorkin, J. R.; Qin, J.; Lam, K.; Rajeev, K. G.; Wong, K. F.; Jeffs, L. B.; Nechev, L.; Eisenhardt, M. L.; Jayaraman, M.; Kazem, M.; Maier, M. A.; Srinivasulu, M.; Weinstein, M. J.; Chen, Q.; Alvarez, R.; Barros, S. A.; De, S.; Klimuk, S. K.; Borland, T.; Kosovrasti, V.; Cantley, W. L.; Tam, Y. K.; Manoharan, M.; Ciufolini, M. A.; Tracy, M. A.; de Fougerolles, A.; MacLachlan, I.; Cullis, P. R.; Madden, T. D.; Hope, M. J. Rational Design of Cationic Lipids for siRNA Delivery. Nat. Biotechnol. 2010, 28 (2), 172– 176,  DOI: 10.1038/nbt.1602
      16
      Rational design of cationic lipids for siRNA delivery
      Semple, Sean C.; Akinc, Akin; Chen, Jianxin; Sandhu, Ammen P.; Mui, Barbara L.; Cho, Connie K.; Sah, Dinah W. Y.; Stebbing, Derrick; Crosley, Erin J.; Yaworski, Ed; Hafez, Ismail M.; Dorkin, J. Robert; Qin, June; Lam, Kieu; Rajeev, Kallanthottathil G.; Wong, Kim F.; Jeffs, Lloyd B.; Nechev, Lubomir; Eisenhardt, Merete L.; Jayaraman, Muthusamy; Kazem, Mikameh; Maier, Martin A.; Srinivasulu, Masuna; Weinstein, Michael J.; Chen, Qingmin; Alvarez, Rene; Barros, Scott A.; De, Soma; Klimuk, Sandra K.; Borland, Todd; Kosovrasti, Verbena; Cantley, William L.; Tam, Ying K.; Manoharan, Muthiah; Ciufolini, Marco A.; Tracy, Mark A.; de Fougerolles, Antonin; MacLachlan, Ian; Cullis, Pieter R.; Madden, Thomas D.; Hope, Michael J.
      Nature Biotechnology (2010), 28 (2), 172-176CODEN: NABIF9; ISSN:1087-0156. (Nature Publishing Group)
      We adopted a rational approach to design cationic lipids for use in formulations to deliver small interfering RNA (siRNA). Starting with the ionizable cationic lipid 1,2-dilinoleyloxy-3-dimethylaminopropane (DLinDMA), a key lipid component of stable nucleic acid lipid particles (SNALP) as a benchmark, we used the proposed in vivo mechanism of action of ionizable cationic lipids to guide the design of DLinDMA-based lipids with superior delivery capacity. The best-performing lipid recovered after screening (DLin-KC2-DMA) was formulated and characterized in SNALP and demonstrated to have in vivo activity at siRNA doses as low as 0.01 mg/kg in rodents and 0.1 mg/kg in nonhuman primates. To our knowledge, this represents a substantial improvement over previous reports of in vivo endogenous hepatic gene silencing.
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      Svedberg, T.; Rinde, H. The Ultra-centrifuge, a New Instrument for the Determination of Size and Distribution of Size of Particle In Amicroscopic Colloids. J. Am. Chem. Soc. 1924, 46 (12), 2677– 2693,  DOI: 10.1021/ja01677a011
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      Kim, J.; Eygeris, Y.; Gupta, M.; Sahay, G. Self-Assembled mRNA Vaccines. Adv. Drug Delivery Rev. 2021, 170, 83,  DOI: 10.1016/j.addr.2020.12.014
      18
      Self-assembled mRNA vaccines
      Kim, Jeonghwan; Eygeris, Yulia; Gupta, Mohit; Sahay, Gaurav
      Advanced Drug Delivery Reviews (2021), 170 (), 83-112CODEN: ADDREP; ISSN:0169-409X. (Elsevier B.V.)
      A review. MRNA vaccines have evolved from being a mere curiosity to emerging as COVID-19 vaccine front-runners. Recent advancements in the field of RNA technol., vaccinol., and nanotechnol. have generated interest in delivering safe and effective mRNA therapeutics. In this review, we discuss design and self-assembly of mRNA vaccines. Self-assembly, a spontaneous organization of individual mols., allows for design of nanoparticles with customizable properties. We highlight the materials commonly utilized to deliver mRNA, their physicochem. characteristics, and other relevant considerations, such as mRNA optimization, routes of administration, cellular fate, and immune activation, that are important for successful mRNA vaccination. We also examine the COVID-19 mRNA vaccines currently in clin. trials. mRNA vaccines are ready for the clinic, showing tremendous promise in the COVID-19 vaccine race, and have pushed the boundaries of gene therapy.
