The DOI matches its Crossref record; 82 of the 83 references it cites were found in a registry or an index.
Visualization of self-delivering hydrophobically modified siRNA cellular internalization
Open accessAbstract
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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“Intracellular distribution followed a perinuclear localization pattern that is characteristic of oligonucleotides11.”
From A divalent siRNA chemical scaffold for potent and sustained modulation of gene expression throughout the central nervous system (2019) · cites this paper as [11]
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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
- 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
- gold
- doi.org
- 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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