Guanidinylated polymers enable safe and efficient dsRNA delivery in plant cells

The widespread use of chemical pesticides in crop protection poses serious environmental risks and has been associated with adverse effects to human health. RNA interference (RNAi) offers a sustainable alternative by enabling species-selective gene silencing through double stranded RNA (dsRNA). However, the practical application is hindered by the instability of dsRNA in vivo and inefficient cellular internalization. To address these challenges, we synthesized a small library of twelve polymethacrylate-based polymers (PAEMA and PGUMA) varying in molar mass (MM) and degree of substitution (DS). Comprehensive structural characterization by ¹H NMR, FT-IR, and SEC confirmed the tunability of MM and DS. Biophysical assays, including nucleic acid condensation, DLS, and z-potential measurement showed efficient formation of small, positively charged polyplexes at low N/P ratios. Stability studies revealed that polymers with higher MM and DS provided superior protection against enzymatic degradation, UV, and alkaline conditions. Cytotoxicity assays in Arabidopsis and tobacco cell cultures indicated that guanidinylated polymers, particularly those with higher DS, exhibited reduced toxicity profiles. Cellular uptake experiments further showed that high DS polymers enhanced delivery efficiency. Furthermore, gene silencing assays in GFP-expressing plant cells demonstrated that siRNA delivered with high MM and DS polymers induced transient gene silencing, outperforming naked siRNA. These findings highlight the potential of guanidinylated polymethacrylates as effective RNAi delivery vehicles in plants, offering a favorable balance between polyplex stability, biocompatibility, and delivery efficiency.