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.
The authors acknowledge the financial support received from MINECO (grant RTI2018-094734-B-C22 and BIGDATASPM PID2019-110210GB-I00), MICIN (grant RTC2019-007260-1), MCIN/AEI /10.13039/501100011033/FEDER, UE (grant PID2021-125910OB-I00) and Generalitat de Catalunya (2017 SGR 1559). A. D-P. received funding from the postdoctoral fellowships program Beatriu de Pinós, funded by the Secretary of Universities and Research (Government of Catalonia), and from the Horizon 2020 program of research and innovation of the European Union under the Marie Skłodowska-Curie grant agreement No 801370. S. G. thanks the Instituto de Salud Carlos III (ISCIII) (CB06/01/1058). CIBER BBN is an initiative funded by the VI National R + D + I Plan 2008–2011, Iniciativa Ingenio 2010, Consolider Program, CIBER actions and financed by ISCIII with assistance from the European Regional Development Fund. The authors want to thank NANBIOSIS ICTS and the Nanostructured Liquid Characterization Unit (U12) for the characterization of materials by high-resolution optical microscopy with spectral analysis. Laura Olmo and Maria Stampa Lopez-Pinto are acknowledged for polymer synthesis. Maria Stampa Lopez-Pinto and Nil González-Rios are acknowledged for the NMR characterization of the polymers. The Nanothemper team is also acknowledged for their complimentary sample processing data of the NPs binding affinity results.