Research ArticleHEALTH AND MEDICINE

Ionizable lipid nanoparticles for in utero mRNA delivery

See allHide authors and affiliations

Science Advances  13 Jan 2021:
Vol. 7, no. 3, eaba1028
DOI: 10.1126/sciadv.aba1028
  • Fig. 1 Overview of LNP formulation and fetal delivery for this study.

    First, ionizable lipid structures were prepared by Michael addition chemistry. Then, the ionizable lipids, PEG-lipid, DOPE phospholipid, and cholesterol were combined into an ethanol phase, and luciferase mRNA was diluted into an aqueous phase. Both phases were mixed at controlled flow rates in microfluidic devices. After LNP formulation, LNPs were injected to individual mouse fetuses through the vitelline vein, which directly delivers to sinusoids in the fetal liver. After 4 or 24 hours, fetuses and tissues were extracted for imaging and further analysis.

  • Fig. 2 Design and characterization of LNPs for fetal mRNA delivery.

    (A) Chemical structures of the polyamine cores (left) and epoxide terminated alkyl tails (right) that were combined to generate the ionizable lipids used in this study. Throughout this paper, LNPs are named for their ionizable lipid component’s alkyl tail length (A = C12, B = C14, and C = C16) as well as their polyamine core (numbered 1 to 5). PA, polyamine. (B) Graphed analyses of LNP pKa for representative NPs A-3 and B-4. The pKa for each LNP was calculated by determining the pH that corresponds to normalized TNS fluorescence at 0.5. (C) LNP characterization table showing hydrodynamic diameter (intensity), encapsulation efficiency, and pKa for each LNP formulation.

  • Fig. 3 LNP-mediated mRNA delivery to fetuses.

    (A) Schematic (left) and photograph (right) showing the vitelline vein injection in a mouse fetus. Photo credit: Andrew Cheng, The Children’s Hospital of Philadelphia. (B) IVIS imaging showing luciferase expression in a representative dam (left) and within the exposed uterine horn (right). (C) IVIS images (left) and quantification (right) of luciferase signal in fetuses following surgical removal from dams. (D) IVIS images (left) and quantification (right) of luciferase signal from livers of fetuses injected with LNPs. Each fetus was injected via the vitelline vein, extracted, and imaged by IVIS 4 hours after injection. Quantifications are the normalized total flux calculated by dividing the luminescence from the area of interest by the background from each individual image. The normalized total flux was averaged across injected fetuses. *P < 0.001 by one-way analysis of variance (ANOVA) with post hoc Tukey-Kramer compared to all other treatment groups, unless indicated otherwise, and outliers were detected using Grubbs’ test and removed from analysis; minimum n = 3 per treatment group; error bars represent SEM.

  • Fig. 4 LNP-mediated mRNA delivery to fetal intestines and lungs.

    (A) IVIS images (left) and quantification (right) of luciferase signal in the lungs. n.s., not significant. (B) IVIS images (left) and quantification (right) of luciferase signal in the intestines. Normalized total flux was averaged across injected fetuses. *P < 0.05 by one-way ANOVA with post hoc Tukey-Kramer compared to all other treatment groups, unless indicated otherwise; minimum n = 3 fetuses per treatment group; error bars represent SEM.

  • Fig. 5 LNPs deliver GFP mRNA and EPO mRNA in utero.

    (A) GFP expression in fetal livers 24 hours after injection with LNPs A-3.GFP or B-4.GFP, or PBS. Scale bars, 150 μm (75×) and 750 μm (16×). eGFP, enhanced GFP. (B) The same fetal livers in (A) were processed into single-cell suspensions and analyzed using flow cytometry to record the percentage of CD45 and GFP+ cells. Dot plots show one liver sample per treatment group to demonstrate the gating and quantification. (C) EPO content in fetal livers (left) at 4 hours (left) or 24 hours (right) after injection of LNPs pA-3.EPO or pB-4.EPO, or PBS. EPO concentrations were averaged across three fetuses per treatment group and analyzed by two-way ANOVA comparing mean EPO concentration among treatment groups; *P < 0.02 and **P < 0.001; error bars represent SEM.

  • Fig. 6 Evaluation of fetal survival and toxicity following LNP injections.

    (A) Percent survival of fetuses injected at E16 and surgically delivered at E19. Survival was determined immediately following extraction. Error bars represent SD from three dams following injection to every fetus in each dam. (B) Liver enzyme analysis from fetal liver tissue collected at E19 following injection at E16. Measured AST or ALT units were normalized by dividing by protein concentration from fetal liver tissue. (C) Cytokine analysis from fetal livers collected immediately following surgical delivery at E19. *P < 0.0001 by two-way ANOVA compared to each treatment group for each cytokine; n = 5 fetuses per treatment group. (D) Liver enzyme analysis, (E) complement system activation, and (F) cytokine analysis from plasma collected from dams at E19 before surgical delivery of the injected fetuses. The following cytokines were out of range of the instrument and is therefore not shown: IFN-𝛾, IL-10, IL-12 (p40), IL-12 (p70), IL-1b, IL-2, IL-4, tumor necrosis factor–α (TNFα), and vascular endothelial growth factor (VEGF); n = 3 dams per treatment group. *P < 0.02 and **P < 0.0001 by two-way ANOVA compared to each treatment group for each cytokine; n = 3 dams per treatment group. Error bars in (B) to (F) represent SEM with outliers detected by Grubbs’ test and removed from analysis.

Supplementary Materials

  • Supplementary Materials

    Ionizable lipid nanoparticles for in utero mRNA delivery

    Rachel S. Riley, Meghana V. Kashyap, Margaret M. Billingsley, Brandon White, Mohamad-Gabriel Alameh, Sourav K. Bose, Philip W. Zoltick, Hiaying Li, Rui Zhang, Andrew Y. Cheng, Drew Weissman, William H. Peranteau, Michael J. Mitchell

    Download Supplement

    This PDF file includes:

    • Figs. S1 to S7

    Files in this Data Supplement:

Stay Connected to Science Advances

Navigate This Article