Research ArticleAPPLIED SCIENCES AND ENGINEERING

Carboxylated branched poly(β-amino ester) nanoparticles enable robust cytosolic protein delivery and CRISPR-Cas9 gene editing

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Science Advances  06 Dec 2019:
Vol. 5, no. 12, eaay3255
DOI: 10.1126/sciadv.aay3255
  • Fig. 1 Design and characterization of self-assembled carboxylated branched PBAE protein nanoparticles.

    (A) Assembly of carboxylated branched PBAEs with proteins. (B) Structures of carboxylate ligands C1 to C10, arranged in order of increasing hydrophobicity. (C) Hydrodynamic diameter and zeta potentials of nanoparticles formulated with BSA (30 w/w) as measured by DLS. Data are presented as means + SD (n = 3). Statistical comparisons of nanoparticle diameter were performed with one-way analysis of variance (ANOVA) with Dunnett’s post hoc tests against the C5 group. *P < 0.05 and **P < 0.01. ns, not significant. Similar statistical comparisons were made with zeta potential data, and no significant differences were observed. (D) Representative transmission electron microscopy (TEM) images of C5/BSA nanoparticles.

  • Fig. 2 Carboxylated PBAE nanoparticles mediate cytosolic protein delivery.

    (A) Average fluorescence intensity of cells treated with carboxylated PBAE nanoparticles encapsulating FITC-BSA (300 ng of FITC-BSA per well, 20 w/w). Data are presented as means + SD (n = 4); statistical significance is determined by one-way ANOVA with Dunnett’s post hoc tests comparing uptake levels to that of the nanoparticle formulation achieving the highest levels of FITC-BSA uptake in each cell line. ***P < 0.001 and ****P < 0.0001. (B) Uptake by HEK cells in the presence of different endocytosis inhibitors. CPZ, chlorpromazine; MCD, methyl-β-cyclodextrin; GEN, genistein; CYD, cytochalasin D. Data are presented as means ± SD; statistical significance is determined by one-way ANOVA with Dunnett’s post hoc tests as compared to the control group (n = 4). *P < 0.05, **P < 0.01, and ****P < 0.0001. (C) Confocal images of HEK cells treated with C5/FITC-BSA nanoparticles or protein alone for 4 hours. Scale bar, 10 μm.

  • Fig. 3 Gal8-GFP recruitment assay to assess nanoparticle-mediated endosomal disruption.

    (A) Gal8 recruitment overview. In cells with intact endosomes, Gal8-GFP is dispersed throughout endosomes with no interactions with intraendosomal glycans. Gal8-GFP binds glycans in disrupted endosomes, resulting in punctate fluorescent dots. (B) Gal8-GFP recruitment was quantified by image-based analysis. Individual cells were identified through nuclear staining (left); Gal8-GFP recruitment could be visualized in the green fluorescence channel (middle); punctate GFP+ spots were identified and counted (red dots). (C) Representative images of Gal8-GFP+ B16 cells treated with carboxylated PBAE/BSA nanoparticles (125-ng BSA per well, 25 w/w; scale bar, 50 μm). (D) Endosomal disruption level quantified by the number of Gal8-GFP spots per cell. Data are presented as means ± SD; statistical significance is determined by one-way ANOVA with Dunnett’s post hoc tests as compared to the C5 group (n = 4). *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

  • Fig. 4 Carboxylated C5 polymeric nanoparticles for cytosolic delivery of different protein types.

    (A and B) Confocal images of HEK cells treated with C5 nanoparticles encapsulating FITC-IgG (A) and GFP (B) for 4 hours; 450 ng of protein was delivered per well at 30 w/w (scale bars, 50 μm). (C) Functional delivery of ribosome-inactivating protein saporin resulted in significant levels of cell death; the final polymer concentration per well was 0.075 μg/μl. Data are presented as means ± SD (n = 4). (D) Representative images of CT-2A cells treated with 10 nM naked saporin or C5/saporin nanoparticles. (E) Molecular weight (MW) and isoelectric point (pI) of proteins delivered by C5 nanoparticles.

