2 research outputs found
Probing the Effect of miRNA on siRNA–PEI Polyplexes
Delivery of small interfering RNA
(siRNA) for silencing of aberrantly
expressed genes is a promising therapy for the treatment of various
genetic disorders. Polymeric carriers have been used in the design
of efficient delivery systems to generate nanoscale siRNA polyplexes.
Despite the great amount of research pursued on siRNA therapeutics,
the underlying mechanisms of polyplex dissociation in cytosol are
still unclear. The fate of siRNA polyplexes during intracellular stages
of delivery and how the endogenous molecules may affect the integrity
of polyplexes remains to be explored. In this study, we have focused
on miRNA-21 as a representative anionic endogenous molecule and performed
gel electrophoresis mobility shift assays, particle size and zeta
(ζ)-potential analyses, and a series of all-atom molecular dynamics
simulations to elucidate the effect of miRNA on siRNA–PEI polyplexes.
We report a slightly better binding to PEI by miRNA than that of siRNA,
and speculated that miRNA may disrupt the integrity of preformed siRNA–PEI
polyplexes. In contrast to our initial speculation, however, introduction
of miRNA to a preformed siRNA–PEI polyplex revealed formation
of a miRNA layer surrounding the polyplex through interactions with
PEI. The resulting structure is a ternary siRNA–PEI–miRNA
complex, where the experimentally determined ζ-potential was
found to decrease as a function of miRNA added
A Delicate Balance When Substituting a Small Hydrophobe onto Low Molecular Weight Polyethylenimine to Improve Its Nucleic Acid Delivery Efficiency
High
molecular weight (HMW) polyethylenimine (PEI) is one of the most versatile
nonviral gene vectors that was extensively investigated over the past
two decades. The cytotoxic profile of HMW PEI, however, encouraged
a search for safer alternatives. Because of lack of cytotoxicity of
low molecular weight (LMW) PEI, enhancing its performance via hydrophobic
modifications has been pursued to this end. Since the performance
of modified PEIs depends on the nature and extent of substituents,
we systematically investigated the effect of hydrophobic modification
of LMW (1.2 kDa) PEI with a short propionic acid (PrA). Moderate enhancements
in PEI hydrophobicity resulted in enhanced cellular uptake of polyplexes
and siRNA-induced silencing efficacy, whereas further increase in
PrA substitution abolished the uptake as well as the silencing. We
performed all-atom molecular dynamics simulations to elucidate the
mechanistic details behind these observations. A new assembly mechanism
was observed by the presence of hydrophobic PrA moieties, where PrA
migrated to core of the polyplex. This phenomenon caused higher surface
hydrophobicity and surface charge density at low substitutions, and
it caused deleterious effects on surface hydrophobicity and cationic
charge at higher substitutions. It is evident that an optimal balance
of hydrophobicity/hydrophilicity is needed to achieve the desired
polyplex properties for an efficient siRNA delivery, and our mechanistic
findings should provide valuable insights for the design of improved
substituents on nonviral carriers