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Starch-based nanoparticles are highly utilized in the realm of drug delivery taking advantage of their biocompatibility and biodegradability. Studies have utilized Quaternized starch (Q-starch) for small interfering RNA (siRNA) delivery, in which quaternary amines enable interaction with negatively charged siRNA, resulting in self-assembly complexation. Although reports present numerous applications, the demonstrated efficacy is nonetheless limited due to undiscovered cellular mechanistic delivery. In this study, a deep dive into Q-starch/siRNA complexes' cellular mechanism and kinetics at the cellular level is revealed using single-particle tracking and cell population level using imaging flow cytometry. Uptake studies depict the efficient cellular internalization via endocytosis while a significant fraction of complexes' intracellular fate is lysosome. Utilizing single-particle tracking, it is found that an average of 15% of cellular detected complexes escape the endosome which holds the potential for the integration in the cytoplasmatic gene silencing mechanism. Additional experimental manipulations (overcoming endosomal escape) demonstrate that the complex's disassembly is the rate-limiting step, correlating Q-starch's structure-function properties as siRNA carrier. Structure-function properties accentuating the high affinity of the interaction between Q-starch's quaternary groups and siRNA's phosphate groups that results in low release efficiency. However, low-frequency ultrasound (20 kHz) application may have induced siRNA release resulting in faster gene silencing kinetics., (© 2024 The Author(s). Small published by Wiley‐VCH GmbH.)

RNAi therapeutics is a powerful tool for treating diseases by sequence-specific targeting of genes using siRNA. Since its discovery, the need for a safe and efficient delivery system for siRNA has increased. Here, we have developed and characterized a delivery platform for siRNA based on the natural polysaccharide starch in an attempt to address unresolved delivery challenges of RNAi. Modified potato starch (Q-starch) was successfully obtained by substitution with quaternary reagent, providing Q-starch with cationic properties. The results indicate that Q-starch was able to bind siRNA by self-assembly formation of complexes. For efficient and potent gene silencing we monitored the physical characteristics of the formed nanoparticles at increasing N/P molar ratios. The minimum ratio for complete entrapment of siRNA was 2. The resulting complexes, which were characterized by a small diameter (~30 nm) and positive surface charge, were able to protect siRNA from enzymatic degradation. Q-starch/siRNA complexes efficiently induced P-glycoprotein (P-gp) gene silencing in the human ovarian adenocarcinoma cell line, NCI-ADR/Res (NAR), over expressing the targeted gene and presenting low toxicity. Additionally, Q-starch-based complexes showed high cellular uptake during a 24-hour study, which also suggested that intracellular siRNA delivery barriers governed the kinetics of siRNA transfection. In this study, we have devised a promising siRNA delivery vector based on a starch derivative for efficient and safe RNAi application., (Copyright © 2014 Elsevier B.V. All rights reserved.)