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    19. 19
      Gilbert, J.; Sebastiani, F.; Arteta, M. Y.; Terry, A.; Fornell, A.; Russell, R.; Mahmoudi, N.; Nylander, T. Evolution of the Structure of Lipid Nanoparticles for Nucleic Acid Delivery: From in Situ Studies of Formulation to Colloidal Stability. J. Colloid Interface Sci. 2024, 660, 66– 76,  DOI: 10.1016/j.jcis.2023.12.165
      There is no corresponding record for this reference.
    20. 20
      Szebeni, J.; Kiss, B.; Bozó, T.; Turjeman, K.; Levi-Kalisman, Y.; Barenholz, Y.; Kellermayer, M. Insights into the Structure of Comirnaty Covid-19 Vaccine: A Theory on Soft, Partially Bilayer-Covered Nanoparticles with Hydrogen Bond-Stabilized mRNA–Lipid Complexes. ACS Nano 2023, 17 (14), 13147– 13157,  DOI: 10.1021/acsnano.2c11904
      There is no corresponding record for this reference.
    21. 21
      Forchette, L.; Sebastian, W.; Liu, T. A Comprehensive Review of COVID-19 Virology, Vaccines, Variants, and Therapeutics. Curr. Med. Sci. 2021, 41 (6), 1037– 1051,  DOI: 10.1007/s11596-021-2395-1
      21
      A Comprehensive Review of COVID-19 Virology, Vaccines, Variants, and Therapeutics
      Forchette, Lauren; Sebastian, William; Liu, Tuoen
      Current Medical Science (2021), 41 (6), 1037-1051CODEN: CMSUCM; ISSN:2523-899X. (Huazhong University of Science and Technology)
      A review. Summary: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative pathogen of the coronavirus disease 2019 (COVID-19), has caused more than 179 million infections and 3.8 million deaths worldwide. Throughout the past year, multiple vaccines have already been developed and used, while some others are in the process of being developed. However, the emergence of new mutant strains of SARS-CoV-2 that have demonstrated immune-evading characteristics and an increase in infective capabilities leads to potential ineffectiveness of the vaccines against these variants. The purpose of this review article is to highlight the current understanding of the immunol. mechanisms of the virus and vaccines, as well as to investigate some key variants and mutations of the virus driving the current pandemic and their impacts on current management guidelines. We also discussed new technologies being developed for the prevention, treatment, and detection of SARS-CoV-2. In this paper, we thoroughly reviewed and provided crucial information on SARS-CoV-2 virol., vaccines and drugs being used and developed for its prevention and treatment, as well as important variant strains. Our review paper will be beneficial to health care professionals and researchers so they can have a better understanding of the basic sciences, prevention, and clin. treatment of COVID-19 during the pandemic. This paper consists of the most updated information that has been available as of June 21, 2021.
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    22. 22
      Heinz, F. X.; Stiasny, K. Distinguishing Features of Current COVID-19 Vaccines: Knowns and Unknowns of Antigen Presentation and Modes of Action. npj Vaccines 2021, 6 (1), 104  DOI: 10.1038/s41541-021-00369-6
      22
      Distinguishing features of current COVID-19 vaccines: knowns and unknowns of antigen presentation and modes of action
      Heinz, Franz X.; Stiasny, Karin
      npj Vaccines (2021), 6 (1), 104CODEN: VACCBC; ISSN:2059-0105. (Nature Research)
      A review. COVID-19 vaccines were developed with an unprecedented pace since the beginning of the pandemic. Several of them have reached market authorization and mass prodn., leading to their global application on a large scale. This enormous progress was achieved with fundamentally different vaccine technologies used in parallel. mRNA, adenoviral vector as well as inactivated whole-virus vaccines are now in widespread use, and a subunit vaccine is in a final stage of authorization. They all rely on the native viral spike protein (S) of SARS-CoV-2 for inducing potently neutralizing antibodies, but the presentation of this key antigen to the immune system differs substantially between the different categories of vaccines. In this article, we review the relevance of structural modifications of S in different vaccines and the different modes of antigen expression after vaccination with genetic adenovirus-vector and mRNA vaccines. Distinguishing characteristics and unknown features are highlighted in the context of protective antibody responses and reactogenicity of vaccines.
