Unravelling the role of individual components in pBAE/polynucleotide polyplexes in the synthesis of tailored carriers for specific applications: on the road to rational formulations.

Oligopeptide end-modified poly(β-amino ester)s (OM-pBAEs) offer a means for the effective implementation of gene therapeutics in the near future. A fine-tuning of OM-pBAEs to meet application requirements is achieved by the proportional balance of oligopeptides used and provide gene carriers with high transfection efficacy, low toxicity, precise targeting, biocompatibility, and biodegradability. Understanding the influence and conformation of each building block at molecular and biological levels is therefore pivotal for further development and improvement of these gene carriers. Herein, we unmask the role of individual OM-pBAE components and their conformation in OM-pBAE/polynucleotide nanoparticles using a combination of fluorescence resonance energy transfer, enhanced darkfield spectral microscopy, atomic force microscopy, and microscale thermophoresis. We found that modifying the pBAE backbone with three end-terminal amino acids produces unique mechanical and physical properties for each combination. Higher adhesion properties are seen with arginine and lysine-based hybrid nanoparticles, while histidine provides an advantage in terms of construct stability. Our results shed light on the high potential of OM-pBAEs as gene delivery vehicles and provide insights into the influence of the nature of surface charges and the chemical nature of the pBAE modifications on their paths towards endocytosis, endosomal escape, and transfection.