Comparison of tyrosine-modified low molecular weight branched and linear polyethylenimines for siRNA delivery

Abstract

Polyethylenimines (PEIs) have been previously introduced for siRNA delivery. In particular, in the case of higher molecular weight PEIs, this is associated with toxicity, while low molecular weight PEIs are often insufficient for siRNA complexation. The tyrosine-modification of PEIs has been shown to enhance PEI efficacy and biocompatibility. This paper evaluates a set of tyrosine-modified low molecular weight linear or branched polyethylenimines as efficient carriers of siRNA. Complexation efficacies and biophysical complex properties were analyzed by zeta potential, dynamic light scattering and circular dichroism measurements as well as gel electrophoresis. Biological knockdown was studied in 2 D cell culture and 3 D ex vivo tissue slice air-liquid interface culture. The results demonstrate that siRNAs were able to form stable complexes with all tested polymers. Complexation was able to protect siRNA from degradation by RNase and to mediate target gene knockdown, as determined on the mRNA level and in PC3-Luc3/EGFP and HCT116-Luc3/EGFP expressing reporter cells on the protein level, using flow cytometry and confocal microscopy. The direct comparison of the studied polymers revealed differences in biological efficacies. Moreover, the tyrosine-modified PEIs showed high biocompatibility, as determined by LDH release and mitochondria integrity (J-aggregate assay) as well as caspase 3/7 (apoptosis) and H₂O₂ levels (ROS). In 3 D tissue slices, complexes based on LP10Y proved to be most efficient, by combining tissue penetration with efficient gene expression knockdown.

Keywords:

• Nanomaterials
• polyethylenimines
• siRNA delivery
• gene knockdown cytotoxicity
• polymeric nanoparticles

Acknowledgments

The authors are grateful to Magdalena Gapińska for expert confocal microscope maintenance. The authors are also grateful for funding by grants from the project “EUROPARTNER” of the Polish National Agency for Academic Exchange (NAWA).

Disclosure statement

The authors declare no conflict of interest.

Additional information

Funding

This work was supported by the grant from the Nacional Science Center of Poland ‘BEETHOVEN LIFE 1’ program, project no: 2018/31/F/NZ5/03454 and Deutsche Forschungsgemeinschaft [DFG; AI 24/21-1; AI 24/24-1].