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    23. 23
      Buschmann, M. D.; Carrasco, M. J.; Alishetty, S.; Paige, M.; Alameh, M. G.; Weissman, D. Nanomaterial Delivery Systems for mRNA Vaccines. Vaccines 2021, 9 (1), 65  DOI: 10.3390/vaccines9010065
      23
      Nanomaterial delivery systems for mRNA vaccines
      Buschmann, Michael D.; Carrasco, Manuel J.; Alishetty, Suman; Paige, Mikell; Alameh, Mohamad Gabriel; Weissman, Drew
      Vaccines (Basel, Switzerland) (2021), 9 (1), 65CODEN: VBSABP; ISSN:2076-393X. (MDPI AG)
      The recent success of mRNA vaccines in SARS-CoV-2 clin. trials is in part due to the development of lipid nanoparticle delivery systems that not only efficiently express the mRNA-encoded immunogen after i.m. injection, but also play roles as adjuvants and in vaccine reactogenicity. We present an overview of mRNA delivery systems and then focus on the lipid nanoparticles used in the current SARS-CoV-2 vaccine clin. trials. The review concludes with an anal. of the determinants of the performance of lipid nanoparticles in mRNA vaccines.
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    24. 24
      Tenchov, R.; Bird, R.; Curtze, A. E.; Zhou, Q. Lipid Nanoparticles─From Liposomes to mRNA Vaccine Delivery, a Landscape of Research Diversity and Advancement. ACS Nano 2021, 15 (11), 16982– 17015,  DOI: 10.1021/acsnano.1c04996
      24
      Lipid Nanoparticles-From Liposomes to mRNA Vaccine Delivery, a Landscape of Research Diversity and Advancement
      Tenchov, Rumiana; Bird, Robert; Curtze, Allison E.; Zhou, Qiongqiong
      ACS Nano (2021), 15 (11), 16982-17015CODEN: ANCAC3; ISSN:1936-0851. (American Chemical Society)
      A review. Lipid nanoparticles (LNPs) have emerged across the pharmaceutical industry as promising vehicles to deliver a variety of therapeutics. Currently in the spotlight as vital components of the COVID-19 mRNA vaccines, LNPs play a key role in effectively protecting and transporting mRNA to cells. Liposomes, an early version of LNPs, are a versatile nanomedicine delivery platform. A no. of liposomal drugs have been approved and applied to medical practice. Subsequent generations of lipid nanocarriers, such as solid lipid nanoparticles, nanostructured lipid carriers, and cationic lipid-nucleic acid complexes, exhibit more complex architectures and enhanced phys. stabilities. With their ability to encapsulate and deliver therapeutics to specific locations within the body and to release their contents at a desired time, LNPs provide a valuable platform for treatment of a variety of diseases. We present a landscape of LNP-related scientific publications, including patents and journal articles, based on anal. of the CAS Content Collection, the largest human-curated collection of published scientific knowledge. Rising trends are identified, such as nanostructured lipid carriers and solid lipid nanoparticles becoming the preferred platforms for numerous formulations. Recent advancements in LNP formulations as drug delivery platforms, such as antitumor and nucleic acid therapeutics and vaccine delivery systems, are discussed. Challenges and growth opportunities are also evaluated in other areas, such as medical imaging, cosmetics, nutrition, and agrochems. This report is intended to serve as a useful resource for those interested in LNP nanotechnologies, their applications, and the global research effort for their development.
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    25. 25
      Kloczewiak, M.; Banks, J. M.; Jin, L.; Brader, M. L. A Biopharmaceutical Perspective on Higher-Order Structure and Thermal Stability of mRNA Vaccines. Mol. Pharmaceutics 2022, 19 (7), 2022– 2031,  DOI: 10.1021/acs.molpharmaceut.2c00092
      25
      A Biopharmaceutical Perspective on Higher-Order Structure and Thermal Stability of mRNA Vaccines
      Kloczewiak, Marek; Banks, Jessica M.; Jin, Lin; Brader, Mark L.
      Molecular Pharmaceutics (2022), 19 (7), 2022-2031CODEN: MPOHBP; ISSN:1543-8384. (American Chemical Society)
      A review. Preservation of the integrity of macromol. higher-order structure is a tenet central to achieving biol. drug and vaccine product stability toward manufg., distribution, storage, handling, and administration. Given that mRNA lipid nanoparticles (mRNA-LNPs) are held together by an intricate ensemble of weak forces, there are some intriguing parallels to biol. drugs, at least at 1st glance. However, mRNA vaccines are not without unique formulation and stabilization challenges derived from the instability of unmodified mRNA and its limited history as a drug or vaccine. Since certain learning gained from biol. drug development may be applicable for the improvement of mRNA vaccines, we present a perspective on parallels and contrasts between the emerging role of higher-order structure pertaining to mRNA-LNPs compared to pharmaceutical proteins. In a recent publication, the location of mRNA encapsulated within lipid nanoparticles was identified, revealing new insights into the LNP structure, nanoheterogeneity, and microenvironment of the encapsulated mRNA mols. We extend those findings by considering the effect of encapsulation on mRNA thermal unfolding with the observation that encapsulation in LNPs increases mRNA unfolding temps.
