Tobramycin crosslinking improves the colloidal stability of arginine chitosan biodynamers for safe and efficient siRNA delivery

We previously reported proteoid biodynamers to form dynamic nanocomplexes (dynaplexes, DPs) with siRNA and their efficient transfection via dynamic responsive changes in endosomal environments. However, their limited colloidal stability requires chemical crosslinking, which may cause toxicity. To address these limitations, we designed biodynamers using positively charged aldehyde chitosan (ACh) as the backbone, which was functionalized with arginine hydrazide (Arg) and/or tobramycin (Tob) to create either Arg-ACh, Tob-ACh or Arg/Tob-ACh, respectively. While the additional positive charges introduced by Arg improved the siRNA complexation, the multiple amine groups in Tob acted as internal crosslinkers. Additionally, the resulting siRNA-loaded DPs were coated with hyaluronic acid (HA), which further enhanced colloidal stability. Compared to proteoid biodynamers, these novel functionalized chitosan biodynamers provided adequate gene silencing efficiency without the need for potentially harmful additional crosslinkers.