Cyclodextrins (CDs) are cyclic oligosaccharides that offer a highly tuneable platform for drug delivery applications, owing to their unique toroidal architecture and modifiable primary and secondary hydroxyl groups. Due to these distinctive features, CDs have emerged as promising non-viral vectors for the delivery of nucleic acid therapeutics, including antisense oligonucleotides (ASOs) and small interfering RNA (siRNA). In this study, we evaluate and compare the physicochemical and biological performance of β-cyclodextrin (β-CD)-based monomer, dimers and cationic polymers engineered for siRNA delivery. The constructs include a head-to-head β-CD dimer linked via a triazole moiety, as well as two β-CD-based polymers functionalized with either quaternary ammonium (QA-polymer) or primary amine (PA-polymer) groups, with degrees of substitution of 3.5 and 7, respectively, for the cationic functionalities. Although the monomer and dimer did not show siRNA complexation under the tested conditions, both QA- and PA-polymers efficiently complexed siRNA, as confirmed by agarose gel, forming stable nanoparticles (NPs) in the 150-200 nm size range at polymer:siRNA mass ratios (MR) of 5:1, 7.5:1, and 10:1. Dynamic light scattering (DLS) revealed narrow size distributions (150 - 200 nm, PDI 0.2 for QA-polymer-NP and 0.4 for PA-polymer-NP) and zeta potentials in the range of 26-37 mV, indicating favourable colloidal stability. Long-term storage at 4 °C and freeze-drying (without the use of cryoprotectants) followed by resuspension demonstrated superior stability of PA-polymer NPs compared to their QA-polymer counterparts. In vitro evaluation, using A549-luciferase (A549luc) lung carcinoma cells showed that both polymeric carriers were non-toxic at working concentrations (20 pmol siRNA/well), maintaining ≥80 % cell viability as demonstrated by CellTiter-Fluor™ Cell Viability Assay. Cellular uptake studies with flow cytometry indicated minimal internalization for the QA-polymer, whereas the PA-polymer achieved up to 40 % uptake, resulting in approximately 40 % gene knockdown, as assessed by ONE-Glo™ EX Luciferase Assay. These findings underscore the influence of polymer architecture and surface charge chemistry on cellular interaction and gene silencing efficiency, with the enhanced performance of the PA-polymer potentially attributable to its protonatable amines and their capacity to promote endosomal escape.
