Dataset Information

Single-Stranded Phosphorothioated Regions Enhance Cellular Uptake of Cholesterol-Conjugated siRNA but Not Silencing Efficacy.

ABSTRACT: Small interfering RNAs (siRNAs) have potential to silence virtually any disease-causing gene but require chemical modifications for delivery to the tissue and cell of interest. Previously, we demonstrated that asymmetric, phosphorothioate (PS)-modified, chemically stabilized, cholesterol-conjugated siRNAs, called hsiRNAs, support rapid cellular uptake and efficient mRNA silencing both in cultured cells and in vivo. Here, we systematically evaluated the impact of number, structure, and sequence context of PS-modified backbones on cellular uptake and RNAi-mediated silencing efficacy. We find that PS enhances cellular internalization in a sequence-dependent manner but only when present in a single-stranded but not double-stranded region. Furthermore, the observed increase in cellular internalization did not correlate with functional silencing improvement, indicating that PS-mediated uptake may drive compounds to non-productive sinks. Thus, the primary contributing factor of PS modifications to functional efficacy is likely stabilization rather than enhanced cellular uptake. A better understanding of the relative impact of different chemistries on productive versus non-productive uptake will assist in improved design of therapeutic RNAs.

SUBMITTER: Ly S

PROVIDER: S-EPMC7452107 | biostudies-literature | 2020 Sep

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Single-Stranded Phosphorothioated Regions Enhance Cellular Uptake of Cholesterol-Conjugated siRNA but Not Silencing Efficacy.

Ly Socheata S Echeverria Dimas D Sousa Jacquelyn J Khvorova Anastasia A

Molecular therapy. Nucleic acids 20200725

Small interfering RNAs (siRNAs) have potential to silence virtually any disease-causing gene but require chemical modifications for delivery to the tissue and cell of interest. Previously, we demonstrated that asymmetric, phosphorothioate (PS)-modified, chemically stabilized, cholesterol-conjugated siRNAs, called hsiRNAs, support rapid cellular uptake and efficient mRNA silencing both in cultured cells and in vivo. Here, we systematically evaluated the impact of number, structure, and sequence c ...[more]

PMID: 32818923

Similar Datasets

Project description:Formation of stable, long-circulating siRNA polyplexes is a significant challenge in translation of intravenously-delivered, polymeric RNAi cancer therapies. Here, we report that siRNA hydrophobization through conjugation to palmitic acid (siPA) improves stability, in vivo pharmacokinetics, and tumor gene silencing of PEGylated nanopolyplexes (siPA-NPs) with balanced cationic and hydrophobic content in the core relative to the analogous polyplexes formed with unmodified siRNA, si-NPs. Hydrophobized siPA loaded into the NPs at a lower charge ratio (N(+):P(-)) relative to unmodified siRNA, and siPA-NPs had superior resistance to siRNA cargo unpackaging in comparison to si-NPs upon exposure to the competing polyanion heparin and serum. In vitro, siPA-NPs increased uptake in MDA-MB-231 breast cancer cells (100% positive cells vs. 60% positive cells) but exhibited equivalent silencing of the model gene luciferase relative to si-NPs. In vivo in a murine model, the circulation half-life of intravenously-injected siPA-NPs was double that of si-NPs, resulting in a >2-fold increase in siRNA biodistribution to orthotopic MDA-MB-231 mammary tumors. The increased circulation half-life of siPA-NPs was dependent upon the hydrophobic interactions of the siRNA and the NP core component and not just siRNA hydrophobization, as siPA did not contribute to improved circulation time relative to unmodified siRNA when delivered using polyplexes with a fully cationic core. Intravenous delivery of siPA-NPs also achieved significant silencing of the model gene luciferase in vivo (∼40% at 24 h after one treatment and ∼60% at 48 h after two treatments) in the murine MDA-MB-231 tumor model, while si-NPs only produced a significant silencing effect after two treatments. These data suggest that stabilization of PEGylated siRNA polyplexes through a combination of hydrophobic and electrostatic interactions between siRNA cargo and the polymeric carrier improves in vivo pharmacokinetics and tumor gene silencing relative to conventional formulations that are stabilized solely by electrostatic interactions.

Project description:The aim of this study was to investigate the fate of curcumin (CUR)-loaded Pickering emulsions with complex interfaces during in vitro gastrointestinal transit and test the efficacy of such emulsions on improving the bioaccessibility and cellular uptake of CUR. CUR-loaded Pickering emulsions tested were whey protein nanogel particle-stabilized Pickering emulsions (CUR-EWPN) and emulsions displaying complex interfaces included 1) layer-by-layer dextran sulphate-coated nanogel-stabilized Pickering emulsions (CUR-DxS+EWPN) and 2) protein+dextran-conjugated microgel-stabilized Pickering emulsions (CUR-EWPDxM). The hypothesis was that the presence of complex interfacial material at the droplet surface would provide better protection to the droplets against physiological degradation, particularly under gastric conditions and thus, improve the delivery of CUR to Caco-2 intestinal cells. The emulsions were characterized using droplet sizing, apparent viscosity, confocal and cryo-scanning electron microscopy, zeta-potential, lipid digestion kinetics, bioaccessibility of CUR as well as cell viability and uptake by Caco-2 cells. Emulsion droplets with modified to complex interfacial composition (i.e. CUR-DxS+EWPN and CUR-EWPDxM) provided enhanced kinetic stability to the Pickering emulsion droplets against coalescence in the gastric regime as compared to droplets having unmodified interface (i.e. CUR-EWPN), whereas droplet coalescence occurred in intestinal conditions irrespective of the initial interfacial materials. A similar rate and extent of free fatty acid release occurred in all the emulsions during intestinal digestion (p > 0.05), which correlated with the bioaccessibility of CUR. Striking, CUR-DxS+EWPN and CUR-EWPDxM significantly improved cellular CUR uptake as compared to CUR-EWPN (p < 0.05). These results highlight a promising new strategy of designing gastric-stable Pickering emulsions with complex interfaces to improve the delivery of lipophilic bioactive compounds to the cells for the future design of functional foods.

Dataset Information

Single-Stranded Phosphorothioated Regions Enhance Cellular Uptake of Cholesterol-Conjugated siRNA but Not Silencing Efficacy.

Publications

Single-Stranded Phosphorothioated Regions Enhance Cellular Uptake of Cholesterol-Conjugated siRNA but Not Silencing Efficacy.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets