Research ArticleBIOCHEMISTRY

A boronic acid–rich dendrimer with robust and unprecedented efficiency for cytosolic protein delivery and CRISPR-Cas9 gene editing

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Science Advances  12 Jun 2019:
Vol. 5, no. 6, eaaw8922
DOI: 10.1126/sciadv.aaw8922
  • Fig. 1 Boronic acid–rich dendrimer with robust efficiency in cytosolic protein delivery.

    (A) Mechanism of boronate-rich dendrimer in complexation with protein complex formation. The dendrimer could bind with both negatively and positively charged proteins via a combination of nitrogen-boronate complexation and cation-π and ionic interactions between the two species. (B) Boronic acid–rich dendrimers with different chemical structures show distinct behaviors in the delivery of proteins into HeLa cells. Fluorescently labeled BSA was used as the model protein here. (C) Model proteins with different molecular weights and pIs in this study.

  • Fig. 2 Boronic acid–rich dendrimer in cytosolic delivery.

    (A) Cytosolic delivery of BSA-FITC into HeLa cells by P0 to P4 for 4 hours. The doses of protein and dendrimer in each well were 6 and 8 μg, respectively. (B) DLS analysis of P0, P1, P2, P3, and P4/BSA complexes, and TEM of P4/BSA nanoparticles. Scale bar, 200 nm. (C) Structure of the PBA analog materials. (D) Confocal images and (E) flow cytometry analysis of HeLa cells treated with P4/BSA-FITC or analog complexes of P5 to P8 for 4 hours. The doses of protein and dendrimer were 6 and 8 μg in each well, respectively. Commercial reagents PULSin and TransEx were used as positive controls. (F) DLS analysis of P4, P5, P6, P7, and P8/BSA-FITC complexes. (G) Time-dependent internalization of P4/BSA-FITC by HeLa cells. (H) Viability of HeLa cells treated with P4/BSA, P4, and BSA complexes. The concentrations of materials were equal to those in cytosolic protein delivery.

  • Fig. 3 Cytosolic delivery of fluorescent proteins.

    (A) P4 in the cytosolic delivery of R-PE into HeLa cells for 4 hours. Commercial reagents PULSin and TransEx were used as positive controls. (B) Quantitative analysis of the protein internalization in (A) by flow cytometry. The dose of R-PE in each well was 0.5 μg. P4 in the cytosolic delivery of (C) negatively charged GFP, YFP, and RFP into HeLa cells for 4 hours. The dose of proteins in each well was 3 μg. P4 in the cytosolic delivery of (D) positively charged Cyt C, lysosome, and trypsin into HeLa cells for 4 hours. The doses of proteins in each well were 6, 6, and 3 μg, respectively, and the dose of P4 for all the experiments was 8 μg.

  • Fig. 4 Cytosolic delivery of native enzymes.

    (A) β-Gal catalyzes the hydrolysis of colorless substrate X-Gal into a blue-colored product. (B) X-Gal staining of the cells treated with β-Gal/P4 for 4 hours. (C) Enzymatic activity of the β-Gal after cytosolic delivery determined by a kit. The dose of β-Gal in each well was 5 μg. (D) HRP catalyzes colorless substrates Amplex Red or TMB into a red fluorescent product, resorufin, or a blue dye, respectively, in the presence of hydrogen peroxide. (E) Confocal microscopy images of HeLa cells treated with P4/HRP and Amplex Red. Commercial reagents PULSin and TransEx were used as positive controls. (F) HRP enzymatic activity in the transduced cell analysis by TMB assay. The dose of HRP in each well was 6 μg. The dose of P4 in the experiments was 8 μg in each well.

  • Fig. 5 Cytosolic delivery of toxic proteins.

    (A) Cytosolic delivery of saporin, RNase A, and trypsin into cancer cells leads to ribosome inactivity, RNA degradation, and intracellular protein hydrolysis, respectively, and cell death. Viability of MDA-MB-231 cells treated with (B) saporin/P4 and (C) RNase A/P4, and (D) HeLa cells treated with trypsin/P4 complexes determined by MTT assay. The concentration of trypsin was 40 μg/ml. Free saporin, RNase A, and trypsin were tested as controls. The dose of P4 in the experiments of cytosolic delivery of saporin, RNase A, and trypsin were 0.6, 0.6, and 2.5 μg in each well, respectively.

  • Fig. 6 Cytosolic delivery of Cas9 RNP for CRISPR-Cas9 gene editing.

    (A) P4-mediated Cas9/sgEGFP delivery for efficient EGFP genome editing. (B) DLS analysis of P4/Cas9 RNP nanoparticles. The inset is the TEM image of P4/Cas9 RNP nanoparticles. Scale bar, 500 nm. (C) Confocal images of 293T-EGFP cells treated with P4/RNP for 4 hours. Scale bar, 50 μm. (D) Flow cytometry analysis of 293T-EGFP cells treated with P4/RNP for 4 hours by means of mean fluorescence intensity. (E) Indel analysis from samples treated by P4/RNP. Commercial reagent CMAX was used as a positive control. T7E1 assay results from the intracellular delivery of P4/RNP targeting (F) AAVS1 and (G) HBB genes in 293T cells. (H) Genome editing efficiency, as revealed by the grayscale density of T7E1 results, after the intracellular delivery of RNP targeting EGFP locus (for 293T-EGFP cells), AAVS1 locus, and HBB locus (for 293T cells). Data represent mean ± SD (n = 3, Student’s t test, **P < 0.01). T7E1 assay results from the intracellular delivery of P4/RNP targeting (I) AAVS1 and (J) HBB genes in HCT-116 and HT-29 cells.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/6/eaaw8922/DC1

    Fig. S1. Characterization of the modified dendrimers by 1H NMR.

    Fig. S2. Cytosolic delivery of BSA.

    Fig. S3. Characterization of the BSA-FITC/dendrimer-RBITC complex by FRET.

    Fig. S4. Confocal images of HeLa cells treated with the BSA-FITC/P4 complex at different times.

    Fig. S5. Characterization of the protein/dendrimer complexes by DLS and TEM.

    Fig. S6. Enzyme activity of β-Gal, HRP, and RNase A.

    Fig. S7. CRISPR-Cas9 gene editing in different cells.

    Table S1. Sequences of sgRNA used in this study.

    Table S2. Primer sequences for PCR amplification of target genes.

  • Supplementary Materials

    This PDF file includes:

    • Fig. S1. Characterization of the modified dendrimers by 1H NMR.
    • Fig. S2. Cytosolic delivery of BSA.
    • Fig. S3. Characterization of the BSA-FITC/dendrimer-RBITC complex by FRET.
    • Fig. S4. Confocal images of HeLa cells treated with the BSA-FITC/P4 complex at different times.
    • Fig. S5. Characterization of the protein/dendrimer complexes by DLS and TEM.
    • Fig. S6. Enzyme activity of β-Gal, HRP, and RNase A.
    • Fig. S7. CRISPR-Cas9 gene editing in different cells.
    • Table S1. Sequences of sgRNA used in this study.
    • Table S2. Primer sequences for PCR amplification of target genes.

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