Developing RNA-interference-based therapeutic approaches with efficient and targeted cytosolic delivery of small interfering RNA (siRNA) is remaining a critical challenge since two decades. Herein, a multifunctional transferrin receptor (TfR)-targeted siRNA delivery system (Tf&INF7) is designed based on siRNA complexes formed with the cationic lipo-oligoamino amide 454, sequentially surface-modified with polyethylene glycol-linked transferrin (Tf) for receptor targeting and the endosomolytic peptide INF7 for efficient cytosolic release of the siRNA. Effective Tf&INF7 polyplex internalization and target gene silencing are demonstrated for the TfR overexpressing tumor cell lines (K562, D145, and N2a). Treatment with antitumoral EG5 siRNA results in a block of tumor cell growth and triggered apoptosis. Tf-modified polyplexes are far more effective than the corresponding albumin- (Alb) or nonmodified 454 polyplexes. Competition experiments with excess of Tf demonstrate TfR target specificity. As alternative to the ligand Tf, an anti-murine TfR antibody is incorporated into the polyplexes for specific targeting and gene silencing in the murine N2a cell line. In vivo distribution studies not only demonstrate an enhanced tumor residence of siRNA in N2a tumor-bearing mice with the Tf&INF7 as compared to the 454 polyplex group but also a reduced siRNA nanoparticle stability limiting the in vivo performance.

Intracellular protein delivery presents a novel promising prospect for cell biology research and cancer therapy. However, inefficient cellular uptake and lysosomal sequestration hinder productive protein delivery into the cytosol. Here, a library of 16 preselected sequence-defined oligoaminoamide oligomers is evaluated for intracellular protein delivery. All oligomers, containing polyethylene glycol (PEG) for shielding and optionally folic acid as targeting ligand, manifest cellular internalization of disulfide-conjugated enhanced green fluorescent protein (EGFP). However, only a PEGylated folate-receptor targeted two-arm oligomer (729) containing both arms terminally modified with two oleic acids shows persistent intracellular protein survival and nuclear import of nlsEGFP (which contains a nuclear localization sequence) in folate-receptor-positive KB carcinoma cells, validating both effective endolysosomal escape and following subcellular transport. Furthermore, using ribonuclease A as a therapeutic cargo protein, among the tested oligomers, the oleic acid modified targeted two-arm oligomers exert the most significant tumor cell killing of KB tumor cells. An investigation of structure–activity relationship elucidates that the incorporated oleic acids play a vital role in the enhanced intracellular protein delivery, by promoting stable formation of 25–35 nm lipo-oligomer protein nanoparticles and by membrane-active characteristics facilitating intracellular cytosolic delivery.

For active cell targeting, viruses frequently capitalize on dual-receptor binding. With the intention to mimic this natural process, a dual peptide-based approach for targeting cancer cells was evaluated. For this purpose, sequence-defined pDNA binding oligo (ethane amino) amides containing a PEG chain with a peptidic targeting ligand at its distal end were applied. Integrin receptor-directed cyclic peptide cRGDfk, transferrin receptor-addressing peptide B6, and epidermal growth factor receptor-targeting peptide GE11 were used in the study in DU145 prostate cancer cells that express all three receptors. Dual-receptor targeted pDNA polyplexes were designed by combining two of the above ligands at various ratios, in order to find an optimal ligand combination. Two polycation/pDNA ratios of nitrogen/phosphate (N/P) 6 and 12 were tested. Dual targeting effects were most pronounced at the lower N/P ratio and found for all three combinations. Cell binding studies and pDNA transfections revealed GE11 plus B6 as the most potent combination. In general, a good correlation of cell binding with gene transfer was observed. Interestingly, GE11 peptide-based polyplexes-mediated bimodal cell association profiles. In contrast, B6 ligand, cRGD ligand, and dual-targeted polyplexes triggered more homogenous monomodal cell binding characteristics. © 2014 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 104:464–475, 2015

The optimization of synthetic carriers for gene transfer remains a major challenge. Cationic polymers such as polyethylenimine (PEI) often show increasing gene transfer activity with increasing molecular weight, but this favorable effect is accompanied by an undesired increase in cytotoxicity. Moreover, the polydispersity of polymers prevents accurate determination of optimum size. Herein we describe the step-by-step elongation of precise linear oligo(ethanamino) amides by making use of the artificial amino acid succinoyl-tetraethylene pentamine (Stp) for solid-phase-assisted synthesis. This procedure enabled us to identify the optimal oligomer Stp30-W (8.4 kDa) with a length of 30 Stp units, with which effective gene transfer occurs in the absence of cytotoxicity. The transfection efficiency of Stp30-W exceeded that of standard linear PEI (22 kDa) by sixfold; nevertheless, Stp30-W exhibited tenfold lower cytotoxicity. In addition to the lower molecular weight, the succinate spacer between the oligoamine units may also contribute to the favorable biocompatibility. The cytotoxicity of the cationic polymer PEI is a major concern for use as a carrier for gene delivery, so this comparison between linear PEI and the new Stp oligomers is particularly relevant.