Functional peptides for siRNA delivery

Abstract

siRNA is considered as a potent therapeutic agent because of its high specificity and efficiency in suppressing genes that are overexpressed during disease development. For nearly two decades, a significant amount of efforts has been dedicated to bringing the siRNA technology into clinical uses. However, only limited success has been achieved to date, largely due to the lack of a cell type-specific, safe, and efficient delivery technology to carry siRNA into the target cells' cytosol where RNA interference takes place. Among the emerging candidate nanocarriers for siRNA delivery, peptides have gained popularity because of their structural and functional diversity. A variety of peptides have been discovered for their ability to translocate siRNA into living cells via different mechanisms such as direct penetration through the cellular membrane, endocytosis-mediated cell entry followed by endosomolysis, and receptor-mediated uptake. This review is focused on the multiple roles played by peptides in siRNA delivery, such as membrane penetration, endosome disruption, targeting, as well as the combination of these functionalities.

Keywords: Cell penetrating peptide; Endosome disruption; Endosomolytic peptide; Multifunctional; Silencing; Targeting; siRNA.

Fig. 1. CPP-mediated siRNA intracellular delivery

A). Schematic illustration of formulation schemes for CPP-mediated siRNA cell entry. (1) CPP-siRNA conjugate; (2) CPP-siRNA nanocomplex; (3) siRNA anchored on an antibody-CPP conjugate; (4) hairpin-structured activatable CPP conjugated with siRNA. B) The helical wheel showing the amphiphilicity of α-helical CPPs: Penetratin, CADY, and KALA. Amino acids are color coded (red: charged; orange: apolar; yellow: polar; gray: hydrophobic).

Fig. 2

Endosome disrupting peptides. A) Conformation-changing fusogenic peptide HA2 exhibits a random coil at neutral pH and adopts a α-helical conformation at lower pH. Adapted with permission from ref. [84]. Copyright 1994 National Academy of Sciences. B) A chemical structure-changing peptide made by CDM modification of the NH₂ groups in melittin (the N-terminus and the Lys side chains indicated by arrows). The membrane activity of melittin is abolished after the modification, but can be recovered in a weakly acidic environment of endosome where it refolds into a homotetramer and disrupts the endosome membrane by pore formation. C) The chemical structure PF6 with a stearyl modified N-terminus and four trifluoromethyl quinoline moieties added onto the TP10 backbone. D) Photo-induced endosomal escape by modifying CPPs with photosensitizers. Light excitation of the sensitizer triggers the generation of singlet oxygen (¹O₂), which lyses the endosome membrane for siRNA escape.

Fig. 3

Gene and siRNA delivery by multifunctional peptides. A) Schematic of the trifunctional peptide for targeted gene delivery. The KALA domain promotes endosome escape; the tandem H1 repeats enable DNA condensation; the NLS sequence enhances nucleus translocation; and the TP1 block serves as a ligand for cell-specific homing. B) Sequence-specific RNA binding peptide U1A-RBD-Tat forms a complex with U1A modified siRNA, which can escape endosome aided by photo-induced endosomolysis. AF546: Alexa Fluor 546. C) Schematic drawing of a siRNA associated with PTD modified dsRBD. Adapted with permission from ref. [139]. Copyright 2009 Nature Publishing Group. D) dsRBD-His₁₈ chimeric peptide as a general platform for targeted delivery of aptamer-siRNA chimeras. Orange: PSAM targeting aptamer; green: siRNA.