Impact of Peptide Sequence on Functional siRNA Delivery and Gene Knockdown with Cyclic Amphipathic Peptide Delivery Agents.

Short-interfering RNA (siRNA) oligonucleotide therapeutics that modify gene expression by accessing RNA-interference (RNAi) pathways have great promise for the treatment of a range of disorders; however, their application in clinical settings has been limited by significant challenges in cellular delivery. Herein, we report a structure-function study using a series of modified cyclic amphipathic cell-penetrating peptides (CAPs) to determine the impact of peptide sequence on (1) siRNA-binding efficiency, (2) cellular delivery and knockdown efficiency, and (3) the endocytic uptake mechanism. Nine cyclic peptides of the general sequence Ac-C[XZ]4CG-NH2 in which X residues are hydrophobic/aromatic (Phe, Tyr, Trp, or Leu) and Z residues are charged/hydrophilic (Arg, Lys, Ser, or Glu) are assessed along with one acyclic peptide, Ac-(WR)4G-NH2. Cyclization is enforced by intramolecular disulfide bond formation between the flanking Cys residues. Binding analyses indicate that strong cationic character and the presence of aromatic residues that are competent to participate in CH-π interactions lead to CAP sequences that most effectively interact with siRNA. CAP-siRNA binding increases in the following order as a function of CAP hydrophobic/aromatic content: His < Phe < Tyr < Trp. Both cationic charge and disulfide-constrained cyclization of CAPs improve uptake of siRNA in vitro. Net neutral CAPs and an acyclic peptide demonstrate less-efficient siRNA translocation compared to the cyclic, cationic CAPs tested. All CAPs tested facilitated efficient siRNA target gene knockdown of at least 50% (as effective as a lipofectamine control), with the best CAPs enabling >80% knockdown. Significantly, gene knockdown efficiency does not strongly correlate with CAP-siRNA internalization efficiency but moderately correlates with CAP-siRNA-binding affinity. Finally, utilization of small-molecule inhibitors and targeted knockdown of essential endocytic pathway proteins indicate that most CAP-siRNA nanoparticles facilitate siRNA delivery through clathrin- and caveolin-mediated endocytosis. These results provide insight into the design principles for CAPs to facilitate siRNA delivery and the mechanisms by which these peptides translocate siRNA into cells. These studies also demonstrate the nature of the relationships between peptide-siRNA binding, cellular delivery of siRNA cargo, and functional gene knockdown. Strong correlations between these properties are not always observed, which illustrates the complexity in the design of optimal next-generation materials for oligonucleotide delivery.

Supramolecular Phenylalanine-Derived Hydrogels for the Sustained Release of Functional Proteins.

Protein-based therapeutics have emerged as next-generation pharmaceutical agents for oncology, bone regeneration, autoimmune disorders, viral infections, and other diseases. The clinical application of protein therapeutics has been impeded by pharmacokinetic and pharmacodynamic challenges including off-target toxicity, rapid clearance, and drug stability. Strategies for the localized and sustained delivery of protein therapeutics have shown promise in addressing these challenges. Hydrogels are critical materials that enable these delivery strategies. Supramolecular hydrogels composed of self-assembled materials have demonstrated biocompatibility advantages over polymer hydrogels, with peptide and protein-based gels showing strong potential. However, cost is a significant drawback of peptide-based supramolecular hydrogels. Supramolecular hydrogels composed of inexpensive low-molecular-weight (LMW) gelators, including modified amino acid derivatives, have been reported as viable alternatives to peptide-based materials. Herein, we report the encapsulation and release of proteins from supramolecular hydrogels composed of perfluorinated fluorenylmethyloxcarbonyl-modified phenylalanine (Fmoc-F5-Phe-DAP). Specifically, we demonstrate release of four model proteins (ribonuclease A (RNase A), trypsin inhibitor (TI), bovine serum albumin (BSA), and human immunoglobulin G (IgG)) from these hydrogels. The emergent viscoelastic properties of these materials are characterized, and the functional and time-dependent release of proteins from the hydrogels is demonstrated. In addition, it is shown that the properties of the aqueous solution used for hydrogel formulation have a significant influence on the in vitro release profiles, as a function of the isoelectric point and molecular weight of the protein payloads. These studies collectively validate that this class of supramolecular LMW hydrogel possesses the requisite properties for the sustained and localized release of protein therapeutics.

RNAi therapeutic strategies for acute respiratory distress syndrome.

Acute respiratory distress syndrome (ARDS), replacing the clinical term acute lung injury, involves serious pathophysiological lung changes that arise from a variety of pulmonary and nonpulmonary injuries and currently has no pharmacological therapeutics. RNA interference (RNAi) has the potential to generate therapeutic effects that would increase patient survival rates from this condition. It is the purpose of this review to discuss potential targets in treating ARDS with RNAi strategies, as well as to outline the challenges of oligonucleotide delivery to the lung and tactics to circumvent these delivery barriers.