In vivo endothelial siRNA delivery using polymeric nanoparticles with low molecular weight

Journal name:
Nature Nanotechnology
Volume:
9,
Pages:
648–655
Year published:
DOI:
doi:10.1038/nnano.2014.84
Received
Accepted
Published online
Corrected online

Abstract

Dysfunctional endothelium contributes to more diseases than any other tissue in the body. Small interfering RNAs (siRNAs) can help in the study and treatment of endothelial cells in vivo by durably silencing multiple genes simultaneously, but efficient siRNA delivery has so far remained challenging. Here, we show that polymeric nanoparticles made of low-molecular-weight polyamines and lipids can deliver siRNA to endothelial cells with high efficiency, thereby facilitating the simultaneous silencing of multiple endothelial genes in vivo. Unlike lipid or lipid-like nanoparticles, this formulation does not significantly reduce gene expression in hepatocytes or immune cells even at the dosage necessary for endothelial gene silencing. These nanoparticles mediate the most durable non-liver silencing reported so far and facilitate the delivery of siRNAs that modify endothelial function in mouse models of vascular permeability, emphysema, primary tumour growth and metastasis.

At a glance

Figures

  1. 7C1 synthesis, characterization and in vivo biodistribution.
    Figure 1: 7C1 synthesis, characterization and in vivo biodistribution.

    a, 7C1 synthesis scheme. b, Target gene expression 24 h following 30 nM treatment with siRNA in human cervical carcinoma (HeLa), human primary endothelial (HMVEC) and murine endothelial (bEnd.3) cells. HeLa target gene expression was measured as Firefly luminescence in HeLa cells expressing Luciferase that were treated with siRNA targeting luciferase. bEnd.3 and HMVEC target gene expression was measured as Tie2 mRNA levels following treatment with siRNA targeting Tie2. c, 7C1 formulation scheme. 7C1 nanoparticles were mixed with C14PEG2000 and siRNA in a high-throughput microfluidic chamber as previously described30. d, 7C1 internal structure characterized by cryo-TEM. Dark bands indicate lipid layers and light bands indicate regions with siRNA. e, Average 7C1 hydrodynamic diameter, measured by dynamic light scattering, and weighted by volume (N = 20 formulations). f, 6,P-toluidinylnapthalene-2-sulfonate (TNS) fluorescence of formulated 7C1 nanoparticles as a function of pH (used to measure 7C1 pKa). g, Representative confocal image of Alexa647-tagged siRNA complexed to 7C1 1 h after intravenous injection. CD31 is a ubiquitous marker for endothelium. h, Serum Cy5.5 concentration following injection with 7C1-Cy5.5 siRNA or naked Cy5.5 siRNA. i, Cy5.5 fluorescence per mg tissue after injection with 7C1-Cy5.5 siRNA. Tissues were removed after injection and weighed individually. Cy5.5 intensity was normalized to each individual tissue. Time points were selected to measure systemic siRNA accumulation after Cy5.5 was cleared from serum. N = 4–5 mice per group. In all cases, data are shown as mean ± s.d. ** <  0.005, ± >  0.75.

  2. 7C1 delivers siRNA to endothelial cells.
    Figure 2: 7C1 delivers siRNA to endothelial cells.

    a, Alexa647 fluorescence uptake in HMVEC cells following 7C1-Alexa647 treatment in the presence of small molecules inhibiting clathrin (Chlorpromazine), caveolin (Fillipin) and both endocytotic pathways (Dynasore). Representative images of cells treated with 7C1 or Dynasore are shown. b, ICAM-2/GapDH mRNA ratios (normalized to PBS-treated mice) following intravenous injection of 7C1-siICAM-2. c, VE-cadherin/GapDH mRNA ratios (normalized to PBS-treated mice) following intravenous injection of 7C1-siVEcad. d, VE-cadherin and β-actin protein expression following treatment with 7C1-siVEcad. e, Evans Blue Dye (EBD) pulmonary absorbance 7 and 14 days following a 0.6 mg kg−1 injection of 7C1-siVEcad. f, Target/GapDH mRNA ratios (normalized to PBS-treated mice) following injection of 7C1 formulated with siCntrol or five siRNAs targeting ICAM-2, Tie2, VE-cadherin, VEGFR2 or Tie1, respectively (siCombination). g, ICAM-2/GapDH mRNA levels as a function of time following a 0.6 mg kg−1 injection of siICAM-2. Data shown as mean ± s.d. N = 4–5 mice per group, * <  0.05, ** <  0.005.

