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Cited by 132RNA Interference and Gene DeliveryCellular transport and secretionNanoparticle-Based Drug Delivery

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Visualization of self-delivering hydrophobically modified siRNA cellular internalization

Open access
Ly, Socheata, Navaroli, Deanna M., Didiot, Marie-Cécile, Cardia, James, Pandarinathan, Lakshmipathi, Alterman, Julia F., Fogarty, Kevin, Standley, Clive, Lifshitz, Lawrence M., Bellve, Karl D., Prot, Matthieu, Echeverria, Dimas, Corvera, Silvia, Khvorova, Anastasia·2017·Nucleic Acids Research
DOI 10.1093/nar/gkw1005PMC PMC5224471
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Abstract

siRNAs are a new class of therapeutic modalities with promising clinical efficacy that requires modification or formulation for delivery to the tissue and cell of interest. Conjugation of siRNAs to lipophilic groups supports efficient cellular uptake by a mechanism that is not well characterized. Here we study the mechanism of internalization of asymmetric, chemically stabilized, cholesterol-modified siRNAs (sd-rxRNAs®) that efficiently enter cells and tissues without the need for formulation. We demonstrate that uptake is rapid with significant membrane association within minutes of exposure followed by the formation of vesicular structures and internalization. Furthermore, sd-rxRNAs are internalized by a specific class of early endosomes and show preferential association with epidermal growth factor (EGF) but not transferrin (Tf) trafficking pathways as shown by live cell TIRF and structured illumination microscopy (SIM). In fixed cells, we observe ∼25% of sd-rxRNA co-localizing with EGF and <5% with Tf, which is indicative of selective endosomal sorting. Likewise, preferential sd-rxRNA co-localization was demonstrated with EEA1 but not RBSN-containing endosomes, consistent with preferential EGF-like trafficking through EEA1-containing endosomes. sd-rxRNA cellular uptake is a two-step process, with rapid membrane association followed by internalization through a selective, saturable subset of the endocytic process. However, the mechanistic role of EEA1 is not yet known. This method of visualization can be used to better understand the kinetics and mechanisms of hydrophobic siRNA cellular uptake and will assist in further optimization of these types of compounds for therapeutic intervention.

