Probing the Effects of Lipid Substitution on Polycation Mediated DNA Aggregation: A Molecular Dynamics Simulations Study

Understanding the molecular mechanism of DNA aggregation and condensation is of importance to DNA packaging in cells, and applications of gene delivery therapy. Modifying polycations such as polyethylenimine with lipid substitution was found to improve the performance of polycationic gene carriers. However, the role of the lipid substitution in DNA binding and aggregation is not clear and remains to be probed at the molecular level. In this work, we elucidated the role of lipid substitution through a series of all-atom molecular dynamics simulations on DNA aggregation mediated by lipid modified polyethylenimine (lmPEI). We found that the lipids associate significantly with one another, which links the lmPEIs and serves as a mechanism of aggregating the DNAs and stabilizing the formed polyplex. In addition, some lipid tails on the lmPEIs stay at the periphery of the lmPEI/DNA polyplex and may provide a mechanism for hydrophobic interactions. The enhanced stability and hydrophobicity might contribute to better cellular uptake of the polyplexes

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

Single and Combinational siRNA Therapy of Cancer Cells: Probing Changes in Targeted and Nontargeted Mediators after siRNA Treatment

Cancer cells are known to be heterogeneous and plastic, which imparts innate and acquired abilities to resist molecular targeting by short interfering RNA (siRNA). Not all cancer cells in a population would show a similar responsiveness to targeting of genes critical for their survival and even the responders could quickly transform and switch to alternative mechanism(s) for their survival. This study was designed to look at this phenomenon by analyzing the effect of siRNA silencing of selected protein mRNAs involved in cell survival and proliferation on other protein mRNAs that could contribute to cell survival. We compared the gene expression profile of the initial population after siRNA silencing to the subpopulation that survived the siRNA silencing, to identify potential overexpressions that might explain the cell survival. Our studies show that silencing well-selected protein mRNAs simultaneously could offer advantages compared to individual siRNA silencing due to an additional impact on the expression level of certain protein mRNAs. We also demonstrate that overexpression of certain protein mRNAs could explain the innate unresponsiveness of a subpopulation of cells. These observations could be a stepping stone for further investigation of the possibility of significant synergistic effect for this combinational RNA interference strategy

Tuning the Potency of Farnesol-Modified Polyethylenimine with Polyanionic Trans-Booster to Enhance DNA Delivery

Low molecular weight polyethylenimine (PEI) based lipopolymers become an attractive strategy to construct nonviral therapeutic carriers with promising transfection efficiency and minimal toxicity. Herein, this paper presents the design and synthesis of novel farnesol (Far) conjugated PEI, namely PEI1.2k-SA-Far7. The polymers had quick DNA complexation, effective DNA unpacking (dissociation), and cellular uptake abilities when complexed with plasmid DNA. However, they were unable to provide robust transfection in culture, indicating inability of Far grafting to improve the transfection efficacy significantly. To overcome this limitation, the commercially available polyanionic Trans-Booster additive, which is capable of displaying electrostatic interaction with PEI1.2k-SA-Far7, has been used to enhance the uptake of pDNA polyplexes and transgene expression. pDNA condensation was successfully achieved in the presence of the Trans-Booster with more stable polyplexes, and in vitro transfection efficacy of the polyplexes was improved to be comparable to that obtained with an established reference reagent. The PEI1.2k-SA-Far7/pDNA/Trans-Booster ternary complex exhibited good compatibility with cells and minimal hemolysis activity. This work demonstrates the exemplary potency of using additives in polyplexes and the potential of resultant ternary complexes for effective pDNA delivery