  3. 7C1 preferentially delivers siRNA to pulmonary endothelial cells in vivo.
    Figure 3: 7C1 preferentially delivers siRNA to pulmonary endothelial cells in vivo.

    a, Alexa647 median fluorescent intensity in pulmonary endothelial (CD31+), haematopoietic (CD45+), epithelial (CD326+), B (CD19+) or T (TCRβ+) cells isolated from mice after treatment with 7C1 formulated with Alexa647-tagged siRNA. Statistical significance calculated between endothelial cells and other pulmonary cell types 1 h after injection. b, ICAM-2 median fluorescent intensity in pulmonary cells (normalized to siCntrol-treated mice) isolated from mice three days following treatment with 7C1-siICAM-2. c, Integrinβ1/β-actin mRNA ratios (normalized to siCntrol-treated mice) in pulmonary endothelial and epithelial cells isolated from mice two days after treatment with siIntegrinβ1. d, Factor 7 serum concentration (normalized to PBS-treated animals) two days following treatment with liver-targeting molecule Hepat01-siFactor7 or 7C1-siFactor7. e, Tie2 and Factor7/GapDH mRNA expression following a 0.15 mg kg−1 injection of 7C1 concurrently formulated with siTie2 and siFactor7. Particles were formulated with different 7C1:cholesterol:C14PEG2000 molar ratios. 7C1 decreased Tie2 mRNA expression in pulmonary, renal and hepatic endothelium without reducing F7 mRNA expression. f, CD45 median fluorescent intensity following treatment with 7C1-siCD45 or positive control C12-200-siCD45. gk, Mean linear intercept (MLI) between alveoli, pulmonary surface/volume ratio, total volume and pulmonary histology following two 0.5 mg kg−1 injections of siCntrol or siVEGFR2. Increased MLI, alveolar volume, decreased surface/volume ratios and constant infiltrating myeloid cells are consistent with an induced emphysema-like phenotype (N = 6–7 animals per group; data shown as average ± s.d.). ** <  0.002, *** <  0.001.

  4. 7C1-mediated mRNA silencing modifies endothelial function in vivo.
    Figure 4: 7C1-mediated mRNA silencing modifies endothelial function in vivo.

    a, Primary Lewis lung carcinoma (LLC) growth following three 1.0 mg kg−1 treatments with PBS, siCntrol, siVEGFR-1 or siDll4 (N = 7–10 animals per group; data shown as average ± s.e.m.). b,c, Representative images (b) and quantification of cleaved caspase 3 (CC3) staining (c), a marker for apoptosis, following treatment with PBS, siCntrol, siVEGFR-1, siDll4. Normalized CC3+ area defined as the total CC3+ surface area divided by the tumour surface area. d, Number of pulmonary surface metastases following four 1.0 mg kg−1 injections with PBS, siCntrol, siVEGFR-1 or siDll4 (N = 4–6 per group; data shown as average ± s.e.m.). To measure effects independent of primary tumour growth, animals were not treated until after primary tumour resection. e, Murine lungs with metastatic lesion removed after treatment with PBS, siLuc, siVEGFR-1 or siDll4. *P < 0.05, ** <  0.002, *** <  0.001.

Change history

Corrected online 20 June 2014
In the version of this Article originally published online, the following authors' names were written incorrectly: Victor Koteliansky, Omar F. Khan and Kamaljeet Singh Sandhu. These have now been corrected in all versions of the Article.