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    26. 26
      Brader, M. L.; Williams, S. J.; Banks, J. M.; Hui, W. H.; Zhou, Z. H.; Jin, L. Encapsulation State of Messenger RNA inside Lipid Nanoparticles. Biophys. J. 2021, 120 (14), 2766– 2770,  DOI: 10.1016/j.bpj.2021.03.012
      26
      Encapsulation state of messenger RNA inside lipid nanoparticles
      Brader, Mark L.; Williams, Sean J.; Banks, Jessica M.; Hui, Wong H.; Zhou, Z. Hong; Jin, Lin
      Biophysical Journal (2021), 120 (14), 2766-2770CODEN: BIOJAU; ISSN:0006-3495. (Cell Press)
      Understanding the structure of mRNA (mRNA) lipid nanoparticles, and specifically the microenvironment of the mRNA mols. within these entities, is fundamental to advancing their biomedical potential. Here, we show that a permeating cationic dye, thionine, can serve as a cryogenic electron microscopy contrasting agent by binding selectively to encapsulated mRNA without disturbing lipid nanoparticle morphol. Cryo-electron microscopy images identify the mRNA location, revealing that mRNA may exist within solvent-filled cavities or may be substantially lipid assocd.
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    27. 27
      Cheng, M. H. Y.; Leung, J.; Zhang, Y.; Strong, C.; Basha, G.; Momeni, A.; Chen, Y.; Jan, E.; Abdolahzadeh, A.; Wang, X.; Kulkarni, J. A.; Witzigmann, D.; Cullis, P. R. Induction of Bleb Structures in Lipid Nanoparticle Formulations of mRNA Leads to Improved Transfection Potency. Adv. Mater. 2023, 35 (31), 2303370  DOI: 10.1002/adma.202303370
      27
      Induction of Bleb Structures in Lipid Nanoparticle Formulations of mRNA Leads to Improved Transfection Potency
      Cheng, Miffy Hok Yan; Leung, Jerry; Zhang, Yao; Strong, Colton; Basha, Genc; Momeni, Arash; Chen, Yihang; Jan, Eric; Abdolahzadeh, Amir; Wang, Xinying; Kulkarni, Jayesh A.; Witzigmann, Dominik; Cullis, Pieter R.
      Advanced Materials (Weinheim, Germany) (2023), 35 (31), 2303370CODEN: ADVMEW; ISSN:0935-9648. (Wiley-VCH Verlag GmbH & Co. KGaA)
      The transfection potency of lipid nanoparticle (LNP) mRNA systems is critically dependent on the ionizable cationic lipid component. LNP mRNA systems composed of optimized ionizable lipids often display distinctive mRNA-rich "bleb" structures. Here, it is shown that such structures can also be induced for LNPs contg. nominally less active ionizable lipids by formulating them in the presence of high concns. of pH 4 buffers such as sodium citrate, leading to improved transfection potencies both in vitro and in vivo. Induction of bleb structure and improved potency is dependent on the type of pH 4 buffer employed, with LNP mRNA systems prepd. using 300 mM sodium citrate buffer displaying max. transfection. The improved transfection potencies of LNP mRNA systems displaying bleb structure can be attributed, at least in part, to enhanced integrity of the encapsulated mRNA. It is concluded that enhanced transfection can be achieved by optimizing formulation parameters to improve mRNA stability and that optimization of ionizable lipids to achieve enhanced potency may well lead to improvements in mRNA integrity through formation of the bleb structure rather than enhanced intracellular delivery.
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    28. 28
      Whitehead, K. A.; Matthews, J.; Chang, P. H.; Niroui, F.; Dorkin, J. R.; Severgnini, M.; Anderson, D. G. In Vitro–In Vivo Translation of Lipid Nanoparticles for Hepatocellular siRNA Delivery. ACS Nano 2012, 6 (8), 6922– 6929,  DOI: 10.1021/nn301922x
      28
      In Vitro-In Vivo Translation of Lipid Nanoparticles for Hepatocellular siRNA Delivery
      Whitehead, Kathryn A.; Matthews, Jonathan; Chang, Philip H.; Niroui, Farnaz; Dorkin, J. Robert; Severgnini, Mariano; Anderson, Daniel G.
