Structure-activity relationship of modified amphiphilic cationic cyclodextrins for enhanced siRNA delivery

The presence of multiple hydroxyl groups, at positions C2, C3 and C6 on the cyclodextrin (CD) ring structure allows for extensive functionalisation, enabling the development of biomaterials with significant potential for therapeutic siRNA delivery. To identify structural modifications that enhance activity, a range of cationic amphiphilic CDs, including both β- and γ-CDs, were synthesised, compared and evaluated. Each CDs incorporated a C₁₂ lipid chain on the primary face of the CD. On the secondary rim, at positions C2 and C3, either primary or tertiary amine groups with different pKa values were introduced via either a triazole or a thiopropyl linker. Nanoparticles (NPs) encapsulating siRNA were formulated at mass ratios 7.5:1 and 10:1 (CD:siRNA) and their physicochemical properties evaluated. A comparative in vitro study was conducted to assess the gene silencing efficacy of these NPs using the luciferase reporter gene in A549-luc cells. Gene silencing levels for both β- and γ-CDs increased when modified with a primary amine compared to a tertiary amine group at position C2. Gene expression inhibition was further improved when the CDs were functionalized with amine functionalities at positions C2 and C3. Modification of the secondary side of γ-cyclodextrins with two sets of primary amine functionalities via a thiopropyl linker, as compared to a triazole linker, achieved up to 80% gene knockdown, regardless of dose. In conclusion, the structure-activity relationship study successfully identified CD modifications that enhance gene silencing efficacy.