DEGylation Enhanced the Stability of Peptide-siRNA Complexes in Serum.

Small interfering RNA (siRNA) shows great therapeutic potential due to its ability to regulate gene expression in a highly selective manner, but this application has been limited by effective delivery, partly because of the low nuclease resistance of siRNA in the presence of serum, and inefficient cellular uptake. We previously reported a library of cell-penetrating and amino acid-pairing peptides that facilitate effective siRNA delivery to mammalian cells without causing cytotoxicity, but they are unstable within serum-containing medium. Here, we investigated the possibility of conjugating the peptide with diethylene glycol to improve its serum stability without compromising its gene-regulation capability. One of the most promising peptides, C6M1, was conjugated with diethylene glycol, and its incorporated siRNA complexes had excellent serum stability and highly efficient cellular uptake with negligible cytotoxicity. The gene-silencing ability of diethylene glycol conjugated-peptide/siRNA complexes was comparable to that of non-conjugated peptide/siRNA at both mRNA and protein levels. Our data demonstrate that conjugating peptides with diethylene glycol is a promising method for improving siRNA delivery by improving its serum stability.

In vitro and in vivo therapeutic siRNA delivery induced by a tryptophan-rich endosomolytic peptide.

At the forefront of medicine, gene therapy provides an effective way to treat a range of diseases by regulating defective genes at the root of the disease. Short interfering RNAs (siRNAs) hold great promise as therapeutic agents in this domain; however, intracellular delivery remains a major obstacle to clinical applications of therapeutic siRNAs. Here we report a peptide designed to mediate siRNA delivery. This peptide, C6M1, is rationally designed to promote the endosomal escape ability of an existing peptide sequence. Formed C6M1-siRNA nanoscale complexes are able to deliver siRNA into cells and induce specific gene knockdown with low toxicity. The increased membrane disruption ability under acidic conditions of the peptide with tryptophan residue substitution may contribute to the enhanced gene silence efficacy. Intratumoral injection of the complexes results in a marked reduction of tumor growth through downregulation of antiapoptotic Bcl-2 protein in mice. In addition, the C6M1-siRNA complex was proven safe at transfection concentration by cytotoxicity assay. These results demonstrate that the C6M1-siRNA complex is a potent system for efficient gene delivery in vitro and in vivo.

Thermodynamic investigation of the binding of dissymmetric pyrenyl-gemini surfactants to DNA.

Gemini surfactants have demonstrated significant potential for use in constructing non-viral transfection vectors for the delivery of genes into cells to induce protein expression. Previously, two asymmetric gemini surfactants containing pyrenyl groups in one of the alkyl tails of the surfactants were synthesized as fluorescence probes for use in mechanistic studies of the transfection process. Here we present the results of a thermodynamic investigation of the binding interaction(s) between the pyrenyl-modified surfactants and DNA. The thermodynamics of the interactions have been examined using isothermal titration calorimetry, light scattering, zeta potential, and circular dichroism measurements. Distinct differences are observed between the interaction of 12-s-12 vs. the pyrene modified py-s-12 surfactants with DNA; an intercalated binding is found for the py-s-12 surfactants that disrupts the typical interactions observed between DNA and gemini surfactants.

A novel synthetic cannabinoid derivative inhibits inflammatory liver damage via negative cytokine regulation.

The therapeutic potential of cannabinoids has been described previously for several inflammatory diseases, but the molecular mechanisms underlying the anti-inflammatory properties of cannabinoids are not well understood. In this study, we investigated the mechanism of action of a novel synthetic cannabinoid, [(+)(6aS,10aS)-6,6-Dimethyl-3-(1,1-dimethylheptyl)-1-hydroxy-9-(1H-imidazol-2-ylsulfanylmethyl]-6a,7,10,10a-tetrahydro-6H-dibenzo[b,d]pyran (PRS-211,092) that has no psychotropic effects but exhibits immunomodulatory properties. Treatment with PRS-211,092 significantly decreased Concanavalin A-induced liver injury in mice that was accompanied by: 1) promotion of early gene expression of interleukin (IL)-6 and IL-10 that play a protective role in this model; 2) induction of early gene expression of the suppressors of cytokine signaling (SOCS-1 and 3), followed by 3) inhibition of several pro-inflammatory mediators, including IL-2, monocyte chemoattractant protein-1 (MCP-1), IL-1beta, interferon-gamma, and tumor necrosis factor alpha. Based on these results, we propose a mechanism by which PRS-211,092 stimulates the expression of IL-6, IL-10 and the SOCS proteins that, in turn, negatively regulates the expression of pro-inflammatory cytokines. Negative regulation by PRS-211,092 was further demonstrated in cultured T cells, where it inhibited IL-2 production and nuclear factor of activated T cells activity. These findings suggest that this cannabinoid derivative is an immunomodulator that could be developed as a potential drug for hepatitis as well as for other short- or long-term inflammatory diseases.