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Cited by 601RNA Interference and Gene DeliveryNanoparticle-Based Drug DeliveryAdvancements in Transdermal Drug Delivery

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Naturally-occurring cholesterol analogues in lipid nanoparticles induce polymorphic shape and enhance intracellular delivery of mRNA

Open access
Patel, Siddharth, Ashwanikumar, N., Robinson, Ema, Xia, Yan, Mihai, Cosmin, Griffith, Joseph P., Hou, Shangguo, Esposito, Adam A., Ketova, Tatiana, Welsher, Kevin, Joyal, John L., Almarsson, Örn, Sahay, Gaurav·2020·Nature Communications
DOI 10.1038/s41467-020-14527-2PMC PMC7033178
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Abstract

Endosomal sequestration of lipid-based nanoparticles (LNPs) remains a formidable barrier to delivery. Herein, structure-activity analysis of cholesterol analogues reveals that incorporation of C-24 alkyl phytosterols into LNPs (eLNPs) enhances gene transfection and the length of alkyl tail, flexibility of sterol ring and polarity due to -OH group is required to maintain high transfection. Cryo-TEM displays a polyhedral shape for eLNPs compared to spherical LNPs, while x-ray scattering shows little disparity in internal structure. eLNPs exhibit higher cellular uptake and retention, potentially leading to a steady release from the endosomes over time. 3D single-particle tracking shows enhanced intracellular diffusivity of eLNPs relative to LNPs, suggesting eLNP traffic to productive pathways for escape. Our findings show the importance of cholesterol in subcellular transport of LNPs carrying mRNA and emphasize the need for greater insights into surface composition and structural properties of nanoparticles, and their subcellular interactions which enable designs to improve endosomal escape.

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Crossref · 10.1038/s41467-020-14527-2