      ACS Nano (2012), 6 (8), 6922-6929CODEN: ANCAC3; ISSN:1936-0851. (American Chemical Society)
      A significant challenge in the development of clin. viable siRNA delivery systems is a lack of in vitro-in vivo translatability: many delivery vehicles that are initially promising in cell culture do not retain efficacy in animals. Despite its importance, little information exists on the predictive nature of in vitro methodologies, most likely due to the cost and time assocd. with generating in vitro-in vivo data sets. Recently, high-throughput techniques have been developed that have allowed the examn. of hundreds of lipid nanoparticle formulations for transfection efficiency in multiple exptl. systems. The large resulting data set has allowed the development of correlations between in vitro and characterization data and in vivo efficacy for hepatocellular delivery vehicles. Consistency of formulation technique and the type of cell used for in vitro expts. was found to significantly affect correlations, with primary hepatocytes and HeLa cells yielding the most predictive data. Interestingly, in vitro data acquired using HeLa cells were more predictive of in vivo performance than mouse hepatoma Hepa1-6 cells. Of the characterization parameters, only siRNA entrapment efficiency was partially predictive of in vivo silencing potential, while zeta-potential and, surprisingly, nanoparticle size (when <300 nm) as measured by dynamic light scattering were not. These data provide guiding principles in the development of clin. viable siRNA delivery materials and have the potential to reduce exptl. costs while improving the translation of materials into animals.
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    29. 29
      Paunovska, K.; Sago, C. D.; Monaco, C. M.; Hudson, W. H.; Castro, M. G.; Rudoltz, T. G.; Kalathoor, S.; Vanover, D. A.; Santangelo, P. J.; Ahmed, R.; Bryksin, A. V.; Dahlman, J. E. A Direct Comparison of in Vitro and in Vivo Nucleic Acid Delivery Mediated by Hundreds of Nanoparticles Reveals a Weak Correlation. Nano Lett. 2018, 18 (3), 2148– 2157,  DOI: 10.1021/acs.nanolett.8b00432
      29
      A direct comparison of in vitro and in vivo nucleic acid delivery mediated by hundreds of nanoparticles reveals a weak correlation
      Paunovska, Kalina; Sago, Cory D.; Monaco, Christopher M.; Hudson, William H.; Castro, Marielena Gamboa; Rudoltz, Tobi G.; Kalathoor, Sujay; Vanover, Daryll A.; Santangelo, Philip J.; Ahmed, Rafi; Bryksin, Anton V.; Dahlman, James E.
      Nano Letters (2018), 18 (3), 2148-2157CODEN: NALEFD; ISSN:1530-6984. (American Chemical Society)
      Endothelial cells and macrophages play active roles in disease and as a result are important targets for nucleic acid therapies. While thousands of chem. distinct lipid nanoparticles (LNPs) can be synthesized to deliver nucleic acids, studying more than a few LNPs in vivo is challenging. As a result, it is difficult to understand how nanoparticles target these cells in vivo. Using high throughput LNP barcoding, we quantified how well LNPs delivered DNA barcodes to endothelial cells and macrophages in vitro, as well as endothelial cells and macrophages isolated from the lung, heart, and bone marrow in vivo. We focused on two fundamental questions in drug delivery. First, does in vitro LNP delivery predict in vivo LNP delivery. By comparing how 281 LNPs delivered barcodes to endothelial cells and macrophages in vitro and in vivo, we found in vitro delivery did not predict in vivo delivery. Second, does LNP delivery change within the microenvironment of a tissue. We quantified how 85 LNPs delivered barcodes to eight splenic cell populations, and found that cell types derived from myeloid progenitors tended to be targeted by similar LNPs, relative to cell types derived from lymphoid progenitors. These data demonstrate that barcoded LNPs can elucidate fundamental questions about in vivo nanoparticle delivery.
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  • Supporting Information

    Supporting Information

    The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsami.4c20077.

    • Mathematical deduction of eq 1; photos of DiD-labeled LNPs before and after ultracentrifugation (Figure S1); RiboGreen assay results show encapsulation efficiency (Figure S2); LNP size distribution by cryoEM (Figure S3); other representative cryoEM images (Figure S4); cellular uptake and functional delivery of mRNA in Huh7 cells (Figure S5); quantification of CLSM images (Figure S6); and colocalization analysis shown as Pearson’s coefficient (Figure S7) (PDF)


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