Multivalent dendritic polyglycerolamine with arginine and histidine end groups for efficient siRNA transfection.

The success of siRNA-based therapeutics highly depends on a safe and efficient delivery of siRNA into the cytosol. In this study, we post-modified the primary amines on dendritic polyglycerolamine (dPG-NH2) with different ratios of two relevant amino acids, namely, arginine (Arg) and histidine (His). To investigate the effects from introducing Arg and His to dPG, the resulting polyplexes of amino acid functionalized dPG-NH2s (AAdPGs)/siRNA were evaluated regarding cytotoxicity, transfection efficiency, and cellular uptake. Among AAdPGs, an optimal vector with (1:3) Arg to His ratio, showed efficient siRNA transfection with minimal cytotoxicity (cell viability ≥ 90%) in NIH 3T3 cells line. We also demonstrated that the cytotoxicity of dPG-NH2 decreased as a result of amino acid functionalization. While the incorporation of both cationic (Arg) and pH-responsive residues (His) are important for safe and efficient siRNA transfection, this study indicates that AAdPGs containing higher degrees of His display lower cytotoxicity and more efficient endosomal escape.

Optimized effective charge density and size of polyglycerol amines leads to strong knockdown efficacy in vivo.

RNA interference (RNAi)-based therapy extends the range of "druggable" targets beyond existing pharmacological drugs and enables the development of new treatment strategies for various diseases. A prerequisite are non-viral polyvalent gene delivery vectors capable for safe and effective siRNA delivery to cells in vivo allowing a broad clinical application. We synthesized hyperbranched polyglycerol amines (hPG amines) which varied in their charge density, multiplicity (absolute frequency of amine groups) and core size to successfully develop potent and safe siRNA transfer vectors. The characterization of hyperbranched polyglycerol amines with an invariable core size (8 kDa) but different amine loading revealed a correlation between the effective charge density and the transfection efficacy without impacting the cell viability in vitro. However, this correlation was not seen in tumor bearing mice in vivo treated with 8 kDa hPG amine-siRNA complexes. Improving the effective charge density and the multiplicity of amine functionalities by increasing the molecular weight (43 kDa) revealed comparable transfection efficacy in vitro but less toxic side effects after systemic administration in vivo compared to the respective hPG amine (8 kDa). In addition, in vivo delivery of 43 kDa hPG amine-siRNA-polyplexes in tumors resulted in a highly specific and significant knockdown effect. These findings demonstrate that hyperbranched polyglycerol amines with a balanced effective charge density, multiplicity and core size are promising gene delivery vectors for siRNA therapy which enable to address so far "undruggable" targets due to high tolerability and effective siRNA delivery.