ABSTRACT
Double pH-responsive xenopeptides comprising polar ionizable succinoyl tetraethylene pentamine (Stp) motifs and lipophilic ionizable lipoamino fatty acids (LAFs) were recently found to efficiently transfect mRNA and pDNA at low doses. However, potency was often accompanied with cytotoxicity at higher doses. Insertion of bioreducible disulfide building blocks (ssbb) or non-reducible hydrophobic spacers between polar and apolar ionizable domains of LAF-Stp carriers should mitigate toxicity of xenopeptides. Carriers showed stable nucleic acid complexation and endosomal pH-dependent lytic activities, both of which were abolished after reductive cleavage of ssbb-containing carriers. For pDNA, U-shaped carriers with one Stp and two LAF units or bundle carriers with two Stps and four LAFs displayed highest potency. For mRNA, best transfection was achieved with bundle carriers with one Stp and four LAFs. Both the ssbb and hydrophobic spacer containing analogs displayed improved metabolic activity, reduced membrane damage, and improved cell growth. The ssbb carriers were most beneficial regarding living cell count and low apoptosis rates. Mechanistically, inserted spacers decelerated the transfection kinetics and altered the requirement of endosomal protonation. Overall, mRNA and pDNA carriers with improved biocompatibility have been designed, with their high potency illustrated in transfection of various cell lines including low passage number colon carcinoma cells.
ABSTRACT
A cationic, dendrimer-like oligo(aminoamide) carrier with four-arm topology based on succinoyl tetraethylene pentamine and histidines, cysteines, and N-terminal azido-lysines was screened for plasmid DNA delivery on various cell lines. The incorporated azides allow modification with various shielding agents of different polyethylene glycol (PEG) lengths and/or different ligands by copper-free click reaction, either before or after polyplex formation. Prefunctionalization was found to be advantageous over postfunctionalization in terms of nanoparticle formation, stability, and efficacy. A length of 24 ethylene oxide repetition units and prefunctionalization of ≥50% of azides per carrier promoted optimal polyplex shielding. PEG shielding resulted in drastically reduced DNA transfer, which could be successfully restored by active lectin targeting via novel GalNAc or mannose ligands, enabling enhanced receptor-mediated endocytosis of the carrier system. The involvement of the asialoglycoprotein receptor (ASGPR) in the uptake of GalNAc-functionalized polyplexes was confirmed in the ASGPR-positive hepatocarcinoma cell lines HepG2 and Huh7. Mannose-modified polyplexes showed superior cellular uptake and transfection efficacy compared to unmodified and shielded polyplexes in mannose-receptor-expressing dendritic cell-like DC2.4 cells.