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Figures

Figure 1. Structure of sd-rxRNA, gene silencing efficacy, and cellular internalization. (A) Chemical modifications of sd-rxRNA include 2′-O-methyl, 2′-fluoro, phosphorothioate linkages, and cholesterol covalently linked at the 3′ end of the sense strand by a tetraethylene glycol (TEG) linker. (B) Dose response was performed in HeLa cells using PPIB-targeting sd-rxRNA (UNT: untreated cells). mRNA levels were measured by QuantiGene Assay (Affymetrix). (C) COS-7 cells were treated with 250 nM sd-rxRNA-DY547 (red) and visualized by total internal reflection (TIRF) microscopy (see Supplementary Video 1). Scale bar = 10 μm.
Figure 1. Structure of sd-rxRNA, gene silencing efficacy, and cellular internalization. (A) Chemical modifications of sd-rxRNA include 2′-O-methyl, 2′-fluoro, phosphorothioate linkages, and cholesterol covalently linked at the 3′ end of the sense strand by a tetraethylene glycol (TEG) linker. (B) Dose response was performed in HeLa cells using PPIB-targeting sd-rxRNA (UNT: untreated cells). mRNA levels were measured by QuantiGene Assay (Affymetrix). (C) COS-7 cells were treated with 250 nM sd-rxRNA-DY547 (red) and visualized by total internal reflection (TIRF) microscopy (see Supplementary Video 1). Scale bar = 10 μm.CC BY · PubMed Central
Figure 2. sd-rxRNA is internalized through a saturable pathway. (A) HeLa cells were treated with 0.5 μM sd-rxRNA-Cy3 and imaged by live confocal microscopy. Uptake was quantified by measuring total fluorescence intensity at specific time points. (B) HeLa cells were either untreated (control) or pre-incubated with 2 μM unlabeled sd-rxRNA for 10 or 60 min. Cells were then treated with 0.5 μM sd-rxRNA-Cy3 and imaged by live cell confocal microscopy. sd-rxRNA fluorescence intensity was measured in a region only 10 pixels from the nucleus. (C) Representative images used for quantification in panels B and D. Blue represents DAPI stain and red represents Cy3-labeled sd-rxRNA. Scale bar = 10 μm. (D) Corresponding plot profiles of lines drawn through images in panel C. Blue lines represent DAPI stain intensity and red lines represent Cy3-labeled sd-rxRNA intensity.
Figure 2. sd-rxRNA is internalized through a saturable pathway. (A) HeLa cells were treated with 0.5 μM sd-rxRNA-Cy3 and imaged by live confocal microscopy. Uptake was quantified by measuring total fluorescence intensity at specific time points. (B) HeLa cells were either untreated (control) or pre-incubated with 2 μM unlabeled sd-rxRNA for 10 or 60 min. Cells were then treated with 0.5 μM sd-rxRNA-Cy3 and imaged by live cell confocal microscopy. sd-rxRNA fluorescence intensity was measured in a region only 10 pixels from the nucleus. (C) Representative images used for quantification in panels B and D. Blue represents DAPI stain and red represents Cy3-labeled sd-rxRNA. Scale bar = 10 μm. (D) Corresponding plot profiles of lines drawn through images in panel C. Blue lines represent DAPI stain intensity and red lines represent Cy3-labeled sd-rxRNA intensity.CC BY · PubMed Central
Figure 3. sd-rxRNA preferentially associates with EGF compared to Tf by live cell TIRF. (A) COS-7 cells were simultaneously treated with 250 nM sd-rxRNA-Cy3, 2 ng/μl EGF-Alexa647, and 25 ng/μl Tf-Alexa488. After 10 min, the cells were washed with fresh media and imaged by TIRF. Representative time-lapse images are shown (see Supplementary Video 3). Scale bar = 10 μm. (B) Enlarged images from panel A. Scale bar = 2 μm.
Figure 3. sd-rxRNA preferentially associates with EGF compared to Tf by live cell TIRF. (A) COS-7 cells were simultaneously treated with 250 nM sd-rxRNA-Cy3, 2 ng/μl EGF-Alexa647, and 25 ng/μl Tf-Alexa488. After 10 min, the cells were washed with fresh media and imaged by TIRF. Representative time-lapse images are shown (see Supplementary Video 3). Scale bar = 10 μm. (B) Enlarged images from panel A. Scale bar = 2 μm.CC BY · PubMed Central
Figure 4. Quantitative analysis of sd-rxRNA co-trafficking with Tf and EGF shows preferential co-localization with EGF by fixed cell structured illumination microscopy. (A) COS-7 cells were grown on cover slips and simultaneously exposed to 25 ng/μl Tf-Alexa647, 250 nM sd-rxRNA-Cy3, and 1 ng/μl EGF-Alexa488 at 37°C and washed at 5 min. Cover slips were transferred to ice-cold PBS and then immediately fixed in 4% paraformaldehyde at the specified time points. Images were acquired by SIM and representative maximum intensity projections of cells fixed at 30 min are shown (see Supplementary Video 4). (B) Co-localization analysis of panel A is presented as mean ± SEM. Scale bar = 10 μm. Dotted line indicates wash with fresh media (*P < 0.05, **P < 0.01, ***P < 0.001, two-tailed Student's t-test).
Figure 4. Quantitative analysis of sd-rxRNA co-trafficking with Tf and EGF shows preferential co-localization with EGF by fixed cell structured illumination microscopy. (A) COS-7 cells were grown on cover slips and simultaneously exposed to 25 ng/μl Tf-Alexa647, 250 nM sd-rxRNA-Cy3, and 1 ng/μl EGF-Alexa488 at 37°C and washed at 5 min. Cover slips were transferred to ice-cold PBS and then immediately fixed in 4% paraformaldehyde at the specified time points. Images were acquired by SIM and representative maximum intensity projections of cells fixed at 30 min are shown (see Supplementary Video 4). (B) Co-localization analysis of panel A is presented as mean ± SEM. Scale bar = 10 μm. Dotted line indicates wash with fresh media (*P < 0.05, **P < 0.01, ***P < 0.001, two-tailed Student's t-test).CC BY · PubMed Central
Figure 5. sd-rxRNA preferentially associates with EEA1- but not RBSN-enriched endosomes. COS-7 cells were grown on cover slips and transfected with either (A) GFP-EEA1 or (B) GFP-RBSN plasmids. All cells were grown on cover slips, kept at 37°C, treated with 250 nM sd-rxRNA-Cy3 and washed with fresh media at 5 min. At the specified time points, cover slips were transferred to ice-cold PBS and then immediately fixed in 4% paraformaldehyde. Images were acquired by SIM and representative maximum intensity projections of cells fixed at 30 min are shown. (C) Co-localization analysis of panels A and B are presented as mean ± SEM. Scale bar = 10 μm (inset scale bar = 500 nm). Dotted line indicates wash with fresh media (*P < 0.05, **P < 0.01, two-tailed Student's t-test).
Figure 5. sd-rxRNA preferentially associates with EEA1- but not RBSN-enriched endosomes. COS-7 cells were grown on cover slips and transfected with either (A) GFP-EEA1 or (B) GFP-RBSN plasmids. All cells were grown on cover slips, kept at 37°C, treated with 250 nM sd-rxRNA-Cy3 and washed with fresh media at 5 min. At the specified time points, cover slips were transferred to ice-cold PBS and then immediately fixed in 4% paraformaldehyde. Images were acquired by SIM and representative maximum intensity projections of cells fixed at 30 min are shown. (C) Co-localization analysis of panels A and B are presented as mean ± SEM. Scale bar = 10 μm (inset scale bar = 500 nm). Dotted line indicates wash with fresh media (*P < 0.05, **P < 0.01, two-tailed Student's t-test).CC BY · PubMed Central
Figure 6. Proposed model of sd-rxRNA internalization and trafficking. EGF traffics through EEA1-enriched endosomes and is sent to the lysosome for degradation while Tf traffics through RBSN-enriched endosomes and is recycled back to the plasma membrane. A fraction of internalized sd-rxRNA preferentially traffics through EEA1-enriched endosomes, similar to EGF. In order to achieve target silencing, sd-rxRNA must undergo endosomal escape in order to reach the cytoplasm where the components of RISC, such as Argonaute 2 (Ago2), are located; however, this mechanism is still not known.
Figure 6. Proposed model of sd-rxRNA internalization and trafficking. EGF traffics through EEA1-enriched endosomes and is sent to the lysosome for degradation while Tf traffics through RBSN-enriched endosomes and is recycled back to the plasma membrane. A fraction of internalized sd-rxRNA preferentially traffics through EEA1-enriched endosomes, similar to EGF. In order to achieve target silencing, sd-rxRNA must undergo endosomal escape in order to reach the cytoplasm where the components of RISC, such as Argonaute 2 (Ago2), are located; however, this mechanism is still not known.CC BY · PubMed Central

