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Quantum dot-mediated delivery of siRNA to inhibit sphingomyelinase activities in brain-derived cells.

ABSTRACT: The use of RNAi to suppress protein synthesis offers a potential way of reducing the level of enzymes or the synthesis of mutant toxic proteins but there are few tools currently available for their delivery. To address this problem, bioconjugated quantum dots (QDs) containing a hydrophobic component (N-palmitate) and a sequence VKIKK designed to traverse across cell membranes and visualize drug delivery were developed and tested on cell lines of brain origin. We used the Zn outer shell of the QD to bind HIS6 in JB577 (W•G•Dap(N-Palmitoyl)•VKIKK•P9 •G2 •H6 ) and by a gel-shift assay showed that siRNAs would bind to the positively charged KIKK sequence. By comparing many peptides and QD coatings, we showed that the QD-JB577-siRNA construct was taken up by cells of nervous system origin, distributed throughout the cytosol, and inhibited protein synthesis, implying that JB577 was also promoting endosome egress. By attaching siRNA for luciferase in a cell line over-expressing luciferase, we showed 70% inhibition of mRNA after 24-48 h. To show more specific effects, we synthesized siRNA for neutral (NSMase2), acid (lysosomal ASMase) sphingomyelinase, and sphingosine kinase 1 (SK1), we demonstrated a dose-dependent inhibition of activity. These data suggest that QDs are a useful siRNA delivery tool and QD-siRNA could be a potential theranostic for a variety of diseases.

SUBMITTER: Getz T

PROVIDER: S-EPMC5570548 | biostudies-other | 2016 Dec

REPOSITORIES: biostudies-other

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Project description:Accumulating evidence underscores the importance of ligand-receptor dynamics in shaping cellular signaling. In the nervous system, growth factor-activated Trk receptor trafficking serves to convey biochemical signaling that underlies fundamental neural functions. Focus has been placed on axonal trafficking but little is known about growth factor-activated Trk dynamics in the neuronal soma, particularly at the molecular scale, due in large part to technical hurdles in observing individual growth factor-Trk complexes for long periods of time inside live cells. Quantum dots (QDs) are intensely fluorescent nanoparticles that have been used to study the dynamics of ligand-receptor complexes at the plasma membrane but the value of QDs for investigating ligand-receptor intracellular dynamics has not been well exploited. The current study establishes that QD conjugated brain-derived neurotrophic factor (QD-BDNF) binds to TrkB receptors with high specificity, activates TrkB downstream signaling, and allows single QD tracking capability for long recording durations deep within the soma of live neurons. QD-BDNF complexes undergo internalization, recycling, and intracellular trafficking in the neuronal soma. These trafficking events exhibit little time-synchrony and diverse heterogeneity in underlying dynamics that include phases of sustained rapid motor transport without pause as well as immobility of surprisingly long-lasting duration (several minutes). Moreover, the trajectories formed by dynamic individual BDNF complexes show no apparent end destination; BDNF complexes can be found meandering over long distances of several microns throughout the expanse of the neuronal soma in a circuitous fashion. The complex, heterogeneous nature of neuronal soma trafficking dynamics contrasts the reported linear nature of axonal transport data and calls for models that surpass our generally limited notions of nuclear-directed transport in the soma. QD-ligand probes are poised to provide understanding of how the molecular mechanisms underlying intracellular ligand-receptor trafficking shape cell signaling under conditions of both healthy and dysfunctional neurological disease models.

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Quantum dot-mediated delivery of siRNA to inhibit sphingomyelinase activities in brain-derived cells.

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