Technologies for Investigating the Physiological Barriers to Efficient Lipid Nanoparticle–siRNA Delivery

Shu, Bin; Abrams, Marc

doi:10.1369/0022155413484152

Cited by 22 publications

(17 citation statements)

References 137 publications

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“…This can be reinforced by the fact that over 85% of the delivered dextran was detected in the vesicles that also contained HRP 5 min after both were simultaneously introduced to cells ( Figure 5B). Both dextran and HRP are similar types of pinocytic endocytosis markers that readily enter cells through multiple pathways [60][61][62]. Nanoparticles formed of cationic polymers, however, are more likely to enter cells through a more specific pathway [63].…”

Section: Discussionmentioning

confidence: 99%

Rapid and facile quantitation of polyplex endocytic trafficking

Lazebnik

Pack

2017

Journal of Controlled Release

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Design of safe and effective synthetic nucleic acid delivery vectors such as polycation/DNA or polycation/siRNA complexes (polyplexes) will be facilitated by quantitative understanding of the mechanisms by which such materials escort cargo from the cell surface to the nucleus. In particular, the mechanisms of cellular internalization by various endocytosis pathways and subsequent endocytic vesicle trafficking have been shown to strongly affect nucleic acid delivery efficiency. Fluorescence microscopy and subcellular fractionation methods are commonly employed to follow intracellular trafficking of biomolecules and nanoparticulate delivery systems such as polyplexes. However, it is difficult to obtain quantitative data from microscopy and subcellular fractionation is experimentally difficult and low throughput. We have developed a method for quantifying the transport of polyplexes through important endocytic vesicles. The method is based on polymerization of 3,3'-diaminobenzidine by endocytosed horseradish peroxidase, causing an increase in the vesicle density, resistance to being solubilized by detergent and quenching of fluorophores within the vesicles, which makes them easy to separate and quantify. Using this method in HeLa cells, we have observed polyethylenimine/siRNA polyplexes initially appearing in early endosomes and rapidly moving to other compartments within 30min post-transfection. At the same time, we observed the kinetics of accumulation of the polyplexes in lysosomes at a similar rate. The results from the new method are consistent with similar measurements by confocal fluorescence microscopy and subcellular fractionation of endocytic vesicles on a Percoll gradient. The relative ease of this new method will aid investigation of gene delivery mechanisms by providing the means to rapidly quantify endocytic trafficking of polyplexes and other vectors.

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Section: Discussionmentioning

confidence: 99%

Rapid and facile quantitation of polyplex endocytic trafficking

Lazebnik

Pack

2017

Journal of Controlled Release

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“…Clinical development of small interfering RNA (siRNA)-based therapeutics outside liver has been limited primarily by poor bioavailability to extra-hepatic tissues and cell types of interest. When delivered systemically, siRNA formulated within a lipid nanoparticle or carrying a hepatocyte-specific targeting ligand can trigger near-complete and highly specific mRNA silencing of numerous targets in the liver at well-tolerated doses 2, 3, 4. For certain indications, local delivery of siRNA (e.g., mucosal, intraocular, intrathecal) may be more desirable than the standard intravenous or subcutaneous administration, as it may enable higher oligonucleotide concentrations in relevant tissues and protection from systemic nucleases 5, 6.…”

Section: Introductionmentioning

confidence: 99%

Intratracheal Administration of siRNA Triggers mRNA Silencing in the Lung to Modulate T Cell Immune Response and Lung Inflammation

Cash-Mason

Wang

et al. 2019

Molecular Therapy - Nucleic Acids

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Clinical application of siRNA-based therapeutics outside of the liver has been hindered by the inefficient delivery of siRNA effector molecules into extra-hepatic organs and cells of interest. To understand the parameters that enable RNAi activity in vivo, it is necessary to develop a systematic approach to identify which cells within a tissue are permissive to oligonucleotide internalization and activity. In the present study, we evaluate the distribution and activity within the lung of chemically stabilized siRNA to characterize cell-type tropism and structure-activity relationship. We demonstrate intratracheal delivery of fully modified siRNA for RNAi-mediated target knockdown in lung CD11c+ cells (dendritic cells, alveolar macrophages) and alveolar epithelial cells. Finally, we use an allergen-induced model of lung inflammation to demonstrate the capacity of inhaled siRNA to induce target knockdown in dendritic cells and ameliorate lung pathology.

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“…159 The efficiency of therapeutic interfering RNAs can be evaluated through biodistribution, cellular and subcellular localization studies, endosomal escape efficiency and RISC-loading efficacy studies. 160…”

Section: ■ Challenges In Delivering Rnasmentioning

confidence: 99%

Cellular Delivery of RNA Nanoparticles

et al. 2016

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RNA nanostructures can be programmed to exhibit defined sizes, shapes and stoichiometries from naturally occurring or de novo designed RNA motifs. These constructs can be used as scaffolds to attach functional moieties, such as ligand binding motifs or gene expression regulators, for nanobiology applications. This review is focused on four areas of importance to RNA nanotechnology: the types of RNAs of particular interest for nanobiology, the assembly of RNA nanoconstructs, the challenges of cellular delivery of RNAs in vivo, and the delivery carriers that aid in the matter. The available strategies for the design of nucleic acid nanostructures, as well as for formulation of their carriers, make RNA nanotechnology an important tool in both basic research and applied biomedical science.

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Technologies for Investigating the Physiological Barriers to Efficient Lipid Nanoparticle–siRNA Delivery

Cited by 22 publications

References 137 publications

Rapid and facile quantitation of polyplex endocytic trafficking

Rapid and facile quantitation of polyplex endocytic trafficking

Intratracheal Administration of siRNA Triggers mRNA Silencing in the Lung to Modulate T Cell Immune Response and Lung Inflammation

Cellular Delivery of RNA Nanoparticles

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