To overcome the main hurdle, we face when it comes to non-viral delivery of molecular therapeutics, a library of eleven branched oligoaminoamides was generated. They comprise of different amino acids and two building blocks that originate from polyethylenimine (PEI). Therefore, they exhibit very efficient nucleic acid binding and endosomal buffering ability. Succinoyltetraethylene pentaamine (Stp) and succinoylpentaethylene hexamine (Sph) that comprise of four and five 1,2-diaminoethane motifs, respectively, and succinic acid coupled on one terminal primary amine which makes oligoaminoamides more biodegradable and suitable for solid-phase-assisted synthesis (SPAS). Branched oligoaminoamides, also called four-arm structures, were assembled by SPAS in a few coupling steps based on C-terminal alanine and two lysine branching points, each coupled with histidine for improved buffering capacity, followed by elongation of the four arms with three Stp and Sph artificial amino acid repeats, respectively, and subsequent additional three repeats of explicit amino acids such as arginine, tyrosine, histidine, tryptophan or lysine, and finally ending with cysteine and N-terminal azido lysine. Arginine and lysine were introduced to improve DNA binding ability, tyrosine, and tryptophan to improve hydrophobic interaction and to stabilize the polyplexes, cysteine to stabilize polyplexes through covalent disulfide cross-linkage that can be reduced in the reductive cytosol and thereby facilitate the payload release, and finally azido lysine for further attachment of shielding agents or ligands using DBCO click-chemistry strategy. These sequence-defined oligomers were purified through size-exclusion chromatography (SEC) and analyzed by 1H-NMR. The polyplexes were formed with pDNA, and evaluated according to their size, zeta potential and binding ability to pDNA. The size of formed polyplexes was in the range of 100 nm, whereas oligomers containing the Sph building block were below 100 nm. For this reason, we can conclude that the Sph building block is superior to the Stp one regarding the formation of small particles. Zeta potential was mostly positive which indicates successful pDNA encapsulation and a beneficial endosomal escape characteristic. Furthermore, all particles bound pDNA very well and after electrophoretic mobility shift assay no pDNA could penetrate out of the particles. For now, we cannot draw any conclusions from the obtained results, regarding the effect of integrated amino acids and their sequences. However, the results of many published studies indicate the beneficial effects that might appear in further in vitro and in vivo evaluations.
|