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Figures

Fig. 1 Cholesterol analogs screened for enhanced mRNA-based gene transfection. a Schematic showing desorption of PEG-lipids from LNPs allows ApoE binding causing LDL-mediated cellular uptake in cells following which a small portion of nucleic acids escape the endosome and the majority are recycled back by lysosomal transporters or directed to degradative endocytic compartments. b IUPAC-IUB nomenclature of (1989) cholesterol ring system with rings (A, B, C, and D), carbon numbering from C-1 to C-29, c the structure of cholesterol, and d schematic diagram showing classification of cholesterol into three regions: head, body, and tail. e Classification of cholesterol analogs- Group I-9,10-secosteroids, Group II-C-24 alkyl steroids, Group III-pentacyclic steroids. Structural variations from cholesterol are highlighted in red. f Cholesterol analogs were screened for particle size (nm), mRNA encapsulation (percent), and transfection efficiency (200 ng mRNA). Transfection experiments were conducted with n = 3. Source data are provided as a Source Data file.
Fig. 1 Cholesterol analogs screened for enhanced mRNA-based gene transfection. a Schematic showing desorption of PEG-lipids from LNPs allows ApoE binding causing LDL-mediated cellular uptake in cells following which a small portion of nucleic acids escape the endosome and the majority are recycled back by lysosomal transporters or directed to degradative endocytic compartments. b IUPAC-IUB nomenclature of (1989) cholesterol ring system with rings (A, B, C, and D), carbon numbering from C-1 to C-29, c the structure of cholesterol, and d schematic diagram showing classification of cholesterol into three regions: head, body, and tail. e Classification of cholesterol analogs- Group I-9,10-secosteroids, Group II-C-24 alkyl steroids, Group III-pentacyclic steroids. Structural variations from cholesterol are highlighted in red. f Cholesterol analogs were screened for particle size (nm), mRNA encapsulation (percent), and transfection efficiency (200 ng mRNA). Transfection experiments were conducted with n = 3. Source data are provided as a Source Data file.CC BY · PubMed Central
Fig. 2 Structural features of C-24 alkyl derivatives that enhance gene transfection. a–d C-24 alkyl derivatives with variations in the tail and body were screened for their effect on size, mRNA encapsulation efficiency (EE), and transfection efficiency of LNPs (dosed at 10–200 ng mRNA per well for C-24 alkyl derivatives in b and 50 ng mRNA per well for sequential addition of double bonds in d). The variations of these structures from cholesterol and β-sitosterol are depicted as red and yellow, respectively. e–f Structural modification of the head group in β-sitosterol with either amino acids (polar) or acetate (non-polar) was also evaluated for its effect on nanoparticle size, encapsulation, and transfection of LNPs (200 ng mRNA per well). The color code is indicative of polarity (green), non-polarity (yellow), and structural difference from cholesterol (red). Horizontal dashed red line represents eLNP expression set to 100 percent. All experiments were conducted with n = 3; mean ± SD. Source data are provided as a Source Data file.
Fig. 2 Structural features of C-24 alkyl derivatives that enhance gene transfection. a–d C-24 alkyl derivatives with variations in the tail and body were screened for their effect on size, mRNA encapsulation efficiency (EE), and transfection efficiency of LNPs (dosed at 10–200 ng mRNA per well for C-24 alkyl derivatives in b and 50 ng mRNA per well for sequential addition of double bonds in d). The variations of these structures from cholesterol and β-sitosterol are depicted as red and yellow, respectively. e–f Structural modification of the head group in β-sitosterol with either amino acids (polar) or acetate (non-polar) was also evaluated for its effect on nanoparticle size, encapsulation, and transfection of LNPs (200 ng mRNA per well). The color code is indicative of polarity (green), non-polarity (yellow), and structural difference from cholesterol (red). Horizontal dashed red line represents eLNP expression set to 100 percent. All experiments were conducted with n = 3; mean ± SD. Source data are provided as a Source Data file.CC BY · PubMed Central
Fig. 3 Impact of β-sitosterol on nanoparticle structure and morphology. a Cryo-TEM showing structural difference between LNP and eLNP (white arrows showing edges not observed in the spherical LNPs). Scale = 100 nm. b Small-angle X-ray scattering profiles of LNPs and eLNPs containing MC3 or Lipid 9.
Fig. 3 Impact of β-sitosterol on nanoparticle structure and morphology. a Cryo-TEM showing structural difference between LNP and eLNP (white arrows showing edges not observed in the spherical LNPs). Scale = 100 nm. b Small-angle X-ray scattering profiles of LNPs and eLNPs containing MC3 or Lipid 9.CC BY · PubMed Central
Fig. 4 Intracellular delivery and endosomal escape of nanoparticles. a HeLa cells were exposed to β-sitosterol containing eLNPs at different doses (1–400 ng) and compared to cholesterol-containing LNPs for cellular uptake. Cy5-EGFP mRNA was used as a tracer, and high-content imaging was performed and quantified using the Cellomics software. (n = 3; mean ± SD. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001. Significance was determined using nonlinear regression with ANOVA.) Source data are provided as a Source Data file. b Uptake kinetics of LNPs and eLNPs at 10 ng mRNA per well (0–24 h) in HeLa cells. c 1st derivative of uptake of LNP and eLNP demonstrates substantially higher rate of uptake and retention in HeLa over the 24 h post-transfection. d Endosomal escape was visualized using smFISH. Representative fluorescent images showing mRNA, LNP, and image analysis after delivery with LNP or eLNP in HeLa cells. e Quantitative image analysis of the number of cytosolic mRNAs (turquoise bars) compared to the number of LNPs per cell (magenta bars) after cellular delivery. f Ratio of cytosolic mRNA delivery to total LNP uptake indicative of endosomal escape. g–j 3D-DyPLoT was performed for high spatiotemporal resolution of nanoparticle transport. g Representative trajectories of internalized LNP and eLNP. The measured 3D position is plotted at 1 ms temporal resolution. h Histograms of the log-linear efficiency for LNP (blue) and eLNP (red), both of which display a bimodal distribution of stationary and mobile. i Population of the stationary and mobile fractions for LNP and eLNP. Population was measured by fitting the histograms in h to a two-term Gaussian and extracting the area under the two modes of the distribution. n = 156 (LNP) and 286 (eLNP); mean ± SD. p = 0.0009. Significance was determined using t-test. j Best fit distributions of LNP and eLNP, showing a clear increase in the mobile fraction for eLNP.
Fig. 4 Intracellular delivery and endosomal escape of nanoparticles. a HeLa cells were exposed to β-sitosterol containing eLNPs at different doses (1–400 ng) and compared to cholesterol-containing LNPs for cellular uptake. Cy5-EGFP mRNA was used as a tracer, and high-content imaging was performed and quantified using the Cellomics software. (n = 3; mean ± SD. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001. Significance was determined using nonlinear regression with ANOVA.) Source data are provided as a Source Data file. b Uptake kinetics of LNPs and eLNPs at 10 ng mRNA per well (0–24 h) in HeLa cells. c 1st derivative of uptake of LNP and eLNP demonstrates substantially higher rate of uptake and retention in HeLa over the 24 h post-transfection. d Endosomal escape was visualized using smFISH. Representative fluorescent images showing mRNA, LNP, and image analysis after delivery with LNP or eLNP in HeLa cells. e Quantitative image analysis of the number of cytosolic mRNAs (turquoise bars) compared to the number of LNPs per cell (magenta bars) after cellular delivery. f Ratio of cytosolic mRNA delivery to total LNP uptake indicative of endosomal escape. g–j 3D-DyPLoT was performed for high spatiotemporal resolution of nanoparticle transport. g Representative trajectories of internalized LNP and eLNP. The measured 3D position is plotted at 1 ms temporal resolution. h Histograms of the log-linear efficiency for LNP (blue) and eLNP (red), both of which display a bimodal distribution of stationary and mobile. i Population of the stationary and mobile fractions for LNP and eLNP. Population was measured by fitting the histograms in h to a two-term Gaussian and extracting the area under the two modes of the distribution. n = 156 (LNP) and 286 (eLNP); mean ± SD. p = 0.0009. Significance was determined using t-test. j Best fit distributions of LNP and eLNP, showing a clear increase in the mobile fraction for eLNP.CC BY · PubMed Central
Fig. 5 Effect of defective cholesterol trafficking on eLNP-mediated gene transfection. LNPs and eLNPs were compared for their ability to deliver mRNA to a wild-type (WT) and NPC1 knockout (NPC1−/−) mouse embryonic fibroblasts (200 ng mRNA per well), and b patient-derived NPC1, and c NPC2 loss-of-function fibroblasts. All experiments were conducted with n = 3; mean ± SD. (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001. Significance was determined using multiple t-test.) Source data are provided as a Source Data file.
Fig. 5 Effect of defective cholesterol trafficking on eLNP-mediated gene transfection. LNPs and eLNPs were compared for their ability to deliver mRNA to a wild-type (WT) and NPC1 knockout (NPC1−/−) mouse embryonic fibroblasts (200 ng mRNA per well), and b patient-derived NPC1, and c NPC2 loss-of-function fibroblasts. All experiments were conducted with n = 3; mean ± SD. (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001. Significance was determined using multiple t-test.) Source data are provided as a Source Data file.CC BY · PubMed Central