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Metadata

Authors
Ly, S., Navaroli, D. M., Didiot, M.-C., Cardia, J., Pandarinathan, L., Alterman, J. F., Fogarty, K., Standley, C., Lifshitz, L. M., Bellve, K. D., Prot, M., Echeverria, D., Corvera, S., & Khvorova, A.
Affiliations
  • University of Massachusetts Chan Medical School (US)
  • Phio Pharmaceuticals (United States) (US)
Corresponding author
Khvorova, Anastasia
ORCID
Venue
Nucleic Acids Research
Publisher
Oxford University Press (OUP)
Published
2016-11-28
Volume
45
Issue
1
Pages
15-25
ISSN
  • 0305-1048 (ISSN-L)
  • 1362-4962
DOI
10.1093/nar/gkw1005
PMC
PMC5224471
OpenAlex
W2557242880
Cited by
132
References
83
Citations by year
2026: 4 · 2025: 13 · 2024: 13 · 2023: 14 · 2022: 9 · 2021: 9 · 2020: 19 · 2019: 22 · 2018: 21 · 2017: 8
FWCI
8.84
Type
journal article
Language
en
Open access
Licence
Locations
  • Nucleic Acids Research (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • The Journal of the American Medical Association (JAMA) Network (American Medical Association) (submittedVersion, cc-by)
  • HighWire Press Open Archive (submittedVersion)
Funders
  • University of Massachusetts (DK32520)
  • University of Oxford
  • National Institutes of Health (1s10od020012-01)
  • National Institute of General Medical Sciences (R01GM108803, R25GM113686)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Interference and Gene Delivery
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Cellular transport and secretion
  • Physical Sciences › Materials Science › Biomaterials › Nanoparticle-Based Drug Delivery
Keywords
siRNA delivery, cellular uptake, endocytosis, early endosomes, transferrin receptor, EEA1, membrane association
MeSH terms
Animals, Biological Transport, Chlorocebus aethiops, Cholesterol, Endocytosis, Epidermal Growth Factor, HeLa Cells, Humans, Microscopy, Fluorescence, Endosomes, Transferrin, Gene Expression, COS Cells, Cyclophilins, Vesicular Transport Proteins, RNA, Small Interfering, Hydrophobic and Hydrophilic Interactions

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