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Introduction: The plasma membrane protects a cell from the extracellular environment. As such it presents an obstacle that therapeutics needs to traverse in order to achieve efficacy. For example, small interfering RNAs (siRNAs) need to be delivered to the cytoplasm, where they can interact with the RNA interference machinery and initiate gene silencing. However, these macromolecules have poor membrane permeability, largely limiting their therapeutic potential. To address this challenge, current strategies involve encapsulating siRNAs into nanoparticles. However, upon cellular uptake, these nanoparticles are trapped in endosomes, which lack access to the cytoplasm. Towards developing an alternative strategy that provides direct access to the cytoplasm, we have been inspired by the unique capabilities of gap junctions to establish passageways between the cytoplasm of neighboring cells. Specifically, six connexins hexamerize to form a connexon hemichannel. Two hemichannels from neighboring cells dock to each other to form a complete gap junction channel, facilitating the exchange of molecular cargoes such as ions and siRNA. Therefore, incorporating the gap junction network into therapeutic delivery materials has the potential to enhance the delivery efficiency of siRNAs by directly depositing siRNAs into the cytoplasm.
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Kinetic analysis of the intracellular processing of siRNAs by confocal microscopy
Abstract Here, we describe a method for tracking intracellular processing of small interfering RNA (siRNA) containing complexes using automated microscopy controls and image acquisition to minimize user effort and time. This technique uses fluorescence colocalization to monitor dual-labeled fluorescent siRNAs delivered by silica nanoparticles in different intracellular locations, including the early/late endosomes, fast/slow recycling endosomes, lysosomes and the endoplasmic reticulum. Combining the temporal association of siRNAs with each intracellular location, we reconstructed the intracellular pathways used in siRNA processing, and demonstrate how these pathways vary based on the chemical composition of the delivery vehicle.
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- PAR ID:
- 10309201
- Date Published:
- Journal Name:
- Microscopy
- Volume:
- 69
- Issue:
- 6
- ISSN:
- 2050-5698
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1093/jmicro/dfaa031
