Increased PIP3 activity blocks nanoparticle mRNA delivery

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Science Advances  22 Jul 2020:
Vol. 6, no. 30, eaba5672
DOI: 10.1126/sciadv.aba5672
  • Fig. 1 PIP3 reduces nanoparticle-mediated mRNA delivery in vitro.

    (A) PIP3 blocks the functional delivery of nanoparticles (NP) carrying GFP mRNA. The data suggest that this is driven in part by reduced endosomal escape. (B) LNP1 and LNP2 chemical composition. Concurrent delivery of PIP3 and 50 ng of LNP1 carrying GFP mRNA led to decreased GFP expression in cells (C) Six hours and (D) 24 hours after transfection. Reduced GFP expression was also observed at (E) 6 hours and (F) 24 hours when mRNA was carried by L2K and administered at a dose of 400 ng per well. (G) Concurrent delivery of PIP3 and varying doses of LNP1 carrying GFP mRNA shows that the percentage of GFP+ cells decreases as the amount of PIP3 increases. (H) PIP3 administered before, concurrently, or after 50 ng of LNP1, 150 ng of LNP2, and 400 ng of L2K carrying GFP mRNA leads to a decrease in GFP expression. (I) PIP3 did not lead to changes in iMAEC cell morphology 24 hours after PIP3 administration. Scale bars, 10 μm. Nuclei stained with DAPI and cytoskeleton (phalloidin) stained with GFP.

  • Fig. 2 Analysis of the effects PIP3 has on LNP uptake and endosomal escape.

    (A) We reasoned that PIP3 could reduce LNP delivery by reducing LNP serum stability, inhibiting cell uptake or endosomal escape, or by altering the metabolic state of the cell. (B to E) LNPs containing GFP mRNA were formulated with fluorescent phospholipids and administered to cells. Cell normalized fluorescence was determined relative to an untreated control at each time point. Normalized fluorescence did not change over time in (B) iMAECs and did change slightly in (C) HEKs after administration of LNP1 and PIP3. Similarly, PIP3 had a minimal effect on LNP2 uptake, measured by MFI, in either (D) iMAECs or (E) HEKs. Normalized AF-647 MFI was also determined relative to an untreated control at each time point. *P < 0.0332, **P < 0.0021, ***P < 0.0002, and ****P < 0.0001. (F) In cells treated with PIP3, LNPs (red) colocalize with endosomes (green), whereas the opposite is observed without PIP3. (G) Colocalization images of endosomes and LNPs can be used to draw (H) representative line profiles. (I) The M1 coefficient shows a significant difference in colocalization of LNP and endosome for cells treated with PIP3 after 30 min and 6 hours. (J) The M2 coefficient is low, indicating that, as expected, there are more endosomes than endosomes colocalized with LNPs. All microscopy images are shown with 10-μm scale bars. Although only a few representative cells are shown, the M1/M2 coefficient generation represents colocalization analysis of more than 30 cells per condition. AU, arbitrary units.

  • Fig. 3 PIP3 changes the transcriptional profile of a cell.

    More genes are up-regulated/down-regulated (A) 6 hours after PIP3 administration than (B) 24 hours after PIP3 administration. Most perturbed genes were unannotated; however, there was a noticeable change in genes associated with metabolism both (C) 6 hours and (D) 24 hours after PIP3 administration. Padj, adjusted P value.

  • Fig. 4 PIP3 changes the metabolic state of the cell.

    (A) PCA revealed distinct grouping between cells 24 hours after they were treated with PIP3 or not treated with PIP3. Similar distinctions between treated and untreated cells at 24 hours were found using (B) joint hierarchical clustering, in this case, displaying metabolites across the seven samples. Significance analysis identified metabolites that were then mapped upon a (C) metabolic pathway diagram, highlighting significantly up-regulated and down-regulated metabolites in the PIP3-positive group compared to the PIP3-negative control at both 6 and 24 hours. Common metabolic pathways in human metabolism are shown, with orange arrows representing multi-reaction steps in these pathways; maroon labels such as “protein synthesis” represent additional cellular metabolic pathways and phenotypes significantly influenced from the canonical metabolites and pathways listed.

  • Fig. 5 PIP3 reduces LNP mRNA delivery in vivo.

    (A) Ai14 mice were injected with PIP3 (10 mg/kg) and then immediately injected with LNP carrying Cre mRNA (1 mg/kg). PIP3 consistently blocks functional LNP1 delivery in (B) lung endothelial cells (ECs) and functional LNP2 delivery in (C) splenic endothelial cells. (D) LNP3 was formulated to deliver Cre mRNA at 0.3 mg/kg. (E) PIP3 blocked LNP-mediated delivery of Cre mRNA to hepatocytes. Statistical analyses are done comparing the positive control (LNP only) to the treatment group (LNP + PIP3) using an unpaired t test. *P < 0.0332, **P < 0.0021, and ***P < 0.0002.

Supplementary Materials

  • Supplementary Materials

    Increased PIP3 activity blocks nanoparticle mRNA delivery

    Kalina Paunovska, Alejandro Da Silva Sanchez, Matthew T. Foster, David Loughrey, Emmeline L. Blanchard, Fatima Z. Islam, Zubao Gan, Athanasios Mantalaris, Philip J. Santangelo, James E. Dahlman

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