Document Detail


Development of siRNA-probes for studying intracellular trafficking of siRNA nanoparticles.
MedLine Citation:
PMID:  23014672     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
One important barrier facing the delivery of short interfering RNAs (siRNAs) via synthetic nanoparticles is the rate of nanoparticle disassembly. However, our ability to optimize the release kinetics of siRNAs from nanoparticles for maximum efficacy is limited by the lack of methods to track their intracellular disassembly. Towards this end, we describe the design of two different siRNA-based fluorescent probes whose fluorescence emission changes in response to the assembly state of the nanoparticle. The first probe design involves a redox-sensitive fluorescence-quenched probe that fluoresces only when the nanoparticle is disassembled in a reductive environment. The second probe design is based on a FRET-labeled siRNA pair that fluoresces due to the proximity of the siRNA pair when the nanoparticle is intact. In both approaches, the delivery vehicle need not be labeled. The utility of these probes was investigated with a lipidoid nanoparticle (LNP) as proof-of-concept in both extracellular and intracellular environments. Fluorescence kinetic data from both probes were fit to a two-phase release and decay curve and subsequently quantified to give intracellular disassembly rate constants. Quantitative analysis revealed that the rate constant of siRNA release measured via the fluorescence-quenched probe was almost identical to the rate constant for nanoparticle disassembly measured via the FRET-labeled probes. Furthermore, these probes were utilized to determine subcellular localization of LNPs with the use of automated high-resolution microscopy as they undergo dissociation. Interestingly, this work shows that large amounts of siRNA remain inside vesicular compartments. Altogether, we have developed new siRNA probes that can be utilized with multiple nanocarriers for quantitative and qualitative analysis of nanoparticle dissociation that may serve as a design tool for future delivery systems.
Authors:
Christopher A Alabi; Gaurav Sahay; Robert Langer; Daniel G Anderson
Publication Detail:
Type:  Journal Article; Research Support, N.I.H., Extramural    
Journal Detail:
Title:  Integrative biology : quantitative biosciences from nano to macro     Volume:  5     ISSN:  1757-9708     ISO Abbreviation:  Integr Biol (Camb)     Publication Date:  2013 Jan 
Date Detail:
Created Date:  2012-12-17     Completed Date:  2013-06-24     Revised Date:  2014-03-20    
Medline Journal Info:
Nlm Unique ID:  101478378     Medline TA:  Integr Biol (Camb)     Country:  England    
Other Details:
Languages:  eng     Pagination:  224-30     Citation Subset:  IM    
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MeSH Terms
Descriptor/Qualifier:
HeLa Cells
Humans
Microscopy, Fluorescence / methods*
Molecular Imaging / methods*
Molecular Probe Techniques*
Molecular Probes / metabolism*,  ultrastructure
Nanoparticles / diagnostic use*
RNA, Small Interfering / metabolism*
Grant Support
ID/Acronym/Agency:
P30 CA014051/CA/NCI NIH HHS; R01 CA115527/CA/NCI NIH HHS; R01 CA132091/CA/NCI NIH HHS; R01-CA115527/CA/NCI NIH HHS; R01-CA132091/CA/NCI NIH HHS; R37 EB000244/EB/NIBIB NIH HHS; R37-EB000244/EB/NIBIB NIH HHS
Chemical
Reg. No./Substance:
0/Molecular Probes; 0/RNA, Small Interfering
Comments/Corrections

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