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Author information

  1. These authors contributed equally to this work

    • James E. Dahlman &
    • Carmen Barnes

Affiliations

  1. Harvard-MIT Division of Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA

    • James E. Dahlman,
    • Robert Langer &
    • Daniel G. Anderson
  2. David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA

    • James E. Dahlman,
    • Omar F. Khan,
    • Siddharth Jhunjunwala,
    • Taylor E. Shaw,
    • Yiping Xing,
    • Gaurav Sahay,
    • Andrew Bader,
    • Roman L. Bogorad,
    • Hao Yin,
    • Yizhou Dong,
    • Shan Jiang,
    • Danielle Seedorf,
    • Apeksha Dave,
    • Kamaljeet Singh Sandhu,
    • Matthew J. Webber,
    • Vera M. Ruda,
    • Abigail K. R. Lytton-Jean,
    • Christopher G. Levins,
    • Robert Langer &
    • Daniel G. Anderson
  3. Institute for Medical Engineering and Science, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA

    • James E. Dahlman,
    • Robert Langer &
    • Daniel G. Anderson
  4. Alnylam Pharmaceuticals, Cambridge, Massachusetts 02139, USA

    • Carmen Barnes,
    • Lauren Speciner,
    • Tim Racie,
    • Tatiana Novobrantseva,
    • Klaus Charisse,
    • Kevin Fitzgerald &
    • Akin Akinc
  5. Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA

    • Omar F. Khan,
    • Robert Langer &
    • Daniel G. Anderson
  6. Department of Microbiology and Immunology, Harvard Medical School, Boston 02115, USA

    • Aude Thiriot,
    • Mark W. Kieran,
    • Ulrich H. von Andrian &
    • Dipak Panigrahy
  7. Center for Systems Biology, Massachusetts General Hospital and Harvard Medical School, Boston 02114, USA

    • Hendrik B. Sager,
    • Partha Dutta &
    • Matthias Nahrendorf
  8. Vascular Biology Program, Children's Hospital Boston, and Division of Pediatric Oncology, Dana Farber Cancer Institute, Harvard Medical School, Boston 02115, USA

    • Brian Kalish &
    • Dayna K. Mudge
  9. Program in Translational Lung Research, Division of Pulmonary Sciences and Critical Care Program, Department of Medicine, University of Colorado School of Medicine, USA

    • Mario Perez,
    • Lynelle Smith &
    • Rubin M. Tuder
  10. Department of Biotechnology and Food Engineering and The Russell Berrie Nanotechnology Institute, Technion Israel Institute of Technology, Haifa 3200, Israel

    • Ludmila Abezgauz &
    • Dganit Danino
  11. Department of Chemical Engineering, Technion Israel Institute of Technology, Haifa 32000, Israel

    • Avi Schroeder
  12. Skolkovo Institute of Science and Technology, Skolkovo 143025, Russian Federation

    • Victor Koteliansky

Contributions

J.E.D., C.B., V.K., R.L. and D.G.A. conceived the experiments. J.E.D., C.B., O.F.K., A.T., S.J., T.E.S., Y.X., H.B.S., G.S., L.S., A.B., R.L.B., H.Y., T.R., Y.D., S.J., D.S., A.D., K.S.S., M.J.W., T.N., V.M.R., A.K.R.L.J., C.G.L., B.K., D.K.M., M.P., L.A., P.D., L.S., K.C., M.W.K., K.F., M.N., D.D., R.M.T., U.H.V.A., A.A., A.S. and D.P. performed experiments. J.E.D., C.B., V.K., R.L. and D.G.A. co-wrote the paper. All authors discussed the results and commented on the manuscript.

Competing financial interests

R.L. is a shareholder and member of the Scientific Advisory Board of Alnylam. R.L and D.G.A have sponsored research grants from Alnylam. Alnylam also has a licence to certain intellectual property that was invented at the Massachusetts Institute of Technology by D.G.A. and R.L.

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