  • Fig. 5 C5 nanoparticle delivery of Cas9 RNPs enables robust CRISPR gene editing in vitro.

    (A) Fluorescence microscopy images of HEK-GFPd2 cells treated with RNPs alone or C5 + RNPs; C5 + RNPs enabled knockout of GFP fluorescence. Scale bar, 50 μm. (B) Flow cytometry quantification of GFP knockout in HEK and GL261 cells. Data are means + SD (n = 4). Editing level of the C5/RNP group for each cell line was compared to that of the corresponding RNP-only group using Holm-Sidak corrected multiple t tests. ****P < 0.0001. (C) Surveyor mutation detector assay of GL261-GFPd2 cells treated with C5 + RNP nanoparticles. (D) Experimental design of HDR assay in the CXCR4 gene; knock-in of a 12-nt insert flanked by homology arms (HA) results in the addition of a Hind III restriction enzyme site. (E) Quantification of total editing (via Surveyor assay) and HDR (via Hind III restriction digest) in HEK cells. Data are means + SD (n = 3). (F) Hind III restriction enzyme assay (top) and Surveyor assay (bottom) of HEK cells treated with different C5/RNP/donor DNA combinations; the orange arrowhead indicates HDR. (G) Inference of CRISPR Edits analysis of Sanger sequencing data from C5 + RNP + donor DNA-treated cells provides a breakdown of different edits. Percentages indicate the percentage of the total DNA population with the indicated genotype. The targeted sequence is highlighted in gray, and the protospacer adjacent motif sequence is highlighted in yellow.

  • Fig. 6 C5/RNP nanoparticles enable CRISPR editing in vivo.

    (A) Schematic of CRISPR-stop gene construct; deletion of a 630-bp expression stop cassette turns on downstream ReNL expression. (B) Direct intracranial administration of C5/RNP nanoparticles to an orthotopic GL261-stop-ReNL tumor enabled CRISPR editing in vivo. Nanoparticles were formulated at 3.5-pmol RNP with C5 polymer (15 w/w). Tumor boundary is outlined in white.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/12/eaay3255/DC1

    Fig. S1. Synthesis and characterization of carboxylated branched PBAE polymers.

    Fig. S2. Synthesis and characterization of carboxylate ligands.

    Fig. S3. Cell viability after treatment with carboxylated branched PBAE protein nanoparticles.

    Fig. S4. Confocal images of cells treated with C5/FITC-BSA nanoparticles.

    Fig. S5. Characterization of polymer pH buffering and endosomal disruption capabilities.

    Fig. S6. C5/RNP nanoparticles enable in vitro gene deletion.

    Fig. S7. C5/RNP nanoparticles are stable in serum-containing media and in lyophilized form.

    Fig. S8. C5/RNP nanoparticle-enabled in vivo CRISPR editing is reproducible.

    Table S1. Characteristics of proteins and encapsulated C5 nanoparticles and optimal nanoparticle formulations used in this study.

    Table S2. DNA sequences.

  • Supplementary Materials

    This PDF file includes:

    • Fig. S1. Synthesis and characterization of carboxylated branched PBAE polymers.
    • Fig. S2. Synthesis and characterization of carboxylate ligands.
    • Fig. S3. Cell viability after treatment with carboxylated branched PBAE protein nanoparticles.
    • Fig. S4. Confocal images of cells treated with C5/FITC-BSA nanoparticles.
    • Fig. S5. Characterization of polymer pH buffering and endosomal disruption capabilities.
    • Fig. S6. C5/RNP nanoparticles enable in vitro gene deletion.
    • Fig. S7. C5/RNP nanoparticles are stable in serum-containing media and in lyophilized form.
    • Fig. S8. C5/RNP nanoparticle-enabled in vivo CRISPR editing is reproducible.
    • Table S1. Characteristics of proteins and encapsulated C5 nanoparticles and optimal nanoparticle formulations used in this study.
    • Table S2. DNA sequences.

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