Effective Cellular Delivery of Antisense Peptide Nucleic Acid by Conjugation to Guanidinylated Diaminobutanoic Acid-Based Peptide Dendrons

Abstract

A series of amino-and guanidino-terminating 3-and 4-generation 2,4-diaminobutanoic acid (Dab) dendrons have been robustly synthesized on a solid phase and characterized as cellular delivery agents in antisense peptide nucleic acid (PNA) conjugates in the pLuc705 HeLa cell splice switching system. The dendron−PNA conjugates exhibited splice correction activity at one digit micromolar concentrations, and guanidino-terminating dendrons were significantly more effective than analogous amine terminating ones. Furthermore, introduction of lipophilic groups such as phenyl, alkyl, or fatty acids increased efficacy, but also increased cellular toxicity. Fluorescence microscopy analyses supported an endosomal uptake mechanism and furthermore predominantly showed colocalization with late endosomes and lysosomes. The robust solid phase synthesis should make such Dab−dendrons a useful platform for further in vitro as well as in vivo optimization.

Antisense Peptide Nucleic Acid–Diaminobutanoic Acid Dendron Conjugates with SbmA-Independent Antimicrobial Activity against Gram-Negative Bacteria

“…The robust synthesis of cationic, amphipathic dendrons consisting of three generations of DAB functionalized with terminal guanidinylated ligands was recently reported. 19 This novel dendrimeric moiety effectively delivers covalently conjugated PNA to eukaryotic cells, and in analogy to certain arginine peptides, these dendrons were anticipated to also interact with Gram-negative membranes and potentially facilitate uptake of antisense PNAs in bacteria.…”

“…This behavior is similar to that previously observed in mammalian cells, showing that the gain in activity achieved by increasing hydrophobicity is commonly accompanied with an increase in non-specific cell toxicity. 19 …”

“…Previously, it was demonstrated that long terminal groups are necessary for efficient delivery of DAB dendron conjugates to eukaryotic cells. 19 Interestingly, identical dendron moieties conjugated to different PNA oligomers result in compounds with different cytotoxicities. Specifically, conjugates designed for eukaryotic antisense targeting (PNA length 18 nt) 19 exhibit higher toxicity than the corresponding bacteria-targeting compounds (PNA length 10 nt), thereby indicating that the PNA oligomer increases the inherent cytotoxicity of the dendron CPP/BPP.…”

Multifunctional Delivery Systems for Peptide Nucleic Acids

“…Interestingly, a similar trivalent adduct was recently found to effectively penetrate into HepG2 cells, but the delivery of the PNA oligomer was improved when the GalNAc groups were placed on three consecutive C(5)-modified PNA monomers instead of a branched spacer at the N-term, thus resulting in a linear orientation of the glycosyl moieties [73]. A structure-dependent activity was also reported for a system based on modified 2, 4-diaminobutanoic acid (Dab) dendrons, conjugated to a PNA to be delivered in HeLa cells [74]. A luciferase assay was used to monitor the internalization of the PNAdendron adducts, reporting a higher performance when guanidinium moieties were used as terminal groups of the dentritic structure in place of ammonium ones, with a further improvement of the uptake upon increase of both the degree of functionalization and the addition of lipophilic units (i.e., longer alkyl spacers, fatty acids or aromatic substituents), although at the expense of a higher toxicity.…”

Peptide nucleic acids and their role in gene regulation and editing

“…[ 82–86 ] Recently, effective cellular delivery of PNA antisense oligomers by conjugating to guanidinylated diaminobutanoic acid‐based peptide dendrons was described. [ 87 ] In addition to traditional CPPs, recent work has introduced a strategy to deliver PNAs using a pH low insertion peptide (pHLIP), which preferentially accumulates in acidic microenvironments and can deliver PNA cargo specifically to cancer cells. [ 28,45,88–89 ] Although pHLIP is useful for delivering PNA into acidic microenvironments, it is not a viable option for delivery into non‐acidic tissues.…”