Gene delivery is a powerful and promising therapeutic tool for treating a wide range of diseases. To ensure the safe and efficient delivery of the genetic material and to prevent degradation, the use of cationic dendritic vectors is particularly popular because of their ability to complex negatively charged genetic material and to form polyplexes. Branched cationic polymers, also called dendrimers, like poly(amidoamine) (PAMAM) have already demonstrated promising results in gene delivery. However, PAMAM has also been associated with cytotoxic effects and heterogeneity. To overcome these limitations, in this thesis, four well- defined polylysine-based dendrons with different functional groups have been synthesised in a reproducible manner using solid-phase peptide synthesis. Targeted gene therapy is a valuable tool for the more specific treatment of cancer or other diseases without the destruction or damage of healthy cells. In this work, as a proof of concept, cancer and liver cells were investigated as targets. For liver targeting, the large surface protein (LSP) derived from hepatitis virus B and its preS1 binding domain, as well as apolipoprotein A1 (ApoA1), which is part of the high-density lipoprotein, were chosen as liver specific targeting moieties. The epidermal growth factor (EGF) was used as a cancer targeting ligand. To avoid cross-linking and to obtain a homogeneous platform, ligand plasmids were designed to have only a single conjugation site. Two ligand variants were successfully recombinantly expressed in Escherichia coli Rosetta (K-EGFRR) and NiCo21 (C-ApoA1) with a yield of 4–6 mg/L expression approach. The ligand-cell interaction was evaluated by flow cytometry and fluorescence microscopy while the ligand-receptor interaction was evaluated by enzyme-linked immunosorbent assay and surface plasmon resonance. The affinity of the EGF variant was even higher than that of wild-type EGF (KD: 5.9 vs 7.3 nM). In addition, heterobifunctional polyethylene glycol linkers were attached to the ligands to allow orthogonal click conjugation to a cargo of choice, in this case the synthesised dendrons. The dendrons were polyplexed with plasmid DNA encoding for enhanced green fluorescence protein (eGFP) or luciferase at different N (dendron amines)/P (DNA phosphates) ratios (0.5–16). In addition, K-EGFRR was either electrostatically incorporated into the polyplex or covalently bound by strain promoted azide-alkyne cycloaddition using different EGF/DNA weight/weight ratios (0.1, 1, 10). The polyplexes were analysed by dynamic light scattering (size: 270–1412 nm and polydispersity index: 0.13–0.99). Transfection efficiency was assessed by measuring the expression levels of eGFP or luciferase. As most of the DNA cargos are trapped and degraded inside endolysosomes, the saponin SO1861 (0.4 μM and 4 μM) was added as an endosomal escape enhancer to the cells either alone (SO1861) or in targeted form conjugated to K-EGFRR (SO1861-EGF). While the polyplexes containing the newly synthesizing polylysine dendrons did not show successful transfection of genetic material, it was demonstrated that PAMAM was able to do so and that the efficiency of PAMAM polyplexes was increased 6-fold by using SO1861-EGF.
