Nanoparticle and targeted systems for cancer therapy.

The lack of safe and efficient gene-delivery methods is a limiting obstacle to human gene therapy. Synthetic gene-delivery agents, although safer than recombinant viruses, generally do not possess the required efficacy. In recent years, a variety of effective polymers have been designed specifically for gene delivery, and much has been learned about their structure–function relationships. With the growing understanding of polymer gene-delivery mechanisms and continued efforts of creative polymer chemists, it is likely that polymer-based gene-delivery systems will become an important tool for human gene therapy.

Design and development of polymers for gene delivery

Drug delivery strategy utilizing conjugation via reversible disulfide linkages: role and site of cellular reducing activities

Poly(ethylenimine) and its role in gene delivery

We investigated the in vitro and in vivo properties of DNA/transferrin-polyethylenimine (800 kDa) complexes before and after covalent coupling of poly(ethylene glycol) (PEG). Upon incubation with plasma, the positively charged non-PEGylated DNA complexes form aggregates. Plasma proteins such as IgM, fibrinogen, fibronectin and complement C3 were found to bind to non-PEGylated DNA complexes. At DNA concentrations relevant for in vivo gene delivery a strong aggregation of erythrocytes was also observed. PEGylation of the complexes strongly reduces plasma protein binding and erythrocyte aggregation. Furthermore, PEGylated complex size was stabilized and had a reduced surface charge. Prolonged circulation in the blood of the PEGylated complexes was also observed when injected intravenously. In tumor bearing mice, application of non-PEGylated complexes through the tail vein resulted in reporter gene expression in tail and lung, but severe toxicity was observed in some mice. In contrast, PEGylated complexes mediated reporter gene transfer to the tumor without significant toxicity.

PEGylated DNA/transferrin–PEI complexes: reduced interaction with blood components, extended circulation in blood and potential for systemic gene delivery

Recently the high transfection potential of the cationic polymer polyethylenimine (PEI) was described (Boussif O et al. Proc Natl Acad Sci USA 1995; 92: 7297-7301). To combine the promising DNA delivering activity of PEI with the concept of receptor-mediated gene delivery, cell-binding ligands (transferrin or antiCD3 antibody) were incorporated by covalent linkage to PEI. DNA complexes of PEI or ligand-PEI conjugates were tested for transfection of cultured neuroblastoma Neuro 2A cells, melanoma B16 or H225 cells, erythroid leukemic K562 cells and T cell leukemia Jurkat E6.1 cells. Depending on the cell line, incorporation of the cell-binding ligand resulted in an up to 1000-fold increased transfection efficiency. This activity depends on ligand-receptor interaction and was observed also at low PEI cation:DNA anion ratios where ligand-free PEI lacks efficiency. Depending on the cell-binding ligand, specific targeting (CD3 antibody, Jurkat cells) can be achieved. Gene transfer can be augmented by the addition of an endosome-destabilizing influenza peptide, but is not dependent on the presence of additional endosomolytic agents. Application of transferrin-PEI for the production of murine interleukin-2 in B16 cells resulted in exceptionally high secretion rates of 19 micrograms IL-2 protein per 10(6) cells per 24 h.

Maria Buchberger

Papers

Coupling of cell-binding ligands to polyethylenimine for targeted gene delivery

Functional maturation of dendritic cells by exposure to CD40L transgenic tumor cells, fibroblasts or keratinocytes.

Xenogenization by tetanus toxoid loading into lymphoblastoid cell lines and primary human tumor cells mediated by polycations and liposomes