Acid-responsive polysaccharide derivatives: Smart particles for therapeutic applications
Zusammenfassung der Projektergebnisse
RNA interference, a technique wherein double stranded, short interfering RNA (siRNA) is used to degrade mRNA before it is translated, holds promise for treating many diseases that are otherwise “undruggable” with current technology. However, the widespread use of siRNA (or shRNA plasmid producing siRNA) for disease prevention and treatment requires the development of clinically suitable, safe, and effective drug delivery vehicles. An acidsensitive acetal-modified dextran (ac-dex) was used as a platform to evaluate a micro- and nanoparticulate siRNA delivery system. In general, ac-dex particles provide biocompatibility, biodegradability, and tunable degradation rates. In order to expand the versatility of ac-dex particles for siRNA delivery, spermine-modified ac-dex was prepared by reductive amination. The grafting of spermine facilitated the preparation of siRNA-loaded particles using a double emulsion technique due to electrostatic interactions between the slightly cationic polymer and the negatively charged genes. Cell experiments were performed with a HeLa cell line stably expressing firefly luciferase and encapsulated siRNA (or shRNA plasmid DNA) knocking down the reporter protein firefly luciferase. The spermine-ac-dex particle provides protection of the encapsulated siRNA against chemical and enzymatic degradation. A positive surface charge of the particles ensures the uptake of particles due to binding to the negatively charged cell membrane. Once inside the cell, hydrolysis of the polymer in the acidic endolysosomal compartment allows the siRNA to be released from the particles. The amines of spermine aid in the transportation of the siRNA out of the endosomal compartments via buffering effects. Degradation of the particles is also expected to supplement the buffering capacity of the amines during endosomal escape by increasing the osmotic pressure and, thus, improve cytosolic release (endosomal escape). This combination of protection and endolysosomal release most likely contributes to the significant knockdown rates observed (60 to 90%) while showing no cytotoxic effects. The parameters influencing the transfection efficiency and the resulting knockdown were evaluated including gene- and particle concentration, blend materials, degradation rate, molar mass, and also other amines or linker groups. The delivery of siRNA revealed to be optimal using a spermine-ac-dex prepared by reductive amination without any further additives. A medium degradation rate should be chosen and molar masses from 6,000 to 60,000 g/mol as they are accessible for FDA approved dextran. In order to prevent aggregation during lyophilization, glucose acts as an efficient cryoprotectant. As a consequence, spermine-ac-dex expands the potential application of ac-dex based particles and represents a promising vehicle for siRNA and shRNA delivery.
