s Gene Transfer with Sequence-Defined Oligo(ethanamino)amides Bioreducibly Attached to a Propylenimine Dendrimer Core

The design of precise and biodegradable gene transfer carriers is one important aim in gene therapy. Here we describe a strategy where we combine sequence-defined oligomers with precise bioreversible coupling to a multivalent dendrimer core. The dendrimer polypropylenimine of generation 2 (PPI G2) was chosen as a core and its eight primary surface amines were modified with 3-nitro-2-pyridinesulfenyl (Npys) modified cysteine. Npys-cysteines react avidly with free thiols forming bioreducible disulfide bonds. By these means, the activated PPI core molecule can be coupled in a directed manner in solution to eight molecules of cysteine-containing sequence-defined oligomers synthesized by solidphase supported synthesis. As proof of concept, oligomers comprising a C-terminal cysteine and one to five succinoyltetraethylene pentamine (Stp) units were conjugated to the dendrimer core. The resulting conjugates were evaluated for plasmid DNA (pDNA) delivery with regard to their biophysical and biological properties. Nanosized polyplexes stable in 90% serum were formed with higher cellular internalization levels of Stp modified conjugates in comparison to unmodified PPI G2. Consistent with protonation capacity at early endosomal pH and endosomal release, enhanced nuclear association of polyplexes and luciferase reporter gene expression was observed, correlating with increasing numbers (≥2) of Stp units per dendrimer arm, revealing PPI-Stp5 conjugate as the most promising conjugate. The new biodegradable conjugates possessed substantially lower cytotoxicity than PPI G2. Intravenous tail-vein injection of PPI-Stp5/pDNA polyplexes as compared with PPI G2 polyplexes revealed superior transgene expression in subcutaneous tumors of mice, but lower gene expression in the lung and liver.