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Authors
Patel, S., Ashwanikumar, N., Robinson, E., Xia, Y., Mihai, C., Griffith, J. P., Hou, S., Esposito, A. A., Ketova, T., Welsher, K., Joyal, J. L., Almarsson, Ö., & Sahay, G.
Affiliations
  • Oregon State University (US)
  • Moderna Therapeutics (United States) (US)
  • Duke University (US)
Corresponding author
Sahay, Gaurav
ORCID
Venue
Nature Communications
Publisher
Springer Science and Business Media LLC
Published
2020-02-20
Volume
11
Issue
1
Pages
983-983
ISSN
2041-1723 (ISSN-L)
DOI
10.1038/s41467-020-14527-2
PMC
PMC7033178
OpenAlex
W3006764150
Cited by
601
References
72
Citations by year
2026: 108 · 2025: 156 · 2024: 127 · 2023: 72 · 2022: 64 · 2021: 50 · 2020: 23
FWCI
16.94
Type
journal article
Language
en
Update notices
correction (2020-07-06)
Open access
Licence
Locations
  • Nature Communications (publishedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • RePEc: Research Papers in Economics (submittedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
  • U.S. Department of Health & Human Services | NIH | National Institute of Biomedical Imaging and Bioengineering (1R15EB021581)
  • U.S. Department of Health & Human Services | NIH | National Heart, Lung, and Blood Institute (1R01HL146736-01)
  • Cystic Fibrosis Foundation (SAHAY18G0)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Interference and Gene Delivery
  • Physical Sciences › Materials Science › Biomaterials › Nanoparticle-Based Drug Delivery
  • Life Sciences › Pharmacology, Toxicology and Pharmaceutics › Pharmaceutical Science › Advancements in Transdermal Drug Delivery
Keywords
cholesterol analogues, lipid nanoparticles, mRNA delivery, endosomal escape, nanoparticle morphology, transfection, cellular uptake, intracellular diffusion, structure-activity relationship
MeSH terms
RAW 264.7 Cells, Animals, Biological Transport, Active, Cell Line, Cholesterol, HeLa Cells, Humans, Lipids, Endosomes, RNA, Messenger, Sitosterols, Transfection, X-Ray Diffraction, Cryoelectron Microscopy, Mice, Nanoparticles, HEK293 Cells

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